IS 6582 (Part 2) : 2001
REAFFIRMED SEP 2007
Indian Standard
BIO-ASSAY METHOD FOR EVALUATING ACUTE TOXICITY OF INDUSTRIAL EFFLUENTS AND WASTE WATERS
PART 2 USING TOXICITY FACTOR TO ZEBRA FISH (BRACHYDANIO RERIO)
(First Revision)
ICS 13.060; 13.060.30
© BIS 2001
BUREAU OF INDIAN STANDARDS
MANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG
NEW DELHI 110002
May 2001MANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG
NEW DELHI 110002
Price Group 3
i
Water Environment Protection Sectional Committee, CHD 12FOREWORD
This Indian Standard (First Revision) was adopted by the Bureau of Indian Standards, after the draft finalized by the Water Environment Protection Sectional Committee had been approved by the Chemical Division Council.The bio-assay method, specified in IS 6582 : 1971 [now proposed to be revised and numbered as IS 6582 (Part 1)], is based on determination of LC50, that is, concentration of sample (of effluent/waste water) on semi-logarithmic scale at which 50 percent of fish could survive. However, this method requires a large number of fish and takes longer time to give result.
Keeping the above in view, for regulatory purposes, present method which is based on toxicity factor (TF), that is, minimum number of times an effluent/waste water is required to be diluted for obtaining no mortality for zebra fish, has been developed. Reproducibility and validity of this method have been found to be acceptable by Central Pollution Control Board, after testing on treated/untreated pesticides/industrial effluents, collected from different parts of the country.
The proposed method, using toxicity factor (TF) will lead to lower concentration level compared to the LC50; however, the former is preferable as it gives toxicity potential of waste water in terms of dilution required to acceptable permissible level.
The composition of the committee responsible for the preparation of this standard is given in Annex A.
In reporting the result of a test or analysis made in accordance with this standard if the final value, observed or calculated, is to be rounded off, it shall be done in accordance with IS 2 : 1960 ‘Rules for rounding off numerical values (revised)’.
ii
Indian Standard
BIO-ASSAY METHOD FOR EVALUATING ACUTE TOXICITY OF INDUSTRIAL EFFLUENTS AND WASTE WATERS
PART 2 USING TOXICITY FACTOR TO ZEBRA FISH (BRACHYDANIO RERIO)
BIO-ASSAY METHOD FOR EVALUATING ACUTE TOXICITY OF INDUSTRIAL EFFLUENTS AND WASTE WATERS
PART 2 USING TOXICITY FACTOR TO ZEBRA FISH (BRACHYDANIO RERIO)
(First Revision)
1 SCOPE
The standard prescribes an alternative method for determination of acute lethal toxicity of waste water and industrial effluents to zebra fish (Brachydaniorerio) under specified conditions.2 REFERENCES
The following standards contain provisions which through reference in this text, constitute provision of this standard. At the time of publication, the editions indicated were valid. All standards are subject to revision and parties to agreements based on this standard are encouraged to investigate the possibility of applying the most recent editions of the standard indicated below:| IS No. | Title |
|---|---|
| 1070 : 1992 | Reagent grade water (third revision) |
| 4733 : 1972 | Methods of sampling and test for sewage effluents (first revision) |
3 TERMINOLOGY
For the purpose of this standard, the following definitions shall apply.3.1 Dilution Water
The dilution water shall have a pH of 7.8 ± 0.2 and a calcium hardness of approximately 200 mg/1 expressed as CaCO3. It shall be prepared in accordance with 7.1.3.2 Test Water
It is the mixture of waste water and dilution water, which is used for testing.3.3 Dilution Factor (T)
Dilution Factor (T) is the numerical expression of volumetric proportion of the waste water in test water. ‘T’ denotes the toxicity unit.3.4 Toxicity Factor, TF
TF is the lowest Dilution Factor T of the Test Water at which all fish survive. TF denotes the toxicity for fish. This is a dimensionless toxicity unit, which is used as standard parameter for compliance monitoring of waste water discharges. Higher the TF, the greater is the toxicity of the tested waste water.3.5 Death of Fish
A fish is declared dead if it does not move when touched.3.6 Range Finding Test
Range finding test is a preliminary test for samples with unknown toxicity to determine the range of dilution within which the TF value is expected.3.7 Standard Test
Standard test is the confirmatory test performed to determine the TF value, under strict compliance with the procedure prescribed in this method.4 GENERAL PRINCIPLE
4.1
Fish are affected by toxic substances chiefly in following two ways:- Epithelia absorb toxic substances, getting damaged in this process, for instance, the gills stick together or get congested with mucus and get destroyed.
