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The influence of time on lead toxicity and bioaccumulation determined by the OECD earthworm toxicity test
Environmental Pollution 121 (2003) 55–61 www.elsevier.com/locate/envpol
The influence of time on lead toxicity and bioaccumulation determined by the OECD earthworm toxicity test Nicola A. Daviesa,b, Mark E. Hodsonb,*, Stuart Blacka 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 LC50s for 14 and 28 days were 5311 1 and 5395 mgPb gsoil respectively. The second experiment was a timed lead accumulation study with worms cultivated in soil con1 taining either 3000 or 5000 mgPb gsoil . The concentration of lead in the worms was determined at various sampling times. Uptake at 1 both concentrations was linear with time. Worms in the 5000 mg g 1 soil accumulated lead at a faster rate (3.16 mg Pb gtissue day 1) 1 1 1 than those in the 3000 mg g soil (2.21 mg Pb gtissue day ). The third experiment was a timed experiment with worms cultivated in 1 soil containing 7000 mgPb gsoil . 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 importance, 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 associated pollution from their previous use and before development can begin, the risk associated with * Corresponding author. Tel.: +44-118-931-6974; fax: +44-118931-6660. E-mail address: [email protected] (M.E. Hodson).
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 promising 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
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test is the OECD draft earthworm reproduction test (OECD, 2000). In this test, a contaminant solution is mixed with the soil to give a range of concentrations. The standard test worms, Eisenia fetida, are then added and the experiment is run for 28 days. Mortality of the worms is checked weekly. However, a number of problems exist with such tests. For example, in a companion paper we describe a series of experiments where we demonstrate that the form of lead in the soil, rather than lead concentration, is an important control on availability and toxicity of lead to worms (Davies et al., 2002). Also, uptake into the animals is not simply a case of bioavailability or the speciation of the metal, but must also be a factor of the biokinetics of uptake, storage and excretion. The rates at which these processes occur will determine the amount of metal in the organisms at any one time and the toxicity of any contaminants (Morgan et al., 1993; Neuhauser et al., 1995). The aim of this study, which forms part of a larger body of work looking at the significance of the OECD earthworm reproduction test, was to determine whether: (i) a specified time was necessary for equilibration of the soil and contaminant solution; (ii) the mortality and accumulation of lead by the worms altered over time and; (iii) toxicity of lead in the earthworm was caused by a breakdown in the regulation of uptake of the metal or whether the lead was simply accumulated to some toxic level at which the animals died.
50% of the population were killed) was determined by Trimmed Spearman–Karber (Finney, 1971) and the NOEC (No Observed Effect Concentration, which is the highest concentration used where no effect is seen) was determined by ANOVA followed by Tukey’s comparison (Williams, 1972). The EC50 (Effect Concentration which caused a 50% reduction in a measured parameter) for growth was determined by the Linear Interpolation Method (USEPA, 1994). Soil samples were collected at times 0, 14 and 28 days after the addition of the worms and chemical extractions were carried out on triplicate sub-samples of the soils using three methods; water, CaCl2 (Houba et al., 1996) and DTPA (diethylenetriaminepentaacetic acid; Quevauviller, 1998). For the water and CaCl2 extractions 25 ml of solution was added to 3 g of soil and shaken for 2 h before being centrifuged and the supernatant filtered through a 0.2 mm filter. The solutions were acidified and kept at 4 C until analysed. The soil: solution ratio for the DTPA extractions were 3 g:20 ml. The samples were then treated as for water and CaCl2. CaCl2 and DTPA extractions have been suggested as standard methods of extraction to correlate bioavailability of lead to plants (Singh et al., 1996; van Raij, 1998; Maiz et al., 2000). The water content of the soil was determined at times 0 and 28 days and the pH of the soil was determined at 0, 14 and 28 days. The extracted samples were analysed for lead content by flame atomic absorption spectrophotometry (AAS). Worms were dissolved in 69% nitric acid overnight, diluted to 25 ml in distilled water and analysed by AAS (Singh et al., 1996).
