N-(5-chloro-2-methoxyphenyl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-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:

2013-01-04 to 2013-01-10

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:

GLP according to German Chemikaliengesetzt and Directive 88/320/EEC

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1 mg/L, 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).

- Eluate: Dilution water
- Differential loading: 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.

*) Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus CHODAT SAG 86.81
- 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 from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.

ACCLIMATION
- Culturing media and conditions (same as test or not): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not measured; the synthetic medium used as dilution water had a nominal hardness of 0.24 mmol Ca+Mg/L

Test temperature:

Mean: 22.5 °C, Min: 22.0 °C, Max: 23.0 °C

pH:

Nominal concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.67 8.29
Control 7.91 8.47

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Nominal limit concentration of test item used to prepare saturated solutions: 1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): sealed with 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: 4332 cells/mL
- Control end cells density: Mean 944265 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: Yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
According to the guideline (OECD)

Composition of Dilution water (OECD)

Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH-value 8.1  0.2
This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.


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



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



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

Nominal Test Item Concentration [mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
Saturated Solution 1% 8%

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

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

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.631 (CI 0.583 - 0.687) mg/L
Yield Inhibition: 0.256 (CI 0.247 - 0.265) mg/L

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.
EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (Limit test design).

Detection of statistically significant differences was considered negligible when inhibition values were ≤ 10 %. These findings were marked as “biologically not significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % are biologically significant.

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

                   based on nominal concentration [mg/L]

	Growth Rate 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 concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
1.00		1		4332		20789	146674	1030721
		2		4332		22044	152279	1037420
		3		4332		19751	155084	1141677
		4		4332		22082	164079	1099880
		5		4332		20887	148630	1047767
		6		4332		20251	167016	1099597
		Mean		4332		20967	155627	1076177
Control		1		4332		23780	173092	980307
		2		4332		23774	162544	938132
		3		4332		23107	156763	936434
		4		4332		23104	157116	988798
		5		4332		19956	151159	998768
		6		4332		20428	160701	823151
		Mean		4332		22358	160229	944265

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

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

Nominal concentration	Replicate	Growth rate		Growth rate inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.00	1		1.82	-1.67		1026389	-9.20
	2		1.83	-1.79		1033088	-9.91
	3		1.86	-3.57		1137345	-21.0
	4		1.85	-2.87		1095548	-16.6
	5		1.83	-1.97		1043435	-11.0
	6		1.85	-2.87		1095265	-16.5
	Mean	(+)^1	1.84	-2.46	(+)^1	1071845	-14.0
Control	1		1.81			975975
	2		1.79			933800
	3		1.79			932102
	4		1.81			984466
	5		1.81			994436
	6		1.75			818819
	Mean		1.79			939933

negative inhibitions = increase of growth - 1) Due to stimulation of growth

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	1.70	1.99	1.73	1.81	0.155	8.56	9.74
	2	1.70	1.92	1.75	1.79	0.115	6.42
	3	1.67	1.92	1.79	1.79	0.120	6.71
	4	1.67	1.92	1.84	1.81	0.124	6.86
	5	1.53	2.03	1.89	1.81	0.257	14. 2
	6	1.55	2.06	1.63	1.75	0.275	15.7
				Mean	1.80
				SD ±	0.02
				CV [%]	1.33

Validity criteria fulfilled:

yes

Conclusions:

In this study no inhibition of 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.
The test item caused a slight stimulation of the algae growth. All effect levels are 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 compliant to GLP (reliability category 1). 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 4332 cells/mL. A saturated solution* with a nominal concentration of 1 mg/L was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

No analytical determination was carried out since no suitable method for determination of the test item could be established: The test item is highly insoluble in water. All effect values of the test item are given based on the nominal concentration.

*) Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6.

