SOP for BIOASSAY METHOD

1.0              Scope:

This standard lays down the bio-assay methods for evaluating the acute toxicity of industrial effluents and waste waters to fish. 

2.0              Apparatus:

2.1       Cylindrical glass jar or wide mouth glass bottle – 25 to 30 cm in diameter of 15 to 20 liters `capacity

2.2       Glass aquarium tanks – 60 to 200 liters capacity provided with arrangement for aeration

3.0       Test animals:

3.3.1    Type of fish: Fresh water fishes are commonly inhabiting unpolluted waters in the locality      

            are preferable. The following fresh water species are recommended:

a)         Cyprinodontidae (Top minnows)

            1) Aplocheilus panchax

b)         Cyprinidae (True minnows)

            1) Nuria darinca

            2) Rasbcra daniconius

            3) Danio Malabaricus

            4) Barbus ticto

            5) Barbus dorsalis

            6) Amblypharyagodon melettinus

            7) Cyprinus carpio

            Fish belonging to the same species shall be used for evaluation of each individual toxicity.

3.3.2    Source: The fish may be obtained from any single common source but preferably from the body of water receiving the pollutant tested.

3.3.3    Size: Specimens of more or less uniform size, averaging 5 to 7.5 cm in length are preferable. The largest shall be not more than 15 times as long as the smallest.

3.3.4    Stocks: Stocks of fish for test shall be kept initially in any suitable enclosures or containers

and in any water of suitable quality and temperature in which they will remain in good condition until needed.

3.3.5        Acclimatization and Feeding: The test fish shall be acclimatized for at least 10 days to laboratory conditions similar to those under which the tests are to be performed. They shall not be fed for about 48 hours before they are used in a test.

3.3.6        Fitness : The incidence of specimens dying or becoming seriously diseased in the acclimatizing aquarium during a period of 4 days immediately preceding a test shall be less than 10 %

4.0       Diluent Water:

4.1       The water to be used as a diluent and acclimatizing medium shall be obtained from the same body of water receiving the sample under investigation.

4.2        The diluent water shall be settled or filtered if excessive amount of suspended matter is     

            present. The dissolved oxygen content of the diluent water shall not be not below 4 mg /l.    

            The diluent water shall be well aerated with compressed air. It shall not contain residual    

             chlorine.

5.0       Number of Test Fish:

 At least 10 fish shall be used for each experimental concentration of the sample under test It is desirable to maintain a ratio of about 1 g of weight of fish per liter of liquid. They may be placed in one container with the test dilution, or preferably divided equally among two or more jars containing solutions of the same strength.

6.0              Dissolved Oxygen Content of Test Samples :

The D.O. content of test solutions or dilutions shall not fall below 4 mg / l throughout the period of the experiment. 

7.0              Procedure :

7.1       Preliminary Test:

Prepare wide ranges of concentrations of the sample under test, for example, 100, 10, 1, 0.1 percent with the diluent water. Place two or more test fishes in an appropriate volume of each concentration contained in wide mouth glass bottles. Observe after 24 hours. Select for full scale test the dilution ranges between the lowest concentration at which all fish survive for 24 hours and the highest concentration at which all or most fish die in 24 hours.

7.2              Preparation o Test Dilutions of the sample :

Shake the sample to disperse suspended matter uniformly. Withdraw measured portions of the sample and add to measured quantity of the diluent water and mix by gentle stirring. Avoid violet agitation.

7.3              Transfer of Test Fish :

Transfer from the acclimatizing aquarium not less than 10 fish into each dilution. This shall be done within 30 minutes after preparation of the dilution. In transferring test fish special care shall be taken to handle them without causing injury.

7.4       Duration of Test and Observation:

7.4.1    The test fish shall not be fed during the course of the test.

7.4.2    Observe the behavior of the fish under test at regular intervals for at least 48 hours.  The test period shall be expanded to 96 hours if more than half of the test fish survive for 48 hours at the lowest concentration tested. The test fish shall be deemed dead if they show no movement even when gently probed with a glass rod.

7.4.3    At the end of the test period transfer the surviving fish from each jar with as little agitation as possible to separate jars containing adequate amounts of the diluent water and observe for a further period of 24 hours. The number of fish which die in excess of those dying in the control within this period shall also be reported separately for the particular dilution.

7.4.4    When required, minimum quantities of the test liquids may be removed for such determination as dissolved oxygen.

7.5       Estimation of TLm :

7.5.1    Plot the percentage of surviving fish in each concentration on semi-logarithmic paper with the sample concentrations laid off on the logarithmic scale and survival percentages on the arithmetic scale.

7.6       Reporting of Results:

7.6.1    If all the test fish survive in the test sample (without any dilution) even after 96 hours the sample shall be reported as free from acute lethal toxicity.

7.6.2    While reporting the TLm value. Mention shall be made of the species of fish used and their origin, weight, and length; the temperature at which the test was carried out; pH and D.O. content of the test and the source of the diluent water and its mineral composition.

METHOD: IS 6582 - 1971