Nitrate Limitation Research Articles

Simultaneous determination of nitrite and nitrate in water by chemiluminescent flow-injection analysis

A sensitive and fully automated flow-injection system for the simultaneous determination of nitrite and nitrate in water is described. The method combines online photolytic conversion of nitrate to nitrite and the chemiluminescent detection of nitrite. Nitrite initially present in the sample is directly determined after oxidation to peroxynitrous acid by the chemiluminescent reaction with luminol. Nitrate in another portion of sample is photolytically reduced to nitrite by absorption of UV light at quartz capillaries and the sum of original and reduced nitrite is detected in the parallel detection system via the same way. Nitrate content is determined from the difference. The detection limit of nitrite (S/N=3) is 2×10 −9 mol/l and the calibration graph is linear in the concentration range 8×10 −9 to 1×10 −5 M NO 2 −. The relative standard deviation is 1.8% for 1×10 −6 M and 2.9% for 3×10 −8 M NO 2 −, respectively. The detection limit of nitrate (S/N=3) is 4×10 −9 mol/l and the calibration graph is linear in the range 8×10 −9 to 1×10 −5 M NO 3 −. The relative standard deviation is 2.1% for 1×10 −6 M and 3.6% for 1×10 −7 M NO 3 −, respectively. The interferences of cations are eliminated passing the sample through a cation-exchange column. Common anions do not interfere. Analysis time is 2.5 min. The concentrations of nitrites and nitrates found by presented method in different water samples were in a good agreement with those obtained by spectrophotometric or chromatographic method, respectively.

Nitrate removal in aquariums by immobilized pseudomonas.

Biological denitrification of nitrate to nitrogen gas was examined in a freshwater and a marine aquarium. Nitrate removal in the aquarium water was accomplished with denitrifiers immobilized in a freeze-dried, alginate-starch matrix. Starch served as a bacterial carbon source and cellular matrix-strengthening filler. Freeze-dried beads were placed in canisters through which nitrate-rich aquarium water was recirculated. The freshwater aquarium (100 L) contained goldfish (Carassius auratus) at a total biomass of 390 g, whereas cichlids (Oreochromis mossambicus) were kept at a similar stocking density in the marine aquarium. Denitrification resulted in low ambient nitrate concentrations in both aquariums. The specific nitrate removal rate of the freshwater beads was significantly higher (50 microg of NO(3)-N/bead/day) than that of seawater beads (5 microg of NO(3)-N/bead/day). Differences in ambient nitrate concentrations between both aquariums and diffusion limitation of nitrate to the active denitrification sites within the beads might explain these observed differences.

Effect of nutrient depletion on β-carotene and glycerol accumulation in two strains of Dunaliella sp.

The response of two strains of Dunaliella, a β-carotene accumulating halotolerant alga, was evaluated under sulphate, nitrate and phosphate limitation. All these factors decreased the growth rate and chlorophyll content but, increased the β-carotene content of the two isolates of Dunaliella, D1, obtained from GTCC and D2 an indigenous strain isolated from Sambhar salt lake, India. Both the strains exhibited accumulation of β-carotene and glycerol under the different nutrient limiting conditions. A maximum accumulation of 3.99 pg/cell of β-carotene was observed under phosphate depletion. However, nutrient depletion did not significantly affect the glycerol accumulation in these cells. D2, the indigenous isolate, was found to be a better accumulator of β-carotene than D1.

Flow-injection analysis method for the determination of nitrite and nitrate in natural water samples using a chemiluminescence NOx monitor.

A flow-injection analysis method for the determination of nitrite and nitrate in natural water samples has been developed that consists of two systems based on their reduction to NO with hydrazine and/or ascorbic acid, followed by chemiluminescence detection. The procedure of sweeping the generated NO into an NOx monitor, by means of a gas-liquid separating coil consisting of microporous polytetrafluoroethylene (PTFE) tubing, offers practical advantages. The adjustment of the carrier gas-flow rates could yield the same calibration graphs for the two measurement systems, and the accumulation sweeping mode provides a higher sensitivity. Chemiluminescence detection allows a wide linear calibration range of 5 x 10(-8) to 5 x 10(-5) M. The detection limits for nitrate and nitrite, defined as three-times the standard deviation of measurement blanks, are 2 x 10(-8) M and 1 x 10(-8) M, respectively, and the average precision was 3.2% at ambient natural concentration levels. Recovery tests were between 94% and 106% for a variety of natural water samples. The method is relatively free from interferences from the substances normally found in natural water, and only ferric ion has an effect for the nitrite determination.

