Environment International 130 (2019) 104848

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Environment International
journal homepage: www.elsevier.com/locate/envint

Impact of nano-sized plastic on the nutritional value and gut microbiota of T

whiteleg shrimp Litopenaeus vannamei via dietary exposure
⁎
Yooeun Chaea, Dasom Kima, Mi-Jung Choib, Youngjae Chob, Youn-Joo Ana,
a
Department of Environmental Health Science, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea
b
Department of Food Science and Biotechnology of Animal Resources, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea

A R T I C LE I N FO A B S T R A C T

Handling Editor: Yong-Guan Zhu Contamination of the world's oceans with plastic waste has attracted increasing attention in recent years.
Keywords: Whereas the ecological consequences of plastic pollution have been the focus of increasing research, the health-
Nanoplastic related implications of plastic pollution have been somewhat overlooked. In this study, we exposed whiteleg
Nutrients shrimp (Litopenaeus vannamei), a widely consumed marine species, to nano-sized plastic (polystyrene) via a
Whiteleg shrimp simulated marine food chain in which mussel (Mytilus edulis) was the food source, and evaluated the effects of
Trophic transfer plastic contamination on shrimp physical, biochemical, and nutritional characteristics over a 21-day exposure
period. We identified the changes in certain important biochemical and nutritional indicators, including changes
in the gut microbiota and contents of amino acids and fatty acids. The biochemical analysis revealed that mi-
crobial activities in the intestine and the glutathione S-transferase and superoxide dismutase activities changed
in L. vannamei exposed to nano-sized plastic. In these individuals, the levels of some essential amino acids and
fatty acids also decreased. Overall, our findings indicate that plastic pollution can directly interfere with nu-
tritional changes in marine food resources, thereby indirectly causing potential health implications for human
consumers.

1. Introduction
Microplastic (< 5 mm) pollution, a major threat to marine en-
vironments (Hämer et al., 2014; Gall and Thompson, 2015), is a source
of growing concern (Andrady, 2011), and it is currently receiving
considerable worldwide attention (Frias and Nash, 2019). Between
1960 and 2015, the global plastic production increased from 0.5 to
322 million tons (PlasticsEurope, 2017). As the plastic production in-
creased, the amounts of plastic wastes discharged into the marine en-
vironment also increased (Kim et al., 2015; Lebreton et al., 2017). In
addition, smaller sized nanoplastics (< 1 μm) (Gigault et al., 2018)
have recently been highlighted as harmful in several studies showing
the degradation or fragmentation of microplastics into nanoplastics in
the environment (Lambert and Wagner, 2016; Hernandez et al., 2017;
Dawson et al., 2018a). Previous studies have revealed that these small
particulate plastics cause potential harm and risks to marine organisms
and ecosystems (Cole et al., 2011; Wright et al., 2013a,; Chae and An,
2017). Scientists have investigated the effects of micro- or nanoplastics
on various marine organisms using histological analysis (Von Moos
et al., 2012) and several indicators, including mortality (Cole et al.,
2015; Bergami et al., 2016), cell viability (Browne et al., 2008), feeding
activity (Wright et al., 2013b; Hall et al., 2015; Watts et al., 2015),
reproduction (Cole et al., 2015), and oxygen consumption rates (Watts
et al., 2015). However, previous studies have generally only considered
the effects on the health of marine organisms and ecosystems from an
ecological perspective. To the best of our knowledge, there has been no
research on the effects of plastic pollution on the quality of seafood
from a nutritional perspective.
The Food and Agricultural Organization (FAO) has reported that, in
2013, approximately 17% of the global consumption of animal protein
was derived from fish, and that this represented 6.7% of the total
protein consumed (FAO, 2016). Additionally, seafood is a source of
valuable and high-quality nutrients (Rupérez, 2002; Mabeau and
Fleurence, 1993; FAO, 2016), including long-chain omega-3 fatty acids,
vitamins, and minerals, which prevent certain diseases and health
problems (Pigott and Tucker, 1987). Thus, seafood is a very important
food resource for humans. Previous studies determined that the nu-
trients in seafood could change in response to several factors such as
climate (Marques et al., 2010), salinity (McCoid et al., 1984), and
contaminants including heavy metals (Jana et al., 1986; Sobha et al.,
2007). These changes can indirectly affect human health through sea-
food ingestion. Thus, we hypothesized that plastic waste in the marine

