Pesticide and nitrate transport in an agriculturally influenced stream in Indiana.

Agrochemicals can be transported from agricultural fields into streams where they might have adverse effects on water quality and ecosystems. Three enrichment experiments were conducted in a central Indiana stream to quantify pesticide and nitrogen transport dynamics. In an enrichment experiment, a compound solution is added at a constant rate into a stream to increase compound background concentration. A conservative tracer (e.g., bromide) is added to determine discharge. Water and sediment samples are taken at several locations downstream to measure uptake metrics. We assessed transport of nitrate, atrazine, metolachlor, and carbaryl through direct measurement of uptake length (S w ), uptake velocity (V f ), and areal uptake (U). S w measures the distance traveled by a nutrient along the stream reach. V f measures the velocity a nutrient moves from the water column to immobilization sites. U represents the amount of nutrient immobilized in an area of streambed per unit of time. S w varied less than one order of magnitude across pesticides. The highest S w for atrazine suggests greater transport to downstream ecosystems. Across compounds, pesticide S w was longest in August relative to October and July. V f varied less than one order of magnitude across pesticides with the highest V f for metolachlor. U varied three orders of magnitude across pesticides with the highest U associate with sediment-bound carbaryl. Increasing nitrate S w suggests a lower nitrate demand of biota in this stream. Overall, pesticide transport was best predicted by compound solubility which can complement and improve models of pesticide abundance used by water quality programs and risk assessments.

Stream denitrification across biomes and its response to anthropogenic nitrate loading.

Anthropogenic addition of bioavailable nitrogen to the biosphere is increasing and terrestrial ecosystems are becoming increasingly nitrogen-saturated, causing more bioavailable nitrogen to enter groundwater and surface waters. Large-scale nitrogen budgets show that an average of about 20-25 per cent of the nitrogen added to the biosphere is exported from rivers to the ocean or inland basins, indicating that substantial sinks for nitrogen must exist in the landscape. Streams and rivers may themselves be important sinks for bioavailable nitrogen owing to their hydrological connections with terrestrial systems, high rates of biological activity, and streambed sediment environments that favour microbial denitrification. Here we present data from nitrogen stable isotope tracer experiments across 72 streams and 8 regions representing several biomes. We show that total biotic uptake and denitrification of nitrate increase with stream nitrate concentration, but that the efficiency of biotic uptake and denitrification declines as concentration increases, reducing the proportion of in-stream nitrate that is removed from transport. Our data suggest that the total uptake of nitrate is related to ecosystem photosynthesis and that denitrification is related to ecosystem respiration. In addition, we use a stream network model to demonstrate that excess nitrate in streams elicits a disproportionate increase in the fraction of nitrate that is exported to receiving waters and reduces the relative role of small versus large streams as nitrate sinks.

Nitrous oxide emission from denitrification in stream and river networks.

Nitrous oxide (N(2)O) is a potent greenhouse gas that contributes to climate change and stratospheric ozone destruction. Anthropogenic nitrogen (N) loading to river networks is a potentially important source of N(2)O via microbial denitrification that converts N to N(2)O and dinitrogen (N(2)). The fraction of denitrified N that escapes as N(2)O rather than N(2) (i.e., the N(2)O yield) is an important determinant of how much N(2)O is produced by river networks, but little is known about the N(2)O yield in flowing waters. Here, we present the results of whole-stream (15)N-tracer additions conducted in 72 headwater streams draining multiple land-use types across the United States. We found that stream denitrification produces N(2)O at rates that increase with stream water nitrate (NO(3)(-)) concentrations, but that <1% of denitrified N is converted to N(2)O. Unlike some previous studies, we found no relationship between the N(2)O yield and stream water NO(3)(-). We suggest that increased stream NO(3)(-) loading stimulates denitrification and concomitant N(2)O production, but does not increase the N(2)O yield. In our study, most streams were sources of N(2)O to the atmosphere and the highest emission rates were observed in streams draining urban basins. Using a global river network model, we estimate that microbial N transformations (e.g., denitrification and nitrification) convert at least 0.68 Tg·y(-1) of anthropogenic N inputs to N(2)O in river networks, equivalent to 10% of the global anthropogenic N(2)O emission rate. This estimate of stream and river N(2)O emissions is three times greater than estimated by the Intergovernmental Panel on Climate Change.

The effects of the pharmaceutical carbamazepine on life history characteristics of flat-headed mayflies (Heptageniidae) and aquatic resource interactions.