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

                   based on nominal concentration [mg/L]

	Growth Rate 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:

2012-11-30 to 2012-12-06

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)

Analytical monitoring:

no

Details on sampling:

No analytical evaluation of the limit test concentration and the control was carried out since no suitable method for determination of the test item could be established due to the test item being highly insoluble in water.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Stock solution: 1 mg/L, freshly prepared with dilution water.
- Method: The stock solution was stirred for 48 hours (1100 rpm, room temperature) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL).
- Eluate: Dilution water
- Differential loading: Saturated solution corresponding to a nominal test item concentration of 1.00 mg/L. Number of replicates: 6
- 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:

0.24 mmol Ca+Mg/l

Test temperature:

min.: 21.5 max.: 23.5, mean value: 22.5

pH:

Nominal concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.97 8.19
Control 8.02 8.30

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

No analytical evaluation of the limit test concentration and the control was carried out since no suitable method for determination of the test item could be established. The saturated solution prepared at and corresponding to a nominal test item concentration of 1.00 mg/L was tested in 6 replicates.

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 4332 cells/mL
- Control end cells density: Mean 555919 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:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH 8.1 +/- 0.2




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: 73.3



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: Yes
Results of the Range Finding Test (0-72 hours)
Saturated solution
1 mg/L [%] Growth Rate Inhibition [%] Yield Inhibition [%]
100 -4 -24
10 -2 -10

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Due to the test item being highly insoluble in water, saturated solutions were tested prepared at the nominal limit concentration of 1 mg/L, and no suitable method for the determination of the test item could be established.
- 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)
Growth Rate Inhibition
ErC50 0.631 0.682 ± 0.452
95 % confidence interval 0.583 - 0.687
Yield Inhibition
EyC50 0.256 0.314 ± 0.144
95 % confidence interval 0.247 - 0.265

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.

Cell Densities

	Nominal concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
1.00		1		4332		13914	87045	562519
		2		4332		13792	87624	671524
		3		4332		10423	78503	677122
		4		4332		11804	76799	640011
		5		4332		12329	100012	657152
		6		4332		12257	92675	760339
		Mean		4332		12420	87110	661445
Control		1		4332		13071	92750	555914
		2		4332		9474	79510	650925
		3		4332		11530	88158	720869
		4		4332		11335	77490	410426
		5		4332		10364	79774	493234
		6		4332		14279	75880	504147
		Mean		4332		11676	82260	555919

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

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

Nominal concentration	Replicate	Growth rate		Growth rate inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.00	1		1.62	-0.60		558187	-1.20
	2		1.68	-4.26		667192	-21.0
	3		1.68	-4.43		672790	-22.0
	4		1.67	-3.27		635679	-15.3
	5		1.67	-3.81		652820	-18.4
	6		1.72	-6.83		756007	-37.1
	Mean	(-)	1.68	-3.87	(-)	657113	-19.1
Control	1		1.62			551582
	2		1.67			646593
	3		1.71			716537
	4		1.52			406094
	5		1.58			488902
	6		1.59			499815
	Mean		1.61			551587

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	1.10	1.96	1.79	1.62	0.453	28.0	34.3
	2	0.783	2.13	2.10	1.67	0.769	46.0
	3	0.979	2.03	2.10	1.71	0.630	36.9
	4	0.962	1.92	1.67	1.52	0.498	32.8
	5	0.872	2.04	1.82	1.58	0.621	39.4
	6	1.19	1.67	1.89	1.59	0.358	22.6
				Mean	1.61
				SD ±	0.07
				CV [%]	4.20

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.
The test item caused a slight stimulation of the algae growth. 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 and compliant to GLP (reliability category 1). 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 4332 cells/mL. Due to the test item being highly insoluble in water a saturated solution* with a nominal concentration of 1 mg/L was tested and no analytical determination was carried out since no suitable method for the determination of the test item could be established. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits. All effect values are given based on the nominal concentration of the test item.

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

                   based on nominal concentration [mg/L]

	Growth Rate 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

*) Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000).

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 13, 2000 to April 27, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guideline and compliant to GLP, well documented translation of original report (in Japanese)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

yes

Remarks:

Deviations are noted in the field 'Principles of method if other than guideline'.

