Maize (Zea mays L.) Plants Alter the Fate and Accumulate Nonextractable Residues of Sulfamethoxazole in Farmland Soil.

The fate of sulfonamide antibiotics in farmlands is crucial for food and ecological safety, yet it remains unclear. We used [phenyl-U-14C]-labeled sulfamethoxazole (14C-SMX) to quantitatively investigate the fate of SMX in a soil-maize system for 60 days, based on a six-pool fate model. Formation of nonextractable residues (NERs) was the predominant fate for SMX in unplanted soil, accompanied by minor mineralization. Notably, maize plants significantly increased SMX dissipation (kinetic constant kd = 0.30 day-1 vs 0.17 day-1), while substantially reducing the NER formation (92% vs 58% of initially applied SMX) and accumulating SMX (40%, mostly bound to roots). Significant NERs (maximal 29-42%) were formed via physicochemical entrapment (determined using silylation), which could partially be released and taken up by maize plants. The NERs consisted of a considerable amount of SMX formed via entrapment (1-8%) and alkali-hydrolyzable covalent bonds (2-12%, possibly amide linkage). Six and 10 transformation products were quantified in soil extracts and NERs, respectively, including products of hydroxyl substitution, deamination, and N-acylation, among which N-lactylated SMX was found for the first time. Our findings reveal the composition and instability of SMX-derived NERs in the soil-plant system and underscore the need to study the long-term impacts of reversible NERs.

Analyzing microbial communities and their biodegradation of multiple pharmaceuticals in membrane bioreactors.

Pharmaceuticals are of concern to our planet and health as they can accumulate in the environment. The impact of these biologically active compounds on ecosystems is hard to predict, and information on their biodegradation is necessary to establish sound risk assessment. Microbial communities are promising candidates for the biodegradation of pharmaceuticals such as ibuprofen, but little is known yet about their degradation capacity of multiple micropollutants at higher concentrations (100 mg/L). In this work, microbial communities were cultivated in lab-scale membrane bioreactors (MBRs) exposed to increasing concentrations of a mixture of six micropollutants (ibuprofen, diclofenac, enalapril, caffeine, atenolol, paracetamol). Key players of biodegradation were identified using a combinatorial approach of 16S rRNA sequencing and analytics. Microbial community structure changed with increasing pharmaceutical intake (from 1 to 100 mg/L) and reached a steady-state during incubation for 7 weeks on 100 mg/L. HPLC analysis revealed a fluctuating but significant degradation (30-100%) of five pollutants (caffeine, paracetamol, ibuprofen, atenolol, enalapril) by an established and stable microbial community mainly composed of Achromobacter, Cupriavidus, Pseudomonas and Leucobacter. By using the microbial community from MBR1 as inoculum for further batch culture experiments on single micropollutants (400 mg/L substrate, respectively), different active microbial consortia were obtained for each single micropollutant. Microbial genera potentially responsible for degradation of the respective micropollutant were identified, i.e. Pseudomonas sp. and Sphingobacterium sp. for ibuprofen, caffeine and paracetamol, Sphingomonas sp. for atenolol and Klebsiella sp. for enalapril. Our study demonstrates the feasibility of cultivating stable microbial communities capable of degrading simultaneously a mixture of highly concentrated pharmaceuticals in lab-scale MBRs and the identification of microbial genera potentially responsible for the degradation of specific pollutants. KEY POINTS: • Multiple pharmaceuticals were removed by stable microbial communities. • Microbial key players of five main pharmaceuticals were identified.

The sulfonamide-resistance dihydropteroate synthase gene is crucial for efficient biodegradation of sulfamethoxazole by Paenarthrobacter species.

