Bioaccumulation and transfer of zinc in soil plant and animal system: a health risk assessment for the grazing animals

Heavy metals pollution has thorough worldwide apprehensions due to the instantaneous growth of industries. Farming regions are irrigated mainly with wastewater which contains both municipal and industrial emancipations. Keeping in view the above scenario, a study was designed in which three sites irrigated with ground, canal, and municipal wastewater in the District Jhang were selected to determine the zinc accumulation and its transfer in the soil, plant, and animal food chain. Zinc concentration was ranged as 18.85–35.59mg/kg in the soil, 26.42–42.67 mg/kg in the forage, and 0.982–2.85mg/kg in the animal samples. Investigated zinc concentration in soil and forages was found to be within the recommended WHO/FAO limits, but blood samples exceed the standards of NRC (2007). The maximum level of pollution load index (0.427–0.805mg/kg) and enrichment factor (0.373–0.894 mg/kg) for zinc was noticed upon wastewater irrigation. Daily intake (0.039 to 0.082 mg/kg/day) and health risk index (0.130 to 0.275 mg/kg/day) of zinc metal was higher in the buffaloes that feed on wastewater-irrigated forages. Bio-concentration factor (0.840 to 2.01mg/kg) for soil-forage was >1 which represents that these plants accumulated the zinc concentration into their tissues and raised health issues in grazing animals on consumption of wastewater-contaminated forages. As animal-derived products are part of human food, then zinc toxicity prevailed in livestock tissues ultimately affects the human food chain. Overall, findings of this study concluded that animal herds should be monitored periodically to devise preventive measures regarding the toxic level of heavy metals availability to livestock.


Introduction
The term "pollution" is derived from "Polluere" which is a Latin word that means "to defile or to make foul." Any substance that deteriorates the eco-quality and disturbs the supremacy of life is called pollutant. Environmental pollution is the occurrence of these defilements in the environment which are detrimental for living creature (Saleem et al. 2020a). Heavy metals pollution in environment is a serious dilemma in almost all states of world, but it is a forefront threat in the emerging countries due to inadequate reserves, standard, strategies, and organizational supervision. Heavy metals are defined as the elements that are dense beyond 4.5g/cm 3 and produce free electrons in a reaction. They are known as good conductor of heat and electricity, having high boiling and melting points, ductile, non-transparent, and shiny in appearance (Szyczewski et al. 2009). They are omnipresent elements and durable and have long biotic half-life. The examples of heavy metals are cobalt, copper, iron, manganese, zinc, lead, cadmium, and vanadium (Duman et al. 2019;Hu et al. 2018) Biological monitoring is a methodical strategy which is practiced to locate the dispersion proliferation and accretion of heavy metals in the natural world. It is centered on the selection of representative tissue and fluids from the living biota and examines them for an extended period of time to determine its status. Mostly sampling of soil, vegetation, and agrarian outputs are obtained from the metropolitan stretch to study heavy metal content in them. It provides better relation among ecological toxins and their consecutive effects on living organisms (Ugulu et al. 2019).
In order to calculate risks associated with the livestock exposure to toxic metals, take into account diet-selecting behaviors, fluctuation in climatic conditions, and herbage growth that affect the metal bioavailability (Fritsch et al. 2011). Livestock is exposed to these toxic metals by drinking polluted water and by ingestion of polluted vegetation. Few elements like Fe, Cu, Zn, and Mn are required by animals as essential constituent, but their high level in the feedstuff imposes health hazards on the yield and developmental process (Khan et al. 2020a;Khan et al. 2019a). Zinc is essential micronutrients for plant and animals. In plants, activity of various enzymes involved in synthesis of auxin, protein, and carbohydrates is regulated by the presence of zinc metal (Zaheer et al. 2020). It also controls the gene expression during high temperature and light intensity. Deficiency of Zn causes stunted growth of plant and yellowing of leaf . In animal and human body, Zn repaired the damage DNA and RNA. Almost 200 enzymes use Zn as a coenzyme. It assists the immune system by controlling the gene expression of specific proteins by defending their conversion to unwanted forms and enhances the metabolic process. Animals especially cattle require zinc concentration between 1.5 and 2.0 g/day. But its excessive uptake damaged reproductive performance and renal and hepatic tissues of animal body. In human body, it plays an important role in the normal functioning of cells especially in the cell division and synthesis of carbohydrates and proteins. In various enzymes, the cadmium swaps up the zinc atom. For that reason, an excessive amount of zinc plays an important role to reduce the cadmium toxicity (Hill and Shannon 2019).
Various researchers reported that health hazards of toxic metal in animal body depend on the metal intake amount, interaction of metal with the internal stability of organism, and category of animate being used Khan et al. 2020a;Nadeem et al. 2020). With the advancement of industries and urbanization, emitted wastewater incorporates the toxic metals into air, water, soil, flora, and fauna which compel the researchers to analyze the various chemical dynamics of environment to detect the degree of deformation in natural environment (Saleem et al. 2020b(Saleem et al. , 2021. The aim of the study was to appraise zinc flow in soil, plant, and animal food chain. The objective of present study was to analyze the effect of various irrigation sources on the accumulation of zinc in soil, forages, and possible health risks assessment in animals via intake of these forages. Various pollution indices were also measured to determine the zinc deficiency or toxicity in grazing animals.

