Appendix B – Ecological Effects Data

This appendix contains toxicity data from three sources:  Ecotoxicology guideline studies in Agency files, data located by ECOTOX in open literature, and data as presented in the registration review ERA.  Reviews for open literature data included in the table, or reviewed and not found valid for quantitative or qualitative use, are also included.

Table   SEQ Table \* ARABIC  1   Ecological Guideline StudiesM for Fomesafen

Guideline #	Requirement Description	Test Product	Data Valid for Risk Assessment?	MRID	Study ClassificationC

71-1	Avian Acute Oral	Technical 	Yes	163168E	Core

71-2	Avian Subacute Dietary	Technical 	Yes

Yes	103022

163384E	Core

Core

71-4	Avian Reproduction	Technical

Technical	Yes	135639E

144862	Core

Core

72-1	FW Fish Acute	Formulation	Yes	103023E	CoreC

72-2	FW Aquatic Invertebrate Acute	Formulation	Yes	163169E	CoreC

72-3 (a)	SW Fish Acute	Formulation	Yes	135651E	CoreC

72-4	SW Aquatic Invertebrate	Formulation	Yes	135647E	CoreC

72-4	FW Fish Early Life Stage	No study located

72-4	SW Fish Early Life Stage	Formulation	Yes	135644E	CoreC

72-4(b)	FW Aquatic Invertebrate Reproduction	Formulation	Yes	135642E CoreC

123-1(a)	Seedling EmergenceA	Formulation

Reflex 2LC	Yes	46673801 E	Acceptable

123-1 (b)	Vegetative VigorA	Formulation

Reflex 2LC	Yes	46673802E	Acceptable

123-2	Aquatic PlantA	Technical	Yes

Yes

Yes

Yes

Yes	46673803E

46673804E

46673805

46673806E

46673807	Acceptable

Supplemental

Acceptable

Acceptable

Acceptable

141-1	Honey Acute Contact	Formulation

Flex 2 LC	YesR	135651	CoreC

	Honey Bee Acute Oral

Non	Earthworm	Technical	Yes	135652	Core

Non	Soil Invertebrates	Technical	Yes	135656	Supplemental

M Not including mammalian studies, which are reviewed by HED

C Studies submitted and classified in 1980’s, when tests on formulated product were permitted

A Not reported in OPPIN

E Used as endpoint in assessment

R Currently, there is no standard method for using this type of data in the risk assessment.Table   SEQ Table \* ARABIC  2   Summary of Registrant Submitted Acute AquaticToxicity Studies

Species	LC50

(mg/L)	Slope	95% C.I.

(mg/L)	NOAEC

(mg/L)	MRID	Toxicity

Category	Study Classification	Notes

Freshwater

Green Alga

(Selenastrum capricornutum)	0.092	2.4	0.078-0.110	0.0095	46673804	NA Supplemental

(72-hr study)	Technical

Freshwater diatom

(Navicula pelliculosa)	3.5	2.4	2.3-5.4	ND

EC05 =0.74	46673805	NA	Acceptable	Technical

Blue-green alga

(Anabaena flos-aquae)	71	5.6	60-84	27.3	46673807	NA	Acceptable	Technical

Duckweed

(Lemna gibba)	0.210	1.9	0.150-0.290	0.064	46673803	NA	Acceptable Technical

Water flea

(Daphnia magna)	376	5.6	323-437	ND	1631169	Practically nontoxic	Core Formulation1,2

Rainbow trout

(Onchorhyncus mykiss)	126	14.6	117-135	ND	103023	Practically nontoxic Core	Formulation1,2

Bluegill sunfish

(Lepomis macrochirus)	1507	ND	ND	ND	163169	Practically nontoxic Supplemental	Formulation1,3

Saltwater

Saltwater diatom

(Skeletonema costata)	0.490	1.6	0.270-0.900	0.360	46673806	NA	Acceptable Technical

Mysid shrimp

(Mysidopsis bahia)	25	ND	19-38	ND	135647	Slightly toxic	Core	Technical

Sheepshead minnow

(Cyrpinodon variegatus)	>163	ND	ND	NR	135644	Practically nontoxic	Core Formulation1,2

1 Data are from studies originally reviewed and classified in 1984, some of which used formulated product. 2  For risk assessment purposes, test concentration were adjusted for percent a.i., if necessary, and endpoints were re-calculated using TOXANAL software, prior to 2006 Section 3 Ecological Risk Assessment.  2  For risk assessment purposes, test concentration were adjusted for percent a.i..  ND-Not determined  NA-Not applicable, no toxicity category scale for aquatic plants.

Table   SEQ Table \* ARABIC  3   Summary of Registrant Submitted Chronic AquaticToxicity Studies

Species	NOAEC

(mg/L)	LOAEC

(mg/L)	Endpoints Affected	MRID	Study Classification	Notes

Freshwater

Water flea

(Daphnia magna)	50	100	Reduced growth, total # of offspring	MRID 1354642 Core	Formulation1,2

Saltwater

Mysid shrimp

(Mysidopsis bahia)	0.7	1.7	Parental mortality	MRID 1354648	Core Technical

Sheepshead minnow

(Cyrpinodon variegatus)	12.2	20.1	Reduced larval survival	MRID 1354644 Core	Formulation1,2

1 Data are from studies originally reviewed and classified in 1984, some of which used formulated product. 2  For risk assessment purposes, test concentration were adjusted for percent a.i., if necessary, and endpoints were re-calculated using TOXANAL software, prior to 2006 Section 3 Ecological Risk Assessment.

Table   SEQ Table \* ARABIC  4   Summary of Registrant Submitted Acute Terrestrial Toxicity Studies

Species	LC50/LD50

(mg/kg)	Slope	95% C.I.	MRID	Toxicity

Category	Study Classification1	Notes

Acute oral dose studies on technical (sodium salt of fomesafen)

Mallard duck

(Anas playrhynchos)	>5,000	ND	ND	163168	Practically non-toxic	Core	none

Guinea pig	(F) 607	ND	ND	164901	Slightly toxic	Minimum	none

Mouse	(F) 745

(M) 766	1.6(0.6-2.6)

1.6(0.6-2.5)	512-1286

525-1341

Rat	(F) 1499

(M) 1858	5.4(2.0-8.9)

2.4(0.8-4.1)	1302-1749

1420-2546

Avian sub-acute dietary on technical (sodium salt of fomesafen)

Mallard duck

(Anas playrhynchos)	>20,000	ND	ND	163384	Practically non-toxic	Core	none

Bobwhite quail (Colinus virginianus))	>20,000	ND	ND	103022	Practically non-toxic	Core	none

Honey bee acute

Honey bee

(Apis mellifera)	>50 mg/bee	ND	ND	135651	Practically non-toxic	Core	Oral dose

	>100 mg/bee	ND	ND

Contact

1Studies originally reviewed and classified in 1980s.

