Next-generation sequencing of microbial communities in the Athabasca River and its tributaries in relation to oil sands mining activities.

The Athabasca oil sands deposit is the largest reservoir of crude bitumen in the world. Recently, the soaring demand for oil and the availability of modern bitumen extraction technology have heightened exploitation of this reservoir and the potential unintended consequences of pollution in the Athabasca River. The main objective of the present study was to evaluate the potential impacts of oil sands mining on neighboring aquatic microbial community structure. Microbial communities were sampled from sediments in the Athabasca River and its tributaries as well as in oil sands tailings ponds. Bacterial and archaeal 16S rRNA genes were amplified and sequenced using next-generation sequencing technology (454 and Ion Torrent). Sediments were also analyzed for a variety of chemical and physical characteristics. Microbial communities in the fine tailings of the tailings ponds were strikingly distinct from those in the Athabasca River and tributary sediments. Microbial communities in sediments taken close to tailings ponds were more similar to those in the fine tailings of the tailings ponds than to the ones from sediments further away. Additionally, bacterial diversity was significantly lower in tailings pond sediments. Several taxonomic groups of Bacteria and Archaea showed significant correlations with the concentrations of different contaminants, highlighting their potential as bioindicators. We also extensively validated Ion Torrent sequencing in the context of environmental studies by comparing Ion Torrent and 454 data sets and by analyzing control samples.

Metatranscriptomic analysis of the response of river biofilms to pharmaceutical products, using anonymous DNA microarrays.

Pharmaceutical products are released at low concentrations into aquatic environments following domestic wastewater treatment. Such low concentrations have been shown to induce transcriptional responses in microorganisms, which could have consequences on aquatic ecosystem dynamics. In order to test if these transcriptional responses could also be observed in complex river microbial communities, biofilm reactors were inoculated with water from two rivers of differing trophic statuses and subsequently treated with environmentally relevant doses (ng/liter to microg/liter range) of four pharmaceuticals (erythromycin [ER], gemfibrozil [GM], sulfamethazine [SN], and sulfamethoxazole [SL]). To monitor functional gene expression, we constructed a 9,600-feature anonymous DNA microarray platform onto which cDNA from the biofilms was hybridized. Pharmaceutical treatments induced both positive and negative transcriptional responses from biofilm microorganisms. For instance, ER induced the transcription of several stress, transcription, and replication genes, while GM, a lipid regulator, induced transcriptional responses from several genes involved in lipid metabolism. SN caused shifts in genes involved in energy production and conversion, and SL induced responses from a range of cell membrane and outer envelope genes, which in turn could affect biofilm formation. The results presented here demonstrate for the first time that low concentrations of small molecules can induce transcriptional changes in a complex microbial community. The relevance of these results also demonstrates the usefulness of anonymous DNA microarrays for large-scale metatranscriptomic studies of communities from differing aquatic ecosystems.

N,N-Diethyl-m-Toluamide Exposure at an Environmentally Relevant Concentration Influences River Microbial Community Development.

Studies of the South Saskatchewan River confirmed that N,N-diethyl-m-toluamide (DEET) is ubiquitous at 10 to 20 ng/L, whereas in effluent-dominated Wascana Creek, levels of 100 to 450 ng/L were observed. Effects of DEET exposure were assessed in microbial communities using a wide variety of measures. Communities developed in rotating annular reactors with either 100 or 500 ng/L DEET, verified using gas chromatography-mass spectrometry analyses. Microscale analyses indicated that both DEET concentrations resulted in significant (p < 0.05) declines in photosynthetic biomass, whereas bacterial biomass was unaffected. There was no detectable effect of DEET on the levels of chlorophyll a. However, pigment analyses indicated substantial shifts in algal-cyanobacterial community structure, with reductions of green algae and some cyanobacterial groups at 500 ng/L DEET. Protozoan/micrometazoan grazers increased in communities exposed to 500 ng/L, but not 100 ng/L, DEET. Based on thymidine incorporation or utilization of carbon sources, DEET had no significant effects on metabolic activities. Fluorescent lectin-binding analyses showed significant (p < 0.05) changes in glycoconjugate composition at both DEET concentrations, consistent with altered community structure. Principal component cluster analyses of denaturing gradient gel electrophoresis indicated that DEET exposure at either concentration significantly changed the bacterial community (p < 0.05). Analyses based on 16S ribosomal RNA of community composition confirmed changes with DEET exposure, increasing detectable beta-proteobacteria, whereas actinobacteria and acidimicrobia became undetectable. Further, cyanobacteria in the subclass Oscillatoriophycideae were similarly not detected. Thus, DEET can alter microbial community structure and function, supporting the need for further evaluation of its effects in aquatic habitats. Environ Toxicol Chem 2019;38:2414-2425. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Effects of experimental nitrogen fertilization on planktonic metabolism and CO2 flux in a hypereutrophic hardwater lake.

