Materials And Methods
3.1 COLLECTION OF SAMPLES
A total of 44 samples (given in appendix) belongs to seven categories (human, water, livestock, agro crop, forest crop, soil and aquatic animal) were collected from Faridpur district in every one month interval. All the samples were chosen randomly. Among these, some samples with GPS position (model- Garmin gps 12xl) were collected (given in appendix).
Figure -3: All prepared samples for arsenic analysis
3.1.1 SOIL SAMPLE
During plant sample collection corresponding soil samples were also collected from 15 cm depth.
Procedure:
§ After collecting the sample, it was broken-up any large clods and spread the entire sample on a clean piece wrapping or waxed paper.
§ Then it was dried (in a fan forced oven at 400C for at least 24 hours) quickly to minimize biological, chemical and physical changes that can occur when stored.
§ The sample was grounded and passed through a 100 mesh sieve having circular holes .the remaining course materials were again grounded and sieved.
§ The ground soil samples were thoroughly mixed on a clean piece of polyethylene sheet.
§ Lastly the samples were Stored in a suitable bottle and level carefully.
3.1.2 AGRO CROP SAMPLE (RICE, VEGETABLES)
Agro crop samples were collected from corresponding field of the study area.
Procedure:
§ The plant samples collected in the field, was first cleaned of soil particles. The deionized water was used for washing to remove foliar spray, fungicides, dusts and soil particles.
§ Then the fresh plant samples washed in sequence in dilute HCl (0.1N) and deionized water.
§ The samples were placed in new paper bag and immediately dried in an oven at 650C to 700C.
§ In the laboratory, the samples were homogenized using a small mill with a sieve of 0.5-1.0 mm.
§ The samples were allowed to cool in desicator before weighing.
3.1.3 WATER SAMPLE (HAND TUBE WELL, RIVER AND POND)
Collection and preparation of water samples play an important role in the results of chemical analysis.
Procedure:
§ Water sample was collected in the polypropylene sample bottle which was previously washed with 1M HCl and rinsed 3-4 times with deionized water and dried.
§ Tube well water (HTW) was collected directly from the outlet point, after continuous discharge of the source for 10–20 minutes.
§ 90 ml of water was taken with a polypropylene syringe fitted with filter holder on its mouth containing >0.45 µm pore size membrane filter paper.
§ 10 ml of 2% HNO3 was added and shaked gently. The final acid concentration of water sample was 0.2% HNO3 and dilution 1.11.
§ After acidification, proper marking and leveling was done.
3.1.4 HUMAN SAMPLE (HAIR AND NAIL)
Hair and nail samples were collected from the tube well water users. Before collecting, scissor for the hair samples and bled for the nail samples were cleaned by acetone to avoid the others contamination.
Procedure:
§ The collected samples were transferred from polyethylene bag to the Petri dish.
§ In order to clean the samples properly the following procedures were followed.
1. Washed with Acetone and dried naturally.
2. Washed with deionized water and then dried.
3. Again washed with Acetone and dried.
§ The dried clean samples were cut into small pieces and took about two gms. of the samples for the final analysis.
3.1.6 LIVESTOCK SAMPLE (LIVER AND MUSCLE FROM GOAT, CHICKEN AND COW)
Procedure:
§ Livestock samples once collected analyzed with fresh weight basis.
3.1.6 AQUATIC ANIMAL SAMPLE (FISH)
Procedure:
§ Fish samples were collected from the river and analyzed with fresh weight basis.
3.1.7 FOREST CROP SAMPLE (WOOD)
The wood sample was collected by using Suunto 300 mm wood increment borer.
Figure -4: Suunto 300 mm wood increment borer
Procedure:
§ The drill applied at a right angle to the axis of the tree and aligned it to the position of the medulla
§ The drill Turned clockwise into the tree with both hands.
§ When the desired drilling depth had reached (position of the medulla), the drill tongue inserted into the drill by sliding it carefully along the interior of the drill in order to move it within the clearance between the drilling core and the drill.
§ The drill turned back anti clockwise by a full or half rotation. Thus the drilling core was torn off the wood.
§ Carefully pulled the drilling core off the drill on the tongue. Then the increment borer removed from the tree. Turned the drill off by applying a slight traction.
§ Lastly the collected samples were preserved in a polyethylene sheet.
3.2 ANALYSIS OF THE SAMPLE
The chemical analysis was carried out in the ‘BARI-CYMMIT ARSENIC LABORATORY, GAJIPUR’. The whole analysis was conducted for three weeks. All the samples were analyzed using Atomic Absorption Spectrophotometer (model- Perkin Elmer AAnalyst 200) equipped with Hydride Generation System (HG-AAS).
Figure -5: BARI-CYMMIT Arsenic Laboratory, Gajipur
Figure -6: Perkin Elmer AAnalyst 200 equipped with HG for As determination
3.2.1 SAMPLE PREPARATION AND MEASUREMENT FOR WATER SAMPLE
5 ml of water sample, 5 ml of concentrated HCl (32%, w/v) and then 5 ml of KI-ascorbic acid reducing agent are added to 20ml polypropylene test tube. The solution is mixed and allowed to react for 1 hour at room temperature to complete the pre-reduction and complexation. The sample is then injected for arsenic analysis. All samples and standards must be prepared in the same analytical matrix.
