The influence of different artificial soil types on the acute toxicity of carbendazim to the earthworm Eisenia fetida in laboratory toxicity tests

Field populations of earthworms have shown a varied response in mortality to the fungicide carbendazim, the toxic reference substance used in agrochemical field trials. The aim of this study was to determine the influence of soil conditions as a potential cause of this variation. Laboratory acute toxicity tests were conducted using a range of artificial soils with varying soil components (organic matter, clay, pH and moisture). Batch adsorption/desorption studies were run to determine the influence of the soil properties on carbendazim behaviour. Adsorption was shown to be correlated with organic matter content and pH and this in turn could be linked to Eisenia fetida mortality, with lower mortality occurring with increased adsorption. Overall while E.fetida mortality did vary significantly between several of the soils the calculated LC50 values in the different soils did not cover a wide range (6.04-16.00 mg kg(-1)), showing that under these laboratory conditions soil components did not greatly influence carbendazim toxicity to E.fietida. (c) 2007 Elsevier Masson SAS. All rights reserved.

The effect of organic materials on the mobility and toxicity of metals in contaminated soils

Organic materials such as compost are often proposed as suitable materials for the remediation of contaminated brownfield sites intended for soft end-use. In addition to vitalising the soil, they are also believed to immobilise metals thereby breaking contaminant-receptor pathways and reducing the ecotoxicity of the contaminants. However, some research has demonstrated contradictory effects between composts on metal immobilisation. In the present study, four different composts and a liming product containing organic matter (LimeX70) were tested to examine both their metal retention and toxicity reduction capabilities on three different metal contaminated soils. Leaching tests, a plant growth test with Greek cress (Lepidium sativum), an earthworm (Eisenia fetida) survival and condition test and a bacterial toxicity test using Vibrio fischeri were carried out. The leaching test results showed that spent mushroom compost caused an increase in metal concentration in the leachates, while LimeX70 caused a decrease. The variation in behaviour between different amendments for each soil was high, so a generic conclusion could not be drawn. Toxicity tests showed significant reduction of metal bioavailability and toxicity for Greek cress, earthworms and bacteria. The results also suggest that more research should be undertaken to understand the mechanisms involved in metal complexation using different types of organic matter, in order to optimise the use of organic materials like compost for soil remediation. Crown Copyright (C) 2007 Published by Elsevier Ltd. All rights reserved.

Is the OECD acute worm toxicity test environmentally relevant? The effect of mineral form on calculated lead toxicity

In a series of experiments the toxicity of lead to worms in soil was determined following the draft OECD earthworm reproduction toxicity protocol except that lead was added as solid lead nitrate, carbonate and sulphide rather than as lead nitrate solution as would normally be the case. The compounds were added to the test soil to give lead concentrations of 625-12500 pg Pb g-1 of soil. Calculated toxicities of the lead decreased in the order nitrate > carbonate > sulphide, the same order as the decrease in the solubility of the metal compounds used. The 7-day LC50 (lethal concentration when 50% of the population is killed) for the nitrate was 5321 +/- 275 mug Pb g(-1) of soil and this did not change with time. The LC50 values for carbonate and sulphide could not be determined at the concentration ranges used. The only parameter sensitive enough to distinguish the toxicities of the three compounds was cocoon (egg) production. The EC50S for cocoon production (the concentration to produce a 50% reduction in cocoon production) were 993, 8604 and 10 246 mug Pb g(-1) of soil for lead nitrate, carbonate and sulphide, respectively. Standard toxicity tests need to take into account the form in which the contaminant is present in the soil to be of environmental relevance. (C) 2002 Elsevier Science Ltd. All rights reserved.

The influence of time on lead toxicity and bioaccumulation determined by the OECD earthworm toxicity test

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 mug(Pb) g(soil)(-1) respectively. The second experiment was a timed lead accumulation study with worms cultivated in soil containing either 3000 or 5000 mug(Pb) g(soil)(-1). The concentration of lead in the worms was determined at various sampling times. Uptake at so' Sol both concentrations was linear with time. Worms in the 5000 mug g(-1) soil accumulated lead at a faster rate (3.16 mug Pb g(tissue)(-1) day(-1)) tiss than those in the 3000 mug g(-1) soil (2.21 mug Pb-tissue g(-1) day(-1)). The third experiment was a timed experiment with worms cultivated in tiss soil containing 7000 mugPb g(soil)(-1). Soil and lead nitrate solution were mixed and stored at 20 degreesC. 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 It 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. (C) 2002 Elsevier Science Ltd. All rights reserved.

