Huron River Nutrient Study:
Laboratory Protocols and SOP
Contents
Filtrate

Alkaline Phosphatase
Alkaline Phosphatase Reagents
NITROGEN
Ammonium (NH4+)
Ammonium Reagents
Nitrate
Dissolved Nitrogen (DN)
Particulate Nitrogen (PN)
DN/PN Reagents
PIGMENTS
Filters for Algal Pigments
Chlorophyll a
Phycocyanin
PHOSPHORUS
Soluble Reactive Phosphorus (SRP)
SRP Color Blanks
Dissolved Phosphorus (DP)
Total Phosphorus (TP)
SRP/DP/TP Reagents
SRP/DP/TP Working Standard
Specific Conductance and pH
SILICON
Soluble Reactive Silicon (SRSi)
Particulate Silicon (Part-Si)

Huron River Nutrient Study
Laboratory Protocols and SOP
Filtrate
1.   Raw water is filtered on site for nutrient analysis using Millipore disposable filter capsules of nominal 0.45 mm pore size.
2.      If filtrate cannot be prepared in the field, prepare 250-ml of filtrate using 4.25 cm GF/C filters immediately on return to the lab.
3.      Rinse filter and flask initially with ca. 100-ml of DI water; discard.
4.      Rinse filter and flask with ca. 50-ml of the water sample; discard.
5.      Filter ca. 300-ml water sample.
6.      Rinse filtrate bottle with ca. 50-ml of the filtrate; discard.
7.      Fill the filtrate bottle with the rest of the filtrate.
Alkaline Phosphatase
1.      Add 4-ml raw water sample to 13-mm fluorometer cuvette (perform in duplicate).
2.      Add 1-ml of 36 mM MUP working reagent; swirl to mix and then read initial fluorescence with CDOM filter combination; record t0
3.      Prepare blank as 4-ml DI water + 1-ml MUP working reagent.
4.   Prepare 4MU standard, 2 mM: 4-ml DI water + 1-ml 4 MU standard.
5.      After at least 1 hour, read fluorescence again; record tf
Alkaline Phosphatase Reagents:
1.      50 mM TRIS buffer- 3 g to 500-ml, adjusted to pH 8.0.
2.      4MU Stock Solution, 50 mM: 99.1 mg 4MU in 10-ml DI water; store at 4 C in the dark for up to two months.
3.   4MU standard, 10 mM: Dilute 0.010 ml 4MU Stock into 50-ml TRIS with 0.1% BSA (bovine serum albumin: 0.1 g/100 ml); store at 4 C in the dark for up to 2 months.
4.      MUP Substrate Stock Solution, 3.6 mM: 9.2 mg MUP in 10-ml TRIS/0.1% BSA; spontaneously hydrolyzes over time.
5.      MUP Working Solution, 36 mM: dilute Stock Solution (4) 1:100 with TRIS/0.1% BSA. Make fresh daily.
NITROGEN
Ammonium (NH4+)
1.      Place 20-ml volumes of standard or samples in plastic scintillation vials. Prepare standards in triplicate and samples in duplicate.
2.      Add 3-ml of OPA working reagent; invert and mix.
3.      Incubate at room temperature in the dark for more than 2 hours but less than 6 h.
4.      Prepare color blanks by adding 3-ml of borate buffer to 20-ml river or lake sample. One color blank per sample is sufficient.
5.      Read flurescence of standards, samples, and color blanks.
6.      Calculate ammonium concentration from sample fluorescence corrected for CDOM fluorescence: Fammonium = Fsample - Fcolor
Ammonium Reagents:
a.       Borate buffer: 80 g of sodium tetraborate to 2-L DI water; dissolve. Stable indefinitely stored at room temperature.
b.      Sodium sulfite: 1 g sodium sulfite to 125-ml DI water. Stable one month in glass stoppered bottle at room temperature.
c.       OPA stock: 2.0 g OPA to 50-ml ethanol. Store in amber glass bottle under refrigeration.
d.      OPA Working Reagent: Combine 500-ml borate buffer (a), 2.5-ml sulfite solution (b), and 25-ml OPA stock (c). Stable for 3 months at room temperature in the dark.