2. Materials and methods 2.1. Experiment 1
2.2. Experiment 2
The toxicity test was carried out according to the OECD draft guideline for the testing of chemicals (OECD, 2000). Sterilised Kettering Loam was dried at 40 C and sieved to < 2 mm. The pH was 6.8 0.4 (ISO, 1998) and the mean water holding capacity was 48 16% (ISO, 1994). Lead nitrate was added in solution form to 500 g subsamples of soil to give concentrations of 100, 400, 1000, 2000, 3000, 5000, 7000 and 10,000 mgPb/gsoil. Sufficient water was used to give a water content of 40% of the water holding capacity. There were four replicates of each concentration and eight controls. The amended soil was left for 7 days before the addition of the worms (10 per replicate; Day 0) and the experiments were left to run for 28 days (Day 28). Adult, clitellate worms weighing 260–350 mg were acclimatised in uncontaminated test soil for 1 week before being introduced to the lead amended soil. The mortality of the worms was assessed each week and the concentration of lead in the survivors measured after 28 days. The LC50 (Lethal Concentration when
An experiment was also set up with concentrations of 1 lead of 3000 and 5000 mgPb gsoil . In the first experiment these concentrations had been determined as being nonlethal and close to the LC50 for lead respectively. The experiments were set up in an identical fashion to the standard toxicity tests (OECD, 2000). Lead was added to the soil in the form of lead nitrate solution to give water contents of 40% of the soil water-holding capacity. Twenty-one 500 g samples of lead amended soil were established at each concentration. The amended soil was left for 7 days before the addition of 10 worms to each 500-g of soil (Day 0). After 3, 7, 10, 14, 17, 24 and 28 days worms from 3 of the 500 g soil samples at each Pb concentration were collected. Numbers of surviving worms were determined and tissue concentration of Pb in these worms was determined by AAS as above. Subsamples of the soil from which the worms were collected were also extracted using water, CaCl2 and DTPA as described previously. The extracts were then analysed for Pb.
N.A. Davies et al. / Environmental Pollution 121 (2003) 55–61
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2.3. Experiment 3
3.2. Experiment 2
Lead nitrate solution was mixed with 15 kg of soil to 1 give a final concentration of 7000 mgPb gsoil and a water content of 40% water holding capacity. The soil was stored at 20 C. At times 0, 7, 10, 14, 17, 21, 28 and 35 days after the addition of the lead, triplicate samples of 600 g of soil were taken and 10 worms added to each replicate. The time to death for all of the worms was determined. Lead extractions, as described before, were carried out on the soil at these time points and also at the time point after 100% mortality of the worms had occurred.
The uptake of lead from the 3000 and 5000 mgPb g 1 soils by the worms over time is shown in Fig. 3. The amount of lead accumulated by the worms over time increased linearly at both lead concentrations. Worms in the 3000 mgPb g 1soil accumulated less Pb, and at a 1 slower rate (2.21 mg Pb gtissue day 1) than the worms in 1 1 the 5000 mgPb g soil (3.16 mg Pb gtissue day 1) (P 40.02). Worm mortality was greater in the 5000 mgPb g 1 soil experiment. 3.3. Experiment 3
3.1. Experiment 1
1 The time to death for the worms in the 7000 mgPb gsoil (Fig. 4) increased from 23 h at the beginning of the experiment to 67 h after the soil and lead nitrate had been equilibrated for 35 days. The extractions of the lead with water, CaCl2 and DTPA over time are shown
The 14-day LC50 for lead nitrate was 5311 (confidence 1 interval, CI, 4975–5649) mgPb gsoil ; the 28-day LC50 was 1 5395 (CI 5087–5722) mgPb gsoil. The NOEC for mortality 1 at 14 and 28 days was 3000 mgPb gsoil . The EC50 for growth in this study was 2015 (CI 1575–2341), the 1 NOEC was 3000 mgPb gsoil . Fig. 1 shows the extraction of lead using the three methods. For each of the solutions, the amount of lead extracted increased with the amount of lead added, but the three types of extraction gave very different results. Water extracted the least Pb (Fig. 1a) with a maximum of 0.5% of the total added lead being extracted. The CaCl2 (Fig. 1b) extracted the next highest with a maximum of 1% extraction. The DTPA (Fig. 1c) was the only method that extracted a high percentage ( > 98%) of the added lead. The results reflect previous studies with water extracting lead only from pore water, CaCl2 extracting pore water plus any exchangeable lead, and DTPA extracting a less mobile, strongly held fraction (Houba et al., 1996; Quevauviller, 1998). For the water and CaCl2 there was no significant difference in the amount of lead extracted over the 28 days 1 at concentrations 45000 mgPb gsoil . However, at the top 1 two concentrations used, 7000 and 10,000 mgPb gsoil , the amount of Pb extracted decreased from Day 0 through to Day 28 (P 40.01). The DTPA extracted close to 100% of the added lead for each of the concentrations used at Day 0, but by Day 14 this was down to c. 50% of the added lead and remained at this level until Day 28. The relationship between both added and extracted lead and the concentration of lead in the surviving worms is shown in Fig. 2. The concentration of lead in the 1 worms did not increase significantly until 5000 mgPb gsoil was added. This was the lowest lead concentration at which mortality was observed.
Fig. 1. Correlation between extracted lead and the concentration of lead added to the soils, (a) extraction with water, (b) extraction with CaCl2, (c) extraction with DTPA. Columns are meansS.D. (n=4).
3. Results
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in Fig. 5. For each of the extractants, the amount of Pb extracted decreased rapidly over the first 5–10 days and then remained relatively constant. At any given time, concentration of extractable Pb decreased in the order DTPA > CaCl2 > H2O.
