Evaluation of transfer of lead in soil plant animal system: assessment of consequences of its toxicity

The instant endeavor was undertaken for determination of lead (Pb) in water, soil, forage, and cow’s blood domesticated in contaminated area of heavy automobiles’ exhaust in Sahiwal town of District Sargodha, Pakistan. Water samples showed that the concentration of Pb ranged from 1.14 to 0.44 mg kg−1 at all sites. It was maximum at site 5 and minimum at site 2. Soil samples showed the concentration of Pb at all sites ranged from 1.58 to 0.279 mg kg−1. It was maximum in soil where Avena sativa was grown at site 5 and was found minimum in soil where Zea mays was grown at site 2. While among samples of forage, the concentration of Pb ranges from 0.048 to 2.002 mg kg−1. The highest Pb amount was found in Brassica campestris at site 1 and the minimum was recorded in Trifolium alexandrinum at site 2. Finally, the blood samples of cow depicted that concentration of Pb ranged from 4.468 to 0.217 mg kg−1. It was the maximum at site 1 and the minimum at site 3. It is recommended that such study should be conducted in other districts for public awareness.


Introduction
Although transport sector has made life luxurious all over the world, but also unfortunately it brought few problems with it. When coming to their roadside effects, one of the hazardous effects noted is that animal forages are contaminated with emission of pollutants from automobiles (Rehman et al. 2019). Different studies have shown that the blood and meat samples of cattle grazing and domesticated on such contaminated forage were found with different heavy metals including lead (Pb). Lead enters in cattle and animals from water, they drink and forage, they eat. It is found that more are heavy automobiles on the roads or highways more is the toxic smoke released into the atmosphere (Javed et al. 2020).
In Pakistan, a relatively short food web of soil + water nutrients/contaminants → forage → cow → milk and meat may be hazardous for the consumers as Codex Alimentarius Commission (CAC) has given allowable limits of Pb in meat of cattle, sheep, and poultry (ML 0.05 mg kg −1 ; lead range 0-0.1 mg kg −1 ) (CAC 1997(CAC -1999. Exposure to high levels of Pb is found hazardous to human body, particularly infants and kids who are more susceptible to the detrimental impacts of lead, which can affect their brain development. Anwar et al. (2020) reported that the plants growing on roadside are deeply contained with heavy metals caused by vehicles' smoke pollution, hence certain physiological disorders and diseases may occur by consumption of vegetation infested with vehicular smoke pollution. Liu et al. (2020) reported that concentration of Pb fluctuated from 0.28 to 1.12 mg kg −1 and 0.31 to 0.83 mg kg −1 in cows and buffaloes, respectively. Among the three organs tested, the highest level of Pb (0.64-0.83 mg kg −1 ) was found in spleen trailed by Pb (0.28-1.12 mg kg −1 ) in lungs and (0.36-0.87 mg kg −1 ) in bones. Additionally, older animals exhibited a significant increase of Pb in them. It was established that Pb accumulation was higher than the standard limit of 0.1 mg kg −1 in both age groups of animals. The above bio-indicators showed environmental pollution a result of presence of hazardous levels of Pb in cattle and apparent risks for human health. Sound management of hazardous waste is needed in Sargodha district due to its high lead concentration in edible commodities. It obviously indicated to address Pb contamination as well as its probable route of entry into the forage of the domestic animals. Nadeem et al. (2020) reported that the quantities for pollution load index were less than legal value in respect of Pb and zinc. Similarly, their transfer had less content indicating that antagonistic effect of metals in soil or plants were resistant to Pb and Zn. Although the metals' daily intake of had lower contents, health risk index values were higher than the legal value that indicated that Pb and Zn were not useful for grazing animals on site of collected samples. EF values were found in order of Pb > Zn and less than standard ones. The results of study showed that irrigation of waste water increased the concentration of both metals at sites of sampling. Jankowski et al. (2019) found that the highest levels of Pb were taken up by Alopecurus pratensis L. (3.843 mg kg -1 DM), and the minimum by A. elatius L. (2.523 mg kg −1 DM). Among these species of plants, the maximum quantity of Cd (0.286 mg kg −1 DM) was found amassed by D. glomerata L., further found that root parts accumulated higher contents of Pb and Cd of the grass than leafy ones. It indicated that substantial contents of Pb and Cd exhausted by highway automobiles polluted the soil. Maximum contents of Pb and Cd were accumulated in the grass germinating at 5-meter distance from the highway edge, and it was applicable on all parts, whether underground or aboveground. Miclean et al. (2019) reported a noncarcinogenic hazardous impact showed