Short-term Toxicity Tests Research Articles

Synergistic Effects of Copper and Butylic Ester of 2,4-Dichlorophenoxyacetic Acid (Esternon Ultra) on Amphibian Embryos

Cu2+ and butylic ester of 2,4-Dichlorophenoxyacetic acid as Esternon Ultra (2,4-D) toxicity on Bufo arenarum embryos were evaluated by means of a short-term chronic toxicity test (AMPHITOX). The NOEC values for Cu and 2,4-D were 0.02 mg/L and 2 mg/L respectively. The toxicity profile curves for Cu and 2,4-D were reported. The interactions of the metal and the herbicide were evaluated by combined treatments with different concentrations of Cu and 2,4-D. Although in all cases, a synergistic effect between these chemicals was observed, the combination of concentrations exerting low level effects in isolated treatments resulted in more adverse embryonic survival. Considering that both products are extensively used in agroecosystems, this fact could be of concern for non target species like amphibians.

Importance of exposure route for behavioural responses in Lumbriculus variegatus Müller (Oligochaeta: Lumbriculida) in short-term exposures to Pb

Lumbriculus variegatus Miller (Oligochaeta), a common freshwater sediment-dweller, has frequently been used in toxicokinetic studies, although has been less used in ecotoxicity tests. For the first time the Multispecies Freshwater Biomonitor (MFB) was applied in a short-term whole-sediment toxicity test. The MFB automatically and quantitatively recorded the spontaneous locomotory behaviour of Lumbriculus variegatus in exposures with two compartments, water and sediment. The study questioned, whether the animals altered their locomotion depending on the compartment which was spiked with lead (Pb). As in the exposures to Pb-contaminated water/clean sediment, the animals exposed to Pb-contaminated sediment/clean water showed higher activities in intermediate Pb-concentrations. This indicates, that spontaneous locomotory activity is affected by Pb-concentrations at sublethal levels regardless of whether the Pb-concentration is found in the water or in the sediment, because these animals use both environmental compartments simultaneously. However, within the same Pb-levels, the animals showed higher locomotory activity in contaminated water compared with contaminated sediment. This indicates a possible tendency to withdraw from ('avoidance') contaminated water into the clean sediment compartment, whereas there was no withdrawal from contaminated sediment into clean water. The latter might be explained by the fact that withdrawal from sediment to water might increase the risk of predation and drift in nature, whereas retracting to sediment might provide shelter, The study showed that spontaneous locomotory responses of L. variegatus to Pb depend on whether the water or sediment is contaminated. The study also concluded that the Multispecies Freshwater Biomonitor can be applied effectively in sediment toxicity testing. More emphasis should be given to the interactions of water/sediment in sediment ecotoxicity tests to better simulate field conditions and increase ecological realism in risk assessment, especially as quantitative recording methods exisit.

Water quality and macroinvertebrate community response following pesticide applications in a banana plantation, Limon, Costa Rica

Pesticides used in banana production may enter watercourses and pose ecological risks for aquatic ecosystems. The occurrence and effects of pesticides in a stream draining a banana plantation was evaluated using chemical characterization, toxicity testing and macrobenthic community composition. All nematicides studied were detected in the surface waters of the banana plantation during application periods, with peak concentrations following applications. Toxicity tests were limited to the carbofuran application and no toxicity was observed with the acute tests used. However, since pesticide concentrations were generally below the lowest LC50 value for crustaceans but above calculated aquatic quality criteria, there remains a risk of chronic toxicity. Accurate ecological assessments of pesticide use in banana plantations are currently limited by the lack of local short-term chronic toxicity tests and tests using sensitive native species. Relatively constant levels of four pesticides (imazalil, thiabendazole, chlorpyrifos and propiconazole), which had toxic effects according to the 96h hydra and 21d daphnia chronic test, were recorded in the effluent of the packing plant throughout the study, indicating that the solid waste trap used in this facility was not effective in eliminating toxic chemicals. Certain taxa, such as Heterelmis sp. (Elmidae), Heteragrion sp. (Megapodagrionidae, Odonata), Caenis sp. (Caenidae, Ephemerotera), and Smicridea sp. (Hidropsychidae, Trichoptera), were more abundant at reference sites than in the banana farm waters, and may be good candidates for toxicity testing. Multivariate analyses of the macroinvertebrate communities clearly showed that the banana plantation sites were significantly different from the reference sites. Moreover, following the pesticide applications, all the banana plantation sites showed significant changes in community composition, with the same genera being affected at all sites and for all pesticides (terbufos, cadusafos and carbofuran). Consequently, the results presented here show that multivariate analysis of community composition was more sensitive in distinguishing pesticide effects than the toxicity tests and richness and composition measures used. We conclude that monitoring macroinvertebrate communities can be a powerful tool in the assessment of ecological effects of banana production.

