Salinity Protocol

Summary: This procedure describes the method for the determination of seawater salinity using a Guildline Portasal™ Salinometer (Model 8410)

1. Principle

During a cruise a 24 bottle rosette is used to collect seawater salinity samples of ~225 mL from all bottles closed from pre-designated depths. A Guildline Instruments Portasal™ Salinometer (8410A) makes the precise conductivity comparisons between the water samples and a reference water standard. From these comparisons salinities are then calculated and logged using PC based software that averages data that meet replicate criteria. Concurrent with the water sampling, a Sea-Bird Electronics CTD (SBE 911 plus) profiles in situ data. Data processing software is used to compare the bottle salts to in situ CTD measurements. This is useful to confirm bottle closure, monitor CTD performance, and to select the best salinity data for future comparison.

2. Sample Bottles

Salinity bottles are collected in ~250 ml borosilicate KIMAX-35 bottles. These square cross sectioned bottles have screw tops and lids with separate plastic thimbles to prevent leakage and evaporation. Numbered stackable divider boxes hold 24 numbered bottles to match the 24 Niskins on the rosette water sampler. Bottles are filled with previously unused sample water to limit salt crystallization and to pre-leech silicate from the glass.

3. Sample Collection

Numbered salt bottles are drawn from corresponding Niskin bottles and filled to the shoulder after three ~40 ml rinses. The last fill is done without interruption until overflowing; then ~10 ml is poured out over the thimble and the bottle is stoppered and capped. Samples are left to equilibrate to room temperate for >8 hours prior to measurement. Before being placed in the salinometer samples are: gently inverted 3 times to remove any possible stratification, wiped around the bottom of the cap to draw out as much water as possible that may be trapped under and around the cap, wiped around the thimble and threaded neck once the cap is removed to eliminate any excess water or salt precipitate, and flushed by dumping out ~10 ml into a collection bucket.

4. Guildline Portasal™ Salinometer (8410A)

Guildline Portasal Salinometer model 8410A is used to make the precise conductivity comparisons between the water samples and reference water standards. Two of these instruments are taken on each cruise. Guildline specifications state an accuracy of ±0.003 PSU (same set point temperature as standardization and within -2°C and +4°C of ambient), and a precision of 0.0003 PSU.

5. Configuring the PORTASAL™

Bath temperature must be within 2 degrees below and 4 degrees above ambient. To check bath temperature set point press the TSET key. Compare set point to actual by pressing ENTER for actual bath temperature. Set point and actual temperature must be within 0.02°C. Press UP ARROW key to view temperatures of the dual bath thermistors (TH1 and TH2). TH1 and TH2 must agree to within 0.04°C.

The Portasal must be powered up for >3 hours to ensure bath temperature regulation has begun before Reference values can be calibrated. While the the FUNCTION switch is in STDBY pressing REF key will display alternating Reference readings plus (+), minus (-), and Reference readings. After several cycles, when + and – values are within 1 unit of each other, pressing the COND key will initiate salinometer self-calibration.

With the FUNCTION switch set to ZERO, pressing the COND key will initiate the ZERO calibration process. When satisfied that the displayed value is stable, press the ZERO key. When the subsequent displayed number is stable (but not necessarily zero) press the COND key. The display should then read 0.00000. Setting the FUNCTION switch back to STDBY will ready the Portasal for Standardization.

6. SubStandard Preparation

Deep sea water is collected on cruises from bottles tripped at depths greater than 300m in 10 liter polyethylene jerry jugs. The seawater is then transferred to 50 liter carboys at the lab for filtration. During the transfer 10 ml hypochlorate (bleach) is added to inhibit biological activity. Concentrated high salinity seawater is prepared by evaporating deep sea water (>300m) by heating the seawater at about 90°C in an oven overnight. A volume of 1800 ml will evaporate down to about 1000 ml in 24 hours, final volume can be adjusted with DI water, this will give a salinity of around 60psu. Filter the concentrate through GF/F filter before using it for adjusting the sub-standard. Filter the deep seawater through GF/F to 46 liter mark. Add 10 ml laundry bleach to inhibit biological activity. Measure the salinity of the filtered water. Adjustment of the substandard salinity is usually done in two steps. For the first adjustment, add concentrate to the filtered sea water to adjust to the desired salinity using the relationship:

(vol. sw)(sal. sw) + (vol. conc.)(sal. conc.) = (vol. sw + vol. conc.)( final sal. sub)

The salinity of the concentrate will be too high to measure on the salinometer. If you have prepared the concentrate as above you can do your initial adjustment using an estimated salinity of 60 for the concentrate. The final salinity of the sub should be close to your standard value ± 0.015 PSU. For the initial adjustment it works well to add about 90% of the concentrate volume calculated above. Be sure you have thoroughly mixed the solution by stirring gently. A motorized paddle wheel is used to thoroughly stir the mixture. Measure the salinity of the sub after the first adjustment. Using the equation above you can now calculate the apparent salinity of the concentrate, by using the salinity of the concentrate as an unknown, along with the known values for the initial salinity of the seawater, and the adjusted value of the substandard as just measured. Applying the calculated salinity value for the concentrate, use the equation again to calculate a new volume of concentrate to be added to get to the final desired salinity of the substandard. If your initial concentrate addition was to large you may have to dilute with DI water using zero for salinity concentrate in the equation. When you have arrived at the desired salinity for the sub-standard add a layer dimethylpolysiloxane to prevent evaporation resulting in salinity changes.

7. IAPSO Standard Seawater

Cut and Paste from WHP Operations and Methods July 1991

Standard Seawater prepared by Ocean Scientific International Ltd. (OSI) is the recognized standard for the calibration of instruments measuring conductivity (salinity). This water is natural surface water which has been collected in the North Atlantic and carefully filtered and diluted with distilled water to yield seawater with a conductivity ratio near unity and a salinity near 35. The seawater is sealed in glass vials and labeled with the date, batch number, K15 value and chlorinity. Mantyla (1987) has compared batches of this seawater (P-29 to P-102) and found inter-batch differences as great as about 0.003 in some of the older batches. Since batch P- 93 however, inter-batch differences have not exceeded about 0.001.

