N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-2-[(2-methoxyphenyl)azo]-3-oxobutyramide

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:

2011-12-02 to 2011-12-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guidelines and compliant to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemikaliengesetzt and directive 88/320/EEC

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The limit test item concentration and control were analytically verified via HPLC-DAD after 0 hours (test start) and 72 hours (test end).
- Sampling method: Samples at test start were taken from additional replicates without algae. After 72 hours samples were taken directly from pooled test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1 mg/L was freshly prepared with dilution water.The stock solution was stirred for 48 hours (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). Centrifugation for 10 min at 10000 rpm was found to be insufficient.
- Eluate: Dilution water
- Differential loading: None, saturated solution corresponding to a nominal test item concentration of 1.00 mg/L.
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

min.: 21.5 max.: 22.0, mean value: 21.8

pH:

pH-value
Start; 0 hours End; 72 hours
Saturated solution prepared
with 1 mg test item/L 7.85 8.51
Control 7.91 8.12

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 103 cells/mL, Actual: mean 4910 cells/mL
- Control end cells density: 792206 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 69.2



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS
- Range finding study:
Results of the Range Finding Test (0-72 hours)

Nominal
test item concentration
[mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 -4 -22
10 -2 -8
1 -1 -5

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to the very poor solubility of the test item, quantitative analytical determination was precluded (below LOQ)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to the very poor solubility of the test item, quantitative analytical determination was precluded (below LOQ)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Due to the very poor solubility of the test item, quantitative analytical determination was precluded (below LOQ)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50 Values of the Reference Item
based on nominal concentrations (mg/L), (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Rate-related inhibition
ErC50 0.488 0.699 ± 0.471
95 % confidence interval 0.466 – 0.512
Yield inhibition
EyC50 0.291 0.318 ± 0.147
95 % confidence interval 0.274 – 0.328

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical
significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) was used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

NOEC, LOEC, EC - values of the test item (0-72 hours)

                   based on the saturated solution prepared with 1 mg test item/L

                  

	Rate-related Inhibition
NOEC	1.00
LOEC	> 1.00
ErC10	> 1.00
ErC20	> 1.00
ErC50	> 1.00
	Inhibition of Yield
NOEC	1.00
LOEC	> 1.00
EyC10	< 1.00
EyC20	> 1.00
EyC50	> 1.00

Cell Densities

			Replicate		Cell density [cells/mL]
		No.		0 hours		24 hours	48 hours	72 hours
Saturated solution prepared with 1 mg test item/L		1		4910		27542	151737	633314
		2		4910		23202	156456	627729
		3		4910		24627	125747	578816
		4		4910		24505	167816	862648
		5		4910		26049	151595	679317
		6		4910		27887	163422	626951
		Mean		4910		25635	152796	668129
Control		1		4910		27183	143295	641574
		2		4910		26059	153978	890710
		3		4910		28533	168486	920227
		4		4910		28232	159570	819489
		5		4910		28814	143119	754531
		6		4910		24262	140709	726707
		Mean		4910		27181	151526	792206

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
	No.	[d^-1]		[%]	[cells/mL]		[%]
Saturated solution prepared with 1 mg test item/L	1		1.62	4.26		628404	20.2
	2		1.62	4.44		622819	20.9
	3		1.59	6.03		573906	27.1
	4		1.72	-1.83		857738	-8.95
	5		1.64	2.88		674407	14.3
	6		1.62	4.46		622041	21.0
	Mean	(-)	1.64	3.37	(-)	663219	15.8
Control	1		1.62			636664
	2		1.73			885800
	3		1.74			915317
	4		1.71			814579
	5		1.68			749621
	6		1.67			721797
	Mean		1.69			787296

Section-by-Section and Average Specific Growth Rates of the Control Group
                   (0-72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.71	1.66	1.50	1.62	0.111	6.84	4.34
	2	1.67	1.78	1.76	1.73	0.0569	3.28
	3	1.76	1.78	1.70	1.74	0.0412	2.36
	4	1.75	1.73	1.64	1.71	0.0609	3.57
	5	1.77	1.60	1.66	1.68	0.0845	5.04
	6	1.60	1.76	1.64	1.67	0.0827	4.97
				Mean	1.69
				SD ±	0.05
				CV [%]	2.66

Validity criteria fulfilled:

yes

Conclusions:

In this study no inhibition on growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item. The NOEC for inhibition of specific growth rate and yield after 72 hours was 1.00 mg/L. All effect levels are given based on the nominal concentration of the test item.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from 2011-12-02 to 2011-12-09, with the definitive exposure phase from 2011-12-06 to 2011-12-09. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5 x 10³ cells/mL. Based on a preliminary range finding the saturated solution* was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

The saturated solution* and the control were analysed by HPLC-DAD after 0 h (fresh media) and 72 h (old media).

