The role of reactive oxygen species (ROS) in copper (Cu) toxicity to two freshwater green algal species, Pseudokirchneriella subcapitata (Korshikov) Hindák and Chlorella vulgaris Beij., was assessed to gain a better mechanistic understanding of this toxicity. Cu-induced formation of ROS was investigated in the two algal species and linked to short-term effects on photosynthetic activity and to long-term effects on cell growth. A light- and time-dependent increase in ROS concentrations was observed upon exposure to environmentally relevant Cu concentrations of 50 and 250 nM and was comparable in both algal species. However, effects of 250 nM Cu on photosynthesis were different, leading to a 12% reduction in photosynthetic activity in P. subcapitata, but not in C. vulgaris. These results indicate that differences in species-specific sensitivities measured as photosynthetic activity were not caused by differences in the cellular ROS content of the algae, but probably by different species-specific ROS defense systems. To investigate the role of ROS in Cu-mediated inhibition of photosynthesis, the ROS scavenger N-tert-butyl-α-phenylnitrone (BPN) was used, resulting in a reduction of Cu-induced ROS production up to control level and a complete restoration of photosynthetic activity of Cu-exposed P. subcapitata. This finding implied that ROS play a primary role in Cu toxicity to algae. Furthermore, we observed a time-dependent ROS release process across the plasma membrane. More than 90% of total ROS were determined to be extracellular in P. subcapitata, indicating an efficient method of cellular protection against oxidative stress.

Mixture toxicity of three herbicides with the same mode of action was studied in a long-term outdoor mesocosm study. Photosynthetic activity of phytoplankton as the direct target site of the herbicides was chosen as physiological response parameter. The three photosystem II (PSII) inhibitors atrazine, isoproturon, and diuron were applied as 30% hazardous concentrations (HC30), which we derived from species sensitivity distributions calculated on the basis of EC50 growth inhibition data. The respective herbicide mixture comprised 1/3 of the HC30 of each herbicide. Short-term laboratory experiments revealed that the HC30 values corresponded to EC40 values when regarding photosynthetic activity as the response parameter. In the outdoor mesocosm experiment, effects of atrazine, isoproturon, diuron and their mixture on the photosynthetic activity of phytoplankton were investigated during a five-week period with constant exposure and a subsequent five-month postexposure period when the herbicides dissipated. The results demonstrated that mixture effects determined at the beginning of constant exposure can be described by concentration addition since the mixture elicited a phytotoxic effect comparable to the single herbicides. Declining effects on photosynthetic activity during the experiment might be explained by both a decrease in water herbicide concentrations and by the induction of community tolerance.

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Effects of three photosystem II inhibitors and of their mixture on a freshwater phytoplankton community were studied in outdoor mesocosms. Atrazine, isoproturon, and diuron were applied as 30% hazardous concentrations (HC30s) obtained from species-sensitivity distributions. Taking concentration addition into account, the mixture comprised one-third of the HC30 of each substance. Effects were investigated during a five-week period of constant concentrations and a five-month posttreatment period when the herbicides dissipated. Total abundance, species composition, and diversity and recovery of the community were evaluated. Ordination techniques, such as principal component analysis and principal response curve, were applied to compare the various treatments on the community level. The three herbicides stimulated comparable effects on total abundance and diversity of phytoplankton during the period of constant exposure because of the susceptibility of the dominant cryptophytes Chroomonas acuta and Cryptomonas erosa et ovata and the prasinophyte Nephroselmis cf. olivacea. Moreover, concentration addition described combined effects of atrazine, isoproturon, and diuron on total abundance and diversity in the constant-exposure period, because their mixture induced effects on abundance and diversity similar to those of the single substances. Principal component and principal response curve analyses revealed that the community structure of diuron- and isoproturon-treated phytoplankton recovered two weeks after constant exposure, which might be related to the fast dissipation of the phenylureas. Species compositions of mixture- and atrazine-treated communities were not comparable to that of the control community five months after the end of constant exposure. This might be explained by the slower dissipation of atrazine relative to the phenylureas and by differences in the species sensitivities, resulting in a different succession of phytoplankton.