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Microplastics in invertebrates on soft shores in Hong Kong: Influence of habitat, taxa and feeding mode
Science of the Total Environment 715 (2020) 136999
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Microplastics in invertebrates on soft shores in Hong Kong: Influence of habitat, taxa and feeding mode Xiaoyu Xu a, C.Y. Wong a, Nora F.Y. Tam a, Hoi-Shing Lo a, Siu-Gin Cheung a,b,⁎ a b
Department of Chemistry, City University of Hong Kong, Tat Chee Avenue, Hong Kong, China State Key Laboratory of Marine Pollution, City University of Hong Kong, Tat Chee Avenue, Hong Kong, China
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Suspected microplastics were found in 32 out of 38 species and 55% of all the organisms. • The three most predominant plastic polymer were cellophane, PET and PA. • Abundance of suspected microplastics was taxon related. • 62.82% of suspected MPs tested were found to be cellulose.
a r t i c l e
i n f o
Article history: Received 25 October 2019 Received in revised form 9 January 2020 Accepted 27 January 2020 Available online 28 January 2020 Editor: Thomas Kevin V Keywords: Microplastics Feeding mode Soft shore habitats Biological taxa
a b s t r a c t Microplastic (MP) pollution in the marine environment has gained much concern in recent years. This study investigated the occurrence of MPs in invertebrates collected on 18 mudflats and sandy beaches in Hong Kong and its relationships to biological taxon, feeding mode and habitat. In total 38 species of gastropods, bivalves and crabs were collected and the mean number of suspected microplastics ranged from 0 to 9.68 particles g−1 wet weight or 0 to 18.4 particles individual−1. Around 26% of the suspected microplastics were confirmed to be synthetic polymers, including CP (cellophane), PET (polyethylene terephthalate), and PA (polyamide). Microplastic fibres were the most abundant type of MPs, followed by pellets. Significantly higher abundance of suspected microplastics was found in gastropods. Since MP abundance might vary with taxon, it is recommended to include different taxonomic groups in any ecological assessment of the impact of MPs. © 2020 Elsevier B.V. All rights reserved.
1. Introduction Microplastics (MPs), which are commonly defined as plastic particles of diameter between 1 μm and 5 mm (Arthur et al., 2009), have become a global environment problem with a growing concern. Their distribution in the marine environment is ubiquitous, ranging from ⁎ Corresponding author at: Department of Chemistry, City University of Hong Kong, Tat Chee Avenue, Hong Kong, China. E-mail address: [email protected] (S.-G. Cheung).
https://doi.org/10.1016/j.scitotenv.2020.136999 0048-9697/© 2020 Elsevier B.V. All rights reserved.
sediments on shores, surface water and water column, to seabed sediments (Jiang, 2018). They are found even in the ice core from the remote Arctic Ocean (Obbard, 2018). The similarity in size between MPs and prey of various aquatic animals increases the bioavailability of MPs (Lusher et al., 2017) and ingestion of MPs has been reported for zooplanktons (Rist et al., 2017), bivalves (Murray and Cowie, 2011; Santana et al., 2016), crabs (Wójcik-Fudalewska et al., 2016), barnacles (Goldstein and Goodwin, 2013), gastropods (Gamage et al., 2017; Naji et al., 2018), fish (Caron et al., 2018; Nadal et al., 2016), and marine mammals (Lusher et al., 2015), although most of the studies were on
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fish in the seawater column. A large number of studies have been reported on MPs in shore sediments, in contrast, studies of their abundance in intertidal zone animals were scarce. The abundance of MPs in mussels and clams ranged from 0.04 particles g−1 body wt in Mytilus galloprocincialis (Vandermeersch et al., 2015) to 12.7 particles g−1 body wt in Amiantis purpuratus (Naji et al., 2018), or 0.6 particles individual−1 in Mytilus edulis (Phuong et al., 2018) to 23.5 particles individual−1 in Crassostrea virginca (Waite et al., 2018). Lower abundances were found in gastropods (0.17 particles g−1 body wt in Littorina sp. (Gamage et al., 2017) to 17.7 particles g−1 body wt in Thais mutabilis (Naji et al., 2018) and the Atlantic mud crab, Panopeus herbstii (15.1–25.8 particles per individual−1) (Waite et al., 2018). A few studies have determined the MP abundance in several species from one habitat, such as five species of mollusks with different feeding modes, including bivalves and gastropods from the northern part of Persian Gulf (Naji et al., 2018), with higher MP abundance being found in the predatory gastropod Thais mutabilis. In another study on three benthic macroinvertebrates in the Rockall Through, Northeast Atlantic Ocean, the MP abundance was related to species, but not feeding mode (CourteneJones et al., 2017). Since ingestion of MPs is species specific and may vary with feeding mode, a comparison of MPs ingested and accumulated in invertebrates from different taxa and with different feeding modes will provide useful information in selecting appropriate organisms as bioindicators of MP pollution and model organisms for studies on trophic transfer of MPs. Once ingested, MPs may transfer from the intestinal tract to body tissue (Collard et al., 2017; Farrell and Nelson, 2013) and elicit various health effects, such as obstruction of feeding organs (Cole et al., 2013), reduction of feeding capacity and energy intake (Xu et al., 2017), enhanced immune response (Lu et al., 2016; Von Moos et al., 2012), reduction in growth rate and reproductive ability (Sussarellu et al., 2015), and even neurotoxicity (Barboza et al., 2018). Even worse is that MPs can also be vectors of hydrophobic organic pollutants, such as PAHs, PCBs and OCPs (Lo et al., 2019) due to their large surface area to volume ratio and lipophilicity. With a population of 7.3 million people and a lack of an efficient plastic recycling industry, Hong Kong produces a huge quantity of plastic waste daily and in the year 2016, 2132 t day−1 of plastic waste were produced (HKEPD, 2017). This served as an important source of MP pollution in local marine waters. Pearl River, the third largest river in China with 60 million people living around the river delta, is considered as a major non-local source of plastic waste in Hong Kong, especially in the western waters of Hong Kong where the river delta is located (Fok and Cheung, 2015). Occurrence of MPs in seawater, as well as in sediments on beaches and the seabed around Hong Kong have been documented (Cheang et al., 2018; Cheung and Fok, 2016; Fok and Cheung, 2015; Tsang et al., 2017). The MPs concentration in Hong Kong coastal water ranged from 51 to 27,909 particles per 100 m3 with PP, HDPE, LDPE, and a mixture of PP, ethylene propylene (EPDM) and styrene acrylonitrile being most common (Tsang et al., 2017). In a study of 25 beaches along the coastline of Hong Kong, the mean MP abundance was found to be 5595 particles per m2 sediment, a value higher than the international average indicating that Hong Kong was a hotspot of MP pollution (Fok and Cheung, 2015). Another study has reported ten times more MPs on mudflats than sandy beaches with PE being most abundant and followed by PP and PET (Lo et al., 2018). The MP abundance in the seabed sediment along the northeastern and eastern shores of Hong Kong ranged between 169 and 221 particles kg−1 ww sediment, with a mean value of 189 particles kg−1 ww sediment (Cheang et al., 2018). This study determined the occurrence of MPs in three major groups of invertebrates, i.e., bivalves, gastropods and crabs, commonly found on sandy beaches and mudflats in Hong Kong. The number of species collected from each shore varied between 2 and 13 species, and in total 38 species were studied (Table S2, S3). The objectives were to understand 1) the differences in abundance and composition of MPs
between organisms from mudflats and sandy shores, and 2) whether MP abundance and composition varied among taxa or feeding modes. 2. Materials and methods 2.1. Study locations and sample collection Eighteen sites were visited with half of them located in the eastern waters and the other half in the western waters. Among the nine sites in each region, four of them were mudflats and the remaining sandy beaches (Fig. 1). The geographic coordinates of these sampling sites are listed in the supplementary information (Table S1). The shores in the eastern waters are facing South China Sea and located in country parks, hence the level of human disturbance is low. In contrast, the shores in the western waters are located near to the Pearl River estuary, hence receive a lot of marine debris through riverine input. All the sampling sites are non-gazetted beaches or mudflats with no frequent cleanup or maintenance. To minimize seasonal effects, the animals were collected during low tides in summer between May and September 2016. Bivalves, gastropods and crabs were collected with the help of stainless-steel shovels, and labour gloves and clothes made of cotton were worn to prevent potential contamination by MPs during the sampling. Animals were placed in labeled cotton bags to minimize contamination during transportation and storage, and transported back to the laboratory within 3 h. Upon arrival at the laboratory, they were stored at −20 °C immediately. The number of species collected from each site was determined not only by the species richness of the sites but also the conditions of the sites. For example, less species were collected from mudflats with very soft mud because of the difficulty in sampling. Information of the species collected from eastern sites is shown in Table S2 and those from western sites in Table S3. 2.2. Extraction of MPs from animals Feeding modes of most of the species were classified according to two references (Chan and Caley, 2003; Williams, 2003). For species which were not included in these references, including Terebralia sulcate (Barnes, 2003), Gaetice depressus (Depledge, 1989), Hemigrapsus penicillatus (Kurihara and Okamoto, 1987), Metopograpsus frontalis and Perisesarma bidens (Poon et al., 2010), their feeding modes were classified according to research reports. All the equipment and glassware were rinsed three times with de-ionized (DI) water before use. The animals were thawed, and shell length of bivalves, shell height of gastropods and carapace width of crabs were measured using Vernier calipers. For species occurring at only one to two sampling sites, the wet weight of each individual was measured directly. For species that occurred in a number of sites, the wet weight of all the individuals (15–30) at one of the sites was measured and a calibration curve relating body size (shell length, shell height or carapace width) and wet weight was computed and used for calculating the wet weight of the same species collected from the other sites. The chemical digestion of the organisms followed the protocol of Karami et al. (2017). Each individual was rinsed with DI water to remove particles attached on the body surface before it was placed in a separate 100 mL beaker. The shell/carapace of the animals was broken to facilitate digestion using 10% potassium hydroxide (KOH) at 40 °C for 48 h in an incubator (ET 618-4 Lovibond Thermostatschrank). Beakers were covered with aluminum foil during digestion to prevent atmospheric contamination. Since the resolution of the FTIR analysis we used in the experiment was around 30 μm, a sieve of 38 μm was used to collect suspected MPs extracted from the animals. To facilitate microscopic examination, particles retained by the sieves were transferred to a 0.45 μm mixed cellulose ester filter paper through vacuum filtration. Beakers and sieves were rinsed thoroughly with DI water to ensure that the suspected MPs attached on the surface were totally
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Fig. 1. 18 sampling sites around Hong Kong. The mudflats at Lai Chi Wo (LCW), Ho Chung (HC), Chek Keng (CK) and Wong Yi Chau (WYC), and sheltered sandy shores at Tai Tan (TT), Lai Chi Chong (LCC), Tai Tam bay (TTB), Tsam Chuk Wan (TCW) and Pak Sha Wan (PSW) are located in the eastern waters. The mudflats at Ha Pak Lai (HPL), Shan Tau (ST), Sham Wat (SW) and Tai O (TO), and sheltered sandy shores at Shui Hau Wan (SHW) Sha Lo Wan (SLW), Yi O (YO), Luk Keng Tsuen (LKT) and Tai Pai Tsui (TPT) are located in the western waters.
