ARTICLE IN PRESS

Deep-Sea Research I 52 (2005) 543–551

www.elsevier.com/locate/dsr

Instruments and Methods

Continuous in situ determinations of nitrite at nanomolar

concentrations
L.R. Adornatoa, E.A. Kaltenbachera, T.A. Villarealb, R.H. Byrnea,
a
University of South Florida, College of Marine Science, 140 7th Ave. South, St. Petersburg, FL 33701, USA
b
University of Texas at Austin, Port Aransas, TX 78373, USA
Received 9 October 2003; received in revised form 28 May 2004; accepted 3 November 2004

Abstract

Sharp gradients of chemical distributions in the nutricline are poorly resolved via conventional sampling techniques.
Resolution of the fine structure of chemical distributions in the water column requires the use of in situ procedures. We
describe here a high-resolution method for the measurement of nitrite concentrations in the upper 200 m of the water
column. A long-pathlength Teflon AF-2400 liquid core waveguide provides the low nanomolar detection limits required
for observations of nitrite in near-surface waters. Our spectrophotometric elemental analysis system (SEAS) has a two
second sampling period. Coupled with an 11 cm/s descent rate, SEAS is able to accurately identify the depth of the
primary nitrite maximum and provide a detailed inventory for nitrite in the upper ocean.
r 2004 Elsevier Ltd. All rights reserved.

Keywords: Nitrite; Sea water; Liquid core waveguide; Spectrophotometry; In situ instrumentation; Oligotrophic; North Pacific
Subtropical Gyre

1. Introduction defined by factors such as light level, nutrient

Recent investigations of phytoplankton niche
partitioning and thin layer formation have chan-
ged the requirements of the oceanographic com-
munity with regard to measurement of chemical
distributions in the water column. Many phyto-
plankton species thrive in narrow environments
Corresponding author. Tel.: +1 727 553 1508;
fax: +1 727 553 1189.
E-mail address: byrne@seas.marine.usf.edu (R.H. Byrne).
availability, temperature, and physical forcing
(Franks, 1995; Cavender-Bares et al., 2001; Dek-
shenieks et al., 2001; DuRand et al., 2001; Rieg-
man and Kraay, 2001). Genomic studies of the
related genera Synechococcus and Prochlorococcus
reveal very different mechanisms for the transport
and usage of nutrients, reflecting adaptation to
specific niches found in the euphotic zone (Rocap
et al., 2003). In conjunction with studies of
microbial distributions, it is also critical to under-
stand both the detailed water column chemical

0967-0637/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dsr.2004.11.008
 ARTICLE IN PRESS

544 L.R. Adornato et al. / Deep-Sea Research I 52 (2005) 543–551

distributions that favor the dominance of certain Concentration (nmol/kg)

0 20 40 60 80 100 120 140 160 180 200
species and the effect of organisms on these 0
distributions. Optimally, a suite of instruments
collecting chemical profiles would serve this 50
purpose; however, only dissolved oxygen is routi-

Pressure (dbar)
nely monitored in situ. 100
Standard sampling protocols require water
collection at discrete depths, and analysis of casts 150
that typically contain from 12 to 36 samples
spanning several hundred to several thousand 200
meters. While some shipboard nutrient analyses Lat 24.2803 N, Lon 133.9883 W
are prompt, a great deal of sample processing
250
occurs either post-cruise or on a time scale that (A)
precludes further adaptive on-site investigation. Concentration (nmol/kg)
Once the samples are analyzed, concentrations are 0 20 40 60 80 100 120 140 160 180 200
0
plotted against depth, and profiles are generated
by linear interpolation. While this method pro- 50
vides general assessments of chemical gradients at
Pressure (dbar)