Principles of method if other than guideline:

Deviating from the guideline, growth rate related inhibition of growth was determined for the interval of 24 to 72 hours only, instead of zero to 72 hours, as recommended by the guideline. From evaluating the raw data, this deviation leads to a slight overestimation of growth rate related inhibition and therefore does not put into question the reliability of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

The study was conducted in accordance with "Eco-toxicity test Standards" by Japanese Ministry of the Environment

Analytical monitoring:

yes

Details on sampling:

At the initiation of exposure, the samplings were made from the remaining test solutions for all groups after distributing to test vessels. At the termination of exposure, the sampling of about the same volume of test solutions was made from three test vessels of each test group for analysis. After sampling, algae were removed by centrifugation (2500 rpm, 10 minutes), the supernatants were analyzed by HPLC.

Vehicle:

yes

Details on test solutions:

After weighing of a necessary amount of test substance using an electronic balance, the same amount of hydrogenated castor oil (HCO-40) was added to the test substance as co-solvent and mixed well. Then, the solution was made up to the volume with culturing medium to prepare the stock solution (100 mg/L). After a necessary aliquot was weighed and sampled from the stock solution for the preparation of each test concentration, the shortfall of co-solvent was added to each solution to adjust the constant concentration of co-solvent (100 mg/L) in the test solution and made up with
medium. In the preparation of test solutions, the amount was considered to cover the necessary solutions for pH measurement and analytical sampling, since the sampling volume for analytical samples at the initiation of the exposure was expected to be more than 5% of test solution volume. The stock solution was used as the test solution of 100 mgIL in the test as it is.
The prepared test solutions showed strong red color due to test substance original in proportion to the test concentration but no visible precipitate was observed. The solution of solvent control was observed to be clear and colorless without visible undissolved test substance.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Scientific name: Selenastrum capricornutum
- Supplier: American Type Culture Collection
- Strain No.: ATCC22662 strain
- Date of receipt: August 1, 1996
- Maintenance after received: Aseptically sub-cultured using agar slant medium.
- Pre-cultivation period (definitive test): January 19, 2001 - January 22, 2001. During this period, exponential growth of algae was observed (the
environmental conditions were the same as in the test).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test temperature:

23 deg C for all test vessels

pH:

7.5 to 7.9 for control and treatment groups during the 72 hours of incubation.

Nominal and measured concentrations:

The following nominal test item concentrations were tested (mg/L):
0.23, 1.0, 3.2, 10, 32, 100;
The measured concentrations of the test substance in the test solution were 94 - 100% and 69 - 96% of the nominal at the initiation of exposure and
termination, respectively. The concentration of test substance in the control and solvent control were less than quantification limit (0.06 mg/L) at the initiation and termination of exposure.
Please see table under section "Any other information on results including tables" for details on analytical results.

Details on test conditions:

- Type of exposure: Static, shaking culture (100 rpm)
- Volume of test solution: 100 mL (OECD medium) / vessel
- Replicates: 3 vessels per test group
- Initial cell concentration: 1 x 10^4 cells/mL
- Lighting: Continuous lighting of 4000 - 5000 lx (within +/- 20%, near the liquid level in flask)
- In both of the pre-cultivation and the study, OECD medium recommended by OECD Guideline was used with sterilization and filtration before use.
- Test vessel: 500 mL glass Erlenmeyer flask (air-permeable silicon stopper)
- Algal culturing apparatus: AGP-50RL (Daikin Plant Co., Ltd.; continuous shaking culture. Able to maintain constant lighting intensity during the culturing)
- Hemocytometer: Burker-Turk
- Particle counter: Coulter Multisizer II (Beckman Coulter, Inc.)
- Electrolyte solution for particle counter: ISOTON II (Beckman Coulter, Inc.)
- Illuminometer: SPI-6A (Topcon Corporation)

- Selection of test concentration:
A range-finding test was conducted at three concentration levels of 1.0 and 10 mg/L, and 100 mg/L (which was the maximum attainable homogeneous dispersion concentration using the co-solvent at 100 mg/L). At the termination (72-hr) of exposure, growth inhibitions (by comparison of areas under the growth curve) were 5.1, ca. 60 and ca. 81% at 1.0, 10 and 100 mg/L treatment level, respectively.
Based on these results, the nominal test concentration in the definitive test was fixed to be performed at six concentration levels of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (common ratio: 3.2). As reference, a control group with dilution water only as well as a solvent control group (with solvent concentration of 100 mg/L identical to the treatment groups) were allocated in the study.