4. Discussion 4.1. Lead mobility The results in Figs. 1 and 5 show that, at least over the first 14 days of the experiments, lead was not in equilibrium with the soil. Results from the extracts show that Pb is partitioning from a mobile, easily extracted phase (H2O, CaCl2 extractable) to a less mobile, less extractable form (DTPA extractable). This has implications for toxicity testing when trying to extrapolate laboratory data to field conditions. Very often laboratory experiments produce LC50s that have a much higher toxicity than similar concentrations found in the field (e.g. Spurgeon and Hopkin, 1995). There are several possible reasons for this but one must be the equilibration of the soil with the contaminants. A contaminant that is added to a test soil in solution form will be (1) not in equilibrium with the soil and, (2) very available to organisms. Calculated toxicities will therefore, be relatively high (low LC50). As the contaminant equilibrates with the soil and becomes less available, calculated toxicities will decrease (leading to higher LC50s). This effect is corroborated by the times to death 1 of the worms in the 7000 mgPb gsoil experiment (Fig. 4) which increased from hours to days as the soil was left for longer periods of time to equilibrate with the Pb before addition of worms. 4.2. Lead bioaccumulation
Fig. 2. Correlation between lead in surviving worms and lead extracted with (a) water and CaCl2, (b) with DTPA and total lead added to soil. Points are means S.D.(n=3). No data is given for 1 1 the 7000 mgPb gsoil and 10 000 mgPb gsoil samples as no worms survived at these concentrations.
From Fig. 2 it can be seen that the amount of lead accumulated by the worms did not change significantly until lead concentration in the soil reached 5000 mgPb gsoil1 — the lowest soil concentration where mortality was seen. This would suggest that Pb accumulation was regulated
1 1 Fig. 3. Lead accumulated in surviving worms over time. Values for 3000 mgPb gsoil are meansS.D. (n=3 replicates). Values for 5000 mgPb gsoil are single measurements due to high worm mortality (i.e. worms died and decomposed before sampling) and therefore no error bars are given.
N.A. Davies et al. / Environmental Pollution 121 (2003) 55–61
by some mechanism up to a certain environmental concentration. Above that concentration, the regulation appears to break down resulting in an influx of lead and mortality of the worms. This is illustrated in Fig. 6; at low Pb concentrations Pb in worms/Pb in soil decreases
1 Fig. 4. Time to death for worms in 7000 mgPb gsoil soil after various times of ageing the soil/contaminant mixture. Points are times to death for all 30 worms used for each sampling time.
1 Fig. 5. Extraction of lead from the 7000 mgPb gsoil soil over time using (a) water, (b) CaCl2 and (c) DTPA. Points are meansS.D. (n=3).
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as the concentration of Pb in soil increases but between 1 soil Pb concentrations of 3000 mgPb gsoil and 5000 mgPb 1 gsoil there is a large increase. Significant worm mortality 1 is first seen at concentrations of 5000 mgPb gsoil . Organisms that can tolerate high environmental concentrations of potentially toxic metals either: (1) do not accumulate the metal; (2) accumulate the metal but in a non-toxic form or accumulate the lead and store it in a non-toxic form; or (3) excrete the metal efficiently. The data in Fig. 3 show that worms do accumulate Pb. In addition, since Pb load increases with time, the Pb is not being excreted efficiently. Thus it would appear that the worms store the Pb in a non-toxic form as suggested by Morgan and co-workers (Morgan and Morgan, 1998; Morgan et al., 1999) who observed Pb-phosphate nodules in worms and postulated that these were a Pb storage mechanism. The continued accumulation of Pb 1 in worms cultured in the 3000 and 5000 mgPb gsoil experiments may imply that excretion of stored Pb is problematic. If this is the case, once the Pb load reaches a critical concentration the worms will die. This is important in toxicity testing as a 28 day exposure period may be insufficient to saturate the Pb storage capacity of the worms giving underestimates (too high LC50) of Pb toxicity. The present experiments produced superficially contradictory evidence as to whether Pb uptake by worms was passive or not. From Fig. 3 it can be seen that metal 1 uptake in the 5000 mgPb gsoil experiment was greater 1 than in the 3000 mgPb gsoil experiment. This suggests that external environmental concentrations control the bioaccumulation of Pb by E. fetida. In contrast, data from the first experiment indicate some degree of regulation. In Fig. 6 the ratio of Pb in surviving worms/Pb added to soil is plotted against soil Pb concentration. The partitioning of Pb into worms relative to soil con1 centrations decreases from c. 0.03 at 100 mgPb gsoil to c. 1 0.001 at Pb concentrations of 3000 mgPb gsoil but then 1 shows a sharp increase at 5000 mgPb gsoil to c. 0.02. This increase coincides with the onset of worm mortality in the experiments. These data imply that accumulation of lead by E. fetida is regulated at low environmental
Fig. 6. Plot of lead in worms (mgPb g 1)/lead in soil (mgPb g 1) against concentration of lead added to soil in experiment 1.
