Acute Toxicity Fish Test Continuous Dosing

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Acute and Chronic Toxicity Testing

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Acute and Chronic Toxicity Testing

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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) • Chronic toxicity testing • 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 0 100 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 24-96 hours • Trying to determine range in which median lethal concentration (LC50) will fall

13. Screening test 0 100 % Responding [X] mg/L # dead none none some all RIP all RIP 30% 100% 100% 0 0 Concen. 10-3 10-2 10-1 100 101

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

16. Set up for definitive test – example 2 low = 101 µg/L high = 103

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. Normal distribution # Responding Log Dose Dose 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

19. Difficult to evaluate a curved line Conversion to a straight line would make evaluation easier Log Dose Log Dose Converting a curvilinear line to straight line Cumulative distribution Probit transformed % Mortality 0 50 100% Probit Units 3 5 7 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% -2 -1 0 1 2 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% 3 4 5 6 7 Probits

23. Example probit analysis 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 1 3 10 30 100 Concentration of Hg (mg/L)

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

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. MATC MATC = √NOEC + LOEC

32. Results of Analysis of Variance test * * * Community Respiration (gC/L/d.) C 1 3 10 30 100 Concentration of Hg (mg/L)

33. If there is magic on earth, it is in water Photo by R. Grippo

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