Microplastics in Small Waterbodies and Tadpoles from YangtzeRiver Delta, ChinaLingling Hu,†,‡ Melissa Chernick,‡ David E. Hinton,‡ and Huahong Shi*,†

†State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai 200062, China‡Nicholas School of the Environment, Duke University, Durham, North Carolina 27708, United States

*S Supporting Information

ABSTRACT: Although microplastic (MP) pollution infreshwater systems is gaining attention, our knowledge of itsdistribution in small waterbodies is scarce. Small waterbodiesare freshwater habitats to many species, including amphibians,that are vulnerable to MP pollution. This study analyzed thedistribution and characteristics of MPs in 25 small water-bodies from the Yangtze River Delta, China. MPs weredetected in surface water, sediment, and tadpoles withabundances ranging from 0.48 to 21.52 items L−1, 35.76 to3185.33 items kg−1, and 0 to 2.73 items individual−1 (0 to168.48 items g−1), respectively. The dominant shape andpolymer of MPs in water and tadpole samples were polyester(PES) fibers, and polypropylene (PP) fibers and fragmentswere dominant in sediment samples. In addition, MPs were primarily <0.5 mm in length in all samples. Tadpole length waspositively correlated to the number of MPs detected. The abundance, shape, and polymer distribution of MPs in tadpolesresembled that of water rather than sediment, suggesting that tadpoles likely take up MPs from the surrounding water. Thisstudy demonstrated that MPs are abundant in these small waterbodies and are ingested by resident tadpoles. This may suggest apathway of MP entry into aquatic and terrestrial food webs.

■ INTRODUCTION

Microplastic (MP) pollution is of growing environmentalconcern and a potential risk to human health.1 In oceans, it hasbeen well documented,2−4 with MPs ubiquitously distributedin all marine realms from beaches to the deep sea.5−7 In recentyears, MPs have also been found in freshwater ecosystems,especially rivers and lakes.8−11

Plastics enter water bodies through land-based activities,especially via runoffs.3,12 MPs are known to enter oceans, largelakes, and rivers as well as small waterbodies.3,11 The term,“small waterbodies” is ambiguous, with no universally acceptedor formal definition. It most often refers to small lakes, ponds,streams, ditches, and springs.13 Here, we use it to refer to smalllakes and rivers, ponds, ditches, puddles, and aquaticfarmlands. Taken together, these small waterbodies comprisethe most numerous freshwater environments globally and arecritical to maintaining freshwater biodiversity and ecosystemservices.13,14 Human development, land use patterns, andclimate change are known to affect small waterbodies,13,15 andrecent studies confirm that these stressors also affect theabundance of MPs.16,17 For example, Zhang et al.10 found thatthe highest average abundances of MPs in surface water fromXiangxi Bay of Three Gorges Reservoir were detected duringthe rainy season and downstream of the largest town.However, the impact of these combined stressors on the

distribution and abundance of MPs in small waterbodiesremains unknown.MPs in aquatic ecosystems can be ingested by a wide variety

of aquatic organisms, including plankton, bivalves, fish, andmammals.18−20 Ingestion has the potential to alter gut functioncausing deleterious effects such as blockage, tissue damage, andfalse sense of satiation that limits nutrient uptake.21−23 Forexample, the food consumption and energy expenditureavailable for growth was reduced after crabs (Carcinus maenas)ingested food containing microfibers for 4 weeks.22 As asecondary effect, ingestion of MPs may facilitate transfer ofpersistent organic pollutants to the organism.24 Medaka(Oryzias latipes) exposed to a mixture of polyethylene andPBTs (persistent bioaccumulative and toxic substances)bioaccumulated the chemicals and suffered liver toxicityincluding glycogen depletion, fatty vacuolation, and singlecell necrosis.24 However, to date, additional researchconnecting laboratory and field studies is needed.Amphibians are an important component of aquatic

ecosystems, especially in small waterbodies such as pondsand farmlands. Owing to their unique life-history and

Received: April 28, 2018Revised: June 23, 2018Accepted: July 11, 2018Published: July 23, 2018

Article

pubs.acs.org/estCite This: Environ. Sci. Technol. 2018, 52, 8885−8893

© 2018 American Chemical Society 8885 DOI: 10.1021/acs.est.8b02279Environ. Sci. Technol. 2018, 52, 8885−8893

