Reaction mass of 4-[[4-(aminocarbonyl)phenyl]azo]-N-(2-ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide and 4-[[4-(aminocarbonyl)phenyl]azo]-3-hydroxy-N-(2-methoxyphenyl)naphthalene-2-carboxamide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

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).

Description of key information

Algae

PR210 

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.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

1 mg/L