Microplastic Extraction from Marine Vertebrate Digestive Tracts, Regurgitates and Scats: A Protocol for Researchers from All Experience Levels
海洋脊椎动物消化道,反流物和粪便中塑料微粒的提取:一个适用于所有经验水平研究者的实验方法   

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Environmental Pollution
Apr 2015

 

Abstract

It is essential to provide a protocol for the separation and identification of microplastics in marine vertebrates (mammals, birds, turtles and fish) that is easy to follow and adaptable depending on research infrastructure. Digesting organic material is an effective way to analyze samples for microplastics. Presented here is an optimized protocol which uses potassium hydroxide (KOH) for processing samples of digestive tracts, scats and regurgitates. KOH is a cheap, effective and simple alkaline digestant that allows extraction of plastics from the sample matrix. Samples are first digested, then filtered before visual and chemical analysis of remaining particle. This allows size, shape, color and polymer of each particle to be ascertained. This protocol has been harmonized with other protocols for the collection of different samples (e.g., diet, parasites, other pathologies). The implementation of this protocol at different levels of economic and/or laboratory resources make information on microplastic incidence available to the entire research community.

Keywords: Microplastics (塑料微粒), Plastic (塑料的), Marine debris (海洋垃圾), Mammals (哺乳动物), Turtle (乌龟), Birds (鸟类)

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.

Materials and Reagents

Note: All materials and reagents can be adapted depending on laboratory resources. For example, specific forceps and glass equipment are not required, but some specific precautions may be performed. See Table 1 for options.


  1. Filter papers (e.g., glass microfibre filters, GF/D, 47 mm, pore size 0.47 microns, Whatman, catalog number: 1823-047)
  2. Aluminum foil lids cut to the required size or non-plastic lids
  3. Laboratory gloves (e.g., blue nitrile gloves, VWR, catalog number: 112-2373)
  4. Mesh (e.g., 200 µm monofilament Nylon fibre mesh) 
  5. Filtered water (e.g., Milli-Q Filtered water, 0.22 μm membrane filter)
  6. Ethanol absolute > 99% (v/v) (VWR, catalog number: 20821.310E) 
  7. Potassium hydroxide, KOH, 85.0%-100.5%, pellets (VWR, catalog number: 26668.296)
  8. Sodium chloride, NaCl, 99.5%-100.5%, pellets (VWR, catalog number: 27810.364)
  9. Sodium iodide, NaI, > 99.5% (VWR, catalog number: 27913.260)
  10. Sodium polytungstate solution, SPT 85% (v/v) (Sigma-Aldrich, catalog number: 80656)

Equipment

  1. 500 ml beakers (Corning, Pyrex®, catalog number: 1395-500)
  2. Magnetic stirrer (Sigma-Aldrich, Benchmark, catalog number: Z742550)
  3. Vacuum filtering flask assembly for 47 mm filters with funnel, filter folder (e.g., 2,000 ml, VWR, catalog number: 511-0264H and Fisher Scientific, KimbleTM, catalog number: K953771-0000) 
  4. Vacuum pump (e.g., mini diaphragm vacuum pump, VP86, VWR, catalog number: 181-0065H)
  5. Stainless steel sieves: 1 mm, 0.5 mm, 0.25 mm, and smaller sieves when a lower size limit is required
  6. Cotton and white laboratory clothes
  7. Non-plastic dissection tools (including scissors, forceps and scalpel)
  8. Stereomicroscope with a camera (e.g., Leica Microsystems, model: MZ6)
  9. FT-IR instrumentation (or similar equipment where possible for chemical analysis) 
  10. Fridge
  11. Incubator and shaker (e.g., New Brunswick Scientific, model: Innova 26R)

Procedure

  1. Sample collection
    Here, we describe a step-by-step procedure for collecting samples from the field (Steps A1-A4, Figure 1). We understand that the methods used for collection can change depending on the samples targeted. The importance of contamination control steps is included as this information is required when collecting samples.


    Figure 1. Procedural steps for extracting plastics and microplastics from vertebrate digestive tracts, scats and regurgitates

    1. Collect digestive tracts following the standard protocols for animal necropsies (e.g., Kuiken and Garcia-Hartmann, 1991). Digestive tracts must be secured with a bridle or a cord to avoid loss of contents. If digestive tracts are not processed immediately, they should be stored in a durable container. It is recommended that when a plastic bag is used, that is made of a bright color. Therefore, in case of accidental contamination such plastic piece will be easy to identify.
    2. Scats and regurgitates are collected in transparent plastic zipped bags or transparent glass containers when possible.
    3. It is recommended that white gloves and white cotton laboratory coats/clothes be used during the collection of all samples. Any other color and polymeric material used must be noted for reference to take potential contamination into account when carrying out analysis.
    4. Samples should be processed fresh, but when this is not possible, samples can be frozen at -20 °C or lower until required.

