Science of the Total Environment 652 (2019) 1209–1218

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Microplastic pollution in rice-fish co-culture system: A report of three

farmland stations in Shanghai, China
Weiwei Lv a,1, Wenzong Zhou a,1, Shibo Lu b, Weiwei Huang a, Quan Yuan a, Minglu Tian c,
Weiguang Lv a,⁎, Defu He b,⁎
a
Eco-environmental Protection Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China
b
School of Ecological and Environmental Sciences, East China Normal University, Shanghai 200241, China
c
Information Research Institute of Science and Technology, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Microplastic pollution was studied in

three rice-fish co-culture ecosystems.
• Microplastics were found in water, soil
and breeding aquatic animal samples.
• An increasing trend of microplastics oc-
curred from non-rice period to rice pe-
riod.
• Major microplastics were small
(b1 mm) polyethylene and polypropyl-
ene fibers.
• Microplastic abundances in aquatic ani-
mals were correlated to abundances in
soils.

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

Article history: Microplastics are emerging contaminants of increasing concern. Despite the occurrence of microplastics in farm-
Received 4 September 2018 land soils, the knowledge on microplastics in rice-fish co-culture ecosystems is limited. In this study, we investi-
Received in revised form 23 October 2018 gated the distribution of microplastics in three rice-fish culture stations in Shanghai. During non-rice and rice-
Accepted 23 October 2018
planting periods, microplastics in water, soils and aquatic animals (eel, loach and crayfish) were systematically
Available online 25 October 2018
assayed using methods of NaCl density extraction, H2O2 digestion and micro-fourier transform infrared spectros-
Editor: Damia Barcelo copy. Results showed that average microplastic abundances were 0.4 ± 0.1 items L−1, 10.3 ± 2.2 items kg−1, 1.7
± 0.5 items individual−1 in water, soils and aquatic animal samples, respectively. We found an increasing trend
Keywords: in microplastic abundances in water, soil and animal samples from non-rice period to rice-planting period. Al-
Microplastics most all of microplastics were found in digestive tracts of animals. Major microplastics were small (b1 mm) poly-
Rice-fish co-culture ethylene and polypropylene fibers, with color of white and translucent. Size, shape, color and polymer type
Aquatic animals distributions of microplastics were similarly found in environmental and animal samples. Moreover, microplastic
Agroecosystem abundances in aquatic animals correlated to abundance in farmland soils. This study, for the first time, reveals the
occurrence and characteristics of microplastic pollution in rice-fish culture ecosystem which suggests the poten-
tial ecological risks of microplastics in the agroecosystem.
© 2018 Published by Elsevier B.V.

