Environ. Sci. Technol. 2007, 41, 8420–8425
Nitrogen Cycling in Natural Waters using In Situ, Reagentless UV Spectrophotometry with Simultaneous Determination of Nitrate and Nitrite RICHARD C. SANDFORD,* ALEXANDRA EXENBERGER, AND PAUL J. WORSFOLD Biogeochemistry and Environmental Analytical Chemistry (BEACh) Group, School of Earth, Ocean and Environmental Sciences, University of Plymouth, Plymouth, PL4 8AA, England
Received June 15, 2007. Revised manuscript received September 07, 2007. Accepted September 12, 2007. Received December 15, 2007
Reliable, high temporal, and spatial resolution data is essential for enhancing our understanding of aquatic nitrogen biogeochemical cycling. This paper describes a novel UV spectrophotometric sensor (ProPS, TriOS GmbH, Oldenburg, Germany) for the real time, in situ, high resolution simultaneous mapping of nitrate/nitrite (linearity 0.01 – 6 mg N L-1, RSD’s NO3-N 4–10%, NO2-N 7–14%) in fresh and estuarine waters. Good agreement (t test at p ) 0.05) was found with MOOS-1 certified reference material and with reference segmented flow analysis data. River Taw deployments identified a diurnal cycle for NO3-N (0.22–0.63 mg L-1, RSD 3.9%) and for NO2-N (0.01–0.28 mg L-1, RSD 12.4%) with the photo-oxidation of dissolved organic nitrogen a source of diurnal nitrate/nitrite, and a large cyclical amplitude (30–62% of mean nitrate/nitrite). In situ Tamar Estuary nitrate/nitrite concentrations, mapped through the salinity gradient, were strongly correlated with suspended particulate material and inversely correlated with dissolved oxygen and pH, indicating midestuarine, bacterially mediated nitrification/denitrification, with the raised estuarine nitrite also significantly correlated with particulate organic nitrogen. Such previously unquantified inputs have important implications for N loadings calculated from coarse scale sampling and laboratory analysis, pollution assessment, and our understanding of the biological rhythms of aquatic organisms.
1. Introduction Riverine, estuarine, and coastal waters are diverse, productive, but ecologically vulnerable ecosystems of significant economic value, with nitrogen (N) playing a key role in their health. Anthropogenic N contamination (eutrophication) is of major concern with the European Environment Agency (1999) recognizing the vital strategic, scientific, and social importance of accurate flux measurements to fully resolve the biogeochemical cycle of N. Aquatic nitrogen loadings are reported principally as nitrate (1), although nitrite and ammonium are also highly * Corresponding author fax: +44 (0) 1752 233035; e-mail:
[email protected]. 8420
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 24, 2007
reactive within estuaries (2) but their fluxes have not been fully quantified and are predicted to increase due to climate change, transport, intensive agriculture, and urbanization (3, 4), Nutrient inputs during ecologically sensitive times, e.g., summer low flows, may be critical in triggering eutrophic related events. High resolution, continuous mapping can reveal new patterns and the nature, magnitude, and frequency of discontinuous nutrient inputs (5) that are due to short-term and seasonal changes in flow regimes and impact on nutrient dynamics, bioavailability, uptake, and ecosystem response, as recently demonstrated for phosphorus (6). Such data will enable accurate modeling of their effect on surface water biology (7), and therefore, there is a clear need for real time, high resolution, in situ measurements of nitrate and nitrite (8). Conventional methods for N (9) use filtered, treated samples, and sample integrity can be compromised due to contamination and chemical, biological, or physical effects during storage. In situ N methods are relatively few and include submersible flow injection analysers for nitrate in estuarine and coastal waters (10), and multiple parametric determinations of nitrate, nitrite, and sulfide (11), and for nitrate, silicate, and phosphate (12). However, such methods require a continuous supply of reagents and have response times not suited to high resolution (10 mg L-1 (field data), the divergencies between the SFA and the real time UV nitrate and especially nitrite data were due to nitrification/denitrification by the UV sensor. These inputs were not identified by SFA analysis, the real time UV sensor measurements excluded the effects of in situ sample filtration and the physical, biological, and chemical changes that can alter species and analyte levels, e.g., oxidation of nitrite to nitrate, during storage (24). This is a significant advantage and has important implications for the interpretation of N loadings and effects on aquatic biological processes in estuarine waters based on laboratory analysis. 3.5. Field Deployments. 3.5.1. Freshwater Diurnal Cycle. Short-term temporal patterns in the freshwater biogeochemical cycling of NO3-N and NO2-N were investigated in the River Taw (3 m from bank, 0.8 m depth, sunrise 0602 h, sunset 2002 h, Figure 3). Diurnal nitrate and nitrite cycles (24 h) were identified with nitrate minima at 1400 and 0400 and maxima at 1930 and at 1000. Nitrite followed a similar pattern with a 1430 minimum, although the 0230 minimum and the 2100 and 0630 maxima were offset in time compared to nitrate which may indicate nitrification/denitrification. The cycles had diurnal amplitudes of 0.4 mg NO3-N L-1(0.22–0.63 mg L-1, RSD 3.9%) and 0.27 mg NO2-N (0.01–0.28 mg L-1, RSD 12.4%) and could be due to biological and/or physical processes coupled to biological activity (25).
