### Data Evaluation Report on the Toxicity of BCS-CO60720 to Aquatic Vascular Plants ( Lemna gibba G3) MRID 49447202

Page 2 of 11 EXECUTIVE SUMMARY

In a 7-day toxicity study, duckweed ( Lemna gibba G3) were exposed to BCS-CO60720 at nominal concentrations of 0 (negative control) and 11.8 mg/L under static conditions. Mean measured concentrations were <0.632 (negative control) and 13.0 mg ai/L. NOAEC and IC 50 values based on all endpoints (frond numbe r yield, frond number growth rate, frond area yield, and fond area growth rate) were ≥ 13.0 and > 13.0 mg ai/L, in terms of mean measured concentrations.

The pH values were not determined on day 7; therefore, it was not known if pH values varied during the study.

This study is scientifically sound and is classified as Acceptable

.

Results Synopsis

Frond number yield IC 50 >13.0 mg ai/L 95% C.I.: N/A NOAEC ≥ 13.0 mg ai/L Probit Slope: N/A

Frond number growth rate IC 50 >13.0 mg ai/L 95% C.I.: N/A NOAEC ≥ 13.0 mg ai/L Probit Slope: N/A

Frond area yield IC 50 >13.0 mg ai/L 95% C.I.: N/A NOAEC ≥ 13.0 mg ai/L Probit Slope: N/A

Frond area growth rate IC 50 >13.0 mg ai/L 95% C.I.: N/A NOAEC ≥ 13.0 mg ai/L Probit Slope: N/A

Endpoints Affected: None

### Data Evaluation Report on the Toxicity of BCS-CO60720 to Aquatic Vascular Plants ( Lemna gibba G3) MRID 49447202

Page 3 of 11 I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: The study was designed to comply with the procedures of the OECD Guideline 221 (2006). The following deviations from the U.S. EPA OCSPP 850.4400 (2012) guideline are noted:

1. The age of the inoculum at test initiation was not reported. 2. The method of cultivation for the stock culture was reportedly missing for this study. 3. The pH values were not determined on day 7; ther efore, it was not known if pH values varied during the study. 4. The pH, total organic carbon (TOC), particulate matter, metals, pesticides, and chlorine concentrations of the dilution water were not reported. 5. The number of plants/replicate and the number of fr onds/plant were not reported; a total of 12 fronds per vessel were used (one contro l replicate contained 15 fronds).

These deviations do not affect the validity of the study.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Data Confidentiality statements were provided. The study was performed in accordance with OECD Principles of GLP and Principl es of GLP according to Annex 1 of the German chemical law (ChemG) with the exception of the stock culture and pre culture documentation as described in chapter 4 (Method of cultivation, Inoculum).

A. MATERIALS

1. Test material BCS-CO60720

Description: White powder

Lot No./Batch No. : SES 10689-26-2; BCS-CO60720-01-01

Purity: 98.3%

Stability of compound under test conditions: Stable. Day 7 measured concentrations ranged from 103 to 104% of their initial measured counterparts.

Storage conditions of test chemicals: Room temperature.

Table 1. Physicochemical properties of BCS-CO60720.

Parameter Values Comments

Water solubility at 20°C Not reported

Vapor pressure Not reported

UV absorption Not reported

pKa Not reported

### Data Evaluation Report on the Toxicity of BCS-CO60720 to Aquatic Vascular Plants ( Lemna gibba G3) MRID 49447202

Page 4 of 11 Parameter Values Comments

Kow Not reported

2. Test organism

Name: Duckweed, Lemna gibb a Strain, if provided: G3 Source: In-house cultures originally obtained from Dr. Janet Slovin, Horticulture Crops Quality Laboratory, U.S. Department of Agriculture, Beltsville, MD 20705 U.S.A. Age of inoculum : Not reported Method of cultivation: Stock culture cultivation methods were reportedly missing for this study.

B. STUDY DESIGN

1. Experimental Conditions

a. Range-finding study

None reported

b. Definitive Study

Table 2. Experimental Parameters

Parameter Details Remarks

Criteria

Acclimation period

Culturing media and conditions

Health Continuously cultured in-house

The culturing conditions were not reported.

