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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)

 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
100

Mean response
Frequency

Protected
5% allowable
impact
0

Resistance (log [X]


Experimental design for toxicity tests

Freg. of response (i.e death)


Percent mortality

Integration
of
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
100
Screening test
% Responding
0

[X] mg/L
# dead none none some all RIP all RIP

0 0 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 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
Normal distribution Cumulative distribution
# Responding

Dose 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


100%

7
Probit Units
% Mortality

5
50

Straight line (easier to


LD50, analyze)
TLM) 3
0

Log Dose Log Dose


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 Deaths %
(mg/L)
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 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
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
Respiration (gC/L/d.)

*
Community

* *

C 1 3 10 30 100
Concentration of Hg (mg/L)
Determination of EC20
Control
Response (growth)

response
10 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”
MATC

MATC = √NOEC + LOEC


Results of Analysis of Variance test
Respiration (gC/L/d.)

*
Community

* *

C 1 3 10 30 100
Concentration of Hg (mg/L)
If there is magic on earth, it is in water

Photo by R. Grippo

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