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physiological characteristics, they are often consideredecological indicator species and have been extensively usedto monitor environmental quality.25,26 As early life stages ofamphibians, tadpoles exhibit extensive feeding activity thatexposes them directly to MPs present in the aquaticenvironment. Native amphibians have oviposition sites inponds or ditches adjacent to farmland, meaning they could beexposed to and incorporate MPs in these waterbodies.Previous studies demonstrated that polystyrene microsphereswere ingested by tadpoles of Xenopus tropicalis and weredetected in the pharynx and eyes of Xenopus laevis embryosand tadpoles.27,28 Based on this, we regard tadpoles as suitableindicator species for the investigation of MP ingestion in fieldstudies.In this study, we investigated MPs in small waterbodies in

the city of Shanghai and Zhejiang province of China. Ourstudy sites were located within the Lower Yangtze River Delta,the most populous and rapidly developing area in China, whereproduction and use of plastics are significant (Table S1).29

Increased anthropogenic activity means it is likely that MPswill be more abundant in small waterbodies within theseareas.17 The aim of this study was to investigate the abundanceand characteristics of MP pollution in water, sediments, andtadpoles of these small waterbodies.

■ MATERIALS AND METHODS

Study Area. From May to August, 2016 and again fromMarch to May, 2017, samples were collected from 25 locations(Shanghai (n = 14) and Zhejiang (n = 11)) in the LowerYangtze River Delta (Figure 1). The sites were mainly infarmland areas (Table S1 and Figure S1) where most wereman-made standing waters such as artificial ponds andfarmlands as well as puddles formed by rainfall.

Methodology Selection. A bulk sampling approach,where the entire volume of the sample was collected withoutreducing it during the sampling process, was used.30,31

Compared with other methods,32 this method can moreaccurately detect the abundance of MPs in samples, especiallythose small in size.For sediment samples, MP density separation can be

performed with sodium chloride (NaCl), sodium bromide(NaBr), sodium iodide (NaI), or zinc bromide (ZnBr2).

33,34

Although the density of saturated NaCl (1.2 g cm−3) is lowerthan partial MP density, we chose it because it is commonlyused,9,35,36 low cost, abundant, and environmentally benign innature. It is also recommended by the Marine StrategyFramework Directive (MSFD) Technical Subgroup on MarineLitter.37

Various methods have been used to digest biologicalmaterial.9,38,39 Nuelle et al.40 found 30% H2O2 (v/v)successfully removed more than 90% of biogenic material.Similarly, our previous study with this method achieved 95%microfiber recovery.4 While this method causes discolorationof the microplastics, it does not affect their number or size.4,40

Sample Collection. The exact location of each site wasrecorded using a hand-held global position system (GPS)device (Table S1), and the type of waterbody (i.e., ditch, pond,river, lake, puddle, farmland) at each site was photographed.Prior to collection, all sampling containers and tools werewashed using filtered tap water and tightly capped. At each site,a 5 L glass bottle was filled with surface water (0−10 cm indepth) using a steel bucket. The exception was with twopuddles, sites S6 and S25, where water volume was restricted. Inthese cases, 5 L glass bottles were filled with available water,resulting in 2 and 3 L from these sites, respectively. Threesamples were collected at each site (n = 3).30,31 Upon reaching

Figure 1. Yangtze River Delta map including sampling sites in Shanghai (site numbers S1-S14) and Zhejiang (site numbers S15-S25). Different colordots represent different small waterbody types. Latitude and longitude are on the edges of each map, and scale bars are in the bottom right corners.

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a sampling site and during the water collection, care was takento avoid disturbing tadpoles. Water samples were taken first,followed by tadpole collection and then sediment (see below).Across collection areas, four different species of frogs

(Microhyla ornata, Rana limnochari, and Pelophylax nigromacu-latus) and/or toads (Bufo gargarizans) were found. In the field,species were collected, photographed, and identified by grosscharacteristics according to Fei et al. (Figure S2).41 Regardingtadpole collection at a particular site, 3 scenarios wereencountered: 1) a single species with individuals of similarbody size from which 30 tadpoles were collected; 2) twospecies with individuals within a species of the same body sizewhere 30 individuals of each species were collected, or 3) asingle species with individuals of two size ranges where 15individuals of larger size and 30 individuals of smaller size werecollected (see Figure S6). Upon collection, tadpoles wereimmediately euthanized with 1 g L−1 3-aminobenzoic acidethyl ester (MS-222, Sigma-Aldrich, St. Louis, MO, USA),then preserved in 70% ethanol in a 250 mL glass bottle, andtransported to the laboratory where they were stored at 4 °Cuntil processing.Sediment samples were collected in triplicate at each site (n

= 3) by use of a clean stainless steel spatula.42 Sampling wasrestricted to the surficial sediment (i.e., top 0−5 cm). Nosediment was collected from S3 due to the presence of a thick

detrital layer or from S5 due to the presence of a rocky bottom.Collected sediment was then transferred to clean, individuallylabeled aluminum foil bags. Each replicate containedapproximately 1 kg of wet sediment. All sediment sampleswere transferred to the laboratory and stored at −20 °C priorto analysis.