transferred. Each filter paper was stored in a clean petri dish for further analysis. 2.3. Qualification and quantification of MPs Visual examination of the filters was performed under a stereomicroscope (Leica M165C) at a magnification of 40× to 120×. Suspected MPs were classified according to their shape (fibre, pellet, fragment, foam and film) (Lo et al., 2018) and colours (white/transparent or other colours). After microscopic examination of all the particles, around 20% of the particles on each filter paper were selected randomly. If there were b5 particles on the filter paper, particles on several filter papers for the same animal species were pooled and then 20% of the particles were selected randomly. In total 280 out of 1284 suspected MPs were selected for chemical composition identification by micro Fourier Transform Infrared Microscope (μFTIR; Thermo Fisher Nicolet iN5, USA) in attenuated total reflection (ATR) mode. The FTIR spectrum of each suspected MP was obtained as an average of 32 scans in the range of 4000–650 cm−1 with a resolution of 4 cm−1. The spectrum was compared with the OMNIC standard spectra library of Hummel Polymer Sample Library and a self-generated library of natural cellulose obtained from pure cotton. A matching with a score N 70% was accepted. 2.4. QA/qc To assess possible contamination from the air or glassware during tissue digestion, a procedural control including all the digestion procedures but without animals was conducted once a week and in total 12 replicates were prepared. To estimate the potential loss of MPs after digestion with KOH, the recovery efficiency of MPs was evaluated in preliminary trails. Clams, gastropods and crabs (n = 5) were placed in beakers with a known number of bright pink polyamide (PA) fibres around 0.2 cm in length being cut from fabric. The spiked MPs were extracted using the methods mentioned above. The filter papers were examined under a microscope and the recovery efficiency was calculated in percentages.
2.5. Statistical analysis The data failed in the Shapiro-Wilk test for normality. Two separate nested generalized linear models (GLM) were used to investigate the effects of region (western and eastern waters), shore type (sandy shores and mudflats), feeding mode and taxon on the abundance of suspected MPs in the organisms. The effect of shore type was nested within region, and the effect of feeding mode or taxa was nested within shore type. According to the results of Pearson chi-square dispersion test, the data were over-dispersed. Therefore, GLM with negative binomial distribution was more appropriate for the analysis, owing to the residual overdispersion. The wet weight of body tissue was used as an offset variable so that the abundance of suspected MPs was mass-adjusted. The similarity in the chemical composition of suspected MPs between shore types (mudflats and sandy shores), and regions (west and east) were analyzed using non-metric multidimensional scaling (NMDS). The correlation between the abundance of suspected MPs in the organisms and sediments was analyzed using Pearson product-moment correlation. The amount of MPs in the sediments was obtained in the same study for the same sites and reported previously (Lo et al., 2018) and Pearson correlation analysis was conducted for the organisms with different feeding modes. The correlation between the wet weight of organisms and quantity of suspected MPs was analyzed using Pearson correlation for each single species. Kruskal-Wallis tests were used to compare the number of suspected MPs among different species. GLM and Pearson correlation were conducted by SPSS, and NMDS by Rstudio.
3. Results 3.1. Abundance of suspected microplastics in organisms The percentage of spiked MPs recovered from bivalve, gastropod and crab samples was 82%, 50% and 70% respectively (see Section 2.4). The extraction efficiency for gastropods was very low as the shell debris remaining on the filter paper disturbed visual observation. The abundance of suspected MPs was not corrected by the recovery efficiencies. In total
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312 individuals (174 gastropods, 68 bivalves and 71 crabs) were collected from 18 sampling sites and the abundance of suspected MPs was determined for each individual. The procedural blanks obtained 2 ± 1.04 fibres on average (n = 12) and the abundance of suspected MPs in each sample was corrected accordingly. After correction for the background contamination, suspected MPs were found in 32 out of 38 animal species and 55% of all the individuals. The mean number of suspected MPs ranged from 0.025 to 9.684 particles g−1 wet weight or from 0.08 to 18.4 particles individual−1. Most of them were fibres (93.3%) and the remaining ones pellets (6.7%). The mean length and diameter of the fibres was 1004.2 ± 464.8 μm and 21.8 ± 7.3 μm, respectively. More than half of the particles were white or transparent (65.1%), while the remaining ones were of other colours (34.9%). Pearson product-moment correlation analysis showed a lack of correlation between the abundance of suspected MPs in sediments and that in organisms with different feeding modes (deposit feeders/ grazers: r = −0.129, p = .079, n = 187; filter feeders: r = −0.232, p = .065, n = 64; predators: r = −0.114, p = .468, n = 43). In the nested GLM with region, shore type and feeding mode as predictive factors, the abundance of suspected MPs varied significantly with all of these factors. Organisms in the eastern waters ingested significantly more suspected MPs than those in the western waters, irrespective of the shore type. For both regions, the sandy shore organisms contained significantly more suspected MPs than the mudflat organisms. The effect of feeding mode varied with habitat (Fig. 2) with the abundance of suspected MPs in the deposit feeders/grazers being significantly higher than that in the filter feeders and predators in both eastern and western waters. For the sandy shores in the eastern waters, the effect of feeding mode was insignificant but the predators in the western waters had a significantly lower abundance of suspected MPs than those with other feeding modes. In the nested GLM with region, shore type and taxon as predictive factors, the organisms from the eastern waters contained significantly
more suspected MPs than those from the western waters. In contrast to the feeding mode, when taxon was used as a predictive factor the effect of shore type was statistically indistinguishable. The abundance of suspected MPs was the highest in the gastropods and followed by bivalves and crabs (Fig. 3) with the differences between the gastropods and crabs being statistically significant irrespective of the shore type and region. The abundance of suspected MPs varied among species in the same taxa (Fig. 4). For the gastropods, the highest abundance of suspected MPs was recorded in the deposit-feeding Batillatira multiformis (5.37 ± 1.24 particles g−1 ww) which was significantly higher than in Nerita chamaeleon (1.50 ± 0.20 particles g−1 ww). For bivalves, the abundance of MPs in Marcia spp. was significantly lower than in Anomalodiscus squamosus (3.48 ± 0.89 particles g−1 ww) and Donax spp. (2.89 ± 0.54 particles g−1 ww). The abundance of suspected MPs in Gafrarium spp. (0.26 ± 0.08 particles g−1 ww) was also significantly lower than in A. squamosus and Donax spp. For the crabs, Metopograpsus frontalis (0.21 ± 0.06 particles g−1 ww) had significantly less suspected MPs than Austruca lactea (2.84 ± 0.44 particles g−1 ww) and Macrophthalmus convexus (2.59 ± 0.73 particles g−1 ww). The abundance of suspected MPs in the organisms with different feeding modes were also compared within individual sampling sites (Fig. 5) where at least three species were collected, i.e., Lai Chi Chong, Pak Sha Wan, Shui Hau Wan, Sha Lo Wan, Luk Keng Tsuen and Tai Pai Tsui. At Lai Chi Chong (LCC), the grazer L. coronata and filter feeder A. squamosus had the highest abundance of suspected MPs, whereas at both Tai Pai Tsui (TPT) and Sha Lo Wan (SLW) the highest abundance of suspected MPs was found in a predatory gastropod R. clavigera. In some species, the particle abundance and wet weight of organisms was negatively correlated, including A. squamosus (r = −0.564, p b .05, n = 19), Batillaria zonalis (r = −0.593, p b .01, n = 20), and L. coronata (r = −0.407, p b .05, n = 31), but the correlation in Donax spp. was positive (r = 0.572, p b .05, n = 15).
