EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 1 of 34 

MEMORANDUM 

TO: 

FROM: 

PEER REVIEW: 

SUBJECT 

UNITED STATES ENVIRONMENTAL PROTECTION AGENC'I 

WASHING' ON DC 2,)460 

AUG 2 "1" i".-li 

/' .' j 

Susanne Cerrelli, Regulatory Action Leader 

Microbial Pesticides Branch 

Biopesticides and Pollution Prevention Division, 751 IC 

Robyn Rose, Entomologist 

Microbial Pesticides Branch 

Biopcsticides and Pollution Prevention Division, 751 IC 

Gail Tomimatsu, Ph.D., Plant Pathologist / ,,' (:'j\, , Microbial Pesticides Branch 

Biopesticides and Pollution Prevention Division, 7511 C 

llFFICE ()\ 

pm 'JFt\ TION, PESTICIDES AND 

I '.<Ie SUBsTANCES 

I 

" 

Technical review summary of non-target, beneficial insect, avian and 

aquatic studies submitted by Kemira Agro Oy in support of the registration 

of Primastop® (EPA Reg. No. 64137-8) Gliocladium calenulatum Cell 

Mass (Technical Product) for outdoor uses. Barcode Nos.: 0244243 & 

0267225; Submission Nos.: S539189 & S580869; Chemical No.: 021009. 

STUDY IDENTIFICATION 

I. M RiD #:445014-0 I. A Dietary LC50 Toxicity and Pathogenicity Study with the 

Northcl11 Boin'.hite, Colinus virginianus. 

2. MR!D II 445014-()2. A Contact and Dietary LC,o Toxicity and Pathogenicity Study 

with the Rainh,m Trout, Oncorhynchus mykiss Under Static Renewal Conditions 

3. M RID #445014-03. A 21-Day Life Cycle LC,o Toxicity Test with the Cladoceran 

Daphma mugllil under Static Renewal Conditions. 

4. M R! [) #445014-04 .A Dietary LC,o Toxicity Test with the Green Lacewing, 

Chrysoperlu I',.' mea 

nlerne1 AddrEhS (Ulil_) • http:-',"w·t .. w.epa.gov 

,:, ·<t'Recyclable ·1 'nnlp,! 'Nitr: I/f'Wotal:ll! 'II 8ihHd Ink·.; ,':\ HH yel"d Paper (M,rmnurTl :JUSc Posl( onO:UfJi','1 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 2 of 34 

5 . .\1f{J[) #445014-05. A Dietary LCso Toxicity Test with the Parasitic Hymenopteran, 

iVason hi vi.1 ri (JCIl n is 

6. M R ID 11445014-06. A Dietary LC,o Toxicity Test with the Ladybird Beetle, 

Hippodal1ll'f ,',mvergens 

7. \1RlD #.445014-07. A Dietary LC,o Toxicity Test with the Honey Bee 

Apis melli/"!',, 

8. MRID #44183-20 . Non-Target Plant Literature Search 

CLASSIFIC,\ liON: Acceptable 

BACKGROliND: 

GliocladiulII (illenulalum cell mass (Registration #64137-1) is currently registered for indoor 

greenhouse W;C. Kemira Agro Oy has submitted ecological effects studies required by EPA to 

register microbial pest control agents for outdoor use .. The end-use-product is called Primastop 

Biofungicidc Powder with the active ingredient listed as 37% CT/iocladium catenu/atum Strain 

J 1446 (contains 1.0x lOX CFU/gram) 63% inert ingredients. 

The intended uses of Prima stop are to control "damping-off, seed rot, root and stem rot, wilt and 

certain foliar dIseases caused by Rhizoctollia, P)'thium, Fusarium, Didymella, Bollyt;s, 

VerticilliulIJ. Aiternaria, Cladosporium, Helminthosporium, Penicillium, and Pi/curia on 

greenhouse or !Ield grown vegetables, herbs, ornamentals, turf, tree and forest seedlings." 

Primastop Illa\' be applied through incorporation in potting media, treatment of cuttings, bulb or 

tubers, as a "Oil drench or foliar spray. 

Three insect species including a neuropteran (Green lacewing, Chrysoperla carneal, parasitic 

hymenopteran I.tvasonia vitripenllis), a coleopteran (Convergent lady beetle, Hippodamia 

convergensl, and honey bees (Apis mellijera) were tested. In addition, potential effects of 

GliocladiulII c,lIcnulatum cell mass on a representative avian species ( northern bobwhite quail; 

Colinlls virgll1,al1us), freshwater fish species (rainbow trout; Oncoryhynchus mykiss) and aquatic 

invertebrate (L Jal'hnia magna) were evaluated. These are appropriate test speci<:s for the 

registration Ilf Illlcrobial pest control products. 

CONCUJSIO'lS: 

Ellvi[QI]lIlelltal Fate 

An AGRlCUi \ search conducted by Kemira Agro Oy using the key words Gliocladium 

catenlilatulII and Illsects resulted in no reports of harmful effects of CTliocladium catenulatum on 

insects (MRID if 441893-21). The only result of this search showed the potential for 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 3 of 34 

Gliocladiufll ,'(/fCllu/alum to degrade the insecticide aldicarb. A second AGRICOLA search was 

conducted IIsi ng the key words Gliocladium catenu/alum and avian, bird, shrimp, mollusk, or 

fish (MRID #441893-19). There were no items found by this search for shrimp or fish. One 

item was ttJlmd tor mollusks. however it addressed Gliocladium Vlrens and Gliocladium 

deliquescell,'; rather than Gliocladium catenulatum. A search using key words "avian" and "bird" 

resulted in 110 items relating to Gliocladiwn catenulalum. However, two items were found 

relating to imoi/avian and Gliocladium, These items did not relate to Gliocladium catenulatum. 

Studies idenli lied from the literature searches (MRID #441891-19 and 441893-21) do not 

suggest any potential for adverse effects presented to non-target species by Gliocladium 

catelllliatwJl applied in the field. 

Environmenlal fate studies are not required at this time since no adverse risks were triggered in 

Tier [Non-target Organisms Studies. Gliocladium catenulatum strain Jl446 is a naturally 

occurring saprophytic soil fungus; as a species, it is found throughout the world in a variety of 

soil types, without pathogenic incidence to plant or animal. The ubiquitous nature of the 

organism suggests that the envirorunental burden is not likely to increase substantial.iy to pose 

adverse risb 10 nontarget organisms as a result of the intended outdoor uses. 

Members ol'th" genus have been reported as fungal biocontrol agents, e.g., Gliocladium virens; 

others may he involved in pathogenesis of some plant species: ornamentals (G. vermoeseni : 

Gliociadiulll seem rot); citrus (Ci. roseum: pink mold) and carrot (G. roseum: hard rot). 

Estimated Environmental Concentration of Gliocladium catenulatllm Cell Mass: 

Based on the Illtended rates of application, the estimated environmental concentration of 

Gliocladium ('(Jtenulatllm Cell Mass is not anticipated to exceed - 500 mg/L for soil 

incorporation applications; and c~200 mg/l. for soil drench applications. 

Ecol.qgjcal Effects 

Results of non-target avian, freshwater fish, daphnia, honey bee and beneficial unseet studies 

submitted to tile Agency generally demonstrated a lack oftoxieity or pathogenicity to the 

indicator species. Results of these studies indicate that minimal toxic or pathogenic effects to 

terrestrial or n";:shwater wildlife are expected from currently intended applications of 

Glioe/adi1lm C,/fclluiat1lm cell mass. Summaries and conclusions of these studies arc presented 

below. 

OPPTS Guideline 885.4050: Terrestrial Avian (MRlD# 445014--01): 

Under condit !OilS of the reported study, no adverse effects were exhibited by juvenile northern 

bobwhlte ann .10 clays when administered (by oral gavage) daily doses of 1 x 1 ()4 Gliocladium 

catel1ulatulll-clllig body weight/day for a five day period. This dosage is approximately 

equivalent to a lotal dosage of7 x I ()O cfu/g body weight tor 5 days. The no observed effect 

dosage of (jiwc/"dium catenll/atum cell mass was determined at approximately 1.4 x 10" cfu/g 

body weight 1;:1' five days. There were no evidences of test substance replication, Icswns, 

3 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 4 of 34 

plaques or other indications of infectivity or pathogenicity for the groups treated with 

Gliocladiul!I ,«tenulatum cell mass in gross necropsies. This study showed a lack oftoxicity or 

pathogenic; I Y 0 r Ciliocladium ciltenulatum cell mass to northern bobwhite quail. 

This study is sCIentifically valid, satisfies current guideline requirements, and is acceptable for 

assessing shoJI -Ierm toxic and pathogenic effects of Glioc/adium catenulatum Strain .11446 to 

terrestrial a\ ;'111 species. 

OPPTS Guideline 885.4200: Freshwater Fish (MRID# 445014,,02): 

Under static-l .. ,newal conditions, the no-observed effect concentration was 179 mg/L (1.3 x 108 

cfu/L) for juvenile rainbow trout dual-exposed via aqueous tcst solutions and in a daily diet for 

30 days. The no mortality concentration was 357 mg/L (2.5 X I 0' dulL). The LC50 was 

calculated as ',04 mg/L (3.5 x 1 OS cfu/L). 

Therc were 110 signs of infection in fish tissues examined for necropsy, and all appeared 

internally and externally normal. Ciliocladium catenulatum was isolated from the hemi, liver and 

brain samples or fish treated with 179 mg/L test substance; and from the kidney, heart, liver and 

brain samples of fish treated with 357 mgiL test substance. Although the test substance was 

present, target organs did not appear to be affected, and there were no apparent effects upon the 

health of the r,,;h. 

This study IS scientifically valid, satisfies current guideline requirements, and is acceptable for 

assessing Sh"'l'.-tcrm toxic and pathogenic effects of Gliocladium catenulatum Strain .11446 to 

freshwater Ii sl, 

OPPTS Guideline 885,4240: Daphnia magna (MRID# 445014-0~: 

Under static'l'cnewal conditions, the 21-day EC,o value for survival of neonate Daphnia magna 

exposed aqucously to Gliocladium catenulatum was 7.8 mg/L (5.5 x 10° cfu/L). The LOEC and 

the NOEC Wei" 11 mg/L and 5.6 mg/L, respectively. 

Of the three p,;:'ameters assessed (survival, growth and reproduction), reproduction proved to be 

the most sellsiHc parameter. The EC,o value was 7.8 mglL (~·5.5 x j06 cfU/L); and the NOEC 

and the LOEC \\"re 2.8 (2.0 x 10" cfu/L) and 5.6 mg/L (3.9 x 10" cfu/L), respectively. 

