Evidence of interregional similarity in crayfish metabolomes at reference sites: Progress towards the metabolome as a biomonitoring tool.

It has been proposed that biomonitoring may benefit from the use of metabolomics (the study of all small molecules in an organism) to detect sub-lethal organism stress through changes in the metabolite profile (i.e., the metabolome). However, to integrate the metabolome into biomonitoring programs the amount of natural variability among and within populations of indicator taxa must be established prior to generating a reference condition. This study determined variation in the metabolome among ecoregion and stream of origin in the northern crayfish (Faxonius virilis) and if that variation inhibited detection of stressor effects at sites exposed to human activities. We collected crayfish from seven minimally disturbed streams (i.e., reference streams), distributed across three level II ecoregions in central Canada and compared their metabolomes. We found ecoregion and stream origin were poor predictors of crayfish metabolomes. This result suggests crayfish metabolomes were similar, despite differing environmental conditions. Metabolomes of crayfish collected from three stream sites exposed to agricultural activity and municipal wastewater (i.e., test sites) were then compared to the crayfish metabolomes from the seven reference streams. Findings showed that crayfish metabolomes from test sites were strongly differentiated from those at all reference sites. The consistency in the northern crayfish metabolome at the studied reference streams indicates that a single reference condition may effectively detect impacts of human activities across the sampled ecoregions.

Evaluating Lower Athabasca River Sediment Metal Concentrations from Alberta Oil Sands Monitoring Programs Using Predevelopment Baselines.

Since 1997, sediment metal concentrations have been monitored in the Alberta Oil Sands Region (AOSR) of the Lower Athabasca River by the Regional Aquatics Monitoring Program (RAMP; 1997-2002), the Joint Oil Sands Monitoring Program (JOSM; 2012-2014), and the Oil Sands Monitoring Program (OSM; 2015-present). However, it has remained difficult to differentiate industrial sources from natural sources and quantify the extent of pollution due to inadequate knowledge of predevelopment reference conditions. Here, baselines were constructed using predevelopment (i.e., pre-1967) sediment concentrations of US EPA priority pollutants (Be, Cr, Cu, Ni, Pb) and V, an element elevated in bitumen and associated waste materials, normalized to Al concentration in cores from floodplain and upland lakes within the AOSR to characterize the natural range of variability. The Lower Athabasca River sediment metal monitoring data were examined in the context of the predevelopment baselines. Most metals are below the threshold for minimal enrichment (<1.5x baseline) except for chromium (up to 4.8x) in some RAMP samples. The predevelopment baselines for sediment metal concentrations will be of particular importance as the oil sands industry potentially shifts from a no-release policy to the treatment and release of oil sands process waters directly to the Lower Athabasca River.

Time-dependent biological responses of juvenile yellow perch (Perca flavescens) exposed in situ to a major urban effluent.

Municipal wastewater treatment plant (WWTP) effluents are significant sources of organic and inorganic pollutants to aquatic ecosystems. Several studies have shown that the health of aquatic organisms can be adversely impacted following exposure to these complex chemical mixtures. The objective of this study was to examine the effects of in situ exposure in the St. Lawrence River (QC, Canada) of juvenile yellow perch (Perca flavescens) to a major WWTP effluent. Perch were caged at a reference site in the St. Lawrence River and downstream of a WWTP effluent-influenced site for one, three, and six weeks. Fish kept in controlled laboratory setting were also examined at the beginning of the experiment to evaluate the potential effect of caging on fish. Liver metabolites and gill oxidative stress biomarkers as well as body condition of perch were investigated at four time points (zero, one, three, and six weeks). Nitrogen (δ15N) and carbon (δ13C) stable isotopes as well as tissue concentrations of halogenated flame retardants and trace metals were also analyzed. Results indicated that body condition of perch caged in the effluent increased after three and six weeks of exposure compared to that of reference fish. Perch caged at the WWTP effluent-influenced site also had higher muscle δ13C and slightly depleted muscle δ15N after three and six weeks of exposure, suggesting differences in sewage-derived nutrient assimilation between sites. Concentrations of Σ34 polybrominated diphenyl ether (PBDE) were 2-fold greater in perch exposed downstream of the WWTP compared to those caged at the reference site. Metal concentrations in kidney of perch after three weeks of exposure were significantly lower at the effluent-influenced site. Kidney concentrations of Cd, Cu, Se, As, Zn and Fe were, however, higher after six weeks of exposure, supporting that metal accumulation is time- and element-specific. The metabolomes of perch from the effluent-influenced and reference sites were similar, but were distinct from the laboratory control fish, suggesting a caging effect on fish. Seven liver metabolites (glucose, malate, fumarate, glutamate, creatinine, histamine, and oxypurinol) were significantly more abundant in perch from cages than in the laboratory control perch. The combination of metabolomics and physiological variables provides a powerful tool to improve our understanding of the mechanisms of action of complex environmental pollutant mixtures in wild fish.

