To incorporate metal mixture toxicity effects into risk-assessment procedures, more information is needed about combined and interactive effects of metal mixtures during chronic exposure. The authors investigated the toxicity of binary Ni-Zn mixtures in 2 independent full-factorial experiments using standard chronic (21-d) Daphnia magna reproduction toxicity tests. Global statistical analysis (i.e., when considering all investigated mixture treatments simultaneously) showed noninteractive effects according to the concentration addition model and significant synergistic effects according to the independent action model. However, treatment-specific statistical analysis revealed that both occurrence and type of interactive effect were dependent on the effect size at which Ni and Zn were combined in the mixture. Only noninteractive or weakly antagonistic effects occurred in mixture treatments in which each of the individual metals produced only weak adverse effects on its own (i.e., ≤20% reduction of reproductive performance). On the other side of the spectrum, synergistic mixture effects occurred in all mixture treatments where both metals already caused a > 20% (for independent action) and a > 40% (for concentration addition) effect on reproduction on their own. Because low effect sizes are the most relevant in most regulatory frameworks, the authors' data suggest that the concentration addition and independent action mixture toxicity models can both serve as conservative models for predicting effects of Ni-Zn mixtures. The present study highlights the importance of investigating metal mixture toxicity at low effect sizes and warns against extrapolating conclusions about metal mixture interactions from high to low effect sizes.

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Although natural populations can harbor evolutionary potential to adapt genetically to chemical stress, it is often thought that natural selection leads to a general reduction of genetic diversity and involves costs. Here, a 10 week microevolution experiment was conducted with a genetically diverse and representative sample of one natural Daphnia magna population that was exposed to copper and zinc. Both Cu- and Zn-selected populations developed a significantly higher metal tolerance (i.e., genetic adaptation), indicated by higher reproduction probabilities of clonal lines in Cu and Zn exposures than observed for the original and control populations. The complete recovery of the population densities after 10 weeks of Zn selection (following an initial decrease of 74%) illustrates an example of evolutionary rescue. Microsatellite genotyping revealed a decrease in clonal diversity but no change in allelic richness, and showed an excess in heterozygosity in the Cu- and Zn-selected populations compared to the control and original populations. The excess heterozygosity in metal-selected populations that we observed has important consequences for risk assessment, as it contributes to the maintenance of a higher allelic diversity under multigenerational chemical exposure. This study is, to our knowledge, the first report of an increase in heterozygosity following multigenerational exposure to metal stress, despite a decline in clonal diversity. In a follow-up study with the Zn-selected populations, we observed no effect of Zn selection on the tolerance to heat and cyanobacteria. However, we observed higher tolerance to Cd in the Zn-selected than in the original and control populations if the 20% effective concentration of Cd was considered (cross-tolerance). Our results suggest only limited costs of adaptation but future research is needed to evaluate the adaptive potential of metal-selected populations to novel stressors and to determine to what extent increased heterozygosity is preserved after genetic recombination following periods of sexual reproduction.

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