DEVELOPMENT OF AN INTEGRATED METHODOLOGY TO ASSESS THE PRESENCE AND THE IMPACT OF (MICRO)PLASTICS ON THE RIVER BENTHIC COMMUNITY

Plastic pollution is one of the main environmental issues of the modern society since the development of synthetic polymers with exceptional chemical/physical characteristics in the 20th century led to a continuous production and use of these materials with a great increase in plastic wastes. Consequently, the mismanagement of the end-of-life plastic products induces a constant flux of these materials into the environment, especially in aquatic ecosystems. In this context, rivers are the main collectors of plastics from the continental areas, representing also an important sink of these contaminants in the sediments, with a possible affection of benthic communities. Since most of the studies about plastic pollution in freshwaters primarily deals with water matrix, the main aim of this project was to develop an integrated methodology to monitor the plastic pollution in several freshwater ecosystems, considering the impact of some variables, as the environmental matrix selection and the temporal variation of the contamination, associated to their toxicity evaluation on the benthic macroinvertebrate Chironomus riparius. Firstly, the method setting was crucial to determine the best sampling and analytical setup for plastic monitoring in sediments. This preliminary step was performed in the Lambro River (Northern Italy), which represents an excellent site for the method development due to its high plastic pollution. In detail, plastics were separated from the collected sediments through two density extraction methods, comparing the efficiency of sodium chloride (NaCl) and zinc chloride (ZnCl2) solutions. Results showed a significant difference (p < 0.05) in the extraction performance, with ZnCl2 extracting more plastics (53.3 ± 16.1 plastics/kg sediments wet weight - w.w.) than NaCl (8.3 ± 10.4 plastics/kg sediments w.w.; see manuscript 1). The study moved subsequently to the comparison of plastic pollution in both waters and sediments in four of the main Po River tributaries (Ticino, Adda, Oglio, and Mincio Rivers), using the ZnCl2 solution in the separation processes. Results showed that contamination in sediments was four times higher than in waters, highlighting heterogeneous pollution from both quantitative and qualitative point of view, with the highest levels in Mincio River (62.9 ± 53.9 plastics/m³ in waters and 26.5 ± 13.3 plastics/kg dry weight - d.w. - in sediments) and the lowest in Ticino River (0.9 ± 0.5 plastics/m3 in waters and 6.8 ± 4.5 plastics/kg d.w. - in sediments; see manuscript 2). This study suggested that the monitoring of both matrices provide complementary information: waters offer a picture of an instantaneous contamination, while sediments, representing a sink of plastics, a “historical” pollution. Since waters represent a snapshot of plastic contamination in a water body, the temporal variation of these contaminants in the water matrix was also assessed. Lake Maggiore, from which Ticino River is the main emissary, was selected as reference site due to the highest plastic contamination compared to the other great sub-alpine Lakes. This work revealed a monthly variation in plastic content, from 0.02 plastics/m3 in September to 0.29 plastics/m3 in December (see manuscript 3). These findings underscore the importance of comprehensive monitoring plans considering both abiotic matrices (waters and sediments), as well as multi-sampling campaigns throughout the year, to better understand the plastic contamination in freshwater ecosystems. Since the most of studies investigated the toxicity of virgin standard plastic particles, ignoring the wide plethora of shapes, sizes and polymers detected in the environment, in this project the sampled plastics in the abovementioned freshwater lotic environments were used in the exposures of Chironomus riparius. Since no normalized methods for the Chironomid exposure to plastics were available, a modification of the Chironomid Life-Cycle Toxicity Test (OECD 233) was performed, and both apical and molecular/cellular multigenerational effects on two generations were evaluated. The toxicity of sampled plastic mixtures from the Po River tributaries (40 plastics/kg d.w. for Ticino, 68 plastics/kg d.w. for Adda, 58 plastics/kg d.w. for Oglio and 420 plastics/kg d.w. for Mincio) were tested in comparison to virgin polystyrene microbeads of 1 μm (PS; 22,400 plastics/kg sediment d.w., 112,000 plastics/kg d.w., 224,000 plastics/kg d.w.). No significant multigenerational effects (p > 0.05) were observed for virgin plastics, despite the highest tested concentrations compared to those used for sampled plastics, while plastics from Adda River significantly decreased (p < 0.05) the Emergence Rate (ER) in the 2nd generation of Chironomus riparius (see manuscript 4). These results suggested that the toxicity of these contaminants could be also related to the physical/chemical characteristics, from shape to polymer composition, instead of the simple plastic concentration. This project contributes to filling the knowledge gap associated to the impact of plastics in water body sediments, suggesting that the monitoring integrated with an ecotoxicological assessment can provide a more complete view of the potential hazardousness of these pollutants.