Thank you to the Research Council of Norway (Oslo Regional Fund, Alcofur rffofjor 269707) and the University of Portsmouth (Faculty of Science PhD research bursary) for providing funding for the studies of Lucy Martin. Also, to Gervais S. Sawyer who provided the wood used to generate the representative results. Turpentine was provided by Prof. Philip Evans of the University of British Columbia.

1. Preparing Test Sticks
<ol>
	<li>After any treatment processes are complete, cut dry wood into test sticks to size 2 mm x 4 mm x 20 mm (Figure 1). Air dry sticks to a constant weight, under laboratory conditions. Use at least 5 replicates of each wood being tested.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62776/62776fig01.jpg" /> 
Figure 1: Tes...

<ol>
	<li>Morrell, J. J., Kutz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+wood-based+materials.">Protection of wood-based materials.</a> Handbook of environmental degradation of materials, 3rd ed. , 343-368 (2018).</li><li>Distel, D. L., Goodell, B., Nicholas, D., Schultz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+marine+wood+boring+bivalves+and+their+bacterial+endosymbionts.">The biology of marine wood boring bivalves and their bacterial endosymbionts.</a> Wood deterioration and preservation. , 253-271 (2003).</li><li>Buslov, V., Scola, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inspection+and+structural+evaluation+of+timber+pier:+case+study.">Inspection and structural evaluation of timber pier: case study.</a> Journal of Structural Engineering. 117 (9), 2725-2741 (1991).</li><li>Registration Eligibility Decision for Chromated Arsenicals. List A, Case No. 0132. US EPA - Office of prevention, pesticides and toxic substances Available from: <a target="_blank" href="https://swap.stanford.edu/20110202084/http://www.epa.gov/oppsrrd1/reregistration/REDs/cca_red.pdf">https://swap.stanford.edu/20110202084/http://www.epa.gov/oppsrrd1/reregistration/REDs/cca_red.pdf</a> (2008)</li><li>Arsenic timber treatments (CCA and arsenic trioxide) review scope document, Review series 03.1. ISSN number 1443. Australian pesticides and veterinary medicines authority Available from: <a target="_blank" href="https://apvma.gov.au/sites/default/files/publication/14296-arsenic-timber-review-scope.pdf">https://apvma.gov.au/sites/default/files/publication/14296-arsenic-timber-review-scope.pdf</a> (2003)</li><li>Commission directive 2003/2/EC of 6 January 2003 relating to restrictions on the marketing and use of arsenic (tenth adaptation to technical progress to Council Deretive 76/769/EEC). 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M., Plarre, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+against+marine+borers:+About+the+revision+of+EN+275+and+the+attempt+for+a+new+laboratory+standard+for+Limnoria.">Resistance against marine borers: About the revision of EN 275 and the attempt for a new laboratory standard for Limnoria.</a> International Research Group on Wood Preservation, Document No. IRG/WP 20-20669. , (2020).</li><li>The European Commission for Standardization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EN+275:1992.+Wood+preservatives-+Determination+of+the+protective+effectiveness+against+marine+wood+borers.">EN 275:1992. Wood preservatives- Determination of the protective effectiveness against marine wood borers.</a> The European Commission for Standardization (CEN). , (1992).</li><li>European Commission. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directive+98/8/EC+concerning+the+placing+of+biocidal+products+on+the+market.">Directive 98/8/EC concerning the placing of biocidal products on the market.</a> Communication and Information Resource Centre for Administrations, Businesses and Citizens. , (2010).</li><li>Mantanis, G. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+screening+of+tropical+hardwoods+for+natural+resistance+to+the+marine+borer+Limnoria+quadripunctata:+The+role+of+leachable+and+non-leachable+factors.">Laboratory screening of tropical hardwoods for natural resistance to the marine borer Limnoria quadripunctata: The role of leachable and non-leachable factors.</a> Holzforschung. 62 (1), 99-111 (2008).</li><li>Cragg, S. M., Pitman, A., Henderson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+the+understanding+of+the+biology+of+marine+wood+boring+crustaceans+and+in+methods+of+controlling+them.">Developments in the understanding of the biology of marine wood boring crustaceans and in methods of controlling them.</a> International Biodeterioration &amp; Biodegradation. 43 (4), 197-205 (1999).</li><li>Cookson, L. J., Vic, M. D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additions+to+the+taxonomy+of+the+Limnoriidae.">Additions to the taxonomy of the Limnoriidae.</a> Memoirs of the Museum of Victoria. 56 (1), 129-143 (1997).</li><li>Cookson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Australasian+species+of+Limnoriidae+(Crustacea:+Isopoda).">Australasian species of Limnoriidae (Crustacea: Isopoda).