### MEMORANDUM UNITED STA TE S ENV I RONMENTAL PROTECTION AGE NCY WASH I NGTON , D

. C

. 2 0460

OFFICE OF CHEMICAL SAFETY AND PO L LUTIO N PREVENTION



## MAR 0 4 2016

SUBJECT: Env ironmental ri s k assess ment for the FIFRA Section 3 reg istrat ion of the TGAI 

### Bacillus amyloliquefaciens strain PTA- 4838 (EPA F il e Symbo l 70664

-T) and two end-use products, Yarnim o® WSP (EPA File Symbo l 70664

- A) and Yarnimo ®ST (EPA File Symbol 70664-L); Submission No

. 958320, Decision No. 495952, DP Barc ode No. 424149 

### FROM: I n-Soon You, Ph.D ., Biologist Microb ia l Pesticide Branch Biop es ticides and Pollution Prevention Di vi s ion , 7511 P THROUGH: Shannon B o c ges , Lead B; o Jo g ; st \__"' ' Microb ial Pesticide Bra nc h

# ~**~'\'** Bi o p es ticides an d P o lluti on Prevention Di v isio n, 7511 P TO: M ike Mendelsohn, R eg ul atory Action Leader Microb ial Pe st icide Branch Bi opest icid es a nd Polluti o n Preve nti o n D iv is ion , 7511 P



### Eco lo gical R i s k Assessment
A. Introduction

L id oC hem, Inc. ha s s ubmitted an application to reg iste r a new active in gre dient , 

### Bacillus amyloliquefac iens strain PTA

- 4838 (hereafter, 

### B. amylol iq uefaciens PTA-4838) , for use as a s uppressant against phytopathogenic fung i a nd nematodes a nd a lso for use as plant growth promoter on bu s hes , crops, he rb s , sp ic es , o rn a m e nt als , s hrub s, trees (fo res t and s hade), turf, vegetabl es a nd vines. 

### B. amyloliquefaciens PT A

- 4 8 38 is the active ingredient (a .i. ) in two endu se products (EP s), Yarnimo ®WSP and Varnimo®ST. Yarnimo ® WSP is a water s u s p e ndabl e powder conta inin g 0.29% a. i. at a minimum of2 x 109 cfu/ g; Yarnimo®ST is a p owder

I nternet Address (URL) • http //www epa gov Recycled/Recyc l ab l e • Pnnted w11h Vegetable Oi l Based Inks on 100% Postconsumcr

. Process Ch l onne Free Recycled Papercontaining 73.4% a.i. at a minimum of 5 x I 0 11 cfulg and is a biological seed treatment that provides early protection against selected parasitic nematodes. Members of the genus 

### Bacillus are endospore forming bacteria. Endospores are formed under adverse growth conditions and help the 

### Bacillus species persist in diverse e nvironments (Logan and de Vos, 2009). 

### B. amyloliquefaciens is a common bacterium present in so i l , water and plant materials and is known to produce many secondary metabolites. EPA registered other strains of 

### B. amyloliquefaciens (strains D747 and FZB24) which were reclassified from 

### B. subt ilis var. 

### amyloliquefaciens (Priest et al., 1987; USEPA , 20 I 0; US EPA, 201 1 ). 

### B. amyloliquefaciens PTA-4838 is a versati le bacteriun1 with multiple and unique modes of action

. Th is strain produces many enzymes with special funct ions (e.g., phytase, chitinase , lipase and protease) and several secondary metabolites including antifunga l li poproteins (MRID No. 494741 02). It is a new a.i., and thus requires a review to assess the potential risks to nontarget organisms that may result from its use. This memorandum contains the ecological risk assessment for the registration of the new a.i. and it s proposed uses.
B. S umm ary o f No nta r ge t E ffec ts
Table I provides the status of the Tier 1 Nontarget Organism Data Requirements for the active ingredient 

### B
. amyloliquefaciens PT A-4838 for ecological risk assessment. N ine original guideline stud ie s with the TGAI and two sets of scientific rationale were submitted to meet data requirements for non-target organisms per 40 CFR Part 158.2150. Relevant information from the subm itted a nd cited studies is included in the discussion of ri sk below (see C. Ecological Expos ure and Rj sk Characterization) and Data Evaluation Records (DERs) for the submitted g uid e lin e st udie s are attached for this memorandum. Table I

. Summary of data subm itted to comply with Nontarget Organism Data Requirements per 40 CFR Part 158.1250 in support of the Section 3 registration of 

### B. amyloliquefaciens PTA4838.

D ata R e qu i r e m e n t OPPTS Res ults S umm ary a nd MRID N o . G uid e li ne C lass ifi cati on Avian o ral 885.4050 Ba sed on the two avian o ral 49474109 toxicity/ pathogenicity studies submitted, 

### B. 49474110 

### amy/oliqu efacie ns strain PTA4838 is not toxic or pathogenic to Northern bobwhite 

### (Colinus virginianus) dosed at 9.46 x l 0 9 cfulml/bird/day. However, the birds were not tested at a maximum hazard dose. C lass ifi ca tio n : S u pp l e m e nta l

2

### Data Requirem e nt OPPTS R es u lts S ummary and 

### MRID N o. Guideline C lassification Av ian Oral 885.4050 Scientific rationale provided is 49474111 sufficient to s upport the requested waiver. 