Fig. 2. The bar charts showing the abundance of suspected MPs in animals with different feeding modes collected from a) mudflats in western waters, b) sandy shores in western waters, c) mudflats in eastern waters, and d) sandy shores in eastern waters. Significant differences according to GLM results ae shown. The number of replicates is shown inside each bar.
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Fig. 3. The bar charts showing the abundance of suspected MPs in animals from different taxa collected on the a) mudflats in western waters, b) sandy shores in western waters, c) mudflats in eastern waters, and d) sandy shores in eastern waters. Significant differences according to GLM are shown. The number of replicates is shown inside each bar.
3.2. Composition of MPs A sub-sample of 280 out of 1284 suspected MPs were analyzed by micro-FTIR. Seventy-four (24.6%) of them were synthetic polymers, 174 (62.82%) cellulose fibres, and the remaining 32 (11.55%) could not be identified (Fig. 6). The three most abundant polymers were cellophane (CP, 43.66%), polyethylene terephthalate (PET, 19.72%) and polyamide (PA, 16.9%). All the pellets were either PP or PE, whereas fibres
belonged to six different kinds of polymer. The length of fibres varied among different types of polymer with CP being significantly longer than PET and PA (Kruskal-Wallis test, n = 5, p b .005). The diameter was also significantly different among different types of fibre (Kruskal-Wallis test, n = 5, p b .05) but no significant difference was found in pair-wise comparisons. The NMDS plots based on the chemical composition of MPs separated mudflats from sandy shores with mudflats characterized by PAN
Fig. 4. The bar charts showing the abundance of suspected MPs in different species of (a) gastropods, (b) bivalves and (c) crabs. The abundance of suspected MPs in each species was calculated from data collected from all the sampling sites. Significant differences according to the Kruskal-Wallis test are represented by different letters. The number of replicates is shown next to each bar. Species with a sample size of b5 were not included in the K-W test.
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Fig. 5. The bar charts showing the abundance of suspected MPs in species collected from 6 representative sampling sites. According to the results of the Kruskal-Wallis test, significant differences among the species from the same sampling sites are represented by different letters. The sample size is shown next to each bar. Species with a sample size of b5 were not included in the K-W test.
and PET and sandy shores by CP and PA (Fig. 7a), except for the mudflat at CK and sandy shore at PSW. However, no regional difference was observed (Fig. 7b). The NMDS plots based on the chemical composition of MPs showed no apparent clustering of the taxa or feeding modes, indicating no polymer preference of each taxonomic group or feeding mode (Fig. 8). 4. Discussion 4.1. Characteristics of the MPs Most of the MPs found in this study were fibres made up of cellophane, polyethylene terephthalate, polypropylene, polyamide and polyethylene. Synthetic fibres occupy N60% of the world fibre consumption (FAO and ICAC, 2013), and their characteristic properties make them suitable materials for sportswear, medical, agricultural and protective systems (e.g., breathing apparatus, cold weather masks, helmets) (ElMogahzy, 2008). Aquaculture and fishing activities are sources of MP fibres in the marine environment as nearly all of the global fishery equipment are made of plastics and a large amount of them is either lost or discarded in the ocean (Andrady, 2011). Laundry also generates a large number of MP fibres from clothing. Studies have shown that N1900 MP fibres were generated in a single washing. Although most of them are trapped in the sewage treatment plant, a small proportion
Fig. 6. MPs of different chemical compositions: CP (cellophane, 43.66%), PET (polyethylene terephthalate, 19.72%), PA (polyamide, 16.9%), PP (polypropylene, 8.45%), PE (polyethylene, 7.04%) and PAN (polyacrylonitrile, 4.23%).
is discharged into the ocean (Browne et al., 2011; Salvador Cesa et al., 2017).In our concurrent study, the microfibre was the most dominant form of MPs in sediments from shores around Hong Kong, accounting for 57.2% of all the MPs collected. Sewage treatment plants were suspected to be a major source of these microfibres because much higher concentrations of them were found at the study sites near the outflow or sewage treatment plants (Lo et al., 2018). In the present study, however, there was no correlation between the MP abundance in the organisms and distance to the nearest sewage treatment plants. The inconsistency in the MP abundance between the shore sediments and intertidal organisms was also found and will be explained later. The three predominant polymers found inside the organisms were CP (43.66%), PET (19.71%) and PA (16.9%), but PE (46.9%), PP (13.8%) and PET (13.5%) in the shore sediments of our concurrent study (Lo et al., 2018). The discrepancy may be due to differences in the forms of MPs analyzed that FTIR instead of μ-FTIR was used in analyzing MPs in sediments with a lowest size limit of 250 μm. Therefore, most of the fibres were excluded from the analysis. In contrast, the use of μ-FTIR in the present study allowed MPs as small as 30 μm, including fibres to be analyzed. Another possibility was feeding selectivity in the organisms. When an amphipod Hyalella azteca was exposed to the same concentration of either PE fragments or PP fibres, PP fibres were preferentially ingested (Au et al., 2015). The study, however, could not identify what the amphipod was selecting for as PE fragments and PP fibres differ not only in shape but also chemical composition, colour and size. CP was the most common polymer type in this study. According to GESAMP (Kershaw and Rochman, 2015), it is defined as bio-derived plastics, where the plastic materials are regenerated from polymers extracted from biomass (Kershaw and Rochman, 2015). CP was also found in many other studies on both sediments and organisms (Alomar and Deudero, 2017; Fang et al., 2018; Gago et al., 2018). In the study of MP contamination in benthic organisms from the Arctic and sub-Arctic region, CP was the fourth common polymer accounting for 13% of all the MPs (Fang et al., 2018) and it was the most common MP (33.33%) ingested by the shark Geleus melastomus in another study (Alomar and Deudero, 2017). Cellophane is used as wrappers in cigarette and food, and as fibreglass products and release agent for the manufacture of rubber (Yang et al., 2015). As most of the suspected MPs in this study were fibres and among them CP fibres were the longest. The longer the fibre, the longer is its retention time in living organisms as demonstrated in mussels in which a lower proportion of the longer fibres was egested in b3 h (Ward et al., 2019). Regardless of the common
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Fig. 7. Non-metric multidimensional scaling (NMDS) ordination of MPs. (a) Separation by chemical composition in relation to shore type (mudflats and sandy shores), and (b) separation by chemical composition in relation to region (west and east). The polymer types are shown in red. The abbreviations of the sampling sites refer to Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
occurrence of CP in the marine environment, the research on its toxicity on living organisms is rare when compared with that on other MPs such as PP and PE (Korez et al., 2018). Synthetic fibres such as rayon are different from cellophane structurally but not chemically. Our FTIR, however, could not differentiate rayon from cellophane, and rayon is commonly moulded into fibres. Therefore, we cannot exclude the possibility of the presence of rayon in our samples. Although criteria such as no visible cellular or organic structure, equally thick or clear and homogeneous colours have been proposed for differentiating synthetic microfibres from natural microfibres using microscopic examination (Hidalgo-Ruz et al., 2012), this is not a very reliable method as it is relatively subjective and relies heavily on the experience of the observer. More importantly, chemical characterization was not employed in a number of studies (Devriese et al., 2015; Waite et al., 2018; Wang et al., 2019). The present study has illustrated this problem as 62% of the suspected microfibres were in fact natural cellulose as confirmed by μFTIR spectrometry. In another study of MPs ingestion by deep-sea invertebrates using protease enzymatic digestion, 165 out of 359 (around 46%) potential MPs were identified as cellulose (CourteneJones et al., 2017). In a study of MPs in mussels, although hydrogen peroxide was used for digestion of organic matter and MPs were extracted