The effect of organic materials on the mobility and toxicity of metals in contaminated soils

Organic materials such as compost are often proposed as suitable materials for the remediation of contaminated brownfield sites intended for soft end-use. In addition to vitalising the soil, they are also believed to immobilise metals thereby breaking contaminant-receptor pathways and reducing the ecotoxicity of the contaminants. However, some research has demonstrated contradictory effects between composts on metal immobilisation. In the present study, four different composts and a liming product containing organic matter (LimeX70) were tested to examine both their metal retention and toxicity reduction capabilities on three different metal contaminated soils. Leaching tests, a plant growth test with Greek cress (Lepidium sativum), an earthworm (Eisenia fetida) survival and condition test and a bacterial toxicity test using Vibrio fischeri were carried out. The leaching test results showed that spent mushroom compost caused an increase in metal concentration in the leachates, while LimeX70 caused a decrease. The variation in behaviour between different amendments for each soil was high, so a generic conclusion could not be drawn. Toxicity tests showed significant reduction of metal bioavailability and toxicity for Greek cress, earthworms and bacteria. The results also suggest that more research should be undertaken to understand the mechanisms involved in metal complexation using different types of organic matter, in order to optimise the use of organic materials like compost for soil remediation. Crown Copyright (C) 2007 Published by Elsevier Ltd. All rights reserved.

Opportunities for reduction in acute toxicity testing via improved design

The conventional method for assessing acute oral toxicity (OECD Test Guideline 401) was designed to identify the median lethal dose (LD50), using the death of animals as an endpoint. Introduced as an alternative method (OECD Test Guideline 420), the Fixed Dose Procedure (FDP) relies on the observation of clear signs of toxicity, uses fewer animals and causes less suffering. More recently, the Acute Toxic Class method and the Up-and-Down Procedure have also been adopted as OECD test guidelines. Both of these methods also use fewer animals than the conventional method, although they still use death as an endpoint. Each of the three new methods incorporates a sequential dosing procedure, which results in increased efficiency. In 1999, with a view to replacing OECD Test Guideline 401, the OECD requested that the three new test guidelines be updated. This was to bring them in line with the regulatory needs of all OECD Member Countries, provide further reductions in the number of animals used, and introduce refinements to reduce the pain and distress experienced by the animals. This paper describes a statistical modelling approach for the evaluation of acute oral toxicity tests, by using the revised FDP for illustration. Opportunities for further design improvements are discussed.

The comparative toxicity to soil invertebrates of natural chemicals and their synthetic analogues

The introduction of Registration, Evaluation and Authorisation of Chemicals (REACH), requires companies to register and risk assess all substances produced or imported in volumes of >1 tonne per year. Extrapolation methods which use existing data for estimating the effects of chemicals are attractive to industry, and comparative data are therefore increasingly in demand. Data on natural toxic chemicals could be used for extrapolation methods Such as read-across. To test this hypothesis, the toxicity of natural chemicals and their synthetic analogues were compared using standardised toxicity tests. Two chemical pairs: the napthoquinones, juglone (natural) and 1,4-naphthoquinone (synthetic); and anthraquinones, emodin (natural) and quinizarin (synthetic) were chosen, and their comparative effects on the survival and reproduction of collembolans, earthworms, enchytraeids and predatory mites were assessed. Differences in sensitivity between the species were observed with the predatory mite (Hypoaspis aculeifer) showing the least sensitivity. Within the chemical pairs, toxicity to lethal and sub-lethal endpoints was very similar for the four invertebrate species. The exception was earthworm reproduction, which showed differential sensitivity to the chemicals in both naphthoquinone and anthraquinone pairs. Differences in toxicity identified in the present study may be related to degree of exposure and/or subtle differences in the mode of toxic action for the chemicals and species tested. It may be possible to predict differences by identifying functional groups which infer increased or decreased toxicity in one or other chemical. The development of such techniques would enable the use of read-across from natural to synthetic chemicals for a wider group of compounds. (C) 2009 Elsevier Ltd. All rights reserved.

