Coral Protocol

ICZMP
Draft Monitoring And Analysis Protocol
DRAFT MONITORING AND ANALYIS PROTOCOL
FOR
COASTAL WATERS UNDER ICZM PROJECT
_______________________________________________________________________________
BACKGROUND
Gujarat is having a coastal belt of about 1600 Kms which is throbbing with variety of
developmental and industrial activities and hence this belt and the nearby area is one of the major
sources of economy of the state. Apart from the primary fishing activities and the ancillary
industries, it also provides employment to the people in the area. Coastal area play vital role for
the growth of fishermen community and also for the growth of developmental projects especially
which are dependent on marine resources. Because of all these activities, there always remain a
pollution potential of the coastal waters. To know the pollution level and its effect on marine life,
monitoring of coastal area is vital. On the basis of analysis and subsequent interpretation of
analytical data, pollution load in coastal area can be estimated. Physico-chemical analysis and
biological analysis is required to assess the quality of coastal water. These parameters are in turn
related to the chemical composition of sea water and its monitoring and analysis help us to control
pollution level at different sources. Deciding suitable and appropriate monitoring locations is
therefore one of the most important aspects for the real evaluation and assessment of
environmental parameters.
MONITORING TEAM
Monitoring of Gulf of Kutch waters is under taken by the Scientists form Regional Offices viz.
Jamnagar, Bhuj and Rajkot in consultation with concerned Regional officers and with the Nodal
Officer ICZMP at Head Office, Gandhinagar.
LOCATIONS
Sampling locations are decided in consultation with concerned Regional Officers of the Board
and the Nodal Officer, ICZMP at Head Office. After completion of selection of all locations, the
details were submitted to S.P.M.U and all PEA’s for their suggestions & recommendation before
implementation of sampling programme under ICZM Project. Following criteria has been
considered for selection of sampling stations:
•
•
•
•
•
•
Industrial activities and developmental activities in surrounding area.
Direct or indirect discharge of waste water into the sea.
Urbanization / human activities in surrounding area.
Approachability to the stations by road in all types of weather.
Availability of boat for sample collection.
Tourism/ Port activities.
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Draft Monitoring And Analysis Protocol
MONITORING AREA
During selection of sampling points, we have considered both ‘Source’ and ‘Sink’ and hence
Source monitoring as well as Sea water monitoring is incorporated in the monitoring protocol. The
‘Source’ sampling locations includes Domestic outfalls / Industrial discharges in the area where as
‘Sink’ sampling locations includes sampling of receiving sea water into which the Domestic out
falls / Industrial discharges have mixed. All, three regions viz. Rajkot, Jamnagar and Kutchh are
included for monitoring.
A. Source Monitoring
Sr.
Longitude &
Industrial Cluster/Area/Other Activity
No.
Latitude
Regional office: Jamnagar
Jamnagar Municipal Corporation Domestic Outfall waste water from 2229’477”N
1
pumping station of Gandhinagar, Jamnagar
7003’318”E
Cooling water return through open channel water sample of GEB TPS – 2225’121”N
2
SIKKA
6949’201”E
2220’606”N
3
Essar Oil Limited, (Sea Water Basin Outlet)
6945’056”E
From Final Outlet of Tata Chemicals, Mithapur, (after dilution after 2224’640”N
4
settling)
6902’066”E
2226’631”N
Reliance Industries Ltd., (Sea water Basin Outlet)
5
6950’621”E
Regional office: Bhuj
22o48’08.82”N
1
Confluence point of Tata Power & Adani Power, Mundra, Tal. : Mundra
69o32’12.38”E
Domestic waste water sample of outlet of KSEZ/Gandhidham city nr. 23o01’03.36”N
2
kidana
70o07’27.28”E
B. Sea Water Monitoring
Sr.
Industrial Cluster/Area/Other Activity
No.
Regional office: Jamnagar
1
Sea Water sample at Rozy Port, Near Light House
2
Sea Water sample at Sachana Ship Breaking Yard
3
Sea water sample @ 2 km. away from RIL Jetty.
4
Sea water sample @ 2 km. away from Essar Jetty.
Longitude
Latitude
&
2234’171”N
7003’421”E
2234’363”N
7010’918”E
2231’277”N
6948’590”E
2226’208”N
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Draft Monitoring And Analysis Protocol
Sea water sample of Tata Chemicals Ltd., Arambhda
6940’515”E
2226’167”N
6902’572”E
Regional office: Bhuj
Sea water sample @ 300 meter away from old mundra port, Tal. :
1
Mundra
Sea water sample @ 300 meter away from new mundra port, Tal. :
2
Mundra
22o43’43.40”N
69o42’10.54”E
22o44’14.37”N
69o43’11.90”E
22o49’21.70”N
3
Sea Water sample at Mandvi Port, Mandavi, Tal. : Mandvi
69o21’04.16”E
Sea water sample @ 300 meter away from Kandala Port, Tal. : 23o00’27.32”N
4
Gandhidham
70o13’21.37”E
Regional office: Rajkot
22o57’33.1”N
1
Sea water sample at Navlakhi port
70o26’38.1”E
23o11’43.2”N
2
Sea water sample of Surajbari creek (Road side)
70o42’59.5”E
The Prominent Sampling Locations thus covers Industrial Outfalls, Treated effluent drainage,
Port activities and other activities in the adjoining area (land mass) of the Gulf of Kutchh.
PARAMETERS MONITORED
Presently, the physico chemical parameters monitored are pH, DO, COD, BOD,
Electrical Conductivity in μmhos/cm, (NO2+NO3)-Nitrogen, Suspended Solids, Faecal Coliform
(MPN/100 ml), Total phosphorous, TKN, Total Ammonia (NH4+NH3)-Nitrogen, Salinity,
Phenols, Oil and Grease and Heavy metals are monitored.
Monitoring and analysis of Parameters like Dissolved / dispersed hydrocarbons in sea
water, Chlorinated hydrocarbons, Pesticide residues, Bottom sediments (for Heavy Metals),
Particulate organic carbon (POC) and particulate nitrogen (PN), Dissolved metals and ions,
Petroleum hydrocarbons and Total organic carbon would be carried out after the Sophisticated
Analytical Instruments are Procured under the Project and Installed at various Laboratories of the
Board.
FREQUENCY OF SAMPLING
Monitoring of coastal waters from above locations has been started from October 2010
with frequency of sampling once in a month for each monitoring location. It has been decided
during meeting with S.P.M.U that first year data will be a base line data.
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METHOLOGY USED FOR SAMPLING
Niskin water samplers are used in the field to collect Surface and Bottom water samples at
each station. Similarly Sediment Grab sampler would be utilized for collection of sediment
samples from the bottom. GPSs are utilized to precisely fix the sampling stations with respective
Latitudes and Longitudes. This helps collecting the water / sediment samples from the same
locations each time. Moreover,
For depth < 5 meters, only surface water sample is to be collected; for depth between 5 to
10 meters both surface and bottom water samples are to be collected whereas for depth > 10
meters surface, middle and bottom water samples are to be collected.
Parameters like B, F and CN are to be analysed only if there is an expected source,
otherwise they can be dropped. COD is to be done only for ‘Source’ stations and not for ‘Sink’
Stations. Silica is to be done for all stations i.e. new parameter Silica/silicates is to be
incorporated for analysis.
METHOLOGY USED FOR ANALYSIS
Methods prescribed in Standards Methods for the Examination of Water and Waste Water
prepared and published by APHA, AWWA are used by the Regional Offices of the Board for
analysis of sea water samples. However, Coastal Water Quality Measurements Protocol for
COMAPS programme prepared by Integrated Coastal and Marine Area Management (ICMAM)
Directorate, Chennai is also referred and incorporated appropriately in this Draft Protocol. The list
of references for various analytical methods appears at the end of this draft protocol.
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COLOR
METHOD:- Visual Comparison Method
COLLECTION OF SAMPLE: Collect the sample in the plastic carbouy.
PRESERVATON : Ice.
APPARTUS:- Color Comparator, Color Disks.
REAGENTS:- Not Applicable
PROCEDURE:- Observe sample color by filling a matched nessler tube to the 50-ml mark with
sample and comparing it with standards. Look vertically downward through tubes toward a white
or spectral surface placed as such an angle that light is reflected upward through the columns of
liquid. If turbidity is present and has not been removed report as “Apparent Color”. If the color
exceeds 70 units, dilute the sample with D.W. in known proportion until the color is within the
range of the standards.
CALCULATION:- Color unit Pt. Co. Scale = Reading x Dilution
INTERFERENCE:- Turbidity
PRECISION & BIAS:- The color value of water is extremely pH dependent and invariably
increases as the pH of the water is raised. When reporting a color value, specify the pH at which
color is determined.
TEMPERATURE oC
METHOD :- A calibrated thermometer is allowed to stand in seawater sample and the reading is
recorded.
APPARTUS :- Certified thermometer: 0-50°C with 0.1° accuracy
Thermometer: 0-50°C having mercury thread, calibrated with the certified thermometer.
PROCEDURE :- Dip the thermometer in the seawater sub-sample drawn for
temperature measurements. This sub-sample to be drawn immediately after retrieving the
samples onboard. Record the temperature after 2 min.
Results: Report the reading in oC.
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TURBIDITY
METHOD:- Nephelometric
This method is based on a comparison of the intensity of light scattered by a sample and a
standard reference under same condition. Higher the intensity of scattered light higher the
turbidity.
COLLECTION OF SAMPLE:- Collect the sample in the plastic carbouy
PRESERVATION: Ice
APPARATUS:- Nephelo Turbidity Meter
REAGENTS:Soultion 1:- Dissolve 1.0 gm Hydrazine Sulfate and dilute to 100 ml with distilled water.
Solution 2:- Dissolve 10 gm hexamethylene tetra mine and dilute to 100 ml.
Solution 3:- Mix 5.0 ml of solution 1 with 5.0 ml of solution 2. Allow it to stand for 24 hours and
dilute to 1000 ml. This will have turbidity of 400 units.
Standards turbidity suspension: Dilute 10ml of solution 3 as prepared above to 100 ml to have
turbidity of 40 units.
