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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:
key study
Study period:
09 Feb 2015 - 13 Feb 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, solvent control, 0.0291, 0.0813, 0.228, 0.638, 1.79, and 5.00 mg a.i./L (nominal)
- Sampling method: samples were taken on Days 0 (from batch preparation for each level) and Days 3 and 4 (composite samples from each level)
Vehicle:
yes
Remarks:
DMF
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: dilution series; for detailed information on the preparation of test solutions see Table 1 in section "Any other information on materials and methods"
- Controls: blank control, solvent control
- Chemical name of vehicle: DMF
- Concentration of vehicle in test medium: 100 μL/L
- Evidence of undissolved material: Precipitates were visible in the 10.0 mg a.i./L solution after 1 hour of stirring. The 5.00 mg a.i./L solution appeared clear and colorless without any visible precipitates. Therefore, the functional limit of solubility was determined to be 5.00 mg a.i./L.
Test organisms (species):
Skeletonema costatum
Details on test organisms:
TEST ORGANISM
- Common name: marine microalga
- Source: Provasoli-Guillard National Center for Marine Algae and Microbiota Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, USA, received January 28, 2014
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: In-house batch culture held under test conditions
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
19.9 - 21.0 °C (0 - 24 h)
20.4 - 20.8 °C (25 - 48 h)
20.4 - 20.8 °C (49 - 72 h)
20.3 - 20.8 °C (73 - 96 h)
19.9 - 21.0 °C (complete exposure period)
pH:
8.0 - 8.6 (control)
8.0 - 8.9 (solvent control)
8.0 - 8.9 (test concentrations)
Salinity:
29 - 30‰ (control)
29 - 30‰ (solvent control)
28 - 30‰ (test concentrations)
Nominal and measured concentrations:
control, solvent control, 0.0291, 0.0813, 0.228, 0.638, 1.79, and 5.00 mg a.i./L (nominal)
<0.0025, <0.0025, 0.020, 0.0516, 0.157, 0.429, 1.26, and 3.71 mg a.i./L (measured)
For more detailed information on measured concentrations see Tables 2 and 3 in section "Any other information on materials and methods"
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: closed
- Material, size, headspace, fill volume: Borosilicate glass, Sterile 250-mL Erlenmeyer flasks covered by inverted glass beakers, Fill Volume: 100 mL
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 90,50 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 (replic ates): 4

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: enriched saltwater
- Culture medium different from test medium: no
- Intervals of water quality measurement: Days 0, 3 and 4

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Adjusted to 8.0 ± 0.1 at experimental start
- Photoperiod: 14 hours light / 10 hours dark
- Light intensity and quality: 4470 to 4950 lux (mean: 4742 lux) from cool white fluorescents
- Salinity: 28 - 30 ‰

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: Manual counts via light microscope and hemocytometer slide

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: (1) 09 - 13 May, 2014: control, 0.010, 0.078, 0.625, 5.00, and 10.0 mg a.i./L. and (2) 19 - 23 Dec, 2014: control, 0.040, 0.20, 1.00, and 5.00 mg a.i./L
- Results used to determine the conditions for the definitive study: growth inhibition for (1) 09 - 13 May, 2014: 9.3, 0.9, 39.4, 97.5, and 95.0% compared to controls; growth inhibition for (2) 19 - 23 Dec, 2014: 4.7, 6.7, 46.6 and 97.8% compared to controls
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.429 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.49 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 1.12 - 1.81 mg a.i./L (95% CI)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.154 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.:
1.36 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 1.32 - 1.40 mg a.i./L (95% CI)
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: Cells at 1.79 and 5.00 mg a.i./L were elongated and deformed.

