Multistress in a changing world: Impacts of pollutant mixture and temperature on stress response, endocrine system, and detoxification mechanisms in the freshwater gastropod Physa acuta (Draparnaud, 1805)

Aquatic ecosystems are vulnerable to climate change and pollution, with individual pollutants and combinations of anthropogenic compounds having a significant impact on them. Mixtures lead to complex interactions with additional stress from climate change, exacerbating temperature increases. Traditional toxicity approaches may not effectively capture the impacts and interactions of these factors, which can put the aquatic invertebrate populations at risk. Therefore, to closely mimic environmental conditions, adults of the freshwater gastropod Physa acuta were exposed to mixtures of three well-known pollutants: Bisphenol A (BPA), Endosulfan (End), and Cadmium (Cd), mimicking the IPCC warming scenario 3 (middle of the road: 2.7°C by 2100) at 18°C and 20.5°C. Samples were collected for gene expression analysis by retrotranscription and Real-Time PCR at 1, 7, and 21 days. Three relevant biological processes — stress response, endocrine regulation, and detoxification mechanisms — were evaluated by analyzing the transcriptional activity of ten genes. Modulation of mRNA levels showed a significant influence of temperature on gene activity, with more pronounced acclimation responses at 18°C compared to 20.5°C. Exposure to Cd and End had the most significant impact, while BPA only showed effects in combination with the two other chemicals. This study shows that mixtures elicit complex and unpredictable responses at varying temperatures. Our findings underscore the importance of studying compound mixtures at different temperatures and highlight the need for standardized molecular toxicity assessments in complex environmental scenarios affected by global change. The information gathered will also benefit other species, helping identify biomarkers that are helpful in ecotoxicological analysis. • Temperature modulates response to chemical mixtures in Physa acuta . • Low temperature (18°C) shows a more pronounced acclimation to pollutants. • Cadmium and Endosulfan were the most toxic, causing significant gene alteration. • Bisphenol A had limited effects but contributed to the mixtures' complex response. • Mixtures caused non-linear and unpredictable patterns of gene expression.