Our understanding of the N cycle is affected by how accurately we can measure NH₄ in natural waters. Measuring NH₄ concentrations requires accounting for matrix effects (ME) that are caused by substances in the sample that attenuate or intensify the signal of the samples relative to the standards. We show that the ME calculation in the recently published fluorometric NH₄ method is mathematically incorrect, producing results that consistently underestimate NH₄ concentration as a nonlinear function of the ME. We provide the correct equation and offer an alternative approach that accounts for ME by using sample water rather than deionized water to make the standards, thereby producing a standard curve that contains the same background chemical properties as the samples. In addition, we show that the previous method for measuring a sample's background fluorescence does not include the background signal of the reagent or its interaction with the matrix constituents of the sample. We provide a new method for measuring a sample's background fluorescence that includes the background fluorescence of the sample, reagent, and their interaction. The simple changes we suggest produce more accurate and precise NH₄ measurements.

Dissolved inorganic N fluxes, including nitrification and denitrification rates, were measured in situ under dark and light conditions using epilithic biofilms from a 6^th to 8^th Strahler-order reach of the River Garonne, France. A broad range of epilithic biofilm biomasses (6–51 g ash-free dry mass [AFDM]/m²) was assessed in 39 benthic chamber experiments. Nitrification was detected in 31 cases (0.1–7.7 mg N m⁻² h⁻¹) and caused a net release of NO₃^– in spite of algal assimilation in light conditions in 5 cases. Denitrification (D_w) (0–8.1 mg N m⁻² h⁻¹), which accounted for 83% of gross NO₃^– removal in the dark, was a significant, permanent N sink. Calculation of mean (±1 SE) N budgets indicated epilithon release of inorganic N in the dark (1.5 ± 0.3 mg N m⁻² h⁻¹) that was significantly related to biofilm mass. Calculation of mean N budgets under light indicated net uptake (1.8 ± 0.4 mg N m⁻² h⁻¹). However, a net release was observed when biomass was >40 g AFDM/m². A calculated daily budget showed evidence for a threshold between photoautotrophy (N assimilation) and heterotrophy (N mineralization) of 23 g AFDM/m². This threshold approximated mean biomass at low water and may correspond to an equilibrium value for epilithic biomass in the river. A conceptual scheme of the epilithic biofilm function and the consequences on the water-column N content in rivers is proposed.

The major ion chemistry of water from an 11.42-km reach of the Pajaro River, a losing stream in central coastal California, shows a consistent pattern of higher concentrations during the 2^nd (dry) half of the water year. Most solutes are conserved during flow along the reach, but [NO₃⁻] decreases by ~30% and is accompanied by net loss of channel discharge and extensive surface–subsurface exchange. The corresponding net NO₃⁻ uptake length is 37 ± 13 km (42 ± 12 km when normalized to the conservative solute Cl⁻), and the areal NO₃⁻ uptake rate is 0.5 μmol m⁻² s⁻¹. The observed reduction in [NO₃⁻] along the reach results from one or more internal sinks, not dilution by ground water, hill-slope water, or other water inputs. Observed reductions in [NO₃⁻] and channel discharge along the experimental reach result in a net loss of 200–400 kg/d of NO₃⁻-N, ~50% of the input load. High-resolution (temporal and spatial) sampling indicates that most of the NO₃⁻ loss occurs along the lower part of the reach, where there is the greatest seepage loss and surface–subsurface exchange of water. Stable isotopes of NO₃⁻, total dissolved P concentrations, and streambed chemical profiles suggest that denitrification is the most significant NO₃⁻ sink along the reach. Denitrification efficiency, as expressed through downstream enrichment in ¹⁵N-NO₃⁻, varies considerably during the water year. When discharge is greater (typically earlier in the water year), denitrification is least efficient and downstream enrichment in ¹⁵N-NO₃⁻ is greatest. When discharge is lower, denitrification in the streambed appears to occur with greater efficiency, resulting in lower downstream enrichment in ¹⁵N-NO₃⁻.

