Ecotoxicology of Marine Organisms

Editors
Bernardo Duarte
and
Isabel Caçador
MARE – Marine and Environmental Sciences Centre
Faculty of Sciences of the University of Lisbon
Campo Grande, Lisbon
Portugal

p,
p,
A SCIENCE PUBLISHERS BOOK
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Library of Congress Cataloging-in-Publication Data

Names: Duarte, Bernardo, 1983- editor.
Title: Ecotoxicology of marine organisms / editors, Bernardo Duarte and
Isabel Caçador, MARE (Marine and Environmental Sciences Centre),
Faculty of Sciences of the University of Lisbon, Campo Grande, Lisbon,
Portugal.
Description: Boca Raton : CRC Press, Taylor & Francis Group, [2019] | “A
science publishers book.» | Includes bibliographical references and
index.
Identifiers: LCCN 2019027243 | ISBN 9781138035492 (hardcover)
Subjects: LCSH: Marine organisms--Toxicology. | Environmental toxicology.
Classification: LCC QH91.5 .E26 2019 | DDC 578.77--dc23
LC record available at https://lccn.loc.gov/2019027243

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 Acknowledgements

The Editors would like to thank all the authors for their important contributions.
The Editors would also like to acknowledge the effort and help provided by the
Reviewers.
The Editors would also like to thank Fundação para a Ciência e Tecnologia
(FCT) for supporting the Editors activity throughout the projects PTDC/CTA-
AMB/30056/2017 (OPTOX), UID/MAR/04292/2013 and SFRH/BPD/115162/2016.
The Editors would also like to thank to Miguel P. Pais, Sofia Henriques and
António Teixeira for their kind donation of the photos present in the cover.
 Preface

Open oceans, coastal zones and transitional waters are key areas for the planet
ecosystem functioning, mostly due to its high productivity and reclining ability.
For decades, these ecosystems have been threatened by multiple stressors, such as
climate change, habitat displacement, bioinvasions, eutrophication and contaminant
disposal. These stressors consequently have severe impacts on all marine organisms,
from bacteria and microalgae to fishes and top predators.
Over recent decades the world has experienced the adverse consequences of
uncontrolled development of multiple human activities (Gavrilescu et al. 2015).
Emerging pollutants include a wide range of man-made chemicals (such as pesticides,
cosmetics, personal and household care products, pharmaceuticals), which are of
worldwide use (Thomaidis et al. 2012). Recent EUROSTAT statistics published in
2013 showed that between 2002 and 2011, over 50 percent of the total production
of chemicals is composed by environmentally harmful compounds, of which over
70 percent have significant environmental impact. It is also striking that the pace
of chemical discovery is growing rapidly, with the Chemicals Abstracts Service
(CAS REGISTRY) reporting in May 2011 the registration of the 60 million chemical
substance. Following CAS REGISTRY 50 million substance registration in only
2009, this second major milestone highlights the continued acceleration of synthetic
chemical innovation globally (Gavrilescu et al. 2015). The relationship between
increased human population density and environmental change in coastal regions
is well known. Coastal water is the ultimate sink for sewage and other by-products
of human activities. The EU’s Task Group recommended that the assessment of
achievement of “Good Environmental Status” under Descriptor 8 should be based
upon monitoring programs covering the concentrations of chemical contaminants
and also biological measurements relating to the effects of pollutants on marine
organisms. They also concluded that the combination of conventional and newer,
effect-based, methodologies, with the assessment of environmental concentrations
of contaminants provides a powerful and comprehensive approach.
Ecotoxicology emerged from this need to understand how contaminants affect
the different organisms. The term ecotoxicology was coined by René Truhaut in 1969
who defined it as “the branch of toxicology concerned with the study of toxic effects,
caused by natural or synthetic pollutants, to the constituents of ecosystems, animal
(including human), vegetable and microbial, in an integral context” (Truhaut 1977).
This becomes particularly important if focusing marine ecosystems and organisms,
highly prone to contaminant inputs.
vi Ecotoxicology of Marine Organisms

Most of the previous publications (with some rare exceptions) focused on

terrestrial or freshwater, rather than marine ecosystems. Moreover, the already
existing publications are mostly concerned with traditional contaminants and model
organisms. Thus, the editors of this book saw the need for a book dedicated to
Marine Ecotoxicology focusing not only non-model but key marine organisms (from
microalgae, to seagrasses, fishes, invertebrates and marine reptiles) and the effects
of several contaminant classes (trace elements, metalloids, pharmaceutical residues,
biotoxins, etc.) on these organisms. With this, the editors intend to provide reference
and recent information that can be used by undergraduate and postgraduate students,
but also by senior researchers, particularly related to future research needs.
This book, which is divided into nine chapters, was developed with the
cooperation and input of several senior researchers’ experts in ecotoxicology. The
chapters in this book proceed address different levels of organism complexity and
different contaminant addressing the wider scope possible.
The Editors hope that you, the readers, learn as much as we did from these nine
chapters and use the knowledge provided herein to further advance the science of
marine ecotoxicology.

References
Gavrilescu, M., Demnerová, K., Aamand, J., Agathos, S. and Fava, F. 2015. Emerging pollutants
in the environment: Present and future challenges in biomonitoring, ecological risks and
bioremediation. N. Biotechnol. 32: 147–156. https://doi.org/10.1016/j.nbt.2014.01.001.
Thomaidis, N.S., Asimakopoulos, A.G. and Bletsou, A.A. 2012. Emerging contaminants: A tutorial
mini-review. Glob. Nest J. 14: 72–79.
Truhaut, R. 1977. Ecotoxicology: Objectives, principles and perspectives. Ecotoxicol. Environ. Saf.
1: 151–173.

Bernardo Duarte and Isabel Caçador

MARE - Marine and Environmental Sciences Centre
Faculty of Sciences of the University of Lisbon
Campo Grande 1749-016 Lisboa, Portugal
 Contents

Acknowledgements iii
Preface v
1. Environmental Disturbance Caused by Stressors: Changing the 1
Focus from Individuals to Habitats
Cristiano V.M. Araújo, Matilde Moreira-Santos and Rui Ribeiro
2. Toxicity of Rare Earth Elements to Marine Organisms 16
Pedro Luís Borralho Aboim de Brito
3. Chemical Contaminants in a Changing Ocean 25
Ana Luísa Maulvault, Patrícia Anacleto, António Marques, Rui Rosa
and Mário Diniz
4. Effects of Harmful Algal Bloom Toxins on Marine Organisms 42
Lopes, V.M., Costa, P.R. and Rosa, R.
5. Overview of Phytoplankton Indicators and Biomarkers as 89
Key-Tools for Trace Element Contamination Assessment in Estuaries
Maria Teresa Cabrita, Bernardo Duarte, Carla Gameiro, Ana Rita Matos,
Isabel Caçador and Rita M. Godinho
6. Phytoremediation and Removal of Contaminants by Algae and 128
Seagrasses
Bouchama Khaled, Rouabhi Rachid and Bouchiha Hanen
7. Ecotoxicology of Pharmaceuticals in Coastal and Marine Organisms 158
Vanessa F. Fonseca and Patrick Reis-Santos
8. Mercury Exposure, Fish Consumption and Provisional Tolerable 185
Weekly Intake: An Overview
Vieira, H.C., Soares, A.M.V.M., Morgado, F. and Abreu, S.N.
9. Contaminants Impact on Marine Turtle Populations Development: 205
An Overview
Patricia Salvarani, Vania C. Foster, Jaime Rendon and Fernando Morgado
Index 235
About the Editors 237
Color Plate Section 239
1 Environmental Disturbance
Caused by Stressors
Changing the Focus from Individuals
to Habitats
Cristiano V.M. Araújo,1,2,* Matilde Moreira-Santos2
and Rui Ribeiro2

INTRODUCTION
The recognition that contamination is a worldwide problem and that a great number
of ecosystems suffer some level of (anthropogenic) disturbance gave rise to
environmental risk assessment (ERA) schemes and resulting environmental decisions
to be strongly based on ecotoxicological assays, designed to accurately evaluate the
effects of the contaminants on organisms (Luoma 1996, Sherman 2000). Therefore,
although the concept of environmental vulnerability needs to be expanded to long-
term effects on ecosystems (De Lange et al. 2010), the traditional ecotoxicological
approach, including mesocosm studies, is focused on how toxic a compound or an
environmental sample is; and toxicity is assayed, that is, measured, from biological
responses at the individual and, in mesocosm studies, at higher levels (Chapman 1995,
Schmitt-Jansen et al. 2008). The fundamental assumption sustaining this approach
is that organisms in ecosystems are directly exposed to contamination and that,
after a specific exposure period, a biological effect is produced. The methodological
procedures to perform such ecotoxicological assays consists in exposing organisms
to compound(s) or environmental sample(s) in a forced exposure test system, in
which organisms are continuously in contact with the stressful agent. However,

1
Institute of Marine Sciences of Andalusia (CSIC), Department of Ecology and Coastal Management,
Puerto Real, Spain.
2
CFE-Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra,
Portugal.
Emails: [email protected]; [email protected]
* Corresponding author: [email protected]
2 Ecotoxicology of Marine Organisms

considering that laboratory ecotoxicological assays intend to simulate the real field
situation, by using a forced exposure system in which organisms are mandatorily
exposed to contaminants, their natural biological possibility to escape from a toxic
effect is not taken into account (Lefcort et al. 2004).
It is a fact that a scenario of forced exposure to contamination under a real situation
is unavoidable for organisms with no ability to move, neither actively nor passively
(e.g., drift). However, theoretically, the same is not expected for mobile organisms.
In fact, many studies have shown the ability of a great number of organisms to detect
environmental disturbers and escape towards less impacted environments (Araújo et
al. 2016a, Tierney 2016). To cite some few examples, species of amphipods (Kravitz
et al. 1999, Hellou et al. 2009), copepods (Ward et al. 2013, Araújo et al. 2014a),
cladocerans (Lopes et al. 2004, Rosa et al. 2008, 2012), snails (Hellou 2010, Araújo
et al. 2016b), decapods (Richardson et al. 2001, Araújo et al. 2016c), amphibians
(Araújo et al. 2014b,c), and fish (Pedder and Maly 1986, Moreira-Santos et al. 2008,
Araújo et al. 2014d) were able to avoid disturbances caused by various types of
contamination. However, contrarily to these observations, some field studies have
reported that under particular circumstances, organisms seem to prefer inhabiting
disturbed areas, rather than avoiding them. For instance, along a mercury gradient
in a shallow coastal lagoon in Portugal, higher density, biomass and growth
productivity of the estuarine mudsnail Peringia ulvae were observed in the area with
intermediate contamination, probably due to resource availability and presence of
refuges (Cardoso et al. 2013). Also, the euryhaline fish Gasterosteus aculeatus, when
exposed simultaneously to a predator chemical signal and toxic cyanobacteria chose
feeding in a potentially toxic environment to reduce the risk of predation (Engström-
Öst et al. 2006). In the same way, tilapia fry performed intermittent displacement
to previously avoidfish farming effluent concentrations when food availability was
increased (Araújo et al. 2016d). A study performed in the estuarine zones of New
South Wales (Australia) showed that fish larvae were most abundant in disturbed
and contaminated areas, probably due to nutrient enrichment (McKinley et al. 2011).
Most probably, in all the latter cases, the final choice for inhabiting the disturbed
habitat was headed by the balance between avoiding the exposure to contamination
or staying in the disturbed habitat to avoid the most aversive/costly environment
(e.g., higher predation risk, limited food resources).
The non-forced exposure approach allows a new vision about the role of
contaminants as habitat disturbers within scenarios where toxic effects at the
individual level are not necessarily expected to occur. In this context, the present
chapter aims to show how risky environmental disturbances can be, if the
focus encompasses also habitats, particularly regarding stressor-driven spatial
displacement. Firstly, since the traditional ecotoxicological methods used to assess
the toxic effects of contaminants on organisms do not consider this approach, we
present an exposure system that allows changing the exposure paradigm: the free-
choice, non-forced exposure system. Secondly, we discuss the ecological relevance
of the spatial avoidance response for assessing the role of the contaminants as habitat
disturbers. Then, an additional very important view-point addressed and discussed
here is how ecotoxicological assays can contribute to assess the habitat selection
process (avoidance/preference) under scenarios in which the co-occurrence of
 Environmental Disturbance Caused by Stressors 3

contamination and other aversive or attractive factors play a crucial role for organisms
to decide whether to move or not from the contaminated habitat. A brief discussion
with reference to moribundity (stupefaction), here defined as the loss of the ability of
organisms to detect and avoid contamination, is also provided as contaminants can
also impair the organism mobility, which in turn can slow down or even prevent their
spatial avoidance. Moreover, the results of a short experiment with marine shrimps
to assess the effect of post-larval stage on avoidance response are presented. Given
that species dispersion patterns in contaminated areas may result, not only from
avoidance, but also from preference responses (Van den Brink 2008), recolonization
is, therefore, an essential response for the recovery of disrupted habitats, which has
been neglected in ecotoxicological studies, but that can potentially be incorporated
into ERA schemes by using the non-forced exposure system. In this context, the
importance of understanding to what extent contaminants regulate organisms’
displacement to avoid contaminant-disturbed habitats and recolonize recovering
habitats and the inclusion of both responses in ERA, to increase ecological realism
and decrease uncertainties in environmental decision making, are also discussed. In
this chapter we did not consider avoidance measured as impairment in the ability
to move, nor studies in which free-choice was restricted to two compartments (one
treated and one control). Only studies using a free-choice, gradient, non-forced
exposure multi-compartmented system were considered.

A Free-choice, Gradient, Non-forced Exposure System

As previously commented, ecotoxicological assays assume that organisms are
forcedly exposed to contamination and consequently, that the possibility to avoid
unfavourable environments is improbable. Indeed, traditional exposure systems
consist in confined environments used to verify the biological effect on organisms
and, in mesocosm studies, on communities and ecosystems resulting from a
continuous and forced exposure to the stressful agent. However, an alternative tool
to this approach, that allows organisms to select different environments, has been
recently developed: the free-choice, gradient, non-forced exposure system (Fig. 1).
This exposure system simulates a gradient or patches of contamination within a
chamber divided into different compartments, through which organisms can freely
move. In this non-forced exposure system, a physiological response cannot be directly
related to a given concentration, as organisms can move across all the different
concentrations or patches being assayed. However, two responses linked to habitat
selection, and thus species dispersion, can be measured: avoidance and recolonization.
An avoidance assay simulates the introduction of a stressor in the habitat inhabited
by a population, assuming that unobstructed connectivity with an undisturbed/clean/
reference habitat exists. Organisms are exposed to all levels of the stressor along
a gradient (from 0 percent—clean compartment—to 100 percent—the opposite
extremity of the system) and may respond by moving away from the most disturbed
compartments. Organisms are, thus, initially distributed along the interconnected
compartments in an uniform manner (equal number of organisms per compartment)
and their final spatial distribution, at the assay end, is registered, allowing one to
calculate the stressor intensity eliciting a fixed amount of avoidance, such as the
4 Ecotoxicology of Marine Organisms

Fig. 1. Schematic representation of a free-choice, non-forced static exposure system.

median avoidance contaminant concentration (i.e., the contaminant concentration

avoided by 50 percent of tested organisms; AC50). A recolonization assay simulates
a recovering habitat being made available to a population inhabiting a clean habitat.
A similar gradient of the stressor is present along the interconnected compartments,
but all the tested organisms are initially introduced in the clean extremity. Those
who are insensitive to the stressor will occupy adjacent compartments. Their final
spatial distribution, along the system, at the end of the assay, grant the possibility
of computing the stressor intensity which was undetected and, thus, colonized by
a given proportion of organisms, such as the median recolonization contaminant
concentration (i.e., the contaminant concentration undetected and colonized by 50
percent of tested organisms: RC50). These responses do not measure the effects of
contaminants at the physiological level, but rather at the habitat level, because the
free-choice, gradient, non-forced exposure system permits individuals to circumvent
toxic effects resulting from continuous exposure to the stressful agent (for more
details see next section).
To ensure that the system design does not influence the avoidance/recolonization
response of the organisms, two factors are of concern: (i) the maintenance through
the time of the linear or patchy gradient across the various compartments during
the entire exposure period and (ii) the absence of a preferential distribution of
the organisms when no contaminant (control) is present. The goal of the system
calibration stage is to ensure that, in the absence of organisms, the system is able
to maintain the different samples inside the interconnected compartments with a
minimal mixture. An easy method to calibrate the free-choice, non-forced system
is to establish a gradient of different concentrations of a reference compound and
assess if and how fast the mixture occurs. Using sodium chloride as the reference
compound, its concentration can be easily and accurately indirectly determined by
measuring conductivity directly inside each compartment with no necessity to take
samples; successive conductivity measurements along the selected exposure period
can be made to verify the pattern of mixture inside the system. This procedure
will allow verifying the maximum exposure period during which the system
maintains the established gradient differences across the compartments. Even if
the calibration indicates the capacity of the system to maintain the gradient across
the compartments, samples can always be taken at the end of the exposure period
to confirm the final concentration in each compartment. The temporal pattern of
contaminant homogenization will depend on the system architecture regarding the
degree of connectivity among compartments. Small distances and large interface
areas between adjacent compartments will dictate a faster mixing. Obviously, the
 Environmental Disturbance Caused by Stressors 5

mixture of the contaminant concentrations among compartments will be accentuated

when organisms are introduced into the system and it will be more intense the
more turbulence the organisms provoke, which is a function of number, speed,
size, shape, surface malleability and other hydrodynamical features. Therefore, the
exposure period may be different for each species, to be able to maintain the desired
contamination gradient at the end of the assay.
Regarding the test to prove the non-preferential distribution of the organisms
along the system, only control medium is used to fill the system, to verify the
possible occurrence of false positive avoidance/recolonization triggered by factors
(confounding factors) other than the contaminant(s) being assayed, such as non-
homogeneous temperature and electromagnetic radiation, including light, inclination,
noise and other mechanical vibrations. These confounding factors resulting from the
laboratory physical conditions can be evaluated and easily eliminated. Furthermore,
behavioural factors (e.g., social attraction, such as schooling or repulsion) can also
generate spatial distribution distortions, which can be compensated through load
decrease and/or replication increase. The former approach reduces the probability of
social interactions among individuals within each replicate, while the latter strategy
eliminates or, at least, strongly smoothes discrepancies among replicates when data
is pooled for the computation of the contaminant concentration eliciting a fixed
amount of avoidance.
For whole-sediment experiments, in which only effects caused by sediment
are targeted, different systems have been used, simulating contamination gradients
(Araújo et al. 2012) or patches (Araújo et al. 2016b), although the goals were similar.
Experiments tackling patchy distributions of sediment contamination have been
performed in dishes divided into fields, so that, in each field, a natural or spiked
sediment sample is deployed. To avoid desiccation, clean water should be carefully
introduced above the sediment. There is no separation among sediment samples, and,
thus, organisms can move freely among the fields.

Contaminants as Habitat Disturbers in Marine/Estuarine Environments

From way back, contaminants have been considered of concern due to the direct
effects they can cause on the health status of organisms. Various potential changes in
the health status of organisms have been proposed as test endpoints, to be considered
in the assessment of risks due to contamination (Newman and Unger 2001, Walker
et al. 2001). This rationale arises from the assumption that once contaminants are
discharged into ecosystems, organisms are exposed to them and, then, a passive
absorption process takes place. As a consequence of this absorption, a cascade of
effects is expected to start taking place, beginning with sub-organismal effects, such
as molecular alterations, and then effects succeed to the cell level, with metabolic and
physiological disruptions, and follow to reach the whole-population level (behaviour,
fecundity, etc.), and eventually community and ecosystem levels (Newman and
Unger 2001, Gerhardt 2007). Although the velocity and intensity (sensitivity) of the
response at high levels of biological organization can change in function of various
factors (e.g., contaminant’s mode of action, concentration, exposure time, species
detoxification ability), such traditional approaches generally assume that the contact
6 Ecotoxicology of Marine Organisms

between organisms and the contaminant is mandatory (Lefcort et al. 2004). This
assumption is correct for organisms that are not able to move, but if organisms with
mobile skills are able to detect aversive agents and interpret such information as
dangerous, it is expected that they try to avoid the stressful agent, escaping towards
more favourable habitats (Hellou 2010, Tierney 2016). This response, here called
avoidance, may prevent organisms to be continuously exposed to contamination
and, therefore, direct deleterious effects at the individual level lose relevance, i.e.,
ecological meaning.
The displacement of individuals escaping from contamination brings serious
consequences for the population, because avoidance consequences are similar to
the death of the organisms: populations may totally, or partially, disappear (Lopes
et al. 2004). Moreover, by triggering avoidance, contaminants can pose serious
environmental risks even when concentrations are not supposed to adversely affect
organisms’ physiology (Rosa et al. 2008). In this context, contaminants, together
with many other stressful agents, may act like habitat disturbers by triggering
avoidance responses that affect species distribution patterns. The most appropriate
way to verify the contaminant-driven spatial displacement of organisms is by using
the free-choice, non-forced exposure system. This novel exposure approach allows
for assessing how the spatial distribution of the organisms would be affected by
the presence of any stressful agent, either along a contamination gradient or in a
patchy contamination distribution (Araújo et al. 2016a). In a coastal habitat,
for instance, if contaminants are discharged from a specific point-source, it is
expected that a linear gradient contamination is formed. Therefore, the closer the
organisms are to the contaminant source, the higher the environmental risk; though
in time, as contamination is diluted with the dispersion, the repulsiveness caused
by contaminants will be reduced and ultimately disappears. Under such scenarios,
it is expected that organisms able to avoid contamination move to less disturbed
areas, in which effects caused by the exposure to contamination are minimal or
inexistent. Theoretically, the preferential spatial distribution of the organisms may
be determined by the contamination gradient, so that lesser biodiversity would be
expected where contamination is higher. Different studies with salmon (Salmo salar)
and trout (S. trutta) have shown that contamination can trigger an early downstream
migration (Saunders and Sprague 1967) and induce a preferential distribution, as the
fish avoid disturbed and contaminated habitats (Ǻtland and Barlaup 1995, Woodward
et al. 1995, Thorstad et al. 2005).
A contamination-driven spatial distribution can also occur in scenarios of patchy
gradients of contamination. If contaminants are not uniformly and linearly dispersed,
but rather form patches with different contamination levels, avoidance can occur
resulting in the changing of the spatial rearrangement of the population (Spromberg
et al. 1998). This is the typical contamination scenario expected for sediments, given
that a homogeneous distribution of the contaminant is not expected to occur, but
instead, patches with different contamination levels may be formed. These patches,
not only create inhabitable areas, but may also prevent the spatial displacement of
the organisms for large distances for less or even uncontaminated patches (Ares
2003). Recently, a few studies with coastal benthic species to assess the avoidance
response in whole-sediment avoidance assays were reported. In a first study, the
 Environmental Disturbance Caused by Stressors 7

estuarine mudsnail P. ulvae was exposed to a linearly-disposed contaminated

estuarine sediment by using a non-forced exposure approach (Araújo et al. 2012).
Four different contamination levels were tested from mixing different proportions
of test and reference sediment samples. Results showed that mudsnails avoided the
most contaminated sediment samples and were able to move towards the reference
sediment. The authors also observed that under extreme toxicity conditions, mudsnails
retracted into their shells and became inactive instead of actively moving to more
favourable locations along the contamination gradient. As discussed above, the
contaminant spatial distribution may be spatially heterogeneous (Swartz et al. 1982,
Lefcort et al. 2004), with the presence of uncontaminated sediment patches favouring
the rearrangement of a population that will use those patches as refuge zones (Swartz
et al. 1982). Motivated by this assumption, an experiment with the same mudsnail
species was designed, aiming to assess its ability to detect different contamination
levels in a heterogeneous contamination scenario with patchy sediment distribution
(Araújo et al. 2016b). It was confirmed that the organisms were able to recognize the
uncontaminated patches in that complex and heterogeneous scenario, changing their
spatial distribution in function of the uncontaminated sediment distribution.
Recently, the approach using the avoidance and recolonization responses was
employed to assess if the spatial distribution could be predicted by non-forced assays
(Araújo et al. 2017). Previous visual observations of the estuarine gastropod Olivella
semistriata along the Ecuadorian coast in Manta (Ecuador) indicated that organisms
were not found close to two zones of untreated effluent discharges. Then organisms
were exposed in the laboratory to different sediment samples and assessed by habitat
preference by using avoidance and recolonization responses. In general, the results
obtained indicated that the laboratory responses were in accordance with the spatial
distribution of the snails densities observed in the field.
To date, the vast majority of the studies on avoidance in non-forced exposure
systems have been carried out with freshwater species (Araújo et al. 2016a). Studies
using estuarine or marine species are scarce. Alongside P. ulvae and O. semistriata,
two other estuarine/marine species were investigated: whiteleg shrimp Litopenaeus
vannamei larval stages and cobia fish Rachycentron canadum fries were exposed to
a copper contamination gradient using a free-choice, gradient, non-forced exposure
system (Araújo et al. 2016c). Concentrations between 0.1 and 1.0 µg Cu L–1 caused
avoidance by around 80 percent of whiteleg shrimp larvae and by 50 percent of cobia
fries.

Avoidance Alongside Preference as Measurements of Habitat Selection

Although contamination can play a crucial role in the habitat selection process by
organisms, many other factors may influence an organism decision of escaping or
not to another environment. Understanding this process is of environmental concern;
however, once again, the forced exposure approach prevents this type of analysis
since the possibility of the organism moving away does not exist. Besides, the free-
choice, non-forced exposure system opens a complementary alternative to apply a
new approach that includes not only avoidance and recolonization responses, but
also habitat selection. An assay on habitat selection simulates the spatial arrangement
8 Ecotoxicology of Marine Organisms

of interconnected adjacent habitats with different characteristics including different

degrees of disturbance (e.g., water pH, food, suspended particles, sediment type,
predator chemical cues, salinity, anthropogenic compounds…), mimicking real
or potential field scenarios of a metapopulation. Field-collected samples, or their
laboratory imitations, are deployed along the system as spatially placed in the field.
A highly disturbed habitat may occur between two reference sites and constitute
a barrier against migration, configuring a habitat fragmentation outline. Such a
situation can be mimicked and its level of obstruction can be preliminarily evaluated
with this novel approach. Organisms are initially distributed along the interconnected
compartments in an uniform manner (equal number of organisms per compartment)
and their final spatial distribution, at the assay end, will no longer reflect, the mere
intensity of a specific fully-controlled stressor, but, instead, all the antagonistic and
synergistic cues that may be present.
If mobile organisms are able to detect contaminants and escape towards less
disturbed areas, then in an area with the different contamination levels it would
be expected a preferential spatial distribution of organisms in habitats with less
disturbance (such as found by Araújo et al. 2017). However, curiously, this expectation
does not always agree with the observed pattern of species distribution. In a shallow
coastal lagoon in Portugal with a mercury gradient, the mudsnail P. ulvae seemed to
prefer inhabiting an area with an intermediate contamination level (Cardoso et al.
2013). Similarly, in New South Wales, Australia, the highest abundance of fish larvae
was observed in the most contaminated estuaries (McKinley et al. 2011). Although
extraordinary, this behavior could be explained by the presence of other factors than the
contamination that attracts organisms to that particular habitat. For instance, the fish
G. aculeatus when exposed simultaneously to two different environments, one with a
chemical predator signal and the other one with toxic cyanobacteria, preferred feeding
in the potentially toxic environment to reduce the risk of predation (Engström-Öst
et al. 2006). Similarly, the fish Danio rerio when exposed to environmentally relevant
herbicide concentrations preferred to move into a plume of contaminants (mixture of
four herbicides) due to an attractive effect caused by herbicides (Tierney et al. 2011).
And recently, a study with tilapia fry (Oreochromis sp.) showed that the intensity of
the avoidance response to an effluent sample was reduced when food availability was
higher in the environments with higher contamination levels (Araújo et al. 2016d).
The possibility to assess the habitat preference selection process by organisms
in an ecotoxicological context changes the paradigm of the exposure focus from
individuals to habitats. The ecological risk posed by contaminants is not only linked
to the effects they can cause on organisms—contaminants as toxicants—but also to
the disturbance they can cause in the dynamic patterns of organisms migration—
contaminants as habitat disturbers—and thus on populations’ spatial distribution.
Moreover, this approach will contribute to understand the interactions between
contaminants and other environmental factors and, then, to assess how determining
such interactions can be for the habitat selection process.
 Environmental Disturbance Caused by Stressors 9

Avoidance and Ontogenesis

There is a curious problem associated with spatial active avoidance: From which
point, during ontogeny (early life stage, juveniles or adult), may avoidance play an
important role in preventing the population from being exposed to a stressor? The
same rationale applies to recolonization and habitat selection.
Although ecotoxicological assays are preferably performed with early life
stages as they are generally more sensitive, in the case of avoidance assays, this
supposition might not be true because the sensory system that detects contamination
can be underdeveloped in juvenile individuals and also because their moving ability
can be insufficient. Therefore, we performed studies to compare the differences in
the sensitivity of different post-larval stages of the estuarine whiteleg shrimp to
avoid contamination. Post-larvae of 5, 10, 20, 45, 60, and 80 days were exposed to
a copper gradient and responded in a different way, so that a slight trend to avoid
more intensively copper gradient was observed for larvae of 10 to 45 days. From
avoidance percentage values (Fig. 2), it was calculated by researchers that the copper
concentration that causes an avoidance by 50 percent of the exposed population

5 days 10 days
100 100
Avoidance (%)

80 80

60 60

40 40

20 20

0 0
0. 00
05
00
56

00

32
00

68

80

00
32

68
34

20
0.
2.
0.

1.

0.
0.

0.

0.

1.
1.

1.
0.

0.

20 days 45 days
100 100
Avoidance (%)

80 80

60 60

40 40

20 20

0 0
0. 0

0. 5
15
0. 7

50

90

30

10
80
20

45
0

1
0

3
0.

0.

0.
0.

0.

1.

1.

0.

1.
0.
0.

60 days 80 days
100 100
Avoidance (%)

80 80

60 60

40 40

20 20

0 0
0. 5
00

63

80
0. 5

70

30

10
20

80

90

20
00

1
1

4
0.
0.

0.

0.
0.

0.

0.

0.

1.
0.

0.

0.

Cu (mg L–1) Cu (mg L–1)

Fig. 2. Avoidance response of different post-larval stages (days) of the whiteleg shrimp Litopenaeus
vannamei exposed to a linear copper gradient in a free-choice, non-forced exposure system.
10 Ecotoxicology of Marine Organisms

Table 1. Values (in mg L–1) of AC50 (avoidance concentration for 50 percent of the exposed population)
and confidence intervals obtained for different post-larval stages (days) of the whiteleg shrimp Litopenaeus
vannamei exposed to a linear copper gradient.

Post-larval AC50 Confidence

stages (days) intervals
5 1.41 0.89–5.09
10 < 0.05 nc
20 < 0.08 nc
45 < 0.15 nc
60 0.86 0.66–1.32
80 0.40 0.27–0.62
nc: not calculated.

(AC50) and the values obtained are presented in Table 1. Our results showed that the
AC for larvae of five days was the highest one, indicating less sensitivity of this stage
to detect and avoid copper. Larvae of 60 and 80 days presented AC50 almost similar
values; however, the lowest AC50 values were recorded for larval stages of 10, 20
and 45 days (lower than the lowest concentration used). Although the mechanism
that can determine these differences was not studied, we hypothesized that younger
larvae (5 days) were less sensitive because they were not physiologically able to
detect as efficiently the copper gradient as did the larvae of 10, 20 and 45 days. On the
other hand, the lower sensitivity of the larvae of 60 and 80 days could be attributed
to the higher larval development that provides higher physiological tolerance to
copper contamination. Our results indicated that, contrary to the assumption used in
traditional toxicity assays, younger organisms are not necessarily more sensitive and,
therefore, more advisable to be used in avoidance assays. The age of the organisms
to be chosen should be carefully assessed using stages to ensure that they are neither
unable to detect contamination or to move away from it, nor that they are less
sensitive to the contamination.

Avoidance and Moribundity

At what concentrations does avoidance cease to play an appreciable role in
preventing toxicity? Many studies have treated avoidance as the loss of ability to
move, focusing on how contaminants impair or provoke changes in the patterns
of movements linked to swimming: velocity, direction and distance. In the non-
forced exposure system, this loss of ability to move is used to characterize a status
of moribundity that slows down or prevents spatial active avoidance. Moribundity,
therefore, can occur when the concentration of the contaminant is so high that severe
toxic physiological effects take place before the reaction to avoid it from being
triggered (e.g., contaminants can act as disruptors of the nervous system impairing
the ability of the organism to detect and escape from contamination). For instance,
moribundity was observed in the above-mentioned avoidance assay with fries of
the estuarine cobia fish, at concentrations of copper of 1.6 and 1.8 µg L–1 (Araújo
et al. 2016c). Also, stream macroinvertebrates of the genus Anomalocosmoecus
 Environmental Disturbance Caused by Stressors 11

exposed to a soluble fraction of crude oil (Araújo et al. 2014e), cladocerans and
copepods exposed to the insecticide endosulfan and to metals (Gutierrez et al. 2012)
and tadpoles exposed to copper (Araújo et al. 2014b) showed signs of moribundity
at the highest tested concentrations. Different contaminants have also been shown
to impair the swimming ability of tadpoles and, therefore, indirectly affected the
avoidance ability (Wojtaszek et al. 2004, Chen et al. 2007, Denoël et al. 2013). In
some cases, impairment on the neuro-muscular function has been attributed as the
cause of decreasing or loss of the organism’s ability to escape (Chen et al. 2007). In
those cases, spatial avoidance is not possible, as mobility of organisms was impaired,
therefore, traditional forced exposure plays a crucial role in assessing the potential
toxicity at the individual level.

Final Considerations: Incorporating Avoidance, Recolonization

and Habitat Selection into ERA Schemes
As discussed above, contaminants can trigger avoidance by organisms from a
disturbed habitat. On the other hand, when a habitat starts to present signs of recovery,
a displacement of the organisms towards the recovering habitat is expected. This
hypothesis has recently been attested through an experiment with fish, in which
the process of recolonization of a habitat recovering from an acid mine drainage
contamination event could be predicted in non-forced exposure assays (Araújo et al.
2018). We observed that the acid mine drainage dilutions that elicited avoidance of 50,
20 or 80 percent of the exposed population were the same, allowing a recolonization
of 50, 80 or 20 percent, respectively, of that same population. These first results point
out the importance of including avoidance and recolonization responses in ERA,
as complementary responses elucidating the role of contaminants in regulating the
patterns of species distribution.
In assessing environmental disturbances caused by stressors, the non-forced
exposure approach is a novel tool allowing a change in the paradigm related to
the forced exposure scenarios traditionally and widely used in ecotoxicological
studies. Although the forced exposure designs lack ecological relevance in many
circumstances, it has its strengths, permitting researchers to establish precise and
accurate cause-effect relationships between contaminant concentrations and a
multitude of organism responses. Therefore, the non-forced exposure approach
should be used as a complementary (not substitutive) tool through which potential
effects on the spatial displacement of the organisms can be assessed.
Besides integrating the biological effects at the habitat level, avoidance,
recolonization and habitat selection assays can help predicting secondary effects
expected to occur as a result of the contamination-driven preferential spatial species
distribution. The immediate effect to be expected is overpopulation in the preferred
habitat and a population reduction or even extinction in the disturbed habitat.
From this spatial rearrangement, changes in inter- and intra-specific relationships
(e.g., competition and predation) may occur (Fleeger et al. 2003). Additionally, at
mid-or long-term, disruption in some biogeochemical processes at the ecosystem
level can be anticipated if the evaded population is not replaced by another
12 Ecotoxicology of Marine Organisms

functionally redundant one (De Laender et al. 2008). Due to the habitat fragmentation
caused by “chemical barriers” and the isolation of the inhabitable habitats, the flux
of individuals and, consequently, of genes may also be reduced, which leads to the
viability decrease of the small-sized populations (Ribeiro and Lopes 2013).
As discussed in the present chapter, contaminants are not, however, the only
factor determinant for the habitat selection by organisms. Many other abiotic and
biotic factors other than contamination present in disturbed habitats may play an
important role on the decision to move from or stay on in a given habitat. Therefore,
once the balance between aversive and attractive parameters is made, if organisms
decide to inhabit (prefer) a disturbed habitat rather than avoiding it, toxic effects at
the individual level are expected to occur. In this case, forced exposure approaches
will become relevant.
The present chapter intended to discuss the importance of adding the non-forced
exposure approach to ERA, highlighting the ecological relevance of avoidance and
recolonization responses (once organisms are able to detect and avoid environmental
disturbers), the biological effects at the ecosystem level (loss of individuals and
population downsizing in disturbed habitats) and how determining the contamination
for habitat selection in complex multi-parameter field scenarios (balance between
aversive and attractive factors) is. The inclusion of the non-forced exposure approach
into ERA should be encouraged because it will bring the disciplines of ecology and
ecotoxicology closer.

Acknowledgments
CVM Araújo is grateful to Spanish Ministry of Economy and Competitiveness for
the Juan de la Cierva contract (IJCI-2014-19318). This study was also partially
funded by the European Fund for Economic and Regional Development (FEDER)
through the Program Operational Factors of Competitiveness (COMPETE) and
National Funds though the Portuguese Foundation of Science and Technology
(postdoctoral fellowship to M. Moreira-Santos—SFRH/BPD/99800/2014, contract
IT057-18_7285).

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 Toxicity of Rare Earth
2 Elements to Marine
Organisms
Pedro Luís Borralho Aboim de Brito

INTRODUCTION
Rare earth elements (REE) comprise the series of Lanthanides1 (atomic numbers
57–71), scandium (atomic number 21) and yttrium (atomic number 39). Lanthanides
are elements of Group IIIA of the periodic table, having similar physical and chemical
properties due to their electronic configurations (IUPAC 2005). In this chapter, only
lanthanides and yttrium were considered as rare earth elements due to the similarities
between these elements, since the lighter element (scandium) has a relatively small
ionic radius, resulting in a different chemistry from the previous elements.
Rare earths elements were first discovered in 1788, in Ytterby, Sweden, in a
black mineral called Ytterbite. Later, in 1794, Professor Gadolin, from the University
of Åbo (Turku), Finland, studied the same mineral and found for the first time a
new kind of “earth” which he called “rare earths” (Greinacher 1981). With the
development in 1885 of the lanthanum and cerium gas mantles (Welsbach mantles),
for the illumination of factories and streets, the exploitation of REE mineral resources
began, and has been increasing significantly until today. The major mining sites of
REE’s mineral ore to feed Welsbach blanket manufacturing were in northern Europe
(mainly Scandinavia) and in the United States (North and South Carolina), and
later in India, Brazil, and China (Inner Mongolia). Over the past 50 years, the two
largest REE mining sites were Mountain Pass, California, USA, and Bayan Obo,

IPMA - Av. Alfredo Magalhães Ramalho, 6, 1495-165 Lisboa.

Email: [email protected]
1
According to IUPAC (IUPAC 2005), the Lanthanides series comprise the elements lanthanum (La),
cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Gd),
terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (er), thulium (Tm), ytterbium (yb) and lutetium
(Lu).
 Toxicity of Rare Earth Elements to Marine Organisms 17

Baotou, Inner Mongolia, China (Klinger 2015). With the crisis in 2010, as a result
of the decline in REE’s export quotas from China, the REE technology-dependent
countries had to find other sources of raw material not only through land mining
(and more recently in the deep ocean), but also through the recycling of end-of-life
products (Eggert 2011).
New REE mining sites around the world have emerged in recent years, from
Strange Lake, in Canada (Gysi and Williams-Jones 2013), to Mount Weld (Lynas
Corporation Ltd 2017), in Western Australia, through the Kvanefjeld (Greenland
Minerals and Energy Ltd 2017), in Greenland, to the seabed of Japan (Takaya et al.
2018), among others.
Due to the unique properties of REE, these elements have become valuable for
the most different industrial applications, being used extensively in cutting edge
technology, but also in the traditional industries such as metallurgy, petroleum
and textiles. The use of REE in the most diversified applications has resulted in
an increase in environmental concentrations and in exposure to these elements (see
Gwenzi et al. 2018 for a review).
The aquatic environments act as REE sinks, through the absorption of these
elements to suspended particulate material that eventually sediment in the bottom
and resuspends because of bioturbation and hydrodynamics effects. The nature of
particles, on the other hand, may promote more or less adsorption/desorption reactions
in the medium. Mineral phases with high surface area, such as Fe-Mn oxyhydroxides
and aluminosilicates, act as efficient REE scavengers (e.g., Johannesson and Zhou
1999, Quinn et al. 2004).
The availability of REE in these environments generally depends on several
processes occurring in mobile fractions such as sorption-desorption reactions
between the water column and the sediment, co-precipitation with particulate matter
and colloids or the complexation with inorganic and/or organic ligands (Migaszewski
and Gałuszka 2015, Quinn et al. 2004), as well as the nature and physic-chemical
properties of these elements and their compounds that influence their behaviour.

REE Toxicity
Over the past three decades, most toxicity studies have relied on heavy metals (e.g.,
cadmium, mercury, chromium, lead, nickel, etc.) due to the high anthropogenic
concentrations found in the environment. With the development of new technologies
and materials, the need for new toxicological studies has significantly increased.
Among other elements, REE (also classified as Technology-Critical Elements or
TCE),2 are part of a vast group of “new elements” that need in-depth studies not only
in material engineering or medicine, but mainly in ecology and toxicity.
In one of the earlier REE toxicity studies, these elements were considered only
slightly toxic (Haley 1965). Although there are some studies on REE toxicity today,

2
These elements are termed “critical” because of their high demand versus their limited supply, and
overall scarcity in economically relevant concentrations.
18 Ecotoxicology of Marine Organisms

the information is still very scarce, and has mainly focused on classic laboratory
tests using rats or fruit flies, and some terrestrial plants, namely those species of
economic interest due to their use in human or animal nutrition (see Rim et al. 2013
for a literature review).
Rare earth elements toxicity is, like other chemical elements, generally
influenced by the characteristics of organisms such as age, size or maturation,
the form of exposure and the concentrations of each element. Dose-response
relationships of REE are generally biphasic, with stimulatory or beneficial effects
at low concentrations, and inhibitory or toxic effects at high concentrations (Pagano
et al. 2015). Several studies have shown that low REE concentrations promote growth
of both aquatic and terrestrial organisms, some of which have been used since 1990
as micronutrients in fertilizers (Zhang and Shan 2001 and references hereafter) and
more recently as livestock feed additives (He and Rambeck 2000, Xun et al. 2014).
As mentioned above, REE toxicity studies have focused particularly on species
of economic interest or those directly related to public health. However, some studies
have been published in the last two decades involving more marine species, from
bacteria to vertebrates, thus covering a greater biodiversity (González et al. 2014 and
references hereafter).

Bacteria
Most studies on REE in bacteria report work on soil species (e.g., Ozaki et al.
2006, Tsuruta 2007) or investigation of alternative industrial methods for extraction
and purification of rare earth oxides (REO) (e.g., Bonificio and Clarke 2016, and
references hereafter, Park et al. 2017), with few studies on freshwater bacteria
(Rodea-Palomares et al. 2011) and even less on marine species (González et al. 2015,
Kurvet et al. 2017).
In a study evaluating the ecotoxicity of cerium (Ce), gadolinium (Gd) and lutetium
(Lu) on Gram-negative bacteria Vibrio fischeri (González et al. 2015), the reduction
of bacterial bioluminescence was observed after 30 minutes of exposure. The half-
maximal effective concentration (EC50) nominal value for Lu was 3,200 mg.L–1,
whereas for Ce and Gd these values were higher than 6,400 mg.L–1. The highest
inhibition was found for Lu, at all concentrations tested, with most significant
differences in concentrations higher than 800 mg.L–1, with toxicity following the
order Lu > Gd > Ce.
Kurvet et al. (2017) studied the effects of five REE (lanthanum, cerium,
praseodymium, neodymium and gadolinium) and nine doped REO on the same
bacteria. The authors observed that all REO produced reactive oxygen species (ROS),
while all soluble REE were toxic to the bacteria under study, with half-effective
concentration, EC50 3.5–21 mg.L–1 and minimal bactericidal concentration, MBC
6.3–63 mg.L–1. However, no REO toxicity was found (EC50 > 500 mg.L–1; MBC >
500 mg.L–1), except for La2NiO4 (MBC 25 mg.L–1), whereas the use of toxic metals
(such as Ni) as REO dopants can significantly decrease their environmental safety.
According to the kinetic acute bioluminescence inhibition assay results, the toxicity
of REE was triggered by disturbing the integrity of the cell membrane. However,
these authors state that REE and REO do not appear to have harmful effects on the
 Toxicity of Rare Earth Elements to Marine Organisms 19

bacteria since they are currently produced in moderate amounts and form insoluble
salts and/or oxides in the environment.

Algae and Other Plants

Most of REE’s ecotoxicity studies in algae refer to freshwater microalgae such as
Chlorella sp. and Raphidocelis subcapitata (Balusamy et al. 2015, Fujiwara et al.
2008, González et al. 2015, Hao et al. 1997, Joonas et al. 2017).
Fujiwara et al. (2008) evaluated the toxicity of certain lanthanides (La and Eu)
in Chlorella kesseleri, estimating an IC50 of 43,475 mg.L–1 for La and 43,612 mg.L–1
for Eu. Trials with Chlorella sp., exposed to high concentrations of La2O3 (1,000
mg.L–1) nanoparticles, did not show significant toxic effects after 72 hours exposure,
but enhanced growth rate and biomass production (Balusamy et al. 2015).
Joonas et al. (2017) in a study on the effects of various REO on the green
algae Raphidocelis subcapitata observed that this species were not viable at REE
concentrations above 1 mg.L–1. In the growth inhibition assays (72 hours) for the
elements Ce, Gd, La and Pr the EC50 values ranged from 1.2 to 1.4 mg.L–1, whereas
for the REO these values were between 1 and 98 mg.L–1. The authors stated that
growth inhibition could be the result of sequestration of nutrients from the growth
medium, while the adverse effects of REO would be due in part to the entrapment
of algae between the particle agglomerates. As a conclusion it is stated that there is
presumably no acute risk for unicellular aquatic algae, since the production rates of
those REO particles are negligible compared to other forms of REE.
Tai et al. (2010) investigated the toxicity of lanthanides in the single-celled
marine algae Skeletonema costatum and observed that each element produced
similar effects in this species. High concentrations of lanthanides (≈ 4.0 mg.L–1)
resulted in a 50 percent reduction in algae growth compared to the controls after a
period of 96 h of exposure. These authors found that a multielement solution with
equivalent concentrations of each, lanthanide had the same inhibitory effect on cells
as each individual element with the same total concentration, suggesting that this
species might not be able to sufficiently differentiate between practically chemically
identical elements.
In the study of exposure of the green seaweed Ulva lactuca to Ce3+, Eu3+, Gd3+
and Yb3+, Stanley and Byrne (1990) observed that the behaviour of REE was strongly
dependent on the complexation of the solution. The affinity of this marine algae for
these elements varied according to the concentrations of the carbonate ion present in
the solution, showing the order Eu3+ > Gd3+ > Ce3+ > Yb3+.
Similarly, REE studies in aquatic plants are mostly found in freshwater species
such as Lemna minor, Hydrilla verticillate or Potamogeton pectinatus, with few
studies focused on the toxicity of lanthanides (Paola et al. 2007, Wang et al. 2007).
Paola et al. (2007) found a growth of Lemna minor roots apexes and yellowing of
leaves when exposed for 5 days to REE nitrate solutions (La, Ce, Pr and Nd). These
authors also verified that the protein content decreased, while an increase of ascorbate
peroxidase, dehydroascorbate reductase and ascorbate free radical reductase
activities was observed. An increase in total ascorbate and glutathione content was
20 Ecotoxicology of Marine Organisms

also observed. These results led the authors to consider that Lemna minor could be a
useful tool to study the biological effects of REE in aquatic environments.
Wang et al. (2007) observed that Hydrilla verticillate, when exposed to
concentrations of La and Ce higher than 10 μM (1,389.05 and 1,401.16 μg.L–1,
respectively), presented oxidative damage evidenced by increased lipid peroxidation
and decreased levels of chlorophyll and protein.

Animals
In a study to measure lanthanum acute and chronic toxicity to Daphnia carinata,
Barry and Meehan (2000) found that, in the medium with the lowest carbonated
hardness (tap water), the 48-h EC50 of La was 43 μg.L–1, contrasting with the value
of 1,180 μg.L–1 for harder water (ASTM standard). In a diluted seawater medium
(DW), the 48-h EC50 was similar to the one of the medium with the lowest hardness
(48 μg.L–1) but a significant precipitation of La was observed. The chronic toxicity
of Daphnia was measured in the DW and ASTM media. The authors observed that
mortality was a more sensitive end-point than growth or reproduction in both chronic
experiments, with 100 percent mortality at concentrations > 80 μg.L–1 by day six of
the experiment using DW media, but no effect on survival growth or reproduction
at lower concentrations. In the ASTM media, a significant mortality to Daphnia at
concentrations > 39 μg.L–1, while no effect of the on growth of surviving daphnids
at concentrations > 57 μg.L–1 was observed. However, the authors found that second
brood clutch sizes were significantly increased at 30, 39 and 57 μg.L–1 compared with
controls. Lanthanum also caused delayed maturation in Daphnia.
Oral et al. (2010), in a study to evaluate the toxicity of La4+ and Ce3+ in sea urchin
Paracentrotus lividus embryos and sperm, observed a mortality of 100 percent in
reared embryos in 1.40 μg.L–1 Ce3+, whereas identical concentrations of La4+ reported
100% percent developmental defects, without causing embryonic mortality. In the
embryos exposed to Ce3+, but not exposed to La4+, the authors observed a significant
concentration-related mitotoxic effect at concentrations between 14 and 420 ng.L–1.
Both elements induced a decrease in the success of sperm fertilization at the highest
concentration (1.40 μg.L–1), while offspring exposed to Ce3+ showed a significant
increase in developmental defects, but not observed in individuals exposed to La4+.
In another study with four species of sea urchins (Paracentrotus lividus, Arbacia
lixula, Heliocidaris tuberculata and Centrostephanus rodgersii), it was observed
that exposure to Gd resulted in inhibition or alteration of skeleton growth in larvae
(Martino et al. 2016). These authors also observed a great variability in sensitivity
to Gd, with the EC50 varying between 8.8 and 21 μg.L–1. Martino et al. (2017) also
found that Gd is able to affect different aspects of sea urchin Paracentrotus lividus
development such as morphogenesis, biomineralization and stress response through
autophagy. In a more recent study, Martino et al. (2018) found a general delay of the
Mediterranean Paracentrotus lividus and of the Australian Heliocidaris tuberculata
embryos development at 24 h post-fertilization, and a strong inhibition of skeleton
growth at 48 h, while total Gd and Ca content in the larvae showed a time and
concentration-dependent increase in Gd, in parallel with a reduction in Ca.
 Toxicity of Rare Earth Elements to Marine Organisms 21

Most REE toxicity studies in fish refer to freshwater species (e.g., Cui et al.
2012, Figueiredo et al. 2018, Hongyan et al. 2002).
Hongyan et al. (2002) investigated physiological and biochemical disturbances
in the liver of Carassius auratus exposed to different concentrations of ytterbium in
solution. The results showed that glutamate-pyruvate transaminase (GPT) activity
in liver was stimulated at 0.05 mg.L–1 and inhibited at higher Yb3+ concentrations,
whereas the activity of the antioxidant enzyme superoxide dismutase (SOD) was
stimulated at Yb3+ > 0.05 mg.L–1, while catalase (CAT) activity was strongly
inhibited after 40 days of exposure. These authors also observed that detoxifying
enzymes glutathione S-transferase (GST) and glutathione peroxidase (GSH-Px)
were stimulated at 0.05 mg.L–1 and inhibited at 0.1 mg.L–1 after 40 days of exposure.
Among the parameters determined, CAT in goldfish liver was most sensitive to Yb3+,
indicating that CAT might be considered a potential tool for Yb3+ biomonitoring
exposure in aquatic ecosystems.
Cui et al. (2012) studied the effects of La and Yb on the morphological and
functional development of zebrafish embryos. The results showed that La3+ and
Yb3+ delayed the development of zebrafish embryos and larvae, reducing survival
and hatchability rates. These authors also found a concentration-dependent tail
malformation and a more severe acute toxicity associated with ytterbium than with
lanthanum.
Exposure of the European eel Anguilla anguilla to environmentally relevant
lanthanum concentrations (120 ng.L–1) for 7 days revealed a significant increase in
acetylcholinesterase (AchE) activity, suggesting that La3+ could inhibit acetylcholine
binding (Figueiredo et al. 2018). A decrease in lipid peroxidation was also observed
in this study, which may indicate an important role of this element in the physical
and functional activities with a free radical scavenger. These authors also observed a
significant inhibition of catalase activity, indicating that the availability of La3+ could
induce physiological impairment.

