Oxygen Deficient Conditions and Nitrate Reduction in the Eastern Tropical North Pacific Ocean

Oxygen Deficient Conditions and Nitrate Reduction in the Eastern Tropical North PacificOceanAuthor(s): Joel D. Cline and Francis A. RichardsSource: Limnology and Oceanography, Vol. 17, No. 6 (Nov., 1972), pp. 885-900Published by: American Society of Limnology and OceanographyStable URL: http://www.jstor.org/stable/2834614 .

Accessed: 23/06/2014 15:34

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].

.

American Society of Limnology and Oceanography is collaborating with JSTOR to digitize, preserve andextend access to Limnology and Oceanography.

http://www.jstor.org

This content downloaded from 62.122.73.30 on Mon, 23 Jun 2014 15:34:48 PMAll use subject to JSTOR Terms and Conditions

http://www.jstor.org/action/showPublisher?publisherCode=limnoc

http://www.jstor.org/stable/2834614?origin=JSTOR-pdf

http://www.jstor.org/page/info/about/policies/terms.jsp


OXYGEN DEFICIENT CONDITIONS AND NITRATE REDUCTION IN THE EASTERN TROPICAL

NORTH PACIFIC OCEAN1

Joel D. Cline2 and Francis A. Richards Department of Oceanography, University of Washington, Seattle 98195

ABSTRACT

Oxygen concentrations in the oxygen-minimum zone of the eastern tropical North Pacific Ocean are at least an order of magnitude lower than previously reported. Concentrations of less than 1l,ug-atom/liter are common throughout the region from below the pycnocline to depths of several hundred meters.

In these nearly anoxic waters, nitrate appears to be reduced to nitrite and free nitrogen. Nitrate deficits, derived from material balance calculations, suggest that up to 13-14 Ag-atoms/liter of nitrate-nitrogen have been so reduced. This is 40-50% of the nitrate normally present in this depth interval. Secondary nitrite concentrations rarely exceed 1.5 Ag-atoms/liter and are generally associated with oxygen concentrations of less than 2 ,ug-atoms/liter. Ammonia concentrations appear to be slightly lower in the region of denitrification, probably because of bacterial assimilation.

If the nitrate reduction rate observed in Darwin Bay in the Galapagos Islands were to apply as a rate of denitrification in the entire oxygen-minimum zone in the eastern tropical North Pacific, the annual loss of 920 X 1012 g of combined nitrogen could be expected. However, considerations of environmental differences suggest that a figure somewhat less than a quarter of this amount is more probable.

INTRODUCTION

In the past 10 years, considerable atten- tion has been focused on the extensive oxygen-minimum3 region of the eastern tropical Pacific Ocean. Of particular interest has been the occurrence of the secondary nitrite maximum below the thermocline and within the upper part of the oxygen-minimum zone. This maximum appears to be associated with oxygen concentrations of less than 0.1 ml/liter

1 This work was supported by National Sci- ence Foundation Grant GA10084 and by Office of Naval Research Contracts NONR-477(37) and N-00014-67-A-0103-0014 with the University of Washington. Contribution No. 670 from the Department of Oceanography, University of Washington.

2 Present address: Institute of Geophysics and Planetary Physics, University of California at Los Angeles 90024.

3 In this paper, "oxygen-minimum" zone, layer, or region will be used generally to refer to the volume or depth interval in which oxygen concentrations determined by the Winkler titration are less than 0.15 ml/liter, or in which the concentrations determined by the colorimetric method of Broenkow and Cline (1969) are less than 0.05 ml/liter.

and suggests anaerobic nitrate reduction (Brandhorst 1959). In contrast, the primary nitrite maximum, usually above or within the thermocline, is not associated with low oxygen concentrations and is believed to result either from ammonia oxidation (Harvey 1955; Wada and Hat- tori 1971, 1972) or excretion by phytoplankton (Vaccaro and Ryther 1960).

Evidence of denitrification in these nearly anoxic waters was provided by Thomas (1966), who demonstrated that significantly more nitrate had been reduced than could be accounted for as nitrite. He suggested that nitrate had been reduced beyond nitrite, probably to free nitrogen or nitrous oxide (denitrification).

Observations by Fiadeiro and Strick- land (1968) off the coast of Peru indicated that the secondary nitrite maximum there is associated with low oxygen concentrations (02 < 0.2 ml/liter) and a deficiency in nitrate ion. They suggested that departures from the expected nitrate concentrations may arise from a slow regeneration rate within the oxygen-minimum region, but they were unable to show this conclu-

LIMNOLOGY AND OCEANOGRAPHY 885 NOVEMBER 1972, V. 17(6)



886 JOEL D. CLINE AND FRANCIS A. RICHARDS

1150 1100 1050 100? 950 900 850 80?

25 2501

200 oTT02629 200

78 27*

0~~~~~~~~~~~~~~

15? I50 0? 0?9?90 5 0

10c o 0 3233 343539 3(3gJ42 TT76 10

5~~~~~~~~~~~~~~5 23

50 [9~~~~~~~~~~2

19~~~~~~~~~~~~4 50~~~~~~~~~~~~~~~~~~~

115 110 050 1Q00 95~ ~~0 9 24 8518

Fic.1. tatin lcatins or PNCH (TT026 and PIC (TT035,TT07)

sively and hence concluded that the observed nitrate deficiencies were probably the result of denitrification.

Observations in the waters off southern Mexico and Central America in 1968 and 1969 have provided additional information on the biochemical processes occurring within this oxygen-deficient environment. Our specific purpose has been to relate the distributions of dissolved oxygen and compounds of nitrogen and phosphorus in the oxygen-minimum zone to the effects of oxygen deficiencies on nitrate reduction, denitrification, and attendant microbiolog- ical processes occurring there. The observations were made aboard the RV Thomas G. Thompson (University of Washington)

in March 1968 (PONCHO cruise TT-026) and in February and April 1969 (PISCO cruise TT-035, 037) (Fig. 1).

We wish to thank W. W. Broenkow, R. C. Dugdale, and J. J. Goering for their criticisms and suggestions concerning this paper. Special thanks are due to S. C. Rit- tenberg for data and valuable discussions, and to R. W. Owens, Jr., and T. S. Mili- lauaopele of the Bureau of Commercial Fisheries, La Jolla, California, who al- lowed us to preview oxygen data from the EASTROPAC expedition. Thanks are also due to J. Anderson, who provided the sta- tistical analyses. We also want to thank the graduate students and technicians who devoted time and effort to the chemical



NITRATE REDUCTION, EASTERN TROPICAL PACIFIC 887

analyses. However, the results and con- clusions are our sole responsibility.