FIGURE 3. Nitrate and nitrite diurnal cycle. In situ UV simultaneous NO3-N (() and NO2-N (9), 24 h, data acquired every 5 min, trendline 20 point moving average overlaid to show diurnal cycle.
FIGURE 4. Estuarine in situ UV, simultaneous (a) NO3-N and (b) NO2-N, correlated to SFA NO3-N and NO2-N and SPM data. Samples 1–6 Towed Tamar Estuary, 7–12 Weir Quay, Tamar Estuary. In situ UV NO3-N (() and NO2-N (red square) (Weir Quay, Tamar Estuary) correlated to salinity (c) and SPM (d). LOD SFA method 0.002 mg N L-1. n ) 3 for UV in situ, n ) 3 for SFA. A significant component of the diurnal nitrate and nitrite cycles could originate from the photo-oxidation of dissolved organic nitrogen (DON), releasing bioavailable nitrogen rich compounds, thereby enhancing further bacterial metabolic mineralization of dissolved organic matter (DOM) (26–28) and the subsequent downstream advection of inorganic N. DON is both a source and sink of inorganic N (29), with Bushaw et al. (26) reporting inorganic N as 0.5 to 2% of DOM by weight and Bronk (30) also reporting freshwater DON as 0.28 – 0.56 mg N L-1. This range of potential N inputs agrees with the amplitude of the observed diurnal nitrate and nitrite cycles. The morning nitrate/nitrite cycles had smaller amplitudes than the evening, which was ascribed to lower photooxidative N inputs in the weaker morning sun, with N uptake (primary productivity) rising, resulting in early afternoon minima. Photooxidative release of N rose as the solar radiation peaked (1300–1500), with the evening nitrate/nitrite maxima resulting from reduced primary productivity, coupled to downstream advection of N released during the afternoon.
The night time nitrate/nitrite minima were due to the cessation of photo-oxidative N inputs, with their onset seen from 0600. The amplitude of the nitrate cycle was similar to that observed by Scholefield (25) (0.3–0.4 mg NO3-N L-1, June 2001). The maximum amplitude of the nitrite cycle (0.27 mg NO2-N) was greater than the 0.020 mg NO2-N L-1 observed by Scholefield (25), although the UV nitrite level did return to a similar 0.01 mg NO2-N L-1 baseline level. The difference was ascribed to photooxidative inorganic N inputs in the strong, prolonged August sunlight, increased denitrification, and to summer, extreme low flow conditions at the field site (0.32–0.41 m3 s-1: 2005 spring mean flow 10 m3 s-1, early summer mean flow 4 m3 s-1, peak flow autumn 80 m3 s-1, Environment Agency yearly median annual flood (QMED) 29.7 m3 s-1). A midwinter (23 February 2005) UV sensor deployment also found a diurnal nitrate cycle (minima at 1100 and 2000, maxima 1500 and 0500), although of smaller amplitude (0.17 mg NO3-N L-1), due to reduced photooxidative N inputs rates coupled to lower water temperature and therefore reduced biological N uptake. VOL. 41, NO. 24, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
8423
To exclude variable instrument response causing the diurnal cycles, the ratio of their amplitudes to their respective means were compared with instrument precision. No relationship was seen, and therefore, instrumental variability was not the cause of the observed diurnal nitrate and nitrite cycles. There were no regular sewage inputs upstream of the IGER site. The raised cyclical concentrations (30–62% of the observed mean nitrate/nitrite concentrations), especially for nitrite, has important implications for the accuracy of N loadings and effects calculated from coarse scale, discrete sampling, storage and laboratory analysis and demonstrates the importance of high resolution, continuous monitoring to accurately determine short-term changes in nitrate/nitrite concentrations. 