Biological results:

Table 1: Mean Growth Rates during the Exposure of Skeletonema costatum to the test item

Nominal Concentrations

(mg a.i./L)

0 – 72 Hour Mean

Growth

Rate

% Inhibition

0 – 96 Hour Mean

Growth

Rate

% Inhibition

Control

0.044416

NA

0.047006

NA

Solvent Control

0.045451

NA

0.048117

NA

Pooled Controls

0.044934

NA

0.047562

NA

0.0291

0.044238

1.5

0.047216

0.7

0.0813

0.046692

-3.9

0.047934

-0.8

0.228

0.045499

-1.3

0.047386

0.4

0.638

0.045506

-1.3

0.045125

5.1

1.79

0.026293

41.5

0.026524

44.2

5.00

-0.012255

127.3

-0.018616

139.1

Observations:

Cells at 1.79 and 5.00 mg a.i./L were elongated and deformed. No cell abnormalities were observed in the controls and all other treatment groups.

Analytical results:

Geometric Mean Measured Recovery Range (Days 0 and 3): 63% - 74% of nominal test concentrations

Mean Measured Recovery Range (Days 0, 3, 4): 57% - 77% of nominal test concentrations

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 - 10 Apr 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
March 2006
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control, Solvent Control, 0.313, 0.625, 1.25, 2.50, and 5.00 mg a.i./L
- Sampling method: Samples were taken on Days 0 (from batch preparation for each level) and Days 3 and 4 (composite samples from each level)
Vehicle:
yes
Remarks:
DMF
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: dilution series
- Controls: blank control, solvent control
- Chemical name of vehicle): DMF
- Concentration of vehicle in test medium: 100 μL/L
- Evidence of undissolved material: No precipitates were observed directly after test solution preparation, however, precipitates were observed in the 10.0 mg/L test solution after 24 hours.
Test organisms (species):
Navicula pelliculosa
Details on test organisms:
TEST ORGANISM
- Common name: freshwater diatom
- Source: University of Texas (UTEX), Austin, Texas, USA, received October 29, 2014
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: In-house batch culture held under test conditions
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
24.4 - 24.8 °C (0 - 24 h)
24.8 - 25.1 °C (25 - 48 h)
24.0 - 24.9 °C (49 - 72 h)
23.6 - 25.2 °C (73 - 96 h)
23.6 - 25.2 °C (complete exposure period)
pH:
7.6 - 9.7 (control)
7.6 - 9.7 (solvent control)
7.6 - 9.6 (test concentrations)
Conductivity:
128 - 138 µmhos/cm (control)
128 - 141 µmhos/cm (solvent control)
128 - 159 µmhos/cm (test concentrations)
Nominal and measured concentrations:
control, solvent control, 0.313, 0.625, 1.25, 2.50 and 5.00 mg a.i./L (nominal)
For mean measured concentrations see Tables 2 and 3 in section "Any other information on materials and methods"
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: closed
- Material, size, headspace, fill volume: Borosilicate glass, Sterile 250-mL Erlenmeyer flasks covered by inverted glass beakers; Volume: 100 ml
- Initial cells density: 1.0 x 10^4 cells/mL
- Control end cells density: 301.25 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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Filter Sterilized 1x AAP medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: Days 0 (from batch preparation for each level) and Days 3 and 4 (composite samples from each level)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Adjusted to 7.5 ± 0.1 at experimental start
- Photoperiod: 24 hours light
- Light intensity and quality: 4620 to 4990 lux (mean: 4772 lux) from cool white fluorescents

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Manual counts via light microscope and hemocytometer slide

TEST CONCENTRATIONS
- Range finding study: yes (17 Mar - 21 Mar, 2014)
- Test concentrations: control, solvent control, 0.080, 0.400, 2.00, and 10.0 mg a.i./L. (nominal)
- Results used to determine the conditions for the definitive study: Percent inhibition for cell density was -6.5, -0.4, -6.3, -11.4% as compared to the controls.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 3.19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 2.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.285 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 0.285 mg a.i./L corresponded to an inhibition of 1.6% as compared to pooled controls
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.266 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: none

Analytical results:

Geometric Mean Measured Recovery Range (Days 0 and 3): 46 to 64% of nominal

Mean Measured Recovery Range (Days 0, 3, 4): 42 to 52% of nominal

Biological results:

Table 1: Calculated Algae Cell Yield, Cumulative Biomass and Growth Rate (mean value of four replicates):

Hour

Nominal

Concentration

(mg a.i./L)

Yield

(cells/mLx104)

