A new simple method with high precision for determining the toxicity of antifouling paints on brine shrimp larvae (Artemia): first results.

The use of antifouling paints is the only truly effective method for the protection of underwater structures from the development of fouling organisms. In the present study, the surface to volume concept constitutes the basis for the development of a new and improved method for determining the toxicity of antifouling paints on marine organisms. Particular emphasis is placed on the attainment of a standardized uniformity of coated surfaces. Failure to control the thickness of the coat of paint in previous studies of this type, has led to inaccurate evaluation of the relative toxicity of samples. Herein, an attempt is made to solve this problem using a simple technique which gives completely uniform and smooth surfaces. The effectiveness of this technique is assessed through two series of experiments using two different types of test containers: 50 ml modified syringes and 7 ml multiwells. The results of the toxicity experiments follow a normal distribution around the average value which allows to consider these values as reliable for comparison of the level of toxic effect detected with the two types of test containers. The mean lethal concentration L(S/V)(50) in the test series conducted in the multiwells (20.38 mm(2)ml(-1)) does not differ significantly from that obtained in the test series using modified syringes (20.065 mm(2)ml(-1)). It can thus be concluded from this preliminary study that the new method and the two different ways of exposing the test organisms to the antifouling paints and their leachates gave reliable and replicable results.

The effects of a copper-based antifouling paint on mortality and enzymatic activity of a non-target marine organism.

Antifouling paints are used on a wide range of underwater structures in order to protect them from the development of fouling organisms. The leaching of the toxic substances from the matrix of the paint causes toxic effects not only to the fouling organisms but also on other "non-target" biota. The present study addresses the impact of the antifouling paint Flexgard VI-II on brine shrimp nauplii selected as convenient test organisms. The surface to volume (S/V) concept developed by Persoone and Castritsi-Catharios (1989) was used to determine S/V-LC50s for the test biota exposed to PVC test panels of 400-1000 mm2 surface coated with the antifouling paint in test vessels containing 20 ml seawater. Total ATPase and Mg2+-ATPase were also analyzed for coated surface areas inducing less than 50% mortality in the brine shrimp nauplii. The calculated S/V-LC50 (24 h) was 24.6 mm2/ml, which shows the high toxic character of the antifouling paint. Decreased enzymatic activities were noted in the brine shrimp nauplii exposed to test panels of 50 and 100 mm2 in 20 ml seawater. The present study indicates that the "surface to volume" concept is an interesting methodology that can be applied with both lethal and sublethal effect criteria for the determination of toxic stress from leaches of painted surfaces.

Development of methods for evaluating toxicity to freshwater ecosystems.

This article presents a summary of a collaborative research program involving five European research groups, that was partly funded by the European Commission under its Environmental Research Program. The objective of the program was to develop aquatic toxicity tests that could be used to obtain data for inclusion at Level 2 of the Risk Evaluation Scheme for the Notification of Substances as required by the 7th Amendment to EC Directive 79/831/EEC. Currently only a very limited number of test methods have been described that can be used for this purpose and these are based on an even smaller number of test species. Tests based upon algae (Chlamydomonas reinhardi, Scenedesmus subspicatus, and Euglena gracilis), protozoa (Tetrahymena pyriformis), rotifera (Brachionus calyciflorus), crustacea (Gammarus pulex), and diptera (Chironomus riparius) were developed. The tests encompassed a range of end points and were evaluated against four reference chemicals: lindane, 3, 4-dichloroaniline (DCA), atrazine, and copper. The capacity of the tests to identify concentrations that are chronically toxic in the field was addressed by comparing the effects threshold concentrations determined in the laboratory tests with those determined for similar and/or related species and end points in stream and pond mesocosm studies. The lowest no-observed-effect concentrations (NOEC), EC(x), or LC(x) values obtained for lindane, atrazine, and copper were comparable with the lowest values obtained in the mesocosms. The lowest chronic NOEC determined for DCA using the laboratory tests was approximately 200 times higher than the lowest NOEC in the mesocosms.

