# Methodology for sampling and measurements

The database has been compiled using the results of field-work carried out by Waterford Institute of Technology forest research department. The data has been collected during numerous studies which have been conducted as part of the Forest Energy Research Programme 2010-2014 and the Shortfor Project.

Presented here is the general methodology which has been refined during the Shortfor project.

## Field-work

#### Inventory and selection of sample trees

Initial site visits involved surveying the forest plot and conducting an inventory of the stand. A site description was recorded including such info as species area, planting year, site accessibility, exposure, slope and elevation. Several inventory plots were taken to measure diameter at breast height (DBH), top height and stocking levels.

Sample trees were selected across the range of diameters present on the site and marked with tape. Where possible, ten trees were sampled.

#### Felling and destructive sampling of selected trees

The selected trees which had been identified for destructive sampling were individually felled, measured and partitioned.

The total stem length and merchantable stem of each felled tree was measured. Stems were measured in length from the base to the tip for tree height; and butt to the height at seven centimetre diameter for length of merchantable stem.

The stem diameter was measured at one metre intervals from the base to the height where the diameter was less than seven centimetres. Diameter at breast height (DBH) was also measured. The diameter of the collar was measured for all branches where the collar diameter was greater than three centimetres.

Felled trees were then partitioned by chainsaw into merchantable stem, top (stem with diameter less than seven centimetres), dead branches, and live branches. The mass of each of these partitions was weighed.

Three samples of live branches were taken per tree; large, medium and small. For most trees, the entire top partition was taken for sampling. However, for the biggest tops a sample was taken. All the dead branches were taken for sampling.

The merchantable stems were sampled by cutting discs, each approximately five centimetres wide, along the length of the stem at three metre intervals, from the base. All samples collected in the field were put into co-extruded, polyethylene sacks and cable-tied at the top in order to limit loss of moisture to the atmosphere between the time of field sampling and the time of analysis work carried out on the samples back at WIT.

#### Further processing of field samples

Following the completion of the on-site destructive sampling procedures, all sample material was taken to West Campus of Waterford Institute of Technology, Carriganore.

#### Branches and Tops

The live branches, tops and dead branches were weighed in their co-extruded sacks. The co-extruded sacks were then emptied and weighed separately in order to determine the net weight of sample material by subtracting the weights of the bags from the total weights.

The foliage was removed from the live branch samples and treated as a separate partition. Leaf material was weighed in order to determine green weight for moisture content samples.

Live branches, dead branches and tops were chipped separately and one to three moisture samples of chip were taken per partition, per tree. Moisture samples were weighed to determine green weight of each sample.

#### Stem, Wood and Bark

The stem sample discs were weighed and a cross was drawn across the longest diameter line through the centre of the discs and at right angles to this. The over-bark and under-bark diameter of all of the discs was measured with an electronic precision calliper along the prepared lines. The mean double bark thickness was calculated by subtracting the averages of the over-bark and under bark diameters. The bark percentage volume was calculated as the ratio of cross-sectional area of wood under-bark to over-bark.

Each disc was then split into two halves and labelled either "A" or "B". Those labelled "A" would be used to determine the basic density and moisture content of the stem material.

The disc sections labelled B were used to determine the moisture content of the wood and bark portions of the stem separately. The bark was removed from the stem using a sharp knife blade and the wood and bark were both weighed.

#### Whole Trees

Trees with a diameter at breast height of less than seven centimetres were not partitioned. These represent the "whole-tree" partition; they were chipped entirely and samples of approximately one kilogramme of chip were collected for moisture content analysis.

#### Basic Density

The volume of the basic density stem sample was determined by submersion. The samples were placed under water for 24 hours in order to become fully saturated. A bucket of water was placed on a mass balance and the weight reading was recorded. The saturated disc piece was then submerged below the water level in the bucket using teasing needles to hold the disc in place just below the surface. The reading on the mass balance was recorded again. The difference between the reading before submersion and during submersion translates from weight to volume as the volume of water displaced in centimetres cubed is reflected by the change in weight on the mass balance in grams. This is because the density of water is approximately one gram per cubic centimetre.

##### Basic Density:

$$D=\frac{m}{V}$$
Where
$D$
is the basic density in kilograms per meter cubed
$m$
is the mass in grams of the dried sample
$V$
is the volume of the sample as determined by immersion technique

#### Partitions Moisture Content

Each of the samples that were prepared for moisture content analysis were placed in a oven at 105°C for 48 hours in order to remove all of the free and bound water from the material. Samples were removed from the ovens individually and immediately weighed to determine the dry weight.

The moisture content was calculated as a percentage of the sample wet-weight basis by subtracting the dry weight from the wet weight and dividing the result by the wet weight.