⁎
Corresponding author.
E-mail address: anyjoo@konkuk.ac.kr (Y.-J. An).

https://doi.org/10.1016/j.envint.2019.05.042
Received 26 February 2019; Received in revised form 15 May 2019; Accepted 16 May 2019
Available online 17 July 2019
0160-4120/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Y. Chae, et al.
environment can represent a serious problem in terms of human health,
not only as a consequence of ingesting plastic-contaminated seafood but
also due to the degradation of seafood quality caused by plastic pol-
lution. However, compared to the attention and concern on the plastic
debris accumulation in the marine environment, research on the ad-
verse effects of these debris on seafood quality and its nutritional values
have been scarcely addressed as many previous studies have only fo-
cused on the environmental and ecological effects of plastic pollution. It
is therefore imperative to investigate both the effects of plastic pollu-
tion on marine ecosystems and on the quality of the food sourced from
the marine environment.
In the present study, we exposed whiteleg shrimp Litopenaeus van-
namei, a marine species that is widely consumed worldwide, to nano-
plastics transferred via the food chain. The mussel Mytilus edulis was
used as prey to evaluate the effects of nanoplastics on physical, bio-
chemical, and nutritional aspects of marine organisms by dietary ex-
posure.
2. Methods
2.1. Test materials
A nano-sized pristine polystyrene sphere (nPs) stock solution
(DS02B-12123, blue dyed, 10% solution) was purchased from Bangs
Laboratories, Inc. (Fishers, IN, USA). According to the manufacturer,
the mean diameter of each nPS was 44 nm and the stock solution (% in
product) contained water (≥89.81%), polystyrene (≤10%), surfactant
(≤0.1%), and sodium azide (≤0.09%). The physical properties of the
nPS particles in distilled water and artificial seawater were analyzed
using dynamic light scattering (DLS) (Zetasizer Nano ZS; Malvern
Instruments, Malvern, UK) and scanning electron microscopy (SEM)
(SUPRA 55VP; Carl Zeiss, Jena, Germany) (Fig. S1).
2.2. Exposure design
Mussel (M. edulis) and whiteleg shrimp (L. vannamei) were pur-
chased from a local market and acclimated in the laboratory (for
10–14 days and for two weeks, respectively) before the experiment.
Before the onset of the experiment, adult L. vannamei (average wet
weight = 25.37 ± 3.54 g, average total length = 160.87 ± 8.26
mm) were maintained in 45-L water tanks containing 30-L of artificial
seawater (30 PSU, pH 8.0–8.5) produced from seawater aquarium salt
(Reef Salt; Aqua Medic, GmbH, Bissendorf, Germany) containing so-
dium, magnesium, calcium, potassium, chloride, sulfate, bicarbonate,
and strontium. Each tank contained five L. vannamei and the seawater
temperature was maintained at 30 ± 1 °C using a heater under a
light:dark cycle of 16:8 h. The seawater was changed every two days
and each shrimp was fed a clean mussel once a day. The sex of L.
vannamei individuals was not determined in this test.
Fifteen M. edulis were maintained in either clean seawater (for
feeding control L. vannamei) or seawater containing nPS (50 μg mL−1)
(for feeding exposed L. vannamei) for 1 h. Exposure concentration
(50 μg mL−1) and time (1 h) were determined after a preliminary test
(Fig. S2) conducted to determine the appropriate conditions to detect
nPS in the body of M. edulis. Only the edible parts of each clean and
contaminated mussel were given to the control and exposed whiteleg
shrimp, respectively. Thus, before the feeding process, mussel shells
were removed and each mussel was gently washed with clean seawater.
After nPS exposure, the organs of M. edulis were observed under an
optical microscope (SMZ-168; Motic Deutschland GmbH, Germany).
The test conditions were identical to those used for acclimation.
Three tanks, each containing five L. vannamei, were used for each
treatment, and the experiment was conducted for 21 days. Because one
shrimp in the exposure group died on the 12th day, n = 15 in the
