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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
11 Feb - 16 Feb 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Limit test concentration is clear below the water solubility.
Qualifier:
according to guideline
Guideline:
EPA OPP 122-2 (Algal Toxicity, Tiers I and II)
Qualifier:
according to guideline
Guideline:
EPA OPP 123-3 (Algal Toxicity, Tiers I and II)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
At test initiation and test termination, triplicate samples from the exposure solution, and a single sample from both the control and the solvent control were analysed for RPA 107382 concentration. The stock solution used to prepare the test solution was also analysed at test initiation.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.1 mL/L
- Method: A 6.0 mg a.i./mL primary stock solution was prepared by placing 0.1517 g (0.1500 g as active ingredient) of RPA 107382 in a 25 mL volumetric flask and diluting to volume with DMF. The 0.60 mg a.i./L test solution was prepared by diluting 0.10 mL of the 6.0 mg a.i./mL primary stock solution with sterile AAP medium to a volume of 1 L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The stock solution was observed to be clear and colourless, with no visible signs of undissolved test substance.

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: 1648
- Source (laboratory, culture collection): Stock culture at Springborn Laboratories, originally obtained from the University of Texas, Austin TX, USA
- Age of inoculum (at test initiation): The inoculum used to initiate the toxicity test was taken from a stock culture that had been transferred to fresh medium six days before testing.
- Cultivation conditions: Stock cultures were maintained in 125 mL glass flasks, covered with stainless steel caps and containing 50 mL of medium, at 24 ± 1 °C, under continuous shaking at 100 ± 10 rpm and continuous illumination with 3200 - 5400 lux.
- Culturing medium (same as test or not): same as test
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
5 d
Hardness:
no data
Test temperature:
23 - 25 °C
pH:
at test start: 7.3 - 7.4
at test end: 10.2 - 10.3
Dissolved oxygen:
no data
Nominal and measured concentrations:
Nominal: 0, 0.60 mg a.i./L
Measured at test start: < 0.054, 0.57 mg a.i./L
Measured at test end: < 0.055, 0.37 mg a.i./L
Mean measured: < 0.055, 0.47 mg a.i./L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): fitted with stainless steel caps
- Material, size, fill volume: glass, 250 mL, 100mL
- Aeration: only by gas exchange
- Initial cells density: 3E03 cells/mL
- Control end cells density: 2.3E06 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, AAP (Algal Assay Procedure) medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile, deionised water
- Total organic carbon: 0.55 mg/L (analysis of Feb 1999)
- Metals: subtoxic levels
- Pesticides: subtoxic levels
- Conductivity: 80 - 100 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was measured continuously in a reference flask next to the test flasks. Conductivity and pH were measured at test start and test end.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous illumination
- Light intensity and quality: 4100 - 4300 lux
- Shaking: constantly at 100 rpm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Determination of cell concentrations: daily using a counting chamber
- Other: daily observations of the health of the cells

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 0.005, 0.05, 0.5 and 5.0 mg a.i./L , undissolved test substance was visible at 5.0 mg a.i./L
- Results used to determine the conditions for the definitive study: At the end of the preliminary test, cell densities for the control averaged 3.16E06 cells/mL and for the respective test concentrations 3.18E06, 3.12E06, 3.10E06 and 3.07E06 cells/mL
Reference substance (positive control):
no
Duration:
5 d
Dose descriptor:
EC50
Effect conc.:
> 0.47 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
5 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.47 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Analytical verification of the test concentration: Compared to nominal values, 95% were measured at test start and 62% at test end (mean: 78%).
- Exponential growth in the control (for algal test): yes, factor of 767
- No abnormalities in cell growth or cell shape are reported.
- Effect concentrations exceeding solubility of substance in test medium: According to the range finding test, the effect concentration exceeds the solubility of the test substance in the AAP medium.
Reported statistics and error estimates:
A t-test (Sokal and Rohlf, 1981, Biometry 2nd edition, W.H. Freeman and Co., New York) was used to compare the cell density of the control to the cell density in the solvent control solution. If no significant difference was determined, control and solvent control data were pooled for further statistical analysis to determine treatment level effects. A t-Test was also conducted to compare the cell density of the treatment level to that of the appropriate control data. Percent inhibition of the cell density of the treatment data were calculated relative to the appropriate control data. Since the test was conducted at only one concentration, EC values were not calculated.

At test termination, cell density in the 0.47 mg a.i./L treatment level averaged 2.30E06 cells/mL which represents -0.14% inhibition relative to the pooled control data. Based on a t-Test, no significant reduction in cell density as compared to the pooled control data was detected.

Table 1: Validity criteria for OECD 201.