Hardwater lakes are common in human-dominated regions of the world and often experience pollution due to agricultural and urban effluent inputs of inorganic and organic nitrogen (N). Although these lakes are landscape hotspots for CO2 exchange and food web carbon (C) cycling, the effect of N enrichment on hardwater lake food web functioning and C cycling patterns remains unclear. Specifically, it is unknown if different eutrophication scenarios (e.g., modest non point vs. extreme point sources) yield consistent effects on auto- and heterotrophic C cycling, or how biotic responses interact with the inorganic C system to shape responses of air-water CO2 exchange. To address this uncertainty, we induced large metabolic gradients in the plankton community of a hypereutrophic hardwater Canadian prairie lake by adding N as urea (the most widely applied agricultural fertilizer) at loading rates of 0, 1, 3, 8 or 18 mg N L-1 week-1 to 3240-L, in-situ mesocosms. Over three separate 21-day experiments, all treatments of N dramatically increased phytoplankton biomass and gross primary production (GPP) two- to six-fold, but the effects of N on autotrophs plateaued at ~3 mg N L-1. Conversely, heterotrophic metabolism increased linearly with N fertilization over the full treatment range. In nearly all cases, N enhanced net planktonic uptake of dissolved inorganic carbon (DIC), and increased the rate of CO2 influx, while planktonic heterotrophy and CO2 production only occurred in the highest N treatments late in each experiment, and even in these cases, enclosures continued to in-gas CO2. Chemical effects on CO2 through calcite precipitation were also observed, but similarly did not change the direction of net CO2 flux. Taken together, these results demonstrate that atmospheric exchange of CO2 in eutrophic hardwater lakes remains sensitive to increasing N loading and eutrophication, and that even modest levels of N pollution are capable of enhancing autotrophy and CO2 in-gassing in P-rich lake ecosystems.

Effects of a herbicide mixture on primary and bacterial productivity in four prairie wetlands with varying salinities: an enclosure approach.

Wetlands in the Prairie pothole region of Saskatchewan and Manitoba serve an important role in providing wildlife habitat, water storage and water filtration. They display a wide range of water quality parameters such as salinity, nutrients and major ions with sulfate as the dominant ion for the most saline wetlands. The differences in these water quality parameters among wetlands are reflected in the composition of aquatic plant communities and their productivity. Interspersed within an intensely managed agricultural landscape where pesticides are commonly used, mixtures of herbicides are often detected in these wetlands as well as in rivers, and drinking water reservoirs. One freshwater and three wetlands of varying salinity in the St. Denis National Wildlife Area, Saskatchewan, Canada were selected to study the effects of a mixture of eight herbicides (2,4-D, MCPA, dicamba, clopyralid, bromoxynil, mecoprop, dichlorprop, and glyphosate) on wetland microbial communities using an outdoor enclosure approach. Six enclosures (three controls and three treatments) were installed in each wetland and the herbicide mixture added to the treatment enclosures. The concentration of each herbicide in the enclosure water was that which would have resulted from a direct overspray of a 0.5-m deep wetland at its recommended field application rate. After herbicide addition, primary and bacterial productivity, and algal biomass were measured in both planktonic and benthic communities over 28 days. The herbicide mixture had a stimulatory effect on primary productivity in the nutrient-sufficient freshwater wetland while no stimulatory effect was observed in the nutrient-deficient saline wetlands. The differences observed in the effects of the herbicide mixture appear to be related to the nutrient bioavailability in these wetlands.

Sub-inhibitory concentrations of different pharmaceutical products affect the meta-transcriptome of river biofilm communities cultivated in rotating annular reactors.

Surface waters worldwide are contaminated by pharmaceutical products that are released into the environment from wastewater treatment plants. Here, we hypothesize that pharmaceutical products have effects on organisms as well as genes related to nutrient cycling in complex microbial communities. To test this hypothesis, biofilms were grown in reactors and subjected low concentrations of three antibiotics [erythromycin, ER, sulfamethoxazole, SL and sulfamethazine, SN) and a lipid regulator (gemfibrozil, GM). Total community RNA was extracted and sequenced together with PCR amplicons of the 16S rRNA gene using 454 pyrosequencing. Exposure to pharmaceutical products resulted in very little change in bacterial community composition at the phylum level based on 16S rRNA gene amplicons, even though some genera were significantly affected. In contrast, large shifts were observed in the active community composition based on taxonomic affiliations of mRNA sequences. Consequently, expression of gene categories related to N, P and C cycling were strongly affected by the presence of pharmaceutical products, with each treatment having specific effects. These results indicate that low pharmaceutical product concentrations rapidly provoke a variety of functional shifts in river bacterial communities. In the longer term these shifts in gene expression and microbial activity could lead to a disruption of important ecosystem processes like nutrient cycling.

Molecular and microscopic assessment of the effects of caffeine, acetaminophen, diclofenac, and their mixtures on river biofilm communities.