Mercury trends in predatory fish in Great Slave Lake: the influence of temperature and other climate drivers.

Here we report on trends in mercury (Hg) concentrations in lake trout (Salvelinus namaycush), burbot (Lota lota), and northern pike (Esox lucius) from Great Slave Lake, located in the Mackenzie River Basin (MRB) and investigate how climate factors may be influencing these trends. Hg concentrations in lake trout and burbot increased significantly over the early 1990s to 2012 in the two major regions of the lake; no trend was evident for northern pike over 1999-2012. Temporal variations in Hg concentrations in lake trout and burbot were similar with respect to timing of peaks and troughs. Inclusion of climate variables based on annual means, particularly temperature, improved explanatory power for variations in Hg over analyses based only on year and fish length; unexpectedly, the temperature coefficient was negative. Climate analyses based on growing season means (defined as May-September) had less explanatory power suggesting that trends were more strongly associated with colder months within the year. Inclusion of the Pacific/North American index improved explanatory power for the lake trout model suggesting that trends may have been affected by air circulation patterns. Overall, while our study confirmed previously reported trends of Hg increase in burbot in the MRB, we found no evidence that these trends were directly driven by increasing temperatures and productivity.

Fatty-acid based assessment of benthic food-web responses to multiple stressors in a large river system.

Rivers are often exposed to multiple stressors, such as nutrients and contaminants, whose impacts on the river food webs may not be distinguished by sole assessment of biological community structures. We examined the benthic algal assemblages and the fatty acids (FA) of benthic macroinvertebrates in the lower Athabasca River in Canada, aiming to assess the changes in algal support and nutritional quality of the benthic food web in response to cumulative exposure to natural bitumen, municipal sewage discharge (hereafter, "sewage"), and oil sands mining ("mining"). Data show that the decline in water quality (increases in nutrient concentrations and total suspended solids) was associated with decreases in benthic diatom abundance, and was driven mainly by sewage-induced nutrient enrichment. Responses in nutritional quality of benthic macroinvertebrates, indicated by their polyunsaturated FA (PUFA) concentrations, were taxon- and stressor-specific. Nutritional quality of the larval dragonfly predator, Ophiogomphus, decreased nonlinearly with decreasing benthic diatom abundance and was lowest at the sewage-affected sites, although exposure to natural bitumen also resulted in reduced Ophiogomphus PUFA concentrations. In contrast, the PUFA concentrations of mayfly grazers/collector-gatherers were not affected by natural bitumen exposure, and were higher at the sewage and sewage+mining sites. The PUFA concentrations of the shredder Pteronarcys larvae did not change with cumulative exposure to the stressors. Sediment metal and polycyclic aromatic compound concentrations were not associated with the macroinvertebrate FA changes. Overall, we provide evidence that sewage induced reduction in trophic support by PUFA-rich diatoms, and was the predominant driver of the observed changes in FA composition and nutritional quality of the benthic macroinvertebrates. Fatty-acid metrics are useful to untangle effects of concurrent stressors, but the assessment outcomes depend on the functional feeding guilds used. A food-web perspective using multiple trophic levels and feeding guilds supports a more holistic assessment of the stressor impacts.

Physical and ecological thresholds for deposited sediments in streams in agricultural landscapes.

