7.12 Subprogramme FC: Foliage chemistry

• 7.12.1 Introduction
• 7.12.2 Methods
• 7.12.2.1 Field methods
• 7.12.2.2 Chemical analyses
• 7.12.2.2.1 Digestion and analysis
• 7.12.2.2.2 Determination
• 7.12.2.2.3 The most frequently used methods for specific elements
• 7.12.2.2.4 Data expression units
• 7.12.3. Validation of the analytical results
• 7.12.4. Data reporting
• 7.12.5 References

7.12.1 Introduction

Foliage analysis provides several types of information. Most importantly, it provides information on the nutritional status of trees. However, the chemical analysis of materials on and in the foliage may under some circumstances provide information on the relative loading of different types of pollutants.

Foliage sampling needs to be undertaken regularly and at the same phenological stage. For example, deciduous species must be sampled in summer, with late summer (after the completion of growth but before the onset of senescence) being best. Evergreen species are best sampled in the dormant season. The chemical composition of foliage needs to be sampled annually if the dynamic nature of the composition is to be fully interpreted.

Elements to be determined: Ca, K, Mg, Na, N, P, S, Cu, Fe, Mn, Zn and TOC
Optional: Al, As, B,Cd, Cl, Cr, F, Mo, Ni and Pb

7.12.2 Methods

7.12.2.1 Field methods

Number of trees
At least 8 trees of each main species (>10% of stems in plot) should be sampled annually. A composite sample for each species should be prepared by mixing equal quantities of foliage from each individual sample. Every five years, the sample trees should be analyzed individually in order to determine the variability of the total element concentrations in the plot.

Selection of sample trees
The number of trees needed for the sampling should be selected in such a way that: 

•  the trees are spread over the total plot area, or around the plot if the stand is homogeneous over a larger area  
•  the trees belong to the predominant and dominant crown classes (forest with closed canopy) or are within ± 20% of the average height of the forest canopy  
•  the trees are in the vicinity of the locations where soil samples were taken for analysis. Care must be taken to ensure that the main roots of the sample trees have not been damaged by the soil sampling  
•  the trees are different from those used for the crown assessment, so as to avoid that successive sampling induces a loss of foliage. If stand and site conditions are homogeneous over an area larger than the plot where crown condition has been assessed, it is advisable to choose the sample trees outside the plot  
•  the trees are representative of the mean defoliation level of the plot (± 5% of the mean foliage loss).

The same trees should be sampled each year; the trees must therefore be numbered. For species with small crowns and few needles (or leaves) in any given year, it is possible (but not recommended) to alternate between two sets of 8 trees, when necessary, to avoid excessive damage to the sample trees. Each set of trees should respond to the above conditions.

Selection of leaves and needles to be sampled
No trees should be felled for foliar sampling.

The sampled leaves or needles should have developed in full light. Usually, the current year needles or leaves of evergreen species are the most suitable for judging the nutrition level but, for a number of elements, comparing element concentrations in older needles with that in the current year may provide more useful results.

The foliage should be sampled from the upper third of the crown, but not from the uppermost (1-4) whorls in conifers. In stands where the different whorls can be clearly identified, it is advisable to sample between the 7th and 15th whorls.

The current year leaves of broad-leaved trees should be sampled.

For evergreen species, the current year, second year and (if present) third year foliage should be sampled.

For all species, only mature leaves should be sampled. Care is therefore required when sampling species with several flushes during the year or with indeterminate growth.

For Larix spp. and Cedrus spp., samples should be taken from the short twigs of the previous year.

Orientation
Sampling should be carried out in such a way that all orientations are represented in the set of sample trees. If necessary, it is possible to sample different orientations on each tree of the sample set. In special sites with evident influence of one orientation (e.g. on steep slopes or with a dominant wind direction), only one orientation should be sampled, and this should always be the same. This should be documented.

Quantity of material to be sampled
The recommended quantities are:

•  20 grams of fresh needles or leaves for each sampled age class, if only major elements and Fe, Mn, Zn and Cu are being analyzed  
•  50 grams of fresh needles or leaves if other elements (e.g. F, Cl, Cd, Pb, Al and B) are being analyzed.

Larger samples should be taken if samples are being archived.

Sampling methods
Any method of sampling is possible, but some basic precautions should be taken to reduce the possibility of sample contamination.

