UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

June 2, 2010

MEMORANDUM

SUBJECT: Environmental risk assessment for the FIFRA Section 3 registration of Typhula phacorrhiza strain #94671 (EPA Reg. Nos. 84888-R and 84888- E) for control of snow mold disease in turf

FROM: Shannon Borges, Biologist / signed / Microbial Pesticides Branch Biopesticides and Pollution Prevention Division, 7511P

THROUGH: Zigfridas Vaituzis, Senior Scientist / signed / Microbial Pesticides Branch Biopesticides and Pollution Prevention Division, 7511P

TO: Denise Greenway, Regulatory Action Leader Microbial Pesticides Branch Biopesticides and Pollution Prevention Division, 7511P

I. Environmental Risk Assessment

A. Introduction

Technology Sciences Group, Inc. has submitted on behalf of Agrium Advanced Technologies RP, Inc. an application to register the microbial pesticide control agent (MPCA), Typhula phacorrhiza strain #94671, for control of pink (Microdochium nivale) and grey ( T. ishikariensis
, T. incarnata) snow mold in turf. T. phacorrhiza strain #94671 is the active ingredient (a.i.) in the manufacturing use product, Nivalis Technical (EPA Reg. No. 84888-R) and the end use product (EP), Nivalis (EPA Reg. No. 84888-E). Both products contain 4% a.i., with 1.861 x 10 5 colony forming units (cfu)/lb product. This memorandum contains BPPD’s ecological risk assessment for the proposed registration of T. phacorrhiza strain #94671.

Typhula phacorrhiza is a clavulate basidiomycete widely reported from northern temperate

zones. T. phacorrhiza is considered to be a psychrophilic saprotroph (Remsburg 1940, Bruehl and Cunfer 1975, Burpee et al. 1987), which means that it thrives in cold temperatures and lives on dead organic matter. While it is related to other Typhula species that are known plant pathogens, it has been shown to be a distinct species (Hsiang and Wu 2000). T. phacorrhiza is often found on corn (Zea mays L.) residues and other organic debris after at least 80 days of2 snow cover (Burpee et al. 1987), but has been collected from dead leaves of other plants, including tree, weed, lawn grass and overwintered leaves (Hsiang et al. 1999, Remsburg 1940). Typhula phacorrhiza strain #94671 was originally isolated from corn stubble in a field near Acton, Ontario. In field testing it showed potential to suppress grey snow mold in creeping bentgrass ( Agrostis palustris) (Hsiang et al. 1999). Cold weather and abundant moisture create optimum conditions for growth and reproduction, and T. phacorrhiza strain #94671 grows optimally at 4oC (Snider et al. 2000). T. phacorrhiza requires the same environmental cues as T. ishikariensis and T. incarnata (high moisture and temperatures near freezing) for sporophore production (Yang et al. 2006); therefore, application of T. phacorrhiza strain #94761 is made at this time of year to optimize biocontrol of pathogenic snow molds.

The mechanism by which T. phacorrhiza suppresses grey snow mold disease is apparently not a direct effect, such as hyperparasitism or cellular lysis, but occurs as a result of resource competition (Burpee et al. 1987, Wu and Hsiang 1997). In other studies, T. phacorrhiza was shown to have a greater ability to utilize carbon sources and to grow and produce sclerotia over a wider range of temperatures compared to other Typhula species, which provides further evidence that T. phacorrhiza is simply a better competitor because it has a greater ability to use resources and is tolerant of a wider range of conditions (Burpee 1994, Wu and Hsiang 1999). Microdochium nivale frequently occurs with pathogenic Typhula species. This pest species also grows and reproduces under conditions similar to those required of Typhula species and it does not require snow cover (Hsiang et al. 1999, Matsumoto 2009). It also has a higher optimum growth temperature (Hoshino et al. 2009) than Typhula phacorrhiza. Therefore, Typhula phacorrhiza would also be able to out-compete M. nivale under the conditions at which the proposed MPCA is applied, so it likely exhibits a similar mode of action against this species.

