Ecological Indicators 133 (2021) 108370

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Ecological Indicators
journal homepage: www.elsevier.com/locate/ecolind

Effects of microplastic fibers on Lates calcarifer juveniles: Accumulation,

oxidative stress, intestine microbiome dysbiosis and histological damage
Mujiao Xie a, c, Lang Lin a, Peng Xu a, c, Weiguo Zhou a, b, Changsheng Zhao a, c, Dewen Ding a, b,
Anning Suo a, b, *
a
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
b
Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 511458, China
c
University of Chinese Academy of Sciences, Beijing 100049, China

A R T I C L E I N F O A B S T R A C T

Keywords: Microplastic fibers originating from fishing ropes and nets are persistent and ubiquitous marine environmental
Microplastic fibers pollutants that pose potentially threat to the health of aquatic species. Therefore, assessing their potential effects
Fish on marine biota has become an urgent research topic. In this study, the fate of fibers ingested by L. calcarifer and
Intestinal microbiome
its consequence on intestine health were analyzed. A total of 150 fish was fed with polyethylene (PE) fibers at 1%
Histopathology
concentration for 56 days. Retention of fibers, oxidative stress indicator malondialdehyde (MDA), intestinal
permeability indicator diamine oxidase (DAO), intestine microbiome modulation and histological damage were
evaluated. The results indicated that the consumption of fibers-contained food did not influence the growth of
the fish. Low retention of fibers was observed, indicating effective elimination of fibers from the body of the fish
and no significant accumulation after successive exposure. However, intestine oxidative stress was observed,
indicated by an up regulation of MDA level. Whereases, DAO level was not influenced by the treatment. Fiber
exposure induced intestine microbiome dysbiosis by decreasing alpha diversity especially community richness
and inhibiting the growth of Lactobacillus and short-chain fatty acid producing bacteria. Lactobacillus reuteri and
L. intestinalis were inhibited significantly, we suggested they can be sensitive indicators for fibers toxicity
evaluation in future studies. Slight intestinal damage was observed after the feeding period. We concluded that
dietary exposure to PE fibers did not induce acute harm to L. calcarifer. However, chronic effects were observed
after the feeding period, including oxidative stress, intestine microbiome alterations, and intestinal damage.
These findings provide valuable data for ecological risks assessment of fishing rope fiber in the marine
environment.

1. Introduction
Plastics pollution has been considered a great environmental prob­
lem and attracted global attention. It was estimated that 4.8 to 12.7
million tons of plastic waste entered the ocean in 2010, and this amount
is predicted to increase significantly by 2025 unless effective waste
management infrastructure and strategies are implemented (Jambeck
et al., 2015). The fishing industry is a major contributor of marine plastic
waste including plastic fishing ropes and nets (Andrady, 2011), and a
study determined that discarded fishing gear accounted for 56% of the
observed plastic items in the Gorringe Bank, northeast Atlantic (Vieira
et al., 2015). A total of 207.8 and 252 tons of derelict fishing gear were
removed in 2009 and 2010, respectively, from the East Sea seabed (Cho,
2011). Fishing gear persists for decades in marine environments and
poses a severe threat to marine species by indiscriminately catching
unintended prey and causing mechanical damage (Bilkovic et al., 2014;
Consoli et al., 2019). Under optimal conditions, fishing rope or nets
discarded in oceans fragment into microplastics (<5 mm) with seawater
immersion and UV radiation (Thomas and Hridayanathan, 2006).
Moreover, recent studies have indicated that fibers originating from
clothes (Browne et al., 2011) and fishing gear (Welden and Cowie, 2017)
represent a harmful and significant marine environment pollutant due to
its high prevalence and risk of uptake by fish. A reduction in mass and
fiber formation has been observed in nylon, polypropylene (PP), and

* Corresponding author at: CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of
Sciences, 164 Xingangxi Road, Guangzhou 510301, China.
E-mail address: san720@sina.com (A. Suo).

