Toxicity testing. Introduction

Transcription

1 Toxicity testing Lab. Objective: To familiarize the student with the concepts and techniques of bioassay utilized in toxicity testing. To introduce students to the calculation of LC 5 values. Toxicity testing involves exposing test organisms for chemical substance/s in order to answer specific questions: Is the substance lethal to test organisms and at what concentration? What are the effects on organisms exposed to low, sublethal concentrations during part or all of its life cycle? Which chemical is most toxic? Which organism is most sensitive? What is the safe concentration of the chemical? Introduction Toxicity tests are an important component for assessing the impact of chemicals on aquatic ecosystems because they indicate toxic effects of single or complex chemical mixtures. In aquatic toxicity tests, groups of selected organisms are exposed to test materials under defined conditions to determine potential adverse effects. A number of standardized toxicity test protocols have been developed for determining toxicity of chemicals to aquatic species. Detailed guidance manuals for marine and freshwater toxicity tests are available from the United States Environmental Protection Agency (U.S. EPA) and other entities such as the American Society for Testing and Materials (ASTM). These protocols provide guidance on application of toxicity tests for assessing toxicity of single chemicals, complex effluents, and ambient samples of water or sediment. The evaluation of acute lethal toxicity in marine animals represents a prior step for studying sublethal effects in the species under study. The median lethal concentration (LC5) is one of the parameters more commonly employed to refer to the acute lethal toxicity of pollutants. From an ecotoxicological point of view, this parameter is often estimated in the more sensitive species and/or stages of a particular aquatic community, and is a potential tool to establish safe concentrations of pollutants in the environment. The LC 5 can be interpolated from the curve by drawing a horizontal line from the 5% mortality point on the y-axis to the concentration response curve and then drawing a line from the point of intersection with the curve to the x-axis. The vertical line intersects the x-axis at the LC 5 value. Although the LC 5 under a defined set of environmental conditions can provide useful information, the numeric value is not necessarily equivalent to acute toxicity in the field. Lethality is only one endpoint in defining acute toxicity. Defining acute toxicity based only on the numeric value of the LC 5 is not appropriate; however, it is widely used in the early phases of most risk assessment.

2 Important definitions Toxicology is the study of the adverse effects of chemicals on humans i.e. focusing on human health. Ecotoxicology is the study of effects of chemical pollutants on natural biota i.e. focusing on free-living flora and fauna and their populations, communities, and supporting ecosystems. Toxicity test a determination of the effect of a material on a group of selected organisms under defined conditions. An aquatic toxicity test usually measures the proportions of organisms affected by their exposure to specific concentrations of chemical, effluent, or receiving water. Static toxicity test - A toxicity test without flowing (exchange) water. Endpoint - A response in a toxicity test such as lethality, growth or behavioral change. Acute toxicity test - A test to determine the short-term toxicity (usually about 4 days) of a chemical or material to an organism. The test usually measures lethality. Lethal means causing death by direct action. Death of organism is defined as the cessation of all visible signs of movement or other activity; no response for gentle probing. LC 5 - The median lethal concentration or the concentration of a substance that kills 5 percent of the organisms tested in a laboratory toxicity test of specified duration (e.g., 96 hour LC 5 ).. Control a treatment in an investigation or study duplicating all conditions of the exposure treatment(s), but must contain no test material. The control is used to determine the absence of measurable toxicity due to basic test conditions (e.g., quality of the control/dilution water, health or handling of test organisms). One of several identical experiments, procedures, or samples. Duplicate is a special case of replicates consisting of two samples or measurements. EC5 - Effect concentration at which 5 percent of the test organisms elicit the defined response (usually a sublethal response). NOEC - No Observable Effect Concentration; in toxicity test, the highest toxicant concentration (resulting in no statistically significant difference from the control in any of the toxicity test endpoints.