PROCEDURE:- Switch on the instrument and select calibration curve for desired range. Place
the filter frame in position. Fill the sample in clean and dry turbid meter tube up to the mark and
then lower the plunger into the sample tube carefully, Place it in the circular groove of the mirror
tube. Close the door of the apparatus. Switch on the ON/OFF switch. Immediately balance the
light intensity of the central spot with the surrounding field with the dial knob and read the scale
on the dial.
CALCULATION:- Determine the turbidity directly from the selected graph and it dilution is
made multiple by dilution factor.
INTERFERENCE:- Dirty glassware, air bubbles, debris and the coarse sediments.
PRECISION & BIAS:- When comparing water treatment efficiencies, do not estimate turbidity
more closely than specified. Uncertainties and discrepancies in turbidity measurement make it
unlikely that result can be duplicated to greater precision than specified.
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pH
METHOD :- Electrometric
The pH is determined by measurement of the electromotive force of a cell comprising an indicator
electrode, an electrode responsive to hydrogen ions (glass electrode) immersed in the test solution
of a reference electrode. Contact between the test solution of the reference solution is achieved by
means of a liquid junction, in the reference electrode. A difference of 1 pH unit produces a
potential change of 58.16 mv at 25°C. The electromotive force is measured with a pH to read
directly as pH value.
COLLECTION OF SAMPLE :- Collect the sample in the plastic carbouy.
PERSERVATION : - Ice
APPARATUS:- pH Meter, Glass Electrode, Reference electrode (Combined electrode).
REAGENTS :- Std. Buffer solution. of pH-7, pH-4 & pH-9. This can be prepared from buffer
tablets, which are commercially available or readymade standard solutions of pH 4, 7 and 9.
PROCEDURE :- Rinse the electrode with Distilled Water and dry by gentle wiping with a soft
tissue paper. Standardize the instrument with the electrodes immersed in a Buffer solution with a
pH close to that of the water sample to be tested.
Take different Samples in beaker and put the electrode in baker and note down the stable reading
at relative temperature.
CALCULATION :- Instrument gives direct reading.
INTERFERENCE :- The glass electrode is relatively free from interference from color, turbidity,
colloidal matter, oxidants, reductions, or high salinity, except for a sodium error at pH >10.
Reduce this error by using special “low sodium error” electrodes. pH measurements are affected
by temperature. The Nernstian slope increases with increasing temperature and electrodes take
time to achieve thermal equilibrium. This can cause long term drift in pH. Because chemical
equilibrium affects pH, standard pH buffers have a specified pH at indicated temperatures.
RESULT :- Report temperature at which pH is measured.
PRECISION & BIAS :- By careful use of a Laboratory pH meter with good electrodes, a
precision of ± 0.02 pH unit and an accuracy of ± 0.05 pH unit can be achieved. However, ± 0.1
pH unit represents the limit of accuracy under normal conditions, especially for measurement of
water samples and poorly buffered solutions. For this reason report pH values to the nearest 0.1
pH unit.
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CONDUCTIVITY
METHOD:- Instrumental Method
PRINCIPLE:- It is defined as the ability of an aqueous solution to carry an electric current. This
Ability depends on the presents of ions, on their total concentration, mobility, and Valance and on
the temperature of instrument.
COLLECTION OF SAMPLE:- Collect the sample in the plastic carbouy.
PRESERVATION:- Ice
APPARATUS:- Conductivity Meter
REAGENTS:1) 0.1 M KCL
2) 0.01 M KCL
3) 0.001 M KCL
7.456 GM 1 Liter distilled water
0.1 Mm KCL 10ml
make up to 100 ml with distilled Water
0.01 M KCL 10 ml
make up to 100 ml with distilled Water.
PROCEDURE:DETERMINATION OF CELL CONSTANT Rinse the conductivity cell with at least three
portions of 0.01 M KCL solution. Adjust the temp of a fourth portion to 25
0.1 ° C. Measure
resistance of this portion and note the temperature. Compute cell consent, C.
CALCULATION:Specific conductance of N/ 100 KCL solutions in µmhos/ cm
Cell Consent C = --------------------------------------------------------------------------------Measured conductance of N /100 KCL solutions in µmhos /cm
It is easy to calculate the cell constant from the measurement of the conductance (or Resistance) of
a solution of known “specific conductance”
Specific conductance =
(Mhos/cm of the
Sample at 25 °C)
Cell Constant
-----------------------------------------measured resistance in ohms
INTERFERENCE:- The Conductivity result mainly depends on the cell constant and hence cell
error in constant determination will cause error in conductivity.
PRECISION & BAIS:- The precision of commercial conductivity meter is commonly between
0.1 to 1.0 % reproducibility of 1 to 2 % is expected after an instrument has been calibrated.
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TOTAL SUSPANDED SOLIDS
METHOD:- Filtration Method
COLLECTION OF SAMPLE :- Collect the sample in plastic Carbouy.
PRESERVATION :- Ice
APPARATUS :a) Conical flasks
b) Funnel
c) Measuring Cylinder
d) Glass fiber filter paper (Whatman GF/c 47 mm diameter circles)
e) Vacuum Assembly
f) Oven
REAGENTS :- Not Applicable
PROCEDURE:- Assemble filtering apparatus & filter and beign suction wet filter with a small
volume of distilled water to seal it. Filter a measured volume well mixed sample through the glass
fiber filter paper. Wash with three successive 10 ml volume of distilled water, allowing complete
drainage between washing and continue suction for about three minutes after filtration is
complete. Carefully remove filter from filtration apparatus with the help of clean forceps and
transfer to Petri dish. Dry for at least 1 hour at 103 °C to 105 °C in an oven, cool in desiccators
before weighing.
Repeat the cycle of drying, cooling, desiccating and weighing until a constant weight is obtained
or until the weight loss is less than 4 % of the previous weight, or 0.5 mg whichever is less.
CALCULATION ;(B - A) 106
TSS mg/l = ----------------------------------Sample taken (ml)
Where,
A = Weight of empty filter paper
B = Weight of filter + dried residue
PRECISION AND BAIS :- Single laboratory duplicate analysis of 41 samples of water and
waste water were made with a standard deviation of differences of 6.0 mg/l.
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TOTAL DISSOLVED SOLIDS
METHOD:- Filtration Method
COLLECTION OF SAMPLE :- Collect the sample in plastic carbouy.
PRESERVATION:- Ice
APPARATUS:1. Conical flask
2. Funnel
3. Beaker
4. Measuring Cylinder
5. Oven
6. Balance
REAGENTS:- Not Applicable
PROCEDURE:- Filter measured volume of well mixed 50 ml sample through glass fiber filter,
wash with three successive 10 ml volumes of distilled water, allowing complete drainage between
washings and continue suction for about 3 minutes after filteration is complete. Transfer filtrate to
a weight evaporating dish or a beaker and evaporate to dryness on a steam bath. If filtrate volume
exceeds dish capacity add successive portions to the same dish after evaporation. Dry for at least
24 hour in an oven at 180 °C
2 °C, cool in a desiccator before weighing. Repeat drying cycle of
drying, cooling, desiccating and weighing until a constant weight is obtained or until weight lost is
less than 4 % of pervious weight or 0.5 mg. whichever is less.
CALCULATION:(A - B) 1000
TDS mg/l= ------------------------------------Sample taken (ml)
Where,
A= Weight of dried residue + beaker or evaporating dish (crusible)
B= Weight of empty beaker or evaporating dish (crusible)
INTERFERENCE:- N.A.
PRECISION AND BAIS:- Single Laboratory analysis of 77 samples of a known of 293 mg/l
were made with a standard deviation of differences of 21.20 mg/l.
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CHLORIDE
METHOD :- Argentometric method (Titrimetric method)
COLLECTION OF SAMPLE :- Collect the sample in the plastic carbouy.
PRESERVATION :- Ice
APPARATUS :- Conical Flasks, Burette, Pipette, Funnels
REAGENTS :1. Standard AgNO3 Solution (Titrant) (0.0282 N) :- 4.79 gm AgNO3 in 1 liter distilled water.
2. Standard NaCl (dried at 140 °C) 1.648 gm in 1000 ml distilled water.
3. Potassium Chromate (K2CrO4) Indicator Solution:- 50 gm K2CrO4 + little volume of distilled
water. Add AgNO3 until definite red precipitates appears, let it stand for 12 hours, then filter &
dilute with 1000 ml distilled water.
PROCEDURE :- Take pretreated sample (Adjust to neutral pH) & add 1 ml Potassium chromate
indicator & titrate against 0.0282 N Silver nitrate.
Also run the Charcoal / reagent blank. Color change – yellow to reddish orange.
In case of coloured sample, give charcoal treatment /3 ml of Al(OH)3 suspension to remove
colour from sample and then filter it.
CALCULATION :B.R. Normality of silver nitrate X 35450
-
Chloride (Cl ) mg/l = ------------------------------------------------------Sample Taken (ml)
Dilution
INTERFERENCE :- Sulfide, Thiosulfate, Sulfite ions, and Orthophosphate in excess of 25 mg/l,
Iron in excess of 10 mg/l interferes.
PRECISION AND BIAS :- A synthetic sample containing 241 mg CI/L, 108 mg Ca/l, 82 mg
Mg/L, 3.1 mg – K/L, 19.9 mg Na/L, 1.1 mg NO3- N/L, 0.25mg NO2-N/L, 259 mg SO4/L and 42.5
mg. Total alkalinity/L (Contributed by NaHCO3) in distilled water was analysed in 41 laboratories
by the Argentometric Method with a relative standard deviation of 4.2 % and a relative error of
1.7%.
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OXYGEN (DISSOLVED)
METHOD :- Winkler ‘s Method – Azide modification
Dissolved Oxygen is the quantity of oxygen dissolved in water/ wastewater
COLLECTION OF SAMPLE :- a) Collect the sample in 300 ml BOD bottle. b) Avoid contact
with air and agitation.
PRESERVATION :a
Add 2 ml MnSO4 (Manganese Sulphate) solution
b
Add 2 ml Alkali iodide azide solution
c
Shake well and allow settling (brown ppt.)
d
Store at low temperature
APPARATUS :- BOD Bottles, Burette, Pipette, 100ml measuring cylinder and 250 ml Flask.
