Acute and Chronic Toxicity Testing

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Presentation transcript:

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 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)

Acute vs chronic toxicity testing (con’t) 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 Mean response 0 100 Frequency Protected 5% allowable impact Resistance (log [X]

Experimental design for toxicity tests Integration of Freg. of response (i.e death) Percent mortality Looking for this area of response Log [X] Log [X] 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 [X] mg/L % Responding 0 100 [X] mg/L # dead none none some all RIP all RIP 30% 100% 100% Concen. 10-3 10-2 10-1 100 101

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

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 2 low = 101 µg/L high = 103 Treatment Division Concentration (µg/L) 1 103 1000 2 102.5 316 3 102 100 4 101.5 31 5 101 10 control

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 Cumulative distribution Dose # Responding Normal distribution Log Dose

Converting a curvilinear line to straight line Difficult to evaluate a curved line Conversion to a straight line would make evaluation easier Cumulative distribution Probit transformed Log Dose Log Dose 0 50 100% % Mortality 3 5 7 Probit Units Straight line (easier to analyze) LD50, TLM)

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% Mean 13.6% 2.13% -2 -1 0 1 2 Standard deviations

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

Example probit analysis Concentration (mg/L) Deaths % Control 0/10 0.3 1 3 1/10 10 4/10 40 30 9/10 90 100 10/10 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. Application factor LC50 x n = ___ = allowable dose Good if do not have better information (chronic tests) Rank hazards  lower LC50 = more toxic 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 * * * Community Respiration (gC/L/d.) C 1 3 10 30 100 Concentration of Hg (mg/L)

Determination of EC20 Response (growth) Control response 10 μg 8 μg 20% reduction relative to control 8 μg Control Dose EC20 eg. 1 mg/L = discharge limit

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”

NOEC LOEC

MATC MATC = √NOEC + LOEC

MATC