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Development of a novel antisapstain product Kovacevic, Snezana B. 2001

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DEVELOPMENT OF A NOVEL ANTISAPSTAIN PRODUCT By S N E Z A N A B. K O V A C E V I C B . A . S c . The University of N o v i Sad, 1981  A T H E S I S S U B M I T E D IN P A R T I A L F U L F I L L M E N T O F T H E REQUIREMENTS FOR THE DEGREE OF M A S T E R OF APPLIED SCIENCE  in T H E F A C U L T Y OF G R A D U A T E STUDIES DEPARTMENT OF CHEMICAL ENGINEERING  We accepted this thesis as conforming toJhjrfeqi)ired standard  T H E UNIVERSITY O F BRITISH C O L U M B I A December, 2000 © S n e z a n a B . Kovacevic, 2000  In  presenting  degree  this  at the  thesis  in  University of  partial  fulfilment  of  of  department  this or  publication of  thesis for by  his  or  that the  her  representatives.  It  this thesis for financial gain shall not  Department The University of British Columbia Vancouver, Canada  for  an advanced  Library shall make  it  agree that permission for extensive  scholarly purposes may be  permission.  DE-6 (2/88)  requirements  British Columbia, I agree  freely available for reference and study. I further copying  the  is  granted  by the  understood  that  be allowed without  head of copying  my or  my written  Abstract  As a biodegradable product, wood is vulnerable to attack by microorganisms that can degrade it and cause loss in value. To prevent their colonization, wood can be protected by the application of chemical preservatives. Environmental and economic pressure on currently used chemicals has led to a situation where considerable effort is now directed toward the development of new preservatives. Among the most promising products are products that contain alkylammonium compounds (AACs). Based on laboratory tests, didecyl dimethyl ammonium chloride (DDAC) has been described as one of the most effective A A C used as wood preservatives. However, there were situations in the practice, where A A C s had problems. Despite investigations into the reasons for these problems, no clarification was accomplished. This thesis describes the process of the development of a new wood preservative that would, while improving the characteristics of AACs, meet the objectives of efficient control of fungal growth, low toxicity, less potential for environmental impact, and easier disposal of treated products withdrawn from service. The project was divided into five general phases.  The experiments of the first phase were designed to test several potential fungicides against moulds and stains in order to eliminate less promising formulations. In Phase 2, the research was extended to other groups of fungi, such as soft rot, brown rot, white rot and D D A C tolerant species. In both phases tests were done on agar plates.  ii  In Phase 3, the most promising formulations from Phase 1 and Phase 2 were tested on a wood substrate to determine the minimum amount of preservative that would be effective against sapstain fungi and moulds. In Phase 4, active components and additives were combined together in larger volumes and product formulation stability was investigated. The potential product solution was then submitted for toxicological testing. In the last (fifth) phase, the relation between efficacy and the required coverage and retention were examined by a simulation of real spraying conditions in wood treatment pilot plant. Samples of lumber were sprayed in the pilot plant with the test product and then stored outside in the yard to see if moulds, fungi or sapstain would develop.  The results of this thesis relate to a synergistic wood preservative composition comprising a quaternary ammonium compound and an additional second active component. The laboratory work on agar plates and the wood substrate, as well as field tests after pilot-plant spraying, have shown that the new product works. It has proved its capability for controlling a large spectrum of wood destroying microorganisms and counteracting the phenomena of the development of fungal resistance toward the applied active components.  iii  Table of Contents Abstract  ii  List of Tables  vii  List of Figures  xii  Abbreviations  xiv  Acknowledgement Chapter 1: Introduction  xv 1  1.1 The Lumber Industry and its Markets  3  1.2 The Wood Preservation Industry  7  1.3 Governmental Regulations and Registrations  11  1.4 Application Methods for Chemical Protection Against Sapstain  13  Chapter 2: Sapstain and Wood Rot 2.1 Factors That Cause Staining and Discoloration of Timber Chapter 3: Anti-Sapstain Protection  16 16 21  3.1 Shortcomings of Traditional Chemical Products  24  3.2  Possible Problem Solution  26  Chapter 4: Fungicides - Literature Rewiew  28  4.1 Pentachlorophenol  29  4.2 Alkyl Ammonium Compounds (Quaternary Ammonium Compounds)  32  4.3 Component 2 (C2)  37  Chapter 5: Development of a Novel Antisapstain Formulation  40  Chapter 6: Objectives of the Research  44  Chapter 7: Methods  48  iv  7.1 Microbiological Screening Tests on Agar Plates  48  7.2 Microbiological Screening Test Using Agar Block Tests  52  7.3 Field Efficacy Test by Using Pilot-Plant Linear Spraying System (Phase 5)  57  7.3.1 Test Parameters and Test Evaluation  61  Chapter 8: Results and Conclusions  63  8.1 Results of Phase 1  63  8.1.1 Microbiological Testing (Bioassay) of Potential Components  63  8.1.2 Conclusions and Comments  71  8.2 Results of Phase 2 8.2.1 D D A C Efficacy  74 ,  8.2.2 C2 Efficacy  77  8.2.3 Resistance to D D A C  78  8.2.4 Synergism in Mixtures of D D A C and C2  80  8.2.5 Conclusions and Comments  84  8.3 Results of Phase 3 8.3.1  Microbiological Screening Test Using Agar Blocks  8.3.2 Conclusions and Comments 8.4 Results of Phase 4  8.5  75  85 86 96 96  8.4.1  Shelf Life  96  8.4.2  Results of Microbiological Screening Test on Wood Samples  98  8.4.3 Definition of DDAC/C2 ratio  101  8.4.4 Results of Toxicity Experiments  104  Results of Phase 5  105  V  1  8.5.1 Field Efficacy Test by Using Pilot-Plant Linear Spraying System  105  8.5.2 Comments and Conclusions  113  Chapter 9: Economic Aspects 9.1 Product Cost Chapter 10: General Cconclusions and Recomendations for Future Work  116 116 119  10.1 Conclusions  119  10.2 Recommendations for Future Work  120  Literature  121  Appendix 1: List of Fungal Cultures  128  Appendix 2: Bardac 2250 & Bardac 2280 Manufacturer Specifications  130  vi  List of Tables  Table 1. Canada Lumber Production - Softwood and Hardwood  5  Table 2. US Softwood Lumber Exports by Volume (1,000 m ) - October 1999  7  3  Table 3. Effectiveness of C2 Against Various Microorganisms  38  Table 4. Summary of Test Parameters  61  Table 5. The growth of Aspergillus niger (I), Gliocladium roseum (2), Aureobasidium pululans (3) ,and Ceratocistis (4), in the agar media containing selected concentrations of C2  71  Table 6. D D A C Efficacy Against Moulds, Stains, Soft, White and Brown Rot  76  Table 7. C2 Efficacy Against Moulds, Stains , Soft, White and Brown Rot  77  Table 8. The Growth Rate of Coniphoraputeana on the Media Containing Selected Combinations of D D A C and C2  80  Table 9-1. Synergistic antx-Aspergilus niger activity of combinations of D D A C (A)  81  Table 9-2. Synergistic anXx-Gliocladium roseum activity of various combinations of DDAC(A) and C2 (B)  81  Table 9-3. Synergistic anti-Aurobasidium pululants activity of various combinations of D D A C (A) and C2 (B)  82  Table 9-4. Synergistic anXx-Ceratocistis activity of various combinations of D D A C (A) andC2(B)  82  Table 9-5. Synergistic anXx-Chaetomium activity of various combinations of D D A C (A) and C2 (B)  83  Table 9-6. Synergistic arxXx-Versicolor activity of various combinations of D D A C (A)  Vll  andC2(B)  83  Table 9-7. Synergistic anti-Coniphora puteana activity of various combinations of D D A C (A) and C2 (B) Table 10. Microorganisms used in the experiments  84 86  Table 11.1. Fungal Growth on Wood Samples Treated with 10 Times Diluted Preservative  88  Table 11.2. Fungal Growth on Wood Samples Treated with 15 Times Diluted Preservative  88  Table 11.3 Fungal Growth on Wood Samples Treated with 20 Times Diluted Preservative  88  Table 11.4 Fungal Growth on Wood Samples Treated with 40 Times Diluted Preservative  88  Table 11.5. Fungal Growth on Wood Samples Treated with 80 Times Diluted Preservative  89  Table 12.1. Fungal growth on Wood Samples Treated with 10 Times Diluted Preservative  90  Table 12.2. Fungal growth on Wood Samples Treated with 15 Times Diluted Preservative  90  Table 12.3. Fungal growth on Wood Samples Treated with 20 Times Diluted Preservative  91  Table 12.4. Fungal growth on Wood Samples Treated with 40 Times Diluted Preservative  91  Table 12.5. Fungal growth on Wood Samples Treated with 80 Times Diluted Preservative  92  viii  Table 13.1. Fungal Growth on Wood Samples Treated with 20 Times Diluted Preservative  93  Table 13.2. Fungal Growth on Wood Samples Treated with 40 Times Diluted Preservative  93  Table 13.3. Fungal Growth on Wood Samples Treated with 80 Times Diluted Preservative  94  Table 13.4. Fungal Growth on Wood Samples Treated with 160 Times Diluted Preservative  94  Table 13.5. Fungal Growth on Wood Samples Treated with 320 Times Diluted Preservative  95  Table 13.6. Fungal Growth on Wood Samples Treated with 640 Times Diluted Preservative  95  Table 14. Efficacy Determination of Preservatives Prepared in Different Ranges of Elapsed Time  97  Table 15.1. Fungal Growth on Wood Samples Treated with 20 Times Diluted Preservative  98  Table 15.2. Fungal Growth on Wood Samples Treated with 40 Times Diluted Preservative  99  Table 15.3. Fungal Growth on Wood Samples Treated with 80 Times Diluted Preservative  99  Table 15.4. Fungal Growth on Wood Samples Treated with 160 Times Diluted Preservative  100  Table 15.5. Fungal Growth on Wood Samples Treated with 320 Times Diluted  ix  Preservative  100  Table 15.6. Fungal Growth on Wood Samples Treated with 640 Times Diluted Preservative  101  Table 16. Titration of D D A C solution (70g Bardac 2280 and 30g water), with 0.007% water solution of C2  102  Table 17. Titration of D D A C solution (30g Bardac 2280 and 70g water), with 0.007% water solution of C2  102  Table 18. Titration of D D A C solution (20g Bardac 2280 and 80g water, with 0.007% water solution of C2)  103  Table 19. Fish Toxicity Test (Rainbow Trout); BCRI Sample #20001089  104  Table 20. Daphnia magna Toxicity Test; BCRI Sample #20001089  105  Table 21. Fungal Growth on Untreated Rough Douglas Fir Samples  107  Table 22. Fungal Growth on Untreated Rough Western Hemlock Samples  107  Table 23. Fungal Growth on Planed Western Hemlock Samples, Treated with 70 ug/cm 108 Table 24. Fungal Growth on Planed Douglas Fir Samples, Treated with 44 ug/cm  108  Table 25. Fungal Growth on Planed Western Hemlock Samples, Treated with 86 ug/cm 109 Table 26. Fungal Growth on Planed Douglas Fir Samples, Treated with 54 ug/cm  109  Table 27. Fungal Growth on Rough Western Samples, Treated with 130 ug/cm  110  2  Table 28. Fungal Growth on Rough Douglas Fir Samples, Treated with 127 ug/cm ..110 2  Table 29. Fungal Growth on Planed Western Hemlock Samples, Treated with 118 ug/cm  2  111  x  Table 30. Fungal Growth on Planed Douglas Fir Samples, Treated with 131 ug/cm ..111 2  Table 31. Fungal Growth on Rough Western Hemlock Samples, Treated with 154 ug/cm  112  2  Table 32. Fungal Growth of Rough Douglas Fir Samples, Treated with 177 ug/cm ... 112 2  Table 33. The Relation Between Preservative Concentration and Achieved Retention For Douglas Fir and Western Hemlock  113  Table 34. Chemical Cost Comparison (per kilogram) of the New Developed Product (NDP) and the Current Major Product (CMP)  117  Table 35. Chemical Cost Comparison (per liter) of NDP {Specific Gravity: 0.96)  117  Table 36. Cost Comparison (per amount of active ingredient) of the New Developed Product (NDP) and the Current Major Product (CMP), per 1,000 F B M  118  Table 37. Toxicity comparison of the New Developed Product (NDP) and the Current Major Product (CMP)  118  xi  List of Figures  Figure 1. World Exports of Softwood Lumber: 1996  5  Figure 2. United States Annual Lumber Production of Softwood and Hardwood, from 1900 to 1998, (in 1,000,000 m ): 3  6  Figure 3. United States Annual Lumber Production of Softwood and Hardwood, from 1990 to 1998, (in 1,000,000 m ): 3  Figure 4. Examples of stain and mould infected lumbers  6 9  Figure 5. Fresh Cut Lumber (Typical settings at a lumber yard)  10  Figure 6. Anti-sapstain Process Steps for the Treatment of Fresh Sawn Lumber  13  Figure 7(a & b). (a): Typical Spray Box; (b): Lumber Coming out from the Spray Box 14 Figure 7 (c). Percentage Use of the Various Application Systems  15  Figure 8. Comparison of Fish Toxicity of Each Active Component  42  Figure 9. Steps in Inoculation Technique:  49  Figure 10. Arrangement of Wood Specimens on Agar Strip Within Petri Dishes  54  Figure 11. Arrangement of Treated Wood Samples Within the Petri Dishes  55  Figure 12. Chemical Feed System  58  Figure 13. Forintek's Linear Spray Process System:  59  Figure 14. Surface Sampling  60  Figure 15. The Growth of G. roseum on media containing only D D A C  64  Figure 16. The Growth of A. niger on media containing only D D A C  65  Figure 17. The Growth of Aurobasidium p. on media containing only D D A C  65  Figure 18. The Growth of Ceratocistis on media containing only D D A C  66  xii  Figure 19. The Growth of A. niger on media containing only C2 Figure 20. The Growth of G. roseum media containing only C2  66 67  Figure 21. The Growth of Aurobasidim p. on media containing only C2  67  Figure 22. The Growth of Ceratocistis on media containing only C2  68  Figure 23. The Growth of A. niger on media containing only Component 3  68  Figure 24. The Growth of G. roseum media containing on Component 3  69  Figure 25. The Growth of Aurobasidium p. on media containing only Component 3  69  Figure 26. The Growth of Ceratocistis on media containing only Component 3  70  Figure 27. Fungal Growth on Control Plates  75  Figure 28. Coniphora Puteana growth on the agar media containing various concentrations of D D A C Figure 29. Rough Wood Samples Ready for Spraying  78 106  Abbreviations  AACs  Alkylammonium compounds  CCA  Chromium Cooper Arsenate  CRC  Carcinogenicity Peer Review Committee  Cu-8  Copper-8- quinolinolate  D. Fir  Douglas Fir  DDAC  Didecyl-dimethyl-ammonium chloride  EPA  Environmental Protection Agency  H.Fir  Western Hemlock or Hem Fir  HPLC  High Pressure Liquid Chromatography  IPBC  3-iodo-3-propanyl butyl carbamate  NP-1  Commercial Antisapstain Product  PCDD  Polychlorinated-p-dioxins  PCDF  Polychlorinated dibenzofurans,  PCP Number Pest Control Products Registration Number PCPs  Polychlorophenols  PMRA  Pest Management Regulatory Agency (PMRA) of Health Canada  S.I.  Synergy Index  TBTO  Tributyltin oxide  TCMTB  2-thiocianomethylthio benzothiazole  TCP  Tetra Chlorophenol  Acknowledgement  I would like to express my true appreciation to Dr. Richard Branion for his critical evaluation, invaluable guidance, and support throughout my research.  I would like to acknowledge the National Research Council of Canada for its help in financing this study. As well, I would like to thank Forintek Canada Corp. and BCRI, from Vancouver, for the use of their facilities for product testing.  I would also like to mention the role of Enviro-Quest Technologies Inc., and its staff in the accomplishment of this study.  Above all I am grateful to my husband Stevo and my daughters Senka and Sanja for their help, encouragement, and infinite patience which made the completion of this work possible.  XV  Chapter 1: Introduction Wood is well known to be one of the most useful raw materials for a large diversity of applications. It is a cellular, fibrous biopolymer, made up of cellulose, hemicellulose, and lignin that is stable and maintains its main characteristics over long time duration. Because of its unique properties, availability in large quantities, its renewability, and its adaptability to modification treatments, wood is a material of ever increasing interest. Today the national and international markets for high quality lumber are greater than ever. Meeting the demand for quality lumber requires attention to a number of key factors. One of these is the need to deliver lumber with the clean, clear appearance of freshly cut timber. In fact, marketplace demand is so strong and the competition so intense for this attractive lumber product, that even one shipment of discolored lumber can result in a major loss of sales.  During years of development, ways have been found to make wood more dimensionally stable, harder and stronger to meet specific needs. However, being of organic origin, wood components are readily degraded by microorganisms, insects, termites, and marine animals that attack wood for food, shelter, or both. Perhaps the most serious threat to wood's high quality appearance is sapstain discoloration caused by moulds and fungi. Within a day of being cut, moulds and fungi that produce a mottled black discoloration can threaten the appearance of quality lumber.  Between cutting and delivery, these fast growing moulds and fungi often have both the time and the necessary environmental conditions to develop to disastrous proportions. Sapstain fungi feed and grow under moist, humid conditions. They can transform high quality lumber into spoiled, discolored shipments before they reach their destination. High demand fir and spruce are among the most susceptible species to such sapstain attack, especially when being transported through tropical conditions such as enroute to overseas markets via the Panama Canal, or during the lengthy Trans-Pacific ocean delivery process.  The history of sapstain protection via application of anti-sapstain chemicals is reviewed below. For many years sapstain was effectively controlled by application of polychlorophenols (PCPs). However the use of these has been abandoned because of their adverse environmental and health risks. This abandonment instigated a search for new, less risky anti-sapstain formulations. None of these new products has been able to meet all of the requirements for being completely safe and effective. Each has had its own specific problems. Some, to varying degrees, are toxic to fish or to humans. Others have failed to adequately protect the wood to which they were applied and some have even resulted in premature deterioration and downgrading of the lumber they were developed to protect. This thesis documents a research and development project aimed at discovering and proving the effectiveness of such a new anti-sapstain formulation.  2  1.1 The Lumber Industry and its Markets  The lumber industry includes the various businesses that convert trees, or timber, into lumber products. Lumber is produced from both hardwood and softwood. Wood from broad-leaved trees is called hardwood, and wood from cone-bearing trees is called softwood, regardless of its actual hardness. Many softwoods are actually harder than some of the so-called hardwoods. Most of the lumber harvested in the United States and Canada is softwood, including such species as southern yellow pine, Douglas fir, ponderosa pine, western red cedar, and the true firs. Most hardwood lumber is cut from species such as oak, gum, yellow poplar, maple, and ash, and is used for miscellaneous industrial applications including wood pallets.  The  lumber industry is of significant economic importance. It is heavily  dependent upon the health of the residential construction and household furniture industries. The sawn lumber softwood markets in 1998 and 1999 directly followed economic developments in North America, Europe, the former USSR and Asia. In North America consumption rose strongly and production reached record levels in 1998, but world trade was also active with a notable exception of a sharp decrease in export to Asia. European exports were also constrained by the Asian economic crisis but as European consumption remained high, exports rose (UN-ECE/FAO 1998/99).  According to the COFI Fact Book (1998), in 1996 the total world roundwood (including softwood and hardwood) harvest totaled 3.2 billion cubic meters. Industrial  3  production of hardwood and softwood,  in 1996, totaled 1.5 billion cubic meters,  accounting for 46.9 percent of the world total.  The Canadian lumber industry is based on an extensive, renewable resource. Canada's annual softwood timber harvest represents 14.5 per cent of the world total and ranks third after the United States and Europe. In 1996 British Columbia's portion of the world's softwood timber harvest was 6.7 %.  Canada is the world's largest exporter of softwood lumber. In 1996 Canada accounted for 51.7 per cent of the world's total export volume of 95.9 million cubic meters, see Figure 1. British Columbia is the largest contributor (57.6%) to Canada's softwood lumber exports representing 30% of the world total. As shown in Table 1, British Columbia plays a major role in Canadian softwood and hardwood production. Table 1 presents Canadian lumber production, in million board feet, of softwood and hardwood from 1988 until 1997:  Most of the United States' wood product industry is concentrated in the Pacific Northwest and the Southeast. Approximately one-third of the U.S. is forested. Of this forested area, two-thirds contain at least 20 cubic feet of commercially usable wood per year per acre. In 1998, United States production of softwood lumber rose to an eight-year high of 120 million cubic meters, while exports declined, primarily due to the Asian economic crisis. See Figures 2 and 3.  4  Former U S S R  o  t  h  e  r  Canada  51.7%  28.4% Figure 1. World Exports of Softwood Lumber: 1996 (Total for 1996: 95.9 million cubic meters)  Table 1. Canada Lumber Production - Softwood and Hardwood Canada Lumber Production Softwood and Hardwood 1988-1997 (Million Board Feet) Year  British Columbia Coast  Alberta Sask. & Ontario Quebec New  Interior Total  Nova  Total  Total  Total  1988 4583  10989  15572 1584  ManiToba 333  1989 4140  11094  15234 1634  369  2083  4336  643  213  24512 523  25035  1990 3798  10400  14198 1618  321  1817  3879  621  218  22673 442  23115  1991 3465  9843  13308 1750  288  1719  3633  572  191  21461 413  21874  1992 3516  10625  14141 1815  273  1851  4197  669  210  23156 470  23626  1993 3585  10796  14381 1851  333  2117  5023  815  249  24769 469  25238  1994 3681  10588  14269 2041  350  2297  5741  937  312  25947 500  26447  1995 3313  10506  13819 2325  375  2367  5842  1040  325  26093 425  26518  1996 3387  10458  13845 2392  384  2363  6323  907  374  26587 437  27024  1997 3032  10344  13376 2553  389  2549  6603  1200  423  27092 354  27446  2266  4470  Brunsw. Scotia Soft- Hard- Canada wood wood 727 215 25167 574 25741  5  1900  1910  1920  1930  1940  1950  1960 1970  1980  1990 1998  Year g Softwood • Hardwood  Figure 2. United States Annual Lumber Production of Softwood and Hardwood, from 1900 to 1998, (in 1,000,000 m ): 3  1990  1991  1992  1993  1994  1995  1996  1997  1998  Year • Softwood • Hardwood  Figure 3. United States Annual Lumber Production of Softwood and Hardwood, from 1990 to 1998, (in 1,000,000 m ): 3  According to FAO's, Forest Product Annual Market Review, 1998-1999, in the first four months of 1999 North American exports to Asia were recovering. For example, United States exports to the Pacific Rim moved up 5.1% in the first quarter of 1999 compared to 1998. The greatest increase appears with China, up to 19%. Table 2 shows U.S. softwood export by destination as it was in October 1999.  