REAGENTS :a. Manganese Sulphate solution: Dissolve 480 gm MnSO4 4H2O or 400 gm MnSO4 or 364
gm MnSO4 in 1 lit distilled water.
b. Alkali – Iodide Azide regent: Dissolve 500 gm NaOH (or 700 gm KOH) + 135 gm NaI (or
150 gm KI) in 1 liter distilled water + 10 ml NaN3 (Sodium Azide) in 40 ml of Distilled
Water.
c. Starch solution:
d. Conc. H2SO4
e. 0.1 N Na2S2O3 (Std. Na2S2O3 Stock solution): 24.82 gm Na2S2O3 5H2O
f. 0.0125 N Na2S2O3 Working Standard Solution: 125 ml stock solution & make upto 1 liter
with distilled water.
PROCEDURE :- Add 2 ml conc. H2SO4 mix to dissolve brown precipitate and take 100 ml out
of 300 ml in 250 ml flask & titrate with 0.0125 N Na2S2O3. When pale straw color resists add 1-2
ml starch solution and continue the titration to the first disappearance of the blue color & read the
final burette reading.
CALCULATION :- 1 ML 0.0125 N Na2S2O3 = 0.1 mg D.O.
D.O. =
0.1 ML reading 1000
------------------------------------100 ml (sample)
INTERFERENCE :- Reducing or oxidizing materials should be absent.
PRECISION AND BIAS :- D.O. can be determined with a precision expressed as a standard
deviation, of about 20 g/L in the distilled water and about 60 g/L in waste water and secondary
effluents. In the presence of appreciable interference, even with proper modifications, the standard
deviation may be as high as 100 g/L. Still greater errors may occur in testing waters having
organic suspended solids or heavy pollution. Avoid errors due to carelessness in collecting
samples, prolonging the completion of test, or selecting an unsuitable modification of the method.
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BIOCHEMICAL OXYGEN DEMAND
METHOD :- Winker’s Azide modification Method.
BOD is defined as the Oxygen required by the living organisms in the utilization and stabilization
of Oxygen matter present in the wastewater.
COLLECTION OF SAMPLE :- Collect the sample in 300-ml BOD bottles. Avoid contact with
air and agitiation.
PRESERVATION :- Ice.
REAGENTS:1. Phosphate Buffer Solution:8.5 gm KH2PO4 (Potassium Dihydrogen Orthophosphate) + 21.75 gm K2HPO4 (Dipotassium Hydrogen Orthophosphate) + 33.4 gm Na2HPO4.7H2O (Disodium hydrogen
phosphate) + 1.7 gm NH4Cl (Ammonium chloride)
Make up volume to 1 liter with
distilled water.
2. Magnesium Sulphate solution (MgSO47H2O):Dissolve 22.5 gm MgSo4 7H2O in 1 liter distilled water.
3. Calcium Chloride Solution (CaCl2):Dissolve 27.5 gm CaCl2 in 1 liter distilled water
4. Feric Chloride Solution (FeCL3 6H2O):Dissolve 0.25 gm FeCl3 6H2O in 1 liter distilled water.
5. Acid – alkali solution 1N:(A) Dilute 28 ml Conc. H2SO4 in 1 liter distilled water.
(B) Dissolve 40 gm NaOH in 1 liter distilled water.
6. Glucose – Glutamic Standard Solution:150 gm Glucose + 150 mg Glutamic acid in 1 liter distilled water.
7. Manganese Sulphate Solution (MnSO4) :480 gm MnSO4 4H2O or 400 gm MnSO4 or 364 gm MnSO4 H2O in 1 liter distilled
Water.
8. Alkali – Iodide – Azide Solution :500 gm NaOH or (700 gm KOH) + 135 gm NaI or (150 gm KI) in 1 liter distilled water +
10 ml NaN3 (sodium azide) in 40 ml distilled water.
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9. Conc. H2SO4:10. Starch Solution:2.0 gm starch + 0.2 gm salicylic acid as a preservative in 100 ml hot distilled water.
11. 0.0125N K2Cr2O7 (Potassium Di- Chromate) :0.613 gm K2Cr2O7 (Potassium Di- Chromate) in 1 liter distilled water.
12. 0.1 N Na2S2O3 (Sodium Thiosulphate) :24.82 gm Na2S2O3 5H2O in 1 liter distilled water.
13. Working Sodium Thiosulphate Solution:0.0125 N Na2S2O3 125 ml stock solution & make upto 1 liter distilled water.
PROCEDURE :- Prepare dilution water by adding 1 ml each reagent 1,2,3,4, per liter in air
Saturated distilled water. Add 2 ml seed per liter, make two sets of several Dilution (800/ COD) of
the prepared samples, on one set determine an immediate oxygen demand. Incubate at 27°C the
blank dilution water and second set after 3 days measure the oxygen left as per dissolve oxygen
estimation method.
CALCULATION :Actual Difference in D.O. 100
BOD mg/l = -----------------------------------------------------------Sample Taken (ml)
Actual Difference = (0 Day D.O. – 3 Day D.O) – Blank difference
INTERFERENCE :- pH of the sample should be adjusted by adding alkali or acid in the range of
6.5 to 8.5. All samples may be sterile and will need seeding. For the purpose of oxidizing the
organic matter where such microorganisms are already present as in surface water, domestic
sewage or unchlorinated effluents etc. seeding is not required.
PRECISION AND BIAS :- The working range is equal to the difference between the maximum
initial D.O. (7 to 9) and minimum D.O. residual of 1 mg/l multiplied by the dilution factor. A
lower detection limit of 2 mg/l is established by the requirement for a minimum D.O. depletion of
2 mg/l.
For the 300 mg/l mixed primary standard, the average 3 Day BOD would be 198 mg/l with a
standard deviation of 30.5 mg/l.
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OIL AND GREASE
METHOD :-Partition – Gravimetric Method
COLLECTION OF SAMPLE :- Collect the sample in glass bottle.
PRESERVATION :- Conc. HCL (to acidity sample)
APPARATUS :- Hot plate , Separating funnel
REAGENTS :1. Hexane :50 ml per sample
2. Sodium Sulphate (anhydrous Na2SO4) :To absorb moisture
3. Sodium Chloride (NaCl):For emulsion.
PROCEDURE :- Take appropriate sample for analysis in the separating funnel (pH of sample
should be less than 2). Add 50 ml / 100 ml hexane to the sample. Mix well the sample in
separating funnel. Hexane soluble Oil & Grease will be separated from wastewater. Filter the
Hexane by 42 no. of filter paper (through Na2So4) in a dry pre weighed (A) beaker. Put on the hot
plate at 65 to 70° C to evaporate the Hexane. Afterwards cool it in desiccators and weight the
beaker having oil & grease residues (B).
CALCULATION :Weight different (B - A) 106
Oil & Grease mg/l = -------------------------------------------Sample Taken (ml)
INTERFERENCE :- Any filterable organic soluble substances will be extracted along with Oil
& Grease. Such as elemental, Sulfur and certain organic dyes.
PRECISION AND BIAS :- This method was tested by a single Laboratory on a raw sewage
sample using both the extraction solvents. By this method the oil & grease concentration was 20.8
mg/L with trichlorotrifluoroethane and 22.4 mg/L. With the 80:20 hexane / methyl – tert-butyl
ether mixture. When sample were dosed with 30 mg Fisher Heavy Mineral Oil, recovery of added
oil was 78.9 % with a standard deviation of 0.8 mg/L. for trichlorotrifluoroethane and 84.2% with
a standard deviation of 1.2 mg/L for hexane / methyl-tert-butyl ether.
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PHENOLIC COMPOUND
METHOD :- Colorimetric method (4- Amino Antipyrine method)
COLLECTION OF SAMPLE :- Collect the sample in the glass bottles.
PRESERVATION :- CuSO4 + Orthophosphoric acid
APPARATUS :- Spectrophotometer, Phenol distillation unit. Nessler tubes
REAGENTS :1) 0.5N Ammonium hydroxide (NH4OH):-Dilute 35 ml fresh Conc. NH4OH & make up to to
1000 ml distilled water.
2) Phosphate Buffer Solution:- Dissolve 104.5 gm K2HPO4 + 72.3 gm KH2PO4 in 1 liter with
distilled water.
3) 4- Aminoantipyrine Solution :-Dissolve 2.0 gm 4 – Aminoantipyrine in 100 ml distilled water.
4) Potassium Fericyanide Solution:- Dissolve 8.0 K3Fe (CN)6 in 100 ml distilled water.
PROCEDURE :- Distillate the preserved samples. Take appropriate ml of distilled sample make
it 100ml. Add 2.5 ml NH4OH soln. and Add 1 ml Phosphate buffer solution. Check the PH@ 7.9
to 9.0 then Add 1 ml 4 – Amino Antipyrine solution & Add 1 ml K3Fe(CN)6, Mix well and let
dark red color developed (residue) for 15 minutes then measure O.D. at 500 nm.
It is necessary to make standard curve before analyzing the sample. Also run the reagent blank.
CALCULATION :O.D Factor
Phenol mg/l = --------------------------------Sample Taken (ml)
Dilution
INTERFERENCE :- Interference such as phenol decomposing bacteria, Oxidizing & reducing
Substances and alkaline pH values are dealt with by acidification. Some highly contaminated
wastewater may require specialized technique for eliminating interference and for quantitative
recovery of Phenolic Compounds.
PRECISION AND BIAS :- Because the ‘ PHENOL’ value is based on C6H5OH, this method
yields only an approximation and represents the minimum amount of phenols present. This is true
because the phenolic reactivity to 4 – aminoantipyrine varies with the types of phenols present.
In a study of 40-refinery wastewater analysed in duplicate at concentration from 0.02 to 6.4 mg/L.
The average relative standard deviation was 12%. Data are not available for precision at lower
concentration.
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HEXAVALENT CHROMIUM
METHOD :- Colorimetric Method (Diphenylcarbazide)
COLLECTION OF SAMPLE :- Collect the sample in plastic carbouy.