Factors affecting epilithon biomass were investigated at 3 sites along a temporary karst stream system on the Mediterranean island of Majorca. Two sites were on the mainstream axis (downstream site [T5], upstream site [T4]) and one site was in a spring-fed tributary (GB). The degree of abiotic control of periphyton biomass was examined at the 3 sites. Mean periphyton biomass during a surficial flow period (as chlorophyll a [chl a] and ash-free dry mass [AFDM]) was nearly 1 order of magnitude higher at T5 than at the other 2 sites. AFDM ranged from low values at GB to intermediate and very high levels at T4 and T5, respectively. Spatial variation in periphyton chl a was related to the coarser, and more stable, karstic substrata and higher discharge at T5 than at GB or T4. A significant proportion of the temporal variation in periphyton chl a at T4 and T5 was related to abiotic factors, but abiotic factors did not explain the temporal pattern at GB. A short-term field experiment was conducted at GB to determine the role of the dominant grazer, the glossosomatid caddisfly Agapetus quadratus, in regulating periphyton biomass. This experiment revealed that densities of A. quadratus may have controlled periphyton biomass at GB (where grazer densities were highest) because the relationship between A. quadratus density and chl a followed a negative exponential function. The grazer prevented an increase in chl a at intermediate and high densities (>4000 ind./m²), but no effect was found at lower grazer densities. Overall, the levels of calcareous deposition over substratum surfaces seemed to influence colonization by grazers, which, in turn, may have influenced periphyton accrual.

The Atlantic Forest of Brazil has been identified as a biodiversity hotspot of global significance. We assessed chironomid (Diptera:Chironomidae) taxa richness in 2 vegetation types in this region: the Atlantic Rain Forest and the Atlantic Semi-deciduous Forest. Taxa were collected from 15 low-order streams across multiple habitats. A total of 191 morphospecies were recognized (125 Chironominae, 28 Tanypodinae, and 38 Orthocladiinae). We estimated chironomid richness using a Bayesian statistical approach. Species-richness estimates ranged from 200 (credibility interval, 195–207) to 267 (248–288). These results place low-order streams from Atlantic Forest among the most chironomid speciose areas in the world.

During the past 30 y, periphyton has been recognized as a key component in the benthos of lake littoral zones. However, the role of meiofauna-sized organisms living in the periphyton and mechanisms regulating and influencing those communities largely have been neglected, and the process of community development and colonization pathways of periphytic meiofauna are unknown in lakes. We studied these processes on littoral hard substrates in an oligotrophic lake. We manipulated the access of meiofauna to artificial hard substrates for a period of 57 d in a field experiment. In one treatment, the direct colonization pathway via active crawling was prohibited by elevating substrates into the water column. Development of the meiofaunal community was compared between elevated and nonelevated (control) treatments. In addition, relationships among the meiofaunal communities on the substrates in the field experiment and those in sediment traps and on natural hard substrates were examined. Periphyton biomass (chlorophyll a, total organic matter) and inorganic matter increased significantly in both experimental treatments throughout the experiment. Meiofauna colonized the substrates quickly and reached maximum densities of 107 ind./cm². The initial colonization phase (2–8 d) was characterized by a great variation in meiofaunal community structure; rotifers and crustaceans were the most abundant groups. Communities became less variable during the experiment and resembled natural communities on hard substrates at the end of the experiment. In general, development of meiofaunal abundance and periphyton biomass was rapid and proceeded similarly in both treatments. Our results show that colonization of littoral hard substrates can be driven by water-column transport of meiofauna and point to the importance of this pathway for dispersal and colonization of hard substrates by meiofauna.

Sand is a common substratum in many streams, especially in lacustrine geologies, but has been less studied as a habitat for invertebrates than other substrata such as gravel and cobble. We hypothesized that benthic organic matter (BOM) content would influence the abundance and community structure of macroinvertebrates in sand habitats. Levels of coarse BOM (no = 0, low = 1%, high = 5% organic matter, as dry mass) were manipulated in 45 colonization chambers (volume = 539 cm³) implanted in a sand-dominated reach of Shane Creek in the Ottawa National Forest, Michigan. Chironomidae, Tipulidae, and Trichoptera were common colonizers of chambers. At the end of the 32-d experiment, invertebrate abundance (F_2,12 = 7.5, p = 0.015) and biomass (F_2,12 = 8.7, p = 0.010) were significantly higher in chambers with low BOM than with no BOM, but the high-BOM treatment did not differ from the no-BOM treatment. Throughout the experiment, functional feeding groups (FFG) were dominated numerically by gathering collectors in all treatments, but predators increased over time in the no-BOM and high-BOM treatments. At the beginning of the experiment, FFG biomass was dominated by shredders, and predator biomass increased in the no-BOM and high-BOM treatments over time. In general, taxon richness was higher in the low-BOM treatment than in the high-BOM treatment (F_2,108 = 4.1, p = 0.025). Overall, the low-BOM treatment supported more invertebrates than the no-BOM and high-BOM treatments, perhaps because of fewer predators. Results suggest that local patterns of BOM accumulation may affect macroinvertebrate abundance and distribution in sand habitats in a nonlinear manner.

