Bottle Sampling Depths, based on downcast profiles

The CalCOFI CTD-rosette is equipped with a Sea-Bird Electronic carousel water sampler (SBE 32), a computer-driven, electro-magnetically-released latch system. The 24 ten-liter plastic (PVC) bottles, equipped with epoxy-coated springs & Viton (non-toxic) O-rings, connect to 24 individual triggers by lanyards which keep the bottle ends open. During the downcast, profiles of different sensor measurements vs depth are displayed real-time on a computer screen. Based on the chlorophyll maximum & mixed layer depths, bottles are closed at specific depths to isolate the seawater. The 10 meter bottle spacing shifts up or down (see table below) to resolve steep gradient features such as chlorophyll, oxygen, nitrite maxima and shallow salinity minimum. Salinity, oxygen and nutrients samples are analyzed at-sea for all depths sampled. Chlorophyll-a and phaeopigments samples from the top 200 meters, bottom depth permitting, are also extracted for 24hrs and analyzed at-sea. Most CTD-rosette casts sample 20 depths to a maximum of 515 meters, bottom depth permitting. Occasionally, additional bottle depths or multiple bottles are tripped at the same depth to provide extra water for ancillary projects or primary productivity incubations. Two basin stations, off Santa Monica & Santa Barbara, are sampled beyond 515m to within 10m of bottom. Wire-length permitting, a 3500m deep cast is performed at sta 90.90.

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Seabird CTD-Carousel Setup & Deck Unit Diagnostics

SIO-CalCOFI’s method of terminating the sea cable uses a custom 4-pin pigtail to terminate a multi-strand conductive wire that allows each individual conductor to be used in any combination. Terminating the sea cable using this technique may be viewed in the CalCOFI Handbook: CTD Termination.

Conventional two-pin Seabird pigtail terminations are standard on NOAA vessels and are usually performed by the ET. So interfacing the CTD deck unit only requires the attachment of the sea cable to the deck unit. This is done by connecting a two-conductor wire, such as Seabird part no. 31371 or a coaxial cable equipped with MS3106A connector, from the winch junction box in the CTD lab to the Sea Cable port on the back of the deck unit (see the deck unit section below).
Once the sea cable is connected to the Seabird 9plus and Seabird 11plus deck unit, the deck unit can be powered on. If cabled properly and all electronics are functioning, the numeric LED panel of the deck unit should display non-zero numbers. These values are different for each channel which can be selected by rotating the WORD SELECT dial to the right of the LED panel.
If the LED panel displays “0.0.0.0.0.0.0.0” then the deck unit and CTD “fish” are not handshaking correctly. Turn the deck unit off and wait 60 seconds before touching any exposed wire. Typically, with only two pins, the problem is reversed polarity so switching the wires in the junction box will correct this issue. Please note that powering on the deck unit with reversed polarity may blow the fuse(s). So after switching the wires and before re-powering on the deck unit, check both the 1/2A and 2A fuses on the back of the deck unit & replace if necessary. It is prudent to keep a good supply of these fuses on hand. Power on the deck unit and hopefully, the LED panel has numbers.

Please note in the photos, yellow tape is used to keep the deck unit Signal Source set to Fish. If the switch is toggled to Tape, the deck unit will not communicate with the CTD. The yellow tape prevents the switch from changing from Fish to Tape unintentionally.

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CalCOFI has always corrected bottom depths reported using Carter Tables 50 & 51 for the CalCOFI operations area, California's west coast. An explanation of the need for this correction can be read below and at:

https://www.ngdc.noaa.gov/mgg/fliers/84mgg18.html

"An echo sounding is measurement of the two-way travel time of an acoustic signal between a shipborne transducer and a submerged reflecting surface, usually the ocean bottom. To convert an echo sounding to depth, the travel-time measurement is commonly halved and multiplied by an assumed speed of sound through seawater, either 4,800 ft/sec, 800 fathoms/sec or 1,500 m/sec. The resulting value must be corrected for variations in the speed of sound because of variations in the temperature, salinity, and pressure of sea water. Traditionally, corrections to echo soundings have been interpolated for various areas of the world's oceans from tables published by D.J. Matthews in 1939.* Though the Matthews tables are long outdated, their continued use allowed new depth values to be merged with the large existing data base of depths derived similarly. In 1980, the United Kingdom's Hydrographic Department published a new edition of the correction tables, based on the work of D.J.T. Carter of the United Kingdom's Institute of Oceanographic Sciences.** The Carter tables were adopted in place of Matthews tables by the Twelfth International Hydrographic Conference in Monaco in 1982."

Sample code illustrating CalCOFI stations from Lines 93-77 may be in Carter Table Zone 50 or 51:

If Line in [77, 80, 82, 83, 87, 90, 93] then
  Case Line of
    77, 80, 82, 83 : Assign(table, 'CARTER50.DAT');
    87 : if Sta in [90, 100, 110] then Assign(table, 'CARTER51.DAT')
               else Assign(table, 'CARTER50.DAT');
    90 : if Sta in [90, 100, 110, 120] then Assign(table, 'CARTER51.DAT')
               else Assign(table, 'CARTER50.DAT');
    93 : if Sta in [55, 60, 100, 110, 120] then Assign(table, 'CARTER51.DAT')
               else Assign(table, 'CARTER50.DAT');
  End

DISSOLVED OXYGEN

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 acicified 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:

1. oceaninformatics.ucsd.edu/calcofi/docs/CCConf06Wolgast.ppt
2. odf.ucsd.edu/index.php?id=56

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.
 

