Evidence of Bacterial Community Coalescence between Freshwater and Discharged tpm-Harboring Bacterial Taxa from Hospital and Domestic Wastewater Treatment Plants among Epilithic Biofilms.

The ability of WWTP outflow bacteria at colonizing rock surfaces and contributing to the formation of river epilithic biofilms was investigated. Bacterial community structures of biofilms (b-) developing on rocks exposed to treated wastewaters (TWW) of a hospital (HTWW) and a domestic (DTWW) clarifier, and to surface waters of the stream located at 10 m, 500 m, and 8 km from the WWTP outlet, were compared. Biofilm bacterial contents were analyzed by cultural approaches and a tpm-based DNA metabarcoding analytical scheme. Co-occurrence distribution pattern analyses between bacterial datasets and eighteen monitored pharmaceuticals were performed. Higher concentrations of iohexol, ranitidine, levofloxacin, and roxithromycin were observed in the b-HTWW while atenolol, diclofenac, propranolol, and trimethoprim were higher in the b-DTWW. MPN growth assays showed recurrent occurrences of Pseudomonas aeruginosa and Aeromonas caviae among these biofilms. An enrichment of multi-resistant P. aeruginosa cells was observed in the hospital sewer line. P. aeruginosa MPN values were negatively correlated to roxithromycin concentrations. The tpm DNA metabarcoding analyses confirmed these trends and allowed an additional tracking of more than 90 species from 24 genera. Among the recorded 3082 tpm ASV (amplicon sequence variants), 41% were allocated to the Pseudomonas. Significant differences through ANOSIM and DESeq2 statistical tests were observed between ASV recovered from b-HTWW, b-DTWW, and epilithic river biofilms. More than 500 ASV were found restricted to a single sewer line such as those allocated to Aeromonas popoffii and Stenotrophomonas humi being strictly found in the b-HTWW file. Several significant correlations between tpm ASV counts per species and pharmaceutical concentrations in biofilms were recorded such as those of Lamprocystis purpurea being positively correlated with trimethoprim concentrations. A tpm source tracking analysis showed the b-DTWW and b-HTWW tpm ASV to have contributed, respectively, at up to 35% and 2.5% of the epilithic river biofilm tpm-taxa recovered downstream from the WWTP outlet. Higher contributions of TWW taxa among epilithic biofilms were recorded closer to the WWTP outlet. These analyses demonstrated a coalescence of WWTP sewer communities with river freshwater taxa among epilithic biofilms developing downstream of a WWTP outlet.

Environmental filtering and mass effect are two important processes driving lake benthic diatoms: Results of a DNA metabarcoding study in a large lake.

Environmental filtering is often found to dominate assembly rules in diatoms. These microalgae are diverse, especially at subspecies level, and tend to exhibit a niche phylogenetic conservatism. Therefore, other rules, such as competition or mass effects, should be detectable when environmental gradients and dispersal barriers are limited. We used metabarcoding to analyse benthic littoral diatom communities in 153 sites in a large lake (Geneva) exhibiting weak geographical barriers and weak environmental gradients outside river estuaries. We assessed assembly rules using variance partitioning, phylogenetic and source tracking analyses. No phylogenetic over-dispersion of communities, indicative of exclusive competition, was detected. Instead, we found these communities to be phylogenetically over-clustered, indicating environmental filtering, which was even stronger near river estuaries where environmental gradients are stronger. Finally, using a Bayesian method (SourceTracker), we found that rivers flowing into the lake bring communities that settle, especially in sites close to estuaries. Rivers with the highest discharges are primarily responsible for immigration, explaining 27% of lake composition. Therefore, despite favourable conditions to observe other rules, our results support that diatom communities are prominently assembled by environmental filtering and immigration processes, in particular from rivers. However, this does not exclude that other assembly rules may be at play at a finer spatial, temporal and/or phylogenetic scale.

SIPIBEL observatory: Data on usual pollutants (solids, organic matter, nutrients, ions) and micropollutants (pharmaceuticals, surfactants, metals), biological and ecotoxicity indicators in hospital and urban wastewater, in treated effluent and sludge from wastewater treatment plant, and in surface and groundwater.

