Wastewater reuse and predicted ecological risk posed by contaminant mixtures in Potomac River watershed streams

A wastewater model was applied to the Potomac River watershed to provide (i) a means to identify streams with a high likelihood of carrying elevated effluent-derived contaminants and (ii) risk assessments to aquatic life and drinking water. The model linked effluent discharges along stream networks, accumulated wastewater, and predicted contaminant loads of municipal wastewater constituents while accounting for instream dilution and attenuation. Simulations using 2016 data suggested that nearly 30% (8281 km) of streams were wastewater impacted. Low- to medium-order streams had the largest range of accumulated wastewater (ACCWW%) values. ACCWW% exceeded a 1% threshold at >39% of drinking-water intakes (varied by temporal condition). Risk assessments of municipal wastewater-contaminant mixtures indicated that 22% (1479 km) of streams impacted by municipal wastewater (5.5% of all reaches modeled) may pose high risk to aquatic organisms under mean-annual conditions, with fish more susceptible to chronic-exposure effects relative to other taxa. Risk varied temporally and by stream order, with the greatest risk occurring in the summer in small streams. These findings suggest that wastewater may be an important factor contributing to environmental degradation in the Potomac River watershed.     

Macroinvertebrate biomonitoring in intermittent coastal plain streams impacted by animal agriculture

Little attention has been given to the ecology of intermittent coastal plain streams in the southeastern United States, and it is not known whether available macroinvertebrate biomonitoring methods reliably detect degradation in these streams. This study compared differences in biomonitoring metrics between reference and agricultural streams, and between the flow period (January-April) and the intermittent flow period (May-December). Percentages of crustaceans, isopods, and Ephemeroptera-Plecoptera-Trichoptera (EPT) were significantly higher at the reference site than the two most impacted sites during the flow period, probably resulting from the abundance of leaf litter and lower temperatures. During this same period, the agriculturally impacted sites had a significantly higher percentage of dipterans--a group that thrives in the silty, nutrient-rich waters. Four metrics (percent Crustacea, Isopoda, Diptera, and EPT) had no overlap between values for the most impacted and the least impacted sites during the flow period, but no metrics were able to detect more discrete differences among sites. Sites were physically and biologically similar during the intermittent period when natural stresses (i.e., stagnant water, high temperatures, low dissolved oxygen) were high, with many metrics, such as percentages of dominant family, burrowers, chironomids, and dipterans becoming similar at all sites. Our findings indicate that development of a better understanding of invertebrate fauna in reference conditions and of the natural variation in intermittent streams is necessary to develop effective biomonitoring programs for these systems.     

DEVELOPMENT OF AN INVERTEBRATE COMMUNITY INDEX FOR AN ALABAMA COASTAL PLAIN WATERSHED1

ABSTRACT: Activities such as agriculture, silviculture, and mining contribute nonpoint pollution to Alabama's streams through polluted runoff and excessive sedimentation. Highly erodible soils characteristic of the Choctawhatchee‐Pea Rivers watershed, combined with intense rainfall and land use practices, contribute large amounts of sediment to streams. Biological monitoring can reflect the acute impacts of pollutants as well as prolonged effects of habitat alteration, and development of biological criteria is important for the establishment of enforceable laws regarding nonpoint source pollution. Macroinvertebrates were collected from 49 randomly selected sites from first through sixth‐order streams in the Choctawhatchee‐Pea Rivers watershed and were identified to genus level. Thirty‐eight candidate metrics were examined, and an invertebrate community index (ICI) was calibrated by eliminating metrics that failed to separate impaired from unimpaired streams. Each site was scored with those metrics, and narrative scores were assigned based on ICI scores. Least impacted sites scored significantly lower than sites impacted by row crop agriculture, cattle, and urban land uses. Conditions in the watershed suggest that the entire area has experienced degradation through past and current land use practices. An initial validation of the index was performed and is described. Additional evaluations of the index are in progress.     