Table 2. US Softwood Lumber Exports by Volume (1,000 m ) - October 1999 3  Softwood Exports  Year to  Last Year  Percentage  By Destination  Date  to Date  Change  Western Hemisphere Europe Asia Oceania Africa  Total Softwood Exports  2,969  2,373  25.1 %  866  822  5.5 %  1,461  1,397  4.6 %  161  165  -2.5 %  12  10  14.7%  5,469  4,766  14.17%  The data in this section indicate the huge magnitude of the amount of lumber produced. Much of this needs to be protected against sapstain and other microbially induced deterioration. Thus there is a large market for protective chemicals.  1.2 The Wood Preservation Industry  Wood preservation extends the service life of timber products by providing effective long-term resistance to attack by fungi, bacteria, insects, and marine borers.  Furthermore, it reduces the need for harvest of already stressed forestry resources, reduces operating costs in industries such as utilities, railroads and construction, and promotes safe working conditions where timbers are used as support structures.  The successful preservation of wood by chemicals was first achieved on a commercial scale about a hundred twenty years ago, and since that time increasingly large quantities of timber have been treated with various wood preservatives (Cartwright etal., 1958).  There are two general classes of wood preservatives: oil and waterborne salts. The effectiveness of the preservatives varies greatly and can depend not only on their composition, but also upon the quantity applied to the wood, the depth of penetration, and the conditions to which the treated material is exposed in service. A great variety of substances have been suggested for use as wood preservatives. However, there can be no universally ideal preservative for all classes of timber, since the timbers to be treated and purposes for which treated wood is required, vary.  Through the sawmilling process, logs are cut into various forms of dimensioned lumber.  Freshly cut green lumber, high in moisture content, sugars and starch levels  provides an ideal environment for infestation by moulds, blue staining fungi, and/or wood destroying fungi. In industry terms, the sapwood of freshly cut, green lumber of most wood species is subject to fungal discoloration commonly known as "sapstain".  While these sapstain organisms usually do not cause considerable strength loss, they can sometimes reduce wood's structural value. Stained wood is generally not  8  recommended for structural purposes where strength is critical. In addition, conditions favorable for stain development are also conducive to decay initiation. Stained wood may also provide an ideal inoculum source for paint-disfiguring fungi (Cartwright 1958).  The individual hyphae of sapstain causing fungi are usually brown in color, however diffraction of light, to which they are exposed, results in an appearance often referred as "blue staining". Staining of this nature results in significant losses. Figure 4 illustrates some sapstained lumber; Figure 5 shows typical outdoor storage of fresh cut lumber.  Figure 4. Examples of Stain and Mould Infected Lumbers  9  Figure 5. Fresh Cut Lumber (Typical settings at a lumber yard)  Sapstain organisms also substantially reduce the lumber's esthetic value and therefore its commercial value. The natural, clean unspoiled beauty of fresh sawn lumber is one of the biggest attributes in maximizing its commercial value and price. As a result, heavy emphasis has been placed on control of sapstain in freshly sawn lumber at the mill  Sawmills perform surface protection operations to protect the lumber against sapstaining that could occur during temporary storage and transit. All green lumber destined for export is protected. There are two principal fungal control processes that have been used in the North American sawmill industry to treat lumber for the control of the fungal sapstain growth. These two processes are drying processes and chemical treatments as described in the following:  10  Kiln and Air Drying - Since high humidity is a favorable condition for fungal growth, protection against it can be by drying the lumber to a moisture content of less than 20%. Kiln drying is utilized in some areas and air-drying in others, but these processes are not always practical or economical and can still result in some staining. Heavy rainfall during staging or high humidity conditions in the hold of a ship during transport may still cause kiln-dried lumber to become susceptible to stain and mould growth.  Additionally,  the  cost  differential  between kiln  drying  lumber  ($50-  100.00/MFBM) and the use of the anti-sapstain formulations ($4-8.00/MFBM) strongly supports  chemical  anti-sapstain  protection as  the  prevalent  method  of  choice  (Kanashevich, 1994).  Chemical Treatment - The most effective and economical way of protecting freshly sawn lumber against mold and fungus decay and discoloration is by the immediate treatment upon sawing with an effective anti-sapstain chemical applied to the wood's surface. Furthermore, for extra protection even kiln-dried lumber is sometimes treated with a chemical anti-sapstain treatment prior to shipping to foreign markets, especially if shipped under high humidity conditions in the hold of a ship.  1.3 Governmental Regulations and Registrations All anti-sapstain pesticide products used for commercial purposes in both the United States and Canada must be registered with and by the appropriate governmental pesticide control agency in that country and be issued a Pest Control Products registration number (PCP Number). Each country has its own unique registration process and  11  operating regulations with which each applicant product must comply. A brief outline is as follows:  1. United States Procedure - In the United States all anti-sapstain products must be registered under the Environmental Protection Agency (EPA). The time frame for product acceptance and registration approval varies from three to nine months.  2.  Canadian Procedure -  In Canada all anti-sapstain products must be  registered with the Pest Management Regulatory Agency (PMRA) of Health Canada. In Canada the registration process usually results in a significant cost. Application fees reflect the amount of work to be performed and the actual cost to conduct the examination that an application entails, and can run into the high tens of thousands of dollars. The minimum time frame for product acceptance and registration approval is one year.  12  1.4 Application Methods for Chemical Protection against Sapstain A general description of anti-sapstain chemical application is illustrated in Figure 6.  Chemical Storage Feed Make-Up  GREEN SAWN LUMBER  Transfer  J-K  VI  AntiSapstain Application (Dipping or Spraying)  Treated Lumber Storage  Figure 6. Anti-sapstain Process Steps for the Treatment of Fresh Sawn Lumber  According to information from Konashewich, (1994) the types and relative percentages of specific application systems that are in use in North America are shown in Figure 7 (c). •  Most of the sawmills treat lumber by using Lineal Spray and Cross Chain spray systems for the application of anti-sapstain chemicals.  13  Variations in equipment types can be found in existing facilities, and are functions of:  •  Site requirements  •  Date of installation  •  Recommendation of the chemical supplier, and  •  Regulatory requirements  14  •  Linear Spray Systems  • Automatic Elevator Dip Tanks  • C r o s s Chain Spray Systems • Forklift-Mechanical Dip Tanks  • Sorting Chain Dip Tanks  Figure 7 (c). Percentage Use of the Various Application Systems  Generally in spray systems, the lumber is moved continuously through a spray chamber or tunnel by a chain, roller, or conveyor belt system. Two types of spray box arrangements are commonly used: linear boxes where the direction of travel is parallel to the longest dimension of the piece of lumber, and the cross chains systems where the direction of travel is perpendicular to the longest dimension.  Spray systems have also been categorized according to the range of pressure applied to the spray nozzles: •  High - pressure systems (72 to 290 psi)  •  Low -pressure systems (22 to 58 psi)  15  Chapter 2: Sapstain and Wood Rot 2.1 Factors That Cause Staining and Discoloration of Timber  When an abnormal discoloration appears on a timber, it could be due to conditions of growth, contact with chemicals or the action of microorganisms. Many timbers exhibit a wide natural variation in color, but abnormal discoloration is usually due to one of the following four main causes:  1. Oxidation or chemical changes of cell contents The cell sap of most plants contains oxidizing enzymes, and their action in the presence of oxygen, gives rise to colored compounds which discolor the wood either superficially or to some depth.  2. Contact with chemicals, especially iron The most important of chemical stains is that caused by contact of timbers containing tannin with iron or salts of iron. This results in the formation of iron tannate, a blue-black compound, which is the principal constituent of ordinary ink. Woods that are naturally rich in tannins (oak and chestnut) are especially liable to develop ink stains when brought into contact with iron or iron rust while in moist condition. Many other timbers, including some softwood species, such as Douglas fir, contain sufficient tannin to react that way  3. Incipient decay caused by a wood-rotting fungus Under favorable environmental conditions many fungi can cause the decay of wood. In most conditions, the white rot and brown rot fungi, which belong to the group of Basydiomicetes, are the most important destroyers of the wood. These  16  fungi produce different types of enzymes, which can cause the decomposition of woody substances. White rot describes a situation in which the lignin component of the wood is attacked by the fungi, leaving the white cellulose. Brown rot occurs when the fungi consume cellulose, leaving behind the brown coloured lignin. The fungal deterioration of wood in contact with water on the ground is mainly due to the attack of soft rot fungi. Their hyphae grow inside the cell wall, forming long tunnels by consuming cellulose. If the timber is moist, its surface becomes soft. These fungi belong to Ascomycetes. 4. Growth on the surface of or throughout, the timber of sap-staining fungi or moulds. Moulds are able to grow mostly on freshly sawn timber, producing masses of colored hyphae and spores. The pigmented hyphae of blue stain fungi can penetrate into sap wood, consuming the carbohydrates located there. As a result of the discoloration, the quality of the timber is diminished. They do not attack the wood so as to affect its strength, but there is a loss of quality as a result of the discoloration. The spores also may be harmful to health. Such moulds belong either to Ascomycetes or to Deuteromycetes.  While there are many individual differences in the physiological requirements of the various species of fungi that cause staining, they react, on the whole, in a similar way to the following involved factors: •  Temperature Staining fungi develop much more rapidly under warm rather than under cool conditions. However, the optimum temperature for the growth of most sapstain  17  species lies somewhat lower than those of the majority of wood-rotting fungi. The optimum and maximum temperatures for growth vary with species. The temperature relations could be classified into three main groups: 1. Low temperature group growing best at 24° C or below. 2. Intermediate temperature group growing best between 24° C and 32° C. 3. High temperature group growing best above 32° C. Moisture and Oxygen Requirements These two factors should be considered together,  since they are closely  interrelated. Wood destroying microorganisms, like other organisms obtain their energy by a process of respiration. Complex organic substances are broken down and energy is thereby released. The process can be illustrated by considering the oxidation of a cellulose unit: C H i O + 6 0 = 5H 0+ 6 C 0 6  0  5  2  2  2  Oxygen may be taken from the atmosphere (aerobic respiration) or energy obtained by intermolecular changes (anaerobic respiration). Wood destroying microorganisms are essentially aerobic organisms, but it is probable that a certain amount of growth can take place under relatively anaerobic conditions in which case the end products are organic substances such as alcohol, oxalic acid, etc., instead of carbon dioxide. Considerable quantities of water are produced by the respiration of certain fungi when in active growth. That means when an outbreak of rot is established, the fungus can continue to develop and spread independently of any external source of moisture. Wood destroying fungi can tolerate a very low partial pressure of oxygen. The optimum moisture content for the growth of any  18  particular fungus cannot be exactly defined, without reference to the particular kind of timber. The minimum moisture content for the growth of most blue-stain fungi is about 25-28 per cent. Some moulds can develop at a lower moisture content than the blue stain fungi. For example Aspergillus glaucus, can grow in wood containing only 19-20 percent moisture. For practical purposes, it can be taken that the minimum moisture in wood for the growth of most wood - rotting fungi is somewhere in the region of 22-24 per cent and that timber containing less than 20 percent moisture is safe from attack by any of the common wood-rotting Basidiomicetes rfCartwright, 1958/ Nutritional Requirements Wood destroying microorganisms can derive energy from a wide range of polysaccharides. Glucose, which is formed in the largest amount when cellulose is broken down, is probably the sugar that is most favorable for their growth. They can also utilize maltose, sucrose and many other sugars, but there has been a report that they cannot utilize lactose (La Fuze, 1937). Starch is readily attacked, as are many of the hemicellulose compounds. Pentoses are also capable of acting as sources of energy, but in general are less favorable media for growth than hexoses. It has also been found that the addition of nitrogen and adequate amounts of minerals, stimulate the decomposition of wood by fungi. The nutritive substances on which the fungi feed occur only in the sapwood.  19  Types of Wood Attacked The specificity of many microorganisms in relation to the hosts on which they grow, can be explained because they find, in the tissue of the particular host, special conditions, which are necessary for their development.  20  Chapter 3: Anti-Sapstain Protection The use of anti-sapstain chemicals has been an integral component of lumber protection in Canada and other lumber producing nations since before the 1930's. The use of anti-sapstain chemicals to control sapstain and mould on lumber was adopted by the forest industry's essential need to satisfy markets that placed a high premium on quality lumber. Lumber producers aim to deliver lumber, which is clear of sapstain, moulds and discoloration, because the presence of any defect can essentially decrease the value of the lumber. The efficacy of anti-sapstain chemicals depends upon their direct toxicity to sapstain and mould fungi and their ability to create an unfriendly environment, which can discourage fungal growth. A n important characteristic of these products is that they, to a degree, could also hinder the growth of wood decay fungi.  The amount of anti-sapstain chemicals used to protect lumber is difficult to determine, but within Canada, it has been calculated that the amount is about 1,100 tons per year.  Worldwide, the amount is more than 5,000 tons per year (Byrne, 1990,  Konashewich, 1994).  British Columbia's annual consumption of anti-sapstains (in terms of active ingredients) in 1987 was estimated at 300,000 to 400,000 kg per year. By 1996, the number of anti-sapstain application facilities had decreased from 100 to 51. However, consumption of fungicide had increased to approximately 846,000 kg, The lower toxicity of currently used anti-sapstains compared to PCPs has resulted in the average antisapstain  average antisapstain facility now applying four times the quantity of active ingredient as they did in 1987 (Krahn, 2000).  Prior to the 1930's, the most common chemicals used for lumber protection were sodium carbonate and bicarbonate solutions. Since the 1930's and until 1987, the protection of lumber was heavily dependent on polychlorophenols  (PCP's). They  provided excellent treatment and protection against sapstains and moulds as exemplified by their acceptance and use for over 50 years. In British Columbia alone more than 700 tons of polychlorophenols per year were used to treat lumber. PCP's were applied to lumber by either dipping lumber in tanks or spraying. Both application techniques produced a thick, toxic, waste sludge that created major disposal problems. PCPs came under increasing scrutiny because of their environmental and health side effects. Environment Canada (Konashevich, 1994) reported that levels of dioxin were abnormally high in workers handling PCP treated lumber. The environmental and health side effects of PCP's, resulted in their ultimate abandonment in the Canadian forest industry. The shift from PCP's to relatively new chemicals has been rapid. The following is a historical review of anti-sapstain agents used in the Fraser River Basin prior to the latest generation of anti-sapstain chemicals (Krahn, 2000: Konashevich, 1994):  •  PCP and T C P (chlorophenols) The use of chlorophenols as anti-sapstains was banned on December 31, 1990. The regulated limits in stormwater runoff are 6 parts per billion (ppb). These compounds are no longer in use in any mills in BC.  22  •  T C M T B (2-thiocianomethylthio benzothiazole) T C M T B is an active ingredient used in commercial formulations Busan 1030 and Busan 30 WB. This chemical was initially used in a significant percentage of B C mills as a replacement for chlorophenols. However, problems with fish toxicity and in worker handling resulted in a rapid phaseout at most facilities. It is currently in very limited use in BC.  •  Cu-8 (Copper-8- quinolinolate) Cu-8 is the active ingredient used in commercial formulations called Nytec G D and PQ-8.  This chemical was used in several mill trials immediately following the  banning of chlorophenates, however now it has been phased out in favor of less toxic chemicals. •  "Borax-sodium carbonate" formulations "Borax" formulations are used individually or in combination with other active ingredients. These compounds have low fish toxicity but they have proven unsatisfactory for such reasons as poor efficacy in wet climates, and rapid leaching due to their high solubility in water.  Because of still growing pressures from environmental groups and government regulators there has been increasing interest in the development of new systems that would present less potential for adverse environmental impact and would allow for easier disposal of treated products. A number of alternative anti-sapstain products that use quaternary ammonium compounds (AACs) as their active component have been developed to meet these objectives. Recently, extensive research has been reported on the  23  effectiveness of these alkylammonium compounds (AACs) as anti-sapstain wood preservatives (Butcher, 1979: Butcher et al., 1979: Cassens et al. 1982). The results have determined that A A C s  are effective  against several wood-destroying fungi and have  suggested that the most effective of the AACs is didecyl-dimethyl-ammonium chloride (DDAC). The new generation of anti-sapstain chemicals that use D D A C as an active component was introduced in New Zealand in 1978 (Lir, 1978). By mid-1993, 80% of the 53 B . C . lumber operations using anti-sapstain chemicals converted to the use of didecyl-dimethyl-ammonium chloride (DDAC) formulations (Konashevich, 1994).  3.1 Shortcomings of Traditional Chemical Products To improve wood's appearance and commercial value, the lumber industry commonly treats freshly cut green lumber with several active chemical ingredients. The u  efficacy of these products depends upon their direct toxicity to sapstain and mould fungi thus they have an ability to create an unfriendly environment that will discourage fungal growth. Among the traditional wood protecting chemicals, the most commonly used for years were fixing salt types based on inorganic arsenic, chromium, cooper, boron and fluorine, and organics such as creosote, pentachlorophenol (PCP's) and its sodium salt (PCP-Na), tributyltin oxide (TBTO) and other T B T formulations. These components were very effective, however they were also highly toxic to the environment. As a result, they have been taken out of use.  24  There have been fluctuations in the effectiveness of some of the chemicals used to control sapstain over the years when applied on lumber at their recommended levels. After extensive research, wood preservatives based on the use of D D A C as the active ingredient were introduced into commercial use. D D A C has shown a strong performance against a large number of staining and wood destroying fungi. Unfortunately, in commercial use, in products where it was the only active ingredient, a number of protection failures occurred. Various reasons for these failures (see Chapter 4) have been determined such as substandard treatment practices, poor distribution of the preservative (coverage of lumber), misuse in service, depletion of the active ingredient, and attack by D D A C tolerant organisms.  It is to be expected that, like many other living organisms, fungi may be able to develop a resistance to a toxicant. Fungicide-resistance may be defined as a stable, inheritable adjustment by a fungus to a fungicide, resulting in a less than normal sensitivity to that fungicide.  The information mentioned above suggests that problems and possible reasons for the failure of the current commercial fungicides can be ascribed to several possible causes. Despite several investigations into possible reasons, no clear, universally applicable explanations as to why some anti-sapstain and anti-fungal formulations failed to protect, have been obtained (Lyr, 1995).  