1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

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

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

Reference 2

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

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

 

 

Reference 3

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

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

July 28, 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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.