UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

JUN 1 5 2015 OFFICE OF CIIEMICAL SAFETY 

AND POLLUTION PREVENTION 

M EMORANDUM 

SUBJECT: 

FROM: 

TO: 

Human Health and Environmental Assessment for the California Section 

18 (Emergency Exemption) request for the usc of AF36, an end use 

formu lation containing the acti ve ingredient, Aspergillusjlavus Strain 

AF36 (PC Code: 006456; EPA File Symbol ISCA03); Decision No. 

505523; Submission No. 969249; DP Barcode No:427607 

Gai l Tomimatsu, Ph.D., Plant Pathologist 

Microbial Pesticides Branch 

Biopesticides and Pollution Prevention Division, 751 I P 

Milutin Djurickovic, M.S., Biolo · t 

Microbial Pesticides Branch 

Biopesticides and Poll ution Prevention Division, 751 1 P 

Shannon Borges, M.S., Lead Biologist 

Microbial Pesticides Branch 

Biopesticides and Pollution Prevention Division, 7511 P 

' 

I. PROPOSED PROGRAM OVERVIEW 

Applicant: CA EPA (California Department of Pesticide Regulation) 

Active Ingredient: non-aflatoxigenic Aspergillusjlavus Strain AF36 

Proposed Usc Sites/Use Pattern and Usc Rate: Four thousand fi ve hundred acres 

(4,500 A) of dried figs in California Central Valley Counties of Fresno, Kern, Madera, and 

Merced are to receive a one-time application of AF36 between June 15 and July 31, 2015. The 

proposed method of application is by grain seed (wheat-infested AF36 inoculum) applied to the 

orchard floor by ground equipment using attached "ant-bait spreaders". Grain seed inoculum is 

to be directed under the tree canopy where soil will be wetted before and after the appl ication. 

The intended rates of application are I 0 lbs product per A (1 0 lbs. product per acre; 0.008 

pounds of a.i. per acre; 36.4 total lbs a.i. for 20 IS applications in listed counties). 

Pest(s): aOatoxigenic Aspergillus spp. (includes: certain strains of Aspergillus jlavus and 

A. parasiticus. eta/.) 
II. BPPD OPINION and CONCLUSIONS: EPA has not identified any human health issues 

that preclude this emergency use. No incremental hazards to nontarget organisms, including 

humans, or to the environment are anticipated as a result of the agricultural applications of AF36 

for the limited scope of a one-time treatment on dried fig acreage as requested. However, it should 

be noted that submitted product performance studies to date have not necessarily demonstrated 

reductions of aflatoxin levels in these crops (except for the submitted data for corn [8]). The 

submitted product performance data for studies conducted in a CA fig research orchard [4] were 

limited also. 

The product is currently registered for agricultural applications to cotton, corn and pistachios, 

therefore our opinion is based on a limited database: mammalian health and environmental hazard 

assessments [1, 11), acceptable data from OPPTS Guideline Studies, as cited in [l , 7J, product 

performance evaluations (efficacy) for EUPs (8, 9], conclusions of peer-reviewed and cogent 

research investigations [2, 3 (, and the proposed use patterns as detailed in the above text. 

Submitted product performance and fungal population monitoring data (of resident and applied 

Aspergillus spp.) from fig research orchards were considered also for the assessment [2, 4]. 

Background and Regulatory Information 

Aspergillus jlavus refers to a diverse group of saprophytic soil fungi, with a cosmopolitan 

distribution and include strains that produce aflatoxin ('toxigenic' strains), as well as strains that 

do not produce aflatoxin (' atoxigenic' strains); as a group, these fungi can cause variable 

aflatoxin contamination of preharvest and postharvest seed, nut or grain crops. The fungus is also 

opportunistic and is considered a human pathogen causing aspergillosis diseases; and is one of 

several species of Aspergillus spp. reported to cause stonebrood in honeybees (6(. The most 

promising strategy currently being used to reduce preharvest contamination of crops with 

aflatoxin is to introduce non-aflatoxin strains of A. jlavus into the crop environment [5]. This 

species of fungus also demonstrates a variety of sclerotia! types, shows variability in response to 

light, variable ability to colonize living plant tissue, and has strains residing in multiple 

vegetative compatibility groups (YCGs). 