Study area
The position of district Jhang is 71°-37°to 73°-13°longitudes toward east as well as 30°-37°to 31°-59°latitude toward North of Punjab, having total area of 8,809 km 2 . Its border touches to Hafizabad and Sargodha District in the North, Toba Tek Singh and Faisalabad District in the East, Muzaffargarh District in the southwest, and Layyah and Bhakkar District toward the west ( Figure 1). In this district, summer duration is prolonged nearly of 7 months, which starts from April and persists until October. May, June, and July are hottest months (Badar et al. 2017).

Sampling locations
Sampling was done during the period of 2019-2020. The collection of soil, fodder crops, and animal samples was done from three different sites of District Jhang. Site-1 was Jhang (Jh-I) irrigated with ground water, Site-2 was Shorkot (Sh-II) where canal water irrigation was in practice, and Site-3 was Ahmad Pur Sial (Aps-III) where municipal wastewater was used for irrigation purpose.

Forage/fodder crops collection
Three replicates of each forage sample (Acacia nilotica L., Capparis decidua Forssk, Zea mays L., Medicago sativa L., and Pennisetum glaucum L.) were taken and kept in polythene bags and transported to lab. All the replicate samples of forages were cleaned with distilled water to remove dust particles and externally deposited contamination. These samples were first air-dried and then oven dried for 7 days at 70-75°C. Then they were ground and kept in labeled sealed bags for digestion (Khan et al. 2019b)

Soil collection
Three replicates of soil samples were collected from depth of 0-20cm. The samples were collected in air-tight bags. Samples were dried in air and then kept in an oven at 70-75°C for approximately 7 days. When all the samples are fully dried, they were stored in labeled sealed bags for next step (Khan et al. 2018) Animal samples collection This experimental study comprised of cow (Bos taurus L.), buffalo (Bubalus bubalis L.), and sheep (Ovis aries L.). Selected animal samples included in this study were blood, hair, and feces of each category. Ten animals of each category were used to collect samples from every site, and these animals mainly feed on the collected forages of pastures.
From the jugular vein of animals, the blood samples were collected in sealed test tubes. The blood samples were centrifuged for 15min at 3500rpm, to separate blood plasma then stored at −20°C, until digestion (Ahmad et al. 2021).
The hair samples were also collected from the bodies of selected animals and are properly labeled. These samples were rinsed with acetone and then followed by distilled water to remove external contamination. Further, they were dried in oven for at least 3-4 days, and then they are ready for digestion (Gabryszuk et al. 2018).
The collected fecal samples are taken directly from the selected animals upon excretion to avoid further contamination (Svane and Karring 2019). Labeled samples are stored in plastic bags. Firstly, they were air dried (3 days) and then kept in oven nearly 3-4 days.

