Analysis of microplastics in the intestines of
stranded cetaceans
1

Liu Yang, 2Ayaka T. Matsuda, 3Toshikazu Kawaguchi, 4Helena Fortunato,
2,5
Takashi F. Matsuishi
1

Graduate School of Global Food Resources, Hokkaido University, Sapporo, Japan;
Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan; 3 Faculty ofEnvironmental Earth Science, Hokkaido University, Sapporo, Japan; 4 Faculty of Science,
Hokkaido University, Sapporo, Japan; 5 Global Centre for Food, Land and Water
Resources, Hokkaido University, Sapporo, Japan. Corresponding author: T. F. Matsuishi,
catm@fish.hokudai.ac.jp
2

Abstract. Microplastics derived from anthropogenic pollution have become a major issue today, asavailable data have shown they are present in all terrestrial and marine ecosystems. These pollutants
originate from multiple sources, from trash dumped in rivers and lakes and travelling to the ocean, tofishing tools (such as nets, ropes, and baskets) abandoned at sea as well as various chemicals releasedin the water. As apex predators, cetaceans are critical in regulating the food web and maintaining
ecosystem balance. They consume lower trophic-level organisms that have ingested microplastics, and,at present, the studies conducted on microplastics in cetaceans are insufficient. Further research on theprevalence and effects of microplastics in these animals is needed to support conservation efforts andshed light on the larger issue of plastic pollution in the oceans. Determining the concentration of
microplastics in cetaceans can inform on the level of microplastic contamination in the entire food web,including seafood that humans consume. This research aimed to assess the presence and accumulationof microplastics in cetaceans to understand better their potential impacts on both these species and themarine environment. Microplastics were detected in 13 out of 17 samples of cetacean intestines; most ofthem were fibres and varied in length, size, and colour. Raman spectroscopy analysis showed that the
microplastics were made of the polymer Nylon 6,6. The results of this study will contribute to the current
knowledge of the impact of microplastics on marine life and ecosystems and can aid in developing
strategies to mitigate and manage this urgent environmental issue.
Key Words: microplastics, plastic fibre, Raman spectroscopy analysis, stranding.

Introduction. Cetaceans are a key indicator of the health of marine ecosystems because
they occupy the highest trophic level (Rogers & Greenaway 2005; Bossart 2006). The
study of these animals can help to identify current or potential threats to marine
ecosystems and define strategies to deal with them (Bossart 2006; Guzzetti et al 2018).
Most cetacean species have been shown to ingest a wide range of plastics (Ryan
2019), from microplastics, such as particles and fibres, to macroplastics, such as plastic
sheets, fishing nets, fishing lines, household items, etc. (Fossi et al 2020). The
International Whaling Commission considers plastic pollution an urgent issue since it may
affect the mortality of cetaceans (IWC 2020).
Two extant cetacean suborders, i.e., baleen whales (Mysticeti) and toothed whales(Odontoceti), greatly differ in terms of their feeding ecology, which affects the evaluationof the impact of microplastics on them. Baleen whales can be exposed to plastic pollution
through the direct uptake of contaminated water and prey as they filter large volumes ofwater while feeding (Bannister 2009; Desforges et al 2015; Germanov et al 2018;
Burkhardt-Holm & N’Guyen 2019; Guerrini et al 2019). By contrast, toothed whales,
which hunt and swallow the prey whole or torn into pieces, should be more prone toincorporate plastics through the ingestion of plastic-contaminated prey, i.e., throughtrophic transfer (Würsig et al 2009; do Sul & Costa 2014; Au et al 2017; Nelms et al
2018; Perez-Venegas et al 2018).

