
Data Requirement:				PMRA Data Code:	9.8.4 (TGAI) or 9.8.6 (EP)
                        EPA DP Barcode:	460199
                        OECD Data Point:	IIA 8.12 (TGAI) and IIIA 10.8.1.1 (EP)
                        MRID:				51235102
                        EPA Guideline:		850.4100

Test material:	TPA Technical								Purity: 97.3% (w/w)
Common name: 	Tetrachloroterephthalic acid
Chemical name:	Tetrachloroterephthalic acid:
				IUPAC:		Not reported
				CAS name:	Not reported
				CAS No.:	Not reported
				Synonyms:	TCTPAc; 2,3,5,6-tetrachloroterephthalic acid

Primary Reviewer:	Joan Gaidos								Signature:  
Senior Scientist, CDM/CSS-Dynamac JV						Date: 01/28/2021
Secondary Reviewer:  Teresa Nelis							Signature:  
Senior Scientist, CDM/CSS-Dynamac JV						Date: 01/29/2021

Primary Reviewer:	Christina M. Wendel						Signature: 
EPA/OPP/EFED/ERB2/Biologist 								Date: 12/10/2021

Secondary Reviewer(s): Michael Wagman						Signature: 
EPA/OPP/EFED/ERB2/Senior Scientist						Date: 12/06/2021


Reference/Submission No.: {.....................}

Company Code: 	{............}	[For PMRA]
Active Code:		{............}	[For PMRA] 
Use Site Category:	{............}	[For PMRA]
EPA PC Code: 		078701

Date Evaluation Completed: 10-12-2021

CITATION: McKelvey, R., J. R. Arnie, G. Sneckenberger, and L. Zhang. 2019. TPA: A Toxicity Test to Determine the Effects on Seedling Emergence of Ten Species of Plants. Final Report. Unpublished study performed by EAG, Inc., Easton, Maryland. EAG Laboratories Project Number: 246P-111. Study sponsored by Amvac Chemical Corporation, Newport Beach, California. Study completed January 4, 2019.


This Data Evaluation Record may have been altered by the Environmental Fate and Effects Division subsequent to signing by CDM/CSS-Dynamac JV personnel. The CDM/CSS-Dynamac Joint Venture role does not include establishing Agency policies.


EXECUTIVE SUMMARY:

The effect of Tetrachloroterephthalic acid (TPA) technical (97.3% (w/w)), a metabolite of Dimethyl Tetrachloroterephthalate (DCPA), on the seedling emergence of four monocot crops (corn, Zea mays; onion, Allium cepa; ryegrass, Lolium perenne; and wheat, Triticum aestivum), and six dicot crops (cabbage, Brassica oleracea; oilseed rape, Brassica napus; soybean, Glycine max; sugarbeet, Beta vulgaris; sunflower, Helianthus annuus; and tomato, Lycopersicon esculentum) was studied. Nominal concentrations ranged from 0.313 to 10 lb a.i./A for all species. 

The test concentrations were analytically confirmed by the reviewer at all treatment levels, and nominal and measured application rates are provided in Table 3. The measured concentrations of TPA were <0.00018 (<LOQ, negative control), 0.31, 0.64, 1.2, 2.2, 4.5, and 9.1 lb a.i./A (onion, ryegrass, wheat, sugarbeet, and cabbage); <0.00018 (<LOQ, negative control), 0.58, 1.1, 2.2, 4.4 and 8.8 lb ai/A (corn, oilseed rape, sunflower, soybean, and tomato). 

The growth medium used in the seedling emergence test was a mixture of kaolinite clay, industrial quartz sand and peat, with limestone added to buffer the pH (loamy sand; pH 6.3; percent organic carbon 1.2%). On day 21, the surviving plants per pot were recorded and cut at soil level for measuring the plant height and dry weight.

Negative control seedling emergence ranged from 80 to 100% for all species tested. When compared to the negative control, there was significant inhibition in cabbage, sugarbeet and sunflower. Inhibition in sugarbeet emergence was significant at 0.64 lb a.i./A and higher test concentrations. Inhibition in cabbage and sunflower emergence was significant only at 0.31 lb ai/A and 2.2 lb a.i./A, respectively, and was not clearly dose-responsive. 

Survival was based on the number planted. Negative control for survival ranged from 75 to 100% for all species tested. However, if survival is based on number planted, as the reviewer calculated it, the mean control seedling survival does not meet the OCSPP 850.4100 test validity element of at least 90% survival at test termination for four species, onion (75%), sunflower (78%), ryegrass (80%), and corn (88%). When compared to the negative control, there was significant inhibition in soybean and sugarbeet. Inhibition in sugarbeet survival was significant at 0.64 lb a.i./A and higher test concentrations. Inhibition in soybean survival was significant only at 8.8 lb a.i./A, the highest test concentration. 

Significant inhibitions in seedling height were found in cabbage, soybean, and tomato. When compared to the negative control, inhibition in cabbage height was significant at 0.64, 2.2 and 4.5 lb a.i./A only and was not dose-responsive. Inhibition was significant in soybean height at 2.2 lb a.i./A only (and were not dose-responsive), and significant in tomato height at 8.8 lb a.i./A only, the highest test concentration when compared to the negative control.

Significant inhibitions in seedling dry weight were found in tomato. When compared to the negative control, inhibition in tomato weight was significant at 8.8 lb a.i./A only, the highest test concentration; however, based on the magnitude (21%) of effect at the second highest test level (4.4 lb a.i./A), in conjunction with consistently lower weights by replicate, although not statistically significant, the reviewer visually determined a lower LOAEC of 4.4 lb a.i./A. This resulted in a lower visually determined NOAEC of 2.2 lb a.i./A. 

The most sensitive dicot species was tomato, based on weight, with NOAEC and IC25 values of 2.2 and 7.76 lb a.i./A, respectively. Based on the reviewer's results, the most sensitive monocot could not be determined. There were no dose-responsive treatment related reductions that were greater than 25% for any monocot species. Therefore, the IC25 was visually estimated as >9.1 lb a.i./A, the highest tested application rate for all monocot species tested, and the NOAEC was 9.1 lb a.i./A for all tested monocot species. 

The following phytotoxic symptoms were noted for all test species: chlorosis, necrosis, and leaf curling. All species showed slight levels of phytotoxic symptoms, except cabbage, onion, sunflower and tomato showed up to moderate levels of phytotoxic symptoms. No species exhibited dose-dependent phytotoxicity. Phytotoxic symptoms were observed in control plants, but these symptoms were slight in severity and were not considered to be related to the treatment.

Seedling emergence studies are designed to capture sub-lethal effects; therefore, survival is not expected to be the most sensitive endpoint and mortality may have confounded growth effects in soybean and sugarbeet, this is considered a study deficiency for the species where survival is the most sensitive endpoint. Therefore, the results for soybean and sugarbeet should be interpreted with caution. For onion, sunflower, and ryegrass, the mean control seedling survival does not meet the OCSPP 850.4100 test validity element is considered to be a study deficiency. In particular for ryegrass there was a potential uncertainty in the effects observed related to the negative control survival (80%) limiting the ability to reliably capture sublethal effects (reductions in dry weight), which were large in some treatments, although not statistically significant (decrease of up to 25% at the lowest concentration). However, any potential effects did not appear to be dose responsive and therefore do not appear to be treatment-related.