Translocations are used increasingly to conserve populations of rare freshwater mussels. Recovery of translocated mussels is essential to accurate assessment of translocation success. We designed an experiment to evaluate the use of passive integrated transponder (PIT) tags to mark and track individual freshwater mussels. We used eastern lampmussels (Lampsilis radiata radiata) as a surrogate for 2 rare mussel species. We assessed internal and external PIT-tag retention in the laboratory and field. Internal tag retention was high (75–100%), and tag rejection occurred primarily during the first 3 wk after tagging. A thin layer of nacre coated internal tags 3 to 4 mo after insertion, suggesting that long-term retention is likely. We released mussels with external PIT tags at 3 field study sites and recaptured them with a PIT pack (mobile interrogation unit) 8 to 10 mo and 21 to 23 mo after release. Numbers of recaptured mussels differed among study sites; however, we found more tagged mussels with the PIT-pack searches with visual confirmation (72–80%) than with visual searches alone (30–47%) at all sites. PIT tags offer improved recapture of translocated mussels and increased accuracy of posttranslocation monitoring.

Freshwater mussels are important constituents of freshwater ecosystems, yet much of their basic biology remains to be examined. The behavior of 3 species of unionid mussels (Unio tumidus, Unio pictorum, and Anodonta anatina) was examined in the lowland River Spree (Germany). Mussels were marked individually, and their positions on the sediment surface and depth below the sediment surface were recorded weekly between May and October 2004. The average rate of horizontal movement was 11 ± 15 cm/wk (mean ± 1 SD). The direction of the movements seemed erratic; however, a significant net shoreward displacement of ~17 cm, possibly caused by rising water levels, was observed during the study. A surprisingly high percentage of the mussels (74 ± 7%) was burrowed entirely in the sediment to depths as great as 20 cm during the summer. Smaller mussels and individuals not infested by the zebra mussel, Dreissena polymorpha, burrowed deeper in the sediments than larger or infested mussels. Burrowing reduced infestation densities in a laboratory experiment. Significantly more U. tumidus individuals were found on the sediment surface during the reproductive period in early summer than in late summer, suggesting that reproductive activity may influence burrowing. Burrowing was significantly related to current velocity (discharge), day length, and water temperature (multiple linear regression, R² = 0.74, p < 0.001), but current velocity appeared to be the dominant factor driving vertical movements (R² = 0.53, p < 0.01). We propose that movement behaviors are important adaptations of unionid mussel populations to the flow and food conditions in rivers. Movement behavior also may help unionids escape predators and control infestation by D. polymorpha.

Intraspecific and interspecific variation in the trophic position of various consumers is central to many theories of aquatic ecosystem functioning and dynamics. In recent years, such issues have been addressed using stable N isotopes (δ¹⁵N) to estimate trophic position of consumers in aquatic ecosystems. However, one needs to correct for variation in baseline δ¹⁵N among sites to estimate the trophic position of any aquatic consumer using δ¹⁵N. We first discuss the steps needed to select a baseline indicator in river ecosystems, where abundant foodweb isotopic data have been published but limited attention has been paid to identification of baseline organisms. We examined within-site differences in δ¹⁵N among primary consumers belonging to different functional and taxonomic groups and found significant differences in δ¹⁵N. Collectors were significantly enriched compared to other primary consumers. We propose scrapers as a baseline δ¹⁵N indicator because they showed low δ¹⁵N values and were more widely distributed than other primary consumers throughout our study sites. Using this baseline δ¹⁵N, we calculated continuous estimates of trophic position of consumers (invertebrates and small fish) for our 87 river food webs. Primary consumers had a significantly lower mean trophic position (2.3) than predatory invertebrates (2.9) and fish (3.5), and these results are concordant with estimates based on traditional studies. However, trophic-position estimates of consumers (both invertebrates and fish) were highly variable across sites with standard deviations spanning up to 0.67 trophic levels, which suggested potential omnivory. Comparison of these trophic-position estimates with estimates based on mean δ¹⁵N of all primary consumers combined (no targeting of scrapers as baseline indicators) suggested that use of a constrained number of groups and corrections for isotopic differences among groups when estimating baseline δ¹⁵N could reduce some biases induced by the use of various functional feeding groups with variable δ¹⁵N values.