ND Not determined  NR Not reported

NA Not applicable, non-definitive endpoint

Table   SEQ Table \* ARABIC  5   Summary of Registrant Submitted Acute Mammal Toxicity Studies on Formulations

Species	LC50/LD50

(mg/kg)	Slope	95% C.I.	MRID	Toxicity

Category	Study Classification	Notes

Mammal acute oral dose on formulations

Rat 	(F) 3863

(M) >5000	NR	NR	44030101	Practically non-toxic	Acceptable	22.1% fomesafen/

Trade: Flexstar HL

Rat	5,000	ND	2,865-8390	46952103	Practically non-toxic	Acceptable	10.1% fomesafen/

46.5%

s-metolachlor

Trade: Prefix

Rat	>1210 and<1780	NR	NR	40031302	Slightly toxic	Core	7.2% fomesafen/

29% Bentazon

Trade:

Faster

Rat	(F) 1210

(M) 1470	NR	NR

1000-2160	158241

also

40340502	Slightly toxic	Core	12.9% fomesafen/

29% Bentazon

Trade:

BASF 530 04

ND Not determined  NR Not reported

Table   SEQ Table \* ARABIC  6   Summary of Registrant Submitted Chronic Terrestrial Toxicity Studies

Species	NOAEC

(mg/kg)	LOAEC

(mg/kg)	Endpoints Affected	MRID	Study Classification1	Notes

Mallard duck

(Anas playrhynchos)	46	ND	No endpoints affected	135639	Core	Technical

Rat	1,000	ND	No reproductive endpoints affected.

Parents and offspring showed modifications in liver histopathology at 1000 ppm	144862	Acceptable	Technical

1Studies originally reviewed and classified in 1980s.

ND Not determined

Table   SEQ Table \* ARABIC  7   Summary of Registrant Submitted Acute Toxicity Studies for Terrestrial Plants

Test	Species	Most Sensitive Endpoint	EC25

(lb ai/A)	95% C.I.

(lb ai/A)	EC05

(lb ai/A)	NOAEC

(lb ai/A)	MRID	Classification

Seedling emergence	Onion	Biomass	0.084	0.024-0.29	0.024	0.030	46673801 Acceptable

	Barnyard grass	Biomass	0.28	Not reported	0.076	0.12

	Corn	None	>0.48	N/A	>0.48	0.48

	Oat	None	>0.48	N/A	>0.48	0.48

	Tomato	% survival	0.0046	0.00048-0.044	0.00021	<0.0038

	Oilseed Rape	% survival	0.073	0.012-0.462	0.028	0.060

	Radish	% survival	0.10	0.004-3.14	0.023	0.060

	Lettuce	Biomass	0.22	0.123-0.444	0.15	0.030

	Sugarbeet	% survival	0.35	Not reported 	0.11	0.12

	Soybean	None	>0.48	N/A	>0.48	0.48

Vegetative vigor	Barnyard Grass	Biomass	0.29	0.14-0.59	0.097	0.24 46673802	Acceptable

	Corn	None	>0.47	N/A	>0.47	0.47

	Oat	Biomass	1.9	0.12-0.30	0.10	0.17

	Onion	Biomass	3.0	0.0007-14,000	0.045	0.47

	Lettuce	Biomass	0.0020	0.0011-0.0035	0.00042	<0.0019

	Radish	Plant height	0.0031	0.003-0.018	0.00037	0.00090

	Oilseed Rape	Biomass	0.0080	0.0050-0.013	0.0036	0.0019

	Sugarbeet	Biomass	0.15	0.12-0.19	0.064	0.0073

	Tomato	Biomass	0.24	0.16-0.36	0.045	0.060

	Soybean	None	>0.47	N/A	>0.47	0.47

Table   SEQ Table \* ARABIC  8   Summary of Valid Aquatic ECOTOX Toxicity Studies on Fomesafen

Species	Measurement	Type of Effect	Endpoint	Concentration 	ECOTOX Ref#

Aquatic

Algae

(Kirchneriella lunaris)

(Scenedesmus obliquus)

(Cryptomonas sp.)	Reduced growth, extent not specified	40 g/L

72 hours	87334

Caquet 2005

Aquatic mesocosm

Plankton

Caldoceran

Common backswimmer

(Notonecta glauca)	Modifications of trophic structure, as evaluated by stable isotope analysis	Community	“perturbation”

~LOAEC	30-60 g/L

15 weeks	87334

Caquet 2006

Aquatic mesocosm

Planktonic community	Decreases in dissolved oxygen	Community	NOAEC	~60 g/L

48 hours	87334

Pershberger 1997

Freshwater snail

(Lymnaea stagnalis)	Reactive oxygen species (ROS)

Phagocytic activity

Lysosomal fragility	Biochemical

Histological	LOAEC	10 g/L

24 hours	E95100

Russo 2007a

	Mortality	Mortality	NOAEC	270 g/L

21 days

Growth	Growth	NOAEC

	Increase in hemocyte density	Biochemical

Histological	LOAEC	10 g/L

24 hours	E95590

Russo 2007b

Table   SEQ Table \* ARABIC  9   Summary of Valid Terrestrial ECOTOX Toxicity Studies on Fomesafen

Species	Measurement	Type of Effect	Endpoint	Concentration 	ECOTOX Ref#

Fungus

(Bipolaris euphobiae)	Reduction in mycelium growth 	Growth	NOAEC	1L/ha

Product

10 days	E95538

Nemoto

	Speed of conidial germination

	1L/ha

Product

12 hours

	Mice

(Mus domesticus)

Various lab strains	Body weight gain	Growth	LOAEC	2,500 mg/kg diet E95026

Krijit 1993

	Increased relative liver weight	Growth	LOAEC	100 mg/kg diet	E95026

Krijit 1993

	Elevated liver and fecal porphyrins	Biochemical	LOAEC	100 mg/kg diet E95026

Krijit 1993

Open Literature Review Summaries for Fomesafen

Studies Classified Valid for Qualitative Use

Open Literature Review Summary

Chemical Name:  Fomesafen

PC Code:  123802

ECOTOX Record Number and Citation:

This review evaluates information from two related aquatic mesoscosm experiments evaluating population- and community-level effect of fomesafen and a fomesafen-surfactant mixture.  The mesocosoms used in this experiment appear to be the same mesocosms descirbed in Jumel et al. 2002 (ECOTOX 64953), although authors focus on different organisms in the mesocosoms.  Other related papers are Russo et. al, 2007 (ECOTOX 95100), and Russo & Medec 2007 (ECOTOX 95590).  All papers have been reviewed.

87334  Caquet, T, Deydier-Stephan, L, Lacroix, G, Le Rouzic, B, and Lescher-Moutoué, F.  (2005)  Effects of fomesafen, alone and in combination with an adjuvant, on plankton communities in freshwater outdoor pond mesocosm.  Environmental Toxicology and Chemistry 24:1116-1124.

95935  Caquet, T.  2006.  Use of carbon and nitrogen stable isotope ratios to assess the effects of environmental contaminants on aquatic food webs.  Environmental Pollution 141:54-59.

