Vol. 3

- ------

AUTUMN 1967

INTERNATIONAL

BIODETERIORATION

BuLLETIN

BIODETERIORATION INFORMATION CENTRE

THE UNIVERSITY OF ASTON IN BIRMINGHAM

ENGLAND

No.2

CATOMANCE LIMITED

manufacturers of

mystox* for the preservation of

timber, textiles paper, cordage

plastics and specialised applications

* mtJsto.'l: is the registered trade mark of

Catomance Limited 94 BRIDGE ROAD EAST, WELWYl'i GARDEN CITY,

HERTS., E:'IIGLA:'IID. Telephone: Welwyn Garden 24373/8

BerkBiocides As is appropriate to the "largest manufacturers of organo-mercurial com­pounds in the U.K., Berk have built up a considerable bank of knowledge on the problems of biodeterioration and a range of outstanding biocides. to surmount them. Included in this range are the following compounds: ORGANO-MERCUR/ALS PHELAM® (Phenylmercuric dimethyldithiocarbamate) which, being ex­tremely insoluble, is particularly suitable for use in anti-fouling paints. Its exceptionally low vapour pressure greatly reduces toxicity hazards both in handling and in domestic paint applications. Our work has also indicated that it is suitable for PVC preservation. Phelam can be supplied in bulk or in Berk Solu-packs*. P.M.A. (Phenylmercuric acetate) which is suitable for use in paper mills, paints, pharmaceuticals, polishes and adhesives. P.M.A. is available in bulk or in Solu-packs* and in either pharmaceutical or technical grades­high quality g6fg8 Benzene soluble with low polymercurates. It can also be obtained in go~ri and JOo/0 solutions. P.M.O. (Phenylmercuric oleate) which, supplied as a so% solution in an aromatic base, is particularly useful in oil based paints and for wood preservation. This range also includes Phenylmercuric chloride and Phenylmercuric naphthenate soo/o solution. * Berk Solu-packs are cold water soluble sachets which offer great ad~·atllages in accurate dosing and safe handling. ORGANOTINS TRIBUTYL TIN OXIDE which, supplied as a liquid, is recommended for use in anti-fouling paints, decorative finishes a'nd for wood preservation. T.B.T.A, T.B.T.C, T.P.T.A, T.P.T.C and T.P.T.H are other compounds obtain­able in this range. If you are interested in any of these compounds or applications please tick the appropriate box and send the coupon below to Berk.

r-----------------------------, 11 ~ Berk Limited Berk House, a Baker Street, London, w.1. I ~ t em interested in: I I 0 PHELAM 0 P.M.A. solid 0 P.M.A. solution 0 P.M.O. 0 T.B.T.O. 0 Olhers I

I My particular field Is I I NAME I I COMPANY I I AOORESS I

~--------------------------~

Ahead with Cl BA

C I B A

Anti-rot Protection

of cotton by

® Arigal

I

Mildew and Rotting

Both can be effectively and durably prevented by

'Arigal C Arigal PMP Suitable for cotton , jute and ramie

After a year 's imm ersion in the Rhine or twenty weeks' burial in soil - not a trace of deterioration on treated fibres .

Arigal also affords protecti on against actinic degradation , chemical corrosion , industrial pollution , exhaust fumes, seawater, maritime atmosphere , manure, etc .

CIBA Limited , Basle , Switzerland CIBA Clayton Limited , Manchester 11

C I B A

Vol. 3 AUTUMN 1967

INTERNATIONAL

BIDDETERIORATION

BuLLETIN

Editor

H. 0. W. Eggins*, Aston in Birmingham, England

Editorial Board

G. Ayerst, Wol.erhampton, England

G. Becker, Berlin, Germany

D. S. Belford, Castleford, Eny'and

D. G. Coursey, London, England

J. J. Elphick, ool.,, England

J .. Garrido, Madrid, Spain

N. E. Hickin, East Grinstead, England

H. J. Hueck, Delft, Holland

A. Kaplan, Natick, U.S.A.

Y. LeGrand, Vert-le-Petit, France

R. W. Traxler, Lafayette, U.S.A.

B. J. Zyska, Katowice, Poland

Publications Editors

M. J. Pennells, Aston in Birmingham, England

M. J. D. Willsher, Aston in Birmingham, England

*Biodeterloration Information Centre, Department of Biological Sciences,

No. 2

University of Aston in Birmingham, Gosta Green, Birmingham 4, England. Telephone 021-359 3611, Ext. 229

N.B.-Full addresses of editorial board members are given on page 89

•

,

INTERNATIONAL BIODETERIORATION BULLETIN

Contents and Summaries } in English

Notes to Contributors

BULLETIN INTERNATIONAL DE LA DETERIORATION BIOLOGIQUE

Table des Matieres et resumes } en Francais

Avis aux collaborateurs

INTERNATIONALES BIODETERIORATIONS BULLETIN

Inhait und Zusammenfaussungen } in Deutscn

Hinweise fur Beitrage

BOLETIN INTERNACIQNAL DE LA BIODETERIORACION

Contenido y Restimenes } , en Espanol

Apuntes para los contribuidores

.,

Page

iii

89

Page

iv

89

Seite

v

89

PAglna

vi

89

CONTENTS AND SUMMARIES

Research Facilities

RESEARCH FACILITIES FOR BIODETERIORATION IN THE U.S.A. R. Traxler and C. Yeager

Review Article

TOXIC MOULDS AND THEIR METABOLITES R. Townsend

The metabolites of toxigenic fungi and their effe~ts on animals and man are reviewed with particular reference to outbreaks of disease associated with the biodeterioration of foods and feedingstuffs. The disease syndromes of the diseases are discussed together with the chemistry of the toxic metabolites responsible for the diseases, attention being given to the relationships between physiological activity and chemical structure.

New techniques and work in progress

AN EVALUATION OF SOME FUNGICIDES FOR OPTICAL INSTRUMENTS P. W. Baker

Common defects in optical instruments under tropical conditions, and preventive measures are briefly analysed. The results are given of comparative tests on four volatile fungicide agents. It is concluded that ethyl mercury chloride could prove an effective fungi~ide for optical instru·

" ments. The primary aim in instrument design, however, should still be the achievement of satisfactory sealing and desiccation.

Page

43-46

47-58

59-64

TESTING CELLULOSE CONTAINING PLASTICS FOR RESISTANCE TO FUNGI 65-66 R. T. Darby

The inclusion of Chaetomlum globosum in the fungus test mixture in ASTM Designation D-1924-63, "Determining Resistance of Plastics to Fungi," is recommended to detect cellniosic constituents of formulations.

STUDIES OF ENZYMIC DEGRADATION OF CELLULOSE: SEPARATION ON DEXTRAN GELS 67-76 OF A CELLULOLYTIC POWDER OF FUNGAL ORIGIN Y. Le Grand and P. Thivend

The cellulolytic preparation is fractionated on Sephadex gels G 25-G 75-G 100. It is shown that the different fractions have different actions on insoluble cellulose and on soluble CMC, supporting the theory of plurality of cellulolytic enzymes.

FUNGI FROM GREEN COFFEE BEANS 77 T. Vincents Nissen

A study has been made of the mould growth on moistened green coffee beans. It is assumed that the moulds mostly originate in the spores of the fermentat!on fungi. A loss in quality may be the result of the growth.

THE TAXONOMY OF POLYPORUS MEGALOPORUS PERS. S. Domanski and A. Orlicz

A taxonomic study is presented of the fungus Polyporus megaloporus which has been reported to cause serious damage to oak wood in buildings, bridges, etc. Alternative nomenclature which has been proposed for the species is discussed and the authors contend that the species should be placed in the genus Fornes (FR.) kickx· emend. teixeira.

Book Reviews

Notices

Acknowledgements to sustaining organisations

Order forms (for I.B.B. and IBBRIS)

Notes for contributors

iii

79-80

81-82

82-83

84

85&87

89

TABLE DES MATIERES ET RESUMES Page

Possibilltes de recherches

POSSIBILITES DE RECHERCHES SUR LA DETERIORATION BIOLOGIQUE AUX U.S.A. 43-46 R. Traxler et C. Yeager

Articles analytiques

LES CHAMPIGNONS TOXIQUES ET LEURS METABOLITES 47-58 R. Townsend

Les metabolites des champignons toxiques sont passes en revue avec leurs effets sur Ies anlmaux et sur l'homme en se referant particuli~rement aux maladies associees a Ia deterioration biologique des produits alimentaires. Les syndromes des maladies sont etudies en meme temps que Ia chimie des metabolites toxiques responsables, !'attention etant attiree sur les relations entre l'activite physiologique et Ia structure chimique.

Nouvelles techniques et travaux en cours

UNE ESTIMATION DE QUELQUES FONGICIDES POUR INSTRUMENTS D'OPTIQUE P. W. Baker

On analyse bri~vement Ies defauts courants des instruments d'optique dans les conditions tropicales ainsi que les mesures preventives. On fournit les resultats d'essais comparatifs de quatre agents fongicides volatils. On en conclut que le chlorure d'ethyle-mercure peut etre un fongicide efficace pour les instruments d'optique. Cependant, le premier objectif dans Ia con­struction d'un instrument doit etre encore de realiser d'une mani~re satisfaisante Ia dessication et les joints hermetiques.

59-64

ESSAIS DE RESISTANCE AUX CHAMPIGNONS DE MATIERES PLASTIQUES CONTENANT DE 65-66 LA CELLULOSE R. T. Darby

II est recommande d'inclure le Chaetomium globosum dans Ia culture mixte fongique d'essai dans Ia specification ASTM D-1924-63 "Determination de Ia resistance des mati~res plastiques aux champignons." II est en effet necessaire de detecter les constituants cellulosiques des formula­tions.

ETUDE DE LA DEGRADATION ENZYMATIQUE DE LA CELLULOSE: FRACTIONNEMENT SUR 67-76 GEL DE DEXTRANES D'UNE POUDRE CELLULOLYTIQUE D'ORIGINE FONGIQUE Y. Le Grand et P. Thivend

La preparation cellulolytique est fractionnee sur gels Sephadex G 25- G 75- G 100. 11 est montre que ies diverses fractions ont des actions differentes sur Ia cellulose insoluble et sur Ia CMC soluble, appuyant ainsi Ia theorie de Ia pluralite des enzymes cellulolytiques.

CHAMPIGNONS DES FRillTS VERTS DU CAFEIER 77 T. Vincents Nissen

Une etude a ete faite sur le developpement de moisissures sur Ies fruits verts humidifies du cafeier. On suppose que Ia plupart des moisissures proviennent des spores de champignons qui font fermenter le cafe. Une baisse de qualite peut etre le resultat de ce developpement.

LA TAXONOM!E DU POLYPORUS MEGALOPORUS PERS. 79-80 S. Domanski et A. Orlicz

Une etude taxonomique est presentee du champignon Polyporus megaloporus qui a ete signale comme provoquant de serieux dommages au bois de chene des habitations, des ponts, etc. Les diverses nomenclatures proposees pour l'esp~ce sont discutees et !'auteur affirme que l'esp~ce devrait etre placee dans le genre Fornes (FR.) Kickx. Emend. Teixeira.

Revue des livres

Avis

Remerclements aux organlsmes de soutler

Les fiches de commande (pour I.B.B. et IBBRIS) Avis aux collaborateurs

iv

81-82

82-83

84 85 & 87 89

INHALT UND ZUSAMMENFASSUNGEN Seite

ForschungsmBgllchkeiten

FORSCHUNGSEINRICHTUNGEN FUR FRAGEN DER MATERIALZERSTORUNG DURCH 43-46 ORGANISMEN IN DEN U.S.A. R. Traxler und C. Yeager

Ubersichtsberlchte

TOXISCH WIRKENDE SCHIMMELPILZE UND IHRE STOFFWECHSELPRODUKTE R. Townsend

Es wird eine Ubersicht gegeben tiber die Stoffwechselprodukte pathogener Pilze und ihre Auswirkungen auf Menschen und Tiere unter besonderer Berticksichtigung von Krankheitsf§llen, die mit der ZerstOrung von Nahrungs- und Futtermitteln durch Organismen zusammenhangen. Die Syndrome der Krankheiten werden besprochen zusammen mit der Chemie der toxisch wirkenden Stoffwechselprodukte, den Erregern dieser Krankheiten, unter Ber!icksichtigung des Verhaltnisses von physiologischer Aktlvitat und chemischer Struktur.

Neue Untersuchungsverfahren und laufende Arbeiten

BEWERTUNG EINIGER FUNGIZIDE FUR OPTISCHE GERATE · P. W. Baker

Es wird ein kurzer Uberblick gegeben tiber haufige ZerstBrungsmerkmale an optischen Geraten unter tropischen Bedingungen und tiber vorbeugende Massnahmen. Die Ergebnisse von vergleichenden Prtifungen von vier fliichtigen Fungiziden werden mitgeteilt. Die Versuche liessen darauf schliessen, dass Athylquecksilberchlorid als wirksames Fungizid In Betracht kommen kBnnte. Das Hauptziel der Gerate-Hersteller sollte jedoch immer noch sein, ihre Gerate so gut wie mBglich abzudichten und gegen Feuchtigkeit zu schtitzen.

47-58

59-64

PRUFUNG CELLULOSE-HALTIGER KUNSTSTOFFE AUF WIDERSTANDSFAHIGKEIT GEGEN 65-66 PILZBEFALL R. T. Darby

Es wird empfohlen, Chaetomium globosum mit in die Pilzmischprtifung der Norm ASTM D-192463 "Bestimmung der Widerstandstahigkeit von Kunststoffen gegentiber Pilzbefall" aufzene­hmen, damit Cellulose-haltige Bestandteile und Verbindungen nachgewiesen werden kBnnen.

UNTERSUCHUNGEN UBER DEN ENZYMABBAU EINES CELLULOLYTISCHEN PULVERS 67-76 PILZLICHEN URSPRUNGS BEl DER CELLUOSE-TRENNUNG AN DEXTRANZELLEN Y. Le Grand und P. Thlvend

Die cellulolytische Praparation wird auf Sephadex Gelen G 25-G 75-G 100 fraktioniert Die Ergebnisse zeigen, dass die verschiedenen Fraktionen eine unterschiedliche Wirkung auf unlBsliche Cellulose und !Osliche CMC haben, was die Annahme mehrerer cellulolytischer Enzyme bestatigt.

PILZE VON GRUNEN KAFFEEBOHNEN 77 T. Vincents Nissen

Das Schimmelpilzwachstum auf angefeuchteten griinen Kaffeebohnen wurde untersucht. Es wird angenommen, dass die Schimmelpilze meist in den Sporen der Fermentationspilze entstehen. Das Schimmelwachstum kann zu einer Qualitatsminderung ftihren.

DIE TAXONOMIE VON POLYPORUS MEGALOPORUS PERS. 79-80 S. Domanski und A. Orlicz

Es wird eine taxonomische Untersuchung des Pilzes Polyporus megaloporus vorgelegt, der schwere Schiiden an Eichenholz in gebauden, Briicken usw. hervorruft. Eine Ersatznomenklatur, die fiir diese Art vorgeschlagen wurde, wird besprochen, und der Verf. empfiehlt, die Art der Gattung Fornes (Fr-) Kicksx. Emend. Teixeira lUZurechnen.

Buchbesprechungen

Notizen

Anerkennung der Sttitzorganisationen

Bestellscheine (fiir I.B.B. und IBBRIS)

Hinwelse fUr mitarbelter

v

81-82

82-83

84 85 & 87

89

CONTENIDO Y RESUMENES Pagina

Facllldades de lnvestlgacl6n

FACIUDADES DE INVESTIGACION DE LA BIODETERIORACION EN LOS ESTADOS UNIDOS 43-46 R. Traxler y C. Yeager

Artlculos de Resefla

MOROS TOXICOS Y SUS METABOUTAS R. Townsend

Se pasa revista a los metabolltas de los bongos toxig6nicos con referencla especial a las epidemias de las enfermedades associadas con Ia biodeterioraci6n de los allmentos. Se dlscuten las sindromias de las enfermedades a Ia par de Ia qulmlca de los metabolltas t6xicos responsables de las enfermedades. Se presta tambi~n atenci6n a! parentesco que exlste entre las actlvidades fisiol6gicas y Ia estructura qulmlca.

T~nlcas nuevas y trabajo que se prosigue

47-58

UNA EVALUACI6N DE ALGUNOS FUNGICIDAS PARA LOS INSTRUMENTOS 6PTICOS 59-64 P. W. Baker

Se analizan en breve algunos defectos corrientes de los instrumentos 6ptlcos en condiciones tropicales, y tambi~n las medidas impeditivas. Se dan los resultados de pruebas comparatlvas en cuatro agentes volatiles fungicidas. Se concluye sugiriendo que el mercurlo etbyl chlorida pudiese demostrarse como fungicida eficaz para los instrumentos 6ptlcos. Con todo, el prop6slto principal en el diseiiar instrumentos deberla ser el consequir una obturacl6n y una desecacl6n adecuadas.

LA PRUEBA DE LOS PLASTICOS QUE CONTIENEN CELLULOSE PARA LA RESISTENCIA A 65-66 LOS HONGOS R. T. Darby

Para descubrir los constituyentes cellul6sicos de las formulaciones se recomlenda el lncluir Chaetomlum globosum en Ia mixtura para Ia prueba de los bongos en ASTM Designaci6n D-1924-63 " Determinaci6n de Ia Resistencla de los Plasticos a los Hongos."

ESTUDIOS DE LA DEGRADACION ENZIMICA DE LA SEPARACION DEL CELLULOSE EN LAS 67-76 CELULAS DEXTRAN DE UNA P6LVORA CELLULOUT!CA DE ORIGEN HONGOSO Y. Le Grand y P. Thivend

La preparaci6n cellulolltlca se fracciona en las gelatinas Sepbadex G 25-G 75-G 100. Se muestra que las fracciones dlferentes tlenen eiectos diversos sobre el CMC soluble y el cellulose insoluble, lo que apoya Ia teorla de Ia pluralidad de los enzymas celulolltlcas.

LOS HONGOS DE LOS GRANOS TIERNOS DE CAFE T. Vincents Nissen

Se ba hecho un estudlo del moho que aparece en los granos tlemos de caf~ cuando h11medos. Se supone que los. mohos nacen por lo mas en las esporas de los bongos de Ia fermentacl6n. Tales mohos pueden resultar en una p~rdida de calidad.

LA TAXONOMIA DE POLYPORUS MEGALOPORUS PERS. S. Domanski y A. Orllcz

Se presenta un estudio taxon6mico del bongo Polyporus megaloporus que, seglln se refire, ha ocasionado daiios graves a Ia madera de encina (roble) de los edificios, puentes y asl suceslva­mente. Se discute Ia propuesta de una nomenclatura altematlva de 1a especle, y el autor afirma que Ia especle deberla clasificarse bajo el g~nero Fornes (FR.) Kickx. Em md. Teixeira.

Revlstas de llbros

Anunclos

Reconoclmlento de las organlzaclones mantenedoras

Las papeletas de pedldo (para I.B.B. y ffiBRIS) Apuntes para los contrlbuldores

vi

77

79-80

81-82

82-83

84 85 & 87

89

,.

Traxler, R. and Yeager, C., Int Biodetn Bull. 3, (2), 43-46, (1967), Research faciliti~s for biodeterioration in the U.S.A.

RESEARCH FACILITIES FOR BIODETERIORATION IN THE U.S.A.

R. Traxler' and C. Yeager'

Effort in the United States in the area of bio­deterioration control dates back to the 1920's. There are now in the U.S. a large number of qualified persons studying a wide assortment of biological deterioration problems. The majority of these scientists are employed by private corporations, a somewhat smaller number by federal or state supported laboratories and a very few in the laboratories of several universities. Because of this distribution of workers it is only natural that

' most of the research is of an applied nature. The primary emphasis is on the prevention of deteriora· tion and little research is devoted to other funda· mental aspects of biodeterioration.

A study of the personnel involved in bio­deterioration research in the U.S. reveals that by far the largest single group identfy themselve~ as microbiologists or chemists. A few entomologists, plant pathologists, biologists, engineers and physicists are found among the ranks. Most of the microbiology deals with mycology with little emphasis on the study of bacteria, algae and related microbes. Few of the U.S. investigators are concerned alone with problems of biodeteriora· tion. More often the investigator has another main research interest or is involved in the sale or service of products.

Typical of early preventive measures in the U.S. were Corrigan's (1920) recommendations of heat, light and such antiseptics as copper sulfate, potassium nitrate, zinc sulfate, zinc chloride, boric acid, formaldehyde and phenols for the destruction of fungal growth on finished material. The exigencies of World War II greatly activated fungicidal programs, and thousands of chemical products were examined for fungistatic and fungi· cidal activity. The methods of Fargher, Galloway and Probert ( 1945) were generally used for screening. These methods determined toxicity but did not consider aspects of the problem which determine a compound's effectiveness for mildew· proofing materials ; thus many fungicides failed to uphold expectations when in use.

During World War II governmental agencies established special organizations to study the effects of different formulations of various fungi­cides on military items subject to microbial attack.

Proper formulation was recognized as being of prime importance and the large chemical houses and formulators coordinated th~ir activities so that sound fungicide programs began to emerge. With the exception of certain recent specialized areas, most of the practices recommended and specified during or shortly after, World War II are still in force today.

Baskin and Chapin (1961) have published a screening technique for predicting the effective· ness of industrial antimicrobial compounds. This technique provides for a simple, reasonably fast procedure that will separate ineffective compounds from those worth further consideration.

Because the United States government has always been a major user of antimicrobial pro­ducts, government laboratories have developed sophisticated techniques and equipment for testing product effectiveness. Today there is an increasing effort in the non-military field due undoubtedly to the vast increase in technology and the adoption by the consumer public of the newer products. It is incumbent upon industry to establish sound programs which result in the sale of effective antimicrobials to both the government and private enterprise.

Textiles

Copper naphthenate was the leading fungicide during the war but is rapidly disappearing from the scene. Today the most prominent fungicides are copper 8-quinolinolate, methylene bis ( 4· chOrophenol), a mixture of zinc dimethyi dithio­carbamate and zinc ~mercaptobenzothiazole,

cuprammonium (mostly for government textiles), various esters of pentochlorophenol, other sub· stituted phenols and dodecyl guanidine tereph· thalate, a relative newcomer, for non-government use. There are a few organiZl_ltions still using some mercurials and alkylated tins, although textile substrates do not lend themselves to relatively fugitive systems. Most of the development work is done for the government at the U.S. Army Natick Laboratories, and at the U.S.D.A. Regional Laboratories at New Orleans. For non-government use the bulk of the work is done by a small handful of fungicide suppliers who have developed extensive facilities for serving the textile industry.

'Department of Microbiology, University of Southwestern Louisiana, Lafayette, Louisiana 70501 (U.S.A.) 'Scientific Chemicals, Inc., Chicago, Illinois 60623 (U.S.A.)

43

Research facilities for biodeterioration in the U.S.A. R. Traxler and C. Yeager

Plastics

The rise of the vinyl industry has created new biodeterioration problems. There are only a few laboratories with the facilities for handling not. only microbial problems, but the accompanying physical problems of light and heat stability, com­patibility testing and compounding. The Natick Laboratories of the U.S. Army and Crane Arsenal of the U.S. Navy represent the government effort while the industrial 'leaders are Scientific Chemicals, Inc., R.T. Vanderbilt, M & T Chemicals and Nuodex Division of Tenneco Chemicals, Inc. There is a rising trend to encourage the growth of biodeterioration research within the vinyl industry, but this is in its infancy and will take years to develop.

Paints, coatings and adhesives

Many organizations are engaged in the study of antimicrobials for use in paints, coatings and adhesives. Mercurials are the most frequently used inhibitors, but. considerable screening is under way to find new and more sophisticated compounds for these industries. A great number of microbiologists are concerned with these prob· !ems because coating systems are changing so rapidly and being applied to many new uses. Buchman Laboratories, Metasol Division of Merck Chemicals, Cosan Chemicals, Nuodex Division of Tenneco Chemicals and most large paint companies and coating manufacturers are active in the preparation of these formulations.

