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.

Periphyton Phosphorus Uptake in Response to Dynamic Concentrations in Streams: Assimilation and Changes to Intracellular Speciation.

Effective modeling and management of phosphorus (P) losses from landscapes to receiving waterbodies requires an adequate understanding of P retention and remobilization along the terrestrial-aquatic continuum. Within aquatic ecosystems, the stream periphyton can transiently store bioavailable P through uptake and incorporation into biomass during subscouring and baseflow conditions. However, the capacity of stream periphyton to respond to dynamic P concentrations, which are ubiquitous in streams, is largely unknown. Our study used artificial streams to impose short periods (48 h) of high SRP concentration on stream periphyton acclimated to P scarcity. We examined periphyton P content and speciation through nuclear magnetic resonance spectroscopy to elucidate the intracellular storage and transformation of P taken up across a gradient of transiently elevated SRP availabilities. Our study demonstrates that the stream periphyton not only takes up significant quantities of P following a 48-h high P pulse but also sustains supplemental growth over extended periods of time (10 days), following the reestablishment of P scarcity by efficiently assimilating P stored as polyphosphates into functional biomass (i.e., phospho-monoesters and phospho-diesters). Although P uptake and intracellular storage approached an upper limit across the experimentally imposed SRP pulse gradient, our findings demonstrate the previously underappreciated extent to which the periphyton can modulate the timing and magnitude of P delivery from streams. Further elucidating these intricacies in the transient storage potential of periphyton highlights opportunities to enhance the predictive capacity of watershed nutrient models and potentially improve watershed P management.

Fish and macroinvertebrate assemblages reveal extensive degradation of the world's rivers.

Rivers suffer from multiple stressors acting simultaneously on their biota, but the consequences are poorly quantified at the global scale. We evaluated the biological condition of rivers globally, including the largest proportion of countries from the Global South published to date. We gathered macroinvertebrate- and fish-based assessments from 72,275 and 37,676 sites, respectively, from 64 study regions across six continents and 45 nations. Because assessments were based on differing methods, different systems were consolidated into a 3-class system: Good, Impaired, or Severely Impaired, following common guidelines. The proportion of sites in each class by study area was calculated and each region was assigned a Köppen-Geiger climate type, Human Footprint score (addressing landscape alterations), Human Development Index (HDI) score (addressing social welfare), % rivers with good ambient water quality, % protected freshwater key biodiversity areas; and % of forest area net change rate. We found that 50% of macroinvertebrate sites and 42% of fish sites were in Good condition, whereas 21% and 29% were Severely Impaired, respectively. The poorest biological conditions occurred in Arid and Equatorial climates and the best conditions occurred in Snow climates. Severely Impaired conditions were associated (Pearson correlation coefficient) with higher HDI scores, poorer physico-chemical water quality, and lower proportions of protected freshwater areas. Good biological conditions were associated with good water quality and increased forested areas. It is essential to implement statutory bioassessment programs in Asian, African, and South American countries, and continue them in Oceania, Europe, and North America. There is a need to invest in assessments based on fish, as there is less information globally and fish were strong indicators of degradation. Our study highlights a need to increase the extent and number of protected river catchments, preserve and restore natural forested areas in the catchments, treat wastewater discharges, and improve river connectivity.

Impaired cellulose decomposition in a headwater stream receiving subsurface agricultural drainage.

Background: Agricultural development of former wetlands has resulted in many headwater streams being sourced by subsurface agricultural drainage systems. Subsurface drainage inputs can significantly influence stream environmental conditions, such as temperature, hydrology, and water chemistry, that drive ecological function. However, ecological assessments of subsurface drainage impacts are rare. We assessed the impact of an agricultural drainage system on cellulose decomposition and benthic respiration using a paired stream study in a headwater branch of Nissouri Creek, in Ontario, Canada. Adjacent first order segments sourced by a spring-fed marsh and a cropped field with subsurface drainage, as well as the adjoining trunk segment, were sampled over a year using the cotton strip assay to measure cellulose decomposition and benthic respiration. Results: Assessments of cellulose decomposition revealed a one-third reduction in the drainage-sourced segment compared to marsh-sourced segment. Between segment differences in cellulose decomposition were associated with reduced summer temperatures in the drainage-sourced segment. Impacts of stream cooling from the drainage-sourced segment were transmitted downstream as cellulose decomposition was slower than expected throughout the drainage-sourced segment and for several hundred meters down the adjoining trunk segment. Benthic respiration only differed between the drainage- and marsh-sourced segments in spring, when stream temperatures were similar. Conclusions: Our findings suggest there may be a widespread reduction in cellulose decomposition in streams across similar agricultural regions where subsurface drainage is prevalent. However, cooling of streams receiving significant amounts of water inputs from subsurface drainage systems may impart increased resiliency to future climate warming.

