Environmental Pollution 269 (2021) 116142

Contents lists available at ScienceDirect

Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Review

Marine mammals and microplastics: A systematic review and call for

standardisation*
Laura J. Zantis a, Emma L. Carroll a, Sarah E. Nelms b, c, Thijs Bosker d, e, *
a
School of Biological Sciences, University of Auckland, Auckland, New Zealand
b
Centre for Ecology and Conservation, University of Exeter, Cornwall, TR10 9EZ, United Kingdom
c
Centre for Circular Economy, University of Exeter, Cornwall, TR10 9EZ, United Kingdom
d
Leiden University College, Leiden University, The Hague, the Netherlands
e
Institute of Environmental Sciences, Leiden University, Leiden, the Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: Microplastics receive significant societal and scientific attention due to increasing concerns about their
Received 8 September 2020 impact on the environment and human health. Marine mammals are considered indicators for marine
Received in revised form ecosystem health and many species are of conservation concern due to a multitude of anthropogenic
17 November 2020
stressors. Marine mammals may be vulnerable to microplastic exposure from the environment, via direct
Accepted 19 November 2020
ingestion from sea water, and indirect uptake from their prey. Here we present the first systematic re-
Available online 28 November 2020
view of literature on microplastics and marine mammals, composing of 30 studies in total. The majority
of studies examined the gastrointestinal tracts of beached, bycaught or hunted cetaceans and pinnipeds,
Keywords:
Marine mammals
and found that microplastics were present in all but one study, and the abundance varied between 0 and
Microplastics 88 particles per animal. Additionally, microplastics in pinniped scats (faeces) were detected in eight out
Best practices of ten studies, with incidences ranging from 0% of animals to 100%. Our review highlights considerable
Plastic pollution methodological and reporting deficiencies and differences among papers, making comparisons and
Standardisation extrapolation across studies difficult. We suggest best practices to avoid these issues in future studies. In
addition to empirical studies that quantified microplastics in animals and scat, ten studies out of 30 (all
focussing on cetaceans) tried to estimate the risk of exposure using two main approaches; i) overlaying
microplastic in the environment (water or prey) with cetacean habitat or ii) proposing biological or
chemical biomarkers of exposure. We discuss advice and best practices on research into the exposure and
impact of microplastics in marine mammals. This work on marine ecosystem health indicator species
will provide valuable and comparable information in the future.
© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

1. Introduction (Brown et al., 2018; Frouin et al., 2012) and climate change (Albouy
et al., 2020; Sanderson and Alexander, 2020), many marine mam-
Marine mammals play key roles in influencing the structure and mals species are of conservation concern (Nelms et al. In prep;
function of the marine environment and are sentinels for Davidson et al., 2012; Pompa et al., 2011).
ecosystem health (Burek et al., 2008; Moore, 2008). However, due Plastic pollution is known to affect marine mammals, through
to an increase in anthropogenic activities, including fishing entanglement (Kraus, 2018), ingestion (Alexiadou et al., 2019; De
(Barcenas-De la Cruz et al., 2018; Ocampo Reinaldo et al., 2016), Stephanis et al., 2013; Unger et al., 2016) and potential habitat
shipping (Halliday et al., 2017; Riley and Hollich, 2018), pollution degradation (Gall and Thompson, 2015; Pawar et al., 2016). One
area of specific concern is the exposure of marine mammals to
microplastics. These small (<5 mm), pervasive and persistent
*
synthetic particles (Moore, 2008) are bioavailable to marine or-
This paper has been recommended for acceptance by Maria Cristina Fossi.
ganisms, through direct ingestion and/or via trophic transfer (Cole
* Corresponding author. Leiden University College, Leiden University, The Hague,
the Netherlands. et al., 2011; Eriksson and Burton, 2003; Nelms et al., 2019a). Mys-
E-mail addresses: [email protected] (L.J. Zantis), [email protected]. ticetes (baleen-whales), for example, are megafilter feeders that
nz (E.L. Carroll), [email protected] (S.E. Nelms), [email protected] engulf large volumes of water alongside their prey, and are
(T. Bosker).

https://doi.org/10.1016/j.envpol.2020.116142
0269-7491/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
L.J. Zantis, E.L. Carroll, S.E. Nelms et al. Environmental Pollution 269 (2021) 116142

