1 Risk Assessment of Trichoderma reesei for Consideration of Addition to the List of Eligible Microorganisms for the Ti ered 5(h)4 Exemptions from MCAN Reporting Requirements

I. INTRODUCTION

Trichoderma reesei is an ascomycetous wood-rot fungus that has been used for decades for the production of enzymes fo r agricultural, food, pharmaceutical, and industrial uses. It is the anamorph (asexual reproduction through mitotic division of conidia) of the teleomorph (sexual reproduction through the formation of ascospores)

known as Hypocrea jecorina . QM6a is the designation giv en to the original strain of T.

reesei isolated in 1944 from decaying tent fabr ic on the Solomon Islands in the South Pacific during World War II. According to Neva lainen et al. (1994) almo st all, if not all,

industrial strains of T. reesei are derivatives of QM6a. Re cent taxonomic studies have shown that actually the species T. reesei consists only of this single isolate and its derivatives. Many other strains of this fungus isolated from other places, which have

sometimes been referred to in the literature as T. reesei, have now been proposed as belonging to the newly named species, T. parareesei (Druzhinina et al., 2010). These

other isolates differ from T. reesei in habitat, sporulation, and metabolic versatility. T.

reesei has been shown to belong to a single species now referred to as H. jecorina/T.

reesei (QM6a) which reflects its re lationship to its teleomorph H. jecorina (Atanasova et al., 2010). The only anamorphic strains within the species H. jecorina/T. reesei are those of QM6a and its derivatives. Thus, giv en these recent taxonomic publications, all

strains of T. reesei are, by definition, QM6a or a derivative (Druzhinina et al., 2010; Atanasova et al., 2010).

T. reesei is not pathogenic to humans, other animals, or plants. However, T.

reesei can produce a secondary metabolite ca lled paracelsin which is a compound known as a peptaibol. Peptaibols are sma ll linear peptides characterized by a high content of the non-prot einogenic amino acid α -aminoisobutyric acid, with an acetylated N terminus, and a C-terminus that is linked to an amino alcohol. Paracelsin, like other peptaibols, modifies cell membranes of vari ous organisms through the formation of ion channels which may result in loss of osmoti c balance and in leakage of cell contents. Paracelsin is inhibitory to the growth of Gr am-positive bacteria and to fungi, but it also has been shown to exhibit cytotoxicity to mammalian cells and to aquatic indicator species. However, paracelsin is apparent ly not produced by the fungus under the conditions of submerged standard industrial fermentation used for enzyme production. There is a long history of safe use of T. reesei for production of enzymes for food and animal feed. Data submitted in Generally Re cognized as Safe (GRAS) petitions to the Food and Drug Administration (FDA) for numerous enzyme preparations from T. reesei

for human and animal consumption dem onstrate that the enzymes are nontoxic. Given the long history of safe use of T. reesei for production of enzymes, it is appropriate to2 consider this microorganism eligible as a recipient microorganism for the 5(h)4 Tiered Exemptions from Microbial Commercial Activi ty Notice (MCAN) reporting requirements if this fungus was to be used in submerged st andard industrial fe rmentation for enzyme production. However, scenarios have been proposed for use of T. reesei in cellulosic

biofuel facilities under conditions not typical of submerged standard industrial fermentation. Publications have suggest ed on-site cellulase production at biofuel facilities by the growth of T. reesei directly on the pre-treat ed cellulosic hydrolysate (i.e., delignified plant material) fo r degradation of the plant material into sugars that are subsequently fermented into ethanol (Wooley et al., 1999; Kazi et al., 2010). The growth of T. reesei directly on plant material in biofuel facilities poses concern because paracelsin may be produced under these conditi ons. Historically, the conditions used by researchers to optimize peptaibol production by Trichoderma species, including T. reesei, have typically involved the addition of insoluble sources of carbon such as

cellulose to the fermentation br oth, the presence of solid surfaces in the fermentation broth, or the growth of the fungus on a solid surface. Presently there is no information on the quantities of paracelsi n that could be formed if T. reesei was grown on pretreated plant material in the saccharificat ion tanks. There is no information on the stability of paracelsin, if formed, during t he entire process stream of ethanol production

in cellulosic biofuel facilities. There is no information available on its persistence or degradation during wastewater treatment of spent ferm entation broth, nor its persistence and resulting potential effects if it made its way into the environment. Therefore, given paracelsin’s demonstrated biological acti vity to bacteria, fungi, mammalian cells, and aquatic indicator species, T. reesei cannot be recommended as a recipient microorganism for the 5(h)4 Tier ed Exemption if used under conditions other than those typical of submer ged standard industrial fermentati on for enzyme production.

History of Commercial Use and Products Subject to TSCA Jurisdiction

There is a long history of safe use of enzymes produced by various species of

Trichoderma in agricultural, food, pharmaceutic al, and industrial ap plications. According to the Enzyme Technical Association (ETA), Trichoderma enzymes are currently used in brewing processes ( β -glucanses), in potable alcohol manufacture (glucoamylases), for maceration in fruit juice production (pectinases, cellulases, hemicellulases), as an additive to lives tock feed (xylanases), and for pet food processing (ETA, 2010). In addition to their use for enz yme production, some species

of Trichoderma are used as biocontrol agents of f ungal plant pathogens, predominately

strains of T. harzianum and T. viride (Verma et al., 2007).

The species T. reesei has been used extensively for enzyme production in both foods and in industrial applications, mainly fo r production of cellulases. Cellulases have been manufactured commercially in Japan sinc e the 1960’s (Toyama, 1969, as cited by Nevalainen et al., 1994). Cellulases are used in the food industry for baking, malting, brewing, and in grain alc ohol production (Nevalainen et al ., 1994; Olempska-Beer et al., 2006). Several cellulases produced by T. reesei have been approved for food use by3 FDA in GRAS petitions (21 CFR §184.1250). GRAS notices for enzymes produced

from T. reesei used in food include cellulase, hemic ellulase, transglucosidase, pectin lyase, and acid fungal protease (http:// www.fda.gov/Food/FoodI ngredientsPackaging/ GenerallyRecognizedasSaf eGRAS/default.htm). Cellulases produced by T. reesei have also been used in the pharmaceutical industry as digestive aids, and in the animal feed industry to increase the digestibility of feed (Malmos, 1977, as cited by Nevalainen et al., 1994).

T. reesei produces a number of different enz ymes that have TSCA uses, especially cellulases. Historically, T. reesei cellulases have been used in the wood processing and textile industrie s (Nevalainen et al., 1994). More recently, however, there has been an enormous interest in producing T. reesei cellulases for use in the saccharification step of plant material in cellulosic biofuel production facilities. T. reesei also produces numerous extracellula r proteases (Kredics et al., 2005). T. reesei may also be used for production of hydrophobins, wh ich are proteins with characteristics for potential use as biosurfactants (Askolin et al., 2001; Wösten, 2001). To date, the Agency has reviewed numerous Microbial Commercial Activity Notices of T. reesei for production of enzymes with TS CA applications such as α -amylase, β -glucosidase, acid protease, glucoamylase, phytase, lacca se, and a number of cellulose-degrading preparations.

II. IDENTIFICATION AND TAXONOMY

A. Characterization of the Genus Trichoderma

The genus Trichoderma, which was first named by Persoon in 1794, is a member

of the Hypocreales (fungi, Ascomycota). It is an anamorphic fungus that reproduces solely asexually through mitotic division of conidia. Hypocrea is the genus name of the teleomorph that reproduces se xually through the formation of ascospores. Members of the genus Trichoderma are ubiquitous in nature. They are geographically widely

distributed and have been observed in a br oad range of habitats because they can colonize a wide variety of substrates. Most Trichoderma are aerobic mesophilic wood rot fungi found in soils and decaying wood and vegetative materials (Gams and Bissett, 1998). Trichoderma spp. are very common in agricultura l, prairie, forest, and desert soils and in salt marshes with a variety of c limates (Klein and Eveleigh, 1998). They are saprophytic fungi that pr oduce copious amounts of degradative enzymes including cellulases, xylanases, and chitinases which enables them to live on a wide variety of organic materials and transform many xeno biotic compounds (Klein and Eveleigh, 1998). The production of cellulases and xyla nases by various species, particularly T. reesei, has made them important industrial mi croorganisms. Mem bers of the genus

Trichoderma produce a vast array of secondary me tabolites such as peptaibols and peptide antibiotics active against fungi and bact eria. Mycotoxins such as trichodermin, trichotoxin A, and trichothecenes are produced by some species of the genus. Some4 Trichoderma strains produce chitinases and possess antagonistic properties to other fungi, and are thus, used as biocontrol agents (J aklitsch, 2009). Species used as fungal biocontrol agents are predom inately strains of T. harzianum and T. viride, but also include T. lignorum, T. virens, T. hamatu m, T. koningii, T. pseudokoningii, T. aureoviride, T. longibrachiatum, and T. asperellum (Verma et al., 2007). The antagonistic properties to fungal plant pathogens by some Trichoderma strains are due to multiple mechanisms such as competit ion for nutrients and space, modification of environmental conditions, promotion of plant growth and plant defense mechanisms, and by mycoparasitism (Haran et al., 1996; Benitez et al., 2004; Harman et al., 2004; Woo et al., 2006; Verma et al., 2007) . Some endophytic species confer plant protection, most likely thr ough the production of secondary metabolites (Degenkolb et al., 2006). There are also Trichoderma biocontrol agents against bacteria, insects and nematodes, and even plants (weeds) (Verma et al., 2007; Bokari, 2009).

Other species of Trichoderma , such as T. aggressivum, T. pleuroticola, T. pleurotom, T. pleuotrophilum, and T. fulvidum are pathogenic to cultivated mushrooms

such as Agaricus bisporus and Pleurotus ostreatus (Samuels et al., 2002; Komo ń

Zelazowska et al., 2007a; Kubicek et al., 2007).

There is one species in the genus that is considered an opportunistic human pathogen, T. longibrachiatum. It has been shown to cause fungal mycosis in immunocompromised individuals (Pfaller and Die kema, 2010; Druzhinina et al., 2005).