Cumulative Biomass

Growth Rate

24

Control

3.90

46.75

0.066080

24

Solvent Control

3.49

41.83

0.061970

24

0.313 mg a.i./L

3.10

37.17

0.058000

24

0.625 mg a.i./L

3.12

37.42

0.058740

24

1.25 mg a.i./L

3.28

39.42

0.060382

24

2.50 mg a.i./L

3.15

37.75

0.059214

24

5.00 mg a.i./L

2.54

30.50

0.052637

 

Hour

Nominal

Concentration

(mg a.i./L)

Yield

(cells/mLx104)

Cumulative Biomass

Growth Rate

48

Control

35.47

519.13

0.074892

48

Solvent Control

28.20

422.04

0.070161

48

0.313 mg a.i./L

24.38

366.83

0.067043

48

0.625 mg a.i./L

28.84

420.96

0.070731

48

1.25 mg a.i./L

29.41

431.71

0.071133

48

2.50 mg a.i./L

30.34

439.63

0.071742

48

5.00 mg a.i./L

25.56

367.75

0.068250

 

Hour

Nominal

Concentration

(mg a.i./L)

Yield

(cells/mLx104)

Cumulative Biomass

Growth Rate

72

Control

158.69

2849.00

0.070458

72

Solvent Control

149.13

2549.92

0.069549

72

0.313 mg a.i./L

135.94

2290.58

0.068320

72

0.625 mg a.i./L

141.63

2466.58

0.068880

72

1.25 mg a.i./L

132.25

2371.58

0.067938

72

2.50 mg a.i./L

99.38

1996.25

0.064006

72

5.00 mg a.i./L

80.31

1638.25

0.061039

 

Hour

Nominal

Concentration

(mg a.i./L)

Yield

(cells/mLx104)

Cumulative Biomass

Growth Rate

96

Control

301.25

8368.25

0.059486

96

Solvent Control

261.00

7471.42

0.057966

96

0.313 mg a.i./L

255.75

6990.83

0.057784

96

0.625 mg a.i./L

254.75

7223.08

0.057728

96

1.25 mg a.i./L

240.50

6844.58

0.057138

96

2.50 mg a.i./L

172.00

5252.75

0.053654

96

5.00 mg a.i./L

148.88

4388.50

0.052169

 

 

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1st Test: 31 Oct 2014 - 07 Nov 2014; 2nd Test: 22 Jun 2015 - 11 Aug 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
July 28, 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, solvent control, 0.625, 1.25, 2.5, 5, 10 mg a.i./L
- Sampling method: samples were taken on experimental day 0 (test initiation), day 3 and day 4 (test termination)
- Sample storage conditions before analysis: freezer
Vehicle:
yes
Remarks:
DMF
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: dilution series; for detailed information on the preparation of test solutions see Table 1 in section "Any other information on materials and methods"
- Controls: control, solvent control
- Chemical name of vehicle: Dimethylformamide (DMF)
- Concentration of vehicle in test medium: 100 µL DMF / L nutrient medium
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Freshwater microalga
- Strain: SAG 61.81
- Source: Collection of Algal Cultures, Institute for Plant Physiology, University of Göttingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: 400 μL of a 7-10 days old stock culture was transferred into a 300 mL cotton plugged Erlenmeyer repl. containing about 100 mL of nutrient medium every 7-10 days. Stock cultures of algae were kept at 22 ± 2 °C with 24 hours light (4.5 – 7.0 klux). Test vessels were placed on a tablet rotating 100 rpm to prevent sedimentation of the cells. All operations were conducted under sterile conditions to handle an axenic algae culture. Pre cultures were prepared from stock cultures 3 days before the start of the test using OECD medium.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
72 hours extended to 96 hours
Test temperature:
22.5 - 23.2 °C (0 - 72 h)
22.4 - 24.0 °C (0 - 96 h)
pH:
7.9 - 8.1 (0 - 72 h)
7.9 - 9.1 (72 - 96 h)
Nominal and measured concentrations:
nominal: control, solvent, 0.625, 1.25, 2.50, 5.00 and 10.0 mg a.i./L
for measured test concentrations at 0, 72 and 96 h see Tables 2-4 in section "Any other information on materials and methods"
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type: closed (sealed with cellulose plugs)
- Material, size, headspace, fill volume: 300 mL Erlenmeyer flasks, filled with 150 mL nutrient medium
- Initial cells density: 1x10^4 cells / mL
- Control end cells density: 1.4x10^6 cells / mL
- No. of organisms per vessel: initial cell number: 10,000 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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: defined nutrient medium according to OECD guideline 202
- Total organic carbon: < 2 mg/L
- Intervals of water quality measurement: samples were taken on experimental day 0 (test initiation), day 3 and day 4 (test termination)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: adjusted to 8.1 ± 0.2 with NaOH
- Photoperiod: 24 h light
- Light intensity and quality: 4400 - 8800 lux (± 15 %); provided by bank light containing cool white fluorescent lamps attached to the ceiling