3.5.2. Tamar Estuary Deployment. Nitrate and nitrite in the Tamar Estuary were monitored during ebb tides, with the in situ nitrate/nitrite concentrations correlated with SPM (Figures 4a, b, and d) and salinity (Figure 4c) to investigate their effects on nitrate/nitrite cycling. At Weir Quay salinity ranged from 12–30, with SPM rising quickly from 7 to >200 mg L-1 during the strong ebb tide. For the towed Tamar and Weir Quay data sets, good agreement (t test at p ) 0.05) was found between the ProPS real time nitrate (p ) 0.07, p ) 0.51) and nitrite (p ) 0.16, p ) 0.15) and the SFA reference data. The in situ UV nitrate and nitrite were inversely correlated with salinity (Figure 4c), reflecting essentially conservative behavior for nitrate, dominated by mixing with the River Tamar. Nitrate and nitrite were also strongly correlated with SPM (Figures 4a, b, and d), ascribed to midestuarine inorganic N inputs derived from bacterially mediated nitrification (20, 31), involving the ammonification of organic N and subsequent conversion to nitrite and nitrate in this high SPM environment. Sediment and fluid mud resuspension/settling resulted in high nitrification and denitrification rates that can impact significantly on N budgets in medium-large macro tidal estuaries such as the Tamar (31), and the change in intensity of these processes over a time scale of hours, with the raised in situ UV nitrite being rapidly oxidized to nitrate. The UV real time nitrite data was strongly correlated with SPM throughout, with, at greater than 10 mg SPM L-1, the in situ UV nitrite diverging from the laboratory SFA nitrite, which, significantly, did not rise with SPM. For the SFA analysis, the relatively consistent nitrite concentrations were due to the in situ sample filtration, which effectively stopped the particulate/bacterially mediated NO2-N production, the low level NO2-N present chemically oxidizing to NO3-N during overnight storage (4 °C) when colloidal and/or intracellular nitrogen could also be released. Furthermore, organic nitrogen has been observed as 0.4% (w/w) of SPM (Tamar Estuary midwinter turbidity maximum zone (32), with good correlation (Weir Quay, R2 ) 0.825) observed between this calculated potential N input and the divergences observed between the real time UV sensor and SFA nitrite concentrations. In addition, and especially evident during high SPM field studies, were inverse correlations of in situ UV nitrate and particularly nitrite with dissolved oxygen, an indicator of ammonification/nitrification (20), and with pH (fluctuated from 7.5 to 7.4 and back to 7.5, not a function of salinity changes), an indicator of nitrification (25). These results support nitrification/denitrification as the source of the raised, in situ UV sensor nitrite data (compared with the SFA data), as significant nitrite/nitrate contributors to the Tamar Estuary and which resulted in nonconservative nitrite behavior (20, 31). In situ N processes can significantly influence macrotidal estuary nitrite/nitrate levels as well as freshwater inputs (33), and therefore using a long-term mean Tamar estuary flow rate of 34 m3 s-1, a TMZ SPM loading of 20 -100 mg m-3 (neap tides) and 200–300 mg m-3 (spring tides) (34) and UV 8424
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 24, 2007
sensor, in situ mean NO2-N data of 0.0031 mg NO2-N mg SPM-1 gives an estuarine input of 2.1–31.6 mg NO2-N m-3 of water. This was not identified by conventional laboratory analysis and, with nitrite peaks in the Tamar midestuary strongly influencing its concentration throughout the lower estuary (20), has significant implications, especially at higher flow rates, for estuarine N loadings and N exports to coastal zones. The ProPS UV sensor is an accurate, robust, stable, relatively low cost reagentless system, deployable to 600 m. This novel, enabling technology was used to quantify unresolved, in situ nitrate and nitrite processes and patterns (diurnal nitrate/nitrite cycling/inputs from photooxidation of DON) and previously little investigated baseline perturbations, e.g., low flow events in the River Taw, and quantified SPM/fluid mud nitrification inputs to estuarine nitrate/nitrite cycling and loadings. These have important implications for freshwater and estuarine nitrate/nitrite flux calculations, aquatic pollution assessment and the biological rhythms of aquatic organisms.
Acknowledgments We gratefully acknowledge the support of TriOS GmbH (Werftweg 15, 26135 Oldenburg, Germany), in particular Ruediger Heuermann and Andreas Weichert, for the loan of the UV sensor and software support. Alan Tappin is thanked for his constructive comments, and the essential role of the Institute of Grassland and Environmental Research (IGER, North Wyke, Devon, England) in supporting field deployments acknowledged.
