JH Sharp et al ESM 1
Electronic Supplementary Material for: “A biogeochemical view of estuarine eutrophication:
lessons from seasonal and spatial trends and correlations in the Delaware Estuary” by
J.H. Sharp, et al.
INTRODUCTION
For the data evaluations in the
publication, we have sampled and
conducted shipboard and laboratory
analyses since 1978. For reference in
discussions of methods, Figure 1 is
included for location of estuarine
regions and stations. Almost all of the
sampling was from dedicated research
cruises on the R/V Cape Henlopen.
Two of the 1978 cruises used alternate
ships and several had supplemental
sampling using U.S. Coast Guard
helicopters (1978-1981) or small boats
with samples returned to the nearby
ship for processing. Many of the
methods were originally designed for
oceanic work and have been applied,
with minor modification, so that they
can be used for samples ranging from
tidal freshwaters to full salinity
oceanic waters. Concentrations of
many of the measured parameters have
broad ranges. Since large
concentration and time scales are
involved, care has been taken for
consistency. This supplement
describes and evaluates consistency
and precision of the methods used for
results in the paper. The data that are
used in the paper are available on the
following url:
Upper River
Urban River
Turbid. Max.
Mid Bay
Lower Bay
Figure 1. The Delaware Estuary showing regular
sampling stations from the head of the tide (Station 1) to
the mouth of the Bay (Station 26). The five regions of
the estuary area shown as well as the km scale down
the axis of the estuary.
http://www.ocean.udel.edu/cms/jsharp/CruiseDatabase.htm
The first author of this paper will see that this is maintained as an accessible web link and
his institution has such a mechanism to keep it available into the future. The first author is
working with a group including state and regional agencies and local offices of federal agencies
to develop and maintain a permanent Delaware Estuary web-based information and data portal
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JH Sharp et al ESM 2
under the title of Delaware Estuary Watershed to Ocean Observing System (DEWOOS). This
database and other data from the Delaware Estuary will be permanently maintained there.
TEMPERATURE AND SALINITY
In early cruises (1978-1985), temperature measurements were made using reversing
thermometers and discrete water samples were collected from the Niskin bottles for salinity
determination using a laboratory salinometer (Industrial Instruments Model RS-7A). Our
estimated precisions, based on 816 duplicate reversing thermometer readings is ±0.04ºC; for the
salinometer, the estimated precision using the measured conductivity ratio and the Cox et al.
(1967) equation is ±0.05 parts per thousand. For the past 20 years, we have used the temperature
and salinity values from the CTD, recorded at the exact time of tripping the Niskin bottle.
Currently, SeaBird instruments are used for the depth profiling CTD measurements, as well as
for the surface mapping system. The R/V Cape Henlopen staff has demonstrated that earlier
versions of the CTD system produced data consistent with that of the current system. Our
periodic checks of the temperature and salinity against the CTD in earlier cruises and the routine
current ones by the ship’s staff indicate that the two methods give values comparable to our
earlier manual measurements. Both temperature (ºC) and salinity (now, unitless on the Practical
Salinity Scale, psu) are reported to two decimal places. We have verified the accuracy of the
correlation between the old and new salinity scales and the algorithms used for conversion of
conductivity to salinity at low salinities (Sharp and Culberson 1982).
LIGHT MEASUREMENTS
Directly before or after the CTD cast, a light meter profile is made using a Biospherical
Instruments light meter. For the light measurements from 1978-1993, a hand-held unit (QSR100) measuring PAR energy was used with manual estimates of depth from cable out; from
1997-2003, a computerized multi-channel instrument (PRR-600) has been used, but only the
downward PAR energy is used for the calculation. In both cases, the natural log of the PAR
energy was plotted against depth. With the earlier instrument, a fairly “clean” plot of 4-10 points
was used. With the later instrument, data returns from the down and up records give many tens
to hundreds of points. Since the plot should be linear, visual evaluation is used to modify the
range for the line to eliminate three causes of inconsistency; ship’s roll, limits of detection of the
light meter, and surface light intensity shift during down and/or upcast.
Near the surface, the “noise” from the ship’s roll creates considerable scatter; these
depths are eliminated when necessary and the remaining depth range often starts around 0.5 to 1
m below the surface. The instrument will reach limits of detection in darker waters and
curvature will occur; these deepest readings are also removed. On occasion, probably due to a
shift in surface light intensity and/or shading from the ship’s superstructure, the down plot is
slightly offset from the up plot, usually with similar slope. In these cases, the down plot is
usually kept and the up plot removed since there is higher density data from the slower lowering
of the meter. With both the old and new meter, linear plot (-ln PAR vs depth) estimates result in
R2 values on the order of 0.99; in very turbid waters and in low light conditions, plots with an R2
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JH Sharp et al ESM 3
on the order of 0.96 to 0.98 have been used. Attenuation coefficients (k), in units of m-1, are
reported to two decimal places. A regression analysis was made of the individual k values
derived from these plots against the ambient suspended sediment concentrations (seston) for all
estimates made with the old and new light meter in the salinity gradient of the estuary (Figure 2).