Development of an Index of Biotic Integrity for a Southeastern Coastal Plain Watershed,USA1

Abstract: This study evaluated biological integrity expectations of fish assemblages in wadeable streams for the Alabama portion of the Choctawhatchee River watershed using a multimetric approach. Thirty‐four randomly selected stream sites were sampled in late spring 2001 to calibrate an index of biotic integrity (IBI). Validation data were collected during the spring 2001, and summer and fall of 2003 from disturbed and least‐impacted targeted sites (n = 20). Thirty‐five candidate metrics were evaluated for their responsiveness to environmental degradation. Twelve metrics were selected to evaluate wadeable streams and four replacement metrics were selected for headwater streams. Scores that ranged from 58 to 60 were considered to be representative of excellent biotic integrity (none found in this study), scores of 48‐52 as good integrity (31% of the sites in this study), 40‐44 as fair (43%), 28‐34 as poor (21%), and 12‐22 as very poor (5%). Of the four stream condition categories (urban, cattle, row crop, and least impacted), the IBI scores for urban and cattle sites differed significantly from least‐impacted sites. Row crop sites, although not significantly different from least‐impacted, tended to have greater variability than the other categories. Lower IBI scores at both urban and cattle sites suggest that the IBI accurately reflects stream impairment in the Choctawhatchee River drainage.     

Headwater Influences on Downstream Water Quality

We investigated the influence of riparian and whole watershed land use as a function of stream size on surface water chemistry and assessed regional variation in these relationships. Sixty-eight watersheds in four level III U.S. EPA ecoregions in eastern Kansas were selected as study sites. Riparian land cover and watershed land use were quantified for the entire watershed, and by Strahler order. Multiple regression analyses using riparian land cover classifications as independent variables explained among-site variation in water chemistry parameters, particularly total nitrogen (41%), nitrate (61%), and total phosphorus (63%) concentrations. Whole watershed land use explained slightly less variance, but riparian and whole watershed land use were so tightly correlated that it was difficult to separate their effects. Water chemistry parameters sampled in downstream reaches were most closely correlated with riparian land cover adjacent to the smallest (first-order) streams of watersheds or land use in the entire watershed, with riparian zones immediately upstream of sampling sites offering less explanatory power as stream size increased. Interestingly, headwater effects were evident even at times when these small streams were unlikely to be flowing. Relationships were similar among ecoregions, indicating that land use characteristics were most responsible for water quality variation among watersheds. These findings suggest that nonpoint pollution control strategies should consider the influence of small upland streams and protection of downstream riparian zones alone is not sufficient to protect water quality.     

Phosphorus transport pathways to streams in tile-drained agricultural watersheds

Agriculture is a major nonpoint source of phosphorus (P) in the Midwest, but how surface runoff and tile drainage interact to affect temporal concentrations and fluxes of both dissolved and particulate P remains unclear. Our objective was to determine the dominant form of P in streams (dissolved or particulate) and identify the mode of transport of this P from fields to streams in tile-drained agricultural watersheds. We measured dissolved reactive P (DRP) and total P (TP) concentrations and loads in stream and tile water in the upper reaches of three watersheds in east-central Illinois (Embarras River, Lake Fork of the Kaskaskia River, and Big Ditch of the Sangamon River). For all 16 water year by watershed combinations examined, annual flow-weighted mean TP concentrations were >0.1 mg L(-1), and seven water year by watershed combinations exceeded 0.2 mg L(-1). Concentrations of DRP and particulate P (PP) increased with stream discharge; however, particulate P was the dominant form during overland runoff events, which greatly affected annual TP loads. Concentrations of DRP and PP in tiles increased with discharge, indicating tiles were a source of P to streams. Across watersheds, the greatest DRP concentrations (as high as 1.25 mg L(-1)) were associated with a precipitation event that followed widespread application of P fertilizer on frozen soils. Although eliminating this practice would reduce the potential for overland runoff of P, soil erosion and tile drainage would continue to be important transport pathways of P to streams in east-central Illinois.     

Regional reference sites: a method for assessing stream potentials

Field assessments of impacted streams require a control or at least an unbiased estimate of attainable conditions. Control sites, such as upstream/downstream or wilderness sites, have proven inadequate for assessing attainable ecological conditions where the control streams differ naturally from the impacted streams to a considerable degree or where different disturbances exist than those being studied. Relatively undisturbed reference sites with watersheds in areas having the same land-surface form, soil, potential natural vegetation, and land use as are predominant in large, relatively homogeneous regions are suggested as alternative control sites. These areas are considered typical of the region and therefore the sites also are considered typical of the region because their watersheds exhibit all the terrestrial variables that make that region a region. The logical basis for developing regional reference sites lies in the ability to group watersheds and common stream types into regions by integrating available maps of terrestrial variables that influence streams. Relatively undisturbed reference sites can be selected from typical areas of the regions and from transition zones where one or two of the terrestrial variables are not the predominant one(s) of the region. These reference sites are useful for estimating attainable conditions, for evaluating temporal and spatial changes in ecological integrity, for classifying attainable uses of streams, and for setting biological and environmental criteria.     

