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Acute and Chronic Toxicity Testing

Acute and Chronic 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)

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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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