  2. Reagent preparation
    Caution: KOH is a caustic and irritant solvent. All researchers must wear laboratory gloves and eye protection at all times. If the solution touches skin, the affected area should be washed immediately with running water.
    1. To make a 10% KOH solution, add 100 g KOH to 1,000 ml of water. Solution volume can be adjusted depending on the sample processing requirements. It is recommended to prepare the solution in a beaker or conical flask with a lid.
    2. Close the lid and gently mix until pellets are dissolved, this can be carried out by shaking by hand or using a magnetic stirrer depending on the volume being prepared. Once pellets have been dissolved, the solution will have turned colorless, leave for at least 15 min to cool.
    3. The final solution should be filtered using a Buchner filter or similar (Table 1) to ensure that there are no residual microplastics in the solution, either from the KOH pellets, water or from procedural contamination.

  3. Laboratory preparation
    1. The laboratory should be scrubbed before use to remove any sources of airborne contamination. There should be limited access to reduce external contamination.
    2. Researchers in the laboratory should wear white cotton laboratory coat and gloves. Any other color and polymeric material used must be noted for reference to take potential contamination into account when carrying out analysis.
    3. All equipment must be rinsed at least three times with pre-filtered water immediately before use. Glass or metal equipment is recommended, however, if unavoidable see Table 1. The water supply used for flushing the samples must be filtered using a sieve with a mesh size smaller than target microplastics. Any variation on the color of the equipment should be noted.
    4. Set up nested metal sieves in a sink connected to the pre-filtered water source. The sieve at the top will have the largest mesh size (e.g., 1 mm) and the bottom one the smaller one (e.g., 50 µm). Using consecutive sieves will allow removal of larger pieces of plastics, food remains and parasites. If researchers wish to target smaller plastics, they can add additional sieves. This allows for size differentiation and speeds up processing in later steps. The sieves should be covered with a lid when not in use to prevent contamination.

      Table 1. Troubleshooting advice and alternative methodological approaches


  4. Sample preparation
    1. If samples have been frozen, it is recommended that samples be defrosted slowly in a fridge (24-36 h), to avoid the fast decomposition of the tissues. It is also possible to defrost samples at room temperature in a clean laboratory.
      Note: Depending on the sample collected there are different initial processing steps required. For digestive tract samples, proceed with Steps D2-D10, for regurgitate and scat samples proceed from Steps D11-13.

    Digestive tracts
    1. Digestive tracts must be rinsed externally before processing and removing the ties to remove external contamination.
      Note: Ties are used to stop contents leaking.
    2. Digestive tracts of marine organisms vary in length and number of compartments (esophagus, stomachs and intestines). Each compartment should be processed and rinsed independently (e.g., Hernandez-Milian et al., 2018).
    3. If different sections of fish digestive tracts cannot be differentiated, it can be analyzed without divisions.
    4. For large intestines (> 1 m, i.e., mammals), we recommend separating into smaller, equally sized pieces. The most appropriate number is 20 equal pieces. The division of this part of the digestive tracts will allow the researcher to investigate if microplastics tend to concentrate in any specific area as some parasites do (Lawlor et al., 1990), as well as make feasible the analysis of a large sample.
    5. Each compartment or intestine section must be opened using clean and sterilized scissors and forceps.
    6. A subsample for genetic analysis should be collected from the middle of the bulk of prey remains and stored frozen or in 70% pre-filtered ethanol.
    7. Transfer the remaining material to the nested sieve column avoiding touching the mucosal surface.
    8. Examination of the gastric and intestinal mucosal surface should be carried out with care to prevent damage to the surface by rubbing it with fingers or other material. If any pathology is detected, a sample of that area should be collected and stored with the fixed chemical required following the standard protocols.
    9. Compartments should be processed individually under the filtered water supply through nested sieves following Step E1 onwards.

    Scats and regurgitates (solid samples)
    1. The container where scats and regurgitates are stored must be rinsed externally with pre-filtered water before processing. This will avoid procedural contamination.
    2. A subsample from the middle of the sample will be collected and frozen or stored in 70% ethanol for genetic analysis.
    3. The rest of the samples should be poured and processed in the nested sieves following Step E1 onwards. If the scat or the regurgitated sample is not fluid, they can be poured into a container with pre-filter water for few hours to hydrate before processing. Samples must always be covered with a lid to prevent procedural contamination.