This study is s,:ientifically valid, satisfies current guideline requirements, and is acceptable for 

assessing shorHenn toxic effects of Giiocladium catenuiatum Strain .11446 to aquatic 

invertebrates The pathogenicity (or infectivity) of Gliocladium catenulatum was not evaluated 

in this stud\' 

OPPTS Guideline 885.4300: Non Target Plants (MRID # 44183-20): 

A review of the literature resulted in the utility of Ciliocladium catenulatum to control plant 

diseases caused by other fungi, Rhizoctonia, PYlhium, Botrytis, Fusarium, Didymella, 

Verti('illiulII, 'ii/crnaria, Cladosporium, Heiminthosporium, Penicillium and Pi/caria. There 

4 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 5 of 34 

were no rcpnn, of pathogenicity to plants .. 

Therefore, C. i'utenulatum is not expected to cause adverse effects to nontarget plants. The 

literature review satisfies current guidelines for non-target plant testing and may be used as the 

basis Jor W31\ ing lurther non-target plant tests, pending appropriate product characterization and 

taxollOlnic ViC!'] ficatioll. 

OPPTS Guideline 885-4380: Honey Bees (MRID# 445014-07): 

This study "'b conducted in compliance with Good Laboratory Practice Standards as published 

by the U.S. I!nvironmental Protection Agency, Office of Pesticide Programs in 40 CFR Part 160. 

Reported be:c mortalities were not dose-responsive nor statistically significant. Therefore the 

dietary Leu v,due for honey bees exposed to G. catenulatum cell mass for 10 days is estimated 

to be greater than 5200 ppm (3.6x I 0" CFlJ/mL), the highest concentration tested, and the no 

observed eficc concentration is estimated to be 5200 ppm (3.6 x 10" CFU/mL). An LD5(J value 

>3.6)( I 0(' en 'tnL and an NOEL of 3.6x I 0' CFU/mL were reported. This study showed a lack 

of toxicity of I ;flOcladium catenuiatum cell mass to honey bees. Mortalities were therefore 

presumed ttl Ix associated with other environmental variables associated with the test arena. 

There were 110 evaluations of infectivity or pathogenicity. 

This study IS scientifically valid, satisfies current guideline requirements, and is acceptable for 

assessing s]l\)Jt-1erm toxic effects of Gliocladium catenulatum Strain Jl446 to honey bees. 

OPPTS Guideline 885.4340: Nontarget Insects - Green Lacewing (MRID# 445014-04): 

The rates of lllllrtality and pupation were similar in the treatment groups as in the untreated 

control group. Reported mortalities were not treatment related. Therefore, the dietary LC50 value 

for green lacewi ng larvae exposed to G. catenulatum cell mass for 12 days is presumed greater 

than 5200 ppm (J.6 xI0" CFUlg) and the no observed effect concentration is ~5200 ppm (3.6x I06 

CFU/g). This study showed a lack of toxicity of Gliocladium catenulatum cell mass to green 

lacewings. There wcre no evaluations of infectivity or pathogenicity. 

This study i" scientifically valid, satisfies current guideline requirements, and is acceptable for 

assessing shm, -term toxic effects of Gliocladiul1l catellulatum Strain .11446 to green lacewings. 

OPPTS Guideline 885-4340: Nontarget Insects - Parasitic Hymenoptera (MRID# 445014-

~: 

Mortalities '"ere related to the test substance in one group--treated with 5,200 ppm G. 

catenlllatlllJl c\.:lllllass. Therefore the dietary LC50 value for parasitic Hymenoptera exposed to 

G. catcnulu/l(lu cel1mass for eight days was calculated approximately 1992 ppm (lAx I 0" 

CFU/lllL) and the no observed effect concentration dctemlined to be 520 ppm (3.6 xlOj 

CFU/mL).T IllS study showed a lack of toxicity of Gliocladium calenulatum cell mass to 

5 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 6 of 34 

parasitic HYlrenoptera. There were no evaluations of infectivity or pathogenicity. 

This study j, ·;cicntifically valid, satisfies current guideline requirements, and is acceptable for 

assessing shor:-term toxic effects of Gliocladium catenu/atum Strain Jl446 to parasitic wasps. 

OPPTS Guideline 885.4340: Nontan~et Insects - Lady Beetles (MRID# 445014-06): 

Mortalities were not dose-responsive, nor associated with Gliocladium catenu/atum cell mass. 

Therefore the dietary LC50 value for ladybird beetles exposed to G. catenu/atum cell mass for 16 

days is estimaled to be greater than 5200 ppm (3.6x I 0" CFU/mL), the highest concentration 

tested, and 1.11(' no observed effect concentration to be 520 ppm (3.6x l 05 CFUlmL). An LDso 

>3.6< 10" Cf-{iimL and an NOEL of 3.6x I OS CFUlmL was reported. This study showed a lack of 

toxicity of lihue/adium catenutatum cell mass to lady beetles. There were no evaluations of 

infectivity (II' pathogenicity. 

This study i," ,'cientifically valid, satisfies current guideline requirements, and is acceptable for 

assessing short -(crm toxic effects of G/iocladium catenu/atum Strain ]1446 to ladybird beetles. 

Imp/!.f:fl· to Endangered Species 

Impacts to endangered species [rom intended exposures of PrimastopTM are anticipated to be 

negligible or nonexistent. This assessment is premised upon a combination of several factors: (I) 

low BEC values; (2) data and evidence from the public literature as summarized in the 

Environmental Fate portion of this assessment; and (3) the practically nontoxic (and noninfective) status ofthe MPCA in surrogate toxicity/pathogenicity studies summarized in the 

Ecological Effects portion. 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 7 of 34 

DATA EVALUATION REPORT 

Reviewed bv: Robyn Rose, Entomologist, BPPD ~t ':? I} \ i :) \ 

"\~' I Secondary Reviewer: Gail Tomimatsu, Plant Pathologist, BPPD"n I 'I; I ' \c' I 

STUDY TY\)I:: 

MRlDNO. 

CHEMICAL ',CL 

TEST MA T!~RIAL: 

STUDY NO,: 

SPONSOR 

TESTING F\CIUTY: 

TITLE OF \,FPORT: 

AUTHORS 

STUDY COMPLETED: 

CLASSIFlCA nON: 

Study Summary; 

Green Lacewing (Chrvsoperla carnea) Larvae Dietary 

Pathogenicity and Toxicity Study (l:54A-23; OPPTS 885.4340) 

445014-()4 

021009 

Gliocladium catellulatum strain J 1446 (Technical Powder) 

2')3-113A 

Kcmira Agro Oy, P.O. Box 330, Helsinki, Finland 

Wildlife International Ltd., 8598 Commerce Dr.. Easton, MD 

21601 

Gliocladium catcnulatum Cell Mass (Tec1mical Product): A 

Dietary Pathogenicity and Toxicity Study With Green Lacewing 

Larvae (Chrvsoperla carnea) 

Kimberly A. Hoxter, Susan J. Palmer, and Henry O. Krueger, PhD. 

2/5/98 

Acceptable 

Objectivc: "The objective of the study was to evaluate the pathogenicity and toxicity of 

Gliociadilllll cutellulalum cell mass administered to green lacewing larvae (Chrvsoperla carneal 

in the diet." 

Test Materi~\1: 

G/ioc/adiufI/ ,urenulalum Cell Mass (Technical Product) Batch No. 44/95111 ; a light brown 

powder with a reported activity of7x 10' colony forming units per gram (cfu/g) was received 

from Kemira Agro Oy (3/26/97; Wildlife International ID #3996). Test substance viability was 

determined 11011] 0, Ig tcst substance + ImL 0.3% TRITON-X sol. + 99mL sterile H20'~JO-J 

dilution. TIll' Illixture was homogenized for ~ 1 0 sec. before preparing final solutions (10-5

, 10-", 

\ 0.7) 10 be pial,xi on potato dextrose agar. The activity of the G. calenulalum was verified at 

Wildlife lntclllatiollal using the equation: Viability (in tenns ofCFU/g) = L:C"IV" x N xL (Dn) 

[cfu/gJ: C:""h ",'colonies on the agar plate, Vn=volume plated(mL), N=# of replicate plates, 

D,,=dilution, 0: the sample(g/mL). 

Tcst Organism. 

Green LacewtJ ':" (Chrysoperlu c({rnea) larvae from Rincon-Vitova Insectaries, Inc., P.O. Box 

1555, Ventura. CA 93002. 

7 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 8 of 34 

Test Methqd,: 

The pathogenicity and toxicity of Gliocladium calenulatum cell mass fed to green lacewing (GL) 

larvae was evaluated. Three nominal concentrations (52, 520, and 5200 ppm) of G. catenulatum 

and a negative control (moth eggs without test substance) were fed to GL larvae via moth 

(Sitotroga sp. From Rincon-Vitova Insectaries, Inc., Ventura, CAl eggs mixed with the test 

substance; the moths were given new diet material weekly ad lihitum. The concentrations were 

equivalent (0 :u,x I 04

, 3.6x 10', and 3.6 x I 0" CFU/g respectively based on a verified activity 

(verifIed 5 tnncs on two dates: May 10 and May 25) of7x 10' colony forming units per gram 

(CFU/g). The test diet was prepared by calculating: Desired Concentration (ppm or fig/g) = Test 

Substance (gj Final Volume of Stock Suspension (mL) x Volume of Stock Suspension Mixed 

With Moth lOggs (mL) -;- Final Weight of Diet (g) x 10" fig/g. Samples were taken from the test 

diets and the control on Day 0 and Day 7 to determine stability and homogeneity of the stock 

suspensiolls hv serial dilution plating onto potato dextrose agar. 

Each concentration and the control received thirty test chambers with one GL in each chamber. 

GLs were reared and fed the test material in the lab. Test chambers consisted of one ounce clear 

semitransparent plastic Solo@ (No. 601 P) cups with clear semitransparent plastic lids. GLs were 

kept in incubators at 20.8 ± 0.2°C (SD) WIth a rangc of20.8 to 21 "C, average relative humidity 

87 ± 6% (Sf) i (range 66 - 90%), and a photoperiod of 12: 12 with overhead fluorescent lighting. 