control group and n = 14 in the exposed group. The nPS in shrimp were
observed under a transmission electron microscope (TEM, Tecnai G2
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Environment International 130 (2019) 104848
Spirit Twin; FEI, Hillsboro, OR, USA).
2.3. Physical and biochemical analysis
After the 21-day experimental period, all L. vannamei were sacri-
ficed and the physical, biochemical, and nutritional effects of nPS were
investigated. For physical analyses, total length, total wet weight, and
body mass index [total wet weight/(total length)2] of shrimps were
measured. The head, internal organs, tail, and exoskeleton of shrimps
were removed to determine the wet weight of edible parts (muscular
and soft parts only), which were then lyophilized for three days before
determining their dry weight and water content.
The modified method of Kim et al. (2016) was used for biochemical
analyses. The activities of gut microorganisms were investigated during
the experiment, at days 7, 14, and 21, to examine the effects of nPS on
the size, granularity, and viability of gut microbiota. Because the ac-
tivities of microbiota are related to health, disease, digestion, and im-
munity performance (Round and Mazmanian, 2009), they can be used
as an indirect indicator for the overall health and nutrition status of
organisms. Shrimp fresh feces were collected (five replicates on day 7
and eight replicates on days 14 and 21 per treatment) and added to
1 mL of filtrated phosphate-buffered saline. After the separation of
microorganisms by vortexing, the samples were centrifuged at 650 ×g
and 4 °C for 5 min. The supernatants (450 μL) were mixed with calcein-
AM solution (50 μM in dimethyl sulfoxide) and incubated at 37 °C for
1 h. After this incubation period, the size, granularity, and viability of
20,000 cells in each sample were analyzed in a flow cytometer
(FACSCalibur; Becton-Dickinson, Franklin Lakes, NJ, USA). Cell gran-
ularity indicates the internal complexity of the cell and it can be used to
identify cellular growth arrest and cell death (Haynes et al., 2009). This
was assessed by side scatter analysis in the flow cytometer. Cell viabi-
lity, which is the ability of the cell to maintain or recover its function
and vitality, was determined by measuring the accumulation of calcein
in the cell using a fluorescent filter in the flow cytometer (FL1;
515–545 nm excitation).
The activities of glutathione-S-transferase (GST), an enzyme in-
volved in detoxification, and superoxide dismutase (SOD), an enzyme
related to antioxidation, in L. vannamei hepatopancreas were also de-
termined. Shrimp hepatopancreas were extracted, homogenized, and
10-fold diluted in 0.1 M potassium phosphate buffer containing 60 mM
ethylenediaminetetraacetic acid and 1 mM phenylmethanesulfonyl
fluoride. The samples were centrifuged at 3000 ×g and 4 °C for 15 min.
The activity of GST was quantified using the method of Habig et al.
(1974), whereas that of SOD was quantified using a SOD Assay Kit
(19160-1KT-F; Sigma-Aldrich, St. Louis, MO, USA).
2.4. Nutritional analysis
For the nutritional analyses, we examined the contents of crude
protein, crude fat, 20 essential amino acids, and 37 fatty acids in the
muscle (edible part) of L. vannamei. The crude protein content was
determined using a slight modification of the semi-micro Kjeldahl
method, as described in the Korean Food Standards Codex of the
Ministry of Food and Drug Safety (KFDA, 2018a). In brief, each lyo-
philized and ground muscle sample (0.1 g) was digested with 2 g of
catalyst and 30 mL of H2SO4 at 420 °C for 1 h to yield a clear solution.
After cooling, the digest was diluted with 100 mL of distilled water in a
Kjeldahl flask connected to a Kjeldahl distillation apparatus. The digest
was then alkalized by the addition of 30% NaOH (30 mL). The am-
monium steam was distilled into 0.05 N H2SO4 (100 mL) supplemented
with three drops of Brunswick solution, and the distilled ammonium
