1. Dra. Argelia Castaño Ministry of Science and Technology National Institute Food and Agrarian Research (INIA) Animal Health Research Center (CISA) Dra. Argelia Castaño Ministry of Science and Technology National Institute Food and Agrarian Research (INIA) Animal Health Research Center (CISA) Ecotoxicology and The Three Rs

2. PREDICTION EVALUATION

3. CHEMICALS: RISK ASSESSMENT OF INDUSTRIAL CHEMICALS: 93/67/CEE 94/1488/CE CLASSIFICATION, PACKAGING AND LABELLING. 67/548/CEE. 99/45/EC PESTICIDES /BIOCIDES 91/414/CE, 98/8/CE VETERINARY MEDICINAL PRODUCTS. 92/18/CEE E cotoxicity Data REQUIRED FOR :

4. 30,000 CHEMICALS (1-100 t):Base set E cotoxicity Data: E C Strategy for a chemicals policy White Paper, 2001 5,600 CHEMICALS (100-1000t): Level 1 2,600 CHEMICALS (+1000t): Level 2

5. W ater Alternatives to vertebrates. Ecotoxicology Alternatives to vertebrates. S oil A ir Terrestrial Aquatic

6. E cotoxicity Data: Vertebrate Terrestial :mammals /birds Avian single-dose oral toxicity test Avian Dietary Toxicity Test Avian Reproduction

7. Fish

8. AQUATIC ACUTE TOXICITY : LC50, NOEC BASE SET REQUIREMENT 7-10 fish /concentration 5 concentration + control 96 h exposure 42-60 x chemical Base Set 1.260 millions (E C w.p. 2001)

9. 14-day prolonged toxicity AQUATIC Bioacumulation: Level 1 80 fish /concentration 4 groups 7-10 fish /concentration 5 concentration + control 96 h exposure 42-60 x chemical 320 x chemical Level 1... 2.024 millions (EC w.p. 2001)

10. P ROBLEMS/ L IMITATIONS OF FISH TESTS. ETHICS COSTS SAMPLE VOLUME EQUIPMENT-FACILITIES

11. Fish Acute Toxicity Test Acute Cytotoxicity Test fish cells R R

12. Alcohols Anilins Phenols Heavy Metals Pesticides PAH PCB in vivo LC 50 /in vitro EC 50 CORRELATION In vitro / in vivo differences in sensitivity ranks between 1 and 3 orders of magnitude In vivo differences in sensitivity ranks between 1 and 4 orders of magnitude ( specie and water quality )

13. Assessment Environmental Processes

14. Combined protocols: T.I.E.

15. Screening of chemicals. Alternatives to acute toxicity fish test Cytotoxicity test with fish cells in ecotoxicology Toxicological assessment of complex mixtures. Assessment of environmental processes.R+DR+D Regulatory guidelines.

16. BIRDs: Egg-injection. Early life stages. Vitellogenin. Androgenicity. Endocrine Disruption Reptiles: vitellogenin. Sex determination (turtles) Amphibian:vitellogenin assay. FETAX. Metamorphosis? Pisces: vitellogenin assay. Early life stage assay. Embrio & sac fry test. Life cycle test

17. BIRDs :avian cell culture. avian plaque assay (brain slices) Endocrine Disruption: alternatives Fish: in vitro vitellogenin (hepatocytes). Transfected fish cell lines. In vitro steroid receptor competition assay

18. Genetic Structure changes. Inability environmental adaptation Population viability

19. Specific metabolic pathways Genetic effects: Different efficency in DNA repair mechanisms. Predictive Assessment of genetic structure changes of wild populations. Carcinogenesis Genetic diversity Carcinogenesis Genetic diversity Comparative toxicogenomics/proteomics

20. Genetic effects: Carcinogenesis Genetic diversity Carcinogenesis Genetic diversity Non-regulatory/ research/diagnosis Non-regulatory/ research/diagnosis R+D Regulatory In vitro AB CD E A B CD E In vivo mw. Primers A (D-4); B ( D-8); C (C-95); D (C-96); E (Trna-1) 1353 1078 603 310

21. CONCLUSIONS 3R Ecotoxicology CONCLUSIONS 3R Ecotoxicology 3R----- Acute 3R......Chronic 3R......ED 3R......Genotoxicity R+DR+D Regulatory guidelines. Fish Birds