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: Test sticks used in short-term laboratory testing to assess gribble feeding rates.&#160;&#160;Test wood sticks sized 2 mm x 4 mm x 20 mm. From left to right: ekki, turpentine, sweet chestnut and beech heartwood and Scots pine sapwood. Scale bar 4 mm.&#160;<a href="https://www.jove.com/files/ftp_upload/62776/62776fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Vacuum Impregnation
	<ol>
		<li>Post wood preparation (i.e., cutting and treatment, if applicable), place sticks under a mesh in a food-safe plastic container, inside the vacuum desiccator and replace lid ensuring there is a tight seal, facilitated by a coating of vacuum grease (Figure 2).</li>
		<li>Attach a three-way valve between the tubing connecting the desiccator and pump, with a third tube leading to open air (Figure 2). Ensure that the three-way valve is closed off to the air and run the pump to achieve a vacuum of between -0.75 to -1.0 bar within the vacuum desiccator and hold this vacuum for 45 minutes - 1 hour.</li>
		<li>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, then 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.</li>
		<li>Then withdraw the tube from the seawater in the container, allowing air to enter, until the desiccator returns to atmospheric pressure. Keep the sticks submerged under the mesh until they sink to the bottom of the plastic container.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62776/62776fig02.jpg" /> 
Figure 2: Equipment used to vacuum impregnate wood sticks with seawater, in preparation for feeding to gribbles during a laboratory feeding assay.&#160; A) Vacuum desiccator; B) Pump; C) Pressure gauge for the vacuum desiccator; D) The three-way valve leading to the vacuum desiccator, pump and to open air or seawater (orange tube).&#160;<a href="https://www.jove.com/files/ftp_upload/62776/62776fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Leaching Wood
	<ol>
		<li>Submerge seawater-saturated test sticks in seawater contained in 50 mL tubes (Figure 3). Replace water regularly for a period of 20 days. 
		NOTE: The leaching process applies to any experimental wood under test, including treated or natural woods.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62776/62776fig03.jpg" /> 
Figure 3: Leachate from wood sticks for preparation for feeding to gribbles during a laboratory feeding assay.&#160; Wood that was fully submerged in seawater contained in a 50 ml Falcon tube, with regular water change (1-3 days), produced distinctly coloured leachate. From left to right leachate from heartwood of; sweet chestnut, turpentine, ekki, and beech and Scots pine sapwood.&#160;<a href="https://www.jove.com/files/ftp_upload/62776/62776fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Extracting Gribble
<ol>
	<li>Extract individual specimens of gribble from an infested wood block. Use a pair of fine forceps and a thin (size 000/0.4 mm or smaller) paintbrush. Carefully peel back any wood that is covering the gribble burrow with the forceps 
	NOTE: Burrows are found on the surface of wood and can be identified by small holes (Figure 4).</li>
	<li>Once gribble have been exposed, use a paintbrush to gently pick up individuals from underneath and deposit in a petri dish filled with seawater. Check gribble under a microscope to identify species and to ensure no damage was caused while extracting. 
	NOTE: Beating pleopods are a sign of vitality.
	<ol>
		<li>Discard any females brooding eggs as gravid females have a reduced feeding capacity.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62776/62776fig04.jpg" /> 
Figure 4: Image of a gribble burrow with two typical ventilation holes.&#160;&#160;L. quadripunctata burrow on a stick of Radiata pine wood, sized 2 mm x 4 mm x 20 mm. Two smaller ventilation holes can be seen next to the burrow entrance. Scale bar 2 mm.&#160;<a href="https://www.jove.com/files/ftp_upload/62776/62776fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Identifying Limnoria quadripunctata
	<ol>
		<li>Identify Limnoria quadripunctata under a stereomicroscope by the four distinct tubercles, arranged in a square pattern, on the animal&#39;s pleotelson in addition to an X-shaped carina on the fifth pleonite19 (Figure 5).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62776/62776fig05.jpg" /> 
