1162 Environmental Toxicology and Chemistry, Vol. 22, No. 5, pp. 1162–1166, 2003  2003 SETAC Printed in the USA 0730-7268/03 $12.00 + .00 INFLUENCE OF AGING ON COPPER BIOAVAILABILITY IN SOILS KOEN LOCK* and COLIN R. JANSSEN Ghent University, Laboratory of Environmental Toxicology and Aquatic Ecology, J. Plateaustraat 22, 9000 Gent, Belgium ( Received 9 July 2002; Accepted 1 November 2002) Abstract—Because of long-term chemical processes, metal bioavailability in field soils decreases with time. Metal toxicity may, therefore, be overestimated if toxicity data with freshly spiked soils are used to derive soil quality criteria, a current practice. In the present study, effects of the long-term processes, called aging, on copper partitioning and ecotoxicity are investigated. Twenty- five field soils contaminated by copper runoff from bronze statues and 25 uncontaminated control soils sampled at 5-m distance from these statues were collected in Flanders (Belgium). The soils were selected so that parameters affecting copper bioavailability (pH, cation-exchange capacity, organic matter content, etc.) varied considerably. To assess the effect of aging on copper toxicity, control soils were spiked at total copper concentrations comparable to those of historically contaminated soils. Pore-water copper concentrations and 0.01 M CaCl 2 –extracted copper concentrations were significantly higher in freshly spiked soils compared to contaminated field soils. However, this could be a pH effect, because pH decreased after spiking. Acute toxicity to Enchytraeus albidus (14 d) as well as chronic toxicity to Folsomia candida (28-d reproduction) and Trifolium pratense (14-d growth) indicated a dose–response relationship between copper toxicity and pore-water copper concentration or the CaCl 2 -extracted copper fraction. Keywords—Ecotoxicology Enchytraeus albidus Folsomia candida Trifolium pratense INTRODUCTION Total metal concentrations in soils are poor predictors of toxicity to terrestrial organisms, because soil characteristics influence metal bioavailability. The importance of considering metal bioavailability in risk assessments of contaminated soils is widely recognized [1–3]. Indeed, not accounting for changes in bioavailability may lead to under- or overestimation of the predicted no-effect concentration, which can have serious con- sequences for the ecological system under consideration. This is especially true when assessing the environmental risks of essential metals such as copper, for which not only toxicity but also possible deficiency effects should be considered [4]. Derivations of soil quality criteria, such as predicted no-effect concentrations or maximum permissible concentrations, are usually based on results of ecotoxicity tests in which freshly metal-spiked soils are used. With these data, species sensitivity distributions are fitted, from which hazardous concentrations and predicted no-effect concentrations are derived [5,6]. How- ever, because of long-term processes, called aging, metal avail- ability decreases with time [3,7,8]. Soil quality criteria based on this type of toxicity data considerably overlap with natural background concentrations of copper, because both toxicity data and background concentrations are expressed in total, not bioavailable, concentrations. Here, we investigated the influ- ence of long-term processes (i.e., aging) on the partitioning and ecotoxicity of copper to enable more realistic effect-based risk assessments of copper-contaminated soils. MATERIALS AND METHODS Test organisms The initial culture of the springtail Folsomia candida was obtained from Aquasense B.V. (Amsterdam, The Netherlands). Animals were cultured on a substrate of plaster of Paris and * To whom correspondence may be addressed (koen.lock@rug.ac.be). pulverized, chemical-activated charcoal in a ratio of 8:1 weight/weight (wt/wt). Granulated dry yeast was added weekly as a food source. The initial culture of Enchytraeus albidus was kindly provided by J. Ro ¨mbke (ECT Oekotoxikologie, Flo ¨rsheim, Germany). The culture substrate of E. albidus con- sisted of artificial soil [9], and animals were fed once a week with ground rolled oats. Both cultures have been maintained in our laboratory for at least six years at 20°C and in complete darkness. Seeds of red clover (Trifolium pratense) were ob- tained from commercial florist (Aveve, Lochristi, Belgium). Toxicity assays Chronic toxicity tests with F. candida were carried out according the International Organization for Standards (Ge- neva, Switzerland) [10]. Ten synchronized springtails (age, 10–12 d) were exposed per glass vessel containing 30 g wet weight of soil. Granulated dry yeast was added weekly on the soil surface as a food source. The reproduction test with F. candida takes four weeks to complete. At the end of the test, juveniles were photographed and counted after flotation. Acute toxicity assays with E. albidus were performed following Or- ganisation for Economic Cooperation and Development (OECD) Guideline 220 [11]. Ten adult worms with a fully developed clitellum were exposed per glass vessel containing 20 g wet weight of soil. Surviving animals were counted after 14 d of exposure. Plant growth tests were performed according to OECD Guideline 208 [12]. Ten seeds were sown in 100 g wet weight of soil. Fourteen days after 50% of the seedlings had emerged in the control soils, all plants were harvested, dried to constant weight at 105°C, and weighed. All inverte- brate and plant tests were carried out at a soil moisture content of 50% of the water-holding capacity. Soil moisture content was adjusted twice a week by replenishing weight loss with the appropriate amount of deionized water. During exposure, vessels were kept at 20  1°C (mean  SEM throughout) and