the nutricline, it cannot resolve important small- 100

scale variations in chemical distributions that are
required to more fully understand phytoplankton
community structure.
Characterization of nitrite distributions in the
upper ocean is particularly challenging. Except for
a narrow concentration spike at the base of the
euphotic zone, nitrite is generally present at low
nanomolar levels. As a result, the distribution of
nitrite obtained by standard sampling techniques
is coarsely represented (Fig. 1). Furthermore, with
conventional spectrophotometric procedures,
wherein pathlengths are less than or equal to
10 cm, near surface concentrations are often
undetectable (Fig. 1).
Methods for the determination of nitrogen species
at low nanomolar concentrations have been devel-
oped only recently (Garside, 1982; Dore and Karl,
1996; Yao et al., 1998). While desktop chemilumi-
nescence analysis and long-pathlength spectrometry
provide the required sensitivity for observations of
low nutrient concentrations, characterizations of
nutrient distributions are still limited by sample
collection methodology (Dore and Karl, 1996). In
addition, nitrite can oxidize to nitrate over time, so
analyses should either be conducted in situ or as
soon as possible after sampling (Goyal and Hafez,
1995; van Standen et al., 1996).
Steimle et al. (2002) described an in situ, long-
pathlength, spectrophotometric elemental analysis
150
200
La 24.2467 N, Lon 151.2533 W
250
(B)
Fig. 1. Profiles obtained on WOCE transect p03hy provided
general upper and lower bounds for the depth of the PNM.
Standard spectrophotometric methods precluded measurement
of low nanomolar concentrations in the upper water column.
system (SEAS) capable of real-time nitrite analysis
at nanomolar levels. Data acquisition was fast,
simple, and free from the nitrox gas contamination
susceptibility that often troubles laboratory ana-
lyses of nitrogen species at low nanomolar
concentrations. In its initial configuration, the
instrument was deployed at a number of depths,
where it flushed itself with ambient seawater,
collected dark and reference spectra, added colori-
metric reagent, allowed 120 s for color develop-
ment, and collected five absorbance spectra. The
data were uploaded post-cast and the apparent
concentrations based on a stopped-flow calibra-
tion line were determined. Although this process
produced prompt results with excellent detection
 ARTICLE IN PRESS
L.R. Adornato et al. / Deep-Sea Research I 52 (2005) 543–551
limits, the profile suffered from the same dearth of
data as previous methods.
Johnson et al. (1986, 1989) developed an in situ
spectrophotometric instrument capable of contin-
uous analysis of various ionic species in seawater.
While the method provided the data density
required to determine detailed nutrient distribu-
tions, the detection limit of 0.1 mM precluded
detection of the low nanomolar nitrite concentra-
tions typically found in oligotrophic waters.
We present here an alternative measurement
procedure that provides a much more thorough
characterization of nitrite distributions in the
upper 200 m of the water column. This method is
applicable to any flow injection colorimetric
analysis, as long as significant color development
occurs within several minutes. Whether or not this
requirement is met for a given analyte is a function
of both the analyte’s in situ concentration and the
inherent reaction kinetics of colored species for-
mation.
2. Methods
Nitrite profiles were obtained at various stations
along a transect on the R.V. Melville during the
Rhizosolenia Mats in the Pacific (RoMP) 2002
cruise (20 June to 16 July 2002) in the North
Pacific Subtropical Gyre (NPSG) (Fig. 2). Inde-
pendent conductivity, temperature, salinity, and
35°N
30°N
25°N
°
20 N
165°W 160°W 155°W 150°W 145°W 140°W 135°W 130°W 125°W 120°W 115°W
Fig. 2. Geographic locations of SEAS casts during the RoMP 2002 cruise between June 20 and July 16, 2002 on the R.V. Melville.
545
fluorescence measurements were obtained with a
Sea-Bird SBE9 attached to the ship’s rosette. PAR
Sensors, models QSP-200L and QSR 240L,
provided photosynthetically active radiance
(PAR) profiles on rosette casts. Location of the
deep chlorophyll maximum (DCM) was deter-
mined from the fluorescence signal, and verified by
chlorophyll a measurements.
Except as noted, reagent and standard prepara-
tion procedures followed those previously de-
scribed by Steimle et al. (after Grasshoff, 1983).
Initial standard dilutions were prepared with
Nanopure Infinity Ultrapure system water
(17.8–18.2 MO), and calibration standards were
made with uncontaminated seawater from the
shipboard flow-through system. A 10.0 mM nitrite
standard was prepared by dissolving 0.345 g of
