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

Dra. Argelia Castaño

Ministry of Science and TechnologyNational Institute Food and Agrarian Research (INIA)

Animal Health Research Center (CISA)

Dra. Argelia Castaño

Ministry of Science and TechnologyNational Institute Food and Agrarian Research (INIA)

Animal Health Research Center (CISA)

Ecotoxicology Ecotoxicology and The Three Rsand The Three Rs

Ecotoxicology Ecotoxicology and The Three Rsand The Three Rs

PREDICTION PREDICTION EVALUATIONEVALUATION

CHEMICALS:CHEMICALS:

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

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

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

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

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

•VETERINARY MEDICINAL PRODUCTS. 92/18/CEE•VETERINARY MEDICINAL PRODUCTS. 92/18/CEE

Ecotoxicity Data REQUIRED FOR:

30,000 CHEMICALS (1-100 t):Base set 30,000 CHEMICALS (1-100 t):Base set

Ecotoxicity Data: E C Strategy for a chemicals policy White Paper, 2001

5,600 CHEMICALS (100-1000t): Level 1 5,600 CHEMICALS (100-1000t): Level 1

2,600 CHEMICALS (+1000t): Level 2 2,600 CHEMICALS (+1000t): Level 2

Water

Ecotoxicology Alternatives to Alternatives to

vertebrates.vertebrates.

Soil

Air

Terrestrial

Aquatic

Ecotoxicity Data: Vertebrate Terrestial :mammals /birds

Avian single-dose oral toxicity test

Avian Dietary Toxicity Test

Avian Reproduction

FishFish

AQUATICAQUATIC

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)

14-day prolonged toxicity 14-day prolonged toxicity

AQUATICAQUATIC

Bioacumulation: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)

PROBLEMS/LIMITATIONS OF FISH TESTS.

ETHICSCOSTSCOSTS

SAMPLE VOLUMESAMPLE VOLUME

EQUIPMENT-FACILITIESEQUIPMENT-FACILITIES

Fish Acute Toxicity Test

Acute Cytotoxicity Test fish cells

RR

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

CORRELATION EC50 "in vitro“ CL50 "in vivo"

0.1 0.2 0.3 0.4

CL50 mM

0

4

8

12

16

r2=0.94y=0.65+0.39x

p<0.0001

EC50

mM

in vivo LC50/in vitro EC50 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)

Assessment

Environmental Processes

Combined protocols: T.I.E.

Screening of chemicals.

Alternatives to acute toxicity fish testAlternatives to acute toxicity fish test

Cytotoxicity test with fish cells in ecotoxicology Cytotoxicity test with fish cells in ecotoxicology

Toxicological assessment of complex mixtures. Assessment of environmental processes.

R+R+DDR+R+DD Regulatory

guidelines.Regulatory guidelines.

BIRDs:Egg-injection. Early life stages. Vitellogenin. Androgenicity.

Endocrine DisruptionEndocrine 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

BIRDs:avian cell culture. avian plaque assay (brain slices)

Endocrine Disruption: alternativesEndocrine Disruption: alternatives

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

Genetic Structure changes.

Inability Inability environmental environmental adaptation adaptation

Population viabilityPopulation viability

Specific metabolic pathways

Genetic effects: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

Genetic effects:Genetic effects:

•Carcinogenesis•Genetic diversity

•Carcinogenesis•Genetic diversity

Non-regulatory/research/diagnosisNon-regulatory/research/diagnosis

R+DR+D

RegulatoryRegulatoryIn vitro

A B C D E A B C D E

In vivo

mw mw

.

Primers A (D-4); B (D-8); C (C-95); D (C-96); E (Trna-1)

1353

CONCLUSIONS3R Ecotoxicology

CONCLUSIONS3R Ecotoxicology

3R----- Acute

3R......Chronic3R......ED 3R......Genotoxicity

R+R+DDR+R+DD

Regulatory guidelines.Regulatory guidelines.

Fish

Birds