⁎ Corresponding authors.
E-mail addresses: lwei1217@sina.com.cn (W. Lv), dfhe@des.ecnu.edu.cn (D. He).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.scitotenv.2018.10.321
0048-9697/© 2018 Published by Elsevier B.V.
1210 W. Lv et al. / Science of the Total Environment 652 (2019) 1209–1218
1. Introduction
After 1950s, plastic has been largely produced and used globally. Up
to 2016, the annual production of plastics has reached 335 million tons
all over the world (Statista, 2018). Thus, a mass of plastic waste has ac-
cumulated in landfills or the natural environments (Geyer et al., 2017).
Microplastics, defined as plastic particles b5 mm, are ubiquitously pres-
ent in the ecosystems as primary microplastics, i.e. manufactured micro
materials, or as secondary microplastics generated by the breakdown of
larger plastic litter (Duis and Coors, 2016). In recent years, microplastics
as emerging contaminants has attracted widespread concerns because
of their frequent occurrence in marine ecosystems (Karthik et al.,
2018; Tang et al., 2018; Zhao et al., 2018; Zhu et al., 2018), freshwater
ecosystems (Di and Wang, 2018; Lin et al., 2018; Rodrigues et al.,
2018; Sighicelli et al., 2018) and terrestrial ecosystems (Horton et al.,
2017; Zhang and Liu, 2018). One of the main threats is that
microplastics may be uptake by human via seafood, such as inverte-
brates, crustaceans, and fish (Rochman et al., 2015; He et al., 2018).
Some aquatic organisms consumed whole, such as farmed shellfish,
are more likely to expose microplastics to the human diet (GESAMP,
2016). However, few studies have explored the occurrence of
microplastics in farmed fish and shellfish.
In the terrestrial ecosystem, agriculture is considered as one of major
sources of microplastic pollution (Ng et al., 2018). In the agro-
ecosystem, microplastics can be either directly generated through the
breakdown of plastic film mulching and greenhouse materials, or indi-
rectly occurred by the use of treated wastewater and biosolids
(Horton et al., 2017). Previous studies devoted more attention to occur-
rence and distribution of microplastics in agricultural soils (Nizzetto
et al., 2016a, 2016b; Rillig et al., 2017; Liu et al., 2018; Zhang and Liu,
2018). Several studies pointed out that wastewater and biosolids used
for irrigation and fertilizer can result in microplastics loading on agricul-
tural lands (Mohapatra et al., 2016; Nizzetto et al., 2016a; Ng et al.,
2018). Each year, approximately 6.3 × 104–4.3 × 105 and 4.4 × 104–
3.0 × 105 tons of microplastics entered in European and north
American farmlands, respectively, through biosolids (Nizzetto et al.,
2016a). In Australia, the mass of microplastics deriving from sludge ap-
plication was about 2.8 × 103–1.9 × 104 tons annually (Ng et al., 2018).
Given the central role of agro-ecosystems in food production,
microplastics pollution in agricultural soils and farmland systems at-
tract significant attention (Rillig et al., 2017).
Rice-fish co-culture, which is practiced in paddy fields over
2000 years in Asian countries (Anita et al., 2014; Islama et al., 2015),
has become an important agro-cultural activity in China (Lu and Li,
2006). In 2005, rice-fish co-culture became a globally important agricul-
tural heritage system (GIAHS) (Hu et al., 2013). Rice-fish co-culture is
considered as a sustainable agriculture type, which can not only opti-
mize the benefits of scare land and water resources, but also reduce
the usage of chemical fertilizers and pesticides relative to intensive,
high-input agriculture (Dwiyana and Mendoza, 2008; Xie et al., 2011).
The usage of plastic film was not common in rice plantation in the
south of China because of abundant rainfall. However, in the paddy
fields of water-stressed areas, such as Central China or the North
China Plain, the plastic film mulching cultivation (MC) method, i.e.
ground cover rice production system, has been widely applied to
adapt to drought conditions and to increase or maintain grain yields
(Qu et al., 2012; Liu et al., 2013; Yao et al., 2014). Nevertheless, so far,
the knowledge on microplastics in rice-fish system is still limited.
Rice-fish farming is of importance for the maintenance of biodiver-
sity due to the unique aquatic environment in this system. Many aquatic
animals have been bred in paddy field areas, including crayfish
Procambarus clarkii (Lu and Li, 2006; Si et al., 2017), crab Eriocheir
sinensis (Li et al., 2007), loach Misgurnus anguillicaudatus (Yang et al.,
2017), eel Monopterus albus (Sow et al., 2013; Ma et al., 2014), carp
Cyprinus carpio (Ren et al., 2018) as well as other species. Of these
aquatic animals, the benthic species, i.e. crayfish, crab, loach and eel,
have stronger bioturbation in the aquatic environment (Sun et al.,
2008; Faller et al., 2016; Alvarez et al., 2018). Activities of these benthic
animals can promote material circulation between surface and deep
soil, and accelerate benthic-pelagic coupling (Wheatcroft, 2006). There-
fore, bioturbation caused by aquatic animals may affect the distribution
of microplastics. Although previous studies demonstrated that
microplastics could be ingested by animals, and even transfer along ma-
rine food-webs (Santana et al., 2017; Lei et al., 2018), little is known
about the potential pollution of microplastics in aquatic animals under
rice-fish culture condition.
In this study, we selected three agricultural experiment stations for
rice-fish co-culture in Shanghai. Environmental samples of water and
soil, and animal samples were respectively collected in non-rice and
rice-planting periods. Microplastics were extracted, and then visually
identified under an optic microscope, followed by confirmation using
micro-Fourier transformed infrared (μ-FT-IR) spectroscopy. The abun-
dance and characteristics of microplastics were compared and analyzed
between environmental and organism samples as well as between in
non-rice and rice-planting periods. We aim to reveal the occurrence of
microplastic contaminations in water, soil and animals, and analyze dis-
tribution characteristics of microplastics in rice-fish co-culture
ecosystems.
2. Materials and methods
2.1. Rice-fish experimental stations
In this study, a total of three rice-fish experimental stations were in-
vestigated, in Shanghai: “rice-eel-loach-crayfish” station in Chongming
(CM), “rice-eel-crayfish” station in Qingpu (QP), and “rice-loach-cray-
fish” station in Fengxian (FX). Fig. 1 shows the location of three experi-
mental stations (Table S1).
