International Journal of

Environmental Research
and Public Health

Review
The Role of the Ecotoxicology Applied to Seafood as a Tool for
Human Health Risk Assessments Concerning Polycyclic
Aromatic Hydrocarbons
Julia Vianna de Pinho 1,2,3 , Paloma de Almeida Rodrigues 1,4,5, * , Ivelise Dimbarre Lao Guimarães 1 ,
Francielli Casanova Monteiro 1 , Rafaela Gomes Ferrari 1,4,6 , Rachel Ann Hauser-Davis 7
and Carlos Adam Conte-Junior 1,2,3,4,5,8,9

1 Center for Food Analysis (NAL), Technological Development Support Laboratory (LADETEC), Federal
University of Rio de Janeiro (UFRJ), Cidade Universitária, Rio de Janeiro 21941-598, RJ, Brazil;
[email protected] (J.V.d.P.); [email protected] (I.D.L.G.);
[email protected] (F.C.M.); [email protected] (R.G.F.);
[email protected] (C.A.C.-J.)
2 National Institute of Health Quality Control, Oswaldo Cruz Foundation, Rio de Janeiro 21040-900, RJ, Brazil
3 Graduate Program in Sanitary Surveillance (PPGVS), National Institute of Health Quality Control (INCQS),
Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro 21040-900, RJ, Brazil
4 Laboratory of Advanced Analysis in Biochemistry and Molecular Biology (LAABBM), Department of
Biochemistry, Federal University of Rio de Janeiro (UFRJ), Cidade Universitária,
Rio de Janeiro 21941-909, RJ, Brazil
5 Graduate Program in Veterinary Hygiene (PPGHV), Faculty of Veterinary Medicine, Fluminense Federal
University (UFF), Vital Brazil Filho, Niteroi 24220-000, RJ, Brazil



6 Agrarian Sciences Center, Department of Zootechnics, Federal University of Paraiba,


Areias 51171-900, PB, Brazil
Citation: de Pinho, J.V.; 7 Environmental Health Assessment and Promotion Laboratory, Instituto Oswaldo Cruz, Fundação Oswaldo
Rodrigues, P.d.A.; Guimarães, I.D.L.; Cruz, Rio de Janeiro 21040-360, RJ, Brazil; [email protected]
Monteiro, F.C.; Ferrari, R.G.; 8 Graduate Program in Food Science (PPGCAL), Institute of Chemistry (IQ), Federal University of Rio de
Hauser-Davis, R.A.; Conte-Junior, Janeiro (UFRJ), Cidade Universitária, Rio de Janeiro 21941-909, RJ, Brazil
9 Graduate Program in Chemistry (PGQu), Institute of Chemistry (IQ), Federal University of Rio de
C.A. The Role of the Ecotoxicology
Applied to Seafood as a Tool for
Janeiro (UFRJ), Cidade Universitária, Rio de Janeiro 21941-909, RJ, Brazil
* Correspondence: [email protected]
Human Health Risk Assessments
concerning Polycyclic Aromatic
Abstract: Background: Polycyclic aromatic hydrocarbons (PAHs) are persistent pollutants routinely
Hydrocarbons. Int. J. Environ. Res.
detected in aquatic ecosystems. It is, therefore, necessary to assess the link between deleterious
Public Health 2022, 19, 1211. https://
marine biota PAH effects, especially in commercialized and consumed animals, environmental health
doi.org/10.3390/ijerph19031211
status, and potential human health risks originating from the consumption of contaminated seafood
Academic Editor: Paul B. Tchounwou products. Thus, this review seeks to verify the relationships of ecotoxicological studies in determining
Received: 15 December 2021 effect and safety concentrations on animals routinely consumed by humans. Methods: A total of
Accepted: 18 January 2022 52 published studies between 2011 and 2021, indexed in three databases, were selected following the
Published: 22 January 2022 PICO methodology, and information on test animals, evaluated PAH, and endpoints were extracted.
Results: Benzo(a)pyrene and phenanthrene were the most investigated PAHs in terms of biomarkers
Publisher’s Note: MDPI stays neutral
and test organisms, and mussels were the most evaluated bioindicator species, with an emphasis on
with regard to jurisdictional claims in
published maps and institutional affil-
reproductive responses. Furthermore, despite the apparent correlation between environmental PAH
iations. dynamics and effects on aquatic biota and human health, few assessments have been performed in a
multidisciplinary manner to evaluate these three variables together. Conclusions: The links between
human and environmental sciences must be strengthened to enable complete and realistic toxicity
assessments as despite the application of seafood assessments, especially to mussels, in bioassays,
Copyright: © 2022 by the authors. the connection between toxicological animal responses and risks associated with their consumption
Licensee MDPI, Basel, Switzerland.
is still understudied.
This article is an open access article
distributed under the terms and
Keywords: PAH; petroleum derivates; organic compounds; marine ecosystem; toxicity assessments;
conditions of the Creative Commons
environmental health; marine biota; fish products; mussels
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Int. J. Environ. Res. Public Health 2022, 19, 1211. https://doi.org/10.3390/ijerph19031211 https://www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW 2 of 26
Int. J. Environ. Res. Public Health 2022, 19, 1211 2 of 25

1. Introduction
1. Introduction
Human, environmental,
Human, environmental, and animal health
and animal health are
are all
all intrinsically
intrinsically correlated
correlated according
according
to the One Health concept [1]. This concept, coined in 2004 through
to the One Health concept [1]. This concept, coined in 2004 through the integration of the integration of
human medicine and veterinary medicine, seeks to incorporate
human medicine and veterinary medicine, seeks to incorporate environmental health environmental health
bases to
bases to establish
establish links
links between
between ecosystem
ecosystem effects
effects and
and thethe triggering
triggering of of conditions
conditions that that
affect human, animal, and environmental health [2]. Within this
affect human, animal, and environmental health [2]. Within this view, it becomes clear view, it becomes clear
that multidisciplinary
that multidisciplinary assessments
assessments are are aa valuable
valuable tool
tool inin the
the environmental
environmental and and Public
Public
Health fields.
Health fields. As
As such,
such, human
human health
health risk
risk assessments
assessments concerning
concerning contaminated
contaminated seafood seafood
consumption should
consumption shouldnot notbebe dissociated
dissociated fromfrom animal
animal healthhealth risk assessments.
risk assessments. In this
In this context,
context, increasing chemical pollution levels worldwide have
increasing chemical pollution levels worldwide have significantly increased concerns significantly increased
concerns regarding
regarding seafood contamination.
seafood contamination.
aromatichydrocarbon
Polycyclic aromatic hydrocarbon (PAH)(PAH) contamination
contamination in marine in marine aquatic
aquatic environ-
environments,
ments, in particular,
in particular, is of significant
is of significant concern as concern as these compounds
these compounds are highly are highly
persistent
persistent
and result and result in deleterious
in deleterious contamination contamination
effects that effects that pose
pose potential potential
risks to bothrisks
animalto both
and
animal and
human healthhuman
[3–6].health [3–6].
Different human activities such as fuel, wood and coal burning, industrial effluents
discharges, oil oil extraction,
extraction, and andshipshiptraffic
traffic[7–11]
[7–11]comprise
comprise thethe
primary
primary PAH PAH sources,
sources,re-
sponsible
responsibleforforthe theinput
inputofofabout
about200 200compounds
compoundsbelonging
belonging to to the
the PAH
PAH group
group into the
environment [12]. [12]. High
HighPAH PAHconcentrations
concentrations have
have beenbeen detected
detected in sediments,
in sediments, rivers,
rivers, and
and
fishesfishes [3]. Moreover,
[3]. Moreover, these these compounds
compounds are known
are known to biomagnify
to biomagnify along along
the foodthe chain
food
chain
[13,14],[13,14],
reachingreaching
humans humans
through through
seafoodseafood consumption,
consumption, potentially
potentially resulting resulting
in several in
several deleterious health effects to both animals and humans,
deleterious health effects to both animals and humans, such as decreased such as decreased immune
function,
function, kidney
kidneyand and liver damage,
liver damage, as well as carcinogenicity
as well as carcinogenicityand genotoxicity [15]. Despite
and genotoxicity [15].
their
Despiteglobal
theirdistribution [16], 16 of
global distribution these
[16], 16 ofsubstances, in particular,
these substances, require require
in particular, prioritypriority
control
due to their
control due tohigh toxicity
their (Figure 1).
high toxicity (Figure 1).

Figure 1.
Figure 1. Chemical
Chemical structure
structure of
of the
the 16
16 priority
priority polycyclic
polycyclic aromatic
aromatic hydrocarbons
hydrocarbons according
according to
to the
the
United States Environmental Protection Agency.
United States Environmental Protection Agency.
Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW 3 of

Int. J. Environ. Res. Public Health 2022, 19, 1211 3 of 25

Due to their high molecular stability, PAHs tend to remain and circulate betwee
environmental compartments for extended periods of time [17–19]. However, the
Due to their high molecular stability, PAHs tend to remain and circulate between envi-
present low compartments
ronmental or no solubility in water,periods
for extended which,ofalongside their
time [17–19]. resistance
However, to biodegradatio
they present low
makes
or no them susceptible
solubility to adsorption
in water, which, ontoresistance
alongside their suspended particles and/or
to biodegradation, makessedimentatio
them
susceptible
[20], creatingtoa adsorption onto suspended
direct exposure route to particles
benthic and/or sedimentation
biota (Figure 2). [20], creating a
direct exposure route to benthic biota (Figure 2).

