Science of the Total Environment 746 (2020) 141289

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Science of the Total Environment

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

Biodegradation and disintegration of expanded polystyrene by land

snails Achatina fulica
Yang Song a, Rong Qiu a,b, Jiani Hu a,c, Xinyu Li a,c, Xiaoting Zhang a,b, Yingxin Chen a,
Wei-Min Wu d,⁎, Defu He a,b,c,⁎⁎
a
School of Ecological and Environmental Sciences, Shanghai Key Laboratory for Urban Ecological Processes and Eco-Restoration, East China Normal University, Shanghai 200241, China
b
Shanghai Engineering Research Center of Biotransformation of Organic Solid Waste, East China Normal University, Shanghai 200241, China
c
Shanghai Key Laboratory for Urban Ecological Processes and Eco-Restoration, East China Normal University, Shanghai 200241, China
d
Department of Civil and Environmental Engineering, William & Cloy Codiga Resource Recovery Center, Center for Sustainable Development & Global Competitiveness, Stanford University,
Stanford, CA 94305-4020, USA

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

• Every land snail A. fulica ingested mean

18.5 mg expanded polystyrene for
4 weeks.
• Snails egested microplastics in feces
with significant mass loss of mean
30.7%.
• GPC showed an increase in PS molecular
weights in feces, confirming depolymer-
ization.
• FTIR, 1HNMR showed oxidation of PS
polymers and chemical modification in
feces.
• Significant shifts in gut microbial com-
munity appeared after the snails
ingested PS.

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

Article history: Despite increasing evidence of widespread plastic pollution in soil, it remains largely unknown about the
Received 9 July 2020 fate of plastic influenced by soil animals. In this study, ingestion and biodegradation capability of expanded
Received in revised form 25 July 2020 polystyrene (PS) foam was investigated in a globally distributed soil invertebrate, Achatina fulica. After 4-
Accepted 25 July 2020
week exposure, 18.5 ± 2.9 mg polystyrene was ingested per snail, and egested microplastics (1.343 ±
Available online 28 July 2020
0.625 mm) in feces with significant mass loss of mean 30.7%. Gel permeation chromatography analysis in-
Editor: Damia Barcelo dicated a significant increase in weight-average molecular weight (Mw) and number-average molecular
weight (Mn) of feces-residual PS, indicating limited extent depolymerization. Fourier transform infrared
Keywords: spectroscopy and proton nuclear magnetic resonance confirmed the formation of functional groups of oxi-
Plastic waste dized intermediates. Suppression of gut microbes with oxytetracycline did not affect the depolymerization,
Polystyrene indicating the independence of gut microbes. High-throughput sequencing analysis revealed significant
Biodegradation shifts in the gut microbiome after ingestion of PS, with an increase of family Enterobacteriaceae,
Achatina fulica Sphingobacteriaceae, and Aeromonadaceae, suggesting that gut microorganisms were associated with PS
Microplastics
biodegradation. These findings suggest that plastic litter can be disintegrated into microplastics and
Soil

⁎ Corresponding author.
⁎⁎ Correspondence to: D. He, School of Ecological and Environmental Sciences, Shanghai Key Laboratory for Urban Ecological Processes and Eco-Restoration, East China Normal
University, Shanghai 200241, China.
E-mail addresses: billwu@stanford.edu (W.-M. Wu), dfhe@des.ecnu.edu.cn (D. He).

https://doi.org/10.1016/j.scitotenv.2020.141289
0048-9697/© 2020 Elsevier B.V. All rights reserved.
2 Y. Song et al. / Science of the Total Environment 746 (2020) 141289
partially biodegraded by A. fulica, which highlights the significance of soil animals for the fate of plastic and
its biodegradation in soil environments.
1. Introduction
Global plastic production has exponentially increased in the past de-
cades, and reached 359 million tons in 2018 (PlasticsEurope, 2019). Al-
though the amount of plastic waste sent to recycling has doubled in
Europe and major industrial counties since 2006, plastic pollution is
still a major environmental concern (Geyer et al., 2017;
PlasticsEurope, 2019; Tokiwa et al., 2009; Xu et al., 2020). Further frag-
mentation of waste plastics produces microplastics (MPs, b 5 mm) and
nanoplastics (NPs, b 100 nm), which have become emerging persistent
contaminants (Lambert and Wagner, 2016; Lindeque et al., 2016; Peters
and Bratton, 2016; Wang et al., 2020; Wu et al., 2017). With global pro-
duction of more than 20 million tons per year, polystyrene (PS),
expressed as [-CH-(C6H5) CH2-]n, is one of the common polymers of
plastic litter detected in the environment. Among PS products, ex-
panded PS foam (EPS), used for food containers, building insulation,
and packing materials, has been widely detected in water environments
including municipal wastewater, rivers, lakes, sludge, and serves as a
source of MPs (Lv et al., 2019; Mani et al., 2015; Zhang et al., 2017;
Zhou et al., 2018).