Final Report on the Safety Assessment of Sodium p -Chloro- m -Cresol, p -Chloro- m -Cresol, Chlorothymol, Mixed Cresols, m -Cresol, o -Cresol, p -Cresol, Isopropyl Cresols, Thymol, o -Cymen-5-ol, and Carvacrol1

Sodium p-Chloro-m-Cresol, p-Chloro-m-Cresol (PCMC), Mixed Cresols, m-Cresol, o-Cresol, p-Cresol, Isopropyl Cresols, Thymol, Chlorothymol, o-Cymen-5-ol, and Carvacrol are substituted phenols used as cosmetic biocides/preservatives and/or fragrance ingredients. Only PCMC, Thymol, and o-Cymen-5-ol are reported to be in current use, with the highest concentration of use at 0.5% for o-Cymen-5-ol in perfumes. The use of PCMC in cosmetics is restricted in Europe and Japan. Cresols can be absorbed through skin, the respiratory tract, and the digestive tract; metabolized by the liver; and excreted by the kidney as glucuronide and sulfate metabolites. Several of these cresols increase the dermal penetration of other agents, including azidothymidine. In acute oral toxicity studies, LD50 values were in the 200 to 5000 mg/kg day-1 range across several species. In short-term studies in rats and mice, an o-Cresol, m-Cresol, p-Cresol or m-Cresol/p-Cresol mixture at 30,000 ppm in the diet produced increases in liver and kidney weights, deficits in liver function, bone marrow hypocellularity, irritation to the gastrointestinal tract and nasal epithelia, and atrophy of female reproductive organs. The no observed effect levels (NOEL) of o-Cresol was 240 mg/kg in mink and 778 mg/kg in ferrets in short-term feeding studies, with no significant dose-related toxicity (excluding body weight parameters). In mice, 0.5% p-Cresol, but neither m-Cresol nor o-Cresol, caused loss of pigmentation. Short-term and subchronic oral toxicity tests performed with various cresols using mice, rats, hamsters, and rabbits resulted in no observed adverse effect levels (NOAELs) for mice of 625 ppm and rats of 50 mg/kg day-1, although the NOEL was 2000 ppm in a chronic study using rats. In rabbits, < or =160 mg/kg PCMC was found to produce irritation and erythema, but no systemic effects. Hamsters dosed with 1.5% p-Cresol in diet for 20 weeks had a greater incidence of mild and moderate forestomach hyperplasia as compared to the control. Acute inhalation toxicity studies using rats yielded LC50 values ranging from >20 mg/m(3) for o-Cresol to >583 mg/m(3) for PCMC. No deaths were recorded in mice given o-Cresol at 50 mg/m(3). Cats exposed (short-term) to 9 to 50 mg/m(3) of o-Cresol developed inflammation and irritation of the upper respiratory tract, pulmonary edema, and hemorrhage and perivascular sclerosis in the lungs. Rats exposed (subchronic) to o-Cresol at 9 mg/m(3) had changes in leukocytes, spinal cord smears, nervous activity, liver function, blood effects, clinical signs, and neurological effects. In guinea pigs, exposure to 9 mg/m(3) produced changes in hemoglobin concentrations and electrocardiograms (EKGs). Rats exposed (subchronic) to 0.05 mg/m(3) Mixed Cresols by inhalation exhibited central nervous system (CNS) excitation, denaturation of lung protein, and decreased weight gain. All cresols appear to be ocular irritants. Numerous sensitization studies have been reported and most positive reactions were seen with higher concentrations of Cresol ingredients. Developmental toxicity is seen in studies of m-Cresol, o-Cresol, and p-Cresol, but only at maternally toxic levels. In a reproductive toxicity study of a mixture of m-Cresol and p-Cresol using mice under a continuous breeding protocol, 1.0% caused minimal adult reproductive and significant postnatal toxicity in the presence of systemic maternal toxicity. The o-Cresol NOAEL was 0.2% for both reproductive and general toxicity in both generations. Cresol ingredients were generally nongenotoxic in bacterial, fruit fly, and mammalian cell assays. Thymol did not induce primary lung tumors in mice. No skin tumors were found in mice exposed dermally to m-Cresol, o-Cresol, or p-Cresol for 12 weeks. In the trypthan blue exclusion assay, antitumor effects were observed for Thymol and Carvacrol. Clinical patch testing with 2% PCMC may produce irritant reactions, particularly in people with multiple patch test reactions, that are misinterpreted as allergic responses. o-Cresol, p-Cresol, Thymol, Carvacrol, and o-Cymen-5-ol caused no dermal irritation at or above use concentrations. In two predictive patch tests, PCMC did not produce a sensitization reaction. Overall, these ingredients are not significant sensitizing or photosensitizing agents. The Cosmetic Ingredient Review (CIR) Expert Panel noted some of these ingredients may increase the penetration of other cosmetic ingredients and advised cosmetic formulators to take this into consideration. The CIR Expert Panel concluded that the toxic effects of these ingredients are observed at doses higher than would be available from cosmetics. A concentration limitation of 0.5% was chosen to ensure the absence of a chemical leukoderma effect. For p-Cresol and Mixed Cresols (which contain p-Cresol), the Panel considered that the available data are insufficient to support the safety of these two ingredients in cosmetics. Studies that would demonstrate no chemical leukoderma at concentrations of use of p-Cresol and Mixed Cresols, or would demonstrate a dose response from which a safe concentration could be derived, are needed.