B. Taxonomy of the genus Trichoderma

Currently, there are more than one hundred different Trichoderma species that have been molecularly characterized. However, the taxonomy of Trichoderma has been problematic and assignment of an accura te species name to an isolate may be challenging. Before the discovery and developm ent of molecular biol ogical identification methods, fungi were classified based sole ly on morphological char acteristics, often incorrectly so. According to Druzhinina et al. (2005), approx imately 50% of the

Trichoderma isolates currently in culture collec tions under names based solely on morphological analysis are wrong. With th e advent of molecular techniques for identification, the internal tr anscribed spacer (ITS) 1 and 2 fragments were first used for assignment of an isolate into a species. Ho wever, Druzhinina et al. (2005) stated that the ITS1 and ITS2 sequences do not provide sufficient phylogenetic resolution even though these sequences are still commonly us ed by some researchers. Proper

identification methods are available however. The currently accepted mode of

identification for Trichoderma and Hypocrea is a more comprehensive approach employing criteria of the Genealogical Concordance Phyloge netic Species Recognition (GCPSR) concept, which often demands the us e of genealogies of three or four genes, not just the spacer regions (Taylor et al ., 2000). The GCPSR was meant to provide a common approach to classifying new strains of Trichoderma . Druzhinina et al. (2005) proposed an online identification tool using an oligonucleotide barcode , i.e., a diagnostic5 combination of several oligonuc leotides specifically alloca ted within the ITS1 and ITS2 sequences of the rDNA repeat which they calle d TrichOKEY. The latest version of this

tool is available via the Internati onal Subcommission on Trichoderma and Hypocrea Taxonomy (ISTH) website at http://www.isth.i nfo/index.php. More recently, Druzhinina et al. (2010) indicated that additional marker s besides those in the GCPSR protocol are essential for differentiating among H. jecorina and its close relatives. In any case, these methods appear to be best suited fo r research laboratories doing Trichoderma systematics rather than routine identificati on methods needed by commercial entities or others. Without these protoc ols, it appears that differentia tion of these closely related species will be difficult. Even though identification of new species of Trichoderma may be challenging, the lineage of all commercial strains of T. reesei from the original QM6a

isolate has been es tablished.

C. The Species Trichoderma reesei, strain QM6a and its Derivatives

1. The species Trichoderma reesei

The history of the well-known T. reesei QM6a strain is presented in Kubicek and Harman (1998). During World War II, because of the military’s interest in determining what was causing the rapid rotting of canvas t ent material and fabric in the South Pacific tropics, research on members of the genus Trichoderma accelerated greatly. A very cellulolytic fungal strain was isolated from an island, reported variously as either Bougainville or Guadalcana l, in the chain known as the So lomon Islands (now politically part of Papua New Guinea). This isolate was designated as Trichoderma viride QM6a

. At that time there was only one species of Trichoderma which was T. viride (Druzhinina et al., 2005). Approximately 20 years la ter, QM6a was re-named by Simmons as

Trichoderma reesei after Elwyn T. Reese who conducted the majority of investigations on this fungal strain (Kubicek and Ha rman, 1988). The Am erican Type Culture Collection designation for th is original strain of T. reesei QM6a (Simmons 1977) is

ATCC13631.

As previously stated, Trichoderma reesei is the anamorphic form (asexual reproduction) of the fungus which also has a teleomorphic form (s exual reproduction)

known as Hypocrea jecorina (Eveleigh, 1985). Kuhls et al. (1996) presented molecular

evidence that T. reesei is indistinguishable from H. jecorina based on sequences of internal transcribed spacer (ITS) regions. In addition, failed attempts to cross T. reesei with wild type H. jecorina strains led Kuhls et al. (1996) to believe that T. reesei was a clonal derivative of H. jecorina

.

Later Lieckfeldt et al. (2000) were successful in isolating anamorphic H. jecorina

from South American soils and vegetation, rather than ju st the usual teleomorphic H. jecorina from ascospores. They demonstrated that H. jecorina was a heterothallic ascomycete having two mating types, with ascospores that exhibited bipolar segregation within the ascus, the sac cont aining the sexual spores during sexual6 reproduction. Using ITS regions, identic al sequences between these new anamorphic H. jecorina isolates and collection-based cultures of H. jecorina and T. reesei QM6a were observed. Thus, it appeared that H. jecorina and T. reesei had a teleomorph/anamorph relationship, shari ng habitats and gene sequences. However, Lieckfeldt et al. (2000) noted differences in metabolic phenotype between strain QM6a and other putative H. jecorina ( T. reesei ) in that all of the new isolates utilized sucrose, nitrate, and nitrite, but QM6a did not.

In the years that followed t here were several other repor ts in the literature on the

isolation of T. reesei from geographical regions other than the Solomon Islands. Kar et al. (2006) reported the isolation of T. reesei from soil in West Bengal, India. Another paper reported the isolation of T. reesei from sea mud in the tideland in Lianyungang, Jiangsu Province, China (Sun et al., 2006). Ho wever, the identification of the fungal

isolate as T. reesei was questionable as the method of identification used was not specified. There is one other ar ticle reporting the isolation of T. reesei from an area south of the Caspian Sea in Iran (Roodsari et al., 2007). Although robust molecular identification methods appear to have been used in the identification of the 22 species

of Trichoderma found on wood or in soils from Iran, this report exists only as an abstract from a presentation at a m eeting, and the data have not been published. It could also be that in these papers the authors us ed the better known species name of T. reesei

rather than H. jecorina.

Later, mating experiments between T. reesei and H. jecorina conducted by Seidl et al. (2009) led to the discovery that T. reesei (strain QM6a) and H. jecorina were not identical in mating types. T. reesei has a single mating type known as MAT1-2,

whereas H. jecorina possesses either a MAT1-1 or a MAT1-2 mating type. Thus, in experiments T. reesei was a successful male donor in mating with opposite type H. jecorina, but was unable to form female st ructures when undergoing mating with H. jecorina, or with an artificially modified T. reesei having opposite mating type. These results led Seidl et al. (2009) to a conclusion different from that of Kuhls et al. (1996)

which was that T. reesei is not a clonal derivative of H. jecorina , but rather a cryptic species. Cryptic species are morphologically similar, or even virtually identical, but have been reproductively isolated. In this case Seidl et al. (2009) speculated that the reproductive isolation may be related to non-functional or absent genes required for female fruiting bodies. Therefore, Seidl et al. (2009) stated that T. reesei is a true anamorph of H. jecorina, i.e., a true member of the same species.

In the literature the names T. reesei and H. jecorina have been used synonymously as some researchers consider them to be identical fungi (except for mating type). Others think that the anamorph and teleomor ph should be referred to as separate species. However, a recent taxo nomic publication has now proposed a split of H. jecorina into as many as four species (Dru zhinina et al., 2010). They proposed the

name T. parareesei for those fungal strains of H. jecorina that were isolated as anamorphs from soils, and showed that the majority of t hese anamorphic strains were7 genetically isolated from H. jecorina/T. reesei. They also suggested that the remaining strains that do not belong to H. jecorina/T. reesei probably belonged to at least one other species, but the number of isolates studied was so limited they did not propose species names for those few isolates. Lat er that year, building upon the work of Druzhinina et al. (2010), Atanasova et al. (2010) indicated that there were actually three species equivalents within H. jecorina sensu lato ( H. jecorina in the broad sense). They provided data further justifying the proposal for the new species T. parareesei based upon differences in habitat, sporulation, and metabolic capabilities of these strains. H. jecorina/T. reesei strains were differentiated from T. parareesei by being more sensitive to light, having a narrower substrate range, gr owing less robustly at higher temperatures on many carbon substrates, and being less compet itive with other fung i. Although more light tolerant, T. parareesei was viewed as primarily a soil fungus while H. jecorina/T.

reesei seemed to be better adapted to plant surfaces. These papers therefore proposed that the species T. parareesei would consist of those strains formerly referred

to as T. reesei from soils in Latin America, Africa, and Southeast Asia. These included fungal isolates from soils of subtropica l and tropical areas in the South American countries of Brazil, Argentina, and Columbia , the Central American country of Mexico, from Ghana and Ethiopia in Africa, and from India (Druzhinina et al., 2010). Atanasova et al. (2010) also presented data showi ng that strains within the species T. parareesei had lost the ability to reproduce sexually

. They stated that although T. parareesei

strains contain both the MAT1-1 and MAT1-2 mating types, the MAT1-2 locus in T. parareesei was altered, and was thus defecti ve compared to that found in H. jecorina.

These recent taxonomic papers also showed that T. reesei sensu stricto (QM6a)

was in the same clade as almo st all teleomorphic strains of H. jecorina (Atanasova et al., 2010; Druzhinina et al., 2010) . T. reesei QM6a was the only anamorphic strain within that clade, termed H. jecorina sensu stricto. Atanasova et al. (2010) stated that the strain QM6a (plus derivativ es) and teleomorphic strains of H. jecorina are a single species now referred to as H. jecorina/T. reesei (QM6a). This, of course, implies that the species T. reesei consists solely of the strain QM6a and its derivatives.

In summary, recently Druzhinina et al. (2010) proposed splitting up some T. reesei/H. jecorina isolates into several new species, one of which was called T. parareesei . Atanosova et al. (2010) continued with that work and characterized the species T. parareesei, which would consist of those H. jecorina strains isolated from soils around the globe as an anamorph which di ffer from the original QM6a strain in sporulation, photosensitivity, substrate utilization, and habitat. Since this new species, T. parareesei, was formally described by Atanasova et al. (2010), it now awaits acceptance from the scientific comm unity. Upon acceptance, the species H. jecorina will consist of the teleomorphic stra ins remaining after those called T. parareesei and some as yet to be named other isolates are re moved, plus the single anamorphic isolate

called T. reesei which encompasses strain QM6a and it s derivatives. Only the latter

has a stable taxonomic status at this ti me and can be unequivocally identified due to well-established culture collection provenance. Therefore, this exemption is being8 considered only for T. reesei strain QM6a and its derivatives , and not for other potential or existing species of Trichoderma or Hypocrea

.

2. Derivatives of QM6a

At the U.S. Army Natick Laboratories in Natick, Massachusetts, researchers
created mutants of QM6a with increased cellul ase production. Conidia of the strain T.

reesei QM6a (called T. viride QM6a at the time) were sus pended in distilled water at

20°C and were irradiated with hi gh energy electrons from a 24-million electron volt, 18- kw linear accelerator (Mandels et al., 1971). A dose of 0.05 megarads killed more than 95% of the conidia, and all we re killed with radiation abov e 0.2 megarads. Survivors (0.05 - 0.2 megarads) formed small colonies on deoxycholat e agar even after extended incubation periods, but then sporulated and were transferred to potato dextrose agar where normal growth resumed. One isolate, designated QM9123 (dose, 0.05 megarads), was found to produce twice as much cellulase as the wild type QM6a strain. According to Morawetz et al. (1992), this T. reesei mutant strain QM9123 was apparently subjected to anot her radiation step to pr oduce the mutant strain T. reesei QM9414. However, the liter ature is not clear in exactly what mutagenesis was

conducted to arrive at stra in QM9414. Nevalainen et al. (1994) says that this mutagenesis also took place at the Natick Laboratory and references the paper by Mandels et al. (1971) for this mutagenesis. The Mandels et al. (1971) paper, however, does not mention strain QM9414, but only QM9123 and another mut ant obtained that was deficient in cellulase production desig nated QM9136. Montenecourt and Eveleigh (1977) stated that QM9414 was derived by a 2- step mutational proc ess from QM6a, but again, there are no specifics regarding the second mutation. Brückner et al. (1984)
stated that QM9414 was obt ained from a second irradiation of the conidia of QM6a, but

these authors reference the Mandels et al. (1971) paper and Montenecourt and Eveleigh (1977), neither of whic h specifically report exactl y what the second mutational step consisted of. Morawetz et al. (1992) fu rther stated that “the nat ure of the mutations leading from the original isolate QM6a (Simmons 1977) to QM9414 have never been elucidated”. It is apparently only in an abs tract of a meeting where Simmons (1977) described QM9414, but this abstract is not available. The American Type Culture Collection (ATCC) gives a history of QM9414 as a mutant of QM9123, and says that it was originally Mandels’ strain 3019. The strain QM9123 from which QM9414 was derived was described by Mandels et al. (1971) as a mutant that appeared to only have an increased quantity of cellulase production and that other carbohydrases were not

affected. Mandels et al. (1971) further reported that QM9123’s increased cellulase activity was stable for more than two y ears in subcultures and through a lyophilized state. Obviously the mutations of QM9123 to arrive at QM9414 resulted in a further increase in cellulase production. ATCC says that QM9414 produces 1.5-2.0 times more

cellulase on cellulose medium than stra in QM9123.