Determination of Nitrites, Nitrates, and Their Mixtures Using Flow Injection Analysis with Spectrophotometric Detection

A new flow injection analysis (FIA) method has been developed for determination of nitrites, nitrates, and their mixtures, based on the reaction of nitrites with rivanol (2-ethoxy-6,9-diaminoacridinium lactate) in HCl medium; the concentration of the diazonium salt formed is evaluated spectrophotometrically (λ = 520 nm). Flow-through determination of nitrates can be carried out by using the same reaction following prior reduction in a flow-through reductor filled with copperised cadmium included in the apparatus. The determination of both ions in a mixture is enabled by the use of a switching solenoid valve, which either excludes the reductor (determination of only nitrites) or connects the reductor in the flow-through system (determination of the sum of nitrites and nitrates). The reaction and flow conditions with the full experimental design were optimized and detection limits for nitrite (0.14 mg L−1) and nitrate (1.2 mg L−1) determination were obtained. Both anions were determined in soil samples and it was found that excellent correlation exists between the proposed method and the standard methods (Griess reaction and ion-selective nitrate electrode).

Transparent exopolymer particle (TEP) dynamics in relation to trophic and hydrological conditions in the NW Mediterranean Sea

Seasonal abundance, volume, size distribution and carbon content of transparent ex- opolymeric particles (TEP) were examined in 2 hydrologically distinct sites in the NW Mediterranean (NWM) Sea: a coastal (Point B, Villefranche Bay) and an offshore (DYFAMED, France, JGOFS) site. TEP concentration varied from 0.2 to 2.2 × 10 5 particles ml -1 , and was higher offshore. The TEP pool was low during the winter mesotrophic period and increased after the spring bloom, remaining rela- tively high throughout summer at both sites. The increase in TEP abundance during the oligotrophic period was relatable to nitrate limitation and a decline in primary production. TEP formation in spring was associated to a nanoflagellate bloom, while the build-up of a large pool of TEP in summer oc- curred in the presence of a phytoplankton community dominated by picoplankters and during strong thermal stratification, limiting vertical sedimentation. In the NWM Sea, when the TEP carbon pool (TEP-C) is high, it may represent up to 22% of the total organic carbon, and reach down to 1% when it is low, suggesting that the particles play a significant role in the carbon cycle. In the NWM Sea, the trophic status of the system and the composition of primary producers control TEP formation. Hydrological processes appear to be of primary importance in governing seasonal TEP distribution.

Chemiluminescent flow-injection analysis of nitrates in water using on-line ultraviolet photolysis

A sensitive, fast and non-toxic flow-injection method for the chemiluminescent determination of trace amounts of nitrate in water is presented. Nitrate is on-line reduced to nitrite due to absorption of UV light at quartz capillaries and formed nitrite is instantly determined as peroxynitrite by the chemiluminescent reaction with luminol. An UV photo-reductor consisting of a high-pressure mercury lamp and two quartz capillaries provides an effective reduction of nitrate even though only deionized water as a carrier is employed. Efficiency of nitrate conversion to nitrite is 56±3%. The detection limit of nitrate is 4×10 −9 M (0.248 μg/l). Linear range of the method is 8×10 −9 to 1×10 −5 mol/l of nitrate. Cations and common anions do not interfere. Analysis time is 6 min. The method was applied to the determination of nitrate in various real water samples. The results are in a good agreement with a reference chromatographic method.

Design of SBR systems for nutrient removal from wastewaters subject to seasonal fluctuations

Designing SBR systems for simultaneous biological nitrogen and phosphorus removal is evaluated and defined for wastewaters from small coastal areas subject to seasonal fluctuations in quantity and quality. A design procedure is developed using basic process stoichiometry and significant operating parameters. A stepwise approach was utilized to secure full nitrification, available nitrate limitation for denitrification, a mixed phase with enough anaerobic fraction for P removal. A fundamental relationship between the effective sludge age, the recycle ratio and the cycle time was developed and used for the determination of physical design parameters.