Soil digestion
The powdered soil (2 g) was taken into the digestion tube. About 20ml concentrated H 2 SO 4 was added into the tube. Run the digestion chamber for 30 min. Further add 10ml H 2 O 2 to assist digestion, and again run the chamber until colorless solution was obtained. Solution is filtered, and final volume (60ml) was adjusted through distilled water addition. The sample was stored in plastic bottles for further metal analysis (Siddique et al. 2014).

Forage/fodder crops digestion
Plants samples were digested through dry ashing process. Firstly, all the crucibles were washed with distilled water and dried in an oven to remove moisture. Ground forage sample (2g) was taken in the crucible and burnt at 550°C, and then let it to cool down at room temperature. Ash content of each crucible was dissolved by adding 2.5ml of hydrochloric acid in it. If some ash remained undissolved, then heat it on the hot plate until complete digestion takes place. The prepared solution was filtered by making a final volume of 60ml and kept in plastic bottles for digestion purpose (Siddique et al. 2014) Animal samples digestion For blood digestion, 5ml of H 2 SO 4 and 2 ml of blood plasma were taken in digestion tube. The mixture was heated to dissolve the organic content until yellow color appears. Further 2ml of H 2 O 2 was added and heated until it turns into colorless solution. This mixture is cooled, filtrated through paper, and stored up to a final volume of 60ml in plastic bottles for metal analysis.
For hair and fecal samples, 2g of each sample was weighted for their digestion. The measured sample were mineralized with H 2 SO 4 and H 2 O 2 (4:1) reagents. After complete digestion, final volume of 60ml was made by adding distilled water. Finally, samples were stored for analysis after their filtration.

Heavy metal analysis
All the processed samples are then analyzed through atomic absorption spectrophotometer having 213.9nm wavelength, and with a hollow cathode lamp, current of 5mA was used to find out Zn concentration in all samples. To ensure the quality of results and to precise the analytical techniques, all the apparatus tools are washed with the distilled water and dried in air prior to usage. For the validation of results, the replicates are analyzed for each sample and compare with the international standards. The concentrations of all the samples are taken in mg/kg (solid sample) or mg/l (liquid sample).

Statistical analysis
The data of soil, forage, and animal are analyzed statistically. The mean concentrations are set up in these samples, and next presentation of data was established through SPSS-16 and two-way ANOVA. The significant probability was observed at 0.05, 0.01, and 0.001 level, respectively.

Bio-concentration factor
The concentrated level of metal uptake in forage tissues was determined by Cui et al. (2004) formula: BCF¼Metal concentration analyzed in forage samples =Metal concentration analyzed in soil samples :

Pollution load index
It is used to determine the contamination of heavy metals in soil. Its formula given by Liu et al. (2005) is: The reference value for Zn is 44.19 mg/kg (Singh et al. 2010).

Enrichment Factor¼
Metal concentration in forage=Metal concentration in soil ð Þ sample Metal concentration in plant=Metal concentration in soil standard

Daily intake of metals
The formula to find the daily intake of metals (DIM) is: DIM ¼ Analyzed metal concentration in forage*Conversion Factor*Daily food intake Average body weight of animals For the conversion factor, value is 0.085 (Jan et al. 2010). For cows, daily intake of food is 12kg and average body weight is 600kg, whereas the daily intake and average weight of sheep are 1.3kg and 75kg, respectively (Johnsen and Aaneby 2019). For buffaloes, average weight is 550kg and their daily intake is 12.5kg (Briggs and Briggs 1980) Health risk index The formula to find the HRI is (USEPA 2002): HRI ¼ Daily intake of metal=Oral reference dose Oral reference dose for Zn is 0.3 mg/kg (USEPA 2010).