Figure
Figure2. 2.PAH
PAHdynamics
dynamics in inmarine
marine ecosystems.
ecosystems. (1) Atmospheric
(1) Atmospheric PAH deposition
PAH deposition and
and (2) oil (2) oil spil
spills
(3) (3)
that may
that maycontaminate aquaticenvironments,
contaminate aquatic environments, (4) followed
(4) followed by adsorption
by adsorption to suspended
to suspended particulateparticula
matter,
matter,and
and(5)(5)sedimentation processes.
sedimentation processes. (6)(6) Benthonic
Benthonic organisms
organisms are exposed
are exposed to PAHthe
to PAH through through th
dietary route, (7) which then bioaccumulate and biomagnify throughout the food
dietary route, (7) which then bioaccumulate and biomagnify throughout the food chain, (8) reachingchain, (8) reachin
high levels
high levelsininlarger
larger fish consumed
fish consumed byby humans,
humans, characterizing
characterizing a potential
a potential human
human health health risk.
risk.

AsAsPAHs
PAHs display the ability to interact with lipid cell membranes, due to their
display the ability to interact with lipid cell membranes, due to the
lipophilicity, they display bioaccumulative properties, and high levels have been reported
lipophilicity, they display bioaccumulative properties, and high levels have been reporte
for different trophic niches, i.e., zooplankton, mussels, fish, aquatic, and terrestrial mam-
formals
different
[21–23].trophic niches,indicate
Several studies i.e., zooplankton, mussels,
negative toxic effects fish, PAH
following aquatic, and interrestri
exposure
mammals [21–23]. groups,
several taxonomic Severalwhich
studiesmay,indicate negative
in turn, alter toxicdynamics
ecosystem effects following
as differentPAH
organ-exposur
in ismic responses
several taxonomicto intoxication,
groups, whether
which may,physiological,
in turn,morphological,
alter ecosystem or biochemical,
dynamicsmay as differen
indicate links between contaminants and ecological effects [24].
organismic responses to intoxication, whether physiological, morphological, o In this regard, in addition
to determining the toxic effects of chemical substances on aquatic organisms themselves,
biochemical, may indicate links between contaminants and ecological effects [24]. In th
human health risks should also be considered [25], as contaminated seafood items are
regard,
considered one of thetomost
in addition determining
severe human thehealth
toxichazards.
effects of chemical substances on aquat
organisms themselves, the
As aforementioned, human
One Health health risks
concept should
indicates also be
that human and considered
animal health [25], a
contaminated seafood
and environmental items are
conditions areconsidered
significantlyone of the most
interrelated [2]. severe
However, human health
obtaining an hazard
equilibrium among these the
As aforementioned, three parameters
One is still a challenge,
Health concept indicateswhich can be adequately
that human and animal healt
andassessed by employing
environmental interdisciplinary
conditions fields in evaluations,
are significantly interrelatedsuch [2].
as ecotoxicology and
However, obtaining a
risk assessments [2,26]. While ecotoxicology makes it possible to assess the impacts of
equilibrium among these three parameters is still a challenge, which can be adequatel
contaminants on organisms in advance by conducting controlled tests with standardized
assessed by employing
test organisms, interdisciplinary
risk assessments fields inwhether
aim to determine evaluations, such
the level as ecotoxicology
of exposure to a an
risk assessments [2,26]. While ecotoxicology makes it possible to assess
certain contaminant to which a population is exposed to for their entire lives, considering the impacts o
contaminants on organisms in advance by conducting controlled tests with standardize
test organisms, risk assessments aim to determine whether the level of exposure to
certain contaminant to which a population is exposed to for their entire lives, considerin
the average life expectancy of the exposed population, is considered safe, by assessin
Int. J. Environ. Res. Public Health 2022, 19, 1211 4 of 25

the average life expectancy of the exposed population, is considered safe, by assessing
xenobiotic concentrations, bioavailability, intoxication pathways, and degree of toxicity, and
are paramount in developing strategies to improve public health status [26]. Ecotoxicology
can, for example, address a series of potential final effects, termed endpoints, such as the
lethal concentration 50% (LC50) of a certain substance, capable of killing 50% of exposed
individuals, or a 50% effect concentration (EC50), which assesses changes in parameters
other than death in exposed organisms, such as growth and reproduction. The application
of different assays, however, evaluating more primary endpoints such as biochemical,
cytological, and histological effects, has been highlighted in recent years, allowing for
assessments on interaction effects between contamination sources [23]. Concerning human
risk assessments, the hazard quotient (HQ) considers factors such as the frequency of
contaminated food consumption, exposure duration, contaminant concentrations, and
maximum permissible reference doses [27,28]. Furthermore, due to the carcinogenic effects
attributed to PAHs, the USA EPA (1991) also establishes a cancer risk assessment model
based on the average lifespan of exposed humans and estimates the probability of an
individual developing cancer over their life history [29].
In this context, this study aims to verify the applicability of ecotoxicological data
concerning contaminated seafood in human health risk assessments by employing a
systematic review.

2. Materials and Methods

2.1. Focus Question
The population, intervention, and comparisons were defined according to the PICO
method. The central question of this study was established as “Do published ecotoxico-
logical studies on PAHs, employing marine biota, enable the assessment of human health
risks associated with contaminated seafood consumption?” This was answered through
sub-items such as “Which organisms have been used in toxicity tests and under which
exposure levels?”, “Which parameters and endpoints were evaluated in these studies?”,
“What is the degree of risk concerning human health by the consumption of marine biota,
and is this risk directly related to the effects observed in contaminated animals?”

2.2. Information Sources

The applied descriptors employed in the data search were selected from the DECS/MeSH
platform. The search was carried out between August and September 2021 at the “Web
of Science”, “PubMed”, and “Embase” databases. To direct the inquiry, four “Search
Components” were defined:
• Search Component 1 (SC1): Population: “Arthropoda” OR “Arthropods” OR “Crus-
tacean” OR “fish” OR “Fish products” OR “Fish culture” OR “Clams” OR “Mussel”
OR “Bivalvia.”
• Search Component 2 (SC2): “Ecotoxicology” OR “Environmental toxicology” OR
“Effects” OR “Toxicity” OR “Toxicology” OR “Bioassay” OR “Lethal concentration”
OR “Effect concentration” OR “Environmental health” OR “Marine toxicity” OR
“Aquatic toxicity” OR “Human health” OR “Human risk”.
• Search Component 3 (SC3): “pah” OR PAH OR “Polycyclic aromatic hydrocarbons”
OR “Polynuclear aromatic hydrocarbon.”
• Search Component 4 (SC4): AND NOT (“detection” OR “collected”)
Following four sequential stages, two authors (J.V.A.d.P. and P.d.A.R.) first conducted
a preliminary selection of the identified abstracts and paper titles independently. Abstracts
were removed if the papers did not investigate associations between the search components.
Articles whose abstracts suggested applying bioassays using water, oil, or sediment contam-
inated by PAHs or that performed the quantification of these elements in animals sampled
from potentially impacted environments were also removed. Studies whose abstracts did
not mention animals or that demonstrate the application of mathematical models were
also removed.
Int. J. Environ. Res. Public Health 2022, 19, 1211 5 of 25

The search was limited to English, and publishing dates were set between 2011 and
2021. Editorials, letters, reviews, mini-reviews, and M.Sc. dissertations and Ph.D. theses
were excluded.
Considering that the use of the descriptors “PAH” and “Risk” could be linked to
the evaluation of oil exploration with metal contamination and the general definition of
dangers from PAH poisoning without actually analyzing these compounds or performing
risk assessments following application models (i.e., USEPA models), some studies were
excluded due to the following:
• Ecological risk assessments based on PAH distributions in the aquatic or
marine environment;
• Oil spills or oil products and their effects on biota;
• Epidemiological studies to determine the incidence/prevalence of diseases associated
with the consumption of contaminated fish;
• Development or applications risk assessment methodologies disregarding the effects
presented by investigated organisms (animals, plants, and humans) subjected to
specific PAH exposure concentrations.
In addition, two screenings were conducted for article selection and extraction to
ensure the removal of studies that did not meet the established criteria, such as the specific
application of a PAH compound or the use of fish products. In the first selection stage, article
suitability was evaluated through reading the abstracts and those fitting the following
inclusion criteria:
• Assessments concerning PAH effects on test organisms under controlled conditions;
• Assessments indicating the tested organism, the PAH, test/effect concentration, and
the evaluated endpoint;
• Evaluations concerning animals sampled from the environment or kept in the labora-
tory for laboratory exposures;
• Indications of assay time and affected biomarkers or physiological or
morphological alterations.
In the second screening (extraction), the articles selected in the first stage were re-
evaluated in their entirety to confirm the initial selection and restrict the analysis to seafood
interest following the previously mentioned exclusion criteria.
The results are reported in agreement with the Preferred Reporting Items for Sys-
tematic Review and Meta-Analyses statement by the SR management StArt tool. After
selection, the end analyses of the results were plotted as column graphs using the ggplot2
package, available in the R software (version 4.0.4) (R Foundation for Statistical Computing,
Vienna, Austria) [30].
Possible bias sources include inclusion/exclusion criteria, the chosen database, date,
language, number of articles, and article types selected for this study.