In an article on the safety of T. reesei in enzyme fermentation, Nevalainen et al. (1994) say that most, if not all, of the T. reesei strains used industrially are derived from9 the T. reesei QM6a originally described by Mandel s and Reese (1957). The authors present a figure showing the genealogy of the different high cellulase-producing T.

reesei strains in use throughout the world. A ll of the following st rains were apparently
derived from the mutant QM 9414: MCG77 (Natick Labor atory), the MG Series (Gulbenkian Institute of Sci ence), the VTT-D Series (Technical Research Center, Finland), the L Series (Cetus Corporation, USA), the MHC Series (Slovak Academy for Sciences, Slovakia), the D1 Series (Indian Institute of Technolog y, India), the Kyowa Series (Kyowa Hakko Kogyo Co. Ltd., J apan), and the CL Series (Societe Cayla, France). The only series of T. reesei that was not derived from this QM9414 mutant is the Rut Series (Rutgers University, USA) which is a derivative directly from the wild type
QM6a strain. The additional mutations a ssociated with these various high cellulaseproducing T. reesei series were not specified.

D. Related Species of Concern

There are a few species within the genus Trichoderma that present human health or ecological concerns . One of these is Trichoderma longibrachiatum, which has been referred to as a serious opportunistic pat hogen that can cause human fungal infections (Pfaller and Diekema, 2010). Druzhi nina et al. (2008) stated that T. longibrachiatum is the species most commonly associated with Trichoderma mycoses, which often has

unfavorable outcomes. In addition to this species, T. citrinoviride is thought of as a potentially pathogenic species (Kuhls et al., 1999) . Earlier reports on the isolation of T. koningii and T. pseudokoningii from clinical samples were found to be incorrect when the sequence analysis of the in ternal transcribed spacer (ITS) region was examined according to Antal et al. (2006). Invasive potential within the genus is apparently

restricted to T. longibrachiatum and T. citrinoviride based on recent molecular taxonomic analyses (Pfaller and Diekema, 2010). Infection by these species has been

associated with immunocom promised individuals, and those undergoing peritoneal dialysis (as summarized by Pfal ler and Diekema, 2010). The species T. longibrachiatum is apparently closely related to Hypocrea orientalis, which is a member

of the H. schweinitzii complex, even though it is not the anamorph of H. orientalis (Samuels et al., 1998, as cited by Antal et al., 2006).

In addition to potential infecti ons from some strains of Trichoderma , other strains present human health concerns because of the production of mycotoxins or other secondary metabolites, some of which ar e highly toxic (Lübeck et al., 2000). The mycotoxin trichodermin is a strong inhibitor of protein synthesis, and t hus, is highly toxic to mammalian cells (Wei et al., 1974). T. viride has been shown to produce a mycotoxin trichotoxin A (Hou et al., 1972) with a median LD 50 (lethal dose required to kill 50% of the population) of 4.36 mg/kg in mice. Degenkolb et al. (2008b) stated that T.

harzianum and T. viride , two biocontrol strains that ar e known to produce trichothecene toxins, have been re-identified as the closely related species T. brevicompactum and T.

arundinaceum. Both of these species belong to the T. brevicompactum complex, a complex with members that have been prov en to produce trichothecene-like mycotoxins10 such as trichodermin and harzianum A (Degenkolb et al., 2008a). Some Trichoderma species and their toxins are associated with “sick building syndrome” from water damaged buildings (Lübeck et al., 2000). T. longibrachiatum, T. citrinoviride, T. harzianum, T. viride, T. hamatum, and T. atroviride were all isolated from waterdamaged building materials in various Dani sh buildings (Lübeck et al., 2000).

In addition to human health concerns, species producing mycotoxins may present concerns for animals other than humans, including aquatic species, and also pose concerns for plants. For instance, tr ichodermin is a potent inhibitor of protein synthesis, so it is toxic to all eukaryotes, both animals and plants.

Some members of the genus Trichoderma are pathogens of commercial mushrooms. Earlier reports in th e literature stated that the species T. harzianum

caused disease in commercial mushroom s (Gams and Bissett, 1998). Later, new names were proposed for the species of Trichoderma that causes green mold of the commercial white button mushroom, Agaricus bisporus (Samuels et al., 2002). The new names proposed were T. aggressivum var. aggressivum , and T. aggressivum var.

europeaeum for the fungi responsible for diseas e of commercial mushrooms in North American and Europe, respectively. The former designations were T. harzianum biotypes Th2 and Th4, respectively (Samuels et al., 2002). More recently, studies have reported that a number of genet ically closely related, but phenotypically divergent species of Trichoderma are responsible for diseas e on the oyster mushroom, Pleurotus
ostreatus, and a number of new species have been described (as summarized by

Komo ń -Zelazowska et al., 2007a). The ability of t hese strains to attack mushrooms is in part due to the production of ch itinase that can degrade chit in, an important component of fungal cell walls, and mush rooms are fungi. Also, the ability of these particular species to cause disease in mushrooms is also due to the production of antifungal compounds. T. aggressivum, T. pleuotrophilum, and T. fulvidum are responsible for significant loss of t he mushroom crops of Agaricus bisporus and Pleurotus ostreatus (Kubicek et al., 2007).

III. HAZARD ASSESSMENT

A. Human Health Hazards

1. Colonization and Pathogenicity

Trichoderma reesei is not a human pathogen. There is only one report in the literature associating T. reesei with an infection in an i mmunocompromised human. T.

reesei is not the species thought to be of in creasing importance with infection of immunocompromised individuals, which is T. longibrachiatum. The other species of the genus thought to have invasive potential is T. citrinoviride (Pfaller and Diekema, 2010).

T. reesei has a long history of safe use for produc tion of enzymes with food, agricultural,11 pharmaceutical, and industrial uses with no in cidences associated with human disease. Because of its long history of enzyme produc tion, and due to the use of some of these enzymes in food and pharmaceuticals, the potential for T. reesei to cause disease and the potential toxicity of its enzyme produc ts has been tested a num ber of times.

An elaborate series of studies designed to elucidate both consumer and occupational health effects were conduc ted over the period of 1978 - 1982 on a cellulase produced by Novo Industries used as a food processing aid (Hjortkjaer et al., 1986). The T. reesei microorganism used to produce an enzyme product known as Celluclast was tested for its pathogenic potent ial and for the production of toxins and antibiotics. Mice, guinea pigs, and rabbits were used for investigating the potential pathogenicity of the T. reesei strain. However, the animals were immunosuppressed first with a high dose of cortisone prior to inoculat ion with either viable or killed spores to facilitate survival and multiplication of t he fungus in the animals. Nonpathogenic microorganisms are typically e liminated readily from a heal thy animal by its immune system defenses. Preliminary studies for dete rmining the lethality of the fungus in mice revealed that the median lethal dose (LD 50 ) was greater than 10 7 viable spores with interperitoneal (ip) injecti on in mice. In the main st udy, the immunosuppressed animals were injected either intravenously (iv) or ip with 10 5 to 10 7 viable spores, or with 10 7
killed spores. No effects were seen with ip injection of 10 5 viable spores, but microorganisms were found in different or gans 40 days after iv injection of 10 5 or 10 7
viable spores or ip injection of 10 7 viable spores. In a ll animals, granulomatous inflammation was observed with histological ex amination. Additional studies on mice

conducted to further elucidate the chronic nature of the lesions led the authors to

conclude that T. reesei was pathogenic to cortisone-treated mice. However, interpretation of these data by Ne valainen et al. (1994) was that T. reesei should be regarded as non-pathogenic because it was only under the circumstances of using high doses of spores in immunocompromised animals that T. reesei behaved as a pathogen.

A pathogenicity study on a strain of T. reesei was conducted by Genencor that was described in a GRAS notice for an enzym e submitted to the FDA (GRN No. 333). With an ip dose of 2.2 x 10 7 viable spores of T. reesei strain A83 in Sprague-Dawley (SD) rats, T. reesei was judged to be non-pathogenic and non- toxic as no animals died, no adverse clinical symptoms were observ ed, and clearance of the microorganism from the rat spleen was observed. A different st udy in the same GRAS notice using an ip

dose of 5 x 10 6 colony-forming units (cfus) in Char les River Sprague-Dawley (CD) rats also demonstrated a lack of pathogenicity and clear ance of the fungus fr om the rats.

Data on the potential pathogenicity of T. reesei are available in another GRAS petition (GRN No. 32) for the en zyme pectin lyase submitted by 

### Röhm Enzyme GmbH.

A genetically modified T. reesei strain, ALKO 2224, and its parental strain were iv injected into Naval Medical Research Institute (NMRI) rats at a dosage of 5 x 10 6 viable spores. Even with the injection of cortis one acetate to one group of animals two days prior to inoculation to immunos uppress them, no pathogenicity of the T. reesei strains12 was observed in any of the rats.

In GRAS notice number 315 from Genencor for the enzyme transglucosidase, data from another pathogenicity study were reported for T. reesei strain EG1-EP9 (GRN No. 315). CD rats were ip injected with 5.6 x 10 6 cfu viable microorganisms or with

heat-killed cells. No deaths occurred and no adverse clinical observations were reported. Initial necro scopy on days 7 and 21 revealed enlarged spleens and decreased liver weight, however, the animals completely recovered thereafter.

There is no information available on the presence of any virulence factors in T.

reesei that would enable it to be pathogenic. Potential fungal virulence factors include adherence factors, penetration factors, necroti c factors (enzymes), melanin pigments, toxins, and the ability to grow at elevated temperatures. T. reesei is a mesophilic fungus that has generally been cultured in vitro in the range of 25-30  C, and been grown between 25 and 28  C for optimal enzyme production. There is one report in the literature stating that T. reesei can grow near human body temperature at 36  C, however, the body of evidence suggests that T. reesei does not pose concerns regarding human pathogenicity.