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concentrations but that at a critical concentration this mechanism breaks down resulting in an influx of metal and death. Although the two sets of data appear to be contradictory, decreased partitioning of Pb into worms at increasing concentrations is consistent with increasing accumulation rates with increasing Pb concentrations provided that accumulation rates increase at a lower rate than the Pb concentration in the soil increases. Thus, combining data from experiments 1 and 2 (Figs. 3 and 6) indicates that although the rate of Pb uptake is controlled by Pb concentration in soils (possibly by a simple diffusion method through the skin of the worms or through the gut wall), at low Pb concentrations worms are able to regulate this uptake. At concentra1 tions above 5000 mgPb gsoil this regulatory mechanism breaks down resulting in widespread worm mortality though individual surviving worms continue to accumulate Pb. The surviving individuals may be especially tolerant to higher Pb concentrations or alternatively have more efficient or robust regulation systems thereby enabling them to survive at higher Pb concentrations. The uptake and excretion efficiency of individuals could be investigated through the use of isotopically labelled metals (e.g. Sheppard et al., 1997).
accumulation of the contaminant and mortality in the test animals. This study has highlighted several problems with applying the OECD worm toxicity test. As stated in Davies et al. (2002) in its present form the test does not permit calculation of environmentally useful contaminant toxicities and the test should not be used for this purpose. However, the test provides a good basis for investigating controls on metal toxicity to worms. Investigation into soil properties may lead to the development of a predictive model for lead bioavailability and toxicity to worms.
Acknowledgements Dave Spurgeon (Department of Biology, University of Cardiff and CEH, Monkswood) and Stephen Hopkin (Department of Animal and Microbial Sciences, University of Reading) are thanked for advice on running ecotoxicological experiments. MEH acknowledges John Duffus for encouraging him to apply a geochemical perspective to ecotoxicology. This research was funded by a grant from the Research Endowment Trust Fund of the University of Reading.
5. Conclusions References Lead initially added to soils as lead nitrate solution becomes less extractable with time, moving from more mobile phases such as pore water and exchange sites to less mobile phases. In the first set of experiments LC50 values were unaffected by this change since death of worms occurred within the first 14 days of the experiment. However, a marked effect was seen in the length 1 of time that worms survived in the 7000 mgPb gsoil experiment in which the soil was left to equilibrate with added Pb for varying lengths of time prior to the addition of worms. This implies that the OECD test should be modified to allow soils to equilibrate with added contaminants prior to addition of test organisms. The equilibration time is likely to be different for different contaminants and different soils. The accumulation of Pb by the worms is partially controlled by external conditions with the rate of Pb accumulation increasing with Pb concentration. Lead load increases with time and, if accumulation continues, it is possible that Pb load may reach toxic levels even at low Pb concentrations. Thus the OECD tests may not run long enough to detect the true toxic effect of lead. Despite evidence for passive uptake of Pb, some regulation of uptake seems to occur at low contamination levels with Pb uptake being limited at Pb concentra1 tions of 43000 mgPb gsoil . This regulation breaks down at higher concentrations resulting in an increase in
Canada Council of Ministers of the Environment. 1992. Report CCME EPC-C534, Winnipeg, Manitoba. Davies, N. A., Hodson, M. E., Black, S. 2002. Is the OECD acute worm toxicity test environmentally relevant? The effect of mineral form on calculated lead toxicity. Environmental Pollution Xref: SO2697491(02)002063. DETR. 1997. Statements of National Planning Policy, Planning Policy Guidance Notes PPG01. General Policy and Principles. London. DoE. 1980. Interdepartmental Committee on the Redevelopment of Contaminated Land (ICRCL), Consultation paper, DOE. Finney, D.J., 1971. Probit Analysis, third ed. Cambridge University Press, London. Giesler, G. 1987. Contaminated Land in the EEC. Dornier System GmbH, Fridrichshafen, FRG. Griffiths, C.M., Board, N.P., 1992. Approaches to the assessment and remediation of polluted land in Europe and America. J. IWEM 2, 720–725. Houba, V.J.G., Lexmond, Th. M., Novozamsky, I., van der Lee, J. J., 1996. State of the art and future developments in soil analysis for bioavailability assessment. Sci. Tot. Environ 178, 21–28. ISO. 1994. Soil Quality—Determination of pH. ISO 10390. ISO. 1998. Soil Quality—Effects of Pollutants on Earthworms (Eisenia fetida). Part 2: Determination of Effects on Reproduction. ISO 11268-2. Maiz, I., Arambarri, I., Garcia, R., Millan, E., 2000. Evaluation of heavy metal availability in polluted sites by two sequential extraction procedures using factor analysis. Environ. Poll 110, 3–9. Morgan, A.J., Morgan, J.E., Turner, M., Winters, C., Yarwood, A., 1993. Metal relationships of earthworms. In: Dallinger, R., Rainbow, P.S. (Eds.), Ecotoxicology of Metals in Invertebrates. Lewis Publishers, Boca Raton, pp. 333–358.