3. Results
A total of 2202 articles were found, as follows: 1213 in Web of Science, 535 in Embase,
and 454 in PubMed. From this total, 565 duplicated assessments were excluded (n = 1637),
as well as studies whose aim was the evaluation of matrices containing PAHs, such as crude
oil, water, contaminated soil, and sediments or even paper, that were not aligned with our
objective (n = 1360). The other 277 papers were fully evaluated in order to disregard studies
with missing data or that did not comply with the criteria established for the “Extraction”
stage (see reasons in the methodology). Of these, 204 were discarded, leaving 73 studies for
analysis. (Figure 3).
From this, 1 study did not make clear the object of analysis (reagent or environmental
sample), and another 12 studies were also excluded because their aim was to test equipment
calibration, develop better quantification and identification methods, and/or apply mathe-
matical models for dispersion calculations and risks using secondary data. Subsequently,
32 extra articles containing essential information such as the dynamics and behavior effects
Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW 6 of 26
Int. J. Environ. Res. Public Health 2022, 19, 1211 6 of 25

behavior effects of PAHs on aquatic biota, factors that influence toxicity, and data
of PAHs on aquatic biota, factors that influence toxicity, and data concerning human health
concerning human health risks were added to the database (Figure 3).
risks were added to the database (Figure 3).

Figure 3. Flowchart indicating the literature search methodology and article selection performed in
the present review.
review.

From thethe5252included
included articles, we we
articles, identified onlyonly
identified studies concerning
studies 6 of the6 16
concerning ofpriority
the 16
PAHs (pyrene (PYR), phenanthrene (PHE), fluoranthene (FLU), benzo(a)pyrene
priority PAHs (pyrene (PYR), phenanthrene (PHE), fluoranthene (FLU), benzo(a)pyrene (BaP),
benzo(a)anthracene (BaA), anthracene (ANT)) with fullerene (C60), and two
(BaP), benzo(a)anthracene (BaA), anthracene (ANT)) with fullerene (C60), and two articles articles regis-
tered ecotoxicological effects from tests performed with complex hydrocarbons
registered ecotoxicological effects from tests performed with complex hydrocarbons mixtures
employingemploying
mixtures both the aforementioned PAHs and naphthalene
both the aforementioned PAHs and(NAP) and chrysene
naphthalene (NAP)(CHR)
and
(Figure
chrysene 4a).
(CHR) (Figure 4a).
Thirty-one animal
Thirty-one animal species
species were
were identified,
identified, grouped
grouped into
into five
five categories:
categories: fish,
fish, shrimp,
shrimp,
bivalves, and crabs/Decapoda, and distributed as animals obtained in
bivalves, and crabs/Decapoda, and distributed as animals obtained in field samplings (n field samplings
(n = 51)
= 51) or or maintained
maintained in the
in the laboratory
laboratory (n (n = 3).
= 3). These
These assessments
assessments were
were carried
carried outout in
in 19
19 countries. The bivalve Mytilus galloprovincialis was the most studied
countries. The bivalve Mytilus galloprovincialis was the most studied test organism (Figuretest organism
(Figure
4b), and4b), andpublished
China China published
the mostthe most studies
studies (n = 32) (n = 32) (Figure
(Figure 4c). 4c).
Res. Public Health 2022, 19, x FOR PEER REVIEW 7 of 26
Int. J. Environ. Res. Public Health 2022, 19, 1211 7 of 25

a.

b.

c.

Figure
Figure 4. Frequency 4. Frequency
of assessed of assessed
PAHs PAHs (a),
(a), species species
(b), and(b), and country
country (c) obtained
(c) obtained ininthis
this systematic
systematic review.
review. 4. Discussion
4.1. PAH Assessments
4. Discussion A total of 274 articles were excluded because, although laboratory exposures with
standardized
4.1. PAH Assessments test organisms were performed, the test agent was a solution obtained from

A total of 274 articles were excluded because, although laboratory exposures with
standardized test organisms were performed, the test agent was a solution obtained from
spill oils or similar (dilbit). In this regard, it is important to note that factors such as
compound bioavailability, solubility, volatilization, and half-life can alter PAH toxicity in
Int. J. Environ. Res. Public Health 2022, 19, 1211 8 of 25

spill oils or similar (dilbit). In this regard, it is important to note that factors such as
compound bioavailability, solubility, volatilization, and half-life can alter PAH toxicity in
aquatic media [31], and the chemical composition of crude oil or oil products may not
reflect the effect of each individual PAH [32].
Phenanthrene was established herein as causing the most significant damage to the
survival of both fish and mussels, while anthracene and naphthalene exposure result in
sublethal effects, such as damage to genetic structures and delayed or reduced reproduction.
Govers et al. (1984) [33] indicated a correlation between the PAH partitioning coefficient
(KOW) and toxicity, while Carls and Mador (2009) [34] report that high molecular weight
soluble PAHs become more toxic and persistent in nature due to extended half-lives.
However, in general, PAHs with lower molecular weights and, thus, fewer aromatic rings
(2–3) tend to cause more significant acute toxicity effects. In contrast, PAHs with higher
molecular weight and more than five rings tend to generate more chronic effects, with
significantly higher carcinogenic potential [35].
In view of these data, it is paramount to define the base toxicity of each PAH in com-
plex mixtures in order to evaluate environmental PAH dispersion, according to chemical,
physical, and toxicological characteristics properties [36] (Table 1). This allows for the
extrapolation of ecotoxicological results based on the amount of each toxic compound in
complex matrixes, such as oil [37,38], by employing mixing models [39].

Table 1. Physical and chemical PAH properties that influence environmental PAH dispersion. Source
(Pubchem, 2012) [40].

Partition Coefficient
Name Structural Formula Molecular Weight Vapour Pressure (25 ◦ C)
(Log KOW)
Anthracene C13 H8 180.20 3.58 5.7 × 10−5
Fluorene C13 H10 166.22 4.18 6.00 × 10−4
Phenanthrene C14 H10 178.23 4.46 1.21 × 10−4
Methylphenanthrene C15 H12 192.25 4.97 1.50 × 10−5
Methylanthracene C15 H12 192.25 - 5.34 × 10−6
Pyrene C16 H10 202.25 4.88 4.50 × 10−6
Fluoranthene C16 H10 202.25 5.16 9.22 × 10−6
Benzo(a)antracene C18 H12 228.3 5.76 2.1 × 10−7
Crysene C18 H12 228.3 5.73 6.23 × 10−9
Benzo(b)fluoranthene C20 H12 252.3 5.78 (0.0015 mg L−1 ) 5.00 × 10−7
Benzo(k)fluoranthene C20 H12 252.3 6.11 9.65 × 10−10
Benzo(e)pyrene C20 H12 252.3 6.44 5.70 × 10−9
Benzo(a)pyrene C20 H12 252.3 6.13 5.49 × 10−9
Indeno[1,2,3-
C22 H12 276.3 6.70 1.3 × 10−10
cd]pyrene
Dibenz(a,h)anthracene C22 H14 278.3 6.50 9.55 × 10−10
Benzo(g,h,i)perilene C22 H12 276.3 6.63 (9.41 mg L−1 ) * 1.0 × 10−10
Coronene C24 H12 300.4 - 2.17 × 10−12
* Solubility in water at 25 ◦ C.