https://doi.org/10.1016/j.ecolind.2021.108370
Received 10 August 2021; Received in revised form 6 November 2021; Accepted 8 November 2021
Available online 11 November 2021
1470-160X/© 2021 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/).
M. Xie et al.
polyethylene (PE) ropes after exposing to benthic conditions (Welden
and Cowie, 2017).
Compared with particle shape microplastics, fiber with high aspect
ratio cannot be completely encapsulated by macrophages, and may
cause more harmful effects to marine species (Cole, 2016). The highest
frequencies of progressive and inflammatory changes attributed to fiber
ingestion have been documented in the freshwater goldfish species
Carassius auratus (Jabeen et al., 2018). Moreover, Carcinus maenas
showed a reduction in food consumption and energy available for
growth after ingestion of (PP) rope fibers (Watts et al., 2015). Other
adverse effects of fiber ingestion on fish species include reproduction
disruption, growth inhibition, and death (Au et al., 2015). Fishing gear
discarded in global marine environments annually (640,000 tons) re­
leases fibers, which pose a severe ecological risk to marine species
(Welden and Cowie, 2017). Therefore, it is very important to investigate
the impacts of fibers ingestion on marine biota.
Lates calcarifer, also known as Barramundi or Asian sea bass, is a
freshwater, marine, and estuarine fish native to the Indo-West Pacific
region from South Asia to Papua New Guinea and Northern Australia.
Barramundi is an important commercial and recreational fish species in
these regions because of its favorable biological characteristics including
high physiological tolerances and rapid growth rate (Venkatachalam
et al., 2018). Due to the habitat transformation, this species is poten­
tially vulnerable to microplastic pollution, particularly in estuarine en­
vironments that are frequently polluted by anthropogenic activity (Alves
and Figueiredo, 2019; Gray et al., 2018). This species has been used as
sensitive biomarkers for indicators of environmental pollution (Guven
et al., 2018; Maharajan et al., 2016). Recently study found the occur­
rence of microplastics both in wild and cage-cultured L.calcarifer (Ibra­
him et al., 2017). A recent study demonstrated that combined
polystyrene microspheres and pyrene exposure affected juveniles L.cal­
carifer swimming behaviors (Guven et al., 2018). To our knowledge, no
research has focused on the effects or biomarkers of ingested fibers on
L. calcarifer. PE fiber was selected in this study because it is widely used
in marine fishing and mariculture (Feng et al., 2020; Moe-Føre et al.,
2016). Plastics or microplastics originated from PE fishing gear attract
our attention increasingly. Most of plastic litter samples correcting from
south west England were constructed of PE, which were likely to be
derived from commercial fishing nets based on appearance analysis
(Turner, 2017). It was estimated that 62.76% of the microplastics
recovered from surface seawater of Sanggou Bay originated from
mariculture facilities such as fishing rope and PE was the most dominant
polymer (Xia et al., 2021). Fiber/line deriving from discarded rope
material comprised 38% of the total microplastics corrected in south­
west coastal water of India and most of them were constructed of PE;
predominant microplastics in marine fishes along the Kerala coast were
also fibres/-lines (Robin et al., 2020). Culture gears (1,037 tons plastics
releasing annually) were the important source of macroalgae micro­
plastics pollution in Haizhou Bay, of which fiber dominating the shape
and PE comprising the material mostly during the culture period (Feng
et al., 2020). Similarly, fibers and fragments recovered from the near­
shore surface waters of Weihai were identifies as PE mostly, which were
mainly delivered from the fishing nets/ropes (Zhang et al., 2021).
Instead of PE microplastic fiber, previous studies reporting the fishing
rope effect on organisms mainly focused on PP fiber (Au et al., 2015;
Watts et al., 2015; Zhao et al., 2021). Synthetic textile fiber toxic effect
has been also widely reported but most of them focused on polyester
fiber (Alnajar et al., 2021; Hu et al., 2020; Jemec et al., 2016; Ziajahromi
et al., 2017). Little is known about toxic effects of PE fiber related to
fishing gear on biota at present. Due to the poor wearability and resis­
tance to conventional dyeing stain, PE fibers are mainly use in bullet-
proof vests, ropes, and fishing nets but rarely in daily clothes (Alber­
ghini et al., 2021). Although PE fiber related to textile industry may
contribute to the microplastics pollution (Browne et al., 2011), it was
not the dominant type and not the real situation related to fishery ac­
tivity. In addition, fiber used in different sector may undergo different
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Ecological Indicators 133 (2021) 108370
manufacturing process and vary in environmental behavior, leading to
different outcome in the environment. Fishing gear is easy to release
microplastics during operation, such as hauling activity (Napper et al.,
2022), which can affect the biota directly (Feng et al., 2020). In order to
simulate the toxicology of microplastics pollution more environmental-
realistically from a fisheries gear standpoint, the ingestion of poly­
ethylene (PE) rope fibers by L. calcarifer was studied to (1) investigate
the accumulation of fibers after exposure and (2) evaluate the intestinal
biological impacts induced by fibers, including oxidative stress
biomarker, intestinal permeability indicator, intestinal microbiome al­
terations, histological structure changes, and mucous cell phenotype
variations.
2. Materials and methods
