Measurement of denitrification in estuarine sediment using membrane inlet mass spectrometry

Abstract Denitrification was measured in intact sediment cores and in homogenised slurries using membrane inlet mass spectrometry. Dissolved concentrations of O₂, N₂, N₂O and CO₂ were simultaneously monitored. Using a 0.8 mm diameter needle probe, a comparison was made of the gas profiles of intact cores obtained under different conditions, i.e. with air or argon as the headspace gas and after the addition of nitrate and/or a carbon source to the sediment surface. O₂ was detectable to a depth of 1 cm under a headspace of air and the depth at which the maxima of denitrification products occurred was 1.5–2 cm. Denitrification products (N₂O, N₂) occurred in the surface layers where O₂ was above the minimum level of detectability (> 0.25 μM): diffusion of N₂ and N₂O upwards from the anoxic zone, local anaerobic microenvironments or aerobic denitrification are alternative explanations for this observation. The addition of nitrate and/or acetate increased the concentrations of N₂, N₂O and CO₂ in the sediment core. In sediment slurries, the pH, nitrate concentration, carbon source and the depth from which the sample was taken affected the rate of denitrification. Nitrogen was the sole detectable end product. Maximum denitrification occurred at pH 7.5 and at 20 mM nitrate. Denitrification was at a maximum in those slurries prepared from sections of core at 1–2 cm depth.     

Direct mass spectrometric measurement of gases in peat cores

Abstract Dissolved gas concentrations (O₂, CH₄, CO₂) in peat cores were monitored simultaneously using a fine (1.56 mm diameter) membrane inlet probe connected to a quadrupole mass spectrometer. This technique allows direct measurements at specific locations within the sample with minimal disturbance. Detailed gas profiles in completely waterlogged peat samples (hollows) and samples in which the water table was several cm below the vegetation surface (hummocks) were compared. The depth of the water table played a central role in the distribution of gases. In a hollow, oxygen was present (90 μM) at the surface but was not detectable (<0.5 μM) at depths greater than 2 cm. Concentrations of CH₄ and CO₂ increased from 6 and 300 μM respectively at the surface to maxima of 450 and 3900 μM at 13 cm depth. At a hummock, O₂ and CO₂ were present above the water table but CH₄ was not detectable. CH₄ was measurable 2 cm below the water table. Both CH₄ and CO₂ concentrations increased with depth but maxima were not attained in the sampled cores.     

Effect of algal deposition on acetate and methane concentrations in the profundal sediment of a deep lake (Lake Constance)

Abstract In the profundal sediment ot Lake Constance (143 m depth) the temperature is constant at 4 °C. Despite the constant temperature, CH₄ concentrations changed with season between about 120 μM in winter and about 750 μM in summer, measured down to 30 cm depth. The acetate concentration profiles also varied between seasons. In summer, acetate concentration reached a maximum at about 100 μM in 2 or 4 cm depth. In winter, maximal concentrations of about 5 μM were observed over the entire depth. Input of organic material in late spring may be the reason for the seasonal change of both compounds. To simulate such a sedimentation event, intact sediment cores were covered with suspensions of Porphyridium aerugenium or Synechococcus sp. The addition of the phytoplankton material resulted in a drastic increase of acetate concentrations with a maximum at 2 cm depth, similar to in situ acetate concentrations measured in summer. The same applies for CH₄ for which increased concentrations were observed down to 6 cm depth. H₂ concentrations, on the other hand, showed no distinct increase. Treatment of intact sediment cores with ¹⁴C-labeled Synechococcus cells resulted in the formation of ¹⁴C-acetate, ¹⁴CH₄ and ¹⁴CO₂. Maximum concentrations of ¹⁴CH₄ were found at 4 cm depth, i.e. just above the depth to which ¹⁴C-acetate penetrated. The results show that phytoplankton blooms may cause a seasonal variation of acetate and CH₄ in profundal sediments of deep lakes despite the constant low temperature. They also indicate that acetate is the dominant substrate for methanogenic bacteria in the profundal sediments of Lake Constance.     