Assessment of chlorophyll-a as a criterion for establishing nutrient standards in the streams and rivers of Illinois

Nutrient enrichment is a frequently cited cause for biotic impairment of streams and rivers in the USA. Efforts are underway to develop nutrient standards in many states, but defensible nutrient standards require an empirical relationship between nitrogen (N) or phosphorus (P) concentrations and some criterion that relates nutrient levels to the attainment of designated uses. Algal biomass, measured as chlorophyll-a (chl-a), is a commonly proposed criterion, yet nutrient-chl-a relationships have not been well documented in Illinois at a state-wide scale. We used state-wide surveys of >100 stream and river sites to assess the applicability of chl-a as a criterion for establishing nutrient standards for Illinois. Among all sites, the median total P and total N concentrations were 0.185 and 5.6 mg L(-1), respectively, during high-discharge conditions. During low-discharge conditions, median total P concentration was 0.168 mg L(-1), with 25% of sites having a total P of > or =0.326 mg L(-1). Across the state, 90% of the sites had sestonic chl-a values of < or =35 microg L(-1), and watershed area was the best predictor of sestonic chl-a. During low discharge there was a significant correlation between sestonic chl-a and total P for those sites that had canopy cover < or =25% and total P of < or =0.2 mg L(-1). Results suggest sestonic chl-a may be an appropriate criterion for the larger rivers in Illinois but is inappropriate for small rivers and streams. Coarse substrate to support benthic chl-a occurred in <50% of the sites we examined; a study using artificial substrates did not reveal a relationship between chl-a accrual and N or P concentrations. For many streams and rivers in Illinois, nutrients may not be the limiting factor for algal biomass due to the generally high nutrient concentrations and the effects of other factors, such as substrate conditions and turbidity.     

Validation of an Invertebrate Community Index for Urban Streams for an Alabama Coastal Plains Watershed1

Abstract: The purpose of this study was to validate the application of an invertebrate community index (ICI) to assess the biological integrity of urban streams. Validation involved comparing chemical and habitat data to ICI scores from 20 urban streams and four least‐impacted streams in the Choctawhatchee and Pea River watersheds located in Southeast Alabama. Chemical and habitat data were collected to support whether the ICI accurately predicts the health of the streams. A significant difference between urban and least‐impacted ICI scores, habitat evaluation scores, chemical variables, taxa richness, and Shannon‐Wiener diversity were observed when urban sites were compared with least‐impacted sites using Mann‐Whitney U‐test. Urban sites having low ICI scores, low species richness and diversity, and poor habitat showed greater impairment than least‐impacted sites. Cluster analysis of macroinvertebrate assemblages indicated two clusters. Significant differences between clusters in habitat evaluations, chemical parameters, and ICI scores showed that some urban sites were more degraded than other urban sites in the study. Differentiation between least‐impacted and urban sites indicated that the ICI provided valid biotic assessments. Therefore, this study validated that the ICI is capable of predicting the biological integrity of urban streams in the Choctawhatchee and Pea River watersheds.     

A morphological comparison of narrow,low-gradient streams traversing wetland environments to alluvial streams

Twelve morphological features from research on alluvial streams are compared in four narrow, low-gradient wetland streams located in different geographic regions (Connecticut, Indiana, and Wisconsin, USA). All four reaches differed in morphological characteristics in five of the features compared (consistent bend width, bend cross-sectional shape, riffle width compared to pool width, greatest width directly downstream of riffles, and thalweg location), while three reaches differed in two comparisons (mean radius of curvature to width ratio and axial wavelength to width ratio). The remaining five features compared had at least one reach where different characteristics existed. This indicates the possibility of varying morphology for streams traversing wetland areas further supporting the concept that the unique qualities of wetland environments might also influence the controls on fluvial dynamics and the development of streams. If certain morphological features found in streams traversing wetland areas differ from current fluvial principles, then these varying features should be incorporated into future wetland stream design and creation projects. The results warrant further research on other streams traversing wetlands to determine if streams in these environments contain unique morphology and further investigation of the impact of low-energy fluvial processes on morphological development. Possible explanations for the morphology deviations in the study streams and some suggestions for stream design in wetland areas based upon the results and field observations are also presented.     