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Both the direct and indirect ingestion of microplastics via filter feeding and prey
consumption, respectively (Fossi et al 2020; Zantis et al 2021) may lead tobioaccumulation (Koelmans 2015; Miller et al 2020; Xu et al 2020). For example, a
relatively high number of synthetic particles (mean value 0.057 particles g-1) was foundin krill ingested by fin whales on the western coast of Iceland (Garcia-Garin et al 2021). A
study found about 45 plastic items in the body of a fin whale floated off conducted off
Jeju Island, South Korea, including fishing lines, parts of fishing nets, plastic filaments,and styrofoam pieces (Im et al 2020). Several polymer types (polyethene, propylene,
nylon, and polyethene terephthalate, among others) were found in humpback whales(Megaptera novaeangliae) in the eastern North Pacific (Besseling et al 2015; Alava 2020;
Zantis et al 2021). In addition, another study found that blue whales (Balaenoptera
musculus) feeding on krill in the Gulf of California are much more exposed to microplastic
contamination through prey transfer than humpbacks, which feed predominantly on fish
(Kahane-Rapport et al 2022).
Toothed whales are also commonly affected by microplastic pollution due to theconsumption of contaminated prey. High amounts of microplastic particles were detectedin the gastrointestinal tracts of beluga whales (Delphinapterus leucas) from Canada’s
Northwest Territories (Jacobsen et al 2010; Moore et al 2020). Similarly, a study ofTrue’s beaked whales (Mesoplodon mirus) stranded on the coasts of Ireland reported alarge number of microplastics mixed with the remains of mesopelagic fish andcephalopods among their stomach contents (Lusher et al 2015).
It is crucial to improve our understanding of microplastic ingestion by cetaceans
for various reasons. First, cetaceans are apex predators in the ocean and can accumulate
high levels of microplastics that can potentially damage their health and threaten theirsurvival (Harlacher 2020; Liu et al 2022). Furthermore, these species are valuable
indicators of the health of marine ecosystems. Therefore, the analysis of microplastics
present in their bodies can provide valuable insights into the extent and impact of marine
plastic pollution (Wilcox et al 2016).
Hokkaido is located in the Northern part of Japan, surrounded by the Pacific Ocean,
the Okhotsk Sea, and the Sea of Japan. The northwestern Pacific Ocean is known for its
high levels of microplastic pollution (Pan et al 2019). On average, 70 cetacean strandings
are observed annually, and the Stranding Network Hokkaido (SNH) collects specimens of
stranded cetaceans and delivers them to various institutions for academic purposes.
The objective of the study was to evaluate the presence of microplastics incetacean intestines collected in Hokkaido, Japan, in order to increase the available
information for the investigation of the potential impacts of these pollutants on marine
ecosystems.
Material and Method
Materials. Specimens collected by SNH were used for this research. The SNH is a
nonprofit organisation that records information related to cetacean strandings, including
beaching, drifting, and bycatch, and collects specimens of the stranded animals. Since its
establishment in 2007, the network has called on the general public, administrative
bodies, fishery officials, and others to report information on strandings to the dedicatedreceiving and reporting desk “IRUKA KUJIRA 110” and recorded an average of about 70
cases per year, with a cumulative total of over 1000 cases by 2022 (Stranding Network
Hokkaido 2023). Upon receiving the information, the SNH investigates theapproachability and condition of the animals and determines the feasibility of conducting
a survey. If a survey is deemed feasible, permission is obtained from the relevant
authorities, and the survey is carried out. Specimens are collected by searching forexternal morphological information and external injuries. The SNH obtains requests inadvance from research institutions in regard to the samples needed for analysis. Whenthe specimens are available, they are sent to the institutes free of charge andunconditionally. The use of stranded specimens allows research to be conducted without
unnecessary collections of living specimens, thus contributing to conservation efforts.