### C la ss ification : Acceptab le Wild mammals 885.4 150 Wi ld mammal test ing was not N / A conducted, but studies required by 40 CFR Part 158.2140 are adequate for assessment of hazards to wild mammals. 

### B. amyloliquefaciens PTA

- 4838 was found not toxic or pathogenic to labo ratory rat s when tested at 3.5 x I 0 8 cfulrat by oral gavage (MRJD No. 49474105) and at 2.7 xI 0 8 cfulrat by intravenous injection (MRID No. 49474 1 07). 

### C lass ificat ion: Acc eptable Freshwater fish 885.4200 A 30-day static renewal test with 49474112 toxicity/ pathogenicity 

### B. amyloliquefaciens PTA-4838 had a negative effect on growth of juvenile Rainbow trout 

### (Oncorhynchus mykiss) dosed at I x I 0 6 cfu/mL and on survival at I x I 0 7 cfulmL. However , fo r l 00% mortalit y observed at 1 x I 0 7 cfu / mL within 15 days of testing , it is not possible to determine whether the effect was see n because of inherent toxicity/ pathogenicity of the a. i. or due to experimental design (e.g., too young fish we re used in the testing ). 

### C lass ific a tion : Supp lemental Freshwater 885.4240 A freshwater invertebrate study 49474113 in vertebrate with 

### B. amyloliquefaciens PTAtoxicity/ pathogen icity 4838 s howed potential adverse effects on reproduction of 

### Daphnia magna at exposure to I x 10 5 cfulmL of test medium in a 21-day static renewal system. Maximum hazard testing was not conducted; however,

3Data Requirem e nt OPPTS Re s ults Su mma ry and MRIDNo. G uid e lin e C lass ifi ca ti on e num e ratio n s indi cated ex p os ure at 10 5 cfulmL. C la ss ifi ca ti on : S upplem e ntal No nt ar ge t In sec t 885.4340 A laborato ry bi oassay with adult 49474117 Test in g ladyb ird be e tl es (Hipp odam ia conver gens) at th e m ax imum hazard do se showed re lat ively h ig h m o rt a lity ( >20%) of B. amyloliqu efac ie ns PTA-4838 to the test organism i n a ll g ro up s in 13 day s of te s tin g . T he res ult s are inconclus ive. C lass ifi cat ion: Unacceptable Non target In se ct 885.4 340 A l aboratory bi oassay w ith adu lt 49474118 Tes tin g parasitic wasp (A phidiu s coleman) at th e m ax imum h azard dose s ho we d tox ic o r path oge nic effects of B. am ylo liquefaciens PTA-48 38 to the test o rg a ni s m in all g roup s w ithin 3 days of tes ting. The diet co n s is ted of 

### l 0 % honey water w ith o r with o ut th e te s t s ub s tan ce. T he s tu dy i s not of suffic ie nt durat io n to be u seful , and resu lt s are inc o nclu s iv e. C la ss ification: Unacceptabl e No ntarge t In sec t 885.4340 A l aboratory bioa ss ay with g ree n 494741 1 6 T es tin g l acew in g larvae (Ch 1 ysoperla rufilabri s) s how e d no tox ic / pathogenic ef f ec t to the tes t organism at 1.1 X I 0 8 cfulc m 2 w ithin 19 day s of tes tin g . The test s ub sta nce a nd in ac tive test s ub stance g ro up s a te > 30% fewe r co rn ea rwo rm eggs (He licoverpa ze a) o n D ay 2 than the un-do sed co ntro l g roup , but the se differences were not rep o rted to be s ig nifi cantl y di fferen t. C lass ifi cati on: Acceptab le H o ney bee tes tin g 885.4380 A 13-day lab o rato ry bioassay 49474 11 9 w ith "yo un g " honey b ees (Apis m e llif e ra ) s howe d a r e latively
4Data Req u irement OPPTS Re s u l ts Summary and 

### MRID N o. Guideline C l assificati on high mortality in all test groups. 

### It was determine d that 

### B. amyloliquefaciens PTA-4838 was not toxic to honey bees at a rate of 1 O

, OOOX MFAR or 1 x 10 11 cfu!mL!sq ft when adm ini stered orally. Classification: Supp l ementa l g iven the duration ( 13 days) of the toxic ity/ pathogenicity testing , the s tated dose rate , the rate of mortality(> 20%) in the control group and the test s ubstance group, and hi g h vari ability shown in the amount of food provided for consumption

. Estuarine/ marin e fish 885.4280 A 30 -day stat ic renewal test w i th 49474114 and invertebrate testing grass shrimp (Paleomonetes vulgaris ) showed no chronic toxic / pathogenic effect of 

