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Heavy metals and antibiotics are growing environmental contaminants leading to public health concern.Poultry manure is enriched with antibiotics and heavy metals that are commonly used as organic fertilizer for agricultural crops. Antibiotics (Suphonamide, lincomycine, erythromycin, oxytetracycline and tylosine) and heavy metals (Pb, Cr, Cd, As Ni, Co and Cu) are used as growth promoters and therapeutic agents in poultry feed. Some of these antibiotics and all heavy metals are not biodegradable in the body tissues of chicken, get deposited in meat as parent compounds and ultimately excreted via poultry droppings into the environment.In the first study an attempt have been done to present a better understanding of emerging contaminants in environment and their associated health effects. In this part we investigated the use of antibiotics in poultry sector and their release into the environment which develop antibiotics resistance bacteria (ARB) along with antibiotics resistance genes (ARGs). The development of ARGs and ARB might lead to huge physical and economic losses, as these bacteria cannot be treated with commonly used antibiotics, proving fatal for thousands of people every year in the world. These antibiotics after entering into food chains seriously affect human immune system, growth and metabolism of the body. Therefore, remediating the ARB, ARGs and antibiotics from the agriculture soil by environmental friendly technique is important to protect the future human health risks. Manure composting and biochar application might be the possible ways to reduce the risk and spread of ARGs in environment. In the second study, heavy metals were quantified in poultry manure, soil and food plants of agriculture fields from eleven districts of Khyber Pakhtunkhwa province. The concentrations of selected metals (Cd, Cr, Ni, Co, Cu, Ba, Pb) in most of the soils (ranged 0.036-0.3±0.086, 1.05-54.1±17.3, 1.8-35.2±9.1, 0.47-16.9±4.1, 0.056-32.5±9.8, 48.5-200.8±45, 0.26-15.7±4.57 mg kg-1, respectively) and manure (ranged 0.1-0.3±0.06, 6.75-52.4±11.9, 9.18 23.6±4.5, 2.86-16.7±4.4, 15.0-82.6±19.7, 24.7-86.9±20.9, 2.67-11.1±2.4 mg kg-1, respectively) samples were observed below the maximum allowable viii limits set by United States, Environmental Protection Agency (USEPA). The concentrations of Cu (0.81 to 247 mg kg-1) and Cr (0.18 to 9.81 mg kg-1) in most of the food plants were significantly (Cu-P<0.05) (Cr-P<0.01) higher and crossed their respective permissible limits (Cu-20 mg kg-1 and Cr-0.5 mg kg-1) set by SEPA, China. The mean concentrations of Pb (ranged 0.41-11.4 mg kg1) in all the food plants were significantly (P<0.01) higher and crossed the limit (0.3 mg kg-1) of FAO and WHO (2001). The values of Cd in radish (0.45 mg kg-1), clover (0.31 mg kg-1), lettuce (0.34 mg kg-1) and argula (0.28 mg kg1) were also above the allowable limits (0.2 mg kg-1) of SEPA. Metals like Pb, Cr, Cd, Cr and Cu were shown higher (>1) bioaccumulation values in the selected plants like radish, garlic, barley and wheat.Among selected heavy metals, Co, Ni and Ba were shown lower BCF values and assumed to have no serious health risk.The health risk index (HRI) values were found >1 in barley (for Cu and Cd), wheat and radish (for Cu, Cd and Pb) and pea and onion (for Cd), which were considered, that may cause potential health risks to the local community. Therefore, heavy metal concentrations in poultry manure must be reduced before its application into cultivated fields through safe and environmentally friendly technology such as biochar and composting. The third study determined the health risk of total As (TAs) and its species in selected food plants grown on poultry manure-based agriculture soils of Khyber Pakhtunkhwa, Pakistan. The concentrations of total arsenic (TAs), arsenobetaine (AsB), monomethyarsenate (MMA), dimethylarsenate (DMA), arsenite (As3+) and arsenate (As5+) were investigated in soil, poultry manure, food plants and poultry feed. The TAs concentration in soil of the study area ranged from 4.56 to 9.98 mg kg−1, showing the high variability in the selected sites. Inorganic arsenic (iAs) was found highest in Charsadda (9.9 mg kg−1) and lowest in district Swat (4.02 mg kg−1) The mean TAs concentrations in the poultry manure and feed ranged from 0.64 to 1.60 mg kg−1 and 0.06 mg kg−1 to 0.13 mg kg−1, respectively. The mean TAs concentrations in the edible parts of the food plants was in range from0.096 to 1.25 mg kg−1, which were found above the safe permissible limits (0.1 mg kg−1) of FAO/WHO (2011) in 83% offood plants, except in pea (0.096 mg kg−1) and mint (0.093 mg kg−1). The ix maximum concentration was observed for wheat (1.25 mg kg−1), cucumber (0.67 mg kg−1) and mallow (0.63 mg kg−1). Furthermore, human health risk was assessed through health risk index (HRI), hazard quotient (HQ), dialy intake rate (ADI) and life time cancer (LTCR). The ADI of As in the different food plants was ranged from 1.42×10−6 mg kg−1 d−1 to 6.62 ×10−5 mg kg−1 d−1 in the adults and 4.07×10−6 mg kg−1 d−1 to 1.89 ×10−4 mg kg−1 d−1 in the children with highest value for Malva neglecta (leafy plants) and lowest for Allium sativum. The total ADI for children (6.33×10-4 mg kg−1day−1) was higher than the USEPA oral reference dose (3 × 10-4 mg kg−1day−1) posing non cancer risk. The HQ values for TAs were less than 1 for the ingestion of the tested food plants, revealing less non-carcinogenic risk in the area. The results revealed that HI value for children (2.1×10+0) were three times higher than the value for adults (7.87×10−1), posing non cancer health risks (HI>1) in the children. The long term potential cancer risk (LTCR) was found slightly higher for children (1.53 × 10-4) than the permissible limits of USEPA, while minimal cancer risk was observed for adults via consumption of selected food plants.This research highlights a need to reduce the level of As in the poultry feed and further suggests to treat poultry manure before their application to the crop field. A fourth study was carried out on the development of ARG and ARB due to manure application in agriculture field. Three different samples of poultry manure (M), soil amended manure (S+M) and soil without manure (S) were collected for assessment of ARGs in the agriculture field area of Khyber Pakhtunkhwa, Pakistan. Total number of 285 ARGs was observed by means of HT-qPCR (High Throghput quantitative PCR), which targeted most common classes of resistance genes. Poultry manure addition significantly (P<0.05) raised the abundance copies of ARGs in manure based soil and have changed composition of bacterial community. The detected resistance genes in manure amended soil gave resistance to antibiotics such as multidrug, aminoglycoside, MLSB, beta-lactmase, tetracycline, Vancomycine, tansposase and sulphoniamide. Total 130 unique ARGs were developed, 76 ARGs were shared by all three groups, 47 x ARGs were transferred from manure to soil and 49 ARGs were shared by manure and manure-based soil. Gene sequencing of 16S rRNA was carried out for the samples, which concluded that Proteobacteria, Bacteroidetes Actinobacteria, Fermicutes, Acidobacteria and Chloroflexi were the common phyla of microbial community, having about 87% of total microbes of 16S rRNA gene sequences. Manure has increased Bacteroidetes, Actinobacteria and Fermicutes in the soil. Hence manure has elevated the ARGs and antibiotic resistance bacteria (ARB) by horizontal gene transfer from manure based soil to indigenous soil microbiota. This study will help us to know the dissemination and fate of ARGs in this ecosystem to efficiently use poultry manure in crop land and highlight antibiotics and ARGs risks managements in the agriculture area of Pakistan.
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