The similarity in the plots illustrates consistency in the estimate of k for the two light meters.
k (m-1)
Relationships between
12
measured light attenuation (k) and
New (n = 143)
seston concentration are similar for
y = 0.071x + 0.48
10
R2 = 0.88
both light meters used during the 258
year dataset (Figure 2). Results during
the period when the new light meter
6
was in use are probably more accurate
4
as individual k values are based on
Old (n = 550)
more data points and many of the
2
y = 0.068x + 0.79
seston measurements were made in
R2 = 0.79
0
duplicate. In addition, there are
0
50
100
150
several data points from the earlier use
of the old light meter in the turbidity
Seston (mg/L)
maximum region (stations 16-18) that
Figure 2. Total suspended sediment concentration
have comparatively lower k values
(seston) plotted against light attenuation coefficient (k)
(indicated by yellow shading of the
estimated with the “old” light meter (open red squares)
symbols in the figure). If these are
and the “new” one (filled blue circles); see text. Only
removed, the slope from the old light
data from the salinity gradient of the estuary are used.
meter becomes higher. While the two
Several of the square symbols have been shaded in
regression lines have similar slopes,
yellow; they are from the turbidity maximum region (see
the new one has a better fit and
text).
smaller intercept (the intercept should
be very low; see discussion about CDOM in Light-seston relationships section of Results and
Discussion in paper).
Through 1988, light attenuation was also approximated using a Secchi disc. A half meter
diameter disc with black and white alternating quarters was lowered over the side of the ship
with a metered rope to measure the disappearance depth. Secchi depths are reported in cm to the
nearest 5 cm. Our primary use of light attenuation measurements is to estimate light depths for
areal primary production calculations. The 1% light level is easily calculated from the k value as
– ln (0.01)/k. By convention, 3 times the Secchi depth is used to estimate of the 1% light level.
The exact origin of the 3X calculation is not clear; however, the relationship most quoted in old
texts comes from Poole and Atkins (1929). Working in coastal waters, they established the
relationship between k and Secchi disappearance depth (D) as 1.7/D, from which a value of
almost 3 would be derived. An empirical relationship has been derived of k = 0.4 + 1.09/(D) for
the turbid San Francisco estuary (Cole and Cloern 1987). Their relationship would also give
close to 3X the Secchi disappearance depth for shallow D values. In the 1970s, we found good
agreement between calculations of the 1% light level with a prototype of the Biospherical
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JH Sharp et al ESM 4
Instruments light meter and Secchi depth in open ocean Pacific waters using 3X Secchi depth for
the 1% light level (Eppley and Sharp 1975; J. H. Sharp and R. W. Eppley, unpublished data).
A comparison of our estuarine
light data from 1978 – 1988 with the
light meter k compared to the 1% light
level from 3X Secchi depth is shown
as Figure 3. For close to 300
individual stations, the slope of the
line is close to unity and there is a
very small intercept. The scatter that
does show is probably primarily
created by the difficulty of measuring
a Secchi depth in shallow turbid
waters. The relatively good R2 value
indicates that careful use of a Secchi
disc gives a reasonable estimate for
the depth of the photic zone in an
estuary.
Fig. 3. The 1% light depth calculated from the light
attenuation coefficient (k) measured with a light meter
versus the depth estimated as 3X the Secchi disc
disappearance depth. Based on samples collected from
1978 – 1988.
DISSOLVED OXYGEN AND DISSOLVED INORGANIC CARBON
Dissolved oxygen (DO) is measured with micro-Winkler titration (Carpenter 1965) using
Brinkman Metrohm digital titrators with 5-ml burets readable to 0.001 ml. From 1978-1988, a
model E535 titrator was used with visual endpoint determination. Samples were collected in
125-ml iodine bottles in triplicate. Based on 954 triplicate samples, the precision at 1 SD was
determined to be ±3 µg-at O/L. Since the mid-1990s, a model 716 DMS Titrino titrator has been
used with automated potentiometric endpoint detection. Our estimated precision for the analysis
of triplicate samples is similar to that with the visual endpoint; for 138 sets of triplicate samples
analyzed in 2003 from estuarine transects, offshore depth profiles, and mesocosms experiments,
the mean precision was also about ±3 µg-at O/L. To estimate relative analytical precision, we use
a CV (coefficient of variation, based on one standard deviation). For our DO measurements, the
CV is ±0.7%. Throughout this paper, all analytical precisions are expressed as %CV. DO
concentrations are reported in units of µg-at O/L (2 µg-at O/L = 1 µM O2) to the nearest whole
number. Percent oxygen saturation is calculated from the measured concentration divided by the
calculated saturation value based on ambient temperature and salinity using the equations of
Kester (1975) and is reported to the nearest whole number.
