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)

All above standardized protocols available from US EPA, Federal Register and researchers that developed the programs

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

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

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

Acute vs. Chronic Toxicity TestsCan 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)

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”

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!

Acute Toxicity Test Organisms Use of test species

based on Lab hardiness Common Known life cycle Cheap Short-lived

Normal distribution of resistance/sensitivity

Fre

quen

cy

5% allowable impact

0

100

Mean response

Protected

Experimental design for toxicity testsPe

rcen

t mor

tali

ty

Log [X] Log [X]

Integration of

Freg

. of

res

pons

e (i

.e d

eath

)

Looking for this area of response

To save money while finding area of mean response use a two step process

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

Screening test

100%30% 100%

% R

espo

ndin

g

[X] mg/L

0

100

# dead none none some all RIP all RIP

0 0

Concen. 10-3 10-2 10-1 100 101

Step 2 – Definitive testFrom previous results

low = 10-2 = 0.01 mg/Lhigh = 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

Set up for definitive test – example 1

Treatment Division Concentration (mg/L)

1 10-2 0.01

2 10-1.5 0.032

3 10-1 0.1

4 10-0.5 0.32

5 100 1.0

Control 0.0

Set up for definitive test – example 2low = 101 µg/Lhigh = 103

Treatment Division Concentration

(µg/L)

1 103 1000

2 102.5 316

3 102 100

4 101.5 31

5 101 10

control 0

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

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

# R

espo

ndin

g

Normal distribution

Log Dose

Cumulative distribution

%

Mor

talit

y0

5

0

100%

Converting a curvilinear line to straight line

Difficult to evaluate a curved line Conversion to a straight line would make evaluation easier

Log Dose

Prob

it U

nits

3

5

7

Straight line (easier to analyze)LD50,

TLM)

Probit transformed

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

Relationship between normal distribution 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 probits

34.13%

13.6%

2.13%

3 4 5 6 7

Probits

Mean

Example probit analysis

Concentration (mg/L)

Deaths %

Control 0/10 0

0.3 0/10 0

1 0/10 0

3 1/10 10

10 4/10 40

30 9/10 90

100 10/10 100

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)

Uses of LC50

1. 1. Application factor LC50 x n = ___ = allowable dose Good if do not have better information (chronic tests)

2. Rank hazards lower LC50 = more toxic3. 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

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

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)

Results of Analysis of Variance test

C 1 3 10 30 100

Com

mun

ity

Res

pira

tion

(gC

/L/d

.) *

**

Concentration of Hg (mg/L)

Determination of EC20

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 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.

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”

MATC

MATC = √NOEC + LOEC

Results of Analysis of Variance test

C 1 3 10 30 100

Com

mun

ity

Res

pira

tion

(gC

/L/d

.) *

**

Concentration of Hg (mg/L)

Photo by R. Grippo

If there is magic on earth, it is in water