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 IC50 values based on all endpoints (frond number 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 

IC50 >13.0 mg ai/L 95% C.I.: N/A 

NOAEC ≥ 13.0 mg ai/L 

Probit Slope: N/A 

Frond number growth rate 

IC50 >13.0 mg ai/L 95% C.I.: N/A 

NOAEC ≥ 13.0 mg ai/L 

Probit Slope: N/A 

Frond area yield 

IC50 >13.0 mg ai/L 95% C.I.: N/A 

NOAEC ≥ 13.0 mg ai/L 

Probit Slope: N/A 

Frond area growth rate 

IC50 >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; therefore, 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 fronds/plant were not reported; a total of 12 fronds 

per vessel were used (one control 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 Principles 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 gibba 

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 

Na2EDTA·2H2O 

NaHCO3 

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 treatment group was 176.3 fronds, yielding -3.5% inhibition 

relative to the negative control. The NOAEC and IC50 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 treatment group was 0.393/day, yielding -2.7% inhibition 

relative to the negative control. The NOAEC and IC50 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 mm2

, yielding -0.1% inhibition relative to 

the negative control. The NOAEC and IC50 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 treatment group was 0.378/day, yielding 1.0% inhibition relative 

to the negative control. The NOAEC and IC50 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 values were not determined on day 7; therefore it was 

not known if pH values varied during the study.

Table 4. Effect of BCS-CO60720 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 calculated >11.8 >11.8 >11.8 >11.8 

IC50 or EC50

(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 for frond number yield, frond number growth rate, frond area 

yield, and frond area growth rate using ToxRat Professional statistical software. The data were assessed for 

normality and homogeneity of variance using Shapiro-Wilk’s test 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 yield, frond number growth rate, frond area yield, and 

frond area growth rate using CETIS version 1.8.7.12 statistical 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 Bartlett’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 values are reported in terms of mean measured exposure 

concentrations. 

Frond number yield 

IC50 > 13.0 mg ai/L 95% C.I.: N/A 

NOAEC ≥ 13.0 mg ai/L 

Probit Slope: N/A 

Frond number growth rate 

IC50 > 13.0 mg ai/L 95% C.I.: N/A 

NOAEC ≥ 13.0 mg ai/L 

Probit Slope: N/A 

Frond area yield 

IC50 > 13.0 mg ai/L 95% C.I.: N/A 

NOAEC ≥ 13.0 mg ai/L 

Probit Slope: N/A 

Frond area growth rate 

IC50 > 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 values were based on mean measured concentrations. The 

reviewer’s results are presented in the Executive Summary 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 

IC50 > 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 

IC50 > 13.0 mg ai/L 95% C.I.: N/A 

NOAEC ≥ 13.0 mg ai/L 

Probit Slope: N/A 

Frond area yield 

IC50 > 13.0 mg ai/L 95% C.I.: N/A 

NOAEC ≥ 13.0 mg ai/L 

Probit Slope: N/A 

Frond area growth rate 

IC50 > 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 Professional”, 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 Program”, Bayer CropScience AG, unpublished report DOM 23038 of 

April 7, 2004.