It is recommended that a single batch of SSW be used during each cruise and that it be identified in the cruise report. It is the responsibility of the salinity analyst and chief scientist to ensure that the quality of the salinity observations are the highest attainable. It follows, therefore, that the inter-batch differences described by Mantyla (1987) should be used to correct the final salinities before they are reported. It has also been noted that the conductivity of some Standard Seawater changes with time. Vials more than 4-5 years old should be compared with fresher Standards to determine possible changes in conductivity due to aging.

8. Wolga Water

In addition to using IAPSO SSW, a batch of aforementioned substandard water has been bottled and sealed in glass vials. This is done to decrease dependence on IAPSO standard while maintaining acceptable standardization accuracies.

9. Standardizing the Portasal with SubStandard

Before standardization can begin the conductivity cell must be flushed rigorously as it had been filled with detergent or 50% ethanol between uses. To flush, attach the peristaltic pump to the Portasal sampling tube. Turn on both the Portasal and peristaltic pumps, and open the valve on the on the substandard feed line. Flush repeatedly (~10 flushes) until the cell is free of contamination. It is convenient to do this during Portasal configuration. Begin the salt program on the salt computer, and complete requisite data fields.

With the conductivity cell full of substandard and a stable value being displayed turn the FUNCTION switch to READ. When displayed value is stable press STD key. When the Portasal display reads STD STANDARDIZE press ENTER key. The display will show the conductivity of the standard being used. Press ENTER key to proceed (if values of substandard need to be adjusted use ARROW keys.) The BATCH # will be displayed and should match what is being used. If values need to be changed modify with the ARROW keys, pressing the ENTER key will ready Portasal for standardization. The display will read ENTER WHEN READY, press ENTER key. The Portasal will begin measurement and display a substandard value. When stable press the COND key. This will terminate the standardization and the displayed conductivity ratio should match that of your sub-standard within ± 0.00001. Pressing the ENTER key on the salt computer will save it to file.

At the beginning of each sample run the Portasal is standardized with substandard. However, every other day IAPSO and Wolga standards are also sampled. All three standards are run before and after the sample run in order to ascertain instrument drift.

10. Salt Data Acquisition Program

The SIO-CalCOFI-authored conductivity recording software, PSal.exe, is a Windows-based data acquisition program that records conductivity values from discreet salinity samples. Averaging five or more conductivity readings from the Guildline Portasal, the operator saves a stable reading. The flow cell is flushed and refilled and additional readings are performed. A pair of conductivity values which agree within 0.0010 standard deviation are recorded and salinity calculated. The software compares this calculated bottle measurement to the matching CTD salinity when available. This comparison is a good indicator of sensor performance and bottle sampling accuracy.

The data are saved as salt run files which may contain more than one station. Keeping the salt runs combined is necessary to calculate the drift-over-time calculated from any change in the end standard or substandard reading. The data are also save in a single, combined, database-friendly csv for database processing (in development).

11. CTD Equipment and Data Processing

A SBE 9ll+ CTD is equipped with dual conductivity and temperature sensors. These are routinely calibrated (~6 months), but because of superior stability the pressure sensor in calibrated much less frequently (~24 months).

The SBE 11plus Deck Unit applies a real time alignment correction to conductivity during data collection, and the accompanying software generates a marker file for each profile containing CTD data at the instant of each Niskin closure.
Seasoft-processed CTD salinities are processed and imported into station csvs for comparison to bottle salinities.

12. DECODR: Data Entry Compiler & Output Data Reports

Dissolved Oxygen Protocol

Summary: The amount of dissolved oxygen in seawater is measured using the Carpenter modification of the Winkler method. Carpenters modification (1965) was designed to increase the accuracy of the original method devised by Winkler in 1889. Using Carpenters modification, the significant loss of iodine is reduced and air oxidation of iodide is minimized. Rather than using the visible color of the iodine-starch complex as an indicator of the titration end-point, we use an automated titrator that measures the absorption of ultraviolet light by the tri-iodide ion, which is centered at a wavelength of 350 nm.

1. Principle

Manganous chloride solution is added to a known quantity of seawater and is immediately followed by the addition of sodium hydroxide iodide solution. Manganous hydroxide is oxidized by the dissolved oxygen in the seawater sample and precipitates forming hydrated tetravalent oxides of manganese.

Mn+2 + 2OH- ———————————————-> Mn(OH)2 (solid)
2Mn(OH)2 + O2 ——————————————–> 2MnO(OH)2 (solid)

Upon acidification of the sample, the manganese hydroxides dissolve and the tetravalent manganese in MnO(OH)2 acts as an oxidizing agent, setting free iodine from the iodide ions.

Mn(OH)2 + 2H+ ——————————————-> Mn+2 + 2H2O
MnO(OH)2 +4H+ +2I ————————————> Mn+2 +I2 +3H2O

The liberated iodine, equivalent to the dissolved oxygen present in the sample, is then titrated with a standardized sodium thiosulfate solution and the dissolved oxygen present in the sample is calculated. The reaction is as follows:

I2 + 2S2O3 ————————————————–> 2I + S4O6

2. Reagent Preparation

2.1. The manganous chloride solution (3M) is prepared by dissolving 600g of reagent grade manganous chloride tetrahydrate, MnCl2•4H20, in Milli-Q water to a final volume of 1 liter. This solution is then filtered using 47mm glass fiber filters (Whatman GF/F).

2.2 The sodium hydroxide (8M)-sodium iodide (4M) solution is prepared by first dissolving 600g sodium iodide (NaI) in approximately 600ml Milli-Q water. After the NaI is dissolved, 320g of NaOH is added slowly (caution-the solution will get hot) and the volume is adjusted to 1 liter with Milli-Q. The solution is then filtered through a GF/F.

