UN I TED STATES ENV IR ONMENTA L PROTECT I ON AGENCY WASHINGTON , 

### D .C. 2 04 60 

## JUN 

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## 5 2015 OFF I CE OF C IIEMI CAL SAFETY AND POLLUTION PREVE NT I ON
M EM ORANDUM SU BJ EC T:

FROM :

TO: 

### Hum an H ea lth and E n v iro nm enta l Assessment for th e Ca lifornia Section 18 (E merge nc y Exemption) r equ es t for the usc of AF36, a n end use formu lat ion containin g th e ac ti ve ingredient , Aspe r g illusjlav us 

### Strain AF36 ( PC Code : 006456 ; EPA File Sy mb o l IS CA03); Dec is io n No. 505523; Submiss ion No. 969249; DP Barcod e N o :4 2 7607 Gai l Tomimatsu , Ph .D., Pl ant P atho logist Mic rob ia l Pe stic ide s Branch Biopes ti cides and Pollution Prevention Di vision , 751 IP Milutin Djurickovic , M .S., Biolo · t Mi crobial Pesticides Branch Bi o pes ticides and Po ll ution Prev ention Di vision, 751 1 P S hann o n Borges , M.S. , Lead Bio logist Microb ia l Pes ticid es Branch Biopesticides a nd Pollution Prevention Di vis io n , 7511 P 

### '

I. PROPOSED PROGRAM OVERVIEW 

### App li cant: CA EPA (Californ i a Departm e nt of Pesticide Regu lat ion) Ac tive In g redi ent: no n-aflatoxi ge nic Aspe r g illusjlav us 

### Strain AF36 Propo sed Usc S ites/Use Pattern a nd Usc Rate: Four th o usand fi ve hundred acres (4,500 A) of dri ed fig s in Ca lif orn ia Ce ntral Valley Count ies of Fres no, Kern , Mad era , and Merc ed are to r ece i ve a one -time application of A F36 between Jun e 1 5 and Jul y 3 1 , 2015. The proposed meth od of application is by grain seed (w he a t-inf ested AF36 in oc ulum ) applied to th e o rchard floor by g ro und e quipm ent using attached " an t-b ait s preader s". Grain see d inoculum is to be directed und e r th e tree canopy where so il w ill be wetted before and after th e appl icat ion. The intended rate s of application a re I0 lb s product pe r A (1 0 lb s. produ c t p er acre; 0

. 008 pounds of a.i

. per acre; 36.4 total lb s a.i. for 20 I S a pplications in listed count ies)

. P est(s): aOatoxigenic Aspergillus 

### s pp. ( includ es: certain s tr a in s ofAspe r gillus jlavus 

### and A. parasiticus. eta/.)II. BP P D OP IN IO N a nd CONCLUS I ONS: E PA has not id e ntified any hum a n hea lth issues that preclude this emergency use. No incremental hazards to nontarget organisms , includin g humans , or to the environment are anticipated as a result of the ag ri cultural applications of AF36 for the limited scope of a one-time treatment on dried fig acreage as requested. However , it should be noted that subm itted product performance stud ies to date have not necessarily demonstrated reductions of aflatoxin levels in th ese crops (except for the submitted data for co rn [8]). The submitted product performanc e data for s tudi es conducted in a CA fi g re searc h o rchard 

### [4] were limit ed also. The product is currentl y registered for agricultural applicati ons to cotton, corn and pistachios , therefore our opinion is based on a limited database: mammalian health and environmental hazard assessmen t s [1 , 11), acceptable data from OPPTS Guid e lin e S tudi es, as cited in [ l , 7J, product performance e valuation s (efficacy) for EU P s 

### (8, 9], conclusions of peer-r ev iewed and cogent re searc h investigations [2 , 3 (, and the proposed u se patterns as detai l ed in the above text. Submitted product performance and fungal populatio n monitoring data (of resident and applied Aspergillus spp.) from fig research orchards were co n sidered a lso for the assessment [2 , 4] . B a ck g ro und a n d R eg ul atory In format i o n Aspergillus jlavus r efe rs to a diverse g r oup of saprop hytic soil fungi, w ith a cosmopol ita n distribution and include strai n s that produce aflatox in ('tox igenic ' strains), as we ll as strains that do not p roduce aflatoxin ( ' atox igenic' strains); as a group, these fungi can cause va riable aflatoxin co n tam in at ion of preha r ves t and postha r vest seed , nut or grai n c rops. The fungus is al s o opportunistic and is considered a hum an pathogen causing asperg ill osis diseases; a nd is one of severa l species o f Aspergillus s pp. re ported to cause stonebrood in hon ey b ees (6 (