O₂(ml/L)=      ---------------------------  -  ---------

Where:
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.

• oceaninformatics.ucsd.edu/calcofi/docs/CCConf06Wolgast.ppt

CalCOFI deploys a Seabird 911+ CTD with 24-place Seabird carousel mounted inside a 24-10L bottle, epoxy-coated rosette frame. Bottle sampling is based on the historical "bottles-hung-on-wire" sampling method utilized by CalCOFI oceangraphers from 1949-1993. Twenty Niskin (5L), Nansen (1.25L), or Wally (2.5L) bottles with messengers were attached to a winch wire and suspended vertically in (typically) 500m of ocean depth. The spring-loaded bottles go into the ocean open so seawater can easily flush through them. Once all 20 bottles were deployed and after a 10+ minute soak for the reversing thermometers to equilibrate. A messenger - 1-2lb metal weight that can clamp & slide freely on the wire - was clamped to the wire and released, dropping onto the first bottle submerged at surface. When closed, the bottles trap seawater from a particular depth with a water-tight seal. This is important since the characteristics of seawater such as salinity and oxygen change with depth. It is critical to the integrity of the measurement that the bottles do not leak and become contaminated by seawater from different depths.

• Nansen bottles: the top end of the Nansen bottle would release from the wire, inverting the bottle, releasing its messenger. The inversion of the thermometers would "break" the mercury in the reversing thermometers, locking in the temperature readings. When inverted, the open bottle ends closed, trapping seawater from that depth for later analyses. Three thermometers - two protected, one unprotected were used on bottles 200m and deeper to derive corrected bottle depth; two thermometers were mounted on bottle shallower than 200m. The released messenger would slide down the wire to trip the next bottle - this would continue until all 20 bottles had closed. Calculating actual bottle depth by comparing protected and unprotected reversing thermometer readings would correct for any inaccuracy due to wire angle or winch-error. In use by CalCOFI 1949 - 1966, and occasionally after until CTD-Rosette implimentation in 1993.
• Niskin bottles: the messenger would strike a plunger, releasing a rotating thermometer rack that would spin 180°, "breaking" the mercury to lock-in the temperature readings of the reversing thermometers. An internal spring would close the o-ringed end caps trapping seawater. CalCOFI Niskins were metal-free & latex-free to allow 14C primary productivity incubation experiments to be conducted with the seawater collected. Epoxy-coated springs and viton o-rings were used to reduce latex toxicity and iron fertilization. After the 1st bottle tripped, the messenger would travel down the wire to the next bottle and so on. The person dropping the messenger would often hold the wire to feel the vibration up the wire of the bottles closing. Eight minutes would elapse for all the bottles to close before the bottle array would be recovered. In use by CalCOFI 1966-1993.
• "Wally" bottles were 2.5L PVC/ABS bottles designed by CalCOFI techs George Anderson & Walt Bryant, similar to Niskins but easier to deploy. They used a single broad wire book-clamp and collected smaller water volumes so were easier-quicker to attach and remove from the wire. They also used epoxy-coated springs and Viton o-rings. During their use, 2.5L of seawater was plenty of volume for the at-sea analyses: salts, nutrients, chlorophylls, oxygens. Like Niskin bottles, after the surface bottle tripped, the messenger would travel down the wire striking the next bottle plunger, closing the bottle, and releasing the next messenger. In use by CalCOFI 1988-1993.
• CTD-rosette: 10L PVC/ABS plastic bottles are equiped with epoxy-spring loaded with Viton o-rings on all surfaces that contact the seawater samples. Unlike a series of bottles clamped to a winch wire, the CTD-rosette is tethered and deployed on a electrically-conductive winch wire. 24-10L bottles encircle the CTD electronics and are rigged open using lanyards attached to the central carousel hub. The CTD sensors display real-time seawater measurements to a computer screen on the ship for the operator & other scientists to see. Based on the downcast profiles of chlorophyll-a and mixed layer depth, as the CTD-rosette is raised, bottles are closed electronically at specific depths by the operator. Reversing thermometers have been replaced by dual CTD temperature sensors. Bottles still trap seawater from specific depths in leak-proof bottles for analyses on-board the ship. In use by CalCOFI 1993-present.

Chlorophyll Determination

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

3. Sample Filtration

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 aliquotted 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 =           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) =   Phaeo (µg/l) =    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

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 p.s.i

· 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. Cons.perm.int 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

Prior to CalCOFI's use of Guildline's Autosal and Portasal, different instrumentation or techniques were used to measure seawater sample salinities. When I first participated in cruises in the mid-1980's, salinities were collected in 250ml sample bottles with wire bail ceramic stoppers with rubber washer.  These samples were run on a Plessey (Hytech) Laboratory Salinometer. I went online to find out more about this particular instrument for our instrumentation timeline and was surprised at how little I found. Fortunately, I have a photocopy of the original Hytech manual so I scanned it to PDF. (JRW 06/2019)

In 1970, this salinometer was relabeled Plessey Model 6230 Portable Laboratory Salinometer by Plessey Environmental Systems.
("In 1970, Bissett-Berman was purchased by the British company Plessey Ltd. and the San Diego Division was renamed Plessey Environmental Systems. Ten years
later, Grundy bought the parent company of Plessey Environmental and a short time later phased out the oceanographic equipment.")

Salinity Sample bottle similar to what was used by CalCOFI in 1970-1990	Plessey/Hytech Laboratory Salinometer Model 6220 Manual (click for PDF of photocopy)