Ex Vivo Measurement of Brain Tissue Nitrite and Nitrate Accurately Reflects Nitric Oxide Synthase Activity In Vivo

Abstract: The ex vivo tissue concentration of nitrite and nitrate (NO_x) was found to correlate closely with the activity of nitric oxide synthase (NOS; EC 1.14.13.39) in various brain regions. Systemic administration of the nonselective NOS inhibitor N ^ω-nitro- l -arginine ( l -NA) at doses that completely inhibited both central and peripheral NOS, depleted whole-brain and CSF NO_x by up to 75% but had no effect on plasma NO_x. Selective inhibition of central NOS by intracerebroventricular administration of l -NA methyl ester produced similar decreases in levels of whole-brain NO_x. A residual concentration of NO_x of 10–15 µ M remained in all brain regions even after complete inhibition of brain NOS. Brain NO_x content decreased rapidly and in parallel with the inhibition of brain NOS. The ex vivo measurement of levels of brain NO_x was found to reflect the in vivo efficacy of several different types of NOS inhibitor: l -NA, N ^ω-monomethyl- l -arginine, and 7-nitroindazole. Intraperitoneal administration of the NOS substrate l -arginine increased brain NO_x concentrations by up to 150% of control values. These results demonstrate that the ex vivo measurement of levels of brain tissue NO_x is a rapid, reliable, and straightforward technique to determine NOS activity in vivo. This method can be used to assess both the regional distribution and the degree of inhibition of NOS activity in vivo.     

Membrane inlet mass spectrometric analysis of N-isotope labelling for aquatic denitrification studies

Abstract: Techniques are described for measuring the isotope distribution in dissolved nitrate and N₂ using membrane inlet mass spectrometry, which allows several gases to be measured in a water sample without the need for any separation steps. The isotope distribution in dissolved nitrate was measured using denitrifying Pseudomonas nautica to reduce the nitrate to N₂ which was then measured by mass spectrometry. Pseudomonas nautica NCIMB 1967 was easily grown in nitrate-limited continuous culture minimising intra- or extracellular nitrate or nitrite pools, and the bioassay was tolerant of a range of salinities. The precision of the bioassay when measuring samples with high ¹⁵NO₃⁻ contents (0.5 μmol) was 0.05 atom%; with 0.1 μmol ¹⁵NO₃⁻, the precision was around 0.2 atom%. Differences in labelling of N₂ in preserved samples obtained from ¹⁵NO₃⁻ incubations of water-covered sediment cores were measured on parallel samples with membrane inlet MS and GC-MS. The membrane inlet technique was accurate but the precision on ratio measurements was lower than by GC-MS.     

Competition between anoxygenic phototrophic bacteria and colorless sulfur bacteria in a microbial mat

Abstract The populations of chemolithoautotrophic (colorless) sulfur bacteria and anoxygenic phototrophic bacteria were enumerated in a marine microbial mat. The highest population densities were found in the 0–5 mm layer of the mat: 2.0 × 10⁹ cells cm⁻³ sediment, and 4.0 × 10⁷ cells cm⁻³ sediment for the colorless sulfur bacteria and phototrophs, respectively. Kinetic parameters for thiosulfate-limited growth were assessed for Thiobacillus thioparus T5 and Thiocapsa roseopersicina M1, both isolated from microbial mats. For Thiobacillus T5, growing at a constant oxygen concentration of 43 μmol l⁻¹, μ_max was 0.336 h⁻¹ and K _s 0.8 μmol l⁻¹. Phototrophically grown Thiocapsa strain M1 displayed a μ_max of 0.080 h⁻¹ and a K _s of 8 μmol l⁻¹ when anoxically grown under thiosulfate limitation. In a competition experiment with thiosulfate as electron donor, Thiocapsa became dominant during a 10-h oxic/14-h anoxic regimen at continuous illumination, despite the higher affinity for thiosulfate of Thiobacillus .     

Sulphate reduction in oxic and sub-oxic North-East Atlantic sediments

Abstract Oxic and sub-oxic N.-E. Atlantic sediments were examined for sulphate-reducing activity. Oxygen and/or nitrate reduction are probably the dominant mineralisation processes in the abyssal plain sediment studied. A low rate of sulphate reduction (0.1 nmol SO²⁻₄/ml/day) was recorded in the surface 5 cm of the continental slope sediment, together with the presence of a range of sulphate-reducing bacteria (SRB). A higher activity of sulphate reduction (2.2 nmol SO²⁻₄/ml/day) occurred in the continental shelf sediment which led to a small decrease in pore water sulphate and an increase in titration alkalinity. This sediment contained approx. 10²–10³ acetate, lactate and propionate oxidising SRB/ml. No low- M _r organic acids were detected in these sediments. However, amendment with 75 μM acetate stimulated sulphate-reducing activity in the shelf sediment.     

