Assessment of sulfamethoxazole and oxytetracycline uptake and transformation in Eisenia fetida earthworms.

The scientific community is becoming increasingly concerned about the recent detection of transformation products (TPs) of antimicrobials (AMs) and their presence in the food chain. There are growing concerns about the potential consequences on food safety and the proliferation of antimicrobial resistance. In this work, the transformation process of sulfamethoxazole (SMX) and oxytetracycline (OTC) in soil was thoroughly evaluated. For that purpose, soils were homogeneously contaminated at three concentration levels of SMX and OTC, independently, and samples were analysed after 7 and 14 days by Ultra High-Performance Liquid Chromatography coupled to a triple quadrupole mass spectrometer (UHPLC-MS/MS). The results have demonstrated a remarkable transformation, particularly noteworthy for SMX, as it exhibited an 89 % - 94 % decrease in concentration within the initial 7 days of the experiment. In addition, to assess whether terrestrial organisms would be able to accumulate the AMs, Eisenia fetida (E. fetida) earthworms were exposed to the above-mentioned concentration levels of AMs in soil. Both AMs were accumulated in the organisms after 14 days, but higher bioaccumulation factor values (BCF) were determined for SMX (0.52-17.84) compared to OTC (0.02-0.21) at all tested concentrations. The analyses were extended to search for TPs in earthworms and soils using a suspect screening approach. Concretely, by means of UHPLC-high resolution mass spectrometry (UHPLC-HRMS) three TPs were identified at 2a and 2b of confidence level. To the best of our knowledge, one SMX-TP and one OTC-TP were identified in earthworms and soil, respectively, for the first time in the present work. Earthworms did not experience weight loss or mortality in the presence of these AMs at levels found in the environment, but there was a decrease in riboflavin levels, which is linked to changes in the immune system. This study represents a significant advancement in understanding the impact of AMs in soil and their subsequent entry into the food chain. It also provides valuable insights into the potential effects of AMs and their TPs on organisms.

Effects of sulfamethazine and tetracycline at molecular, cellular and tissue levels in Eisenia fetida earthworms.

Soil contamination by antibiotics is a global issue of great concern that contributes to the rise of bacterial antibiotic resistance and can have toxic effects on non-target organisms. This study evaluated the variations of molecular, cellular, and histological parameters in Eisenia fetida earthworms exposed to sulfamethazine (SMZ) and tetracycline (TC), two antibiotics commonly found in agricultural soils. The earthworms were exposed for 14 days to a series of concentrations (0, 10, 100, and 1000 mg/kg) of both antibiotics. SMZ and TC did not affect the survival of E. fetida, however, other effects at different levels of biological complexity were detected. The two highest concentrations of SMZ reduced the viability of coelomocytes. At the highest TC concentration, there was a noticeable decline in cell viability, acetylcholinesterase activity (neurotoxicity), and the relative presence of mucopolysaccharides in the epidermis (mucous production). Glutathione S-transferase activity decreased in all TC treatments and at the highest SMZ concentration. However, levels of malondialdehyde and protein carbonyls did not change, suggesting an absence of oxidative stress. Tetracycline was neurotoxic to E. fetida and changed the integrity of the epidermis. Both antibiotics altered the intestinal microbiota of E. fetida, leading to a reduction in the relative abundance of bacteria from the phyla Proteobacteria and Bacteroidetes, while causing an increase in the phylum Actinobacteroidota. All observed changes indicate that both SMZ and TC can disrupt the earthworms' immune system and gut microbiome, while fostering the growth of bacteria that harbour antibiotic resistance genes. Finally, both antibiotics exerted additional metabolic and physiological effects that increased the vulnerability of E. fetida to pathogens.

Antimicrobials in Eisenia fetida earthworms: A comprehensive study from method development to the assessment of uptake and degradation.

In this work, an accurate analytical method was developed for the simultaneous analysis of twenty-seven antimicrobials (AMs) in earthworms using liquid chromatography coupled to a triple quadrupole mass spectrometry detector (UHPLC-MS/MS). Adequate apparent recoveries (80-120 %) and limits of quantification (LOQ) (1 µg·kg-1 - 10 µg·kg-1) were obtained, with the exception of norfloxacin (34 µg·kg-1). The method was applied to evaluate the accumulation of sulfamethazine (SMZ) and tetracycline (TC) in earthworms after performing OECD-207 toxicity test, in which Eisenia fetida (E. fetida) organisms were exposed to soils spiked with 10 mg·kg-1, 100 mg·kg-1 or 1000 mg·kg-1 of SMZ and TC, individually. The results confirmed the bioaccumulation of both AMs in the organisms, showing a greater tendency to accumulate SMZ since higher bioconcentration factor values were obtained for this compound at the exposure concentrations tested. In addition, the degradation of both AMs in both matrices, soils and earthworms was studied using liquid chromatography coupled to a q-Orbitrap high resolution mass spectrometry detector. Thirteen transformation products (TPs) were successfully identified, eight of them being identified for the first time in soil/earthworm (such as 4-Amino-3-chloro-n-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide or 4-(dimethylamino)-1,11,12a-trihydroxy-6,6-dimethyl-3,7,10,12-tetraoxo-3,4,4a,5,5a,6,7,10,12,12a-decahydrotetracene-2-carboxamide, among others) and their formation/degradation trend over time was also studied. Regarding the biological effects, only SMZ caused changes in earthworm growth, evidenced by weight loss in earthworms exposed to concentrations of 100 mg·kg-1 and 1000 mg·kg-1. Riboflavin decreased at all concentrations of SMZ, as well as at the highest concentration of TC. This indicates that these antibiotics can potentially alter the immune system of E. fetida. This research represents a significant advance in improving our knowledge about the contamination of soil by AM over time. It investigates the various ways in which earthworms are exposed to AMs, either by skin contact or ingestion. Furthermore, it explores how these substances accumulate in earthworms, the processes by which earthworms break them down or metabolise them, as well as the resulting TPs. Finally, it examines the potential effects of these substances on the environment.