Purpose of Review (DP Barcode or Litigation):  Registration Review

Date of Review:  4/24/08

Summary of Study Findings:

Caquet, et. al 2005.  Authors evaluated the effects of fomesafen and a mixture of fomesafen and Agral-90 (a polyethoxylated nonylphenol surfactant) on planktonic communities in outdoor mesocosms.  Mesoscosms were treated with fomesafen (nominal concentration 40 g/L) or fomesafen/Agral-90 mixture (nominal concentrations 40 g/L and 90 g/L respectively) every three weeks, with a total of five treatments.  Water concentrations of fomesafen measured at the end of the treatment period were 62.5 g/L in the fomesafen only treatments, and 19.4 g/L in the mixture treatments.  Phytoplankton and zooplankton were collected, enumerated and identified at regular intervals throughout the experiment, which included a pre-treatment period, a treatment period, and a post-treatment period..  For algae, abundance data were used to calculate  Margalef’s diversity index and Simpson’s dominance index.  Concurrent laboratory toxicity tests were conducted on six algal species: Chlorella vulgaris, Kirchneriella lunaris, Scenedesmus obliquus,  and Staurastrum polymorphum (Chlorophyceae), Cryptomonas sp. (Cryptophyceae) and Cyclotella pseudoselligera (Bacillariophyceae).

g/L fomesafen (nominal) had a negative effect on the growth of 3 of the 6 species tested (K. lunaris, S. obliquus, and Cryptomonas sp.).  Authors do not specify the extent of the effect, merely noting it was statistically significant.

Authors conclude that Chlororphyceae were slightly inhibited in the fomsafen only treatments, and this may be due to the fact that a greater number of species in this class are more sensitive to fomesafen.  However, they were apparently replaced in the ecosystem by another less sensitive species.  They also note the between pond heterogeneity, which was high, may have decreased the statistical sensitivity of the study.  Fomesafen alone had no significant effect on zooplankton.

Caquet (2006).  In this study, author used stable isotopes of carbon and nitrogen to evaluate potential trophic effects associated with application of fomesafen to outdoor mesocosms.  The organism evaluated was 4th and 5th instars of the common backswimmer, Notonecta glauca L., which is an aquatic predator that feeds on zooplankton.  Upper level predators can integrate changes occurring in lower trophic levels, and the stable isotope ratios can be used to track these changes.  Authors measured stable isotopes of carbon and nitrogen in the backswimmer, but not lower trophic levels such as their cladoceran and copepod prey, or the primary producers.  The level of enrichment in 13C can be used to trace the origin of carbon in the food chain, and enrichments in 15N indicate trophic level.

Experimental design of the mesoscosms is as described above.  N. glauca nymphs were collected at two different times: once four weeks after initiating treatments (following two applications of fomesafen) and once seven weeks after initiating treatments (following three applications).  All nymphs collected the first time were 4th instar, and those collected the second time were 5th instar.  Instars were processed separately for stable isotopes.  Treatments did not have any effect on size or abundance of the organisms.

Based on information from the plankton study, summarized above, there were some changes in the composition of the plankton communities in the treated ponds, although no change in abundance.  Data on zooplankton communities presented in this paper (Table 2, pg 57) show an increase in rotifers and a decrease in calanoid copepods in both the fomesafen only and the mixture ponds, with the shift being more apparent in the mixture ponds.  According to author, copepods are a prey of the nymphs, along with cladocerans (no significant differences were found in cladoceran populations).

Comparisons of the stable isotope ratios (Fig 1, pg 56) shows two distinct groupings, comprised of the 4th instar and the 5th instar.  There was a significant effect of larval instar on 15N, indicating a shift in trophic level.  Post-hoc tests comparing the controls and the treatments showed fomesafen had an effect on 13C (indicating a potential shift in primary production) and the mixture had effects on both 13C and 15N ( indicating potential shifts in both primary production and trophic level).  [note:  author does not specify if post hoc test described were for both 4th and 5th instar, or just for 5th instar where differences were more discernable or if there was any interaction effect between treatment and instar.]

Information Applicable to Fomesafen Ecological Risk Assessment

Based on the Work of Caquet et. al.

g/L(“maximum mean”) in the fomesafen only ponds and as 19.4 ±7.6 g/L (“maximum mean”) in the fomesafen-Agral 90 ponds.  Although concentrations are reported only in Caquet et al., 2005, reviwer has assumed they are valid for both papers.  Authors make no mention of measuring concentrations in other compartments in the mesocosm or performing any mass balance calculations to determine fate of fomesafen in the mixture mesocosms, although they speculate (Caquet et al., 2005) it may have accumulated at the interfaces.

g/L) and 60 -day (6-13 g/L) EECs predicted by PRZM-EXAMS when fomesafen is applied in accordance with current labels (0.375-0.5 lb ai/A) (Fomesafen ERA, DP 302766, 314014, 314112) is lower than the concentrations evaluated in the mesocosm tests.  The most sensitive freshwater plant based on guideline tests was a green alga (Selenastrum capricornutum), with an LC50 of 92 g/L and an NOAEC of 9.5 g/L (MRID 46673806).

Description of Use in Document (QUAL, QUAN, INV):  QUAL

Rationale for Use:  Provides insight into potential effects on phytoplankton and zooplankton communities exposed to fomesafen, although concentrations used in the study are above modeled EECs.

Limitations of Study:  Measured concentrations of fomesafen are only explicitly stated for the end of the treatment period.  None of the papers associated with this mesocosm study have presented the water concentrations in a form that allows determination of a mean-measured or time-averaged concentration.

Primary Reviewer:

Paige D. Doelling, Ph.D., Fisheries Biologist, ERB1

Secondary Reviewer

Christine Hartless, Ph. D., Wildlife Biologist, ERB1

Open Literature Review Summary

Chemical Name:  Fomesafen

PC Code:  123802

ECOTOX Record Number and Citation:

This review includes a summary of data from five papers written by the same first author (Jan Krijit) describing and evaluating effects of fomesafen on mice liver hepatocytes.  Papers were published in various journals over a period extending from 1993-2003.  Evaluations of the papers are presented in chronological order.

E95026  Krijit, J, van Hosteijn, I, Vokurka, M, and Blaauboer, B. (1993)  Effect of diphnyl ether herbicides and oxadiazon on porphyrin biosynthesis in mouse liver, rat primary hepatocyte culture and HepG2 cells.  Archives of Toxicology 67:255-261.

E95399  Krijit, J, Vokurka M, Sanitrák, J, Janousek, V, van Hosteijn, I, and Blaauboer, B. (1994a)  Effect of the protophyrinogen oxidase-inhibiting herbicide fomesafen on liver uroporphyrin and heptacarboxylic prophyrin in two mouse strains.  Food Chemical Toxicology 32(7): 641-649.

E95589  Krijit, J, Vokurka M, Sanitrák, J, and Janousek, V. (1994b)  Effect of Protophyrinogen Oxidase Inhibitors on Mammalian Porphyrin Metabolism.  American Chemical Society Symposium Series 559:247-254.

E95400  Krijit, J, Stránska, P, Sanitrák, J, Chlumská, and Fakan, F. (1999)  Liver preneoplastic changes in mice treated with the herbicide fomesafen. Human and Experimental Toxicology 18:338-344.