Damaged and immature specimens often result in macroinvertebrate data that contain ambiguous parent–child pairs (i.e., abundances associated with multiple related levels of the taxonomic hierarchy such as Baetis pluto and the associated ambiguous parent Baetis sp.). The choice of method used to resolve ambiguous parent–child pairs may have a very large effect on the characterization of invertebrate assemblages and the interpretation of responses to environmental change because very large proportions of taxa richness (73–78%) and abundance (79–91%) can be associated with ambiguouparents. To address this issue, we examined 16 variations of 4 basic methods for resolving ambiguous taxa: RPKC (remove parent, keep child), MCWP (merge child with parent), RPMC (remove parent or merge child with parent depending on their abundances), and DPAC (distribute parents among children). The choice of method strongly affected assemblage structure, assemblage characteristics (e.g., metrics), and the ability to detect responses along environmental (urbanization) gradients. All methods except MCWP produced acceptable results when used consistently within a study. However, the assemblage characteristics (e.g., values of assemblage metrics) differed widely depending on the method used, and data should not be combined unless the methods used to resolve ambiguous taxa are well documented and are known to be comparable. The suitability of the methods was evaluated and compared on the basis of 13 criteria that considered conservation of taxa richness and abundance, consistency among samples, methods, and studies, and effects on the interpretation of the data. Methods RPMC and DPAC had the highest suitability scores regardless of whether ambiguous taxa were resolved for each sample separately or for a group of samples. Method MCWP gave consistently poor results. Methods MCWP and DPAC approximate the use of family-level identifications and operational taxonomic units (OTU), respectively. Our results suggest that restricting identifications to the family level is not a good method of resolving ambiguous taxa, whereas generating OTUs works well provided that documentation issues are addressed.

We investigated whether 2 stream sampling protocols collected different diatom assemblages within sampling reaches and, consequently, influenced the outcome of bioassessment. We analyzed data collected at 71 sites by both reach-scale (Environmental Monitoring and Assessment Program [EMAP]) and targeted-habitat (US Environmental Protection Agency [EPA] Science to Achieve Results [STAR]) sampling protocols as part of the US EPA EMAP survey. Overall, diatom assemblages generated by the 2 protocols were similar. Median Bray–Curtis (BC) similarity between EMAP and STAR diatom counts was 70% (range 19–91%). Taxon richness (r² = 0.7), autecological metrics (e.g., siltation index: r² = 0.9, trophic diatom index: r² = 0.8), and morphological metrics (e.g., % erect taxa: r² = 0.7, % prostrate taxa: r² = 0.8) were comparable between the 2 protocols. Relationships between diatom assemblages (summarized as nonmetric multidimensional scaling ordination axes) and environmental variables were similar between the 2 protocols, with diatom assemblages relating more to instream water-quality variables (e.g., total P, conductivity) than to physical-habitat or watershed characteristics. Diatom assemblages generated by the 2 protocols did diverge under a specific set of environmental conditions. Sites with the lowest BC similarities between EMAP and STAR counts tended to be larger and less shaded and to have higher values of water-quality variables affected by human disturbance (e.g., conductivity, total P, % fine sediment) than sites with higher values of BC similarity between protocols. We conclude that EMAP and STAR sampling protocols, in general, collect similar diatom assemblages. However, researchers should exercise caution when combining diatom data sets collected with different sampling protocols, particularly in large streams, where the potential for sampling of multiple habitats is increased.