The Bellecombe pilot site - SIPIBEL - was created in 2010 in order to study the characterisation, treatability and impacts of hospital effluents in an urban wastewater treatment plant. This pilot site is composed of: i) the Alpes Léman hospital (CHAL), opened in February 2012, ii) the Bellecombe wastewater treatment plant, with two separate treatment lines allowing to fully separate the hospital wastewater and the urban wastewater, and iii) the Arve River as the receiving water body and a tributary of the Rhône River and the Geneva aquifer. The database includes in total 48 439 values measured on 961 samples (raw and treated hospital and urban wastewater, activated sludge in aeration tanks, dried sludge after dewatering, river and groundwater, and a few additional campaigns in aerobic and anaerobic sewers) with 44 455 physico-chemistry values (including 15 pharmaceuticals and 14 related transformation products, biocides compounds, metals, organic micropollutants), 2 193 bioassay values (ecotoxicity), 1 679 microbiology values (including microorganisms and antibioresistance indicators) and 112 hydrobiology values.

Cyprus' diatom diversity and the association of environmental and anthropogenic influences for ecological assessment of rivers using DNA metabarcoding.

Human activities are the leading cause of environmental impairments. Appropriate biomonitoring of ecosystems is needed to assess these activities effectively. In freshwater ecosystems, periphytic and epilithic biofilms have diatom assemblages. These assemblages respond rapidly to environmental changes, making diatoms valuable bioindicators. For this reason, freshwater biomonitoring programs are currently using diatoms (e.g., Water Framework Directive). In the past ten years, DNA metabarcoding coupled with next-generation sequencing and bioinformatics represents a complementary approach for diatom biomonitoring. In this study, this approach is used for the first time in Cyprus by considering the association of environmental and anthropogenic pressures to diatom assemblages. Statistical analysis was then applied to identify the environmental (i.e., river types, geo-morphological) and anthropogenic (i.e., physicochemical, human land-use pressures) variables' role in the observed diatom diversity. Results indicate differences in diatom assemblages between intermittent and perennial rivers. Achnanthidium minutissimum was more abundant in intermittent rivers; whereas Amphora pediculus and Planothidium caputium in perennial ones. Additionally, we could demonstrate the correlation between nutrients (e.g., nitrogen, phosphorus), stations' local characteristics (e.g., elevation), and land use activities on the observed differences in diatom diversity. Finally, we conclude that multi-stressors and anthropogenic pressures together as multiple stressors have a significant statistical relationship to the observed diatom diversity and play a pivotal role in determining Cyprus' rivers' ecological status.

Diatom DNA metabarcoding for ecological assessment: Comparison among bioinformatics pipelines used in six European countries reveals the need for standardization.

Ecological assessment of lakes and rivers using benthic diatom assemblages currently requires considerable taxonomic expertise to identify species using light microscopy. This traditional approach is also time-consuming. Diatom metabarcoding is a promising alternative and there is increasing interest in using this approach for routine assessment. However, until now, analysis protocols for diatom metabarcoding have been developed and optimised by research groups working in isolation. The diversity of existing bioinformatics methods highlights the need for an assessment of the performance and comparability of results of different methods. The aim of this study was to test the correspondence of outputs from six bioinformatics pipelines currently in use for diatom metabarcoding in different European countries. Raw sequence data from 29 biofilm samples were treated by each of the bioinformatics pipelines, five of them using the same curated reference database. The outputs of the pipelines were compared in terms of sequence unit assemblages, taxonomic assignment, biotic index score and ecological assessment outcomes. The three last components were also compared to outputs from traditional light microscopy, which is currently accepted for ecological assessment of phytobenthos, as required by the Water Framework Directive. We also tested the performance of the pipelines on the two DNA markers (rbcL and 18S-V4) that are currently used by the working groups participating in this study. The sequence unit assemblages produced by different pipelines showed significant differences in terms of assigned and unassigned read numbers and sequence unit numbers. When comparing the taxonomic assignments at genus and species level, correspondence of the taxonomic assemblages between pipelines was weak. Most discrepancies were linked to differential detection or quantification of taxa, despite the use of the same reference database. Subsequent calculation of biotic index scores also showed significant differences between approaches, which were reflected in the final ecological assessment. Use of the rbcL marker always resulted in better correlation among molecular datasets and also in results closer to these generated using traditional microscopy. This study shows that decisions made in pipeline design have implications for the dataset's structure and the taxonomic assemblage, which in turn may affect biotic index calculation and ecological assessment. There is a need to define best-practice bioinformatics parameters in order to ensure the best representation of diatom assemblages. Only the use of similar parameters will ensure the compatibility of data from different working groups. The future of diatom metabarcoding for ecological assessment may also lie in the development of new metrics using, for example, presence/absence instead of relative abundance data.

