Protocol

Author

Yusuke Onoda

Overview

Leaf mechanical resistance is important for ecological processes such as leaf lifespan, plant-animal interactions and leaf litter decomposition. This protocol explains several leaf fracture properties and outlines how to measure them.

Background

Here we focus on leaf fracture properties and do not mention other mechanical properties such as elastic properties and dynamic mechanical properties. There are three methods widely used to measure leaf fracture properties;

(1) Shearing tests, also called scissor, cutting and guillotine tests (Atkins & May 1979; Lucas & Pereira 1990; Wright & Illius 1995; Darvell et al. 1996; Henry 1996; Aranwela et al. 1999; Wright & Cannon 2001), which measure the work required to cut a leaf;

(2) Punch tests, including punch-and-die and penetrometer tests (Williams 1954; Cherrett 1968; Feeny 1970; Coley 1983; Aranwela et al. 1999; Onoda et al. 2008), which measure the maximum load required for the punch rod to penetrate a leaf; and

(3) Tearing tests, also called tensile tests (Vincent 1992; Hendry & Grime 1993; Aranwela et al. 1999; Cornelissen et al. 2003), which measure the breaking force required to tear a strip of leaf lamina. Each method has its own relative merit.

The most appropriate method is dependent on the purpose of the study and/or the mode of fracture imposed on the leaf. Shearing tests can measure heterogeneity of fracture resistance along a transect of leaf (e.g. veins and lamina). Punch tests typically measure fracture resistance of a small area (normally lamina). Tearing method can measure leaf resistance to tensile force. This method is often used in studies on grasses, where large herbivores gather leaves by pulling and tearing. In the tearing test, force is applied parallel to the surface, while in the shearing and punch tests, force is applied perpendicular to leaf surface.

Table 1. Three major measuring methods

^*1

Test	Direction of force	Where to measure	What to measure
Shear	perpendicular to leaf surface	Traverse lamina at the middle of a leaf	Work
Punch	perpendicular to leaf surface	Lamina between the secondary veins	Maximum force (and work)*2
Tear	parallel to leaf surface	Lamina parallel to midrib	Maximum force (and work)*2

^*1 This table explains common practice of measurements in comparative ecology and may not be applicable for studies with a specific purpose. ^*2 Work can be measured when force and displacement are simultaneous measured.

Materials/Equipment

General statement Leaves are generally soft material, thus required devices must be able to measure small forces ( 0.01 N precision). In all methods, the best way to measure mechanical properties is to extend (or compress) the material at a constant speed and measure both force and displacement simultaneously because the results obtained in this way (force-displacement curve) provide a series of different mechanical properties (strength, toughness and stiffness etc). This is easily achieved when a material testing machine (Instron 5542 etc) is available. However, such machines are expensive and not always available for field research. Field biologists have made original devices to measure leaf fracture properties especially by punch and tearing tests. To enable data comparable across studies, care must be taken in building these measuring devices (see below).

Shearing test This method requires a machine to measure force and displacement simultaneously. There are two types used in shearing a leaf: one with a pair of blades (i.e. instrumented scissors; Darvell et al. 1996), and another with a single blade and an anvil (Aranwela et al. 1999). In the former method, the angle of the two blades becomes narrower as the point of fracture moves away from the pivot, while in the latter method, the angle between the blade and anvil is kept constant because the blade is mounted on upper moving arm with a constant angle. In both, the total work required to cut a specimen is given by the area under the force-displacement curve. Results are sensitive to blade angle, sharpness and clearance, and data measured by different devices can differ by 2 fold. See Darvell et al. (1996), Henry (1996), Aranwela et al. (1999) and Wright & Cannon (2001) for details of this type of test.

Punch test Ecological use of this method was once criticized by a few reasons: (1) incorrect use of terms, (2) friction was not accounted in most studies and (3) fracture in this test was not controlled in terms of shearing, compression and tension (Vincent 1992). Aranwela et al (1999) though a careful methodological study, developed several ways to improve this technique. The punch should have a flat-end and sharp-edge, and go through the middle of the hole of the die without any friction. The clearance between the punch and die should be as small as possible, so that leaf is fractured by shear force rather than compression, bending and tension. However, clearance smaller than 0.05 mm causes friction between the punch and die. Therefore, 0.05-0.1 mm clearance is ideal. The diameter of punch differed 0.5 – 5.5 mm across past studies. A smaller punch rod can measure an almost homogenate, specific part of leaf while a larger punch rod captures more heterogeneity of tissue. The size of the leaf measured also limits the size of the punch rod. When the device is properly made and leaf is fractured by mainly shear force, data obtained from different diameters of punch rod are roughly comparable when data are expressed per unit circumference of punch (Onoda, unpublished result). However, a consistent use of the same punch rod is strongly recommended. In my experience, a punch rod with 2 mm diameter is a good option since it can be used for relatively small leaves and punch out lamina without major veins.

