Standard Toxicity Tests Research Articles

Acute toxicity of cerium oxide, titanium oxide and iron oxide nanoparticles using standardized tests

In this study, several standard toxicity tests have been performed on selected inorganic nanoparticles. Acute toxicity tests were selected according to their extensive use in toxicological studies and included phytotoxicity using several seeds, Daphnia magna and a bioluminescent test (Microtox®). All of them have been used in several international regulations as toxicity assays. In the case of nanoparticles (NPs), we have studied those of cerium oxide, titanium dioxide and iron oxide. Iron oxide NPs are well known and broadly used and were selected because of their low toxicity. Titanium dioxide and cerium oxide NPs are currently being used in several fields such as photocatalysis and medical applications, but their toxicity effects have been scarcely studied. Our results revealed that cerium NPs are extremely toxic in the entire set of tests conducted (inhibition higher than 80% at very low concentrations for the bioluminescence test and LC50=0.012mg/ml of mortality in the assays of D. magna), whereas titanium NPs were practically inert in terms of toxicity (values similar to those of controls). The possible toxicological effect of the solvents necessary to stabilize NPs in liquid medium for the three cases (stabilizers) has been also studied. Only in the germination test (phytotoxicity) of some seeds they showed some detrimental effect to germination. In general, the standardized tests proposed in this study have proved to be very useful in the determination of NPs toxicity when no or few data are available, although further work is necessary in the case of the germination test.

Effect of the exposure to metal lead on the regenerative ability of Lumbriculus variegatus (Oligochaeta)

Lumbriculus variegatus is a recommended species for use in sediment toxicity tests and is known to have a remarkable power of segmental regeneration. Here, we tested the effects of a chemical stressor on the regenerative ability of L. variegatus and investigated the potential of regenerative ability as an additional new parameter in standard toxicity tests. The worms were cut into two equal segments, and exposed to various concentrations of lead. Two assays were performed: one with sediment spiked with lead and the other with water spiked with lead. The endpoints were segmental regeneration, survival and behaviour. Regenerative ability was clearly affected by exposure to lead-contaminated sediment and lead-contaminated water. Organisms exposed to lead grew more slowly than those not exposed; worms exposed to contaminated water showed higher mortalities than those exposed to contaminated sediment. Results showed that L. variegatus’ regenerative ability, as a developmental test parameter, is more sensitive than mortality.

Assessing water quality suitability for shortnose sturgeon in the Roanoke River, North Carolina, USA with an in situ bioassay approach

Summary The aim of this study was to determine the suitability of water quality in the Roanoke River of North Carolina for supporting shortnose sturgeon Acipenser brevirostrum, an endangered species in the United States. Fathead minnows Pimephales promelas were also evaluated alongside the sturgeon as a comparative species to measure potential differences in fish survival, growth, contaminant accumulation, and histopathology in a 28-day in situ toxicity test. Captively propagated juvenile shortnose sturgeon (total length 49 ± 8 mm, mean ± SD) and fathead minnows (total length 39 ± 3 mm, mean ± SD) were used in the test and their outcomes were compared to simultaneous measurements of water quality (temperature, dissolved oxygen, pH, conductivity, total ammonia nitrogen, hardness, alkalinity, turbidity) and contaminant chemistry (metals, polycyclic aromatic hydrocarbons, organochlorine pesticides, current use pesticides, polychlorinated biphenyls) in river water and sediment. In the in situ test, there were three non-riverine control sites and eight riverine test sites with three replicate cages (25 × 15-cm (OD) clear plexiglass with 200-μm tear-resistant Nitex® screen over each end) of 20 shortnose sturgeon per cage at each site. There was a single cage of fathead minnows also deployed at each site alongside the sturgeon cages. Survival of caged shortnose sturgeon among the riverine sites averaged 9% (range 1.7–25%) on day 22 of the 28-day study, whereas sturgeon survival at the non-riverine control sites averaged 64% (range 33–98%). In contrast to sturgeon, only one riverine deployed fathead minnow died (average 99.4% survival) over the 28-day test period and none of the control fathead minnows died. Although chemical analyses revealed the presence of retene (7-isopropyl-1-methylphenanthrene), a pulp and paper mill derived compound with known dioxin-like toxicity to early life stages of fish, in significant quantities in the water (251–603 ng L−1) and sediment (up to 5000 ng g−1 dry weight) at several river sites, no correlation was detected of adverse water quality conditions or measured contaminant concentrations to the poor survival of sturgeon among riverine test sites. Histopathology analysis determined that the mortality of the river deployed shortnose sturgeon was likely due to liver and kidney lesions from an unknown agent(s). Given the poor survival of shortnose sturgeon (9%) and high survival of fathead minnows (99.4%) at the riverine test sites, our study indicates that conditions in the Roanoke River are incongruous with the needs of juvenile shortnose sturgeon and that fathead minnows, commonly used standard toxicity test organisms, do not adequately predict the sensitivity of shortnose sturgeon. Therefore, additional research is needed to help identify specific limiting factors and management actions for the enhancement and recovery of this imperiled fish species.