Each co-culture station included two paddies. One was rice-fish ex-
perimental paddy bred with eel, loach and/or crayfish; another was
the control rice paddy without aquaculture. The experimental and con-
trol paddies had a similar structural configuration which was composed
of rice growing area (80% of total area), aquaculture area (10%) and the
ridge area (10%). A lateral trench with width of 0.3 m was dug in aqua-
culture area for aquatic cultivation (Fig. 2). The ridge was wrapped with
high density polyethylene film, which is slippery enough to prevent
aquatic animals from escaping. Eel (M. albus), loach
(M. anguillicaudatus) and/or crayfish (P. clarkii) were artificially re-
leased into experimental paddies in July 2017. In the three culture sta-
tions, breeding densities of eel, loach and crayfish were 2000, 10,000,
and 200 individuals per 667 m2, respectively. During a 150-day rice
cycle (from June 10th to November 10th), fertilizers and fish diets
were added into these co-culture systems at irregular intervals. For all
the three stations, base fertilizers (organic fertilizer 400 g, compound
fertilizer 27.2 g, calcium super-phosphate 36.5 g, KCl 7.3 g per
667 m2) and tillering fertilizers (urea 8.2 g per 667 m2) were applied
in each experimental or control paddy during May to June. Every two
days, 1.0–2.0 kg fish diets were added into each experimental paddy.
2.2. Sample collection
Water, soil and organism samples (crayfish, eel and/or loach) were
respectively collected from three rice-fish experimental stations in
April 2018 (non-rice period) and July 2018 (rice-planting period). In
each station, three 5 L duplicate water samples were collected from 0
to 10 cm below the surface with a stainless steel bucket and preserved
in glass bottles. A total of three soil samples were collected from rice
growing area in the volume of 0.006 m3 (0.25 m × 0.25 m × 0.10 m);
and another three duplicate samples from aquaculture area in the vol-
ume of 0.002 m3 (0.15 m × 0.15 m × 0.10 m) using a Lenz sampler
(Hydro-Bios, Germany). More than 1 kg of wet soils were collected
from each replicate and stored in aluminum pots. In the three stations,
 W. Lv et al. / Science of the Total Environment 652 (2019) 1209–1218
Fig. 1. The location of three rice-fish co-culture stations (FX, CM, and QP) in Shanghai.
water and soil samples were simultaneously collected in experimental
(FX-exp, CM-exp, QP-exp) and control paddies (FX-con, CM-con, QP-
con). During the sampling of aquatic animals, the rice field was briefly
adjusted to a low water level. The organism samples were collected
with a ground cage. In each experimental paddy, three eels, eight
loaches and eight crayfish were collected in non-rice or rice-planting
period. Water and soil samples were sealed and kept at 4 °C, and the an-
imal samples were kept at −20 °C until further analysis.
2.3. Sample treatment
The method of two-step filtration was used to extract microplastics
from water (Su et al., 2016). Each sample was measured and filtered
through 20-μm (pore size) nylon net filter (Millipore, MA, USA) by a
vacuum system. After that, 100 mL of 30% H2O2 was used to wash the
particles into a glass flask from the net filter, and then, the sealed flasks
were placed in an oscillation incubator to digest organic substances at
65 °C and 80 rpm for 72 h. The digested liquid was filtered again through
20-μm filters. Finally, the filters were covered and stored in dry petri
dishes for further observation.
A density separation method was used to extract microplastics from
soil samples according to our previous method (Liu et al., 2018). Each
soil sample was packed with aluminum foil and dried at 70 °C for
24 h. The dry soil was weighted and moved into clean glass beakers,
and then, added into saturated NaCl solutions (1.24 ± 0.05 g mL−1).
The mixtures were stirred 30 min and stood for 24 h, and then, the su-
pernatants were collected into clean glass bottles. The process of density
separation was repeated three times to fully extract plastic from soils.
After that, the suspended liquids were filtered with 20-μm pore size fil-
ter to obtain the supernatants (Liu et al., 2018). After the above steps,
the supernatants on the filter were washed into a glass flask with
100 mL of 30% H2O2, and this digestion process continued for 72 h at
65 °C and 80 rpm. Until all organic matters digested, the liquids were
1211
filtered again through 20-μm net filters. At last the filters attached
microplastics were collected and dried under room temperature before
observation.
To isolate microplastics, the organism samples were first rinsed with
deionized water to remove microplastics on the skin or carapace. Then,
dissection was conducted to remove digestive tissues from crayfish
(stomach and gut) and eel (foregut and hindgut) (Table S2). After
that, the above tissues and surplus bodies of crayfish (except for cara-
pace) and eels were transferred to the clean flask, and then 30% H2O2
was added for digestion. The loaches were directly digested without dis-
section due to their small-size body. The volume of H2O2
(10–15 mL g−1) was adjusted based on the mass of tissue/organism
samples. Then, the flask was covered and placed in oscillation incubator
at 65 °C and 80 rpm for 72 h. After digestion, the liquid was filtered with
5-μm nylon filter (Millipore, MA, USA), and the filter was stored in dry
petri dishes for further observation (Li et al., 2015; Su et al., 2018).
2.4. Observation and identification of microplastics
Microplastics were examined using a microscope (Olympus, BX43).
Photographs of the suspected microplastics were captured by a high
resolution camera (Olympus, DP73), and then, sizes of these particles
on the filters were measured using an image analysis software. Subse-
quently, some microplastics were picked out under a dissecting micro-
scope (Nikon, SMZ745) and confirmed by μ-FT-IR (Yang et al., 2015).
A total of 97 particles, including 23 items in water samples, 61 items
in soil samples and 13 items in organism samples, were randomly se-
lected for confirmation using the transmittance mode of μ-FT-IR. The se-
lected particles included each morphotype of visually identified
microplastics. Briefly, μ-FT-IR was used under the transmittance mode
(Peng et al., 2017), and the spectrum range was set in the range of
4000–675 cm−1 with a collection time of 3 s and scanned 32 times.
The detailed procedure for the spectra of microplastics was based on
1212 W. Lv et al. / Science of the Total Environment 652 (2019) 1209–1218