Landscape Character and Fish Assemblage Structure and Function in Western Lake Superior Streams: General Relationships and Identification of Thresholds

As part of a comparative watershed project investigating land-cover/land-use disturbance gradients for streams in the western Lake Superior Basin, we examined general relationships between landscape character and fish assemblage structure and function. We also examined the shape of those relationships to identify discontinuity thresholds where small changes in landscape character were associated with marked shifts in the fish assemblages. After completing a geographic analysis of second- and third-order watersheds in the western Lake Superior drainage, we randomly selected 48 streams along mature forest and watershed storage gradients in 2 hydrogeomorphic regions as our study sites. During the summers of 1997 and 1998, we used electrofishing to sample fish assemblages from each stream. Each of the landscape factors was significantly associated with fish assemblage structure and function based on analysis of covariance. Watershed storage was related to the greatest number of fish assemblage characteristics, but hydrogeopmorphic region and mature forest cover were strongly associated as well. The hydrogeomorphic region also mediated relationships between watershed character and fish assemblages. Discontinuity thresholds for our fish assemblages averaged 11% for watershed storage and 50% for watershed mature forest cover based on piecewise regression analysis. Although many of the landscape–fish relationships might have been manifest through effects on in-stream habitat, our results highlight the importance of management and land-use planning decisions at the watershed and landscape scales.Published online     

Evaluating the Eco‐Geomorphological Condition of Restored Streams Using Visual Assessment and Macroinvertebrate Metrics

The Stream Performance Assessment (SPA), a new rapid assessment method, was applied to 93 restored, 21 impaired, 29 reference, and 13 reference streams with some incision throughout North Carolina. Principal component analysis (PCA) indicated restored streams align more closely with reference streams rather than impaired streams. Further, PCA‐based factor analysis revealed restored streams were similar to reference streams in terms of morphologic condition, but exhibited a greater range of scores relative to aquatic habitat and bedform. Macroinvertebrate sampling and GIS watershed analyses were conducted on 84 restored streams. SPA and watershed data were compared to Ephemeroptera, Plecoptera, and Trichoptera (EPT) taxa to determine which factors indicate stream health. SPA and watershed factors were used in least squares, ridge, and principal component regression (PCR) to develop a prediction model for EPT taxa. All three methods produced reasonable predictions for EPT taxa. Cross‐validation indicated ridge regression resulted in the lowest prediction error. The ridge model was then used to predict EPT taxa numbers for 21 impaired and 25 reference streams in addition to the 84 restored streams. Statistical comparisons of the predicted scores indicated urban streams (>10% impervious watershed cover) have lower expected numbers of EPT taxa. Rural restored streams have macroinvertebrate metric scores similar to those predicted for rural reference streams.     

Effects of agricultural runoff on vegetation composition of a priority conservation wetland, Vermont, USA

This study examined the effects of agricultural runoff on the vegetation structure of Franklin Bog, a priority conservation area located in a rapidly developing region of northwestern Vermont. Forested and agricultural runoff from the mixed land use watershed created differential vegetation patterns in the wetland, including weedy species introductions. Concentrations of nitrogen and phosphorus were measured in the stream runoff from four forested subwatersheds and two agricultural subwatersheds. Nutrient concentrations were significantly higher for agricultural vs. forested runoff for all measured parameters. Nitrate and total phosphorus concentrations in agricultural runoff ranged from 0.62 to 1.35 mg L(-1) and 0.07 to 0.37 mg L(-1), respectively. Forested runoff values were less than 0.37 mg L(-1) nitrate and 0.09 mg L(-1) total phosphorus. Significantly higher proportions of weedy species occurred at impacted vs. reference sites (46 +/- 5% vs. 23 +/- 4%). Furthermore, significantly higher total percent vegetated cover occurred at impacted vs. reference sites (116 +/- 11% vs. 77 +/- 9%) suggesting nutrient induced plant growth. Of the nine frequently occurring species categorized as bog species, only one was found within impacted sites while all nine were found at the reference sites. This suggests that the wetland's distinctive native flora is being replaced by widespread, vigorous species enhanced by agricultural nonpoint pollution in the watershed of Franklin Bog. Protection of wetlands requires attention to conservation measures throughout the entire watershed.     

