Biosolids for safe land application: does wastewater treatment plant size matters when considering antibiotics, pollutants, microbiome, mobile genetic elements and associated resistance genes?

Soil fertilization with wastewater treatment plant (WWTP) biosolids is associated with the introduction of resistance genes (RGs), mobile genetic elements (MGEs) and potentially selective pollutants (antibiotics, heavy metals, disinfectants) into soil. Not much data are available on the parallel analysis of biosolid pollutant contents, RG/MGE abundances and microbial community composition. In the present study, DNA extracted from biosolids taken at 12 WWTPs (two large-scale, six middle-scale and four small-scale plants) was used to determine the abundance of RGs and MGEs via quantitative real-time PCR and the bacterial and archaeal community composition was assessed by 16S rRNA gene amplicon sequencing. Concentrations of heavy metals, antibiotics, the biocides triclosan, triclocarban and quaternary ammonium compounds (QACs) were measured. Strong and significant correlations were revealed between several target genes and concentrations of Cu, Zn, triclosan, several antibiotics and QACs. Interestingly, the size of the sewage treatment plant (inhabitant equivalents) was negatively correlated with antibiotic concentrations, RGs and MGEs abundances and had little influence on the load of metals and QACs or the microbial community composition. Biosolids from WWTPs with anaerobic treatment and hospitals in their catchment area were associated with a higher abundance of potential opportunistic pathogens and higher concentrations of QACs.

Adsorption of the hydrophobic organic pollutant hexachlorobenzene to phyllosilicate minerals.

Hexachlorobenzene (HCB), a representative of hydrophobic organic chemicals (HOC), belongs to the group of persistent organic pollutants (POPs) that can have harmful effects on humans and other biota. Sorption processes in soils and sediments largely determine the fate of HCB and the risks arising from the compound in the environment. In this context, especially HOC-organic matter interactions are intensively studied, whereas knowledge of HOC adsorption to mineral phases (e.g., clay minerals) is comparatively limited. In this work, we performed batch adsorption experiments of HCB on a set of twelve phyllosilicate mineral sorbents that comprised several smectites, kaolinite, hectorite, chlorite, vermiculite, and illite. The effect of charge and size of exchangeable cations on HCB adsorption was studied using the source clay montmorillonite STx-1b after treatment with nine types of alkali (M+: Li, K, Na, Rb, Cs) and alkaline earth metal cations (M2+: Mg, Ca, Sr, Ba). Molecular modeling simulations based on density functional theory (DFT) calculations to reveal the effect of different cations on the adsorption energy in a selected HCB-clay mineral system accompanied this study. Results for HCB adsorption to minerals showed a large variation of solid-liquid adsorption constants Kd over four orders of magnitude (log Kd 0.9-3.3). Experiments with cation-modified montmorillonite resulted in increasing HCB adsorption with decreasing hydrated radii of exchangeable cations (log Kd 1.3-3.8 for M+ and 1.3-1.4 for M2+). DFT calculations predicted (gas phase) adsorption energies (- 76 to - 24 kJ mol-1 for M+ and - 96 to - 71 kJ mol-1 for M2+) showing a good correlation with Kd values for M2+-modified montmorillonite, whereas a discrepancy was observed for M+-modified montmorillonite. Supported by further calculations, this indicated that the solvent effect plays a relevant role in the adsorption process. Our results provide insight into the influence of minerals on HOC adsorption using HCB as an example and support the relevance of minerals for the environmental fate of HOCs such as for long-term source/sink phenomena in soils and sediments.

A fast and robust method for the extraction and analysis of quaternary alkyl ammonium compounds from soil and sewage sludge.

Alkyltrimethylammonium compounds (ATMACs), dialkyldimethylammonium compounds (DADMACs) and benzylalkyldimethylethylammonium compounds (BACs) are quaternary alkylammonium compounds (QAAC), which are released into the environment in large quantities after their use in cleaning agents and disinfectants. Despite their potential role as selective agents promoting resistance against QAACs as well as antibiotics, there is a lack of data for QAACs in soil due to the lack of sensitive analytical methods. Therefore, we present a robust and fast method for the extraction and quantification of concentrations of these compounds in soil and sewage sludge. The method is based on ultrasonic extraction (USE) with a mixture of acetonitrile and HCl followed by a solid phase extraction (SPE) cleaning step and a subsequent quantification of concentrations with high performance liquid chromatography with mass spectrometry (HPLC-MS/MS) in multi mass reaction mode (MRM). The proposed method is suitable for the quantification of ATMACs (chain length C-8 to C-16), BACs (C-8 to C-18) and DADMACs (C-8 to C-16). The achieved limits of quantification (LOQ) range from 0.1 µg kg-1 to 2.1 µg kg-1. The recovery rates of spiked soil samples for non-deuterated homologues were between 47% and 57%. The analysis of sewage sludge samples and soil samples revealed that BAC-C12 was the most abundant QAAC with concentrations up to 38600 µg kg-1 in sewage sludge and up to 81 µg kg-1 in a Mexican soil that was irrigated with wastewater. Overall, the presented methods open perspectives for effectively studying fate and effects of QAACs in soils.

Smectite clay minerals reduce the acute toxicity of quaternary alkylammonium compounds towards potentially pathogenic bacterial taxa present in manure and soil.

Quaternary alkylammonium compounds (QAACs) are a group of cationic surfactants which are disinfectants with numerous industrial and agricultural applications and frequently released into the environment. One recent hypothesis is that bacteria present in soil will be protected from acute toxic effects of QAACs in the presence of expandable layer silicates due to interlayer sorption. We therefore studied bacterial growth kinetics with high temporal resolution and determined minimal inhibitory concentrations (MICs) of two QAACs, benzyldimethyldodecylammonium chloride (BAC-C12) and didecyldimethylammonium chlorid (DADMAC-C10), for eight strains of different bacterial taxa (Escherichia coli, Acinetobacter, Enterococcus faecium, Enterococcus faecalis, and Pseudomonas fluorescens) in relation to QAAC sorption to smectite and kaolinite. The MICs of BAC-C12 and DADMAC-C10 were in the absence of smectite and kaolinite in the order of 10 to 30 µg mL-1 and 1.0 to 3.5 µg mL-1 for all strains except the more sensitive Acinetobacter strain. For all tested strains and both tested QAACs, the presence of smectite increased apparent MIC values while kaolinite had no effect on MICs. Sorption curves without bacteria showed that smectite sorbed larger amounts of QAACs than kaolinite. Correcting nominal QAAC concentrations employed in toxicity tests for QAAC sorption using the sorption curves explained well the observed shifts in apparent MICs. Transmission electron microscopy (TEM) demonstrated that the interlayer space of smectite expanded from 13.7 ± 1 Å to 19.9 ± 1.5 Å after addition of BAC-C12. This study provides first evidence that low charge 2:1 expandable layer silicates can play an important role for buffering QAAC toxicity in soils.