2. Mycotoxin Production by T. reesei

Many fungi produce mycotoxins, and more produce other secondary metabolites that are inhibitory to various organisms but are typically less toxic. Mycotoxins are highly toxic secondary metabolites that are thought to provi de the fungus with a competitive edge against other microorganisms, including other fungi. There is one report in the lit erature that T. reesei produces the mycotoxin trichodermin (Watts et al., 1988). Trichodermin is a member of a group of 12,13-epoxytrichothecenes that are potent inhibitors of protein synthesis in euk aryotes, and is therefore highly toxic to animals, including humans, and to plants (Wei et al., 1974). According to Blumenthal (2004) trichodermin is a member of this family of potent food contamin ation toxins but it is much less toxic than some mycotoxins.

Watts et al. (1988) reported that tr ichodermin and another secondary metabolite with antifungal activity designated as Tx were isolated from a mycelial extract of T.

reesei strain P-12. They described t he strain P-12 as a mutant of T. reesei strain QM9414 although the nature of the mutagenesis t hat resulted in their strain was not specified. As stated above, QM9414 is a r adiation mutant of strain QM9123 (Morawetz et al., 1992) which is a radiation mutant of T. reesei QM6a (Mandels et al., 1971). In her paper on mycotoxin testing in f ood grade enzyme preparat ions produced by A. niger, A. oryzae, and T. reesei, Blumenthal (2004) questioned the identity of the strain used by Watts et al. (1988). She suggested that it was more likely a strain of T.

harzianum (based upon a personal communication). However, Watts et al. (1988) specifically stated t hat they obtained the T. reesei strain QM9414 directly from the Army13 Laboratories in Natick, Massachusetts where the QM9414 strain was or iginally created. Thus, it would appear that there should be no question as to the proper identity of the

strain as T. reesei . Rather than the strain used by Wa tts et al. (1988) being incorrectly identified, it may be possible that t he unspecified mutagenesis that QM9414 was subjected to produce the mut ant strain P-12 resulted in trichodermin production even though trichodermin is not typically produced by T. reesei . There are no other reports in the literature suggesting that T. reesei is capable of producing trichodermin. On the contrary, more recent papers have demonstrat ed that mycotoxin production is restricted to only certain species of the Trichoderma genus that are not closely related to T.

reesei

.

In a review of peptaibiotics and myco toxins from species in the genera

Trichoderma and Hypocrea, Degenkolb et al. (2008b) stated that T. harzianum and T.

viride , two biocontrol strains that are known to produce trichothecene toxins, have been re-identified as the closely related species T. brevicompactum and T. arundinaceum. Both of these species belong to the T. brevicompactum complex, a complex with members that have been proven to produce trichothecene-like mycotoxins such as trichodermin and harzianum A (Degenkolb et al ., 2008a). Degenkolb et al. (2008b) specifically stated “only T. brevicompactum and three closely related species within the T. brevicompactum complex have been proven to produce trichothecenes-type mycotoxins (trichodermin, harzianum A)”. They further stated t hat two studies have

shown that trichothecenes were not produced by any species outside the T. brevicompactum complex. These more current papers suggest that T. reesei is not capable of producing the m ycotoxin trichodermin.

According to Nevalanein et al . (1994) industrial strains of T. reesei are routinely checked by enzyme producers to confirm the absence of antibiotic activity and toxins including aflatoxin B, ochratoxin A, st erigmatocystin, T-2 toxin, and zearalenone according to the recommendations of the Joint FAO/WHO Expert Committee on Food Additives (JEFCA, 1981). Given that T. reesei has a long history of safe use in the production of food enzymes wher e there is a need to routinel y check for the absence of toxins and harmful metabolites, T. reesei strains utilized for production of enzymes used in industrial applications likely also do not produce these compounds under the growth conditions used in submerged standard i ndustrial enzyme fermentation.

3. Production of Secondary Metabolites by T. reesei

Secondary metabolites, like mycotoxi ns, may also provide the fungus with competitive mechanisms against other microor ganisms. Martinez et al. (2008) stated

that T. reesei has numerous genes that code for bi osynthetic pathways of secondary metabolites, and the authors spec ulated that these may help it survive in its natural soil

habitat. T. reesei has been shown to produce a se condary metabolite known as paracelsin which is a compound known as a peptaibol (Brückner and Graf, 1983). Peptaibols are small (up to 20 amino acid s) linear peptides of 1000-2000 Daltons14 characterized by a high content of the non-proteinogenic amino acid α -aminoisobutyric acid (Aib), with an N-terminus that is typica lly acetylated, and a C-terminus that is linked to an amino alcohol which is usually pheny lalaninol, or sometime s valinol, leucinol, isoleucinol, or tryptophanol (Szekeres et al., 2005). The term peptaibol was formed

from the names of its three characteri stic components, pept ide, Aib , and amino alcohol

. Peptaibiotics (peptide antibiotics) are sim ilar secondary metabolites but the N-terminus is rarely acetylated (Szekeres et al., 2005) . Peptaibols are associated with a wide variety of biological activities as they modify membranes and cause pore formation, resulting in leakage from t he cell. They can have antifungal, antibacterial, sometimes antiviral, antiparasitic, and neurotoxic ac tivity, and many fungal strains producing peptaibols are used as biological contro l agents against plant pathogens (Szekeres et al., 2005). Part of their role as biocontro l agents is that they induce plant defense mechanisms. They are also being invest igated as therapeutic agents because of their anti-cancer activity (Mukherjee et al., 2010).

It is known that peptaibols are synt hesized by large non-ribosomal peptide synthetases by a gene referred to as Tex1 , and two other genes that code for different size peptaibols bearing high homology to Tex1, referred to as Tex2 and Tex3 (Kubicek et al., 2007; Mukherjee et al., 2010). However, peptaibol formation by Trichoderma is still apparently poorly understood. According to K ubicek et al. (2007) in a review article on peptaibols in Trichoderma , there are numerous data gaps regarding how peptaibols are actually produced. Kubicek et al. (2007) point out that in most studies investigating peptaibol formation, surface cultures were used, but with the same fungus under submerged conditions, peptaibols were not form ed. There were a few exceptions to this, however, as peptaibol production in submerged culture was observed when an insoluble component, for instance, a carbon source such as cellulose, was added to the culture medium (Brewer et al., 1987). T. viride was shown to produce the peptaibol alamethicin in culture when the growth m edium was supplemented with one of several insoluble components such as dextrin, pharmam edia, or fish meal. Yields of the peptaibol were greatly reduced in the absence of these solid s (Brewer et al., 1987). These authors reported that a number of in soluble polysaccharides added to the culture medium increased the production of the peptaibol alamethicin, and that methylcellulose resulted in the largest increase. T he production of the co mpound paracelsin by T.

reesei as originally discovered by Brückner and Graf (1983) was accomplished by the solid phase fermentation of the fungus on bagasse and other cellulose-containing substrates. Schirmböck et al. (1994) also got peptaibol product ion with addition of a solid component to a T. harzianum culture. However, in this study, the solid surface was cells walls of the fungus Botyrtis cinerea. Further investigation revealed it was the presence of the cell wall materi al from the other fungus t hat triggered both peptaibol production and production of hydrolytic enzym es including chitinase, glucanase, and protease to degrade the fungal cell walls. Song et al. (2007) used solid-state fermentation (SSF) to optimiz e production of the peptaibols known as trichokonins from T. koningii.15 Kubicek et al.’s (2007) summary of t he literature also re ported that peptaibol production in Trichoderma occurred in older cultures, usually more than 15 days old. They speculated that with older cultures spor ulation was likely occurring. However, they

stated that there was no definitive link between conidiat ion and peptaibol production. Later that year, researchers in Kubi cek’s laboratory published a paper that

demonstrated that the formation of the peptaibol atroviridin by Hypocrea atroviridis was conidiation-associated, and was regulated by both blue light and by a protein known as GNA3 (Komo ń -Zelazowska et al., 2007b). It was s hown that light tr iggered peptaibol formation, but it was only because the light tr iggered conidiation. No atroviridin was produced during vegetative gr owth of the fungus.

Stress conditions have also been shown to result in peptaibol production by

Trichoderma. An earlier study by Vizcaino et al. (2006) showed that the T. harzianum peptaibol synthetase transcrip t could only be detected under nitrogen starvation. To date more than 900 peptaibol sequenc es have been published (http://www. cryst.bbk.ac.uk/ peptaibol/sequence. htm). However, Mukherjee et al. (2010) stated that despite the knowledge of the sequences, t he genetics of peptaibol formation are not

well-understood.

Peptaibols have been shown to be produced in very high amounts in cultures of

Trichoderma strains in some studies. Oo ka et al. (1966) showed that T. viride produced a peptaibol in the extremely high concentration of 20 g/L ov er the course of 70 - 90 hours (< 3 - < 4 days) with shak e-flask culture of a broth supplemented with 4% soluble starch and 1% Polypepton at 30°C. Meyer and Reusser (1967) used submerged culture supplemented with mola sses, dextrin, fish meal, and Pharmamedia to get production of 8 - 10 mg/L of a peptaibol from a strain of T. viride grown at 25°C for 4 - 5 days. Song et al. (2007) obtained the ma ximum production of tr ichokonin VI of 4.07 mg/g dry substrate under optimized condition s in solid-state fermentation.

Several different paracelsins have been discovered since the term was first used by Brückner and Graf (1983) for t he toxic secondary metabolite produced by T.

reesei . Paracelsins have 20 amino acids (Brückner and Graf, 1983), and are characterized by the presence of phenylalani nol as the C-terminal amino alcohol, and by the specific amino acid content and sequence (Ritieni et al., 1995). The structures of paracelsin A, B, C, and D (Brückner et al ., 1984), which were isolated from both the fungal mycelium and the culture broth of T. reesei QM9414, were elucidated by Fast Atom Bombardment Mass Spectro scopy (fabms) by Przybylski et al. (1984). These four paracelsins, A, B, C, and D, were i ndividual components in a microheterogeneous paracelsin mixture. Przybylski et al. (1984) stated that a char acteristic of peptaibols is their microheterogeneity which is a resu lt of their non-ribosomal biosysthesis

mechanism. Paracelsin E was later isolated from T. saturnisporum which also produced paracelsin A, B, C, and D (Ritieni et al., 1995).

Brückner and Graf (1983) demonstrated that parac elsin extracted from T. reesei16 QM9414 had antibacterial action against Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Streptococcus faecalis, Streptococcus lactis, and Streptococcus thermophilus. It was not inhibitory to Candida albicans, Escherichia coli, Porteus vulgaris, Serratia marcescens , Salmonella typhimurium, Pseudomonas aeruginosa, or Enterobacter aerogenes. Paracelsin was shown to have membranemodifying properties and antibiotic activity, particularly against Gram-positive bacteria.