Maximum Labeled Rate: Not reported 

Results Synopsis

	Monocot
	Most sensitive monocot:	Could not be determined
	EC50/IC50: >9.1 lb a.i./A			95% C.I.: N/A 
   EC25/IC25: >9.1 lb a.i./A				95% C.I.: N/A 
   EC05/IC05: NC 						95% C.I.: N/A 
   NOAEC: 9.1 lb a.i./A
   LOAEC: >9.1 lb a.i./A
   Slope: N/A							95% C.I.: N/A
   
Dicot
	Most sensitive dicot: Tomato, based on weight
   EC50/IC50: NC						95% C.I.: N/A
   EC25/IC25: 7.76 lb a.i./A				95% C.I.: 1.85-21 lb a.i./A
   EC05/IC05: 0.778 lb a.i./A				95% C.I.: N/A-3.21 lb a.i./A
   NOAEC: 2.2 lb a.i./A
   LOAEC: 4.4 lb a.i./A
   Slope: N/A							95% C.I.: N/A

Table 1. Summary of most sensitive parameters by species (lb a.i./A).
Species
                                   Endpoint
                                     NOAEC
                                     IC05
                                     IC25
                                     IC50
Cabbage*
                                    Height
                                     0.31
                                    0.0279
                                      NC
                                      NC
Corn1
                                     None
                                      8.8
                                      NC
                                      NC
                                      NC
Oilseed rape
                                     None
                                      8.8
                                      NC
                                      NC
                                      NC
Onion1
                                     None
                                      9.1
                                      NC
                                      NC
                                      NC
Ryegrass1
                                     None
                                      9.1
                                      NC
                                      NC
                                      NC
Soybean*2
                                   Survival
                                      4.4
                                     1.14
                                    >8.8
                                    >8.8
Sugarbeet*2
                                   Survival
                                     0.31
                                     0.191
                                     8.95
                                    >9.1
Sunflower1
                                     None
                                      8.8
                                      NC
                                      NC
                                      NC
Tomato3
                                    Weight
                                      2.2
                                     0.778
                                     7.76
                                      NC
Wheat4
                                     None
                                      9.1
                                      NC
                                    >9.1
                                    >9.1
NC  -  not calculable
*Endpoints and/or confidence intervals are outside tested range of concentrations and should be interpreted with caution.
[1] If survival is based on number planted, as the reviewer calculated, the mean control seedling survival does not meet the OCSPP 850.4100 test validity element of at least 90% survival at test termination for onion (75%), sunflower (78%), ryegrass (80%), and corn (88%). Studies are designed to capture sub-lethal effects, and for ryegrass, this uncertainty may impact the reliability of the other growth endpoints (i.e., dry weight) which although not statistically significant had relatively high differences between several treatment groups and controls (i.e., up to 25% decrease at the lowest concentration). However, any effects on ryegrass dry weight did not appear to be dose responsive and therefore do not appear to be treatment related.
2 Studies are designed to capture sub-lethal effects; therefore, survival is not expected to be the most sensitive endpoint and low survival may impact the validity and reliability of the other growth endpoints for soybean and sugarbeet.
[3] The reviewer visually determined a lower LOAEC of 4.4 lb a.i./A, based on the magnitude (21% effect at this level), in conjunction with consistently lower weights by replicate, therefore, the NOAEC was also lowered and visually determined to be 2.2 lb a.i./A.
[4] Due to the lack of a dose response, likely attributed to low performing replicates across the test levels, (Dunnett's test, p<0.05), and the overlap of the confidence intervals (CIs) (the CIs around the nominal 5.0 lb a.i./A treatment level appear to encompass the control mean), the single significant difference observed at the nominal 5.0 lb a.i./A level, 15%, was discounted, and the NOAEC was visually determined to be the highest test concentration.

This study is scientifically sound and is classified as supplemental overall; individual species are classified as follows: it is classified as acceptable for all species except soybean, sugarbeet, onion, sunflower, and ryegrass. For soybean and sugarbeet, onion and sunflower, the study is considered supplemental and may be used to calculate risk quotients (for endpoints with survival as most sensitive endpoint) and for ryegrass, the study is considered supplemental and may be used for risk characterization only.

I. MATERIALS AND METHODS

   GUIDELINE FOLLOWED:		This study was conducted in compliance with U.S. EPA OCSPP 850.4100 (2012). The reviewer evaluated the study methods according to OCSPP Guideline 850.4100. There were some deficiencies and deviations noted by the reviewer:

 The study author determined the most sensitive dicot was tomato, based on dry weight, with an IC25 of 9.12 lb a.i./A. Corresponding confidence intervals for this IC25 were not reported in the study report. The reviewer was able to determine an IC25 and associated confidence intervals for this endpoint and this is considered a minor deviation.
 Survival was the most sensitive endpoint for soybean and sugarbeet. Soybean mortality was 15% at the highest test concentration and sugarbeet mortality was 13, 18, 28 and 18% at the four highest test concentrations. Terrestrial plant studies are designed to capture sub-lethal effects; therefore, survival is not expected to be the most sensitive endpoint and mortality may have confounded growth effects in soybean and sugarbeet. Therefore, the results for soybean and sugarbeet should be interpreted with caution. The reviewer determined survival based on the number planted; the negative control for survival ranged from 75 to 100% for all species tested. The study author determined survival based on number emerged; the negative control for survival ranged from 93 to 100% for all species tested. The study author's survival results differed from the reviewer's as the study author found no significant inhibitions in survival for any species. However, if survival is based on number planted, as the reviewer did, the mean control seedling survival does not meet the OCSPP 850.4100 test validity element of at least 90% survival at test termination for four species; onion (75%), sunflower (78%), ryegrass (80%), and corn (88%). This is considered a study deficiency for the species where survival is the most sensitive endpoint (soybean and sugarbeet), and where the mean control seedling survival does not meet the OCSPP 850.4100 test validity element (onion, sunflower, and ryegrass).
 The number of seedlings per replicate was five for all test species. OCSPP guidance recommends at least 10 seeds per replicate. However, the total number of seeds for each treatment group for each species was 40 (5 seeds per 8 replicate pots), which does meet the OCSPP guideline of 40 seeds per treatment level.  This is a minor deficiency.
 Humidity for onion, ryegrass, wheat, sugarbeet and cabbage was a mean +- standard deviation (SD) (range) of 60.57% +- 6.21% (SD) (23.71-90.50%), and for corn, oilseed rape, soybean, sunflower and tomato was a mean +- SD (range) of 59.90% +- 5.91% (SD) (23.71-90.50%), which includes humidity measurements lower than OCSPP recommended humidity of 70% (daytime) +- 15%. However, overall this was within the recommended range per the OCSPP guideline and is considered to be a minor deficiency.
 CEC and percent moisture were not reported. This is a minor deficiency.
 The physico-chemical properties of the test material were not reported. This is a minor deficiency.
 The total volume of water applied was not reported. This is a minor deficiency.
 The test substance was supposed to be stored refrigerated but was inadvertently stored under ambient conditions for about 2 months before the mistake was corrected (May 1, 2018 to June 28, 2018, p. 13). A percent area analysis was performed at beginning and end of sample analysis to verify stability of test substance during improper storage.  Based on the information provided in the study report, the improper storage did not appear to adversely impact the stability of the test substance. This is a minor deficiency.

The deficiencies did not have an impact on the acceptability of this study, with the exception of two of the test species, soybean, and sugarbeet, only.

     COMPLIANCE:			Signed and dated Good Laboratory Practices (GLP), Quality Assurance, and No Data Confidentiality statements were provided. This study was conducted in compliance with U.S. EPA 40 CFR Pat 160 which is compatible with OECD Principles of GLP with the following exceptions: (1) periodic analyses of water and soil for potential contaminants were not performed according to GLP standards but were performed using a certified laboratory and standard U.S. EPA analytical methods and (2) characterization and stability of the test substance under conditions of storage at the test site was not determined in compliance with GLP standards. 
   A. MATERIALS:

   	1. Test Material: 			TPA Technical, a metabolite of DCPA
      Description: 				Solid
      Lot No./Batch No.: 			T/H-1-166 (Lot No.); expiration date December 8, 2019
      Purity: 						97.3% (w/w)
      Stability of compound 
      under test conditions:	Analytical confirmation of test concentrations in the spray solutions yielded recoveries of 86-101% of the nominal concentrations (n=20). Stability of test substance under test conditions was not determined. However, the substance was found to be stable a two month storage period (see below).
      							(OECD recommends chemical stability in water and light) 
      Storage conditions of 
      test chemicals: 				The test substance was supposed to be stored refrigerated but was inadvertently stored under ambient conditions for about 2 months before the mistake was corrected (May 1, 2018 to June 28, 2018, p. 13). A percent area analysis was performed at beginning and end of sample analysis to verify stability of test substance during improper storage. Initial and final average percent area was 99.7 and 99.4%, respectively. The improper storage did not appear to adversely impact the stability of the test substance. 

