1. Toxicity Test Methods Chapter 4

2. Introduction Working knowledge of standard test methods very important in understanding the field of environmental toxicology Both strengths and weaknesses needed to interpret test results Methods were mostly developed in the ’80’s with some modifications since then Most “old” tests have not been replaced with new tests but rather have been “tweaked” over the years

3. Overview Toxicity tests can range from cell cultures to ecosystem function Most tests fall under two general classifications –Single-species Relatively cheaper, shorter, low ecological realism –Multi-species Tend to be longer, more expensive and have higher ecological realisms

4. Single species toxicity testing Daphnia 45-h Acute Toxicity Test –Most widely used test for evaluating toxicity of water –Three primary species Daphnia magna – general testing Ceriodaphnia dubia – chronic testing over an acute time period Daphnia pulex – evaluation of stormflow toxicity –All are easy to culture when published guidelines are followed but some differences in testing procedure –Note: algal tests are most likely to show hormesis effect

5. D. magna vs. C. dubia Type of testAcute 48 hStatic renew/chronic Age of organisms< 24 h old # orgs per chamber101 Test vessel250 ml beakers30 ml containers Test sol’n volume200 ml15 ml # of replicates/treat.310 Feeding regimenDo not feedYCT Test duration48 h5 - 7 d Renew test water?NoYes Temperature (°C)20 ± 225 ± 1 EndpointImmobilizationreproduction Major useTest xenobioticsTest effluent

6. Ceriodaphnia dubia toxicity test

7. Daphnia magna toxicity test

8. Single species toxicity testing (con’t) Algal 96-h growth toxicity test –Examines toxicity to freshwater and marine algae –Algae are extremely important because they generate most of the primary productivity in aquatic and marine sytems  effects on algae can have high impact at higher trophic levels in ecosystem

9. Microalgae 96-h toxicity test Static OrganismsVarious freshwater and saltwater species # orgs per chamber2 - 5 x 10 4 cells/ml Test vesselSterilized Erlenmeyer flask, any size Test solution volumeNot to exceed 50% of flask volume # of replicates/treat.2 or more Feeding regimen Nutrients in test water Test duration96-h Renew test water?No (would lose algae!) Temperature (°C)20-24 ± 2 EndpointBiomass, cell number, area underneath growth curve, chlorophyll content Major useTest toxicants and effluent

10. Algal toxicity test

11. Single species toxicity testing (con’t) Acute toxicity tests with aquatic organisms –Multiple tests using a variety of fish, amphibians and macroinvertebrates –Endpoint is either death or immobilization –More difficult to culture or obtain as test organisms so often use animals collected from the wild or available commercially as “bait” Test organisms may need to be acclimated to laboratory conditions More variability in response because of increased heterogeneity of gene pool Locally collected organisms may provide better indication of effect of xenobiotics on local ecosystems See table 4.5 (p. 80-82) for specific test organisms and test conditions

12. Fathead minnow culture

13. Single species toxicity testing (con’t) Terrestrial vertebrate toxicity test –Mammals, birds –Toxicant usually introduced in food (gelatin capsule or gastric lavage) –Mammals  surrogate for human health effects –Birds  effect of pesticides on non-target species –Both animal groups are usually tested for 90 days –Endpoint death, test may include urinalysis, hematology, necropsy

14. Small mammals collected from the wild may not be suitable for toxicity testing

15. Beware of using large wild rats!

16. Single species toxicity testing (con’t) Frog Embryo Teratogenesis Assay: Xenopus (FETAX) One of few standardized amphibian-based toxicity tests Designed to test teratogenicity of chemicals or effluent using the African Clawed frog as a surrogate for humans Good correlation between known human teratogens and FETAX results

17. Xenopus life cycle

18. Multi-species toxicity test Artificially contained communities Must contain two or more interacting species Trying to simulate environmental realism but often want to reduce heterogeneity in test conditions Wide range of size and complexity in multi-species test protocols

19. Microcosms and Mesocosms –Microcosms Small (can usually be picked up) Assembled to include specific components Highly defined artificial ecosystem Easy, cheap to replicate Moderate level of environmental realism –Mesocosms Larger (kiddie wading pool to 3 acre ponds) May include volunteer components so system is less defined Expensive, hard to maintain High level of environmental realism

20. Microcosms

21. Mesocosms

22. Uses of microcosms and mesocosms 1° - Test for ecosystem level effects of pesticides (FIFRA microcosms) Effects of oil spills on marine ecosystem Effect of draining aquaculture ponds into adjacent stream Any ecosystem level test