Combined developmental toxicity of bisphenol A and genistein in micromass cultures of rat embryonic limb bud and midbrain cells

Bisphenol A (BPA), widely used in industry and dentistry, and genistein (GEN), the predominant component of soy product, are both known environmental estrogen. In the present study, we investigated the developmental toxicity of BPA and GEN and their combined effect using micromass test, which is one of three standard alternative developmental toxicity tests recommended by European Center for the Validation of Alternative Methods (ECVAM). The results showed that IC50-P (cell proliferation) and IC50-D (cell differentiation) of BPA and GEN were approximately 20 and 5 μg/ml, respectively. No observed adverse effect level (NOAEL) of BPA and GEN were 10 and 0.94 μg/ml, respectively. The manifestation of BPA as a teratogen was insufficient, although the “low dose” effect should be paid attention to. While the evidence of GEN as a teratogen was solid, especially with the consideration of “high dose” application in clinical treatment. The combined effect of BPA and GEN was generally additive action except that in MB proliferation.

Use of vegetated agricultural drainage ditches to decrease toxicity of irrigation runoff from tomato and alfalfa fields in California, USA

The current study investigated the potential of vegetated drainage ditches for mitigating the impact of agricultural irrigation runoff on downstream aquatic ecosystems. Water column toxicity to larval fathead minnow (Pimephales promelas),and the amphipod Hyalella azteca was measured for 12 h or less at the ditch inflow and outflow, using custom-built in situ exposure systems. In addition, water and sediment samples were subject to standard toxicity tests with Ceriodaphnia dubia and H. azteca, respectively. No acute toxicity to larval fathead minnow was observed; however, runoff was highly toxic to invertebrates. Passage through a 389- to 402-m section of vegetated ditch had a mitigating effect and reduced toxicity to some degree. However, runoff from an alfalfa field treated with chlorpyrifos remained highly toxic to both invertebrate species, and runoff from a tomato field treated with permethrin remained highly toxic to H. azteca after passage through the ditch. Predicted toxic units calculated from insecticide concentrations in runoff and 96-h median lethal concentration (LC50) values generally agreed with C. dubia toxicity measured in the laboratory but significantly underestimated in situ toxicity to H. azteca. Sediments collected near the ditch outflow were toxic to H. azteca. Results from the current study demonstrate that experimental vegetated ditches were unable to eliminate the risk of irrigation runoff to aquatic ecosystems. In addition, protective measures based on chemical concentrations or laboratory toxicity tests with C. dubia do not ensure adequate protection of aquatic ecosystems from pyrethroid-associated toxicity.

