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Appendix F: Groundwater Analysis Materials and Methods

Construction and Installation of Groundwater samplers

An area of Kikuyu grass was chosen which appeared fairly uniform and had little foot traffic. This was important so the experiment would not be in the way or disturbed.

Core samples of the soil were taken using a soil probe (Forestry Suppliers, Inc. Stock #77451). Examination of the soil core samples revealed that grass roots were present no deeper than 9". From this it was decided that the groundwater samplers would be constructed such that the ceramic cup would be about 12" deep. This would ensure that any nutrients found in the groundwater had already passed by the grass roots unused.

Diagram of lysimeter

Construction of Groundwater samplers

Three groundwater samplers were constructed. A 12' length of 1.5"-diameter PVC was glued to a porous ceramic cup (Soilmoisture Equipment Corp. Item #0653X01-BO2M2) using an all-purpose weather-resistant glue. The inside diameter of the PVC was bored out to allow the male piece of the ceramic cup to fit. PVC grinding was done using a hand-held router.

Rubber stoppers (size #9) were used to seal the top. Glass tubes were inserted in the stoppers one of which was connected to plastic tubing reaching all the way to the bottom of the groundwater sampler, the other went just far enough to clear the bottom of the stopper. Both tubes reached above the top of the stopper and were connected to plastic tubing that could be sealed off in order to create a vacuum. A diagram of a groundwater sampler in the ground can be seen above.

Installation

The three groundwater samplers were buried just to the east of Thomas Garrett Hall. Each was buried so that just the rubber stopper was above ground. This allowed lawnmowers to pass over without disturbing the experiment, and also ensured the ceramic cups were below the measured root depth.

Sampling

Groundwater samples were taken for approximately 1 month starting one week before fertilizer application (03-16-01) and continuing for three weeks after application. Samples were collected once every 3-4 days during this period. Two 3-mL (minimum) samples were collected from each groundwater sampler, one for a nitrate test and the other for ammonium.

A vacuum of approximately 60k Pa was created in the groundwater samplers using a MightyVac hand powered pump (Cole-Parmer Instrument Co. #79301-00). The vacuum was allowed to sit for a minimum of four hours, at which point groundwater samples were drawn from the groundwater samplers, again using the MightyVac. In general, there was enough water in each groundwater sampler to provide two 3-mL samples.

All samples were stored and analyzed at the end of the sampling period. The nitrate samples were frozen immediately. Fifty microliters of 6N HCL was added to the ammonium samples, which were then refrigerated.

Sample Analysis

Nitrate Analysis

Nitrate samples were analyzed using a 'Standard Range Field Test Kit' (The Nitrate Elimination Co., Inc. catalog #F-NTK-101). Nitrate reductase in the test kit reduces the nitrate to nitrite, which can then react with a color reagent to produce a pink color. The amount of pink is directly proportional to the amount of nitrate. The directions of the kit were followed with the following exception. Since the analysis was performed in a laboratory, we were able to use a spectrophotometer to read the absorbance instead of merely visually comparing our colors to those on a standard card. Three standards were made: 1.0, 5.0, and 10.0 ppm nitrate-N. Absorbance of the nitrate samples was read at 540 nm.

Ammonium Analysis

No test kit was available to easily test for ammonium. The following method, provided by Pat Micks of the Marine Biological Laboratory in Woods Hole, MA, was used. This analysis uses the phenol-hypochlorite method with nitroprusside as a catalyst (Solorzano). Ammonium reacts with phenol and hypochlorite under alkaline conditions to form indophenol blue with the color intensity being proportional to the concentration of ammonium. The indophenol blue can be read on a spectrophotometer with absorbance at 640 nm.

All work with phenol reagents should be conducted in a vented hood.

 

REAGENTS

  1. Phenol solution (In a HOOD)

  2. Na nitroprusside solution

  3. Alkaline Reagent

  4. Na hypochlorite

  5. Oxidizing solution

 

PROCEDURE (In a HOOD)

For a 3-mL sample in a (pre-reacted) 10-mL borosilicate culture tube add the following reagents in order with each addition followed by mixing with the Vortex mixer:

  1. 0.12 mL phenol

  2. 0.12 mL Na nitroprusside

  3. 0.30 mL oxidizing solution

  4. Develop indophenol blue color in the dark for at least 1 hour (20o-27o C) with tubes covered with parafilm.

  5. Read absorbance on spectrophotometer at 640 nm. Make sure waste line from sipper unit empties into waste bottle

  6. Pour out remaining solution into labeled hazardous waste bottle, rinse tubes thoroughly with deionized H2O and dry tubes in oven while preventing rims from touching any surfaces

 

STANDARDS

Ammonium Chloride: NH4Cl FW 53.49
Primary Standard:
  10 mM N as NH4Cl Using dry NH4Cl add 0.534 g to 1000 mL volumetric and dilute to 1000 mL with deionized H2O
Secondary Standard:
  1 mM N as NH4Cl
(= 1000 mM N)		 10 mL of primary std diluted to 100 mL with deionized H2O
Working Standards:
Add the following volumes of secondary standard to 100 mL volumetrics and dilute to 100 mL with deionized H2O:
 
0.00 mL per 100 mL 0.0 mM N
0.10 mL per 100 mL 1.0 mM N
0.20 mL per 100 mL 2.0 mM N
0.50 mL per 100 mL 5.0 mM N
1.00 mL per 100 mL 10.0 mM N
2.00 mL per 100 mL 20.0 mM N

 

PRE-REACTED CULTURE TUBES

Culture tubes are often contaminated and can be cleaned to keep blanks low by pre-reacting the tubes the day before the analysis.

 

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