Approved Abstracts

Testing comparability of existing and innovative bioassays for sediment quality assessment using sea urchins and polychaetes



Author(s): Garcia-Velasco N; Carrero JA; Urionabarrenetxea E.; Zaldibar B; Izagirre U; Soto M;
Presenter: Nerea Garcia-Velasco

Sediment toxicity testing has become a crucial component for assessing the risks posed by contaminated sediments and for the development of Sediment Quality Guidelines and dredged material regulations. The most commonly used organisms for bioassays with estuarine sediments are amphipods, Arenicola marina polychaetes and echinoids, being the Sea Urchin Embryo test the most widely used. However, the main limitation of this bioassay is the unavailability of gametes all year-round, particularly outside the natural spawning seasons. As an alternative, reliable assessment of the hazards resulting from pollutants in sediments can be achieved with more ecologically relevant species in which biomarkers at different levels of biological complexity can be integrated. Among organisms inhabiting sediments, the polychaete Hediste diversicolor is widespread in estuaries and has the capacity to accumulate pollutants. Moreover, in vitro approaches with their immune cells (coelomocytes) could be useful and sensitive tools to determine sediment pollution effects. The aim of this work was to develop reliable in vivo and in vitro sediment toxicity tests with H. diversicolor and its coelomocytes compared with toxicity values obtained with species included in regulatory guidelines (sea urchin). First, experimental units were designed for the in vivo assays, consisting in tanks filled with spiked sediments in a layer and seawater column. Polychaetes (n=5) were maintained for 7 days in reference sediments (collected in Plentzia) and artificially polluted sediments (CuCl2, 0-300 mg/kg) in continuous air flow, 18 ºC, 33 salinity, pH 7.5 and photoperiod (12 h:12 h light: dark). Three replicates were done for each treatment. After exposure, survival, weight loss, metal accumulation in tissues, histopathology and antioxidants activities (CAT, SOD) were recorded. Regarding in vitro approaches, the procedure for primary cultures of polychaete coelomocytes was optimized (cell extrusion method, culture medium, gamete avoidance) and validated by exposing cells to CuCl2 and AgNPs (0-50 mg/L). After 2 and 24h, coelomocyte viability was determined in microplate (Calcein-AM assay) and with the aid of flow cytometry (PI). In parallel, Sea Urchin Embryo Test (ICES, 2012) was performed, exposing fertilized eggs to the same reference toxicants. After 48h, embryo size increase and developmental abnormalities were measured according to Carballeira et al. 2012. In vivo exposure of polychaetes to Cu caused mortality at 150 mg Cu/Kg and dose-response weight loss. In addition, polychaetes maintained in spiked sediments accumulated Cu and Cd in tissues. After exposure to the highest concentration (300 mg Cu/Kg) detached cuticles and a major blood irrigation and brown deposits were observed in digestive epithelia. Thus, metal exposure posed organism level effects, histopathological alterations and oxidative stress in H. diversicolor. The best procedure for H. diversicolor coelomocytes primary culture was extrusion by electric shock (9V) using PBS adjusted to the salinity of ragworms coelomic fluid (0.17M NaCl) and passing the cell solutions through 40μm cell strainer to avoid gametes. After exposure to reference toxicants significant dose-response decrease in the calcein retention of coelomocytes was recorded. In flow cytometry an enhancement in mortality as increasing Cu and AgNPs concentrations was obtained. Sea urchin embryos appeared to be more sensitive to metal exposure than H. diversicolor coelomocytes, probably due to the longer exposure time of the test. However, after the optimization of the methodology for primary cultures of H. diversicolor coelomocytes, in vitro approaches (viability assay in microplate and flow cytometry) were successfully used to test the toxicity of reference toxicants. The in vivo and in vitro approaches developed herein for H. diversicolor appeared to be accurate to assess pollution effects in sediments and demonstrate great potential for estuarine sediment ecotoxicology. Acknowledgements: Basque Gov. (IT1302-19, IT1213-19), U. Basque Country and MINECO (seaDIMENTOX CTM2017-87766-R).

Keywords: Sediment ecotoxicology; Bioassays; In vitro toxicity tests

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