From 1978 - 1985, dissolved inorganic carbon (DIC) was calculated from precise pH and
alkalinity measurements (Culberson 1988). Since then, DIC has been measured directly with
methods that involve acid sparging of samples in an oxygen gas stream and analysis of the
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JH Sharp et al ESM 5
resultant CO2 with non-dispersive infrared analysis. Samples are collected directly from the
Niskin bottles in 25 ml scintillation vials that have inverted cones in the caps for displacing
water, leaving no air. Vials are filled with overflow similar to the procedure for DO and then 0.2
ml of 5% HgCl2 is added to inhibit microbial activity. Samples are stored refrigerated until
analysis. Analysis is usually conducted at sea within 10 hours of collection. Samples stored for
up to several weeks show no change in DIC concentration when compared to those run fresh at
sea. For the samples from 1987-1999, a relatively crude DIC setup was used for the analysis
(Sharp 1973a). This old method (old instrument) was standardized with liquid standards made
from NaHCO3; estimates of precision for analysis at sea were on the order of ±3%.
Since 2000, a high precision MBARI-clone DIC instrument has been used (new
instrument). This instrument was constructed in our shop based on the original developed at the
Monterey Bay Aquarium Research Institute, MBARI (Walz and Friederich 1996; G. Friederich,
personal communication). This instrument has the O2-carrier gas metered with a mass flow
controller. The sample injections are performed with a Kloehn syringe pump: 1.25 ml injections
are used; two rinses precede injection of the sample into the sparging column. After the rinses,
but prior to the sample injection, 100 µL of 5% H3PO4 is added to the column. Then, the sample
is slowly injected into the sparging cell, resulting in good Gaussian peaks. The gas is dried by
passing through an anhydrone (MgClO4) drying column and then the CO2 is measured using a
Licor LI-6252 analyzer with pressure compensation. Control of the instrument and logging of the
CO2 peak areas (area integrated by the Licor software) are performed by computer using a visual
basic program (written by Gernot Friederich and modified by Charles Culberson).
Standardization is done directly using
the SIO (Scripps Institution of
Oceanography) certified reference
materials (Johnson et al. 1998).
Results are converted to µM C units
by density correction based on
measured laboratory temperature at
the time of analysis. With all three
methods, DIC concentrations are
reported as µM C to the nearest
whole number. Using the new
MBARI-clone instrument, we have
demonstrated a consistent precision
with four replicate injections (using
the best 3) of ± 0.07 % for oceanic
Fig. 4. Dissolved inorganic carbon (DIC) versus salinity
samples; all with DIC concentrations
for samples within the salinity gradient (salinity > 1).
around 2000 µM. For the range of
Data the cruises in 1978-1985 (Yellow diamonds) based
salinities in a normal estuarine
on calculation from pH and alkalinity (n=327); data from
sampling, the precision is not as
1987-1999 (open red circles) represent use of the old
good. With 63 samples analyzed in
DIC analyzer (n=299); data from 2000-2003 (filled blue
2003 from a salinity range of 0.5 to
squares) represent use of the new DIC analyzer (n=77).
30 (psu) and DIC concentration range
of 900 – 1900 µM, the average
precision was ±0.18 %CV.
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JH Sharp et al ESM 6
Plotting DIC versus salinity for all data from a salinity of 1 to the bay mouth (salinity ~
34) for the three periods provides an assessment of the consistency between the three methods
used (Figure 4). We have shown that it is reasonable to consider the major ion chemistry of the
Delaware Estuary to represent dilute seawater to salinity as low as 0.5 (Sharp and Culberson
1982). The slopes, intercepts, and regression coefficients for the three plots are similar;
especially those from the two direct instrument methods. The 2000-2003 plot with the new
analyzer (y=36.2x + 812, r2 of 0.96) compares with the 1987-1999 plot using the old DIC
analyzer (y= 35.1x + 811, r2 of 0.87) and with a regression of estuary through adjacent surface
ocean waters (y = 35.6x + 819, r2 of 0.97). We have verified consistency between the two direct
methods also by analyzing NaHCO3 standards made as in the earlier method against the SIO
reference materials. The older DIC values from the alkalinity calculations show a higher slope;
all of the lowest DIC values at low salinity come from before 1982. There was a strong pH and
pCO2 anomaly in the tidal river in the past (Culberson 1988) that may have intruded enough into
the upper bay to make those low salinity DIC concentrations in the late 1970s anomalously low.