</a> Memoirs of the Museum of Victoria. 52 (2), 137 (1991).</li><li>Jones, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+geographical+and+vertical+distribution+of+British+Limnoria+[Crustacea:+Isopoda].">The geographical and vertical distribution of British Limnoria [Crustacea: Isopoda].</a> Journal of the Marine Biological Association of the United Kingdom. 43 (3), 589-603 (1963).</li><li>Borges, L. M. S., Cragg, S. M., Busch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+laboratory+assay+for+measuring+feeding+and+mortality+of+the+marine+wood+borer+Limnoria+under+forced+feeding+conditions:+A+basis+for+a+standard+test+method.">A laboratory assay for measuring feeding and mortality of the marine wood borer Limnoria under forced feeding conditions: A basis for a standard test method.</a> International Biodeterioration &amp; Biodegradation. 63 (3), 289-296 (2009).</li><li>BSI Standards Publication. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BS+EN+350:2016.+Durability+of+wood+and+wood-based+products+-+Testing+and+classification+of+the+durability+to+biological+agents+of+wood+and+wood-based+materials.">BS EN 350:2016. Durability of wood and wood-based products - Testing and classification of the durability to biological agents of wood and wood-based materials.</a> BSI Standards Publication. , (2016).</li><li>Menzies, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The phylogeny, systematics, distribution, and natural history of limnoria. , 196-208 (1951).</li><li>Palanti, S., Feci, E., Anichini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+four+tropical+wood+species+for+their+resistance+to+marine+borers+(Teredo+spp+and+Limnoria+spp)+in+the+Strait+of+Messina.">Comparison between four tropical wood species for their resistance to marine borers (Teredo spp and Limnoria spp) in the Strait of Messina.</a> International Biodeterioration &amp; Biodegradation. 104, 472-476 (2015).</li><li>Delgery, C. C., Cragg, S. M., Busch, S., Morgan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+epibiotic+heterotrich+ciliate+Mirofolliculina+limnoriae+and+moulting+on+the+faecal+pellet+production+by+the+wood-boring+isopods+Limnoria+tripunctata+and+Limnoria+quadripunctata.">Effects of the epibiotic heterotrich ciliate Mirofolliculina limnoriae and moulting on the faecal pellet production by the wood-boring isopods Limnoria tripunctata and Limnoria quadripunctata.</a> Journal of Experimental Marine Biology and Ecology. 334 (2), 165-173 (2006).</li><li>Morrell, J. J., Helsing, G. G., Graham, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+wood+maintenance+manual:+a+guide+for+proper+use+of+Douglas-fir+in+marine+exposures.">Marine wood maintenance manual: a guide for proper use of Douglas-fir in marine exposures.</a> Forest Research Laboratory. , (1984).</li><li>Slevin, C. R., Westin, M., Lande, S., Cragg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+and+marine+trials+of+resistance+of+furfurylated+wood+to+marine+borers.">Laboratory and marine trials of resistance of furfurylated wood to marine borers.</a> Eighth European Conference on Wood Modification. , 464-471 (2015).</li><li>Westin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+borer+resistance+of+acetylated+and+furfurylated+wood+-+results+from+up+to+16+years+of+field+exposure.">Marine borer resistance of acetylated and furfurylated wood - results from up to 16 years of field exposure.</a> International Research Group on Wood Preservation. , (2016).</li><li>Westin, M., Rapp, A., Field Nilsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+test+of+resistance+of+modified+wood+to+marine+borers.">Field test of resistance of modified wood to marine borers.</a> Wood Material Science and Engineering. 1 (1), 34-38 (2006).</li><li>Borges, L. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradation+of+wood+exposed+in+the+marine+environment:+Evaluation+of+the+hazard+posed+by+marine+wood-borers+in+fifteen+European+sites.">Biodegradation of wood exposed in the marine environment: Evaluation of the hazard posed by marine wood-borers in fifteen European sites.</a> International Biodeterioration &amp; Biodegradation. 96 (1), 97-104 (2014).</li><li>Treu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Durability+and+protection+of+timber+structures+in+marine+environments+in+Europe:+An+overview.">Durability and protection of timber structures in marine environments in Europe: An overview.</a> BioResources. 14 (4), 10161-10184 (2019).</li><li>Williams, J. R., Sawyer, G. S., Cragg, S. M., Simm, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+questionnaire+survey+to+establish+the+perceptions+of+UK+specifiers+concerning+the+key+material+attributes+of+timber+for+use+in+marine+and+freshwater+engineering.">A questionnaire survey to establish the perceptions of UK specifiers concerning the key material attributes of timber for use in marine and freshwater engineering.</a> Journal of the Institute of Wood Science. 17 (1), 41-50 (2005).