Microplastic Extraction from Marine Vertebrate Digestive Tracts, Regurgitates and Scats: A Protocol for Researchers from All Experience Levels

Background

Effectively monitoring plastic pollution in the environment has become a priority for scientists, non-governmental organizations (NGOs) and stakeholders. Standardized or harmonized protocols are required to allow comparisons between research groups from around the world (e.g., Taylor et al., 2016; Lusher et al., 2017; Unger et al., 2017). Microplastics and mesoplastics (plastics generally < 1 mm and < 5 mm respectively), as a form of marine litter, are now of major concern and have been included in international directives to monitor environmental health. Microplastics have been identified from surface waters to sediments, and from coastal areas to deep sea regions (Lusher, 2015); and, they might have physical and chemical effects on aquatic and terrestrial environments. Most worryingly, microplastics have been found in digestive tracts of aquatic organisms, including vertebrates (e.g., Tanaka and Takada, 2016; Karami et al., 2018). The presence of microplastics raises concern for cellular, individual level, food chain and ecosystem effects (Galloway et al., 2017).

Investigations on microplastics in marine vertebrate digestive tracts have increased over the last ten years. Methods include visual searching and manipulation of digestive tracts, digestion of organic material and sieving to separate contents (Lusher et al., 2017). Generally, these methods are focused on the collection of macro- and microplastics, and sometimes food remains (e.g., Fukuoka et al., 2016), however, additional material such as parasites or samples for histological examination and other pathologies are usually not considered. Currently utilized method studies do not consider a holistic view including other pathologies which might have an amplified effect on organisms. Thus, by building on microplastic research and utilizing other disciplines, we present a more simple and practical protocol that can be adapted depending on the research question being addressed and the resources available.

Utilizing a range of disciplines can deliver a thorough investigation on effects of microplastics as a pollutant. A standardized protocol to acquire samples for different disciplines (e.g., trophic ecology, parasitology, diseases) is necessary to establish a true understanding of the transfer and effects of this pollutant. This will contribute to comparative results within the scientific community whilst obtaining results from multidisciplinary approaches (e.g., Jepson et al., 2016).

The general approach to investigate macroplastics is focused either to extract microplastics directly, use density separation, or digest organic material and examining potential particles (Lusher et al., 2017). Chemical identification methods require costly and advanced instrumentation, with tedious protocols that are not commonly accessible to all researchers (e.g., focal plane array Fourier transform infrared spectrometry -FPA-FT-IR, for review see Löder and Gerdts, 2015).

Within current microplastic research, potassium hydroxide (KOH) is being pursued as the most effective technique for extracting microplastics from biotic samples (Rochman et al., 2015; Dehaut et al., 2016). It is cost efficient, utilizes accessible chemicals and requires a simple sampling procedure. What has been missing from previous research is the inclusion of other biological and ecological disciplines which can provide much more information on the health of organisms, thus leading to a better interpretation of results.

Some research groups collect dietary remains using a form of elutriation, or density separation (e.g., Bigg and Olesiuk, 1990). During this process, dietary contents are placed in a container with water overflooding and the material that sinks (hard remains such as otoliths, bones) are collected for dietary analysis; if the predator feeds on crustaceans, they will be collected from the surface. However, microplastics might be lost during this method. Extraction efficiencies of elutriation devices have been demonstrated to be inconsistent (Zobkov and Esiukova, 2017). In addition, dissection and visual inspection of digestive tracts contents are usually carried out under conditions in which airborne contamination may occur, especially on large samples (e.g., marine mammal digestive tracts). On the other hand, dissolving biotic material using chemicals is a more appropriate approach to obtaining microplastics from samples. There have been many different protocols developed to digest biotic material including: acid (e.g., HNO₃, HClO₄, CH₂O₂), alkaline (e.g., KOH and NaOH), oxidizing (e.g., H₂O₂), and enzymatic treatments (e.g., Proteinase K, Lipex and Savinase). Many of these protocols have been found to affect polymers, require expensive chemicals and have complex and time-consuming extraction (Lusher et al., 2017). Of these protocols, KOH, potassium hydroxide, has been identified as the most appropriate strategy because it is economically cost efficient, utilizes easily accessible chemicals, and requires a simple sampling procedure (e.g., Rochman et al., 2015; Dehaut et al., 2016; Kühn et al., 2017).

Here we present a protocol that has been developed for the extraction and identification of microplastics from digestive tracts, scats and regurgitates of marine vertebrates (marine mammals, seabirds, sea turtles and fish). The protocol has been optimized and established over the last five years to provide a feasible microplastic analysis to research teams which may not have access to more sophisticated techniques. Using this protocol researchers can acquire results that are comparable to more advanced research teams, while collecting other type of samples (e.g., diet, parasites, exudate) is feasible. The protocol proposed for microplastic extraction is not subject to the number of samples or sample size. Generally, the more samples available, the better interpretation of the results. However, access to samples from large vertebrates is not always easy or possible. This protocol will allow researchers from locations worldwide to use a flexible approach with different levels of complexity depending on their disciplines, facilities and available resources. Therefore, this protocol will provide a full understanding of microplastic presence in biota using a combination of different disciplines.