  5. Sample processing
    1. Rinse each sample or compartment into the nested sieves separately (Figure 1).
    2. Transfer the non-plastic material collected in both top sieves to containers with 70% ethanol for between 2 and 24 h to sterilize the material and prevent mold and odor.
    3. Store hard food remains (e.g., otoliths, bones, shells) dry in bags or containers, while cephalopod beaks, and other soft remains (e.g., crustaceans, worms, parasites) should be stored in 70% ethanol for further identification (Figure 1).
    4. Rinse the plastic material (> 1 mm) found in the larger sieve with pre-filtered water and store dry. Material kept in the smaller sieve will be transferred to a glass container with pre-filtered water to obtain a suspension for the microplastic sample (Figure 1).
    5. The suspension should be as concentrated as possible. This will reduce the volume of KOH solution required in the following step.
    Note: Eye protection must be worn from this step.
    1. Add 10% KOH solution to the suspension in a ratio of 3:1 (KOH:suspension) (Figure 1).
    2. Cover samples loosely with aluminum foil, or screw top lids to prevent contamination and evaporation.
    3. Incubate samples at 60 °C for 24 h with continuous agitation at 125 rpm; alternatively, heat can be forgone but the reaction will take longer (e.g., 3 weeks, Foekema et al., 2013).
    4. The solution will turn transparent/slight yellow in coloration when all biological material has been digested.
    5. Remove samples from the incubator and leave to cool before further processing.
    6. After cooling, filter solution under vacuum using equipment such as a glass Buchner filter with a microfiber filter (GF/D or alternative). Alternatively, if this equipment is unavailable, glass funnels with a microfiber filter covered during the filtration can be used.
      Note: Filter papers must be checked under a microscope for contamination before use.
    7. When large amounts of undigested organic material (e.g., bones) remain after filtering, density flotation can be used to separate undissolved organic material from low density plastics which will float. Great care is required when using density separation as there are some less dense dietary remains that can float, such as crustacean carapaces. NaCl is the recommended density separation solution (1.2 g cm-3). More costly solutions include NaI (1.8 g cm-3) and SPT (2.8 g cm-3).
    8. Another option to remove undigested organic matter involves rinsing the solution through a sieve once more before filtering; this reduces the likelihood of filter papers clogging.

  6. Visual characterization
    Visual characterization is valuable when researchers are categorizing and sorting samples. Visual characterization of plastics must be used with other more robust identification techniques such as chemical analysis (e.g., Steps F4-F5, G1-G7), even if large plastics can be recognized. If more robust techniques are not available, Steps F4-F5 can aid researchers in reducing the likelihood of misidentification.
    1. Use a stereomicroscope to investigate particles retained on the filter paper. It is recommended that a camera be attached to the microscope to allow researchers to record visual images of all particles.
    2. Visually inspect each filter paper for potential plastic microparticles.
    3. Carry out visual characterization following existing criteria (Lusher et al., 2014):
      1. Measured longest dimension (mm) and smallest aperture lengths.
      2. Record shape and color (Figure 1).
        1. Shape categories include: fiber, fragment and spherical (beads). Fragment can be further divided into films and foams depending on research objectives, but this division is not always necessary.
        2. Color is subjective and therefore not recommended as a stand-alone classification, but it does help researchers when categorizing samples.
    4. Particles observed need to be visually inspected for the following characteristics, otherwise they should be rejected or tested with other chemical techniques (Lusher et al., 2014):
      1. Homogeneous color.
      2. No natural structures (such as cells).
      3. Unnatural bending.
      4. Fibers should have a consistent thickness throughout length.
      5. There should be no fraying at ends of fibers.
    5. A hot needle can be used during visual characterization to aid in the presence of plastic particles, which in case of plastic will react to the heat through bending or melting. This method has been recommended by National Oceanographic and Atmospheric Administration (NOAA) and the European Union under the Marine Strategy Framework Directive (EU-MSFD) however the hot needle is not suitable for semi-synthetic fibers as they do not react. Therefore, some research groups are advised to take this step with caution.