Tearing test The tensile test is one of the most common methods in material testing, thus many commercial products are available. Some biologists have also made hand-made devices to measure the force required to tear a leaf sample (see Hendry & Grime 1993). The tearing method may be straightforward as the device just measures force (and displacement) to stretch a strip of leaf. It is important to use suitable grips that reduce damage the specimen by clamping pressure and avoid the specimen slipping out from the clamps under insufficient clamping pressure. Standardizing clamp pressure across species is impossible as species differ in material properties. Pneumatic grips with adjustable air pressure may be useful to choose and apply a suitable pressure for samples that have similar mechanical properties. The “glue-and-screw” technique has also been used for mounting specimens (see Aranwela et al. 1999).

Units, terms, definitions

It is important to know some basic terms in engineering (Some mechanical terms have been misused in past ecological papers which brought extra-confusions in plant biomechanics study. Should use proper terms following the engineering concept as much as possible.)

Stress: the force per unit area applied to a material (N m^-2)
Strain: increase in length of a material under load per unit of original length (dimensionless)
Stiffness: the resistance a material to deformation (N m^-2)
Strength: the stress at which the material fractures (N m^-2)
Fracture toughness: the work (energy) required to fracture the material (J m^-2)

Table 2. Data normalization of three measuring methods. Abbreviations: W, work; F

_max, the maximum force; l, fracture length; c, circumference of the punch rod; w, width of lamina strip; t, thickness.

Methods	At structure level (per fracture length)		At material level (per fracture area )
	Calculation	Name	Calculation	Name
Shear	x²W/l -1	Work to shear	x²W/(l x t) –2	Specific work to shear *1
Punch	x²Fmax/c -1	Force to punch	x²Fmax/ (l x c) -2	Specific force to punch *2
Tear	x²Fmax /w -1	Force to tear	x²Fmax/ (w x t) –2	Specific force to tear *3

^*1 Also called fracture toughness (e.g. Lucas & Pereira 1990) ^*2 Also called shear strength (Gere & Timochenko 1999) ^*3 Also called tensile strength (Gere & Timochenko 1999).

Procedure

Sample condition Since leaf mechanical properties change with hydraulic status, fresh leaf samples are normally used. Leaf samples are stored in a sealed plastic bag at 4 degree with a wet paper towel to maintain hydraulic level until measurement. Herbaceous leaf samples should be measured as soon as possible (i.e. within one day after the harvest). Mechanical properties of schlerophyll leaves can stay unchanged for several days (I. J. Wright, personal communication). A preliminary study is highly recommended to check whether mechanical properties change with time after the harvest. Mechanical properties also depend on leaf developmental stages as young leaves are much softer than old leaves. For cross-species comparison, young, fully-matured leaves are normally used.

Shearing test (single blade type)

