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.

The effects of complex metal oxide mixtures on three soil invertebrates with contrasting biological traits.

For regulatory purposes, the concentration addition model is the default first tier for assessing joint-action toxicity of metal mixtures. Although many researchers have evaluated binary and ternary mixtures, fewer have investigated joint-action toxicity in more complex mixtures, where deviations from additivity are more likely due to the greater number of potential interactions. In this study, we tested fixed ratios of five metals (lead, copper, nickel, zinc, cobalt) as metal oxide mixtures on three soil invertebrate species (Enchytraeus crypticus, Folsomia candida, Oppia nitens) at different dose effect levels (EC10-EC90) in an acid sandy forest and a loamy soil. Total metal concentrations for mixture ratios in soil did not explain or correlate with species responses. For F. candida, toxicity was linked to metal solubility, while for O. nitens and E. crypticus, toxicity did not correlate with total or extractable metals. In O. nitens and E. crypticus, however, soil ingestion could be an important route of exposure. Analysing the joint effect of metal mixtures, F. candida response was globally additive, while E. crypticus and O. nitens both presented synergistic effects at low-dose effect levels. Estimations at the EC50 level underestimated the deviations from additivity which were larger at higher and especially lower effect levels. Testing across different effect concentrations (EC10-EC90) was an important tool allowing the identification of these larger deviations from additivity outside the EC50 threshold. Considering most protection thresholds are set below the EC50 level, and it was in this low effect range where the highest synergisms were observed, risk assessment schemes should test additivity at the target protection level using representative test organisms.

Metal oxides and annealed metals as alternatives to metal salts for fixed-ratio metal mixture ecotoxicity tests in soil.

In soil metal ecotoxicology research, dosing is usually performed with metal salts, followed by leaching to remove excess salinity. This process also removes some metals, affecting metal mixture ratios as different metals are removed by leaching at different rates. Consequently, alternative dosing methods must be considered for fixed ratio metal mixture research. In this study three different metal mixture dosing methods (nitrate, oxide and annealed metal dosing) were examined for metal concentrations and toxicity. In the nitrate metal dosing method leaching reduced total metal retention and was affected by soil pH and cation exchange capacity (CEC). Acidic soils 3.22 (pH 3.4, CEC 8 meq/100g) and WTRS (pH 4.6, CEC 16 meq/100g) lost more than 75 and 64% of their total metals to leaching respectively while Elora (6.7 pH, CEC 21 meq/100g) and KUBC (pH 5.6, CEC 28 meq/100g) with higher pH and CEC only lost 13.6% and 12.2% total metals respectively. Metal losses were highest for Ni, Zn and Co (46.0%, 63.7% and 48.4% metal loss respectively) whereas Pb and Cu (5.6% and 20.0% metal loss respectively) were mostly retained, affecting mixture ratios. Comparatively, oxide and annealed metal dosing which do not require leaching had higher total metal concentrations, closer to nominal doses and maintained better mixture ratios (percent of nominal concentrations for the oxide metal dosing were Pb = 109.9%, Cu = 84.6%, Ni = 101.9%, Zn = 82.3% and Co = 97.8% and for the annealed metal dosing were Pb = 81.7%, Cu = 80.3%, Ni = 100.5%, Zn = 89.2% and Co = 101.3%). Relative to their total metal concentrations, nitrate metal dosing (lowest metal concentrations) was the most toxic followed by metal oxides dosing while the annealed dosing method was generally non-toxic. Due to the lack of toxicity of the annealed metals and their higher dosing effort, metal oxides, are the most appropriate of the tested dosing methods, for fixed-ratio metal mixtures studies with soil invertebrates.

Single metal and metal mixture toxicity of five metals to Oppia nitens in five different Canadian soils.