Isolation of Microplastics. To minimize contamination, a100% cotton laboratory coat was worn during all steps ofanalytical procedures. Liquid solutions, including tap water,saturated sodium chloride solution (1.2 g cm−3), and hydrogenperoxide (H2O2, 30%, v/v), were filtered prior to use(Millipore TMTP04700, filter pore size = 5 μm). Allcontainers and devices were washed using filtered tap waterand covered until time of use. Procedural blanks were includedfor water, sediment, and tadpole samples. These blanksincluded chemicals used for the processing of samples andwere taken through the same isolation and digestionprocedures.The protocol for extraction of MPs from water and sediment

followed procedures of Su et al.9 Briefly, water volume wasrecorded, and suspended substances in water were removed byfiltration with a 47 mm diameter Nylon membrane filter and apore size of 20 μm (Millipore, Burlington, MA, USA,NY2004700) facilitated with a vacuum pump (FY-2C-N,VALUE, China). Next, substances on the filter, includingorganic matter, were rapidly removed by washing with 100 mLof H2O2 (30%, v/v) into a 250 mL glass flask. For digestingorganic material, flasks were covered with glass Petri dishes andheated in an oscillating incubator (HZ-9612K, Taicang, China)at 65 °C at 80 rpm for no more than 72 h, and resultantdigestate was filtered again using a 47 mm diameterpolycarbonate filter with a pore size of 5 μm (MilliporeTMTP04700). Filters were placed in a glass Petri dish and air-dried overnight. Material on filters was then observed under amicroscope as described below.Sediment samples were placed in an aluminum pot, capped,

and dried in an oven (DHG-9848A, Shanghai Jinghong Co.,Ltd., Shanghai, China) at 65 °C for 1 week. Then, 300 g of drysediment was mixed with 1.2 g cm−3 saturated NaCl at a ratioof 1:2 (v/v) in a 2-L glass container of 30 cm depth. Themixture was stirred and then allowed to settle overnight, theaqueous layer was collected and filtered, and the process wasrepeated. Filtrate was digested using 30% (v/v) H2O2 anddried as described above.The mouth width (mm), body length (mm), and weight

(mg) of each tadpole specimen were measured (Table S2).Next, they were washed three times with filtered water anddigested using H2O2 (30%, v/v) for no more than 72 h.Approximately 5 or 10 tadpoles were pooled in each of threereplicates per field site. The digested solutions were filteredthrough 47 mm diameter polycarbonate filters with a pore sizeof 5 μm (Millipore TMTP04700). All filters were stored in dryglass Petri dishes until further observation.

Observation and Validation of Microplastics. Tadpolesand filters were observed under a Carl Zeiss Discovery V8stereomicroscope (MicroImaging GmbH, Goottingen, Ger-many), and all images were taken with an AxioCam digitalcamera (MicroImaging GmbH, Goottingen, Germany). First,any substances that were not plastics were disregarded asdescribed in our previous studies.4,19,43 Second, all itemssuspected of being MPs were placed on cellulose nitrate gridmembrane filters (Whatman WME, 0.45 μm pore size, 47 mmdiameter) and examined under the attenuated total reflection

Figure 2. Analysis with micro-Fourier Transform Infrared Spectros-copy (μ-FT-IR) (A-D) and images (E-H) of the most prevalent typesof microplastics found in samples. Microplastics consisted of fibers (E,F), fragments (G), and granules (H) consisting of A, E) polyester(PES); B, F, G) polypropylene (PP); C) polyethylene (PE); and D,H) polystyrene (PS). Note: white spots in (H) are due to reflectedlight.