was titrated with 0.05 N NaOH.
Crude fat content was determined using the Rose-Gottlieb method,
as described in the Korean Food Standards Codex with minor mod-
ifications (KFDA, 2018b). Briefly, 1 g of lyophilized and ground shrimp
muscle was transferred to a Mojonnier fat extractor and 11 mL of
Y. Chae, et al.
distilled water was added. The solution was then mixed with 2 mL of
28% ammonia and 10 mL of 95% ethanol, and gently mixed with 15 mL
of ethyl ether for 30 s. After evaporating the ether, the solution was
thoroughly mixed with petroleum ether for 30 s and, after the ether
evaporated, the supernatant of the mixture was transferred to a
weighed flask through filtration. The steps from mixing with ethyl ether
to filtration were repeated three times. The solvent in the flask was
evaporated, and the residue (fat content) was weighed.
Fatty acid content was determined using the following method. To
extract lipids, shrimp muscle samples were mixed with 10 mL of 8.3 M
HCl at 70 °C for 40 min with shaking. After cooling, the mixture was
sequentially mixed with 25 mL diethyl ether and 25 mL dehydrated
petroleum ether for 5 min. The mixture was then centrifuged at
4000 rpm for 10 min and evaporated using a TurboVap LV evaporation
system (JYSCO, Seoul, Korea). The concentrated lipid solution was se-
quentially mixed with 2 mL chloroform and 3 mL diethyl ether by
vortexing and then evaporated at 40 °C. The resulting solution was
mixed with 2 mL of 7% BF3-methanol and 1 mL of toluene by vor-
texing. After heating, the lipid solution was mixed with 5 mL distilled
water, 1 mL hexane, and 1 g dehydrated Na2SO4. This solution was then
centrifuged at 4000 rpm for 10 min, and the resulting supernatant was
filtered and used for gas chromatography (GC) in an Agilent 7890
system (Agilent, Santa Clara, CA, USA) equipped with a flame ioniza-
tion detector (FID) and a SP-2560 column (100 m × 0.25 mm, i.d., 0.2-
μm film thickness; Supelco Inc., Bellefonte, PA, USA). The column
temperature was maintained at 100 °C for 4 min, and then ramped to
240 °C (3 °C min−1) and maintained for 20 min. The injector and de-
tector temperatures were set at 225 and 285 °C, respectively, and the
gas flow rate was 0.75 mL min−1. The sample size was 1 μL and the
split used was 100:1.
For the essential amino acid analysis, approximately 1 g of finely
ground freeze-dried shrimp muscle was transferred to a conical tube
followed by the addition of 5 mL of 0.1 N HCl. This mixture was soni-
cated for 30 min, centrifuged at 5000 rpm for 10 min, and filtered using
a polyvinylidene fluoride syringe filter. Thereafter, the filtrate was
frozen at −20 °C until use. High-performance liquid chromatography
(HPLC) was performed in an Agilent 1100 series fluorescence detector
(Agilent Technologies, Böblingen, Germany) equipped with a G1313A
auto sampler, a G1311A pump, a G1316A column oven, and a G1314A
UV detector. Chromatographic analysis was performed using an Agilent
ZORBAX Eclipse Plus C18 column (4.6 mm × 150 mm, 3.5 μm; Agilent
Technologies). The HPLC conditions were as follows: mobile phase A:
10 mM Na2HPO4:10 mM Na2B4O7, pH 8.2:5 mM NaN3; and mobile
phase B: 45:45:10 (v/v/v) acetonitrile: methanol:water. The flow rate
was constant at 1.2 mL min−1 and the column was maintained at 40 °C.
The sample was detected using excitation and emission wavelengths of
262 and 338 nm, respectively, whereas the reference sample was de-
tected at excitation and emission wavelengths of 324 and 390 nm, re-
spectively.
2.5. Statistical analyses
All data were analyzed using SPSS statistical package software
(SPSS Version 24.0 for Windows; SPSS Inc., Chicago, IL, USA). To
confirm the reliability of the data, we initially checked for normality
using the Shapiro–Wilk test. To check variance homogeneity, the
Levene test for the equality of variances was conducted. The t-test was
used to identify significant differences between the unexposed and nPS-
exposed groups. Significance levels of 95%, 99%, and 99.9% (for
