• Dose response relationships

– A graph describing the response of an organism, population, or biological community to a range of concentrations of a xenobiotic

– Usually shown as a curve or histogram describing enzyme inhibition, DNA damage, death, behavioral changes or any other measurable/quantifiable parameter

• dose ≠ concentration

– Concentration is the relative concentration of a chemical with respect to water/air e.g. concentration of DDE in Lake Apopca water

– Dose is the amount of a chemical that actually gets into an organism / actually reaches the active site e.g. concentration of DDE in Alligator tissue

• Additional mortality is seen as dose is increased

• The first derivative of this dose response curve is often a normal distribution

– This implies that there is a concentration range that has a greater effect on mortality than the range above and below

– Typically observed for multigenic responses

• Other distributions can be observed. For example a bimodal distributions would result, if the toxicant had a greater effect on male than on females

• There are several parameters that are commonly estimated by graphical or computation means from dose response plots :

– LD50 Dose that is Lethal in 50% of individuals tested;

– LC50 Concentration that is Lethal in 50% of individuals tested; estimated by graphical or computation

means

– EC50 Concentration that has a measurable Effect on 50% of individual tested. This is the

parameter that is used estimate effects that are not lethal.

– IC50 Concentration that Inhibits 50% of a particular process e.g. enzyme activity, growth rate in

bacteria

• How do we rank the toxic effect of chemical ? / How do we compare the toxicity of two chemicals ?

– Run an experiment with both chemicals through the same concentration range and using the same tester species (all else being equal)

– Plot the data together on the same Percent mortality plot

• Slopes of lines are similar• Midpoint (LC50) is different

• Problem with the LD50 method

– Quoting LC50 only can be misleading because the slope of two plots can be very different, but result in the same LC50 (ignores kinetic differences)

– LC50 Experiments are typically done for short periods of time (typically 96 hours). The risk of slow acting chemicals, or chemicals that move into tissue slowly may be underestimated

– chronic exposure

– how to you measure dose ?

• LD50 plots with similar slopes may suggest a similar mode of action

• To describe the effect of chemicals that do not have an immediate lethal effect other terminology is used:

– NOEC No Observed Effect Concentration; determined by statistical hypothesis testing

– NOEL No Observed Effect Level; determined by statistical hypothesis testing; reported as a dose

– NOAEC No Observed Adverse Effect Concentration; chosen for its’ impact on a species

– NOAEL No Observed Adverse Effect Level; chosen for its’ impact on a species – reported as dose

– LOEC Lowest Observed Effect Concentration

– LOEL Lowest Observed Effect Level (dose)

– MTC Minimum Threshold Concentration

– MATC Maximum Allowable Toxicant Concentration

• Threshold concentration :

– concentration below which no effect is observed

– A : no threshold– B : threshold– C : hormetic response

• Over the years test methods have been standardized. Protocols are available from

– American Society for Testing and Materials (ASTM)– Organization for Economic Cooperation and Development

(OECD)– National Toxicology Program (NTP)

• Protocols are available as

– U.S. EPA publications – The federal register– From researchers that pioneer and develop standard

methodology

• Assumptions of standard methods:

– The response observed is due to the chemical administered

– The magnitude of the response is related/proportional to the amount of chemical administered

• Assumes there is a molecular target / receptor for the chemical in an organism

• Assumes that the concentration of the chemical at the receptor site is related to the dose administered

– There is a method for accurately and precisely quantifying the response/toxicity

– Endpoints are known i.e. toxicity assay needs to be run within the range of the toxic effect of a chemical – too little, no effect; too much, 100% mortality at lowest dose

• Advantages of standard methods

– 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 methods handbook

– Methods can be critically investigated and changed based on best available science

– Provides guidelines on how to collect data and perform statistical analysis

• Types of toxicity tests :

type ofstudy

temporal/spatial scale

immediate reproduction Interspeciesinteraction

Communityabiotic factors

ecosystem succession

acutechronic

microcosm

mesocosm

field study

• Types of tests:

– Single species or community– Aquatic

• Usually whole body exposure• Design variable usually relate to test chamber

– Static tests– Static renewal– Recirculation– Continuous or intermittent flow-through

– Terrestrial• Intravenous (mouse, rat, rabbit, cat, dog)• Intromusuclar(“”)• Intraperitoneal(“”)• Gavage (tube into stomach)• Feeding studies• Inhalation• Dermal (rabbit)• Spraying (community/field study)• Soil addition

• Standard test organisms

– Should be widely available– Easily maintained in the lab– History of genetic composition (known mutations)– Must be sensitive to a toxicant– Must be representative of a particular taxonomic class

• Mayer et. Ellersieck 1986

– Examined 5000 end-point tests on 66 species for 410 chemicals – Found that 88% of test on Daphnia, Gammarus, and rainbow trout

provided the lowest indication of toxicity