Ecotoxicology and The Three Rs

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Ecotoxicology and The Three Rs
Dra. Argelia Castaño Ministry of Science and
Technology National Institute Food and Agrarian
Research (INIA) Animal Health Research Center
(CISA)
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PREDICTION
EVALUATION
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Ecotoxicity Data REQUIRED FOR
CHEMICALS

• CLASSIFICATION, PACKAGING AND LABELLING.
  67/548/CEE. 99/45/EC

• PESTICIDES /BIOCIDES 91/414/CE , 98/8/CE

• RISK ASSESSMENT OF INDUSTRIAL CHEMICALS
  93/67/CEE 94/1488/CE

• VETERINARY MEDICINAL PRODUCTS. 92/18/CEE

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Ecotoxicity Data
E C Strategy for a chemicals policy White Paper,
2001
30,000 CHEMICALS (1-100 t)Base set
5,600 CHEMICALS (100-1000t) Level 1
2,600 CHEMICALS (1000t) Level 2
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Air
Ecotoxicology Alternatives to vertebrates.
Terrestrial
Soil
Aquatic
Water
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Ecotoxicity Data Vertebrate Terrestial
mammals /birds
Avian Dietary Toxicity Test
Avian Reproduction
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Fish
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AQUATIC
BASE SET REQUIREMENT
ACUTE TOXICITY
LC50, NOEC
42-60 x chemical

• 7-10 fish /concentration
• 5 concentration control
• 96 h exposure

Base Set 1.260 millions (E C w.p. 2001)
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AQUATIC
Level 1
42-60 x chemical
14-day prolonged toxicity

• 7-10 fish /concentration
• 5 concentration control
• 96 h exposure

Bioacumulation
320 x chemical

• 80 fish /concentration
• 4 groups

Level 1... 2.024 millions (EC w.p. 2001)
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PROBLEMS/LIMITATIONS OF FISH TESTS.
ETHICS
COSTS
SAMPLE VOLUME
EQUIPMENT-FACILITIES
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Fish Acute Toxicity Test
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Acute Cytotoxicity Test fish cells
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in vivo LC50/in vitro EC50 CORRELATION

• Alcohols
• Anilins
• Phenols
• Heavy Metals
• Pesticides
• PAH
• PCB

• 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)

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Assessment Environmental Processes
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Combined protocols T.I.E.
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Alternatives to acute toxicity fish test
Cytotoxicity test with fish cells in
ecotoxicology

• Screening of chemicals.

• Toxicological assessment of complex mixtures.

• Assessment of environmental processes.

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Endocrine Disruption

• BIRDsEgg-injection. Early life stages.
  Vitellogenin. Androgenicity.

• Reptiles vitellogenin. Sex determination
  (turtles)

• Amphibianvitellogenin assay. FETAX.
  Metamorphosis?

• Pisces vitellogenin assay. Early life stage
  assay. Embrio sac fry test. Life cycle test

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Endocrine Disruption alternatives

• BIRDsavian cell culture. avian plaque assay
  (brain slices)

• Fish in vitro vitellogenin (hepatocytes).
  Transfected fish cell lines. In vitro steroid
  receptor competition assay

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Genetic Structure changes.
Inability environmental adaptation
Population viability
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• Carcinogenesis
• Genetic diversity

Genetic effects

• Specific metabolic pathways

• Different efficency in DNA repair mechanisms.

• Predictive Assessment of genetic structure
  changes of wild populations.

• Comparative toxicogenomics/proteomics

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• Carcinogenesis
• Genetic diversity

Genetic effects
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CONCLUSIONS 3R Ecotoxicology
RD
Fish
3R----- Acute
3R......Chronic
3R......ED
3R......Genotoxicity
Regulatory guidelines.
Birds