Response addition is more protective of biogeochemical cycles of carbon and phosphorus compared to concentration addition

In soils, enzymes are crucial to catalyzing reactions and cycling elements such as carbon (C), nitrogen (N), and phosphorus (P). Although these soil enzymes are sensitive to metals, they are often disregarded in risk assessments, and regulatory laws governing their existence are unclear. Nevertheless, there is a need to develop regulatory standards for metal mixtures that protect biogeochemical cycles because soil serve as a sink for metals and exposures occur as mixtures. Using a fixed ratio ray design, we investigated the effects of 5 single metals and 10 quinary mixtures of Zn, Cu, Ni, Pb, and Co metal oxides on two soil enzymes (i.e., acid phosphatases [ACP] and beta glucosidases [BGD]) in two acidic Canadian soils (S1: acid sandy forest soil, and S2: acid sandy arable soil), closely matched to EU REACH standard soils. Compared to BGD, ACP was generally the more sensitive enzyme to both the single metals and the metal mixtures. The effective concentration inhibiting 50% enzyme activity (EC₅₀) estimates for single Cu (2.1–160.7 mmol kg⁻¹) and Ni (12–272 mmol kg⁻¹) showed that those were the most toxic to both enzymes in both soils. For metal mixtures, response addition (RA) was more conservative in predicting metal effects compared to concentration addition (CA). For both additivity models, antagonism was observed except at lower concentrations (≤10,000 mg/kg) where synergism was observed. At higher concentrations (>10,000 mg/kg), free and CaCl₂ extractable Cu protected both enzymes against the toxicity of other metals in the mixture. The results suggest that assuming CA at concentrations less than EC₅₀ does not protect biogeochemical cycling of C and P. And Cu in soil may protect soil enzymes from other toxic metals and thus may have an overall positive role.