The Model of D-Galactosamine-Induced Injury of Rat Hepatocytes in Primary Culture

D-galactosamine (GalN) is a highly selective hepatotoxin that causes liver damage similar to human viral hepatitis via depletion of uridine nucleotides, which subsequently diminishes synthesis of RNA and proteins. Model of galactosamine hepatotoxicity is frequently used in animal experiments in vitro. The purpose of our study was to establish the model of GalN-induced hepatocyte injury in in vitro conditions using primocultures of rat hepatocytes as an important pre-requisite for further experiments in which we would like to study potential hepatoprotective effect of various substances. Rate of hepatocyte injury was evaluated by morphological changes, changes in cell viability, albumin production, mitochondrial membrane potential, activity of mitochondrial dehydrogenases and glutathione content. Marked dose dependent hepatocyte injury was found after 24-hour incubation with GalN. Based on the results we suggest as an optimal model for short-term toxicity test exposure to GalN for 24 hours in dose of 40 mM.

Acute Effects of Hexavalent Chromium on Survival, Oxygen Consumption, Hematological Parameters and Some Biochemical Profiles of the Indian Major Carp, Labeo rohita

An attempt has been made in the present investigation to determine the acute toxicity of hexavalent chromium and its toxicological effects on survival, physiological, hematological and biochemical parameters of the widely consumed Indian major carp, Labeo rohita. Short-term acute toxicity tests were performed adopting renewal bioassay technique (USEPA, 1975) over a period of 96 h, using different concentrations of potassium dichromate to the fish and the 96 h LC[50] value was found to be 111.45 mg/l (Cr(+6) as 39.40 mg/l). ANOVA results showed that the normal respiratory activity of the fish was significantly affected and there is a depression in the metabolic rate at the end of 24, 48, 72 and 96 h exposure. The metal also induced significant decrease (p<0.001) in the hematological parameters of the fish like total erythrocyte count, hemoglobin percent and absolute value Mean cell hemoglobin (MCH) both at the end of 24h and 96 h exposure indicating anemia. Appreciable decline in the biochemical profiles such as total glycogen, total lipids and total protein contents of the fish was also observed. However, the decrease in protein content was significant only at the end of 96 h. This study reflects the extent of the toxic effects of hexavalent chromium and the metal induced cumulative deleterious effects at various functional levels in the widely consumed freshwater fish, Labeo rohita.