3.2.2 DIGESTION PROCEDURE FOR TOTAL ARSENIC
3.2.2.1 REAGENTS
♣ Trace element grade concentrated HNO3 (65-69) %, mass density 1.40 mol/L
♣ Trace element grade H2O2 (30) %
Reagent should be analyzed to determine the level of impurities. If method blank is less than the minimum detection limit, then the reagent can be used.
3.2.2.2 PROCEDURE
Step-1: The sample should be air dried/ oven dried and thoroughly homogenized, then ground to pass a 100 mesh in-1 nominal pore-size sieve.
Step-2: Weigh an approximately 0.5000g of soil into the 100 ml digestion tube. Record the exact sample weight to the nearest 0.001g.
Step-3: Add 10.0 ml of 1:1 HNO3 (trace element grade), Mix the slurry and cover with watch glass or vapor recovery device and allow the sample for overnight at room temperature. Heat the sample to 1000C for 10 to 15 minutes without boiling. Allow the sample to cool, add 5 ml of Conc. HNO3 and heat to 1200C. If the brown fume is generated, indicating oxidation of the sample by HNO3, repeat this step (addition of 5 ml HNO3) over and over until no brown fumes are given off by the sample, indicating the complete reaction of HNO3.
Step-4: After step-3 has been completed, cool the sample and slowly add 2.0 ml of water and 3 ml of 30% H2O2 and shake gently. Return the sample to the heat source for starting the peroxide reaction. Care must be taken to ensure that losses do not occur due to excessive vigorous effervescence. Repeat this step (addition of 2 ml 30% H2O2) over and over until the effervescence is minimal or the sample appearance is unchanged. Do not add more than a total of 10 ml of 30% H2O2.
Step-5: Cool the sample and volume with de-ionized distilled water to 50 ml flask. Filter the extract with Whatman-42 or equivalent. The sample is now ready for analysis of total arsenic by HG-QT-AAS.
3.2.3 ARSENIC ANALYSIS
Arsenic hydride formation, which involves the reaction of arsenic compounds with NaBH4 to produce the corresponding arsines is a well documented method for the determination of arsenic at trace levels.
3.2.3.1 METHODS
A. Hydride generation method for determination of total arsenic in Soil/Biological Materials:
Determination of total arsenic first involves decomposition of the minerals and organic components that are responsible for binding arsenic in soil or any biological materials. Decomposition of the arsenic binding agents in soil or any biological material is usually accomplished by digestion with concentrated acids with an external heat source. Several concentrated acids are commonly utilized in acid digestion procedures. Using the above digestion procedure, all organic arsenic and As (III) oxidized and utilized as As (V). In that case, the digested samples with proper dilution injected for arsenic analysis directly. All samples and standards must be prepared in the same analytical matrix
B. Pre-reduction method for determination of total Arsenic in water
Water samples contain As(III) and As(V) mostly. The different rates of arsination of As(III) versus As(V) can be overcome by pre-reduction of As(V) to As(III), so that all As is present as As(III). KI-ascorbic acid used as pre-reducing agent. With this method, total As of water sample can be measured.
3.2.3.2 REAGENTS
♣ NaBH4 1.5% + NaOH 0.5% (Reductant): Weigh 7.5g of AR grade sodium boro-hydride (should be moisture free) in 500 ml volumetric flask. Add about 100 ml of deionized distilled water. Dissolve the salts, add 2.5 g of NaOH. Added deionized distilled water to volume. Prepare this reagent daily as required.
♣ KI-ascobic acid(5%. m/v each) pre-reducing solution: 5gm of KI and 5 gm of ascorbic acid were dissolved in 100 ml of water. This reagent should be prepared fresh before use.
♣ 5M HCl (carrier solution): trace element grade
♣ Secondary Stock Solution (4 mg As L-1): Transfer1000 µl of 1000 mg As L-1 in 250 ml volumetric flask with 1% of HNO3.
♣ Working Standard Solution: Transfer 0,125, 250, 375, 500, 625, 750, 875 and 1000 µl of 4 mg As L-1 (secondary stock solution) into 50 ml volumetric flask to get 0,10, 20, 30, 40, 50, 60, 70 and 80 µg As L-1 As respectively. Made volume with 1% HNO3 and shaked thoroughly.
In recent years, the problem of Arsenic (As) contamination of groundwater in Bangladesh has received considerable attention as a serious health risk for the population, drinking water containing Arsenic above the maximum national permissible level of 50 m g per litre of water (= 50 ppb). A large number of patients with Arsenic related symptoms have already been identified by the physicians. This situation has given rise to the apprehension that ingestion of Arsenic through food is adding to the problem, as Arsenic contaminated groundwater is widely used for irrigation of crop lands, particularly rice fields. (Khaliquzzaman, 2003).