Fig. 2. Real picture of one rice-fish co-culture system (FX station) (A), and schematic overview and sampling sites (B) of the experimental design. Red dots show sampling points in the
system. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the method previously descripted (Liu et al., 2018). The microplastics 3. Results
identified by μ-FT-IR were calculated and further classified by their
shapes, colors and sizes. Basing on their shapes, microplastics were clas- 3.1. Microplastics in water
sified into four types: fibers, fragments, films and granules. Basing on
their sizes, microplastics were divided into small (b1 mm), medium Microplastics were found in all water samples from the three rice-
(1–3 mm) and large (3–5 mm) microplastics. fish culture stations except for control paddy of Qingpu (Fig. 3 and
Table S3). There were no significant differences in microplastic abun-
2.5. Quality control of experiments dances between in experimental and control paddies (Independent-
samples t-test, F = 5.5, P = 0.08). The average abundance of
All sampling tools and containers were rinsed three times with Milli
Q water before sample collection and treatment. During the sampling, 2.5
Non-rice period
the tools were washed using filtered water, which was filtered with Rice-planting period
0.45-μm nylon filter (Whatman, Maidstone, UK) between two collec-
Abundance of microplastics in water (items L-1)

tions. The samples were kept in enclosed space to isolate plastic pollu- 2.0
tion from air. During treatment, three blank samples were set as
background without water, soil and organism in each batch samples;
and no microplastics were observed in all blank groups. 1.5

