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Toxicity Testing II P erforming a toxicity test

Toxicity Testing II P erforming a toxicity test. Toxicity testing. Acute toxicity test Short time frame exposure (96h) “kill ‘em and count ‘em” LC 50 , TLM (median tolerance limit EC 50 (effective concentration) Chronic toxicity test Longer time frame exposure (1 week to 3 years)

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Toxicity Testing II P erforming a toxicity test

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  1. Toxicity Testing II Performing a toxicity test

  2. Toxicity testing Acute toxicity test • Short time frame exposure (96h) • “kill ‘em and count ‘em” • LC50, TLM (median tolerance limit • EC50 (effective concentration) Chronic toxicity test • Longer time frame exposure (1 week to 3 years) • Endpoints are reproduction (brood size) physiology, behavior, biochemistry • More ecologically relevant

  3. Analysis • 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)

  4. 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

  5. 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.

  6. 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

  7. Log Dose • Difficult to evaluate a curved line • Conversion to a straight line would make evaluation easier Cumulative distribution # Responding

  8. Relationship between normal distribution and standard deviations 34.13% Mean 13.6% 2.13% -2 -1 0 1 2 Standard deviations

  9. 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

  10. Probits • 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

  11. Cumulative distribution Probit transformed # Responding Log Dose Log Dose • Cumulative distribution is curvilinear • Probit transformation straightens the cumulative distribution line • Easier to calculate LC50 50% response LC50

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

  13. Aquatic toxicity testing Daphnia (water flea) “White rat of aquatic toxicity testing” Daphnia toxicity test

  14. Example problem Dose% alive 0 mg/L (control) 100 1 100 3 90 10 30 30 20 100 0

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

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  17. Today’s laboratory exerciseAcute toxicity test – Daphnia pulex • Use neonates (<24 h old) • All females (parthenogenetic) • Class will break up into 3 groups of 2 • 1 person in group will calculate dilutions, other will select neonate Daphnia

  18. Calculation of Dilutions • Have stock solutions at a given concentration (10 mg/L Zn, 1 mg/L Cu, 100% effluent) • Want to make 100 ml test solutions at correct concentrations • Use C1 V1 = C2 V2 10 mg/L changes 100 ml Where C1 = concentration of stock solution V1 = volume needed of stock solution for ea. concentration C2 = desired concentration of test solution V2 = final volume needed in test chamber

  19. To make up solutions in cups • Put correct volume of toxicant into graduated cylinder • Add MH water to ~98-99 ml • Top off with squirt bottle of MH water (bottom of meniscus) • Pour into plastic cup • Start with lowest concentration 1st, work up to highest (why?) • Rinse graduated cylinder with MH water when done

  20. To count out Daphnia • Work on light table, easier to see. • Arrange 6 small plastic sample cups. • Put 2-3 ml in each sample cup (large drop). • Suck up Daphnia from brood container (2-3 at a time, neonates only). • Express Daphnia into large drop. Note: take care to release Daphnia under water  if just drop in may get caught in surface tension and die • Put 2-3 in 1st cup, 2-3 in next cup, until all cups have 10  random distribution will prevent putting all easiest to capture in 1st cup, next easiest all in 2nd cup, etc • After have 10 in ea. Cup and test chambers are ready, carefully lower cup into test chamber and release animals under water. • Record # dead/alive 24 and 48 hrs

  21. Estimate LC50 • If have at least two partial kills  use computer program • If one or less partial kills  use probit paper

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