1875 Environmental Toxicology and Chemistry, Vol. 24, No. 8, pp. 1875–1885, 2005  2005 SETAC Printed in the USA 0730-7268/05 $12.00 + .00 EFFECT OF SOIL PROPERTIES AND AGING ON THE TOXICITY OF COPPER FOR ENCHYTRAEUS ALBIDUS, ENCHYTRAEUS LUXURIOSUS, AND FOLSOMIA CANDIDA MO ´ NICA JOA ˜ O DE BARROS AMORIM,*² J ORG RO ¨ MBKE,‡ HANS-JOACHIM SCHALLNAß,‡ and AMADEU MORTA ´ GUA VELHO MAIA SOARES² ²Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal ‡ECT Oekotoxikologie, Boettgerstrasse 2-14, D-65439 Flo ¨rsheim, Germany ( Received 7 October 2004; Accepted 24 January 2005) Abstract—In the present study, the effect of the heavy-metal salt copper chloride (CuCl 2 ·2H 2 O) in soils freshly spiked (3 d) and aged (70  10 d; mean  SD) was studied in the test species Enchytraeus albidus, E. luxuriosus, and Folsomia candida. Up to nine soils were used: Besides the Organisation for Economic Co-operation and Development (OECD) artificial soil and the Ag- ricultural Testing and Research Agency (Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Speyer, Germany) 2.2 natural standard soils, the others were selected based on the EURO Soil approach, taking into account the effect of different soil parameters (pH, organic matter, grain size distribution, and carbon to nitrogen ratio). Additionally, the effect of the chloride ions was studied separately. The results revealed the following: First, a soil effect was observed; for example, in F. candida, median effective concentrations (EC50s) varied between 262 mg/kg in a sample from the same site as the original EURO Soil 5 soil and greater than 1,000 mg/kg in OECD soil. Second, an aging effect was observed, mainly in F. candida. For example, toxicity of offspring survival was increased twofold in the OECD soil and approximately eightfold with aging in the EURO Soil 7 soil, whereas the enchytraeid species did not react differently after aging. Third, an effect of chloride ions on reproduction of the animals was found; however, this effect was independent of the aging period. Fourth, species variation was seen in terms of sensitivity (EC50), decreasing in the following order: E. luxuriosus  E. albidus  F. candida. Differences in toxicity of offspring survival between enchytraeids and F. candida might be explained by the different routes of uptake. Keywords—Natural soils Laboratory tests EURO-Soils INTRODUCTION Terrestrial toxicity not only varies between species, the soil characteristics also greatly influence the effect concentration of metals by altering, for instance, the bioavailability [1]. For reasons of practicability and comparability, it is most common in toxicity testing to use the Organisation for Economic Co- operation and Development (OECD) artificial soil [2] or the standard natural Landwirtschaftliche Untersuchungs- und For- schungsanstalt (LUFA; Speyer, Germany) 2.2 soil. Neverthe- less, current risk-assessment procedures ignore that variation in soil properties results in substantial differences regarding uptake in and effects on organisms in different soils [3]. Aging is an important issue to take into account: Laboratory tests should mimic the most realistic situation, and freshly spiked soils do not allow the equilibration time that is required to resemble the common field situation. Incorporating the effect of aging in the environmental risk assessment of metal-con- taminated soils may contribute to a more realistic assessment regarding the impact of metals on terrestrial ecosystems [4]. One of the problems is in terms of feasibility of the tests: The recommended aging period is a minimum of 60 d [5], a very long period considering the urgent demand for results and the need for experimental repetition. Therefore, the assurance of the need for such periods is very relevant and should be in- vestigated. In the present study, we tested the effects of the heavy- metal copper on two groups of organisms, enchytraeids and collembolans. Organisms with different exposure routes, such * To whom correspondence may be addressed (mjamorim@bio.ua.pt). as oligochaetes and arthropods, should be used simultaneously to assess the environmental risk of metal-contaminated soils [6]. Additionally, standardized test procedures are available for the selected test species: Enchytraeus albidus, E. luxurio- sus, and Folsomia candida. The common long-term applica- tion of copper fungicides against pests results in soil contam- ination [7]. Despite the fact that copper is an essential metal, at high dosages it becomes toxic to soil invertebrates. Other reasons for selecting copper as a test substance include that this is a well-known substance and that results from our studies can be compared with the Dutch (see, e.g., [3]) and Belgian [8] data. Therefore, the main aims were to study the effects of the characteristics of different soils in terms of the suitability of the test species and the influence on the toxicity of a certain toxic substance. MATERIALS AND METHODS Test species Two groups of organisms were used, enchytraeids and col- lembolans. The test species selected among the enchytraeids were E. albidus Henle 1837 and E. luxuriosus [9]. Enchy- traeus albidus is one of the largest species of the genus En- chytraeus (adults reach 15–40 mm), whereas E. luxuriosus is much smaller (adults reach 8–13 mm). Both species were maintained in laboratory cultures, being bred in moist soil (50% OECD soil, 50% natural garden soil) at 20°C in the dark and fed once a week with finely ground and autoclaved rolled oats (Cimarron, Portugal). Details of the culturing process are given in Ro ¨mbke and Moser [10].