Paper Industry

Slime control in the paper industry has been a problem for many years, but this industry has shown little interest in establishing their own research or testing facilities. Therefore, they are dependent upon the servicing facilities of a few suppliers of antimicrobial agents. Extensive microbial facilities are required as well as field service involving isolations in the field and laboratory identification of the organisms involved in these slime problems. The service responsibility is so extreme that only a limited number of organizations can effectively compete in this field.

Wood and wood products

Entire industries have been born out of the requirements of the wood and lumber field for preservatives for wood and wood products. Numerous companies are engaged in the applica­tion of creosote or pentachlorophenol to wood by pressure treating and a smaller number of organizations are engaged in the development of formulations of pentachlorophenol and copper naphthenate for application to wood by dip­treating. Similarly, many organizations such as utility companies are engaged in testing applica· lions of fungicides, above and below ground, for such products as utility poles, railroad ties, structural timbers, etc. The National Woodwork Manufacturers Association oversees the use of

44

fungicidal formulations for the door and sash industry. The American Wood Preservers' Asso· ciation has the responsibility for certifying the use of antimicrobial agents for all wood uses ami preparing standards which are faithfully followed by the industry. It is unlikely that any antimicro· bial substance could be adopted by these industries without first gaining the approval of these highly regarded organizations. Recently a solubilized version of copper 8-quinolinolate was adopted in a standard by the AWPA for use on wood in areas which might be in contact with food products. Years of testing preceded this adoption. Other antimicrobials such as water-born inorganic salt formulations are permitted for special uses under standards issued by the AWPA. Because of the tremendous time period involved, the appearance of new formulations is slow. It is probable that work is underway with other antimicrobials, many of which will never reach the stage where a standard will be written.

. One of the leading research organizations for wood products in the United States is the Forest Products Laboratories of the United States Depart­ment of Agriculture. A staff of microbiologists is engaged in constant laboratory and field tests. It is th~ usual practice for manufacturers to submit new products to this laboratory for comparison with accepted compounds. These laboratories are also engaged in research on new uses for wood products and new preservatives for wood products.

Rubber Industry

This industry is concerned with biodeterioration of several of their industrial products. Although there are many arguments pro and con regarding the deterioration of natural and synthetic rubbers by microorganisms, many items are produced con­taining susceptible entities such as cellulosic fibers or jackets which may be preserved by the addition of antimicrobials to the rubber formulation. Con­veyor belts and fire hoses are excellent examples. In the former the antimicrobial agent is added to the rubber formulation, whereas in the latter the fungicide is added to the jacket surrounding the rubber lining. Most of the major rubber pro­ducers contribute to this program. Both R. T. Vanderbilt and Scientific Chemicals, Inc. maintain extensive service facilities to assist this industry.

Industrial Cooling water

The treating of industrial cooling water is an area which requires extensive servicing. For this reason the number of organizations engaged in this effort is limited. Most of the leaders are also active in slime control for the paper industry, and control work in secondary recovery for the petroleum industry.

Petroleum Products

Numerous products are used in the control of bacterial contamination of cutting or soluble oils.

Research facilities for biodeterioration in the U.S.A. R Traxler and C. Yeager

Dr. E. 0. Bennett, the University of Houston; has developed excellent techniques for the· testing of soluble oil formulations and the screening of antimicrobials for use in cutting oils. This prob­lem involves not only the biodeterioration of the cutting oils but also the medical hazard to the cutting tool operators who come in contact with the contaminated materials.

On the production side of the petroleum industry water flooding as used in secondary recovery creates problems of bacterial fouling and corrosion which are overcome by the addition of antimicro­bial compounds to large quantities of recycled water. Organizations active in this area include most of the major oil companies, as well as com­panies devoted to water treatment systems.

Drilling muds which are complex formulations often including starch, casein and other organic materials, are formulated for their specific gravity and viscosity characteristics. Bacterial contamina­tion can drastically modify these compounds so that they are unfit for use in drilling. Various inhibitors can be added to the muds to retard this biodeterioration. This problem does require con­siderably more ·study than it receives at the present time.

A problem of more recent importance is that of jet aircraft and diesel fuel contamination. The presence of small amounts of water condensate, particularly in fuel tanks, allows the growth of a wide variety of hydrocarbon utilizing micro­organisms. These microbial systems and their by­products are responsible for severe corrosion problems, causing sludge formation which fouls motor parts. Work in this area was instigated by the U.S .. Air Force in cooperation with the U.S. Army Biological Laboratories. An extensive investi­gation of this problem has been performed by Melpar, Inc. under contract to the U.S. Air Force. Antifreeze additives of the type developed by Phillips are found to be effective in the control of this problem.

Miscellaneous

There are numerous agricultural uses for anti­microbal compounds for preservation purposes. Since the Food and Drug Administration controls products going into or coming in contact with food, few products are permitt~d. The product most frequently used is solubilized copper 8-quinolinolate in such items as berry boxes, lug boxes, potato storage areas, pea viners, food storage areas and a host of other uses.

Many fringe areas exist which have not been discussed. The general field of plant sanitation concerns itself with problems bordering on bio­deterioration. Numerous organizations are con­cerned with sanitation of wearing apparel although they are really interested in odor control rather than preservation of the item. The. development of new resin systems such as the polyurethanes wjll create· new problems of biodeterioration, re-

45

quiring additional effort on the part of existing organizations or encourage the growth of new ones.

An area of biodeterioration studies which seldom receives attention is the beneficial side of deterioration. In this regard, micro-organisms are being used to degrade solid and liquid wastes and in the process produce products of commercial value. A project under study at the Gulf South Research Institute illustra~es this concept. Studies are underway on the biodegradation of Ligno­Cellulose in sugar cane waste in the hope of increasing digestibility and the use of the waste as a forage crop. A similar study at the Bio­deterioratlon Center of the University of South­western Louisiana is the conversion of sweet­potato waste and other food wastes into usable products. This area of biodeterioration study is particularly important because of the growing problem of water pollution by solid and liquid wastes.

-The American universities do not generally

train students in the specific field of biodeteriora­tion. There is a growing tendency in the univer­sities for fundamental research problems in biodeterioration to be undertaken as a part of graduate training. The specific information on antimicrobial compounds and testing methods are too specialized for the university purpose which is to teach concepts. If the students know the fundamentals of their particular science the specific information which will change in each different organization can be readily learned on the job.

Each year the problems in biodeterioration become more complex and the research effort more sophisticated as new products and materials reach the consumer market. As national boundaries shrink and world trade becomes more extensive new problems will .arise and better control will be required. More accurate investigation of the economic significance of biodeterioration will be necessary before many industries can be con­vinced of its importance. One obvious need is for the coordination of the biodeterioration studies and exchange of information.

The coverage of biodeterioration problems in the U.S. is very broad with investigators being concerned with textiles, plastic, polymers, wood products, protective coatings, paints, detergents and corrosion. Large groups are interested in antimicrobial agents for the protection of various products and _considerable emphasis is placed on testing methods and assessing protective quality of chemical agents. Some studies are directed towards the marine environment and others toward space systems. The largest effort in the United States is associated with industry and a smaller group in government requirements.

Research facilities for biodeterioration in the U.S.A. R. Traxler and C. Yeager

Bibliography

(1) Corrigan, V. F. J. Soc. Dyers Colourlsts, 36, 198 (1920).

(2) Fargher, R. G. et al. Incidence and Control of Mould and Bacterial Attack on Textiles ; British Cotton Industry Res. Assoc. (March, 1945).

(3) Baskin, A. D., et al. Developments In Industrial Microbiology, 2, 215, (1961) .

•

46

~~--------------------------------------------------------------

Town~end, R. J., Int. Biodetn Bull., 3, (2), 47·58, (1967). Toxic moulds and their metabolites.

TOXIC MOULDS AND THEIR METABOLITES

R. J. TOWNSEND'

The biodeterioration of perishable goods and materials has been known to man for centuries, and an infinite variety of raw materials, manu· factored goods and commodities are involved. These range from cotton, paper and timber pro· ducts to cereals and foodstuffs.

The best known cases of biodeterioration, how­ever, are to be found among the farm produce and foodstuffs. Plant diseases, insect pests, rodents, bacteria and fungi are the principal agents and all contribute to the loss in both quantity and quality of many millions of pounds worth of food.

Some examples are : Plant diseases -Mildew of tomato plants, potato

blight. Insect pests- Green fly on fruit trees, codling

moth lavae in apples . . Rodents- Consumption of cereals and grain by

rats and mice. Bacteria- Contamination of meat and meat

products by pathogenic organisms, e.g., Salmonella typhl. A health hazard.

Storage fungi- Destruction of cereals and hay by Alternaria and Fusaria spp., Aspergilli on oilseeds and nuts, Penicillia on rice, citrus fruits and dairy produce, Mucorales on baked products such as bread.

The total cost to the world economy must run to several thousand billion pounds. It has been estimated (Losses in Agriculture, 1954) that the annual loss of fruit and vegetables in the United States alone amounts to over 300 million dollars!

In the last 30 years an increasing number of reports have appeared on diseases in animals of unknown etiology which have been associated with the ingestion of mouldy food. Investigations have now clearly shown that many of these diseases are caused by the ingestion of toxigenic fungi or their metabolites, through the consumption of mouldy

. feed or pasture. It is indeed surprising that the significance of mouldy food in the etiology of · animal disease should have been overlooked for so long, especially since ergotism, the " St. Anthony's Fire " of the Middle Ages, a fungal toxicosis due to the ingestion of barley or rye bread infected with Clavlceps purj>Urea, has been so well documented. Until recently the subject of

fungal poisoning was studied by only a few veterinary workers and mycologists, mainly in Japan and the U.S.S.R. where outbreaks have been more frequent. Consequently our knowledge In Britain of diseases caused by toxigenic fungi has been patchy and disconnected, due in the main to the difficulty of obtaining satisfactory abstracts and translations of the relevant literature. Even now considerable difficulty is experienced in obtaining original papers, and satisfactory trans­lations.

The veterinary and mycological aspects of fungal poisoning among farm animals in countries with mainly temperate climates have been critically· reviewed by Sarkisov (1954) and Forgacs and Carll (1962).

In this paper an attempt will be made to review briefly the metabolites of the toxigenic fungi which are responsible for the various mycotoxi­coses repQrted in the literature and which are associated with the blodeterioration of foods and feedingstuffs in both temperate and tropical climates.

The disease syndromes will be discussed, together with the toxic metabolites which produce similar or the same physiological effects, attention being given to the relationships between chemical structure and physiological activity.

Mycotoxicoses may be defined (Forgacs and Carll, 1962) as the poisonings of animals and man by the ingestion of toxigenic fungi or their metabolites.

Outbreaks of such food poisonings are characterised by several unusual features (Towns­end, 1964; Coveney, 1966).

a) The onset of the disease is associated with a particular food or feedingstuff, e.g., rice, groundnutmeal, and rye-grass ;

b) Examination of the suspected food shows • evidence of fungal activity;

c) In field outbreaks the diseases are often found to be seasonal ;

d) The diseases are not . transmissible from animal to animal, i.e., the disease is neither contagious nor infectious ;

e) Treatment of the affected animals with drugs

'Dept. Food Science and Technology, Borough Polytechnic, Borough Road, London S.E.l., England· (Copy received 13th April, 1967.)

47

Toxic moulds and their metabolites. R. J. Townsend

or antibiotics has little effect upon the course of the disease ;

f) Avitaminosis or vitamin deficiency syndrome is a common feature of mycotoxicoses, which may be distinguished from the genuine vitamin deficiency diseases by the fact that vitamin therapy has little or no effect upon the toxicoses ; .

g) Immunisation is ineffective, since mycotoxins, as distinct from bacterial toxins, are not complex protein molecules and consequently have no antigenic action.

Fungal poisons may be roughly classified into four categories according to the type of lesions or pathological damage they produce and the particular organ or organs affected. These are :

The Hepatotoxins or Liver poisons. These produce severe liver cirrhosis, hepatoma

and necrosis of the liver cells. Many of these metabolites are also potent carcinogens, e.g., aflatoxins, the toxic principles of Aspergillus flavus, Link. and the phalloidines from the "Death Cap " fungus, Amanita phalloides.

The Nephrotoxins or Kidney poisons. Produce chronic and acute nephrosis, eventually

leading to complete renal failure. Citrinin, a metabolite of the fungus Penicillium citriJ!Uffi Thorn., is a good example of this class of poison.

The Neurotoxins or Brain and Central Nervous System poisons.

These metabolites act principally upon the brain and central nervous system, producing severe haemorrhages and degeneration of the nerve tissues. An example of this type of poison is the substance patulin, a metabolite of Penicillium patulum Bainter.

Finally there are the Photodynamic Dermatotoxic Metabolites.

These produce dermatitic-like skin lesions, in man and animals, on contact with food or crops infected with the toxigenic fungus. 8-methoxy psoralen and 4.5'.8 trimethyl psoralen are two powerful dermatitic substances which are pro­duced by toxic strains of the organism Sclerotinla sclerotiorum, the causative agent in " Pink Celery Rot " disease.

This classification is somewhat artificial since many outbreaks. of mycotoxicosis exhibit . more than one type of disease syndrome. This is often due to the presence of two or more toxic agents acting together or separately, a complication which often makes diagnosis difficult.

The study of mycotoxins and their effects is well illustrated by the recent works on " Turkey X disease" which brought into focus, in this country, the importance of mycotoxins to all food and feedingstuffs manufacturers, to farmers •and the veterinary profession.

Outbreaks of this disease occurred (Blount, 1961 ; Asplin and Carnaghan, 1961 ; Allcroft, Carnaghan, Sargeant and O'Kelly, 1961) through­out British poultry farms in 1960 and over

48

100,000 turkey poults perished as a result of this new disease of unknown etiology, which was given the name " Turkey X disease."

The disease is characterised by inappetance, depression, staggering· gait and sudden death of the affected birds. At death the birds adopted a characteristic position with the neck arched, the head drawn back and the legs fully extended back­wards. Post-mortem examination on the affected birds revealed liver haemorrhages and severe liver necrosis. Histological examination of the livers showed degeneration of the liver parenchyma cells and proliferation of the bile duct epithelium, with concomitant bilary stasis. Ducklings and pheasants were similarly affected, and Loosmore and co­workers (1961) showed that pigs and calves were poisoned by feeding rations containing 17%-20% of a toxic Brazilian groundnut meal, which had been shown to be associated with Turkey X disease by Blount (1961). Other consignments of groundnut meal were soon found to be toxic and the disease was also to become known as " Ground­nut Poisoning."

Post-mortem examination (Harding, Done, Lewis and Allcroft, 1963) of pigs poisoned by toxic groundnut meal showed liver changes identical to those observed in affected turkeys, namely bile duct proliferation, disorganisation of the parenchyma cells and fibrosis.

Ducklings were found to be especially susceptible to the toxin and the characteristic proliferation of the liver bile duct cells in these animals formed the basis of a useful bioassay (Ailcroft, Carnaghan, Sargeant and O'Kelly, Hi61 ; Sargeant, O'Kelly et a!, 1961), which proved of great value in the isolation and purification of the toxin from the groundnut meal. The bioassay was carried out as follows:

Toxic groundnut meal was defatted with petroleum and then extracted with methanol. The extracted material was then suspended in water and fed orally to day-old Khaki Cambell ducklings over a period of four days. The number of ducklings dying within seven _days of the start of the test is then recorded. Any survivors were then sacrificed and the level of toxicity of the meal determined by the extent of the bile duct proliferations in the livers of all the ducklings. In this way it was possible to estimate concentrations of toxin of the order of 0.1 ppm.

The methanol extracts from batches of toxic groundnut meal were found to be very fluorescent in ultra-violet light, the intensity of the blue fluorescence being proportional to the toxicity of the meal, as determined by the bioassay. From the blue fluorescent methanol extracts of toxic Brazilian groundnut meal Sargeant and co­workers ( 1961) isolated a white crystalline mixture of toxins by column chromatography on alumina using chloroformjmethanol solvent 98.5 : 1.5. The toxin was extremely potent 20/'g proving fatal to the day-old ducklings in 24 hours.

Toxic moulds- and their metabolites. R. J. Townsend

Mycological examination of toxic grounrlnuts from· Uganda showed them to be heavily con­taminated with fungi. Isolation of 'the fungi showed that Aspergillus llavus Link was the most abundant organism present, accounting for over 60% of the isolates. Cultures of Aspergillus flavus grown on Czapek-Dox media for seven days at 27'C were extracted with chloroform. The chloroform extracts possessed the same blue fluorescence as the toxin, previously isolated, from the Brazilian meal and from these extracts a crystalline material was isolated. (Nesbitt, O'Kelly, Sargeant and Sheridan, 1962). This material was lethal to day-old ducklings, prod'ucing characteristic liver lesions identical to those produced by the toxic Brazilian groundnut meal and seen in the livers of turkey poults from field outbreaks of "Turkey X disease." This material was called aflatoxin.

Thin layer chromatography of the crude toxin resolved the material into two components. A blue-violet fluorescent substance Rf = 0. 7 aflatoxin B, and a green fluorescent material RF = 0.5 aflatoxin G.

These were further resolved on silica gel G chromatoplates with chloroform 2% methanol mixture into 4 compounds. (Hartley, Nesbitt and O'Kelly, 1963). These are:

Aflatoxin B, CnHnO• M.Wt = 312 Aflatoxin B, CuHnOe M.Wt = 314 Aflatoxin G, CnH120, M.Wt = 328 Aflatoxin G, CuH,.O, M.Wt = 330

I

H-,-,--,(''J/

H"---/'-

li

H'..---r----,t...-.....'(" '"" HI"-..../'-..../'-.J~,

The structural formulae of these aflatoxins have been determined by Asao and Buehl et a! (1963). The aflatoxins B, and G, have the same structures as B, and G, except that the double bond of the outside furan ring is reduced. The four aflatoxins occur in varying proportions in synthetic media depending upon the conditions of incubation and the strain of the organism used. Cultures_ of A. flavus grown on Czapek-Dox media produced aflatoxins in the following ratios: B,: B,: G, : G, 40 : 1 : 50 : 1. In groundnut meal aflatoxin B, is the major metabolitP., little or no G, or G, is observed. (Coomes, 1963).

49

Rabie ( 1965) has determined the effect of temperature on the growth of Aspergillus flavus and on the production of the aflatoxins, using cultures of A. llavus grown on Czapek-Dox broth at 18'C, 24'C, 30'C, 36'C and 42'C.

The aflatoxins were assayed by thin layer chromotagraphy and by bioassay, using white day­old Pekin ducklings. The fungus was found to have a wide growth range, growing well at 24'C, 30'C, 36'C, and 42'C with maximum growth at 18'C. The maximum production of B, was observed at 24'C, no B, was produced at 36'C or at 42' C. The production of G, was best at 30'C, no G, was detected at 18'C and only small amounts at 36'C. These results indicate that the amount of fungal material present does not indicate the amount of toxin in the culture media.

The aflatoxins are extremely potent substances, the L.DH for each compound in ducklings is 18.2/'g B, = 3641'g/Kg for B., 84.81'g B, = 1696/'g/Kg of B,, 39.21'g G, = 7841'gfKg for G, and 172.51'g/G, 3450/'g/Kg for G,. The presence of only a few parts per million of aflatoxin B, in groundnuts constitutes a danger to the health of man and animals, as can be seen from the fact that the toxic groundnut meal· implicated in outbreaks of Turkey X disease con· talned only 7-10ppm. Even '!ower concentrations

m.pt. m.pt. m.pt. m.pt.

265'-270'C 305'-309'C 247'-250'C 237'-240'C

(I) (II) (Ill) (IV)

are dangerous. 200 grms of groundnut meal con­taining only 0.1 ppm. would contain enough aflatoxin B, to kill the average duckling.

It was, therefore, necessary to devise methods of assay for the aflatoxins and the intense fluorescence of these compounds provided a basis for the analytical procedures for the detection and estimation of the aflatoxins. The recommended procedures for the detection and estimation of aflatoxin B,, the most potent of the toxins, are published in two reports, No. G6 and No. G13, by the Tropical Products Institute, London.

The methods of assay all employ the following essential steps :

1. Extraction of fat~ and lipids by petroleum or hexane;

2. Extraction of the aflatoxins with methanol and/or chloroform;

3. Partial purification of the aflatoxin extracts by partition between chloroform and aqueous methanol;

4. Thin layer chromatography on silica gel G chromatoplates using 5% methanol chloro­form.

The concentration of the aflatoxins is estimated by dilution until visual extinction of the fluorescence, under U.V. light, due to the afla­toxins Is achieved. Comparison of the fluorescence of the toxic extracts with that of a standard

Toxic moulds imd their metabolites. R. J. Townsend

solution or' aflatoxin B, containing 0.11'gfml B, in plates of silica gel G is also used. The standard solution of aflatoxin B, containing 0.11'gfml B, in pure ChCI,. By these procedures the smallest weight of aflatoxin B, giving an observable fluorescence is 0.0004/'g = 4 x 10~'grms of B,. The visual limit of sensitivity for aflatoxin G, is found to be 0.0003/'g = 3 x lO..,'grms of G,. Results are normally expressed In ppm or as l'gfKg = parts/1000 million·

More important still are the chronic effects. It has been clearly established by several worker3 that aflatoxin can produce liver carcinoma in rats. Lancaster and coworkers ( 1961) showed that rats fed a 20% toxic groundnut ration over six months developed multiple hepatoma. These results were confirmed by Butler and Barnes (1964), who dosed rats with 2.5 mgs of aflatoxin B, over a period of three months and showed that multiple liver tumours were produced, even though the rats were maintained on a normal diet for up to a year after the administration of the aflatoxin was stopped. Latest figures show that as little as lOI'g per day fed over a six-week period can have this effect. This makes aflatoxin the most potent carcinogen known, at least as far as the rat is concerned, being 75 times more active than dimethyl nitro­samine (V) and 900 times more active than Butter Yellow (VI), which has been a prohibited food colour for many years.

Aflatoxin has been shown to be carcinogenic to trout (Halver, 1964). In this, fish levels of the order of a few parts per 10", i.e., from 2ppb to 320ppb in diet, produce hepatoma and it has been established that cyclopropenoid fatty acids synergise this action to such an extent that only 4ppb of aflatoxin B, can produce severe liver damage in trout fry after three months.

Allcroft and Carnaghan (1962) showed that dairy cows can metabolise the aflatoxins present in toxic peanut meal and to secrete a milk toxin, now known as aflatoxin M, which produces liver lesions in ducklings identical to those caused by aflatoxin B,. Extraction of dried cows milk, obtained from dairy cows fed upon highly toxic groundnut rations, followed by thin layer chroma· tography of the extracts has shown (de Iongh, Vies and van Pelt, 1964) that the milk toxin M is different from the aflatoxins B., B, G, or G,.

The milk toxin has been resolved into two

compounds, aflatoxin M, and aflatoxin M,, which have been shown to be hydroxylated derivatives of aflatoxin B, and B, by Holzapfel, Steyn and Purchase (1966). The molecules being, hydro­xylated at the junction of the two furan rings as shown.

50

Aflatoxin M, = VII Aflatoxin M, = VIII

The biochemical effects of aflatoxin B, on the metabolism of liver tissue, from ducklings fed rations containing 0.5ppm aflatoxin B, have been studied by Brown and Abrams (1965). Severe inhibition of the mitochondrial enzyme systems occurs, particularly of the dehydrogenase, succinate dehydrogenase, the cytochrome electron transport system, and the oxidative phosphoryla· tion system. Such inhibitory actions result in a large reduction in ATP synthesis and the con­sequent reduction of protein biosynthesis, since the high energy ATP is necessary to drive the biosynthetic reactions of the cell.

Similar effects have been observed by several workers (Theron, Liebenburg and Joubert, 1965 ; Gallagher, 1964) who noted the inhibition of adenosine triphosphatase, inosine diphosphatase and thiamine pyrophosphatase by aflatoxin B,. Severe physical disruption of the mitochondria and lysis of the cell wall and mitochondrial mem· branes are the characteristic cellular lesions of the tQxicosis. These are also produced by spori· desmin, the toxic metabolite of the fungus Pithomyces chartarum.