NaNO2 in 500 ml of deionized water. A pellet of
NaOH was added to avoid production of nitrous
acid, and 1 ml of chloroform was added as a
preservative. The solution was refrigerated
(t ¼ 4 1C) in a brown glass bottle. A 2 mM nitrite
standard was prepared by dilution prior to each
calibration. Calibration standards, prepared by
spiking nitrite-depleted surface seawater to 25, 50,
75, 100, and 150 nM nitrite concentrations, were
run immediately after preparation. Deionized
water and surface seawater blanks, run prior to
each calibration, were indistinguishable. Surface
seawater nitrite concentrations were consistently
below the 1 nM detection limit, defined as three
 ARTICLE IN PRESS
546 L.R. Adornato et al. / Deep-Sea Research I 52 (2005) 543–551
times the standard deviation of the blank signal.
Standards were analyzed at surface seawater
temperatures, which averaged 23.471.5 1C in the
gyre and 18.870.4 1C in the California Current.
The minor influence of temperature on nitrite
measurements is discussed in the results section.
The analyses utilized the Griess method, in
which nitrite reacts with sulfanilamide to form a
diazonium ion, which subsequently reacts with
N(1-naphthyl)ethylenediamine dihydrochloride
(NED) to form a pink azo dye (maximum
absorbance at 541 nm) (Fox, 1979). A 5:1 ratio
of sulfanilamide to NED was selected for the color
reagent solution. Transmitted light at a non-
absorbing wavelength (700 nm) was monitored to
reduce potential error created by lamp intensity
fluctuations or changes in optical cell performance.
Because of the relationship between concentration
and pathlength in the Beer–Lambert Law,
A ¼
bc;
where A is the wavelength dependent absorbance,
e is the molar absorptivity (M1 cm1), b is
pathlength (cm), and c is analyte concentration
(M); the 97 cm pathlength used for this analysis
extended the detection limit by an order of
magnitude compared to a standard 10 cm spectro-
photometric cell. The detection limit, defined at
the 95% confidence level, was 1.0 nM, and the
linear dynamic range exceeded 500 nM (Skoog et
al., 1998).
The Type I LCW employed in this work is made
of a flexible AF-2400 fluoropolymer (Dupont)
with a refractive index (n ¼ 1:29) smaller than that
of pure water (1.33) and seawater (1.34) (Byrne
and Kaltenbacher, 2001; Callahan et al., 2002).
Light introduced axially at an angle equal to or
smaller than 151 relative to the waveguide/water
boundary is internally reflected through the water-
filled waveguide and detected by a spectrometer
coupled to the waveguide via an optical fiber
(Callahan et al., 2002). To lessen possible changes
in signal due to movement of the waveguide as the
system traverses the water column, the waveguide
is immobilized in a teflon case (i.d. 7.6 cm) during
deployment. An opaque cover over the top of the
instrument minimizes stray light.
Signal quality was maintained between deploy-
ments by flushing the waveguide sequentially with
DI water, dilute Micro 90 surfactant/cleaner
(Cole-Parmer), DI water, 1.0 M HCl, and DI
water. In addition to removing any adsorbed
material from the waveguide, the surfactant aided
in the removal of microbubbles trapped in the
system. In order to ensure optimal performance of
the waveguide, a custom designed interface pro-
gram monitored absorbance spectra throughout
the cleaning procedure.
The instrument (Fig. 3) was powered from a
12 V battery, interfaced with a Falmouth Scientific
Inc. CTD (Model MDTD-DBP-D), and secured
on a custom-made frame. During a programmed
5-min delay time, the instrument was lowered to
10 m depth, where it pumped ambient seawater for
270 s. The pressure at 10 m collapsed any bubbles
remaining in the waveguide. After dark and
reference spectra were taken, the reagent pump
was activated for 80 s prior to initiation of data
collection. After this period, the instrument was
lowered at a speed of seven meter per min while
data were collected every 2 s. To ensure consistent
Sample
Chemical Liquid
Reservoir Core
Waveguide
Mixing Column
Dye Main Waste
Pump Pump
Optical
Fibers
Lamp
Electronics
Spectrometer
CTD Battery
Fig. 3. Schematic diagram of the SEAS instrument (11.5 cm
diameter, 50 cm long). The pressure housing (rated to 500 m)
contains the pump motors, lamp, spectrometer, and electronics.
The reagent reservoir, sample intake, pump heads, and
waveguide are exterior to the pressure housing. Two optical
fibers are used to transmit light from the lamp into the
waveguide, and from the waveguide to the spectrometer. A 12-
V battery and Falmouth CTD are connected to SEAS by a
waterproof cable. Specifications and additional operational
characteristics of the SEAS instrument have been previously
detailed by Kaltenbacher et al. (2000) and Steimle et al. (2002).
 ARTICLE IN PRESS