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Fig. 8. Non-metric multidimensional scaling (NMDS) ordination of MPs. (a) Separation by chemical composition in relation to taxa, and (b) separation by chemical composition in relation to feeding modes. The polymer types are shown in red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
using saline (NaCl) solution, natural cellulose fibres were still present in the final samples (Li et al., 2018). Therefore, a more cautious approach should be adopted in the future including the development of more effective digestion methods and more importantly the chemical characterization using μ-FTIR, Raman spectroscopy or other analytical methods. According to the results of NMDS (Fig. 7a), mudflats were separated from sandy beaches and characterized by PAN (polyacrylonitrile) and PET. PAN fibres are usually used in hot gas filtration systems, outdoor awnings, sails, fibre-reinforced concrete (Nithya et al., 2017) and knitted clothing, such as sweaters and socks (Choe et al., 2006). N60% of the global production of PET is used as synthetic fibres, others include films, bottles and other moulded products (Jankauskait et al., 2008). A high abundance of PET and PAN near Shan Tau (ST) and Ho Chung (HC) may be due to the densely-populated residential area near these two sampling sites and land-based contamination of MPs through laundry. 4.2. Influence of feeding mode and taxon on the abundance of suspected MPs As shown in Figs. 2 and 3, the abundance of suspected MPs was related to both feeding mode and taxon with deposit feeders/grazers
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and gastropods being more vulnerable to MP ingestion. Contradictory results, however, are shown in Fig. 5 when results of different animal species at the same sites were compared. A significantly higher number of suspected MPs was found in the predatory gastropod R. clavigera at Tai Pai Tsui and Sha Lo Wan, and in both the deposit feeding gastropod L. coronata and filter feeder bivalve A. squamosus at Lai Chi Chong. The apparent relationship between feeding mode and microplastics abundance was probably due to sampling bias as filter feeders were absent on some of the mudflats where MPs were more abundant (Lo et al., 2018). This would underestimate the abundance of suspected MPs in filter feeding bivalves, resulting in higher concentrations of suspected MPs in deposit feeders/grazers. The effect of feeding mode on MP abundance in animals is inconsistent in the literature. For example, Bour et al. (2018b) showed that a lower frequency of MP ingestion was observed for filter-feeders compared to deposit-feeders and predators, and in another study a significantly lower number of particles per individual was observed for filter-feeders compared to predators (Bour et al., 2018a). Other studies, however, have documented that filter feeders are most prone to ingesting MPs (Scherer et al., 2017; Setälä et al., 2015). In the present study, irrespective of the feeding mode, higher abundance of suspected MPs was found in gastropods and probably related to the morphology of the digestive system which follows the spiral shape of the shell (Lőw et al., 2016), and torsion of the body ends up with a more convoluted digestive tract (Page, 2003). This complicated gut morphology may increase the retention time of MPs and facilitates its accumulation, regardless of the feeding mode of the gastropods. When comparing compositions of MPs from different taxa or feeding modes, the results of NMDS (Fig. 8) did not reveal any pattern, indicating that feeding selectivity may be species specific and depend on the shape of the particles rather than chemical composition. Correlation between the abundance of suspected MPs and wet weight of organisms was observed in some species. The results, however, were inconsistent among species with negative correlations in gastropods B. zonalis and L. coronata and in a bivalve A. squamosus, but a positive correlation for another bivalve, Donax sp. This indicates that MP ingestion and retention is species specific and independent of the feeding mode. 4.3. Influence of region and shore type on the abundance of suspected MPs A higher abundance of suspected MPs was found in the organisms from both the eastern waters and sandy shores. The results were contradictory to those obtained in our concurrent study on MPs in sediments in which higher concentrations of MPs were obtained on the mudflats as well as in the western waters. Several reasons can explain the discrepancies. Firstly, the species composition on the mudflats and sandy beaches was different with the number of gastropod species collected from the sandy shores doubled that from the mudflats. As shown in the above, the gastropods contained more suspected MPs, probably due to their modes of feeding and complicated digestive tracts, hence resulted in a higher abundance of suspected MPs in the organisms on the sandy beaches. Secondly, the abundance of suspected MPs varied among species and those with a higher abundance occurring mostly in the eastern waters, e.g., a clam A. squamosus was found at 3 sites in the eastern waters but 1 site in the western waters, and a gastropod B. multiformis was found in 2 sites in the eastern waters but not found in the western waters. As the overall abundance of suspected MPs in organisms from a habitat is influenced by the species composition, species of different taxa should be included when inter-site comparisons of the ecological impact of MPs is made.
freshwater fish Orechromis niloticus (Ding et al., 2018). The present study, however, did not find any such correlation between the sediments and organisms. Similar results were obtained in other studies. In Northeastern Pacific Ocean, no systematic relationship was found between the prevalence of MPs in the fish stomach and the waters around the study colonies (Hipfner et al., 2018). Even though a higher MP abundance in the habitats increases the opportunity of the organisms in encountering MPs, avoidance of MP ingestion may occur as shown in the collembolans (Zhu et al., 2018). Filter feeders such as bivalves can avoid ingesting MPs through the production of pseudofeces. In the blue mussel Mytilus edulis, 71% of the MP fibres were rejected as pseudofeces and only 9% was ingested (Woods et al., 2018). Depuration through faeces production was also effective in removing MPs in the Mediterranean mussel Mytilus galloprovincialis in which around 85% of the MPs were eliminated after 6 days of depuration (Fernández and Albentosa, 2019). The lack of correlation between abundance of MPs in sediments and in animals could also be due to different size fractions of particles analyzed as only MPs between 5 mm and 250 μm were studied in Lo et al. (2018) on sediments but between 5 mm and 30 μm in the present study. 4.5. Limitation and future studies Since this study involved collecting a large number of animals from a number of beaches and mudflats, among which some of them did not have many animals while others were too muddy for sample collection, only a small number of individuals were examined for each species. Moreover, very muddy sampling sites do not favor the survival of filter feeding bivalves, hence only gastropods were collected from several sampling sites including Tai O, where the sediment contained the highest abundance of suspected MPs compared to all the other sites (Lo et al., 2018). This inevitably introduced biases in the comparisons of the results among different feeding modes or taxonomic groups as well as the representativeness of the data. The tremendous amount of work also limited the percentage of suspected MPs (20%) analyzed by μ-FTIR. A lower recovery efficiency of MPs from the gastropods than from the bivalves and crabs would underestimate the differences in the abundance of suspected MPs between these taxonomic groups but the conclusion of the study was not affected as even though the data of the abundance of suspected MPs were not corrected for the differences in the recovery efficiency, the gastropods still had the highest MP abundance. 5. Conclusion Suspected MPs were found in 32 out of 38 animal species in the present study with more being found in gastropods. This indicated that the ingestion of MPs was common in intertidal animals and taxon related. The three most predominant types of plastic polymer were cellophane, PET and PA. However, a very high percentage of fibres (62.82%) were found to be cellulose. This highlights the significance of chemical analysis of the suspected MPs using μ–FTIR or other analytical method such as Raman Spectroscopy. Since MP abundance might vary with taxon, it is recommended to include different taxonomic groups in any ecological assessment of the impact of MPs. 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.
