Assessment of Lead and Zinc Profile from Vehicle Emission in Roadside Fodder Plants

Due to increasing population and increasing number of automobiles the fodder samples were collected from Khushab District along the roadside to examine the heavy metal contamination. It was a serious issue because animals consume these contaminated fodders and then they enters the food chain where they cause toxicity. Five fodder plants Pennisetum glaucum, Trifolium alexandrinum, Saccharum officinarum, Zea mays and Brassica compestris were selected for sampling. The sampling of selected fodder plants and their soil was done during November which was the driest season of Khushab city and the dust and smoke was present on the fodder plants. The samples were air and oven dried. Then subjected to digestion. Acid digestion was done with Nitric acid (HNO3), Hydrogen peroxide (H2O2) and Hydrogen chloride (HCL). The samples become transparent and the heavy metal analysis of both soil and fodder samples was done through Atomic absorption spectrophotometer (AASP). Lead and zinc heavy metals were analyzed. The pollution load index was lower than 1 which indicates that there was low concentration of metal in soil of the collected samples of the current study. It was obtained that the soil was less polluted. The daily intake of metals was lower than 1 that concluded that they were at safe limit during consumption. The present study showed the enrichment concentration of metals greater than 1 it means these metals were present in both soil and fodder samples growing near roadside.


Introduction
The word pollution is derived from Latin word "Pollutioneum" meaning to make dirty. Atmospheric pollution can be defines as change in the air constituent due to human activity [1]. According to National Environmental Research Council, pollution can be defined as, release of wasteful substances and energy through human activities that cause harmful changes in the natural environment. Health Effects Institute reported that 95% of world's population breathe dangerous and polluted air. The presence of one or more contaminants such as dust, smoke, smog that cause injurious effects on human health, plants and animal life, or which interferes with enjoyment of life and property is called Air pollution. The atmospheric pollution is the serious problem of today. It is the more severe type of pollution, it causes declines in environmental conditions. According to this report Pakistan, Bangladesh, India have contributing steepest in increasing air pollution level since 2010. In Pakistan tremendous increase in motor vehicles is 37% per year. Vehicles emit 20-90% of cadmium that have negative impacts on green pigment in plants [2].
Plants are very important to enhance the air quality by absorbing gases and particles from the air especially in urban areas [3]. The effective indicator of overall air pollution are plants. Plants are mostly used for the determination of air pollutants in a very short time. So that, the trees and Shrubs are used as dust filters to determine the increasing heavy metal an d dust pollution level of atmosphere [4]. collected in polythene bags by digging soil about 15-30 cm deep with the help of shovel. The 0-25 cm depth was considered to represent the plough layer and average root zone for nutrients uptake and heavy metals burden by plants.

Sample Preparation Preparation of Fodder samples
Plants were first placed in open air and then transferred to oven at 72ºC till all the moisture contents are removed. Then these dried samples were converted into powder form and stored in plastic bags until used for digestion. Wet digestion method was used for the digestion of fodder samples. Samples were digested in 5mL of 68%HNO3 for 24 h. Then put into digestion chamber and heat it and removed until when the evaporation of fumes stops. Then 5mL of 30% H2O2 were added and again heated until it becomes colorless. By adding the filtered distilled water the digested samples were made up to 50 mL and kept in labeled plastic bottles [20]. The solution was then taken toatomic absorption Spectrophotometer for heavy metal analysis. The sample preparation procedure was performed according to [20].

Preparation of Soil samples
Soil samples was air dried at room temperature and then oven dried at 70ºC. 0.5g of soil samples were subjected to digestion chamber. 0.5g of sample and 20mL of HNO3 was added in digestion tubes. Heat the tubes in digestion chamber. Add 5mL of H2O2 was added and then heated again until it becomes colorless [20]. By adding distilled water the samples were made up to 50mL. Then the samples were stored in plastic bottles. These solutions were then subjected to Atomic absorption spectrophotometer for analysis of heavy metals.