More recent work (Recondo, Fraysslnet and le Breton, 1966) has shown that aflatoxin B, rapidly inhibits RNA and DNA synthesis and acts directly upon the DNA molecule. Further work (Sporn, Dingman, Phelps and Wogan, 1966) has shown that aflatoxin B, binds to both natural and de­natured DNA and inhibits the incorporation of cytidine'H into rat liver RNA. The biochemical effects of aflatoxin B, are apparently diverse. However, since the biosynthesis of RNA, DNA and cytidine triphosphate require the active participa­tion of ATP, the inhibition of ATP synthesis by the disruption of the mitochondrial enzyme system provides an explanation and would bring about the cessation of RNA and DNA synthesis and also the incorporation of cytidine into RNA.

The binding of the toxin to DNA appears to be a separate issue and this would provide an ex­planation for inhibition of RNA and protein synthesis, since the binding of aflatoxin B, to the DNA molecule could render the DNA molecule unsuitable for use as a template for RNA synthesis. This is comparable with the inhibitory . action of actinomycin D.

Another toxicosis of importance is facial eczema disease which occurs in sheep and is very similar to aflatoxicosis.

Facial Eczema Disease. Facial eczema disease in sheep is a serious

problem to the New Zealand sheep farmer. Out-

Toxic moulds and their metabolites. R. J. Townsend

breaks occurring in 1928 were particularly severe and were responsible for the loss of over £9million worth of sheep. The disease is now known to be caused by the ingestion of perennial ryegrass infected with the ubiquitous saprophytic fungus Pithomyces chartarum (Burk and Curt) Ellis. formerly known as Sporidesmium bal<eri Sydow. (Symposium on Facial Eczema disease. Proceed· ings of the New Zealand Society of Animal Production. 99 44-90).

Outbreaks occur in the autumn after a dry and hot summer. There are several climatic factors which operate together to provide the ideal con­ditions for facial ezcema disease to develop. During the dry summer months the amount of dead plant material, or litter, amongst the ryegrass pasture increases. This provides an abundance of potential substrate for the growth of the fungus. If the dry period is followed by a period of heavy rain or warm humid weather then the fungus proliferates rapidly throughout the plant litter. Conditions are especially dangerous if the minimum grass temperature is greater than 54 "F, when the fungus soon becomes the dominant organism on the pasture. In these conditions sheep grazing upon ryegrass heavily infected with Pithomyces chartarum ingest the highly toxic spores, which contain the toxic metabolite sporidesmin (IX).

This substance is a powerful liver poison and

rapidly produces proliferation of the bile ducts and chronic liver cirrhosis. (Mortimer and Taylor, 1962; Done, Mo•·timer and Taylor, 1962; Mortimer, 1963.)

Such liver damage leads to the accumulation of bile substances, bilirubin and phylloerythrin, pro­ducing jaundice and photosensitization of the skin. This photosensitization shows itself in the development of eczema like lesions, on those parts of the animals' skin which are not covered with wool or hair, on exposure to sunlight.

Sporidestnin has been Isolated (Hodges, Ronald­son, Taylor and White, 1963) from potato-carrot cultures of Plthomyces chartarum by ether extraction of the dried mycelium. Fractionation of the extracts by column chromatography followed by sequential partition chromatography using two solvent systems gave sporidesmin (IX) a white crystalline compound C,.H~O,N,S,Cl.

X

Sporidesmin (IX) Gliotoxin (X)

This metabolite was shown by Mortimer (1963) and by Mortimer and Taylor ( 1962) to produce liver lesions in experimental animals identical to

51

those found in sheep suffering from facial eczema disease in the field. The L.D~ was found to be = 1.5 to 2.0 mgjKg in sheep.

The structure of sporidesmin (IX) was deter­mined by Fridrichsons and Mathieson (1962) by X-ray analysis of the methylene dibromide adduct. Hodges and coworkers ( 1963) showed the presence of an N-methyl !satin nucleus containing a chloro-dimethoxy N-methyl aniline moiety. On the evidence of the ultra-violet, infra red and N.M.R. spectral data of sporidesmin, and its close resemblance to gliotoxin (X) they concluded that sporidesmin contained a disulphide bridge system across a diketopiperazine ring. It is of interest to note that gliotoxin itself is a potent antibiotic and toxic fungal metabolite, L.D~ = 45mgjKg orally In mice, produced by a number of moulds including Trichoderma viride, Aspergillus fumi­gatus, Penicillium terlikowskii, and Peniclilium clnerascens.

Destruction of the disulphide bridge destroys the toxicity of both sporidesmin and gliotoxin.

Attempts to control facial eczema disease by the application of fungicides, such as copper sulphate dressings and mixtures of fatty acids, have shown promise; (Thornton, 1963; Campbell, 1965). Thornton has shown the antifungal activity of an homologous series of straight chain fatty acids towards Pithomyees chartarum to. vary with the chain length and concentration. In practice, since normal pasture contains sufficient dead plant litter in the summer to support massive growth of the mould, the growth of Pithomyces chlirtarum will depend upon two factors :

1. The presence of spores ; 2. The weather conditions.

Periods of rain after a dry summer with a minimum grass temperature of 54"F, as measured by a grass minimum thermometer in contact with the blades of grass, will certainly produce dangerous conditions.

New Zealand farmers now receive weather fore­casts and warnings, by the local Facial Eczema Disease Committees, of potential outbreaks of the disease. Two courses of action have been · recommended by Campbell (1965). These are:

a) Feeding the ·sheep on well grown clean crops of turnips, swedes, cabbages, etc., which must be free of dead weeds and grass ;

b) Enclosing the sheep in paddocks and feeding hay. Particular care must be taken that the hay is clean, since It is an ideal substrate for the growth of Plthomyces chartarum.

These precautions do not constitute a solution to the problem of facial eczema disease, but they do prevent outbreaks of clinical facial eczema among flocks of sheep.

A common toxicosis is that of mushroom poisoning. The agaric Amanita phalloides, com­monly known as the " Death Cap " fungus, is noted for its poisonous properties and has been

Toxic moulds and their metabolites. R. J. Townsend

responsible for the death of several people who have eaten it by mistake 'or the edible mushroom Psalliota campestris. Liver haemorrhage, necrosis of the parenchyma cells, bilirubinaemia, and hepatic coma characterise the course of the poisoning by this fungus and is typical of many fungal toxicoses in animals.

Wieland ( 1962) isolated several toxic cyclopeptides from Amanita phalloides, by mel~anol extractic:1 of the dried mycelium, which could be classified into groups, the amanitins, which give a violet colour reaction, and the phalloidines, which give a blue colour reaction with cinnamaldehyde and hydrochloric acid (Wieland, 1963).

The amanitins, ·~amanitin L.D~ ~ O.lmg/Kg : {3 amanitin L.D,.. ~ 0.4mg/Kg, and amanitin L.D.. ~ 0.15mg/Kg are extremely toxic com­pounds but slower acting (20 hours) than the phalloidines (I hour), phalloidin (XI), phalloin (XII) and phallacidin (XIII) which are less potent with L.D • .'s ~ 1.9mg/Kg, 1.4mg/Kg and 2.5mg/Kg, intraperitoneal in mice, respectively.

~2 ---cH--

TH2RI /NH co, PH

CH-C-CH /CO ~ /CH-3 1 ~H CH R3

OH I 'c tc: 0 ~~ I _j}_ I ~H-CH2 S-CHrfH

NH CO

'co rf "-._CH-NH-CO~

R1 R2 R3 I CH

CH3 H Xi- OH, CH3 , 3 ,. H

XI] - H , CH3

, CH3

,

XIn- OH, CH(CH3) ;COOH. 2

The toxicity of these substances manifests itself by immediate disruption of the hepatic functions. Investigations by Wieland and Rehbinder (1959), using isolated perfused rat liver preparations, have shown that glycogen synthesis is stopped or im­paired, although glycogenolysis is unaffected. There is, however, an immediate stoppage in the secretion of bile, inhibition of bilirubin and bromo­sulphthalein elimination, together with a very rapid rise in the serum glutamic/oxalacetic transaminase level. Liver damage is often res­ponsible for avitaminosis A and K, as well as a fall in prothrombin concentration in the plasma. These symptoms are frequently reported in the literature on the various mycotoxicoses.

The phalloidines posses a cyclopeptide structure with a sulphur bridge structure between the L-tryptophan and L-cystein moieties. The struc­ture of the amanitins are similar.

The effect of structural changes in these cyclopeptides on their pharmacological action has been studied by Wieland ( 1963). Removal of the thioether bridge, by treatment with Rayney nickel, gave the dethio-monocyclic peptide which

52

was completely atoxic. Toxicity was found to depend upon the bicyclic thio-ether bridge, since the monocyclic thioethers prepared from the dethiopeptides are also nontoxic. Cleavage of the peptide ring of phall9idin, with cold 507- tri­ftuoroacetic acid gave the seco-phalloidin which is also innocuous.

A similar cyclopeptide has been found to be one of a series of fungal metabolites involved in the "Yellow Rice" problem in Japan, which has been studied by Japanese scientists for the past 70 years.

The toxicity of mouldy " Yellow Rice " was first established by Sakaki in 1891 as a result of his attempts to establish the cause of a beri-beri outbreak at that time. The outbreaks occurred after a long hot rainy season and was associated with poor quality rice which was often mouldy. He showed that certain samples of mouldy rice pro­duced a central paralysis in experimental animals. Further investigations by Miyake and Tsunoda (1940) led to the isolation of several toxigenic strains of fungi, namely : Penicillium toxicariurn Miyake, Penicillium islandicurn Sopp., Penicillium cltrinurn Thorn., Penicillium rugulosurn Thorn and Penicillium citreoviride Biourge.

Penicillium islandicurn was isolated from samples of "Islandia" yellowed rice imported from Spain. The organism is ubiquitous and has been found in rice samples from Burma, Thailand, Pakistan, Vietnam, China, Turkey, Egypt, Peru, Argentina, and Ethiopia.

Feeding experiments by Miyake et al. (1940) showed that cultures of Penicillium islandicurn Sopp., grown on rice, when administered to rats and mice produced acute and chronic liver damage depending upon the amount of mouldy rice con­sumed. Post mortem examination revealed the presence of hepatic carcinoma, proliferation of the bile ducts and cirrhosis of the liver. A study of the survival curves showed that the life time of the experimental animals was clearly divided into two groups, of the order of 100 days and 300 days, indicating a two stage toxicosis and the existence of two or more poisonous substances in the moulded rice. This was confirmed by the isolation of islanditoxin and luteoskyrin from cultures of Penicillium islandicurn grown on Czapek-Dox medium.

rH20H -CH-....._

/NH C\ /co NH

CH=C~H \ 3 2 1 CH-CH-OH

I 2 NH CO

to ~ \ ~ c~

CH __..co Cl 0 "--NH Cl

Toxic moulds and their metabolites. R. J. Townnend

Islanditoxin (XIV) is. isolated from the metabolism solution concentrate by extraction with butanol. Recrystallisation from methanol and then acetone gave colourless crystals, C,,H,O,N,Cl, of ·a chlorine containing cyclopeptide. Hydrolytic studies showed .islanditoxin to consist of two molecules of L-serine and one molecule each of ,..amino-butyric acid, D.·/3 phenyl-f3-amino propionic acid and L-dichloroproline. The last two amino acids had not been found previously in natural products. On the basis of this and the infra-red and ultra-violet spectral data Marumo (1959) showed islanditoxin to be L-seryl-L-seryl-L­d)chloroprolyl-D·f3·phenyl- f3·amino propionyl- L-a· amino butyric acid anhydride. (XIV).

lslanditoxin is an extremely powerful hepato­toxin, L.D,. = 475f'g/Kg s.c., 335mg/Kg i.v., and 655ug/Kg orally in mice. Death is rapid, (2 · 3 hours) and post-mortem examination showed severe destruction of the liver cells with massive heamorrhage. The destruction of the pancreas was also observed. Removal of the chlorine atoms from the L-dichloroproline moiety of the islandi­toxin molecule destroys its physiological activity.

Luteoskyrin (XV), the other toxic metabolite of Penicillium islandicum Sopp. was isolated together with six other fungal metabolites, namely: rubros­kyrin, islandicin, iridoskyrin, catenarin, skyrin, and erythroskyrin, by ether extraction of the defatted mycelium. All these compounds, with the exception of luteoskyrin, had been described previously by Howard and Raistrick (1954). A comparative study of the chemistry, together with the ultra-violet and infra-red spectral data, of luteoskyrin (XV), rubroskyrin (XVI) and rugulo­sin (XVII) a metabolite of Penicillium rugulosum, Thorn, was carried out by Shibata and coworkers (Shibata et al, 1956; Shibata and Kitagawa, 1956 and 1960). They showed luteoskyrin (XV) and rugulosin (XVII) to have the following structures:

OH 0 0

OHO

m Luteoskyrin (XV) = 1.1 bis (2:4:5:8 tetrahydroxy Rugulosin (XVII) = 1.1 bis (2:4:5: trihydroxy 7-met Rubroskyrin (XVI) = 1.1 bis (2:5:8 trihydroxy 7-m

Both luteoskyrin and rugulosin have been shown to produce liver damage. (Uraguchi, 1961). With luteoskyrin, L.D~ = 147 mgs/Kg s.c., and 221 mgsjKg orally in mice, marked centrolobular necrosis was produced after 48 hours, together with fatty degeneration of the liver cells. Rugulo­sin was also found to produce similar liver damage on prolonged administration.

The L.D~ = 1070mgjKg in mouse. The other six related compounds isolated from Penicillium islandicum Sopp. were found to be atoxic. Buii-Hoi and Zajdela ( 1962), however, report that purified luteoskyrin does not produce hepatotoxic effects, eve.n in rats fed on a low protein diet; these workers attribute the reported toxicity to impurities, perhaps islanditoxin. It is of interest to note that only rugulosin and luteoskyrin do not possess a true p-quinonoid structure.

53

Two fungi, Penicillium citrinum Thorn and Penicillium toxicarium Miyake are frequently isolated from samples of " Thai " yellow rice. Feeding experiments (Sakai, 1955) have shown that rice cultures of Penicillium citrinum produce a toxicosis in mice, which was found to be due to citrinin (XVIII). This common fungal metabolite has been isolated from cul)ures of Penicillium expansum., Penicillium implicatum., Penicillium chrzaszszi, Penicillium citreosulphuratum., Penicil· lium lividum, Aspergillus terreus, Aspergillus candidus and Aspergillus niveus.

HOOC~OH O~CH

~c H 3

HOW HO

COOHO

CH3

The structure of citrinin (XVIII) was established by Robertson and coworkers ( 1949), who assigned to it a fused quinonoid pyran ring structure. Sakai ( 1955) studied the toxicology of citrinin (XVIII) and showed it to produce acute or chronic nephrosis in which the reabsorption of water by the kidneys is inhibited. Citrinin has an L.D~ of 35 mgsjKg s.c. or i.p. in mice.

Citreomycetin (XIX) has a double pyran ring structure, and has been isolated from several fungi, e.g., Penicillium frequentans, Penicillium glabrum, and many Citreomyces strains. Although not acutely toxic, citreomycetin, is an accumula-

· 7- methyl 2-3-dihydro-anthraquinone). hyl 2-3 dihydro-anthraquinone). ethvl 1·2 dihyro-4-9-10 anthracene trione).

Toxic moulds and their metabolites. R. J. Townsend

tivc poison which causes chronic kidney damage on prolonged administration. Similar effects are produced by kojic acid (XX), a metabolite of the Aspergillus oryzae-tamarll group of fungi. (Fried· mann, 1934). This metabolite was first isolated by Saito (1907) and was shown by Yabuta (1924) to be· 5-hydroxy 2-hydroxymethyl pyrone. It is toxic to dogs, rats and rabbits and has an L.D •. = 1500 mgsjKg orally in mice.

In 1952 an outbreak of food poisoning among dairy cattle occurred in Japan. The mass death of 118 cows was attributed to the consumption of mouldy malt feed infected with a ·toxigenic strain of Penicillium urticae Bainicr. (Yamamoto, Yamamoto and Ukai, 1954 ; Yamamoto, 1954 ; Yamamoto, 1954a). This organism was shown to produce patulin (XXI). a metabolite frequently found in cultures of Penicilium patulum, Penicil· lium expansum, Penicillium griseofulvum, Penicil· lium claviforme, as well as Aspergillus clavatus, Aspergillus terreus, and Aspergillus giganteus.

!1 0 OH

m.

The structure of patulin (XXI) has been estab· lished as a fused pyrone tetronic acid ring system by Woodward and Singh (1950), who confirmed the structure by the synthesis of patulin and patulin monoacetate.

Yamamoto investigated the conditions for the growth of Penicillium urticae, and the production of patulin on various media. He concluded that substrates containing glucose or maltose with a pH range of 4.5 to 6.6 favoured the growth of the fungus and the formation of patulin. Further studies showed patulin (XXI) to be a neurotoxic substance, with an L.D.. = 35 mgsjKg orally in mouse. Post-mortem examination showed the presence of haemorrhagic foci in the brain and central nervous system.

The toxic properties of Penicillium toxicariurn Miyake have been studied by Uraguchi et ai. (1942), who showed that alcoholic extracts from rice cultures of this organism produced an acute ascending paralysis in higher vertebrates, the toxicosis resembling the disease syndrome of acute beri-beri in man.

Penicillium toxicarium Miyake is a frequent contaminant of Japanese rice, as is Penicillium citreoviride Biourge, and these fungi may have some etiological significance to diseases of un· known origin in Japan.

54

From rice cultures of a toxigenic strain of Penicillium toxicarium, Hirata (1947, 1957) isolated citreoviridin (XXII), a yellow toxic polyene compound which was also produced by toxic strains of Penicillium citreoviride and Penicillium ochrosalmoneum Udagawa. Citreo­viridin (XXII) was isolated by acetone extraction of the culture, followed by column chroma· tography on alumina using a methanol-ether mixture. Recrystallization from methanol gave a yellow crystailine compound C,.H~O,.CH,OH. m.pt 107'C·ll1 'C. The structure of this compound has been elucidated by Hirata and coworkers in 1964, who have shown it to consist of a 4-methoxy 5 methyl n-pyrone group linked to a 2-4·5· trimethyi 3·4 dihydroxy tetrahydrofuran moiety by a con· jugated polyene chain. This structure may be compared with that of aurethin (XXIJJ), a toxic metabolite of the organism Streptomyces thio­luteus (Hirata, Nakata, Yamada et ai., 1961) which aiso contains a substituted pyrone and tetrahydro­furan groups.

Earlier studies, by Uraguchi (1947), on the physiological effects of citreoviridin, showed that it produced paralysis and respiratory failure, on oral administration, in rats. The minimum lethal dose levels were found to be 10 mgsjKg s.c., 8 mgsjKg i.p., and 30 mgsjKg orally in rats.

Citreoviridin gives a strong yellow fluorescence in ultra-violet light and post-mortem examination under ultra-violet light showed that citreoviridin was localised in the central nervous system, the adrenal cortex, the liver and the kidneys.

Another outbreak of toxicosis in dairy cattle occurred in Japan in 1954, and was found to be due to the consumption of malt sprout fe~d

infected with the fungus Aspergillus oryzae var. microsporus (Ohkubo, lizuka and !ida, 1955). This fungus was grown on Czapek-Dox broth and from the metabolism solution concentrate a new metabolite, maltoryzine (XXIV), was obtained by ether extraction. Column chromatography on ceilulose powder using a butanoljwater system gave a yellow crystalline substance, m.pt. 69'C, CuH1:01.

Toxic moulds and their metabolites. R. J. Townsend

The ultra-violet and infra-red spectra indica­ted that maltoryzine (XXIV) contained a benzoyl group, an hydroxyl group, a carbonyl group and a non-conjugated double bond. Ozono­lysis of maltoryzine trimethyl ether gave acetic acid and a f:l-keto acid, which on acid hydrolysis gave 2-3-6 trimethoxy benzoic acid and another molecule of acetic acid. Ketonic hydrolysis of the f:l-keto acid gave carbon dioxide and 2-3-6 tri­methoxy acetophenone. On this evidence Iizuka and !ida (1962) showed the structure of maltoryzine (XXIV) to be: 1-(3-pentenoyl) 2-3-6 trihydroxy benzene.

Intraperitoneal injection of this metabolite, L.D~ = 3 mgsjKg in mice, produced tremors, acute muscular paralysis and anorexia. Post­mortem examination showed extensive haemorrhage of the brain and serous membranes. It is of interest to note that a similar tremorgenic sub­stance has been isolated from the closely related fungus Aspergillus Oavus by Wilson and Wilson (1962).

Ochratoxin A (XXV), a new fungal toxin, has rece:1tly been isolated by Van der Merwe et a!. ( 1965) from maize cultures of the fungus Aspergillus ochraceus Wilhelm. Chloroform/ methanol extraction of the moulded maize meal cultures and chromatography on silica gel, followed by bicarbonate extraction and ion ex­change chromatography gave ochratoxin A (XXV) C,H,.O.NCI. Its infra-red and ultra-violet spectra indicated the presence of a carboxyl group, a secondary amide group and a lactone ring. This new chlorine containing metabolite gave phenyl­alanine and 5-chloro 7-carboxy 8-hydroxy 3-methyl 3-4 dihydro iso coumarin on acid hydrolysis. On the basis of this data the South African workers concluded that ochratoxin A is the amide formed from one molecule of phenylalanine and one molecule of the 5-chloro 7-carboxy 8-hydroxy 3-methyl 3-4 dihydro iso coumarin, i.e., (XXV).

Ochratoxin A is an extremely powerful poison with an L.D~ = 5oo,.gjKg orally in ducklings and is comparable in potency with the aflatoxins. Post­mortem examination of the affected birds showed gross fatty infiltration of the liver cells. No liver necrosis or proliferation of the bile ducts was observed, a feature which distinguishes this toxicosis from aflatoxicosis or groundnut poisoning. Two other non-toxic metabolites, ochratoxin B and ochratoxin C, were also isolated from cultures

55

of Aspergillus ochraceus. These two compounds are related to ochratoxin A, being the dechloro­ochratoxin A and ochratoxin A ethyl ester respectively. This relationship suggests that the toxicity of ochratoxin A is dependent upon the presence of the 5-chloro group and on the free carboxyl group in the phenylalanine moiety. It is of interest to note that ochracin (XXVI) or 8-hydroxy 3-methyl 3-4 dihydro-isocoumarin was isolated from cultures of Aspergillus ochraceous in 1933 by Yabuta and Sumiki.

Another type of mycotoxicosis was reported in 1961 by Birmingham and coworkers, who observed a blistering dermatitis amongst celery harvesters. The disease was found to run concurrently with a fungal infection of the celery plants, called "Pink Rot." This is caused by the mould Scerotinium sclerotiorum (Lib.) de Bary which is often present in the soil and attacks the celery plants at the base line.

Extracts of the dried, diseased celery plants were shown to produce phototoxic lesions, when applied topically to albino rabbit skin and exposed to an ultraviolet light source emitting light at 3650 J!.. Using this bioassay technique to follow the isolation of the phototoxic agents, Perone (1961), isolated two substituted psoralen com­pounds. These were identified, by comparison of their ultra-violet and infra-red spectra, with those of known psoralen compounds, as 8-methoxy psoralen (XXVII) and 4-5'-8 trimethyl psoralen (XXVIII). The activity of these compounds was found to be 1.5 l'gfsq. ins. and 0.1 f'gfsq. ins·, respectively.

Furocoumarins are found in many plants, e.g., Psoralae corylifolla. They are often photodynamic toxins, producing photosensitization of the skin. Musajo (1962) has studied the relationship between chemical structure and photosensitization activity, which is due to the furocoumarin nucleus, since reduction of the double bonds of the furan or pyran rings completely destroys the activity. A linear configration was found to be more active than an angular one, e.g., psoralen (XXIX), bergapten (XXX), angellicin (XXXI) and allo­bergapten (XXXII).

