An informative short-term study on the impacts of a triclocarban/weathered multi-walled carbon nanotube-adsorbed complex to benthic organisms.

Freshwater organisms are suitable models to study the fate of environmental pollutants. Due to their versatile and everyday use, many environmental pollutants such as triclocarban (TCC) or multi-walled carbon nanotubes (MWCNTs) enter environmental compartments very easily. TCC is known as a disinfectant and is declared as a highly aquatic toxicant. Multi-walled carbon nanotubes are used, e.g., in the automotive industry to improve plastic properties. Both TCCs and MWCNTs can pose major pollution hazards to various organisms. In addition, these substances can bind to each other due to their tendency to interact via strong hydrophobic interactions. Therefore, a short-term test was conducted to investigate the effects of the individual chemicals TCC and weathered MWCNTs (wMWCNTs) on a benthic biofilm and a grazing organism, Lymnaea stagnalis. Furthermore, the two compounds were coupled by an adsorption experiment resulting in a coupled complex formation (TCC + wMWCNTs). L. stagnalis showed no effects in terms of mortality. For benthic biofilm, the coupling test (TCC + wMWCNTs) showed a decrease of 58% in chlorophyll a (Chl-a) concentration. The main effect could be attributed to the wMWCNTs' exposure alone (decrease of 82%), but not to presence of TCC. The concentration range of Chl-a upon TCC exposure alone was comparable to that in the control group (32 and 37 µg/cm2). With respect to the particulate organic carbon (POC) concentration, very similar results were found for the solvent control, the TCC, and also for the TCC + wMWCNTs group (3, 2.9, and 2.9 mg/cm2). In contrast to the control, a significant increase in POC concentration (100%) was observed for wMWCNTs, but no synergistic effect of TCC + wMWCNTs was detected.

Impact of weathered multi-walled carbon nanotubes on the epithelial cells of the intestinal tract in the freshwater grazers Lymnaea stagnalis and Rhithrogena semicolorata.

Freshwater grazers are suitable organisms to investigate the fate of environmental pollutants, such as weathered multi-walled carbon nanotubes (wMWCNTs). One key process is the uptake of ingested materials into digestive or absorptive cells. To address this, we investigated the localization of wMWCNTs in the intestinal tracts of the mud snail Lymnaea stagnalis (L. stagnalis) and the mayfly Rhithrogena semicolorata (R. semicolorata). In L. stagnalis, bundles of wMWCNTs could be detected in the midgut lumen, whereas only single wMWCNTs could be detected in the lumina of the digestive gland. Intracellular uptake of wMWCNTs was detected by transmission electron microscopy (TEM) but was restricted to the cells of the digestive gland. In larvae of R. semicolorata, irritations of the microvilli and damages in the apical parts of the epithelial gut cells were detected after feeding with 1 to 10 mg/L wMWCNTs. In both models, we detected fibrillar structures in close association with the epithelial cells that formed peritrophic membranes (PMs). The PM may cause a reduced transmission of wMWCNT bundles into the epithelium by forming a filter barrier and potentially protecting the cells from the wMWCNTs. As a result, the uptake of wMWCNTs into cells is rare in mud snails and may not occur at all in mayfly larvae. In addition, we monitor physiological markers such as levels of glycogen or triglycerides and the RNA/DNA ratio. This ratio was significantly affected in L. stagnalis after 24 days with 10 mg/L wMWCNTs, but not in R. semicolorata after 28 days and 10 mg/L wMWCNTs. However, significant effects on the energy status of R. semicolorata were analysed after 28 days of exposure to 1 mg/L wMWCNTs. Furthermore, we observed a significant reduction of phagosomes per enterocyte cell in mayfly larvae at a concentration of 10 mg/L wMWCNTs (p < 0.01).

Municipal Wastewaters Carry Important Carbapenemase Genes Independent of Hospital Input and Can Mirror Clinical Resistance Patterns.

