Guido Persoone

Proposal for a short-term toxicity test with Artemia nauplii.

Abstract Although standardization of toxicity tests on aquatic organisms is an urgent necessity, very little has yet been achieved for the marine environment. As a first step in this direction, a simple, inexpensive and reliable short-term routine test with Artemia larvae is proposed. This test is the result of an extensive study in our laboratory, taking into account the advantages and disadvantages of Artemia bioassays published by various authors. The major advantage of the brine shrimp as a test species is its continuous availability under the form of dry cysts which can be hatched very easily; this eliminates all biological, technological, and financial problems of stock recruitment and/or culturing. The acute test presented is based on the determination of the LC50-24 hr of instar II–III nauplii of a specific Artemia strain. Presently this test is the subject of an intercalibration exercise in North America; an analogous exercise is now in progress in the European Economic Community countries.

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 LC50s. The LC50s 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 LC50s 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.

Decapsulation of Artemia cysts: A simple technique for the improvement of the use of brine shrimp in aquaculture

Abstract Although it is a common practice in different disciplines of fundamental research on the brine shrimp, and despite the very interesting applications that it offers for the use of Artemia in aquaculture, the “decapsulation” technique, which removes the outer layer of the cyst shell of Artemia , is not known to shrimp and fish aquaculturists. The present paper describes the technology developed by the authors for the routine decapsulation of Artemia cysts. The advantages which result from the use of decapsulated cysts in aquacultural hatcheries are discussed.

New microbiotests for routine toxicity screening and biomonitoring

1. Reviews on Toxicity Tests. 2. Toxicity Testing for Regulatory Purposes. 3. New Microbiotests and Specific Test Criteria. 4. Sensitivity Comparisons of Toxicity Tests. 5. Toxicity Testing of Natural Waters. 6. Toxicity Testing of Wastes, Waste Waters and Leachates. 7. Toxicity Testing of Soils and Sediments. 8. Toxicity Testing of Air Pollution. 9. Toxicity Testing of Specific Chemicals. 10. Testing for Mutagenicity and Genotoxicity. 11. Microbiotests for Toxicity Evaluation of Biotoxins. Subject Index: Listing for major subject classes. Subject Index: Alphabetical listing.

Predictive value of laboratory tests with aquatic invertebrates: influence of experimental conditions

Abstract Considering the difficulty of making meaningful extrapolations of laboratory bioassay data to real world situations, short-term tests have been carried out in a factorial pattern to determine the magnitude of effect variation resulting from changes in experimental abiotic conditions. Three selected zooplankton species (the rotifer Brachionus plicatilis, the brine shrimp Artemia salina and the waterflea Daphnia magna) have been exposed to increasing concentrations of two chemicals (one inorganic and one organic) in different combinations of two major environmental variables. For the brackish water rotifer B. plicatilis the acute toxicity of potassium dichromate and sodium laurylsulphate was determined in 16 different combinations of temperature and salinity (10–17–24–31°C and 5–20–35–50‰). For the marine crustacean A. salina, the acute toxicity of the same two chemicals was determined in 20 temperature-salinity combinations (10–15–20–25–30°C and 5–20–35–50‰) and for the freshwater crustacean D. magna, 16 combinations of temperature and water hardness (7–14–21–28°C and 80–320–560–800 mg/1 CaCO3) were assayed. The entire study comprised nearly 300 complete toxicity tests. 24-h LC50 values (for Artemia and Brachionus) and 24-h EC50 values (for Daphnia) revealed that the variation in toxicity resulting from changing environmental conditions, is both species- and chemical-specific and (within the limits of this study) ranged from a minimum of a factor 2.5 to a maximum exceeding a factor of 100. The necessity to take such variations into consideration in predictive hazard assessment studies is underlined.

Acute toxicity bioassays using rotifers. I. A test for brackish and marine environments with Brachionus plicatilis

Abstract A standardized 24-h acute toxicity test for the marine environment using the rotifer Brachionus plicatilis is described. Test animals are obtained by hatching cysts, thus eliminating the need for stock cultures. Since animals hatching from cysts are of similar age, genotype and physiologically condition, test variability is greatly reduced. Controlled cyst hatching is achieved by transferring to lower salinity, warmer temperature and light. After 23 h at 25°C and 15 ppt salinity, hatching begins and proceeds rapidly. By 28 h, 90% of the cysts have hatched. A protocol is outlined to collect the neonates and use them in a simple acute toxicity test to calculate a 24 h LC 50 . A reference test using sodium pentachlorophenate (NaPCP) is described as well as a range-finding test and a definitive test for unknown toxicants. The toxicity of six compounds to B. plicatilis was examined with the following decreasing sensitivities: copper > NaPCP > SDS > free NH 3 > cadmium > malathion. For 3 of the 6 compounds tested, salinity increase from 15 to 30 ppt resulted in higher sensitivity, whereas for the other 3 compounds there was no effect. In comparison with current test organisms B. plicatilis is either more, equal or less sensitive depending on the compound, confirming the species-chemical specificity of mode of action of toxicants. The repeatability of the rotifer test is 5–6 times better than that reported for Daphnia tests and twice as good as the Artemia nauplii bioassay. Like the standard brine shrimp nauplii acute test, the cyst-based rotifer test is an important advance in acute toxicity testing since it eliminates stock cultures, is rapid, sensitive, highly repeatable, easy to execute and cost effective.