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

 approx. 766.7

 yes

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

 Not reported

 Not applicable

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

 Not reported

Not applicable 
Validity criteria fulfilled:
not specified
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
01 Feb - 04 Feb 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Undissolved test material was visible during the test. The maximum pH variation in any test solution was 1.2.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted 1984
Deviations:
yes
Remarks:
The maximum pH variation observed in any test solution was 1.2
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
adopted 1992
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations were analysed for the presence of the test substance at the beginning and end of the test period.
- Sample storage conditions before analysis: Samples were analysed within 24 h after preparation or appropriately preserved and stored until analysis
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide (DMF)
- Method: A stock solution of the highest test substance concentration was prepared by dissolution of 200 mg of test substance per mL DMF. Stock solutions of the four lower concentration levels were prepared by serial 1 in 2 dilutions of the first stock solution in DMF. Each stock solution was manually agitated.
- Concentration of vehicle in test medium (stock solution and final test solution): 0.1 mL/L
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: 86.81 SAG
- Source: Laboratory culture, originally obtained from the University of Göttingen, Germany
- Age of inoculum (at test initiation): The algal cells used for the test cultures were taken from a preculture which was initiated 96 hours prior to the test under the same conditions as in the test.
- Method of cultivation: Laboratory cultures were maintained in 500 mL glass vessels containing 300 mL of culture and fitted with stainless steel caps, at 23 ± 2°C, with a 16 h light/8 h dark photoperiod, 1000 lux light intensity and manual agitation at least once per working day.
- Culturing media and conditions (same as test or not): same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
no data
Test temperature:
21.0 - 21.7 °C at test start
23.0 - 24.4 °C at test end
pH:
7.5 - 8.8
Dissolved oxygen:
no data
Nominal and measured concentrations:
Nominal: 0, 1.3, 2.5, 5.0, 10.0 and 20.0 mg/L
Mean measured: < LOQ, 1.1, 2.3, 4.4, 8.6 and 16.4 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): fitted with stainless steel caps
- Material, size, fill volume: glass, 250 mL, 100 mL
- Aeration: only by gas exchange
- Initial cells density: 2E04 cells/mL
- Control end cells density: approx. 2.22E06 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: culture medium was from Bio Media, Boussens, France
- Detailed composition if non-standard medium was used: medium composition equals OECD TG 201 medium as stated in the guideline with one exception: FeCl3*6H2O was used at final 0.08 mg/L (OECD TG 201: 0.064 mg/L).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water
- Total organic carbon: < 1 mg/L
- Particulate matter: < 1 mg/L
- Metals (mg/L): Ca 6.2, Mg 2.6, K 0.5, Na 13.5, all other metals tested < 0.005 to 0.1
- Pesticides: < 0.05 to 50 µg/L
- Chloride: 23.5 mg/L
- Ca/Mg ratio: 2.4:1
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature and pH were measured from bulk preparations at test start and in each vessel at test end.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 16 h light/8 h dark
- Light intensity and quality: 9400 - 9850 lux
- Shaking: 99 rpm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Determination of cell concentrations: At test start and every 24 h using a counting chamber, microscope and image analyser.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2x
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 0, 0.26 and 10.0 mg/L
- Results used to determine the conditions for the definitive study: Following 72 h of exposure no inhibition of algal growth was observed
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 16.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
15.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Details on results:
- Exponential growth in the control (for algal test): yes, factor of 114
- Analytical measurements: 92 - 100% of nominal values at test start and 71 - 83% of nominal values at test end.
- Effect concentrations exceeding solubility of substance in test medium: EC50 based on both biomass and growth rate is above the visual limit of water solubility of the test item.
Reported statistics and error estimates:
Determination of the EC50: Four linear regression curves were computed based on (1) untransformed data, (2) untransformed response vs. logarithm-transformed concentration, (3) logarithm-transformed response vs. untransformed concentration, (4) logarithm-transformed response vs. untransformed concentration. The regression line which provided the best fit of the transformed or untransformed data was selected based on the highest coefficient R2. This regression equation was then applied to calculate EC50 values.
Determination of the NOEC: The NOEC was estimated by statistical analyses of the mean cell culture densities in each test group at test termination. The dilution water control group was compared to the solvent control group using an F-test. If the F-test was not significant, a t-test was performed, if the F-test was significant, a modified t-test was performed. Whether or not, a significant difference was observed, the exposed groups were compared to the solvent control group for subsequent comparisons. The exposed groups were then compared to solvent control group by use of Bartlett's test for homogeneity of variances, analysis of variance (ANOVA). If Bartlett's test indicated homogeneous variances and the ANOVA was significant, the exposed group means were intercompared to the solvent control group using the Dunnett's test.

Additional effect concentrations based on biomass:

24 h EC50: 17.1 mg/L

48 h EC50: 16.8 mg/L

Table 1: Cell culture densities of S. subspicatus during the 72 h exposure to RPA 107382. Values at time 0 were measured from bulk preparations. For time +72 h mean values with SD in brackets are given.