The concentration (fresh media, 0 h) of the test item in the freshly prepared saturated solution* and in the old media (72 h) was , therefore all effect values of the test item are given based on the saturated solution prepared with 1 mg/L.

Table 1:   NOEC, LOEC, EC - values of the test item (0-72 hours)

                   based on the saturated solution prepared with 1 mg test item/L

                  

	Rate-related Inhibition
NOEC	1.00
LOEC	> 1.00
ErC10	> 1.00
ErC20	> 1.00
ErC50	> 1.00
	Inhibition of Yield
NOEC	1.00
LOEC	> 1.00
EyC10	1.00
EyC20	> 1.00
EyC50	> 1.00

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-28 to 2011-11-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guidelines and compliant to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemikaliengesetzt and directive 88/320/EEC

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The limit test item concentration and control were analytically verified via HPLC-DAD after 0 hours (test start) and 72 hours (test end).
- Sampling method: Samples at test start were taken from additional replicates without algae. After 72 hours samples were taken directly from pooled test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 C° until analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1 mg/L was freshly prepared with dilution water. The stock solution was stirred for 48 hours (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). Centrifugation for 10 min at 10000 rpm was found to be insufficient.
- Eluate: Dilution water
- Differential loading: Saturated solution* corresponding to a nominal test item concentration of 1.00 mg/L, six replicates
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

min.: 21.5 max.: 22.5, mean value: 22.0

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.86 8.32
Control 7.92 8.72

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 4910 cells/mL
- Control end cells density: Mean 876660 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 69.2



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS
- Range finding study:
Results of the Range Finding Test (0-72 hours)

Nominal
test item concentration
[mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 -1 -3
10 2 11
1 3 14

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to very poor solubility of test item, concentrations in water were below LOQ of analytical method (0.1 mg/L)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to very poor solubility of test item, concentrations in water were below LOQ of analytical method (0.1 mg/L)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50 Values of the Reference Item
based on nominal concentrations (mg/L), (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Rate-related inhibition
ErC50 0.488 0.699 ± 0.471
95 % confidence interval 0.466 – 0.512
Yield inhibition
EyC50 0.291 0.318 ± 0.147
95 % confidence interval 0.274 – 0.328

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical
significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s Test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

NOEC, LOEC, EC - values of the test item (0-72 hours)

                   based on nominal concentration [mg/L]

                               

	Rate-related Inhibition
NOEC	1.00
LOEC	> 1.00
ErC10	> 1.00
ErC20	> 1.00
ErC50	> 1.00
	Inhibition of Yield
NOEC	1.00
LOEC	> 1.00
EyC10	> 1.00
EyC20	> 1.00
EyC50	> 1.00

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
1.00		1		4910		24018	175395	899328
		2		4910		24065	170213	829699
		3		4910		28137	174989	949731
		4		4910		22779	156551	815888
		5		4910		27027	172230	820796
		6		4910		26983	145096	824520
		Mean		4910		25502	165746	856660
Control		1		4910		26330	176776	968484
		2		4910		26404	174874	908298
		3		4910		26699	176620	887413
		4		4910		22674	191186	822996
		5		4910		25392	166953	756650
		6		4910		29583	169072	916118
		Mean		4910		26180	175914	876660

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.00	1		1.74	-0.55		894418	-2.60
	2		1.71	1.00		824789	5.39
	3		1.76	-1.61		944821	-8.38
	4		1.70	1.33		810978	6.97
	5		1.71	1.21		815886	6.41
	6		1.71	1.12		819610	5.98
	Mean	(-)	1.72	0.420	(-)	851750	2.30
Control	1		1.76			963574
	2		1.74			903388
	3		1.73			882503
	4		1.71			818086
	5		1.68			751740
	6		1.74			911208
	Mean		1.73			871750