Healthy and suitable plants were used.

Test system Static/static renewal

Renewal rate for static renewal Static

N/A

Incubation facility Growth incubator

Duration of the test 7 days

### Data Evaluation Report on the Toxicity of BCS-CO60720 to Aquatic Vascular Plants ( Lemna gibba G3) MRID 49447202

Page 5 of 11 Parameter Details Remarks

Criteria

Test vessel Material: Size: Fill volume: Glass 470 mL 200 mL Glass dishes (10 cm i.d., 6 cm height) covered with glass lids to permit gas exchange

Details of 20X AAP medium pH in new solutions: pH in old solutions: Chelator used: Carbon source: 7.5 Not determined Na 2 EDTA · 2H 2 O NaHCO 3 EPA recommends the following culture media: 20X-AAP for Lemna gibba. Chelating agents (e.g. EDTA) are recommended in the nutrient medium for optimum cell growth. Lower concentrations of chelating agents (down to one-third of the normal concentration recommended for AAP medium) may be used in the nutrient medium used for test solution preparation if it is suspected that the chelator will interact with the test material. ASTM reference, E1415-91and D 3978-80 (reapproved 1987).

If non-standard nutrient medium was used, detailed composition provided (Yes/No) A standard medium was used. A detailed composition was provided.

Dilution water source/type: pH: water pretreatment (if any): Total Organic Carbon: particulate matter: metals: pesticides: chlorine: Ultra-pure water Not reported Purified (Milli-Q-water) Not reported Not reported Not reported Not reported Not reported pH of 7.5 ± 0.1 is acceptable if 20XAAP nutrient media is used.

Indicate how the test material is added to the medium (added directly or used stock solution) 15 mg of the test substance was dissolved in 1250 mL of test medium. The resulting solution was stirred and sonicated.

Aeration or agitation None

### Data Evaluation Report on the Toxicity of BCS-CO60720 to Aquatic Vascular Plants ( Lemna gibba G3) MRID 49447202

Page 6 of 11 Parameter Details Remarks

Criteria

Sediment used (for rooted aquatic vascular plants) Origin: Textural classification (%sand, silt, and clay): Organic carbon (%): Geographic location: N/A

Number of replicates Negative control: Solvent control: Treatments: 6 N/A 6 4 minimum

Number of plants/replicate Not reported

3-5 plants

Number of fronds/plant Not reported (total of 12 fronds per vessel except 15 fronds in one control replicate) 3-4 fronds per plant

Test concentrations Nominal:

Mean measured: 0 (negative control) and 11.8 mg/L

<0.632 (negative control) and 13.0 mg ai/L Mean measured concentrations were determined by the reviewer as the mean of day 0 and day 7 measured concentrations.

The study author reported that test material was not detected at a concentration higher than the standard concentration (0.632 mg ai/L) used in this study. This concentration is high compared to the LOD (0.003 mg ai/L) reported for a separately conducted method validation study. Nonetheless, visual inspection of the control chromatograph indicates that test material was not present in the control.

EPA requires at least 5 test concentrations except for limit tests.

Solvent (type, percentage, if used) N/A

### Data Evaluation Report on the Toxicity of BCS-CO60720 to Aquatic Vascular Plants ( Lemna gibba G3) MRID 49447202

Page 7 of 11 Parameter Details Remarks

Criteria

Method and interval of analytical verification Test concentrations were measured in newly prepared solutions on day 0 and from expired solutions on day 7 using HPLC-UV.

Test conditions Temperature: Photoperiod: Light intensity and quality: 23.5 to 23.7oC Continuous 7240 to 8250 lux (quality not reported) Light intensity measured at 9 locations.

Reference chemical (if used) Name: Concentrations: N/A

Other parameters, if any N/A

2. Observations

Table 3. Observation parameters

Parameters Details Remarks/Criteria

Parameters measured (e.g., number of fronds, plant dry weight or other toxicity symptoms) Frond number
Frond area
Growth rate

Measurement technique for frond number and other end points Counting of fronds and determination of total frond area was carried out using a LemnaTec Scanalyzer machine.