Observations of mortality and pupation were made within four hours (- 3/4 of an hour) after test 

initiation and Ihen daily for 12 days. Observations were discontinued after the 12th day when 

pupation exceeded 50% and mortality exceeded 20% in the negative control. The LC,O values 

were determined by the cumulative mortality percentages observed in the treatment groups. The 

concentration resulting in no effects was determined through mortality, pupation, and 

observation, _ 

Reported Res"l1~: 

Cumulative percent mortality of green lacewing (GL, Chrysoper/a carnea) larvac exposed to 

Giiocladiulli ,-"renulalum cell mass and percent pupation of green lacewing larvae exposed to G. 

catenu/atuII! cell mass were' 

Control 52 ppm 520 ppm 5200 ppm - Percent M ()] 1 a lity 27 20 13 13 

Percent Pupat 1011 57 67 70 70 

The results showed the dietary LC,o value for green lacewing larvae exposed to G. carenulatum 

cell mass for 12 days to be greater than 5200 ppm (3.6x I 0" CFUlg) and the no observed effect 

concentration to be 5200 ppm (3.6x 1 0' CFU/g). There were no signs of overt toxicity noted in 

the surVIving larvae in the untreated control or treatmcnt groups. The rates of mortality and 

pupation werc similar in the treatment groups as to the untreated control group. Percent 

mortality and percent pupation were respectively slightly higher and slightly lower than groups 

exposed to'; c"tenu/alum. 

8 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 9 of 34 

For nominal stc)Ck solution concentrations of 312, 3,120 and 31,200 mg/L; mean viable counts of 

G. calenu/aillfl' were reported: from Day 0 verification samples: 7,5 x 104

, 1.8 x 10" and >2.0 x 

10" CFU/mL. respectively; and from Day 7 stability samples: 5.4 x 104 ,8.3 x 10' ami 1.5 x 107 

CFU/mL. Ua:- () verification and Day 7 stability samples each demonstrated mean value of 0.0 

CFU/mL. 

Reviewer C()Jll)11ents: 

A. Methods 

This study \\a,; conducted in compliance with Good Laboratory Pf31ctice Standards as published 

by the U.S. hlvironmental Protection Agency, Office of Pesticide Programs in 40 CFR Part 160. 

I. Thi; study did not include two controls: an infectivity control group (inactivated 

Gliocladium e,denu/alum) and a treatment in which lacewings are dosed with sterile filtrate 

from produclion cultures. However, absence of these treatments did not influence the overall 

conclusions 01 the study. 

B. ConcluslOllS 

The results of Ihis study showed the dietary Le,o value for grecn lacewing larvae exposed to G. 

catenulatulI! ("II mass for 12 days is greater than 5200 ppm (3.6 x 106 CFU/g) and the no 

observed eflccl concentration is 5200 ppm (3.6 x I 0" CFU/g). This study showed a lack of 

toxicity of (,'lll)ciadium catenulatum cell mass to green lacewings. The rates of mortality and 

pupation were similar in the treatment groups as (0 the untreated control group. Percent 

mortality and percent pupation were respectively slightly higher and slightly lower than groups 

exposed to (, ,·alenu/atum. 

9 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 10 of 34 

DATA EVALUATION REPORT 

Reviewed by: Robyn Rose, Entomologist, BPPD l~ i:"J(J I (co I 

Secondary RC\lCwer: Gail Tomimatsu, Plant Pathologist, BPPD:;~:~ ,;/? i i t'l 

STUDY TYPI.: 

MRID NO. 

CHEMICAL 1\0.: 

TEST MATFR1AL: 

STUDY NO .. 

SPONSOR 

TESTING F,\,'IUTY: 

TITLE OF REPORT: 

AUTHORS 

STUDY COMPLETED: 

CLASSIFIC ATJON: 

Study Summan: 

Parasitic Hymenoptera (Nasonia vi/rtpenl1is) Dietary Pathogenicity 

and Toxicity Study (154A-23; OPPTS 885.4340) 

445014-05 

021009 

Gliocladiurn catenulaturn strain .11446 (Technical Powder) 

293-112 

Kcmira Agro Oy, P.O. Box 330, Helsinki, Finland 

Wildlife International Ltd., 8598 Commerce Dr., Easton, MD 

21601 

Gliocladiurn catenulaturn Cell Mass (Technical Product): A 

Dietary Pathogenicity and Toxicity Study With Parasitic 

Hymenoptera (Nasonia vi/ripennis) 

Kimberly A. Hoxter, Susan 1. Palmer, and Joann B. Beavers. 

2/5/98 

Acceptable 

Objective: "The objective of the study was to evaluate the pathogenicity and toxicity of 

Gliocilldiulli ( illel/ulaturn cell mass administered to parasitic Hymenoptera (Nasonia l'itripel1l1is) 

in the JieL" 

Test Matena!: 

GliocladiullI uucilulatum Cell Mass (Technical Product) Batch No. 44/95JJl; a light brown 

powder with ;" reported activity of7x 108 colony fomling units per gram (cfu/g) was received 

from Kemira .\gro Oy (3/26/97; Wildlife International ID #3996). Test substance viability was 

determined Ir,Jln O.lg test substance -+ I mL 0.3°1t. TRITON-X soL -+ 99mL sterile H20=]()·3 

dilution. The mixture was homogenized for -10 sec. before preparing final solutions (10-', ]()-", 

J 0-7). The activity of the G. catenulatum was verified at Wildlife International using the 

equation: V labi illy (in tenus of CFU/g) .~ [C./V" x N x L (D,,) [cfu/g]; C,,=# of colonies on the 

agar plate, \,.\·olume plated(mL), N=# of replicate plates, D,,=dilutions of the sample(g/mL). 

Test OrgamslI\: 

Parasitic HYI11~noptera (Nasonia vitripcnnis) obtained from Carolina Biological Supply 

Company, :C/[)O York Rd., Burlington, NC 27215-3398 as larvae in blowfly (Sarcophaga sp.) 

pupae Adulh were obtained from pupae after 3 days in an incubator set to maintain a 

10 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 11 of 34 

temperature t) f . HI to 28°C, with relative humidity above ~40%. 

Test Method~: 

The pathogenIcity and toxicity of Gliocladiwn catenulatum cell mass fed to Nasonia vitripennis 

was evaluated Three nominal concentrations (52, 520, and 5200 ppm) of Gliocladium and a 

negative control were presented to N vifripennis twice each week via a honey and deionized 

water diet cOal'cd on a cotton swab. Samples were taken from the tcst diets and the control on 

Day 0 and Dll', 7 to determine stability and homogeneity of the stock suspensions by serial 

dilution platll1g on potato dextrose agar. The nominal concentrations were equivalent to 3.6x 10\ 

3 .6x I 0" and} (" I 0" respectively based on an activity of 7x 10' colony forming units per gram 

(CFUig). The test substance was combined with 10mL deionized water and honey was added 

until I OOml. ",as reached; diets were prepared immediately before test initiation and on Day 7. 

Twenty five wasps ornearly identical size were immobilized with nitrogen gas and impartially 

distributed am,lIlg the test chambers. Wasps were allowed ad lihitum access to diet and water in 

test chambers. 

Test chambel" consisted of one-pint rolled paper containers (9cm diameter and 9 cm high) 

covered with" disposable plastic petri dish (~ 10cm diameter) with an inverted 20mL glass vial 

containing dci''Ilized water inserted through it. Test dosages and the negative control were 

replicated in lilplicate; 75 adult wasps were tested per treatment group. N. vitripennis were kept 

in incubators ,,'. 27.2 ± 1.3°C (SD) with a range of24.9 to 28.9°C, average relative humidity was 

82 ± 8'% (range: 7() - 93%), and a photoperiod of 12: 12 with overhead fluorescent lighting. 

Observations ,)1' mortality and clinical signs of toxicology were made one and 1.5 hours after the 

initiation of thl: test and then daily for eight days. Observations were discontinued after the 

eighth day \'. h,:n mortality exceeded 20% in the negative control. 

The C. E. SlCP'1a1l computer program was used to analyze mortality results at test termination. 

The to,,, vallie and the 95% confidence interval (by probit analysis), and the moving average 

method or \)]nomial probability with nonlinear interpolation were calculated via the C..E. Stephan 

computer prnl:ram. The concentration resulting in no effects was determined through visual 

inspection 0[' mortality and clinical observation. 

Reported Resu It,;: 

Cumulative perccllt mortality of parasitic Hymenoptera (N. vitripennis) exposed to Gliocladium 

catenulatulJI cell mass were: - Control 52 ppm 520 ppm 5200 ppm 

Percent IVI mta lity 23 35 25 68 - # thal did 17175 26/75 119175 51/75 - Mortality Il·om the 52 ppm and 520 ppm treatment groups were comparable to the negative 

control group i'ercent mortality at test termination in the 5200 ppm treatment group was 68% 

(51 of 75) \. sl11all number of wasps exhibited a loss of equilibrium or were immobile during 

11 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 12 of 34 

the test. All olher surviving wasps in this treatment group were normal in appearance and 

behavior throughout the test period. Mortality in the 5200 ppm treatment group began to 

increase on da\" 2 and continued until test termination; and in this treatment mortalities appeared 

to be related I" treatment with the test substance. 

A small numb"r of wasps were immobile or lethargic in the 52 ppm treatment group during the 

test. All other surviving wasps in the 52 and 520 ppm treatment groups were normal in 

appearance an,1 behavior throughout the test. Mortality at test termination in the 52 and 520 ppm 

treatment grolips were comparable to the negative control group. Therefore, the mortalities and 

clinical signs observed were not considered to be related to treatment with the test substance. 

The results showed the dietary LC50 value for parasitic Hymenoptera exposed to G. 

calenu/aluill cdlmass for eight days to be approximately 1992 ppm (I AxIO" CFUlmL) and the 

no ohserved ·;::Te,:t concentration to be 520 ppm (3.6>< 10' CFU/mL). 

Viahle populations of G. calenu/alllm from verification and stability samples of nominal 

concentration" of 52, 520 and 5200 mg/L were as follows: Day 0 verification samples 

demonstrated mean values of2.3 x 104,2.7 X 10' and 1.3 x 10" CFU/mL respectively. Day 7 

stability samples demonstrated mean values of 2.2 x 103,4.0 x 10', and 1.5 x 105 CFU/mL 

respectively. Day 0 verification and Day 7 stability samples from Ih<: negative control diet 

showed 0.0 ('U/mL. The number of plates used to determine mean values was not reported. 

Reviewer C'2!nments: 

A. Method, 

This study \va" conducted in compliance with Good Laboratory Practice Standards as published 

by the U.S. L:llvironmental Protection Agency, Office of Pesticide Programs in 40 CFR Part 160. 

1. '11m study did not include two controls: an infectivity control group (inactivated 

Gliocllldiulil ,,,{clIu/alum) and a treatment in which parasitic wasps are dosed with sterile filtrate 

from productun cultures. However, absence of these treatments did not influence the overall 

conclusions o! tile study. 