Soil analysis
The Zn analysis of variance showed that significant effect of treatments (p<0.001) was found on sites, while reverse (p˃0.05) was true for soil and site*soil (Table 1). The Zn concentration in soil samples varied from 18.85-35 to 59 mg/kg ( Table 2). The minimum concentration was observed in soil of C. decidua at site Jh-I, whereas P. glaucum soil showed maximum concentration at the site Aps-III.
Present concentration of zinc in soil was lesser than acceptable limit (300mg/kg) of WHO/FAO (2007). Similarly, Narwal et al. (2013) stated that Zn deficiency was prevailed in almost 50% of the world soil. The predominant climatic conditions, source of irrigation, and the applied practices brought differences in the content of heavy metals. The reported zinc level of Eissa and Almaroai (2019) in soil was 600 mg/kg which was found to be higher than the current results. Pathak et al. (2010) also recorded the higher level of zinc (211.96 mg/kg) than the present study. The findings of Murtaza et al. (2012) and Orisakwe et al. (2017) were lowered than the present values. In China, Lu et al. (2015) reported that wastewater-irrigated sites showed higher concentration of zinc as compared to area that employs groundwater source for irrigation which was in accordance with present findings. Normally the Zn concentration in the agricultural soil must be placed between 10 and 100mg/kg (Mertens and Smolder 2013). Many factors such as the category of soil (saline, sandy, calcareous in nature), distribution of phosphorous and nitrogen, and the content of organic matter are associated with insufficient amount of zinc in present soil (Saleem et al. 2020c;Sadeghzadeh 2013).

Forage Analysis
According to variance of analysis, non-significant difference (p˃0.05) for the zinc metal was noticed in the site, forage and site*forage treatments (Table 1). In collected forage samples, Zn concentration was present in the ranged of 26.42-42.67 mg/kg (Table 2). Wastewater-irrigated Z. mays presented the maximum zinc concentration, but ground water-irrigated M. sativa presents minimum concentration of this metal.
The present outcomes of zinc were lower than 60 mg/kg value given by WHO/FAO (2007) and recommended that deficient amount of Zn metal was present within the plants. Ogundiran et al. (2012) and Udiba et al. (2013) evaluated the higher zinc content than present forages. They documented that either plants absorb this metal from the polluted soil directly or the deposition take place in those organs that are exposed to the polluted air (Khan et al. 2019d). The recorded values were closer to the results of Raja et al. (2015) that utilize wastewater to irrigate crops of Faisalabad. In turn, Orisakwe et al. (2017) had reported the lower concentration of Zn (3.205-6.910mg kg −1 ) in Zamfara state of Nigeria. Normally plants ranged the Zn content between 30 and 100mg/kg on the basis of dry matter, but the toxicity occurs when it surpassed the limit of 300mg/kg (Noulas et al. 2018). Maximum concentration of Zn was examined in the forage Z. mays because its roots were scattered in the top soil which effectively absorb the content of zinc, chromium, nickel, and lead from the contaminated soil and accrete them in various parts (Khan et al. 2019c;Lu et al. 2015). Moreover, Karyotis et al. (2011) acknowledged that the annual plants accrete 4 times higher amount of Zn, in contrast to perennial grazing land.

