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Principles of Bioassay
A biological assay is a procedure involving use of the responses of aquaticorganisms to detect or measure the presence or effect of one or more sub-stances, wastes, or environmental factors, alone or in combination.
Short-term. This type of bioassay reveals in relatively less time (usually 8 daysor less) the relative toxicity of different toxicants to a selected test organism. It shows the relative sensitivity of various organisms to different conditions or variables like temperature and pH. It also determines the median lethal con-centration (LC50), or the effective concentration values.
Intermediate. This bioassay is used when LC50 determination requires addi-tional time (usually 8-90 days) for studies of the life stages of organisms with long life cycles, and to indicate toxicant concentrations for life cycle tests.
Long-term.This bioassay procedure is almost always a flow-through test. Itdetermines the maximum allowable toxicant concentration, or safe concentra-tion, for indicating water quality standards;
Static.The test animals remain in the same test concentration for the durationof the test.
Renewal.This is a static test where the test animals are transferred to a freshtest solution of the same composition at periodic intervals, usually every 24 h.
Re-circulation.This static test involves the circulation of test solution throughtest chambers. The test solution may be treated by aeration, filtration, steriliza-tion or other means to maintain water quality.
Flow-through.Measured quantities of dilution water and the stock toxicant so-lution are mixed and delivered periodically to the test containers to provide continuous flow-through of the test toxicant.
Criteria for selecting and preparing test animals:
Sensitivity to the materials under consideration;
Availability and abundance;
Recreational, economic and ecological importance;
Adaptability to laboratory conditions;
Suitability for bioassay tests;
Originating from a single common source; Uniform in size and of the same stage of maturity;
Healthy, free from disease and parasites;
No previous exposure to heavy metals, pesticides, and other substances;
Acclimation to laboratory conditions for at least 10 days;
During acclimation, provide for daily feeding. Mortality should be less than 10% of the total population.
Experimental water. The water should not be polluted or contaminated withwastes from any source.
Experimental design.There should be a minimum of 5 test concentrations andcontrol in at least duplicate containers.
Test concentrations. Express liquid waste concentrations as percent on a vol-ume to volume basis. Express concentrations of non-aqueous wastes and of individual chemical composition by weight. Indicate whether the LC value is based on concentration of total material or active ingredient.
Test containers. Glass aquaria should be clean and of uniform size and shape.They should measure 15-30 cm deep and should be arranged at random in the testing area. If replicates are used, the series of test containers should be ran-domized separately.
Number of test animals or biomass. (1) There should be at least 10 animals perconcentration. (2) Less than 10 animals may be used for the range finding test. (3) There should be a maximum of 1 g fish/ liter of water. (4) Distribute ani-mals at random by adding one at a time to each container
Preparing test solutions.Test solutions must be freshly prepared. Also avoidunnecessary exposure to air and light.
Feeding.Do not feed for at least 2 days before the test. Do not feed during theentire experimental period for short-term tests (96 h or less).
Biological data and observations. Observe and record mortality at periodic in-tervals. The usual indicator for death is no movement, especially no gill move-ment in fish, and no reaction to gentle prodding. Remove dead organisms as soon as observed.
Report effects using terms like erratic swimming, loss of reflex, discoloration, changes in behavior, excessive mucus production, hyperventilation, opaque eyes, curved spine, and hemorrhaging.
Physical and chemical water quality.Measure temperature, salinity, hardness,alkalinity, pH, dissolved oxygen, ammonia and nitrite at the beginning of the test, and daily thereafter.
Calculation, analysis, and reporting of results. The recommended measures orindices of relative toxicity are 48 and 96 h LC50. Always compute for 95% confidence limits for LC50 and EC50 values.
The most widely used methods for calculating an LC50 and confidence limit are probit, logit, moving average and Lithcfield-Wilcoxon (1949). Other meth-ods are straight line interpolation and the Reed-Muench method (1938).
Report the LC50 with specified exposure time and the confidence limits of LC50.
Provide descriptions of test organisms (species, source, size, weight), proce-dures for acclimation to test conditions and observations on behavior during the test). Describe also the source of experimental water and its characteristics, source and properties of tested material and concentrations of the test solu-tions. Indicate also the experimental temperature, test method, test conditions (type of container with volume and depth of solution, number of organisms), and the criterion of response.
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