Kinetics of leaf oxygen uptake represent in planta activities of respiratory electron transport and terminal oxidases

We present, for the first time, the oxygen response kinetics of mitochondrial respiration measured in intact leaves (sunflower and aspen). Low O₂ concentrations in N₂ (9–1500 ppm) were preset in a flow-through gas exchange measurement system, and the decrease in O₂ concentration and the increase in CO₂ concentration as result of leaf respiration were measured by a zirconium cell O₂ analyser and infrared-absorption CO₂ analyser, respectively. The low O₂ concentrations little influenced the rate of CO₂ evolution during the 60-s exposure. The initial slope of the O₂ uptake curve on the dissolved O₂ concentration basis was relatively constant in leaves of a single species, 1.5 mm s⁻¹ in sunflower and 1.8 mm s⁻¹ in aspen. The apparent K _0.5(O₂) values ranged from 0.33 to 0.67 μ M in sunflower and from 0.33 to 1.1 μ M in aspen, mainly because of the variation of the maximum rate, V _max (leaf temperature 22°C). The initial slope of the O₂ response of respiration characterizes the catalytic efficiency of terminal oxidases, an important parameter of the respiratory machinery in leaves. The plateau of the response characterizes the activity of the mitochondrial electron transport chain and is subject to regulations in accordance with the necessity for ATP production. The relatively low oxygen conductivity of terminal oxidases means that in leaves, less than 10% of the photosynthetic oxygen can be reassimilated by mitochondria.     

A method for long-term measurement of respiration in intertidal fishes during simulated intertidal conditions

Aquatic and aerial respiration of the amphibious fishes Lipophrys pholis and Periophthalmus barbarus were examined using a newly designed flow-through respirometer system. The system allowed long-term measurements of oxygen consumption and carbon dioxide release during periods of aquatic and aerial respiration. The M o ₂ of L. pholis , measured at 15° C, was 2·1 μmol O₂ g^–1 h^–1 during aquatic and 1·99 μmol O₂ g^–1 h^–1 during aerial exposure. The corresponding values of the M co₂ were 1.67 and 1.59 μmol O₂ g^–1 h^–1 respectively, giving an aquatic respiratory exchange ratio (RER) of 0·80 and an aerial RER of 0·79. The M o₂ of P. barbarus , measured at 28°C, was 4·05 μmol O₂ g^–1 h^–1 during aquatic and 3·44 μmol O₂ g^–1 h^–1 during aerial exposure. The corresponding values of the Mco₂ were 3·29 μmol CO₂ g^–1 h^–1 and 2·63 μmol CO₂ g^–1 h^–1 respectively, giving an aquatic RER of 0·81 and an aerial RER of 0·77. While exposed to air for at least 10 h, both species showed no decrease in metabolic rate or carbon dioxide release. The RER of these fishes equalled their respiratory quotient. After re-immersion an increased oxygen consumption, due to the payment of an oxygen debt, could not be detected.     

Development of a gas diffusion probe for the determination of methane concentrations and diffusion characteristics in flooded paddy soil

Abstract A probe for the measurement of dissolved CH₄ in anoxic methanogenic environments was developed. The probe was based on the diffusion of dissolved CH₄ through a silicone membrane into a gas space at the end of the probe. This gas space was flushed with N₂ and analyzed gas-chromatographically for CH₄. The probe had a spatial resolution of < 1.3 mm, the detection limit was about 20 μM CH₄, the precision of the measurement was 9%, and consecutive measurements could be made every 4 min. Memory effects after analysis of high CH₄ concentrations could be avoided by flushing the probe with N₂ between each measurement. The probe was sensitive for water movement and, therefore, was calibrated in an artificial sediment of glass beads (100 μm diam.) immersed by aqueous solutions of known CH₄ concentrations. Sensitivity of the probe for changes in the sediment's porosity could not presently be excluded. The probe was used to measure vertical profiles of dissolved CH₄ in microcosms of anoxic paddy soil. The vertical CH₄ profiles measured with the probe compared fairly well with those measured after an extraction procedure. The profiles clearly showed that CH₄ was produced in deeper layers and diffused upwards to be consumed in the oxic top 2 mm soil layers. The probe was also used to determine the diffusion coefficient of CH₄ in an inactivated paddy soil microcosm using a set-up which allowed modelling of a measured CH₄ concentration profile using Fick's 2nd law.     