Standard Preparation:
The standards were prepared by using following methods ➢ To make the standard solution glass were used and it must be clean. 100mL volumetric flask and funnel were washed with de-ionized water.
➢ Analytic balance was used to weight the sample in a beaker. ➢ Small volume of sample or another solvent can be used to dissolve the sample ➢ The sample was completely dissolved in solution. To speed the process of dissolution hot plate was used.
➢ Volumetric flask was used to pour the sample quantitatively. Beaker, funnel and stirring rod was washed completely with de-ionized water. Sample was transferred to the flask.
➢ The beaker was washed with de-ionized water for few minutes ➢ Made a mark below 100mL on volumetric flask and then filled it with few mL. ➢ By using dropper add few drops of de-ionized water so that meniscus touched the mark that was placed on the flask.
➢ Shake the flask for few minutes to mix it thoroughly ➢ Make sure that solution becomes homogeneous ➢ At the end again checked that meniscus touch the mark of 100mL at volumetric flask

Mineral analysis:
Plants and soil samples were directed towards the Atomic Absorption spectrophotometer for heavy metal analysis. The Atomic absorption spectrophotometer Perkin-Elmer AAS-5000 (Perkin-Elmer Corp, 1980), was used after acid digestion ( Table 2). It was used to analyze the heavy metals lead (Pb) and Zinc (Zn) in the present study. As was analyzed through using graphite furnace.

Statistical analysis
SPSS (Special Programme for social sciences) was used for the analysis of variance and correlations. Variance analysis of heavy metals in soil and fodder plants was determined by two way ANOVA. The soil to fodder correlation was determined. Significance of mean values was at 0.05, 0.001, 0.01probability levels reported by [21].

Bio-Concentration Factor (BCF)
The concentration of heavy metal in fodder sample from soil to fodder is called as bio concentration factor (on the basis ofdry weight).

Bio-concentration factor=Cfodder/Csoil
Cfodderindicates the metal concentration present in the fodder sample while the Csoilindicates the metal concentration present in the soil, mg/kg dry weight [22].

Daily Intake of Metals (DIM)
The formula to determine the daily intake of metal was given by [23].

DIM= Cmetal× Cdaily food intake × Conversion factor / Baverage weight
Cmetal indicates the concentration of metals, Cdaily food intake indicates the consumption of fodder by cattle per day in Kg, the value of Conversion factor was 0.085, the Baverage weight represents the average body weight of buffalo of the selected sampling site. The average consumption of fodder by animal was 12.5Kg and average body weight was 550Kg per cattle [24].

Health Risk Index (HRI)
To determine the health risk of heavy metals through ingestion of contaminated fodders health risk index was calculated. This can be calculated by dividing the daily intake of metal to oral reference dose [25].

HRI=DIM/RfD
DIM indicates the daily intake of metal while RfD indicates the oral reference dose of animal.

Enrichment Factor: (EF)
It can be defined as the withholding of metal in soil and it depends upon the availability of metal in soil. It was used to find the concentration of metal present in the soil.

EF=concentration of metals in the amended soil/concentration
of metals in the controlled soil [26] Pollution Load Index The pollution load index helps us to determine the concentration of heavy metal in the soil.

PLI= Csoil/C reference value
Csoil denotes investigated concentration of metal in the soil and Creference value indicates the reference value of soil for each metal [27].

Correlation
The correlation coefficient of metal concentration in soil and fodder samples were determined by using (SPSS) abbreviated as Statistical program for social sciences. To determine the correlation of metal from soil to fodder one way ANOVA was used in the current study.

Results and discussions 3.1. Soil
The analysis of variance of Pb in soil showed significant effect on Sites, Treatment, Fodders, Sites * Treatment, Sites * Fodders, Treatment * Fodders, Sites * Treatment * Fodders ( Table 3). The significant effect was showed in analysis of variance of data for Zn Sites, Treatment, Fodders, Sites * Treatment, Sites * Fodders, Treatment * Fodders, Sites * Treatment * Fodders (Table 4).

Fodder
The analysis of variance of data for Pb showed significant effect on Sites, Sites* Treatment, and significant effect on Treatment, Sites* Fodders and non-significant effect on Fodders, Treatment * Fodders, Treatment* Fodders and Sites * Treatment * Fodders. The analysis of variance for Zn showed significant effect on Sites, Fodders, Sites * Treatment, Sites * Fodders, Treatment * Fodders, Sites * Treatment * Fodders while non-significant effect on Treatment (