METHODS AND PROCEDURES

Chemical and physical observations included salinity, temperature, dissolved oxygen (Winkler and colorimetric), inorganic phosphate, nitrate, nitrite, nitrous oxide, ammonia, silicate, pH, alkalinity, total carbon dioxide, dissolved hydrogen sulfide, sulfite, thiosulfate, and soluble and particulate iron. During the PISCO cruise, nitrate, nitrite, ammonia, phosphate, and silicate determinations were carried out on a Technicon AutoAnalyzer according to the recommendations of Hager et al. (1968).

Salinity was determined from conduc- tivity measurements.

Temperature was observed using deep- sea reversing thermometers.

Dissolved oxygen was determined two ways: by the Winkler titration using reagents recommended by Carpenter (1966) and by a colorimetric procedure designed for low oxygen concentrations (Broenkow and Cline 1969).

Inorganic phosphate was determined by the ascorbic acid-potassium antimonyl tar- trate method of Murphy and Riley (1962).

Nitrate ion was estimated by the cadmium-copper reduction method of Wood et al. (1967) adapted for use on the Auto- Analyzer.

Nitrite was determined by the method of Bendschneider and Robinson (1952).

Nitrous oxide was tested for chromato- graphically using a Barber Colman series 5000 gas chromatograph, equipped with a model 5120 electron capture detector and model 5041-1 electrometer. The detector was operated in the helium ionization mode, with 1-mm electrode spacing and a potential field of 2,000 V/cm. The output signal was recorded on a series 8000, 1-mV recorder.

The standard procedure was to remove the dissolved gases from a 5-ml aqueous sample in a gas stripper, similar to the type described by Swinnerton et al. (1962),

and separate the N20 from the air gases on a 2- x 0.5-cm column of silica gel, 60/80 mesh (Appl. Sci.). Carbon dioxide was absorbed on Ascarite (A. H. Thomas Co.) and water vapor on Drierite (Ham- mond Drierite Co.). The calibration gas (51 ppm N20 in He) and sweep gas (ul- trapure He) were prepared by Air Prod- ucts and Chemicals.

The detection limit of N20 is 0.5 Ztmole /liter, using a 5-ml aqueous sample, equivalent to a signal amplitude of twice the nominal noise level.

Ammonia was determined by the method of Richards and Kletsch (1964).

Reactive silicate was determined by the method of Mullin and Riley (1955), modified by using Na2SiF6 as a primary standard.

Dissolved hydrogen sulfide was estimated by a colorimetric methylene-blue method (Cline 1969).

Sulfite and thiosulfate concentrations were determined colorimetrically, using the procedures of West and Gaeke (1956) and Urban (1961).

Water samples were collected in 5.6-liter all-plastic Niskin bottles and 6-liter high- impact polyvinyl chloride samplers, modified from a design by Carruthers et al. (1950).

All nutrient samples were stored at 2C until analyzed, usually within 4 hr of sampling. Oxygen and other gasometric analyses were carried out immediately.

OBSERVATIONS

The distribution of oxygen along a section normal to the southern coast of Mex- ico is shown in Fig. 2 (PONC'HO cruise, stations 16-24). When the oxygen concentration fell below 25 jug-atoms/liter, colorimetric oxygen values were used; the Winkler values were consistently higher by 8-10 jug-atoms/liter (-0.1 ml/liter).

Anoxic or nearly anoxic conditions were found between 200 and 800 m. In many cases, oxygen was undetectable (02 < 1 jug-atom/liter). Iodine blanks determined on all samples showed the presence of




STATIONS 016 017 018 019 020 021022023 024

I ~ ~~~~ ~ 2- 0

200 ? 200 200 2 20 2 . ,

15

_10\ 5 0. . 2 lss

5. 400

E 3 4 2-.

FIG. 0. Lug- atoms giter

600 27

2

800 10 "

1000, ___ 400 300 200 100 0

DISTANCE OFFSHORE, nautical miles

FIG. 2. Oxygen concentrations (ALg-atoms/liter) along a section normal to the southern coast of Mexico (PONCHO, stations 16-24, TT-026). Ox- ygen concentrations less than ca. 25 ALg-atoms/liter were measured by the colorimetric procedure of Broenkow and Cline (1969).

compounds that reduced iodine. The maximum observed concentration of these substances was approximately 2 pt-equivalents /liter as iodine. Interference by nitrite was eliminated by the addition of sodium azide to the KOH-KI reagents.

Two distinct features of the oxygen-minimum zone are the apparent deficits in nitrate and the unusually high concentrations of nitrite. It is generally accepted that an apparent loss of nitrate in these nearly anoxic waters arises from respiratory nitrate reduction in which nitrate ion serves as the terminal electron acceptor in the oxidation of organic material (Richards 1965). Nitrite ion presumably is an intermediate product in the overall process leading to free nitrogen (denitrification).

NITRATE DEFICITS (ANOMALIES)

Investigations in the eastern tropical Pacific Ocean have shown that reduction

beyond nitrite, presumably to free nitrogen, has occurred. This raises questions concerning the amount of nitrate reduction and the rates of these processes.

As an initial step in examining these questions, we estimated the amount of nitrate reduction from the following equation:

AN = (NO3)exp- (NO3) obs- (NO2)obs (1)

where (NO3 ) exp is the amount of nitrate4 expected from both oxidative and preformed sources and represents the concentrations expected to be present before the onset of nitrate reduction. The other terms on the right-hand side of equation 1 are observed quantities. Ammonia-nitrogen was not included in the mass balance because of its low concentration (<1 ,g- atom/liter) throughout most of the water column; only the near-surface layers con- tained higher concentrations. The quan- tity ( NO3) exp was estimated from the apparent oxygen utilization (AOU), nitrate, and phosphate relationships estab- lished in or near the survey area. The use and validity of these relationships have been discussed previously (Redfield 1934; Redfield et al. 1963). Observed AOU: NO3: P04 ratios for the region are shown in Fig. 3; the slopes, determined by two- way least-squares regression analysis, are compared with the values obtained by Broenkow (1965) for the Costa Rica Dome (Table 1).

The AAOU: AP04 ratio in the upper 100-200 m differs significantly from that in the water below 900 m (Antarctic Inter- mediate Water), but it is nearly the same as the theoretical value given by Redfield et al. (1963) for the decomposition of marine plankton. The smaller value in the

4 N03, NO2, P04, and NH4 will be used as ab- breviations for NO3-, NO2-, PO4-, and NH4+.

FIG. 3. Observed AOU: NO3: P04 ratios for PONCHO stations 7-40 (TT-026) and stations 16-24 PISCO (TT-037). For clearer presentation, only a portion of the data in Table 1 is shown here. Indi- cated ratios are from Table 1. The changes in slope occur at about 150-200 m and 900-1,000 m.




