MEASUREMENT OF TOXICITY By, Dr. M. David Department of Zoology, Karnatak University Dharwad

MEASURING TOXICITY Animal Testing – Most commonly used and widely accepted toxicity test is to expose a population of laboratory animals to measured doses of specific toxins. Sensitivity differences pose a problem. – Dose Response Curves – LD50 - Dose at which 50% of the test population dies. (lethal dose of 50% of the population)

Population Sensitivity Variations Probable variations in sensitivity to the toxin within a population. Some members of a population may be very sensitive to a given toxin, while others are much less sensitive. The majority of the population falls somewhere between the two extremities

Cumulative population response to increasing doses of a toxin. The LD 50 is the dose that is lethal to half of the population

Three possible dose response curves at low doses (a). Some individuals respond, even at zero dose, indicating that some other factors must be involved. (b). Response is linear down to the lowest possible dose ©. Threshold must be paused before any response is seen.

Acute vs. Chronic Effects Acute Effects - Caused by a single exposure and result in an immediate health problem. Chronic Effects - Long-lasting. Can be result of single large dose or repeated smaller doses. – Very difficult to assess specific health effects due to other factors

Test Types Acute and Short-term Chronic Tests – Static non-renewal – Static renewal – Flow through Test Species dependent Use dependent

Acute Toxicity Tests Test Procedures – 96 hours or less (species specific) – Mortality is the measured endpoint – For daphnia mortality determined by immobilization Advantages – less expensive and time consuming than chronic – endpoint is easy to quantify Disadvantages – indicates only lethal concentrations – only the effects of fast acting chemicals are exhibited

Acute Test Acceptability Criteria Minimum control survival at least 90% Temperature 20 +/- 1 o C Maximum test organism age at start: – 14 days for fish – 5 days for shrimp – 24 hours for Daphnids

Short-term Chronic Toxicity Tests Test Procedures – typically 4-10 days – Mortality, growth, fecundity, reproduction Advantages – more sensitive than acute, assess parameters other than lethality – may better reflect real world Limitations – more costly and time intensive than acute – more sensitive to low level contamination

Chronic Test Acceptability Criteria Minimum control survival 80% Minimum control dry weight (average): – 0.25 mg for fish – 0.20 mg for Mysid shrimp Minimum of 15 young (average) for control C. dubia Temperature 25 +/- 1 o C Maximum test organism age at start: – 48 hours for fish – 7 days for Mysid shrimp – 24 hours for daphnids

Factors affecting Toxicity of Chemical Toxicity of various chemicals are under the influence of number of factors which are as follows; – Duration of exposure and concentration of chemicals. – Species – Environmental factors – Nature of toxicants

Duration of exposure and concentration of chemicals First a chemical must come in contact with the receptors of organisms at an adequate concentration and duration in order to react and to cause adverse effect. That is the concentration of chemicals and duration of exposure are two important factors to have a toxic effect on organism

Species Susceptibility to toxicant varies from species to species. Within the species, the size, age, sex, food, physiological condition, health etc. influence the toxicity of chemicals. – Eg; small sized individuals, immature young ones, ill healthy organisms and females are more prone to toxicants.

Environmental factors. Many biotic and abiotic factors affect the toxicity of chemicals. Biotic factors include; – Population density – Competition etc Abiotic factors; Temperature pH Salinity etc.

Nature of toxicant The toxicity of chemicals is also influenced by the composition and physicochemical properties of the toxicant as well as there mode of administration and nature of activity.

Evolution of toxicity Today both developing and developed countries face ecological and toxicological problems due to indiscriminate release of pollutant in to the environment. Toxicity is nothing but a chemicals potency to cause an adverse impact on living organism and is a dose and duration dependent.

In evaluating the toxicity of pollutants the following terms are used. – Acute toxicity : a short term exposure of organism (up to 96 hrs) to relatively high doses of a substance.

– Chronic toxicity; long term exposure of organism to the pollutants of relatively low doses. – Lethal concentration (LC); a concentration of a pollutant which will cause mortality in a particular proportion of the experimental animal. Eg; LC 50 of 24, 48, 72 & 96 Hrs.

– Lethal dose; the dose which will cause mortality in a particular proportion of the experimental animal. Eg; LD 50 for 24, 48, 72 & 96 hrs – Lethal time; the time required to kill the organisms at certain dose/ concentration and time. LT 50

Effective concentration; the concentration which will cause a desired effect, usually sub lethal in a particular proportion of test animals. – EC 50 for 24, 48, 72 & 96 hrs.

Effective dose; the dose of the toxicants which will cause mortality in a particular proportion and exposure time – ED 50 for 24, 48, 72 & 96 hrs

Knockdown dose; (KD), the dose which will cause the knockdown of animals in a particular proportion and exposure time. – KD 50 for 24, 48, 72 & 96 hrs.

Knockdown time; (KT), the time required to cause knockdown of animals in a particular proportions and exposure time. – KT 50 for 24, 48, 72 & 96 hrs.

Inhibiting concentration; (IC), the concentration of the toxicant which will cause inhibition of biological function in a specific percentage. Medium tolerance limit (TLM); the concentration at which 50% of test animals survive for a specific exposure time.

Static bioassay Renewal bioassay Flow through bioassay Synergism; the combined effect of substance is greater than the individual effect of the toxicants.

Antagonism; is the induction of an effect in which one toxicant will induce the effect of another, in a mixture of pollutants.

RISK ASSESSMENT Factors influencing risk perception: – Rating risks based on agendas. – Most people have trouble with statistics. – Personal experiences can be misleading. – We have an exaggerated view of our abilities to control our fate. – News media sensationalizes rare events. – Irrational fears lead to overestimation of certain dangers. Fear of the unknown.

Accepting Risks Most people will tolerate a higher probability of occurrence of an event if the harm caused by that event is low. – Harm of greater severity is acceptable only at low levels of frequency. EPA generally assumes 1 in 1 million is acceptable risk for environmental hazards.

ESTABLISHING PUBLIC POLICY It is difficult to separate the effects of multiple hazards and evaluate their risks accurately, especially when exposures are near the threshold of measurement and response. – May not be reasonable to mandate protection, no matter how small the risk, from every potentially harmful contaminant in our environment. – EPA’s standards for toxic exposure does not take into account the natural env.

In setting standards for environmental toxins, we need to consider: – 1) combined effects of exposure to many different sources of damage – 2) different sensitivities of members of the population – 3) effects of chronic and acute exposure