What are the flaws of this article?

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I posted an article about microplastic plastic pollution. I need help figuring out the flaws in this study such as what they should have done or what assumptions they overlooked or how could this study be improved.

Just simply read it and I need to know what you understand in the results and just critical thinking analysis from the methods or results section or anything that you find interesting about the article using your basic knowledge of things.

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Environmental Pollution 234 (2018) 347e355 Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/locate/envpol Using the Asian clam as an indicator of microplastic pollution in freshwater ecosystems* Lei Su a, Huiwen Cai a, Prabhu Kolandhasamy a, Chenxi Wu b, Chelsea M. Rochman c, Huahong Shi a, * a b c State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai 200062, China State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Ontario, Canada a r t i c l e i n f o a b s t r a c t Article history: Received 7 August 2017 Received in revised form 22 November 2017 Accepted 23 November 2017 Available online 28 November 2017 Bioindicators play an important role in understanding pollution levels, bioavailability and the ecological risks of contaminants. Several bioindicators have been suggested for understanding microplastic in the marine environment. A bioindicator for microplastics in the freshwater environment does not exist. In our previous studies, we found a high frequency of microplastic pollution in the Asian clam (Corbicula fluminea) in Taihu Lake, China. In the present study, we conducted a large-scale survey of microplastic pollution in Asian clams, water and sediment from 21 sites in the Middle-Lower Yangtze River Basin from August to October of 2016. The Asian clam was available in all sites, which included diverse freshwater systems such as lakes, rivers and estuaries. Microplastics were found at concentrations ranging from 0.34.9 items/g (or 0.4e5.0 items/individual) in clams, 0.5e3.1 items/L in water and 15e160 items/kg in sediment. Microfibers were the most dominant types of microplastics found, accounting for 60e100% in clams across all sampling sites. The size of microplastics ranged from 0.021-4.83 mm, and microplastics in the range of 0.25e1 mm were dominant. The abundance, size distribution and color patterns of microplastics in clams more closely resembled those in sediment than in water. Because microplastic pollution in the Asian clam reflected the variability of microplastic pollution in the freshwater environments, we demonstrated the Asian clam as an bioindicator of microplastic pollution in freshwater systems, particularly for sediments. © 2017 Elsevier Ltd. All rights reserved. Keywords: Microplastic Freshwater Asian clam Bioindicator Sediment 1. Introduction Plastic pollution in the oceans has been an issue of concern since the first report on the subject appeared in the 1970s (Carpenter and Smith, 1972). In recent years, the focus has shifted to small-sized plastic pollutants, called microplastics (plastic items < 5 mm). Global investigations on microplastics have been conducted in a diversity of marine habitats (Cole et al., 2011; Thompson et al., 2004). The occurrence of microplastic pollution has been confirmed in organisms (Gall and Thompson., 2015), water (Van Sebille, 2014) and sediments (Browne et al., 2011) globally. The interactions of microplastics throughout the marine ecosystem have become one of the primary concerns associated * This paper has been recommended for acceptance by Maria Cristina Fossi. * Corresponding author. E-mail address: hhshi@des.ecnu.edu.cn (H. Shi). https://doi.org/10.1016/j.envpol.2017.11.075 0269-7491/© 2017 Elsevier Ltd. All rights reserved. with microplastic pollution (Galloway et al., 2017; Wang et al., 2016). Using field studies, the uptake and ingestion of microplastics has been demonstrated in a wide diversity of marine organisms, including plankton, fish, and mammals (Desforges et al., 2015; Fossi et al., 2014; Wesch et al., 2016). The transfer of microplastics from one trophic level to another has been demonstrated €l a € et al., 2014; Van Franeker et al., 2011). in the laboratory (Seta Animals represent an important transport mechanisms for microplastics in the environment (Clark et al., 2016; Hu et al., 2016). In the oceans, marine vertebrate animals, including fish, seabirds, fin whales and turtles have been suggested as good bioindicator for marine plastic debris due to their life-history strategies (Fossi et al., 2014; Jabeen et al., 2017; Mascarenhas et al., 2004; Provencher et al., 2015). These bioindicators can provide information about microplastic pollution concentrations in their habitats (Wesch et al., 2016). 