Mean concentration of heavy metals in fodder Plants
The highest and lowest mean concentration of Pb showed that the B.compestris was facing highest contamination of Pb through vehicles near and even at a distance from the road ( Table 7). The highest concentration of Zn was present in B.compestris of site 1 and lowest ( Figure 1) value was present in S.officinarum of site 2 present at some distance from the road ( Table 8, Figure 2). The present study showed that the mean concentration Pb, Zn of fodder samples collected from roadside were higher as compared to the mean concentration given by [28]. The work done by [28]. The lead concentration was 0.0520mg/kg which was lower than the present study. Their work was done along major roads and control sites of Dibete Area. He also analyzed the heavy metal concentration along roadside forage samples. In Nigeria the same work was done by Ogandele, he collected forage sample along major roadsides. The samples were acid digested and analysis of metals were done by Atomic Absorption Spectrophotometer. According to present study range of Pb was 13.0352 mg/kg to 2.2152 mg/kg, which was within the range (24 to 397mg/kg) as proposed by [29]. The mean concentration of Ni and Zn were higher as compared to present study while the concentration of Cd and Pb was lower [30]. According to Ahmad [31], the levels of heavy metals concentration for Pb was lower in their forage samples as compared to the current study.

Mean concentration of heavy metals in soil
The soil of P.glaucum had highest concentration of Pb while the Z.mays showed the lowest concentration facing high traffic density (Table 9, Figure 3). The Zn showed highest and lowest concentration in the samples B.compestris and Z. mays fodder samples facing highly traffic pollutants (Table 10, Figure 4). The mean concentration of the zinc is lower as compared to present study [30]. The trend of heavy metal concentration of soil taken roadsides was given as Fe > Zn >Mn>Pb> Cd [32]. The concentration of metals were lower as compared to the present study. The higher Zn and Cd level along roadside soil samples were due to high traffic density and lubricating oils which contains Zn in the form of Zn dithiophosphates [33]. Opaluwa [34] reported the mean concentration of Fe, As, Pb, Zn, Ni was lower as compared to current study. Mmolawa [35] worked on soil samples along major roadsides of Botswana. He selected soil samples from five sites and analyzed the heavy metals in them.

Bio concentration Factor
The highest mean concentration of Bio concentration factor was 5.646365mg/kg in S.officinarumat a distance from the road while lowest in Z.mays 0.214276mg/kg present near the road edge (Table 11). The Highest bio concentration of Zn was present in Z.mays samples of near roadside while lowest in S.officinarum samples collected away from the road ( Table 12). The order of Bio concentration factor of the present study was Pb> Zn. The BCF concentration of Zn was (0.349) lower as compared to the present study [36]. The bio concentration depends upon the pH of the soil and they do not easily transfer in the fodder plants [22,37] Soil pH effects the mobility of metals in soil. Higher the soil pH causes low mobility of metals in soil [38]. It was reported that if the bio concentration factor is less than 1 no accumulation of metals occurred in soil. The higher bio concentration of metal indicates the higher range of its accumulation in plants [39].

Correlation
The positive and non-significant correlation was found in Soil-P. glaucum and Soil-B. compestris and the negative non-significant correlation was present in Soil-T.alexandrinum, Soil-S.officinarum and Soil-Z.mays (Table 13). The Zn correlation coefficient showed the negative non-significant correlation in Soil-S.officinarum, Soil-Z.mays and Soil-B. compestris (Table 14). The positive non-significant correlation was present in Soil-P.glaucum and Soil-T.alexandrinum. The current study showed the nonsignificant correlation of Pb and Zn. According to Onjefu et al., [53] Fe, Mn, Ni, Zn Cr showed the strong (r > 0.5 to 0.9) and extremely strong (r > 0.9) positive correlation while the Pb showed weak positive correlation (r=0.3524). According to Nazzal et al. [54] Pb, Mn, Ni, Fe, Cd have showed the significant positive correlation As compared with current study in which the heavy metal Pb showed negative non-significant correlation.