##### Moisture Content:

$$M\%=\frac{m1-m2}{m1}\times\frac{100}{1}$$
Where
$_M$$\% is the moisture content expressed as a precentage of green weight m$$1$
is the mass in grams of the green sample
$m$$2 is the mass in grams of the dried sample #### Preparing ground sample material Following the oven-drying process of each partition as part of the moisture content analysis, sample material was available in a pre-dried state for further analysis. This pre-dried material was ground to a dust with a nominal top size of less than one millimetre, using a Fritsch Universal Cutting Mill Pulverisette 19. Stem discs and some branch material which were too large to grind in the cutting mill were first reduced in particle size by splitting and chipping before grinding. ## Internal Laboratory Analysis #### Moisture content of general analysis sample Biomass materials had all been pre-dried in an oven before being reduced to a ground sample but due to the hygroscopic nature of wood, materials would have absorbed some moisture from the atmosphere in the time between removal from the oven and arriving at the lab in a ground state. In order to determine the ash content and gross calorific value on a dry basis, it was first necessary to determine the moisture content of the general analysis sample. Determination of the moisture content of the general analysis sample was carried out in accordance with I.S. EN 14774-3:2009, described briefly here: Approximately one gram of general analysis sample was weighed in an oven-dried, porcelain crucible, this was the initial weight. The sample in the crucible was then placed in an oven at 105°C for 4 hours or until it reached a constant weight. The oven-dried crucible and sample material were then re-weighed to determine the dry weight of the sample. ##### Moisture content of general analysis sample:$$Mad=\frac{m_2-m_3}{m_2-m_1}\times\frac{100}{1}$$Where Mad is the moisture content of the test sample used for the determination expressed as a precentage m1 is the mass in grams of the empty dish plus lid m2 is the mass in grams of the dish plus lid and sample before drying m3 is the mass in grams of the dish plus lid plus sample after drying #### Ash content Determination of the ash content of sample material was carried out in accordance with I.S. EN 14775:2009. A porcelain crucible was first exposed to 550°C in a furnace in order to volatilise any contaminants and evaporate moisture. The weight of the clean, dry crucible was first recorded. Approximately one gram of general analysis sample was then weighed out in the crucible and the new weight was recorded. The crucible, containing sample, was then put in an electric muffle furnace (Carbolite OAF 11/1). The furnace was set to ramp up to 250°C from room temperature at 6°C per minute, then left at 250°C for one hour to allow all volatiles to leave the sample before ignition. The furnace was then set to ramp up to 550°C at a ramp rate of 10°C per minute and left at 550°C for two hours. Following controlled combustion of the sample in the furnace, the crucible containing residual ash was then transferred to a heat resistant plate for five to ten minutes, then to a desiccator without desiccant to allow cooling to ambient temperature. When the crucible was cool, it was reweighed a final time and recorded. Ash content was then calculated using the following equation: ##### Ash content:$$A_d=(\frac{m_2-m_1}{m_2-m_1}\times100\times(\frac{100}{100-Mad}))$$Where Mad is the moisture content of the test sample used for the determination expressed as a precentage m1 is the mass, in grams, of the empty dish m2 is the mass, in grams, of the dish plus test sample m3 is the mass, in grams, of the dish plus ash #### Calorific value Calorific value was determined in accordance with I.S. EN 14918:2009 and expressed as gross calorific value on a dry basis, including ash. Approximately one gram of the general analysis sample was pressed into a pellet with a 2811 Pellet Press. The sample pellet was then combusted under controlled conditions with a Parr 6300 automated isoperibol bomb calorimeter. The instrument automatically records the change of enthalpy in the system as the calorimeter jacket receives heat from the bucket following combustion. Gross calorific value as received is automatically calculated and printed by the instrument. Using the moisture content values calculated from the test on the general analysis sample and the gross calorific value as received determined by calorimetry, the gross calorific value on a dry basis can be calculated using the following equation: ##### Determination of gross calorific value (GCV)$$q_Vgr,d=qV,_gr\times\frac{100}{100-Mad}$$Where qV, gr, d is the gross calorific value at constant volume of the dry (moisture free) fuel, in megajoules per kilogram Mad is the moisture content in the analysis sample, in percentage by mass qv, gr is the gross calorific value at constant volume of the fuel as analysed, in joules per gram ## External Laboratory Analysis #### Determination of carbon, hydrogen, nitrogen (CHN), chlorine (Cl) and sulphur (S) Samples were sent to the Microanalytical lab in University College Dublin, Ireland for CHN, Cl and S analysis. CHN were measured using an Exeter Analytical CE 440 elemental analyser. Cl and S were determined through a titrimetric method. #### Determination of metal content The samples were analysed for minor elements arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), nickel (Ni), lead (Pb) and zinc (Zn) and were prepared by using an adapted method of EN 15297:2011. 400–500 mg of the ground (≤ 1 mm nominal top size) sample was weighed and mixed with 8 ml 69% Puriss HNO3 and 2 ml 35% H2O2. Samples were digested in a closed vessel using a CEM Mars 5 Station using a specific temperature profile. • Temperature increased to 190°C over 15 minutes • Temperature held at 190°C for 15 minutes • Samples were allowed to cool for 20 minutes Samples were filtered using Whatman 42 70 mm filter paper and diluted up to 25 ml using 2% HNO3 and stored at 4°C in polypropylene sample bottles. Metal analysis was carried out using a Varian 710-ES Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES). Single element standards (Inorganic ventures 1,000 µg/ml)–Arsenic (As), Cadmium (Cd), Chromium (Cr), Copper (Cu), Lead (Pb), Mercury (Hg), Nickel (Ni) and Zinc (Zn) were prepared using 2% HNO3. Concentrations of the standards solutions are listed in the table below. Wavelengths were chosen for each element according to the following: • Best interference free line • Greatest intensity with lowest background noise • The best correlation coefficient (≥ 0.9950). #### Metal standard concentrations (mg/L) with selected wavelengths (nm). Element Wavelength λ (nm) Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 As 188.980 0.1 1.0 10.0 Cd 226.502 0.0007 0.0015 0.004 0.007 0.01 Cr 267.716 0.001 0.005 0.01 0.025 0.04 Cu 327.395 0.005 0.1 0.2 0.3 0.4 Hg 253.652 0.1 1.0 10.0 Ni 231.604 0.004 0.02 0.04 0.05 Pb 220.353 0.1 1.0 10.0 Zn 213.857 0.01 0.05 0.1 1.0 2.0 3.0 Values determined are the mean metal content of three replicates (mg/kg) (db) ± standard deviation which were calculated using the following equation: #### Determination of metal content:$$w_i=\frac{V(ci-ci,o)}{m}\times\frac{100}{100-Mad}$$Where$w$i is the concentration of the element in the sample, on a dry basis, in mg/kg$c$i is the concentration of the element, in the diluted sample digest, in mg/l$c$i,o is the concentration of the element, in the solution of the blank experiment, in mg/l$V$is the volume of the diluted sample digest solution, in ml, m is the mass of the test portion used, in g$M\$ad
is the moisture content in the analysis test sample in % m/m

