Microplastic fragments and microbeads in digestive tracts of planktivorous fish from urban coastal waters

Abstract

We investigated microplastics in the digestive tracts of 64 Japanese anchovy (Engraulis japonicus) sampled in Tokyo Bay. Plastic was detected in 49 out of 64 fish (77%), with 2.3 pieces on average and up to 15 pieces per individual. All of the plastics were identified by Fourier transform infrared spectroscopy. Most were polyethylene (52.0%) or polypropylene (43.3%). Most of the plastics were fragments (86.0%), but 7.3% were beads, some of which were microbeads, similar to those found in facial cleansers. Eighty percent of the plastics ranged in size from 150 μm to 1000 μm, smaller than the reported size range of floating microplastics on the sea surface, possibly because the subsurface foraging behavior of the anchovy reflected the different size distribution of plastics between surface waters and subsurface waters. Engraulis spp. are important food for many humans and other organisms around the world. Our observations further confirm that microplastics have infiltrated the marine ecosystem, and that humans may be exposed to them. Because microplastics retain hazardous chemicals, increase in fish chemical exposure by the ingested plastics is of concern. Such exposure should be studied and compared with that in the natural diet.

Full text

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Scientific RepoRts | 6:34351 | DOI: 10.1038/srep34351
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Microplastic fragments and
microbeads in digestive tracts
of planktivorous sh from urban
coastal waters
Kosuke Tanaka & Hideshige Takada
We investigated microplastics in the digestive tracts of 64 Japanese anchovy (Engraulis japonicus)
sampled in Tokyo Bay. Plastic was detected in 49 out of 64 sh (77%), with 2.3 pieces on average and up
to 15 pieces per individual. All of the plastics were identied by Fourier transform infrared spectroscopy.
Most were polyethylene (52.0%) or polypropylene (43.3%). Most of the plastics were fragments (86.0%),
but 7.3% were beads, some of which were microbeads, similar to those found in facial cleansers. Eighty
percent of the plastics ranged in size from 150 μm to 1000 μm, smaller than the reported size range
of oating microplastics on the sea surface, possibly because the subsurface foraging behavior of the
anchovy reected the dierent size distribution of plastics between surface waters and subsurface
waters. Engraulis spp. are important food for many humans and other organisms around the world. Our
observations further conrm that microplastics have inltrated the marine ecosystem, and that humans
may be exposed to them. Because microplastics retain hazardous chemicals, increase in sh chemical
exposure by the ingested plastics is of concern. Such exposure should be studied and compared with
that in the natural diet.
e plastic use continues to increase worldwide, and some waste plastics are released into the oceans1,2. Plastic
debris is ubiquitous in the oceans around the world; it is estimated that at least 5.25 × 1012 plastic particles weigh-
ing 2.7 × 105 t are currently oating at sea3. In particular, microplastics (dened as plastics < 5 mm4) are the most
common size fraction in seawater3,5,6. ey are divided into primary and secondary microplastics by their sources.
Primary microplastics are plastic particles originally manufactured at those sizes. Secondary microplastics are
fragments generated by the breakdown of larger pieces. Fragmentation of plastics at sea occurs through photo-
degradation, physical impacts, and other processes, and results in the generation of a larger number of smaller
particles2. Most microplastics in the marine environment are secondary3,7,8.
To the best of our knowledge, this is the rst report that chemically identied microbeads, which are one of
the major primary microplastics, in sh. Microbeads are spherical or amorphous particles used in personal care
and cosmetic products9. ey are made most commonly of polyethylene (PE), followed by nylon, polypropylene
(PP), poly(methyl methacrylate) (PMMA), and poly(ethylene terephthalate) (PET)10. Microbeads are discarded
down the drain and carried to sewage treatment plants (STPs). Although they are eciently removed by settling
during treatment, a small but signicant proportion is discharged in nal euent11. In addition, combined sewer
overows introduce microbeads into receiving waters. Aer their discharge, microbeads stay in water bodies for
a long time because of their non-biodegradable nature. Microbeads have been reported in surface waters12,13. e
other primary microplastics include plastic resin pellets, industrial scrubbers, and plastic powder. e pellets are
feedstock for consumer products, and are generally cylindrical or disk-shaped14. Industrial scrubbers include
synthetic ‘sandblasting’ media made from polymers such as acrylic, polystyrene, melamine, and polyester15.