Observation intervals Fronds were counted on days 0, 2, 5, and 7.

Other observations, if any Visual observations were made on study days 2, 4, and 7.

Indicate whether there was exponential growth in the control Yes, the doubling time in the negative control was 1.8 days.

Were raw data included? Yes

### Data Evaluation Report on the Toxicity of BCS-CO60720 to Aquatic Vascular Plants ( Lemna gibba G3) MRID 49447202

Page 8 of 11 II. RESULTS and DISCUSSION

A. INHIBITORY EFFECTS

After 7 days, the mean frond number of the treatment group was 188.3 fronds, yielding -3.0% inhibition relative to the negative control. The study author did not assess frond number data.

The mean 0-7 day frond number yield of the treatme nt group was 176.3 fronds, yielding -3.5% inhibition relative to the negative control. The NOAEC and IC 50 values reported by the study author based on frond number yield were ≥ 11.8 and >11.8 mg/L, respectively, in terms of nominal concentrations.

The mean 0-7 day frond number growth rate of the tr eatment group was 0.393/day, yielding -2.7% inhibition relative to the negative control. The NOAEC and IC 50 values reported by the study author based on frond number growth rate were ≥ 11.8 and >11.8 mg/L, respectively, in terms of nominal concentrations.

The mean 0-7 day frond area yield of the treatment group was 1229.7 mm 2 , yielding -0.1% inhibition relative to the negative control. The NOAEC and IC 50 values reported by the study author based on frond area yield were ≥ 11.8 and >11.8 mg/L, respectively, in terms of nominal concentrations.

The mean 0-7 day frond area growth rate of the treatme nt group was 0.378/day, yielding 1.0% inhibition relative to the negative control. The NOAEC and IC 50 values reported by the study author based on frond area growth
rate were ≥ 11.8 and >11.8 mg/L, respectively, in terms of nominal concentrations.

No morphological abnormalities were observed. The pH va lues were not determined on day 7; therefore it was not known if pH values varied during the study.

Table 4. Effect of BCS-CO 60720 on frond number of duckweed, Lemna gibba

Treatment
Mean measured

and (nominal) concentrations mg ai/L Initial frond number/test

solution Frond number

2 days 4 days 7 days

Frond number % Inhibition a

Negative control 12.5 26.3 59.2 182.8 N/A 13.0 (11.8) 12 27.3 67.5 188.3 -3.0

a Calculated by the reviewer relative to the negative control.

### Data Evaluation Report on the Toxicity of BCS-CO60720 to Aquatic Vascular Plants ( Lemna gibba G3) MRID 49447202

Page 9 of 11 Table 5. Effect of BCS-CO60720 on frond number yield of duckweed, Lemna gibba Treatment
Mean measured

and (nominal) concentration mg ai/L Initial frond number/test

solution Frond number growth rate (day -1 ) Frond number yield a

0-7 days % Inhibition 0-7 days % Inhibition

Negative control 12.5 0.383 N/A 170.3 N/A 13.0 (11.8) 12 0.393 -2.7 176.3 -3.5

a Calculated by the reviewer as final minus initial frond number N/A = Not applicable

Table 6. Effect of BCS- CO60720 on biomass of duckweed, Lemna gibba

Treatment
Mean measured and

(nominal) concentrations mg ai/L Frond area Frond area growth rate (day -1 ) Frond area yield a

day 7 % Inhibition b 0-7 days % Inhibition 0-7 days % Inhibition

Negative control 1318.7 N/A 0.382 N/A 1228.2 N/A

13.0 (11.8) 1323.3 -0.3 0.378 1.0 1229.7 -0.1

a Calculated by the reviewer as the final minus initial frond area.

b Calculated by the reviewer relative to the negative control. N/A = Not applicable

Table 7. Statistical endpoint values (calculated by the study author based on nominal concentrations)