2.3. Sulfuric acid solution (10N) is prepared by slowly adding 280ml of reagent grade concentrated sulfuric acid, H2SO4, to 770ml of Milli-Q water. This should be prepared with caution as is gets very hot.

2.4. The sodium thiosulfate solution (0.2N) is prepared by dissolving 50g sodium thiosulfate pentahydrate (Na2S2O3.5H20) and 0.1g anhydrous sodium carbonate (Na2CO3) in Milli-Q water to a final volume of 1 liter. This solution is prepared approximately 2 weeks before use and stored in an amber glass bottles.

2.5. The potassium iodate standard (0.0100N) is prepared by first drying potassium iodate(KIO3) in a drying oven for approximately one hour. Once the KIO3 is dried, carefully measure out 0.3567g, using a 5-place balance, and dissolve in Milli-Q water to a final volume of 1 liter.

3. Sample Drawing

3.1. Oxygen samples are always drawn first from the Niskin bottles and should be drawn as soon as possible to avoid contamination from atmospheric oxygen. Approximately 6 inches of Tygon tubing connected to a temperature probe via a y-connector is slipped onto the discharge valve of the Niskin.

3.2. A calibrated volumetric flask is rinsed three times, then with the seawater still flowing, the end of the tygon tubing is placed into the flask nearing the bottom. The sample is then overflowed with twice the sample volume while making sure that there are no bubbles in the tubing during the overflow process. The tubing is then carefully removed from the sample flask to prevent the influx of bubbles.

3.3. Immediately after drawing the sample, 1 ml of manganous chloride solution is added into the flask. This is followed by the addition of 1ml of sodium hydroxide-sodium iodide solution to the sample. Both dispensers should be purged to remove air bubbles prior to the addition of these reagents.

3.4. The stopper is then carefully placed in the bottle to avoid the trapping of air and the temperature of the seawater at the time of sample draw is recorded.

3.5. After all samples are drawn, they are shaken vigorously to disperse the precipitate uniformly through the flask. This process is repeated again after the precipitate has settled to the bottom of the flask, or after at least ten minutes.

4. Standardization of thiosulfate

4.1. Proper care in the setup of the auto-titrator is required before running blanks, standards and samples. The UV lamp is turned on at least 30 minutes prior to the run and should have a stable voltage of 2.4-2.5 volts for the run. The dosimat tubing is carefully purged so that the lines are completely free of air bubbles, and the water bath inside the auto-titrator is clean and filled with Milli-Q water prior to analysis. Make sure, prior to your first run, any thiosulfide solution is rinsed off the tip of the line with Milli-Q after purging.

4.2. Using a Metrohm 655 Dosimat, dispense 10 ml of the standard potassium iodate solution into a clean oxygen flask. Add a stir bar and rinse down the sides with a small amount of Milli-Q water. Add 1 ml of the 10N sulfuric acid solution and swirl to ensure the solution is well mixed before adding the pickling reagents.

4.3. Add 1 ml sodium hydroxide-sodium iodide solution to the acidified flask and swirl gently. Then add 1ml of Manganous chloride solution, swirl gently, and fill the solution to the neck of the flask with Milli-Q water.

4.4. The UV detector on the auto-titrator measures the transmission of ultra-violet light through the standard (as well as seawater sample and blank) as a Metrohm 665 Dosimat dispenses thiosulfate at increasingly slower rates. The endpoint is reached when no further change in absorption is detected by the detector. At this point all of the iodine has been consumed.

5. Blank determination

5.1. Using the Metrohm 655 Dosimat, dispense 1 ml of the standard Potassium Iodate solution into a clean oxygen flask. Add a stir bar and rinse down the sides with a small amount of Milli-Q water. Add 1 ml of the 10N sulfuric acid solution and swirl to ensure the solution is well mixed before adding the pickling reagents.

5.2. Add 1 ml sodium hydroxide-sodium iodide solution to the acidified flask and swirl gently. Then add 1ml of Manganous chloride solution, swirl gently, and fill the solution to the neck of the flask with Milli-Q water. The solution is then titrated to the end-point as described in section 4.3 above.

5.3. A second 1 ml aliquot is added to the same solution which is then titrated to a second end-point. The difference between the first and second titration is used as the reagent blank.

6. Sample analysis

6.1. Samples are analyzed after all of the precipitate settles to the bottom of the flask, after the second shake. The top of the flask is wiped with a kimwipe to remove moisture containing excess reagent around the stopper and then the stopper is carefully removed.

6.2. 1ml of 10N sulfuric acid is added to the sample and a stir bar is placed inside the flask. The flask is then secured inside the clean water bath. The tip of the thiosulfate dispenser is placed inside the sample flask and the automated titration can begin with the use of an auto-titrator program.

7. Certified Standard Comparison

7.1 Presented here are the results of a comparison of several certified standards with a CalCOFI prepared standard (weighed and diluted up to set normality in 1 liter volumetric)standard. Three certified iodate solutions, (Table 1) were tested using the same 0.2N thiosulphate solution. Iodate concentrations are back calculated from defined thiosulphate concentrations for comparison purposes. It is noteworthy that the Acculute and Fisher solutions had to be diluted before use. The same volumetric was used for all dilutions, the same Dosimat and piston was used for all titrations, extensive rinsing between uses. Titration n=9 as dictated by the maximum number of samples that could be acquired from the 100ml portion for the OSIL standard. CalCOFI values are a result of cruise and shore based titrations to demonstrate an integrated real use sampling. Differences between all standards represent less than +/- 0.5 percent of the signal.

7.2 These results are consistent with previously published methods comparison for precision of oxygen measurements1 (WOCE Report 73/91) and previous replicate analysis to verify precision with auto-titration methods2. Although the Ocean Data Facility (ODF) performed the 1991 comparisons for Scripps and was different than CalCOFI, the Winkler techniques used then and now are the same. Presently, CalCOFI methods employ a UV end point auto-titrator made by ODF that produces a precision of 0.005-.01 ml/L. The difference between high and low samples in replicate analysis equals ~0.5% with a standard deviation typically under 0.010 ml/L.

