Biodegradation of Pesticides at the Limit: Kinetics and Microbial Substrate Use at Low Concentrations.

The objective of our study was to test whether limited microbial degradation at low pesticide concentrations could explain the discrepancy between overall degradability demonstrated in laboratory tests and their actual persistence in the environment. Studies on pesticide degradation are often performed using unrealistically high application rates seldom found in natural environments. Nevertheless, biodegradation rates determined for higher pesticide doses cannot necessarily be extrapolated to lower concentrations. In this context, we wanted to (i) compare the kinetics of pesticide degradation at different concentrations in arable land and (ii) clarify whether there is a concentration threshold below which the expression of the functional genes involved in the degradation pathway is inhibited without further pesticide degradation taking place. We set up an incubation experiment for four weeks using 14C-ring labeled 2-methyl-4-chlorophenoxyacetic acid (MCPA) as a model compound in concentrations from 30 to 20,000 µg kg-1 soil. To quantify the abundance of putative microorganisms involved in MCPA degradation and their degradation activity, tfdA gene copy numbers (DNA) and transcripts (mRNA) were determined by quantitative real-time PCR. Mineralization dynamics of MCPA derived-C were analyzed by monitoring 14CO2 production and 14C assimilation by soil microorganisms. We identified two different concentration thresholds for growth and activity with respect to MCPA degradation using tfdA gene and mRNA transcript abundance as growth and activity indices, respectively. The tfdA gene expression started to increase between 1,000 and 5,000 µg MCPA kg-1 dry soil, but an actual increase in tfdA sequences could only be determined at a concentration of 20,000 µg. Accordingly, we observed a clear shift from catabolic to anabolic utilization of MCPA-derived C in the concentration range of 1,000 to 5,000 µg kg-1. Concentrations ≥1,000 µg kg-1 were mainly associated with delayed mineralization, while concentrations ≤1,000 µg kg-1 showed rapid absolute dissipation. The persistence of pesticides at low concentrations cannot, therefore, be explained by the absence of functional gene expression. Nevertheless, significant differences in the degradation kinetics of MCPA between low and high pesticide concentrations illustrate the need for studies investigating pesticide degradation at environmentally relevant concentrations.

Temporal and spatial variable in-stream attenuation of selected pharmaceuticals.

Organic micropollutants enter rivers mainly with discharges of wastewater treatment plants (WWTP) and pose a risk to aquatic ecosystems and water quality. A considerable knowledge gap exists for disentangling overlapping processes and driving conditions that control the fate of these pollutants. Thus, the aim of this study was to identify the driving parameters for attenuation of selected pharmaceuticals (carbamazepine, diclofenac, tramadol and venlafaxine) under field conditions. The presented study was performed at a small river (Ammer River, mean discharge 0.87 m3 s-1) which is hydrologically complex due to karstification, numerous artificial discharges, and engineered modifications of the channel. We applied a Lagrangian sampling scheme at two sequential river reaches. In general, for the investigated compounds and over the length of the tested reaches, the absolute net attenuation representative for 24 h was low (≤ 23% net attenuation), yet calculated half-lives were comparable to literature. Photodegradation is specifically relevant for the first river reach characterized by a higher net attenuation of the photosensitive compound diclofenac (14.5% ±11.3%) compared to the second section (9.8% ±13.7%). This is likely due to a spatial difference in canopy shading, which is supported by significant correlations (R2 ≥ 0.8) of the temporally changing 'temperature' and 'solar radiation' with time-specific degradation rate constants of photosensitive compounds for consecutive hourly water parcels. In general, the presented spatially and temporally resolved approach is a suitable tool to determine the attenuation of organic micropollutants and to narrow down the interpretation of net attenuation to a few reasonable processes.

Influence of Emission Sources and Tributaries on the Spatial and Temporal Patterns of Micropollutant Mixtures and Associated Effects in a Small River.

Organic micropollutants of anthropogenic origin in river waters may impair aquatic ecosystem health and drinking water quality. To evaluate micropollutant fate and turnover on a catchment scale, information on input source characteristics as well as spatial and temporal variability is required. The influence of tributaries from agricultural and urban areas and the input of wastewater were investigated by grab and Lagrangian sampling under base flow conditions within a 7.7-km-long stretch of the Ammer River (southwest Germany) using target screening for 83 organic micropollutants and 4 in vitro bioassays with environmentally relevant modes of action. In total, 9 pesticides and transformation products, 13 pharmaceuticals, and 6 industrial and household chemicals were detected. Further, aryl hydrocarbon receptor induction, peroxisome proliferator-activated receptor activity, estrogenicity, and oxidative stress response were measured in the river. The vast majority of the compounds and mixture effects were introduced by the effluent of a wastewater-treatment plant, which contributed 50% of the total flow rate of the river on the sampling day. The tributaries contributed little to the overall load of organic micropollutants and mixture effects because of their relatively low discharge but showed a different chemical and toxicological pattern from the Ammer River, though a comparison to effect-based trigger values pointed toward unacceptable surface water quality in the main stem and in some of the tributaries. Chemical analysis and in vitro bioassays covered different windows of analyte properties but reflected the same picture. Environ Toxicol Chem 2020;39:1382-1391. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Combining in vitro reporter gene bioassays with chemical analysis to assess changes in the water quality along the Ammer River, Southwestern Germany.