**, mean densities are significantly different (α = 0.01) from the mean of the solvent control values.

Mean measured concentration (mg/L)

Cell culture density x 10,000 (cells/mL)

Time 0

Time +72 h

Control < LOQ

1.95

222.46

(3.05)

Solvent control < LOQ

2.05

225.25

(1.95)

1.1

2.10

222.17

(0.63)

2.3

2.00

221.42

(0.63)

4.4

1.90

221.50

(1.75)

8.6

2.05

199.92**

(3.17)

16.4

2.15

109.33**

(3.21)

Table 2: Validity criteria for OECD 201.

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

Control: 222.46

Solvent control: 225.25

 yes

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

 Not reported

 Not applicable

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

 Not reported

 Not applicable

Table 3: Percentages of inhibition based on area under the growth curve (AUC) and growth rate after 24, 48 and 72 h of exposure

Mean measured concentration (mg/L)

% inhibition based on AUC

% inhibition based on growth rate

24 h

48 h

72 h

24 h

48 h

72 h

1.1

2.2

1.2

0.5

4.9

2.5

1.6

2.3

2.9

0.6

0.4

2.8

0.7

0.6

4.4

2.2

0.3

0.3

0

0

0

8.6

4.8

4.1

8.2

5.0

2.7

3.3

16.4

59.3

59.5

54.1

44.5

28.8

17.1
Validity criteria fulfilled:
not specified
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
27 - 31 Jan 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
adopted 2006
Deviations:
yes
Remarks:
control pH increase: 1.9 units (recommendation < 1.5)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Each concentration level (day 0 and 4)
- Sampling method: 10 mL smaples were taken
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solution of 50.0 g/L prepared by diluting 0.5188 g test item into 10 mL dimethylformamide (DMF). 100 µL of the stock solution were transferred into 1 L distilled water to obtain the desired highest test concentration test solution. Lower test concentration solutions were made via serial dilution from the next higher test concentration. All vessels were brought to a volume of 1 L with filter sterilized 1 X AAP Media after adding the appropriate volume of the next higher test solution. 50 µL of DMF were added to these test solutions.
- Differential loading: no
- Controls: dilution water only (control) and dilution water containing 100µL DMF/L (solvent control)
- Chemical name of vehicle: dimethylformamide (DMF)
- Concentration of vehicle in test medium: stock solution: 100%, final test solutions: 100µL/L, control: 0%, solvent control: 100µL/L
- Evidence of undissolved material: All prepared test solutions were clear and colorless with no visible precipitates.
Test organisms (species):
Anabaena flos-aquae
Details on test organisms:
TEST ORGANISM
- Common name: canobacteria
- Source: in-house laboratory culture, orinially obtained from University of Texas, Austin, Texas, USA in 2007
- Age of inoculum: 3 d batch culture in log phase growth
- Method of cultivation: According to Stein (1973), 24 h light photoperiod, 2200 lux (± 15%), 24 ± 2.0°C
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
23.2 - 24.6°C
pH:
7.4 - 9.4
Conductivity:
88 - 93 µmhos/cm
Nominal and measured concentrations:
Nominal: 0 (control), 0 (solvent control), 0.313, 0.625, 1.25, 2.50 and 5.00 mg a.i./L
Mean measured: < LOQ (control), < LOQ (solvent control), 0.316, 0.634, 1.25, 2.47, and 4.10 mg a.i./L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250-mL Erlenmeyer flasks covered by inverted glass beakers
- Material: borosilicate glass, fill volume: 100 mL
- Aeration: no
- Initial cells density: 1.0 x 10^4 cells/mL
- Control end cells density (72 h): control: 55.84 x 10^4 cells/mL, solvent control: 56.28 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

GROWTH MEDIUM
- Standard medium used: yes: Filter Sterilized 1 x AAP Media (0.2 µm filter) medium according to ASTM

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 1 x AAP Media (0.2 µm filter) medium according to ASTM
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH and conductivity: day 0, 3 and 4, temperature: continuously

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 4690 - 4990 lux (mean: 4854 lux)

EFFECT PARAMETERS MEASURED: cell densitiy (daily)
- Determination of cell concentrations: manual counts via light microscope and hemocytometer slide