600 0 o B

0 . 0 J

L. 0 0~~~~~~~~~~0 0 500 X? _D 0 *0 0 =

. AAOU 100 _ . A*..SHALLOWSAMPLESN03168 E

o300 * g

:D200* 0

0~~~~~~~~~~

100 a SHALLOW SAMPLES

. DEEP SAMPLES

*0

0 5 10 15 20 25 30 35 40 45 50

NO3 ,,ug-atoms / h ter

I I I I I ,1 1 1 600 -

500

0200 r 400 0

10 0. 2

O 1.0 3-2 0 3 0 4.0 5.C




deep water suggests that phosphate has been released early in the decomposition of marine plankton. A low ratio could also arise from a decrease in preformed phosphate below the Antarctic Intermediate Water, but this seems unlikely.

A similar change in the AAOU: ANO3 ratio is observed in the same depth inter- vals, but it appears that less nitrate relative to phosphate has been liberated in the deep water (ANO3: AP04 = 10) .

In the water between the salinity maximum of the Equatorial Pacific and the Antartcic Intermediate Water, the AAOU: ANO3: AP04 ratios are substantially altered by mixing of these two water types (Table 1). Consequently, any estimate of the nitrate loss in this depth interval calculated from a nitrogen mass balance will depend on both the in situ oxidative ratios and the degree of mixing that has occurred.

The first term on the right of equation 1 for the nitrate anomaly is:

(NO3)exp = (NO3)0Ox + (NO3) p, (2) where (NO3)ox and (NO3) p are the oxidative and preformed nitrate concentrations present before the onset of nitrate reduction. The oxidative nitrate is assumed to be related to the apparent oxygen utilization (AOU) by the following equation:

(NO3)ox = [ANO3/AAOU]z(AOU). (3)

The subscript z implies that the oxidative ratio: [AN03/AAOU], may be depth de- pendent. Preformed nitrate is assumed to be some function of preformed phosphate.

1.4 I l I

ao SALINITY MINIMUM 1.2 *7

STA 16 0 cn 0 ~~~~~17o

E 1.0 - 18* _ o 0\ 19 A

\ o 20v

e08: X ~~~~~21 0 0.8

0 OA\

0.6

SALINITY MAXIMUM o0

0.4l l ' _ 34.6 34.8 35.0

SALINITY /o

FiG. 4. Preformed phosphate vs. salinity in the depth interval 150-900 m at stations 16-22, PONCHO (TT-026).

To test the constancy of the oxidative ratios in the depth interval of about 150- 900 m, preformed phosphate (PO4)p was calculated from the expression:

(P04)p =(P04)ObS-[(AOU)/263], (4) where (AAOU: APO4) 150-900 m = 263 (from Table 1), and plotted against salinity. The results are shown in Fig. 4 for stations 16-22 (TT-026). The relationship between salinity and temperature is also linear in this depth interval. Inorganic phosphate liberated during nitrate reduction was estimated to be no greater than 0.15 yug-atom /liter, assuming that the ratio ANO3: APO4 = 94.5 (Richards 1965) obtains during denitrification and that a maximum of 14

TABLE 1. AOU:NO8:PO4 relationships* in the northeastern tropical Pacific Ocean

1 2 3 4 5 6 7

Depth b a Sb r n Slope Theoretical interval ratios

(m)

AAOU 0-150 263 0.57 ?3.3 0.97 294 274 276 APO4 >900 231 0.87 ?4.6 0.98 95

AAOU 0-150 16.8 2.3 ?0.2 0.99 299 16.5 17.2 ANO3 >900 23.0 18.4 ?0.9 0.98 102

* Key: 1-2-The quantities a and b are the average intercept and slope, determined by two-way linear regression analysis; 3-standard error in the slope; 4-correlation coefficient; 5-number of values; 6-from Broenkow (1965); 7-from Redfield et al. (1963).




STATI ON S 016 017 018 019 020 021022023 024

C ' I ,1 I1 ,

2000

0

400 5 2

E /0 m

PONCH (TT ~-02) Nirt anml / n

600 05

5

800

0

400 300 200 100 0 DISTANCE OFFSHORE, nautical miles

FxG. 5. Calculated nitrate anomaly and observed nitrite distributions for stations 16-24, PONCHO (TT-026). Nitrate anomaly (-~) and dissolved nitrite (---) concentrations are given in /ug-atoms/liter.

yug-atoms/liter of nitrate ion had been reduced. No attempt was made to correct equation 4 for this contribution to the phosphate pQol.

The linearity of the relationship between preformed phosphate and salinity between 150 and 900 m implies that the AAOU: APO4 ratio was constant over this interval. On this basis, we assume that the AAOU: ANO3 ratio was also constant from 150 to 900 m, and that any departures from the expected AAOU: ANO3 ratio arise from changes in preforned nitrate concentrations, or nitrate reduction, or both.

The sensitivity of equation 4 to changes in the AAOU:APO4 oxidative ratio is approximately +10, that is a systematic change in this ratio of more than ?10 in the depth interval of interest would have produced discernible curvature in the (PO4)p vs. S%oO plot (Fig. 4).

Oxidative ratios in the upper 900 m were assumed to be constant at the values given in Table 1. The relationship between preformed nitrate and phosphate was estimated from the AOU: NO3: P04 intercepts (Table 1) and the average preforned nutrient concentrations at 900-100 m from 22 stations within the oxygen-minimum region. The straight line, defined by these

SN , g- atoms/ I iter 0 4 8 12 16

0 I I

2004

400 -

a)

E 5 600 -

LiI

800 -

1000 _

1200 I FIG. 6. Reconstructed nitrate anomaly profile

for PONCHO station 19. Procedure used in the reconstruction is given in the text.

two points, is described by the equation: (NO3)p = 13.1 (P04)p

- 5.1 [yug-atoms/liter]. (5) The slope of this line is close to the value 12.5 used by Fiadeiro and Strickland (1968) for the waters off Peru.

Using equations 1-5, we can reconstruct nitrate-depth profiles through the oxygen- minimum zone in a manner similar to that of Fiadeiro and Strickland, except that the estimates of preformed nutrient concentrations and the individual oxidative ratios are empirical rather than arbitrary. The results of these calculations are shown in longitudinal section for stations 16-24, along with the relative position of the secondary nitrite maximum in Fig. 5. Nearly perfect matching of the nitrate concentration profiles above 200 m and below 1,000 m suggest that the modeling procedure (equations 1-5) gave a reasonable estimate of the amount of nitrate lost, presumably by denitrification. An example of com- puted nitrate anomalies is shown in Fig. 6.




