• Why are standard test methods important ?

– 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

• 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– 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 :

– pH– Dissolved oxygen– Optical density– Nutrient levels– Count live animals and algae

• Common species used in toxicity testing:

Fish

– Coho salmon– Rainbow trout– Brook trout– Gold fish – Fathead Minnow – Channel Catfish– Bluegill– Green sunfish

Invertebrates

– Daphnids– Amphipods (Gammarus)– Crayfish– Stoneflies– Mayflies– Midges– Snails (Physa sp., Amnicola sp.)– Planaria (Dugesia tigrina)– Copepods (Acartia sp.)

Algae

– Chlamydomonas reinhardi

– Ulothrix sp.

– Microcystis

– Anabaena

Avian species

– Mallard– Northern bobwhite– Ring-necked pheasant

• Factors influencing the activity of toxicants

– There are many pollutants in the environment – Their toxicity is influenced by

• 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)

– 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:

– 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:

– 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

CHLORDANE

• Synergism

– The effect of chemical A in the presence of chemical B is greater than the sum of their individual effects

(96 hours) chemical A chemical B % mortality

A 0.1 ug L-1 - 12%

B - 5 ug L-1 15%

A+B 0.1 ug L-1 5 ug L-1 56%

chemical A chemical B % mortality

A 0.1 ug L-1 - 15 %

B - 5 ug L-1 0 %

A+B 0.1 ug L-1 5 ug L-1 35 %

• Potentiation

– The effect of chemical A is increase by the presence of chemical B, where chemical B has not measurable effect by itself