N.A. Davies et al. / Environmental Pollution 121 (2003) 55–61 Morgan, A.J., Sturzenbaum, S.R., Winters, C., Kille, P., 1999. Cellular and molecular aspects of metal sequestration. Invertebr. Reprod. Dev. 36, 17–24. Morgan, J.E., Morgan, A.J., 1998. The distribution and intracellular compartmentation of metals in the endogeic earthworm Apporectodea calignosa sampled from an unpolluted and a metal-contaminated site. Environ. Poll. 99, 167–175. Neuhauser, E.F., Cukic, Z.V., Malecki, M.R., Loehr, R.C., Durkin, P.R., 1995. Bioconcentration and biokinetics of heavy metals in the earthworm. Environ. Poll. 89, 293–301. OECD. 2000. Draft Guideline for the Testing of Chemicals. Earthworm Reproduction Tests (Eisenia fetida/andrei). Quevauviller, P., 1998. Operationally defined extraction procedures for soil and sediment analysis. 1. Standardisation. Trends in Analytical Chemistry 17, 289–298. Sheppard, S.C., Evenden, W.G., Cornwell, T.C., 1997. Depuration and uptake kinetics of I, Cs, Mn, Zn, and Cd by the earthworm (Lumbricus terrestris) in radiotracer-spiked litter. Environmental Toxicology and Chemistry 16, 2106–2112.
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Singh, S.P., Tack, F.M.G., Verloo, M.G., 1996. Extractability and bioavailability of heavy metals in surface soils derived from dredged sediments. Chemical Speciation and Bioavailability 8, 105– 110. Spurgeon, D.J., Hopkin, S., 1995. Extrapolation of the laboratorybased OECD earthworm toxicity test to metal-contaminated field sites. Ecotoxicology 4, 190–205. Tadesse, B., Donaldson, J.D., Grimes, S.M.J., 1994. Contaminated and polluted land: a general review of decontamination management and control. Chem.Tech. Biotech 2, 227–240. USEPA. 1994. In: Lewis, P.A., Klemm, D.J., Lazorchak, J.M., Norberg-King, T.J., Peltier, W.H., Heber, M.A. (Eds.), Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, third ed. EPA/600/491/002. van Raij, B., 1998. Bioavailable tests: alternatives to standard soil extractions. Commun. Soil Sci. Plant Anal. 29, 1553–1570. Williams, D.A., 1972. The comparison of several dose levels with a zero dose control. Biometrics 28, 519–531.
The influence of time on lead toxicity and bioaccumulation determined by the OECD earthworm toxicity test Nicola A. Daviesa,b, Mark E. Hodsonb,*, Stuart Blacka 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 LC50s for 14 and 28 days were 5311 1 and 5395 mgPb gsoil respectively. The second experiment was a timed lead accumulation study with worms cultivated in soil con1 taining either 3000 or 5000 mgPb gsoil . The concentration of lead in the worms was determined at various sampling times. Uptake at 1 both concentrations was linear with time. Worms in the 5000 mg g 1 soil accumulated lead at a faster rate (3.16 mg Pb gtissue day 1) 1 1 1 than those in the 3000 mg g soil (2.21 mg Pb gtissue day ). The third experiment was a timed experiment with worms cultivated in 1 soil containing 7000 mgPb gsoil . 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 importance, 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 associated pollution from their previous use and before development can begin, the risk associated with * Corresponding author. Tel.: +44-118-931-6974; fax: +44-118931-6660. E-mail address: [email protected] (M.E. Hodson).
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 promising 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
56
N.A. Davies et al. / Environmental Pollution 121 (2003) 55–61
test is the OECD draft earthworm reproduction test (OECD, 2000). In this test, a contaminant solution is mixed with the soil to give a range of concentrations. The standard test worms, Eisenia fetida, are then added and the experiment is run for 28 days. Mortality of the worms is checked weekly. However, a number of problems exist with such tests. For example, in a companion paper we describe a series of experiments where we demonstrate that the form of lead in the soil, rather than lead concentration, is an important control on availability and toxicity of lead to worms (Davies et al., 2002). Also, uptake into the animals is not simply a case of bioavailability or the speciation of the metal, but must also be a factor of the biokinetics of uptake, storage and excretion. The rates at which these processes occur will determine the amount of metal in the organisms at any one time and the toxicity of any contaminants (Morgan et al., 1993; Neuhauser et al., 1995). The aim of this study, which forms part of a larger body of work looking at the significance of the OECD earthworm reproduction test, was to determine whether: (i) a specified time was necessary for equilibration of the soil and contaminant solution; (ii) the mortality and accumulation of lead by the worms altered over time and; (iii) toxicity of lead in the earthworm was caused by a breakdown in the regulation of uptake of the metal or whether the lead was simply accumulated to some toxic level at which the animals died.