potentially exposed to microplastics via both pathways; direct

uptake of microplastics from the environment (environmental (Microplastic* OR “Plastic particle*" OR “Marine Debris*")
exposure, e.g. Germanov et al., 2018; Guerrini et al., 2019), and AND (Whale* OR Cetacean* OR Dolphin* OR Delphinid* OR
indirect ingestion, from consuming contaminated prey (trophic Pinniped* OR Seal* OR Manatee* OR “Polar bear*" OR
transfer exposure, e.g. Burkhardt-Holm & N’Guyen, 2019; Desforges Mysticete* OR Odontocete* OR Porpoise* OR Phocid* OR
et al., 2015). In comparison, odontocetes (toothed-whales) and Otariid* OR “Sea lion*")
pinnipeds (seals, sea lions and walruses) are most likely to be
exposed through trophic transfer (Au et al., 2017; Ivar Do Sul and
Costa, 2014; Nelms et al., 2018; Perez-Venegas et al., 2018).
Studies on other taxa indicate that microplastics may present a 2.2. Screening process
number of potential impacts, acting as a vector for pathogens or
chemical contaminants (Prinz and Korez, 2020). Articles found during the searches were assessed for inclusion
Though the impact of microplastics on marine mammals is using a two-step screening process:
relatively understudied compared to other taxa, research on the Step 1: Study inclusion criteria
uptake and exposure of marine mammals to microplastics has The title and abstract of each publication were evaluated for
increased in recent years. Studies have investigated microplastic relevance using a number of inclusion criteria;
abundance and exposure risk in marine mammals using gut con-
tent analysis (e.g. Lusher et al., 2015; Nelms et al., 2019b), faecal o Subject: Discusses link between microplastic pollution and ma-
analysis (e.g. Hudak and Sette, 2019; Nelms et al., 2018; Ryan et al., rine mammals, including pinnipeds, cetaceans, manatees or
2016) as well as indirectly by measuring levels of chemical bio- polar bears.
markers, such as phthalates (e.g. Baini et al., 2017; Fossi et al., 2014). o Results: Presents information on the interaction between marine
Importantly, a wide range of microplastic identification and mammals and microplastic. For a detailed list of variables, we
contamination prevention methods are used within these studies, searched for and minimum requirements see Table S1.
highlighting the need for standardized protocols for robust and o Type of study: Empirical study published in a peerereviewed
comparable microplastic analysis (Panti et al., 2019; Stock et al., journal
2019).
Reviews on plastic ingestion and entanglement by marine Step 2: Data extraction and presentation
mammals (e.g. Baulch and Perry, 2014; Simmonds, 2012) have Potentially relevant papers were read in full, and information
highlighted the abundance of interactions of marine mammals and data which were relevant for this review were extracted from
with plastic debris. Given the growing interest in this field, the the eligible papers. When available, information on study type,
objective of this study was to conduct the first systematic literature target species, study location, method, abundance of microplastics,
review on microplastics and marine mammals. We sought to syn- polymer identification protocol, polymer characteristics and
thesize and summarize the existing literature on the topic, high- contamination identification protocol were collected (See Table S1
light knowledge gaps and recommend avenues for future research, for extracted information).
and suggest best practices to move the field forward. In the results we summarize and discuss the results focussing on
digestive tracts (section 3.1) and scat samples (section 3.2). Next,
we summarize and discuss methodological differences (section 3.3)
followed by suggestion on best practices (section 3.4). In section
2. Materials and methods 3.5, we will discuss inferential studies in which biomarkers or levels
of microplastics in prey are linked to risk of exposure.
2.1. Literature search parameters
3. Results and discussion
The design of this systematic literature review follows the
guidelines of Siddaway et al. (2019). The main search for literature Searches with the main search terms in two databases returned
was conducted in September 2019, and an update was made in May a total of 297 articles. Three additional articles were found through
29, 2020. Searches for relevant peer-reviewed literature were made other sources. After removing duplicates, 219 articles were left.
using two online publication databases; Web of Science and Title and abstract screening further excluded 156 articles. A
PubMed. The selection process of articles is summarized according remaining 63 publications were then screened based on their full
to the PRISMA approach (Moher et al., 2009; Fig. S1). The bibliog- text, resulting in 30 articles, which were finally included in this
raphies of peer-reviewed publications were also explored, and review (Table S2).
potentially relevant studies not found in online databases were Most of the scat and gut studies on microplastics and marine
recorded. mammals were conducted in Europe (47%; n ¼ 10) e mostly in the
The following search terms were utilised during a first scoping United Kingdom and in Italy, followed by North America (19%;
exercise and resulted in a selection of relevant articles: n ¼ 4), Sub Antarctic and Antarctica (14%; n ¼ 3 pinniped studies),
Latin America (10%; n ¼ 2) and Asia (10%; n ¼ 2; Fig. 1).

Subject: Microplastic*, “Plastic particle*”, “Marine Debris*” The majority of papers on gut content analyses focussed on

Target: Whale*, Cetacean*, Dolphin*, Delphinid*, Mysticete*, cetaceans, particularly odontocetes (Fig. S2). In contrast, all studies
Odontocete*, Porpoise*, Phocid*, Otariid*, Pinniped*, Seal*, on microplastics in faeces used scat from pinnipeds, mostly otariids
“Sea lion*”, Manatee*, “Polar bear*”. (eared seals). No studies on sirenians and polar bears were iden-
tified (Fig. S2).
The terms within each category (“subject” and “target”) were
combined using the Boolean operator “OR”. The two categories 3.1. Microplastics in digestive tracts
were then combined using the Boolean operator “AND”. An Asterix
(*) is a wildcard that represents any group of characters, including In total, 12 publications were identified that examined digestive
no characters. The full search string thus reads as follows: tracts for microplastics using samples from beached (n ¼ 8
2
L.J. Zantis, E.L. Carroll, S.E. Nelms et al. Environmental Pollution 269 (2021) 116142

Fig. 1. The global distribution and focus of studies on microplastics and marine mammals. Note: modelling studies were not included.