Excessive sedimentation in streams and rivers remains a pervasive problem for the protection of aquatic habitat and the sustainability of aquatic communities. Whereas water quality criteria have been determined for suspended sediments in many jurisdictions across North America, comparably little has been done for deposited (also known as bedded) sediments. Through Canada's National Agri-Environmental Standards Initiative, assessment techniques and analytical tools were developed for estimating environmental thresholds for deposited sediments in agricultural watersheds in New Brunswick (NB) and Prince Edward Island (PEI) in the Atlantic Maritimes of Canada. Physical thresholds were developed through assessment of geomorphic metrics, which were then analyzed using y-intercept and 25th percentile approaches. For NB, there was strong agreement in physical thresholds for both analytical approaches (e.g., percent fines <2 mm were 7.5 for y-intercept and 6.9 for 25th percentile approaches). In contrast, physical thresholds for PEI differed considerably between approaches (e.g., percent fines <2 mm were 6.1 for y-intercept and 19.6 for 25th percentile approaches), likely due to a narrower range in agricultural land cover. Cross-calibration of our provisional physical thresholds for NB with ecological (i.e., benthic macroinvertebrate) assessments show that ecological thresholds, calculated as change-points in relationships between Ephemeroptera-Plecoptera-Trichoptera relative abundance or Modified Family Biotic Index and geomorphic criteria, were more liberal than physical thresholds. These results suggest that provisional thresholds developed using geomorphic criteria should demarcate change from the least disturbed condition and reduce the risk of sedimentation degrading benthic ecosystems.

Development of environmental thresholds for nitrogen and phosphorus in streams.

Inputs of nutrients (P and N) to freshwaters can cause excessive aquatic plant growth, depletion of oxygen, and deleterious changes in diversity of aquatic fauna. As part of a "National Agri-Environmental Standards Initiative," the Government of Canada committed to developing environmental thresholds for nutrients to protect ecological condition of agricultural streams. Analysis of data from >200 long-term monitoring stations across Canada and detailed ecological study at ~70 sites showed that agricultural land cover was associated with increased nutrient concentrations in streams and this, in turn, was associated with increased sestonic and benthic algal abundance, loss of sensitive benthic macroinvertebrate taxa, and an increase in benthic diatom taxa indicative of eutrophication. Chemical thresholds for N and P were defined by applying five approaches, employing either a predetermined percentile to a water chemistry data set or a relationship between water chemistry and land cover, to identify boundaries between minimally disturbed and impaired conditions. Comparison of these chemical thresholds with biological thresholds (derived from stressor-response relationships) produced an approach for rationalizing these two types of thresholds and deriving nutrient criteria. The resulting criteria were 0.01 to 0.03 mg L(-1) total P and 0.87-1.2 mg L(-1) total N for the Atlantic Maritime, 0.02 mg L(-1) total P and 0.21 mg L(-1) total N for the Montane Cordillera, ~0.03 mg L(-1) total P and ~1.1 mg L(-1) total N for the Mixedwood Plains, and ~0.10 mg L(-1) total P and 0.39-0.98 mg L(-1) total N for the interior prairies of Canada. Adoption of these criteria should result in greater likelihood of good ecological condition with respect to benthic algal abundance, diatom composition, and macroinvertebrate composition.

Metabolomic Analysis of Hexagenid Mayflies Exposed to Sublethal Concentrations of Naphthenic Acid.

The oil sands region in northeastern Alberta, Canada contain approximately 165 billion barrels of oil making it the third largest oil reserves in the world. However, processing of extracted bitumen generates vast amounts of toxic byproduct known as oil sands process waters. Naphthenic acids and associated sodium naphthenate salts are considered the primary toxic component of oil sands process waters. Although a significant body of work has been conducted on naphthenic acid toxicity at levels comparable to what is observed in current oil sands process waters, it is also important to understand any impacts of exposure to sublethal concentrations. We conducted a microcosm study using the mayfly Hexagenia spp. to identify sublethal impacts of naphthenic acid exposure on the survival, growth, and metabolome across a concentration gradient (0-100 µg L-1) of sodium naphthenate. Nuclear magnetic resonance-based metabolomic analyses were completed on both the polar and lipophilic extracted fractions of whole organism tissue. We observed a positive relationship between sodium naphthenate concentration and mean principal component score of the first axis of the polar metabolome indicating a shift in the metabolome with increasing naphthenic acid exposure. Eleven metabolites correlated with increased naphthenic acid concentration and included those involved in energy metabolism and apoptosis regulation. Survival and growth were both high and did not differ among concentrations, with the exception of a slight increase in mortality observed at the highest concentration. Although lethal concentrations of naphthenic acids in other studies are higher (150-56,200 µg L-1), our findings suggest that physiological changes in aquatic invertebrates may begin at substantially lower concentrations. These results have important implications for the release of naphthenic acids into surface waters in the Alberta oil sands region as an addition of even small volumes of oil sands process waters could initiate chronic effects in aquatic organisms. Results of this research will assist in the determination of appropriate discharge thresholds should oil sands process waters be considered for environmental release.