p < 0.05, 0.01, and 0.001, respectively) were used for all comparisons.
The flow cytometric data were analyzed using FlowJo single-cell ana-
lysis software (Treestar, Inc., San Carlos, Northampton, CA, USA) using
event values as histogram statistics.
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Environment International 130 (2019) 104848
3. Results
3.1. Exposure of M. edulis to nano-sized polystyrene
According to the results of the DLS and SEM analyses, the nPS ag-
gregated with salt particles in the artificial seawater and their size in-
creased up to about 1.4 μm (Fig. S1B, D). We exposed M. edulis daily to
nPS in artificial seawater for 1 h and then removed their shells before
providing them as food to L. vannamei. The observation of M. edulis
under the optical microscope revealed that the nPS particles were ad-
sorbed on or attached to the surfaces of mussel organs (particularly gill
surfaces) and foot, and thus transferred to L. vannamei through inges-
tion of these parts (Fig. S3). No mussel mortality was observed due to
nPS exposure. The nPS transferred to shrimp were observed as spherical
particles attached to gut tissues (Fig. S4).
3.2. Physical effects of nPS on shrimp
Each shrimp was fed one unexposed or exposed mussel per day for
21 days. After this period, the total length and total wet weight, and the
wet weight, dry weight, and water content of edible parts were de-
termined and the body mass index was calculated (Table S1). No sta-
tistically significant differences in these physical indices were detected
between the unexposed (control) and exposed shrimp groups
(p > 0.05).
3.3. Biochemical effects of nPS on shrimp
There were several differences in gut microbial activities between
unexposed and exposed shrimp (Figs. 1 and S5). After the first week
(day 7), cell size (p < 0.01), cell granularity (p < 0.05), and cell
viability (p < 0.01) significantly increased for the gut microbiota of
shrimp exposed to nPS compared to that of control shrimp (Fig. 1A, B).
After the second week (day 14), there were no significant differences in
cell size and cell granularity between the two groups but there was a
significant difference (p < 0.001) in cell viability (Fig. 1C, D). The
results obtained at the end of the experiment (21st day) were similar to
those recorded on day 14, with the only significant difference between
the experimental groups being cell viability (p < 0.01). Overall, the
differences in cell size and cell granularity between the unexposed and
exposed groups decreased throughout the experiment (no significant
differences in days 14 and 21 in contrast to that observed in day 7)
(Fig. 1E, F).
The activities of GST and SOD in the hepatopancreas of shrimp
exposed to nPS were slightly higher (p < 0.05) than in control shrimp
(Fig. 2).
3.4. Nutritional effects of nPS on shrimp
After 21 days, no significant changes were detected in the crude
protein and crude fat contents between control and exposed shrimp
(Table S2). However, significant changes were detected in the contents
of essential amino acids after 21 days of nPS exposure (Fig. 3). Notably,
the contents of asparagine (Asn; 13.05 mg 100 g−1 in control and
2.76 mg 100 g−1 in exposed shrimp), glutamine (Gln; 406.58 mg
100 g−1 in control and 111.93 mg 100 g−1 in exposed shrimp), and
proline (Pro; 1227.42 mg 100 g−1 in control and 575.02 mg 100 g−1 in
exposed shrimp) (p < 0.01), and histidine (His; 67.57 mg 100 g−1 in
control and 28.57 mg 100 g−1 in exposed shrimp), cysteine (Cys;
46.91 mg 100 g−1 in control and 14.79 mg 100 g−1 in exposed
shrimp), and arginine (Arg; 777.11 mg 100 g−1 in control and
284.12 mg 100 g−1 in exposed shrimp) (p < 0.05) decreased after nPS
exposure (Fig. 3A, B). In contrast, glutamic acid (Glu; 18.53 mg
100 g−1 in control and 73.38 mg 100 g−1 in exposed shrimp) and
isoleucine (Ile; 6.95 mg 100 g−1 in control and 128.44 mg 100 g−1 in
exposed shrimp) (p < 0.01), and aspartic acid (Asp; 0.83 mg 100 g−1
Y. Chae, et al. Environment International 130 (2019) 104848