- Test operation procedure:
The number of cells of pre-cultivated algae was counted with a particle counter and an aliquot of pre-cultivated solution was added to each test solution to make 1 x 10^4 cels/mL of cell concentration. After mixing well, 10 mL of the solution, each was transferred into three test vessels per group.
The test was started by placing each of the test vessels in a culturing apparatus maintained at 23 +/- 2 deg C. After 24, 48 and 72 hours of exposure, cell concentrations in all three vessels of all groups were measured. A part of test solution was sampled from each test vessel and the cell concentrations were measured with a hemocytometer.
The pH at the initiation of exposure was measured for the remaining test solutions after distributing into test vessels. At the termination of exposure, pH was measured for one vessel (No. 1) out of three vessels in each test group, since difference in growth rate of each test group were within double. The temperature, lighting intensity and rotation speed in algal culturing apparatus were measured once a day during the exposure period.

Growth curve
The growth curve was drawn by plotting the mean cell concentration of each test group against the time.
Calculation of growth inhibition concentration

Growth inhibition rates were calculated in accordance with the methods described below (area method and growth rate method).
1) Growth inhibition rates (IA) by comparison of areas under the growth curve (Area-based method)
Area under the growth curve was calculated using the following equitation:
A = [(N_1 - N_0)/2]*t_1 + [(N_1 + N_2 - 2*N_0)/2]*(t_2 - t_1) + ... + [(N_n-1 + N_n - 2*N_0)/2]*(t_n - t_n-1)
whith
N_0: Nominal cell concentration at the initiation of exposure (cells/mL)
N_i: Measured cell concentration at t_i (cells/mL)
N_n: Measured cell concentration at to (cells/mL)
t_1: Time of first measurement of cell concentration after initiation of exposure
t_n: Time of nth measurement of cell concentration after initiation of exposure
Calculation of inhibition based on area under the growth curve [%]:
I_A = [(A_c - A_t)/A_c]*100
where A_c: Area under the growth curve for solvent control; A_t: Area under the growth curve for the treatment group.
Calculation of growth rate based inhbition was performed according to formulas given in OECD 201.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

corresponding to 97 mg/L gemetric mean measured concentration

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

corresponding to 2.85 mg/L gemetric mean measured concentration

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Test item concentration analytically verified

Basis for effect:

biomass

Remarks:

determined from area under the growth curve

Remarks on result:

other: 95%CI: 14-20

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

corresponding to 0.92 mg/L gemetric mean measured concentration

Basis for effect:

biomass

Remarks:

determined from area under the growth curve

Details on results:

Based on results of the preliminary verification for the difference of means of growth in two-sample (Student's t-test) between control and solvent control test group, the solvent control vales were used for statistical analysis, since the growth in solvent control group was increased compared to control group, significantly.
During 72 hours of incubation, the cell concentrations in the control and the solvent control group increased to 305 and 331 fold on average, respectively.
It was observed that the growth in the 0.32 mg/L test group was almost the same as that in the solvent control group. However, in the other test groups, decreased growth was observed depending upon the test concentration. In the comparison of 24-48 hours growth rate, 40% of inhibition was observed at the highest attainable concentration (100 mg/L) compared to solvent control. In case of growth rate during 24 - 72 hours, statistically significat inhibition between 14 and 34% was observed in the test groups exposed to 10 mg/L and above.
The prepared test solutions showed strong red color in proportion to test item concentration, but no visible precipitate was observed. The solution of solvent control was observed to be clear and colorless.