Contribution of nitrogen sources to streams in mixed-use catchments varies seasonally in a cold temperate region.

Intensive agriculture and growing human populations are important nitrogen (N) sources thought to be associated with eutrophication. However, the contribution and seasonality of N delivery to streams from human activities is poorly understood and knowledge of the role of stream communities in the assimilation of N from human activities is limited. We used N and oxygen stable isotope ratios of dissolved inorganic N (DIN) and concentrations of artificial sweeteners to identify the relative contribution of key sources of anthropogenic N (i.e., fertilizers, human, and livestock waste) to tributaries of the Red River Valley (RRV), Manitoba, Canada. Water and algae were sampled in 14 RRV tributaries during snowmelt, spring, summer, and autumn; and water was sampled at three locations in the Red River in spring, summer, and autumn. δ15N values of DIN in tributary water differed seasonally and were greatest during snowmelt. Incorporation of ammonium δ15N provided evidence for the importance of manure N to tributaries during snowmelt. Fertilizer and municipal lagoons served as principal sources of N to streams in spring and summer. Human and livestock waste sources of N were the dominant contributor to algae at greater than 90% of sites and algae δ15N was greatest at sites downstream of municipal lagoons. We also showed that the tributaries contribute human and livestock waste N to the Red River, though much of the nitrate in the river originates outside of Manitoba. Overall, our study determined that the anthropogenic sources of N to RRV streams vary seasonally, likely due to regional hydrologic conditions. Our study also showed the potential of artificial sweeteners and ammonium δ15N as tools for identifying N sources to rivers. Moreover, we demonstrate the need for the management of N sources and the protection of stream function to control downstream transfer of N from landscapes to waterbodies.

Crayfish tissue metabolomes effectively distinguish impacts of wastewater and agriculture in aquatic ecosystems.

Environmental metabolomics has been proposed as a tool for biomonitoring because organisms regulate production or consumption of metabolites in response to environmental conditions. We evaluated the efficacy of the metabolome of three tissues (hepatopancreas, gill, and tail muscle) from the northern crayfish (Faxonius virilis) to detect and differentiate between impacts of human activities (i.e., reference, municipal wastewater, and agriculture). We conducted a reciprocal transfer study exposing crayfish for 1 or 2 weeks in three streams with different amounts and types of human activities in southern Manitoba, Canada. Tissue samples were analyzed using nuclear magnetic resonance spectroscopy to generate a metabolic profile. Findings indicated the gill tissue metabolome best detected and differentiated between human activities. In particular, the gill metabolome was able to rapidly integrate abrupt changes in environmental conditions associated with municipal wastewater activity. In contrast, the tail metabolome best differentiated between crayfish collected at the reference site from those collected at the two impacted sites. Metabolites extracted from hepatopancreas tissue showed limited and inconsistent detection of among site differences. Based on our findings, we conclude that the metabolome of the northern crayfish can be an effective biomonitoring tool, but monitoring purpose will dictate tissue selection. Indeed, we recommend the gill metabolome be used for short-term assays aimed at detecting acute effects, whereas the tail be applied for survey monitoring aimed at detecting deviations in ecological condition at test sites from reference site conditions.

Ecological causal assessment of benthic condition in the oil sands region, Athabasca River, Canada.

