Water Toxicity Study

Sea Urchin Fertilization Test

Procedure:

Collect Gametes: Induce spawning in 4-6 sea urchins by injecting 2 mL KCl into the soft area surrounding the mouth. Place the urchins with the mouth facing down on a clean petri dish. If a yellow discharge is seen, eggs are being shed and this is a female urchin. Turn the urchin over to collect the eggs on the dish. If a white discharge is present, sperm are being produced. Turn this male urchin over to collect the sperm.

Eggs: Take the eggs produced from 1-2 (or more) urchins and put them together in a clean petri plate. Place 10 drops of this concentrated egg solution in 10 mL of the test (control) solution in a test tube. Allow the eggs to sit in the test (control) solution for at least 20 minutes before introducing sperm. All solutions should be kept at 12°C during the experiment.

Sperm: Combine the sperm from 1-2 (or more) urchins on a clean petri plate. Prepare a slide of the sperm. Under a low power lens of a compound mircrcope, the sperm should appear to be concentrated and have good motility. Place 10 drops of pooled, good sperm into a test tube containing 10 mL of 12°C test (control) solution for fertilization experiment. The sperm will remain in the test (control) solution for a 20 minute exposure time before the sperm is added to the egg solutions.

Fertilization: Add1 mL of exposed (20 minutes exactly) sperm solution into the corresponding tubes containing exposed (at least 20 minutes) egg solution.

End Test: The test ends with the addition of a preservative (10% formaldehyde) exactly 20 minutes after the addition of the sperm. Add 2 drops of the 37% formaldehyde to the sperm/egg solution.

Count Fertilized Eggs: Prepare depression slides of each solution. Count the number of fertilized and unfertilized eggs. The total number counted should be 100. A fertilized egg will have a complete fertilization membrane (figure 1). Note the eggs which have an incomplete or deformed fertilization membrane (figure 2).

Data Sheet

Water Toxicity Study

Sea Urchin Fertilization Test

Data Sheet
Date:
Investigator:

Record the spawning record in Table 1 below.

Number of females with useable eggs:
Number of males with useable sperm:

Table 1. Spawning Record

Figure 1. Fertilized egg with complete fertilization membrane	Figure 2. Fertilized egg with incompete fertilization membrane

Keep an accurate record of the seawater source for each test tube in the table below.

Test Tube Number	Water Source
Test Tube 1
Test Tube 2
Test Tube 3
Test Tube 4
Test Tube 5
Test Tube 6
Test Tube7
Test Tube 8
Test Tube 9
Test Tube 10

Table 2. Test Tube number correlated with water source.

Test Tube #	Sample ID	Time of sperm addition	Time of egg addition	Time of Preservative addition

Table 3. Record of time of exposure and fertilization.

Count 100 eggs from each test tube. Record the number of fertilized eggs out of each batch of 100.

Test Tube #	Sample ID	Number fertilized (formed fertilization membrane)	Number not fertilized	Comments

Table 4. Record of fertilization rate.

Results

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