</li><li>Purnell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+carbon+footprint+of+reinforced+concrete.">The carbon footprint of reinforced concrete.</a> Advances in Cement Research. 25 (6), 362-368 (2013).</li><li>Hill, C. A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+environmental+consequences+concerning+the+use+of+timber+in+the+built+environment.">The environmental consequences concerning the use of timber in the built environment.</a> Frontiers in Built Environment. 5, 129 (2019).</li><li>Mercer, T. G., Frostick, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaching+characteristics+of+CCA-treated+wood+waste:+a+UK+study.">Leaching characteristics of CCA-treated wood waste: a UK study.</a> Science of the Total Environment. 427, 165-174 (2012).</li><li>Brown, C. J., Eaton, R. A., Thorp, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chromated+copper+arsenate+(CCA)+wood+preservative+on+early+fouling+community+formation.">Effects of chromated copper arsenate (CCA) wood preservative on early fouling community formation.</a> Marine Pollution Bulletin. 42 (11), 1103-1113 (2001).</li><li>Brown, C. J., Eaton, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+chromated+copper+arsenate+(CCA)-treated+wood+to+non-target+marine+fouling+communities+in+Langstone+Harbour,+Portsmouth,+UK.">Toxicity of chromated copper arsenate (CCA)-treated wood to non-target marine fouling communities in Langstone Harbour, Portsmouth, UK.</a> Marine Pollution Bulletin. 42 (4), 310-318 (2001).</li><li>Brown, C. J., Albuquerque, R. M., Cragg, S. M., Eaton, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+CCA+(copper-chrome-arsenic)+preservative+treatment+of+wood+on+the+settlement+and+recruitment+of+wood+of+barnacles+and+tube+building+polychaete+worms.">Effects of CCA (copper-chrome-arsenic) preservative treatment of wood on the settlement and recruitment of wood of barnacles and tube building polychaete worms.</a> Biofouling. 15 (1-3), 151-164 (2000).</li><li>Lebow, S. T., Foster, D. O., Lebow, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+copper,+chromium+and+arsenic+from+treated+southern+pine+exposed+in+seawater+and+freshwater.">Release of copper, chromium and arsenic from treated southern pine exposed in seawater and freshwater.</a> Forest Products Journal. 49 (7), 80-89 (1999).</li><li>Smith, P. 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Prior to selecting gribble specimens to be used in the feeding experiment, individuals should be screened to assess their suitability. There can be some variation in feeding rate between individuals due to differences in size, so only fully grown adult specimens should be selected.&#160;No significant difference between feeding rate of individuals between 1.5 mm and 3 mm length was detected by Borges et al., 200917. Female Limnoria brood their eggs, during which time have a reduced feedin...

Wood-borers can cause extensive damage to marine wooden structures, such as sea defences, piers, and aquaculture structures; the replacement or restoration of which costs billions of dollars per annum worldwide1,2,3. In order to protect these structures, timber is often treated to reduce biodegradation. However, due to the restriction of use of broad-spectrum biocides in Australia, EU, UK, and USA, in the marine environment, new modification techniques and species of wood that are naturally durable to borers are sought after4,5,6,7. Novel techniques for the preservation of wood in the marine environment require thorough testing in order to meet regulatory standards and limit environmental impacts from hazards such as leaching of any chemical preservative. For example, the European standard, EN 275, which is the current European standard from 1992, is used to evaluate wood preservation treatments against marine wood-borer damage8,9. This standard, along with other legislations against the use of biocidal compounds, such as CCA4,5,6,7 and creosote10, necessitates sustainable, non-toxic methods of wood protection and the use of naturally durable timber species to replace biocidal treatments11,12. Marine trials, such as those specified in EN 275, require long exposure periods and are thus expensive and slow to yield meaningful results. Laboratory tests, however, provide a much quicker alternative to test methods of preserving timber products against marine wood-borer attack, allowing rapid evaluation of adjustments to treatment schedules13. Results from this rapid laboratory experiment are designed to inform novel modification processes of wood and to identify timber species with natural durability to borer damage. A low feeding rate and vitality can indicate increased resistance in potential products and this information can then be fed back to industry partners to allow them to improve designs. Our method allows a nimble and rapid response, that is desirable in industry, and once promising products have been identified, results can be supplemented with those from marine trials.