### B. amyloliquefaciens PTA

- 4838 when tested at concentrations of I 0 5 cfu/g diet and 10 6 cfu/g diet. T he ECso for Varnimo ®Technical was determined to be > 10 6 cfu/g, based on mmtality observations. While there was no s ignificant difference in the weight or length of th e sh rim p among the test g roup s, contami n ation of the controls makes the results less use f ul fo r a risk assessment. Classification: Supplemental Estuarine/ marine fish 885.4280 A 30 -day laboratory test with 494741 1 5 and invertebrate test in g inland si lverside (Menidia beryl/ina) dosed at 10 6 cfu/mL and 10 7 cfu/mL showed potential chronic toxic and/or pathogenic effect at 10 7 cfu/ mL in a 30-day stat ic renewal system. Based on mmtality data (40% mortality at 107 cfu/ mL) , the LCs o for Varnimo ®Technca l is > 1x 10 6 cfu/ m L. However, the su bmitted
5D a ta R e quire m e nt OP PT S R es u l t s S umm ary and M RID N o. G ui d e lin e C l ass ificati o n study is insufficient to detennine whether other sublethal effects may result from exposure to the active ingred ient. C l ass ificati on: S u pp l e mental Nontarget plant 885.4300 A data waiver from nontarget 49474309 Testing plant testing requirement for 

### B. amyloliquefaciens PTA

- 4838 was submitted. Scientific rationale based on pub li shed literature is not suffic i ent to support no adverse effects to a ll plants

. C l ass ifica t ion : Unacc e pta b l e Additional information is needed to suppo rt claims of nonpersistence and lack of phytotoxicity.
C. Ecolo g i c a l E xpo s u re and R i s k C ha rac te riza ti on
The EP Varnimo®WSP is a water suspendab le powder that is intended for use as a suppressant against certain fungal pathogens and/or parasitic nematodes on bushes , crops , herbs , spices , ornamentals , shrubs , trees , turf, vegetables and vines , and/ or as plant growth promoter. Th is EP can be applied alone or in combination and/or rotati on with chemical fungicides as a tool for integrated disease management i n agricultural crops, ornamentals , nu rsery plants and turfgrass

. Varnimo ® WSP may be used at the following rates : For fo li ar app lications , 3-32 ounces Varnimo ®WSP m ixed in a minimum of25 gallons of water can be app li ed per acre (1.9 x 10 7 cfulmL)

. For drench/drip irrigation applications, 3

- 32 ounces Varn imo® WSP in a minimum of 25 gallons of water can be applied

. The EP Varnimo® ST is a b iological seed treatment that provides early season protection aga inst selected plant parasitic nematodes. For seed treatment, it can be app li ed up to 0

. 09 ounces per 100 lbs seed, resu lting in ca.l x 10 6 cfu/g seed. The EP Varnimo®WSP is recommended to be applied in pre - emergence (e

. g. , 2-7 days pr i or to planti ng) as sprays, by chemigation (e

. g. , sprinkler , drip , drench , flood , i n-furrows and borders), or by direct applicat ions (e

. g . , as pre-planting, at-planting and post-planting amendments)

. There is not much drift from sprinkler irrigation, and in most chemigation applications , watering in is required, which would reduce runoff. All of the chemigation app lications are soil directed and soi l-directed applicati ons w i th Varnimo ® WSP are generally expected to have less potential for nontarget exposure compared to other app li cations (e.g

. , aerial sprays). Additionally, for preemergence uses, the labe l d irects the user to irrigate immediately after treatment and to keep soil mo i st until plant ing

.
6

### For post-emergence applications (e.g., foliar and soil-directed applications), Varnimo® WSP is expected to have more potential for nontarget exposure, since nontarget organisms and/ or their food items may be directly sprayed with the product. However, it is noted that irrigation immediately after application is required on the label to move the product into the root zone, so exposure on foliar surfaces within the treated area will be sho rt liv ed. This does not eliminate exposure to soil-dwelling organisms. Additionally, some drift is expected from foliar sprays, and the label is not explicit about whether aerial applications may be made. Aerial spray applications would be expected to result in greater amounts of the product drifting off-site compared to gro und sp r ay applications. The EP Vamimo® ST is intended for seed treatment which is likely to result in low exposure of nontarget organisms to the product. Birds may be exposed by direct consumption of seeds , and soil-dwelling organisms may also be exposed to some degree. It is noted , however, that 

### B

. 

### amyloliquefaciens occurs naturally in soil, water and plant materials, so so il-, water- , and plantborne non-target organisms in those areas currently have some exposure to the existing B. 

