Performance of Chlorella Vulgaris Exposed to Heavy Metal Mixtures: Linking Measured Endpoints and Mechanisms.

Microalgae growth inhibition assays are candidates for referent ecotoxicology as a fundamental part of the strategy to reduce the use of fish and other animal models in aquatic toxicology. In the present work, the performance of Chlorella vulgaris exposed to heavy metals following standardized growth and photosynthesis inhibition assays was assessed in two different scenarios: (1) dilutions of single heavy metals and (2) an artificial mixture of heavy metals at similar levels as those found in natural rivers. Chemical speciation of heavy metals was estimated with Visual MINTEQ software; free heavy metal ion concentrations were used as input data, together with microalgae growth and photosynthesis inhibition, to compare different effects and explain possible toxicity mechanisms. The final goal was to assess the suitability of the ecotoxicological test based on the growth and photosynthesis inhibition of microalgae cultures, supported by mathematic models for regulatory and decision-making purposes. The C. vulgaris algae growth inhibition test was more sensitive for As, Zn, and Pb exposure whereas the photosynthesis inhibition test was more sensitive for Cu and Ni exposure. The effects on growth and photosynthesis were not related. C. vulgaris evidenced the formation of mucilaginous aggregations at lower copper concentrations. We found that the toxicity of a given heavy metal is not only determined by its chemical speciation; other chemical compounds (as nutrient loads) and biological interactions play an important role in the final toxicity. Predictive mixture effect models tend to overestimate the effects of metal mixtures in C. vulgaris for both growth and photosynthesis inhibition tests. Growth and photosynthesis inhibition tests give complementary information, and both are a fast, cheap, and sensitive alternative to animal testing. More research is needed to solve the challenge of complex pollutant mixtures as they are present in natural environments, where microalgae-based assays can be suitable monitoring tools for pollution management and regulatory purposes.