Gribbles (Limnoria) are a genus of isopod crustacean in the family Limnoriidae. There are over 60 species of Limnoria worldwide13,14,15, with three common species found in the UK, Limnoria lignorum, Limnoria tripunctata and Limnoria quadripunctata16.&#160;They bore tunnels on the surface of wood that is submerged in seawater, often causing economically significant damage. Gribbles are highly abundant in coastal UK waters and are easy to maintain under laboratory conditions, making them ideal organisms for the study of wood biodegradation by marine wood-boring invertebrates. Evaluating the feeding rates and vitality of gribbles on different timber species and wood preservation methods can determine the efficacy of their resistance to biodegradation. The following protocol sets out a standard method for measuring gribble feeding rates, developed from that described by Borges and colleagues12,17, in addition to streamlining the introduction of image analysis to make the process operable in non-specialist labs. Image analysis is also used to reduce the practical limitations of manually counting large number of samples. Durability in long-term marine testing, according to the British Standard EN350-1:1994, are graded in reference to Pinus sylvestris sapwood18. In the short-term laboratory testing presented here, we use Scots pine (Pinus sylvestris L) sapwood as a control to testing heartwood of the species ekki (Lophira alata Banks ex C.F Gaertn), beech (Fagus sylvatica L), sweet chestnut (Castanea sativa Mill) and turpentine (Syncarpia glomulifera (Sm.) Nied). Average faecal pellet production and vitality among eight replicates per wood species was used as an indicator of durability. We provide illustrative data collected from a typical evaluation, using the gribble species Limnoria quadripunctata and a range of naturally durable timber species. Limnoria quadripunctata, identified by the keys provided by Menzies (1951), was selected as the optimal species for biodegradation trials due to the fact that it is the most well-studied member of the family and is well-established as a model species for use in biodegradation trials. This protocol is also applicable for testing woods of different treatments although the control used should be untreated replications of the same species.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12-well cell culture plates</td>
			<td>ThermoFisher Scientific</td>
			<td>150200</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Falcon tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable volume pipette</td>
			<td>Fisher Scientific</td>
			<td>FBE10000</td>
			<td>1-10 ml</td>
		</tr>
		<tr>
			<td>Beech</td>
			<td>G. Sawyer (consultant in timber technology)</td>
			<td>Fagus sylvatica</td>
			<td>Taxonomic authority: L</td>
		</tr>
		<tr>
			<td>Ekki</td>
			<td>G. Sawyer (consultant in timber technology)</td>
			<td>Lophira alata</td>
			<td>Taxonomic authority: Banks ex C. F. Gaertn.</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>10098140</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>LMS LTD</td>
			<td>INC5009</td>
			<td></td>
		</tr>
		<tr>
			<td>Microporous specimen capsules</td>
			<td>Electron Microscopy Sciences</td>
			<td>70187-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>Scots Pine</td>
			<td>G. Sawyer (consultant in timber technology)</td>
			<td>Pinus sylvestris</td>
			<td>Taxonomic authority: L.</td>
		</tr>
		<tr>
			<td>Size 00000 paintbrush</td>
			<td>Hobby Craft</td>
			<td>5674331001</td>
			<td>Size 000 or 0000 also acceptable</td>
		</tr>
		<tr>
			<td>Sweet Chestnut</td>
			<td>G. Sawyer (consultant in timber technology)</td>
			<td>Castanea sativa</td>
			<td>Taxonomic authority: Mill</td>
		</tr>
		<tr>
			<td>Turpentine</td>
			<td>P. Evans (Professor, Dept. Wood Science, University of British Columbia)</td>
			<td>Syncarpia glomulifera</td>
			<td>Taxonomic authority: (Sm.) Nied.</td>
		</tr>
		<tr>
			<td>Vacuum desiccator</td>
			<td>Fisher Scientific</td>
			<td>15544635</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid testing of resistance of timber to biodegradation by marine wood-boring crustaceans

Wood-boring invertebrates rapidly destroy marine timbers and wooden coastal infrastructure, causing billions of dollars of damage around the globe every year. As treatments of wood with broad spectrum biocides, such as creosote and chromated copper arsenate (CCA), are now restricted in marine use by legislation, naturally durable timber species and novel preservation methods of wood are required. These methods undergo testing in order to meet regulatory standards, such as the European standard for testing wood preservatives against marine borers, EN 275. Initial investigation of durable timbers species or wood preservative treatments can be achieved quickly and inexpensively through laboratory testing, which offers many advantages over marine field trials that are typically costly, long-term endeavours. Many species of Limnoria (gribble) are marine wood-boring crustaceans. Limnoria are ideal for use in laboratory testing of biodegradation of wood by marine wood-borers, due to the practicality of rearing them in aquaria and the ease of measuring their feeding rates on wood. Herein, we outline a standardizable laboratory test for assessing wood biodegradation using gribble.