	Table 2.  Physical/chemical properties of TPA
Parameter
Values
Comments
Water solubility at 20˚C
Not reported
Not reported
Vapor pressure
Not reported
Not reported
UV absorption
Not reported
Not reported
pKa
Not reported
Not reported
Kow
Not reported
Not reported

   	2. Test organism:

      Monocotyledonous species: Onion (Allium cepa, Liliaceae; Yellow Granex Hybrid); Ryegrass (Lolium perenne, Poaceae; Gator 3 Perennial); Wheat (Triticum aestivum, Poaceae; Glenn Hard Red Spring Wheat OG); and Corn (Zea mays, Poaceae; Nothstine Dent OG). EPA recommends four monocots in two families, including corn.
      Dicotyledonous species: Sugarbeet (Beta vulgaris, Chenopodiaceae; Non-GMO Variety); Oilseed rape (Brassica napus, Brassicaceae; Dwarf Essex); Cabbage (Brassica oleracea, Brassicaceae; All Seasons); Soybean (Glycine max, Fabaceae; Viking 2265 OG); Sunflower (Helianthus annuus, Asteraceae; Royal Hybrid 1121 F1); and Tomato (Lycopersicon esculentum, Solanaceae; Rutgers). EPA recommends six dicots in four families, including soybean and a root crop.
      OECD recommends a minimum of three species selected for testing, at least one from each of the following categories: Category 1: ryegrass, rice, oat, wheat, and sorghum; Category 2: mustard, rape, radish, turnip, and Chinese cabbage; Category 3: vetch, mung bean, red clover, fenugreek, lettuce, and cress.
      Seed source: Onion obtained from Park Seed Co. (Greenwood, SC); ryegrass and tomato obtained from Meyer Seed Company (Baltimore, MD); sugarbeet obtained from Neseed (Hartford, CT); cabbage obtained Sustainable Seed Co. (Covelo, CA); and wheat, corn, oilseed rape, soybean and sunflower obtained from Johnny's Selected Seeds (Winslow, ME).
      Prior seed treatment/sterilization: Not treated with fungicides, insecticides or repellants prior to test initiation.
      Historical % germination of seed: Onion: 94%; Ryegrass: 90%; Wheat: 94%; Corn: 90%; Sugarbeet: 85%; Oilseed rape: 96%; Cabbage: 90%; Soybean: 88%; Sunflower: 90%; and Tomato: 85% (percent germination as reported by supplier or determined by EAG laboratories).
      Seed storage, if any: Not reported.

   B. STUDY DESIGN:

      1. Experimental Conditions

         a. Limit test: None.
         b. Range-finding study: A preliminary non-GLP range-finding seedling emergence test was conducted exposing onion, wheat, corn, sugarbeet, oilseed rape, soybean, sunflower, and tomato to nominal application rates of 0.016, 0.08, 0.4, 2, and 10 lb a.i./A, and a control (three replicates per application rate, five seeds per replicate, yielding 15 seeds per treatment or control). Fresh weight inhibitions maximum of 36% were observed; height inhibitions were a maximum of 23% were observed; survival inhibitions were maximum of 16%; and emergence was a maximum of 25% were observed. The test concentrations selected for use in the definitive study for each of the species were based on the results of this non-GLP range-finding study.
         
         c. Definitive Study

Table 3. Nominal and Analytically Confirmed Test Application Rates (lb a.i./A).
                                    Nominal
                                  Test Rates
                    Analytically Confirmed Rates of TPA[1]
                             (Percent of Nominal)
                                       
                  Onion, Ryegrass, Wheat, Sugarbeet, Cabbage
                Corn, Oilseed Rape, Sunflower, Soybean, Tomato
                             0 (negative control)
                             <0.00018 (<LOQ)
                             <0.00018 (<LOQ)
                                     0.313
                                  0.31 (98%)
                                  0.29 (93%)
                                     0.625
                                  0.64 (101%)
                                  0.58 (93%)
                                     1.25
                                   1.2 (97%)
                                   1.1 (89%)
                                      2.5
                                   2.2 (89%)
                                   2.2 (88%)
                                      5.0
                                   4.5 (90%)
                                   4.4 (88%)
                                      10
                                   9.1 (91%)
                                   8.8 (88%)
 LOQ = limit of quantification.
 [1] Measured rates and recoveries are rounded rates (Appendix 1 below).
 
Table 4:  Experimental Parameters - Seedling Emergence.
                                  Parameters
                              Seedling Emergence
                                       
                                    Details
                                    Remarks
                                       
                                       
                                   Criteria
Duration of the test
21 days



Recommended test duration is 14-21 days.
OECD recommends that the test be terminated no sooner than 14 days after 50 percent of the control seedlings have emerged
Number of seeds/plants/species/ replicate
Five seeds per replicate pot. Eight replicate pots per treatment level.



Ten seeds per replicate should be used.
OECD recommends a minimum of five seeds planted in each replicate within 24 hours of incorporation of the test substance. All seeds of each species for each test should be of the same size class. The seed should not be imbibed.
Number of replicates
Control:
Adjuvant control:
Treated:

8
N/A
8



Four replicates per dose should be used.
OECD recommends a minimum of four replicates per treatment
Number of test concentrations
6 treatment levels per species, plus the negative control
Negative control = municipal water purified by reverse osmosis (R/O) water.


Five test concentrations should be used with a dose range of 2X or 3X progression
OECD recommends three concentrations, preferably with application rates equivalent to 0.0 (control), 1.0, 10.0 and 100 mg substance per kg of oven-dried soil.
Method and interval of analytical verification

LOQ:
LOD:
Samples of the spray solutions were analyzed by LC/MS/MS coupled with HPLC.
<0.00018 lb a.i./A 
Not reported
Measured concentrations in test samples ranged from 88.7 to 101% of nominal on August 13, 2018, from 87.6 to 93.3% of nominal on August 16, 2018.
Adjuvant (type, percentage, if used)
N/A

Test container (pot)
Size/Volume:
Material: (glass/polystyrene)

Pots (16 cm diameter; 12 cm deep)
Plastic



Non-porous containers should be used.
OECD recommends that non-porous plastic or glazed pot be used.
Growth facility
Greenhouse

Method/depth of seeding
A template was used to gently compact the soil and leave five uniform, ca. equally spaced holes for planting. Onion, ryegrass, sugarbeet, oilseed rape, cabbage and tomato were planted at a depth of 10 mm, and wheat, corn, sunflower and soybean were planted at a depth of 20 mm. One indiscriminately selected seed was then planted in each hole, for a total of five seeds in each pot. Holes were closed by depressing the soil surface.

Test material application
Application time including the plant growth stage

Number of applications 

Application interval

Method of application

Seeds were planted on the day of application

1

N/A- single application

The test material was applied using a DeVries Research Track Sprayer (spray booth). The spray mixture was placed into a 500 mL polyethylene spray bottle pressurized to 20 psi (1.4 bar) with nitrogen that was connected to a moveable spray nozzle (Teejet 8002 E). The nozzle was placed ca. 41 cm above the soil surface during delivery of the spray mixture. Applications were made to the control first, followed by the treatment groups in order of increasing concentration to avoid cross-contamination.
On August 13 and August 16, 2018 spray mixtures were prepared by diluting 28.7871g of the test substance to a final volume of 500 mL. The spray mixture was described as an opaque milky white suspension. On each day of preparation, proportional dilutions from the highest-concentration spray mixture were used to prepare spray mixtures for the lower test rates, (i.e., aliquots of 100, 50, 25, 12.5 and 6.25 mL were diluted to 200 mL with municipal water purified by reverse osmosis) for the 5.0, 2.5, 12.5, 0.625, and 0.313 lb a.i./A levels, respectively. 
Details of soil used
Geographic location
Depth of soil collection


Soil texture
% sand
% silt
% clay
pH:

% organic carbon
CEC (meq/100g)
Moisture at 1/3 atm (%)

Artificial soil mixture of kaolinite clay, industrial quartz sand and peat with limestone added to buffer pH.

Loamy sand
87
4
9
6.3 (water)
6.1 (0.01M CaCl2)
1.2
Not reported
Not reported
Organic matter content: 2.0%
Slow-release fertilizer was added to provide nutrients. Samples of soil representative of that used in this study were sent to Agvise Laboratories Inc. in Northwood, North Dakota, for analysis of the particle size distribution and organic matter content of the soil. Additionally, the soil used for plant studies are periodically screened for pesticides and metals. No analytes were measured at levels that were expected to have an impact on the study. Although the complete reports were filed in the archives at EAG Laboratories the results of these analyses were not provided in the study report for reference. 