Nitrate
1.   Use DI water for 0 mM NO3 blank.
2.   Use 100 mM HNO3 as standard (duplicate).
3.      Use HR filtrate for nitrate determination.
4.      Scan and record the UV spectrum of each sample from 260 to 200 nm at 0.5 nm intervals; use 1-cm quartz cuvettes; use DI water for baseline scan.
DN (Dissolved Nitrogen)
1.      Prepare all samples and standards 10-ml volumes dispensed by Eppendorf pipette into borosilicate tubes.
2.      Use filtered DI water for DN blanks: triplicate.
3.   Use 100 mM HNO3 for DN standard: triplicate.
4.      Dispense HR DN samples in duplicate.
5.      Add 1.5-ml alkaline persulfate oxidant to samples and standards.
6.      Oxidize in dry block heater at 105 C for 6 hours; cool to room temperature.
7.      Neutralize and dissolve carbonate precipitate by adding 0.5-ml 33% HCl.
8.      Scan and record UV spectrum of each sample from 260 to 200 nm at 0.5 nm intervals; use 1-cm quartz cuvettes.
PN (Particulate Nitrogen)
1.      Prepare all samples and filter blanks as 100-ml raw water (or filtered DI for filter blanks) filtered through 2.5-cm GF/C filters; place filters in borosilicate tubes.
2.      Prepare filter blanks in quadruplicate.
3.      Prepare PN samples in duplicate.
4.      Add 10-ml filtered DI water to samples and filter blanks.
5.      Add 1.5-ml alkaline persulfate oxidant to samples and filter blanks.
6.      Oxidize in dry block heater at 105 C for 6 hours; cool to room temperature.
7.      Neutralize by adding 0.5-ml 33% HCl.
8.      Filter each sample to remove glass fiber debris.
9.      Scan and record the UV spectrum of each sample from 260 to 200 nm at 0.5 nm intervals; use 1-cm quartz cuvettes.
DN/PN Reagents
(a)    Alkaline persulfate: Add 6 g NaOH to 100-ml filtered DI water, dissolve. Then add 6 g low-N potassium persulfate, dissolve.
(b)    33% HCl: Add 100-ml concentrated HCl to 150-ml DI water; dilute to 300-ml total volume with DI water.
PIGMENTS
Filters for Algal Pigments
1.      Collect algae on glass fiber filters- either 2.5-cm or 4.25-cm depending on cell density and ease of filtration.
2.      Filter 100-ml samples for Chlorophyll a determination by fluorometry.
3.      Filter 250-ml samples for Phycoyanin determination and Chlorophyll by spectrophotometry.
4.      Place filters in aluminum foil envelopes; label with date, station, volume; freeze under desiccant until extraction.
Chlorophyll a
1.      For spectrophotometric analysis, filter 250-mL of raw water through Whatman AH filters and freeze filters over silica gel desiccant until extraction. For fluorometric assays, use 100-mL samples.
2.      Macerate filters in ice-cold 90% v/v acetone by tissue grinder.
3.      Filter resulting slurry through a Whatman GF/D filter.
4.      Record extract volume.
5.      Chlorophyll is measured fluorometrically using a Turner Designs TD700 fluorometer with 436 nm excitation filter and 680 nm emission filter; spectrophotometric determination uses the trichromatic method and 5-cm optical path length.
Phycocyanin
Preparation of PC Standard and calibration:
1.      Add ca. 0.2 g freeze-dried bluegreen algae powder (preferably Aphanizomenon) to ca. 12 ml pH 7 buffer in capped centrifuge tube.
2.      Extract in refrigerator for up to 48 h.
3.      Centrifuge and decant the blue supernatant.
4.      Set or record blank absorbance (optical density) at 750 nm and 620 nm using 0.05M pH 7 buffer.
5.       Use spectrophotometer to record A750 nm (turbidity blank) and A620 nm (Phycocyanin absorbance peak) for the sample extract.