- Besides the above immediate effects, harmful substances are adsorbed through the gills, skin or intestine, thereby impairing physiological functions. These effects may ultimately lead to the death of fish.
4.2
These toxic effects are determined in terms of the death of the test organism in standard dilution series of waste water samples with standard dilution water.5 SAMPLING
5.1 Point of Sampling
In those cases where the effluent at a specific point is to be tested, the question of choosing the point of sampling does not arise. However, where the composition of an effluent as finally discharged by the sewage treatment plant is to be ascertained, the point of sampling shall be the final outlet of the treatment plant.5.2 Frequency of Sampling
When it is required to find out variations in the composition of the effluent during a specified period, such as that of peak discharge, the samples shall be taken at short and appropriate intervals, say, every 5, 10, 15 or 30 min and analysed. To study the average conditions over a cycle of operations or a period (usually 24 hours) or during the daily working period of the treatment plant, the collection of composite sample shall be adopted. The composite sample shall be made by collecting at appropriate intervals samples from the common channel or drain at a point where the flow of the effluent is likely to be most representative of the entire volume, and mixing. The volume of the individual samples shall be a fixed proportion of the volume of the effluent flowing at that time. The interval should depend upon the frequency of variation in the nature of the effluent and the volume of flow. Care shall be taken to take the samples in such a way as to maintain the true proportion of suspended solids. Samples shall not be taken by skimming the top or scraping the bottom. A point about one-third of the way from the bottom shall normally be selected. The samples shall be drawn gently without unnecessary aeration. In most cases, collection of samples every hour would be sufficient.5.3 Sampling Instrument
Porcelain-lined or enamelled pails, in which the lining is unbroken, or glass vessels shall be used for taking samples. The vessels used for taking the sample shall be wide-mouthed and small enough for the contents to be transferred quickly to the sample container without leaving behind any deposit or scum. Automatic sampling devices, if available, may be used.5.3.1
Each individual sample shall be deposited in a receptacle of sufficient size to hold the entire composite sample. Clean and dry carboys, other large glass containers or enamelled buckets with lids without chipping may be used for pooling the sample.5.4 Sample Containers
5.4.1
The quantity of sample required for analysis shall be taken from the composite sample after thorough mixing in order to keep the solids in suspension.5.4.2
The sample for analysis shall be drawn in clean glass-stoppered bottles, which shall be rinsed with a portion of the sample. New bottles shall be washed with acid and thoroughly rinsed with distilled water before being brought into use. About 2 to 3 litres of the sample will be required for analysis. The bottle containing the final sample shall be filled so that a small air bubble is present after closure to prevent leakage or even breakage arising from any subsequent changes in temperature. The stopper shall be firmly inserted and, if the sample is to be transported some distance, tied down to keep in position.5.4.3
The label on the bottle shall bear the name of the sampling authority, details of the type of sample, place, date and time of sampling.5.5 Preservation of Samples
5.5.1
The samples shall be kept at a low temperature (about 4°C) during collection and thereafter.5.5.2
No single method of preservation is applicable for the sample for all the tests. The analysis shall be carried out, preferably, immediately after collection. Storage at 3° to 4°C in a well insulated ice box or refrigerator is the best way to preserve most samples till the next day. Where chemical preservatives are used as specified for individual tests, these shall be added to each portion of sample taken for the particular test and not to the entire sample.6 MATERIAL
6.1 Test-Fish Species
Brachydanio rerio (Hamilton Buchanan), having a size1 of 30 ± 5 mm, corresponding to approximately 0.2 to 0.3 g of mass.