PRESERVATION :- Conc. HNO3
APPARATUS :-Spectrophotometer
REAGENTS :1. Stock Chromium Solution :- Dissolve 141.4 mg K2Cr2O7 in distilled water & make 100 ml
with distilled water.
2. Standard Chromium Solution :- Dilute 1 ml stock solution to 100 ml. i.e. 1 ml = 5.00 g Cr
3. Sulphuric Acid :- 1:1 (i.e. 1 ml H2SO4 + 1 ml distilled water)
4. Phosphorbic Acid : Concentrated
5.Diphenyl Carbazide :- Dissolve 250 mg 1, 5 Diphenyl Carbazide in 50 ml acetone. Store in a
brown bottle.
PROCEDURE :- Remove the color of the sample. Take 50 ml sample, add 1:1 Sulfuric Acid,
3 to 5 drops of Orthophosphoric acid Dilute it to 100 ml with distilled water then add 2 ml D.P.C.
mix and let stand 5 to 10 minutes for full color development then take O.D.at 540 nm.
It is necessary to make a Standard Curve before the analysis of sample. Also run to the reagent
blank.
CALCULATION :O.D. Factor
100
Hexavalent Chromium (Cr ) mg/l = ----------------------------------------Sample Taken (ml)
+6
INTERFERENCE :- Hexavalent molybdenum and mercury salts, Vanadium iron, Ferric ion and
Copper etc. are also interferences.
PRECISION AND BIAS :- The dissolved Chromium was determined in 31 Laboratories with
synthetic sample containing 110 mg Cr/L, 500 mg Al/L, 50 mg Cd/L, 470 mg Ca/L, 300 mg Fe/L,
70 mg Pb/L, 120 Mn/L, 150 mg Ag/L and 650 mg Zn/L in the distilled water. The relative
standard deviation was 47.8% and relative error was 16.3%.
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CHEMICAL OXYGEN DEMAND
METHOD :- Open Reflux Method - Followed by Titrimetric Method
Chemical Oxygen Demand test determines the oxygen required for chemical oxidation of organic
matter with help of strong chemical oxidant.
COLLECTION OF SAMPLE :- Collect the sample in plastic carbouy.
PRESERVATION :- Conc. Sulfuric Acid
APPARATUS :- COD Digestion Apparatus
REAGENTS:a) Potassium DI- Chromate k2Cr2O7 (0.25 N):- 12.259 gm dried k2Cr2O7 in 1 liter distilled
water.
b) Ferrous Ammonium Sulphate :- 0.1 N: 39.2 gm FAS + 20 ml conc. H2SO4 & make upto 1
liter with distilled water.
c) Ferroin Indicator Solution :- 1.485 gm 1.10 phenenthroline monohydrate + 0.695 gm FeSO4
7H2O in 100 ml distilled water.
d) Potassium Hydrogen Phthalate (KHP) : 0.425 gm dried KHP in 1 liter distilled water.
PROCEDURE :- Take 10 ml Potassium dichromate in COD tube add 30 ml Conc. Sulfuric Acid
containing Ag2SO4, add 1 gm Mercuric Sulphate and appropriate ml of sample (maximum 20 ml
sample). Shake well the mixture and reflux for about 2 hours at 150 °C. Titrate against 0.1 N
Ferrous Ammonium Sulphate using Ferroin indicator. Note down the burette reading. Also run the
reagent blank.
CALCULATION :N of potassium Dichromate Vol. of K2Cr2O7
Normality of FAS = -----------------------------------------------------------------ml of FAS used
Actual. Difference Factor
(Factor = N of FAS 8000)
COD mg/l = ----------------------------------------------------------------------------Sample taken (ml)
INTERFERENCE :- Volatile straight – chain aliphatic compounds, Presence of halogens may
interfere. Chloride, Bromide and Iodide ions. Nitrite (NO2)
HALIDE INTERFERENE REMOVAL :- (A) By adding HgSO4 .(B) By calculation – 1 mg
Chloride contribute 0.23 mg COD. (C) Removal of Halide by adding AgNO3 & Ag2SO4. (D)
NO2 by adding sulfamic acid in K 2Cr2 O7.
PRECISION AND BIAS :- A set of synthetic samples containing KHP and NaCl was tested by
74 laboratories at a COD of 200 mg O2/l in the absence of Cl, the standard deviation was 13
mg/l (Coefficient of variation 6.5%) at COD of 160 mg O2/l and 100 mg Cl/l. The standard
deviation was
14 mg/l (Coefficient of variation 10.8 %) .
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SULPHATE
METHOD :- Turbidimetric Method
COLLECTION OF SAMPLE :- Collect the sample in the plastic carbouy
PRESERVATION :- Ice
APPARATUS :Conical flask, pipette
Magnetic stirrer
Spectrophotometer
REAGENTS :1. Conditioning Reagent:- 30 ml Conc. HCl + 300 ml distilled water + 100 ml 95% Ethanol or
Isopropyl Alcohol + 75 gm NaCl + 50 gm Glycerol.
2. BaCl2 powder or saturated solution of BaCl2
3. Standard Sulphate Solution:- Dissolve 0.1509 gm anhydrous Na2SO4 in distilled water & dilute
it to 1000 ml with distilled water.
PROCEDURE :- Take 50 ml Distilled water in the flask and add 5 ml Conditioning Reagent and
one spatula BaCl2 powder ( or 2 ml of saturated BaCl2 solution ). Put the prepared flask on
magnetic stirrer and add the sample until turbidity appears. Then add distilled water (Total volume
of sample and distilled water must be equal to 50ml) & Measure turbidity on 420 nm.
It is necessary to make a Standard Curve before the analysis of samples. Also run the reagent
blank.
CALCULATION :O.D. Factor 100
SO4 MG/L = ----------------------------------Sample Taken (ml)
Dilution
INTERFERENCE :- Silica in excess of 500 mg/l will interfere and large quantities of organic
materials do not give satisfactory precipitation of BaSO4.
PRECISION AND BIAS : With a turbidimeter in a single laboratory, with a sample having a
mean of 7.45 –mg SO4/L a standard deviation of 0.13 mg/L and a coefficient of variation of 1.7
were obtained. Two sample closed with sulfate gave recovery of 85% & 91%.
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CYANIDE
METHOD :- Colorimetric
COLLECTION OF SAMPLE :- Collect the sample in plastic carbouy or glass bottle.
PRESERVATION :- NaOH palates
APPARATUS :- Cyanide distillation unit, Spectrophotometer, Specific Ion meter.
REAGENTS :a)
b)
c)
d)
e)
f)
Sodium Hydroxide (1.25%) :- 50 gm NaOH in 1 liter distilled water (for distilation)
Magnesium Chloride :- 510 gm MgCl2 in 1 liter distilled water.
Sulfuric Acid :- 1:1 (i.e. 1 ml Conc. H2SO4 + 1 ml distilled water)
Chloramine –T : 1 gm Chloramine – T in 100 ml distilled water.
Sodium Dihydrogenphosphate (1 M) :- 138 gm NaH2PO4H2O in 1 liter distilled water
Pyridine –Barbituricacid:- 15 gm Barbituricacid + 75 ml pyridine + 15 ml Conc. HCl &
make upto 250 ml with distilled water.
PROCEDURE :1.PRELIMINARY DISTILLATION METHOD :- Take 100 ml preserved (in NaOH) sample in
the boiling (250 ml round bottom) flask. Add 20 ml of MgCl2 solution. Then add 50 ml of 1:1
H2SO4. Take 50 ml 1.25 N of NaOH solution. to the absorbing tubes. (Impingers 2 Nos. in series).
Adjust suction so that approximately 1 air bubbles enters the boiling flask. This air rate will carry
HCN gas from above flask to alkali absorber to convert in NaCN. Then heat the solution in the
round bottom flask for one & half hour. After that mix all above impingers absorber soln. in 250
ml volumetric flask and make the volume 250 ml by D.W. Afterwards if CN is above 1 mg than
analyse it by titrimetric method using rhodamine indicator against 0.0193 N AgNO3 soln. If
concentration is lower; then follow below mentioned colorimetric method or selective Ion
electrode method.
2. COLORIMETRIC METHOD :- Cyanide in the alkaline distillate from preliminary treatment
is converted to CNCl by reaction with Chloramine – T at pH less than 8 without hydrolyzing to
CNO. After the reaction is complete CNCl forms red blue dye on addition of a pyridine Barbituric
Acid. The absorbance is read at 578 nm.
Take distilled 50 ml sample and make volume 250 ml, from this take 50 ml diluted sample add 4
ml buffer soln. & 1 ml Chloramine – T soln. Let it stand for 2 minutes and add 4 ml of pyridine
barbituric acid soln. Take O.D. at 578 nm, after 8 minutes and before 15 minutes.
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CALCULATION :O.D. Factor Total vol. of abs. media
1000 for ltr
CN mg/L = ----------------------------------------------------- ---------------------------Original volume of sample distilled
Taken for distillation
INTERFERENCE :- All known interference are eliminated or reduced to a minimum by
Distillation.
PRECISION AND BIAS :- As a quality control measure, periodically test apparatus, reagents
and other potential variable in the concentration range of interest. As an example at least 100 4%
recovery from 1 mg CN/l standard should be obtained ( Total CN after distillation ).
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FLUORIDE
METHOD :- Ion – Selective Method
The classical ion- selective electrode consists of a tube made of a good electrical insulator, which
is closed at its lower end by a sensing membrane. Within the tube there is a filling solution known
as internal filling solution containing a fixed column of the ion to which the membrane is
sensitive.
COLLECTION OF SAMPLE :- Collect the sample in plastic carbouy.
PRESERVATION :- Ice
APPARATUS :1. Ion Selective Meter
2. Fluoride Electrode
3. Magnetic Stirrer
REAGENTS :For TISAB :- Take 500 ml distilled water + 170 gm NaNO3 + 68 gm Sodium acetate trihydrate +
92.4 gm Tri Sodium Citrate and make the volume upto 1 litre with distilled water.
PROCEDURE :Adjust the pH of sample between 7 to 8 and take 20 ml of sample, add 20 ml of TISAB reagent.