See references:


8. Calculation and Expression of Results

8.1. The auto-titrator uses a UV detector that detects changes in voltage as thiosulfate is added to the sample. The volume of thiosulfate added is recorded at an endpoint once there is no change in voltage. The end point is determined by a least squares fit using a group of data points just prior to the end point, where the slope of the titration curve is steep, and a group of data points just after the endpoint, where the slope of the curve is close to zero. The intersection of the two lines is taken as the endpoint.

8.2. The calculation of dissolved oxygen follows the same principle oulined by Carpenter (1965). Our results are expressed in mL/L.

(R-Rblk)VIO3*NIO3*E DOreg
O2(ml/L)= ————————— – ———

(Rstd-Rblk)*(Vb-Vreg) Vb

R= Sample titration (mL)
Rblk = Blank value (mL)
VIO3 = Volume of KIO3 standard (mL)
NIO3 – Normality of KIO3 standard
E = 5,598 mL O2/equivalent
Rstd = Volume used to titrate standard
Vb – Volume of sample bottle
Vreg = Volume of reagents
DOreg = Oxygen added in reagents

9. Equipment/Supplies

Volumetrically calibrated 100ml Glass Erlenmeyer flasks with paired ground glass stoppers
3 – 1ml Brinkman reagent dispensers
Tygon tubing
Fisher Scientific UV Longwave Pencil Lamp, 365 nm and power supply, 115 VAC
2 Metrohm Dosimat 665 Automatic Burets
10ml Metrohm Dosimat Exchange unit
1ml Metrohm Dosimat Exchange unit
Metrohm Dosimat Keypad
Spare 1ml and 10ml dispensor pistons for the Metrohm Dosimat Exchange units
Scripps/STS Auto-titrator Unit and Software
PC Computer
Waterproof sampling thermometer
Concentrated Sulfuric Acid, H2SO4, ACS Grade
Manganous Chloride Tetrahydrate, MnCl2•4H20, ACS Grade
Sodium Hydroxide, NaOH, ACS Grade
Sodium Thiosulfate, Na2S2O3•5H20, ACS Grade
Potassium Iodate, Dry high purity KIO3, Alfa Aesar
Granular Sodium Iodide, EMD Chemicals via VWR
Magnetic Stir Bars

10. References

Anderson, G. C., compiler, 1971. “Oxygen Analysis,” Marine Technician’s Handbook, SIO Ref. No. 71-8, Sea Grant Pub. No. 9.
Carpenter, J. H., 1965. The Chesapeake Bay Institute technique for the Winkler dissolved oxygen method. Limnol. Oceanogr., 10: 141-143.
Culberson, C. H. 1991. Dissolved oxygen. WHP Operations and Methods — July 1991.
Parsons, T. R., Y. Maita, C. M. Lalli, 1984. A Manual of Chemical and Biological Methods for Seawater Analysis. Pergamon Press Ltd., 3-28.

Nutrient Protocol

Summary: The phytoplankton macro nutrients nitrate, nitrite, silicate, and phosphate in seawater are analyzed using colorimetric assays. Ammonium concentrations are determined using a fluorometric assay.

1. Principle

Nutrient analysis is performed on a QuAAtro continuous segmented flow autoanalyzer (SEAL Analytical). A sample of seawater enters a reagent stream within a manifold on the analyzer where it undergoes a series of reactions that ultimately produce a colored compound. These compounds absorb light at a specific wavelength. A monochromatic beam of light is passed through the sample and the absorbance is measured. The machine is calibrated with a series of known standards and a standard curve is produced. The intensity of the color produced by the unknown sample is proportional to the concentration of the analyte present. The product of the ammonia method is a fluorescent species; however the same basic principle applies, where the intensity of the fluorescence is directly related to concentration. The methods for silicate and total oxidized nitrogen (TON) are modified versions of those described by used Armstrong et al. (1967) and Gordon et al. (1992). The phosphate determination employs a modification of the method described by Murphy and Riley (1962), and ammonia is analyzed based on the Kerouel and Aminot (1997) fluorometric method.

2. Method Description

Silicate (SiO2)

Silicate is analyzed using a modified technique of Armstrong (1967). An acidic solution of ammonium molybdate is added to a seawater sample to produce silicomolybdic acid which is then reduced to a blue silicomolybdous acid following the addition of ascorbic acid. The amount of blue color produced is proportional to the amount of dissolved reactive silicate in the sample. Oxalic acid is added to inhibit PO4 color interference. The sample is passed through a 10mm flow cell and the absorbance is measured at 820nm.

Nitrate and Nitrite (NO3 and NO2)

A modification of the Armstrong (1967) procedure is used for the analysis of nitrate plus nitrite, or total oxidized nitrogen (TON). For this analysis, the seawater sample is passed through a cadmium reduction coil where nitrate is reduced to nitrite. The efficiency of this reduction is determined by running two equimolar solutions, one containing only nitrate and one containing only nitrite, through the coil. The percent of nitrate converted to nitrite yields the coil efficiency, a factor used to calculate the nitrate concentration. Sulfanilamide is introduced to the sample stream followed by N-(1-naphthyl) ethylenediamine dihydrochloride which complexes with nitrite to form a red azo dye. The stream is then passed through a 10 mm flowcell and the absorbance measured at 520nm.

The same method is employed for nitrite analysis, except the cadmium column is not present. Consequently, only nitrite already present in the sample is measured.

The nitrate concentration is calculated on a “virtual channel” by the following equation, using the coil efficiency, TON, and NO2:

TON equation

NO3=Nitrate (in sample)

NO2= Nitrite (in sample)

TON=Total oxidized nitrogen (NO3+NO2) in sample

A= NO3 concentration in mixed calibrant

B= NO2 concentration in mixed calibrant

Recovery = Coil efficiency (expressed as a decimal)

Phosphate (PO4)

Phosphate is analyzed using a modification of the Murphy and Riley (1962) technique. Similar to the silicate method, an acidic solution of ammonium molybdate is added to the sample to produce phosphomolybdic acid that is subsequently reduced to blue phosphomolybdous acid following the addition of ascorbic acid. Color intensity is directly related to the concentration of dissolved phosphate in the sample. The reaction product is then passed through a 10mm flow cell and the absorbance measured at 880nm.

