1. Marine Biotechnology Lab
Toxicity testing

2. 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.

3. 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?

4. 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).

5. The median lethal concentration (LC50) is one of the parameters more commonly employed to refer to the acute lethal toxicity of pollutants.
The median lethal concentration or the concentration of a substance that kills 50 percent of the organisms tested in a laboratory toxicity test of specified duration (e.g., 96 hour LC50).

6. The LC50 can be interpolated from the curve by drawing a horizontal line from the 50% 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 LC50 value.

7. Materials and Method Test organism. Test substances.
Stock solutions: Prepare stock solutions of 10,000 mg/l each by dissolving 1 gm of the test substance in 100 ml of distilled water.
استخدم مذوب في حالة عدم دوبان المذاب في الماء

8. Make serial dilutions of the test toxic substance.
Procedure
Find the proper range of concentrations. Conduct an initial test with 10-fold dilutions.
Low Concentration is the Concentration in the original range-finding test that produced mortality closest to 0. High Concentration is the Concentration that caused mortality closest to 100%.
Make serial dilutions of the test toxic substance.
الوفيات mortality

9. All Organisms used in a test should be
Transfer a number of test organism into each beaker. Higher numbers are of course better but as few as 10 (or more) per concentration group can be used.
All Organisms used in a test should be
In the same age.
From the same source.
Organisms of the same species from different sources may produce different test results.

10. Start with the control (0% toxicant) and end with the highest toxicant concentration.
Set up each concentration group in triplicate (or more).
The test begins when test organism are first exposed to the toxicant and ends 96 hours later. Control test chambers must show 90 percent survival or greater for test acceptance or validity.
Observe the test organisms in the test and control chambers periodically during the test.

11. Remove dead organisms as soon as they are observed.
For each group, count the number of dead and live organism, including the control group every 24 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.

12. The test is terminated on day 4 at the same hour of the day that the test was begun
Use Abbot's Formula, to find the corrected percentage mortality of organism in each group

13. Calculation
Use the mortality data collected during the test to calculate a 96–h LC50 value. The 24–, 48–, and 72–h values should also be calculated whenever there is sufficient mortality data to determine such values.
Calculate the mean number of deaths every 24 h at each concentration and write down your results in the following tables.

14. 24 hour toxicity test results represents Corrected % Mortality

15. 48 hour cumulative toxicity test results represents Corrected % Mortality

16. 72 hour cumulative toxicity test results represents Corrected % Mortality

17. 96 hour cumulative toxicity test results represents Corrected % Mortality

18. 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 LC50
Chart this on an x-y scatter plot (x-axis conc, y-axis percent deaths).

19. Make a normal scatter chart

20. Double click the X-axis to change the maximum value to 1000, and check the logarithmic scale box. Click ok.

21. Adjust the Y-axis scale to be maximum 100, major 10, and minor 5.
Right click on the dots and select add trendline.
From Type window select Logarithmic.
From Options window check both Display equation on chart, and Display Rsquared value on chart boxes, and click Ok.
Delete the series box.

23. 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 LC50 value. In this case 41.5 mg/l