Results with reference substance (positive control):

The 50% algal growth inhibition concentration (EbC50) at 72 hr with the reference substance (potassium dichromate, guaranteed grade, Lot No. ACQ2610, Wako Pure Chemical Industries) was 0.50 mg/L, which was comparable to the historical EbC50 of 0.42 - 0.50 mg/L (n=6) obtained at the testing facility since August, 1996.

Reported statistics and error estimates:

The EC50 was determined by the Logit method (biomass based value only).
The no-observed-effect concentration (NOEC) was determined to be the highest test concentration which had no significant difference (a = 0.05) compared with the solvent control group using statistical analysis (homogeneity of variance (Bartlett), one-way analysis of variance and multiple comparison analysis (Dunnett).

Growth rates and rate related inhibition of growth: Selenastrum capricornutum during 72 hour exposure.

Nominal	Vessel	Growth rate per day
Concentr.	No.				Mean	SD	CV		% Inhibition
mg/L		0 to 24 h	24 to 48 h	48 to 72 h	0 to 72 h			24 to 72 h	24 to 72 h
	1	2.0229	1.8014	1.8260	1.8834	0.1214	6.4448	1.8137
	2	2.1725	1.7111	1.8954	1.9263	0.2322	12.0549	1.8033
Control	3	2.2690	1.6430	1.8124	1.9081	0.3238	16.9703	1.7277
	Average	2.1599	1.7141	1.8453	1.9064	Mean CV	11.8233	1.7797
	SD			SD	0.0215
				CV	1.1295
	1	1.8976	2.0381	1.8671	1.9343	0.0912	4.7150	1.9526
	2	2.0516	1.9300	1.8446	1.9421	0.1040	5.3545	1.8873
Solvent Control	3	1.9459	2.0541	1.7741	1.9247	0.1412	7.3359	1.9141
	Average	1.9671	2.0057	1.8285	1.9338	Mean CV	5.8018	1.9180
	SD			SD	0.0087
				CV	0.4492
	1	2.0229	2.0025	1.7193	1.9149			1.8609
	2	2.2332	1.6748	1.8806	1.9295			1.7777
0.32	3	1.9459	2.0812	1.7851	1.9374			1.9332
	Average	2.0748	1.9135	1.7939	1.9274			1.8572	3.2
	SD
	1	2.2214	1.7163	1.9068	1.9481			1.8115
	2	2.0069	1.9858	1.5776	1.8568			1.7817
1	3	1.8810	2.1263	1.5689	1.8587			1.8476
	Average	2.0464	1.9333	1.6924	1.8907			1.8136	5.4
	SD
	1	2.0373	2.1058	1.4679	1.8704			1.7869
	2	1.9459	1.8585	1.5977	1.8007			1.7281
3.2	3	1.9769	1.8733	1.6837	1.8446			1.7785
	Average	1.9874	1.9567	1.5752	1.8398			1.7645	8.0
	SD
	1	2.1199	1.5256	1.3051	1.6502			1.4153
	2	1.7156	1.9454	1.4487	1.7032			1.6971
10	3	1.8810	1.6857	1.4543	1.6737			1.5700
	Average	1.9194	1.7059	1.4040	1.6764			1.5608	18.6**
	SD
	1	1.4907	2.0119	1.2718	1.5915			1.6419
	2	1.4907	1.8903	1.4117	1.5976			1.6510
32	3	1.4398	1.9171	1.3661	1.5743			1.6416
	Average	1.4740	1.9415	1.3482	1.5879			1.6448	14.2**
	SD
	1	1.4398	1.1174	1.4206	1.3259			1.2690
	2	1.4398	1.1328	1.3253	1.2993			1.2290
100	3	1.2698	1.3905	1.2377	1.2993			1.3141
	Average	1.3863	1.2114	1.3275	1.3084			1.2707	33.7**
	SD							1.8137

% Inhibition is related to solvent control, as growth was increased compared to control.