2.0

1.6

9zj0.8 ; ;*

0~~~~~~

0. .

0

0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

02, Xg-oatoms/ liter

FIG. 7. Secondary nitrite vs. oxygen concentrations for stations 10-40, PONCHO (TT-026). Oxy- gen concentrations were determined colorimetrically (Broenkow and Cline 1969).

The core of maximum nitrate loss is at about 500 m along the entire section, with maximum nitrite concentrations at about 400 m. There is little horizontal change in either nitrate anomaly or nitrite concentration out to about 600 km from the coast. At that point, the values both of nitrite and of nitrate anomaly decrease, presumably in response to an increase in oxygen (Fig. 2) or a decrease in biological activity at that point. The similarity in the shapes of the contours suggests that nitrite and nitrate anomaly are biologically related.

Maximum concentrations of nitrite and nitrate anomaly along this section were 1.34 and 13.6 )ug-atoms/liter, both values occurring at station 20. In a section farther north, near Acapulco (TT-026, stations 10- 15), the maximum nitrite concentrations were 1.5-1.6 )ug-atoms/liter, but the nitrate anomaly values were not significantly larger in this part of the area.

The dependence of nitrite and the nitrate anomaly on the oxygen concentration is shown in Figs. 7 and 8. Except for a few isolated points, nitrite concentrations

in excess of 0.1 [ig-atom/liter occurred only at oxygen concentrations below 2 yug- atoms/liter (ca. 0.02 ml/liter). However, low nitrite values also are evident at low oxygen concentrations, suggesting that nitrite may be transient in the absence or near absence of oxygen.

Nitrate anomalies showed a dependence on low oxygen concentrations as did nitrite, but differed slightly in their relation to oxygen. Nearly zero values of nitrate anomaly were not observed at low oxygen concentrations as was the case with nitrite, suggesting that the product of denitrification, as represented by the nitrate anomaly, is both chemically and biochemically stable with respect to oxygen. Relatively high values of the nitrate anomaly occurring at high oxygen concentrations may have arisen from vertical mixing with oxygenated waters at the boundaries, or analytical uncertainties, or both. These observations tend to confirm our interpre- tation that nitrate anomaly represents the product of denitrification: free nitrogen.

The distribution of ammonia in the oxygen-poor water is of particular importance




20

16

0)

4-. 0

,- 0 u12 000..

E 0

0

4.0

00

A 80 2.0 - 0

FbIG * . Nirt aolyv. oxgncnetainfrsains1I2 OCO(T06.Oye

ws4 .0 a

000 * *0 0

0 . I. 1 I I.* I * 0 2.0 4.0 6.0 8.0 0o.0 12.0 14.0 16.0 18.0 02, ,ug-atoms/ liter

FIG. 8. Nitrate anomaly vs. oxygen concentration for stations 16-22, PONCHO (TT-026). Oxygen was determined colorimetrically.

because of its involvement in biochemical processes. A typical distribution of ammonia, along with comparative oxygen and nitrite concentrations, is shown for station 028 (PONCHO) in Fig. 9. Characteris- tically, ammonia concentrations reached maximum values of 2-3 [ig-atoms/liter NH4-N in the surface layers, then de- creased sharply to zero or nearly zero in the core of the oxygen minimum; below this the ammonia concentration appears to increase slightly, although it does not al- ways do so. Ammonia and nitrite seem sometimes to be inversely related (Fig. 9).

Although nitrous oxide (N20) has been suggested as a possible product of marine denitrification (Thomas 1966), it was not observed in samples taken from six stations within the oxygen-poor zone (stations 12-17, TT-026) at the level of detection (0.5 jtmole/liter). Craig and Gordon (1963) have reported an average N20 concentration for seawater of 0.2 ppmv or about 0.009 itmole/liter, a value well below our stated level of detection.

No specific tests were made for hydroxylamine, but colorimetric iodine analyses

revealed the presence of unknown reducing substances in the water of the oxygen minimum. If the reaction between hydroxylamine and iodine in acid solution can be represented by the equation

NH20H +2I2+1H20= NO2- + 4I- + 5H+, (6)

the maximum concentration of NH20H present would have been about 0.5 ug- atom/liter, assuming that all the observed iodine reduction was by hydroxylamine. If present at all, hydroxylamine should be restricted to anoxic or nearly anoxic waters because of its reactivity with dissolved oxygen (Fiadeiro et al. 1967). Deter- mination of ammonia by the method of Richards and Kletsch (1964) includes hydroxylamine, indicating that if any was present, its concentration was less than 0.3 [Lg-atom/liter.

Tests were made for sulfite (SO32-) and thiosulfate (S2032-) at selected stations within the core of the oxygen minimum and for hydrogen sulfide at all sampling depths within the oxygen minimum. None of these was observed at the levels of




NH3 and NO, vg -atoms /liter 0 04 08 1 2 16 20 24

200 ___ , g -_ AA

400

6E i / ,- <

?^ 600 H dk,-~ _

800 2

I1000, _V

200 0 l00 200 300 400 500 600 02, L9 - atoms /I iter

FIG. 9. Ammonia, oxygen, and nitrite concentrations at station 028, PONCHO (TT-026). Note the near-zero oxygen gradient between 100 and 800 m.

detection, which were 0.5, 6, and 1 /tg- atom/liter for SO32-, S2032-, and H2S.

DISCUSSION

It appears that nitrate reduction in the northeastern tropical Pacific Ocean is largely an anaerobic process. Oxygen concentrations throughout most of the oxygen minimum are no greater than 1 Jug-atom /liter and may be much lower because of the increasing probability of air contam- ination at these concentrations. The oxygen levels at which nitrate becomes the preferred electron acceptor for the oxidation of organic material in the ocean are not known, but they may be reached in large parts of the study area. Longmuir (1954) reported respiration rates for a number of aerobic bacteria at low concentrations in terms of the Michaelis-Menten constant, K., the substrate level at which the respiration rate is 50% of the maximum rate. In this case, oxygen was analogous to the limiting substrate. The values of Km ranged from 0.022 yug-atom/liter for small bacteria (0.5 u) to 6.2 [ig-atoms/liter for the larger microbes (4.0 FL). Although these Km values were determined under laboratory conditions at about 20C, the

oxygen concentrations are similar to those in the region where nitrate reduction is suggested. The low oxygen concentrations and the lack of significant oxygen gradients in the oxygen-minimum zone strongly suggest that nitrate reduction is the dom- inant respiratory process, except possibly for bacterial fermentation. Also, it is unlikely that any organisms, except bacteria, actively respire in these waters. Although lantern and hatchet fish, euphausiids, and squid are reported in waters containing from 0.5 to <0.1 ml/liter of dissolved oxygen (Richards 1957), it seems to us that oxygen concentrations as low as 0.01-0.02 ml/liter would place severe limitations on the metabolic process of such animals.