Effect of organic loading on nitrification and denitrification in a marine sediment microcosm

Abstract The effects of organic additions on nitrification and dentrification were examined in sediment microcosms. The organic material, heat killed yeast, had a C/N ratio of 7.5 and was added to sieved, homogenized sediments. Four treatments were compared: no addition (control), 30 g dry weight (dw) m⁻² mixed throughout the 10 cm sediment column (30M), 100 g dw m⁻² mixed throughout sediments (100M), and 100 g dw m⁻² mixed into top 1 cm (100S). After the microcosms had been established for 7–11 days, depth of O₂ penetration, sediment-water fluxes and nitrification rates were measured. Nitrification rates were measured using three different techniques: N-serve and acetylene inhibition in intact cores, and nitrification potentials in slurris. Increased organic additions decreased O₂ penetration from 2.7 to 0.2 mm while increasing both O₂ consumption, from 30 to 70 mmol O₂ m⁻² d⁻¹, and NO₃⁻ flux into sediments. Nitrification rates in intact cores were similar for the two methods. Highest rates occurred in the 30M treatment, while the lowest rate was measured in the 100S treatment. Total denitrification rates (estimated from nitrification and nitrate fluxes) increased with increased organic addition, because of the high concentrations of NO₃⁻ (40 μM) in the overlaying water. The ratio of nitrification: denitrification was used as an indication of the importance of nitrification as the NO₃⁻ supply for denitrificaion. This ratio decreased from 1.55 to 0.05 iwth increase organic addition.     

Photosynthesis and photoassimilation of glucose during photomixotrophic growth of Marchantia polymorpha cells in suspension culture

Even in the presence of glucose the growth of Marchantia polymorpha L. (cell line HYH-2F) requires light, and growth is more sensitive to 10⁻⁶ M 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea than to 10⁻⁴ Antimycin A. The inability of the cells to grow in the dark is due to the low level of respiration. The respiration rate under light increased to four times the dark value. The values of the compensation ratio (the photosyntehtic rate/the respiration rate) for the oxygen exchange were below 1.0 daring the growth period, although oxygen evolution was found. At the early exponential phase, oxygen evolution was 0.373 μmol (mg cell dry weight)⁻¹ h⁻¹ [61.7 μmol (mg chlorophyll)⁻¹ h⁻¹]. M. polymorpha cells are unable to grow anaerobically in the light without a supply of carbon dioxide. When 1% carbon dioxide in nitrogen is supplied, photochemically produced oxygen and energy are sufficient for sustained growth although at significantly reduced yields in both cell dry weight and chlorophyll. Photosyntehtic CO₂ assimilation rate was 0.13 μmol (mg cell dry weight)⁻¹ h⁻¹[11.3 μmol (mg chlorophyll)⁻¹ h⁻¹]. At least one-third of the carbon atoms in cellular constituents seem to be derived from atmospheric carbon dioxide, which indicates that M. polymorpha cells grow photomixotrophicaily.     

Hydrogen-dependent control of the continuous thermophilic anaerobic digestion process using membrane inlet mass spectrometry

The control of a thermophilic continuous anaerobic digestion system when subjected to potential inhibitory shock loadings was achieved through the regulation of dissolved H₂, monitored using membrane inlet mass spectrometry, by the controlled addition of carbon source. At a feed pump switching threshold equivalent to 1 μmol/1 H₂ a steady state rate of methanogenesis of approximately 40 μmol/1/min was obtained. Higher H₂ thresholds resulted in an inhibition of methanogenesis, but precise control of H₂ concentration was demonstrated with an oscillatory response of period 2·5–5·0 min.     