that milk consumption has no risk for human health brought from local markets, but the average cancer risk was potentially existed. Bhuiyan et al. (2016) studied results of vehicle smoke on the environment as many researchers had been already working upon it. The extended vehicle smoke affect used to be observed on Malabar spinach plant and specific forages. Sajid et al. (2017) found that the strength of pathologic adjustments was without delay and associated to the quantitative accumulation of Pb in a range of organs of the animals. The minerals were related with each existence functioning by the manufacturing of hormone, enzymes and alter metabolism due to their movements as coworker. The complexities associated to mineral diet and metabolism might be based on the cure of a mineral connected disorder (Khan et al. 2006). Nazir et al. (2015) found that industries manufacturing, mining, farming, and transportation sectors resulting from human activities had been releasing high quantities of toxic heavy metals on soils and drinking and irrigation water and eventually to the environment. People drinking contaminated water such cases have to get through certain ailments including of kidney, hair, and gastrointestinal tract. Pb had direct function in metabolism, however, it has created toxicity when exceeded from its necessary levels as properly as due to the deficiencies of other hint elements (Farmer and Farmer 2000; Bibi et al. 2014). Barik et al. (2014) found that due to vehicular emissions soil porosity diminished up to 10% while aggregate ability lowered up to 23%. Zhang et al. (2013) found that roots hold most of the heavy metals that a plant absorbs. Foliage of a plant was once believed to accumulate lead and it might get transferred to different components of plant. Khan et al. (2013) reported that Pb amounts were found from 1.2 to 3.5 mg kg −1 in soil, 0.33 to 0.70 mg kg −1 in forage and it was from 0.018 to 0.050 mg L −1 in milk. Its intensity gradually reduced in samples of soil, forage, and milk with increasing intervals of sampling. Pb amounts were lower than the allowable limits in both soil and forage as compared to as reported in some studies. Pb amount in cow milk was slightly 1.14 ± 0.034 6 0.832 ± 0.045 ns, p˂ 0.01 more than the allowable limit that could be risky for human health and to the health of animal's calves mostly perhaps due to environmental pollution including water, air, and soil. Viard et al. (2004) found that the expressway tempted the pollution of the nearby environment, and it was found near to 320 m, and the highest contamination was detected 5-20 m. The amounts calculated in plants in the neighborhood of the expressway were found 2.1 mg Pb kg −1 DW, 0.06 mg Cd kg −1 DW, and 62 mg Zn kg -1 DW and the amounts calculated among snails were found 21.3 mg Pb kg −1 DW, 5.7 mg Cd kg −1 DW, and 510.8 mg Zn kg −1 DW. The concentrations were found with decreasing trend with distance increasing away from the expressway. The results of the three above metals showed that Pb was appeared the finest metal for contamination evaluation or pollution caused by road transport. Parkpian et al. (2003) showed their findings that plants growing adjoining the expressway were exposed frequently to more accumulations of heavy metal than the plants grown away from the expressway. However, their analysis confirmed that improved farm management practices resulted in substantial reduction of Pb and Cd levels in soil and green forage and ultimately to reduce the Pb and Cd contents in the milk. Lokhande and Bathe (2001) investigated that the waste water application and the vehicle smoke have brought on soil air pollution of two heavy metals which change the houses of soil. The changed houses of soil motive easy mobility of heavy metals into the plants. Farmer and Farmer (2000) discovered that heavy metals entered into our bodies and milk of the cattle by means of the water they drink and fodder they eat. Fodder acquired contaminated by way of the application of more than a few fungicides, fertilizers, industrial effluents, etc.
Lead increased concentrations in soil may be due to lead contaminated air, excessive application of industrial effluents, and smoke from vehicle and automobiles. And as a result, an excessive concentration of lead gets accumulated in animals' body and also in that of humans. Animals feeding on pastures situated near industrial units, mining communities, and road sides get more exposed to heavy metals. Airborne lead and lead taken up by roots of plants affect grazing animals. Major risk on soil contaminated plants was dust from roadside rather than metal uptake.
The current endeavor was designed to determine lead concentration in soil, forage, water, and cow's blood serum of domesticated near automobile transportation and to correlate its results with biochemical and hematological parameters in Sahiwal, District Sargodha, Pakistan, with objective to evaluate addition and transfer of Pb in water, soil, forage, and animals' blood.

