Toxicity Test Methods
Chapter 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
Age of organisms
Static renew/chronic
< 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
5-7d
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 test
Static
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