Influence of dissolved organic matter on nickel bioavailability and toxicity to Hyalella azteca in water-only exposures

Dissolved organic matter (DOM) is known to reduce the bioavailability of metals in aquatic systems. This study evaluated the effects of DOM from various sources (e.g., Little Bear Lake sediment, Suwannee River, peat moss) and various DOM fractions (humic acids, HA; fulvic acids, FA) on the bioavailability of nickel (Ni) to Hyalella azteca, a common freshwater benthic invertebrate. In particular, this study was conducted to evaluate the effect of surficial sediment DOM on Ni bioavailability. Short-term (48 h) acute toxicity tests with H. azteca conducted in synthetic water demonstrated that the aqueous Ni concentrations required for lethality were greater than what could be significantly complexed by environmentally relevant concentrations of dissolved organic carbon (DOC: 0.6–30.4 mg/L). At Ni concentrations sublethal to H. azteca (500 μg/L), the bioavailability of Ni was significantly reduced in the presence of representative surface water DOC concentrations regardless of DOC source or fraction. DOC fraction (i.e., FA and HA) differentially affected Ni speciation, but had little or no effect on Ni accumulation by H. azteca. Tissue Ni was found to be strongly dependent upon the Ni 2+ concentration in the exposure solutions and the Ni:DOC ratio. Overall, the concentration of DOC played a greater role than either DOC source or fraction in determining Ni speciation and hence bioavailability and toxicity to H. azteca.

Gammarus aequicauda(Crustacea: Amphipoda): A potential test species in marine sediment toxicity assessment

In this study, the amphipod Gammarus aequicauda was evaluated as a test organism for use in sediment toxicity bioassays. Sensitivity to noncontaminant variables, to the reference toxicants and to some contaminated field sediments was analysed. Amphipods were tolerant to various salinity and temperature combinations during a ten-day assay. The organisms tested with different type of diet showed highest survival on the natural diet.The organism density effect on survival and growth of Gammarus aequicauda during a 28-day assay was examined. The results indicated that the density did not affect survival and production, but the effect of density was significant on the average weight.No effect on survival was observed on three sediment types during the 10-day exposure. Sensitivity to contaminants was assessed using cadmium chloride and copper chloride as reference toxicants in a 96 h water-only test. Methods were developed for conducting a short-term toxicity test with cadmium chloride-spiked sediment using this species. Water-only testing revealed high sensitivity of amphipods to reference toxicants. Experiments conducted with organisms of four different size classes demonstrated no significant differences in sensitivity.

Ecological risk assessment on a cadmium contaminated soil landfill—a preliminary evaluation based on toxicity tests on local species and site-specific information

In recent years, methodology of ecological risk assessment has been developed and applied frequently for addressing various circumstances where ecological impacts are suspected or have occurred due to environmental contamination; however, its practice is very limited in Taiwan. In 1982, brown rice from rice paddy fields in Da-Tan, Tau-Yuan, was found to be contaminated with Cd and Pb due to illegal discharges of wastewater, known as the “Cd rice” incidence. Cadmium laden soil was transferred to a constructed landfill in an industrial park 15 years after the incident. Possible leakage of the landfill was suspected by committee members of a supervising board for the remediation process, and a preliminary ecological risk evaluation was requested. A possible risk scenario was that groundwater contamination due to the leachate containing Cd and Pb from the landfill could result in pollution of coastal water, and subsequently produce toxic effects to aquatic organisms. Chemical dissipation in groundwater systems was simulated and short-term chronic toxicity tests on larvae of three local aquatic species were also performed to determine the no-observed adverse-effect concentrations (NOAECs), as well as the predicted no effect concentrations (PNECs), of the two metals in the organisms tested. The hazard quotient (HQ), the ratio of predicted environmental concentrations (PECs) to PNECs, was used for risk characterization. A worst-case-scenario calculation showed that the maximum Cd concentration at 60 m and farther downstream from the site in the groundwater system would be 0.0028 mg l − 1 with a maximum initial concentration of 0.65 mg l − 1 in the leachate, while for Pb, the highest concentration of 0.044 mg l − 1 would be reached at a distance of 40 m and farther, which was based on an initial concentration of 4.4 mg l − 1 in the leachate; however, both cases would only occur 80 years after the initiation of leakage. A presumed dilution factor of 100 was used to calculate the PECs based on the maximum initial concentrations. The Cd and Pb's PNECs for three aquatic animals were 0.01∼0.1 mg l − 1 , higher than their PECs in coastal water, resulting in HQs less than 1 for the scenario described. We concluded that if a leakage does occur, the ecological impact would be minimal because a majority of the metals will be retained in the soil phase and the remaining, if released into the aquatic system, cannot reach high enough concentrations to produce toxicity to marine animals.

