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PubMed2002

Cyst-based toxicity tests XII--development of a short chronic sediment toxicity test with the ostracod crustacean Heterocypris incongruens: selection of test parameters.

Experiments were carried out with neonates of the freshwater ostracod Heterocypris incongruens hatched from cysts in order to develop a new culture/maintenance-free solid-phase microbiotest for the toxicity assessment of contaminated sediments. Based on preliminary investigations, a number of test parameters were investigated for a short-chronic assay: hatching time, size of the cups of the multiwell test plates, feeding of the test organisms prior to the test, amount of supplemental algal food, volume of sediment, and duration of the test. On the basis of the findings, a test protocol was formulated for a 6-day assay in 12-cup multiwell plates with 10 organisms per cup and 3 replicates. The test organisms were collected 52 h after the start of the incubation of the cysts in standard freshwater at 25 degrees C under continuous illumination after a 4-h prefeeding with 1.3 mg/mL Spirulina. The test biota in the cups were exposed to 300 microL of test sediment in 2 mL of standard freshwater with 3 x 10(7) live algal cells (Raphidocelis subcapitata) as food supplement. Calibrated sand was used as a reference sediment. Mortality and growth of the ostracods were determined after 6 days' incubation at 25 degrees C in darkness. The selected test parameters for the new microbiotest were found adequate for toxicity determination of natural sediments compared with the 10-day contact test with the amphipod Hyalella azteca.

Animals↗
PubMed2002

Cyst-based toxicity tests XIII--development of a short chronic sediment toxicity test with the ostracod crustacean Heterocypris incongruens: methodology and precision.

Experiments were carried out with neonates of the freshwater ostracod Heterocypris incongruens to verify and complete previous choices of test parameters for a new culture/maintenance-free solid-phase microbiotest for freshwater sediments. From trials with increasing volumes of reference sediment, it was concluded that 300 microL was the most appropriate amount of substrate to be put in 12-cup multiwell plates with 2 mL of standard freshwater. Tests in 3-9 replicates eventually showed that six parallels were needed to have good assay precision (repeatability). Application of the final test protocol to oil-contaminated sediments from the St. Lawrence River in Canada revealed that the 6-day chronic ostracod microbiotest had less variation in repeated tests than did the 10-day contact assay with Hyalella azteca and hence can be considered more precise. Based on the 95% confidence intervals for mortality and growth of the ostracods in the controls (reference sediment) of the 56 tests carried out for the Canadian project, a validity threshold of 20% for mortality was eventually selected, in analogy with the acceptability limits applied in many chronic bioassays. A minimum length of 600 microm in the control sediment after 6 days' exposure was also taken as the threshold for good health of the test organisms and for reliable test conditions. The new microbiotest has been tailored in a handy and user-friendly new toxkit, the Ostracodtoxkit, which is particularly suited for cost-effective routine monitoring.

Animals↗
PubMed2005

A novel algal toxicity testing technique for assessing the toxicity of both metallic and organic toxicants.

This study presents a closed-system algal toxicity test technique that is capable of detecting the effects of both organic and metallic toxicants. Toxicity testing was conducted by transferring adequate amounts of algal suspension, dilution water (with culture growth medium), and toxicants into 300-mL BOD bottles. The BOD bottles were completely filled up with no head-space left. The initial cell density and the exposure time were 15,000 cells/mL and 48 h, respectively. The performance of the above test method was evaluated using three heavy metals and six organic toxicants based on three different test endpoints, i.e., dissolved oxygen production, algal growth rate, and cell density. The proposed test revealed excellent test sensitivity and reproducibility. Currently, none of the existing algal toxicity test protocols is adequate for assessing the toxicity of organic chemicals. The closed-system algal toxicity tests developed by previous researchers also may not be ideal because the enlarged headspace and/or enriched bicarbonate buffer may result in either underestimations of the exposure concentrations or insensitive responses to both heavy metals and organic toxicants. Compared to the aforementioned algal toxicity test methods, the proposed technique in the present study has a more general applicability under conditions such as effluent samples containing both metals and organic toxicants or samples with unknown compositions.

Benzene Derivatives↗
PubMed1987

Test guideline. Behavioral toxicity testing in animal experiments according to section 9, para. 1, No. 2 of the Chemicals Act (Chemikaliengesetz) of the Federal Republic of Germany.