In a study on peptaibols produced by 12 different species of Trichoderma , most of which had potential as bi ocontrol agents, a strain of T. reesei was shown to produce paracelsin (Solfrizzo et al., 1994). Pa racelsin production was common among the

Trichoderma species studied as it was also observed in T. citrinoviride, in one of two

strains of T. hamatum, in T. inhamatum, T. koningii, T. longibrachiatum, T. parceramosum, T. polysporum, and T. pseudokoningii, in both strains of T. saturnisporum, and in T. viride . The only species in which paracelsin production was

not observed was in the two strains of T. harzianum.

Paracelsin has been shown to have toxici ty toward mammalian cells. Brückner et al. (1984) reported that par acelsin exhibited hemolytic ac tivity on human erythrocytes. They stated that erythrocyte hemolysis is a common characte ristic of other peptaibols such as alamethicin, suzukacillin, and trichotoxin. The in vitro hemolytic activity of paracelsin was reported as C 50 = 3.7 x 10 -5 mol/liter (where C 50 is the concentration that causes 50% hemolysis). Paracelsin exhibite d lesser hemolytic activity than trichotoxin (C 50 = 2.1 x 10 -5 mol/liter) and alamethicin F-50 (C 50 = 1.6 x 10 -5 mol/liter). Brückner et al. (1984), when discussing the hemolytic activity of parac elsin, also stated that “ in vivo hemolysis is also suspected to be the reason why the lethal dose is about 5 mg/kg mice when paracelsin is administered intraperitoneally ”. However, the authors do not present any data for determination of this lethal dose, nor do they cite a reference for this study. Therefore, it is unknown whether this val ue is from unpublished dat a from the authors or

whether these are data from anot her study that cannot be loca ted in the literature.

Abu Raya et al. (1993) repor ted that paracelsin showed to xicity to PC12 cells (a cell line derived from a pheochromocytoma of the rat adrenal medulla) with a CC 50 (cytotoxicity concentration of 50%) of 21.8 μM. Grigoriev et al . (2003) showed that paracelsin A was toxic to mice at an intr aperitoneal dosage of 20 mg/kg body weight. In this study, all of the highly membrane acti ve peptaibols which included alamethicin F30, paracelsin A, chrysospermin C, and the lipopeptaibol texenomycin A were toxic to mice. Long lasting hypothermia in mice, whic h was an indicator of neuroleptic activity (affecting the brain, especially by reduci ng the intensity of ner ve function, e.g., tranquilizing) was not observed in this st udy because the mice died shortly after administration of the compounds (Grigoriev et al., 2003).

The toxicity of paracelsin reported in the two studies above seems quite high, particularly in comparison to the reported to xicity of trichodermin, an actual mycotoxin with high toxicity to both animals and pl ants (Wei et al., 1974). Blumenthal (2004)17 stated that trichodermin had an LD 50 of 500 mg/kg of body weig ht with intraperitoneal or

subcutaneous administration in mice accordi ng to the Registry of Toxic Effects of Chemical Substances. This Registry has other toxicity values for trichodermin in addition to this one reported value. The LD 50 ‘s of trichodermin were 500 and 354 mg/kg in mice with intraperitoneal injection, and 500 mg/kg in mice with subcutaneous injection. LD 50 values were approximately two or ders of magnitude lower with oral administration of trichodermin. The Registry of Toxic Effects of Chemical Substances reported an LD 50 of 6.2 mg/kg with oral administrat ion of trichodermin to mice, and an

LD 50 of 7.5 mg/kg with oral administr ation of trichodermin to rats.

In addition to paracelsin, other secondary metabolites have been reported from

T. reesei . Trichodermatides, which are polyke tide compounds that exhibit weak cytotoxicity against the A 375-S2 melanoma cell line, were reported by Sun et al. (2006) and Sun et al. (2008) as bei ng from the marine fungus T. reesei . As stated previously, these two papers do not specify how the particular strain of Trichoderma was identified.

T. reesei is not known as a marine fungus, so it is unknown whether the fungus studied in these two papers was actually T. reesei. There apparently ar e, however, marine

strains of T. longibrachiatum (Mohamed-Benkada et al., 2006; Ruiz et al., 2007) and T. koningii (Landreau et al., 2002), the former of which was isolated from blue mussel and the latter from a shellfish (cockle) farm.

4. Test Data on Toxin Production

A number of different studies have been conducted assessing the potential for T.

reesei to produce toxins during submerged ferm entation for production of enzymes for food, pharmaceutical, or industrial uses. In addition to the pathogeni city testing of the T.

reesei strain used to produce the enzyme Cellu last as discussed above, the enzyme product itself has also been tested for toxicity (H jortkjaer et al., 1986). The test material,

referred to as the “tox- batch” was tested for general oral toxicity, effects on reproduction, mutagenic potential, inhalation toxi city, irritation to skin and to eyes, and

for skin sensitization. Acute oral toxici ty studies were conducted in mice, rats, and dogs. No toxicity was observed in any species and the acute oral toxicity was > 8 g/kg in rats, >16 g/kg in mice, and >5 g/kg in dogs (the highest dose tested in each species). Subchronic toxicity studies, which consisted of feeding the tox batch for 91 days at 0, 1, 2, or 5%, were conducted in rats and dogs. There was no evidence of systemic effects in dogs up to 3.0 g/kg of bodyweight (bw) per day, the highest dose tested. In the 91- day subchronic toxicity study in rats, the hi ghest dietary level of the Celluclast enzyme product (tox-batch) of 5% depre ssed body weights in male rats but not female rats.

Rats tolerated the tox-batch at the 1% or 2% dietary level (~ 1000 mg/kg/bw).

Additional toxicity studies have been conducted on other enzymes produced by

T. reesei. In GRAS petition No. 315, a 91-day subc hronic feeding study with a cellulase was conducted in CD rats to assess the potent ial toxicity of the enzyme product. No mortalities were recorded throughout the entire investigation. There were no treatment-18 related adverse effects on any of the param eters measured (systemic toxicity, food consumption, opthalmology, clinical chem istry, body weight, organ weight, urine analysis, hematology, necropsy, and histop athology). It was concluded that the

cellulase from T. reesei did not result in toxicity up to and including a dose level of 5%

w/w in the diet. A No Observable Effect Level (NOEL) was established at 5% in the diet corresponding to 3.35 and 4.05 g cellulase/kg bw/day for male and female rats, respectively (GRN No. 315).

Data from a number of ot her studies are also pres ented in this GRAS petition (GRN No. 315) for different enzymes from the same T. reesei strain including a high pI xylanase, a low pI xylanase, endoglucan ase III, endoglucanase I, and protease. A 91- day subchronic feeding study in CD rats at concentrations up to 50,000 mg/kg of a high pI xylanase reported two deaths, but they were not attributed to the treatment. No other

treatment-related adverse effects were obser ved on any of the parameters measured (organ weight, urine analysis, hematology, nec ropsy, and histopathology) with the high pI xylanase indicating a lack of toxicity of the enzyme. Likewise, a 91-day subchronic feeding study was conducted with a low pI xylanase produced by T. reesei with feedingconcentrations up to 3000 mg/kg/day. There were no deaths and no adverse treatmentrelated effects on any of the same paramet ers measured as listed previously. The NOEL was established at 3000 mg/k g for the low pI xylanase.

Also presented in GRAS Notice 315 is dat a from a 28-day oral toxicity study in

rats for a T. reesei endoglucanse III administered at concentrations up to 1000 mg/kg/day for four weeks. No mortalitie s were observed in the Charles River rats during the study. There we re no treatment-related adver se effects either, which established a NOEL of 1000 mg/kg/day (GRN No. 315). For another T. reesei enzyme, endoglucanase I, there was a 14-day oral feeding study and a 91-day subchronic feeding study conducted. There were no ad verse effects reported for any parameter monitored in the 14-day oral study in Charles River CrlCD ® BR rats dosed with up to 1000 mg/kg/day by oral gavage using a dosage volume of 10 ml/kg bw. Likewise, there was no toxicity observed in the 91-day subchronic feeding test. There were no mortalities, and no adverse treatment-relat ed effects were observed on any of the parameters measured (organ weight, urine analysis, hematology, necropsy, and histopathology). At study termination, there were no differences in the behavioral test (open field) and functional tests (gripping refl ex, startle reflex) be tween the control and treatment groups. A NOEL was establishe d for this enzyme of 1000 mg/kg/day.

A protease produced by T. reesei was also tested for toxicity with a 91-day subchronic feeding test (GRN No. 315). Ther e were no treatment-related deaths or

adverse effects observed. A NOEL of 31.25 mg total protei n/kg bw/day was established (the highest dosage studied).

Data from an acute to xicity test for another T. reesei enzyme

, glucoamylase, are also presented in GRAS petition 315. Under t he conditions of this study where the rats19 received a single oral dose followed by a 14-day period of obser vation, the oral LD 50 was > 1996 mg total protein/kg bw which was the highest possible dose volume of 10 ml/kg. This same enzyme was also tested in a 91-day subchronic feeding test in SD rats. There were no overt signs of systemic toxicity or adverse clinical observations attributable to treatment (GRN No. 315).

5. Allergic Reactions/Sensitization to T. reesei and its Enzymes

Although fungi in general can be allergen s, there are no reports in the literature specifically suggesting that T. reesei causes allergenicity in humans but there are reports for other Trichoderma species. There are two repo rted cases of hypersensitivity pneumonitis, also known as extrinsi c allergic alveolitis, caused by T. koningii and by T.

viride (Halpin et al., 1994; Enrique z-Matas et al., 2009). In addi tion, there is one report

of T. longibrachiatum causing allergic fungal sinusit is (Tang et al., 2003).

Enzymes themselves are known allergens. However, there are no allergenicity issues specifically attri butable to enzymes produced by T. reesei vs. enzymes produced by other fungi.

6. Human Health Hazard Conclusions

There is only one potential concern fo r human health hazards associated with T.

reesei and that is paracelsin prod uction. There is no concer n for potential pathogenicity

of T. reesei. Test data on various industrial strains typically do not show adverse effects, except in one case where pathogeni city to mice was demonstrated, but only

under the conditions where the mice were intentionally imm unosuppressed and then subjected to high inoculation rates

. Toxicity testing on a number of enzymes produced by T. reesei indicates that the fungus does not pr oduce toxins under the conditions used in submerged standard industr ial fermentation for enzym e production. There is, however, concern for the production of the secondary metabolite paracelsin if T. reesei was to be grown under certain other condition s. Paracelsin has been shown to be capable of hemolysis of human er thyrocytes, and has been reported to be toxic to mice at a concentration of 20 mg/kg (Grigoriev et al., 2003) which is a lower amount than that of an actual mycotoxin known as trichodermin that is produced by some Trichoderma

spp.