Removing the five lowest DIC values from that period does give a lower slope, closer to that of
the other two data sets, suggesting a deviation in the oldest data set due to the riverine anomaly.
LABORATORY SAMPLE PREPARATION
Within a few minutes of collection in the Niskin bottle, samples are drawn into 4 L
polypropylene bottles and transferred to our portable laboratory van for processing. The
laboratory van has been used for the entire period of this database. It has a large volume air
handling system and is kept clean and partially isolated from the ship’s internal atmosphere and
the outside air. Blanks prepared in the van for nutrient and particulate carbon analyses, as well as
consistent low dissolved organic carbon concentrations measured in deep water offshore samples
prepared in the lab van show that this environment is “clean”.
Relatively large volumes of water and abundant rinses are used to further minimize
potential contamination problems. Water for all “dissolved samples” (i.e., nutrients, dissolved
organic carbon, nitrogen, and phosphorus) is filtered through 47 mm diameter GF/F filters (prior
to 1988, GF/C filters were used). As a precaution to remove potential DOC contamination,
filters used for particulate analysis are rendered organic-free by baking in a muffle furnace (450º
C for 2 hours) in Petri dishes; they are then stored in a dessicator prior to use. To process an
individual water sample, three aliquots of about 100 ml each are used to rinse the filter apparatus
and flask and then water is collected for samples. A volume considerably larger than needed for
all samples is collected in the filter flask so that sample bottles can be rinsed before filling.
NUTRIENTS
For nutrient analyses, samples are collected in polyethylene bottles (either HDPE or
LDPE). After three rinses, bottles are filled sufficiently below the top for expansion when
frozen. Usually, two samples are collected in 250 and 125 ml bottles so that a backup archive
sample is available for repeat nutrient analyses. Samples are then quick-frozen in dry ice, stored
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JH Sharp et al ESM 7
in the ship’s freezer, and transferred to
a clean freezer in our laboratory as
soon as the ship returns. In comparing
these quick frozen samples to those
run at sea, we have found excellent
agreement at most concentrations (L.
Solorzano and J.H. Sharp, unpublished
data). Efforts are made to ensure that
the ship’s freezer remains free of
biological samples and food to avoid
airborne contamination; this is most
critical for ammonium (NH4) analysis.
Comparison of NH4 analyses done at
sea immediately following collection
to those on frozen samples shows that
there is not a major contamination
problem for samples in the normal
concentration range of the estuary
(Figure 5a). For lower NH4
concentrations in offshore samples,
there is a significant contamination
problem, likely from the shipboard
freezer (Figure 5b). Since most of the
estuarine samples have concentrations
> 0.5 µM, we interpret the sample
handling adequate for accurate
analyses of frozen samples. This
comparison does indicate a serious
problem if one wishes to perform
accurate NH4 analyses on frozen
coastal or oceanic samples.
Fig. 5. Comparison of ammonium nitrogen analyses
performed at sea immediately and later on land using
samples quick-frozen in plastic bottles. 5a. Estuarine
samples with concentrations in 0.1-17 µM range. 5b.
Continental shelf and slope samples with
concentrations below 0.5 µM. Regression lines (red
dashed) and coefficients are shown on each graph and
the 1/1 line (solid green) is shown in 5b. for contrast.
Nutrient analyses are
conducted by standard colorimetric
methods (Strickland and Parsons 1972)
using the Liddicoat et al. (1975)
modification for NH4 and modified as
manual methods for small volumes
(Sharp et al. 1982). Ammonium (NH4), nitrite (NO2), phosphate (PO4), and silicate (Si) methods
have been essentially unchanged during the entire period of the database. The nitrate (NO3)
method has changed slightly. Small gravity columns were used originally (Sharp et al. 1982)
with chipped cadmium prepared as in Strickland and Parsons (1972). Now, a small straight glass
column (6 mm OD by about 15 cm length) is used packed with reagent grade granular Cd metal.
A peristaltic pump is used with a flow rate of about 4ml/min to pump the sample through the
column. Routinely, a reduction efficiency of about 95% is attained with the columns. For best
consistency of reduction efficiency, a narrow concentration range is maintained by diluting high
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JH Sharp et al ESM 8
NO3 concentration samples; 5, 10, and 20 fold dilutions are used to maintain concentrations in
the 1-5 µM range. All other nutrients are analyzed at ambient concentrations with no dilution.