. The mo st promising strategy currently be in g used to red u ce preharvest con t am in ation of c r ops with aflatoxin is to introduce non-aflatoxin strains ofA. jlavus into the crop environment [5]

. This s p ec ies of fungus also demonstrates a variety of scle rotia! types , shows variability in response to li ght, variab le ability to co lon ize liv i ng plant tissue, and has strains residing in multiple vege t a ti ve co mpatibility g roups (YCGs). Aspe r gillusjlavus Strain AF36 (AF36) is registered as a microbial pesticide to reduce the overa ll aflatoxin levels o n aflatoxin-contaminated crops (E PA Reg. No. 071693-1). The microbial active ingredient is a naturally occ urring soil co lonizer , does n ot produce aflatoxins , cyclopiazo ni c acid , or known intermediates in the aflatoxi n biosynthesis pathway and was originally isolated from Arizona cotto n see d (I , 7 ]

. The pesticidal mode of actio n i s by co mp et iti ve "d isp lace m e nt " of aflatoxin-producing Aspergillus s pp [1 , 5 ] ; and is d ete rmin ed by exte n s iv e p op ulation sa mplin g (p roduc t pe rf onnance data) wit h follow-up ve rifi cation of representative microbial iso lates by specialized microbiological ana l yses 

### [1 , 3 J . Th is mode of biological co ntro l is currently the most wide l y used b iocontrol method for reducing aflatoxin contamination in world-wide crops such a s maize and cotto n seed where aflatoxin con tami n at ion is a persistent problem for human and ani mal hea lth (W u and Khlangwiset, 20 I Oa , as cited in (51).

2Product Performance (Efficacy Assessme nt) for Usc on D ried Fig Acreage (Attach m e nt N, [41) Population data were submitted for the use ofAspergillus jlavus AF36 to reduce aflatoxi n contamination of figs in California [4]

. The submitted data are li mited in scope, since discussion of results and information pertaining to methodology (e.g., applicat ion equ ipm ent, application rates and sampling procedures, and specialized microbiological ana lyses [1 , 3]) , experimental design , and other environmental paramete rs (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 samp lin g (conducted 2003

- 2004) of Aspergillus spp. fungal populations 1 (so il , dropped leaves and fig samples) showed trends of successful colonization by AF36 \· V

ithin the research orchard fo llo w in g earl y summer applications of AF36 in 2003 and 2004 at th e rate of ( 10 lbs product / A) to the o rchard floor. A steady increase in percentage r ecovery of i solates belonging to the AF36 group in annual soil samp les was also noted for acreage not treated w ith 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 gro up ; these data are partially r eflected in Table 8 and F ig. 2; Attachment N. BPPD notes also that there was not a s ignificant presence ofA. jlavus S stra in s 2 in the sampled orchard , and there was not a stat i st ica ll y significant displacement ofA. jlavus S and L stra in s or A. parasiticus by AF36 [4].
Product performance could be affected by availab l e soil moisture and temperatures
. Product that is applied in areas that have " moderately wet" so il h as opt im al conditions for sporulation and co lonization as opposed to "very wet" (i.e., directly under drip line emitters) areas, "s lightly wet" areas, or "always dry" areas . Quantitative data to show levels of so il moisture ( i. e . , measured so il moisture tensions) were not provided in Attachment N. Optimal temperatures for sporu lati on 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 obse r ved to peak at 95°F and decline within I to 2 days of the initial peak. Assessment for the 20 15 Emergency Usc of AF36 on Dried F ig s Human/Mammalian Health Assessme n t EPA has not identified any human h ealth issues that preclude the use of AF36 in a Section 18 Emergency Exemption in fig orchards at the prescribed rate of 10 lb/a cre. 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 r ev iews and published Biopesticide Registration Action Documents for AF36. Reviewed studies and rationales by EP

### NBPPD

1 Data were ba sed on quantitative sampling, monitoring and microbiolo g ical ana lyses such as rnorphotyping and tes tin g vegetat i ve compatibility for AF36 )I , 3) grouping for the years 2002-2007 in one CA resea rch ftg orchard. Some of the re sea rch was pub li shed in a se parate paper 121, not includ ed in Attachment N. 2 A