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 0 (control), 0 (solvent control), 0.040, 0.20, 1.0 and 5.0 mg a.i./L
- Results used to determine the conditions for the definitive study:
- Growth rate inhibition compared to controls: 0.2, 0.0, 0.0 and 0.2%
- Cells in all test levels appeared to be normal compared to the control.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 4.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 4.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 4.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 4.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: No cell abnormalities were observed in the control and in the treatment groups.
- Effect concentrations exceeding solubility of substance in test medium: no precipitates observed
Reported statistics and error estimates:
Prior to the statistical analysis of each endpoint, the control and solvent control groups were compared to evaluate if they could be pooled. For all endpoints, performance of control and solvent control did not differ statistically significant (two sided t-test; p ≤ 0.05). Thus, controls were pooled for further statistical evaluation. Raw or transformed data from treatment groups were compared to controls for normality and homogeneity of variance using the Shapiro-Wilks test and Bartletts's equality of variance test, respectively. If normality and homogeneity of variance were demonstrated for the raw or transformed values, then parametric analyses were conducted using analysis of variance (ANOVA) followed by Dunnett's test. If normality and/or homogeneity of variance were not demonstrated on raw or transformed values, nonparametric procedures were used. The EC10, EC20, and EC50 values, and the respective 95% confidence intervals, were calculated with help of linear interpolation or regression analysis for cell yield, cumulative biomass, and growth rate.

Table 1: Measured Algae Cell Densities, Calculated Cumulative Biomass and Growth Rate after 72 Hours in the Ethiprole Technical Anabaena flos-aquae Growth Test

 

 

Hour

Nominal Concentration (mg a.i./L)

 

 

Replicate

Yielda (cells/mL) x 10,000

 

Cumulative Biomass b

 

Growth Rate c

72

Control

A

41.33

843.75

0.052022

72

Control

B

62.50

1046.75

0.057653

72

Control

C

58.88

1030.42

0.056837

72

Control

D

60.63

1030.33

0.057237

72

Control

Mean

55.83

987.81

0.055937

72

Solvent Control

A

54.13

989.50

0.055689

72

Solvent Control

B

52.75

889.67

0.055338

72

Solvent Control

C

58.88

1010.83

0.056837

72

Solvent Control

D

59.38

949.83

0.056952

72

Solvent Control

Mean

56.28

959.96

0.056204

72

0.313

A

45.58

850.33

0.053351

72

0.313

B

56.75

985.17

0.056335

72

0.313

C

57.38

980.83

0.056485

72

0.313

D

61.50

1014.67

0.057433

72

0.313

Mean

55.30

957.75

0.055901

72

0.625

A

48.83

818.67

0.054287

72

0.625

B

65.25

1050.33

0.058242

72

0.625

C

57.63

996.42

0.056544

72

0.625

D

65.75

1062.33

0.058347

72

0.625

Mean

59.36

981.94

0.056855

72

1.25

A

40.25

884.52

0.051662

72

1.25

B

61.25

1031.00

0.057377

72

1.25

C

56.25

973.00

0.056214

72

1.25

D

46.92

917.00

0.053743

72

1.25

Mean

51.17

951.38

0.054749

72

2.50

A

51.25

906.33

0.054945

72

2.50

B

56.50

968.67

0.056275

72

2.50

C

48.33

834.67

0.054147

72

2.50

D

62.75

1047.00

0.057708

72

2.50

Mean

54.71

939.17

0.055769

72

5.00

A

56.00

977.33

0.056153

72

5.00

B

49.83

885.92

0.054563

72

5.00

C

61.00

1022.00

0.057321

72

5.00

D

60.00

1042.00

0.057095

72

5.00

Mean

56.71

981.81

0.056283

a Samples for Yield are collected approximately every 24 hours throughout the duration of the test.

b Cumulative biomass is equal to the area under the growth curve (statistics were conducted using a CETIS program)

c Growth rate [ 1/h ] is calculated from the cell density data (statistics were conducted using a CETIS program)

Table 2: Measured Concentrations of Ethiprole Technical During the 96-hour Exposure of Anabaena flos-aquae

 

Nominal Concentration(mg a.i./L)

Day 0 Measured Concentration (mg a.i./L)

 

Day 0 % Nominal

Day 4 Measured Concentration (mg a.i./L)

 

Day 4 % Nominal

Mean Measured Concentration (mg a.i./L)

 

Percent Mean Measured Concentration

Control

< LOQ

NA

< LOQ

NA

NA

NA

S. Control

< LOQ

NA

< LOQ

NA

NA

NA

0.313

0.319

102%

0.312

100%

0.316

101%

0.625

0.647

103%

0.621

99%

0.634

101%

1.25

1.28

102%

1.23

99%

1.25

100%

2.50

2.52

101%

2.42

97%

2.47

99%

5.00

4.48

90%

3.71

74%

4.10

82%

 Limit of quantification = (0.025 mg a.i./L) NA = Not Applicable

Calculations for mean, standard deviation, and percent of nominal concentration are based on recoveries from Day 0 and Day 4.

Table 3: Validity criteria for OECD 201.