Put two appropriate range of standards. The slope should be between 50 to 60. Then analyze the
sample. The instrument shows results directly in mg/l.
CALCULATION :Fluoride concentration can be measured in units of moles per liter, equivalents per liter, parts per
million, or any convenient concentration unit.
INTERFERENCE :Fluoride forms complexes with several polyvalent cations notably aluminium and iron. In acid
solution fluoride forms a poorly ionized HF. HF complex but the buffer maintains a pH above 5 so
as to minimize hydrogen fluoride complex formation. In alkaline solution hydroxide ion also can
interfere with electrode response to fluoride ion whenever the hydroxide ion concentration is
greater than one tenth the concentration of fluoride ion.
PRECISION AND BIAS :A synthetic sample containing 0.850 mg/L, F- in distilled water was analysed in 111 laboratories
by the electrode method, with a relative standard deviation of 3.6% and a relative error of 0.7%.
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BORON
METHOD :- Curcumin Method (Colorimetric Method)
COLLECTION OF SAMPLE :- Collect the sample in plastic carbouy.
PRESERVATION :- Ice
APPARATUS :a) Spectrophotometer
b) Porcelain evaporating dish
c) Water bath
REAGENTS :1. Curcumin Reagent :Dissolve 40 mg Curcumin + 5 gm Oxalic Acid + 80ml Conc. HCL in 100 ml ethyl alcohol.
2. IPA (Isopropyl Alcohol)
PROCEDURE :- Take the sample in evaporating dish & add 4 ml Curcumin reagent - Evaporate
in a water bath at 55° C For about 80 minutes. Dissolve the residue in 95 % Isopropyl alcohol and
make up the volume 25 ml with it. Measure O.D. at 540 nm within 1 hour after red color is
developed. Prepare a standard curve before analysis.
CALCULATION :O.D. Factor
Boron mg/l = -----------------------------Sample Taken (ml)
INTERFERENCE :- NO3-N Concentration above 20 mg/l and Hardness above 100 mg/l will
interference.
PRECISION AND BIAS :- A synthetic sample containing 240 Boron/L, 40 µg As/L, 250 µg
Be/L, 20 µg Se/Land 6 µg V/L in distilled water was analyzed in 30 Laboratories by the Curcumin
method with a relative standard deviation of 22.8 % and a relative error of 0%.
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MANGANESE (Mn)
METHOD :- Colorimetric (perssulfate method)
COLLECTION OF SAMPLE :- Collect the sample in the plastic carbouy.
PRESERVATION :- Ice
REAGENTS :1. Special Reagent:- Dissolve 75 gm mercuric sulphate + 400 ml Conc. HNO3 + 200 ml Distilled
Water + 200 ml 85% Phosphoric Acid + 35 gm Silver nitrate & make up 1 litter with distilled
water.
2. H2O2:- 2 drops per sample.
3. Ammonium Persulfate : 2 gm per sample.
APPARATUS :- Spectrophotometer, conical flask - 100 ml capacity, Nessler’s tubes – 100 ml
capacity.
PROCEDURE :- Take sample of 50 ml or 100 ml in conical flask. Add 5 ml Special reagent & 1
drops H2O2 and concentrated to 90 ml by boiling or dilute to 90 ml. Add 1 gm (NH4)2S2O8 bring to
a boil and boil for 1 minute (do not heat on water bath), cool it, dilute it 100 ml after taking it in
nessler tube and after reddish pink color is developed, Compare it with blank and take reading at
525 nm.
It is necessary to make a std. Curve before the analysis of sample. Also run the reagent blank.
CALCULATION :O.D. Factor
Manganese (Mn ) mg/l = ----------------------------dilution
Sample Taken (ml)
++
INTERFERENCE :- Bromide and Iodide, Air interference makes Mn to MnO2.
PRECISION AND BIAS :- A synthetic sample containing 120 µg Mn/L, 500 µg Al/l, 50 µg
Cd/L, 110 µg Cr/L, 470 µg Cu/L, 300 µg Fe/L, 70 µg Pb/L, 150 µg Ag/L and 650 µg Zn/L. in
distilled water was analyzed in 33 Laboratories by Persulfate method with relative standard
deviation of 26.3% and a relative error of 0%. A second sample similar in all respect except for 50
µg Mn/l and 1000 µg Cu/L was analyzed in 17 Laboratories by same method with a relative
standard deviation of 50.3% and relative error of 7.2%.
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AMMONICAL NITROGEN & TOTAL KJELDAHL NITROGEN
METHOD :- Distillation / Nasslerisation
COLLECTION OF SAMPLE :- Collect the sample in the plastic carbouy.
PRESERVATION :- Conc. Sulfuric acid
APPARATUS :- Ammonia distillation apparatus
REAGENTS :1. Sodium Hydroxide (NaOH) 6 N :- 240 gm NaOH in 1 liter distilled water
2. Boric Acid Solution :- 20 gm H3BO3 in 1 liter Ammonia Free distilled water
3. Mixed Indicator Solution :- Dissolve 200 mg (0.2 gm) methyl red indicator in 100 ml
95% ethyl or isopropyl alcohol. Dissolve 100 mg (0.1 gm)
methylene blue in 50 ml 95% ethyl or ethyl alcohol,
combine above two indicator solutions. Prepare monthly.
4. 0.1 N H2SO4 (Stock) : Take 2.8 ml H2SO4 in 1 liter distilled water.
5. 0.02 N H2SO4 :- Take 200 ml stock solution & make up 1000 ml with distilled water.
6. 0.02 N Na2CO3 :- Take 1.065 gm Na2CO3
dissolve in 1 liter distilled water to
standardize with 0.02 N H2SO4
PROCEDURE :FOR NH3-N :Take 50 ml Sample. Add 2 drops phenopthalein indicator and 6N NaOH – distilled in
Boric acid added with mix indicator. If ammonia is present Boric acid will be convered into blue
to green. Titrate against 0.02N H2SO4 till the color change from green to blue.
FOR T.K.N.:Take appropriate volume of water sample. Add 10 ml sulfuric acid, add 5 gm CuSO4, 5 gm
K2SO4 and digest upto 370° C for @ 2 hours and follow procedure as per NH3- N.
FOR NESSLERISATION:Take 50 ml sample and 1 ml Nessler reagent. Wait for 10 minute until colorless to yellow
color is developed and take O.D. at 425 nm. It is necessary to make a Standard Curve before the
analysis of sample. Also run the reagent blank.
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CALCULATION :-
For NH3- N mg/l
& T.K.N.
=
B.R. 14 0.02 1000
---------------------------------------------------Sample Taken (ml)
O.D. Factor
For Nesslerisation method NH3- N = -----------------------------------Sample Taken (ml)
INTERFERENCE :- Glycine urea, Glutamic acid and acetamide hydrolyze very slowly in
solution on standing but, of these, only urea and cyanates will hydrolyze on distillation at pH of
9.5. Hydrolysis amounts to about 7 % at this pH for urea and about 5% for cynates. Volatile
alkaline compounds such as hydrazine and amines will influence titrimetric results. Residual
chloride reacts with ammonia; remove by sample pretreatment. If a sample is likely to contain
residual chlorine, immediately upon collection, treat with dechlorinating agent. (Dissolve 3.5 gm
Sodium Thiosulfate in 1000 ml distilled water.)
PRECISION AND BIAS :- Synthetic samples containing ammonia and other constituents
dissolved in distilled water were analysed by titration method.
Sample 1 contained 200 g NH3- N/L, 10 g Cl/L, 1 g NO3- N/L, 1.5 g organic – N/L, 10 g
PO4/L and 5 g silica/L. The relative std. Deviation and relative error for the 21 participating
Laboratories were 69.8% and 20% respectively.
Sample 2 contained 800 g NH3- N/L, 200 g Cl -/L, 1.0 mg NO3-N/L, 0.8 g organic N/L, 0.5
g PO4 3/L, and 15.0 g silica /L. The relative standard deviation and relative error for the 20
participating laboratories were 28.6% and 5% respectively.
Sample 3 contained 1500 g NH3- N/L, 400 g Cl/L, 1.0 g NO3- N/L, 0.2 g organic N/L, 0.5
g PO4 3/L, and 30.0 g silica/L. The relative standard deviation and relative error for the 21
participating laboratories were 21.6% and 2.6% respectively.
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NITRATE – NO3-N
METHOD :- Colorimetric Method (Phenol Di-Sulphonic Acid Method)
COLLECTION OF SAMPLE :- Collect the sample in plastic carbouy.
PRESERVATION :- Ice
APPARATUS :a) Beaker – 100ml cap.
b) Nessler Tube – 100-ml cap
c) Hot air oven
d) Spectrophotometer
REAGENTS :a) P.D.A.(Phenol Disulphonic Acid)
b) Ammonia Solution
PROCEDURE :- If necessary decolorize the sample and take 50 ml sample in 100 ml capacity
beaker and dry at 105°C in oven for 12 hrs. After cooling add 2 ml PDA solution and rub the
residue thoroughly. Dilute with 20ml distilled water. Transfer in 100 ml. Nesseler tube then add
10 ml Liquor Ammonia solution and make up final volume 100 ml with distilled water. Measure
O.D. at 410nm.
It is necessary to make a std. Curve before the analysis of sample. Also run the reagent blank.
CALCULATION :O.D. Factor
NO3-N mg/l = ------------------------------Sample Taken (ml)
INTERFERENCE :- Chlorides interfere seriously with the determination of nitrates because of
their recycling action.
PRECISION AND BIAS :- Accuracy of the order of 0.1 mg/l NO3-N can be obtained only by
the proper treatment of the chloride and nitrite interference.
A synthetic unknown sample containing 1 mg/L NO3-N, 10 mg/L Cl, 200 mg/L NH3-N, 1.5 mg/L
Organic-N, 10 mg/L PO4 and 5 mg/L silica in the distilled water was determined by
phenoldisulfonic method with a relative standard of 74.4% and relative error of 38% in 46
Laboratories. A second synthetic unknown sample containing 1 mg/L, Nitrate – N, 200 mg/L, Cl,
800 mg/L NH3-N, 800 mg/L organic-N, 5 mg PO4 and 15 mg/L, silica in distilled water was
determined by the Phenoldisulfonic acid method with a relative standard deviation of 57.9% and a
relative error of 31% in 46 Laboratories.