Supporting Information Available Details of the Tamar Estuary and R Taw (S. W England) field deployment sites used for the in situ, real time mapping of nitrate and nitrite and associated DON and nitrification inputs by the fully optimized, novel UV sensor. This material is available free of charge via the Internet at http://pubs. acs.org.
Literature Cited (1) Van Breemen, N. Nitrogen cycle: Natural organic tendency. Nature. 2002, 415, 381–382. (2) Middelburg, J. J.; Nieuwenhuize, J. Nitrogen uptake by heterotrophic bacteria and phytoplankton in the nitrate-rich Thames estuary. J. Mar. Ecol. Prog. Ser. 2000, 203, 13–21. (3) Nixon, S. W. Coastal marine eutrophication: A definition, social causes, and future concerns. Ophelia. 1995, 41, 199–219. (4) Howarth, R. W. An assessment of human influences on fluxes of nitrogen from the terrestrial landscape to the estuaries and continental shelves of the North Atlantic Ocean. Nutr. Cycling Agroecosyst. 1998, 52, 213–223. (5) Haygarth, P. M.; Condron, L. M.; Heathwaite, A. L.; Turner, B. L.; Harris, G. P. The phosphorus transfer continuum: Linking source to impact with an interdisciplinary and multi-scaled approach. Sci. Total Environ. 2005, 344 (1–3), 5–14. (6) Jordan, P.; Arnscheidt, A.; McGrogan, H.; McCormick, S. Characterising phosphorus transfers in rural catchments using a continuous bank-side analyser. Hydrol. Earth Syst. Sci. 2007, 11 (1), 372–381. (7) Hanrahan, G.; Gledhill, M.; Fletcher, P. J.; Worsfold, P. J. High temporal resolution field monitoring of phosphate in the River Frome using flow injection with diode array detection. Anal. Chim. Acta 2001, 440, 55–62. (8) Kirchner, J. W.; Feng, X.; Neal, C.; Robson, A. J. The fine structure of water-quality dynamics: the (high-frequency) wave of the future. Hydrol. Process. 2004, 18 (7), 1353–1359. (9) Mordy, C. W.; Stabeno, P. J.; Ladd, C.; Zeeman, S.; Wisegarver, D. P.; Salo, S. A.; Hunt, G. L. Nutrients and primary production along the eastern Aleutian Island Archipelago. Fish. Oceanogr. 2005, 14 (1), 55–76. (10) David, A. R. J.; McCormack, T.; Morris, A. W.; Worsfold, P. J. A submersible flow injection-based sensor for the determination of total oxidised nitrogen in coastal waters. Anal. Chim. Acta 1998, 361 (1–2), 63–72.
(11) Le Bris, N.; Sarradin, P. M.; Birot, D.; Alayse-Danet, A. M. A new chemical analyzer for in situ measurement of nitrate and total sulfide over hydrothermal vent biological communities. Mar. Chem. 2000, 72 (1), 1–15. (12) Thouron, D.; Vuillemin, R.; Philippon, X.; Lourenco, A.; Provost, C.; Cruzado, A.; Garcon, V. An autonomous nutrient analyzer for oceanic long-term in situ biogeochemical monitoring. Anal. Chem. 2003, 75 (11), 2601–2609. (13) Winkler, S.; Rieger, L.; Saracevic, E.; Pressl, A.; Gruber, G. Application of ion-sensitive sensors in water quality monitoring. Water Sci. Technol. 2004, 50 (11), 105–114. (14) Moorcroft, M. J.; Davis, J.; Compton, R. G. Detection and determination of nitrate and nitrite: a review. Talanta. 2000, 54 (5), 785–803. (15) Le Goff, T.; Braven, J.; Ebdon, L.; Scholefield, D. Automatic continuous river monitoring of nitrate using a novel ion-selective electrode. J. Environ. Monit. 