Modelling of human acute toxicity from physicochemical properties and non-vertebrate acute toxicity of the 38 organic chemicals of the MEIC priority list by PLS regression and neural network.

Linear and non-linear modelling of human acute toxicity (as human lethal concentrations; HLCs) of the 38 organic chemicals from the 50 priority compounds of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme was investigated. The models obtained were derived either from a set of 23 physicochemical properties of the compounds or from their acute toxicities to five aquatic non-vertebrates together with the physicochemical properties. For the linear type, modelling was performed using a partial least square projection to latent structures (PLS) regression method; for the non-linear models, both PLS regression and neural network were utilized. A neural network using a combination of backpropagation and cascade-correlation algorithms was applied in this study. The results generally reveal a slightly better predictive performance of the models obtained from PLS regression than those obtained from neural networks. However, the model composed of physicochemical properties (PC-model) from the trained neural network using a back propagation algorithm with pruning technique proved superior to that trained with a combination of backpropagation and cascade-correlation algorithms after leave-one-out cross-validation. The predictive power of the PC-models, whether linear or non-linear, was comparable with that of the corresponding models consisting of both structural descriptors and the ecotoxicological tests (ECOPC-models), except for the battery (ECOPC-model) from the neural network. The composition of the 'best' PLS and neural network models points to the importance of the combination of physicochemical properties reflecting lipophilicity, size, volume, intermolecular binding forces and electronic properties of the molecule. All the aquatic non-vertebrate tests are shown to be essential in explaining human acute toxicity. However, the degree of contribution differed, with the crustacean (Artemia salina) and the bacterial (Microtox) bioassays being more important to the linear and non-linear PLS models, whereas the crustacean (Artemia salina and Streptocephalus proboscideus) tests, and the rotifer (Brachionus calyciflorus) assay were important to the neural network models. The organochlorine (lindane) and bipyridinium (paraquat) pesticides were common outliers in all the models. Moreover, the latter two compounds and the organophosphate (malathion) pesticide were also common outliers in all ECOPC-models. Other types of pesticides, however, fit the models. The predicted HLCs of a number of non-pesticides, including some chlorinated compounds, also deviated from the observed HLCs by more than one order of magnitude.

Ecotoxicological studies with the freshwater rotifer Brachionus calyciflorus. IV. Rotifer behavior as a sensitive and rapid sublethal test criterion.

The swimming behavior of the freshwater rotifer Brachionus calyciflorus exposed to copper (Cu), pentachlorophenol (PCP), 3,4-dichloroaniline (DCA), and lindane, for periods ranging from 5 min to 5 hr, was examined. A swimming behavior test is described which is based on the rotifers' movement rates as they swim over a grid. For all four toxicants a clear dose-response was observed, with the swimming activity decreasing with increasing toxicant concentrations. For Cu the EC50's, the concentration that reduced the swimming activity to 50% of that of the control value, sharply decreased from 0.22 mg/liter after an exposure of 5 min to 0.068, 0.038, and 0.014 mg/liter after exposures of 30, 60, and 300 min, respectively. PCP affected the rotifers' swimming behavior more gradually, with EC50's decreasing from 7.0 mg/liter after an exposure of 5 min to 5.9, 5.4, and 1.5 mg/liter after 30, 60, and 300 min, respectively. A similar pattern was found for DCA with EC50's ranging from 193 to 45.5 mg/liter for the 5-min and 3-hr exposures, respectively. Exposed to lindane however, B. calyciflorus swimming activity exhibited a different response, and the EC50's gradually increased from 13.7 mg/liter after an exposure of 5 min to significantly higher values of 16.4 and 18.5 mg/liter after periods of 1 and 5 hr, respectively. The results of the swimming activity assays were compared to those of acute and chronic toxicity tests performed with the same test species. The potential use and relevance of this behavioral test criterion were evaluated and discussed.

