Chlorimuron ethyl

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 122-2 (Algal Toxicity, Tiers I and II)

Deviations:

yes

Remarks:

Refer 'Any other information on results incl. tables'

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: U.S. EPA-FIFRA, 40 CFR part 158.540 Pesticide Assessment Guidelines Subdivision J, 123-2

Deviations:

yes

Remarks:

Refer 'Any other information on results incl. tables'

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 2-({[(4-chloro-6-methoxypyrimidin-2-yl)carbamoyl]amino}sulfonyl)benzoate

EC Number:

618-690-2

Cas Number:

90982-32-4

Molecular formula:

C15H15ClN4O6S

IUPAC Name:

ethyl 2-({[(4-chloro-6-methoxypyrimidin-2-yl)carbamoyl]amino}sulfonyl)benzoate

Test material form:

solid

Details on test material:

Purity: 98.2%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sample storage conditions before analysis: Frozen with dry ice

Test solutions

Vehicle:

yes

Remarks:

Well water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
•Selenastrum capricornutum
The test substance was dissolved in well water purified by reverse osmosis to provide a primary stock solution with a concentration of 10 µg a.i./mL. In order to solubilize the test material, the pH of the stock solution was adjusted to approximately 8.0 using 0.1 N NaOH. Aliquots of the primary stock solution were individually diluted to one liter with synthetic algal assay procedure (AAP) medium to prepare the 0.31, 0.63, 1.3, 2.5, 5.0 and 10 µg a.i./L nominal test concentrations. The test solutions were clear and colorless.
•Anabaena flos-aquae
The test substance was dissolved in well water purified by reverse osmosis to provide a primary stock solution with a concentration of 9.9 µg a.i./mL. In order to solubilize the test material, the pH of the stock solution was adjusted to approximately 8.0 using 0.1 N NaOH. Aliquots of the primary stock solution were individually diluted to one liter with AAP medium to prepare the 3.5, 7.0, 14, 28 and 56 µg a.i./L nominal test concentrations. All test solutions were clear and colorless.
•Navicula pelliculosa
The test substance was dissolved in well water purified by reverse osmosis to provide a primary stock solution with a concentration of 298 µg a.i./L. In order to solubilize the test material, the pH of the stock solution was adjusted to approximately 8.0 using 0.1 N NaOH. A 198 µL aliquot of the primary stock solution was diluted to one liter with AAP/Si (AAP medium with approximately 20 mg/L Silicon) medium to prepare the 58.8 µg a.i./L nominal test concentration. The test solution was clear and colorless.
•Skeletonema costatum
The test substance was dissolved in well water purified by reverse osmosis to provide a primary stock solution with at concentration of 297 µg a.i./mL. In order to solubilize the test material, the pH of the stock solution was adjusted to approximately using 8.0 using 0.1 N NaOH. A 198 µL aliquot of the primary stock solution was diluted to one liter with MAA medium to prepare the 58.8 µg a.i./L nominal test concentration. The test solution was clear and colorless.

Test organisms

Test organisms (species):

other: Selenastrum capricornutum, Anabaena flos-aquae, Navicula pelliculosa & Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Selenastrum capricornulum, a freshwater, unicellular, non-motile, green alga
- Anabaena flos-aquae, a freshwater, filamentous, blue-green alga
- Navicula pelliculosa, a freshwater, unicellular, non-motile diatom
- Skeletonema costatum, a marine diatom of ecological importance and wide distribution
- Source: UTEX - The Culture Collection of Algae at the University of Texas at Austin, The Department of Botany.
- Conditions: Prior to the study, algal cultures were held under the same environmental conditions used in the study, with the exception that Anabaena flos-aquae and Skeletonema costatum cultures were stirred continuously on a gyratory shaker. The culture organisms were maintained in 250-mL Erlenmeyer flasks containing approximately 100 mL of the appropriate medium and were aseptically transferred to fresh medium every three to five days.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

120 h

Test conditions

Test temperature:

Selenastrum capricornutum: 24.0-25.1°C
Anabaena flos-aquae: 23.5-24.5°C
Navicula pelliculosa: 23.5-24.6°C
Skeletonema costatum: 20.1-21.7°C

pH:

Selenastrum capricornutum: 7.3-8.3
Anabaena flos-aquae: 7.5-8.1
Navicula pelliculosa: 7.4-7.8
Skeletonema costatum: 7.9-8.6

Nominal and measured concentrations:

Nominal Concentrations:
Selenastrum capricornutum: 0.31, 0.63, 1.3, 2.5, 5.0 and 10 µg a.i./L
Anabaena flos-aquae: 3.5, 7.0, 14, 28 and 56 µg a.i./L
Navicula pelliculosa: 58.8 µg a.i./L
Skeletonema costatum: 58.8 µg a.i./L

Measured concentrations:
Selenastrum capricornutum: 0.28, 0.52, 1.1, 2.2, 4.3 and 7.4 µg a.i./L
Anabaena flos-aquae: 3.1, 6.5, 12, 26 and 52 µg a.i./L
Navicula pelliculosa: 51.9 µg a.i./L
Skeletonema costatum: 49.4 µg a.i./L

Details on test conditions:

TEST SYSTEM
- Test vessel: Flasks
- Cells density: 3000 cells/mL for Selenastrum capricornutum, Anabaena flos-aquae and Navicula pelliculosa and 10000 cells/mL for Skeletonema costatum
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Well water purified by reverse osmosis
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Photoperiod: 24 hrs light for Selenastrum capricornutum, Anabaena flos-aquae and Navicula pelliculosa; 16 day/8 night for Skeletonema costatum
- Light intensity: Selenastrum capricornutum: 3650-4950 lumens/m² (average: 4421 lumens/m²)
Anabaena flos-aquae: 2070-3010 lumens/m² (average 2367 lumens/m²)
Navicula pelliculosa: 3710-4870 lumens/m² (average 4343 lumens/m²)
Skeletonema costatum: 3660-4810 lumens/m² (average 4139 lumens/m²)

TEST CONCENTRATIONS
- Range finding study: The range finding test for Selenastrum capricornutum was conducted to define the range for EC25 and EC50 values, with the high concentration not exceeding the solubility limit of the test substance. The range finding tests for Anabaena flos-aquae, Navicula pelliculosa and Skeletonema costatum were conducted with a high dose level equivalent to the maximum labelled use rate for a single application rate of the test substance.
If growth inhibition at that maximum rate was greater than or equal to 50% during the range finding test, a Tier II dose-response test was triggered for that species. Otherwise, a Tier I test, where exposure is limited to a single concentration equivalent to the maximum labelled use rate of the test substance, was conducted.
A dose-response test with six non-zero rates was conducted for the freshwater alga, Selenastrum capricornutum. The nominal concentrations for the definitive test were 0.31, 0.63, 1.3, 2.5, 5.0 and 10 µg a.i./L.
The freshwater alga, Anabaena flos-aquae, exhibited growth inhibition greater than 50% at the maximum labelled use rate during the range- finding test. Therefore, a dose-response test with five non-zero rates was conducted for this species. The nominal concentrations for the definitive test were 3.5, 7.0, 14, 28 and 56 µg a.i/L.
The freshwater diatom, Navicula pelliculosa, and the marine diatom, Skeletonema costatum, exhibited growth inhibition less than 50% at the maximum labelled use rate during the range finding tests. These organisms were exposed to a nominal concentration of 58.8 µg a,i./L, which is representative of the concentration resulting from a direct overspray of a body of water 6" in depth with the maximum labelled use rate for a single application of the product.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

120 h

Dose descriptor:

other: EC25

Remarks:

Selenastrum capricornutum

Effect conc.:

0.62 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95%CI = 0.41 and 0.80 µg a.i./L

Effect concentrations 2

Key result

Duration:

120 h

Dose descriptor:

EC50

Remarks:

Selenastrum capricornutum

Effect conc.:

1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95%CI = 0.81 and 1.4 µg a.i./L

Effect concentrations 3

Key result

Duration:

120 h

Dose descriptor:

NOEC

Remarks:

Selenastrum capricornutum

Effect conc.:

0.28 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Effect concentrations 4

Key result

Duration:

120 h

Dose descriptor:

other: EC25

Remarks:

Selenastrum capricornutum

Effect conc.:

0.42 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Area under growth rate

Remarks on result:

other: 95%CI = 0.27 and 0.62 µg a.i./L

Effect concentrations 5

Key result

Duration:

120 h

Dose descriptor:

EC50

Remarks:

Selenastrum capricornutum

Effect conc.:

0.77 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Area under growth rate

Remarks on result:

other: 95%CI = 0.35 and 1.1 µg a.i./L

Effect concentrations 6

Key result

Duration:

120 h

Dose descriptor:

NOEC

Remarks:

Selenastrum capricornutum

Effect conc.:

0.28 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Area under growth rate

Effect concentrations 7

Key result

Duration:

120 h

Dose descriptor:

other: EC25

Remarks:

Selenastrum capricornutum

Effect conc.:

2.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 8

Key result

Duration:

120 h

Dose descriptor:

EC50

Remarks:

Selenastrum capricornutum

Effect conc.:

4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 9

Key result

Duration:

120 h

Dose descriptor:

NOEC

Remarks:

Selenastrum capricornutum

Effect conc.:

0.52 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 10

Key result

Duration:

120 h

Dose descriptor:

other: EC25

Remarks:

Anabaena flos-aquae

Effect conc.:

6.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95%CI = 4.5 and 19 µg a.i./L

Effect concentrations 11

Key result

Duration:

120 h

Dose descriptor:

EC50

Remarks:

Anabaena flos-aquae

Effect conc.:

18 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95%CI = 13 and 21 µg a.i./L

Effect concentrations 12

Key result

Duration:

120 h

Dose descriptor:

NOEC

Remarks:

Anabaena flos-aquae

Effect conc.:

3.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Effect concentrations 13

Key result

Duration:

120 h

Dose descriptor:

other: EC25

Remarks:

Anabaena flos-aquae

Effect conc.:

12 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Area under growth curve

Remarks on result:

other: 95%CI = 0.66 and 16 µg a.i./L

Effect concentrations 14

Key result

Duration:

120 h

Dose descriptor:

EC50

Remarks:

Anabaena flos-aquae

Effect conc.:

19 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Area under growth curve

Remarks on result:

other: 95%CI = 15 and 21 µg a.i./L

Effect concentrations 15

Key result

Duration:

120 h

Dose descriptor:

NOEC

Remarks:

Anabaena flos-aquae

Effect conc.:

3.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Area under growth curve

Effect concentrations 16

Key result

Duration:

120 h

Dose descriptor:

other: EC25

Remarks:

Anabaena flos-aquae

Effect conc.:

27 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95%CI = 25 and 29 µg a.i./L

Effect concentrations 17

Key result

Duration:

120 h

Dose descriptor:

EC50

Remarks:

Anabaena flos-aquae

Effect conc.:

45 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95%CI = 44 and 47 µg a.i./L

Effect concentrations 18

Key result

Duration:

120 h

Dose descriptor:

NOEC

Remarks:

Anabaena flos-aquae

Effect conc.:

3.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 19

Duration:

120 h

Dose descriptor:

other: Percent inhibition value

Remarks:

Navicula pelliculosa

Effect conc.:

-29 other: %

Basis for effect:

other: Inhibition of growth and reproduction

Effect concentrations 20

Duration:

120 h

Dose descriptor:

other: Percent inhibition value

Remarks:

Skeletonema costatum

Effect conc.:

-11 other: %

Basis for effect:

other: Inhibition of growth and reproduction

Reported statistics and error estimates:

Selenastrum capricornutum and Anabaena flos-aquae:
The means and standard errors of the cell densities for each treatment and control group were calculated using standard procedures. The densities, growth rates, and areas under the growth curve were used to calculate each interval's EC25 and EC50 values. These "effective concentrations" are defined as the concentrations producing an inhibition of growth, relative to the control, of 25% and 50%, respectively. The EC values and associated 95% confidence limits were determined by linear interpolation of the initial measured test concentrations against measured parameter. The NOEC, defined as the highest concentration of material tested that had no statistically significant effect on the measured parameter relative to the control, was determined when possible using Williams's test. All tests of significance are at α = 0.05.
Navicula pelliculosa and Skeletonema costatum:
The means and standard errors of the cell densities for each treatment and control group were calculated using standard procedures. The percent inhibition and its 95% confidence interval were calculated using Welch's t' statistic for ratios. The null hypothesis of 50% or greater inhibition was tested at the 95% confidence level using Welch's t' test for ratios.

Any other information on results incl. tables

Deviations:

The light intensity for Selenastrum capricornutum ranged from 3650 to 4950, which was slightly outside of the desired range for this test (3660 - 4960 lumens/m²).

In addition to Days 0 and 5 of the test, the light intensity for Anabaena flos-aquae was measured at the end of the recovery phase.

The light intensity for Anabaena flos-aquae ranged from 2070 to 3010 lumens/m², which was slightly outside of the desired range for this test (1830 to 2470 lumens/m²).

Samples for the determination of cell density for Navicula pelliculosa were collected 27 hours after test initiation rather than approximately 24 hours (24 hours ± 10%).

The well water was analyzed once during 1994, rather than twice, to measure the concentrations of selected organic and inorganic constitutents.

The nominal concentration of CoC12•6H20 in the MAA medium for Skelelonema Costatum was 21.5 µg/L, rather than 6.06 µg/L.

No protocol deviations existed that compromised the validity of the study.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Selenastrum capricornutum: The 1.1, 2.2, 4.3 and 7.4 µg a.i./L treatments exhibited inhibition of cell density greater than 50%. The effects upon algal growth and reproduction were found to be algistatic at those levels.
Anabaena flos-aquae: The 26 and 52 µg a.i./L treatments exhibited inhibition of cell density greater than 50%. The effects upon algal growth and reproduction were found to be algistatic at those levels.
Navicula pelliculosa: The test substance appeared to have no inhibitory effect on the growth and reproduction of Navicula pelliculosa exposed to an initial measured concentration of 51.9 µg a.i./L for 120 hours.
Skeletonema costatum: The test substance appeared to have no inhibitory effect on the growth and reproduction of Skeletonema costatum exposed to an initial measured concentration of 49.4 µg a.i./L for 120 hours.

Executive summary:

The study was conducted to determine the effects of the test substance on the growth and reproduction of two freshwater algae (Selenastrum capricornutum and Anabaena flos-aquae), one freshwater diatom (Navicula pelliculosa) and one marine diatom (Skeletonema costatum) according to the guideline EPA OPP 122-2 and 123-2. The definitive test concentrations were selected in consultation with the Sponsor on the basis of range finding test results. The range finding test for Selenastrum capricornutum was conducted to define the range for EC25 and EC50 values, with the high concentration not exceeding the solubility limit of the test substance. A Tier II dose-response test was conducted for that species. The range finding tests for Anabaena flos-aquae, Navicula pelliculosa and Skeletonema costatum were conducted with a high dose level equivalent to the maximum labeled use rate for a single application rate of the test substance. If growth inhibition at that maximum rate was greater than or equal to 50% during the range finding test, a Tier II dose-response test was triggered for that species. Otherwise, a Tier I test, where exposure is limited to a single concentration equivalent to the maximum labelled use rate of the test substance, was conducted.

A dose-response test with six non-zero rates was conducted for the freshwater alga, Selenastrum capricornutum. The nominal concentrations for the definitive test were 0.31, 0.63, 1.3, 2.5, 5.0 and 10 µg a.i./L. The 0-hour measured concentrations were 0.28, 0.52, 1.1, 2.2, 4.3 and 7.4 µg a.i./L.

The freshwater alga, Anabaena flos-aquae, exhibited growth inhibition greater than 50% at the maximum labelled use rate during the range- finding test. Therefore, a dose-response test with five non-zero rates was conducted for this species. The nominal concentrations for the definitive test were 3.5, 7.0, 14, 28 and 56 µg a.i/L. The 0-hour measured concentrations were 3.1, 6.5, 12, 26 and 52 µg a.i./L.