Pretreatment of samples prior to despatch for analysis
For foliage from broadleaves, it is advisable to detach the leaves from the twigs (and even, in certain species, the small leaves from the axis) but this is not necessary for conifer needles. The shoots of the current year and those of the second year are separated and preserved in separate bags. The use of high density polyethylene bags is recommended. If possible, samples should be dried in a clean room and stored in a cool, dark place in perforated polyethylene bags.

7.12.2.2 Chemical analyses

Only the total element concentration should be determined. More detailed information is available if separate fractions are analyzed, and these can be of considerable value. However, such studies lie outside the scope of the standard IM work.

Treatment before analysis
The mass of 100 leaves or 1,000 needles, should be determined.

It is not necessary to cut the petioles off the leaves but in the case of compound leaves, the small leaves should be detached from the axis if this has not been done in the forest. Avoid contamination, use plastic gloves.

It is not necessary to systematically wash the samples, but it may be advisable in regions with a high level of air pollution or near the sea. The samples should be washed in water without any additions. Once a decision to wash or not to wash has been taken, this should be adhered to in subsequent years.

Oven drying should be done at 80ºC for 24 hours. The needles should be removed from the twigs using the same precautions as for detaching small leaves from their axis.

Dry samples should be ground in order to obtain a fine powder, as homogeneous as possible. There will always remain some fibers, depending on the tree species, this is not a major inconvenience if they are small and if the powder is mixed carefully before taking samples for analysis. When determining Mn, Fe, Cu, Cd, Al and Pb, check that the grinder does not contaminate the samples. The grinder can be tested by grinding dried fibrous cellulose and analysing it for these elements before and after the grinding.

7.12.2.2.1 Digestion and analysis

There is no single way in which analyses should be taken. Rather, the best way is to use the standard procedures for the laboratory and calibrate the results through the use of ring tests. As an indication, the four main groups of methods for wet digestion or dry ashing are given below.

Wet digestion in acid and/or oxidizing conditions
For instance:

1. Kjeldahl method for N Organic N is digested in concentrated H₂SO₄, in the presence of catalysts and converted into NH₄⁺. N eventually present in NO₃^- or NO₂^- form is not transformed into NH₄⁺ and therefore not determined by methods specific for NH₄⁺.

2. Digestion by oxidants and hot acids at atmospheric pressure.

•  H₂SO₄ + H₂O₂ (for N and P analysis);  
•  H₂SO₄ + HNO₃;  
•  HNO₃;  
•  H₂O₂+ HNO₃
•  HNO₃ or H₂O₂ followed by HClO₄. Perchloric acid is very efficient but dangerous (risk of explosion on contact with organic material, or drying and heating perchlorates). The storage and manipulation must be done very cautiously. HClO₄ digestion must be preceded by cold attack of the sample powder by H₂O₂ 110 V or concentrated HNO₃ during 24 hours in order to digest most of the organic tissues before adding HClO₄.  
•  HNO₃ + HF, teflon vessels; after digestion the HF should be dispersed with HNO₃.  These methods may be used for N (H₂SO₄ + H₂O₂), P, S, Ca, Mg, K, Na, Mn, Zn and Cu.

3. Digestion with H₂O₂ + HNO₃ in a microwave oven.

4. Pressurized digestion with HNO₃, or HNO₃ + H₂O₂ in teflon bombs at 180. 200 mg vegetal powder + 3 ml concentrated HNO₃. This method may be used for the same elements as for A2 methods except N: S, P, Ca, Mg, K, Na, Mn, Zn, Cu and for Al, Cd, Pb, Cl and B.

5. Digestion in HNO₃ under a backward column. This method is used for Ca and Pb determination, but may be used for other elements with the exception of N.

Dry ashing

The sample powder is heated at 450-600ºC for 4 hours and ashes are dissolved in water or dilute acids (HCl, HNO₃, HClO₄).

1. Dry ashing at atmospheric pressure in a furnace at 450-600ºC, according to the element, in platinum, porcelain, quartz or Ni crucibles. Ash dissolution may be made with acids, such as HNO₃, HCl, or HClO₄.

This method is convenient for P, K, Na, Ca, Mg, Fe, Mn, and Zn. For P, the analysis should be delayed for 24 hours in order to ensure complete oxidation of P in to PO₄^-.