Daily intake and health risk index
The Highest value of Daily intake of Pb was found in nearby road sample of Z.mays of site 4 while the lowest value was recorded in T.alexandrinum of site 2 ( Table 15). The Highest value of Health risk of Pb was also found in the Z.mays of site 4 collected near the road while the lowest value was recorded in the B.compestris of site 3 collected near the road ( Table 16). The highest daily intake concentration of Zn was calculated in site 1 T.alexandrinum sample taken far away from the road. The lowest value was found in the away road sample of S.officinarum of site 2. The highest concentration of Zn in health risk index was found in the B.compestris of site 1 of away road sample while the lowest value was found in the away road sample of S.officinarum of site 2. The order of Daily intake of metals in animals was in the order Pb> Zn in the current study. The daily intake of Pb was lower in the present study as compared to given by Guerra et al., [42]. The daily intake of metals in the present study lower than 1, indicates no risk of health for livestock via consumption as reported by Radwan and Salama [43]. Our present study showed the order of health risk index as Pb> As > Cd > Fe > Zn > Ni >Mn> Cr. The health risk index was obtained by using the oral reference dose of the metals. The present study showed the health risk index of Pb, Cr, Cd, Zn were higher as compared to Ali et al., [44]. but the health risk of Ni was lower in the present study. According to Khan et al., the health risk of Mn was higher as compared to present study. The concentration of heavy metal greater than 1 showed high risk of health for livestock Sajjadet al., [45]. The metal accumulation even at very low level in liver and kidney cause abnormality of metabolism and health risk for both wildlife and livestock [46,47]. Forages growing in polluted environment such as along roadsides accumulates toxic metals in high concentration which poses danger to the life of animals and enters the food chain and cause detrimental effects [48]. Increase concentration of lead in the forage samples along roadsides cause restriction of root expansion and cell division and cell elongation [49]. The highest mean concentration of lead from vehicle exhaust enters the food chain and cause serious problems in kidneys, liver, central nervous system, reproductive system and anemia in humans [50].

Pollution Load index
The highest pollution load index of Pb was examined in P.glaucum growing near road of site 3 and lowest was found in away road sample of Z.mays of site 4 ( Table 17). The pollution load index of Zn in fodders soil was found highest in T.alexandrinum of site 1 collected near the road while the lowest concentration was found in the Z.mays of site 4 taken near the road side (Table 18). The pollution load index of Zn, Pb, Mn and Fe was higher as compared to the present study. The pollution load index of Pb and Zn in the current study is lower as obtained by Mmolawa [35]. The pollution load index will be equal to 1 than plants are contaminated by pollutants, if PLI <1 it shows excellence level and if PLI >1 it shows the site is deteriorated with anthropogenic activities [40]. As in the present study the highest concentration of pollution load index Of Zn showed that it directly relates with traffic density and distance from the road. As the distance of soil sample from the road decreases the level of its concentration becomes low [41].

Enrichment factor
The highest value of Pb concentration was observed in S.officinarum of site 4 collected away from the road, while the lowest value was observed in Z.mays of site 3 collected from the roadside. The highest enrichment factor of Zn metal was determined in near road sample of Z.mays while the lowest concentration was present in away road sample of B. compestris of site 2. Sulaiman and Hamzah, reported the enrichment factor of Pb and Cd which was lower as compared to the current study [51]. The enrichment factor of Pb in the present study was higher. Their study showed the range of enrichment factor from moderate contamination to considerable contamination. Enrichment factor is the concentration of heavy metal present in the soil. The present study showed the order of enrichment factor as Zn>Pb. The study reported by Amieret al., the concentration of Pb showed extremely high enrichment as compared to present study which showed the lower enrichment concentration [52].

Conclusions
Atmospheric pollution is the major cause of these metals in soil and plants. Vehicle exhaust is the source of atmospheric pollution. The current study reported that the natural weathering of building materials, emissions from industries, bricks kilns, and heating systems burning of fuel also cause the heavy metal pollution but the vehicles are also the source of its accumulation in plant and animal bodies.
The pollution of heavy metals from automobiles is of serious environmental issue. Due to increase in population the traffic burden is also increasing day by day. The present study was done on the fodder samples along roadside environment. Fodder plants and soil are facing high concentration of heavy metal https://doi.org/10.37358/RC.20.12.8393 pollution originating from busy roads in the vicinity of suburbs and contribution of high traffic density to high level of exhaust emissions.
Samples analyzed in the current study have low and high values of heavy metals from the permissible limits. The heavy metal concentration is strongly and inversely correlated with distance from the road. Lead and Zinc are major metals present in roadside environment and they are released from burning of fuels, tyres and through leakage of oils. Heavy metal from vehicles exhaust are very toxic for living organisms. Even some trace elements are essential for plants but they become toxic if their occurrence exceed the limit.