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(ATR) mode of micro-Fourier Transform Infrared Spectros-copy (μ-FT-IR; Bruker, LUMOS, Beijing, China). All datawere collected at a resolution of 4 cm−1 with a 32 s scan time.All spectra were compared with a database from Bruker toverify the polymer type.19 Finally, 523 MPs in total for allsamples from all sites were randomly selected to examine thepolymer types: 206 particles from water samples, 128 particlesfrom tadpole samples, and 189 particles from sedimentsamples. The spectrum matches were at least 60% for mostof the identified particles, and the wavenumber regions ofdifferent polymer types were also used for identification.44

These results were used to remove any remaining substancesthat were not MPs. To aid in the identification of particles,their surface structure and qualitative elemental compositionwere examined using a scanning electron microscope (SEM;Hitachi S-4800, Japan) with an energy dispersive spectrometer(EDS, EMAX).4 For example, compared with the PS granule(Figure 2D, H), microbeads identified as silicon compoundswere smaller and whiter with a rougher surface (Figure S4A).MPs were classified based on Qu et al.19 and categorized by

type according to their morphological characteristics: fibers(elongated, Figure 2E-F), fragments (small irregular pieces,Figure 2G), or granules (spherical and ovoid pieces, Figure2H). The size of all particles was determined by measuring thelongest dimension with ImageJ 1.48 software.45 To facilitatelater comparisons, MP particles were classified into groupsaccording to the mouth sizes of tadpoles: <0.5 mm, 0.5−1 mm,1−2 mm, or 2−5 mm (Table S2).Data Analysis. Statistical analyses were performed using

SPSS 22.0 (IBM Armonk, NY, USA), GraphPad Prism 5(GraphPad Software, La Jolla, CA, USA), and Origin 9.0(OriginLab Corporation, Northampton, MA, USA) software.Student’s t tests were used to determine the differences in thequantities of MPs among different size tadpoles. A linearregression and Pearson’s coefficient were used to test whetherthere was a significant correlation among the abundance of

MPs in tadpoles, water, and sediment. A p < 0.05 wasconsidered to be statistically significant.

■ RESULTSIdentification and Validation of Microplastics. In the

blanks for water, tadpoles, and sediment, 0.45 ± 0.69, 0.27 ±0.47, and 0.64 ± 0.67 MP items filter−1 were found,respectively. When visually inspected, the average number ofMPs for blanks after filtration was less than one, with thebackground contamination equal to 0.7−4.3% of theabundance of MPs in all of the samples.Of the randomly selected particles in different samples,

93.2%, 81.3%, and 81.0% were identified as MPs in water,tadpoles, and sediment, respectively. Fifteen different polymertypes were identified in total (Figure S3). Overall, thedominant polymer was polyester (PES), followed bypolypropylene (PP), polyethylene (PE), and polystyrene(PS) (Figure 2A-D).The dominant polymers were determined in each of the

environmental samples. The most abundant MPs in watersamples were PES fibers (78.5%) followed by PP fibers (3.4%)and fragments (4.0%). In sediment samples, PP fibers (39.6%)dominated but were followed closely by PP fragments (36.4%),then PE fragments (11.0%), and PES fibers (5.2%) (FigureS3). Tadpole samples were most similar to water samples inthat the prevalent polymer was PES fibers (67.6%) followed byPP fibers (6.7%) (Figure S3). In Microhyla ornata tadpoles atS12, the abundance of “microfibers” reached 91.7 ± 26 itemsindividual−1. However, within these organisms, some micro-fibers were too small to identify using μ-FT-IR and were laterdetermined to be diatoms when analyzed with SEM-EDS(Figure S4B). This finding emphasizes the need for multipleforms of analysis beyond visual identification to reduce errorsin identification.46

Microplastics in Water and Sediment. While MPabundance differed among the 25 sampling small waterbodies

Figure 3. Abundance and spatial distribution of microplastics detected in surface water (A, B) and sediment (C, D) samples collected fromShanghai (A, C) and Zhejiang (B, D). Increasing height and deepening color of bars indicate increasing numbers of items according to the scales inthe top right corners of A and C.