Human acute toxicity prediction of the first 50 MEIC chemicals by a battery of ecotoxicological tests and physicochemical properties.

Five acute bioassays consisting of three cyst-based tests (with Artemia salina, Streptocephalus proboscideus and Brachionus calyciflorus), the Daphnia magna test and the bacterial luminescence inhibition test (Photobacterium phosphoreum) are used to determine the acute toxicity of the 50 priority chemicals of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. These tests and five physiocochemical properties (n-octanol-water partition coefficient, molecular weight, melting point, boiling point and density) are evaluated either singly or in combination to predict human acute toxicity. Acute toxicity in human is expressed both as oral lethal doses (HLD) and as lethal concentrations (HLC) derived from clinical cases. A comparison has also been made between the individual tests and the conventional rodent tests, as well as between rodent tests and the batteries resulting from partial least squares (PLS), with regard to their predictive power for acute toxicity in humans. Results from univariate regression show that the predictive potential of bioassays (both ecotoxicological and rodent tests) is generally superior to that of individual physicochemical properties for HLD. For HLC prediction, however, no consistent trend could be discerned that indicated whether bioassays are better estimators than physicochemical parameters. Generally, the batteries resulting from PLS regression seem to be more predictive than rodent tests or any of the individual tests. Prediction of HLD appears to be dependent on the phylogeny of the test species: cructaceans, for example, appear to be more important components in the test battery than rotifers and bacteria. For HLC prediction, one anostracan and one cladoceran crustacean are considered to be important. When considering both ecotoxicological tests and physicochemical properties, the battery based on the molecular weight and the cladoceran crustacean predicts HLC substantially better than any other combination.

QSAR models for predicting the acute toxicity of selected organic chemicals with diverse structures to aquatic non-vertebrates and humans.

The linear and non-linear relationships of acute toxicity (as determined on five aquatic non-vertebrates and humans) to molecular structure have been investigated on 38 structurally-diverse chemicals. The compounds selected are the organic chemicals from the 50 priority chemicals prescribed by the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. The models used for the evaluations are the best combination of physico-chemical properties that could be obtained so far for each organism, using the partial least squares projection to latent structures (PLS) regression method and backpropagated neural networks (BPN). Non-linear models, whether derived from PLS regression or backpropagated neural networks, appear to be better than linear models for describing the relationship between acute toxicity and molecular structure. BPN models, in turn, outperform non-linear models obtained from PLS regression. The predictive power of BPN models for the crustacean test species are better than the model for humans (based on human lethal concentration). The physico-chemical properties found to be important to predict both human acute toxicity and the toxicity to aquatic non-vertebrates are the n-octanol water partition coefficient (Pow) and heat of formation (HF). Aside from the two former properties, the contribution of parameters that reflect size and electronic properties of the molecule to the model is also high, but the type of physico-chemical properties differs from one model to another. In all of the best BPN models, some of the principal component analysis (PCA) scores of the 13C-NMR spectrum, with electron withdrawing/accepting capacity (LUMO, HOMO and IP) are molecular size/volume (VDW or MS1) parameters are relevant. The chemical deviating from the QSAR models include non-pesticides as well as some of the pesticides tested. The latter type of chemical fits in a number of the QSAR models. Outliers for one species may be different from those of other test organisms.

Ecotoxicological studies with the freshwater rotifer Brachionus calyciflorus. III. The effects of chemicals on the feeding behavior.

The effect of short-term exposure to xenobiotics on the feeding behavior of the freshwater rotifer Brachionus calyciflorus was studied. The filtration and ingestion rates of this rotifer decreased after an exposure of 5 hr to sublethal concentrations of copper, pentachlorophenolate, 3,4-dichloroaniline, and lindane. The effective concentrations at which feeding rate was reduced to 50% of that in controls (EC50) for the respective chemicals are 0.032, 1.85, 41.2, and 8.5 mg/liter. The potential use of feeding behavior as test criterion for toxicity screening tests with aquatic invertebrates is discussed.