The German Chemicals Act requires that chemicals are tested for behavioral toxicity at stage 2 of the testing procedure, i.e. if more than 1000 annual tons are produced. For this purpose a guideline was developed according to which data on behavioral toxicity are to be collected, which are based on cageside observations during longterm exposure. The protocol covers outer appearance, as well as motor, sensory, autonomic and central nervous system functions. Data are to be reported in tabular form and should be evaluated by taking all aspects of the toxicological profile into account.

Animals↗
PubMed1998

Validation of alternative methods for toxicity testing.

Before nonanimal toxicity tests may be officially accepted by regulatory agencies, it is generally agreed that the validity of the new methods must be demonstrated in an independent, scientifically sound validation program. Validation has been defined as the demonstration of the reliability and relevance of a test method for a particular purpose. This paper provides a brief review of the development of the theoretical aspects of the validation process and updates current thinking about objectively testing the performance of an alternative method in a validation study. Validation of alternative methods for eye irritation testing is a specific example illustrating important concepts. Although discussion focuses on the validation of alternative methods intended to replace current in vivo toxicity tests, the procedures can be used to assess the performance of alternative methods intended for other uses.

Animal Testing Alternatives↗
PubMed2002

An investigation of new toxicity test method performance in validation studies: 2. Comparison of three measures of toxicity test performance.

An area that requires further research is how best to measure test method performance in validation studies and how to set criteria that should be used to judge the adequacy of this performance. The studies reported here were designed to begin an investigation of these questions. Computer simulations were used to generate data sets similar to those that might be obtained from a large validation study. These data were then analysed using three procedures including determination of the 95% prediction interval (PI), calculation of Pearson's correlation coefficient and calculation of the contingent probability statistics (CPS), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The results of this work suggest that of the three approaches examined, quantitative measurements with calculation of the 95% PI provide the most information to allow discrimination between the performance of several different NTMs. The results also suggest that dividing data sets into positive and negative toxicity classifications followed by the calculation of CPS leads to considerable information loss. This loss of information may be so significant that it is not possible in certain circumstances to distinguish between NTMs that are adequate and those that are not.

Predictive Value of Tests↗
PubMed1996

[OECD ist accepting test guidelines for validated in vitro toxicity tests in 1996]

Since 1990 in Europe a scientific concept for the validation of in vitro toxicity tests has been developed to facilitate regulatory acceptance of the new methods at the international level. ERGATT and ECVAM have promoted the concept of validation on two workshops in 1990 and 1994. A pre-validation stage is an essential part of this validation concept to achieve a better standardisation of in vitro tests before entering formal validation. Within the NTP in 1995/96 Federal Agencies of the USA represented by the validation center ICCVAM have accepted a validation concept, which basically agrees with the essentials of ECVAM"s European validation concept. Subsequently, in January of 1996 the major industrial nations have at the OECD level agreed to the European/US validation concept. This will at the international level allow mutual acceptance of data obtained with in vitro toxicity tests rather than with animal tests. It seems very likely that in 1996 in vitro testing for skin penetration with human skin will be the first in vitro toxicity test accepted by the OECD. Several in vitro tests for local irritancy testing will follow in 1997/98, since they are currently undergoing validation according to OECD criteria.

Journal Article↗
PubMed2004

Toxicity of the 13 priority pollutant metals to Vibrio fisheri in the Microtox chronic toxicity test.

The Microtox Acute Toxicity Test has been successfully used to measure the toxicity of metals and other pollutants at high concentrations (ppm) in selected environmental samples. However, metals and other toxicants are often found in much lower concentrations (ppb) in many municipal wastewaters and receiving waters. In order to assess the toxicity of these pollutants in these samples, a more sensitive toxicity assay is needed. The Microtox chronic toxicity test has been developed to measure the sublethal effect of toxicants over multiple generations of the test species, Vibrio fisheri. In this study, the toxicity of the 13 priority pollutant metals [i.e. As, Se, Cd, Cr (III and VI), Cu, Pb, Sb, Ag, Tl, Zn, Be, Hg and Ni] to V. fisheri was evaluated using the Microtox chronic toxicity test. In this test, the inhibitory concentration (IC), lowest observable effect concentration (LOEC), and no observable effect concentration (NOEC) were obtained after 22-h of incubation at 27+/-1 degrees C, by comparing the light output of the control to that of the test sample. Among the 13 priority pollutant metals, beryllium (Be) was found to be the most toxic in the test (LOEC=0.742-1.49 microg/l) while thallium (Tl) was the least toxic (LOEC=3840-15300 microg/l). The LOECs for copper (as Cu) and lead (Pb) in reagent (ASTM Type I) water were 6.78-13.6 microg/l and 626-1251 microg/l, respectively. The toxicity of copper sulfate (as Cu) in reagent water was shown and significantly reduced with the addition of natural organic matter (fulvic acid) or EDTA to the sample. The LOEC values for the 13 priority pollutant metals in this test were comparable to or lower than those reported for commonly used aquatic toxicity tests, such as the Ceriodaphnia dubia assay.