Figure 5: Limnoria quadripunctata identifying features.&#160;&#160;Image of dorsal surface Limnoria quadripunctata, taken on a stereomicroscope at x20 magnification. Identifying features shown by red arrow - indicates the X- shaped carina and blue arrow - indicates four tubercles on pleotelson. Scale bar 1 mm.&#160;<a href="https://www.jove.com/files/ftp_upload/62776/62776fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Preparing Well Plates
<ol>
	<li>In multi-well plates with wells of diameter 20 mm, place one test stick and 5 mL of unfiltered seawater, between 32-35 PSU, per well (Figure 6).</li>
	<li>Place treatments/species of wood systematically throughout the well plate so that each type of wood is represented at least once per plate. Add one gribble per well. 
	&#8203;NOTE: Temperature should be kept stable in an incubator at 20 &#176;C &#177; 2 &#176;C for the species L. quadripunctata, other species of Limnoria can be used with adjustments to the temperature made to suit the specific species.</li>
	<li>Keep plates in constant dark conditions as the photoperiod does not have an effect on gribble feeding rate15.</li>
</ol>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62776/62776fig06.jpg" /> 
Figure 6: Experimental set up for gribble feeding assay.&#160; An example of a 12 multi-well plate used in the laboratory testing of gribble feeding rate. Each well contains 5 ml seawater and one test stick (20 mm x 4 mm x 2 mm) of different wood species; Scots Pine sapwood and ekki, beech, sweet chestnut, and turpentine heartwood. Scale bar 20 mm.&#160;<a href="https://www.jove.com/files/ftp_upload/62776/62776fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Collecting and Counting Faecal Pellets and Assessing Vitality.
<ol>
	<li>Twice per week, remove the test stick and each gribble (one per well) from the well plate and place into a freshly pre-prepared well plate (containing 5 mL of seawater per well [32-35 PSU, 18-22 &#176;C]).</li>
	<li>Use a paintbrush to gently brush off any faecal pellets from the stick before transferring and retain the faecal pellets within the original well. 
	NOTE: Prior to transferring the gribble to a fresh well plate, vitality can be assessed on a scale of 1-5; 1= dead, 2 = passive, not on the wood, 3 = actively swimming or beating pleopods, not on the wood, 4 = crawling on the surface of the wood, 5 = burrowed into the wood.</li>
	<li>Image Processing
	<ol>
		<li>Use a fine paintbrush to separate any clumps so that individual pellets are visible and brush pellets away from the very edges of the well. Take a detailed&#160;photograph under a stereo microscope, at magnification x4 and upload to a computer (Figure 7). 
		&#8203;NOTE: Ensure the pellets are in focus and the background is uniform, with no shadows or light reflections on the surface of the water.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62776/62776fig07.jpg" /> 
Figure 7: Image of gribble faecal pellets.&#160;&#160;L. quadripunctata faecal pellets (small, cylindrical, brown pellets) from feeding on Radiata pine wood in one well of a multi-well plate. Taken at x4 magnification. Images prior to manipulation for image analysis (see Figure 7).&#160;A) Example of a suitable image to be used for automated counting in ImageJ. Pellets are sufficiently spread out and away from the edges of the well. The well is centred and there are no obstructions or reflections. B) An example of an image that is unsuitable for image analysis. The well is off-centre, cutting off the bottom half. Blue (dotted) circle shows light reflection off the surface of the water. Orange (solid) circle shows pellets that are clumped too closely together and too near the edge of the well. Red (dashed) circle shows a wood chip that was not removed. Scale bar 10&#160;mm.&#160;<a href="https://www.jove.com/files/ftp_upload/62776/62776fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2" style="margin-left: 40px;">
	<li>Process to Generate Faecal Pellet Count Using ImageJ.
	<ol>
		<li>Download ImageJ (latest version as of 03/08/21, 1.8.0_172) from&#160;https://imagej.nih.gov/ij/download.html or run from the computer&#8217;s browser.&#160;</li>
		<li>Upload a stack of images by dragging and dropping or by selecting File | Import | Image sequence | Browse. Do not change any parameters then select Okay.</li>
		<li>Next, use the circle tool to select the bottom section of the well containing the faecal pellets. Remove the well edges, select Edit | Clear outside. Make the image binary, select Process | Make binary.</li>