Toxic moulds and their metabolites. R. J. Townsend

Q):)o

.XXXII

The presence of an OH group was found to destroy the activity, while methylation partially restores it. Two methoxyl groups in the 5 and 8 oositions leads to inactivation. It appears that the presence of C-methyl groups does not lead to a loss of activity. Masajo has observed the forma­tion of complex products from the photodyn~mic psoralen compounds and flavin mononucleotide, on irradiation of aqueous ethanolic solutions of flavin mononucleotide and the furocoumarins with ultra­violet light, at 3655A. No reaction was observed with the inactive compounds. Ether extraction of the reaction mixtures gave degradation products of psoralen compounds, which had undergone oxidation of the furan ring, as well as some new yellow flavin derived substances. For example, the photoreaction between psoralen and flavin mononucleotide produced 7-hydroxy-coumarin 6-acetic acid methyl ester and 7-hydroxy 6 formyl coumarin, which have been identified by chroma­tography. The nature of the flavin derived pro­ducts is not yet known.

This paper has only touched upon part of a large and increasingly important field of research in the biodeterioration of foods and feeding stuffs, and of veterinary and medical science. The importance of mycotoxins in foods and feeding stuffs cannot be over estimated in view of the evidence that fungal toxins are the causative agents of many and varied diseases of previous unknown etiology. The high incidence of liver disease and liver cancer in tropical countries, where rice, groundnuts, and maize are the staple diets, may not be coincidental. Uraguchi reports that the amount of mouldy rice condemned as unfit for consumption during the period 1950-1954 exceeded 100,000 tons in Japan alone (Uraguchi, 1961). Interesting case histories of food poison­ings have been cited by the same author, who mentions the poisoning of Korean immigrants by mouldy corn in 1941, and an outbreak of food poisoning among youths who had eaten rice infected with Fusarium spp. Uraguchi concludes that "From the toxicological point of view we hold the assumption that there must be some unknown substances with liver InJUrious or carcinogenic action in the foods of the above

56

areas." (He was referring to the high incidence of primary carcinoma of the liver in the tropical countries of Asia.)

Oettle has reviewed the epidemiology of liver carcinoma and has also concluded that mycotoxins offer a plausible explanation, showing that the observed distribution of the disease correlates better with areas of high humidity and defective food storage, than the incidence of malnutrition, senecio poisoning, infectious hepatitis, bilharzia or chronic alcoholism.

Much remains to be done and learnt in the field of mycotoxins, not only in the detection and isolation of the fungi and fungal metabolites, but in the fields of animal husbandry, agricultural techniques, storage and food preservation. Much more could be done, by the application of present knowledge and techniques, to prevent fungal and bacterial contamination of foods and feedingstuffs.

In this respect the further education of agricultural workers, food producers and manu­facturers, in the developing areas of the world, in the agricultural sciences, food science and technology, and applied biology, is an essential step in the fight against the biodeterioration of the world's food and feedingstuffs.

References : Allcroft, R., Carnaghan, R. B. A. (1962). Vet. Rec.,

74, 863-864. Groundnut toxicity, Aspergillus llavus toxin (aflatoxin) in animal products.

Allcroft, R., Carnaghan, R. B. A. (1963). Chern. Ind. Loud., 50. Toxic products in groundnuts. Biological Effects.

Allcroft, R., Carnaghan, R. B. A., Sargeant, K., and O'Kelly, J. (1961). Vet. Rec., 73, 428-29. A toxic factor in Brazilian groundnut meal.

Asao, T., BUchi, G., Abdel-Kedder, M. M., Chang, S. B., Wick, E.L., and Wogan, G. N. (1963). J. Amer. Chern. Soc., 85, 1706-07. Aflatoxins Band G.

Asplin, F. D., and Carnaghan, R. B. A. (1961). Vet. Rec., 73, 1215·19. The toxicity uf certain groundnut meals for poultry with special reference to their effects on ducklings and chickens.

Birmingham, D. J., Key, M. M., Tubich, G. E., Perone, V. B. (1961). Arch. Derma!, 83, 73. Dermatitis among celery harvesters

Blount, W. P. (1961). Turkeys., 9, (2), 52. Turkey X disease.

Brown, J. M. M., and Abrams, L. (1965). Onder­stcpoort. J. Vet. Res., 32, (1), 119-146. Bio­chemical studies on afiatoxicosis.

Butler, W. H., and Barnes, J. M. (1964). Nature, 202, 1016. Carcinogenic activity of aflatoxin to rats.

Buii-Hoi, P., and Zajdela, F. (1962). Med. exp. 6, 29-32. La luteoskyrine est elle le principe lepato-toxique du riz " Jauni " ?

Campbell, A. G. (1965). New Zealand J. Agric., 111, 23. Facial eczema.

Toxic moulds and their metabolites. R. J. Townsend

Coomes, T. J., and Sanders, J. C. ( 1963). Analyst, 88, 209-213. The detection and estimation of aflatoxin in groundnuts and groundnut materials.

Coveney, R. D., Peck, H. M., and Townsend, R. J. ( 1966). S.C.!: Monograph No. 23. Recent Advances in Mycotoxicoses, p.31.

Done, J., Mortimer, P. H., and Taylor, A. (1962). Res. Vet. Sci., 3, 147. The experimental intoxi· cation of sheep with sporidesmin, a metabolic product of Pithomyces chartarum. II. Changes in some serum constituents, after oral adminis· !ration of sporidesmin.

Forgacs, J., and Carll, W. T. (1962). Advan. in Vet. Sci., 7, 273. Mycotoxicoses.

Fridrichsons, J. and Mathieson, Me. L. A. ( 1962). Tetrahadron letters, 26, 1265. The structure of sporidesmin. Causative agent of facial eczema in sheep.

Friedmann, T. E. (1934). Science, 80, 34. Gallagher, C. H. (1964). Nutritional fac.tors am'

enzymological disturbances in animals. London. Crosby Lockwood and Sons.

Halver, J. E. (1964). Aflatoxicosis and trout hepatoma in Wogan, G·N., Mycotoxins in Food· stuffs, Cambridge, Mass., M.I.T. Press, p209.

Harding, J. D. J., Done, J. T., Lewis, G., and Allcroft, R. (1963). Res. Vet. Sci., 4, (2), 217. Experimental groundnut poisoning in pigs.

Hartley, R. D., Nesbitt, B. F., and O'Kelly, J. (1963). Nature, 198, ( 4885), 856. Toxic metabolites of Aspergillus flavus.

Hirata, Y. ( 1947). Bull. Chern. Soc. Japan, 68, 64, 74, 106. On the products of mold.

Hirata, Y.,. Nakata, H., Yamada, K., Okuhara, K., and Naito, T. (1961). Tetrahedron. 14, 252.

Hirata, Y. ( 1957). Ann. Rep. Coope. Res. Minis!. Educ. Japan, p.129. Studies on a polyene fungal metabolite.

Hirata, Y., Sakabe, N., and Goto, T. (1964). Tetrahedron letters. (27), 1825-30. The structure of litreoviridin, a toxic compound produced by F. citreovirlde molded on rice.

Hodges, R., Ronaldson, J. W., Taylor, A., and White, E. P. (1963). Chern. and Ind., (1), 42-43. Sporidesmin and Sporidesmin B.

Holzapfel, C. W., Steyn, P. S., and Purchase, I. F. M. ( 1966). Tethedron letters. (25), 2799· 2803. Isolation and structure of aflatoxins M, and M,.

Howard, B. H., and Raistrick, H. (1954). Blochem. J., 57, 212. Studies in the biochemistry of microorganisms. The colouring matter of Penicillium islandicum Sopp.

Iizuka, H., and !ida, M. (1962). Nature, 196, 681. Maltoryzine, a new toxic metabolite produced by a strain of A. oryzae var. microsporus, isolated from the poisonous malt sprout.

long, de H., VIes, R. 0., and Pelt, van J. G. (1964). Nature, 202, 466-467. Milk of mammals fed on aflatoxin containing diet.

57

Lancaster, M. C., Jenkins, F. P., and Philp, J. Me. L. (1961). Nature, 192, 1095-1097. Toxicity associated with certain samples of groundnuts.

Loosmore, R. M., and Harding, J. D. J. (1961). Vet. Rec., 73, 1362·1364. A toxic factor in Brazilian groundnuts causing liver damage to pigs.

Loosmore, R. M., and Markson, L. M. ( 1961) Vet. Rec., 73, 813-814. Poisoning of cattle by Brazilian groundnut meal.

Losses in Agriculture. ARS-20-1, June, 1954 Agricultural Research Service, U.S. Department of Agriculture.

Marumo, S. (1959). Bull. agric. Chern. Soc. Japan, 23, (5), 428. lslanditoxin, a toxic metabolite produced by Penicillium islandicurn Sopp. Pt. III. Structure of islanditoxin.

Miyake, 1., Naito, H., and Tsunoda, H. (1940). Beikoku. Riyo. Kenkyuto. Hokoku., 1, 1-29. Studies on the production of toxin in the sapro· phyte growing rice grains under storage.

Miyake, I. (1947). Nishin lgaku, 34, 167. Penicil· lium toxicarium growing on the yellow mouldy rice.

Mortimer, P. H. (1963). Res. Vet. Sci., 4, (1), 166 The experimental intoxication of sheep with sporidesmin, a metabolic product of Pithomyces chartaru11,1. IV. Histological and histochemical examinations of orally dosed sheep.

Mortimer, P. H. (1962). Res. Vet. Sci., 3, (3), 269. The experimental intoxication of sheep with sporidesmin, a metabolic product of Pithomyces chartarum. Ill. Some changes in cellular com­ponents and coagulation properties of the blood, in serum proteins and in liver function.

Mortimer, P. H., and Taylor, A. (1962). Res. Vet. Sci., 3, (2), 147. The experimental intoxication of sheep with sporidesmin, a metabolic product of Pithomyces chartarum. I. Clinical observa· tions and post mortem findings.

Musajo, A. ( 1962). Pure and Applied Chern., 5, 415.

Nesbitt, B. F., O'Kelly, J., Sargeant, K., and Sheridan, A. (1962). Nature, 195, 1062. Toxic metabolites of Aspergillus flavus.

Ohkubo, Y., Iizuka, H., and lida, M. (1955). Jap. J. Vet. Sci., 17, (4), 145.

Perone, V. B. (1961). Report. Occupation~! Health. Res. and Training Faculty, Cincinnati, U.S.A.

Rabie, C. J. (1965). Symposium on Mycotoxins. Agricultural Aspects. University of Pretoria, Pretoria. I, 18-30. Department of Agricultural and Technical Services, Pretoria, S. Africa. Influence of temperature on the production of aflatoxin by Aspergillus Oavus.

Rabie, C. J., Klerl, de W. A., and Terblanche, M. (1964). S. African. J. Agrlc. Sci., 7, 341-346. Toxicity of Aspergillus amstelodami to poultry and rabbits.

Toxic moulds and their metabolites. R. J. Townsend

Recondo, de A. M., Frayssinet, C. H., Lafarge, C., and Breton, le E. (1966). Biochim. Biophys. Acta, 119, 322-330. Action de l'aflatoxins sur le metobolismedu DNA au cours de I' hypertrophie compensatrice du foie apres hcpatectomie portielle.

Robertson, A., Cartwright, N. J., and Walley, W. B. ( 1949). Nature, 163, 94.

Saito, K. (1907). Bot. Mag. (Tokyo), 21, 249. Uber die saurebildung bei Aspergillus oryzae. ( Vorlaiifige mitteilling).

Sakai, F. (1955). Folia Pharm. Japan, 51, 431. Experimental studies on rice yellowsis caused by P. citrinum Thorn and toxicity especially kidney damage ~effect of citrinin pigment pro­duced by the fungus.

Sargeant, K., O'Kelley, J., Carnaghan, R. B. A., and Allcroft, R. (1961). Vet. Rec., 73, (46), 1219-1223. The assay of a toxic principle in certain groundnut meals.

Sargeant, K., Sheridan, A., O'Kelly, J., and Carnaghan, R. B· A. (1961). Nature, 192, 1096. Toxicity associated with certain samples of groundnuts.

Sarkisov, A. Kh. (1954). ~likotokochsikozi gosurdarstvennoe izdatelstuo selskohoziastvennoi literatury. Moskva.

Shibata, S., Murakami, T., Kitagawa, 1., and Kishi, T. (1956). Pharm. Bull. (Japan), 4, 111-116. Metabolic products of fungi. VIII. Rugulosin I. The structure of dianhydrorugulosin and its re­lation to the structure of iridoskyrin.

Sporn, M. B., Dingman, C. W. and Phelps, H. L. ( 1966). Science, 151, 1539-1541. Aflatoxin B. binding to DNA in vitro and alteration of RNA metabolism in vivo.

Symposium on Facial Eczema Disease. (1962). Proc. N.Z. Soc. Animal. Production, 99, 44-90.

Theron, J. J., Liebenberg, N., and Joubert, H. J. B. Symposium on Mycotoxins (1965). Pretoria. Veterinary Aspects. II, 43, Department of Agricultural and Technical Services, Pretoria. Acute liver injury in ducklings as a result of aflatoxin and ochratoxin poisoning.

Thornton, R. H. (1963). N.Z.J. agric. Res., 6, 469-483. Antifungal activity of fatty acids to Pithomyces chartarum (Burk and Curt) M. B. Ellis, and other fungi.

Townsend, R. J., Moss, M. 0., and Peck, H. M (1966). J. Pharm, Pharmac., 18, 472. Isolation and characterisation of hepatotoxins from Penicillium rubrum.

Uraguchi, K. (1942). Folia. Pharm. Japan, 34, 39 Pharmacological studies on the extract of the yellow mouldy rice 0-Henmai.

Uraguchi, K., Tatsuno, T., Sakai, F., Tsukioka, M., Sakai, Y., Yonemitsu, 0., Ito, H., Miyake, M., Saito, M., Enomoto, M., Shikata, T., and Ishiko, T. ( 1961). Japan J. Exper. Med., 31, 1-18. Toxico­logical approach to the metabolites of Penicil·

58

lium islandicum Sopp. growing on the yellowed rice.

Uraguchi, K., (1947). Nisshin. lgaku., 34, 155. Existence of toxic substances in the mouldy rice. Pharmacological studies on the toxicity of the yellowed rice 0-Henmai.

Van der Merwe., K. J., Steyn, P. S., Fourie, L .. Scott, de B., and Theron, J. J. (1965)· Nature, ~Lon d., 205, 1112. · Ochratoxin A, a toxic metabolite produced by Aspergillus ochraceous Wilh.

Wieland, T. ( 1963). Pure and Applied Chern., 6, 339. Chemical and toxicological studies with cyclopeptides of Amanita phalloides.

Wieland, T., and Schnabel, H. W. (1962). Annalen, 657, 218. Cyclopeptides of Amanita pballoides.

Wieland, T., and Wieland, 0. (1959). Pharmacal. Revs., 11, 87.

Wilson, B. J., and Wilson, C. H. (1962). J. Bact., 83, 693. Hepatotoxic substance from Penicillium rubrum.

Wilson, B. J., and Wilson, C. H. (1962a). J. Bact., 84, 283-290. Extraction and preliminary characterisation of a hepatotoxic substance from cultures of Penicillium rubrum.

Woodward, R. B., and Singh, G. (1950). J. Amer. Chern. Soc., 72, 1428. Synthesis of patulin acetate.

Woodward, R. B., and Singh, G. (1949). i. Amer. Chern. Soc., 71, 758. Structure of patulin and patulin acetate.

Yabuta, T. (1924). J. Chern. Soc. Japan, 125, 575. The constitution of kojic acid, a ll-pyrone derivative formed by Aspergillus oryzae from carbohydrates.

Yabuta, T., and Sumiki, Y. (1933). J. Agric. Chern. Soc. Japan, 9, 1264. Isolation of ochracin from A. ochraceous.

Yamamoto, T., Yamamoto, Y., and Ukai, T. (1954). J. Pharm. Soc. Japan, 74, 450. Studies on the poisonous substance from a strain of Penicillium II. Culture methods and chemical structure.

Yamamoto, T. (1954a). J. Pharm. Soc. Japan, 74, 787. Studies on the poison producing mold isolated from dry malt. I. Morphology and identification. P. urticae.

Yamamoto, T. (1954b). J. Pharm. Soc. Japan, 74, 810. Studies on the poison producing mold isolated from dry malt. IV. On the toxicity.

Yamamoto, T. (1957). Ann. Rep. Hug. Lab. Kobe. Municipal. Hyg. Lab., 43-48. Biochemical studies on the mold fungus isolated from foodstuff.

Baker, P. W., Int. Blodetn Bull., 3, (2), 59-64, (1967). An evaluation of some fungicides for optical instruments.

AN EVALUATION OF SOME FUNGICIDES FOR OPTICAL INSTRUMENTS

P. W. BAKER'

Summary Common defects in optical instruments under

tropical conditions and preventive measures are briefly analysed. The results are given of com· parative tests on four volatile fungicidal agents. It is concluded tha_t ethyl mercury chloride could prove an effective fungicide for optical instru­ments. The primary aim in instrument design, however, should still be the achievement of satisfactory sealing and desiccation.

List of contents 1 Introduction 2 Preventive Measures 3 Fungicides Evaluated 4 Method of Evaluation 5 Test Results 6 Conclusions

Ust of lllustratlons Plate 1 Fungus growth on glass sample­

Penicillium eapsulatum ( approx. mag­nification x 25).

Plate 2 Fungus growth on glass sample­Aspergillus glaucus ( approx. magnifica· lion x 25).

Plate 3 Test rack in plastic tray, with glass cover removed.

Plate 4 Test rack. in plastic tray, with glass cover in place, showing method of sealing by the salt solution used to control humidity.

1. Introduction 1.1. Extreme difficulty is frequently experi­enced in maintaining optical instruments serviceable and free from fungus attack in tropicaljjungle areas. Many types of instru­ments can become unserviceable after only three months' use, whilst storage facilities are often primitive, resulting in new instruments deteriorating rapidly 'on the- shelf' because of fungal attack. 1.2. The deterioration of optical instruments in tropical areas can be attributed almost entirely, either directly or indirectly, to the very high humidity conditions prevailing. Trouble commonly arises from one or more of the following effects :

(i) Temporary obscuring of vision by

moisture condensation. (ii) Blurring and obscuring by fungal

growth promoted by the high humidity conditions. This can cause : (a) the temporary problem of obscuring

vision (b) permanent damage by acids and

alkalis, released by the mould fila­ments, attacking bloomed surfaces and etching the glass.

(iii) Corrosion of metal components which can result in loose particles of corrosion residue becoming deposited on glass surfaces causing local pitting of the glass or promoting centres of fungus attacli.

2. Preventive Measures 2.1. The ideal solution to the problem is obviously to seal (hermetically if possible, where this is not excluded by servicing re­quirements) and fill (desirably pressurise) the instrument with an inert gas or dry air: Current design in the U.K. and U.S.A. Is in gllneral directed to this end : however, for this approach to be successful, the sealing of the instrument . must be 100% effective, which involves very careful attention to the mechanical design of an instrument at an early stage in its deveropment.

The number of exposed. joints, access panels and rotating members must be. kept to a minimum. Screws securing access panels should not penetrate through the main body casing and should be located outside the sealed area. Exposed focussing eyepieces are a potential source of leakage and should be avoided. For instance, in one Admiralty instrument, the adjustable binocular focussing elements are sealed within an extension of the main body of the instrument, behind a one piece eye-glass cover, which is relatively simple to seal. 2.2. However, even assuming that a satisfactory standard of sealing is achieved in new instru­ments, it is quite likely that, because of servicing difficulties, possible lack of spares, etc., the initial standard cannot always be maintained. It is desirable, therefore, to incorporate some form of fungicidal agent to

'Ministry of Aviation, Royal Radar Establishment, St. Andrews Road, Great Malvern, Worcestershire, England. (Copy received 6th February, 1967).

59

An evaluation of some fungicides for optical instruments.

provide a ' second line of defence ' against mould attack. ' 2.3. Considerable effort was expended in the U.K., Australia and the U.S.A. during the latter half of the 1939-'45 war to find. a satisfactory fungicide for optical instruments, with a certain measure of success (Turner et al., 1946 ; Vickland, 1946 ; Teitell and Berk, 1952). However, work on the problem was virtually abandoned, both in this country and Australia, at the end of hostilities. In the U.S.A. some further work was carried out until 1953-54, see Teitell and Berk (1954), but no investigations have apparently been made since. More recent work in Germany by Carl-Zeiss-Siftung (1960), KaUer (1962) and KaUer ( 1966), in Czechoslovakia by Minar ( 1963) and in Russia by Troshchenke and Bourgozhin (1958) has been directed chiefly at evolving protective treatments that can be applied direct to the glass surfaces.

3. Fungicides Evaluated 3.1. In this series of tests, four different volatile fungicide agents were evaluated for :

(i) effectiveness in preventing mould growth

( ii) adverse side-effects, such as corrosion of finishes and other items (e.g., elec­tronic components, wiring, etc.) which may be within the same casing

The four fungicides tested were : (a) Meta-cresyl-acetate (Cresatin) (b) Sodium ethylmercurithiosalicylate

(M.T.S. or Merthiosal) (c) Ethyl mercury chloride (E.M.C.) (d) Tri-iodo-chloro-ethylene (T.I.C.E.).

3.2. The fungicides were used as follows : (a) Cresatin, (a liquid) mixed with ethyl

cellulose powder (roughly 50-50 by volume) to form a stiff gel or "taffy," approx. 15 gm. of the mixture, placed in a small jar, being used within a test volume of 6000 c.c., i.e., 2.5 mg.jc.c.

(b) Merthiosal, (a white crystalline solid) was obtained ready mixed with a matt black lacquer (Parsons No. 8687C). It was painted on to a piece of aluminium sheet 12 em. x 10 em., giving approx. 4 sq. em. per 100 c.c. of test volume.

(c) E.M.C. (a white flaky crystalline powder) was used in a small jar, approximately 3 gm., within a test volume of 6000 c.c., i.e., 0.5 mg/c.c.

(d) T.I.C.E. (a fine powder) was again placed directly ,in a small jar, approximately 10.5 gm., within a test volume of 6000 c.c., i.e., 1.75 mg.jc.c. (In practice, Cresatin has been fixed to a

suitable point in an instrument as a small block (mixed, of course, with ethyl cellulose) or contained in a porous capsule : see Teitell and Berk (1952). Merthiosal doped lacquer has been used as the internal finish of instru­ments in lieu of the normal finish : see Turner

60

P. W. Baker

et. al. (1946). E.M.C. can also be used mixed with paint: see Dieter (1965), but its use in some form of small capsule or cartridge with a suitable filter under a removable seal or cap is proposed. The cap or seal could be quickly removed aud the cartridge screwed into the instrument from outside in the same manner as an instrument type desiccator. T.I.C.E. could be used in the same manner. I

4. Method of Evaluation 4.1. Preliminary mould growth te~ts were made on a wide variety of optical glasses using four different mould cultures to assess the likely extent of damage in the absence of a fungicidal agent: The glass samples were polished blanks If' diameter x f" thick, with bloomed surfaces. Three compound assemblies, each comprising two blanks cemented together with a proprietary epoxy resin cement to form a ! " thick sample, were also tested. 4.2. Five sets of glass samples were then sub­jected to mould growth test to evaluate the four fungicides, one set being a control. Included with each set were the following items:

. (i) Sample of printed-circuit board (" Vero· board") 4f' x 4".

{ii) An enscapsulated type silver mica capacitor ( 470 pF) with silver plated leads, soldered to ( i)

(iii) A t watt H.S. resistor ( 47K 5•.;) with tinned leads, soldered to (i)

(iv) A 2!in. length of P.V.C. sheathed 22 S.W.G. tinned copper wire, with bared ends soldered to (i)

(v) Two "Terry" spring clips (size 80/3) with passivated cadmium finish, secured to (i) with cadmium plated screws, nuts and washers, and supporting (vi) and (vii)

(vi) A B9G screening can -aluminium with matt black paint finish inside and out (except for the inside skirt, left bare

aluminium) (vii) A B9G screening can- brass with black

copper oxide finish. 4.3. Each glass blank was inoculated in two positions on each face with cultures obtained from the Commonwealth Mycological Institute. Kew, as follows : (a) Front, top left- CMI 53242, Aspergillus ·

glaucus group (b) Front, top right- CMI 53243, Aspergillus

glaucus group. (c) Rear, top left-CMI 40576, Penicillium

capsula tum (d) Rear, top right- CMI 108299, Eurotium

tonophilum. (N.B. The electronic component/material

samples detailed in para. 4.2. were not inoculated with mould spores.)