publications), by-caught (n ¼ 2) or hunted (n ¼ 2) marine mammals type for all, or a sub-sample of, particles using analytical polymer
(Table 1; Fig. S2). characterisation techniques, such as Fourier-transform spectros-
All of the studies found suspected microplastics in at least one copy (FTIR) or Near Infrared Spectroscopy (NIR; Table S3). The
animal examined (Table 1), with the exception of Bourdages et al. proportion of suspected microplastics analysed for polymer type
(2020), who reported none in the stomach contents of 142 hun- varied from 19%e100% among studies and of those particles ana-
ted arctic seals (ringed seals; Phoca hispida; n ¼ 135, bearded seals; lysed, the proportion that were confirmed as synthetic ranged from
Erignathus barbatus; n ¼ 6, and one harbour seal; Phoca vitualina; 16% to 77% per study. The remaining particles were either natural,
n ¼ 1). Drawing direct comparisons among studies is challenging semi-synthetic or too degraded/dirty to obtain reliable spectra
due to differences in the amount of digestive tract content analysed, matches. Of the confirmed microplastics, sixteen main polymer
and the lack of information provided about the analysed amount. types were reported, but the composition varied considerably
For example, some studies examined all content from the whole among studies (Table S2). This variation is likely due to the het-
digestive tract and reported the number of suspected microplastics erogeneity of plastic pollution sources as well as lack of uniformity
per animal (Lusher et al., 2015, 2018; Nelms et al., 2019b). This in polymer analysis techniques and equipment (e.g. polymer li-
ranged from three in a white-beaked dolphin (Nelms et al., 2019b) braries, interpretation of spectral matches, confidence criteria). For
to 88 in a True’s beaked whale (Mesoplodon mirus) (Lusher et al., example, four of the studies accepted FTIR spectra matches with
2015, Table 1). This information on microplastic abundance per confidence levels of between 70% and 80% but the remaining three
animal, coupled with information on animal size, age-class, sex and studies do not specify their accepted confidence thresholds.
species, allows for further investigation into potential drivers any
observed trends in microplastic load. 3.2. Microplastics in scat samples
Where sub-samples were taken from the digestive tract, some
studies report the number of microplastics per animal without In total, nine peer-reviewed papers have analysed marine
reporting the volume of content examined, making it impossible to mammal scats for the presence of microplastics (Table 2; Fig. S2).
calculate total microplastic load. Another approach involved All of these examined scats originate from pinnipeds, likely because
extrapolating the number of microplastics found within sub- of i) ease of collection compared with cetaceans due to use of
samples, to estimate the microplastic abundance range for the terrestrial habitats (e.g. haul out sites) and ii) access to long-term
whole animal. For example, Moore et al. (2020) found 81 micro- datasets where scat was collected for other purposes (e.g. diet
plastics in digestive tract sub-samples of seven Beluga whales analyses).
(Delphinapterus leucas) and estimated that each whale contained 18 In the six studies for which microplastics in scat were reported,
to 147 microplastics (average of 97 ± 42 per individual) by esti- the occurrence varied from 1% in scats collected in 2016/2017 from
mating the intestinal length and calculating the potential micro- grey seals (Halichoerus grypus atlantica) on the Atlantic coast of the
plastic abundance throughout. Though this approach is useful USA (n ¼ 129, Hudak and Sette, 2019) to 100% in scats collected in
where no other means of garnering such information exist, it 1996/1997 from Sub Antarctic and Antarctic fur seals (Arctocephalus
should be used with caution. tropicalis; A. gazella) on Marion Islands (n ¼ 100, Eriksson and
Fibres were the predominant particle shape for the majority of Burton, 2003, Table 2). The reporting of microplastic load varied,
studies (Table S2). However, Moore et al. (2020) found that as some studies reported it as a mean or incidence for all scats
approximately half of microplastics in Beluga whales were frag- analysed (all), while some reported statistics only for those scats in
ments and half were fibres (51% and 49%, respectively; Table S2). In which microplastics were detected (positives). This also could have
addition, three studies also reported foam, sheet and bead-shaped contributed to increased variance, ranging from a mean of
particles (Besseling et al., 2015; Hernandez-Gonzalez et al., 2018; 0.87 ± 1.09 in 31 grey seal scats collected from captive animals
Xiong et al., 2018). Due to concerns regarding air-borne contami- (Nelms et al., 2018: all scats) to a mean of 37.3 ± 38.1 per positive
nation, some studies did not seek to extract microfibres or excluded scat in the 34 scats found to have microplastics in Perez-Venegas
them, or particles below a certain size limit, from their results et al. (2018) (Table 2). The numbers reported by Perez-Venegas
(Besseling et al., 2015; Bourdages et al., 2020; Hernandez-Milian et al. (2018) need to be interpreted with caution, as several mis-
et al., 2019; van Franeker et al., 2018). Only five studies presented takes were found in their supplementary information. The authors
information on the colour of particles detected, of which blue and have been notified, and will produce a correction, but this was not
black were the most common (Table S2). available at the time of printing of this article.
Of the 11 studies that report the presence of suspected micro- The route of exposure was also examined, with the study by
plastics in digestive tracts, seven presented information on polymer Nelms et al. (2018) being a key paper as this is the only controlled
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L.J. Zantis, E.L. Carroll, S.E. Nelms et al. Environmental Pollution 269 (2021) 116142

Table 1
Summary of results of studies investigating microplastic (MPs) in the gastrointestinal track of bycaught, hunted or beached marine mammals. N/R means not recorded within
the study.

Species Sample Sample Number of particles (confirmed or suspected Size of particles Source
origin size microplastics)

Total % samples “All” mean MPs Range MPs Mean size (±SD) Size range (mm)
MPs with MPs per animal per animal (mm)
a

