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Interlaboratory comparison of a reduced volume marine sediment toxicity test method using the amphipod Ampelisca abdita.

The U.S. Environmental Protection Agency has standardized methods for performing acute marine amphipod sediment toxicity tests. A test design reducing sediment volume from 200 to 50 ml and overlying water from 600 to 150 ml was recently proposed. An interlaboratory comparison was conducted to evaluate the precision of this reduced sediment volume toxicity test method using the marine amphipod Ampelisca abdita. A negative control and three sediment samples of varying degrees of toxicity ranging from low to high were tested by six laboratories. Complete agreement was reached in rank of relative toxicity for all samples tested by five out of six laboratories. Test acceptability for control survival was achieved by all laboratories, and 69% agreement in classification of the sediments as toxic or nontoxic was documented. Coefficients of variation in all test samples were similar to those reported in other interlaboratory studies using marine amphipods. Results of this study indicate that the reduced sediment volume test using A. abdita is a reliable and precise measure of acute toxicity in marine sediment samples.

Amphipoda↗

Heavy metal toxicity testing in environmental samples.

The toxicity of heavy metals in the environment depends on a number of physicochemical and biological factors. The complexity of these relationships has encouraged the use of bioassays for direct measurement of the [table: see text] impact of toxic metals on selected test species. Fish and daphnid bioassays are well accepted by the scientific and regulatory communities, but their length (48 h or more) and the considerable time and effort needed to culture the test organisms make their application to sample screening problematical. Microbial and biochemical assays based on the inhibition of bioluminescence, enzyme activity, enzyme biosynthesis, growth, respiration, and heat production are typically faster and less expensive than the traditional and fish bioassays. Some of these tests approach or equal the sensitivity of daphnids to heavy metals. Since the soil acts as a sink for airborne and waste-applied metals, the uptake of metals by plants and the associated toxic impacts are important. Growth inhibition, enzyme induction, and production of stress proteins have been considered as toxicity end points. Enzymatic tests have been developed that are specific for heavy metal toxicity. Such tests can facilitate toxicity reduction evaluations. Detection of individual metals in the environment may eventually be possible using biosensors consisting of genetically engineered microorganisms. Direct solid-phase tests for soil, sediment, or sludge toxicity, using bacterial bioluminescence or enzyme activity as end points, have been developed. Such tests may complement traditional solid-phase toxicity tests using nematodes or earthworms as indicator organisms. Based on the work reviewed, we draw the following conclusions: 1. The Microtox assay is sensitive to mercury but would fail to detect the toxicity of certain metals, such as cadmium. Among all the microbial assays reviewed, the bioassay based on growth inhibition of the alga Selenastrum capricornutum appears to give the lowest EC50s, similar to those seen for daphnid bioassays. 2. Biosensors, using genetically engineered microorganisms, offer an elegant means of detecting the presence of specific heavy metals in environmental samples. However, at the present time, they are not designed for assessing heavy metal toxicity. 3. The use of bioassays specific for heavy metal toxicity can be useful for directly assessing the bioavailability of these toxicants in environmental samples, thus avoiding the need for fractionation.+4

Animals↗

Toxicity testing and chemical analyses of recycled fibre-based paper for food contact.