4.2. Volatility
PAH volatility influences the occurrence of these compounds in different environ-
mental compartments, with more volatile substances becoming more representative in
the atmosphere due to burning processes. Naphthalene is one of the most volatile PAHs,
leading to uncertainties concerning its toxicity in aquatic organisms [41], as it is easily lost
Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW

Int. J. Environ. Res. Public Health 2022,is19,

the1211simplest
non-linear PAH, and this type of arrangement leads to differences 9 of 25 b
forces of attraction between the atoms that make up these molecules, making them
or less stable [43].
Despite the
by volatilization andgeneric
sorptionrelationship between
processes. However, mostmolecular weight and
PAHs are relatively volatility, PAH
non-volatile
and poorly soluble in the aquatic environment, leading to high chances
as naphthalene may be harder to apply in bioassays due to the loss of these ag of incorporation
into bottom sediments [42].
photodegradation adsorption, mainly volatilization [44]. Similar losses hav
As indicated in Table 1, low molecular weight PAHs (LMW) also tend to exhibit the
observed
lowest KOW, forexcept
BaP for[14], PHE, ANT
isomerism cases,[45],
such and
as thefluoranthene [46]. BaP 500 and 1000 µg
linear isomers 1-methylphenanthrene
trigger histological and
and 1-methylanthracene DNA
(Figure damage
5). Both exhibitinthe
the
samebivalve Mytilus
structural formulagalloprovincialis
and have one with
methyl
of radical (CH
exposure 3 ) butalthough
[47], different angulation and doubleup
concentrations bondtoarrangements.
10-fold lower For example,
already aff
while anthracene (ANT) is a linear PAH, phenanthrene (PHE) is the simplest non-linear
detoxification system of these bivalves [48]. At 100 µg L , anthracene and phenan
−1
PAH, and this type of arrangement leads to differences between forces of attraction between
alter boththat
the atoms enzymatic
make up theseand non-enzymatic
molecules, makingantioxidants
them more or lesssuch as AChE
stable [43]. and triglycerid

Figure 5. Example of two linear isomers (a) 1-methylphenanthrene and (b) 1-methylanthracene, with
Figure
changes 5. Example
in the of two linear
angular orientation of theisomers (a) 1-methylphenanthrene
molecule and and (b)
position of double bonds indicated 1-methylant
in red.
with changes in the angular orientation of the molecule and position of double bonds indi
red. Despite the generic relationship between molecular weight and volatility, PAHs such
as naphthalene may be harder to apply in bioassays due to the loss of these agents by
photodegradation adsorption, mainly volatilization [44]. Similar losses have been observed
4.3. Molecular
for BaP Weight
[14], PHE, Influence
ANT [45], and fluoranthene [46]. BaP 500 and 1000 µg L−1 can trigger
histological
PAHsand areDNA damage
often in the bivalve
categorized Mytilus to
according galloprovincialis
the number within 72 h of ex- rings a
of aromatic
posure [47], although concentrations up to 10-fold lower already affect the detoxification
molecular weight (HMW, 4–6 rings) and low molecular weight (LMW, 2–3
system of these bivalves [48]. At 100 µg L−1 , anthracene and phenanthrene alter both
compounds [49]. HMW antioxidants
enzymatic and non-enzymatic compounds are
such usually
as AChE more associated
and triglycerides [45]. with geno
because of their higher chronic effects [50]. This occurs due to their degradation pro
4.3.HMW
as Molecular Weight
PAHs areInfluence
more persistent, directly correlated to fugacity and vaporizatio
PAHs are often
due to their higher categorized
KOW [31]. according to the number of aromatic rings as high molecu-
lar weight (HMW, 4–6 rings) and low molecular weight (LMW, 2–3 rings) compounds [49].
The effects of acute and long-term exposure to PAHs in marine animals as a f
HMW compounds are usually more associated with genotoxicity because of their higher
of theireffects
chronic molecular mass
[50]. This are commonly
occurs generalized
due to their degradation between
processes, assevere
HMW PAHs physiological
are d
and
moremortality
persistent, for LMW
directly compounds
correlated andand
to fugacity cancer, although
vaporization rates[51]
dueindicated that PAHs
to their higher
KOW [31].
act as direct carcinogens, with their metabolites being, instead, responsible f
The effects of acute and long-term exposure to PAHs in marine animals as a function of
deleterious process. Thus, HMW PAH genotoxicity is, in fact, caused b
their molecular mass are commonly generalized between severe physiological damage and
biotransformation processes.
mortality for LMW compounds andAccording to Baird
cancer, although et al. (2005)
[51] indicated that PAHs[52],
docancer associate
not act as
PAHs is due to the
direct carcinogens, witheffects of products
their metabolites being,ofinstead,
the reaction of for
responsible thethis
K-region epoxide o
deleterious
compounds,
process. Thus,such HMWasPAH 7-methylbenz[a]anthracene
genotoxicity is, in fact, caused (7-MeBa)
by their and BaP with DNA, al
biotransformation
processes. According to Baird et al. (2005) [52], cancer
the form of these products may vary depending on the assessed PAH. associated with PAHs is Indue
fish, for ex
to the effects of products of the reaction of the K-region epoxide of these compounds,
both LMW and HMW compounds are routinely detected, although LMW compou
such as 7-methylbenz[a]anthracene (7-MeBa) and BaP with DNA, although the form of
generally
these productspresent in higher
may vary concentrations
depending on the assessed[53]PAH.
as these animals
In fish, easilyboth
for example, metaboliz
compounds compared to other organisms such as mollusks [54]. Assessments con
LMW and HMW compounds are routinely detected, although LMW compounds are
generally present ingenotoxicity
PAH-associated higher concentrations
in animals[53] must,
as thesetherefore,
animals easily metabolize
consider that these
following e
exposure, i.e., contact with assimilable PAH forms; these compounds undergo act
in animal metabolism by oxidative processes (epoxide formation) that prom
formation of adducts between these substances and DNA, leading to mutations [5
Int. J. Environ. Res. Public Health 2022, 19, 1211 10 of 25

compounds compared to other organisms such as mollusks [54]. Assessments concerning

PAH-associated genotoxicity in animals must, therefore, consider that following effective
exposure, i.e., contact with assimilable PAH forms; these compounds undergo activation in
animal metabolism by oxidative processes (epoxide formation) that promote the formation
of adducts between these substances and DNA, leading to mutations [52].
The retrieved papers in our systematic search indicate that anthracene (ANT) is a
common contaminant in estuaries and coastal areas, having been reported at concentrations
of up to 35 µg L−1 , despite being reported as not resulting in lethal effects in fish T. carolinus
(Florida pompano) at this concentration. However, this estuarine fish feeds on molluscs,
crustaceans, and other small fish [55], potentially bioaccumulating this PAH, indicating
that even as an LMW compound, ANT can affect organisms at environmentally realistic
concentrations. This PAH has also been associated with increased enzymatic activity,
such as catalase (CAT), and protein concentrations, such as glutathione (GSH) in Chanos
chanos (milkfish) tissues [56]. This fish typically inhabits the coastal zone and may even
enter streams due to a diversified diet, ranging from phytoplankton to invertebrates and
small fish. Furthermore, ANT has been reported as lethal for milkfish at 0.30 mg L−1
after 96 h of exposure [56], 8.57-fold higher than that for T. carolinus [55], demonstrating
different tolerances between species and test concentrations, which can be explained by
their different trophic chain positions.
In turn, phenanthrene (PHE), also an LMW PAH; it can linearly affect the cardio-
vascular system of Navaga cod fish (Eleginus navaga) through changes in cellular ion
fluxes, resulting in cardiac dysfunction at doses ranging between 1 and 30 µmol L−1 [57],
also leading to hormetic spermatogenesis responses in the fish Sebastiscus marmoratus
(sea ruffe) [11], with lower PHE concentrations inhibiting the development of testes, while
a higher concentration (6 µg L−1 ) was less inhibitory concerning testicular development.
The fish S. maroratus (sea ruffe) is also sensitive to BaP, PYR, and PHE at environmen-
tally relevant concentrations, undergoing expressive mortality at 0.1 µg L PHE exposure
after eight days in tanks with water and PAH. Although BaP is an HMW compound and
the most addressed PAH in the studies included in this systematic review, mortality was
not indicated as the primary endpoint for this substance in this species, even the following
exposure at 1 µg L−1 , with 10% of the exposed animals dying, while for PHE, this same
percentage was exceeded in a 10-fold lower concentration, although BaP affects certain
endpoints, such as craniofacial skeletal development, more than PHE [58]. Although these
facts are not explained based on eventual differences of these two PAH mechanisms, this
indicates that molecular mass alone cannot define PAH lethality, although the mechanism
for these effects was not investigated in the retrieved assessments.
The importance of assessing primary toxicity endpoints, such as histological and
biochemical changes, as a response to PAH toxicity becomes clear as lethality is not
always observed.

4.4. Lipophilicity
Lipophilicity is one of the main physicochemical PAH properties that result in seafood
contamination [46,59], as lipid affinity makes PAHs likely to interact with lipid contents,
such as animal cell membranes. For example, Honda et al. [60] point out that increased
lipid content in marine fish may indicate the specific accumulation of PAH in particular
tissues, altering the lipid metabolism, resulting in endoplasmic reticulum dysfunctions,
among others.
Concerning benzo(a)pyrene (HMW PAH), several effects of this PAH on DNA have
been reported, although the selected articles in our systematic review assessed mainly
cytological and histological alterations as well as Phase I biotransformation biomark-
ers. Concerning anthracene, an LMW PAH, two studies evaluated fish (Trachinotus caroli-
nus—Florada pompano and Chanos chanos—milkfish) and one, the Mediterranean mussel
M. galloprovincialis. In the fish assessments, DNA alterations were verified employing the
Int. J. Environ. Res. Public Health 2022, 19, 1211 11 of 25

Comet assay [55], and survival rates were assessed [56], while oxidative stress biomarkers
and filtration rates were the evaluated endpoints concerning the bivalve study [44,61].
In a comparative analysis for these two PAH, Palanikumar et al. [56] reported that
C. chanos exposed to anthracene and benzo(a)pyrene (BaP) exhibited a directly proportional
increase in accumulation levels as a function of PAH concentrations. Furthermore, BaP
was found to be more toxic to C. chanos compared to anthracene, confirming the linearity
between molecular mass, lipophilicity, and toxicity, as even at 3-fold higher concentrations,
ANT exposure resulted in minor survival effects, acetylcholinesterase activity alterations,
and Phase 2 detoxification enzymes compared to the higher mass compound [50,61].