There may be Si and Al precipitates which are insoluble in HCl and which may absorb a small quantity of elements; but the associated error is usually very small. It can be avoided by filtration of the ash in a HCl solution, calcination of the filter in a platinum crucible, dissolution of the filter ashes in HF, dry evaporation, dissolution of the residue in HCl, and addition of this solution to the first filtrate.

The latter procedure is necessary for Al analysis, the solubilization of which is not complete without using HF, and for elements present in very small quantities in the plant tissue (Cu). A platinum crucible is necessary when using HF, although teflon and carbon are viable alternatives.

For several elements (Cl, S, Pb, Cd), dry ashing at atmospheric pressure causes loss by volatilization. In order to prevent this, stabilizers should be added: Mg(NO₃)₂ (for S), NH₄NO₃ (for Cd and Pb), Na₂CO₃ (for Cl), CaO for (B, F) and NaOH (for F).

2. Low temperature (50-60ºC) ashing in O₂ atmosphere excited by a radiofrequency magnetic field over 8 hr can be used for F analysis.

3. Combustion in a Schöniger flask, a closed flask in which plant powder is burnt in O₂ atmosphere and directly dissolved in acidic or alkalic solution. This method is used for many elements, including S, P, and Cl, but is very time-consuming because only one sample can be ashed and digested at a time.

Integrated oxidation and detection

More and more frequently, special apparatus are being used, performing automatically, in a closed circuit, detection and quantification of gases released during oxidation. Several firms sell this CHN or NS equipment.

Analysis of N and S by these methods is likely to become increasingly frequent. Unlike the Kjeldahl method, these methods give total N concentration, including NO₂^- and NO₃^- forms. When reporting results for N, it is therefore important to state which method was used.

X-ray fluorescence

All metals and non-metals down to F can be determined with X-ray fluorescence, without digesting or ashing, in compacted vegetal powder.

7.12.2.2.2 Determination

Many ways for the determination of the various elements are possible.

Titration

•  NH after digestion by the Kjeldahl method and distillation of NH₃ in HBO₃;  
•  Cl: by AgNO₃ in the presence of CrO (the end of precipitation of AgCl is detected by the red colour of Ag₂CrO₄;  
•  Cl: by micro-titration with AgNO₃ in acetonic solution and potentiometric indication;  
•  SO₄ by titration with BaCl₂ and thorine as an indicator.

Colorimetry

•  NH₄: indophenol blue; or FIA method (diffusion of NH₃ through a teflon membrane, and colorimetry in a solution of phenol + ethanol + NaCl + NaOH);  
•  P: phosphovanadomolybdate (yellow) or molybdene blue;  
•  Cl: colorimetry of Fe(SCN)₃ after reaction of Hg(SCN)₂;  
•  S: metorine (8); DMSA III and other colour indications;  
•  B: 1-1'dianthrimide.

Turbidimetry

•  S: turbidimetry of a suspension of insoluble BaSO₄ with a tensioactive agent (Tween 80).

Ionic chromatography for P, S, Cl, F.

Specific ion electrodes for F, Cl.

Capillary electrophoresis for Cl, S, P and N as NO₃ or NH₄.

Flame emission spectrometry for K, Na.

Atomic absorption spectrometry

1. Flame AAS: Na, K, Ca, Mg, Fe, Mn, Zn, Al and Cu.

2. Graphite furnace AAS: Pb, Cd and Cu.

ICP (Inductively coupled plasma) after atomic emission spectrometry

1. Without ultrasonic nebulisation: Na, K, Ca, Mg, Fe, Mn, Zn, Al, Cu, P, S, Cl and B.

2. With ultrasonic nebulisation: Pb, Cd and Cu.

Processes (ashing with direct determination, in CHN or NS apparatus) and (direct determination by X-ray fluorescence) can be combined.

7.12.2.2.3 The most frequently used methods for specific elements

Nitrogen

a) N organic + NH₄:

Digestion
Kjeldahl method: concentrated H₂SO₄ with K₂SO₄ and Se as catalysts Methods derived from the Kjeldahl method: catalysts other than Se, which is toxic in the environment, such as Ti or Cu; H₂SO₄ + H₂O₂ without a catalyst.

Determination
NH₄⁺ colorimetry (indophenol blue or FIA method)
NH₃ distillation and titration.