Benthic Diatom Communities in an Alpine River Impacted by Waste Water Treatment Effluents as Revealed Using DNA Metabarcoding.

Freshwater ecosystems are continuously affected by anthropogenic pressure. One of the main sources of contamination comes from wastewater treatment plant (WWTP) effluents that contain wide range of micro- and macropollutants. Chemical composition, toxicity levels and impact of treated effluents (TEs) on the recipient aquatic ecosystems may strongly differ depending on the wastewater origin. Compared to urban TEs, hospital ones may contain more active pharmaceutical substances. Benthic diatoms are relevant ecological indicators because of their high species and ecological diversity and rapid response to human pressure. They are routinely used for water quality monitoring. However, there is a knowledge gap on diatom communities' development and behavior in treated wastewater in relation to prevailing micro- and macropollutants. In this study, we aim to (1) investigate the response of diatom communities to urban and hospital TEs, and (2) evaluate TEs effect on communities in the recipient river. Environmental biofilms were colonized in TEs and the recipient river up- and downstream from the WWTP output to study benthic diatoms using DNA metabarcoding combined with high-throughput sequencing (HTS). In parallel, concentrations of nutrients, pharmaceuticals and seasonal conditions were recorded. Diatom metabarcoding showed that benthic communities differed strongly in their diversity and structure depending on the habitat. TE sites were generally dominated by few genera with polysaprobic preferences belonging to the motile guild, while river sites favored diverse communities from oligotrophic and oligosaprobic groups. Seasonal changes were visible to lower extent. To categorize parameters important for diatom changes we performed redundancy analysis which suggested that communities within TE sites were associated to higher concentrations of beta-blockers and non-steroidal anti-inflammatory drugs in urban effluents vs. antibiotics and orthophosphate in hospital effluents. Furthermore, indicator species analysis showed that 27% of OTUs detected in river downstream communities were indicator for urban or hospital TE sites and were absent in the river upstream. Finally, biological diatom index (BDI) calculated to evaluate the ecological status of the recipient river suggested water quality decrease linked to the release of TEs. Thus, in-depth assessment of diatom community composition using DNA metabarcoding is proposed as a promising technique to highlight the disturbing effect of pollutants in Alpine rivers.

River biofilm community changes related to pharmaceutical loads emitted by a wastewater treatment plant.

Wastewater treatment plants (WWTP) are the main sources of a broad spectrum of pharmaceuticals found in freshwater ecosystems. These pollutants raise environmental health concerns because of their highly bioactive nature and their chronic releases. Despite this, pharmaceuticals' effects on aquatic environments are poorly defined. Biofilms represent a major part of the microbial life in rivers and streams. They can drive key metabolic cycles and their organizations reflect exposures to changing chemical, physical, and biological constraints. This study estimated the concentrations, over a 3-year period, of ten pharmaceuticals and five nutrients in a river contaminated by a conventional WWTP fed by urban and hospital wastewaters. Variations in these concentrations were related to biofilm bacterial community dynamics. Rock biofilms had developed over defined periods and were harvested at four locations in the river from the up- and downstream WWTP discharge point. Pharmaceuticals were found in all locations in concentrations ranging from not being detected to 192 ng L-1. Despite the high dilution factor of the WWTP effluents by the receiving river, pharmaceuticals were found more concentrated downstream than upstream the WWTP. Shifts in bacterial community structures linked to the environmental emission of pharmaceuticals were superior to seasonal community changes. A community structure from a site located downstream but close to the WWTP was more strongly associated with high pharmaceutical loads and different from those of biofilm samples from the WWTP upstream or far downstream sites. These latter sites were more strongly associated with high nutrient contents. Low environmental concentrations of pharmaceuticals can thus be transferred from WWTP effluents to a connected stream and induce bacterial aquatic community changes over time.

The SIPIBEL project: treatment of hospital and urban wastewater in a conventional urban wastewater treatment plant.