6.       Calculate PC (micrograms per ml solvent for 1-cm pathlength):
PC= (A620 - A750)/0.00073.
7.       Calculate phycocyanin concentration in original sample of standard extract by dividing by optical path length (if different from 1-cm), and by multiplying by the ratio of extraction volume to filtered volume. Concentrations are usually expressed as micrograms per liter.
8.       Dilute 0.2 ml PC standard to 10-ml with pH 7 buffer.  This is PC working standard.
9.        Use phycocyanin filter set in TD700 fluorometer.
10.    Calibrate TD700 to read 200 with PC working standard.
11.      Record sensitivity factor and actual fsu of PC working standard.
12.         Record fsu of pH 7 buffer blank.
Preparation of Lake Water Samples:
1.      Filter 250-ml samples of raw water through Whatman AH glass fiber filters to collect algae.
2.      Place filters in aluminum foil envelopes; labeled with date, station, and volume; freeze under desiccant if immediate extraction is not practicable.
3.      Submerse each filter in ca. 5-ml of 0.05 M pH 7 buffer and fit the extraction vessel with a cap. Extract in a refrigerator for 24 to 48 hours.
4.      Macerate filter using a tissue grinder and 0.05M pH7 buffer as needed.
5.  Filter the created slurry using a Whatman GF/D filter and 0.2 mm membrane filter. Rinse grinding vessel and filtration setup twice with small volumes of phosphate buffer.
6.      Measure final extraction volumes including combined rinses.
7.      Measure fluorescence in TD700 calibrated to read 200 with PC working standard.
PHOSPHORUS
Soluble Reactive Phosphorus (SRP): 40-ml Samples
1.      Prepare all standards (triplicate) and samples (duplicate) as 40-ml volumes measured by graduated cylinder and dispensed into borosilicate tubes.
2.      Pour DI water blanks in triplicate.
3.   Pour 1.61 mM P standards in triplicate.
4.      Pour filtrate samples in duplicate.
5.      Calculate required volume of mixed reagent: 4 ml per sample tube.
6.      Prepare required volume of mixed reagent by combining, in order, 2 parts (a) 3% ammonium molybdate, 5 parts (b) SRP sulfuric acid, 2 parts (c) ascorbic acid, 1 part (d) potassium antimonyl tarttrate.
7.      Set spectrophotometer wavelength to 885 nm; use 10-cm cylindrical cells and zero with DI water.
8.      Dispense 4-ml mixed reagent to samples. Make additions only to enough samples at a time that you can read within 30 minutes.
9.      Wait 5 minutes for color development, then record absorbance values.
SRP Color Blanks: 40-ml Samples
1.      Prepare all samples as 40-ml volumes in duplicate, measured by graduated cylinder, and dispensed into borosilicate tubes.
2.      Prepare mock mixed reagent by combining equal volumes of SRP sulfuric acid solution and DI water.
3.      Dispense 4-ml mock mixed reagent to samples. Samples are ready to read immediately and are stable indefinitely.
4.      Set spectrophotometer wavelength to 885 nm; use 10-cm cylindrical cells and zero with DI water.
5.      Record absorbance values using DI water as the instrument blank.
Dissolved Phosphorus (DP)/Total Phosphorus (TP): 40-ml Samples
1.      Prepare all standards (quadruplicate) and samples (duplicate for DP or triplicate for TP) as 40-ml volumes measured by graduated cylinder and dispensed into borosilicate tubes.
2.      Pour DI water blanks in quadruplicate.
3.   Pour 1.61 mM P standard in quadruplicate.
4.      DP = filtrate; TP = raw water.
5.      Pour DP (duplicate) and TP (triplicate) samples.
6.      Add 0.4 g of potassium persulfate to each tube using a calibrated scoop.
7.      Oxidize in dry block heater at 105 C for 2 hours; cool to room temperature.
8.      Calculate required volume of mixed reagent: 4-ml per tube.
9.      Prepare required volume of mixed reagent by combining, in order, 2 parts (a) 3% ammonium molybdate, 5 parts (b) SRP sulfuric acid, 2 parts (c) ascorbic acid, 1 part (d) potassium antimonyl tarttrate.