Food-contact materials, including paper, have to comply with a basic set of criteria concerning safety. This means that paper for food contact should not give rise to migration of components, which can endanger human health. The objectives of this pilot study were, first, to compare paper of different qualities as food-contact materials and to perform a preliminary evaluation of their suitability, from a safety point of view, and, second, to evaluate the use of different in vitro toxicity tests for screening of paper and board. Paper produced from three different categories of recycled fibres (B-D) and a raw material produced from virgin fibres (A) were obtained from industry, and extracts were examined by chemical analyses and diverse in vitro toxicity test systems. The products tested were either based on different raw materials or different treatments were applied. Paper category B was made from 40% virgin fibres, 40% unprinted cuttings from newspapers, and 20% de-inked newspapers and magazines. Paper categories C and D were based on newspapers and magazines. However, paper D was de-inked, whereas C was not. To identify constituents of the papers with a potential to migrate into foodstuff, samples of the paper products were extracted with either 99% ethanol or water. Potential migrants in the extracts were identified and semiquantified by GC-IR-MS or GC-HRMS. In parallel to the chemical analyses, a battery of four different in vitro toxicity tests with different endpoints were applied to the same extracts. (1) a cytotoxicity test using normal human skin fibroblasts. The test was based on measurements of the reduction of resazurin to resorufin by cellular redox processes and used as a screening test for acute or general toxicity; (2) a Salmonella/microsome assay (Ames test) as a screening test for mutagenic and potentially carcinogenic compounds; (3) a recombinant yeast cell bioassay as a screening test for compounds with oestrogenic activity; (4) an aryl hydrocarbon (Ah)-receptor assay (CALUX assay) as a screening test for compounds with dioxin-like activity. In addition, the papers were testedfor microbial content and, in general, the microbiological load was quite low. The following microorganisms were counted and identified on both surface and homogenized pulp samples: the total number of aerobic bacteria, the number of aerobic and anaerobic spore formers, the number of Bacillus cereus/thuringiensis, and the number of yeast and moulds. The chemical analyses showed a significantly higher amount and different composition pattern of chemicals extracted with ethanol compared with water. Analyses of the ethanol extracts showed a distinctly smaller number and lower concentrations of chemicals in extracts prepared from sample A compared with extracts of samples B-D. The compounds identified in B-D were similar, but the amounts were lower in B compared with C and D. In accordance with the chemical analyses, the water extracts were less cytotoxic than the ethanol extracts. The extract prepared from virgin fibres was less cytotoxic than the extracts prepared from paper made from recycled fibres, and extracts prepared from C was the most cytotoxic. None of the extracts showed mutagenic activity. No conclusion about the oestrogenic activity could be made, because all extracts were cytotoxic to the test organism (yeast cells). Ethanol extracts of A and B showed a negligible positive response in the Ah-receptor assay at the highest nontoxic concentration, whereas C and D showed a more pronounced effect with C being the most potent. A comparable weak effect of water extracts of samples B-D was observed, too. However, the active compound(s) was not identified by chemical analyses.

Biological Assay↗

A comparison of acute and chronic toxicity tests used to examine the temporal stability of a gradient in copper tolerance of Hediste diversicolor from the Fal estuary, Cornwall, UK.

The aim of this study was to use two different toxicity tests to verify the existence of a gradient in tolerance along Rostronguet Creek. Hediste diversicolor was collected from five populations in the Fal estuary previously shown to vary in copper tolerance. Exposure to 4 mgL(-1) of copper in an acute assay demonstrated that Mylor Creek worms were sensitive (LT(50) 86 h) and the tolerance of Rostronguet Creek worms increased moving upstream from the mouth of the creek (LT(50)s 100-258 h). There was no significant difference in tolerance between Mylor worms and worms from the mouth of Rostronguet Creek. This is in agreement with a previous study [Grant, A., Hateley, J.G., Jones, N.V., 1989. Mapping the ecological impact of heavy metals on the estuarine polychaete Nereis diversicolor using inherited metal tolerance. Marine Pollution Bulletin 20, 235-238] and demonstrates temporal stability of the gradient. Copper tolerance was also measured using a chronic toxicity test run for 90 d using step-wise increases in challenge concentration. A significant difference in tolerance was shown between populations from Mylor Creek and those at the mouth of Rostronguet Creek, which has not been reported previously. Experimental protocol was therefore an important factor in detecting population variation in tolerance.

Animals↗

Evaluation of a vero cell toxicity test to detect EHEC infection.