Statistical Endpoint Frond number Frond number yield Frond number growth rate Frond area yield Frond area growth rate NOAEC (mg/L) Not calculated ≥ 11.8 ≥ 11.8 ≥ 11.8 ≥ 11.8 LOAEC (mg/L) Not calculate d >11.8 >11.8 >11.8 >11.8
IC 50 or EC 50 (mg/L) (95% C.I.) Not calculated >11.8 (N/A) >11.8 (N/A) >11.8 (N/A) >11.8 (N/A)

N/A = Not applicable

B. REPORTED STATISTICS

The study author statistically analyzed the endpoints fo r frond number yield, frond number growth rate, frond area yield, and frond area growth rate using ToxRat Professi onal statistical software. The data were assessed for normality and homogeneity of variance using Shapiro-Wilk’s te st and Student’s t-test, respectively. The ECx values were empirically determined. All analyses were based on nominal concentrations.

### Data Evaluation Report on the Toxicity of BCS-CO60720 to Aquatic Vascular Plants ( Lemna gibba G3) MRID 49447202

Page 10 of 11 C. VERIFICATION OF STATISTICAL RESULTS

The reviewer assessed the endpoints for frond number yi eld, frond number growth rate, frond area yield, and frond area growth rate using CETIS version 1.8.7.12 st atistical software using backend database settings implemented by EFED on 25 March 2014. All data were confirmed to be normally distributed and have homogeneous variances using Shapiro-Wilk’s and Bartle tt’s tests, respectively. Hypothesis testing was conducted using an equal variance two-sample t-test. The ICx values were empirically determined to be greater than the highest concentration tested. All toxicity va lues are reported in terms of mean measured exposure concentrations.

Frond number yield IC 50 > 13.0 mg ai/L 95% C.I.: N/A NOAEC ≥ 13.0 mg ai/L Probit Slope: N/A

Frond number growth rate IC 50 > 13.0 mg ai/L 95% C.I.: N/A NOAEC ≥ 13.0 mg ai/L Probit Slope: N/A

Frond area yield IC 50 > 13.0 mg ai/L 95% C.I.: N/A NOAEC ≥ 13.0 mg ai/L Probit Slope: N/A

Frond area growth rate IC 50 > 13.0 mg ai/L 95% C.I.: N/A NOAEC ≥ 13.0 mg ai/L Probit Slope: N/A

D. STUDY DEFICIENCIES

See guideline deviations.

E. REVIEWER’S COMMENTS

The reviewer’s results were in agreement with those of the study author. The study author’s toxicity values were based on nominal concentrations whereas the reviewer’s va lues were based on mean measured concentrations. The reviewer’s results are presented in the Executive Su mmary and Conclusions sections of this report.

The laboratory portion of the definitive test was conducted from 08 to 15 November 2010.

F. CONCLUSIONS

This study is scientifically sound and is classified as Acceptable

. Frond number yield IC 50 > 13.0 mg ai/L 95% C.I.: N/A NOAEC ≥ 13.0 mg ai/L Probit Slope: N/A

### Data Evaluation Report on the Toxicity of BCS-CO60720 to Aquatic Vascular Plants ( Lemna gibba G3) MRID 49447202

Page 11 of 11 Frond number growth rate IC 50 > 13.0 mg ai/L 95% C.I.: N/A NOAEC ≥ 13.0 mg ai/L Probit Slope: N/A

Frond area yield IC 50 > 13.0 mg ai/L 95% C.I.: N/A NOAEC ≥ 13.0 mg ai/L Probit Slope: N/A

Frond area growth rate IC 50 > 13.0 mg ai/L 95% C.I.: N/A NOAEC ≥ 13.0 mg ai/L Probit Slope: N/A

Endpoints Affected: None.

III. REFERENCES

OECD Guideline 221 “Lemna sp. Growth Inhibition Test” (March 23, 2006).

Statistical Software “ToxRat Professi onal”, version 2.10.05, produced by ToxRat  Solutions GmbH, 52477 Alsdorf, Germany (February 20, 2010).

ToxRat  Solutions GmbH (2009) ToxRat  Validation Report, release December 01, 2009, valid for ToxRat  Versions 2.10.

M. Dorgerloh, “LemnaTec Scanalyzer Validation Progr am”, Bayer CropScience AG, unpublished report DOM 23038 of April 7, 2004.