Animal analysis
Analysis of variance showed that site (p< 0.001) and source (p<0.05) were significantly varied with the presence of Zn, but the opposite trend (p˃0.05) was observed within animal, s i t e * a n i m a l , s i t e * s o u r c e , a n i m a l * s o u r c e , a n d site*animal*source (Table 1). Zn concentration varied in the animal samples as 0.982-2.85mg/kg (Table 3). The blood samples were varied in concentration of Zn as 1.37-2.53 mg/l. The minimum concentration was present in the sheep blood that grazed on site Jh-I, while buffalo blood of Aps-III  exhibited maximum concentration of Zn metal. The samples of hair were ranged for Zn concentration between 1.25 and 2.29mg/kg. The maximal concentration of hair was examined in the sheep at Aps-III, while minimal concentration was found in the buffalo of Jh-I. The feces samples were differed from 0.982 to 2.85 mg/kg in the Zn concentration. The lowest concentration was found in the cow feces at site Jh-I, and the buffalo feces of Aps-III showed the highest concentration of this metal. NRC (2007) described that the normal blood level of Zn lies within the range of 0.8-1.20mg/l which was lesser than observed range. In Nigeria, Milam et al. (2017) stated the mean value of Zn in blood samples of sheep as 1.115 mg/l which was found to be lowered, but a high level of zinc (20.58 mg/kg) was noticed in Egypt by Diab and Donia (2018). Olmedo-Juárez et al. 2012) study revealed the poorer level of Zn metal with reference to this study. Orisakwe et al. (2017) studied the mean level of Zn in the blood cattle as 2.0400mg/l that was within this range. The depleted level of zinc in blood demonstrates that its poorer absorption takes place. The absorption of Zn was hindered in the presence of secondary metabolites (oxalic acid, tannins) because they act as chelating agents. Exposure of cows to the higher Pb concentration can also demote the absorption of Zn (Zhang et al. 2010). Hashem et al. (2017) presented the range of zinc as 85.7-141.1 mg/kg in the animal hairs collected from the cows, buffalo, goat, and sheep. Szczegielniak et al. (2012) documented the high Zn content fluctuated from 124 to 215 mg/kg of animal hairs assembled from different centers. The results of these two studies were placed beyond the analyzed range. The existing values were lower than the obtained results of various authors (Stoklasova et al. 2020;Pieper et al. 2017). As compared to the observed values, a prominent difference of elevated Zn concentration was studied by Ogundiran et al. (2012) in the cow feces of control (56.5 mg/kg) and contaminated sites (83.6 mg/kg). The enhanced level of Zn also showed correspondence with the findings of Svane and Karring (2019), whereas the Omonona et al. (2019) studied the 0.04-0.17 mg/kg value in the dry season that was identified as a lowered one as compared to examined range of this study. Animal body receives the Zn metal mainly from soil-plant system. However its concentration varies with the age factor (Szczegielniak et al. 2014). Normally Zn is added to the feed of animals to enhance its nutritional value (Lu et al. 2015), but too much profusion of its concentration can contaminate the dairy products derived from these animals (Omonona et al. 2019;Zhang et al. 2012). That is why animal samples surpassed the standard permissible limits in present study.

Bio-concentration factor
The observed BCF values for Zn were varied from 0.840 to 2.01 (Table 4). In the present study, the C. decidua was exposed to the maximum concentration of zinc at Jh-I, while the minimum value was present in the P. glaucum of Aps-III. These recorded values were lower than the detected amount (0.10-0.84) of Balabanova et al. (2015). On the other hand, Mahmoud and Ghoneim (2016) depicted the higher BCF values of this metal as 1.32-2.82 in the Zefta drain of Egypt. Alrawiq et al. (2014) stated that the observed BCF ˃1 showed that the forages accumulated the absorbed Zn content in their tissues. Metal uptake capacity of plant directly depends upon the metal interaction with soil and plant root (Saleem et al. 2020d).

Pollution load index
The PLI results for Zn metal ranged from 0.427-0.805 in the soil samples (Table 4). The minimum value was practiced in the C. decidua of ground watered soil, while the maximum value was represented in the P. glaucum cultivated at wastewater site.
Overall, the values of pollution load index were observed to be less than 1 which indicated that all the sites are unpolluted (Khan et al. 2019e). Singh et al. (2010) reported the reference content of Zn in soil as 44.19 which were seemed to be higher than the present range. Similarly, Bao et al. (2014) also studied the higher PLI values (1.03-1.14) in the soil that is irrigated with the sewage water for 40 years. The values reported by Ezemokwe et al. (2017) and Khan et al. (2020b) were 0.05 and 0.0649 mg/kg, which are observed to be lowered than the observed concentration. Observed PLI range was less than 1 in all sites, which represent that these sites are unpolluted in present research (Khan et al. 2019e).