1. Set up the machine on a stable level table. Replace blade if necessary.
2. Make sure that all accessories are firmly fixed.
3. Turn on the machine.
4. Adjust a position of the blade so that the blade passes the anvil with a clearance of ca. 0.1 mm (or lower if possible). Be careful that the blade does not catch the anvil during the cut, otherwise it may cause permanent damage to the machine.
5. Set the speed of vertical blade displacement in a range of 0.1-1 mm s^-1.
6. Conduct a dry run (without a sample) to check there is no friction between the blade and anvil. (In scissor systems, friction is inevitable. Thus it is necessary to measure friction along the cut every time after cutting a specimen).
7. (It is recommended that some standard materials filter paper, PPT plastic etc are used to check blade sharpness at a regular basis.)
8. Prepare samples. If leaf samples are stored at cool temperature, allow some time for the sample to reach to room temperature (ca. 20^∘C).
9. For cross-species comparison, lamina between secondary veins is used unless a leaf is too small (midrib and secondary veins are generally not obvious in small leaves anyway). Samples are taken (excised) from the middle point between the tip and base of leaf. The size of samples is usually 1-2 cm width (along the direction of midrib).
10. Width and thickness of the sample is measured by calipers (0.1 mm precision) and a thickness gauge (0.01 mm precision).
11. Place the sample on the anvil with the longitudinal axis perpendicular to the cut and with >5 mm extended from the anvil edge so that the blade cut across the specimen.
12. The sample should be fixed with a weight or double-sided tape between the sample and anvil.
13. Start measuring. The blade descends and traverses a leaf from one end to another. The machine measures force and displacement at constant time intervals (e.g. 0.05 second)
14. Stop the measurement when the blade has cut across the whole sample.
15. Save the data (if the machine does not do this automatically)
16. To examine whether friction between the blade and sample is significant, repeat the processes of 13-15 with the specimen untouched to measure friction force. If the friction force is significant, subtract this friction from the force-displacement curve obtained during leaf cut (this is normally not required for single blade type since friction is very small and thus ignored).
17. Remove the specimen, and clean any leaf sap/secretions from the blade and edge of anvil with wet paper in order to maintain the sharpness of the cutting edge.
18. Place a new specimen. And repeat the process 8-17.
19. Data calculation. Calculate the work required to cut the specimen which is given by the area of force-displacement curve.
20. Work to shear, which indicates fracture resistance per unit fracture length, is calculated as the total work required to cut divided by width of the sample.
21. Specific work to shear (fracture toughness) is calculated as work to shear divided by lamina thickness.

Punch test (punch-and-die measurement combined with a general testing machine)

1. Set up the machine on a stable level table.
2. Make sure that all accessories are firmly fixed.
3. Turn on the machine.
4. Adjust the position of the punch and die so that the punch goes through the die without any friction.
5. Set the speed of the punch slower than 1 mm s^-1 (e.g. 0.1 mm s^-1). Lamina thickness is normally 0.1-0.5 mm so measurements finish within 1 second if the speed of the punch is fast. At a high-speed punch, the material cannot respond rapidly enough the load.
6. Dry run (without a sample) to check if there is no friction between the punch and die.
7. (It is recommended that some standard materials filter paper etc are tested on a regular basis to check whether the edges of punch and die are not worn.)
8. Prepare samples. If leaf samples are stored at cool temperatures, allow some time for the sample to reach to room temperature (ca. 20 degree Celsius)
9. Measure the thickness of the sample with a thickness gauge (0.01 mm precision) at the intended punch location. Punch tests are typically done on the lamina between secondary veins at the middle part of leaf.
10. Place a sample on the die with the target punch location immediately below the punch.
11. For curled leaves, some weight or double-sided tape may be required to flatten the leaf on the die.
12. Start the measurement. The punch goes down at a constant speed and the machine measures force and displacement at constant time intervals (e.g. 0.05 second). (For penetrometer test, weight may be gradually increased by water, sand etc. until the punch rod goes through the leaf.)
13. Stop measurement when the punch have completely cut out the sample.
14. Save the data (if the machine does not do this automatically)
15. Remove the specimen and clean any leaf debris from the die.
16. Put a new specimen in place and repeat the process 6-15.
17. Data calculation. The maximum force (F_max) is normally recorded just before the fracture initiates, thus used for calculation for leaf resistance for punch force. F_max divided by the circumference of the punch is called “force to punch”. “Force to punch” divided by lamina thickness indicates material resistance for shear stress.

Tearing test

1. Set up the machine on a stable level table.
2. Make sure that all accessories are firmly fixed.
3. Turn on the machine.
4. Set the speed of tension in a range of 0.1-1 mm s^-1.
5. Prepare samples. If leaf samples are stored at cool temperatures, allow some time for the sample to reach to room temperature (ca. 20^∘C)
6. Excise a strip of lamina from a leaf, parallel to the leaf main axis and excluding the midrib. The strip should be long enough (preferred length-width ratio is >10) so that force can be applied along its main axis. For small leaves or narrow leaves where a lamina strip cannot be made, a whole leaf blade may be used.
7. Measure the width and thickness of the lamina strip with calipers ( 0.1 mm precision) and a thickness gauge ( 0.01 mm precision) respectively, at the middle of lamina strip.
8. Mount the strip in the pair of grips. Take care not to squash the lamina.
9. Measure the length of strip between the ends of the grips. This is not necessary if only breaking force is of interest but it is necessary for stiffness measurements. It is recommended to measure this, however, as the effect of the aspect ratio on strength can be tested.
10. Stretch the strip at a constant speed. The machine measures force and displacement at constant time intervals (e.g. 0.1 second). (For devices which only measure force, the maximum force to tear the strip is measured.)
11. If the lamina strip is torn close to the either of the grips, discard the data because tear resistance is likely to be underestimated due to compression damage caused by the clamping pressure.
12. Remove the sample from the grips.
13. Mount a new sample and repeat the process 5-12.