2.1. Ethics approval and consent to participate
This study was approved by the Animal Care and Use committee of
South China Sea Institute of Oceanology, Chinese Academy of Sciences.
All experiments were carried out in accordance with the Guide for the
Care and Use of Laboratory Animals of China.
2.2. Fish collecting and acclimation to laboratory conditions
Three hundred juvenile L. calcarifer were purchased from Zhuhai,
Guangdong province in China. The experimental procedure was carried
out at the Key Laboratory of Tropical Marine Bio-resources and Ecology,
South China Sea Institute of Oceanology, Chinese Academy of Sciences.
Fish were acclimated in the laboratory for 30 days by feeding them with
a basal diet daily. The basal diet (Zhongshan President Enterprises
Brand) contained crude protein ≥ 41%, crude lipids ≥ 5%, crude fiber ≤
4%, crude ash ≤ 18%, water ≤ 12.9%, and total phosphorus ≥ 0.8%. In
order to detect whether the feed is contaminated by microplastics or not,
virgin fish fodder was digested with 10% KOH for 24 h and filtered
through a 0.45 μm pore size filter (MF-Millipore, HAwp04700, Ger­
many). Fodders were detected to be free from fiber contamination by
observation under a stereoscopic microscope (Olympus, SZX16).
2.3. Fiber contaminated food preparation and feeding trail
PE (diameter 200 μm) ropes were used to create approximately 2–3
mm long fibers by cutting sections of the original rope. Fibers were then
soaked in ethanol (70%) overnight to remove possible contamination,
washed with deionized water and dried at room temperature. This fiber
size was common seen in wild fish gastrointestinal tract (GIT) (Davison
and Asch, 2011; Ory et al., 2018) and could be ingested by juvenile
L. calcarifer in our experiment.
Fibers and the basal diets were then mixed with water (250 g water
per 1000 g food), pressure-pelleted using a pelletizer (F-26, South China
University of Technology, Guangzhou, China) with a diameter and
length of 2.5 mm and 1–1.2 cm, respectively, air-dried at room tem­
perature, and stored in sealing bags. Two different diets were prepared
for the study: one experimental diet supplemented with 10 g of fibers
from PE ropes per kg of basal feed (PE group) and a control diet (CN
group), which contained the basal feed only. The Fourier transform
infrared spectroscopy analysis (Thermo Fisher, Nicolet iN10, USA) re­
sults of the plastic ropes and food pellets used in this study are provided
in the Supplementary Figs. S1–S2. A total of 150 healthy fish was
selected and then randomly distributed into six glass tanks (200 L/tank,
three tanks per diet, 25 fish per tank). The mean mass of the individual
fish in the six tanks ranged from (12.43 ± 5.09) g to (13.95 ± 5.59) g
(details in Supplementary Table S1). Fish were fed by hand at a rate of
2% of their body weight for 56 days. The initial exposure concentrations
translated to 330.44 ± 19.12 μg/L water, which was higher than
microplastics concentrations (250 μg/L) reported in the North Pacific
subtropical gyre (Goldstein et al., 2012) but lower than that used in
M. Xie et al.
sheepshead minnow (Cyprinodon variegatus) study (50 and 250 mg/L)
(Choi et al., 2018). Every two weeks, fish from each tank were weighed
to adjust the daily amount of feed given (2% body weight). The water
quality was monitored during the experiment and the results are pre­
sented in Supplementary Table S2.
2.4. Growth performance measurement and sampling
At the end of the feeding trial, the fish were fasted for 24 h before
sampling and were anesthetized with 100 mg/L eugenol subsequently.
The final weights of all the fish in each tank were determined. Intestine
(divided into proximal intestine, mid intestine, and distal intestine)
sampled from 15 fish in each tank were frozen in liquid nitrogen for 72 h
and then stored at − 80 ◦ C for further analysis. In addition, three in­
testine samples obtained from three fish of each tank were preserved in a
10% formaldehyde solution for segment preparation. Also, five fish from
each tank were obtained and stored at − 20 ◦ C for fiber retention
detection. The following metrics were calculated:
Viscerosomatic index(VSI%) = 100
× viscera weight(g)/whole body weight (g)
Hepatosomatic index(HSI%) = 100 × liver weight (g)/whole body weight(g)
Survival rate(%) = 100 × (finial number of fish)/(initial number of fish)
2.5. Fibers extraction and analysis
Fifteen fish per group were used for fibers extraction and analysis.
The fish were dissected, liver, esophagus, stomach, pyloric caecum,
whole intestine, and gill were collected. All tissues were used for fibers
extraction following the adjusted protocol (Jovanovic et al., 2018).
Briefly, tissues were placed in 50 mL glass bottles and treated with 30 mL
of 10% KOH for 24 h at 60 ◦ C in a water bath. After 24 h, the samples
were washed with distilled water and filtered through a filter with 0.45
μm pore size and 47 mm diameter. Each filter was placed into a clean
glass Petri dish with a cover for observation. The fibers were counted
under a stereoscopic microscope using a digital camera (Olympus,
SZX16). In order to minimize microplastic cross contamination, nitrile
gloves and cotton lab coats were worn during the experiments. Liquids
used for analysis were filtered with 0.45 μm pore size filters. Vessels and
tools were cleaned with filtered alcohol and covered with tin foil before
and after use. Three procedural blanks without tissue sample were
performed simultaneously during microplastic extraction. Clean Petri
dish with a wetted membrane filter (0.45 μm pore size) inside was
placed on the ground at each site during experiment with an aim to
collect potential airborne microplastics. Area of stereoscopic microscope
was cleaned before use. No microplastic was found under stereoscopic