Inoculation was by a dry method, using a small spatula made from 22 S.W.G. nickle· chromium wire to deposit a very small spot of

An evaluation of some fungicides for optical instruments. P. W. Baker

agar nutrient and mould spores on the glass surface. Usual sterilisation precautions were taken to prevent contamination of the cultures. 4.4. During test, the samples were .held in a perspex rack (see Plate 3). A saturated salt solution of potassium sulphate in a shallow tray was used to maintain a constant R.H. of 97%, the samples (and fungicide agent where used) being sealed in by an inverted glass tank resting on the bottom of the tray. The tray ;tank combinations were stored in a humidity cabinet with the temperature con­trolled at 29"C and a R.H. of 95% approx. to limit the rate of evaporation from the salt solution.

5. Test Results 5.1. Tests on Glass Samples only (without

Fungicides) 5.1.1. In these preliminary tests, the integrated light transmission through the individual samples was measured, using a collimated tungsten filament lamp source in combination with a 15 em. water cell as an infra-red filter. Measurements were made:

( i) Before tests commenced. (ii) After three months mould growth

test at 29"C 97% R.H. (iii) After washing the samples in distilled

water to remove the mould hyphae from the glass, thus leaving only the results of any permanent damage, such as removal of the blooming and etching of the glass itself.

5.1.2. The transmission was measured through the centre area of the blank only, thereby excluding the original inoculation points. Unfortunately, microscopic exami· nation showed that on several samples the mould had not yet spread extensively into the centre area of the blank (the inocula­tion points being near the edge of the blank) but would obviously have done so given, say, a further three to six months exposure. The results of the light trans­mission measurements (see Table I) do not, therefore, give a true indication of the extent of obscuration by mould in the individual samples, as had been hoped. However, they do demonstrate that infec­tion of an optical instrument by mould ~ould easily result in a 2% to 3% reduction in transmission for each glass/air surface, e.g., roughly -equivalent to removing the an~i-reflection (bloomed surface) coatings.

TABLE I- Results of Tests without Fungicide

Extent of Mould Growth after

Chance Type % 3 months

Sample Glass No. Transmission (Microscopic examination)

No. Type (or ~qui valent) Before After After Front Rear

test 3 mo!lths Wash L.H. R.H. L.H. R.H.

1 sc 515564 96 92 94 s L s L .. 2 sc 515564 96 92 94 M s M L

6 zc 508612 95.5 95 95 L L s M

7 zc 508612 95.5 95 95 s M L L

11 DEDF 748278 97.5 96 96 s L L N

12 DEDF 748278 98 92 95 M L M L

17 DBC 606600 98 92 94 s M s M

18 DBC 606600 97.5 96 96 M M s M

21 MBC 572577 95.5 93 94 M s s L

24 LF 579411 95.5 94 94.5 M M M s 27 LBF not known 96 94 95 M M L L

30 Green - 81.5 79 81.5 M M s L Plate

40 HC/DF 519604/623360 96 90 94 M s M M

42 EDF/HC 651336/519604 95.5 92 94 s L M M

48 MBC/ 572577/- - - - L M - -Dural

SC- Soft Crown ZC- Zinc Crown S --Severe HC -· Hard Crown . M -- Moderate DF --Dense Flint L- Light EDF- Extra Dense Flint N -NIL DEDF- Double Extra Dense Flint DBC- Dense Barium Crown LF- Light Flint LBF -·Light Barium Flint MBC Medium Barium Crown

61

An evaluation of some fungicides for optical in>truments. P. W. Baker

5.1.3. After one month, mould growth was visible on most samples by viewing through the glass tank with the aid of a torch. Microscopic examination, after three months, showed that all the samples had been damaged to a varying extent, ranging from removal of the blooming to severe etching of the glass. One inocula· tion only, on one sample (No. 11- DEDF), had not resulted in growth.

5.2. Tests Using Fungicides 5.:!.1. The results of tests using fungicides are detailed in Table II. It can be seen that only ethyl mercury chloride (E.M.C.) was completely effective in preventing mould growth on the glass samples. 5.2.2. With Cresatin, mould growth occurred on four samples out of five to a light or moderate extent. With tri-iodo-

chloro-ethylene (T.I.C.E.), two samples were affected by mould -and with Merthiosal (M.T.S.) only one glass sample was affected, but this to a relatively severe extent. Mould growth had occurred on all the glass samples in the Control set, mostly to a moderate or severe extent. 5.2.3. T.I.C.E. had no significant corrosion effect on the other materials included. With Merthiosal,- there was extensive mould growth and corrosion spotting on the aluminium screening can {although this had not been deliberately inoculated with mould spores). Cresatin had caused slight corrosion spotting of the black copper oxide finish of the brass screening can and also its cadmium plated spring. With E.M.C. there was a very slight green deposit on the black copper oxide finish.

TABLE II- Results of Tests using Fungicides

Extent of Mould Growth after 3 Months

Sample Glass Chance Type Comments on Condition of No. Type No. Front Rear Electronic Items, etc.

(or equivalent) L.H. R.H. L.H. R.H.

SET A- With CRESA TIN 3 sc 515564 L L L N

13 DEDF 748278 L L M M Slight corrosion spotting of

19 DBC 606600 N L N N black copper oxide {screening can), also cadmium plated

22 MBC 572577 L L L M spring. 49 HC/Dural 519604/- N N - -

SET B-With E.M.C. 4 sc 515564 N N N N

8 zc 508612 N N N N No corrosion, except for very 14 DEDF 748278 N N N N slight green deposit on black

23 MBC 572577 N N N - copper oxide {screening can).

46 DF/Dural .623360/- N N - -SET C -· With MERTHIOSAL

5 sc 515564 N N N N 9 zc 508612 N N N N Extensive mould growth and

15 DEDF 748278 N N s M corrosion spotting on 25 LF 579411 N N N N aluminum screening can.

44 LBF/Dural Not known N N - -SET D- With T.I.C.E.

10 zc 508612 N N N N 16 DEDF 748278 N N N N 20 DBC 606600 L N N N No significant corrosion 26 LF • 579411 L M M L 47 EDF/Dural 651336/- N N - -

SET E- CONTROL- no Fungicide 43 SC/LF 515564/579411 L L M s 36 EDF/ZC 651336/508612 s M M M 37 DBC/LF 606600/579411 s M s s No significant corrosion

45 Green - M M - -Plate; Dural

N.B.: Symbols as for Table I

62

An evaluation of some fungicides for optical inslruments.

PLATE 1 Fungus growth on glass sample, penicillium

capsula tum (a pprox. magnification x 25)

PLATE 3 Test rack in plastic tray, with glass cover

removed.

63

P. W. Baker

PLATE 2 Fungus growth on glass sample, aspergillus

glaucus ( approx . magnification x 25)

PLATE 4 Test rack in plastic tray, with glass cover

in place, showing method of sealing by the salt solution used for humidity control.

An evaluation of some fungicides for optical instruments. P. W. Baker

6. Conclusions 6.1. Of the four fungicides. evaluated, only ethyl mercury chloride (E.M.C.) was com­pletely effective as a fungicide, but caused very slight corrosion of the black copper oxide finish.

6.2. Cresatin proved relatively ineffective in these tests and caused slight corrosion of the black copper oxide finish.

6.3. Neither Merthiosal (M.T.S.) nor tri-iodo­chloro-ethylene (T.I.C.E.) were completely effective. But, whereas Merthiosal caused corrosion of aluminium, T.I.C.E. appeared to have no significant corrosion effect. 6.4. The corrosion of black copper oxide finishes by Cresatin and that of aluminium by Merthiosal have been observed by previous investigators (1,3).

6.5. It is proposed to carry out a further evaluation of E.M.C., in particular the possible corrosion hazards at higher temperatures. Meanwhile, it may be concluded that E.M.C. is likely to prove much more effective as a fungicide for optical instruments, than either Cresatin or Merthiosal and probably presents less of a· corrosion risk. Its use must, however, always be regarded as a secondary measure, the primary aim in instrument design being to produce an instrument capable of being properly sealed and desiccated.

Acknowledgement Crown Copyright, reproduced with the permis·

sion of the Controller, H.M.S.O.

64

References

Carl·Zeiss-Siftung (1960). British Patent No. 848349. An article of light transmitting material and process for the manufacture thereof.

Dieter, E. (1965). East German Patent No. 38393. Fungicidal paint.

Kaller, A. (1962). Fejngeratetechnik, 11, 23-24. Weatherproofing optical instruments-some new ideas.

KaUer, A. (1966). East German Patent No. 45885. Prevention of fungus growth in optical instru­ments.

Minar, S. (1963). Jemna Mechanlka a Optlka, 8, 69-73. Protection of optical surfaces against deterioration effects by climatic factors.

Teitell, L. and Berk, S. (1952). Ind. Eng. Chern., 44, 1088-1095. Prevention of mould growth in optical instruments.

Teitell, L. and Berk, S. ( 1954). Ind. Eng. Chern., 46, 778-784. Radioactive materials in prevention of mould growth in optical instruments.

Troshchenke, A. T. and Bourgozhin, A. (1958). U.S.S.R. Patent No. 116493. A process for protecting the optical parts of apparatus from biological damage.

Turner, J.S., McLennan, E.!., Rogers, A.S. and Matthaei, E. (1946). Nature, 158, 469-473. Tropical proofing of optical instruments by a fungicide.

Vickland, R. E. ( 1946). Ind. Eng. Chern., 38, 774-779. Preventing the fungus fouling of optical instruments.

Darby, R. T., Int. Biodetn Bull., 3, (2), 65-66 (1967). Testing cellulose-containing plastics for resistance to fungi.

TESTING CELLULOSE-CONTAINING PLASTICS FOR

RESISTANCE TO FUNGI

R. T. DARBY'

The widely used ASTM ( 1963) mixed culture petri dish method for the determination of the susceptibility of plastics and plastic-like materials to mold growth makes use of a combination of species of fungi. The mixture contains Aspergillus niger ATCC 9642, A. ftavus ATCC 9643, A. versi­color ATCC 11730, Penicillium funlculosum ATCC 9644, Trichoderma sp. ATCC 9645, and Pullularia pullulans ATCC 9348.

The original reasons for the choice of these particular organisms have been lost in history, but it is probable that their choice was largely on empirical grounds rather than on research de­signed to discover the best organisms for the tests. There are, in fact, few data to rationalize the use of any of the organisms in the mixture over equally serviceable organisms, and probably some of them never, or rarely, contribute to the efficacy of the mixture.

Conversely, work in these laboratories has shown that the mixture is deficient in at least one major capability, namely the ability to detect reliably the susceptibility of a cellulose-containing plastic material to cellulose decomposition, cellulose frequently being a component of plastic formulations or laminated with synthetic materials. The absence of a dependable cellulose utilizing organism in the mixture has been a fault which has led to erroneous evaluations of the resistance of some materials to mold growth. This note demonstrates one such case.

A routine test of a sample of a recently intro­duced synthetic, laminated polymeric material, widely used for better grade shoes, indicated that it was not subject to mold growth. When tested according to ASTM (1963) Designation D-1924-63,. the results showed that there was a trace of transient superficial growth of fungus on the surface of the material after one week incubation but that this was not noticeable at the end of the three week test. The material was considered to have passed the test.

Repetition of the test some time later, this time

with the addition of spores of the strongly cellulo­lytic fungus Chaetomlum globosum, QM 459 ( = ATCC 6205) to the inoculum produced the opposite result. In this second test. the material supported various amounts of gro-.. th as shown in Figures 1 and 2. After one week of incubation, the lower side showed light growth and fruiting heads of Chaetomlum globosum, and this increased to the state shown in Figure 1 by the end of the third week. The cut edge of the piece, however, supported heavy growth of the fungus· as shown in Figures 1 and 2. No other fungus grew on the material. The upper surface of the material was free of growth.

The significance of these. findings in the reliability of the present ASTM test is obvious. Since there is always the possibility that cellulose may be part- of some plastic materials, either as a component of the formulation or as a laminate (as in this case) the susceptibility of the material may depend on this ingredient alone. Although the ASTM mixture does already contain known cellulose decomposers, notably Trichoderma, ex­perience has shown that this organism is highly unpredictable in its behaviour in the mixture and cannot be relied on to detect the presence of cellulose.

Modification of the ASTM method to include' the more reliable Chaetomium globosum seems to be needed so that this fungus may seek out any susceptible cellulosic constituents which may be present.

References :

American Society for Testing and Materials ( 1963), 1916 Race St., Philadelphia, Pennsylvania, 19103; Recommended Practice for Determining Resistance of Plastics to Fungi. ASTM Designation D-1924-63.

Abstract:

The inclusion of Chaetomlum globosum in the fungus test mixture in ASTM Designation D-192463, " Determining Resistance of Plastics to Fungi," is recommended to detect cellulosic constituents of formulations.

'Pioneering Research Division, U.S. Army Natick Laboratories, Natick, Mass., U.S.A.

(Copy received 4th March, 1967) 65

Testing cellulose-containing plastics for resistance to fungi. R. T. Darby

Upper Lower

Figure I.-Appearance of material after 3 weeks in mixed

culture petri dish test, using ASTM D-1924-63 organisms plus Chaetomium globosum QM 459 ( = ATCC 6205) .

Left : upper surface. Right : lower surface.

Figure 2.-Appearance of cut edge of material after 1 week

incubation in mixed culture petri dish test, showing fruiting heads (perithecia )of Chaetomium globosum along the edge. Actual length of piece approx. lf'.

66

LeGrand, Y. et Thivend, P. Int. Blodetn Bull., 3, (2), 67-76 (1967). Etude de Ia degradation enzymatique de Ia cellulose : fractionnement sur Ia gel de dextranes, d'une

poudre cellulolytique d'origine fongique.

ETUDE DE LA DEGRADATION ENZYMATIQUE DE LA CELLULOSE: FRACTIONNEMENT SUR GEL DE DEXTRANES D'UNE POUDRE CELLULOLYTIQUE

D'ORIGINE FONGIQUE

Y. LEGRAND' et P. THIVEND'

Introduction :

Le mecanisme de Ia degradation enzymatique de Ia cellulose est un phenomlme qui interesse de nombreux domaines de Ia science ou de l'industrie. Le chimiste etudie Ia structure des fibres de cellulose en ultilisant des enzymes celluloly­tiques don! !'action est plus specifique et moins brutale que celle des agents chimiques. L'agronome cherche a connaitre le mode de degradation de Ia matiere organique dans le sol ; le zootechnicien etudie Ia digestion de Ia cellulose des fourrages ou de Ia paille consommee par le ruminant. Les industries textiles ou papetieres exigent des donnees de plus en plus precises sur Ia fa~on

dont ont peut degrader ou au contraire proteger Ia matiere premiere qu'elles utilisent.

La degradation enzymatique de Ia cellulose a fait !'objet de nombreux travaux mais elle demeure mal connue et surtout mal expliquec. Deux theories contradictoires s'affrontent quant au nombre d'enzymes eellulolytiques responsables de cette degradation.

-La theorie de Reese (1960) qui propose une attaque en trois temps : unc cellulase C1 trans­formerait Ia cellulose native en chaines lineaires d'anhydroglucose; celles-ci seraient hydrolysees par une ou plusieurs cellulases Cx, en produits solubles a courtes chaines qui eux-memes subiraient !'action d'une {3 glucosidase pour etre transformes en glucose.

-La theorie de Whitaker (1953) qui affirme qu'un seul enzyme assure Ia totalite de Ia degrada­tion a partie des fibres cellulosiques.

D'une f~~on generale, on admet qu'il y a plusieurs enzymes en cause. Marsh, Merola et Simpson (1953) puis Youatt et Jermyn (1956) ont mis en evidence un enzyme, le " S factor " au facteur de gonflement, qui correspondrait a Ia cellulase C1 de Reese. II reduirait Ia structure cristalline de Ia flbrille de cellulose et faciliterait ainsi !'action ultericure des autres enzymes eellulolytiques. Grimes, Duncan et Hoppert (1957)

separent, par electrophorese, six fractions cellu­lolytiques differentes a partir d'une culture de Myrotheelum verruearla. Utilisant Ia meme technique, Sison, Schubert et Nord ( 1958) dis­Unguent une cellulase vraie et une cellobiase dans un flltrat de culture de Poria valllantil. Selby, Maitland, et Thompson ( 1963) admettent Ia presence de deux enzymes A et B, !'enzyme. A

attaquant Ia cellulose fibreuse et !'enzyme B agissant sur Ia cellulose degradee. Plus recemment, ces auteurs (Selby et Maitland, 1965) ontdedouble !'enzyme A en deux eomposants de poids moleculaire tres differents "mais actifs l'un et !'autre sur les memes substrats. Entin,. Iwasaki, Hayashi et Funatsu ( 1965) on! mis en evidence a partir d'un filtrat de culture de ·Trichoderma konlngll, deux cellulases distinctes l'une attaque les substrats cellulosiques insolubles (cellulose fibreuse) tandis que !'autre agit essentiellement sur les substrats solubles (glycol cellulose, cello­biose).

Cependcnt Whitaker, Hanson et Datta ( 1963) semblent avoir !sole un seul enzyme, homogene a !'electrophorese et a !'ultra centrifugation et qui de plus, agit sur tous les substrats cellulosiques. Jermyn ( 1955, 1962) appuie eette theorie en affirmant que les divers enzymes eellulolytiques mis en evidence dans un meme preparation ne soot que des complexes stables formes entre une cellulase unique et divers composes glucidiques du milieu.

Le probleme reste done tres contreverse et mal eonnu. Les divergences semblent provenir a le fois des sources d'eniymes · et des methodes de purification utilisees. II apparait necessaire, avant d'etudier Ia degradation enzymatique de Ia cellulose, d'isoler et de definir les differentes cellulases, si elles existent. C'est !'objet du present travail au cours duquel nous avons fractionne sur gel de dextranes, une preparation cgllulolytique d'origine fongique. Nons rapportons ici les premiers resultats relatifs a eette separation.

'Service de Biologie et d'Entomologie, Centre d'Etudes du Bouchet, 91, Vert-le-Petit, France. (Copy received 8th February, 1967).

67

Etude de Ia degradation enzymatique de Ia cellulose : fractionnement sur Ia gel de dextranes, d'une poudre cellulolytique d'origine fongique. Y. le Grand et P. Thivend

Material et methodes :

ENZYME: Nous avons utilise une preparation enzymatique

commerciale ; d'origine fongique, provenant d'une culture d'enrichiesement a partir d'une terre naturelle. L'espece, non determinee, est un basidiomycete. Cette preparation e!lzymatique se presente sous Ia forme d'une poudre blanchii.tr&, dont Ia partie soluble (60 p.100) renferme Ia totalite de l'activite cellulolytique.

SUBSTRATS:

a) Substrats insolubles dans l'eau-*Poudre de eaton (DP 2400), obtenue par

broyage des fibres de colon prealablement degraissees, sechees puis impregnees d'eau et plongees dans !'azote liquide.

*Alpha-cellulose (DP 2100) obtenue a partir de eaton ecru, par extraction au benzene et a l'alcool puis par debouillissage a 105"C dans un bain de 15 gjl de soude et de 5 gjl de bisulfite de sodium.

*Poudres de linters de colon don! le degre de polymerisation (DP) est ega! a:

--1500 (Linters BUCKEYE) 800 (Linters FORAY MESCHENMOSER}

•Cellulose de bois (DP 415)

b) Substrat soluble dans l'eau­Carboxymethyl cellulose (C.M.C.)- Reference

I.C.I. Cellofas B.50, en granules.

MESURE DE L'ACTIVITE CELLULOLYTIQUE:

a) Cas des substrats insolubles-Nous avons utilise une methode permettant de

determiner Ia perle de matiere seche d'un substrat donne SOUmis a !'action de !'enzyme dam des conditions bien definies :

300 mg de substrat insoluble son! mis en sus­pension dans un erlenmeyer contenant 25 ml d'un melange de tampon acetique (pH 4,6) et de nitrure de sodium M/200, ce dernier etant utilise comme antiseptique. On ajoute 5 ml d'une solution de cellulase ; Ia concentration en azote total est de 0,2 p.100 pour Ia poudre utilisee. Le melange enzyme-substrat est alors agile dans un bain-marie a 40°C pendant 24 heures. A Ia fin de l'essai, l'activite enzymatique est bloquee par addition de 10 ml d'une solution d'acide acetique 5 M. Le suspension est filtree sur verre fritte, prealablement tare. On lave abondement a l'eau distillee puis on porte a l'etuve a 105°C jusqu'a poids constant.

b) Cas des substrats solubles-Nous avons determine ]'augmentation de

fluidite d'une solution de C.M.C. par une methode viscosimetrique. La reaction a lieu pendent 5 minutes, a 28oC avec 1 ml de solution enzymatique melangee a 25 ml de C.M.C. a 0,3 p,100 et 4 ml de tampon acetique 0,05 M La mesure de fluidite se fait dans un viscosimetre type OSWALD (figure 1) muni de tubulures (A, B, C, D) permettant des

68

rin~ages rapides et ]'aspiration sous vide du melange enzyme-substrat a partir d'une fiole demontable a diverticule. Celle-ci permet le rechaffement prealable de !'enzyme et Je reglage exact du temps d'incubation par deversement de l'enzyme a partir du diverticule dans le substrat. L'unite cellulosique viscosimetrique (U.C.V.) est definie comme etant Ia quantile d'enzyme qui fait augmenter de 0,1 Ia vitesse d'accroissement de Ia fluidite specifique.

DOSAGE DES SUCRES:

La determination des produits d'hydrolyse de Ia cellulose a ete effectuee :

-soil par Ia methode a !'anthrone (Loewus, 1952) pour Ia mesure des sucres totaux ;

- soit par Ia methode a Ia glucose-oxydase (Thivend, Mercier, Guilbot, 1965) pour Je !osage du glocose.

FRACTJONNEMENT DES ENZYMES CELLULOLY­TIQUES:

Principe : Nous avons utilise a Ia fois pour Ia deminerali­

sation des solutions enzymatiques et pour le fractionnement des proteines, Ia chromatographie sur colonne de gel de dextranes SEPHADEX. Les proteines apparaissant dans les eluats sont reperees par spectrophotometrie d'adsorption a 254 nanometres, en enregistrement continu au moyen d'un appareil LKB Uvicord. Des fractions (2 ou 5 ml) soot recueillies dans un collecteur et l'activite cellulolytique est mesuree salon les methodes exposee precedemment. Malgre Ia plus faiblc adsorption des proteines a 254 nanometres, Jes courbes d'enregistrement de l'appareil LKB Uvicord soot semblables ii celles obtenues a 280 nanometres. Elles sont en outre plus dctaillees car le mesure a lieu par fractions d'environ 0,1 ml au lieu de fractions de 2 ml dans Je cas d'une mesure faite au moyen d'un spectrophotometre non enregistreur.

Preparation des colonnes de fractionnement­Trois types de Sephadex ont ete employees

G 25-G 75-G 100. Pour chaque type, le poudre de Sephadex est mise en suspension dans une solution de tampon acetique (0,05 M a pH 4,6) jusqu'a ce que les grains soient suffixamment hydrates. On Jaisse decanter puis on elimine les fi!les particules surnageantes. Cette suspension est ensuite deversee dans des colonnes parfaite­ment verticales. Le remplissage doit etre regulier. L'elimentation des colonnes est realisee par des flacons de Mariotte dont le hauteur est reglee par rapport a Ia sortie des collones de fa~on a assurer un debit constant de l'eluat. Les colonnes utilisees ont un diametre de 3 em et Ia hauteur des gel est approximativement de 80 em. Les vitesses d'elution varient de 30 ml/h pour les Sephadex G 25 a 15 ml/h pour les Sephadex G 75.