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.

The Biological Assessment and Rehabilitation of the World's Rivers: An Overview.

The biological assessment of rivers i.e., their assessment through use of aquatic assemblages, integrates the effects of multiple-stressors on these systems over time and is essential to evaluate ecosystem condition and establish recovery measures. It has been undertaken in many countries since the 1990s, but not globally. And where national or multi-national monitoring networks have gathered large amounts of data, the poor water body classifications have not necessarily resulted in the rehabilitation of rivers. Thus, here we aimed to identify major gaps in the biological assessment and rehabilitation of rivers worldwide by focusing on the best examples in Asia, Europe, Oceania, and North, Central, and South America. Our study showed that it is not possible so far to draw a world map of the ecological quality of rivers. Biological assessment of rivers and streams is only implemented officially nation-wide and regularly in the European Union, Japan, Republic of Korea, South Africa, and the USA. In Australia, Canada, China, New Zealand, and Singapore it has been implemented officially at the state/province level (in some cases using common protocols) or in major catchments or even only once at the national level to define reference conditions (Australia). In other cases, biological monitoring is driven by a specific problem, impact assessments, water licenses, or the need to rehabilitate a river or a river section (as in Brazil, South Korea, China, Canada, Japan, Australia). In some countries monitoring programs have only been explored by research teams mostly at the catchment or local level (e.g., Brazil, Mexico, Chile, China, India, Malaysia, Thailand, Vietnam) or implemented by citizen science groups (e.g., Southern Africa, Gambia, East Africa, Australia, Brazil, Canada). The existing large-extent assessments show a striking loss of biodiversity in the last 2-3 decades in Japanese and New Zealand rivers (e.g., 42% and 70% of fish species threatened or endangered, respectively). A poor condition (below Good condition) exists in 25% of South Korean rivers, half of the European water bodies, and 44% of USA rivers, while in Australia 30% of the reaches sampled were significantly impaired in 2006. Regarding river rehabilitation, the greatest implementation has occurred in North America, Australia, Northern Europe, Japan, Singapore, and the Republic of Korea. Most rehabilitation measures have been related to improving water quality and river connectivity for fish or the improvement of riparian vegetation. The limited extent of most rehabilitation measures (i.e., not considering the entire catchment) often constrains the improvement of biological condition. Yet, many rehabilitation projects also lack pre-and/or post-monitoring of ecological condition, which prevents assessing the success and shortcomings of the recovery measures. Economic constraints are the most cited limitation for implementing monitoring programs and rehabilitation actions, followed by technical limitations, limited knowledge of the fauna and flora and their life-history traits (especially in Africa, South America and Mexico), and poor awareness by decision-makers. On the other hand, citizen involvement is recognized as key to the success and sustainability of rehabilitation projects. Thus, establishing rehabilitation needs, defining clear goals, tracking progress towards achieving them, and involving local populations and stakeholders are key recommendations for rehabilitation projects (Table 1). Large-extent and long-term monitoring programs are also essential to provide a realistic overview of the condition of rivers worldwide. Soon, the use of DNA biological samples and eDNA to investigate aquatic diversity could contribute to reducing costs and thus increase monitoring efforts and a more complete assessment of biodiversity. Finally, we propose developing transcontinental teams to elaborate and improve technical guidelines for implementing biological monitoring programs and river rehabilitation and establishing common financial and technical frameworks for managing international catchments. We also recommend providing such expert teams through the United Nations Environment Program to aid the extension of biomonitoring, bioassessment, and river rehabilitation knowledge globally.

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.

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.

Episodic loadings of phosphorus influence growth and composition of benthic algae communities in artificial stream mesocosms.

