1.0
Scope:
The
standard prescribes an alternative method for determination of acute toxicity
of waste water and industrial effluents to zebra fish ( Brachydaniorerio) under
specified conditions.
2.0
Principle:
Fish
are affected by toxic substances chiefly in following two ways:
2.1 Epithelia absorb
toxic substances , getting damged in this process , for instance , the gills,
skin together or get congested with mucus and get destroyed.
2.2 Besides the above
immediate effects, harmful substances are adsorbed through the gills, skin, or
intestine , thereby impairing physiological functions.These effects may lead to
the death if fish.
3.0
Material:
3.1 Test
– Fish Species
Brachydanio rerio ( Hamilton
Buchanan ) having a size of 30± 5mm, corresponding to
approximately 0.2 to 0.3 g of mass.
3.2
Stock of Fish:
3.2.1
The fish shall be kept at
temperature of approximately 25°C in aerated chlorine free water of
roughly similar characteristics as the
dilution water . The population density of fish shall
not
exceed 1 g per litre.
3.2.2
The daily illumination shall be
in the range of 12 to 16 h. The stock shall be kept in a normal diet. The fish
shall be free of manifest disease or visible malformation. The minimum
acclimatization 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 % per week.
3.2.3
For each test, fish shall be
selected from the same stock tank.
3.3
Equipment:
All
equipment shall be of inert material , preferably of stainless steel or
borosilicate glass.
3.3.1
Glass beaker – 31 capacity
3.3.2
Measuring pipettes – 5 & 10
ml
3.3.3
Volumetric pipettes –
10,20,& 50 ml
3.3.4
Volumetric flask – 100 ml &
1 litre
3.3.5
Measuring cylinder – 100ml&
1 litre
3.3.6
Beakers – 100 , 200 , 500 ,
& 1 litre
3.3.7
Thermometer
3.3.8
Oxygen Meter
3.3.9
PH Meter
3.3.10
Conductivity Meter
3.3.11
Tanks for Fish Stock and
Dilution Water
3.3.12
Water Baths
3.3.13
Aquaria
3.3.14
Aerators
3.3.15
Thermostats
3.3.16
Air & Water Filter with
Activated Charcoal
3.3.17
Handnets
3.4
Reagents:
3.4.1
Calcium Chloride
3.4.2
Magnesium Sulphate
3.4.3
Sodium Bicarbonate
3.4.4
Potassium Chloride
4.0
Procedure:
4.1 Preparation
of Dilution Water:
4.1.1 Dilution water is
prepared by mixing 25ml 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.
4.1.1.1 Calcium
chloride solution – Dissolve 11.76g of calcium chloride dihydrate ( CaCl2 ,
2H2O) in water and dilute to 1
litre .
4.1.1.2 Magnesium sulphate solution – Dissolve 4.93 g of magnesium
sulphate heptahydrate (MgSO4,7H2O) in water and dilute 1 litre.
4.1.1.3 Sodium bicarbonate solution – Dissolve 2.59g
of sodium bicarbonate (NaHCO3) in water
and dilute to 1
litre.
4.1.1.4 Potassium chloride
– Dissolve 0.23 g of potassium chloride (KCL) in water and dilute to 1 litre.
4.1.2 Aeration of the Dilution Water
The dilution water
shall be aerated for 24 hr. prior to the test.
4.2 Preparation
of Test Solutions:
4.2.1 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
|
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
|
9
|
|
100
|
1 : 100
|
99
|
1000
|
1 : 1000
|
999
|
4.2.2 Standard Test:
4.2.2.1 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
|
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
|
15
|
|
32
|
31
|
|
64
|
1 : 64
|
63
|
125
|
1 : 125
|
124
|
250
|
1 : 250
|
249
|
500
|
1 : 500
|
499
|
1000
|
1 : 1000
|
999
|
4.2.2.2 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.
4.3 Test Conditions:
Following test
conditions shall be maintained:
a) 24 hr prior to the test, feeding shall be stopped.
b) Fish for a single
test, shall be selected from a tank with population of the same stock.
c) 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.
d) 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.
e) The samples and test
solutions shall not be aerated or treated; else extreme BOD content or extreme
pH values may influence the result.
f) The duration of the
test shall be 96 h.
g) 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.
h) In case of fish
dying in the control vessel, the test shall be discarded and fresh testing
shall be repeated.
i) 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.
j) 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 behavior
of fish shall be avoided.
k) All tests shall be
carried out under normal laboratory illumination with natural photo period.
l) The temperature
shall be maintained at 25 + or – 1.0 C.
5.0 Validity:
5.1 The results shall be
considered valid if the following requirements are met.
5.1.1 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/l potassium do chromate,
all fish should survive (that is TF= 1)
5.1.2 All conditions defined
in the test method are fulfilled.
REFERNCE: IS 6582 (PART 2):
2001
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