Mysticete
Part of GIT 1 16 100% 16 16 N/R 1.1e4.7 x Besseling et al. 2015
Humpback whale 0.4e 2.4
Odontocete
Atlantic white- GIT 1 8 100% 5.5 ± 2.7b 3-12b Fib: 2.0±2.3 Frag: Fib: 0.1- 20, Frag: 0.1-4b Nelms et al. 2019b
sided dolphin 0.9±1.1b
GIT 7 81 100% 11.6 ± 6.6 3-24 <1mm (87%), 1- N/R Moore et al. 2020
Beluga whale 2mm (20%)
GIT 1 6 100% 5.5 ± 2.7b 3-12b Fib: 2.0±2.3 Frag: Fib: 0.1- 20, Frag: 0.1-4b Nelms et al. 2019b
Bottlenose dolphin 0.9±1.1b
GIT 2 39 100% 25.5b 1-88b N/R 0.3 - 16.7b Lusher et al. 2018
GIT 16 91 100% 5.5 ± 2.7b 3-12b Fib: 2.0±2.3 Frag: Fib: 0.1- 20, Frag: 0.1-4b Nelms et al. 2019b
Common dolphin 0.9±1.1b
Stomach 35 411 94% 12 ± 8 3-41 Fib: 2.11±1.26, Fib: 0.29-4.92 Frag: Hernandez Gonzalez
Frag: 1.29±0.93 0.49-4.07 Bead: 0.95 et al. 2018
GIT 9 187 100% 25.5b 1-88b N/R 0.3 - 16.7b Lusher et al. 2018
GIT 1 53 100% 25.5b 1-88b N/R 0.3 - 16.7b Lusher et al. 2018
Cuvier’s beaked
whale
Intestine 7 134 100% 19.1 ± 7.2 10-32 N/R N/R Xiong et al. 2018
Finless porpoise
b b b
GIT 21 110 100% 5.5 ± 2.7 3-12 Fib: 2.0±2.3 Frag: Fib: 0.1- 20, Frag: 0.1-4 Nelms et al. 2019b
Harbour porpoise 0.9±1.1b
Stomach 654 71 7% 0.11 ± 0.02 1-5 0.009 ± 0.004 0.2-2.6g van Franeker et al.,
2018
GIT 5 103 100% 25.5b 1-88b N/R 0.3 - 16.7b Lusher et al. 2018
Intestine 3 77 100% 0.2-0.6 items/g 2-45 2.2± 0.4 0.1-4.8 Zhu et al. 2019
Indo-Pacific
humpbacked
dolphin
GIT 1 39 100% 25.5b 1-88b N/R 0.3 - 16.7b Lusher et al. 2018
Killer whale
GIT 1 4 N/R 5.5 ± 2.7b 3-12b Fib: 2.0±2.3 Frag: Fib: 0.1- 20, Frag: 0.1-4b Nelms et al. 2019b
Pygmy sperm 0.9±1.1b
whale
GIT 1 9 N/R 5.5 ± 2.7b 3-12b Fib: 2.0±2.3 Frag: Fib: 0.1- 20, Frag: 0.1-4b Nelms et al. 2019b
Risso’s dolphin 0.9±1.1b
GIT 1 7 N/R 5.5 ± 2.7b 3-12b Fib: 2.0±2.3 Frag: Fib: 0.1- 20, Frag: 0.1-4b Nelms et al. 2019b
Striped dolphin 0.9±1.1b
GIT 1 88 100% N/R 88 2.2±1.4 0.3 e 7 Lusher et al. 2015,
True’s beaked Lusher et al. 2018
whale
GIT 1 3 100% 5.5 ± 2.7b 3-12b Fib: 2.0±2.3 Frag: Fib: 0.1- 20, Frag: 0.1-4b Nelms et al. 2019b
White-beaked 0.9±1.1b
dolphin
Phocidae
Bearded seals Stomach 6 0 0 0 0 0 0 Bourdages et al. 2020
Intestine 13 363 100% 27.9 ± 14.7 13-71 N/R N/R Hernandez-Milian
Grey seal et al. 2019
GIT 3 18 100% 5.5 ± 2.7b 3-12b b
Fib: 2.0±2.3 Frag: Fib: 0.1- 20, Frag: 0.1-4 Nelms et al. 2019b
0.9±1.1b
Stomach 1 0 0 0 0 0 0 Bourdages et al. 2020
Harbour seal
GIT 4 17 100% 5.5 ± 2.7b 3-12b Fib: 2.0±2.3 Frag: Fib: 0.1- 20, Frag: 0.1-4b Nelms et al. 2019b
0.9±1.1b
Stomach and Stom: 107, Stom: Stom: Stom: 0.26 Int: 0-8 N/R N/R Bravo Rebolledo et al.
Intestine Int: 100 28, Int: 7 11.2% Int: 0.07 2013
1%
Stomach 135 0 0 0 0 0 0 Bourdages et al. 2020
Ringed seals
a
# all suspected microplatics: some studies did not confirm whether observed particles were actual plastic polymers, or analyzed a subset
b
average within study including multiple species

study on microplastic and marine mammals to date. In this study, results support the hypothesis of trophic transfer. In field experi-
the microplastic load of both prey and scat was directly measured, ments, the authors typically either did not specifically hypothesise
and a similar incidence, type and colour of microplastic was found about the route of exposure (Donohue et al., 2019; Hudak and Sette,
in the fish used to feed captive grey seals and their scat. These 2019; Perez-Venegas et al., 2018) or suggested trophic transfer
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L.J. Zantis, E.L. Carroll, S.E. Nelms et al. Environmental Pollution 269 (2021) 116142

Table 2
Summary of results of studies investigating microplastics (MPs) in scat of pinnipeds. N/R means not recorded within the study.

Species Sample Number of particles Size of particles Author

size
Total MPa % samples with “All” mean MPs per Range MPs per Mean size Size range (mm)
MPs scat±SD scat (mm)

Otariidae
Antarctic fur seal 145 164c 100% 1.13 ± 0.43c 1e4c 4.1  1.9c 89%: 2e5c Eriksson and Burton
(2003)
42 0 0 0 0 0 0 Garcia Garin et al.
(2020)
Juan Ferna
ndez 40 Unknownb Fib: 62.5%; Fib: 30; Fib: 0e200; N/R N/R Perez-Venegas et al.,
fur seal Frag: 12% Frag: 2 Frag: 0-30 (2020)¥
Northern fur seals 44 584 Frag: 55%; Fib: Frag: 16.6 ± 19.1, Fib: Frag: 1e86; N/R Frag: 82%: <1, Fib: 70%: <2, Donohue et al. (2019)
41% 3.8 ± 3.4 Fib: 1-18 28%: 2-10
Sub Antarctic 4905 0 0 0 0 0 0 Ryan et al. (2016)
fur seals
145 164c 100% 1.13 ± 0.43c 1e4c 4.1  1.9c 89%: 2e5c Eriksson and Burton
(2003)
South American 79 Unknownb Fib: 65%; Frag: Fib: 16.5; Frag: 1 Fib: 0e182; N/R N/R Perez-Venegas et al.,
fur seal 6% Frag: 0-32 (2020)
c
51 1268 67% 37.26 ± 38.08 3e182 N/R Fibres: 67% > 0.1 Perez-Venegas et al.
(2018)
South American 36 Unknownb Fib: 86%; Frag: Fib: 43; Fib: 0e267; N/R N/R Perez-Venegas et al.,
sea lion 11% Frag: 1 Frag: 0-18 (2020)
Phocidae
Grey seals 129 2 1% 0.02 ± 0.12 0e1 N/R 1.9  0.8-2.6  1.1 Hudak and Sette
(2019)
31 Prey: 18, seal 48% 0.87 ± 1.09 0e4 1.5 ± 1.2 Scat: Frag: 0.4e5.5, Fib: 0.6 Nelms et al. (2018)
scat: 26 e3.5.
Harbor seal 32 2 6% 0.06 ± 0.25 0e1 N/R 1.19  0.58 - 3.45  1.81 Hudak and Sette
(2019)
125 0 0 0 0 0 0 Bravo Rebolledo et al.
(2013)
a
All suspected microplastics: some studies did not confirm whether observed particles were actual plastic polymers, or analysed a subset.
b
Authors classify all particles found as MPs but state they only tested the contents of 6 scats for each seal population (number of particles unknown). Of the particles tested
30% were confirmed as polymers (PET and Nylon).
c
Average within study including multiple species