2.6. Statistical analysis

1.0
In this study, all the data were shown as mean ± standard error of
the mean (SEM). t-Test was used to determine abundance differences
between rice-planting period and non-rice period, and between exper-
0.5
imental paddy and control paddy at 95% confidence level using SPSS
16.0 (P value b 0.05 was considered statistically significant). The corre-
lations of microplastic pollution among aquatic animals and environ-
0.0 ☆
ment were evaluated by a linear regression analysis. The average
FX-exp FX-con CM-exp CM-con QP-exp QP-con
microplastic abundances in water, soils and animals collected from the
three experimental paddies were pooled to calculate the correlation. Fig. 3. The average abundances of microplastics detected in water samples (n = 3) from
The Pearson coefficient was used to determine the significance of three rice-fish culture stations. The bars and whiskers represented mean and standard
correlation. error. ☆ expresses 0 items L−1.
 W. Lv et al. / Science of the Total Environment 652 (2019) 1209–1218
microplastics was 0.4 ± 0.1 items L−1. In the three experimental
paddies, the abundances of microplastics were 0.5 ± 0.1 and 0.9 ±
0.2 items L−1 in non-rice and rice-planting periods, respectively. How-
ever, the abundances of microplastics in control paddies were relatively
low, and their average values were 0.1 ± 0.1 and 0.2 ± 0.1 items L−1 in
non-rice and rice-planting periods, respectively. In the three rice-fish
culture stations, Chongming station showed the highest abundance of
microplastics in both non-rice and rice-planting periods. The lowest
abundance of microplastics was detected in QP-con during non-rice pe-
riod and CM-con during rice-planting period. Fig. 4A and B showed typ-
ical images of microplastics found in water from rice-fish culture
stations (Fig. 4).
Size, shape and color distributions of microplastics in rice-fish cul-
ture water are presented in Fig. S1. The vast majority were small and
medium size microplastics. Of four shapes, film and fiber were most
dominant types of microplastics detected from water in non-rice period
or rice-planting period. Of the four colors, white microplastics presented
the highest percentage in non-rice period; but the dominant
microplastics were transparent in rice-planting period.
3.2. Microplastics in soil
In all paddies of the three rice-fish culture stations, the average
abundance of microplastics was 10.3 ± 2.2 items kg−1 in soils. Further-
more, microplastic abundances were comparatively analyzed between
the rice soils and aquaculture soils (Fig. 5A, B and Table S3). We found
that the average abundance in rice soils was 16.1 ± 3.5 items kg−1,
and significantly higher than that in aquaculture soils (4.5 ±
1.2 items kg−1) (Independent-samples t-test, F = 16, P b 0.001). In
the rice-planting soils of experimental paddy, the average abundances
were 4.1 ± 1.8 and 20.8 ± 3.9 items kg−1 in non-rice and rice-
planting periods, respectively (Fig. 5A). However, in rice-planting soils
of the control paddies, the average abundances were 12.1 ± 2.5 and
27.6 ± 5.9 items kg−1 in non-rice and rice-planting periods, respec-
tively. In FX-con, CM-con, QP-exp and QP-con, the average abundances
in rice-planting period were significantly higher than that in non-rice
period (Paired-samples t-test, P b 0.05, Fig. 5A), but no significant vari-
ations were detected between experimental and control paddies in the
three stations (Independent-samples t-test, P N 0.05, Fig. 5A).
Fig. 4. Photographs of microplastics in rice-fish co-culture systems of Shanghai. Microplastics (red arrows) in water (A, blue polyethylene film and B, while polyethylene fiber), in soil (C,
translucent polyvinylchloride granules and D, white polypropylene fiber), and in aquatic animals (E, translucent polyethylene film and F, black polyethylene film). (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of this article.)
1213
In aquaculture soils of the three experimental paddies, the average
abundances of microplastics were 3.0 ± 0.7 and 10.1 ± 1.6 items kg−1
in non-rice and rice-planting period, respectively (Fig. 5B); and were 3.0
± 0.7 and 2.6 ± 0.6 items kg−1 in control paddies. In CM-exp and QP-
exp sites, microplastic abundances in rice-planting period were signifi-
cantly higher than that in non-rice period (Paired-samples t-test, P b
0.05, Fig. 5B). Moreover, in Chongming and Qingpu stations, the abun-
dances of microplastics in experimental paddies were significantly
higher than that in control paddies during rice-planting period (Inde-
pendent-samples t-test, F = 0.3, P = 0.003; F = 0.4, P = 0.02, Fig. 5B).
Fig. 4 shows some real images of microplastics assayed in soils of the
rice-fish co-culture system (Fig. 4C, D). The detailed information of size,
shape and color distributions of microplastics is shown in Fig. S2. There
are similar characteristics of microplastics between in rice-planting and
aquaculture soils. The dominant microplastics are in the size of b1 mm.
Meanwhile, microfibers are the largest proportion. White, translucent
and black are dominant colors of microplastics in the soils.
3.3. Microplastics in aquatic animals
Microplastics were detected in the three aquatic animals, i.e. eel,
loach and crayfish (Fig. 6 and Table S4). The abundances of microplastics
were respectively analyzed across the three rice-fish culture stations.
There were no significant changes in microplastic abundances of three
animals from non-rice to rice-planting period (Paired-samples t-test, P
N 0.05, Fig. 6). The total average abundance of microplastics in the
three animals was 1.7 ± 0.5 items individual−1. In Chongming station,
the microplastic abundances were 1.5 ± 0.3 items individual−1 in
non-rice period, and 3.2 ± 0.5 items individual−1 in rice-planting pe-
riod. Basing on individual analysis, the abundances are relatively high
in eel (3.3 ± 0.5 items individual−1), following crayfish (2.5 ±
0.6 items individual−1) and then loach (1.8 ± 0.5 items individual−1)
(Fig. 6A). All of microplastics were found in the digestive organs of
eels and crayfish (Fig. 6B). In Qingpu station, the average abundances
of microplastics were 0.9 ± 0.3 items individual−1 in non-rice period,
and 4.6 ± 0.6 items individual−1 in rice-planting period. The abun-
dances in eels (4.2 ± 0.9 items individual−1) were relatively higher
than in crayfish (2.2 ± 0.7 items individual−1) (Fig. 6C). No
microplastics were detected in the hindgut. The average abundances
1214 W. Lv et al. / Science of the Total Environment 652 (2019) 1209–1218

Non-rice period Rice-planting period

80 20
A B
*
Abundance of microplastics
*

In rice-planting soil (items kg-1)

* # #

In aquaculture soil (items kg-1)

60 15

*
40 10
*
*

20 5

0 0
FX-exp FX-con CM-exp CM-con QP-exp QP-con FX-exp FX-con CM-exp CM-con QP-exp QP-con

Fig. 5. The average abundances of microplastics in rice-planting (A) and aquaculture soils (B) (n = 3) in three rice-fish systems. The bars and whiskers represented mean and standard
error. #, P b 0.05 compared with experimental and control paddy. *, P b 0.05 compared with non-rice and rice-planting period.