Soil mixes containing sandy loam, loam, or clay loam soil with no greater than 2% organic matter are preferable. Glass beads, rock wool, and 100% acid washed sand are not preferred.
OECD prefers the soil to be sieved (0.5 cm) to remove coarse fragments. Carbon content should not exceed 1.5% (3% organic matter). Fine particles (under 20um) makeup should be between 10 and 20%. The recommended pH is between 5.0 and 7.5.
Details of nutrient medium, if used
None reported

Watering regime and schedules
Water source/type:



Volume applied:
Interval of application:
Method of application:


EAG Laboratories municipal water purified by reverse osmosis (R/O), well water, and greenhouse well water
Not reported
Every 1 to 5 days. 
Top watered on day of application; sub-irrigation as needed thereafter.
Approximately 59-65 passes of the spray nozzle in the spray booth using the rain-fall cycle, immediately after test substance application. The days on which watering occurred are listed in Appendix 5 (pgs. 67-68) of the study report. The well water used for plant studies are periodically screened for pesticides and metals. No analytes were measured at levels that were expected to have an impact on the study. Although the reports were filed in the archives at EAG Laboratories the results of these analyses were not provided in the study report for reference.


EPA prefers that bottom watering be utilized for seedling emergence studies so that the chemical is not leached out of the soil during the test.
Any pest control method/fertilization, if used
A slow-release fertilizer was added to the soil to provide nutrients essential for plant growth.

Test conditions

Temperature:
Photoperiod:
Light intensity and quality:

Relative humidity:



Temperature:
Photoperiod:
Light intensity and quality:

Relative humidity:
Onion, ryegrass, wheat, sugarbeet, cabbage
Mean: 30.09°C; Range: 20.52-42.15°C
16L:8D
Mean: 13.8 moles PAR; Range: 10.3-15.4 moles PAR
Mean: 60.57% +- 6.21% (SD); Range: 23.71-90.50%

Corn, oilseed rape, soybean, sunflower, tomato
Mean: 30.64°C; Range: 20.52-42.85°C
16L:8D
Mean: 13.8 moles PAR; Range: 10.3-15.4 moles PAR
Mean: 59.90% +- 5.91% (SD); Range: 23.71-90.50%
Light source is natural daylight supplemented with artificial light (high pressure sodium).  Summary of the daily environmental conditions are listed in Appendix 5 (pgs. 67-68) of the study report.


EPA prefers that the cold vs warm loving plants be tested in two separate groups to optimize plant growth.

OECD prefers that the temperature, humidity and light conditions be suitable for maintaining normal growth of each species for the test period.
Reference chemical (if used)
Name:
Concentrations:
Other parameters, if any

N/A

Other parameters, if any
None


	2. Observations:

Table 5:  Observation Parameters - Seedling Emergence.
                                  Parameters
                              Seedling Emergence
                                       
                                    Details
                                    Remarks
Parameters measured (e.g., number of germinated seeds, emerged seedlings, plant height, fresh weight or other endpoints)
- Emergence
- Survival
- Phytotoxicity
- Dry weight
- Height

Measurement technique for each parameter 
Phytotoxicity (seedling condition) was visually determined. Emergence was defined as the presence of visible plant tissue at the surface of the soil. Survival was defined as the number of emerged seedlings in each pot at test termination. Height was measured with a ruler to the nearest whole centimeter from the surface of the soil to the apical meristem or to the tip of the longest leaf. Dry weight was measured by clipping the seedlings at soil level, then drying and weighing.
Seedling condition was described by noting the presence or absence of visible signs of phytotoxicity (i.e., necrosis, leaf curling, or stem curling), and employed a rating system as described below.

Observation intervals 
Emergence were recoded weekly (days 7, 14, & 21). Survival, height, and dry weight were recorded at study termination (Day 21). Seedling condition (phytotoxicity) was assessed at test termination when seedlings were collected. 

Other observations, if any
None

Were raw data included?
Yes

Phytotoxicity rating system, if used
Rating system where 0 = no effect; 10 to 30 = slight effect; 40 to 60 = moderate effect; 70 to 90 = severe effect; 100 = complete effect.


II. RESULTS and DISCUSSION:

       INHIBITORY EFFECTS:
      
      The reviewer determined the negative control for emergence ranged from 80 to 100% for all species tested. When compared to the negative control, the reviewer found statistically significant inhibitions in cabbage, sugarbeet and sunflower (Table 6a). Inhibition in sugarbeet emergence was significant at nominal 0.625 lb a.i./A and higher test concentrations (Jonckheere-Terpstra Step-Down test, p<0.05). Inhibition in cabbage and sunflower emergence was significant only at nominal 0.313 lb a.i./A and 2.5 lb a.i./A, respectively (Mann-Whitney Two-Sample test, p<0.05). 
      
      The study author determined the negative control for emergence ranged from 80 to 100% for all species tested. The study author emergence results differed from the reviewer's for cabbage, sugarbeet and sunflower. The study author did not find statistically significant inhibition in cabbage and sunflower emergence at nominal 0.313 and 2.5 lb a.i./A, while the reviewer did. The study author also found significant inhibition in sugarbeet emergence at nominal 2.5 lb a.i./A and higher test concentration (Dunnett's test, p<0.05), whereas the reviewer found significance at nominal 0.625 lb a.i./A and higher.

Table 6a: Percent Inhibition of Plant Emergence
                                    Nominal
                                     Rate
                                 lb a.i./A[2]
                             Percent Inhibition[1]
                                       
                                     Corn
                                     Onion
                                   Ryegrass
                                     Wheat
                                    Cabbage
                                 Oilseed rape
                                    Soybean
                                  Sugar beet
                                   Sunflower
                                    Tomato
                                     0.313
                                      -6
                                      -9
                                       0
                                      -6
                                    15*[3]
                                       8
                                       5
                                       5
                                      -15
                                      -6
                                     0.625
                                      -9
                                      -6
                                       0
                                      -3
                                       3
                                       0
                                       3
                                     8*[3]
                                      -6
                                       0
                                     1.25
                                      -6
                                       3
                                      -9
                                      -8
                                       3
                                       3
                                       3
                                    10*[3]
                                      -6
                                      -8
                                      2.5
                                      -6
                                      -6
                                       6
                                      -6
                                       5
                                       3
                                       5
                                      18*
                                    18*[3]
                                      -8
                                      5.0
                                      -6
                                      -9
                                      -3
                                       3
                                       5
                                      -3
                                       8
                                      28*
                                       0
                                      -6
                                      10
                                      -6
                                       3
                                      -9
                                      -8
                                      -3
                                       0
                                      10
                                      18*
                                      -6
                                      -3
* Statistically significant when compared to the negative control.
[1] Treatment groups compared to the negative control.
[2] Analytically measured rates confirmed by the reviewer were 0.31, 0.64, 1.2, 2.2, 4.5, and 9.1 lb a.i./A for onion, ryegrass, wheat, sugarbeet, and cabbage, and 0.29, 0.58, 1.1, 2.2, 4.4 and 8.8 lb a.i./A for corn, oilseed rape, sunflower, soybean, and tomato.
[3] The reviewer identified this response as significantly different from the control, while the study author did not. 

      The reviewer determined survival based on the number planted; negative control for survival ranged from 78 to 100% for all species tested. When compared to the negative control, the reviewer found significant inhibition in soybean and sugarbeet survival (Table 6b). Inhibitions in sugarbeet survival were significant at nominal 0.625 lb a.i./A and higher test concentrations (Jonckheere-Terpstra Step-Down test, p<0.05). Inhibition in soybean survival was significant only at nominal 10 lb a.i./A, the highest test concentration (Jonckheere-Terpstra Step-Down test, p<0.05). 
      
      The study author determined survival based on number emerged; negative control for survival ranged from 93 to 100% for all species tested. The study author's survival results differed from the reviewer's as the study author found no significant inhibitions in survival for any species. 
      