#### Instrumentation

A Varian 710-ES Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES) equipped with a SPS3 autosampler was used for the analysis of the samples. Instrumental conditions and sample introduction settings are listed in the table below:

Varian 710-ES operating parameters.

Operating parameters Value
Power (kW) 1.0
Plasma flow (L/min) 15.0
Auxiliary flow (L/min) 1.5
Nebuliser pressure (kPa) 200.0
Instrument Stabilisation delay (s) 15.0
Sample introduction settings
Sample uptake delay (s) 30.0
Pump rate (rpm) 15.0
Rinse time (s) 10.0
Fast pump (sample delay/rinse) On
General settings
Replicates 3
Correlation coefficient ≥0.9950

#### Limit of detection and limit of quantification determination

The limit of detection (LOD) and limit of quantification (LOQ) for each element was determined by analysing ten blank solutions with the standards and wavelengths listed in the table above. LOD and LOQ values were determined using the calculations below provided by the Varian ICP instrument:

LOD = (3 times the average of the standard deviation results of 10 blank replicates)

LOQ = (10 times the average of the standard deviation results of 10 blank replicates)

#### LOD and LOQ determined for each element analysed (mg/kg).

Element Wavelength, λ (nm) LOD (mg/kg) LOQ (mg/kg)
As 188.980 0.1297 0.4323
Cd 226.502 0.0018 0.0062
Cr 267.716 0.0034 0.0114
Cu 327.395 0.0040 0.0135
Hg 253.652 0.0019 0.0066
Ni 231.604 0.0043 0.0143
Pb 220.353 0.0370 0.1230
Zn 213.857 0.0019 0.0066

#### Certified reference material

The use of a certified reference material (CRM) allowed the determination of the accuracy of the method. Measured metal content was compared to the certified value and was expressed as a recovery percentage. A combination of Tomato leaves (1573a), Sea lettuce (BCR–279) and Aquatic plant (BCR–060) were used. CRM were digested and analysed in the same manner as the samples.

Element CRM (mg/kg) Obtained (mg/kg db) % Recovery
As 0.112 ± 0.004 Not detected 0.00
Cd 1.52 ± 0.04 1.18 ± 0.14 77.42
Cr 1.99 ± 0.06 2.11 ± 0.05 106.00
Cu 4.7 ± 0.14 3.66 ± 0.13 77.88
Hg 0.034 ± 0.004 Not detected 0.00
Ni 1.59 ± 0.07 1.53 ± 0.20 96.38
Pb 64 ± 4 43.08 ± 3.55 67.32
Zn 30.9 ± 0.7 29.18 ± 2.46 94.42