To be able to avoid the development of fungicide failures in the future, it is important that a high level of efficacy be available, so that flexibility exists in the design  25  of counter measures. Several reports from the literature (Ward, 1988, Hsu, 1990) mention synergistic action between two fungicides. This phenomenon might also appear to have value for the avoidance of resistance if the second fungicide interferes with the fungi's resistance mechanism towards the first fungicide.  3.2 Possible Problem Solution On account of the problems that may occur with the majority of commercial antisapstain products that use D D A C as an active component, the possible solution of the problem appears to be the use of a second active component for improving long-term performance and broadening the spectrum of activity. Generally, the expected response of a mixture of two chemicals is the sum of the effects of the components separately. However there is the possibility that these two chemicals can react synergistically against certain microorganisms which would result in achieving the additional advantages of the mixture which might enhance the efficiency of the individual components. At the same time, the amount of active ingredients would be reduced.  The  industry resolved this situation through the addition of a second active  component into. formulations that had D D A C as the primary active ingredient. This represented a major improvement and broadening of the spectrum of activity. This generation of products captured the market due to their effectiveness. However there still remains a concern regarding the toxicity levels of the secondary active ingredients. As an example, the anti-sapstain product that currently retains the dominant North American  26  market share position with approximately 90% of the market is NP-1; NP-1 uses D D A C as its primary active ingredient and 3-iodo-3-propanyl butyl carbamate (IPBC) as its secondary active component.  However as previously mentioned, there is a degree of  uncertainty about IPBC and its toxicity levels to fish. The challenge has been to find a second active component that, when combined in solution with D D A C , would increase the effectiveness and lower the toxicity level of the product  27  Chapter 4: Fungicides - Literature Review Under favorable environmental conditions, many fungi can cause decay and/or discoloration of wood. In most conditions white rot and brown rot, which belong to the Basidiomycetes, are the most important destroyers of wood. These fungi produce different types of enzymes, which can cause decomposition either of all the wood substances, with the cellulose part remaining (white rot), or vice versa (brown rot) with the lignin remaining. The fungal deterioration of wood in contact with the ground or water is mainly attack by soft rot fungi, whose hyphae form long tunnels by consuming cellulose.  Apart from these wood-destroying fungi, there are two other groups, which can cause discoloration of timber to a greater or lesser degree: blue stain fungi and moulds. The blue stain fungi develop on the surface of timber, and their pigmented hyphae can penetrate into the sapwood, consuming the carbohydrates located there. Moulds are able to grow mostly on freshly sawn timber, producing masses of colored hyphae and spores. They do not attack the wood, but there is a loss of quality as a result of the discoloration.  Many uses of wood, which has no or insufficient resistance to fungal attack, dictate that it must be protected by wood preservatives. This protection may be temporary on freshly sawn timber in order to prevent the growth of moulds and blue stain fungi (anti-sapstain protection), or to prevent the growth of wood-rotting (wood destroying) fungi (Lyr 1995).  28  The successful preservation of wood by chemicals was first accomplished on a commercial scale about a hundred twenty years ago (Cartwright, 1958). Since then hundreds of chemicals in various combinations have been proposed but of all of them, comparatively few have been found to be practicable and effective and at the same time devoid of objectionable properties. There can be no universally ideal preservative for all classes of timber, since the timbers to be treated and the purposes for which treated wood is required, vary (Cartwright 1958). Nevertheless  it's important to underline the  significance of wood degradation caused by sapstaining fungi and moulds. For example, it has been estimated that in New Zealand export losses of radiata pine are connected to an annual loss in revenue of more than 100 million NZ$ (Wakeling 1996)  A wood preservative, to be appropriate for commercial use, should be toxic to wood destroyers, permanent, penetrative, safe to handle and use, harmless to wood and metal, unlimited in availability and economic in cost. Furthermore, for the treatment of building lumber or for other special purposes it may also need to be clean, colorless, odorless, paintable or have certain combinations of these properties.  4.1 Pentachlorophenol  Production of chlorinated phenols in the United States for wood preserving experiments did not begin until about 1930.  The first field tests started with  tetrachlorophenol in 1931. A year later pentachlorophenol was produced in limited quantities. The penta form appeared to have better properties than the tetra form and,  29  since its introduction, has been the principal chlorinated phenol used in wood preservation. In 1965, a total of more than 20 million pounds was reported to be used by the organized wood-preservative industry (Cartwright, 1958)  Pentachlorophenol is a crystalline chemical compound (C6CI5OH), which is sufficiently  soluble, for wood-preserving purposes, in heavy petroleum oils. The  corresponding structural formula is:  OH  CI  CI  CI  The character of solvent used with chlorinated phenols may have some effect on the performance of the preservative. Actually, changing the solvent and also the concentration of the dissolved toxic chemical can vary the preservative value of pentachlorophenol solutions.  Pentachlorophenol (PCP) is of environmental concern because of its immediate toxicity to aquatic organisms (Konashevich, 1994) Associated with PCP preservatives are such  chemical  impurities  as  polychlorinated-p-dioxins  and  polychlorinated  dibenzofurans, which are toxic, accumulate in aquatic organisms and persist in the  30  environment with long-term consequences. The effects of occupational exposure to PCP and its PCDD and PCDF contamination have become very important topics in many recent research investigations. There are papers that discuss potential occupational exposures of workers. For example Hryhorczuk et al. (1998) in their study concluded that occupational exposure to PCP is associated with biochemical abnormalities which may persist years after exposure. Treated wood has been even hypothesized as an important source of PCP in milk and beef (Fries et al., 1999). A survey of reported data relating exposure to PCP and its derivatives in occupational settings has indicated that exposure via the food chain is judged to be the most significant intake route for PCP, PCDD, and PCDF (Eduljee, 1999). Investigation of the adverse neurobehavioral effects of long-term exposure to wood preserving chemicals containing PCP showed that long-term low-dose exposure could be related to subtle alterations of neurobehavioral performance in women and also might cause mutations in living cells, and might damage a developing fetus (Peper 1999). Short-term exposure to elevated levels of pentachlorophenol can lead to poisoning that is rapidly fatal.  Increasing  environmental  concerns  initiated  research  towards  developing  alternatives that were more environmentally acceptable than those once used like pentaclorophenol and chromated copper arsenate.  31  4.2  Alkyl  Ammonium  Compounds  (Quaternary  Ammonium  Compounds) In 1890 Menschutkin reported the first preparation of quaternary ammonium compounds by the treatment of an alkyl halide with a tertiary amine. Following Oertel's publication in 1965, quaternaries were used as wood preservatives (Pernak et al, 1998). Wood preservatives based on alkyl ammonium compounds (AAC) were introduced into commercial use in New Zealand in 1978 (Butcher, 1977 and Drysdale 1978). Due to their generally low toxicity to mammals and fish, as well as for economical reasons, they are extensively used in the wood preserving industry.  There are two basic types of A A C s that are of interest to wood preservation: quaternary ammonium compounds and tertiary amine salts. A l l of these compounds can be solubilized in water, and for most applications aqueous solutions would be used. However, some of the A A C s could be formulated as organic solvent based systems if desired. Alkyl chain length and type of anion are two major factors that appear to effect the efficacy of the A A C s against wood decay fungi (Nicholas and Preston, 1980).  AACs  are synthesized by step-wise alkalization of ammonia to give an  intermediate tertiary amine, which is further reacted to the end products. The hydrophobic carbon chain (s) is (are) derived from either natural fatty acids (tallow, soya, coconut) or synthetically from ethylene to give a wide range of compounds, depending on the type and length of the alkyl chain. Butcher et al. (1977) suggested that  32  didecyldimethyl ammonium chloride was the most effective of the A A C s against Basidiomycotina, being equivalent to chromated-copper-arsenate wood preservatives.  A A C solutions are stable and can be used in presently operating treatment plants without modification of the equipment. Because of the A A C s surfactant characteristics some foaming problems may be experienced, but experience has shown that these are readily corrected by the use of suitable anti-foams (Nicholas and Preston 1980).  Didecyldimethyl ammonium chloride (DDAC) belongs to the group of quaternary ammonium  compounds.  It  is  currently  being  used  as  a  replacement  for  polychlorophenols. Its structural formula is:  cm  +  (CHi) CH,  I  9  N  cm  cf  cm The application of quaternary ammonium compounds has led to a demand by their users  for effective  methods  Association  recommends  standards  Didecyldimethylammonium  Chloride  of control. The American Wood-Preservative A16-93  (Standard for  Determination in  HPLC  Method For  Treated Wood),  A17-93  (Standard for Determination of Quaternary Ammonium Compounds in A C Q Solutions), and A18-93 (Standard for Determination of Quaternary Ammonium Compounds in Wood by 2-Phase Titration).  33  Biotransformation is expected to be the main route of disintegration of D D A C in the environment. Earlier studies had indicated that rapid and complete degradation of D D A C occurred when low concentrations were exposed to mixed bacterial cultures (Resuggan, 1951).  Alkyl ammonium compounds belong to a group of disinfectants having almost the same degree of effectiveness against bacteria, yeasts, moulds, fungi and protozoa. It is very likely that when they are adsorbed onto the cell wall, they form un-ionized complexes, which are responsible for the death of the cell, however, there are also many things as yet undecided about their exact mode of action (Resuggan, 1951).  Rosen (1975) outlined a mechanism for improved preservative efficacy on wood substrates by variation of the treating solution's acidity. At low levels of acid addition to the treating solutions, the added protons competed with alkylammonium cations for fixation sites. Thus they prevented the preferential adsorption of the alkylammonium cations by the first cell walls contacted during the liquid uptake phase of preservative treatment. This resulted in a uniform distribution of alkylammonium compound throughout the wood and improved effectiveness against Basidiomicetes. As the addition of acid to the treating solution increased, more protons were present to compete for fixation sites. This resulted in a decrease in cation exchange in the cell wall layers adjacent to the cell lumens, and a greater penetration of cell walls as "free" alkylammonium cations were adsorbed on the less readily available fixation sites within the S2 layer. This explanation was supported by biological evidence, for soft-rot control  34  improved in all timbers as the acid levels in the treating solutions increased. At the same time, control of Basidiomicete attack was reduced. These results support the general view that cell wall loading of a toxicant is most efficacious against soft-rot fungi, and lumen surface deposits are best against Basidiomycetes.  The probable effect of adding alkali to treating solutions is the removal of protons from the wood substance, creating negative sites, which would aid adsorption of alkylammonium cations. The result of treating with such solutions was the preferential uptake and fixation of quaternary ammonium cations in peripheral zones of the wood. This led to increased losses of wood substance during fungal exposure as all the test wood blocks were first cross-cut sawn to expose the central (untreated) zones.  Nicholas and Preston (1980) also examined the modification of the effects of alkylammonium compounds on wood protection by the addition of acid or alkali at various levels of acid (HC1) or alkali (NaiCOs). The effect was determined through an increase or decrease in decay after treated wood was incubated with brown rot, white-rot, and soft-rot organisms. Addition of low levels of acid (0.025-0.1%W/W HCL) improved performance against Basidiomycetes, and to a lesser extent soft-rot fungi. Addition of alkali to treating solutions generally led to increased loss of wood substance when treated wood was exposed to fungal attack. Observations during the preliminary work indicated that treatment with acidic solutions resulted in an even distribution of alkylammonium compounds through the wood, whereas treatment with alkaline solutions resulted in preferential adsorption in the surface layers of wood samples. The importance of this  35  work has been to show how simple modification of treating solutions may influence the distribution of A A C in wood, with consequent increases and decreases in preservative efficacy. Such simple modifications are not possible with C C A preservatives.  While laboratory tests have consistently provided positive results, field testing of unmodified A A C s has failed to confirm their efficacy. Rudick (1986) in his paper considered two possible causes for declining A A C activity. These were leaching of A A C from the wood after application and the influence of non-wood destroying fungi. Since his results eliminated leaching as a primary cause, further study was conducted to determine whether the colonization of the treated wood stakes by staining fungi, could cause a reduction in A A C effectiveness. The results suggested that degradation of the A A C was taking place rather than rupture of the AAC-wood substrate bonding. Lee et al. (1992) suggested that wood cell wall components might react with fungicides under direct contact situations to deactivate their fungicidal properties.  There is also the possibility that uneven protection of different parts of the wood, may result in unsatisfactory fungicidal performance . Since the effect of solution pH on the adsorption of quaternary ammonium compounds (for example DDAC), onto cellulose and lignin may effect uneven protection of different parts of the wood, adsorption was found to be predominantly onto lignin with much lower adsorption onto cellulose. Consequently, D D A C by itself provides little protection against cellulose degradation by brown rot fungi (Jin and Preston, 1991).  36  Didecyldimethylammonum chloride has a proven history as a successful antisapstain chemical, but performance failures mentioned in the research works initiated the idea to broaden its spectrum of activities by adding a second active component.  4.3 Component 2 (C2)  Since the results of this research are the basis for a patent currently pending, the fungicide chosen to be a second active component, in a preservative combination with D D A C , in this study will be called Component 2 (C2).  C2 is widely known as an industrial biocide which is strongly antagonized by exogenous thiol-containing agents and which interacts oxidatively with accessible thiols, such as glutathione, within the cell.  This study examined the possibility of adding C2 in combination with D D A C for wood protection. The C2 used was a 3:1 mixture of two components that belong to the same group of chemicals.  This mixture is non foaming and stays effective over a broad pH range. It is easy to disperse it into formulations in the water phase, where the microorganisms live. Since C2 has lower adsorptivity than anionic or cationic materials, it stays available for use (Manufacturer of C2). When diluted below the concentrations in which they are used in industrial applications, according to a manufacturer study that used radioassay studies to  37  follow the biodegradation of the active ingredient in natural river water, C2 is biodegradable.  The mode of action of C2 biocides is based on a reaction with nucleophilic entities such as enzymes, proteins and amino acids. According to manufacturer's information the C2 used in this study has shown outstanding antimicrobial characteristics in the process of killing prokaryotic (bacteria) and eukaryotic (algae and fungi) microbes.  Table 3 gives the minimum levels of active ingredient that inhibited growth of various microorganisms in test tube cultures. These data demonstrate the broad-spectrum of activity of C2 and are useful tools for screening antimicrobial materials (Manufacturer ofC2).  Table 3. Effectiveness of C2 Against Various Microorganisms Organism Type Active Ingredient (ppm) Aspergillus niger  Fungi  9  Aspergillus  foetidus  Fungi  8  Aspergillus  oryzae  Fungi  5  Chaetomium globosum  Fungi  9  Gliocladium fimbr latum  Fungi  9  Penicillium  Fungi  5  Candida albicans (yeast)  Fungi  5  Lentinus lepideus  Fungi  4  Bacteria Gram-Positive  2  Steptococcus pyogenes  Bacteria Gram-Positive  9  Eschericia  Bacteria Gram-Negative  9  Bacteria Gram-Negative  5  Bacillus  funiculosum  subtilis  coli  Salmonella typhosa  38  Since this study emphasizes the importance of adding a second active component to D D A C it is important to note that the literature reports that the C2 is very active in the control of wood destroying fungi.  The analytical techniques recommended for the chemical analysis of aqueous C2 solutions  are  High  Pressure  Liquid  Chromatography (HPLC)  and Ultraviolet  Spectroscopy (Manufacturer of C2).  39  Chapter 5: Development of a Novel Antisapstain Formulation Antisapstain chemicals are products that are supposed to be toxic to wood destroying microorganisms, but at the same time, they must be environmentally responsible and safe for the people that are in contact with them. A review of literature and consideration of all the known products that are currently registered for the Canadian market gave rise to an idea for improving these products.  Since 90% of the Canadian market uses a combination of D D A C and IPBC, we analyzed the advantages and disadvantages of those formulations and tried to identify possible reasons for customers' dissatisfaction. Even though D D A C already had a proven history in the industry, the possibility of developing resistance by some microorganisms and DDAC's greater affinity for lignin than for cellulose opened the door to considering the possibility of the addition of a different (than IPBC) second active component which could counteract these problems.  On the other hand, the second active component of many major use commercial products, IPBC, does not show favorable fish toxicity characteristics (Konashevich, 1994). There are also some studies that show IPBC's weaknesses against some wood destroying microorganisms. Plackett (1982), claimed that a 1% IPBC based treatment provided protection of hem-fir timber comparable to that of a treatment containing 0.65% PCP. He also showed that a 0.5% level of IPBC was ineffective. On the contrary, the manufacturer of C2 (1989), stated that the efficacy of 0.3% to 0.4% of C2 was superior to  40  that of 4% PCP. It also showed complete protection of Pinus sylvestris panels that were exposed for eighteen (18) months to heavy rainfalls.  One compound that came to mind as a potential addition to D D A C for wood protection was C2. It is less toxic to fish than IPBC; see below.  From a health and safety point of view, D D A C and C2 are both classified in: Group D - Not Classifiable As To Human Carcinogenicity. By contrast, there are certain questions and controversies as to the carcinogenicity of IPBC. The Carcinogenicity Peer Review Committee (CRC) classified (June 16,1993) IPBC as a Group C - "Possible Human Carcinogen". On September 18, 1996, the CRC, at the request of a registrant, concluded  that  the  additional  evidence  provided by  the  registrant  supported  reclassification of IPBC as "not likely" to be carcinogenic. Concern over IPBC however still remains.  Keeping in mind that currently the most used preservative on the market uses D D A C and IPBC as its active components, the toxicological characteristics of C2 are compared with those two chemicals. The charts below compare the concentration of effluent in dilution water for IPBC, and C2 that causes mortality to 50 percent of a Rainbow Trout test population (96 hour LC50). Each of these components is also compared with D D A C , the primary active ingredient used with both IBPC and C2 .(Konashevich, 1994, and Manufacturer of C2)  LC50 (96 hours) Rainbow Trout (ppm) All Active Ingredient Comparison  All Active Ingredient  LC50 (96 hours) Rainbow Trout (ppm) Second Active Component Comparison C  0.2  IPBC  C2  Figure 8. Comparison of Fish Toxicity of Each Active Component  42  LC50 is the concentration of fungicide, in dilution in water that causes mortality of 50 percent of the fish in a test population. The higher the LC50 number is the less toxic the fungicide. Thus the amount of the chemical in the water that it takes to cause mortality in 50 percent of the test population is also higher.  As can be seen from Figure 8, C2 has a toxicological advantage over IPBC.  In the summer of 1995, a questionnaire designed to determine the use pattern of antisapstain chemicals was mailed to all producing members (sawmills) of the Western Woods Products Association. In the questionnaire, the respondents were asked to provide their level of agreement with a series of statements regarding their selection of antisapstain chemicals. The opinions expressed were neither strongly positive nor negative, indicating that users are not totally convinced of the effectiveness of existing treatments. This convinced us that it would be desirable to conduct research toward developing a new anti-sapstain product that would satisfy consumers' desire for: •  efficient control of fungi,  •  low toxicity,  •  less potential for environmental impact (low leachability), and  •  easier disposal of treated products withdrawn from service.  