The influence of time on lead toxicity and bioaccumulation determined by the OECD earthworm toxicity test Nicola A. Davies a,b , Mark E. Hodson b, *, Stuart Black a a Postgraduate Research Institute for Sedimentology, University of Reading, PO Box 227, Whiteknights, Reading, Berkshire RG6 6AB, UK b Department of Soil Science, University of Reading, PO Box 233, Whiteknights, Reading, Berkshire RG6 6DW, UK Received 17 September 2001; accepted 3 April 2002 ‘‘Capsule’’: Timing of lead addition and worms to soil affects the response of the worms to soil affects the response of the worms to lead. Abstract Internationally agreed standard protocols for assessing chemical toxicity of contaminants in soil to worms assume that the test soil does not need to equilibrate with the chemical to be tested prior to the addition of the test organisms and that the chemical will exert any toxic effect upon the test organism within 28 days. Three experiments were carried out to investigate these assumptions. The first experiment was a standard toxicity test where lead nitrate was added to a soil in solution to give a range of concentrations. The mortality of the worms and the concentration of lead in the survivors were determined. The LC 50 s for 14 and 28 days were 5311 and 5395 mg Pb g 1 soil respectively. The second experiment was a timed lead accumulation study with worms cultivated in soil con- taining either 3000 or 5000 mg Pb g 1 soil . The concentration of lead in the worms was determined at various sampling times. Uptake at both concentrations was linear with time. Worms in the 5000 mgg 1 soil accumulated lead at a faster rate (3.16 mgPbg 1 tissue day 1 ) than those in the 3000 mgg 1 soil (2.21 mgPbg 1 tissue day 1 ). The third experiment was a timed experiment with worms cultivated in soil containing 7000 mg Pb g 1 soil . Soil and lead nitrate solution were mixed and stored at 20  C. Worms were added at various times over a 35-day period. The time to death increased from 23 h, when worms were added directly after the lead was added to the soil, to 67 h when worms were added after the soil had equilibrated with the lead for 35 days. In artificially Pb-amended soils the worms accumulate Pb over the duration of their exposure to the Pb. Thus time limited toxicity tests may be terminated before worm body load has reached a toxic level. This could result in under-estimates of the toxicity of Pb to worms. As the equilibration time of artificially amended Pb-bearing soils increases the bioavailability of Pb decreases. Thus addition of worms shortly after addition of Pb to soils may result in the over-estimate of Pb toxicity to worms. The current OECD acute worm toxicity test fails to take these two phenomena into account thereby reducing the environmental relevance of the contaminant toxicities it is used to calculate. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Metals; Bioavailability; Earthworms; Ecotoxicological testing; Eisenia fetida 1. Introduction The toxicity of substances on contaminated land is becoming an issue of economic and political impor- tance, due to the reduction in available pristine land for development and the increasing need to re-use brown field sites (DETR, 1997). These sites often have asso- ciated pollution from their previous use and before development can begin, the risk associated with contaminants in the soil to both the environment in general and human health must be assessed accurately (Griffiths and Board, 1992). However, levels of metals in soils that cause harm to ecosystems are generally poorly understood, and current legislation and assessment of toxic metal concentrations in soils is based on the total concentration of metals present in the soil (DOE, 1980; Giesler, 1987; Canada Council of Ministers of the Environment, 1992; Tadesse et al., 1994). One promis- ing way forward in the assessment of acceptable levels of metals in soils, in terms of ecosystem health, is through the use of standardised toxicity tests following internationally agreed protocols. An example of such a 0269-7491/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0269-7491(02)00207-5 Environmental Pollution 121 (2003) 55–61 www.elsevier.com/locate/envpol * Corresponding author. Tel.: +44-118-931-6974; fax: +44-118- 931-6660. E-mail address: m.e.hodson@reading.ac.uk (M.E. Hodson).