were 4.2 ± 0.9 in foregut of eels, and 1.3 ± 0.5 and 0.9 ± 0.4 items were in small size, and with shape of fiber, and mostly in color of
individual−1 in stomach and gut of crayfish (Fig. 6D). In FX-exp, the av- white (Fig. S3).
erage abundances were 1.1 ± 0.3 items individual−1 in non-rice period
and 4.1 ± 0.6 items individual−1 in rice-planting period (Fig. 6E). 3.4. Identification of microplastics
Microplastic abundances were 3.0 ± 0.8 items individual−1 in crayfish,
and 2.2 ± 0.6 items individual−1 in loach. In digestive organs, the aver- Of the 97 randomly selected particles, 87 items were confirmed as
age abundances were 2.2 ± 0.8 items individual−1 in stomach and 0.6 plastics using μ-FT-IR, including 19 items in water samples, 57 items
± 0.2 items individual−1 in gut of crayfish (Fig. 6F). Totally in the in soil samples and 11 items in organism samples. A total of three
three aquatic organisms, there is an increase trend of microplastic abun- types of polymers were identified, including polyethylene (PE), poly-
dances from non-rice period to rice-planting period; however these propylene (PP), and polyvinylchloride (PVC). In water samples, the pro-
changes were not significant between two assay periods. Some images portions of PP and PE were 68.4% and 31.6%, respectively. In soil
of microplastics found in aquatic organisms were shown in Fig. 4E and samples, PE (61.4%) had the highest proportion, followed by PP
F. Analysis demonstrated that the major proportions of microplastics (35.1%) and PVC (3.5%). In animal samples, ten PE and one PP

Non-rice period Rice-planting period

12 8
In total (items individual-1)

In tissues (items individual-1) In tissues (items individual-1)

A B
9 6
In Chongming

6 4

3 2

0 0
Abundance of microplastics

Eel Crayfish Loach Foregut Hindgut stomach Gut

Eel Crayfish
12 12
-1
In total (items individual )

C D
9 9
In Qingpu

6 6

3 3

0 0 ☆☆ ☆
Eel Crayfish Foregut Hindgut Stomach Gut
Eel Crayfish
In tissues (items individual-1)

10 8
-1
In total (items individual )