L.R. Adornato et al. / Deep-Sea Research I 52 (2005) 543–551 547

mixing between sample and reagents, the main The time required for a sample to travel from
pump and reagent pump were programmed to the sample inlet to the waveguide inlet was 7874 s.
operate at constant speeds for the duration of the The overall 7.7 cm3/min (sample plus reagent) flow
deployment. rate included a 0.6 cm3/min (7.5%) contribution
Since the instrument was deployed in a con- from the reagent. Since the internal volume of the
tinuous-flow mode, the in situ nitrite concentration 97 cm waveguide was 0.50 cm3, the nominal fill-
was determined by running a calibration in the time of the waveguide was 4 s. At the conclusion of
laboratory immediately before or after each each cast, the data were immediately uploaded and
deployment (Fig. 4). As full development of the plotted against depth. Because the instrument was
azo dye requires several minutes (Fox, 1979), either descending or ascending throughout the
the slope of the calibration line reflected both the period of sample collection, the depth of each
fractional completion of the reaction under the measurement was adjusted based on the observed
chosen experimental conditions and the molar 78-s delay between sample acquisition and mea-
absorbance of the azo dye (Steimle et al., 2002). surement. Thus, the location of each concentration
The calibration slope provides a multiplicative observation reflected the depth of each water
constant that relates the extent of color develop- sample as it entered the in situ instrument.
ment in the continuous flow mode to the full color
development that occurs after the reaction pro-
ceeds to completion. During the course of the 28
day cruise, the calibration slope averaged 3. Results and discussion
0.5970.05. Calibration slopes obtained early in
the cruise using laboratory-composed sulfanila- 3.1. SEAS performance characteristics
mide solutions averaged 0.6370.04 with an inter-
cept of 8.372.3. A mid-cruise change to A nitrite profile acquired with the SEAS
commercially produced sulfanilamide solution instrument is compared in Fig. 5 with measure-
(LabChem) provided an average calibration slope ments performed on samples obtained from a
of 0.5770.03 and intercept of 2.770.9. The rosette cast 2 h later. The rosette samples were
commercially made sulfanilamide was used analyzed in the lab immediately following the cast
throughout the remainder of the cruise due to
the reduced reagent blank and improved calibra- Concentration (nM)
tion stability. 0 20 40 60 80 100 120 140 160 180
0

20

100 40
90 60
y = 0.558x + 2.639
Apparent Concentration (nM)

Depth (m)

80 R2 = 1.000 80
70
100
60
120
50
140
40
30 160

20 180

10
Fig. 5. Comparison of in situ data and laboratory measure-
0 ments of bottle samples for station 14. The upcast for the SEAS
0 20 40 60 80 100 120 140 160
Added Nitrite (nM) instrument preceded the rosette cast by about two hours.
Diamonds represent in situ data and circles represent data from
Fig. 4. A typical continuous flow calibration obtained using the bottle samples. The four discrete points below 100 m differ in
SEAS instrument with commercial sulfanilamide solution depth from the corresponding in situ concentrations by an
(LabChem). average of 1.370.9 m.
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548 L.R. Adornato et al. / Deep-Sea Research I 52 (2005) 543–551