The production of methane under these quasi-anoxic conditions was also consid- ered as a possible respiratory process. Gas chromatographic tests for dissolved methane were made in November 1965 during the first voyage of the RV Thomas G. Thompson (TT-001, OPERCAT). The tests indicated that methane was absent in concentrations greater than the limit of detection, 0.1 )umole/liter (Atkinson and Rich- ards 1967). In contrast, the occurrence of methane with hydrogen sulfide in some anoxic environments is well documented (Atkinson and Richards 1967), suggesting that the Eh in these waters is not low enough to permit the formation of CH4 or H2S. In effect these waters are Eh-buf- fered by the dissolved nitrate ions.

The amounts of denitrification, estimated from numerical integration of the nitrate anomaly profiles (Fig. 6), tend to increase as the thickness of the oxygen-minimum layer increases. They also decrease offshore (Table 2), except for station 21, where the oxygen-minimum zone is also anomalously thin. Comparison of the thickness of the oxygen-minimum zone with the integrated nitrate anomalies suggests that relatively more denitrification occurs near the coast than away from it (Table 2). Because nitrate is present at all depths along the section, it is probable that if nitrate reduction is more intense along the coast, it is because of a higher rate of




TABLE 2. The nitrogen deficit (integrated value of the nitrate anomaly) and thickness of the oxygen-minimum layers in a section normal to the

coast of Acapulco, Mexico

Sta. No. 6N Thickness of 02 min (g-atoms/m2) (m)

16 2.0 530 17 3.9 600 18 4.3 710 19 4.5 850 20 5.7 820 21 4.5 600 22 5.9 880

supply of organic matter there. If the principal locus of nitrate reduction is along the coast, the offshore decrease in the integrated anomaly values may simply result from dilution as the coastal water moves offshore and spreads. It is difficult to say whether circulation or the supply of organic matter predominates in determin- ing the distribution of the nitrate anomaly and the oxygen-minimum layer, but both must be important.

The simultaneous occurrence of nitrite and the apparent loss of nitrate suggest that nitrite is an intermediate in the reduction of nitrate to free nitrogen. In a study of Darwin Bay, a flooded caldera on Isla Genovesa in the Galapagos Archipelago, Richards and Broenkow (1971) found that nitrate reduction was in progress below sill depth and that most of the reduced nitrate appeared as nitrite. In fact, the ratio ANO3: ANO2 was 1:1, with an intercept of 4 pg-atoms/liter of nitrate-N, indicating that reduction beyond nitrite had occurred.

In an attempt to determine the significance of nitrite in the overall process of denitrification, we collected a sample of water from 500 m in the oxygen-minimum zone and isolated it in an airtight reaction chamber (Cline and Richards 1969). Dur- ing a 10-day experiment, the time rate of change of various nitrogenous products under anaerobic conditions was observed. The details of this study were published elsewhere (Goering and Cline 1970), but nitrate was initially reduced to nitrite, and only after the nitrate concentration de-

creased to about 8 [ig-atoms/liter from an initial value of 30 yug-atoms/liter did nitrite decrease with time. At the end of the experiment, all the original nitrate and transitory nitrite were reduced to unde- tectably low concentrations. These observations are supported by the work of Carlucci and Schubert (1969), who noted the initial production of nitrite from nitrate-reducing bacteria isolated from the oxygen-deficient waters off Peru. The overlap in these two processes suggests that under suitable environmental conditions, nitrate reduction to nitrite and nitrite reduction to free nitrogen may occur simultaneously.

Carlucci and McNally (1969) demonstrated that nitrite can arise also from the biochemical oxidation of ammonia in the presence of low oxygen concentrations. However, the oxygen levels in the region of the secondary nitrite maximum probably are too low (-0.01 ml/liter) for this process to be important. The best explana- tion for the existence of the secondary nitrite maximum still seems to be the reduction of nitrate to nitrite, followed by the terminal reduction of nitrite to free nitrogen. In the open sea where steady- state conditions are assumed to prevail, it appears that the reduction of nitrate to nitrite and of nitrite to free nitrogen occur simultaneously, that is, as coupled reactions, which may be carried on by the same or different organisms. The reduction of nitrite to ammonia appears to be unimportant in this region in view of the low observed ammonia concentrations.

An unresolved question concerning respiratory processes at low oxygen concentrations is the rate of ammonia released during bacterial degradation of organic material. During sulfate reduction, amine nitrogen accumulates as ammonia, but during denitrification ammonia may either be oxidized or simply accumulate (Richards 1965; Richards et al. 1965). Let us first assume that amine nitrogen is not oxidized by any process and that it accumulates in the water column according to the equation (Richards 1965):




TABLE 3. Gibbs standard free energy change for the oxidation of one mole of glutamic acid, utiliz-

ing various electron acceptors

*~~~~~~~~~~~~~~~~~~~~~~~~ Electron AG 1 Nitrogen acceptor (kcal mole ) product

02 -483 NH4

02 -568 N03 NO3 -469 NH4, N2

NO3 -545 N2

4O -19 NH4

SO?4 -19 N2

* Standard chemical potentials for all dissolved substances except glutamic acid were obtained from Pourbaix (1966). Thermodynamic properties of glutamic acid were obtained from Greenstein and Winitz (1961).

(CH20)106 (NH3)16 H3PO4 + 84.8 HN03 = 106C02 + 42.4N2 + 148.4H20

+ 16NH3 + H3P04. (7)

If 10 pg-atoms/liter N03-N were lost by denitrification, equation 7 would predict the accumulation of about 2 ,ug-atoms/liter of ammonia. This clearly does not occur. Ammonia concentrations are generally less than 0.5 ,tg-atom/liter throughout the oxygen minimum and may be much lower since there are analytical difficulties at this level of concentration. The other alterna- tives are that the amine nitrogen is oxidized, either chemically or biochemically, or that it is assimilated by organisms.