Metal mixture toxicity across soil types is a daunting challenge to risk assessment. Here, we evaluated metal mixture toxicity in Oppia nitens, using ten fixed metal mixture ratios in five Canadian soils that closely matched some of the EU PNEC reference soils. Soils were dosed with five metals (Cu, Zn, Pb, Co, Ni) as single metals (ten concentrations) and as mixtures (eight concentrations). Synchronized adult mites were exposed to metals, with survival and reproduction assessed after 28 days. We found out that (i) the differences among soils in mite sensitivity and single metals were not consistent when mites were exposed to metal mixtures, (ii) assuming concentration addition, the mixture interaction factor (MIF) showed that single metal low effect levels excessively underestimated low level metal mixture effects (iii) Zn emerged as a protective metal in most mixtures, and (iv) Soil properties such as CEC, independent of effects on metal speciation, explained more of the variation than measured metals. This study suggests that metal risk assessment should be done on a case by case basis. Further work is needed to ensure that by protecting soil-dwelling organisms from single metals, the risk from metal mixtures is appropriately protected for.

Toxicity assessment of metal mixtures to soil enzymes is influenced by metal dosing method.

Metals are present as mixtures in the environment, yet testing such complex mixture poses design and technical challenges. One possible solution is the use of fixed ratios, i.e. rays of increasing metal concentrations. But fixed ratios rays are compromised when soils dosed with metal salts are leached due to metal-soil selectivity rules. Two alternative metal forms, metal oxides and spinel minerals of quinary metal mixtures (Pb, Cu, Co, Ni, Zn), were evaluated for their toxicity to soil microorganisms measured by the activity of ammonia monooxygenases and acid-phosphatases in three soils. Leaching, a required step for salts, had a larger effect on ammonia monooxygenases than metals. Generally, metal salts were the most toxic form, while the spinel minerals were the least toxic form. Two extractants, CaCl2 and DTPA, were evaluated for their ability to link toxicity to metals across all three metal forms. Salt toxicity was closely linked to CaCl2 extractable concentrations but DTPA was the most appropriate for oxides. We strongly recommend combining fixed ratio rays with metal oxides for metal mixture studies, since soil ratios created using oxides were more precise and required less experimental effort compared to salts and spinel minerals. Furthermore, because DTPA and CaCl2 closely tracked the toxicity of more realistic metal forms (i.e. oxides), we recommend that field studies investigating metal mixtures use both DTPA and CaCl2.

Petroleum hydrocarbon mixture toxicity and a trait-based approach to soil invertebrate species for site-specific risk assessments.

Although petroleum hydrocarbons released to the environment typically occur as mixtures, petroleum hydrocarbon remediation guidelines often reflect individual substance toxicity. It is well documented that groups of aliphatic petroleum hydrocarbons act via the same mechanism of action (nonpolar narcosis) and, theoretically, concentration addition mixture toxicity principles apply. To assess this theory, 10 standardized acute and chronic soil invertebrate toxicity tests on a range of organisms (Eisenia fetida, Lumbricus terrestris, Enchytraeus crypticus, Folsomia candida, Oppia nitens, and Hypoaspis aculeifer) were conducted with a refined petroleum hydrocarbon binary mixture. Reference models for concentration addition and independent action were applied to the mixture toxicity data with consideration of synergism, antagonism, and dose level toxicity. Both concentration addition and independent action, without further interactions, provided the best fit with observed response to the mixture. Individual fraction effective concentration values were predicted from optimized, fitted reference models. Concentration addition provided a better estimate than independent action of individual fraction effective concentrations based on comparison with available literature and species trends observed in toxic responses to the mixture. Interspecies differences in standardized laboratory soil invertebrate species responses to petroleum hydrocarbon-contaminated soil was reflected in unique traits. Diets that included soil, large body size, permeable cuticle, low lipid content, lack of ability to molt, and no maternal transfer were traits linked to a sensitive survival response to petroleum hydrocarbon-contaminated soil in laboratory tests. Traits linked to sensitive reproduction response in organisms tested were long life span and small clutch size. By deriving single-fraction toxicity endpoints considerate of mixtures, we can reduce the resources and time required to conduct site-specific risk assessments for the protection of a soil organism's exposure pathway. Environ Toxicol Chem 2018;37:2222-2234. © 2018 SETAC.