2.1 Study Area:
District Mianwali is in south-western region of province Punjab. This district has plains of western area of salt range. It is situated near the Sakesar hill. Mianwali district has boundaries with Khushab, D.I Khan, Bhakkar and Bannu districts. Canal irrigation system is very less developed, only a little area is irrigated with Indus river irrigation system. The native of this district is affiliated with bordering plant resources. District Mianwali is divided into three Tehsils such as Piplan, Mianwali and Esakhel. Mianwali city is the larger city. Its human population is about 140000. One of the popular areas is Kalabagh, brown salt hills, Kalabagh Dam, Indus river and Nawab of Kalabagh. At the south side of district tehsil Piplan is situated and towads north-west tehsil Esakhel is located. These areas are economic and commercial zones of the district (Ghani et al., 2016; Qureshi et al., 2007).
2.2 Sample collection from sites
In district Mianwali four sites were selected for sampling. Three samples of agricultural soil, forages and animal blood, hair and feces were taken to examine the metal profile of soil-forage-animal continuum. The samples were taken from Wan bhachran site, Mianwali, Esakhel and Piplan. The summer, autumn and winter seasons were selected for sampling. The samples were taken randomly from sites.
2.3 Soil sample collection
In the district Mianwali four sites were selected to collect the samples. 3 samples of soil were collected with equal distances in the field. Stainless steel auger was used to dig up the upper layer of soil about 12-15 cm (Siddique et al., 2019). These samples were packed into plastic bags to avoid the mixing of other chemical compounds into it. Samples were stored in laboratory and labeled then metal analysis was performed. For each sample three composite samples were made. The collected samples were firstly air then oven drying at 72℃ for 2 days. The samples were placed in incubators at 70℃ temperature for 5 days.
2.4 Forage sample collection
Sterilized apparatus was used to collect the forage samples. Forage and soil were collected from same field and place. Only those forages were selected for taking samples that are used as common feed of livestock. 3 samples of each forage plant were taken from the sampling area. The samples were washed with distilled water to clear impurities and dirt. These samples were dried to eliminate moisture in the freshly collected samples. The collected samples were dried for further process.
These are following species that were selected for sampling. Calotropis procera (Apocynaceae), Dactyloctenium aegyptium (Poaceae), Parthenium hysterophorus (Asteraceae), Rumex dentatus (Polygonaceae) and Ziziphus jujube (Rhamnaceae).
2.5 Animal blood plasma, hair and feces sample collection
Blood samples of cow, buffalo and sheep of Mianwali was taken in 2020. Young animals within age of two years were selected for sampling. Blood was collected from four sites of district Mianwali. Animal blood was calculated from 10 animals (Cow, buffalo and sheep) each from each sampling site and heavy metal evaluation was done. Sterilized syringe was used to obtain the blood samples. The grazing ruminant’s blood was taken from the vein. The vacuum was created in evacuated tubes while collecting blood to minimize the extent of clotting. The blood was collected in heparinized Na-citrate voiles quickly. For 15 minutes blood was centrifuged at 3000 rpm and blood plasma was separated. Polyethylene tubes were used to store the blood plasma and frozen at -20°C. Hair and feces samples were also collected and stored for the further digestion process.
2.6 Sample measurement and preparation
Arrangement and preparation of samples involves the digestion process. This method of digestion is called wet digestion. It has following steps. Acid and hydrogen per oxide is used for complete digestion process. Distilled water is added after digestion into prepared samples for dilution purpose. After that filtration of samples occur. In next step Atomic Absorption Spectrophotometer (AAS) is an apparatus through which metal analysis is done.
2.7 Apparatus and chemicals for digestion
Chemicals that are used for digestion process includes the 10 ml Nitric acid ,70% Sulphuric acid (H2SO4), 50% Hydrogen peroxide (H2O2) and newly synthesized condensed water or distilled (H2O). The apparatus for digestion includes digestion flasks of 100 ml, measuring cylinder (50 ml), beakers (50ml) and (100ml), pipette (10 ml), filter paper, stirrer, hotplate and gloves.