To test the possibility of biochemical ammonia oxidation, a thermodynamic ex- amination was made of the glutamic acid- nitrate ion system, using glutamic acid (C5H904N) to represent an oxidizable organic substrate and nitrate ion the terminal electron acceptor. Glutamic acid was selected because its molecular composition resembles the elemental ratios of average marine plankton (C: H :0: N) = (106:212:106:16). For the oxidation of glutamic acid at the expense of nitrate ion, in which ammonia is oxidized or released, the reactions would be:

5 C5H904N + 21 N03- + 21 H+ = 25C02 + 13N2 + 33 H20; (8)

5C5H904N + 18N03- + 23H+ -25CO2+5NH4++9N2+24H20. (9)

All the ammonium ion shown in equation 9 results from deamination of glutamic acid. The Gibbs standard free energy change (per mole of glutamic acid) was calculated from the following equation:

AGOR= (1/vg) E vjFL*, (10)

where /* is the standard chemical potential of the ith species (complete with sign), and vg is the stoichiometric coefficient of glutamic acid in the balanced equation, i.e. 5. The free energy changes accompanying equations 8 and 9 are shown in Table 3, along with free energy changes that would occur if molecular oxygen and sulfate ions were the primary electron acceptors. It is evident thermodynamically that the oxidation of the amine nitrogen to molecular nitrogen at the expense of nitrate is ener- getically favorable, although less so than with molecular oxygen. In contrast, there is little or no thermodynamic advantage to the oxidation of ammonia during sulfate reduction. It should be stressed that this calculation does not imply that ammonia oxidation during denitrification is inevita- ble, only that it is thermodynamically possible. Contrary to these calculations is the fact that there is little, if any, experimental evidence for the biological oxidation of ammonia to free nitrogen (S. C. Ritten- berg, personal communication). Goering (1968) detected no oxidation of ammonia during studies of natural bacterial flora from the oxygen-deficient waters of the eastern tropical Pacific.

Inorganic oxidation of ammonia is thermodynamically possible, but we have no evidence suggesting its occurrenoe. One such reaction might be the van Slyke reaction:

NH4++ NO2-= N2+2H20, (11)

but the rate of reaction is not known. In the absence of a suitable catalyst and at low temperatures, this reaction is presumably slow; its probable significance is further diminished by the preferential ex- traction of both N02- and NH4+ by organisms for growth.

The accumulation of ammonia may be




prevented by bacterial assimilation. Be- cause neither the bacterial populations nor their growth rates are known, the maximum amount of ammonia lost through assimilation might be estimated from the energy yield of nitrate reduction. Hadji- petrou and Stouthamer (1965), working with Aerobacter aerogenes, found that 3 moles of ATP were produced for each mole of nitrate reduced. The yield of ATP was about the same for several different carbon sources. They also reported that reduction of nitrate beyond nitrite yielded no more ATP, i.e. the only ATP-producing reaction was:

2e- + 2H+ + NO3- = NO2- + H20. (12)

Growth yield measurements for the same organism show that an average of 10.8 g of cell material (dry weight) is produced for each mole of ATP (Forrest 1969). This average was based on glucose, fructose, mannitol, and generated gluconate as carbon sources. We will also assume that about 10% of the cell dry weight is nitrogen (Luria 1960).

The reduction of 10 jtg-atoms/liter of nitrate would produce 320 pg/liter of dry weight cell material, equivalent to 32 utg /liter of nitrogen, or about 2.3 jug-atoms /liter of ammonia. This value is strikingly close to our estimate of the amount of ammonia that should have been released according to equation 7. We suggest that the near absence of ammonia from the oxygen-minimum zone can be attributed principally to assimilatory processes.

The oxygen-minimum zone in the eastern tropical Pacific Ocean is extensive and probably represents an important site of denitrification (Brandhorst 1959). The amount of free nitrogen produced by denitrification in the world oceans is not known, but several estimates based on mass balance considerations place the value in the range of 7-10 x 1013 g/yr (Emery et al. 1955; Eriksson 1959). The budget by Emery et al. (1955) is repro- duced in Table 4. The amount of nitrogen necessary to bring the budget into balance is 69.4 X 1012 g/yr, which they attribute

TABLE 4. Oceanic nitrogen balance*

Reserves Amount (g) Inorganic combined nitrogen 5.8 x 1012 (NH+, NO2, NO3)

Dissolved organic nitrogen 3.4 x 1012

Total 9.2 x 1012

Gains Rate (g/yr) Land drainage (inorganic and 19 x 1012 organic) Precipitation on sea surface 59 x 1012

Total 78 x 1012

Losses Rate (g/yr) Permanent burial in sediments 8.6 x 1012 Unknown (by difference) 69.4 x 1012

Total 78 x 1012

* After Emery et al. (1955).

to marine denitrification. However, their material balance does not include marine nitrogen fixation (if it should occur), which will tend to raise the amount of denitrified nitrogen if the system is to remain in steady state. R. Dugdale (personal communication) has modified the original mass balance calculations of Emery et al. (1955) by assuming that 1% of the nitrogen re- quired by photosynthetic organisms in the tropical and subtropical ocean is supplied by nitrogen fixation. This will increase the amount of denitrified nitrogen by 8 x 1012 g/yr to a total of 77.4 x 1012 g/yr. Major unknowns in the budget are the amounts of nitrogen fixed photosynthetically by marine bacteria and algae and the rates at which fixed nitrogen compounds are in- corporated into marine sediments. In view of these uncertainties, it is worthwhile to estimate the rate of denitrification in the eastern tropical Pacific and compare it with other entries in the budget.

In situ rates of denitrification in the eastern tropical Pacific are not available, but in vitro rates were determined on natural denitrifying bacteria by Goering (1968), using labeled nitrate (15N-NO3-) (Table 5). These rates are probably much higher than in situ rates because of the proliferation of bacteria during incubation




TABLE 5. Rates of denitrification

Location Depth Rate* Observer+ interval

(m)

14055'N,100004'W 149-347 2,940 G

19002'N,105041'W 294-417 2,755 G

0018'N, 89 057'W 40-120 60 R&B (Darwin Bay)

* The rate (in Ag-atoms liter-1 yr-') represents an arith- metic average over the indicated depth interval with each value receiving equal weight.

t G = Goering (1968); R&B = Richards and Broenkow (1971). Temperatures in both studies were about 20 ? 2C.

in small volumes. The probably more real- istic rate of denitrification obtained for Darwin Bay, Galapagos Islands, from two sets of observations at different times, 60 pg-atoms liter-' yr1 (Richards and Broen- kow 1971), represents the minimal average rate of nitrate reduction for the anoxic part of the water column and is about 1/50th the rates determined by Goering (1968).

