Effect of leaching and aging on the bioavailability of lead to the springtail Folsomia candida

Because it is unclear if leaching can account for differences in metal bioavailability observed between metal-spiked soils and historically contaminated field soils, we simultaneously assessed Pb toxicity to the springtail Folsomia candida in three transects of Pb-contaminated soils and in leached and unleached soils spiked at similar total Pb concentrations. Total Pb concentrations of 3,877 mg/kg dry weight and higher always caused significant effects on F. candida reproduction in the spiked soils. In the transects, only the soil with the highest Pb concentration of 14,436 mg/kg dry weight significantly affected reproduction. When expressed as pore-water concentrations, reproduction was never significantly affected at Pb concentrations of 0.539 mg/L, whereas reproduction was always significantly affected at Pb concentrations of 0.678 mg/L and higher, independent of the soil treatment. These results indicate that pore-water Pb concentrations can explain, at least in part, the observed differences in the toxicity data expressed as total Pb concentrations. Leaching after the spiking procedure only caused small differences in Pb toxicity and, therefore, cannot account for toxicity differences between laboratory-spiked soils and historically contaminated field soils.     

Analysis of arsenic bioavailability in contaminated soils

The bioavailable arsenic (As) content of contaminated soils was determined by joint analyses of acid-soluble, total water-soluble, and biovailable As by using a luminescent bacterial sensor, Escherichia coli MC1061(pTOO31). According to the results of this study, a significant positive correlation was found between the concentration of total water-soluble As and the bioavailability of As. However, the bioavailability of As in soil varied between sampling sites and was not equal when compared to the concentration of total water-soluble As; bioavailable As was 3 to 77% of total water-soluble As in soil. Our experiments also showed that aging and sequestration of As occurs in contaminated soils and As compounds thus become progressively less bioavailable with time. As a consequence, the bioavailability and toxicity of As should be considered when evaluating the ecological risks of contaminated soils.     

Influence of aging on copper bioavailability in soils

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 CaCl2-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 CaCl2-extracted copper fraction.     

The relative sensitivity of growth and reproduction in the springtail, Folsomia candida, exposed to xenobiotics in the laboratory: an indicator of soil toxicity

The Folsomia candida reproduction test [ISO, 1998. Soil quality--Inhibition of reproduction of Collembola (Folsomia candida) by soil pollutants. International Standard Organization Report 11267, 1998, Geneva] is used to evaluate the ecotoxicological risks of contaminants in soils. The aim of this study was to compare the sensitivity of growth and reproduction of F. candida to four xenobiotics: two metals (Cd, Al), one metalloid (As), and one organic compound (pentachlorophenol). We showed that reproduction is a slightly more sensitive parameter than growth: EC(20) for reproduction was 1.25 microg/g dry soil for arsenic, 56 microg/g for cadmium, 97.5 microg/g for aluminum, and 41.7 microg/g for pentachlorophenol. The corresponding EC(20) values for growth were 2.8, 65, 630, and 94.6 microg/g. Keeping in mind that a growth test needs fewer juveniles and less time than a reproduction test, we conclude that the two parameters are complementary and could be used for a better ecotoxicological evaluation of contaminants. However, the relative growth and reproduction sensitivities should be tested with more chemicals before growth could be considered as a good alternative for a faster sublethal test.     

Assessing the toxicity of contaminated soils using the nematode Caenorhabditis elegans as test organism