Global estimates of net carbon production in the nitrate‐depleted tropical and subtropical oceans

Nitrate availability is generally considered to be the limiting factor for oceanic new production and this concept is central in our observational and modeling efforts. However, recent time‐series observations off Bermuda and Hawaii indicate a significant removal of total dissolved inorganic carbon (CT) in the absence of measurable nitrate. Here we estimate net carbon production in nitrate‐depleted tropical and subtropical waters with temperatures higher than 20°C from the decrease in the salinity normalized CT inventory within the surface mixed layer. This method yields a global value of 0.8 ± 0.3 petagrams of carbon per year (Pg C yr−1, Pg = 1015 grams), which equates to a significant fraction (20–40%) of the recent estimates (2.0–4.2 Pg C yr−1) of total new production in the tropical and subtropical oceans [Emerson et al., 1997; Lee, 2001]. The remainder is presumably supported by upward flux of nutrients into the euphotic zone via eddy diffusion and turbulent mixing processes or lateral exchange. Our calculation provides the first global‐scale estimate of net carbon production in the absence of measurable nitrate. We hypothesize that it is attributable to dinitrogen (N2) fixing microorganisms, which can utilize the inexhaustible dissolved N2 pool and thereby bypass nitrate limitation.

The large capacity for dark nitrate-assimilation in diatoms may overcome nitrate limitation of growth.

• The ability of the diatom Thalassiosira weissflogii to assimilate inorganic N in darkness is compared with that seen in flagellates. • Experiments were conducted with T. weissflogii grown in N-replete and in N-limiting cultures and the rates and capacity for ammonium and nitrate assimilation were determined. • High daily growth rates in the diatom under high-light nitrate-replete conditions are only attainable by continuing nitrate assimilation in darkness using excess C accumulated in the light when nitrate assimilation cannot match C-fixation. The ability to use ammonium in darkness is greater than for nitrate but the ratio of dark to light assimilation for each N source is similar over a wide range of cellular N:C ratios. These capabilities are in strong contrast with those in the flagellates Heterosigma carterae and Heterocapsa illdefina, which are incapable of high nitrate use in darkness. • While the possession of large capacity for dark nitrate-assimilation in diatoms may provide a mechanism that overcomes nitrate limitation of growth, the explanation for the lower capabilities exhibited by flagellates is less clear.

Efflux of organic acids in Penicillium simplicissimum is an energy-spilling process, adjusting the catabolic carbon flow to the nutrient supply and the activity of catabolic pathways.

Continuous cultivation was used to study the effect of glucose, ammonium, nitrate or phosphate limitation on the excretion of tricarboxylic acid (TCA) cycle intermediates by Penicillium simplicissimum. Additionally, the effect of benzoic acid, salicylhydroxamic acid (SHAM) and 2,4-dinitrophenol on TCA cycle intermediates was studied. The physiological state of the fungus was characterized by its glucose and O(2) consumption, its CO(2) production, its intra- and extracellular concentrations of TCA cycle intermediates, as well as by its biomass yield, its maintenance coefficient and its respiratory quotient. The excretion of TCA cycle intermediates was observed during ammonium-, nitrate- and phosphate-limited growth. The highest productivity was found with phosphate-limited growth. The respiratory quotient was 1.3 under ammonium limitation and 0.7 under phosphate limitation. Citrate was always the main excreted intermediate. This justifies calling this excretion an energy-spilling process, because citrate excretion avoids the synthesis of too much NADH. The addition of benzoic acid further increased the excretion of TCA cycle intermediates by ammonium-limited hyphae. A SHAM-sensitive respiration was constitutively present during ammonium-limited growth of the fungus. The sum of the excreted organic acids was negatively correlated with the biomass yield (Y(GlcX)).

Screening lettuce cultivars for low nitrate content during summer and winter production