Ammonia (NH3)

Ammonia is measured fluorometrically using a modification of the method described by Kerouel and Aminot (1997). In the presence of a borate buffer, samples are reacted with o-pthalaldehyde (OPA) to form a fluorescent complex that is excited at 370nm and emits at 460nm. The reaction takes place at 75°C. Sodium sulfite is added to the working reagent to reduce sensitivity to dissolved amino acids.

3. Method Notes

Ammonium is a difficult parameter to measure accurately due to its insidious nature and problems with contamination. Phosphorus and nitrogen compounds are also potential sources of contamination with poor sampling technique. Care must be taken during sampling to insure there is no contamination (e.g. touching the inside of the tube or the cap with fingers, smoking near rosette).

4. Water Sampling

Nutrient samples are drawn into 30 ml polypropylene screw-capped centrifuge tubes.

The tubes and caps are cleaned with 10% HCl and rinsed 3 times with sample before filling.

Samples that are not analyzed immediately are refrigerated and analyzed within 16 hours of collection. All samples are allowed sufficient time to reach room temperature. The centrifuge tubes fit directly onto the sampler.

5. Calculations

All data is reported in micro-moles/liter. The main calculations for concentration on the QuAAtro are run through the required AACE software interface. These calculations still follow the principle of other instruments, where:

[X] micro moles/liter =(Absorbance-blank) x F1 (Response Factor)

Values are corrected for drift based on changes in beginning and end standards, ultra pure water and the relative position of samples in the run. Corrections for linearity are performed, if necessary, based on a set of absorbances and concentrations; deviations from Beer’s law can be plotted to reveal a polynomial function that can be applied to correct sample values accordingly. Improvements in optics in the QuAAtro instrument have resulted in marked improvement in linearity and reduction of blank values for nitrate and silicate and phosphate.

6. Quality Control

A sample of reference material for nutrients in seawater (RMNS), produced by KANSO technos ( is included in every run and those data are monitored for consistency.
An aliquot from a large volume of stable deep seawater is run once a day as an additional check. The stability of the deep seawater check is aided by the addition of mercuric chloride as a poison.

The efficiency of the cadmium column used for nitrate reduction is monitored throughout the cruise and usually ranges from 97.0-100.0%.

NO3, PO4, NO2, and NH4 are reported to two decimals places and SiO2 to one.

Accuracy is based on the quality of the standards; the levels in micro moles/liter (µM ) are:

NO3 = 0.05
PO4 = 0.004
SiO2 = 2-4
NO2 = 0.05
NH3 = 0.03
The precision of the instrument for NO3, PO4, and NH4 is 0.01 μM and 1.0μM for silicate and 0.01μM for NO2.

The detection limits in micro moles/liter for the instrumentation are:

NO3+NO2 = 0.02
PO4 = 0.02
SiO2 = 0.5
NO2 = 0.02
NH3 = 0.04

7. Equipment/Supplies

Seal Analytical continuous-flow QuAAtro run by IOD since CalCOFI 1203SH; AutoAnalyzer 3 (AA3) run by ODF on cruises prior to 1203SH. Distributed by Bran and Luebbe,

30 ml centrifuge tubes and test tube racks, 8 sets color coded and numbered

Barnstead Nanopure purified water system or equivalent polished water source

Sundry laboratory glassware

8. References

Armstrong, F.A.J., C.R. Stearns, and J.D.H Strickland, (1967). “The measurement of upwelling and subsequent biological processes by means of the Technicon Autoanalyzer and associated equipment,” Deep-Sea Research, 14, pp.381-389.

Atlas, E.L., S.W Hager, L.I. Gordon, and P.K. Park, (1971). “A Practical Manual for Use of the Technicon AutoAnalyzer in Seawater Nutrient Analyses Revised,” Technical Report 215, Reference 71-22, p.49, Oregon State University, Department of Oceanography.

Gordon, L.I., J.C. Jennings, A.A. Ross, J.M. Krest, (1992). “A suggested Protocol for Continuous Flow Automated Analysis of Seawater Nutrients in the WOCE Hydrographic Program and the Joint Global Ocean Fluxes Study,” Grp. Tech Rpt 92-1, OSU College of Oceanography Descr. Chem Oc.

Hager, S.W., E.L Atlas, L.I Gordon, A.W. Mantyla, and P.K. Park, (1972). ” A comparison at sea of manual and autoanalyzer analyses of phosphate, nitrate, and silicate ,” Limnology and Oceanography, 17, pp.931-937.

Keuroul, R. and A. Aminot, (1997). “Fluorometric determination of ammonia in sea and estuarine waters by direct segmented flow analysis,” Marine Chemistry Vol. 57, no. 3-4, pp.265-275.

Murphy, J. and J.P. Riley, (1962). A modified single solution method for the determination of phosphate in natural waters. Analytica Chimia Acta, Vol. 27 pp.31-36.

Chlorophyll-a & Phaeopigment Protocol

Summary: Chlorophyll-a is extracted in an acetone solution. Chlorophyll and phaeopigments are then measured fluorometrically using an acidification technique.

1. Principle

Seawater samples of a known volume are filtered (< 10 psi) onto GF/F filters. These filters are then placed into 10ml screw-top culture tubes containing 8.0ml of 90% acetone. After a period of 24 to 48 hours, the fluorescence of the samples is read on a fluorometer. Then samples are acidified to degrade the chlorophyll to phaeopigments (i.e. phaeophytin) and a second reading is taken. The readings prior to and after acidification are then used to calculate concentrations of both chlorophyll a and ‘phaeopigment’. The method used today is based on those developed by Yentsch and Menzel (1963), Holm-Hansen et al. (1965) and Lorenzen (1967). Note that concentrations of ‘phaeopigments’ are not a good measure of Chl-a degradation products present in the sample since Chl-b present in the sample will be measured as ‘phaeopigments’.