43  Chapter 6: Objectives of the Research A lot of information is required to fully evaluate a chemical as a wood preservative. In general, the basic areas that must be addressed are: •  Chemical properties  •  Biocidal effectiveness  •  Treating solution characteristics  •  Properties of treated wood  •  Toxicological properties  The objectives for this research are to address the above mentioned areas and to provide results adequate to support the development of new anti-sapstains formulations that would meet the objectives of efficient control of fungal growth, low fish toxicity, less potential for environmental impact (low leachability), and easier disposal of treated products (e.g. discarded lumber treated by anti-sapstains) withdrawn from service. The research was separated into several phases:  Phase 1  Preliminary Efficacy Evaluation of Potential Fungicides Against Molds and Stains  The experiments of the first phase were designed to test several potential fungicides against moulds and stains in order to provide results that would eliminate less promising formulations and help focus our research for a new, multi-active agent,  44  chemical  formulation. A  number of  commercial fungicides,  with toxicological  characteristics that would satisfy the safe handling and environmental requirements, were mixed in different ratios with D D A C  and water and submitted to preliminary  microbiological testing. The bioassays  were carried out to estimate the relative  effectiveness of biocides against the moulds and staining fungi and to indicate the possible resistance to such chemicals by these organisms. The organisms were grown on an artificial substrate, agar, and exposed to various potential fungicides.  Phase 2  Efficacy Evaluation of the Most Promising Fungicidal Combinations  In Phase 2, the research was extended to other groups of fungi, such as soft rot, brown rot, white rot and D D A C tolerant species. Again the tests were done on agar plates.  Phase 3  Efficacy Evaluation of the Most Promising Fungicidal Combinations on Wood Substrate  The objective of Phase 3 was to test the most promising formulations discovered in Phase 1 and Phase 2 by using the U.S. Standard for Testing Fungicides for Controlling Sapstain and Mould on Unseasoned Lumber (D445). The results narrowed the search for appropriate chemicals and suggested a range of possible concentration ratios between the  45  active components. They also determined the minimum amount of preservative that would be effective against sapstain fungi and moulds on different kinds of wood (obtained from the different sawmills in British Columbia). In Phase 3 the various microorganisms were grown on wood samples rather than on agar plates.  Phase 4  Determination of product stability and ecotoxicological characteristics  In Phase 4, active components and additives were combined together in larger volumes and product formulation stability was investigated. After ensuring physical and chemical stability, in order to decide whether the product met the levels accepted by Environment Canada, the potential product solution was submitted for toxicological testing to a Vancouver based laboratory BCRI.  Phase 5  Pilot-Plant Spray System Trial (Forintek Laboratory)  Wood protection can only be ensured if the required coverage and retention are achieved on the wood. Prior to going to a mill trial, the product was examined by a simulation of real spraying conditions in the Forintek's wood treatment pilot plant. In Phase 5  commercial samples of lumber were sprayed in the pilot plant with the test product and then stored to see if moulds, fungi or sapstain would develop.  47  Chapter 7: Methods 7.1 Microbiological Screening Tests on Agar Plates All organisms have the potential to grow or increase in mass by cell division, cell enlargement, or both. In simple organisms such as nonmycelial fungi, cell enlargement, accompanied by nuclear division and synthesis of cytoplasm is primarily responsible for growth. Mycelial fungi grow through a combination of cell division and enlargement. The simplest method of assessing fungal growth is by linear measurement. The change in the radius of a developing colony on agar is observed over a period of time.This method is very simple, but is of much value in making rough estimates of growth. It is a nondestructive method, which allows repeated observations of the same mycelium (MooreLandecker, 1982).  The selection of a suitable substratum is very important, since it should simulate fungal growth in nature. A disadvantage of natural media is that they can never precisely duplicated as they are of an unknown composition in each separate case (MooreLandecker, 1982). Alternatively, a completely synthetic medium, such as malt agar that is used in this experiment, can be precisely duplicated.  Use of microorganisms to determine an amount of biocide that is necessary to suppress fungal growth, is called a bioassay. The chosen organisms should be sensitive enough to the presence of the test substance to show a growth response to dosage increases. These would be plotted in linear fashion over some concentration range.  48  Prior to use as inoculum, the fungi were grown on a medium which provided adequate amounts of all factors needed (malt agar). The amount of inoculum used must be carefully standardized to give repeatable results, see Figure 9:  (A)  (B)  Figure 9. Steps in Inoculation Technique: (A) Small piece of colony is removed from Petri dish (B) Small piece of colony transferred to a new dish of agar  In general, myceliar growth may be qualitatively divided into the following (Moore-Landecker, 1982):  Stage 1 (lag phase) with no growth Stage 2 (linear phase) with rapid and approximately linear growth Stage 3 (decline phase) in which there no growth occurs  In order to examine the biocidal toxic effects of a test preservative, solutions of 4% malt agar were prepared and sterilized at 121°C for 20 minutes (at 103 KPa). Once the  49  malt agar cooled to "hand hot" in which state it was still liquid, the previously prepared biocidal solutions were added in to obtain the range of different concentrations. For example: in order to produce a concentration of 500 ppm D D A C in the media, 7.5ml of the 0.4 % stock solution of D D A C was added to 112.5 ml of 4% agar. Ten ml of each solution was transferred into petri plates and allowed to solidify.  Specific fungi from pure culture plates were placed in the center of each plate. Fungal cultures were obtained from Forintek Canada, see Appendix 1. Fungal growth was evaluated by monitoring the diameter of a developing colony on agar over a period of time and then calculating the percent coverage of the agar plate surface by the colony. Three replicas of every fungicide were prepared and measured. The measurements were converted to express the percentage of the plate's surface.  The  above procedure was repeated for the toxicological evaluation of each  concentration of each component or mixture of components. Generally, the expected response of a mixture of two chemicals is the sum of the effects of the components separately. Additional advantages of the mixture could be obtained due to synergistic interactions by which the efficiency of the individual components is increased and at the same time the amount of active ingredients is reduced. In this case, the effectiveness of the mixture cannot be computed from that of the individual ingredients.  To show more graphically any possible synergy in the fungicidal mixtures, the results from the agar-plate test were used to calculate a Synergy Index (S.I.). The S.I. used,  50  shares similarities with the Synergy Index used by Hsu modified  for our particular research,  since  the  (1988).  activities  However, it was  of wood  destroying  microorganisms were being considered.  In order to reflect the long-term effects of a potential preservative, the 22  nd  day of the  experiment was chosen to define a reference point (RP). The minimum biocidal concentration of the potential wood preservative (mixtures of D D A C and ITA) were analyzed using the following equation:  1/2 (MCA'/MCA) + 1/2 (MCB'/MCB) = SI  (1)  1/2 SI + 1/2 SI =SI  (2)  A  B  wherein: (MCA'/MCA) = SI and  (MCB'/MCB) = SI  A  B  (3) (4)  M C A = Concentration of compound A in parts per million, acting alone, which prevents fungal growth at the reference point M C A ' = Concentration of compound A in parts per million, in the mixture, which prevents fungal growth at the reference point M C B - Concentration of compound B in parts per million, acting alone, which prevents fungal growth at the reference point MCB' = Concentration of compound B in parts per million, in the mixture, which prevents fungal growth at the reference point  51  When the sum of ratios 1/2 (MCA'/MCA) = SI and 1/2 (MCB'/MCB) = SI A  B  is  greater than one, antagonism is indicated. When the sum of SIA and SIB is equal to one, compatibility is demonstrated. When the sum of SU and SIR is less than one, synergy is demonstrated. The smaller the sum, the higher the synergistic effect. The synergy index is represented by the formula: S.I.= Log(SI)  (5)  Since in the case of additivity SI=1, than S.I. = Log (SI) = 0. Consequently, SI>1 (antagonism) give positive value for S.I. and values SI<1 (synergism) give negative values for S.I. Synergism and antagonism are phenomena that attract attention from a practical point of view. The directed use of synergists is useful not only because they lower the use-level of biocide, but also because it can be very favorable for increasing the antifungal spectrum, or decreasing the danger of resistance formation in the fungal population. This is especially useful in situations where one compound does not achieve the best results due to weak activity against certain organisms.  7.2 Microbiological Screening Test Using Agar Block Tests  Modified Standard method A S T M D445 was used for determining the minimum concentration of our formulation that was effective in preventing biodeterioration by sapstain fungi and moulds in selected samples of wood. The wood specimens were treated by immersing them in solutions of a fungicide formulation prepared at five concentration levels. The toxicity to fungicides was tested against various spore  52  suspensions. The intensity of surface fungal growth was estimated after incubation and the results were used to determine the chemical treatment concentration that gives zero growth. Apparatus: •  Incubation Cabinet, Maintained at a temperature of 25+/- 1°C and high humidity,  •  Petri dishes and aluminum pans with aluminum foil cover Wood Species:  •  Locally available commercial species selected on the basis of their susceptibility to staining  •  fungi (Douglas fir and Western Hemlock) .  The dimension of the specimens were 7 by 20 mm in cross section and 7 cm long. Culture Media:  •  Malt Agar Substrate consisting of 2% malt extract and 2% agar Preparation of Inoculum:  •  The inoculum was prepared from cultures grown on petri dishes. For the preparation of a spore suspension, 10 ml of sterile water was added to each petri dish. Then the spores were loosened from the malt agar culture using a blunt glass rod. After that they were combined with other similarly collected spores.  Preparation of Test Chambers •  A l l specimens were autoclaved before treatment at 121°C, at 1 MPa for 20 min. then two samples of wood were placed in a petri dish. One of them was treated, the other was just immersed in water and served as a control for comparison. The temperature  53  was set at 25°C. To maintain high humidity during the test period; wet absorbent paper was placed on the bottom of each dish. Inoculation •  Inoculation of the microorganisms and arrangement of test species was performed in two ways:  1. Test microorganisms that were previously grown on a nutrient medium consisting of 2% malt agar were placed on the absorbing paper in the bottom of the petri dish used as the test chamber. Then the two specimens, treated and untreated were placed on the top of them. These two specimens were adjacent, almost attached, to each other (see Figure 10).  Test Wood Specimens Absorbent Paper  Petri Dish  Agar Strip with Microorganisms  •  Figure 10. Arrangement of Wood Specimens on Agar Strip Within Petri Dishes  2. Two adjacent wood samples were placed on the absorbent paper. Then, about 1ml of spore suspension was streaked along the length of one flat side of each sample (see Figure 11).  54  55  Test Evaluation  Spores of fungi germinate more or less readily, according to the particular species involved, when they come into contact with a moist substratum. The spore germinates by bursting the external cell wall and pushing out a germ tube which soon branches to form a mycelium, which is possible to see on the wood surface about 3-4 weeks after germination (Moore-Landecker, 1982).  The intensity of the surface fungal growth was estimated after incubation and the results used to determine the chemical treatment concentration giving zero growth. Evaluations were made visually, using a scale from 0 to 5. Five (5) indicates a sample covered 80-100 % with mycelium, one  (1) indicates a sample covered 10-20% with  mycelium etc. Values between 0.1 and 0.9 indicate that it was not possible to identify mycelium on the wood surface, but it was possible to identify germ tubes by using a microscope.  The estimate was based on observations of the intensity of growth and discoloration.  Data Analysis  In most of the tests reported in this thesis the value of the reported results lies in a comparison of situations in which no growth of fungi occurred compared to situations were growth did occur. Thus if no growth occurred the result was assigned a non-zero value depending on the measure of growth used as described above. No attempt has been  56  made to compare relative amounts of fungal growth when it did occur. The comparisons, as said are in terms of no growth (no, 0) or growth ( yes, positive number).  7.3 Field Efficacy Test by Using Pilot-Plant Linear Spraying System (Phase 5) Objective: To define the efficacy of fungicide depending on different chemical retention. Wood Samples Used for Testing 220 pieces of long (2x4 green, rough-cut untreated ), hem-fir and Douglas fir lumber, were supplied by a local mill. 130 pieces were planed in Forintek to dimensions of: 96 mm X 46 mm, and 90 pieces were used unplaned (rough cut). Wood Species: 110 pieces of Douglas fir and 110 pieces of Western Hemlock were used. By visual estimate, the approximate breakdown of Western Hemlock and amabilis fir in the hem-fir lumber supplied appeared to be about 80% hemlock and 20% amabilis fir Each piece was labeled with a treatment code (Table 5) and numbered 1-20. Antisapstain Chemicals Used in Test -  Concentrate was prepared to have 55% D D A C (Source Bardac 2280) Prior to treatment the concentrate was analyzed for its D D A C content as was a sample of the parent D D A C source Bardac 2280 using an HPLC method. HPLC analysis showed that the actual concentration of D D A C was 49.5% D D A C w/w. The lower than anticipated level of D D A C was attributable to  57  the fact that the Bardac 2280 contained 72.4% D D A C and not the 80% D D A C marked on the label. Samples of each diluted control product were also analyzed to check that they were correct.  Figure 12. Chemical Feed System  The concentrated chemical was diluted to concentrations shown in Table 5, and applied to the wood using Forintek's linear spray system. Figure 12 shows chemical feed passing trough the screen to be filtered and defomed. The feed speed of lumber through the spray was adjusted to a level, which combined with the specific chemical concentration would achieve the target chemical retention. Figure 13 displays steps from the spraying experiment that was done in Forintek.  58  (c)  (d)  Figure 13. Forintek's Linear Spray Process System: (a) Lumber is coming into Linear Spray System; (b) System Inlet; (c) Lumber Coming out of Spray Box; (d) System Outlet  59  Following treatment, f i v e 6.4 cm wood surface samples were taken with a specially 2  designed punch (Figure 14 a) from each species-target retention combination and stored in petri dishes (Figures 14 b and c). These samples were individually extracted with acetonitrile containing an internal standard and the extract was analyzed using the same HPLC analytical method.  (a)  (b)  (c)  Figure 14. Surface Sampling (a) Wood Surface Samples Taken by a Specially Designed Punch (b) Five Samples Taken from the One Lumber Piece (c) Samples Taken from the Four Different Species-Target Groups  60  7.3.1 Test Parameters and Test Evaluation A summary of the test parameters is shown in Table 4. Samples were labeled by a code, from 1 to 5 (A or B), for each species-target retention combination, while control samples were coded as 6 for Douglas fir, and 7 for Hem-fir. From this table it is also obvious that target retention and actual retention sometimes differed from each other. Since this experiment was done for the first time using this spraying system, it was not unexpected that lumber speed and chemical concentration could not exactly predict the resulting retention.  Table 4. Summary of Test Parameters  c o  Wood  Wood  #  Species  Surface  of  D E  7 6 5A 5B 4B 4A 3A 3B 2A 2B IB 1A  Boards  Rough Rough Planed Planed Planed Douglas fir Planed Rough Hem-fir Douglas fir Rough Planed Hem-fir Douglas fir Planed Rough Hem-fir Douglas fir Rough  Hem-fir Douglas fir Hem-fir Douglas fir Hem-fir  9 9 20 20 20 20 20 20 20 20 20 20  Speed (ft/ min)  Spray Solution Target (and Analysed) Concentration ( % D D A C w/w)  Target Retention (Ug/cm )  Actual Retention (ug/cm )  0 0 400 400 240 240 467 467 400 400 400 400  0 (0) 0 (0) . 2.2 (2.17) 2.2 (2.17) 2.2 (2.11) 2.2 (2.11) 5.2 (4.99) 5.2 (4.99) 5.2 (4.77) 5.2 (4.77) 5.9 (5.96) 5.9 (5.96)  0 0 60 60 100 100 120 120 140 140 160 160  0 0 70 44 86 54 130 127 118 131 154 177  2  Mean  2  57 70 128 125 166  Wood Samples sprayed by using Forintek's spray system were stored in piles (4X5), and placed outside. Every pile had 20 samples that belonged to the same species, and had been sprayed with same concentration of the preservative (same species-target retention  61  combination). All together there were 20 piles with 20 samples and two piles with nine control samples. Sources of Microorganisms: The yard were the samples were stored was near to a forest and also near a waste water plant, so that there was high probability of infestation by different kinds of moulds and stains. Test Evaluation The top, bottom, left and right sides of every piece were examined weekly and the percentage of fungal growth coverage was scaled from 1 to 10, where 1 means 10% and 10 means 100%.  62  Chapter 8: Results and Conclusions 8.1 Results of Phase 1 Objective; To develop guidance in making decisions about which chemicals had potential to be used in combination with D D A C against moulds and stains. The results related to: •  microbiological testing (bioassay) of potential components  •  preliminary microbiological testing of different component combinations comprising D D A C and one or more additional biocides.  Since the results of this phase will be used by Enviro-Quest Technologies Inc. in the future and represent the basis for other product patents, the information will stay confidential and all other components (not relevant for this particular study) will be called C2, Component 3, Component 4, and Component 5.  8.1.1 Microbiological Testing (Bioassay) of Potential Components  Each fungal group was represented by two species: Mold Fungi: Aspergillus niger and Gliocladium roseum and Sapstain Fungi: Aereobasidium pullulans and Ceratocystis adiopsa  63  Component 4 (an adhesive) and Component 5 (a water repellent) did not prevent fungal growth in the range of concentrations in which they were tested, but since they can improve product performance they could be used in the future, as potential additives in some new products. The bioassay results of the fungicidal components: D D A C , C2 and Component 3 are presented in Figures 15-26.  Figure 15. The growth of G. roseum on media containing D D A C  64  Figure 16. The growth of A. niger on media containing DDAC  5  o  100 95 90 85 80 75 70 65 60 55 50  i I-  /  500 ppm  r I  •:4— 1000 ppm  —1 i  45  40 35 30 25 20 15 10 5 0 —1  -*—100 ppm  /  2500 ppm -©—3000 ppm  i  - f l — Control  / (  / I—J !• 1 r 1 f—8  1  2 3 4  B—15  5 6  —iS  — «  7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Incubation Time  (day)  Figure 17. The growth of A. pullulans. on media containing DDAC  65  o O  100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0  3 3  m— 50 ppm — 100 ppm -A— 500 ppm -X  1000 ppm  —jK,—  3000 ppm  —#— Control  0  1  2 3 4  9 10 11 12 13 14 15 16 17 18 19 20 21 22  5 6 7  Incubation Time (day)  Figure 18. The growth of C. adiopsa on media containing D D A C  100  o  1—  CJ  95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0  -7 • — i  / /  — *  j—i  -#— 10 ppm - 0 — 100 ppm -E3—  250 ppm  A  500 ppm  —Q—Control  /  s  i  i fl 1 3 4  1I  5 6  ii  I 7 8  i  9 10 11 12 13 14 15 16 17 18 19 20 21 22  Incubation Time (day)  Figure 19. The growth of A. niger on media containing C2  66  •X— 10 ppm -0—100 ppm -S— 250 ppm A  500 ppm  -e—Control  0  1 2  3  4  5 6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22  Incubation T i m e (day)  Figure 20. The growth of G. roseum media containing C2  1 A A  QR 3 J  -  Q A 3 U  -  O J  -  an ou 75 70 (RR O J 1 •  -  -  >  ou RR 0 0-  o  5 0  O  45  ^  40 35 30 25 ?n  /  1 X  /  10 ppm  — $ — 1 0 0 ppm —Q—250 ppm A  500 ppm  — • — Control -  15 10 o ()  I"' 1 ! I i 1 i 1I r i i i L :I 3 < 5 f5 7  i 8  ..............J 1 i ii i i 4 1 5 16 17 18 19 20 21 22 9 10 1 I I 2 13 1  Iricubati on Tir ne (d<ay)  Figure 21. The growth of A. pullulans on media containing C2  67  -v->  o  #  100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0  1-1  >—  /  rf T  r ,r  /  ~T  /  y  ~X—10 ppm -0—100 ppm ~B— 250 ppm A  /  /  y  f r"  r;  i -iri 1 2 3 4  H i  ii  i3  f1  5 6 7  iI  11  8 9 10 11 12 13 14 15 16 17 18 19 20 21 22  Incubation Time (day)  Figure 22. The growth of C. adiopsa on media containing C2  Figure 23. The growth of A. niger on media containing Component 3  500 ppm  - © — Control  Figure 24. The growth of G. roseum media containing Component 3  Figure 25. The growth of A. pullulans on media containing Component 3  69  0  25ppm  -a—100 ppm -A— 500 ppm  5  o  —0— 1000 ppm —*—2500 ppm —Q—Control  0  1  g g g I 2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22  Incubation Time (day)  Figure 26. The growth of C. adiopsa on media containing Component 3  As it can be seen from Figures 15-26, C2 completely inhibited growth of all four test fungi at a concentration of 100 ppm. A second experiment was designed to determine C2 activity in the following range of concentrations: 10 ppm, 15 ppm, 25 ppm, and 50ppm. The results are presented in Table 5:  70  Table 5. The growth of Aspergillus niger (1), Gliocladium roseum (2), Aureobasidium pullulans (3) ,and Ceratocistis adiopsa (4), in the agar media containing selected concentrations of C2. Concentration (ppm) 10  Fungi  1  Incubation 2 3 4  (#)  P e r i o d (days) 5 6 7 11 Growth (%)  22 86.7  1  0  1.2  3.01  7.23  9.64  9.64  16.9  43.4  15  1  0  0  0  0  0  0  0  0  0  25  1  0  0  0  0  0  0  0  50 10  1 2  0 0  0 0  0  0  0  0  0  0 0  0 0  4.22  13.3  18.1  24.1  28.9  69.9  100  15  2  0  0  4.82  9.64  12  16.9  24.1  48.2  62.7  25  2  0  0  0.8  1.61  3.21  4.02  6.43  12.9  20.9  50  2  0  0  0  0  0  0  0  0  0  10  3  0  0  0  0  0  0  0  7.23  22.5  15  3  0  0  0  0  0  0  0  25  3  0  0  0  0 0  0  0  0  0  0 0  50  3  0  0  0 .  