microscope, indicating the environment in the laboratory was consid­
ered clean.
2.6. Enzymes biochemical analysis
Intestine samples, comprising a mix of the proximal, mid, and distal
intestine from one fish of each tank, were homogenized in an ice-cold
phosphate buffer (1:10 dilution). The homogenate was then centri­
fuged for 20 min (4 ◦ C, 3,000 r/min) and aliquots of the supernatant
were used for the following analysis. Intestine malondialdehyde (MDA)
level and diamine oxidase (DAO) level were measured using commercial
assay kits (Jiancheng, Ltd, Nanjing, China) following the manufacturer’s
instructions with a spectrophotometer (xMark, Bio-Rad, USA). MDA
level was determined by Thibabituric Acid (TBA) method at 532 nm.
DAO was determined by measuring Nicotinamide adenine dinucleotide
decrease per minute at 340 nm. Each assay measured in three replicates
intestine samples per group.
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Ecological Indicators 133 (2021) 108370
2.7. Intestine microbiome analysis
Six replicate samples from each group and totally 12 mid-intestine
samples were used for intestine microbiome analysis. Microbial com­
munity genomic DNA was extracted from each intestine sample using
the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) ac­
cording to the manufacturer’s instructions. The hypervariable region
V3–V4 of the bacterial 16S rRNA gene was amplified with primer pairs
338F (5′ -ACTCCTACGGGAGGCAGCAG-3′ ) and 806R (5′ -GGAC­
TACHVGGGTWTCTAAT-3′ ) using an ABI GeneAmp® 9700 PCR ther­
mocycler (ABI, CA, USA). The PCR amplification of 16S rRNA gene was
performed as follows: initial denaturation at 95 ℃ for 3 min, followed by
29 cycles of denaturing at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s and
extension at 72 ℃for 45 s, and single extension at 72 ℃ for 10 min, and
end at 4 ℃. Purified amplicons were paired-end sequenced on an Illu­
mina MiSeq PE300 platform (Illumina, San Diego, USA) according to the
standard protocols of Majorbio Bio-Pharm Technology Co. Ltd.
(Shanghai, China). Operational taxonomic units (OTUs) with a 97%
similarity cutoff were clustered using UPARSE version 7.1. The taxon­
omy of each OTU representative sequence was analyzed by RDP Clas­
sifier version 2.2 against the 16S rRNA database using a confidence
threshold of 0.7. The alpha diversity was calculated with Mothur soft­
ware (version v.1.30.1).
2.8. Intestine histopathological and histochemical analysis
Nine intestine (divided into proximal intestine, mid intestine, and
distal intestine) samples per group were used for histopathological and
histochemical analysis. Segments of 5 µm were stained with hematox­
ylin and eosin (HE) for histopathological analysis. Histopathological
alterations of intestines were assessed using a score from 0 to 4 (0:
normal structure; 1: slight structural alteration; 2: moderate structural
alterations; 3: pronounced structural alterations; 4: severe structural
alterations) (Peda et al., 2016). For mucous cell determination, seg­
ments were stained with alcian blue (pH 2.5) - periodic acid-Schiff (AB-
PAS) reagent, whereby red indicated PAS-positive neutral mucosub­
stances, blue indicated PAS-negative acid mucosubstances, and purple-
reddish signified a combination of neutral and acid mucosubstances
(Carim and Mahomed, 2013). All segments were analyzed under a light
microscope (Nikon Eclipse E100). Three mucosal folds viewed at a 100
× magnification were used for mucous cells counting. Mucous cells per
mucosal fold were calculated.
2.9. Statistical analyses
The data obtained were analyzed using SPSS software (version 22.0,
IBM, USA). Normality of the variables and variance homogeneity were
confirmed by the Shapiro–Wilk and Levene tests, respectively. Growth
parameters, enzymes activity and gut microbiome diversity were
assessed by Student’s t-test. The relative abundance of different species
and the mucous cell number in the different groups were statistically
compared using the Mann-Whitney U test. Significance was set at p <
0.05 or p < 0.01.
3. Results
3.1. Growth performance
All fish consumed the contaminated food actively and appeared
healthy during the exposure. After 56 days, fish survival rate ranged
from 92 to 100% in all tanks. The final weight, survival rate, VSI index,
and HIS index showed no difference respect to CN group (Student’s t-
test, p > 0.05, Supplementary Table S3).
M. Xie et al.
3.2. Retention of fibers
No fiber was detected in the CN group. Also, no fiber was detected in
the livers or gills of the fish from PE group. However, fibers were
observed in the gastrointestinal tract (GIT) of 13.33% (two out of 15
fish) for fish in PE group. Fish with fibers detected in the intestine alone
accounted for one fish, whereas those with fibers in both stomach and
intestine accounted for one fish in the PE group. Furtherly, three items of
fiber were detected in the stomach and one item of fiber was detected in
the intestine.
3.3. Intestine MDA and DAO level
The MDA level was significantly higher (fold change 4.84) in the PE
group than in the CN group (Student’s t-test, p < 0.01, Fig. 1A). DAO
level was not influenced by fiber exposure (Student’s t-test, p > 0.05,
Fig. 1B).
3.4. Intestine microbiome profile
Fiber exposure altered the fish gut microbiome alpha diversity. Sobs
index decreased significantly after fiber exposure (Student’s t-test, p <
0.05, Fig. 2A). The Shannon index also decreased after treatment but
without statistical difference (Student’s t-test, p > 0.05, Fig. 2B). Both
Ace and Chao indexes decreased significantly in PE group (Student’s t-
test, p < 0.05, Fig. 2C, D). Sobs, Ace, Chao indexes all decreased
significantly, indicating intestinal microbiome community richness