An assessment of Hyalella azteca burrowing activity under laboratory sediment toxicity testing conditions

Burrowing of the freshwater amphipod Hyalella azteca was evaluated under laboratory conditions similar to those recommended for standard sediment toxicity testing in Canada (EPS 1/RM/33; Environment Canada, 1997) and the United States (EPA/600/R-99/064; US EPA, 2000). Sediment type, time of day (light versus dark), size of animal, and the presence or absence of food were varied to assess their effects on burrowing activity. Hyalella azteca were found to burrow rapidly in fine, organic-rich sediments, but were slower to burrow in a sandy sediment. There was no increase in the number of animals occupying the sediment surface of a fine, organic-rich sediment after 4 h of darkness compared to the previous 4 h of light. Over a 9- to 10-d duration, a higher percentage of animals occupied the surface of the sandy sediment. The addition of food promoted burrowing in sandy sediment, as did using smaller animals. Overall, longer-duration tests involving older animals and coarse sediments may require formal observation to confirm burrowing and ensure adequate sediment exposure. The addition of food during a test may promote the burrowing of larger animals in coarse sediments, but may not be necessary in field-collected sediments that are not excessively sandy.

Impact of environmental chemicals on lung development.

BackgroundDisruption of fundamental biologic processes and associated signaling events may result in clinically significant alterations in lung development.ObjectivesWe reviewed evidence on the impact of environmental chemicals on lung development and key signaling events in lung morphogenesis, and the relevance of potential outcomes to public health and regulatory science.Data sourcesWe evaluated the peer-reviewed literature on developmental lung biology and toxicology, mechanistic studies, and supporting epidemiology.Data synthesisLung function in infancy predicts pulmonary function throughout life. In utero and early postnatal exposures influence both childhood and adult lung structure and function and may predispose individuals to chronic obstructive lung disease and other disorders. The nutritional and endogenous chemical environment affects development of the lung and can result in altered function in the adult. Studies now suggest that similar adverse impacts may occur in animals and humans after exposure to environmentally relevant doses of certain xenobiotics during critical windows in early life. Potential mechanisms include interference with highly conserved factors in developmental processes such as gene regulation, molecular signaling, and growth factors involved in branching morphogenesis and alveolarization.ConclusionsAssessment of environmental chemical impacts on the lung requires studies that evaluate specific alterations in structure or function—end points not regularly assessed in standard toxicity tests. Identifying effects on important signaling events may inform protocols of developmental toxicology studies. Such knowledge may enable policies promoting true primary prevention of lung diseases. Evidence of relevant signaling disruption in the absence of adequate developmental toxicology data should influence the size of the uncertainty factors used in risk assessments.

Effects of the Veterinary Pharmaceutical Ivermectin in Indoor Aquatic Microcosms

The effects of the parasiticide ivermectin were assessed in plankton-dominated indoor microcosms. Ivermectin was applied once at concentrations of 30, 100, 300, 1000, 3000, and 10,000 ng/l. The half-life (dissipation time 50%; DT50) of ivermectin in the water phase ranged from 1.1 to 8.3 days. The lowest NOECcommunity that could be derived on an isolated sampling from the microcosm study by means of multivariate techniques was 100 ng/l. The most sensitive species in the microcosm study were the cladocerans Ceriodaphnia sp. (no observed effect concentration, NOEC = 30 ng/l) and Chydorus sphaericus (NOEC = 100 ng/l). The amphipod Gammarus pulex was less sensitive to ivermectin, showing consistent statistically significant reductions at the 1000-ng/l treatment level. Copepoda taxa decreased directly after application of ivermectin in the highest treatment but had already recovered at day 20 posttreatment. Indirect effects (e.g., increase of rotifers, increased primary production) were observed at the highest treatment level starting only on day 13 of the exposure phase. Cladocera showed the highest sensitivity to ivermectin in both standard laboratory toxicity tests as well as in the microcosm study. This study demonstrates that simple plankton-dominated test systems for assessing the effects of ivermectin can produce results similar to those obtained with large complex outdoor systems.

