Toxicity Test MethodsChapter 4

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

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

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

D. magna vs. C. dubia

Type of test Acute 48 h Static renew/chronic

Age of organisms < 24 h old < 24 h old

# orgs per chamber 10 1

Test vessel 250 ml beakers 30 ml containers

Test sol’n volume 200 ml 15 ml

# of replicates/treat. 3 10

Feeding regimen Do not feed YCT

Test duration 48 h 5 - 7 d

Renew test water? No Yes

Temperature (°C) 20 ± 2 25 ± 1

Endpoint Immobilization reproduction

Major use Test xenobiotics Test effluent

Ceriodaphnia dubia toxicity test

Daphnia magna toxicity test

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

Microalgae 96-h toxicity testStatic

Organisms Various freshwater and saltwater species

# orgs per chamber 2 - 5 x 104 cells/ml

Test vessel Sterilized Erlenmeyer flask, any size

Test solution volume Not to exceed 50% of flask volume

# of replicates/treat. 2 or more

Feeding regimen Nutrients in test water

Test duration 96-h

Renew test water? No (would lose algae!)

Temperature (°C) 20-24 ± 2

Endpoint Biomass, cell number, area underneath growth curve, chlorophyll content

Major use Test toxicants and effluent

Algal toxicity test

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

Fathead minnow culture

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

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

Beware of using large wild rats!

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

Xenopus life cycle

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

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

Microcosms

Mesocosms

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