Cyst-based toxicity tests. VI: Toxkits and Fluotox tests as cost-effective tools for routine toxicity screening.

During the last two decades microbiotests have been developed which are independent of recruitment, maintenance and/or culturing of live stocks of test organisms. "Culture and maintenance free" microbiotests have been worked out in the Laboratory for Biological Research in Aquatic Pollution at the University of Ghent, with selected aquatic invertebrates. The new approach is based on the use of "resting stages" (cysts) as inert biological material from which live test organisms can be hatched "on demand". The "cyst-based" bioassays have recently been miniaturized in Toxkits. Four cyst-based screening tests have reached the stage of commercialization: two freshwater 24h-LC50 tests (Rotoxkit F and Streptoxkit F) and two estuarine/marine 24h-LC50 bioassays (Rotoxkit M and Artoxkit M), based on cysts of rotifer and crustacean species, respectively. Recently, the same laboratory has also been focusing on the development of a "rapid" (one hour) sublethal bioassays with aquatic invertebrates. The so called "Fluotox" screening test is based on the visual observation of in vivo inhibition of an enzymatic process, using a fluorescent substrate. Besides the advantages of rapidity and cost-effectiveness, the Fluotox assay also appears to have a remarkable predictive potential for mortality, as displayed by the remarkable correlation between the Fluotox 1h-EC50's and conventional 24h-LC50's with the same species.

Acute toxicity tests using rotifers. IV. Effects of cyst age, temperature, and salinity on the sensitivity of Brachionus calyciflorus.

Several aspects of the response to toxicants using a standardized toxicity test with the freshwater rotifer Brachionus calyciflorus are described. Test animals are obtained by hatching cysts which produce animals of similar age and physiological condition. The acute toxicity of 28 compounds is described with 24-hr LC50's. The LC50's span five orders of magnitude, from silver at 0.008 mg.liter-1 to benzene at more than 1000 mg.liter-1. Control mortality in 84 tests averaged 2% with a standard deviation of 3%, indicating very consistent test sensitivity. Only once in 84 trials did a test fail because of excessive control mortality, yielding a failure rate of 1.2%. Cyst age from 0 to 18 months had no effect on the sensitivity of neonates to reference toxicants. Both high and low temperatures increased rotifer sensitivity to reference toxicants. Copper sensitivity was greater at 10, 25, and 30 degrees C compared with results at 20 degrees C. Likewise, sodium pentachlorophenol toxicity was greater at 10 and 30 degrees C compared with results at 20 degrees C. Survivorship curves at 25 degrees C of neonates under control conditions indicated that mortality begins at about 30 hr. This places a practical limit on toxicant exposure for the assay of 24 hr. B. calyciflorus cysts hatch at salinities up to 5 ppt and acute toxicity tests using pentachlorophenol at this salinity yielded LC50's about one-half those of standard freshwater. B. calyciflorus is preferred over Brachionus plicatilis for toxicity tests in salinities up to 5 ppt because it is consistently more sensitive.

Toxicity tests on ciliates--a short review.

A variety of methods has been proposed to study the toxicity of chemicals or polluted waters on ciliates as representative test organisms of the microfauna of aquatic ecosystems. These methods are based on morphological, ultrastructural, ethological, or metabolic criteria. The techniques proposed are mostly species dependent and so are their applications and restrictions. In view of a future standardization of protozoan toxicity tests a selection has been made by the authors on the basis of the applicability of the tests for routine analysis. The methods withheld are briefly described and comments are made on their advantages and limitations. The equipment involved, the operational complexity, and the accuracy of the results and their interpretation are listed in a synoptic table.

New type of turbidostat with intermittent determination of cell density outside the culture vessel.

An inexpensive turbidostat featuring intermittent determination of optical density in an interchangeable measuring tube and incorporating electronic circuits for the automated mechanics is described.