### amyloliquefaciens strains.

Birds and Mammals The guideline study s ubmitted for the avian oral toxicity/pathogenicity testing requirement showed no adverse effects in birds (Northern bobwhite , Colinus virginianus) 

### when tested at 9.46 x I 09 

### cfu/ mllquaillday. 

### B. amyloliquefaciens strain PTA-4838 is not known to be pathogenic to birds. The spray concentration resulting from the hi ghest appl ication rate (32 oz) mixed in the minimum amount of water (25 gallons) based on the Varnimo WSP label is 1.9 x 107 

### cfu/mL (the product contains at least 2 x I 0 9 

### cfu/g). This concentration is below the level at which birds were tested and shown to have no adverse effects. Exposure by direct consumption of treated seed is also expected to be below this level, since a bird would need to consume> 1 OOOg of seed per day to achieve the same level of exposure as the birds in the study describe above. Based on expected field application rate and lack of adverse effects observed in birds exposed to 

### B. 

### amyloliquefaciens stra in PTA-4838 at the level tested, ri sk to nontarget birds resulting from the proposed registration is not expected. Since birds were not tested at the maximum hazard dose, any future increases in application rate of B. 

### amyloliquefaciens PTA-4838 would need to be evaluated fo r bird risks. An acute oral toxicity/pathogenicity study with laboratory rats indicated no adverse effects of 

### B. 

### amyloliquefaciens strain PTA-4838 when administered at a dose of 3.5 x 10 8 

### cfu/rat (MRlD No. 49474105). An 

### acute intravenous inj ection study (MRlD No. 49474107) was also s ubmitted, which s howed that B. 

### amyloliquefaciens strain PTA-4838 was non-toxic and not infective to rats at a dose of2.7 x 10 8 

### cfulanimal (USEPA , 2016).

7Some wild mammals may be exposed to 

### B. amy/oliquefaciens s train PT A-4838 in treated areas ; however, adverse effects wer e not observed in the mammalian stud ies. Therefore , risk to w ild mammals is expec ted to be low from the proposed reg is tration of th e new a .i. Freshwate r F i s h and Inv e rtebrate s A g uide line s tud y s ubmitted for the non-targe t fres hwater fi s h testin g requirement showed an effect of 

### B. amy loliquefaciens PT A-4838 on 

### Oncorhynchus mykiss ( rai nbo w trout) when te sted at I 

### x I 0 6 cf u

/ mL and I x 107 cfulrnL. The a .i. was fo und to have an a dv e rse effect on survival (e.g., 1 00% m o rt ali ty within 15 days oftesting) ofthe tes t o rgani s m s at 1 0 7 cfulmL, and an adverse effect on growth (e.g. , weight and length ) of the test organisms at 10 6 cfulmL. 100 % m o rtality see n at I 0 7 cfu/ mL , but not at 10 6 cfulmL, app ears to be dose-dependent, hence the a.i. s hows a toxic effect und e r the conditions tested. Howeve r, for o th er observati o ns (e .g. , growth rate , ab no rm a l behavior / appearance , and weig ht), it is not po ss ibl e to determine whether the effec ts we re see n because of inherent toxicity/ pathogenicity of the a. i

. or due to experimenta l de s ig n. The study report indicates that the water in th e 107 cfu/ mL test s ubstance group was heav il y cloudy with no v isibility on many days. The nec r o p sy s howed that man y fish i n this group s howed discolo ration , indicating st r ess. Many of t he fi sh a lso died s uddenly ( e.g. , 10 died in o ne repli cat e o n day 5 due to loss of air supply , 19 died o n day 15 for reaso n s not explained). No follow-up work was performed to determin e i f the m orta lit y was the res ult of pathogenic effects. It i s p ossi bl e th at the effect s observed were due to toxicity and/o r pathogenicity , but may also hav e been the re s ult of the physical co nditi o n s produced by the high conce ntratio n of th e test s ubstan ce . A g uid e line s tudy fo r the non-target fres hw at e r inverte brate t es tin g indicated no sig nificant effect o n m ob ility ( indicato r of mortality), but showed a p o t entia l toxic/ pathogenic effec t of 

### B. amy loliqu efacie n s s train PT A-4838 on reproduction of 

### Daphnia magna whe n tested at 1 x 10 5 cfu/ mL

. The maximum haza rd dose (testing at 1 x 106 cfu a.i. / mL ) was not u se d in the study , and old a nd n ew te s t media were contaminated in so m e cases. However, the contamination was not d esc rib e d as the test s ubstanc e, and it is po ss ible th at other microbes entered th e non-ste ril e test e nv iron m ent. It is unclear whether there may be effects from the contamination that was de t ec ted ; however , conclu s ions r egardin g lack of m or ta lity are cons ide red reliable.
T he EPA Standard Wet land , which i s a we tl and of l ac re in s ize and 15 em in de pth , can be u sed to determine a worst-case sce n ari o esti mate for EEC in water . T he EEC for water is calculated to b e 3 x I 0 3 cfu/ml ((32 ozs (product/ac re) X (28.35 g/oz) X (2 X I 0 9 cf u/g pro duct) X (1 ac re/40 , 468 ,5 64 c m 2 ) ] ..;- 15 e m )