Vertical distribution and abundance of invertebrates within the sandy substrate of a low-gradient headwater stream

SUMMARY. 1 The vertical distribution of invertebrates (>0.053 mm) was studied in a sandy-bottomed, first-order stream on the Coastal Plain of Virginia, U.S.A. Invertebrate species composition, abundance and bio-mass were determined monthly over one year at sediment depth intervals of 0–1, 1–5, 5–15,15–30 and 30–40 cm.
2. The subsurface community was numerically dominated by species of Chironomidae, Nematoda and Crustacea, while much of the biomass was due to early instars of several species of Trichoptera.
3. Invertebrate density and biomass decreased significantly with depth in the substrate (ANOVA; P <0.05). Annual mean density decreased from 1,346,844 individuals m⁻³ at the surface to 13,578 individuals m⁻³ at 15–30 cm. Annual mean biomass decreased from 66.30 g m⁻³ at the surface to 0.44 g m⁻³ at 15–30 cm.
4. Dissolved oxygen decreased markedly from the surface to the 5 cm depth in the substrate, anaerobic conditions often occurring below 10 cm. Density and biomass both showed a significant positive relationship with dissolved oxygen concentration (Linear regression; P <0.05).
5. Physical forces were important in structuring the subsurface invertebrate community. Besides low dissolved oxygen concentration, sediment scouring resulting from storm discharge dramatically reduced density and biomass     

THE SURVIVAL OF RAINBOW TROUT (SALMO GAIRDNERII RICHARDSON) AND PERCH (PERCA FLUVIATILIS L.) AT VARIOUS CONCENTRATIONS OF DISSOLVED OXYGEN AND CARBON DIOXIDE

The relative importance of dissolved oxygen and dissolved carbon dioxide in determining the lethal effect of an environment for rainbow trout and perch has been investigated with an apparatus which controls the concentrations of these gases in a body of water. It is shown that concentrations of carbon dioxide which sometimes occur in polluted streams can more than double the minimum concentration of dissolved oxygen necessary for the survival of half a population of rainbow trout fingerlings for 24 hr. Increase in temperature between 12·5 and 19·5°C. shortens period of survival in solutions containing up to 67 p. p. m. CO₂. Within the range of dissolved oxygen concentration which is lethal in the presence of 59 p. p. m. CO₂ or more, perch are more resistant than rainbow trout in the lower, but less resistant in the higher, oxygen concentrations. The relation between carbon dioxide concentration and the oxygen tension at which rainbow trout blood is half saturated with oxygen is similar to the relation between carbon dioxide concentration and the oxygen tension at which the median period of survival of this species is I hr.     

Spatial variation of dissolved gas concentrations in a willow vegetation filter treating landfill leachate

Membrane inlet mass spectrometry (MIMS) was used to monitor continuously and simultaneously the concentrations of dissolved gases (O₂, CO₂, CH₄) within the treatment bed of a willow vegetation filter treating leachate at a landfill site in mid Wales. The distribution of dissolved gasses within the bed was shown to be highly heterogeneous at the small spatial scale with considerable variation between vertical profiles measured simultaneously at different locations. In general, aerobic conditions were observed above the water table with reduced levels of oxygen and increasing levels of carbon dioxide and methane below it. Distinct pockets of oxygen (up to 200 μM) were observed in anaerobic zones and pockets of reduced oxygen and elevated carbon dioxide were observed in the aerobic zone. Pockets of methane were observed in some profiles coexisting with up to 200 μM oxygen at 5 cm depth. These observations confirm the hypothesis that micro-sites exists within the soil/root matrix where aerobic organic matter decomposition and anaerobic processes such as methanogenesis can occur in relatively close proximity to each other. We hypothesise that the distribution of dissolved gases is determined by rapid diffusion of air maintaining aerobic conditions above the water table, removal of oxygen by microbial processes creating anaerobic conditions below the water table and the distribution of willow roots in the soil which create local aerobic zones by oxygen release.     

Oxygen Perception and O2 Toxicity in the Freshwater Ciliated Protozoon Loxodes

ABSTRACT. Loxodes reached peak abundance close to the oxic-anoxic boundary (O₂ 5% atm) in two lakes, in test tube cultures, and in glass chambers with horizontal O₂ gradients. Vertical profiles of CO₂, pH, sulfide, and Fe²⁺ in a lake were not closely related to Loxodes abundance. In a laboratory experiment, Loxodes followed a retreating source of O₂ and was repelled by a high pO₂. This behavior was sustained when cells simultaneously swam up or down gradients of both CO₂ and pH. Aggregation of cells was abolished by KCN (10^-4-10^-6 M). Sodium azide (10^-1-10^-4 M) had no effect and 2,4-DNP sharpened the aggregation. Rotenone, Antimycin A, and HOQNO had no obvious effect. Cytochrome oxidase is probably the oxygen receptor. Loxodes striatus contained low activities of superoxide dismutase and catalase. Extracellular production of superoxide (O_-²) and hydrogen peroxide (H₂O₂) were probably not responsible for the exclusion of Loxodes from water with a high pO₂. Continuous exposure of Loxodes to oxygen at normal atmospheric pressure at 10°C led to 50% mortality in 10 days. Cells left free to swim in an oxygen gradient doubled their number in the same period. Light exacerbated the toxic effects of O₂. Behavioral responses to the dissolved oxygen tension probably controlled the spatial distribution of Loxodes.     