Materials and methods
The study area selected was Sahiwal, District Sargodha Pakistan. This is an agricultural area where most important crops are grown. The forage chosen for learn about had been Zea mays L., Avena sativa L., Trifolium alexandrinum L. and Brassica campestris L. Samples of water, soil, forage and cow's blood were collected.

Water samples
All of the samples of water measuring 100 ml were collected by following the method given by Association Francaise de Normalisation (AFNOR) (1997).

Forage samples
A total of one hundred twenty samples of 4 forages had been composed of maize, oat, berseem, and Brassica from winter season. The sampling procedure was executed as 4 replicates of every forages from each place. Samples of maize, berseem, oat, and Brassica were collected from the roadsides of Dera Jara, Radhan, Majoka, Sial Sharif, and Nehang. The samples from the distant avenue had been amassed from Vijh. All of the samples of forages were subjected to air drying followed by oven drying at 70-75°C for 7 days. From these samples about 2 g (Nascimento et al. 2014) with the objective to decide Pb availability in the soil and forage by the use of the Mehlich 1, DTPA, and USEPA 3051 and 3052 methods of extraction. The weighed samples were saved for the additional procedure (Zhang et al. 2010). Forage (0.5 g) used to be digested by means of the usage of 5 ml of HNO 3 (70%) and 1.5 ml of HCIO 4 (60%). It was heated until the evaporation of brown fumes (Jones 1991).

Soil samples
The samples of soil were amassed as two hundred and forty replicates of soil from six websites along with 120 samples from winter season. Each of these (1 kg sample of soil) had been gathered in polythene luggage by digging soil about 15-30-cm deep with the help of shovel. Followed by way of series of samples about 10 g of each sample was once air dried. After air drying samples were followed oven dried for about 70-75°C . From the oven, the soil samples were eliminated and after 7 days and till then all the moisture contents from the soil samples have been removed. Then these samples have been beaten using pestle mortar and about 2 g of every sample used to be saved for the additional technique after sieving of all samples. Total contents of heavy metals in soil samples were studied after the digestion of samples. Approximately, 2.5 ml nitric acid, 0.5 ml hydrogen peroxide (30%), and 7.5 ml of hydrochloric acid were applied (Kilburn n.d.).

Blood samples
A total of 60 cow blood samples were collected, 12 from Radhan, 12 from Nehang, 12 from Dera Jara, 12 from Majoka, and 12 from Sial Sharif facet. Samples had been gathered in 16 × 150 mm sealed test tubes. Blood samples of cow have been collected from the major veins of cow in standing condition by using sanitized needle. After collection, blood was positioned in heparinized Na-citrate vials quickly for halting clotting. Blood serum was separated from plasma by using a centrifuge running at 3000 rpm for 15 to 30 min. The serum was used placed in small labeled vials and stored in freezer at 200°C.
All blood serum samples were organized by wet digestion according to the process of Richards (1968). Briefly, 0.5 ml of serum was used for digestion with 10 ml focused nitric acid in a 100-ml digestion flask initiating at low temperature for 15 to 20 min until the contents were clear and followed with 5 ml perchloric acid for 15 min. Resultant in the flask was heated strongly until 2-3 ml colorless solution was obtained. After cooling, the contents were diluted to 20 ml by adding redistilled water in volumetric flask and finally preserved for further analysis.

Apparatus and chemicals and instrument
Measuring cylinder (10 ml), salts, pipette (10 ml), volumetric flasks (1000 ml and 100 ml), and beakers (500 ml) were washed with distilled water. The chemical substances used in this manner have been of MERCK Company. The digestion of the forage, soil, and blood samples used to be followed by using the heavy metal analysis of samples. This analysis was performed after the samples have been diluted the usage of freshly prepared distilled water. Heavy metal analysis used to be performed via the use of Flame Atomic Absorption

Statistical analysis
Analysis of variance and correlations was observed by SPSS (special program for social sciences) software program model No. 20. Variance for lead in soil, feed, and water was found through making use of one-way ANOVA. Correlations with Pb concentrations of forage and soil were accounted with mean value at 0.05, 0.001, and 0.01 chance stages represented by Steel and Torrie (1980).