with the same instrumental procedure that had
been employed in situ. There was excellent
agreement between the two data sets.
In continuous flow instruments, inter-sample
mixing can become problematic. However, the
comparisons shown in Fig. 6 indicate that sample
carryover was insignificant in our system. Were
this not the case, upcast and downcast concentra-
tion discrepancies would be particularly evident at
both the inflection depth and at the peak. It should
be noted, as well, that significantly greater resolu-
tion can be obtained via more slowly descending
and ascending instrumental systems. Ultimately
this would involve the use of untethered systems
that are not influenced by ship’s motions.
Nitrite depth profiles often reflected the move-
ment of internal waves, whereby apparent peak
depths were somewhat offset for downcasts and
upcasts. In order to remove this influence on the
0 20 40
0
50
Depth (m)
profiles, nitrite distributions were plotted against
density. The resultant upcast and downcast
profiles aligned well along isopycnals (Fig. 6B).
It has been demonstrated that the reaction rate
of the Griess reaction decreases at temperatures
below 20 1C (Fox, 1979). As such, one factor that
potentially affects the profiles is water-column
thermal gradients. The temperature at the primary
nitrite maximum (PNM) was 17.471.4 1C in the
gyre, with the first seven stations averaging
18.670.2 1C and the next six averaging
16.070.7 1C. Maxima for stations 24 and 26 in
the California Current occurred at 15.1 and
12.0 1C. However, using a 3-min dwell time,
Steimle et al. (2002) reported only a 4.5%
difference in calculated nitrite concentrations
between 16.8 and 21.2 1C. Given the small range
of temperatures at the PNM across the gyre, errors
attributable to reaction kinetics should therefore
be minimal. Inclusion of a thermostatted heating
unit on future versions of the instrument should
Concentration (nM)
60 80 100 120 140 160 180 alleviate potential problems with temperature-
dependent reaction kinetics that may result from
deployment in colder regimes.

100
3.2. Nitracline measurements

150 In terms of peak concentration and depth, the

nitrite profiles obtained in this work correspond
200 well with previously published profiles for the
North Pacific Subtropical Gyre (NPSG) (Dore and
250 Karl, 1996). It is evident, however, that the profiles
(A)
Concentration (nM) obtained in situ exhibit far greater detail. In
0 20 40 60 80 100 120 140 160 180 general, the upper water column nitrite gradient
1023.5
(between the surface and the depth of a sharp
1024
inflection) was 0.0870.03 nM/m. The nitrite in-
1024.5
flection depth is well defined by the intersection of
Density (kg/m3)

1025 the upper water column gradient and the much

1025.5 steeper gradient of the primary nitrite maximum
1026 (PNM) (Fig. 7). The average PNM nitrite con-
1026.5 centration gradient (inflection to peak) for sixteen
1027 stations in the North Pacific was 1073 nM/m, and
(B) the average distance between the inflection depth
Fig. 6. (A) The nitrite profile for station 8, including upcast and
and the nitrite maximum was 2578 m. Below the
downcast data, shows a slight depth discrepancy caused by peak, nitrite concentrations decreased to low
internal waves. Fig. 6. (B) Profiles plotted vs. density remove nanomolar levels, albeit at slower rates than
the influence of internal waves on the nitrite distribution. observed for the shallower inflection-to-peak
 ARTICLE IN PRESS

L.R. Adornato et al. / Deep-Sea Research I 52 (2005) 543–551 549

Concentration (nM) Voituriez, 1979; Dore and Karl, 1996; Letelier et

0 20 40 60 80 100 120 140 160 180 al., 2004). An in situ nitrate instrument, currently
0

20
under development, will facilitate investigations of
the relationship between the nitrite inflection and
40
the nitracline.
60
Depth (m)

Because of incomplete reduction of nitrate by

80
phytoplankton at low irradiance, the primary
100
nitrite maximum is often located only slightly
120 inflection below the deep chlorophyll maximum (Kiefer et
140 al., 1976; French et al., 1983; Brzezinski, 1988;
160 Collos, 1998). A comparison of the nitrite and
180 fluorescence distributions relative to density for
station 7 (Fig. 8) shows the close correlation often
Fig. 7. Nitrite inflection depths are well defined by the
intersection between the best-fit upper water column gradient
observed between the two profiles over the course
(0.0870.03 nM/m) and that of the PNM gradient (1073 nM/ of the cruise. For all stations in the NPSG and
m). California Current transect, the fluorescence max-
imum was observed at depths intermediate to the
well-defined PNM and nitrite inflection depths.
gradient. All but two casts of seventeen contained The average PNM depth determined with SEAS in
a single, well-defined peak.
The nitracline has variously been defined as (1)
the depth of the first detectable nitrate concentra- Concentration (nM)
0 10 20 30 40 50 60 70 80 90 100
tion by standard spectrophotometric methods 1023.5

