1. Acute and Chronic Toxicity Testing

2. 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

3. 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

4. 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

5. 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

6. 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)

7. Acute vs chronic toxicity testing (con’t)
Growth, reproduction
More ecologically relevant data but takes longer, more expensive
Shows effect at much lower dose
Test requires much more “baby-sitting”

8. Acute Testing - theory
Population of organisms has normally distributed resistance to toxicants  acute toxicity test designed to identify mean response
Regulations allow 5% of species to be impacted
Most tests only use 2-3 species (up to 6)  not really enough to protect 95% of all species!

9. Acute Toxicity Test Organisms
Use of test species based on
Lab hardiness
Common
Known life cycle
Cheap
Short-lived

10. Normal distribution of resistance/sensitivity
Mean response
Frequency
Protected
5% allowable impact
Resistance (log [X]

11. Experimental design for toxicity tests
Integration of
Freg. of response (i.e death)
Percent mortality
Looking for this area of response
Log [X]
Log [X]
To save money while finding area of mean response use a two step process

12. Step 1 – Screening test
Expose 5–10 organisms to 10x increasing [ ] for hours
Trying to determine range in which median lethal concentration (LC50) will fall

13. Screening test [X] mg/L % Responding
[X] mg/L
# dead none none some all RIP all RIP
30%
100%
100%
Concen

14. Step 2 – Definitive test From previous results low = 10-2 = 0.01 mg/L
high = 100 = 1.0 mg/L
Run test using logarithmic scale of concentrations because organisms usually respond logarithmically to toxicants
Usually use at least 5 concentrations + control
Control – checks toxicity of dilution water, health of test organisms, stress level of testing environment (test chambers, lighting, temperature, etc)
If >10% of control organisms die  throw out test!
Use 10 – 30 organisms  randomly split up among tanks

15. Set up for definitive test – example 1
Treatment
Division
Concentration (mg/L) 1
10-2
0.01 2
10-1.5
0.032 3
10-1
0.1 4
10-0.5
0.32 5
100
1.0
Control
0.0

16. Set up for definitive test – example 2 low = 101 µg/L high = 103
Treatment
Division
Concentration
(µg/L) 1
103
1000 2
102.5
316 3
102
100 4
101.5 31 5
101 10
control

17. Analysis of Toxicity Tests
Based on hypothesis that resistance to toxicants is normally distributed
Use a probit transformation to make data easier to analyze
Based on SD so each probit has a percentage attached to it
Mean response defined as probit = 5 so all probits are positive  easier to visualize
Can use probit analysis to calculate LC50 because probit transformation will straighten the cumulative distribution line

18. Probit Analysis
Response of organisms to toxic chemicals = normal distribution
Cannot measure normal distribution directly because effect is cumulative, so graph as cumulative distribution
Cumulative distribution
Dose
# Responding
Normal distribution
Log Dose

19. Converting a curvilinear line to straight line
Difficult to evaluate a curved line
Conversion to a straight line would make evaluation easier
Cumulative distribution
Probit transformed
Log Dose
Log Dose %
% Mortality
Probit Units
Straight line (easier to analyze)
LD50, TLM)

20. Note: probit forces data towards middle of distribution  good because most organisms are “average” in their response

21. Relationship between normal distribution and standard deviations
34.13%
Mean
13.6%
2.13%
Standard deviations

22. Difficult to deal with SD (34.13, 13.6, etc) so rename SD to probits
34.13%
Mean
13.6%
2.13%
Probits

23. Example probit analysis
Concentration (mg/L)
Deaths %
Control
0/10
0.3 1 3
1/10 10
4/10 40 30
9/10 90
100
10/10
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)

24. Uses of LC50 1. Application factor
LC50 x n = ___ = allowable dose
Good if do not have better information (chronic tests)
Rank hazards  lower LC50 = more toxic
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

25. 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

26. 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)

27. Results of Analysis of Variance test* * *
Community Respiration (gC/L/d.) C
Concentration of Hg (mg/L)

28. Determination of EC20 Response (growth) Control response 10 μg 8 μg
20% reduction relative to control
8 μg
Control
Dose
EC20 eg. 1 mg/L = discharge limit

29. Ecosystem Tests (microcosms, mesocosms)
AOV design (4 reps X 3 treat., 3 rep X 4)
Time = 1 – 2 years
$106 /year
Endpoints are
Biomass
Diversity
Species richness
Etc.

30. 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”

31. NOEC LOEC

32. MATC
MATC = √NOEC + LOEC

33. MATC