A feeding experiment of L. quadripunctata was conducted over 20 days, using five different wood types (Scots pine (Pinus sylvestris L) sapwood, and heartwood of beech (Fagus sylvatica L), ekki (Lophira alata Banks ex C. F Gaertn), sweet chestnut (Castanea sativa Mil), and turpentine (Syncarpia glomulifera (Sm.) Neid)) (See Table of Materials), in November 2020. Eight replicate sticks were used per wood species and one specimen of Limnoria...

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Rapid Testing of Resistance of Timber to Biodegradation by Marine Wood-Boring Crustaceans

This protocol presents a method for assessing the feeding rate of the wood-boring crustacean, Limnoria, by measuring faecal pellet production. This method is designed for use in non-specialist labs and has potential for incorporation into standard testing protocols, to evaluate enhanced wood durability under marine conditions.

Wood-boring invertebrates rapidly destroy marine timbers and wooden coastal infrastructure, causing billions of dollars of damage around the globe every ...

Wood is an excellent material for construction, widely used for marine structures, such as piers, jetties, wharves and coastal defenses, such as groynes. So really excellent for these structures because of the strength, the impact resistance, the ability of people to shape, repair, and modify structures, all of those things are good. However, there's a major problem that results in damage that must run into the billions of dollars per year in terms of damage to marine structures.This damage is caused either by ship worms, which tunnel through the word and create quite large tunnels and as we will see in this particular presentation, a small crustacean. So here's a tube with some of them in it just about visible, the size of a small ant, but they occur in large numbers. They erode away the surface of wood and, eventually, cause collapse.What are we going to do about that problem? Well, the traditional approach is effective. It employs broad spectrum biocides, but as their name implies, these biocides are chemicals which do not distinguish between the different sorts of organisms.So a marine structure may be emitting chemicals that are damaging to the wider environment. With these concerns in mind, legislation has shifted what is permitted for marine use in North America, in the European waters, and in Australian waters. There's a major shift which requires innovation where the methods of protecting the wood are directed specifically at the borders and are not things which are going to cause problems in the broader environment.So we are setting out in this presentation to demonstrate how we test wood protection methods against the gribble, the tiny crustacean, and what we're presenting is a rapid response test. The traditional methods of testing wood materials for use in the sea require a five-year testing period and companies need to be much more nimble than that allows them to be. So we are looking and have developed a rapid method of evaluating preservation methods.This allows us to interact with the engineers producing the new methods, allows them to modify and us to test again. So here in this presentation, we'll be looking at a rapid testing method for evaluating ways of protecting wooden structures from the wood boring crustacean, the gribble. This is a bucket laboratory assessment to test for resistance of timber to biodegradation by marine wood borers.We are using the standard method to assess the feeding rate of a wood boring crustacean, the gribble, by measuring its fecal pellet production as well as assessing its vitality and mortality. After any treatment processes are complete, cut dry wood into test sticks to size two millimeters by four millimeters by 20 millimeters. Add dry sticks to a constant weight under laboratory conditions.At least five rep occurs of each wood being tested should be used. Post wood preparation. Place sticks under mesh in a food safe plastic container inside the vacuum desiccator and replace the lid, ensuring that there is a tight seal facilitated by a coating of vacuum grease.Attach three-way valve between the tubing connecting the desiccator and pump with the third tube leading to open air. Ensure that the three-way valve is closed off to air and run the pump to achieve a vacuum of between 0.75 to minus one bar within the vacuum desiccator and hold this vacuum for 45 minutes to one hour. Submerge the open end of the third tube into a container of seawater.Switch the pump off and close the valve leading to the pump and slowly open the valve until seawater is drawn by the vacuum into the desiccator. Allow the water to flow until it fills the plastic container above the level of the mesh. Then withdraw the tube from the seawater in the container, allowing air to enter until the desiccator attends to atmospheric pressure.Keep the sticks submerged under the mesh until they sink to the bottom of the plastic container. Submerge seawater saturated test sticks