Le colmatage des Sephadex G 100 etant tres rapide au-deJa d'une hauteur de gel de 60 em, il

Etude dt Ia degradation enzymatique de Ia cellulose : fractionnement sur Ia gel de dextranes, d'une poudre cellulolytique d'origine fongique. Y. le Grand et P. Thivend

s'est avere necessaire d'employer un systeme d'elution de bas en baut (Rothstein, 1965) permettant d'utiliser des colonnes plus tongues (85 a. 90 em) et d'ameliorer ainsi le fractionne· ment.

L'eluant est un melange de tampon acetiquc 0,05 M et de nitrure de sodium M/200 (antisep· tique) ainsi con~ues, les differentes colonnes ant ete utilisees pendant plusieurs semaines, sans qu'il y ait eu de variations debit.

Resultats :

JUSTIFICATION DE LA METHODE GRAVI· METRIQFF. UTILISEE POUR LA MESURE DE L'ACTIVITE CELLULOLYTIQUE:

Nous avons compare Ies valeurs obtenues en mesurant le quantile de substrat hydrolyse, soit par gravimetrie, soit en dosant les sucres formes (sucres totaux solubles dans l'eau, determines par !'anthrone, glucose determine par Ia glucose· oxydase, tableau 1). Le pourcentage de cellulose degradee, mesure par gravimetrie est toujours superieur a celui obtenu par les deux autres methcdcs, lesquelles donnent des resultats sen· siblemcnt equivalents pour une durce d'hydrolyse de 24 heures. Pour des temps d'attaque plus courts, nous avons observe, sur un substrat donnli (linters D.P. 800) un ecart maximum, au debut de !'hydrolyse (tableau 2).

MISE EN EVIDENCE DE L'ACTIVITE CELLUL­LOLYTIQUE DE LA POUDRE ENZYMATIQUE UTILISEE:

L'activite cellulolytique de Ia poudre enzymatique utilisee est ties generale, contrairement a celle de certaines cellulases precedement etudiees. En faisant agir sur differents substrats cellulosiques, J'extrait aqueux obtenu a partir de Ia poudre brute, nous avons observe, soit une diminution de Ia matiere seche initiale (substrats insolubles, tableau 3), soit une baisse de viscosite (substrat soluble, C.M.C.). Cette diversite d'action nous a conduits a examiner le nombre et la nature des prot6inc::. de cet cxtrait aqueux, pour Ctudier si son activite cellulolytique est diie a un ou plusieurs enzymes.

FRACTIONNEMENT SUR GEL DE DEXTRANES SEPHADEX:

a) Sephadex G 25 -Les sepha\]ex G 25 (separant les substances

ayant un poids moleculaire inferieur a 5000) ont permis de recueillir, a partir de l'extrait aqueux initial, un premier pic qui contient !'ensemble des proteines a haut poids moh\culaire (fig. 2). Seul ce premier pic possede une activite a Ia fois sur des substrats insolubles (Linters) et solubles (C.M.C.). Une electrophorese sur gel d'amidon indique que cette premiere ·fraction active renferme un nombre important de pro· teines. 11 apparait parmi elles, une fraction amylolytique, peu apres Ia ligne de depart (observee par destruction du gel d'amidon). Son

action sur !'amidon de mais est tres importante (70 p.100 sont hydrolyses en 24 heures a pH 6,9; 30 p.100 a pH 4,6).

69

b) Sephadex G 75 et G 100 -La fraction active, isolee par passage sur

Sephadex G 25 est ensuite concentree par evaporation a sec puis reprise dans 10 ml de tampon et chromatographiee :

- sur Sephadex G 75 ou sur Sephadex G 100, dont les gels permettent de separer les substances ayant un poids moleculaire

·inferieur a 50 000 (G 75) ou a 100000 (G 100). - sur Ies deux types de Sephadex simultane·

ment, en utilisant deux colonnes montees en serie.

Les Sephadex G 75 et G 100 utilisees separement ont donne des resultats sensiblement analogues (fig. 3 et 4). Cinq pies dis tincts apparaissent sur Ies chromatogrammes. Les mesures d'activite cellulolytique mettent en evidence une fraction tres active sur poudre de Linters (DP 800 et DP 1500) mais totalement inactive sur Ia viscosite· de C.M.C. (pic no. 3). Par contre, trois fractions (pies 2, 4 et 5) sont actives sur C.M.C. alors que leur action sur les substrata insolubles est tres faible (pies 2 et 4). Nous avons constate en ultilisant simultanement les Sephadex G 75 et G 100 que les differents pies precedemment determines etaient constitues de plusieurs pro· tcnes (fig 5) mais il n 'a pas ete possible de mieux les isoler.

Discussion :

La chromatographie sur gel de dextranes est une technique seduisante pour demineraliser, con· centrer et fractignner les enzymes cellulolytiques de Ia poudre etudiee. Nous avons ecarte les methodes classiques de dialyse (difficilement applicables aux cellulases par suite de Ia nature cellulosique de Ia plupart des membrane• dialysantes) ainsi que celles par precipitation fractionnee utilisant les solvants mineraux ou organiques, difficiles a eliminer . sans perle d'activite. 11 semble cependant qu'il soit. diflicile d'ameliorer Ia separation des differents enzymes par !'utilisation exclusive des Sephadex et que pour mieux isoler et purifier ces proteines, il faille faire appel a des techniques complemen· !aires (electrophorese).

La methode gravimetrique que nous avons utilisee pour mesurer l'activite cellulolytique sur les substrats insolubles permet de determiner globalement !'importance de l'attaque enzymatique. Elle est en outre d'executimi facile et suffisamment rapide pour permettre des dosages repetes. Les differences observees entre les resultats fournis par cette methode et deux obtenus par dosage des sucres sont vraisemblablement dOs a !'action d~ certains enzymes glycolytiques (glucose-oxydase) presents dans Ia poudre enzymatique, ou a un manque de concordance entre les diverses methodes ultilisees (filtration dans un cas, centri-

Etude de Ia degradation enzymatique de Ia cellulose : fractionnement sur Ia gel de dextranes, d'une poudre cellulolytique d'origine fongique. Y. le Grand et P. Thivend

fugation dans !'autre). Les resultats entre le dosage des sucres totaux et celui du glucose sont sensiblement equivalents; ceci confirme 1es donnees des chromatogrammes qui semblent indiquer une conversion quasi-totale du cellobiose en glucose pour une dun;e d'hydrolyse de 24 heures. En effet, les hydrolysats contiennent du glucose et du cellobiose, ce dernier etant pro­gressivement transforme en glucose au cours du temps.

II apparait a Ia suite des divers fractionnements de le poudre enzymatique utilisee, qu'il existe plusieurs enzymes cellulolytiques agissant sur des substrats tres different. L'enzyme correspondent au pic 3 semble analogue a Ia cellulase C1 de Reese (1950) ou a !'enzyme A de Selby (1963, 1965) ou encore a le cellulase II d'Iwasaki, Hayashi et Funatsu ( 1965). Les autres composants eel· lulolytiques pourraient correspondre au deuxieme type de cellulase mis en evidence par ces divers auteurs. Les deux groupes d'enzymes que nous avons determines se differencient par leur affinite differente vis-a-vis des substrats etudies et par leurs caracteristiques chimiques et biochimiques (poids moleculaire, pH optimum d'action fig. 6). il est difficile d'expliquer, dans l'etat actuel de nos travaux, les differences qui existent entre les pies 2, 4 et 5. On peut considerer ces proteines, comme autant de cellulases Cx qui agiraient sur des substrats plus ou moins degrades. Peut etre, les divers composants cellulolytiques agissant sur C.M.C. ne sont qu'un seul enzyme associe a differentes substances inactives (Petterson, 1963). Seule.. Ia separation complete de ces differentes proteines permettra de determiner leurs divers constiluants; mais d'ores-et-deja, il semble tres peu probable, contairement a ce que pens• Jermyn (1955, 1962) que ces cellulases soient le resultat de dlfferents complexes d'unememe enzyme avec les composants glucidiques du milieu puisque Ia teneur en glucides totaux de Ia poudre utilisee est de l'ordre de 2 a 3 p.100.

En co'nclusion, Ia filtration sur gel de dextranes nous apermis d'isoler des fractions ayant des activites cellulolytiques differentes, ce qui semble confirmer Ia these de Ia pluralite des cellulases. Nous poursuivens cette etude en essayant de separer et d'isoler ces divers composants celluloly· tiques afin d'etudier leur nature et leur mode d'action sur une gamme de substrats plus etendue et bien determines.

Remerclements : Nous addressons nos vifs remerciements a la

Societe KE.AB.*, qui nous a gracieusement fourni Ia poudre enzymatique etudiee.

References bibllographiques:

Grimes, R M., Duncan, C. W., Hoppert, C. A. (1957). Arch. Biochem. Biophys., 68, 412-24.

Multiplicity of cellulolytic enzymes of Myrothe· cium verrucaria.

Iwasaki, T., Hayashi, K., Funatusu, M. (1965)· J. Biochem., 57, (4),467-477 Biochemical Studies on cellulase- I- Purification and characteriza­tion of two types of cellulase from Trichoderma lmningl.

Jermyn, M. A. (1952). Aust. J. Bioi. Sci., 5, (3), 433-443. Fungal cellulases- II- The com· plexity of enzymes from Aspergillus orizae that split p glucosidic linkages and their practical separation.

Jermyn, M. A. (1955). Aust. J. Bioi. Sci., 8, 541-562. Fungal cellulases- IV -Production a!ld purification of an extra cellulase glucosidase of Stachybotrys atra.

Jermyn, M. A. (1962). Aust. J. Bioi. Sci., 15, 769-786. Fungal cellulases- X- Further purifi·

. cation of the {3 glucosidase of Stachybotrys atra.

Loewus, F. A. (1952). Anal. Chern., 24, 219. Improvement in the anthrone method for de­termination of carbohydrates.

Marsh, P. B., Merola, G. V., Simpson, M. E. (1953). Textile Res. J., 23, 831-41. Experiments with an alkali swelling centrifuge test applied to cotton fiber.

Petterso:1, G. (1963). Biochim. Biophys. Acta, 77, 665-667. Separation of cellulases on Shephadex G 100.

Reese, E. T., Siu, R G. H., Levison, H. S. (1950). J. Bacteriol., 59, 485-97. The biological degrada­tion of soluble cellulose derivatives and its relationship to the mechanism of cellulose hydrolyses.

Rothstein, F. (1965). J. Chromatog., 18, 36-41. A column design for reverse flow sephadex gel permeation chromatography.

Selby, K., Maitland, C. C., Thompson, K.V.A. (1963). Biochem. J., 88, 288-96. The degrada· tion of cotton cellulose by the extracellular cellulase of Myrothecium verrucaria. 2. The existence of an " exhaustible " cellulase.

Selby, K., Maitland, C. C. (1965). Biochem. J., 94, 578-83. The fractionation of Myrotheciunt verrucaria cellulase by gel filtration.

Sison, B. C., Jr., Schubert, W. J. and Nord, F. ·F. (1958). Arch. Biochem. Biophys., 75, 260-272. On the mecanism of enzyme action LXV -A cellulolytic enzyme from the mold Poria vaillantiL

Thivend, P., Mercier, c.,.. Guilbot, A. (1965). Ann. Bioi. Anim ... Bloch. Biophys, 5, ( 4) 513-526. Dosage de !'amidon dans les milieux complexes.

Van Soest, P. J. (1963). Journal of the A.O.A.C., 46, (5) 825-829. Use of detergents in the analyses of fibrous feeds. I -Preparation of fiber residues of low nitrogen content.

• Societe d'Etudes et d'Applications Biologiques, Route de Versailles· 78 • JOUY-en-JOSAS.

70

Etude de Ia degradation enzymatique de Ia cellulose: fractionnement sur Ia gel de dextranes, d'une poudre cellulolytique d'origine fongique. Y. le Grand et P. Thivend

Whitaker, D. R. (1953)· Arch. Biochem. Biophys., 43, 253·68. Purification of Myrotheclum verrucrla Cellulase.

Whitaker, D. R., Hanson, K. R., Datta, P. C. (1963). Can. J. Biochem. Physiol., 41, 671-96. Improved procedures for preparation and characterization of Myrothecium cellulase. Part 2 -Purification procedures.

Youatt, G., Jermyn, M. A. Enzymes splitting {J glucosidic linkages in Stachybotrys atra, In Symp, Marine Boring and Fouling Organisms, Friday Harbour 397-409 (Ray, D. L., Ed., Univ: of Washington Press, Seatle, 1959-).

71

Resume

La preparation cellulolytique est fractionnee sur gels Sephadex G 25- G 75- G 100. II est montre que les diverses fractions out des actions differentes sur Ia cellulose insoluble et sur Ia C.M.C. soluble, appuyant ainsi Ia theorie de Ia pluralite des enzymes cellulolytiques.

Etude de Ia degradation enzymatique de Ia cellulose : fractionnement sur Ia gel de dextranes, d'une poudre cellulolytique d 'origine fongique . Y. le Grand et P. Thivend

72

Etude de Ia degradation en~ymatique de Ia cellulose : fractionnement sur Ia gel de dextranes, d'une poudre cellulolytique d'origine fongique. Y. Je Grand et P. Thivend

D.O. (/)

" .... D c. w " w +' ... w c.

0 0 ~ 10 c.

~ E -..... > u ::J

:\ 50 :; ' .

I' I : . . \

25 i . . 'I\: ' \ ~ I ' ' r \ •..,.

1 DO

Volume de l 1 eluant (ml) 200 300 400

Figure 2 : 'Distribution des proteines .et des activites cellulolytiques apres chromatographie sur Sephadex G 25, de Ia partie soluble de Ia poudre enzymatique etudiee.

Adsorption a 254m p. ---Activite cellulolytique mesuree par gravimetrie sur poudre de linters .

• .. .. ....... Actiyite cellulolytique mesuree par viscosimetrie sur C.M.C.

"' ~ D.O.~

40

20

600

Molume de 1 1 eluant (ml.

Figure 3 : Distribution des proteines et des activites cellulolytiques apres chromatographie sur Sephadex G 75 des proteines du premier .pic isole sure Sephadex G 25.

--- Adsorption a 254m

............ Activite cellulolytique mesuree par viscosi.metrie sur C.M.C.

Etude de Ia degradation enzymatique de Ia cellulose : fractionnement sur Ia gel de dextranes, d'une poudre cellulolytique d'origine fongique. · Y. le Grand et P. Thivend

.;

n.o ~ u :::> • "C ... 40 c

"-~ 2

"C

~ +' 20 ~

1!_1

0 0 . "- 100

2

I

' ' ..... ,,-, ,' ' ,.: ,~ .. -~ , '•

200

'

300

\ ··· ...... .

400

\ \ ..

500 600 Volume de

1' ~luant (ml)

·Figure 4 : Distribution des proteines et des activites cellulolytiques apres chromatographie sur Sephadex G 100 des proteines du premier pic isoh\ sur Sephadex G 25.

"' "0 ... 0 0.

D.O.

Ill 20 "0

Ill .... " Ill a.

D D ~

• 0.

Adsorption a 254 m p.

Activite cellulolytique mesuree par gravimetric sur poudre de Linters

Activite cellulolytique mesuree par viscosimetrie sur C.M.C.

...., E

' > u ::::>

30

zoo 300

·'· .. : \ : \ ,. ... . . ,•' '\ ...... :"'-,\ ········· . ... _, : \ \\

... ...... ... .. 400 500

Volume de l 1 8luant (ml)

Figure 5 : Distribution des proteines et des activites cellulolytiques apres chromatographic, sur Sephadex G 75 puis G 100 des proteines du premier pic isole sur Sephadex G 25.

---- Adsorption a 254 m p. ---Activite cellulolytique mesuree par gravimetric sur poudre de Linters.

Activite cellulolytique mesuree par viscosimetrie sur C.M.C.

. ~-

Etude de Ia degradation enzymatique de Ia cellulose: fractionnement sur Ia gel de dextranes, d'une poudre cellulolytique d'origine fongique. Y. le Grand et P. Thivend

..... E

' > u :::>

{'l 20 ... 0 a. Ql 20 "0

Ql 10 ....

" Ql a.

0 10

0 ~ . a.

pH

Figure 6 : Infuence du pH sur l'activite cellulolytique de Ia poudre enzymatique etudiee.

---- Activite cellullolytique mesun\e par gravimetrie sur poudre de Linters

• • Activite cellulolytique mesuree par viscosimetrie sur C.M.C.

Remarque sur Ia figure 6 :

L'activite croissante vers les pH has de Ia carboxymethyl- cellulase semble inhabituel pour une enzyme: cette courbe d'activite a cependant ete confirmee avec les diverses fractions actives isolees.

Cette activite est mesuree par Ia formule suivante: le nombre d'unites cellulasiques viscosimetriques UCV = 1 x _1_ x difference entre Ia fluidite specifique de Ia solution enzymatique et celle

0,1 5 min. d'un temoin sans enzyme soit UCV = ....L x _1_ / __L._ -·

0,1 5 min. \T~- To oil T, est le temps d'ecoulement de l'eau distillee (36 sec.)

T, est le temps d'ecoulement de Ia solution tamponnee de C.M.C. (variant de 90 sec. a 146 sees.) T, est le temps d'ecoulement de Ia solution tamponnee de C.M.C. additionnee d'enzyme. La fluidite specifiques des temoins se situe toujours entre 0,36 et 0,66 tandis que celle de Ia solution

enzymatique varie entre 0,61 et 15.

A pH 2,2 qui est le pH extreme possible, le temps d'ecoulement de Ia solution enzymatique etait de 38,4 sec., c'est a dire tres proche de celui de l'eau distillee, ce qui montre que le maximum d'activite se situe a un pH tres voisin de 2.

75

Etude de Ia degradation enzymatique de Ia cellulose : fractionnement sur Ia gel de dextranes, d'une poudre cellulolytique d'origine fongique. Y. le Grand et P. Thlvend

Tableau I

COMPARISON DE DIFFERENTES METHODES UTILISEES POUR LA MESURE DE L'HYDROLYSE ENZYMATIQUE DE LA CELLULOSE

(temps d'incubation 24h. a 40'C, pH 4,6)

~ cellulose Source d'enzyme co ton a cellulose linters I linters II dl! bois

e e

(2400) (1) (2100) (1500) (800) (415)

poudre brute anthrone . 1.74 0.26 2.17 14.84 8.48 (2)

glucose-oxydase 1.04 1.00 2.47 12.08 7.99

extrait aqueux de Ia gravimetrie 3.68 3.46 5.62 23.54 16.80 poudre brute

anthrone 1.81 3.40 6.16 .21.00 10.80 (2) glucose-oxydase 2.00 3.40 4.90 19.60 11.00

fraction active isolee gravimetrie 3.95 4.50 7.31 31.54 15.25 sur Sephadex G 25

anthrone 2.62 2.89 5.47 31.02 12.50

glucose-oxydase 1.17 2.65 4.80 30.61 -(1) les valeurs correspondent au degre de polymerisation (DP) des differents substrats (2) les mesures ont ete eflectuees a taux d'azote constant quelle que soit Ia source d'enzyme

Tableau 2

COMPARAISON DE DIFFERENTES METHODES UTJLISEES POUR LA MESURE DE L'HYDROLYSE ENZYMATIQUE DE LA CELLULOSE

(Etude sur poudre de linters DP 800- Resultats exprimes en p.100 de cellulose degradee- temps d'incubation 24h. a 40'C, pH 4,6)

e

e 30 90 24 source d'enzyme ~ e minutes 1heurE minutes 2 heures heure ~ heures heures

fraction active isolee gravimetrie 4.22 7.01 9.65 10.29 15.95 sur Sephadex G 25 anthrone 2.40 4.60 7.53 9.40 14.12

glucose-oxydase 0.86 3.26 7.20 - 13.30

Tableau 3

INFLUENCE DE LA NATURE DU SUBSTRAT SUR L'IMPORTANCE DE L'ATTAQUE CELLULOLYTIQUE

(temps d'incubation 24h. a 40' C, pH 4,6)

17.83 31.54

16.08 31.02

15 66 30.61

~ cellulose de plantes

~ cellulose fourrageres ( 1)

coton cellulose linters linters de bois h!gumineuse gramlnee

D.P. 2400 2100 1500 800 415

perte de rna tiere seche (en p.100 de cellulose 3.70 3.46 5.62 23.54 16.80 4.16 14.70 degradt\e)

(1) La cellulose a ete extraite de Ia plante fourragere par Ia methode de Van Soest (1963).

76

Vincents Nissen, T·, Int. Blodetn Bull., 3, (2), 77, (1967) Fungi from green coffee beans

FUNGI FROM GREEN COFFEE BEANS

T. VINCENTS NISSEN'

Green coffee beans (samples of Brasilian, Javanese and Arabian origin) were slightly moistened and held under humid conditions at 27"C. After 24 -36 hours mould colonies were observed on the ·surface of the beans; the beans were overgrown by moulds in three days, dis­colouration of the beans frequently taking place.

Pure cultures of the different organisms were obtained on an oatmeal-yeast extract- agar (pH 5.7). Dominating fungi were Aspergillus ochraceus, A. tamaril, A. ftavus and A. niger. It is known that some of these fungi occur in the coffee fermentation process (Thorn and Raper, 1945) and it is probable that many of the fungus spores present on the beans originate jn the fermentation.

A sporeforming bacterium (Bacillus sp.) was also isolated ; at the drying up of the cultures an actinomycete (Streptomyces sp.) now became very prominent as a white covering of the beans.

The Isolated moulds were able to break down pectin as tested on a pectin-mineral gel in petri dishes. A fair growth of the isolates also was obtained in a sterile infusion of ground green coffee beans.

The loss of quality of green coffee that is often seen with rising moisture content may, in part, be the result of mould growth on the beans. During storage in a warm, damp climate or during trans­portation by ship from warm to colder climates, the moisture may condense on the surface of the beans. The experiments indicate that growth of moulds arising from spores of the fungi present in the fermentation very soon may take place under such circumstances.

Literature :

Thorn, C. and Raper, K. B. (1945). A Manual of the Aspergilli. Baltimore. The Williams and Wilkins Co.

Summary:

A study has been made of the mould growth on moistened green coffee beans. It is assumed that the moulds mostly originate In spores of fungi associated with the fermentation. A loss In quality may be the result of such growth.

'Royal TechJ!fcal University of Denmark, 83 Solvgade, Copenhagen K., Denmark. (Copy received 12th January, 1967)

77

Domanski, S. and Orlicz, A. Int. Biodetu Bull., 3, (2), 79-80 (1967). Taxonomy of Polyporus megaloporus Pers.

THE TAXONOMY OF POLYPORUS MEGAWPORUS PERS.

S. DOMANSKI and A. ORLICZ'

The fungus Polyporus megaloporus Pers (1825) is common mostly in England and France and has been reported as the cause of serious qamage of oak wood in buildings, bridge construction and so on. It is also commonly known as Poria megalopora (Pers.) Sacc. (1888) Phellinus cryptarum Karst. (Cartwright) and Findlay (1946) and Phellinus megaloporus (Pers.) Bond. (1953). Such a choice of names is troublesome to the mycologist, who intends placing the fungus in one of the families in the natural system of the polypores (Kotlaba and Pouzar, 1957; Donk, 1964). The genera Polyporus (Mich.) Fr. and Poria Pers. ex S.F. Gray do belong to the family Poly­poraceae s. str. (Donk, 1964 : 278), while the genus Phellinus Que!. to the family Hymeno­chaetaceae (Donk, 1964: 274).

To explain these differences of opm1on it was necessary to test the cultures and the carpophores from the point of view of their microscopic characters, with special attention to the presence or Jack of clamp connections. In the complex of characters for the carpophores of separate families of the order Aphyllophorales (Donk, 1964) the lack of clamp connections in the hyphae of the carpophores of the family Hymeno· chaetaceae becomes most visible and distinguishes the polypores belonging to that family, from the brown coloured carpophores of the species of the Polyporaceae. The same also concerns the cultures, where the presence or absence of clamp connections is easily proved.