B. Conclusio"s 

Mortality ohs,:rvcd at the 52 and 520 ppm were comparable to the control and, therefore, not 

considered to be treatment related. Mortality in the 5200 ppm treatment group began to increase 

on day 2 and ,:ontmued until test termination; and in this treatment mortalities appeared to be 

related to treatll1ent with the test substance. Therefore the dietary LC50 value for parasitic 

Hymenoptera 'exposed to G. ealenu/alum cell mass tilr eight days was calculated as .- 1992 ppm 

(lAx I 0" CHIllL) and the no observed elfect concentration was calculated as 520 ppm (3.6 x I 0' 

CFU/mL).1 his study showed a lack of toxicity of Glioc/adiun1 c"tenu/atum cell mass to 

parasitic H \'!1 ",noptera. 

12 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 13 of 34 

DATA EVALUATION REPORT 

Reviewed by: Robyn Rose, Entomologist, BPPD {L Ii 8/:21 (0\,,~, I 

Secondary Re"iewer: Gail Tomimatsu, Plant Pathologist, BPPDk)' I, I -: 

STUDY TYPf: 

MRID NO .. 

CHEMICAL ,',0: 

TEST'vIATERIAL: 

STUDY NO : 

SPONSOR 

TESTING FACILITY: 

TITLE OF RFPORT: 

AUTHORS. 

STUDY COMPLETED: 

CLASSIFIC'l.T10N: 

Study Summar~': 

" 

Ladybird Beetle (Hippodamia convergens) Dietary Pathogenicity 

and Toxicity Study (154A-23; OPPTS 885.4340) 

445014-06 

021009 

Gliocladiul11 catenu/atum strain J1446 (Technical Powder) 

293-11 I 

Kemira Agro Oy, P.O, Box 330, Helsinki, Finland 

Wildlife International Ltd., 8598 Commerce Dr., Easton, MD 

21601 

Gliocladiwn catenu/atul11 Cell Mass (Technical Product): A 

Dietary Pathogemcity and Toxicity Study With The Ladybird 

Beetle (Hipl'odamia convergel1s) 

Kimberly A. Hoxter, Susan 1. Palmer, and Henry O. Krueger, PhD, 

2/5/98 

Acceptable 

Objective: "fhe: objective of the study was to evaluate the pathogenicity and toxicity of 

G/ioc/adilllll UI{Cl1u/atum cell mass administered to the ladybird beetle (Hippodmnia convergens) 

in the diet." 

Test Material: 

G/iocladiul1l (Olcflu/alum Cell Mass (Technical Product) Batch No. 44/95 111 ; a light brown 

powder with a reported activity of7x I 08 colony forming units per gram (cfu/g) was received 

from Kemira·\gro Oy (3/26/97; Wildlife International ID #3996), Test substance viability was 

deternlined limn O.lg test substance + ImL 0.3% TR1TON-X sol. + 99mL sterile H,O=IO'] 

dilution. The :ni xture was homogenized for -10 sec. before preparing final solutions (10-5

, 10-6, 

10,7). The actlvity of the G. catenu/alum was verified at Wildlife International using the 

equation: y",hdity (in terms ofCFU/g) = ~C,/V, x N x ~ (0,) [cfu/g]; C,=# of colonies on the 

agar plate, V"l'olume plated(mL), N=# ofreplicate plates, D,=dilutions of the sample(g/rnL). 

Test Orgamsill. 

Adult Ladybird Beetles (Hipl'odamia convergens) obtained from A-I Unique Insect Control, 

5504 Sperrv L}.'i\'e, Citrus Heights, CA 95IJ2L 

13 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 14 of 34 

Test Methods 

The pathogenicity and toxicity of Gliocladium catenulatum cell mass fed to adult ladybird 

beetles (Hippodamia convergen.\') was evaluated. Three nominal concentrations (52, 520, and 

5200 ppm) 0 I' Gliocladium and a negative control were fed to N. vitripennis twice each week via 

a honey and d"ionized water diet coated on a cotton swab .. The test substance was combined 

with I OmL deionized water and honey was added until I OOmL was reached. The concentrations 

were equivaiclilio 3.6x I 04 , 3.6X 105, and 3,6x I 0" respectively based on an activity of 7x 1 08 

colony fomling units per gram (CFU/g). All treatments and the negative control were replicated 

three times (7' ladybird beetles per treatment). Samples werc taken from the tcst diets and the 

control on Da\' () and Day 7 to determine stability and homogeneity of the stock suspensions 

Test chambers consisted of one-pint rolled paper containers (<Jcm diameter and 9 cm high) 

covered willi :.1 disposable plastic petri dish (~I Ocm diameter) with an inverted 20mL glass vial 

containing deionized water inserted through it. Adult ladybird beetles were kept in an incubator 

for four days pl'ior to test initiation; temperature was sct at 26 to 28°C and the relative humidity 

was above approximately 40%. During the test, the temperature in chambers was 27.2 ± 1.3°C 

(SD) \\'ith a r,mgc of24.9 to 28.9°C, relative humidity was 82 ± 8% (SD) with a range 000 to 

93%, and the photoperiod was 12: 12 with overhead floureseent lighting. Each test chamber 

consisted of 2'i IJ. convergen.\'. Observations ol'mortality were made approximately one hour 

and 1.5 hours ;Iner test initiation and then daily for 16 days (except Day 12); negative control 

mortality reach 20% on Day 16. Samples were taken from the test diets and the control on Day 0 

and Day 7 to t!\~lcrmine stability and homogeneity of the stock suspensions. 

An estimation '.l r mortality patterns were determined by visual inspection of data. The 

concentration ,csL!lting in no effects was determined through mortality and clinical observation 

data. The LC 5, was determined by nominal concentrations. 

Reported Results: 

At telmination or the test, the percent mortalities were: -

Control 52 ppm 520 ppm 5200 ppm -

Percent M on ality 23 17 19 17 -

/I that die d 17175 13175 14/75 13175 .. Mortalities were not the result of the treatment substance sll1ce the mortahtles did not occur 111 a 

dose-responSive pattern and the percent mortality in all treatment groups was less than that of the 

control on the last 2 days of the test. A small number of immobile beetles were observed during 

the tests. All other surviving beetles in the 52, 520, and 5200 ppm treatment groups were 

normal in app<:arance and behavior throughout the study. 

Result:; () r plate counts for nominal test concentrations used in the study were 3.6 x 104

, 

3.6 x 105, alld .l,b x 10" CFU/mL, respectively. Samples collected on the day aftest initiation 

(Day 0) demonstrated mean values of2.3 x 104

.2.7 X lOS, and 1.3 x 10" CFU/mL, respectively. 

14 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 15 of 34 

Stability samples from Day 7 demonstrated mean values of2.2 x 103,4.0 X 103 , and 1.5 x 105 

CFUimL. Dav 0 verification and Day 7 stability samples from the negative control diet showed 

0.0 CHJ/mL 

Reviewer C:OI)ll11Cnts: 

A. Methods 

This study "as conducted in compliance with Good Laboratory Practice Standards as published 

by the U.S. i-:t'vironmental Protection Agency, Office of Pesticide Programs in 40 CFR Part 160. 

I. Till> study did not include two controls: an infectivity control group (inactivated 

GliocladiulIl cUlcnulatum) and a treatment in which ladybird beetles are dosed with sterile filtrate 

from producti,)]) cultures. However, absence of these treatments did not influence the overall 

conclusions oC the study. 

B. Results and Discussion 

The pattem 01 mortalities exhibited did not establish that expected ofa typical dosage response. 

Ladybird bectle mortalities were noted in all treatments, including the negative control; 

cumulative murtalities were slightly greater in the negative control group than in any of the 

groups treated with logarithmic increments of Gliocladium catcnulatum cell mass. The dietary 

LC50 value I,)r ladybird beetles exposed to G. catenulatum cell mass for 16 days was estimated 

to bc greater tnan 5200 ppm (3.6x I 0" CFt;/mL), the highest concentration tested, and the no 

observed elTcd concentration estimated to be 520 ppm (3.6x 105 CFU/mL). An LD,o >3.6x I 0" 

CFU!mL and an NOEL of 3.6x 105 CFU/mL was reported When compared to the negative 

controls, resul1s showed a lack of toxicity of Gliocladium catcnulatum cell mass to lady beetles. 

15 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 16 of 34 

DA T A EVALUATION REPORT 

Reviewed by: Robyn Rose, Entomologist, BPPD (.~ t? IJ: IA 

Secondary Reviewer: Gail Tomimatsu, Plant Pathologist, BPPD~~,\"_'''':+v' i, (', 

STUDY TYPL: 

MRlDNO,. 

CHEMICAL NO.: 

TEST MATERIAL: 

STUDY NO . 

SPONSOR 

TESTING FACIUTY: 

TITLE OF REPORT: 

AUTHORS 

STUDY COMPLETED: 

CLASSIFIC.A.T10N: 

Study Summary: 

Honey bee (Apis mellifera) Dietary Pathogenicity and Toxicity 

Study (154A-23; OPPTS 885.4380) 

445(J14-07 

021009 

Gliocladium catenu/alum strain J1446 (Technical Powder) 

293-110 

Kemira Agro Oy, P.O. Box 330, Helsinki, Finland 

Wildlife International Ltd., 8598 Commerce Dr., Easton, MD 

21601 

Gliocladium catenuialum Cell Mass (Technical Product): A 

Dietary Pathogenicity and Toxicity Study With The HOlley Bee 

(Api.\' melli{era) 

Kimberly A. Hoxter, Susan .r. Palmer, and Henry O. Krueger, PhD, 

2/5/08 

Acceptable. 

Objective: "The objective of the study was to evaluate the pathogenicity and toxicity of 

Glioe/adium (cilcl1uiatum cell mass administered to the honey bee (Apis mellifera) in the diet." 

Test Materi al: 

GliocladiulII (il/cl1uiatum Cell Mass (Technical Product) Batch No. 44/95,,1; a light brown 

powder with a reported activity of 7x 108 colony fonning units per gram (cfu/g) was received 

from Kemira ·\gro Oy (3/26/97; Wildlife InternationallD #3996). Test substance viability was 

determined I'rom 0.1 g test substance + I mL 0.3% TRITON-X sol. + 99mL sterile H"O=I 0-3 

dilution. The mixture was homogenized for -10 sec. before preparing final solutions (105, 1O-", 

10-7 serial dilutions). The activity of the G. catcnuiatum was verified at Wildlife International 

using the equation: Viability (in terms ofCFU/g) = LC,IV, x N x L (0,) [cfu/g]; c,~# of 

colonies 011 the agar plate, V",ovolume plated(mL), N=# of replicate plates, D"o'dilutions of the 

samplc(g/mL, 

Test Organisn! 

Honey bee (Al'is melli/era), an important plant pollinator, obtained as capped brood from hives 

maintained b\ Wildlife Intemational Ltd. and maintained according to practices recommended 

by the State ,)!' Maryland. 