A Quantitative Tool for Assessing the Integrity of Southern Coastal California Streams

We developed a benthic macroinvertebrate index of biological integrity (B-IBI) for the semiarid and populous southern California coastal region. Potential reference sites were screened from a pool of 275 sites, first with quantitative GIS landscape analysis at several spatial scales and then with local condition assessments (in-stream and riparian) that quantified stressors acting on study reaches. We screened 61 candidate metrics for inclusion in the B-IBI based on three criteria: sufficient range for scoring, responsiveness to watershed and reach-scale disturbance gradients, and minimal correlation with other responsive metrics. Final metrics included: percent collector-gatherer + collector-filterer individuals, percent noninsect taxa, percent tolerant taxa, Coleoptera richness, predator richness, percent intolerant individuals, and EPT richness. Three metrics had lower scores in chaparral reference sites than in mountain reference sites and were scored on separate scales in the B-IBI. Metrics were scored and assembled into a composite B-IBI, which was then divided into five roughly equal condition categories. PCA analysis was used to demonstrate that the B-IBI was sensitive to composite stressor gradients; we also confirmed that the B-IBI scores were not correlated with elevation, season, or watershed area. Application of the B-IBI to an independent validation dataset (69 sites) produced results congruent with the development dataset and a separate repeatability study at four sites in the region confirmed that the B-IBI scoring is precise. The SoCal B-IBI is an effective tool with strong performance characteristics and provides a practical means of evaluating biotic condition of streams in southern coastal California.     

Relationships among nutrients, chlorophyll-a, and dissolved oxygen in agricultural streams in Illinois

A better understanding of the controls on algae and dissolved O2 in agricultural streams of Illinois is needed to aid in development of nutrient standards. We investigated the relationships between dissolved nutrients, algal abundance, and dissolved O2 in five streams in east-central Illinois from March through November 2004. The streams drained watersheds from 25 to 777 km2 that were dominated by row crop agriculture. Three sites had open canopies and two were bordered by a narrow forest of deciduous trees. Algal abundance was measured as chlorophyll-a (chl-a) concentration in the water column (sestonic) and on the streambed (periphytic). Mean NO3-N concentrations ranged from 5.5 to 8.8 mg N L(-1) and did not relate to algal abundance. Sestonic chl-a values ranged from nearly zero to >15 mg m(-3) with no differences between open and shaded streams and only a weak correlation with dissolved reactive P (mean concentrations were 44-479 microg L(-1)). The results suggest that sestonic chl-a is a poor criterion for assessing nutrient-related problems in these streams. Greatest periphytic chl-a occurred during low flow from August through October, but periphyton occurred consistently in only two of the five streams. The abundance of filamentous algae explained 64% of the variation in diel O2 saturation, but was not correlated with nutrients. Currently it appears that hydrology and light, rather than nutrients, control algal abundance in these streams, and in the agricultural landscape of east-central Illinois, it may not be possible to reduce nutrient concentrations sufficiently to limit filamentous algal blooms.     

A GIS-based technique for linking landscape characteristics to non-point source nitrogen export potential: implications for contributing areas management

A Nitrate-N Leaching Index (NLI) is calculated and the results indicate that nitrogen loss in the study area occurs through both leaching and surface runoff. A non-linear regression model of trapping efficiency was combined with a first order decay model to examine the impact of soil characteristics, slope, vegetative cover, land use and distance to streams on the spatial pattern of non-point source nitrogen inputs to streams. The model evaluates the statistical significance of each landscape factor and provides an easy interpretation of the landscape delivery ratio of nitrogen based on a pixel-based characterisation of the watershed. The model was validated by comparing the distributions of the observed and estimated monthly nitrogen concentrations. The exploratory GIS-based method presented here can improve the understanding of the impact of landscape characteristics on nitrate-nitrogen contributing areas and therefore assist watershed management efforts.     

Broad-host-range plasmids in treated wastewater effluent and receiving streams

The occurrence of broad-host-range (BHR) plasmid amplicons belonging to incompatibility (Inc) groups IncA/C, IncN, IncP, and IncW in two wastewater treatment plant (WWTP) effluents and effluent-receiving streams in Northwest Arkansas, Mud Creek and Spring Creek, was determined. Community DNA captured on filter membranes and plasmid DNA extracted from antibiotic-resistant Escherichia coli isolated from Mud Creek was used for polymerase chain reaction at amplification of partial gene sequences specific to BHR plasmids. IncP plasmid amplicons were detected in effluent and downstream sites in both streams, while IncN and IncW plasmid amplicons were detected in Spring Creek in effluent and downstream but not upstream. IncA/C plasmid amplicons, in contrast, were detected at all sites, including upstream in most samples in Spring Creek and in one sample from Mud Creek. One IncP and two IncN were the only BHR plasmid amplicons found in 85 screened antibiotic-resistant E. coli isolates, and were detected only in isolates from effluent and downstream samples. Broad-host-range plasmids frequently carry antibiotic-resistance genes and can facilitate horizontal transfer of those genes. While BHR plasmids have been detected in WWTPs, WWTPs do not target these genetic elements for destruction. This study indicates that BHR plasmids are in WWTP effluent and are introducing BHR plasmids into streams. Additionally, species other than E. coli may be better targets as indicator bacteria for future studies of the impact of treated effluent on environmental dissemination of BHR plasmids.     