rather than environmental exposure (Eriksson and Burton, 2003; among studies (Table S3). For example, some studies inspected
Perez-Venegas et al., 2020). whole, or sub-samples of, single digestive tract sections (e.g.
The majority of studies reported fragments as the most domi- stomach or intestines only; Bourdages et al., 2020; Hernandez-
nant particle shape (Table S4). However, two studies only found Gonzalez et al., 2018; Hernandez-Milian et al., 2019; van Franeker
fibres in scat samples (Table S4; Perez-Venegas et al., 2018, 2020). et al., 2018; Xiong et al., 2018; Zhu et al., 2019). Others examined
Most studies presented information on the colour of particles all, or sub-samples of, the whole digestive tract (Bravo Rebolledo
detected, of which white, blue and black were the most common et al., 2013; Lusher et al., 2015, 2018; Moore et al., 2020; Nelms
(Table S4) (Donohue et al., 2019; Eriksson and Burton, 2003; Nelms et al., 2019b). The volume and origin of gut content analysed is
et al., 2018; Perez-Venegas et al., 2018, 2020). However, Hudak & likely to affect the abundance of microplastics detected due to
Sette (2019) mostly observed red and purple fragments in their variation in sampling effort and the uneven distribution of micro-
study on grey seals. Of the six studies that report the presence of plastics throughout the digestive tract (Lusher et al., 2015; Moore
suspected microplastics, five presented information on polymer et al., 2020; Nelms et al., 2019b).
type for all, or a sub-sample of, particles using analytical polymer There was limited variation in collection of scat samples, as they
characterisation techniques, such as Fourier-transform spectros- were all taken from haul out sites, although these did vary between
copy (FTIR; Table S3). Of the confirmed microplastics, five main coastal and offshore locations. The amount of scat analysed varied
polymer types were reported (polyethylene, nylon/polyamide, among studies and was often not reported. The impact of the age
polypropylene, phenoxy resin and rubber; Table S4). One study also (i.e. time since deposition) of the scats was investigated in one
identified semi-synthetic particles, such as cellophane (Hudak and study, but no statistically significant difference in microplastic load
Sette, 2019). between fresh or aged scats was found (Perez Venegas et al., 2018).

3.3. Differences in methodological approaches 3.3.2. Extraction protocols

Once the gut content was extracted, potential microplastics were
There are three key steps in the determination of microplastic in isolated from organic material using a range of techniques,
scat and digestive tracts: 1) collection, 2) extraction and 3) identi- including physical separation (e.g. sieving and/or filtering), diges-
fication. In addition, the prevention of contamination is a key part tion (e.g. using chemicals or enzymes), or a combination of both
of determining microplastics levels. However, there are consider- (Table S3). Potassium hydroxide (KOH; usually a 10% concentration
able methodological differences across studies, preventing com- applied for a range of durations) was the most commonly used
parisons among studies. chemical digestion technique (Table S3), while Nelms et al. (2019b)
used enzymatic digestion with Proteinase K. Finally, the range of
3.3.1. Collection of samples filter and sieve mesh sizes (20 mme1000 mm) used to extract
The amount and origin of the gut content differed significantly microplastics also varied considerably (Table S3). This likely
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L.J. Zantis, E.L. Carroll, S.E. Nelms et al. Environmental Pollution 269 (2021) 116142