decreased respect to CN group. Although no significant difference,
community diversity showed downward trend indicating by Shannon
index.
Moreover, fiber exposure altered the fish gut microbiome community
composition. At the phylum level, Proteobacteria, Firmicutes, Actino­
bacteriota were the dominant taxa (Fig. 3A). At the class level, Actino­
bacteria, Gammaproteobacteria, Fusobacteria, and Clostridia were the
dominant taxa (Fig. 3B). After fiber exposure, Bacilli decreased signifi­
cantly (Mann-Whitney U test, p < 0.05, Fig. 3C). At genus level,
Burkholderia-Caballeronia-Paraburkholderia and Cetobacterium were
the dominant taxa (Fig. 4A). Relative abundance of Lactobacillus
decreased significantly respect to CN group (Mann-Whitney U test, p <
0.05, Fig. 4B).
At species level, several species beneficial for host were inhibited by
the fibers exposure. The relative abundance of Lactobacillus reuteri, L.
intestinalis L.johnsonii decreased significantly respect to CN group
(Mann-Whitney U test, p < 0.01 for the first two species and p < 0.05 for
the last one, Fig. 5A-C). Especially, relative abundance fold change of L.
reuteri, and L.intestinalis reached to 98.84, 85.30 respectively (Supple­
mentary Table S4). Unclassified genus including Succiniclasticum, Veil­
lonella, Odoribacter all decreased significantly respect to CN group
(Mann-Whitney U test, p < 0.05 for the first two species and p < 0.01 for
the last one, Fig. 5D-F).
Fig. 1. Oxidative stress and intestine injuries caused by fiber exposure. A:
malondialdehyde (MDA) level; B: diamine oxidase (DAO) level. “**” indicates p
< 0.01, Student’s t-test.
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Ecological Indicators 133 (2021) 108370
3.5. Histological damage and mucous cell phenotype
All the scored histopathology features were combined. No structural
alterations of intestine were observed in the CN group (Fig. 6A); how­
ever, 22.22% (two out of nine fish) in the PE treatment showed slight
structural alterations (score 1). Histological damage including lamina
propria widening and epithelium breakage were observed in the mid
intestine and distal intestine of the PE group fish (Fig. 6B, C). Red, blue,
and purple-reddish mucous cells were observed in the intestine epithe­
lium and blue mucous cells were the dominant type (Fig. 6D–F). After
fiber exposure, the number of mucous cells in proximal intestine and
mid-intestine did not differ from the CN group (Mann-Whitney U test, p
> 0.05, Fig. 7A, B); however, this number increased significantly in the
distal intestine (Mann-Whitney U test, p < 0.01, Fig. 7C).
4. Discussion
4.1. Fiber retention and impacts on fish growth
Previous studies have demonstrated that the ingestion of micro­
plastic fibers by fish is an energy-consuming activity. For example,
plastic particles bind to apolipoproteins, preventing fish from properly
utilizing their fat reserves (Cedervall et al., 2012). Moreover, managing
the inflammation or damage caused by microplastic ingestion requires
further energy (Jabeen et al., 2018). Microplastic accumulation in the
digestive tracts of fish may cause false satiety, furtherly affecting the
optimal growth (Cole and Galloway, 2015). It has been determined that
the growth of Ambassis dussumieri (Naidoo and Glassom, 2019) and
C. auratus (Jabeen et al., 2018) are inhibited by microplastics ingestion
for 95 and 42 days, respectively. However, present study demonstrated
that the ingestion of fibers for 56 days had no significant effects on the
growth and survival of L.calcarifer. Similarly, growth of gilt head seab­
ream (Sparus aurata) was not influenced by the 45 days’ exposure at
concentration of 3.33 g kg− 1 feed to microplastics (Jovanovic et al.,
2018). The shape and exposure concentrate of microplastics are
important factors determining the biological effects involved with their
ingestion. Microplastics with small sizes and particle shapes can be
easily removed from the intestines of fish. For example, when goldfish
(C. auratus) ingested fibers with length between 50 μm and 500 μm, 50%
and 90% of these microplastics were evacuated after 10 h and 33.4 h,
respectively (Grigorakis et al., 2017). Low microplastics concentration
and the smoother morphology making it an effective elimination, which
could limit the negative effects on fish described by Jovanovic et al.
(2018). Even a higher exposure concentration (diet enriched with10%
low-density PE particles between 200 and 500 μm) also did not report
significant effects on fish’s survival and growth after 90 days’ treatment
due to the effective elimination (Solomando et al., 2020). Although
microplastics did not impact the growth rate of Dicentrarchus labrax
larva after 37 days’ exposure, ingestion of PE microbeads (10–45 μm) at
high concentration (12 mg/g feed) increased the mortality (Mazurais
et al., 2015). Such result might be attribute to the fish in their sensitive
period, large amounts of microplastics ingestion causing intestinal
obstruction and leading to this acute toxic effect. Different from the
study of Jabeen et al. (2018), microplastics with bigger size (0.7 mm-5
mm length) or with sharp edges might be easy to injure the tissue then
affecting fish’s growth indirectly. In the present study, fiber concen­
tration in the feed was 1% by weight and the fish were fed 2% of their
body mass daily. Low fiber retention was observed in the PE group
(13.33%) and the fiber items were relatively low (one or three items per
tissue), indicating that both the long-term and short-term ingestion of
fibers could be rapidly excreted by the body. As a result, the potential for
bioaccumulation was relatively low, which could limit the adverse ef­
fects of fibers.
M. Xie et al. Ecological Indicators 133 (2021) 108370