348 L. Su et al. / Environmental Pollution 234 (2018) 347e355 Among invertebrates, bivalves are valuable sentinel organisms for indicating levels of different pollutants in the environment (Boening, 1999). They have the ability to concentrate and accumulate pollutants substantially above background environmental levels. Filter feeder organisms act as a trap, accumulating pollutants because of their low excretion rates (Jara-Marini et al., 2013). Such advantages allow the use of bivalves as a tool to biomonitor organic contaminants and metals (Koch et al., 2007). The uptake of microplastics in marine bivalves (e.g. blue mussel) has been well documented (Li et al., 2015, 2016; Van Cauwenberghe and Janssen, 2014). As such, mussels have been proposed as a bioindicator of microplastics. Bivalves make a good bioindicator because of their ability to ingest microplastics, but also because of their relevance to the issue of seafood safety (Rochman et al., 2015). Because humans consume bivalves whole, they are a direct route of exposure via a seafood diet. Although current research cannot provide an accurate dose of microplastics that will pose direct harm to human health, concerns related to microplastic-associated risk to humans is increasing (Seltenrich, 2015). Microplastics may accumulate and cause a potential health risk once they are ingested (Wright and Kelly, 2017). In addition to risk from the physical particle, the chemicals bound to microplastics may be transferred to humans (Browne et al., 2013). Because the level of health risk from microplastics remains unclear, more efforts to address the interaction between microplastics and biota are critical. Measuring the pollutants inside bivalves is a direct way to assess internal exposure levels and to begin to link the bioavailability to effects (Escher and Hermens, 2004). More recently, researchers have begun to investigate microplastic pollution in freshwater and terrestrial ecosystems which are recognized as a major source and transport pathways of plastics to the ocean (Eerkes-Medrano et al., 2015; Horton et al., 2017; Rillig, 2012). Today, the study of microplastics in freshwater systems remain at an early stage in comparison with the indepth studies that have been conducted in the marine environment. Microplastic contamination in freshwater and terrestrial environments deserves further investigation and should be considered as a separate issue rather than as supplementary to marine microplastic research. In our previous study, we found microplastic pollution in a freshwater bivalve, the Asian clam (Corbicula fluminea), in all of our sampling sites in Taihu Lake, China (Su et al., 2016). Populations of Asian clams are widely distributed across China and globally. They are also abundant across a diversity of freshwater systems. For the same reasons as stated above for marine bivalves, Asian clams are successfully used to monitor various contaminants (e.g., nanoparticles) and to study toxicological effects of microplastics in the laboratory (Cid et al., 2015; Sousa et al., 2008; Rochman et al., 2017). A high level of contamination including nitrogen, heavy metals and emerging organic pollutants, have been reported in many parts of the Yangtze River (Chen et al., 2000; Dai et al., 2011; Floehr et al., 2013). An increase in the concentrations of these pollutants has also been reported over decades (Michishita et al., 2012). This area has been polluted for a long period of time. Recently, there have been several studies demonstrating microplastic pollution in fish, water and sediment from the Middle-Lower Yangtze River Basin (Zhang et al., 2017; Zhao et al., 2015). Here, we carried out a large-scale investigation of microplastics in the Middle-Lower Yangtze River Basin sampling Asian clams, water and sediments. The relation of microplastic in the Asian clam to those in water and sediment was also analyzed. Based on our results, we propose that the Asian clam can be used as a bioindicator of microplastic pollution in freshwater systems. 2. Materials and methods 2.1. Survey sites and areas Our field survey was conducted in the Middle-Lower Yangtze River Basin from August to October 2016 (Fig. 1). Lakes, rivers and estuarine areas in the Yangtze catchments were selected as study areas (S1-S21). The sampling areas and individual sampling sites were located in urban as well as rural areas, which are impacted by different sources of pollutants. The sources of these pollutants include agriculture, river traffic, industry and tourism. Detailed information on the sampling area is provided in Supplementary Materials Table 1. During sampling, large plastic debris were commonly observed. In addition, Asian clams were successfully acquired in all of the sampling sites. 2.2. Sample collection Water samples were collected prior to sediments and Asian clams to avoid collecting suspended solids from the bottom of sampling sites. We collected approximately 5 L of water by dipping a steel bucket from a boat. Water was collected from 0-12 cm below the surface, based on the diameter of the bucket. Three samples were collected at each site (n ¼ 3). Three samples of sediment were collected at each site (n ¼ 3) with a Peterson sampler from the boat (Hosseini Alhashemi et al., 2012). The top 10 cm of sediment was collected. Each replicate contained approximately 2 kg of wet sediment. Three samples of Asian clams were collected at each site using bottom fauna trawls from the boat (n ¼ 3). Each replicate consisted of at least 10 living clams of similar sizes. Sediment and water samples were sealed and kept at 4  C, and the clam samples were kept at 20  C until further analysis. 