0  0  0  0  0  0  10  4  0  0  0  0  0  0  0  0  0 0  15  4  0  0  0  0  0  0  0  0  25  4  0  0  0  0  0  0  0  0  0  50  4  0  0  0  0  0  0  0  0  0  8.1.2 Conclusions and Comments  8..1.2.1 Efficacy Against Moulds  Aspergillus niger and Gliocladium roseum were the moulds utilized in this study. Aspergillus  niger is commonly used in bioassays. It can be seen from the results that as  the concentrations of D D A C , C2, and Component 3, increased, colony quantity and size were effectively reduced. However, the control plates showed normal growth and were totally covered after 10 days. This indicated that the inoculum used was viable and the lack of growth on the treated plates can only be attributed to the presence of biocides. The extensive sporulation, observed visually, at the lower concentrations could be an  71  indication of the fungus being under stress due to the presence of biocides. As the concentration increased, either the spore dispersal became inhibited or the germination of spores was inhibited, this resulted in fewer colonies. It was also observed that as concentration of the fungicides changed, the morphology of the colony also changed. That is often associated with the interaction of a fungus and its surrounding medium. From results obtained in this bioassay (graphs 16, 19 and 23) Aspergillus niger was completely controlled by: DDAC  2500 ppm  C2  100 ppm  Component 3  1000 ppm  Gliocladium roseum is the mould recommended by A S T M D 4445. Doyle and Rudick (1993) also suggested this fungus because it had the capability of degrading Alkyl Ammonium Compounds in a wood matrix. The control plates indicated that the inoculum used in this test was viable. The results (Graphs 15, 20 and 23) show that as the concentrations of D D A C , C2, and Component 3 were increased, colony sizes were effectively reduced. Gliocladium. roseum was completely controlled by: DDAC  2500 ppm  C2  100 ppm  Component 3  2500 ppm  72  8.1.2.2 Efficacy Against Sapstain Fungi  The sapstain fungi utilized in this study were Aerobasidium pullulans and Ceratocistis adiopsa. It was observed that most of the biocides used in the bioassay appeared to be very effective against them. The results (Graphs 17, 21, 25, 18, 22 and 26) show that Aerobasidium pullulans and Ceratocistis adiopsa were completely controlled by:  DDAC C2 Component 3  Aerobasidium pullulans  Ceratocistis adiopsa  500 ppm  500 ppm  10 ppm  100 ppm  2500 ppm  500 ppm  8.1.2.3 C2 Efficacy •  C2 shows excellent fungicidal characteristics. In the first run we tested it in the range of concentrations of 10 ppm, 100 ppm, 250 ppm and 500 ppm. Protection was complete at 100 ppm so we repeated the test over the range of lower concentrations: lOppm, 15ppm, 25 ppm, and 50 ppm. The results show complete protection at the level of 15-25 ppm for all fungi except Gliocladium roseum, which was protected against by 50 ppm. Incorporation of this component in very low concentrations could result in the definition of a cost-effective and more environmentally acceptable antisapstain product.  73  •  Since C2 showed excellent protection against moulds and stains, the conclusion at this stage was that it should be considered as a possible second active component in combination with D D A C .  8.2 Results of Phase 2 • Objective: To estimate the relative effectiveness of D D A C and C2 against, Soft rot, Brown rot, White rot and D D A C tolerant organisms, and to examine how they functioned in combination.  The results related to: •  microbiological testing (bioassay) of potential components  •  preliminary microbiological testing of different component combinations comprising of a D D A C and C2.  Each fungal group was represented by one fungi: •  Soft rot fungus: Chaetomium globosum  •  Brown rot fungus: Coniphora puteana (this is also a D D A C tolerant organism)  •  White rot fungus: Coriolus versicolor  74  To evaluate the viability o f each inoculum, the control plates were examined in each test for each microorganism. A s seen in Figure 27, they showed normal growth, and they indicate that the inoculum used was viable. Consequently, any lack o f growth on the treated plates can only be attributed to the presence o f fungicides.  •  A. niger.  G. Roseum  • K — C . adiopsa H  A. pullulans.  —$K—C.globosum  #  C. versicolor  C.puteana.  Figure 27. Fungal Growth on Control Plates  8.2.1 DDAC Efficacy  Efficacy Aureobasidium Coriolus  results  against  pullulans  Versicolor  (3),  (6), and  Aspergillus Ceratocistis Coniophora  niger  (1),  Gliocladium  roseum  (2),  adiopsa (4),  Chaetomium  globosum  (5),  Puteana  (7) in the agar media containing  selected concentrations o f D D A C are presented in Table 6. A l l o f these tests were run at concentrations o f 250, 500, 750, 1000, 2000, 2250 and 2500 ppm. A t some level in each  75  case there was no growth. While tests above this no growth level were done, they are not included in Table 6, since they merely continue to show no growth.  Table 6. DDAC Efficacy Against Moulds, Stains, Soft, White and Brown Rot Concentration Fungi (ppm) (#) 250 500 750 1000 . 2000 2500 250 500 750 1000 2000 2500 250 500 750 2500 250 1000 2500 250 500 750 1000 2000 2500 250 500 750 1000 2000 2500 250 500 750 1000 1250 1500 2000 2250 2500  1 1 1 1 1 • 1 2 2 2 2 2 2 3 3 3 3 4 4 4 5 5 5 5 5 5 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7  I n c u b a t i o n P e r i o d (d ays) 3 4 5 6 7 11 G r o w t h (%)  1  2  0.24 0.24 0.24 0.24 0 0 0.24 0.24 0.24 0 0 0 0 0 0 0 0 0 0 4.82 0.8 0.8 0 0 0 1.2  1.2 0.48 0.48 0.32 0 0 0.48 0.24 0.24 0.24 0 0 0 0 0 0 0 0 0 5.62 0.8 0.8 0 0 0 1.2  2.41 1.2 0.64 0.32 0 0 2.01 1.12 2.01 1.2 0 0 0 0 0 0 0 0 0 5.62 1.2 2.01 0.8 0 0 3.61  3.61 2.17 0.88 0.4 0 0 7.63 6.02 4.42 2.81 0 0 0 0 0 0 0 0 0 6.43 2.41 2.01 0.8 0 0 6.83  6.02 3.21 1.2 0.72 0 0 10.4 6.83 5.22 3.61 0 0 0 0 0 0 0 0 0 7.23 2.81 2.81 1.2 0 0 9.24  9.64 5.22 2.41 1.2 0 0 13.7 7.63 6.02 4.82 0 0 0 0 0 0 0 0 0 8.03 2.81 3.61 1.2 0 0 10.4  11.2 7.23 3.61 2.41 0 0 17.3 11.6 8.03 6.83 0 0 0 0 0 0 0 0 0 10 2.41 4.42 1.2 0 0 11.2  20.9 16.9 9.24 28.1 0 0 16.1 11.2 10 10 0 0 0 0 0 0 0 0 0 10 4.82 4.42 1.2 0 0 15.3  45.8 35.7 23.7 16.9 0 0 49.8 36.9 27.9 24.3 0 0 4.82 2.41 0 0 0 0 0 13.3 6.83 4.82 2.81 0 0 36.9  0 0 0 0 0 0 0 0 0 0 0 0 0 0  1.2 0 0 0 0 0 0 0 0 0 0 0 0 0  1.2 0 0 0 0 1.2 0.8 1.61 0 0 0 0 0 0  1.2 0 0 0 0 1.2 1.2 2.41 0 0 0 0 0 0  2.41 0 0 0 0 1.2 1.2 2.01 0.4 4.82 1.2 1.2 0 0  4.02 0.8 0.4 0 0 1.2 1.2 3.21 1.61 9.64 4.82 2.41 0 0  4.82 0.8 0.4 0 0 1.2 6.02 3.21 3.21 14.5 9.64 4.82 1.2 0  8.84 0.8 1.2 0 0 1.2 7.23 5.22 6.43 19.3 16.9 9.64 2.41 0  18.7 4.82 1.2 0 0 1.2 8.84 12.9 25.7 38.6 27.7 12 4.82 0  22  76  Test plates that had 2500 ppm of D D A C in the agar solutions did not show growth of any of seven tested microorganisms. The most resistant fungus to D D A C alone was  Coniphora puteana.  8.2.2 C2 Efficacy Table 7. C 2 Efficacy Against Moulds, Stains, Soft, White and Brown Rot Concentration (ppm) 10 15 25 50 10 15 25 50 10 15 25 50 10 15 25 50 10 15 25 50 10 15 25 50 10 15 25 50  Fungi (#) 1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4 5 5 5 5 6 6 6 6 7 7 7 7  I n c u b a t i o n P e r i o d (days) 7 6 4 5 1 2 3 G r o w t h Rate (%)  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  1.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.6 0 0 0 0 0 0 0 0 0 0  °  3.01 0 0 0 4.22 4.82 0.8 0 0 0 0 0 0 0 0 0 1.2 0 0 0 0 0 0 0 0 0 0 0  7.23 0 0 0 13.3 9.64 1.61 0 0 0 0 0 0 0 0 0 2.41 0 0 0 0 0 0 0 0 0 0 0  9.64 0 0 0 18.1 12 3.21 0 0 0 0 0 0 0 0 0 2.41 0 0 0 0 0 0 0 0 0 0 o  9.64 0 0 0 24.1 16.9 4.02 0 0 0 0 0 0 0 0 0 3.01 0 0 0 0 0 0 0 0 0 0 0  16.9 0 0 0 28.9 24.1 6.43 0 0 0 0 0 0 0 0 0 3.01 0 0 0 8.03 1.61 0 0 0 0 0 0  11  22  43.4 0 0 0 69.9 48.2 12.9 0 7.23 0 0 0 0 0 0 0 8.43 0 0 0 17.7 8.03 0 0 0 0 0 0  86.7 0 0 0 100 62.7 20.9 0 22.5 0 0 0 0 0 0 0 13.3 . 0 0 0 100 33.3 0 0 0 0 0 0  Table 7 show the growth of Aspergillus niger (1), Gliocladium roseum (2), Aureobasidium pullulans (3), Ceratocistis adiopsa (4), Chaetomium globosum (5),  77  Coriolus versicolor (6), and Coniphora puteana (7) on agar media containing selected concentrations of C2 . 50 ppm inhibited all growth of all the fungi tested. Gliocladium roseum was the most resistant. For all the other fungi, 25 ppm inhibited all growth.  8.2.3 Resistance to DDAC  According to the results from Table 6, Coniophora puteana showed increasing growth as the concentration of D D A C increased, implying that either D D A C was ineffective or that Conoiphora puteana could metabolize it This phenomena is graphically presented in Figure 28:  Coniophora  puteana  3  4  (DDAC)  5  6  Elapsed Time (days) 250 ppm  500 ppm  750 ppm  1000 ppm  Figure 28. Conoiphora puteana growth on the agar media containing various concentrations of DDAC  78  Since according to Table 7, C2 showed excellent protection against Coniophora puteana at very low concentrations, the next step was to try to counteract this resistance to D D A C and broaden the activity spectrum of the preservative by adding C2. A selected amount of C2 was mixed with D D A C . The results, which represent the growth of Coniophora puteana on the  agar media containing combinations  of  selected  concentrations of D D A C and C2 are presented in Table 8.  These results indicate that low concentrations of C2 (5 and 7.5 ppm) were ineffective even after combined with a D D A C concentration of 1000 ppm. 10 ppm of C2 with 250 ppm of D D A C was also ineffective but all other combinations were completely inhibitory to the growth of Coniophora puteana. The amount of C2 required to inhibit growth was relatively low compared to the amount of D D A C used. Thus C2 was able to broaden the spectrum of the antifungal action of D D A C .  79  Table 8. The Growth Rate of Coniophora puteana on the Media Containing Selected Combinations of DDAC and C2 Mixture Concentration DDAC C2 (ppm) (ppm) 2500 750 750 750 500 500 500 1000 1000 1000 250 250 250 100 1000 100 750 1000 500  150 10 15 25 10 15 25 10 15 25 10 15 25 25 5 15 7.5 7.5 7.5  1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  I n c u b a t i o n P e r i o d (days) 2 3 4 5 6 7 Growth (%) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0.8 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 1.61 0 0 0 2.41 0 0.4 0 0  0 0 0 0 0 0 0 0 0 0 2.41 0 0 0 4.02 0 0.8 0.4 2.41  0 0 0 0 0 0 0 0 0 0 2.41 0 0 0 4.82 0 3.21 2.41 4.02  0 0 0 0 0 0 0 0 0 0 3.21 0 0 0 9.24 0 6.02 3.21 4.82  11  22  0 0 0 0 0 0 0 0 0 0 6.43 0 0 0 18.1 0 10.4 7.23 6.43  0 0 0 0 0 0 0 0 0 0 16.1 0 0 0 39 0 22.5 17.7 27.1  8.2.4 Synergism in Mixtures of DDAC and C2  The next experiment was designed to examine how different combinations of D D A C and C2 functioned together in mixtures to explore their possible synergistic effects. The data presented in Tables 9.1 to 9.7 are examples of combinations of D D A C and  C2 that anticipated a synergistic effect.  They indicate that relatively low  concentrations of C2 enhanced the fungicidal activity of D D A C enhancement extended over a wide range of concentrations.  and that this  Table 9-1. Synergistic anti-Aspergilus niger activity of combinations of DDAC (A) and C2 (B)  Quantity producing end point S QA (ppm) QB (ppm) Ratio Total 0 100 250 500 500 750 750 750 1000 1000 1000 2500  50 25 25 15 25 10 15 25 10 15 25 0  50  4 10 33.33 20 75 50 30 100 66.66 40 0  125 275 515 525 760 765 775 1010 1015 1025 2500  Mixture  Synergy Index  Ratios  % A  B  0 100 80 20 90.9 9.09 97.1 2.91 95.2 4.76 98.7 1.32 98 1.96 96.8 3.23 99 0.99 98.5 1.48 97.6 2.44 100 0  QA/Qa QB/Qb 0 0.04 0.1 0.2 0.2 0.3 0.3 0.3 0.4 0.4 0.4 1  1 0.5 0.5 0.3 0.5 0.2 0.3 0.5 0.2 0.3 0.5 0  SUM 1 0.54 0.6 0.5 0.7 0.5 0.6 0.8 0.6 0.7 0.9 1  0 -0.27 -0.22 -0.3 -0.15 -0.3 -0.22 -0.1 -0.22 -0.15 -0.05 0  Table 9-2. Synergistic anti-Gliocladium roseum activity of various combinations of DDAC(A) and C2 (B)  Quantity producing end points QA (ppm) QB (ppm) Ratio Total 0 100 250 250 500 750 750 1000 2500  50 25 15 25 25 15 25 25 0  Mixture % A B  0 100 4 125 80 20 16.667 265 94.3 5.66 10 275 90.9 9.09 20 525 95.2 4.76 50 765 98 1.96 30 775 96.8 3.23 40 1025 97.6 2.44 0 0 2500 100 50  Synergy Index  Ratios QA/Qa QB/Qb 0 0.04 0.1 0.1 0.2 0.3 0.3 0.4 1  1 0.5 0.3 0.5 0.5 0.3 0.5 0.5 0  SUM 1 0.54 0.4 0.6 0.7 0.6 0.8 0.9 1  0 -0.27 -0.4 -0.22 -0.15 -0.22 -0.1 -0.05 0  81  Table 9-3. Synergistic waii-Aurobasidium pullulants activity of various combinations of DDAC (A) and C 2 (B)  Quantity producing end poinl s QA (ppm) QB (ppm) Ratio Total 0  50  50  Ratios  Mixture  Synergy Index  % QA/Qa QB/Qb  SUM  A  B  0  100  0  1  1  0  0.3  0.34  -0.47  115  87  13  0.04  16.667  125 265  80 94.3  20 5.66  0.04 0.1  0.5 0.3  0.54 0.4  -0.27 -0.4  25  10  275  90.9  9.09  0.1  0.5  0.6  -0.22  10  50  510  98  1.96  0.2  0.2  0.4  -0.4  15  33.333  515  2.91  0.2  20  525  4.76  0.2  0.3 0.5  0.5  25  97.1 95.2  -0.3 -0.15  750  10  75  760  98.7  1.32  0.3  0.2  0.5  -0.3  750  15  50  765  98  1.96  0.3  0.3  0.6  -0.22  100  15  100 250  • 25 15  250 500 500 500  6.6667 4  0.7  750  25  30  775  96.8  3.23  0.3  0.5  0.8  -0.1  1000  10  100  1010  99  0.99  0.4  0.2  0.6  -0.22  1000  15  66.667  1015  98.5  0.4  0.3  0.7  -0.15  1000  25  40  1025  97.6  1,48 2.44  0.4  0.5  0.9  -0.05  2500  0  0  2500  100  0  1  0  1  0  Table 9-4. Synergistic anti-Ceratocistis adiopsa activity of various combinations of DDAC (A) and C 2 (B)  Quantity producing end poinl s QA (ppm) QB (ppm) Ratio Total 0  50  100  15  6.6667  Mixture  Synergy Index  Ratios  % QA/Qa QB/Qb  SUM  A  B  50  0  100  0  1  1  0  115  87  13  0.04  0.3  0.34  -0.47  100  25  4  125  80  20  0.04  0.5  0.54  -0.27  250  25  10  275  90.9  9.09  0.1  0.5  0.6  -0.22  500  10  50  510  98  1.96  0.2  0.2  0.4  -0.4  500  15  33.333  515  97.1  2.91  0.2  0.3  0.5  -0.3  500  25  20  525  95.2  4.76  0.2  0.5  0.7  -0.15  750  10  75  760  98.7  1.32  0.3  0.2  0.5  -0.3  750  15  50  765  98  1.96  0.3  0.3  0.6  -0.22  750  25  30  775  96.8  3.23  0.3  0.5  0.8  -0.1  1000  10  100  1010  99  0.99  0.4  0.2  0.6  -0.22  1000  15  66.667  1015  98.5  1.48  0.4  0.3  0.7  -0.15  1000  25  40  1025  97.6  2.44  0.4  0.5  0.9  -0.05  2500  0  0  2500  100  0  1  0  1  0  82  Table 9-5.  Synergistic anti-Chaetomium globosum activity of various combinations of D D A C (A) and C2  Quantity producing end point s QA (ppm) QB (ppm) Ratio Total 0 100 250 500 500 750 750 750 1000 1000 2500  T a b l e 9-6.  50 25 25 15 25 10 15 25 15 25 0  Mixture  Ratios  Synergy Index  % A  B  0 50 4 125 80 10 275 90.9 33.333 515 97.1 20 525 95.2 75 760 98.7 50 765 98 30 775 96.8 66.667 1015 98.5 40 1025 97.6 0 2500 100  100 20 9.09 2.91 4.76 1.32 1.96 3.23 1.48 2.44 0  QA/Qa QB/Qb 0 0.04 0.1 0.2 0.2 0.3 0.3 0.3 0.4 0.4 1  1 0.5 0.5 0.3 0.5 0.2 0.3 0.5 0.3 0.5 0  SUM 1 0.54 0.6 0.5 0.7 0.5 0.6 0.8 0.7 0.9 1  0 -0.27 -0.22 -0.3 -0.15 -0.3 -0.22 -0.1 -0.15 -0.05 0  Synergistic unti-Coriolus versicolor activity of various combinations of D D A C (A) and C2  Quantity producing end points QA (ppm) QB (ppm) Ratio Total 0 100 100 250 250 500 500 500 750 750 750 1000 1000 1000 2500  (B)  50 15 25 15 25 10 15 25 10 15 25 10 15 25 0  6.6667 4 16.667 10 50 33.333 20 75 50 30 100 66.667 40 0  50 115 125 265 275 510 515 525 760 765 775 1010 1015 1025 2500  (B)  Mixture  Ratios  Synergy Index  cVo  A  B  0 87 80 94.3 90.9 98 97.1 95.2 98.7 98 96.8 99 98.5 97.6 100  100 13 20 5.66 9.09 1.96 2.91 4.76 1.32 1.96 3.23 0.99 1.48 2.44 0  QA/Qa QB/Qb SUM 0 0.04 0.04 0.1 0.1 0.2 0.2 0.2 0.3 0.3 0.3 0.4 0.4 0.4 1  1 0.3 0.5 0.3 0.5 0.2 0.3 0.5 0.2 0.3 0.5 0.2 0.3 0.5 0  1 0.34 0.54 0.4 0.6 0.4 0.5 0.7 0.5 0.6 0.8 0.6 0.7 0.9 1  0 -0.47 -0.27 -0.4 -0.22 -0.4 -0.3 -0.15 -0.3 -0.22 -0.1 -0.22 -0.15 -0.05 0  83  Table 9-7.  Synergistic anti-Coniophora puteana activity of various combinations of DDAC (A) and C2 (B)  Quantity producing end points QA (ppm) QB (ppm) Ratio Total 0  50  100 100  15 25  Mixture % A B  Synergy Index  Ratios QA/Qa QB/Qb  SUM  1  1  0  50  0  100  0  13 20  0.04 0.04  0.3 0.5  0.34 0.54  250  15  100/25 250/25  265  94.3  5.66  0.1  0.3  0.4  -0.47 -0.27 -0.4  250  25  250/25  275  90.9  9.09  0.1  0.5  0.6  -0.22  500  10  500/10  510  98  1.96  0.2  0.2  0.4  -0.4  500  15  500/15  515  97.1  2.91  0.2  0.3  0.5  -0.3  500  25  500/25  525  95.2  4.76  0.2  0.5  0.7  -0.15  750  10  750/10  760  98.7  1.32  0.3  0.2  0.5  -0.3  750  15  750/15  765  98  1.96  0.3  0.3  0.6  -0.22  750  25  750/25  775  96.8  3.23  0.3  0.5  0.8  -0.1  0.2  0.6  -0.22  100/15  115 125  87 80  1000  10  1000/10 1010  99  0.99  0.4  1000  15  1000/15 1015  98.5  1.48  0.4  0.3  0.7  -0.15  1000  25  1000/25 1025  97.6  2.44  0.4  0.5  0.9  -0.05  2500  0  100  0  1  0  1  0  0  2500  8.2.5 Conclusions and Comments  Table 9.1 shows that all of the combinations of C2 and D D A C used displayed synergistic effects against Aspergilus niger, since all of the values of the synergy index (see Section 7.1 for definition) were negative. Table 9.2 shows that all of the combinations of C2 and D D A C displayed synergistic effects against Gliocladium roseum since all of the values of the synergy index were negative. Table 9.3 shows that all of the combinations of C2 and D D A C displayed synergistic effects against Aurobasidium pullulans since all of the values of the synergy index were negative.  84  Table 9.4 shows that all of the combinations of C2 and D D A C displayed synergistic effects against Ceratocistis adiopsa since all of the values of the synergy index were negative. Table 9.5 shows that all of the combinations of C2 and D D A C displayed synergistic effects against Chaetomium globosum since all of the values of the synergy index were negative. Table 9.6 shows that all of the combinations of C2 and D D A C displayed synergistic effects against Coriolus versicolor since all of the values of the synergy index were negative. Table 9.7 Coniophora puteana shows that all of the combinations of C2 and D D A C displayed synergistic effects against Aurobasidium pullulans since all of the values of the synergy index were negative.  In the summary all of the presented combinations of C2 and D D A C resulted in synergy. Thus the combinations were more effective than the individual components at the same concentrations as used in the mixtures.  8.3 Results of Phase 3  Objective: To define the product dilution (minimum preservative concentration) that would be effective against sapstain fungi and moulds on wood.  85  The results were obtained by using: •  A modified A S T M D445 test on commercial wood (Douglas Fir) obtained from a local saw-mill  •  The microorganisms tested in these experiments ar listed in table 10 :  Table 10. Microorganisms used in the experiments  # 1 2  MICROORGANISM Moulds Aspergillus Gliocladium  niger roseum  Sapstains 3 4  Aerobasidium  5  Chaetomium  6  Coriolus  Ceratocistis  pullulans addiopsa.  Soft rot globosum  White rot versicolor  Brown rot (DDAC tolerant microorganism) 7  Conoiphora  puteana  8.3.1 Microbiological Screening Test Using Agar Blocks  8.31.1 Efficacy of the Preservative Containing 20.1% Active Ingredient •  Ten specimens per concentration of formulation for each fungus tested were used.  •  Ten untreated control specimens were used for each fungus tested.  •  The lowest concentration of formulation was selected to be the concentration of the product diluted in the ratio 1:80 (it was expected that the concentration of active component when the product was diluted 80 times would not provide satisfactory  fungal protection).  Each of the following concentrations was twice the following  concentration (1:10, 1:20, 1:40, 1:80). •  The concentrations of active ingredient for the tests on wood samples were about ten times higher than the concentrations of active ingredient when the experiments were done on agar plates.  •  The wood samples were immersed into a prepared solution of the preservative for 15 sec. Similarly, untreated control specimens were treated with water. ( A S T M D445)  8.3.1.1.1 Inoculated A g a r Strips Placed on Absorbent Paper  Tables 11.1 to 11.5 show fungal growths on wood samples treated with various concentrations of preservative. There were two wood samples in every petri dish (see Figure 10, section 7.2). Samples that were just immersed in water and placed near samples treated with preservative were called "blank"; samples that were immersed in various preservative dilutions were called 'treated". For every microorganisms group that was examined, there were three replicas of "control" samples. Two control samples were immersed in water, infested with certain microorganisms, placed in a petri dish and examined in the same manner as all of the other samples.  87  Table 11.1. Fungal Growth on Wood Samples Treated with 10 Times Diluted Preservative Concentrated 20.1 % /Dillution: 1:10/Samples placed on agar strips Active ingredient after dilution: 20,100 ppm Type of Sample Type in Blank Treated Control Fungi Petri Dish (% Growth) (% Growth) (% Growth) 2 Moulds Blank +Treated 0 0 4.2 2 Stains Blank +Treated 0 0 4.8 2 Moulds 2 Treated N/A 0 4.75 2 Stains 2Treated N/A 0 4.5  No. of Replicas £ 3 3 3 3  Table 11.2. Fungal Growth on Wood Samples Treated with 15 Times Diluted Preservative Concentrated 20.1 % /Dillution: 1:15/Samples placed on agar strips Active ingredient after dilution: 13,400 ppm Type of Sample Type in Blank Treated Control Fungi Petri Dish (% Growth) (% Growth) (% Growth) 2 Molds Blank +Treated 0 0 4.2 2 Stains Blank+Treated 0 0 4.8 2 Molds 2 Treated N/A 0 4.75 2 Stains 2 Treated N/A 0 4.5  No. of Replicas II 3 3 3 3  Table 11.3. Fungal Growth on Wood Samples Treated with 20 Times Diluted Preservative Concentrated 20.1% /Dillution: 1:20/Samples placed on agar strips Active ingredient after dilution: 10,500 ppm Type of Sample Type in Blank Treated Control Fungi Petri Dish (% Growth) (% Growth) (% Growth) 2 Molds Blank +Treated 0 0 4.2 2 Stains Blank +Treated 0 0 4.8 2 Molds 2 Treated N/A 0 4.75 2 Stains 2 Treated N/A 0 4.5  No. of Replicas C_  3 3 3 3  88  Table 11.4. Fungal Growth on Wood Samples Treated with 40 Times Diluted Preservative Concentrated 20.1 % /Dillution: 1:40/Samples placed on agar strips Active ingredient after dilution: 5,250 ppm Type of Sample Type in Blank Treated Control Fungi Petri Dish (% Growth) (% Growth) (% Growth) 2 Molds Blank +Treated 0 0 4.2 2 Stains Blank +Treated 0 0 4.8 2 Molds 2Treated N/A 0 4.75 2 Stains 2Treated N/A 0.3 4.5  No. of Replicas 3 3 3. 