Microplastic sample purification methods - Assessing detrimental effects of purification procedures on specific plastic types.

In search of effective, fast, and cheap methods to purify environmental samples for microplastic analysis, scientific literature provides various purification protocols. However, while most of these protocols effectively purify the samples, some may also degrade the targeted polymers. This study was conducted to systematically compare the effects of purification protocols based on acidic, alkaline, oxidative, and enzymatic digestion and extraction via density separation on eight of the most relevant plastic types. It offers insights into how specific purification protocols may compromise microplastic detection by documenting visible and gravimetric effects, analyzing potential surface degradation using Fourier transform infrared spectroscopy (FTIR) and bulk erosion on a molecular level using gel permeation chromatography (GPC). For example, protocols using strong acids and high temperatures are likely to completely dissolve or cause strong degradation to a wide range of polymers (PA, PC, PET, PS, PUR & PVC), while strong alkaline solutions may damage PC and PET. Contrarily, Fenton's reagent, multiple enzymatic digestion steps, as well as treatment with a zinc chloride solution frequently used for density-separation, do not degrade the eight polymers tested here. Therefore, their implementation in microplastic sample processing may be considered an essential stepping-stone towards a standardized protocol for future microplastics analyses.

Effects of microplastic particles and leaching additive on the life history and morphology of Daphnia magna.

Plastic waste is continuously introduced not only into marine, but also freshwater environments, where it fragments into microplastics. Organisms may be affected by the particles themselves due to ingestion and indirectly via incorporated additives such as plasticizers, since these substances have the ability to leach out of the polymer matrix. Although it has been indicated that the likelihood of additives leaching out into the gut lumen of organisms exposed to microplastics is low, studies distinguishing between the effects of the synthetic polymer itself and incorporated additives of the same polymer are scarce. Since this is obligatory for risk assessment, we analyzed the chronic effects of flexible polyvinylchloride (PVC), a widely used polymer, containing the plasticizer diisononylphthalate (DiNP) on morphology and life history of the freshwater crustacean Daphnia magna and compared these effects with the effects of rigid PVC, lacking DiNP, as well as a glass bead control. After up to 31 days of exposure, rigid PVC and glass beads did not affect body length and relative tail spine length of D. magna, whereas flexible PVC led to an increased body length and a reduced number of offspring. None of the treatments increased the mortality significantly. We were able to show that 2.67µg/L DiNP leached out of the flexible PVC into the surrounding medium using GC-MS. Yet, we were not able to measure leachate inside the gut lumen of D. magna. The effects emerged towards the end of the experiment, due to the time dependent process of leaching. Therefore, the results highlight the relevance of long-term chronic exposure experiments, especially as leaching of additives takes time. Further, our study shows the importance to distinguish between microplastics containing leachable additives and the raw polymer in ecotoxicological testing.

Enzymatic Purification of Microplastics in Environmental Samples.

Micro-Fourier transform infrared (micro-FTIR) spectroscopy and Raman spectroscopy enable the reliable identification and quantification of microplastics (MPs) in the lower micron range. Since concentrations of MPs in the environment are usually low, the large sample volumes required for these techniques lead to an excess of coenriched organic or inorganic materials. While inorganic materials can be separated from MPs using density separation, the organic fraction impedes the ability to conduct reliable analyses. Hence, the purification of MPs from organic materials is crucial prior to conducting an identification via spectroscopic techniques. Strong acidic or alkaline treatments bear the danger of degrading sensitive synthetic polymers. We suggest an alternative method, which uses a series of technical grade enzymes for purifying MPs in environmental samples. A basic enzymatic purification protocol (BEPP) proved to be efficient while reducing 98.3 ± 0.1% of the sample matrix in surface water samples. After showing a high recovery rate (84.5 ± 3.3%), the BEPP was successfully applied to environmental samples from the North Sea where numbers of MPs range from 0.05 to 4.42 items m-3. Experiences with different environmental sample matrices were considered in an improved and universally applicable version of the BEPP, which is suitable for focal plane array detector (FPA)-based micro-FTIR analyses of water, wastewater, sediment, biota, and food samples.

Spatial and temporal variation of macro-, meso- and microplastic abundance on a remote coral island of the Maldives, Indian Ocean.

Plastic debris is ubiquitous in the marine environment and the world's shores represent a major sink. However, knowledge about plastic abundance in remote areas is scarce. Therefore, plastic abundance was investigated on a small island of the Maldives. Plastic debris (>1mm) was sampled once in natural long-term accumulation zones at the north shore and at the high tide drift line of the south shore on seven consecutive days to quantify daily plastic accumulation. Reliable identification of plastic debris was ensured by FTIR spectroscopy. Despite the remoteness of the island a considerable amount of plastic debris was present. At both sites a high variability in plastic abundance on a spatial and temporal scale was observed, which may be best explained by environmental factors. In addition, our results show that snapshot sampling may deliver biased results and indicate that future monitoring programs should consider spatial and temporal variation of plastic deposition.

Interclonal proteomic responses to predator exposure in Daphnia magna may depend on predator composition of habitats.

Phenotypic plasticity, the ability of one genotype to express different phenotypes in response to changing environmental conditions, is one of the most common phenomena characterizing the living world and is not only relevant for the ecology but also for the evolution of species. Daphnia, the water flea, is a textbook example for predator-induced phenotypic plastic defences; however, the analysis of molecular mechanisms underlying these inducible defences is still in its early stages. We exposed Daphnia magna to chemical cues of the predator Triops cancriformis to identify key processes underlying plastic defensive trait formation. To get a more comprehensive idea of this phenomenon, we studied four genotypes with five biological replicates each, originating from habitats characterized by different predator composition, ranging from predator-free habitats to habitats containing T. cancriformis. We analysed the morphologies as well as proteomes of predator-exposed and control animals. Three genotypes showed morphological changes when the predator was present. Using a high-throughput proteomics approach, we found 294 proteins which were significantly altered in their abundance after predator exposure in a general or genotype-dependent manner. Proteins connected to genotype-dependent responses were related to the cuticle, protein synthesis and calcium binding, whereas the yolk protein vitellogenin increased in abundance in all genotypes, indicating their involvement in a more general response. Furthermore, genotype-dependent responses at the proteome level were most distinct for the only genotype that shares its habitat with Triops. Altogether, our study provides new insights concerning genotype-dependent and general molecular processes involved in predator-induced phenotypic plasticity in D. magna.