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PHOSPHATE
METHOD :- Stannous Chloride Method
COLLECTION OF SAMPLE :- Collect the sample in the plastic carbouy
PRESERVATION :- Ice
APPARATUS :- Spectrophotometer
REAGENTS :1. Strong Acid :- 300ml. Conc. H2SO4 600 ml Distilled water. 4 ml Conc. HNO3. Make up 1 liter
with distilled water.
2. Ammonium Molyblate : 25 gm Ammonium Molyblate + 175 ml distilled water + 280 ml Conc.
H2SO4 + 400 ml Distilled water & make up 1 liter with distilled water.
3. Stannous Chloride :- Dissolve 2.5 gm SnCl2 2H2O
100 ml Glycerol.
PROCEDURE :- Take 100 ml of sample in conical flask. Add 4 ml strong acid & digest it on hot
plate upto 15 to 20 ml and cool it. Then maske volume to 100 ml in nesseler tube with distilled
water and add 4 ml ammonium molybdate and add 10 drops SnCl2 Now put it 10 minutes for color
development. Blue color will obtain from colourless. It PO4 is present measure O.D. at 690 nm
It is necessary to make a Standard Curve before the analysis of sample. Also run the reagent blank.
CALCULATION :-
PO4 mg/l =
O.D. Factor
------------------------------Sample Taken (ml)
INTERFERENCE :- Silica and Arsenate cause positive interference. Only if the sample is heated
Arsenite Flouride, Thorium, Bismuth, Sulfide, and Thiosulphatethiocynate or excess molybdate
cause negative interferences.
PRECISION AND BIAS :- The minimum detectable concentration is about 3
sensitivity at 0.3010 absorbance is about 10 g/L for an absorbance change of 0.009.
g /L. The
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HEAVY METALS (Cu, Pb, Ni, Zn, Fe, Total Cr, Cd, Hg,).
There are several methods available for analysis of trace Metals Heavy Metals covering both
Chemicals and Instrumental Methods. Some of them are as under:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Colourimetry.
Atomic Emission Spectrometry.
Atomic Absorption Spectrometry.
U.V. Spectrometry.
Inductive Couple Plasma Atomic Absorption Spectrometry.
Infrared Spectrometry.
Electro-Chemical Method (Electrometry)
Neutron activation
Fluorimetry.
The above analytical methods are being used for the metal – trace metal analysis in the field of
Environmental for Water, Waste water, air, solid waste - hazardous waste etc.
1. SAMPLE COLLECTION, PRESERVATION AND PREPARATION
One of the most important part if trace metal-metal analysis is the sample collection, preservation
and preparation of the sample
The objective of sampling is to collect a portion of material small enough in volume to be
transported conveniently and yet large enough for analytical purpose, while still accurately
representing the material being sampled.
The sample should be handled in such a way that no significant changes in the composition occur
before the tests are made.
General Requirement:
Obtain a sample that meets the requirements of sampling programme and handle it so that is does
not deteriorate or become contaminated before it is analysed.
Ensure that all sampling equipments ( containers- bottles, buckets, rnug, funnel etc.) is clean and
quality assured before use.
Use sample container that are clean and free of contaminants and also should be free from the
contamination of the metals to be analysed.
Fill the samples in cleaned and well washed containers before that rinse the sample container.
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Moreover one important factor which effect the analysis is that you have to clean your sample
container and the glassware with the tape water, neutral detergent, distilled water and with the
dilute Nitric Acid.
Prior to sampling it is essential to rinse the sample containers and other material to be used for the
sample collection should be rinsed with the 1+1 HNO3
Generally trace-metal samples except mercury should be collected in the plastic good quality, PEPloy Ethylene containers. The plastic material should not react with sample of metals i.e with the
analyte (the metal to be analysed) which are to be analyse.
For General metals-Minimum sample size-volume should be 1000 ml should be collected.
To analyse the dissolved metals is the sample of water and waste water sample should be filtered
immediately.
For Chromium VI the sample should be collected in the same type of container but the volume of
the sample to be collected is recommended is 100 ml and maximum storage period is 24 hours.
For Mercury follow the same procedure for sample collection as described for general other metal
moreover, one important factor which effect the analysis is that you have to clean your sample
container and the glassware with the tape water, neutral detergent , distilled water and with the
dilute Nitric Acid.
All the samples for which the metal analysis is to carried out should be preserved with Con. HNo 3
(Nitric Acid) to pH <2
By preserving the sample you can minimize the precipitation and adsorption of the metals on the
container’s wall.
Generally 1.5 ml/Lit. Conc. HNO3 or 3 ml/Lit. 1+1 HNO3 is to be used for the preservation of the
sample for metal analysis.
After the acidification of the homogenised sample preferably should stored in a refrigerator at
approx. 4 0C to prevent changes in volume due to evaporation.
To minimize the potential for volatization or bio degradation between sampling and analysis keep
samples as cool as possible
The best sample containers are made up of quartz or TFE. These containers are very expensive; it
is preferred to collect the samples in PE polyethylene container.
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2. PRELIMINARY TREATMENT OF THE SAMPLE:( Sample Preparation)
Samples containing organic or particulate material, generally required pre-treatment before
analysis “Total Metals” includes all metals, inorganically and organically bound, both dissolved
and particulate.
Colorless, transparent samples, turbidity of 1 NTU no odour and single phase may be analysed
directly by Atomic Absorption spectrometry or other method without digestion.
If the dissolved or suspended metals are to be determined, filter the sample at the time of
collection.
Extractable metals are lightly absorbed on particulate material, Because some sample digestion
may be unavoidable use rigidly controlled condition to obtain meaningful and reproducible
results. Maintain constant sample volume and contact time. Express results as extractable metals
and specify extraction. During preliminary treatment of samples care should be taken so that
introduction of other metal cannot take place.
Reagents, acids and chemicals which are used in the analysis should be of ultra pure quality.
The distilled water to be used for the analysis should be metal free deionised water.
3. PRELIMINARY DIGESTION OF THE SAMPLE FOR METALS:
 Digestion for metal sample is quite necessary to remove organics and to get free metal ions;
also to get dissolve the metals and can concentrate the metal in the sample if it is in very less
quantity.
 With the help of digestion you can reduce the remaining oxidising agents and also you can
reduce the interferences of the organic matter.
 By digestion you can do pH adjustment.
 Sometime it is essential and useful to use buffer solution plus indicator. Add buffer solution
and indicator to the sample solution in normal extraction procedure it should be carried out
at 3 to 3.5 pH.
 Separation of metals can be done and also you can enrich the metals
Separation of Metal extraction can be also done with the help of digestion- Microseparator,
separating funnel, volumetric flask are used.
Requirements for Digestion & Extraction
1. Erlenmeyer-conical flask 250 ml cap. Or digestion tubes, measuring cylinders
25.50, 100 ml cap. Fynnel.
2. Dispensers, Pipettes, Micropipettes.
3. Digestion Unit/Heating Block or Hot plate.
4. Con. HNO3, H2 SO4, HclO4, H2O2, KMNO4, K2S2O8, HCl,
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5. Good quality of distilled water / Double distilled water.
6. 50,100 ml cap. Volumetric Flask, filter paper
7. Beakers, Hand-gloves Acid Alkali proof, Asbestos Hand gloves, Safety goggles.
And other required material.
Always prepare an acid blank for each type of digestion performed.
Experience indicates that a blank made with the same acids and subjected to the same digestion
procedure as the sample can correct for impurities present in acids, in reagent water. Here some
digestion procedures are given.
NOTE: Always make up the same volume of the digested sample for which you have taken for
digestion (before digestion).
I.
Nitric Acid Digestion:
Mix well (homogenised) sample and transfer a suitable volume (50 to 100 ml) to a 250 ml conical
flask or in a digestion tube). Add 5 ml Conc. HNO3 and few boiling chips, or glass beads. Bring to
a slow boiling and evaporate on a hot plate or on the digestion block- Digestion unit to the lowest
possible volume (about 10 to 20 ml) before precipitation occur. Continue heating and adding nitric
acid (Con HNO3) as necessary until digestion complete. Do not let sample dry during digestion.
Wash down conical flask or digestion tubes (which used for the digestion) well with distilled
water then filter if necessary. Transfer the filtrate to a volumetric flask of 50 or 100 ml cap. Rinse
the digestion vessel and make up the volume to the make with distilled water. Mix thoroughly.
Take the portion of the digested sample for the analysis of directly can be used for the
determination of metal in atomic Absorption spectrophotometric analysis.
Here we use Con. HNO3 Nitric Acid for digestion because it is a strong oxidizing agent and most
of the metals are soluble in it.
II. NITRIC ACID-HYDROCHLORIC ACID DIGESTION:
Transfer a measured (50 or 100 ml) volume of well mixed homogenised and acid preserved
sample in to a conical flask 250 ml cap, or in digestion vessel-tubes. Add 3 ml Con. HNO3
evaporate it on a hot plate or on heating block or on a digestion unit, add 1+1 HCL and heat again
the sample to dissolve any precipitate or residue. Give distilled water wash and filter if necessary
to a 100 ml volumetric flask and make up the volume up to the mark.
Use this digested sample for analysis.
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ATOMIC ABSORPTION SPECTROPHOTOMETER
Scientist Walsh, Alkemade, and finally Winefardener mainly contributed for the establishment and
demonstration in the year 1962 for the trace metal analysis.
Now a days Atomic Absorption Spectrophotometer has become one of the most extensively
employed technique for determination of trace metals. It is a high attainable sensitivity for a wide
range of element, and a high sensitivity for the analyte element which is to be analyse.
Atomic Absorption Spectroscopy is the term used when the radiation absorb by atoms is
measured. Atomic Absorption is a part of Spectroscopy. AAS- Atomic Absorption
Spectrophotometer follows the Lambert & Beer’s law like other Spectrometer of Spectroscopy.