  7. Chemical characterization
    Note: Chemical characterization should only be carried out on clean and dry suspected plastic particles.
    1. Chemical characterization should be carried out on a representative subsample. MSFD recommends 10% although as many particles as possible should be analyzed chemically to reduce identification errors.
    2. The specific instrumentation used for chemical classification will depend on the research facilities available. Here we describe FT-IR because this instrumentation is available at most of research laboratories (Figure 1).
    3. Trained personnel should conduct chemical analysis. Sufficient knowledge of polymer identification and spectra interpretation is required.
    4. Spectral analysis should follow the protocols available at individual laboratories to produce an output spectrum.
    5. All output spectra should be compared to a polymer library database.
    6. Only polymers which matched reference spectra with a high level of certainty (> 70% match) should be accepted as correct identification.
    7. Researchers with sufficient knowledge of polymers may also consider visual inspection of produced spectra that have a lower level of certainty (60%-70%). Caution should be taken for such interpretation. One example is water absorbance which can alter spectra and that have clear polymeric characteristics.

Data analysis

This protocol is a standard protocol for the collection of microplastics. Data analysis is subject to the specific user requirements of the protocol. It is recommended that researchers assess the number of particles in each identified compartment (stomach, intestine section or individual scat and/or regurgitate). Values obtained can be compared with other samples from the same research area or further afield. Specifically dividing the digestive tract and intestines allows researchers to compare microplastic levels between and within species (Lusher et al., 2018).

Acknowledgments

The authors obtained no external funding for the elaboration of this protocol, A.L.L. and G.H.M. acknowledge their previous funders which supported their earlier investigations. A.L.L. was funded by an Irish Research Council Postgraduate Scholarship and a GMIT 40th anniversary studentship between 2012-2015. G.H.M. was supported by a Beaufort Ecosystem Approach to Fisheries Management award, as part of the Irish Government’s National Development Plan (NDP) between 2013-2015. Both authors thank National Parks and Wildlife Service (NPWS) for reporting the strandings to the Irish Whale and Dolphin Group (IWDG).
Author Contributions: A.L.L. and G.H.M. conceived and designed the protocol; performed the experiments, analyzed the data; and wrote the paper equally.
This protocol is the culmination of adaptions from previous work by both the authors.

Competing interests

The authors declare no conflict of interest.

References

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简介

必须提供一种分离和鉴定海洋脊椎动物(哺乳动物,鸟类,海龟和鱼类)中的微塑料的方案,该方案易于遵循和适应性,这取决于研究基础设施。消化有机物质是分析微塑料样品的有效方法。这里介绍的是一种优化的方案,它使用氢氧化钾(KOH)处理消化道,粪便和反流的样本。 KOH是一种廉价,有效且简单的碱性消化剂,可以从样品基质中提取塑料。首先消化样品,然后在剩余颗粒的视觉和化学分析之前过滤。这允许确定每个颗粒的尺寸,形状,颜色和聚合物。该协议已与其他协议协调,用于收集不同的样本(例如,饮食,寄生虫,其他病症)。在不同经济和/或实验室资源水平上实施该协议使得整个研究界可以获得关于微弹性发生率的信息。

【背景】有效监测环境中的塑料污染已成为科学家,非政府组织和利益攸关方的优先事项。需要标准化或协调的协议,以便在世界各地的研究小组之间进行比较(例如,Taylor 等。,2016; Lusher 等。,2017; Unger et al。,2017)。微塑料和中间塑料(塑料通常分别<1毫米和<5毫米)作为海洋垃圾的一种形式,现在是主要关注的问题,已被纳入监测环境健康的国际指令中。已经确定了从地表水到沉积物,从沿海地区到深海地区的微塑料(Lusher,2015);它们可能对水生和陆地环境产生物理和化学影响。最令人担忧的是,在水生生物的消化道中发现了微塑料,包括脊椎动物(例如,Tanaka和Takada,2016; Karami et al。,2018)。微塑料的存在引起了对细胞,个体水平,食物链和生态系统影响的关注(Galloway et al。,2017)。

在过去的十年中,对海洋脊椎动物消化道微塑料的研究有所增加。方法包括视觉搜索和操纵消化道,消化有机物质和筛分以分离内容物(Lusher et al。,2017)。一般来说,这些方法主要集中在宏观和微塑料的收集上,有时候还有食物残留(例如,Fukuoka et al。,2016),但是,其他材料如通常不考虑用于组织学检查和其他病理的寄生虫或样品。目前使用的方法研究不考虑包括可能对生物体具有放大效应的其他病理的整体观点。因此,通过建立微弹性研究和利用其他学科,我们提出了一个更简单实用的协议,可根据所研究的研究问题和可用资源进行调整。