**: Significant difference from solvent control (Dunnett´s Test, alpha = 0.01)

Solvent control: HCO-40 (100 mg/L)

Nominal and measured test item concentrations:

Nominal		Measured Concentration (mg/L)
Concentration (mg/L)	0 Hour	Percent of Nominal	72 Hours	Percent of Nominal
Control	<0.06	--	<0.06	--
Solvent Control	<0.06	--	<0.06	--
0.32	0.32	100	0.22	69
1.0	0.99	99	0.86	86
3.2	3.0	94	2.7	84
10	9.8	98	7.2	72
32	30	94	29	91
100	98	98	96	96

Validity criteria fulfilled:

yes

Conclusions:

In a reliable study (reliability category 1) on green alga Selenastrum capricornutum growth inhibition (static, 72 hours, analytical verification of test item concentration) performed according to GLP the following growth rate related (no)effect concentrations were determined for the test item:
EC50 (72 hours) > 100 mg/L (97 mg/L geometric mean measured concentration);
NOEC (72 hours) = 3.2 mg/L (2.85 mg/L geometric mean measured concentration).
These values were determined in relation to the solvent control (100 mg/L HCO-40, equal concentration to treatment groups), as growth was significantly enhanced compared to the control without solvent.
Tested concentrations were far above water solubility of the test item (11.8 µg/L) and were achieved by use of hydrogenated castor oil (HCO-40) at equal weight as co-solvent. The strong red coloring of test solutions might have resulted in light absorption causing reduced growth of algae due to reduced effective illumination strength.
Thus, observed effects are to be regarded as not relevant because they are
1) most probably not due to intrinsic toxicity of the test item and
2) far above the true water solubility of the test item (NOEC is a factor of 270 above water solubility).
Thus, the NOEC is not appropriate to derive a PNEC, as light absorption due to the coloring of the test item is not relevant in the range of actual water solubility.
The test item is to be regarded as non-toxic to freshwater green algae.

Executive summary:

In a reliable study (reliability category 1) on green alga Selenastrum capricornutum (new name: Pseudokirchneriella subcapitata) growth inhibition (static, 72 hours, analytical verification of test item concentration) performed according to GLP the following growth rate related (no)effect concentrations were determined for the test item:

EC50 (72 hours) > 100 mg/L (97 mg/L geometric mean measured concentration);

NOEC (72 hours) = 3.2 mg/L (2.85 mg/L geometric mean measured concentration).

These values were determined in relation to the solvent control (100 mg/L HCO-40, equal concentration to treatment groups), as growth was significantly enhanced compared to the control without solvent.

Tested concentrations were far above water solubility of the test item (11.8 µg/L) and were achieved by use of hydrogenated castor oil (HCO-40) at equal weight as co-solvent. The strong red coloring of test solutions might have resulted in light absorption causing reduced growth of algae due to reduced effective illumination strength.

Thus, observed effects are to be regarded as not relevant because they are

1) most probably not due to intrinsic toxicity of the test item and

2) far above the true water solubility of the test item (NOEC is a factor of 270 above water solubility).

Thus, the NOEC is not appropriate to derive a PNEC, as light absorption due to the coloring of the test item is not relevant in the range of actual water solubility.

The test item is to be regarded as non-toxic to freshwater green algae.

All validity criteria of the guideline are fulfilled: The average biomass increase in the control culture ant the solvent control was 305 and 331 -fold, respectively. The mean coefficient of variation for section-by-section specific growth rates in control cultures were below 35% (11.8 and 5.8 % for control and solvent control, respectively) and the coefficient of variation of average specific growth rate of replicate control cultures was below 7 % (1.13 and 0.45 % for control and solvent control, respectively).

The robust study summary is based on the translation of the original study report performed by the same company involved with performance of the test (Sumika TechnoService Corporation, Japan).