FUNDAMENTAL FLAWS IN EPA'S WET VALIDATION APPROACH REVEALED: WET TEST VARIABILITY IS FAR WORSE THAN CHEMICAL METHODS

The United States Environmental Protection Agency (EPA) has adopted short-term chronic whole effluent toxicity test methods (the chronic WET test) for use in monitoring compliance with effluent limitations. These methods are reported by EPA to be similar in inter-laboratory test variability to the test methods adopted for chemical parameters (see Final Report: Interlaboratory Variability Study of EPA Short-term Chronic and Acute Whole Effluent Toxicity Test Methods, Vol. 1). This paper reviews in part the technical basis upon which EPA made its assertion and demonstrates test variability far exceeds that estimated by EPA.

Acute morphological and physiological effects of lead in the neotropical fish Prochilodus lineatus

The present study investigated lead effects on gill morphology, hematocrit, blood sodium, glucose, lipids, protein, and cholesterol of Prochilodus lineatus exposed to two sublethal lead concentrations for 96 h. Preliminary series of short-term static toxicity tests were run to determine LC50 (96 h) of lead in P. lineatus, which was 95 mg Pb.L-1. Therefore, lead concentrations tested in the sublethal experiments were 24 and 71 mg Pb.L-1, which correspond to 25% and 75% of the LC50 (96 h), respectively. Gills of P. lineatus exposed to both lead concentrations during 96 h presented a higher occurrence of histopathological lesions such as epithelial lifting, hyperplasia, and lamellar aneurism. P. lineatus did not show significant alterations in hematocrit during exposure to both lead concentrations. Fish exposed to the highest lead concentration showed a significant decrease in Na+ plasma concentration after 48 h, possibly reflecting a sodium influx rate decrease. P. lineatus exposed to both lead concentrations presented a "classical general adaptation syndrome to stress", as hyperglycemia associated with lowered lipids and proteins was reported. Stress-response magnitude was dose-dependent. While the response to the lowest lead concentration might represent adaptation, the highest concentration seems to characterize exhaustion.

Toxicity of octameric elemental sulphur in aquatic sediments

The proportions of elemental sulphur species in sediment from a contaminated area and a reference sediment sample were compared with the acute toxicity measured in short-term toxicity tests. Octameric elemental sulphur was separated into three operationally defined fractions. Here we show that octameric elemental sulphur contributes to the acute toxicity measured by bioluminescence inhibition. Our findings show that only a small part of extractable elemental sulphur was effective during exposure to the test organisms. A firmly bound fraction of sulphur was found in a contaminated sediment. This information has to be taken into account while interpreting the acute sediment toxicity.

Spirotox-Spirostomum ambiguum acute toxicity test-10 years of experience.

Spirotox is a short-term acute toxicity test with a large ciliated protozoan Spirostomum ambiguum. The test was created in the Department of Environmental Health Sciences, Medical University of Warsaw, Poland, during the early 1990s. It was presented for the first time during the 6th International Symposium on Toxicity Assessment, in Berlin in 1993. S. ambiguum was very resistant to a wide range of environmental conditions, especially to pH and dissolved oxygen. Over the last 10 years the sensitivity of S. ambiguum to many classes of toxicants was evaluated. Spirotox was found to be very sensitive to heavy metals, fungicides, and pharmaceuticals used to cure diseases of the human nervous system. On the other hand, it was generally less sensitive than standard bioassays to simple organics. Spirotox was also used for analysis of cyanobacterial blooms. Though it was moderately sensitive to hepatotoxins, the test seems to be a good tool for evaluation of the entire toxicity of blooms. The last but not the least of the applications of the Spirotox test was evaluation of the toxicity of extracts from medical devices. Protozoa, which are the simplest eukaryotes, seem to be good screening bioassays for monitoring the technology of medical device production.