3  Table 11.5. Fungal Growth on Wood Samples Treated with 80 Times Diluted Preservative Concentrated 20.1 % /Dillution: 1:10/Samples placed on agar strips Active ingredient after dilution: 2,625 ppm Type of Sample Type in Blank Treated Control Fungi Petri Dish (% Growth) (% Growth) (% Growth) 2 Molds Blank +Treated 3.5 2 4.2 2 Stains Blank +Treated 4 3.5 4.8 2 Molds 2 Treated N/A 1.75 4.75 2 Stains 2 Treated N/A 0.3 4.5  No. of Replicas 3 3 3 3  Tables 11.1-11.5 show that the growth of the test organisms was completely inhibited at active ingredient concentrations greater than 5250 ppm. At 2625 ppm growth occurred on the treated samples  8.3.1.1.2 Samples inoculated by suspension of spores Tables 12.1 ~ 12.5 present the results of test done of wood block specimens using inoculation with spore suspension (see  Section 7.2, Figure 11). The results show that  89  complete protection against all of the organisms tested occurred at active ingredient concentrations of 13,400 ppm or greater.  Table 12.1. Fungal growth on Wood Samples Treated with 10 Times Diluted Preservative Concentrated 20.1 % /Dillution: 1:10/Active ingredient: 20100 ppm Type Sample Type Average Average Average of in Growth on Growth on Growth on Fungi. Petri Dish Blank Treated Control  No. of Replicas  2 Moulds  Blank +Treated  0  0  4  3  2 Stains  Blank +Treated  0  0  4.5  3  2 Moulds +2 Stains All from Table 10  Blank +Treated  N/A  0  4  3  2 Treated  N/A  0  4.5  3  Table 12.2. Fungal growth on Wood Samples Treated with 15 Times Diluted Preservative Concentrated 20.1% /Dillution: 1:15/Active ingredient: 13400 ppm Type Sample Type Average Average Average of in Growth on Growth on Growth on Fungi. Petri Dish Blank Treated Control  No. of Replicas  2 Moulds  Blank +Treated  0  0  4  3  2 Stains  Blank +Treated  0  0  4.5  3  2 Moulds +2 Stains All Fungi from Table 10  Blank +Treated  N/A  0  4  3  2 Treated  N/A  0  4.5  3  90  Table 12.3. Fungal growth on Wood Samples Treated with 20 Times Diluted Preservative Concentrated 20.1 % /Dillution: 1:20/ Active inj^redient: 1050C ppm Type Sample Type Average Average Average of in Growth on Growth on Growth on Fungi. Petri Dish Blank Treated Control  No. of Replicas  2 Moulds  Blank +Treated  0  0  4  3  2 Stains  Blank +Treated  0  0  4.5  3  2 Moulds +2 Stains All Fungi from Table 10  Blank +Treated  0  4  3  0.2  4.5  3  N/A  N/A 2 Treated  Table 12.4. Fungal growth on Wood Samples Treated with 40 Times Diluted Preservative Concentrated 20.1% /Dillution: 1:40/ Active ingredient: 5250 ppm Type Sample Type Average Average Average of in Growth on Growth on Growth on Fungi. Petri Dish Blank Treated Control  No. of Replicas  2 Moulds  Blank +Treated  4.2  2.5  4  3  2 Stains  Blank +Treated  4.5  3  4.5  3  2 Moulds +2 Stains All Fungi from Table 10  Blank +Treated  2  1.7  4  3  2 Treated  N/A  0.2  4.5  3  91  Table 12.5. Fungal growth on Wood Samples Treated with 80 Times Diluted Preservative Concentrated 20.1% /Dillution: 1:80/ Active ingredient: 2625 ]ppm Average Type Sample Type Average Average Growth on of In Growth on Growth on Control Fungi. Petri Dish Blank Treated  No. of Replicas  2 Moulds  Blank +Treated  3.9  3.5  4  3  2 Stains  Blank +Treated  4.2  3.9  4.5  3  2 Moulds +2 Stains All Fungi from Table 10  Blank +Treated  2  1.7  4  3  1.9  4.5  3  2 Treated  8.3.1.2 Efficacy  of  N/A  57%  Active  Ingredient  Freshly Prepared  Preservative Inoculation: by suspension of spores along the length of one flat side of each sample. •  Ten specimens per concentration of formulation for each fungus tested were used.  •  Ten untreated control specimens were used for each fungus tested.  •  The lowest concentration of formulation was selected to be the concentration of the product diluted in the ratio 1:640  (it was expected that the  concentration of active component when the product was diluted 640 times would not provide satisfactory fungal protection). •  Each of the following concentrations was twice the following concentration (1:20, 1:40, 1:80, 1:160, 1:320, 1:640,).  •  Microorganisms used in this test were the same as presented in Table 10.  Tables 13.1 ~ 13.6 present the results of test done of wood block specimens immersed the various dilutions of 57% concentrated preservative.  Table 13.1. Fungal Growth on Wood Samples Treated with 20 Times Diluted Preservative Concentrated 57% /Dillution: 1:20/Active ingredient: 28500 ppm Number of Average Fungal Treated and Growth on Microorganisms Average Fungal Control Growth on Treated Control Samples Type Samples Samples Molds  10  0  5  Stains  10  0  5  All Fungi from Table 10  10  0  5  Table 13.2. Fungal Growth on Wood Samples Treated with 40 Times Diluted Preservative Concentrated 57% /Dillution: 1:40/Active ingredient: 14250 ppm Number of Average Fungal Treated and Microorganisms Growth on Average Fungal Control Type Growth on Treated Control Samples Samples Samples Molds  10  0  5  Stains  10  0  5  All Fungi from Table 10  10  0  5  Table 13.3. Fungal Growth on Wood Samples Treated with 80 Times Diluted Preservative Concentrated 57% /Dillution: 1:80/Active ingredient: 7125 ppm Number of Treated and Average Fungal Microorganisms Control Growth on Treated Type Samples Samples Molds  10  0  Stains  10  0  Average Fungal Growth on Control Samples  5  5 r  All Fungi from Table 10  10  0  5  Table 13.4. Fungal Growth on Wood Samples Treated with 160 Times Diluted Preservative Concentrated 57% /Dillution: 1:160/Active ingredient: 3562.5 p]Dm Average Fungal Number of Growth on Treated and Average Fungal Microorganisms Control Samples Control Growth on Treated Type Samples Samples Molds  10  0.4  5  Stains  10  0.1  5  All Fungi from Table 10  10  1.05  5  94  Table 13.5. Fungal Growth on Wood Samples Treated with 320 Times Diluted Preservative Concentrated 57%/Dillution: l:320Active ingredient: 1781.25 ppm Average Fungal Number of Average Fungal Treated and Growth on Microorganisms Growth on Treated Control Control Samples Type Samples Samples Molds  10  1.1  5  Stains  10  0.25  5  All Fungi From Table 10  10  0.5  5  Table 13.6. Fungal Growth on Wood Samples Treated with 640 Times Diluted Preservative Concentrated 57%/Dillution: 1:640/Active ingredient: 890.63ppm Number of Average Fungal Average Fungal Microorganisms Treated and Growth on Treated Growth on Control Control Samples Samples Type Samples Molds  10  1.1  5  Stains  10  0.45  5  All Fungi From Table 10  10  0.7  5  95  8.3.2 Conclusions and Comments  The results show that complete protection against all of the organisms tested occurred at active ingredient concentrations of 7125 ppm or greater.  8.4 Results of Phase 4 8.4.1 Shelf Life Objective: To confirm that the biological activity of fungicidal combinations did not deteriorate after various elapsed times  8.4.1.IResults of Microbiological Screening Test on Agar Plates Since the results from the previous research phases suggested that a concentration of 2500 ppm of active ingredient completely prevented fungal growth on agar plates, a number of samples (prepared in different time frames) were diluted until 2500 ppm concentration was reached in the agar solution. They were tested against the three most resistant fungi used in previous tests. Results are presented in Table 14:  96  Table 14. Efficacy Determination of Preservatives Prepared in Different Ranges of Elapsed Time A.I. #  (%)  1 20.1 2 26.7 20.4 3 4 20.1 5 20.1 6 20.1 7 25.1 20.1 8 70.3 9 10 54.9 11 57 12 57 13 57 14 57  Time Elapsed from Preparation Date to Test 27 months 15 months 14 months 13 months 10 months 10 months 10 months 9 months 9 months 7 months 6 months 5 months 4 days 4 days  Gliocladium roseum (2500 ppm) Fungal Growth  Aurobasidium pullulants (2500 ppm) Fungal Growth  Coniophora puteana (2500 ppm) Fungal Growth  0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%  0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%  0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%  Color Change  No Yes Yes No No No Yes No No Yes No No No No  8.4.1.2 Conclusions and Comments From the results presented in Table 14 it can be seen that the products stayed biologically active for all samples even though they were used at different lengths of time after their preparation. Activity was retained for periods of storage up to more than two years. The difference was that some samples of the preservatives, after 1 -3 weeks, slightly changed color. After contacting the manufacturer of C2, they suggested adding copper nitrate (1% of the amount of second active component) to ensure its stability. After additions of copper nitrate (samples: #11, #12, #13, and #14), there was no change in color. Since a very  97  small amount of copper nitrate was recommended, it did not actually effect any other product characteristics (for example, did not effect the fish toxicity).  8.4.2 Results of Microbiological Screening Test on Wood Samples  The purpose of this test was to ensure that the 57% concentrated product, after three months of storage, could protect wood against fungi. A three month old chemical composition, containing 57% active ingredient, was diluted in a same way that was done with freshly prepared product ( see section 8.3.1.2 ). The results are presented in Tables 15.1- 15.6:  Table 15.1. Fungal Growth on Wood Samples Treated with 20 Times Diluted Preservative Concentrated 57% /Dilution: 1:20/Active ingredient: 28500 ppm Number of Average Fungal Microorganisms Treated and Control Growth on Type Samples Treated Samples  Average Fungal Growth on Control Samples  Molds  10  0  5  Stains  10  0  5  All Fungi from Table 10  10  0  5  98  Table 15.2. Fungal Growth on Wood Samples Treated with 40 Times Diluted Preservative Concentrated 57%/Dilution: 1:40/Active ingredient: 14250 ppm Number of Average Fungal Microorganisms Treated and Control Growth on Type Samples Treated Samples  Average Fungal Growth on Control Samples  Molds  10  0  5  Stains  10  0  5  All Fungi from Table 10  10  0  5  Table 15.3. Fungal Growth on Wood Samples Treated with 80 Times Diluted Preservative Concentrated 57% /Dilution: 1:80/Active ingredient: 7125 ppm Number of Average Fungal Microorganisms Treated and Control Growth on Type Samples Treated Samples  Average Fungal Growth on Control Samples  Molds  10  0  5  Stains  10  0  5  All Fungi from Table 10  10  0  5  99  Table 15.4. Fungal Growth on Wood Samples Treated with 160 Times Diluted Preservative Concentrated 57% /Dilution: 1:160/Active ingredient: 3562.5 ppm Number of Average Fungal Microorganisms Treated and Control Growth on Samples Treated Samples Type  Average Fungal Growth on Control Samples  Molds  10  1  5  Stains  10  0.15  5  All Fungi from Table 10  10  0.85  5  Table 15.5. Fungal Growth on Wood Samples Treated with 320 Times Diluted Preservative Concentrated 57% /Dilution: l:320Active ingredient: 1781.25 ppm Number of Average Fungal Microorganisms Treated and Control Growth on Samples Type Treated Samples  Average Fungal Growth on Control Samples  Molds  10  1.1  5  Stains  10  0.25  5  All Fungi from Table 10  10  1.1  5  100.  Table 15.6. Fungal Growth on Wood Samples Treated with 640 Times Diluted Preservative Concentrated 57% /Dilution: 1:640/Active ingredient: 890.63ppm Number of Average Fungal Microorganisms Treated and Control Growth on Samples Treated Samples " Type  Average Fungal Growth on Control Samples  Molds  10  1.2  5  Stains  10  0.35  5  All Fungi from Table 10  10  1.7  5  8.4.2.2 Conclusions and Comments Comparison of the results from Tables 14.1 - 14.6 and 15.1 ~ 15.6 show that the product did not lose its ability to protect wood samples against all of the microorganisms tested, even though the set of results presented in Tables 15.1- 15.6 present the  fungal  protection ability of a 3 months old preservative. These results also show that complete protection against all of the organisms tested occurred at active ingredient concentrations of 7,125 ppm or greater (same as the fresh product).  8.4.3 Definition of DDAC/C2 ratio  Objective: To have both active components in the same liquid phase, and to incorporate as much as possible of the second active component. Thus increasing amounts of a solution of C2 were added to see if a two-phase mixture was created.  101  8.4.3.1 Titrations  A range of D D A C solutions in water were titrated with 0.007 % active C2. The results are presented in Tables 16, 17 and 18.  Table 16. Titration of DDAC solution (70g Bardac 2280 and 30g water), with 0.007% water solution of C2 C2 Increnented (ml)  Total Volume (ml)  % of C2  % of DDAC  25  125  0.140  135  4  Mixing Time (sec)  Ratio DDAC/ C2  Mixture Appearance  44.8  320  Clear  0.181  41.5  229  Clear  139  0.196  40.3  205  Clear  4  143  0.210  39.2  186  Clear  1  144  0.214  38.9  182  Cleared after a while  0.5  144.5  0.216  38.8  180  Milky  10  ,  10 sec  From the results presented in Table 16 it can be seen that the mixture stayed in the same liquid phase (clear liquid) if the addition of the second active component was less than 0.214 %, or if the DDAC:C2 ratio was higher than 186.  Table 17. Titration of DDAC solution (30g Bardac 2280 and 70g water), with 0.007% water solution of C2 C2 Incremented (ml) 4 10.8 3.2 1 0.5 0.5  Total Volume (ml)  % of C2  104 114.8 118 119 119.5 120  0.043 0.144 0.171 0.179 0.183 0.187  % of DDA C 23.1 20.9 20.3 20.2 20.1 20.0  Mixing Time (sec)  10 sec  Ratio DDAC/ C2  Appearance of the Mixture  536 145 119 113 110 107  Clear Clear Clear Clear Cleared after a while Milky  102  From the results presented in Table 17 it can be seen that the mixture stayed in the same liquid phase (clear liquid) if the addition of the second active component was less than 0.179%, or if the D D A C : C2 ratio was higher than 112. Table 18. Titration of DDAC solution (20g Bardac 2280 and 80g water, with 0.007% water solution of C2) C2 Incremented (ml)  % of C2  % of DDAC  3 6.5 4.5 1.5 1  Total Volum e (ml) 103 109.5 114 115.5 116.5  0.033 0.097 0.138 0.150 0.159  15.5 14.6 14.0 13.9 13.7  0.5  117  0.163  13.7  Mixing Time (sec)  Ratio DDAC/ C2  Appearance of the Mixture  20 sec  476 150 102 92 87  Clear Clear Clear Clear Cleared after a while Milky  84  From the results presented in Table 18 it can be seen that the mixture stayed in the same liquid phase (clear liquid) if the addition of the second active component was less than 0.159%, or if the D D A C : C2 ratio was higher than 87.5.  8.4.3.2 Conclusions and Comments  According to Tables 16, 17 and 18 it can be concluded that it was possible to incorporate more C2 when DDAC/Water solution was more concentrated, before creating an emulsion. For example, the results show that in the case where the most concentrated D D A C solution (20g Bardac 2280 and 80g water) was titrated, it was possible to add to the mixture the highest amount of C2, while mixture still remained clear.  8.4.4 Results of Toxicity Experiments  LC50 is the concentration of fungicide in dilution in water that causes mortality of 50 % of the fish or daphnia in the test population after a 96 hour exposure. If the LC50 number is high, it means that the amount of the chemical in the water that it takes to cause mortality in 50 percent of the test population is also high. In other words, the higher the LC50 number the less toxic the chemical is.  8.4.4.1 96-h LC50 Rainbow Trout Bioassay on 57.5% Active Product  Method  : A S T M STP 634.1077  96-h LC50 mg/L: 1.41  Table 19. Fish Toxicity Test (Rainbow Trout); BCRI Sample #20001089  Control  Test Concentration (mg/1)  Percent Survival 24 h  Percent Survival 48 h  Percent Survival 72 h  Percent Survival 96 h  0  100  100  100  100  5 2 1 0.5 0.1  0 0 100 100 100  0 0 100 100 100  0 0 100 100 100  0 0 100 100 100  8.4.4.2 48-h LC50 Bioassay for Daphnia Magna on 57.5% Active Product  Method : EPS l/RM/14  104  48-h LC50 mg/L: 0.13 Table 20. Daphnia Magna Toxicity Test; BCRI Sample #20001089 Test Concentration (mg/L) Control  Percent Survival 24 h  Percent Survival 48h  0  100  100  2 1 0.5 0.25 0.1 0.05 0.025  0 0 0 10 100 100 100  0 0 0 0 100 100 100  The LC o for a 57.5% active ingredient was 1.41 mg/1 for fish and 0.13 mg/1 for daphnia 5  (an aqutic insect). The fish toxicity results show that this formulation was less toxic than IPBC and C2 alone (0.049 mg/lit and 0.12 mg/1 respectively; see section 5, Figure 8).  8.5  Results of Phase 5  8.5.1 Field Efficacy Test by Using Pilot-Plant Linear Spraying System  Objective: To find the optimal chemical coverage capable of protecting wood samples under real outside conditions.  Wood samples were transported from a mill one week after cutting, and they stayed another week in the Forintek's yard before being treated. Prior to spraying, all samples were inspected. Most of the samples were free from fungi. A l l samples numbered as 3B were exceptionally dirty. Furthermore, prior to spraying, samples 3B/3,  3B/13 and 3b/15 were also probably infected with fungi (see Figure 29). These three samples were monitored weekly in order to determine whether microorganisms continued to grow or not.  Samples were carefully inspected and the results presented as percentage of fungal growth on the top, bottom, left and right sides of the test specimens. In Tables 21 to 32 the abbreviations T, B, L and R represent top, bottom, left and right side of the wood sample, respectively.  Figure 29. Rough Wood Samples Ready for Spraying (It is obvious that two samples were infected)  Tables 21 and 22 present the results of samples coded as 6 (Rough Douglas Fir) and 7 (Rough Hem Fir) in Table 4 on page 61. There were nine samples of each species group, and they were coded as A.B,C, D, E, F ,G, H, and I. Tables 23-32 present fungal growth  106  on the wood species sprayed over a range of preservative chemical retention  The test  parameters and conditions are also shown in Table 4 on page 61.  Table 21. Fungal Growth on Untreated Rough Douglas Fir Samples S p e c i e s D. Fir  |Rough  Retention: 0  Speed: 0  S a m p l e C h e c k e d after 1 w e e k C h e c k e d after Code T B L R T B A 0 0 0 0 0 0 0 0 0 0 0 0 B  Concentration: N/A  3 weeks L R  I Code:  C h e c k e d after 5 w e e k s T B L R  Control 6 C h e c k e d after 6 w e e k s T B L R  0  0  0  0  0  0  0  1  0  0  0  0  0  2  0  0  0  4  7  0.1  C  5  0  0  0  6  0  1  0  g  0  5  1  9  0  5  1  D  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  E  0  0  0  0  0  0  0  0  0.1  0  0  0  0 0.2  2  0  0  F  0  0  0  0  0  0  0  0  1.5  0  0  0  3  0  0.2  0.2  G H  6  0  0  0  7  0  0  0  8.5  0  0  0  9  0  0  0  0  0  0  0  0  0  0  0  1  0  0  0  0  0  0  0  1  0  0  0  0  0  0  0  0  1  0  0  0  0  0  0  0  Table 22. Fungal Growth on Untreated Rough Western Hemlock Samples Species H. Fir  |Rough  Retention: 0 | S p e e d : 0  Sample C h e c k e d after 1 w e e k C h e c k e d after # T B L R T B A 0 0 0 0 0 0 B 6 0 0 0 8 0  Concentration: N/A  3 weeks  I Code  C h e c k e d after 5 w e e k s  Control 7 C h e c k e d after 6 w e e k s  L  R  T  B  L  R  T  B  L  R  0  0  1  3  0  0  1  6  2  0.4  0  0  9  0  2  3  9  0  2  3  C  0  0  0  0  0  0  0  0  1  0  0  0  2.5  0  0  1.5  D  0  0  2  2  0.5  2  5  5  1  4  8  7  2  5  8  7  E  0  5  0  2  0  6  1  4  1  10  2  6  2  10  2  7  F  0  0  0  0  0  0  0  0  2  0  0  1  4  0  0  2  G  0  0  0  0  0  0  0  0  2  0  4  0  2  0  6  0  H  0  0  0  0  0  0  0  0  2  0  6  0  4  0  8  0  I  0  0  0  0  0  0  0  0  0  0  6  4  0  0  6  4  107  Table 23. Fungal Growth on Planed Western Hemlock Samples, Treated with 70 ug/cm Species H. Fir Sample # 1 2 3 4  |Planed Retention:70 | S p e e d : 400 C h e c k e d after 1 w e e k C h e c k e d after 3 w e e k s T B L R T B L R 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  5  0  0  0  0  0  0  0  Concentration: 2.17  CODE:5A C h e c k e d after 5 w e e k s C h e c k e d after T B L R T B 0 0 0 0 0 0 0 0 0 0 0 0  0  6 weeks L  R  0  0  0  0 0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0 0  6  0  0  0  0  0  0  0  0  0  7  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  8  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  9  0  0  0  0  0  0  0  0  0  10  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  11  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  12  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  13  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  14  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  15  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  16  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  17  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  18  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  19  0  0~  0  0  0  0  0  0  0  0  0  0  0  0  0  20  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  Table 24. Fungal Growth on Planed Douglas Fir Samples, Treated with 44 ug/cm  2  S p e c i e s D. Fir Sample # 1 2 3  |Planed Retention:44 | S p e e d : 400 C h e c k e d after 1 w e e k C h e c k e d after 3 w e e k s T B L R T B L R 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  Concentration: 2.17  ICODE: 5B C h e c k e d after 5 w e e k s C h e c k e d after 6 w e e k s T B L R T B L R 0 0.5 1 05 0 4 0 0 0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  4  0  0  0  0  0  0  0  0  0  0  5  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  6  0  0  0  0  0  0  0  0  2  1  7  0  0  0  0  0  0  3  1  0  0  0  0  0  0  0.5  0  0  0  0.5  0  0  0 0  8  0  0  0  0  0  0  0  0  0  9  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  10  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  11  0  0  0  0  0  0  0  0  0  12  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  13  0  0  0  0  0  0  0  0  0  14  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0.2  0  0.5  0.5  0.2  0  0.5  0.5 0  15  0  0  0  0  0  0  0  0  0  0  16  0  0  0  0  0  0  0  0  0  0  0  0  0  0  17  0.1  0  0  0  0  0  0  0  0  0.2  0  0  0  0  0  0  0  18  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  19  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  20  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  108  Table 25. Fungal Growth on Planed Western