E95588  Krijit, J, Pšenák, O, Vokurka, M, Chlumská, A, and Fakan, F. (2003)  Experimental hepatic uroprophhyria induced by the diphnyl-ether herbicide fomesafen in male DBA/2 mice. Toxicology and Applied Pharmacology 189:28-38.

Purpose of Review (DP Barcode or Litigation):  Registration Review

Date of Review:  4/14/08

Summary of Study Findings:

Fomesafen is a peroxidizing enzyme, inhibiting the enzyme protoporphyrin-III oxidase, which catalyzes the formation of protoporphyrin IX.  Protoporphyrin IX is a precursor molecule common to both plants and animals.  In plants, it is a step in chlorophyll biosynthesis, and in animals, it is a step in hemoglobin biosynthesis.  Some chemicals of this class (diphenyl-ether herbicides, which also includes aciflourfen, oxyflourfen, lactofen, and nitrofen) have been associated with peroxisome proliferation, which can induce hepatocellualr carcinomas in rodents.  (Smith and Elcombe 1989, Ashby et al., as cited in Krijit et al., 1999).

Krijit, et. al, 1993:  Authors investigated the effects of fomesafen ( and other chemicals) in vivo (mouse livers), and in vitro (rat primary hepatocyte culture and Hep G2 cells).  Based on a range-finding test, authors tested “0.25% (w/w) concentration of the herbicides in the diet” for a 10-day period.  Conversion of 0.25% to ppm in diet yields 2,500 ppm.

For the in vivo tests each herbicide and the control were tested with 4 male C57B1/6J mice per treatment.  Authors report statistically significant effects (p<0.05, student t-test) in the fomesafen-treated group for the endpoints of body weight (decrease) , relative liver weight (increase), liver  porphyrins (increase), fecal porphyrins (increase), liver microsomal ethoxyresorufin and pentoxyresorufin O-dealkylation (EROD and PROD) activity (increase), and peroxisomal β-oxidation activity( increase).  Effects on cytochrome P-450 level were not significant.  Raw data were not available to confirm statistical analysis.

Based on this study, there appears to be in decrease in body weight and a modification in liver activity for mouse fed a diet containing 2,500 ppm fomesafen for 10 days.

(Note:  The ECOTOX database lists an LOAEL of 100 ppm on mouse growth-weight for this paper.  This appears to be based on a change in relative liver weight (Table 1, pg 257) and has not been used as an endpoint.)

Krijit et. al.,1994a.  Authors investigated effects of long-term (5 months) dietary exposure to fomesafen on the enzymes protoporphyrin-II oxidase (enzyme 7 in heme biosynthesis) and uroporphyrinogen-III decarboxylase (enzyme 5 in heme biosynthesis).  They also evaluated the effects of administering iron separately and in conjunction with fomesafen on the biochemical endpoints.

Male ICR mice (n=4) were fed 0.25% w/w (2,500 ppm) fomesafen in diet for 5 months.  Treatment groups were a control, a group treated with just iron (subcutaneaous injection, 1 time, prior to feeding trial), a group treated with fomesafen (in diet), and a goup pre-treated with iron and fed fomesafen.  Authors note iron pretreatment can induce experimental prophyria.  Fomesafen decreased body weight, increased relative liver weight, and bile prophyrin content by a statistically significant amount (p<0.05, test not specified, Table 1).Some of the mice exhibited dark-colored livers.  Serum alanine aminotranferase (ALT), and liver uroporphyrinogen decarboxylase (UROD) activity and liver porphyrins were also affected.  Elevated ALT and UROD activities indicated hepatotoxicity.

A similiar experiment was conducted with male C57Bl/6J mice, although in this case they were fed 0.20% w/w (2,000 ppm) in diet, and oxyfluorfen and an oxyfluorfen-iron combination were tested in addition to fomesafen.  Results for body weight, relative liver weight, liver porphyrin and bile porphyrin were similar.  In this group of mice, authors also measured acitivity of liver cytochrome P-450 and associated enzymes, as well as peroxisomal β-oxidation activity.  Peroxisomal β-oxidation activity was affected in all treatment groups, and enzyme activity levels were elevated in most treatment groups, including those treated with fomesafen..

Authors concluded that it “appears fomesafen . . . can block two enzymes in the porphyrin biosynthetic pathway-protoporphyrinogen oxidase and uroporphyrinogen decarboxylase.  Whereas the inhibition of proyoporphyrinogen oxidase requires relatively low doses of the herbicide (Krijit et al., 1993) [100 to 2,500 ppm in diet for 10 days] the inhibition of URO-D can be observed only after feeding of high doses of fomesafen for prolonged periods [2,000 to 2,500 ppm for 3 to 5 months, depending on strain of mouse].” (pg 648)

Krijit et. al, 1994b.  Authors investigated the effects of fomesafen administered in the diet on biochemical endpoints (primarily prophyrin concentrations) in the liver, kidney, and bile of male ICR mice.  In all cases, treatment groups appeared to consist of 3 or 4 mice.  Results are presented in what appears to be (but is not specifically noted as) mean ± SD.  Table III (pg 249) presents statistical comparisons (p<0.05, test not specified), but the others do not.  A diet including 2,500 ppm of fomesafen fed to male ICR strain mice increases liver, bile, and fecal porphyrin content compared to controls (Table I, pg 248).  Smaller increases in liver and bile porphyrin content were noted in the same strain of mice fed 100 ppm fomesafen for 10 days.  In an experiment where the male ICR mice were fed 5000 ppm fomesafen in the diet for 10 days, followed by a 5-day period of control diet, porphyrin concentrations in the liver, bile and feces returned to levels not different than the controls (authors do not specify if these results were compared statistically.  Fomesafen fed to male C57Bl/6J for 3 months at a concentration of 2,500 ppm did not cause a change in total microsomal cytochrome P-450 or EROD, but did cause an increase in PROD as compared to controls.  Raw data were not available to confirm statistical analysis.

Authors conclude “rather high doses are needed to produce relatively slight changes in porphyrin concentrations in vivo.  Also, the accumulation of liver porphyrins induced by fomesafen treatment is clearly reversible.”  (pg 253)

Krijit, et al. 1999.  Authors investigated effects of long-term (50 weeks to 14 months), high-dose (2,300-3,000 ppm in diet) consumption of fomesafen on biochemical and histological endpoints in two strains of mice (C57Bl/6J mice and ICR mice).  Biochemical endpoints measured were associated with porphyrin biosynthesis and histological endpoints evaluated were precursors to hepatocellular carcinomas.  Fomesafen caused preneoplastic changes in both strains of mice, as well as modifications in enzyme activity.  Only the ICR mice developed uroporphyria.  Iron injections increased response in both strains of mice.  In a portion of the study which included a recovery period after long-term treatment with a high dose(3,200 ppm for 9 months, followed by a 3 month recovery), authors noted that liver histology returned to normal, and liver porphyrin content decreased, although still remaining above control levels.

