Toxicity Testing II Toxicity Testing II PPerforming a toxicity testerforming a toxicity test

Toxicity testingToxicity testing

Acute toxicity test Acute toxicity test – Short time frame exposure (96h)Short time frame exposure (96h)– ““kill ‘em and count ‘em”kill ‘em and count ‘em”– LCLC5050, TLM (median tolerance limit, TLM (median tolerance limit– ECEC5050 (effective concentration) (effective concentration)

Chronic toxicity test Chronic toxicity test – Longer time frame exposure (1 week to 3 years)Longer time frame exposure (1 week to 3 years)– Endpoints are reproduction (brood size) Endpoints are reproduction (brood size)

physiology, behavior, biochemistryphysiology, behavior, biochemistry– More ecologically relevant More ecologically relevant

AnalysisAnalysis

Analysis of Variance (hypothesis testing)Analysis of Variance (hypothesis testing)– Test for significant difference from control (C Test for significant difference from control (C

+ 5 doses) + 5 doses)

Regression analysisRegression analysis– EC20 (concentration that causes 20% EC20 (concentration that causes 20%

reduction relative to control)reduction relative to control)

Determination of ECDetermination of EC2020

10 μg

8 μg

Control EC20 eg. 1 mg/L = discharge limit

Res

pons

e (g

row

th) Control

response

20% reduction relative to control

Dose

Ecosystem TestsEcosystem Tests((microcosms, mesocosms)microcosms, mesocosms)

AOV design (4 reps X 3 treat., 3 rep X 4)AOV design (4 reps X 3 treat., 3 rep X 4)

Time = 1 – 2 yearsTime = 1 – 2 years

$10$1066 /year /year

Endpoints areEndpoints are– BiomassBiomass– DiversityDiversity– Species richnessSpecies richness– Etc.Etc.

Probit AnalysisProbit Analysis

Response of organisms to toxic chemicals = normal Response of organisms to toxic chemicals = normal distributiondistribution

Cannot measure normal distribution directly because effect is Cannot measure normal distribution directly because effect is cumulative, so graph as cumulative distributioncumulative, so graph as cumulative distribution

Log Dose

Cumulative distribution

Dose

# R

espo

ndin

g

Normal distribution

Log Dose

Cumulative distribution

# R

espo

ndin

gDifficult to evaluate a curved lineDifficult to evaluate a curved line

Conversion to a straight line would make Conversion to a straight line would make evaluation easierevaluation easier

Relationship between normal Relationship between normal distribution and standard deviationsdistribution and standard deviations

34.13%

13.6%

2.13%

-2 -1 0 1 2

Standard deviations

Mean

Difficult to deal with SD (34.13, 13.6, etc) so rename SD to probitsDifficult to deal with SD (34.13, 13.6, etc) so rename SD to probits

34.13%

13.6%

2.13%

3 4 5 6 7

Probits

Mean

ProbitsProbits

Based on SD so each probit has a Based on SD so each probit has a percentage attached to itpercentage attached to it

Mean response defined as probit = 5 so all Mean response defined as probit = 5 so all probits are positive probits are positive easier to visualize easier to visualize

Can use probit analysis to calculate LCCan use probit analysis to calculate LC50 50

because probit transformation will because probit transformation will straighten the cumulative distribution linestraighten the cumulative distribution line

Log Dose

Cumulative distribution

# R

espo

ndin

gCumulative distribution is curvilinearCumulative distribution is curvilinearProbit transformation straightens the Probit transformation straightens the cumulative distribution linecumulative distribution line

Easier to calculate LCEasier to calculate LC5050

Log Dose

Probit transformed

LC50

50% response

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

Aquatic toxicity testingAquatic toxicity testing

Daphnia (water flea)

“White rat of aquatic toxicity testing”

Daphnia toxicity test

Example problemExample problem

DoseDose % alive% alive

0 mg/L (control)0 mg/L (control) 100100

1 1 100100

33 90 90

1010 30 30

30 30 20 20

100100 0 0

Photo by R. Grippo

If there is magic on earth, it is in water

This slide purposely left blankThis slide purposely left blank

Today’s laboratory exerciseToday’s laboratory exerciseAcute toxicity test – Acute toxicity test – Daphnia pulexDaphnia pulex

Use neonates (<24 h old)Use neonates (<24 h old)

All females (parthenogenetic)All females (parthenogenetic)

Class will break up into 3 groups of 2Class will break up into 3 groups of 2

1 person in group will calculate dilutions, 1 person in group will calculate dilutions, other will select neonate other will select neonate DaphniaDaphnia

Calculation of DilutionsCalculation of Dilutions

Have stock solutions at a given concentration (10 mg/L Zn, 1 mg/L Have stock solutions at a given concentration (10 mg/L Zn, 1 mg/L Cu, 100% effluent)Cu, 100% effluent)Want to make 100 ml test solutions at correct concentrationsWant to make 100 ml test solutions at correct concentrations

Use Use CC1 1 VV11 = C = C2 2 VV22

10 mg/L changes 100 ml10 mg/L changes 100 ml

Where Where CC1 1 = = concentration of stock solutionconcentration of stock solution

VV11 = = volume needed of stock solution for ea. concentrationvolume needed of stock solution for ea. concentration

CC2 2 = = desired concentration of test solutiondesired concentration of test solution

VV2 2 = = final volume needed in test chamberfinal volume needed in test chamber

To make up solutions in cupsTo make up solutions in cups

1.1. Put correct volume of toxicant into graduated cylinderPut correct volume of toxicant into graduated cylinder

2.2. Add MH water to Add MH water to ~98-99 ml~98-99 ml

3.3. Top off with squirt bottle of MH water (bottom of meniscus)Top off with squirt bottle of MH water (bottom of meniscus)

4.4. Pour into plastic cupPour into plastic cup

5.5. Start with lowest concentration 1Start with lowest concentration 1stst, work up to highest (why?), work up to highest (why?)

6.6. Rinse graduated cylinder with MH water when doneRinse graduated cylinder with MH water when done

To count out To count out DaphniaDaphnia

1.1. Work on light table, easier to see.Work on light table, easier to see.2.2. Arrange 6 small plastic sample cups.Arrange 6 small plastic sample cups.3.3. Put 2-3 ml in each sample cup (large drop).Put 2-3 ml in each sample cup (large drop).4.4. Suck up Suck up Daphnia Daphnia from brood container (2-3 at a time, neonates from brood container (2-3 at a time, neonates

only).only).5.5. Express Express DaphniaDaphnia into large drop. into large drop.Note: take care to release Daphnia under water Note: take care to release Daphnia under water if just drop in may if just drop in may

get caught in surface tension and dieget caught in surface tension and die6.6. Put 2-3 in 1Put 2-3 in 1stst cup, 2-3 in next cup, until all cups have 10 cup, 2-3 in next cup, until all cups have 10

random distribution will prevent putting all easiest to capture in 1random distribution will prevent putting all easiest to capture in 1stst cup, next easiest all in 2cup, next easiest all in 2ndnd cup, etc cup, etc

7.7. After have 10 in ea. Cup and test chambers are ready, carefully After have 10 in ea. Cup and test chambers are ready, carefully lower cup into test chamber and release animals lower cup into test chamber and release animals under water.under water.

8.8. Record # dead/alive 24 and 48 hrsRecord # dead/alive 24 and 48 hrs

Estimate LC50Estimate LC50

If have at least two partial kills If have at least two partial kills use use computer programcomputer program

If one or less partial kills If one or less partial kills use probit use probit paperpaper