Recent studies have shown increasing evidence of (micro)plastic
pollution in soil (Bläsing and Amelung, 2017; He et al., 2018; Rillig,
2012; Scheurer and Bigalke, 2018; Zhang and Liu, 2018). According to
estimates, annually 63,000–430,000 tons of MPs litter enters European
farmlands from sludge application alone (Nizzetto et al., 2016). Farmers
use plastics for multiple purposes, resulting in accumulation of plastic
waste in soil. Other entry paths include plastic encapsulated with pesti-
cides; organic fertilizers from urban waste recycling, such as compost,
sewage sludge, wastewater for irrigation; as well as dust from indoor
and outdoor air (Hurley et al., 2018; K. Liu et al., 2019; C. Liu et al.,
2019; Wang et al., 2019). The presence of (micro)plastics in soil envi-
ronments creates opportunity for interaction between plastic and soil
biota. Ubiquitous soil animals likely possess various capacity, such as in-
gestion and transportation of plastics (Helmberger et al., 2020; Ng et al.,
2018; Krueger et al., 2015; Zhu et al., 2018a). For example, Rillig et al.
(2017) demonstrated that microplastic polyethylene particles could
be transported from the soil surface down the soil profile via earth-
worms. Collembolans (Arthropoda) can transport microplastics in soil
(Maass et al., 2017). Another study showed Lumbricus terrestris could
cause significant incorporation of the small-fraction MPs from surface
litter into burrow walls in soil (Lwanga et al., 2017). In addition,
Lwanga et al. (2018) revealed that earthworm could uptake LDPE
microplastics and decrease particle sizes thus facilitating the decay of
LDPE, which even appeared in bacteria extracted from earthworm's
guts. It indicates soil animals have impacts on (micro)plastics, however,
the biodegradation by soil biota has been not addressed so far.
Although resistant to microbial biodegradation (Ho et al., 2017; Mor
and Sivan, 2008) in a wide range of habitats, PS has been known to be
susceptible to biodegradation in the gut of the larvae of darkling beetles
Tenebrio monitor and Tenebrio obscurus (Brandon et al., 2018; Ho et al.,
2017; Peng et al., 2019; Yang et al., 2015a, 2018a, 2018b) as well as
Zophobas atratus (Peng et al., 2020; Yang et al., 2020). These insects
chewed and ingested EPS and converted it to CO2 and water (Yang
et al., 2015a). Analysis of residual PS in egested feces using gel perme-
ation chromatography (GPC) indicated that PS was broad
depolymerized in the gut, i.e. significant decrease in both the number-
averaged molecular weight (Mn) and the weight-averaged molecular
weight (Mw). Other studies have demonstrated that the PS biodegrada-
tion in Tenebrio larval guts is gut-microbe-dependent because the depo-
lymerization was inhibited after these larvae were fed antibiotics to
© 2020 Elsevier B.V. All rights reserved.
depress gut microbes (Peng et al., 2019; Yang et al., 2015b, 2018a,
2018b). Despite that several studies have revealed biodegradation of
PS by some particular insects (Yang et al., 2015a, 2020; Lou et al.,
2020), it remains largely unknown about the ability of soil animals to
degrade PS.
Achatina fulica, one of the most globally distributed invertebrates
(Lange and Mwinzi, 2015), and invasive species of land snail, belongs
to Class Gastropoda, Family Achatinidae. This snail is originally native
in East Africa, and currently found as a pet and commercial food source
in Asia and Europe. A. fulica inhabits the top layer of soil and can be
widely found around gardens, fields and agricultural areas. The snail
A. fulica is a macrophytophagous herbivore, and eats a wide range of
plant material, fruit, vegetables, and even cardboard using the radula in-
side its mouth to break up food. The radula is a chitinous ribbon-like
structure containing rows of microscopic teeth. A. fulca snail matures
in 5–6 months and can live five to six years in captivity (Li et al., 2015;
Song et al., 2019; Wang et al., 2014). In the environment, A. fulica fre-
quently moves on the surface of soil, touching and ingesting litter that
may contain plastics.
Concerning the soil pollution of plastics including PS, it is of real en-
vironmental implication to explore the possibility of biodegradation by
the particular soil animal A. fulica, but none research has involved in this
field. In this study, the consumption of EPS was weighed after plastic-
feeding; mass balances were systemically analyzed. Compared to origi-
nal EPS, the residual PS extracted from feces was assayed by GPC to de-
termine changes of Mn and Mw; oxidized functional groups in the
residual PS was confirmed using proton nuclear magnetic resonance
(1H NMR) and Fourier transform infrared (FTIR). Antibiotic suppression
test and gut microbiome analysis were further performed to check the
involvement of gut microbiota in the PS depolymerization of the snails.
The objective of this study is to determine the ingestion and biodegrada-
tion capability of A. fulica for polystyrene.