6.2 Stock of Fish
6.2.1
The fish shall be kept at temperature of approximately 25°C in aerated chlorine free potable water of roughly similar characteristics as the dilution water. The population density of fish shall not exceed 1 g per litre.6.2.2
The daily illumination shall be in the range of 12 to 16 h. The stock shall be kept on a normal diet. The fish shall be free of manifest diseases or visible malformations. The minimum acclimatisation period shall be 10 days prior to test under conditions of water quality and illumination similar to those used in the test. Mortality shall not exceed 1 percent per week.6.2.3
For each test, fish shall be selected from the same stock tank, the population of which is under conditions of water quality and illumination similar to those applied in the test.6.3 Equipment
All equipment shall be of inert material, preferably of stainless steel or borosilicate glass.6.3.1
Glass Beaker—3 1 capacity.6.3.2
Measuring Pipettes—5 and 10 ml.6.3.3
Volumetric Pipettes—10, 20 and 50 ml.6.3.4
Volumetric Flask—100 ml and 1 litre.6.3.5
Measuring Cylinders—100 ml and 1 litre.6.3.6
Beakers—100, 200, 500 ml and 1 litre.6.3.7 Thermometer
6.3.8 Oxygen Meter
6.3.9 pH Meter
6.3.10 Conductivity Meter
6.3.11 Tanks for Fish Stock and Dilution Water
6.3.12 Water Baths
6.3.13 Aquaria
6.3.14 Aerators
6.3.15 Thermostats
6.3.16 Air and Water Filter with Activated Charcoal
6.3.17 Handnets
6.4 Reagents
6.4.1
Calcium Chloride (CaCl2, H2O)6.4.2
Magnesium Sulphate (MgSO4, 7 H2O)6.4.3
Sodium Bicarbonate (NaHCO3)6.4.4
Potassium Chloride (KCl)6.5 Quality of Reagents
Unless specified otherwise, pure chemicals and distilled water (see IS 1070) shall be employed in the tests.NOTE—‘Pure chemicals’ shall mean chemicals that do not contain impurities which affect the results of analysis.
7 PROCEDURE
7.1 Preparation of Dilution Water
7.1.1
Dilution water is prepared by mixing 25 ml each of the following four stock solutions and diluting to 1 litre with water. Thereafter the pH is adjusted using sodium hydroxide or hydrochloric acid solution.7.1.1.1
Calcium chloride solution—Dissolve 11.76 g of calcium chloride dihydrate (CaCl2, 2H2O) in water and dilute to 1 litre.7.1.1.2
Magnesium sulphate solution—Dissolve 4.93 g of magnesium sulphate heptahydrate (MgSO4, 7H2O) in water and dilute to 1 litre.7.1.1.3
Sodium bicarbonate solution—Dissolve 2.59 g of sodium bicarbonate (NaHCO3) in water and dilute to 1 litre.7.1.1.4
Potassium chloride—Dissolve 0.23 g of potassium chloride (KCl) in water and dilute to 1 litre.7.1.2 Aeration of the Dilution Water
The dilution water shall be aerated for 24 h prior to the test.7.2 Preparation of Test Solutions
7.2.1 Range Finding Test
In case the range of toxicity of an effluent is unknown, a range-finding test should be performed prior to the standard test to determine the concentration range within which 0 percent and 100 percent mortality is observed within 24 h. For example, in case of an effluent with unknown toxicity, the following dilution series with a dilution factor of 10 may be used :| Dilution Factor(T) | Ratio of Waste Water to Test Water (by Volume) | Parts of Dilution Water to be Added to one Part of Waste Water (by Volume) |
|---|---|---|
| 1 | 1:1 | 0 |
| 10 | 1:10 | 9 |
| 100 | 1:100 | 99 |
| 1 000 | 1:1 000 | 999 |
7.2.2 Standard Test
7.2.2.1
The test water is prepared by adding the effluent to the dilution water in a rounded logarithmic (geometric) progression scale as given below:| Dilution Factor(T) | Ratio of Waste Water to Test Water (by Volume) | Parts of Dilution Water to be Added to one Part of Waste Water (by Volume) |
|---|---|---|
| 1 | 1:1 | 0 |
| 2 | 1:2 | 1 |
| 4 | 1:4 | 3 |
| 8 | 1:8 | 7 |
| 16 | 1:16 | 15 |
| 32 | 1:32 | 31 |
| 64 | 1:64 | 63 |
| 125 | 1:125 | 124 |
| 250 | 1:250 | 249 |
| 500 | 1:500 | 499 |
| 1 000 | 1:1 000 | 999 |
7.2.2.2
Up to five consecutive concentrations have to be selected based on previous experience with regard to potential toxicity of the effluent. Each test vessel is filled with 2 litres of test water. One control vessel containing only dilution water is provided for each test. The test may be performed with one replicate.7.3 Test Conditions
Following test conditions shall be maintained:- 24 h prior to the test, feeding shall be stopped.
