• Why are standard test methods important ?
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Results are comparable between different labs
Results can be reproduced by other labs
Data can be compiled from the literature and comparisons drawn
Provides criteria for decision making
Logistically simplified – hire technicians that can perform many
assays with little training
Standard ASTM (American Society for Testing of Materials) and
EPA methods handbooks
Methods can be critically investigated and changed based on
best available science
Provides guidelines on how to collect data and perform statistical
analysis
Allows you estimate cost and required personal
Methods can be modified in ways to test specific hypotheses
about xenobiotics
• Single species Toxicity tests
– Most commonly used is the
Daphnia magna or Daphnia pulex
48-hour acute toxicity test
– For chromic tests a 21-day
timeframe is used with Daphina
– Daphnids are ‘Cladoceran’ waster
fleas
– 1-2 mm long
– Over 100 species known
– Fresh water
– Easy to culture
– Require hard water
• Water quality is a major factor in performing the
Daphnia acute toxicity test.
– other sources of mortality
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Chlorine
Chlorinated organics
Heavy metals
Organics from groundwater (if using well water)
– 40 to 50 mg/L CaCO3 is recommended for D. pulex ;
twice that for D. magna
• Test includes both positive and a negative control
– Sodium pentachlorophenate (NaPCP) = positive control
• Animal care considerations
– Strict guidelines for humane treatment of animals by government
agencies – National Institutes of Health
– More regulated for vertebrates than for invertebrates
– Research is reviewed by ‘Animal Use Committees’
– Strict protocols
– Efficient use of laboratory animals i.e. use as few animals as possible to
collect as much data as possible
– Reduce pain and suffering
– If possible the test should be replaced with an alternative methodology
e.g. tissue culture, QSAR
– Reducing the power of the statistical test slightly can dramatically
decrease the number of animals needed
– Refining the method e.g. using biochemical stress indicators
• Multi-species tests
– At least two or more interacting species
– Assumes that an ecosystem is more than the sum of the parts
– Emphasizes environmental heterogeneity i.e. the goal of the
experiment is to reduce this heterogeneity for hypothesis testing
(field test would be the alternative)
– Volumes can be as little as a one liter for bacterial communities
to 800 liter or even larger
– Controversial because of small scale and low diversity in the
system – does not represent real systems
– Two types
• Aquatic mesocosms
• Terrestrial mesocosms
• Standard Aquatic Microcosm
– Developed by Frieda Taub and her colleagues
• Multi species chronic toxicity test
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63 days
10 algae
4 invertebrates
1 bacterial species
3 liters of defined media in 4 liter jar + sand + chitin
• Effect on respiration as well as primary production
(photosynthetic rate). Measurements are taken every three
days :
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pH
Dissolved oxygen
Optical density
Nutrient levels
Count live animals and algae
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Common species used in toxicity testing:
Fish
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Algae
Coho salmon
Rainbow trout
Brook trout
Gold fish
Fathead Minnow
Channel Catfish
Bluegill
Green sunfish
Invertebrates
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Daphnids
Amphipods (Gammarus)
Crayfish
Stoneflies
Mayflies
Midges
Snails (Physa sp., Amnicola sp.)
Planaria (Dugesia tigrina)
Copepods (Acartia sp.)
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Chlamydomonas reinhardi
Ulothrix sp.
Microcystis
Anabaena
Avian species
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Mallard
Northern bobwhite
Ring-necked pheasant
• Factors influencing the activity of toxicants
– There are many pollutants in the environment
– Their toxicity is influenced by
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Partitioning, fate, and transport
physiochemical properties
mode of exposure
Time
Environmental factors
Interaction
Biological factors
Nutrition, starvation
Genetics
Proteins (Mixed Functional Oxidases)
Lipids
vitamins
Sex / effect on males vs. females
Disease
Behavior
Chemodynamics
Bio-availabiltiy
• The physiochemical properties of a pollutant determine
it’s fate and transport in the environment
– Is the pollutant solid, liquid, gaseous ?
• does it evaporate, dissolve in ground water, or stick to particles ?
– Water solubility ?
• What concentration does it reach in solution ? Toxicity is
concentration dependent e.g. carbon monoxide
– Organic for inorganic ?
• Can it be mineralized by bacteria ? radionuclide ?
– Ionic or neutral ?
• Membrane permeability
Fate and transport of a xenobiotic chemical - an example:
• Trichloroethylene (TCE)
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Colorless, odorless, and sweet tasting
Degreaser
Paints, adhesives, paint and spot removers
Non-flammable
Dissolves little in water i.e. goes mostly to air through
evaporation
– Can be trapped in soil / soil particles
– found in water attached to particles
Health effects:
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Headaches, breathing problems, dizziness
Nerve, kidney, and liver damage
Birth defects
Skin rashes
cancer
•If emitted to air, TCE will end up mostly in the gaseous phase (atmosphere)
Distribution would be Air > Water > Soil
•This would be very different, if TCE was leaking from a buried drum, for example.
In this case the distribution would end up being Soil > Water > Air
• Bioavailability
– Is the chemical in a toxic or inert form ?
– For example, total mercury in sediment does not correlate with
toxicity / concentration of mercury in midge larvae
– Bioavailability of mercury is controlled by mercury oxidation
state, whether it is complexed with methyl groups, and pH
• Another example:
CHLORDANE
– hydrophobic pesticide
– Partitions into DOC (dissolved organic carbon)
– Solubility of chlordane in groundwater is increased 5x by
the presence of DOC
– But, the mobilized fraction has little effect on soil
microorganisms
• Synergism
– The effect of chemical A in the presence of chemical B is greater
than the sum of their individual effects
(96 hours)
A
chemical A
0.1 ug L-1
chemical B
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% mortality
12%
B
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5 ug L-1
15%
A+B
0.1 ug L-1
5 ug L-1
56%
• Potentiation
– The effect of chemical A is increase by the presence of chemical
B, where chemical B has not measurable effect by itself
A
chemical A
0.1 ug L-1
chemical B
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% mortality
15 %
B
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5 ug L-1
0%
A+B
0.1 ug L-1
5 ug L-1
35 %