An epidemiologic investigation was carried out in Modena (Italy) to evaluate the prevalence of faecal VEROtoxin (FVT) in diarrhoeal stool specimens. One thousand and sixty-six stool specimens, submitted to the Clinical Microbiology Laboratory of the University Hospital of Modena, were collected and faecal filtrates tested for neutralizable cytotoxin by a toxicity test on VERO cells. Cytopathic effect on VERO cells was produced by 301 stool specimens (28%); neutralizable VT was detected in 40 (13%) out of 301 positive samples (3.7% of 1066 specimens). The prevalent FVT type was VT2 (50%), followed by VT1 (32.5%) and VT1+2 (17.5%). We evaluated an assay that detects both VTs directly from stool specimens to demonstrate that enterohemorrhagic strains (EHEC) should be considerated a causative agent of sporadic non-bloody diarrhoea. Our results suggest that toxin neutralization assay is a sensitive and specific technique and may be used as an alternative method to diagnose diarrhoeal infections caused by EHEC.

Adolescent↗

Significance of the dog as 'second animal species' in toxicity testing for establishing the lowest 'no-toxic-effect level'.

The toxicity data of 66 compounds adequately tested in the rat (as 'prime animal species') and in the dog (as 'second animal species') were studied to find out for how many of these compounds the study in dogs led to a lower 'no-toxic-effect level' (NTEL) than that found in the rat. From a quantitative point of view such information would contribute to a better insight into the desirability of toxicity studies in dogs, because acceptable daily intakes and permissible exposure levels in man are generally based on the lowest NTEL. For 44% of the compounds the NTEL in the dog was lower than that in the rat. When rat NTEL was divided by the arbitrary factor of 10, the adjusted NTEL was lower than the dog NTEL for 86% of the compounds tested in sub-chronic studies, and for 80% of the compounds tested in chronic studies. Whether it is justifiable to substitute such an adjusted NTEL for a study in dogs should be judged from compound to compound. It mainly depends on the size of the margin between the NTEL in the rat (as 'prime animal species') and the (expected) actual exposure level in humans, and also on the acceptability of loosing additional qualitative toxicological information. When this margin is large enough (e.g. 1000 or more), a study in dogs might be superfluous, despite the loss of qualitative data. Such an approach could lead to a reduction of the number of animals in toxicity testing and also to a reduction of the costs.

Animals↗

Using transgenic Caenorhabditis elegans in soil toxicity testing.

Soil bioassays are important tools for evaluating toxicological effects within the terrestrial environment. The American Society for Testing and Materials E2172-01 Standard Guide outlines a method for conducting laboratory soil toxicity tests using the nematode Caenorhabditis elegans. This method is an efficient tool for extracting C. elegans from soil samples and can be carried out after a 24-h exposure period using relatively small amounts of soil. Drawbacks of this method include problems with (1) recovery of nematodes from soils containing a high percentage of organic matter, and (2) distinguishing indigenous nematode species from nematodes added for the laboratory test. Due in part to these issues, C. elegans has not been extensively accepted for use in soil testing. To address these concerns and improve upon the American Society for Testing and Materials method, this project focused on using transgenic strains of C. elegans carrying a GFP-expressing element. Lethality and behavior tests revealed that the transgenic nematodes respond similarly to the wild-type N2 strain, indicating that they can be used in the same manner in soil testing. The GFP marker is easily identifiable not only within soils containing a large amount of organic matter, but also in field-collected soils containing indigenous nematodes. These results support the use of transgenic GFP C. elegans in soil bioassays as a tool to further the reliability of laboratory toxicity tests.

Aluminum Silicates↗

Sediment toxicity testing using large water-sediment ratios: an alternative to water renewal.