. Us in g the EP A Standard P ond , wh ich is a pond of 1 acre in size and 6ft ( 1 82.88 em) deep , the EEC wou ld be 2.5 xl0 2 cfu/mL. To gain a more realistic EEC based on the pote nti a l for sp r ay drift , the T i er I aeria l agricu ltural app li cation sce nari o in the Ag DRI FT s pray drift m ode l can be applied to these numb e rs. Under that scenario assuming fine to m ed ium droplet size , a maximum of 12 .5% of the amount applied is expected to reach aquatic ar eas at th e water ' s edge. The refore , th e EEC for th e wetland and pond can be refi ned to 3.8 x
8102 cfu/mL and 3.1 x 1 0 1 cfu/mL , respectively, and this exposure is expected to be even lower with ground spray applications. Since the potential for exposure falls three orders of magnitude or more below the 10 5 cfu/ mL from the 

### Daphnia study described above, and the I 0 6 and I 0 7 cfu/mL in the rainbow trout study above , adverse effects to aquatic animals in freshwater systems are not expected.


### B. amyloliquefaciens PT A

- 4838 produces a biosurfactant (surfactin), chitinase and other secondary metabolites (MRID No. 494741 02). Surfactants are reported to be toxic to fish and invertebrates (Abel , 1974; Abe l and Ski dmore, 1975) and chitinase can be toxic to invertebrates containing chitin as a structural component. Some adverse sublethal effects were observed in the stud ies describ ed above , and there is some uncertainty as the level of exposure at which such effects might occur. These metabolites likely were included in the test material used in the studies above , and they may have contributed to the effects observed. However , as described above , exposure to the a.i. in aquatic environments is expected**to~** 1 OOOX below the levels tested. Therefore , any effect of these metabolites on aquatic organ i sms is expec ted to be minimal. Since the maximum hazard dose was not tested in the 

### Daphnia study, any futu re increases in the applicat ion rate would need to be reevaluated for potential effects o n these aquatic organi sms. Mar ine/Estuari ne Fish and Invertebrates The app li cant submitted two guideline studies to meet the requirement for nontarget marine / estuarine fish and inver1ebrate testi ng. This testing is cond it io nally required if significant exposure of nontarget marine/ estuarine fish and invertebrates to 

### B. 

### amyloliquefaciens strain PTA-4838 is expected; however, EPA did not spec ifically requ ire these studies. A 30-day static renewa l test was conducted with grass shrimp 

### (Paleomonetes vulgaris) to determ in e the chron ic toxicity/pathogenicity of 

### B. amyloliquefaciens PTA-4938 at concentrations of 10 5 cfu/g diet or I 0 6 cfu/g diet. The active ingredient was shown to have no significant difference in mortality and in the we ight/length of the shr imp among different test gro up s under the cond itions tested. The EC50 was determined to be > 10 6 cfu/ g , based on mortality observations. However, because of contan1ination in the un-dosed contro l and the steri le filtrate contro l , it is not possible to discern whether the test material had an effect on weight or length. A 30-day static renewa l test was conducted with inland si lve rside 

### (Menidia beryllina) to detennine the chronic tox icity/pathogenicity of 

### B. amyloliquefaciens PTA-4938 at concentrations of 106 cfu/ mL or 10 7 cfu/mL in the test water (these groups were also exposed via d iet at 10 5 cfu/g and 10 6 cfu/g diet , respectively). A mortality rate of 40% was shown in the test organisms when exposed at 10 7 cfu/mL, but no s ign ifi cant mortality (96.7%) was seen whe n exposed at 10 6 cfu/mL. Based on mortality observations, the LC50 is determ ined to be> 10 6 cfu/mL. 

### B. 

### amyloliquefaciens PTA-4938 s howed no statistically s ignificant difference in the we ight or length of the s il verside among different test groups when tested at! 0 6 cfu/mL or l 0 7 cfu/mL. The test organisms in all groups lost 40-60% in weight and 9- 1 3% in length

. It is not clear
9whether these are sublethal effects that resulted from exposu re; however, si nce the fish were shown to have shrunk in length (an unusual result for studies with these an imal s). These observat ions suggest a husbandry issue or poor measu r eme nt technique at the testing laboratory. Contaminat ion with potential test substance in the un- dosed control and ste rile filtrate groups , introduces uncertainty in the observations of subletha l effects, so these results are not considered reliable Similar to a case with the nontarget organisms in f reshwater envi ronmen ts, 

### B. amyloliquefaciens PTA-48 38 is not expecte d to reach marine/estuarine env ironme nt s in s ig nificant quantities , and exposu re is ex p ected to be > 1,OOOX below the levels at which effec ts were tested in the above studi es . Eve n thou g h there is uncertainty related to th e s ubl ethal observations in these studies, adverse effects are expected to be very minimal , gi ven the low level of exposure in these e nvironm e nt s . Nontarge t In sects and Honey Bees A guideli ne study submitted for the requireme nt for nontarget in sec t testing showed an effect to ladybird beetles 

