Five water samples per village (at a distance of at least ½ Km from each other) were collected through field staff of Local Govt. at Bahawalpur, Rahim Yar Khan and Multan Districts. Out of five, one sample was sent to PCRWR. In total 2395 samples were received by PCRWR instead of 2500. While 106 samples could not be collected by UNICEF team due to the reason that water of hand pumps or tube wells was not available in running condition. The sampling details are given in Table-1.

Table-1 Sample Collection Details for Bahawalpur, Rahim Yar Khan & Multan Districts

Water samples were collected in ½ liter polyethylene bottles according to standard methods. A distance of half kilometer was maintained between the two sampling points. The preservative (1% concentrated HCl) was used in the samples collected for arsenic analysis.

Three analytical methods for arsenic (inorganic) analysis are reported in standard methods. These methods are; 1) Atomic Absorption Spectrometric (AAS) method, 2) Silver Diethyldithiocarbamate method and 3) Inductive Coupled Plasma (ICP) Spectrometric method. Atomic Absorption Spectrometer (Hydride Generation mode) was used for the analysis of arsenic in water samples. All samples were analysed on HS 55 Mercury/Hydride system, an accessory (AAS, Vario 6 Analytik Jena AG) for the matrix free determination of the hydride forming elements such as As, Bi, Sb, Se, Sn and Te.

The Hydride technique makes use of fact that hydrogen liberated in the reaction of the weakly acidic sample solutions with sodium boro-hydride which combines with metal ions to form gaseous hydrides. These are carried to the hot quartz cell by the carrier gas and decomposed by collision processes in a series of steps, until free As atoms were obtained.

2.3.1 Equipment and Apparatus

For the analysis of arsenic the Atomic Absorption Spectrophotometer (AAS Vario 6 Analytik Jena AG), Mercury/Hydride System HS55 (Analytik Jena AG), and Argon Gas with 99.99% purity were used.

2.3.2 Reagents and Their Preparation

The following common reagents were used for the analysis;

1. Sodium borohydride (NaBH_4, 98% purity)

2. Sodium hydroxide, NaOH

3. Hydrochloric Acid (Concentrated 37% HCl)

4. Arsenic Standard (1007 mg/ml, As in 2% HNO_3, BDH)

In order to make reducing solution (Reductant), 15 g sodium borohydride (NaBH₄) and 5 g of sodium hydroxide were dissolved in 500 ml distilled water. This reagent was used as reducing agent for Arsenic analysis.

2.3.3 Procedure of Analysis

The HS 55 Mercury/Hydride system consisting of a basic unit and the cell unit was operated and controlled from PC. Basic unit consists of three accessories. These include batch module, single channel-peristaltic pump and gas valve box. The gas valve box supplied argon gas for scavenging and for transporting the metal hydrides to the system.

Fig.-1: Mercury Hydride System of Atomic Absorption Spectrophotometer (AAS)

Pressure of the argon gas cylinder was adjusted at 3-5 bars. After attaining the necessary temperature (950 ^oC) reducing agent was fed by the peristaltic pump. A 10 ml sample was taken into reaction cell and 0.8 ml of concentrated HCl was dispensed into sample and reaction cell was adjusted properly at its place. Calibration standards of arsenic with concentrations (0,10,20,30,40,50 ppb) and (50,60,70,80,90,100 ppb) were prepared. New method of calibration was developed using these standards under the operation of software, and then the method developed was loaded for analysis of actual samples. HS 55 hydride system analyzes the water samples in the following sequences:

• Pre-wash time

• Reaction time

• Rewash time

The detection limit of this method is 0.1 ppb.

Fig.-2: Sample Induction in the Reaction Cell of Mercury Hydride System of AAS

Strict quality control measures were observed in the laboratory by adopting calibration of instruments with known standards and reproducibility checks. Instrument was calibrated with multiple arsenic standards using standard stock solution. The standard stock solution was certified by BDH laboratory supplies LE 17 4XN England. According to certificate of analysis, standard was prepared gravimetrically to contain the elemental concentration of 1007 ppb. The solution was assayed titrimetrically and also concentration was verified by Inductive Coupled Plasma Spectrophotometer (ICP). All the glassware used for preparation of standard solution were acid washed with 1:1 HCl and rinsed three times with deionized water prior to use. Deionized water used for analytical work was freshly prepared having EC of >19 Mohm/cm.

Calibration was evaluated by the value of R² (Degree of fitness) between concentration and absorbance i.e. 0.9995356. After calibration, the analysis of all ground water samples was carried out. During arsenic analysis, a blank and a known arsenic standard was run after every 10 samples for accuracy assurance. After every 10 samples, one pre-analyzed sample was rechecked randomly for reproducibility evaluation. So 10.02% samples were rechecked and results showed the %age of reproducibility less than 10% which is recognized internationally within the range of quality control standards. The results of blank and standards run after every 10 samples are shown at Annexure-I. Results of reproducible samples are given as Annexure-II.

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