Sulfonamide antibiotics (SAs) are serious pollutants to ecosystems and environments. Previous studies showed that microbial degradation of SAs such as sulfamethoxazole (SMX) proceeds via a sad-encoded oxidative pathway, while the sulfonamide-resistant dihydropteroate synthase gene, sul, is responsible for SA resistance. However, the co-occurrence of sad and sul genes, as well as how the sul gene affects SMX degradation, was not explored. In this study, two SMX-degrading bacterial strains, SD-1 and SD-2, were cultivated from an SMX-degrading enrichment. Both strains were Paenarthrobacter species and were phylogenetically identical; however, they showed different SMX degradation activities. Specifically, strain SD-1 utilized SMX as the sole carbon and energy source for growth and was a highly efficient SMX degrader, while SD-2 did could not use SMX as a sole carbon or energy source and showed limited SMX degradation when an additional carbon source was supplied. Genome annotation, growth, enzymatic activity tests, and metabolite detection revealed that strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation and a pathway of protocatechuate degradation. A new sulfonamide-resistant dihydropteroate synthase gene, sul918, was identified in strain SD-1, but not in SD-2. Moreover, the lack of sul918 resulted in low SMX degradation activity in strain SD-2. Genome data mining revealed the co-occurrence of sad and sul genes in efficient SMX-degrading Paenarthrobacter strains. We propose that the co-occurrence of sulfonamide-resistant dihydropteroate synthase and sad genes is crucial for efficient SMX biodegradation. KEY POINTS: • Two sulfamethoxazole-degrading strains with distinct degrading activity, Paenarthrobacter sp. SD-1 and Paenarthrobacter sp. SD-2, were isolated and identified. • Strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation. • A new plasmid-borne SMX resistance gene (sul918) of strain SD-1 plays a crucial role in SMX degradation efficiency.

Long-Term Field Study on Fate, Transformation, and Vertical Transport of Tetrabromobisphenol A in Soil-Plant Systems.

Soil contamination with tetrabromobisphenol A (TBBPA) has been an environmental concern for many years, but in situ studies of the fate and potential risk of TBBPA are lacking. In this study, we investigated the dissipation, metabolism, strong alkali-hydrolytic (SAH-TBBPA), and vertical movement of TBBPA in the field with and without rice-wheat rotation and reed growth for 1225 days. After 342 days of incubation, 21.3% of the TBBPA remained in the surface soil accompanied by obvious leaching to deeper soil layers in the first 92 days. By day 1225, TBBPA was nearly absent from the surface soil layer. A very low amount of SAH-TBBPA (2.31-3.43 mg/kg) was detected during the first 342 days of incubation. In the surface soil, five metabolites were identified that represented four interconnected pathways: oxidative skeletal cleavage, O-methylation, type II ipso-substitution, and reductive debromination. Both rice-wheat rotation and monocultural reed growth accelerated TBBPA removal in the field by stimulating the anaerobic debromination and aerobic O-methylation, especially the oxidative skeletal cleavage of TBBPA in the rhizosphere soil. Though far from comprehensive, our study investigated the natural attenuation and metabolism of TBBPA in situ and the influence by crops to estimate the environmental risk of TBBPA in a field scale.

Arsenic Mobilization from Historically Contaminated Mining Soils in a Continuously Operated Bioreactor: Implications for Risk Assessment.

Concentrations of soil arsenic (As) in the vicinity of the former Zloty Stok gold mine (Lower Silesia, southwest Poland) exceed 1000 µg g(-1) in the area, posing an inherent threat to neighboring bodies of water. This study investigated continuous As mobilization under reducing conditions for more than 3 months. In particular, the capacity of autochthonic microflora that live on natural organic matter as the sole carbon/electron source for mobilizing As was assessed. A biphasic mobilization of As was observed. In the first two months, As mobilization was mainly conferred by Mn dissolution despite the prevalence of Fe (0.1 wt % vs 5.4 for Mn and Fe, respectively) as indicated by multiple regression analysis. Thereafter, the sudden increase in aqueous As[III] (up to 2400 µg L(-1)) was attributed to an almost quintupling of the autochthonic dissimilatory As-reducing community (quantitative polymerase chain reaction). The aqueous speciation influenced by microbial activity led to a reduction of solid phase As species (X-ray absorption fine structure spectroscopy) and a change in the elemental composition of As hotspots (micro X-ray fluorescence mapping). The depletion of most natural dissolved organic matter and the fact that an extensive mobilization of As[III] occurred after two months raises concerns about the long-term stability of historically As-contaminated sites.