2. Materials and methods
2.1. Test materials
Expanded polystyrene foam was purchased from Kunshan Kesun
High Polymer Materials Co., Ltd. (Suzhou, China). The polystyrene pu-
rity was higher than 98% according to the manufacturer. GPC analysis
showed that Mn was 92,000 Da and Mw was 234,500 Da. The EPS was
produced according to manufacturing standards in China (QB/T 4009-
2010) without added catalysts or other additives. Other chemicals and
reagents were purchased from either Sinopharm Chemical Reagent
Co., Ltd. or Thermo Fisher Scientific.
2.2. Achatina fulica
A. fulica snails (3–4 months old) were purchased from Jiaxing Hong-
Fu Breeding farm (Zhejiang, China), where they are commercially
reared with vegetables as feedstock. The snails were in their growing
period with average weight of 26.08 ± 0.98 g, shell diameter size of
3.46 ± 0.05 cm and shell length of 6.02 ± 0.09 cm. The snails were ac-
climatized in the laboratory for 3 weeks prior to tests at an air humidity
of 60%. A. fulica snails were treated with an 8 h light and 16 h dark cycle
per day at 25 ± 1 °C and were fed with a normal diet of lettuce (Song
et al., 2019; Chen et al., 2017).
 Y. Song et al. / Science of the Total Environment 746 (2020) 141289
2.3. EPS consumption tests
A. fulica snails were placed in glass jars, and fed an EPS block which
was pre-brushed with lettuce juice. Lettuce juice was brushed on the
surface of EPS block once a week. A co-diet of lettuce leaves was supple-
mented every 2 days. The test lasted for 4 weeks. At the end of each
week, the snails were removed to a clean glass jar for 12 h to collect
their feces for analysis of residual PS for fragmentation, depolymeriza-
tion and biodegradation. A total of 150 snails were used for the test, in-
cluding 76 snails fed EPS, 24 snails in the control group fed a normal
lettuce-only diet, 44 snails fed EPS plus antibiotics and 6 snails used
for gut microbial community analysis, respectively. The survival rate
(SR) was calculated based on live snails versus the initial number of
snails. The number of snails, average weight, length and diameter of
shells were measured weekly. All these parameters were recorded, to
compare between the PS-fed group and Control, referring to previous
tests with PET (Song et al., 2019).
2.4. Size of microplastics in feces
In order to measure the size of residual MPs in feces, fecal sample
were collected, and mixed with 30% H2O2 solution in a conical flask
for 72 h at 60 °C to digest/oxidize organic matters. PS were then ex-
tracted using saturated sodium chloride solutions as previously re-
ported (Liu et al., 2018; Lu et al., 2020). After digestion, MPs were
rinsed and then vacuum filtered through a 8-μm glass filter paper
(Whatman GF/B). The filters attached MPs were stored in glass culture
dish, and dried at room temperature. The MPs were observed under
an optical microscope; their morphotype and particle sizes were deter-
mined and analyzed.
2.5. Extraction of residual PS
Fecal samples of the snails fed with EPS plus lettuce was rinsed in de-
ionized water and then ethyl alcohol absolute (purity N99.99%) to re-
move dissolved organics, residual chlorophyll-like compounds and
other impurities. After the pretreatment, tetrahydrofuran (THF, purity
≥99.8%, GC) was used to extract residual PS polymer for 2 h at ambient
temperature. The extracted solution was filtered with a 0.20 mm PVDF
sterile syringe filter (Thermo Fisher Scientific), transferred to a clean,
pre-weighed glass vial, and then evaporated to collect extracted PS
polymer (Yang et al., 2015a; Peng et al., 2019). The glass vial was then
weighed to calculate the weight of extracted polymer for GPC, 1H
NMR, and FTIR analysis.
To determine the mass balance of PS degraded, a group of snails
(n = 24) was fed EPS. After four weeks, all feces were collected, and
then extracted by THF to calculate the mass of residual PS in feces. In
the meantime, the EPS block was weighed weekly. The mass reduction
of EPS was calculated using the initial weight of the EPS block minus the
weight of remaining EPS block and the residual PS in feces. The THF ex-
tractable ratio was calculated from the weight of extracted polymer di-
vided by the dry weight of the feces.
2.6. Characterization and analysis of residual plastic in feces
GPC (Agilent 1260, Agilent Technologies Inc., USA) was applied to
analyze the molecular weights (Mw and Mn) and molecular weight dis-
tribution (MWD) of the polymer. PS extracted from the EPS feedstock or
feces using THF was filtered through 0.20 mm PVDF filters. The THF so-
lution was mixed on a magnetic stirrer with a heater. After that, the ex-
tracted solution was concentrated to 5 mg/mL for GPC analysis at a flow
rate of 0.7 mL/min at 40 °C with THF as eluent to determine Mn and Mw
and plot MWD (Yang et al., 2018a; Peng et al., 2019). All GPC analyses
were performed in duplicate. Polydispersity Index (PDI) was calculated
as PDI = Mw/Mn.