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

 56

yes 

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

 20%

yes 

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

 3.2%

yes 
Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
23 - 27 Mar 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
adopted 2006
Deviations:
yes
Remarks:
control pH increase: 2.1 units (recommendation < 1.5)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0.188, 0.375, 0.750, 1.50 and 3.00 mg a.i./L (day 0, 3 and 4)
- Sampling method: 10 mL samples were taken
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solution of 30.0 g/L prepared by diluting 0.3113 g test item into 10 mL dimethylformamide (DMF). 100 µL of the stock solution were pipetted into 1 L distilled water. Lower stock solutions were made via serial dilution of 5 mL from the next stock solution with 5 mL DMF. Test solutions were prepared by adding 100 µL of the respective stock solution into 1 L filter Sterilized 1x AAP medium.
- Differential loading: no
- Controls: dilution water only (control) and dilution water containing 100µL/L DMF
- Chemical name of vehicle: dimethylformamide (DMF)
- Concentration of vehicle in test medium: stock solution: 100%, final test solution: 100µL/L, control: 0%, solvent control: 100µL/L
- Evidence of undissolved material: All prepared test solutions were clear and colorless with no visible precipitates.
Test organisms (species):
Navicula pelliculosa
Details on test organisms:
TEST ORGANISM
- Common name: diatom algae
- Source: in-house laboratory culture, originally obtained from University of Texas, Austin, Texas, USA in 2014
- Age of inoculum: 3 d
- Method of cultivation: held unter test conditions
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
23.4 - 24.0°C
pH:
7.4 - 9.6
Conductivity:
106 - 150 µmhos/cm
Nominal and measured concentrations:
Nominal: 0 (control), 0 (solvent control), 0.188, 0.375, 0.750, 1.50, and 3.00 mg a.i./L
Mean measured: < LOQ (control), < LOQ (solvent control), 0.193, 0.386, 0.651, 1.42 and 2.70 mg a.i./L (Days 0, 3 and 4: 87% - 103% of nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250-mL Erlenmeyer flasks
- Type: closed with inverted glass beakers
- Material: borosilicate glass, fill volume: 100 mL
- Aeration: no
- Initial cells density: 1.0 x 10^4 cells/mL
- Control end cells density: 123.93 x 10^4 cells/mL (control), 112.79 x 10^4 cells/mL (solvent control)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates):4

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: Filter Sterilized 1 x AAP Media (0.2 µm filter) medium according to ASTM

TEST MEDIUM / WATER PARAMETERS
- Conductivity: 106 - 150 µmhos/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature: measured continuously throughout the exposure in a centrally located substitute test vessel, pH and conductivity: day 0, 3 and 4

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous light
- Light intensity and quality: 4450-4890 lux (mean: 4664 lux)

EFFECT PARAMETERS MEASURED: cell density
- Determination of cell concentrations: Manual counts via light microscope and hemocytometer slide and Counts with Z Series Dual Beckman Coulter Counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0 (control), 0 (solvent control), 0.040, 0.20, 1.0 and 5.0 mg a.i./L
- Results used to determine the conditions for the definitive study: Cell density inhibition as compared to the pooled controls: -5.2, 8.7, 5.7 and 27%
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.75 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 3 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 3 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: No cell abnormalities were observed in the control and in the treatment groups during the study.
- Other: 96 hour growth rate was calculated based on mean measured concentrations . The LOEC (0.651 mg a.i./L) corresponded with a 1% inhibition of growth rate compared to the controls, which is not a biologically relevant finding for an exponentially growing population. The NOEC for growth rate is considered > 2.70 mg a.i./L, as up to the functional limit of solubility, growth rate inhibition was less than 5% in all levels tested.
Reported statistics and error estimates:
Raw or transformed data from treatment groups were compared to controls for normality and homogeneity of variance using the Shapiro-Wilks test and Bartletts's equality of variance test, respectively. If normality and homogeneity of variance were demonstrated for the raw or transformed values, then parametric analyses were conducted using analysis of variance (ANOVA) followed by Dunnett's test. If normality and/or homogeneity of variance were not demonstrated on raw or transformed values, nonparametric procedures were used.

Table 1: Measured algae cell densities, calculated cumulative biomass and growth rate in the Navicula pelliculosa growth test after 72 hours

 

 

Hour

Nominal concentration (mg a.i./L)

 

 

Replicate

Yield (cells/mL) x 10,000

 

Cumulative biomass

 