Aspergillusjlavus Strain AF36 (AF36) is registered as a microbial pesticide to reduce the overall 

aflatoxin levels on aflatoxin-contaminated crops (EPA Reg. No. 071693-1). The microbial active 

ingredient is a naturally occurring soil colonizer, does not produce aflatoxins, cyclopiazonic acid, 

or known intermediates in the aflatoxin biosynthesis pathway and was originally isolated from 

Arizona cottonseed (I, 7]. The pesticidal mode of action is by competitive "displacement" of 

aflatoxin-producing Aspergillus spp [1, 5]; and is determined by extensive population sampling 

(product perfonnance data) with follow-up verification of representative microbial isolates by 

specialized microbiological analyses [1, 3J. This mode of biological control is currently the most 

widely used biocontrol method for reducing aflatoxin contamination in world-wide crops such as 

maize and cottonseed where aflatoxin contami nation is a persistent problem for human and 

animal health (Wu and Khlangwiset, 20 I Oa, as cited in (51). 

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Product Performance (Efficacy Assessment) for Usc on Dried Fig Acreage (Attachment N, 

[41) 

Population data were submitted for the use of Aspergillus jlavus AF36 to reduce aflatoxin 

contamination of figs in California [4]. The submitted data are limited in scope, since discussion 

of results and information pertaining to methodology (e.g., application equipment, application 

rates and sampling procedures, and specialized microbiological analyses [1, 3]), experimental 

design, and other environmental parameters (e.g., rainfall and temperature data) were missing in 

the attachment [4]. Raw data were not reported to support the statistical analyses. Aflatoxin 

measurements were not reported in Attachment N [4]. 

Results of semi-annual sampling (conducted 2003-2004) of Aspergillus spp. fungal populations 1 

(soil, dropped leaves and fig samples) showed trends of successful colonization by AF36 \·Vi thin 

the research orchard following early summer applications of AF36 in 2003 and 2004 at the rate of 

( 10 lbs product/ A) to the orchard floor. A steady increase in percentage recovery of isolates 

belonging to the AF36 group in annual soil samples was also noted for acreage not treated with 

AF36 between October 2003, September 2004 and September 2005. In October, 2006 and in 

September 2007, there were notable declines in the percent recovery of soil isolates belonging to 

the AF36 group; these data are partially reflected in Table 8 and Fig. 2; Attachment N. BPPD 

notes also that there was not a significant presence of A. jlavus S strains2 in the sampled orchard, 

and there was not a statistically significant displacement of A. jlavus S and L strains or A. 

parasiticus by AF36 [4]. 

Product performance could be affected by available soil moisture and temperatures. Product that 

is applied in areas that have "moderately wet" soil has optimal conditions for sporulation and 

colonization as opposed to "very wet" (i.e., directly under drip line emitters) areas, "slightly wet" 

areas, or "always dry" areas. Quantitative data to show levels of soil moisture (i.e., measured soil 

moisture tensions) were not provided in Attachment N. Optimal temperatures for sporulation and 

subsequent colonization of treated acreage were shown to be within a range of ~ 77°F to 86 , 

based on laboratory data represented in Fig 3[4]. Aspergillusjlavus AF36 population levels on 

·wheat seed were also observed to peak at 95°F and decline within I to 2 days of the initial peak. 

Assessment for the 2015 Emergency Usc of AF36 on Dried Figs 

Human/Mammalian Health Assessment 

EPA has not identified any human health issues that preclude the use of AF36 in a Section 18 

Emergency Exemption in fig orchards at the prescribed rate of 10 lb/acre. Additionally, EPA 

does not expect adverse human health effects as long as the product is applied according to the 

label requirements and precautionary statements. 

This determination is based on previously written reviews and published Biopesticide 

Registration Action Documents for AF36. Reviewed studies and rationales by EP NBPPD 

1 Data were based on quantitative sampling, monitoring and microbiological analyses such as rnorphotyping and 

testing vegetative compatibility for AF36 )I , 3) grouping for the years 2002-2007 in one CA research ftg orchard. 

Some of the research was published in a separate paper 121, not included in Attachment N. 