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The intestines of 11 cetacean specimens were obtained during 10 January 2019
and 14 April 2022 from different locations in Hokkaido. Table 1 shows the detailed
information for each individual used in this study. A total of 17 samples were preparedand screened for the presence of microplastics. A 5-cm sample was taken from each
received intestine. In three cases, multiple samples were prepared: five for individual
SNH21007 and two each for individuals SNH20091 and SNH190032. To avoid
contamination, samples were wrapped in aluminium foil and frozen before the analysis.
The study of carcasses can be complicated due to uncertainties associated with thedetermination of the stage of decomposition. However, it is crucial to have a system inplace to evaluate the quality of the material being studied. A code-based system wasestablished by the Smithsonian Institution’s Scientific Event Alert Network. In Table 1,
CODE 2 defines a carcass in good condition, while CODE 3 means that the carcass isdecomposed, but the organs are basically intact (Geraci & Lounsbury 2005). In thepresent study, the experimental intestines were considered adequate.
An experiment was conducted to ascertain if all plastics emitted fluorescent lightand could thus be observed under a fluorescence microscope. Small pieces (less than 5mm) were cut from several plastic items commonly used in households and industries,
and they were marked and placed in one intestinal sample. This was then examined
under a fluorescence microscope using the same conditions as those used for the othersamples in this study. The materials used for this test were: polyurethane (PU),polyethylene terephthalate (PET), poly (methyl methacrylate), i.e., acrylic plastic andplexiglass (PMMA), and polystyrene, i.e., foams (PS). The observed microplastic fibers
were classified based on their size, color, shapes, and possible origins. Furthermore, theirchemical composition (polymer types) was identified using Raman analysis.
Methods. Raman spectroscopy is a useful tool for the analysis of microplastics. It can
provide specific information about the chemical composition and structure of a sample
without the need for sample preparation or destruction. This makes it a non-destructive
and efficient method for identifying microplastics (Shim et al 2017). Another merit ofRaman spectroscopy is its ability to differentiate between different types of polymers
based on their unique Raman spectral signatures (Vankeirsbilck et al 2002). Based on
their characteristic peaks, the spectra of polyethene, polypropylene, and polystyrene canbe easily separated. Here, Raman spectra (RENISHAW inVia Raman Microscope) wereused to identify the microplastics found in the samples. The measured spectra were
uploaded to Open Specy and compared with those in the database library. Open Specy
automatically displays the highest matching polymer spectra, thus allowing theidentification of microplastics.
To detect microplastics more efficiently in the studied samples, the following threeobservation methods were adopted:a). Digestion: microplastics were extracted from the intestines following Chen’s
procedure (Chen et al 2020). After lightly washing their surface with Mili-Q water, thesamples were placed into a 10% KOH solution, and a water bath was used to accelerate
the digestion process. With the help of a vacuum pump, the solution was filtered throughfilter paper (20-25 µm). Labelled plastic disks were placed into a labelled Petri dish andsealed with sticky tape. An optical microscope (Nikon ECLIPSE 50i) was used topreliminarily identify the particles and assess if they were made of plastic. Observations
were made under 100x magnification. The obtained images were used to evaluate theshapes and sizes of the observed microplastics.
b). Direct observation: after the intestines were washed with Mili-Q ultrapure
water, they were opened and flattened with the help of clips to expose the inner wall
(Figure 1). A digital microscope was used to search for larger plastic fragments in thesample under a magnification of 200x.
c). Fluorescence microscopy: this method was used to reduce the likelihood of
overlooking microplastics present in the studied samples. A cryostat (Leica CM 3050S)
was used to slice the samples for further observation under a fluorescence microscope.
Figure 2 shows the stages of this process.

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Table 1
Stranding locations and species of sampled cetaceans
Sample ID

Strandedlocation

SNH20091

41.986064N 140.906982E

SNH19001

41.868298N 140.115493E

SNH20032

41.940520N 143.242260E

SNH21060

42.389176N 141.084797E

SNH21052

42.600722N 141.488556E

SNH22001

42.010660N 140.104330E

SNH22006

41.468139N 140.029861E

SNH21066

42.619847N 141.564035E

SNH21007

42.697500N 140.051944E

SNH22016

42.331027N 141.022174E

SNH21036

42.393920N 140.906860E

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Species name
Balaenoptera
acutorostrata
Lagenorhynchus
obliquidens
Stenella
coeruleoalba
Stenella
coeruleoalba
Stenella
coeruleoalba
Lagenorhynchus
obliquidens
Phocoenoides
dalli
Stenella
coeruleoalba
Phocoena
phocoena
Phocoena
phocoena
Stenella
coeruleoalba

CommonnameMinke whale

SuborderBody length

Sex

Mysticeti

495.0 cmMale

Decomposition
stage
CODE 2

Pacific white-sided dolphin

Odontoceti

219.9 cm

Male

CODE 3

Striped dolphin

Odontoceti

176.0 cm

Male

CODE 2

Striped dolphin

Odontoceti

191.1 cm

FemaleCODE 2

Striped dolphin

Odontoceti

196.9 cm

FemaleCODE 2

Pacific white-sided dolphin

Odontoceti

202.4 cm

Male

CODE 3Dall’s porpoise

Odontoceti

196.5 cm

Male

CODE 3

Striped dolphin

Odontoceti

215.7 cm

Male

CODE 2

Harbour porpoise

Odontoceti

178.6 cm

FemaleCODE 3

Harbour porpoise

Odontoceti

136.1 cm

FemaleCODE 2

Striped dolphin

Odontoceti

186.8 cm

FemaleCODE 22749

Figure 1. Dissection of the intestine to expose the inner wall.

