Variation in sulfamethoxazole and its metabolites in pig manure Variation in sulfamethoxazole and its metabolites during pig manure fermentation

Pork production is often required for a large number of pigs in stock, particularly in a wide range of breeding areas. The annual total amount of livestock and poultry manure has reached approximately 3.05 billion tons in China. A large amount of pig manure accounts for a relatively high proportion of the total manure. Therefore, the disposal of pig manure can also dominate in the pollution control and prevention of the breeding industry. Among them, antibiotics are widely used in the livestock and poultry breeding industry for therapeutic and disease prevention. Furthermore, the veterinary antibiotics administered to animals in the breeding industry cannot be fully metabolized by the animals. Approximately 20% to 97% of these antibiotics are excreted in their active forms via manure, leading to antibiotic residues in the environment and also posing serious threats to ecosystems and human health. Such as, sulfamethoxazole (SMX) has been widely used as a veterinary antibiotic in the livestock and poultry breeding industry. It can exist in the environment long-term, after it is discharged via livestock manure, due to its stable chemical properties. Furthermore, some degradation metabolites of the SMX in the environment also exhibit significantly higher toxicity than the SMX itself. Among them, the toxicity of 4-hydroxyl-N-(5-methyl-1,2-oxazole-3-yl) benzene-1-sulfonamide (4-OH-SMX) and 4-nitro-sulfamethoxazole (4-NO₂-SMX) is 5.7 times and 21.4 times that of SMX, respectively. Previous researches have been focused primarily on the residual levels of the SMX in the environment, with less attention given to its higher toxic metabolites. Therefore, anaerobic composting can be expected for the harmless treatment of the pig manure. This research aims to investigate the degradation dynamics of the SMX and its seven important metabolites during the facultative anaerobic composting of the pig manure. A systematic evaluation was carried out to explore the influence of the varying ambient temperatures (35 , 25 , and 15 ℃) and initial moisture contents of the raw manure substrate (70%, 75%, and 80%) over a defined experimental period. A 90-day facultative anaerobic composting was conducted under all tested conditions. The results demonstrated that the substantial degradation efficiency of the SMX ranged from 64.82% to 78.04%. Notably, the most effective SMX removal was achieved under the combination of 35 °C composting temperature and 80% moisture content. At the same time, the SMX degradation was synergistically enhanced by elevated temperatures coupled with the high moisture levels. Metabolic transformation pathways were determined to detect the six SMX metabolites during the facultative anaerobic composting. Among them, 3-amino-5-methylisoxazole emerged as the predominant transformation product, thereby reaching a peak concentration of 4.13 mg/kg. Its formation pathway was predicted by structural characterization. The enzymatic cleavage of the S-N bond was involved within the parent SMX molecule, which was likely mediated by specific bacterial metabolic activities inherent to the composting microbiome. Critically, the 2 highly toxic metabolites (4-OH-SMX and 4-NO₂-SMX) were also identified after composting. The maximum concentrations of the 4-OH-SMX and 4-NO₂-SMX were detected as 0.12 and 0.03 mg/kg, respectively. The 4-NO₂-SMX also exhibited a temperature dependency, where the highest formation concentration occurred specifically at 25 °C. Comparatively, the 4-OH-SMX showed lower sensitivity to the fluctuations in either composting temperature or the initial moisture content of the manure. The key biochemical pathways to the 4-OH-SMX formation were remarkably constant under standard composting conditions. They cannot be effectively suppressed or avoided solely after temperature or moisture adjustments in the tested ranges. Consequently, the unavoidable presence of the 4-OH-SMX was often required to implement the additional and targeted post-composting treatment. The residual toxicity was effectively avoided to significantly enhance the overall environmental safety and suitability of the final compost product for subsequent agricultural utilization. Therefore, the compost piles were turned or aerated during facultative anaerobic composting. The temperature above 30 °C can also be used to reduce the production of the highly toxic transformation products. In conclusion, the empirical findings and mechanistic insights can offer the actionable technical support to optimize the treatment protocols of the pig manure. The finding can provide a scientific basis for the decision-making on the safe utilization of the pig manure. The degradation efficiency of the residual SMX antibiotics was maximized during composting, in order to simultaneously minimize the accumulation and potential environmental risks posed by its highly toxic transformation products.