		<li>Calibrate by selecting Analyse | Set scale and choose the number of pixels per millimeter for the image (for example 10 pixels = 1 mm). Count the pellets, select Analyse | Analyse particles.</li>
		<li>In the box next to Size (unit2), select a lower threshold that is the same as the smallest size pellet, using the unit scale set earlier (for example, if 10 pixels = 1 mm and the smallest pellet is 0.5 mm, choose 5-infinity).</li>
		<li>In the Show drop down box, select Outlines and then tick Summarise and press Okay (Figure 8). 
		NOTE: Further information can be found at&#160;https://imagej.nih.gov/ij/docs/guide/index.html</li>
	</ol>
	</li>
</ol>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62776/62776fig8.1v2.jpg" />
<img alt="Figure 8.1" src="/files/ftp_upload/62776/62776fig8.2v2.jpg" /> 
Figure 8: A flow diagram of the process used in ImageJ to count faecal pellets.&#160;&#160;A) Importing an image sequence in the File tab of ImageJ. B) The browse button in the &#39;Import Image Sequence&#39; dialog box to import a sequence of images from a local device. C) Using the circle tool to select area containing faecal pellets D) Clear outside button in the edit tab area to remove outside of selected area. E) Make binary button in the process tab. F) Set scale button in the Analyse tab. Distance in pixels is equivalent to the number of pixels to one unit of measurement (mm). G) Analyse particles button in the Analyse tab. Size (unit^2) set to the lower threshold of faecal pellet size, in pixels, to infinity. Show &#39;outlines&#39; and &#39;summarise&#39; are selected.&#160;<a href="https://www.jove.com/files/ftp_upload/62776/62276fig8v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Data Analysis 
	<ol>
		<li>Convert pellet counts to pellets per day, which gives and indirect measure of feeding rate. Discard data from any moulting individuals on days that moulting occurred (Figure 9). 
		NOTE: Moulting occurs over 1-3 days and can be identified when a full moult of the exoskeleton can be seen.</li>
	</ol>
	 </li>
</ol>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62776/62776fig09.jpg" /> 
Figure 9: Example of a gribble moult.&#160;&#160;Gribble (L. quadripunctata) moulting, on a Radiata pine wood test stick sized 20 mm x 4 mm x 2 mm. Moults are indicated by red circles. Scale bar 2 mm.&#160;<a href="https://www.jove.com/files/ftp_upload/62776/62776fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<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). Official Journal of the European Communities Available from: <a target="_blank" href="https://www.legislation.gov.uk/eudr/2003/2/adopted">https://www.legislation.gov.uk/eudr/2003/2/adopted</a> (2003)</li><li>The Hazardous Waste (England and Wales) Regulations 2005 No.894. Environmental Protection England and Wales Available from: <a target="_blank" href="https://www.legislation.gov.uk/uksi/2005/894/contents/made">https://www.legislation.gov.uk/uksi/2005/894/contents/made</a> (2005)</li><li>Palanti, S., Cragg, S. 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. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modification+of+wood+by+acetylation+or+furfurylation:+A+review+of+the+present+scaled-up+technologies.">Chemical modification of wood by acetylation or furfurylation: A review of the present scaled-up technologies.</a> BioResources. 12 (2), 4478-4489 (2017).</li><li>Borges, L. M. S., Cragg, S. M., Bergot, J., Williams, J. R., Shayler, B., Sawyer, G. 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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+to+human+health+and+estuarine+posed+by+pulling+out+creosote-treated+timber+on+oyster+farms.">Risk to human health and estuarine posed by pulling out creosote-treated timber on oyster farms.</a> Aquatic Toxicology. 86 (2), 287-298 (2008).</li><li>Brown, C. J., 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=Assessment+of+Effects+of+Chromated+Copper+Arsenate+(CCA)-Treated+Timber+on+Nontarget+Epibiota+by+Investigation+of+Fouling+Community+Development+at+Seven+European+Sites.">Assessment of Effects of Chromated Copper Arsenate (CCA)-Treated Timber on Nontarget Epibiota by Investigation of Fouling Community Development at Seven European Sites.</a> Archives of Environmental Contamination and Toxicology. 45 (1), 0037-0047 (2003).</li></ol>