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Determination of cell concentrations: spectrophotometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Range finding study: yes
- Test concentrations: 0.1, 0.5, 1.0 and 5.0 mg a.i./L in the first test and 1.0, 5.0, 8.0 and 10.0 mg a.i./L in the second test
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.97 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 1.73 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 1.97 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 1.73 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
- Unusual cell shape: larger cells in test conc. 10.0 mg a.i./L at 24h
- Aggregation of algal cells: clumping of cells at 48, 72 and 96 h in treatment 5.00 and 10.0 mg a.i./L

Analytical results:

The analytical findings of the test item in the treatment levels found on day 0 were 78.9 % to 92.4 % of nominal (average 87.3 %). After 72 hours analytical findings of the test item were 36.3 % to 67.2 % of nominal (average 57.9 %) and after 96 hours 32.4 % to 47.6 % of nominal (average 43.5 %) were found. Results are based on geometric mean measured or initial measured test concentrations of the test item.

Table 1: The static algae growth inhibition test provided the following tabulated effects after 72 hours:

nominal concentration [mg a.s./L]

cell number after 72 h (means) per mL

(0-72h)-average specific growth rates [days-1]

inhibition of average specific growth rate [%]

pooled control

684 000

1.405

0.0

0.625

551 000

1.334

5.1

1.25

648 000

1.386

1.4

2.50

650 000

1.384

1.5

5.00●

--

--

--

10.0●

--

--

--

test initiation with 10,000 cells/mL

● counting of algae cells was not possible due to cell clumping

Table 2: The static algae growth inhibition test provided the following tabulated effects after 96 hours:

nominal concentration [mg p.m./L]

cell number after 96 h (means) per mL

(0-96h)-average specific growth rates [days-1]

inhibition of average specific growth rate [%]

pooled control

1 394 000

1.233

0.0

0.625

1 276 000

1.211

1.8

1.25

1 289 000

1.215

1.5

2.50

1 240 000

1.201

2.6

5.00●

--

--

--

10.0●

--

--

--

test initiation with 10,000 cells/mL

● counting of algae cells was not possible due to cell clumping

Description of key information

Freshwater:

ErC50 (72 h) >3.19 mg a.i./L (Key, M-535793-01-1, Navicula pelliculosa, OECD 201, geometric mean measured)

NOEC (72 h) = 0.285 mg a.i./L (Key, M-535793-01-1, Navicula pelliculosa, OECD 201, geometric mean measured)

Marine water:

ErC50 (72 h) = 1.49 mg a.i./L (Key, M-527594-01-1, Skeletonema costatum, OECD 201, geometric mean measured)

NOEC (72 h) = 0.429 mg a.i./L (Key, M-527594-01-1, Skeletonema costatum, OECD 201, geometric mean measured)

Key value for chemical safety assessment

EC50 for freshwater algae:
3.19 mg/L
EC50 for marine water algae:
1.49 mg/L
EC10 or NOEC for freshwater algae:
0.285 mg/L
EC10 or NOEC for marine water algae:
0.429 mg/L

Additional information

Three experimental studies are available investigating the toxicity of the test item to aquatic algae (M-535793-01-1, M-553842-02-1, M-527594-01-1). All studies were performed according to OECD 201 and GLP with analytical monitoring of the test item concentration. Two studies were performed with freshwater species: one diatom species (Navicula pelliculosa; M-535793-01-1) and one green algae species (Pseudokirchneriella subcapitata; M-553842-02-1). One study was performed with a saltwater species (Skeletonema costatum; M-527594-01-1). The test item was dissolved in dimethylformamide to prepare the test solutions. In all studies an additional solvent control was tested to ensure that the solvent has no influence on growth of algae cells (maximum concentration of 100 µL/L in test solutions).