in sea water contained in 50 millimeter Falcon tubes. Replace water regularly for a period of 20 days.Extract individual specimens of gribble from an infested woodblock. Use a pair of fine forceps and a thin paintbrush. Carefully peel back any wood that is covering the gribble burrow with the forceps.Once the gribble have been exposed, use a paintbrush to gently pick out individuals from underneath and deposit them in a Petri dish filled with seawater. Check the gribble under a microscope to identify the species and ensure that no damage was caused while extracting. Any females brooding eggs should be discarded as gravid females have a reduced feeding capacity.Limnoria quadripunctata can be identified under a stereo microscope by the four distinct tubercules arranged in a square pattern on the animal's pleotelson in addition to an on the fifth In well plates with wells of diameter 20 millimeters, place one test stick in five millimeters of unfiltered seawater between 32 and 35 PSU per well. Place treatments or species of woods randomly throughout the well plate. Add one Gribble per well.Twice per week, remove the test stick and each gribble, one per well from the plate and place into a freshly prepared well plate containing five millimeters of sea water per well. Use a paintbrush to gently brush off any fecal pellets from the stick before transferring and retain the fecal pallets within the original well. Plates can be kept in constant dark conditions as photo period does not have an effect on gribble feeding rate.For changing the well plate and collecting the fecal pellets, vitality of individual gribble should be assessed. If a gribble has died, this cause a vitality of one. A score of two is given to gribble that are no longer on the wood and lethargic or slowly moving.Three is given to gribble that are actively swimming or moving, but not on the wood. Gribble that are crawling on the wood's surface are given a vitality score of four. Finally, the highest score of five is given to gribble who have created burrows within the wood.Use a fine paint brush to separate any clumps so the individual pellets are visible and brush any pellets away from the very edges of the well. Take a detailed photograph under a stereo microscope at one times magnification and upload to a computer. Ensure the pellets are in focus in the background as uniform with no shadows or light reflection on the surface of the water as this can interfere with imaging.Upload a stack of images by dragging and dropping or by selecting File, Import, Image Sequence, and Browse. Do not change any parameters and then select OK.Next, use the circle tool to select the bottom section of a well containing the fecal pellets. Remove the well edges and select Edit, Clear Outside.Make the image binary by selecting Process, Make Binary. Calibrate by selecting Analyze, Set Scale, and choose the number of pixels per millimeter for your image. Count the pellets and select Analyze, Analyze Particle in the box next to the size unit squared.Then select a lower threshold that is the same as the smallest size of a pellet using the unit scale set earlier. Invert show dropdown box, select Outlines, and then tick Summarize and press OK.Convert pellet counts to pellets per day, which give an indirect measure of feeding rate. Discard data from any molting individuals on days that molting occurred.Our representative results look to the species beech, Scots pine, turpentine, Ekki, and sweet chestnut on the feeding rate and vitality of gribbles. Daily fecal pellet production was calculated and averaged among eight replicates. Counts from individuals that were molting or had previously died were not included in the averages.The two control species beech and Scots pine saw the highest fecal pellet production while the hardwood Ekkis were below it. The highest vitality of five, shown in dark blue, is seen only on beech and Scots pine wood. Mortality represented by black, a vitality of one, was highest on sweet chestnut.The majority of remaining living individuals stayed a vitality of four in sweet chestnut, Ekki, and turpentine. The benefits of laboratory trials is that we can rapidly assess really promising modified woods for their durability against marine wood boring organisms and biodegradation in the ocean. I have some woods right here that has been really heavily degraded by these wood boring organisms.If you imagine, this was quite a decent sized piece of wood and it has been really, really eaten and chewed up. We can see how damaging these organisms are. By testing woods in our laboratory, it is cheaper, quicker, and more efficient than by simply going straight to trials in the ocean and we can actually get results really, really quickly.We can start to see whether or not the organisms can destroy and eat and survive on the wood and then we can start to pick really, really promising, modified wood that can then progress towards more expensive marine trials.

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