3
The fecal samples were analyzed using Fourier Transform Infrared
Spectroscopy (FTIR, Thermo Fisher Scientific, Inc., Pittsburgh, PA) in
comparison of the EPS feedstock and feces of snails fed only lettuce at
a range of 4000–500 cm−1. Prior to the analyses, samples were freeze
dried for at least 36 h, and then ground with KBr to prepare a homoge-
nous KBr pellet for scanning (Peng et al., 2019).
Proton nuclear magnetic resonance (1H NMR) analysis was adopted
to characterize the degradation by the PS-feeding groups. The EPS feed-
stock and residual polymers from feces were comparatively analyzed.
Samples extracted from the feces of snails fed only lettuce were used
as a control. The PS was extracted from fecal samples (50 mg) as de-
scribed above, and then re-suspended in chloroform-D (purity
≥99.9%). Proton-NMR spectra were obtained on a Varian Inova 500-
MHz NMR spectrometer at 55 °C (Agilent Technologies, Inc., Santa
Clara, CA). The 1H-spectra were measured on a 500-MHz spectrometer
(32 scans, delay time d1 = 0.0 s) and referenced to the residual
deuterated-chloroform peak (1H-7.26 ppm). MestReNova software
(version 10.0.2) was used to analyze spectra, and the value was
shown in parts per million (ppm) (Yang et al., 2018a).
2.7. Antibiotic suppression test
To test the effect of antibiotic suppression on PS depolymerization
and biodegradation, the snails were fed oxytetracycline prior to EPS. A
total of 44 snails were divided into two groups. The antibiotic sup-
pressed group (30 snails) was fed with lettuce containing oxytetracy-
cline (100:1, w/w) for 5 days while the control group was fed with
lettuce only. Subsequently, they were fed EPS treated with lettuce
juice, and then fecal samples were collected for GPC analysis. This pro-
cedure was repeated every two weeks. In order to check the effect of
suppressing gut microbes, six snails were randomly taken from each
group to count the intestinal bacteria. Then snail shells were cut off, eu-
thanized with 80% ethanol, and then rinsed with Milli-Q water. After-
wards, the gut contents of the snails were then submerged and
suspended in 2 mL sterile saline water (7.5% NaCl). The gut suspension
was serial-diluted from 10−1 to 10−6 and spread on tryptic soy agar
plates. After incubation at 37 °C for 48 h, the number of colonies was
counted.
2.8. Microbial community analysis
After starting EPS-feeding, six snails were randomly selected at day 0
(before), day 14 and day 28 for gut microbial community analysis. The
snails were quickly dissected under sterile conditions. The entire intes-
tine was removed with a sterile scalpel under sterile conditions, and the
intestinal wall was cut up and put into a 2 ml sterile centrifuge tube con-
taining sterile saline. The saline was homogenized using a laboratory
vortex and centrifuge to enrich the pellet at 4 °C for further DNA analy-
sis. The samples were named W0 (wa0, wb0), W2 (wa2, wb2), and W4
(wa4, wb4) to represent the samples from day 0, day 14 and day 28,
respectively.
After extraction of total DNA from the gut samples, universal primers
linked with indices and sequencing adaptors were used to amplify the
V3-V4 regions of the bacterial 16S ribosomal RNA (16S rRNA) genes.
The amplicons were sequenced on an Illumina Miseq platform to obtain
250-bp paired-end reads and taxonomic assignment. The DNA extrac-
tion and PCR amplification methods were similar to previous reports
(Lin et al., 2019). The detailed procedures are presented in Supporting
Information.
In order to investigate the species composition and microbial diver-
sity, clean tags were clustered to generate an OTU (Operational Taxo-
nomic Unit) after splicing and filtering of all samples and removing
the barcodes and primers simultaneously. Representative sequences of
OTU were selected and compared with existing databases to obtain spe-
cies annotation information. Sequence analysis was performed by
USEARCH software (V10). Sequences with ≥97% similarity were
4 Y. Song et al. / Science of the Total Environment 746 (2020) 141289
assigned to the same OTU. An OTU was considered as a possible repre-
sented species. The most frequently occurring sequence was extracted
as a representative sequence for each OTU and was screened for further
annotation. During the clustering, USEARCH could remove the chimera
sequence and singleton OTU at the same time. Finally, microbial com-
munity composition, hierarchical cluster analysis, principal coordinate
analysis (PCoA), and ternary analysis were run on the online platform,
MagicHand (Magigene, Guangzhou, China).
3. Results and discussion
3.1. Consumption and disintegration of EPS
The A. fulica snails actively ate the EPS block covered in lettuce juice
(Fig. 1a and b). Despite also eating some of the EPS block without lettuce
juice prior to the test, the brushed lettuce juice on EPS block enhanced
the appetite of snails for the EPS block. They egested green-colored
feces containing residues of lettuce and PS within 12 h after feeding
(Fig. 1c). The snails especially preferred to chew the edge of EPS block
and use their radula to scrape deeply into the surface of the block,
resulting in a number of hollows up to 2.0 cm in width and
2.0–4.0 mm in depth. After passing through the intestine, a part of
ingested EPS foam was disintegrated or fragmented into PS MPs
(Fig. 1c). Based on microscopic observation and statistical analysis, the
mean size of the MPs was 1.343 ± 0.625 mm, ranging from 0.146 to
3.094 mm (Fig. 1d).