.jlavus S s train s are considered 'toxigenic' and L strains are considered "atoxigen ic" 

### Ill


### 3

### scientists led to the recommendation of AF36 for registrat ion (10 , 11 , 12J

. The conclusions from these stud ie s indi cate that acute ora l and pulmonary toxicity/ pathogenicity studies in rats confirmed th e clearance of Af36 with no evidence of toxici ty or infectivity (l O J. Additionally , intratracheal dosing showed clearance by day eight. These results supported waivers for intravenous , intracerebral , intraperitoneal injection s tudi es , acute dermal toxici ty /pathogenicity , primary dermal irritation , primary eye irritation , and hypersensit i vi ty. I mmune response was also tested in rats, indicating that the tested rat s can clear AF36 from their bodies without compromising their immune system. No hypersensitivit y incidents hav e been reported to date from wo r kers who have been exposed. Overall , no problems with AF36 have been reported , but accord in g to FIFRA 6(a)(2) a ll hypersensitivity prob lems or incident s must be reported by the registrant iJOJ

. To l e ranc e Exe mption 

### According to 40 CF R 1 80.1206 there are tolerance exemptions for a range of crops for Af36 includin g; cotton, pistachio, and corn. Based o n EPA's r eco mme ndation this can be temporaril y extended to include dried figs from June 15, 2015 to Jul y 31 , 20 15 in Fresno, Kern, Madera, and Merced counties in the s tate of California. Envi r o nm e n ta l Assess m e nt for t he 

### 2015 E m e r ge ncy Use of AF36 o n Dri e d Figs 

### The Agency has previously determined that agricultural uses of Af36 will have no adverse effects on terres trial and aquatic vertebrate and invertebrate species, including birds , insect s, honey bees and other pollinators , es tuarine and marine animals , and non-target plant species (lJ

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

### stra in s known to cause honeybee pathogenicity , and avian and mammalian respiratory infections. The expos ure and potential hazard of AF36 colonized-wheat seed to honey bees was assessed for 30 days fo ll ow in g a single aerial application on b looming cotton in Arizona (MRID 45739102 , as cited in [1, 7J). No significant difference in mort a lity between the tes t and control hiv es was see n. No larva l disease 's tonebrood ' 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 pa th ogen icity to rodents from acu te ora l and pu lmonary test in g at a maximum h azard dose. Subm it ted avian inh a lat io n tox ic ity and pathogenicity s tudie s (MR ID No. 45798 102, as cited in ( 1, 71) are s ufftcient to demonstrate that there would be no hazards to endangered birds from the proposed uses ofA

.. flavus 

### AF36

. The ag ricultura l uses of AF 36 are not expected to increase th e t o tal A. jlavus 

### content in the env ironment (1 , 7J. Results of soil and air mon itoring stud ies s howed that a one-time seasonal application of Af36 does not appreciably change the overa ll quantity of A .

. flavus 

### spore load on the environment , within the range of natura l variat io ns (MR ID 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 ofAsp ergillus 161 : A

. jlavus has mo s t frequently been reported , followed by A

. fumigatus , A

. nig e r and other species can kill honey bees (Gilliam and Vandenberg, 1997 , as cited in 1 61): 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 aflatox in- producing strain s (Vojvodic, unpublished , as cited in 16 1).



### 4

### 0.5 to > I 0 5 

### cf u

/g of soil and may vary s ig ni fica ntl y greater th an I 0-fold w ithin a s ingle field in a yea r. A three-year field s tud y co ndu cted by th e USDA demons trated that whi le th e t otal A. jlavus 

### concentration in creased w ith the app li ca ti o n of non-t oxigenic A. jlavus , 

### it declined by the spring of the following year [1 , 7). 

### The total amoun t ofA.jlavus 

### in the so il d id not increase in the long term. S ince the conce ntr ation of natural populations ofA. jlavus 

### were s hown to fluctu ate w ide ly, th e temporary increase in A. jlavus 

### AF36 post a pplication is no t ex pected to produc e effec ts not a lready see n from th e na tura l non-to xigen ic and toxin produ c in g A. jlavus 

### population fluctuation s. Endangered Species Assessment No incremen t a l ex pos ures o f AF36 for e nd a nge r e d wildlife are a nticipated for thi s use, as there we r e no in c r e me ntal haza rd s to nontarg et wild lif e in prev ious assessments 

## p, 2, 7 ]

. Data from so il and ai r populat ion samp lin g s tudie s for AF36 and Aspergillu s 

### conducted in Arizona [1 , 

### 8 ] , California ( 2, 4 , 7 , 9] , and Texas [8 ] demonstrated that th e total am oun t of A. jlavus 

### in th e soil did not increase in the long term, and cou ld fluctuate wide ly. Tempora ry in creases in A.jlavus 

### AF36 post app li cations are not expected to produce effects not a lre ady seen from natural populati ons o f A. jlavus. 