The spatiotemporal variation of several carbapenemase-encoding genes (CRGs) was investigated in the influent and effluent of municipal WWTPs, with or without hospital sewage input. Correlations among gene abundances, bacterial community composition, and wastewater quality parameters were tested to identify possible predictors of CRGs presence. Also, the possible role of wastewaters in mirroring clinical resistance is discussed. The taxonomic groups and gene abundances showed an even distribution among wastewater types, meaning that hospital sewage does not influence the microbial diversity and the CRG pool. The bacterial community was composed mainly of Proteobacteria, Firmicutes, Actinobacteria, Patescibacteria, and Bacteroidetes. Acinetobacter spp. was the most abundant group and had the majority of operational taxonomic units (OTUs) positively correlated with CRGs. This agrees with recent reports on clinical data. The influent samples were dominated by blaKPC, as opposed to effluent, where blaIMP was dominant. Also, blaIMP was the most frequent CRG family observed to correlate with bacterial taxa, especially with the Mycobacterium genus in effluent samples. Bacterial load, blaNDM, blaKPC, and blaOXA-48 abundances were positively correlated with BOD5, TSS, HEM, Cr, Cu, and Fe concentrations in wastewaters. When influent gene abundance values were converted into population equivalent (PE) data, the highest copies/1 PE were identified for blaKPC and blaOXA-48, agreeing with previous studies regarding clinical isolates. Both hospital and non-hospital-type samples followed a similar temporal trend of CRG incidence, but with differences among gene groups. Colder seasons favored the presence of blaNDM, blaKPC and blaOXA-48, whereas warmer temperatures show increased PE values for blaVIM and blaIMP. IMPORTANCE Wastewater-based epidemiology has recently been recognized as a valuable, cost-effective tool for antimicrobial resistance surveillance. It can help gain insights into the characteristics and distribution of antibiotic resistance elements at a local, national, and even global scale. In this study, we investigated the possible use of municipal wastewaters in the surveillance of clinically relevant carbapenemase-encoding genes (CRGs), seen as critical antibiotic resistance determinants. In this matter, our results highlight positive correlations among CRGs, microbial diversity, and wastewater physical and chemical parameters. Identified predictors can provide valuable data regarding the level of raw and treated wastewater contamination with these important antibiotic resistance genes. Also, wastewater-based gene abundances were used for the first time to observe possible spatiotemporal trends of CRGs incidence in the general population. Therefore, possible hot spots of carbapenem resistance could be easily identified at the community level, surpassing the limitations of health care-associated settings.

A workflow to investigate the impacts of weathered multi-walled carbon nanotubes to the mud snail Lymnaea stagnalis.

Although the development and application of nanomaterials is a growing industry, little data is available on the ecotoxicological effects on aquatic organisms. Therefore, we set up a workflow to address the potential uptake of weathered multi-walled carbon nanotubes (wMWCNTs) by a model organism, the pulmonary mud snail Lymnaea stagnalis (L. stagnalis), which plays an important role in the food web. It represents a suitable organism for this approach because as a grazer it potentially ingests large amounts of sedimented wMWCNTs. As food source for L. stagnalis, benthic biofilm was investigated by the use of a transmission electron microscope (TEM) and a scanning electron microscope (SEM) after exposure with wMWCNTs. In addition, isotopic labeling was applied with 14C-wMWCNTs (0.1 mg/L) to quantify fate, behavior, and enrichment of 14C-wMWCNTs in benthic biofilm and in L. stagnalis. Enrichment in benthic biofilm amounted to 529.0 µg wMWCNTs/g dry weight and in L. stagnalis to 79.6 µg wMWCNTs/g dry weight. A bioconcentration factor (BCF) for L. stagnalis was calculated (3500 L/kg). We demonstrate the accumulation of wMWCNTs (10 mg/L) in the digestive tract of L. stagnalis in an effect study. Moreover, the physiological markers glycogen and triglycerides as indicators for the physiological state, as well as the RNA/DNA ratio as growth indicator, were examined. No significant differences between exposed and control animals were analyzed for glycogen and triglycerides after 24 days of exposure, but a decreasing trend is recognizable for triglycerides. In contrast, the significant reduction in the RNA/DNA ratio of L. stagnalis indicated an inhibition of growth with a following recovery after depuration. The described workflow enables a comprehensive determination of the fate and the behavior of wMWCNTs specifically and in general all kinds of CNTs in the aquatic environment and therefore contributes to a holistic risk assessment of wMWCNTs.

Origin and evolution of antibiotic resistance: the common mechanisms of emergence and spread in water bodies.

The environment, and especially freshwater, constitutes a reactor where the evolution and the rise of new resistances occur. In water bodies such as waste water effluents, lakes, and rivers or streams, bacteria from different sources, e.g., urban, industrial, and agricultural waste, probably selected by intensive antibiotic usage, are collected and mixed with environmental species. This may cause two effects on the development of antibiotic resistances: first, the contamination of water by antibiotics or other pollutants lead to the rise of resistances due to selection processes, for instance, of strains over-expressing broad range defensive mechanisms, such as efflux pumps. Second, since environmental species are provided with intrinsic antibiotic resistance mechanisms, the mixture with allochthonous species is likely to cause genetic exchange. In this context, the role of phages and integrons for the spread of resistance mechanisms appears significant. Allochthonous species could acquire new resistances from environmental donors and introduce the newly acquired resistance mechanisms into the clinics. This is illustrated by clinically relevant resistance mechanisms, such as the fluoroquinolones resistance genes qnr. Freshwater appears to play an important role in the emergence and in the spread of antibiotic resistances, highlighting the necessity for strategies of water quality improvement. We assume that further knowledge is needed to better understand the role of the environment as reservoir of antibiotic resistances and to elucidate the link between environmental pollution by anthropogenic pressures and emergence of antibiotic resistances. Only an integrated vision of these two aspects can provide elements to assess the risk of spread of antibiotic resistances via water bodies and suggest, in this context, solutions for this urgent health issue.