Marine plastics aect a wide range of species, from invertebrates to seabirds and whales16. Microplastics are
bioavailable to many species16, and are easily ingested by planktivorous or smaller organisms17. Physical impacts
such as injury or clogging of the digestive tract18 and impairment of feeding capacity have been observed19.
Moreover, because microplastics contain hazardous chemicals sorbed from seawater as well as retained addi-
tives20, their toxicological impacts are of concern. An increasing number of reports document the ingestion of
Laboratory of Organic Geochemistry, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509,
Japan. Correspondence and requests for materials should be addressed to H.T. (email: shige@cc.tuat.ac.jp)
Received: 15 March 2016
Accepted: 13 September 2016
Published: 30 September 2016
OPEN

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plastics, including microplastics, by sh species21–26. However, only a few studies22,25,26 reported polymer types.
is information is essential to assessing the risks of chemicals associated with ingested plastics, because the com-
position and magnitude of the chemicals vary among polymer types; for example, PE absorbs more hydrophobic
chemicals, such as polychlorinated biphenyls (PCBs), than other polymers20. In addition, specic additives are
compounded into specic polymers; for example, phthalates in PVC or hexabromocyclododecane (HBCDD) in
polystyrene27,28. e identication of polymer types is important also to identifying the sources of microplastics,
as are detailed observations of shape and size; for example, microbeads are made mostly of PE, and some of
them are spherical. In particular, the discrimination between primary and secondary microplastics is essential
for source control. We investigated the plastics in Japanese anchovy (Engraulis japonicus) caught in Tokyo Bay,
o the Tokyo metropolitan area (Fig.1). e population in the drainage basin is 29 million, which accounts
for one-fourth of the total population of Japan29. Tokyo Bay receives river water, sewage, industrial wastewater,
and surface runo from the city. We assumed that domestic and industrial activities are important sources of
microplastics in Tokyo Bay. Sewage from a large proportion of the population in the catchment is treated by com-
bined sewer systems, so untreated wastewater is occasionally discharged into the bay during heavy rain.
Results
Plastics in sh. We found plastics in the digestive tracts of 49 of the 64 anchovies (77%). Each sh had an
average of 2.3 (± 2.5) pieces of ingested microplastic, and from 0 to 15 pieces of debris. All of the plastics were
photographed (Supplementary Table S1) and identied by FT-IR (Fig.2). Among all 150 pieces found, there were
129 fragments (86.0%), 11 beads (7.3%), 8 laments (5.3%), and 2 foams (1.3%) (Table1, Fig.3a). e beads
comprised 6 spherical PE beads, 1 granular PE bead (an aggregation of 1 large sphere and some small spheres),
Figure 1. Sampling location in Tokyo Bay. Sixty-four Japanese anchovy (Engraulis japonicus) were caught
on 23 August 2015. Map created using QGIS47 and data provided by the Geospatial Information Authority of
Japan48.
Figure 2. Spectra of FT-IR analysis and photographs of plastic fragments. All suspected plastics were
identied by FT-IR and photographed. Two examples are shown.

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2 granular PP beads (shaped like a bunch of grapes), and 2 spherical white PS beads (Fig.4). e longest length of
the pieces ranged from 150 to 6830 μ m (average 783 μ m ± 1020) (Fig.5), and the width ranged from 68 to 1880 μ m
(average 345 μ m ± 272). e plastics consisted primarily of PE (52.0%) and PP (43.3%), followed by PS (2.0%),
ethylene/propylene copolymer (2.0%), and ethylene/propylene/diene terpolymer (0.7%) (Fig.3b). Most plastics
were white (40.0%) or transparent (31.0%), with a lower presence of green (12.3%), yellow and yellowed (12.3%),
black (2.6%), brown (1.3%), and pink (0.6%).