4.5. Environmental Chemistry and PAH Dispersion

Environmental factors can significantly alter PAH toxicity [62]. Although heat and light
incidence can increase their degradation [63], higher temperatures favor increased toxicity
of certain PAHs such as phenanthrene, naphthalene, and anthracene due to increased
kinetics, accelerating intoxication processes [64]. Moonfish exposed to anthracene, for
example, exhibited increased mortality due to higher water temperatures and oxygen
levels [65].
Furthermore, environmental characteristics such as geomorphology, environment,
and ecology also significantly influence ecotoxicological endpoints. For example, in Gulf
killifish, increased mortality and development alterations in the larval stage were observed
as a function of temperature and hypoxia, with both variables highlighted as potential
detoxification pathway inhibitors [66]. Climatic conditions, solar incidence, and seasonal
changes can also generate toxicity differences concerning xenobiotic dispersion in aquatic
media [28].
HMW PAHs are more abundant in the atmosphere and high-temperature environ-
ments due to combustion processes [50]. High temperatures also favor sedimentary particle
sorption and desorption as well as toxicity [67]. Furthermore, environmental temperatures
can influence individual tolerance through physiological strategies developed for acclimati-
zation to extreme temperature conditions, such as extreme cold. Arctic cod, for example,
present low gastric evacuation rates and high lipid assimilation efficiency, which favor PAH
tissue distribution [68].
Figure 6 displays the different taxonomic groups evaluated by region/country and
applied temperature ranges in the studies retrieved in this systematic review.
Some differences between response patterns were observed concerning the wide PAH
concentration ranges applied at different temperatures (Figure 5). In studies conducted in
China, for example, Ruditapes philippinarum—Manila clam—(n = 5 studies) was subjected
to a range of 12 to 25 ◦ C, while Chlamys farreri (n = 4 studies) underwent bioassays from
15 to 31 ◦ C. (Figure 5). Despite the high amplitude for this condition, only one study
recorded significant temperature effects on PAH toxicity [68].
The bivalve Ruditapes decussatus can respond differently to the same chemical com-
pounds (FLU, PHE, and PYR) at different temperatures (20, 24, 28, and 30 ◦ C), even altering
their defense mechanisms [69]. A significant effect on total hemocyte counts was observed
only for FLU at different temperatures and concentrations, while PYR at 20 ◦ C also in-
creased this parameter, confirming that PAH effects depend on both the evaluated PAH
and temperature.
PAH effects may also vary with oxygen and salinity variations [66,70], with decreased
salinity values increasing toxic PAH effects [70]. Several studies indicate that salinity fluctu-
ations promote a need for physiological compensation, such as gill activity. These structures
are responsible for ion regulation and comprise the first barrier against xenobiotics, and
salinity variations can result in PAH damage to the gill epithelium.
 environmental temperatures can influence individual tolerance through physiological
strategies developed for acclimatization to extreme temperature conditions, such as
extreme cold. Arctic cod, for example, present low gastric evacuation rates and high lipid
assimilation efficiency, which favor PAH tissue distribution [68].
Figure 6 displays the different taxonomic groups evaluated by region/country and
Int. J. Environ. Res. Public Health 2022, 19, 1211 12 of 25
applied temperature ranges in the studies retrieved in this systematic review.

Figure
Figure6.6. Different
Different taxonomic groups by
taxonomic groups byregion/country
region/countryand
andapplied
appliedtemperature
temperature ranges
ranges in the
in the
studies evaluated in our systematic review.
studies evaluated in our systematic review.

Despite variations in temperature, salinity was constant in the selected studies, ranging
between 30% and 37.5% without restriction regarding species or location. Only two papers
differed, one establishing a salinity value of 20% when assessing blue mussel M. edulis [46]
and another evaluating the effect of increased salinity values on the Crassostrea brasiliana
transcriptome, which reported that PHE exhibited decreasing levels in tanks containing
only water, even without animals, probably due to its low solubility in saline solutions [49].
In turn, oxygen was determined in 6 of the 32 analyzed articles, ranging from
6.5 to 8.6 mg L−1 [43,45,56,59,71–73]. Besides its possible participation in PAH trans-
formation processes, oxygen plays a vital role in osmoregulation, and its consumption can
be interrupted by toxicant action in gill tissues, affecting osmoregulation [43].

4.6. PAH Toxicity in Mixtures

Laboratory exposures of aquatic organisms to combined contaminants (xenobiotic
cocktails) and adsorbed to other materials, such as plastic or carbon nanotubes and mi-
croplastics, may lead to additive, synergistic or antagonistic effects [48], indicating environ-
mentally realistic situations [74,75], sometimes at concentrations below individual contam-
inant lowest observed effect concentration (LOEC) [76]. For example, PAH absorbed by
these materials became bioavailable and toxic to the algae Pseukichneriella subcapitata [77]).
As indicated in Table 2, nine of the selected articles exposed PAHs as mixtures to
marine organisms. Of these, four involved PAH coexposures, two were based on binary
mixtures and two on complex mixtures, in addition to studies conducted employing metals
(copper—Cu), nanoparticles (TiO2 NP), microplastics (MPs), and organic pesticides (i.e.,
dichloro-diphenyl-trichloroethane, DDT).
Int. J. Environ. Res. Public Health 2022, 19, 1211 13 of 25

Table 2. Binary and complex PAH exposure mixtures reported in the selected articles in this
systematic review.

PAH Compound
Common Total Mixture
Species Compound Concentrations in Concentrations in Reference
Name Concentrations
Single Treatments Single Treatments
100 µg L−1
Mytilus gallo- Mediterranean PHE 100 µg L−1
- (50 µg L−1 [45]
provincialis Mussel ANT 100 µg L−1
each)
NAP,
12.64 µg kg−1
PHE,
8.38 µg kg−1
dibenzothiophene
0.58 µg kg−1
Gadus morhua Atlantic cod (DBT), - - [78]
1.45 µg kg−1
PYR
1.93 µg kg−1
BaP,
15.03 µg kg−1
FLU
NAP, 10,600 mg L−1
ANT, 10,200 mg L−1
PHE, 7500 mg L−1
Scophthalmus
Turbot FLU, - - 13,300 mg L−1 [44]
Maximus
PYR, 3300 mg L−1
CHR, 15,500 mg L−1
BaP 5200 mg L−1
1000 µg L−1 of
C60 + 5 µg L−1
of BaP
1000 µg L−1 of
C60 +
Mytilus gallo- Mediterranean BaP 5, 50, and 10, 100, and
50 µg L−1 of [48]
provincialis Mussel C60 100 µg L−1 of BaP 1000 µg L−1 of C60
BaP
1000 µg L−1 of
C60
+100 µg L−1 of
BaP
10 µg L−1 of
Mytilus gallo- Mediterranean BaP
10 µg L−1 of BaP 10 µg L−1 of Cu BaP + 10 µg [62]
provincialis Mussel (Cu)
L−1 of Cu
100 nM of PYR
PYR
Lates calcarifer Barramundi 100 nM of PYR 100 MP L−1 + [73]
MPs
100 particles L−1
100 µg L−1 of
FLU +
FLU 50, 10 µg L−1 of 1000 MP mL−1
Mytilus edulis Blue mussel 100, 1000 MP mL−1 [46]
MPs FLU 50 µg L−1 of
FLU +
100 MP mL−1
20 µg L−1 BaP +
0.2 mg L−1
BaP TiO2 NP
Mytilus edulis Blue mussel 20 µg L−1 of BaP 0.2, 2 mg L of TiO2 [79]
TiO2 NP 20 µg L−1 BaP +
2 mg L−1
TiO2 NP
20 µg L−1
BaP
Perna viridis Green mussel 10 µg L−1 of BaP 10 µg L−1 of DDT (10 µg L−1 of [80]
DDT
each one)
Int. J. Environ. Res. Public Health 2022, 19, 1211 14 of 25