Conclusions
Further ecotoxicity studies of REE in marine organisms are necessary to better
understand the effects and mechanisms of these elements at different trophic levels.
As discussed in this chapter, most published studies refer to terrestrial or freshwater
organisms. Data for marine organisms is still very scarce. However, the information
obtained in freshwater species is generally protective because these organisms tend
to be more sensitive than marine organisms. These differences in sensitivity may be
related to interspecies variations rather than to the speciation of REE in the different
exposure matrices.
In terms of spatial distribution, most REE derives from continental contributions
and are retained in coastal transition zones, such as estuaries, with sediments being
the main sinks of these elements. In this way, and from the point of view of REE
ecotoxicity, these transition areas should be the focus for future studies, aiming to
broaden the diversity of species studied.
22 Ecotoxicology of Marine Organisms

Acknowledgments
The author thanks Prof. Isabel Caçador and Dr. Bernardo Duarte for the opportunity
to collaborate in this chapter.

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3 Chemical Contaminants
in a Changing Ocean
Ana Luísa Maulvault,1,* Patrícia Anacleto,1
António Marques,1 Rui Rosa2 and Mário Diniz3

INTRODUCTION
The human footprint on the planet has dramatically increased since the Industrial
Revolution due to the world population’s constant growth, excessive use of natural
resources and massive production of pollutants. This has contributed to one of the
greatest environmental concerns of our time: climate change. Over the last decade,
greenhouse gas (GHG; e.g., CO2, CH4, N2O) emissions have reached unprecedented
values, unequivocally contributing to a warmer atmosphere and ocean (Solomon
et al. 2007). Apart from increased average surface seawater temperatures (up to +4ºC,
according to the Intergovernmental Panel for Climate Change [IPCC] for the worst-
case predicted scenario; IPCC 2014), some of the most notorious climate changes
affecting marine ecosystems include alterations of wind and precipitation patterns/
intensity, diminished snow cover and rising sea level with consequent changes in
seawater salinity, ocean acidification (–0.4 pH units) due to the disturbance of the
carbon cycle and reduced levels of dissolved oxygen (IPCC 2014). Depending on the
region, each effect can occur alone or in combination with other effects, representing
additional challenges to the resilience of marine ecosystems. Moreover, one climate
change effect may act in synergism or potentiate the occurrence of another. Regardless
of whether acting alone or combined, climate change effects will certainly have an
impact on marine biota, affecting their fitness, metabolism, reproduction, recruitment
and distribution, among other ecological features (Rosa et al. 2014, 2016), raising

1
Division of Aquaculture and Seafood Upgrading. Portuguese Institute for the Sea and Atmosphere, I.P.
(IPMA), Rua Alfredo, Magalhães Ramalho, 6, 1495-006 Lisboa Portugal.
2
MARE – Marine and Environmental Sciences Centre, Laboratório Marítimo da Guia, Faculdade de
Ciências da Universidade de Lisboa, Av. Nossa Senhora do Cabo, 939, 2750-374 Cascais, Portugal.
3
UCIBIO, REQUIMTE Chemistry Department, Centre of Fine Chemistry and Biotechnology, Faculty of
Sciences and Technology, Nova University of Lisbon (CQFB-FCT/UNL), 2829-516 Caparica, Portugal.
Emails: [email protected]; [email protected]; [email protected]; [email protected]
* Corresponding author: [email protected]
26 Ecotoxicology of Marine Organisms

the need for species to adapt to the new prevailing environmental conditions or, in
extreme cases, leading to extinction.
In marine ecosystems, particularly those more vulnerable to anthropogenic
impacts, such as estuaries and coastal areas, biota are exposed to several stressors,
as they are surrounded by an array of chemical contaminants and depend on food
availability and fluctuations of environmental conditions. Such stressors can
further induce physiological stress, emphasise contaminants’ toxicity or even
determine species’ success in a changing climate (Marques et al. 2015, Jager 2016,
Maulvault et al. 2016, 2017). As will be discussed later in this chapter, chemical
contaminants’ availability in marine sediments/water column and toxicity to
biota are strongly influenced by environmental drivers, such as temperature, pH,
upwelling and stratification events (e.g., Noyes et al. 2009, Marques et al. 2010).
By altering species’ physiological status and, at the same time, exacerbating many
forms of water pollution, deleterious impacts on marine organisms can be expected
if the climate continues changing as forecasted, including changes in contaminants’
uptake, retention and detoxification rates (e.g., Maulvault et al. 2016, 2017).
Given the current lack of empirical data, with most available information being
based on mechanistic approaches, the interaction between environmental conditions
and pollution is still unclear. Thus, there is an urgent need to further explore this
research topic to better forecast the ecological consequences of climate change. In
this way, the main aim of this chapter is to provide an overview of the expected
challenges that marine species will face in tomorrow’s ocean, based on mechanistic
toxicological models as well as on findings from the most recent laboratory and
field ecotoxicological studies. Despite a wide variety of environmental factors,
acting alone or combined with other stressors, which also deserve attention, the
impacts of ocean warming, acidification, changes of salinity regimes and hypoxia
on contaminants’ toxicity will be primarily addressed here, given the prevalence of
these variables in the current state of the art with respect to climate change’s effects
on marine ecosystems.

Fig. 1. Interaction between climate change effects and chemical contaminants.

 Chemical Contaminants in a Changing Ocean 27

Chemical Contaminants in Tomorrow’s Ocean

Contaminants’ fate and bioavailability

Organic compounds (OCs)
This group of chemical compounds includes pesticides, polychlorinated biphenyl
(PCBs), flame retardants, dioxins and polycyclic aromatic hydrocarbons (PAHs),
among others. Temperature is one of the most relevant factors influencing their
distribution, half-life in biological compartments, volatilisation and re-emission
(Gouin and Wania 2007, Teran et al. 2012), altering OC’s partitioning into the different
phases (solid, liquid and gas) (Macdonald et al. 2002). Increased temperatures
can likely enhance OCs’ volatilisation and, consequently, the exchanges between
the ocean and atmosphere, which can result in slight reductions in contaminant
exposure by marine biota (Armitage et al. 2011, Nadal et al. 2015). Depending on
the geographic area, increased temperatures and precipitation can directly influence
salinity levels in aquatic systems. Given their lower solubility in saltwater, OCs’
bioavailability and toxicity can be enhanced at higher salinities (Noyes et al. 2009).
Conversely, the opposite effects in OCs’ bioavailability can be expected in regions
of the planet where precipitation and snow/ice melting will increase, resulting in the
high input of freshwater into the ocean.

Toxic metals (e.g., Hg, Pb, Cd, and As)

Solubility and speciation of toxic metals are largely dependent on seawater temperature
and pH (Hoffmann et al. 2012). Thus, environmental variations associated with
climate change will certainly have preponderant effects on metals’ availability and
concentration in the marine environment by altering their behaviour and transfer from
sediments into the water column and vice versa, as well as their toxicity (Marques
et al. 2010). Additionally, since metal inputs into aquatic systems are strongly linked
to climate events, such as snow melting and precipitation, alterations in elemental
profiles, distribution and concentrations due to climate change are expected (Marques
et al. 2010, Hoffmann et al. 2012). Due to metals’ ability to precipitate, bind, or
be released from sediments is largely determined by environmental characteristics,
such as pH, cation exchange capacity, organic matter content, redox conditions and
chloride content, varying salinities can likely affect metal mobility in intertidal
sediments, bioavailability and toxicity to biota, as demonstrated in previous studies
(Du Laing et al. 2002, 2008, Hatje et al. 2003). Low dissolved oxygen levels
(hypoxia) can also facilitate the release of metals from sediments to the water column
(Schiedek et al. 2007). Apart from changes in bioavailability and mobility, increased
seawater temperatures can also have a prevalent role in elements’ speciation and,
consequently, their toxicity to biota (Booth and Zeller 2005, Maulvault et al. 2016).
28 Ecotoxicology of Marine Organisms

Pharmaceuticals and personal care products (PPCPs)

These compounds are currently among the least studied groups of contaminants,
especially within a climate change context. Despite being relatively new pollutants,
for which the available data are still limited, recent studies suggest that environmental
conditions play a key role in the chemical behaviour, degradation and metabolisation
of such pollutants, emphasising the need to consider and further assess the potential
effects of climate change when investigating the toxicological risks of PPCPs (e.g.,
Azzouz and Ballesteros 2013). Since most substances are extremely sensitive to
light, heat and surrounding pH conditions (Moreno et al. 2009, Welankiwar et al.
2013, Gul et al. 2015), the expected warming, increased UV radiation due to a
depleted ozone layer and acidification can likely exacerbate PPCPs’ degradation in
the aquatic environment, depending on the degree of stability of each compound, and
enhance their toxicity to biota (Macdonald et al. 2005, Schiedek 2007, Azzouz and
Ballesteros 2013).

Contaminants’ Carry Over and Toxicity

When assessing the effects of climate change from an ecotoxicological point of
view, two aspects should be taken into account: (i) bioaccumulation and (ii) toxicity.
Adapting to one set of environmental stressors implies great physiologic efforts and,
therefore, increased susceptibility to other existing stressors. For those reasons, the
interactions between climate change and chemical contaminants can be looked at
from two different angles. On one hand, by affecting biota’s metabolism, climate
changes can result in altered bioaccumulation/detoxification mechanisms and
toxicological responses to contaminant exposure (e.g., Noyes et al. 2009, Marques
et al. 2010, Maulvault et al. 2016, 2017). On the other hand, organisms already living
on the edge of their physiological capacities due to constant inputs of pollutants will
certainly exhibit lower tolerance to environmental changes and will struggle to adapt
to the new prevailing conditions (Noyes et al. 2009, Marques et al. 2010, Maulvault
et al. 2016). The following section will present an overview of the available data,
which were gathered from recent field and laboratory studies and reports concerning
the ecotoxicological responses of marine species subjected to the combination of
climate change effects and pollution.

Ocean warming
Out of the environmental variables affected by climate change, to date, temperature
is one of the best documented parameters from a marine and ecological point of
view (e.g., Madeira et al. 2012, 2013, Anacleto et al. 2014, Maulvault et al. 2017).
Since most marine species are ectothermic, temperature is a crucial variable to their
physiological functioning. Although many organisms have evolved to cope with daily
or seasonal temperature variations, when multiple environmental stressors take place
concurrently, species’ resilience to temperature peak events or drastic season changes
may be surpassed, thus compromising their survival (e.g., Madeira et al. 2012).
 Chemical Contaminants in a Changing Ocean 29

Fig. 2. Summary of climate change effects and observed ecotoxicological responses of marine species
exposed to climate change and contaminants.

Metabolic changes induced by thermal stress are among the most studied
physiological responses in marine biota (e.g., Neuheimer et al. 2011, Holt and
Jørgensen 2015, Madeira et al. 2016). In general, organisms subjected to warmer
temperatures exhibit enhanced metabolism, accompanied by increased ventilation
and feeding rates in response to higher metabolic demands. Such changes can
translate into higher contaminant bioaccumulation (contaminants dissolved in the
water column, i.e., via respiration, or present in feeds or natural preys, i.e., via
ingestion) and elimination rates (i.e., contaminant metabolisation and excretion)
(e.g., Maulvault et al. 2016, Sampaio et al. 2016). Following the current lack of
empirical data on this subject, and within the framework of the European project
ECsafeSEAFOOD (reference no. 311820), the bioaccumulation of several emerging
contaminants was evaluated in marine bivalves (Mytilus galloprovincialis and
Ruditapes philippinarum) exposed to different climate change scenarios (warming
and acidification). Thus, results gathered in this pilot laboratory study showed that
bioaccumulation patterns are largely dependent on the behaviour and chemical
properties of each compound, as warming induced higher bioaccumulation of some
contaminants (e.g., sotalol, carbamazepine, triclosan, and TBBPA), but also lower
bioaccumulation of others (e.g., PFOA and PFOS) (Maulvault, unpublished data).
Furthermore, this study revealed that the interaction of warming and acidification
can either exacerbate contaminants’ bioaccumulation (e.g., sulfamethoxazole
and TBBPA) or impair it (e.g., venlafaxine, citalopram, methylparaben and iAs;
Maulvault et al. 2018; Serra-Compte et al. 2018).
30 Ecotoxicology of Marine Organisms

Fig. 3. MeHg concentrations in muscle, liver and brain of D. labrax exposed to warming and dietary
MeHg during 56 days of experiment (28 days of exposure followed by 28 days of elimination) (adapted
from Maulvault et al. 2016).

To adequately assess the relation between warming and contaminants’

bioaccumulation/elimination patterns, animal growth efficiency is a parameter that
deserves careful consideration when interpreting data, because (i) an enhanced
metabolism also implies high energetic costs to biota, often leading to lower animal
condition and fitness (Johnston and Dunn 1987, Stauber et al. 2016), (ii) animal
feeding rates cannot be seen as directly proportional to their metabolic demands, since
the amount of ingested food is largely determined not only by prey/feed availability
but also by predator behaviour (Dijkstra et al. 2013). In this context, bioenergetics
models become a crucial tool, although currently poorly developed, towards a more
accurate and consistent estimation of contaminants’ bioaccumulation rates in climate
change scenarios (Dijkstra et al. 2013).
In addition to changes in contaminant bioaccumulation patterns, alterations in
animals’ metabolism and condition induced by warming can also exacerbate compound
toxicity, lowering the ability of an organism to successfully respond to contaminants
and/or to detoxify them (Sampaio et al. 2016, Maulvault et al. 2017) or increasing
their biotransformation into more toxic compounds (Stauber et al. 2016). Such trends
were observed in two recent studies performed on juvenile seabass (Dicentrachus
labrax) exposed to warming and MeHg acting alone or in combination (Maulvault
et al. 2016, 2017). In the former study, animals subjected to warmer temperatures (22ºC
versus 18ºC) exhibited higher MeHg bioaccumulation, but also lower elimination of
 Chemical Contaminants in a Changing Ocean 31

Fig. 4. GST, CAT and SOD activities in the liver of D. labrax after 28 days of exposure to warming and/
or dietary MeHg (adapted from Maulvault et al. 2017).

this compound in the liver, that is, the primary organ responsible for contaminant
metabolisation and subsequent transport to other organs or hepato-biliary excretion
(Maulvault et al. 2016). As for the latter work, contaminated specimens subjected to
increased seawater temperatures revealed, overall, depressed enzymatic activity, for
instance, in terms of glutathione S-transferase (GST), which is a major second phase
detoxification enzyme, and catalase (CAT) and superoxide dismutase (SOD), which
play key roles in cell defence against the formation of reactive oxygen species (ROS)
induced by stress (Maulvault et al. 2017).

Ocean acidification
At first glance, the effects of ocean acidification seem to be more noticeable and
mostly dramatic in calcified organisms, to which the development and growth are
intrinsically dependent on the equilibrium of the calcium carbonate cycle. For
this reason, over the years, great research efforts have been channelled towards
32 Ecotoxicology of Marine Organisms

the assessment of potential ecological threats of ocean acidification to marine

invertebrates, where most of the studies have been focused on coral reefs, followed
by bivalves and crustaceans (e.g., Kleypas et al. 2006, Hoegh-Guldberg et al. 2007,
Cohen and Holcomb 2009). Nevertheless, although fish species are known to be
relatively tolerant to pH variations, since they are capable of adjusting their internal
pH according to the surrounding levels (Fabry et al. 2008), recent studies have
revealed that hypercapnia can lead to body malformations, as well as changes in
buoyancy and loss of spatial orientation (Gutowska et al. 2010, Pimentel et al. 2014).
As previously discussed, contaminants’ chemical properties are largely
influenced by environmental conditions, with metals and other ionic compounds
being particularly affected by the surrounding seawater pH levels. Such is the case
of the PPCP triclosan (TCS), which becomes increasingly protonated and loses its
negative charge as pH decreases, which, on the other hand, translates into enhanced
compound availability and toxicity to biota (Orvos et al. 2002, Rowett et al. 2016).
Indeed, the recent study of Rowett et al. (2016), using the freshwater amphipod
Gammarus pulex as a model organism, evidenced increased TCS toxicity under
lower pH levels. As argued by these authors, such a trend is explained by the fact
that lipid membranes are generally impermeable to ionised molecular forms, and
that TCS requires a pH value around 8.0 pH units to become ionised (i.e., in its less
toxic form) (Lyndall et al. 2010, Rowett et al. 2016). Similarly, two recent studies
using marine bivalves (i.e., Scrobicularia plana, Freitas et al. 2016, Ruditapes
philippinarum, Munari et al. 2016) also reported synergistic interactions between the
surrounding pH level and the PPCPs carbamazepine and diclofenac, with specimens
exhibiting higher mortality and oxidative stress when exposed to the combination of
acidification plus PPCPs than those exposed to each stressor separately.
When addressing the toxicological impacts of acidification, it is also worthwhile
to consider its effects on biota’s behaviour. Such effects are mostly attributed to
the disruption of the ionic balance in proton-based neurotransmitter receptors, such
as the GABAA neurotransmitter, which can likely translate into increased animal
anxiety and boldness (Hamilton et al. 2014, Munday et al. 2014, Sampaio et al. 2016,
Lai et al. 2017). Furthermore, recent studies have evidenced that, given the high
conservation of the nervous system throughout evolution within vertebrates, water
pollutants that are neurotoxic to humans (e.g., MeHg) or were designed to modulate
specific human behaviours (i.e., psycho-active drugs, such as antidepressants,
anxiolytics and anticonvulsants) may also promote similar responses in fish (Valenti
et al. 2012, Brooks 2014, Fong and Ford 2014, Sampaio et al. 2016). Despite the recent
findings consistently suggesting that ocean acidification can potentially promote
neurotoxicological aspects of these contaminants (Bisesi et al. 2014, Sampaio et al.
2016, Maulvault et al. 2017), further research on this topic should be carried out to
accurately explore different fish behavioural cues, neurological functioning and the
ecotoxicological impacts of climate change.

Salinity
Salinity constitutes a critical environmental variable vis-à-vis marine biota, both to
stenohaline organisms that exhibit a narrow tolerance range (thereby requiring stable
 Chemical Contaminants in a Changing Ocean 33

salinity regimes) as well as euryhaline species (since osmoregulatory mechanisms

involved in the maintenance of the internal homeostasis are energy-demanding)
(Sampaio and Bianchini 2002). Hence, because salinity variations lead to metabolic
changes and great energy costs, bioenergetic models can provide accurate estimations
of the physiological and ecological impacts of salinity fluctuations (e.g., Normant
et al. 2012, Chen et al. 2017). Moreover, the use of such models becomes further
relevant when chemical contaminants are also involved in order to determine
compound toxicokinetics under osmotic stress, adding to the equation important
variables such as feed intake, feed efficiency, growth, excretion and ventilation (e.g.,
Chen et al. 2017).
Interactions between salinity and chemical contaminants are complex, since
salinity by itself is able to largely influence marine species’ physiological status and
affect compound speciation and toxicity (Marques et al. 2010). In this sense, several
studies, not necessarily focusing on climate change effects, have been conducted
to assess the effects of different salinity levels on metal bioaccumulation and
toxicity (e.g., Yung et al. 2015, Piazza et al. 2016, Zhou et al. 2017). By becoming
increasingly more complexed, metals tend to be less toxic as salinity increases (e.g.,
Modassir 2000, Yung et al. 2015, Piazza et al. 2016, Zhou et al. 2017). Such was
the case of Zn oxide nanoparticles’ toxicity to the diatom Thalassiosira pseudonana
(Yung et al. 2015). In this study, increased compound aggregation and decreased ion
release were observed at higher salinity, thus leading to lower bioavailability and
toxicity associated to Zn+2 ions (Yung et al. 2015). Similarly, early life stages of the
barnacle Amphibalanus amphitrite exposed to Cd also revealed increased animal
mortality, immobilisation, and changes in swimming speed at lower salinity ranges
(Piazza et al. 2016). In opposition to this trend, an earlier study using mangrove clams
(Polymesoda erosa) showed lower Hg toxicity at lower salinity levels, which could
be related to enhanced detoxification (i.e., increased ventilation rates, metabolisation,
and excretion) of this contaminant, as well as changes in this element’s speciation
and bioavailability (Modassir 2000). It is also noteworthy that salinity changes may
additionally affect the internal transport of metals through ionic channels. Zhang and
Wang (2007) observed in the seabream Acanthopagrus schlegeli that the calcium
channel was actively involved in the uptake of Cd and Zn (transcellular uptake) at
low salinity ranges, but not at high salinity levels.
As opposed to metals, and despite the limited information available, overall,
persistent organic pollutants’ (POPs) toxicity seems to be enhanced at elevated
salinities (Noyes et al. 2009). For instance, Song and Brown (1998) observed
increased mortality in brine shrimp Artemia sp. exposed to the organophosphate
pesticide dimethoate when salinity increased three to four times in relation to the
iso-osmotic salinity value of this species. In an earlier study focused on the impacts
of severe droughts due to climate change, physiological and histological alterations
observed in the gills of the fish species Sarotherodon melanotheron after short-term
waterborne DDT contamination were potentiated by fluctuating salinities (Riou
et al. 2012).
As for PPCPs, distinct and controversial ecotoxicological responses have
been observed when marine biota was subjected to different salinity regimes. For
instance, similarly to metals, sulfapyridine, sulfamethoxazole, sulfadimethoxine and
34 Ecotoxicology of Marine Organisms

trimethoprim showed to be less toxic to the green microalgae Chlorella vulgaris at

higher salinities (Borecka et al. 2016). In this study, the authors argued that such
toxicity decrease could be associated with the reduced permeability of algal cell
walls to PPCPs, as higher salinities promoted elevated concentrations of inorganic
monovalent cations, which, in turn, showed the ability to bind to algal surfaces’
countercharges (hydroxyl functional groups, Borecka et al. 2016). Following this
pattern, González-Ortegón et al. (2013) observed reduced growth (about 18 percent)
in larvae of the marine shrimp Palaemon serratus exposed to clotrimazole and low
salinity in comparison to specimens subjected to reference salinity levels.
Finally, and particularly to marine bivalve species, increased contaminant toxicity
at lower salinity can also be associated to the fact that hypohaline conditions promote
valve closing. As argued by Correia et al. (2015) in their study on R. philippinarum
exposed to paracetamol, by having such a defense strategy (i.e., valve closing reflex
under stress), bivalves can prevent the absorption of more contaminants from the
external media, but the drawback is that they are unable to excrete metabolised forms
of the contaminants already bioaccumulated.

Hypoxia
Hypoxic and anoxic zones in the ocean can occur as a result of (i) increased
eutrophication (nutrients and pollutants), particularly in coastal areas, which leads to
biomass increase and, consequently, to enhanced respiration rates (Nixon and Buckley
2002, Keeling et al. 2010), or (ii) increased seawater stratification due to warming
and/or salinity changes (Diaz and Rosenberg 2008). So, when exploring potential
physiological and ecotoxicological responses associated to hypoxia, it is of utmost
importance to assess interdependence relations between hypoxia and other abiotic
factors such as temperature, salinity, nutrients and suspended organic particles. In
this sense, the interdependent impacts of hypoxia and increased temperature are the
most widely described in the literature, suggesting that oxygen availability plays
a key role in marine organisms’ thermal tolerance limits (e.g., Anttila et al. 2013,
Artigaud et al. 2014, Pédron et al. 2017). Interactive effects between acidification
and hypoxia were observed in the physiological status and energy budget of
M. coruscus, clearly evidencing further metabolic depression when both stressors
were combined (Sui et al. 2016). Rosa et al. (2013) also observed that during
cuttlefish (Sepia officinalis) embryogenesis, the already harsh conditions inside
the egg capsules can be further exacerbated by hypoxia acting in synergism with
warming and acidification, likely leading to premature hatching and smaller post-
hatching animal size, and, consequently, compromising the survival of this species
and the successful development of posterior ontogenetic stages.
As for the effects of hypoxia from a toxicological perspective, the information
available to date is extremely limited. However, the few available studies, which
were mostly performed on freshwater species, suggest that hypoxia potentially
enhances contaminants’ toxicity, mostly because it can significantly impair biota’s
metabolism and detoxification mechanisms (Marques et al. 2010). Mustafa et al.
(2012) observed enhanced oxidative DNA damage, histopathological alterations
in different tissues and changes in feed conversion efficiency and growth rates in
 Chemical Contaminants in a Changing Ocean 35

European carp (Cyprinus carpio) exposed to hypoxia and dietary Cu. Wang et al.
(2014) reported interactive effects of hypoxia and titanium dioxide nanoparticles
(nano-TiO2) in the immune system of the mussel Perna viridis, with increased ROS
formation, and lower total hemocyte counts in specimens subjected to low dissolved
oxygen levels. Conversely, recent studies have also evidenced that pollutants can, on
the other hand, hamper biota’s ability to cope with the physiological stress induced
by hypoxic events (e.g., Negreiros et al. 2011, Gorokhova et al. 2013). Importantly, a
recent study regarding the interactions between hypoxia and Cu exposure suggested
that hypoxia may act in opposite ways during the embryonic development of three-
spined stickleback (Gasterosteus aculeatus), that is, having a protective effect on
metal toxicity before embryo hatches, but exacerbating Cu toxicity after hatching
(Fitzgerald et al. 2017). The authors further argued that this pattern can occur in
teleost species, as these toxicological interactions were also observed in zebrafish
embryos (Fitzgerald et al. 2016).

Conclusions and Perspectives

Although many possible interactions between climate change and chemical
contaminants have been pointed out, to date, little research has been conducted
addressing such interactions in either laboratory or field conditions, and even fewer
studies address marine species. Thus, the current lack of empirical data clearly
suggests that greater efforts are needed to understand how multiple environmental
stressors interact with each other. This will enable researchers to develop broader
management and efficient mitigation strategies in tomorrow’s ocean.
Future research should take into consideration regional trends (i.e., abiotic
factors, pollution levels and types of contaminants) when addressing potential
ecotoxicological responses to climate change, as the alterations of prevailing
environmental conditions will certainly not affect marine ecosystems in the same
way across the planet. Furthermore, because environmental stressors will unlikely
occur in isolation, or all at once, different combinations of contaminant mixtures
and abiotic conditions (exploring less pronounced to more severe climate change
scenarios) should be investigated to gain a broader view on ecotoxicological impacts
of climate change. Finally, apart from empirical toxicological data, and given the
multiple physiological and metabolic alterations involved, bioenergetic models
should be further explored in climate change contexts, as they provide accurate
insights on biota’s welfare and are resourceful tools to understand contaminants’
toxicokinetics in a changing climate.

Acknowledgements
The authors would like to thank the Fundação para a Ciência e Tecnologia (FCT)
through the strategic projects UID/MAR/04292/2013 granted to MARE and UID/
Multi/04378/2013 granted to UCIBIO, the contracts of AM and RR in the framework
of the IF program, as well as, the PhD Grant of ALM (SFRH/BD/103569/2014) and
post-PhD Grant of PA (SFRH/BPD/100728/2014).
36 Ecotoxicology of Marine Organisms

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4 Effects of Harmful Algal
Bloom Toxins on Marine
Organisms
Lopes, V.M.,1,2,* Costa, P.R.2,3 and Rosa, R.1

INTRODUCTION
Phytoplanktonic communities are vital to marine ecosystems. These communities
constitute the basis of marine food webs throughout the planet, providing food for
filter-feeding organisms, such as bivalves and planktivorous fish and also a number
of vertebrate and invertebrate larval stages. Algal blooms are natural occurrences,
defined as the sudden overgrowth of microscopic algae under optimal environmental
conditions, reaching up to millions of cells per litre (Hallegraeff 1993). These
blooms are typically beneficial for the ecosystem, increasing feeding opportunities
for countless organisms. However, if toxin-producing microalgae undergo this
sudden overgrowth, it can lead to harmful algal blooms (HABs). Despite the fact that
approximately 2 percent of microalgae species produce toxins (Hallegraeff 2014,
Smayda 1997), HABs can significantly impact marine communities.
In the marine realm, the majority of HAB-toxins are produced by dinoflagellates
and diatoms (Table 1). Biochemically, phycotoxins are secondary metabolites that can
have a wide range of effects. They can act on the nervous system (brevetoxins), which
can induce permanent short-term memory loss (domoic acid) or cause sensorimotor
impairment, leading to death (paralytic shellfish toxins) and act on the digestive tract,
inducing gastrointestinal distress (diarrhetic shellfish toxins). During the last decades,
several new toxins and new toxin derivatives, such as gymnodimines, azaspiracids,
pterotoxins, pinnatoxins and hydroxybenzoate saxitoxin, okadaic and domoic acid

1
MARE – Marine Environmental Sciences Centre, Laboratório Marítimo da Guia, Faculdade de Ciências
da Universidade de Lisboa, Portugal.
2
IPMA – Portuguese Institute for the Sea and Atmosphere, Avenida de Brasília, 1449-006 Lisboa,
Portugal.
3
CCMAR – University of Algarve, Campus of Gambelas, 8005-139 Faro, Portugal.
* Corresponding author: [email protected]
 Effects of Harmful Algal Bloom Toxins on Marine Organisms 43

Table 1. The most common toxins produced by marine phytoplankton.

Toxic phytoplankton
Toxin Mode of action Toxin family
species
Alexandrium sp., Inhibition of voltage-gated
Paralytic
Saxitoxins Gymnodinium catenatum, sodium channels in neural
shellfish toxins
Pyrodinium bahamense cells
Pseudo-nitzschia spp., Binding to glutamate receptors
Amnesic
Domoic acid Amphora coffaeaiformis, in neural cells causing
shellfish toxins
Nitzschia sp., constant influx of Ca2+
Karenia brevis, Karenia sp.,
Chatonella cf. verrucosa, Binding to voltage-sensitive
Neurotoxic
Brevetoxins C. antiqua, C. marina, sodium channels causing
shellfish toxins
Fibrocapsa japonica, membrane depolarization
Heterosigma akashiwo
Okadaic acid and Dinophysis sp., Inhibition of activity of Diarrhetic
dinophysistoxins Prorocentrum sp. protein phosphatase 1 and 2 shellfish toxins

analogues have been described, mostly due to scientific and technological advances
(Cruz et al. 2006, Miles et al. 2000, Negri et al. 2003, Satake et al. 1998, Takada
et al. 2000, Zaman et al. 1997). In addition, changes on global climate conditions
and anthropogenic pressures have been conducting several tropical and subtropical
endemic HAB-toxins, namely ciguatoxins, palytoxins and brevetoxins to expand
their geographical range into temperate waters (Botana et al. 2015, Villareal et al.
2007).
There is a great body of available information regarding the effects of these
toxins in marine organisms, although, the information is much dispersed. Therefore,
and with recent increases in HAB frequency and intensity, this chapter aims to update
and summarize the available information on the effects of different phycotoxins in
marine organisms.

Routes of Toxin Exposure

Toxin transfer can be foodborne or waterborne, that is via food web transfer or
through exposure to toxins dissolved in the water after their excretion or cell release
(Fig. 1). The most likely pathway of toxin transfer is when toxin-producing species
bloom, thus achieving massive concentrations in the water column. However, there
are many potential toxin vectors (Fig. 2), depending mostly on the ecology of the
toxin producer (pelagic or epibenthic) and the organism’s likelihood of exposure to
the toxin.
If an organism is exposed to a sudden bloom of toxin-producing microalgae, the
toxin concentrations will certainly trigger immediate physiological and behavioural
alterations and ultimately cause the death of the organism. In addition, the continuous
exposure to low HAB-toxin concentrations can lead to chronic effects.
Here, the pathways of exposure will be divided into direct and indirect contact
with the toxin-producer. Through ingestion of toxic phytoplanktonic cells by filter-
feeding organisms, such as bivalve molluscs, zooplankton and planktivorous fish, the
toxins present inside the cell can accumulate in the predator’s viscera. This can create
44 Ecotoxicology of Marine Organisms

Fig. 1. Foodborne and waterborne exposure to HAB-toxins. Solid lines illustrate the well documented
route of dietary exposure; dashed lines illustrate the less studied routes of dissolved toxins exposure.

Fig. 2. Schematic view of HAB-toxins food web transfer.

a chain of vectors throughout the food web, potentially eliciting adverse effects in
marine communities. Depending on the vector, these toxins can be transferred to
humans and cause a variety of shellfish poisonings, due to the ingestion of contaminated
shellfish, such as Amnesic Shellfish Poisoning (ASP), Paralytic Shellfish Poisoning
(PSP), Neurotoxic Shellfish Poisoning (NSP), Diarrhetic Shellfish Poisoning (DSP)
and other syndromes (Table 1).
Some microalgae species produce exotoxins, or exudates, that are released into
the water column, causing other organisms to come inadvertently in contact with
these compounds. Similarly, when the bloom becomes senescent, the cells lyse and
release the toxins to the surrounding environment (Lefebvre et al. 2008), opening
another possible pathway to the organisms’ direct contact with the toxins. Lastly,
there are other HAB-species which segregate the toxin on the outer surface of their
 Effects of Harmful Algal Bloom Toxins on Marine Organisms 45

cells, potentially inducing damage upon contact. These toxins’ effects on marine
organisms will be discussed in detail in the following sections.

HAB-toxin Effects on Marine Organisms

Paralytic shellfish toxins

Paralytic shellfish toxins (PSTs) are one example of phycotoxins produced by
dinoflagellates, and one of the most abundant and toxic phycotoxins in oceans
worldwide. Dinoflagellates from three genera, Alexandrium, Gymnodinium and
Pyrodinium, produce saxitoxin or a suite of over 50 derivatives (Anderson et al.
2012), which the most frequent can be divided according to their chemical structure
into carbamoyl, decarbamoyl and sulfamate toxins. These compounds block the
conduction of electrical impulses in neural cells through the inhibition of voltage-
gated sodium channels on these cell’s membranes. This leads to membrane
hyperpolarization and results in paralysis in muscle cells as determined in laboratory
animals (Kao and Nishiyama 1965, Ritchie and Rogart 1977). PSTs have shown to
elicit a wide range of effects on marine organisms, from sublethal and recoverable
effects to events of mass mortality in fish, marine mammals and seabirds (Tables 2a
and 2b).
PSTs producers inhabit the pelagic realm, the same habitat as many planktonic
species. Therefore, plankton species come in contact with PSTs through contact
with PST-producing cells or their exudates. It has been shown that many planktonic
organisms can be affected by these toxins. In some cases, PSTs inhibit the growth of
diatoms, haptophytes and heliozoans, and induce disruptions in swimming behaviour
leading to death in ciliates (Table 2a). Diatoms and haptophytes had reduced growth
rates after being placed in water previously conditioned by the PSTs producer
A. lusitanicum, presumably releasing the watersoluble toxins into the culture medium
(Blanco and Campos 1988).
Regarding the effects of PSTs on planktonic grazers, there are several studies
indicating that some species can selectively avoid ingestion of toxic dinoflagellates,
while others do not (Turner and Tester 1997). The latter group can present different
effects, with some species being less affected by the toxins, namely Euterpina
acutifrons, and other species, such as Acartia grani, presenting high mortality rates
(Costa et al. 2008, 2012).
Direct exposure of bivalve molluscs to PST-producers has been shown to
elicit negative effects, as summarized in Table 2a. Exposure to dinoflagellate cells
increased shell valve closure in many bivalve species (e.g., Crassostrea virginica,
Mytilus edulis), leading to decreased filtration rates, potentially impacting the
animal’s normal feeding behaviour. A. minutum and purified STX exposure in
C. gigas resulted in decreased phagocytic activity and ROS production in oyster
hemocytes (Mello et al. 2013), leading to higher susceptibility of contracting an
infection. Also, the presence of the toxic dinoflagellates decreased byssus production
in M. edulis and Geukensia demissa. However, byssus production in mussels
(M. edulis) that have been previously exposed to these toxins was less affected.
Table 2a. Documented cases of marine invertebrates exposed to and affected by paralytic shellfish toxins (PSTs).
46

Target species PST source Route of exposure Levels of Tissues Effects Observations References
exposure analysed
Phytoplankton Isochrysis A. lusitanicum Exposure in pre- - - Reduced growth rates Seawater (Blanco and
galbana conditioned seawater previously Campos 1988)
with A. lusitanicum containing
A. lusitanicum
cultures
Pavlova A. lusitanicum Exposure in pre- - - Reduced growth rates - (Blanco and
lutheri conditioned seawater Campos 1988)
with A. lusitanicum
Skeletonema A. lusitanicum Exposure in pre- - - Reduced growth rates - (Blanco and
costatum conditioned seawater Campos 1988)
Ecotoxicology of Marine Organisms

with A. lusitanicum
Ciliates Favella A. tamarense Exposure to 4 × 106 cells - Disruption of - (Hansen 1989)
ehrenbergii A. tamarense cells L–1 swimming patterns,
immobilization,
swelling and death
A. ostenfeldii Exposure to Higher than - Backward - (Hansen et al.
A. ostenfeldii cells 2 × 106 cells swimming, swelling 1992)
L–1 and death
Crustaceans Acartia clausi A. lusitanicum Exposure to Up to 1600 Egg production - (Dutz 1998)
A. lusitanicum cells µg C L–1 limited
 A. minutum Exposure to Up to 0.7 µg A. minutum Increased feeding - (Frangopulos et
A. minutum cells C ml–1 cultures rates with increasing al. 2000)
dinoflagellate
densities, lower
hatching success and
nauplii production
A. hudsonica Alexandrium spp. Exposure to Up to 1 × 103 - Non-selectively fed - (Teegarden
Alexandrium spp. cells cells L–1 on Alexandrium spp. et al. 2001)

A. fundyense Exposure to - - Naïve populations - (Colin and Dam

A. fundyense cells had decreased 2004)
somatic growth,
size at maturity,
egg production and
survival
Protogonyaulax Exposure to 25 × 104–30 x - Lower activity, - (Ives 1987)
tamarensis (now A. tamarense cells 104 cells L–1 reduced feeding rates
A. tamarense)
A. tonsa Alexandrium spp. Exposure to Up to 25 pg Dinoflagellate Avoided feeding on - (Teegarden
Alexandrium spp. cells STX eq cell–1 cultures toxic Alexandrium 1999)
spp.
Calanus G. tamarensis Exposure to - - Reduced fecundity - (Gill and Harris
helgolandicus (now A. tamarense cells 1987)
A. tamarense)
Table 2a contd. ...
Effects of Harmful Algal Bloom Toxins on Marine Organisms
47
...Table 2a contd.
48

Target species PST source Route of exposure Levels of Tissues Effects Observations References
exposure analysed
Calanus A. excavatum Exposure to 45 µg STX Copepods Avoided feeding on - (Turriff et al.
finmarchicus A. excavatum cells kg–1 toxic A. excavatum 1995)
Centropages Alexandrium spp. Exposure to Up to 31 pg Dinoflagellate Avoided feeding on - (Teegarden
hamatus Alexandrium spp. cells STX eq cell–1 cultures toxic Alexandrium 1999)
spp.
Eurytemora Alexandrium spp. Exposure to Up to 31 pg Dinoflagellate Non-selectively fed - (Teegarden
herdmani Alexandrium spp. cells STX eq cell–1 cultures on Alexandrium spp. 1999)
Euterpina A. minutum and Exposure to Up to 106 - Reduced naupliar - (Bagøein et al.
acutifrons G. catenatum A. minutum and G. cells L–1 (A. activity at low cell 1996)
catenatum cells minutum) and densities, immobility
Ecotoxicology of Marine Organisms

17.5 × 104 at higher cell

cells L–1 (G. densities
catenatum)
Palaemonetes Gonyaulax Exposure to Up to 1.2 × - High mortality rates - (Sievers 1969)
pugio monilata (now A. monilatum cells 106 cells L–1 following molting
A. monilatum)
Pseudocalanus P. tamarensis Exposure to Up to 6 × 105 - Lower activity, - (Ives 1987)
sp. (now A. tamarense cells cells ml–1 reduced feeding rates
A. tamarense)
Nauplii of Alexandrium spp. Exposure to Up to 1 × 103 - Avoided feeding on - (Teegarden et
Semibalanus Alexandrium spp. cells cells L–1 toxic Alexandrium al. 2001)
balanoides spp.
Temora G. tamarensis Exposure to - - Reduced fecundity - (Gill and Harris
longicornis (now A. tamarense cells 1987)
A. tamarense)
Annelids Neanthes Gonyaulax Exposure to Up to 1.2 × - High mortality rates - (Sievers 1969)
succinea monilata (now A. monilatum cells 106 cells L–1 following spawning
A. monilatum)
Polydora Gonyaulax Exposure to Up to 1.2 × - High mortality rates - (Sievers 1969)
websteri monilata (now A. monilatum cells 106 cells L–1 following spawning
A. monilatum)
Molluscs Larvae of A. tamarense Exposure to Up to 11 pg A. tamarense Activity and growth - (Yan et al.
Argopecten A. tamarense cells STX eq cell–1 cultures inhibition, lower 2003)
irradians attachment rates and
concentricus reduced climbing
rates
A. ventricosus G. catenatum Exposure to Up to 4 × 106 - Lower feeding Paralysis was (Escobedo-
G. catenatum cells cells L–1 activity, paralysis, found to be Lozano et al.
increase in reversible 2012)
hemocytes, epithelial
melanisation,
increase in
pseudofaeces
production
Larvae of A. tamarense Exposure to Up to 5.9 × A. tamarense High mortality rates, - (Yan et al.
Chlamys A. tamarense cells 109 g STX L–1 cultures lower hatching rates 2001)
farreri
Crassostrea G. Exposure to 5 × 104 cells - Reduced pumping - (Dupuy and
gigas washingtonensis A. catenella cells L–1 activity, increased Sparks1967)
(now valve activity
A. catenella)
A. minutum Exposure to Up to 12 × - Decreased valve (Lassus et al.
A. minutum cells 104 cells L–1 activity, clearance 1999)
and filtration rates
Effects of Harmful Algal Bloom Toxins on Marine Organisms

Table 2a contd. ...

49
...Table 2a contd.
50

Target species PST source Route of exposure Levels of Tissues Effects Observations References
exposure analysed
A. tamarense and Exposure to Up to 12 × - Reduced clearance (Laabir and
A. minutum dinoflagellate cells 108 cells L–1 rates Gentien 1999)
A. minutum Exposure to 5 × 106 cells - Mono and - (Haberkorn et
A. minutum cells L–1 diacylglycerols al. 2010)
reduced in digestive
gland, inflammation
of gastrointestinal
tract, modified
spermatozoa and
mitochondria
Ecotoxicology of Marine Organisms

A. minutum Exposure to 15 × 106 cells - Altered circadian - (Tran et al.

A. minutum cells L–1 rhythm 2015)
A. minutum Exposure to 2 × 104 cells - Reduced hemocyte Results (Mello et al.
A. minutum cells and L–1 and 0.05 phagocytic activity, obtained in both 2013)
purified STX µg STX L–1 decreased ROS treatments
production
Larvae of A. tamarense Exposure to Up to 108 - High mortality rates - (Matsuyama et
C. gigas A. tamarense cells cells–1 al. 2001)
A. taylori Exposure to A. taylori Up to 108 - High mortality rates - (Matsuyama et
cells cells–1 al. 2001)
C. virginica G. monilata (now Exposure to Up to 1.2 × - Inhibition of byssus - (Sievers 1969)
A. monilatum) A. monilatum cells 106 cells L–1 production and shell
closure
Brachiodontes G. monilata (now Exposure to Up to 1.2 × - Inhibition of byssus - (Sievers 1969)
recurvus A. monilatum) A. monilatum cells 106 cells L–1 production and shell
closure
 Geukensia P. tamarensis Exposure to 2.5–5.5 × 105 - Inhibited cardiac - (Gainey and
demissa (now A. tamarense cells cells L–1 activity Shumway 1988)
A. tamarense)
A. tamarense Exposure to 105 cells L–1 - Reduced clearance - (Lesser and
A. tamarense cells rates Shumway 1993)

P. tamarensis Exposure to 5 x 105 cells - Reduced clearance - (Shumway and

(now A. tamarense cells L–1 rates, increased Cucci 1987)
A. tamarense) mucus production
P. tamarensis Exposure to 106 cells L–1 - Inhibition of byssus - (Shumway et al.
(now A. tamarense cells production 1987)
A. tamarense)
Mercenaria A. tamarense Exposure to 105 cells L–1 - Reduced clearance - (Lesser and
mercenaria A. tamarense cells rates Shumway 1993)

Mya arenaria P. tamarensis Exposure to 2.5-5.5 × 105 - Inhibited cardiac - (Gainey and
(now A. tamarense cells cells L–1 activity Shumway 1988)
A. tamarense)
A. tamarense Exposure to Up to 77 × Viscera and Naïve populations - (MacQuarrie
A. tamarense cells 104 µg STX other tissues had higher toxicity and Bricelj
eq kg–1 in and mortality, 2008)
viscera reduced clearance
rates, oxygen
consumption rates
and burrowing
capacity
A. tamarense Exposure to 105 cells L–1 - Reduced clearance - (Lesser and
A. tamarense cells rates Shumway 1993)
Effects of Harmful Algal Bloom Toxins on Marine Organisms

Table 2a contd. ...

51
...Table 2a contd.
52

Target species PST source Route of exposure Levels of Tissues Effects Observations References
exposure analysed
P. tamarensis Exposure to 5 × 105 cells - Reduced clearance - (Shumway and
(now A. tamarense cells L–1 rates Cucci 1987)
A. tamarense)
A. excavata and Exposure to Up to 30.4 × Soft tissue Burrowing incapacity - (Bricelj et al.
A. tamarense Alexandrium spp. cells 104 µg STX 1996)
eq kg–1
Larvae and A. tamarense Exposure to Up to 64–69 Soft tissue Larvae not affected, Naïve (Bricelj et al.
post-larvae A. tamarense cells pg STX eq post larvae exhibited populations were 2010)
M. arenaria cell–1 high mortalities, more severely
paralysis, burrowing affected
Ecotoxicology of Marine Organisms

incapacity
Mytilus edulis P. tamarensis Exposure to 2.5–5.5 × 105 - Inhibited cardiac Transient (Gainey and
(now A. tamarense cells cells L–1 activity inhibition, long Shumway 1988)
A. tamarense) term inhibition
and long-term
excitation
Dissolved STX Intramuscular injection 3330 µg STX Digestive Higher GST activity - (Gubbins et al.
kg–1 glands 2001)
A. tamarense Exposure to 105 cells L–1 - Reduced clearance - (Lesser and
A. tamarense cells rates Shumway 1993)

P. tamarensis Exposure to 5 × 105 cells - Increased mucus - (Shumway and

(now A. tamarense cells L–1 production Cucci 1987)
A. tamarense)
P. tamarensis Exposure to 106 cells L–1 - Inhibition of byssus - (Shumway et al.
(now A. tamarense cells production 1987)
A. tamarense)
 M. chilensis A. catenella Exposure to Up to 7240 Soft tissue Clearance rates, Effects (Navarro and
A. catenella cells µg STX eq ingestion of reversible after Contreras 2010)
kg–1 organic matter, and three days
absorption efficiency
decreased at the start
of the experiment.
Nodipecten G. catenatum Intramuscular injection Up to 0.18 µg - Paralysis, mantle (Estrada et al.
subnodosus of GTX 2/3 STX retraction, inhibition 2010)
of shell closure,
hemocyte reduction
N. subnodosus G. catenatum Exposure to 2 × 106 cells - Production of - (Estrada et al.
G. catenatum cells individual–1 pseudofaeces, 2007)
partial shell
closure, increase
in melanisation,
increased activity
of GPx and lipid
peroxidation in gills,
decrease in SOD
activity in gills
Ostrea edulis P. tamarensis Exposure to 2.5–5.5 × 105 - Decreased in heart Effects (Gainey and
(now A. tamarense cells cells L–1 rate reversible Shumway 1988)
A. tamarense)
Perna A. tamarense Exposure to 12950 µg Soft tissue No mortalities, Oxygen (Marsden and
canaliculus A. tamarense cells STX eq kg–1 normal byssus consumption rate Shumway 1992)
production and increased after
oxygen consumption 1 h of exposure
rates but normalized
after 24 h
recovery
Effects of Harmful Algal Bloom Toxins on Marine Organisms

Table 2a contd. ...

53
...Table 2a contd.
54

Target species PST source Route of exposure Levels of Tissues Effects Observations References
exposure analysed
P. viridis A. tamarense Exposure to 105 cells L–1 - Reduced clearance - (Lesser and
A. tamarense cells rates Shumway 1993)

Placopecten P. tamarensis Exposure to 5 × 105 cells - Increased clearance - (Shumway and

magellanicus (now A. tamarense cells L–1 rates Cucci 1987)
A. tamarense)
Ruditapes A. tamarense Exposure to Up to 170 µg Soft tissue Decreased absorption - (Li et al. 2002)
phillipinarum A. tamarense cells STX eq kg–1 efficiency and
reduced scope for
growth with higher
Ecotoxicology of Marine Organisms

toxin concentration,
decreased clearance
and growth rates
A. tamarense Exposure to 2 × 105 cells - Hepatic GPx, gill - (Choi et al.
A. tamarense cells L–1 LPO were positively 2006)
correlated with PSP
concentrations, GST
presented negative
correlation
Spisula A. tamarense Exposure to 105 cells L–1 - Reduced clearance - (Lesser and
solidissima A. tamarense cells rates Shumway 1993)
Table 2b. Documented cases of marine vertebrates exposed to and affected by paralytic shellfish toxins (PSTs). IP – Intraperitoneal; IC – Intracoelomic.

Target species PST source Route of exposure Levels of Tissues Effects Observations References
exposure analysed
Fish Chanos chanos A. minutum Dinoflagellate cells - - Damage to the gills Exposure to (Chen and
fingerlings and extracts (hyperplasia, edema different cell Chou 2001)
and necrosis) densities

Clupea harengus PSTs extracted from Oral and IP Up to 5240 µg Viscera Irregular swimming Death (White 1981)
harengus G. excavata (now (intraperitoneal STX eq kg–1 behaviour, loss of occurred after
A. tamarense) injection) balance, shallow and 20–60 min of
arrhythmic breathing exposure in
both routes
Larval Clupea P. tamarensis (now Dinoflagellate cells Exposure up to - Paralysis and erratic Heart stopped (Gosselin et
harengus harengus A. tamarense) and extracts, via 15 × 105 cells swimming, increased 20 min after al. 1989)
prey L–1 mortality complete
immobilization
Larval Clupea Dissolved STX Uptake from 4000 µg STX - Reduced spontaneous Effects (Lefebvre et
harengus pallasi surrounding eq kg–1/day for and touch-activated reversible al. 2005)
seawater 7 days swimming
Cyprinodon G. monilata (now A. Exposure to Up to 1.2 × 106 - 100 percent mortality - (Sievers1969)
variegatus monilatum) A. monilatum cells cells L–1 rate
Larval C. variegatus A. fundyense Prey (copepod 9–12 µg STX Whole body Death after Reduced (Samson et al.
Coullana eq kg–1 consuming 6–12 prey capture 2008)
canadensis) contaminated and predator
copepods avoidance
Diplodus sargus PSTs extracted from Intracoelomic 15.2 µg STXeq Liver Increased GST - (Costa et al.
G. catenatum injection (IC) kg–1 activity and 2012)
erythrocyte nuclear
abnormalities
Effects of Harmful Algal Bloom Toxins on Marine Organisms

Table 2b contd. ...

55
Table 2b contd. ...
56

Target species PST source Route of exposure Levels of Tissues Effects Observations References
exposure analysed
Fundulus Dissolved STX Uptake from 0, 75, - Decreased c-Fos - (Salierno et al.
heteroclitus surrounding and 150 ppb, expression, paralysis, 2006)
seawater nominal decreased activity,
concentrations floating
Larval Fundulus A. fundyense Prey (copepod 17–25 µg STX Whole body Death after Reduction in (Samson et al.
heteroclitus Coullana eq kg–1 consuming 6–12 prey capture, 2008)
canadensis) and copepods and swimming
A. fundyense following direct performance
ingestion of
dinoflagellate cells
Ecotoxicology of Marine Organisms

Larval Mallotus P. tamarensis (now Exposure to Up to 15 × 105 - Paralysis and erratic Heart stopped (Gosselin et
villosus A. tamarense) A. tamarense cells cells L–1 swimming, increased 20 min after al. 1989)
mortality complete
immobilization
Pollachius virens PSTs extracted from Oral and IP Up to 3430 µg Viscera Irregular swimming Death (White 1981)
G. excavata (now STX eq kg–1 behaviour, loss of occurred after
A. tamarense) balance, shallow and 20–60 min of
arrhythmic breathing exposure in
both routes
Pseudopleuronectes PSTs extracted from Oral and IP Up to 8370 µg Viscera Irregular swimming Death (White 1981)
americanus G. excavata (now STX eq kg–1 behaviour, loss of occurred after
A. tamarense) balance, shallow and 20–60 min of
arrhythmic breathing exposure in
both routes
Recently-settled A. fundyense Prey (copepod 41–58 µg STX Whole body Death after Reduced (Samson et al.
P. americanus Coullana eq kg–1 consuming 6–12 swimming 2008)
canadensis) contaminated abilities
copepods
 Onchorhynchus Dissolved STX IP injection 1.752 µg kg–1 - Elevated levels of - (Bakke et al.
mykiss cortisol, decreased 2010)
attack latency times
Salmo salar Dissolved STX IP injection 10 µg STX - Loss of balance, - (Bakke et al.
kg–1 decreased respiration 2010)
rate, muscle bursts
and twitching
PSTs extracted from Oral and IP Up to 9410 µg Viscera Irregular swimming Death (White 1981)
G. excavata (now STX eq kg–1 behaviour, loss of occurred after
A. tamarense) balance, shallow and 20–60 min of
arrhythmic breathing exposure in
both routes
Seabirds Uria aalge Gonyaulax Prey (small fish and - - Death - (Mckernan
californica, Gavia catenella (now crustaceans) and Scheffer
arctica pacifica, A. catenella) 1942)
Melanitta fusca
deglandi, Lunda
cirrhata, Puffinus
griseus, Fulmarus
glacialis, Diomedea
nigripes
Larus argentatus G. catenella (now Prey (small fish and - - Death - (Mckernan
A. catenella) crustaceans) and Scheffer
1942)
A. tamarense Likely Ammodytes 1100 Intestine and Death - (Levasseur et
hexapterus (intestine) and brain of dead al. 1996)
480 (brain) µg birds
STX eq kg–1
G. excavata (now Likely A. hexapterus 970 µg STX eq Prey Death - (Nisbet 1983)
A. tamarense) kg–1 in
Effects of Harmful Algal Bloom Toxins on Marine Organisms

A. hexapterus
57

Table 2b contd. ...