The authors examined effects of three common contaminants, caffeine (CF), acetaminophen (AC), and diclofenac (DF), as well as their mixtures on the development, functioning, and biodiversity of river biofilm communities. Biofilms were cultivated in rotating annular reactors. Treatments included AC, CF, DF, AC + CF, AC + DF, CF + DF, AC + CF + DF at 5 µg/L, and their molar equivalent as carbon and nutrients. Incubations using ¹4C-labeled AC, DF, and CF indicated that 90% of the CF, 80% of the AC, and less than 2% of the DF were converted to CO2. Digital imaging revealed a variety of effects on algal, cyanobacterial, and bacterial biomass. Algal biomass was unaffected by AC or CF in combination with DF but significantly reduced by all other treatments. Cyanobacterial biomass was influenced only by the AC + DF application. All treatments other than AC resulted in a significant decrease in bacterial biomass. Diclofenac or DF + CF and DF + AC resulted in increases in micrometazoan grazing. The denaturing gradient gel electrophoresis of Eubacterial community DNA, evaluated by principal component analysis and analysis of similarity, indicated that relative to the control, all treatments had effects on microbial community structure (r = 0.47, p < 0.001). However, the AC + CF + DF treatment was not significantly different from its molar equivalent carbon and nutrient additions. The Archaeal community differed significantly in its response to these exposures based on community analyses, confirming a need to integrate these organisms into ecotoxicological studies.

Effluent-dominated streams. Part 1: Presence and effects of excess nitrogen and phosphorus in Wascana Creek, Saskatchewan, Canada.

Regina, Saskatchewan, Canada (population 190,400) treats its sewage at a modern sewage treatment plant (STP) on Wascana Creek. In the winter, treated sewage effluent makes up almost 100% of stream flow. Four surveys conducted from 2005 to 2007, in differing seasons, indicated significantly higher nitrogen (N) and phosphorus (P) concentrations at sites downstream of the STP compared to an upstream control site. Downstream, Wascana Creek is N hypersaturated (total dissolved N >3 mg/L) and soluble reactive phosphorus (SRP) makes up a greater percentage of total P (TP). Diminished nutrient retention capacities for both N and P are directly attributable to STP effluent. Creek SRP concentrations are less than estimates of equilibrium P concentrations (EPC(o)), indicating that creek sediments may be a source of P, further exacerbating hypereutrophic ambient SRP concentrations. As well, NO(2) + NO(3)-N concentrations far surpass World Health Organization limits for drinking water (10 mg/L) and sensitive taxa, while NH(3)-N, NH(4)-N, and NO(2) + NO(3)-N exceed Canadian Water Quality Guidelines for Protection of Aquatic Life and those for the U.S. Environmental Protection Agency. High NH(4)-N concentrations may be responsible for depressions not only in algal biomass and production observed downstream but reductions in primary to bacterial production ratios (PP:BP). In spring and fall, these reductions push PP:BP from net autotrophy to heterotrophy. The Wascana Creek study highlights the considerable problems associated with excess nutrients in effluent-dominated ecosystems (EDS). It also underlines the need for better controls on NH(4)-N additions from STPs in such EDS, especially in a day and age when freshwater supplies are dwindling and negative effects of climate change are expected.

Effluent-dominated streams. Part 2: Presence and possible effects of pharmaceuticals and personal care products in Wascana Creek, Saskatchewan, Canada.

Recent worldwide surveys have not only established incomplete removal of pharmaceuticals and personal care products (PPCPs) by sewage treatment plants, but also their presence in surface waters receiving treated sewage effluent. Those aquatic systems where sewage effluent dominates flow are thought to be at the highest risk for ecosystem level changes. The city of Regina, Saskatchewan, Canada (population 190,400) treats its sewage at a modern tertiary sewage treatment facility located on Wascana Creek. The Wascana Creek hydrograph is dominated by one major event: spring snow melt. Thereafter, creek flow declines considerably and in winter treated sewage effluent makes up almost 100% of stream flow. Four water surveys conducted on the creek from winter 2005 to spring 2007 indicated that PPCPs were always present, in nanogram and sometimes microgram per liter concentrations downstream of the sewage treatment plant. This mixture included antibiotics, analgesics, antiinflammatories, a lipid regulator, metabolites of caffeine, cocaine and nicotine, and an insect repellent. Not surprisingly, concentrations of some PPCPs were highest in winter. According to hazard quotient calculations and homologue presence, ibuprofen, naproxen, gemfibrozil, triclosan, erythromycin, trimethoprim, and sulfamethoxazole were present in Wascana Creek at concentrations that may present a risk to aquatic organisms. The continual exposure to a mixture of pharmaceuticals as well as concentrations of un-ionized ammonia that far exceed Canadian and American water quality guidelines suggests that Wascana Creek should be considered an ecosystem at risk. Although the Wascana Creek study is regional in nature, the results highlight the considerable risks posed to aquatic organisms in such effluent-dominated ecosystems.