Section-by-Section and Average Specific Growth Rates of the Control Group
                   (0 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.68	1.90	1.70	1.76	0.124	7.04	9.78
	2	1.68	1.89	1.65	1.74	0.131	7.55
	3	1.69	1.89	1.61	1.73	0.142	8.17
	4	1.53	2.13	1.46	1.71	0.370	21.6
	5	1.64	1.88	1.51	1.68	0.189	11.2
	6	1.80	1.74	1.69	1.74	0.053	3.04
				Mean	1.73
				SD ±	0.03
				CV [%]	1.70

Validity criteria fulfilled:

yes

Conclusions:

In this study no inhibition on growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item. The NOEC for inhibition of specific growth rate and yield after 72 hours was 1.00 mg/L. All effect levels are given based on the nominal concentration of the test item, which has been analytically verified by HPLC-DAD.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5 x 10³ cells/mL. Based on a preliminary range finding the saturated solution* was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

The saturated solution* and the control were analysed by HPLC-DAD after 0 h (fresh media) and 72 h (old media).

The concentration (fresh media, 0 h) of the test item in the freshly prepared saturated solution* and in the old media (72 h) was< LOQ. All effect values of the test item are given based on the nominal concentration.

Table 1:   NOEC, LOEC, EC - values of the test item (0-72 hours)

                   based on nominal concentration [mg/L]

                               

	Rate-related Inhibition
NOEC	1.00
LOEC	> 1.00
ErC10	> 1.00
ErC20	> 1.00
ErC50	> 1.00
	Inhibition of Yield
NOEC	1.00
LOEC	> 1.00
EyC10	> 1.00
EyC20	> 1.00
EyC50	> 1.00

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2011-12-02 to 2011-12-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guidelines and compliant to GLP

Justification for type of information:

Justification for type of information: see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Acetolone Pigments – Nanoforms and Bulk Forms (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemikaliengesetzt and directive 88/320/EEC

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The limit test item concentration and control were analytically verified via HPLC-DAD after 0 hours (test start) and 72 hours (test end).
- Sampling method: Samples at test start were taken from additional replicates without algae. After 72 hours samples were taken directly from pooled test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1 mg/L was freshly prepared with dilution water.The stock solution was stirred for 48 hours (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). Centrifugation for 10 min at 10000 rpm was found to be insufficient.
- Eluate: Dilution water
- Differential loading: None, saturated solution corresponding to a nominal test item concentration of 1.00 mg/L.
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

min.: 21.5 max.: 22.0, mean value: 21.8

pH:

pH-value
Start; 0 hours End; 72 hours
Saturated solution prepared
with 1 mg test item/L 7.85 8.51
Control 7.91 8.12

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 103 cells/mL, Actual: mean 4910 cells/mL
- Control end cells density: 792206 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 69.2



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS
- Range finding study:
Results of the Range Finding Test (0-72 hours)

Nominal
test item concentration
[mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 -4 -22
10 -2 -8
1 -1 -5

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to the very poor solubility of the test item, quantitative analytical determination was precluded (below LOQ)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to the very poor solubility of the test item, quantitative analytical determination was precluded (below LOQ)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Due to the very poor solubility of the test item, quantitative analytical determination was precluded (below LOQ)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50 Values of the Reference Item
based on nominal concentrations (mg/L), (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Rate-related inhibition
ErC50 0.488 0.699 ± 0.471
95 % confidence interval 0.466 – 0.512
Yield inhibition
EyC50 0.291 0.318 ± 0.147
95 % confidence interval 0.274 – 0.328

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical
significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) was used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

NOEC, LOEC, EC - values of the test item (0-72 hours)

                   based on the saturated solution prepared with 1 mg test item/L

                  

	Rate-related Inhibition
NOEC	1.00
LOEC	> 1.00
ErC10	> 1.00
ErC20	> 1.00
ErC50	> 1.00
	Inhibition of Yield
NOEC	1.00
LOEC	> 1.00
EyC10	< 1.00
EyC20	> 1.00
EyC50	> 1.00