4.4. Correlation with the MP abundance in shore sediments Acknowledgement It is intuitive that the higher the concentration of MPs in the sediment, the higher is that in the organisms. Such a concentrationdependent effect was observed in the accumulation of PS-MPs in a
The work described in this paper was supported by the Research Grants Council of Hong Kong SAR (CityU 11302815). We are grateful
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to Ng Wing Yi, Fung Chun Kit Edmond and So Lai Hong for their help in sample collections. Supplementary data Brief descriptions in nonsentence format listing the contents of the files supplied as Supporting Information. Supplementary data to this article can be found online at https://doi.org/10.1016/j.scitotenv.2020. 136999. References Alomar, C., Deudero, S., 2017. Evidence of microplastic ingestion in the shark Galeus melastomus Rafinesque, 1810 in the continental shelf off the western Mediterranean Sea. Environ. Pollut. 223, 223–229. https://doi.org/10.1016/J.ENVPOL.2017.01.015. Andrady, A.L., 2011. Microplastics in the marine environment. Mar. Pollut. Bull. https:// doi.org/10.1016/j.marpolbul.2011.05.030. Arthur, C., Baker, J., Bamford, H., 2009. Proceedings of the International Research Workshop on the Occurrence, Effects, and Fate of Microplastic Marine Debris. NOAA. Au, S.Y., Bruce, T.F., Bridges, W.C., Klaine, S.J., 2015. 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Microplastics in invertebrates on soft shores in Hong Kong: Influence of habitat, taxa and feeding mode Xiaoyu Xu a, C.Y. Wong a, Nora F.Y. Tam a, Hoi-Shing Lo a, Siu-Gin Cheung a,b,⁎ a b
Department of Chemistry, City University of Hong Kong, Tat Chee Avenue, Hong Kong, China State Key Laboratory of Marine Pollution, City University of Hong Kong, Tat Chee Avenue, Hong Kong, China
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Suspected microplastics were found in 32 out of 38 species and 55% of all the organisms. • The three most predominant plastic polymer were cellophane, PET and PA. • Abundance of suspected microplastics was taxon related. • 62.82% of suspected MPs tested were found to be cellulose.
a r t i c l e
i n f o
Article history: Received 25 October 2019 Received in revised form 9 January 2020 Accepted 27 January 2020 Available online 28 January 2020 Editor: Thomas Kevin V Keywords: Microplastics Feeding mode Soft shore habitats Biological taxa
a b s t r a c t Microplastic (MP) pollution in the marine environment has gained much concern in recent years. This study investigated the occurrence of MPs in invertebrates collected on 18 mudflats and sandy beaches in Hong Kong and its relationships to biological taxon, feeding mode and habitat. In total 38 species of gastropods, bivalves and crabs were collected and the mean number of suspected microplastics ranged from 0 to 9.68 particles g−1 wet weight or 0 to 18.4 particles individual−1. Around 26% of the suspected microplastics were confirmed to be synthetic polymers, including CP (cellophane), PET (polyethylene terephthalate), and PA (polyamide). Microplastic fibres were the most abundant type of MPs, followed by pellets. Significantly higher abundance of suspected microplastics was found in gastropods. Since MP abundance might vary with taxon, it is recommended to include different taxonomic groups in any ecological assessment of the impact of MPs. © 2020 Elsevier B.V. All rights reserved.
1. Introduction Microplastics (MPs), which are commonly defined as plastic particles of diameter between 1 μm and 5 mm (Arthur et al., 2009), have become a global environment problem with a growing concern. Their distribution in the marine environment is ubiquitous, ranging from ⁎ Corresponding author at: Department of Chemistry, City University of Hong Kong, Tat Chee Avenue, Hong Kong, China. E-mail address: [email protected] (S.-G. Cheung).
https://doi.org/10.1016/j.scitotenv.2020.136999 0048-9697/© 2020 Elsevier B.V. All rights reserved.
sediments on shores, surface water and water column, to seabed sediments (Jiang, 2018). They are found even in the ice core from the remote Arctic Ocean (Obbard, 2018). The similarity in size between MPs and prey of various aquatic animals increases the bioavailability of MPs (Lusher et al., 2017) and ingestion of MPs has been reported for zooplanktons (Rist et al., 2017), bivalves (Murray and Cowie, 2011; Santana et al., 2016), crabs (Wójcik-Fudalewska et al., 2016), barnacles (Goldstein and Goodwin, 2013), gastropods (Gamage et al., 2017; Naji et al., 2018), fish (Caron et al., 2018; Nadal et al., 2016), and marine mammals (Lusher et al., 2015), although most of the studies were on
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fish in the seawater column. A large number of studies have been reported on MPs in shore sediments, in contrast, studies of their abundance in intertidal zone animals were scarce. The abundance of MPs in mussels and clams ranged from 0.04 particles g−1 body wt in Mytilus galloprocincialis (Vandermeersch et al., 2015) to 12.7 particles g−1 body wt in Amiantis purpuratus (Naji et al., 2018), or 0.6 particles individual−1 in Mytilus edulis (Phuong et al., 2018) to 23.5 particles individual−1 in Crassostrea virginca (Waite et al., 2018). Lower abundances were found in gastropods (0.17 particles g−1 body wt in Littorina sp. (Gamage et al., 2017) to 17.7 particles g−1 body wt in Thais mutabilis (Naji et al., 2018) and the Atlantic mud crab, Panopeus herbstii (15.1–25.8 particles per individual−1) (Waite et al., 2018). A few studies have determined the MP abundance in several species from one habitat, such as five species of mollusks with different feeding modes, including bivalves and gastropods from the northern part of Persian Gulf (Naji et al., 2018), with higher MP abundance being found in the predatory gastropod Thais mutabilis. In another study on three benthic macroinvertebrates in the Rockall Through, Northeast Atlantic Ocean, the MP abundance was related to species, but not feeding mode (CourteneJones et al., 2017). Since ingestion of MPs is species specific and may vary with feeding mode, a comparison of MPs ingested and accumulated in invertebrates from different taxa and with different feeding modes will provide useful information in selecting appropriate organisms as bioindicators of MP pollution and model organisms for studies on trophic transfer of MPs. Once ingested, MPs may transfer from the intestinal tract to body tissue (Collard et al., 2017; Farrell and Nelson, 2013) and elicit various health effects, such as obstruction of feeding organs (Cole et al., 2013), reduction of feeding capacity and energy intake (Xu et al., 2017), enhanced immune response (Lu et al., 2016; Von Moos et al., 2012), reduction in growth rate and reproductive ability (Sussarellu et al., 2015), and even neurotoxicity (Barboza et al., 2018). Even worse is that MPs can also be vectors of hydrophobic organic pollutants, such as PAHs, PCBs and OCPs (Lo et al., 2019) due to their large surface area to volume ratio and lipophilicity. With a population of 7.3 million people and a lack of an efficient plastic recycling industry, Hong Kong produces a huge quantity of plastic waste daily and in the year 2016, 2132 t day−1 of plastic waste were produced (HKEPD, 2017). This served as an important source of MP pollution in local marine waters. Pearl River, the third largest river in China with 60 million people living around the river delta, is considered as a major non-local source of plastic waste in Hong Kong, especially in the western waters of Hong Kong where the river delta is located (Fok and Cheung, 2015). Occurrence of MPs in seawater, as well as in sediments on beaches and the seabed around Hong Kong have been documented (Cheang et al., 2018; Cheung and Fok, 2016; Fok and Cheung, 2015; Tsang et al., 2017). The MPs concentration in Hong Kong coastal water ranged from 51 to 27,909 particles per 100 m3 with PP, HDPE, LDPE, and a mixture of PP, ethylene propylene (EPDM) and styrene acrylonitrile being most common (Tsang et al., 2017). In a study of 25 beaches along the coastline of Hong Kong, the mean MP abundance was found to be 5595 particles per m2 sediment, a value higher than the international average indicating that Hong Kong was a hotspot of MP pollution (Fok and Cheung, 2015). Another study has reported ten times more MPs on mudflats than sandy beaches with PE being most abundant and followed by PP and PET (Lo et al., 2018). The MP abundance in the seabed sediment along the northeastern and eastern shores of Hong Kong ranged between 169 and 221 particles kg−1 ww sediment, with a mean value of 189 particles kg−1 ww sediment (Cheang et al., 2018). This study determined the occurrence of MPs in three major groups of invertebrates, i.e., bivalves, gastropods and crabs, commonly found on sandy beaches and mudflats in Hong Kong. The number of species collected from each shore varied between 2 and 13 species, and in total 38 species were studied (Table S2, S3). The objectives were to understand 1) the differences in abundance and composition of MPs
between organisms from mudflats and sandy shores, and 2) whether MP abundance and composition varied among taxa or feeding modes. 2. Materials and methods 2.1. Study locations and sample collection Eighteen sites were visited with half of them located in the eastern waters and the other half in the western waters. Among the nine sites in each region, four of them were mudflats and the remaining sandy beaches (Fig. 1). The geographic coordinates of these sampling sites are listed in the supplementary information (Table S1). The shores in the eastern waters are facing South China Sea and located in country parks, hence the level of human disturbance is low. In contrast, the shores in the western waters are located near to the Pearl River estuary, hence receive a lot of marine debris through riverine input. All the sampling sites are non-gazetted beaches or mudflats with no frequent cleanup or maintenance. To minimize seasonal effects, the animals were collected during low tides in summer between May and September 2016. Bivalves, gastropods and crabs were collected with the help of stainless-steel shovels, and labour gloves and clothes made of cotton were worn to prevent potential contamination by MPs during the sampling. Animals were placed in labeled cotton bags to minimize contamination during transportation and storage, and transported back to the laboratory within 3 h. Upon arrival at the laboratory, they were stored at −20 °C immediately. The number of species collected from each site was determined not only by the species richness of the sites but also the conditions of the sites. For example, less species were collected from mudflats with very soft mud because of the difficulty in sampling. Information of the species collected from eastern sites is shown in Table S2 and those from western sites in Table S3. 2.2. Extraction of MPs from animals Feeding modes of most of the species were classified according to two references (Chan and Caley, 2003; Williams, 2003). For species which were not included in these references, including Terebralia sulcate (Barnes, 2003), Gaetice depressus (Depledge, 1989), Hemigrapsus penicillatus (Kurihara and Okamoto, 1987), Metopograpsus frontalis and Perisesarma bidens (Poon et al., 2010), their feeding modes were classified according to research reports. All the equipment and glassware were rinsed three times with de-ionized (DI) water before use. The animals were thawed, and shell length of bivalves, shell height of gastropods and carapace width of crabs were measured using Vernier calipers. For species occurring at only one to two sampling sites, the wet weight of each individual was measured directly. For species that occurred in a number of sites, the wet weight of all the individuals (15–30) at one of the sites was measured and a calibration curve relating body size (shell length, shell height or carapace width) and wet weight was computed and used for calculating the wet weight of the same species collected from the other sites. The chemical digestion of the organisms followed the protocol of Karami et al. (2017). Each individual was rinsed with DI water to remove particles attached on the body surface before it was placed in a separate 100 mL beaker. The shell/carapace of the animals was broken to facilitate digestion using 10% potassium hydroxide (KOH) at 40 °C for 48 h in an incubator (ET 618-4 Lovibond Thermostatschrank). Beakers were covered with aluminum foil during digestion to prevent atmospheric contamination. Since the resolution of the FTIR analysis we used in the experiment was around 30 μm, a sieve of 38 μm was used to collect suspected MPs extracted from the animals. To facilitate microscopic examination, particles retained by the sieves were transferred to a 0.45 μm mixed cellulose ester filter paper through vacuum filtration. Beakers and sieves were rinsed thoroughly with DI water to ensure that the suspected MPs attached on the surface were totally
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Fig. 1. 18 sampling sites around Hong Kong. The mudflats at Lai Chi Wo (LCW), Ho Chung (HC), Chek Keng (CK) and Wong Yi Chau (WYC), and sheltered sandy shores at Tai Tan (TT), Lai Chi Chong (LCC), Tai Tam bay (TTB), Tsam Chuk Wan (TCW) and Pak Sha Wan (PSW) are located in the eastern waters. The mudflats at Ha Pak Lai (HPL), Shan Tau (ST), Sham Wat (SW) and Tai O (TO), and sheltered sandy shores at Shui Hau Wan (SHW) Sha Lo Wan (SLW), Yi O (YO), Luk Keng Tsuen (LKT) and Tai Pai Tsui (TPT) are located in the western waters.