Fate of Tetrabromobisphenol A (TBBPA) and Formation of Ester- and Ether-Linked Bound Residues in an Oxic Sandy Soil.

Bound-residue formation is a major dissipation process of most organic xenobiotics in soil. However, both the formation and nature of bound residues of tetrabromobisphenol A (TBBPA) in soil are unclear. Using a 14C-tracer, we studied the fate of TBBPA in an oxic soil during 143 days of incubation. TBBPA dissipated with a half-life of 14.7 days; at the end of incubation, 19.6% mineralized and 66.5% formed bound residues. Eight extractable metabolites were detected, including TBBPA methyl ethers, single-ring bromophenols, and their methyl ethers. Bound residues (mostly bound to humin) rapidly formed during the first 35 days. The amount of those humin-bound residues then quickly decreased, whereas total bound residues decreased slowly. By contrast, residues bound to humic acids and fulvic acids increased continuously until a plateau was reached. Ester- and ether-linked residues accounted for 9.6-27.0% of total bound residues during the incubation, with ester linkages being predominant. Residues bound via ester linkages consisted of TBBPA, TBBPA monomethyl ether, and an unknown polar compound. Our results indicated that bound-residue formation is the major pathway of TBBPA dissipation in oxic soil and provide first insights into the chemical structure of the reversibly ester-linked bound residues of TBBPA and its metabolites.

Enhanced transformation of tetrabromobisphenol a by nitrifiers in nitrifying activated sludge.

The fate of the most commonly used brominated flame retardant, tetrabromobisphenol A (TBBPA), in wastewater treatment plants is obscure. Using a (14)C-tracer, we studied TBBPA transformation in nitrifying activated sludge (NAS). During the 31-day incubation, TBBPA transformation (half-life 10.3 days) was accompanied by mineralization (17% of initial TBBPA). Twelve metabolites, including those with single benzene ring, O-methyl TBBPA ether, and nitro compounds, were identified. When allylthiourea was added to the sludge to completely inhibit nitrification, TBBPA transformation was significantly reduced (half-life 28.9 days), formation of the polar and single-ring metabolites stopped, but O-methylation was not significantly affected. Abiotic experiments confirmed the generation of mono- and dinitro-brominated forms of bisphenol A in NAS by the abiotic nitration of TBBPA by nitrite, a product of ammonia-oxidizing microorganisms (AOMs). Three biotic (type II ipso-substitution, oxidative skeletal cleavage, and O-methylation) and one abiotic (nitro-debromination) pathways were proposed for TBBPA transformation in NAS. Apart from O-methylation, AOMs were involved in three other pathways. Our results are the first to provide information about the complex metabolism of TBBPA in NAS, and they are consistent with a determining role for nitrifiers in TBBPA degradation by initiating its cleavage into single-ring metabolites that are substrates for the growth of heterotrophic bacteria.

Degradation and metabolism of tetrabromobisphenol A (TBBPA) in submerged soil and soil-plant systems.

Contamination by tetrabromobisphenol A (TBBPA), the most widely used brominated flame retardant, is a matter of environmental concern. Here, we investigated the fate and metabolites of (14)C-TBBPA in a submerged soil with an anoxic-oxic interface and planted or not with rice (Oryza sativa) and reed (Phragmites australis) seedlings. In unplanted soil, TBBPA dissipation (half-life 20.8 days) was accompanied by mineralization (11.5% of initial TBBPA) and the substantial formation (60.8%) of bound residues. Twelve metabolites (10 in unplanted soil and 7 in planted soil) were formed via four interconnected pathways: oxidative skeletal cleavage, O-methylation, type II ipso-substitution, and reductive debromination. The presence of the seedlings strongly reduced (14)C-TBBPA mineralization and bound-residue formation and stimulated debromination and O-methylation. Considerable radioactivity accumulated in rice (21.3%) and reed (33.1%) seedlings, mainly on or in the roots. While TBBPA dissipation was hardly affected by the rice seedlings, it was strongly enhanced by the reed seedlings, greatly reducing the half-life (11.4 days) and increasing monomethyl TBBPA formation (11.3%). The impact of the interconnected aerobic and anaerobic transformation of TBBPA and wetland plants on the profile and dynamics of the metabolites should be considered in phytoremediation strategies and environmental risk assessments of TBBPA in submerged soils.