Hemlock Samples, Treated with 86 ug/cm 2  Retention:86 | S p e e d : 240  iPlaned  Species: H. Fir  S a m p l e C h e c k e d after 1 w e e k C h e c k e d after 3 w e e k s R L R T B # T B L  Concentration: 2.11  |CODE : 4B C h e c k e d after 6 w e e k s  C h e c k e d after 5 w e e k s  T  B  L  R  1  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  2  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  3  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  4  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  5  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  6  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  7  0  0  0  0  0 0  0  0  0  0  0  0  0  0  0  0  0  8  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  9  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  10  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  11  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  12  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  13  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  14  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  15  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  16  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  17  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  18  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  19  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  20  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  T  B  L  R  Table 26. Fungal Growth on Planed Douglas Fir Samples, Treated with 54 ug/cm  2  Retention:54 | S p e e d : 240 Concentration: 2.11  | Planed  Species: D. Fir  S a m p l e C h e c k e d after 1 w e e k C h e c k e d after 3 w e e k s T L R # T B L R B  CODE : 4A  C h e c k e d after 5 w e e k s  C h e c k e d after 6 w e e k s T  B  L  R  1  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  2  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  3  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  4  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  5  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  6  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  7  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  8  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  9  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  10  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  11  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  12  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  13  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  14  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  15  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  16  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  17  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  18  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  19  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  20  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  T  B  L  R  109  Table 27. Fungal Growth on Rough Western Hemlock Samples, Treated with 130 ug/cm 2  S p e c i e s : H. Fir  Rough Retention:130|Speed: 467 Sample C h e c k e d after 1 w e e k C h e c k e d after 3 w e e k s # T B L R T B L R 1 0 0 0 0 0 0 0 0  Concentration: 4.99 | C O D E : 3 A C h e c k e d after 5 w e e k s  C h e c k e d after 6 w e e k s  T  B  L  R  T  B  L  R  0  0  0  0  0  0  0  0  2  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  3  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  4  0  0  0  0  0  0  0  0  0  0  0  0  0  0 0  0  0  0 0  0  0  0 0  0  0  0 0  0  5  0 0  0  0  6  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  7  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  8  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  9  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  10  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  11  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  12  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  13  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  14  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  15  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  16  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  17  0  0  0  0  0  0  b  0  0  0  0  0  0  0  0  0  18  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  19  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  20  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  Table 28. Fungal Growth on Rough Douglas Fir Samples, Treated with 127 ug/cm  2  S p e c i e s D. Fir  Rough  Retention: 127 Speed:467  Sample C h e c k e d after 1 w e e k C h e c k e d after 3 w e e k s # T B L R T B L R 1 0 0 0 0 0 0 0 0  Concentration: 4.99  CODE:3B  C h e c k e d after 5 w e e k s  C h e c k e d after 6 w e e k s  T  B  L  R  T  B  L  R  0  0  0  0  0  0  0  0 0  2  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  3  0  3  1  1  0  3  1  1  3  0  1  1  0  3  1  1  4  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  5  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  6  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  7  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  8  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  9  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  10  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  11  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  12  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  13  2  0  5  0.5  2  0  5  0.5  2  0  5  0.5  2  0  6  0.5  14  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  15  0.2  0  0.1  0  0.2  0  0.1  0  0.2  0  0.1  0  0.2  0  0.1  0  16  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  17  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  18  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  19  0  0  0  . 0  0  0  0  0  0  0  0  0  0  0  0  0  20  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  110  Table 29. Fungal Growth on Planed Western Hemlock Samples, Treated with 118 ug/cm 2  Species: H. Fir Planed Retentions 18|Speed:400 S a m p l e C h e c k e d after 1 w e e k C h e c k e d after 3 w e e k s # T B L R T B L R 1 0 0 0 0 0 0 0 0  Concentration 4.77  CODE:2A  C h e c k e d after 5 w e e k s  C h e c k e d after 6 w e e k s  T  B  L  R  T  B  L  R  0  0  0  0  0  0  0  0 0  2  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  3  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  4  0  0 0  0 0  0 0  0 0  0 0  0  0 0  0  0 0  0  0  0  5  0  0 0  0  0 0  0  0 0  0  0  0  6  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  7  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  8  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  9  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  10  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  11  0  0  0  0  0  0  0  0  0  0  0  0  0  0  12  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  13  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  14  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  15  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  16  0  0  0  0  0  • 0  0  0  0  0  0  0  0  0  0  0  17  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  18  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  19  0  0  0  0  0  0  0  0  0  0  0  0  0  0  20  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  Table 30. Fungal Growth on Planed Douglas Fir Samples, Treated with 131 ug/cm  2  Species D. Fir  Planed Retention:131 Speed:400 S a m p l e C h e c k e d after 1 w e e k C h e c k e d after 3 w e e k s # T B L R T B L R 1 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0  Concentration: 4.77  C O D E : 3A  C h e c k e d after 5 w e e k s  C h e c k e d after 6 w e e k s T B L R  T  B  L  R  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  3  0  0  0  0  0  0  0  0  0  0  0  0  0  0  4  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  5  0  0  0  0  0  0  0  0  0  0  0  0  0  0  6  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  7  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  8  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  9  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  10  0  0  0  0  0  0  0  0  0  0  0  0  0  0  11  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  12  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  13  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  14  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  15  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  16  0  0  0  0  0  0  0  0  0  0  0  0  0  0  17  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  18  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  19  0  0  0  0  0  0  0  0  0  0  0  0  0  0  20  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  111  Table 31. Fungal Growth on Rough Western Hemlock Samples, Treated with 154 ug/cm 2  S p e c i e s : H. Fir  |Rough  Retention:154 Speed:400  S a m p l e C h e c k e d after 1 w e e k C h e c k e d after 3 w e e k s  Concentration: 5.96  C O D E : 1B  C h e c k e d after 5 w e e k s  C h e c k e d after 6 w e e k s  #  T  B  L  R  T  B  L  R  T  B  L  R  T  B  L  R  1  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  2  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  3  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  4  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  5  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  6 7  0 0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0 .  0  0  0  0  0  0  0  0  0  0  8  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  9  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  10  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  11  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  12  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  13  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  14  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  15  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  16  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  17  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  18  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  19  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  20  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  Table 32. Fungal Growth of Rough Douglas Fir Samples, Treated with 177 ug/cm  2  Rough  S p e c i e s : D. Fir  Retention :177 Speed:400  S a m p l e C h e c k e d after 1 w e e k C h e c k e d after 3 w e e k s  Concentration: 5.96  C O D E : 1A  C h e c k e d after 5 w e e k s  C h e c k e d after 6 w e e k s  #  T  B  L  R  T  B  L  R  T  B  L  R  T  B  L  R  1  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  2  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  3  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  4  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  5  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  6  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  7  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  8  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  9  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  10  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  11  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  12  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  13  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  14  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  15  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  16  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  17  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  • 18  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  19  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  20  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  112  8.5.2 Comments and Conclusions  The results of these field tests contributed towards the answering of important questions that could not be answered under laboratory conditions. There were several elements, which contributed to the complexity of this test:  Wood Species The two most common wood species from B.C. mills were used. This was done to determine the amount of preservative necessary for application and to compare which species better absorbs the preservative.  Table 33, a summary of data provided from  Forintek's pilot-plant tests, demonstrates that for a planed surface of the wood, and lower concentrations of chemical (~2 %), Western Hemlock absorbed the preservative more readily. Since rough samples have much larger and varying surface areas, it was not possible to come to a conclusion about which species absorbed chemical in the rough condition easier.  Table 33. The Relation Between Preservative Concentration and Achieved Retention For Douglas Fir and Western Hemlock Concentration (%) 2.17 2.11 4.99 4.77 5.96  D. Fir Retention (ug/cm2) 44 54 127 131 177  W.H. Retention (ug/cm2) 70 86 130 118 154  Surface Type Planed Planed Rough Planed Rough  113  Number and Positions of Samples  All samples of the same species that were sprayed with the same concentration were stored in piles. There were all together 20 piles with 20 pieces (4X5) plus two piles with nine wood samples (control samples). A l l sprayed and control piles were placed close to natural fungal sources (forest and wastewater treatment plant), and exposed to natural weather conditions.  During the experiment, samples were carefully examined from all sides (top, bottom, left and right). To ensure that every sample had a chance to be reached by fungal spores, after examination, their position in the pile was randomly changed.  Due to natural weather and field conditions, every sample had a unique opportunity to interact with fungal infestation. This is the reason why the results are not presented as an average. If fungal growth was detected in any of twenty samples from the same pile, it could mean that after a change in position or weather conditions other samples could also become infected.  For example in Table 24, the preservative retention on Douglas Fir samples was undesirably two times lower than was targeted. The targeted retention of 60 ug/cm was 2  not obtained, instead the actual retention was 44 ug/cm . Consequently, samples #1, #7, 2  #14 and #16 showed some fungal growth. In Table 27, rough Douglas Fir samples #3, #13 and #15 were sprayed with 127 ug/cm2, very close to the targeted retention (120  114  ug/cm ), but still fungal growth appeared. It is important to emphasize that these samples 2  were very dirty before spraying and also might have been infested before spraying. As shown in Tables 21 and 22, there was some infestation on both control groups, so the absence of fungal growth on all other samples could be attributed to the presence of the right amounts of preservative.  Active Ingredient Concentration  The recommended active ingredient concentration for most commercial antisapstain products is about -100 ug/cm2 (Konashhevich, 1994). The results of this field test open the door to the possibility of applying a lower amount of chemical (60-90 ug/cm2) while still providing satisfactory fungal protection. Since in comparison with other commercial products, the fish toxicity of this preservative has had encouraging results, using lower amounts of chemicals would contribute to better compliance with environmental regulations.  115  Chapter 9: Economic Aspects The primary strategy in marketing a new antisapstain product is to emphasize the features, benefits and advantages as compared to the competitors' product(s). The specific product features and benefits are the following:  •  Broader spectrum of activity  •  Combating resistant fungi  •  Lower fish toxicity  •  Environmentally and user friendly  •  Cost Effective  The first four aspects are illustrated in details in previous sections of this thesis. The final consideration, which will decide whether or not the potential new formulation can be competitive on the market, is an economical analysis and cost effectiveness.  9.1 Product Cost  According to information from the literature (Konashevich, 1994), the required fungicide coverage, for the lumber protected by D D A C is 100 micrograms per square centimeter (100 ug/cm ) of active ingredients. This coverage concentration is the critical element for  116  both effective protection and the cost of chemicals to the mill. Therefore competitive price comparisons were done on this basis.  Tables 34-35 show the difference in the cost of active chemicals between the New Developed Product  (NDP) and the Current Major Product (CMP). The prices of  chemicals were obtained from the manufacturers of the active components. As the Table shows, there is a difference of approximately 20.7% in the cost of chemicals per kilogram of product.  Table 34. Chemical Cost Comparison (per kilogram) of the New Developed Product (NDP) and the Current Major Product (CMP) Product: CMP NDP  Product Cost: ($/kg) C D N 8.55 C D N 6.78  Table 35. Chemical Cost Comparison (per liter) of NDP (Specific Gravity: 0.96) and CMP (Specific Gravity: 0.93) Product: CMP NDP  Product Cost: ($/liter) C D N 9.19 C D N 7.06  Table 36 shows the calculated difference in price when compared on the basis of chemical coverage per 1,000 board feet ("FBM"). This difference is 5%, a definite price advantage for the New Developed Product (NDP) due primarily to a lower chemical cost of goods per unit volume of product.  117  Table 36. Cost Comparison (per amount of active ingredient) of the New Developed Product (NDP) and the Current Major Product (CMP), per 1,000 F B M . Chemical Cost per 1,000 F B M (in Canadian Funds) Desired Coverage 100 ug/cm 80 ug/cm 120 ug/cm 2  CMP NDP NDP's Advantage:  2  6.43 6.11  5.15 4.89  7.72 7.34  5.0%  5.0%  5.0%  2  The cost calculation comparisons presented in Table 36, are based on coverage costs on 2" x 4" dimensional lumber with desired coverage ratios of 80, 100, and 120 micrograms per square centimeter (ug/cm2) of active ingredients. These calculations are presented on a per 1,000 foot board measurement (FBM or fbm) basis.  Table 37 shows the summary of Toxicological Characteristics of New Developed Product (NDP) and the Current Major Product (CMP) and their calculated chemical costs. While maintaining better fish toxicity characteristics, NDP has an important advantage in the chemical cost.  Table 37. Toxicity comparison of the New Developed Product (NDP) and the Current Major Product (CMP) Product  CMP NDP  Chemical Cost Per l K g 8.55 6.78  LC50 (mg/1) (Rainbow Trout) 0.68-1.3 1.41  Recommended Dilutions in Water 40-50 40-50  ** Konashevich, 1994 provides these two values for L C  5 0  Rainbow Trout  118  Chapter 10: General Conclusions and Recommendations for Future Work  10.1 Conclusions  The process of the development of a new antisapstain product based on the screening of potential fungicides suitable to be incorporated as a second active component in combination with D D A C , was investigated in the present study. The main observations and conclusions from this research are:  •  The research resulted in the successful development of a new antisapstain product, which is capable of protecting freshly cut wood from moulds, stains, and white, soft and brown rot fungi.  •  The additional biocide, which has been combined with D D A C , showed a synergistic effect with D D A C and proved that it could significantly broaden the spectrum of DDAC's fungicidal activity.  •  The combination of D D A C and C2 demonstrated the capability of counteracting any resistance of tolerant microorganisms toward D D A C .  119  •  The new antisapstain product was designed as a highly dispersible combination, which allows D D A C to serve as a vehicle, enabling full C2 coverage of, and a strong adherence to, the wood's surface.  •  The combination of  active  components  resulted  in a formulation that  is  environmentally friendly (less toxic and damaging to the environment than the leading competitor products).  •  Extremely low concentrations of the second active component C2 not only enhance the fungicidal activity of D D A C , but due to its effectiveness, reduced the amount of D D A C required, which also improves the toxicological characteristics of the composition when D D A C and C2 are combined.  •  While maintaining better fish toxicity characteristics, the New Developed Product is more cost effective than the leading product in the B.C. market.  •  A patent for this process has been applied for and subject to some revisions, it appears that it will be granted.  10.2 Recommendations for Future Work The main recommendations for future work are: •  To continue field-tests through the spring, when intensive fungal growth is expected at higher humidity and elevated temperatures.  120  Literature Amburgey T., Observation on the Soil-Block Methods of Evaluating Wood DecayTests, Material und Organismen 11/4, 1976  Archer Kevin and Nicholas Darrel D., Screenings of Wood Preservatives: Comparison of the Soil-Block, Agar-Block, and Agar-Plate Tests, Forest Product Journal 45, 1995.  Byrne Tony, Lumber Protection in the 90's, Proceedings of meeting held at: FORINTEK C A N A D A CORP., Western laboratory, 1992.  Butcher John A., Serial Exposure Technique for Assessing Performance of Wood Preservatives, Forest Research Institute, New Zealand, 1979.  Butcher John A., Preston Alan F. and Drysdale Jeanette, Potential of Unmodified  Alkylammonium Compounds as Groundline Preservatives, Forest Research Institute, New Zealand, 1979.  Butcher John A . and Drysdale Jeanette, Relative Tolerance of Seven Wood-destroying  Basidiomicetes to Quaternary Ammonium Compounds and Copper-Chrome-Arsenate Preservative, Forest Research Institute, New Zealand, 1978.  121  Butcher John A . and Drysdale Jeanette, Efficacy of Acid and Alkaline Solutions of Alkylammonium Compounds as Wood Preservatives, Forest Research Institute, New Zealand, 1978.  Cassens D.L. and Eslyn W.E., Fungicides to Prevent Sapstain and Mold on Hardwood Lumber, Forest Product Journal 31(9), 1982.  Cartwright K . and Findlay W, Decay of Timber and its Prevention, Her Majesty's Stationery Office, 1958.  COF/Fact Book 98, http//www.cofi.org/factbook98/one/l-5,htm.  Cserjesi A.J. and Johnson E.L., Mold and Sapstain Control: Laboratory and Field Tests of 44 Fungicidal Formulations, Forest Product Journal 31(9), 1982.  De Groot Rodney C , Alternative Species and Preservatives for Wood Roofing: Laboratory Decay Studies, Forest Product Journal 42 (11/12), 1992.  Eduljee G., Secondary Exposure to Dioxins Through Exposure to PCP and its Derivatives, Science of the Total Environment 232: (3), 1999.  Henderson, N . D., A Review of the Environmental Impact and Toxic Effects of DDAC, B C Environment Ministry of Environment Land and Parks, Victoria, 1992.  