| China,as the world’s foremost producer of livestock and poultry,employs heavy metal trace elements extensively as additives to prevent diseases and enhance animal growth.Annually,China generates approximately 3.8 billion tons of livestock and poultry manure,contributing significantly to the introduction of substantial amounts of heavy metal elements into the soil environment when left untreated.Moreover,the presence of heavy metals in the gastrointestinal tracts of livestock and poultry exerts selective pressure on microbial communities,resulting in the emergence of Metal resistance genes(MRGs).Despite these concerns,research on the accumulation of heavy metals in soil,associated pollution risks,and their impact on soil sustainability due to continuous livestock and poultry manure application is limited.Additionally,the effects of agricultural activities on soil microbial metal resistance and the unclear understanding of the distribution and transmission mechanisms of MRGs in soil further compound these challenges.In this study,a combination of outdoor field surveys and indoor analyses was employed as the research approach.The research was conducted using Chongqing City in the southwestern region of China as the study area.The study primarily focused on analyzing the spatial distribution characteristics of heavy metals in livestock and poultry manure from large-scale livestock and poultry farms within the research region,as well as in the soil of vegetable fields surrounding these farms.The analysis also included an assessment of ecological pollution risks.Additionally,long-term monitoring data from designated research sites were used in conjunction with various methods such as 16 S rRNA sequencing,metagenomics,and quantitative real-time polymerase chain reaction(q PCR).These methods were applied to investigate changes in the accumulation of heavy metals in vegetable field soils over a continuous six-year period of applying livestock and poultry manure.The study also examined the characteristics of vertical migration,alterations in microbial community structures,and the presence,transmission,and environmental impact factors of mobile resistance genes(MRGs)within the soil.The main findings of this study are as follows:1.This study selected 17 large-scale pig farming operations and 15 large-scale chicken farming operations covering a wide range of districts and counties in the study area for field sampling and indoor determination of heavy metal concentrations,as well as pollution risk assessment.The results show that the average contents of copper(Cu),zinc(Zn),and mercury(Hg)in pig manure from livestock and poultry breeding in the study area are higher than those in chicken manure,being 1.77 times higher for Cu,1.31 times higher for Zn,and 1.12 times higher for Hg,respectively.Meanwhile,the average contents of nickel(Ni)and lead(Pb)in chicken manure are higher,being 1.25 times higher for Ni and 1.10 times higher for Pb than in pig manure.The coefficients of variation for heavy metals in pig manure and chicken manure are 41.60% to 129.02% and27.06% to 70.64%,respectively.There are significant differences in the heavy metal content in pig manure.In addition to mercury(Hg),the heavy metal content in livestock and poultry manure exceeds the standard to varying degrees.Among them,pig manure and chicken manure have the highest exceedance rate for Zn,both exceeding 80%,followed by cadmium(Cd)with an exceedance rate of more than 50%.The heavy metal contents in pig manure and chicken manure exhibit certain regional trends.Specifically,the Cu,Zn,Ni,and Hg contents in pig manure are highest in western Chongqing,Cd is highest in southeast Chongqing,and Pb is highest in northeast Chongqing.In chicken manure,the Cu,Cd,and Hg contents are highest in western Chongqing,while Ni and Pb are highest in southeast Chongqing,and the Zn content is highest in northeast Chongqing.2.A total of 233 samples from surface soil in vegetable fields(<100 m)around largescale farms with a history of manure application and typical vegetable bases were collected in the study area.Compared with the vegetable field soil near the farms,the average values of Cu,Zn,Cd,and Hg in the pollution-free vegetable bases were lower,but the average values of Ni and Pb were higher.Among them,the coefficient of variation in heavy metal content in the vegetable field soil around the farms was relatively high,and the average content of Cu,Zn,Cd,Pb,and Hg were 1.47 times,1.32 times,6.25 times,1.07 times,and 1.16 times higher than the background values of Chongqing soil,respectively.Referring to the "Soil Environmental Quality Agricultural Land Soil Pollution Risk Management and Control Standards(Trial)"(GB 15618-2018),the point exceeding standard rate of Cd is as high as 93.33%,followed by Zn(8.7%)and Cu(3.81%).The average contents of Ni,Pb,and Cd in the soil of the pollution-free vegetable bases exceeded the Chongqing soil background values by 1.03 times,1.14 times,and 5.75 times.Among them,the Cd point exceedance rate was 92.19%,and the contents of the remaining five heavy metals did not exceed the soil pollution risk screening values for agricultural land.The results of single-factor and Nemerow comprehensive pollution indices showed that Cd pollution existed in the soil of vegetable fields around the farms and pollution-free vegetable bases.The combined heavy metal pollution in the vegetable fields around the farms is even greater.In accordance with China’s food safety standards(GB 2762-2022)and the international food safety standards(CXS 193-1995)formulated by FAO,The content of Pb in the edible parts of leafy vegetables,solanaceous fruits,beans,melons,and root vegetables in the study area exceeded the standards.Among different types of vegetables,leafy vegetables pose the highest risk.The non-carcinogenic risk index(HI)and total carcinogenic risk index(TCR)of vegetables consumed by local children are higher than those of adults.Overall,the study area exhibits carcinogenic health risks within the range considered acceptable for human exposure.Cadmium(Cd)and lead(Pb)are the primary heavy metals of concern for the local residents in