Haem peroxidase activity in Daphnia magna: A biomarker for sub-lethal toxicity assessments of kerosene-contaminated groundwater

A novel biomarker was developed in Daphnia magna to detect organic pollution in groundwater. The haem peroxidase assay, which is an indirect means of measuring oxidase activity, was particularly sensitive to kerosene contamination. Exposure to sub-lethal concentrations of kerosene-contaminated groundwater resulted in a haem peroxidase activity increase by dose with a two-fold activity peak at 25%. Reproduction in D. magna remained unimpaired when exposed to concentrations below 25% for 21 days, and a decline in fecundity was only observed at concentrations above the peak in enzyme activity. The measurement of haem peroxidase activity in D. magna detected sublethal effects of kerosene in just 24 h, whilst offering information on the health status of the organisms. The biomarker may be useful in determining concentrations above which detrimental effects would occur from long-term exposure for fuel hydrocarbons. Moreover, this novel assay detects exposure to chemicals in samples that would normally be classified as non-toxic by acute toxicity tests.

A comparison of the relative toxicity of bone meal and other P sources used as remedial treatments to the earthworm Eisenia fetida

It has been suggested that sources of P could be used to remediate metal-contaminated soil. The toxicity of four potential P sources, potassium hydrogen phosphate (PHP), triple superphosphate (TSP), rock phosphate (RP) and raw bone meal (RBM) to Eisenia fetida was determined. The concentration of P that is statistically likely to kill 50% of the population (LC50) for PHP, TSP and RBM was determined in OECD acute toxicity tests. 14 day LC50s expressed as bulk P concentration lay in the range 3319–4272 mg kg−1 for PHP, 3107–3590 mg kg−1 for TSP and 1782–2196 mg kg−1 for RBM (ranges present the 95% confidence intervals). For PHP and TSP mortality was significantly impacted by the electrical conductivity of the treated soils. No consistent relationship existed between mortality and electrical conductivity, soil pH and available (Olsen) P across the PHP, TSP and RBM amendment types. In RP toxicity tests mortality was low and it was not possible to determine a LC50 value. Incineration of bone meal at temperatures between 200 and 300 ◦C, pre-washing the bone meal, co-amendment with 5% green waste compost and delaying introduction of earthworms after bone meal amendments by 21 days or more led to significant reductions in the bone meal toxicity. These results are consistent with the toxicity being associated with the release and/or degradation of a soluble organic component present in raw bone meal. Bone meal can be used as an earthworm-friendly remedial amendment in metal-contaminated soils but initial additions may have a negative effect on any earthworms surviving in the contaminated soil before the organic component in the bone meal degrades in the soil.

A comparative study of the effects of metal contamination on collembola in the field and in the laboratory

We examined the species diversity and abundance of Collembola at 32 sampling points along a gradient of metal contamination in a rough grassland site ( Wolverhampton, England), formerly used for the disposal of metal-rich smelting waste. Differences in the concentrations of Cd, Cu, Pb and Zn between the least and most contaminated part of the 35 metre transect were more than one order of magnitude. A gradient of Zn concentrations from 597 to 9080 mug g(-1) dry soil was found. A comparison between field concentrations of the four metals and previous studies on their relative toxicities to Collembola, suggested that Zn is likely to be responsible for any ecotoxicological effects on springtails at this site. Euedaphic ( soil dwelling) Collembola were extracted by placing soil cores into Tullgren funnels and epedaphic ( surface dwelling) species were sampled using pitfall traps. There was no obvious relationship between the total abundance, or a range of commonly used diversity indices, and Zn levels in soils. However, individual species showed considerable differences in abundance. Metal "tolerant'' (e.g., Ceratophysella denticulata) and metal "sensitive'' (e.g., Cryptopygus thermophilus) species could be identified. Epedaphic species appeared to be influenced less by metal contamination than euedaphic species. This difference is probably due to the higher mobility and lower contact with the soil pore water of epedaphic springtails in comparison to euedaphic Collembola. In an experiment exposing the standard test springtail, Folsomia candida, to soils from all 32 sampling points, adult survival and reproduction showed small but significant negative relationships with total Zn concentrations. Nevertheless, juveniles were still produced from eggs laid by females in the most contaminated soils with 9080 mug g(-1) Zn. Folsomia candida is much more sensitive to equivalent concentrations of Zn in the standard OECD soil. Thus, care should be taken in extrapolating the results of laboratory toxicity tests on metals in OECD soil to field soils, in which, the biological availability of contaminants is likely to be lower. Our studies have shown the importance of ecotoxicological effects at the species level. Although there may be no differences in overall abundance, sensitive species that are numerous in contaminated sites, and which may play important roles in decomposition("keystone species'') can be greatly reduced in numbers by pollution.