利用一系列学科可以对微塑料作为污染物的影响进行全面调查。需要一个标准化的协议来获取不同学科的样本(例如,营养生态学,寄生虫学,疾病),以便真正了解这种污染物的转移和影响。这将有助于科学界的比较结果,同时从多学科方法中获得结果(例如,Jepson 等。,2016)。研究宏观塑料的一般方法主要是直接提取微塑料,使用密度分离,或消化有机物质和检查潜在的颗粒(Lusher et al。,2017)。化学鉴定方法需要昂贵且先进的仪器,所有研究人员都不能使用繁琐的方法(例如,焦平面阵列傅里叶变换红外光谱仪-FPA-FT-IR,有待审查,请参阅Löder和Gerdts ,2015)。

在目前的微塑性研究中,氢氧化钾(KOH)正被用作从生物样品中提取微塑料的最有效技术(Rochman et al。,2015; Dehaut et al。 ,2016)。它具有成本效益,利用可获得的化学品,需要简单的取样程序。以前的研究遗漏的是包含其他生物学和生态学学科,这些学科可以提供关于生物健康的更多信息,从而更好地解释结果。

一些研究小组使用淘洗或密度分离的方式收集膳食残留物(例如,Bigg和Olesiuk,1990)。在此过程中,将饮食内容物放入盛有水过量的容器中,收集下沉的物质(诸如耳石,骨头等硬物残留物)用于饮食分析;如果捕食者以甲壳类动物为食,它们将从地表采集。但是,在此方法中可能会丢失微塑料。淘洗装置的提取效率已被证明是不一致的(Zobkov和Esiukova,2017)。此外,消化道内容物的解剖和目视检查通常在可能发生空气污染的条件下进行,特别是在大样本(例如,海洋哺乳动物消化道)上。另一方面,使用化学品溶解生物材料是从样品中获得微塑料的更合适的方法。已经开发了许多不同的方案来消化生物材料,包括:酸(例如,HNO 3,HClO 4,CH 2 < / sub> O 2),碱性(例如,KOH和NaOH),氧化(例如,H 2 O 2)和酶处理(例如,蛋白酶K,Lipex和Savinase)。已经发现许多这些方案影响聚合物,需要昂贵的化学品并且具有复杂且耗时的提取(Lusher 等人,,2017)。在这些方案中,KOH,氢氧化钾,已被确定为最合适的策略,因为它在经济上具有成本效益,利用易于获取的化学品,并且需要简单的取样程序(例如,Rochman et al。,2015; Dehaut et al。,2016;Kühn et al。,2017)。

在这里,我们提出了一个协议,该协议是为从海洋脊椎动物(海洋哺乳动物,海鸟,海龟和鱼类)的消化道,粪便和反流中提取和鉴定微塑料而开发的。该协议在过去五年中已经过优化和建立,为研究团队提供了可行的微弹性分析,这些研究团队可能无法获得更复杂的技术。使用该协议,研究人员可以获得与更高级研究团队相当的结果,同时收集其他类型的样本(例如,饮食,寄生虫,渗出物)是可行的。建议用于微弹性提取的方案不受样品数量或样品量的影响。通常,可用的样本越多,对结果的解释就越好。然而,从大型脊椎动物获取样本并不总是容易或可能的。该协议将允许来自世界各地的研究人员使用灵活的方法,具有不同的复杂程度,具体取决于他们的学科,设施和可用资源。因此,该协议将使用不同学科的组合提供对生物群中微弹性存在的充分理解。

关键字:塑料微粒, 塑料的, 海洋垃圾, 哺乳动物, 乌龟, 鸟类

材料和试剂

注意:所有材料和试剂均可根据实验室资源进行调整。例如,不需要特定的钳子和玻璃设备,但是可以执行一些特定的预防措施。有关选项,请参阅表1.


  1. 滤纸(例如,玻璃微纤维滤光片,GF / D,47 mm,孔径0.47微米,Whatman,目录号:1823-047)
  2. 铝箔盖切成所需尺寸或非塑料盖
  3. 实验室手套(例如,蓝色丁腈手套,VWR,目录号:112-2373)
  4. 网眼(例如,200μm单丝尼龙纤维网)&nbsp;
  5. 过滤水(例如,Milli-Q过滤水,0.22μm膜过滤器)
  6. 乙醇绝对值> 99%(v / v)(VWR,目录号:20821.310E)&nbsp;
  7. 氢氧化钾,KOH,85.0%-100.5%,颗粒(VWR,目录号:26668.296)
  8. 氯化钠,NaCl,99.5%-100.5%,颗粒(VWR,目录号:27810.364)
  9. <碘化钠,NaI>, 99.5%(VWR,目录编号:27913.260)
  10. 聚钨酸钠溶液,SPT 85%(v / v)(Sigma-Aldrich,目录号:80656)