Phenotypic yeast growth analysis for chronic toxicity testing

In Saccharomyces cerevisiae the pH-dependent growth inhibition of the heavy metals Cu 2+, Cr6+, Zn2+, Co2+, and Cd2+ was examined in comparison to that of organic solvents and pure compounds DMSO, MNNG, 4-NQO, MTBE, ethanol, and 2-AA. The assay was based on both S. cerevisiae wild-type and genetically modified cells deleted in the transporters Pdr5, Snq2, and Yor1 that facilitate pleiotropic drug resistance to explore the potential for short-term chronic aquatic toxicity tests. The strain deleted in the proteins that mediate the efflux of structurally diverse hydrophobic compounds exhibited high sensitive growth inhibition at low (0.04mg/L 4-NQO) to moderate (5.5mg/L DMSO) organic compound exposure. At pH 6.4 the EC50's, for all tested heavy metals were significantly low, in contrast to acidic pH conditions, in which both strains were able to grow in the presence of high concentrations of the transition metals Cu2+, Zn2+, and Co2+, with the pdr5 yor1 snq2 mutant being more tolerant. Cd2+ exerted the highest toxicity, with an EC50 of 0.49mg/L. Obtained results were compared with data determined from growth-inhibition tests involving other unicellular species. The comparison provided evidence that yeast is a sensitive and practical model system for toxicological risk assessment.

Modeling impacts on populations: fathead minnow ( Pimephales promelas) exposure to the endocrine disruptor 17 β-trenbolone as a case study

Evaluation of population-level impacts is critical to credible ecological risk assessments. In this study, a predictive model was developed to translate changes in fecundity of the fathead minnow ( Pimephales promelas) in a short-term laboratory toxicity test to alterations in population growth rate. The model uniquely combines a Leslie population projection matrix and the logistic equation. Application of the model requires only a life table for the organism of interest, a measure of carrying capacity for the given population, and an estimation of the effect of a stressor on vital rates. The model was applied to investigate population dynamics for fathead minnow exposed to the androgen receptor agonist 17 β-trenbolone. Organismal-level responses for fathead minnows exposed to varying levels of 17 β-trenbolone were used to determine projected alterations in a population existing in a small body of water containing varying concentrations of the androgen. Fathead minnow populations occurring at carrying capacity and subsequently exposed to 0.027 μg/L of 17 β-trenbolone exhibited a 51% projected decrease in average population size after 2 years of exposure. Populations at carrying capacity exposed to concentrations of 17 β-trenbolone ⩾0.266 μg/L exhibited a 93% projected decrease in average population size after 2 years of exposure. Overall, fathead minnow populations exposed to continued concentrations of 17 β-trenbolone equal to or greater than 0.027 μg/L were projected to have average equilibrium population sizes that approached zero.

Bioassay selection, experimental design and quality control/assurance for use in effluent assessment and control.

In the UK Direct Toxicity Assessment Programme, carried out in 1998-2000, a series of internationally recognised short-term toxicity test methods for algae, invertebrates and fishes, and rapid methods (ECLOX and Microtox) were used extensively. Abbreviated versions of conventional tests (algal growth inhibition tests, Daphnia magna immobilisation test and the oyster embryo-larval development test) were valuable for toxicity screening of effluent discharges and the identification of causes and sources of toxicity. Rapid methods based on chemiluminescence and bioluminescence were not generally useful in this programme, but may have a role where the rapid test has been shown to be an acceptable surrogate for a standardised test method. A range of quality assurance and control measures were identified. Requirements for quality control/assurance are most stringent when deriving data for characterising the toxic hazards of effluents and monitoring compliance against a toxicity reduction target. Lower quality control/assurance requirements can be applied to discharge screening and the identification of causes and sources of toxicity.

Comparison of laboratory single species and field population-level effects of the pyrethroid insecticide lambda-cyhalothrin on freshwater invertebrates.