Total N: CHNS apparatus.

Sulphur

a) Digestion or ashing.

•  Wet acidic and oxidizing digestion: HNO₃; H₂O₂ + HNO₃; H₂O₂ + HClO₄; HNO₃ + HClO₄
•  HNO₃ in teflon bombs  
•  dry ashing with addition of Mg(NO₃)₂ and MgO; ash dissolution in HCl or water  
•  Schöniger flask

b) Determination

•  Turbidimetry by BaSO₄ (preferably with dissolution by HCl and filtration before BaSO₄ precipitation)  
•  ICP  
•  Ionic chromatography (after ash dissolution in water)  
•  Colorimetry

c) Direct determination in CNS apparatus.

d) Direct determination by X-ray fluorescence.

Phosphorus

a) Digestion and ashing

•  wet acidic and oxidizing digestion: H₂SO₄ + H₂O₂; H₂SO₄ + HNO₃; HNO₃; HNO₃ + H₂O₂; H₂O₂ + HClO₄; HNO₃ + HClO₄
•  HNO₃, or HNO₃ + H₂O₂, in teflon bombs  
•  dry ashing at 450-500ºC at atmospheric pressure (wait for 24 h after ash dissolution in HCl or HClO₄ before determining PO₄^2-)
• Schöniger flask

b) Determination

•  Colorimetry: phosphovanadomolybdate or molybdene blue  
•  ICP-AES  
•  Ionic chromatography  
•  X-ray fluorescence (direct determination)

Calcium, Magnesium, Iron, Manganese, Zinc

a) Digestion or ashing

•  Wet acid and oxidizing digestion at atmospheric pressure (HNO₃, H₂SO₄ + HNO₃, HNO₃ + H₂O₂; H₂O₂ + HClO₄, HNO₃ + HClO₄)  
•  HNO₃ or HNO₃ + H₂O₂, in teflon bombs  
•  Dry ashing at atmospheric pressure, ash dissolution in HCl or HClO₄
•  Schöniger flask

b) Determination

•  Atomic absorption spectrometry  
•  ICP-AES  
•  X-ray fluorescence (direct determination in ash)

Potassium, Sodium

a) Digestion or ashing

b) Determination

•  Flame emission spectrometry  
•  Atomic absorption spectrometry  
•  ICP-AES  
•  X-ray fluorescence (direct determination in ash)

Chloride

a) Digestion or ashing

•  HNO₃ or HNO₃ + H₂O₂, in teflon bomb  
•  Dry ashing at 450-550ºC after addition of Na₂CO₃ and ash dissolution in hot water  
•  Schöniger flask

b) Determination

•  Titrimetry with AG(NO₃)₂
•  Colorimetry of Fe(SCN)₃ after reaction with Hg(SCN)₂
•  Ionic chromatography  
•  Capillary electrophoresis  
•  Specific electrode  
•  ICP  
•  X-ray fluorescence

Boron

a) Digestion or ashing

•  HNO₃ or HNO₃ + H₂O₂, in teflon bomb  
•  Dry ashing at 450-500ºC after addition of CaO, and ash dissolution in 25% acetic acid  
•  Schöniger flask

b) Determination

•  Colorimetry by 1-1'dianthrimide  
•  Direct determination by X-ray fluorescence  
•  ICP

Fluoride

a) Dry ashing

•  At 600ºC for 16 hours (after progressive temperature increase) with addition of CaO (1 g sample + 0.2 g CaO); ash dissolution in 4N HClO₄
•  At 450ºC for 16 hours in Ni crucible, with addition of NaOH; ash dissolution by pH 5.7 buffered acid (Buffer HCl) acetic or HCl/citric acid). The muffle must be covered with Ni.  
•  Low temperature ashing in O₂ atmosphere  
•  Schöniger flask

b) Determination

•  Specific electrode (eventually after micro-diffusion procedure of trimethylfluorosilane (TMFS) in 0.1 M NaOH, after ash dissolution in HClO₄
•  Ionic chromatography