2. Sample Drawing

Chlorophyll bottles should be rinsed three times with sample prior to filling. The bottles are calibrated for volume, so the sample drawer must insure that air bubbles are not clinging to the sides of the bottle and it is filled completely. The sensitivity of the fluorometric method allows for sample bottles of ~50 to 250 ml.

3. Sample Filtration

3.1. Check that the filtration funnels are well seated on the base, and be sure that the filters (Whatman GF/F) are in place. Improperly placed filters or loose funnels will result in loss of sample. The chlorophyll samples are volumetric and should sample loss occur, replace the filter with a new one and redraw the sample.

3.2. Turn on the vacuum pump, pour the sample into the filter funnel, and open the valve. Check the vacuum pressure to see that it does not exceed 10 psi or ~500mm Hg. Generally samples are filtered in such a way as to insure that the deepest samples (i.e. those typically containing less chlorophyll) are filtered at the same manifold positions in each time. When a shallow cast is performed and a reduced number of samples is taken, it is advisable to filter them on positions typically used for those approximate depths. This reduces the potential for contaminating filter funnels used for filtering deep samples that in general contain low levels of chlorophyll.

3.3. When a sample has finished filtering, turn off the valve; once all the samples have filtered, turn off the pump; use designated sample forceps to pick off the filter and place it in the appropriate numbered tube containing 8 mL 90% acetone. Make sure that the filter is completely submerged in the acetone.

3.4. Cap TIGHTLY but be aware that tube tops can break off, then place the sample tubes in a rack. The sample rack is then placed in a refrigerator and the filtration time is recorded.

4. Standardization of Fluorometer

4.1. A commercially available chlorophyll standard (e.g. Anacystis nidulans, Sigma Aldrich) should be used to calibrate the fluorometer, preferably before and after each cruise. The Chl-a standard is dissolved in 100% acetone to yield approximately 0.1mg-Chl per ml solution. 1ml of this solution can then be diluted in 100ml 100% acetone and read in a spectrophotometer at 664nm. A second reading at 750nm is also recorded as a blank value to correct for sample turbidity. The remainder can be aliquoted into cryo-tubes and stored in liquid N2 for future use. Chl-a standards such stored are stable for years. The initial dilution is made with 100% acetone because it stores better in liquid N2 than those made with 90%. However, since 90% acetone is used for the extraction, it is also used for dilutions when generating a standard curve.

4.2 The concentration (mg l-1) of the standard is determined by the following equation: Chl-a = Equation1 A664 = absorption at 664nm A750 = absorption at 750nm E = Extinction coefficient (100% acetone = 88.15, 90% acetone = 87.67) from Jefferies and Humphrey (1975) l = cuvette path length (cm)

4.3. A series of dilutions using 90% acetone (N> 5) are then made and read, recording both Rb and Ra values. Blank values should be subtracted from the Rb and Ra prior to performing calculations. If using a fluorometer with multiple sensitivity and range settings such as a Turner model 10, then the proper blank value must be subtracted for readings taken at a given setting.

4.4. A calibration factor (F) must be calculated for each fluorometer. It is the slope of the line resulting from plotting the fluorometer reading (x-axis) vs. chlorophyll concentration (y-axis). This line is forced through zero. An acidification coefficient (τ) is the average acid ratio (Rb/Ra) for the pure chlorophyll standards used in the calibration.

4.5. Calculating chlorophyll and phaeopigment concentration in a sample is accomplished by using the following equations (Knap et al., 1996):

Chl (µg/l) = Equation2

Phaeo (µg/l) = Equation3

F = Linear calibration factor (see 4.4)

τ = Average acid ratio (Rb/Ra) – Note that these are actually corrected values, with the blank readings already subtracted.

Ve = Volume of extract (ml)

Vf = Volume of sample filtered (l)

S = Sensitivity setting of fluorometer (Applicable to Turner model 10. If using a model 10AU or another fluorometer, use a value of “1”)

Rng = Range setting of fluorometer (Applicable to Turner model 10. If using a model 10AU or another fluorometer, use a value of “1”)

There are variations of this equation that can be used and other factors that can affect chlorophyll measurements. More detailed descriptions can be obtained in Strickland and Parsons (1968) and Holm-Hansen and Riemann (1978).

Note: After a cruise, the fluorometer is calibrated again and the calibration factors and average acid ratios obtained from pre and post-cruise calibrations are averaged for final data processing.

5. Reading Samples on the Fluorometer

5.1. The fluorometer should be allowed to warm up for approximately 1/2 hour before using it. Samples must extract in acetone for at least 24 hours prior to reading on the fluorometer and should be read before 48 hours.

5.2. Samples must be at room temperature prior to reading. One hour before samples are to be read, they should be removed from the refrigerator and allowed to warm up in a dark place.

5.3. A blank tube containing the same acetone batch used for the extractions should be prepared and read prior to reading samples. This blank should be read before and after every sample run and after door setting have been changed (Turner model 10 fluorometer)

5.4. A coproporphyrin standard should be read prior to reading samples (D’Sa et al., 1997). While not used in any calculation, it is useful to monitor the performance of the fluorometer over time between calibrations. Significant changes in coproporphyrin readings may indicate a problem with the fluorometer.

5.5. Remove the filter, shake the sample to insure that it is well mixed, and use a Kimwipe to remove fingerprints from the exterior of the tube prior to running samples.

5.6. Read the sample and record the number (Rb). Add 100µl of 10% HCl and wait approximately 30 seconds for the number to stabilize and record the value (Ra).