50% of the population were killed) was determined by Trimmed Spearman–Karber (Finney, 1971) and the NOEC (No Observed Effect Concentration, which is the highest concentration used where no effect is seen) was determined by ANOVA followed by Tukey’s comparison (Williams, 1972). The EC50 (Effect Concentration which caused a 50% reduction in a measured parameter) for growth was determined by the Linear Interpolation Method (USEPA, 1994). Soil samples were collected at times 0, 14 and 28 days after the addition of the worms and chemical extractions were carried out on triplicate sub-samples of the soils using three methods; water, CaCl2 (Houba et al., 1996) and DTPA (diethylenetriaminepentaacetic acid; Quevauviller, 1998). For the water and CaCl2 extractions 25 ml of solution was added to 3 g of soil and shaken for 2 h before being centrifuged and the supernatant filtered through a 0.2 mm filter. The solutions were acidified and kept at 4 C until analysed. The soil: solution ratio for the DTPA extractions were 3 g:20 ml. The samples were then treated as for water and CaCl2. CaCl2 and DTPA extractions have been suggested as standard methods of extraction to correlate bioavailability of lead to plants (Singh et al., 1996; van Raij, 1998; Maiz et al., 2000). The water content of the soil was determined at times 0 and 28 days and the pH of the soil was determined at 0, 14 and 28 days. The extracted samples were analysed for lead content by flame atomic absorption spectrophotometry (AAS). Worms were dissolved in 69% nitric acid overnight, diluted to 25 ml in distilled water and analysed by AAS (Singh et al., 1996).
2. Materials and methods 2.1. Experiment 1
2.2. Experiment 2
The toxicity test was carried out according to the OECD draft guideline for the testing of chemicals (OECD, 2000). Sterilised Kettering Loam was dried at 40 C and sieved to < 2 mm. The pH was 6.8 0.4 (ISO, 1998) and the mean water holding capacity was 48 16% (ISO, 1994). Lead nitrate was added in solution form to 500 g subsamples of soil to give concentrations of 100, 400, 1000, 2000, 3000, 5000, 7000 and 10,000 mgPb/gsoil. Sufficient water was used to give a water content of 40% of the water holding capacity. There were four replicates of each concentration and eight controls. The amended soil was left for 7 days before the addition of the worms (10 per replicate; Day 0) and the experiments were left to run for 28 days (Day 28). Adult, clitellate worms weighing 260–350 mg were acclimatised in uncontaminated test soil for 1 week before being introduced to the lead amended soil. The mortality of the worms was assessed each week and the concentration of lead in the survivors measured after 28 days. The LC50 (Lethal Concentration when
An experiment was also set up with concentrations of 1 lead of 3000 and 5000 mgPb gsoil . In the first experiment these concentrations had been determined as being nonlethal and close to the LC50 for lead respectively. The experiments were set up in an identical fashion to the standard toxicity tests (OECD, 2000). Lead was added to the soil in the form of lead nitrate solution to give water contents of 40% of the soil water-holding capacity. Twenty-one 500 g samples of lead amended soil were established at each concentration. The amended soil was left for 7 days before the addition of 10 worms to each 500-g of soil (Day 0). After 3, 7, 10, 14, 17, 24 and 28 days worms from 3 of the 500 g soil samples at each Pb concentration were collected. Numbers of surviving worms were determined and tissue concentration of Pb in these worms was determined by AAS as above. Subsamples of the soil from which the worms were collected were also extracted using water, CaCl2 and DTPA as described previously. The extracts were then analysed for Pb.
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2.3. Experiment 3
3.2. Experiment 2
Lead nitrate solution was mixed with 15 kg of soil to 1 give a final concentration of 7000 mgPb gsoil and a water content of 40% water holding capacity. The soil was stored at 20 C. At times 0, 7, 10, 14, 17, 21, 28 and 35 days after the addition of the lead, triplicate samples of 600 g of soil were taken and 10 worms added to each replicate. The time to death for all of the worms was determined. Lead extractions, as described before, were carried out on the soil at these time points and also at the time point after 100% mortality of the worms had occurred.