Results
ANOVA showed a non-significant impact of sites on the Pb concentration in soil grown with all forages, maize, oat, berseem, and Brassica, and blood and water samples while significant influence of sites was detected on the concentration of Pb in maize (Table 1). Water samples showed Pb concentration in all sites that ranged from 1.14 to 0.44 mg kg −1 . Pb contents were maximum at site 5, and minimum at site 2 (Table 2 and Fig. 1). Soils samples of all the forages also recorded Pb concentration at all sites and it ranged from 1.58 to 0.279 mg kg −1 . The concentration was the maximum in A. sativa grown at site 5, and it was the minimum in Z. mays at site 2 (Table 3 and Fig. 2). Samples of forages also contained concentration of Pb and it ranged from 0.048 to 2.002 mg kg −1 . The maximum value of concentration of Pb was in B. campestris at site 1, while the minimum value of concentration of Pb was in T. alexandrinum at site 2 (Table 4 and Fig. 3). Similarly, samples of cow blood showed Pb concentration at all sites that ranged from 4.468 to 0.217 mg kg −1 . Pb concentration was the maximum at site 1, while it was minimum at site 3 (Table 5 and Fig. 4).

Pollution load index
Pollution load index (PLI) for Pb at six sites of irrigation in all forages ranged from 0.03 to 9.19. It was the maximum for A. sativa at site 4, while the minimum for Zea mays at site 2. The descending order of PLI at site 1 in all the given forages was B. campestris < Z. mays < T. alexandrinum < A. sativa. The descending order of PLI at site 2 was Z. mays < B. campestris < T. alexandrinum < A. sativa. For site 3, the order of PLI was Z. mays < B. campestris < T. alexandrinum < A. sativa. For site 4, the order of PLI was B. campestris < Z. mays < T. alexandrinum < A. sativa. The order of PLI at site 5 was T. alexandrinum < B. campestris < Z. mays < A. sativa. The order of PLI at site 6 was Z. mays < B. campestris < T. alexandrinum < A. sativa (Table 6).

Bioconcentration factor
Bioconcentration factor (BCF) for Pb at six sites of irrigation in all forage samples ranged from 0.03 to 9.95. It was found highest for A. sativa at site 4 of irrigation, while it was lowest for T alexandrinum at site 2. The order of BCF for Z. mays at different sites was site 4 < site 5 < site 1 < site 6 < site 3 < site 2. For A. sativa, BCF at different sites was in the order site 2 < site 1 < site 6 < site 5 < site 3 < site 4. The order of BCF for T. alexandrinum at different sites was in the order site 4 < site 2 < site 3 < site 1 < site 5 < site 6. For B. campestris, order of BCF at different sites was site 5 < site 4 < site 3 < site 6 < site 2 < site 1 (Table 7).

Daily intake of metal and health risk index
Daily intake of metal (DIM) was the highest for B. campestris at site 1 while it was the lowest for Z. mays at sites 1, 2, 4, and 6, for A. sativa at sites 1, 2, 3, and 6, for T. alexandrinum at sites 2, 3, and 6, and for B. campestris at sites 1, 3, 5, and 6.
Health risk index (HRI) was the highest for B. campestris at site 1, while it was the lowest for Z. mays at site 4. The order of health risk index for Z. mays at different sites was site 4 < site 5 < site 1 < site 6 < site 3 < site 2. For A. sativa, DIM at different sites was in the order site 2 < sites 1 and 6 < site 5 < site 3 < site 4. For T. alexandrinum at different sites, the order was site 2 < site 1 < site 3 < site 6 < site 5 < site 4. For B. campestris, order of DIM at different sites was site 5 < site 4 < sites 3 and 6 < site 2 < site 1 (Table 8).

Discussion
To keep protection against exposures of lead, the Codex has framed latest limits of contamination of lead found in food. Exposure to high levels of lead is hazardous to human body, particularly infants and children are susceptible to the detrimental effects of lead, which can impact their brain development. In Pakistan a relatively short food web of soil + water nutrients/contaminants → forage → cow → milk and meat may be hazardous for the consumers as Codex Alimentarius Commission (CAC) has given permissible limits of lead in meat of cattle, sheep, and poultry (ML 0.05 mg kg −1 ; lead range 0-0.1 mg kg −1 ) (CAC 1997(CAC -1999. The instant endeavor has given Pb values to higher sides in blood of cow as compared to CAC which can be associated with meat of cow. However, it depends on the daily intake of the meat. Hence care is mandatory to keep avoid risks to human health. Additionally, most of the results were in conformity with the earlier works with few exceptions, conducted by above authors viz., Viard et al. (2004), Saleem et al. (2020), Parkpian et al. (2003), Kodrik et al. (2011), Miclean et al. (2019, and Liu et al. (2020).

Conclusion
Lead found in current study is the hazardous metal for human and animal health but luckily its contents are below the dangerous levels on the basis of daily intake as described by CAC. However further studies are imperative to investigate harmful effects of Pb transferred from animals and their products in local conditions.