Based on information contained in this paper, authors conclude the liver porphyrin biosynthesis pathway is the most sensitive biomarker for fomesafen exposure.  They also note the toxicological significance of liver porphyrin accumulation is uncertain.  The extent of the response differs depending on the strain of mouse.

Krijit et al., 2003  Authors evaluated the effects of dietary fomesafen on two strains of male mice (C57Bl/6N and DBA/2N).  These strains of mice were selected because “PAH-induced uroporphyria readily develops in animals of the C57Bl6 strain, whereas DBA/2N mice are almost completely resistant” (pg 35).  Fomesafen content in the diet was 0.25% w/w (equivalent to 2,500 ppm, author converted to a daily dose of 350 mg/kg for DBA/2N group) for 18 weeks.

In the DBA/2N mice, administration of induced hepatic uroporphyria, indicating there was some inhibition of the uroporphyrinogen III decarboxylase enzyme (5th enzyme in the heme biosysthesis pathway) in addition to inhibition of protophyrinogen oxidase (7th enzyme in the heme biosysthesis pathway).  The C57Bl/6N mice showed a slight (although statistically significant) elevation in liver porphyrins.  In this study, authors also measured cytochrome P-450 activities, performed histological examinations, and looked at messenger-RNA in an attempt to elucidate biochemical processing differences between the two strains.

Based on comparisions between biochemical endpoints modified by exposure to fomesafen, and the known uroporphyria inducers TCDD and PCBs, authors concluded that there may be a “yet unknown mechanism of uroporphyria induction (p28).”  Extent and nature of the response to fomesafen differs depending on the strain of mouse.

Information Applicable to Fomesafen Ecological Risk Assessment

Based on the Work of Krijit et. al.

The work of Krijit et. al., demonstrates that fomesafen ingested by mice at concentrations ranging from 100-2,500 ppm diet for 10 days causes elevated liver and fecal porphyrins, primarily via inhibition of the 7th enzyme of the heme biosynthesis protoprophyrin III oxidase (Krijit et. al,1993).  Body weight gain is statistically significantly affected when fomesafen is administered at concentrations of 2,500 ppm in diet for 10 days (Krijit et. al,1993).  The treatment groups in all investigations are small (n=3 or 4).  Mice used in the experiments were generally in the size range of 30-35 grams.  In some cases, fomesfen may also inhibit uroporphyrinogen III decarboxylase, but specific biochemical responses may vary dependent on the strain of mouse (Krijit et. al,1994b, Krijit et. al, 2003).  .  Consumption of high doses (2,300-3,000 ppm) for extended periods of time (50 weeks to 14 months) caused development of precancerous cells in the liver (Krijit et. al,1999).  Even with extended exposure , mice appeared to recover following a period of clean diet (3months) with liver porphyrins decreasing, and liver histology not noticeably different from controls (Krijit et. al,1999).  In both studies that included a recovery period, liver porphyrin concentrations decreased rapidly following withdrawal from fomesafen, but did not return to the same levels as controls during the observation period (Krijit et. al,1999).

Description of Use in Document (QUAL, QUAN, INV):  QUAL

Rationale for Use:

Provides insight as to how chronic effects on mammals (and by extension, other animals) are mediated.  Subacute (10 day) growth endpoint derived from Krijit et. al 1993 (LOAEC decreased weight at 2,500 ppm diet) is higher than current guideline-derived NOAEC of 250 ppm and LOAEC of 1000 ppm and higher than acute dose LD50 (607 mg/kg).

(Note:  The ECOTOX database lists an LOAEL of 100 ppm on mouse growth-weight for this paper.  This appears to be based on a change in relative liver weight (Table 1, pg 257) and has not been used as an endpoint.)

Limitations of Study:

It is uncertain how modifications in liver porphyrin levels might affect wild mammals at the individual or population level.

Treatment groups were small (n=3or 4).

Strains of laboratory mice are inbred, and lack genetic variability found in wild populations that may affect response to the toxicant.  Different strains of mice were shown to react to a different extent, but all exhibited some type of negative effects.

Doses used in laboratory testing were generally an order of magnitude above what animals are anticipated to encounter in a treated field based on TREX calculations.  Kenaga upper bound EEC for short grass at 0.5 lb ai/A (highest application rate as of May 2008) is 118 mg/kg.  Kenaga upper bound dose for short grass-consuming 35 g mammal is 78 mg/kg bwt.

Primary Reviewer:

Paige D. Doelling, Ph.D., Fisheries Biologist, ERB1

Secondary Reviewer

Christine Hartless, Ph. D., Wildlife Biologist, ERB1

Open Literature Review Summary

Chemical Name:  Fomesafen

PC Code:  123802

ECOTOX Record Number and Citation:

95538 Nemoto,MCM, Nahas, E, Pitelli, RA, and Coelho, L.  (2002)  Germination and mycelial growth of Bipolaris euphorbiae Muchovej & Carvalho as influenced by herbicides and surfactants.  Brazilian Journal of Microbiology 33:352-357.

Purpose of Review (DP Barcode or Litigation):  Registration Review

Date of Review:  4/25/08

Summary of Study Findings:

Authors investigated the effects of herbicides and surfactants on the mycelium growth and conidial germination of Bipolaris euphorbiae, a fungus which serves as a biological control for milkweed in soybean fields.  Authors describe test concentrations of 1L/ha for fomesafen, but do not specify the commercial product used in the test, thus a determination of dose in terms of active ingredient (ai) is not possible.  Fomesafen tested at this concentration had no effect on either mycelium biomass or conidial germination of the test organism.  Authors conclude tank mixes of Bipolaris euphorbiae and fomesafen are not contraindicated.

Description of Use in Document (QUAL, QUAN, INV):  QUAL

Rationale for Use:  Provides information of effects on underrepresented taxa.

Limitations of Study:  Determination of dose in terms of active ingredient (ai) is not possible.  Test quantity should be presented in terms of L/ha (end-use product).

Primary Reviewer:

Paige D. Doelling, Ph.D., Fisheries Biologist, ERB1

Secondary Reviewer

Christine Hartless, Ph.D., Wildlife Biologist, ERB1

Open Literature Review Summary

Chemical Name:  Fomesafen

PC Code:  123802

ECOTOX Record Number and Citation:

E53095  Perschbacher, PW, Stone, N, Ludwig, GM, Guy, Jr. CB. (1997) Evaluation of effects of common aerially-applied soybean herbicides and propanil on the plankton communities of aquaculture ponds.  Aquaculture 157:117-122.

Purpose of Review (DP Barcode or Litigation):  Registration Review

Date of Review:  4/25/08

Summary of Study Findings:

Authors investigated the effects of spray drift deposition on aquatic mesocosms for herbicides associated with soybean production.  Herbicides were sprayed across the surface of outdoor 500-L fiberglass pools at concentrations of full label rate, 1/10 label rate (described as “high drift”) and 1/100 label rate (described as “low drift”).  Pools were filled with water drawn from nearby catfish aquaculture ponds, but authors do not specify total water volume or note whether water was tested for any other toxicants.  Following treatment, authors evaluated phytoplankton productivity, morning dissolved oxygen (DO) levels chlorophyll a, pheophytin a, total ammonia nitrogen, nitrite nitrogen, and performed a light bottle/dark bottle estimation of phytoplankton productivity and respiration.  Zooplankton and phytoplankton were apparently identified and enumerated for concurrent investigations, but results are not reported in this paper.  Authors do note cyanobacteria dominated the phytoplankton population.