In this study, nine uncontaminated reference soils and 22 contaminated soils with different physico-chemical properties and contamination patterns were tested with a standardized toxicity test, using the nematode, Caenorhabditis elegans, as test organism. Fertility, growth and reproduction of C. elegans in the soils were compared with the exposure in standard soil Lufa St.2.2. C. elegans showed 100% fertility and a very low variability of growth in the reference soils. Although, reproduction varied considerably between the various reference soils, validity criteria (>30 offspring per test organism) were met in all reference soils. Moreover, Lufa St. 2.2 turned out to be a suitable and representative control soil. In order to clearly classify the effects of the polluted soils on C. elegans, toxicity thresholds were derived for nematode fertility (20% inhibition), growth (10% inhibition) and reproduction (40% inhibition) on the basis of the test inherent variability (MDD=minimal detectable difference), as well as their variability between the uncontaminated reference soils (MTI=maximal tolerable inhibition). The contaminated soils showed clear toxic effects on the nematodes, whereas the toxicity was better correlated to organic than to heavy metal contamination in bulk soil. Interestingly, the results of the nematode toxicity test were not well correlated with those of tests with oligochaetes, collembolans and plants, performed with the same soils, showing that the results are not redundant. The toxicity test using C. elegans turned out to be suitable for testing the toxicity of field collected soils and might by a valuable addition to soil test batteries.     

Ecotoxicity of a polycyclic aromatic hydrocarbon (PAH)-contaminated soil

Soil samples from a former cokery site polluted with polycyclic aromatic hydrocarbons (PAHs) were assessed for their toxicity to terrestrial and aquatic organisms and for their mutagenicity. The total concentration of the 16 PAHs listed as priority pollutants by the US Environmental Protection Agency (US-EPA) was 2634+/-241 mg/kgdw in soil samples. The toxicity of water-extractable pollutants from the contaminated soil samples was evaluated using acute (Vibrio fischeri; Microtox test, Daphnia magna) and chronic (Pseudokirchneriella subcapitata, Ceriodaphnia dubia) bioassays and the EC values were expressed as percentage water extract in the test media (v/v). Algal growth (EC50-3d=2.4+/-0.2% of the water extracts) and reproduction of C. dubia (EC50-7d=4.3+/-0.6%) were the most severely affected, compared to bacterial luminescence (EC50-30 min=12+/-3%) and daphnid viability (EC50-48 h=30+/-3%). The Ames and Mutatox tests indicated mutagenicity of water extracts, while no response was found with the umu test. The toxicity of the soil samples was assessed on the survival and reproduction of earthworms (Eisenia fetida) and collembolae (Folsomia candida), and on the germination and growth of higher plants (Lactuca sativa L.: lettuce and Brassica chinensis J.: Chinese cabbage). The EC50 values were expressed as percentage contaminated soil in ISO soil test medium (weight per weight-w/w) and indicated severe effects on reproduction of the collembola F. candida (EC50-28 d=5.7%) and the earthworm E. fetida (EC50-28 d=18% and EC50-56 d=8%, based on cocoon and juvenile production, respectively). Survival of collembolae was already affected at a low concentration of the contaminated soil (EC50-28 d=11%). The viability of juvenile earthworms was inhibited at much lower concentrations of the cokery soil (EC50-14 d=28%) than the viability of adults (EC50-14 d=74%). Only plant growth was inhibited (EC50-17d=26%) while germination was not. Chemical analyses of water extracts allowed us to identify inorganic water-extractable pollutants as responsible for toxicity on aquatic species, especially copper for effects on D. magna and C. dubia. The soil toxicity on collembolae and earthworms could be explained by 4 PAH congeners-fluorene, phenanthrene, pyrene, and fluoranthene. Yet, toxicity of the cokery soil as a whole was much lower than toxicity that could be deduced from the concentration of each congener in spiked soils, indicating that pollutants in the soil became less bioavailable with ageing.     

Arsenic accumulation and toxicity in the earthworm Eisenia fetida affected by chloride and phosphate

Chloride and phosphate (PO4) were added to field soils contaminated with arsenic and heavy metals, and Eisenia fetida were inoculated in these treated soils for 70 d. After 28 d, earthworm survival, body weight, and cocoon production were measured. During the exposure period, the accumulation of As and four metals (Cu, Zn, Cd, and Pb) in E. fetida was analyzed. Bulk soils and soil solutions were characterized for the contents of As, metals, organic matter, and major cations and anions; cation exchange capacity; and pH. Although addition of Cl to the soil did not impact As toxicity, it significantly reduced As uptake. The addition of PO4 induced changes in As toxicity and reduced As accumulation even though the As concentration in the soil pore water increased because of the substitution of As by P at soil sorption sites. These results imply that the addition of Cl induced the formation of an As-Cl complex, thereby resulting in decreased As bioavailability; they also imply that monovalent phosphate (H2PO4(-)) competes with As for uptake through transporter-mediated mechanisms in cells.     