B. Summary of Nontarget Effects Data

Table 1 provides the status of the data requirements for the active ingredient (TGAI) of Typhula phacorrhiza strain #94761 for ecological risk assessment. Data waiver rationale were submitted to satisfy data requirements for nontarget organisms as published in 40 CFR § 158.2150. A plant pathogenicity study was also reviewed, and an oral toxicity study with laboratory mammals has been submitted. Both of these studies were also used for risk assessment. Information from the data waiver rationale is included in the discussion of risk below (see section C), and Data Evaluation Records (DERs) of the data waiver rationale are attached.

Review of the rationale and data submitted to support the registration of T. phacorrhiza strain #94761 was completed jointly with Health Canada’s Pesticide Management Regulatory Agency (PMRA). The information provided is sufficient to satisfy the Tier I nontarget organism data requirements and for nontarget organism risk assessment for the active ingredient. Further testing of nontarget organisms at higher tier levels is not required.3 Table 1 . Summary of data submitted to comply with nontarget organism data requirements published in 40 CFR § 158.2150 for support of the registration of Typhula phacorrhiza strain #94671. Data Requirement OPPTS Guideline No. Results Summary and Classification MRID No. 1

• Avian oral toxicity/pathogenicity • Avian inhalation toxicity/pathogenicity • Freshwater fish toxicity/pathogenicity • Freshwater invertebrate toxicity/pathogenicity • Estuarine/marine fish and invertebrate testing • Nontarget insect testing • Honey bee testing 885.4050

885.4100

885.4200

885.4240

885.4280

885.4340

885.4380 Data waiver rationale provides sufficient information to determine that toxicity/pathogenicity to avian wildlife, freshwater and marine/estuarine fish and invertebrates, honey bees, and nontarget insects is not expected as a result of the proposed uses of Typhula phacorrhiza strain #94671. Classification: Acceptable 478021-07 478021-08

Wild mammal toxicity/pathogenicity 885.4150 Tests required by 40 CFR § 158.2140 are adequate and appropriate for assessment of hazards to wild mammals. An acute oral toxicity test with albino rats indicates no adverse effects at 5000 mg/kg-bw. Data waiver rationale also support the conclusion that adverse effects to wild mammals are not expected as a result of the proposed label uses. Classification: Acceptable for wild mammal risk assessment 478021-07 478021-08

Nontarget plant testing 885.4300 Plant pathogenicity data show no evidence of adverse effects on two monocotyledonous plant species and three dicotyledonous plant species. The study was classified as supplemental . Additional data waiver rationale support the conclusion that adverse effects to nontarget plants are not expected from the proposed uses. Classification: Acceptable data waiver rationale 478021-07 478021-08

1 Publications referenced in the data waiver rationale were contained in MRID 478021-09.4 C. Ecological Exposure and Risk Characterization

1. Terrestrial Animals and Plants

The proposed EP is a granular product that will be applied with ground equipment or by hand (aerial applications are prohibited), which will limit application of the EP to the intended treatment area. Applications will be made after the last mowing of the season, but before the first snowfall. Conditions typical of this time of year (high moisture and low temperatures) favor the production of Typhula growth and reproduction (Yang et al. 2006), but would limit dispersal by wind of basidiospores produced during this time. Hsiang et al. (1999) also state that the primary inoculum is believed to be mycelia that originate from sclerotia instead of basidiospores. Since T. phacorrhiza strain #94671 sclerotia are several orders of magnitude heavier than other propagules that spread through airborne movement (e.g., pollen) (MRID 478021-09), movement of propagules off the treatment site by wind is not expected. Some movement via runoff may occur with heavy rain, but the sclerotia produced on treated plants are usually attached to plant material (e.g., see Schneider and Seaman 1986), which also reduces off-site movement. Therefore, movement of T. phacorrhiza strain #94671 from the treatment site is not expected to be great, and nontarget exposure is expected to be limited to the treated area and its immediate margins

Growth of the T. phacorrhiza strain #94761 population after application is expected (MRID 478021-09). These increases primarily would occur under snow cover, which would limit nontarget exposure and off-site movement. Following snow melt, increasing temperatures will create conditions that are unfavorable for growth and reproduction. Therefore, opportunity for exposure to actively growing T. phacorrhiza strain #94671 will be limited in the terrestrial environment. Hsiang et al. (1999) showed that a single application of T. phacorrhiza strain #94671 applied at approximately 2x the proposed application rate controlled Typhula blight in experimental turf grass plots for three years (each year new inoculations of T. incarnata and T. ishikariensis were applied to the treatment plots, but not T. phacorrhiza). Therefore, T. phacorrhiza strain #94671 populations may persist for some time; however, based on these data the population will eventually fall to levels at which biocontrol is unsuccessful, which are expected to be comparable to natural concentrations.