Fig. 1. Size, granularity, and viability of microbial cells in the guts of controls (CON, gray columns) and shrimps exposed to nanoplastic (EXP, blue columns) on days
7 (A, B), 14 (C, D), and 21 of exposure (E, F). The gray- and blue-colored contour plots show the distributions (cell viability in the y axis and cell size in the x axis) of
microbial cells from control and exposed shrimp, respectively (B, D and F). Asterisks indicate significant differences between treatments (*p < 0.05; **p < 0.01;
***p < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

in control and 3.25 mg 100 g−1 in exposed shrimp), tryptophan (Trp;
1.78 mg 100 g−1 in control and 11 mg 100 g−1 in exposed shrimp), and
leucine (Leu; 23.18 mg 100 g−1 in control and 29.7 mg 100 g−1 in
exposed shrimp) (p < 0.05) increased after nPS exposure (Fig. 3A, B).
Although the major amino acids (Gln, Arg, and Pro) increased after nPS
exposure, the total content of amino acids in the exposed shrimp de-
creased to approximately 60.5% that of the control shrimp (3798.27 mg
100 g−1 in control and 2297.78 mg 100 g−1 in exposed shrimp)
(Fig. 3C). For fatty acids, only the content of eicosapentaenoic acid
(EPA, C20:5(n-3), polyunsaturated fatty acid) showed a significant
decrease (p < 0.05) after nPS exposure (1 g 100 g−1 in control and
0.96 g 100 g−1 in exposed shrimp) (Fig. 4).
4. Discussion
We detected no significant differences between changes in the
physical characteristic of control and exposed shrimp after the 21-day
exposure to nanoplastic-contaminated mussels. Previous studies have
investigated the effects of micro- and nanoplastics on the growth, bio-
mass, and physical condition of organisms. Although some of these
studies detected significant adverse effects of micro- and nanoplastics
(Au et al., 2015; Ziajahromi et al., 2017), others did not (Green, 2016;
Sussarellu et al., 2016; Lo and Chan, 2018). Such inconsistencies might
reflect the fact that the manifestation of the physical effects of micro-
and nanoplastics depends on the concentration and duration of the
exposure, on the life stage of organisms, and on various environmental