Paracelsin production is not expect ed to occur under the conditions of submerged standard industria l fermentation for enzyme production. However, according to information available in t he literature, extended fermentations, or

fermentations that contain insoluble carbon sources such as cellulose may result in paracelsin production. Ther efore, the potential for par acelsin production during fermentation of plant material in a cellu losic biofuel facility, and the potential environmental introduction of paracel sin resulting from the use of T. reesei in cellulosic material degradation warrants fu rther investigation. Even before interest in using T.20 reesei for biofuels, Brückner et al. (1984) ca me to a similar conclusion years ago with

other uses of Trichoderma, and it was Brückner and Graf (1983) that first isolated paracelsin. Brückner et al. (1984) stated “With regar d to the most intensive efforts for a biotechnological application of Trichoderma – both in submerged and solid-substrate fermentation – for the enzymat ic conversion of cellulosic materials used for animal feed or human food and, moreover, the contribution of these microfungi and their relatives to the moldiness, spoilage and deterioration of organic matter, the excretion of paracelsin and related metabolites should be seriously taken into account. These mycotoxins exhibit a great variety of biological activity and are sometimes produced in extraordinarily high amounts”.

B. Environmental Hazards

1. Hazards to Animals

T. reesei is not pathogenic to animals, and there are no reports in the literature suggesting it can cause infection or disease in animals. In a summ ary of the literature Klein and Eveleigh (1998) stated that some of the secondar y metabolites produced by

some Trichoderma spp. such as glioviridin (a di ketopiperazine), sesquiterpenoids, trichothecenes, cyclic peptides, and isocyanide- containing metabolites (trichoviridin) may be inhibitory to organisms other than microorganisms. Some of these compounds may result in ill-thrift of sheep due to thei r inhibitory action on cellulolytic rumen microflora (Brewer et al., 1982). It is know n that paracelsin, the peptaibol produced by

T. reesei , is inhibitory to Gram-p ositive bacteria such as Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus faecalis, Streptococcus lactis, and Streptococcus thermophilus (Brückner and Graf, 1983). Brü ckner et al. (1984), in a discussion of the potential effects of paracel sin, stated “the increas e of the digestion of

starch and cellulose in rumen liquor by the addition of low concentrations of paracelsin

should be considered in the discussion of the association of metabolites from

Trichoderma with the ill-thrift of past ure fed ruminants, and may be explained by the suppression of various feed rivals of the cellulolytic activ(e) microorganisms”.

Paracelsin has been shown to exhibit toxi city to mammalian ce lls. Data from mice and rat studies were previously presen ted in the human health effects section. The toxicity of paracelsin to aquatic species has also been directly tested. Solfrizzo et al. (1994) studied the toxicity of paracelsi n and other compounds extracted from six

Trichoderma species to Artemia salina (brine shrimp) larvae. Paracelsin was shown to

have an LC 50 (concentration that is lethal to 50% of the or ganisms) of 2.2 μ M which was equivalent to approximately 4 μg/ml (Solfrizzo et al., 1994). The toxicity of the extracts

of five of the six Trichoderma strains to A. salina could be attributed to the production of paracelsin by these strains, whereas in T. harzianum, toxicity was attributed to an unidentified peptaibol(s).

Favilla et al. (2006) tested the toxicity of various secondary metabolites of21 biocontrol fungi to both Artemia salina and Daphnia magna (water flea). The three peptaibols tested included alam ethicin, paracelsin, and anti amoebin, the first two of which are produced by some Trichoderma species. The paracelsin used in these studies was a mixture of paracelsin homologs (purity 88.4%) purchased from a chemical supply company. With 24 hr. exposure to A. salina , the LC 50 of paracelsin was 21.26 μM (equivalent to 40.84 μg/ml). The LC 50 decreased with 36 hr. exposure to 9.66 μM (equivalent to 18.56 μg/ml). One of the most toxic fungal compounds was alamethicin, which is produced by some Trichoderma species including all the members of the Trichoderma brevicompactum clade that previously included strains of T. viride (Degenkolb et al., 2008a). For comparison, the LC 50 of alamethicin was 5.32 μM (10.43 μg/ml) and 1.96 μM (3.84 μg/ml) for 24 and 36 hr . exposure, respectively, so it is approximately four to five times more toxic to brine shrimp than paracelsin. However, paracelsin was about twice as toxic to brine shrimp as gliotoxin, an actual mycotoxin produced by several fungi such as Aspergillus, Penicillium, and Trichoderma. In experiments with D. magna, the LC 50 of paracelsin was 7.70 μM (equivalent to 14.79 μg/ml) with a 24 hr. exposure, and 5.60 μM ( equivalent to 10.76 μg/ml) with a 36 hr. exposure (Favilla et al., 2006). These LC 50 values for D. magna are considered moderately toxic. Favilla et al . (2006) stated that the biological activity of peptaibols is a result of their ability to form pores in lip id membranes by forming gated ion channels. The pores lead to the loss of osmo tic balance from leakage.

Poirier et al. (2007a) reported that peptaibols and peptaibol-producing

Trichoderma were detected in marine sediments that exhibited a high toxicity to mussel larvae from a shellfish farming area in Franc e. Peptaibols were also detected in the marine bivalves. Poirier et al. (2 007b) conducted bioassays with oyster ( Crassostrea gigas ) embryos with alamethicin and different groups of peptaibols that were produced by a strain of T. longibrachiatum that was isolated from the mussels. They found that peptaibols of both long and short sequence disturbed embryogenesi s of oysters at nanomolar concentrations. Toxicity of the peptaibols was between 1.3 and 8 times greater than the toxicity induced by copper which was used for reference, and the longer peptaibols were found to be more toxi c than the shorter ones. The toxicity was suggested to be associated with the memb rane-modifying properties of peptaibols wherein they interact with biological me mbranes and modify their permeability by forming voltage-dependent membrane ion channel s through which cytoplasmic contents can leak out (Poirier et al., 2007b).

In an earlier study, Sallenave et al . (1999) examined the experimental bioaccumulation in filter-feeding mollusks of fungal “mycotoxins”, which were later referred to as peptaibols rather than mycotoxi ns by Poirier et al. (2007b). Sallenave et al. (1999) exposed mussels to culture filtrates of Trichoderma koningii that was previously isolated from cockles ( Cerastiderma edule ). The toxic metabolites bioaccumulated in the mussels, and the digest ive gland of the mussels had the greatest toxicity indicating that contam ination was from filtration. There are no reports in the literature of bioaccumulation of peptaibols by aquatic species in the environment.22 However, it was not until 2007 when peptaibo ls were actually detected in the

environment in marine sediments and in shellfis h (Poirier et al., 2 007a).

In a review of peptaibioti cs and mycotoxins in the genera Trichoderma and Hypocrea, Degenkolb et al. (2008b) commented that the high toxicity reported for the peptaibols by Poirier et al. (2007b) (alameth icin in oysters) and Favilla et al. (2006) (alamethicin, paracelsin, and others in A. salina and D. magna ) in aquatic invertebrate studies may be a result of contamination of the alamethicin used in these experiments. Degenkolb et al. (2008b) suggest ed that the alamethicin st andard used in these studies (Sigma-Alrich A-4665) may have been contam inated with the tric hothecene mycotoxin

harzianum A which was shown to be the case in some of their own studies. Harzianum A has been shown to be highly toxic to A. salina . However, this pos sible explanation for the toxicity of alamethicin in these two studies has no relevance to the toxicity values of paracelsin to brine shrimp or D. magna presented by Favilla et al. (2006), nor on the toxicity values of paracelsin to brine shrimp reported by Solfrizzo et al. (1994).

Landreau et al. (2002) speculat ed that the neurot oxicity observed with peptaibols

from T. koningii was related to the amphiphilic nature of the molecules that form pores in phospholipid membranes. In addition to pore formation, peptaibols have been shown to uncouple oxidative phosphorylat ion of mitochondria, stimulat e catechol secretions of adrenal cells, activate membrane-bound enzymes, and impart cytotoxicity, neurotoxicity, and neuroleptic effects (as summarized in Poir ier et al., 2007b; Szekeres et al., 2005).

2. Hazards to Plants

Trichoderma reesei is not a pathogen of plants. Although it is capable of degrading cellulose and hemicellulose due to the copious quantities of these enzymes it can produce, it is not a primary colonizer on plant tissue. Genetic studies have shown that it does not contain any genes for lignina ses that are required for initial breakdown of plant material (Maheshwari, 2008). T. reesei is known as a wood rot fungus, but it apparently attacks decaying plant material, not live plants. This species does not appear to pose a hazard to plants.

3. Effects on Other Organisms

As previously mentioned, peptaibols, incl uding paracelsin, ar e toxic to Grampositive bacteria and to some fungi. Peptai bols are apparently not inhibitory to Gramnegative bacteria, and it assumed t hat this is a result of the differences in the cell walls of Gram-positive and Gram-negative bacteria. Paracelsin has also been shown to be inhibitory to the fungus Phoma destructiva (Grigoriev et al., 2003). They measured differences in membrane pore formation by a number of peptaibols produced by a number of different fungi including paracelsin isolated from an unspecified Trichoderma sp. The 19-20mer peptaibols such as alamethicin F-30, paracelsin A, and chrysospermin C all had very high membrane activity, and the larger peptaibols23 exhibited higher activity by several or ders of magnitude com pared with the smaller compounds, the 11-17mer peptaibols. Watts et al. (1988) reported th at two metabolites produced by T. reesei, trichodermin, and another compound designated as Tx, were inhibitory to species of Aspergillus, Cladosporium, Fusarium, Rhizoctonia, and Pythium

. However, as mentioned previously, the identity of that fungus as T. reesei has been questioned. Other studies have invest igated the biocont rol potential of T. reesei. Harjono and Widyastuti (2001) found that hi gh concentrations of an endochitinase produced by T. reesei lysed the hyphae of the plant pathogenic fungus Ganoderma philipii

. Seidl et al. (2004) reported that the teleomorph of T. reesei, H. jecorina, was antagonistic to the plant pathogen Pythium ultimatum in plate confront ation tests. Apparently T. reesei can be antagonistic to other fungi , but it has not been developed commercially as a biocontrol agent because its ant ifungal activity is inferior to that of other strains of the genus.

As previously mentioned, some species of Trichoderma, specifically T. aggressivum, T. pleuotrophilum, and T. fulvidum are pathogens of mushrooms, which of course, are fungi. However, T. reesei is not a pathogen of mushrooms.

4. Stability of Peptaibols in the Environment

There is no information in the literature on the stability of the paracelsin molecule

in the environment. There is li ttle literature on the stability of other peptaibols either, but it is known that peptaibols form helical stru ctures in phospholipid membranes. In a paper on the structure-functi onal relationship of model α -aminoisobutyric acid peptides, Higashimoto et al. (1999) st ated that “ the presence of α -aminoisobutyric acid, because of its preference for certain di hedral angles most suitable for 3 10 – or α -helical structures, gives a particular st ability to their conformations in membranes in a variety of

environments”.