Fomesafen (as the product Reflex) was applied at the rate of 0.43 kg ai/ha (equivalent to 0.38 lb ai/A; probably based on the aerial application rate for soybeans of 0.375 lb ai/A).  Authors note “the highest rate of application of 0.57 kg/ha was equivalent to 0.06 mg/L (pg 121)”, but do not specify if this value was calculated or measured.

Dissolved oxygen was found to be the most sensitive parameter.  For fomesafen, there was no significant difference in dissolved oxygen content in the treatments as compared to the control for any concentration.

Information Applicable to Fomesafen Ecological Risk Assessment

Based on the Work of Pershbacher et. al.

work, one could interpret that short-term effects on the phytoplankton abundance and production from concentrations of fomesafen ≤0.06 mg/L are not anticipated.  This should be caveated with the fact that the mesocosm community was dominated by cyanobacteria (which appear to be less sensitive to fomesafen than green algae based on registrant-submitted studies: MRID 46673806, green alga EC50 92 g/L; MRIDs 46673807, blue-green alga EC50 710 g/L).  Also, measurement endpoints reported by authors evaluate overall primary productivity of the system, rather than community structure, which may be more responsive.

Description of Use in Document (QUAL, QUAN, INV):  QUAL

Rationale for Use:  Confirmatory data for other non-vascular aquatic plant information.  Exposure scenario closely mimics current EFED spray drift evaluation.

Limitations of Study:  Aquatic concentrations were not measured, and can only be estimated.  Effects should be associated with technical end-use product Reflex, rather than technical fomesafen.

Primary Reviewer:

Paige D. Doelling, Ph.D., Fisheries Biologist, ERB1

Secondary Reviewer

Christine Hartless, Ph. D., Wildlife Biologist

Open Literature Review Summary

Chemical Name:  Fomesafen

PC Code:  123802

ECOTOX Record Number and Citation:

This review evaluates two papers regarding fomesafen effects on the hemocytes of the freshwater snail Lymnaea stagnalis.

E95100  Russo, J, Lefeuvre-Orfila, l, and Lagadic, L.  (2007) Hemocyte-specific responses to the peroxidizing herbicide fomesafen in the pond snail Lymnaea stagnalis (Gastropoda, Pulmonata). Environmental Pollution 146:420-427.

E 95590  Russo, J, Madec, L.  (2007)  Hemocyte apoptosis as a general cellular immune response of the snail, Lymnaea stagnalis, to a toxicant.  Cell Tissue Research 328:431-441.

Purpose of Review (DP Barcode or Litigation):  Registration Review

Date of Review:  4/23/08

Summary of Study Findings:

Fomesafen acts by inhibiting protoporphyrinogen oxidase (Grimm 1999, as cited in Russo et. al, 2007a) an enzyme in the porphyrin biosynthesis pathway for chlorophyll (plants) and hemoglobin (animals).  This results in production of reactive oxygen species (ROS), which can disrupt cellular membranes in plants (Wakabayashi and Böger 1999, as cited in Russo et. al, 2007b) and in animals Elcombe et. al, 1996, Krijit et. al, 1999 as cited in Russo et. al, 2007b).  The papers evaluated in this review are concerned with effects on the circulating hemocytes of freshwater snails exposed to fomesafen in aqueous solution.

Russo et. al, 2007  Snails were exposed to concentrations of 10, 30, 90, and 270 g/L fomesafen (nominal)  for 21 days in a laboratory setting.  Fomesafen was solubilized in acetone, with the concentration of solvent in exposure media maintained below 1 L/mL.  Authors maintained both solvent and neutral controls.  Endpoints measured included intracellular production of reactive oxygen species (ROS), phagocytic activity, and membrane integrity.  Measurements were made at 24 hours and 504 hours of exposure.  Results were evaluated with two-way ANOVA for time and treatment, and one-way ANOVA to compare different exposure regimens for each exposure duration.

●  Even at the highest concentration (270 g/L), there were no observable effects on snail mortality or hemocyte cell viability.

	●  Intracellular production of ROS was enhanced in all concentrations at both time durations except for the 270 g/L-21 day group.  Based on results from the two-way ANOVA, exposure time was significant (p=0.0001).  Results were not presented for the interaction term.

	●  Phagocytic activity followed approximately the same pattern as proportion of ROS-producing hemocytes, with reductions in activity even at the 10 g/L concentration, and greater inhibition of phagocytic activity in the hemocytes from snails exposed for 504 hours.  Based on two-way ANOVA analysis, the concentration of fomesafen was significant (p=0.049), but the effects of time, and the interaction of time and concentration were not significant.

	●  Authors evaluated the “oxidative burst,” a cellular immune defense, in snail hemocytes stimulated by E. coli (low stimulation) and phorbol 12-myristate 13-acetate (PMA, high stimulation).  In the E. coli stimulated cells, the oxidative burst was statistically significantly reduced in all treatment groups when measured at 504 hours, but not when measured at 24 hours.  In the PMA stimulated cells, the reverse situation occurred, where the burst was statistically significantly reduced in all treatment groups when measured at 24 hours, but not when measured at 504 hours.

●    Lysosomal fragility (as indicated by neutral-red dye retention time) was statistically significantly decreased in all treatment groups, and length of exposure also had a significant effect, with great fragility noted in the 504 hour exposure.  Authors estimate the LOAEC for lysosomal fragility is 10 g/L for both exposure periods (Williams test, p=0.05, pg 423).  In their discussion, they note tha “lysosome stability represents a key component of cell responses to chemical stress (page 426) but caution “the significance of physiological changes in the snails needs to be carefully defined (p 426.)”

Russo & Medec 2007

(Reveiwer’s note:  Snails described in this paper may be the same snails described in Russo et al., 2006, with the second paper focused on different endpoints.)

Authors investigated the effect of exposure to fomesafen on apoptosis (programmed cellular death) in freshwater snail circulating hemocytes.  Exposure to fomesafen can generate reactive oxygen species (ROS) in the cells, which may initiate the apoptosis sequence as cellular membranes are compromised.  Exposure periods were 24 hrs, 96 hrs, and 504 hrs (21 days).  The experiment evaluated two events which occur in apoptosis:  the disruption of the mitochondrial transmembrane potential, and membrane asymmetry with phophatidylserine (PS) release.

g/L).  Cell viability was consistently 94%.  Authors state exposure to fomesafen at 30 g/L and higher concentrations, had a significant effect on hemocyte density compared to the control (Dunnett’s test, p=0.01 to 0.0003).  ,Authors note time of exposure did not affect hemocyte density (p=0.08), and for this endpoint, comparisons appear to have been averaged over time.  Based on Figure 1 (pg 434, tables with specific values not included), exposure to acetone increased the hemocyte density, although authors do not note if the increase is significantly different from the neutral control.