Cell Densities

			Replicate		Cell density [cells/mL]
		No.		0 hours		24 hours	48 hours	72 hours
Saturated solution prepared with 1 mg test item/L		1		4910		27542	151737	633314
		2		4910		23202	156456	627729
		3		4910		24627	125747	578816
		4		4910		24505	167816	862648
		5		4910		26049	151595	679317
		6		4910		27887	163422	626951
		Mean		4910		25635	152796	668129
Control		1		4910		27183	143295	641574
		2		4910		26059	153978	890710
		3		4910		28533	168486	920227
		4		4910		28232	159570	819489
		5		4910		28814	143119	754531
		6		4910		24262	140709	726707
		Mean		4910		27181	151526	792206

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
	No.	[d^-1]		[%]	[cells/mL]		[%]
Saturated solution prepared with 1 mg test item/L	1		1.62	4.26		628404	20.2
	2		1.62	4.44		622819	20.9
	3		1.59	6.03		573906	27.1
	4		1.72	-1.83		857738	-8.95
	5		1.64	2.88		674407	14.3
	6		1.62	4.46		622041	21.0
	Mean	(-)	1.64	3.37	(-)	663219	15.8
Control	1		1.62			636664
	2		1.73			885800
	3		1.74			915317
	4		1.71			814579
	5		1.68			749621
	6		1.67			721797
	Mean		1.69			787296

Section-by-Section and Average Specific Growth Rates of the Control Group
                   (0-72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.71	1.66	1.50	1.62	0.111	6.84	4.34
	2	1.67	1.78	1.76	1.73	0.0569	3.28
	3	1.76	1.78	1.70	1.74	0.0412	2.36
	4	1.75	1.73	1.64	1.71	0.0609	3.57
	5	1.77	1.60	1.66	1.68	0.0845	5.04
	6	1.60	1.76	1.64	1.67	0.0827	4.97
				Mean	1.69
				SD ±	0.05
				CV [%]	2.66

Validity criteria fulfilled:

yes

Conclusions:

In this study no inhibition on growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item. The NOEC for inhibition of specific growth rate and yield after 72 hours was 1.00 mg/L. All effect levels are given based on the nominal concentration of the test item.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from 2011-12-02 to 2011-12-09, with the definitive exposure phase from 2011-12-06 to 2011-12-09. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5 x 10³ cells/mL. Based on a preliminary range finding the saturated solution* was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

The saturated solution* and the control were analysed by HPLC-DAD after 0 h (fresh media) and 72 h (old media).

The concentration (fresh media, 0 h) of the test item in the freshly prepared saturated solution* and in the old media (72 h) was , therefore all effect values of the test item are given based on the saturated solution prepared with 1 mg/L.

Table 1:   NOEC, LOEC, EC - values of the test item (0-72 hours)

                   based on the saturated solution prepared with 1 mg test item/L

                  

	Rate-related Inhibition
NOEC	1.00
LOEC	> 1.00
ErC10	> 1.00
ErC20	> 1.00
ErC50	> 1.00
	Inhibition of Yield
NOEC	1.00
LOEC	> 1.00
EyC10	1.00
EyC20	> 1.00
EyC50	> 1.00

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2011-10-28 to 2011-11-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guidelines and compliant to GLP

Justification for type of information:

Justification for type of information: see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Acetolone Pigments – Nanoforms and Bulk Forms (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemikaliengesetzt and directive 88/320/EEC

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The limit test item concentration and control were analytically verified via HPLC-DAD after 0 hours (test start) and 72 hours (test end).
- Sampling method: Samples at test start were taken from additional replicates without algae. After 72 hours samples were taken directly from pooled test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 C° until analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1 mg/L was freshly prepared with dilution water. The stock solution was stirred for 48 hours (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). Centrifugation for 10 min at 10000 rpm was found to be insufficient.
- Eluate: Dilution water
- Differential loading: Saturated solution* corresponding to a nominal test item concentration of 1.00 mg/L, six replicates
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

min.: 21.5 max.: 22.5, mean value: 22.0

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.86 8.32
Control 7.92 8.72

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 4910 cells/mL
- Control end cells density: Mean 876660 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 69.2



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS
- Range finding study:
Results of the Range Finding Test (0-72 hours)

Nominal
test item concentration
[mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 -1 -3
10 2 11
1 3 14