The contribution of rare taxa to bioassessments based on multispecies assemblages is the subject of continued debate. As a result, users of predictive models such as River InVertebrate Prediction and Classification System (RIVPACS) disagree on whether to exclude locally rare taxa from the O/E index, where O is the number of taxa observed in a sampled assemblage, and E is the number that would be expected if the site were in a minimally disturbed reference condition. We assessed how the bioassessment performance of O/E was affected by the exclusion of taxa with site-specific, model-predicted occurrence probabilities that did not exceed thresholds of P_T = 0 , 0.1, 0.2, … , 0.7. We assessed O/E performance for each of 10 predictive models applied to a total of 5685 stream and lake samples from throughout the contiguous USA. For 5 of the 10 cases, the standard deviation (SD) of O/E across reference sites was reduced by at least 0.02 O/E units when locally rare and uncommon taxa were excluded (P_T = 0.5) from O/E, as compared with all taxa being included (P_T = 0 ). These reductions in SD denote increases in precision of the O/E index. We also assessed the sensitivity of O/E, measured by the % of test sites (that is, sites independently assessed as not being in reference condition) that were declared to be outside the reference distribution of O/E scores. Five of our 10 cases showed increases in sensitivity of ≥10% at P_T = 0.5 as compared with P_T = 0 . All cases that did not show increases in sensitivity or precision also showed no decrease in either of these performance measures as P_T increased. A comparison of observed occurrence frequencies of taxa at reference and test sites qualitatively explained the size of the O/E sensitivity response in all 10 cases. This result suggests that effects of rare-taxa exclusion are a direct consequence of these occurrence frequencies rather than of predictive-model structures. Thus, we predict that other assemblage-based bioassessment tools are likely to show improved sensitivity when rare taxa are excluded.

Recent comparisons of benthic macroinvertebrate (BMI) sampling protocols have shown that samples collected from different habitat types generally produce consistent stream classifications and assessments. However, these comparisons usually have not included biological endpoints used by monitoring agencies, such as multimetric indices (e.g., benthic index of biotic integrity [B-IBI]) or observed-to-expected (O/E) indices of taxonomic completeness, as target variables, and estimates of method precision are rarely provided. Targeted-riffle (TR) and reach-wide (RW) benthic samples have been collected at thousands of sites across the western USA, but little guidance is available for understanding: 1) the extent to which raw data sets can be combined in regional or large-scale analyses, 2) the degree of precision afforded by each method, or 3) the efficacy of cross-application of biological indicators derived from one sample type to the other. To address these issues, we used data from 193 sites in California where the Environmental Monitoring and Assessment Program (EMAP) collected the 2 samples side by side. We also conducted a separate study wherein 3 replicates of each sample type were collected from 15 streams to estimate minimum detectable difference (MDD) as a measure of each method's precision. Metrics calculated from TR and RW samples showed similar dose–response relationships to stressor gradients and similar raw scoring ranges. Biological indices (B-IBI, O/E₀, and O/E₅₀) derived from RW samples were more precise than those derived from TR samples, but precision differences were not substantial. On average, pairwise differences in any index between TR and RW sample types were much less than the MDD associated with either sampling method. We observed a weak but consistent bias toward higher O/E₅₀ scores from TR samples than from RW samples at the highest elevations and in the largest watersheds. Broad-scale condition assessments were nearly identical when B-IBI and O/E₀ were used as endpoints, and assessments based on O/E₅₀ were only slightly less similar. Our analyses indicate that raw data sets and biological indicators derived from TR and RW samples may be generally interchangeable when used in ambient biomonitoring programs.

Selection of reference sites is a critical component in the process of developing and applying biological indicators of ecological condition. Some evidence suggests that despite its importance the rules by which reference sites are selected have not always been evaluated critically to assure that the sites represent least-disturbed conditions. We present a comparison of physical and chemical disturbance measures and biotic indices at handpicked reference sites provided by resource agencies and at sites selected by a probability design from a 12-state survey of western streams and rivers. In most cases, the distributions of disturbance measures and biotic index scores were essentially the same for both types of sites; that is, only a subset of the handpicked reference sites represented least-disturbed conditions. We recommend that all agencies that use reference sites critically review those sites with a set of explicit criteria, using field-collected data as well as mapped information.