Deterioration of overlying water quality during toxicity tests with benthic invertebrates is a serious problem with some sediments. One solution is periodic renewal of overlying water. However, this is either labour intensive or requires construction and maintenance of special equipment. Furthermore, water renewal has the potential for flushing toxic chemicals out of the test chamber and establishes nonequilibrium conditions between the water and sediment. An alternative is testing under static conditions using atypical test vessels (e.g. Imhoff settling cones) with a large water volume (1 l) overlaying a much smaller sediment volume (e.g. 15 ml). This results in dramatic improvement of overlying water quality compared to standard static toxicity tests. Compared to water renewal, the test method is much simpler, all toxic substances leached from the sediment are retained in the test vessel, and contaminant concentrations in water and sediment have more time to equilibrate. Chronic sediment toxicity tests (10-28 days) have been conducted successfully under these conditions with Chironomus riparius, Hexagenia sp., Hyalella azteca and Tubifex tubifex.

Journal Article↗

An evaluation of a short-term chronic effluent toxicity test using sheepshead minnow (Cyprinodon variegatus) larvae.

In response to recent changes in National Pollutant Discharge Elimination System (NPDES) permit regulations, rapid (7-day) static renewal toxicity tests have been developed to detect chronic (sublethal and lethal) effect concentrations of municipal and industrial effluents on freshwater and marine/estuarine organisms. This paper evaluates results from short-term (7-day) chronic effluent tests measuring effects on the growth and survival of the larvae of estuarine sheepshead minnow (Cyprinodon variegatus). Growth was the more sensitive endpoint in 52% of the effluent tests; survival was most sensitive in 7% of the tests. Growth and survival were equally sensitive endpoints in 26% of the tests, while there was no effect on either endpoint at the highest test concentration in 15% of the tests. Four short-term chronic tests were conducted concurrently with early life stage (ELS) tests using a common effluent. Effect concentrations were generally (two of three) within one dilution (a factor of three). Comparison of short-term chronic and ELS tests were also conducted with reference single chemicals and the results were slightly better than the effluent comparisons. A series of ten intralaboratory tests and seven interlaboratory tests were conducted to assess the variability among tests results. Toxicity in these tests varied by no more than one test concentration interval. We concluded that this sheepshead minnow test is suitable to estimate chronic values for complex effluents discharged to marine/estuarine environments within the NPDES permit regulations.

Journal Article↗

Embryo-larval toxicity tests with the African catfish (Clarias gariepinus): comparative sensitivity of endpoints.

Embryo-larval toxicity tests with the African catfish (Clarias gariepinus) were performed to assess the comparative sensitivity of different endpoints. Measured test responses included embryo and larval survival, hatching, morphological development, and larval growth. Chromium, cadmium, copper, sodium pentachlorphenol (NaPCP), and malathion were used as model toxicants. Hatching was not affected by any of the chemicals tested, and embryo survival was only affected by chromium at > or = 36 mg/L. The growth of larvae was significantly reduced at > or = 11 mg/L Cr, > or = 0.63 mg/L Cu, > or = 0.03 mg/L NaPCP, and > or = 1.25 mg/L malathion. Morphological development of C. gariepinus was affected by all of the toxicants tested. Different types of morphological aberrations were observed, i.e., reduction of pigmentation in fish exposed to cadmium and copper, yolk sac edema in fish exposed to NaPCP and malathion, and deformation of the notochord in fish exposed to chromium and malathion. The sensitivity of the endpoints measured can be summarized as follows: growth > abnormality > larval survival > embryo survival > hatching.

Animals↗

Development and application of a sediment toxicity test using the benthic cladoceran Chydorus sphaericus.

This study reports on the development and application of a whole sediment toxicity test using a benthic cladoceran Chydorus sphaericus, as an alternative for the use of pelagic daphnids. A C. sphaericus laboratory culture was started and its performance under control conditions was optimised. The test was firstly validated by determining dose-response relationships for aqueous cadmium and copper and ammonia, showing a sensitivity of C. sphaericus (96 h LC(50) values of 594 microg Cd/L, 191 microg Cu/L and 46 mg ammonia/L at pH 8) similar to that of daphnids. Next, sediment was introduced into the test system and a series of contaminated sediments from polluted locations were tested. A significant negative correlation between survival and toxicant concentrations was observed. It is concluded that the test developed in the present study using the benthic cladoceran C. sphaericus is suitable for routine laboratory sediment toxicity testing.