2.3. Quality control of experiments All the containers (glass bottle, aluminum pot and aluminum foil bag) and sampling tools were washed using tap water, which was filtered prior to use (pore size of filter was 0.45 mm). The tools were sealed in an aluminum foil bag and kept clean before using. During the sampling procedure, the tools were prewashed using water in situ to avoid contamination. In the laboratory, blanks were run (51 blank samples in total) without water, sediment or clam tissue and were performed simultaneously to correct and evaluate background contamination. Procedural contamination ranged from 0.19 to 0.62 items per treatment group (0e3 particles per sample) for water, clam and sediment samples. All the microplastics in blank samples were microfibers. The background contamination was equal to 4.9e6.9% of the abundance of microplastics in all of the samples. The background contamination was not subtracted from the final results in the current study, but should be taken into consideration for interpretation. 2.4. Isolation of microplastics A two-step filtration process was used to extract microplastics from the water and sediment samples (Su et al., 2016). Briefly, the volume of water was first recorded and particles in the water were filtered onto nylon net filter using a vacuum system. The pore size of filter was 20 mm (Millipore Nylon NY2004700). Any particles on the filter were washed into a glass flask using 100 mL of hydrogen peroxide (30%, V/V) to digest the organic substances. The flasks were covered and placed in an oscillation incubator at 65  C and 80 rpm for no more than 72 h. The liquid in the flask was filtered again, and the filter was covered and stored in dry Petri dishes for further observation. L. Su et al. / Environmental Pollution 234 (2018) 347e355 349 Fig. 1. Locations of sampling areas and sites. The wet sediment was pooled in an aluminum pot with a cap and dried in an oven at 65  C. Approximately 300 g of dry sediment was weighed and mixed with saturated sodium chloride solution (1.2 g/mL) at a ratio of 1:2 (V/V) in a 2-L glass container with a 30cm depth. It should be noted that the use of saturated sodium chloride solution might lead to the loss of some material with densities greater than 1.2 g/mL. The mixture was stirred and settled for 24 h. The supernatant was then transferred onto nylon filters with a 20-mm pore size. All particles on the filter were washed into a glass flask with 100 mL of hydrogen peroxide for digestion. The digestion and filtration processes were performed using the same method for the water samples. The isolation of microplastics from Asian clams was based on our previous study (Li et al., 2015; Su et al., 2016). Briefly, we recorded the clam's total weight, soft tissues weight and shell length (Supplementary materials Table 1). In the present study, the abundance of microplastics in Asian clam was based on the weight of soft tissue. For each sampling site, three replicates of 2e4 clams were extracted and analyzed. The clams were transferred to a flask and 200 mL of hydrogen peroxide was added to digest them. The flask was then covered and placed in an oscillation incubator at 65  C and 80 rpm for no more than 72 h. After digestion, the liquid in the flask was filtered, and the filter was covered and stored in dry Petri dishes for further observation. 2.5. Observation and validation of microplastic All suspected plastic particles on the filters were observed and photographed using a microscope (MicroImaging GmbH, Goottingen, Germany) with 25e80x magnification. We used visual assessments to quantify and sort the suspected microplastics based on their properties (Yang et al., 2015). Visually identified microplastics from water, sediment and clam samples were classified into four groups: fiber, pellet, film and fragment (Su et al., 2016). Fibers were rod-like and flexible strips; pellets were items with a spherical shape; films were very thin and small layers; fragments were incomplete or isolated parts of large plastic debris. The color and size of the microplastics were also measured and recorded in visual assessments. From the 1303 particles, 150 particles were selected for validation using micro-Fourier Transform Infrared Spectroscopy (m-FT-IR). To verify the accuracy of our visual identification of microplastics, we randomly selected 1e2 particles from the central area of each filter. The polymer composition was measured under the attenuated total reflection mode of an m-FT-IR (Bruker, LUMOS). All data were collected at a resolution of 4 cm1 with a 32-s scan time. All spectra were compared with a database from Bruker to verify the polymer type (Güven et al., 2017). The spectra matching with a quality index 70 were accepted. Finally, the number of microplastics reported was recalculated by excluding the verified non-plastic items. 