### The r efo re a No Effec t (NE) d etermin a tion was opined in 2007 

### P I to an y e nd an ge r ed/threate ned t e rrestri a l or aq ua ti c s pecies as li s t e d by the U

. S. Fis h and Wildlife Serv ice (USF WS ) and the Nat io na l Oceani c and Atmospheric Adm inistrat io n (NOAA). The NE detennination applies fo r d irec t and indir ect effects to li sted spec ies and th ei r designated critical habitats. For th e c urren t reque s t ed emergency use on dried figs , a No Effect find in g is also supported by the da ta eva lua ti o ns as sta t ed above

. R eferenc es: [ 1). 

### Tomimatsu, G

. and Vait uzis , Z. 2003. E nvi r o nmental Haza rd Assessme nt f or the Microb ia l Pes ti cide , Aspe r g illusjlavus 

### AF36 for Co nditi o nal Regi s trati o n in Arizo na and EU P Exten s io n in Texa s; EPA Reg. No. 07 1693- R; Ba r code Nos. D288777; 0288782 ; 0286708 (2]

. D oster , M.A

. ,T.J. Michailides , L. 

### Doster , P.J

. Cotty. 2008. Biocontrol of aflatox i ns in fi gs. Acta Hort. ( I S H S) 798:223-226. ( 3 ]. Baym a n, P. and P. J. 

### Co tt y. 1 99 1. V ege t ative co mpatibility and genetic varia ti on in the Aspergillusjlavus 

### popul ati o n of a sing le fie ld

. Can. 1. 

### Bot. 69: 1707-17 11. ( 4). 

### CA Dept. of Pe sticide Regulation. 2015. Attachmen t N. Appli catio n of AF36 in a Research Fig Orchard (2002-2007). 14 p. Californ ia autho rization for pesticide use unde r U

. S.E PA Sec tion 18 Spec ifi c Exe mption for Distribut ion a nd Use only w ithin Ca liforni a. (S J. Eh rli c h, K.C

. 20 1 4.Non-afla t oxige ni c Aspe r g illusjlavus 

### to prev ent afla tox in co ntaminati o n in crops: adva nt ages and limita tion s . Front. Micro. http : //dx.doi.o rg/t 0.3389 / fmicb.20 14. 00050

5[ 6 ] Jensen , A.B., K.Aronst e in , J.M.Fiores , S.Yojvodic, M.A. Palacio and M. Spivak. 2013. Standard methods for fungal brood disease re sea rch. J .Ap ic Re s. 52( l ). http: //www. ncbi. nlm.ni h.gov/ pmc/articles/PMC3 816652 / [ 7 ]

. Yaituzis, 

## z
. 2007. Endangered Species Risk Assessment for Aspe r gillusjlavus AF36 Expe rim e ntal Use on Pi s tachio in California (71693-EUP-R). Memo to S. Bacchus. DP Barcode: 335793; D ecis io n No.:371294. No MRID. [8 ]. Gagliardi , J. and J. Kough. 2010 and 201 1. Aspergil/usjlavus AF36 (E PA Reg No. 71693- EUP-2) usc on corn. Memos to S.Bacchus. Deci s ion No.:4 24829; 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 .B acchus. Deci s io n Nos: 444580, 444582; D ata Eva lu a tion R eco rd MRID: 48363701 [ 10 ]. BPPD R ev iew o f Product Che mi st r y and Efficacy D ata to satisfy conditions of Registration for Aspergillus jlavus AF36 [EPA Reg. No. 71693-1 ], September 13, 2007. [11]. BPPD Review of Data Waiver Request s Subm itted by IR-4 on beha l f of th e Arizona Cotton and Re sea rch Co uncil in s upport of the H ea lth Effects ofAspergillus jlavus J\F36 for us e 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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