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sampled, differences were not significant for MP abundance,shape, or size (Figure S1 and Figure S8). The counts forvarious MPs were averaged for water, tadpoles, and sedimentat each site. Across sites, MPs in surface water samples were0.48−21.52 items L−1. Spatially, relatively greater MPabundances were observed at S19 (21.06 ± 5.36 items L−1)and S20 (21.52 ± 1.24 items L−1) in Zhejiang (Figure 3B). Theabundance of MPs in most of the water samples was in therange of 1−5 items L−1 (Figure 3A-B). The abundance of MPsin sediments varied from 35.76 to 3185.33 items kg−1. Thegreatest MP abundances were found in S7 sediments inShanghai (3185.33 ± 871.28 items kg−1) (Figure 3C) and inS18 sediments in Zhejiang (3059.70 ± 1786.57 items kg−1),followed by S20 in Zhejiang (1022.91 ± 114.05 items kg−1)

(Figure 3D). The abundance of MPs in most of the sedimentsamples was dichotomous, either in the range of 100−200items kg−1 or 500−1000 items kg−1 (Figure 3C−D).Overall, the predominant MP in water samples was fibers

(87.8%) (Figure 4A). Conversely, sediment samples containedan almost equal amount of fragments (55.4%) and fibers(42.6%) (Figure 4B). MPs that were <0.5 mm in size were themost abundant in both water and sediment samples, with thenumber of particles decreasing with increasing size (Figure 4A-B).

Microplastics in Tadpoles. MPs were observed intadpoles belonging to 4 species (Figure S2 and Table S2):Bufo gargarizans, Microhyla ornata, Rana limnochari, andPelophylax nigromaculatus. MPs were found in all tadpoleswith the exception of Rana limnochari in S14. The averageabundance of MPs was highest in Rana limnochari byindividual in S10 (2.73 ± 0.78 items individual−1) and byweight in S25 (168.48 ± 51.99 items g−1). The abundance ofMPs in Bufo gargarizans, Microhyla ornata, and Pelophylaxnigromaculatus was in the range of 0.17−1.89 itemsindividual−1 (2.44−56.88 items g−1), 0.53−2.60 itemsindividual−1 (35.21−157.88 items g−1), and 1.27−1.80 itemsindividual−1 (3.01−4.46 items g−1), respectively (Figure 5).Sites S10, S12, S22, and S25 fell into the second scenario (twospecies in the same sampling site), and MP abundances indifferent species were significantly different, with the exceptionof S22 (Figure 5). There was a weak relationship between MPabundances in tadpoles and in surrounding water (p = 0.046,Figure S5A). Of the different MP morphologies, only fibers<0.5 mm (58.6%, Figure 4C) were found in all tadpoles.Within tadpoles, MP lengths were shorter than those in watersamples, more closely resembling those in sediment (FigureS5B).Rana limnochari proved interesting as individuals fell into

our third scenario (one species with differing sizes at the samesite). Tadpoles at these sites presented a bimodal distributionthat could be placed into two categories based on both lengthand weight: greater or less than 20 mm in length and 200 mgin weight (Figure S6). The number of MPs found wassignificantly greater in the larger tadpoles when quantified byindividual (Figure 6A), except at S23, while significantly morein smaller tadpoles when quantified by weight (Figure 6B),with the exception of S2. Pearson coefficients (pc) of thosetadpoles indicated that there was a strong positive correlationbetween body length and weight (pc = 0.895, p < 0.001), andother species were analogous (Figure S7A). This was similar tolength of the tadpole and the number of MPs by individualfound (pc = 0.589, p < 0.001) (Figure S7B).

■ DISCUSSIONMicroplastics in Small Waterbodies. In this study, we

identified high abundances of MPs in water and sedimentsamples collected from various types of small waterbodies(Table S3). Interestingly, we also observed that sizes, shapes,and quantities of these polymers varied based on the field siteand sample type (i.e., surface water or sediment). To date,what is known about MPs in freshwater is mainly from studiesof rivers and lakes, with this being the first study of MPs insmall waterbodies.11

Overall, the amounts of MPs we detected in smallwaterbodies were greater than abundances reported in mostlarge-scale freshwater systems (Table S3). For example, wefound MPs in surface water and sediment samples to be higher

Figure 4. Percent microplastics by shape and size distribution in water(A), sediment (B), and tadpole (C) samples from all sites.

