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Toxicity Testing
Standard Methods
Multiple methods have been standardized
(certified) by multiple organizations
American Society for Testing and Materials (ASTM)
Organization for Economic Cooperation and
Materials (OECD) – (Europe based)
National Toxicology Program (NTP)
All above standardized protocols available from
US EPA, Federal Register and researchers that
developed the programs
Advantages of Standard Methods
Tests are uniform and comparable to previous results
within the same or other laboratories
Can be replicated (confirmed) by other laboratories
Makes it easier for decision makers to accept test
results
Logistics are simplified, developmental work already
done
Methods establish baseline from which
modifications can be made if necessary
Data generated can be combined with those from
other laboratories for use in QSAR, ERA’s
Advantages of Standard Methods (con’t)
Detailed listing of
apparatus, dilution
water, test material, test
organisms, etc
Experimental,
analytical and
documentation
procedures are detailed
Acceptability criteria
are listed
Disadvantages of Standard Methods
Often very specific hard to apply to other
situations or answer other questions
Tend to be used in inappropriate situations
(research, cause and effect evaluation)
May not be applicable to natural environment
Acute vs. Chronic Toxicity Tests
Can broadly classify toxicity tests based on length of
exposure
Acute Toxicity test
Drop dead testing
Time = 2 days (invertebrates) to 4 d. (fish)
LD50
LC50
TLm (median tolerance dose)
EC50 (effective concentration)
Lose equilibrium, sit on bottom “ecologically” dead
Not very ecologically relevent but quick, relatively cheap
(but still ~$700-1,200 per test)
Acute vs chronic toxicity testing (con’t)
Mean response
Frequency
Protected
5% allowable
impact
0
Integration
of
Looking for
this area of
response
To save money while finding area of mean response use a two step process
Step 1 – Screening test
[X] mg/L
# dead none none some all RIP all RIP
2 10-1.5 0.032
3 10-1 0.1
4 10-0.5 0.32
5 100 1.0
Control 0.0
Set up for definitive test – example 2
low = 101 µg/L
high = 103
7
Probit Units
% Mortality
5
50
13.6%
2.13%
-2 -1 0 1 2
Standard deviations
Difficult to deal with SD (34.13, 13.6, etc) so rename SD to probits
34.13%
Mean
13.6%
2.13%
3 4 5 6 7
Probits
Example probit analysis
Concentration Deaths %
(mg/L)
Control 0/10 0
0.3 0/10 0
1 0/10 0
3 1/10 10
10 4/10 40
30 9/10 90
100 10/10 100
Look at data should be able to tell immediately that LC50 should be between 10 and 30 mg/L
Graph fit line by eye (approximately equal number above and below line)
Uses of LC50
1. 1. Application factor
LC50 x n = ___ = allowable dose
Good if do not have better information (chronic tests)
2. Rank hazards lower LC50 = more toxic
3. Lead to chronic testing
Remember: LC50 does not provide an ecologically
meaningful result bad because trying to protect
ecosystem need more ecosystem level testing
Probit is trade-off between cost and getting
sufficient data to make a decision about the
environmental toxicity of a chemical
Chronic toxicity testing
Sublethal
Time = 7d. to 18 months
Endpoints are
growth
Reproduction
brood size (Ceriodaphnia dubia can have 2-3 broods
in seven days)
Hatching success
Analysis of chronic tests
Analysis of Variance (hypothesis testing)
Test for significant difference from control (C + 5
doses)
Regression analysis
EC20 (concentration that causes 20% reduction
relative to control)
Results of Analysis of Variance test
Respiration (gC/L/d.)
*
Community
* *
C 1 3 10 30 100
Concentration of Hg (mg/L)
Determination of EC20
Control
Response (growth)
response
10 μg
$106 /year
Endpoints are
Biomass
Diversity
Species richness
Etc.
All toxicity tests try to determine level of
toxicant which will or will not cause an effect
NOEC – No Observable Effect Concentration
Highest conc not signficantly different from control
LOEC – Lowest Observable Effect Concentration
Lowest test concentration that is significantly different
from control
MATC – Maximum Allowable Toxicant
Concentration
Geometric mean of NOEC and LOEC
Often called the “chronic value”
MATC
*
Community
* *
C 1 3 10 30 100
Concentration of Hg (mg/L)
If there is magic on earth, it is in water
Photo by R. Grippo