The uptake of lead from the 3000 and 5000 mgPb g 1 soils by the worms over time is shown in Fig. 3. The amount of lead accumulated by the worms over time increased linearly at both lead concentrations. Worms in the 3000 mgPb g 1soil accumulated less Pb, and at a 1 slower rate (2.21 mg Pb gtissue day 1) than the worms in 1 1 the 5000 mgPb g soil (3.16 mg Pb gtissue day 1) (P 40.02). Worm mortality was greater in the 5000 mgPb g 1 soil experiment. 3.3. Experiment 3
3.1. Experiment 1
1 The time to death for the worms in the 7000 mgPb gsoil (Fig. 4) increased from 23 h at the beginning of the experiment to 67 h after the soil and lead nitrate had been equilibrated for 35 days. The extractions of the lead with water, CaCl2 and DTPA over time are shown
The 14-day LC50 for lead nitrate was 5311 (confidence 1 interval, CI, 4975–5649) mgPb gsoil ; the 28-day LC50 was 1 5395 (CI 5087–5722) mgPb gsoil. The NOEC for mortality 1 at 14 and 28 days was 3000 mgPb gsoil . The EC50 for growth in this study was 2015 (CI 1575–2341), the 1 NOEC was 3000 mgPb gsoil . Fig. 1 shows the extraction of lead using the three methods. For each of the solutions, the amount of lead extracted increased with the amount of lead added, but the three types of extraction gave very different results. Water extracted the least Pb (Fig. 1a) with a maximum of 0.5% of the total added lead being extracted. The CaCl2 (Fig. 1b) extracted the next highest with a maximum of 1% extraction. The DTPA (Fig. 1c) was the only method that extracted a high percentage ( > 98%) of the added lead. The results reflect previous studies with water extracting lead only from pore water, CaCl2 extracting pore water plus any exchangeable lead, and DTPA extracting a less mobile, strongly held fraction (Houba et al., 1996; Quevauviller, 1998). For the water and CaCl2 there was no significant difference in the amount of lead extracted over the 28 days 1 at concentrations 45000 mgPb gsoil . However, at the top 1 two concentrations used, 7000 and 10,000 mgPb gsoil , the amount of Pb extracted decreased from Day 0 through to Day 28 (P 40.01). The DTPA extracted close to 100% of the added lead for each of the concentrations used at Day 0, but by Day 14 this was down to c. 50% of the added lead and remained at this level until Day 28. The relationship between both added and extracted lead and the concentration of lead in the surviving worms is shown in Fig. 2. The concentration of lead in the 1 worms did not increase significantly until 5000 mgPb gsoil was added. This was the lowest lead concentration at which mortality was observed.
Fig. 1. Correlation between extracted lead and the concentration of lead added to the soils, (a) extraction with water, (b) extraction with CaCl2, (c) extraction with DTPA. Columns are meansS.D. (n=4).
3. Results
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in Fig. 5. For each of the extractants, the amount of Pb extracted decreased rapidly over the first 5–10 days and then remained relatively constant. At any given time, concentration of extractable Pb decreased in the order DTPA > CaCl2 > H2O.
4. Discussion 4.1. Lead mobility The results in Figs. 1 and 5 show that, at least over the first 14 days of the experiments, lead was not in equilibrium with the soil. Results from the extracts show that Pb is partitioning from a mobile, easily extracted phase (H2O, CaCl2 extractable) to a less mobile, less extractable form (DTPA extractable). This has implications for toxicity testing when trying to extrapolate laboratory data to field conditions. Very often laboratory experiments produce LC50s that have a much higher toxicity than similar concentrations found in the field (e.g. Spurgeon and Hopkin, 1995). There are several possible reasons for this but one must be the equilibration of the soil with the contaminants. A contaminant that is added to a test soil in solution form will be (1) not in equilibrium with the soil and, (2) very available to organisms. Calculated toxicities will therefore, be relatively high (low LC50). As the contaminant equilibrates with the soil and becomes less available, calculated toxicities will decrease (leading to higher LC50s). This effect is corroborated by the times to death 1 of the worms in the 7000 mgPb gsoil experiment (Fig. 4) which increased from hours to days as the soil was left for longer periods of time to equilibrate with the Pb before addition of worms. 4.2. Lead bioaccumulation
Fig. 2. Correlation between lead in surviving worms and lead extracted with (a) water and CaCl2, (b) with DTPA and total lead added to soil. Points are means S.D.(n=3). No data is given for 1 1 the 7000 mgPb gsoil and 10 000 mgPb gsoil samples as no worms survived at these concentrations.
From Fig. 2 it can be seen that the amount of lead accumulated by the worms did not change significantly until lead concentration in the soil reached 5000 mgPb gsoil1 — the lowest soil concentration where mortality was seen. This would suggest that Pb accumulation was regulated
1 1 Fig. 3. Lead accumulated in surviving worms over time. Values for 3000 mgPb gsoil are meansS.D. (n=3 replicates). Values for 5000 mgPb gsoil are single measurements due to high worm mortality (i.e. worms died and decomposed before sampling) and therefore no error bars are given.
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by some mechanism up to a certain environmental concentration. Above that concentration, the regulation appears to break down resulting in an influx of lead and mortality of the worms. This is illustrated in Fig. 6; at low Pb concentrations Pb in worms/Pb in soil decreases
1 Fig. 4. Time to death for worms in 7000 mgPb gsoil soil after various times of ageing the soil/contaminant mixture. Points are times to death for all 30 worms used for each sampling time.
1 Fig. 5. Extraction of lead from the 7000 mgPb gsoil soil over time using (a) water, (b) CaCl2 and (c) DTPA. Points are meansS.D. (n=3).