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than those in the lakes and rivers from Middle-Lower YangtzeRiver Basin of China reported by Su et al.9,47,48 Compared tothe rivers and lakes investigated in those studies, waterbodiesin our study had a considerably smaller total water volume, andthey function as receiving bodies. By virtue of their small size,they accumulate terrestrial runoff, pollutants, and stressorswithout the potential for dilution present in larger catch-ments.14 This might explain the high concentrations of MPs weobserved. For example, Hurley et al.49 found that flooding canexport approximately 70% of the MP load stored in river bedsin northwest England and remove microbead contaminationpreviously seen at 7 sites, i.e., fluvial flushing process efficientlydilutes MPs.Small waterbodies provide several ecosystem services

including outputs for human generated wastewater andmaterials, regulation and mediation of water and nutrients(e.g., water flow and storage; nutrients and carbon transfer),and ponds which provide cultural services (e.g., fishing, boatingetc.).13,50 The close proximity of the small waterbodies in thepresent study to human population centers is worthy ofconsideration. The Lower Yangtze River Valley has a higherpopulation density and more anthropogenic activity comparedto the Middle Yangtze River Valley.29 For example, sites S20 toS25 in Zhejiang had high levels of MPs, specifically fibers, whichwould be expected as all are located near residential areas andtextile processing plants (Table S1). Also, although the smallwaterbodies in our study were of differing sizes and land uses(e.g., the large landscape lake in S16, the small rain puddle in S6,and the rice farmland in S8), they were all located in or nearvery large cities. A potential input of MPs to these smallwaterbodies may be through the discharge of sewagecontaining synthetic fibers and microbeads from personalcare products, laundry wastewater, or textile processing plants.

Many of the small waterbodies in this study were also adjacentto roadways where they were likely affected by illegal trashdisposal. Furthermore, MPs could be released into theenvironment during storm events via surface runoff or aerialtransport.12,51,52

Small waterbodies are the most numerous freshwaterenvironments globally and are facing all the threats affectinglarger waters.13 Our study suggests that MP contamination ispervasive in small waterbodies, yet they remain the leastinvestigated part of the aquatic environment and are largelyexcluded from water management planning. More studies areneeded to address the potential aggregation of plastics in thesesystems.

Microplastics in Tadpoles. Small waterbodies areespecially important for amphibians, which are sensitive tovarious environmental stressors.26 Our results suggest that MPcontamination in tadpoles is widespread, with the averageabundance in tadpoles greater than that of other freshwaterorganisms (Table S3) and other filter feeders (e.g., mussels).4

In the laboratory, filter feeding tadpoles of Xenopus tropicaliswere exposed to polystyrene microspheres at differentconcentrations: 0.1−105 particles mL−1. The results showedthat the amount of microspheres in tadpoles significantlyincreased in a concentration dependent manner.27 It has beenshown that filter feeders and omnivores are more likely toingest MPs than carnivores.53 Mizraji et al.53 observed thatomnivorous fish had a greater number of MP fibers thanherbivores or carnivores. They suggested this to be due to thewider range of food sources for omnivores compared to theothers. Considering the relatively high MP abundance in thesmall waterbodies we sampled, resident tadpoles likely have anincreased probability of ingesting MPs.

Figure 5. Abundance and spatial distribution of microplastics detected in tadpole samples collected from Shanghai (A, C) and Zhejiang (B, D).Abundance of microplastics in tadpoles was calculated by individual (A, B) and by weight (C, D). Increasing height and deepening color of barsindicate increasing numbers of items according to the scales in the top right corners of A and C. Different species represented by different patternedbars seen along the bottom of the figure. Sites S10, S12, S22, and S25 had two different species, and the remaining sites had one species. Student’s ttests were used to calculate significant differences in abundance of microplastics between different species. *p < 0.05; **p < 0.01; ***p < 0.001.

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In this study, MP abundances in Rana limnochari tadpoles inthe same sampling sites were different from that in Microhylaornata tadpoles but were not different from Bufo gargarizanstadpoles (Figure 5). Differences in feeding strategy may explainthese differences in MP ingestion between species. Microhylaornata tadpoles are filter feeders that constantly filter largeamounts of water to obtain food, greatly increasing theprobability of MP ingestion. The other three tadpole speciesare omnivores that graze on periphytic algae and prey oninvertebrates. Water column position may also be ofimportance. Microhyla ornata tadpoles sustain midstreamswimming activity with little resting at the bottom, whilethe other species stay along the bottom or occasionally swim atthe surface, making it possible that their uptake of MPs willdiffer.41 Setala et al.54 linked ingestion of plastic microspheresto particle concentration and encounter rates indicative of apatchy environment. Additionally, Hu et al.27 observed thatwhen food was absent, tadpoles ingested more and egested lessmicrospheres. In a rapidly growing tadpole, the patchiness ofthe normal food source, the movement and timing of MPs intothese waterbodies, bioavailability, feeding strategies, watercolumn positions, etc. likely affect ingestion amounts and ratesand, in turn, growth. These contributing factors should all beconsidered important in MPs ingestion.We also found a correlation between increasing tadpole

length and increased MPs in their tissues, i.e., larger tadpolesingest more MPs, a pattern also observed in freshwater fish.17