Birds and Mammals

Birds and mammals may be exposed to T. phacorrhiza strain #94671 through direct consumption of the granules at application. They may also incidentally consume the fungus during its times of growth just before and after snowfall, as well as the sclerotia during times of warmer and drier weather. Some mammals occupying treated areas under snow could also be exposed during winter. However, the optimal growth temperature for T. phacorrhiza strain #94671 was determined to be around 4°C, and it does not grow at 30°C (Snider et al. 2000), so it is not able to grow at normal mammalian and avian body temperatures. Additionally, mammals dosed with 5000 mg/kg-bw T. phacorrhiza strain #94671 in an acute oral toxicity test showed no adverse effects over 14 days of observation (MRID 478021-02). Therefore, if exposure were to occur to birds and mammals as a result of the proposed labeled applications, no adverse effects are expected to result. *Inert ingredient information may be entitled to confidential treatment*5 Nontarget Insects and Honey Bees

Toxicity/pathogenicity data were not submitted for nontarget insects or honey bees; however, T. phacorrhiza is not known to be toxic or pathogenic to insects. Since T. phacorrhiza is known to be saprotrophic, it growth is limited to dead organic material. Therefore, it is not expected to affect insects that may be overwintering in treated areas. During the time of year when insects are active, T. phacorrhiza exists in a dormant stage and would not affect nontarget insects, honey bees, or other invertebrates.

Nontarget Plants

Several members of Typhula spp. are known to cause disease of cereals and grasses at low temperatures. However, there is no known relationship reported in the published scientific literature between T. phacorrhiza strain #94671 and known plant pathogens, and an investigation into the genetic variation of T. phacorrhiza from snow moulds caused by T. incarnata and T. ishikariensis supported the distinction of these three main Typhula species (Hsiang and Wu 2000). In the published scientific literature, a strain of T. phacorrhiza (DAOM 195241) was found to be pathogenic to winter wheat (Schneider and Seaman 1986). The author reported sclerotia of T. phacorrhiza (DAOM 195241) were found firmly attached to the surface of necrotic leaves by a well developed stalk. Based on these results, T. phacorrhiza (DAOM 195241) may pose a hazard to non-target plants; however the strain being proposed for registration failed to infect plants in laboratory testing. The applicant submitted a laboratory infection study to address phytopathogenic effects of T. phacorrhiza strain #94671 on non-target terrestrial plants, including monocotyledonous plants (wheat [ Tricum aestivum]

, rice [ Oryza sativa]) and dicotyledonous plants (tomato [Solanum lycopersicum]

, geranium [Pelargonium hortorum]

, and Nicotiana benthamiana) (data provided in MRID 47802109). The leaves of each test subject were exposed to a five millimeter agar disk from an active growing culture of T. phacorrhiza strain #94671. During the incubation period of 7-8 days at 4°C and 25°C, the leaf surfaces were evaluated daily for the presence of lesions or other signs of pathogenicity. There were no treatment related effects reported from the exposure of either monocots or dicots to disk containing the MPCA. Toxic/pathogenic endpoints such as mortality, seedling emergence, growth inhibition, and vegetative vigor were not reported. While the concentration of the MPCA to which test subjects were exposed was not provided in the report, the study does show that exposure to an active, growing culture of T. phacorrhiza strain #94671 did not cause adverse effects in the species tested, including wheat.