Table 6b: Percent Inhibition of Plant Survival
                                    Nominal
                                     Rate
                                 lb a.i./A[2]
                             Percent Inhibition[1]
                                       
                                     Corn
                                     Onion
                                   Ryegrass
                                     Wheat
                                    Cabbage
                                 Oilseed rape
                                    Soybean
                                  Sugar beet
                                   Sunflower
                                    Tomato
                                     0.313
                                      -6
                                      -13
                                       0
                                      -3
                                      13
                                       8
                                       5
                                       5
                                      -10
                                      -3
                                     0.625
                                      -9
                                      -7
                                       3
                                      -3
                                       0
                                      -3
                                       3
                                     8*[3]
                                      (0)
                                      -3
                                       0
                                     1.25
                                      -6
                                      10
                                      -9
                                      -8
                                       3
                                       0
                                       3
                                    13*[3]
                                      (3)
                                      -13
                                      -8
                                      2.5
                                      -3
                                      -13
                                       3
                                      -6
                                       2
                                       0
                                       5
                                    18*[3]
                                      (0)
                                      19
                                      -8
                                      5.0
                                      -6
                                      -13
                                      -6
                                       3
                                       5
                                      -6
                                      10
                                    28*[3]
                                      (0)
                                       0
                                      -6
                                      10
                                      -6
                                       0
                                      -13
                                      -8
                                      -3
                                       3
                                    15*[3]
                                      (6)
                                    18*[3]
                                      (0)
                                      -6
                                      -3
* Statistically significant when compared to the negative control.
[1] Treatment groups compared to the negative control. Reviewer survival is based on number planted; study author's survival is based on number emerged. Study author inhibitions that are significant and differ from the reviewer's inhibitions are shown in parentheses.
[2] Analytically measured rates confirmed by the reviewer were 0.31, 0.64, 1.2, 2.2, 4.5, and 9.1 lb a.i./A for onion, ryegrass, wheat, sugarbeet, and cabbage, and 0.29, 0.58, 1.1, 2.2, 4.4 and 8.8 lb a.i./A for corn, oilseed rape, sunflower, soybean, and tomato.
[3] The reviewer identified this response as significantly different from the control, while the study author did not. 

      When compared to the negative control, the reviewer found significant inhibitions in seedling height in cabbage, soybean, and tomato (Table 6c). Inhibition in cabbage height was not dose responsive and was significant at nominal 0.625, 2.5 and 5.0 lb a.i./A only (Mann-Whitney Two-Sample test, p<0.05). Inhibition in soybean height did violate assumptions of monotonicity, was not dose-responsive, and was significant at the nominal 2.5 lb a.i./A level only (Dunnett Multiple Comparison test, p<0.05). Inhibition in tomato height did not violate assumptions of monotonicity and was significant at nominal 10 lb a.i./A only, the highest test concentration (Williams Multiple Comparison test, p<0.05).
      
      The study author's seedling height results differed from the reviewers' for cabbage for the following reason. The study author found the results of the Dunnett's test for cabbage height to be ambiguous and a trend test not warranted, and as a result, determined none of the inhibitions in height for cabbage were significant. The reviewer however used the non-parametric, Mann-Whitney U Two-Sample test (p<0.05) as the data failed the assumption of normality. The study author did consider the inhibitions in soybean and tomato height to be a dose response; the William's test for soybean and tomato showed significant inhibitions at the same treatment levels as the reviewer. 
      
Table 6c: Percent Inhibition of Seedling Height
                                    Nominal
                                     Rate
                                 lb a.i./A[2]
                             Percent Inhibition[1]
                                       
                                     Corn
                                     Onion
                                   Ryegrass
                                     Wheat
                                    Cabbage
                                 Oilseed rape
                                    Soybean
                                  Sugar beet
                                   Sunflower
                                    Tomato
                                     0.313
                                      -1
                                      -6
                                       3
                                      -2
                                       2
                                       6
                                       2
                                      -1
                                      12
                                       5
                                     0.625
                                       1
                                      -4
                                      -4
                                      -6
                                    10*[3]
                                      -2
                                       3
                                       1
                                       8
                                      17
                                     1.25
                                      -4
                                      -17
                                       0
                                      -3
                                      10
                                      -3
                                       0
                                      -3
                                       8
                                       9
                                      2.5
                                      -3
                                      -17
                                       8
                                      -3
                                     9*[3]
                                      -1
                                      10*
                                       1
                                       3
                                       5
                                      5.0
                                      -6
                                       1
                                      -4
                                      -4
                                    13*[3]
                                      -3
                                       4
                                      -5
                                       9
                                      10
                                      10
                                      -1
                                      -13
                                       3
                                      -6
                                       4
                                      -1
                                       6
                                      -2
                                       3
                                      22*
* Statistically significant when compared to the negative control.
[1] Treatment groups compared to the negative control. Study author inhibitions that are significant and differ from the reviewer's inhibitions are shown in parentheses.
[2] Analytically measured rates confirmed by the reviewer were 0.31, 0.64, 1.2, 2.2, 4.5, and 9.1 lb a.i./A for onion, ryegrass, wheat, sugarbeet, and cabbage, and 0.29, 0.58, 1.1, 2.2, 4.4 and 8.8 lb a.i./A for corn, oilseed rape, sunflower, soybean, and tomato.
[3] The reviewer identified this response as significantly different from the control, while the study author did not.

      When compared to the negative control, the reviewer found significant inhibitions in seedling dry weight of wheat and tomato (Table 6d). Both the reviewer and study author found that the data for wheat did not show a monotonic trend and used Dunnett's Multiple Comparison Test. Although there was a significant effect in wheat weight inhibition at nominal 5.0 lb a.i./A only (Dunnett Multiple Comparison Test, p<0.05), the effect was discounted due to a lack of a dose response, and in tomato weight was significant at nominal 10 lb a.i./A only, the highest test concentration (Jonckheere-Terpstra Step-Down test, p<0.05). The reviewer visually determined a lower LOAEC of 4.4 lb a.i./A, based on the magnitude (21% effect at this level), in conjunction with consistently lower weights by replicate.
      
Table 6d: Percent Inhibition of Seedling Dry Weight
                                    Nominal
                                     Rate
                                 lb a.i./A[2]
                             Percent Inhibition[1]
                                       
                                     Corn
                                     Onion
                                   Ryegrass
                                     Wheat
                                    Cabbage
                                 Oilseed rape
                                    Soybean
                                  Sugar beet
                                   Sunflower
                                    Tomato
                                     0.313
                                      -4
                                      -4
                                      25
                                      -3
                                      -12
                                       3
                                       9
                                      -2
                                       2
                                       5
                                     0.625
                                       1
                                      -7
                                      27
                                      -6
                                      15
                                      -24
                                       5
                                      -4
                                      -1
                                      15
                                     1.25
                                      -12
                                      -16
                                      -4
                                       8
                                      15
                                      -12
                                      -2
                                      -11
                                      14
                                      10
                                      2.5
                                      -12
                                      -12
                                      28
                                       7
                                      21
                                      10
                                       9
                                      -20
                                       1
                                       7
                                      5.0
                                      -12
                                       3
                                       8
                                    15*[3]
                                      10
                                       4
                                      -1
                                      -14
                                      10
                                     21[4]
                                      10
                                      -4
                                      -16
                                      31
                                      -2
                                      -10
                                      -5
                                      -6
                                      -15
                                      -7
                                      31*
* Statistically significant when compared to the negative control
[1] Treatment groups compared to the negative control. 
[2] Analytically measured rates confirmed by the reviewer were 0.31, 0.64, 1.2, 2.2, 4.5, and 9.1 lb a.i./A for onion, ryegrass, wheat, sugarbeet, and cabbage, and 0.29, 0.58, 1.1, 2.2, 4.4 and 8.8 lb a.i./A for corn, oilseed rape, sunflower, soybean, and tomato.
[3] The reviewer identified this response as significantly different from the control, while the study author did not. This was not dose-responsive. Although statistically significant when compared to the negative control; however, due to the lack of a dose response, likely attributed to low performing replicates across the test levels, (Dunnett's test), and the overlap of the confidence intervals (CIs) (the CIs around the nominal 5.0 lb a.i./A treatment level appear to encompass the control mean), the single significant difference observed at the nominal 5.0 lb a.i./A level, 15%, was discounted, and the NOAEC was visually determined to be the highest test concentration.
[4] Although not statistically significant when compared to the negative control (Jonckheere-Terpstra Step-Down test, p<0.05); however, the reviewer visually determined it to be, based on the magnitude (21% effect at this level), in conjunction with consistently lower weights by replicate.
   