Figures (from left to right): Shearing test (courtesy: Ian. J. Wright), punch-and-die test and tensile test.

Notes and troubleshooting tips

Some plants contain much silica (e.g. grass species) which decreases sharpness of the blade very quickly. For shearing test that do not use disposable blade, high-silica species may be avoided. Some leaves are not in flat structure, and their cross-sections cannot be assumed to be rectangular shape. In such cases, some modification is needed to calculate cross-sectional area.

Links to resources and suppliers

Instron: http://www.instron.com

Literature references

Aranwela N., Sanson G. & Read J. (1999). Methods of assessing leaf-fracture properties. New Phytologist, 144, 369-383.

Atkins A.G. & Mai Y.W. (1979). On the guillotining of materials. Journal of Materials Science, 14, 2747-2754.

Choong M.F., Lucas P.W., Ong J.S.Y., Pereira B., Tan H.T.W. & Turner I.M. (1992). Leaf fracture toughness and sclerophylly: Their correlations and ecological implications. SO – New Phytologist. 121(4). 1992. 597-610.

Coley P.D. (1983). Herbivory and Defensive Characteristics of Tree Species in a Lowland Tropical Forest. Ecological Monographs, 53, 209-234.

Cornelissen J.H.C., Lavorel S., Garnier E., Diaz S., Buchmann N., Gurvich D.E., Reich P.B., ter Steege H., Morgan H.D., van der Heijden M.G.A., Pausas J.G. & Poorter H. (2003). A handbook of protocols for standardised and easy measurement of plant functional traits worldwide. Australian Journal of Botany, 51, 335-380.

Darvell B.W., Lee P.K.D., Yuen T.D.B. & Lucas P.W. (1996). A portable fracture toughness tester for biological materials. Measurement Science & Technology, 7, 954-962.

Feeny P. (1970). Seasonal changes in oak leaf tannins and nutrients as a cause of spring feeding by winter moth caterpillars. Ecology, 51, 565-581.

Gere J.M. & Timoshenko S.P. (1999). Mechanics of Materials 4th SI edition. Tanley Thornes (Publisher) Ltd, Cheltenham.

Hendry G.A.F. & Grime J.P. (1993). Methods in comparative plant ecology : a laboratory manual. Chapman & Hall, London, UK.

Henry D., Macmillan R. & Simpson R. (1996). Measurement of the shear and tensile fracture properties of leaves of pasture grasses. Australian Journal of Agricultural Research, 47, 587-603.

Lucas P.W. & Pereira B. (1990). Estimation of the Fracture Toughness of Leaves. Functional Ecology, 4, 819-822.

Onoda Y., Schieving F. & Anten N.P.R. (2008). Effects of Light and Nutrient Availability on Leaf Mechanical Properties of Plantago major: A Conceptual Approach. Ann Bot, 101, 727-736.

Read J. & Sanson G.D. (2003). Characterizing Sclerophylly: The Mechanical Properties of a Diverse Range of Leaf Types. New Phytologist, 160, 81-99.

Vincent J.F.V. (1992). Plants. In: Biomechanics-materials: a practical approach (ed. Vincent JFV). Oxford University Press Oxford, pp. 165-191.

Wright I.J. & Cannon K. (2001). Relationships between leaf lifespan and structural defences in a low-nutrient, sclerophyll flora. Functional Ecology, 15, 351-359.

Wright W. & Illius A.W. (1995). A comparative study of the fracture properties of 5 grasses. Functional Ecology, 9, 269-278.

Health, safety & hazardous waste disposal considerations

High pressure is applied to samples during measurement, thus be careful as fingers can be squashed, cut, punched or torn!