Contaminant loads to rivers of the Canadian oil sands region are linked to industrial and natural sources. To date, biomonitoring studies have been unable to unequivocally assess potential environmental impacts associated with this development. As part of the Joint Alberta-Canada Oil Sands Monitoring initiative, we aimed to assess cumulative effects of anthropogenic activities and exposure to natural bitumen geology on benthic macroinvertebrate assemblages in the lower Athabasca River. We examined associations among macroinvertebrates and environmental correlates, such as nutrients, ions, metals, polycyclic aromatic compounds, and total suspended solids. The study design included sites within and outside the mineable bitumen deposits, within and outside of the active mining and extraction area, and above and below municipal sewage effluents. We predicted observing a negative association between ecological condition of the river and exposure to natural bitumen and oil sands activity. However, contaminant concentrations in water and sediment were far below known toxicity thresholds, and benthic macroinvertebrate assemblages in sites exposed to oil sands mining activities appeared more affected by nutrient enrichment from the MSE than contaminants from mining or natural bitumen. Although sites within the area of intense oil sands activity showed signs of mild environmental stress, assemblage pattern was more strongly associated with MSE nutrient enrichment than to diffuse contamination from either natural bitumen or oil sands mining. Enrichment likely increases food resources available to consumers, thereby potentially masking toxic responses of consumers to contaminants. Current regulations prohibit the direct release of oil sands contaminants to waterways, with diffuse atmospheric deposition of aerial emissions and fugitive dust the main contaminant pathways to freshwaters. As the storage capacity of tailings ponds is reached, this nutrient-contaminant pattern could change if the river receives the proposed direct release of treated oil sands process water. Focused investigation-of-cause studies are required to better assess the consequences of cumulative interactions and ecological effects of nutrients and contaminant exposure in this system.

Fate of bioavailable nutrients released to a stream during episodic effluent releases from a municipal wastewater treatment lagoon.

Municipal wastewater lagoons are common across North America and, unlike larger mechanical wastewater treatment plants, typically release nutrient-rich effluent directly to rivers in intermittent pulses. However, little is known about the fate of nutrients from these episodic events, which may happen under varying hydrologic or thermal conditions. We assessed fate of nitrogen (N) and phosphorus (P) from lagoon effluent during three releases to Deadhorse Creek, Manitoba, Canada. Using net nutrient uptake lengths and natural abundance stable isotope ratios of dissolved inorganic nitrogen (DIN) and primary producers, we found that DIN was processed during the summer releases though the dominant mechanism was unclear. However, nitrate was largely exported in autumn. Primary producers assimilated lagoon N but did not appear to reduce DIN concentrations. The longitudinal pattern of soluble reactive phosphorus (SRP) varied between releases and in summer 2019 the stream became a net source of SRP despite concomitant processing of DIN. We hypothesize that low demand for P in Deadhorse Creek, as suggested by upstream SRP > 0.05 mg P L-1, and nutrient ratios indicative of N limitation, reduced instream processing of P. Furthermore, our results indicated that cool or high flow conditions may result in the export of much of the lagoon nutrient load downstream. Our findings suggest the processes that transform wastewater nutrients are overwhelmed during effluent releases. Managers should consider increasing effluent dilution via continuous release of effluent rather than pulsed delivery. However, management of upstream nutrient supply may also be needed when relying upon the self-purifying capacity of rivers.

Persistence of the antimicrobials lincomycin, chlortetracycline, and sulfamethazine in prairie wetlands.

Antimicrobials used in livestock production can be present in manure via excretion in the feces and/or urine. Application of raw or processed (composted or stockpiled) manure to crop and pasture land as a plant nutrient source can result in antimicrobial transport to surface waters via rainfall or snowmelt runoff. Little is known regarding antimicrobial persistence in aquatic ecosystems. Consequently, dissipation of environmentally relevant concentrations of three veterinary antimicrobials (lincomycin, chlortetracycline, and sulfamethazine) was studied in three wetlands on the Canadian Prairies. Study wetlands were fortified in the fall to simulate antimicrobial transport via rainfall runoff from fall manure applications to the wetland catchments. After fortification, water column concentrations of all three antimicrobials decreased through September and October. Plotting natural logarithm values of antimicrobial concentration against time resulted in linear relationships for all three antimicrobials, indicating that the summation of all dissipation processes for each antimicrobial could be described by first-order kinetics. The slopes of the three plots were significantly different, indicating that the order of dissipation was lincomycin < sulfamethazine < chlortetracycline. Consequently, the dissipation DT50 (time required for 50% antimicrobial dissipation) values for lincomycin (14.0 d), sulfamethazine (7.0 d), and chlortetracycline (3.3 d) were significantly different. The longer DT50 values of lincomycin and sulfamethazine suggest that environmentally relevant concentrations of these antimicrobials may affect bacterial production in prairie wetlands.