Figure 2. Images illustrating the preparation of an intestinal sample for observation under
the fluorescence microscope.
Results. Microplastics were found in 13 out of the 17 samples of cetacean intestines
examined (Table 2). The two samples without microplastics were from individuals
SNH20032 and SNH22006 (Table 2). The observed particles showed differences in length,size, and colour (Figure 3), and most were fibres (Figure 4). Only Nylon 6,6 was
identified by Raman spectroscopy (Figure 5). Figure 6 shows the test experiment results
using common household items made of different plastic polymers.
Table 2Presence of microplastics in stranded cetaceans from various locations in Japan (forspecies names and detailed location information, refer to Table 1)
Sample ID
SNH20091
SNH19001
SNH20032
SNH21060
SNH21052
SNH22001
SNH22006
SNH21066
SNH21007
SNH22016
SNH21036

Number ofsamples taken
2
1
2
1
1
1
1
1
5
1
1

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Observation of microplastics
in the samples
Presence
Presence
Absence
Presence
Presence
Presence
Absence
Presence
Presence
Presence
Absence2750

Figure 3. Typical microplastics found in cetacean intestines: a) green fragment; b) red
fragment; c) pink fiber; d) green fiber; e) green fiber; f) yellow fiber.

Figure 4. Detailed fluorescence microscope images of microplastic fragments and fibers
found in cetacean intestines.