Benzopyrans↗
PubMed2003

Assessment of the freshwater annual fish Cynopoecilus melanotaenia as a toxicity test organism using three reference substances.

This study presents a preliminary evaluation of the use of the Brazilian fish Cynopoecilus melanotaenia as a test organism in toxicity tests. The cryptobiotic stage presented by the eggs of fish C. melanotaenia can overcome the difficulty of continuously keeping cultures and recruiting healthy animals in sufficient numbers to be used in toxicity tests. In order to determine the applicability of this species as a test organism, three different reference substances were evaluated in 96-h acute toxicity tests: Copper sulfate (CuSO4 x 5H2O), sodium dodecil sulfate (C12H25NaO4S), and sodium chloride (NaCl). Sensitivity ranged as follows: copper sulfate (0.05-0.13 mg/L), sodium dodecil sulfate (10.7-19.0 mg/L), and sodium chloride (1.44-1.96 g/L). We conclude that C. melanotaenia shows potential as a test organism in toxicity tests; however, further research should be conducted with other substances and should be compared with the research on other species before we can reach more conclusive results.

Animals↗
PubMed1975

The lysosomal permeability test modified for toxicity testing with cultured heart endothelioid cells.

A modified lysosomal fragility test is described which is suitable for use with cultured cells. The permeability (fragility) of the lysosomal membranes of the cells to the substrate beta-glycerophosphate is measured by assessing the degree of particulate lysosomal straining seen after exposing the cells to the Gomori acid phosphatase staining reaction under carefully controlled conditions. Monolayer cultures of endothelioid cells from the hearts of neonatal rats have been used in all experiments. The time-course of lysosomal straining for cells exposed to various treatments (normal saline, isotonic sucrose, 0.25 m sucrose, distilled water, acetate buffer pH 5.0, cold acetone, neutral formalin, acetic-ethanol, Triton X-100, hydrocortisone, choloroquine and vitamin A) was compared with that of control cells stained under identical conditions. Statistical differences in staining between the test and control cells were determined by the Wilcoxin Signed Rank Test and also by regression analysis following a transformation designed to allow for the saturation character of the reaction. The success of the modified technique depends upon meticulous methodology. It is capable of demonstrating both lysosomal membrane labilization and stabilation, second- and third-stage lysosomal activation, and apparent lysosomal enzyme loss or destruction in situ. The technique also allows the degree of reversible or first-stage lysosomal activation to be subdivided on an almost continous basis and is suitable for investigating the effects of drugs and other agents on the integrity of the lysosome in situ.

Animals↗
PubMed1980

[Safety evaluation of DE-020 eye drops--eye irritation test and subacute toxicity test in rabbits (author's transl)].

Basic experiments on safety evaluation of 0.3%, 1% and 3% solution of KW-1062 (DE-020 eye drops) were carried out. These eye drops applied topically to rabbits eyes had no undesirable local or systemic effects as follows: 1. Repeated instillations of DE-020 eye drops (One drop every 1 hr for 4 hrs) showed no significant irritation except only a temporary and slight congestion localized at the conjunctivae. 2. Daily instillation of DE-020 eye drops (3 times daily for 28 days) showed no abnormal findings in the eye mucosa, clinical signs, body weight, hematological examination, biochemical examination, autopsy, organ weight and histopathological examination.

Aminoglycosides↗
PubMed2003

Cyst-based toxicity tests. XI. Influence of the type of food on the intrinsic growth rate of the rotifer Brachionus calyciflorus in short-chronic toxicity tests.