122  Hryhorczuk et al, A morbility Study of Former Pentachlorophenol-production workers, Environmental Health Perspectives 106 (7), 1998.  Hsu Jemin C , Synergistic Microbicidal Combinations, U.S. Patent No. 289066, 1988.  Dubois J.W., Byrne A . Clark J.E., Canadian Bluestain Fungi: Variation in Tolerance to Sapstain Control Biocides, Forest Product Journal 50 (1), 2000.  Ince P. J., Industrial Wood productivity in the United States, 1900-1998, USDA, United States Department of Agriculture, March 2000.  Jin L . H , Preston A.F., The Interaction of Wood Preservatives with Lignocellulosic Substrates, I. Quaternary Ammonium compounds, Holzforschung 45, 1991.  Lyr Horst, Modern Selective Fungicides, Gustav Fisher Verlag, Jena, 1995.  Konashewich  D.E., Anti-Sapstain  Wood Protection,  Environment Canada B C  Environmennnt, 1994.  Krahn P.K., Antisapstain Protection, Environment Canada Pacific and Yukon Region, Wsiwyg://127/htp://vvww.pyr.ec.gc.ca/ep/enforcement/96a-l.htm.  123  Kreber B., Formation of Brownstain in Inoculated Western Hemlock Sap, Material und Organismen 30 (1), 1996.  Lee D., Takahasgi M . and Tsunoda K., Fungal Detoxification of Organoiodine Wood Preservatives Part 2. Fungal Metabolism in the Decomposition of the Chemicals, Holzforshung46(1992).  May  Oscar  W.,  Synergistic  Compositions  Containing  Hydroxypropyl  Methanethiolsulfonate and Methods of Using Same, U.S. Patent No.4650808, 1987.  McCullagh Karen, Regulation of Wood Preservatives in Canada Proceedings Canadian Wood Preservation Association, pg.95, 1991.  Nicholas Darrel D and Preston Alan F., Evaluation of Alkyl Ammonium Compounds as Potential Wood Preservatives, American Wood Preservatives' Assotiation, 1980.  Peper M.., Ertl M . , and Gerhard I, Long Term Exposure to Wood-Preserving Chemicals Containing Pentachlorophenol is Related to Neurobehavioral Performance in Women, American Journal of Industrial Medicine 35: (6), 1999.  Pocious C. Frances, C , Synergistic Blend of Biocides, U.S. Patent No. 4295932, 1981.  124  Preston A.F.,- and Chittenden C. M , Alkyylammonium Wood Preservativess,  Compounds  as  Above-Ground  New Zealand Journal of Forestry Science 12 (1), 1982.  Nicholas D.D., Williams A.D., Preston A.F., Zhang S.,Distribution and Permanency DDAC  in Southern  Pine Sapwood  Treated by the Full-Cell  Process,  of  Forest Product  Journal 41, 1991.  Preston Alan, F., Wood preservation  to the year 2000, Proceedings Canadian Wood  Preservation Association, 1988.  Rosen, M . J., Relationship of Structure to Properties in Surfactants: (1) Adsorption of the Solid-Liquid  Interface from Aqueous Solutions, J. Amer. Oil. Chem. Soc. 52 (11),  Ruddick John N.R., The influence of Staining Fungi on the Decay Resistance Treated with Alkyldimethylbenzylammonium  Chloride,  1975.  of Wood  Material und Organismen 21/2,  1986.  Ruddick  John N.R. and Sam Anthony R.H., Didecyldimethylammonium  Quaternary Ammonium  Wood Preservative:  Chloride-  Its Leachability from, and Distribution  a in,  Four Softwoods, Material und Organismen 17/4, 1982.  Smith, R. S. Chemical  Opportunities for Lumber Protection,  Proceedings of a Joint  Forintek Canada Corp. and COFI Workshop, 1985.  125  U N - E C E / F A O , Forest Products Annual Market Review 1998-1999, Timber Bulletin, VolXii,ECE/TIM/BULL/52/3,http://wwwunece.org/trade/timber/docs/rev-99/rev99.htm.  USDA, United States Department of Agriculture, Industrial Wood Productivity in the  United States, 1900-1998.  Vyas Subhash C , Nontarget Effects of Agricultural Fungicides, CRC Press Inc. Florida, 1988.  Wakeling R.N., Protection  of Export Logs from Fungal degrade, Wood processing  Newsletter Issue No. 19, 1996.  Ward Hans A., Wood Preservatives, U.S. Patent No.397,692, 1989.  Webster John, Introduction to Fungi, Alden & Mowbray L T D , Oxford, 1970.  Xiao Y. and Kreber B., Effect of IPBC/DDAC on Spore Germination and Hyphal Growth  of the Sapstaining Fungus Ophistoma piceae, Holzforschung 53, 1999.  Zwick, Robert, International Market Potential for Treated Wood Products from B.C. Proceedings Canadian Wood Preservation Association, 1989.  126  Appendix 1 List of Fungal Cultures  List of Fungal Cultures Obtained from FORJNTEK C A N A D A CORP.  1. Aspergillus niger 2. Coniophora puteana 9B 3. Coriolus versicolor 105E 4. Aureobasidium pullulans 13 2B 5. Chaetomium globosum 172B 6. Gliocladium roseum 32IF 7. Ceratocystis adiopsa  Appendix 2 Bardac 2250 & Bardac 2280 Manufacturer Specifications  L O N Z A INC. Corporate Headquarters 17-17 Route 208 Fair Lawn. N . J . 07410 (201) 794-2400  Specialty Chemicals  ( v  ^  INTRODUCTION Bardac 2250/2280, didecyl dimethyl ammonium chloride represents a class of germicidally active quaternary ammonium compounds that is radically different from the traditional alkyl benzyl type of quaternary. The Bardac "twin chain" type of structure has produced quaternary germicides with greatly improved performance characteristics over quaternary products with a benzene ring structure. M a d e by Lonza patented process technology, the Bardac products, with their superior performance, have had significant impact on the germicide industry. The Bardac quaternary products are dialkyl dimethyl ammonium chlorides in which two alkyl groups in the C to C range are attached directly to the nitrogen atom. These alkyl chains were f o u n d to maximize germicidal performance; Bardac 2250/2280 has the chemical structure shown to the right.  CI  8  1 0  Where R.= n-decyl  Chemical Composition - Typical Active Ingredients  Bardac 2280  Bardac 2250  Didecyl dimethyl ammonium chloride ............ 50.0% Inert Ingredients:  80.0%  . u . „ . ^ 50.0% .:  20.0%  Specifications % Quaternary @ MW=361 Color (APHA) pH (10% Active Solution)  50-52 200Max ,6.5 - 9.0  80 - 82 250 Max. ....6.5 - 9.0  Specifications are based upon Lonza's Analytical Test Methods, copies of which are available upon request..  Physical Properties Physical State Flash Point (Seta Flash) Specific Gravity at 2 5 ° C Density (Ibs./gallon) EPA Registration N u m b e r CAS N o  Liquid 109°F 0.927 7.73  107°F 0.891 7.43  6836-51. v.... 6836-53 :.. 7173-51-5  130 The seller makes no warranty, expressed or implied, concerning the accuracy ol any results to be obtained Irom the use ol any inlormation and no warranty expressed or implied concerning the use of the products other than indicated above. The buyer assumes all risks of use and/or handling. No statement is intended or should be construed as a  GERMICIDAL ACTIVITY  The germicidal activity of Bardac 2250/2280 is substantiated by an extensive series of generally recognized microbiological tests including those required for EPA registration. Standard laboratory evaluations indicate that Bardac 2250/2280 has superior over-all germicidal activity when compared to quaternaries with other structures. This activity is also exhibited under use conditions once considered detrimental to the performance of quaternaries. The following summarizes the advantages of Bardac 2250/2280. . . . Broad spectrum biocidal activity against both gram positive and gram negative organisms. . . . Better disinfectant performance at lower use concentrations. . . . Greater hard water tolerance for sanitizing activity at lower use concentrations. . . . Superior fungicidal performance. . . . Substantial organic soil tolerance. . . . Greater tolerance for anionic contaminants than previously possible.  Disinfectant Activity Determined by A O A C Use-Dilution Tests  The minimum concentration of Bardac 2250/2280 required for effective disinfection is determined by the A O A C , commonly known as the Use-Dilution Test.  ATCC Strain No.  Test Organism  Staphylococcus aureus Salmonella choleraesuis Pseudomonas aeruginosa (PRD-10)  Quaternary concentration on 100% active basis  6538 10708. 15442  300 ppm 300 ppm 500 ppm  Organic Matter^Tblerance and Residual Anionic Tolerance Determined by the A O A C Use-Dilution Test . • :  Organic matter normally interferes with the biocidal activity of quaternary compounds. When compared to other quaternary compounds; Bardac 2250/2280 maintains an unusually high level of activity in the presence of proteinaceous soil. This is substantiated by the fact that Bardac 2250/2280 at 400 ppm active concentration in the presence of 5% blood serum is effective against Staphylococcus aureus accordingto the AOAC Use-Dilution Test.  Test Organism  Staphylococcus aureus  ATCC Strain No.  Contaminent  6538  Blood Serum 5%  Quaternary concentration on 100% active basis  400 ppm  Quaternaries, which are cationic surface active agents, are deactivated by anionic surface active agents with the formation of an insoluble cationic-anionic complex. The ability of Bardac 2250/2280 to maintain bactericidal activity in the presence of anionics is important because residual amounts of anionics are often present in hard surface disinfection applications. When Bardac 2250/2280 is evaluated at a concentration of 400 ppm active against Staphylococcus aureus in the presence of 300 ppm sodium lauryl sulfate, it passes the AOAC Use-Dilution test whereas other quaternaries fail.  Test Organism  Quaternary concentration on 100% active basis  Strain No.  Contaminent  Staphylococcus aureus  6538  300 ppm sodium lauryl sulfate  Sanitizing Activity Determined Method  by the A O A C Germicidal and Detergent Sanitizer  400 ppm  The germicidal activity of quaternary products decreases in the presence of hard water. The hard water tolerance of Bardac 2250/2280 is measured by the AOAC Germicidal and Detergent Sanitizer Method, commonly called the Hard Water Tolerance test. Exposure of 100 million organisms of Escherichia Coli (#11229) to 150 ppm of Bardac 2250/2280 for 30 seconds at 25°C in 7,000 ppm of" water hardness results in the required reduction of 99.999% of the bacteria. Calcium and magnesium salts are typical hard water components; however, other electrolytes may be present during actual field applications. The unusually high hard water tolerance of Bardac 2250/2280 affords users a margin of safety over a range of quaternary concentrations. .  . Test Organism  Concentration of Bardac 2250/2280 Required for 99.999% Reduction  Hard Water Ceiling -  50 ppm 100 ppm 150 ppm 200 ppm  .250 ppm hard water 1600 ppm.hard water 1,000 ppm hard water 1,500 ppm.hard water  Escherichia Coli #11229  • .  Fungicidal Performance as Determined by the A O A C Fungicidal Test  Possessing superior fungicidal activity, Bardac 2250/2280 effectively passes the AOAC Fungicidal test at one-fifth to one-seventh the concentration required for alkyl benzyl, quaternaries. Ten Minute Killing Dilution (100% Active)  Bardac 2250/2280  Ten Minute Killing Dilution In Use Dilution =  P. ovale #12098  1:4845 200 ppm  T. C. mentagrophytes albicans C D C #-X-32 #14053  1:7350 150 ppm  1:7350 150 ppm  Phenol Coefficient as Determined by the A O A C Phenol Coefficient Test  The phenol coefficient of a germicide is measured by its relative effectiveness in comparison to phenol. The bactericidal activity of Bardac 2250/2280 produces phenol coefficient results that surpass the alkyl benzyl quaternaries. The test protocol is.AOAC.  Test Organism  ATCC Strain N o .  Phenol Coefficient 100% active quaternary  Staphylococcus aureus Salmonella typhosa  6538 6539  1050 1050  Bactericidal Efficiency as Determined by the M i n i m u m Inhibitory Concentration Test  This tube-dilution test determines the minimum concentration of Bardac 2250/2280 which will reduce the bacterial count of a suspension by at least 99.999% from a level of 100 million organisms per ml. in 30 seconds at 25°G The test method is a broth dilution test with 18 hour immersion at 37°C. Minimum inhibitory concentration test results indicate that Bardac 2250/2280 offers superior performance. A T C C Strain N o . Test Organism  (unless otherwise noted)  Staphylococcus aureus Escherichia Coli Pseudomonas aeruginosa Proteus vulgaris  6538 11229 15442 9920  Gram Stain  M i n i m u m Inhibitory Concentration (100% active)  + -  0.5 ppm 5.0 ppm 50.0 ppm 20.0 ppm  Fungi  A. niger T. menragrophytes C. albicans  16404 (SWRI) Enmons 10231  5.0 ppm . 5.0 ppm 1-5 ppm  Summary of Germicidal Activity  Th e test da t a p res en t ed substantiates the over-allsuperior.germicidabperforrnance of Bardac 2250/2280. APPLICATIONS  '  The unique performance characteristics of Bardac 2250/2280 have been used to develop a series of disinfectants, sanitizers, and cleaners that are in commercial use in homes, , hospitals, and institutions. These systems range from simple dilutions to more •complex blends of quaternaries, surfactants and detergent builders. Summary of Applications and R e c o m m e n d e d Use Levels Application Areas  General-Hospital Disinfection Sanitizing Water Treatment/Cooling Towers Water Treatment/Secondary Oil Recovery Laundry Mildew Preventative/Sanitizer Bacteriostat/Preservative/Fungicide  R e c o m m e n d e d Use-Levels on 100% Active Basis  300-500 ppm ' 150 ppm 5-20 ppm 5-20 ppm 630 ppm 5-1000 ppm  Wood Preservation B a r d a c 2250/2280 will effectively c o n t r o l the d e c a y of w o o d c a u s e d by a b r o a d s p e c t r u m of fungi w h e n utilized in e x t e r i o r a b o v e g r o u n d situations s u c h as f e n c e a n d p o r c h rails, d e c k s , r o o f i n g , s i d i n g , w i n d o w a n d d o o r f r a m e s , etc. T o a c h i e v e d e c a y c o n t r o l , l a b o r a t o r y studies utilizing a v a c u u m i m p r e g n a t i o n m e t h o d of a p p l i c a t i o n i n d i c a t e that r e t e n t i o n levels of 0.06 to 0.4 lbs. per c u b i c foot (1.0 to 6.4 K g / m ) are r e q u i r e d to c o n t r o l all of the test f u n g i . 3  S o l u t i o n s of B a r d a c 2250/2280 are stable a n d c a n b e u s e d in p r e s e n t l y available t r e a t m e n t plants w i t h o u t e q u i p m e n t Lumber  treated  with  modifications.  s o l u t i o n s of  B a r d a c 2250/2280 s h o w  no color change  after  t r e a t m e n t . Light c o l o r e d paints a n d c o a t i n g s also s h o w n o d i s c o l o r a t i o n o n treated surfaces. O p e r a t o r s h a n d l i n g freshly t r e a t e d wet l u m b e r s h o u l d w e a r r u b b e r g l o v e s , but d r y t r e a t e d l u m b e r can b e h a n d l e d w i t h o u t any d e r m a l p r o t e c t i o n . In d e v e l o p i n g y o u r final p r o d u c t a n d label b a s e d o n B a r d a c 2250/2280, y o u s h o u l d s p e c i f y m e t h o d s of a p p l i c a t i o n w h i c h will p r o v i d e sufficient r e t e n t i o n levels (as n o t e d a b o v e ) to p r e v e n t d e c a y . If b r u s h or soak a p p l i c a t i o n s are r e c o m m e n d e d , the t h o r o u g h n e s s of s u c h a p p l i c a t i o n s r e q u i r e d to o b t a i n t h e d e s i r e d r e t e n t i o n level must b e i n d i c a t e d (i.e., two f l o w i n g b r u s h coats o r 30 m i n u t e s soak, etc.).  GENERAL  INFORMATION  P r o d u c t Registration B i o c i d e s a n d p r o d u c t s with b i o c i d a l c l a i m s r e q u i r e registration b y the E n v i r o n m e n t a l P r o t e c t i o n A g e n c y u n d e r the F e d e r a l I n s e c t i c i d e , F u n g i c i d e a n d R o d e n t i c i d e A c t . (n a d d i t i o n , most state authorities r e q u i r e s e p a r a t e registration. T h e E P A N U M B E R S F O R B A R D A C 2250 A N D B A R D A C 2280 are 6836-51 a n d 6836-53 r e s p e c t i v e l y , a n d m a y b e r e f e r r e d to b y c o n s u m e r s o f this p r o d u c t . A s a t e c h n i c a l s e r v i c e , L O N Z A I N C . p r o v i d e s - a d v i c e o n t h e registration of B a r d a c 2250/2280 b a s e d p r o d u c t s .  iSafety and Handling T h e toxicity of B a r d a c 2250/2280 is of the s a m e o r d e r of m a g n i t u d e as o t h e r c o m m e r c i a l q u a t e r n a r i e s . B a r d a c 2250/2280 at an " a s - i s " c o n c e n t r a t i o n m a y b e c o n s i d e r e d a p r i m a r y skin a n d e y e irritant. T h e data b e l o w s u m m a r i z e s the a c u t e oral a n d d e r m a l toxicity of B a r d a c 2250/2280 o n a 80% activity basis as d e t e r m i n e d in m i c e a n d rabbits r e s p e c t i v e l y . Oral L D  5 0  = 450 m g / k g  Acute Dermal L D For  5 0  - 3342 m g / k g  d e t a i l e d h a n d l i n g i n f o r m a t i o n c o n s u l t t h e B a r d a c 2250/2280 M a t e r i a l Safety Data  S h e e t w h i c h is available u p o n r e q u e s t  —  Packaging B a r d a c 2250 is available in 425 l b . net w e i g h t d r u m s . B a r d a c 2280 is available in 410 lb.- net w e i g h t d r u m s .  General and Hospital Disinfection A g e n e r a l p u r p o s e D i s i n f e c t a n t - S a n i t i z e r - F u n g i c i d e - D e o d o r i z e r p r o d u c t may b e p r o d u c e d by d i l u t i n g B a r d a c 2250/2280 to 7.5% a c t i v e l e v e l . This p r o d u c t - w i l l a f f o r d e f f e c t i v e d i s i n f e c t i o n a n d / o r s a n i t i z i n g in h o s p i t a l s , s c h o o l s , h o m e s , d a i r y , farm a n d i n d u s t r i a l areas w h e n u s e d at a p p r o p r i a t e use d i l u t i o n s . A s a m p l e l a b e l for a D i s i n f e c t a n t - S a n i t i z e r - F u n g i c i d e - D e o d o r i z e r b a s e d o n B a r d a c 2250/2280 is a t t a c h e d . It may b e u s e d as a g u i d e i n r e g i s t e r i n g y o u r p r o d u c t w i t h the E n v i r o n m e n t a l P r o t e c t i o n Agency. Sanitizing B a r d a c 2250/2280 has b e e n c l e a r e d by the F D A , Title 21, C o d e of F e d e r a l R e g u l a t i o n s , S e c t i o n 178.1010, F o o d A d d i t i v e s , f o r s a n i t i z i n g at a c o n c e n t r a t i o n of 150ppm without the requirement of a potable water rinse. In a d d i t i o n , the use of this s a n i t i z i n g s o l u t i o n is consistent w i t h the c u r r e n t p r a c t i c e s of t h e Grade "A" Pasteurized Milk Ordinance, 1978 Recommendations of the U n i t e d States P u b l i c H e a l t h S e r v i c e . T h e h i g h h a r d water t o l e r a n c e of B a r d a c 2250/2280 a l l o w s the use o f a l o w e r c o n c e n t r a t i o n (150 p p m uselevel) at a h a r d water c e i l i n g of 1,000 p p m . In c o n t r a s t / t h e alkyl b e n z y l q u a t e r n a r i e s w h e n u s e d as sanitizers must b e u s e d at 200 p p m . This l o w e r use c o n c e n t r a t i o n f o r B a r d a c 2250/2280 results in a m a t e r i a l s a v i n g of 25%.  Water Treatment Microbiocide/Cooling Tower B a r d a c 2250/2280 p r o v i d e s the f o r m u l a t o r w i t h a s u p e r i o r m i c r o b i o c i d e for b u i l d i n g a n d i n d u s t r i a l c o o l i n g t o w e r s at use levels o f 5 - 20 p p m .  Water Treatment Microbiocide/Secondary Oil Recovery M a n y w a t e r f l o o d s in the s e c o n d a r y r e c o v e r y of o i l c o n t a i n b a c t e r i a i n c l u d i n g t h e sulfate r e d u c i n g b a c t e r i a D e s u l f p v i b r i o d e s u l f u r i c a n s . This m i c r o - o r g a n i s m p r o d u c e s c o r r o s i v e d e g r a d a t i o n p r o d u c t s . B a r d a c 2250/2280 w i l l i n h i b i t t h e g r o w t h of D. d e s u l f u r i c a n s , a n d thus r e d u c e t h e c o r r o s i v e n a t u r e of t h e w a t e r f l o o d . W h e r e oil f i e l d f l o o d waters a n d salt w a t e r d i s p o s a l systems r e q u i r e t h e use o f a m i c r o b i o c i d e , B a r d a c 2250/2280 m a y - b e a d d e d to t h e s y s t e m at a level of 5.0 to 20,0 p p m . B a r d a c 2 2 5 0 / 2 2 8 0 is r e g i s t e r e d w i t h t h e E n v i r o n m e n t a l P r o t e c t i o n A g e n c y (file*6836-32) for b o t h c o o l i n g t o w e r a n d s e c o n d a r y Oil r e c o v e r y a p p l i c a t i o n s of w a t e r t r e a t m e n t u s e ; a s a m p l e l a b e l is a v a i l a b l e for t h e s e a p p l i c a t i o n s . : ^V :  ;  Laundry Mildew Preventative/Sanitizer S o i l e d c o m m e r c i a l l i n e n s are p r o n e t o m o l d a n d m i l d e w attack c a u s i n g p e r m a n e n t s t a i n i n g . B a r d a c 2250/2280 at use level.630 p p m b a s e d o n the d r y w e i g h t o f the f a b r i c is r e c o m m e n d e d in the final rinse of t h e l a u n d r y c y c l e to r e d u c e the loss of l i n e n s d u e to s t a i n i n g . A t this l e v e l , B a r d a c 2250/2280 p r o v i d e s r e s i d u a l b a c t e r i o s t a t i c a n d self s a n i t i z i n g p r o t e c t i o n . For these s a n i t i z i n g p r o c e s s p u r p o s e s , B a r d a c 2250/2280 is EPA r e g i s t e r e d as a L o n z a l a u n d r y m i l d e w p r e v e n t a t i v e (file #6836-30); a s a m p l e label is a v a i l a b l e as a t e c h n i c a l s e r v i c e .  Bacteriostat/Preservative/Fungicide B a r d a c 2250/2280 is a h i g h l y e f f e c t i v e b r o a d s p e c t r u m bacteriostat for a variety of i n d u s t r i a l a p p l i c a t i o n s . For situations w h e r e c o m p a t i b i l i t y w i t h a c a t i o n i c material has b e e n e s t a b l i s h e d , t h e a c t u a l use levels f o r the q u a t e r n a r y s h o u l d b e d e t e r m i n e d for e a c h application.  IMPORTANT — PLEASE READ CAREFULLY!  Production Formula and Procedure of Preparation for 7.5% Active Bardac 2250/2280 Dilution  The formula you are manufacturing is EPA registered, thus specifying by law the correct amounts of active ingredients to be present in your finished product. Therefore, the amounts and directions for the proper production of the formula given below must be followed explicitly. The greatest accuracy in preparing this formulation is achieved when all ingredients are added by weight. If this isnot possible, measure the liquidsand weigh the solids. The amounts given are for a production batch of 1,000 lbs. In order to produce largeror smaller batches, merely add multiples or fractions of the amounts listed. The specific gravity/density of the ingredients required to correctly produce a 7.5% active Bardac 2250/2280 dilution have been taken into account in the amounts given below: Bardac 2250/2280 /7.5% Dilution 1,000 lb. batch  Ingredients  Bardac 2250 Bardac 2280 Water Total  Density Ib./gal.  ; % wt/wt 2250 2280  If Addition is by Weight, add in lbs. 2250 2280  7.73 7.43 8.34  15.0 — — 9.38 85.0 90.62 100.0 100.00  150.0 — — 93.8 850.0 906.2 1,000.01,000.0  If Addition is by Volume, add in gallons 2250 2280  19.4 101.92 123.32  — 12.62 108.66 121.28  • Specific Gravity at room temperature = of Bardac 2250/2280 - 7.5% active dilution  0.974  • Density at rOom temperature of Bardac 2250/2280 - 7.5% active dilution  8.12 lbs./gal.  =  Production Procedure for Bardac 2250/2280 - 7.5% Active Dilution  In a suitable blending vessel add together the water and Bardac 2250/2280. Mix for 15 minutes, making sure a clear, uniform solution has been achieved. 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