Interactions between BaP and fullerene (C60) generated antagonistic effects concerning
genotoxic and proteome expressions, significantly increasing DNA strand breaks following
three days of exposure to 0.1 mg L of a mixture of both compounds when compared to
the control and individual treatments [48]. On the other hand, individual and combined
PHE and ANT treatments led to total thiol status alterations, which may result in physio-
logical and morphological mussel gill alterations [45]. The observed antagonistic effects
between BaP and C60 cannot be explained by B[a]P sorption onto C60 but rather by the
free radical scavenging property of C60, as single and combined exposures resulted in
common response mechanisms of transcriptomic alterations related to genotoxic mech-
anisms [48]. Concerning the mixture between PHE and ANT, the authors indicate that
the absence of observed additive effects may be due to exposure adaptation during the
7-day exposure period.
Song et al. [80] recorded changes in the levels of some metabolites associated with
the single exposure to 10 µg L−1 BaP in Perna viridis mussel gills, with some amino acids
from energy metabolism, such as BCAAs, dimethylamine, and dimethylglycine, signifi-
cantly reduced while proteins involved in cytoskeleton organization, catabolic protein, and
apoptosis were increased. However, no metabolic changes in a 1:1 mixture with DDT, a
pesticide of global concern due to its high persistence in environmental compartments,
were observed, suggesting antagonistic effects between BaP and DDT that may be linked
to their different metabolic pathways [80].
However, for exposure to BaP + C60, ANT + PHE, and BaP + DDT mixtures, the
concentration of the second toxicants was set as a constant, modifying only the investigated
PAH concentrations. This experimental design is relevant for understanding the inter-
action between substances but can also be restrictive and non-environmentally relevant.
Thus, non-additive and antagonistic interactions may occur at different doses intervals
than those tested in the laboratory. For example, different sets of proteins and comple-
mentary modes of action were observed when analyzing mussel gills exposed to BaP, Cu,
and their mixture [62]. Unlike other studies, no BaP accumulation was observed, which
may be associated with competitiveness between the tested compounds and consequent
greater metal absorption, although the interaction between PAH and Cu leads to common
response mechanisms.

4.7. Factors That Can Affect the Toxicological Response of Animals to PAH
Laboratory tests aim to verify contaminant effects on test organisms in a controlled
manner. However, the response may vary between different taxonomic groups due to
varying tolerances and a significant variety of laboratory protocols. Thus, this topic will
address the main differences in conducting ecotoxicity bioassays with PAHs in the papers
selected in this systematic review.
Two studies in our systematic review [46,73] (Table 2) aimed to evaluate the effect
of combined exposure to PAHs and microplastics. Pyrene (PYR) is commonly associated
with adverse outcomes in fish, but when associated with microplastics (MPs), sublethal
damage or no effects have been reported in studies when each compound was tested
individually [73]. However, the mixture between these agents quickly affected juvenile
barramundi (Lates calcarifer) predatory performance [73]. In turn, single fluoranthene
exposure resulted in bioaccumulation in both the gills and digestive gland of the blue
mussel (Mytilus eduli), which was not observed when adding MPs [46], even though MPs
are considered an important vector for many pollutants.
Interestingly, mussels previously kept in clean water subjected to FLU and MP coexpo-
sure bioaccumulated more FLU than animals subjected to a single FLU exposure. However,
despite the clear hypothesis of the additional effect of PAH adsorption to particles, an
mRNA analysis suggested that the presence of MPs alters detoxification activity [46]. This
information is vital for understanding multiple-effect pathways since it is suggested that
extra-biological interaction, i.e., a crude mixture between toxic agents, is the main path-
Int. J. Environ. Res. Public Health 2022, 19, 1211 15 of 25

way of cointoxication. Thus, as observed between DDT and BaP [80], different xenobiotic
binding sites can generate complimentary or overlapping biological response pathways.
Furthermore, the potential for adsorption onto surfaces and particles can make it
difficult to accurately assess effects under controlled conditions. The tendency of BaP
to adsorb to aquaria walls, for example, may explain changes in PAH concentrations in
exposure bioassays, although the composition of these tanks (plastic or glass) was not
indicated in the studies investigated herein [81]. Different solvents and tank compositions
should, therefore, be tested. For example, the use of acetone as a solvent for BaP in
some tanks may favor BaP losses through evaporation and adsorption, which seems to be
associated with a high abrasive potential that can also remove lipids and proteins [81].

4.8. Aquatic Biota PAH Exposure Studies

The ecotoxicological studies obtained in our systematic review (Table 3) mostly focused
on the evaluation of effects on aquatic invertebrates, such as zooplankton and small fish.
However, bivalves like the blue mussel were also noteworthy and frequently employed as
a model species due to their wide geographic distribution and filter-feeding characteristics,
making them susceptible to bioaccumulation processes, thus demonstrating their adequacy
as PAH contamination bioindicators or sentinel species [82]. It is also important to note the
important bivalve role as a significant food source worldwide. For example, from 2009 to
2018, global mussel aquaculture production increased by 8%, driven mainly by the growth
of the Spanish output. In 2018, for example, the EU provided 84% of the global production
of Mytilus galloprovincialis, represented by both live and fresh mussels (44%) and prepared
and preserved mussels (39%) [83].

Table 3. Employed test species and their respective effect concentrations for the endpoints evaluated
in exposures to different PAHs obtained in our systematic review.

Species Common Name Reference Compound Concentrations Exposure Time

Trachinotus
Florida pompano [55] Anthracene 8–32 µg L−1 24 h *
carolinus
Girella punctata Largescale blackfish [84] Benzo(a)anthracene 1 and 10 ng/d dose 10 days
Chanos chanos Milkfish [56] Benzo(a)pyrene 0.002–0.031 mg L−1 96 h
Chlamys farreri Farrer’s scallop [85–87] Benzo(a)pyrene 0.025–10 µg L−1 10 days
Crassostrea gigas Pacifi cupped oyster [88] Benzo(a)pyrene 0.2–5 µg L−1 15 days
Dicentrarchus labrax Sea bass [72] Benzo(a)pyrene 2–256 µg L−1 96 h
Gadus morhua Common cod [89] Benzo(a)pyrene 2.52–252.3 µg L−1 48 h
Litopenaeus
White shrimp and
vannamei and [90] Benzo(a)pyrene 0.03–3 µg L−1 21 days
Korean mussel
Mytilus coruscus
Mytilus Mediterranean
[48] Benzo(a)pyrene 5–100 µg L−1 3 days
galloprovincialis mussel
Mytilus Mediterranean
[47] Benzo(a)pyrene 0.5 and 1 mg L−1 72 h
galloprovincialis mussel
Oreochromis
Tilapia [91] Benzo(a)pyrene 20 mg kg−1 120 h
niloticus
Pachycara
- [92] Benzo(a)pyrene 10 and 100 mg L−1 10 days
brachycephalum
Brown mussel and
Perna viridis and
Japanese [81] Benzo(a)pyrene 2–16 µg L−1 14 days *
Pinctada martensii
Pearl-oyster
Int. J. Environ. Res. Public Health 2022, 19, 1211 16 of 25

Table 3. Cont.

Species Common Name Reference Compound Concentrations Exposure Time

Planiliza klunzinger Klunzinger’s mullet [93] Benzo(a)pyrene 5–50 mg kg−1 14 days
Portunus
gazami crab [94,95] Benzo(a)pyrene 0.1–2.5 µg L−1 10 days
trituberculatus
Ruditapes
Manila clam [96] Benzo(a)pyrene 0.03–3 µg L−1 21 days
philippinarum
Ruditapes
Manila clam [97,98] Benzo(a)pyrene 4 µg L−1 5 and 15 days
philippinarum
Ruditapes
Manila clam [99] Benzo(a)pyrene 0.02 and 0.2 µmol L−1 96 h
philippinarum
Sebastes schlegelii Korean rockfish [100] Benzo(a)pyrene 2–200 µg g bw−1 48 h
Sebastiscus
Sea ruffle [58] Benzo(a)pyrene 0.01–1 µg L−1 6 days
marmoratus
Sparus aurata Gilt-head [59] Benzo(a)pyrene 2 mg L−1 72 h
Sparus aurata Gilt-head [101] Benzo(a)pyrene 10−4 to 106 µg L−1 72 h
Trachinotus
Florida pompano [102] Benzo(a)pyrene 1–8 mg L−1 10 days
carolinus
Crassostrea
Mangrove oyster [43] Phenanthrene 100 µg L−1 96 h
brasiliana
Chlamys farreri Farrer’s scallop [103] Benzo(a)pyrene 1–8 mg L−1 10 days
Chlamys farreri Farrer’s scallop [104] Benzo(a)pyrene 1–8 mg L−1 29 days
Meretrix meretrix Asiatic hard clam [14] Benzo(a)pyrene 1–8 mg L−1 24 h
Mytilus edulis Blue mussel [46] Fluoranthene 50 and 100 µg L−1 96 h
Ruditapes
Carpet shell [69] Fluorene 0.1–1 mg L−1 24 h
decussatus
Boreogadus saida Polar cod [80] Benzo(a)pyrene 0.1 and 480 µg L−1 14 days
Crassostrea
Mangrove oyster [105] Phenanthrene 100 and 1000 µg L−1 24 h
brasiliana
Eleginus navaga Atlantic navaga [57] Phenanthrene 1–30 µmol L−1 -
Epinephelus
Dusky grouper [71] Phenanthrene 0.47–3.76 mg L−1 96 h
marginatus
Nodipecten nodosus Lions-paw scallop [106] Phenanthrene 50 and 200 µg L−1 96 h
Sebastiscus
Sea ruffle [10] Phenanthrene 0.06–6 µg L−1 50 days
marmoratus
Lates calcarifer Barramundi [73] Pyrene 1–275 nM 24 h
Sebastiscus
Sea ruffle [107] Pyrene 10.2–102 mg L−1 5 days
marmoratus
Mytilus Mediterranean
[82] Anthracene 0.05, 0.15, 0.4 µg L−1 8 days
galloprovincialis mussel
* indicates the exposure tests followed by “clearance” of, respectively, 144 h and 14 days.