Like the Flame Photometer AAS consists of a Flame, a Grating, a Monochromater to isolate
emission line. Detector and an Amplifier. In addition to that the absorption system is a light source
– Hollow Cathode Lamp of Electron Discharge Lamp which emits a stable and intense light
particular wavelength.
Each element has characteristic wave length. A radiation – light source with wave length readily
absorbed by the element to be determined is directed through the flame and measure of it’s
intensity of the sample introduced in the Flame. The Decrease in the intensity of the light observed
with the sample is a measure of the concentration of the element.
PRINCIPLE
The AAS method for metal analysis is based on the fact that atoms in their ground state can absorb
light of a particular energy (frequency). In AAS light of a different wavelength radiates through
the atomizer system ( i.e. flame or graphite furnace- cuvette) and is absorb by atoms in the ground
stats. The quantity of absorbed light is proportional to the concentration of non-excited atoms
(ground state atoms). It is measured as resonance in a detector.
Light is emitted at the source of radiation and absorbed by atoms in the atomizer system at exactly
defined wavelength and within strictly limited spectral ranges. Each spectrum line is specific for
different element. As every element has it’s own characteristic wavelength (which is indicated to
the absorption wavelength in the ground state)
TYPES OF TECHNIQUES FOR ATOMIC ABSORPRION SPECTROMETRY
Following techniques are generally being used for the analysis of Metal analysis.



Flame Atomic Absorption Spectrophotometry- Flame AAS
Graphite Tube- Furnace AAS
Vapour Generation AAS,Hydride AAS, Cold Vapour
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Flame AAS
In flame AAS you can determine –analyse the concentration of metal in the mg/l or ppm level.
Here Air – Acetylene Flame, N2O- acetylene flame is being used Generally Air Acetylene flame is
widely being used.
Air is used as an Oxidant or fuel supporting gas. As a fuel Acetylene or Nitrous Oxide is used.
Both the gases as well as Air should be of good quality i.e. it should be interferences free. At the
time of operating certain pressure is required of the gases or Air. Mixture of Air Acetylene and
Nitrous oxide Acetylene is generally used. Temperature of Air Acetylene is less than the
temperature of N2- Acetylene flame. Nitrous Oxide – Acetylene is used when the oxides of
elements like Mo. Be, and V are thermally too stable to be dissolve in the Air Acetylene flame.
The temperature can be reached up to 31500 in the N2O- Acetylene flame where as in Air –
Acetylene flame the temperature will reach up to 25000C Different burners are used for Air
Acetylene and Nitrous Oxide- Acetylene flame.
Nebulization-Atomisation:
To atomize the liquid sample in a flame AAS the sample is sprayed via a pneumatic sprayer into a
mixing chamber where it is mixed with a combustible gas (Acetylene) and Oxidant Air or Nitrous
Oxide. Then reaches to the flame through Burner. As a result of the heat dissociation takes place
in the Atoms. These atoms absorbs the light radiation at a define wavelength from the Hollow
Cathode Lamp- HCL. Sample introduction should also be maintain to have better atomization and
also you will get fine size of aerosol for atomisation. Monochromator/gratting is isolating the
emission lines of the element. The isolated light is directed on the detector. This is a Photomultiplier Tube (PMT) which produces electric current which is depends on the intensity of the
light. The current is then amplified and processed by a electronic system to produce a signal which
is a measure of light attenuation occurring in the sample cell. This signal can be further processed
to produce on instrument readout directly in the concentration units.
GRPHITE FURNACE AAS:
In this AAS technique Graphite furnace and graphite tube is used. The generation of atoms by
means of an electrically heated graphite furnace atomizer. This technique is used for measuring
elements metals concentrations in ppb- Parts Per Billion level with better accuracy. Graphite
furnace AAS contributed by L’vov and Marzmann who had developed this technique.
In graphite furnace , HCL Hollow cathode Lamps, graphite tubes (Pyrolytic-coated and non
coated) platform furnace are being used.
It is also a part of AAS but for the analysis of trace metals in ppb conc. Where Flame AAS can not
be advisable to use, it is applicable. In this graphite furnace AAS, furnace has a very high
temperature to generate a population of free atoms so that the atomic absorption can be measured.
This is generally achieved in three stages.
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 A Drying
 An Ashing and
 Atomization
Inert gas like Nitrogen/Argon is used to protect the incent-decent graphite from excessive
corrosion. Here is a upward flow of N2/Ar which surrounds the graphite- heater grahite. N2/Ar gas
also sweeps away any product from the light path.
Compare to Flame and Vapour generation – Hydride generation techniques here in the Graphite
Tube AAS very small quantity of sample is required and the measurement can be done in ppb
level. The graphite AAS is 100 times more sensitive than flame. Sample volume ineeded is 5 to
100 microliter.
VAPOUR GENERATION/ HYDRIDE GENERATION SYSTEM-AAS
It is also one of the techniques of AAS. Some elements which generates their hydride and cold
vapour , are measured by this method. As , Se, Bi, Te, Sn, Sb, and Hg are the elements which can
be measured. In acidic condition above elements produced their hydrides with reducing agents
NaBH4 – Sodium Boro Hydride. Then it is treated in the absorption cell which is specially made
of Silica glass. Then atomic absorption is measured. In cold vapour generation AA system
reagents are the same but it’s concentration is different than the Hydride generation. Mercury can
be measured with this technique. Mercury can be brought in to vapour phase without flame or
heating or temperature. Due to it’s sensitivity flame methods are not used. Chemical reduction
method is used. Mercury ions are reduced to metallic mercury with NaBH4 & HCL with the inert
gas N2.The cold vapour of mercury swept in the absorption cell from which the radiation passed
and measures the absorption. Finally we can get the concentration.
SAFETY & MAINTENANCE OF THE INSTRUMENT:
 Here are some the important points for safety.
 Along with the Analysis you have to take extreme care of your safety and the safety of the
instrument.
 Always operate the instrument at required voltage.
 Always use the correct burner and gas regulators.
 Drain the Air tank of the compressor (if provided) intermittently.
 Replace-change the Acetylene gas cylinder when the min. pressure is left (mentioned in the
instruction manual of the instrument supplier).
 In the case of Varian Spectra AA-20 min at a pressure of Acetylene 7 kg/Cm2 never inst.
Can be operated. Otherwise there will be a chance of an accident.
 Always use safety wares while analysing on AAS.
 Never keep the flame of AAS un attainable.
 Check Exhaust system always. Check the pipes and as pressure regularly.
 Inst. Should be serviced and clean regularly. Routine maintenance should be done
periodically.
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SILICATE IN SEAWATER
The element silicon is the most abundant element in the universe . During weathering
of rocks, silicate is brought into solution and thus is present in sea water. The average
concentration is around 1 mg/l, much below its saturation value of 50 mg/l. In addition
silica is also present in particulate, in varying quantities.
Many believe that the distribution of silica in sea water is controlled by processes involving
organisms. Thus, hydrated silica is a major constituent of diatoms, which form a large
proportion of phytoplankton. When the organisms die, silicon is liberated. The element passes
through t hese cycles many times, in one season. It is estimated that approximately 120
million tonnes of silicon is removed from sea per annum by the growth and sedimentation of
phytoplankton.
DETERMINATION OF REACTIVE SILICATE: The determination is based on the
formation of a yellow silicomolybdic acid when an acidified solution of the sea water is treated
with molybdate. This complex exists in two isomeric forms, depending on pH, which differ in
their hydration. The α isomer is formed at pH 3.5 – 4.5 and is very stable, once formed. On
the other hand, the β form is rapidly formed in the pH range 0.8 – 2.5, but it is much less stable.
However, the latter has a higher molar absorptive.
Since both the isomeric forms have only low intensity absorbance, several methods have been
developed to reduce the complexes to intensely coloured blue complexes. For the purpose,
several organic and inorganic reducing agents have been used. In the present method, the use
of oxalic acid has been recommended.
SENSITIVITY :- The molar absorptive is around 19,000 in sea water, lower than that in
distilled water of 22,000.
PRINCIPLE: The seawater sample is allowed to react with molybdate under conditions
which result in the formation of silicomolybdate, phosphomolybdate and arsenomolybdate
complexes. A reducing solution, containing metol and oxalic acid, is then added which
reduces the silicomolybdate complex to give a blue reduction compound and simultaneously
decomposes the phosphomolybdate or arsenomolybdate eliminates the phosphate and arsenate
interference.
REAGENTS :1. Molybdate reagent: Dissolve 4 gm of ammonium paramolybdate
(NH4Mo7O24.4H2O) in about 300 ml of MQ water and add 12.0 ml of conc.HCl,
mix well and make the volume to 500 ml with MQ water. Store it in a polythene
bottle.
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2 . Metol – Sulphate solution: Dissolve 1.5gm of anhydrous sodium sulphite
(Na2SO3), in 125ml of MQ water and then add 2.5gm of metal (pmethylaminophenol sulphate). Store in a plastic bottle.
3. Oxalic acid solution: Prepare a saturated oxalic acid solution by shaking 10g of
oxalic acid dehydrate (COOH)2, 2H2O (AR) with 100 ml of MQ water; decant the
solution from the crystals for use.
4. Sulphuric acid solution (50%V/V): Add slowly 100ml of conc. H2SO4 into 250
ml of MQ water and allow to cool.
5. Reducing reagent: Mix 100 ml of metol - sulphate solution with 60 ml of oxalic
acid solution. Add 60 ml of 50% sulphuric acid solution slowly with mixing and
make the mixture to 300 ml with MQ water.
6. Preparation of synthetic sea water: Add 25 g NaCl + 8 g MgSO4.7H2O in
1000 ml MQ water.
APPARATUS :-Spectrophotometer: With 1 cm path length cell. Plastic containers: With
marking at 25 ml volume. Standard flasks: 100ml and 25ml. Standard pipettes: 5ml and 10ml.
PROCEDURE :Preparation of standard solution
1. Dissolve 0.0188 g of sodium Silicofluoride (Na2SiF6) in 100 ml of synthetic
seawater and store this stock solution in a polythene container. This solution
contains 1000 µmol Si/L.