Pharmaceutical pollutants are commonly detected in freshwater ecosystems around the world and have biological effects on aquatic organisms. However, current understanding of the influence this contaminant class has on freshwater communities and ecosystems is lacking. Recently the scientific community has called for research focusing on certain pharmaceuticals due to their ubiquity and potential toxicity. Carbamazepine is one of these pharmaceuticals. To better understand the effect carbamazepine has on life history characteristics of aquatic organisms and consumer-resource interactions, we quantified the influence of carbamazepine on the development, growth and behavior of mayfly nymphs (Stenonema sp.) and the alterations in food consumer-resource interactions between Stenonema and algae (Chaetophora). Microcosms were assembled in a factorial design containing algae and mayfly nymphs native to central Indiana and dosed with environmentally relevant concentrations of carbamazepine. From this ecotoxicological experiment we were able to infer that carbamazepine at 2,000 ng/L influenced the development and behavior of Stenonema nymphs and the body dimensions of adult individuals. However, it appears that carbamazepine does not influence consumer-resource interactions at concentrations found in surface waters. The pharmaceutical carbamazepine may influence the behavior, growth and development of mayflies, which could have significant consequences at the population, community and ecosystem level.

The influence of six pharmaceuticals on freshwater sediment microbial growth incubated at different temperatures and UV exposures.

Pharmaceutical compounds have been detected in freshwater for several decades. Once they enter the aquatic ecosystem, they may be transformed abiotically (i.e., photolysis) or biotically (i.e., microbial activity). To assess the influence of pharmaceuticals on microbial growth, basal salt media amended with seven pharmaceutical treatments (acetaminophen, caffeine, carbamazepine, cotinine, ibuprofen, sulfamethoxazole, and a no pharmaceutical control) were inoculated with stream sediment. The seven pharmaceutical treatments were then placed in five different culture environments that included both temperature treatments of 4, 25, 37°C and light treatments of continuous UV-A or UV-B exposure. Microbial growth in the basal salt media was quantified as absorbance (OD(550)) at 7, 14, 21, 31, and 48d following inoculation. Microbial growth was significantly influenced by pharmaceutical treatments (P < 0.01) and incubation treatments (P < 0.01). Colonial morphology of the microbial communities post-incubation identified selection of microbial and fungal species with exposure to caffeine, cotinine, and ibuprofen at 37°C; acetaminophen, caffeine, and cotinine at 25°C; and carbamazepine exposed to continuous UV-A. Bacillus and coccus cellular arrangements (1000X magnification) were consistently observed across incubation treatments for each pharmaceutical treatment although carbamazepine and ibuprofen exposures incubated at 25°C also selected spiral-shaped bacteria. These data indicate stream sediment microbial communities are influenced by pharmaceuticals though physiochemical characteristics of the environment may dictate microbial response.

The influence of TCS on the growth and behavior of the freshwater snail, Physa acuta.

Triclosan (TCS) is among the top 10 most persistent contaminants found in U.S. rivers, streams, lakes, and aquifers. Although TCS has not been found to be hazardous to humans, it can be toxic to aquatic environments and animals. The effects of TCS on growth rates and the locomotive behavior of the freshwater snail, Physa acuta, were studied by exposing snails to a range of environmentally-relevant trace concentrations previously documented in freshwater ecosystems. Effects of TCS on snail growth were calculated using a non-linear regression model, and effects on behavior were determined using a two-way analysis of variance. Environmentally relevant concentrations of TCS (0.5 to 1.0 µg/L) enhanced Physa growth rates at low concentrations, but slowed growth rates at concentrations greater than 5 µg/L. Acute exposure did not affect immediate snail behavior; however, chronically exposed snails moved more slowly than naïve snails. These data indicate that concentrations of TCS currently found in freshwater ecosystems can potentially affect the growth and behavior of snails.

Distribution of nonprescription pharmaceuticals in central Indiana streams and effects on sediment microbial activity.