affected the number and sizes of particles detected in each study Therefore measures to limit and account for contamination are
(Lindeque et al., 2020). necessary for obtaining accurate estimates of microplastics
Similarly, for the scat samples, the digestion and filtration steps (Hidalgo-Ruz et al., 2012). Out of the 20 studies we reviewed that
differed significantly among studies (Table S3). Three studies did quantified microplastics in scat or gut content, there was a wide
not use or specifically detail a digestion step (e.g., Eriksson and range of contamination prevention protocols, ranging from absent
Burton, 2003; Hudak and Sette, 2019; Ryan et al., 2016), one pa- to extensive (Table S5). Five papers did not describe a contamina-
per physically degraded scat samples via homogenization tion protocol, and we assume they did not have any methods to
(Donohue et al., 2019), while the remaining four studies used limit or control for contamination in place (Table S5). However,
chemical digestion with KOH (Garcia-Garin et al., 2020; Perez- three of these five studies did not include fibres as they were seen
Venegas et al., 2018, 2020) or enzymatic digestion with protein- as a potential contamination source (Besseling et al., 2015; Bravo
ase K (Nelms et al., 2019a) (Table S3). The remaining paper used an Rebolledo et al., 2013; van Franeker et al., 2018).
alternative enzymatic digestion approach where scats were During sample preparation and analysis, the most common
machine-washed in fine-mesh laundry bags with washing deter- methods used to prevent contamination were to cover samples
gent (Bravo Rebolledo et al., 2013). The size of the mesh used during when not used (n ¼ 14 publications), the use of clean equipment
the filtration step likely influences the findings, as highlighted in (e.g. wiped with ethanol and Milli-Q water; n ¼ 12), to work under
the previous section. For example, Perez-Venegas et al. (2018) used appropriate conditions that minimize environmental contamina-
fine mesh (0.7 mm) which was several orders of magnitude finer tion in the laboratory (e.g. positive pressure laminar flow hood;
than that used by Ryan et al. (2016; 0.5 mm). The ability to detect n ¼ 6) and to wear non-synthetic clothing (e.g. cotton lab coats;
smaller microplastics will likely increase the detectable amount in n ¼ 7). Finally, to account for possible airborne contamination some
the scat (Huvet et al., 2016; Lenz et al., 2016). studies (n ¼ 5 publications) exposed a wet filter in a Petri-dish to
the same conditions as the samples and examined them for parti-
3.3.3. Identification of potential microplastics cles. Negative controls or blanks were also used to determine any
There is a wide range of approaches used to identify potential background contamination (n ¼ 11 publications). Four studies also
microplastics extracted from samples (Table S3). The simplest and sampled equipment for further analysis to compare with their
cheapest form is visual identification of potential microplastics, findings, three sampled plastic equipment used in the laboratory
however, it is important to note that this method could give high (Donohue et al., 2019; Hudak and Sette, 2019; Nelms et al., 2019a)
error rates of up to 70% (Hidalgo-Ruz et al., 2012). Therefore it is and one took clothing samples during sample collection (Moore
highly recommended for microplastics to undergo further analysis et al., 2020).
and identification (Dekiff et al., 2014). A variety of more precise Of the 16 papers with contamination control measures in place,
methods are available to characterise the microplastic polymer, only four had a very detailed protocol, which accounted for
ranging from thermal analysis to spectroscopy (Hidalgo-Ruz et al., contamination during all stages of sample processing, from
2012; Shim et al., 2017). Additional analysis is important as it collection to analysis. In these studies, control samples from
gives more information on whether a particle is an actual micro- clothing were taken during animal sample collection and blanks
plastic, while providing additional information on the type of were used during the microplastic analysis to monitor potential
plastic and, potentially, its origin and source (Dioses-Salinas et al., contamination. In addition, the analysis was done inside a positive
2020; Schwarz et al., 2019). pressure laminar flow hood, equipment was cleaned in advanced, if
Of the studies that directly measured microplastics from scat or possible plastic material was avoided and cotton lab coats and
inside organisms (n ¼ 20), four studies used visual identification gloves were worn (Nelms et al., 2018, 2019b; Donohue et al., 2019;
under a microscope only (Table S3). As indicated above, these re- Moore et al., 2020). However, most papers had a much less elabo-
sults need to be treated with caution due to potential high error rate protocol, and often only checked for a limited number of
rates in the identification process (Lusher et al., 2020). The majority contamination sources (Table S5). Moreover, some contamination
of studies did perform further analyses to characterise the type of protocols might not be very effective, or could actually introduce
polymer found, with 12 using (micro-)Fourier transform infrared microplastics (for example, rinsing with tap water without col-
(FTIR) analysis, one Raman Spectroscopy and one a Phazir (NIR) to lecting the residues, Bourdages et al., 2020). Importantly, as some
characterise the type of polymers found (Table S3). In addition, studies had no or limited measures in place, it is difficult to be
three studies did not use or define any methods to confirm that the confident that the suspected microplastics are actual microplastics
particles found were microplastics (Table S3). from collected samples. Several studies without a protocol to
Encouragingly, more recent studies (i.e., publication from 2019 determine air contamination excluded microfibres from their re-
to 2020) are more likely to use FTIR spectrometry to identify sults and considered them all as airborne contamination (Table S5).
polymer types. However, FTIR identification is an expensive pro- This method, however, might underestimate the presence of
cess, and most studies only analyse a subset of their suspected microplastic in animals, as the majority of microplastic detected in
particles. Importantly, when using techniques such as FTIR it is key samples are microfibres (see Tables S2 and S4).
to have clear QA/QC protocols in place, for example a threshold for Several of the more recent papers had more detailed and elab-
matching, to minimize misclassification (Kühn et al., 2020). orate protocols for contamination prevention compared to papers
Furthermore, terminology varies significantly among studies and if which were published 3e15 years ago (Table S5), highlighting the
polymer types are not confirmed, terminology needs to include increased awareness among scientists about the risk of contami-
caveat, e.g. “suspected”, “putative” or “potential” microplastics. nation (Hidalgo-Ruz et al., 2012; Lo € der and Gerdts, 2015; Nore

n,
Determining the colour of a potential microplastic can be very 2007).
subjective, depending on the viewer’s perception of a colour and
can be influenced by background colour of the filter or light used
during microscopic analysis for example. 3.4. Best practices for future studies

3.3.4. Contamination prevention As highlighted in previous sections, the differences in contam-

The contamination of samples with microplastics during ination protocols among studies make comparing results across
collection, preparation and analysis, can alter the results of a study. species difficult. In order to facilitate harmonisation across studies,
6
L.J. Zantis, E.L. Carroll, S.E. Nelms et al. Environmental Pollution 269 (2021) 116142

Fig. 2. Recommended standardised protocol for limiting and accounting for potential environmental and laboratory contamination during microplastic analyses of gut content and
scat analyses of marine mammals.

we have developed a standardized protocol to limit and account for 2019a).