- Fish for a single test, shall be selected from a tank with population of the same stock.
- After obtaining the correct temperature, 5 fishes shall be placed in each of the vessels. The fishes shall be selected randomly from the stock population and distributed at random in the various vessels with the help of a small fine-mesh dip net of soft inert material.
- Any fish dropped or otherwise harmed during the transfer shall be discarded. All the fish, for a single test shall be introduced to test vessels within a period of 30 min.
- The samples and test solutions shall not be aerated or treated; else extreme BOD content or extreme pH values may influence the result.
- The duration of the test shall be 48 h.
- Count of the dead fish in each vessel shall be taken each after 2 h, 6 h, 24 h and 48 h; the dead fish shall be removed from the vessels.
- In case of fish dying in the control vessel, the test shall be discarded and fresh testing shall be repeated.
- The dissolved oxygen concentration, the pH and temperature in each vessel shall be measured at the beginning of the test and each after 24 h and 48 h.
- The handling of fish, solutions and all test procedures shall be carried out in premises free of harmful concentrations of vapours. Any disturbances that may change the behaviour of fish shall be avoided.
- All tests shall be carried out under normal laboratory illumination with natural photo period.
- The temperature shall be maintained at 25 ± 1.0°C.
8 CALCULATIONS
8.1
The dilution factor of the test solution with the highest concentration of effluent in which all fish survive shall be recorded in rounded numbers as TF.Example : If TF = 8, it shows that all or some of the fish die in the test solution for a T value, less than 8 (1 part waste water +7 parts of dilution water) and all are alive in test solutions for a T value of 8 and above, after 48 h.
9 VALIDITY
9.1
The results shall be considered valid if the following requirements are met.9.1.1
The TF value of the reference chemical for each stock of fish should be in agreement with results obtained previously in the same laboratory, as a reference material potassium dichromate is used. In dilution water containing 100 mg/1 potassium dichromate, all fish should survive (that is TF = 1).9.1.2
All the conditions defined in the test method are fulfilled.10 REPORT
10.1
The test report shall include the following information.10.1.1
The specification of the test effluent and full information for identification of test samples.10.1.2
Any deviation from the procedure specified in this standard and the reason for this, including a description of the circumstances which could have influenced the results.3
ANNEX A
COMMITTEE COMPOSITION
(Foreword)
Water Environment Protection Sectional Committee, CHD 12
| Chairman | Representing |
|---|---|
| Shri D. K. Biswas | Central Pollution Control Board, Delhi |
| Members | |
| Dr P. K. Mathur | Bhabha Automic Research Centre, Mumbai |
| Dr T. N. Mahadaven (Alternate) | |
| Shri Imranur Rahman | Central Mechanical Engineering Research Institute, Durgapur |
| Shri B. Ruj (Alternate) | |
| Shri A. K. Gupta | Bharat Heavy Electricals Ltd, Haridwar |
| Shri N. G. Srivastava (Alternate) | |
| Dr P. S. Ramanathan | Gharda Chemicals Ltd, Maharashtra |