The rate of nitrate reduction in Darwin Bay cannot be assumed to apply to the oxygen-deficient waters of the eastern tropical Pacific because of a variety of environmental differences. For example, the average temperature below sill depth in Darwin Bay was 19.4C, but in the oxygen-minimum zone ('500 m), it is 9 ? IC. If a twofold decrease in the biological rate results from a 100 decrease in temperature, the rate should be adjusted to 30 pg-atoms liter-1 yr-1. This value probably represents a maximum, because Dar- win Bay-is shallower (max depth '200 m) and presumably more productive than the oxygen-minimum zone of the open ocean.

Because of the depth differences, it is necessary to estimate an average rate of denitrification for the oxygen-deficient water column. To make this estimate, we assumed: 1) that the rate for Darwin Bay was a maximum, 2) that there is no significant amount of denitrification in the presence of oxygen, 'and 3) that the dependence of the rate on depth is of the idealized form:

R=Rosin2iVz' O<z'<1, (13) where z' is the normalized depth interval and Ro is the maximum denitrification rate.

This idealized rate function generates a curve that is reasonably close in shape to the observed AN distribution, but it does not take into account a variable vertical eddy diffusion coefficient nor a reasonable in situ distribution of oxidizable organic material. The actual distribution of the rate of denitrification is probably similar in shape to the above model, but with the maximum skewed toward the upper boundary of the oxygen-poor layer. This is near the base of the pycnocline where particulate organic material would be expected to be most plentiful, hence the site of the most active organic decomposition. The average of R is 0.5 Ro, so the average rate for the oxygen-deficient part of the water column would be 15 pg-atoms liter-1 yr-1. This rate is probably also too high; the rate of denitrification should also depend on the rate at which oxidizable carbon reaches the site of denitrification, and on the average, this latter rate would be expected to be higher in the 40-120-m depth interval in Darwin Bay than it is in the approximately 300-800-m depth interval in the open eastern tropical Pacific. Also it is likely that surface productivity decreases with increasing distance from shore, resulting in a lower rate of denitrification toward the western boundaries of the region. This would also cause our estimate to be excessive.

The area and volume of the oxygen- minimum zone were calculated from observed oxygen profiles taken from the Scripps expeditions EASTROPIC in 1955 (EASTROPIC Exped. 1956) and EAST- ROPAC in 1969 (unpublished) and from the University of Washington expeditions PONCHO (in 1968) and PISCO (in 1969). The oxygen data supplied by Scripps came from standard or modified Winkler titra- tions, those of the University of Washing- ton from colorimetric measurements. Be- cause the oxygen gradients at the upper and lower boundaries are relatively large, no important differences in the estimates of thickness result from the different analytical methods and sensitivity limits. The thickness of the oxygen-minimum zone was




TABLE 6. Estimates of oceanic denitrification

Amount Region Investigators (g/yr)

7 x 1013 world ocean Emery et al. 1955

10 x 1013 world ocean Eriksson 1959

23 x 1013 eastern tropical This work north Pacific

1 x 1o13 eastern tropical Goering et al. north Pacific (in press)

plotted on an equal area map and subdi- vided into 20 squares. The average thickness of the oxygen-minimum layer was determined by averaging the data for each square. In a few-locations data were sparse or lacking and interpolation was necessary. If the oxygen-minimum zone was less than 150 m thick, the square was not included in the numerical integration. The boundaries of the above-defined oxygen-minimum zone are about 200 N lat, 1170 W long, 90 N lat, and the coast of Central America. The total surface area is about 3.1 x 106 kMi2, a region nearly equal in area to the Mediterranean and Black Seas combined (Sverdrup et al. 1942). The volume is about 1.1 x 1015 m3. This volume and the estimated denitrification rate lead to the annual production of biological nitrogen of 230 x 1012 g. Comparison of this value with other total oceanic estimates is shown in Table 6. It should be stressed that this value probably represents an absolute upper limit on the amount of nitrogen being supplied through denitrification in the eastern tropical North Pacific Ocean. Even though our calculations do not include our possible regions of denitrification such as the coastal waters of Peru, portions of the Arabian Sea, Black Sea, and the Cariaco Trench, the agree- ment is quite good. It is also apparent that denitrification in the northeastern tropical Pacific Ocean is enough to account for discrepancies in the oceanic budget (Table 6), making it unnecessary to invoke aerobic denitrification to explain the differences (Eriksson 1959). Even if our estimate is an order of magnitude too high, denitrification in the oxygen-defi-

cient regions of the world oceans is a significant factor in the oceanic nitrogen budget.

REFERENCES

ATKINSON, L. P., AND F. A. RICHARDS. 1967. The occurrence and distribution of methane in the marine environment. Deep-Sea Res. 14: 673-684.

BENDSCHNEIDER, K., AND R. J. ROBINSON. 1952. A new spectrophotometric method for the determination of nitrite in sea water. J. Mar. Res. 11: 87-96.

BRANDHORST, W. 1959. Nitrification and denitrification in the eastern tropical north Pa- cific. J. Cons., Cons. Perm. Int. Explor. Mer 25: 187-193.

BROENKOW, W. W. 1965. The distribution of nutrients in the Costa Rica Dome in the eastern tropical Pacific Ocean. Limnol. Ocean- ogr. 10: 40-52.

, AND J. D. CLINE. 1969. Colorimetric determination of dissolved oxygen at low concentrations. Limnol. Oceanogr. 14: 450- 454.

CARLUCCI, A. F., AND P. M. McNALLY. 1969. Nitrification by marine bacteria in low concentrations of substrate and oxygen. Limnol. Oceanogr. 14: 736-739.

- AND H. R. SCHUBERT. 1969. Nitrate reduction in seawater of the deep nitrite maximum off Peru. Limnol. Oceanogr. 14: 187-193.

CARPENTER, J. H. 1966. New measurements of oxygen solubility in pure and natural water. Limnol. Oceanogr. 11: 264-277.

CARRUTHERS, J. N., H. G. STUBBINGS, AND A. L. LAWFORD. 1950. Water sampling in estu- arial waters. Dock Harbour Authority, De- cember, p. 2-12.

CLINE, J. D. 1969. Spectrophotometric determination of hydrogen sulfide in natural waters. Limnol. Oceanogr. 14: 454-458.

, AND F. A. RICHARDS. 1969. Oxygena- tion of hydrogen sulfide in seawater at constant salinity, temperature, and pH. Environ. Sci. Technol. 3: 838-843.

CRAIG, H., AND L. I. GORDON. 1963. Nitrous oxide in the ocean and the marine atmosphere. Geochim. Cosmochim. Acta 27: 949-955.