设备

  1. 500毫升烧杯(Corning,Pyrex®,目录号:1395-500)
  2. 磁力搅拌器(Sigma-Aldrich,Benchmark,目录号:Z742550)
  3. 用于47 mm过滤器的真空过滤瓶组件,带有漏斗,过滤器夹(例如,2,000 ml,VWR,目录号:511-0264H和Fisher Scientific,Kimble TM,目录号:K953771-0000)&nbsp;
  4. 真空泵(例如,迷你隔膜真空泵,VP86,VWR,目录号:181-0065H)
  5. 不锈钢筛:1 mm,0.5 mm,0.25 mm和较小的筛子,当需要较小的尺寸限制时
  6. 棉和白色实验室服装
  7. 非塑料解剖工具(包括剪刀,镊子和手术刀)
  8. 带摄像头的立体显微镜(例如,徕卡显微系统,型号:MZ6)
  9. FT-IR仪器(或可能用于化学分析的类似设备)&nbsp;
  10. 冰箱
  11. 孵化器和振动器(例如,New Brunswick Scientific,型号:Innova 26R)

程序

  1. 样品采集
    在这里,我们描述了从现场采集样品的逐步程序(步骤A1-A4,图1)。我们知道用于收集的方法可能会根据目标样本而改变。包括污染控制步骤的重要性,因为收集样品时需要此信息。


    图1.从脊椎动物消化道,粪便和反流中提取塑料和微塑料的程序步骤

    1. 按照动物尸检的标准方案收集消化道(例如,Kuiken和Garcia-Hartmann,1991)。消化道必须用缰绳或绳索固定,以避免内容物损失。如果消化道不立即处理,应将它们储存在耐用的容器中。当使用塑料袋时,建议使用明亮的颜色。因此,在意外污染的情况下,这种塑料件将易于识别。
    2. 在可能的情况下,粪便和回流物收集在透明塑料拉链袋或透明玻璃容器中。
    3. 建议在收集所有样品时使用白手套和白色棉质实验室外套/衣服。必须注意使用的任何其他颜色和聚合物材料以供参考,以便在进行分析时考虑潜在的污染。
    4. 样品应该是新鲜加工的,但是如果不能这样做,样品可以在-20°C或更低温度下冷冻直至需要。

  2. 试剂准备
    注意:KOH是一种腐蚀性和刺激性的溶剂。所有研究人员必须始终佩戴实验室手套和眼睛保护装置。如果溶液接触皮肤,应立即用流水冲洗受影响的区域。
    1. 要制备10%KOH溶液,将100克KOH加入1,000毫升水中。可根据样品处理要求调整溶液体积。建议在带盖的烧杯或锥形瓶中制备溶液。
    2. 关闭盖子并轻轻混合直至颗粒溶解,这可以通过手动摇动或使用磁力搅拌器进行,这取决于所制备的体积。一旦颗粒溶解,溶液就会变成无色,静置至少15分钟。
    3. 应使用布氏过滤器或类似方法(表1)过滤最终溶液,以确保溶液中没有残留的微塑料,无论是来自KOH颗粒,水还是来自程序污染。

  3. 实验室准备
    1. 实验室应在使用前进行清洗,以清除任何空气污染源。应尽可能减少外部污染。
    2. 实验室的研究人员应穿白色棉质实验室外套和手套。必须注意使用的任何其他颜色和聚合物材料以供参考,以便在进行分析时考虑潜在的污染。
    3. 所有设备必须在使用前立即用预过滤水冲洗至少三次。建议使用玻璃或金属设备,但如果不可避免,请参见表1.用于冲洗样品的供水必须使用筛孔尺寸小于目标微塑料的筛网过滤。应注意设备颜色的任何变化。
    4. 在与预过滤水源连接的水槽中设置嵌套金属筛。顶部的筛网具有最大的筛孔尺寸(例如,1mm),而底部的筛网具有较小的筛网尺寸(例如,50μm)。使用连续的筛子可以去除更大的塑料,食物残渣和寄生虫。如果研究人员希望针对较小的塑料,他们可以添加额外的筛子。这允许尺寸区分并加速后续步骤中的处理。不使用时,应用盖子盖上筛子以防止污染。