potential contamination sources in different key steps of the
collection and extraction process (Fig. 2). By using this proposed 3.5. Microplastics exposure assessment
standardized protocol, we can improve comparability, reproduc-
ibility and transparency across studies. Aside from quantifying levels of microplastics in organisms and
In addition, there is a wide range in reporting of results scat, a total of ten studies attempted to infer exposure (and
(Table S3). In order to facilitate meaningful comparisons across sometimes risk) levels of microplastics to marine mammals, all
studies, we have also developed guidelines for the collection and focussing on cetaceans (Table S6). Six of these studies linked habitat
reporting of qualitative and quantitative metrics during micro- or prey species to exposure risk, while four studies attempted to
plastic studies (Fig. 3). We also recommend defining colour cate- use chemical and biological markers to assess exposure levels.
gories (e.g. making “orange, yellow, gold” one category) to make
results more consistent (Gauci et al., 2019; Wright et al., 2013, 3.5.1. Linking habitat and prey to exposure risk
Fig. 3). Adoption of these guidelines will enable future work to be The linking of habitat and prey to exposure risk has been done,
synthesised to facilitate comparisons across studies, comparisons both on a global scale (Germanov et al., 2018; Burkhardt-Holm &
by taxa, and to identify species or regions with highest levels of N’Guyen, 2019), as well as a more regional scale (Fossi et al.,
exposure. Moreover, to ensure transparency and reproducibility in 2017; Guerrini et al., 2019). A broad scale study was conducted by
science, raw data per sample should be made available as supple- Germanov et al. (2018) in which baleen whale distribution was
mentary material or as online dataset (see https://www.nature. combined with recognized microplastic hot-spots. Not only did the
com/sdata/policies/repositories#other for suggested databases). paper provide some insight into the overlap between whale habitat
To allow for better comparison across studies, we suggest and microplastic hotspots, it also highlights how the biology of
reporting i) total number of microplastics found and total number individual species needs to be adequately accounted for in broad-
of samples (scat or GIT) analysed; ii) proportion of samples which scale assessments and modelling exercises. For instance, hump-
had at least one microplastic, and iii) the microplastic load on a per back whales were considered to have a presence in all key buoyant
gram basis, clearly stated as wet or dry weight. In addition to microplastic pollution hotspots bar one (Mediterranean Sea) by
reporting, the identification of prey species or trophic level of the Germanov et al. (2018). However, exposure risk might not be high
prey species within a study would be a major step towards un- in each of the microplastics hotspots. For example, satellite
derstanding microplastic exposure from trophic transfer. However, telemetry work in the South Atlantic shows that humpback whales
most studies did not determine prey species or trophic level in their migrate through the South Atlantic gyre, likely with minimal
studies, even though well-developed protocols are available. In feeding (Zerbini et al., 2006, 2011), and therefore the actual expo-
pinnipeds, identification of otoliths or other hard parts in scat has sure is most probably minimal as foraging is unlikely to occur here.
been a common method of assessing diet for decades (Bowen and The approach by Burkhardt-Holm & N’Guyen (2019) did include the
Iverson, 2012; Tollit et al., 2009). DNA diet methods are also feeding biology of whales, and this approach is therefore, in our
becoming more common and affordable (Pompanon et al., 2012), opinion, a better approach to estimate levels of exposure. However,
and have been used in both cetaceans (Carroll et al., 2019; de Vos uptake via seawater was not included in the assessment, even
et al., 2018; Jarman et al., 2002) and pinnipeds (Casper et al., though that is a likely important source for mega-filter feeders
2007; Deagle et al., 2009; Hardy et al., 2017). Concurrent assess- (Burkhardt-Holm & N’Guyen, 2019).
ment of diet and microplastic load per scat/GIT sample should be In contrast to the previous two studies, more detailed and
encouraged in future studies to start building a picture of exposure complex modelling studies were conducted by Fossi et al. (2017)
from environmental and trophic transfer routes (Nelms et al., and Guerrini et al. (2019). Fossi et al. (2017) conducted a study in
7
L.J. Zantis, E.L. Carroll, S.E. Nelms et al. Environmental Pollution 269 (2021) 116142

Fig. 3. Key information to report in any marine mammal study on microplastics.

which field measurements of zooplankton, microplastic abundance model.

and cetacean survey data were combined with models on ocean Importantly, both Fossi et al. (2017) and Guerrini et al. (2019)
circulation and potential fin whale habitat. This resulted in a pre- highlight that their approach could be used in risk assessment.
liminary risk assessment for whales, highlighting that areas with However, in our opinion it provides a confirmation that there is risk
high levels of microplastic overlap with fin cetacean habitat and of exposure of fin whales within the area but falls short of a risk
several sightings (Fossi et al., 2017). Guerrini et al. (2019) used a assessment. In risk assessment there is a need to determine the
model to track particles from release points (sources) to estimate severity and the probability of adverse effects (Suter II, 2016), not
the hazard. This approach does allow for identifying areas where just exposure to a contaminant. In both cases the adverse effects of
exposure might be relatively high. However, there currently is microplastics on whales were not assessed, only the likelihood of
limited data on the contribution of microplastics from different exposure. Additionally, to conduct a risk assessment future
sources, and this data is needed to improve the accuracy of the research should focus on i) how long microplastics remain inside