E F
8
6
In Fengxian

6
4
4

2 2

0 0
Crayfish Loach Stomach Gut
Crayfish

Fig. 6. The abundances of microplastics in aquatic animals (n = 3–8) in Chongming (A–B), Qingpu (C–D) and Fengxian (E–F) rice-fish culture stations in Shanghai. The bars and whiskers
represented mean and standard error. ☆ expresses 0 items individual−1.
 W. Lv et al. / Science of the Total Environment 652 (2019) 1209–1218
microplastics were identified. Representative microplastics and their
transmittance spectrums were shown in Fig. S4.
3.5. Relationship of microplastics in animals, water and soils
Correlation of microplastics abundances were analyzed between
water, soils and aquatic animals. There were not significant correlation
between microplastic abundance in animals and water (regression
analysis, P = 0.215, Fig. 7A). However, we found that the abundance
of microplastics in aquatic animals were significantly correlative with
that in rice-planting soils (regression analysis, P = 0.003, Fig. 7B) and
that in aquaculture soils (regression analysis, P = 0.011, Fig. 7C).
4. Discussion
4.1. The occurrence of microplastics in rice-fish co-culture system
This study provides the first evidence of the occurrence of
microplastics in rice-fish co-culture systems. Microplastics were found
in both environmental samples (water and soil) and aquatic animals.
Our results showed that the abundances of microplastics in water of
these co-culture paddies (0.4 ± 0.1 items L−1) were generally lower
than that in other natural freshwater environments, such as Taihu
Lake (0.5–3.1 items L−1) (Su et al., 2018) and small waters of Yangtze
River Delta (0.5–21.5 items L−1) (Hu et al., 2018). Additionally, the
abundances of soil microplastics in this study were significantly lower
than that found in vegetable soils in Shanghai (Liu et al., 2018) or in sed-
iments of natural rivers (Peng et al., 2017). The difference in levels of
microplastic pollution among these studies may be due to hydrody-
namic conditions and application of plastic products in different envi-
ronments. In this study, the rice-fish culture paddies were usually
isolated from natural water column by the ridges. Under this condition,
(micro)plastics can be blocked for the entry into aquaculture paddies. In
addition, the irrigation water was often used in agricultural effluent in
other farmlands, which was considered as the main source of
microplastics in terms of previous studies (Kong et al., 2012; Zhang
and Liu, 2018). However, irrigation water was rarely utilized in rice-
fish co-culture environments in rainy season of Shanghai. On the con-
trary, the applications for plastic products such as organic fertilizer
and commercial fish diets could serve as a vehicle for the entry of
microplastic into the rice-fish culture environment.
In this study, microplastics were found in all three aquaculture ani-
mals; however, the abundances were relatively low and in the range
of 0.91–4.55 items per individual. In three aquaculture animals, eel
Fig. 7. The relations of microplastic abundances in aquatic animals and that in water (A), rice soils (B) and aquaculture soils (C) in three rice-fish culture stations. The circles represented
three experimental paddies in two sampling periods: ●, non-rice period; ○, rice-planting period.
1215
showed higher abundances of microplastics than crayfish and loach.
This may be relative to trophic transference of microplastics along
food chain (Santana et al., 2017; Diepens and Koelmans, 2018). How-
ever, this viewpoint was still controversial in the research of freshwater
and marine microplastics. Some scholars believed that microplastic in-
gestion by animals poses a significant risk for biomagnification of
microplastics up the aquatic food chain (Anastasopoulou et al., 2013;
Hurley et al., 2017; Rochman et al., 2017; Pegado et al., 2018). However,
another study found the reduced likelihood of trophic cascading of par-
ticles during the transference of microplastics from prey to predators
(Santana et al., 2017). As the top predator in “rice-eel-loach-crayfish”
and “rice-eel-crayfish” system, eel could feed on abundant prey, such
as loach, crayfish and other benthos. The predation may cause the sec-
ondary accumulation of microplastics, however, it needs further
research.
This study found that the abundances of microplastics in aquatic an-
imals were positively related with microplastic level in aquaculture and
rice soil. Moreover, size distributions, shape compositions and polymer
types of microplastics in aquatic animals were similar to that in soils of
experimental paddies. Therefore, it can be deduced that microplastic
content in aquatic animals may be closely dependent on the abundance
of soil microplastics.
The ingestion of microplastics in animals may be related to their
habits. In the current study, three aquatic animals were considered as
typical bottom-dwelling creatures. Given that the concentration of
microplastics was relatively high in surface soil of aquaculture area,
the ingestion may be more likely to be realized. Moreover, microplastics
particles of 0.02–1 mm and fibers had the greatest contribution, which
may be more easily ingested by aquatic animals (Bellas et al., 2016;
Huerta-Lwanga et al., 2016; Mizraji et al., 2017). Meanwhile, the excre-
ments of aquatic animals entered soils not only as rice fertilizer, but also
as the carrier of microplastics (Ory et al., 2018), which indirectly in-
creased the concentrations of water and soil microplastics. Therefore,
microplastics could cycle between surface soil and aquatic animals in
benthic environments.
This study demonstrated that microplastic abundances in experi-
mental paddies were generally higher that in control paddies. It indi-
cates that the bioturbation transports of aquatic animals can
contribute to distributions of microplastics in the rice-fish system.
Näkki et al. (2017) verified that secondary microplastics can be trans-
ferred to deep layers of soft marine sediments by Polychaete worms
(Marenzelleria spp.), amphipods (Macoma balthica) and bivalves
(Monoporeia affinis). In this study, the three aquatic animals, i.e. crayfish,
eel and loach, had an enhanced ability of burrowing and swimming.
1216 W. Lv et al. / Science of the Total Environment 652 (2019) 1209–1218
Therefore, microplastics can probably be brought into water from deep
soil or transferred to the surface of soil from the water by these animals.
Moreover, the skins of eels and loaches were covered by a lot of mucus,
which may be an important carrier of microplastics. Sun et al. (2008) in-
dicated that 39.7% of the tracer particles were transferred upward from
six-centimeter deep under the bioturbation of loaches. Therefore, the
bioturbation may be an important driver for the spatial distribution of
microplastics in rice-fish system.
4.2. The possible sources of microplastics in the agro-ecosystem of rice-fish