The toxicity of the pyrethroid insecticide lambda-cyhalothrin to freshwater invertebrates has been investigated using data from short-term laboratory toxicity tests and in situ bioassays and population-level effects in field microcosms. In laboratory tests, patterns of toxicity were consistent with previous data on pyrethroids. The midge Chaoborus obscuripes was most sensitive (48- and 96-h EC50 = 2.8 ng/L). Other insect larvae (Hemiptera, Ephemeroptera) and macrocrustacea (Amphipoda, Isopoda) were also relatively sensitive, with 48- and 96-h EC50 values between 10 and 100 ng/L. Generally, microcrustacea (Cladocera, Copepoda) and larvae of certain insect groups (Odonata and Chironomidae) were less sensitive, with 48-h EC50 values higher than 100 ng/L. Mollusca and Plathelminthes were insensitive and were unaffected at concentrations at and above the water solubility (5 microg/L). Generally, the EC50 values based on initial population responses in field enclosures were similar to values derived from laboratory tests with the same taxa. Also, the corresponding fifth and tenth percentile hazard concentrations (HC5 and HC10) were similar (laboratory HC5 = 2.7 ng/L and field HC5 = 4.1 ng/L; laboratory and field HC10 = 5.1 ng/L), at least when based on the same sensitive taxonomic groups (insects and crustaceans) and when a similar concentration range was taken into account. In the three field enclosure experiments and at a treatment level of 10 ng/L, consistent effects were observed for only one population (Chaoborus obscuripes), with recovery taking place within 3 to 6 weeks. The laboratory HC5 (2.7 ng/L) and HC10 (5.1 ng/L) based on acute EC50 values of all aquatic arthropod taxa were both lower than this 10 ng/L, a concentration that might represent the "regulatory acceptable concentration." The HC5 and HC10 values in this study in The Netherlands (based on static laboratory tests with freshwater arthropods) were very similar to those derived from a previous study in the United Kingdom (1.4 and 3.3 ng/L). This suggests that for pesticides like lambda-cyhalothrin, HC5 values based on static laboratory tests may provide a conservative estimate of the potential for community-level effects under field conditions. While these HC5 values are conservative for initial effects, they do not provide information on recovery potential, which may be important for regulatory decision-making.

Short-term algal toxicity test based on phosphate uptake

In order to develop a short-term algal toxicity test, the growth of and the phosphate uptake by the green alga Selenastrum capricornutum during batch culture were observed. In the control medium, S. capricornutum took up phosphate earlier than it grew. It was also observed that the phosphate uptake was inhibited by the presence of a toxicant. From these results, phosphate uptake was considered as one of the useful effect parameters for a short-term algal toxicity test. As the removal rate of phosphate from the medium is a function of the amount of algal cell initially inoculated, the test period is variable. The relationship between the amount of inoculation and phosphate uptake was examined and the test conditions suitable for a 3-h toxicity test were established as one example. According to this test procedure, the inhibitory effect of some toxicants on the phosphate uptake was determined. For comparison, a conventional algal assay based on algal growth was also performed. The EC50 s for both tests were close. This indicated that the algal toxicity test method proposed in this paper would be useful for the uses where rapidity is required.

Final Report of the Amended Safety Assessment of Dioscorea Villosa (Wild Yam) Root Extract1