In table I of Nobles' paper ( 1965) the species codes of cultures of fungi which belong to the family Hymenochaetaceae are characterized by the presence of the code symbol 6, which means a complete Jack of clamp connections on the hyphae.

The authors had two cultures FPRL 178 E and FPRL 178 F, received by courtesy of the Forest Products Research Laboratory, Princes Risborough, England, and two following specimens of carpo­phores:

1. Berlin-Wannensee, Germany, oak timbering, 1958, det. A. Pilat (PR)

2. Oxford, England, roof of college chapel, V. 1946, det. J. S. Hughes (Herb. Hort. Bot. Reg. Kew.)

These cultures and carpophores had been obtained in Poland in order to find the possible relationship of P. megaloporus with the newly described fungus Poria albidofusca Doman. from Bialowieza in Poland by Domanski (1966).

For both cultures the following species codes have. been put together following the system de­tailed by Nobles (1965) 2, 3, 8, 34, 36,.37, 38, 39, 44, -45, ( 48), 53, 54, 60. Both cultures are positive in the test for extracellular oxidase, both of them have thin-walled hyphae with clamp connections from 1 to 4 microns in diameter or, especially in the substrate mycelium, they have membered hyphae from 3.5 to 9 microns in diameter. Only in the culture of 178 E was the appearance of thick-walled, yellow-brownish fibre hyphae from 1 to 2.5 microns in diameter observed. Both cultures produced chlamydospores measuring up to 10- 12.5 x 3.5- 7 microns. The mat of 178 F was unchangeable white with hyaline hyphae with the reverse hyphae unchanged in colour ; the mat of 178 E changed later to yellow-brown, and the reverse coloured partly brown. Both cultures had an unpleasant smell. Only in culture 178 E did the carpopbores appear before the end of 6 weeks, with basidia measuring 6.5 - 15 x 4- 6.5 microns and cystidiols measuring 13 - 15 x 2- 4 microns. The basidiospores are ellipsoid, some­times truncated on one end, measuring 4.5 - 7 x 3 - 3.5 microns, hyaline, yellowish at the end, not amyloid. Single-spore cultures have been obtained from the spores and by the method of pairings it was stated that the fungus is heterothallic and tetra polar.

As one may see from the code above, in culture there are clamp connections on thin·walled hyphae (code symbol 3) which places the fungus into the family Polyporaceae s. str. rather than into the family Hymenochaetaceae. When testing the microscopic details of the carpophores the authors found that in all parts of the carpophores of P. megaloporus the hypha! system is trimitic and contains :

'Research Institute of Forest Pathology, High Technical School of Agriculture (Wyzsza Szkola Rolnicza), Krakow, Poland. (Copy, translated into English by Dr. B. J. Zyska, received March, 1967).

79

The taxonomy of Polyporus megalporus Pers. S. Domanski and A. Orlicz

1. hy11line, thin-walled generative hyphae with clamp connections, or some thick-walled generative hyphae, from 2 to 6.5 microns in diameter, stained blue in cotton blue, cresyl blue and in giemsa ;

2. fibre Skeletal hyphae, from 1.5 to 4.5 microns in diameter, olive-brownish, not stained in cotton blue, but dark brown In cresyl blue and in giemsa ;

3. hyaline binding hyphae, from 0.5 to 1.5 microns in diameter, flexuous and frequently branched, hyaline in cotton blue and giemsa, light blue in cresyl blue.

This confirmed in the carpophore above all the presence of clamp connections in the generative hyphae which definitely excludes, in the authors' opinion, Polyporus megaloporus from the genus Phellinus Que!. in the family Hymenochaetaceae. The fungus has instead all the characters of a species of the family Polyporaceae s. str. 1n the Artificial System of classification of this family It may of course belong there to the residual genus Poria Pers. ex. S.F. Gray s.l. under the name Poria megalopora (Pers.) Sacc. In the natural system of the family Polyporaceae s. str. the fungus Polyporus megaloporus has all the characters for belonging to the genus Fornes (Fr.) Klckx. emend. Teixeira (1958: 173; 1962: 67). It develops a perennial carpophore of a corky-woody context of a more or less brown colour, never white ; the generative hyphae with clamp connec­tions develop Into two kinds of special thick­walled or full branches without clamp connections, light or olive-brown under the microscope ; the hymenium without setae and cystldla, with clavate basidia ; spores hyaline smooth, delicate-walled, not amyloid. Apart from this the context becomes black in a solution of KOH, similar to Fornes fomentarlus (L.ex.Fr.) Klckx. The decomposition of wood by this fungus is in the form of a white rot and in culture in the test for extracellular oxidase it gives a positive result, similar to F. fomentarlus. In the examined cultures of P. megaloporus and in the culture of Fornes fomentarius the authors found some similarities of the morphology of thin-walled hyphae in the advancing zone. Taking this all Into account the authors' proposal "is to P.Ut the fungus into the genus Fornes (Fr.) Klckx. emend. Teixeira in the following nomenclatural combil)ation Fornes megaloporus (Pers.) Doman & Orlicz (1967).

80

References :

Bondarcew, A. S. (1953). Trutowyje griby ewrope­jskoj tshasti SSSR l Kawkaza, Moskwa­Leningrad.

Cartwright, K. S. G. and Findlay, W. P. K. (1946). Decay of timber and its prevention, London : Her Majesty's Stationery Office.

Department of Scientific and Industrial Research. Forest Products Research Laboratory, Princes Risborough, Aylesbury, Bucks ( 1961). The list of cultures In the Mycological Section.

Domanski, S. (1966) Acta Soc. Bot. Pol., 35, 485-502. Grzyby zasledlajace drewno w Puszczy Bialowleskiej. IV. Poria albldofusca, sp. nov. i jej rozpoznanie.

Domanski, S. and Orlicz, A. (196.7). Acta Soc. Bot. Pol. 36 (in Press). Grzyb Polyporus megaloporus Pers. w. rodzinle Polyporaceae s. str.

Donk, M. A. ( 1964). Persoonla 3, 199-324. A conspectus of the families of Aphyllophorales.

Kotlaba, F. and Pouzar, Z. (1957). Ceska Mykol., ll, 152-170. Poznamky · k trideni evropskyh chorosu.

Nobles, M. K. (1965). Can J. Bot., 43, 1097-1139. Identification of cultures of wood-inhabiting Hymenomycetes.

Teixeira, A. R. (1958). Arquiv. Bot. Estado S. Paulo, 3, 165-174. Tiplficacao do genero Fornes (Fries) Klckx.

Teixeira, A. .R. (1962). Rlckla, 1, 13-93 Mlcroestruturas do basidiocarpo e sistematica do genero Fornes (Fries) Klckx.

Summary:

A taxonomic study Is presented of the fungus Polyporus megaloporus Pers. which is reported to cause serious damage to oak wood in buildings, bridges, etc.

Alternative nomenclature which has been pro­posed for the species is discussed and the authors contend that the species should be placed in the genus Fornes (Fr.) Klckx. emend. Teixeira.

BOOK REVIEWS

CONSERVATION OF BUILDING TIMBERS

N. E. Hickin (Hutchinson, London, 1967, 144pp., price 35/·)

Dr. Hickin's books are not easy to review, especially when one has known, respected and admired the author for many years. Like its predecessors in this series, it contains so much information that is first class, presented in an authoratative and erudite way, that one ought to be inisting on it being compulsory reading for every student of wood in the country and of excellent value to the "general reader. Instead, one is put in a quandary by the unhappy insertion of a chapter or two, which, for commercial reasons, do not give the complete story and therefore may leave the uninformed reader with the wrong impression-

On the one hand this book is not a treatise on the conservation of timber in buildings, but more an analysis of the state of the timber in buildings which have been inspected by a specialist remedial treatment company because some degradation of the timber has been sus­pected. The discussion of wood preservatives and methods of treatment is limited to one type only and omits reference to several major parts of the industry and processes that are both important and widely employed. On the other hand, much useful i'nd interesting Information and ideas are to be found within its pages, that would perhaps not have been published at all without the inclusion of these commercial chapters.

One is therefore left as a reviewer with n~ alternative but to say "If you have a critical faculty, read this book, it has some excellent p1rts, but do not forget to use your critical faculty."

J. Levy.

LABORATORY METHODS IN MICROBIOLOGY

W. F. Harrigan and Margaret E. McCance (Academic Press, London & New York, 1966, 362 pp., price 79/·)

This is a laboratory handbook based on courses in microbiology run at the West of Scotland Agricultural College, and is intended mainly for students following· food science, dairying, agriculture and allied courses to degree and diploma level. It is in two main parts, Part 1 (87 pages) covering a general introduction to preparation, cultivation and identifica­tion methods for bacteria, moulds and yeasts, with a mention of serological, bacteriophage and disinfectant evaluation tests: and Part II (111 pages) which is concerned with the examination of soil, water, silage, foods, liquid milk, dairy utensils and equipment, starter cultures, cheese, butter, milk powder, canned milk, ice cream and fresh cream. There are also three appendices (128 pages) giving (1) diagnostic tables for Gram negative bacteria that can be grown on agar; (2) some formulae for stains, reagents and media; and (3) probability tables for the estimation of bacterial numbers by the dilution tube technique. In addition, there is a • selected ' bibliography of 48 book titles, and about 250 references, plus a useful index.

Part I appears to consist of notes possibly as they should be ideally written up by

students attending the lectures at the West of Scotland College of Agriculture during their first year. In Part II the authors are obviously most at home with the methods given for the examination of milk and dairy products in accordance with the current beliefs and practices approved in Scotland. The three appendices might be considered the best part of the book since it is most concise and clearly written consisting mainly of Information gained from other authoritative works.

Many departments giving instruction in applied microbiology are somewhat shy at publishing in book form an outline of what methods they are actually teaching their students. Even if their approach is somewhat diffuse, the authors here are to be congratulated in making this very useful contribution and for braving the risk of adverse criticism from the less adventurous in the same field. The book is impeccably printed and produced.

A. Harry Walters..

'

f·

·!f

THE USE OF BIOLOGICAL LITERATURE

Edited by R. T. Bottle and H. V. Wyatt (Butterworths, London, 1966, ix + 286pp., price 58/·)

This book consists of a series of essays, by both research workers and librarians, on several aspects of biological literature including pointers on where to find it and how to use it. About half the chapters are dedicated to describing literature resources in various sub-disciplines and the remainder are on more general aspects (e.g. primary sources, libraries, etc.). It is these latter chapters that will probably be of the greatest value to practising biologists and I would especially recommend Dr. Wyatt's stimulating "Epilogue," and Mr. Gollin's introduction to the use of the patent literature- this is a source of information that is all too frequently ignored, particularly by academic researchers.

A major application for this work is claimed in the planning of courses on the use of biological literature, and an unusual and useful inclusion is a series of test exercises in handling the literature in response to set questions (teachers who contemplate using such a method will be relieved to know that a guide to answers is also included ! ) . On the whole, it meets this claim quite well and the book goes a long way towards filling a long standing need for a reliable general introductory work in this field. However it is also claimed that the volume would find use as a reference work and this I am inclined to dispute- few of the chapters are comprehensive enough to be of great value for reference purposes, several standard reference works are omitted (this is not surprising- to be fully comprehensive, the book would probably have to be at least three times its present size) and the index is not detailed enough. The volume could perhaps be of reference value to specialists moving into a new area of research.

Stronger editorial control of the styles of presentation could have made this book easier to use, but in general it is a very useful addition to the scanty literature of biological infor· mation and it is a pity that the fairly high price will probably deter all but the most dedicated biologists from buying his own copy.

B. J. Perry.

NOTICES

PUBLICATIONS NOW AVAILABLE FROM THE B.I.C.

1. INTERNATIONAL BIODETERIORATION BULLETIN (I.B.B.)

Commenced 1965. Two issues per year (Spring and Autumn). 1965 and Autumn 1966 issues out of print. Price £1 per issue. Order forms appear in current issues.

2. INTERNATIONAL BIODETERIORATION BULLETIN REFERENCE INDEX SUPPLEMENT (IBBRIS)

Commenced 1967. Four issues per year (Spring, Summer, Autumn and Winter). Price 5/· per issue. Order forms appear in each issue.

3. BIODETERIORATION THESAURUS

Containing a list of the terms used at the B.l.C. for indexing papers dealing with biodeterioration. In press.

4. B.I.C. ANNUAL REPORT 1966-67

A limited supply of copies available free of charge on application to the B.I.C.

82

g)

1st International Biodeterioration Symposium

PURPOSE, OF THE SYMPOSIUM

Today the volume of complex manufactured products is higher than it has ever been and the number that have to conform to narrowly defined standards is increasing. Biodeterioration is among the factors that can cause a product to be sub­standard, and the more highly-processed a product is, the more serious are the economic effects of biodeterioration.

Over the past few years there has been a con­siderable re-awakening of interest in the biodeterior­ation of materials. In spite of the extreme diversity of the organisms involved-bacteria, fungi, algae, insects, etc.-and the vast range of potentially susceptible materials, quite different problems of biodeterioration frequently possess many features in common.

One major obstacle in the path of many workers in the field is that they of necessity have to restrict their investigations to limited aspects of the subject, and have difficulty in familiarising themselves with techniques and other developments in related aspects.

It is felt that an International Symposium covering the field of biodeterioration of materials would serve a most useful function by presenting, to biologists and materials scientists, a broad review of present thinking in the fundamentals of the subject, and more detailed treatments of many of the currently important aspects of biodeteriora­tion.

In order to emphasise the overall unity of the subject, the programme will attempt a division into basic topics rather than deal with problems material by material.

NATURE OF THE MEETING

It is hoped that major .topics will be introduced by specially invited speakers. The proceedings of the meeting will be arranged in six broad sections as detailed below.

I. Fundamentals of biodeterioration: philosophy, ecology, taxonomy.

II. Mechanisms of biodet~rioration: mechanisms of enzyme action, bacterial corrosion, marine fouling, etc.

III. Control of biodeterioration: protection of materials, biocidal action, testing procedures.

IV. Enzymic attack of materials: attack on textiles, plastics, etc. etc.

V. Corrosion of materials: bacterial and fungal corrosion proolems, including fuel tank corrosion.

VI. Mechanical damage or functional impairment of materials:

insect and rodent damage, marine fouling, etc.

TRANSLATION

Speakers are encouraged to give their papers in English. However, papers may be given in any other language and where possible an English translation should be available for distribution.

PROCEEDINGS OF THE SYMPOSIUM

It is planned to publish the proceedings of this Symposium to make available the most recent information on biodeterioration.

HEADQUARTERS OF THE MEETING

The Meetings will be held at the University of Southampton, England.

ACCOMMODATION

Accommodation will be available in Halls of Residence at a cost of approxifJiately £12.10.0. ($37 .00) for the period of the Symposium, excluding lunches.

EXCURSIONS

It is intended to devote one day (Thursday) during the Symposium to excursions and in addition to arrange a tour, for those who may be interested, starting immediately after the Symposium, and including visits to several laboratories and to the Biodeterioration Information Centre at the Uni­versity of Aston in Birmingham.

PRESENTATION OF PAPERS

Intention to offer a paper should be indicated on the provisional application form as requested.

Abstracts will be required by the 30th September, 1967 and completed manuscripts by the 31st March, 1968.

REGISTRATION FEE

The Symposium Registration Fee which will be £9.0.0.($26.00) will include a copy of the advance papers and a copy of the proceedings when pub­lished.

Full details and application form can be obtained from : Hon. Secretary, 1st International Biodeterioratlon Symposium, 14, Belgrave Square, London, S.W.l., England.

ACKNOWLEDGMENTS TO SUSTAINING ORGANISATIONS

Financial support for the International Biodeterioration Bulletin from the following organisations is gratefully acknowledged : AVON RUBBER COMPANY LIMITED, Melksham,

Wiltshire ; manufacturers of tyres and miscel­laneous rubber products.

BEECHAM GROUP LIMITED, Toiletry Division, Beecham House, Great West Road, Brentford, Middlesex.

"BORAX CONSOLIDATED LIMITED, Borax House, Carlisle Place, London, S.W.1. (Tel. VICtoria 9070) ; miners, refiners and suppliers of borax, boric acid, boron ores, boron products and allied chemicals.

THE BRITISH DRUG HOUSES, LIMITED, B.D.H. Laboratory Chemicals Division, Poole, Dorset ; manufacturers of laboratory chemicals, bio­chemicals, industrial fine chemicals and micro­hiocides.

BRI1J'ISH INSULATED CALLENDERS CABLES LIMITED. 38 Wood Lane, London, W.12.

THE BRITISH PETROLEUM COMPANY LIMITED. B.X.L. PLASTICS MATERIALS GROUP LIMITED,

Tyseley, Birmingham. CADBURY BROTHERS LIMITED, Bournville,

Birmingham ; manufacturers of cocoa, chocolate, cake and milk products.

CATOMANCE LIMITED, Welwyn Garden City, Hertfordshire ; manufacturers of speciality chemicals for the textile, paper, timber, leather industries, etc., including fungicides, bactericides and insecticides.

CENTRAL LABORATORY TNO, Delft, The Nether­lands ; research, analysis and testing facilities in materials science and technology, including hiodeterioration and marine research. Sponsored by government agencies and by international industries.

CIBA LIMITED, 4000 Basle 7, Switzerland. COALITE AND CHEMICAL PRODUCTS LIMITED,

P.O. Box No. 21, Chesterfield, Derbyshire : manufacturers of chlorinated and alkylated phenols, phosphate plasticisers and bactericides. Extensive research work and development carried out In all these fields and cus~omer

service facilities are available for bactericides. COMMERCIAL PLASTICS GROUP OF COM-

PANIES, Industrial Division, Berkeley Square House, Berkeley Square, London, W.1.; specialists in the production of film and sheeting in PVC, polythene and high impact polystyrene fOr all types of applications.

COURTAULDS LIMITED, Coventry. CUPRINOL LIMITED, Adderwell, Frome, Somerset;

manufacturers of preservatives for wood, textiles, cordage, paper, adhesives, paints, leather, plastics, and paint driers.

THE DUNLOP COMPANY LIMITED. FARBENFABRIKEN BAYER A.G., Leverkusen,

Germany ; manufacturers of dyestuffs, industrial chemicals, synthetic fibers, pharmaceutical and agricultural chemicals and preservatives for wood, foodstuffs and technical products.

FISONS PEST CONTROL LIMITED, Saffron Walden, Essex; makers of agricultural chemicals. Research on control of weeds, plant ciseases,

84

spoilage organisms, and agricultural and live­stock pests.

GAGLIARDI RESEARCH CORPORATION, East Greenwich, Rhode Island, U.S.A.; sponsored industrial research in textile chemical dyeing and finishinl! nroducts and orocesses.

GALLOWAY & BARTON-WRIGHT, Haldane Place, London, S.W.18.; consultants in industrial microbiology and microbiological deterioration.

GEIGY (U.K.) LIMITED, Simonsway, Manchester, 22.

;r, R. GEIGY S.A., Basle 21, Switzerland ; manu· facturers of dyestuffs, industrial chemicals, pharmaceuticals and agricultural chemicals.

GLAXO LABORATORIES LIMITED, Greenford Road, Greenford, Middlesex; manufacturers of pharmaceuticals, industrial enzymes and fine chemicals.

ARTHUR GUINESS SON & COMPANY (DUBLIN) LIMITED.

IDCKSON & WELCH (HOLDINGS) LTD., Ings Lane, Castleford.

IMPERIAL CHEMICAL INDUSTRIES LIMITED, Agricultural Division, Billingham, Co. Durham.

ARTHUR D. LITTLE LIMITED, with offices at Berkeley Square House, Berkeley Square, London, W.1., and offices and laboratories at Inversk Gate, Musselhurgh, Midlothian ; inde­pendent sponsored research organisation.

MAY & BAKER LIMITED, Dagenham, Essex; chemical manufacturers.

RECKITT & SONS LIMITED, Hull. RENTOKIL LABORATORIES LIMITED, East

Grinstead, Sussex. REVERTEX LIMITED, Harlow, Essex. SCIENTIFIC CHEMICALS INC., 1637 South Kil­

bourne Ave., Chicago, Illinois, 60623, U.S.A.: leading manufacturers of industrial fungicides and bactericides who maintain substantial re­search and development facilities to assist customers in the development of final products geared to meet government and industry standards.

SHELL INTERNATIONAL CHEMICAL COMPANY LIMITED

STAUFFER CHEMICAL COMPANY RICHMOND RESEARCH CENTER, 1200 South 47th Street, Richmond, California 94804, U.S.A.

TELEPHONE MANUFACTURING COMPANY LIMITED ; head office : Martell Road, West Dulwich, London, S.E.21 ·; manufacturers of telecommunication apparatus and equipment, relay and switching ~omponentry, professional grade capacitors, marine and private telephone apparatus and master and slave clocks.

VINYL PRODUCTS LIMITED, Butter Hill, Carshalton, Surrey ; manufacturers of synthetic resins, specialising in emulsion polymers for adhesives, building, paper, surface coating and textile appllca tions.

YARSLEY LABORATORIES LIMITED, Clayton Road, Chessington, Surrey ; Independent re­search and testing facilities.

I.B.B. ORDER FOR~!

Please supply me with copies of the International Biodeterioration Bulletin (I.B.B.) at a cost

of £2 ($6, 28NF, 22DM, 334pesetas,-3486lire) per annum.

Signed ..................... ' ............. ·- ....... ·- .......... .

Mailing address (block capitals please)

Address for receipt of invoices (block capitals please)

Please complete and return to :- Biodeterioration Information Centre, Dept. Biological Sciences, University of Aston in Birming!lam, Gosta Green, Birmingham 4. England.

N.B. The present price of the I.B.B. is £1 p.a. The price of £2 p.a. will take effect from January 1st, 1968.

85

IBBRIS ORDER FORM

Please supply me with copies of the International Biodeterioration Bulletin Reference Index

Supplement (IBBRIS) at a cost of £1 ($3, 14NF, llDM, 167 pesetas, 1743lire).

Signed

Mailing address (block capitals please)

Address for receipt of invoices (block capitals please)

Please complete and return to :- Bwdeterioration Information Centre, Department of Biological Sciences, University of Aston in Birmingham, Gosta Green, Birmingham 4, England.

87

NOTES FOR CONTRIBUTORS

1be BolleUa Is produe.d twice yearly 1D the Sprilla: and AutumD.. Typescript c:untributl011.1 In trlpllate ahDU.lcl be Rill to the Editor, DT.ll 0. W. E!Wru, Bl<XIeterlontlOD Information Centre, De· part:ment or Blologle&l Sciences, The UniV~~nib' of Allon ID Blnnlngham, Goata Green, Blnnl.Dghl.m, 4, ERdaad. or to one of the followln&: : llr. G. Ayent, Dept.Blology,College of Technotoc',

Wolverhamptoo, Elld&ad. Prof. G. Bectu, Bunde:sanstal!H. fur MI.W'W­

prufq, Berlln-Dahlern e, Unter dm Elchen 8&/87, Gemw17.

Mr, D, G. Coursey, Tropltal Prod.utta IDIUtute, IS&--62 Gnys 1Im Road, Lcmdcm W.C.l~ f'.IIPud,

Mr. J. J. Elphkt, AdmlnJty lllt.erbk Llboraloey, Holtoa. Heath, JWle, Donet, Enclad-

Dr. J. Garrido, Deputmento de Fermmtadones lii4Uittl.lles, CU1ello 25, Madrid, SpaiD.

Dr. H. J. Hueck, Ceutral Llbon.tory TNO, Schoe­matentrut 117, P.O.B. 217, Delft, The Nethu­......

Dr. A. K. K&p!m, Bw:l t'lmKtddes & Germ1ddes Llboraloey, PloDeer1D3 Reteueb DlvliSo!l, u.s. AnDy N~tkt Labontone., Natlek,Uaaaeh\Uetb, U.8.A.

MoiiSieur v: LeGrand, Cezltre d'etudes du Bouchet. Vert·le-PeUt, (S & 0), Fnaee.