Growth rate

72

Control

A

119.37

2046.81

0.066536

72

Control

B

119.54

2048.78

0.066555

72

Control

C

124.17

2047.85

0.067078

72

Control

D

132.64

2118.25

0.067988

72

Control

Mean

123.93

2065.42

0.067039

72

Solvent Control

A

107.32

1827.24

0.065071

72

Solvent Control

B

122.26

1978.64

0.066865

72

Solvent Control

C

115.11

2042.46

0.066035

72

Solvent Control

D

106.46

1828.29

0.064960

72

Solvent Control

Mean

112.79

1919.16

0.065733

72

0.188

A

127.35

2080.27

0.067428

72

0.188

B

128.02

2208.51

0.067500

72

0.188

C

120.49

2057.12

0.066664

72

0.188

D

115.86

1953.26

0.066125

72

0.188

Mean

122.93

2074.79

0.066929

72

0.375

A

104.04

1901.86

0.064644

72

0.375

B

127.12

2051.80

0.067402

72

0.375

C

113.82

1966.88

0.065881

72

0.375

D

96.12

1664.87

0.063554

72

0.375

Mean

110.27

1896.35

0.065370

72

0.750

A

97.02

1625.40

0.063683

72

0.750

B

102.91

1707.40

0.064494

72

0.750

C

107.73

1788.44

0.065123

72

0.750

D

119.72

1934.17

0.066576

72

0.750

Mean

106.85

1763.86

0.064969

72

1.50

A

98.13

1615.76

0.063839

72

1.50

B

80.36

1410.01

0.061096

72

1.50

C

99.50

1656.14

0.064030

72

1.50

D

85.40

1519.90

0.061931

72

1.50

Mean

90.85

1550.45

0.062724

72

3.00

A

83.15

1391.33

0.061564

72

3.00

B

87.64

1428.32

0.062285

72

3.00

C

70.45

1225.58

0.059291

72

3.00

D

76.11

1932.16

0.060350

72

3.00

Mean

79.33

1494.35

0.060872

Table 2: Measured concentrations of test item during the 96-hour exposure of Navicula pelliculosa

 

Nominal concentration(mg a.i./L)

 

Day 0 measured concentration (mg a.i./L)

 

Day 0 % Nominal

 

Day 3 measured concentration (mg a.i./L)

 

Day 3 %Nominal

 

Day 4 measured concentration (mg a.i./L)

 

Day 4 % Nominal

 

Mean measured concentration (mg a.i./L)

 

Percent mean measured concentration

Control

<LOQ

NA

<LOQ

NA

<LOQ

NA

NA

NA

Solvent control

<LOQ

NA

<LOQ

NA

<LOQ

NA

NA

NA

0.188

0.212

113%

0.195

104%

0.172

91%

0.193

103%

0.375

0.435

116%

0.375

100%

0.348

93%

0.386

103%

0.750

0.754

101%

0.648

86%

0.550

73%

0.651

87%

1.50

1.57

105%

1.46

97%

1.24

83%

1.42

95%

3.00

3.12

104%

2.65

88%

2.34

78%

2.70

90%

Limit of quantification (LOQ) = (0.0125 mg a.i./L)

NA = Not Applicable

Table 3: Validity criteria for OECD 201.

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

 113

yes 

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

 11%

 yes

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

 1.5%

 yes
Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 - 12 Jun 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
yes
Remarks:
control pH increase: 2.5 units (recommendation < 1.5)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0.188, 0.375, 0.750, 1.50, and 3.00 mg/L (day 0, 3 and 4)
- Sampling method: 10 mL samples
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution of 30.0 g/L prepared by diluting 0.3118 g test item into 10 mL dimethylformamide (DMF). 100 µL of the stock solution were pipetted into 1 L distilled water. Lower test concentration solutions were made via serial dilution from the next higher test concentration. All vessels were brought to a volume of 1 L with filter sterilized 1 X AAP Media after adding the appropriate volume of the next higher test solution. 50 µL of DMF were added to the 1.50, 0.750, 0.375 and 0.188 mg a.i./L test solutions.
- Differential loading: no
- Controls: dilution water only (control) and dilution water containing 100µL/L DMF
- Chemical name of vehicle: dimethylformamide (DMF)
- Concentration of vehicle in test medium: stock solution: 100%, final test solution: 100µL/L, control: 0% , solvent control: 100µL/L
- Evidence of undissolved material: All test solutions were clear and colorless with no visible precipitates
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Source: in-house laboratory culture, orinially obtained from University of Texas (UTEX #1648A), Austin, Texas, USA in 2014
- Age of inoculum: 4 d batch culture in log phase growth
- Method of cultivation: According to Stein (1973), 24 h light photoperiod, 4300 lux (± 15%), 24 ± 2.0°C
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
23.8 - 24.5°C
pH:
7.4 - 10.0
Conductivity:
65.2 - 86.4 µmhos/cm
Nominal and measured concentrations:
Nominal: 0 (control), 0 (solvent control), 0.188, 0.375, 0.750, 1.50, and 3.00 mg a.i./L
Mean measured: < LOQ (control), < LOQ (solvent control), 0.156, 0.319, 0.653, 1.29 and 2.73 mg a.i./L (Days 0, 3, 4, 83 - 91% of nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile 250-mL Erlenmeyer flasks
- Type: closed with inverted glass beakers
- Material: Borosilicate glass, fill volume: 100 mL
- Aeration: no
- Initial cells density: 1.0 x 10^4 cells/mL
- Control end cells density: 135.6 x 10^4 cells/mL (72 h), 271.16 cells/mL (96 h)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