Ammonia↗

A field assessment of long-term laboratory sediment toxicity tests with the amphipod Hyalella azteca.

Response of the amphipod Hyalella azteca exposed to contaminated sediments for 10 to 42 d in laboratory toxicity tests was compared to responses observed in controlled three-month invertebrate colonization exposures conducted in a pond. Sediments evaluated included a sediment spiked with dichlorodiphenyldichloroethane (DDD) or dilutions of a field sediment collected from the Grand Calumet River (GCR) in Indiana (USA) (contaminated with organic compounds and metals). Consistent effects were observed at the highest exposure concentrations (400 microg DDD/goc [DDD concentrations normalized to grams of organic carbon (goc) in sediment] or 4% GCR sediment) on survival, length, and reproduction of amphipods in the laboratory and on abundance of invertebrates colonizing sediments in the field. Effect concentrations for DDD observed for 10-d length and 42-d reproduction of amphipods (e.g., chronic value [ChV] of 66 microg DDD/goc and 25% inhibition concentration [IC25] of 68 microg DDD/ goc for reproduction) were similar to the lowest effect concentrations for DDD measured on invertebrates colonizing sediment the field. Effect concentrations for GCR sediment on 28-d survival and length and 42-d reproduction and length of amphipods (i.e., ChVs of 0.20-0.66% GCR sediment) provided more conservative effect concentrations compared to 10-d survival or length of amphipods in the laboratory or the response of invertebrates colonizing sediment in the field (e.g., ChVs of 2.2% GCR sediment). Results of this study indicate that use of chronic laboratory toxicity tests with H. azteca and benthic colonization studies should be used to provide conservative estimates of impacts on benthic communities exposed to contaminated sediments. Bioaccumulation of DDD by oligochaetes colonizing the DDD-spiked sediment was similar to results of laboratory sediment tests previously conducted with the oligochaete Lumbriculus variegates, confirming that laboratory exposures can be used to estimate bioaccumulation by oligochaetes exposed in the field.

Amphipoda↗

The timing of preclinical toxicological studies: pharmaceutical company approaches to toxicity testing in support of initial clinical investigations.

The completion of preclinical toxicity studies to support the first administration to humans is a time-critical step in the clinical development of medicines, and is complicated by differences in national regulatory requirements. A questionnaire-based study was carried out in 1994 to ascertain pharmaceutical companies' actual practices and views on an ideal approach to the timing of different types of nonclinical safety studies in relation to clinical investigation. Forty-one companies or their subsidiaries from Europe, Japan, and the United States responded by providing data. A range of preclinical packages were indicated as being used by companies prior to initiating Phase I clinical trials. The selection of studies tended to be based on the recommendations of the regulatory authority of the region in which the respondents were located. Differences were evident regarding the extent of genetic toxicity testing, the duration of repeat-dose toxicity studies, and the need for male fertility testing to support the first single administration of a compound to humans. In an ideal situation, the respondents would have preferred to conduct shorter duration repeat-dose toxicity studies prior to the first single administration to humans than was their actual practice in 1994. A harmonized guideline on the timing of toxicity studies in relation to clinical trials will allow better integration between clinical and nonclinical studies in an international development program. However, the diversity in the responses has demonstrated the need for flexibility in any future guideline.

Animals↗

A comparison of in situ and laboratory toxicity tests with the estuarine amphipod Eohaustorius estuarius.