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-01-08 to 2010-01-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study meets requirements of OECD Guideline 211 and GLP requirements with no deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

No analytical determination of the test item was carried out due to the low water solubility of the test item.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution* was prepared. A dispersion of 1 mg/L prepared in dilution water was shaken with 20 rpm for 48 h at ambient temperature and filtrated with 0.45 µm RC filter.
- Eluate: Dilution water
- Differential loading: The saturated solution was tested as limit concentration.
- 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 from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

The test medium had a nominal hardness of 0.24 mmol CaMg/L

Test temperature:

Mean: 21.5 °C, Min: 21 °C, Max: 22 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
Saturated solution (1 mg/L) 8.04 8.35
Control 8.13 8.34

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

See above

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- 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: 5074 cells/mL
- Control end cells density: Mean 636336 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


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



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Spacing factor for test concentrations: None, limit test
- Range finding study:
Results of the Range Finding Test (0-72 h)

Saturated solution [mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
Filtration of the saturated solution
20 - 3 - 19
1 - 3 - 19

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 other: mg/L (saturated solution)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

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:
Rate-related inhibition: 0.60 (CI 0.545 - 0.653) mg/L
Yield Inhibition: 0.28 (CI 0.266 - 0.299) mg/L

Reported statistics and error estimates:

The NOEC and LOEC were determined by calculation of
statistical analyses statistical significance of growth rates and yield.
t-test was carried out for the determination of statistically significant differences compared to control replicates. When running a t-test a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. The a-value was a=0.05. Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. These findings were marked biological not significant.

NOEC, LOEC and EC - values and 95 % Confidence Intervals of the test item (0-72 h)

                   based on nominal test item concentration [mg/L]

                               

	Rate-related Inhibition
NOEC	Saturated solution (1 mg/L)
LOEC	> Saturated solution (1 mg/L)
ErC10	> Saturated solution (1 mg/L)
ErC20	> Saturated solution (1 mg/L)
ErC50	> Saturated solution (1 mg/L)
	Inhibition of Yield
NOEC	Saturated solution (1 mg/L)
LOEC	> Saturated solution (1 mg/L)
EyC10	> Saturated solution (1 mg/L)
EyC20	> Saturated solution (1 mg/L)
EyC50	> Saturated solution (1 mg/L)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
Saturated solution              (1 mg/L)		1		5074		19924	172088	811286
		2		5074		22729	170534	778264
		3		5074		24729	147336	721561
		4		5074		22023	140071	772979
		5		5074		23323	172006	761032
		6		5074		21941	177028	809346
		Mean		5074		22445	163177	775745
Control		1		5074		22589	146618	743013
		2		5074		22778	126409	607983
		3		5074		21527	162481	677384
		4		5074		19227	151563	600533
		5		5074		25234	125864	562982
		6		5074		23782	161637	626122
		Mean		5074		22523	145762	636336

Evaluation after 72 h

              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]		[%]
Saturated solution              (1 mg/L)	1		1.69	-5.12		806212	-27.7
	2		1.68	-4.26		773190	-22.5
	3		1.65	-2.69		716487	-13.5
	4		1.68	-4.11		767905	-21.7
	5		1.67	-3.79		755958	-19.8
	6		1.69	-5.07		804272	-27.4
	Mean	(+)*	1.68	-4.17	(+)*	770671	-22.1
Control	1		1.66			737939
	2		1.60			602909
	3		1.63			672310
	4		1.59			595459
	5		1.57			557908
	6		1.61			621048
	Mean		1.61			631262

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

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 h	24 - 48 h	48 - 72 h
Control	1	1.49	1.87	1.62	1.66	0.192	11.5	14.4
	2	1.50	1.71	1.57	1.60	0.108	6.78
	3	1.45	2.02	1.43	1.63	0.338	20.7
	4	1.33	2.07	1.38	1.59	0.411	25.8
	5	1.60	1.61	1.50	1.57	0.062	3.95
	6	1.55	1.92	1.35	1.61	0.286	17.8
				Mean	1.61
				± SD	0.03
				CV [%]	2.04

Validity criteria fulfilled:

yes

Conclusions:

In this study the test item was found not to inhibit the freshwater green algae Desmodesmus subspicatus after 72 h at the saturated solution (prepared with 1 mg/L). All effect levels are given based on the nominal concentration of Pigment Red 112.