      表1.故障排除建议和替代方法


  4. 样品制备
    1. 如果样品已冷冻,建议将样品在冰箱中缓慢解冻(24-36小时),以避免组织快速分解。也可以在干净的实验室中在室温下对样品进行解冻。
      注意:根据收集的样本,需要不同的初始处理步骤。对于消化道样本,继续执行步骤D2-D10,进行反流和散布样本从步骤D11-13开始。

    消化道
    1. 消化道必须在处理之前进行外部清洗,并去除带子以去除外部污染。
      注意:Ties用于阻止内容泄漏。
    2. 海洋生物的消化道长度和隔室(食道,胃和肠)的数量不同。每个隔室应独立处理和冲洗(例如,Hernandez-Milian 等。,2018)。
    3. 如果鱼消化道的不同部分无法区分,则可以不分割地进行分析。
    4. 对于大肠(> 1米,即,哺乳动物),我们建议分成较小的,同等大小的碎片。最合适的数字是20个相等的部分。这部分消化道的划分将允许研究人员研究微塑料是否倾向于像某些寄生虫一样集中在任何特定区域(Lawlor et al。,1990),并使其可行分析大样本。
    5. 必须使用干净且经过消毒的剪刀和镊子打开每个隔室或肠道部分。
    6. 应从大量猎物残骸的中间收集用于遗传分析的子样本并冷冻储存或在70%预过滤的乙醇中储存。
    7. 将剩余材料转移到嵌套筛柱中,避免接触粘膜表面。
    8. 应小心检查胃和肠粘膜表面,以防止用手指或其他材料摩擦表面。如果检测到任何病理,应收集该区域的样本,并按照标准方案使用所需的固定化学品进行储存。
    9. 在步骤E1之后,应通过嵌套筛网在过滤水供应下单独处理隔室。

    粪便和反流(固体样本)
    1. 储存粪便和反流的容器必须在加工前用预过滤的水冲洗外部。这将避免程序污染。
    2. 将收集来自样品中间的子样品并冷冻或储存在70%乙醇中用于遗传分析。
    3. 在步骤E1之后,应将其余样品倒入并在嵌套筛中处理。如果粪便或回流的样品不是流体,则可以在加工前将它们倒入具有预过滤水的容器中几个小时以进行水合。样品必须始终盖上盖子以防止程序污染。

  5. 样品处理
    1. 将每个样品或隔室分别冲洗到嵌套筛中(图1)。
    2. 将收集在两个顶筛中的非塑料材料转移到含70%乙醇的容器中2至24小时,以对材料进行消毒并防止霉菌和气味。
    3. 储存硬质食物残留物(例如,耳石,骨骼,贝壳)干燥在袋子或容器中,而头足类动物喙和其他软遗骸(例如,甲壳类动物,蠕虫,寄生虫)应储存在70%乙醇中进行进一步鉴定(图1)。
    4. 用预过滤的水冲洗较大筛子中的塑料材料(> 1mm)并储存干燥。将保存在较小筛网中的材料转移到具有预过滤水的玻璃容器中,以获得微塑料样品的悬浮液(图1)。
    5. 悬浮液应尽可能浓缩。这将减少后续步骤中所需的KOH溶液的体积。
    注意:此步骤必须佩戴眼睛。
    1. 以3:1(KOH:悬浮液)的比例向悬浮液中加入10%KOH溶液(图1)。
    2. 用铝箔或螺旋盖盖松散地覆盖样品,以防止污染和蒸发。
    3. 将样品在60℃下孵育24小时,同时以125rpm连续搅拌;或者,可以放弃加热,但反应需要更长的时间(例如,3周,Foekema 等,2013)。
    4. 当所有生物材料都被消化后,溶液将变成透明/淡黄色。
    5. 从培养箱中取出样品并在进一步处理前冷却。
    6. 冷却后,使用诸如玻璃布氏过滤器和微纤维过滤器(GF / D或替代)的设备在真空下过滤溶液。或者,如果此设备不可用,可以使用在过滤期间覆盖微纤维过滤器的玻璃漏斗。
      注意 : 使用前必须在显微镜下检查滤纸是否有污染。
    7. 当过滤后仍留有大量未消化的有机物质(例如,骨头)时,密度浮选可用于将未溶解的有机物质与将漂浮的低密度塑料分离。使用密度分离时需要非常小心,因为有一些不太密集的饮食残骸可以漂浮,例如甲壳类甲壳。 NaCl是推荐的密度分离溶液(1.2 g cm -3)。更昂贵的溶液包括NaI(1.8g cm -3)和SPT(2.8g cm -3)。
    8. 除去未消化的有机物质的另一种选择包括在过滤之前再次通过筛子冲洗溶液;这减少了滤纸堵塞的可能性。