Fig. 2. Community diversity of fish gut microbiome. A: Sobs index; B: Shannon index; C: Ace index; D: Chao index. “*” indicates p < 0.05, Student’s t-test.

Fig. 3. Gut microbiome composition of fish at different level. A: phylum level; B: class level; C: relative abundance of Bacilli. “*” indicates p < 0.05, Mann-Whitney
U test.

Fig. 4. Gut microbiome composition of fish. A: genus level; B: relative abundance of Lactobacillus. “*” indicates p < 0.05, Mann-Whitney U test.

4.2. Fiber exposure induced oxidative stress and intestine microbiome
alteration
Microplastics negatively impact organisms directly or indirectly as
observed in studies where microplastics that triggered ROS generation
in Paracyclopina nana (Jeong et al., 2017). Additionally, microplastics
cause organ damage and inflammation via direct contraction (Jabeen
et al., 2018). Particulate matters can increase intestinal permeability
when transported across the intestine barrier (Lerner and Matthias,
2015). Generally, abnormal intestine permeability increase is related to
inflammatory disease (Breslin et al., 2001). Exposure of the zebrafish
D. rerio to microplastics resulted in permeability increase and intestine
injury (Qiao et al., 2019). Here, the MDA level increased significantly,
suggesting that fiber exposure induced oxidative damage. The results
agree with previous study in S.aurata exposed to microplastics for 90
days with MDA level increasing and antioxidant enzymes activity
modulation (Solomando et al., 2020). Microplastics causing oxidative
stress and antioxidant response were also observed in other fish tests
including D.labrax juveniles (Barboza et al., 2018) and red tilapia
(Oreochromis niloticus) (Ding et al., 2018). DAO level was not influenced
after treatment in the present work, indicating that intestine mucosa
permeability variation was not involved in this process. These results
might be attributed to fibers being eliminated effectively, protecting the
fish from more severe damage. In contrast, the ingestion of microplastics
by D. rerio for 21 days caused significant oxidative stress and intestinal
permeability increase (Qiao et al., 2019). However, the smaller-sized
microplastics (5 μm) used in the study (Qiao et al., 2019) may have
travelled to different tissues and induced inflammation. Fibers used in
this study were 2–3 mm long, it is more likely that oxidative stress
resulted from contraction after direct exposure to fibers. Despite the
effective elimination of fibers, daily feeding ensured that the fish
interacted with fibers continuously, resulting in chronic oxidative
damage. In natural environments, microplastics do not readily degrade
and persist in marine environment for long periods. It has been reported
that an estimated 3968 tons of microplastics are generated from fishing
gear monthly (Welden and Cowie, 2017). Also, trawl nets and other
ropes may release microplastics into the environment. Therefore, fish
are likely to be exposed to various types of microplastics over their
lifetimes. The chronic effects caused by microplastic ingestion threat­
ening fish health and ecosystem stability in the long term cannot be