Executive summary:

The toxicity of Pigment Red 112 (batch number 2468) to the unicellular freshwater green algae Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from January 08 to 14, 2010 with the definitive exposure phase from January 11 to 14, 2010. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

The study was conducted under static conditions with an initial cell density of approximately  2 – 5 x 10³cells/mL. Based on a preliminary range finding test, a limit test with a saturated solution*  was carried out. The saturated solution was prepared out of the dispersion of 1 mg/L. Six replicates were tested for the limit concentration and control. Environmental conditions were determined to be within the acceptable limits.

All effect values are given based on the nominal concentration of the test item.

NOEC, LOEC and EC - values and 95 % Confidence Intervals of the tets item (0-72 h)

                   based on nominal test item concentration [mg/L]

                               

	Rate-related Inhibition
NOEC	Saturated solution (1 mg/L)
LOEC	> Saturated solution (1 mg/L)
ErC10	> Saturated solution (1 mg/L)
ErC20	> Saturated solution (1 mg/L)
ErC50	> Saturated solution (1 mg/L)
	Inhibition of Yield
NOEC	Saturated solution (1 mg/L)
LOEC	> Saturated solution (1 mg/L)
EyC10	> Saturated solution (1 mg/L)
EyC20	> Saturated solution (1 mg/L)
EyC50	> Saturated solution (1 mg/L)

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Refer to the analogue approach provided in the CSR.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: results are based on a saturated solution

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

corresponding to 97 mg/L geometric mean measured

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

Sumika, 2001

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

Noack, 2012

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

Noack, 2013

Description of key information

NOEC / EC₁₀(72 hours) for freshwater algae > solubility limit

Additional information

All four available tests were performed according to OECD 201 and compliant to GLP, meeting the validity criteria of the guideline. 

Pigment Red 22 was tested on Selenastrum capricornutum (new name: Pseudokirchneriella subcapitata) using a pigment dispersion. This allowed for testing concentrations (0.23 to 100 mg/L) far above the solubility limit in water and avoiding at the same time formation of (visible) precipitate (solvent control included, all treatment groups were tested with the same concentration of dispersant HCO-40). Test item concentrations were analytically verified. With relation to growth rate, a NOEC (72 hours) of 3.2 mg/L was determined. Strong red coloring was observed depending on test item concentration. An observed inhibition of growth rate at 100 mg/L after 72 hours of approximately 34% is thus most probably caused by light absorption due to the strong coloring, causing reduced growth of algae due to reduced effective illumination strength. As even the NOEC is a factor of 270 above water solubility (11.8 µg/L) and effects above this concentration are most probably caused by light absorption and not toxicity, this value is not appropriate for derivation of a PNEC. Rather, Pigment Red 22 must be regarded as non-toxic to freshwater green algae. 

This is in agreement with the testing results for Pigments Red 112, Red 210 and Red 2 on Desmodesmus subspicatus, where saturated solutions (water accommodated fractions) were tested. No toxicity was observed for all of these pigments at nominal concentrations well above their respective solubility limits. The very poor solubility of the test items (9.8, 34 and 5.4 µg/L, respectively) precluded the analytical quantification of the test items in the saturated solution (below limit of quantification; 1 mg/L nominal test item concentration). At the same time in these cases, no coloration of test item solutions was observed. Therefore, in contrast to the study on Pigment Red 22 where an intensely colored dispersion was tested and growth reduction due to reduced effective illumination strength was observed at high concentration, this was not the case in the algae studies on Pigments Red 112, Red 210 and Red 2 using saturated solutions.  

Therefore, Naphtol-AS Pigments should be regarded as acutely and chronically non-toxic to aquatic plants / algae.