The general procedure is to thaw the larger sample (250 ml) and analyze for all five
nutrients. On the first day, NH4, NO2, NO3, and PO4 are analyzed; the sample is then left
refrigerated until the next day to allow slow depolymerization of Si (e.g., Macdonald et al. 1986)
which is analyzed on the second day. The second, smaller sample (125 ml), is used for backup
in case of analytical difficulties with the original sample. We conduct manual analyses for the
nutrients usually because of the large range of concentrations in a single cruise sampling and the
periodic large number of samples followed by months of no samples. We have used a nutrient
autoanalyzer on rare occasions in the past, but find that it is not feasible to have it maintained for
our demands of large concentration ranges with the irregular periodic availability. Over the
years, several spectrophotometers have been used for the colorimetric readings, ranging from a
Beckman DU used in the late 1970s to a Milton-Roy Spectronic 601 used today. In all cases,
good stable readings have been obtainable in 1 cm and 10 cm pathlengths, including stability at
the longer wavelengths for PO4 and Si analyses.
Currently each nutrient method is calibrated with standards prepared in Milli-Q TOC+
water (Gradient A10); previously, regular Milli-Q water or a large volume building DI system
water had been used. Care was taken to make sure NO3 and Si blanks were kept low; deionization columns, as they age, will allow passage of these salts prior to showing decreased
readings on resistivity meters. Primary standards are made for each nutrient and when
demonstrated to be accurate, these are stored refrigerated and used for up to a year. Secondary
standards are made periodically and also stored refrigerated; daily standards are made fresh from
the secondary standards.
Concentrations for nutrients are reported in µM of the element; for NO3, NO2, NH4, and
PO4, values are reported to two significant figures, and Si to one significant figure. All analyses
are done with duplicate subsamples; this is for confirmation of measurement rather than a
thorough statistical analysis. However, by averaging the CV values of the duplicates, an
estimate of precision can be made. For the three estuarine cruises in 2003, the estimated
precisions are based on 43 sets of duplicate analyses for each of the nutrients (See Table 1). For
PO4, the range of concentrations was 0.04 – 6.7 µM P and the average precision was ± 1.7%. A
10 cm pathlength cell was used for the lowest concentrations (generally those with < 1 µM); the
average precision for that subset of samples was ± 2.9%. For NO2, the concentration range was
0.1 -7.2 µM N and the average precision was ± 1.7%. For the low concentration subset (<1 µM),
the precision was ± 3.3%. For Si, the range of concentrations was 1-75 µM Si and the average
precision was ± 2.7%. For NH4, the range of concentrations was 0.1 – 37 µM N and the average
precision was ± 4.8%. For NO3, the range of concentrations encountered was 2 – 144 µM N.
The higher concentration samples were diluted 5, 10, or 20 fold for an analysis range of 0.4 –
12.5 µM N. The average precision for the analysis of nitrate plus nitrite was ± 0.3%. Since many
of the samples were diluted prior to analysis and NO2 must be subtracted to derive NO3, the
propagation of errors makes the NO3 precision to be about ± 5%. For undiluted lower NO3
concentration coastal samples (0.2 – 6.5 µM) analyzed in this same time period (2003), the
average CV was ± 1.4%. Earlier nutrient analyses had similar precisions; concentration ranges
measured were higher with NO3 up to 250 µM N, NO2 up to 25 µM N, NH4 up to 175 µM N,
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JH Sharp et al ESM 9
and Si up to about 100 µM Si; PO4 concentrations from the earlier estuary transects were in the
same range as recent ones.
DISSOLVED ORGANIC CARBON AND NITROGEN
For dissolved organic carbon (DOC) and total dissolved nitrogen (TDN), the current
preparation involves pouring 50 ml of the sample filtrate into a glass bottle to which 50 µl of
concentrated “trace metals grade” HCl is added. In all cases, glass ampoules were used that had
been baked for at least 2 hrs at 450º C to render them organic-free. Currently, 12 ml aliquots are
measured out into 20 ml glass ampoules and the ampoules are sealed immediately with a propane
torch and quick frozen in dry ice. Currently, as well as with the prior methods, silicone rubber
tubing “chimneys” are attached to the tip of the ampoule before sealing to keep combustion
products from contaminating the samples. Two to three replicate aliquots of each sample are
prepared to allow for duplicate measurement and as backup for potential ampoule breakage.
This procedure has been used since 2002; from the mid-1990s, 5 ml aliquots were placed in 10
ml ampoules for DOC analysis only. Prior to the mid-1990s, 5 ml of decarbonated samples
(acidified with conc. H3PO4, and sparged by bubbling for 10 min with O2 or N2) were placed in
10 ml ampoules with 200 g K2S2O8, the ampoules sealed immediately, and autoclaved at sea.
They were then stored at room temperature until later analysis on land; usually 3 replicates were
prepared (Sharp 1973b). Prior to the 2002 samples, the wet chemical TDN method was
performed on filtrate samples that were essentially identical to nutrient samples.