Integrating contributing areas and indexing phosphorus loss from agricultural watersheds

Most states in the USA have adopted P Indexing to guide P-based management of agricultural fields by identifying the relative risk of P loss at farm and watershed scales. To a large extent, this risk is based on hydrologic principles that frequently occurring storms can initiate surface runoff from fields. Once initiated, this hydrological pathway has a high potential to transport P to the stream. In regions where hydrologically active areas of watersheds vary in time and space, surface runoff generation by "saturation excess" has been linked to distance from stream, with larger events resulting in larger contributing distances. Thus, storm-return period and P loss from a 39.5-ha mixed-land-use watershed in Pennsylvania was evaluated to relate return-period thresholds and distances contributing P to streams. Of 248 storm flows between 1997 and 2006, 93% had a return period of 1 yr, contributing 47% of total P (TP) export, while the largest two storms (10-yr return period) accounted for 23% of TP export. Contributing distance thresholds for the watershed were determined (50-150 m) for a range of storm-return periods (1-10 yr) from hydrograph analysis. By modifying storm-return period thresholds in the P Index and thereby contributing distance, it is possible to account for greater risk of P loss during large storms. For instance, increasing return period threshold from 1 (current P indices) to 5 yr, which accounted for 67% of TP export, increased the P-management restricted area from 20 to 58% of the watershed. An increase in impacted area relative to a decreased risk of P loss creates a management-policy dilemma that cannot be ignored.     

Development of Macroinvertebrate-Based Index for Bioassessment of Idaho Rivers

Theoretical constructs, such as the river continuum concept, predict that the composition of benthic fauna in rivers will be different from that of headwater streams. There exists a need to modify, for use on larger rivers, the bioassessment techniques commonly used on small streams. Using aquatic macroinvertebrates and the “reference condition” approach, we developed and tested a multimetric index for use on the rivers of Idaho. Reference sites were selected to represent the best current conditions (i.e., least impacted) among Idaho rivers. The index performed well in distinguishing reference sites from sites displaying some form of anthropogenic impairment. Individual metrics used in the index included: number of EPT taxa, total number of taxa, percent dominant taxon, percent Elmidae, and percent predators. The index we developed for Idaho rivers was essentially a modification of a framework designed for small streams, suggesting that techniques, including data analysis, currently used for streams can be adapted for use on larger rivers. Adapting these methods for use in rivers is primarily a matter of (1) selecting metrics relevant to the rivers of interest; (2) expanding the field sampling to encompass the greater habitat area and, potentially, heterogeneity of rivers; and (3) selecting an appropriate form of data analysis. The approach we describe here should be applicable to geographic regions other than Idaho.     

Instream Large Wood: Denitrification Hotspots with Low N2O Production

We examined the effect of instream large wood on denitrification capacity in two contrasting, lower order streams — one that drains an agricultural watershed with no riparian forest and minimal stores of instream large wood and another that drains a forested watershed with an extensive riparian forest and abundant instream large wood. We incubated two types of wood substrates (fresh wood blocks and extant streambed wood) and an artificial stone substrate for nine weeks in each stream. After in situ incubation, we collected the substrates and their attached biofilms and established laboratory‐based mesocosm assays with stream water amended with ¹⁵N‐labeled nitrate‐N. Wood substrates at the forested site had significantly higher denitrification than wood substrates from the agricultural site and artificial stone substrates from either site. Nitrate‐N removal rates were markedly higher on woody substrates compared to artificial stones at both sites. Nitrate‐N removal rates were significantly correlated with biofilm biomass. Denitrification capacity accounted for only a portion of nitrate‐N removal observed within the mesocosms in both the wood controls and instream substrates. N₂ accounted for 99.7% of total denitrification. Restoration practices that generate large wood in streams should be encouraged for N removal and do not appear to generate high risks of instream N₂O generation.