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Fig. 2. Changes in the activities of glutathione-S-transferase (GST, top panel)

and superoxide dismutase (SOD, bottom panel) in the hepatopancreas of control
(CON, gray columns) and shrimps exposed to nanoplastic (EXP, blue columns)
after the 21 days of treatment. The asterisk indicates a significant difference
between treatments (*p < 0.05). (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)

conditions that tend to differ among different studies.

On days 7, 14, and 21 of exposure to nPS, differences in the char-
acteristics of gut microorganisms were detected between control and
exposed shrimp; however, these tended to stabilize over the 21 days of
exposure. The gut microbiota of shrimps play important roles in their
nutrition, immunity, and health status, and on their development and
resistance to external factors (Ley et al., 2005; Sanz et al., 2010; Wang
et al., 2017; Zhu et al., 2018a). The results found here indicate that
although the characteristics (cell size, granularity, and viability) of the
gut microbiome were initially affected by the exposure to nPS, the
microorganisms adapted to the prolonged exposure. Because the pre-
sent study did not concern the study of gut microbiota composition, the
direct effect of nPS on microbiome diversity was not assessed. However,
the morphology (size and granularity) and viability of microorganisms
Fig. 3. Contents of the 20 essential amino acids in the edible parts of controls
were evaluated before and after nPS exposure. In recent studies, several
(gray columns) and shrimps exposed to nanoplastic (blue columns) after
researchers have investigated the gut microbiome of various organisms 21 days of treatment. (A) Aspartic acid, glutamic acid, asparagine, serine, his-
after exposure to plastics. For example, based on 16S rRNA sequencing, tidine, threonine, alanine, tyrosine, cysteine, valine, methionine, tryptophan,
Jin et al. (2018) observed microbial dysbiosis in the gut of zebrafish phenylalanine, isoleucine, leucine, and lysine. (B) Glutamine, glycine, arginine,
(Danio rerio) after exposure to polystyrene microplastics, and Lu et al. and proline. (C) Total amino acids. Asterisks indicate significant differences
(2018) observed the same phenomenon in the gut of mice. In the soil between treatments (*p < 0.05; **p < 0.01). (For interpretation of the re-
environment, researchers have investigated disturbances in the gut ferences to colour in this figure legend, the reader is referred to the web version
microbiota of the collembolan Folsomia candida (Zhu et al., 2018b) and of this article.)
of the oligochaete Enchytraeus crypticus (Zhu et al., 2018c). The com-
position of an organism's microbial population is determined by the et al., 2018) and affect their health and nutrition status (Flint et al.,
surrounding environment (feeding and media) and can be influenced by 2012; Nicholson et al., 2012).
diet, foreign matter, and age (Dehler et al., 2017; Lu et al., 2014; Wang Regarding biochemical effects, there were significant disturbances
et al., 2017). The dysbiosis of microbiota in response to microplastics in microbiota morphology (size and granularity) and viability on day 7
may lead to metabolic abnormalities in organisms (Ba et al., 2017; Wu and changes in viability on days 14 and 21. Additionally, we observed