Particle-specific sorption/desorption properties determine test compound fate and bioavailability in toxicity tests with Chironomus riparius-high-resolution studies with lindane

We studied the sorption (batch equilibrium experiments) and desorption (consecutively harsher supercritical fluid extractions) of lindane to different types of sediment and food particles, as well as larval uptake in standardized peat-based artificial sediment toxicity tests with the midge Chironomus riparius. Lindane sorption to organic particles was fast and efficient, reaching 98+/-0.1 and 97+/-0.1% of added compound in 48 h for peat and Tetraphyll(R), respectively, and 77+/-0.2% in whole sediment. Sorption to inorganic particles, that is, sand and kaolin clay, was much lower, 9.6+/-1.3% and 8.3+/-0.8%, respectively. Supercritical fluid extractions showed that most of the lindane sorbed to organic particles and sediment was loosely bound, as only 9 to 14% remained associated with particles after weak and intermediate extractions strengths. Larval uptake of dissolved lindane was 4.9+/-0.71 and 10.8+/-1.2 microg/g wet weight in 22 and 68 microg/L treatments, respectively, and four to five times higher than that of particle-associated lindane, ranging 1.0+/-0.15 to 2.7+/-0.21 microg/g in the above treatments. Surprisingly, larval uptake of lindane was similar from refractory peat and the more labile Tetraphyll particles. Despite an efficient larval uptake of dissolved lindane, sorption/desorption of lindane to/from Tetraphyll particles will facilitate digestive uptake in toxicity tests, particularly in spiked-water scenarios where food particles may act as vectors. Our results show that the exposure scenario is an important determinant for the behavior and bioavailability of test compounds in standardized toxicity tests.

A new method for early assessment of effects of exposing two non-target crustacean species, Asellus aquaticus and Gammarus fossarum, to pesticides, a laboratory study

A reliable method is needed for assessing the condition of aquatic animals and their resistance to toxic pollutants. The physiological responses of two freshwater crustaceans, Asellus aquaticus and Gammarus fossarum, following in vitro exposure to two pesticides (atrazine and imidacloprid), were measured by a combination of electron transport system (ETS) activity and respiration (R). Short-term exposure concentrations were selected according to standard toxicity tests and ranged from 0.01 mg L(-1) to 10 mg L(-1). When pesticide concentration was greater than 1 mg l(- 1) (which is below the LC(50) [48 hours] determined for both species), A. aquaticus and G. fossarum responded to short-term exposure with elevated levels of R and/or lower levels of ETS activity. One hour exposure to concentrations of up to 10 mg L(-1) showed an effect in both test species. Laboratory tests confirmed that G. fossarum is more sensitive to short-term pesticide exposure than A. aquaticus. The combination of these two methods provides a useful and effective tool for assessing the general condition of aquatic animals. It also enables to determine toxic effects on freshwater biota of specific or combined pollutants. ETS/R ratio may be used as a quick predictor of effects on organisms exposed to pesticides and other stress factors such as changes in temperature, light, salinity, oxygen concentration and food.

Saltatory ontogeny of fishes and sensitive early life stages for ecotoxicology tests

Fish display a wide range of developmental ontogenies. These distinctions have taxonomic, evolutionary, and ecological importance in addition to practical implications on the use of fish in aquatic toxicity tests. With respect to animal welfare, vertebrates are afforded protected or non-protected status in the European Union based upon whether they feed endogenously off the yolk or exogenously by procurement and ingestion of food. The concept of saltatory ontogeny suggests development is not gradual but proceeds in leaps separated by a series of stable developmental states. In this context, endogenous/exogenous feeding also distinguishes the developmental phases of embryo (egg), eleutheroembryo (feeding off the yolk sac) and larvae (exogenous feeding) in fish. The recent proposal for the Fish Embryo Test (FET) as an animal alternative to the standard fish acute toxicity test (OECD 203 and equivalent tests) puts a clear focus on the need to identify the non-protected and protected life intervals in test species as well as their sensitivities which coincides with the developmental phases identified in saltatory ontogeny. In this paper we described a method to quantify embryo, eleutheroembryo, and larva phases in Danio rerio, the zebrafish. Danio eleutheroembryos preyed upon 5 different protozoan species ( Euglena, Euplotes, Paramecium aurelia, Paramecium bursaria and Paramecium multimicronucleatum) between 24 and 48 hr following hatching (85–95% of fish, n = 20 per species, 25 °C). Based upon these data it is recommended that testing of developing zebrafish embryos should be terminated between 24 and 48 hr after hatching in order to be compliant with existing animal welfare legislation within Europe.