Synthesis of typical sulfonamide antibiotics with [14C]- and [13C]-labeling on the phenyl ring for use in environmental studies.

Background: Due to their widespread use, sulfonamide antibiotics (SAs) have become ubiquitous environmental contaminants and thus a cause of public concern. However, a complete understanding of the behavior of these pollutants in complex environmental systems has been hampered by the unavailability and high cost of isotopically labeled SAs. Results: Using commercially available uniformly [14C]- and [13C]-labeled aniline as starting materials, we synthesized [phenyl-ring-14C]- and [phenyl-ring-13C]-labeled sulfamethoxazole (SMX), sulfamonomethoxine (SMM), and sulfadiazine (SDZ) in four-step (via the condensation of labeled N-acetylsulfanilyl chloride and aminoheterocycles) or five-step (via the condensation of labeled N-acetylsulfonamide and chloroheterocycles) reactions, with good yields (5.0-22.5% and 28.1-54.1% for [14C]- and [13C]-labeled SAs, respectively) and high purities (> 98.0%). Conclusion: The synthesis of [14C]-labeled SAs in milligram amounts enables the preparation of labeled SAs with high specific radioactivity. The efficient and feasible methods described herein can be applied to the production of a variety of [14C]- or [13C]-labeled SAs for studies on their environmental behavior, including the fate, transformation, and bioaccumulation of these antibiotics in soils and aqueous systems. Supplementary Information: The online version contains supplementary material available at 10.1186/s12302-022-00598-z.

Release of tetrabromobisphenol A (TBBPA)-derived non-extractable residues in oxic soil and the effects of the TBBPA-degrading bacterium Ochrobactrum sp. strain T.

Tetrabromobisphenol A (TBBPA) forms large amount of non-extractable residues (NER) in soil. However, the stability of TBBPA-NER with TBBPA degrader in soil had not been determined. In this study, a 14C-tracer was used to follow the release and alteration of TBBPA-derived NER during 214 days of incubation in oxic soil and in the presence or absence of the TBBPA-degrading bacterium Ochrobatrum sp. strain T. In the absence of strain T, 1.89% of the TBBPA and its metabolites were slowly released from the NER, with TBBPA as the predominant component, accompanied by 2.47% mineralization by day 91 of the incubation. The addition of active cells strongly stimulated the release and mineralization of NER (10.93% and 4.64%, respectively), reduced the amount of the ester-linked fraction, and transformed NER from humin-bound to HA-bound forms. Cells added to the soil in sterilized form had much smaller effects on the stability and internal alterations of NER. Among the ester-linked compounds, 47.4% consisted of TBBPA; two metabolites were so detected. These results provide new information on the stability and internal transformation of TBBPA-NER in soil during its long-term incubation and underlines the importance of microbial TBBPA degraders in determining the composition of NER in soil.

Influence of the geophagous earthworm Aporrectodea sp. on fate of bisphenol A and a branched 4-nonylphenol isomer in soil.