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as the concentration of Pb in soil increases but between 1 soil Pb concentrations of 3000 mgPb gsoil and 5000 mgPb 1 gsoil there is a large increase. Significant worm mortality 1 is first seen at concentrations of 5000 mgPb gsoil . Organisms that can tolerate high environmental concentrations of potentially toxic metals either: (1) do not accumulate the metal; (2) accumulate the metal but in a non-toxic form or accumulate the lead and store it in a non-toxic form; or (3) excrete the metal efficiently. The data in Fig. 3 show that worms do accumulate Pb. In addition, since Pb load increases with time, the Pb is not being excreted efficiently. Thus it would appear that the worms store the Pb in a non-toxic form as suggested by Morgan and co-workers (Morgan and Morgan, 1998; Morgan et al., 1999) who observed Pb-phosphate nodules in worms and postulated that these were a Pb storage mechanism. The continued accumulation of Pb 1 in worms cultured in the 3000 and 5000 mgPb gsoil experiments may imply that excretion of stored Pb is problematic. If this is the case, once the Pb load reaches a critical concentration the worms will die. This is important in toxicity testing as a 28 day exposure period may be insufficient to saturate the Pb storage capacity of the worms giving underestimates (too high LC50) of Pb toxicity. The present experiments produced superficially contradictory evidence as to whether Pb uptake by worms was passive or not. From Fig. 3 it can be seen that metal 1 uptake in the 5000 mgPb gsoil experiment was greater 1 than in the 3000 mgPb gsoil experiment. This suggests that external environmental concentrations control the bioaccumulation of Pb by E. fetida. In contrast, data from the first experiment indicate some degree of regulation. In Fig. 6 the ratio of Pb in surviving worms/Pb added to soil is plotted against soil Pb concentration. The partitioning of Pb into worms relative to soil con1 centrations decreases from c. 0.03 at 100 mgPb gsoil to c. 1 0.001 at Pb concentrations of 3000 mgPb gsoil but then 1 shows a sharp increase at 5000 mgPb gsoil to c. 0.02. This increase coincides with the onset of worm mortality in the experiments. These data imply that accumulation of lead by E. fetida is regulated at low environmental
Fig. 6. Plot of lead in worms (mgPb g 1)/lead in soil (mgPb g 1) against concentration of lead added to soil in experiment 1.
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concentrations but that at a critical concentration this mechanism breaks down resulting in an influx of metal and death. Although the two sets of data appear to be contradictory, decreased partitioning of Pb into worms at increasing concentrations is consistent with increasing accumulation rates with increasing Pb concentrations provided that accumulation rates increase at a lower rate than the Pb concentration in the soil increases. Thus, combining data from experiments 1 and 2 (Figs. 3 and 6) indicates that although the rate of Pb uptake is controlled by Pb concentration in soils (possibly by a simple diffusion method through the skin of the worms or through the gut wall), at low Pb concentrations worms are able to regulate this uptake. At concentra1 tions above 5000 mgPb gsoil this regulatory mechanism breaks down resulting in widespread worm mortality though individual surviving worms continue to accumulate Pb. The surviving individuals may be especially tolerant to higher Pb concentrations or alternatively have more efficient or robust regulation systems thereby enabling them to survive at higher Pb concentrations. The uptake and excretion efficiency of individuals could be investigated through the use of isotopically labelled metals (e.g. Sheppard et al., 1997).
accumulation of the contaminant and mortality in the test animals. This study has highlighted several problems with applying the OECD worm toxicity test. As stated in Davies et al. (2002) in its present form the test does not permit calculation of environmentally useful contaminant toxicities and the test should not be used for this purpose. However, the test provides a good basis for investigating controls on metal toxicity to worms. Investigation into soil properties may lead to the development of a predictive model for lead bioavailability and toxicity to worms.
Acknowledgements Dave Spurgeon (Department of Biology, University of Cardiff and CEH, Monkswood) and Stephen Hopkin (Department of Animal and Microbial Sciences, University of Reading) are thanked for advice on running ecotoxicological experiments. MEH acknowledges John Duffus for encouraging him to apply a geochemical perspective to ecotoxicology. This research was funded by a grant from the Research Endowment Trust Fund of the University of Reading.
5. Conclusions References Lead initially added to soils as lead nitrate solution becomes less extractable with time, moving from more mobile phases such as pore water and exchange sites to less mobile phases. In the first set of experiments LC50 values were unaffected by this change since death of worms occurred within the first 14 days of the experiment. However, a marked effect was seen in the length 1 of time that worms survived in the 7000 mgPb gsoil experiment in which the soil was left to equilibrate with added Pb for varying lengths of time prior to the addition of worms. This implies that the OECD test should be modified to allow soils to equilibrate with added contaminants prior to addition of test organisms. The equilibration time is likely to be different for different contaminants and different soils. The accumulation of Pb by the worms is partially controlled by external conditions with the rate of Pb accumulation increasing with Pb concentration. Lead load increases with time and, if accumulation continues, it is possible that Pb load may reach toxic levels even at low Pb concentrations. Thus the OECD tests may not run long enough to detect the true toxic effect of lead. Despite evidence for passive uptake of Pb, some regulation of uptake seems to occur at low contamination levels with Pb uptake being limited at Pb concentra1 tions of 43000 mgPb gsoil . This regulation breaks down at higher concentrations resulting in an increase in
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