(Herbland and Voituriez, 1979), (2) the depth that 1024

is halfway between the first depth at which nitrate
1024.5
Density (kg/m3)

plus nitrite is greater than or equal to 100 nM and

the depth of the sample immediately above it 1025

(Dore and Karl, 1996), (3) the shallowest depth at 1025.5

which nitrate reaches micromolar concentrations
1026
(Villareal et al., 1999), and (4) the depth at which
1026.5
the nitrate plus nitrite concentration gradient
surpasses 2 nmol kg1 m1 (Letelier et al., 2004). (A)
1027
Letelier et al. (2004) found that the depth of the Fluorescence
nitracline was 11775 m during the summer 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07
1023.5
months from 1998 to 2001 at Station ALOHA in
the NPSG. With SEAS, the nitrite inflection depth 1024
across the NPSG in the summer of 2002 was
Density (kg/m3)

1024.5
117710 m, and 116711 m including two casts in
1025
the California Current. Given the sharp inflections
that were typically observed with SEAS in the 1025.5

North Pacific, the nitrite inflection depth may 1026

serve as a well-defined maximum depth for the 1026.5
nitracline. As the nitrite maximum is tightly
1027
coupled with, and situated just below the top of (B)
the nitracline, and the PNM is observed only in the Fig. 8. Comparison of nitrite and fluorescence profiles for
presence of nitrate, the nitrite inflection must be station 7, one of two stations containing multiple nitrite peaks.
located at or just beneath the top of the nitracline Close correlations between nitrite and fluorescence profiles were
as defined by Letelier et al. (Herbland and observed throughout the cruise.
 ARTICLE IN PRESS
550 L.R. Adornato et al. / Deep-Sea Research I 52 (2005) 543–551
-20
-40
Nitrite
(nM)
-60
-80
Depth(m)
-100
-120
-140
-160
-180
-200
-155 -150 -145 -140 -135 -130
West Longitude
Fig. 9. Contour plot of nitrite distribution vs. longitude and
depth. Diamonds represent the depths of fluorescence maxima.
For the sake of clarity, only fluorescence data collected within
several hours of nitrite casts are included. Shoaling of the nitrite
maximum occurred at a front located between 1501W and
1401W, and in the California Current.
the NPSG in the summer of 2002 was 138710 m.
The average depth of the fluorescence maximum
was 125711 m in the gyre, and 123712 m includ-
ing stations in the California Current (Fig. 9). This
is similar to the average depth of the summer
fluorescence maximum, 12579 m, reported by
Letelier et al. (2004) for Station ALOHA.
4. Conclusions
We have demonstrated the utility of in situ,
continuous flow measurements for characteriza-
tion of nitrite distributions in the natural environ-
ment. SEAS-nitrite instruments are sufficiently
compact to allow deployment on either a ship’s
rosette sampling system or autonomous profiling
systems, and can be used to specify both the nitrite
inflection and nitrite maximum depths with great
precision. The resolution that can be achieved
using this instrumental system should greatly
facilitate studies of upper-ocean nitrite dynamics.
Accurate integration of nitrite concentrations over
a given depth range is now possible, making
240 between-cast comparisons much more meaningful.
225
210 The method described here can be adapted to any
195
180 colorimetric analysis, provided that the rate of
165
color development is sufficiently rapid to allow a
150
135 continuous flow configuration.
120
105
90
75
60
45
30 Acknowledgements
15
0
The authors wish to thank Xuewu Liu and Kelly
Quinn (University of South Florida) for facilitat-
ing the collection of nitrite profiles and for many
valuable discussions; the officers and crew of the
R.V. Melville for their professionalism and ex-
pertise during the cruise; and two anonymous
reviewers for thoughtful suggestions that greatly
enhanced the manuscript. This work was funded
by the Office of Naval Research under contract
number N00014-96-1-5011 (R.H.B.), and by the
National Science Foundation under grant number
OCE–0099015 (T.A.V.).
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