Table 2b contd. ...
58

Target species PST source Route of exposure Levels of Tissues Effects Observations References
exposure analysed
L. dominicus G. catenella (now Prey (mussels) - - Death Some bird (Shumway
A. catenella), populations et al. 2003)
G. grindleyi (now decreased
Protoceratium drastically
reticulatum) following
dinoflagellate
outbreak
L. hartlaubii G. catenella (now Prey (mussels) - - Death Some bird (Shumway
A. catenella), populations et al. 2003)
G. grindleyi (now decreased
Protoceratium drastically
Ecotoxicology of Marine Organisms

reticulatum) following
dinoflagellate
outbreak
L. occidentalis G. catenella (now Prey (small fish and - - Death - (Mckernan
A. catenella) crustaceans) and Scheffer
1942)
Haematopus moquini G. catenella (now Prey (mussels) - - Death Some bird (Shumway
A. catenella), populations et al. 2003)
G. grindleyi (now decreased
Protoceratium drastically
reticulatum) following
dinoflagellate
outbreak
L. atricila G. excavata (now Likely A. hexapterus 970 µg STX eq - Death - (Nisbet 1983)
A. tamarense) kg–1 in
A. hexapterus
 Phalacrocorax G. tamarensis (now - Not determined - Intestinal Drastic (Armstrong
aristotelis A. tamarense) in birds. Up hemorrhaging; population et al. 1978)
to 6000 MU loss of balance and decline
(mouse units) stagger, vomiting,
100 g–1 in death
mussel meat
Anas rubripes G. tamarensis (now Prey (mussels) 20000–40000 - Death Other bird (Shumway
A. tamarense) µg STX eq species et al. 2003)
kg–1 affected nearby
Sterna hirundo, G. excavata (now Likely A. hexapterus 970 µg STX eq - Death - (Nisbet 1983)
S. paradisaea, A. tamarense) kg–1 in
S. dougallii A. hexapterus
Marine Monachus monachus A. minutum - 3030–3390 µg Liver Death Analysis (Costas and
Mammals STX eq kg–1 on stranded Lopez-Rodas
animals 1998)
G. catenatum Prey 6–900 µg Liver, kidney, Death PST producer (Reyero et al.
or Pyrodinium dcSTX kg–1 muscle, brain uncertain, 2000)
bahamense var. analysis on
compressa stranded
animals
A. minutum, Prey 2–280 µg Liver, kidney, Lethargy, PST producer (Hernández et
G. catenatum, dcSTX kg–1 muscle, brain sensorimotor uncertain al. 1998)
D. acuta liver 8–64 µg impairments,
dcSTX kg–1 paralysis, death
brain
Megaptera - Horse mackerel - - Death PST producer (Geraci et al.
novaeangliae (Scomber scombrus) uncertain, 1989)
analysis on
stranded
animals
Effects of Harmful Algal Bloom Toxins on Marine Organisms
59
60 Ecotoxicology of Marine Organisms

Similar experiments performed on greenshell mussels (Perna canaliculus) showed

that mussels presented oxygen consumption and clearance rates similar to the control
group after 24 h exposure to A. tamarense (Marsden and Shumway 1992).
Exposure of M. chilensis to A. catenella for 21 days (Navarro and Contreras 2010)
resulted in lowered clearance rates, organic matter intake and absorption efficiency
at the start of the experiment, followed by an increase to levels similar to the ones
presented in the control group. The STX uptake steadily increased throughout the
experiment, similarly to the mussels’ excretion rates. Oxygen consumption rates
seemed unaffected by the ingestion of this toxic species, revealing that this species
may possess defence mechanisms that allow them to feed safely on this dinoflagellate.
On the other hand, some scallop and clam species presented negative effects
when exposed to this toxin. The scallop Nodipecten subnodosus presented paralysis of
the adductor muscle while maintaining the digestive tract functioning after receiving
intramuscular injections of GTX 2/3. Also, the hemocyte number decreased and they
presented mantle retraction and their shells remained open up to 40 days after the
exposure (Estrada et al. 2010). In a different study on the same species (Estrada
et al. 2007), exposure to G. catenatum cells resulted in the increased production
of mucus, pseudo-faeces, melanisation and hemocyte aggregation in gill tissue.
Biochemically, an antioxidative stress response to the toxin was shown in gill tissue,
this tissue being the first to come in contact with the toxin. There was an increase in
glutathione peroxidase (GPx) activity and lipid peroxidation, along with a decrease
in superoxide dismutase activity, indicating oxidative and cellular damage. Similar
results were obtained when feeding G. catenatum cultures to scallops (Argopecten
ventricosus) (Escobedo-Lozano et al. 2012). The scallops presented paralysis of
the adductor muscle, lower feeding activity, increased pseudo-faeces production,
increased number of hemocytes in gill, mantle and adductor muscle tissue and
epithelial melanisation in gill and mantle tissue. These results indicate that scallops
have efficient mechanisms that protect them against lethal effects from external
toxicants. Studies conducted in Ruditapes phillipinarum feeding on A. tamarense
cultures for 6 (Li et al. 2002) and 15 days (Choi et al. 2006) showed reduced scope
for growth, decreased absorption efficiency, clearance and growth rates after six
days of exposure, and increased activity of GPx in hepatopancreas and gill lipid
peroxidation with increasing toxin burden after 15 days of exposure.
PST effects on bivalve early stages are comparatively less understood. Bricelj et
al. (2010) addressed this issue by exposing larvae, post-larvae and juveniles of Mya
arenaria to A. tamarense. They showed that larvae were not significantly affected by
the dinoflagellate due to the fact that the cells were too large for prey capture; thus,
the larvae did not accumulate the toxin or displayed any intoxication symptoms. On
the other hand, the post-larvae presented decreased burrowing capacity, here used
as proxy of sensitivity to PSTs. Also, the post-larvae had increased mortality rates,
especially in populations that are not usually exposed to A. tamarense blooms, while
juvenile were less susceptible.
PST effects on bivalves are species-specific and seem to differ geographically
within the same species and life stages. Species that are usually exposed to toxic
dinoflagellate blooms seem to be more resistant, and appear to have developed
 Effects of Harmful Algal Bloom Toxins on Marine Organisms 61

defence mechanisms that allow them to cope with high PST levels, unlike other
species in areas less affected by blooms.
These studies reveal that bivalves are not immune to the effects of PST
contamination, and there are some species with higher sensitivity to these toxins.
This may pose additional concerns over the ecosystem’s health and elicit negative
economic impacts, since some of these species are commercially farmed in shellfish
aquacultures, and blooms may occur in farmed areas.
PSTs have long been associated with fish kills. Fish can be directly exposed to the
toxins, as is the case of planktivorous fish such as sardines, herring and anchovies, or
indirectly through feeding on vectors, affecting many levels of the marine food web,
from groupers and hake to sturgeons and artificially fed fish, such as farmed salmon.
Only a few events have been directly linked to PST contamination, since these
events are unpredictable and sporadic, many times leading to inconclusive data. For
a complete list of fish kills associated with PSTs refer to Costa (2016, Table 1).
When studying PST’s effect on fish, it is procedurally simpler to inject the toxin
intracoelomically (IC), in order to closely control the given concentration. Standard
STX is the toxin most commonly administered. However, despite the benefits, these
methods are less ecologically relevant, since the toxins do not enter directly in
the coelom, and STX is but a fraction of the toxins produced by the dinoflagellate
species. Nevertheless, these studies provide windows into the symptoms presented
by fish and insight into the effects of these neurotoxins.
STX’s effects on killifish (Fundulus heteroclitus) were quantified regarding the
expression of c-Fos protein (Salierno et al. 2006), responsible for regulating neural
cells’ survival, and is associated with long term memory (Sadananda and Bischof
2002). It was shown that the expression of this protein decreased, and the fish
presented behavioural alterations including paralysis, lethargy and loss of balance.
STX most likely affects the neural pathways responsible for swimming. In Atlantic
salmon (Salmo salar), it was shown that STX crosses the blood-brain barrier and
that sublethal doses of this toxin affect the activity of brain subregions in the central
nervous system (CNS), possibly affecting the organism’s cognitive abilities (Bakke
and Horsberg 2007). Intracoelomic injections of STX in white seabream (Diplodus
sargus) resulted in an increase of glutathione-S-transferase (GST) activity, an
enzyme responsible for removing xenobiotics, among many other roles. STX also
induced DNA damage (chromosome breaks or loss) and increased erythrocyte
nuclear abnormalities (Costa et al. 2012).
In order to simulate bloom conditions, milkfish (Chanus chanus) fingerlings were
exposed to STX extract and A. minutum cells in increasing concentrations and cell
density, respectively (Chen and Chou 2001). After 24 h, the fish presented oedema,
hyperplasia and necrosis in gill lamellae. The exposure also resulted in increased
mortality rates in the treatments with higher cell density and STX concentrations, due
to increasing oxygen demand following gill damage. Similar results reporting gill
damage following fish exposure to PST-producing dinoflagellate cells were found in
salmon and trout (Mortensen 1985). White (1981) reported high mortality rates after
20 to 60 minutes in Atlantic herring (Clupea harengus harengus), American pollock
(Pollachius virens), winter flounder (Pleuronectes americanus), Atlantic salmon
(S. salar) and cod (Gadus morhua) when dosed intraperitonially (IP) or orally with
62 Ecotoxicology of Marine Organisms

toxin extracts from A. tamarense cultures. Prior to death, the fish presented loss of
balance, immobilization and arrhythmic breathing, consistent with the symptoms
described here in adult fish.
In early stages of development, some fish species present different ecologies than
the adults, starting out as planktonic larvae, and thus occupying the same niche as the
pelagic dinoflagellate PST-producers. Also, earlier stages of development are likely
more vulnerable to the effects of these toxins as they possess higher mass-specific
metabolic rates and they lack fully developed detoxification systems (Vasconcelos
et al. 2010).
Overall, when fish early stages are exposed to bloom simulations in experimental
conditions, it resulted in extremely high mortality rates, nearing the totality of
the experimental population, besides the sublethal effects displayed by the young
(Table 2b). Fish early stages can be exposed to the toxin through feeding on
zooplanktonic vectors, such as copepods, or through direct exposure to the dissolved
toxins. Recently settled flounders (P. americanus), sheepshead minnow (Cyprinodon
variegatus) and mummichog larvae (F. heteroclitus) were fed with contaminated
copepods, acting as vectors of A. fundyense (Samson et al. 2008). After consuming
6–12 contaminated copepods, the fish died. In this study, the fish were also fed with
fewer copepods, resulting in a variety of effects, such as reduced swimming abilities,
prey capture success, predator avoidance and overall activity (Table 2b).
Gosselin et al. (1989) exposed capelin and herring larvae to three different
treatments to ascertain the effects of PSTs through different routes of exposure,
recurring to both direct exposure through feeding the larvae with A. tamarense cells
in increasing densities, and placing toxin extracts in the experimental tanks. Indirect
exposure was achieved by feeding the larvae with contaminated microzooplankton.
Capelin and herring larvae fed on A. tamarense swam erratically, lost motility and
sank to the bottom paralysed, dying after 20 minutes of exposure, contrary to the
lack of effect when exposed directly to the toxin dissolved. Feeding these larvae
with contaminated zooplankton elicited similar results as feeding directly on the
dinoflagellate, resulting in paralysis and high mortality rates. However, exposure
of herring larvae to dissolved STX resulted in a reversible dose-dependent suite of
sensorimotor impairments (Lefebvre et al. 2005), such as spontaneous swimming and
tactile response inhibition. Also, it was shown that older larvae were more susceptible
to the dissolved toxin, likely due to the degree of gill and body maturation leading to
higher toxin uptake.
Monk seal populations have been greatly impacted by PST outbreaks. In
the late 1990s in Cape Blanc Peninsula over 100 monk seals died following PST
intoxication. Tissue analysis revealed PSTs in brain tissue, suggesting that these
toxins were present in the seal’s nervous system (Costas and Lopez-Rodas 1998,
Reyero et al. 2000). The cause has been attributed to PSTs since there were high
levels of these toxins in many fish species that the seals prey upon (Reyero et al.
2000). Dying organisms presented many behavioural alterations, lethargy, paralysis
and sensorimotor discoordination (Hernández et al. 1998).
Earlier, in 1987, over a dozen humpback whales washed ashore dead along
Nantucket Sound. The cause of the stranding was ascertained by analysing fish
and whale tissues. It was determined that one of the fish species analysed, Atlantic
 Effects of Harmful Algal Bloom Toxins on Marine Organisms 63

mackerel (Scomber scombrus), presented high levels of STX and stomachal content
analysis revealed that the whales were previously feeding on this species. It was
worth noting that the time-lapse between the onset of the first symptoms and death
(approximately 90 minutes, Geraci et al. 1989), suggesting a very quick process,
characteristic of severe STX intoxication.
Seabird deaths due to HAB-toxins have been comparatively overlooked.
However, there have been countless events where many seabird species died
following the ingestion of contaminated fish and shellfish (Table 2b). Shumway
et al. (2003) extensively reviewed all the registered seabird deaths that were linked
with HAB-toxins, including PSTs. PSTs were reported to cause loss of motor
coordination and paralysis, resulting in the bird’s inability to feed and thus, causing
death by starvation. Female terns presented an inability to lay eggs due to sublethal
onset of paralysis, resulting in the egg breaking inside the body and causing fatal
haemorrhages. Other species presented severe inflammation of the gastro-intestinal
tract and haemorrhages in the intestines and brain.
Understanding the effects of PSTs, which are produced worldwide, is of vital
importance, since, as reviewed here, the range of possible consequences is very
wide. In some cases, the toxins directly affect the species, causing high mortalities,
and in other cases the toxin accumulates and is transferred throughout many levels
of the marine food web, causing indirect damage to the ocean’s health, communities
and human populations.

Amnesic shellfish toxins

Domoic acid (DA) is a potent neurotoxin produced by some species belonging to
two genera of diatoms, Pseudo-nitzschia and Nitzschia. This toxin is known to cause
Amnesic Shellfish Poisoning (ASP), and the attention on this toxin and its possible
consequences was focused after an incident involving the death of three people in
1987 following the ingestion of mussels contaminated with DA. Afterwards, most
coastal countries developed monitoring programs, regularly analysing bivalve
tissue for DA and other phycotoxins in order to prevent foodborne illnesses. These
monitoring programs have been successful at avoiding further human casualties.
Nevertheless, there have been many events of DA intoxication in marine organisms.
DA acts in neural cells, competing for the same receptors as glutamate, an
excitatory neurotransmitter. By having less affinity for these receptors, glutamate
fails to bind normally, causing excessive concentrations of glutamate outside the
synapses, triggering AMPA, kainate and NMDA receptors’ activation, permanently
opening the neural cell’s membrane, leading to excessive influx of Ca2+ (Berman and
Murray 1997). This causes membrane depolarization and subsequent degeneration of
neural cells. The higher concentration of glutamatergic receptors in the hippocampus,
the brain region responsible for memory acquisition and learning, is the cause for the
memory loss.
In Table 3 we summarized the effects of DA in marine organisms. Bivalves
are common vectors of this toxin and the effects of this toxin seem somewhat
overlooked. DA seems to affect haemolymph chemistry, increase the number of
hemocytes as well as increase cholinesterase activity and DNA damage. On the other
Table 3. Documented cases of marine organisms exposed to and affected by domoic acid (DA). IP – Intraperitoneal; IC – Intracoelomic.
64

Target species DA source Route of Toxicity Tissues Effects Observations References

exposure analysed
Crustaceans Tigriopus Dissolved DA Uptake from 8.62 µM - Death - (Shaw et al.
californicus seawater 1997)
Molluscs C. gigas P. pungens Exposure to Up to 0.86 µg Soft tissue Increased number and Effects (Jones et al.
f. multiseries diatom cells DA g–1 activity of hemocytes reversible 1995)
P. pungens Exposure to 36.3 µg DA g–1 Soft tissue Respiratory acidosis, Haemolymph (Jones et al.
f. multiseries diatom cells haemolymph PO2 and pH 1995)
hypercapnia and returned to
increased bicarbonate, normal
low haemolymph PO2
levels
Ecotoxicology of Marine Organisms

M. P. pungens Exposure to 3.6 µg DA g–1 Soft tissue Increased haemolymph Effects (Jones et al.
californianus f. multiseries diatom cells pH, decreased PO2, reversible 1995)
decreased PCO2
M. edulis Dissolved DA Intramuscular Up to 50 µg DA g–1 - Cholinesterase activity, - (Dizer et al.
injection DNA damage and 2001)
number of hemocytes
increased, phagocytic
activity decreased
Larvae of Dissolved DA Uptake from 5.21 pg DA Whole body Decreased growth, Exposure of (Liu et al.
P. maximus seawater individual–1 shell length and 25 days 2007)
survival
P. maximus Dissolved DA Feed Up to 302.5 ng Whole body Decreased growth and - (Liu et al.
incorporated DA g–1 survival 2008)
with DA
Fish O. kisutch Dissolved DA IC injections Up to 34 µg DA g–1 - Circle, spiral and - (Lefebvre
upside-down et al. 2007)
swimming
O. mykiss Dissolved DA IP injections 0.75 mg DA kg–1 - Increased cortisol - (Bakke et al.
levels, decreased 2010)
attack latency time
Engraulis Dissolved DA IC injections Up to 14 µg DA g–1 - Spinning, - (Lefebvre
mordax disorientation, inability et al. 2001)
to school, death

S. salar Dissolved DA IP injections 6 mg DA kg–1 - Increase in metabolic - (Bakke and

activity Horsberg
2007)
F. heteroclitus Dissolved DA IP injections 5 mg DA kg–1 - Increased c-Fos - (Salierno et al.
activity expression in 2006)
optic brain regions

Sparus aurata Dissolved DA IP injections Up to 9 mg DA kg–1 - Death at higher Sublethal (Nogueira

concentrations, spiral, effects et al. 2010)
circle and upside-down (swimming
swimming anomalies)
reversible
after 24 h
Siganus Dissolved DA IC injections 2 x 103 µg DA kg–1 - Increased CYP1A - (Wang et al.
oramin activity (protein 2008)
involved in xenobiotic
metabolism)

Table 3 contd. ...

Effects of Harmful Algal Bloom Toxins on Marine Organisms
65
...Table 3 contd.
66

Target species DA source Route of Toxicity Tissues Effects Observations References

exposure analysed
Sea birds Callithrix Dissolved DA IP injection 4 mg DA kg–1 - Epileptic seizures, - (Perez-
jacchus death Mendes et al.
2005)
Pelecanus Pseudo-nitzschia Prey 37.17 × 103 µg Digestive tract Death 150 birds dead (Sierra-
occidentalis sp. (S. scombrus) DA kg–1 in 5 days Beltrán et al.
1997)
P. australis Prey 27.9 × 103 µg Pelican stomach Hemorrhage, tissue Death (Work et al.
(E. mordax) DA kg–1 necrosis, death occurred after 1993)
~ 60 min of
exposure
Ecotoxicology of Marine Organisms

P. australis Prey 45–48 × 103 µg Pelican stomach Death (Fritz et al.

(E. mordax) DA kg–1 1992)
Phalacrocorax P. australis Prey 27.9 × 103 µg Cormoran Hemorrhage, tissue Death (Work et al.
penicillatus (E. mordax) DA kg–1 stomach necrosis, death occurred after 1993)
~ 60 min of
exposure
P. australis Prey 45-48 × 103 µg Cormoran Death (Fritz et al.
(E. mordax) DA kg–1 stomach 1992)
Marine Balaenoptera P. australis Prey 258 × 103 µg Whale feces Death - (Fire et al.
mammals acutorostrata (E. mordax) DA g–1 2010)

Callorhinus Pseudo-nitzschia Likely through 18600 × 103 ng Seal’s feces Ataxia, seizures, - (Lefebvre et
ursinus sp. prey DA kg–1 lesions in brain and al. 2010)
heart, death
Kogia Pseudo-nitzschia Likely through 13.56 × 103 ng Whale feces Death - (Fire et al.
breviceps sp. prey DA kg–1 2009)
 K. sima Pseudo-nitzschia Likely through 967 × 103 ng Whale feces Death - (Fire et al.
sp. prey DA kg–1 2009)
Zalophus Pseudo-nitzschia Likely through 96.8 × 103 µg Sea lion’s feces Ataxia, head weaving, - (Bargu et al.
californianus sp. prey DA kg–1 disorientation, seizures 2011)
and death
P. australis Likely through Up to 182.01 × 103 Sea lion’s feces Ataxia, head weaving, - (Scholin et al.
prey µg DA L–1 disorientation, seizures 2000)
and death
Pseudo-nitzschia Maternal 44 × 103 ng DA L–1 Stomach Premature births, - (Goldstein et
sp. transfer contents of abortions, reproductive al. 2009)
premature pups failure and brain
edema
Pseudo-nitzschia Maternal 261 × 103 ng DA Maternal urine Premature births, - (Brodie et al.
sp. transfer L–1 abortions and 2006)
reproductive failure
P. australis Prey - - Seizures, hippocampal - (Silvagni et al.
(E. mordax) atrophy, neural 2005)
necrosis
- - - - Heart and brain - (Zabka et al.
lesions, death 2009)
P. australis Prey 136.5 × 103 µg DA Sea lion’s feces Death - (Lefebvre et
(E. mordax) kg–1 al. 1999)
Effects of Harmful Algal Bloom Toxins on Marine Organisms
67
68 Ecotoxicology of Marine Organisms

hand, exposure to this toxin decreased phagocytic activity, growth and survival rates.
In some cases, the effects of exposure to the DA-producing diatoms were reversible
and the organisms recovered after a short period of time (up to 24 h), emphasizing
the notion that bivalves are quite resilient to DA and other toxins.
Information regarding the effects of DA in wild animals is very scarce and
limited to marine mammals and seabirds. Domoic acid’s effects on fish have been
studied through IC injection (Table 3). This technique allows the use of known
DA concentrations without dispersal throughout the organism’s body. However,
this method does not always allow for ecologically relevant DA concentrations to
be used or for natural DA uptake and transfer between body tissues to take place
naturally. Regarding the effects of DA in fish, most studies concluded that it causes
abnormal swimming behaviour, including spiral, circle and upside-down swimming,
and ultimately death. Other effects that can escalate DA toxicosis are inability to
school in Engraulis mordax (Lefebvre et al. 2001), possibly making the fish easier
targets for predators, disrupting the balance of the food web during diatom blooms.
Killifish (F. heteroclitus) IC injected with up to 9 mg DA kg–1 showed that c-Fos
activity, a protein associated with long term memory (Sadananda and Bischof 2002),
increased in several brain regions, indicating neuronal stress following exposure.
Variations in c-Fos expression can lead to effects at the behavioural levels, as
observed in Salierno et al. (2006), such as disorientation and loss of equilibrium.
One of the main groups affected by domoic acid are marine mammals, more
specifically sea lions. There is an extensive record of sea lion deaths going back
nearly two decades, when over 400 sea lions (Zalophus caifornianus) were found
stranded or displayed neurological symptoms associated with DA intoxication, later
confirmed by detecting DA in sea lions’ tissues (Scholin et al. 2000). The cause
of death was attributed to ingestion of contaminated anchovies, a common food
source for these mammals. Behavioural tests and magnetic resonance imaging
(MRI) performed on sea lions displaying intoxication symptoms revealed abnormal
behaviours, such as head weaving, ataxia and severe disorientation. The MRIs
showed hippocampal lesions damaging hippocampal-thalamic networks. Also, DA
has been detected in the stomach contents of premature pups and shown to elicit
premature births, abortions and death of pregnant sea lions (Brodie et al. 2006)
due to the consumption of contaminated prey, possibly endangering this species’
populations. Throughout the years, many other events of sea lion mortality have been
attributed to DA intoxication (Table 3).
Studies regarding the interaction of HAB-toxins occurring simultaneously is very
scarce. However, between February–April 2008, over 100 dolphins (T. truncatus)
were found stranded along the coast of Texas, and their tissues were positive for DA,
brevetoxins and okadaic acid (Fire et al. 2011), although in different proportions and
only a small percentage was positive for more than one toxin. The mass stranding
may be linked to an interaction of these three toxins, but without historical data and
means of comparison, it may not be safe to conclude so.
DA, as mentioned above, acts on glutamatergic receptors, mainly present in
organisms with developed brains, possibly explaining the discrepancy between
the effects caused in vertebrates and invertebrates. Invertebrates are likely less
affected by this toxin, since they mostly lack complex brains and possess effective
 Effects of Harmful Algal Bloom Toxins on Marine Organisms 69

elimination systems, as in the case of bivalve molluscs. The fact that bivalves seem
to be less affected and are efficient at eliminating DA does not exclude the sublethal
effects that it may cause in other organisms higher up the food web, through chronic
ingestion of contaminated prey.

Brevetoxins
Brevetoxins (BTXs—PbTx1-10, BTX 1-4) are produced by dinoflagellates and
raphidophytes and can cause Neurotoxic Shellfish Poisoning (NSP). These toxins
are a group of complex polycyclic polyether compounds that alter the properties
of membranes in excitable cells by binding to voltage sensitive sodium channels in
nerve cells, leading to membrane depolarization and disrupting normal processes
in nerve cells (Landsberg 2002, Lopes et al. 2013). The term “red tides” is mainly
associated with blooms of Karenia brevis (= Gymnodinium breve), which proliferate
in great concentrations and their pigments discolour the surrounding seawater. This
dinoflagellate is mainly responsible for causing many events of mass mortality in
marine organisms, with reports dating back to the 19th century (Glennan 1887).
Although no human deaths have been related to brevetoxins, human populations are
affected at the sublethal level, mostly through consumption of contaminated shellfish
or aerosol inhalation during red tide events (Landsberg 2002). Effects of brevetoxins
in marine organisms, from copepods to marine mammals, are summarized in Table 4.
Brevetoxins can have allelopathic effects on other species of phytoplankton,
as shown in Prince et al. (2008), where cultures of Asterionellopsis glacialis,
Prorocentrum minimum and Skeletonema costatum presented decreased growth rates
when their individual cultures were mixed with K. brevis cultures and exudates.
Studies on copepods revealed that these organisms are very sensitive to
K. brevis, by presenting accelerated heart rates, loss of motor control, suppressed
swimming behaviour, lethargy, paralysis, regurgitation and decreased survival and
growth (Cohen et al. 2007, Huntley et al. 1987, Huntley et al. 1986, Sykes and
Huntley 1987, Turner et al. 1996). Other BTXs producers have elicited negative
impacts upon feeding in copepods (Uye 1986, Uye and Takamatsu 1990).
Bivalves, as one of the main vector of phycotoxins, also accumulate high
concentrations of BTX, with very few studies regarding its effect. Leverone et al.
(2007) showed that when exposed to K. brevis cells and extracts caused reduced
clearance rates in A. irradians, C. virginica, Mercenaria mercenaria and P. viridis.
Recruitment of bay scallop (A. irradians concentricus) in North Carolina was greatly
affected by K. brevis blooms, jeopardizing the sustainability of scallop beds in the
region (Summerson and Peterson 1990).
Red tides can also affect the species’ abundance and richness of the impacted
areas, in some cases nearly wiping out many important benthic infaunal species
(Simon and Dauer 1972), decreasing richness in fish species by 50 percent and
causing a decrease in invertebrate species’ abundance in general (Dupont et al. 2010).
Knowledge on the effects of BTXs in fish is very scarce beside the numerous
accounts of fish kills associated with BTXs (Gunter et al. 1948, 1947, Rounsefell and
Nelson 1966, Thronson and Quigg 2008). Flaherty and Landsberg (2011) reported
reduced annual recruitment in Cynoscion nebulosus, C. arenarius, and Sciaenops
Table 4. Documented cases of marine organisms exposed to and affected by okadaic acid (OA) and dinophysistoxins (DSTs).
70

Target species OA source Route of Toxicity Tissues Effects Observations References

exposure analysed
Molluscs M. edulis Dissolved OA Mixed with 2.5 nM OA - Increased DNA - (McCarthy et al.
algal diet fragmentation 2014)
M. Dissolved OA In vitro exposure Up to 500 nM - DNA damage - (Prego-Faraldo
galloprovincialis OA leading to necrosis et al. 2015)
and apoptosis in gill
tissue
Dissolved OA Mixed with Up to 6.5 µg - Up-regulation of - (Manfrin et al.
mussel feed OA gene transcripts 2010)
associated with
stress response
Ecotoxicology of Marine Organisms

C. gigas Dissolved OA Mixed with 2.5 nM OA - Increased DNA - (McCarthy et al.

algal diet fragmentation 2014)
C. virginica P. minimum Exposure to 3.9 × 106 cells - Larvae presented - (Wikfors and
dinoflagellate ml–1 lower growth Smolowitz 1995)
cells rates, and slower
development
P. perna P. lima Exposure to Up to 10000 - Higher incidence - (Carvalho Pinto-
dinoflagellate cells mussel–1 of micronuclei and Silva et al. 2005)
cells nuclear lesions in
hemocytes
Dissolved OA Uptake from 0.3 µg OA - Higher incidence - (Carvalho Pinto-
seawater of micronuclei in Silva et al. 2003)
hemocytes
 R. decussatus P. lima extracts Exposure to Up to 100 nM - DNA damage in gill - (Flórez-Barrós et
and cells extracts and OA and 20000 tissue al. 2011)
dinoflagellate cells ml–1
cells
Fish Dicentrarchus P. lima pre- Exposure to Previously - Reduced feeding - (Ajuzie 2007)
labrax conditioned pre-conditioned with 9 × 103 reflexes, abnormal
seawater sewater with cells ml–1 swimming patterns
P. lima
P. lima Exposure to 4.5 × 103 cells - Death - (Ajuzie 2007)
dinoflagellate ml–1
cells
Effects of Harmful Algal Bloom Toxins on Marine Organisms
71
72 Ecotoxicology of Marine Organisms

ocellatus and Riley et al. (1989) revealed that upon hatching, larvae of S. ocellatus
were negatively affected by BTXs, developing deformities, swimming erratically and
being paralysed before dying. Seabird deaths attributed to brevetoxicosis date back
to the early 70’s, when thousands of lesser scaup (Athya affinis) died concurrently
in a red tide event (Forrester et al. 1977), along with many other seabird species
throughout the years (Landsberg et al. 2009). Also, a number of sea turtles strandings
have been positively linked to red tide events, as the number of strandings increases
during red tides (Landsberg et al. 2009).
The highly endangered Florida manatees (Trichechus manatus latirostris) have
suffered great impacts from red tides. In 1996, over a hundred manatees died following
a red tide caused by K. brevis (Bossart et al. 1998), likely through prolonged exposure
to BTX aerosols or ingestion of contaminated seawater. Recently, Flewelling et al.
(2005) showed that seagrass (Thalassia testudinum) accumulates high concentrations
of BTXs, the main manatee food source, opening a likely pathway for manatees’
BTX uptake. Since 1996, many other events of marine mammal mortality have been
attributed to BTXs intoxication (reviewed in Landsberg et al. 2009). What is worth
noting is the death of 107 bottlenose dolphins (Tursiops truncatus), following a red
tide in 2002. Again, Flewelling et al. (2005), analysed dolphin tissues and undigested
fish remains, and concluded that both had very high levels of BTXs, enough to
cause brevetoxicosis and death. Until then, fish were not considered likely vectors
of this toxin, as it caused the fish to die in a very short period of time, limiting the
toxin transfer higher up the food chain. Several mass fish kills followed red tides
(Landsberg 2002, 2009, Steidinger et al. 1973), drawing attention to this toxin’s
ichthyotoxic potential. Many studies have revealed that fish are more sensitive to
BTXs dissolved in seawater than ingestion of K. brevis cells. It was shown that fish
survive direct exposure to the dinoflagellate cells, whereas exposure to the dissolved
toxin leads to death (Landsberg et al. 2009).
The effects of BTXs are still poorly understood, especially in marine
invertebrates, despite the great impact red tides have on ecosystems worldwide.

Diarrhetic shellfish toxins

Diarrhetic Shellfish Toxins (DSTs) are produced by many species of Dinophysis
and Prorocentrum, both cosmopolitan genera occurring worldwide. Dinophysis are
pelagic species, whereas Prorocentrum are typically epibenthic. DSTs are lipophilic
toxins, comprising okadaic acid (OA) and dinophysistoxins (DTX).
OA was first isolated from the marine sponge Halichondria melanodocia. It was
later discovered that this toxin was produced by a dinoflagellate that was accumulated
in the sponge through filter-feeding. OA specifically inhibits the activity of protein
phosphatase 1 and 2, two of the main protein phosphatases in mammals, increasing
protein phosphorylation. Gastrointestinal distress symptoms may arise from the loss
of balance in membrane transport and substance secretion, resulting in loss of fluids.
The effects of OA and dinophysistoxins on marine organisms are summarized
in Table 5. DSP outbreaks are not caused any human fatalities (Hallegraeff et al.
2003), and of all the other shellfish poisonings, it can be considered the mildest,
with patients fully recovering after few days. However, OA has been identified as a
 Effects of Harmful Algal Bloom Toxins on Marine Organisms 73

tumour promoting compound (Suganuma et al. 1988), posing additional threats to

human health and marine life alike.
OA has been documented to be actively accumulated in sponges, which increases
the sponge’s immune system against parasites and bacterial infections (Schröder
et al. 2006, Wiens et al. 2003).
It is worth noting that, although greatly lacking supportive evidence, P. lima has
been shown to elicit allelopathic effects when grown in culture with other microalgal
species. OA and DTX-1 produced by P. lima inhibit the growth of the other species
present, and, at lower concentrations it enhanced growth in P. lima cultures (Windust
et al. 1996). Similarly, when exposed to OA, Dunaliella tertiolecta cultures decreased
cell density and increased oxidative stress response. Additionally, OA inhibited the
ability for electron transport in photosystem II, impairing photosynthesis (Perreault
et al. 2012).
Regarding other marine organisms, OA has been found to accumulate in
numerous shellfish species, the main vectors of this toxin. Blue mussels have been
reported to accumulate high concentrations of OA for long periods of time (up to
five months, Shumway 1990). Typically, bivalves are not directly affected by the
toxins, most likely due to their rapid clearance rates and the fact that many species
can convert the parent compound into less toxic derivatives (Suzuki et al. 1999).
Most studies regarding OA toxicity beyond DSP symptoms have been performed in
mice and human cell lines. The toxic effects of OA on marine organisms is still fairly
unknown. Recently it was shown that OA induced genotoxic and cytotoxic effects
on bivalves (McCarthy et al. 2014). OA has been shown to elicit the formation of
micronuclei and nuclear lesions in mussel hematocytes (Carvalho Pinto-Silva et al.
2003, 2005) and up-regulation of gene transcripts associated with stress response
(Manfrin et al. 2010). In clams (R. decussatus), OA induced higher DNA damage to
clams’ gills than in hemocytes when exposed to lower OA concentrations, as opposed
to when they were exposed to high OA concentrations for a shorter period of time
(Flórez-Barrós et al. 2011). Mussels (M. galloprovincialis) presented similar results,
with hemocytes having less DNA damage than gill tissue when exposed to OA, and
gills presenting increased DNA damage at lower OA concentrations (Prego-Faraldo
et al. 2015). Gills are the first tissue to come in contact with the toxin, and the lack of
response when exposed to higher concentrations suggests: (i) very efficient defence
mechanism in bivalves and (ii) detoxification pathways by metabolizing OA into less
toxic compounds.
While the target protein phosphatases are as sensitive to OA in vitro exposure
in blue mussels (M. edulis) as they are in other organisms (Svensson and Förlin
1998), there are no records of mussel mortalities due to OA exposure. Mussels can be
exposed to OA throughout the year; thus, it is proposed that they possess detoxification
mechanisms. Svensson et al. (2003), found that M. edulis can accumulate OA in the
lysosomal system, therefore, preventing cellular damage in hematocytes.
Marine turtles have been found to accumulate OA in their tissues, produced by
the epibenthic Prorocentrum spp., likely present on the surface of the algae that the
turtles consume. Coincidentally, two DSP-producing Prorocentrum species (P. lima
and P. concavum) occur where there is high risk of fibropapillomatosis, a neoplastic
Table 5. Documented cases of marine organisms exposed to and affected by brevetoxins (BTXs).
74

Tissues
Target species Brevetoxin source Route of exposure Toxicity Effects Observations References
analysed
Phytoplankton K. brevis
Asterionellopsis Exposure to Up to 55 ng Indicates (Prince et al.
extracellular - Inhibition of growth
glacialis exudates L–1 PbTx-2 allelopathy 2008)
exudates
K. brevis
Skeletonema Exposure to Up to 55 ng Indicates (Prince et al.
extracellular - Inhibition of growth
costatum exudates L–1 PbTx-2 allelopathy 2008)
exudates
K. brevis
Prorocentrum Exposure to Up to 55 ng Indicates (Prince et al.
extracellular - Inhibition of growth
minimum exudates L–1 PbTx-2 allelopathy 2008)
exudates
Crustaceans A. tonsa Exposure to Up to 2400 Decreased survival (Prince et al.
Ecotoxicology of Marine Organisms

K. brevis - -
dinoflagellate cells cells ml–1 and egg production 2006)
Ptychodiscus brevis Exposure to Up to 19567 Increased feeding (Turner and Tester
- -
(now K. brevis) dinoflagellate cells cells ml–1 rates 1989)
Up to 1 × 107
Exposure to Increased
cells L–1 and (Cohen et al.
K. brevis dinoflagellate cells - mortality at higher -
15 µg PbTx-2 2007)
and brevetoxins concentrations
L–1
C. pacificus Loss of motor
P. brevis (now Exposure to 0.68 ng C control, increased (Huntley et al.
- -
K. brevis) dinoflagellate cells cell–1 heart rate and 1987)
lethargy
Avoided feeding,
P. brevis (now Exposure to 0.68 ng C increased heart rate (Huntley et al.
- -
K. brevis) dinoflagellate cells cell–1 and loss of motor 1986)
control
Increased heart rates
P. brevis (now Exposure to Up to 1000 ng (Sykes and
- and loss of motor
K. brevis) dinoflagellate cells C cell–1 Huntley 1987)
control
 Centropages P. brevis (now Exposure to Up to 19567 Decreased feeding (Turner and Tester
- -
typicus K. brevis) dinoflagellate cells cells ml–1 rates 1989)
Up to 1 × 107
Exposure to Increased
cells L–1 and (Cohen et al.
K. brevis dinoflagellate cells - mortality at higher -
15 µg PbTx-2 2007)
and brevetoxins concentrations
L–1
Labidocera P. brevis (now Exposure to Up to 19567 Increased feeding (Turner and Tester
- -
aestiva K. brevis) dinoflagellate cells cells ml–1 rates 1989)
P. brevis (now Exposure to Up to 19567 Increased feeding (Turner and Tester
Oncaea venusta - -
K. brevis) dinoflagellate cells cells ml–1 rates 1989)
Paracalanus P. brevis (now Exposure to Up to 845 Decreased feeding (Turner and Tester
- -
quasimodo K. brevis) dinoflagellate cells cells ml–1 rates 1989)
Up to 5 × 106
Exposure to
Temora cells L–1 and Suppressed (Cohen et al.
K. brevis dinoflagellate cells - -
turbinata 15 µg PbTx-2 swimming behaviour 2007)
and brevetoxins
L–1
Molluscs A. irradians P. brevis (now Exposure to Decreased (Summerson and
- - -
K. brevis) dinoflagellate cells recruitment Peterson 1990)
Exposure to
Up to 22000 Reduction in (Leverone et al.
K. brevis dinoflagellate cells -
cells ml–1 clearance rates 2007)
and extracts
Exposure to
Up to 24600 Reduction in (Leverone et al.
C. virginica K. brevis dinoflagellate cells -
cells ml–1 clearance rates 2007)
and extracts
Exposure to
Up to 23100 Reduction in (Leverone et al.
M. mercenaria K. brevis dinoflagellate cells -
cells ml–1 clearance rates 2007)
and extracts
Table 5 contd. ...
Effects of Harmful Algal Bloom Toxins on Marine Organisms
75
...Table 5 contd.
76

Tissues
Target species Brevetoxin source Route of exposure Toxicity Effects Observations References
analysed
Exposure to
Up to 23800 Reduction in (Leverone et al.
P. viridis K. brevis dinoflagellate cells -
cells ml–1 clearance rates 2007)
and extracts
Fish Larval Deformities,
P. brevis (now Exposure to Up to 2040
Sciaenops - abnormal swimming, - (Riley et al. 1989)
K. brevis) dinoflagellate cells cells ml–1
ocellatus paralysis and death
Sea turtles Likely through (Landsberg et al.
Caretta caretta K. brevis - - Death -
prey 2009)
Likely through (Landsberg et al.
Chelonia mydas K. brevis - - Death -
prey 2009)
Ecotoxicology of Marine Organisms

Lepidochelys Likely through (Landsberg et al.

K. brevis - - Death -
kempii prey 2009)
Seabirds Aythya affinis Over 6000
Prey
G. breve (now Lethargy, ataxia, specimens (Forrester et al.
(contaminated - -
Karenia brevis) death found dead or 1977)
clams)
moribund
Weakness,
Prey inability to dive,
G. breve (now (Quick and
(contaminated - - unresponsiveness, -
K. brevis) Henderson 1974)
clams) loss of reflexes,
death
Prey
G. breve (now (Schreiber et al.
(contaminated - - Dead or weakened
K. brevis) 1975)
clams)
Prey
Phalacrocorax Cerebellar ataxia, (Kreuder et al.
K. brevis (contaminated - - -
auritus death 2002)
fish)
 Kidney,
liver, testes,
Analysis
heart
Up to 33.1 performed
Sterna maxima K. brevis - muscle, Death (Vargo et al. 2006)
PbTx ng g–1 on beached
stomach,
specimens
intestines,
lung
Analysis
16.2 PbTx performed
Larus atricilla K. brevis - Kidney Death (Vargo et al. 2006)
ng g–1 on beached
specimens
Marine Trichechus Death following
Mammals manatus pulmonary oedema
Aerosols or Over 100 (Bossart et al.
latirostris K. brevis - - and hemmorhage,
ingested seawater manatees died 1998)
congestion of liver
and kidneys
Contaminated Up to 300 ~ 30 manatees (Flewelling et al.
K. brevis Liver Death
seagrass ng g–1 died 2005)
Possibly ingestion Congestion and
G. breve (now ~ 40 manatees (O’Shea et al.
of contaminated - - hemorrhage in brain
K. brevis) died 1991)
ascidians tissue, death
Tursiops Up to 613 Over 100 (Flewelling et al.
K. brevis Prey Feces Death
truncatus ng g–1 dolphins died 2005)
P. brevis (now Likely through Up to 15820 Over 700
Liver Death (Geraci 1989)
K. brevis) prey ng g–1 dolphins died
G. breve (now Over 100
Prey - - Death (Mase et al. 2000)
K. brevis) dolphins died
Up to 2896 Stomach
K. brevis Prey Death - (Fire et al. 2007)
ng g–1 contents
Effects of Harmful Algal Bloom Toxins on Marine Organisms
77
78 Ecotoxicology of Marine Organisms

disease specific to sea turtles. Therefore, OA may play an important role in this
disease’s etiology (Landsberg 2002, Landsberg et al. 1999).
Although not positively linked to DSTs, many seabird deaths occurred after DSP
and other toxin’s outbreaks (Shumway et al. 2003). The presence of DSTs is likely
to decrease the organism’s fitness and well-being, making them more vulnerable to
other toxicants.
Despite being regarded as a less dangerous toxin, OA has been shown to cause a
wide array of responses and effects in marine organisms, highlighting the potential of
this toxin to affect many other organisms, including human populations chronically
ingesting low doses of a tumour promoting toxin.

Future Directions and Concluding Remarks

It is predicted that many changes in the world’s oceans will occur. Increasing
temperature and CO2 concentrations are but two of the many factors affecting HAB
distribution, frequency and intensity. HAB ecology is complex, and it is dependent
on the interaction of many factors, including ocean stratification, oceanic currents,
nutrient availability and precipitation (Wells et al. 2015). Currently, there are a
number of studies on the effect of climate change in HABs. However, the interactions
simulated are scarce and do not allow for species adaptation and plasticity.
Temperature fluctuations directly affect phytoplankton communities. Typically, with
increasing temperatures, phytoplanktonic species tend to have higher growth rates
until a species-specific temperature threshold is met (Wells et al. 2015). There is
growing evidence that HABs are increasing in frequency and intensity throughout
the globe (Dolah 2000), and further studies are needed to better understand the shifts
in HAB ecology and physiology in these new conditions.
It is evident from the present work that there is a great lack of knowledge on
the effects that HAB toxins have on early stages of development, a very sensitive
and critical stage in an organism’s life. Moreover, most toxins may be chronically
accumulated and, in many cases, not elicit outward signs of toxicity. Here, we
reviewed the effects that HAB toxins have on marine organisms, more specifically,
the four main groups of toxins (PSP, ASP, NSP and DSP). Still, there has been growing
evidence that in addition to the increasing intensity and toxicity of these blooms, new
and emerging toxins, such as palytoxins, cyclic imines, tetrodotoxins and ciguatoxins,
are occurring in regions previously undetected (Soliño et al. 2014). Also, the co-
occurrence of emerging toxins with endemic HAB-toxins may lead to additive,
synergistic or antagonistic effects on marine organisms; however, the available data
is not sufficient to confirm and characterize such effects. Multidisciplinary studies
are necessary to comprehensively understand the effects of exposure to multiple
toxins. Thus, there is great need to reach out to policy makers and work alongside
with the monitoring programs already implemented in many countries worldwide, to
better understand the risk faced by organisms exposed to marine toxins in the natural
environment, the consequences on an ecosystem’s stability and to develop models of
biotoxins kinetics useful to predict the toxic effects highlighted in this study.
 Effects of Harmful Algal Bloom Toxins on Marine Organisms 79

Acknowledgements
This study had the support of Fundação para a Ciência e Tecnologia (FCT),
through the strategic project UID/MAR/04292/2013 granted to MARE and UID/
Multi/04326/2019 granted to CCMAR. The research leading to these results has
received funding from the project Cigua (PTDC/CTA-AMB/30557/2017) supported
by the Portuguese Foundation for Science and Technology (FCT) and FEDER.
The authors would like to thank the Portuguese Foundation for Science and
Technology for the “Investigador FCT” grants to RR for a project grant PTDC/BIA-
BMA/28317/2017, and the Ph.D. and PRC and the Ph.D. scholarship to V.M. Lopes
(SFRH/BD/97633/2013).

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5 Overview of Phytoplankton
Indicators and Biomarkers
as Key-Tools for Trace
Element Contamination
Assessment in Estuaries
Maria Teresa Cabrita,1,* Bernardo Duarte,2 Carla Gameiro,2 Ana
Rita Matos,3 Isabel Caçador2 and Rita M. Godinho1

INTRODUCTION
Estuaries are among the most productive ecosystems on Earth, providing a wide
range of resources, benefits and natural services. Recycling of nutrients and other
materials, such as pollutants, is the main natural service provided by estuaries,
placing these ecosystems among the most valuable on a global scale (Constanza
et al. 1997, Millennium Ecosystem Assessment 2005). As major centres of human
population and pivotal points of trade, industry, energy production, fisheries and
tourism, estuaries are severely impacted by human activities leading to a decline
in the water quality, consequently threatening estuarine organisms as a whole. This
is particularly relevant in estuaries where human populations rely on estuarine
resources for food and recreation. Trace elements, such as chromium (Cr), cobalt
(Co), nickel (Ni), copper (Cu), zinc (Zn), cadmium (Cd), mercury (Hg) and lead (Pb)
are among the most persistent pollutants found in estuaries (Deforest et al. 2007).

1
Portuguese Institute of Sea and Atmosphere (IPMA), Av. de Brasília, 1449-006 Lisboa, Portugal;
Present affiliation: Centro de Estudos Geográficos (CEG), Instituto de Geografia e Ordenamento do
Território (IGOT), University of Lisbon, Rua Branca Edmée Marques, 1600-276 Lisbon, Portugal.
2
MARE – Marine and Environmental Sciences Centre, Faculty of Sciences of the University of Lisbon,
Campo Grande 1749-016 Lisboa, Portugal.
3
BioISI—Biosystems and Integrative Sciences Institute, Plant Functional Genomics Group,
Departamento de Biologia Vegetal, Faculdade de Ciências da Universidade de Lisboa, Campo Grande,
1749-016 Lisboa, Portugal.
* Corresponding author: [email protected]
90 Ecotoxicology of Marine Organisms

The main sources of trace element pollution in estuaries, linked to anthropogenic

activities, are industrial, wastewater and domestic effluents (Fu and Wang 2011),
atmospheric inputs, boating and dredging activities (Förstner and Wittmann 1979,
Eggleton and Thomas 2004). Impacts of these activities mainly include the transfer
of trace elements to the water column (Nriagu 1990, Nayar et al. 2004). The impact
of this mobilisation on estuaries is of particular concern due to the persistence,
biogeochemical recycling and toxicity of several elements (Deforest et al. 2007, Pan
and Wang 2012). Although dissolved trace element concentrations in estuarine waters
are relatively low due to particle adsorption (Hoffmann et al. 2012), contamination
has been detected in different trophic levels of estuarine food webs (Wang 2002).
This may have dramatic consequences for primary productivity and commercially
important fish stocks.
Phytoplankton are considered a promising indicator tool for trace elements, as
they can accumulate considerable amounts of these elements (González Dávila 1995),
and are at the base of estuarine food webs. Because of their ubiquitous presence in
estuaries, phytoplankton play an important role in trace element bioaccumulation,
transport and recycling in these systems. Recently, some estuarine phytoplankton
species have been identified as effective indicators of trace element contamination
under both natural conditions and extreme contamination events (Cabrita et al. 2013,
2014, 2016), due to their rapid response to changes in element availability in the
water column (GonzálezDávila 1995, Sunda and Huntsman 1998, Rainbow 2006).
In particular, the diatom Phaeodactylum tricornutum has been widely used as a
model species in several ecotoxicology and trace element assessment works (e.g.,
Horvatić and Peršić 2007, Cabrita et al. 2013, 2014, 2016) because this species reacts
rapidly to trace element fluctuations and is representative of estuarine and coastal
phytoplankton communities which are frequently dominated by diatoms.
Once incorporated into phytoplankton cells, trace element have been found to
cause impairment of fundamental physiological processes (Anderson and Morel
1978, Shaw 1990, Sunda and Huntsman 1998, Küpper et al. 2002, Cabrita et al.
2016), affecting cell growth (Thomas et al. 1980, Brand et al. 1986, Cabrita et
al. 2016) and photosynthetic performance (Cid et al. 1995, Küpper et al. 1996,
Cabrita et al. 2016), thus providing the opportunity to identify specific indicators
and biomarkers of trace element stress, in representative estuarine phytoplankton
species. Besides these physiologically-based indicators, the phytoplankton
community as a whole and individual taxa, known to be altered by trace elements
(Hollibaugh et al. 1980, Sunda 1989), can also be used as sensitive and efficient
indicators of trace element contamination (Cabrita et al. 2014), although these have
been generally disregarded within the scope of element pollution assessments. The
variety of phytoplankton responses to trace element loading may thus be used to
extract adequate indicators and biomarkers to be applied for the early detection of
trace element contamination in estuaries. Phytoplankton indicators and biomarkers
effectively integrate both physical-chemical status and biological quality elements,
effectively enabling a holistic perspective of the adverse impact of pollution on the
health status of the estuarine organisms, populations and ecosystem. In estuaries
and coastal waters where trace element contamination is an enduring problem, a
comprehensive identification of phytoplankton indicators and biomarkers as key-
 Overview of Phytoplankton Trace Element Indicators and Biomarkers 91

tools for the early detection of element stress is an emerging requirement. Moreover,
the value of phytoplankton biomarkers has been increasingly emphasised within the
scope of the Water Framework Directive (WFD, European Commission 2000) and
the Marine Strategy Framework Directive (MSFD, European Commission 2008).
This offers the opportunity to promote an ecosystem-based approach including both
phytoplankton indicators and biomarkers of element stress as valuable integrative
key-tools to monitoring programmes in the future.
This chapter provides an integrated overview of the overall alterations in the
water column induced by trace element contamination in estuarine systems, using the
Tagus Estuary as a case-study, in order to identify reliable and efficient phytoplankton
indicators and biomarkers of element stress to be employed for the early detection
and monitoring of trace element contamination in estuarine systems. Impacts of
trace element contamination on phytoplankton are addressed using dredging events
such as drivers of trace element contamination in estuaries, as well as laboratory
experiments simulating in situ conditions of trace element loading. Suitable sentinel
and biomonitoring phytoplankton species are also suggested along with techniques
and methodologies able to address field experimental constraints.
We present a straightforward and logical step-by-step approach for setting up a
successful plan to obtain suitable phytoplankton indicators and biomarkers of element
stress, following the pathway of trace elements from their sources in the water column
to their allocation within phytoplankton cells, and subsequent effects (Fig. 1). The
first step is the chemistry of trace elements in the contaminated estuarine environment
which controls their bioavailability. The second step is the trace element accumulation
into phytoplankton to provide proof that the bioavailable trace elements are actually
taken up and in what relative proportions, and the evaluation of phytoplankton as a
biomonitoring tool. Next, intracellular trace element partitioning in phytoplankton
is tackled to understand consequences to cellular toxicity, tolerance mechanisms,
and element fate. The following step is the overall effect of trace element overload
on phytoplankton fundamental processes, such as growth and photosynthesis, to
determine potential physiologically-based indicators. As the trace element combined
effects on individual cells define the overall effect on phytoplankton populations and
communities, community and individual taxa indicators can also be extracted. Finally,
phytoplankton biomarkers are investigated and identified, providing further evidence

Fig. 1. Step-by-step approach to obtain suitable phytoplankton indicators and biomarkers of trace element
stress.
92 Ecotoxicology of Marine Organisms

supporting the use of phytoplankton as key-tool for the early detection of element
stress. Steps are illustrated with relevant results obtained from our research work,
providing other detailed sources supporting our findings. The phytoplankton indicators
and biomarkers of element stress herein identified can potentially be applied to element
contaminated estuarine and coastal systems worldwide.