Microbeads in personal care products. We investigated plastic particles in four brands of facial cleansers
(labeled M1, M2, R1, S1) manufactured by three companies. All four products contained plastic particles, iden-
tied by FT-IR as PE, as stated in the list of ingredients. e size of all microbeads ranged from around 10 μ m
to 500 μ m. M2 and S1 contained spherical microbeads of PE, with average diameters of 314 ± 120 μ m (M2) and
188 ± 80 μ m (S1). M1, M2, and R1 contained amorphous particles (irregular shapes, including thread-like) of
PE, with average lengths of 247 ± 96 μ m (M1), 295 ± 54 μ m (M2), and 117 ± 58 μ m (R1) (Fig.6). e spherical
microbeads in M2 were blue and transparent, and those in S1 were transparent. All of the amorphous particles
were transparent or white.
Discussion
e frequency of occurrence of plastics in the digestive tract of Japanese anchovy, at 77%, is one of the highest
recorded in shes22,24. Most of the ingested plastics were fragments, followed by beads and laments derived from
shing gear, and most were PE or PP (Table1, Fig.3). is proportion of plastics is consistent with those in previ-
ous studies of plastic debris in surface seawater, which were dominated by fragments3,7,8 and by PE or PP7,30,31. e
fragments have various surface features, such as sharp edges with cracks, rounded shapes with smooth surfaces,
or degraded rough surfaces (Supplementary Table S1). Although we couldn’t identify the sources of the frag-
ments, their appearance may relate to their origin or history of degradation in the environment. Further study for
their source identication is needed. Among the major polymers, only PE and PP are less dense than seawater,
and therefore they predominate in surface water2. Japanese anchovy are known to stay in pelagic shallow water
at around 10 m depth32, and thus the proportion of ingested plastics reects that of the water. e predominance
of fragments has been also observed in sh from the North Pacic Central Gyre21 and in sh at a market in
Indonesia24. On the other hand, bers accounted for 68.3% of plastics in sh from the English Channel, which
were identied as rayon, polyamide, or polyester22. Fibers were also predominant in sh from the USA24. is
dierence may be due to regional source dierences or feeding habits of sh species; further studies are needed.
To the best of our knowledge, our study provides the rst evidence of the ingestion of microbeads, suspected of
being derived from personal care products, by sh. Beads accounted for 7.3% of the plastics in the anchovies, and
most were PE (Table1, Fig.4). ese articial shapes strongly indicate that they are manufactured as micro-sized
products. Over 90% of products with microbeads list polyethylene in the ingredients, and the others include PP
(calculated from lists provided by Beat the Microbead10). Our results conrm the presence of PE particles in four
brands of facial cleansers popular on the Japanese market, and spherical microbeads in two of them (Fig.6). e
spherical PE beads detected in the sh were similar in size and appearance to those in the facial cleansers (Figs4
and 6). One of PE beads in the anchovy was shaped like a single large sphere aggregated with some small spheres
Fragment Bead Filament Foam T OTA L
PE 70 7 1 78
PP 54 2 7 2 65
PS 1 2 3
Others 4 4
TOTAL 129 11 8 2 150
Table 1. Total number of plastic pieces in the digestive tract of Japanese anchovy (Engraulis japonicus) by
shape and polymer. PE, polyethylene; PP, polypropylene; E/P, ethylene/propylene copolymer; PS, polystyrene.
“Others” include ethylene/propylene copolymer and ethylene/propylene/diene terpolymer.
Figure 3. Types of plastics recovered from digestive tracts of Japanese anchovy (Engraulis japonicus).