Amphipod survival in laboratory and in situ exposures was assessed using the eastern Pacific Haustoriid species Eohaustorius estuarius. Toxicity test results were compared using intact (unhomogenized) and homogenized sediment samples in both field and laboratory exposures. Experiments were conducted in Moss Landing Harbor, California, an impaired waterbody under 303 (d) of the US Clean Water Act. Synoptic laboratory and in situ sediment toxicity tests were conducted at two stations: Sandholdt Bridge (SB), the most contaminated station in the harbor, and at the South Jetty (SJ), a more marine station near the mouth of the harbor. We found that Eohaustorius is amenable to in situ testing. Despite highly variable field salinity regimes (6-32 PSU at SB and 21-34 PSU at SJ), in situ control survival was 87% and 84% at SB and SJ, respectively. Amphipod survival was lower in the in situ exposures relative to the laboratory exposures at both sites. Survival at SB was 30% and 76% in the homogenized in situ and laboratory samples, respectively, and 40% and 64% in the intact (unhomogenized) in situ and laboratory samples, respectively. Neither the homogenized or intact samples from the SJ station were toxic in laboratory experiments, but amphipod survival was only 40% in the intact in situ exposure at this station, possibly due to predation. These experiments suggest that the interaction of contaminants and variable physical parameters such as salinity and temperature may have resulted in lower survival in the in situ exposures. Sediment homogenization prior to in situ deployment may have reduced effects of predators in some samples.

Amphipoda↗

A new interpretation of avian and Mammalian reproduction toxicity test data in ecological risk assessment.

The long-term risks of pesticides to wildlife in the EU currently are assessed by comparing the lowest no-observed-effect concentration (NOEC) determined from the suite of endpoints measured in existing avian and mammalian laboratory reproduction tests with estimated exposure concentrations by calculating Toxicity to Exposure Ratios (TERs). Regulatory authorities experience difficulties when assessing long-term risks because of the lack of accepted methods to improve the ecological realism of exposure and toxicity estimates and understand risks at a population level. This paper describes an approach for interpreting existing avian and mammalian toxicity test data that divides breeding cycles into several discrete phases and identifies specific test endpoints as indicators of direct pesticide effects possible at each phase. Based on the distribution of breeding initiation dates for a species of concern and the dates of pesticide applications, this approach compares the phase-specific toxicity endpoint with the expected pesticide exposure levels during each of the breeding phases. The fate of each breeding attempt is determined through a series of decision points. The cumulative reproductive response of individuals in a breeding population based on this decision framework provides a means of examining the estimated risks over the course of the breeding season and deriving an overall metric of the impact of the pesticide on reproduction. Research needed to further improve the approach is discussed.

Animals↗

Xenobiotic organic compounds in leachates from ten Danish MSW landfills--chemical analysis and toxicity tests.

A monitoring program comprising chemical analysis and biological toxicity testing of leachate samples from 10 Danish landfills (six engineered and four uncontrolled) revealed the presence of 55 different xenobiotic organic compounds (XOCs) and 10 degradation products of XOCs. The compounds belong to the following groups: BTEX, C3-benzenes, bicyclo compounds, napthalenes, chlorinated aliphatics, phenols (chloro-, methyl-, dimethyl, nonyl-), pesticides, and phthalates. Concentrations of single XOCs ranged from <0.1 to 2220 microg/L. A pesticide screening including 101 different compounds resulted in detection of 18 pesticides and three degradation products. The findings of degradation products of toluene, phenols, phthalates, pesticides, and nonylphenol ethoxylates show that degradation occurred inside the landfills. In biotests with bacteria and algae it was found that the non-volatile organic compounds were toxic as the samples only needed to be pre-concentrated from 1.3 to 9.4 times to give 50% inhibition of the test organisms. One of the ten samples proved to be genotoxic in the umuC test after 141 times pre-concentration. A major part of the organic chemicals causing toxicity remains unknown and it is recommended to combine chemical analyses and biotests in future monitoring programs.

Bacteria↗

Food and Drug Administration guidelines for reproductive toxicity testing.

The Food and Drug Administration generally requires reproductive toxicity testing of all new drugs to be used by pregnant women or women or men of reproductive potential. These requirements may vary among the centers within the FDA. Reproductive and developmental toxicity is usually tested in one or two animal species and is divided into three segments to represent treatment throughout the reproductive process. The FDA monitors adverse drug effects on human reproduction through postmarketing surveillance.

Clinical Trials as Topic↗