As important members of zooplankton communities worldwide, rotifers are used extensively in ecotoxicological research. Chronic rotifer tests are, however, dependent on live algal food which adds to the complexity, the variability and the costs of these bioassays. To bypass the former problem, experiments have been undertaken with the freshwater rotifer Brachionus calyciflorus, to determine their intrinsic growth rate (r) when fed for 48 h on a mixture of green algae (Raphidocelis subcapitata recently renamed Pseudokirchneriella subcapitata) obtained from algal beads stored for different periods of time, and other inert foods. All tests have been performed in disposable multiwells, in 1 ml cups each inoculated with 1 rotifer freshly hatched from dried cysts. The majority of the growth tests was performed in eight replicates. The investigations revealed that microalgae from algal beads stored for up to one year, in darkness, at 4 degrees C, supplemented with dried blue-green algae (Spirulina) gave satisfactory rotifer reproduction. The intrinsic growth rates of the rotifers, were, however, dependent on the storage time of the algal beads; the highests r's (0.7-0.8) were obtained with algae from beads not older than four months. Growth tests with combinations of P. subcapitata and other inert feeds revealed that the enrichment food Selco used in aquaculture, also gave the same reproductive output as the combination microalgae/Spirulina. A rotifer growth experiment with 18 replicates showed that the variation coefficient is below 20% when the tests comprise eight replicates. This study demonstrated that microalgae from beads, supplemented by other inert food, open the door for a practical and cost-effective short-chronic rotifer test, which is totally independent of the culturing of both the test species and its live food.

Animals↗
PubMed2006

Temperature-based rapid toxicity test using Ceriodaphnia dubia.

Toxicants, which may cause the inhibition to the biological process in sewage/wastewater treatment plant, need to be monitored within short time to prevent from serious damage. A new method, Temperature-based rapid toxicity test (TempTox test) using Ceriodaphnia dubia, was developed and compared with the standard 48 hr acute bioassay (Std. 48-hr test). Inorganic toxicants of cadmium, zinc, copper, cyanide, chromium (III), chromium (VI) and organic toxicants of phenol, PCP and pesticides of BPMC, Diazinon, Fenitrothion were tested for TempTox test and Std. 48-hr test. Because the TempTox test is based on just temperature control, C. dubia neonates were exposed to toxicants under high temperature (35.5 degrees C) condition without any complicated pretreatment. After given exposure time of 1, 1.25, 1.5 hours, the number of the live (no toxic effect) or the dead (toxic effect) was counted with eye without the aid of any microscope and median effective concentrations (EC50 values) were determined. From the results for all toxicants, the TempTox test was proved to be as sensitive as the Std. 48-hr test with shorter-time of just 1.25-1.5 hours. Moreover, the TempTox test was further much more sensitive than alternative bioassays such as the 1-hour l.Q. test and 30-minute Microtox. The TempTox test showed a high applicability of toxicity bioassay for real sewage/wastewater treatment plant by its easiness, rapidity and sensitivity. Finally, the prototype for short-term TempTox test was introduced.

Animals↗
PubMed2002

Incorporation of toxicity tests into the Turkish industrial discharge monitoring systems.

A toxicity evaluation is an important parameter in wastewater quality and in the monitoring of discharged effluents. Some organic and inorganic compounds at toxic levels have been detected in industrial discharges, resulting in plant upsets and discharge permit violations. In some cases, even though the effluent does not exceed the chemical-specific discharge limits, the results of toxicity tests show potential toxicity. Knowledge of the toxicity of effluents can benefit treatment plant operators in optimizing plant operation, protecting receiving water quality, and establishing sewer discharge permits. In the Turkish regulations only toxicity dilution factor with fish is part of the toxicity monitoring program of permissible wastewater discharge. This study investigated the acute toxicity of pulp-paper, leather, and petrochemical industry wastewaters by traditional and enrichment toxicity tests and emphasized the importance of toxicity tests in wastewater discharge regulations. The enrichment toxicity tests are novel applications indicating whether there is potential toxicity or stimulation conditions. Different organisms were used, including bacteria (Floc and Coliform bacteria) algae (Chlorella sp.), fish (Lepistes sp.), and protozoan species (Vorticella sp.) to represent four trophic levels. The toxicity test results were compared with chemical analyses to identify the pollutants responsible for the toxicity in the effluent wastewater samples. Toxicity of the effluents could not be explained by using physicochemical analyses in five cases for the leather and four cases for the pulp-paper and petrochemical industries. The results clearly showed that the use of bioassay tests produce additional information about the toxicity potential of industrial discharges and effluents.

Animals↗