(a) Percentage by shape. (b) Percentage by polymer.

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(Fig.4e). Because it was reported that some facial cleansers contain granular PE microbeads as well as spherical
ones33, the non-spherical PE bead in the anchovy is likely to be originated from such granular microbeads in the
personal care products. Both PP beads we found in anchovy were granular and were shaped like a bunch of grapes
(Fig.4j,k), and may also be derived from personal care products. However, personal care products that contain
PP beads are uncommon on the Japanese market based on our survey. It is also reported that only a few percent of
products contains PP beads while PE accounts for > 90 percent on the world market10. e detection frequency of
PP beads relative to PE beads seems higher than expected from the current market share, so PP beads in the sh
may be derived from other sources. Finally, the white polystyrene beads that we found accorded with the shape
and size of pre-expanded polystyrene beads, which are spherical and measure 0.1 to 2 mm34.
Eriksen et al.12 found many multi-colored spherical polymers of < 1 mm in surface waters of the Laurentian
Great Lakes of North America12. Mani et al.13 detected opaque spherules identied as PE on the surface of the
Rhine River13. ese studies identied them as microbeads used in consumer products such as facial cleans-
ers12,13. Some studies found spherical microplastics in some organisms, such as in commercial bivalves from
China35 and in zooplankton samples from the English Channel36, but the polymer types were not reported. It is
Figure 4. Photographs of microbeads ingested by Japanese anchovy (Engraulis japonicus). Scale bar
represents 500 μ m. All photographs were taken in reected light, but in the case of d, only photographs taken
with transmitted light are available.
Figure 5. Size distribution of plastics in Japanese anchovy (Engraulis japonicus).

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dicult to discuss sources without information on polymer types, because there are other sources of spherical
microplastics than personal care products, as indicated by the presence of many non-PE spherical beads (such as
acrylic, polyurethane, and polyester copolymers) on the sea surface in a South Korean bay37.
Microbeads in personal care and cosmetic products are discarded down the drain aer use and go through
several treatment processes. Although 95% to 99.9% of them are removed by settling at STPs, the remainder are
discharged with euent and end up in the aquatic havitats11. More importantly, combined sewer overows can
brings large amounts of untreated wastewater containing microbeads to coastal waters; in Tokyo, about half of
the population in the catchment (29 million) is served by combined sewer systems38. When heavy rain (gener-
ally > 5 mm) falls, sewer overows occur and could ush microbeads out. On the basis of the capacity of STPs
and precipitation patterns in Tokyo, overows occur around 50 times a year39, implying that ~7% ([50 days/365
days] × 0.5) of microbeads used in the catchment are discharged into aquatic environments without settling dur-
ing treatment.
We found spherical microbeads in sh stomachs. As we could not distinguish amorphous microbeads (Fig.6)
from degraded microplastic fragments, the “fragments” may include amorphous microbeads, and the distribution
of microbeads may be underestimated.
e size distribution of microplastics in the anchovies was dierent from those reported in surface seawaters
around Japan6. Over 80% was < 1000 μ m and more than half was < 500 μ m in size (Fig.5), although anchovy
can ingest prey from several tens of μ m40 to > 5 mm (such as zooplankton we found in the gut of some shes).
In the surface waters around Japan, however, microplastics (< 5 mm) larger than 1 mm dominate smaller ones,
which account for around 20% of all microplastics, and the size distribution peaked around 1 mm6. Although
the number of smaller microplastics in seawater samples is underestimated owing to sampling bias due to net
size (350-μ m mesh), the dierence in the size distribution of microplastics between the anchovies and surface
seawaters is clear only in the comparison of microplastics of > 400 μ m. is dierence is probably due to the
feeding characteristics of Japanese anchovy, which are pelagic lter feeders and ingest suspended particles in
subsurface waters. Because smaller plastic particles have lower rise velocities, smaller plastics (0.5–1.0 mm) were
more abundant in subsurface water than in surface water in the North Atlantic Gyre5. If the same vertical prole
of microplastics occurs in Tokyo Bay and smaller plastics are more abundant than larger plastics in the subsurface
water, by feeding in subsurface water the anchovies could accumulate smaller microplastics in their digestive
tract. More studies are necessary to understand the vertical proles of microplastics of various size ranges.