8
L.J. Zantis, E.L. Carroll, S.E. Nelms et al. Environmental Pollution 269 (2021) 116142

the digestive tract and whether there is transfer to the tissue of mesopelagic fish were determined within the North Atlantic, and
marine mammals (Perez-Venegas et al., 2018) and ii) whether these levels were linked to dietary composition. Lusher et al. (2016)
microplastic exposure results in any effects on animal health (Claro estimated that a single individual could be exposed to 1.3 million
et al., 2019; Panti et al., 2019). particles day1, or 463 million particles year1. As far as we are
aware, these are the only studies that attempt to quantify uptake
3.5.2. Phthalates and other persistent contaminants as biomarkers through trophic transfer in wild marine mammals. In addition, two
Four papers that investigated the use of biomarkers to predict studies (Fossi et al., 2014, 2016) attempted to quantify the levels of
marine mammal exposure to microplastics. The studies focus on microplastic taken up by fin whales, based on microplastic abun-
phthalate levels [predominantly mono(2-ethylhexyl) phthalate dances recorded for seawater and the whales’ filtering capacity.
(MEHP) and bis(2-ethylhexyl) phthalate (DEHP)], within the envi- Uptake was estimated to be 3653 particles day1 (Fossi et al., 2014)
ronment, in zooplankton and/or in whale blubber. Phthalates are and “thousands of particles” per day (Fossi et al., 2016).
added to plastics to increase plasticity and can leach from plastic Although this could be an interesting and illustrative approach
into the environment (Hermabessiere et al., 2017; Teuten et al., to quantify uptake of microplastics from the water column, it is
2009). In addition, phthalates can bioaccumulate in organisms, over-simplified and significant improvements are needed. We
and can cause potential adverse effects, including effects on embryo highlight this point, as an extreme example, by taking the blue
development and reproduction, and the disruption of endocrine whale (Balaenoptera musculus) feeding of the Coast of British
functioning (Gunaalan et al., 2020; Hermabessiere et al., 2017). Columbia in the Northeast Pacific Ocean. The blue whale can engulf
However, we want to highlight several issues with these studies 83 m3 of sea water per mouthful (Goldbogen et al., 2011). Desforges
which need to be addressed before this approach can be used to et al. (2014) conducted a study on microplastics in the size range
determine exposure levels. First of all, in all these studies the 62e5000 mm and found an average level of 279 particles m3, but a
variance was often (very) high making meaningful statistics diffi- range from 8 to 9200 particles m3. This means that, based on this
cult to perform. In many cases the coefficient of variance (CoV; reported range, a blue whale feeding of the coast of British
standard deviation/mean x 100%) exceeded 100% for key mea- Columbia could engulf anywhere between 663 and 763600 parti-
surements (e.g. microplastic levels and DEHP and MEHP levels in cles per mouthful. However, there is considerable uncertainty
zooplankton and whale blubber). Secondly, phthalates (including about levels of microplastics in surface waters, especially at lower
MEHP) are used in a range of different products and industrial size ranges of plastics (Huvet et al., 2016; Lenz et al., 2016). A recent
processes, and therefore can enters the environment from different study of the coast of British Columbia using advanced quantifica-
sources, including wastewater (Jiang et al., 2018). This makes the tion techniques to detect particles as small as 5 mm estimated
direct linkage between MEHP levels in organisms and microplastic average levels of ~4 million microplastic m3 in the open ocean and
exposure difficult to establish. Finally, these studies had low sample 15 million microplastic m3 in coastal waters (empirical findings;
sizes (for example Baini et al. (2017) sampled between n ¼ 1 and Brandon et al., 2020). Using this range, it can best estimated that
n ¼ 3 animals per species), and therefore can only be used as blue whales could be exposed to between 332 and 1245 million
preliminary studies (which was also highlighted by the authors). microplastics per mouthful. Clearly, given this range between
For these reasons, significant further work is needed to validate and studies, significant work needs to be done to estimate exposure
optimize this approach. levels of marine mammals to microplastics.
In addition, the level of other organochlorine contaminants
(HCB, DDT and its metabolites and PCBs) were determined in Fossi 4. Conclusion
et al. (2016), as well as certain biomarkers, including CYP1a and
CYP2b (CYP family of enzymes, responsible for the metabolism of Charismatic megafauna such as marine mammals can help bring
organic contaminants) and lipid peroxidation (LPO: indicator of the public’s attention to anthropogenic impacts. However, to fully
oxidative stress). The organochlorine contaminants were included assess risk of exposure to threats, and how they vary across species
based on the Trojan Horse hypothesis, which is centred around the and ecosystems, standardised analysis and reporting protocols are
idea that microplastic can be a vehicle for the transfer of other required. Therefore, a key output of this paper is a framework to
organic contaminants into organisms (Burns and Boxall, 2018). improve consistency across studies that examine the incidence of
However, this hypothesis is widely debated (Burns and Boxall, microplastics in marine mammal gut and scat. We strongly urge
2018), and there is no consensus in the scientific community that scientists working in this field to adopt our protocols where
microplastics are a major source of transfer of organic contami- possible. However, if not possible, for example due to financial or
nants into organisms (Bakir et al., 2016; Burns and Boxall, 2018; technical constrains, transparency about study constraints is
Lohmann, 2017). Therefore, this approach should also be used with essential. Alternatively, increased collaborations between partners
caution. and institutions with access to advanced equipment would help
optimize the quality of reported data. In addition, a continuous
3.5.3. Total exposure search to develop improved and more affordable technology to
Though the papers above attempt to determine risk of exposure extract and identify microplastics is needed, but this is important
and identify markers of exposure, only very few studies have for all studies focussing on microplastic pollution, as this research
attempted to quantify total exposure levels. A first attempt was field seems likely to continue to burgeon in the future.
made by Desforges et al. (2015) which estimated levels of micro- Overall, it is encouraging to see the marine mammal community
plastics in two foundation zooplankton prey species (Neocalanus produce a rapidly growing body of work on the exposure of these
cristatus and Euphausia pacifia) in the Northeast Pacific. They taxa to microplastics. Microplastics were detected in most marine
encountered microplastics in 2.9% and 5.9% in N. cristatus and mammals samples analysed, with large variation among samples,
E. pacifia, respectively. Using these results, the authors estimated even within studies. A key next step is to try and understand im-
that a humpback whale in coastal British Columbia is exposed to pacts of microplastics on marine mammal health, for example by
300 000 microplastics d1 (assuming it consumes 1.5% of its body using marine mammal cell lines linked directly to empirical mea-
weight in krill and zooplankton every day). In a similar way, Lusher surements of microplastic exposure. The use of biological or
et al. (2016) attempted to determine microplastic exposure of chemical markers was suggested in several preliminary studies, but
striped dolphins through trophic transfer. Levels of microplastic in significant work is needed to confirm that these markers can be
9
L.J. Zantis, E.L. Carroll, S.E. Nelms et al. Environmental Pollution 269 (2021) 116142

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