Phosphorus (P) is an essential macronutrient for algal communities, but in excess can exacerbate stream eutrophication. However, P loadings to streams vary temporally from continuous to episodic as a result of inputs from point and non-point sources, respectively. P loading pattern can thus alter the temporal availability of P and may influence effects of P enrichment on algal communities. We assessed how P loading pattern influences algal biomass and composition by conducting a 29-day P enrichment experiment in nine artificial streams exposed to either: (1) continuous P enrichment; (2) episodic P enrichment, or; (3) no P enrichment. P enrichment increased algal biomass accrual, but peak biomass did not differ between continuously and episodically enriched treatments. Maximum absolute growth rates were also comparable between P enriched treatments. However, episodic P additions sustained elevated rates of biomass accrual, whereas absolute growth rates in the continuously enriched communities declined towards the end of the experiment. P enrichment resulted in comparable increases in relative abundance of chlorophytes and decreased proportions of bacillariophytes and charophytes in algal communities for continuously and episodically enriched treatments. However, composition of bacillariophyte (diatom) assemblages differed significantly among all P enrichment treatments in accordance with species autecological attributes for P. Our results demonstrate that episodic and continuous P enrichment may augment algal biomass similarly. Yet, P loading pattern regulated the composition of algal communities. Thus, remedial management strategies for the control of nuisance algae production may require focus on the predominant source of P to streams. Finally, species specific responses of diatom assemblages to P enrichment and associated loading patterns suggests this taxonomic group may have potential as diagnostic indicators for identifying the presence of key nutrient sources associated with eutrophication of stream ecosystems.

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.

Intra-annual variation of the association between agricultural best management practices and stream nutrient concentrations.

Temporal variation may influence the ability of best management practices (BMPs) to mitigate the loss of agricultural pollutants to streams. Our goal was to assess variation in mitigation effects of BMPs by examining the associations between instream nutrient concentrations and the abundance and location of four structural BMPs over a hydrologic year. Water samples were collected monthly (Nov. 2013-Oct. 2014) in 15 headwater streams representing a gradient of BMP use in Southern Ontario, Canada. Partial least squares (PLS) regression models were used to associate two groups of collinear nutrient forms with the abundance and location of BMPs, antecedent precipitation and time of year. BMP metrics in PLS models were associated with instream concentrations of major phosphorus forms and ammonium throughout the year. In contrast, total nitrogen and nitrate-nitrite were only associated with BMPs during snowmelt. BMP metrics associated with reductions of phosphorus and ammonium included greater abundances of riparian buffers and manure storage structures, but not livestock restriction fences. Likewise, the abundance and location riparian vegetation in areas capturing more surface runoff were associated with decreased stream nitrogen concentrations during snowmelt. However, the amount of tile drainage was associated with increased nitrogen concentrations following snowmelt, as well as with greater phosphorus and ammonium concentrations throughout the year. Overall, our findings indicate that increasing the abundance of riparian buffers and manure storage structures may decrease instream nutrient concentrations in agricultural areas. Additionally, the implementation of these structural BMPs appear to be an effective year-round strategy to assist management objectives in reducing phosphorus concentrations in small agricultural streams and thus loadings to downstream tributaries. Further mitigation measures, such as managerial BMPs and controlled tile drainage, may be required to further reduce instream nutrient concentrations during baseflow periods and snowmelt events.

Effects of Best Management Practice on Ecological Condition: Does Location Matter?

Best management practices (BMPs) are increasingly being promoted as a solution to the potentially adverse effects agriculture can have on aquatic systems. However, the ability of BMPs to improve riverine systems continues to be questioned due to equivocal empirical evidence linking BMP use with improved stream conditions, particularly in regard to ecological conditions. Explicitly viewing BMP location in relation to hydrological pathways may, however, assist in establishing stronger ecological linkages. The goal of this study was to assess the association between water chemistry, benthic macroinvertebrate community structure, and the number and location of agricultural BMPs in a catchment. Macroinvertebrate and water samples were collected in 30 small (<12 km(2)) catchments exhibiting gradients of BMP use and location in the Grand River Watershed, Southern Ontario, Canada. Stepwise regression analysis revealed that concentrations of most stream nutrients declined in association with greater numbers of BMPs and particularly when BMPs were located in hydrologically connected areas. However, BMPs were significantly associated with only one metric (%EPT) describing macroinvertebrate community structure. Furthermore, variance partitioning analysis indicated that less than 5% of the among site variation in the macroinvertebrate community could be attributed to BMPs. Overall, the implemented BMPs appear to be achieving water quality improvement goals but spatial targeting of specific BMP types may allow management agencies to attain further water quality improvements more efficiently. Mitigation and rehabilitation measures beyond the BMPs assessed in this study may be required to meet goals of enhanced ecological condition.