16 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 17 of 34 

Test Methods: 

The pathogenicity and toxicity of Gliocladium catenulatum cell mass fed to honey bees was 

evaluated. Three nominal concentrations (52,520, and 5200 ppm) of Gliocladium and a negative 

control (honey and water) were fed to honey bees ad libitum via a honey and deionized water (20 

mL) diet coated on a cOllon swab. The concentrations were equivalent to 3.6x I04, 3.6x l05, and 

3.6x 10" respectively based on an activity of7x 10' colony forming units per gram (CFU/g). 

Viability ol'the test substance was determined prior to test initiation in CFUlmL. The test 

substance Vias combined with 20mL deionized water and honey was added unlil200mL was 

reached; diets were prepared fresh and presented to the bees immediately before test initiation 

and on Da}' 7 

Twenly five apparently healthy bees (approximately 1 to 3 days old) were aclded to each 

test chamber. The bees were obtained by removing frames containing pupae from hives on site. 

During a 3-d"y acclimation period before test initiation, the [rames were housed in clear acrylic 

boxes in an environmental chamber at an averag,~ temperature of -32°C.; emerging bees were 

allowed to feed ad libitum on honey and pollen remaining in the cells of the frames. 

Three test chambers per treatment were evaluated. Test chambers consisted of one-pint 

rolled papel" containers (9cm diameter and 9 cm high) covered with a disposable plastic petri dish 

("I Oem diameter) with an inverted 20 mL glass vial containing the appropriate diet inserted 

through it, and another glass vial containing deionized water was also inserted through the lid. 

Honey bees were kept in tcst chambers at 26°C to 28°C (x'~ 27.2 ± 0.2°C), rellativc humidity 

ranging from b2 to 96% (x = 77 ± 85), and total darkness except for when diet was replaced and 

observations were: made. Diets were available to the bees to feed ad libitum throughout the test 

perioa. Ohservations of mortality and clinical signs oftoxicology were made y, and I 3/4 hours 

after the initiation of the test and then daily for ten days. Observations were di.seontinued after 

the tenth day IYhen mortality exceeded 20'Yo in the negative control. 

Samples of the diets for all treatment grollpS and the negative control were collected after 

preparation 011 Day 0 and after diets had been stored under test conditions on Day 7. These 

samples were assayed to determine homogeneity, test concentration measurements and establish 

Day 0 and Dav 7 values for evalluating stability of the test substancein the diets, and under test 

conditions. 

The C "E'. Stephan computer program was used to analyze mortality results at test 

termination. The LOS II value and the 95% confidence interval (hy probit analysis), and the 

moving average method or binomial probability with nonlinear interpolation were calculated via 

the C. E. Slephan computer program. The concentration resulting in no effects was determined 

through v'lsual inspection of mortality and clinical observation. 

17 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 18 of 34 

Rep0l1ed Results: 

At termination (lfthe test (after 10 days), the percent cumulative mortalities were: 

Control 52 ppm 520 ppm 5200 ppm 

Percent lV!ortalit) I 25 37 37 36 

Ii that died 19175 28/75 28175 27175 

Bee mortalttlcs were noted first in the highest nominal concentration tested (5200 mg/L) on Day 

1 of the teS1. Before the end of Day 3, bee mortalities were noted in all treatments, mc luding the 

negative control. Percent mortalities increased erratically in all groups through Day 10. 

Mortalities were not the result of the treatment substance since the mortalities did not occur in a 

dose-reSpOnS1YC pattern and the percent mortality in all treatment groups was comparable to that 

of the control. A small number of bees (l per treatment) exhibited lethargy or loss of equilibrium 

(unbalanced) III all groups on the last day of the test. Lethargic or unbalanced bees were noted 

also in the group treated with 520 mg/L on Day 5 and in the group treated with 5200 mg/L on 

Day 7. Durill.~ the test, a small number of the bees in all treatments and one bee in the control 

group appeared lethargic or exhibited a loss of equilibrium. 

The results of this study showed the dietalY LCso yalue for honey bees exposed to G. 

ca/enu/alum celi mass for 10 days to be greater than 5200 ppm (3.6 x 1 0" CFU/mL), the highest 

concentration tested and the no observed effect concentration to be 5200 ppm (3.6 x 10" 

CFU/mL). ;\11 LDso >3.6x 10" CFUlmL and an NOEL of 3.6x 10' CFU/mL was reported. These 

values are not statistically significant. 

Viable plate counts for homogeneity/stability samples on Day 0 were 2.1 x 102 , 

4.8 x 103

, and (,.4 x 104 CFU/mL for nominal concentrations 52 (3.6 x 10" CFU/mL diet) , 520 

(3.6 x \ 0' cn 'irnL diet) and 5200 mg/L (3.6 x 10" CFU/mL diet) respectively. The Day 0 

verification samples and tbe Day 7 stability samples for the negative control on Day 0 were 0.0 

CFUhnL. 

Reviewer C\'lll!!l<:!ltS: 

A. Methods 

This study was conducted in compliance with Good Laboratory Practice Standards as published 

by the u.s. Environmental Protection Agency, Office of Pesticide Programs in 40 CFR Part 160. 

1 .. J hi., study did not include two controls: an infectivity control group (inactivated 

Glivcladium (-ulel/ulalum) and a treatment in which honey bees are dosed with sterile: nltrate 

from producll()n cultures. However, absence of these treatments did not influence the overall 

conclLlsions Of1ilC study 

J 8 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 19 of 34 

2. ViabilIty determination, and stability/homogeneity oftest substance: The viability 

counts appear remarkably low for results of the nominal test concentrations. 

B. Conclusiolls 

The reported percent mOlialities did not reveal a pattern in response to logarithmic 

increments 01' test substances dosages, and although percent mortalities in the treated groups 

were slightly higher than in the negative control on the last 3 days of the test, they were not 

statistically Significant. Mortalities were therefore presumed to be associated with other 

environmental variables associated with the test arena. 

The dietary LCIO value for honey bees exposed to C. catenuiatlll1J cell mass for 10 days is 

greater than 520() ppm (3.6x 10" CFU/mL), the highest concentration tested and the no observed 

etTect concentration to be 5200 ppm (3.6 X I 06 CFUlmL). An LOso value >3.6x ilO" CFU/mL and 

an NOEL 01' 3.6'; 10' CFUlrnL were reported. These values are not statistically significant. 

There wcre no other observations of infectivity or pathogenicity. 

19 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 20 of 34 

DATA E:VALUATION REPORT 

Reviewed by: Robyn Rose, Entomologist, BPPD (L d~ 61 Jlivl 

Secondary Reviewer: Gail Tomimatsu, Plant Pathologist, BPPo; ~:t,.·,\,-·c!" ,I .,,, 

STUDY TYPI-. 

MRID NO. 

CHEMICAL NO.: 

TEST MATI~RIAL: 

STUDY NO.: 

SPONSOR 

TESTING FACILITY: 

TITLE~ OF RHORT: 

AUTHORS: 

STUDY COMPLETED: 

CLASSIFICA liON: 

Study Summllr~':: 

Avian Oral Pathogenicity and Toxicity Study (154A-23; OPPTS 

885.4050) 

445014-0 I 

021009 

Gliac/adium ('{{Ienu/alum strain Jl446 (Technical Powder) 

293-109 

Kemira Agro Oy, P.O. Box 330, Helsinki, Finland 

Wildlife International Ltd., 8598 Commerce Dr., Easton .. MD 

21601 

Gilacladiwn calenu/alum Cell Mass (Technical Product): An 

A vian Oral Pathogenicity and Toxicity Study With The Northern 

Bobwhite Quail 

Kimberly A. Hoxter, Susan .T. Palmer, and Henry O. Krueger, PhD. 

5/29/97 

Acceptable 

Objective: "The objective of this study was to evaluate the acute toxicity and pathogenicity of 

GliacladiufII ""lcl/u/alum cell mass when administered orally to northern bobwhite (Colinus 

virginianusi. 

Test Materia!: 

Gliacladiufll c:llellulalum Cell Mass (Technical Product) Batch No. 44/95; a light brown powder 

with a reported activity of7xl0s colony forming units per gram (cfu/g) was received from 

Kemira Agro Uy Ireported 3/26/97; Wildlife International TD #3723), and stored under 

refrigeration c,mditions. The viability of the test substance was verified by Wildlife International 

Ltd. and was 6 % )< 10' cfu/g. 

Test Organism. 

Seemingly healthy northern bobwhite quail (Calinus virginian us) from a f10ck maintained by 

Wildlife International Ltd. that hatched September 9, 1996. Chicks were fed a vitamin 

supplement daily until test initiation. Water from the local town (Easton) supply and feed were 

provided ad Iifoillll1l hefore and dunng the test. Prior to testing, birds were acclimated (the 

precise duration of the acclimation period was not reported) and observed daily for behavior or 

physical injuries. Only apparently healthy .2 I-day old birds were included in the test. 

20 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 21 of 34 

Test l\1etho"lf,: 

The pathogenicity and toxicity of Gliocladium catenulatum cell mass fed to juvenile bobwhite 

quail was evaluated based upon procedures specified in OPPTS 885.4050 (previously the 

Pesticide Assessment Guidelines, FIFRA Subdivision 1\1, Section 154A-16). 

Birds were dosed daily for 5 days with a mixture of the test substance and reverse-osmosis (RO) 

water; ncgati\'c control birds were given only RO (1 % v/w) water. The dosing suspensions for 

the treatment group were prepared daily by measuring a calculated volume ofthe test substance 

and mixing thoroughly with RO water. Dosing stocks were prepared by weighing I () g oftest 

substance in 'I 100 mL beaker and sufficient amount of RO water was added to bring the final 

volumc to 50 lilt and blended with a glass stir rod. During dosing, the stock was stin'ed 

constantly by placing on a magnetic stir plate. 

Eight pens With five birds each were evaluated. Two pens were used as negative controls (ten 

birds total) and six pens were used as treatments. A total of 30 birds were individually treated 

with l.4x 1 ()" cfulg body weight over a five day dosing period; the resultant total dosage was 

approximated at 7 x 10" cfu/g of body weight over the five day period. All birds were weighed 

individually and administered the appropriate dosing suspension directly into the crop or 

proventriculus using alec tubcrculin syringe. Treatment and control groups were given a total 

of 1 () mLlkg hody weight solution. No antibiotic medications were administered to the chicks 

during the 5t lIdv. 

Birds of unknown sex were randomly assigned to pens that measured approximately 72x90x23 

cm high. A II sides consisted of galvanized wire mesh and the walls and ceiling also had 

galvanized siIeeting. Floors of the pens consisted of galvanized wire mesh. Conditions during 

testing were 21.5o, 1.8°C (SO), with an average relative humidity of 62%H2°"'i. A photoperiod 

of 1 () hours 0 r flourescent light 1.0 approximate noon day sunlight was maintained by a time clock 

during acclimation and throughout the study. The birds were exposed to approximately 177 ± 55 

lux of illurt1lf1ation. In general, the National Institutes of Health recommended housing and 

husbandry practices were followed. 