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M. Xie et al. Ecological Indicators 133 (2021) 108370

Fig. 5. Effects of PE fiber exposure on the microbiome composition. A: Lactobacillus reuteri B: L. intestinalis; C: L.johnsonii; D: Succiniclasticum E: Veillonella; F:
Odoribacter. “*” indicate p < 0.05, “**” indicates p < 0.01, Mann-Whitney U test.

Fig. 6. Transverse sections of the intestine of

fish exposed to fibers, scale bar represents
200 μm. A–C were stained with HE regent
and D–F were stained with an AB-PAS
regent. A: normal structure of distal intes­
tine in CN group; B: structure alterations
observed in mid intestine of the PE group; C:
structure alterations observed in distal in­
testine of the PE group; D: mucous cells dis­
tribution in the distal intestine of CN group;
E: mucous cells distribution in mid intestine
of the PE group; F: mucous cells distribution
in distal intestine of PE group; BEP: epithe­
lium breakage, BMC: blue mucous cell, EP:
epithelium, LP: lamina propria, LPW: lamina
propria widening, PRMC: purple-reddish
mucous cell, RMC: red mucous cell.

6
M. Xie et al.
Fig. 7. Mucous cell number alteration after fiber exposure. A: proximal intestine; B: mid intestine; C: distal intestine. “**” indicates p < 0.01, Mann-Whitney U test.
ignored.
Intestinal microbiome alpha diversity especially community richness
decrease and composition structure alteration were observed in the
present study. Specially, relative abundance of Lactobacillus including
Lactobacillus reuteri, L. intestinalis and L.johnsonii decreased significantly
after fibers exposure. Lactobacillus is a probiotic that helps to maintain
the health status of the intestine. For insistence, L. reuteri activates the
key signaling pathway and accelerates the regeneration of intestinal
stem cells to maintain intestinal mucosal integrity (Hou et al., 2018).
Additionally, Lactobacillus inhibit the production of pro-inflammatory
factors and prevent excessive proliferation of pathogenic microorgan­
isms by producing antibacterial substances (Cleusix et al., 2008). In
addition, fiber exposure inhibited the short chain fatty acid (SCFA)
producing bacteria, including Succiniclasticum, Veillonella, Odoribacter.
SCFA are the primary energy sources for intestine epithelial cells,
including acetic acid, propionic acid, and butyric acid. Veillonella me­
tabolizes lactate into propionate in the colon, which in turn promotes
athletic performance by increasing the heart rate and maximum rate of
oxygen consumption. Moreover, Veillonella also reduces levels of in­
flammatory cytokines (Scheiman et al., 2019). Similarly, Odoribacter is a
maker of butyrate and has been described to contribute to the inflam­
matory process (Jiang et al., 2018). It has been suggested that intestine
barrier function might be compromised with decreasing levels of
Lactobacillus and SCFA producing bacteria after fiber exposure. The
mechanism involved in the alteration of microbiome from microplastic
ingestion remains unclear. Previous studies have demonstrated that the
close relationship between intestinal oxidative stress level and the in­
testine microbiome (Li et al., 2020). Alleviated oxidative stress may
induce intestinal environment imbalance and down regulates the
expression of key proteins, leading to the alteration of mucus pheno­
types and the enhancement of intestinal barrier permeability (Tomas
et al., 2016). Also, inflammation may create a unique nutritional envi­
ronment that favors the growth of bacterial species such as Enterobac­
teriaceae, whose genomes encode the capability of utilizing
inflammation by-products including reactive oxygen and nitrogen spe­
cies (Winter and Bäumler, 2014). As MDA level increased significantly in
the present study, one possible mechanism is that the fibers induced
oxidative stress and altered micro-environment in the fish, inhibiting the
growth of sensitive bacterial especially L.reuteri and L.intestinalis.
Therefore, community diversity decreased and community composition
was altered. Microbial community alteration and alpha diversity
reduction are common observation in other microplastics tests (Huang
et al., 2020; Qiao et al., 2019). A balanced intestine microbiome com­
munity and diversity are pivotal for maintaining healthy intestinal
barrier. The present results indicated that the ingestion of microplastic
fibers by L. calcarifer resulted in intestinal microbiome dysbiosis, a
possible mechanism leading to intestinal toxicities in this fish species.
The growth of L.reuteri and L.intestinalis were inhibited significantly,
which might be sensitive indicators for fibers toxicity evaluation.
7
Ecological Indicators 133 (2021) 108370