Fig. 1. Polystyrene Styrofoam were uptake by A. fulica and fragmented into microplastics. (a) The exposure scene of Styrofoam and A. fulica. (b) Mark on Styrofoam of being gnawing (the
top right showing enlarge image in the blue box). (c) Microplastics in feces. (d) The distribution of microplastic sizes.
Despite a growing body of research about the ingestion of (micro)
plastics by aquatic animals (Jâms et al., 2020), and even turning MPs
into NPs through digestion of Antarctic krill (Dawson et al., 2018), di-
gestive fragmentation of plastic litter has rarely before been reported
in other soil animals. This study reveals that a major species of land
snails does ingest and digest PS, and turn it into microplastics as plastic
residue, which is subsequently discharged into soils. Plastic pollution is
of growing concerns; soil may represent a large reservoir for plastics in
the environment (Chae and An, 2018; He et al., 2018; Nizzetto et al.,
2016; Steinmetz et al., 2016). Plastic fragments are thus likely wide-
spread, irrespective of their origin, and are expected to arrive at the
soil surface, and further exert adverse consequences (Cao et al., 2017;
Kim and An, 2019; Zhu et al., 2018b); earthworms and springtails can
experience critical damages following microplastic exposure (Ju et al.,
2019; Lwanga et al., 2016). Soil animals like land snails could contact
and ingest plastics on the surface of soil. Our results reveal that digestive
disintegration or fragmentation of land snails can produce a mass num-
bers of MPs. The number and size of MPs might be dependent on the
radula, intestinal structures, and biodegrability of snails. The upper
size of MPs is likely limited by the size of their mouth. It implies that
soil animals can become one of main drivers of MP production in soil
environments.
Further analysis showed that the accumulated EPS consumption and
residual PS mass in feces increased as feeding time progressed (Fig. 2a).
The average EPS consumption rate was 18.5 ± 2.9 mg per snail at the
end of the 28 d test. During the same period, the snails consumed
5.6 g lettuce (dry weight/snail) resulting in a ratio of 0.06 (g/g, w/w)
 Y. Song et al. / Science of the Total Environment 746 (2020) 141289
Fig. 2. Polystyrene consumption, survival rate and weight of A. fulica. (a) The PS consumption and residual PS in feces during PS-feeding test period. N = 8, *p b 0.05, ingested PS compared
to residual PS in feces. (b) The survival rate and changes in weight of A. fulica. No significant difference was observed (p N 0.05).
PS/lettuce consumed, indicating that the snails primarily consumed let-
tuce with limited consumption of EPS. In the feces, the average extract-
able fraction by THF (residual PS) was 3.65% (w/w) on the basis of
average of 4 weeks (Fig. S1). The mass of residual PS in feces was signif-
icantly lower than the amount of PS consumed for both 2 and 4 weeks
(Fig. 2a). In the experiment group, the mass ratio of ingested EPS and
egested PS in feces was 1.4:1.0 (g/g), indicating that approximately
30.7% of ingested EPS was removed in the digestive system of the snails.
The survival rates of the EPS-fed group versus the control were sim-
ilar (Fig. 2b). The weight change of PS-fed group was the same as the
control group (Fig. 2b). Both length and diameter of shells followed an
increasing trend without significant difference between PS-fed and con-
trol groups (p N 0.05, Fig. S2). These results indicated that the ingestion
of EPS did not have any negative impact on the survival and develop-
ment of the snails.
This study is the first to confirm the ability of consumption of PS by
A. fulica. According to previous studies, MPs pollution is associated with
the activity of terrestrial animals (Chae and An, 2018; Helmberger et al.,
2020). For instance, Allasino et al. (2019) found that bees living alone
could break large plastics into small fragments and use them as nesting
materials. Similarly, terrestrial isopods (Procellio scaber) were proved to
crush PE bags into small fragments (Jemec Kokalj et al., 2018). Our pre-
vious investigation found 20 types of small animal-based medicinal ma-
terial and available corresponding fresh terrestrial animals contained
MPs (Lu et al., 2020). Among them, earthworms, slugs, mole crickets,
cockroaches, centipedes, eupolyphaga and grubs have a high probability
of ingesting soil contaminated with MPs. Based on the findings in the
present study, it is speculated that land snails could ingest PS and
other plastics via contaminated plant leaves, litter or soil (Song et al.,
2019); however, more research need to determine the feasibility of
fragmentation and degradation for other plastic wastes such as PE, PP
and PVC.
3.2. Depolymerization of PS
Fecal samples of the PS-fed group were collected at end of week 2
and week 4, and analyzed in comparison to EPS feedstock (or week 0).