Trace Element Bioavailability in Estuaries

Estuaries receive trace elements, mainly through atmospheric inputs, industrial,
wastewater and domestic effluents, boating, harbour and dredging activities
(Eggleton and Thomas 2004, Fu and Wang 2011). Despite rigorous environmental
regulations requiring improved effluent quality, and recent developments of
innovative, cheaper and more effective technologies for reducing the amount of
trace elements in effluents (Barakat 2011), historical contamination is still a serious
threat to estuarine ecosystems (Johnston and Roberts 2009). Estuarine sediments
have accumulated trace elements mainly from historical industrial pollution sources
for long periods of time and are still being contaminated by those elements that are
currently used in industrial processes and remain in the effluents produced (Förstner
and Wittmann 1979, Cundy et al. 2003). Disturbance resulting from storms, strong
currents, tides, bioturbation, trawling and dredging can cause periodic remobilisation
of contaminated sediments into the estuarine water column (Calmano et al. 1993,
Eggleton and Thomas 2004) where sediment particles rapidly bind to particulate
matter and ultimately sink again into the estuarine bottom sediments (Cundy et al.
2003). However, during resuspension, trace elements are released by sediments into
the water column (Eggleton and Thomas 2004). For instance, dredging activities,
which are recurrent operations to maintain the water depth of navigation and access
channels (Kenny and Rees 1994), are frequently a major source of trace elements in
estuaries. Large variations in trace element concentrations can be observed during
dredging events in the dissolved and suspended particulate fractions (Cabrita et al.
2014). The exchanges of trace elements between dissolved and particulate fractions
may occur in short-time scales, namely within the first few minutes following
sediment resuspension (Vale et al. 1998, Caetano et al. 2003). The exposure of
the dredged anoxic sediments to a different chemical environment, such as the
oxygenated estuarine waters, can induce several reactions on particle surfaces,
resulting in desorption and transformation of trace elements into more bioavailable
forms, depending on their relative solubility in such conditions (Zhuang et al. 1994,
Caetano et al. 2003). In particular, sulphide-bound complexes tend to be rapidly
oxidised when particles are exposed to oxygen (Calmano et al. 1993). The result
is the release of cations to solution, followed by the formation of fresh iron-oxides
concomitantly with the incorporation of other elements that have been mobilised to
the solution. The desorption rates of elements adsorbed to sulphides may vary. For
instance, Cu, Hg and Pb are released faster than Zn to the water column (Caille et al.
2003). The enhanced dissolved trace elements transferred to the water column may
then accumulate in the estuarine biota, including phytoplankton.
A prerequisite to adequately assessing impacts of trace element loading on
phytoplankton is the measurement of the variation of trace element bioavailability
 Overview of Phytoplankton Trace Element Indicators and Biomarkers 93

in the water column over space and time, during disturbance events. Only
environmentally bioavailable trace elements in the estuarine water column, rather
than total element concentrations, are relevant to accumulation and have the
potential for toxicity in phytoplankton (Rainbow 2006). Diffusive gradients in thin
films (DGTs) have been recently used as a possible approach to directly assess
in situ environmental bioavailability of trace elements in estuarine waters (INAP
2002), minimising problems associated with extremely low concentrations typical
of some trace elements and contamination risks during collection and analysis. The
DGT passively accumulates labile element species from solution while deployed in
situ, avoiding contamination problems related to conventional water collection and
filtration procedures. Labile element species are trapped in binding agents (Chelex100
resins) after diffusion through a diffusive gel (type APA, 0.8 mm thickness, open
pore) (Zhang and Davison 1999). The Chelex resin used in DGT is selective for
free or weakly complexed species, thus providing a proxy for the element labile
fraction in solution and consequently, bioavailability (further details in INAP 2002).
Trace element concentrations are then directly quantified in resin eluates, typically
by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The DGT exposure
period typically varies between hours to days, providing a time-integrated measure
of labile-element species over the chosen deployment time interval (Davison and
Zhang 1994). This is particularly relevant as bioavailability of trace elements
commonly fluctuates over time in estuaries due to differential inputs of river and
sea water. Extremely low trace element bioavailability (close to detectability limits)
may occur, particularly for some elements, shifting to much higher levels during
the sediment disturbance period (Cabrita et al. 2014), depending on the source of
contamination (e.g., amount of suspended sediments, and the degree, frequency and
duration of contamination) and the physical, chemical and biological characteristics
of the estuarine area (Vale et al. 1998). Particularly concerning dredging events,
operational conditions and rhythm of dredging, sediment composition and trace
element fractionation (Newell et al. 1998, Vale et al. 1998), coupled with fluctuations
of the currents with the tide (Vale and Sundby 1987), all contribute to large variations
of total trace element concentrations and bioavailability found during the dredging
period. Difficulties in recording trace element transformations during resuspension
events have been identified by Vale et al. (1998), highlighting the complexity
and quickness of fractionation of trace elements in solids, via dredging simulated
laboratory experiments. Therefore, appropriate time-scales must be chosen in order
to assess the trace element contamination event as accurately as possible, and in
agreement with the particular objectives of each study. It is important to bear in
mind that each DGT measurement represents a sole time-integrated measure of
labile-element species over a chosen deployment time interval, and that extended
measurements in time are a fundamental requirement in order to have a realistic
picture of the impact of sediment disturbance. In our studies investigating trace
element bioavailability changes resulting from dredging conducted in the Tagus
Estuary (Cabrita et al. 2014), DGTs were used with a 48-h exposure period, three
times before dredging, weekly during the dredging period and twice a few months
after dredging had stopped, for an accurate comparison of water column changes.
A significant increase of trace element bioavailability was found during dredging
94 Ecotoxicology of Marine Organisms

in comparison with non-dredging periods, indicating the escape of trace elements

as contaminated sediments were dredged and transferred to the dredger vessel
(Fig. 2). Comparison between non-dredging and dredging conditions in terms of
concentration medians, highlighted increases of 12 times for Cr (0.046 to 0.56 µM),
10 times for Hg (0.0050 to 0.051 µM), 6 times for Pb (0.021 to 0.13 µM), 5 times
for Cu (0.33 to 1.6 µM), and less than 3 times for Zn (2.8 to 8.9 µM) and Cd (0.016
to 0.027 µM) (Cabrita et al. 2014). Despite the differences between these contrasting
conditions, levels of dissolved elements like Cr, Cu, Cd and Pb varied within broader
intervals during dredging (10 times for Cu, 8 times for Hg and Pb and 3 times for
Zn) than under no dredging conditions (less than 5 times for the same elements).
This was most likely due to rapid chemical interactions between the particle surface
and dissolved fraction, in addition to rapid sedimentation of dredged sediments
and dilution due to water tidal renewal occurring between consecutive dredging
procedures, as mentioned above. These results are consistent with previous research
that identified that the dissolved fraction only indicate punctual alterations of the
water quality, failing integration in time (Zhou et al. 2008). Even tough trace element
bioavailability only provides a rough indication of the potential for ecotoxicological
impact, it should not be disregarded as a valuable complementary measure providing
chemical environmental context, as long as natural drivers (typical of impacted
estuaries) are taken into account.

Fig. 2. Mean and standard deviation (n = 3) of dissolved Cr, Cu, Zn, Cd, Hg and Pb (µg L–1) in the water
column, at an experimental site on the Tagus Estuary, during non-dredging and dredging conditions. Grey
area indicates the dredging period (modified from Cabrita et al. 2014).
 Overview of Phytoplankton Trace Element Indicators and Biomarkers 95

Accumulation of Trace Elements by Phytoplankton

Trace element environmental bioavailability does not directly translate into
accumulated element levels in the receiving phytoplankton (Sunda 1989, Rainbow
2006), because the way trace elements exert a biological effect is largely dependent
on the environmental chemical conditions and on the organisms themselves
(Campbell et al. 2006). Therefore, the next logical step to evaluate impacts of trace
element contamination on phytoplankton, is to measure the amount of trace elements
accumulated within the phytoplankton cells. This will provide evidence that
bioavailable trace elements are taken up and serves as the foundation for understanding
potential toxicological impacts of trace elements. Because accumulation of trace
elements in immobilised cells is a time-integrated process, phytoplankton species
are considered preferable and valuable indicators of anthropogenic disturbances
in estuarine systems (Reynolds 1998). Interaction between trace elements and
phytoplankton is a two-way process where trace elements control phytoplankton
growth, biomass and species composition, and concurrently phytoplankton regulate
the distribution, chemical speciation, and cycling of these elements in the water
through cell uptake and recycling processes, downward flux of biogenic particles,
release of organic chelators and mediation of redox reactions (Sunda and Huntsman
1998, Vijver et al. 2004, Sunda 2012) (Fig. 3). These complex relationships have a
profound influence on the biogeochemistry, distribution and fate of trace elements in
the estuaries (Beardall and Stojkovic 2006). Trace element content of phytoplankton
thus allows for the anticipation of the consequences of anthropogenically driven

Fig. 3. Conceptual diagram of the interactions between trace element chemistry and phytoplankton in
estuarine and coastal areas.
96 Ecotoxicology of Marine Organisms

element enhancement to phytoplankton organisms and community, and to trace

element biogeochemistry in disturbed estuaries.
Phytoplankton can accumulate considerable amounts of trace elements, whether
essential or not, due to their high binding affinity to elements (GonzálezDávila 1995).
Trace elements are required by phytoplankton to maintain crucial cell biochemical
and physiological processes. A thorough review of trace element functions in
phytoplankton was recently produced by Quigg (2016). For instance, Mn is involved
in the O2-evolving complex of photosystem II (PS II), and is part of the superoxide
dismutase usually found in diatoms (Wolfe-Simon et al. 2005). Cobalt is component
of vitamin B12 with functions on C and H transfer reactions, while Ni is involved
in the hydrolysis of urea, a cofactor in enzymes and in the Ni form of superoxide
dismutase (Wolfe-Simon et al. 2005). Cu is associated with both photosynthetic and
respiratory electron transport chains, and is also a component of the Cu-Zn form of
superoxide dismutase. Zinc has many important metabolic functions, as constituent
of more than 150 enzymes, due to its ability to function as a Lewis acid. The only
known biological function of Cd in phytoplankton is being an element cofactor in
carbonic anhydrase in diatoms (Lane et al. 2000, 2005). Despite their metabolic role
on phytoplankton, essential trace elements, particularly Zn, Cu and Ni, are known
to become toxic to phytoplankton cells at elevated concentrations (Sunda 1989).
Other trace elements are nonessential, such as Hg and Pb with unknown metabolic
roles, and have been found to be exceedingly bioaccumulative and extremely toxic
even in very low concentrations (Sunda 1989, Jaishankar et al. 2014). These toxic
elements enter phytoplankton cells using the same transport systems as essential
elements (Sunda and Huntsman 1998), displacing essential elements from their
metabolic sites, causing cell malfunctioning and toxicity. The mechanisms of trace
element uptake and accumulation in phytoplankton have been thoroughly presented
and explained (e.g., Sunda 1989, GonzálezDávila 1995, Sunda and Huntsman
1998, Wang and Chen 2006, Sunda 2012). Briefly, trace elements occurring as
element cations in the estuarine water column are passively adsorbed and actively
assimilated by phytoplankton. Adsorption to the phytoplankton cell surface takes
place through interactions between element ions and element-functional groups
(for instance carboxyl, phosphate, hydroxyl, amino and sulphur) located in the cell
wall. Subsequently, trace elements are transferred across the cell membrane, and
typically enter the cells by binding to receptor sites of membrane transport proteins
responsible for the transport of essential elements (e.g., Mg, Fe, Mn, Zn, Co and
Cu) (Wang and Chen 2006). Once inside the cell cytoplasm, trace element ions
are compartmentalized into different subcellular organelles (Twining et al. 2003,
Godinho et al. 2014).
To date, most studies are carried out under laboratory controlled conditions and
mostly focus on trace element accumulation in individual phytoplankton species
(e.g., Fisher et al. 1984, Jin et al. 2012) and more rarely on natural phytoplankton
assemblages (e.g., Connell and Sunders 1999) exposed to one element in concentrations
that frequently lack environmental and ecological relevance. Furthermore, in
disturbed estuaries, contamination rarely occurs for single trace elements, being
commonly due to mixtures of elements. Effects of combined elements under natural
environmental conditions in phytoplankton are also few (e.g., Braek et al. 1976,
 Overview of Phytoplankton Trace Element Indicators and Biomarkers 97

Thomas et al. 1980, Cabrita et al. 2016). Surprisingly, field studies monitoring in situ
accumulation of trace elements in phytoplankton in element contaminated areas (e.g.,
Dwivedi et al. 2010) are, to the best of our knowledge, insufficient. This is probably
because measuring trace element accumulation in phytoplankton during sediment
disturbance events (e.g., dredging) may itself be far more complex than originally
expected. The sizes of resuspended sediment particles are frequently similar to those
of phytoplankton cells which makes the collection of phytoplankton samples, with
sediment content at the lowest possible level, extremely difficult to obtain during
those events.
Immobilisation of phytoplankton cells may partially overcome this problem
providing an indication of trace element accumulation in phytoplankton species
representative of the phytoplankton community (Cabrita et al. 2013, 2014). This
technique is extremely useful because it allows for the easy manipulation and
maintenance of the microalgae under in situ conditions (Bozeman et al. 1989,
Twist et al. 1997) and avoids sediment particle accumulation in the cell samples.
Immobilised cells retain their respiratory and photosynthetic activities, maintain
contact with the surrounding water environment, are able to accumulate available
trace elements, and simultaneously, are prevented from being washed away or
grazed on by herbivores during in situ exposure. Calcium alginate is an ideal gel for
entrapment of phytoplankton cells. Immobilisation in calcium alginate gel is a simple,
inexpensive, rapid and nontoxic method for immobilisation of phytoplankton cells.
However, the use of alginate-immobilised species has proven to be a challenging
task in estuaries due to the gel instability occurring with exposure to saline waters
(Cabrita et al. 2013). The main reason appears to be the presence of non-gelling
cations (Na+, Mg+ and K+), which are more abundant in estuarine and seawater
than in freshwater, causing mechanical instability of the alginate matrix (Fraser
and Bickerstaff 1997). Additionally, water turbulence has also been found to be an
influencing factor (Cabrita et al. 2013). Consequently, successful immobilisation of
phytoplankton cells in Ca-alginates for application in estuaries requires a tailormade
approach tackling restrictions associated mainly with salinity and turbulence and
concomitantly ensuring sustained cell growth. Chemical characteristics of alginate
beads must be modified to ensure gel stability in estuarine water, namely the
guluronic (G):mannuronic (M) acid ratio of the alginate polymer chain, and the
type and concentration of the hardening agent (Martinsen et al. 1989). Moreira
et al. (2006) have shown that a guluronic acid rich alginate was crucial to ensure bead
stability of Phaeodactylum tricornutum in a saltwater milieu. The hardening agent
for microalgae immobilisation purposes is usually CaCl2, used as a cation source
(Ca2+) (Cabrita et al. 2013) because it provides no toxicity and produces more stable
alginate gels (i.e., Cu, Sr, Ba and Pb). Increasing Ca2+ concentration strengthens the
linkage of Ca2+ to the alginate chains, and increases gel thickness (Chai et al. 2004).
However, compactness of the gel structure should be balanced with suitability for cell
growth. Too much compactness leads to the decrease of cell growth rates (Cabrita
et al. 2013) and to the formation of higher pore sizes within the gel structure (Gåserød
et al. 1998) increasing the risk of cell leakage. Our studies have demonstrated that
CaCl2 concentration of 5 percent (w/v) provided the best conditions for alginate beads
hardening in order to maintain the gel stability in estuarine water for time periods
98 Ecotoxicology of Marine Organisms

long enough to detect environmental impacts (e.g., eight days) and concomitantly to
avoid cell growth inhibition (Cabrita et al. 2013). Beads produced with this CaCl2
concentration were more resistant to salinity and better withstood natural turbulence
conditions than beads gelled with lower CaCl2 concentrations. The replacement of
Ca2+ in the gel matrix by Na+, Mg+ and K+ present in the estuarine water was slow
enough to avoid loss of the alginate matrix integrity and thus decreased the risk of gel
dissolution. This is particularly relevant for in situ assays designed for the detection
of trace element impacts in mesotidal and macrotidal estuaries where tidal currents
are a continuing source of water turbulence. Furthermore, alginate beads hardened
with CaCl2 concentration of 5 percent (w/v) maintained a high diffusional resistance
(Donati and Paoletti 2009) which is fundamental for contact of immobilised cells
with the surrounding water environment during exposure throughout disturbance
events. In fact, free ions and elements bonded to organic ligands in the water column,
easily and quickly diffuse through the alginate gel matrix and actually reach the
immobilised cells (Chen et al. 1993, Cabrita et al. 2013). A possible experimental
design for this type of in situ assessment is schematically presented in Fig. 4, and has
been thoroughly explained in Cabrita et al. (2013, 2014). Briefly, beads are placed in
modified 24-well plates (PBEs, plates for bead exposure), with the bottom of each
well and the top of the plate lids replaced by a 50 μm size nylon mesh net to ensure

Fig. 4. Schematic drawing of the experimental design for the in situ assessment of trace element
accumulation in phytoplankton exposed to dredging in estuarine and coastal areas, using immobilised
Phaeodactylum tricornutum in alginate beads, showing the construction of plates for bead exposure
(PBE), of test and control PBEs and incubation devices and in situ incubation device setup (modified from
Cabrita et al. 2013).
 Overview of Phytoplankton Trace Element Indicators and Biomarkers 99

full water circulation within the PBEs. Control and test devices must be produced.
The PBEs are inserted in transversely cut 5 L polyethylene bottles which are placed
into 200 µm nylon mesh bags closed with nylon thread in order to simultaneously
allow entrance of estuarine water and prevented damage. The bags with the 5 L
bottles inside are fastened to a buoy attached to a fixed platform to keep them at sub-
surface depth regardless of water level tidal fluctuations during the entire exposure
period. The beads are then recovered and the cells demobilised (for further details,
see Cabrita et al. 2013). The obtained cell biomass is free of sediments which enables
the accurate determination of the accumulation of trace elements in the cells.
Results obtained from the application of this technique are relevant if the chosen
phytoplankton species are considered as good proxies of the estuarine phytoplankton
communities concerning element accumulation efficiency. For instance, the model
diatom P. tricornutum was used in our studies on the accumulation of trace elements
in phytoplankton during dredging events, because diatoms are a major component
of the phytoplankton communities in estuaries and other coastal systems, and
also this ubiquitous marine species has been shown to rapidly respond to trace
element changes in the environment (Cabrita et al. 2014). This species accumulated
considerable amounts of trace elements, such as Cr, Cu, Zn, Cd, Hg and Pb, during
the dredging period in comparison with periods where dredging was not taking place
(Cabrita et al. 2014), providing the much-needed indication that elements were being
consumed and, furthermore, accumulated in relatively high concentrations (Fig. 5).

Fig. 5. Mean and standard deviation (n = 3) of concentration of Cr, Cu, Zn, Cd, Hg and Pb
(µg g–1 d.w.) in non-exposed (open box) and exposed (shaded box) immobilised Phaeodactylum
tricornutum, incubated in situ in the water column, at an experimental site on the Tagus Estuary, during
non-dredging and dredging conditions. Grey area indicates the dredging period (modified from Cabrita
et al. 2014).
100 Ecotoxicology of Marine Organisms

These results also allowed for the identification of the differences in element relative
accumulation, and connection with their bioavailability in the water column. For
instance, some trace elements, such as Cr, Hg and Pb, were highly accumulated in
the cells although their bioavailability in the water column was relatively low in
comparison with that of other elements. Zinc was, by far, the highest accumulated
element, as well as the most abundant element in the water. This shows that
accumulation of trace elements in immobilised cells is a time-integrated process,
and further highlights phytoplankton species as valuable indicators of trace element
changes, as previously pointed out by Reynolds (1998). Additionally, internal
concentrations of trace elements in phytoplankton were also found to be good
indicators of element exposure, as highlighted by Luoma and Rainbow (2005).

Intracellular Trace Element Partitioning in Phytoplankton

Once evidence has been obtained that trace elements are accumulated in the
phytoplankton cells, the question remains whether elements are simply being
adsorbed to the cell wall or whether are they actively internalized into the cells.
This is an important issue to further evaluate the impacts of trace elements on
phytoplankton. The intracellular elemental distribution provides information about
the physiological state and biogeochemical role of diatoms, and reflects biochemical
demands and environmental availability (Twining et al. 2003, Godinho et al. 2014,
2015). Understanding trace element uptake, accumulation and compartmentalization
in whole phytoplankton cells exposed to element overload will allow for the
examination of cellular toxicity and tolerance mechanisms (Godinho et al. 2014,
2015). Furthermore, the trace element content of phytoplankton constitutes a
direct measurement of environmental changes, and influences the distribution and
geochemical fate of various trace elements (Beardall and Stojkovic 2006), which has
implications for broader estuarine biogeochemical cycles.
The traditional bulk size-fractionation techniques used to measure
phytoplankton elemental composition may present ambiguous results due to the
presence of considerable detrital matter (Twining et al. 2003). The determination
of intracellular elemental distributions in isolated cells is challenging, involving
extraction and purification of different organelles and the use of specific labelling
probes, with associated risks of modifying the cell physiological state and production
of artefacts (Fahrni 2007). Alternatively, nuclear microprobe techniques are a
powerful tool that allows for qualitative imaging of the distribution and quantitative
determination of intracellular concentrations (Ortega et al. 2009). The high trace
element sensitivity and spatial resolution of the microprobe has been shown to be
well-suited for studying the interactions of trace elements and single cells (Ortega
et al. 2009, Godinho et al. 2014). The simultaneous assessment of cell morphology
and elemental partitioning enables to identify boundaries that allow distinguishing
between adsorption to the cell walls from absorption into compartments, and to infer
element function and imbalances in exposure condition. The analysis of morphology,
elemental concentrations and their distribution in individualized phytoplankton cells
with nuclear microscopy techniques requires cellular integrity to be maintained and
no interference from culture media. Major obstacles to the analysis of estuarine
 Overview of Phytoplankton Trace Element Indicators and Biomarkers 101

or marine diatoms are the high medium salinity and the presence of extracellular
exudates, mainly polysaccharides, produced by microalgae in both natural media
and in vitro, which severely hamper cell identification and element analysis. Cells
must be completely isolated from the surrounding media components. Furthermore,
the use of fixative agents, such as glutaraldehyde, to avoid cell disruption, have
been proved inappropriate for elemental determination and for intracellular
morphological analysis, as trace elements are washed out from cells (Godinho et al.
2015). Methodological protocols for suitable sample preparation of diatom cells to
elemental analysis have been thoroughly established to overcome these problems
(Godinho et al. 2014, 2015).
Response to trace element exposure, namely Ni, Cu and Zn, has been
evaluated in two diatom species Coscinodiscus eccentricus and Coscinodiscus
wailesii, representative of estuarine phytoplankton, simultaneously using Particle
induced X-ray emission (PIXE), Rutherford backscattering spectrometry (RBS)
and scanning transmission ion microscopy (STIM) to obtain morphological and
quantitative elemental distribution data (Godinho et al. 2014, 2015). Results showed
that elements were actually internalised into the cells and not only adsorbed to the
cell walls. Concentrations of Cu and Zn in exposed cells were higher than in non-
exposed cells, and Ni was virtually absent in controls. The compartmentalisation
of these trace elements also contrasted between exposed and control cells (Fig. 6),
highlighting their role in several metabolic pathways. Ni was mainly stored in the
vacuole of exposed cells which may indicate the ability of these diatom species to
remove excess Ni efficiently. Although marine phytoplankton has been shown to
require Ni for specific enzymatic processes, such as nitrogen recycling from urea
carried out by ureases (Follmer 2008), absence of this element in controls merely
indicates that levels of Ni in the cells may have been much lower relative to the
concentrations of the other measured elements and therefore difficult to detect.

300 Non-exposed cells

Trace element concentration (g g-1)

200

100
nd
0
Wall Citoplasm Vacuole
300 Ni Exposed cells
Cu
Zn
200

100

0
Wall Cytoplasm Vacuole
Fig. 6. Trace element concentration values (µg g–1) in different cell locations (wall, cytoplasm and
vacuole) of Coscinodiscus eccentricus, incubated in the lab, under control and exposed conditions. The
notation “nd” means not detected (modified from Godinho et al. 2014).
102 Ecotoxicology of Marine Organisms

Copper was significantly accumulated in the cytoplasm of exposed cells but not
mobilized to the vacuole suggesting cell susceptibility to Cu toxicity, which is in
line with other studies reporting Cu toxicity to phytoplankton (e.g., Thomas et al.
1980). In particular, Cu has been shown to inhibit photosystem II performance and
cell growth (Reiriz et al. 1994), and cause chloroplast damage and oxidative stress
(Wang and Zheng 2008). Unlike Ni and Cu, Zn was equally distributed in the cell
wall, cytoplasm and vacuole in exposed cells, whereas in controls, this element was
essentially associated with the cytoplasm. Furthermore, the concentration of Zn in
cytoplasm was not significantly different in exposed cells. This feature denotes a
tight control of the cell on maintaining Zn levels in the cytoplasm and an ability of
the cell to mobilize this element to the vacuole. This could mean that cells had a
mechanism of tolerance to Zn. It may have been a way for the cell to reduce toxicity
associated with this element as high concentrations have been shown to inhibit cell
growth and have a toxic effect (Sunda and Huntsman 2000). Alternatively, Zn has
been shown to be a high demand element for the cells (Sunda and Huntsman 2005)
and vacuole compartmentalisation may just constitute a way to store this element
for later use (i.e., luxury uptake). The significant enrichment of the cell wall with Zn
in exposed diatoms, indicates that Zn may also be adsorbed or retained in frustules.
The differentiated intracellular accumulation patterns, namely, Ni and Cu in the
cell organic fraction, and Zn in the cell inorganic fraction (diatom frustule), imply
distinct environmental fates. Cu and Ni may be recycled in the water column and
remain available with potential for biomagnification through the estuarine food web,
whereas Zn may be deposited in the bottom sediments through frustule sedimentation
(Safi and Hayden 2010). The latter mechanism may constitute a considerable
biological sink of Zn in estuaries, due to the short life and large abundance of diatoms
and the elevated amounts of internalised Zn.
Although these results are promising, further work is needed to obtain quantitative
profiles of the distribution of other trace elements in exposed phytoplankton cells.
This will clearly define trace element signatures of cellular contents and exudates
that will contribute to the understanding of the complex relationships between the
phytoplankton and the trace element impacted estuarine environments.

Phytoplankton Indicators of Trace Element Pollution

With evidence that trace elements are internalised in different phytoplankton cell
compartments, the following step is to evaluate the effects of element overload in
phytoplankton cells. Several phytoplankton processes are affected by internalised
trace elements which enable phytoplankton to be used as sensitive, informative,
efficient and reliable bioindicators. The use of phytoplankton as bioindicators offers
many advantages, mostly related to the fact that they are efficient scavengers of trace
elements (Gonzalez-Dávila 1995), rapidly respond to environmental trace element
changes (Cabrita et al. 2014), are present throughout the water column and are
relatively tolerant to a wide range of temperature, salinity and eutrophic conditions.
Biological processes within individual phytoplankton cells, as well as species or
communities may all potentially act as bioindicators and be successfully used to
assess changes in environmental conditions over time. These responses at different
 Overview of Phytoplankton Trace Element Indicators and Biomarkers 103

levels of phytoplankton organization are time-integrated, reflecting present and past

environmental conditions, and, moreover, multi-effect integrated because in estuaries,
the source of contamination is commonly a mixture of trace elements. In fact, the sum
of the effects of trace elements on individual microalgal cells determines their overall
effect on phytoplankton populations and communities. The phytoplankton responses
to enhanced trace elements can be both quantitative (e.g., reduction in phytoplankton
growth) or qualitative (e.g., absence of a trace element sensitive microalgae species
from the phytoplankton standing stock) and should be concurrently used to provide a
more integrated insight on the effects of trace element overload.

Growth and Photosynthesis as Indicators of Trace Element Pollution

Among biological processes within phytoplankton individual cells, cell growth and
photosynthesis have been shown to be highly effective indicators of trace element effects
(Cid et al. 1995, Cabrita et al. 2014, 2016). Among the effects causing impairment of cell
growth (Thomas et al. 1980, Brand et al. 1986, Cabrita et al. 2016) and photosynthetic
performance (Cid et al. 1995, Küpper et al. 1996, Cabrita et al. 2016), are the disturbance
of cell membrane permeability (De Filippis 1979), disruption of nutrient uptake processes
(Shaw 1990, Sunda and Huntsman 1998), inhibition of oxygen consumption in respiration,
reduction of photosynthetic electron transport and carbon fixation (Anderson and Morel
1978, Küpper et al. 1996), and inhibition of enzyme reactions or protein synthesis (Sunda
and Huntsman 1998) are among the effects causing impairment of cell growth (Thomas
et al. 1980, Brand et al. 1986, Cabrita et al. 2016) and photosynthetic performance (Cid
et al. 1995, Küpper et al. 1996, Cabrita et al. 2016).
Growth responses to trace elements vary between different phytoplankton species
(Whitton 1968, Stauber and Florence 1985, Kumar et al. 2014) due to different
uptake and absorption efficiencies, cell compartmentalization strategies and tolerance
and detoxification mechanisms, which are dependent on the trace element type and
concentration, as well as the cell physiological status (Sunda and Huntsman 1998).
In general, trace elements have been found to suppress phytoplankton growth to
various degrees (Torres et al. 1997, Markina and Aizdaicher 2006, Horvatić and Peršić
2007). Our research regarding trace element effects was focused on P. tricornutum
used as a sentinel and model phytoplankton species for trace element contamination
(Cabrita et al. 2013 and 2014, Cabrita et al. 2016). Results obtained under both in situ
(Cabrita et al. 2014) and laboratory simulated element exposure conditions (Cabrita
et al. 2016), generally corroborated those previous findings, showing an accentuated
decrease in P. tricornutum growth rates unequivocally attributed to a combination of
trace elements (Fig. 7). Regarding the effect of each trace element acting individually
on growth, Hg was the most toxic element causing growth inhibition, followed by
Pb, Co, Cr and all elements combined (Mix) (Cabrita et al. 2016) (Fig. 7). Although
Pb and Hg do not take part in the phytoplankton metabolic pathways, P. tricornutum
cells incorporated these elements (Cabrita et al. 2014, 2016). Internalized Hg in cells
can have harmful repercussions to cell growth and division (Hannan and Patouillet
1972, Deng et al. 2013), by binding to cytosolic ligands, blocking functional groups
of essential biomolecules (e.g., enzymes), and allocating into organelles (Le Faucheur
et al. 2014). Lead is also a severe cell toxicant, even in low concentrations (Moreira et
104 Ecotoxicology of Marine Organisms

Fig. 7. Mean and standard deviation (n = 3) of specific growth rate (day–1) of immobilised Phaeodactylum
tricornutum, incubated in situ in the water column, at the experimental site on the Tagus estuary, during
no dredging and dredging conditions, and of P. tricornutum non-exposed and exposed cells to Cr, Co, Ni,
Cu, Zn, Cd, Hg, Pb and Mix (mixture of all trace elements combined). Grey area indicates the dredging
period (modified from Cabrita et al. 2014, 2016).

al. 2001) it is able to reduce cell growth (Irmer 1985). As for other elements, such as
Ni, Zn and Cu, their effect on growth was minor or even undetectable in P. tricornutum,
as has been shown for several other phytoplankton species (Fisher 1981), possibly
because they are essential components of microalgae metabolic pathways (Sunda
1989), and levels of these elements found during dredging were not apparently within
toxic levels able to induce severe cell damage. Tolerance to Zn, Cu and Ni, as reported
for several other phytoplankton species (Fisher 1981), comply with these elements
being essential components in many diatom metabolic pathways (Sunda 1989). In
the case of Ni, tolerance may be explained by the low cell binding capacity of Ni
found in several microalgae species (Horvatić and Peršić 2007), preventing Ni to reach
toxic concentrations within the cells. Nevertheless, high levels of these elements have
been found to be toxic to microalgae, as previously reported for P. tricornutum and
other microalgae under high levels and chronic conditions (Sunda 1989, Horvatić and
 Overview of Phytoplankton Trace Element Indicators and Biomarkers 105

Peršić 2007). The values of “no observable effect concentration” (NOEC) and “lowest
observable effect concentration” (LOEC) obtained from previous chronic toxicity tests
with P. tricornutum, were < 1.5 µg L–1 and 1.5 µg L–1, for Cu, respectively (Levy et al.
2007, 2008), and a NOEC value of 2700 µg L–1 was reported for Zn (Bodar 2007). For
Ni and the other elements, no NOEC or LOEC values were found for P. tricornutum in
the available literature. Copper and Zn levels in the water during exposure experiments
were lower than these reported chronic threshold levels which further suggests that the
observed effects of trace elements on P. tricornutum cell growth were probably not
triggered by Zn and Cu, and also possibly not by Ni due to its low cell binding capacity,
but rather by the other elements present in the water (e.g., Cr, Hg and Pb). Regarding Cd,
the effects on growth ranged from a significant stimulation at the beginning of exposure
(Cabrita et al. 2016), to no detectable or insignificant decrease after prolonged exposure
(Cabrita et al., unpublished data). Growth enhancement with Cd addition has also been
previously observed for P. tricornutum (Lee and Morel 1995). Stimulation triggered
by Cd may be associated with a requirement of P. tricornutum cells for Cd, reportedly
necessary for the formation cadmium carbonic anhydrase in diatoms (Lane et al. 2000,
2005) which plays a key role in the acquisition of inorganic carbon for photosynthesis
in phytoplankton. Gene sequences for Cd carbonic anhydrase were found in
P. tricornutum (Park et al. 2007). Carbonic anhydrase can either use Zn or Cd as its
element centre (Xu et al. 2008), and the expression of cadmium carbonic anhydrase
was controlled by pH changes, and induced by the addition of Cd (Park et al. 2008),
which were conjoint features of trace element contamination in the estuarine waters and
may have been the reason for growth enhancement found in P. tricornutum. Although
Cd levels may have increased within the cells with continued exposure, tolerance to
Cd toxicity appeared to be a feature of P. tricornutum (Torres et al. 1997), mainly due
to the ability of this species to incorporate Cd induced sulfide ions in Cd-phytochelatin
complexes and produce nanometer sized phytochelatin-coated CdS nanocrystalites
(Scarano and Morelli 2003).
Regarding photosynthesis, degradation and biosynthesis inhibition of
photosynthetic pigments (De Filippis and Pallaghy 1994), structural changes in
the chlorophyll a (Chl a) molecule (Küpper et al. 1996, Aggarwal et al. 2012),
changes in pigment profiles (Duarte et al. 2012), and inhibition of photosystem II
(PS II) (Küpper et al. 2002) are triggered by some elements. These effects have
been found in several plant species, including phytoplankton. These alterations
dramatically affect the overall photosynthetic performance of phytoplankton cells
(Cid et al. 1995, Küpper et al. 1996, Cabrita et al. 2016), which will inevitably
have consequences for the primary productivity of estuarine systems impacted by
trace element contamination. Chlorophyll fluorescence intensity measurements
have been extensively used to understand plant photosynthetic responses to trace
element induced stress (Duarte et al. 2012, Santos et al. 2014, Cabrita et al. 2016,
Anjum et al. 2016 and references herein). Furthermore, Chl a fluorescence is a
sensitive indicator of the status of photochemical reactions (Buschmann 2007).
Among several fluorescence techniques, Laser-Induced Fluorescence (LIF) and
Pulse Modulated Amplitude (PAM) Fluorometry have been shown to be highly
sensitive, time-saving and powerful non-destructive techniques, enabling direct
106 Ecotoxicology of Marine Organisms

optical probing of phytoplankton cells. In vivo assessment of trace element stress

in phytoplankton is thus possible without requiring the addition of reagents or cell
fixatives. Adding to that, these techniques yield nearly instant results so that many
replicate measurements can be performed on the same sample. The possibility for
measurements to be performed under natural conditions may be an advantage to get
rapid large-scale assessment of an entire region under surveillance and to identify
stressed zones, which can be studied in detail afterwards, utilizing more expensive
and time-consuming methods.
In our studies, the application of LIF technique to the assessment of the effect of
trace elements in P. tricornutum provided reliable and suitable Chl a fluorescence-
based indicators for trace element stress (Cabrita et al. 2016). In vivo Chl a
fluorescence spectra of this diatom typically included two maxima, one in the red
(685 to 690 nm) and one in the far-red (710 to 740 nm) regions of the spectrum,
comparable to fluorescence emission bands already observed in other phytoplankton
species (Barbini et al. 1998), intertidal microphytobenthos (Vieira et al. 2011), and
higher plants (Lichtenthaler and Rinderle 1988). Most of this emission is linked to the
pigment protein PS II-associated Chl a (Govindjee 1995), with the red band arising
from the main electronic transitions, and the far-red band resulting from vibrational
sublevels whose relative intensities are increased in vivo through self-absorption
(Franck et al. 2002). Additionally, some fluorescence of the photosystem I (PS I),
which is highest around 730 nm, also contributes to the fluorescence emitted at the
far-red band (Govindjee 1995, Pfündel 1998). Exposure to trace elements induced
alterations in the fluorescence emission intensity of Chl a as well as deviations in
the maximum-emission wavelength, in comparison to non-exposed P. tricornutum
cells. These alterations indicating Chl a concentration variations have been pointed
as one of the earliest indicators of physiological status in microalgae (Baker 2008),
as shown for Hg as an example (Fig. 8). The significant decrease in fluorescence
intensity (mostly in the far-red compared to the red region) combined with large
deviations in wavelength emission maxima allocated in the red as compared to the
far-red region, observed in exposed cells, suggest Chl a damage, structural changes
in Chl a with the possibility of formation of low fluorescent or non-fluorescent trace
element-substituted Chl a (Küpper et al. 1996), a decrease in re-absorption due
to the decline in Chl a, and also imply that PS II chlorophylls were more affected
by trace elements than the PS I chlorophylls. These alterations were observed for
most of the trace elements tested (Co, Ni, Cu, Zn, Cd, Hg, Pb and all elements
combined), in particular for Co, Hg and all elements combined, supported by similar
findings observed in higher plants and microalgae (Küpper et al. 1996, Pandey and
Gopal 2011). For instance, sublethal Cd and Hg levels strongly inhibit chlorophyll
biosynthesis (De Filippis and Pallaghy 1976) and induce a Chl a decline in several
microalgae species (Maurya et al. 2008). Cobalt, Cd, Hg and Pb have also been
shown to cause structural and functional damage in photosynthesis (Aggarwal et
al. 2012), due to substitution of magnesium (Mg) in the Chl a molecule by Ni,
Cu, Zn, Cd, Hg and Pb that caused a decline in the net photosynthesis (Küpper et
al. 1996, Küpper et al. 2002). The red/far red emission fluorescence ratio (F685/
F735 ratio) obtained from the fluorescence spectra significantly increased for all
elements, in particular for Co, Hg and all elements combined (Mix), except for Zn,
 Overview of Phytoplankton Trace Element Indicators and Biomarkers 107

Wavelength (nm)

Fig. 8. Absolute fluorescence spectra (counts) of non-exposed (black line) and Hg-exposed (gray line)
Phaeodactylum tricornutum cells, between 650 and 790 nm, maximum fluorescence deviation (counts)
and wavelength fluorescence maxima deviations (nm) from control (non-exposed cells), and red/far red
fluorescence ratio (F685/F735 ratio) in non-exposed and exposed cells to Co, Ni, Cu, Zn, Cd, Hg, Pb
and Mix (mixture of all trace elements combined) (mean ± standard deviation, n = 6). Different letters
above standard deviation bars indicate values significantly different (p < 0.05) from non-exposed values
(modified from Cabrita et al. 2016).

mostly due to a decline in fluorescence intensity at 735 nm as illustrated here for Hg

(Fig. 8). This suggests that trace elements incorporated in the cells not only induced
alterations in the PSII efficiency, but also affect the distribution of excitation energy
between the two photosystems (state transitions) (Wollman 2001). The increase in
red/far red fluorescence ratios in response to trace element toxicity is comparable
to different types of stress inflicted on many plant species showing breakdown or
reduced synthesis of Chl a and damage in the photosynthetic apparatus, caused by
nutrient stress (Subhash and Mohanan 1997), Cd exposure (Maurya and Gopal 2008),
108 Ecotoxicology of Marine Organisms

herbicides and senescence processes (D’Ambrosio et al. 1992). Overall, these results
clearly showed deviation in wavelength emission maxima and F685/F735 ratio as
reliable and suitable Chl a fluorescence-based indicators that can be used for trace
element stress detection and monitoring in estuarine systems, using P. tricornutum.
Furthermore, these indicators discriminated the toxic action of the different trace
elements, highlighting Co and Hg as the most damaging elements to this species.
Pulse Modulated Amplitude (PAM) Fluorometry was used in parallel in
experiments to provide further insight on trace element induced changes throughout
the photosynthetic pathway, namely in the PS II activity and overall efficiency during
light harvesting, the behaviour of the Electron Transport Chain (ETC) and also the
entire energetic transduction pathway from the PS II-captured photons to the PS
I acceptor side. This complementary approach allowed for the extraction of other
efficient and instantaneous indicators that can potentially be employed for the early
detection of trace element stress in phytoplankton. The stepwise flow of energy
through PS II reflects the alterations of fluorescence response over time, in the shape
of a curve (known as the Kautsky curve, the chlorophyll fluorescence transient or the
OJIP-transient curve). The Kautsky curve provides an overall imaging of the energetic
processes occurring inside the cells. The chlorophyll fluorescence intensity rises from
a minimum level (the O level, O is for origin), in less than 1s, to a maximum level
(the P-level, P is for Peak) and J and I are two intermediate levels. From this curve,
specific OJIP-test parameters were extracted and investigated as potential indicators
of element stress that can be employed for the early detection of trace element
contamination in estuarine systems (Strasser and Strasser 1995). Table 1 summarizes
the parameters computed from the fluorometric data. Corroborating LIF results;
PAM Fluorometry data also highlighted the damaging effects of Hg, Co, Cr, Pb, Hg
and all trace elements combined (Mix) and pointing to Hg as the most deleterious
element to P. tricornutum, affecting all fluorometric parameters. The other elements
had more restricted effects. Here we explain the damaging effects of these elements,
using Hg as an example (Fig. 9). Mercury induced a distinct decrease on the PS II
activity of cells and a striking decline in the reaction centres (RC) closure net rate
and, consequently, a decrease in the light absorption energy flux, affecting negatively
the partial performance due to the light reactions for primary photochemistry and
shifting the equilibrium constant for the redox reactions between PS II and PS I.
Considering a lower light absorption by the RC, a rise in the dissipation energy was
still apparent. Additionally, a concurrent shift in the balance towards the PS I activity
was found, triggered by an enhancement in the electron transport from formation of
plastoquinol (PQH2) to the reduction of PS I end electron acceptors, reducing them
efficiently, although an increase in the number of oxidized RC and in the electronic
transport energy flux was noticed. The lack of efficiency verified in the PS II in
Hg-exposed cells was mostly due to the low connectivity between PS II antennae,
leaving a large part of the quinone pool (Q) in the oxidized state. This excessive
energy dissipation was visible in the appearance of the K-step in the Kautsky curve.
The K step in the OJIP curves, at about 300 μs, is always associated with the damage
to the PS II donor side (Strasser et al. 2000, Chen and Cheng 2009). Strasser et al.
(2000) found that changing the oxygen evolving complex (OEC) enables alternative
internal electron donors to donate electrons to PS II, creating a short-lived increase
 Overview of Phytoplankton Trace Element Indicators and Biomarkers 109

Table 1: Fluorometric analysis parameters and their description.

Photosystem II Efficiency Description

F’0 and F0 Basal Fluorescence under weak actinic light in light and dark-adapted
samples.
F’M and FM Maximum Fluorescence measured after a saturating pulse in light and
dark-adapted samples.
F’v and Fv Variable fluorescence light (F’M – F’0) and dark (FM – F0) adapted
samples.
PSII Operational and Light and dark-adapted Quantum yield of primary photochemistry,
Maximum Quantum Yield equal to the efficiency by which an absorbed photon trapped by the
PSII reaction centre will result in reduction of QA to Q-A.
Rapid Light Curves (RLCs)
rETR Relative electron transport rate at each light intensity (rETR = QY ×
PAR × 0.5).
ETRmax Maximum ETR obtained from the RLC after which photo-inhibition
can be observed.
Ek The onset of light saturation.
a Photosynthetic efficiency, obtained from the initial slope of the RLC.
Energy Fluxes (Kautsky
curves)
Area Corresponds to the oxidized quinone pool size available for reduction
and is a function of the area above the Kautsky plot.
W Variable fluorescence intensity normalized to the
J-step (W = Ft – F0)/(FJ – F0).
WK Amplitude of the K-step (WK = VK – VJ).
φP0 Maximum Yield of Primary Photochemistry.
φE0 Probability that an absorbed photon will move an electron into the ETC.
φD0 Quantum yield of the non-photochemical reactions.
ψ0 Probability of a PS II trapped electron to be transported from QA to QB.
Mo Net rate of PS II RC closure.
PG The Grouping Probability is a direct measure of the connectivity
between the two PSII units (Strasser and Stribet 2001).
ABS/CS Absorbed energy flux.
TR/CS Trapped energy flux.
ET/CS Electron transport energy flux.
DI/CS Dissipated energy flux.
RC/CS Number of available reaction centres per leaf cross section.
RE0/RC Electron transport from PQH2 to the reduction of PSI end electron
acceptors.
δR0 Efficiency with which an electron can move from the reduced
intersystem electron acceptors to PSI to reduce the end electron
acceptors.
TR0/DI0 The contribution or partial performance due to the light reactions for
primary photochemistry.
(ψ0/1-ψ0) Contribution of the dark reactions from QA− to primary
photochemistry.
(δR0/1-δR0) Contribution of PSI, reducing its end acceptors.
(ψE0/1- ψE0) Equilibrium constant for the redox reactions between PSII and PSI.
 110 Ecotoxicology of Marine Organisms

Fig. 9. PS II variable fluorescence and quantum yields, Kautsky curves and associated parameters, in light and dark-adapted Phaeodactylum tricornutum non-exposed and
exposed cells to Hg (mean ± standard deviation, n = 3), * above standard deviation bars indicates values significantly different (p < 0.05) from control (modified from Cabrita
et al. submitted).
 Overview of Phytoplankton Trace Element Indicators and Biomarkers 111

in the fraction of Pheo– and QA–, which causes an increase in the K fluorescence step.
Krüger et al. (2014) suggest that this K-band indicates accessibility of internal non-
water electron donors to PS II (e.g., ascorbate red) competing with the OEC. The
difference in fluorescence at the J-step between exposed and control cells lower than
zero was probably due to decelerated electron donation from H2O to an increased
activity of PS I or stimulated reactive oxygen compounds (ROS) formation, as
suggested by Krüger et al. (2014). Similarly, the 30 ms (Iband) was significantly
reduced in exposed cells compared to control ones, suggesting an enhancement of
the final reduction of end electron acceptors, such as Fdred and NADP+, due to a higher
oxidized state of the pool mixture of plastoquinone, cytochrome b/f and plastocyanin
(Yusuf et al. 2010) and higher activity of PS I (Krüger et al. 2014). The significant
alterations found in these fluorometric parameters with relevant physiological and
ecological meaning indicate that they can be used as a useful suite of indicators for
future trace element contamination assessment programmes.

Phytoplankton Community and Individual Taxa as Indicators of

Trace Element Pollution
The structure of the phytoplankton community as well as several phytoplankton
species have been widely used as indicators of ecological condition for the evaluation
of a range of impacts, for instance, eutrophication (Tas et al. 2009) and climatic
change (Paerl et al. 2010), but has seldom been applied in trace element contamination
assessments. Previous studies showed that phytoplankton indicators provided valid
information on the level of eutrophication in a dredged lake (Wang et al. 2012, Xu
and Pan 2013). Our studies have identified much needed phytoplankton indicators,
at the community and individual taxa levels, for the monitoring of estuarine systems
contaminated with elements, taking into account the local hydrologic conditions and
the natural variability of the estuarine phytoplankton assemblages in a given area
(Cabrita 2014).
Our results indicated diatom:other groups ratio and benthic:pelagic diatom ratio
as efficient and reliable indicators of trace element contamination (Cabrita 2014)
(Fig. 10). The consistent raise in the diatom:other groups ratio found during increased
trace element in the water column, triggered by dredging, was mostly due to benthic
diatoms, as highlighted by the benthic:pelagic diatom ratio, and seemed to have
been generated by inputs of suspended particulates that occurred during dredging.
Similarly, De Jonge (2000) reported enhanced contribution of resuspended benthic
cells to phytoplankton related to increased background turbidity in the Ems Estuary,
during dredging events. High contribution of benthic diatoms to the phytoplankton
community was also observed during periods of high perturbation in shallow areas
(Lucas 2003). This implies that benthic microalgae were not settling on the bottom
sediments, but otherwise remained in the water column, growing and even blooming,
in spite of the elevated levels of trace elements triggered by dredging. Trace element
resistance has been reported for benthic diatoms growing in contaminated sediments
(Tuovinen et al. 2012), with diatom density decreasing only under considerably severe
contamination (Cattaneo et al. 2008). Adaptation strategies to high trace element
112 Ecotoxicology of Marine Organisms

Fig. 10. Diatom:other groups ratio and benthic:pelagic diatom ratio in surface water, at an experimental
site on the Tagus Estuary, before, during and after dredging periods, and projection of Cr, Ni, Cu, Cd,
Hg, Pb dissolved concentrations and abundance of Asterionellopsis glacialis, Coscinodiscus sp.,
Cylindrotheca closterium, Detonula pumila, Guinardia sp., Navicula sp., Nitzschia sp., Skeletonema
costatum, Surirella sp. and Thalassiosira sp., obtained from the Principal Component Analysis (PCA).
Two diatom assemblages are highlighted in light grey and dark grey. Percentage of total variance is
indicated in brackets close to principal component axes (modified from Cabrita 2014).

concentrations occurring in benthic diatoms that inhabit contaminated sediments

were possibly a competitive advantage of the suspended diatoms during dredging
in relation to the pelagic phytoplankton adapted to a relatively less contaminated
environment.
Species richness (Margalef’s index) and species diversity indices (Simpson’s
diversity, Shannon-Wiever’s, and Warwick and Clarke’s taxonomic diversity and
distinctness indices) failed to discriminate variations in the phytoplankton community
induced by dredging, possibly because the species richness was maintained
regardless of occurrence of dredging (Cabrita 2014). Similar results were obtained
by Chadwick and Canton (1984) for a stream invertebrate community subject to
element mine drainage, suggesting that those indices can be useful for detecting
environmental perturbations that favour tolerant species over more sensitive ones with
resulting reduction in species richness. Although sensitive species reduced or even
 Overview of Phytoplankton Trace Element Indicators and Biomarkers 113

disappeared due to increasing availability of trace elements driven by dredging, the

displacement of the benthic species from the bottom sediments into the water column
surely contributed to sustain phytoplankton species diversity. Even the distinctness
index, found to be a more sensitive indicator of community perturbation in relation
to anthropogenic disturbances (e.g., Brown et al. 2002), was unable to highlight
community changes. This points to the importance of a comprehensive analysis of
the phytoplankton community when assessing pollution impacts, particularly those
driven by dredging, that seem to not compromise species richness.
Identifying phytoplankton taxa as potential indicators of trace element changes
induced by dredging was also carried out, requiring care in order to correctly
ensure that changes in species individual contributions associated with dredging are
discriminated from seasonal variations. In our studies, dredging coincided with the
spring and summer period when pelagic species such as Asterionellopsis glacialis
and Thalassiosira spp. have been found to occur in relatively high concentration, or
even to bloom. Nevertheless, trace elements were found to promote the shaping of the
phytoplankton community composition, as already found in other studies (Hollibaugh
et al. 1980, Horvatić and Peršić 2007). Phytoplankton responded essentially through
a shift in the microalgae composition towards species less susceptible to trace
elements. This response was in line with previous findings in other systems subject
to trace element enhancement (Moore et al. 1979, Monteiro et al. 1995, Jing et al.
2011). In fact, most of the species developing highest abundance during dredging
have been found resistant to elevated trace element levels. The pelagic Skeletonema
costatum has been shown to be tolerant to Cu, Zn and Pb (Fisher 1981, Rukminasari
and Sahabuddin 2012), Coscinodiscus sp. to a wide range of trace elements (Rick
and Dürselen 1995), and the benthic diatoms, Navicula sp. was shown resistant to
Cu (Thomas and Seibert 1977, Ivorra et al. 1999) and Nitzschia sp. to Cu, Zn, Cd
and Pb (Thomas and Seibert 1977, Moore 1981, Santos et al. 2013). The increased
abundance of those benthic diatoms in the water column during dredging (Fig. 10)
suggests that these species may be suitable indicators of dredging induced changes.
An additional outcome of the trace element enhancement during dredging was the
decrease and even the disappearance of the most sensitive microalgae species. The
dissociation of the pelagic diatoms Asterionellopsis glacialis, Thalassiosira spp.,
Guinardia sp. and Surirella sp. from the trace element enhancement generated by
dredging (Fig. 10) implied low tolerance of these species to elements, as reported for
some of these taxa (A. glacialis, Thalassiosira spp.) in preceding studies (Tortell and
Price 1996). Specifically, the decline of A. glacialis and Thalassiosira spp. and the
fact that they are part of the phytoplankton standing stock and may form blooms in the
Tagus Estuary (Cabrita 1997, Gameiro et al. 2004, Brogueira et al. 2007) suggest that
the absence of these species would be considered a good indicator of trace element
changes associated with the occurrence of dredging in this estuarine area in particular.
However, the wide range of trace element sensitivity among phytoplankton species
and the varying sediment contamination levels from different areas requires site-
specific evaluations for the identification of the best taxa indicators, bearing in mind
the species composition of the local phytoplankton assemblages and their temporal
variation patterns.
114 Ecotoxicology of Marine Organisms

Overall, these results point to diatom:other groups ratio, benthic:pelagic diatom

ratio and individual taxa as reliable and sensitive indicators of trace element stress
that can be used, in combination with the other formerly mentioned indicators, for
the early detection of element impacts during disturbance events in estuaries.