 

In the key study Navicula pelliculosa was exposed under static conditions (shaken cultures) for 96 h (in total) to nominal concentrations of 0.313, 0.625, 1.25, 2.50, and 5.00 mg a.s./L. Analytical verification of test solutions revealed a range of recovery of 46% to 64% (mean measured concentration up to 72 h) and 42% to 52% (mean measured concentration up to 96 h). The results after 72 h are reported based on geometric mean measured concentrations from days 0 and 3 (i.e. control (<LOQ), solvent control (<LOQ), 0.149, 0.285, 0.661, 1.29, and 3.19 mg a.s./L). In contrast the results after 96 h are reported based on mean measured concentrations from days 0, 3 and 4 (i.e. control (<LOQ), solvent control (<LOQ), 0.133, 0.266, 0.553, 1.15, and 2.60 mg a.s./L). The ErC50 (72 h) was > 3.19 mg a.s./L with LOEC (72 h) and NOEC (72 h) values of 0.661 and 0.285 mg a.s./L, respectively. The ErC50 (96 h) was > 2.60 mg a.s./L with LOEC (96 h) and NOEC (96 h) values of 0.553 and 0.266 mg a.s./L, respectively.

The supporting study was conducted using Pseudokirchneriella subcapitata. Algae cells were exposed in a static system over a period of 72 h with a prolongation to 96 h to nominal concentrations of 0.625, 2.50, 5.00 and 10.0 mg a.s./L (corresponding to geometric mean measured concentrations (0-72 h) of 0.440, 0 851, 1.97, 3.90 and 5.77 mg a.s./L and (0-96 h) of 0.390, 0.772, 1.73, 3.39 and 5.35 mg a.s./L).. The analytical findings of the test item in the treatment levels found on day 0 were 78.9% to 92.4% of nominal (average 87.3%). After 72 h analytical findings of 36.3% to 67.2% of nominal (average 57.9%) were found. After 96 h analytical findings of 32.4% to 47.6% of nominal (average 43.5%) were found. Based on analytical findings the biological endpoints are reported as geometric mean measured (72 h results) and mean measured test concentrations of the test item (96 h results). The ErC50 (72 h) was >1.97 mg a.s./L (based on geometric mean measured concentrations), the ErC50 (96 h) was >1.73 mg a.s./L (based on mean measured concentrations). The NOErC values after 72 and 96 h were determined to be ≥1.97 and ≥1.73 mg a.s./L, respectively.

The marine species Skeletonema costatum (M-527594-01-1) was exposed to nominal concentrations of 0.0291, 0.0813, 0.228, 0.638, 1.79 and 5.00 mg a.s./L in a static system for a total period of 96 h. Analytical verification of test solutions revealed a range of recovery of 63% to 74% (days 0 and 3) and 57 to 77% (days 0, 3, 4). The results after 72 h are reported based on geometric mean measured concentrations from days 0 and 3 (i.e. control (<LOQ), solvent control (<LOQ), 0.020, 0.0516, 0.157, 0.429, 1.26, and 3.71 mg a.s./L). The results after 96 h are reported based on mean measured concentrations from days 0, 3 and 4 (i.e. control (<LOQ), solvent control (<LOQ), 0.0175, 0.0462, 0.154, 0.398, 1.20, and 3.86 mg a.s./L). Inhibition of growth was recorded resulting in an ErC50 (72 h) of 1.49 mg a.s./L with corresponding LOEC (72 h) and NOEC (72 h) values of 1.26 and 0.429 mg a.s./L, respectively. After a test duration of 96 h an ErC50 of 1.36 mg a.s./L with corresponding LOEC and NOEC values of 0.398 and 0.154 mg a.s./L, respectively were derived.