Since the discovery of trace concentrations of pharmaceuticals in streams and treated drinking water around the world, a call has been made by both the scientific community and the general public to increase understanding of the potential effects these compounds may have on freshwater integrity. We measured abundance and distribution of pharmaceuticals in headwater streams across the Upper White River Watershed of central Indiana. Four nonprescription pharmaceuticals (1,7-dimethylxanthine, caffeine metabolite; acetaminophen; caffeine; cotinine, nicotine metabolite) were found at one or more sites with mean concentrations of 0.038, 0.109, 0.057 and 0.041 µg/l, respectively. Caffeine was measured at trace concentrations at all sites sampled. Higher pharmaceutical concentrations were associated with streams having >90% agricultural land use in the sub watershed, suggesting that nonpoint sources, such as septic tanks, may contribute to stream pharmaceutical contamination. To assess the influence of these pharmaceuticals on stream microbial activity, we measured changes in sediment respiration and nutrient uptake in response to pharmaceuticals using both in vitro and in situ techniques. For in vitro experiments, respiration rates were not significantly different from controls with pharmaceutical exposure. However, net NO(3) (-)-N uptake increased significantly with nicotine concentrations. Net NH(4)(+)-N uptake was reduced in response to caffeine and nicotine exposure. In situ experiments indicated nicotine exposure increased microbial respiration. Our data show pharmaceuticals are ubiquitous in headwater streams of central Indiana and likely influence stream microbial activity depending on the pharmaceutical compound and history of exposure.

A national reconnaissance of trace organic compounds (TOCs) in United States lotic ecosystems.

We collaborated with 26 groups from universities across the United States to sample 42 sites for 33 trace organic compounds (TOCs) in water and sediments of lotic ecosystems. Our goals were 1) to further develop a national database of TOC abundance in United States lotic ecosystems that can be a foundation for future research and management, and 2) to identify factors related to compound abundance. Trace organic compounds were found in 93% of water samples and 56% of sediment samples. Dissolved concentrations were 10-1000× higher relative to sediment concentrations. The ten most common compounds in water samples with detection frequency and maximum concentration were sucralose (87.5%, 12,000ng/L), caffeine (77.5%, 420ng/L), sulfamethoxazole (70%, 340ng/L), cotinine (65%, 130ng/L), venlafaxine (65%, 1800ng/L), carbamazepine (62.5%, 320ng/L), triclosan (55%, 6800ng/L), azithromycin (15%, 970ng/L), diphenylhydramine (40%, 350ng/L), and desvenlafaxine (35%, 4600ng/L). In sediment, the most common compounds were venlafaxine (32.5%, 19ng/g), diphenhydramine (25%, 41ng/g), azithromycin (15%, 11ng/g), fluoxetine (12.5%, 29ng/g) and sucralose (12.5%, 16ng/g). Refractory compounds such as sucralose may be good indicators of TOC contamination in lotic ecosystems, as there was a correlation between dissolved sucralose concentrations and with the total number of compounds detected in water. Discharge and human demographic (population size) characteristics were not good predictors of compound abundance in water samples. This study further confirms the ubiquity of TOCs in lotic ecosystems. Although concentrations measured rarely approached acute aquatic-life criteria, the chronic effects, bioaccumulative potential, or potential mixture effects of multiple compounds are relatively unknown.

Effects of atrazine, metolachlor, carbaryl and chlorothalonil on benthic microbes and their nutrient dynamics.

Atrazine, metolachlor, carbaryl, and chlorothalonil are detected in streams throughout the U.S. at concentrations that may have adverse effects on benthic microbes. Sediment samples were exposed to these pesticides to quantify responses of ammonium, nitrate, and phosphate uptake by the benthic microbial community. Control uptake rates of sediments had net remineralization of nitrate (-1.58 NO3 µg gdm⁻¹ h⁻¹), and net assimilation of phosphate (1.34 PO4 µg gdm⁻¹ h⁻¹) and ammonium (0.03 NH4 µg gdm⁻¹ h⁻¹). Metolachlor decreased ammonium and phosphate uptake. Chlorothalonil decreased nitrate remineralization and phosphate uptake. Nitrate, ammonium, and phosphate uptake rates are more pronounced in the presence of these pesticides due to microbial adaptations to toxicants. Our interpretation of pesticide availability based on their water/solid affinities supports no effects for atrazine and carbaryl, decreasing nitrate remineralization, and phosphate assimilation in response to chlorothalonil. Further, decreased ammonium and phosphate uptake in response to metolachlor is likely due to affinity. Because atrazine target autotrophs, and carbaryl synaptic activity, effects on benthic microbes were not hypothesized, consistent with results. Metolachlor and chlorothalonil (non-specific modes of action) had significant effects on sediment microbial nutrient dynamics. Thus, pesticides with a higher affinity to sediments and/or broad modes of action are likely to affect sediment microbes' nutrient dynamics than pesticides dissolved in water or specific modes of action. Predicted nutrient uptake rates were calculated at mean and peak concentrations of metolachlor and chlorothalonil in freshwaters using polynomial equations generated in this experiment. We concluded that in natural ecosystems, peak chlorothalonil and metolachlor concentrations could affect phosphate and ammonium by decreasing net assimilation, and nitrate uptake rates by decreasing remineralization, relative to mean concentrations of metolachlor and chlorothalonil. Our regression equations can complement models of nitrogen and phosphorus availability in streams to predict potential changes in nutrient dynamics in response to pesticides in freshwaters.