350 L. Su et al. / Environmental Pollution 234 (2018) 347e355 2.6. Data analysis A one-way analysis of variance (ANOVA) was used to determine the differences in the quantities of microplastics among individual sampling sites and the distribution of microplastics shape, size and color. To test for multiple comparisons, post-hoc Tukey's HSD test (homogeneous variances) and the Tamhane-Dunnett test were applied (heterogeneous variances). A 0.05 and 0.01 significance level was chosen. A linear regression analysis was used to test whether there was a significant correlation among the abundance of microplastic in clams, water and sediment. The Pearson correlation coefficient determined the goodness of fit and significance of the correlation. The size distribution of microplastics in different fractions was plotted using a heat map and a cumulative curve to compare different fractions. In the heat map, the depth of gray in a size interval represented the percentage of microplastic in that size interval. The process of degradation in the environment might result in a loss of color. Hence, the transparent and white items in samples were marked as “colorless”, and items of other colors were marked as “colored”. The digestion of hydrogen peroxide could also have resulted in the bleaching of microplastics and a subsequent overestimation of the number of colorless particles. However, our current digestion process was not strong enough to lead to a complete bleaching of microplastics, and only parts of individual items were discolored (Li et al., 2016). To avoid an overestimation of colorless items, only whole white and transparent items were considered to be “colorless”. Principal component analysis was then used to analyze the variance of data and identify the independent principal components (PC). The PC bi-plots were created to describe similar or dissimilar patterns of variance for the colorless and colored group from different samples. The data analysis in the current study was processed using SPSS 22.0 and GraphPad Prism 5.0. 3. Results Of the 150 randomly selected items, 122 items were confirmed as plastics using m-FT-IR. As such, the success rate of our visual identification was 81%. We identified fourteen polymer types (Supplementary materials Table 2). The dominant polymer was polyester (33%), followed by polypropylene (19%) and polyethylene (9%). The selected particles represented the most common types of visually identified particles. 3.1. Microplastic pollution in Asian clams The average abundance of microplastic in clams from each site ranged from 0.3-4.9 items/g and 0.4e5.0 items/individual (Fig. 2). The abundance differed significantly among 21 sampling sites (p < 0.01). The lowest abundance of microplastics was found in Poyanghu Lake (S9) by items/g and in Chaohu Lake (S8) by items/ individual. The highest abundance of microplastics in clams was in S3 by items/g and in S5 in Gaoyouhu Lake by items/individual. For the clams, fibers were most dominant (p < 0.01), accounting for 60e100% of particles across all sampling sites (Fig. 3A). The size of microplastics in clam samples ranged from 0.021-4.02 mm, with the 0.25e1 mm size was dominant (p < 0.01) (Fig. 3B). Blue and transparent items made up more than 30% of all particles and were significantly more abundant than other colors (p < 0.05) (Fig. 3C). 3.2. Microplastic pollution in water and sediment The average abundance of microplastics in samples across all sites ranged from 0.5-3.1 items/L in water and 15e160 items/kg in sediment (Fig. 4). The microplastic abundance differed significantly Fig. 2. The abundance of microplastics in the Asian clam by the terms of items/g (A) and items/individual (B). among the 21 sampling sites (p < 0.01). The lowest abundance of microplastics was found in S17 in water and in S15 in sediment in Dianshanhu Lake, and the highest was found in South Branch of the Yangtze Estuary (S13) in water and Gaoyouhu Lake (S3) in sediment. The size of microplastics ranged from 0.022-4.83 mm in water and sediment samples. Fibers were dominant among all types (p < 0.01), and particles that were 0.25e1 mm in size dominated most water and sediment samples (p < 0.01) (Supplementary materials S1 and 2). Again, the transparent and blue items were most commonly found, and the transparent items dominated in all color classes in water and sediment (p < 0.01) (Supplementary materials S1 and 2). 3.3. The relationship of microplastics in clams, water and sediments Based on the regression analysis, the abundance of microplastics in clams significantly depended on the microplastic pollution in water (p < 0.05) and sediment (p < 0.01) (Fig. 5A). According to the heat map and the cumulative curve of microplastic size di ...
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Article Analysis
The report is destined to locate and identify the presence of environmental pollution in
the water bodies through the Asian clam to prove the microplastic pollution. When conducting a
research, there are specific objectives that are intended. The objective of the research was to
prove the existence of mi...

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