transferred. Each filter paper was stored in a clean petri dish for further analysis. 2.3. Qualification and quantification of MPs Visual examination of the filters was performed under a stereomicroscope (Leica M165C) at a magnification of 40× to 120×. Suspected MPs were classified according to their shape (fibre, pellet, fragment, foam and film) (Lo et al., 2018) and colours (white/transparent or other colours). After microscopic examination of all the particles, around 20% of the particles on each filter paper were selected randomly. If there were b5 particles on the filter paper, particles on several filter papers for the same animal species were pooled and then 20% of the particles were selected randomly. In total 280 out of 1284 suspected MPs were selected for chemical composition identification by micro Fourier Transform Infrared Microscope (μFTIR; Thermo Fisher Nicolet iN5, USA) in attenuated total reflection (ATR) mode. The FTIR spectrum of each suspected MP was obtained as an average of 32 scans in the range of 4000–650 cm−1 with a resolution of 4 cm−1. The spectrum was compared with the OMNIC standard spectra library of Hummel Polymer Sample Library and a self-generated library of natural cellulose obtained from pure cotton. A matching with a score N 70% was accepted. 2.4. QA/qc To assess possible contamination from the air or glassware during tissue digestion, a procedural control including all the digestion procedures but without animals was conducted once a week and in total 12 replicates were prepared. To estimate the potential loss of MPs after digestion with KOH, the recovery efficiency of MPs was evaluated in preliminary trails. Clams, gastropods and crabs (n = 5) were placed in beakers with a known number of bright pink polyamide (PA) fibres around 0.2 cm in length being cut from fabric. The spiked MPs were extracted using the methods mentioned above. The filter papers were examined under a microscope and the recovery efficiency was calculated in percentages.
2.5. Statistical analysis The data failed in the Shapiro-Wilk test for normality. Two separate nested generalized linear models (GLM) were used to investigate the effects of region (western and eastern waters), shore type (sandy shores and mudflats), feeding mode and taxon on the abundance of suspected MPs in the organisms. The effect of shore type was nested within region, and the effect of feeding mode or taxa was nested within shore type. According to the results of Pearson chi-square dispersion test, the data were over-dispersed. Therefore, GLM with negative binomial distribution was more appropriate for the analysis, owing to the residual overdispersion. The wet weight of body tissue was used as an offset variable so that the abundance of suspected MPs was mass-adjusted. The similarity in the chemical composition of suspected MPs between shore types (mudflats and sandy shores), and regions (west and east) were analyzed using non-metric multidimensional scaling (NMDS). The correlation between the abundance of suspected MPs in the organisms and sediments was analyzed using Pearson product-moment correlation. The amount of MPs in the sediments was obtained in the same study for the same sites and reported previously (Lo et al., 2018) and Pearson correlation analysis was conducted for the organisms with different feeding modes. The correlation between the wet weight of organisms and quantity of suspected MPs was analyzed using Pearson correlation for each single species. Kruskal-Wallis tests were used to compare the number of suspected MPs among different species. GLM and Pearson correlation were conducted by SPSS, and NMDS by Rstudio.
3. Results 3.1. Abundance of suspected microplastics in organisms The percentage of spiked MPs recovered from bivalve, gastropod and crab samples was 82%, 50% and 70% respectively (see Section 2.4). The extraction efficiency for gastropods was very low as the shell debris remaining on the filter paper disturbed visual observation. The abundance of suspected MPs was not corrected by the recovery efficiencies. In total
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312 individuals (174 gastropods, 68 bivalves and 71 crabs) were collected from 18 sampling sites and the abundance of suspected MPs was determined for each individual. The procedural blanks obtained 2 ± 1.04 fibres on average (n = 12) and the abundance of suspected MPs in each sample was corrected accordingly. After correction for the background contamination, suspected MPs were found in 32 out of 38 animal species and 55% of all the individuals. The mean number of suspected MPs ranged from 0.025 to 9.684 particles g−1 wet weight or from 0.08 to 18.4 particles individual−1. Most of them were fibres (93.3%) and the remaining ones pellets (6.7%). The mean length and diameter of the fibres was 1004.2 ± 464.8 μm and 21.8 ± 7.3 μm, respectively. More than half of the particles were white or transparent (65.1%), while the remaining ones were of other colours (34.9%). Pearson product-moment correlation analysis showed a lack of correlation between the abundance of suspected MPs in sediments and that in organisms with different feeding modes (deposit feeders/ grazers: r = −0.129, p = .079, n = 187; filter feeders: r = −0.232, p = .065, n = 64; predators: r = −0.114, p = .468, n = 43). In the nested GLM with region, shore type and feeding mode as predictive factors, the abundance of suspected MPs varied significantly with all of these factors. Organisms in the eastern waters ingested significantly more suspected MPs than those in the western waters, irrespective of the shore type. For both regions, the sandy shore organisms contained significantly more suspected MPs than the mudflat organisms. The effect of feeding mode varied with habitat (Fig. 2) with the abundance of suspected MPs in the deposit feeders/grazers being significantly higher than that in the filter feeders and predators in both eastern and western waters. For the sandy shores in the eastern waters, the effect of feeding mode was insignificant but the predators in the western waters had a significantly lower abundance of suspected MPs than those with other feeding modes. In the nested GLM with region, shore type and taxon as predictive factors, the organisms from the eastern waters contained significantly
more suspected MPs than those from the western waters. In contrast to the feeding mode, when taxon was used as a predictive factor the effect of shore type was statistically indistinguishable. The abundance of suspected MPs was the highest in the gastropods and followed by bivalves and crabs (Fig. 3) with the differences between the gastropods and crabs being statistically significant irrespective of the shore type and region. The abundance of suspected MPs varied among species in the same taxa (Fig. 4). For the gastropods, the highest abundance of suspected MPs was recorded in the deposit-feeding Batillatira multiformis (5.37 ± 1.24 particles g−1 ww) which was significantly higher than in Nerita chamaeleon (1.50 ± 0.20 particles g−1 ww). For bivalves, the abundance of MPs in Marcia spp. was significantly lower than in Anomalodiscus squamosus (3.48 ± 0.89 particles g−1 ww) and Donax spp. (2.89 ± 0.54 particles g−1 ww). The abundance of suspected MPs in Gafrarium spp. (0.26 ± 0.08 particles g−1 ww) was also significantly lower than in A. squamosus and Donax spp. For the crabs, Metopograpsus frontalis (0.21 ± 0.06 particles g−1 ww) had significantly less suspected MPs than Austruca lactea (2.84 ± 0.44 particles g−1 ww) and Macrophthalmus convexus (2.59 ± 0.73 particles g−1 ww). The abundance of suspected MPs in the organisms with different feeding modes were also compared within individual sampling sites (Fig. 5) where at least three species were collected, i.e., Lai Chi Chong, Pak Sha Wan, Shui Hau Wan, Sha Lo Wan, Luk Keng Tsuen and Tai Pai Tsui. At Lai Chi Chong (LCC), the grazer L. coronata and filter feeder A. squamosus had the highest abundance of suspected MPs, whereas at both Tai Pai Tsui (TPT) and Sha Lo Wan (SLW) the highest abundance of suspected MPs was found in a predatory gastropod R. clavigera. In some species, the particle abundance and wet weight of organisms was negatively correlated, including A. squamosus (r = −0.564, p b .05, n = 19), Batillaria zonalis (r = −0.593, p b .01, n = 20), and L. coronata (r = −0.407, p b .05, n = 31), but the correlation in Donax spp. was positive (r = 0.572, p b .05, n = 15).