Large amounts of endocrine disrupting chemicals (EDCs) including bisphenol A (BPA) and nonylphenol (NP) are released into the soil due to the application of biosolids. Earthworms are the predominant biomass in many terrestrial ecosystems and profoundly influence the physico-chemical and biological properties of soils. However, information about the effects of earthworm activities on the behaviors of EDCs in soil is still limited. Here, the effects of earthworms on mineralization, degradation, and bound residue formation of BPA and NP were investigated using the 14C tracer technique. The results showed that earthworms did not affect mineralization of BPA, but significantly inhibited bound residue formation of BPA and changed the size distribution of BPA residues within humic substances. Regarding NP, earthworms significantly inhibited mineralization and bound residue formation, and thus significantly promoted the degradation of NP and NP's metabolites in soil. After nine days of incubation, 75% and 46% of the initially applied 14C-BPA and 14C-NP were already present in bound residues, respectively, indicating that the major route of degradation of BPA and NP in soil was bound-residue formation. Among total 14C-BPA or 14C-NP residues accumulated in earthworms, bound residues were also predominant (>50%), implying that risk assessment of EDCs based on their concentrations of free form in earthworms might be significantly underestimated. Taken together, our results suggest that fate of EDCs in soil not only depended on their physico-chemical properties but also was intensively affected by earthworm activities, underlining that effects of earthworms should be considered when evaluating environmental behavior and potential risk of EDCs in soil.

Mineralisation of 14C-labelled polystyrene plastics by Penicillium variabile after ozonation pre-treatment.

Large amounts of polystyrene (PS), one of the most widely used plastics in the world, end up in the environment through industrial discharge and littering, becoming one of the major components of plastic debris. Such plastics, especially the small-sized microplastics and nanoplastics, have received increasing concerns in terms of their potential environmental risks. Feasible approaches for the degradation of PS in waste materials and in the environment are highly desirable. Physicochemical pretreatments of PS may be applied to enhance biological degradation. In the present study, we synthesized 14C-labelled PS polymers, either uniformly labelled on the ring ([U-ring-14C]-PS) or labelled at the ß-carbon position of the alkyl chain ([ß-14C]-PS), and investigated the mineralisation of the 14C-PS polymers by the fungus Penicillium variabile CCF3219 as well as the effect of ozonation as a physico-chemical pre-treatment on the mineralisation by the fungi. Biodegradation of the 14C-PS polymers was studied in liquid medium (pH 7.5, without additional carbon substrate) with P. variabile for 16 weeks. During the incubation time, 14CO2 was captured to calculate the mineralisation of 14C-PS and the remaining polymers were analysed by means of scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectrometry and gel-permeation chromatography (GPC). The results showed that the fungi mineralised both labelled polymers, and that the [U-ring-14C]-PS with a lower molecular weight led to a higher mineralisation rate. Ozonation pre-treatment strongly enhanced mineralisation of [ß-14C]-PS. SEM analysis showed that the surface of the ozonated [ß-14C]-PS became uneven and rough after the incubation, indicating an attack on the polymer by P. variabile. FT-IR analysis showed that ozonation generated carbonyl groups on the [ß-14C]-PS and the amount of the carbonyl groups decreased after incubation of the [ß-14C]-PS with P. variabile. GPC analysis showed that the molecular weights of the ozonated [ß-14C]-PS decreased after incubation. The present data suggest that ozonation pretreatment could be a potential approach for degradation of PS waste and remediation of PS-contaminated sites.

Formation, characterization, and mineralization of bound residues of tetrabromobisphenol A (TBBPA) in silty clay soil under oxic conditions.