2 A. jlavus S strains are considered 'toxigenic' and L strains are considered "atoxigenic" Ill 

3 
scientists led to the recommendation of AF36 for registration (10, 11 , 12J. The conclusions from 

these studies indicate that acute oral and pulmonary toxicity/pathogenicity studies in rats 

confirmed the clearance of Af36 with no evidence of toxicity or infectivity (l OJ. Additionally, 

intratracheal dosing showed clearance by day eight. These results supported waivers for 

intravenous, intracerebral, intraperitoneal injection studies, acute dermal toxicity/pathogenicity, 

primary dermal irritation, primary eye irritation, and hypersensitivity. Immune response was 

also tested in rats, indicating that the tested rats can clear AF36 from their bodies without 

compromising their immune system. No hypersensitivity incidents have been reported to date 

from workers who have been exposed. Overall, no problems with AF36 have been reported, but 

according to FIFRA 6(a)(2) all hypersensitivity problems or incidents must be reported by the 

registrant iJOJ. 

Tolerance Exemption 

According to 40 CFR 180.1206 there are tolerance exemptions for a range of crops for Af36 

including; cotton, pistachio, and corn. Based on EPA's recommendation this can be temporarily 

extended to include dried figs from June 15, 2015 to July 31 , 20 15 in Fresno, Kern, Madera, and 

Merced counties in the state of California. 

Environmenta l Assessment for the 2015 Emergency Use of AF36 on Dried Figs 

The Agency has previously determined that agricultural uses of Af36 will have no adverse 

effects on terrestrial and aquatic vertebrate and invertebrate species, including birds, insects, 

honey bees and other pollinators, estuarine and marine animals, and non-target plant species (lJ. 

Also, particular emphasis was given to determining whether AF36 is one of the 

A. jlavus strains known to cause honeybee pathogenicity, and avian and mammalian respiratory 

infections. The exposure and potential hazard of AF36 colonized-wheat seed to honey bees was 

assessed for 30 days following a single aerial application on blooming cotton in Arizona (MRID 

45739102, as cited in [1, 7J). No significant difference in mortality between the test and control 

hives was seen. No larval disease 'stonebrood'

3 was observed (MRlD No. 46196806, as cited in 

(71). The AF36 toxicity data for Human Health Assessment indicate that there is no significant 

toxicity nor pathogenicity to rodents from acute oral and pulmonary testing at a maximum hazard 

dose. Submitted avian inhalation toxicity and pathogenicity studies (MRID No. 45798 102, as 

cited in (1, 71) are sufftcient to demonstrate that there would be no hazards to endangered birds 

from the proposed uses of A .. flavus AF36. 

The agricultural uses of AF 36 are not expected to increase the total A. jlavus content in the 

environment (1 , 7J. Results of soil and air monitoring studies showed that a one-time seasonal 

application of Af36 does not appreciably change the overall quantity of A .. flavus spore load on 

the environment, within the range of natural variations (MRID Nos. 4530720 I, 45307202; as 

cited in 1, 7). Naturally occurring populations of total A. jlavus have been reported to vary from 

3 Stonebrood is a very rare honey bee brood disease caused by several different species of Aspergillus 161 : A.jlavus 

has most frequently been reported, followed by A. fumigatus, A. niger and other species can kill honey bees (Gilliam 

and Vandenberg, 1997, as cited in 161): adults, larvae and pupae can become infected. In addition, aflatoxins have 

been suggested to be the primary cause of death in stonebrood infected honey bees (Burnside. 1930, as cited in 161). 

A non-aflatoxin producing A. jlavus strain has been observed to induce stone brood symptoms equally well in in 

vitro- reared honey bee larvae as aflatoxin-producing strains (Vojvodic, unpublished, as cited in 161). 

4 
0.5 to > I 05 cfu/g of soil and may vary significantly greater than I 0-fold within a single field in a 

year. A three-year field study conducted by the USDA demonstrated that while the total A. 

jlavus concentration increased with the application of non-toxigenic A. jlavus, it declined by the 

spring of the following year [1 , 7). The total amount of A.jlavus in the soil did not increase in 

the long term. Since the concentration of natural populations of A. jlavus were shown to 

fluctuate widely, the temporary increase in A. jlavus AF36 post application is not expected to 

produce effects not already seen from the natural non-toxigenic and toxin producing A. jlavus 

population fluctuations. 

