Disentangling the effects of low pH and metal mixture toxicity on macroinvertebrate diversity * Riccardo Fornaroli a, * , Alessio Ippolito b, 1 , Mari J. Tolkkinen c , Heikki Mykr € a d , Timo Muotka c, e , Laurie S. Balistrieri f , Travis S. Schmidt g a Department of Earth and Environmental Science (DISAT), University of Milano-Bicocca, Milan, Italy b European Food Safety Authority (EFSA), Via Carlo Magno 1, 43126 Parma, Italy c Department of Ecology and Genetics, University of Oulu, Oulu, Finland d Finnish Environment Institute, Freshwater Centre, Oulu, Finland e Finnish Environment Institute, Natural Environment Centre, Oulu, Finland f US Geological Survey, and University of Washington, School of Oceanography, Seattle, WA, USA g US Geological Survey, Colorado Water Science Center, Fort Collins, CO, USA article info Article history: Received 1 September 2017 Received in revised form 19 December 2017 Accepted 24 December 2017 Keywords: Stream invertebrates Metal accumulation pH Biodiversity Limiting effect Quantile regression abstract One of the primary goals of biological assessment of streams is to identify which of a suite of chemical stressors is limiting their ecological potential. Elevated metal concentrations in streams are often asso- ciated with low pH, yet the effects of these two potentially limiting factors of freshwater biodiversity are rarely considered to interact beyond the effects of pH on metal speciation. Using a dataset from two continents, a biogeochemical model of the toxicity of metal mixtures (Al, Cd, Cu, Pb, Zn) and quantile regression, we addressed the relative importance of both pH and metals as limiting factors for macro- invertebrate communities. Current environmental quality standards for metals proved to be protective of stream macroinvertebrate communities and were used as a starting point to assess metal mixture toxicity. A model of metal mixture toxicity accounting for metal interactions was a better predictor of macroinvertebrate responses than a model considering individual metal toxicity. We showed that the direct limiting effect of pH on richness was of the same magnitude as that of chronic metal toxicity, independent of its influence on the availability and toxicity of metals. By accounting for the direct effect of pH on macroinvertebrate communities, we were able to determine that acidic streams supported less diverse communities than neutral streams even when metals were below no-effect thresholds. Through a multivariate quantile model, we untangled the limiting effect of both pH and metals and predicted the maximum diversity that could be expected at other sites as a function of these variables. This model can be used to identify which of the two stressors is more limiting to the ecological potential of running waters. © 2018 Elsevier Ltd. All rights reserved. 1. Introduction One of the primary goals of biological assessment of streams is to identify which of a suite of chemical stressors is limiting their ecological potential. Quantitative relationships between species richness and environmental gradients are needed to better predict biodiversity patterns, to develop increasingly accurate biotic indices, and to set environmental quality standards. However, describing these relationships is not straightforward, because species richness is typically dependent on a multitude of environ- mental gradients (Friberg et al., 2011; Ormerod et al., 2010) such as water pollution (Kail et al., 2012), nutrient supplies (Wagenhoff et al., 2012), habitat quality (Dunbar et al., 2010) and changes in climate and land use (Kuemmerlen et al., 2015). Elevated concentrations of metals are common in streams draining mineralized and especially mined basins (Clements et al., * This paper has been recommended for acceptance by Prof. W. Wen-Xiong. * Corresponding author. Department of Earth and Environmental Science (DISAT), University of Milano-Bicocca, Piazza della Scienza 1, 20126, Milan, Italy. E-mail address: riccardofornaroli@gmail.com (R. Fornaroli). 1 DisclaimerdA. Ippolito is employed by the European Food Safety Authority. The positions and opinions presented in this article are those of the authors alone and do not necessarily represent the views or scientific works of the European Food Safety Authority. Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/locate/envpol https://doi.org/10.1016/j.envpol.2017.12.097 0269-7491/© 2018 Elsevier Ltd. All rights reserved. Environmental Pollution 235 (2018) 889e898