Birds were evaluated for illness,. abnomlal behavior, and mortality at test initiation and twice 

daily until tcnnlllation on day 30. Body weights were determined after birds were dosed on days 

(), 1, 2, 3, 4 .. I ;. 18,25, and 30. Feed consumption was determined on days 4, 11, 18, 25, and 30. 

Attes! termination, birds were euthanized using carbon dioxide gas and subjected to gross 

necropsy.·j IS';UCS were examined for abnormalities, lesions, and any sign of pathogenicity. 

Reported Rcsult~: 

There were 110 Illortalities nor abnormalities observed in the negative control group. There were 

no mortalil:!cs. obvious signs of toxicity, pathogenicity, abnormal appearance or behavior 

demonstrated hy Gliocladium-treated birds. Of the treated birds, one bird fractured a wing on 

day 25 and ()i' bird demonstrated subcutaneous emphysema ( a pocket of air under the skin) from 

21 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 22 of 34 

days 15-30. l\"ither of these occurrences were treatment related. There was also no effect of 

GliociadiulII clltenulatum cell mass treatment on body weight or feed consumption of1:he birds. 

Gross necropsy ill1dings included a bird in the negative control group with a fractured right 

humenls; and a bird in the Gliocladium-treated group had subcutaneous emphysema in the 

abdominal and thoracic regions and pale breast muscle with petechial hemorrhaging. These 

results were considered unrelated to treatment. There were no evidences of test substance 

replication, lesions, plaques or other indications of pathogenicity for the groups treated with 

GliocladiulII ('iI{('lmlatum cell mass . 

Reviewer COml1lS'Ilts: 

A. Method" 

1. This study was conducted in compliance with Good Laboratory Practice Standards as 

published by the U.S. Environmc:ntal Protection Agency, Office of Pesticide Programs in 40 

CFR Part lbO, except: Verification of the test concentrations, stability, and homogeneity of the 

tcst substance i 11 deionized water were not determined. 

2. This study did not include two controls: an ini,;ctivity control group (inactivated Gliocladium 

catenuiatul1I) ancl a treatment in which birds arc dosed with sterile filtrate from production 

cultures. Hnwcvcr, absence of these treatments did not influence the overall conclusions of the 

study 

B. Results and Conclusions 

Under conditions of the reported study, no adverse effects were exhibited by juvenile northern 

bobwhite aficr .10 clays when administered daily doses (by oral gavage) of 1 x 104 (;{iocladium 

catenuiatul1l··cftug body weight/day for a five day period. This dosage is approximately 

equivalent to a total dosage of 7 :< 10" cfu/g body weight for 5 days. The no observed effect 

dosage of Gliucl"dium catellulatum cell mass was determined at approximately 1.4 x 10" cfulg 

body \Veight t,)r tive days. There were no evidences of test substance replication, lesions, plaques 

or other indlcallons of infectivity or pathogenicity for the groups treated with Gliocladium 

cl1tenuil1tul1l cell mass in gross necropsies. This study showed an lack of toxicity or pathogenicity 

of GllOc!adillll, (utenullltum cell mass to northern bobwhite quail. 

22 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 23 of 34 

DA TA EVALUATION REPORT 

Reviewed by: Robyn Rose, Entomologist, BPPD fI,IL 'J, :;;1.\1 ,j~ " Ie' 

Secondary Reviewer: Gall T0I11Imatsu, Plant PathologIst, BPPD:'l~"h \ ,I 

STUDY TY!'!·: 

MRIDNO.: 

CHEMICAL "-0.: 

TEST MATERIAL: 

STUDY NO.: 

SPONSOR 

TESTING i',\CILlTY: 

TITLE OF REPORT: 

AUTHORS 

STUDY COMPLETED: 

CLASSIFICA nON: 

STUDY SUMMARY: 

Rainbow Trout Toxicity and Pathogenicity Test (154A-19; OPPTS 

885.4200) 

445014-02 

021009 

(;lioc/adium catenulatum strain Jl446 (Technical Powder) 

293A-I04 

Kcmira Agro Oy, P.O. Box 330, Helsinki, Finland 

Wildlife International Ltd., 8598 Commerce Dr., Easton, MD 

21601 

Gliocladium catenalatum Cell Mass (Technical Product): A FiveConcentration Toxicity and Pathogenicity Test With the Rainbow 

Trout (Oncoryhynchus mykiss)-Static Renewal 

Susan J. Palmer. and Henry O. Krueger, PhD. 

211 0/98 

Acceptable 

Objective: "The objective of this study was to evaluate the toxic and pathogenic ct1ixts of 

Gliocladiw." c "tenulatum cell mass (technical product) to the rainbow trout (OnchoIJ}/lchus 

mykiss) dun 1lf! a 30-day aqueous and dietary exposure period under static-renewal tcst 

conditions .. ' 

Test Material: 

Glioc/adnan Ccilcl1ulatum Cell Mass (Technical Product) Batch No. 44/95:; a light brown powder 

witb a reporrcd activity of 7x I OX colony fonning units per gram (cfu/g) was received from 

Kemira Agro Oy (3/26/97; Wildlife International ID #3996). Jest substance viability was 

determined 11'0111 0.1 g test substance + I mL 0.3°;', TRITON-X sol. + 99mL sterile H,o=1 0-3 

dilution. The mixture was homogenized for ~ I 0 sec. before preparing final solutions (10-', 10-", 

10'). The activity of the G. eatenu/alum was verified at Wildlife International using the 

equation: Viahllity (in tenns ofCFU/g) = IC/V, x N x I (D,,) [cfll/g]; C,,=# of colonies on the 

agar plate, 'v.- volume plated(mL), N=# of replicate plates, D,,=dilutions of the sample(g/mL). 

Test Organism: 

Juvenile rainhow trout (Onehorvnchus mykiss) obtained as green eggs and sperm from Mt. 

Lassen Trout I ann, Red Bluff; California on Aprill )(), 1997 were used in this tcst. '\iegative 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 24 of 34 

control fish measured at the end of the test were 36 to 40 mm (x = 39 mm) in length and weighed 

0.70 to 1.3 g !< .0 I. I g). Fish were held for 40 dlays after hatching and allowed to acclimate to 

test conditions li)r 52 hours prior to testing. For 14 days prior to testing, fish were held at 11.8°C 

- 12.5"C, 8.~ ... S.4 pH, and 9.0 - 9.9 mg/L dissolved oxygen. Prior to a 7(, hour starvation period 

before test lIlitiation, rainbow trout were fed salmon mash and/or salmon starter supplied by 

Zeigler Bros. Inc., Gardners, PA. Apparently disease and stress lree fish that were from the 

same source. vear class, and approximate standard length were used for testing. 

Test Methods: 

This test was conducted according to guidelines outlined in OPPTS 885.4200 (previously OPP 

Section ]S4A·] (), Subdivision M of the Pesticide Testing Guidelines) and ASTM Standard 

E729-88a, Standard Guide for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates 

and Amphlbi'lI1s. 

Test chambers consisted of 38-L glass aquaria containing 15 L of test solution measuring 12 em 

deep. Chambers were kept in a controlled room at 12± 1°C, 16 hours of flourescent light similar 

to natural sunlight and eight hours of dark regulated by a timer, and gently aerated throughout the 

test. This 1.es! was conducted for 30 days. A YeUow Springs Instrument Model 51 B dissolved 

oxygen meter and a Fisher Accumet Model 915 pH meter were used to measure dissolved 

oxygen and pH respectively in each test chamber at the beginning and end of observations. A 

Yellow Springs Instrument Model 33 Salinity-Conductivity- Temperature meter was llsed to 

detemline specllic: conductance and hardness and alkalinity measurements, and were based on 

procedures in Standard Methods for the Examination of Water and Wastewater. 

The routes or ({'st substance administration were aqueous and dietary. For the aqueous 

exposures, the lest material was pre-welted and mixed thoroughly in dilution water on a weight 

to volume b,,,,s hefore adding to the test chambers. After mixing, it was reported that test 

Solutl()nS appeared tan in color and were cloudy with particulate matter throughout the water 

column; and lhat the cloudiness increased [rom the lowest to higher concentrations. Test 

solutions were prepared and renewed every Monday, Wednesday, and Friday in separate aquaria 

that trout were transferred into. Water was obtained from a 40 m deep well located at Wildlife 

International Ltd and charactenzed as moderately-hard. Pmticlcs larger than 2511m were 

removed by" tiing through a sand filter. 

Diet was prcpared by adding 6.6 mg of Glioc/adium catenu/atum to 100g of trout chow. The 

diet was mixed for 1.5 hours on a jar mill rotating at 50 rpm. Trout chow and 4.62x 107 cfu/kg 

was the resullant mixture. The diet was provided in each tcst chamber twice daily. Control fish 

were provided with untreated trout chow. One test chamber containing ten trout were evaluated 

for each concentration and control. Nominal concentrations ofthe technical product were 45,89, 

179, :l57, anO 714 mg/L. Based on an activity of 7:< 108 cfu/g (verified by laboratory), these 

concentratlon-. clrc equivalent to 3 15 x 1 07

, b.23>: 107

, 1.25 x I 0', 2.50X 10', and 5.0x 11)" cfu/L, 

rcspectivel v. 

24 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 25 of 34 

Water samples of new and old test solutions werc collected from each test chamber during the 

first week of the study; and then at weekly intervals for the remainder of the study. Samples 

were placed In sterile glass scintillation vials and analyzed immediately or stored refi-igerated 

(Oto SOC) up to 3 weeks until they could be processed and analyzed by dilution plating for 

viabi lily. Serial dilutions of the Ilest solution were prepared before spreading 0.1 mL onto plates 

containing potato dcxtrose agar. After 3 days of incubation at 28°C, all plates were examined for 

growth and enumerated for viable Gliocladium cateuu/atum growth (CFU/mLl. 

Observations of mortality and clinical signs of toxicology were made daily throughout the test 

by sampling for dead or abnonmtl fish by running a dip net through the tank no more than three 

times. Watel· Illr all treatments was somewhat cloudy. Obscrvations on day I for the 179 mg/L 

concentration and on all days the 357 and 714 mg/L treatment groups were difficult because the 

water was panicularly cloudy. Therefore, a dip net was used to observe fish in these treatments. 