GPC results showed that the Mn values of residual PS extracted from
the feces gradually increased from 92,000 ± 850 (prior to test) to
106,000 ± 80 on day 14 and 113,000 ± 4100 on day 28; an increase
by 15.3% and 22.9%, respectively (Fig. 3a). The Mw increased from
234,500 ± 2200 to 250,300 ± 400 by 6.7% on day 14 and shifted to
227,000 ± 17,000, statistically unchanged (p N 0.05), on day 28
(Fig. 3a). MWD analysis indicated that the molecular weight of the
5
residual polymers shifted towards higher molecular weights (Fig. 3b,
red and black lines). According to Mn and Mw values, polydispersity
index (PDI) was determined as polymer diversity indicator. Results
showed that PDI of EPS feedstock was 2.55, and PDI of residual PS de-
creased progressively to 2.36 on week 2 and 2.01 on week 4.
GPC provides Mn and Mw, which have been considered as major in-
dication of depolymerization of polymers (Albertsson et al., 1998; Yang
et al., 2015a). Our results showed significant increase in Mw and Mn and
MWD shift towards higher molecular of PS in the digestion of the snails
after 2 and 4 weeks (Fig. 3). The changes in Mn and Mw indicated the de-
crease in lower molecular weight portion and increase in higher molec-
ular weight portion in the egested feces. PDI results confirmed
reduction of broadness of polymer chains in the egested PS. This indi-
cates a specially limited extent depolymerization of plastic polymer.
The similar depolymerization pattern was also observed during biodeg-
radation of polyurethane (PUR) by a landfill microbial culture, which
showed that Mw of PUR increased from 208,500 to 229,400 on day 15
and then decreased to 169,900 on day 25 during a 25 day batch test
(Gaytán et al., 2020). In addition, PS degradation in Galleria mellonlla lar-
vae resulted in an increase in Mn from 132,1000 to 146,900 and Mw
from 361,700 to 377,800 in a recent study (Lou et al., 2020). Peng
et al. (2020) also showed that biodegradation of low-density polyethyl-
ene and PS in superworms, larvae of Zophobas atratus from a source in
the USA with characteristics of decrease in Mn and Mw. Nevertheless, a
commonly observed PS depolymerization pattern during PS biodegra-
dation in the larvae of Tenebrio genus (Brandon et al., 2018; Peng
et al., 2019; Yang et al., 2015a, 2015b; Yang et al., 2018a, 2018b; Yang
et al., 2020) and bacterial culture (Syranidou et al., 2019) was a broad
depolymerization pattern, i.e. the decrease in both Mn and Mw of the
plastic polymer, as well as a shift of MWD towards lower molecular
weight (Peng et al., 2019). The limited extent depolymerization of PS
may be related to the selective depolymerization/biodegradation of
lower molecular polymers at higher rate than longer chain polymers.
Further research is needed to investigate the mechanism of the different
depolymerization patterns.
3.3. Evidence of biodegradation and oxidization
Evidence of biodegradation and oxidation of PS were obtained by
using FTIR and 1H NMR analyses to confirm the formation of oxidized
intermediation and modification of residual PS. Based on comparisons
of the FTIR spectra of the EPS feedstock and PS extracted from the
snail feces, there was an obvious occurrence of chemical changes and
the incorporation of oxygen (Fig. 4a). Peaks at 625–970 cm−1 (ring-
6 Y. Song et al. / Science of the Total Environment 746 (2020) 141289

Fig. 3. Molecular weight changes of PS through the digestion of A. fulica. (a) Mn and Mw of EPS feed, and feces-residual PS extracted from normal, prior antibiotics (gentamicin)-treatment
and after antibiotics treatment. (b) Molecular weight distribution shifts of original PS, and feces-residual PS after 2 and 4 weeks with and without antibiotics treatment. N = 8, * p b 0.05, **
p b 0.01, compared to primary PS.

bending vibration) were high in the EPS feedstock but lower in fecal
samples. Characteristic peaks of the PS benzene ring (C_C stretch,
1550–1610 and 1800–2000 cm−1) were dampened in PS-fed fecal sam-
ples, indicating evidence of ring cleavage. New functional group peaks
were observed at 1075–1150 cm−1 (−C−O−), 1650 cm−1 (−C=C−
stretch), and 1700 cm−1 (−C=O stretch) in the PS-fed feces (Fig. 4a),
indicating oxidation of the PS polymer.
The 1H NMR spectra of PS-fed feces showed new peaks related to
oxidation (Fig. 4b), i.e. the new peaks were detected in chemical shift
regions associated with -CH=CH-, carbonyl (H2C=O), and hydroxyl
(-OH) groups, which appeared in PS-fed feces, but not in the EPS feed-
stock or control feces, indicating obvious evidence of transformations
and modifications in molecular structure of PS.