  6. 视觉表征
    当研究人员对样品进行分类和分类时,视觉表征很有价值。塑料的视觉表征必须与其他更强大的识别技术一起使用,例如化学分析(例如,步骤F4-F5,G1-G7),即使可以识别大型塑料。如果没有更强大的技术,步骤F4-F5可以帮助研究人员减少错误识别的可能性。
    1. 使用立体显微镜研究保留在滤纸上的颗粒。建议将相机连接到显微镜上,以便研究人员记录所有颗粒的视觉图像。
    2. 目视检查每个滤纸上是否有潜在的塑料微粒。
    3. 按照现有标准进行视觉表征(Lusher et al。,2014):
      1. 测量的最长尺寸(mm)和最小孔径长度。
      2. 记录形状和颜色(图1)。
        1. 形状类别包括:纤维,碎片和球形(珠子)。根据研究目标,碎片可以进一步分为薄膜和泡沫,但这种划分并不总是必要的。
        2. 颜色是主观的,因此不建议作为独立分类,但它在分类样本时确实有助于研究人员。
    4. 观察到的颗粒需要在视觉上检查以下特征,否则应该拒绝或使用其他化学技术进行测试(Lusher et al。,2014):
      1. 均匀的颜色。
      2. 没有天然结构(如细胞)。
      3. 不自然的弯曲。
      4. 纤维在整个长度上应具有一致的厚度。
      5. 纤维末端不应有磨损。
    5. 在视觉表征期间可以使用热针以帮助存在塑料颗粒,在塑料的情况下,塑料颗粒将通过弯曲或熔化对热量起反应。该方法已被国家海洋和大气管理局(NOAA)和欧盟根据海洋战略框架指令(EU-MSFD)推荐,但热针不适用于半合成纤维,因为它们不反应。因此,建议一些研究小组谨慎采取这一步骤。

  7. 化学特性
    注意:化学表征只能在清洁干燥的可疑塑料颗粒上进行。
    1. 化学表征应在代表性的子样品上进行。 MSFD推荐10%,尽管应尽可能多的颗粒进行化学分析,以减少识别错误。
    2. 用于化学分类的具体仪器将取决于可用的研究设施。在这里我们描述了FT-IR,因为这种仪器可以在大多数研究实验室获得(图1)。
    3. 训练有素的人员应进行化学分析。需要足够的聚合物识别和光谱解释知识。
    4. 光谱分析应遵循各个实验室可用的协议来产生输出光谱。
    5. 应将所有输出光谱与聚合物库数据库进行比较。
    6. 只有与高参考光谱匹配且具有高确定性(> 70%匹配)的聚合物才应被接受作为正确的鉴定。
    7. 具有足够聚合物知识的研究人员也可以考虑对产生的光谱进行目视检查,这些光谱具有较低的确定性(60%-70%)。应谨慎对待这种解释。一个例子是吸水率,它可以改变光谱并具有明显的聚合物特性。

数据分析

该协议是用于收集微塑料的标准协议。数据分析取决于协议的特定用户要求。建议研究人员评估每个确定的隔室(胃,肠切片或个体粪便和/或反流)中的颗粒数量。获得的值可以与来自相同研究区域或更远地区的其他样品进行比较。特别划分消化道和肠道使研究人员能够比较物种之间和物种内的微弹性水平(Lusher et al。,2018)。

致谢

作者没有为制定该协议获得外部资金,A.L.L。和G.H.M.承认他们以前的资助者支持他们早先的调查。所有。由爱尔兰研究理事会研究生奖学金和2012-2015学年GMIT 40周年学生资助。 G.H.M.作为爱尔兰政府2013 - 2015年国家发展计划(NDP)的一部分,该项目得到了博福特生态系统渔业管理奖的支持。两位作者都感谢国家公园和野生动植物管理局(NPWS)向爱尔兰鲸鱼和海豚集团(IWDG)报告了这些情况。
作者贡献: A.L.L。和G.H.M.构思并设计了协议;进行实验,分析数据;并平等地写了这篇论文。
该协议是作者对以往工作的适应性的最终结果。

利益争夺

作者宣称没有利益冲突。

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引用:Lusher, A. L. and Hernandez-Milian, G. (2018). Microplastic Extraction from Marine Vertebrate Digestive Tracts, Regurgitates and Scats: A Protocol for Researchers from All Experience Levels. Bio-protocol 8(22): e3087. DOI: 10.21769/BioProtoc.3087.
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