2. Prepare 5, 10, 15, 20 and 25 µmol Si/L by transfer 0.5, 1, 1.5, 2.0 and 2.5 ml of
the stock solution and make up the volume up to 100ml with same synthetic
seawater. These solutions contain 5, 10, 15, 20 and 25 µmol Si/L concentrations.
0.0188 g in 100 ml = 1000 µmol Si /L
1 ml from 1000 µmol solution in 100 ml = 1 µmol Si /L.
5 ml from 1000 µmol solution in 100 ml = 5 µmol Si /L.
10 ml from 1000 µmol solution in 100ml = 10 µmol Si /L.
15 ml from 1000 µmol solution in 100ml = 15µmol Si /L.
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Calibration of standards and blank: Measure out 25 ml of MQ water for blank in triplicate.
Similarly measure out 25 ml of working standard solutions in clean plastic container in
triplicate. Add 10 ml of molybdate solution to each tube mix well and allow to stand for 10
min, add 15 ml of reducing reagent rapidly and mix immediately. After 2 hours incubation
measure the absorbance of blank A(b) and standard solutions A (st) in a spectrophotometer
using 1 cm cell at 810 nm against MQ water as reference.
Sample analysis: Measure out 25ml of the sample (triplicate) in a clean plastic container and
add 10 ml of molybdate solution to each tube, mix well and allow to stand for 10 min, then
add 15 ml of reducing reagent and mix immediately. Measure the absorbance A (s) of the
sample in 1 cm cell at 810 nm.
CALCULATION :Calculation for Factor value (F):
F=
Conc. of standard solution
A(st) - A(b)
Where A (st) = Mean absorbance of standards.
A (b) = Mean absorbance of blanks.
Calculation the amount of reactive silicate present in the sample
Silicate – Si µmol/L = F x A (s) – A (b)
Where A (s) = Mean absorbance of samples.
A (b) = Mean absorbance of blanks.
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SALINITY
METHOD :- Mohr-Knudson argentometric titration method.
Chlorinity: Chlorinity is defined as the mass in grams of pure silver necessary to precipitate the
halogens in 0.3285255 kg of seawater (All weights are vacuum weights).
Salinity: Salinity is defined as the weight in grams (in vacuo) of solids that can be obtained
from 1 kg of seawater (also measured in vacuo), when all of the carbonate has been
converted to oxide, the bromine and iodine replaced by chloride, all organic matter oxidized,
and the residue dried at 480°C to constant weight.
Outline of the method
Standard solution of silver nitrate is used to precipitate halide ions in seawater using potassium
chromate as an indicator, to form silver halides. When a slight excess of silver ions are
present, red silver chromate is formed.
REAGENTS :1.Standard solution of seawater (SSW): Use known chlorinity (19.375×10-3) / salinity
(34.99 ppt) or as quoted for seawater (SSW) supplied by the institute of oceanographic science
in Wormley, Godalming, Surray, (U.K.) in sealed glass ampules for standardizing silver
nitrate solution.
2.Silver nitrate solution: Dissolve 25 g silver nitrate (AR) in1000 ml DW. Store in an amber
glass bottle.
3.Potassium chromate solution: Dissolve 8 g potassium chromate (AR) in 100 ml DW. Store
in a stoppered glass bottle.
APPARATUS :1.
2.
3.
4.
5.
Burette: 25 ml, accuracy 0.1 ml
Bulb pipette: 5 ml, Accuracy 0.1 ml
Conical flask: 50 ml
Magnetic stirrer
Magnetic needle
PROCEDURE :Standardisation of silver nitrate solution : Pipette out 5.0 ml SSW into a clean conical
flask, add 6 drops of potassium chromate indicator and titrate with silver nitrate solution
from the burette while stirring vigorously on a magnetic stirrer. Clean the inner wall of the
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flask with a jet of distilled water frequently and continue the titration. When colour change is
observed, slow down the addition of titrant to drop by drop till colour change is observed from
yellow to dirty orange. Repeat the standardization at least thrice and find out the mean of
burette readings [BR (SSW)]. Find out the standardization factor F as follows:
F =
Chlorinity of SSW
Mean BR (SSW) (ml)
SAMPLE ANALYSIS :Pipette out 5.0 ml sea water sample into a clean conical flask. Add distilled water (25 ml
along the wall of the flask). Add 6 drops of indicator. Titrate against silver nitrate in the same
manner as described as above. Obtain the reading (ml) [BR(s)].
CALCULATIONS :Calculate the “normalised volume” (V) from the Equation
V = BR(s) × F
Obtain the correction factor (k) corresponding to V from established table and then calculate the
chlorinity (Cl) and salinity (S) by using the relations
Cl = V + k
S = 1.80655 × Cl
If salinity of SSW is given, calculate salinity of samples as follows:
F =
Salinity of SSW
.
Mean BR (SSW) (ml)
Salinity (ppt) = F × BR(s) (ml)
Note: The salinity can be measured insitu by CTD probes/ dedicated salinometer / hand
held refractometer etc. However, the argentometric, titrimetric procedures can be employed
for finding out the instrumental error if any. The titrimetric procedure can be improved by using
autotitrators.
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TOTAL COLIFORM & FAECAL COLIFORM
METHOD :- Multiple tube fermentation technique
COLLECTION OF SAMPLE :- Collect the sample in sterile glass bottle.
PRESERVATION :- Ice
APPARATUS :- Autoclave, Incubator controlled at 37°C and 44°C Gas burner, Nichrome wire
loop (diameter of 3 mm), Sterile pipettes and sterile dilution water bottles.
REAGENTS :a) Mac conkey’s broth. Single & Double strength
b) Brillient green bile lactose broth
c) Covav’s reagent and peptone water
PROCEDURE :- Inoculate 10 ml of sample in three double strength (each of 10 ml) fermentation
tubes, add 1 ml of sample in three single strength (each of 5 ml) fermentation tubes and 0.1 ml in
three single strength (each of 5 ml) fermentation tube near the flame. Then incubate at 37°C for 24
hours and note down the positive tubes showing acid or gas Production. Reincubate the tubes
which shows negative results.
CONFIRM TEST:- Transfer 1-2 loopful of culture from the presumptive Positive tube in each of
two brilliant green broth each of 5 ml fermentation tube and incubate One tube at 37°C for 24 hr
and other tubes at 44°C for 24 hr. The formentation of gas in the tube after 24 hr, 48 hr. shows
positive test. Also inoculate 1-2 loopful of culture in peptone water tube (each of 5 ml) and
incubate at 44°C for 24 hours.
CALCULATION :- Find out the MPN/100ml by the help of MPN index. Tubes which are
Showing positive (gas formation) result at 37°C are Coliform organism and positive at 44°C are
Fecal Coliform organisms. While addition of Covav’s reagent in peptone water tube, the red color
is developed it indicates positive result.
INTERFERENCE :- To avoid contamination procedure should be done in sterile condition.
PRECISION AND BIAS :- Not Applicable
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ANALYTICAL METHOD REFERENCE LIST
Sr.
No.
PARAMETER
1
Colour
2
Temperature oC
3
Turbidity.
4
pH Value
5
Conductivity
6
Suspended Solids
7
Total Dissolved Solids
8
Chloride
9
Dissolved O2
10
BOD
11
Oil & Grease
12
Phenolic Compound
13
Hexavalent Chromium
14
COD
15
Sulphate
16
Cyanide
17
Fluoride.
18
Boron
19
Manganese
TEST METHOD / STANDARD AGAINST WHICH
TESTS ARE PERFORMED
[IS: 3025 (Part – 4) – 1983 (Reaffirmed 2002) Pt-co. Method
Visual Comparison Method]
ICMAM Protocol Method
Nephelometric method.
(2130 B APHA Standard Methods 21st Edition.)
Electrometric Method
IS: 3025 (Part – 11) – 1983 (Reaffirmed 2002)
2510 B APHA Standard Methods 21st Edition.
Gravimetric method.
(2540 D APHA Standard Methods 21st Edition.)
Gravimetric method.
(2540 C APHA Standard Methods 21st Edition.)
Argentometric method.
(4500 Cl― B APHA Standard Methods 21st Edition.)
Winkler method – Azide modification.
(4500-O – C APHA Standard Methods 21st Edition.)
3 – day BOD test.
(IS 3025 (Part 44) 1993 Reaffirmed 1999)
Liquid – Liquid Partition Gravimetric method.
(5520 B APHA Standard Methods 21st Edition.)
4 Amino Antipyrene method without Chloroform Extraction
(Direct Photometric method)
(5530 D APHA Standard Methods 21st Edition.)
Colorimetric method
APHA (21st Edition) – 3500 – Cr B :
APHA (21st Edition)- 5220 B
Open Reflux Method
Turbidimetric method
APHA(21st Edition) 4500 SO4 E
Preliminary Distillation treatment followed by Colorimetric
method.
(4500 - CN― E APHA Standard Methods 21st Edition.)
Ion Selective Electrode method. (4500 - F― C APHA
Standard Methods 21st Edition.)
Colorimetric Curcumin method. (4500-B B. APHA Standard
Methods 21st Edition.)
Colorimetric ( Persulfate Method ) (3500 – Mn B. APHA
Standard Methods 21st Edition.)
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20
Ammonia Nitrogen.
21
Nitrate Nitrogen.
22
Phosphate
23
Heavy Metals
24
25
Reactive silicate
Salinity
Total Coliform
Faecal Coliform
26
(Cu, Pb, Ni, Zn, Fe, Total Cr, Cd, Hg,).
1).Titrimetric method
2).Nesslerization method. (4500 NH3 B & C APHA Standard
Methods 18st Edition. And IS: 3025 [Part 34]-1988 Method)
1)Spectrophotometric method. (213 D APHA Standard
Methods 13th Edition.) OR
2) Spectrophotometric (Cadmium Reduction method 4500 –
NO3 - E APHA standard methods 21st Edition.)
Stannous Chloride method. (4500 – P D APHA Standard
Methods 21st Edition.)
Flame Atomic Absorption Spectrometr. (3111 B APHA
Standard Methods 21st Edition.)
ICMAM Protocol Method
ICMAM Protocol Method
Multiple Tube Fermentation (MTF)Method
***
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