788 Environmental Toxicology and Chemistry, Vol. 25, No. 3, pp. 788–796, 2006  2006 SETAC Printed in the USA 0730-7268/06 $12.00 + .00 EFFECT OF pH ON METAL SPECIATION AND RESULTING METAL UPTAKE AND TOXICITY FOR EARTHWORMS DAVID J. SPURGEON,*² S TEPHEN LOFTS,‡ PETER K. HANKARD,² M ARK TOAL,² D ONNA MCLELLAN,² SAMANTHA FISHWICK,§ and CLAUS SVENDSEN² ²Centre for Ecology and Hydrology, Monks Wood, Abbots Ripton, Huntingdon, Cambridgeshire PE28 2LS, United Kingdom ‡Centre for Ecology and Hydrology, Library Avenue, Bailrigg, Lancaster LA1 4AP, United Kingdom §Environment Agency, Block 1 Government Buildings, Burghill Road, Westbury-on-Trym, Bristol BS10 6BF, United Kingdom ( Received 18 January 2005; Accepted 3 May 2005) Abstract—In the present study, relationships between changes in the solubility and speciation of metals in contaminated soils under different pH regimes and their toxicity to earthworms were investigated. Earthworms (Lumbricus rubellus) were exposed in a laboratory bioassay to metalliferous soils under three pH regimes: Unamended pH, pH lowered by one unit (pH -1), and pH increased by one unit (pH +1). In each soil, total (hot nitric acid–extractable) and 0.01 M CaCl 2 –extractable metal concentrations were measured and soil pore-water chemistry analyzed to allow metal speciation to be modeled using the Windermere Humic Aqueous Model. Earthworm metal accumulation was determined and toxicity assessed by measuring survival and reproduction and at the molecular level by recording expression of the gene encoding metallothionein-2 (MT-2) using quantitative reverse transcriptase– polymerase chain reaction. Both metal solubility and speciation were found to be highly pH dependent. Metal accumulation in earthworms was influenced by soil concentration and, in some cases (e.g., Cd), by pH. Reproduction was affected (reduced up to 90%) by soil metal level, pH, and their interaction. Relationships between analyzed and calculated Zn concentrations and toxicity and between analyzed and calculated Cd concentrations and tissue accumulation and MT-2 expression were compared by fitting dose–response models and assessing the fit of the data. This analysis indicated that values based on a pH-adjusted free ion concentration best explained toxicity (r 2 = 0.82) and accumulation (r 2 = 0.54). Expression of MT-2 was, however, poorly correlated ( p  0.05) with all analyzed and modeled soil metal concentrations. Keywords—Zinc Cadmium Biological availability Toxicological availability Free ion activity INTRODUCTION Variations in soil properties can alter the partitioning of chemicals [1,2]. As a result, uptake and toxicity of chemicals in soil-dwelling organisms are governed not only by the total concentration present but also by the soil conditions [3–6]. An understanding of the influence of soil properties on chemical toxicity is, thus, a prerequisite for accurate prediction of the site-specific risks of toxic chemicals in soil ecosystems. The importance of soil properties in modulating bioavail- ability has led to the development of modeling approaches that describe the important soil–chemical interactions. For lipo- philic organic chemicals, the equilibrium-partitioning concept has been applied. This approach assumes that dermal uptake of the fraction of the chemical present in pore water is the main route of exposure [7]. Recently, Jager et al. [1] dem- onstrated that intestinal uptake contributes significantly to the accumulation of organic chemicals in earthworms; neverthe- less, the equilibrium-partitioning concept remained valid. For metals, a diversity of modeling approaches have been developed to assess partitioning and speciation in soils. All are based on simulation of interactions between the metal ion and ligands on particles and in soil solution. One of the most well-developed speciation models is the Windermere Humic Aqueous Model (WHAM). Designed to calculate equilibrium chemical speciation in surface waters and groundwaters, sed- * To whom correspondence may be addressed (dasp@ceh.ac.uk). Presented at the Symposium on Risk Assessment of Metals in Soils, 14th Annual Meeting, SETAC Europe Meeting, Prague, Czech Republic, April 18–22, 2004. iments, and soils, WHAM combines a humic ion–binding mod- el with a simple inorganic speciation code for aqueous solu- tions. The model is designed to take into account the precip- itation of Al and Fe oxides, cation exchange on an idealized clay mineral, and adsorption–desorption reactions of fulvic acid. The model was developed initially by Tipping [8], and it has been used to study speciation under a range of water and soil conditions. Recently, attempts have been made to establish a link between metal speciation and toxicity for soil species. That work has indicated the important role that the free metal ion may play in defining toxic effect [2,9,10]. To investigate how metal speciation chemistry influences toxicity for soil species, the present study uses WHAM to interpret how changes in soil conditions alter speciation of metals and their uptake and toxicity for earthworms (Lum- bricus rubellus). For the present study, worms were exposed to metalliferous-contaminated field soils under three pH treat- ments: Unamended pH, pH lowered by one unit (pH -1), and pH increased by one unit (pH +1). In each soil, environmental availability was assessed by measuring total and extractable metal concentrations and by analyzing soil pore-water prop- erties to allow metal speciation to be modeled. Earthworm metal accumulation was analyzed to determine biological availability and the effects of exposure on the induction of the intracellular metal detoxification system assessed by recording changes in the expression of a gene that encodes the metal- binding protein metallothionein-2 (MT-2). Toxicity also was assessed at the whole-organism level by measuring survival and reproductive rate.