      Based on the reviewer's results, the most sensitive monocot could not be determined. There were no dose-responsive treatment related reductions that were greater than 25% for any monocot species. Therefore, the IC25 was visually estimated as measured >9.1 lb a.i./A (nominal 10 lb a.i./A), the highest tested application rate for all monocot species tested, and the NOAEC was measured 9.1 lb a.i./A (nominal 10 lb a.i./A) for all tested monocot species. The most sensitive dicot species was tomato, based on weight, with NOAEC and IC25 values of 2.2 and 7.76 lb a.i./A, respectively. 
      
      The following phytotoxic symptoms were noted for all test species: chlorosis, necrosis, and leaf curling. All species showed slight levels of phytotoxic symptoms, except cabbage, onion, sunflower and tomato showed up to moderate levels of phytotoxic symptoms.  No species exhibited dose-dependent phytotoxicity. Phytotoxic symptoms were observed in control plants, but these symptoms were slight in severity and were not considered to be related to the treatment.

       REPORTED STATISTICS BY THE STUDY AUTHOR:

      The following tables are the study author reported statistical analysis table summaries. Mean seedling emergence on day 21, survival at day 21, shoot dry weight, and shoot height on day 21, of the controls and treatment groups were compared with a one-tailed Dunnett's t-test, using the DUNNETT option of the GLM (general linear model) procedure of SAS (version 9.4). Significance was determined at the level of 0.05 (Dunnett's test, p<0.05). Dunnett's test was used to help establish the LOER and NOER by determining which treatment groups differed significantly (p< 0.05) from the control group.
      
      When possible, ER25 and ER50 and their confidence limits were estimated using the non-linear regression analysis of Bruce and Versteeg. Where reductions in endpoints of at least 25% and 50% relative to the control were not observed in treatment groups of a species, the ER25 and ER50 were considered greater than the highest test rate.

Table 7a: Effect of TPA on 21 Day Seedling Emergence.
Species
Results summary for height (lb a.i./A)

Mean height (cm)
                                     NOER
                                     ER05
                                    95% CI
                                     ER25
                                    95% CI
                                     ER50
                                    95% CI
                                     slope
                                     95%CI
Cabbage
12.5-14.5
                                     10[1]
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Corn
93.6-100.1
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Oilseed rape
27.1-29.7
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Onion
16.7-19.7
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Ryegrass
9.6-10.9
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Soybean
56.4-63.0
                                    1.25[2]
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Sugarbeet
20.6-21.8
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Sunflower
39.6-44.8
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Tomato
16.9-21.6
                                     5[2]
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Wheat
36.8-39.1
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
ND- Not determined. N/A- Not applicable.
All NOER and LOER values were determined by means comparison with the Negative Control using Dunnett's Test (α=0.05)
unless otherwise noted
1 Based on Dunnett's test and lack of significant trend the NOER is considered to be 10 lb a.i./A.
2 Based on William's Test.

Table 7b: Effect of TPA on 21 Day Seedling Emergence.
Species
Results summary for weight (lb a.i./A); dry weight

Mean weight (g)
                                     NOER
                                     ER05
                                    95% CI
                                     ER25
                                    95% CI
                                     ER50
                                    95% CI
                                     slope
                                     95%CI
Cabbage
0.221-0.314
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Corn
2.74-3.11
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Oilseed rape
0.540-0.742
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Onion
0.026-0.031
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Ryegrass
0.028-0.040
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Soybean
1.49-1.75
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Sugarbeet
0.588-0.703
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Sunflower
1.33-1.65
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Tomato
0.179-0.258
                                     5[2]
                                      ND
                                      N/A
                                     9.12
                                      NR
                                    >10
                                      N/A
                                      N/A
                                      N/A
Wheat
0.368-0.454
                                     10[1]
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
ND- Not determined. N/A- Not applicable. NR- Not reported. 
All NOER and LOER values were determined by means comparison with the Negative Control using Dunnett's Test (α=0.05)
unless otherwise noted.
1 Based on William's Test.
2 Based on Jonckheere-Terpstra Test for trends. 


Table 7c: Effect of TPA on 21 Day Seedling Emergence.
Species
Results summary for emergence (lb a.i./A)

%
                                     NOER
                                     ER05
                                    95% CI
                                     ER25
                                    95% CI
                                     ER50
                                    95% CI
                                     slope
                                     95%CI
Cabbage
83-100
                                     10[1]
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Corn
88-95
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Oilseed rape
85-95
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Onion
78-88
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Ryegrass
78-90
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Soybean
90-100
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Sugarbeet
73-100
                                     1.25
                                      ND
                                      N/A
                                     11.3*
                                  2.34-54.98
                                    >10
                                      N/A
                                      N/A
                                      N/A
Sunflower
68-95
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Tomato
90-98
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Wheat
88-98
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
ND- Not determined. N/A- Not applicable.
All NOER and LOER values were determined by means comparison with the Negative Control using Dunnett's Test (α=0.05)
unless otherwise noted.
*Value is extrapolated beyond the range of rates tested.
1 Results based on Jonckheere-Terpstra Test for trends.

Table 7d: Effect of TPA on 21 Day Seedling Emergence.
Species
Results summary for survival (lb a.i./A); based on # emerged

%
                                     NOER
                                     ER05
                                    95% CI
                                     ER25
                                    95% CI
                                     ER50
                                    95% CI
                                     slope
                                     95%CI
Cabbage
97-100
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Corn
98-100
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Oilseed rape
94-100
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Onion
86-100
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Ryegrass
94.4-100
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Soybean
94-100
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Sugarbeet
98-100
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Sunflower
89-100
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Tomato
98-100
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
Wheat
97-100
                                      10
                                      ND
                                      N/A
                                    >10
                                      N/A
                                    >10
                                      N/A
                                      N/A
                                      N/A
ND- Not determined. N/A- Not applicable.
All NOER and LOER values were determined by means comparison with the Negative Control using Dunnett's Test (α=0.05) unless otherwise noted.

Mid-study emergence
                                    Control
                                    Cabbage
                                     Corn
                                 Oilseed rape
                                     Onion
                                   Ryegrass
                                    Soybean
                                   Sugarbeet
                                   Sunflower
                                    Tomato
                                     Wheat
                               Formulation Blank
                                    78-100
                                    83-100
                                     88-95
                                     85-95
                                     78-88
                                     78-90
                                    90-100
                                    73-100
                                     68-95
                                     90-98
                                     88-98
                                      N/A
N/A - Not applicable.
   


   C. VERIFICATION OF STATISTICAL RESULTS BY THE REVIEWER:

   All analyses were conducted comparing treated to the negative control. These analyses were conducted using CETIS version 1.9.6.12 with database backend settings implemented by EFED on 7/25/2017. Data for each endpoint were tested to determine if their distributions were normal and if their variances were homogeneous using Shapiro-Wilk's and Levene's tests, respectively. Data that satisfied these assumptions were subjected to Dunnett's and William's tests, and data that did not satisfy these assumptions were subjected to the non-parametric Mann-Whitney U and Jonckheere's tests. Linear (emergence and survival) and non-linear (dry weight and height) regression models were used to interpret ICx values. Analytically measured concentrations confirmed by the reviewer were used in the statistical analyses.
   
   Mortality in emergence and survival controls was adjusted using Abbott's Correction and Optimization for probit regression in CETIS according to EFED guidance dated June 9, 2020. In the event that CETIS doesn't allow application of Abbott's Correction and Optimization to these controls, linear regression without Abbott's Correction and Optimization will be performed and noted in the CETIS comments.
   
Table 8a: Effect of TPA on 21 Day Seedling Emergence.
Species
Results summary for height (lb a.i./A)

Mean height (cm)
                                     NOAEC
                                     IC05
                                    95% CI
                                     IC25
                                    95% CI
                                     IC50
                                    95% CI
                                     slope
                                     95%CI
Cabbage1
12.5-14.5
                                     0.31
                                      ND
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
Corn
93.6-100.1
                                      8.8
                                      ND
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Oilseed rape
27.1-29.7
                                      8.8
                                      ND
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Onion
16.7-19.7
                                      9.1
                                      ND
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
Ryegrass
9.59-10.9
                                      9.1
                                      ND
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
Soybean2
56.4-63.0
                                      1.1
                                     3.15
                                  0.355-13.4
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Sugarbeet
20.6-21.8
                                      9.1
                                      ND
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
Sunflower
39.6-44.8
                                      8.8
                                      ND
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Tomato3
16.9-21.6
                                      4.4
                                     0.159
                                   N/A-2.09
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Wheat
36.8-39.1
                                      9.1
                                      ND
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
ND- Not determined. N/A- Not applicable.
1 Significant decrease in cabbage height, inhibition of 10, 9 and 13% at the 0.64, 2.2, and 4.5 lb a.i./A treatment levels, respectively, compared to the negative control (Mann-Whitney U Two-Sample test, p<0.05). Not dose-responsive. The reviewer used the non-parametric, Mann-Whitney test as the data failed the assumption of normality
2 Significant decrease in soybean height, inhibition of 10, 4, and 6% at the 2.2, 4.4 and 8.8 lb a.i./A treatment levels, respectively, compared to the negative control (Jonckheere-Terpstra Step-Down test, p<0.05).
3 Significant decrease in tomato height, inhibition of 22% at the 8.8 lb a.i./A treatment level, respectively, compared to the negative control (William's test, p<0.05).