Both FTIR and 1H NMR spectra showed the formation of new oxi-
dized functional groups of PS-fed feces as evidence of formation of
biodegraded intermediates as similar to that previously observed dur-
ing PS biodegradation in Tenebrio genus (Brandon et al., 2018; Peng
et al., 2019; Yang et al., 2018a, 2018b). This provided strong evidence
of PS biodegradation in A. fulica snails. Despite considerable knowledge
gaps of mechanisms about the limited biodegradation, the increase in
Mw and Mn means that the ingested PS was modified and could be selec-
tively depolymerized for lower molecular weight polymers at a higher
rate than higher molecular polymers or the presence of crosslinking re-
actions during biodegradation or both. In addition, the time-dependent
decrease of PDI indicated a notable reduction in the broadness of poly-
mer molecular weight distributions after the digestion of snails. It is
consistent with significant changes in PDI in the previous studies
about the biodegradation of PS in T. molitor (Peng et al., 2019; Yang
et al., 2015a; Yang et al., 2018a; Yang et al., 2020). This study discloses
the ability of PS biodegradation by the soil animal A. fulica snails. After
extensive initial photo- or thermal-oxidative degradation, biodegrada-
tion may play an important role in the ultimate fate of plastics in the soil.
3.4. Antibiotics suppression effects
We used oxytetracycline, a broad-spectrum antibiotic, active against
a wide variety of bacteria by interfering with the ability of bacteria to
produce essential proteins. After pre-treatment of oxytetracycline for
5 day, the gut bacteria in snails decreased from 5.81 × 107 to
0.93 × 105 CFU per gut (Fig. S3). In the control (no antibiotics), the num-
ber of gut bacteria remained unchanged. After the snails were fed with
EPS foam, Mw increased slightly from 234,500 ± 2200 to 246,000 ± 400
and 251,000 ± 3200; while Mn increased from 92,000 ± 850 to
100,900 ± 400 and 109,700 ± 1900 on day 14 and day 28, respectively
(Fig. 3a). Both Mw and Mn of PS-fed feces with oxytetracycline were sig-
nificantly higher than those of EPS feedstock (p b 0.05); but there were
no significant differences between normal and antibiotic-treated groups
(Fig. 3a). The MWD shift was also similar with the normal group
(Fig. 3b). PDI decreased from 2.55 to 2.43 (week 2) and then 2.28
(week 4), which was not significantly different from those without an-
tibiotics (2.36 and then 2.01) as described in the above section. These
results indicate that the limited extent depolymerization of PS still

Fig. 4. Depolymerization characteristics of EPS through the digestion of A. fulica. (a) FTIR spectra of primary PS, feces-residual PS, and control feces (without EPS feed). (b) 1H NMR spectra
of primary PS, feces-residual PS, and control feces. The appearance of alkene derivatives is highlighted in gray box.
 Y. Song et al. / Science of the Total Environment 746 (2020) 141289
occurred under the oxytetracycline-induced suppression of gut bacteria,
i.e. the decrease of polymer broadness at lower rate than that without
antibiotics. It might be due to the special or limited depression on gut
bacteria induced by oxytetracycline, which is obviously different from
previous observations using the antibiotic gentamicin in darkling bee-
tles T. molitor (Peng et al., 2019; Yang et al., 2015b; Yang et al., 2018a,
2018b). Based on antibiotics suppression effects of oxytetracycline
treatment, the present study indicates that PS depolymerization is inde-
pendent of the gut-microbe to a certain degree, which implies that the
digestive enzyme(s) in intestinal tracts of snails can perform breaking
down of PS polymers, especially with lower molecular weight.
3.5. Gut microbial communities
Using high-throughput sequencing of the V3–V4 regions of the 16S
ribosomal RNA genes, the gut microbiomes were assayed before
(week 0) or after EPS feeding for 2/4 weeks. A total of 212,518 se-
quences were obtained, with an average length of 474 bps; sampling
coverage was above 0.99. This indicated that Illumina Miseq sequencing
was detectable. Rarefaction curves with a Shannon Index at the opera-
tional taxonomic unit (OTU) level were generated for all samples at
0.97. The rarefaction curves of all samples achieved a plateau at about
4000 reads, implying that the amplicons were enough for sequencing
(Fig. S4). Compared to the control group (mean OTUs number of 694),
the PS-fed groups had lower taxonomic richness, with mean OTUs num-
ber of 511 or 452 after 2 or 4 week- exposure. The results indicated that
the gut microbiome dropped after EPS feeding with co-diet of lettuce.
The difference in initial diversity could be related to their normal
diets, as well as gut eco-physiology. As mentioned above, A. fulica snails
ingested a relatively small amount of PS in week 2, which might be
linked to the diversity of gut microbiome in the second week. In addi-
tion, the change of microbial structure was investigated using Principal
coordinate analysis (PCoA) at the OTU level (Fig. S5). Results showed
the microbial community structure was dispersed among three groups.
On the basis of relative abundance, the gut microbiome in the
snails contained 20 main families (Fig. 5a). Before PS-feeding, the
dominant families included Enterobacteriaceae, Sphingobacteriaceae,
Aeromonadaceae, Porphyromonadaceae and Shewanellaceae. After
PS-feeding, the relative abundances of Enterobacteriaceae and
Sphingobacteriaceae increased, but Aeromonadaceae and Shewanellaceae
decreased. The ingestion of PS resulted in the increase in relative abun-
dance of Enterobacteriaceae from 11.9% to 64.4% and 32.2% on week 2
Fig. 5. Changes of gut microbial community of A. fulica after feeding EPS. (a) Relative bacterial abundances at family level. (b) Gut bacterial communities at genus level in A. fulica, before
feeding (PS-0W), and after feeding for 2 (PS-2W) or 4 weeks (PS- 4W). The color intensity of the scale indicates the relative abundance.