The nature and stability of bound residues (BRs) derived from the widely used brominated flame retardant tetrabromobisphenol A (TBBPA) in fine-textured soil is unknown. We incubated 14C-labeled TBBPA in silty clay rice paddy soil for 93days under oxic conditions. TBBPA dissipated with a first-order kinetic constant kd of 0.0474±0.0017day-1 (t1/2 14.6±0.3days) and mineralized with a km of 0.0011±0.00002day-1. At the end of the incubation, four metabolites, including two methylation products (TBBPA monomethyl and dimethyl ether), accounted for 7.9±0.1% of the initial TBBPA. The BRs continuously increased in amount to a maximum of 80.1±3.6%. About 86.3±0.9% of the BRs localized in the humin fraction and 55.9±1.5% was hydrolyzable with strong alkali (SAH-BRs), which represents reversible BRs. Together with results previously reported for coarse-textured soil, these results indicate that the absolute amounts of both BRs and SAH-BRs of TBBPA as well as the relative contribution of SAH-BRs to total BRs in fine-textured soil are markedly higher than in coarse-textured soil. When BRs-containing soil was incubated with fresh soil for 231days, 9.2±0.3% was mineralized (km 0.00047±0.00002day-1) and SAH-BRs decreased to 34.1±1.1%, accompanied by transformation into other BR forms. These indicate that BRs are bioavailable in the soil. Amendment with rice root exudates did not effectively affect the mineralization, release, and distribution of BRs, suggesting that bioavailability of BRs but not microbial activity limits the degradation of BRs in the silty clay soil. This study provides first insights into the nature and stability of TBBPA-derived BRs in fine-textured soil under oxic conditions and indicates the significant role of reversible BRs in the environmental risk of TBBPA.

Rhizobacteria and plant symbiosis in heavy metal uptake and its implications for soil bioremediation.

Certain species of plants can benefit from synergistic effects with plant growth-promoting rhizobacteria (PGPR) that improve plant growth and metal accumulation, mitigating toxic effects on plants and increasing their tolerance to heavy metals. The application of PGPR as biofertilizers and atmospheric nitrogen fixators contributes considerably to the intensification of the phytoremediation process. In this paper, we have built a system consisting of rhizospheric Azotobacter microbial populations and Lepidium sativum plants, growing in solutions containing heavy metals in various concentrations. We examined the ability of the organisms to grow in symbiosis so as to stimulate the plant growth and enhance its tolerance to Cr(VI) and Cd(II), to ultimately provide a reliable phytoremediation system. The study was developed at the laboratory level and, at this stage, does not assess the inherent interactions under real conditions occurring in contaminated fields with autochthonous microflora and under different pedoclimatic conditions and environmental stresses. Azotobacter sp. bacteria could indeed stimulate the average germination efficiency of Lepidium sativum by almost 7%, average root length by 22%, average stem length by 34% and dry biomass by 53%. The growth of L. sativum has been affected to a greater extent in Cd(II) solutions due its higher toxicity compared to that of Cr(VI). The reduced tolerance index (TI, %) indicated that plant growth in symbiosis with PGPR was however affected by heavy metal toxicity, while the tolerance of the plant to heavy metals was enhanced in the bacteria-plant system. A methodology based on artificial neural networks (ANNs) and differential evolution (DE), specifically a neuro-evolutionary approach, was applied to model germination rates, dry biomass and root/stem length and proving the robustness of the experimental data. The errors associated with all four variables are small and the correlation coefficients higher than 0.98, which indicate that the selected models can efficiently predict the experimental data.

Degradation of sulfonamide antibiotics by Microbacterium sp. strain BR1 - elucidating the downstream pathway.

Microbacterium sp. strain BR1 is among the first bacterial isolates which were proven to degrade sulfonamide antibiotics. The degradation is initiated by an ipso-substitution, initiating the decay of the molecule into sulfur dioxide, the substrate specific heterocyclic moiety as a stable metabolite and benzoquinone imine. The latter appears to be instantaneously reduced to p-aminophenol, as that in turn was detected as the first stable intermediate. This study investigated the downstream pathway of sulfonamide antibiotics by testing the strain's ability to degrade suspected intermediates of this pathway. While p-aminophenol was degraded, degradation products could not be identified. Benzoquinone was shown to be degraded to hydroquinone and hydroquinone in turn was shown to be degraded to 1,2,4-trihydroxybenzene. The latter is assumed to be the potential substrate for aromatic ring cleavage. However, no products from the degradation of 1,2,4-trihydroxybenzene could be identified. There are no signs of accumulation of intermediates causing oxidative stress, which makes Microbacterium sp. strain BR1 an interesting candidate for industrial waste water treatment.