Effect of soil properties on lead bioavailability and toxicity to earthworms

Soil properties are important factors modifying metal bioavailability to ecological receptors. Twenty-one soils with a wide range of soil properties (USA; http://soils.usda.gov/technical/classification/taxonomy/) were amended with a single concentration of Pb (2,000 mg/kg) to determine the effects of soil properties on Pb bioavailability and toxicity to earthworms. Earthworm mortality ranged from 0 to 100% acute mortality following exposure to the same total concentration of Pb (2,000 mg/kg) in amended field soils. Internal Pb concentrations in earthworms ranged from 28.7 to 782 mg/kg, with a mean of 271 mg/kg. Path analysis was used to partition correlations in an attempt to discern the relative contribution of each soil property. Results of path analysis indicated that pH was the most important soil property affecting earthworm mortality (p < 0.01) and internal Pb (p < 0.05). Soil pH was related inversely to mortality and internal Pb, soil solution Pb, and Pb bioavailability. The most important soil property modifying reproduction was amorphous iron and aluminum oxides (FEAL). Because FEAL is rich in pH-dependent cation-exchange sites, several soil properties, including pH, FEAL, and cation-exchange capacity, have a causal effect on Pb adsorption and soluble Pb. Path analysis is useful for assessing contaminated soils with a wide range of soil properties and can assist in ecological risk assessment and remediation decisions for contaminated sites. Soil properties are important factors modifying metal bioavailability and toxicity and should be considered during the ecological risk assessment of metals in contaminated soils.     

The effect of counterion and percolation on the toxicity of lead for the springtail Folsomia candida in soil

In standard soil toxicity tests, heavy metals are amended as water-soluble salts. The role of the counterion in metal salt toxicity is scarcely looked into. In this study, we assessed the contribution of nitrate and chloride to the toxicity of lead to Folsomia candida in a natural standard soil. Both lead salts were tested according the standard test protocol as well as after percolation of the soil with deionized water. Lead nitrate was more toxic than lead chloride for survival as well as reproduction. Percolation proved to be an effective method to remove counterions from the soil. Survival of F. candida increased for both metal salts when percolation was included. Percolation reduced the reproduction toxicity of lead, the effect of which was largest for the nitrate salt. In percolated treatments, the nitrate and chloride lead salts did not differ in toxicity. It is concluded that counterions contribute to metal toxicity and that nitrate is more toxic to F. candida than chloride.     

Comparative ecotoxicity of three polluted industrial soils for the Collembola Folsomia candida

We tried to quantify the ecotoxicity of three industrial soil samples contaminated by different metals, using the Collembolan Folsomia candida as a biological model and mortality, growth and reproduction as parameters. The observed ecotoxicities are rather normal for the first such soil sample (aluminum factory) but are high for the second sample (ore treatment) and relatively low for the third one (zinc factory) considering its high metal concentrations. For these last two soil samples, an unusual ecotoxicity plotting is observed: two high ecotoxicity recordings fit with a low and high percentage of polluted soil to non-polluted soil and noticeably lower ecotoxicity recordings are observed between them. Chemical analyses of metals in pore waters show that arsenic probably explains part of such an unusual ecotoxicity curve. Otherwise, mortality and growth of the animals are less sensitive parameters than reproduction. Our experiments show that the results of the ecotoxicological assays of polluted soils are complex and difficult to interpret.     