Hospital wastewater (HWW) receives increasing attention because of its specific composition and higher concentrations of some micropollutants. Better knowledge of HWW is needed in order to improve management strategies and to ensure the preservation of wastewater treatment efficiency and freshwater ecosystems. This context pushed forward the development of a pilot study site named Site Pilote de Bellecombe (SIPIBEL), which collects and treats HWW separately from urban wastewater, applying the same conventional treatment process. This particular configuration offers the opportunity for various scientific investigations. It enables to compare hospital and urban wastewater, the efficiency of the two parallel treatment lines, and the composition of the resulting hospital and urban treated effluents, as well as the evaluation of their effects on the environment. The study site takes into account environmental, economic, and social issues and promotes scientific and technical multidisciplinary actions. ᅟ.

Two-year survey of specific hospital wastewater treatment and its impact on pharmaceutical discharges.

It is well known that pharmaceuticals are not completely removed by conventional activated sludge wastewater treatment plants. Hospital effluents are of major concern, as they present high concentrations of pharmaceutically active compounds. Despite this, these specific effluents are usually co-treated with domestic wastewaters. Separate treatment has been recommended. However, there is a lack of information concerning the efficiency of separate hospital wastewater treatment by activated sludge, especially on the removal of pharmaceuticals. In this context, this article presents the results of a 2-year monitoring of conventional parameters, surfactants, gadolinium, and 13 pharmaceuticals on the specific study site SIPIBEL. This site allows the characterization of urban and hospital wastewaters and their separate treatment using the same process. Flow proportional sampling, solid-phase extraction, and liquid chromatography coupled with tandem mass spectrometry were used in order to obtain accurate data and limits of quantification consistent with ultra-trace detection. Thanks to these consolidated data, an in-depth characterization of urban and hospital wastewaters was realized, as well as a comparison of treatment efficiency between both effluents. Higher concentrations of organic carbon, AOX, phosphates, gadolinium, paracetamol, ketoprofen, and antibiotics were observed in hospital wastewaters compared to urban wastewaters. Globally higher removals were observed in the hospital wastewater treatment plant, and some parameters were shown to be of high importance regarding removal efficiencies: hydraulic retention time, redox conditions, and ambient temperature. Eleven pharmaceuticals were still quantified at relevant concentrations in hospital and urban wastewaters after treatment (e.g., up to 1 µg/L for sulfamethoxazole). However, as the urban flow was about 37 times higher than the hospital flow, the hospital contribution appeared relatively low compared to domestic discharges. Thanks to the SIPIBEL site, data obtained from this 2-year program are useful to evaluate the relevance of separate hospital wastewater treatment.

Separate treatment of hospital and urban wastewaters: A real scale comparison of effluents and their effect on microbial communities.

Hospital wastewaters (HWW) contain wider spectrum and higher quantity of pharmaceuticals than urban wastewaters (UWW), but they are generally discharged in sewers without pretreatment. Since traditional urban wastewater treatment plants (WWTP) are not designed to treat HWWs, treated effluents may still contain pollutants that could impair receiving aquatic environments. Hence, a better understanding of the effect of pharmaceuticals in the environment is required. Biofilms are effective "biological sensors" for assessing the environmental effects of pharmaceuticals due to their ability to respond rapidly to physical, chemical and biological fluctuations by changes in their structure and composition. This study evaluated the efficiency of biological treatment with conventional activated sludge system performed parallel on HWW and UWW. Furthermore, six successive monthly colonizations of biofilms were done on autoclaved stones, placed in grid-baskets in the hospital treated effluents (HTE) and urban treated effluents (UTE). The biomass of these biofilms as well as the structure and diversity of their bacterial communities were investigated. Results showed better treatment efficiency for phosphate and nitrite/nitrate during the treatment of UWW. Pharmaceuticals from all investigated therapeutic classes (beta-blockers, nonsteroidal anti-inflammatory drugs, antibiotics, analgesics and anticonvulsants) were efficiently removed, except for carbamazepine. The removal efficiency of the antibiotics, NSAIDs and beta-blockers was higher during the treatment of HWW. HTE and UTE shaped the bacterial communities in different ways. Higher concentrations of pharmaceuticals in the HTE caused adapted development of the microbial community, leading to less developed biomass and lower bacterial diversity. Seasonal changes in solar irradiance and temperature, caused changes in the community composition of biofilms in both effluents. According to the removal efficiency of pharmaceuticals, the separate treatment was beneficial. However, their high concentrations in the HTE and the following adaptations of biofilm communities identify the importance of adapting wastewater treatment to specific hospital pollutants.