Ring test for whole-sediment toxicity assay with -a- benthic marine diatom

This work presents the results of an interlaboratory proficiency exercise for whole-sediment toxicity assays with the benthic marine diatom Cylindrotheca closterium. An assay protocol was established and followed by all participating laboratories. Cell growth after 72 h exposure was the endpoint used. Four sediment samples of unknown toxicity were assayed. The main problem encountered during this exercise was the differences in the cell growth of algae exposed to reference sediment. Those differences may be associated with changes in the physiological status of the initial culture due to temperature changes during transport to the other laboratories. In general, the method proposed presented good replicability (precision between replicates) and reproducibility (interlaboratory precision). Around 80% (17 out of 21) of results obtained were classified as satisfactory ( Z-scores < 2). The whole-sediment assay with C. closterium presented here can be considered sufficiently successful for possible use as a standard toxicity test. The assay is simple to perform, the proposed species is ecologically relevant as an integral component of microphytobenthos, and is widely distributed around the world. These positive factors suggest that the whole-sediment assay with the benthic marine diatom C. closterium can be used as a reliable tool in marine sediment quality assessment.

Toxicity assessment of sequential leachates of tire powder using a battery of toxicity tests and toxicity identification evaluations

Approximately 460 000 ton of rubber are dispersed annually along the European roads due to tire wear. Tire rubber is known to leach compounds that are toxic to aquatic organisms. However, the potential effects of tire wear material on aquatic organisms at environmental relevant concentrations, and over time have so far not been extensively studied. In this study, rubber from three different tires was abraded and the powder leached in deionised water. The rubber powder was leached six times sequentially. All leachates were tested for toxicity using standardized toxicity tests including green algae ( Pseudokirchneriella subcapitata, 72 h growth inhibition), crustaceans ( Daphnia magna, 24 and 48 h immobility and Ceriodaphnia dubia, 48 h survival and 9 d reproduction and survival), and zebra fish eggs ( Danio rerio, 48 h lethality). The reproduction of C. dubia was the most sensitive endpoint tested, with an EC50 of 0.013 g L −1 up to the third leaching of the most toxic tire, which is similar to a predicted concentration in road runoffs. The toxicity of all tires was reduced by the sequential leachings and after the sixth leaching the EC50s were >0.1 g L −1 for all endpoints. Toxicity identification evaluations indicated that the toxicity was caused by zinc and organic compounds.

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.

Development of an alternative artificial soil for earthworm toxicity testing in tropical countries

The standard soil invertebrate toxicity tests developed by OECD and ISO use an artificial soil as the test substrate, which contains sphagnum peat as a component. This type of peat is not widely available. Investigation of possible alternative substrates using locally available materials therefore is vital for performing such ecotoxicity tests, particularly in the tropics. We studied the suitability of paddy husk (PH), saw-dust (SD), non-composted (NCCP) and composted coco peat (CCP) as a replacement for sphagnum peat. Artificial soil (AS) was prepared by mixing 70% sand and 20% kaolin clay with 10% PH, SD, NCCP or CCP. First, the reproduction potential of the earthworm Eisenia andrei was investigated in modified artificial soil (MAS) using the original OECD AS as the control. The number of juveniles produced in OECD AS, MAS PH and MAS CCP was not significantly different but it was significantly reduced ( p < 0.05) in MAS SD and MAS NCCP. The toxicity of chlorpyrifos, carbendazim and carbofuran for E. andrei was determined to validate the substrates. The 28-day LC 50s for the three pesticides in original AS, MAS CCP and MAS PH were not significantly different, but the EC 50 for effects on reproduction in the MAS PH was significantly lower ( p < 0.05) compared to OECD AS and MAS CCP. We conclude that composted coco peat might be a suitable replacement for sphagnum peat in AS for soil ecotoxicity studies.