7
and week 4, respectively. The relative abundance of Sphingobacteriaceae
also increased from 6.7% to 25.5%. But the relative abundance of
Aeromonadaceae decreased from 9.2% to 1.7% after PS feeding. A hierar-
chical clustered heatmap analysis showed that microbial communities
in W2 and W4 groups exhibited many similarities but were different
from the control group (Fig. 5b). This is consistent with the relative
abundance analysis and ternary analysis of gut microbial community
(Fig. S6). Recently, a similar increase in Enterobacteriaceae was observed
in the ingestion of PS or PE/PS by the beetle T. molitor and T. obscurus
(Brandon et al., 2018; Peng et al., 2019). These results are consistent
with the present study, which indicates that the family Enterobacteria-
ceae might be specially associated with PS degradation. In addition,
Xanthomonas and Pseudomonas were reported to be related to PS degra-
dation (Mohan et al., 2016; Sekhar et al., 2016). It reflects the gut-
microbe-independence of degradation of beeswax and LDPE (Kong
et al., 2019), and also containing PE-degrading bacteria in gut (Ren
et al., 2019).
According to these results, the ingestion of EPS by the snails caused
the shift of gut microbiota and the established new microbiota was as-
sociated with the PS biodegradation. Despite further research needed
for the in-depth mechanism of PS biodegradation by A. fulica, this
study suggests that gut microbiota is likely involved in the biodegrada-
tion of PS in A. fulica snails. The ingestion of PS by the snails caused the
shift of gut microbiota and the established new microbiota was associ-
ated with the biodegradation. We hypothesize that gut microbes, such
as Enterobacteriaceae and Sphingobacteriaceae with increased abun-
dance after PS-feeding, could play a role in the biodegradation of inter-
mediates in snail guts.
In our recent study, the digestion of snails can cause cracks and de-
terioration of the surface of polyethylene terephthalate (PET)
microfibers, which was mostly dependent on the action of the intestine
(Song et al., 2019). In this study, A. fulica snails used were commercially
reared animals which had been fed with clean artificial feed such as veg-
etables. Their history may limit their gut microbiome for plastics degra-
dation. The presence of high-efficient PS-degrading gut microbes in
wild snails cannot be ruled out. The snail radula also helps to change
plastics into small fragments with high specific surface area, which ben-
efits enzymatic depolymerization and biodegradation. PS biodegrada-
tion could be more efficient due to synergistic reactions if a proper gut
microbiome were selected or adopted. More research is still needed to
identify the ubiquity of plastic degradation using A. fulica and other spe-
cies of snails from various geographical locations and functional gut-
8 Y. Song et al. / Science of the Total Environment 746 (2020) 141289
microbial species that are capable of PS biodegradation of major
intermediates.
4. Conclusion
For the first time, this study reveals that land snails Achatina fulica
has the capacity to depolymerize and biodegrade polystyrene. Mass bal-
ance, GPC, FTIR and 1H NMR analyses confirmed the limited extent de-
polymerization and oxidation of PS polymers, which supported the
occurrence of biodegradation. A. fulica snails ingested EPS and also gen-
erated numerous microplastics via excretion, and further adapted to the
occurrence of (micro)plastics in soil environments. Concerning land
snail was one of the mostly popular and rapidly proliferated terrestrial
animals, these findings are significant in regards to the fate of plastic lit-
ter and its biodegradation in soil environments.
CRediT authorship contribution statement
Yang Song: Conceptualization, Formal analysis, Writing - original
draft. Rong Qiu: Methodology, Formal analysis. Jiani Hu: Methodology,
Investigation. Xinyu Li: Investigation. Xiaoting Zhang: Methodology,
Investigation. Yingxin Chen: Methodology, Formal analysis. Wei-Min
Wu: Conceptualization, Methodology, Writing - review & editing.
Defu He: Conceptualization, Methodology, Investigation, Validation,
Supervision, Funding acquisition, Project administration, Writing -
review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This work was supported by the National Key Research and Develop-
ment of China (No. 2018YFC1901004), Joint Project of National Natural
Science Foundation of China (No. U19A2095), National Science and
Technology Major Project of the Ministry of Science and Technology of
the People's Republic of China (No.2018ZX07208008), and funded by
Shanghai Engineering Research Center of Biotransformation of Organic
Solid Waste (SERC2020A01). Dr. W.-M. Wu appreciates the financial
support by the Woods Institute for Environment at Stanford University
(Award 1197667-10-WTAZB). The authors thank Ms. Julia T. Wu, Uni-
versity of Wisconsin, Madison for the assistance of English editing and
preparation.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2020.141289.
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