Table 8b: Effect of TPA on 21 Day Seedling Emergence.
Species
Results summary for weight (lb a.i./A); dry weight

Mean weight (g)
                                     NOAEC
                                     IC05
                                    95% CI
                                     IC25
                                    95% CI
                                     IC50
                                    95% CI
                                     slope
                                     95%CI
Cabbage
0.221-0.314
                                      9.1
                                      ND
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
Corn
2.74-3.11
                                      8.8
                                      NC
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Oilseed rape
0.54-0.742
                                      8.8
                                      NC
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Onion
0.026-0.031
                                      9.1
                                      NC
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
Ryegrass
0.028-0.040
                                      9.1
                                      ND
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
Soybean
1.49-1.75
                                      8.8
                                      NC
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Sugarbeet
0.588-0.703
                                      9.1
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
Sunflower
1.33-1.65
                                      8.8
                                      ND
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Tomato1*
0.179-0.258
                                      2.2
                                     0.778
                                   N/A-3.21
                                     7.76
                                    1.85-21
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Wheat2
0.368-0.454
                                      9.1
                                      NC
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
ND- Not determined. N/A- Not applicable.
*Endpoints and/or confidence intervals are outside tested range of concentrations and should be interpreted with caution. 
1 Significant decrease in tomato dry weight, inhibition of 31% at the 8.8 lb a.i./A treatment levels, respectively, compared to the negative control (Jonckheere-Terpstra Step-Down test, p<0.05). The reviewer visually determined a lower LOAEC of 4.4 lb a.i./A, although not statistically significant (Jonckheere-Terpstra Step-Down test, p<0.05), based on the magnitude (21%) of effect at the second highest test level (4.4 lb a.i./A), in conjunction with consistently lower weights by replicate, therefore, the NOAEC was also lowered and visually determined to be 2.2 lb a.i./A. 
2 Significant decrease in wheat dry weight, inhibition of 15% at the 4.5 lb a.i./A treatment level, respectively, compared to the negative control (Dunnett's test, p<0.05). However, due to the lack of a dose response, likely attributed to low performing replicates across the test levels, (Dunnett's test, p<0.05), and the overlap of the confidence intervals (CIs) (the CIs around the nominal 5.0 lb a.i./A treatment level appear to encompass the control mean), the single significant difference observed at the nominal 5.0 lb a.i./A level, 15%, was discounted, and the NOAEC was visually determined to be the highest test concentration.

Table 8c: Effect of TPA on 21 Day Seedling Emergence.
Species
Results summary for emergence (lb a.i./A)

%
                                     NOAEC
                                     IC05
                                    95% CI
                                     IC25
                                    95% CI
                                     IC50
                                    95% CI
                                     slope
                                     95%CI
Cabbage[1]
83-100
                                      9.1
                                      ND
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
Corn
88-95
                                      8.8
                                      NC
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Oilseed rape
85-95
                                      8.8
                                      NC
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Onion
78-88
                                      9.1
                                      ND
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
Ryegrass
78-90
                                      9.1
                                      ND
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
Soybean
90-100
                                      8.8
                                     1.48
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Sugarbeet2
73-100
                                     0.31
                                     0.226
                                 0.00107-0.69
                                     9.15
                                   3.87-302
                                    >9.1
                                      N/A
                                     0.604
                                  0.185-1.02
Sunflower3
68-95
                                      9.1
                                      ND
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Tomato
90-98
                                      8.8
                                      ND
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Wheat
88-98
                                      9.1
                                      ND
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
ND- Not determined. N/A- Not applicable.
1 Due to the lack of a dose response and the lack of significance at all higher concentrations (Mann-Whitney U Two-Sample test, p<0.05), the single significant difference observed in the lowest concertation, 15%, was discounted, and the NOAEC was visually determined to be the highest test concentration. Inverse relationship
2 Significant decrease in sugarbeet emergence, inhibition of 8, 10, 18, 28, and 18% at the 0.64, 1.2, 2.2, 4.5 and 9.1 lb a.i./A treatment levels, respectively, compared to the negative control (Jonckheere-Terpstra Step-Down test, p<0.05). Endpoints (IC50) and/or confidence intervals are outside tested range of concentrations or not calculated and should be interpreted with caution.
[3] Due to the lack of a dose response, likely attributed to low performing replicates across the test levels, (Mann-Whitney U Two-Sample test, p<0.05), the single significant difference observed at the 2.2 lb a.i./A level, 18%, was discounted, and the NOAEC was visually determined to be the highest test concentration.
Table 8d: Effect of TPA on 21 Day Seedling Emergence.
Species
Results summary for survival (lb a.i./A); based on # planted

%
                                     NOAEC
                                     IC05
                                    95% CI
                                     IC25
                                    95% CI
                                     IC50
                                    95% CI
                                     slope
                                     95%CI
Cabbage
83-98
                                      9.1
                                      ND
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
Corn
88-95
                                      8.8
                                      ND
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Oilseed rape
83-95
                                      8.8
                                      ND
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Onion
68-85
                                      9.1
                                      ND
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
Ryegrass
78-90
                                      9.1
                                      ND
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
Soybean*1
85-100
                                      4.4
                                     1.14
                                 0.000431-3.25
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Sugarbeet*2
73-100
                                     0.31
                                     0.191
                                0.000378-0.626
                                     8.95
                                   3.72-401
                                    >8.8
                                      N/A
                                     0.58
                                  0.167-0.994
Sunflower
63-85
                                      8.8
                                      ND
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Tomato
90-98
                                      8.8
                                      ND
                                      N/A
                                    >8.8
                                      N/A
                                    >8.8
                                      N/A
                                      N/A
                                      N/A
Wheat
88-98
                                      9.1
                                      NC
                                      N/A
                                    >9.1
                                      N/A
                                    >9.1
                                      N/A
                                      N/A
                                      N/A
ND- Not determined. N/A- Not applicable.
*Endpoints and/or confidence intervals are outside tested range of concentrations and should be interpreted with caution.
1 Significant decrease in soybean survival, inhibition of 15% at the 8.8lb a.i./A treatment level compared to the negative control (Jonckheere-Terpstra Step-Down test, p<0.05).
2 Significant decrease in sugarbeet survival, inhibition of 8, 13, 18, 28, and 18% at the 0.64, 1.2, 2.2, 4.5 and 9.1 lb a.i./A treatment levels, respectively, compared to the negative control (Jonckheere-Terpstra Step-Down test, p<0.05). Mortality in emergence control was adjusted using Abbott's Correction and Optimization for probit regression.

Mid-study emergence
                                    Control
                                    Cabbage
                                     Corn
                                 Oilseed rape
                                     Onion
                                   Ryegrass
                                    Soybean
                                   Sugarbeet
                                   Sunflower
                                    Tomato
                                     Wheat
                               Formulation Blank
                                    78-100
                                    83-100
                                     88-95
                                     85-95
                                     78-88
                                     78-90
                                    90-100
                                    73-100
                                     68-95
                                     90-98
                                     88-98
                                      N/A
N/A - Not applicable.

