1. Marine Biotechnology Lab
Preparation Of The Most Commonly Used Chemicals In Aquaculture Processes

2. Determination of active chlorine content in commercial grade bleach (hypochlorite solution)
Preparation of a disinfecting 500 ppm hypochlorite solution from commercial grade bleach
Chemical methods to sterilize seawater (for algal, rotifer and artemia cultures)

3. Determination of active chlorine content in commercial grade bleach (hypochlorite solution)
The active ingredient in most chlorine bleaches is sodium hypochlorite, NaOCl.
The oxidizing action of hypochlorite ion, OCl-, kills germs and also decolorizes many stains and dyes.

4. Principle:
In acid solution, hypochlorite ions oxidize iodide ions to form iodine, I2.
The amount of hypochlorite ion present in a solution of bleach is determined by finding out how much iodine, I2, is produced by oxidizing iodide ion, I .
The released iodine is thus titrated with a standard solution of sodium thiosulphate, Na2S2O3, using starch as the indicator, which converts the colored iodine to colorless iodide ion.

5. I3-(aq) + 2 S2O32-(aq) 3I-(aq) + S4O62-(aq)
The analysis takes place in a series of steps:
Acidified iodide ion is added to hypochlorite ion solution, and the iodide ion is oxidized to iodine.
2 H+(aq) + ClO-(aq) + 2 I-(aq) Cl-(aq) + I2(aq) + H2O(l)
Iodine is only slightly soluble in water. It dissolves very well in an aqueous solution of iodide ion, in which it forms a complex ion called the triiodide ion. The triiodide ion is yellow in dilute solution, and dark red-brown when concentrated.
I2(aq) + I-(aq) I3-(aq)
The triiodide is titrated with a standard solution of thiosulfate ions, which reduces the iodine back to iodide ions:
I3-(aq) + 2 S2O32-(aq) I-(aq) + S4O62-(aq)

6. During this last reaction the red-brown color of the triiodide ion fades to yellow and then to the clear color of the iodide ion.
It is possible to use the disappearance of the color of the I3- ion as the method of determining the end point, but this is not a very sensitive procedure.
Addition of starch to a solution that contains iodine or triiodide ion forms a reversible blue complex.

7. The disappearance of this blue colored complex is a much more sensitive method of determining the end point.
The quantity of thiosulfate used in the titration is directly related to the amount of hypochlorite initially present.
Caution! Undiluted bleach and hydrochloric acid can cause chemical burns and ruin your clothes. Bleach is also irritating to the eyes.

8. Reagents: Acetic acid (glacial, concentrated).
Potassium iodide (KI) crystals.
Sodium thiosulphate (Na2S2O3 .5 H2O, i.e g/mol) 0.1 N standard solution:
dissolve 25 g of sodium thiosulphate in 1 liter of freshly boiled distilled water.
improve its stability by adding 0.1 g of Na2CO3.
Add 0.5 ml of chloroform (CHCl3 ) as preservative to prevent growth of Thiobacillus thioparus and store in a clean glass bottle in the dark.

9. Starch indicator solution:
Mix 5 g starch with a little cold water and grind in a mortar, pour into 1 liter of boiling distilled water, stir and let settle overnight.
Use the clear supernatant preserved with 1.25 g salicylic acid and store in a dark bottle.
The solution needs refrigeration to avoid decomposition of starch which results in a vague end point. Keep the solution cool and use within a week.

10. Procedure:
In 250-ml Erlenmeyer flask dissolve 0.5 to 1 g KI in 50 ml distilled water, add 5 ml acetic acid.
Add 1 ml from sample to be checked, a red-brown iodine color will appear.
Titrate away from direct sunlight: add 0.1 N thiosulphate from a burette until the solution is pale yellow color.
Add 1 mL starch indicator solution to the flask; a starch-iodine complex will form which has an intense blue color.

11. Immediately continue titration until the blue color formed is disappeared for at least 30 seconds.
Calculation:
1 ml of 0.1 N thiosulphate equals to 3.54 mg active chlorine.

12. Preparation of a disinfecting 500 ppm hypochlorite solution from commercial grade bleach
Disinfection has widespread application as a method to destroy pathogenic microorganisms associated with inanimate objects.
Chemical agents called disinfectants kill the vegetative stage of pathogenic microorganisms, but not necessarily their spore stage.
Chlorine is widely used as disinfectant agent.

13. A concentration of 500 ppm active chlorine is widely used to disinfect objects and equipment .
Water discharged from hatcheries may be also chlorinated to destroy endemic or exotic pathogens.
After disinfection, the equipment and tank must be thoroughly rinsed with chlorine-free water, and any residual chlorine should be neutralized with a sodium thiosulfate before it is used to handle fish.

14. Procedure
Check the content of free chlorine in the available bleach following the abovementioned method.
Say the content found is 4% free chlorine (typical range: 5 to 15% in commercial bleach).
A free chlorine 500 ppm solution means 500 ml of free chlorine in milliliters, or 0.5 ml in 1 liter.
0.5 ml of free chlorine are found in 12.5 ml of 4% bleach (0.5/0.04);
to prepare say 1 liter of a 500 ppm hypochlorite solution add 12.5 ml of 4% active chlorine bleach to 1 liter of water.

15. Disinfection procedure
When still wet rinse tank and all equipment with tap water to eliminate the bulk of organic debris
Wash it thoroughly with brush and detergent and rinse it again;
For tanks wash or spray the tank walls or fill the tank with 500 ppm active chlorine solution
For small tools and equipment dip in a container filled with a 500 ppm hypochlorite solution.

16. Keep in a hypochlorite solution overnight (at least 2 hours).
Rinse the tank and equipment with tap water well until the chlorine smell is gone;
let the tank to dry and use when needed.

17. Large containers with hypochlorite solution are used to keep cleaned small equipment
Chlorine container for laboratory use

18. Chemical methods to sterilize seawater (for algal, rotifer and artemia cultures)
The standard disinfecting concentration for culture seawater is 10 ppm of free chlorine and the corresponding safe concentration of sodium thiosulphate to deactivate residual chlorine is 12 ppm.
Say the content of free chlorine in the available bleach is 4%.
A free chlorine 10 ppm solution means 10 ml of free chlorine in milliliters, or 0.01 ml in 1 liter.
0.01 ml of free chlorine are found in 0.25 ml of 4% bleach (0.01/0.04).

19. to disinfect each liter of culture water add 0
to disinfect each liter of culture water add 0.25 ml of 4% active chlorine bleach (final chlorine concentration: 0.01 ml/l).
provide a gentle aeration for 30 minutes to mix well, then let stand overnight.
add 12 mg of sodium thiosulphate to each liter of seawater (to have a 12 ppm solution, i.e. 12 mg in mg) and aerate strongly for one hour,

20. Identification of active chlorine residue
Check for residual chlorine and use only if no blue stain develops.
Identification of active chlorine residue
To one ml sample add 1 ml of KI solution, 1 ml of 0.1 N thiosulphate solution and 1 ml of starch indicator. If a blue color develops it is a proof of presence of active chlorine residues.