The current DOC/TDN method uses the Shimadzu TOC-V instrument; from 1992 –
2002, the Shimadzu TOC-5000 was used for DOC analysis only (see Sharp et al. 2002a). The
methods and comparison of the two instruments are discussed in (Sharp et al. 2004). From 1978
– 1988, the persulfate oxidation method was used for DOC analysis; this method and evaluation
of blanks are discussed in Sharp, 1997. DOC analysis by the wet chemical and high temperature
methods are considered comparable (Sharp 1997). In the Correlations and Trends section of this
paper, Figure 9a also helps illustrate that the older persulfate oxidation and newer high
temperature combustion (HTC) methods give comparable DOC numbers. The 78 and 88 period
data sets used the same older DOC method. In comparing these two sets, significant differences
were seen in averages for 4 of the 5 estuarine regions. This dataset, using the same older method
was evaluated for consistency of standardization and blanks (Sharp 1997). The modern period
(98) used the new DOC method. There was no difference in average values for 3 of the 5
regions when comparing the 98 period with the new method to the 88 period where the older
DOC method was used. From 1980 - 2002, the persulfate oxidation method was used for TDN
(Solorzano and Sharp 1980a). Rough comparisons of estuarine distribution and of deep offshore
waters indicate that our analyses by wet chemical and high temperature combustion methods are
comparable; this is consistent with our intercalibration effort (Sharp et al. 2002b). Also, as is
shown in Figure 9c of the paper, the DOC/DON ratio is relatively similar for the two time
periods in most of the estuary; the 1980s analysis used persulfate oxidation for both DOC and
TDN while the 2002-2003 DOC/TDN analysis was performed with the HTC method.
With our current DOC/TDN method, we estimate the DOC precision to be ± 1.6%. The
instrument program is set with 5 injections of each sample; data are screened manually and the 4
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JH Sharp et al ESM 10
areas that give the closest fit are used for calculations. The precision estimate is based on
analysis in 2003 of 107 samples from estuarine cruises with DOC concentrations ranging from
110 – 300 µM; for the same 2003 cruise analyses, 23 oceanic samples with DOC concentrations
in the 41 – 75 µM range also gave a precision of ± 1.6%. DOC analysis from 1992-2002 had a
precision on the order of ± 2%. The precision of the earlier wet chemical DOC method was on
the order of ± 3-5%.
The precision of DON measurement depends on the precision of the TDN measurement
and the composite DIN (NO3 + NO2 plus NH4) measurements. For the current method, the
precision in the estuary is based on analysis from 2003 when the new Shimadzu TOC-V
instrument was used; only the 43 estuarine samples are used for the precision estimate. The
measured TDN concentrations ranged from 9 – 194 µM and the estimated precision is ± 3.2%.
With propagation of errors based on TDN, NH4 and NO3 + NO2 analysis, the estimated precision
is ± 12%; this is for a DON range of 7.5 – 63 µM. An international intercalibration exercise for
oceanic DON measurements is underway, supervised in this laboratory, with hopes of improving
DON precision. For the earlier wet chemical DON method, the precision for our samples is
probably on the order of ± 10-20%. In 1988, triplicate samples were taken on three cruises for
TDN analysis; the average precision for these triplicates (44 estuarine and coastal samples
ranging from 6-217 µM TDN) is ± 7.6%. While this type of error analysis is less conservative, it
does indicate that replicate precision of the persulfate TDN analysis is probably not as good as
that from the HTC method.
DISSOLVED ORGANIC PHOSPHORUS
From 1978 – 1988, dissolved organic phosphorus (DOP) analysis was performed using
the high temperature combustion method of Solorzano and Sharp (1980b) with the modifications
later published in Lebo and Sharp (1993). This method measures total dissolved phosphorus
(TDP); DOP = TDP – PO4. Duplicate 10 ml samples were placed in liquid scintillation vials and
dried at sea in an oven; samples were stored in desiccators until later analysis back in the
laboratory. From frozen nutrient samples, duplicate aliquots were used in analysis. From three
cruises in 1988, the precision of the method has been estimated. For a total of 100 sets of
duplicates of TDP and PO4, 7 did not have pairs due to elimination of clear outliers in the pair.
For the other 93 sets of pairs, the TDP concentrations ranged from 0.37 to 5.41 µM P and
derived DOP ranged from 0.05 – 1.08 µM P (PO4 ranged from 0.01- 4.82). The average
precision for TDP was ± 1.40% and, by propagation of error, the average precision for DOP was
± 7.5 %.
TOTAL SUSPENDED SEDIMENTS
Total suspended sediment concentration (seston) is determined by gravimetric analysis.