The freshwater diatom, Navicula pelliculosa, and the marine diatom, Skeletonema costatum, exhibited growth inhibition less than 50% at the maximum labelled use rate during the range finding tests. These organisms were exposed to a nominal concentration of 58.8 µg a.i./L, which is representative of the concentration resulting from a direct overspray of a body of water 6" in depth with the maximum labeled use rate for a single application of the product. The 0-Hour measured concentrations for the Navicula pelliculosa and Skeletonema costatum treatment groups were 51.9 and 49.4 µg a.i./L, respectively.

The organisms were exposed for 5 days without test medium renewal. Cell densities and percent inhibition values relative to those cell densities were determined for each 24-hour interval. For the dose- response tests, cell densities were used to calculate growth rates and area under the growth curve values for each interval. From the data obtained in the dose-response tests, the concentration producing 50% inhibition relative to the control (EC50), the concentration producing 25% inhibition relative to the control (EC25), and the no observable effect concentration (NOEC) were determined where statistically appropriate. The 120-hour test results were as follows.

 

•Selenastrum capricornutum

Cell Density

EC25 0.62 µg a.i/L (95% CI = 0.41 and 0.80 µg a.i./L)

EC50 1.0 µg a.i/L (95% CI = 0.81 and 1.4 µg a.i./L)

NOEC 0.28 µg a.i/L

 

Area Under the Growth Curve

EC25 0.42 µg a.i/L (95% CI = 0.27 and 0.62 µg a.i/L)

EC50 0.77 µg a.i/L (95% CI = 0.35 and 1.1 µg a.i/L)

NOEC 0.28 µg a.i/L

 

Growth Rate

EC25 2.4 µg a.i/L (95% CI = 2.1 and 2.6 µg a.i/L)

EC50 4.0 µg a.i/L (95% CI = 3.7 and 4.5 µg a.i/L)

NOEC 0.52 µg a.i/L

 

At the end of the exposure period, samples from those treatment groups showing a 50% or greater inhibition of cell density were diluted with untreated algal medium to a concentration of the test substance active ingredient that was not expected to inhibit algal growth and reproduction. The 1.1, 2.2, 4.3 and 7.4 µg a.i./L treatments for Selenastrum capricornutum exhibited greater than 50% inhibition of cell density compared to the control. The effects upon growth and reproduction of Selenastrum capricornutum were found to be algistatic.

 

•Anabaena flos-aquae

Cell Density

EC25 6.4 µg a.i./L (95% CI = 4.5 and 19 µg a.i./L)

EC50 18 µg a.i./L (95% CI = 13 and 21 µg a.i./L)

NOEC 3.1 µg a.i./L

 

Area Under the Growth Curve

EC2S 12 µg a.i./L (95% CI = 0.66 and 16 µg a.i./L)

EC50 19 µg a.i./L (95% CI = 15 and 21 µg a.i./L)

NOEC 3.1 µg a.i./L

 

Growth Rate

EC25 27 µg a.i./L (95% CI = 25 and 29 µg a.i./L)

EC50 45 µg a.i./L (95% CI = 44 and 47 µg a.i./L)

NOEC 3.1 µg a.i./L

 

At the end of the exposure period, samples from those treatment groups showing a 50% or greater inhibition of cell density were diluted with untreated algal medium to a concentration of the test substance active ingredient that was not expected to inhibit algal growth and reproduction. The 26 and 52 µg a.i./L treatments for Anabaena flos-aquae exhibited greater than 50% inhibition of cell density compared to the control. The effects upon growth and reproduction of Anabaena flos-aquae were found to be algistatic.

 

•Navicula pelliculosa

The 120-hour percent inhibition value for the treatment was as follows:

-29% (95% CI = -257 to 21)

 

•Skeletonema costatum

The 120-hour percent inhibition value for the treatment was as follows:

-11% (95% CI = -28 to 3.2)