SummaryConcerns about high nitrate levels in vegetable produce have led the European Union to introduce limits on nitrate concentrations in some salad crops including lettuce (Lactuca sativa L.), with the aim of decreasing nitrate intake by consumers. These limits are likely to create problems for lettuce growers in northern European countries such as the UK, where leafy vegetables can accumulate high nitrate concentrations in leaf tissues due to low light levels, especially during the winter. One option to overcome this problem is the use of low nitrate-accumulating genotypes. The objective of this work was to screen a selection of soil-grown glasshouse lettuce cultivars for head weight and nitrate concentration during the summer and the winter seasons. Two pairs of trials were carried out using a Trojan square design, following commercial cropping practices. Eight long-day commercial lettuce cultivars were grown for both early- and late-summer harvest, and eight short-day commercial cultivars for both early- and late-winter harvest. Both pairs of experiments included examples of cultivars belonging to the following types: Butterhead, Cos, Batavia, Curly, Oakleaf, Lollo Rosso and Lollo Bionda. It was found that the commercial fresh weight of the heads depends on season (summer or winter) and cultivar, with significant interaction between cultivar and experiment (i.e. harvest date within season). By comparison, nitrate concentration showed not only great variability between cultivars in general, but also between the main lettuce types and between cultivars within the Butterhead type. The data for the summer crops suggest that ‘Vegus’ (in particular) tended to accumulate less nitrate than the other cultivars of the Butterhead type. For the winter crops, although no single cultivar was found to exhibit consistently lower nitrate concentrations at harvest, the means for the Butterhead group were generally lower than the average of the cultivars for the other types. Several of the long-day cultivars were found to exceed the EU maximum summer nitrate limit of 3500 mg kg–1 fresh weight, whereas relatively few of the short-day cultivars had nitrate concentrations greater than the corresponding 4500.mg kg–1 limit for winter harvested crops.

Changes in the development of the winter-spring phytoplankton bloom in the Bay of Calvi (NW Mediterranean) over the last two decades: a response to changing climate?

The development of the winter-spring phytoplankton bloom was investigated in the Bay of Calvi (Corsica, Ligurian Sea, northwestern Mediterranean) in 1979, 1986, 1988, 1997 and 1998. A drastic reduction of phytoplankton biomass was evidenced over the last 2 decades, in relation to long- term changes in climatic and environmental conditions. Between 1979 and 1998, the monthly aver- aged chlorophyll a concentrations at 1 m decreased by about 80% during February, March and April. Simultaneously, major changes to hydrodynamic conditions include warmer water, overall decrease of salinity at 10 m depth, longer periods of bright sunshine and lower wind stress. The changes in environmental conditions were large enough to affect the vertical stability of the water column dur- ing the winter-spring period and to reduce nutrient replenishment of the surface layer prior to the usual period of phytoplankton growth. Until 1986, the main factor driving nutrient replenishment was the winter upward mixing of nutrient-rich deep waters, while the progressive reduction of mixing from 1988 induced nutrient limitation of surface waters in the last decade. The following hypotheses on changes in the development of the winter-spring phytoplankton bloom are made: (1) Until 1986, phytoplankton peaks took place in relatively high-nutrient waters and were diatom-dominated. (2) Between 1986 and 1988, decreasing Si availability led to Si limitation which caused a reduction in diatom abundance. This resulted in the disappearance of the diatom-dominated pulses and in lower phytoplankton biomass and was accompanied by a shift toward non-siliceous phytoplankton. (3) In 1988, 1997 and 1998, decreasing nitrate availability led to nitrate limitation, thus explaining the pro- gressive reduction in non-siliceous phytoplankton biomass. Other, associated changes in benthos assemblages and ichthyofauna are documented. The conclusions from the Bay of Calvi are extended to the whole western Corsican coast. This confirms that the Mediterranean reacts rapidly to external perturbations, which are driven by climate change in that particular area.

Sediment denitrification in the Gulf of Mexico zone of hypoxia

The largest zone of anthropogenic bottom water hypoxia in the Western Hemisphere occurs seasonally in the northern Gulf of Mexico between the Mississippi River delta and the coast of eastern Texas. This zone of hypoxia reaches its greatest extent in the summer months and is a conse- quence of seasonal stratification of the water column com- bined with the decomposition of organic matter derived from accelerated rates of primary production. This enhanced pro- ductivity is driven primarily by the input of inorganic nitrogen from the Mississippi River. There are 3 likely sinks for fixed nitrogen within this zone of hypoxia: sequestration in the sediment, dispersion and dilution into the Gulf of Mexico, and denitrification. We assessed potential denitrification rates at 7 stations in the zone of hypoxia during the summer of 1999. Those data are compared with bottom water nitrate, ammo- nium and dissolved oxygen (DO) concentrations. No denitri- fication was observed in the water column. Denitrification potential rates in the surface sediments were unexpectedly low and ranged between 39.8 and 108.1 µmol m -2 h -1 . The highest rates were observed at stations with bottom water DO concentrations between 1 and 3 mg l -1 . Denitrification activity was significantly lower at stations where DO was lower than 1 mg l -1 or higher than 3 mg l -1 . Nutrient data for these sta- tions demonstrate that as anoxia is approached, the dominant species of nitrogen shifts from nitrate to ammonium. The shift in nitrogen species suggests competition between microbial populations in the sediment community. The lower denitrifi- cation rates at stations with bottom water DO <1 mg l -1 may be due to nitrate limitation or an increase in the competitive advantage of microorganisms capable of dissimilatory nitrate reduction to ammonium (DNRA). Suppression of denitrifica- tion at low DO by any mechanism will increase the residence time of bioavailable nitrogen. This trend could act as a posi- tive feedback mechanism in the formation of hypoxic bottom waters.