The actual number of mortalities was determined at test tennination. A sterile necropsy was 

perfornled on tllree fish from each treatment and control group to evaluate external or internal 

signs of lesions inflammation, areas of discoloration or other abnonnalities. Fins, eyes, nares, 

oral cavity, anus, and gills were externally examined. Liver, kidney, heart, and brain were 

evaluated fOI·necropsy. Samples from these organ tissues were streaked onto plates ofpotato 

dextrose agar lor presence of Gliocladiwn catenulatum. 

The CE. Stephan computer program was used to analyze mortality results at test tern1ination. 

The LD", vallie and the 95% con:fidence interval (by probit analysis), and the moving average 

method or binomial probability with nonlinear interpolation wcre calculated via the C. E. Stephan 

computer program. The concentration resulting in no effects was detennined through visual 

inspection ,",1" mortality and clinical observation. 

A. Test Subsl,lllCC Viability and Verification of Test Concentrations 

The aVGrage concentration from each group measured during the test were 8.3x 105

, 

9.76 X J()5, 2.32·!O", 5.58 x lO", and2.08x 107 CFUiL (for nominal test concentrations 45, 89,179, 

357 and 714 mgiL). "[TJhere was no measured activity in the negative controls indicating that 

the negative l·omrol solutions were free of contamination." 

B. Water Chell1lstry 

Temperatures (measured continuously) ranged from approximately 11.5 to l3.0 D C; dissolved 

oxygcn levels ranged from 10.2 to 1.9 mg dissolved oxygen/L (~94% saturation in freshwater at 

l2°C), and pH ranged Irom 8.1 to 8.6 during the 30 day study. Specific conductance of the 

negative comrol ranged from 290 to 320 1l11lhos/cl1l (x ~ 303); alkalinity of the negative control 

solution ranged from 178 to 182 mg/L as CaCO) (x '0 180); and values for hardness ranged fonn 

132 to do ( 134). 

25 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 26 of 34 

C. Mortality and Clinical and Necropsy Observations 

There was 110 mOliality and fish appeared and behaved normal in the negative control and in the 

groups receiving 4:5,89, and 179 mg test substanc:e/L treatments gletll's. There was no mortality 

in thc 357 mgl l treatment group but observations of sublethal signs such as lethargy and lying 

on the bottom of the test chamber with only gill movement were made the second week after test 

initiation. The 714 mg/L treatment resulted in 100% mortality eight days after test initiation. Of 

the surviving lIsh examined for necropsy (3 from each of the treatment groups and negative 

control), all appeared internally and externally normal and there were no signs of infection. The 

30 day LC,,, was 504mg/L (3.5 xlO8 cfu/L). The lower 95% confidence limit was 357 mg/l 

(2.5 x I OS cfull.) clnd upper confidence limit was 714 mg/L (5.0x I08cfu/L). Tissue samples 

collected from [ish in the negative control, 45 andl 89 mg/L treatment groups demonstrated no 

Gliocladium ('(/tenulatum cell mass (technical product) colony growth. The 179 mg/l treatment 

group showed () to 16 colonies forming (from heart, liver and brain tissues sampled) and 1 to 24 

colonies (from samples of kidney, heart, liver and brain tissues) werc observed from lish tissues 

sampled from the 357 mg/L treatment group. Although the test substance was present, target 

organs did not appear to be affected, and there were no apparent effects upon the health of the 

fish. 

Reviewer Comments: 

A. Methods 

This study was conducted in compliance with Good Laboratory Practicc Standards as published 

by the U.S. EllVlronmental Protection Agency, Office of Pesticide Programs in 40 C'FR Part 160. 

This study was conducted according to EPA guidelines outlines in OPPTS 885.4200 titled 

Freshwater Fish Testing, Tier I. 

Inclusion of a control group of fish exposed (aqueous and dietary) to sterile filtrates from 

production cultures of Gliac/adium catenulatum might supplement the negative pathologies 

noted in the studv; however absence of this control does not significantly alter the conclusions. 

B. Conclusion" 

The 1(1 day LC", was calculated as 504 mg/L (3.5 x I ()< cfu/L). The no-observed effect 

concentration W,IS 179 mg/l (1.3 x 10' cfu/L). Th" 30 day no mortality concentration was 357 

mg/L (2.5 x II)' cfu/l). 

Ther,: were no signs of infection in fish tissues examined for necropsy, and all appeared 

internally and ~xtemally normal. Gliocladium cotcnulatum could not be recovered from tissue 

samples collected Irom fish in the negative control, 45 and 89 mg/L treatment groups; however 

several colony forming units of G. catenulatum were recovered from the heart, liver and brain 

samples of fish treated with 179 mg/L test substance, and from the kidney., heart, liver and brain 

samples of Cish treated with 357 mg/L test substance. Although the test substance was present, 

target organs did not appear to be affected, and there were no apparent effects upon the health of 

the fish. 

2() 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 27 of 34 

DATA EVALUATION REPORT 

Reviewed by: Robyn Rose, Entomologist, BPPD I~J,- ':";(:,J.,l lD \, 

Secondary Reviewer: Gail Tomimatsu, Plant Pathologist, BPPD ",-,~, 1-, III:>( ! 

STUDY TYPE 

MRIDNO: 

CHEMICAL NO.: 

TEST MATERIAL: 

STUDY NO.' 

SPONSOR. 

TEST1NG FACILITY: 

TITLE OF REPORT: 

AUTHORS 

STUDY COMPLETED: 

CLASSIFICATION: 

STUDY StlMMARY: 

Daphnia 21-Day Life-Cycle Toxicity and Pathogenicitv Test 

(154A-20; OPPTS 885.4240) 

445014-03 

021009 

Gliociadiul1I catenulatum strain J1446 (Technical Powder) 

293A-I03 

Kemira Agro Oy, P.O. Box 330, Helsinki, Finland 

Wildlife International Ltd., 8598 Commerce Dr., Easton, MD 

21601 

Gliocladium catenulalum Cell Mass (Technical Product): A 21-

Day Life-Cycle Toxicity and Pathogenicity Tesl! With The 

Cladoceran (Daphnia magna) 

Susan J. Palmer, and Henry 0. Krueger, PhD. 

211 0/98 

Acceptable 

Objective: "[he objective of this study was to evaluate the toxic and pathogenic etTects of 

Giiociadiwil ( ([Ienulalum cell mass (technical product) on the Cladoceran, Daphnia magna, 

during a 21 ··day exposure period under static-renewal test conditions." 

Test Materi.;ll: 

Gliodadiwl1 I <ltenulatum Cell Mass (Technical Product) Batch No. 44/95; a light brown powder 

with all activity nf7x108 colony forming units per gram (cfu/g) was received from Kemira Agro 

Oy (3/26/97: Wildlife InternationallD #3996). Test substance viability was determined from 

O.lg (cst substance + ImL 0.3% TRITON-X sol. + 99mL sterile H20=IO~' dilution. The mixture 

was homogelllw:i for -10 sec. before preparing final solutions (10.5, I O~", 10"). The activity of 

the U catenllilllllnt was verified at Wildlife Intemational using the equation: Viability (in terms 

of CFU/g) . ::.: C/V" x N x L (D,,) [cfu/g]; C" =# of colonies on the agar plate, V" =volume 

platedlmL). N'iI of replicate plates, D,,=dilutions ofthe sample(g/mL). The test concentration 

was verified weekly by taking samples from alternating replicates from each treatment level. 

Test Orgal}ism: 

Neonate 1),("/1111([ magna «24 hOllrs old) obtained from cultures maintained by Wildlife 

Internatiol1.dl. [.ld .. Easton, MD were tested as a representative aquatic invertebrate. Neonate 

27 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 28 of 34 

juveniles were rcared and collected from 15 to 21 day old adult daphnids and used in this test. 

The adult daplmids were reared in water supplemented with selenium that was 20.6 to 2l.5 D C, 

8.2 to 8.6 pH, and 8.4 to 9.0 mg dissolved oxygenJL, and showed no signs of disease or stress 

during the 14-day period preceding the test. 

Test Methods: 

At test initiation, neonate daphnids were col1ected from cultures and transferred to I O-mL glass 

beakers contall1l1lg a small amount of dilution water. Progeny from six adults were used in the 

study. Ten daphnids were addeclto 12 separate 10 mL glass beakers containing a small amount 

of dilution water. These daphnids were then transferred into a test chamber. 

Test chambers were 21 L glass aquaria containing 5 L of test solution. Each test chamber 

consisted 01 Iwo compartments constructed from 500 mL glass beakers (,·8 em in diameter and 

13 em in height) containing five daphnids for the first seven days of the study. A nylon mesh 

screen attached to the side oftest compartments allowed water was to flow through. On the 

seventh day, dapl111ids were individually placed in a compartment in the test chamber. These 

compartments were -60 mm in diameter and 15 mm high culture dishes with 350 11m nylon 

screen attached to the sides (,,1 () cm in height). 

Test solution was added to chambers to a height of -5.7 cm. Daphnids were fed three times 

daily throughout the test and once on the last day. A mixture of yeast, Cerophyll®, and trout 

chow were feti to daphnids during culture and testing in addition to Selenastrum caprlcornutum, 

freshwater algae. During the test, the target temperature was 20,,,1 DC (measured by a hand·held 

thernlOmetcr) and the photoperiod was 16 hours of light and 8 hours of dark, controlled by an 

automatic tltner. The temperature was measured continuously in one negative control replicate 

using a Fulsco]Je ERJC Recorder. To aVOid sudden changes in lighting, a 30·min transition 

period oflow light intensity was provided: light intensity during the test was approximately 350 

lux at the wakr surface and provided by fluorescent tubes (Colortone®). Dissolved oxygen and 

pH were measured. in each lest chamber al the beginning and end of the test, daily for the first 7 

days, and at the beginning and end of one renewal period each week. Measurements of pH were 

made using a Fisher Accumet Model 915 pH meter, and dissolved oxygen was measured using a 

Yellow Sprin,>;s Instrument Model 51 B dissolved oxygen meter. Hardness and alkalinity and 

specitic conductance were measured in the dilution water at the beginning and end of the test, 

and at appruxllnately weekly intervals during the test. Test solutions were renewed OIl a 

Monday/W cdJlC'sday/Friday schedule. Nominal concentrations of the test substance tested were 

2.0x 1O(', 3,).jO". 7.7 x IO", 1.6x IO', and 3.Z<I07 CFU/L(2.8, 5J), 11,23, and 45 mg/L). 

The MlUrc'~ of water used for testing was freshwater obtained from an approximately 40 

m deep well located on Wildlifc International Ltd. site; and characterized as moderately·hard. 

The W.lter WCI' passed through a sand tilter to remove particles> 25 11m, pumped in 1.0 a 37,800 L 

storage tan k ,lI1d aerated with spmy nozzles. Befbre testing, the water was filtered again and 

passed througl': all ultraviolet sterilizer and aerated overnight. 