Influence of pH, Hardness, Dissolved Organic Carbon Concentration, and Dissolved Organic Matter Source on the Acute Toxicity of Copper to Daphnia Magna in Soft Waters: Implications for the Biotic Ligand Model

The influence of pH, dissolved organic carbon (DOC) concentration, water hardness, and dissolved organic matter (DOM) source on the acute toxicity of copper were investigated with standardized 48-h Daphnia magna toxicity tests. Toxicity tests were conducted according to a four-factor complete factorial design. Nominal factor levels were as follows: pH 6 and 8; DOC, 2.5 and 10 mg/L; hardness, 10, 20, and 40 mg/L as CaCO3; and two DOM sources (collected from the Black River and Edisto River, SC, USA). The experimental design resulted in 24 different factor level combinations. Results indicated that all factors had significant effects on copper toxicity. Furthermore, a strong interactive effect of DOC concentration and pH was detected. Because the biotic ligand model (BLM) has become a widely used tool for predicting toxicity and interpreting toxicity test results, its performance with these data was evaluated. Seventy percent of BLM predictions were within twofold of the observed median lethal concentrations. However, BLM parameters could be adjusted to improve model performance with this data set. This analysis suggested that in soft waters, the CuOH+ complex binds more strongly with the biotic ligand and that the competitive effect of hardness cations should be increased. The results of the present study may have implications for application of the BLM to some types of surface waters. Furthermore, a comprehensive analysis of BLM performance with all available data should be performed, and necessary updates to model parameters should be made to produce the most robust and widely applicable model.

Temperature and salinity effects on the toxicity of common pesticides to the grass shrimp, Palaemonetes pugio

This study investigated the effects of increased temperature and salinity, two potential impacts of global climate change, on the toxicity of two common pesticides to the estuarine grass shrimp, Palaemonetes pugio. Larval and adult grass shrimp were exposed to the fungicide chlorothalonil and the insecticide Scourge® under standard toxicity test conditions, a 10°C increase in temperature, a 10 ppt increase in salinity, and a combined increased temperature and salinity exposure. Toxicity of the fungicide chlorothalonil increased with temperature and salinity. Toxicity of the insecticide Scourge® also increased with temperature; while increased salinity reduced Scourge® toxicity, but only in adult shrimp. These findings suggest that changes in temperature and salinity may alter the toxicity of certain pesticides, and that the nature of the effect will depend on both the organism's life stage and the chemical contaminant. Standard toxicity bioassays may not be predictive of actual pesticide toxicity under variable environmental conditions, and testing under a wider range of exposure conditions could improve the accuracy of chemical risk assessments.

Toxicity of copper in natural marine picoplankton populations

Standard microalgae toxicity tests should be able to establish responses in real ecosystems. Natural marine picoplankton samples collected during the months of March, June, August, October 2007 and January 2008, where exposed to 72 h copper toxicity tests. Results analysed by flow cytometry distinguished two groups, with different cytometric characteristics that can match with two of Synechococcus populations. EC(50) values for these two populations resulted low, ranging from 0.62 to 26.28 microg L(-1), this converts copper in a very powerful contaminant and Synechococcus in one of the most sensitive groups of phytoplankton. Differences in EC(50) values for a same population can be related to the month of collection including different initial cellular densities and oceanographic parameters that can affect the picoplankton's tolerance and distribution.

ECOLOGY AND WILDLIFE: Pharmed Fish

ECOLOGY AND WILDLIFE: Pharmed Fish

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.