Plant carbohydrate limitation on nitrate reduction in wetland microcosms

Although nitrate limitation in most natural wetlands results in pseudo-first-order reductions, large site-to-site variations in apparent denitrification rates cannot be easily explained by water quality (e.g., pH, Temp, DOC) or plant productivity. Our microcosm results show increasing nitrate removal efficiencies at higher ratios of total applied plant carbon to nitrate reduced, suggesting that denitrification rates may be limited by the rates of supply of both electron donor or acceptor, described by an applied carbon to nitrate (C App : N Red) ratio. However, the observed first-order rate constants varied more strongly ( r 2=0.77, p⪡0.0001) with the acid-soluble carbohydrates to nitrate (CH 2O App : N Red) ratio than the total C App : N Red ratio. Although observed rate constants for bulrush ( Scirpus sp .) were significantly lower (0.01< p<0.06) than for other plant sources ( Hydrocotyle sp., Lemna sp., Typha sp .), there were no significant differences in the plant-specific rate constants when compared at the same CH 2O App : N Red ratio. In full-scale wetlands, this suggests that either high plant productivity or low NO 3 − loading (high CH 2O App : N Red) may contribute to a high effective denitrification rate. In contrast, low productivity or a high NO 3 − loading (low CH 2O App : N Red) would promote a lower denitrification rate. This co-limitation between plant carbon and nitrate may confound first-order rate comparisons in full scale denitrification wetlands since highly N-loaded systems may become carbon limited, requiring higher order reaction kinetics to better describe performance variations. In addition to hydraulic residence time, temperature and nitrate removal data, extending these results to compare large wetlands will require estimation of the CH 2O App : N Red ratio from an inventory of plant species, productivity estimates and carbon quality.

Identifying riparian sinks for watershed nitrate using soil surveys.

The capacity of riparian zones to serve as critical control locations for watershed nitrogen flux varies with site characteristics. Without a means to stratify riparian zones into different levels of ground water nitrate removal capacity, this variability will confound spatially explicit source-sink models of watershed nitrate flux and limit efforts to target riparian restoration and management. We examined the capability of SSURGO (1:15 840 Soil Survey Geographic database) map classifications (slope class, geomorphology, and/or hydric soil designation) to identify riparian sites with high capacity for ground water nitrate removal. The study focused on 100 randomly selected riparian locations in a variety of forested and glaciated settings within Rhode Island. Geomorphic settings included till, outwash, and organic/alluvial deposits. We defined riparian zones with "high ground water nitrate removal capacity" as field sites possessing both >10 m of hydric soil width and an absence of ground water surface seeps. SSURGO classification based on a combination of geomorphology and hydric soil status created two functionally distinct sets of riparian sites. More than 75% of riparian sites classified by SSURGO as organic/alluviumhydric or as outwash-hydric had field attributes that suggest a high capacity for ground water nitrate removal. In contrast, >85% of all till sites and nonhydric outwash sites had field characteristics that minimize the capacity for ground water nitrate removal. Comparing the STATSGO and SSURGO databases for a 64000-ha watershed, STATSGO grossly under-represented critical riparian features. We conclude that the SSURGO database can provide modelers and managers with important insights into riparian zone nitrogen removal potential.

Simultaneous determination of nitrate and nitrite in saliva and foodstuffs by non-suppressed ion chromatography with bulk acoustic wave detector.