Incorporating traits in aquatic biomonitoring to enhance causal diagnosis and prediction.

The linkage of trait responses to stressor gradients has potential to expand biomonitoring approaches beyond traditional taxonomically based assessments that identify ecological effect to provide a causal diagnosis. Traits-based information may have several advantages over taxonomically based methods. These include providing mechanistic linkages of biotic responses to environmental conditions, consistent descriptors or metrics across broad spatial scales, more seasonal stability compared with taxonomic measures, and seamless integration of traits-based analysis into assessment programs. A traits-based biomonitoring approach does not require a new biomonitoring framework, because contemporary biomonitoring programs gather the basic site-by-species composition matrices required to link community data to the traits database. Impediments to the adoption of traits-based biomonitoring relate to the availability, consistency, and applicability of existing trait data. For example, traits generalizations among taxa across biogeographical regions are rare, and no consensus exists relative to the required taxonomic resolution and methodology for traits assessment. Similarly, we must determine if traits form suites that are related to particular stressor effects, and whether significant variation of traits occurs among allopatric populations. Finally, to realize the potential of traits-based approaches in biomonitoring, a concerted effort to standardize terminology is required, along with the establishment of protocols to ease the sharing and merging of broad, geographical trait information.

Snowmelt and its role in the hydrologic and nutrient budgets of prairie streams.

Small watersheds in the Canadian Prairies are characterized by seasonally disconnected hydrologic networks whereby stream channels are hydrologically connected during snowmelt but have disconnected reaches throughout the remainder of the year. Snowmelt is the most significant hydrological event in the Canadian Prairies, yet few studies have investigated the role of snowmelt in the nutrient budget of prairie streams. We quantified hydrologic and nutrient dynamics during snowmelt for ten agricultural subwatersheds distributed along a gradient of human activity in the Red River Valley, Canada, to evaluate the timing of nitrogen (N) and phosphorus (P) export. Elevated concentrations of total P (TP) and total N (TN) were observed during the snowmelt peak, with maximum concentrations reaching 3.23 mg TP L(-1) and 18.50 mg TN L(-1). Dissolved P and N dominated the total nutrient pool throughout snowmelt, likely due to reduced erosion and sediment transport resulting from the combination of the flat topography, frozen soil and stream banks, and gradual snow cover melt. Significant correlations were observed between snowmelt N load (r=0.91; p<0.05) and both agricultural land cover and fertilizer usage, with a weaker correlation between snowmelt P load (r=0.81; p<0.05) and agricultural area. Our results showed that snowmelt plays a key role in nutrient export to prairie aquatic ecosystems and this may have serious impacts on downstream ecosystems. Land use management practices need to consider the snowmelt period to control nutrient loads to Lake Winnipeg and other waterbodies in the Great Plains.

The stream and its altered valley: integrating landscape ecology into environmental assessments of agro-ecosystems.

Little is known about the importance of landscape and land cover to the implementation and performance of agricultural conservation projects designed to improve stream quality. In our study, we addressed the potential importance of landscape and land cover to conservation projects by measuring variation across 191 mu-basins (100-2400 ha) and integrating the observed variation into a study design aimed at determining the effectiveness of conservation projects. Our findings indicate that there are strong gradients across which landscape and land cover attributes vary. Land cover varied along a gradient of agricultural intensity, basin morphometry across gradients of stream closure and basin size, basin substrate was described by variation in drumlin formation, glacial landform type, and soil drainage, while agricultural conservation projects varied according to the level of project implementation. Correlation of these gradients found several associations between landscape and land cover, indicating that agricultural intensity was being constrained predominantly by drumlin formation and glacial landform type. Landscape and land cover did not appear to be determining factors in the implementation of conservation projects by land owners. Based on these findings we chose 32 mu-basins which represented the variability along each of the defined gradients for further study. We conclude that landscape scale variables demonstrate important variation and covariation that can and should be integrated into study designs for the assessment of streams and human activities affecting streams.