5
Y. Chae, et al.
Fig. 4. Contents of the 37 fatty acids in the edible parts of control (gray columns) and exposed to nanoplastic (blue columns) shrimp after 21 days of treatment. The
asterisk indicates a significant difference between treatments (*p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
slightly increased enzymatic activities of GST and SOD in the hepato-
pancreas of L. vannamei exposed to nPS. Whereas GST generally plays a
role in detoxification, SOD plays an antioxidant role by catalyzing the
dismutation of the superoxide radical into oxygen or hydrogen per-
oxide. In previous studies, researchers have confirmed the effects of
microplastics on the enzymatic activities of fish (Lu et al., 2016; Ding
et al., 2018), mussel (Magni et al., 2018), and crab (Yu et al., 2018),
thereby indicating that microplastics are a potential source of toxic and
oxidative stress in organisms. Thus, although there were only slight
changes in GST and SOD activities in the present study, nanoplastic
particles might have various adverse effects on the activities of enzymes
involved in detoxification and antioxidation in organisms.
Many researchers have examined the nutrition status of shrimps and
have attempted to improve and enhance the quality of these important
food resources or to determine the factors that affect nutrient compo-
sitions, including season (Bottino et al., 1980; Rosa and Nunes, 2004;
Yanar et al., 2004), environmental conditions (Bono et al., 2012; Li
et al., 2017), disease (Mugnier et al., 2013), food (Bottino et al., 1980;
Mourente et al., 1995; Tacon et al., 2002), and pollutants (Wu and
Chen, 2005). Various environmental factors can induce changes in the
nutrient contents of organisms, particularly food resources, and these
can be directly related to human health (Della Penna, 1999; Grusak and
Della Penna, 1999; Welch, 2002). To the best of our knowledge, the
present study is the first to demonstrate a relationship between ex-
posure to nanoplastic particles and the nutritional components of a
marine food resource. Although we detected no significant changes in
the amount of crude protein and crude fat, there were significant de-
creases in certain amino acids and fatty acids in shrimp exposed to nPS
compared to control shrimp. Especially, total amino acid and EPA
contents significantly decreased. Amino acids are the basic components
of proteins, which constitute the human body (Yu et al., 2007), and EPA
is an important nutrient that prevents cardiovascular disease in humans
(Bhatt et al., 2019). The decreased contents of these essential nutrients
due to nanoplastics, allied to the fact that amino acids and fatty acids
play very important functions in organisms, imply that plastic pollution
of the marine environment directly affects the quality and nutrition
status of food resources derived from marine organisms, which in-
directly has potential health-related consequences for human con-
sumers.
To date, only a few studies have examined the contamination of
crustaceans by plastic waste or the effects of microplastics on crusta-
cean species in marine ecosystems. Several researchers have also
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Environment International 130 (2019) 104848
detected microplastic contamination in shrimps in their field studies.
About 63% of brown shrimp (Crangon crangon) from the southern North
Sea and 20% of krill species (Euphausia spp.) from the South China Sea
contained microplastics in their bodies (Devriese et al., 2015; Sun et al.,
2017). Similarly, the present study confirmed that L. vannamei con-
tained nanoplastic particles in their bodies. Other researchers have
examined the ingestion, depuration, and effects of microplastics in
shrimp species under laboratory conditions. The kinetics, bioaccumu-
lation, and toxicity of microplastics transferred via the food chain
consisting of zooplankton and mysids (Setälä et al., 2014) to Antarctic
krill (Euphausia superba) were evaluated (Dawson et al., 2018b). Na-
noplastics with two different functional groups (carboxylated and
amino-modified) were provided to brine shrimp (Artemia franciscana)
and it was confirmed that these particles impaired the food uptake,
behavior, and physiology of brine shrimp (Bergami et al., 2016). Si-
milarly, a study using grass shrimp (Palaemonetes pugio) exposed to 11
different microplastics with various shapes and sizes showed that mi-
croplastics can be accumulated in shrimps and cause mortality de-
pending on their physical properties (Gray and Weinstein, 2017). These
studies directly or indirectly demonstrated the potential effects of mi-
croplastics on shrimp species. However, although the shrimp species
used in the studies mentioned above are ecologically important com-
ponents of complex marine food webs in marine ecosystems, they are
not common food resources for humans. Moreover, researchers have
not considered the indirect effect of microplastics on human health
linked to the consumption of shrimp with low nutritional status due to
plastic pollution. In addition to L. vannamei, used in the present study,
other shrimp species are commonly used by humans as food sources,
including the coonstripe shrimp (Pandalus hypsinotus), black tiger
shrimp (Penaeus monodon), kuruma shrimp (Marsupenaeus japonicus),
and Chinese shrimp (Fenneropenaeus chinensis). However, these shrimps
have received little attention regarding the problem of marine pollution
caused by plastic waste. In the present study, we examined the adverse
effects of nanoplastics on the physical, biochemical, and nutritional
characteristics of the whiteleg shrimp L. vannamei, an important human
food resource from the marine environment. Although some of these
characteristics were essentially unaffected by nanoplastic pollution,
there were changes in certain important indicators, notably biochem-
ical and nutritional factors, in response to nanoplastic exposure. Thus,
future studies should utilize these indicators to evaluate the quality of
food resources contaminated by plastic waste or other pollutants. In the
light of the growing concerns regarding marine pollution caused by
Y. Chae, et al.
plastic waste, the disturbance and contamination of marine ecosystems
are of increasing importance; however, in future studies, we should also
devote attention to the potential threats to human health posed by
marine plastic pollution.
5. Conclusions
In summary, we evaluated the effects of nanoplastic particles on L.
vannamei, an important human food resource from the marine en-
vironment, contaminated via the food chain. Because shrimp species
are widely consumed food resources, it is very important to evaluate the
potential effects of plastic pollution on the nutritional quality of shrimp
species. Although we did not conduct a qualitative assessment of the
transfer of nanoplastic particles from mussel (prey) to shrimp (pre-
dator), we did observe the adsorption of these particles on the mussel
and observed significant biochemical and nutritional changes on the
shrimp, which derived from the ingestion of nanoplastics. Thus, these
nanoplastics might be transferred from seafood to human through food
ingestion. Further researches addressing the effects of plastic pollution
on food quality and on its indirect effects on human health should be
conducted.
Declaration of Competing Interest
The authors declare no conflicts of interest.
Acknowledgments
This research was supported by a Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded by
the Ministry of Science, ICT and Future Planning
(2016R1A2B3010445). This study was also funded by the Ministry of
Environment as the Graduate School of Specialization for managing
information of chemical risk. We thank the Korean Basic Science
Institute (KBSI) for their assistance with our dynamic light scattering,
TEM, and SEM analyses.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.envint.2019.05.042.
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