The study performed by Speciale et al. (2018) [47] best describes the One Health
concepts mentioned previously, associating animal and human health risk endpoints.
The authors report that the acute exposure of blue mussels to BaP is capable of causing
pathological changes in gills, which confirms the biotransformation activity of this tissue
due to PAH intoxication. In addition, PAH CYP1A bioactivation is associated with DNA
damage and carcinogenic potential [90]. Thus, CYP1A may comprise a valuable tool as a
human health risk assessment biomarker. Furthermore, exposure of mononuclear cells to
Int. J. Environ. Res. Public Health 2022, 19, 1211 17 of 25

contaminated products by BaP at levels similar to human ingestion rates demonstrated

toxic potential with morphological alteration to mussels at 0.5 mg L−1 , which effectively
indicates that the consumption of contaminated mollusks constitutes a significant risk to
human health [47].

4.9. Ecotoxicological Responses

Even though environmental chemistry can assess PAH effects under different condi-
tions, ecotoxicology establishes the flow of these contaminants in the biota and the effects
on individuals’ interaction populations. Furthermore, this field is essential for understand-
ing environmental and ecological scenarios and evaluating the effects of chemical and
xenobiotic substances on food chains as well as being economically relevant [108], as these
data contribute to public health maintenance through risk assessments.
Several trials selected in our systematic review evaluated the deleterious effects of
exposures in different media (sediment and/or water), simulating what naturally occurs in
the aquatic environment. For mollusks and crabs, exposure to a stock solution of the test
PAH in tanks/aquaria containing water and sediment was the most commonly applied
method, followed by exposure to PHE, BaP, FLU, ANT, or BaA for intervals ranging from
24 h to 50 days (Table 3).
In one of the assessments, the BaP did not significantly bioaccumulate in exposed
Klunzinger’s mullet tissues, despite intraperitoneal injection applications increasing the
activity of superoxide dismutase (SOD) and cytochrome P450 enzymes [83]. In turn, when
subjected to this PAH, bivalves not only accumulated this compound in tissues, especially
gills, but also suffer reproductive organ damage, i.e., ovarian development inhibition and
damage to ovarian envelope connective tissues [86], indicating the magnitude of different
responses observed in this type of assessment.

4.10. Biomarker Evaluations

Only two studies efficiently addressed the relationship between ecotoxicology and
potential human health risks [47,59], while the other 51 selected studies and the extra refer-
ences added to this study contributed mainly to parameter definitions and endpoints from
the intoxication by different PAHs in animals that constitute the food base of many human
populations. As discussed throughout this document, the two aforementioned studies
evaluated the effects of BaP in marine animals (blue mussel and sea bream, respectively)
and possible implications for human health. While [47] exposed mollusks to 1 mg L−1 of
pyrene 72 h, [59] subjected the marine fish Sparus aurata to double this amount (2 mg L−1 )
for the same period and time, also exposing human peripheral blood mononuclear cells.
The findings obtained in these assessments may, therefore, be useful in indicating common
enzyme markers for both animals and humans, which may, in turn, indicate an evaluation
route for concerning human exposure due to contaminated seafood diets.
However, a common mechanism of action has not yet been well-explored, even though
PAH effects on Phase 1 and 2 biotransformation detoxification markers are clear (Figure 6).
This process, essential in xenobiotic detoxification, is initiated by the transcription of en-
zymes by the aryl hydrocarbon receptor (AhR) pathway, which can also be applied as a
PAH toxicity biomarker [92]. The enzymes involved in this process, such as the cytochrome
P450 enzymes that act in Phase 1, can be classified as a function of the biotransformation
stage during which they act, with polarity being responsible for transforming liposoluble
compounds into more water-soluble forms, easier to excrete [86], typically generating differ-
ent metabolites. Some enzymes also act in the conjugation of these metabolites (Phase II),
resulting in xenobiotic conjugates, which are more readily excreted (Figure 7) [59,92].
Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW 18 of 26
Int. J. Environ. Res. Public Health 2022, 19, 1211 18 of 25

Figure 7. PAH
PAHbiotransformation
biotransformationpathway
pathwayscheme
scheme indicating
indicating intoxication
intoxication steps,
steps, CYP1A-mediated
CYP1A-mediated bio-
biotransformation phase I, antioxidation enzymes that act against ROS during Phase II, and water-
transformation phase I, antioxidation enzymes that act against ROS during Phase II, and water-soluble
soluble conjugate
conjugate excretion.
excretion. AHH—aryl AHH—aryl hydrocarbon
hydrocarbon hydroxylase;
hydroxylase; EROD—ethoxyresorufin
EROD—ethoxyresorufin O-
O-deethylas;
deethylas; SOD—superoxide dismutases; CAT—catalase; GPx—glutathione peroxidase;
SOD—superoxide dismutases; CAT—catalase; GPx—glutathione peroxidase; GSH—reduced glu- GSH—
reduced glutathione; GSR—glutathione reductase; GSSg—glutathione disulfide.
tathione; GSR—glutathione reductase; GSSg—glutathione disulfide.

The
The CYP1A
CYP1A subfamily
subfamily (Figure (Figure 7) 7) is
is the
the most studied among
most studied among the cytochrome P450
the cytochrome P450
family of enzymes as it is significantly induced by PAHs.
family of enzymes as it is significantly induced by PAHs. The activity of this The activity of this marker
marker in
in fish
fish and shellfish has been applied in many assessments to verify
and shellfish has been applied in many assessments to verify PAH effects, especially BaP, PAH effects, especially
BaP,
which which significantly
significantly increases
increases the expression
the expression of CYP1A
of CYP1A as a function
as a function of exposure
of exposure timetime
and
and
dosedose [47,59,85–89,92,93,100].
[47,59,85–89,92,93,100].
Despite
Despite CYP1A
CYP1A mediation,
mediation, metabolite
metabolite formation
formation cancan bebe harmful
harmful to to many
many organisms,
organisms,
even
even though these products can also be applied to assess intoxication. Thus, the metabolic
though these products can also be applied to assess intoxication. Thus, the metabolic
mechanism ofofPAHs PAHs is also
is also an essential
an essential point ofpoint of attention
attention for the ecotoxicological
for the ecotoxicological evaluation
evaluation of these compounds.
of these compounds. For example, For example,
concerning concerning Phase I enzymes,
Phase I enzymes, the oxidation
the oxidation process
process
of BaP toofepoxides
BaP to epoxides
and phenols and hasphenols
a betterhasdescription.
a better description.
FollowingFollowing
this phase, this phase,
epoxides
epoxides
undergo one undergo
to twoone to two hydrolysis
hydrolysis by epoxide by epoxideand,
hydrolase hydrolase and, after
after second second CYP-
CYP-mediated oxi-
mediated
dation, areoxidation,
convertedare intoconverted into di-olepoxides
di-olepoxides with high potential
with high carcinogenic carcinogenic [80]potential [80]
Furthermore,
Furthermore,
metabolites such metabolites such as B
as di-hydrodiols di-hydrodiols
can be oxidized B can be oxidized
to quinones by to quinones by
di-hydrodiol di-
dehy-
hydrodiol
drogenase dehydrogenase
[96]. This process [96]. This process
commonly leadscommonly
to reactive leads
oxygen to species
reactive(ROS)
oxygen species
formation,
(ROS)
which formation,
is detoxified which
by the is protective
detoxified antioxidant
by the protective
system. antioxidant
This system system. This several
comprises system
enzymes and
comprises proteins,
several enzymessuch andas superoxide dismutase
proteins, such (SOD), catalase
as superoxide dismutase (CAT) and reduced
(SOD), catalase
glutathione
(CAT) (GSH), glutathione
and reduced glutathione-S-transferase (GST), and glutathione
(GSH), glutathione-S-transferase (GST), peroxidase (GPx),
and glutathione
which accumulate
peroxidase (GPx), inwhich
important protective
accumulate in metabolic
importantpathways
protective and serve as oxidative
metabolic pathwaysstress
and
biomarkers [56,80].
serve as oxidative stress biomarkers [56,80].
GSH, a non-enzymatic
non-enzymatic biomarker, biomarker, has hasbeen
beenthethetarget
targetof ofseveral
several PAHPAH exposure
exposure as-
sessments, including
assessments, includingFLU, FLU,ANT, ANT,PHE,PHE,and andBaP
BaP[45,53,55,82,96].
[45,53,55,82,96]. Decreased
Decreased GSH levels
were reported
were reported for for gills
gills inin M.M. edulis
edulis subjected
subjected to to FLU
FLU individually
individually and and combined
combined withwith
microplastics [46]
microplastics [46] and
and inin M.M. galloprovincialis
galloprovincialis exposed
exposed to to single
single treatments
treatments and and aa mixture
mixture of of
ANT and PHE, while increased GSH levels were observed
ANT and PHE, while increased GSH levels were observed in milkfish following in milkfish following anthracene
exposure. On
anthracene the otherOn
exposure. hand,
the decreased
other hand, GSH levels were
decreased GSHreported
levels werefollowing BaPfollowing
reported exposure
in milkfish [56] which reinforces the different responses between
BaP exposure in milkfish [56] which reinforces the different responses between species species and, above all, the
effects of different doses and exposure durations (Table 3).
and, above all, the effects of different doses and exposure durations (Table 3).
In addition to biotransformation responses, which comprise quick response biochem-
ical pathways, PAH can also alter morphological and physiological components in ad-
Int. J. Environ. Res. Public Health 2022, 19, 1211 19 of 25