In the present paper, nitrate and nitrite in foodstuffs and saliva were simultaneously determined using a non-suppressed ion chromatography (IC) method with a bulk acoustic wave sensor (BAW) as detector, and 1.5 mmol/L potassium hydrogenphthalate (KHP) as mobile phase. The IC-BAW method is simple, rapid and accurate. The determination limits for nitrite and nitrate are 0.20 and 0.30 mg/L, respectively. The IC-BAW is comparable and agrees with the conventional spectrophotometric method for nitrite and nitrate determination.

Temporal variations in benthic communities and their response to physicochemical forcing: a numerical approach

A generic complex ecological model, the European Regional Seas Ecosystem Model (ERSEM), was applied to a shallow lagoon system in the Eastern Mediterranean. Model results depicting the seasonal variation of nutrients and Chl-α in the water column, as well as three benthic functional groups (suspended feeders, deposit feeders, and benthic carnivores), are validated with in situ data. The likely effect of a technical intervention (river input) increasing the freshwater nutrient inputs on ecosystem functioning is also investigated. Detailed annual carbon fluxes and benthic fauna biomasses are calculated, before and after the river input. The importance of external physical/chemical forcing on the pelagic system and its subsequent effect on the benthic system are demonstrated. Model experiments indicate the shift of the ecosystem from nitrate limitation to predator control with external inputs. Model experiments also show a significant increase in the amount of carbon entering the benthic system through the activity of filter feeders when river inputs are implemented.

Phytoplankton, nutrients and hydrography in the frontal zone between the Southwest Indian Subtropical gyre and the Southern Ocean

A survey was made of the Southwest Indian Ocean frontal region between 30 and 50°E containing the Agulhas Return, Subtropical and Subantarctic Fronts. From CTD, SeaSoar and extracted samples the distribution of nitrate, silicate and chlorophyll a is shown to be strongly linked to the front and water mass structure, varying zonally and meridionally. Surface chlorophyll a concentrations were low to the north and south leaving a band of elevated chlorophyll between the Subtropical and Subantarctic Fronts. The low concentration of chlorophyll a to the north, in Subtropical Water, was clearly due to nitrate limitation. Between the Subtropical and Subantarctic Fronts, where the chlorophyll a concentrations were highest, the surface layer showed silicate depletion limiting diatom growth. South of the Subantarctic Front there were deep extending, low concentrations of chlorophyll a, but despite plentiful supplies of macro-nutrients and a well-stratified surface layer, high concentrations of chlorophyll a were absent. Changes from west to east were associated with the meandering of the Southern Ocean Fronts, especially the Subtropical Front, and their strength and proximity to each other. Concentrations of chlorophyll a peaked where the Agulhas Return, Subtropical and Subantarctic Fronts were in close proximity. Combined frontal structures appear to have particularly pronounced vertical stability and are associated with enhanced upwelling of nutrients and leakage of nutrients across the front. Light levels are high within the shallow stable layer. Such conditions are clearly favourable for biological growth and support the development of larger-celled phytoplankton communities.

Rapid determination of nitrite by reversed-phase high-performance liquid chromatography with fluorescence detection

Measurement of nitrite and nitrate, the stable oxidation products of nitric oxide (NO), provides a useful tool to study NO synthesis in vivo and in cell cultures. A simple and rapid fluorometric HPLC method was developed for determination of nitrite through its derivatization with 2,3-diaminonaphthalene (DAN). Nitrite, in standard solution, cell culture medium, or biological samples, readily reacted with DAN under acidic conditions to yield the highly fluorescent 2,3-naphthotriazole (NAT). For analysis of nitrate, it was converted to nitrite by nitrate reductase, followed by the derivatization of nitrite with DAN to form NAT. NAT was separated on a 5-μm reversed-phase C 8 column (150×4.6 mm, I.D.) guarded by a 40-μm reversed-phase C 18 column (50×4.6 mm, I.D.), and eluted with 15 m M sodium phosphate buffer (pH 7.5) containing 50% methanol (flow-rate, 1.3 ml/min). Fluorescence was monitored with excitation at 375 nm and emission at 415 nm. Mean retention time for NAT was 4.4 min. The fluorescence intensity of NAT was linear with nitrite or nitrate concentrations ranging from 12.5 to 2000 n M in water, cell culture media, plasma and urine. The detection limit for nitrite and nitrate was 10 pmol/ml. Because NAT is well separated from DAN and other fluorescent components present in biological samples, our HPLC method offers the advantages of high sensitivity and specificity as well as easy automation for quantifying picomole levels of nitrite and nitrate in cell culture medium and biological samples.