Long-term responses of snails exposed to cadmium-contaminated soils in a partial life-cycle experiment

Juvenile snails were exposed during their growth period to Cd-contaminated field and artificial soils and then transferred to uncontaminated soil to assess the sequels of previous exposure on adult reproduction. Growth modelling highlighted growth inhibitions of 5% and 10% after 70 and 84 days of exposure to 20 and 100 mg Cd kg(-1) in artificial soils, respectively. Growth disruption was accompanied by a decrease in the clutch number and a 4-week delay in the egg-laying cycle. Although it was also contaminated at 20 mg Cd kg(-1), the contaminated field soil did not lead to detectable effects in snails, suggesting a lower Cd bioavailability confirmed by the bioaccumulation analysis. We demonstrated that the 28-day growth test, as advised by the ISO-guideline, may not be sufficient to assess sublethal toxic effects of realistically contaminated soils. For this purpose, a life cycle experimental set-up is proposed, allowing a thorough assessment of toxicity during successive life stages.     

Modeling zinc toxicity for terrestrial invertebrates

Acute and chronic ecotoxicity tests with zinc were performed with the earthworm Eisenia fetida, the potworm Enchytraeus albidus, and the springtail Folsomia candida. To assess the influence of the soil type on zinc toxicity for these soil invertebrates, these tests were carried out in a standard artificial soil, a sandy and a loamy field soil. Based on the results of this experimental work and data taken from literature, models were developed relating the ecotoxicity of zinc to the most important parameters controlling bioavailability: pH and cation exchange capacity. Models were developed for E. fetida and F. candida using the regression technique partial least squares projection to latent structures (PLS). Acute as well as chronic toxicity data of both organisms could be normalized on the basis of the pH and the cation exchange capacity of the test soils. For other terrestrial invertebrates, not enough data were available to develop reliable models.     

Assessing 2,4,6-trinitrotoluene (TNT)-contaminated soil using three different earthworm test methods

Within the scope of a phytoremediation project, the toxicity of 2,4,6-trinitrotoluene (TNT) contaminated soil (and its toxic metabolites) on earthworms was assessed. In addition to the standard acute and reproduction tests (ISO 11268), an avoidance response test was applied. The test methods covered all important ecological relevant endpoints (acute, chronic, behavioral). At a concentration of 1142 mg/kg, TNT caused significant toxic effects in all test methods, but at lower test concentrations no significant acute or reproduction toxic effects could be observed. The avoidance response test, however, showed significant repellent effects at a concentration of 29 mg/kg TNT and therefore proved to be more sensitive than the other tests in this case. Results of the earthworm tests compared well with results of an ecotoxicological biotest battery. Thus, earthworm toxicity tests are useful tools for terrestrial risk assessment but require a hierarchy of test designs that differ in effect levels (behavior, sublethal, lethal). Whereas higher concentrations of a pollutant can easily be assessed with the acute test (which requires lethal concentrations to show an effect), contaminated soils with lower (sublethal) pollutant concentrations require more sensitive test methods such as reproduction or behavioral tests in their risk assessment.     

Effect of soil properties and aging on the toxicity of copper for Enchytraeus albidus, Enchytraeus luxuriosus, and Folsomia candida

In the present study, the effect of the heavy-metal salt copper chloride (CuCl2.2H2O) 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 Agricultural 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.     

Feeding inhibition in the soil collembolan Folsomia candida as an endpoint for the estimation of organic waste ecotoxicity

Despite the increasing quantities of organic wastes that are being reused in soils, there are few studies that focus on the selection of bioassays for the ecotoxicological risk assessment of organic wastes to soils. In the present study, differences in feeding inhibition in the soil collembolan Folsomia candida were evaluated as an ecotoxicological endpoint for the assessment of risk to soils amended with polluted organic wastes. Seven organic wastes (dewatered sewage sludges, thermally dried sewage sludges, composted sewage sludges, and a thermally dried pig slurry) were tested. These wastes had different origins, treatments, and pollutant burdens, and were selected as a representative sample of the wide variety of wastes currently generated. A clear dose response was observed for this parameter, with an increase in percentage of individual feeding inhibition with increased doses of organic wastes. More significantly, feeding inhibition correlated highly with mortality and reproduction inhibition in the different wastes. Composted sludges displayed the lowest toxicity, followed by thermally dried sludge and dewatered sludge. Thermally dried pig slurry showed the highest toxicity for feeding, with lower median effective concentration (EC50) values than the lowest dose tested. Among waste physicochemical parameters and pollutants, low organic matter stability appeared to be the main predictor of potential adverse effects on soil fauna, because it correlated significantly with feeding inhibition and mortality. Furthermore, feeding inhibition tests were run over a short exposure time (less than 7 d), which, together with the results obtained, makes this bioassay a good screening tool for organic waste toxicity.     

