Increased occurrence of heavy metals, antibiotics and resistance genes in surface soil after long-term application of manure

The purpose of this study was to investigate the impact of long-term application of pig manure on the accumulation of heavy metals, antibiotics and ARGs in surface soil sampled from the Jiaxing long-term field experimental site with three manure treatments, N-PM (0 kg/ha/y, dw), L-PM (7720 kg/ha/y, dw), and H-PM (11,580 kg/ha/y, dw), in 2013 and 2014. The results showed that most serious metal pollution of Zn and Cu was recorded in all manured samples in both years, and their contents exceeded the soil quality standards. Among the three tetracyclines, chlortetracycline was the predominant antibiotic detected with a range of 3.04-98.03 μg·kg-1 in 2013 and 28.67-344.74 μg·kg-1 in 2014 after long-term pig manure application. Q-PCR results showed that the average accumulation of ribosomal protection protein genes (tetM, tetO, tetQ and tetW) was lower than most of the efflux pump genes (tetA and tetG). The abundance of tet and sul genes of those sites with manure application was significantly higher than that of sites without manure application in both years. Metagenomics analysis of ARGs revealed that the abundance of multidrug resistance genes was the most abundant subtype, followed by fluoroquinolone, bacitracin, sulfonamide and tetracycline. There was a positive correlation between the levels of ARGs; soil organic matter, antibiotics, Cu, As, and Zn levels in both years. These results may shed light on the mechanism underlining the effects of long-term manure application on the occurrence and dissemination of ARGs in surface soil.