The effects of the psychiatric drug carbamazepine on freshwater invertebrate communities and ecosystem dynamics.

Freshwater ecosystems are persistently exposed to pharmaceutical pollutants, including carbamazepine. Despite the ubiquity and recalcitrance of carbamazepine, the effects of this pharmaceutical on freshwater ecosystems and communities are unclear. To better understand how carbamazepine influences the invertebrate community and ecosystem dynamics in freshwaters, we conducted a mesocosm experiment utilizing environmentally relevant concentrations of carbamazepine (200 and 2000 ng/L). Mesocosms were populated with four gastropod taxa (Elimia, Physa, Lymnaea and Helisoma), zooplankton, filamentous algae and phytoplankton. After a 31 d experimental duration, structural equation modeling (SEM) was used to relate changes in the community structure and ecosystem dynamics to carbamazepine exposure. Invertebrate diversity increased in the presence of carbamazepine. Additionally, carbamazepine altered the biomass of Helisoma and Elimia, induced a decline in Daphnia pulex abundance and shifted the zooplankton community toward copepod dominance. Lastly, carbamazepine decreased the decomposition of organic matter and indirectly altered primary production and dissolved nutrient concentrations. Changes in the invertebrate community occurred through both direct (i.e., exposure to carbamazepine) and indirect pathways (i.e., changes in food resource availability). These data indicate that carbamazepine may alter freshwater community structure and ecosystem dynamics and could have profound effects on natural systems.

Relationships between the psychiatric drug carbamazepine and freshwater macroinvertebrate community structure.

Pharmaceutical pollutants are commonly detected in surface waters and have the potential to affect non-target organisms. However, there is limited understanding of how these emerging contaminants may affect macroinvertebrate communities. The pharmaceutical carbamazepine is ubiquitous in surface waters around the world and is a pollutant of particular concern due to its recalcitrance and toxicity. To better understand the potential effects of carbamazepine on natural macroinvertebrate communities, we related stream macroinvertebrate abundance to carbamazepine concentrations. Macroinvertebrate and water samples were collected from 19 streams in central Indiana in conjunction with other stream physiochemical characteristics. Structural equation modeling (SEM) was used to relate macroinvertebrate richness to carbamazepine concentrations. Macroinvertebrate richness was positively correlated with increasing concentrations of carbamazepine. From the SEM we infer that carbamazepine influences macroinvertebrate richness through indirect pathways linked to Baetidae abundance. Baetidae abundance influenced ephemeropteran abundance and FBOM percent organic matter, both of which altered macroinvertebrate richness. The pharmaceutical carbamazepine may alter freshwater macroinvertebrate species composition, which could have significant consequences to ecosystem processes.

Human and veterinary pharmaceutical abundance and transport in a rural central Indiana stream influenced by confined animal feeding operations (CAFOs).

Previous research has documented the ubiquity of human and veterinary pharmaceuticals and personal care products (PPCPs) in freshwater, though their persistence and transport is relatively unknown. The objective of this study was to quantify the abundance and transport of human and veterinary PPCPs in a rural, central Indiana stream influenced by confined animal feeding operations (CAFOs). Research objectives also aimed to identify mechanisms controlling abundance and transport. PPCP concentrations and stream physicochemical characteristics were measured monthly over one year at multiple sites along a 60 km reach. Overall, human PPCPs were more abundant and measured at higher concentrations than veterinary pharmaceuticals. Veterinary pharmaceutical concentrations (lincomycin, sulfamethazine) were greatest in stream reaches adjacent to CAFOs. No distinct spatial variation was observed for human PPCPs. However, caffeine and paraxanthine had significant temporal variation with higher concentrations in winter. In contrast, DEET had higher concentrations in summer. Pharmaceutical load (µg/s) ranged from<0.005 to 1808 µg/s across sites, sampling events and pharmaceutical compounds with human PPCPs having higher loads relative to veterinary pharmaceuticals. Reach input ranged from net retention (sulfamethazine in August) to 1667 µg/m/d paraxanthine in March. Triclosan had the highest measured mean input into the reach (661 µg/m/d) and sulfamethazine had the lowest mean input (32 µg/m/d). Across measured compounds, input of PPCPs into the reach was two orders of magnitude lower than nitrate-N input (57,000 µg/m/d). Transport metrics indicated acetaminophen and caffeine are transported farther than triclosan though had lower loss velocities (loss relative to abundance). Loss rate of PPCPs was an order of magnitude lower than nitrate-N loss rate. Human PPCPs were more abundant than veterinary pharmaceuticals in this rural watershed influenced by CAFOs. Further, concentrations had significant temporal and spatial variation highlighting differential sources and fates. Thus, mechanisms driving PPCP retention and transport need to be identified to aid management of these emerging contaminants.