Toxicity benchmarks for antimony, barium, and beryllium determined using reproduction endpoints for Folsomia candida, Eisenia fetida, and Enchytraeus crypticus

The U.S. Environmental Protection Agency is developing ecological soil screening levels (Eco-SSLs) for the ecological risk assessment of contaminants at Superfund sites. The Eco-SSLs for several soil contaminants have been developed from toxicity benchmarks for soil invertebrates in the existing literature. Insufficient information to generate Eco-SSLs for Sb, Ba, and Be necessitated toxicity testing to fill the data gaps. We used standardized toxicity tests with the earthworm Eiseniafetida, enchytraeid Enchytraeus crypticus, and collembolan Folsomia candida in the present study. These tests were selected on the basis of their ability to measure chemical toxicity to ecologically relevant test species during chronic assays and their inclusion of at least one reproduction component among the measurement endpoints. Tests were conducted in Sassafras Sandy Loam soil, which supports relatively high bioavailability of metals. Weathering and aging procedures for metals in amended soil were incorporated into these studies to better reflect exposure conditions in the field. The relative toxicity of metals to the soil invertebrates tested was Be > Sb > Ba based on the median effective concentration values for reproduction. These studies produced toxicological data that can contribute to the development of Eco-SSLs for Sb, Ba, and Be for soil invertebrates.     

Relative bioavailability and bioaccessibility and speciation of arsenic in contaminated soils

Background: Assessment of soil arsenic (As) bioavailability may profoundly affect the extent of remediation required at contaminated sites by improving human exposure estimates. Because small adjustments in soil As bioavailability estimates can significantly alter risk assessments and remediation goals, convenient, rapid, reliable, and inexpensive tools are needed to determine soil As bioavailability.Objectives: We evaluated inexpensive methods for assessing As bioavailability in soil as a means to improve human exposure estimates and potentially reduce remediation costs.Methods: Nine soils from residential sites affected by mining or smelting activity and two National Institute of Standards and Technology standard reference materials were evaluated for As bioavailability, bioaccessibility, and speciation. Arsenic bioavailability was determined using an in vivo mouse model, and As bioaccessibility was determined using the Solubility/Bioavailability Research Consortium in vitro assay. Arsenic speciation in soil and selected soil physicochemical properties were also evaluated to determine whether these parameters could be used as predictors of As bioavailability and bioaccessibility.Results: In the mouse assay, we compared bioavailabilities of As in soils with that for sodium arsenate. Relative bioavailabilities (RBAs) of soil As ranged from 11% to 53% (mean, 33%). In vitro soil As bioaccessibility values were strongly correlated with soil As RBAs (R² = 0.92). Among physicochemical properties, combined concentrations of iron and aluminum accounted for 80% and 62% of the variability in estimates of RBA and bioaccessibility, respectively.Conclusion: The multifaceted approach described here yielded congruent estimates of As bioavailability and evidence of interrelations among physicochemical properties and bioavailability estimates.     