Aluminium

a) Digestion or dry ashing

•  Dry ashing at 450-500ºC in platinum crucible, ash dissolution by HCl, filtration, collection of the filtrate, calcination of the filter, digestion of ash by HF, dry evaporation, dissolution of the residue in HCl and addition to the first filtrate  
•  Dry ashing at 450-500ºC in platinum or Ni crucible, followed by alkaline fusion of the ashes in lithium borate LiBO₂, and uptake in diluted HNO₃
•  HNO₃, or HNO₃ + H₂O₂, in teflon bomb

b) Determination

•  Flame atomic absorption spectrometry  
•  ICP  
•  X-ray fluorescence

Copper

a) Digestion or ashing

•  Wet acid digestion (HNO₃, HNO₃ + H₂O₂, HNO₃ + HClO₄, H₂O₂ + HClO₄)  
•  HNO₃, or HNO₃ + H₂O₂, in teflon bomb  
•  Dry ashing and ash dissolution in HCl or HClO₄
•  Dry ashing, dissolution in HCl, filtration, calcination of the filter, HF, dry evaporation, dissolution in HCl (as for Al determination)

b) Determination

•  Flame atomic absorption spectrometry  
•  ICP  
•  ICP with ultrasonic nebulisation
• Lead and Cadmium (5g sample)

a) Digestion or ashing

•  HNO₃ or HNO₃ + H₂O₂, in teflon bomb  
•  Acid extraction by HNO₃ in a flask with a backward column  
•  Dry ashing at 450-550ºC after addition of NH₄NO₃, ash dissolution in HCl (for determination by ICP) or HNO₃ (for determination by atomic absorption)

b) Determination

•  Electrothermic atomic absorption spectrometry (after ash dissolution in HNO₃)  
•  ICP with ultrasonic nebulisation (after ash dissolution in HCl)  
•  Flame atomic absorption spectrometry

7.12.2.2.4 Data expression units

The total concentration in needles and leaves should be given by reference at 105ºC dried material. The residual water content after drying at 60-80ºC must be determined by drying at 105ºC and weighing, and the results given by the analysis performed on 65-80ºC dried powder must be corrected. Samples should be dried at 65-80ºC immediately before weighing them for analysis.

Major elements (N, P, S, K, Mg, Ca and TOC) must be expressed in mg/g dry powder.

Trace elements (Fe, Mn, Na, Zn, Cu, Al, As, B, Cd, Cl, Cr, F, Mo, Ni and Pb) must be expressed in µg/g dry powder.

7.12.3. Validation of the analytical results

The total element concentrations obtained by the laboratories' standard methods need to be checked in order to determine the accuracy of these methods. Two steps of quality assurance are recommended:

Comparison of the results of the national methods with the concentrations of reference standard samples. These reference standard samples, with certified total element concentrations supplied e.g. by the Central Bureau of References of the EC or by ISO (International Standard Organization), or by the US group of foliar analysis, will be sent to participating laboratories for analysis. The certified concentrations will be supplied to individual laboratories once a sufficient amount of laboratories have submitted their results.

In order to permanently check the accuracy of the analyses, it is also recommended that each laboratory provides several of its own standard samples for analysis in each batch of samples. The data should only be accepted if the analyses of the known samples match the reference results.

See also Chapter 8 for data quality management.

7.12.4. Data reporting

Values are reported on oven dry basis (105ºC).

Example files

FC example Excel file
FC example ASCII file

• File identifier SUBPROG states the subprogramme.   
• MEDIUM refers to the dominant tree species of the stand. (from NCC code list B4 (=LISTMED), see Annex 6 and TF Chapter for a list of common tree species).   
• LEVEL is given as the sampling height from the ground (cm).   
• Spatial pool SPOOL refers to the number of individual samples taken for pooled analysis.   
• Please NOTE! the pretretament codes and determination codes in use are listed in Code list DB.   
• C in the last field NEEDLES indicates results from current year needles, P= previous year's needles. General information on flags is given in Chapter 4.   
• Sampling year and month are given as YYYYMM, day field is left blank.

7.12.5 References

ICP Forests Manual
http://icp-forests.net/page/icp-forests-manual
(2010, 2016)

ICP Forests manual, 1997. Manual on methods and criteria for harmonized sampling, assessment, monitoring and analysis of the effects of air pollution on forests, 4th edition. Edited in 1997 by the Programme Coordination Centre Federal Research Centre for Forestry and Forest Products (BFH), Hamburg, Germany.

The most up-to-date methods of analysis are available from Official Methods of Analysis of AOAC International, http://www.eoma.aoac.org/