10.  Set spectrophotometer wavelength to 885 nm; use 10-cm cylindrical cells and zero with DI water.
11.  Dispense 4-ml mixed reagent to samples. Make additions only to enough samples at a time that you can read within 30 minutes.
12.  Wait 5 minutes for color development, then record absorbance values.
SRP/DP/TP Reagents:
(a)    Ammonium Molybdate: 15 g to 500-ml DI water.
(b)    SRP Sulfuric Acid: 140-ml concentrated sulfuric acid to 900-ml DI water.
(c)    Ascorbic Acid: 27 g to 500-ml DI water.
(d)    Potassium Antimonyl Tartrate: 0.34 g to 250-ml DI water.
Mix in order and in volume ratio: 2(a) : 5(b) : 2(c) : 1(d)
SRP/DP/TP Working Standard = 1.61 mM P:
Dilute 1.0-ml P Stock (1-ml = 0.050 mg P) to 1000-ml with DI water.
Specific Conductance and pH
1.      Turn on water bath heater and confirm temperature is regulated at 25 C.
2.      Pour river samples after one rinse into clean borosilicate tubes (1 for each sample) and place in water bath.
3.      Turn on conductivity meter and perform 2-point calibration.
4.      Turn on pH meter and perform 2-point calibration.  Calibrate with pH 7 buffer first, then pH 4; then confirm by reading pH 7 again.
5.   As samples reach bath temperature, record K25 and temperature for a 1413 mS KCl standard at 25 C and all water samples.
6.   At completion of measurements, re-measure K25 for the 1413 mS conductivity standard and record the final value.
SILICON
Soluble Reactive Silicon (SRSi)
1.   Place 5-ml of 50 mM standard in polyethylene vessels in triplicate; use 5-ml of DI water in triplicate for blanks.
2.      Place 5-ml of sample filtrate in polyethylene vessels in duplicate.
3.      If sample dilution is necessary, place 1-ml sample plus 4-ml DI water in polyethylene vessels (N.B. this is a dilution to 20% full strength)
4.      Add 0.3 ml of mixed reagent (3); swirl to mix and wait for 15 minutes.
5.      Add 0.2 ml of Tartaric acid (4); swirl to mix and wait 2 minutes
6.      Add 0.1 ml reductant (6); swirl to mix.
7.      Read at 815 or 660 nm in 1-cm cell after 5 minutes.
SRSi Reagents:
1.      Ammonium molybdate, 10% w/v.
2.      HCl, 1N: Dilute 83 ml HCl to 1 L.
3.      SRSi Mixed reagent: mix 1 part (1) with 1 part DI water and 3 parts (2); prepare fresh daily.
4.      Tartaric Acid, 10% w/v.
5.      Stannous Chloride stock, 3.5 N: dissolve 40 g SnCl2•2H2O in 100-ml HCl.
6.      Working Stannous Chloride, 0.05 N: dilute 1-ml (5) to 70-ml with DI water; prepare fresh daily.
SRSi Standards:
Stock standard: 10 mM Si (1 ml = 10 mmol Si)
50 mM Si: dilute 0.5 ml Stock to 100 ml.
Particulate Silicon (Part-Si)
1.      Fill 15-ml polystyrene centrifuge tubes with raw water in duplicate.
2.      Add 2 drops of Lugol’s iodine.
3.      Centrifuge at top speed in clinical centrifuge for 10 minutes.
4.      Decant supernatant.
5.      Add 4-ml of 0.2 N NaOH.
6.      Heat at ca. 85 C for 1 hour.
7.      Cool; add 1-ml of 0.8 N HCl.
8.      Prepare Blanks: 4-ml of 0.2 N NaOH + 1-ml of 0.8 N HCl.
9.   Prepare 24 mM standard: 4-ml 0f 30 mM Si/0.8 N NaOH + 1-ml of 0.8 N HCl.
10.  Run analysis as SRSi.