Other information available for T. phacorrhiza indicates a lack of effects on plants. The results of winter trials published in the scientific literature were submitted to PMRA to demonstrate the efficacy of several isolates of T. phacorrhiza in controlling snow molds on turf. A variety of formulations were applied at rates up to 200 g of inoculated mixed grain/m2 to 12 species of turf grasses and two cultivars of winter wheat. The results of this testing showed lack of pathogenicity towards the turf grasses and winter wheat cultivars by any of the isolates tested; however, confirmation that the proposed MPCA was among these isolates was not provided (Wu and Hsiang 1998). Nonetheless, the results of this study are in contrast to those seen in testing conducted using T. phacorrhiza isolates obtained from winter wheat fields (Schneider and6 Seaman 1986), and the author of the current study proposed that there may be differences in pathogenicity potential between isolates collected from different types of plants. Additionally, no information was found in the literature that indicated any effects of Typhula phacorrhiza on other plant species (see below). Therefore, based on the information presented above, proposed applications of T. phacorrhiza strain #94671 are not expected to result in adverse effects to plants.

2. Aquatic Animals and Plants

As discussed above, off-site movement of T. phacorrhiza strain #94671 is not expected to be great, and direct applications to water are prohibited on the proposed label. Therefore, concentrations of T. phacorrhiza in surface water nearby to treated areas are not expected to increase above current inputs resulting from the natural distribution of T. phacorrhiza in the environment and natural processes of movement. T. phacorrhiza is not known to proliferate in water, and has not been reported as a pathogen of aquatic animals or plants. Studies with plants as described above have shown that T. phacorrhiza strain #94671 is not pathogenic to terrestrial plants, and T. phacorrhiza apparently does not produce toxins or other chemical products. Therefore, since exposure to T. phacorrhiza strain #94671 is expected to be minimal and because T. phacorrhiza is not a known pathogen in aquatic environments, adverse effects to aquatic animals and plants (both freshwater and marine/estuarine) are not expected to result from the proposed applications of T. phacorrhiza strain #94671 as labeled.

3. Open Literature Information

Literature searches were performed to determine if other effects have been reported that were not contained within the information provided by the registrant. Initially, the Science Citation Index Expanded database (1970 – present) was searched using the term ‘ Typhula phacorrhiza,’ and 19 records were returned. Additional refined searches with ‘ Typhula ’ and other search terms did not yield additional information. A search was also performed with the Environmental Information Search service available to the Agency. This search tool simultaneously queries the Agricola; Biosis Previews; CAB Abstracts; Energy, Science, and Technology, General Science Abstracts, National Technical Information Service, and Waternet databases within the years of 1964 – present. A search was performed with the term ‘ Typhula phacorrhiza,’ which returned 55 records. A search with the genus name returned 1600+ records, many of which were not relevant to nontarget effects. Therefore, refined searches were conducted with ‘ Typhula ’ combined with the terms, ‘bird,’ ‘mammal,’ insect,’ ‘arthropod,’ ‘aquatic,’ ‘invertebrate,’ ‘fish,’ ‘reptile,’ ‘amphibian,’ ‘toxic,’ and ‘toxin,’ and these searches returned a total of 16 records. None of the records found in any of the searches described here reported effects on any nontarget species or additional information useful to this risk assessment.

D. Threatened and Endangered Species Assessment

EPA has determined that adverse effects will not occur to nontarget organisms as a result of the proposed labeled applications of Typhula phacorrhiza strain #94671. Additionally, T. phacorrhiza is a saprotrophic fungus that competes specifically with other snow mold fungi, and7 reduction or loss of these fungi will not affect habitat or other resources required by threatened and endangered species.

Since EPA has determined that no effects are anticipated for any non-target species exposed to Typhula phacorrhiza strain #94671 as a result of the proposed labeled applications, effects to federally-listed threatened and endangered species and their designated critical habitats are also not expected. Therefore, a “No Effect” determination is made for direct and indirect effects to listed species and their designated critical habitats resulting from the proposed uses of Typhula phacorrhiza strain #94671, as labeled.

II. Environmental Assessment Summary

The Agency has performed an environmental risk assessment based on the data, literature citations, and data waiver rationale provided by the registrant, and has determined that the proposed use of Typhula phacorrhiza strain #94671 does not pose risk to nontarget organisms. Typhula phacorrhiza is a saprotrophic snow mold that is an obligate psychrophile (i.e., it requires low temperatures for growth and reproduction and lives on dead organic matter). It is abundant within its range and is widely distributed in areas with persistent snow cover. Typhula phacorrhiza is recognized as a species distinct from the target Typhula species that are recognized plant pathogens. It is not known to produce toxins or other chemicals as a means of competing with other fungi. Exposure of nontarget organisms to actively growing Typhula phacorrhiza strain #94671 is also expected to be limited due to the conditions it requires for growth.