Nuclepore polycarbonate filters of 47 mm diameter with 1.0 µm pore size are used. Filters in
numbered Petri dishes are kept in a vacuum desiccator after taring. The volume of water filtered
(between 50 and 250 ml) is gauged so that filtration with mild vacuum (not exceeding 250 mm
Hg) is brief; thus, the volume depends on seston concentration. Filters are weighed after
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JH Sharp et al ESM 11
sufficient drying to give constant weight. Currently a Cahn model 29 Electrobalance is used.
We now take duplicate seston samples; the average CV for duplicate filters from three estuarine
transects in 2003 (43 stations) was ± 5.3%. Values are reported as mg/L to two decimal places.
PARTICULATE CARBON AND NITROGEN
Simultaneous particulate carbon and nitrogen (PC/N) analysis has been performed on
samples retained on 25 mm diameter GF/F filters (through 1988, GF/C filters were used instead
of GF/F). Prior to cruises, filters are rendered organic-free by baking in glass Petri dishes in the
muffle furnace (at least 2 hrs at 450ºC) and are then stored in the same dishes in a vacuum
desiccator. After sample collection, they are stored in the desiccator until later analysis on land.
In all cases, particulate matter on the filter has been analyzed without correction for carbonates;
while this correction is of concern with oceanic samples that may contain coccolithophorids (von
Bodungen et al. 1991), we do not consider this to be important for estuarine samples. For
samples prior to 1990, the Hewlett Packard CHN analyzer was used for the analysis (Sharp
1974). Since then, the Europa Tracermass GC-MS system has been used for all particulate C/N
analysis.
Filter volumes, usually a volume of 50 to 250 ml, are determined visually by coloration
on the GF/F filter. This provides enough particulate matter for analysis, but avoids clogging and
excessive filtration times. The same sample volumes are used for the particulate PC/N and P, and
chlorophyll analysis (on glass fiber filters) and seston (on polycarbonate filters).
For most of the period of this database, single PC/N samples have been used; in the past
several years, duplicate filters have been prepared. With duplicate samples from the estuarine
gradient analyzed in 2003, the mean precision for PC was ± 5.4% based on 38 paired samples
(with two outliers removed). For PN, the similar 2003 paired sample analysis gave a precision of
± 6.1% based on 39 pairs (with one outlier removed). Similar analysis of precision for lower
concentration duplicate samples from the continental shelf and slope gave ± 12.1% CV for PC
and ± 14.0% for PN.
PARTICULATE PHOSPHORUS
Particulate phosphorus was analyzed from 1978-1988 using the high temperature
combustion method (Solorzano and Sharp 1980b). Samples were filtered on baked 25 mm
diameter GF/C glass fiber filters that were pretreated and handled in the same way as for the
PC/N analysis. Single filters were used and no estimate was made of replicate precision. In
method development, it was estimated that the precision of the particulate P was similar to that
of the total dissolved phosphorus; in the analysis of DOP precision above, the TDP precision was
estimated at ± 1.4%.
11
JH Sharp et al ESM 12
CHLOROPHYLL
Chlorophyll is measured with fluorometric methods using acetone extracts (Strickland
and Parsons 1972; Parsons et al. 1984). Samples are filtered on 25 mm diameter GF/F glass
fiber filters; earlier sampling used GF/C filters. Currently, the filters are then placed in a mixture
of 90% acetone and dimethyl sulfoxide (6 parts to 4 parts; respectively) in liquid scintillation
counter vials and kept in the dark in the freezer until analysis. Usually, fluorescence is read
within 2-15 hours of collection. When it is not feasible to complete the analyses at sea, samples
are placed in the vials without the acetone mixture and stored in the freezer until later analysis on
land (usually stored for less than 1 week). Prior to 1990, the extract solvent was 90% acetone,
without the DMSO, and samples were stored in the freezer for at least 24 hours prior to analysis.
Several instruments have been
used for the chlorophyll analysis
throughout the 27 years. Initially the
older GK Turner Associates model III
fluorometer was used with each door
calibrated separately. Since the early
1990s, we have used several of the
Turner Designs Model 10 instruments
with a single calibration factor applied
to all four door factors; improved
instrument design gives precise
correlation among doors. This
fluorometer is calibrated with pure
chlorophyll against the
Fig. 6. Chlorophyll concentration (acetone extract)
spectrophotometer with recalibration
measured with a Turner Designs Model 10 fluorometer
checks usually about once a year. A
with readings before and after acidification (Old
check of the calibration factors used
Fluorometer) compared to the same samples measured
with a single instrument over a period
on the Turner Designs Model AU-10 fluorometer with
of 6 years shows a drift of 2%. We
readings only before acidification (AU-10). A series of
used the newer Turner Designs model
samples from a mesocosm experiment run in 2001 were
-1
AU-10 for several cruises between
used with a chlorophyll range of 0.3 to 30 µg L (n=28).