6. Equipment/Supplies

  • Whatman 25mm GF/F filters (Fisher Scientific)
  • Volumetric sample bottles (~130-150ml)
  • Vacuum filtration apparatus with vacuum pump capable of maintaining 10 psi
  • Fluorometer and proper filter kit for measuring chlorophyll-a/phaeophytin with acidification method (Turner model 10AU uses a 10-037R optical kit)
  • Pipet (or re-pipet) capable of delivering 100µl
  • Personal protection equipment (PPE) consisting of gloves and safety glasses
  • Kimwipes or equivalent laboratory wipes
  • 10ml screw-top sample tubes (Fisher Scientific)
  • Two sets of forceps (one for sample manipulation and one for replacing clean filters)
  • Assorted laboratory glassware, including volumetric flasks for diluting calibration standards

7. Reagents

  • Milli-Q or equivalent polished water source
  • HPLC-grade or equivalent low-fluorescing acetone. Note that volume is not conserved when preparing solution of water and acetone. The addition of 413ml Milli-Q water to 3800ml of acetone results in 4130ml of 90% acetone.
  • 10% HCl solution
  • Chlorophyll-a (Sigma Aldrich catalog number C6144)
  • Coproporphyrin III tetramethyl ester (Sigma Aldrich catalog number C7157)

8. References

  • D’Sa, E.J., Lohrenz, S.E, Asper, V.L., and Walters, R.A. (1997). Time Series Measurements of Chlorophyll Fluorescence in the Oceanic Bottom Boundary Layer with a Multisensor Fiber-Optic Fluorometer. , 167: 889–896. DOI: 10.1175/1520-0426(1997)0142.0.CO;2
  • Holm_Hansen, O., Lorenzen, C.J., Holms, R.W., Strickland, J.D.H. (1965). Fluorometric Determination of Chlorophyll. J. Explor. Mer. 30: 3-15.
  • Holm-Hansen, O., and B. Riemann. (1978). Chlorophyll a determination: improvements in methodology. Oikos, 30: 438-447.
  • Jeffery, S.W. and Humphrey, G.F. (1975). New spectrophotometric equations for determining chlorophylls a, b, c1, and c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol. Pflanz. 167: 191-194.
  • Knap, A., A. Michaels, A. Close, H. Ducklow and A. Dickson (eds.). (1996). Protocols for the Joint Global Ocean Flux Study (JGOFS) Core Measurements.
  • JGOFS Report Nr. 19, vi+170 pp. Reprint of the IOC Manuals and Guides No. 29, UNESCO 1994.
  • Lorenzen, C. J. (1967) Determination of chlorophylls and phaeopigments: spectrophotometric equations. Limnol. Oceanogr. 12: 343–346.
  • Strickland J. D. H., Parsons T. R., (1968). A practical handbook of seawater analysis. Pigment analysis, Bull. Fish. Res. Bd. Canada, 167.
  • Yentsch, C.S., Menzel, D.W. (1963). A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep-Sea Res. 10: 221-231

Primary Productivity Protocol

Summary: Primary production is estimated from 14C uptake using a simulated in situ technique in which the assimilation of dissolved inorganic carbon by phytoplankton yields a measure of the rate of photosynthetic primary production in the euphotic zone.

1. Principle

Seawater samples are incubated with a radioactive substrate to determine the incorporation of inorganic carbon into particulate organic carbon due to photosynthesis at selected light levels. The data have units of mg-carbon per m3 per half day.

2. Productivity Cleaning Procedures

2.1. Micro-90 Cleaning solution is diluted to 2% solution using de-ionized water (DW). Hydrochloric acid (HCl) Trace Metal Grade, Fisher Scientific, solution (1.2M) diluted with DW. Acid-washing of Teflon should be done with great care as Teflon is porous to HCl which can compromise dilute basic stock solutions of 14C -bicarbonate.

2.2. 250 ml polycarbonate incubation bottles are filled to capacity with 2% MICRO for 3 days with the cap on in an inverted position. Next, rinse all Micro away and then rinse down the walls with 20 -30mls 10%HCl and recap and shake to acid rinse inside bottle. This should be left overnight 12-16 hours. The acid is removed by rinsing the bottles three times with milliQ clean water before air drying.

2.3. 10 liter rosette sample bottles are cleaned with a 2% MICRO soak for 3 days, rinsed with de-ionized water and then dipped in 10% metals free HCl. Caps, special coated springs and valve assemblies are also cleaned with a 2% MICRO soak for 3 days and then rinsed with de-ionized water and dried.

2.4. All lab ware to be used is cleaned in this manner.

3. Preparation of Isotope Stock

3.1. To prevent contamination of self or solutions, work with the isotope stock is performed wearing vinyl gloves.

3.2. A solution of 0.3 g of Na2CO3 anhydrous (ALDRICH 20,442-0, 99.995%) per liter Milli-Q filtered DW in a Micro cleaned 1 liter Teflon bottle to yield a concentration of 2.8 mM Na2CO3. This solution is filtered through 0.2µM Nucleapore filter to remove particulate carbonate.

3.3. Concentrated stock, 50ml of NaH-14CO3 (~50-57 mCi mmole; MP Biomedicals LLC.) was diluted with 350 ml the 2.8mM Na2CO3 solution in productivity-cleaned 1 liter polycarbonate graduated cylinder. It has become necessary to pH this up with an ultra clean 1N NaOH solution to raise the pH to ~10.

3.4. Specific activity can be checked by diluting the above made solution to working concentrations, ie 50-200µl added to 250ml polycarbonate centrifuge bottle and measuring out triplicate 1ml portions into beta ethanolamine spiked (1.5%v/v) Ecolume scintillation cocktail.

3.5. To check for 14C-organic carbon contamination another working aliquot of 200µl can be placed into a scintillation vial and acidified with 0.5ml 10% HCl and placed on a shaker overnight. This is done in the hood as it liberates 14C-CO2. The acidified dpm should be <0.0001% of the total dpm of the 14C preparation.

4. Incubation Systems: Situ Incubation Techniques

4.1. Incubation apparatus consists of seawater-cooled, temperature monitored incubator tubes wrapped with neutral-density screens which simulate in situ light levels.

4.2. Six incubation depths are selected, they represent 56, 30, 10, 3, 1 and ~0.3 % light level. These values are estimated using a wand type PAR meter after cleaning tubes and screens covering them. The near surface light level is reduced to 56% using common plastic screening to prevent a lens effect and subsequent cooking of the surface samples.