Phytoplankton Biomarkers of Trace Element Pollution

During the past 25 years, several biomarkers for trace element pollution have
been developed with the aim of supporting and complementing more conventional
environmental biomonitoring. In Europe, conventions and legislation have been
developed, aiming to prevent deterioration or restore adversely affected coastal and
marine areas, although the potential of biomarkers is not always stressed. The Water
Framework Directive (WFD, European Commission 2000) provides a major driver
for achieving sustainable management and use of water bodies in the EU Member
States, to ensure the progressive reduction of aquatic pollution. The directive,
focusing on the two main elements “good ecological status” and “good chemical
status”, offers a key legislative opportunity to promote and implement an integrated
approach including biomarkers, for the protection of estuarine and coastal waters.
The Marine Strategy Framework Directive (MSFD, European Commission 2008)
already promotes the application of an integrated ecosystem-based approach, with
the incorporation of biomarkers. Furthermore, the use of biomarkers as a tool for
the assessment of ecosystem health is being increasingly endorsed by the OSPAR
Commission of Western Europe and also by Baltic Marine Environment Protection
Commission (HELCOM). Biomarkers are thus envisioned as valuable integrative
tools that will become fully integrated in the monitoring programmes in future.
The value of biomarkers relies on their capacity to effectively integrate both
physical-chemical status and biological quality elements, which may provide
an early warning of trace element induced stress in contaminated estuaries and a
holistic perspective of pollution adverse consequences on the health of the estuarine
organisms, populations and ecosystem (Depledge et al. 1993, Moore et al. 2004). A
wide array of biomarkers have been proposed to assess trace element exposure and
effects in estuarine waters, focused on possible sentinel organisms, mostly invertebrate
and fish species (e.g., Vieira et al. 2009, Caçador et al. 2012, Vasanthi et al. 2012).
Considered as effective biomarkers of trace element contamination (Cabrita et al.
2013, 2014, 2016), and placed at the base of the estuarine food web, phytoplankton
can provide sensitive and effective biomarkers that can be employed for the early
detection of trace element contamination in estuarine systems (Volterra and Conti
2000, Torres et al. 2008). Trace elements internalised into phytoplankton cells are
known to cause impairment of fundamental physiological processes (Overnell 1975,
Sunda and Huntsman 1998, Cabrita et al. 2016) which may be seen as an opportunity
to identify specific biomarkers of element stress, in representative estuarine
phytoplankton species. Phytochelatins (Perales-Vela et al. 2006), heat-shock proteins
(Spijkerman et al. 2007) and a wide range of oxidative stress response and defence
molecules (Pinto et al. 2003), have been highlighted as powerful phytoplankton
biomarkers for trace element contamination (see review by Torres et al. 2008). Our
work focused on P. tricornutum as a model sentinel species has identified trace
 Overview of Phytoplankton Trace Element Indicators and Biomarkers 115

element substituted chlorophyll a (Me-Chl a) molecules as unequivocal biomarkers

of element stress (Cabrita et al. submitted). Exposure to Cu, Zn and Cd induced the
formation of Cu-, Zn- and Cd-Chl a in the cells exposed to those elements (Fig. 11).
Results showed that Me-Chl a accounted for approximately 40 percent of the total
Chl a cell content, promoting energy dissipation which caused impairment of the
photosynthesis energetic pathway in stressed cells compared to non-exposed ones.
Trace elements, such as Ni, Cu, Zn, Cd, Hg or Pb, have been found to replace the
central atom of the chlorophyll molecule, magnesium, under in vivo conditions, in
element exposed plant cells (Küpper et al. 1996). Most of the Me-Chls are unsuitable
for photosynthesis because the first excitation state becomes unstable, preventing
the resonance energy transfer from the antenna pigment complexes to the reaction
centres in the thylakoids. As a result, in vitro fluorescence quantum yields are much
lower in Me-Chls than in Mg-Chls (Watanabe and Kobayashi 1988). Furthermore, the
Me-Chls’ capacity to release electrons from the singlet excited state is considerable
decreased compared to all Mg-Chls (Watanabe et al. 1985). A significant fraction of
Me-Chls in total photosynthetic pigments, occurring during trace element exposure,
will thus lead to a complete breakdown of photosynthesis (Küpper et al. 1996).
The concentrations of Me-Chl a also allowed researchers to identify differences
in the element relative toxicity when cells were exposed to a mixture of trace elements,
and highlighted the complexity of element combined effects on phytoplankton.

Fig. 11. Trace element substituted chlorophylls (Cu-, Zn- and Cd-Chl a) cellular content in Phaeodactylum
tricornutum non-exposed and exposed cells to Cu, Zn, Cd and Mix (mixture of all trace elements
combined), and pigment concentration patterns in cells exposed to trace elements (Cr, Co, Ni, Cu, Zn, Cd,
Hg, Pb and Mix) (modified from Cabrita et al. submitted).
116 Ecotoxicology of Marine Organisms

In estuaries, elements are commonly discharged as mixtures, and synergistic and

antagonistic interactions between multiple trace elements are expected, with each
element showing a different relative toxicity (Thomas et al. 1980, González-Dávila
1995). In cells exposed to element mixture containing Cu, Zn and Cd, only Zn- and
Cd-Chl a were found (Fig. 11), illustrating that Cu was only toxic when tested singly
and toxicity was mainly due to Zn and Cd. Zinc induced a lower concentration of
Zn-Chl a when acting alone than in cells exposed to element mixture and Cd-Chl
a concentrations were similar regardless of single or combined element exposure
(Fig. 11). These effects suggests competition for the chlorophyll Mg binding site
and a prevalent effect of Zn and Cd over Cu, in P. tricornutum. These results are in
line with previous studies. For instance, Kawakami et al. (2006) showed similar Cu,
Zn and Cd combined effects on the production of phytochelatins and glutathione in
Phaeodactylum tricornutum. Similarly, competitive antagonism has been found for
Cu and Zn in another diatom species (Rueter and Morel 1981). The application of trace
element substituted chlorophylls as sensitive biomarkers of element stress allows to
identify which elements are most likely to produce synergistic or antagonistic effects
and points out to the most critical elements in the mixture of elements which may be
extremely useful in the assessment of element contamination in natural conditions.
Alongside trace element substituted chlorophylls, naturally occurring
phytoplankton pigments can also be used as suitable biomarkers of trace element
contamination, contributing to the better understanding of the effects of trace
elements on the photosynthetic features of phytoplankton. Trace elements have been
found to affect pigment content and profile in phytoplankton because they inhibit
photosynthetic pigments biosynthesis and cause pigment degradation (De Filippis
and Pallaghy 1994) resulting in changes in pigment profiles (Duarte et al. 2012).
These changes may occur very fast and the evaluation of the pigment content
after trace element exposure generally reflects integration in time, so that element-
regulated pigment interconversion intermediate steps may be overlooked. Of course,
this could be minimized with a continuous sampling plan which may not always be
applicable. Otherwise, interpretation of pigment profiles should be made with extreme
caution which makes naturally occurring phytoplankton pigments less unequivocal
biomarkers than Me-Chls. Furthermore, photosynthetic pigments in phytoplankton
also change rapidly during growth (exponential, stationary and declining phases). In
P. tricornutum, a rise in the carotenoid:chlorophyll a ratio, associated with variations
in the percentage of individual carotenoids occurs with the shift from logarithmic to
the stationary phase (Carreto and Catoggio 1976). The fucoxanthin content decreases
whereas diadinoxanthin content is enhanced and β-carotene remains nearly constant
with cell aging. Therefore, experiments regarding phytoplankton responses to trace
element stress in terms of pigment variations should only be performed during the
growth exponential phase to ensure that observed changes in pigments are essentially
associated with responses to trace element stress rather than to other factors such
as cell aging or culture nutrient depletion. In our previous studies, cell aging has
been detected in P. tricornutum cultures after six days, obscuring the physiological
responses to environmental stressors (data not published). Our research conducted
with P. tricornutum as a sentinel species, to investigate pigments as biomarkers
of element stress, showed significant changes in the levels of several pigments,
 Overview of Phytoplankton Trace Element Indicators and Biomarkers 117

including Chl a and c, Pheophytin a, β-carotene, Fucoxanthin, Diadinoxanthin and

Diatoxanthin (Fig. 11), which were induced by trace elements internalised in the
phytoplankton cells. Under trace element stress conditions, changes in the pigment
profiles of P. tricornutum occurred. A common feature found for most trace elements,
particularly detected for Cu, Cr and Mix, was the decrease in Chl a and other pigments
involved in photosynthesis, in particular β-carotene, concomitant with elevated
levels of Fucoxanthin, suggesting that the Xanthophyll cycle was triggered with the
conversion of β-carotene into Fucoxanthin to allow for thermal dissipation of the
excess energy at the antenna side of the photosynthetic apparatus (Xia et al. 2013). In
addition, an enhancement of Diadinoxanthin and Diatoxanthin levels was also found,
more visible for cells exposed to Cr, Co, Cu, Hg, Pb and all elements combined
(Mix), possibly related to decrease in the effective absorption cross section of PS
II as a result of energy dissipation in the reaction centre. The apparent activation
of the diadinoxanthin cycle involving the interconversion of two carotenoids,
Diadinoxanthin and Diatoxanthin, has been observed in several species of diatoms
(Xia et al. 2013, Masmoudi et al. 2013), including P. tricornutum (Olaizola et al.
1994, Grouneva et al. 2009). For instance, in P. tricornutum subject to dithiothreitol
stress, changes in the non-photochemical quenching appeared to result from energy
dissipation in the reaction centre and were found to be associated with decreased
photochemical efficiency (Olaizola et al. 1994). This defense feature underlies
P. tricornutum strategy to cope with the damaging effect of Cr, Co, Cu, Hg, Pb and all
trace elements combined (Mix) on the photochemical apparatus. These results point
to pigments as sensitive biomarkers of trace element stress that can be extrapolated
to other phytoplankton species, with application to efficient biomonitoring programs
in estuaries.
Our search for efficient phytoplankton biomarkers led us to investigate fatty
acids (FA) as potential biomarkers of trace element stress. Phytoplankton are major
producers of fatty acids in diverse lipid classes (Guschina and Harwood 2009). They
are able to synthetize linoleic—(C18:2) and linolenic—(C18:3) acids (C18:3), of
the classes omega 6 (ɷ-6) and omega-3 (ɷ-3), respectively, which are essential
fatty acids (EFA) for vertebrates. Since most organisms at higher trophic levels
of estuarine food webs, such as fish and humans, have limited ability to produce
long chain polyunsaturated fatty acids (LC-PUFA) from EFA, they rather obtain
them from the diet, relying on their de novo production by estuarine phytoplankton
(Parrish 2009). Also, the FA composition of cell membranes is a key factor allowing
cells to deal with changes in environmental conditions (Upchurch 2008). Therefore,
we hypothesised that trace elements could potentially modify phytoplankton fatty
acid composition, which could then be used as potential biomarkers, and thus acquire
reinforced relevance in future biomonitoring studies. As a first approach, the effect
of Ni on P. tricornutum fatty acid composition was investigated, and a reduction of
polyunsaturated fatty acid eicosapentaenoic acid (C20:5, EPA) percentage induced
by this element was found (Fig. 12) (Matos et al. 2016). In spite of this promising
result pointing to EPA as a sensitive biomarker of trace element overload, further
research is needed to obtain quantitative profiles of fatty acids in phytoplankton cells
exposed to other trace elements, individually and combined, in order to undoubtedly
118 Ecotoxicology of Marine Organisms

Fig. 12. Relative abundance of fatty acids of Phaeodactylum tricornutum non-exposed and exposed cells
to Ni, namely saturated and mono-unsaturated fatty acids (white, and lighter shades of grey), plastidial
fatty acids (vertical and oblique stripe patterns and darker shades of grey), omega-6 fatty acids (black
and dense dotted pattern), omega-3 fatty acids (checkerboard pattern) (modified from Matos et al. 2016).

assert fatty acids as trace element biomarkers to be incorporated in estuarine and

coastal biomonitoring programs in the future.

Conclusions
Capitalising our ongoing effort to provide suitable phytoplankton indicators and
biomarkers of element stress with potential application to element contaminated
estuarine and coastal systems worldwide, a step-by-step approach was set, following
the pathway of trace elements from their sources in the water column to their
allocation within phytoplankton cells, and subsequent effects.
A trace element-specific suite of phytoplankton indicators and biomarkers
was produced. Trace element accumulation and intracellular partitioning, growth,
photosynthesis (Chl a fluorescence, F685/F735 ratio and OJIP-test parameters),
diatom:other groups ratio, benthic:pelagic diatom ratio and individual taxa, were
found effective and sensitive indicators of trace element stress alongside trace
element substituted chlorophylls and pigments as reliable biomarkers that can be used
in combination for the early detection of trace element impacts during disturbance
events in estuarine and coastal waters. The potential of fatty acids as efficient
phytoplankton biomarkers of trace element stress was disclosed, highlighting the need
for further research before fatty acids can be proclaimed as efficient phytoplankton
biomarkers of trace element stress. This overview pave the way for further, improved
assessments of trace element contamination events in estuarine and coastal systems,
which, hopefully, will necessarily lead to more robust decision-making regarding
effective environmental monitoring and protection programmes.

Acknowledgments
The authors would like to thank to the “Fundação para a Ciência e Tecnologia (FCT)”
for funding the research in the Marine and Environmental Sciences Centre (MARE)
throughout the project UID/MAR/04292/2013 and the Biosystems and Integrative
 Overview of Phytoplankton Trace Element Indicators and Biomarkers 119

Sciences Institute (BioISI) throughout the project UID/MULTI/04046/2013.

B. Duarte and M.T. Cabrita investigation were supported by FCT through Posdoctoral
grants SFRH/BPD/115162/2016 and SFRH/BPD/50348/2009, respectively.

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6 Phytoremediation and
Removal of Contaminants
by Algae and Seagrasses
Bouchama Khaled,1,4,* Rouabhi Rachid2 and Bouchiha Hanen3

INTRODUCTION
The marine environment is a complex system which is mainly influenced by various
physical, chemical and biological processes (Bandekar and Haragi 2017). The open
ocean is rather stable compared to the near shore waters, where the interaction with
the terrestrial zone results in variations of different physic-chemical parameters near
the shore waters (Bhadja and Kundu 2012). The high human population density
in coastal regions has produced many economic benefits, including improved
transportation links, industrial and urban development, revenue from tourism and
food production, but the combined effects of booming population growth and
economic and technological development are threatening the ecosystems that
provide these economic benefits (Creel 2003). Now, one of the major concerns
of the industrialized world is the high level of marine pollution with hazardous
compounds (Naik and Dubey 2017). Pollutants are responsible for significant lethal
and sub-lethal effects on marine life; pollutants impacts all trophic levels, from
primary producers to apex predators, and thus interfere with the structure of marine
communities and consequently, ecosystem functioning (Todd et al. 2010). Many
pollutants are susceptible to interact with the physiological processes such as growth

1
Ecology and environment department, Khenchela University, Algeria.
2
Applied biology department, Tebessa University, Algeria.
3
Biology department, El Tarf University, Algeria.
4
Cellular Toxicology Laboratory, Annaba University, Algeria.
* Corresponding author: [email protected]
 Phytoremediation and Removal of Contaminants by Algae and Seagrasses 129

and reproduction of marine biota; in fact, pollutants can alter their life and lead to
serious disruptions such as reduction of populations and changes of the reproductive
functions (Hamza-Chaffai 2014). In contaminated marine environments, several
plant species have shown the ability to tolerate and accumulate organic and non-
organic pollutants. Many of them can accumulate various contaminants and their
concentrations are much higher than present in their environments (Fernandez and
Gardinali 2016, Navarro et al. 2016, Qiu et al. 2017).

Pollution and Marine Biota

Marine and aquatic environment are reservoirs of biological diversity with an extreme
importance for several animal species and plants; for a considerable number of them,
water and wetlands are absolutely necessary for life cycle achievement (Gayet et
al. 2016). However, they are severely affected by human activity and pollution,
which greatly affect the aquatic environments that are very sensitive to different
contamination typologies (Mille et al. 1998). Various forms of physical, chemical
and biological contaminants are reported in polluted waters (Dhir 2013a). The
major part of all pollution comes from terrestrial and coastal areas, either through
runoff and discharges via water ways including the river influx (44%) or through the
atmosphere, mainly due to flaring from offshore oil and gas activity and the entering
to sea through atmospheric transport (33%) (IMO Center 2012). Only 12 percent of
all pollution are due to maritime activity and shipping accidents, garbage and sewage
dumping (10%), as well as the consequences of offshore drilling and mining (1%)
(Clark et al. 1989, Roose et al. 2011, IMO Center 2012).
Many harmful contaminants find their way into the marine environment, being
ecotoxicologically the most significant substances harmful to marine ecosystems
(Tornero and Hanke 2016). It should be kept in mind that very few substances are
added to the sea in a chemically pure state, but most are part of complex liquid
or gaseous solutions (Kachel 2008). There are thousands of chemicals present
in the marine environment due to anthropogenic activities (Gioia et al. 2011).
Contaminants generally change marine ecosystem functioning by reducing
productivity and increasing respiration; however, these effects vary according to
the type of contaminant and the components of the system studied (e.g., particular
trophic levels, functional groups or taxonomic groups) (Johnston et al. 2015). These
xenobiotics can be divided into those organic and inorganic contaminants, according
to their composition (Newman 2014).

Organic contaminants
Organic contaminants discharged to the aquatic environment are characterized by
a high diversity with respect to their molecular structures and the resulting physic-
chemical properties (Schwarzbauer 2006). Organic compounds are crudely divided
into aromatic compounds include benzene or similar compounds in their structure
and aliphatic compounds composed of carbon chains with various bonds and other
non-aromatic structures (Newman 2014). Aquatic plants irrespective free-floating,
130 Ecotoxicology of Marine Organisms

submerged and emergent including seagrasses and algae possess immense potential
for remediation of various organic contaminants (Dhir 2013b).

Polycyclic aromatic hydrocarbons (PAHs)

The PAHs are classified depending on their origin (Dahle et al. 2003, Hylland 2006).
There are three major types: petrogenic, biogenic and pyrogenic (Dahle et al. 2003).
Pyrogenic PAHs are formed by incomplete combustion of organic compounds, such
as fossil fuels (Abdel-Shafy and Mansour 2016), while petrogenic PAHs are present
in oil and by-products, closely related to shipping activities and sewage input (Dahle
et al. 2003, Li et al. 2015b). Although petrogenic PAHs appear to be bioavailable
to a large extent, pyrogenic PAHs are often associated with soot particles and less
available for uptake by organisms (Hylland 2006). The third type, biogenic PAHs
can be produced biologically by certain plants, fungi and bacteria or formed during
the organic matter decomposition, and include perylene and retene (Blackman
1986, Abdel-Shafy and Mansour 2016). Polycyclic aromatic hydrocarbons have a
relatively low solubility in water, but are highly lipophilic, and therefore interfere
with cell membrane fluidity (Van Brummelen TC 1998, Boehm 2004). Consequently,
the concentration of these compounds in seawater and sediments undoubtedly carries
toxicological significance for both benthic and pelagic marine organisms (Wetzel and
Van Vleet 2004). In ecotoxicologically terms, PAHs are carcinogenic and mutagenic
to aquatic animals and humans (Pampanin and Sydnes 2013, Rubio-Clemente
et al. 2014). Petrogenic, biogenic and pyrogenic entering the water system can first
accumulate in sediments (Meador et al. 1995, Abdel-Shafy and Mansour 2016).
They remobilize later in seawater, becoming bioavailable and finally accumulated in
some marine biota such as gelatinous zooplankton (Almeda et al. 2013) and corals
Acropora sp. and Montipora sp. (Ko et al. 2014). Inevitably polycyclic aromatic
hydrocarbons will also be bioaccumulated through the food chain (Li et al. 2015a).
Despite the toxic effect of those compounds, some species of algae and seagrasses
have shown the ability to accumulate and biodegrade those contaminants; this
capacity could play an important role in the fate of PAHs in coastal and estuarine
systems. Algae are widely distributed in aquatic environments which may be a major
sink for degradation and/or transformation of PAHs. These organisms oxidize PAHs
under photoautotrophic conditions to form hydroxylated intermediates (Mueller
et al. 1996). According to Hong et al. (2008), the brown diatom algae Skeletonema
costatum and Nitzschia sp. are capable of accumulating and degrading phenanthrene
and fluoranthene (two typical PAHs). Also, in a recent study, for the first time,
the simultaneous removal, bioaccumulation and degradation of a mixture of three
PAHs (phenanthrene, fluoranthene and pyrene) by the marine algae Rhodomonas
baltica were observed (Arias et al. 2017). The transformation of PAHs in marine
and freshwater algae is species specific and depends on the presence and activity
of enzymes localized in the plant cells. The most important enzyme systems for
detoxification are o-diphenol oxidase, cytochrome P450 and peroxidase (Kirso and
Irha 1998). Previous studies on bioconcentration and transformation of PAHs by
algae, have demonstrated that the removal of those organic contaminants results from
various physicochemical processes, transformation, accumulation, metabolization,
 Phytoremediation and Removal of Contaminants by Algae and Seagrasses 131

abiotic transformation through photodegradation, volatilization, sorption and

adsorption (Kirso and Irha 1998, Matamoros et al. 2015, Ghosal et al. 2016, Gupta
et al. 2017). Concerning seagrasses in general, when exposed to petrochemicals
and PAHs at sub-lethal concentrations, the contaminants are incorporated into the
tissue, resulting in reduced tolerance capacities to other stress factors, suffocation,
reduce growth rates and lower flowering rates (Zieman et al. 1984, Kenworthy
et al. 1993, Dean et al. 1998). Several studies were performed to investigate the
PAHs concentration in tissues of different seagrass species and evaluated the use of
these organisms as bioindicators, as well as for biodegradation and bioaccumulation
purposes: Posidonia oceanica (Pergent et al. 2011, Apostolopoulou et al. 2014),
Thalassia testudinum and Halodule wrighti (Lewis et al. 2007), Syringodium
filiforme and Thalassia testudinum (Bouchon et al. 2016). Despite the toxic effects
of PAHs, these studies revealed that the accumulation of those contaminants does
not seem to be an environmental threat for seagrass beds, with some species showing
a good potential as bioindicator. Effectively, there are numerous examples of PAHs
contamination having no significant impact on the various seagrass after several
years (see Kenworthy et al. (1993) and references herein).

Persistent organic pollutants (POPs)

Persistent organic pollutants (POPs) constitute a group of organic chemicals that
contain bound chlorine or bromide atoms (Haynes and Johnson 2000). Among the
most important classes of persistent organic pollutants are many families of chlorinated
and brominated aromatics, including polychlorinated biphenyls (PCBs), dioxins as
polychlorinated dibenzo-p-dioxins and-furans (PCDD/Fs) and different organochlorine
pesticides (DDT and it’s metabolites, toxaphene) (Jones and de Voogt 1999). Their
synthesis is mostly directed towards industrial or agrochemicals uses (Jones and de
Voogt 1999), while few may have natural origin, for example from volcanic eruptions
(WHO 2009). Persistent organic pollutants enter the marine realm by two main
ways: either by atmospheric deposition by way of dry or wet sedimentation or via
surface effluent release, for the offshore areas, this being the primary route by which
POPs enter the marine environment through riverine input (Song 2011). Under the
Stockholm Convention, 90 signatory countries have agreed to reduce and/or eliminate
the production, use, and release of the 12 POPs (e.g., aldrin, chlordane, DDT, dieldrin,
heptachlor, mirex, etc.) of greatest concern to the global community (USEP Agency
2002). This refers to a group of substances that to varying extents resist photolytic,
biological and chemical degradation, characterized by low water solubility and high
lipid solubility, leading to their bioaccumulation in fatty tissues (IPCS 1995). For
example, the accumulation of PCBs and DDT in Selenastrum capricornutum increases
with the increase in total algal lipid content (Halling-Sørensen et al. 2000). The
potential collateral effect of POPs to the nontarget organisms is not well understood
as such contaminants behave differently individually, whereas the toxicity increases/
decreases several folds due to synergistic and antagonistic effects of co-occurring POPs
(Newman 2014). Also, they possess a very high bioaccumulation potential in aquatic
and terrestrial organisms including humans (Beek 1999). Persistent organic pollutants
(POPs) could hardly be degraded by microorganism under natural circumstances, since
132 Ecotoxicology of Marine Organisms

are biodegraded at very slow rates (Song 2011). In marine environment, different living
species possess the ability to accumulate POPs, such as the two species of seagrasses
Thalassia testudinum and Halodule wrighti (Lewis et al. 2007). As well Chlorella sp.
(Geyer et al. 2000). Marine diatoms (Phaeodactylum tricornutum, Thalassiosira
nordenskio, Skeletonema costatum) and algae (Emiliana huxleyi) as a consequence
of their large surface area and significant organic carbon content, they sorb PCBs
(Gerofke et al. 2005).
Several factors influence the amount of POPs accumulated in seagrasses and
algae. Climate change will have an effect on the environmental fate and behaviour
of contaminants by altering physical, chemical and biological drivers of partitioning
between atmosphere, water, soil and biota, including reaction rates. For example,
increasing temperatures and subsequent melting of snow and ice can remobilize
POPs into water and the atmosphere (Noyes et al. 2009).

Emerging organic contaminants (EOCs)

Emerging and newly identified contaminants are a major concern for marine
ecosystem (Jiang et al. 2014, Geissen et al. 2015). This term is used to cover not
only newly developed compounds, but also newly compounds discovered in the
environment often due to analytical developments (Richardson and Ternes 2011,
Sauvé and Desrosiers 2014). The contaminants of emerging concern (CEC) can occur
naturally, be manufactured or manmade or can be new materials which have now been
discovered or are suspected to be present in various environmental compartments
and whose toxicity or persistence are likely to significantly alter the metabolism
of a living being (Sauvé and Desrosiers 2014). A diverse array of synthetic organic
compounds are used by society in high amounts for a range of purposes including
the production and preservation of food, industrial manufacturing processes and for
human and animal healthcare (Lapworth et al. 2012). With effects ranging from acute
to chronic in aquatic organisms, its accumulation in the ecosystem leads inevitably to
losses of habitats and biodiversity, as well as threats to the human health (Sánchez-
Avila et al. 2012). An increasing number of emerging contaminants studies are
arising in the ecology and ecotoxicology literatures (Geissen et al. 2015, Thomaidi
et al. 2015, Hurtado et al. 2017, Pintado-Herrera et al. 2017).

Endocrine-disrupting chemicals (EDCs)

According to the World Health Organization, endocrine disrupting chemicals
EDCs and potential EDCs are mostly man-made, found in various materials such
as pesticides, metals, additives or contaminants in food and personal care products
(WHO 2013). They include insecticides, fungicides, herbicides, pharmaceuticals
and industrial contaminants (Hotchkiss et al. 2008). Endocrine-disrupting chemicals
are defined as contaminants with the ability to interfere with the endocrine system
of different organisms causing important alterations in their normal development
(Álvarez-Muñoz et al. 2016). Numerous studies on aquatic organisms such as algae
and different types of seagrass have been recently explored for the removal of organic
pollutants possessing endocrine disrupting capacity. Eliana Gattullo et al. (2012)
observed that after 2–4 days of bisphenol exposure at low concentrations, there were
 Phytoremediation and Removal of Contaminants by Algae and Seagrasses 133

no toxic effects detectable for the green alga Monoraphidium braunii, whilst a good
removal efficiency was detected. In addition, the study of Solé and Matamoros (2016)
indicated that the use of co-immobilized microalgae-based wastewater treatment
systems increased the removal efficiency for nutrients and some EDCs from
wastewater effluents. In recent years, several species of marine mammals and birds
have been affected by EDCs (Tanabe 2002). Mangroves and seagrasses are known
to support very high densities juvenile reef fish species and likely to offer enhanced
survival for these individuals compared to those living in other habitats (Nagelkerken
2009). Seagrassbeds help to trap sediments as they settle out in the calm waters
among shoots and roots, and can also play a role in the active removal of pollutants
dispersed into adjacent waters (Waycott et al. 2009). For example, the accumulation
of Diuron (herbicide used to control a wide variety of annual and perennial broadleaf
and grassy weeds) by Zostera capricorni and Halodule uninervis, oftenly occurs in
higher concentrations in plant tissues than sediment concentrations (Haynes et al.
2000). This filtration potential is thus recognised as an essential ecosystem service
for protecting juvenile fish, benthic organisms and nursery areas (Burke et al. 2011).

Pharmaceuticals and personal care products (PPCPs)

Pharmaceuticals and personal care products (PPCPs) is currently one of the
most concerned new forms of pollution (Xie 2017). Many (PPCPs) were found
in aquatic environment, including personal care products (e.g., detergents and
musks), pharmaceuticals (e.g., drugs, veterinary and human antibiotics and birth
control substances) (Claessens et al. 2013, Dhir 2013b, Newman 2014). Personal
care products, pharmaceuticals compounds and their bioactive metabolites are
continually introduced to the aquatic environment as complex mixtures via a number
of routes, but mostly by both untreated and treated sewage (Daughton and Ternes
1999). An increasing number of studies has confirmed the presence of various PPCPs
in different environmental compartments, which raises concerns about the potential
adverse effects to humans and wildlife (Ebele et al. 2017). Simvastatin, clofibric
acid, diclofenac, carbamazepine, fluoxetine and triclosan represent some of the
most commonly used and/or detected PPCPs in aquatic environments (DeLorenzo
and Fleming 2008). Biodegradation, photodegradation and sorption are the main
processes involved in PPCP removal during treatment but also depending on
the properties of the compounds (Wang et al. 2017). There is a growing body of
literature reporting the effects of PPCPs on freshwater organisms, but studies on the
effects of PPCPs to marine and estuarine organisms are more limited (Prichard and
Granek 2016). The study of Bai and Acharya (2017) evaluated the removal of five
common PPCPs (trimethoprim, sulfamethoxazole, carbamazepine, ciprofloxacin
and triclosan) from Lake Mead water mediated by the green alga Nannochloris sp.,
showed that the algae-mediated sorption contributed to 11 percent of the removal of
trimethoprim and sulfamethoxazole, 13 percent of carbamazepine and 27 percent
of the removal of triclosan from the lake water. Similar research studies were
performed to investigate the capability and mechanisms of PPCPs removal by algae
in laboratory conditions which showed a high ability to remove many PPCPs; for
example, the biodegradation of 17b-estradiol by Selenastrum capricornutum and
134 Ecotoxicology of Marine Organisms

Chlamydomonas reinhardtii (Hom-Diaz et al. 2015), biodegradation of Norgestrel

by Scenedesmus obliquus (Peng et al. 2014), the biotransformation of Estriol by
Scenedesmus dimorphus (Zhang et al. 2014) and the hydrolysis, photolysis and
adsorption of 100 percent of 7-amino cephalosporanic acid by Mychonastes sp. in
five days (Guo et al. 2016). However, several PPCPs exhibited low removal rates
(carbamazepine, lorazepam, trimethoprim and verapamil) which can be attributed to
their low biodegradability (Wang et al. 2017).

Surfactants
Surfactants are one of the most ubiquitous families of organic compounds (Cirelli
et al. 2008). Although their main application is the formulation of household and
industrial detergents, they are also used in the manufacturing of other products
such as cosmetics and personal care products, paints, textile, dyes, polymers,
agrochemicals and petroleum (Jackson et al. 2015, Álvarez-Muñoz et al. 2016). The
global surfactant market volume size is more than 18 million tons per year; large
quantities are continuously released into the environment, where they can or cannot
be degraded depending on their structure (Cirelli et al. 2008). Surfactants contain
both hydrophobic groups (their “tails”) and hydrophilic groups (their “heads”)
making them soluble in both organic solvents and water; they are classified into
nonionic, anionic, cationic or zwitterionic by the presence or absence of formally
charged hydrophilic head groups (Cowan-Ellsberry et al. 2014). These compounds
enter the aquatic environment after use, often going via sewage treatment works,
where they may undergo biological degradation before entering rivers and other
waters (Sumpter et al. 1996). The most-used surfactants and their acronyms are
shown in Table 1.
Table 1. Acronyms of the most widely used surfactants (Ying 2006, Cirelli et al. 2008).

Common name Acronym

Anionic surfactants
Linear alkyl benzene sulfonates LAS
Secondary alkane sulfonates SAS
Alcohol ether sulfates (Alkyl ethoxy sulfates) AES
Alcohol sulfates (Alkyl sulfates) AS
Nonionic surfactants
Alkylphenol ethoxylates APE (or APEO)
Nonyl phenol ethoxylates NPE (or NPEO)
Octyl phenol ethoxyales OPE (or OPEO)
Alcohol ethoxyaltes (Alkyl ethoxyaltes) AE (or AEO)
Cationic surfactants
Quaternary ammonium-based compounds QAC
Alkyl dimethyl (and trimethyl) ammonium halides DMAC (and TMAC)
Alkyl benzyl dimethyl ammonium halides BDMAC
Dialkyl dimethyl ammonium halides DADMAC
Dihydrogenated tallow dimethyl ammonium chloride DHTDMAC or DTDMAC
Ditallow trimethyl ammonium chloride DTTMAC
Diethyl ester dimethyl ammonium chloride DEEDMAC
Zwitterionic surfactants
Cocamidopropyl betaine CAPB
Cocamidopropyl hydroxysultaine CAHS
 Phytoremediation and Removal of Contaminants by Algae and Seagrasses 135

It is also likely that surfactants, and their degradation products, bioaccumulate

and/or bioconcentrate in aquatic organisms (Sumpter et al. 1996). Various species
of algae (Scendesmus quadriauda, Chlorella vulgaris, Ankistrodesmus acicularis,
Chroococcus minutes and the diatom Navicula incerta) have shown a ability for
the removal and biodegradation of nonylphenol (NPs) (Liu et al. 2010, He et al.
2016). Also, the seagrass Posidonia oceanica biomass showed a high potential
for adsorptive removal of anionic (NaDBS) and non-ionic (TX-100) surfactants
from aqueous solutions. Ncibi et al. (2008) found that as a biological adsorbent to
remove anionic and non-ionic surfactants, Posidonia oceanica biomass seems to be
a promising technique.

Inorganic contaminants
The presence of inorganic contaminants is very common in the marine environment
and polluted waters; these mainly include metals, ions/nutrients and radionuclides
(Dhir 2013b). Metals are present in the marine ecosystems within its different
compartments: water column, sediments and marine biota, where they are accumulated
directly from water or food (Barka 2012). Nevertheless, many metals are released
naturally into the environment through weathering and leaching processes, with
human activities inducing severe shifts in these elements estuarine and coastal budgets
(Karouna-Renier et al. 2007). Two metals types can be distinguished according
to their physiological and toxic effects: the essential elements as macroelements
(N, P, K, Mg, S, Ca) or microelements (Zn, Fe, Mn, Cu, B, Mo, Co, Ni, Cl, V),
which are essential for plant growth in low concentrations (Prasad 2001, Mitra
2017); however, beyond certain threshold concentrations they become toxic (Mitra
2017). Some common micronutrients required by both plants and animals include
aluminum (Al), boron (B), bromine (Br), chromium (Cr), cobalt (Co), copper (Cu),
fluorine (F), gallium (Ga), iodine (I), iron (Fe), manganese (Mn), molybdenum (Mo),
selenium (Se), silicon (Si), strontium (Sr), tin (Sn), titanium (Ti), vanadium (V), and
Zinc (Zn) (Pidwirny 2017). Other elements are non-essential, namely arsenic (As),
cadmium (Cd), mercury (Hg) and lead (Pb) and are not required but they have been
studied extensively due to their potentially hazardous effects to plants, animals and
microorganisms (Stevenson 1999, Adriano 2001). In fact, beyond a given threshold,
all metals, whether essential or not, may have toxicological and ecotoxicological
effects (Barka 2012). In different marine organisms, the behaviour of heavy metals is
described in terms of their absorption, storage, excretion and regulation when different
concentrations are available in the environment (Bryan 1971) as well as the route and
duration of exposure, that is, acute or chronic exposures (Jaishankar et al. 2014). In
polluted marine ecosystems, some types of seagrass and algae show a high capacity
to absorb heavy metals as results of tolerance mechanisms such as the uptake of (Cd,
Cu, Mn, Ni, Pb, Zi and Hg) by marine algae (Centrocerous clavutum, Sargassum
wightii, Colpemenia sinosa, Spyridia hypnoides, Valoniopsis pachynema, Ulva
reticulata, Gelidialla acerosa and Turbinaria ornate) and seagrasses (Syringodium
isoetifolium & Cymodocea serrulata) (Sudharsan et al. 2012). Also Halodule pinifolia
showed the capacity to absorb nickel (1.1 ppm) higher than the threshold value limit
(0.02 ppm) for water medium (Espiritu and Paz-Alberto 2018). The uptake of metals
136 Ecotoxicology of Marine Organisms

in seaweeds and seagrass depends on the surface reaction in which metals absorbed
through electrostatic attraction to negatives sites, this being independent of factors
influencing metabolism such as temperature, light, pH or age of the plant, but it is
dependent on the virtual abundance of elements in the surrounding water (Sánchez-
Rodrıguez et al. 2001). Concerning ions/nutrients, although in a strict sense not as
toxic as the pollutants discussed above, nutrients can have severely damaging effects
on the marine environment (Kachel 2008). The major ions present in contaminated
water are ammonia, nitrite, nitrate, chloride, sulphate, phosphorus and cyanide (Dhir
2013b). These result mostly from nutrient application as fertilizers in agriculture and
correspondent runoff to the sea due by riverine inputs (Kachel 2008). Contributions
of high levels of nitrogen and phosphorus compounds, in particular, often result
in eutrophication (Kachel 2008, Chislock et al. 2013). In India, the seagrass
(Cymodocea rotundata) was used as biosorbent to remove excessive N and P from
aqueous solution and dye from the textile wastewater. This study revealed that this
species has a high potential in removing N and P from aqueous solution (Vasanthi
et al. 2015).
Recently, there has been an enrichment of specific radionuclides in the
environment due to manufacture and testing of nuclear weapons, extensive
construction of nuclear power plants, commercial fuel reprocessing, nuclear waste
disposal, uranium mining and enrichment and nuclear accidents (Mitra 2017). The
term radiation refers to the propagation of energy through space, this being in the
form of photons, ejected atomic nuclei or their fragments or subatomic particles.
Additionally, radiation can also be generated within the nuclei of unstable elements:
such nuclei are called radionuclides (Newman 2014). Exposure of biota to radiation
and transfer of radionuclides in the environment are intimately linked, and occurs
when radionuclides, present naturally in the environment or released through man’s
activities, decay releasing radiation of various types and energies (Brown et al. 2009).
There are three natural and one artificial series of radionuclides in which one (parent)
radionuclide decays to another (progeny) in a series of steps until the most stable
element is formed, the series include the uranium–radium, thorium, actinium and
neptunium series (Newman 2014). After the Fukushima disaster in Japan in 2011,
many studies discussed the effects of radionuclides releases from the accident site
in marine biota (Buesseler et al. 2012). Some of these studies detected Cs isotopes
in zooplankton and mesopelagic fish, and also 131I and 134,137Cs in seaweed, mollusks
and fish (i Batlle and Vandenhove 2014). According to Moss (1973), the presence of
radionuclides in the marine environment is possibly due to radioactive discharges
resulting from the practice of nuclear medicine.

Biology and Ecology of Seagrasses and Algae

Seagrass
Seagrass are a functional group of about 60 species of underwater marine flowering
plants monocotyledons. They often grow in dense beds and extensive meadows
creating a productive and diverse habitat used as shelter, nursery, spawning
or food area by a large variety of animal species (Green and Short 2003). These
 Phytoremediation and Removal of Contaminants by Algae and Seagrasses 137

Fig. 1. Composite illustration demonstrating morphological features used to distinguish main seagrass
taxonomic groups (McKenzie et al. 2009).

plants occupy sandy, muddy and rocky substrates in every sea in the world (Denny
and Gaines 2007). As reported by Duarte (2002), seagrasses cover about 0.1–0.2
percent of the global ocean. They range from the tiny, 2–3 cm, rounded leaves of
sea vine (e.g., Halophila decipiens) to the strap-like blades of eelgrass (e.g., Zostera
caulescens) at more than 4 meter long (Green and Short 2003), the world’s longest
seagrass reaching more than 7 metres (Aioi et al. 1998). Like terrestrial plants,
seagrass have leaves, roots, conducting tissues, flowers and seeds. Unlike terrestrial
plants, however, seagrasses do not possess the strong, supportive stems (Fig. 1).
Rather, seagrass blades are supported by the natural buoyancy of water, remaining
flexible when exposed to waves and currents (Denny and Gaines 2007). Seagrass
accomplish their underwater reproduction by producing filamentous pollen grains
that can be transported by water currents (Mitra and Zaman 2015). Due to their
morphology, seagrasses are sometimes confused with marine macroalgae (seaweeds)
(Levinton 1995, Mitra and Zaman 2015). In contrast to seagrass categorized as
vascular, seaweeds have little or no vascular tissues and they lack a true root system
as they have holdfasts (Lobban and Harrison 1994). Additionally, seaweeds have
spores and do not flower or produce fruit, while seagrasses produce flowers, seeds
and fruit (Denny and Gaines 2007, Larkum et al. 2007). Seagrass provide a habitat
for fish and shellfish and nursery areas to the larger ocean, and perform important
physical functions of filtering coastal waters, dissipating wave energy and anchoring
sediments (Green and Short 2003). A reduction in seagrass vigorous growth and
production, and consequent major reductions in their area, will result in serious
changes to coastal ecosystems. Artificial seagrass restoration projects increased fish
and shrimp in number and diversity and provided feeding areas, highlighting their
essential ecosystem role (Talbot and Wilkinson 2001).

Algae
Seagrasses do not grow in isolation but form an integral and often defining part of
highly complex ecosystems. Although they themselves are an important standing
stock of organic matter, the productivity of these ecosystems is usually enhanced by
other primary producers, including macroalgae epiphytic and free-living microalgae
138 Ecotoxicology of Marine Organisms

(Green and Short 2003). There is considerable variability in the organization of algae,
from unicellular organisms only a few microns in size to thalli of complex structure,
such as the large phaeophycean kelp Macrocystis, which can reach 100 meters in
length. Nevertheless they all have rudimentary conducting tissues (Gallardo 2014).
Algae can be classified into two main groups; first one is the microalgae, which
includes blue green algae, dinoflagellates, bacillariophyta (diatoms), etc., and second
one composed by macroalgae (seaweeds) which includes green, brown and red algae
(El Gamal 2010). In contrast to seagrass, marine algae supports a great diversity
of species. They have been estimated to include anywhere from 30,000 to more
than one million species, most of which are marine algae (Guiry 2012). However,
they are more primitive than seagrasses (Mellors and McKenzie 2008). Algae are
biochemically and physiologically very similar to what is known as higher plants:
they essentially have the same metabolic pathways, possess chlorophyll and produce
similar proteins and carbohydrates (Gallardo 2014).

Accumulation of Contaminants
Aquatic plants have been shown to play important roles in wetland biogeochemistry
through their active and passive circulation of elements (Vymazal and Kröpfelová
2008). In contaminated marine environment, several flora species have shown to
have the ability to tolerate and accumulate organic and non-organic pollutants, and
in some cases present concentrations much higher than those present in water and
sediments (Singh et al. 2002, Ayangbenro and Babalola 2017). Contaminants are
deposited in the various components of the aquatic environment (water, sediment
and biota) and can be accumulated through the food chain (Binelli and Provini 2003).
In natural environments, organisms living in chronically polluted sites are exposed to
low concentrations of contaminants for long periods; in the other cases, the organism
may be abruptly exposed to high levels contaminants upon the outfall of a pollutant
in coastal waters (Torres et al. 2008). The adaptability of these species depends
mainly on the type of pollutant, its concentration, duration of exposure and some
physicochemical parameters of water (Pérez-Ruzafa et al. 2000, Lawson 2011).

Bioconcentration and Bioaccumulation Factor

Bioconcentration is the result of the direct uptake of a chemical by an organism only
from water and the result of such a process is measured by the bioconcentration factor
(BCF), which represents the ratio of steady state concentration of the respective
chemical in the biota (mass of chemical per kg of organism dry weight) and the
corresponding freely dissolved chemical concentration in the surrounding water
(mass of chemical per unit of volume) (Geyer et al. 2000). Concerning the Biota-
Sediment bioaccumulation factor (BAF or BSAF) is defined as the ratio between the
contaminant concentration in the biota (mass of chemical per kg of biota dry weight)
and that in the sediment (mass of chemical per kg of sediment dry weight) (Nenciu
et al. 2014, Newman 2014). According to the definition given by the USEPA, a
substance is considered bioaccumulative when it has a BCF ranging between
 Phytoremediation and Removal of Contaminants by Algae and Seagrasses 139

1000 and 5000 and very bioaccumulative if it has a BCF greater than 5000 (TSCA
2012). However, the European Chemicals Legislation (Registration, Evaluation,
Authorization and Restriction of Chemicals, R.E.A.CH) stipulates that a substance
fulfils the bioaccumulation criterion when the bioconcentration factor (BCF) is
higher than 2000 (EC 2006). Regarding (BAF or BSAF) the different species of
marine organisms can be classified into three groups, such as macroconcentrator
(BSAF > 2), microconcentrator (1 < BSAF < 2) or deconcentrator (BSAF < 1)
(Dallinger 1993). Several studies, generally in situ, have been carried out concerning
the use of seagrasses and marine algae for the decontamination of marine and aquatic
environment (Table 1). The bioconcentration factor (BCF) and the biota-sediment
bioaccumulation factor (BAF) cited in Table 2, show that some species of seagrass
and algae represent an enormous potential for removal of a variety of contaminants,
including heavy metals, inorganic/organic pollutants by phytoremediation.

Phytoremediation
The use of plants directly or indirectly to remediate contaminated water is known
as phytoremediation. This technology has emerged as a cost effective, noninvasive
and publicly acceptable way to address the removal of environmental contaminants.
Plants can be used to accumulate inorganic and organic contaminants, metabolize
organic contaminants and encourage microbial degradation of organic contaminants
in the root zone (Arthur et al. 2005). Different techniques can be distinguished within
phytoremediation of polluted water according to their objectives, the characteristics
of the environment, the contaminants and the used species: phytoextraction,
phytodegradation (rhizodegradation), phytostabilization rhizofiltration (Brooks
1998, Salt et al. 1998, Kirkham 2006). Treatment of organic contaminants mainly
involves phytostabilization, phytodegradation (rhizodegradation) and rhizofiltration.
While phytostabilization, rhizofiltration and phytoaccumulation mechanisms are
involved in the treatment of inorganic contaminants (Dhir 2013b). Rhizofiltration is a
form of phytoremediation is a combination of phytoextraction and phytostabilisation,
which refers to the approach of using hydroponically cultivated plant roots to
remediate contaminated water through absorption, concentration and precipitation
of pollutants. Due to their morphology, seagrass are more adapted for rhizofiltration
(Salt et al. 1998, Alkorta et al. 2004). Phycoremediation is defined as the “use of algae
to treat wastes or wastewaters”. The algae comprise both the microalgae as well as
the marine macroalgae, more commonly known as the seaweeds (Phang et al. 2015).
The phytoremediation in marine ecosystem is affected by several factors including,
species, biomass (the high-biomass possess higher contaminant removal potential)
and physicochemical parameters of water and contaminant (Dhir 2013a).

Tolerance and Detoxification Mechanisms of Inorganic Contaminants

Plants have many endogenous genetic, biochemical and physiological properties
to tolerate pollution, that make them ideal agents for soil and water remediation
(Meagher 2000). In aquatic environments, the contaminant uptake from water
140 Ecotoxicology of Marine Organisms

Table 2. Bioconcentration factors (BCFs) and bioaccumulation factors (BSAF) biota-sediment

accumulation factor of contaminants in seagrasses and algae.

Species Contaminant BAF and BCF References

Seagrasses
Zostera japonica As, Cd and Mn BAFAs = 3.43; BAFMn = 6.43; (Lin et al. 2016)
BAFCd = 30.95
Halodule wrightii Triazine BCFmax = 21634 (Fernandez and
BCFaverage = 6681 Gardinali 2016)
Syringodium filiforme Triazine BCFaverage = 6809 (Fernandez and
Gardinali 2016)
Posidonia oceanica Ni BCFNi = 59000 (Joksimovic and
BAFNi = 2,7 Stankovic 2012)
Mytilus galloprovincialis As BCFAs = 88000 (Joksimovic and
BAFAs = 1,7 Stankovic 2012)
Holodule uninervis PCDD BAF = 1.73 (McLachlan et al.
2001)
Zostera marina Total PAHs Total BAFroot = 3 (Huesemann et al.
PCBs BAFroot = 4.2 2009)
Zostera marina Triazine BCF = 25000 (Scarlett et al.
1999)
Algae
Phytoplankton (eight Cr, Pb and Cu BCFCr = 4810 (Tao et al. 2012)
species) BCFPb = 24500
BCFCu = 1710
Ulva spp. Pb BCFPb = 0,41–6,05 (Jitar et al. 2015)
(macroalgae) BAFpb = 1,45–1,82
Ulva fasciata DDTs BCFDDTs = 17,2.103 (Qiu et al. 2017)
(macroalgae) and BCFPBDEs = 6.103
PBDEs BAFDDTs = 0.25
BAFPBDEs = 0.26
Phytoplankton species DDTs BCFDDTs = 1642,7.103 (Qiu et al. 2017)
(Chaetoceros and BCFPBDEs = 575,2.103 (Arias et al. 2017)
lorenzianus, Nitzschia, PBDEs BAFDDTs = 23.5
Bacteriastrum hyalinum BAFPBDEs = 24.6
and Thalassionema PAHs:
nitzschioide) Phenanthrene BCF = 7.51
Fluoranthene BCF = 12,89
Rhodomonas baltica Pyrene BCF = 16.51

(directly or from adsorption/accumulation in food) is counteracted by endogenous

enzymatic biotransformation and elimination processes. Hydrologic, geochemical
and environmental conditions can also strongly affect bioaccumulation of a particular
contaminant (either inorganic or organic) by interfering in its bioavailability in the
aqueous phase (Guha 2004). According to Dhir (2013b), the presence of inorganic
contaminants is very common in marine environment and polluted waters; these
mainly include metals, ions/nutrients and radionuclides. Reactive oxygen species
(ROS) generation derived from contaminant exposure leads to the inhibition or
retardation of growth and cell division, impairment of photosynthetic activity
 Phytoremediation and Removal of Contaminants by Algae and Seagrasses 141

and cell death (Di Toppi and Gabbrielli 1999). Bacteria and higher organisms
have developed resistance mechanisms to toxic metals to make them innocuous.
Organisms respond to heavy metal stress using different defence systems, such as
exclusion, compartmentalization or complexation with binding proteins such as
metallothioneins (MTs) or phytochelatins (PCs) and consequent translocation into
vacuoles (Mejáre and Bülow 2001). Metal removal mechanism is a complex process
that depends on the chemistry of metal ions, cell wall compositions, physiology
of the organism as well as physicochemical factors like pH, temperature, time of
exposure, ionic strength and metal concentration (Mishra et al. 2010). In this part, we
paid a special attention to tolerance and detoxification mechanisms of heavy metals
in seagrasses and marine algae.