EASTROPIC EXPEDITION. 1956. Data collected by Scripps Institution vessels on EASTROPIC Expedition (September-December 1955). Univ. Calif., Scripps Inst. Oceanogr. Ref. 56- 28. 156 p.

EMERY, K. O., W. L. ORR, AND S. C. RITTENBERG. 1955. Nutrient budgets in the ocean, p. 299-310. In Essays in the natural sciences in honor of Captain Allan Hancock. Univ. S. Calif.




ERIKSSON, E. 1959. The circulation of some atmospheric constituents in the sea, p. 147- 157. In B. Bolin [ed.], The atmosphere and the sea in motion (Rossby Mem. Vol.). Rockefeller Inst. 509 p.

FIADEIRO, M., L. SOLORZANO, AND J. D. H. STRICKLAND. 1967. Hydroxylamine in seawater. Limnol. Oceanogr. 12: 555-556.

- AND J. D. H. STRICKLAND. 1968. Ni- trate reduction and the occurrence of a deep nitrite maximum in the ocean off the west coast of South America. J. Mar. Res. 26: 187-201.

FORREST, W. W. 1969. Energetic aspects of microbial growth, p. 65-86. In P. M. Meadow and S. J. Pirt [eds.], Microbial growth. Cambridge.

GOERING, J. J. 1968. Denitrification in the oxygen minimum layer of the eastern tropical Pacific Ocean. Deep-Sea Res. 15: 157-164.

, AND J. D. CLINE. 1970. A note on denitrification in seawater. Limnol. Oceanogr. 15: 306-308.

, F. A. RICHARDS, L. A. CODISPOTI, AND R. C. DUGDALE. IN PRESS. Nitrogen fixation and denitrification in the ocean: biogeo- chemical budgets. In Int. Symp. Hydrogeo- chem. Biogeochem. Tokyo, 1970.

GREENSTEIN, J. P., AND M. WINITZ. 1961. Chem- istry of the amino acids, v. 1. Wiley. 760 p.

HAGER, S. W., L. I. GORDON, AND P. K. PARK. 1968. A practical manual for use of the Technicon AutoAnalyzer in seawater nutrient analysis. Rep. B.C.F. Contract 14-17-0001- 1759, Oregon State Univ. Ref. 68-33.

HADJIPETROU, L. P., AND A. H. STOUTHAMER. 1965. Energy production during nitrate respiration by Aerobacter aerogenes. J. Gen. Microbiol. 35: 29-34.

HARVEY, H. W. 1955. Chemistry and fertility of sea water. Cambridge Univ. 224 p.

LONGMUIR, I. S. 1954. Respiration rate of bacteria as a function of oxygen concentration. Biochem. J. 57: 81-87.

LURIA, S. E. 1960. The bacterial protoplasm: Composition and organization, p. 1-34. In I. C. Gunsalus and R. Y. Stanier [eds.], The bacteria, v. 1. Academic. 513 p.

MULLIN, J. B., AND J. P. RILEY. 1955. The colorimetric determination of silicate with special reference to sea and natural waters. Anal. Chim. Acta 12: 162-176.

MURPHY, J., AND J. P. RILEY. 1962. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta 27: 31-36.

POURBAIX, M. 1966. Atlas of electrochemical equilibria in aqueous solutions. Pergamon. 644 p.

REDFIELD, A. C. 1934. On the proportions of organic derivatives in seawater and their rela-

tion to the composition of plankton, p. 176- 192. In James Johnstone Mem. Vol. Univ. Liverpool.

, B. H. KETCHUM, AND F. A. RICHARDS. 1963. The influence of organisms on the composition of sea water, p. 65-77. In M. N. Hill [ed.], The sea, v. 2. Interscience.

RICHARDS, F. A. 1957. Oxygen in the oceans, p. 185-238. In J. W. Hedgpeth [ed.], Trea- tise on marine ecology and paleoecology, v. 1. Geol. Soc. Amer. Mem. 67.

. 1965. Anoxic basins and fjords, p. 611- 645. In J. P. Riley and G. Skirrow [eds.], Chemical oceanography, v. 1. Academic.

,AND W. W. BROENKOW. 1971. Chem- ical changes, including nitrate reduction, in Darwin Bay, Galapagos Archipelago, over a 2-month period, 1969. Limnol. Oceanogr. 16: 758-765.

- J. D. CLINE, W. W. BROENKOW, AND L. P. ATKINSON. 1965. Some consequences of the decomposition of organic matter in Lake Nitinat, an anoxic fjord. Limnol. Oceanogr. 10(Suppl.): R185-R201.

, AND R. A. KLETSCH. 1964. A spectrophotometric determination of ammonia and labile amino compounds in fresh and seawater by oxidation to nitrate, p. 65-81. In Y. Miyake and T. Koyama [eds.], Recent re- searches in the fields of hydrosphere, atmosphere and nuclear geochemistry. Maruzen.

SVERDRUP, H. U., M. W. JOHNSON, AND R. H. FLEMING. 1942. The oceans. Prentice-Hall. 1087 p.

SWINNERTON, J. W., V. J. LINNENBOM, AND C. H. CHEEK. 1962. Determination of dissolved gases in aqueous solutions by gas chromatog- raphy. Anal. Chem. 34: 483-485.

THOMAS, W. H. 1966. On denitrification in the northeastern tropical Pacific Ocean. Deep- Sea Res. 13: 1109-1114.

URBAN, P. J. 1961. Colorimetry of sulfur anions, part 1 and 2. Z. Anal. Chem. 179: 415-426.

VACCARO, R. F., AND J. H. RYTHER. 1960. Ma- rine phytoplankton and the distribution of nitrite in the sea. J. Cons., Cons. Perm. Int. Explor. Mer 25: 260-271.

WADA, E., AND A. HATTORI. 1971. Nitrite me- tabolism in the euphotic layer of the central North Pacific Ocean. Limnol. Oceanogr. 16: 766-772.

, AND . 1972. Nitrite distribution and nitrate reduction in deep-sea studies. Deep-Sea Res. 19: 123-132.

WEST, P. W., AND G. C. GAEKE. 1956. Fixation of sulfur dioxide as disulfitomercurate (II) and subsequent colorimetric estimation. Anal. Chem. 28: 1816-1819.

WOOD, E. D., F. A. J. ARMSTRONG, AND F. A. RICHARDS. 1967. Determination of nitrate in sea water by cadmium-copper reduction to nitrite. J. Mar. Biol. Ass. U.K. 47: 23-31.



Documents

Oxygen Deficient Conditions and Nitrate Reduction in the Eastern Tropical North Pacific Ocean