The registrant has submitted data waiver rationale to satisfy data requirements for nontarget organism risk assessment with the active ingredient (TGAI). Review of the rationale and data submitted to support the registration of T. phacorrhiza strain #94761 was completed jointly with Health Canada’s Pesticide Management Regulatory Agency. Submitted data, data waiver rationale, and a search by BPPD of several literature databases provides sufficient information to determine that toxicity/pathogenicity to wild mammals, avian wildlife, freshwater and marine/estuarine vertebrates and invertebrates, honey bees, nontarget insects, and terrestrial and aquatic plants is not expected from the proposed label uses. As a result, a “No Effect” determination is also made for direct and indirect effects to listed species and their designated critical habitats resulting from the proposed use of Typhula phacorrhiza strain #94671, as labeled.

The information provided is sufficient to satisfy the Tier I nontarget organism data requirements and for nontarget organism risk assessment for the active ingredient. Further testing of nontarget organisms at higher tier levels is not required.

References Cited

Bruel, G.W. and B. M. Cunfer. 1975. Typhula species pathogenic to wheat in the Pacific Northwest. Phytopathology 65: 755-760. Contained in MRID 478021-09.8 Burpee, L.L. 1994. Interactions among low-temperature-tolerant fungi: prelude to biological control. Canadian Journal of Plant Pathology 16: 247-250.

Burpee, L.L., L.M. Kaye, L.G. Goulty, and M.B. Lawton. 1987. Suppression of gray snow mold on creeping bentgrass by an isolate of Typhula phacorrhiza. Plant Disease 71: 97-100. Contained in MRID 478021-09.

Hsiang, T. and C. Wu. 2000. Genetic relationships of pathogenic Typhula species assessed by RAPD, ITS-RFLP and ITS sequencing. Mycology Resistance 104: 16-22. Contained in MRID 478021-09.

Hoshino, T., N. Xiao, and O.B. Tkachenko. 2009. Cold adaptation in the phytopathogenic fungi causing snow molds. Mycoscience 50: 26-38.

Hsiang, T., N. Matsumoto, and S. Millet. 1999. Biology and management of Typhula snow molds of turfgrass. Plant Disease 83: 788-798. Contained in MRID 478021-09.

Hsiang, T., C. Wu, and S. Cook. 1999. Residual efficacy of Typhula phacorrhiza as a biocontrol agent of grey snow mold on creeping bentgrass. Canadian Journal of Plant Pathology 21: 382-387. Contained in MRID 478021-09.

Matsumoto, N. 2009. Snow molds: a group of fungi that prevail under snow. Microbes in the Environment 24: 14-20.

Remsburg, R. 1940. Studies in the Genus Typhula. Mycologia 32: 52-96. Contained in MRID 478021-09.

Snider, C.S., T. Hsiang, G. Zhao, and M. Griffith. 2000. Role of ice nucleation and antifreeze activities in pathogenesis and growth of snow molds. Phytopathology 90: 354-361. Contained in MRID 478021-09.

Wu, C. and T. Hsiang. 1997. Mechanisms involved in biological control of grey snow mold of turfgrass with Typhula phacorrhiza. Phytopathology 87: S104. Contained in MRID 478021- 09.

Wu, C. and T. Hsiang. 1998. Pathogenicity and formulation of Typhula phacorrhiza, a biological agent of gray snow mold. Plant Disease 82: 1003-1006. Contained in MRID 478021-09.

Wu, C. and T. Hsiang. 1999. Mycelial growth, sclerotial production and carbon utilization of three Typhula species. Canadian Journal of Botany 77: 312-317. Contained in MRID 478021-09.

Yang, Y., F. Chen, and T. Hsiang. 2006. Fertile sporophore production of Typhula phacorrhiza in the field is related to temperatures near freezing. Canadian Journal and Microbiology 52: 9-15.