Biosorption
Biosorption is a promising technology which pays attention to fabricate novel, cheap
(low-cost) and highly-effective materials to apply in wastewater purification technology
(Anastopoulos and Kyzas 2015). The term biosorption is employed to describe the range
of processes by which biomass removes metals and other substances from solution, yet
it can also be used in a stricter sense to mean uptake by dead or living biomass by purely
physico-chemical processes such as adsorption or ion exchange (White et al. 1995).
Biosorption is a method that can be used for the removal of pollutants from wastewater,
especially those that are not easily biodegradable like heavy metals and industrial dyes
(Mata et al. 2008). The use of a biosorbent such as dried algae and seagrass biomass
in wastewater treatment often depends not only on the biosorptive capacity, but also
on how well the biosorbent can be regenerated and recycled (Pennesi et al. 2015).
The biosorbent ability of macrophytes (i.e., seaweeds and seagrasses) resides in the
structure of the cell walls and cuticles of seagrasses and macroalgae (Flouty and
Estephane 2012, Wang et al. 2013). Also micro-algal cell wall have a high binding
affinity for metal cations via counterion interactions (Crist et al. 1981). The algal cell
wall matrix contains different functional groups, such as, hydroxyl (OH), phosphoryl
(PO3O2), amino (NH2), carboxyl (COOH), sulphydryl (SH) and other charged groups,
which are generated by complex heteropolysaccharides and lipid components which
favor sequestration of positively charged molecules (Crist et al. 1981, Niu and Volesky
2000, Mohan et al. 2002, Daneshvar et al. 2007) (Table 3). The biosorption capacity
of different chemical groups depends on several factors: the number of sites on the
adsorbent material, accessibility, their chemical status (availability) and the affinity
between site and metal and contaminants (bond strength) (Vieira and Volesky 2000).
Proteins, lipids and occasionally, nucleic acids may also be present on the surface of
the cell walls. These molecules, however, occur mainly in the plasma membrane and
in the cytoplasm and therefore, are bound to metal ions through their functional groups
(aminic, carboxylic, imidazolic, thiolic, thioesteric, nitrogen and oxygen in peptidic
bonds) mostly within the cell (Majidi et al. 1990). The accumulation of heavy metals
involves two processes: an initial rapid (passive) uptake (its a physical adsorption
between the metal ions and the algae surface), followed by a much slower (active)
uptake (chemical adsorption) (Bates et al. 1982, Garnham et al. 1992). During the
passive uptake, metal ions adsorb onto the cell surface within a relatively short span of
142 Ecotoxicology of Marine Organisms

Table 3. Binding groups for biosorption in some seaweeds and seagrasses (Pennesi et al. 2015).

Binding Chemical Ligand Occurrence in biomolecules Phylum

group formula atom
Amine – NH2 Hydrogen Protein Chlorophyta
Metallothionein Magnoliophyta
Carboxyl – COOH Oxygen Cutin Magnoliophyta
Alginic acid Heterokontophyta

Carbonyl >C=O Oxygen Alginic acid Heterokontophyta

Protein Chlorophyta
Alginic acid Heterokontophyta
Hydroxyl – OH Oxygen Polysaccharides Chlorophyta
Cutin Magnoliophyta
Agarose (agar), carrageenan Rhodophyta
Fucoidan (sulfated polysaccharide) Heterokontophyta
Agaropectin (agar), carrageenan
Sulfonate – SO2=O Oxygen (Sulfated polysaccharides), Rhodophyta
porphyran, furcellaran
(sulfated galactans)
Sulfhydryl – SH Sulfur Metallothionein Magnoliophyta
(thiol)

time (few seconds or minutes), and the process is metabolism independent. However,
the active uptake is metabolism-dependent, causing the transport of metal ions across
the cell membrane into the cytoplasm (Gadd 1988). Biosorption of metals by algae
may be affected by several factors, including concentration of metals and biomass, pH,
temperature, the presence of competing ions and contact time (Dixit and Singh 2015).
Feng and Aldrich (2004) indicated that marine alga Ecklonia maxima can be used as an
efficient biosorbent material for the treatment of aqueous waste streams contaminated
with Cu, Pb and Cd. Also, the Sargassum species showed a high efficiency of copper
biosorption (Carsky and Mbhele 2013). Cystoseira crinita for Ni (II) and Cystoseira
barbata for Cu (II) (Simeonova and Petkova 2007). The principal mechanism of
metallic cation sequestration involved the formation of complexes between a metal ion
and functional groups (carboxyl, carbonyl, amino, amido, sufonate, phosphate, etc.)
present on the surface or inside the porous structure of the biological material (Fourest
and Volesky 1997). It was shown that the presence of alginic acid in the cell wall
played a major role in the complexation of Cd and Pb in Sargassum fluatan (Fourest
and Volesky 1997), and also for Pb2+, Cu2+, Zn2+ and Cd2+ in Sargassum filipendula
(Kleinübing et al. 2013). Effectively, the higher removal performance of those species
is mainly explained by the presence of alginate (anionic polysaccharides) as one of
the major components of brown algae (Kawai and Murata 2016). In red algae, the
amorphous portion is formed by a number of sulfated galactans such as carrageenan,
agar, furcellaran and porphyran. On the other hand, green algae may have an external
capsule that is composed of protein or polysaccharides or both (Graham and Wilcox
2000). For seagrass, the surface of the leaves is covered by a layer of polymeric material
called cuticle (cutin and cuticular waxes) (Smith et al. 2010). The thin cuticle facilitates
uptake of ions and carbon; seagrasses are able to uptake nutrients and carbon directly
 Phytoremediation and Removal of Contaminants by Algae and Seagrasses 143

through the leaves. In order to facilitate the exchange of solutes and gases, the cuticle
of seagrasses is porous and perforated (Hemminga and Duarte 2000). The cuticle of
seagrasses is also rich in carboxyl groups, which are involved in the bond with the
protonated metal, while red and green algae have a lower capacity to immobilize
protonated metals due to the presence of groups other than carboxylic ones (Table 2)
(Pennesi et al. 2015). The recent research of Pennesi et al. (2013) suggest that
biomass of Posidonia oceanica can be used as an efficient biosorbent for removal of
vanadium (III) and molybdenum (V) from aqueous solutions. According to Pennesi
et al. (2015), its assumed that the number of binding sites identified decreases in the
order: brown algae > seagrasses > green algae > red algae. Biosorption is generally used
for the treatment of heavy metal pollutants in wastewater; application of biosorption
for organic and other pollutants could also be used for the treatment of wastewater
(Tsezos and Bell 1989). An example of this is the bisorption of phenol by Posidonia
oceanica fibres (Ncibi et al. 2006), and of 2–4 dichlorophenol onto Posidonia oceanica
(Demirak et al. 2011). There are also reports of adsorption of phenol by Sargassum sp.
and Chaetomorpha algae (Navarro et al. 2016) and the biosorption of methylene blue
dye from wastewater by Sargassum hemiphyllum (Liang et al. 2017).

Phytochelatin and metallothionein

The consequences of xenobiotic bioaccumulation in a biological system are revealed
at multiple hierarchical levels: from single organism effects to cross-linked trophic
connections in the ecosystem as a whole (Newman and Unger 2003). Plants and
algae have the ability to hyper accumulate various heavy metals by the action of
phytochelatins and metallothioneins forming complexes with heavy metals and
translocate them into vacuoles (Suresh and Ravishankar 2004). The mechanism of
accumulation and adsorption of metals by algae involve adsorption onto the cell
surface and binding to cytoplasmic ligands, phytochelatins and metallothioneins
(Fig. 2) (Mehta and Gaur 2005). Phytochelatins are intracellular metal-binding
peptides produced enzymatically by higher plants, fungi and algae in response
to many metals (Ahner et al. 1995). Phytochelatins are polypeptides with the
amino acid structure (y-glu-cys),-gly, where n ranges from 2 to 11 (Grill et
al. 1985). They chelate metals through coordination with the reduced sulfur
in cysteine (Ahner et al. 1995). Metallothioneins resemble phytochelatins
in many ways structurally and functionally (Salt et al. 1995). However,
metallothioneins are characterized by lower molecular weight (6–7 kDa)
and result from mRNA translation, with a distinct enzymatic synthesis when
compared with phytochelatins synthesis (Cobbett and Goldsbrough 2002,
Romero-Isart and Vašák 2002). In plants, these molecules bind to free metal
ions that carry them into vacuoles, where their toxicity is reduced, staying
available for metabolic functions if the target metals are essential for growth
(Murphy et al. 1997). Verbruggen et al. (2009) suggested that cellular vacuoles
are the target compartments for sequestration phytochelatin-conjugated metals.
Metal compartmentalization has been reported for several algae and seagrasses,
for example, compartmentalization of Pb and Zn in Chlorella saccharophila,
Navicula incerta and Nitzschia closterium (Jensen et al. 1982). Other chelation
144 Ecotoxicology of Marine Organisms

Exopolysaccharides and cell wall

M M M

Plasma membrane
M
M M M SH
M
M Poly-p M SH
M
body M
Phytochelatin/
M Metallothionein
M SH
Vacuole M M
M SH

M M

Fig. 2. Metal-binding sites of a typical algal cell. The alphabet M represents the metal species (independent
of its oxidation state) (Mehta and Gaur 2005).

process is based in the ability of polyphosphate bodies to accumulate a number

of heavy metals like Fe, Zn, Cd, Cu and Pb and may serve as protection of algal
cells from metal toxicity (Pawlik-Skowronska and Skowrori 2001). The marine
alga Dunaliella tertiolecta has been shown to have high phytochelatin content
attributed to its capability to hyperaccumulate Zn and Cd (Tsuji et al. 2003). Ahner
et al. (2002) reported that upon exposure to Cd or Cu, there was a significant
increase in the intracellular concentration of phytochelatin in diatoms (Emiliania
huxleyi, Phaeodactylum tricornutum, Thalassiosira pseudonana, Thalassiosira
weissflogii), and green alga (Dunaliella sp.). Also in some seagrass species
such as Enhalus acoroides, a strong correlation between phytochelatin and Pb
tissue concentration was found in the root organ collected from sediment highly
contaminated with Pb (Nguyen et al. 2017). The same could be verified in roots and
leaves of Thalassia hemprichii treated with Pb (Tupan et al. 2014). Same findings
were found in the seagrass Posidonia oceanica exposed to Cu and Cd, with the
synthesis of metallothionein leading to a reduction of the oxidative stress caused
by metals (Cozza et al. 2006). Another study showed that Cd may be transformed
by microalgae to form sulphides, which have low solubilities and thus, lower
toxicity due to its low bioavailability (Edwards et al. 2013).

Antioxidant response
The toxic effect of heavy metals appears to be related to production of reactive
oxygen species (ROS) and the resulting unbalanced cellular redox status (Pinto et al.
2003b). It is known that heavy metals cause oxidative stress on the cell, which
generates reactive oxygen species “ROS” (O2−, H2O2….) that can react with cellular
components and cause oxidation of lipids, carbohydrates, proteins, pigments and
DNA. These results in growth inhibition and photosynthesis (Shamsi et al. 2008,
Bouchama et al. 2016). These are specific but complex mechanisms involving
morphological, physiological and biochemical adaptation. In particular, the ROS-
 Phytoremediation and Removal of Contaminants by Algae and Seagrasses 145

combating antioxidant system, including diverse enzymes such as superoxide

dismutase, catalase, glutathione peroxidase and ascorbate peroxidase, and non-
enzymatic system consists of glutathione (GSH) and ascorbic acid (Pinto et al.
2003a, Gill and Tuteja 2010). An increasing activity of superoxide dismutase (SOD)
and guaiacol peroxidase (POD) was founded in Thalassia hemprichii exposed to
various concentrations of Zn2+, Cd2+, Pb2+ and Cu2+ (Li et al. 2012). Also all tissues
of live sheaths and root/rhizomes experienced an increase in cysteine, glutathione
(GSH), γ-glutamylcysteine (γ-EC) and phytochelatin-like peptides as a response
to Cd exposure (Alvarez-Legorreta et al. 2008). An increase in catalase and GST
activities was observed in Posidonia oceanica exposed to low concentrations of
mercury chloride (Ferrat et al. 2002). The research of Lee and Shin (2003) showed
an increase of guaiacol and ascorbate peroxidases and catalase activity whereas
superoxide dismutase and glutathione reductase activity markedly decreased, in the
marine alga Nannochloropsis oculata exposed to Cd. This study suggested that the
ability of this marine algae to tolerate the harmful effects of cadmium is probably due
to the modification of antioxidant enzyme levels.

Tolerance and Detoxification Mechanisms of Organic Contaminants

In recent years, various studies have demonstrated the bioaccumulation,
biotransformation and biodegradation potential of several algal and seagrasses species
for various organic contaminants, such as the bioaccumulation and biodegrading of
two polycyclic aromatic hydrocarbons, phenanthrene (PHE) and fluoranthene (FLA)
in diatoms Skeletonema costatum and Nitzschia sp. (Hong et al. 2008). Also the uptake
of polychlorinated biphenyls (PCBs) by Emilliana huxleyi, Skeletonema costatum,
Thalassiosira nordenskioidii Bacillariophyceae Phaeodactylum tricornutum
(Gerofke et al. 2005). Selective phytoplankton, diatoms and microalgal species
have shown the potential of biodegradation of organic contaminants, especially
biotransformation of the complex organic compounds in lower carbon compounds
(Singh et al. 2015). Akin to the biodegradation of endocrine disrupting chemicals
including nonylphenols (NPs), bisphenol (BPA), 17 alpha-ethynylestradiol (EE2)
and estradiol (E2) in marine diatom Navicula incerta (Liu et al. 2010). In another
study, the bioaccumulation of PAHs and PCBs in roots and leaves of Zostera marina
without major physiological disturbance (Huesemann et al. 2009). In a recent
study, Fernandez and Gardinali (2016) have demonstrated the ability of Halodule
wrightii to accumulate irgarol, a triazine herbicide. The biodegradation processes
of PCBs, including dechlorination, can transform PCBs, altering their potential
toxicity effectively (Borja et al. 2005). Although some of the organic pollutants
may not be completely mineralised, they may be converted to less toxic or nontoxic
compounds, which is an important bioremediation strategy (Subashchandrabose et al.
2013). It is clear that although the complete degradation of aromatic pollutants is
rare, algae are capable of carrying out biotransformations on aromatic pollutants,
such as the hydroxylation of naphthalene and benzo[a]pyrene to their hydroxylated
intermediates. These initial biotransformation may be extremely important in the
overall degradation of pollutants in the environment by other microorganisms like
white rot fungi (Meulenberg et al. 1997). Subashchandrabose et al. (2013) reported
146 Ecotoxicology of Marine Organisms

that several factors such as algal size, density, morphology and metabolic activities
are important in affecting the uptake and removal of the toxicants. According to
Wolff (1976), the low decomposition rates of seagrass have an important role in the
maintenance of the standing stock of organic matter in the coastal environment and
even in deep sea environment.

Conclusion
Pollution problems greatly affect the marine ecosystem which is mainly sensitive
to several types of contaminants; harmful organic and inorganic contaminants can
be present at different levels in the marine environment. Increased consciousness of
the necessity to safeguard those environments had motivated a search for alternative
technologies to remove pollution from this sensible ecosystem. The present review
emphasises the role of seagrass and algae in phytoremediation and phycoremediation
technologies. Different techniques can be distinguished within phytoremediation of
polluted water according to their objectives, the characteristics of the environment,
the contaminants and the used species. Several species of seagrasses and algae have
been reviewed in this chapter. Some of them represent an immense potential for
removal of a variety of contaminants including heavy metals, inorganic/organic,
by bioaccumulation, biodegradation, biotransformation and biosorption using the
binding groups for biosorption, ROS-combating antioxidant system, phytochelatin
and metallothionine.

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7 Ecotoxicology of
Pharmaceuticals in Coastal
and Marine Organisms
Vanessa F. Fonseca,1,* and Patrick Reis-Santos1,2,*

INTRODUCTION
For as long as they have been produced, pharmaceutical compounds have been released
in the environment. And although these compounds and other personal care products
are classified as contaminants of emerging concern, this term does not necessarily
imply their occurrence in the environment as recent. It rather alludes to contaminants
from multiple sources (domestic, industrial or agricultural) that escaped prior notice
and classically were not monitored in spite of their potential to cause adverse effects
to the environment; or to compounds for which only recently have environmental
concerns been fully raised (Glassmeyer et al. 2007, Sauvé and Desrosiers 2014). In
the end, the use of the term ‘emerging contaminants’ has the intention to highlight
the largely unregulated nature of the presence in the environment of substances
such as pharmaceutical compounds, but also others such as cosmetics, UV blocker
agents (sunscreens) or fragrances (Daughton 2016). Furthermore, the continuous and
rapid technological development in highly sensitive analytical instrumentation has
enabled the discovery and quantification of numerous compounds and substances
in the aquatic environment, and from complex matrices, which had previously been
undetected (Pérez and Barceló 2007, Sanderson and Thomsen 2009, Klosterhaus et
al. 2013).
Pharmaceuticals have come under particular scrutiny regarding their occurrence
and effects on aquatic environments due to a few key features. Firstly, both

1
MARE – Marine and Environmental Sciences Centre, Faculdade de Ciências, Universidade de Lisboa,
Campo Grande, 1749-016 Lisboa, Portugal.
2
Southern Seas Ecology Laboratories, School of Biological Sciences, The University of Adelaide, South
Australia 5005, Australia.
* Corresponding authors: [email protected]; [email protected]
 Ecotoxicology of Pharmaceuticals in Coastal and Marine Organisms 159

human and veterinary pharmaceutical compounds are continuously released into

the environment worldwide, resulting in their ubiquitous and persistent presence.
Moreover, their concentrations in aquatic ecosystems are projected to continue to
rise, with mounting environmental concerns, due to an expected increase in both the
access and the widespread use of medication by a growing global population (Kuster
and Adler 2014). Additionally, unlike several chemical contaminants, pharmaceutical
compounds are biologically active and target particular metabolic pathways that in
many cases are evolutionary conserved (Gunnarsson et al. 2008, Furuhagen et al.
2014), eliciting effects at very low environmental concentrations (e.g., ng/L), and
shown to specifically affect multiple algae and animal functions (e.g., Franzellitti et
al. 2013, Aguirre-Martínez et al. 2015, Minguez et al. 2016). However, it is important
to notice that the term pharmaceuticals does not refer to a specific or unambiguous
class of molecules sharing an a priori defined set of chemical, physical or biological
similarities, but to a varied group of therapeutic compounds used for human or
veterinary treatment encompassing a wide range of kinetics, metabolism, modes
of action (MOA), and ultimately, an array of potential underlying effects to the
environment (Taylor and Senac 2014).
In this context, over the last couple of decades, growing attention has been given
to monitoring and evaluating the presence and the ecotoxicology of pharmaceutical
compounds in the aquatic environment (Daughton 2016). Yet, in comparison
to freshwater systems, where studies on the occurrence and potential effects of
pharmaceuticals are manifold, transition and coastal marine environments have
been comparatively overlooked or poorly investigated. In part, this is likely due to
the assumption that dispersion and dilution processes, including from freshwater
sources to estuarine and coastal environments, would be suffice to lessen or cancel
any potential effects. Only recently has this trend begun to be reversed, with research
gradually focusing towards coastal areas and showcasing that pharmaceuticals
are present throughout transition and marine environments at levels potentially or
effectively adverse to different levels of biological complexity (e.g., Fatta-Kassinos
et al. 2011, Klosterhaus et al. 2013, Gaw et al. 2014, Aminot et al. 2016, Arpin-Pont
et al. 2016, Du et al. 2016, Fabbri and Franzellitti 2016). Moreover, it is important to
highlight that presumed impacts on transition and coastal environments are expected
to continue to increase allied to population growth and coastal settlement, as well as
from accessory human activities such as aquaculture (Burridge et al. 2010, Gaw et al.
2014, Tornero and Hanke 2016). Overall, the increase in research and literature since
2014 regarding the occurrence, fate and ecotoxicology of pharmaceuticals in coastal
and marine environments may, at least in part, be attributed to a review by Gaw et al.
(2014), and the call for research prioritization. At that time, Gaw et al. (2014) found
49 studies from the year 2000 onwards reporting concentrations of pharmaceuticals
in marine and coastal environments. A number that has since raised considerably, and
we were able to compile information from 124 studies (since the year 2000) focusing
on the occurrence and effects of pharmaceuticals in transition and coastal marine
environments [Web of Science search in February 2017 with the terms: Marine AND
pharmaceutical AND (occurrence OR effect* OR toxicity)].
In the present chapter, we aim to provide a brief overview of the most
recent advances in the literature regarding the occurrence and ecotoxicology of
160 Ecotoxicology of Marine Organisms

pharmaceuticals in coastal and marine environments. We critically assess recent

research and provide an integrative analysis focusing on the sources of major
therapeutic classes of pharmaceuticals to transition and coastal marine environments,
their pathways and ecotoxicology to different levels of biological complexity,
highlighting reported adverse effects of pharmaceuticals exposure in coastal and
marine organisms. In the interest of a focused approach, the scope of the current
chapter has been restricted to major therapeutic pharmaceutical compounds, excluding
natural and synthesized hormones. Overall, we will prioritize in situ evaluations
of effects of environmentally relevant concentrations, highlight knowledge gaps
and present-day challenges, and provide an outline of key areas and opportunities
where future research should be prioritized to underpin the delineation of effective
management options. Ultimately, understanding the effects of pharmaceuticals on the
marine environment and unraveling their ecotoxicology, MOA and bioaccumulation
rates, together with research on their occurrence and fate, is key to safeguarding
potential threats to environmental and human health and supporting effective risk
management strategies.

Sources and Occurrence of Pharmaceuticals in Coastal and Marine

Environments
In this section, we outline the major sources of pharmaceutical contaminants in
marine environments as well as their contamination pathways, highlighting ranges
of concentrations found, and then briefly refer the physical and chemical processes
that may influence the environmental concentrations of these contaminants in both
water and sediments.
The sources and pathways of pharmaceutical contaminants in coastal and marine
environments are manifold (Kummerer 2009c, Gaw et al. 2014). However, estuarine
and coastal areas receive a complex mixture of pharmaceutical contaminants from a
set of overarching key origins, namely: (i) human household use; (ii) hospital use; (iii)
veterinary applications, via aquaculture or from the terrestrial environment, including
livestock production or household pets’ care; (iv) and industrial and commercial
activities linked to the production of pharmaceuticals (Fig. 1). All these produce large
amounts of waste that via a multitude of entwined pathways result in the presence of
pharmaceutical compounds, their metabolites and transformation by-products, both
directly or via diffuse routes. Assumptions that pharmaceuticals would be negligible
due to hydrodynamics or dilution processes in coastal and marine environments
are by large currently refuted (Gaw et al. 2014, Fabbri and Franzellitti 2016). In
fact, pharmaceutical contaminants have been detected in marine environments at
distances that exceed tens and even hundreds of kilometers from what would be
their anticipated sources (e.g., WWTP marine outfalls, coastal areas) (Wille et al.
2010, Zhang et al. 2013, Alygizakis et al. 2016). Even when contaminants are not
found in water or sediments, or detected only sporadically, pharmaceuticals are still
detected in marine organisms such as bivalves and fish, showcasing their potential
for bioconcentration (Wille et al. 2011, Maruya et al. 2012, Klosterhaus et al. 2013).
 Ecotoxicology of Pharmaceuticals in Coastal and Marine Organisms 161

Sources
Personal use Hospital use Veterinary use Pharmaceutical industry
Livestock Aquaculture

Pathways

Fate and Transformation

Biodegradation and transformation

Dilution & Diffusion

Landfill waste
Hydrolysis

Untreated Photolysis
sewage
WWTP Volatilization to atmosphere

Deposition and accumulation

Other uses
e.g. sludge, manure, fertilizers

Leaching Sorption to sediments

Deposition and resuspension

Groundwater
Bioaconcentration

Rivers
Estuaries

Marine environment

Fig. 1. Major sources and pathways of pharmaceutical contamination into coastal and marine
environments. Also shown, the main fate and transformation processes that affect the presence and
concentration of pharmaceutical compounds.

The major route of entry for pharmaceuticals and their by-products in natural
aquatic environments are point source wastewater discharges of treated [i.e., outflow
of waste water treatment plants (WWTP) and septic tanks] and untreated sewage
(Glassmeyer et al. 2007, Fatta-Kassinos et al. 2011). Estuarine and coastal marine
environments, particularly those near urban clusters, receive large volumes of these
effluent discharges both directly via coastal or offshore underwater outfalls (e.g.,
Togola and Budzinski 2008, Alygizakis et al. 2016), and indirectly via loadings from
streams and rivers where wastewater discharges have taken place (Xu et al. 2013,
Cantwell et al. 2016). For instance, the annual loads of pharmaceuticals flushed out to
sea from the Yangtze estuary are estimated to surpass 150 metric tons, as a result of the
discharge of c. 50 × 106 m3 of sewage (Qi et al. 2014). In another study in South-west
France, an assessment of 53 compounds produced an estimated influx of c. 10 kg per
day of pharmaceuticals to the Garonne estuary (Aminot et al. 2016). It is important
to highlight that even in peak conditions, treatment plants are unable to remove all
pharmaceutical contaminants from wastewaters, and that performance efficiency and
WWTP removal rates of pharmaceuticals vary significantly from 100 percent to < 1
percent, depending on the type of treatment, operating conditions, chemical loads and
the specific physico-chemical properties of the different pharmaceutical compounds
(Kim et al. 2007, Gros et al. 2010, Luo et al. 2014, Silva et al. 2014). As a result, over
the years, different human and veterinary pharmaceuticals have been found in coastal
and marine waters over a wide range of concentrations: for example, 0.01 ng/L
(e.g., roxithromycin—antibiotic; Yan et al. 2013) to 6800 ng/L (e.g., norfloxacin—
antibiotic; Zou et al. 2011) and even above 200000 ng/L in areas closely affected
by WWTP effluents (e.g., paracetamol—analgesic; Togola and Budzinski 2008); as
well as in sediments (e.g., from 0.01 to c. 17 ng/g dry weight metoprolol—β-blocker;
Cantwell et al. 2016) (see also Gaw et al. 2014, Arpin-Pont et al. 2016, Fabbri and
Franzellitti 2016). It is worth highlighting that the contamination and persistence of
pharmaceuticals in some transition and coastal environments such as bays, inlets,
estuaries and coastal lagoons where water residency and flushing times are reduced,
162 Ecotoxicology of Marine Organisms

or with periodic connections to the sea, may be of added concern. Many of these
coastal areas are favored human settlement or seasonal holiday hubs, and in addition
to direct sewage discharges and other local loadings (e.g., river input, groundwater
contamination) there is an increased potential risk hazard associated to the confined
nature and the distinctive physico-chemical properties of these systems, where
dilution and dispersion of contaminants is likely reduced and changes in sorption
kinetics will affect the accumulation of pharmaceuticals over time (Dougherty et al.
2010, Liu et al. 2013, Moreno-González et al. 2015, Aminot et al. 2016).
Other sea based human activities such as shipping, particularly cruise and large
passenger ships may have a significant impact on specific coastal and marine areas
as a result of wastewater discharges (Alygizakis et al. 2016, Westhof et al. 2016).
Over 20 million passengers board cruise ships every year (Cruise Line International
Association Industry Outlook), with individual liners that hold passenger and crew
numbers above those of small townships regularly visiting highly sought confined
or sensitive areas, and though wastewater discharges are regulated (Annex IV of the
MARPOL convention on pollution prevention), treatment performance still lacks
effective administrative regulation or monitoring, so the potential for contamination
is substantial. For instance, Westhof et al. (2016) estimated annual loads of ibuprofen
exceed 3.3 Kg for a ship with 4000 persons on board.
Wastewaters from healthcare and pharmaceutical production facilities are
other key sources of pharmaceutical compounds to estuarine and coastal marine
environments (Fig. 1). By their very nature, hospital activities generate a sizable
quantity of contaminated effluents. These are dependent on numerous factors which
include, but are not limited to, bed density, number of patients or medical specialties,
with several studies characterizing pharmaceutical residues in hospital wastewaters
in different regions worldwide (e.g., Santos et al. 2013, Herrmann et al. 2015, Oliveira
et al. 2015, Azuma et al. 2016). Compiling information on hospital and healthcare
facility effluents, Oliveira et al. (2017) showcased that many pharmaceuticals were
present at concentrations below 10 µg/L, though for several of the most common
active ingredients, values were significantly higher (e.g., paracetamol 1368 µg/L,
ciprofloxacin 125 µg/L). Overall, though healthcare facilities have been pointed out
as key contributors, everyday household discharges are still generally acknowledged
as the main contributor of human use pharmaceuticals to the environment (see
Kummerer 2009c, Le Corre et al. 2012, Herrmann et al. 2015). In part, this is due to
the sheer number of users and the amount of pharmaceutical consumption that takes
place in the domestic context, with many outpatients also continuing treatment or
receiving palliative care outside hospital facilities.
Regarding drug manufacturing, a number of studies have also reported
environmental contamination as well as the damaging effects of exposure to
effluents from pharmaceutical production sites, with several evidences of high
concentrations in effluents, with contamination values reaching tens of mg/L (Fick
et al. 2009, Cardoso et al. 2014, Larsson 2014). Remarkably, and for purposes of
management and supervision, it is possible to reconstruct exposure pathways and
disentangle factory source contamination from human use by evaluating the ratio
of pharmaceutical precursor and of its human metabolites (e.g., Prasse et al. 2010).
Overall, drug factory discharges, environmental risk and contamination patterns are
 Ecotoxicology of Pharmaceuticals in Coastal and Marine Organisms 163

not specifically linked to use patterns or seasonality, and will mainly affect coastal
and marine environments via their localization, or via loading of rivers and streams
with subsequent contamination downstream. Though pharmaceutical industries are
mostly located in south east Asia (e.g., Bangladesh, China, India and Pakistan),
production sites elsewhere (e.g., Europe, US) are also identified as significant
contamination sources (see Cardoso et al. 2014, Larsson 2014, Rehman et al. 2015).
Veterinary applications of pharmaceuticals in both aquaculture and land
based animal husbandry or livestock productions are also known contributors
of pharmaceuticals to natural environments (Fig. 1). In response to the rising
demand in seafood products worldwide, aquaculture has been seeing a continued
boost in both the number of farms as well as production yield, and this is in part
associated to the availability of an array of pharmaceutical compounds that
enhance productivity (Sapkota et al. 2008, Tornero and Hanke 2016). The range of
veterinary pharmaceuticals accessible to fish farmers include antibiotics, analgesics
and antiparasitics, among others, some of them of generic human use, with many
compounds applied prophylactically (Cabello 2006, Burridge et al. 2010, Tornero
and Hanke 2016). Thus, any sea (coastal or estuarine) based aquaculture activities
are direct entry points of pharmaceuticals into the marine environment. By large,
pharmaceuticals are incorporated into feed, though other administration routes such
as dilution and immersion in baths are available. In any case, these pharmaceutical
compounds, as well as their excreted metabolites and transformation products will
fuel environmental contamination and elicit impacts on non-target organisms (see
Sapkota et al. 2008, Burridge et al. 2010, Chen et al. 2015). Other key pathways
for pharmaceuticals to enter estuarine and coastal environments are wastewater
discharges from land based aquaculture activities (Le and Munekage 2004, Zou et al.
2011). Though best practices vary worldwide, pond or tank based aquaculture of
multiples species of crustaceans and fish is extensive throughout estuarine and coastal
environments, with discharges made directly to these areas. Leakage from ponds is
also an acknowledged pathway for veterinary pharmaceuticals to reach estuarine and
coastal waters. Over the years, the range of concentrations in wastewater discharges
or water and sediments surrounding aquaculture activities has been found to vary
widely from a few ng/L to 2.5 mg/L (i.e., oxolinic acid) (Le and Munekage 2004,
Chen et al. 2015, Kim et al. 2017). In some cases, the effect of released pharmaceutical
loads may be aggravated by a combination of low flow conditions and the local
abundance of juveniles of many species, as aquaculture farms are established in areas
(e.g., estuarine habitats, mangroves) renowned to have a key nursery role (Beck
et al. 2001) where potential effects on juvenile biota may be subsequently exported to
adult populations (Rochette et al. 2010, Vasconcelos et al. 2011, Fonseca et al. 2015).
Discharges of both treated and untreated wastewaters from land based agricultural
and livestock productions may also contribute to the presence of pharmaceuticals in
estuaries and coastal environments (e.g., Lim et al. 2013, Awad et al. 2014, Paíga et
al. 2016) (Fig. 1). Furthermore, fecal excretion of metabolites over pastoral lands
can contaminate the groundwater via leaching or run-off, as can the use of waste
products as fertilizers (e.g., manure). Overall, the presence of pharmaceuticals in
groundwater can originate from many sources, including sewage contamination (e.g.,
via septic tanks or sewer leakage), contaminated leachate from landfill (e.g., animal
164 Ecotoxicology of Marine Organisms

carcasses, pharmaceutical waste products), use of contaminated sludge as fertilizer,

as well as by the use of grey waters for irrigation (see review by Sui et al. 2015). The
latter can all be relevant sources of pharmaceutical contamination to coastal marine
environments, due to discharge and connectivity between groundwater and coastal
systems (Dougherty et al. 2010, Sui et al. 2015) (Fig. 1). Overall, due to the added
risk of contamination to public drinking water, evaluating pathways of groundwater
contamination is also paramount (e.g., Fick et al. 2009).
Upon pharmaceutical intake, significant fractions of parent compound are
excreted unprocessed as well as in the form of metabolites and transformation by-
products. Irrespective of their sources, the fate and persistence of pharmaceuticals
in coastal environments, as well as their subsequent potential to affect biota
or bioconcentrate, are linked to key physico-chemical processes: transport,
biodegradation, transformation and sequestration (Fig. 1). Overall, pathways and
routes of exposure rely on both dissolved and particle transport (upon sorption
to particulate matter or sediments), with the fate of individual pharmaceuticals
influenced by environmental conditions (e.g., salinity, suspended particulate matter,
hydrodynamics, water column mixing, pH, turbidity or light penetration) as well
as by their own physical and chemical properties (Glassmeyer et al. 2007). Thus,
information collated for degradation or sorption of pharmaceuticals in freshwater
environments may not be directly applicable in estuarine and marine contexts (see for
instance Fenet et al. 2014, Gaw et al. 2014, Zhao et al. 2015, Fabbri and Franzellitti
2016). Nonetheless, hydrolysis, photolysis, biodegradation and adsorption are
recognized as the most likely to alter pharmaceutical compounds. Understanding the
complex interactions among sorption kinetics, potential resuspension and transport
is crucial; all these processes play a key role in the fate of pharmaceuticals along the
interface between estuarine and coastal marine environments, and will determine
the persistence of these compounds in the environment, their availability for
bioaccumulation and exchange between environments (Kummerer 2009c, Liu et al.
2013). For instance, sorption to colloids can represent a sink for pharmaceuticals,
increasing persistence but decreasing bioavailability (pending resuspension) whilst
pH and salinity variations can also affect the ionization and solubility of different
compounds. In transition areas, changing environmental conditions as well as major
seasonal variations in both environmental conditions and contaminant inputs (e.g., in
recreational and holiday areas) may have significant repercussions in the occurrence
of pharmaceuticals, bioavailability and transfer to the marine environment (Liu et al.
2013, McEneff et al. 2014, Moreno-González et al. 2015, Zhao et al. 2015).

Ecotoxicological Effects of Pharmaceutical Exposure on Coastal and

Marine Organisms
Pharmaceuticals are designed to elicit biological effects at low doses, targeting
specific metabolic and physiological pathways to achieve the desired therapeutic
effects in human and veterinary medicine. In addition to high specificity at low
concentrations, the evolutionary conservation of most molecular targets across taxa
implies that environmental concentrations of pharmaceuticals have the potential
 Ecotoxicology of Pharmaceuticals in Coastal and Marine Organisms 165

to chronically impact non-target aquatic organisms, with several adverse effects

of pharmaceutical exposure reported in organisms at different levels of biological
organisation (e.g., Huerta et al. 2012).
The majority of ecotoxicological data on pharmaceutical compounds pertains
to the freshwater environment (reviews by Crane et al. 2006, Fent et al. 2006), yet
scientific contributions on occurrence and effects of pharmaceuticals on coastal and
marine biota are increasing. For the current chapter, we found 124 studies focusing
on ecotoxicology of pharmaceuticals in coastal and marine biota, as well as on
bioaccumulation in wild coastal and marine organisms. Six major therapeutic classes,
namely analgesic non-steroid anti-inflammatory drugs (NSAIDs), antidepressants,
antibiotics, anticonvulsants, antihypertensives and lipid regulators, clearly stood out
and encompassed c. 91 percent of all studies. This is likely due to their frequent
detection in the marine environment as well as to their higher sales and consumption.
Nonetheless, we cannot exclude a bias towards better-known or more-established
compounds and MOA, with researchers favoring the possibility of data comparison
with available information.
Concerning major taxonomic groups, mollusks are the most frequent group
of organisms in pharmaceutical accumulation and toxicity studies (69 studies),
followed by crustaceans (32 studies) and fish (27 studies) (Fig. 2). Research with
marine microorganisms, specifically microalgae (11 studies) and bacteria (six
studies), were predominantly standard toxicity tests. Overall, research on the effects
of the major therapeutic classes is well distributed among taxonomic groups. Though
there is less data available for antihypertensives and lipid regulators on mollusks,
in comparison to other therapeutic classes (Fig. 2). In terms of study type, most of
the data relate to molecular changes (71 studies), which include gene and protein
expression as well as other biochemical changes (e.g., biomarkers of oxidative stress
and xenobiotics biotransformation) (Fig. 2). Molecular endpoints are ubiquitous
to all therapeutic classes and are the primary endpoint in analgesic and NSAIDs
toxicity studies, whereas behavior endpoints are particularly associated with
antidepressant exposures. Effects on development, mortality and reproduction of
marine biota are also important endpoints in pharmaceutical toxicity assessments
(27, 21 and 17 studies, respectively). Twenty-two studies reported the accumulation
of pharmaceuticals in finfish, crustaceans and shellfish tissues (Fig. 2). This is a
noteworthy increase from the 14 studies identified in Gaw et al. (2014). Overall,
antibiotics are the main therapeutic class investigated in bioaccumulation studies
of marine and coastal organisms, with many studies linked to major aquaculture
production. For instance, quinolones, sulfonamides and macrolides were detected in
wild mollusk species collected along the coast of the Bohai Sea in China, with the
highest concentrations ranging from 36 to 1575 µg/kg dw (Li et al. 2012). Evaluating
bioaccumulation and biomagnification of several antibiotic agents in a marine trophic
web in Laizhou Bay (China), trimethoprim, nine sulfonamide, five fluoroquinolone
and four macrolide antibiotics were all detected in marine invertebrates and fish (Liu
et al. 2017). Additionally, sulfonamides and trimethoprim were found to biomagnify
along the food web, whilst fluoroquinolones and macrolides were biodiluted.
Nonetheless, local seafood consumption was considered unlikely to pose a major
human health risk, regarding antibiotic concentrations. Other studies have focused
166 Ecotoxicology of Marine Organisms

Antidepressants (64) Antibiotics (48)

Analgesics and NSAIDs (58)

Antihypertensives (34)

Lipid regulators (33)

Anticonvulsants (40)

Fig. 2. Tree map representation of studies on the effects of pharmaceutical exposure in coastal and
marine organisms per therapeutic class, biological endpoints and major taxonomic groups. Therapeutic
classes are antidepressants, analgesics and non-steroid anti-inflammatories (NSAIDs), anticonvulsants,
antibiotics, antihypertensives and lipid regulators. Biological endpoints and respective abbreviations are
molecular changes, accumulation (accumul), development (develop), mortality, reproduction (repro)
and behavior (behav). Major taxonomic groups and respective abbreviations are fish, tunicates (tun),
echinoderms (echi), mollusks (moll), crustaceans (crust), rotifers (rot), annelids (ann), nematods (nem),
cnidarians (cni), algae (alg) and bacteria (bact). Individual box sizes are proportional to number of entries,
and total number of entries per therapeutic class is shown (n). Note that a single study may have multiple
entrances per therapeutic class (total number of studies 124).
Color version at the end of the book
 Ecotoxicology of Pharmaceuticals in Coastal and Marine Organisms 167

on field monitoring of several pharmaceutical compounds in coastal waters via

long term caging experiences with marine bivalves. Wille et al. (2011) detected five
pharmaceuticals in caged mussels along the Belgium coast, namely salicylic acid,
paracetamol, propranolol, ofloxacin and carbamazepine (highest concentrations
ranging from 11 to 490 ng/g dw). McEneff et al. (2014) quantified carbamazepine,
mefenamic acid and trimethoprim (peak concentrations of 7.28 to 9.22 ng/g dw) in
Mytilus spp. following a one year experiment in the Irish coast. Yet, knowledge on
the fate, biotransformation and bioaccumulation of pharmaceutical compounds in
the marine environment is still insufficient, as evidenced by the lack of concordance
between field-derived bioaccumulation factors for ribbed horse mussels (Geukensia
demissa) and model-predicted bioconcentration factors (Klosterhaus et al. 2013),
as well as the lack of correlations between accumulation and observed molecular
effects of pharmaceuticals (Mezzelani et al. 2016b).
Analgesics and NSAIDs reduce pain and inflammation and are amongst the
highest consumed pharmaceuticals worldwide (Fent et al. 2006). Representative
compounds include acetaminophen, diclofenac, ibuprofen, ketoprofen and
indomethacin. Biological targets are cyclooxygenase isoforms (Cox1 and
Cox2) and these drugs act by non-specific inhibition of the synthesis of various
prostaglandins from arachidonic acid (Vane and Botting 1998). Besides being
involved in inflammation and pain responses, prostaglandins also play important
roles in various physiological functions, including reproduction processes, reducing
hypertension, fatty acids metabolism and synthesis of the protective gastric mucosa
(Jones 1972). In fish and marine invertebrates, prostaglandins have been related with
reproduction, ion regulation and immune responses (Sorbera et al. 2001, Rowley
et al. 2005). Accordingly, the marine clam, Ruditapes philippinarum, exhibited
significant immunological alterations following a seven day exposure to ibuprofen,
particularly at the highest concentrations tested (500 and 1000 ug/L) (Matozzo et al.
2012). In the crustacean Carcinus maenas, osmoregulatory capacity was impaired
with environmentally relevant concentrations of diclofenac over 7 days (10 ng/L and
100 ng/L), although no stress-related effects were observed in these individuals
(Eades and Waring 2010). The potential of NSAIDs for endocrine disruption has been
suggested in mussels Mytilus galloprovincialis exposed to 250 ng/L of ibuprofen or
diclofenac for two weeks, with males and females presenting elevated levels of gonad
vitellogenin-like proteins (Gonzalez-Rey and Bebianno 2012, 2014). However, a
shorter time frame study with diclofenac (1 to 1000 ug/L, 96 h) and bivalves Mytilus
spp. did not show differences in the expression of these vitellogenin-like proteins
(Schmidt et al. 2011). Akin to endocrine disruption, variable neurotoxic responses
have been reported in mussels in response to analgesics and NSAIDs, namely via
tissue specific inhibition and the increased activity of acetylcholinesterase (AChE)
(e.g., Milan et al. 2013, Mezzelani et al. 2016a, 2016b). Multibiomaker approaches
highlighted changes in immunological responses, lipid metabolism and DNA integrity
in M. galloprovincialis exposed separately to various analgesics and NSAIDs
(acetaminophen, diclofenac, ibuprofen, ketoprofen or nimesulide) at 25 µg/L and
0.5 µg/L concentrations for 14 days (Mezzelani et al. 2016a, 2016b). Subsequent
gene transcription analysis, via DNA microarrays, corroborated biomarker responses,
highlighting the similarities on proposed MOA of NSAIDs between bivalves and
168 Ecotoxicology of Marine Organisms

vertebrate species (Mezzelani et al. 2016b). However, the lack of a significant change
in oxidative stress biomarkers (catalase, glutathione peroxidase and glutathione
reductase activities, total glutathione, and total oxyradical scavenging capacity) or
recovery of the antioxidant system indicate that prooxidant response is not a key
target in the pharmacology of these compounds (Gonzalez-Rey and Bebianno 2014,
Mezzelani et al. 2016a, 2016b).
To date, the effects of analgesics and NSAIDs in coastal and marine fish species
have only been evaluated in vitro, and to test their effects on the activities of several
enzymes related to xenobiotic and steroid metabolism. Ribalta and Solé (2014)
reported that diclofenac significantly interfered in the CYP1A and CYP3A systems
of Mediterranean fishes, particularly in the middle slope gadiform Trachyrincus
scabrous. Ibuprofen exposure (100 µM concentration) also inhibited the activity of
the CYP3A4 enzyme, benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxylase
(BFCOD) in the liver microsomal fraction of Solea solea, whilst acetaminophen had
no effects on measured enzyme activities (Crespo and Solé 2016).
Antidepressants are neuroactive drugs for the treatment of depression and related
psychiatric disorders (e.g., anxiety, obsessive-compulsive disorder, post-traumatic
stress disorder). Selective serotonin reuptake inhibitors (SSRIs, such as fluoxetine,
sertraline, citalopram) and serotonin and norepinephrine reuptake inhibitors (SNRIs,
such as venlafaxine) are some of the most prescribed antidepressants. Their highly
specific MOA is based on the modulation of neurotransmission in the human brain,
targeting and blocking serotonin and norepinephrine reuptake proteins which leads
to increased levels of these neurotransmitters in the synaptic cleft (Hiemke and
Härtter 2000). Serotonin is also present in lower vertebrates and invertebrates, and as
in humans, this biogenic monoamine appears to be involved in various physiological
functions and behaviors interacting with reproduction and neuroendocrine processes
(e.g., Winberg and Nilsson 1993, Winberg et al. 1997, Fong 1998). A review of the
effects of antidepressant exposure on mollusks and crustaceans outlined the impacts
of antidepressants on the organisms’ metabolism, growth, reproduction, feeding,
locomotion and behavior, yet the bulk of information was related to freshwater
invertebrates (Fong and Ford 2014). Noteworthy, changes to spawning and larval
release in bivalves as well as impaired locomotion and fecundity in snails occurred at
environmentally relevant concentrations of antidepressants; although the occurrence
of non-monotonic dose response curves were also reported with significant biological
effects at lower but not at higher concentrations (Fong and Ford 2014). Regarding
toxicity to marine invertebrates, altered cognitive capacities (learning and memory
retention) and less efficient cryptic behaviors were observed in cuttlefish Sepia
officinalis following fluoxetine exposure at hatchling stages (1 ng/L to 100 ng/L)
(Di Poi et al. 2013, 2014). Fluoxetine at concentrations ranging from 43 µg/L to
4.34 mg/L, also induced foot detachment from the substrate in five species of marine
snails from different habitats (Fong and Molnar 2013). This potentially lethal outcome
was also observed in two marine snail species exposed to venlafaxine, albeit different
locomotion behaviors at the onset of foot detachment suggest that venlafaxine and
fluoxetine have different physiological mechanisms of action (Fong et al. 2015).
In another study, long-term exposure to low concentrations of fluoxetine (0.3 ng/L
to 300 ng/L) diminished algal clearance rates, growth and gonadosomatic index
 Ecotoxicology of Pharmaceuticals in Coastal and Marine Organisms 169

in California mussel M. californianus (Peters and Granek 2016). Pharmacological

effects of fluoxetine and trait-based sensitivity have also been described for the
marine worm Hediste diversicolor, based on increased serotonin levels in the
coelomic fluid and tissues. Fluoxetine effects on H. diversicolor included weight
loss (up to 2 percent at 500 μg/L), decreased feeding rates (68 percent at 500 μg/L)
and increased oxygen consumption and ammonia excretion (from 10 μg/L), but only
limited influence on predator avoidance behaviors (Hird et al. 2016). Regarding
sertraline, an early life-stage bioassay with sea urchin embryos found it to be highly
toxic given the development of significant abnormalities at ng/L range concentrations
(Ribeiro et al. 2015).
As for invertebrates, information on the toxicity of antidepressants to fish stem
mainly from freshwater species. These include deleterious effects on physiology,
reproduction, behavior (e.g., reproductive, predator avoidance, territorial and
defensive behaviors) and the potential of SSRIs as endocrine disruption compounds
in various fish species (e.g., Mennigen et al. 2010a, 2010b, Schultz et al.
2011, Weinberger and Klaper 2014). Few studies have evaluated the effects of
antidepressants on coastal and marine fish species. Two in vitro studies reported
species specific responses when assessing the impact of multiple pharmaceuticals
on various enzyme activities in coastal and deep-sea fishes: (1) In Solé and Sanchez-
Hernandez (2015), fluoxetine had no effect on carboxylesterase (CbE). The latter
is involved in metabolism and activation of numerous exogenous and endogenous
compounds in humans and has been associated with pesticide detoxification in fish
(Wheelock et al. 2008). In Ribalta and Solé (2014), fluoxetine inhibited cytochrome
P450, which are enzymes linked to phase I of xenobiotic metabolism as well as
to the metabolism of endogenous compounds (e.g., steroids). Moreover, high
concentrations of fluoxetine, administered intraperitoneally to the gulf toadfish
Opsanus beta, affected the branchial urea excretion and intestinal osmoregulation and
resulted in a severe stress response with high levels of plasma cortisol (Morando et
al. 2009). Additionally, fluoxetine has also been shown to affect marine fish behavior
by reducing locomotor activity (EC50 155 µg/L at 32 h of exposure) (Winder et al.
2012).
Beta-adrenergic receptor antagonists or β-blockers are antihypertensive drugs,
commonly used to treat high blood pressure, angina, arrhythmias and other cardiac
conditions. The MOA of blockers such as propranolol, atenolol and metoprolol
consists in their specific binding to adrenoreceptors, competing with β-adrenergic
agonists, decreasing resting heart rate, cardiac output and cardiac muscles
contractibility, among others (Bourne 1981). A comparative physiology review
described the similarity in beta-adrenergic receptors between mammals and fish,
highlighting the diversity of physiological processes mediated by these receptors, and
proposed biomarkers for β-blockers exposure included cardiovascular dysfunction,
with subsequent potential negative effects on fish growth and fecundity (Owen et al.
2007). Only recently have the effects of β-blockers been evaluated in marine fish.
Both studies were in vitro 100 µM propranolol exposures and described decreased
CbE and BFCOD activities in the microsomal fraction of liver of coastal and deep-
sea fishes (Solé and Sanchez-Hernandez 2015, Crespo and Solé 2016).
170 Ecotoxicology of Marine Organisms