Atmospheric nitrogenous compounds and ozone – is NOx fixation by plants a possible solution?

Air quality thresholds for O₃ for the protection of human health and vegetation set by the European Union (EU) have been exceeded in Europe regularly in the 1990s. Because target reductions for oxides of nitrogen (NO_x) set for the year 2000 are unlikely to be achieved, these O₃ exceedances are likely to continue into the next millenium. Improvements of plant tolerance towards O₃ are being investigated but very little work has been done to explore NO_x tolerance and plant acclimation to NO₂ and NO. However, it is clear that within the populations of some plant species there is wide variation, and some individuals can fix NO_x and use the nitrogen directly from the atmosphere, rather than rely upon, for example, root uptake of nitrate. It is possible that individuals capable of fixing NO_x could be selected for a range of species, and genotypes with high rates of uptake could be of value as crops or for forestation in polluted areas (e.g. landscaping in the vicinity of motorways) to reduce tropospheric concentrations of NO_x significantly and also to decrease the potential for O₃ production.     

Activity of methanotrophic bacteria in Green Bay sediments

Abstract Sediment pore water samples obtained from a 19 m station in Green Bay in Lake Michigan were examined for levels of ambient dissolved methane and copper, and for the potential for in situ methane oxidation by methanotrophs found within surface sediments. The in situ methane concentration in the upper oxic sediment layer ranged from 20–150 μmol · 1⁻¹ at this station. The activity of methanotrophs and the kinetics of methane oxidation in these sediments were demonstrated by the uptake of radiolabeled methane. K_s values varied between 4.1–9.6 nmol · cm³ of sediment slurry. High V_max values (12.7–35.2 nmol · cm⁻³ · h⁻¹) suggest a large population of methanotrophs in the sediments. An average methane flux to the oxic sediments of 0.24 mol · m⁻² · year⁻¹ was calculated from the pore water methane gradients. Pore water concentrations of copper in the upper sediment layer ranged from 10–120 nmol · 1⁻¹. Based upon the copper concentration, other measured parameters, and equilibrium conditions defined by WATEQF4, an estimate for dissolved free Cu²⁺ concentration of 5–38 nmol · 1⁻¹ pore water was obtained. Several factors control the rate of methane oxidation, including oxygen, methane, and the bioavailability of free Cu²⁺.     

Characterization of Serotonin N-Acetyltransferase in the Lateral Eye of the Green Frog Rana perezi: Protective Action of EGTA

Abstract: The kinetics of seRotonin N -acetyltransferase (NAT) from the lateral eye of Rana perezi have been characterized. NAT from ocular tissue reached maximal activity at a phosphate buffer concentration of 250 m M and a pH of 6.5. Reaction linearity was highly conserved within the homogenate fraction range tested (0.033-0.33). The time course of ocular NAT reaction showed a high linearity at 25 and 35°C. K _m and V_max estimations for acetyl-CoA at a 10 m M tryptamine concentration were 63.3 μ M and 4.42 nmol/h per eye, respectively. Regardless of the acceptor amine (tryptamine or serotonin), the K _m was not affected by the acetyl-CoA concentration (50 or 250 μ M ), whereas the V _max was significantly increased at a 250 μ M acetyl-CoA concentration. Ocular NAT showed a higher affinity for serotonin ( K _m= 20.7 μ M ) than for tryptamine ( K _m= 48-60 μ M ); V _max however, was similar for both substrates. Acetyl-CoA does not protect ocular NAT; in contrast, the use of EGTA (4 m M ) in the assay is essential to protect the enzyme because NAT in ocular crude homogenate shows rapid inactivation. This result suggests that intracellular calcium levels are involved in the NAT inactivation mechanisms in frog ocular tissue.