Oxidative stress (ROS production) was measured by exposing the hemocytes to a fluorescing dye, and then measuring the response using flow cytometry.  Cells from the treatment groups were compared to the neutral control and to a positive control.  Flow cytometry showed “two functional hemocyte populations,” distinguished by the the wavelength at which they fluoresced.  Authors state that “according to the four criteria used for the multivariate test (criteria not specified) the effect of fomesafen exposure on ROS production was highly significant.”  Both populations of hemocytes from exposed snails displayed an increased magnitude of flouresence compared with control and PMA-induced samples.  At 504 hours there was a marked decrease in ROS production.

g/L treatment groups, but not in the 90 and 270 g/L groups.

Membrane asymmetry, which the authors describe as an indicator of intermediate stage apoptosis, was evaluated by using two flourescing dyes, one of which binds to a cellular surface marker, and one of which intercalates DNA.  Hemocytes were placed into groups based on the proportions of these dyes present in the cells.  The groups were viable cells, apoptic cells, necrotic cells, and dead cells.  At 96 hours, the samples from fomesafen-treated snails included a higher percentage of cells in the apoptic and necrotic stage than the control.  Authors note “the subsequent increase of necrotic cells did not imply that these cells passed into the population of dead cells.”  At 504 hours, there were no significant differences between any of the treatment groups.

Authors conclude fomesafen is “responsible for increasing plasma membrane permeability and affects cyltoplamic manifestations but does not provoke an advanced stage of the [cellular] death process.”

Information Applicable to Fomesafen Ecological Risk Assessment

Based on the Work of Russo et. al.

≤270 g/L for durations ≤504 hours (21 days) these was no observable abnormal behavior, mortality, of reduction of hemocyte cell viability in the snails (Russo et. al 2007, Russo and Madec 2007).  Based on this work, it appears that fomesafen induces an immune response in freshwater snails at concentrations that may occur in the environment, but how these responses translate to measurable effects at an organism level (e.g. reduced growth, tumor activation, increase susceptibility to disease) are unknown.

Description of Use in Document (QUAL, QUAN, INV):  QUAL

Rationale for Use:  Provides insight into mechanisms whereby fomesafen affects aquatic wildlife.

Limitations of Study:  Concentrations were nominal, but fomesafen is relatively persistent in aquatic systems.  Effects at biochemical level are not readily translated to the growth, survival, and reproduction endpoints used in the EFED risk assessment process.

Primary Reviewer:

Paige D. Doelling, Ph.D., Fisheries Biologist, ERB1

Secondary Reviewer

Christine Hartless, Ph.D., Wildlife Biologist, ERB1

Open Literature Review Summaries for Fomesafen

Studies Classified Invalid

Open Literature Review Summary

Chemical Name:  Fomesafen

PC Code:  123802

ECOTOX Record Number and Citation:

64953  Jumel, A. Coutellec, M-A, Cravedic J-P, and Lagadic, L.  (2002)  Nonylphenol polyethoxylate adjuvant mitigates the reproductive toxicity of fomesafen on the freshwater snail Lymnaea stagnalis in outdoor experimental ponds.  Environmental Toxicology and Chemistry 21:1876-1888.

Purpose of Review (DP Barcode or Litigation):  Registration Review

Date of Review:  4/22/08

Summary of Study Findings:

Background:  Nonylphenol, a common surfactant has been associated with endocrine-disrupting effects.  Fomesafen is a diphenyl ethers (DPE) herbicide, which are protopophyrinogen oxidase inhibitors.  DPEs induce production of reactive oxygen species (ROS), resulting in cellular membrane disruption.  This investigation was “designed to study the influence of polyethoxylated nonylphenols formulated as Agral 90 on the toxicity of fomesafen in the pond snail L. stagnalis, with particular attention to the reproductive performance and underlying energetic and hormonal processes (pg 1877).

Tests were preformed in both the laboratory and in mesocosms.

Snails tested in the laboratory, were housed in stainless steel tanks with a density of  1.75-2.5 snails/L.  For the laboratory experiment, test concentrations were 22 g/l technical (99% purity) fomesafen and a combination of 22 g/l technical (99% purity) fomesafen and 50 g/l Agral 90.  No solvent was used.

Outdoor mesocosms contained phytoplantkton, zooplankton, aquatic invertebrates and plants.  Snails were caged within the mesocosms. Mesocosms were treated five times with either 40 g/l formulated fomesafen or 40 g/l formulated fomesafen and 90 g/l Agral 90 at three week intervals beginning in April.  Three replicates were used for each treatment.  Fomesafen concentrations in both the water and the snails was analytically confirmed.  Limit of detection was 1g/L for water and 2 ng/g for snails.  Limit of quantitation was 5g/L for water and 10 ng/g for snails.

Endpoints evaluated were:

Organism level: survival, fecundity, egg development and hatching rate, and F1 growth rate.

!

"

%

&

,

-

.

2

3

4

5

9

:

W

"

&

.

4

5

Ff

 5

:

X

d

h

p

v

w

€

Ž

š

ž

¦

¬

²

Ê

Ö

Ú

â

è

é

 W

X

a

c

d

g

h

n

o

p

t

u

v

w

Ž

™

š

ž

¤

¥

¦

ª

«

¬

Ê

Õ

Ö

Ù

Ú

à

á

â

æ

ç

è

é

í

î

0é

î

V

0

1

=

>

Q

R

S

\

]

`

a

q

y

z

{

ƒ

$

(

2

=

>

D

S

]

a

e

i

m

q

{

…

Ž

˜

¡

¬

¹

Ä

Ï

ƒ

„

…

‹

Ž

”

–

—

˜

ž

¡

«

¬

Ù

Ú

Û

à

á

ô

õ

-Ï

Ú

Û

á

õ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

전・ý尀

전・ý尀

摧紻Æ

hM

hM

hM

 h*

 h*

 h*

hM

hM

hM

hM

hM

hM

hM

hM

hM

hM

hM

hM

hM

hM

hM

hM

hM

\

gdM

hM

hM

hM

hM

 hŸE

hM

hM

 h

hM

hM

hM

hM

hM

hM

hM

hM

hM

h¥

h¥

h¥

h¥

h¥

h¥

h¥

h¥

hM

hM

hM

hM

hM

hM

hM

hM

hM

hM

전・ý尀

h¥

h¥

h¥

h¥

h¥

h¥

h¥

h¥

전・ý尀

전・ý尀

Æ

\

전・ý尀

\

4s; glycogen phosphorylase, UDP-galactose 4-epimerase, polysaccharide content (glycogen and galactogen), protein, and gonad steroids (testosterone and estradiol-17

Results:

Authors state intermesocosm variability for several endpoints was significant, and was taken into account in the data analysis

g/L in water (estimated from Fig 1) to a peak of 60 g/L at the conclusion of the exposure period (reported in text).  Concentrations of fomesafen in the mixture treatment were consistently lower than in the pesticide only treatment throughout the exposure period.  Concentrations ranged from a low ~5 g/L in water (estimated from Fig 1) to a peak of 30 g/L at the conclusion of the exposure period (reported in text).  Authors did not measure concentration of nonylphenol.