Enrichment factor
The zinc outcomes for enrichment factor were present between 0.373 and 0.894 mg/kg (Table 4). The Jh-I site exhibited the maximum enrichment in the C. decidua fodder, while the minimum EF was found in the P. glaucum of Aps-III. Alghobar and Suresha (2015) declared the values of EF in the WW (0.67) and TWW (0.80) that were lower than the present range. As compared to present results, a higher level of Zn in Giza Governorate (1.18-1.71) and Sudan (37.9&12.1) was described by Sherif et al. (2015) and Taha et al. (2013), respectively. According to Barbieri (2016) standards, the values (EF<2) showed that insufficient enrichment of zinc was observed at study sites.

Daily intake of metal and health risk index
The Zn daily intake varied from 0.039 to 0.082 mg/kg/day. Minimum intake was studied in the sheep that are feed on the M. sativa of Jh-I site, while the buffaloes of Aps-III perceive the maximum concentration by grazing on the Z. mays. The HRI value for Zn was amounting from 0.130 to 0.275 mg/kg/ day. Maximum value of HRI was analyzed in the buffaloes browse on the wastewater-irrigated forages, while the minimum level was assessed in the sheep of Jh-I (Table 5).
Present values of daily intake were lowered than the range (0.041-0.115mg/kg/day) found by Ahmad et al. (2020). Khan et al. (2020b) observed that the DIM range (0.00164-0.0813mg/kg/day) is similar to present results. The obtained Zn concentration was greater in response to DIM values (0.039-0.769mg/kg/day) suggested by Nadeem et al. (2020) at different wastewater-irrigated sites. When the present HRI values for Zn were related with the work of these researchers, it can be concluded that the higher zinc range was reported by Ahmad et al. (2020) (0.19-0.72 mg/kg/day) and Nadeem et al. (2020) (0.13-2.67 mg/kg/day) in the contaminated sites of Sargodha region. Khan et al. (2020b) reported the lowered HRI range (0.0054-0.965mg/kg/day) in sewage-irrigated sites. Although, there was no health risk associated with animals by browsing on these contaminated sites (Khan et al. 2019a) because HRI values in this research study was observed to be less than unity.

Conclusion
It is concluded from this research work that the maximum concentration of Zn in forages and soil was examined mainly at wastewater-irrigated site but lesser than WHO/FAO limits. Animal samples absorb the zinc concentration beyond their standards. Thus, zinc supplements in the animal feed should be avoided. Results from bio-concentration index determined that a large amount zinc metal was transferred from the soil to forage tissues due to wastewater irrigation. Therefore, proper manageable use of wastewater can be consumed by local farmers for irrigation purpose. Although wastewater is a power supply of organic matter that enhances soil fertility but the presence of heavy metals and soluble salts can contaminate the food chain. That's why recommended that wastewater effluent must be treated before their appliance on farming lands as heavy metals are present in it and these metal contaminated forages affect the grazing animals and ultimately the human health. Government should organize campaigns to educate the farmers regarding heavy metal toxicity in soil, plant and human food chain.
Author contribution ZIK and KA supervised the study. FC, FGM, and JM were responsible for writing the manuscript. AA, MN, and SM1 were responsible for conducting the experiments and the data analysis. ISM, MUFA, SM2, and MN were responsible for analyzing and interpreting the data. All authors read and approved the final manuscript.
Funding This work was supported by the National Natural Science Foundation of China (Nos. 51974313 and 41907405) and the Natural Science Foundation of Jiangsu Province (BK20180641).
Data Availability All data generated or analyzed during this study are included in this published article.

Declarations
Ethical approval The authors declare that the manuscript has not been published previously.
Consent to participate All authors voluntarily participate in this research study.
Consent to publish All authors consent to the publication of the manuscript.

Conflict of interest
The authors declare no competing interests.