Acute and Chronic Toxicity Testing

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Acute and Chronic

Toxicity Testing

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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

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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

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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

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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)

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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”

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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!

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Acute Toxicity Test Organisms

 Use of test species based on

 Lab hardiness

 Common

 Known life cycle

 Cheap

 Short-lived

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Normal distribution of resistance/sensitivity

Frequency

5% allowable impact

0 100

Mean response

Protected

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Experimental design for toxicity tests

Percent mortality

Log [X] Log [X]

Integration of

Freg. of response (i.e death)

Looking for this area of

response

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

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Step 1 – Screening test



Expose 5–10 organisms to 10

^x

increasing [ ] for 24-96 hours



Trying to determine range in which median

lethal concentration (LC

₅₀

) will fall

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Screening test

100%

30% 100%

% Responding

[X] mg/L

0 100

# dead none none some all RIP all RIP

0 0

Concen. 10-3 10^-2 10^-1 10⁰ 10¹

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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

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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

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Set up for definitive test – example 2

low = 10¹ µg/L high = 10³

Treatment Division Concentration (µg/L)

1 10³ 1000

2 10^2.5 316

3 10² 100

4 10^1.5 31

5 10¹ 10

control 0

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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 LC₅₀because probit transformation will straighten the cumulative distribution line

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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

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Log Dose

Cumulative distribution

% Mortality 0 50 100%

Converting a curvilinear line to straight line

 Difficult to evaluate a curved line

 Conversion to a straight line would make evaluation easier

Log Dose Probit Units 3 5 7

Straight line (easier to analyze)

LD₅₀, TLM)

Probit transformed

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Note: probit forces data towards middle of

distribution  good because most organisms

are “average” in their response

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Relationship between normal distribution and standard deviations

34.13%

13.6%

2.13%

-2 -1 0 1 2 Standard deviations

Mean

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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

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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)

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Uses of LC

₅₀

1. 1. Application factor

 LC₅₀ x n = ___ = allowable dose

 Good if do not have better information (chronic tests)

2. Rank hazards  lower LC₅₀ = more toxic

3. 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

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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

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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)

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Results of Analysis of Variance test

C 1 3 10 30 100 Community Respiration (gC/L/d.)

*

* *

Concentration of Hg (mg/L)

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Determination of EC

₂₀

10 μg

8 μg

Control EC₂₀ eg. 1 mg/L = discharge limit

Response (growth) ^Control_response

20% reduction relative to control

Dose

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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.

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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”

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NOEC LOEC

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MATC

MATC = √NOEC + LOEC

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