### (Hippodamia convergens) upon a 13-day dietary exposure to corn earworn1 

### (Helicoverpa zea) eggs sp rayed with 

### B. amylofiquefaciens PTA-4838 at the maximum hazard dose. Adult ladybird beetles were used and the ir age was not specified. The use of the adult insects of unknown age can affect the stud y results. Relatively high mortality rates(> 20%) were shown in a ll groups, including the control group, in 1 4 days , but toxicity of the test substance could not be clearly demonstrated. The results of th e study are inconclusive and the study is unacceptable.
A guideline stud y with g reen lacewing 

### (Ch1ysoperla rufllabris) larvae did not show a toxic/ pathogenic effect to the test organ ism in a 19-day dietary ex po s ure at the maximum hazard do se of 

### B. a m ylo liqu efac iens PT A -48 38

. There appears to be mu c h less (30%, Tab le 3 of the MR lD No. 49474116) amount of eggs consumed in th e inactiv e and active test substance groups than in th e un-do sed control group, but these differences we r e not re ported to be sig nificantly different. A 3-day bioassay stud y with parasitic wasp 

### (Aph idius colemani) showed toxic effects of 

### B. amyloliquefaciens PTA-4838 to the test organism when t ested at the maximum hazard dose. The study ended in 3 days due to high mortality observed in the control group (77%) and >90% in both the test substance and inactive test substance groups. The results are inconclusive as no differences in mortality were found among different test groups. A guideline s tudy with h oney bees 

### (Apis mellifera ) was conducted for 13 days. 

### B. amyloliquefaciens PTA-4838 was determined to be not toxic to honey bees when admi ni stered orally at a stat ed concentration of 1 O,OOOX the maxirmun field appl icati on (MFAR) or 1 x I 0 11 cfulml/s q 

### ft in 50% s u gar solution

. 

### It is noted that th e Agency ca lculat ion indicate s the do se rate is mu ch low e r than the state d 1 O,OOOX MFAR, but was s till we ll above the maximum hazard 10

### dose. The mortality observed in the control group and the test substance group on day 13 were 23% and 25% , respectively. It was stated in the study (MRID No

. 47474119) that "young" bees were tested, but spec ific in formation regarding age was not provided. When young bees are used , th e bees need be newly emerged in o r der to obtain the results useful for a risk assessment. Exposure to honey bees may occur with applications to foliage; however , based on the study, potential hazard to bees is not expected. Exposure to nontarget insects is expected in the treated areas. Nontarget in sects may be directly sprayed within treated areas, or they may contact treated surfaces

. Exposure through contact with treated foliage is expected to be short- li ved within areas targeted for treatment, since the label requires i mm ed iate irri gation of treated p lants to move the product off of foliar s urfaces and into the root zone . T herefore, insect exposure in the treated area is primarily expected to occur through direct contact w ith spray during app li cation or through contact with soil in treated areas. Within so il , exposure is expected to be somewhat spatially limited , since 

### B. amyloliquefaciens PTA-4838 is expected to inhabit plant roots once applied. 

### B. amyloliquefaciens PTA-4838 may persist with formation of endospo r es; however, it is noted that 

### B. amyloliquefaciens is a common inhabitant of soi l and nontarget insects likely are already exposed at some leve l to 

### this bacterium. Foliar sprays are likely to result in exposure to nearby areas through drift of the applied product. In these areas , exposure w ill be lower , since a fraction of the sprayed amoun t is expected to drift. However, unlike treated areas that will be immediately irrigated after treatme nt , 

### B. amyloliquefaciens PTA-4838 is expected to remain on fol iar surfaces for a l onger period of ti me after app li cation. Therefo r e, exposure at some level cannot be avo ided in these areas. While exposure is expected to be somewhat lim ited in treated areas , the potential for exposure to nontarget in sects cannot be ruled out. Data available for other nontarget insects are incomplete, since two of the subm itted stud ies were inconclusive. One study ind icates th at applications of 

### B. amyloliquefaciens PTA-4838 may be relatively safe for certa in nontarget in sects. However, as described above, 

### B. amyloliquefaciens is known to produce secondary metabolites and/ or enzymes that may adverse ly affect in sects, and several recent reports in the li terature indicate the potential for use of this bacterium or its biosurfactants in insect contro l (Gadhave and Gange, 2016; Khedher et al., 2015; Li et al., 2015; Yun et al. 20 1 3). On the other hand , 

### Bacillus amyloliquefaciens has been isolated from the gut of certain insects (see US EPA, 201 0). Concerns for nontarget in sects had previ ou s ly been raised for a currently registered strain of 

### B. amyloliquefaciens, but EPA determined that the weight of the evidence indicated t hat adverse effects were not expected (US EPA , 201 0)

. However, given the vary in g information regardi n g the potential for adve rse effects on nontarget insects, including more recent reports, the lack of acceptab le data on this spec ific strain, and the potential for exposure particularly associated with fo li ar sprays , adverse effects to nontarget in sects cannot be rul ed out with high certai nty . Remova l of fo liar sprays from the proposed label would s i gnificantly reduce exposure to nontarget insects bot h w ithin the treated areas and in adjacent areas. This applicat ion method may be better supported in the future with additional acceptable tes tin g on insects.