Figure 5. Raman spectra of Nylon 6,6.
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Figure 6. Images of the experiment testing different plastic materials. a) PU(polyurethane) and b) PET (polyethylene terephthalate) are clearly identified under the
fluorescence microscope, while c) PS (polystyrene) and d) PMMA (poly (methyl
methacrylate)) cannot be distinguished from the surrounding tissue under the
fluorescence microscope.
Discussion. Our study aimed to ascertain the presence of microplastics in the intestinesof stranded cetaceans in several areas of Japan and characterise the detected particles.
Plastic production and its use are continuously increasing (Rhodes 2018) despite thealarming data reported in numerous studies (Thushari & Senevirathna 2020). Indeed,plastic pollution and its consequences for marine diversity have been widely documented(CBD 2012; IWC 2020; Zantis et al 2021). Although observations of microplastic
ingestion in cetaceans have increased (Besseling et al 2015), they are still lagging behindthose in other marine groups. Being top predators, cetaceans are key sentinel species toevaluate the health of the world’s oceans (Rogers & Greenaway 2005; Bossart 2006;
Guzzetti et al 2018). Thus, it is crucial to increase our knowledge of the consequences ofplastic pollution to develop better strategies and guidelines for the protection of thisimportant group of marine organisms.
As previously mentioned, toothed whales (Odontoceti) and baleen whales(Mysticeti) have different feeding habits, the former being top carnivore species while thelatter obtaining nourishment through filter feeding (IWC 2020; Zantis et al 2021).
Microplastic particles were found in most analysed samples through fluorescence andoptical microscopy. The small amounts detected may be related to the fact that theintestines examined derived primarily from toothed cetacean species. Indeed, only one
baleen species was examined in this study. Baleen whales are more exposed tomacroplastics because of their feeding ecology (Jacobsen et al 2010; Unger et al 2016).
Predictions based on observations and modeling have shown that toothed cetaceans
feeding on fish and cephalopods would be less impacted by microplastics than krillfeeding baleen species (Kahane-Rapport et al 2022). The most numerous microplastics
observed in this study were fibres, which is in line with the results of several previous
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studies of cetaceans and other marine vertebrates (Lusher et al 2015; Lusher et al 2018;
Nelms et al 2019).
In order to compare data, the same methodology should be followed, if at all
possible. Unfortunately, it was not possible to access the instruments normally used forthis type of study. Consequently, it was decided to use fluorescence microscopy. The useof this method to observe microplastics in cetacean intestines has not been reported
before, so this analysis also has value as a methodological first trial. Even thoughfluorescence microscopy could clearly capture microplastic images, sample processing
was long and inefficient. Therefore, only one sample was used for this trial to confirm
that fluorescence microscopy produces clear images of microplastic particles. We would
recommend this method for studies investigating smaller samples.
The small amounts of microplastics found in this study could be due to several
reasons. First, fluorescence microscopy may not be the most efficient method to observe
microplastics in the intestines of marine mammals, as it is normally used to detectmicroplastics in water (Qiu et al 2015; Dehghani et al 2017; Scircle & Cizdziel 2020). In
addition, many microplastics have low intrinsic fluorescence signals or lack them together(Yan et al 2012; Spizzichino et al 2016; Karakolis et al 2019), making their observation
quite challenging. Figure 6 shows that while PU and PET particles have strong
fluorescence emission signals, the signals are very weak and cannot be observed under
the fluorescence microscope. One way to improve the fluorescence emission signals is tostain the samples before observation using fluorescent dyes such as Nile Red (Maes et al2017). It was our intention to check the reliability of this method in tissues.
Unfortunately, our samples were very brittle after being processed for the microtome, sothey could not be further used to separate the microplastics from the medium. Therefore,
we relied on the digestion method to assess the presence of microplastics in the samples.
It is generally difficult to compare studies of microplastic contents in cetaceans
due to methodological issues. As explained above, fluorescence microscopy was chosenbecause the instruments normally used in other studies were not available. Unfortunately,as our experiments with this method did not yield reliable results, we turned to lesscommonly used methods, such as the digestion of tissues. Furthermore, some previous
studies examined both the stomach and intestines (Lusher et al 2015; Lusher et al 2018),
while others used subsamples to extrapolate the total potential amounts of microplastics(Moore et al 2020). This study examined only the intestines, which was possibly not
sufficient to detect the full extent of microplastic contamination. Several studies havereported blue and black as the most frequent microplastic colours (Nelms et al 2019). In
contrast, this study found mostly green fibres and fragments (Figure 3), as well as anumber of red and yellow fragments.
Conclusions. This study reports for the first time the presence of Nylon 6,6 in cetaceans.
This robust plastic, often used to manufacture pelagic fishing nets, is considered acommon source of microplastics in the ocean. More studies are necessary to assess its
possible transfer to the animal food chain in the marine environment.
As plastic pollution increases in the oceans, more data from ecologically criticalprey species are needed to accurately assess the risks posed by microplastics. New dataobtained from different species at all trophic levels would allow to achieve a complete
understanding of plastic distribution in marine food webs and its consequences for apex
predators, including predatory fish, seabirds, and marine mammals.
Acknowledgements. Prof. Takahashi Masashi provided a fluorescence microscope andmicrotome and the Stranding Network Hokkaido provided the cetacean samples for thisstudy. This work was partly supported by the GCF, Hokkaido University, and JST SPRING,
Grant Number JPMJSP2119.
Conflict of interest. The authors declare that there is no conflict of interest.

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Received: 31 August 2023. Accepted: 09 October 2023. Published online: 29 October 2023.
Authors:
Liu Yang, Graduate School of Global Food Resources, Hokkaido University, Sapporo, Hokkaido 060-0809 Japan,
e-mail: yangliu20190101@yahoo.co.jp
Ayaka T. Matsuda, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611 Japan,
e-mail: matsuda@fish.hokudai.ac.jp
Toshikazu Kawaguchi, Faculty of Environmental Earth Science, Hokkaido University, Sapporo, Hokkaido 0600810 Japan, e-mail: t_kawa@ees.hokudai.ac.jp
Helena Fortunato, Faculty of Science, Hokkaido University, Sapporo, Hokkaido 060-0810 Japan, e-mail:helenaf@sci.hokudai.ac.jp
Takashi Fritz Matsuishi, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611
Japan, e-mail: catm@fish.hokudai.ac.jp
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution and reproduction in any medium, provided the original author and source
are credited.
How to cite this article:
Yang L., Matsuda A. T., Kawaguchi T., Fortunato H., Matsuishi T. F., 2023 Analysis of microplastics in theintestines of stranded cetaceans. AACL Bioflux 16(5):2746-2756.

AACL Bioflux, 2023, Volume 16, Issue 5.
http://www.bioflux.com.ro/aacl

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