Structure and Functioning of a Managed Grassland Ecosystem
Exercise D: Nutrient pools in roots and above-ground vegetation

EQUIPMENT

Field

• tapes and pins for positioning sampling sites
• random number table
• soil corers (see Exercise B for details)
• knife
• polythene bags

Laboratory

• paper bags
• foil trays
• balance
• volumetric glassware (including safety pipettes)
• drying oven
• flame photometers (or atomic absorption spectrophotometer)
• continuous flow auto-analyser
• digestion block

Reagents

• 1000 mg l^-1 K, Na and Ca standard solutions
• 100 mg N l^-1 standard solution (any N salt, e.g. NH₄NO₃, but calculate N concentration.)
• 2000 mg l^-1 lanthanum chloride solution (CARE: POISON)
• Digestion mixture (350ml 100% H₂O₂, 0.42 g selenium metal powder, 14 g lithium sulphate, 420 ml conc. H₂SO₄ added with cooling) (CARE NEEDED)
• Digest Acid Blank (DAB)

METHOD

Field procedure

• Five vegetation samples, located at random, are taken from each side (mown and unmown) of the research area. The vegetation is cut off at ground level and placed in polythene bags.
• Five cores, each 10 cm deep and located at random from each side (mown and unmown) of the research area, are taken and placed in labelled bags.

Laboratory procedure

• The vegetation samples and soil cores are weighed.
• The vegetation samples and soil cores are oven dried at 85°C for 24 hours or overnight.
• The dried samples are weighed.
• The dried samples are analysed for K, Na and Ca using flame photometers. (Alternatively, an atomic absorption spectrophotometer may be used.) For Ca, lanthanum chloride is added to suppress interference by phosphate and aluminium ions.
• Samples are analysed for N using a continuous flow auto-analyser. (Alternatively, N could be determined volumetrically.)
• The setting up and use of the analytical instruments need to be demonstrated.
• A series of standard dilutions for each of the elements is made up to calibrate the instruments.

Making up standards for Na, K, Ca and N

Sodium

• A 1000 mg l^-1 Na standard solution is diluted to 100 mg l^-1 10 ml are pipetted into a 100 ml volumetric flask, made to volume with Digest Acid Blank (DAB), labelled and mixed well.
• 0, 2, 4, 6, 8 ml are pipetted into volumetric flasks, made to volume with DAB and mixed well.
• The standard series will then be: 0, 2, 4, 6 and 8 mg l^-1 Na. Flasks are labelled with the concentrations and marked DAB.

Potassium

• A 1000 mg l^-1 K standard solution is diluted to 100 mg l^-1
• 10 ml are pipetted into a 100 ml volumetric flask, made to volume with DAB, labelled and mixed well.
• 0, 2, 5, 10, 15 ml are pipetted into 100 ml volumetric flasks, made to volume with DAB and mixed well.
• The standard series will then be: 0, 2, 5, 10 and 15 mg l^-1 K. Flasks are labelled with the concentrations and marked DAB.

Calcium

• A 1000 mg l^-1 Ca standard solution is diluted to 200 mg l^-1
• 20 ml are pipetted into a 100 ml volumetric flask, made to volume with DAB, labelled and mixed well.
• A series of standards is prepared by pipetting 0, 5, 10, 15, 20 ml of 200 mg l^-1 into 100 ml volumetric flasks.
• For Ca analysis by flame photometer (or atomic absorption spectrophotometer AAS): to each 200ml flask 20 ml of 2000 mg l^-1 lanthanum chloride solution are added (CARE: POISON), made to volume with DAB and mixed well.
• The standard series will be 0, 10, 20, 30, 40 mg l^-1 Ca, each one having a 400 mg l^-1 concentration of lanthanum chloride. Flasks are labelled with the concentrations and marked DAB.

Nitrogen

• A series of standards is prepared by pipetting 0, 0.25, 0.5, 1, 1.5, 2 ml of N Standard (100 mg N l^-1) into 50 ml volumetric flasks.
• To each flask 5 ml of DAB are added, made to volume with de-ionised water and mixed well.
• The standard series will be: 0, 0.5, 1, 2, 3, 4 mg l^-1 N, each one being amended with the correct amount of DAB solution. Flasks are labelled with the concentrations and marked DAB.

Calibration of instruments

• The standards for Na, K and Ca are run on flame photometers and calibration curves are constructed on an Excel spread sheet or on graph paper. As a check on the standard preparations, students are provided with quality control samples for each element. These are run on the flame photometers to find whether the results tally with their standard preparations.
• The data output from the N auto-analyser is a series of traces with the height of the peaks proportional to N concentrations (as NH₄⁺-N). The series of standard dilutions is required to calibrate these peak heights.

Sample analysis

• A wet digestion is carried out on the weighed samples of oven-dry sward and roots. This procedure can be hazardous and is best demonstrated unless carried out under close 1:1 supervision by an experienced person. The digestion mixture is based on sulphuric acid and hydrogen peroxide oxidants, with the addition of lithium sulphate to elevate the digestion temperature and selenium metal as a catalyst. This oxidation procedure destroys the organic structure of the sample, allowing for the determination of mineral nitrogen, phosphorus (not measured here) and most mineral cations. The digestion takes a minimum of 2 hours on a digestion block and the mixture is then cooled and diluted (CARE NEEDED) to 100 ml in a volumetric flask.
• For Na and K the digests of plant and root samples are run directly on the flame photometers. If samples are over range they are diluted using the DAB solution.
• Samples for Ca analysis require amending with lanthanum chloride, as for the standards. 8 ml of the sample are pipetted into a ‘3 x 1’ specimen tube and 2 ml of 2000 mg l^-1 lanthanum chloride solution are added, giving a final concentration of 400 mg l^-1 lanthanum (so the sample has been diluted by a factor of 1.25 - i.e. to x 0.8 of original concentration). If the samples are over range they are diluted with DAB solution and amended with lanthanum to give a 400 mg l^-1 concentration.
• Samples are analysed for N using the continuous flow auto-analyser.
• The concentrations in the extracts are read from the calibration curves.

Data handling and statistical techniques

• The general formula used to calculate the concentration (in mg gl^-1 of oven-dry plant material) for all the elements:
  
  

  sample reading (mg l-1) x volume of extractant (ml/1000) x [dilution factor]

  dry weight sample (g)*

   

* to standardise dry weights a sub-sample of material should be oven dried at 105°C and a correction factor applied.

• Means ± standard errors of the results from mown and unmown areas are calculated.

Timetable

The length of the digestion procedure makes it difficult to schedule this exercise into one day. Sampling and sample preparation is usually carried out during one practical exercise, then the digestate prepared during the week in preparation for a laboratory practical.

SPECIMEN RESULTS

Nutrient concentrations

DATA INTERPRETATION

• Na is not a plant nutrient and is present in low concentrations in mown and unmown vegetation.
• Except in halophytes, Na is not a plant nutrient and is present in low concentrations in the shoots. Higher Na concentrations in roots reflect contamination with mineral soil.
• Both Na and K are present in cells as free cations and are rapidly leached from senescent material.
• Calcium concentrations are similar in mown and unmown vegetation because this element is largely structural. Ca is present mainly in cell walls and is mobilised on decomposition. Hence the small differences in concentrations between living and dead materials.
• N is mainly in Rubisco (Ribulose bisphosphate carboxylase/oxygenase, which catalyses the initial step of CO2 fixation), hence N concentrations are higher in green leaves than in roots.
• The higher N concentrations in living mown compared with unmown material reflect the more dynamic N cycling produced by mowing.

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