Endangered Species Assessment 

No incremental exposures of AF36 for endangered wildlife are anticipated for this use, as there 

were no incremental hazards to nontarget wildlife in previous assessments p, 2, 7]. Data from 

soil and air population sampling studies for AF36 and Aspergillus conducted in Arizona [1 , 8], 

California (2, 4, 7, 9], and Texas [8] demonstrated that the total amount of A. jlavus in the soil 

did not increase in the long term, and could fluctuate widely. Temporary increases in A. jlavus 

AF36 post applications are not expected to produce effects not already seen from natural 

populations of A. jlavus. Therefore a No Effect (NE) determination was opined in 2007 PI to 

any endangered/threatened terrestrial or aquatic species as listed by the U.S. Fish and Wildlife 

Service (USFWS) and the National Oceanic and Atmospheric Administration (NOAA). The NE 

detennination applies for direct and indirect effects to listed species and their designated critical 

habitats. 

For the current requested emergency use on dried figs, a No Effect finding is also supported by 

the data evaluations as stated above. 

References: 

[1). Tomimatsu, G. and Vaituzis, Z. 2003. Environmental Hazard Assessment for the Microbial 

Pesticide, Aspergillusjlavus AF36 for Conditional Registration in Arizona and EUP Extension 

in Texas; EPA Reg. No. 07 1693-R; Bar code Nos. D288777; 0288782; 0286708 

(2]. Doster, M.A.,T.J. Michailides, L. Doster, P.J. Cotty. 2008. Biocontrol of aflatoxins in figs. 

Acta Hort. (ISHS) 798:223-226. 

(3]. Bayman, P.and P. J. Cotty. 1991. Vegetative compatibility and genetic variation in the 

Aspergillusjlavus population of a single field. Can. 1. Bot. 69: 1707-17 11. 

(4). CA Dept. of Pesticide Regulation. 2015. Attachment N. Application of AF36 in a 

Research Fig Orchard (2002-2007). 14 p. California authorization for pesticide use under 

U.S.EPA Section 18 Specific Exemption for Distribution and Use only within California. 

(SJ. Ehrli ch, K.C. 20 14.Non-aflatoxigenic Aspergillusjlavus to prevent aflatoxin contamination 

in crops: advantages and limitations. Front. Micro. http://dx.doi.org/t 0.3389/fmicb.20 14.00050 

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[6] Jensen,A.B., K.Aronstein, J.M.Fiores, S.Yojvodic, M.A. Palacio and M. Spivak. 

2013. Standard methods for fungal brood disease research. J .Apic Res. 52( l ). 

http://www. ncbi. nlm.ni h.gov/pmc/articles/PMC3 816652/ 

[7]. Yaituzis, z. 2007. Endangered Species Risk Assessment for Aspergillusjlavus AF36 

Experimental Use on Pistachio in California (71693-EUP-R). Memo to S. Bacchus. DP 

Barcode: 335793; Decision No.:371294. No MRID. 

[8]. Gagliardi, J. and J. Kough. 2010 and 201 1. Aspergil/usjlavus AF36 (EPA Reg No. 71693-

EUP-2) usc on corn. Memos to S.Bacchus. Decision No.:424829; Data Evaluation Records 

MRlDs: 4793510 1 and 48330101 

[9]. Gagliardi, J. and J. Kough. 201 1. Aspergillus.flavus AF36 use on pistachio. Memo to 

S.Bacchus. Decision Nos: 444580, 444582; Data Evaluation Record MRID: 48363701 

[10]. BPPD Review of Product Chemistry and Efficacy Data to satisfy conditions of Registration 

for Aspergillus jlavus AF36 [EPA Reg. No. 71693-1 ], September 13, 2007. 

[11]. BPPD Review of Data Waiver Requests Submitted by IR-4 on behalf of the Arizona Cotton 

and Research Council in support of the Health Effects of Aspergillus jlavus J\F36 for use on 

cotton food/feed commodity, May 22, 2003. 

[12]. U.S. EPA. 2003. Biopesticides Registration Action Document: Aspergillusjlavus strain 

AF36 (PC Code 006456), July 3, 2003. 

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