2003, 5, 353–358. (16) Karlsson, M.; Karlberg, B.; Olsson, R. J. O. Determination of nitrate in municipal waste water by UV spectroscopy. Anal. Chim. Acta 1995, 312 (1), 107–113. (17) Finch, M. S.; Hydes, D. J.; Clayson, C. H.; Weigl, B.; Gwilliam, P. A low power ultra violet spectrophotometer for measurement of nitrate in seawater: introduction, calibration and initial sea trials. Anal. Chim. Acta 1998, 377, 167–177. (18) Clayson, C. H. Sensing of nitrate concentration by UV absorption spectrophotometry. In Chemical Sensors in Oceanography; Varney, M., Ed.; Gordon and Breach: London, 2000. (19) Johnson, K. S.; Coletti, L. J. In situ ultraviolet spectrophotometry for high resolution and long term monitoring of nitrate, bromide and bisulphide in the ocean. Deep-Sea Res. 2002, 1 (49), 1291– 1305. (20) Uncles, R. J.; Fraser, A. I.; Butterfield, D.; Johnes, P.; Harrod, T. R. The prediction of nutrients into estuaries and their subsequent behaviour: application to the Tamar and comparison with the Tweed, U.K. Hydrobiologia 2002, 475–476, 239– 250. (21) Collos, Y.; Mornet, F.; Sciandra, A.; Waser, N.; Larson, A.; Harrsion, P. J. An optical method for the rapid measurement of micromolar concentrations of nitrate in marine phytoplankton cultures. J Appl. Phycol. 1999, 11, 179–184. (22) Berger, A. J.; Koo, T. W.; Itzkan, I.; Feld, M. S. An enhanced algorithm for linear multivariate calibration. Anal. Chem. 1998, 70, 623–627. (23) Thomas, O.; Gallot, S.; Mazas, N. Ultraviolet multi-wavelength absorbtiometry (UVMA) for the examination of natural waters
(24)
(25)
(26)
(27)
(28) (29) (30) (31)
(32) (33)
(34)
and waste waters, Part II: determination of nitrate. Fresenius′ J. Anal. Chem. 1990, 338 (3), 238–240. Gardolinski, P. C. F. C.; Hanrahan, G.; Achterberg, E. P.; Gledhill, M.; Tappin, A. D.; House, W. A.; Worsfold, P. J. Comparison of sample storage protocols for the determination of nutrients in natural waters. Water Res. 2001, 35 (15), 3670–3678. Scholefield, D.; Le Goff, T.; Braven, J.; Ebdon, L.; Long, T.; Butler, M. Concerted diurnal patterns in riverine nutrient concentrations and physical conditions. Sci. Total Environ. 2005, 344 (1– 3), 201–210. Bushaw, K. L.; Zepp, R. G.; Tarr, M. A.; Schulz-Jander, D.; Bourbonniere, R.; Hodson, R. E.; Miller, W. L.; Bronk, D. A.; Moran, A. M. Photochemical release of biologically available nitrogen from aquatic dissolved organic matter. Nature. 1996, 381. Vahalato, A. V.; Salonen, K.; Munster, U.; Jarvinen, M.; Wetzel, R. G. Photochemical transformation of allochthonous organic matter provides bioavailable nutrients in a humic lake. Archiv fur Hydrobiogie 2003, 156 (3), 287–314. Vahalato, A. V.; Zepp, R. G. Photochemical mineralisation of dissolved organic nitrogen to ammonium in the Baltic Sea. Environ. Sci. Technol. 2005, 39 (18), 6985–6992. Berman, T.; Bronk, D. A. Dissolved organic nitrogen: A dynamic participant in aquatic ecosystems. Aquat. Microbiol. Ecol. 2003, 31, 279–305. Bronk, D. A.; Dynamics of DON. In Biogeochemistry of marine dissolved organic matter; Hansell D. A., Carlson C. A. Eds.; Academic Press: London, 2002. Abril, G.; Riou, S. A.; Etcheber, H.; Frankignoule, M.; de wit, R.; Middleburg, J. J. Transient, tidal scale, nitrogen transformations in an estuarine turbidity maximum-fluid mud system (the Gironde, South-west France). Estuarine Coastal Shelf. Sci. 2000, 50, 703–715. Tappin, A. D. School of Earth Ocean and Environmental Sciences, University of Plymouth, Devon, England. 2006, Personal communication. Trimmer, M. A.; Nedwell, D. B. A.; Sivyer, D. B. B.; Malcolm, S. J. B. Nitrogen fluxes through the lower estuary of the river Great Ouse, England: The role of the bottom sediments. J. Mar. Ecol. Progr. Ser. 1998, 163, 109–124. Grabermann, I.; Uncles, R. J.; Krause, G.; Stephens, J. A. Behaviour of Turbidity Maxima in the Tamar (UK) and Weser (F.R.G.) Estuaries. Estuarine Coastal Shelf. Sci. 1997, 45, 235–246.
ES071447B
VOL. 41, NO. 24, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
8425