system
This study found that the abundance of microplastics was noticeably
increased in the rice-fish agro-ecosystem from non-rice period to rice-
planting period. Moreover, in most experimental paddies the rice-
planting soils contained higher concentrations of microplastics than
the aquaculture soils. It indicates that the activity of rice-planting is
one of the sources of microplastics in rice-fish co-culture system. During
the plantation of rice, striking input of plastic particles can stem from
the consequence of the application of chemical fertilizer and fish diets.
In the agro-ecosystem, primary microplastics were considered as an im-
portant part of total plastic debris (Ng et al., 2018). A recent study has
verified that organic fertilizer can serve as a vehicle for the entry of
microplastic into the environment (Weithmann et al., 2018). Beyond
that, the major ingredients of commercial fish diets are fishmeal,
which may become an important source of microplastics. Many studies
have demonstrated the occurrence of microplastics in wild fishes and
macroinvertebrates (Neves et al., 2015; Phillips and Bonner, 2015;
Cannon et al., 2016; McGoran et al., 2017; Kolandhasamy et al., 2018;
Windsor et al., 2018), which were often used as the raw materials of
fishmeal. Some scholars have reported the microplastics pollution
from atmospheric deposition (Dris et al., 2016; Cai et al., 2017), and
rainfall was an inescapable source of microplastics in rice-fish co-
culture system. In Shanghai, the rainy season usually occurs from June
to July, and the accumulation of microplastics may be accelerated dur-
ing this period.
In our study, there was an increasing trend in plastic pollution of
three aquatic animals from non-rice period to rice-planting period.
Moreover, the abundance of microplastics in the digestive tissues of
crayfish and eel accounted for 93.8–100% of total microplastics. The
above results prove that the transportation of microplastics may mainly
be occurred in the gastrointestinal tracts of eel and crayfish. Ory et al.
(2018) found that a ‘gustatory trap’ may be produced by fish food to
allow the fish to discriminate and reject the microplastics. Combined
with previous studies, our results imply that agriculture fertilizers, fish
diets and atmospheric deposition may be major sources of microplastic
pollution in the rice-fish co-culture systems.
4.3. The possible risks of microplastics pollution in rice-fish system
The rice-fish co-culture is a common agriculture activity all over the
world. Recently, several researchers demonstrated high levels of
microplastic pollution around aquaculture farms (Kazmiruk et al.,
2018; Wang et al., 2018; Xu et al., 2018). Compared with these natural
farms, the rice-fish co-culture may be more protected against
microplastic pollution. Moreover, compared to the monoculture of fish
or rice, the co-culture needs fewer applications of fertilizers in the sys-
tem (Hu et al., 2013; Feng et al., 2016). In addition, (micro)plastics
also can enter into paddies when waste water were drained in the co-
culture field. However, we found that most of microplastics were settled
on the surface of soils and did not spread out through water draining.
The levels of microplastic pollution were not high enough to profoundly
influence external water environment around the paddies. Therefore,
this study reveals actual pollution of microplastics under condition of
rice-fish co-culture environments.
Aquatic products intended for human consumption, such as crayfish,
loaches and eels, were another channel that microplastics can be spread
out from rice-fish system. Through ingestion, microplastics in aquatic
animals may eventually enter into the human food chain. Theoretically,
animals in aquaculture have less opportunity for microplastic exposure,
because the breeding conditions in paddies are strictly controlled.
Moreover, the lifespans of aquatic animals are generally short in breed-
ing aquaculture. However, Mathalon and Hill (2014) showed that the
mean abundance of microfibers in farmed mussels was 178 items
individual−1, and higher than that in wild mussels (126 items
individual−1). Nevertheless, there was no significant difference of
microplastics abundances between in cultured and wild clams
(Venerupis philippinarum) in another study (Davidson and Dudas,
2016). The occurrence of microplastics was also demonstrated in mus-
cle of commercial fish and crustacean, which fit for human consumption
(Abbasi et al., 2018; Akhbarizadeh et al., 2018). In contrast, microplastic
abundances of three aquatic animals in this study were actually at low
levels. Additionally, almost all of microplastics were found in digestive
organs of eels (foregut and hindgut) and crayfish (stomach and gut).
Therefore, the risk of microplastic pollution for human can be reduced
by the removal of the digestive tract when we eat these aquatic animals.
However, the most troublesome problem is that the intake opportunity
of microplastics cannot be accurately estimated according to normal
food detection. Further studies need to be performed in order to fully
explore microplastic pollution and its risks in agriculture.
5. Conclusion
This study revealed microplastic pollution in water, soil and aquatic
animals of rice-fish co-culture system. We found an increasing trend in
microplastic abundances in water, soil and animal samples from non-
rice period to rice-planting period. In rice-fish co-culture paddies,
microplastics level in rice-planting soils was generally higher than
that in aquaculture soils. Most of microplastics were found in digestive
tracts of eels, loach and crayfish. The majority of microplastics were PE
and PP in small size (b1 mm), with shape of fiber and fragment, and
in color of white and transparent. Size, shape, color and polymer type
distributions of microplastics were similarly found in environmental
and animal samples. Moreover, we found that microplastics abundance
in aquatic animals was correlated with that in farmland soils. Collec-
tively, our results reveal the occurrence and characteristics of
microplastics in rice-fish paddies and provide important data for subse-
quent research on the ecological risks and food security within rice-fish
co-culture systems.
Acknowledgements
This study was supported by a grant from Shanghai Municipal Sci-
ence and Technology Commission (15391912200), Shanghai Municipal
Agricultural Commission [(2016) 2-2-4] and [(2018) 1-28], the National
Natural Science Foundation of China (51708352), the Shanghai Rising-
Star Program (18QB1403000), the National Key Research and Develop-
ment of China (NO. 2016YFC1402204), the SAAS Program for Excellent
Research Team [nong ke chuang 2017 (A-03)].
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2018.10.321.
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