Nutrient Protocols

Christopher Garside



Discrete samples will be taken from all hydrographic casts employing 24x5 1 Niskin bottles. Samples will be collected immediately after the gas samples have been collected. 20 ml samples will be taken into 25 ml high density linear polyethylene scintillation vials fitted with conical polyethylene cap liners, after three rinses of c.a. 5 ml each. Between sampling, the vials will contain the previous nutrient sample. Samples will be stored refrigerated in the dark, and will be analyzed directly from the vials as soon as possible after collection, and no more than 24 hours thereafter.


Nutrient profiles will be made daily or as frequently as possible to 100--125 m. We will deploy a 2" i.d. PVC hose connected to a well de-watering pump delivering 500 l min into an on deck 190 l reservoir. The intake to the pumping system will be attached to a CTD/ BOPS package. A second pump in the reservoir will deliver 50 l min through a garden hose to the continuous flow analyzer (CFA) in the laboratory. The flowing seawater in the garden hose will be sub-sampled through a solenoid valve alternating between sample and artificial seawater.


All nutrient analyses will be done using a six channel CFA to measure ammonium, urea, nitrite, nitrate + nitrite, phosphate, and silicate colorimetrically. Deep samples with nutrient concentrations greater than the analytical range will be precisely diluted with low nutrient artificial seawater (ASW) of known nutrient content to bring their nutrient concentrations within the analytical range.


Ammonium (NH) will be measured using the indophenol blue method (Berthelot's reaction). NH reacts with alkaline hypochlorite in the presence of nitroprusside, and is complexed with alkaline phenol. Precipitation of magnesium and calcium is prevented by complexing with a strong citrate buffer. The reaction mixture is heated to 80 C to accelerate the reaction, and absorbance is measured at 660 nm in a 50 mm flow cell.


Urea will be measured using a modification of the method first described by DeManche et al. ( Limnol. Oceanog., 1973 (18) 686--689). The sample is made strongly acid with a mixture of sulfuric and phosphoric acids containing trace amounts of ferric ion. A pink chromophore is formed by reaction with a color reagent containing diacetylmonoxime and thiosemicarbazide at 95 C, and absorbance is measured at 520 nm in a 50 mm flow-cell.


Nitrite will be measured using the method of Bendschneider and Robinson ( J. Mar. Res., 1952 (11) 87--96). Nitrite reacts with sulfanilamide in strong acid medium to form a diazonium salt which is then coupled with N-(1-napthyl)ethylenediamine dihydrochloride to form a magenta diazo dye. Absorbance is measured at 540 nm in a 50 mm flow cell.

Nitrate plus Nitrite

Nitrate is first reduced to nitrite using a heterogeneous reaction on a copperized cadmium column based on the method of Wood et al. ( J. Mar. biol. Ass. U.S., (47) 23--31). Nitrite is then determined as above.


Phosphate is determined using an automated version of the method described by Murphy and Riley ( Anal. Chim. Acta, 1962 (12) 162--176). Phosphate reacts with acid molybdate to form phosphomolybdic acid. The phosphomolybdic acid is reduced to a phosphomolybdenum blue complex by ascorbic acid with mild heating (38 C), and the resulting absorbance is measured at 880 nm in a 50 mm flow cell. Silicate does not interfere because of the strongly acid conditions used for the reaction.


Molybdate reacts with silicate to form silicomolybdic acid which is then reduced to a silicomolybdenum blue complex which is measured at 660 nm in a 50 mm flow cell. The method differs from that described by Armstrong et al. ( Deep Sea Res., 1967 (14) 381--389) in that ascorbic acid is used as the reductant rather than stannous chloride. Oxalic acid is used to prevent the interference of phosphate.

Artificial Seawater

All samples will be run with an alternating ASW containing an isotonic sodium chloride and magnesium sulfate mixture, and this will also be the matrix for working standards. The ASW will be introduced to the CFA after a DIW baseline on reagents has been established. This will permit detection of any ASW contamination offsetting the baseline from true zero (the ASW offset).


Standards will be prepared at 1 mM from pre-weighed salts or compound. Primary standards will be prepared in freshly drawn de-ionized water (DIW) using class A volumetric glassware. They will be stored in high density polyethylene, refrigerated and in the dark. A calibration working standard will be prepared in each new batch of ASW, and will be monitored as described below (QA/QC). Analytical ranges will be 5, 5, 5, 40, 5 and 40 µM respectively, with a precision of ±0.5 -- 1.0 % of full scale. Calibration will generally be made using a single top working standard.

Computation of Sample Concentrations

Sample peak heights will be corrected for ASW offsets and for interpolated baseline. Standards will be corrected for baseline only since they are made in ASW. Mean calibration factors (concentration per unit peak height) will be computed for each block of standards, and interpolated calibration factors will be used to compute individual sample peak heights.



Linearity will be checked periodically using a range of working standards prepared by dilution of the calibration working standard into the same ASW. Linearity will also be monitored by running one or more standard dilutions as samples within the run from time to time.

Standard Integrity

New working standards will be made for each new batch of ASW. Old working standards will be run as samples in runs calibrated with new working standards. This procedure will maintain standard continuity and provenance, and check for possible standard degradation with time.


Analytical precision will be estimated from replicates of standards and diluted standards during normal running.


Accuracy involves many more factors than precision, not all of which can be tested. Replication of sampling into nutrient vials will be examined, and time permitting, replicate bottles may be tripped at the same depth to test replication of water sampling. There will be many replicate casts at each station which will provide a measure of station variance, but physical processes can change water column distributions, so that this comparison is not a totally satisfactory measure of accuracy. Comparison with historical data will be equally useful, but less than perfect because of temporal change, although the time and space scales of change of deep water nutrient properties are large and make these comparisons more rigorous and they also will be part of the QA/QC procedure.