Japanese anchovy is widely distributed around Japan and is a common food in Japan. It is one of the most
caught fish species in Japan41, and is typically eaten without removal of the digestive tract. Nine species of
Engraulis are distributed in coastal waters around the world and are ecologically important because of their huge
Figure 6. Photographs of polyethylene microbeads in four brands of facial cleansers. (a) Brand M1,
irregular shapes. (b) Brand M2, blue and transparent spheres and irregular shapes. (c) Brand R1, irregular
shapes. (d) Brand S1, transparent spheres.

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biomass and their central role in the diet of many sh, birds, and marine mammals42. Our observations further
conrm that microplastics have inltrated marine ecosystems globally, and humans are now exposed to them,
although plastics within the observed size range (0.2–5 mm) would be excreted if we ate contaminated anchovies.
But plastics in the marine environment contain various hazardous chemicals, both additives compounded during
manufacture and hydrophobic chemicals adsorbed from seawater20. e ingestion of microplastics by anchovies
may increase the body burden of the hazardous chemicals to both anchovies and humans. Regarding chemicals
sorbed to microplastics, their transfer and accumulation in sh tissue upon ingestion were demonstrated by labo-
ratory exposure experiments43,44. On the other hand, anchovies and humans are exposed to hazardous chemicals
through natural prey, too. e low number of microplastics that we found in the anchovies (~2 pieces per sh on
average) suggests that chemical exposure through the ingested microplastics is minor compared with that from
the natural diet. However, inputs of plastics into the oceans and number of ingested plastics in sh will continue to
increase if no action is taken, and exposure of additive-derived chemicals may become more important in future.
Once plastics are discharged into the marine environment, they are dicult to recover, especially if they are
small. erefore, reducing the amount discharged from land to the oceans is the rst priority. To the best of
our knowledge, this is the rst study to identify and conrm microbeads in sh by examining polymer types
and shapes. e USA and some other countries now regulate microbeads from rinse-o personal care products.
Japanese cosmetics companies have just started voluntary elimination of microbeads from their products. While
laudable, it is important to note that these regulations do not cover all products containing microbeads and other
forms of microplastics. Global controls on microbeads should be considered, with restrictions on a wider range of
products. More importantly, however, plastic fragments should be regulated with rst priority, because a majority
of the microplastics in the sh were fragments. For the eective regulation, identication of the origins or origi-
nal products of individual fragments are necessary. eir appearance (shape) and color can be connected to the
original products, though both, especially the former, are altered by photo-oxidation. Identication of additive
chemicals specic to specic original products or their usage may help source identication. Control of plastic
products which can be easily fragmented is important to solving pollution of the marine environment by plastics.
Methods
Sampling and processing. e Japanese anchovy (Engraulis japonicus) were caught by shing using Sabiki
rigs from a pier in Tokyo Bay (35°25′ 43″ N 139°41′ 15″ E) (Fig.1) from 07:00 to 14:00 on 23 August 2015. e
depth of the water there was around 15 to 20 m, and the sh were caught at a range of 5 to 10 m from the surface.
We collected 64 anchovy. e sh were put in iced water and dissected at the laboratory the same day. Aer
measurement of their body length (112.5 mm ± 6.4 mm), we removed the whole of the digestive tract (from top
of the esophagus to the anus) and put it into a 10-mL glass vial that had been baked for 4 h at 550 °C in advance.