11

### Nontarget Plants A guideline study for Nontarget Plant Testing, Tier I was not submitted, but a data waiver was requested for the new a.i. 

### B. amyloliquefaciens grows in association with plants (Alvarez et al. , 20 II) and is a plant growth-promoting bacterium (Chen et a l. , 2007), but is not a known plant pathogen. Few adverse effects of 

### B. amyloliquefaciens on plants have been reported by the applicant from the published literat ure on existi ng 

### B. amyloliquefaciens stra in s on greenho u se, growth chamber or field studie s (M RJD No

. 49474309). Sc ie ntifi c rationale submitted for a waiver of the req uireme nt for Nontarget Plant Testing s hould be strengthene d as some referenc es were e ithe r too ge neral or outdated to be useful. The rational e provided was in s uffic i ent to co nc lud e that the proposed use s of 

### B. amyloliquefaciens PTA-48 38 are not expected to result in increase d expo s ure to, or adverse effects in nontarget p lant s of eco nom ic i mportance. 

### Bacillus amyloliquefaciens produces secondary metabolites that may have adverse effects on certa in nontarget organisms (e.g., fish, aquatic in vertebrates, in sects
- see discussion above). Its reported biology (e

. g., an endospore former) and th e w i de range of use si tes and application methods may argue again st the applicant's claim of no adverse effects and nonp ersistence of the active ingredient in all environments
. Additionally, Ta lboys et al. (2014) reported reductions in inorganic phosphorus (Pi) uptake by 

### Triticum aestivium seeds treated with 

### B. amyloliquefaciens FZB42, resulting from the act ion of auxin produced by this st rain in low Pi enviro nments. These effects were not observed in high Pi environments, where the effect was observed to be beneficial. Whi le this paper may not signal potential fo r adverse effects in all conditions, the auU1ors conclude the imp ortance of under standi ng bac t eria-p lant interactions when deciding among pesticide alternatives. More information like this wou ld better i nform users of biopesticide products and also future pesticide risk assessments. Exposure to nontarget plants is expected with the proposed reg is tration of 

### B. amyloliquefaciens PTA-4838 , particularly w ith folia r sprays that may cause dri ft o r to nontarge t plants on the field that may co ntac t 

### B. amylol iquefaciens in the soil. 

### B. amylolique.faciens is not a known plant pathogen and is a common soil bacterium that ha s a close assoc i ati on with many plant species

. Adverse effec ts to nontarget plants also have not been documented. Th eref o re, adverse effects are not anti c ip ated from the proposed re g istrati on of the products containing this a . i.
D . E nv iro nm e n ta l Pe rs is te nce 

### Bacillus amyloliquefaciens is a common bacterium found in soil , water and plant materials. Like other 

### Bacillus species, it fo rm s endospores und er adverse growth conditions. Endospo re s can survive extreme env ironmental conditions (e.g. , heat , desiccati on and chem ic als) that would normally kill the bacterium , wh ich makes the bacterium persistent in diverse envi ronment s . It 

### is also known to produce spec ia l enzymes and secondary metabolites , inc luding antibiotics and ant ifungal lip oprote in s (Alvarez et al., 2011; Chen et a l. , 2007).



### 12

### Bacillus amyloliquefaciens PT A-4838 is a nutritionally and biochemically versatile bacterium with multiple and unique modes of action. This strain produces many enzymes with special functions (e.g

. , phytase, chitinase, lipase and protease) and several secondary metabolites such as fengycin, iturin, and surfactin (MRID No. 494741 02). The EPs likely contain both cells with endospores and vegetative cells. Therefore, the active ingredient is expected to persist in the environment for a prolonged period once introduced. Whether it persists at levels that are likely to have pesticidal benefits or adverse effects is uncertain.


### E. Fe d era ll y L i s ted Threa ten ed an d E nd a n gere d S p ecies
Based on available information, EPA has determined that no adverse effects are anticipated for birds, mammals, fish , aquatic invertebrates , and plants exposed to 

### B. amylofiquefaciens PTA4838 as a result of the proposed labeled applications

. Therefore , a "No Effect" determination is made for direct and ind irect effects to listed species of these taxa and their designated critica l habitats resulting from the proposed registration of 

### B. amyfoliquefaciens PTA-4838. Concerns were raised for nontarget insects for the proposed registration of 

### B. amyloliquefaciens PT A

- 4838 as labeled, due to uncertainties related to lack of acceptable data and reports of potential effects in the literature. Therefore, direct effects to listed insects cannot be precluded , nor can indirect effects to listed obligate insectivores whose food supply may be affected. However , adverse effects are not expected to nontarget insects if foliar applications are removed from the proposed Yarnimo ® WSP label , as this is expected to significantly reduce exposure to nontarget insects. In such case, EPA could make a "No Effect " detennination for direct effects to listed insects and for indirect effects to listed obligate insectivores.