Sublethal toxicology of propranolol on marine invertebrates include molecular,

physiological and behavior changes. Motor activity of amphipod Gammarus sp.
has been shown to decrease even in the presence of predator cues, with respiration
rate and feeding rate increasing with propranolol concentrations (100 µg/L to
5 mg/L), probably to compensate for higher energy requirements (Wiklund et al.
2011). However, another study documented a decreased feeding rate with associated
oxidative damage and neurotoxicity in mussels (147 µg/L propranolol) (Solé
et al. 2010). Exposure has also been linked with lower scope for growth, byssus
strength and byssus abundance, potentially reducing substrate fixation ability
in blue mussels, albeit at remarkably high propranolol concentrations (1 to 10
mg/L) (Ericson et al. 2010). A series of complementary experiments with mussels
M. galloprovincialis, evaluated the MOA, molecular targets and associated endpoints,
as well as unspecific effects of exposure to pharmaceuticals interacting with the
cAMP-dependent pathway. cAMP cell signaling influences various physiological
functions of mussels, namely their reproduction, metabolic regulation, and filtering
efficiency (Fabbri and Capuzzo 2010). Overall, exposure to environmentally-
relevant concentrations of propranolol revealed differences on cAMP-related
endpoints, suggesting differential expression of molecular targets in digestive glands,
mantle/gonads and gill tissues (Franzellitti et al. 2011). Furthermore, coexposure to
fluoxetine and propranolol suggested adrenergic regulation in the digestive gland,
whereas serotonergic prevailed in the mantle/gonads of exposed mussels (Franzellitti
et al. 2013). A multibiomarker approach revealed altered lysosomal parameters in
mussels exposed to low propranolol concentration (0.3 ng/L), but other oxidative
stress responses were only observed in the combined fluoxetine and propranolol
treatment (Franzellitti et al. 2015). Furthermore, transcriptional and functional
regulation of genes (e.g., ABCB) and transporters (e.g., P-glycoprotein) related to the
multixenobiotic resistance (MXR) system highlighted the potential of propranolol
to impair immunotoxic response in mussels, thus potentially affecting the ability to
extrude contaminants and cope with environmental stressors in general (Franzellitti
and Fabbri 2013, Franzellitti et al. 2016).
Anticonvulsants, also termed antiseizure or antiepileptics, are neuroactive drugs
that interact with the central nervous system to treat epilepsy, bipolar disorder and
are increasingly used as mood-stabilizers. Several compounds lead to decreased
neuronal activity through different MOA. For example, benzodiazepines (such as
diazepam or lorazepam) enhance the γ-aminobutyric acid (GABA) neurotransmitter
affinity for its receptor increasing chloride channel opening frequency, whilst
carbamazepine acts via the blockage of sodium voltage-dependent channels
of excitatory neurons inhibiting their sustained firing. Both result in lower cell
excitation (Rang et al. 1999). A high degree of evolutionary conservation in
GABA receptors (e.g., in fish) whose functions are related with reducing neuronal
excitability and muscle tension has been reported (Carr and Chambers 2001).
Even if carbamazepine’s MOA is not fully understood, molecular targets appear
to be conserved in mussels M. galloprovincialis following in vivo exposure, with
reduction of the second messenger cyclic AMP and cAMP-dependent protein kinase
(PKA), akin to responses in mammals (Martin-Diaz et al. 2009). Follow-up studies
have described transcriptional and functional impairment of the MXR system in
 Ecotoxicology of Pharmaceuticals in Coastal and Marine Organisms 171

this species, highlighting the potential of carbamazepine, and others (i.e., fluoxetine
and propranolol), in inducing immunotoxicological effects in marine bivalves at
environmental relevant concentrations (Franzellitti et al. 2010, 2014, 2016). Other
recent studies have focused on the effects of carbamazepine exposure on biomarker
responses in several marine invertebrate species. Biomarkers of cellular health (e.g.,
lysosomal membrane stability, LMS), xenobiotic metabolism (e.g., EROD, GST),
oxidative stress (e.g., CAT, SOD, LPO), neurotoxicity (AChE) and genotoxicity
(DNAd) have all been induced by varying exposure concentrations of carbamazepine
in crab C. maenas (Aguirre-Martínez et al. 2013a, 2013c), clams R. philippinarum
(Aguirre-Martínez et al. 2013b, 2016, Almeida et al. 2014) and Scrobicularia plana
(Freitas et al. 2015), and in the polychaetes H. diversicolor (Pires et al. 2016) and
Diopatra neapolitana (Freitas et al. 2015). Toxicity of anticonvulsants in coastal and
marine fish has seldom been reported. Reduced oxidative stress response, increased
swimming lethargy and abnormal posture were observed in the euryhaline fish
Gambusia holbokrii following acute diazepam exposure (in mg/L range) (Nunes
et al. 2008), with acute toxicity LC50 estimated at 12.7 mg/L (Nunes et al. 2005).
In vitro assays confirmed inhibitory action of carbamazepine on CbE and BFCOD
activity in coastal and deep-sea fish species (Solé and Sanchez-Hernandez 2015,
Crespo and Solé 2016).
Lipid regulators or antilipidemic drugs include two major groups of lipid
lowering agents: statins (e.g., simvastatin) and fibrates (e.g., bezafibrate, gemfibrozil).
Their therapeutic role is to decrease the concentration of cholesterol and triglycerides
(fibrates only) in blood plasma. Statins, such as simvastatin and atorvastatin, inhibit
the activity of the enzyme HMG-CoA (3-hydroxymethylglutaryl coenzyme A
reductase), which is responsible for feedback control of cholesterol synthesis. As a
result of decreased intracellular cholesterol concentration, there is an over expression
of low-density lipoprotein (LDL) receptors in hepatocyte membranes which leads
to the resorption of circulating LDL cholesterol. Recent findings suggest that the
MOA of statins is highly conserved, and hypothesized that all metazoan taxa might
be susceptible to the effects of statins at environmentally relevant concentrations
(Santos et al. 2016). Fibrates are peroxisomal proliferators whose MOA is not yet
fully described. Their action is mediated through changes in the expression of the
genes involved in lipoprotein metabolism. Fibrates bind to nuclear transcription
factors of peroxisome proliferator activated receptors (PPARs), which then interacts
with various cellular pathways determining hepatic lipid uptake and the metabolism
of free fatty acids (Rang et al. 1999).
Antilipidemic toxicity data in marine organisms is limited, nonetheless recent
studies have reported a variety of effects on the development and reproduction
of invertebrates, whereas in fish, responses have been mainly assessed through
molecular and biochemical changes. Chronic exposure to low levels of simvastatin
(64 ng/L to 8 µg/L) in the marine amphipod G. locusta ensured severe impacts
on growth, gonad maturation and fecundity, the latter at relevant environmental
concentrations (Neuparth et al. 2014). In sea urchin Paracentrotus lividus, Ribeiro
et al. (2015) described delayed embryo development and increased percentage of
embryo abnormalities when exposed to simvastatin (5 and 2 mg/L, respectively).
Accordingly, another study considering a range of realistic environmental
172 Ecotoxicology of Marine Organisms

concentrations of simvastatin (0.16 and 1.6 µg/L), reported a decrease in development

time and a concomitant increase in body length and growth rate of copepods Nitokra
spinipes (Dahl et al. 2006). Regarding gemfibrozil, exposure at 1 mg/L induced
vitellogenin-like proteins (ALP) in Mytilus spp., which may be indicative of the
potential for endocrine disruption by this fibrate (Schmidt et al. 2011). Concerning
lipid regulators’ toxicity to fish, gemfibrozil exposure (150 µg/L) upregulated
PPAR-related genes transcription in juvenile Sparus aurata, albeit no concomitant
activation of PPAR pathways was observed (Teles et al. 2016). Activation of immune
responses was also suggested following increased mRNA levels of genes linked with
pro-inflammatory processes at 15 ug/L gemfibrozil. Increase in cortisol, as evidence
of stress related effects from gemfibrozil exposure were also observed, even if only at
a concentration of 1.5 mg/L (Teles et al. 2016). Gemfibrozil (injected at 1 mg/kg body
weight in Solea senegalensis) also induced the activity of CYP-related and phase II
(UDPGT) biotransformation enzymes, whilst inhibiting antioxidant defenses (Solé
et al. 2014). Furthermore, simvastatin and fenofibrate have been shown to inhibit
CbE activity in various coastal and deep-sea fishes (Solé and Sanchez-Hernandez
2015), with simvastatin exposure also decreasing AChE levels in estuarine Fundulus
heteroclitus, (1.25 mg/L and LC50 of 2.68 mg/L) (Key et al. 2009).
Antibiotics are used in both human and veterinary medicine to treat bacterial
infections, but may also be used as animal growth promoters. This group
encompasses compounds derived from natural products (e.g., secondary metabolites
of bacterial origin), semi-synthetic derivatives or completely synthetic compounds
which act through various mechanisms, such as suppression of bacterial cell wall or
of protein synthesis and growth (Kummerer 2009a). Penicillins (e.g., penicillin and
amoxicillin), macrolides (e.g., erythromycin), quinolones (e.g., ciprofloxacin) and
tetracyclins (e.g., tetracycline) are amongst the most common types of antibiotics.
As antibiotics are designed to target microorganisms, their toxicity on bacteria
and microalgae is commonly 2 to 3 orders of magnitude above effect levels
reported for higher trophic groups (Kummerer 2009a). Accordingly, exposure to
clarithromycin and clindamycin induced significant growth inhibition in the marine
diatom Skeletonema marinoi at very low concentrations (EC50 of 156 and 154 ng/L,
respectively) (Minguez et al. 2016). In contrast, Aguirre-Martínez et al. (2015)
reported an EC50 of 400 mg/L for inhibition of bacterial luminescence in Vibrio
fischeri, after 15 min of exposure to the antibiotic novobiocin, and an IC50 of 72.8
mg/L for growth inhibition in the algae Isochysis galbana (96 h exposure period).
This study also reported effects for other pharmaceuticals in the mg/L range, yet
novobiocin showed the highest toxicity for microorganisms when compared with
IC50 values determined for carbamazepine, ibuprofen and caffeine. Growth of
marine microalgae (I. galbana and Tetraselmis chui) was inhibited by three different
antibiotics not usually found in the environment (chloramphenicol, florfenicol
and thiamphenicol) with EC50 values ranging from 1.3 to 158 mg/L. Concerning
other phototrophs, one study reported that sulfathiazole exposure, in concentrations
commonly used in aquaculture (25 to 50 mg/L), induced growth inhibition on
macroalgae Ulva lactuca (Leston et al. 2014).
In marine bivalves, exposure to trimethophin (300 to 900 ng/L) and to amoxicillin
(100 to 400 µg/L) affected haemocyte parameters in both R. philippinarum and
 Ecotoxicology of Pharmaceuticals in Coastal and Marine Organisms 173

M. galloprovincialis (Matozzo et al. 2015, Matozzo et al. 2016). The genotoxicity

of amoxicillin was also confirmed via increased micronucleus frequency in both
species’ haemolymph (Matozzo et al. 2016). Similarly, exposure to environmental
concentrations of oxytetracycline resulted in decreased lysosomal membrane stability
in mussels (Banni et al. 2015). Regarding crustaceans, Han et al. (2016) described
several toxicity effects of trimethophin exposure (in the mg/L range) in copepod
Tigriopus japonicas, including increased ROS levels, upregulation of antioxidant
and xenobiotic detoxication-related genes, delayed development time and impaired
reproduction. Antibiotic toxicity in marine fish, encompasses thus far, feeding
behavior and biomarker responses in juveniles of the common goby Pomatoschistus
microps exposed to cefalexin (from 1.3 to 10 mg/L) (Fonte et al. 2016). At 20ºC
and over four days exposure, predation performance was significantly impaired
(> 5 mg/L) and lipid peroxidation levels increased (at 10 mg/L). At 25ºC, the
cefalexin toxicity increased with a decrease of predation performance at 2.5 mg/L
(Fonte et al. 2016).
Antibiotics could also have relevant ecosystem level effects through changes
to microbial communities and their functions (e.g., denitrification, organic matter
decomposition), compromising ecosystem health (Kummerer 2009a, Caracciolo
et al. 2015). Furthermore, constant environmental exposure could promote the
development of antibiotic resistance (Kummerer 2009b), which is a public health
issue if resistance is transferred to human pathogens (Baran et al. 2011).
Despite the limited number of studies, in comparison to freshwater systems, the
information currently available on the ecotoxicity of pharmaceuticals to coastal and
marine species can already be taken into consideration for management and regulation
purposes. The examples highlighted in this chapter clearly demonstrate that multiple
pharmaceutical compounds have adverse effects on coastal and marine organisms at
environmentally relevant concentrations (e.g., Franzellitti et al. 2016, Minguez et al.
2016). However, current legislation is still mostly based on freshwater toxicity
data, even though the marine environment may be more sensitive to pharmaceutical
residues than freshwater (Minguez et al. 2016—based on a comparative toxicity
analyses of 48 pharmaceuticals in both marine and freshwater microalgae and
crustacean species). Ultimately, there are still multiple shortcomings in the evaluation
of pharmaceutical contamination in coastal and marine environments that we should
aim to resolve.

Knowledge Gaps, Current Challenges and Futures Perspectives

There is still a lack of information regarding concentrations, fate and ecotoxicology of
pharmaceuticals in coastal and marine environments (Brausch et al. 2012, Fabbri and
Franzellitti 2016). Additionally, there is also a clear disparity of information among
regions worldwide which we should tackle. For developing regions, where population
increase, higher standards of living and improved access to pharmaceuticals will
likely contribute to increased environmental contamination; this could be a key
moment to start early monitoring schemes to evaluate environmental accumulation,
and to develop associated strategies to minimize detrimental impacts both from
household use and commercial enterprise (e.g., aquaculture, industry). In developed
174 Ecotoxicology of Marine Organisms

countries, mitigation plans are necessary as the environmental pressure exerted by

pharmaceuticals will continue to rise linked to population ageing and prevalence
of chronic diseases. However, up to now most approaches are limited to spatial
or temporal isolated data, lacking long-term aims, rather than encompassing large
regional and temporal coverage. The latter is particularly important in coastal and
transition systems, where variations in loadings are associated to natural fluctuations
in physical and chemical conditions (e.g., salinity, river flow, temperature, water
chemistry), which may imply significant changes to the fate of pharmaceuticals in
the environment (Glassmeyer et al. 2007, Zhao et al. 2015).
In the long run, management strategies for contamination by pharmaceuticals
should aim to act in advance of ensuing adverse effects, promote the development of
a suit of best practices to reduce their occurrence in the environment, and drive the
improvement of systems that constrain potential contamination sources or increase
the effectiveness of the removal and degradation of these compounds from the
environment. The first line of action to reduce the potential entry of pharmaceuticals
in the environment are WWTP, with continued research on the behavior, degradation
and varying removal efficiencies of different WWTP treatments for multiple
therapeutic classes still required. Developing novel methodologies that enhance
the efficacy of WWTPs tertiary treatment to specifically remove or degrade
pharmaceutical compounds is an acknowledged path for reducing the potential
impact of pharmaceuticals (Margot et al. 2013, Calisto et al. 2017). In fact, Directive
2013/39 EU (European Parliament 2013) underlines the importance of finding new
ways of tackling water pollution by pharmaceuticals, and unravelling the physico-
chemical processes that determine degradation and transformation of pharmaceutical
compounds, their metabolites and by-products that will further contribute to resolving
these issues.
Different pharmaceuticals have been shown to bioaccumulate (Klosterhaus et al.
2013) and even biomagnify (Liu et al. 2017), yet in general, there is insufficient
information on bioaccumulation and the impacts of pharmaceutical residues across
the trophic web, namely for top-predators (Gaw et al. 2014). Likewise, given the
effects of pharmaceuticals on bacteria and algae (Backhaus et al. 2011, Minguez et al.
2016) and the high degree of homology between chloroplasts and bacteria as well as
among other metabolic pathways across multiple phyla (Brain et al. 2008), the lack
of research on higher marine phototrophs (e.g., halophytes, plants) is conspicuous.
Compiling information on bioaccumulation, effects and understanding MOA
and adverse outcomes of pharmaceuticals are critical for the effective management
of pharmaceutical contamination and to safeguard coastal and marine biota.
Thousands of different active pharmaceutical ingredients are available for human
and veterinary use, which impedes assessing the full spectrum of contaminants in
any given monitoring scheme. Furthermore, the consumed amount and toxicity
of individual drugs varies greatly; thus it is key to prioritize research directives,
monitoring and regulation. Several options have been forwarded over the years (e.g.,
Schreiber et al. 2011, Caldwell et al. 2014, Rudd et al. 2014), though three main
aspects to take into consideration are generally consumption levels, ecotoxicological
risk and persistence in the environment. Rather than in isolation, these facets should
be evaluated simultaneously, as directing resources to higher risk but low use or
 Ecotoxicology of Pharmaceuticals in Coastal and Marine Organisms 175

persistence pharmaceuticals may not prove a good investment of time and resources.
Approaches based on MOA take into consideration the evolutionary and functional
conservation of molecular targets of pharmaceuticals (e.g., receptors, enzymes), and
cellular and physiological processes across species, which enables the identification of
relevant endpoints and experimental conditions to determine drug toxicity (Christen
et al. 2010, Fabbri and Franzellitti 2016, Santos et al. 2016). Furthermore, chronic
exposure assessments at environmentally significant concentrations are central to
evaluate the risk posed by pharmaceutical substances (Fabbri and Franzellitti 2016).
Acute testing has several limitations that can compromise resulting environmental
regulation. Yet, contamination thresholds are still mostly based on acute standard
toxicity tests. Even though, they are less sensitive than other endpoints in non-
model species (e.g., Aguirre-Martínez et al. 2015), and neglect potential long-term
effects from chronic exposures, which are more representative of the persistent
contamination organisms experience in their natural environment (Crane et al. 2006,
Fent et al. 2006).
Ecotoxicological assessments should strive to fill the gap between sub-cellular
endpoints and adverse individual or population level effects. This is a major challenge
and requires the development of frameworks that synthesize data at many levels of
biological organization. The adverse outcome pathways (AOP) is a good example of
this, and several studies have illustrated the potential of AOP for population-modelling
and predictive ecotoxicology (Ankley et al. 2010, Franzellitti et al. 2014, Hird
et al. 2016). Furthermore, the utility of the AOP approach has been demonstrated for
cross species extrapolation and integrating life-history theory (Groh et al. 2015). One
of the key issues is ensuring baseline toxicity studies produce robust and accurate
quantitative data that can be subsequently integrated in population modelling
approaches. Ideally dose-response or concentration-response relationships for both
lethal and sub-lethal effects should be defined allowing response curves, effect-
thresholds and the probability of effects occurring at different levels of biological
organization to be estimated (Kramer et al. 2011).
Ultimately, monitoring of prioritized pharmaceuticals, metabolites and by-
products in coastal environments should complement risk assessment and is
integral to current European policy. The EU dynamic watch list for emerging
contaminants under the Water Framework Directive (WFD—Directive 2000/60/
EC) includes pharmaceutical diclofenac, and two hormones 17-beta-estradiol (E2),
and 17-alpha-ethinylestradiol (EE2), with three additional antibiotics proposed for
inclusion (erythromycin, clarithromycin and azithromycin). In addition to consistent
water collections and analysis, monitoring strategies can build upon the success of
programs such as Mussel Watch (Goldberg and Bertine 2000), which would allow
for both bioaccumulation (Wille et al. 2011, McEneff et al. 2014) and monitoring
of effects and ecotoxicology via a standardized set of biomarkers (Franzellitti et al.
2015, Mezzelani et al. 2016a). Other prospective monitoring tools for baseline
concentration data include the use of passive sampling devices (Martínez Bueno
et al. 2016) or the use of unmanned automated sampling devices in ships and marine
platforms of opportunity (Brumovsky et al. 2016).
176 Ecotoxicology of Marine Organisms

Acknowledgments
This work had the support of the Fundação para a Ciência e a Tecnologia (FCT) via
UID/MAR/04292/2013 and project grant PTDC/MAR-EST/3048/2014. PRS was
funded with FCT postdoctoral grant SFRH/BPD/95784/2013.

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8 Mercury Exposure, Fish
Consumption and Provisional
Tolerable Weekly Intake
An Overview
Vieira, H.C.,* Soares, A.M.V.M., Morgado, F. and Abreu, S.N.

INTRODUCTION
Mercury (Hg) appears at the top of the priority list of hazardous substances published
by the US Agency for toxic substances and disease registry (ATSDR 2013) due to its
persistence, ability to enter into biological systems (Wolkin et al. 2012, Liao et al.
2016) and its high toxicity even at low concentrations (Hajeb et al. 2009).
Originating from natural sources (Siegel and Siegel 1984, Gustin et al. 2000,
Coolbaugh et al. 2002) such as volcanic emissions, geothermal releases, and biomass
burning (Renzoni et al. 1998, Pirrone et al. 2010), and anthropogenic sources
(Pacyna et al. 2001, Dommergue et al. 2002) as mining, chloro-alkali production and
fossil fuels combustion (Renzoni et al. 1998, Pirrone et al. 2010), Hg can be found
either in aquatic or terrestrial environments (Gochfeld 2003) in three oxidation states
(Hg0, Hg+1 and Hg+2) as well in various forms (Ullrich et al. 2001, Rasmussen et al.
2005). All forms of Hg are toxic, however, the level of toxicity is strongly related to
the chemical properties of each form (Lindqvist and Rodhe 1985, Gochfeld 2003,
Harris et al. 2003). For example, inorganic forms may be transformed into organic
forms (methylated species). Methylmercury (MeHg), the most toxic form of Hg (due
to the neurotoxic effects), can be processed through bacteria, like sulfate-reducing
bacteria (Compeau and Bartha 1985), and accumulate in the food chain, resulting in
a process of bioaccumulation and biomagnification, leading to high concentrations

Department of Biology & CESAM, University of Aveiro, 3810-193 Aveiro, Portugal.

* Corresponding author; [email protected]
186 Ecotoxicology of Marine Organisms

of Hg in fish. Ultimately, fish is consumed by humans, resulting in an increased risk

of adverse effects to human health. Human exposure to MeHg is almost exclusively
a result of the consumption of fish and shellfish (Mergler et al. 2007, Hajeb et al.
2009). In predatory marine fish, about 90 percent of the Hg exists in the methylated
form (WHO 2008).
Apart from being pointed as the main route of MeHg to humans, fish
consumption has also been recognised as an important component of a healthy diet
(Malm et al. 1995, Mergler et al. 2007). Due its high nutritional value, fish is rich
in protein with essential amino acids, macroelements (calcium and phosphorus),
microelements (iodine, selenium and zinc), vitamins and unsaturated fatty acids. On
the other hand, fish is considered an important source of n-3 fatty acids such as
docosahexaenoic acid (22:6, n-3, DHA) and eicosapentaenoic acid (20:5, n-3, EPA)
(Egeland and Middaugh 1997, FAO 2012, WHO 2003). All these elements play an
important role preventing the development of some diseases, especially regarding
cardiac and circulatory disorders, and reducing mortality in patients with coronary
diseases (Kris-Etherton et al. 2002).
The concern of Hg pollution arises from a human health problem observed in
various parts of the world, resultant from Hg exposure through the consumption
of marine and freshwater seafood (Lin and Pehkonen 1999, Baeyens et al. 2003,
Carrasco et al. 2011, You et al. 2014). The effects of Hg in human health have gained
more importance essentially since the Hg poisoning incident occurred in the 1950s at
Minamata Bay in Japan (Ullrich et al. 2001, Rasmussen et al. 2005). Following this
episode, some international agencies such as United States Environmental Protection
Agency (USEPA) and World Health Organization (WHO), have established reference
doses (RfD) to the mercury tolerable intake levels, aiming to prevent health risks.
Currently, Food and Agriculture Organization (FAO)/WHO Joint Expert Committee
on Food Additives (JECFA) established the RfD “provisional tolerable weekly
intake” (PTWI) for MeHg at 0.23 mg kg bw–1 day–1 (EFSA 2012) and USEPA fixed
the RfD in 0.1 mg MeHg kg bw–1 day–1 (USEPA 1997a). Recently, PTWI suggested
by JECFA for MeHg was revised by the European Food Safety Authority (EFSA) to
0.19 µg MeHg kg bw–1 day–1 (EFSA 2012).
Hair has been used in several studies as a bioindicator of Hg exposure for human
populations (Dorea et al. 2003, Agusa et al. 2005). Hair grows approximately 1 cm
per month, during hair formation, Hg present in the circulating blood is incorporated
into the hair follicles, where it becomes stable, registering and providing the
accumulation pattern and history of exposure (Dolbec et al. 2001, Agusa et al. 2007,
Díez et al. 2008, Freire et al. 2010).
There are several reasons to use hair as bioindicator for mercury exposure: (1) it
is easy to collect in a noninvasive manner, (2) it captures temporal exposure history
as Hg is incorporated into growing hair and (3) it does not require special facilities for
transport or storage (Cizdziel and Gerstenberger 2004). PTWI and RfD correspond
to a hair Hg concentration of 2.2 and 1.0 μg g−1, respectively. WHO, through analysis
of neurotoxicological data, considered Hg concentration of 50 μg g−1 in human hair
as a “no observed adverse effect level” (NOAEL) value for MeHg (WHO 1990).
 Mercury Exposure, Fish Consumption and Provisional Tolerable Weekly Intake 187

Interspecific Hg Variation in Fish

Having the highest seafood consumption per capita in Europe per year, Portugal also
occupies the top rank in countries with the highest seafood consumption per capita in
the world (Failler et al. 2007, FAO 2010). The Azores archipelago is the Portuguese
region with the highest consumption rate per capita of fishery products, where each
Azorean consumes about 80 kg of fish per year (Megapesca 2007).
In the Portuguese Mainland, anthropogenic sources, mainly chlor-alkali plants
(Mil-Homens et al. 2008) are the highest Hg contributors. One important example
is the case of Laranjo basin in Ria de Aveiro, that received a highly contaminated
effluent discharge from a chlor-alkali plant Hg cell located in the Estarreja industrial
complex, from the 1950s until 1994 (Pereira et al. 2009). On the other hand, the
volcanic Portuguese archipelagos, particularly the Azores, are mostly exposed to Hg
from natural sources (e.g., volcanic activity) linked with geochemical anomalies and
hot springs (Loppi 2001), being a remote area and away from large anthropogenic
sources, the Azores have no significant discharge of Hg from industrial sources
(Rodrigues et al. 2004).
The interspecific Hg variation in fish is the result of its intrinsic trophic position,
growth rate, specimen age and food web complexity (Magalhães et al. 2007,
Koenig et al. 2013). Thus, different fish may contain very different Hg content.
Hg concentrations presented in Table 1 were based on seven studies that evaluated
concentrations found in 28 fish species caught near the Azores archipelago.
Sparisoma cretense was the fish species with the lowest Hg concentration, on
the other hand, the species Mora moro presented the highest Hg concentration.
Hg concentration of each species was compared to permissible limits set forth
by Commission Regulation (EC) no. 1881/2006 of December 2006. This regulation
establishes 0.5 µg g–1 as the maximum Hg concentration for most fish species, with
an “exception list” of species whose maximum Hg concentration allowed increases
to 1 µg g–1. Regarding the permissible limits for fish consumption, only Mora moro
exhibited higher values than those allowed (0.81 µg g–1 vs. 0.5 µg g–1). Higher values
for Hg concentrations in Mora moro were also observed by Koening et al. (2013) in
a study on deep-sea organisms from the NW Mediterranean.

Fish Consumption and Hg Exposure Assessment Tools

Food frequency questionnaires (FFQ) are tools designed to evaluate the usual diet
of a population. FFQ have the advantages of being easy and quick to apply, and
practical to identify usual food consumption, in addition to its low cost (Ocké et al.
1997). Over the years, the use of FFQ have been increasingly used in studies where
human exposure to Hg is assessed through fish consumption (MacIntosh et al. 1997,
Björnberg et al. 2003, Passos et al. 2003, Oken et al. 2016). On the other hand, it
is possible to reconstitute recent past exposure history (Cernichiari et al. 1995) by
cutting the hair into centimeter-long samples and analyzing the Hg content of each
centimeter independently (Passos et al. 2003).
The information related to the fish consumption (number of meals per week)
was assessed during hair sampling, using a FFQ where each individual was asked to
188 Ecotoxicology of Marine Organisms

Table 1. Hg concentrations (µg g–1) in 28 fish species caught near the Azores archipelago and maximum
Hg level set forth by Commission Regulation (EC) no. 1881/2006 of December 2006 for fish consumption.

Scientific name [Hg](µg g–1) Maximum level References

Aphanopus carbo 0.89 1.0 Afonso (2007)
Aphanopus carbo 0.71 1.0 Costa (2009)
Capros aper 0.05 0.5 Monteiro (1996)
Ceratoscopelus maderensis 0.12 0.5 Monteiro (1996)
Chelon labrosus 0.20 0.5 Andersen (1997)
Conger conger 0.25 0.5 Andersen (1997)
Conger conger 0.41 0.5 Magalhães (2007)
Diplodus sargus cadenati 3.41 0.5 Andersen (1997)
Diplodus sargus cadenati 0.30 0.5 Andersen (1997)
Electrona rissoi 0.10 0.5 Monteiro (1996)
Helicolenus dactylopterus 0.29 0.5 Monteiro (1991)
Helicolenus dactylopterus 0.26 0.5 Andersen (1997)
Katsuwonus pelamis 0.19 1.0 Andersen (1997)
Katsuwonus pelamis 0.04 1.0 Torres (2016)
Lepidopus caudatus 0.28 1.0 Andersen (1997)
Lepidopus caudatus 0.32 1.0 Magalhães (2007)
Macrorhamphosus scolopax 0.02 0.5 Monteiro (1996)
Maurolicus muelleri 0.11 0.5 Monteiro (1996)
Mora moro 0.81 0.5 Magalhães (2007)
Mullus surmuletus 0.12 1.0 Andersen (1997)
Muraena helena 0.05 0.5 Andersen (1997)
Myctophum punctatum 0.10 0.5 Monteiro (1996)
Pagellus acarne 0.20 1.0 Magalhães (2007)
Pagellus bogaraveo 0.26 1.0 Andersen (1997)
Pagrus pagrus 0.47 0.5 Andersen (1997)
Phycis blennoides 0.15 0.5 Magalhães (2007)
Phycis phycis 0.10 0.5 Andersen (1997)
Phycis phycis 0.13 0.5 Magalhães (2007)
Polyprion americanus 0.27 0.5 Magalhães (2007)
Pontinus kuhlii 0.16 0.5 Monteiro (1991)
Scomber japonicus 0.03 0.5 Monteiro (1996)
Thunnus alalunga 0.37 1.0 Andersen (1997)
Thunnus obesus 0.14 1.0 Torres (2016)
Trachurus picturatus 0.05 0.5 Monteiro (1996)
Trachurus picturatus 0.04 0.5 Andersen (1997)
Trachurus picturatus 0.16 0.5 Magalhães (2007)
 Mercury Exposure, Fish Consumption and Provisional Tolerable Weekly Intake 189

complete a questionnaire detailing age, gender, body weight, height, smoking habits
and frequency of fish consumption.
In an initial study, the hair samples were obtained randomly from 29 males and
81 females in Terceira Island, Azores (Fig. 1) who had been fully informed about the
purpose of the study through a descriptive document.
In Terceira Island, the average of Hg concentration (n = 110) was 0.86 ± 0.05
μg g–1. These Hg concentrations can be considered relatively low, when compared
with the normal values established by international agencies for fish consumption
population, once the USEPA established as normal levels the concentration of 1 μg g–1
of Hg in hair (USEPA 1997a), or can be assumed to be even lower when related
with the 2 μg g–1 assumed by WHO as normal level of Hg in hair (WHO 1990).
Having stated that, about 33 percent of the population in Terceira Island exceeded
the RfD indicated by USEPA, whereas 5.9 percent of the same population exceeded
the normal level set forth by WHO (Fig. 2).
The volunteers were then grouped according to the fish consumption habits and
levels of consumption. In the initial approach, the volunteers were divided into two
groups according to their fish-eating habits (consume and not consume). The highest
Hg level 0.89 ± 0.05 μg g–1 was obtained in the first group that admitted eating fish
(Consume), as opposed to 0.39 ± 0.08 μg g–1 of the second group that said they did
not eat fish (Not Consume) (Fig. 3).
In the second approach, the volunteers were grouped into four categories: never
(0 fish meals per week), 1–2 meals per week, 3–4 meals per week and 5 or more
meals per week according the frequency of fish consumption (Fig. 4). A significant
increase (p < 0.001) was found between the volunteers who claimed that they do not
eat fish and the volunteers who admitted having five or more meals of fish per week. A
significant increase was also observed between all groups of fish consumption except
when comparing Hg concentration between the group who admitted eating fish 3–4
idge

Corvo
N
tic R

Flores
Atlan

Graciosa
Mid-

Terceira
São Jorge
Faial

Pico

São Miguel

Atlantic ocean
Santa Maria

35 km

Fig. 1. Map of study area.

190 Ecotoxicology of Marine Organisms

Fig. 2. Distribution of Hg concentration in hair of the 110 inhabitants of Terceira Island.

Fig. 3. Hg concentration in the hair of the inhabitants of Terceira Island in relation to fish consumption.

meals per week and the group that consumed 3 or 4 fish meals per week. Similar results
have been reported by Al-Majed and Preston (2000) and Díez et al. (2008).
Comparing Hg in the hair of the residents and their habits of fish consumption
it was found a positive statistical correlation in the first approach where the study
group was divided into two groups according to their fish-eating habits (R = 0.267,
 Mercury Exposure, Fish Consumption and Provisional Tolerable Weekly Intake 191

Fig. 4. Hg concentration in the hair of the inhabitants of Terceira Island in relation to fish consumption
(meal week–1).

p < 0.05) and also in the second approach where the volunteers were grouped into
four categories (R = 0.415, p < 0.001). Other authors like Holsbeek et al. (1996) and
Hajeb et al. (2008), have also associated the consumption of fish as a potential route
of uptake of Hg.
In a second study, hair samples were obtained randomly from 157 young
volunteers of two different high schools, 84 inhabitants (46 females and 41 males)
from Angra do Heroísmo (Azores) and 73 inhabitants (41 females and 32 males)
from Murtosa (Mainland), with ages between 14 and 18 years. Volunteers were
invited to participate in the study after a detailed explanation of the main objectives.
Considering the Hg concentration of 1 μg g–1 in hair established as RfD by
USEPA (USEPA 1997c), 24.4 percent of the Azorean volunteers and 52.9 percent
of the sampled population in the Mainland exceeded this safety standard (Fig. 5).
These results suggest higher Hg levels in the Mainland when compared to data from
the Azores.
Hg hair concentration in the Azores ranged from 0.03 to 2.13 μg g–1 and the
average was 0.82 ± 0.06 μg g–1, on the other hand, the average in the Mainland was
1.13 ± 0.07 μg g–1 varying from 0.03 µg g–1 to 2.60 µg g–1. Significant differences
in average of Hg concentrations present in hair between sampling site were found.
Fish consumption can be the key to these differences, since there is a significant
difference (p < 0.05) when we compare the number of fish meals per week of the
volunteers from the Mainland and the Azorean volunteers, 3.31 ± 0.22 meals week–1
and 2.08 ± 0.18 meals week–1, respectively.
Hg accumulation in the hair was also evaluated separately for both sampling
sites (Azores and Mainland), and the volunteers of both sites were grouped, in the
initial approach, into two categories (Fig. 6a) according to their fish-eating habits
192 Ecotoxicology of Marine Organisms

Fig. 5. Distribution of Hg concentration in the hair obtained in both sampling sites (Azores and Mainland).

(consume and not consume). In the second approach, the group was divided into four
categories: never (0 meals per week), 1–2 meals per week, 3–4 meals per week and
5 or more meals per week (Fig. 6b).
Volunteers from both sites that admitted consuming fish present higher Hg
concentrations than volunteers who did not consume fish (Fig. 6a). This difference is
statistically significant for both sites (p < 0.05).
In the second approach, where volunteers were grouped into four categories of
fish consumption rates (Fig. 6B), the volunteers who assume that they do not have
fish meals were 6.4 percent in the Azores and 4.3 percent of the Mainland. On the
other hand, 66.7 percent assumed to have a maximum of two meals of fish per week
in the Azores, making the category of 1–2 meals per week the most representative for
the Azorean volunteers; however, this category represents only 31.4 percent of the
Mainland volunteers. In contrast to what was previously observed, the category of
3–4 meals per week, Mainland was the most representative, where 40 percent of the
individuals assumed to eat fish 3 or 4 times per week in opposition to the 21.8 percent
found in the Azores. In the category with higher fish consumption (5 or more meals
of fish per week), 5.1 percent of the Azores volunteers were represented as opposed
to 24.3 percent in the Mainland. The range of Hg concentration for each category was
0.03 – 0.49 μg g−1 (never), 0.13 – 2.12 μg g−1 (1–2 meal per week), 0.28 – 2.07 μg g−1
(3–4 meals per week) and 0.95 – 2.00 μg g−1 (≥ 5 meals per week) in the Azores while
in the Mainland it ranged between 0.03 – 0.32 μg g−1 (never), 0.27 – 1.77 μg g−1,
(1–2 meal per week) 0.41 – 2.47 μg g−1 (3–4 meals per week) and 0.93 – 2.60 μg g−1
(≥ 5 meals per week).
The Hg concentration in both sampling sites (Azores and the Mainland) showed
an increment with the increase of fish meals and are in accordance with Al-Majed
and Preston (2000), Díez et al (2008), Vieira et al. (2013) and Shao et al. (2013).
The levels of Hg concentration indicated a significant increase in the case of
the individuals who claimed to never eat fish in relation to those who admitted to
consume fish 3–4 meals per week and five or more meals per week (p < 0.05).
Fig. 6. Hg concentration in hair in the volunteers of both sites in relation to (a) fish consumption habits and (b) fish consumption (meals per week).
Mercury Exposure, Fish Consumption and Provisional Tolerable Weekly Intake
193
194 Ecotoxicology of Marine Organisms

Hg Intake Levels, Through the Application of Formulas Established by

the World Health Organization
In humans, the ingestion of Hg is related to the concentration of Hg found in the
bloodstream (Sherlock et al. 1984). On the other hand, the concentration of Hg in
human hair is assumed to be 250 times the concentration in blood (USEPA 1997c).
From the concentration present in the hair, it is possible to estimate the MeHg
intake (µg MeHg kg body weight–1 day–1) by conversion of the Hg concentration in
the hair (µg g–1) to that in the blood (µg L–1), according the following equation a)
(USEPA 1997c):

Hghair(μgg–1)
× 1000 a)
250
And the conversion of the Hg blood concentration into MeHg intake can be
estimated from hair Hg concentration using the following formula b) (USEPA 1997c):
c×b×V
d= b)
A × f × bw

Where d = dose (µg MeHg kg body weight–1 day–1)

C = mercury concentration in blood (µg l–1)
b = elimination rate constant (0.014 per day–1)
V = blood volume (5 liters)
A = fraction of the dose absorbed (0.95)
f = the absorbed fraction distributed to the blood (0.05)
bw = body weight (60 kg)
and to extrapolate the MeHg intake, the following formula was used c) (WHO 2008):

MeHg intake
Amount of fishing gested (gweek –1) × [MeHg] in fishing gested (μgg–1)
= c)
Kilogram body weight (Kgbw)

Through the MeHg intake obtained with the formula b), the amount of fish consumed
per week and body weight, the concentration of MeHg in fish was calculated
following the equation d)

[MeHg] in fish ( ppm)

MeHg intake (μgMeHgkg body weight day)
= × 60 (Kgbw) d)
Amount of fishing gested (gweek)
The potential levels of Hg uptake inferred from PTWIs formulas were evaluated
in adolescents of two distinct locations (Angra do Heroísmo and Murtosa), based on
the concentrations of mercury present in the human hair.
 Mercury Exposure, Fish Consumption and Provisional Tolerable Weekly Intake 195

The levels of MeHg exposure for Azores and Mainland were estimated through
formulas a) and b). A body weight of 60 kg was used as denominator of the equation
in the USEPA guidelines to calculate the Hg daily dose (USEPA 1997c). In our case
study, this value was assumed for both sampling sites, since the average body weight
in Azores and Mainland was 60.5 kg and 60.8 kg, respectively.
The exposure level (μg kg–1 bw week–1) results showed that adolescents from the
Mainland present a higher exposure to the MeHg than the adolescents from Azores,
0.77 μg kg–1 bw week–1 against 0.57 μg kg–1 bw week–1. The difference between
sampling sites is statistically significant (p < 0.001).
The majority of Azorean adolescents (77.6 percent) had exposure levels
below the USEPA RfD, in contrast with the 52.2 percent observed in the Mainland
adolescents.
MeHg intake values calculated based on the concentration of Hg present in hair,
showed obviously identical trends to Hg concentration in (Fig. 7), increasing with
the fish consumption. Similar results were found in other studies such as Bjornberg
et al. (2005) and Rubio et al. (2008).
Both sites presented values above the RfD; however, in the Azores these values
were only found in volunteers who consumed five or more meals per week, while in
the Mainland, the volunteers that had three or more meals of fish per week already
exceeded this reference value.
On the other hand, considering fish consumption as the main cause of Hg
concentration present in the hair, the application of formula (d) would allow
researchers to estimate [MeHg] in the fish.
The calculated MeHg in fish (Fig. 8) show levels of 0.16 μg g–1 and 0.13 μg g–1
(Azores and Mainland, respectively) for the 1–2 meals of fish per week groups.
However, with the increase of fish consumption (3–4 and 5 or more fish meal per
week) in both sampling sites, a surprising decrease in the levels of calculated MeHg
in fish (< 0.1 μg g–1) was observed.
Higher fish consumption rates led to an increase in the amount (mass) of fish
consumed, but the corresponding Hg burden is not linearly assimilated; in this way,
MeHg concentrations determined in the ingested fish (source) by volunteers from
Azores and Mainland indicate a decrease of Hg concentration in the fish in the
highest rates of fish consumption. However, assuming fish as the common source of
MeHg for all fish consumption categories, this occurrence could not be acceptable.
This is because consuming one fish meal per week or five fish meal per week would
have had no effect on the Hg concentration in the fish specimens. Accordingly,
considering fish consumption as the main route of Hg in humans, the levels of MeHg
in fish should be identical regardless of the category of consumption (1–2, 3–4 and
5 or more than 5 meals per week). Thus, assuming a common level of MeHg in fish
for every consumption category and having the MeHg level in fish obtained for the
lowest fish meal per week (0.16 μg g–1 for Azores and 0.13 μg g–1 for Mainland)
as reference, the potential levels of Hg in hair would have to be higher than the
determined [Hg] (found in hair) that our results had first indicated.
The extrapolated intake (Fig. 9) increased linearly with the fish consumption.
The differences between intakes are approximately the same when compared with the
real intake for the fish consumption of 1–2 meals per week, approximately 2-fold in
196 Ecotoxicology of Marine Organisms

Fig. 7. MeHg intake (µg MeHg kg body weight–1 day–1) in volunteers of Azores and Mainland.

Fig. 8. MeHg concentration in fish ingested in volunteers from Azores and Mainland.

the volunteers that consume 2–3 fish meals per week in both sites and approximately
3-fold and 2-fold in the category of 5 or more fish meals per week in the Azores and
the Mainland, respectively.
Metals, such as Hg, are known to induce metallothioneins (MT) (Bucheli and
Fent 1995, Yasutake et al. 1998) that have high cysteine content, acting similarly
to metal chelators, providing heavy metal tolerance and regulating Hg distribution
and retention (Yoshida et al. 1997, Klaassen and Liu 1998, Yoshida et al. 1999).
Chelators of Hg (e.g., glutathione and N-acetylcysteine) have been shown to
 Mercury Exposure, Fish Consumption and Provisional Tolerable Weekly Intake 197

Fig. 9. Real MeHg and potential MeHg intake versus fish consumption for (a) Azores and (b) Mainland.

enhance the clearance of Hg from the blood perhaps by reducing metal uptake by
erythrocytes (Gochfeld 1997). According to Gundacker et al. (2007), an increment in
fish consumption per week (higher Hg intake), may lead to higher levels of MT in the
bloodstream. Since MT are important detoxification proteins, an increase of this type
of proteins may result in the reduction of Hg concentration in the blood at the time
of hair formation, contributing to the differences observed between Hg concentration
measured in the hair of the volunteers and the extrapolated Hg concentration after
applying the formula c). Micronutrients such as selenium, methionine, cysteine and
vitamin E can also protect against Hg bioactivity, likely via antioxidant responses
(e.g., bcl2 gene induction in the kidney, free radical scavenging) trapping Hg and it
may also aid to explain Hg detoxification (Drasch et al. 1996, Patrick 2002, Battin
and Brumaghim 2009), leading to a lower Hg concentration compared to the expected
(extrapolated) values.

Isocurves of the Maximum Number of Fishmeal per week Without

Exceeding the MeHg RfD
Epidemiological studies in New Zealand, Seychelles and the Faeroe are the basis of
the safety levels of daily Hg exposure established by international agencies such as
the USEPA and JECFA (Burger et al. 2001, Rice 2004, Maycock and Benford 2007).
USEPA fixed the guideline values for maximum exposure limits (RfD) of 0.1 μg
MeHg kg bw–1 day–1 (USEPA 1997a) and JECFA established PTWI equivalent to
0.19 μg kg–1 day–1 for MeHg (EFSA 2012).
Uncertainty factors (UF) are used for the calculation of RfD. The USEPA
RfD was established by applying a 10-fold Uncertainty Factor (UF) to explain the
variation between prolonged healthy human populations exposed to the consumption
of contaminated fish (USEPA 1997b).
The reference dose setting established by the USEPA was suggested by
Grandjean and Budtz-Jørgensen (2007) based on developmental neurotoxicity
studies at exposure levels close to 50 percent below USEPA RfD (Jedrychowski et al.
2007, Lederman et al. 2008, Suzuki et al. 2010). With this adjustment, the reference
dose would be reduced from 0.1 to 0.05 μg MeHg kg bw–1 day–1.
198 Ecotoxicology of Marine Organisms

The level of exposure to MeHg depends on (i) type and amount of ingested fish
per unit time (such as day or week); (ii) MeHg concentrations in fish; and (iii) the
body weight of the fish consumers. Thus, MeHg intake (µg MeHg kg bw–1 week–1 for
individuals or populations) can be calculated by the following the formula e) (WHO
2008):

MeHg intake
Amount of fishing gested (gweek –1) × [Hg] in fishing gested (μgg–1)
= e)
Kilogram body weight (Kgbw)

Therefore, the maximum number of fish meals per week can be calculated using
formula f) by considering a reference dose for MeHg as the maximum exposure
value, the body weight of 60 kg based on USEPA guidelines (USEPA 1997c), the
amount (g) of fish ingested per meal and MeHg concentration present in ingested
fish:
(RfD × bw) × 7 days
Fish meal week = f)
(Fish meal size (g) × [MeHg] in fish) ÷ 1000

The results for the application of the formula f) are shown as trend lines (isocurves)
(Fig. 10) indicating the maximum number fish meals per week allowed without
exceeding the RfD (0.1 μg MeH g kg bw–1 day–1 or 0.05 μg MeHg kg bw–1 day–1),
combining fish meal size of 50 g (Fig. 10a), 100 g (Fig. 10b), 150g (Fig. 10c), 170 g
(Fig. 10d), 200 g (Fig. 10e) and 250 g (Fig. 10f) under different MeHg concentrations.
Regarding a RfD of 0.1 μg MeHg kg bw–1 day–1, the isocurves demonstrate
that the RfD is exceeded if we consume a 50 g fish meal once a week with MeHg
concentrations above 0.84 μg g–1 (Fig. 9a), whereas when considering a RfD of 0.05 μg
MeHg kg bw–1 day–1 the MeHg concentrations should not exceed 0.42 μg of MeHg g–1
of fish (Fig. 9a). As expected, as the portion of fish per meal increases the values of
mercury in fish would have to decrease in order to obtain a specific level of exposure.
For example, to the RfD of 0.1 μg MeHg kg bw–1 day–1 and the fish size of 100, 150,
170, 200 and 250 g (Fig. 9b–9f), the MeHg concentration in fish would not have to
exceed the 0.42, 0.28, 0.24, 0.21 and 0.17 µg g–1, respectively, following that RfD.
Regarding the RfD of 0.05 μg MeHg kg bw–1 day–1 and for the fish size of 100, 150,
170, 200 and 250 g, the values related to MeHg in fish would obviously have been
decreased to half when compared to the values of fish meal size using USEPA RfD.

Conclusion
The concentration of Hg found in the hair of the two populations studied (Azores and
Mainland) indicates that individuals with higher fish consumption per week generally
have higher concentrations of Hg, which makes fish consumption the main route of
exposure for these populations. Overall data indicate relatively low concentration of
mercury in hair despite the high fish consumption per capita.
Hg concentration in human hair is assumed to reflect MeHg intake through fish
consumption, allowing researchers to evaluate the risk associated with the MeHg
 Mercury Exposure, Fish Consumption and Provisional Tolerable Weekly Intake 199

Fig. 10. Fish meal week–1 in relation to the [MeHg] in fish (µg g–1) for: (a) RfD of 0.1 µg MeHg kg bw–1
day–1 and (b) ½ RfD (0.05 µg MeHg kg bw–1 day–1).

Color version at the end of the book

intake for the human population. The results of the Hg concentration of adolescents
from the Azores and the Mainland indicate that the effective Hg uptake becomes lower
than expected as the rate of fish consumption increases. These results accentuate the
capacity of the human body to induce a self-protection response, minimising MeHg
assimilation probably by detoxification mechanisms, thereby mitigating Hg levels in
the blood when experiencing increasing Hg exposure.
200 Ecotoxicology of Marine Organisms

Finally, even a single meal (per week) of 50 g fish with 0.84 μg g−1 of MeHg
would reach the USEPA RfD maximum levels, despite the 1.0 μg g−1 of MeHg in fish
which is being allowed for fish consumption.

Acknowledgements
This work was supported by the Portuguese Science Foundation (FCT) through
a PhD grant awarded to Vieira, H.C. (PD/BD/127808/2016). FCT also financed
CESAM: UID/AMB/50017/2013.

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9 Contaminants Impact
on Marine Turtle
Populations Development
An Overview
Patricia Salvarani,1 Vania C. Foster,2 Jaime Rendon3
and Fernando Morgado1,*

INTRODUCTION
There are seven sea turtle species in the world, distributed across almost all the
oceans (Caretta caretta, Chelonia mydas, Dermochelys coriacea, Eretmochelys
imbricata, Lepidochelys olivacea, Natator depressus and Lepidochelys kempii).
With the exception for Natator depressus, all others are catalogued as vulnerable,
endangered or critically endangered in the IUCN list (IUCN 2016). Currently, six
species of sea turtles are included in the lists of endangered species worldwide
(MTSG 1995, Lutcavage et al. 1997) and on the Red List of Threatened Animals
(IUCN). According to the Marine Turtle Specialist Group (MTSG), currently, the
main threats to sea turtles are coastal development, incidental capture by fisheries,
direct use for human consumption, climate change, pollution and biotic threats.
During their life cycle, sea turtles face various challenges in a fight for survival
and are subject to many biotic, abiotic and anthropogenic threats. Biotic threats are
related to diseases and parasites such as fibro papillomatosis and spiroquidiasis,
bacterial or fungi infections in eggs (Aguirre 1995, 2000, Rossi et al. 2009,
Rodenbusch et al. 2014), presence of other turtles spawning (Eckrich and Owens
1995), eggs and offspring predation (Fowler 1979) and also vegetation growth around

1
Department of Biology & CESAM, University of Aveiro, 3810-193 Aveiro, Portugal.
Email: [email protected]
2
Programa de Pós-graduação em Ecologia e Conservação, Universidade Federal de Mato Grosso do Sul,
79070-900 Campo Grande, Mato Grosso do Sul, Brazil.
3
Instituto Epomex, Universidad Autónoma de Campeche, Av Augustin de Melgar y Juan de la Barrera
s/n, 24039 Campeche, México.
* Corresponding author
206 Ecotoxicology of Marine Organisms

the nests, since roots can invade nests and cause offspring mortality (Godfrey et al.
1995). Abiotic threats are related to strong rains, erosions, heat stress and climate
change (Hawkes et al. 2009). The eggs absorb moisture from the environment around
them and can change the morphological and embryonic and hatchling physiological
features (Hewavisenthi and Parmenter 2001). Such adsorbed substances may affect
the process of hatching nests and influence the development and survival of the
offspring since the early stages of embryonic development are the most vulnerable
to toxic exposure (Bishop et al. 1991, Alam and Brim 2000, Hewavisenthi and
Parmenter 2001).
In addition to effects on phenology, according to Walther et al. (2002), climate
change may have more direct impacts on thermally sensitive organisms, and under
future scenarios of climate warming, and given the conservation concern regarding
the sea turtle population, understanding of the potential effects of temperature
increases on sea turtle populations and their potential to cope with such changes is
important. Sea turtles have temperature-dependent sexual determination, where the
sex ratio is influenced by the temperature of the sand during incubation. In these
organisms, sex is determined by temperature in the middle third period of incubation
with males offspring occurring when the eggs are exposed to lower temperatures and
females under higher temperatures, within a thermal tolerance range of 25 to 35ºC
(Ackerman 1997, Hawkes et al. 2007).
Threats associated with human activities are related to population increase
(Marcovaldi and Marcovaldi 1999), hunt and egg collection (Márquez and Doi 1973,
Wetherall et al. 1993, Gallo 2006, Pupo et al. 2006), erosion and accretion of beaches
(Witherington and Martin 2000), artificial lighting (Marcovaldi and Marcovaldi
1999, Witherington and Martin 2000), fishing nets (Spotila et al. 2000, Heppel
et al. 2003) and shading (Van De Merwe et al. 2006).
Sea turtles assume a very important ecological role in trophic nets of the coastal
ecosystem, either as consumers (seaweed, seagrass, sponges, tunicates, crustaceans,
cnidarians) or as prey (eggs, youth and adults). Their movements during spawning
and feeding between different habitats (seagrass beds, coral reefs, ocean waters,
sandy beaches) are important in energy transfer and nutrient recycling (Bjorndal
1997). As species with slow growth and a long-life cycle, they are often used as
models for evolutionary studies of adaptation to different environmental conditions,
since they are extremely susceptible to the action of man in all phases of its life cycle
(Dodd and Dreslik 2008).
Organochlorine contaminants (OCs) cause a strong impact on the environment
due to three basic characteristics: environmental persistence, bioaccumulation and
high toxicity. These characteristics may affect the health of marine animals, including
sea turtles. The bioaccumulation of these pollutants in tissues and organs, influence
the growth and development of natural populations of sea turtles worldwide, leading
to mortality in various stages of development (egg, juvenile or adult) (Lake et al.
1994, von Osten 2005, de Andréa 2008, Marcovecchio and Freije 2013, Guerranti
et al. 2014).
These contaminants are transferred to the offspring via eggs, with serious
implications for embryonic development and health once outside the shell (Russell
 Contaminants Impact on Marine Turtle Populations Development 207

and Haffner 1999, Alava et al. 2006). Environmental contaminants of chemical origin
are chlorinated hydrocarbons synthesised by man, not occurring naturally in the
environment and can be divided into two groups: low molecular weight (industrial
solvents and chlorofluorocarbon (CFCs)) and high molecular weight (organochlorine
pesticides (OCPs) and polychlorinated biphenyls (PCBs)) (Clark 1992). The
bioaccumulation of these substances due to transfer in the food chain has become a
reason for concern about the health of these communities (Storelli et al. 2003, Alava
et al. 2006, da Silva et al. 2014). Because they are threatened with extinction, it is
extremely important to understand how sea turtle population responds to impacts
and what are the required conservation measures needed in a long-term perspective.
Heavy metals as Hg, Pb, Cd, Cr, As, Se, Fe, Mn, Zn, Cu, Ni and Co in tissues,
eggs, and blood can contribute to the contamination of sea turtle populations. Some
of them are essential to the life processes of organisms, but others are toxic, even at
low concentrations, they accumulated in organisms via food, respiratory pathways or
the dermal contact (Sakai et al. 1995, Becker et al. 2002, Innis et al. 2008, Jakimska
et al. 2011) that may affect, for example, the reproductive system, gastrointestinal,
respiratory, immunologic and renal (Godley et al. 1999, Sakai et al. 2000, Franzellitti
et al. 2004, Cortés-Gómez et al. 2014).