1998-2000. We made a direct
comparison of chlorophyll
measurements with the two instruments. A series of samples were collected and after extraction,
the samples were read in the AU-10 and immediately read in the Model 10 (with before and after
acidification readings, for pheophytin correction; in the AU-10, narrower pass filters allow
skipping the acidification step). A comparison of calculated chlorophyll from the two
instruments is shown in Figure 6. Although the linearity of the regression is very good, the
readings from the AU-10 are consistently slightly high when compared to the Model 10. The
Model 10 fluorometer was calibrated within less than one month of this comparison with
readings at all four door factors, an average ratio of Rb/Ra of 2.01, and a CV of ± 2.85% for the
estimate of the door factor; thus, we suspect the accuracy of the calibration of the AU-10 at the
time we used it. The very good correlation between the two fluorometers with an offset does
12
JH Sharp et al ESM 13
indicate that they should be comparable with good standardizations. Since we did not maintain
the AU-10 in our laboratory with control of the calibration, we have returned to use of the Model
10 with readings before and after acidification.
We routinely filter duplicate aliquots for chlorophyll. On the basis of an n of 96 from
duplicates run in 2003, the average CV value is ± 4.8%. Occasionally (about 1 in 20 samples),
duplicates give an unresolvable difference of greater than ± 10%, but most of the time the
individual pairs have CV values in the range of 0 – 5%.
13
JH Sharp et al ESM 14
Table 1. Estimated precisions for parameters recorded in the Delaware Estuary Database. The concentration range and method with
period used are given in the second and third columns. The number of sets of replicates is given as n with period for the estimate of
precision where appropriate in parentheses. Analytical precision estimated for replicate analyses of a method is given in units as
averages of one standard deviation; where appropriate, %CV is given instead of or in addition to units (%CV = 1SD/concentration
averaged for n sets of replicates).
Parameter
Temperature
Salinity
DO
DO
DIC
DIC - estuary
DIC- offshore
PO4
NO3
NO3
NO2
NO2
NH4
Si
DOC
DOC
DOC - estuary
DOC -offshore
DON
DON
DOP
Seston
Particulate C
Particulate N
Chlorophyll
Concentration range
-0.5 – 29 ºC
0 – 33 ppt
40 – 1030 µg-at O L-1
270 – 890 µg-at O L-1
280 -2000 µM C
755 – 1955 µM C
2050 – 2055 µM C
0.04 – 6.7 µM P
2 – 144 µ M N
0.2 – 6.5 µM N
0.1 – 7.2 µM N
< 1 µM N
0.1 – 37 µM N
1 – 75 µM Si
40 – 300 µM C
40 – 300 µM C
40 – 75 µM C
10 – 80 µM
7.5 – 63 µM N
0.05 – 1 µM P
1.5 – 170 mg L-1
10 – 300 µM C
1 – 60 µM N
0.5 – 180 µg L-1
Method (estimate period)
Thermometer (1978 - 1985)
Salinometer (1978 - 1985)
Metrohm E535 (1978 - 1988)
Metrohm 716 Titrino (1990- present)
Old method (1987 - 1999)
MBARI analyzer (2000 - present)
MBARI analyzer
Manual colorometric (1978 – present)
Manual colorometric (1978 – present)
Manual colorometric (1978 – present)
Manual colorometric (1978 – present)
Manual colorometric (1978 – present)
Manual colorometric (1978 – present)
Manual colorometric (1978 – present)
Wet Chemical method (1978 - 1988)
Shimadzu TOC 5000 (1992 - 2002)
Shimadzu TOC –V (2002 - present)
Shimadzu TOC-V
Wet chemical (1980 - 2002)
Shimadzu TOC/(TN) (2003 - present)
HTC analysis (1978 – 1988)
Gravimetric (1978 – present)
HTC instruments (1978 - present)
HTC instruments (1978 - present)
Fluorometric (1978 – present)
14
n
816
816
954
138 triplicates (2003)
80 (2003)
24 (2003)
43 duplicates (2003)
43 duplicates (2003)
43 duplicates (2003)
43 duplicates (2003)
43 duplicates (2003)
107 (2003)
23 (2003)
43 (2003)
93 (1988)
43 duplicates(2003)
38 duplicates (2003)
38 duplicates (2003)
96 duplicates (2003)
Precision
± 0.04º C
± 0.05 ppt
± 3 µg-at O L-1
± 3 µg-at O L-1 (0.7%)
ca ± 3%
± 0.19%
± 0.07%
± 1.7%
± 0.4%
± 1.4%
± 1.7%
± 3.3 %
± 4.8%
± 2.7%
± 3 – 5%
± 2%
± 1.6%
± 1.6%
± 10 -20%
± 12%
± 7.5%
± 5.3%
± 12%
± 14%
± 4.8 %
JH Sharp et al ESM 15
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