GROWTH MEDIUM
- Standard medium used: yes: Filter Sterilized 1 x AAP Media (0.2 µm filter) medium according to ASTM

TEST MEDIUM / WATER PARAMETERS
- Conductivity: 65.2 - 86.4 µmhos/cm
- Intervals of water quality measurement: temperature: measured continuously throughout the exposure, pH: day 0, 3 and 4

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 4690 - 4990 lux (mean: 4854 lux)

EFFECT PARAMETERS MEASURED: cell density (day 0, 1, 2, 3 and 4)
- Determination of cell concentrations: Z Series Dual Beckman Coulter Counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0 (control), 0 (solvent control), 0.024, 0.120, 0.600, and 3.00 mg a.i./L
- Results used to determine the conditions for the definitive study: Percent inhibition for cell density compared to controls: 0.4, -2.1, 1.2, and 32.5%
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.74 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 236 - not applicable)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 3 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits not determinable
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.375 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: No cell abnormalities were observed in the controls or in any other treatment groups during the study.
Reported statistics and error estimates:
Raw data from the control and solvent control groups were compared for equal variance using the Equal Variance Two-Sample t test to determine if the data sets were poolable. Raw or transformed data from treatment groups were compared to controls for normality and homogeneity of variance using the Shapiro-Wilks test and Bartletts's equality of variance test, respectively. If normality and homogeneity of variance were demonstrated for the raw or transformed values, then parametric analyses were conducted using analysis of variance (ANOVA) followed by Dunnett's test. If normality and/or homogeneity of variance were not
demonstrated on raw or transformed values, nonparametric procedures were used. The EC10, 20 and 50 values, and the respective 95% confidence intervals, were calculated using linear interpolation for cell growth rate after 72 hours.

Table 1: Calculated algae cell yield, cumulative biomass and growth rate during the Pseudokirchneriella subcapitata growth test after 72 h

 

 

Hour

Nominal concentration (mg a.i./L)

 

 

Replicate

 

Yield a (cells/mLx10^4)

 

Cumulative biomass b

 

Growth rate c

72

Control

A

141.30

2635.87

0.06886

72

Control

B

146.56

2631.02

0.06936

72

Control

C

128.21

2405.38

0.06752

72

Control

D

126.37

2337.64

0.06732

72

Control

Mean

135.61

2502.48

0.06827

72

Solvent Control

A

143.02

2536.37

0.06903

72

Solvent Control

B

131.37

2281.98

0.06786

72

Solvent Control

C

138.90

2392.37

0.06862

72

Solvent Control

D

127.82

2245.44

0.06748

72

Solvent Control

Mean

135.28

2364.04

0.06825

72

0.188 mg a.i./L

A

124.76

2185.41

0.06714

72

0.188 mg a.i./L

B

139.90

2393.53

0.06872

72

0.188 mg a.i./L

C

115.03

1989.78

0.06603

72

0.188 mg a.i./L

D

133.25

2248.54

0.06805

72

0.188 mg a.i./L

Mean

128.24

2204.31

0.06749

72

0.375 mg a.i./L

A

132.60

2257.45

0.06798

72

0.375 mg a.i./L

B

126.82

2200.11

0.06737

72

0.375 mg a.i./L

C

115.64

2028.39

0.06610

72

0.375 mg a.i./L

D

121.29

2066.07

0.06676

72

0.375 mg a.i./L

Mean

124.09

2138.00

0.06705

72

0.750 mg a.i./L

A

128.53

2197.20

0.06755

72

0.750 mg a.i./L

B

129.75

2213.90

0.06768

72

0.750 mg a.i./L

C

116.00

2036.76

0.06614

72

0.750 mg a.i./L

D

112.11

1996.64

0.06567

72

0.750 mg a.i./L

Mean

121.60

2111.12

0.06676

72

1.50 mg a.i./L

A

119.88

2046.54

0.06659

72

1.50 mg a.i./L

B

114.36

1999.15

0.06594

72

1.50 mg a.i./L

C

121.76

2065.43

0.06681

72

1.50 mg a.i./L

D

115.95

1954.77

0.06614

72

1.50 mg a.i./L

Mean

117.99

2016.48

0.06637

72

3.00 mg a.i./L

A

70.81

1279.11

0.05936

72

3.00 mg a.i./L

B

82.54

1500.67

0.06146

72

3.00 mg a.i./L

C

84.20

1535.79

0.06174

72

3.00 mg a.i./L

D

68.79

1263.08

0.05896

72

3.00 mg a.i./L

Mean

76.58

1394.66

0.06038

a Samples for Yield are collected approximately every 24 hours throughout the duration of the test.