Poirier et al. (2007a) were the first to detect the presence of peptaibols produced by Trichoderma in the environment, in marine sediment s. Their isolation of peptaibols

from the bivalve mussels cultivated in those s ediments led them to st ate that theirs was

“the first observation of contamination of the marine human-food chain by fungal

metabolites”. The fact that they could still detect peptaibo ls in sediments samples that had been frozen for years led Poirier et al. (2 007a) to suggest that peptaibols could be

considered as stable markers for fungal presence in the mari ne environment.

Song et al. (2006) described the stability of secondary metabolites produced by T. koningii called trichokonins, which they referr ed to as peptaibol-like antimicrobial compounds. These trichokonins were refe rred to as peptaibols, rather than just peptaibol-like compounds, in a review article by Samuels et al. (2006). They are also considered to be peptaibols by others as they are included in the peptaibol database (http://www.cryst.bbk.ac.uk/pept aibol/home.shtml). Trichokoni ns also contains the nonproteinogenic α -aminoisobutyric acid as do peptaibols. Song et al. (2006) reported that24 trichokonins were inhibitory to Gram-pos itive bacteria and a number of plant pathogenic fungi. To evaluate their stability to tem perature, a solution of the antimicrobial compounds dissolved in buffer was heated for 30 minutes at 50, 70, 90, or 100  C, and was also subjected to autoclaving. In addition, the solution was stored at 4  C for 10 days, at -20  C for 30 days, and the frozen solu tion was then lyophilized. The trichokonins were found to be extremely heat resistant, with no loss of activity at any temperature tested, in cluding autoclaving at 121  C and 15 psi for 15 minutes. Cold storage and lyophilization did not affect their acti vity either. Their bi ological activity was retained over a pH range of 3.0 to 10.0 as well. The sensitivity of trichokonins to proteolytic enzymes such as trypsin, neut ral protease, basic pr otease, and multiplex protease was also tested by Song et al. ( 2006). The trichokonins were insensitive to treatment with proteolyt ic enzymes, and the majority of t heir antimicrobial activity was

retained.

5. Environmental Hazard Conclusions

The issues for environmental hazards ar e similar to those for human health hazards. The primary hazard concern for T. reesei is for the production of paracelsin, the moderately toxic secondary metabolite. Under usual conditions of submerged culture for standard enzyme pr oduction, well-established co mmercial strains of this species do not seem to produc e paracelsin. However, fungal secondary metabolites can be produced after extended fermentation, par ticularly when sporulation occurs after depletion of nutrients. It has also been demonstrated that these peptaibol compounds may be produced in the presence of an inso luble carbon source such cellulose. Therefore, its growth on plant material as proposed for on-site production of enzymes in cellulosic biofuel facilities by NREL (Wooley et al., 2009; Kazi et al., 2010) may potentially result in the production of si gnificant quantities of paracelsin in the

fermentation broth. Large quantities of paracelsin could be possible with extended

fermentations and/or the absenc e of exponential growth (i.e ., the stationary growth phase) in the presence of a surplus of carb on with a shortage of nitrogen, conditions

that are known to stimulate the formati on of secondary metabo lites by fungi.

There are two issues warranti ng consideration with the use of T. reesei cells in cellulosic biofuel plants. One is the potential for cells of T. reesei to escape containment, and T. reesei is expected to survive in t he environment if inadvertently released. However, the releases expected from closed system use are expected to be

low. It is unknown whether T. reesei would produce any quantity of paracelsin in the environment that would be of any concern. However, there needs to be a consideration of the concentrations of paracelsin that could be released in wastewater from a cellulosic biofuel facility. It is unknown whet her paracelsin, if it was formed, would be denatured, with a resulting loss of toxicity, at some point in the process stream, such as with high temperatures used for ethanol distill ation. It is also not known whether paracelsin would be rapidly degr aded during wastewater treatm ent, or whether it could25 possibly remain intact in the stable α -helical form known to occur in membranes. There

are no data in the literature on the degradation of peptaibols in the environment, except for the one paper of Poirier et al. (2007a) that repor ted the persistence of peptaibols in marine sediments and stability of the compounds for years w hen in frozen sediments. Extraordinary stability was observed for the peptaibols known as trichokonins, as extremes in temperature, in cluding autoclaving, and freezing followed by lyophilization, did not denature the compounds. The co mpounds also functioned over a wide pH range. In addition, they also were fairly resistant to attack by trypsin and proteases as judged by the retention of their antimicrobial ac tivity. It is unknown if paracelsin would be similarly resistant to temperature extrem es, pH levels, or to enzymatic degradation as are the trichokonin peptaibols

.

IV. EXPOSURE ASSESSMENT

Estimates of occupational exposure and re leases of the microorganism from an enzyme fermentation facility with the resultin g exposures to the environment and to the general human population are given below. Note that these numbers have been revised since the worker exposure and envir onmental release and exposure estimates used in the risk assessments of the original t en eligible recipient microorganisms listed at §725.420 in the Final Microbial Biotechnology Rule promul gated in 1997. Information gleaned from Microbial Commercial Activity No tices received over the years since the rule was published has allowed for better estimates of exposures using a generic scenario (Chemical Engineering Branch, 2011). For this generic scenario, it is assumed that a large-scale closed system enzyme fermentation pl ant operates 24 hours per day for 350 days/yr and that t here are 100 batches/yr.

A. Worker Exposure

No data were available for assessing the releases and exposures specifically for enzyme fermentation facilities using T. reesei. Therefore, the potential worker exposures and routine releases to the env ironment from large- scale, conventional fermentation processes were estimated usi ng information available from a number of

submissions to EPA under TSCA Section 5 and from published information collected from non-engineered microorgani sms. These values are based on reasonable worstcase scenarios and typical ranges or va lues are given for comparison.

During fermentation processes, worker exposure is possible during laboratory pipetting, inoculation, sampling, harvesting, extraction, processing, and decontamination procedures. A typical fermentat ion facility has a total of 36 workers with four workers per shift involving fermentation, four worker s per shift for recove ry operations, three workers per shift for laboratory procedures, one supervisory worker, all with three shifts per day.26 The National Institute for Occupat ional Safety and Health (NIOSH) has conducted walk-through surveys of several ferm entation facilities in the enzyme industry

and monitored for microbial air contamination. These particular facilities were not using recombinant microorganisms, but the processes were considered typical of fermentation process technology. Typically, area moni toring data would not be used to estimate occupational exposure levels since the co rrelation between area concentrations and worker exposure is highly uncertain. Howe ver, personal sampling da ta are not available at the present time. Thus, area sampli ng data have been the only means of assessing exposures for previous microbial biotechno logy submissions. Applying these NIOSH data to the workers in the various plant areas (laboratory, fermentation, recovery, control room) yields a range of potential inhalation exposures of 81 to 9800 cfu/day. The uncertainty associated with this esti mated range of exposures is not known.

The potential dermal exposure for workers in the various plant areas ranges from

6.0 x 10 6 to 1.8 x 10 7 cfu/day. However, the typica l personal protection equipment (PPE) used in these types of facilities (i.e ., gloves and lab coats) is expected to be adequate to protect against dermal exposure to the microorganism. In the unlikely event that dermal exposure was to occur, un broken skin is considered to be a protective barrier to entry of the microorganism into t he body. Even in the event of dermal contact with broken skin, there is little risk of infection because this microorganism is not pathogenic to humans.

B. Releases and Environmental and General Population Exposure

Estimates of the number of T. reesei cells released during typical enzyme production are tabulated in Table 1. T he uncontrolled/untreated scenario assumes no

control features for the fe rmentor off-gases, and no inacti vation of the fermentation broth for the liquid and solid waste releases. The containment criteria required for the full exemption scenario assume the use of features or equipment that minimizes the

number of viable cells in the fermentor off-gases. They also assume inactivation procedures resulting in a validated 6- log reduction of th e number of viable microorganisms in the liquid and solid wastes re lative to the maximum cell density of the

fermentation broth.27 Table 1. Estimated Numbers of Viable T. reesei Cells Released During Production

________________________ _____________________ ________________________ Uncontrolled/ Full Release Media Untreated Exemption Release (c fu/day) (cfu/day) (days/yr)

AIR

Fermentor exhaust 2 x 10 5 < 2 x 10 5 350 Rotary Drum Filter 7 x 10 5 < 7 x 10 5 100

WATER - to WWT Fermentor cleaning 1.4 x 10 13 1.4 x 10 7 100

LAND - to landfill

Filter cakes 7 x 10 14 7 x 10 8 100

________________________ _____________________ _______________________ These are "worst-case" estimates for releases from the facility that assume a maximum cell density in the fermentation broth of 10 7 cfu/ml for fungi, a fe rmentor size of 70,000 liters, and a separation efficiency for the rotary drum filter of 99 percent. For the Full Exemption there is also a release to wast ewater treatment (WWT ) 100 days/yr of 6.9 x

10 5 cfu/day each from treatment of ferm entor and rotary drum exhausts.

1. Releases to Air

Specific data which indi cate the survivability of T. reesei in the atmosphere after release are currently unavailable. Survival of vegetative cells during aerosolization is typically limited due to stresses such as shear forces, desiccation, temperature, and UV light exposure. Human exposure may o ccur via inhalation if the organisms are dispersed in the atmosphere attached to dust particles, or lofted through mechanical or

air disturbance.

Air releases from fermentor off-gas and rotary drum filtrati on could potentially result in nonoccupational inhalation exposu res due to point source releases. To estimate exposures from these sources, the sector averaging form of the Gaussian algorithm described in Turner (1970) was used. For purposes of this assessment, a release height of 3 meters and downward co ntact at a distance of 100 meters were assumed. Assuming that there is no remova l of organisms by controls/equipment for aerosol releases, the potentia l human inhalation dose rates are estimated to be < 1 cfu/yr from both the fe rmentor off-gas and from the rotary drum filtration exhaust, and less than that for systems meeting the full exem ption criteria. It should be noted that28 these estimates represent hypothetical exposures under reasonable worst case

conditions.

2. Releases to Water

The concentrations of T. reesei in surface water were estimated using stream flow values for water bodies receiving pr ocess wastewater discharges from facilities within SIC Code 283 (drugs, medi cinal chemicals, and pharma ceuticals). The surface water release values (cfu/day) tabulated in Table 1 were divided by the stream flow values to yield a surface water concentration of the organism (cfu/l). The stream flow

values for SIC Code 283 were based on disc harger location data retrieved from the Industrial Facilities Dischargers (IFD) database on December 5, 1991, and surface

water flow data retrieved from the RXGAGE database. Flow values were obtained for water bodies receiving wastewater discharges fr om 154 indirect (facilities that send their waste to a POTW) and direct dischargers facilities that have a NPDES permit to discharge to surface water). Tenth percentile values indicate flows for smaller rivers within this distribution of 154 receiving wate r flows and 50th percentile values indicate flows for more average rivers. The flow value expressed as 7Q10 is the lowest flow observed over seven consecutive days durin g a 10-year period. The use of this methodology to estimate concentrations of T. reesei in surface water assumes that all of the discharged organisms survive wastewater treatment and that growth is not enhanced by any component of the treatment process. Estimated concentrations of T.

reesei in surface water for the uncontrolled/ untreated and the full exemption scenarios

are tabulated in Table 2.