A comparative study of the effects of metal contamination on collembola in the field and in the laboratory

We examined the species diversity and abundance of Collembola at 32 sampling points along a gradient of metal contamination in a rough grassland site ( Wolverhampton, England), formerly used for the disposal of metal-rich smelting waste. Differences in the concentrations of Cd, Cu, Pb and Zn between the least and most contaminated part of the 35 metre transect were more than one order of magnitude. A gradient of Zn concentrations from 597 to 9080 mug g(-1) dry soil was found. A comparison between field concentrations of the four metals and previous studies on their relative toxicities to Collembola, suggested that Zn is likely to be responsible for any ecotoxicological effects on springtails at this site. Euedaphic ( soil dwelling) Collembola were extracted by placing soil cores into Tullgren funnels and epedaphic ( surface dwelling) species were sampled using pitfall traps. There was no obvious relationship between the total abundance, or a range of commonly used diversity indices, and Zn levels in soils. However, individual species showed considerable differences in abundance. Metal "tolerant'' (e.g., Ceratophysella denticulata) and metal "sensitive'' (e.g., Cryptopygus thermophilus) species could be identified. Epedaphic species appeared to be influenced less by metal contamination than euedaphic species. This difference is probably due to the higher mobility and lower contact with the soil pore water of epedaphic springtails in comparison to euedaphic Collembola. In an experiment exposing the standard test springtail, Folsomia candida, to soils from all 32 sampling points, adult survival and reproduction showed small but significant negative relationships with total Zn concentrations. Nevertheless, juveniles were still produced from eggs laid by females in the most contaminated soils with 9080 mug g(-1) Zn. Folsomia candida is much more sensitive to equivalent concentrations of Zn in the standard OECD soil. Thus, care should be taken in extrapolating the results of laboratory toxicity tests on metals in OECD soil to field soils, in which, the biological availability of contaminants is likely to be lower. Our studies have shown the importance of ecotoxicological effects at the species level. Although there may be no differences in overall abundance, sensitive species that are numerous in contaminated sites, and which may play important roles in decomposition("keystone species'') can be greatly reduced in numbers by pollution.

The influence of mineral solubility and soil solution concentration on the toxicity of copper to Eisenia fetida Savigny

The OECD 14 d earthworm acute toxicity test was used to determine the toxicity of copper added as copper nitrate (Cu(NO3)(2)), copper sulphate (CuSO4) and malachite (Cu-2(OH)(2)(CO3)) to Eisenia fetida Savigny. Cu(NO3)(2), and CuSO4 were applied in both an aqueous (aq) and solid (s) form, Cu-2(OH)(2)(CO3) was added as a solid. Soil solution was extracted by centrifugation, and analysed for copper. Two extractants [0.01 M CaCl2 and 0.005 M diethylenetriminpentaacetic acid (DTPA)] were used as a proxy of the bioavailable copper fraction in the soil. For bulk soil copper content the calculated copper toxicity decreased in the order nitrate > sulphide > carbonate, the same order as decreasing solubility of the metal compounds. For Cu(NO3)(2) and CuSO4, the LC50s obtained were not significantly different when the compound was added in solution or solid form. There was a significant correlation between the soil solution copper concentration and the percentage earthworm mortality for all 3 copper compounds (P less than or equal to 0.05) indicating that the soil pore water copper concentration is important for determining copper availability and toxicity to E. fetida. In soil avoidance tests the earthworms avoided the soils treated with Cu(NO3)(2) (aq and s) and CuSO4 (aq and s), at all concentrations used (110-8750 mug Cu g(-1), and 600-8750 mug Cu g(-1) respectively). In soils treated with Cu-2(OH2)CO3, avoidance behaviour was exhibited at all concentrations greater than or equal to3500 mug Cu g(-1). There was no significant correlation between the copper extracted by either CaCl2 or DTPA and percentage mortality. These two extractants are therefore not useful indicators of copper availability and toxicity to E. fetida.

