Uptake, fate, and cellular effects of natural and synthetic microparticles in marine invertebrates

Petroleum-based plastics are integral components of daily life. Huge quantities of mostly disposable plastic products are in use. Low environmental consciousness and inappropriate waste management causes drastic plastic littering in terrestrial and aquatic environments. Plastics are very persistent, but UV radiation and mechanical stress induce degradation of larger plastic objects into numerous so-called microplastics. Increasing public awareness raises concern about microplastic pollution. However, research on the effects of microplastics on organisms often provides inconclusive results. Comparing how organisms cope with natural microparticles in their habitats allows drawing conclusions on the susceptibility to the potential effects of synthetic microparticles. Therefore, the objective of this thesis is to investigate the uptake, the internal allocation, and the cellular effects of natural and synthetic microparticles in marine invertebrates. This study contributes to a better understanding and distinction between the reactions to natural microparticles and the effects induced by anthropogenic microparticles. Coastal and estuarine regions are characterized by turbid waters and may contain up to one gram per litre of suspended particulate matter (SPM) in the µm-size range. Ingestion of natural indigestible microparticles is common in marine invertebrates. Stomach content analysis of brown shrimp, Crangon crangon, collected in their natural environment revealed quantities of up to several hundred natural microparticles, mainly sand grains and fragments of bivalve shells, per individual. Most indigestible items leave the body through the gut as faecal pellets within 24 hours. Larger particles are regurgitated through the flexible esophagus. Experimentally administered fluorescent microbeads of 2.1, and 9.9 μm diameter passed through the stomach and gut of the shrimp. Only the smallest particles of 0.1 μm entered the midgut gland, which is the principal site of nutrient resorption in crustaceans. A fine-meshed chitinous filter system in the stomach of the shrimp prevents the passage of particles larger than about 1 μm into the midgut gland, where particles can be absorbed and interact with the cells of the midgut gland epithelium. Indigestible material, including particulate matter, which enters the midgut gland, is presumably deposited in vacuoles of specific digestive cells, the B cells, and released by cell rapture into the lumen of the digestive tract to be evacuated with the rest of faeces. These results suggest that accumulation of microparticles in shrimp is unlikely. Incorporation of microparticles in the cells of the midgut gland may entail various cellular reactions. A frequently reported effect is the induction of oxidative stress due to the production of reactive oxygen species (ROS), but the underlying mechanisms are not sufficiently identified. A suggested source of ROS is the NADPH-oxidase enzyme complex, which is well investigated in vertebrate neutrophils. NADPH-oxidase is also present in shrimp midgut gland tissue as revealed by transcriptome analysis and immunological verification. Excess formation of ROS is counterbalanced by a complex cascade of antioxidants. The induction of superoxide dismutase (SOD) is an important indication of oxidative stress. Significant SOD induction appeared in the Atlantic ditch shrimp, Palaemon varians, within few hours upon microplastic ingestion, but was lacking in C. crangon. Moreover, overall SOD-activities were significantly lower in C. crangon than in P. varians. This result is surprising, but clearly indicate that even closely related species may react differently to microplastic exposure. Moreover, C. crangon showed no differential oxidative stress response after administration of synthetic or natural microparticles at environmentally relevant SPM-concentrations of 20 mg per litre. The differences between species may be related to differential formation of ROS or differential protection against ROS. Both aspects demand further research in marine invertebrates, particularly the NADPH-oxidase and the enzymatic and non-enzymatic anti-oxidative defence system. In summary, this study provides important insight into the interaction of marine invertebrates with natural or anthropogenic microparticles. Marine invertebrates are exposed to much higher concentrations of natural microparticles than anthropogenic microplastics. Microparticles are ingested, as shown for brown shrimp, but most of these particles cannot advance into sensitive organs due to anatomical barriers. Smaller particles in the sub-micrometer size range may enter the cells of the midgut gland. However, they seem to cause differential biochemical stress reaction between species. C. crangon did not show any stress response following the ingestion of natural or anthropogenic microparticles suggesting that species from habitats with high natural particle load have evolved specific anatomical and biochemical properties to cope with a high SMP-load in turbid waters. These species will likely be less affected by anthropogenic microplastics and, thus, may be favored in future scenarios of continuously increasing environmental microplastic pollution