: Sampling, Isolating and Identifying Microplastics Ingested by Fish and Invertebrates
A. L. Lusher, N. A. Welden, P. Sobral and M. Cole
- 8.1 Introduction 120
- 8.2 Methodological Review 121
- 8.2.1 Sampling 122
- 22.214.171.124 Field-Collected Organisms 122
- 126.96.36.199 Laboratory-Exposed Organisms 123
- 8.2.2 Isolating Microplastics 123
- 188.8.131.52 Dissection 124
- 184.108.40.206 Depuration 124
- 220.127.116.11 Digestion 124
- 18.104.22.168 Density Separation 129
- 8.2.3 Microplastic Identification 129
- 22.214.171.124 Visual Identification 130
- 126.96.36.199 Polymer Verification 131
- 8.2.4 Contamination 132
- 188.8.131.52 Contamination during Field Sampling 132
- 184.108.40.206 Contamination during Sample Processing and Analysis 132
- 8.2.5 Data Analysis 133
- 8.3 Discussion 133
- 8.3.1 Controlling Sampling Bias 134
- 8.3.2 Effective Plastic Isolation 135
- 8.3.3 Polymer Verification 135
- 8.3.4 Mitigating Contamination 136
- 8.3.5 Data Analysis 136
- 8.3.6 Recommendations for Future Work 136
- 8.4 Concluding Remarks 138
References 138 
Article is licensed under a Creative Commons Attribution 3.0 Unported Licence.
Over the past century, there has been an exponential increase in plastic demand and production . Concurrently, improper disposal, accidental loss and fragmentation of plastic materials have led to an increase in tiny plastic particles and fibers (microplastic, <5 mm) polluting the environment [2,3]. Microplastics have been observed in marine , freshwater [5,6] and terrestrial  ecosystems across the globe, and biotic interactions are widely evidenced (Figure 8.1). Microplastics can be consumed by a diverse array of marine organisms across trophic levels, including protists , zooplankton [9-17] annelids [18-26], echinoderms [27-31], cnidarian , amphipods [19,26,33], decapods [34-41], isopods , bivalves [43-60], cephalopods , barnacles , fish [58,66-94] turtles , birds  and cetaceans [97,98]. Over 220 different species have been found to consume microplastic debris in natura. Of these, ingestion is reported in over 80% of the sampled populations of some invertebrate species [34,38,41]. Interactions between microplastics and freshwater invertebrates, fish and birds are increasingly reported [99-107], although some researchers are focusing on model species such as Dapbnia magna [108-111]. The consumption of microplastics by terrestrial organisms is poorly documented; however, laboratory studies indicate earthworms (Lumbricus terrestris) can consume plastic particles present in soil .
There are a number of exposure pathways by which organisms may interact with microplastic debris. Direct consumption of microplastic is prevalent in suspension feeders, including zooplankton , oysters  and mussels [43-58,60-63], and deposit feeders, such as sea cucumbers , crabs [35-37,39,40] and Nephrops, [34,41] owing to their inability to differentiate between microplastics and prey. Predators and detritivores may indirectly ingest plastic while consuming prey (i.e., trophic transfer) or scavenging detrital matter (e.g., marine snows, fecal pellets, carcasses) containing microplastic [13,34,35,41,113-115]. Micro- and nanoplastics can adhere to external
FIGURE 8.1 Publication trend of studies investigating biota interactions with microplastics through June 30, 2016.
appendages, including the gills of the shore crab (Carcinus maenas)  and mussels (.Mytilus edulis)  and setae of copepod swimming legs and antennules . Other studies have identified that microplastics can bind to microalgae [116-118] or macroalgae . Microplastic exposure has been associated with a suite of negative health effects, including increased immune response , decreased food consumption [20,22], weight loss , decreased growth rate , decreased fecundity , energy depletion  and negative impacts on subsequent generations [59,104]. Microplastics have also been shown to readily accumulate waterborne persistent organic pollutants including pesticides, solvents and pharmaceuticals, which may pose further health effects such as endocrine disruption and morbidity [106,120,121].
The United Nations Environment Programme (UNEP) has identified plastic pollution as a critical problem; the scale and degree of this environmental issue is comparable to that of climate change . There is currently much public and political debate surrounding the issue of microplastics as additives to household and industrial products and the methods by which impacts of said microplastics on the environment are to be measured. Determining the degree to which biota consume microplastics is essential to determine and monitor “good environmental status” for plastic pollution (e.g., EU Marine Strategy Framework Directive, 2008/56/EC; UNEA, US EPA). Equally, the development of robust environmental legislation is reliant on toxicological studies with ecological relevance, requiring an accurate measure of microplastic loads in natura . As such, it is imperative that researchers are able to accurately isolate, identify and enumerate microplastic debris consumed by or entangled with biota. Here, systematically and critically review methods employed in the extraction, identification and quantification of microplastic particles ingested by biota are reviewed. The effectiveness and limitations of a range of field sampling, laboratory exposure, extraction, and analytical techniques, and steps for mitigating contamination, are considered. This review primarily focuses on peer-reviewed publications that have investigated the interactions between invertebrates and fish from the wild and following controlled laboratory exposure.
-  Previously published in Anal. Methods, 2017, 9,1346-1360 (DOI: 10.1039/C6AY02415G). This Open Access