以下协议描述的方法:海面上的塑料微粒采样,颗粒的微型和化学鉴定分离。该协议是由MSFD技术小组对海洋垃圾公布了塑料微粒监测的建议。
Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an »A frame« attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.
Microplastic pollution in the sea represents a growing concern to contemporary society, due to the constant increase in plastic production and its subsequent disposal and accumulation in the marine environment1. Even if plastic macro litter would no longer enter the seas, microplastic pollution would continue to grow due to fragmentation of already existing plastic litter in the sea2. The majority of microplastic pollution studies were carried out in marine and fresh water ecosystems and mainly addressed sea surface pollution3.
The term microplastic refers to plastic particles smaller than 5 mm in size4. This term describes a heterogeneous mixture of particles, which can differ in size (from a few microns to several millimeters), color and shape (from very different shapes of fragments to long fibers). Microplastic particles can be of a primary or secondary origin5. Microplastic of primary origin is manufactured as small particles used in the cosmetics industry (pilling crème etc.) or chemical industry as precursor for other plastic products (e.g. plastic pellets used in plastic industry). Microplastic of secondary origin arise via the degradation of larger plastic pieces in the environment due to physical and chemical processes, induced by light, heat, oxygen, water and organisms6. In 2015, four types of microplastic sources were defined: larger plastic litter, cleaning products, medicines and textiles6. The main source (80 %) of larger plastic litter is assumed to be land based7. Microplastic from cosmetic products, medicines and textile enters water ecosystems through sewage and storm waters6. Microplastic particles most frequently found in water ecosystems are fragments from larger plastic litter and textile fibers8.
Microplastics have several negative effects on the environment. Their small size allows them to enter the food web through ingestion by marine organisms9, 10. Ingested particles can cause physical damage or block the digestive system of animals11. Particles can also be carriers of persistent organic pollutants (POPs). Their hydrophobic surface and favorable ratio of large surface area to small volume, enables POPs to adsorb onto the microplastics12. In the environment or digestive systems of animals who ingest them, POPs and other plastic additives can be leached from microplastic particles13.
Previous studies reported the ubiquitous presence of microplastics in the marine environment3, from the water column to the bottom sediments. The threat of microplastic pollution was already identified by the Marine Strategy Framework Directive in the EU and, consequently, mandatory monitoring of microplastics was advised14. Accordingly, the EU Technical Subgroup on Marine Litter (TSG-ML) prepared recommendations for monitoring of microplastics in the European seas15. Thus, the video guidelines for microplastics sampling are of high importance, as they support comparative monitoring and a coherent management process all over the world.
This protocol was developed within the DeFishGear project for the first monitoring of microplastic pollution in the Adriatic Sea. Recommendations from the document “Guidance on Monitoring of Marine Litter in European Seas” by TSG-ML15 were taken into account. This protocol describes the methodology for microplastics sampling on the sea surface, separation of microplastics from the samples, and chemical analysis of microplastic particles to confirm that particles are from plastic material and to identify the type of plastic. Sampling was done by the use of a manta net, which is the most suitable equipment for sampling in calm waters16. Separation of microplastics from the samples was carried out by visual identification using a stereomicroscope. Isolated particles were later chemically identified using Fourier transform infrared (FTIR) spectroscopy and micro FTIR spectroscopy.