### Refe r en ces Abel , P

. O. 1974. Toxicity of synthetic detergents to fish and aquatic invertebrates. Journal of Fish Biology , 6: 279-298. Abel, P.D. and J.F. Skidmore. 1975. Toxic effect s of an anionic detergent on the gills of rainbow trout. Water Research, 9: 59-65. Alvarez , F

. , M. Castro, A. Princ ipe, G. Borio li , S. Fisher , G. Fischer, G

. Mori and E. Jofre. 2011. The plant-associated 

### Bacillus amyloliquefaciens strains MEP218 and ARP23 capable of producing the cyclic lipopipetides iturin or surfactin and fengycin are effective in biocontrol of sclerotinia stem rot disease. Journal of App lied Microbiology, 112: 159

- 174. Chen , X.H. , A. Koumoutsi, R. Scholz, A. Eisenreich, K. Schneider, I. Heinemeyer, B. Morgenstern, B

. Voss, W.R

. Hess, 

### 0

. Reva, H. Junge, B. Voigt, P

. R. Jungblut, J. Yater, 

### R. Sussmuth, H. Liesegang, A. Strittmatter, G

. Gottscha lk and R. Borriss

. 2007

. Comparative

13analysis of the complete genome sequence of the plant growth -promotin g bacterium 

### Bacillus amyloliquefaciens FZB42. Nature Biotechnology , Advance Online Publication pages 7-14.
Gandhave, K.R. and A.C. Gange. 2016. Plant-a ssociated 

### Bacillus spp. A l ter lif e-history traits of the specia list insect 

### Brevicoryne brassicae L. Agricultural and Forest Entomo lo gy. 18:35-42. Khedher, S

. B

. , H. Boukedi, 0. Kilani-Feki , I. Chaib , A. Laarif , L. Abdelkefi-Mesrati and S. Tounsi. 2015

. 

### Bacillus amyloliquefaciens AG 1 biosurfactant: Putative receptor diversity and hi stopathological effects on 

### Tufa absoluta midgut. Journal oflnvertebrate Patholo gy , 132 : 42- 47.
L i, H. , M.A. Soares , m . S. Torre s, M. Bergen and J.F. White. 2015. E ndoph yt ic bacterium , 

### Bacillus amyloliquefaciens, enhances ornamental hosta resistance to disease and insect pests. Journal of Plant Interactions , 10: 224-229. Logan, N.A

. and P. de Vos. 2009

. Geneus 

### I. Bacillu s . Pp. 2l-128ln: P.de Vos, G.M. Garrity , D

. Jones, N. R. Krieg, W. Ludwig, F.A

. Raine y, K.H

. Schleifer, and W. Whitman (Eds.) Bergey's Manual of Systematic Bacteriology, Volume 3, 2nd Ed. Springer , New York.
Priest , F.G

. , M. Goodfellow , L.A

. Shute , and R.C.W. Berleley. 1987. 

### Bacillus amyloliquefaciens sp. nov., nom. Rev. Intern ational Journ a l of Systematic Bacteriology , 37: 69- 71. Talboys P.J., Owen D.W. , Healey J. R

. , Withers P.J. , and D.L. Jon es. 20 14. Auxin secretion by 

### Bacillus amyloliquefaciens FZB42 both s timul ates root exudation and limits phosphorus uptake in 

### Triticum aeslivum. BMC plant biology, 14: 51 US EPA. 2010

. 

### Bacillus subtilis Final Registration Review Decision Case 6012. Docket No. EPA- .HQ-OPP- 2007-1 026. Washington , D.C

. US E P A. 201 1. Memorandum from S. Borges (OPP/BPPD) to S. Cerrelli , Re: Envi ronmental risk assessment for the FIFRA Section 3 registration of B. amyloliquefaciens strain D474 (EPA File Symbols 70051-RNI and -RNT); Subm ission No. 879171, Decision No

. 437753 , DP Barcode No. 382915. D ated October 3 , 2011. US EPA. 2016

. Memorandum from M. Djurickovic (OPP/BPPD) toM

. Mendelsohn, Re: R eview of 5 B atch E num eration , Product Chemistry , Manufacturin g Process, Storage Stability, Acute Toxicity Studies , and Tolerance Exemption for Varn imo®Technica l / ST (EPA Reg. No. 760644-T) submitted by Lidochem Inc. Draft dated March 4, 2016. Yun , D.C., S.I. Yang, Y

. C

. Kim, I.S. Kim and Y.H. Kim. 2013

. Identification of surfactin as an aphic id a l metabolite produced by 

### Bacillus amyloliquefaciens G 1. Journal of Korean Society for App li ed Biolo gical Chemistry, 56: 751-753.
14