dition to impairing survival. PHE, for example, is known for its potential to induce
DNA damage and disturb aquatic organism behavior in addition to affecting the hepato-
cyte area and resulting in lethality for certain fish species, such as Epinephelus marginatus
(LC50 1.51 mg L−1 ) [71]. ANT has been shown to compromise the swimming behavior of
Palaemon serratus fish by reducing swimming speeds at environmentally relevant concen-
trations from 128 ug L−1 [72,109] (Table 3). From 150 nmol L−1 PYR, the fish Lates calcarifer
exhibited increased immobility and decreased survival rates to decreased feeding rates [73]
(Table 3).
Thus, in addition to direct animal health effects, PAHs, even though distinct in terms
of chemical structures, environmental dispersion, and metabolism pathways, can alter
ecosystem dynamics through damage to key species and even indirect damage to trophic
interactions [14]. Therefore, ecotoxicological studies are paramount to determining PAH
levels resulting from human–environment interactions, establishing no-effect values that
also indicate no risks to human health [59].

4.11. The Relationship between Toxic Limits and Different Risk Concepts
The consumption of contaminated fish is a potential source of risk, increasing the
chances of several deleterious effects in humans [110]. Risk assessment articles usually
focus on human effects and do not evaluate environmental impacts, while ecotoxicolog-
ical studies, even if carried out in controlled environments, tend to conclude that their
findings constitute a basis for developing quality control standards for public manage-
ment. However, it is important to note that joint efforts in both areas should be carried
out as a theoretical basis can contribute to decision-making aiming at decreasing aquatic
contamination effects and improving human and environmental quality.
In our systematic review, only papers assessing the interface between toxicity values
and risk of exposure to biological matrices able to trigger disorders in human beings
were assessed, while articles associated with mathematical models from meta-analyses or
development studies regarding purely risk calculation methodologies or risk models were
excluded. This led to the selection of only two articles that evaluated the link between
ecotoxicological methods and human health effects, although with no risk assessment
modeling efforts carried out.
According to the model developed by the US Environmental Protection Agency [111],
direct particle ingestion, inhalation, and dermal contact as exposure routes should be con-
sidered. For non-carcinogenic compounds, the risk is estimated using a hazard index (HI),
which is equal to the sum of hazard quotients, calculated as HI = HQing + HQinh + HQdermal ,
as follows:
lnR × EF × ED
HQing = C × × 10−6 (1)
BW × AT
lnhR × EF × ED
HQinal = C × (2)
PEF × BW × AT
SL × SA × |×| EF × ED
HQdermal = C × × 10−6 (3)
BW × AT
where HQing corresponds to the toxicant ingestion (mg kg−1 day−1 ), HQinal refers to
inhalation, HQdermal is the dose associated with dermal contact, C is the concentration of
the contaminant agent in the exposure matrix, in mg kg−1 . InhR is the mean ingestion
rate of the contaminated matrix; while EF, ED, BW, and AT are the exposure frequencies
(180 days year−1 ), duration of exposure (years of consumption), average body weight
(15.0 kg3 for children and 70 kg for adults), and average time (DE × 365 days). These
equations can also be simplified as:

D
HQ = (4)
RfD
Int. J. Environ. Res. Public Health 2022, 19, 1211 20 of 25

where HQ is the hazard quotient obtained by the ratio between the dose of the contaminant
in mg kg−1 (D) and its reference dose (RfD). HQ values are obtained through the quotient
between the maximum concentrations of the studied substance, and the predicted no-effect
concentration (PNEC), considering the effect concentration (EC50 ), lethal concentration
(LC50 ), or even the non-observed effect concentration (NOEC), which are usually available
in the literature or can be discovered employing bioassays, further linking the ecotoxicology
and human health risk assessment fields.
A fundamental difference is noted, however, between theoretical and modeled risks.
While the theory involves calculating the probability of effect from the ratio between
potential exposures and effect concentrations [112], in practice, the risk quotient is obtained
from predictive models, which uses the bases of toxicology, also applied to define legislation
levels, in comparison to predicted environmental concentrations [113].
In turn, ecological risk assessments aim to characterize the probability of occurrence
of environmental effects resulting from human actions. This field of research favors effect
assessments on organisms (animals and plants) that make it possible to verify xenobiotic
ecological toxicity. This index can be estimated by comparing the studied substance’s
hazard quotient (HQ) with its corresponding environmental quality value. However, as
the lack of data on the individual toxicities of PAH can be a challenge in this regard, some
researchers have agreed that a PAH toxicity equivalence factor (TEF) can be used in blank
dates due to similar ecological and human health effects [114].

Cm
CQ = (5)
Cqv

where CQ is the risk quotient provided by the ratio between Cm, the PAH concentration in
the studied matrix (e.g., water), and Cqv , the quality value that considers the permissible
concentrations.
Environmental risk assessment is, therefore, essential to determine whether pollutants
present in water bodies threaten aquatic biota and human beings. Thus, concentrations in
the marine environment and their toxicity data concerning different organisms become
paramount in determining the risks of these compounds.

5. Conclusions
Even with the increasing number of studies aimed at the applicability of environmental
and public health concepts in conjunction with the One Health concept and the urgent
need for information to aid in the determination of safe levels of toxic agents, laboratory
tests and field studies still appear to be the greatest source of data for conducting human
health risk assessments.
Temperature, dissolved oxygen, and salinity fluctuations, as well as intrinsic physico-
chemical properties, can affect PAH availability and toxicity. Their interactions with each
other and with other contaminants of anthropic origin are also of note, with different effects
on marine organisms that could, possibly, affect human health. This should be further
addressed by ecotoxicology assessments.
Tests on commercially important organisms such as bivalve mollusks, crabs, and fish
tend to compose most risk assessments. Studies concerning fishery products have, in fact,
increasingly evaluated several PAH intoxication markers as putative indicators for animal
health problems, despite a significant lack of investigations concerning the potential associ-
ations to human health effects being noted. However, our systematic review still makes it
clear that environmental and ecological aspects are still mainly studied separately, demon-
strating that multidisciplinary assessments regarding PAH toxicities are urgently required.
This can be evidenced by the fact that only 2 studies among the 1360 selected studies make
the connection between animal and human health in a connected way, highlighting a gap
in knowledge.
Int. J. Environ. Res. Public Health 2022, 19, 1211 21 of 25

Author Contributions: Conceptualization, J.V.d.P. and P.d.A.R.; methodology, J.V.d.P. and R.G.F.;
software, J.V.d.P., I.D.L.G. and F.C.M.; data curation, J.V.d.P.; writing—original draft preparation,
J.V.d.P. and P.d.A.R.; writing—review and editing, R.G.F., R.A.H.-D. and C.A.C.-J.; supervision,
P.d.A.R., R.A.H.-D. and C.A.C.-J.; project administration, C.A.C.-J. All authors have read and agreed
to the published version of the manuscript.
Funding: This research was funded by Fundação de Amparo a Pesquisa do Rio de Janeiro (FAPERJ)
grant numbers E-26/201.167/2020, E-26/210.442/2021, and E-26/200.891/2021”. The APC was
funded by FAPERJ.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing not applicable.
Acknowledgments: The authors are thankful for the financial support provided by the Fundação de
Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) Brazil—grant numbers [E-26/203.049/2017];
[E-26/201.167/2020]; [E-26/001.442/2021] and [SEI-260003/002211/2021], the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq)—grant numbers [313119/2020-1] and [150450/2020-
6], and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Brazil—Finance
Code [88887.518753/2020-00].
Conflicts of Interest: The authors declare no conflict of interest.

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