Manganese toxicity in soil for Eisenia fetida, Enchytraeus crypticus (Oligochaeta), and Folsomia candida (Collembola)

The U.S. Environmental Protection Agency is developing Ecological Soil Screening Level (Eco-SSL) benchmarks for ecological risk assessment (ERA) of contaminants at Superfund sites. Eco-SSLs are developed from published values whenever sufficient quantity and quality of data exist. Because insufficient information was available to generate an Eco-SSL for Mn, standardized toxicity testing was undertaken to fill the data gaps. Tests included the earthworm (Eisenia fetida) cocoon production test, the enchytraeid (Enchytraeus crypticus) reproduction test, and the collembolan (Folsomia candida) reproduction test, all conducted in Sassafras sandy loam soil that supports a relatively high bioavailability of metals. Weathering and aging of manganese-amended soil were carried out to more closely simulate exposure effects at Superfund sites on soil invertebrates. Data were analyzed by nonlinear regression to determine EC20 and EC50 values based on concentration-response relationships. The toxicity order for manganese in Sassafras sandy loam was E. crypticus>E. fetida>F. candida, with EC20 values of 116, 629, and 1209 mg kg(-1), respectively. The Eco-SSL requirement for the testing of multiple representative species is well justified. All study results will be submitted to the Eco-SSL Task Group for quality control review prior to inclusion in the Eco-SSL database.     

Influence of the Activity of Allobophora molleri in Microbial Activity and Metal Availability of Arsenic-Polluted Soils

We investigate the use of Allolobophora molleri as a biomarker of arsenic (As)-polluted soils and study the influence of A. molleri on the metabolic activity and microbial biodiversity of soil polluted with As. Because there are no experimental data available regarding the effect of the pollutant rate of As on A. molleri, we determined the LC₅₀ that was 143.5 mg As kg^?1. Sodium arsenite was added at two rates, equivalent to 143.5 and 71.8 mg As kg^?1 soil, to a soil that was then maintained with and without worms for 120 days. In addition, a nonpolluted soil without and with earthworms was used as the control. The As concentration in the soil was measured after 7 and 120 and the worm weight and As concentration after 120 days of exposure. Soil enzymatic activities and the structure of the soil microbial community, by analysis of phospholipid fatty acids, were determined. At the end of the experiment, the highest earthworm As contents were found in soils polluted with the highest rate of As. Earthworm weights significantly decreased in soil polluted with 143.5 or 71.8 mg As kg^?1, by 49.9 and 29.8 % of initial weight, because the worm consumption rate decreased. These results suggest that A. molleri can be used as a good biomarker of the As toxicity. The As available fraction decreased in polluted soil with worms because the metal was accumulated in worm tissues. However, this assimilation was lower than other worms such as L. rubbellus or L. terrestris. Soil enzymatic activities were decreased in As-polluted soils but were increased significantly by the presence of earthworms. The earthworms modified the soil microbial diversity. In this respect, A. molleri significantly increased (p < 0.05) the bacterial and fungal populations. Soil As pollution decreased microbial biodiversity but to a lesser extent in the presence of A. molleri.     

Effects and risk assessment of linear alkylbenzene sulfonates in agricultural soil. 4. The influence of salt speciation, soil type, and sewage sludge on toxicity using the collembolan Folsomia fimetaria and the earthworm Aporrectodea caliginosa as test organisms

Sewage sludge applied to agricultural soils often contains linear alkylbenzene sulfonates (LAS) in the range of 1 to 10 g/kg dry weight, and their toxicity to relevant soil organisms should, therefore, be assessed to ensure safe use of sewage sludge as a fertilizer. Studies of LAS toxicity to soil organisms are few, and to our knowledge, factors that may influence the toxicity in the field have not been studied in detail. In this paper, we report on the influence of speciation of LAS in the test solution added to soil (soluble Na-LAS vs poorly soluble Ca-LAS or Mg-LAS), the influence of soil type, and the modifying effects of sludge amendment on the toxicity of LAS. These issues were investigated using reproduction of Collembola and growth of juvenile earthworms as test parameters. Speciation of the LAS added to test soil did not have any influence on toxicity for any of the test species. Likewise, in three different agricultural soils (sand, loam, and clay), we found almost equal toxicities. The LAS added to test soil in a sludge-water suspension was equally toxic as when it was added in an aqueous solution. However, anaerobic incubation for 7 and 14 d of the LAS-sludge suspension (with no decay of LAS) caused the toxicity to increase almost threefold in both collembolan and earthworm. The relationships between soil constituents, bioavailability, and toxicity are also discussed.