b Cumulative biomass is equal to the area under the growth curve

c Growth rate [ 1/h ] is calculated from the cell density data

Table 2    Measured concentrations of the test item during the 96-hour exposure of Pseudokirchneriellasubcapitata 

 

Nominal concentration (mg a.i./L)

Day 0 Measured concentration (mg a.i./L)

 

Day 0 %Nominal

Day 3 Measured concentration (mg a.i./L)

 

Day 3 %Nominal

Day 4 Measured concentration (mg a.i./L)

 

Day 4 %Nominal

Mean Measured concentration (mg a.i./L)

 

Standard deviation

Percent mean measured concentration

Control

<LOQ

NA

<LOQ

NA

<LOQ

NA

NA

NA

NA

Solvent Control

<LOQ

NA

<LOQ

NA

<LOQ

NA

NA

NA

NA

0.188

0.153

82%

0.164

87%

0.150

80%

0.156

0.00757

83%

0.375

0.312

83%

0.336

89%

0.307

82%

0.318

0.0152

85%

0.750

0.670

89%

0.672

90%

0.620

83%

0.654

0.0292

87%

1.50

1.24

82%

1.35

90%

1.28

85%

1.29

0.0571

86%

3.00

2.73

91%

2.79

93%

2.68

89%

2.73

0.0528

91%

 Limit of quantification = (0.005 mg a.i./L) NA = Not Applicable

Calculations for percent of nominal concentration are based on recoveries from Day 0, 3 and 4.

Table 3: Validity criteria for OECD 201.

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

 134

 yes

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

 16%

yes 

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

 1.2%

yes 
Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.

Description of key information

ErC50 (72h) > 3 mg/L (nominal, Pseudokirchneriella subcapitata, OECD 201)

NOEC = 0.375 mg/L (nominal, Pseudokirchneriella subcapitata, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
3 mg/L
EC10 or NOEC for freshwater algae:
0.375 mg/L

Additional information

The toxicity of the substance to aquatic algae was investigated in four GLP studies.

In the key study, green alga Pseudokirchneriella subcapitata were exposed under static conditions for 96-hours according to OECD 201. Nominal test item concentrations were 0.188, 0.375, 0.750, 1.50, and 3.00 mg a.i./L. Furthermore, a control containing only test medium and a solvent control which contained test medium and 100 mL N, N-dimethylformamide (DMF)/L were tested. Mean measured concentrations from days 0, 3 and 4 ranged from 83% to 91% of nominal concentrations. Derived 72-h ErC50 was > 3 mg/L (nominal). The NOEC (72 h) was determined to be 0.375 mg/L.

This result is supported by another study performed with freshwater diatom Navicula peliculosa according to OECD 201. Diatoms were exposed under static conditions to a control, a solvent control (100 µL DMF/L) and nominal test item concentrations between 0.188 and 3.00 mg a.i./L. Mean measured concentrations ranged from 101% to 116%. Growth based ErC50 (72 h, nominal) value was > 3.00 mg a.i./L. The 72 h NOEC was determined to be 0.750 mg a.i./L (nominal).

A further study performed according to OECD 201 determined the toxicity of the test item on the cyanobacterium Anabaena flos-aquae. The test was performed under static conditions and the cyanobacteria were exposed to nominal test item concentrations in a range of 0.313 and 5.0 mg a.i./L, a control and a solvent control containing 100 µL/L DMF as solvent. Measured test item concentrations were between 74% and 103% of nominal concentrations. Therefore, growth rates were calculated based on mean measured concentrations. The 72-hour EC50 value for growth rate (ErC50) is > 4.10 mg a.i./L with LOEC and NOEC values of > 4.10 and ≥ 4.10 mg a.i./L, respectively.

Another study according to OECD 201 was performed with Scenedesmus subspicatus as test organism. In this test, the ErC50 (72h) exceeded 16.4 mg/L (only 17% inhibition after 72 h), while the EbC50 (72h) was 15.8 mg/L. The NOEC (72h) is reported as 4.4 mg/L. In the analytical verification of the nominal test substance concentrations, 92 – 100% was measured at test start and 71 – 83% at test end. In the two highest test concentrations no clear solution was observed (particles were observed at 20 mg/L and transient precipitation was observed at preparation for 10 mg/L). The reported effect concentrations were determined based on the mean measured concentrations of the test substance.

Another supporting study, a limit test with Pseudokirchneriella subcapitata, was carried out according to guidelines EPA OPP 122-2 and 123-3. No adverse effects were observed when P. subcapitata was exposed to a nominal concentration of 0.6 mg/L for 5 days. The measured concentrations were 95% at test start and 62% at test end, the mean measured concentration is given as 0.47 mg/L. Therefore, the reported effect concentrations are EC50 (5 d) > 0.47 mg/L (arithm. mean measured) and NOEC (5d) ≥ 0.47 mg/L (arithm. mean measured).