Each vial then received 7–8 mL (> 3× the volume of the gut) of 10% KOH solution to digest organic material23,24.
e vials were incubated at 40 °C for 10 days, during which digestion was observed to be completed in 3 to 4
days. Each vial was then shaken around 20 times to break up the mass of indigestible materials such as shells of
zooplankton, and all oating material was collected in another vial. Pieces larger than 200 μ m were clearly visible.
e precipitate that remained in the vial was put on a glass Petri dish and examined under a microscope, but no
particles not resembling natural prey were observed. Because our target sh (anchovy) is commonly caught by
recreational-shing and eaten by people and our procedure of shing and dissection is exactly same as what peo-
ple sh and cook it by, our procedure has no ethical problem. Our procedure of measurement of microplastics in
the digestive tract does not conict with ethical rules for animal experiment of our university.
Classication and identication of plastics. All oating items suspected to be plastic polymers were
photographed individually (Supplementary Table S1), and the color and shape were recorded. e denition of
the shape of microplastics is as follows: fragments: particles produced by fragmentation of larger materials, beads:
particles manufactured as micro-sized products, either spherical or an aggregate of spheres, pellets: granules
manufactured as a raw material of larger plastic products, generally in the size range of 2–5 mm with shape of a
cylinder or a disk, foams: foams made from polymer, lms: so fragments of thin polymers derived from plastic
bags or wrapping paper and so on, sheets: hard fragments of thin polymers, laments: thread-like polymers pro-
duced by fragmentation of ropes or lines used in shing, > 50 μ m, and bers: thread-like polymers derived from
textiles, including clothing and furnishings, ≤ 50 μ m. e 50-μ m threshold for bers was chosen because typical
textile bers have a diameter of 10 to 20 μ m (up to 50 μ m)45, and the diameter of monolaments used in shing
ropes or lines is larger than several hundred μ m46.
We analyzed all of the pieces of suspected plastic (n = 173) by Fourier transform infrared (FT-IR) spectroscopy
(Nicolet iS10, ermo Scientic) to identify polymer types. e IR absorbance from 450 to 4000 cm−1 was compared
with spectra in the soware database, with a similarity threshold of > 70%. Twelve particles were identied as having
a natural origin, 11 could not be identied, and the others (i.e., 150 pieces) were identied as synthetic polymers.
To avoid contamination, we kept samples sealed in a vial or Petri dish at all times except when picking out the
suspected plastics. A procedural blank analysis found no plastics. To estimate airborne contamination, we put
Petri dishes (total 17 000 mm2) on a table near the work bench for 1 day to collect airborne particles. We collected
3 polymer bers (PET, polyamide [similarity < 70%], and polyolen [similarity < 70%]), and more than 10 cot-
ton or wood bers, but no other bers. is means that procedural contamination did not signicantly aect the
results in the present study.
Microbeads in personal care and cosmetic products. We examined plastics in personal care products
to determine the features of the plastic beads used in them. We bought four major brands of facial cleansers that
listed polyethylene as an ingredient. ey were labeled M1, M2, R1, and S1. We mixed several grams of product
in distilled water and identied oating solid particles by FT-IR.

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8
Scientific RepoRts | 6:34351 | DOI: 10.1038/srep34351
Acknowledgements
We are grateful to Dr. Hiroaki Furumai for providing information on the population served by combined
sewer systems and Dr. Satoshi Nakaba for providing microscope. We thank Dr. John Farrington for invaluable
comments on the manuscript. e present study was supported by a Grant-in-Aid from the Ministry of Education
and Culture of Japan (Projects No. 26-8120 and No. 26550038) and the Environment Research and Technology
Development Fund (project no. 4-1502).
Author Contributions
K.T. designed the study, conducted the eld sampling and experimental work and wrote the paper. H.T. advised
on experimental design, provided laboratory facilities (FT-IR) and supervised the paper.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Tanaka, K. and Takada, H. Microplastic fragments and microbeads in digestive tracts of
planktivorous sh from urban coastal waters. Sci. Rep. 6, 34351; doi: 10.1038/srep34351 (2016).
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