Dioscorea Villosa (Wild Yam) Root Extract is an extract of the rhizomes of the wild yam, D. villosa. A manufacturing process was described in which cut up and ground rhizomes are combined with an eluant (e.g., oleyl alcohol), the plant material precipitated with addition of a miscible solvent, washed, and redissolved in the original eluant. The extract contains glycoside and steroidal saponins (< or =0.4%), diosgenin (< or =3.5%), alkaloids, tannins, phytosterols, and starch. Levels of heavy metals, 1,4-dioxane, chloroform, methylene chloride, trichloroethylene, and benzene are reported to be below limits of detection. Although only one use was reported to the U.S. Food and Drug Administration (in a body and hand preparation), industry reported uses in body and hand creams, lotions, powders, and sprays at a concentration of 0.00001% (equivalent to 0.000002% plant solids), and in moisturizing creams, lotions, powders, and sprays at concentrations up to 15% (equivalent to 0.5% plant solids). Preparations fromD. villosaare used in herbal medicine for treatment of a variety of ailments and by the pharmaceutical industry in the preparation of steroids. Using Dioscorea Villosa (Wild Yam) Root Extract prepared via a specified process, it is possible to produce a stable extract with a narrow range of diosgenin content. The extract produced using this methodology was tested in acute and short-term toxicity tests, dermal irritation tests, a sensitization test, an ocular irritation test, a rat uterotropic assay, and genotoxicity tests. An acute oral toxicity test produced hypoactivity, piloerection, and dyspnea and a death in 1 of 10 rats at 2 g/kg using the specified extract, but no toxicity in rats given 0.5 g/kg. A dermal toxicity test using the specified extract demonstrated no acute toxicity in rats. Both a 7-day local tolerance test and a 28-day dermal toxicity test in rats produced no significant adverse effects at the maximum tested concentration of 10%. A single application of undiluted extract to the intact and abraded skin of rabbits produced sufficient irritation for the test material to be rated"irritant,"but a 10% dilution was not irritating. Undiluted extract was only mildly irritating to the conjuctiva of the rabbit eye; irritation in the iris and cornea was mild and transient. Undiluted extract was not irritating during the induction phase of a guinea pig sensitization study, nor did challenge with a 25% dilution elicit any sensitization. The specified extract at concentrations up to 500 mg/kg/day did not have any estrogenic activity in the juvenile rat uterotrophic assay. Genotoxicity assays in bacterial and mammalian systems were negative, except that Ames test strain TA 1537 was positive at one dose level using the plate incorporation method, but not using a preincubation method. Although the concentration at which the actual plant extract is used in cosmetic products is low, one of the primary safety concerns with this plant extract is the possible metabolic/endocrine activity, e.g., estrogen-like or progesterone-like activity as a result of the presence of small amounts of plant phytosterols such as diosgenin. Extracts prepared as described in this safety assessment, with an upper limit of 3.5% diosgenin, did not have any estrogenic activity, demonstrating that it is possible to produce material that does not present this specific safety concern. Although extracts from pesticide-free plants were not considered genotoxic and it was the view of the Cosmetic Ingredient Review (CIR) Expert Panel that there do not appear to be any components that could be carcinogenic, pesticide residues could raise this issue. It was urged that manufacturers limit pesticide residues to the limit previously used for lanolin of not more than 40 ppm (with not more than 10 ppm for any one residue). Based on these data, it was concluded that Dioscorea Villosa (Wild Yam) Root Extract is safe as used in cosmetic formulations. This conclusion regarding safety, however, is valid only for extracts prepared in a manner that produces a similar chemical profile as that described in this report, particularly as regards diosgenin. Extracts not prepared in a manner that produces a similar chemical profile would be considered safe if they have a similar safety test profile.

Recommendations for the assessment of TNT toxicity in sediment.

Previous investigations of the ecotoxicity of TNT in spiked sediments noted the rapid degradation and disappearance of the toxicant, yet little is understood regarding the effects of this process on toxicity and subsequent derivation of toxicity reference values. We conducted environmental fate studies and 28-d sediment toxicity tests with benthic oligochaete worms (Tubifex tubifex) with sediments spiked at three different TNT concentrations (440, 1,409, and 4,403 nmol/g dry wt) aged for 1, 8, and 29 d. Because of rapid degradation of TNT, disappearance of degradation products, and partitioning to overlying water, only 25 to 40% of the added nitroaromatic mass balance was associated with sediment immediately after spiking. Lethal toxicity decreased with aging time and was best described by measured sediment nitroaromatic concentrations (sum of TNT and degradation products) at the beginning of exposure, with a median lethal concentration of nitroaromatic compounds of 184 nmol/g dry weight. To accurately describe the ephemeral exposure doses of TNT and its degradation products during toxicity tests with spiked sediments, we suggest that sediments should be aged at least 8 to 14 d after spiking, exposure should be based on measured sediment concentrations or chemical measures of availability, exchange of overlying water should be avoided or minimized, and short-term toxicity tests should be considered.