TABLE 2. T. reesei Concentrations in Surface Water

________________________ _____________________ ____________________

Receiving

Flow Stream Flow Organisms (MLD*) (cfu/l)

__________________________ ____________________ Mean 7Q10 Mean 7Q10

__________________ __________________ __________________ ___________

Uncontrolled/Untreated

10th Percentile 156 5.60 8.97 x 10 4 2.50 x 10 6

50th Percentile 768 68.13 1.82 x 10 4 2.05 x 10 5

Full Exemption

10th Percentile 156 5.60 8.97 x 10 -2 2.50

50th Percentile 768 68.13 1.82 x 10 -2 2.05 x 10 -1

__________________ __________________ __________________ ___________ *MLD = million liters per day29 C. Release and Exposure Summary

Although direct monitoring data are unav ailable, worst-case estimates do not suggest high levels of exposure of T. reesei to either workers or the public resulting from typical enzyme fe rmentation operations.

D. Survival of the Or ganism in the Environment

The species T. reesei is known only from the singl e original isolate QM6a from

the Solomon Islands in the 1940s . Therefore, there is littl e information on its prevalence

or behavior in the environment. Howeve r, microcosm studies conducted on T. reesei suggests that it would survive in the environ ment. Providenti et al. (2004) studied the
survival of a derivative of QM6a marked with a recombinant hygromycin-B (HygB) resistance gene, designated as QM6a#4, added to laboratory contained intact soil-core microcosms containing three different textur ed soils. In one of those soils, survival was also tested with or without a pl ant rhizosphere (bush lima beans, Phaseolus limensis ).

Soil cores were incubated at 22  ̊ C and were sampled periodi cally over a 4-month period. Then the soil cores were subj ected to a simulated winter treatment (approximately 2 weeks at 4  ̊ C, 3 weeks at -20  ̊ C, and 2 weeks at 4  ̊ C), and then

returned to 22  ̊ C for another 2 weeks. The levels and viability of T. reesei were determined by quantitative polym erase chain reaction (PCR) of total soil DNA extracts and by dilution-plating of soil on a semiselect ive growth medium, respectively. QM6a#4

was added at a rate of 2.0 ml of inocula (consisting of 0.35 g of wet fungal mycelia per ml that had been processed at high speed in a Waring blender) onto the top 1 cm of the soil core. In Soil 1 (sandy loam), QM 6a#4 numbers decreased over the first 2 - 3 months (40- to 170-fold for the soil with or without a plant rhizosphere, respectively), after which a steady state was reached. In Soil 2, (medium texture, soil only), the
decrease in QM6a#4 was observed within the fi rst month of incubation (80-fold) after which a steady state was reached . In Soil 3 (clay loam, soil only), no clear trend was apparent, but there was an approximate 4-fo ld decrease observed by day 114.

After the cores were subj ected to the simulated ov erwinter treatment, QM6a#4 levels did not change in any of the three soils without the plant rhizosphere. In the one soil with the bean rhizosphere (Soil 1), there wa s a statistically significant approximately 3-fold increase in QM6a#4 levels, reflecting a “bloom” associated most likely with an increase in nutrients from the decaying dead plant roots after thawing of the cores. In all three soils, QM6a#4 remained viable and cu lturable, even after the simulated winter

treatment.

From this data given in Providenti et al. (2004) above, it appears that even though T. reesei was originally isolated from a tropica l climatic region, it is able to persist in soils for ex tended periods of time. T. reesei survived in soil even after colder temperatures of a simulated overwintering treatment of 4  ̊ C for 2 weeks, -20  ̊ C for 3 weeks, followed by 4  ̊ C for 2 weeks. In addition to mere persistence, fungal growth30 actually occurred after the winter treatm ent in the soil with the bean rhizosphere, presumably due to the nutrient releas e from the dead roots.

V. INTEGRATION OF RISK

The fungus T. reesei has a long history of safe use in submerged standard

industrial fermentation for enzyme production with food, feed, pharmaceuticals and industrial uses. It is not pathogenic to humans , animals, or to plant s. The fungus has been shown to be nonpathogenic to healthy rats in a number of acute and chronic pathogenicity tests. The en zyme fermentation industry r outinely tests for toxin production in their enzyme products, and T. reesei has not been associated with mycotoxin production during typical enzyme ferm entation conditions. Acute and chronic toxicity tests on a number of enzymes for which GRAS petitions were made to the FDA have demonstrated the lack of toxicity of T. reesei enzymes such as cellulase, xylanase, endoglucanase, glucanase, protease, and pectin lyase. The use of T. reesei in submerged standard industrial fe rmentation poses low risk. T. reesei has a long history of safe use in enzyme production and is a good c andidate for the list of eligible recipient microorganisms in the Tiered Ex emptions if used for production of enzymes under the conditions of submerged standard industrial fermentation.

However, T. reesei is not an appropriate recipient microorganism for the 5(h)4 Tiered Exemptions for any other closed syst em use or under conditions unlike those used in standard submerged enzyme fermentation. T. reesei is known to produce a secondary metabolite known as paracelsin in submerged fermentation in the presence

of insoluble substrates such as cellulose. Cellulose is, of course, the main component of plant material, and in some form or ot her, the substrate of choice for biofuel production. Cellulosic biofuel fa cilities pre-treat various types of plant material such as sugarcane, switchgrass, corn stover, wood chip s, etc. with acid or other chemicals to initiate breakdown of the lignin in the plant material. Then, the fungus might actually be grown on the plant hydrolysate in the seed fe rmentor, which would t hen be transferred to a cellulase fermentor, for on-site en zyme production. One approach would be to subsequently transfer both the br oth containing high levels of cellulases and live cells to

the saccharification tank wher ein the cellulases would brea kdown the cellulose in the pretreated plant material into sugars which c ould then be fermented into ethanol.

Given this knowledge that an insolubl e substrate such as cellulose could stimulate peptaibol synthesis, t here are a number of differ ent scenarios to consider. The first of which is whether paracelsin would actually be produced in a cellulosic biofuel facility in the plant biomass breakdow n step, and if so, at what levels. It is possible that paracelsin would not be produc ed in submerged fermentation even in the presence of plant material, particularly if other conditions known to be conducive to peptaibol formation such as the use of older cultures, exposure to light, exposure to stress such as nitrogen limitation, and sporulation are all avoide d. It is known that, in31 general, that production of secondary metabolites in fungal liquid culture usually occurs late in the growth stage, i. e., the stationary phase, after exponential growth has ceased and when there is a nitrogen limitation in t he presence of excess carbon substrates. These conditions are known to cause funga l metabolism to shift into production of secondary metabolites.

If in fact paracelsin was produced in the plant biomass breakdown tank, the ethanolic fermentation step follo wing degradation of the plant ma terial into sugars could be inhibited because paracelsin is inhibitory to the growth of other fungi, and a yeast (a fungus) is typically used for the ethanolic conv ersion step. However, a particular yeast,
Candida , studied by Brückner and Graf (1983) was not inhibited by paracelsin, so it is not known whether the particular yeasts used for ethanol production would be inhibited. If paracelsin was produced at high c oncentrations with the growth of T. reesei on the plant material rather than using purified enz ymes void of the f ungus, the manufacturer may find that efficient ethanol fermentation was not possible. However, microorganisms other than yeast, such as the Gram-negative bacteria Escherichia coli or Zymonomas mobilis, could alternatively be used as the ethanologen. Gram-negative microorganisms would not be inhibited by parac elsin, so paracelsin production could possibly go undetected.

Another scenario for consideration is t hat the production of paracelsin in the fermentation tanks used for pl ant material breakdown may be inconsequential if at some other point in the ethanol production process t he compound is denatured. It is possible that the high temperatures (78°C) used fo r ethanol distillation could break down the peptide. However, other peptaibols have been s hown to retain their biological activity after high temperature treatm ent. It is unknown if par acelsin would be denatured or

whether it would retain its conformation and biol ogical activity. Anothe r possibility is that if paracelsin is not degraded during the latter st ages of the ethanol pr oduction process, it could be broken down during wastewater treatment of t he spent fermentation broth, and thus, would not enter the environment. However, there is no information available

in the literature that addre sses the potential breakdown of paracelsin in a wastewater treatment system, nor is there informa tion on the breakdown or persistence of paracelsin in the environment. There is some indication that other peptaibols appear to be relatively stable due to formation of α -helical structures resulting from the presence

of the α -aminoisobutyric acid which favors that structure. There is one article in the

literature that detect ed peptaibols in marine sediments t hat were causing toxicity to shellfish. The peptaibols were apparently rather persistent in the environment, and the authors suggested that peptaibols could be used as stable markers for the presence of

Trichoderma fungi in the marine environment (Poirier et al., 2007a). There is also the one article on the stability of pept aibol-like compounds produced by Trichoderma koningii called trichokonins that also contain high proportions of α -aminoisobutyric acid and have antimicrobial properties like other peptaibols. These trichokonins, which now are recognized as peptaibols, were found to be stable at high temperatures, up to 100°C, and even after autoclaving . They retained their biol ogical activity after low32 temperature treatment as well, including freezing followed by lyophilization, and over a pH range of 3.0 to 10.0. They were also found to be resistant to enzymes such as trypsin and other proteases that normally br eak down proteinaceous materials (Song et al., 2006).

In the absence of any dat a or literature specifica lly on paracelsin and its degradation or persistence in the environment, the cons equences of paracelsin exposure to environmental receptors must be considered. At the far extreme, it is possible that large quantities of paracelsi n could be produced wit h the growth of T.

reesei on plant material, and that paracelsin w ould not be denatured/inactivated at any other point in the ethanolic fermentation process stream. Under this scenario, paracelsin could adversely affect the functioning of wastewater treatment by inhibition of growth of Gram-positive bacteri a in the POTW or on-site tr eatment facility with disposal of liquid wastes. There is no informati on available in the literature on whether paracelsin would be rapidly broken down in the wastewater tr eatment system, or

whether the molecule is resist ant to proteases or other enzym es present in wastewater. If the paracelsin remains intact through wa stewater treatment, then the potential consequences of exposure of paracelsin to the environment must be considered.

Recommendations

T. reesei (QM6a and its derivatives) is reco mmended as an eligible recipient microorganism for the 5(h) 4 Tiered Exemption when us ed with submerged standard industrial fermentation conditions for enzym e production where no solid material is present in the fermentation broth. Ho wever, in the absenc e of data on potential production of paracelsin, T. reesei is not recommended for an ex emption from review when used in closed systems under conditions unlike those encountered in submerged standard enzyme fermentation, such as the growth directly on plant material.33 VI. REFERENCES

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