Fig. 2. The bar charts showing the abundance of suspected MPs in animals with different feeding modes collected from a) mudflats in western waters, b) sandy shores in western waters, c) mudflats in eastern waters, and d) sandy shores in eastern waters. Significant differences according to GLM results ae shown. The number of replicates is shown inside each bar.
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Fig. 3. The bar charts showing the abundance of suspected MPs in animals from different taxa collected on the a) mudflats in western waters, b) sandy shores in western waters, c) mudflats in eastern waters, and d) sandy shores in eastern waters. Significant differences according to GLM are shown. The number of replicates is shown inside each bar.
3.2. Composition of MPs A sub-sample of 280 out of 1284 suspected MPs were analyzed by micro-FTIR. Seventy-four (24.6%) of them were synthetic polymers, 174 (62.82%) cellulose fibres, and the remaining 32 (11.55%) could not be identified (Fig. 6). The three most abundant polymers were cellophane (CP, 43.66%), polyethylene terephthalate (PET, 19.72%) and polyamide (PA, 16.9%). All the pellets were either PP or PE, whereas fibres
belonged to six different kinds of polymer. The length of fibres varied among different types of polymer with CP being significantly longer than PET and PA (Kruskal-Wallis test, n = 5, p b .005). The diameter was also significantly different among different types of fibre (Kruskal-Wallis test, n = 5, p b .05) but no significant difference was found in pair-wise comparisons. The NMDS plots based on the chemical composition of MPs separated mudflats from sandy shores with mudflats characterized by PAN
Fig. 4. The bar charts showing the abundance of suspected MPs in different species of (a) gastropods, (b) bivalves and (c) crabs. The abundance of suspected MPs in each species was calculated from data collected from all the sampling sites. Significant differences according to the Kruskal-Wallis test are represented by different letters. The number of replicates is shown next to each bar. Species with a sample size of b5 were not included in the K-W test.
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Fig. 5. The bar charts showing the abundance of suspected MPs in species collected from 6 representative sampling sites. According to the results of the Kruskal-Wallis test, significant differences among the species from the same sampling sites are represented by different letters. The sample size is shown next to each bar. Species with a sample size of b5 were not included in the K-W test.
and PET and sandy shores by CP and PA (Fig. 7a), except for the mudflat at CK and sandy shore at PSW. However, no regional difference was observed (Fig. 7b). The NMDS plots based on the chemical composition of MPs showed no apparent clustering of the taxa or feeding modes, indicating no polymer preference of each taxonomic group or feeding mode (Fig. 8). 4. Discussion 4.1. Characteristics of the MPs Most of the MPs found in this study were fibres made up of cellophane, polyethylene terephthalate, polypropylene, polyamide and polyethylene. Synthetic fibres occupy N60% of the world fibre consumption (FAO and ICAC, 2013), and their characteristic properties make them suitable materials for sportswear, medical, agricultural and protective systems (e.g., breathing apparatus, cold weather masks, helmets) (ElMogahzy, 2008). Aquaculture and fishing activities are sources of MP fibres in the marine environment as nearly all of the global fishery equipment are made of plastics and a large amount of them is either lost or discarded in the ocean (Andrady, 2011). Laundry also generates a large number of MP fibres from clothing. Studies have shown that N1900 MP fibres were generated in a single washing. Although most of them are trapped in the sewage treatment plant, a small proportion
Fig. 6. MPs of different chemical compositions: CP (cellophane, 43.66%), PET (polyethylene terephthalate, 19.72%), PA (polyamide, 16.9%), PP (polypropylene, 8.45%), PE (polyethylene, 7.04%) and PAN (polyacrylonitrile, 4.23%).
is discharged into the ocean (Browne et al., 2011; Salvador Cesa et al., 2017).In our concurrent study, the microfibre was the most dominant form of MPs in sediments from shores around Hong Kong, accounting for 57.2% of all the MPs collected. Sewage treatment plants were suspected to be a major source of these microfibres because much higher concentrations of them were found at the study sites near the outflow or sewage treatment plants (Lo et al., 2018). In the present study, however, there was no correlation between the MP abundance in the organisms and distance to the nearest sewage treatment plants. The inconsistency in the MP abundance between the shore sediments and intertidal organisms was also found and will be explained later. The three predominant polymers found inside the organisms were CP (43.66%), PET (19.71%) and PA (16.9%), but PE (46.9%), PP (13.8%) and PET (13.5%) in the shore sediments of our concurrent study (Lo et al., 2018). The discrepancy may be due to differences in the forms of MPs analyzed that FTIR instead of μ-FTIR was used in analyzing MPs in sediments with a lowest size limit of 250 μm. Therefore, most of the fibres were excluded from the analysis. In contrast, the use of μ-FTIR in the present study allowed MPs as small as 30 μm, including fibres to be analyzed. Another possibility was feeding selectivity in the organisms. When an amphipod Hyalella azteca was exposed to the same concentration of either PE fragments or PP fibres, PP fibres were preferentially ingested (Au et al., 2015). The study, however, could not identify what the amphipod was selecting for as PE fragments and PP fibres differ not only in shape but also chemical composition, colour and size. CP was the most common polymer type in this study. According to GESAMP (Kershaw and Rochman, 2015), it is defined as bio-derived plastics, where the plastic materials are regenerated from polymers extracted from biomass (Kershaw and Rochman, 2015). CP was also found in many other studies on both sediments and organisms (Alomar and Deudero, 2017; Fang et al., 2018; Gago et al., 2018). In the study of MP contamination in benthic organisms from the Arctic and sub-Arctic region, CP was the fourth common polymer accounting for 13% of all the MPs (Fang et al., 2018) and it was the most common MP (33.33%) ingested by the shark Geleus melastomus in another study (Alomar and Deudero, 2017). Cellophane is used as wrappers in cigarette and food, and as fibreglass products and release agent for the manufacture of rubber (Yang et al., 2015). As most of the suspected MPs in this study were fibres and among them CP fibres were the longest. The longer the fibre, the longer is its retention time in living organisms as demonstrated in mussels in which a lower proportion of the longer fibres was egested in b3 h (Ward et al., 2019). Regardless of the common
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Fig. 7. Non-metric multidimensional scaling (NMDS) ordination of MPs. (a) Separation by chemical composition in relation to shore type (mudflats and sandy shores), and (b) separation by chemical composition in relation to region (west and east). The polymer types are shown in red. The abbreviations of the sampling sites refer to Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
occurrence of CP in the marine environment, the research on its toxicity on living organisms is rare when compared with that on other MPs such as PP and PE (Korez et al., 2018). Synthetic fibres such as rayon are different from cellophane structurally but not chemically. Our FTIR, however, could not differentiate rayon from cellophane, and rayon is commonly moulded into fibres. Therefore, we cannot exclude the possibility of the presence of rayon in our samples. Although criteria such as no visible cellular or organic structure, equally thick or clear and homogeneous colours have been proposed for differentiating synthetic microfibres from natural microfibres using microscopic examination (Hidalgo-Ruz et al., 2012), this is not a very reliable method as it is relatively subjective and relies heavily on the experience of the observer. More importantly, chemical characterization was not employed in a number of studies (Devriese et al., 2015; Waite et al., 2018; Wang et al., 2019). The present study has illustrated this problem as 62% of the suspected microfibres were in fact natural cellulose as confirmed by μFTIR spectrometry. In another study of MPs ingestion by deep-sea invertebrates using protease enzymatic digestion, 165 out of 359 (around 46%) potential MPs were identified as cellulose (CourteneJones et al., 2017). In a study of MPs in mussels, although hydrogen peroxide was used for digestion of organic matter and MPs were extracted
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Fig. 8. Non-metric multidimensional scaling (NMDS) ordination of MPs. (a) Separation by chemical composition in relation to taxa, and (b) separation by chemical composition in relation to feeding modes. The polymer types are shown in red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