3 Materials and Method Test organism: Ask your instructor. Test substances: Ask your instructor Stock solutions: Prepare stock solutions of, mg/l each by dissolving gm of the test substance in ml of distilled water. If the substance does no properly dissolve in water, use a specific volume of appropriate solvent first, and then adjust the volume to ml by distilled water. Procedure. Find the proper range of concentrations. Conduct an initial test with -fold dilutions.. Low Concentration is the Concentration in the original range-finding test that produced mortality closest to. High Concentration is the Concentration that caused mortality closest to %.. Make serial dilutions of the test toxic substance. All reagents and solutions in the laboratory areas shall be labeled to indicate identity, concentration, storage requirements, and expiration date. Deteriorated or outdated reagents and solutions shall not be used 4. Transfer a number of test organism into each beaker. Higher numbers are of course better but as few as (or more) per concentration group can be used. All Organisms used in a test should be of the same age and from the same source. Organisms of the same species from different sources may produce different test results. 5. Start with the control (% toxicant) and end with the highest toxicant concentration. 6. Set up each concentration group in triplicate (or more). 7. Always include a control group that is treated in an identical manner except that they receive no toxic substance. Do not feed the test organisms during acute toxicity tests or for a minimum of 48 hours before the test. If they fed less than 48 hours before test initiation, fecal matter and uneaten food may decrease the dissolved oxygen concentration and otherwise affect the biological activity of some toxicants. 8. The test begins when test organism are first exposed to the toxicant and ends 96 hours later. Control test chambers must show 9 percent survival or greater for test acceptance or validity. 9. Observe the test organisms in the test and control chambers periodically during the test.. For each group, count the number of dead and live organism, including the control group every 4 hours after starting the test.. Remove dead organisms as soon as they are observed. The criterion for death is lack of movement and lack of reaction to gentle probing.. The test is terminated on day 4 at the same hour of the day that the test was begun.

4 . Use Abbot's Formula, to find the corrected percentage mortality of organism in each group (% alive control % alive treated ) Corrected % Mortality = X% (% alive control) Plot the concentration and corrected % mortality and determine the LC5 for the toxicant on organism, and comment on the toxicity of the organism (for example at which concentrations it was more or less toxic? Should more testing be done,or we had done enough, etc?). 4. Use the mortality data collected during the test to calculate a 96 h LC5 value. The 4, 48, and 7 h values should also be calculated whenever there is sufficient mortality data to determine such values. 5. Calculate the mean number of deaths every 4 h at each concentration and write down your results in the following tables. a. 4 hour toxicity test results represents Corrected % Mortality b. 48 hour cumulative toxicity test results represents Corrected % Mortality c. 7 hour cumulative toxicity test results represents Corrected % Mortality

5 d. 96 hour cumulative toxicity test results represents Corrected % Mortality Analyze Toxicity Data (Example) The completed chart for each time interval should look something like the example below. Use the 96 h mortality test results in the next table to calculate the 96 h LC 5 Concentration (mg/l) cumulative Corrected % Mortality Chart this on an x-y scatter plot (x-axis conc, y-axis percent deaths).. Make a normal scatter chart Series 5

6 . Double click the X-axis to change the maximum value to, and check the logarithmic scale box. Click ok. Series 4. Adjust the Y-axis scale to be maximum, major, and minor Right click on the dots and select add trendline. 5. From Type window select Logarithmic. 6. From Options window check both Display equation on chart, and Display R- squared value on chart boxes, and click Ok. 7. Delete the series box y = 4.78Ln(x) R =.994 6

7 8. Select Chart from the main menu and then select chart options, under Value (x) axis check minor gridline box, and Ok. This will result in figure as the same as lower graph picture. From which you can easily calculate the LC5 value. In this case 4.5 mg/l y = 4.78Ln(x) R =.994 Now we can calculate the LC5 from the equation y = 4.78Ln(x) y = 4.78Ln(x) (for LC 5 ) = 4.78Ln(x) = 4.78Ln(x) 4.78Ln(x)= Ln(x)=54.899/4.78 = Since Ln (a)=b is equivalent to a=e b, thus x = e = 4.45 Laboratory Report Each student is expected to write a laboratory report. Lab report should include the following: o a: Introduction o b: Material and Methods o c: Result o d: Discussion o e: Bibliography Do not retype the handout. In your introduction, refer to websites or other sources. Be sure to cite any sources and include them in the bibliography. Write the objective of this experiment in your own words. 7