APPARATUS:
Eppendorfpipettes (10-100 µl, 100-1000 µl und 1-5 cm³)
Various volumetric flasks
Various cylinders und beakers
Various bottles to store the reagents
Photometer

REAGENTS:
Phenol solution: Dissolve 8.0 g phenol in 30 cm³ ethanol and 60 cm³ pure water. Dissolve 60 mg disodium nitroprusside in 10 cm³ pure water and add to phenol solution. Store refrigerated and tightly closed in glass bottle. Stable for several months.
WATCH OUT: Phenol  is toxic and corrosive. Protect yourself and collect the waste for special disposal.

Sodium hydroxide: Dissolve 20 g NaOH in 1 dm³ pure water. Store in PE-bottle.
Citrate buffer: Dissolve 24 g trisodium citrate in 50 cm³ pure water to which 2 cm³ NaOH-solution is added. Store in PE-bottle at room temperature. Stable for several months.

DTT reagent: Dissolve 50 mg dichloro isocyanuric acid in 20 cm³ NaOH-solution. Unstable. Prepare immediately before use. NH4-standard (MERCK) 1 g NH₄Cl per liter
Salt water 36 g/l NaCl in pure water

STANDARDS:
Stock standard: NH₄+-standard (MERCK) = 55.56 mmol/l
Work standard: Dilute the stock standard with salt water in different relations. To obtain standards with different concentrations for a calibration (5-6 calibration points).
Calibration range: 16.67 - 83.55µmol/l (for pore water samples) and 1.667 -16.67 µmol/l (for samples from the water column)
 

PROCEDURE:
1 cm³ of sample or standard is made up to 5 cm³ with pure water and 0.2 cm³ phenol solution is added. After 2 minutes 0.1 cm³  citrate buffer and 0.2 cm³ DTT reagent are added. After mixing, the samples are kept protected from sunlight for about 10 hours under room temperature, before the absorbance is measured at 630 nm.
To speed up the development of the indophenol blue, the samples can alternatively be kept at 30-40°C for 1-2 hours. (WATCH OUT: lids might pop up !)

INTERFERENCES:
High concentrations of H₂S (> 1mM) in the sample have a negative influence on the measurements. Therefore these samples should be acified with 10 µl HCl (30%/suprapur) per cm³ sample and then be stored for 1-2 days, without lid, in a cool room. (Don`t forget the dilution factor of the acid when you calculate the final result!) It is also possible to displace the H₂S by Argon.

Bacterias can oxidate NH₄ rapidly. Samples should be measured as quick as possible after sampling. Avoid pollution.