using saline (NaCl) solution, natural cellulose fibres were still present in the final samples (Li et al., 2018). Therefore, a more cautious approach should be adopted in the future including the development of more effective digestion methods and more importantly the chemical characterization using μ-FTIR, Raman spectroscopy or other analytical methods. According to the results of NMDS (Fig. 7a), mudflats were separated from sandy beaches and characterized by PAN (polyacrylonitrile) and PET. PAN fibres are usually used in hot gas filtration systems, outdoor awnings, sails, fibre-reinforced concrete (Nithya et al., 2017) and knitted clothing, such as sweaters and socks (Choe et al., 2006). N60% of the global production of PET is used as synthetic fibres, others include films, bottles and other moulded products (Jankauskait et al., 2008). A high abundance of PET and PAN near Shan Tau (ST) and Ho Chung (HC) may be due to the densely-populated residential area near these two sampling sites and land-based contamination of MPs through laundry. 4.2. Influence of feeding mode and taxon on the abundance of suspected MPs As shown in Figs. 2 and 3, the abundance of suspected MPs was related to both feeding mode and taxon with deposit feeders/grazers
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and gastropods being more vulnerable to MP ingestion. Contradictory results, however, are shown in Fig. 5 when results of different animal species at the same sites were compared. A significantly higher number of suspected MPs was found in the predatory gastropod R. clavigera at Tai Pai Tsui and Sha Lo Wan, and in both the deposit feeding gastropod L. coronata and filter feeder bivalve A. squamosus at Lai Chi Chong. The apparent relationship between feeding mode and microplastics abundance was probably due to sampling bias as filter feeders were absent on some of the mudflats where MPs were more abundant (Lo et al., 2018). This would underestimate the abundance of suspected MPs in filter feeding bivalves, resulting in higher concentrations of suspected MPs in deposit feeders/grazers. The effect of feeding mode on MP abundance in animals is inconsistent in the literature. For example, Bour et al. (2018b) showed that a lower frequency of MP ingestion was observed for filter-feeders compared to deposit-feeders and predators, and in another study a significantly lower number of particles per individual was observed for filter-feeders compared to predators (Bour et al., 2018a). Other studies, however, have documented that filter feeders are most prone to ingesting MPs (Scherer et al., 2017; Setälä et al., 2015). In the present study, irrespective of the feeding mode, higher abundance of suspected MPs was found in gastropods and probably related to the morphology of the digestive system which follows the spiral shape of the shell (Lőw et al., 2016), and torsion of the body ends up with a more convoluted digestive tract (Page, 2003). This complicated gut morphology may increase the retention time of MPs and facilitates its accumulation, regardless of the feeding mode of the gastropods. When comparing compositions of MPs from different taxa or feeding modes, the results of NMDS (Fig. 8) did not reveal any pattern, indicating that feeding selectivity may be species specific and depend on the shape of the particles rather than chemical composition. Correlation between the abundance of suspected MPs and wet weight of organisms was observed in some species. The results, however, were inconsistent among species with negative correlations in gastropods B. zonalis and L. coronata and in a bivalve A. squamosus, but a positive correlation for another bivalve, Donax sp. This indicates that MP ingestion and retention is species specific and independent of the feeding mode. 4.3. Influence of region and shore type on the abundance of suspected MPs A higher abundance of suspected MPs was found in the organisms from both the eastern waters and sandy shores. The results were contradictory to those obtained in our concurrent study on MPs in sediments in which higher concentrations of MPs were obtained on the mudflats as well as in the western waters. Several reasons can explain the discrepancies. Firstly, the species composition on the mudflats and sandy beaches was different with the number of gastropod species collected from the sandy shores doubled that from the mudflats. As shown in the above, the gastropods contained more suspected MPs, probably due to their modes of feeding and complicated digestive tracts, hence resulted in a higher abundance of suspected MPs in the organisms on the sandy beaches. Secondly, the abundance of suspected MPs varied among species and those with a higher abundance occurring mostly in the eastern waters, e.g., a clam A. squamosus was found at 3 sites in the eastern waters but 1 site in the western waters, and a gastropod B. multiformis was found in 2 sites in the eastern waters but not found in the western waters. As the overall abundance of suspected MPs in organisms from a habitat is influenced by the species composition, species of different taxa should be included when inter-site comparisons of the ecological impact of MPs is made.
freshwater fish Orechromis niloticus (Ding et al., 2018). The present study, however, did not find any such correlation between the sediments and organisms. Similar results were obtained in other studies. In Northeastern Pacific Ocean, no systematic relationship was found between the prevalence of MPs in the fish stomach and the waters around the study colonies (Hipfner et al., 2018). Even though a higher MP abundance in the habitats increases the opportunity of the organisms in encountering MPs, avoidance of MP ingestion may occur as shown in the collembolans (Zhu et al., 2018). Filter feeders such as bivalves can avoid ingesting MPs through the production of pseudofeces. In the blue mussel Mytilus edulis, 71% of the MP fibres were rejected as pseudofeces and only 9% was ingested (Woods et al., 2018). Depuration through faeces production was also effective in removing MPs in the Mediterranean mussel Mytilus galloprovincialis in which around 85% of the MPs were eliminated after 6 days of depuration (Fernández and Albentosa, 2019). The lack of correlation between abundance of MPs in sediments and in animals could also be due to different size fractions of particles analyzed as only MPs between 5 mm and 250 μm were studied in Lo et al. (2018) on sediments but between 5 mm and 30 μm in the present study. 4.5. Limitation and future studies Since this study involved collecting a large number of animals from a number of beaches and mudflats, among which some of them did not have many animals while others were too muddy for sample collection, only a small number of individuals were examined for each species. Moreover, very muddy sampling sites do not favor the survival of filter feeding bivalves, hence only gastropods were collected from several sampling sites including Tai O, where the sediment contained the highest abundance of suspected MPs compared to all the other sites (Lo et al., 2018). This inevitably introduced biases in the comparisons of the results among different feeding modes or taxonomic groups as well as the representativeness of the data. The tremendous amount of work also limited the percentage of suspected MPs (20%) analyzed by μ-FTIR. A lower recovery efficiency of MPs from the gastropods than from the bivalves and crabs would underestimate the differences in the abundance of suspected MPs between these taxonomic groups but the conclusion of the study was not affected as even though the data of the abundance of suspected MPs were not corrected for the differences in the recovery efficiency, the gastropods still had the highest MP abundance. 5. Conclusion Suspected MPs were found in 32 out of 38 animal species in the present study with more being found in gastropods. This indicated that the ingestion of MPs was common in intertidal animals and taxon related. The three most predominant types of plastic polymer were cellophane, PET and PA. However, a very high percentage of fibres (62.82%) were found to be cellulose. This highlights the significance of chemical analysis of the suspected MPs using μ–FTIR or other analytical method such as Raman Spectroscopy. Since MP abundance might vary with taxon, it is recommended to include different taxonomic groups in any ecological assessment of the impact of MPs. 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.
4.4. Correlation with the MP abundance in shore sediments Acknowledgement It is intuitive that the higher the concentration of MPs in the sediment, the higher is that in the organisms. Such a concentrationdependent effect was observed in the accumulation of PS-MPs in a
The work described in this paper was supported by the Research Grants Council of Hong Kong SAR (CityU 11302815). We are grateful
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to Ng Wing Yi, Fung Chun Kit Edmond and So Lai Hong for their help in sample collections. Supplementary data Brief descriptions in nonsentence format listing the contents of the files supplied as Supporting Information. Supplementary data to this article can be found online at https://doi.org/10.1016/j.scitotenv.2020. 136999. References Alomar, C., Deudero, S., 2017. Evidence of microplastic ingestion in the shark Galeus melastomus Rafinesque, 1810 in the continental shelf off the western Mediterranean Sea. Environ. Pollut. 223, 223–229. https://doi.org/10.1016/J.ENVPOL.2017.01.015. Andrady, A.L., 2011. Microplastics in the marine environment. Mar. Pollut. Bull. https:// doi.org/10.1016/j.marpolbul.2011.05.030. Arthur, C., Baker, J., Bamford, H., 2009. Proceedings of the International Research Workshop on the Occurrence, Effects, and Fate of Microplastic Marine Debris. NOAA. Au, S.Y., Bruce, T.F., Bridges, W.C., Klaine, S.J., 2015. 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