Dr, B, J, Zyata. Glowll7 lnltytut <klm!CUn, Katowice, Plae. Gwarbnr I, l'elancl. 1be Bulletl!r. wW ad. u a nhkla for the ptib­

licatl.cm of wort em all aspect.~ cl blodeterloraUon generally, I.e. the deterlonUcm or mat.erlab of ec:un~ fmportuu:e b7 mlcroorpnisms. inlecb, J'lldentt. et.e. It ia pl"imal'tl1 1 new1 buDeUn md thenfon II permlsdon to quote frpm artlelu is to be obtllned rrom autluml th1.l should be lndkatecl. on the manlllttipt b)' 111 uterlsk. An

approprkte IDdkaUcm will theD be made wllen pllblhbed.

F.atb eontJ'fbuUon is to be .ubm!Ued on lntu­natlonal A4 me pape:r (R.27l.n. by lUiiln. or 21.0 em. by 29.7 em.) •nil, e:c.eept for the remw •l'llcle, Ia not to ueeed five pap• contaiDlnc 1ppi'O%!mately liOO woflil: pe:r page, lDcludiJii: tables lllld Lllilltn.Uons..

llmlllttiptl mould be t)'p(lwrltteD ID triplicate, doubiHplleed. 011 0111! lllde of the papar only. They m1y be wbmltted !or publltatlon in oalther E:Dgllm, French, Guman or Spanllh, w:Ub a brld IIIDIIIW')' hi Eq!llb of lppi"'XXmmtely S5 wonb.

DlusUitlona Jbould be clearly drawn In lndll!l lDk or lhoulcl be pbotogr.Jpbed. Tbe reducUoll dulred lhould be dearly IDdluted ; lllustnUOm wMn redueed •re not to aceed 17cm. by 211cm.

For ~ to be ill&erled. In the tu. tlw 1pproxtmate p11ce ror eatb one lbould bll nwUII lD the tJpelc:rlpt.

'1'111 blbl.l.tlppb.l~ reference~ U'tl to be Indicated u <~~.& .. nee- and Lerinlon (am) lD t!le ten. and in th! blblloKnphJ : Rftte, E. T. md Lftln.. .em, H. G. (Jg,s2), l'll)'lloL Plaat., I, !M-3!11. CompanUn dudJ' ol the bR&kdOWD of eelluJoM

""""""'""' Prooll will not be Rill to ·•utbora before flDI]

"'"""""' 25 reprlntl will be RDt fn:e ol clwp to eaeb author, Additional reprlntl lt8 obtal.Dihle DD 1ppllaitlon to the Publlca~ Editor at 1 tbarp of !4, 511, per llundrecl.

Copies ol the Bulletl!r. wW t.e RDt to III1VM lnterested lD the l!eld of blodeterloratlon; It Is hoped that the redpieat. wt1l JeDd to tlw Editor ll.llles of 1111 other peopill who may be intenlted.

AVIS AUX COLLABOBATBUBS

Le buDelln m publl6 deux foil p1t 111. au prlnlemp11 et 1 r•utoDme. Les tm8 d&ety~ uaphlh doJV~~nt entre enroy&l m lrlpill aempl&lre 111 RM&cteur, DT. H. 0. W, Egglna.Biodeterloratlon lnlormatlan centte, Department of BUllog:lell Sdenco, Tbe Uninnlty ol Alton ID Blrmlnibam, Gosta Green, BJrminghlm, 4, E:J1C11D4 (Allgleterre)

ou l ruu d• pera:~1111u llllvantu : Mr. G. AJent. ~pt. BioloEy, ColltEeolTftbnoloo',

Wol..ethunptoll. EIIPad. Prof, G. Beder, Bundeaumaldt tur lLlterial­

prllfung, Berla-Dm!em 45., U!l.tu deD Elcbm Uj81, Gernw!¥. ,

Jlr. D. G. eo.u.,; Tropkal Produdl lnltltute, M-82 GriJI. 11m Road, Londim. W.C.I., Eapu4.

Jlr. J. J. Elphict, Adm1n.I.IJ' Materials Llbontory, Haltoa. Heath. Poole, Dollet. Ell&bJIIl.

Dr. J. Gmido, Departmmto de Fmnrntadones lDchutrlales, CUte11o 25,. Mldri4, Spa.la.

Dr. H. J. Bueek, Ce11tn!' Llibontory TNO, Schoe­maUntrut 17, P.O.B. 217, Delfl. '1'111! Hei.II.Ri. ......_

Dr. A. ll. &plan. Hnd, f'lmdclde. & Germicides Llbontoty, Plonee:rtna: Re:Jurcb Dlvldcm,. u.s. Army Natkk Labontone., Natkk.llallaclwJetb. U.s.A.

lloDJieor Y. LeG ~"~~~d. Ceutre d'etuda du Bouc:het, Veft.le.htlt, (S & 0), FraDce.

Dr. B. J, Z;rW. GIITII'Jl7 liwtJtlrt Gomktin, Kalolriee, Plae. Gwntow t. Polud. Le bulletin Rnln d'0q111e de ~lllloll des

tranm: tn.l.tant tou.Jles upedl de II d~Urloration blologtque en gU.uaJ. e'est-1-dite 11 d~tulontlon pu lei mkroorpnlmJ.a_ les lnRct., les ron&eW"'

etc ••• de m.tt&tau ayant unt tmporlance f.conomlque.

Now up&ona qu'll IBI. poG(ble da llgDalel'

lea IID!m des eollaborateu~ nee lea riffrences dana dLaque bulletin..

C'ert m pnmler lieu liD bulldln d'lnformatioll et par coi!Mquent. •'ll f1ut obtenir du wtot!P­tlona de dtu des art1des de II put de leurll

•uteun, U r.auudno l'ID.d.lquer p1t un ut&1que sur ID texte et IIDfl lDdkatlcm approprlh Hft h.tte 1 II publl~:~Uon.

Cbaque tutl! devra dra pns.!nte mr Aol papier (2t.Ocm. s ~.'l'cm.) et, 11111 pour rlrlicie analytlque, ne dema pas dtpuaer dnq JII&MI eomportllll 500 mob el!.l'lrDn pu pap 7 eomprb 1fll. tableau d Ia ~

Lea tuta dlvrout dra dAetflognpbl& en tro11 e:rempla!ra nee double ·e$p;I~DI d ta~ 1111r UD. Jeul. e.:!t6 seulment. 111. denont etre p!Ueetu pour ta publlcltlon en anatats, franC~!~. allriiJIDd OD ~ ITeC 1111 bvef rinlllfl fll u.a:w. ... ZJ met. eulroll.. .

~~ fllurM dlin'OIIt etre dalremont deA!Mes 1 l'e~~.ere de ehiDio au pbotogn.phl&t. Ll rfd.uo. tlon dN:trie dnn ell'<~~ d&lrmonl lnd(quk; •Prill ffiludlon, lei llgw'el ne dem~nf pu dtpuRr 17tm. pu Utm.

Lanqoe da fl&urea devront etre ~ dana le tate. Ia plae. de chl.amt d'ellt dena etre appnrdm&Uument mmtu~e dan• le texte dactyJo. grapbl6.

Lei riffrencet: blblopgraphlquu denont eire indlquEes selcm l'eum.ple ~t: lteele et LI'I'IMon (llnl2) dana le lute et dana j& lllb~ KnPiilil Ree111, E. T. et Lerilllon, H. G. (I~~) -Etude CIJIIIp&liUYe da ta ·do!grad.IUo:n de II relluloee par les mlaoorpnlanea : I'IIJ'doL l'lut. ,,~tiiJ.

Lea fpreDTeJ ne RrOD.t pu en¥071!.11 aw: auteur~ IY&D.t la publltatlon dftl.nltl«.

25 uem.pl&lfu RrOD.t eni'O)'& gn.tultemmt l d!.aque auteur, Du uemplairt1 wppl6mentaires pruYeDt etre obtenUJ mr demande l rEdltenr .w pr!J: de 55 NF. par centalae.

Des e:c.emplairu du buDetln HIWit envoy& • ~ qula'ln~resRJJt lll cW~rlon.Ucm Moiodqoe; IIDIIS ~ que lei deJtlnatalrl!ll I'Oildrant bleD upb:iie:r au lledf;cteur lea 11om1 da peri01111es mseeptibles d'etre lDUnsRes.

89

BINWEISE FUR HITARBEITEft

Du Bulldln erscbelnt :rwdmal jll!rlkh, UDd lll'&r 1m Frtlhjlllr und Herblt. Jtkhtm Sle bltte lhre maschln~ebrnen Ikltrl,i:e an dm Hen.UI(l!ber, Dr. H. 0. W. Et:giDs. Billdetertora­tlov. lnform~Uon Ceutre, DepartmeDt of Blolocleal Sciences, The Uni\'ttll!y of AltoD in Blrmlngliun. Goate Green, B!rmlnabam. 4, Eqlalld, oclw 111 elne der foliemlen uuehrlften : Mr. G. Ayu~t.Dept. Bioloil', Collrp oiTedllloloil',

WolYerhampton. EaPaL l'J,)f, G. Becku, Bundeamttalilt lllr llltertal­

prillUlli', Bt!rlln-Dihlem .-,, Unter den EleheD M/87, Germuy.

llt. D. G. Collrte7, Troplell Produeb. IDditute. ~ 01'1J'1: ·lim Road. Lamlou W.C.t., ~

Jlr. :J. J. Elpbkt. Admiralty KataUll Llbon!My, BoHtm Heath, Poole, Donet, Ellltud.

DT. J. Garrtda, Departlil.eDto de Fermell.l.ldoael Imlllltrlalel, Cutello 25, )ladrl4, Spa.la.

Dr. H. J. Botek, Central Llbon.ton TNO. Schoe­

matentraat 117, P.O.B. :U7, DeJtt, fte Ndkro ..... Dl. A. 14. Kaplan. Head. Fllllglc:idel & Germlc:idel

Llboratoty, Pioneering ReRarm D!Ylllan., U.S. Almy Natlct Llboratorlea,NaUek,Jb.Aac:bu:setU. U.8A.

MoiU!eur Y.l.eGl'I.Dd, Centte d'etlldea du Bouehet, Vf.rt.t..PeUt. (S & 0), l'r&Dce.

Dr, B. J. l!:,U... Glo'IIJl\)' lnatytut <klmiCUft. Katowice. Pile. Gwuli:011' 1, Palud. Du .Bullettn 1011 du Vu!iffmtllehung YOII.

Arbe.lten Dbu all! Alpette des Abbaues dureh Otaanlsmm fm aJJa:emdnen dlenen, wte E. B. die ZmlDI'llllZ Yon lLllerllllea YOD 111'lrtP::haftikber Badeutuq durc:b Hlt:fwrgmlmltna, J4Rkten. Nagetlue 111'1', Dill Bulletin lrird YOrwlegend Neues bJfn&m. Desbalb, lftDD d1e Erlaubnll. IIIII Arbdtm m dt!eren, 'JODI Autor elngdlolt werden miiJI', 151. diel durch e1n Studlen •uf dem Manu·

U:rlpt :ru. Kenm:elthen, 10 da/J elne dielbntl&llo:be Allmc!rkulli: bel der VerlllfentllthUlli' gematbt werdmka.JID,

:Ieder Ikltag KID. 1m A4 papler elperelcht wellien (it.Ocm. s 29.7cm.) UDd etn.eb1lRlltb del: Tlbellen und BUder 5 Sellen mlt je IID&eflhr liOO Wllrtem nleht tibenchrelten.

Die MIDusl:rlptl aallm in Uac:her Aus:futl&unr, JDI.Khlneii&U(hrleben mit heJlliem Abltlnd UDd nnr auf dner Sclte del Plplen, rln.gesan(lt WU'den. Zum Abdrud: ~1111en de ln Eaglisdl, Fnn!illildl. Deubcb <XIer Span!Jch YO~~ werden, mllfRD • lhr 8tH brall ea&llac:be ZIIIUIIIIWit'UIDD& YOil

qettllr 25 Wl!rten enthaheD. AbblldUlli'ell werden mit Aw:debtu.Jdle pzelcb­

net ocler •II Photognphlen erbetell- Du gtwll!lscllte Vuk~e~DeruiJi:IIIDjf stab lrt deutlleh llllUieben. Ver.tlelnerte Abblldunpn. aollten elD

Format YOD 17cm. s 26CIII. Dleht ~ten. Ftlr tn den Test dllzufllgenda BUder· bt der ungeflhre 'f'lW ftlr jedea e!Dzclne in dem Yuwlkrl.pt two Zll temu:l.eclmm.

PUr die billllographiscben HIInrebe liltfolgemla Bdsplel: Reese UDd Lmnsun (11") 1m Te:d. lm Uten.turven:elthnla: Reeae, E. T. UDd LevfD. IOD, H. G. (1952). VarglakhE:Dde untemlchiiiJZ llber den Al>bau YOD CelhiiOII! dun:h ~ orpniameD : l'h1sloL PlaaL I, S54-3118.

Vor dm. enllgllltlgen Verl!lfentnclmDg erb.alten die Autoren kelne Karretlurb:llge.

Jeder Au tor entbllt 25 Freluemptan. Zudtzl.ltbe Sonderdnleke liDd auf Anfnge bel dem H~ber der Zettsebrtft seJt!D eiDe ~bllhr YOII oUI JUl. je 100 Stlld: erbaltllcb.

Jed!JI', dtt &leh fur du Gebld del Abbaues dureh OrpnlmJ.en lntereAlert. b.n.n den BulletlD erhlltm. Die empflllgc!r .Adrasell. 1lll1 ll!dmm IDteraslertOD Penonen 111 dell Herauaa:ebel'

""'"""--~

APUNTES PABA LOS CONTBIBUIDORES

El Boletln 111 produce dol •- Ill &DO, en II prlmiYera y til d OtoiiD (lbr11 7 DCtubre). Lu eonlrlbudo:nes acrltu a II mtqulna 1 por t:rlplle.da debet~ IU mNdu Ill Redactor DT. H. .0. W. Ealns. BiodeterloraUoe Jn!orm~Uo:n

Ceutre. ~t ol Bloloilcal Sciences, The Un!YI!nity of Astoll. 1n lllrmlDglllm. GOita Green, B~ham, 4, Eq:lalld, 0 I uno de los ilgulentea: lll!".O.Ayrrat_Dept. Bloiou, ColltEe of TedtnallliJ',

WolYerbamptoD. Eq:lu4. Prof. G. Becter, BuDdeaaMtaldt tur Uatarb.l·

prilluna. Berllii-Dahlem u. U11ter deD E.ltben U/87, Gernwl¥.

llt. D. G. Coarsey, Trop1cal Produeb. lnst1tu~ 15-6~ G1'1J'1: 1Im Road, LDndcm W.C.I., EDz)uu!..

Mr. J. J. Elpbkk, Admlnltr lfateriall Laboratory, Holton Heatb, Poole, Donet. EDz)uu!..

m. :1. Garrido, ~partmeDto de l'mneDtldonu llldustrlales, Cutello 25, Madrid, SplllL

Dr, lL J. Hueck. Centn.l. Llboratol")' TNO, Sdlo. matmtraal 07, P.O.B. 217, Delft, 'l1le Nr&llrr­...._

Dr. A. 14. K&plan, Had, FwlilddeJ & Germlddu Laboratory, l'1oneetUIJ Raleltth D!Ylllon, U.S. Almy N1ijd; Lllboratortea, Natkt, M&suehutett:f,.

U.s.A. Monsleor Y, LeGrmd, centre d'etudes du Bouchet.·

Vert-le-PeUt, (S & 0), Frlllce. Dr. B. J, Zylb. Glown:r 1tut7tnt GorDictln.

K&towlee, P1ae. G\Yirtow 1, P8bnd. El Boletio. sen1rl de fthkulo p11ra Ia publleAd6D

de obru de todll diM aalae todoa los upeetool de II blodeterlondOll er1 genenl, et dedr, li. deterlond6u de Ina JDI.terlales de fm~ eam6mlca por lol mlcroozpnl.am.os, iNectol. roedarea (n.tu) ete. F.a et~' pr1m!i lllpr 1111 baletfn de DUI!'YU y por eoD.Jlguler~te, d 111 ne«ss.ta obtmer de c£erto. 111\orea el peru!lso d1

dtar de 1111 lrUeulos. ae debert lndlculo por liD

uterbco rn el manlllttipto y • lllrl Dill

obsen'ad6n aproprilda a1 publkulo. Cad& contrlbudOll debe estar preamtada m

ptglnu de papel en A4 (21.0cm. pot ~.7cm.) y, COD ueepdon del artkulo de reruta. debe llmltane a dneo ptglnu.de IIIWI ~ plllabm por ~ intluldu Ju tablu e llUJtrldones.

Lu cont:rlbudane:t debm estar acrltu a II miqutna por trlpllndo, 1 do1 espedos, en 1111 l&llo de II -boj1 aalamente. Pueden M presmtad.u pall II pub\k:ldolD m lql&_ filnri.s, alemtn 0 I!:I)W!Ot. eGD liD lime l'ftUDIN N lql& de lliiU .. ........

Hay qua pruentu Ju l!ustradoDea ~tam.eDte truadu eD tlnta c:biDa 0 fotanJlldu. Ll d~ reducd6n debe IDdltarJe. ellri.mmte, :r tlllllda redoddu lu llulltaclonea no (Ieben 8%eeder 17 em. ,... .. ~

CUando har que lntrodDC!t clh'u en 1!1 l8%to, tuoy que IDdleli en el docum~ el tupr •proxlmado donde deben pon.ene.

L11 nlerenclu blbllogriflcu deben tndleuse u.l-poi rjrmplo "ReeJe and Lerilllon (1"~)" ell el tuto, l' ell II blbllografla "llee5D. E. T. l!ld Lnlnzlan., H. G. (l~~J. CompantJye Study ol the breakdown' of eellulott by ml~ PIIJIIoL PIID.t. $, ~f-3611. ~

No 1e devo\Ytrill 111 prubu a lo1 autores mte. de 11 publlcacl6n defin!Uya.

Se rnl'il.rlin JTIUI Ill autor ~ tlndes &parte de ~lmpresi<Sn. Se puedtD obtenu mJ.J Undes dirl&tbd0$e al ~ctor de PubUCaclOll • ru6n d8 670 paset.a. por dento.

Se ml!ldarhl ejempllrea del boletrn a qu!enquircra que II! interese por los ertudlo1 de II blodeterlarad6n. Se eapua qua las l'Wban envlar.ln a1 redactor lOll nombns de otros lndlridua• qua puedlll lnteresarse por este lema.

Have you placed your

order for IBBRIS?

(INTERNATIONAL BIODETERIORATION BULLETIN REFERENCE

INDEX SUPPLEMENT)

IBBRIS IS A NEW JOURNAL FOR DISSEMINATING REFERENCES TO MATERIAL PUBLISHED ON ALL ASPECTS OF BIODETERIORATION AND

CONTAINS A MINIMUM OF 2,400 REFERENCES PER ANNUM.

SAMPLE PAGE:

Gl443-Gl463 METALS (M8) PAPER (MIO), RUBBER, PLASTICS, RESINS, BITUMEN & ASPHALT (Mil)

G1JM

METALS (M8) (Coat.)

D~WCCIA, J • J,

l!at~rioh Protection, ~· (6), 49-51 1 (1966)

Evalur.H:;!ll of J.:etal15 in Deop OaeB# Environment,

TIPPS, .c. "1,

Jlateriab Protection. ~~ (9), 9-11, (1966)

t1Ddersroun4 COlTOllion

G1445 llcDOUGALL, J,

J.nti-Corrollion llethode llaterlale. !• (B), 9-13, (1966)

lliorobial Corroaicn ot Jletale

HAGGm, J. H. S.

lllduatrlal Endneerl.nrj Chemiat:r;y, ~ (B), 27-341 (1966)

Corrosion Activity in 1965

G1455

JOHNSON, A, H, c! STOKES, J, L,

J, Bacterio1og. ~· (4.), 151+3-151+-71 (1966)

lle.ngenne Oxidati.on by Sphaerotilua dieoophorua

PAPER (M10)

PlmXlSS, B, E,

~temational Paper Boatd lndustn; 2• (10) 1 1.5-18, (1966)

Slhe Control in the Paper lnd\l#try

Cll.AVE:IS, B, B,

!!.e2!. !<2. (8), 53A-55A, (1966)

Stabilbed Chlorine Dioxide tor Jlicroorganiam Contl'Ol

G1.457 SETH, H. K.

Trsnsaotiona British Llyco1odoal. Sooiet:y. ~ (1), 4.5-47,

See page 87 of this bulletin for full details and order form.

THE BIODETERIORATION

INFORMATION CENTRE

NOW OFFERS THE FOLLOWING

SERVICES INTERNATIONALLY

1. QUM;TION ANSWERING SERVICE (free of charge)

Detailed information is supplied to enquiries in all fields of bio­deteriqration. Question forms appear in IBBRIS or can be obtained from the B.I.C. Telephone enquiries are also accepted. A return of post service operates wherever possible.

2. ALERTING SERVICE (free of charge)

The B.I.C. can supply you regularly with references applicable to your field of interest. This service is at present confined to special cases where the reference classification scheme published in IBBRIS is inadequate to particular individuals' needs. Please apply to the B.I.C. for further details.

3. PHOTOCOPY REQUEST SERVICE

Copies of papers listed in IBBRIS are loaned free Of charge or supplied at a cost of 6d. (7c.) per page. Photocopy request forms appear in IBBRIS or can be obtained on application to the B.I.C. A return of post service operates wherever possible.

Biodeterioration Information Centre, Department of Biological Sciences, University of Aston in Birmingham, Gosta Green, Birmingham 4, England.

When you need a paint fungicide, you should look to N uodex.

Why?

Because we have the most versatile line around.

SUPER AD-IT The recommended universal paint fungi· cide for both oil and aqueous systems.

PM0-10 For mildew resistant oil, oleoresinous and alkyd paints.

PMA-18 For use in standard aqueous systems as a fungi· cide and/or preservative.

PMA-60 For use in emulsion paint systems to prevent bacterial spoilage during liquid paint storage and to pro· teet the applied paint film against mildew attack. Packaged in 4 oz. and 8 oz. water-soluble packages.

FUNGITROL 11 Fungicide-bactericide for non-aqueous paint systems. Effective for meat-packing plants, breweries, dairies, etc. Also imparts fungus resistance to baking enamels.

NUODEX products are available from the following affilia!es and licensees:

ARGENTINA Plastica Bernabo S.A., Terrada 658!664, Buenos Aires; AUSTRALIA · Nuodex IAust.l Pty. ltd .. 49-61 Stephen Road, Botany, N. S. W.; BRAZIL - Nuodex S. A, Industria, E, Comercio de Secantes, Rua Dam Gerardo 80-1" and., Rio de Janeiro: ENGLAND -Nuodex Limited, Birtley, County Durham: FRANCE - Nuodex France S. A. R.l., 14, Rue de Moscou, Paris 8e, France; WEST GERMANY­Gebr. Borchers A. G., Elizabethstr. 14, Dusseldorf; G. Siegle & Co, GmbH, Sieglestrausse 25, Stuttgart-Feuerbach (Stabilizers onlyl; HOLLAND- N. V. Chemise he Fabriek Servo, Delden; ITALY- Nuodex ltaiJana, S. P. A .. Piazza Della Republica ll/A, Milan; JAPAN -Harima, Kasei Kogya Ltd., Mizuashi, Noguchi-cho, Kakogawa, Hyago; MEXICO - Nuodex Mexicana. S. A., Durango 209, Desp. 101, Me:-:ico 7 D. F.; NEW ZEALAND . A. C. Hatrick IN. Z.l Ltd., 66 Main Road, Tawa, Wellington: SOUTH AFRICA Poly-Resin Products, Ltd., Durban; SPAIN- Nuodex Espanola, S. A., Av. Jose Antonio 55, Madrid.

In any language NUODEX is your symbol for quality and service.

e TENNECO CHEMICALS, INC. NUODEX DIVISION INTERNATIONAL DEPT.

P.O. Box 2. Piscataway, N.J.

Published by the B;odeterioration Information Centre. COST PRICE I 0/- Printed by Harris and Sheldon Ltd.