5. Sampling

5.1. Primary productivity samples are taken each day shortly before local apparent noon (LAN). Light penetration was estimated from the Secchi depth (Using the definition that the 1% light level is three times the Secchi depth). The depths with ambient light intensities corresponding to light levels simulated by near surface and the on-deck incubators were identified and sampled on the rosette up-cast. Extra bottles were tripped in addition to the usual 20 levels sampled in the combined rosette-productivity cast in order to maintain the normal sampling depth resolution.

5.2. Using a dark sleeve to subdue the light, water samples are transferred to the incubation bottles (250 ml polycarbonate bottles) and stored in a dark box until inoculation. Triplicate samples (two light and one dark control) were drawn from each productivity sample depth.

6. Isotope Addition and Sample Incubation

6.1. Samples are inoculated with 50-200 µl of 14C as NaHCO3 stock solution of sodium carbonate (Fitzwater et al., 1982).

6.2. Samples are incubated from LAN to civil twilight in a surface seawater-cooled incubators with neutral-density screens which simulate in situ light levels, corresponding to those from which samples were taken (see 4.2).

6.3. At civil twilight the incubation is terminated and the time noted. Sea state and safety is the only exception accepted to delay the end time.

7. Filtration

7.1. At the end of the incubation, all bottles have subsamples of 10mls removed for DO14C analysis. The LTER DOC filtrate apparatus consists of a plexi-glass filtration manifold to hold up to 18 scintillation vials over which syringe needles with 0.45um equivalent micro-syringe filters can be passed through stoppers with 25 ml syringe bodies serving as filter funnels. The exception to this is dark bottles are only sampled for DO14C on two each high and low chlorophyll stations.

7.2. Additionally, from dark bottles a 1ml sample is placed into beta mercapto-ethanol spiked (1.5%v/v) Ecolume scintillation cocktail to determine the specific radioactivity in the samples. These values are used to calculate an average cruise value after removing outliers.

7.3. Finally the samples are filtered onto Millipore HA filters and placed in scintillation vials. One half ml of 10% HCl was added to each sample. The samples are then allowed to sit, without a cap, at room temperature for at least 3 hours (after Lean and Burnison, 1979).

8. 14C Sample Processing

8.1. After addition of 10mls of Ecolume cocktail, vials are tightly capped and mixed before vials are counted for up to 10 minutes each for 14C on a Beckman 6100LC liquid scintillation counter set to 1.0% counting precision.

8.2. Data are captured to a flat file using Beckman data capture software for Windows in ASCII format. This format is then used to integrate productivity depths into the CalCOFI data processing flow.

9. Calculations

Data are presented as mean mg Carbon assimilated per meter cubed of seawater for one half light day. mgC/m3 per one half light day = ((Sampledpm – Blankdpm) x W)/R, where W = 25200 = 12,000 x A x FT x 1.05 12,000= molecular weight of carbon in milligrams A = carbonate alkalinity (milliequivalents/liter) FT = Total carbon dioxide content/ carbonate alkalinity 1.05 is the 14C isotope fractionation factor, reflecting preferential use of C12 over C14 by a factor of 5% R = dpm added to sample (µCi/200µl x 2.2 x 106) To better understand this equation and variables see Strickland and Parsons (1968).

10. Equipment/Supplies

  • 10 liter primary productivity cleaned sampling bottles
  • Secchi disk
  • Re-pipet dispensers for delivering 20µl, 200µl, 0.5ml
  • Pipets able to measure 1ml and 10ml
  • 250 ml polycarbonate centrifuge bottles
  • Liquid scintillation counting (LSC) vials
  • Seawater-plumbed incubation rack with neutral density screening
  • Par meter, wand type (Biospherical Instruments)
  • 14C sodium bicarbonate stock solution (MP Biomedicals, LLC)
  • Millipore Type HA filters (Fisher Scientific)
  • Vacuum filtration system, including separate device for DOC filtrate capture
  • Polycarbonate centrifuge bottles
  • Teflon laboratory wares
  • Vortex mixer
  • Liquid scintillation counter (LS 6000LC Beckman Instruments, Inc.)

11. Reagents

  • Milli-Q filtration/anion exchange water purifier
  • Micro-90 Cleaning solution, Cole Palmer Instrument Co.
  • HCl for trace metal analysis (Fisher Chemical)
  • Na2CO3 (99.995%) Aldrich Chemical
  • NaH-14CO3 solution (cat #17441H MP Biomedicals, LLC.)
  • 2-amino ethanol (ethanolamine) ACS grade
  • Aquasol-II (Dupont)
  • Ecolume (MP Biomedicals, LLC.)

12. Re-count

Check 14C scintillation counts were checked for accuracy by re-counting an entire cruise (n>200) of vials 9 months after original counting. Depletion due to half life was ignored due to the long half life of 14C. Results for samples greater than 1000dpms were averaged resulting in a return of counts equal to 101.3%. Efficiencies had a similar recount statistic of 100.9%. The exercise lead to evaluating cruise counts where the source of some replicate inconsistency was the result of chemiluminescence problems in which the counter displays a “lumex %”. It is important to monitor for higher lumex numbers which result in elevated counts due to a chemiluminescent reaction. Samples were dark adapted and recounted resulting in much better replicates.

13. References

  • Fitzwater, S. E., G. A. Knauer and J. H. Martin, 1982. Metal contamination and its effect on primary production measurements. Limnol. Oceanogr., 27: 544-551.
  • Lean, D. R. S. and B. K. Burnison, 1979. An evaluation of errors in the 14C method of primary production measurement. Limnol. Oceanogr., 24: 917-928.
  • Steeman-Nielsen, E. (1951). “Measurement of production of organic matter in sea by means of carbon-14”. Nature 267: 684–685.
  • Strickland, J. D. H. and Parsons, T. R. 1968. A Practical Handbook of Seawater Analysis pp. 267-278.