BACKGROUND: Rivers receive water and associated organic micropollutants from their entire catchment, including from urban, agricultural and natural sources, and constitute an important environmental component for catalyzing pollutant turnover. Environmental removal processes were extensively investigated under laboratory conditions in the past but there is still a lack of information on how organic micropollutants attenuate on the catchment scale. The aim of this study was to describe the chemical and toxicological profile of a 4th order river and to characterize in-stream processes. We propose indicator chemicals and indicator in vitro bioassays as screening methods to evaluate micropollutant input and transport and transformation processes of the chemical burden in a river. Carbamazepine and sulfamethoxazole were selected as indicators for dilution processes and the moderately degradable chemicals tramadol and sotalol as indicators for potential in-stream attenuation processes. The battery of bioassays covers seven environmentally relevant modes of action, namely estrogenicity, glucocorticogenic activity, androgenicity progestagenic activity and oxidative stress response, as well as activation of the peroxisome proliferator-activated receptor and the aryl hydrocarbon receptor, using the GeneBLAzer test battery and the AhR-CALUX and AREc32 assays. RESULTS: Both approaches, targeted chemical analysis and in vitro bioassays, identified a wastewater treatment plant (WWTP) as a major input source of organic micropollutants that dominantly influenced the water quality of the river. Downstream of the WWTP the amount of detected chemicals and biological effects decreased along the river flow. The organic indicator chemicals of known degradability uncovered dilution and potential loss processes in certain river stretches. The average cytotoxic potency of the river water decreased in a similar fashion as compounds of medium degradability such as the pharmaceutical sotalol. CONCLUSIONS: This study showed that the indicator chemical/indicator bioassay approach is suitable for identifying input sources of a mixture of organic micropollutants and to trace changes in the water quality along small rivers. This method forms the necessary basis for evaluating the natural attenuation processes of organic micropollutants on a catchment scale, especially when combined with enhanced sampling strategies in future studies.

Improved synthesis of (+/-)-trichodiene--a volatile marker for trichothecene mycotoxins.

Trichodiene is the first and only volatile intermediate in the biosynthesis of Fusarium mycotoxins and its detection in the gas-phase might therefore be of potential interest as a marker for food safety analysis. We herein present an improved diastereoselective synthesis of trichodiene which can be used as an analytical standard for a headspace gas chromatography / mass spectrometry method to be developed.

Inhibition of lipopolysaccharide transport to the outer membrane in Pseudomonas aeruginosa by peptidomimetic antibiotics.

The asymmetric outer membrane (OM) of Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet and phospholipid in the inner leaflet. During OM biogenesis, LPS is transported from the periplasm into the outer leaflet by a complex comprising the OM proteins LptD and LptE. Recently, a new family of macrocyclic peptidomimetic antibiotics that interact with LptD of the opportunistic human pathogen Pseudomonas aeruginosa was discovered. Here we provide evidence that the peptidomimetics inhibit the LPS transport function of LptD. One approach to monitor LPS transport involved studies of lipid A modifications. Some modifications occur only in the inner membrane while others occur only in the OM, and thus provide markers for LPS transport within the bacterial envelope. We prepared a conditional lptD mutant of P. aeruginosa PAO1 that allowed control of lptD expression from the rhamnose promoter. With this mutant, the effects caused by the antibiotic on the wild-type strain were compared with those caused by depleting LptD in the mutant strain. When LptD was depleted in the mutant, electron microscopy revealed accumulation of membrane-like material within cells and OM blebbing; this mirrored similar effects in the wild-type strain caused by the antibiotic. Moreover, the bacterium responded to the antibiotic, and to depletion of LptD, by introducing the same lipid A modifications, consistent with inhibition by the antibiotic of LptD-mediated LPS transport. This conclusion was further supported by monitoring the radiolabelling of LPS from [¹4C]acetate, and by fractionation of IM and OM components. Overall, the results provide support for a mechanism of action for the peptidomimetic antibiotics that involves inhibition of LPS transport to the cell surface.

Exploiting enzymatic promiscuity to engineer a focused library of highly selective antifungal and antiproliferative aureothin analogues.

Aureothin is a shikimate-polyketide hybrid metabolite from Streptomyces thioluteus with a rare nitroaryl moiety, a chiral tetrahydrofuran ring, and an O-methylated pyrone ring. The antimicrobial and antitumor activities of aureothin have caught our interest in modulating its structure as well as its bioactivity profile. In an integrated approach using mutasynthesis, biotransformation, and combinatorial biosynthesis, a defined library of aureothin analogues was generated. The promiscuity of the polyketide synthase assembly line toward different starter units and the plasticity of the pyrone and tetrahydrofuran ring formation were exploited. A selection of 15 new aureothin analogues with modifications at the aryl residue, the pyrone ring, and the oxygenated backbone was produced on a preparative scale and fully characterized. Remarkably, various new aureothin derivatives are less cytotoxic than aureothin but have improved antiproliferative activities. Furthermore, we found that the THF ring is crucial for the remarkably selective activity of aureothin analogues against certain pathogenic fungi.

Peptidomimetic antibiotics target outer-membrane biogenesis in Pseudomonas aeruginosa.

Antibiotics with new mechanisms of action are urgently required to combat the growing health threat posed by resistant pathogenic microorganisms. We synthesized a family of peptidomimetic antibiotics based on the antimicrobial peptide protegrin I. Several rounds of optimization gave a lead compound that was active in the nanomolar range against Gram-negative Pseudomonas spp., but was largely inactive against other Gram-negative and Gram-positive bacteria. Biochemical and genetic studies showed that the peptidomimetics had a non-membrane-lytic mechanism of action and identified a homolog of the beta-barrel protein LptD (Imp/OstA), which functions in outer-membrane biogenesis, as a cellular target. The peptidomimetic showed potent antimicrobial activity in a mouse septicemia infection model. Drug-resistant strains of Pseudomonas are a serious health problem, so this family of antibiotics may have important therapeutic applications.