由蝠净海面上塑料微粒采样为海面上塑料微粒的采样一种广泛使用的方法,但迄今还没有统一的方法。大体积的水可通过蝠网进行过滤,从而捕集塑料微粒的相关数的可能性高,结果被认为是可靠的。不同的样品中结果的可比性是通过规范化放心。在我们的情况下,该浓度通过由净开口的水平宽度拖网距离乘以相关的取样区。另一种选择是使用一个流量计,固定在网的开口。使用流量计是可能的,因为净曼塔其侧翼是海面上非常稳定,因此在跳浪是最小的。流量计记录的过滤水的体积,从而使每水样16卷结果的正常化。
<p class="jove_content">最常用蝠网具有大约300微米的筛目大小和3 – 4.5米长。这些尺寸进行优化,以避免网的堵塞,并允许一定量的水的采样尽可能大。拖网速度推荐为介于2 – 结3,但它是依赖于波高度,风速和海流。这是很重要的,该蝠网是在监督下采样期间的全部时间,如果它开始跳频,在拖网速度必须减小。该拖网时间,建议大约30分钟,而是取决于悬浮物的浓度。它可能发生浮游物有时会堵塞蝠网。在这种情况下,拖网,必须立即停止,否则微型塑料颗粒可能丢失和净可能会损坏。蝠网是最经常从容器的侧面固定。这也是最合适的选择,而蝠鲼网肯定是出了醒区。在一些调查曼塔网从船的船尾固定17,18,但你必须要确保网是出来之后区域的情况下。的距离,在其上拖网设置为取样,应单独确定,因为所引起的容器湍流的区域从容器的尺寸和从船上19,20的速度而变化。从海面样本的微型颗粒分离是最常见的视觉识别21刚刚完成。颗粒大于1毫米可以很容易地用肉眼识别,而颗粒小于1mm需要使用体视显微镜。以减少混淆非塑料颗粒与塑料的,使用上立体显微镜偏振的光的可能性,建议。塑料颗粒误认的可能性变得更小颗粒的高。由此颗粒> 0.5,MM可以仅通过使用立体显微镜来识别视觉上21。为颗粒小于0.5mm额外的,更精确的方法是必需的如微ATR-FTIR光谱仪21。
在塑料微粒分离来自样品的处理样品污染与空气长丝的可能性非常高。出于这个原因,控制悬空在工作台上培养皿强烈建议潜在的污染物空气中颗粒物的鉴定。即,数据的质量强烈依赖于:1)用样品,2)的质量和立体显微镜的放大倍数,以及3)的有机物质的量的样品16中工作的人的精度。视觉识别后,强烈建议进行分析排序的颗粒为材料8的化学鉴定可用的技术之一。
几种方法聚合物鉴定存在,其中FTIR光谱和拉曼光谱是最frequenTLY使用22。红外和拉曼光谱是互补的技术以及其准确性是相似的。在我们的协议中,红外光谱和显微红外光谱与“衰减全反射”(ATR)介绍。它们是使用简单,它们能够快速和准确的结果。塑料聚合物具有与不同的带图案高度特异性的红外(IR)光谱,从而使红外光谱的最佳技术用于塑料微粒21的识别。与样品,使特征IR光谱22的测量交互时的IR辐射的能量激励一个特定的分子的振动。红外光谱还可提供关于颗粒,如氧化23和降解24的水平的强度的额外信息。而ATR-FTIR适合较大颗粒的化学识别(> 0.5mm)的,微ATR-FTIR光谱可以提供关于颗粒&#的化学结构信息60 0.5毫米,因为它结合了显微镜和红外光谱仪的功能。
使用FTIR和微FTIR光谱之前,微型塑料颗粒必须预先干燥的,因为水强烈吸收IR辐射22,和纯化的,在情况下,它们都覆盖着生物膜和/或其它的有机和无机粘剂,其可以影响红外光谱。以纯化样品的大多数非侵入性的方式是通过搅拌,并用新鲜水25漂洗。如果这还不够,则建议使用30%的过氧化氢。所有其它的方法可对微型塑料颗粒的负面影响(如超声清洗可以进一步细分颗粒,强酸性或碱性溶液可能会损坏几个塑料聚合物等),因此不推荐使用。更有前途的是采用了连续酶促消化,为塑料友好纯化步骤。使用不同技术的酶纯化(例如脂肪酶,mylase,蛋白酶,几丁质酶,纤维素酶,蛋白酶K)已成功地应用于减少浮游生物的生物基质,从而被证明是一个有价值的技术,以尽量减少在红外光谱测量22矩阵文物。
通过视觉识别和选出的颗粒的化学鉴定塑料微粒的分离都非常耗时的过程。这项工作必须由一个准确和耐心的人谁具有立体显微镜,不仅在识别塑料粒子,而且在识别生物物质的经验来进行。即使是有经验的人无法从甲壳素或硅藻片段22歧视明确所有潜在的微型颗粒。因此,视觉分拣的误差率范围为20%26至70%21,并与减小粒径增大。
The authors have nothing to disclose.
此协议的发展由IPA亚得里亚海跨境合作计划2007 – 2013年成立后,DeFishGear项目(1°STR / 00010)之内。