Detection of pharmaceuticals and personal care products (PPCPs) in near-shore habitats of southern Lake Michigan.

Pharmaceuticals and personal care products (PPCPs) have been documented throughout the United States freshwaters but research has focused largely on lotic systems. Because PPCPs are designed to have a physiological effect, it is likely that they may also influence aquatic organisms. Thus, PPCPs may negatively impact aquatic ecosystems. The objectives of this research were to quantify PPCP abundance in near-shore habitats of southern Lake Michigan and identify factors related to PPCP abundance. Stratified sampling was conducted seasonally at four southern Lake Michigan sites. All sites and depths had measurable PPCP concentrations, with mean individual compound concentrations of acetaminophen (5.36 ng/L), caffeine (31.0 ng/L), carbamazepine (2.23 ng/L), cotinine (4.03 ng/L), gemfibrozil (7.03 ng/L), ibuprofen (7.88 ng/L), lincomycin (4.28 ng/L), naproxen (6.32 ng/L), paraxanthine (1,7-dimethylxanthine; 46.2 ng/L), sulfadimethoxine (0.94 ng/L), sulfamerazine (0.92 ng/L), sulfamethazine (0.92 ng/L), sulfamethoxazole (26.0 ng/L), sulfathiazole (0.92 ng/L), triclocarban (5.72 ng/L), trimethoprim (5.15 ng/L), and tylosin (3.75 ng/L). Concentrations of PPCPs varied significantly among sampling times and locations (river mouth vs offshore), with statistical interactions between the main effects of site and time as well as time and location. Concentrations of PPCPs did not differ with site or depth. Temperature, total carbon, total dissolved solids, dissolved oxygen, and ammonium concentrations were related to total pharmaceutical concentrations. These data indicate that PPCPs are ubiquitous and persistent in southern Lake Michigan, potentially posing harmful effects to aquatic organisms.

Nitrate and ammonium uptake by natural stream sediment microbial communities in response to nutrient enrichment.

Anthropogenic activities have increased nitrogen concentration in many ecosystems. Because microbes have higher metabolic rates relative to larger organisms, microbial activity may influence nitrogen movement and degradation significantly in ecosystems. Thus, the ability for microorganisms to adapt to increasing nitrogen concentrations is essential to ecosystem sustainability. We measured sediment microbial community nitrogen assimilation after sustained nitrogen enrichments using in vitro and isotopic techniques. Mixed-microbial communities were exposed to-enriched concentrations of NO(3)-N (1 mg l(-1)) and NH(4)-N (30 µg l(-1)) for four weeks. Each week, filtered water samples were collected from each mesocosm and sediment was removed to quantify rates of nitrogen assimilation by the sediment microbial community. During the fourth week, isotopic tracers (15)NO(3) and (15)NH(4) were added to mesocosms to directly measure nitrogen incorporation into microbial cells as organic (15)N. Initial microbial responses to nitrogen enrichment were distinctly different from the sustained microbial community responses. NH(4)-N uptake was initially stimulated with NH(4)-N enrichments but increased uptake rates were not sustained over time. Sustained responses to changing nitrogen availability equilibrated within 1-3 weeks (depending on nitrogen form), indicating that even though microbial communities can respond to increased availability, potential for increased assimilation is limited.

Temporal variation of pharmaceuticals in an urban and agriculturally influenced stream.