2.8 Digestion of soil, forages and Animal samples
Digestion of soil and forages and animal samples (blood, hair and faeces) include various steps. First, the samples are air dried and followed by oven dried process at 72°C for 5 days until the moisture content is removed. When plants are completely dried, they weighed with electrical balance. Standard procedure of digestion was applied to digest the samples (Siddique et al. 2019). 1gm sample was weighed by electrical balance and placed in a beaker of 50ml. A 10 ml nitric acid was added to beaker and was kept overnight. Hot plate was used for digestion of sample by pouring H2O2 drop wise until solution becomes transparent. Cooling at room temperature was done. For dilution 50 ml distilled water added to solution. To filter the solution Whatman filter paper of 42 µm was used. Then this prepared solution was kept in plastic bottles for metal profile evaluation.
Blood samples collected from the Mianwali district were stored and freeze at -20°C. For digestion process the samples were from freezer and digested with same standard procedure as applied to soil and forages (Siddique et al., 2019). Hair sample was sun dried and was cut into pieces of 1.0-2.9 cm. De-ionized water was used to wash the samples and ethanol was also applied to wash. Oven drying process was carried out and for 4 hours and then desiccator cooling was performed (Hashem et al., 2017). Feces samples were collected from cow, buffalo and sheep after air drying and oven dry the samples were submitted for digestion (Nicholson et al.1999).
2.9 Metal profile evaluation analysis
The prepared samples were then analyzed for metal contents by Atomic Absorption Spectrophotometer (Perkin-Elmer Corp, 1980). Nutritional minerals that were evaluated in the sample were Co and Pb. Standard solution was prepared to get the standardized curve. The metal analysis was done by running the samples through Atomic Absorption Spectrophotometer. This apparatus is equipped with a graphite furnace. Each metal is measured according to value of standard solution. The amount of each metal occurring in the sample is obtained in absolute farm. While sample is run through the Atomic Absorption Spectrophotometer the little quantity of sample is sprayed at the flame. Atomic resonance absorption line by element is calculated and measured. The apparatus is convenient for analysis. Any radiation that is emitted by flame had no effect on the working of apparatus. The absorption method is independent of the excitation potential of the spectral line used.
2.10 Evaluation Indices:
2.10.1 Bio concentration Factor (BCF):
For assessment of metal (mg/kg) transport from agricultural soil and forages that are growing on this soil, a BCF is applied (Cui et al., 2004).
BCF for soil to forage
(BCF) = Level of metal in forage /Level of metal in soil
2.10.2 Pollution load index (PLI)
Liu et al. (2005) described a formula which was used to find these indices.
(M)IS = (mg/kg) Concentration of metal that occurs in soil to investigate
(M)RS= Soil reference value of metal (Table 1).
2.10.3 Enrichment factor (EF)
Formula for Enrichment factor is described by Buat-Menard and Chesselet (1979). See the Table 2.
2.10.4 Daily intake of metals (DIM)
Daily intake of metal (DIM) can be calculated by following equation from Sajjad et al. (2009).
Cmetal is the concentration of metals in forages,
Dfood intake is the daily intake of forages,
Baverage weight is the average body weight.
For calculating this daily intake of metal, the conversion factor was taken 0.085 (Jan et al., 2010). Daily intake metal for cow was calculated by using animal body weight 600 kg and daily forage intake 12 kg while for sheep body weight was taken 75 kg and daily forage intake 1.3 kg (Johnsen and Aaneby, 2019). To calculate the DIM for buffalo body weight was taken 550 kg and daily forage intake (TDI) was taken 12.5 kg (Yang et al., 2020).
Health Risk Index (HRI)
Health risk index is the ratio of daily intake of metals in the forages to oral reference dose (RfD) and was calculated by the help formula (USEPA, 2002).
DIM = Daily intake of heavy metal
RfD = Oral reference dose (Table 3)
An HRI > 1.0 for any single metal indicates that the health of consumer population is at risk, or it is carcinogenic (USEPA, 2013).