Single versus multiple occupancy - Effects on toxicity parameters measured on Eisenia fetida in lead nitrate-treated soil

The mortality (7 and 14 d), weight change (7 and 14 d), and metal uptake of Eisenia fetida (Savigny, 1826) kept in Pb(NO3)(2)-treated Kettering loam soil in single- and multiple-occupancy (10 earthworms) test containers were determined. The number of earthworms to dry mass (g) ratio of soil was 1:50 in both sets of test containers. Lead concentrations were in the nominal range of 0 to 10,000 mg Pb/kg soil (mg/kg hereafter). Levels of mortality at a given concentration were statistically identical between the single- and multiple-occupancy tests, except at 1,800 mg/kg, at which significantly (p less than or equal to 0.05) more mortality occurred in the multiple-occupancy tests. Death of individual earthworms in the multiple-occupancy tests did not trigger death of the other earthworms in that soil. The LC50 values (concentration statistically likely to kill 50% of the population) were identical between the multiple- and single-occupancy soils: 2,662 mg/kg (2,598-2,984, 7 d) and 2,589 mg/kg (2,251-3,013, 14 d) for the multiple-occupancy soils and 2,827 mg/kg (2,443-3,168, both 7 and 14 d) for the single-occupancy soils (values in brackets represent the 95% confidence intervals). Data were insufficient to calculate the concentration statistically likely to reduce individual earthworm mass by 50% (EC50), but after 14 d, the decrease in earthworm weight in the 1,800 and 3,000 mg/kg tests was significantly greater in the multiple- than in the single-occupancy soils. At 1,000, 1,800, and 3,000 mg/kg tests, earthworm Pb tissue concentration was significantly (p less than or equal to 0.05) greater in earthworms from the multiple-occupancy soils. The presence of earthworms increased the NH3 content of the soil; earthworm mortality increased NH3 concentrations further but not to toxic levels.

Toxicity testing of groundwater quality

Groundwater is an important resource in the UK, with 45% of public water supplies in the Thames Water region derived from subterranean sources. In urban areas, groundwater has been affected by onthropogenic activities over 0 long period of time and from a multitude of sources, At present, groundwater quality is assessed using a range of chemical species to determine the extent of contamination. However, analysing a complex mixture of chemicals is time-consuming and expensive, whereas the use of an ecotoxicity test provides information on (a) the degree of pollution present in the groundwater and (b) the potential effect of that pollution. Microtox (TM), Eclox (TM) and Daphnia magna microtests were used in conjunction with standard chemical protocols to assess the contamination of groundwaters from sites throughout the London Borough of Hounslow and nearby Heathrow Airport. Because of their precision, range of responses and ease of use, Daphnia magna and Microfox (TM) tests are the bioassays that appear to be most effective for assessing groundwater toxicity However, neither test is ideal because it is also essential to monitor water hardness. Eclox (TM) does not appear to be suitable for use in groundwater-quality assessment in this area, because it is adversely affected by high total dissolved solids and electrical conductivity.

Is there a relationship between soil and groundwater toxicity?

Part IIA of the Environmental Protection Act 1990 requires environmental regulators to assess the risk of contaminants leaching from soils into groundwater (DETR, 1999). This newly introduced legislation assumes a link between soil and groundwater chemistry, in which rainwater leaches contaminants from soil into the saturated zone. As the toxicity of both groundwater and overlying soils is dependent upon the chemicals present, their partitioning and their bioavailability, similar patterns of soil, leachates and groundwater toxicity should be observed at contaminated sites. Soil and groundwater samples were collected from different contaminated land sites in an urban area, and used to determine relationships between soil chemistry and toxicity, mobility of contaminants, and groundwater chemistry and toxicity. Soils were leached using water to mimic rainfall, and both the soils and leachates tested using bioassays. Soil bioassays were carried out using Eisenia fetida, whilst groundwater and leachates were tested using the Microtox(TM) test system and Daphnia magna 48 h acute tests. Analysis of the bioassay responses demonstrated that a number of the samples were toxic to test organisms, however, there were no significant statistical relationships between soil, groundwater and leachate toxicity. Nor were there significant correlations between soil, leachates and groundwater chemistry.