To meet the increasing demand for energy, development of alternative and renewable energy sources, such as bioenergy, has accelerated during the last decade. In this context, biofuels are one potential replacement for fossil fuels, although their impact on the environment has not been widely studied. Only a few studies are available on toxicity of biofuels and biofuel combustion. Furthermore, for a complete understanding of the environmental impact, the entire life cycle of a biofuel has to be analyzed. This study is an exemplary ecotoxicological investigation of a biomass-to-biofuel production process with respect to the generation of environmentally relevant contaminants either by means of biomass pretreatment or microbial activity. Our aim is the demonstration of the suitability of ecotoxicological biotests as part of a comprehensive hazard assessment of biofuels and related samples or processes. Five ecotoxicological endpoints were assayed to determine the impact of four different biomass pretreatments on process substrates and effluent toxicities. Four different test organisms (bacterium, yeast, fish cell line, and fish embryo) from different trophic levels as well as a combination of acute and mechanism-specific biotests were applied to strengthen the ecotoxicological relevance of this investigation. Biotest results revealed cytotoxic, acute embryotoxic and mutagenic effectiveness, and weak estrogenic activity, with biomass toxicity depending on the mechanism of substrate pretreatment. Open microbial communities (reactor microbiomes) involved in the production process decreased the toxicity considerably to levels of the product n-butyric acid due to degradation of inhibiting by-products, verifying their simultaneous biomass conversion and detoxification potential. Our results demonstrate that ecotoxicological biotests are useful tools for the biofuel industries to gain environmental friendliness as a selling point.