| Dr S. R. Jakali (Alternate) | |
| Shri R. A. Ramanujam | Central Leather Research Institute, Chennai |
| Shri N. Samivelu (Alternate) | |
| Shri S. K. Jain | Department of Industrial Development, Ministry of Industry, Govt of India. New Delhi |
| Shri AnilJain | Engineers India Ltd, Gurgaon |
| Dr (Ms) B. Swaminathan | The Fertilisers Association of India, New Delhi |
| Dr S. Nand (Alternate) | |
| Dr S. D. Machijani | Central Pollution Control Board, Delhi |
| Dr R. C. Trivedi (Alternate) | |
| Shri Satinder Kumar Mehra | ABB Alstom Power India Ltd, West Bengal |
| Shri Subrato Kumar Basu (Alternate) | |
| Shri Bakul B. Dave | Hindustan Lever Ltd, Mumbai |
| Shri U. P. Singh (Alternate) | |
| Dr Anil Bhasker | ICMA, Mumbai |
| Shri B. B. Narnla (Alternate) | |
| Dr K. R. P. Singh | Industrial Toxicology Research Centre, Lucknow |
| Dr Krishna Gopal (Alternate) | |
| Shri M. N. Vyas | Indian Petro Chemicals Corpn Ltd, Vadodra (Gujarat) |
| Dr J. D. Desai (Alternate) | |
| Dr V. K. Verma | Shriram Institute for Industrial Research, Delhi |
| Shri M. S. Dhingra (Alternate) | |
| Dr M. P. Singh | Indian Oil Corporation Ltd, Faridabad |
| Dr A. S. Mathur (Alternate) | |
| Dr P. S. Minhas | Central Soil Salinity Research Institute, Kamal |
| Shri P. M. Khanderia | Tata Chemicals Ltd, Gujarat |
| Shri J. M. Khan (Alternate) | |
| Shri Sunil Sachdeva | Thermax Limited, Pune |
| Shri S. S. Basargekar (Alternate) | |
| Shri A. K. Jindal | Therman Fuji Electric Ltd, Pune |
| Dr S. K. Ghosh | National Institute of Occupational Health, Ahmedabad |
| Shri S. B. Sahay | National Thermal Power Corpn Ltd, West Bengal |
| Shri R. L. Srivastava (Alternate) | |
| Shri S. M. Shingote | National Council for Cement and Building Materials, New Delhi |
| Shri V. P. Chatterjee (Alternate) | |
| Dr G. K. Gureja | Thermax Limited (CII) |
| Shri S. K. Chopra (Alternate) | |
| Dr S. K. Kapoor | DRDO, New Delhi |
| Dr (Smt) Kshipra Mishra (Alternate) | |
| Shri M. R. Rajput | Regional Labour Institute, Kanpur |
| Dr S. N. Banerjee (Alternate) | |
| Shri D. K. Dua | Punjab Pollution Control Board, Punjab |
| Dr (Mrs) H. K. Parwana (Alternate) | |
| Representative | The Dharamsi Morarji Chemical Co Ltd, Mumbai |
| Representative | Indian Meteriological Department, Safdarjung Airport, New Delhi |
| Dr Dipak Chakraborty | West Bengal Pollution Control Board, Calcutta |
| Dr Ujjal Mukherjee (Altenate) | |
| Representative | Maharashtra State Pollution Control Board, Mumbai 4 |
| Shri Thiru S. Balakrishnan | Tamilnadu Pollution Control Board, Chennai |
| Dr A. K. Saxena | |
| Shri M. A. Patil (Alternate) | National Productivity Council, New Delhi |
| Representative | Department of Rural Development, New Delhi |
| Representative | Punchayat Raj Department, Hyderabad |
| Dr C. M. Agrawal | Directorate General of Health Services, New Delhi |
| Shri Ashok Bhatia | Ministry of Environment & Forests, New Delhi |
| Dr S. P. Pande | Water Technology Engineering Division. Nagpur |
| Representative | RDSO, Ministry of Railways, Lucknow |
| Shri A. K. Dey | Central Fuel Research Institute, Dhanbad |
| Shri M. M. Malhotra, Director (Chem) | Director General, BIS (Ex-officio Member) |
| Dr R. K. Singh Additional Director (Chem), BIS | |
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