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to very poor solubility of test item, concentrations in water were below LOQ of analytical method (0.1 mg/L)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to very poor solubility of test item, concentrations in water were below LOQ of analytical method (0.1 mg/L)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50 Values of the Reference Item
based on nominal concentrations (mg/L), (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Rate-related inhibition
ErC50 0.488 0.699 ± 0.471
95 % confidence interval 0.466 – 0.512
Yield inhibition
EyC50 0.291 0.318 ± 0.147
95 % confidence interval 0.274 – 0.328

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical
significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s Test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

NOEC, LOEC, EC - values of the test item (0-72 hours)

                   based on nominal concentration [mg/L]

                               

	Rate-related Inhibition
NOEC	1.00
LOEC	> 1.00
ErC10	> 1.00
ErC20	> 1.00
ErC50	> 1.00
	Inhibition of Yield
NOEC	1.00
LOEC	> 1.00
EyC10	> 1.00
EyC20	> 1.00
EyC50	> 1.00

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
1.00		1		4910		24018	175395	899328
		2		4910		24065	170213	829699
		3		4910		28137	174989	949731
		4		4910		22779	156551	815888
		5		4910		27027	172230	820796
		6		4910		26983	145096	824520
		Mean		4910		25502	165746	856660
Control		1		4910		26330	176776	968484
		2		4910		26404	174874	908298
		3		4910		26699	176620	887413
		4		4910		22674	191186	822996
		5		4910		25392	166953	756650
		6		4910		29583	169072	916118
		Mean		4910		26180	175914	876660

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.00	1		1.74	-0.55		894418	-2.60
	2		1.71	1.00		824789	5.39
	3		1.76	-1.61		944821	-8.38
	4		1.70	1.33		810978	6.97
	5		1.71	1.21		815886	6.41
	6		1.71	1.12		819610	5.98
	Mean	(-)	1.72	0.420	(-)	851750	2.30
Control	1		1.76			963574
	2		1.74			903388
	3		1.73			882503
	4		1.71			818086
	5		1.68			751740
	6		1.74			911208
	Mean		1.73			871750

Section-by-Section and Average Specific Growth Rates of the Control Group
                   (0 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.68	1.90	1.70	1.76	0.124	7.04	9.78
	2	1.68	1.89	1.65	1.74	0.131	7.55
	3	1.69	1.89	1.61	1.73	0.142	8.17
	4	1.53	2.13	1.46	1.71	0.370	21.6
	5	1.64	1.88	1.51	1.68	0.189	11.2
	6	1.80	1.74	1.69	1.74	0.053	3.04
				Mean	1.73
				SD ±	0.03
				CV [%]	1.70

Validity criteria fulfilled:

yes

Conclusions:

In this study no inhibition on growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item. The NOEC for inhibition of specific growth rate and yield after 72 hours was 1.00 mg/L. All effect levels are given based on the nominal concentration of the test item, which has been analytically verified by HPLC-DAD.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5 x 10³ cells/mL. Based on a preliminary range finding the saturated solution* was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

The saturated solution* and the control were analysed by HPLC-DAD after 0 h (fresh media) and 72 h (old media).

The concentration (fresh media, 0 h) of the test item in the freshly prepared saturated solution* and in the old media (72 h) was< LOQ. All effect values of the test item are given based on the nominal concentration.

Table 1:   NOEC, LOEC, EC - values of the test item (0-72 hours)

                   based on nominal concentration [mg/L]

                               

	Rate-related Inhibition
NOEC	1.00
LOEC	> 1.00
ErC10	> 1.00
ErC20	> 1.00
ErC50	> 1.00
	Inhibition of Yield
NOEC	1.00
LOEC	> 1.00
EyC10	> 1.00
EyC20	> 1.00
EyC50	> 1.00

Description of key information

In this study (PY120 Nanoform) no inhibition on growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item. The NOEC for inhibition of specific growth rate and yield after 72 hours was 1.00 mg/L. All effect levels are given based on the nominal concentration of the test item, which has been analytically verified by HPLC-DAD.

In this study (PY154 bulk) no inhibition on growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item. The NOEC for inhibition of specific growth rate and yield after 72 hours was 1.00 mg/L. All effect levels are given based on the nominal concentration of the test item.