Comparative acute toxicity of the first 50 Multicentre Evaluation of In Vitro Cytotoxicity chemicals to aquatic non-vertebrates

The acute toxicity data of the first 50 chemicals of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme is compared for three “cyst-based toxicity tests” (Artoxkit M with Artemia salina, Streptoxkit F with Streptocephalus proboscideus, and Rotoxkit F with Brachionus calyciflorus), and two other tests (the Daphnia magna and the Photobacterium phosphoreum Microtox™ tests) commonly used in ecotoxicology. The difference in sensitivity for the 50 chemicals was as high as 19 orders of magnitude (on a molecular weight basis) between the most and least sensitive species. Generally, a similar toxicity ranking of the 5 test species was found for most of the chemicals and the interspecies correlations were high. Results from Principal Component Analysis (PCA) and cluster analysis indicated that the groupings are not related to a clear and defined chemical structure. However, the loading plot of the first two principal components may aid in selecting the minimum number and type of tests that have to be included in a battery which encompasses a broad spectrum of toxicity levels. Consequently, this study supports the use of a selected battery of tests to evaluate ecotoxicity and suggests its possible importance for screening of biologically-active compounds from natural sources.

Acute toxicity bioassays using rotifers. II. A freshwater test with Brachionus rubens

Abstract A 24-h acute toxicity test for freshwater is described using the rotifer Brachionus rubens hatched from cysts. Hatchlings are used in a simple protocol that provides for LC50 calculation and yields highly repeatable results. Hatching is initiated by transferring cysts to warmer temperatures and light. At 25°C, hatching commences after 17 h and by 25 h, 40% of cysts have hatched. The average hatching percentage for B. rubens cysts was 53%. A reference test using sodium pentachlorophenate (NaPCP) yielded an LC50 of 0.62 mg/l, with a coefficient of variation of 9.7%. The no observed effect concentration (NOEC) for NaPCP was 0.28 mg/l. Protocols for range-finding and definitive tests of unknown toxicants are also described. Six compounds were assayed and had the following toxicity rankings: copper > NaPCP > cadmium > SDS > free NH3 > malathion. B. rubens was at least twice as sensitive as Brachionus plicatilis to all toxicants tested except malathion. The precision of the B. rubens acute toxicity test is about 3 times better than that of Daphnia. Like its marine counterpart with B. plicatilis, the B. rubens test for fresh water has a major advantage over current aquatic tests in that it eliminates culturing and maintenance of live stocks. Test animals are obtained from dormant eggs which have a shelf life of at least 1 yr. Moreover, the rotifer test proposed is fast, convenient, sensitive and repeatable, making it a useful new tool for routine assessment of aquatic toxicity of chemicals and effluents.

The use of Artemia nauplii for toxicity tests--a critical analysis.

Because of their easy hatching from dry cysts and their year-round availability, nauplii of the brine shrimp Artemia salina are most convenient test organisms for toxicity studies. The bioassays mentioned in literature have, however, mostly been carried out with larvae of which neither the exact instar stage nor the geographical origin is known. The incubation of the cysts should always be carried out under strictly controlled temperature conditions since both the onset of hatching and the length of the hatching period are temperature-dependent. Moreover, the experiments should only be carried out with populations of nauplii of the same stage of development since it is demonstrated that second and third instars are significantly more sensitive to toxicants than first instar larvae. It is demonstrated that the sensitivity to chemicals varies from one geographical Artemia strain to another.

Cyst-based toxicity tests. VIII. Short-chronic toxicity tests with the freshwater rotifer Brachionus calyciflorus

Abstract The development and potential use of a 4-day static renewal test [4-day Life Table (LT) test] and a 3-day static test [3-day Population Growth (PG) test] with the freshwater rotifers Brachionus calyciflorus are described. For both bioassays, test animals are obtained by hatching cysts which eliminates the need for the culturing and maintenance of the organisms. The toxicity of copper (Cu), pentachlorophenol (PCP), 3,4-dichloroaniline (DCA) and lindane was assessed using the developed methods. The NOECs, based on the test endpoint rm, obtained with the 4-day LT test were 0.0025, 0.4, 5 and 20 mg/l for Cu, PCP, DCA and lindane, respectively. Similar results were obtained with the 3-day PG tests for which NOECs of 0.005, 0.8, 20 and 10 mg/l, respectively, were recorded. The mean cv between replicated 3-day PG tests was 10%, indicating a good intra-laboratory reproducibility of the test results. For Cu and PCP, the sensitivity of B. calyciflorus compared favourably to chronic toxicity tests with Daphnia magna, while for the other two compounds B. calyciflorus proved to be rather insensitive. Considering the increasing need for relatively short toxicity tests, the two described short-chronic bioassays could be valuable new tools for routine toxicity evaluations. The major advantages associated with these tests are: they are less labour-intensive than existing chronic tests, they can be completed within one work week, and do not require stock culturing of test organisms.