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) – Endpoints are reproduction (brood size) physiology, behavior, biochemistry –More ecologically relevant

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)

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

Ecosystem Tests ( microcosms, mesocosms) AOV design (4 reps X 3 treat., 3 rep X 4) Time = 1 – 2 years $10 6 /year Endpoints are –Biomass –Diversity –Species richness –Etc.

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

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

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

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

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 LC 50 because probit transformation will straighten the cumulative distribution line

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

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

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

Example problem Dose % alive 0 mg/L (control)100 0 mg/L (control)

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

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Today’s laboratory exercise Acute 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

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 C 1 V 1 = C 2 V 2 10 mg/L changes 100 ml 10 mg/L changes 100 ml Where C 1 = concentration of stock solution V 1 = volume needed of stock solution for ea. concentration V 1 = volume needed of stock solution for ea. concentration C 2 = desired concentration of test solution C 2 = desired concentration of test solution V 2 = final volume needed in test chamber V 2 = final volume needed in test chamber

To make up solutions in cups 1. Put correct volume of toxicant into graduated cylinder 2. Add MH water to ~98-99 ml 3. Top off with squirt bottle of MH water (bottom of meniscus) 4. Pour into plastic cup 5. Start with lowest concentration 1 st, work up to highest (why?) 6. Rinse graduated cylinder with MH water when done

To count out Daphnia 1. Work on light table, easier to see. 2. Arrange 6 small plastic sample cups. 3. Put 2-3 ml in each sample cup (large drop). 4. Suck up Daphnia from brood container (2-3 at a time, neonates only). 5. 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 6. Put 2-3 in 1 st cup, 2-3 in next cup, until all cups have 10  random distribution will prevent putting all easiest to capture in 1 st cup, next easiest all in 2 nd cup, etc 7. After have 10 in ea. Cup and test chambers are ready, carefully lower cup into test chamber and release animals under water. 8. Record # dead/alive 24 and 48 hrs

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