Metal toxicity assessment in soils using enzymatic activity: Can water be used as a surrogate buffer? Isabelle Lessard a, * , Giancarlo Renella b , Sébastien Sauvé c , Louise Deschênes a a CIRAIG, Chemical Engineering Department, Polytechnique Montréal, P.O. Box 6079, Montréal, Quebec H3C 3A7, Canada b Department of Plant, Soil and Environmental Sciences, University of Florence, P.le delle Cascine,18 50144 Florence, Italy c Department of Chemistry, Université de Montréal, P.O. Box 6128, Centre-Ville, Montréal, Quebec H3C 3J7, Canada article info Article history: Received 1 May 2012 Received in revised form 7 September 2012 Accepted 8 September 2012 Available online 23 September 2012 Keywords: Enzymes Enzymatic activity Ecotoxicity Buffer pH Speciation Metal Zinc abstract Ecotoxicological tests based on soil enzyme activity are widely used to assess the terrestrial ecotox- icology of metals in soils. However, several standard enzymatic methods use buffers that may alter the chemical pseudoequilibrium of soils and affect metal speciation, and, in turn, the metal effects on enzymes and enzyme kinetics. Researchers have suggested the use of H 2 O as a solvent rather than chemical buffers, but opponents are concerned about pH fluctuations during incubation and the resulting difficulty in comparing enzymatic studies. Enzyme assays were conducted on 10 pairs of Zn- contaminated soils to evaluate 1) the buffer effect on Zn lability 2) the pH fluctuation during enzy- matic assays conducted in water and 3) the comparison of enzymatic results obtained using chemical buffers versus water. Four standard enzymatic methods covering the major biogeochemical cycles were targeted: arylsulfatase (acetate pH 5.8), urease (borate pH 10), acid phosphatase (modified universal buffer pH 6.5) and protease (THAM pH 8.1). Furthermore, deionized water was tested in parallel as a surrogate solvent for these four methods. With the exception of the acetate buffer, the tested solvents did not significantly change the labile Zn concentration in the soil samples. The pH slightly fluctuated by þ0.57 pH unit, corresponding to the intrinsic variability of soils. Enzymatic methods using buffers showed similar results compared to those using water, except for urease. These observations suggest that enzymatic methods setting alkaline conditions should be used with caution and that H 2 O could be used as surrogate solvent in this context. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction The ecotoxicological assessment of metal-contaminated field- collected soils based on the estimated aggregated activity of several soil enzymes appears to be a suitable approach to develop routine terrestrial ecotoxicological tests (Epelde et al., 2008). Soil enzy- matic activity (EA) reflects the rate-limiting step of biogeochemical decomposition required for microbial processes and is reproduc- ible, affordable and easy to execute (German et al., 2011). Aggre- gating the results of many enzyme activities covering the C, P, N, and S cycles yields unique quantitative indices of soil functional diversity that could be used as bioindicators for soil health and metal toxicity (Bastida et al., 2008). For example, an assessment of the functional diversity of metal-contaminated soils using a modi- fied Shannon’s diversity index adapted for soil enzymes (acid and alkaline phosphatase, urease, arylsulfatase and b-glucosidase) showed that the presence of metal significantly affects the index (Epelde et al., 2008). Sunray plots were also used as schematic representations of enzymatic activities in metal-contaminated soils (dehydrogenase, urease, acid and alkaline phosphatase, arylsulfa- tase and b-glucosidase) compared to an uncontaminated soil (Hinojosa et al., 2004; Bastida et al., 2008). Biochemical analyses of metal-contaminated field-collected soils constitute a powerful approach to integrate the in situ partitioning of trace elements, thus considering metal speciation and bioavailability at pseudoequilibriumdtwo critical concepts for the assessment of metal toxicity in ecosystem studies (Sauvé, 2001). The labile (reactive) and free metal concentrations are the most widely- accepted metal fractions estimating metal bioavailability. The labile metal in the soil solution includes the free metal and metal ion-pair (mainly inorganic but also some organic ligands) showing a rapid dissociationeassociation kinetics (Nolan et al., 2005). Most of the soil enzymatic activity measurement methods recommend running assays in soils using optimal parameters such * Corresponding author. Tel.: þ1 514 340 4711x4794; fax: þ1 514 340 5913. E-mail address: isabelle.lessard@polymtl.ca (I. Lessard). Contents lists available at SciVerse ScienceDirect Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio 0038-0717/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.soilbio.2012.09.009 Soil Biology & Biochemistry 57 (2013) 256e263