28 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 29 of 34 

Water samples of new and old test solutions were collected from each test chamber 

during the lI .. s! week of the study; and then also at weekly intervals from alternating replicates of 

new test solutions for the remainder of the study, Samples were placed in sterile glas~; 

scintillation vials and analyzed immediately or stored refrigerated (0 to 8°C) up to 3 weeks until 

they could be processed and analyzed by serial dilution plating for viability onlo plates 

containing potato dextrose agar. After 3 days of incubation at 28°C, all plates were examined for 

growth and enumerated for viable Gliocladiul11 catCl1u/atlll11 growth (CFU/mLl, 

Obs,ervations of mortality, immobility, or clinical signs of toxicity for first generation 

daphnids were recorded every Monday, Wednesday, and Friday during the test Daphnids were 

recorded as dead when a heartbeat was absent, coloration was white opaque, there was a lack of 

movement and response to gentle prodding. Signs or toxicity included inability to maintain 

position in the water column, uncoordinated swimming, lethargy, opaque color and ceased 

feeding. Nl:Ollate daphnids were discarded when observations were made and at test termination. 

Length (from the apex of the head to the base onhe spine) and dry weight of the daphnids were 

measured at the end of the 21-day exposure period. 

Statistical analyses were performt'd on the survival of the first-generation daphnids on 

the 7"' .. 14'h. and 21" days as well as on the length and dry weight, and the number oflive young 

produced by each surviving first-generation daphnids. A 2 X 2 contingency table was used to 

analyze survival data and significant differences between the treatment and control groups were 

detennined bv a chi-square test. The CB. Stephan program was used to calculate a 21-Day EC,O 

value. Reproduction and growth were evaluated for normality via the Shapiro Wilds test and 

homogeneity or variance was evaluated using Bartlett's test. Bonferroni tcst determined 

statistical di ITcrences. 

Reported Results:: 

A. Test Suhslance Viability 

ActivJ[Y of the test substance, Gliocladiunl catenu/alum cell mass was measured prior to 

the test; and all nominal concentrations (see Methodology) were based 011 a verified and 

Sponsor-approved average of 4 x lOx CFU/g test substance. The average Gliocladillin 

catenulalum concentration from each group measured during the test (average of new solutions 

for Days 2. 7 and 14) were 3.0>< 104, Ll X I 0', 7,9x I 05

, 9.4x 10', and 3.3x 1 0" CFUlL (0.04, 0,07, 

0.16. i,l, I,. and4.7mg/L). 

B. Welter ('Ill'l11istry Measurements 

Measurements of temperature were well within the range 0[20 ± 1°C (range: 20.1 to 

20.6°('); dissllived oxygen levels were within the range of8.5 to 9.0 mg/L (? 93 % saturation at 

200 e); and water pH ranged from 8.1 to 8 6. 

29 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 30 of 34 

C. Mortality alld Survival, and Other Clinical Observations 

There were no significant differences between the percent mortality in any of the 

treatment groups and negative control on Day 7 Crable I). On Day 14 there were no significant 

differences bel ween the 2.8 and 5.6 mg/L treatment groups and the control; however there was 

significantly more mortality in the 11, 23, and 45 mg/L treatment groups than the negative 

control group (Table I). The test was terminated after 21 days because mortality in the negative 

control group exceeded 20%. There were no signs of immobility or unusual behavior in 

surviving organisms through test termination. Therefore, Wildlife International concluded that 

the 2 I-day IT," value for Daphnia magna exposed to Gliocladium catenuiatum cell mass 

(technical product} was 7.8 mg/L. The 95% confidence limits were 5.2 and II mg/L. The 

LOEC was] I mg/L and the NOEC was 5.6 mg/L. 

Table 1. Summary of Cumulative Percent Mortality 

Nominal Tesi Cur' cent 

IlL ) 

raliOn 

mg/L i ( 'F I 

:'\.egati\(' (,,1 ntro I 

2.8 (2.0:< II)" I 'FU/ L) 

,'i.e) (3.9>< 1 n{' I TI II L) -

; I (7.7 x \.)1' { II! L) 

2~(1'()xl'J ( r't il I.) 

4\ (3.2"' ).' ( lUI .) -. 

Day 7 (% mortalit~,) 

0 

0 

0 

15 

0 

5 

Day 14 (~':) mortality) Day 21 (%, mortality) 

5 ,W 

5 :20 

j J5 

4() * 70 * 

()O * 75 * 

85 * 95 * 

*Percent mortalities significantly different (p s OJJ5) fro111 the negative controL 

Reproduction 

The ncgati ve control group produced an average of 59 live young. The 2.8 mg/L 

Glioe/adiulII nllelluiatum cell mass treatment group produced 57 live young which was not 

statistically dtiTcrent from the control group. With 51 young adults produced, the 5.6 mg/L 

GliocladiulII ,·,tfenuiatum cell mass treatment group was statistically less (p :> 0.05) than the 

control group. Significant mortalities were noted in groups treated withi 1,23, and 45 mg test 

substancc/L, therefore, reproduction results were not analyzed for these groups. The 21·day Ee,O 

value was 7.8 mg/L (-5.5 x IOI'du/L) w 95% confidence intervals of5.2 and II mgiL (3.6 x 10" 

and 7.7 x 10" dulL). The NOEC and the LOEC were 2.8 (2.0 x 10" cfu/L) and 5.6 mg/L (3.9 x 

10" cfu/L), m;pectively. 

Growth 

Dapillll(is in the groups treated with 2.8 and 5.6 mg test substanceiL grew to a similar size 

as the dapbnids in the untreated group. The mean length and mean dry weight for adults in the 

control group were 4.0 mm and 0.649 mg. Significant effects on survival were noted in groups 

30 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 31 of 34 

recclving 11,23., and 45 mg test substancc/L treatments, therefore, growth results were not 

analyzed statlsl1cally. The LOEC for growth was cstimated to be> 5.6 mg/L and the NOEC was 

5.6 Illg/L. 

Reviewer Comments: 

A. Methods 

This study was conducted in compliance with Good Laboratory Practice Standards as 

published by the U.S. Environmental Protection Agency, Office of Pesticide Programs in 40 

CFR Part I (,0 This study meets the guidelines outlined in OPPTS 885.4340 (updated from 

FIFRA Subdivision M, Series 154A-23). 

There were no verifications or observatiOons of infectivity. The inclusion of a control 

group using sterile filtrate from production cultures might have aided in assessing pathogenesis. 

The absence () f this treatment docs not significantly alter the overall conclusions, however, for 

hazard testing of Gliocladium catenulatum for the current intended uses. 

B. Conclm.ions 

Under conditions of the reported study, the 21-day ECsll value for survival fOor Daphnia 

magna exposed 10 Gliocladium catenulatum cell mass (technical product) was 7.8 mglL (5.5 x 

10' dulL). The LOEC was 11 mg/L (3.6 x 10" cfu/L) and the NOEC was 5.6 mg/L. 

Of the three parameters assessed (survival, growth and reproduction), reproduction 

proved to he the most sensitive parameter in this study. The 21-day EC,o vaille was 7.8 mg/L 

(~5.5 li. IO"clu/L); and the NOEC and the LOEC were 2.8 (2.0 x 10" cfu/L) and 5.6 rng/L (3.9 x 

10" cfu/L), Ic,;pcctively. 

31 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 32 of 34 

DATA EVALUATION REPORT 

Reviewed by Robyn Rose, Entomologist, BPPD f2,t ':)(;! ( I,) I 

Secondary Reviewer: Gail Tomimatsu, Plant Pathologist, BPPD'lf &/ z-' /of 

STUDY TYPE: 

MRIDNO.: 

CHEMICAL NO.: 

TEST MATFRIAL: 

STUDYNO .. 

SPOJ\SOR 

TESTING 1·'i\ClLITY: 

TITLE OF REPORT: 

AUTHORS. 

STUDY COMPLETED: 

CLASSIFI c;\ nON: 

Study Summary:: 

Non-Target Plant Literature Search (OPPTS Guideline 885.4300) 

44183-20 

021009 

Gliocladium calenu/alum strain J 1446 (Technical Powder) 

NIA 

Kemira Agro Oy, P.O. Box 330, Helsinki, Finland 

'vVildlife International Ltd .. 8598 Commerce Dr., Easton, MD 

21:601 

Gliocladium catenu/atum - Literature Search for Nontarget Plants 

Oili Mohammadi 

11122/96 

Acceptable 

ObjC.f.tivc: Summary of the literature search on Gliocladiwn catenu/atum and plants 

Results of ;~carcll 

A search on AGRICOLA, CA SEARCH, CAB Abstracts, BIOSIS PREVIEW. EMBASE, 

Enviroline, and Life Sciences Collection with the key words Gliocladium catenu/atum and plants 

resulted in ,21 items found. 

Discussion: Of the 21 abstracts found, 14 related to the biological control of Gliocladium 

catenu/atunl against plant diseases, one related to nematode control, and two articles addressed 

pine seed stimulation. Four abstracts reported Gliocladium catcnulatum isolated from diseased 

or dead planh. Two abstracts reported damping,·off of coffee in Cuba, on reported a member of 

the pea rool rot pathogen complex, and an isolation from dead birch trees was reported. 

However, these articles did not show that Gliocladium catenulatum was the causal agent of the 

disease. Prc,~nce of Glioc/ariium calenu{alum is usually a result rather than a cause of the 

infection. 

Reviewer ( .·omments: 

Giiociadiwil calcllulatum is a fungal pathogen that controls plant diseases such as damping-off, 

seed rot, root and stem rot, wilt and certain foliar diseases caused by Rhizoctonia, Pythium, 

Fusarium, /){{(nnella, Botrytis, Vcrticilliumc Alternaria, Cladosporium, Helminthosporium, 

Penicillium. and Pilcaria. Therefore, Gliocladium catenulatum is not expected to adversely 

32 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 33 of 34 

effect non-target plant species. In addition a literature search conducted by Kemira Agro Oy did 

not show any potential adverse effects of Ciliocladium catenulatum on non-target plants. This 

literature review of studies relating to potential adverse effects of Gliocladium catenulatum on 

no-target planl species is sufficient to satisfy current guidelines for non-target plants a\ this time. 

33 
EPA's Records Disposition Schedule PEST 361 Scientific Data Reviews HED Records Center - File R139870 - Page 34 of 34 

~ 13544 

• 

R139870 

Chemical: Gliocladium ('atenulatuUl strain J 1446 

HED File Code: 

l\1emo nate: 

File ID: 

Accession #: 

PC Code: 

021009 

41300 BPPIl Eco Elfects 

812112001 

DPD244243 

DPD267225 

000-00-900 I 

HED Records Reference Center 

3/2212007 

• 

•