4.3. Intestine histology and mucus phenotype in response to fiber exposure
Fish intestine is vulnerable to invaders as it provides an absorption
pathway for a variety of toxic substances including microplastics. In this
study, only slight pathological damage including lamina propria
widening and epithelium breakage was observed, which might have
occurred from direct contact with the fibers. Although no imminent
harm was observed, the intestinal histological structure was influenced
by chronic and continuous fiber exposure. A previous study reported
that D. labrax chronically exposed to polyvinyl chloride microplastics at
0.1% concentrate also induced pathological alterations by increasing
mucus secretion as a defense strategy; however, severe inflammatory
changes including mucosa layers edematous, vessels ectasia and leuko­
cyte infiltration were detected after 90 d exposure (Peda et al., 2016).
Notably, microplastics used in the above study were deployed in a ma­
rine environment for three months and grounded before exposure.
Therefore, the pollutants that were absorbed in the marine environment
and the sharp edges of the microplastics might have contributed to in­
testine inflammation. Virgin polystyrene microplastics, with particle
sizes of 10 μm at concentration of 500, 1000, or 2000 μg/g feed, also
have been reported to cause glomerulopathy and nephrogenesis in head
kidney of Japanese medaka (Oryzias latipes) (Zhu et al., 2020). Similarly,
histology alteration originated from microplastics exposure has been
reported in Cyprinus carpio (Xia et al., 2020) and C.auratus (Jabeen et al.,
2018). Correspondingly, mucous cells increased significantly in distal
intestine in the present study. Mucus secreted by mucous cells serves as
the defensive line of the epithelium, which can lubricate the intestinal
tract and accelerate the fibers excretion especially in distal intestine
being connected to the rectum. Inflammation or stress can lead to in­
creases in mucus. In such case, increasing mucus resist the enhancement
of mucosal permeability and reduce toxic metabolites penetrating the
mucosa (Johansson and Hansson, 2016). As fibers caused intestine
damage in present study, fish provided the first defense strategy by
increasing mucus to protect the intestine from further damage.
5. Conclusion
The diet comprising of 2–3 mm long fibers showed no significant
effect on the L. calcarifer juveniles’ growth. The fibers could be elimi­
nated effectively. However, fibers cause oxidative stress as MDA level
increased significantly. Also, fibers cause intestinal microbiome alter­
ation by decreasing alpha diversity especially community richness and
inhibiting the growth of several crucial species. Slight histological
damage and mucus secreting phenotypes alteration in distal intestine
were found. Overall, dietary exposure of fibers at 1% concentrate by
weight did not induce acute harm to juvenile L. calcarifer, but long-term
exposure lead to chronic harm in this species including oxidative stress,
intestine microbiome dysbiosis and intestine histological damage.
Microplastics pollution will become more and more severe in the future
with the increasing consumption of plastics and plastic products. Fish
are likely to be exposed to various types of microplastics over their
lifetimes in the natural environment. Therefore, what we should aware
M. Xie et al.
of is that fish may be facing an even greater threat of microplastics than
in laboratory trial. These findings provide valuable data for ecological
risks assessment of fishing rope fiber in the marine environment.
Furthermore, growth of Lactobacillus reuteri and L. intestinalis were
inhibited significantly after treatment, we suggested they can be sensi­
tive indicators for fibers toxicity evaluation in future studies.
CRediT authorship contribution statement
Mujiao Xie: Methodology, Investigation, Data curation, Writing –
original draft. Lang Lin: Methodology, Investigation. Peng Xu: Formal
analysis, Software. Weiguo Zhou: Resources. Changsheng Zhao:
Formal analysis. Dewen Ding: Supervision. Anning Suo: Conceptuali­
zation, Funding acquisition, Data curation, 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 influence
the work reported in this paper.
Acknowledgements
This work was supported by the Key Special Project for Introduced
Talents Team of Southern Marine Science and Engineering Guangdong
Laboratory (Guangzhou) (No. GML2019ZD0402). We are deeply
indebted to those anonymous reviewers for their valuable comments
and suggestion.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ecolind.2021.108370.
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