Sea Turtles Classification

The sea turtles are classified into two families: Cheloniidae and Dermochelyidae.
The first family has six species (C. caretta, C. mydas, N. depressus, E. imbricata,
L. olivacea, L. kempii), with a carapace covered with plates, variable in numbers for
each species. This family is characterized by having a strong skull and secondary
palate. Their edges are shaped as nonretractable fins covered with numerous small
plates; elongated fingers and tightly stuck for connective tissue. Their claws are
reduced to one or two fins (Pritchad et al. 1997). The second family includes only
the species D. coriacea in which instead of having a shell covered with plates, it
has a skin similar to leather. This species is characterized by an extreme reduction
of shell and cravats; development of a dorsal surface composed of a mosaic with
millions polygonal bones; lack of claws and plates in the shells (there are plates until
the juvenile stage); and a skeleton full of fat with extensive areas of vascularised
cartilage in the vertebras and fin joints. Its skull does not have nasal bones and its
surface mandibular is covered with keratin. This species is considered the largest one
among all the species (MOLL et al. 1979).
From seven species listed in the world, most of the species are distributed in
the tropical seas (C. mydas, E. imbricata, C. caretta, L. olivacea, D. coriacea), and
two species have restricted distribution, N. depressus in northeast of Australia and
L. kempii in the Gulf of Mexico and North Atlantic (IUCN 2016) (Table 1). Turtles
are identified for many characteristics such as inframarginal scutes (womb), costal
scutes (back), size and shape of the head, the shape of heads scutes and the formation
and colour of costal scutes.
Table 1. Main characteristics of sea turtles.
208

Species Key Features Habitat Feeding Spawning Length/ Weight Location Status Iucn
Caretta caretta large size of the shallow crustaceans, molluscs, from 110 to 110 cm Florida, South Africa, Vulnerable
(Linnaeus 1758) head, proportionally depth, jellyfish, hydrozoans, 130 eggs carapace/150 kg Greece, Turkey, Indian
larger than the other about 20 m fish eggs Ocean, Australia, Japan,
species United States, Mediterranean,
Brazil
Eremochelys hull is formed by shallow fish eggs, crustaceans, from 110 to 110 cm Atlantic, Indo-Pacific, the Critically
imbricata (Linnaeus overlapping scales, depth, molluscs, bryozoans, 180 eggs carapace/120 kg Caribbean Islands, Australia endangered
1766) corneum beak about 40 m coelenterates, and Oceanic Islands
hedgehogs Brazilian
Chelonia mydas color is gray-green; shallow puppies are omnivorous, from 110 to 120 cm Globally distributed and Endangered
(Linnaeus 1758) the belly is white depth, becoming herbivorous 130 eggs carapace/230 kg generally found in tropical
about 20 m in adulthood and subtropical waters
Ecotoxicology of Marine Organisms

Lepidochelys one of the smallest shallow fishes, molluscs, from 100 to 70 cm Central America, Mexico, Vulnerable
olivácea (Escholtz species, greenish depth, crustaceans, bryozoans, 110 eggs carapace/70 kg India, Suriname, French
1829) gray about 20 m jellyfish, fish eggs Guiana, Brazil
Dermochelys color is black depth ctenophores and from 80 to 180 cm French Guiana Pacific and Vulnerable
coriácea (Linnaeus with white spots, between cnidarians 90 eggs carapace/700 kg Costa Rica, Atlantic, South
1758) carapace has 50 and Africa
seven longitudinal 80 m
keels
Lepidochelys kempii endemic to the depth crabs, fishes, jellyfish, from 100 to 60–70 cm Gulf of Mexico Critically
(Garman 1880) Gulf of Mexico, about 50 m molluscs 120 eggs carapace/45 kg endangered
least of all species,
arribadas
Natator depressus carapace flattened, depth of squid; sea cucumbers; from 50 to 100 cm Australia Data deficient
(Garman 1880) oval and smooth about 10 m soft corals; molluscs 70 eggs carapace/90 kg
 Contaminants Impact on Marine Turtle Populations Development 209

Ecotoxicological Studies on Sea Turtles

Bioaccumulation of heavy metals in sea turtles

Sea turtle characteristics offer many specific advantages as pollution in dicators by
heavy metals and other kinds of marine environment contaminants, as their long
life spans and migratory cycles provide more time to integrate environmental
exposure. The concentration of a metal in the tissues of a predator may be higher
than in the tissues of its victims, because of bioaccumulation, resulting from trophic
transfer factor (DeForest et al. 2007, Jakimska et al. 2011). Metal bioaccumulation
in an animal depends on many biotic factors such as size, weight, age, gender, diet,
metabolism and the position in the food chain and abiotic factors such as metals
distribution in their environmental, salinity, temperature, pH, habitat type, water and
the interactions with other metals (de Souza 2008).
The organisms that live near the coast are the most susceptible to exposure
to all types of pollutants, due to proximity to areas of human occupation. The
bioaccumulation of contaminants in the tissues of these animals is considered
a major problem for growth and development factors (Jakimska et al. 2011). The
metals present in an animal may have no effect on its health, but they can also be
harmful. Some metals in small concentrations allow the metabolic system to function
normally, but if these levels are exceeded, they become toxic and can cause death
(Bianchini 2008).
Many works have been conducted on determining heavy metals in tissues and
sea turtle organs (Caurant et al. 1999, Godley 1999, Franzellitti 2004, Maffucci et al.
2005, Lam et al. 2006, Storelli et al. 2008, Andreani et al. 2008, Silva 2011). Agusa
et al. (2008) carried out analyses of concentrations of total arsenic (As) in liver,
kidney, muscle and stomach content of green turtles (C. mydas) and hawksbill turtle
(E. imbricata), where the concentrations in hawksbill turtles were higher than those
in green turtles, indicating that hawksbill turtles may have a specific accumulation
mechanism for As.
Anan et al. (2001), carried out analyses in the liver, kidney, and muscle to determine
the concentration of 18 trace elements in turtles in Japan. The accumulation features
of trace elements in the three tissues were similar between green and hawksbill turtles,
and no gender differences in trace element accumulation in liver and kidney were found
for most of the elements. Sakai et al. (1995), carried out analyses on sea turtle eggs to
determine the concentration of heavy metals concentration and thus develop a non-
lethal technique method. Also, Hg concentrations also were measured in keratinized
fragments (shells) and in internal tissues of green turtles, is the use of shell fragments
considered a non-invasive and non-lethal technique (Bezerra et al. 2012, 2013).
Lam et al. (2004) analyzed the concentrations of 19 individuals trace elements
and heavy metals in various tissues and organs to establish a baseline dataset for
chemical contaminants in green turtles nesting in South China, thus provide data
for comparison with previous studies. The results showed that As levels in different
turtle tissues of South China were similar to those reported from Japan and Hawaii.
Other studies focused in tissues and organs of sea turtle population focusing heavy
metals and organochlorine compounds concentrations in the Pacific Ocean (Sakai
210 Ecotoxicology of Marine Organisms

et al. 2000, Lam et al. 2004, Malarvannan et al. 2011), Mediterranean Sea (McKenzie
et al. 1999, Storelli et al. 2005, Storelli et al. 2014) and in the Atlantic Ocean (Casal
et al. 2007, Monagas et al. 2008, Oros et al. 2009, Rossi 2014).

Bioaccumulation of persistent organic pollutants (POPs) in sea turtles

POPs are chemicals that do not break down easily in the environment, being
resistant to chemical, photolytic and biological degradation (Clark 1992). As a
result of their generalized use, ability to be transported long-distance and due to
their bioaccumulative and biomagnifiable characteristics in the food chain, these
compounds are present in all the sea systems of the world (Hamann et al. 2010).
Bioaccumulation of these toxic substances has become a cause of concern for the
likelihood of its possible transfer through the food chain, causing an impact on many
species of animals in the marine environmental (Marcotrigiano and Storelli 2003,
Keller et al. 2004, Ogata et al. 2009). The pollution of the marine system is one of
the investigation priorities by researchers who work in areas related to turtle biology
and its conservation (Hamann et al. 2006).
Pursuant to a decision in the Stockholm Convention on Persistent Organic
Pollutants, control measures focused on reducing and eliminating releases of
12 POPs which are grouped into three categories. The list includes eight pesticides
(aldrin, chlordane, DDT, dieldrin, endrin, heptachlor, mirex, and toxaphene), two
industrial chemicals (PCBs and hexachlorobenzene, also used as a pesticide) and two
involuntary by-products from the industrial combustion process (dioxins and furans).
Organochlorine contaminants, such as OCPs and PCBs, due to their toxicity and
higher persistence in the environment may be absorbed and accumulated by living
organisms and through the bloodstream, can accumulate in the fat tissue (Corsolini
et al. 2000). Many studies have reported contamination levels in sea turtles in the
whole world in the past decade (Cobb and Wood 1997, Keller et al. 2006, Alava
et al. 2006, Orós et al. 2009, D’Ilio et al. 2011, Camacho et al. 2014, De Andrés
et al. 2016).
Gardner et al. (2003), measured organochlorine residues in three species of sea
turtles from the Baja California peninsula, Mexico. Seventeen of 21 organochlorine
pesticides analysed were detected in the 35 tissue samples, and PCBs were detected
in all but one of the nine turtles studied. The researchers concluded that the levels
of organochlorines detected in the study were low, possibly to the feeding habits of
the predominant species collected that are herbivorous or by their samples have been
collected in an unindustrialized region.
Studies that analysed pollutants concentration in sea turtles using tissues
collected from dead animals (Malarvannan et al. 2011, Guerranti et al. 2014, Storelli
et al. 2014), as adipose tissue (Lazar et al. 2011) or in more than one tissue (Corsolini
et al. 2000a, Miao et al. 2001, Gardner et al. 2003, Keller et al. 2004b, Monagas et al.
2008, da Silva 2009, Oros et al. 2009) are represented in the Table 2.
Certain levels of POPs may have negative effects on sea turtle health parameters,
as shown by some studies (Cobb et al. 1997, De Andres et al. 2016, Keller et al.
2004, Storelli et al. 2014). These contaminants are maternally transferred and may
adversely affect normal embryonic development and hatchling sizes. Guirlet et al.
 Contaminants Impact on Marine Turtle Populations Development 211

Table 2. Published studies on persistent organic pollutant concentrations in sea turtle tissues.

Location Species Tissue (n) ΣPCBs ΣDDTs Reference

Hawaii C. mydas liver/adipose 52.1 ± 664.7* Miao et al.
tissue (3) (2001)
Mexico L. olivacea liver (1) 58.1 10.4 Gardner
kidney (1) 63.4 18.3 et al. (2003)
adipose (1) 18.4 5.1
muscle (1) 16.9 8.6
Florida C. caretta adipose tissue 2.01 ± 2.96 452 ± 643 Keller et al.
(44) (2004b)
Japan C. mydas liver (5) 0.04 ± 0.92 0.008 ± 0.27 Malarvannan
E. imbricata liver (3) 2.5 ± 13 0.56 ± 9.0 et al. (2011)
C. caretta liver (2) 1.3 ± 3.8 2.0 ± 3.8
Eastern C. caretta yellow fat (27) 474 ± 547 112.5 ± 72.3 Lazar et al.
Adriatic Sea (2011)
Southeast C. mydas adipose tissue Adipose Sarmiento
Coast of (51)/hepatic (8.11 ± 13.72); (2013)
Brazil tissue (64) Hepatic
(4.25 ± 8.53)
Southeast C. caretta liver (12) Males (1255.39 ± Storelli et al.
Italy 702.10); Female (2014)
(1497.16 ± 1671.50)
PCBs (ng/g−1 lipid weight); *female.

(2010) examined the OCs, pesticides (DDTs and HCHs), PCBs and the temporal
variation of blood and eggs concentrations in leatherback turtles, where all OCs
detected in leatherback blood were detected in eggs, suggesting a maternal transfer
of OCs. This transfer was shown to depend on female blood concentration for ΣDDTs
and for the most prevalent PCB congeners since significant relationships were found
between paired blood–egg concentrations.
van de Merwe et al. (2010a) investigated the maternal transfer and effects of
POPs on embryonic development in a green sea turtle, C. mydas population in
Peninsular Malaysia. They found a significant correlation between increasing egg
POP concentration and decreasing hatchling mass: length ratio. POPs may, therefore,
have subtle effects on the development of C. mydas eggs, which may compromise
offshore dispersal and predator avoidance. Similar results have been found in the
study by Stewart et al. (2011), where concentrations were lower than those shown to
have acute toxic effects in other aquatic reptiles but may have sub-lethal effects on
the hatchling body condition and health.
It is for this reason that fat tissues and blood are being used to verify a possible
maternal transfer in female turtles as shown by some studies (Cobb et al. 1997,
McKenzie et al. 1999, Alava et al. 2006, D’Ilio et al. 2011, García-Besné et al.
2014). Blood samples were successfully used to measure organochlorine pollutant
concentrations, being considered a non-lethal collect technique in different species
of sea turtle (Table 3). Other analyses were also carried out through blood tests with
the Comet assay in erythrocytes of C. caretta resulting in a useful method to detect
Table 3. Published studies on persistent organic pollutant concentrations in blood and eggs of sea turtles.
212

Location Species Matrix (n) ΣPCBs ΣDDTs ΣChlordane ΣHCHs ΣOCPs ΣPOPs (OC+ ΣPAHs Reference
PCB+PBDE)
Merritt Island, Cc, Cm eggs 78 66* Clark and
Florida (Cm-2/Cc-9) Krynitsky (1980)
Merritt Island, Cc eggs (56) dw 99 Clark and
Florida Krynitsky (1985)
South Carolina Cc eggs (16) lw 1188 ± Cobb (1997)
311
Hernon Island, Cm eggs (15) 1.7 ± 0.3* Podreka et al.
Queensland (1998)
Mar Medit., Cc, Cm, eggs (1) Cc-89/ Cc-155/ McKenzie et al.
Escocia Dc Cm-6.1 Cm-4.3 (1999)
Ecotoxicology of Marine Organisms

Northwest Cc eggs (20) dw 753–800 Alam and Brim

Florida (2000)
North Carolina Cc blood (12) ww 5.14 ± 0.58 ± 0.30 0.26 ± 0.18 Keller et al.
3.95a (2004a)
North Carolina Cc blood (48) ww 5.56 ± 0.65 ± 0.68 0.22 ± 0.20 6.55 ± Keller et al.
5.28 6.14 (2004)
North Carolina Cc, Lk blood (Cc-44/ Cc-0.002 Cc-0.30 ± Cc-0.10 ± Keller et al.
Lk-10) lw ± 0.003/ 0.57/Lk- 0.15/Lk-0.07 (2004b)
Lk-0.98 ± 0.17 ± 0.15 ± 0.08
0.001
Southern Cc eggs (22) ww 144 ± 280 50.2 ± 92.4 25.5 ± 46.7 0.258 ± 0.508 Alava et al.
Flórida (2006)
North Carolina Cc blood (27) ww 6.25 ± 0.721 ± 0.253 ± Keller et al.
1.14 0.152* 0.048 (2006)
Cape Cod, Lk blood (31) ND Innis et al.
Massachusetts (n = 13)/ (2008)
<10 (n =5)*
Australia Cm eggs (10) PCB 18- trans-0.02 ± endolsufan van de Merwe et
0.016 ± 0.0004 I-0.20 ± al. (2009a)
0.0004 0.005

Malaysia Cm eggs (55) ww 0.47 ± 0.083 ± 0.057 ± 0.069 ± 0.009 0.39 ± 1.09 ± 0.43 van de Merwe et
0.083 0.018 0.009 0.04 al. (2009)

French Guiana Dc eggs/blood B-1.26 ± B-0.31 ± B-0.15 ±

(38) ww 0.71/E- 0.22/E-1.44 0.16/E- 0.41 Guirlet et al.
6.98 ± ± 1.26 ± 0.26 (2010)
5.02
Gulf of Lk, Cm blood (Cm-9/ Cm-0.53 Cm-0.12 Cm-0.01 ± Cm-0.014 ±
Mexico Lk-46) ww ± 0.70/ ± 0.11/Lk- 0.020/Lk- 0.011/ Swarthout et al.
Lk-0.42 ± 0.68 ± 0.65 0.11 ± 0.10 Lk-0.015 ± (2010)
0.36 0.014

Australia Cm blood (16) ww 0.68 ± 0.93 ± 0.17 van de Merwe et

0.15 al. (2010)
Malaysia Cm eggs (33)/ E-0.55 ± N.A. E-0.17 ± E-1.29 ± 0.07/ van de Merwe et
blood (11) ww 0.05/B- 0.007 **/ B-1.38 ± 0.19 al. (2010a)
0.58 ± B-0.50 ± 0.06
0.09
Southeastern Cc eggs–WF-11/ WF-32.4 WF-23.8 ± WF-20.8 ± WF-0.449 Alava et al.
United States EF-24/NC-9 ± 14.1/ 7.1/EF-136 9.6/EF-113 ± 0.017/EF- (2011)
Lw EF-372 ± 56/NC- ± 31/NC- 1.21 ± 0.49/
± 148/ 694 ± 251 375 ± 146 NC-3.15 ±
NC-1460 1.39
Contaminants Impact on Marine Turtle Populations Development

± 493

Table 3 contd. ...

213
...Table 3 contd.
214

Location Species Matrix (n) ΣPCBs ΣDDTs ΣChlordane ΣHCHs ΣOCPs ΣPOPs (OC+ ΣPAHs Reference
PCB+PBDE)
San Diego Bay Cm blood (20) 0.736 0.554 ± 0.915 ± 0.092 Komoroske
± 0.097* 0.073 (n=12) (yHCH) (2011)
Eastern, Cc blood (19) ww 0.005 ± 0.005 ± 0.002 Ragland et al.
Florida 0.002 (2011)
Eastern, Dc eggs (34) 8.45 ± 3.1 1.87 ± 0.4 < LOD Stewart et al.
Florida (2011)

Canary Cc blood IC-6.38 Camacho et al.

Islands/Cape (IC-162/ ± 4.95/ (2012)
Verde CV-205) CV-5.95
Ecotoxicology of Marine Organisms

± 4.88

Canary Cc blood (IC- IC-3.77 IC-0.78 ± IC-1.98 Camacho et al.

Islands/Cape 162/CV-197) ± 5.90/ 1.22/CV- ± 2.58/ (2013)
Verde CV-0.15 ± 0.21 ± 0.30 CV-0.77 ±
0.40 1.21
Cape Verde Cc blood (50) 0.17 ± 0.075 ± 0.095 ± 1.64 ± Camacho et al.
0.19 0.07* 0.08 1.14 (2013a)
South Carolina Cc eggs (10) lw b 659 ± 216 325 ± 185 94.9 ± 41.2 Keller (2013)

Cape Verde Cm, Ei Blood (Cm- Cm-0.53 Cm-0.33 ± Cm-12.06 Camacho et al.
21/Ei-13) ± 1.05/ 0.75/Ei-0.2 ± 0.61/ (2014)
Ei-0.19 ± ± 0.37 Ei-2.95 ±
0.56 2.90
Canary Islands Cc blood (56) 27.06 ± 1.16 ± 6.00 ± Camacho et al.
42.09 1.79 8.26 (2014a)
 Mexico Cm, Ei eggs/blood Cm-B-3.38 García-Besné et
(30) lw ± 3.08/E- al. (2014)
38.72 ± 0 //
Ei – B-3.55
± 3.52/E-
410.5 ±
379.7*

Hawaii Cm blood (13) 0.144 ± 0.013 ± 0.013 ± Keller et al.

wwc 0.165 0.008* 0.004 (2014)

Adriatic Sea/ Cc blood (MA- MA-1.15 ± MA-20.1 Bucchia et al.

Canary Islands 35/IC-30) 0.96/C-0.17 ± 30.89/ (2015)
± 0.33 IC-6.29 ±
4.54

Caribbean Cc, Cm eggs Cm-4.57 Cm-0.17 Cm-0.17 ± Dyc et al. (2015)

(Cm-11/Ei-4) ± 0.47/ ± 0.04/Ei- 0.007/
Ww Ei-1.72 0.19 Ei-0.47

Costa Rica Dc eggs (18) lw 102.3 ± 113.9 ± 96.5 De Andrés et al.

93.3 (2016)
Loggerhead (Cc), green (Cm), leatherback (Dc), Kemp’s ridley (Lk), olive ridley (Lo), hawksbill (Ei), flatback (Nd) sea turtles;
Mean ± SD in ng/g; * for only p,p′ -DDE; ** only yHCH; dw - dry weight; lw – lipid weight; ww - wet weight;
ND = not detected; N.A. = not availabre; < LOD = below detection; B – blood; E – egg; a - whole blood - technique A; b - Botany Bay Island, South Carolina; c - Kailua Bay;
MA - Adriatic Sea; IC- Canary Islands; CV = Cape Verde; WF = Western Florida; EF = Eastern Florida; NC = North Carolina.
Contaminants Impact on Marine Turtle Populations Development
215
216 Ecotoxicology of Marine Organisms

possible genotoxic effects, increasing knowledge about the state of ecotoxicological

healthy of this species (Caliani et al. 2014).

Biochemical Biomarkers in Sea Turtles

Biochemical biomarkers are essential for assessing health risks from exposure to
potentially chemical toxic products (Timbrell 1998). Biomarkers are used in a broad
meaning to include any measure which shows an integration between a biological
system and a potential danger which may be chemical, physical and biological
(Organization 1993). It may be used for various purposes depending on study aim and
on chemical exposure and can be classified into three types: exposure biomarkers,
affect biomarkers and susceptibility (Amorin 2003, von Osten 2005). The use of
biochemical biomarkers is being used by some studies, to analyse the level of several
indicators of oxidative and pollutant stress in the tissue and blood of the sea turtle
population (Yoshida 2012, Labrada-Martagón et al. 2011, Richardson et al. 2009).
According to Agarwal et al. (2005), oxidative stress is a situation created by
an imbalance between oxidants and antioxidants, in favour of oxidants. In certain
physiological, environmental, psychological and social situations, our organism goes
to alert state, when oxygen consumption and enzymatic activities of some oxygenases
and oxidases are high. Enzymes from oxidative stress represented by catalase (CAT),
superoxide dismutase (SOD), glutathione reductase (GR) and peroxidase (GPx) are
necessary for the maintenance of life as they are associated with the detoxification
process of compounds formed in living beings and are considered important for
allowing the survival of environmental impacted organisms (Cogo et al. 2009). An
analysis of the activities of these enzymes allows environmental control and works
as a contamination alert signal. Researchers applied these analyses in blood (Table 4)
and in several tissues of sea turtle population (Table 5).
A need for sublethal detection of toxic effects on the organisms led to biomarkers
indicators. They can be used as early indicators of environmental changes before
happening permanent damages happens in an ecosystem (Huggett et al. 1992).
Use of biochemical biomarkers in monitoring programs of sea turtle offers some
advantages since they are normally the first to be changed and show good sensibility
to contaminants, relative specificity and low-cost analysis when compared to
chemical analysis (Stegeman and Hahn 1994).
Glutathione-S-transferase (GST), for instance, develops a critical role in the
electrophiles exogenous and endogenous detoxification and acts just as well as
products of oxidative stress. Considering the knowledge available on the natural
and anthropogenic threats to sea turtles, the chemical and environmental effects and
biochemical mechanisms related such as GST detoxification catalyzed is probably
least understand. In the study carried out by Richardson et al. (2009), GST activity
was characterized in four species of sea turtles with varied life histories and feeding
strategies. Results of this study provided information about differences regarding
the biotransformation potential and the possible impacts of the studied contaminants
on the health and biotransformation ability of sea turtles. The results of the study
indicate that hawksbill, loggerhead, olive ridley, and green sea turtles possess
functional GST enzymes with similar kinetic parameters; however, rates of catalytic
Table 4. Published studies on antioxidant enzyme activity (GST; CAT; Mn-SOD; t-SOD; GR; GPx; U mg Hb−1) in blood of sea turtles.

Location Species Matrix (n) GST CAT Mn-SOD t-SOD GR GPx Reference
Baja Cm blood PA-0.99 ± 0.17*/ PA-223.29 ± 19.77*/ PA-0.27 ± 0.04*/ PA-0.43 ± 0.06*/ PA-12.39 ± 0.97*/ Labrada-
California, (PA-39/BM- BM-0.62 ± 0.11* BM-92.22 ± 14.07* BM-1.99 ± 0.37* BM-2.95 ± 0.32* BM-12.50 ± 2.68* Martagón
Mexico 11) et al.
(2011)
Brazil Cm blood(27)** 78.39 ± 5.73 3.66 ± 0.35 74.66 ± 7.56 0.65 ± 3.52 ± 0.16 Yoshida
0.12 (2012)
Southwest Lk blood (13) 48.0 ± 10.3*** Perrault et
Florida al. (2014)
Mean ± SD; *SE = standard error; **group 2 (n=10); ***SOD: U/ml; PA-Punta Abreojos; BM-Bahía Magdalena; kemp’s ridley (Lk), green turtle (Cm); GST (glutathione
S-transferase); CAT (catalase); t-SOD, Mn-SOD (total and mitochondrial superoxide dismutase); GR (glutathione reductase); GPx (glutathione peroxidase).
Contaminants Impact on Marine Turtle Populations Development
217
Table 5. Published studies on antioxidant enzyme activity (GST; CAT; t-SOD; U mg Hb−1) in tissue of sea turtles.
218

Location Species Tissue (n) Enzymes GST CAT t-SOD Reference

h (102.6 ± 18.1); h (8.9 ± 1.2); h (22.05 ± 3.7);
k (457.7 ± 49.4); k (63.6 ± 8.5); k (3.1 ± 3.4); li
Baja California, heart, kidney, liver, lung, GST*/SOD*/
C. mydas agassizii li (1032.2 ± 147.5); li (78.1 ± 12.2); (35.1 ± 4.7); lu Valdivia et al. (2007)
Mexico pectoral muscle (19) CAT*
lu (68.6 ± 9.2); lu (3.7 ± 0.4); (12.1 ± 1.5); pm
pm (210.3 ± 17.5) pm (5.6 ± 0.8) (24.1 ± 2.5)

Cc-7.52 (3.89)/
liver (Cc-4/
Baja California, Lo-4.68 (1.26)/
Cc, Lo, Ei, Cm Lo-4/Ei-3/ GST** Richardson et al. (2009)
Mexico Ei-6.16 (1.16)/
Cm-4)
Cm-7.70 (1.89)
Baja California,
Cc, Lo, Cm liver (Cc-4/Lo-4/Cm-4) PCB/GST *** Richardson et al. (2010)
Mexico
Ecotoxicology of Marine Organisms

*Mean ± S.E.M.; **Mean ± SD, catalytic efficiency (Vmax/Km) as nmol/min/mg protein/μM; ***no significant differences were observed in cytosolic GST activity;
Loggerhead (Cc), green turtle (Cm), olive ridley (Lo), hawksbill (Ei); GST (glutathione S-transferase); CAT (catalase); SOD (superoxide dismutase).
 Contaminants Impact on Marine Turtle Populations Development 219

activities using class-specific substrates show inter and intraspecies variation. GST
from the herbivorous green sea turtle shows 3–4.5 fold higher activity with the
substrate ethacrynic acid than the carnivorous olive ridley sea turtle.
Hematologic and biochemical standards analyses, including oxidative stress
indicators were carried out in a subspecies green turtle group of Oriental Pacific
(C. mydas agassizii) from a relatively undisturbed habitat in Mexico. Antioxidant
enzymes, SOD, CAT, and GST were determined as a defense mechanisms indicator
against reactive oxygen species (ROS) in the study carried out by Valdivia et al.
(2007), where overall levels of all found enzymes were within ranges reported for
other reptile species. Nevertheless, these authors suggest differences in oxidative
metabolism among tissues. For example, liver and muscle showed the highest
SOD activity, while kidney had the highest CAT and GST activities. This study has
achieved information on an oxidative stress indicators base, providing important
insights on the evaluation of health wildlife, especially for threatened species.
Perrault et al. (2014), analysed plasma brevetoxin concentrations and superoxide
dismutase (SOD) in sea turtles Ridley Kemp at Florida. No significant correlations
were observed between plasma brevetoxin concentrations and plasma proteins or
SOD activity, however, alpha-globulins tended to increase with increasing brevetoxin
concentrations in the bloom group, where Smaller (carapace length and mass) bloom
turtles had higher plasma brevetoxin concentrations than larger bloom turtles.
In order to determine potential contaminant effects in young turtles, C. mydas
from East Pacific, Labrada-Martagón et al. (2010), has analysed environmental
concentrations organochlorine of pesticides, trace metals and correlation to body
condition, measuring the activity of antioxidant enzymes and lipid peroxidation
levels, using spectrophotometric analyse, where the results indicate that correlations
between antioxidant enzyme activities and concentration of xenobiotics suggest
physiological sensitivity of East Pacific green turtles to chemicals. Analysis of
hematological and biochemical parameters, including oxidative stress indicators, is a
valuable tool in assessing wildlife health.

Hematological biochemical parameters in sea turtles

In the clinical investigation of reptiles, blood samples can be easily obtained and
are of great diagnostic value, where hematological evaluation has great importance.
Serum biochemistry represents an important tool for monitoring the health and
physiological status of sea turtles, due to the increasing need to evaluate their health
status, for their possible maintenance as healthy animals in captivity as well as
rehabilitation of free-living individuals (Pires et al. 2006).
The comparison of the data obtained is, however, limited due to possible
differences among populations, as well as variations in the techniques used and
factors such as age, size, seasonality, health, habitat, and diet, which may also
influence in hematological parameters. On the other hand, the descriptions of the
morphological characteristics of blood cells of marine chelonians are limited (Pires
et al. 2009).
Due to chemical pollution exposure, the sea turtle population face of numerous
environmental challenges, which may contribute to an immune system failure
220 Ecotoxicology of Marine Organisms

resulting in increasing diseases. Therefore, it is necessary to implement a complete

evaluation of the immune status of these threatened animals that are in danger of
extinction, and these hematological analysis are being successfully used in several
studies and are used to evaluate immune functions in a range of aquatic species
(Rousselet et al. 2013).
In the last years, blood sampling has been used as a non-destructive technique
to monitor and evaluate clinically the condition of the organism (Table 6). As in
the results obtained by Keller et al. (2004), the study provides the first evidence,
although strictly correlative, that OCs contaminant may be affecting sea turtle health.
Although the concentrations of OCs are relatively low compared with other species,
significant correlations between OCs levels and health indicators as homeostasis of
proteins, carbohydrates, and ions were observed, according to the above mentioned
study.
Hamann et al. (2006), presented comparative data on population demographics
and biochemical blood parameters for green sea turtles C. mydas in their foraging
grounds in the Gulf of Carpentaria, Australia. Based on the biochemical analysis of
blood, green turtles appeared to be healthy. However, their mean levels of glucose
and magnesium were generally lower than the ranges observed in other studies of
clinically healthy green turtles. This study was the first to describe the population
structure and provide a biochemical assessment of C. mydas for this population and
it will provide an important dataset for assessing future impacts such as cyclone
damage to seagrass communities.
Plasma samples collected from fed and fasting C. mydas turtles to
analyses Triglycerides, β-hydroxybutyrate, and glycerol concentration using
spectrophotometric assays, for determining recent feeding history using a single
blood sample (Hamann et al. 2006). Serum triglyceride and glycerol concentrations
decreased during fasting periods, while serum ß-hydroxybutyrate concentration
increased during fasts. For triglyceride and glycerol, this decrease apparently
occurred in the first five days of fasting and was unaltered by further fasting (Price
et al. 2013). Blood analyses were also measured in studies with L. kempii to measure
plasma corticosterone, glucose, and testosterone concentrations, to determine the
effects of acute handling stress in Cedar Keys, Florida, showing that mean plasma
corticosterone and glucose concentrations increased significantly with time (Gregory
et al. 2011). No significant difference was observed over time for mean testosterone
concentrations. Approximately half of the turtles demonstrated an increase in plasma
testosterone after 60 min of captivity while the others demonstrated a decrease
(Gregory et al. 2011).
As shown by Innis et al. (2010), health evaluations of 19 leatherback turtles in the
northwestern Atlantic were undertaken, where relatively high blood concentrations
of selenium and cadmium in all turtles were found. Blood samples too were
successfully used to measure heavy metals and metalloids concentration due to vital
functions carried out by blood cells and their susceptibility to intoxication (Innis
et al. 2008, Jerez et al. 2010, van de Merwe et al. 2010).
Hematological values and plasma biochemistry in the D. coriacea species were
determined in some studies (Deem et al. 2006, Perrault et al. 2012) and the OCs
concentrations found in leatherback in these studies were lower than concentrations
Table 6. Published studies on hematological parameters in blood matrices of sea turtles.

Species Location Blood analysis (n) Variables Reference

Cayman Hematological parameter PVC; Hb; RBC; WBC Wood and Ebanks (1984)
Islands (51)
Bahamas Blood biochemical (100)/ PVC; GLC; Na; K; Cl; CO2; IB; BUN; CR; BUN/CR; UA; Ca; P; TP; Alb; Bolten and Bjorndal (1992)
Packed cell volume (106) Glob; A/G; IC; TB; ALP; LDH; AST; ALT; CHOL; TC; Fe
Hawaii Morphology and blood Lymp; MO; Het; Eos; Bas; WBC Work et al. (1998)
cytochemistry (26)
Australia Blood biochemical CR; U; GLC; ALT; AST; CPK; TP; Alb; Glob; A/G; Ca; P; Mg; TSI; UA Hamann et al. (2006)
Brazil Blood biochemical (33) ALT; AST; ALP; CPK; CR; U; UA; TP; Alb; Glob; A/G; GLC; TC; P; Cl; Leite (2007)
Na; K;
Chelonia
mydas Brazil Hematological parameter Hct; RBC; Hb; RBC; MCH; MCHC; WBC; Mo; Lymp; Het; Eos; Bas, THB de Deus Santos et al. (2009)
(60)
Australia Hematological parameter/ PCV; THB; Lymp; Het; Eos; Mo; Bas; WBC/Alb; ALP; AST; Ca; Cl; CR; Flint et al. (2010)
Blood biochemical (290) CPK; Glob; GLC; LDH; Mg; P; K; TP; Na; TB; U; UA
USA Hematological parameter/ WBC; Het; Lymp; Eos; Bas; Mo; Hct/ ALP; ALT; CR; LDH; TP; Alb; Glob; Komoroske et al. (2011)
Blood biochemical (41) CHOL; GLC; Ca; P; Ca; K; Na; UA; U; Cl
Cayman Blood biochemical (5) GLYC; TRIG; BUTY Price et al. (2013)
Islands
Atol Hematological parameter/ Bas; Eos; Het; Lymp; Mo; WBC; PCV/CPK; ALP; AST; LDH; TP; Alb; McFadden et al. (2014)
Palmyra Blood biochemical (157) Glob; CHOL; TRIG; Ca; GLC; K; P; Na; UA

Table 6 contd. ...

Contaminants Impact on Marine Turtle Populations Development
221
...Table 6 contd.
222

Species Location Blood analysis (n) Variables Reference

USA Hematological parameter/ RBC; Hb; Hct; Het; Lymp; Mo; Eos; Azu; Bas; Het/GLS; TP; Alb; Glob; Keller et al. (2004)
Blood biochemical (48) A/G; BUN; UA; CR; SBR; AST; ALP; LDH; CPK;
USA Plasma Protein plasma protein fractions Gicking et al. (2004)
Electrophoresis (41)
Brazil Hematological parameter/ PVC; Hb; RBC; WBC; Het; Eos; Bas; Lymp; Mo; THB; TP Pires et al. (2006)
Plasma Protein (8)
Spain Hematological parameter Ery; Het; Eos; Lymp; Mo; THB Casal and Orós (2007)
(35)
Japan Hematological parameter/ RBC; WBC; PVC; Het; Bas; Lymp; Mo/SBR; GOT; GPT; GGT; ALP; Amy; Kakizoe et al. (2007)
Blood biochemical (5) LDH; CPK; TP; Alb; CR; BUN; UA; GLC; TC; CHOL; Ca; P; K; Na; Cl
Ecotoxicology of Marine Organisms

Spain Ultrastructural Ery; Het; Eos; Lymp; Mo; THB Casal et al. (2007)
characterization of blood
Caretta
cell (15)
caretta
Brazil Hematological parameter/ Hct; RBC; Hb; THB; MCH; MCHC; WBC; Het; Lymp; Eos/ TP; Alb; Glob; Pires et al. (2009)
Blood biochemical (27) A/G; GLC; CHOL; TC; CR; UA; AST; ALP
USA Hematological parameter/ PCV; RBC; WBC; Het; Lymp; Mo; Eos; Bas; Azu/GLC; Na; K; Cl; CO2; U; Deem et al. (2009)
Blood biochemical (83) CR; TP; Alb; Glob; CHOL; TC; Ca; P; UA; ALT; AST; LDH; CPK; Amy;
Lip; GGT
Spain Hematological parameter/ PCV; RBC; THB; WBC; Het; Lymp; Eos; Bas; Mo/TP; Alb; Glob; CR; UA; Casal et al. (2009)
Blood biochemical (103) U; SBR; CHOL; TC; GLC; Ca; AST; ALT; ALP; LDH
USA Hematological parameter/ PVC; WBC; Het; Lymp; Eos; Bas; Mo/TP; Alb; Glob; Alb; CR; UA; BUN; Rousselet et al. (2013)
Blood biochemical (85) GLC; SBR; CHOL; ALP; CPK; AST; ALT; GGT; Amy; Ca; Na; K; Cl
Africa Hematological parameter/ PVC; RBC; WBC; THB; Het; Lymp; Eos; Bas; Mo/TP; Alb; Glob; GLC; Camacho et al. (2013)
Blood biochemical (50) CR; UA; U; CHOL; SBR; TC; ALT; AST; ALP; LDH; GGT; CPK; Amy;
Lip; Na; K; Cl; Ca; P; Mg
 USA Hematological parameter Ery; Het; Eos; Lymp; Mo Cannon (1992)
(100)
Lepidochelys USA Hematological parameter/ WBC; Hct; Het; Lymp; Mo; Eos; Bas/ALT; AST; CPK; LDH; GGT; Alb; Innis et al. (2009)
kempii Blood biochemical (176) TP; Glob; SBR; BUN; CR; CHOL; GLC; Ca; P; Cl; K; Na; UA; A/G;
Mexico Hematological parameter corticosterone; GLC, and testosterone Gregory et al. (2011)
(44)
Africa Hematological parameter/ PCV; RBC; WBC; Het; Lymp; Mo; Eos/GLC; Na; K; CO2; BUN; CR; TP; Deem et al. (2006)
Blood biochemical (35) CHOL; TC; Ca; P; UA; ALT; AST; LDH; CPK; Amy; Lip; GGT
Canada Hematological parameter/ Lymp; Het; Eos; Mo; Bas; WBC/ALT; AST; ALP; LDH; CPK; Alb; CR; Innis et al. (2010)
Dermochelys
Blood biochemical (12) CHOL; GLC; Ca; P; K; UA; TP; Glob; Cu; Se; Hg; A; E
coriacea
USA Hematological parameter/ PVC; RBC; WBC; THB; Het; Lymp; Eos; Bas; Mo/TP; GLC; CR; UA; Perrault et al. (2012)
Blood biochemical BUN; CHOL; ALT; AST; ALP; LDH; CPK; Amy; Lip; Na; K; Cl; Ca; P;
(60–70)
Lepidochelys Mexico Hematological parameter/ WBC; Lymp; Neo/ TP; GLC; Alb; Glob; BUN; UA; AST; CPK; Na; K; Ca; Swarthout et al. (2010)
kempii/ Blood biochemical (Lk P; Cl
Chelonia (46); Cm (9)
mydas
PCV = packed cell volume; Hb = hemoglobin level; RBC = red blood cell count; WBC = white blood cell count; MCH = Mean corpuscular hemoglobina; MCHC = Mean
corpuscular hemoglobin concentration; Amy = Amylase; Alb = Albumin; ALP = Alkaline phosphatase; AST = Aspartate Aminotransferase; ALT = Alanine Aminotransferase;
BUN = Urea nitrogen; BUN/CR = creatinine ratio; Ca = Calcium; Cl = Chloride; CR = Creatinine; CPK = Creatine phosphokinase; CO2 = Carbon dioxide; CHOL =
Cholesterol; Fe = Iron; Glob = Globulin; GLC = Glucose; IC = Ionized calcium; IB = Ion balance; K = Potassium; Lip = Lipase; LDH = Lactic dehydrogenase; Mg =
Magnesium; Na = Sodium; P = Phosphorus; TP = Total protein; TB = Total bilirubin; A/G = Albumin/globulin ratio; TC = Triglycerides; TSI = Total serum iron; U = Urea;
UA = Uric acid; SBR = Bilirubin; GOT = glutamic oxaloacetic transaminase; GPT = glutamate pyruvate transaminase; GGT = gama -glutamyl transferase; Cu = copper; Se =
selenium; Cad = Cadmium; Hg = mercurium; A = vitamin A; E = vitamin E; Neo = Neutrophis; Ery = Erythrocyte; Lymp = Lymphocyte; Mo = Monocyte; Eos = Eosinophiles;
Bas = Basophiles; WCC = Total WBC; Hct = Hematocrit; Het = Heterophiles; THB= Thrombocytes; TRIG = triglycerides; BUTY = ß-hydroxybutyrate; GLYC = glycero;
Azu = Azuroph; Lepidochelys kempii (Lk); Chelonia mydas (Cm).
Contaminants Impact on Marine Turtle Populations Development
223
224 Ecotoxicology of Marine Organisms

measured in other sea turtles, which might be due to the lower trophic position (diet
based on gelatinous zooplankton) and to the location of their foraging and nesting
grounds. All OCs detected in leatherback blood were detected in eggs, suggesting
a maternal transfer of OCs in the studies carried out by Guirlet et al. (2008). This
method was also used by Páez-Osuna et al. (2010) to validate the maternal transfer
of lead (Pb) via egg-laying.
Ley-Quiñónez et al. (2011) determined baseline concentrations of zinc, cadmium,
copper, nickel, selenium, manganese, mercury and lead in the blood of 22 clinically
healthy, loggerhead turtles (C. caretta), captured for several reasons in Mexico. That
concluded that blood is an excellent tissue to measure in relatively non-invasive way
baseline values of heavy metals. In the study located in the Republic of Cape Verde,
almost all of the samples showed detectable levels of the 11 elements (Cu, Mn, Pb,
Zn, Cd, Ni, Cr, As, Al, Hg and Se), strengthening the usefulness of blood for the
monitoring of the levels of contaminating elements and their adverse effects on blood
parameters in sea turtles (Camacho et al. 2013a).
Blood tests were carried out in sea turtles from the C. caretta species with
a goal to obtain values from the hemogram standards and plasma protein of this
species (Camacho et al. 2013, Rousselet et al. 2013). Nonlethal fat biopsies and
blood samples were collected from live turtles for OC contaminant analysis, and
concentrations were compared with clinical health assessment data, including
hematology, plasma chemistry, and body condition. Blood concentrations of
∑Chlordanes were negatively correlated with red blood cell counts, hemoglobin,
and hematocrit, indicative of anemia. Positive correlations were observed between
most classes of OC contaminants and white blood cell counts and between mirex and
∑TCDD-like PCB concentrations and the heterophil: lymphocyte ratio, suggesting
modulation of the immune system. These correlations suggest that OC contaminants
may be affecting the health of loggerhead sea turtles (Keller et al. 2004).
A total of 5 millilitres of blood was collected from captive C. caretta sea turtles
in order to obtain hemoglobin and plasma protein parameters, whereof the analysed
variables such as erythrocyte count, mean globular volume, the mean globular
hemoglobin and total and differential counts of leukocytes presented different values
for the species in question when compared with the literature consulted (Pires et al.
2006, 2009). Hematologic characteristics and plasma chemistry values of juvenile
loggerhead turtles (C. caretta), was analysed, and the results of these studies were
used to establish a hematology and blood chemistry baseline for captive juvenile
loggerhead turtles and will aid in their medical management (Casal et al. 2007,
Kakizoe et al. 2007, Deem et al. 2009). de Deus Santos et al. (2009), has set young
C. mydas turtles hematological values in captivity, concluding his results vary in
relation to other authors, due to different methodologies with the same species.
Hamann et al. (2006) found mean levels of glucose and magnesium were generally
lower than the ranges observed in other studies of clinically healthy green turtles.
Wood and Ebanks (1984), analysed distinguishable blood cells, and Differential
counts of white blood cells showed age dependent differences and the hemoglobin
level and packed cell volume of the green sea turtle apparently increase with age.
Similar to the study reported by Bolten and Bjorndal (1992) where there was a
significant correlation of the body size to the blood parameters measured. Thus,
 Contaminants Impact on Marine Turtle Populations Development 225

research dealing with this issue will have increasing importance due to the use of
this non-lethal technique, which helps in with wildlife contaminant monitoring. The
information obtained will be used to monitor any changes caused by potentially
inorganic pollutants and may help in the clinical diagnosis of diseases that affect
these sea turtles (D’Ilio et al. 2011, Ikonomopoulou et al. 2011, Cortés-Gómez et al.
2014, McFadden et al. 2014, Villa et al. 2015).

Conclusions
Sea turtle population conservation strategies must include research, strengthening
environmental education, local management strategies, interaction with local
fishermen, encouraging turtle safe release when accidentally caught in their fishing
net, discussion programs for wildlife conservation threatened by extinction, and
voluntaries training courses. Worldwide there are many studies that address the
issue of contaminants and their effects on sea turtles and the conservation plans
that include management programs for the protection of nests and nesting females,
the nest translocation to protected hatcheries and protection against poaching. It
is also important to emphasize the information exchange between science, policy,
and public participation in the design and implementation of conservation actions.
Because contaminants are maternally transferred to eggs or by bioaccumulation,
information about contamination by heavy metals, POPs or PAHs in nesting females
are crucial for assessing the state of health of marine turtles. Also, help to evaluate and
prevent malformations in embryos and hatchlings. Despite there being conservation
programs for sea turtles, still, there is still a need to strengthen these programs and
to raise awareness in the communities surrounding major beaches in order to involve
them in the work of conservation protection in the long term.

Acknowledgements
This work was supported by Coordination for the Improvement of Higher Education
Personnel (CAPES Brazil) (1201/2013-01).

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 Index

A L
algae 19 lanthanides 16, 19
animals 20
avoidance 2–12 M
Marine 158–175
B
marine organisms 26, 34
bacteria 18, 19 Marine pollution 12
bioaccumulation 28–30, 33, 185 Mercury 185, 186, 194, 198
biomagnifications 185 Mercury tolerable intake 186
Biomarkers 216
brevetoxins 42, 43, 68, 69, 74, 75 N

C non-forced exposure 2–4, 6, 7, 9–12

chemical contaminants 25–28, 33, 35 P

climate change 25–30, 32, 33, 35
clinical plasma chemistry 224 paralytic shellfish toxins 42, 43, 45, 46, 55
coastal and transition environments 174 pharmaceutical 158–167, 169, 170, 172–175
coastal systems 92, 99, 118 phytoplankton indicators and biomarkers 89–92,
contaminants 205–207, 209, 210, 216, 224, 225 118
Phytoremediation Bioaccumulation factors
D (BSAF) 138–140
plants 18, 19
Detoxification mechanism 139, 141, 145 preference 2, 3, 7, 8
diarrhetic shellfish toxins 42, 43, 72
domoic acid 42, 43, 63, 64, 68 R

E REE 16–21
Reference dose 186, 197, 198
effects 158–160, 162–175
environmental disturbance 1, 2, 11 S
estuaries 89–99, 102, 103, 114, 116, 117
exposure 160, 162, 164–173, 175 Sea turtle 205–212, 215–221, 224, 225

F T

Fish consumption 185–193, 195, 197–200 therapeutic class 160, 165, 166, 174
Food webs 42–44, 61, 63, 68, 69 toxicity 16–21, 26–28, 30, 32–35
trace element pollution 90, 102, 103, 111, 114
H
Y
habitat selection 2, 3, 7–9, 11, 12
yttrium 16
 About the Editors

Bernardo Duarte graduated in 2008 in Cellular Biology and Biotechnology (Faculty

of Sciences of the University of Lisbon), followed by a Master degree in 2011 in
Management and Conservation of Natural Resources (University of Évora) and by
the PhD in Biology and Ecology (Faculty of Sciences of the University of Lisbon) in
2016. He is an Investigator of the Faculty of Sciences of the University of Lisbon and
of MARE – Marine and Environmental Sciences Centre. In the last 15 years developed
his research mostly focusing on contaminant biogeochemistry, ecotoxicology and
ecophysiology of marine organisms. This work resulted in more than 70 publications
in international ISI Journals (h-index 18), 13 book chapters and over 1,300 citations.
He is routinely reviewing papers for several international journals in these fields of
expertise and acts as a reviewer for several funding bodies in USA, South Africa and
Europe. He is presently Reviewing Editor (Marine Pollution section) at Frontiers in
Marine Science and Frontiers in Environmental Science. He currently leads several
projects focusing on bio-optical techniques and its application in the development of
new ecotoxicological tests.
Isabel Caçador graduated in Biology in 1978, finished her PhD in 1995 and her
Habilitation in 2008. She is presently a Professor in Plant biology Department at the
Faculty of Sciences of the University of Lisbon, Portugal and a senior investigator
at MARE – Marine and Environmental Sciences Centre. Her major expertise is in
marine and estuarine ecology and ecotoxicology. Her 40 years of expertise in the
field have resulted in 200 publications, of which 121 publications in international
peer reviewed journals, resulting in over 3,100 citations and h-index of 29. In these
areas she has obtained international recognition in the arbitration of scientific articles
and books, collaborations in European projects, establishment of cooperation actions,
invitation to participate in international juries of merit to award prizes and also, have
seen award winning work of which was co-author. She participated in more than 50
national and international projects, being coordinator of 16. Isabel Caçador is also the
author of about 170 international communications (40 by invitation). Additionally,
she supervised more than 15 PhD thesis. She is regularly reviewing papers for over
50 international journals and has been evaluator for several funding agencies.
Color Plate Section
240 Ecotoxicology of Marine Organisms

Chapter 7
Antidepressants (64) Antibiotics (48)

Analgesics and NSAIDs (58)

Antihypertensives (34)

Lipid regulators (33)

Anticonvulsants (40)

Fig. 2. Tree map representation of studies on the effects of pharmaceutical exposure in coastal and
marine organisms per therapeutic class, biological endpoints and major taxonomic groups. Therapeutic
classes are antidepressants, analgesics and non-steroid anti-inflammatories (NSAIDs), anticonvulsants,
antibiotics, antihypertensives and lipid regulators. Biological endpoints and respective abbreviations are
molecular changes, accumulation (accumul), development (develop), mortality, reproduction (repro)
and behavior (behav). Major taxonomic groups and respective abbreviations are fish, tunicates (tun),
echinoderms (echi), mollusks (moll), crustaceans (crust), rotifers (rot), annelids (ann), nematods (nem),
cnidarians (cni), algae (alg) and bacteria (bact). Individual box sizes are proportional to number of entries,
and total number of entries per therapeutic class is shown (n). Note that a single study may have multiple
entrances per therapeutic class (total number of studies 124).
 Color Plate Section 241

Chapter 8

Fig. 10. Fish meal week–1 in relation to the [MeHg] in fish (µg g–1) for: (a) RfD of 0.1 µg MeHg kg bw–1
day–1 and (b) ½ RfD (0.05 µg MeHg kg bw–1 day–1).