Ingestion of MPs by tadpoles may lead to false satiation thatlimits nutrient uptake, which in turn could reduce growth and

the ability to evade predators. Ingested MPs may releaseplasticizers or other persistent organic pollutants, slowingdevelopment and delaying reproduction in frogs and toads.55,56

We think strongly that laboratory studies are needed to reallyaddress MP ingestion as related to tadpole size and growth.

Relationship of Microplastics in Environmentand Tadpoles. The polymer distributions and the propor-tions of MP morphologies found in tadpoles were most similarto that found in water. This would be expected if tadpolesingest MPs as they swim through the water column. Forinstance, Microhyla ornata tadpoles sustain midstreamswimming, and Bufo gargarizans tadpoles swim and forage inthe surface layer of the water.41 Tadpoles may be capturingsuspended MPs in the water column as they feed and asthey respire via gills. These routes of uptake were demonstratedwith microspheres in our previous laboratory study of Xenopustadpoles.27

It was interesting that morphology and polymer in tadpoleswere similar to water, whereas MP size was most similar to thatfound in sediment. Su et al.9 similarly found that MPs inAsian clams (Corbicula f luminea) and sediments weresignificantly smaller than those in water. Previous studiesillustrate that MPs ingested by organisms would be brokendown during passage through the gut. Watts et al.22 found thatthe size of polypropylene microfibers was reduced between theplastic present in food and fecal pellets of shore crabs (Carcinusmaenas). Cole et al.57 showed plastics floating in the watercolumn can be redistributed when they are ingested and thenbecome embedded in feces that sink to the bottom. Wehypothesize that MPs ingested from the water column werebroken down during digestion and sank to the sediment afterexcretion. This would explain the patterns we observed intadpoles related to MPs found in water and sediment.Tadpoles had the highest amount of MPs < 0.5 mm across

all types, but predominantly fibers. This high concentration offibers combined with feeding strategy also indicates that thesource of MPs in tadpoles was water. The lack of larger MPs intadpoles (>0.5 mm) combined with the range of mouthopenings (0.5−3.2 mm) suggests that they can consume mostsizes and that they are either selecting smaller MPs or the gut isbreaking down larger MPs.It is important to keep in mind that we recovered MPs from

whole individuals. Follow-up work is needed to localize variouspolymers and morphologies of MPs to specific organs anddetermine whether MPs are translocated within the organisms.We also did not characterize specific toxicity (e.g., changes ingrowth or metamorphosis, tissue specific alterations); addi-tional, controlled laboratory studies should be done to carefullydetermine such changes. This study illustrates the need forsuch studies as MPs are both abundant in these smallwaterbodies and in the organisms that inhabit them.In conclusion, this study is the first to report MP distribution

and characteristics in small waterbodies and tadpoles fromYangtze River Delta, China. MP pollution was widespread inwater and sediment, particularly in the form of fibers, likely theresult of nearby human activities. It demonstrated the need forreducing inputs of plastic waste and supporting conservationand effective management of small waterbodies. Correspond-ing high abundances of MPs in resident tadpoles stronglysuggest that MPs may transport through the food chain tohigher aquatic or terrestrial trophic levels.

Figure 6. Comparison of abundance of microplastics in different sizesof Rana limnochari tadpoles collected from the same sites: A) byindividual and B) by weight. Tadpoles from the same sites weredivided into two groups based on length and weight: greater (whitebars) or less (gray bars) than 20 mm in length and 200 mg in weight.Bars represent mean ± SD of three replicates (n = 3). Student’s t testswere used to calculate the significant differences in the abundance ofmicroplastics among different size tadpoles. *p < 0.05; **p < 0.01;***p < 0.001.

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■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.est.8b02279.

Figures S1−S8 and Tables S1−S3 (PDF)

■ AUTHOR INFORMATIONCorresponding Author*Phone: +86 21 62455593. E-mail: hhshi@des.ecnu.edu.cn.ORCIDHuahong Shi: 0000-0003-2978-0680NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by grants from the Natural ScienceFoundation of China (41776123) and China ScholarshipCouncil ([2017]3109).

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