Developing metabolomics-based bioassessment: crayfish metabolome sensitivity to food and dissolved oxygen stress.

There is a need to develop bioassessment tools that can diagnose the effects of individual stressors that can have multiple ecological effects. Using nuclear magnetic resonance (NMR)-based metabolomics, our experiments aimed to identify the sensitivity of metabolites to changes in food availability and dissolved oxygen (DO) concentrations, and compare these results to identify metabolites that may differentiate between the effects of these two stressors. Forty-eight, laboratory-raised, red swamp crayfish (Procambarus clarkii) were randomly assigned and exposed to one of three food availability or DO treatment levels (high, normal, low). Starved crayfish had lower amounts of amino acids than fed crayfish, suggesting catabolic effects of starvation on tail muscle tissue for energy requirements. In contrast, crayfish exposed to hypoxic conditions experienced changes in abundance of metabolites primarily associated with energy metabolism. Tail muscle was the only tissue sensitive to food and DO stress, suggesting the need to select tissues for monitoring appropriately. Our evaluation of environmental metabolomics as a tool for bioassessment indicates that several identified metabolites in crayfish tail muscle may be able to diagnose food and oxygen stress. Further study is required to determine if these metabolic effects are linked with changes of individual fitness and higher levels of biological organization, such as population size.

Effects of pulp mill effluent on benthic assemblages in mesocosms along the Saint John River, Canada.

We used mesocosms to examine the impact of different concentrations of pulp mill effluent (PME) on structural and functional endpoints of a benthic assemblage in the Saint John River (NB, Canada) during 1999 and 2000. Previous studies on this effluent's effects produced conflicting results, with field surveys suggesting a pattern of mild nutrient enrichment, while laboratory toxicity tests linked effluent exposure to moderate contaminant effects. Experimental treatments included three concentrations of sulfite pulp mill effluent (0, 5, 10% v/v PME). Endpoints for the assessment included algal biomass and taxonomic composition, benthic invertebrate abundance and composition, and insect emergence. Low concentrations of PME increased periphyton biomass and caused changes in community structure within the diatom-dominated community. Pulp mill effluent addition had little effect on several structural endpoints measured for benthic invertebrates, including abundance and taxonomic richness, but significantly changed community composition. For both periphyton and benthic invertebrates, community composition endpoints were more sensitive indicators of PME exposure. Insect emergence was a highly relevant functional endpoint. When benthic and emerged insects were combined, total abundance increased with PME addition. Results from two trophic levels, which provided multiple lines of evidence, indicated that the main impact of these PME concentrations is nutrient enrichment rather than effluent toxicity. Our findings also suggest that benthic invertebrate and periphyton assemblages, algal biomass production, and insect emergence are sensitive response measures. Future studies may confirm this observation. The consideration of both functional and structural endpoints at different trophic levels can greatly improve our understanding the effects of discharges to rivers. Such an understanding could not have been obtained using standard assessment techniques and illustrates the value of mesocosms and the benthic community assemblage approach in environmental assessment.

Toward a knowledge infrastructure for traits-based ecological risk assessment.

The trait approach has already indicated significant potential as a tool in understanding natural variation among species in sensitivity to contaminants in the process of ecological risk assessment. However, to realize its full potential, a defined nomenclature for traits is urgently required, and significant effort is required to populate databases of species-trait relationships. Recently, there have been significant advances in the area of information management and discovery in the area of the semantic web. Combined with continuing progress in biological trait knowledge, these suggest that the time is right for a reevaluation of how trait information from divergent research traditions is collated and made available for end users in the field of environmental management. Although there has already been a great deal of work on traits, the information is scattered throughout databases, literature, and undiscovered sources. Further progress will require better leverage of this existing data and research to fill in the gaps. We review and discuss a number of technical and social challenges to bringing together existing information and moving toward a new, collaborative approach. Finally, we outline a path toward enhanced knowledge discovery within the traits domain space, showing that, by linking knowledge management infrastructure, semantic metadata (trait ontologies), and Web 2.0 and 3.0 technologies, we can begin to construct a dedicated platform for TERA science.