this region.3.Long-term monitoring data from designated test sites reveal that continuous application of livestock and poultry manure has led to an accumulation of copper(Cu),zinc(Zn),and cadmium(Cd)at monitoring points in WZ,HY,and BS,with the most pronounced growth observed in the high-dosage manure treatment.In the BS trial monitoring site,pig manure treatment resulted in higher increases in surface soil Cu,Zn,and Hg content compared to chicken manure,while soil Cd content was higher in the chicken manure treatment.In the 0-60 cm soil layers,significant increases in Cu,Zn,Pb,Cd,chromium(Cr),and Hg content were observed in the high-dosage manure treatment compared to the control.Each heavy metal element exhibited varying degrees of vertical migration within the soil layers.Cu and Zn primarily accumulated in the 0-20 cm soil layer,with higher manure application leading to downward migration of Zn from chicken manure to the sub-surface layer(20-40 cm).Hg,Cd,Pb,nickel(Ni),and Cr displayed a trend of downward vertical migration,with Hg,Cd,and Pb primarily accumulating in the 20-40 cm layer,while Ni and Cr mainly accumulated in the 40-60 cm layer.Chicken manure demonstrated a stronger downward migration capacity for introducing heavy metals compared to pig manure.According to the "Management Control Standard for Soil Environmental Quality on Agricultural Land"(GB 15618-2018),Cu and Zn content in the 0-20 cm soil layer of the high-dosage pig manure treatment,as well as Cd content in all treatments within the 0-60 cm soil layer,exceeded the risk screening values,indicating a potential risk of heavy metal pollution.Assessment results based on the single-factor pollution index and the geo-accumulation index consistently identify Cd as the primary pollutant among the vegetable field soils at the designated monitoring points in WZ,HY,and BS.HY exhibited severe Cd pollution,followed by BS and WZ monitoring points,with the possibility of Cd pollution increasing over time with extended manure application.The comprehensive pollution index,as indicated by the Nemerow Comprehensive Pollution Index,revealed that all soil sites in the HY and BS monitoring points exhibited severe pollution,while the WZ site predominantly had light pollution(accounting for 77.78% of the locations).Soil heavy metal ecological risk assessments showed that soil at the HY monitoring site is subject to high comprehensive ecological risks,with 75% of the soil at the BS monitoring site falling into the category of moderate ecological risk.Meanwhile,77.78% of the soil at the WZ monitoring site experienced relatively lower ecological risks.In summary,the application of livestock and poultry manure has overall increased the soil heavy metal pollution risk and potential ecological risks.Furthermore,with an increase in the duration of manure application,pollution and risk indices continued to rise,with Cd contributing the most to soil pollution in the surveyed region.4.The application of livestock and poultry manure significantly altered the microbial community structure in vegetable field soils at the designated monitoring points in WZ,HY,and BS.The diversity and abundance of soil microbes in these monitoring points responded differently to varying doses of livestock and poultry manure.Specifically,in WZ’s vegetable field soil,microbial diversity and abundance exhibited an increasing trend with higher chicken manure application.In HY’s vegetable field soil,microbial diversity and abundance were highest with low-dose pig manure application and gradually decreased with increasing pig manure application.In BS’s soil,bacterial diversity and abundance increased in response to pig manure treatment but displayed an initial increase followed by a decrease in response to chicken manure treatment.At all three monitoring points,the dominant bacterial phyla were Proteobacteria(17.86%-45.19%)and Actinobacteria(15.11%-41.60%),while the dominant fungal phylum was Ascomycota.Soil microbes exhibited preferences for different types of livestock and poultry manure.Chicken manure treatment notably increased the relative abundance of Actinobacteria in BS’s soil,while pig manure treatment more significantly increased the relative abundance of Firmicutes.The microbial community structure in vegetable field soils was influenced by heavy metals and pH,with Cu,Zn,and pH being the environmental factors exerting a greater impact.5.Continuous application of livestock and poultry manure,especially pig manure,has led to an increase in the relative abundance of soil Metal Resistance Genes(MRGs)and Mobile Genetic Elements(MGEs).Specifically,the relative abundance of Cu-MRGs(copA and pcoC)increased by 1.13-13.37 times and 1.41-2.56 times,respectively,compared to the control(CK).Zn-MRGs(zraR)increased by 1.15-6.67 times.Hg-MRGs(merA)increased by 1.02-17.60 times.Ni-MRGs(nikE)increased by 2.19-5.90 times.Notably,zraR,nikE,and zntA exhibited a stronger preference for pig manure.In the tested soils,a total of 279 subtypes of metal resistance genes were detected,including Class Ⅰ multidrug resistance genes(39.0%)and Class Ⅱ single metal resistance genes(71.0%).Cu resistance genes were the predominant single metal resistance genes in the soil samples.Network analysis(Pearson correlation,r>0.9,p<0.01)revealed that plasmids such as pSCATT,pRHL1,pSCL4,integrons intl and intl1,and insertion plasmids IS630 and IS110 were major mediators of horizontal gene transfer and dissemination of MRGs.The main host bacteria for MRGs transitioned from Sphingomonas in the soil prior to livestock and poultry manure application to norank＿f＿＿Bacillaceae and Conexibacter after application.Cu,Zn,and pH significantly influenced bacterial communities and typical MRGs(cop A,pco C,mer A,zraR,nikE,and znt A).Bacterial communities,MGEs,heavy metals,and MRGs were all significantly correlated(p<0.05),with bacterial communities being the primary factor affecting MRGs’ variations.While fertilization treatments did not have a direct and significant impact on soil MRG abundance,they influenced the presence and dissemination of MRGs by altering bacterial community abundance,MGEs,and heavy metal content. |