The authors have no conflicts of interest related to this study.

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><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>&lt;em&gt;Fagus sylvatica&lt;/em&gt;</td><td>Taxonomic authority: L</td></tr><tr><td>Ekki</td><td>G. Sawyer (consultant in timber technology)</td><td>&lt;em&gt;Lophira alata&lt;/em&gt;</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>&lt;em&gt;Pinus sylvestris&lt;/em&gt;</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>&lt;em&gt;Castanea sativa&lt;/em&gt;</td><td>Taxonomic authority: Mill</td></tr><tr><td>Turpentine</td><td>P. Evans (Professor, Dept. Wood Science, University of British Columbia)</td><td>&lt;em&gt;Syncarpia glomulifera&lt;/em&gt;</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...

Watch this Scientific Journal Video about Rapid Testing of Resistance of Timber to Biodegradation by Marine Wood-Boring Crustaceans at JoVE.com

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 ...

rapid-testing-resistance-timber-to-biodegradation-marine-wood-boring

Research

JoVE Journal

Environment

3.7K Views.  University of Portsmouth. Hout is een uitstekend materiaal voor de bouw, veel gebruikt voor maritieme structuren, zoals pieren, steigers, werven en kustverdedigingen, zoals kribben. Dus echt uitstekend voor deze structuren vanwege de sterkte, de slagvastheid, het vermogen van mensen om structuren te vormen, te repareren en aan te passen, al die dingen zijn goed. Er is echter een groot probleem dat resulteert in schade die in de miljarden dollars per jaar moet lopen in termen van schade aan mariene structuren.De...

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

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of the woodboring beetle, Chlorophorus caragana Xie &#38; Wang (2012), for both types of electron microscopy. The scanning electron microscopy (SEM) sample preparation protocol was based on sample chemical fixation, dehydration in a series of ethanol baths, drying, and sputter-coating. By adding Tween 20 (Polyoxyethylene sorbitan laurate) to the fixative and the wash solution, the insect body surface of woodboring beetle was washed more cleanly in SEM. This study&#39;s transmission electron microscopy (TEM) sample preparation involved a series of steps including fixation, ethanol dehydration, embedding in resin, positioning using fluorescence microscopy, sectioning, and staining. Fixative with Tween 20 enabled penetrate the insect body wall of woodboring beetle more easily than it would had been without Tween 20, and subsequently better fixed tissues and organs in the body, thus yielded clear transmission electron microscope observations of insect sensilla ultrastructures. The next step of this preparation was determining the positions of insect sensilla in the sample embedded in the resin block by using fluorescence microscopy to increase the precision of target sensilla positioning. This improved slicing accuracy.

In order to observe ultrastructure of insect sensilla, scanning and transmission electron microscopy (SEM and TEM, respectively) sample preparation protocol were presented in the study. Tween 20 was added into the fixative to avoid sample deformation in SEM. Fluorescence microscopy was helpful for improving slicing accuracy in TEM.

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of ...

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

Biology

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

Bioengineering

Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the adhesive cure, and the effect of adhesive penetration on the wood interphase; thus, normal testing of a neat adhesive film is generally not useful. Most tests of wood adhesive bond strength are slow, laborious, can be strongly influenced by the wood and do not provide information on the kinetics of cure. Test method ASTM D 7998-19, however, can be used for fast evaluation of the strength of wood bonds. The use of a smooth, uniform, and strong wood surface, like maple face-veneer, and sufficient bonding pressure reduces the adhesion and wood strength effects on bond strength. This method has three main applications. The first is to provide consistent data on bond strength development. The second is to measure the dry and wet strengths of bonded lap shear samples. The third is to better understand the adhesive heat resistance by quickly evaluating thermal sensitivity and distinguishing between thermal softening and thermal degradation.

We present a procedure, ASTM D7998-19, for a rapid and more consistent evaluation of both dry and wet strength of adhesive bonds on wood. The method can also be used to provide information on strength development as a function of temperature and time or strength retention up to 250 &#176;C.

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.

Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an ...

Exploring Entomopathogenic Fungi Resources from Forest Wood Borers

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence period is considered an acceptable alternative to chemical control. However, EPF resources have been significantly less explored for FWBs, in contrast to agricultural insect pests. This paper presents a protocol for exploring EPF resources from FWBs using wild Monochamus alternatus populations as an example. In this protocol, the assignment of traps baited with M. alternatus attractants to different populations guaranteed the collection of adequate samples with natural infection symptoms, during the emergence periods of the beetle. Following finely dissecting integuments and placing them onto a selective medium, fungal species were isolated from each part of beetle bodies and identified based on both molecular and morphological traits.
Several fungal species were certified as parasitic EPFs via re-infection of healthy M. alternatus with spore suspensions. Their behavioral phenotypes on M. alternatus were observed using scanning electron microscopy and further compared with those on the Coleopteran model insect Tribolium castaneum. For EPFs that present consistent parasitism phenotypes on both beetle species, evaluation of their activities on T. castaneum provided valuable information on lethality for future study on M. alternatus. This protocol helped the discovery of EPF newly reported on M. alternatus populations in China, which could be applied as an efficient approach to explore more EPF resources from other FWBs.

Author Spotlight: Evaluation of Entomopathogenic Fungi in Wild Monochamus alternatus Populations for Biocontrol Applications in Forest Wood Borers

Here we present a protocol for obtaining entomopathogenic fungi from a forest wood borer and a substitutive way to evaluate their entomopathogenic activities using a Coleopteran model insect. This method is efficient and convenient for exploring entomopathogenic fungal resources from wood-boring insect pests in natural forests.

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence ...

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

Samenvatting

Meer video's verkennen