Pharmaceuticals have become ubiquitous in the aquatic environment. Previous studies consistently demonstrate the prevalence of pharmaceuticals in freshwater but we do not yet know how concentrations vary over time within a given system. Two sites in central Indiana with varying land use in the surrounding watershed (suburban and agricultural) were sampled monthly for pharmaceutical concentrations and stream physiochemical parameters. Sediment samples were also collected at each sampling event for measurement of δ(15)N natural abundance and sediment organic content. Across sites and sampling events, twelve pharmaceuticals were detected including acetaminophen, caffeine, carbamazepine, cotinine, N,N-diethyl-meta-toluamide (DEET), gemfibrozil, ibuprofen, sulfadimethoxine, sulfamethazine, sulfamethoxazole, triclosan, and trimethoprim. Sulfathiazole, lincomycin, and tylosin were not detected at either site at any time. The agriculturally-influenced site had comparable pharmaceutical concentrations to the urban-influenced site. In general, pharmaceutical concentrations increased during winter at both sites and decreased during spring and summer. Multiple regression analyses indicated that water column dissolved oxygen, the number of days since precipitation, and solar radiation influenced total pharmaceutical concentration in the urban-influenced site; whereas pH, chlorophyll a concentration, and total amount of rainfall in the previous 10 days influenced total pharmaceutical concentrations in the agriculturally-influenced site. Pharmaceutical concentrations were not correlated with sediment δ(15)N across or within sites. However, sediment in the urban-influenced site had higher mean δ(15)N signatures relative to sediment in the agriculturally-influenced site. These data indicate pharmaceuticals are persistent in aquatic ecosystems influenced by both agricultural and suburban activity. Pharmaceuticals are designed to have a physiological effect; therefore, it is likely that they may also influence aquatic organisms, potentially threatening freshwater ecosystem health.

Factors controlling sediment denitrification in midwestern streams of varying land use.

We investigated controls on stream sediment denitrification in nine headwater streams in the Kalamazoo River Watershed, Michigan, USA. Factors influencing denitrification were determined by using experimental assays based on the chloramphenicol-amended acetylene inhibition technique. Using a coring technique, we found that sediment denitrification was highest in the top 5 cm of the benthos and was positively related to sediment organic content. To determine the effect of overlying water quality on sediment denitrification, first-order stream sediments were assayed with water from second- and third-order downstream reaches, and often showed higher denitrification rates relative to assays using site-specific water from the first-order stream reach. Denitrification was positively related to nitrate (NO3 ) concentration, suggesting that sediments may have been nutrient-limited. Using stream-incubated inorganic substrata of varying size classes, we found that finer-grained sand showed higher rates of denitrification compared to large pebbles, likely due to increased surface area per volume of substratum. Denitrification was measurable on both inorganic substrata and fine particulate organic matter loosely associated with inorganic particles, and denitrification rates were related to organic content. Using nutrient-amended denitrification assays, we found that sediment denitrification was limited by NO3- or dissolved organic carbon (DOC, as dextrose) variably throughout the year. The frequency and type of limitation differed with land use in the watershed: forested streams were NO3- -limited or colimited by both NO3- and DOC 92% of the time, urban streams were more often NO3- -limited than DOC-limited, whereas agricultural stream sediments were DOC-limited or co-limited but not frequently limited by NO3- alone.

Comparing denitrification estimates for a Texas estuary by using acetylene inhibition and membrane inlet mass spectrometry.

Characterizing denitrification rates in aquatic ecosystems is essential to understanding how systems may respond to increased nutrient loading. Thus, it is important to ensure the precision and accuracy of the methods employed for measuring denitrification rates. The acetylene (C2H2) inhibition method is a simple technique for estimating denitrification. However, potential problems, such as inhibition of nitrification and incomplete inhibition of nitrous oxide reduction, may influence rate estimates. Recently, membrane inlet mass spectrometry (MIMS) has been used to measure denitrification in aquatic systems. Comparable results were obtained with MIMS and C2H2 inhibition methods when chloramphenicol was added to C2H2 inhibition assay mixtures to inhibit new synthesis of denitrifying enzymes. Dissolved-oxygen profiles indicated that surface layers of sediment cores subjected to the MIMS flowthrough incubation remained oxic whereas cores incubated using the C2H2 inhibition methods did not. Analysis of the microbial assemblages before and after incubations indicated significant changes in the sediment surface populations during the long flowthrough incubation for MIMS analysis but not during the shorter incubation used for the C2H2 inhibition method. However, bacterial community changes were also small in MIMS cores at the oxygen transition zone where denitrification occurs. The C2H2 inhibition method with chloramphenicol addition, conducted over short incubation intervals, provides a cost-effective method for estimating denitrification, and rate estimates are comparable to those obtained by the MIMS method.