• 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

replace

d 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

– Planaria (

Dugesia tigrina

) – Copepods (

Acartia

sp.) 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:

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 B A+B chemical A 0.1 ug L -1 0.1 ug L -1 chemical B 5 ug L -1 5 ug L -1 % mortality 12% 15% 56%

A B • Potentiation – The effect of chemical A is increase by the presence of chemical B, where chemical B has not measurable effect by itself A+B chemical A 0.1 ug L -1 0.1 ug L -1 chemical B 5 ug L -1 5 ug L -1 % mortality 15 % 0 % 35 %