Reproductive performance:  Trends in reproductive effects appeared similar between the laboratory and mesocosm experiments, although specific values are difficult to compare given both the exposure concentrations and exposure times were different.  There was no significant effect on either number of egg masses or number of eggs per mass in the laboratory experiment.  In the mesocosm experiment, there was a significant effect on number of egg masses in the fomesafen-only treatment after the first application, (Fig 4, pg 1881), but data presented in Table 1 (pg 1882) indicate the snails in the fomesafen-only group were depositing less egg masses even before treatment.  The decrease in egg masses may not be treatment related.  There are no significant differences between either of the treatments and the controls at any other time period.  There was no significant differences in development of egg masses, hatching rate, or growth of the F1 generation in the treatment groups.

Energetic biomarkers:  Laboratory exposures to fomesafen and the fomesafen-Agral 90 mixture did not affect glycogen or glycogen phosphorylase activity.  In the mesocosms there were significant differences in glycogen content (at 12 days, Fig 5, pg 1883) and glycogen phosphorylase (at 8 days and 35 days, Fig 5, pg 1883) in the fomesafen treated group, but these reduced responses did not correlate with one another in a time sequence, and trends were not consistent over the exposure period.  There was no significant response in galactogen content or UDP-galactose 4-epimerase activity in the treatment groups as compared to the controls.

Hormonal biomarkers:  There were no significant differences in testosterone-like or estradiol-like steroids in the  treatment groups as compared to the control groups in the laboratory experiment.  Mean steroid concentrations in the gonadodigestive complex were slightly lower in the laboratory treatment groups than in the controls.  In the mesocosms, authors state “steroid-like level values were significantly lower in fomesafen-exposed snails as compared to the controls and the snails exposed to the fomesafen-Agral 90 mixture (treatment effect F[2,24]=3.00 p=0.069, and F[2,24]=5.04, p=0.015 for testosterone-like and estradiol-17-like, respectively, Tukey =0.05). (pg 1884).”

g/L at the end of the contamination period) and the snails strongly suggest that the surfactant properties of the adjuvant were responsible for such an overwhelming effect (pg 1886).”

Reviewer’s Note:  In both laboratory and mesocosm experiments, control mortality was high (20% and 29% respectively), indicating potential problems with either viability of the organisms and/or husbandry issues.  Given that both exposure durations and concentrations varied between the laboratory experiment and the mesocosm experiment it is difficult to correlate specific effects.  However, the glycogen content effects that were significantly different in the mesocosms were not observed in the laboratory as well as not occurring consistently over time or (based on data presented in Fig 5, pg 1883) or occurring in any obvious time sequence associated with the applications.  Although no variability information was provided, based on the values in Table 1 (pg 1882), it appears that the snails in the fomesafen treatment group evidenced depressed egg production compared to the other groups even prior to fomesafen exposure.  Authors do not provide any explanation as to why this may have occurred.  Thus, any conclusions regarding fomesafen effects on the snail reproduction based on these endpoints or related endpoints, such as the lower steroid levels in the gonadodigestive complex are questionable.   It is the reviewer’s opinion that no firm conclusions regarding the effect of fomesafen on L. stagnalis can be drawn based on the data presented in the paper.  Reviewer also believes that application of the surfactant Agral 90 reduces exposure in the dissolved phase (as evidence by the much lower measured water column concentrations of fomesafen in the mixture mesocosm) rather than necessarily reducing effects.

Description of Use in Document (QUAL, QUAN, INV):  INV  (Due to high control mortality and depressed reproductive performance in fomesafen treatment group prior to exposure.)

Rationale for Use:  Not used

Limitations of Study:  High control mortality and depressed reproductive performance in fomesafen treatment group prior to exposure.  Mean-measured value for mesocosm exposures can only be estimated from figure, and analytical verifications do not appear to have been spaced equally over time.  Fomesafen concentrations in laboratory experiment do not appear to have been analytically verified.  Thus it cannot be determined if fomesafen concentrations in the mixture treatment were the same as the fomesafen only treatment or if the surfactant also reduced dissolved phase fomesfen in the lab experiment.

Primary Reviewer:

Paige D. Doelling, Ph.D., Fisheries Biologist, ERB1

Secondary Reviewer

Christine Hartless, Ph.D., Wildlife Biologist, ERB1

Open Literature Review Summary

Chemical Name:  Fomesafen

PC Code:  123802

ECOTOX Record Number and Citation:

95404  Singh, KJ, Thakur RC, and Singh, OP.  (1988)  Toxicity of insecticides, on eggs, nymphs, and adults of tur-pod bug (Calvigralla gibbosa) and its ovipositional behavior on pigeonpea (Cajanus cajan).  Indian Journal of Agricultural Sciences 58(8): 621-623.

Purpose of Review (DP Barcode or Litigation):  Registration Review

Date of Review:  4/25/08

Summary of Study Findings:

전・ý尀

resumably of the 1,200L/ha applied).  They report significant mortality of nymphs (70-100%) and adults (67-90%) in the 72 hour observation period following treatment.

Reviewer’s Note:  Fomesafen is not generally classed as an insecticide.  Authors do not report specific source of pesticides used in experiment.  An internet search for the product the authors describe using, PP 21 5 EC, returned no hits, although references to PP 021 were located.  PP 021 is not currently registered for use in the United States.  Registrant-submitted data regarding fomesfen effects on terrestrial insects (MRID135656), honeybees (ACC 135651), and earthworms (ACC 135652) indicate relatively low or no toxicity for these classes of organisms.  It is the reviewer’s opinion that either 1) the product used was a multiple active ingredient product containing an insecticidal active ingredient, or 2) the plants in the fomesafen plot were contaminated by one of the other active ingredients evaluated, some of which are insecticides toxic at low concentrations.

Given these uncertainties, and information contradictory to the bulk of the available data, this study is classified invalid.

Description of Use in Document (QUAL, QUAN, INV):  INV

Rationale for Use:  Not used.

Limitations of Study:  Actual treatment concentration cannot be calculated.  Cross-contamination by other test materials may have occurred.

Primary Reviewer:

Paige D. Doelling, Ph.D., Fisheries Biologist, ERB1

Secondary Reviewer

Christine Hartless, Ph.D., Wildlife Biologist, ERB1

B-  PAGE  5


[Federal Register: March 19, 2008 (Volume 73, Number 54)]
[CORRECTIONS]
[Page 14880]
[DOCID:fr19mr08-142]

-----------------------------------------------------------------------

SECURITIES AND EXCHANGE COMMISSION

Proposed Collection; Comment Request

Correction

    In notice document E8-3665 appearing on page 10501 in the issue of
Wednesday, February 27, 2008, make the following correction:
    In the seventh line, ``1-2 Rule 17a-2;'' should read ``Rule 17a-
2;''.

[FR Doc. Z8-3665 Filed 3-18-08; 8:45 am]

BILLING CODE 1505-01-D