Monocot
	Most sensitive monocot:	Could not be determined
	EC50/IC50: >9.1 lb a.i./A			95% C.I.: N/A 
   EC25/IC25: >9.1 lb a.i./A				95% C.I.: N/A 
   EC05/IC05: NC 						95% C.I.: N/A 
   NOAEC: 9.1 lb a.i./A
   LOAEC: >9.1 lb a.i./A
   Slope: N/A							95% C.I.: N/A
   
Dicot
	Most sensitive dicot: Tomato, based on weight
   EC50/IC50: NC						95% C.I.: N/A
   EC25/IC25: 7.76 lb a.i./A				95% C.I.: 1.85-21 lb a.i./A
   EC05/IC05: 0.778 lb a.i./A				95% C.I.: N/A-3.21 lb a.i./A
   NOAEC: 2.2 lb a.i./A
   LOAEC: 4.4 lb a.i./A
   Slope: N/A							95% C.I.: N/A

   D.  STUDY DEFICIENCIES:

 The study author determined the most sensitive dicot was tomato, based on dry weight, with an IC25 of 9.12 lb a.i./A. Corresponding confidence intervals for this IC25 were not reported in the study report. The reviewer was able to determine an IC25 and associated confidence intervals for this endpoint and this is considered a minor deviation.
 Survival was the most sensitive endpoint for soybean and sugarbeet. Soybean mortality was 15% at the highest test concentration and sugarbeet mortality was 13, 18, 28 and 18% at the four highest test concentrations. Terrestrial plant studies are designed to capture sub-lethal effects; therefore, survival is not expected to be the most sensitive endpoint and mortality may have confounded growth effects in soybean and sugarbeet. Therefore, the results for soybean and sugarbeet should be interpreted with caution. The reviewer determined survival based on the number planted; the negative control for survival ranged from 75 to 100% for all species tested. The study author determined survival based on number emerged; the negative control for survival ranged from 93 to 100% for all species tested. The study author's survival results differed from the reviewer's as the study author found no significant inhibitions in survival for any species. However, if survival is based on number planted, as the reviewer did, the mean control seedling survival does not meet the OCSPP 850.4100 test validity element of at least 90% survival at test termination for four species; onion (75%), sunflower (78%), ryegrass (80%), and corn (88%). This is considered a study deficiency for the species where survival is the most sensitive endpoint, (soybean and sugarbeet), and where the mean control seedling survival does not meet the OCSPP 850.4100 test validity element (onion, sunflower, and ryegrass).
 The number of seedlings per replicate was five for all test species. OCSPP guidance recommends at least 10 seeds per replicate. However, the total number of seeds for each treatment group for each species was 40 (5 seeds per 8 replicate pots), which does meet the OCSPP guideline of 40 seeds per treatment level.  This is a minor deficiency.
 Humidity for onion, ryegrass, wheat, sugarbeet and cabbage was a mean +- standard deviation (SD) (range) of 60.57% +- 6.21% (SD) (23.71-90.50%), and for corn, oilseed rape, soybean, sunflower and tomato was a mean +- SD (range) of 59.90% +- 5.91% (SD) (23.71-90.50%), which includes humidity measurements lower than OCSPP recommended humidity of 70% (daytime) +- 15%. However, overall this was within the recommended range per the OCSPP guideline and is considered to be a minor deficiency.
 CEC and percent moisture were not reported. This is a minor deficiency.
 The physico-chemical properties of the test material were not reported. This is a minor deficiency.
 The total volume of water applied was not reported. This is a minor deficiency.
 The test substance was supposed to be stored refrigerated but was inadvertently stored under ambient conditions for about 2 months before the mistake was corrected (May 1, 2018 to June 28, 2018, p. 13). A percent area analysis was performed at beginning and end of sample analysis to verify stability of test substance during improper storage.  Based on the information provided in the study report, the improper storage did not appear to adversely impact the stability of the test substance. This is a minor deficiency.

   E.  REVIEWER'S COMMENTS:
      
      The reviewer and study author results were in agreement. Both the study author and reviewer determined there was no most sensitive monocot species. The study author determined the most sensitive dicot species was tomato based on weight, with NOAEC and IC25 values of 5.0 and 9.12 lb a.i./A, respectively. Based on the reviewer's results, the most sensitive monocot could not be determined. There were no dose-responsive treatment related reductions that were greater than 25% for any monocot species. Therefore, the IC25 was visually estimated as measured >9.1 lb a.i./A (nominal 10 lb a.i./A), the highest tested application rate for all monocot species tested, and the NOAEC was measured 9.1 lb a.i./A (nominal 10 lb a.i./A) for all tested monocot species. The most sensitive dicot species was tomato, based on weight, with NOAEC and IC25 values of 2.2 and 7.76 lb a.i./A, respectively. 
      
      Most differences in the study author and reviewer conclusions were a result of differences in statistical analyses (parametric vs. non-parametric tests), the study author's use of nominal concentrations and the reviewer's use of analytically measured concentrations, and the study author basing survival on number emerged, while the reviewer-based survival on number planted. For tomato dry weight endpoint, the reviewer visually lowered the LOAEC to 4.4 lb a.i./A, although not statistically significant (Jonckheere-Terpstra Step-Down test, p<0.05), based on the magnitude (21%) of effect at the second highest test level (4.4 lb a.i./A), in conjunction with consistently lower weights by replicate, therefore, the NOAEC was visually determined to be 2.2 lb a.i./A.
      
      The reviewer's results are presented in the Executive Summary and Conclusions sections of this DER. 
      Seedling emergence studies are designed to capture sub-lethal effects; therefore, survival is not expected to be the most sensitive endpoint and mortality may have confounded growth effects in soybean and sugarbeet, this is considered a study deficiency for the species where survival is the most sensitive endpoint. Therefore, the results for soybean and sugarbeet should be interpreted with caution. For onion, sunflower, and ryegrass, the mean control seedling survival does not meet the OCSPP 850.4100 test validity element is considered to be a study deficiency. In particular for ryegrass there was a potential uncertainty in the effects observed related to the negative control survival (80%) limiting the ability to reliably capture sublethal effects (reductions in dry weight), which were large in some treatments, although not statistically significant (decrease of up to 25% at the lowest concentration). However, any potential effects did not appear to be dose responsive and therefore do not appear to be treatment-related.  
      
      The in-life portions of this study were conducted as follows: initiation for onion, ryegrass, wheat, sugarbeet and cabbage on August 13, 2018 and completion on September 3, 2018; initiation for corn, oilseed rape, sunflower, soybean and tomato on August 16, 2018 and completion on September 6, 2018. Final dry weight measurements were completed on September 12, 2018. 
      
   F. CONCLUSIONS:
   
      This study is scientifically sound and is classified as supplemental overall; individual species are classified as follows: it is classified as acceptable for all species except soybean, sugarbeet, onion, sunflower, and ryegrass. It is classified as supplemental and may be used to calculate risk quotients (for soybean and sugarbeet survival endpoints) and onion and sunflower, and supplemental and may be used for risk characterization only (for ryegrass). The most sensitive monocot could not be determined. There were no dose-responsive treatment related reductions that were greater than 25% for any monocot species. Therefore, the IC25 was visually estimated as >9.1 lb a.i./A, the highest tested application rate for all monocot species tested, and the NOAEC was 9.1 lb a.i./A for all tested monocot species. The most sensitive dicot species was tomato, based on weight, with NOAEC and IC25 values of 4.4 and 7.76 lb a.i./A, respectively. The IC25 confidence intervals were outside the tested range of concentrations and the IC50 was not calculable; therefore, these results for tomato should be interpreted with caution.
      
   	Most sensitive monocot and EC25/IC25: None; the IC25 was visually estimated as >9.1 lb a.i./A, the 				highest tested application rate for all tested monocot species.
   	Most sensitive dicot and EC25/IC25: Tomato (weight); 7.76 lb a.i./A

III.  REFERENCES:

U.S. Environmental Protection Agency. 2012. Series 850- Ecological Effects Test Guidelines, OCSPP Number 850.4100: Seedling Emergence and Seedling Growth.

Frans, Robert E. and Ronald E. Talbert. 1977. Design of Field Experiments and the Measurement and Analysis of Plant Responses. Pages 15-23 in B. Truelove, ed. Research Methods in Weed Science. Southern Weed Science Society, Auburn University, Alabama.

Bruce, Robert D. and Donald J. Versteeg. 1992. A Statistical Procedure for Modeling Continuous Data. Environmental Toxicology and Chemistry. 11: 1485-1494.

SAS Institute, Inc. 2002-2012. SAS Proprietary Software Version 9.4, Cary, NC, SAS Institute, Inc.



APPENDIX 1. APPLICATION RATE CONVERSIONS from EXCEL FILE


