2-[(4-methoxy-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

effects on growth of green algae

Data waiving:

other justification

Justification for data waiving:

other:

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-04-20 to 2012-04-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg/L was freshly prepared with dilution water. The stock solution was stirred for 72 hours (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by centrifugation for 2 x 10 min at 10000 rpm in plastic centrifuge tubes.
- Eluate: Dilution water
- Differential loading: None, saturated soklution corresponding to a nominal test item concentration of 100 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.: 21.0 max.: 21.5, mean value: 21.3

pH:

pH-value
Start; 0 hours End; 72 hours
Saturated solution
prepared with 100 mg test item/L 7.91 8.01
Control 7.90 8.02

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

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: 979354 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:
No

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 other: mg/L (saturated solution)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

& yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 other: mg/L (saturated solution)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

& yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

& 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
Current Study Valid Range (average ± 3 x SD)
Rate-related inhibition
ErC50 0.540 0.686 ± 0.470
95 % confidence interval 0.528 - 0.551
Yield inhibition
EyC50 0.324 0.319 ± 0.140
95 % confidence interval 0.304 - 0.347
SD = Standard deviation

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 100 mg test item/L

                               

	Rate-related Inhibition
NOEC	100
LOEC	> 100
ErC10	> 100
ErC20	> 100
ErC50	> 100
	Inhibition of Yield
NOEC	100
LOEC	> 100
EyC10	> 100
EyC20	> 100
EyC50	> 100

Cell Densities

			Replicate		Cell density [cells/mL]
		No.		0 hours		24 hours	48 hours	72 hours
Saturated solution prepared with 100 mg test item/L		1		4910		29190	195702	1088814
		2		4910		24400	189406	1131566
		3		4910		30896	202993	1133022
		4		4910		29478	198944	945086
		5		4910		26465	178127	872174
		6		4910		25798	180605	1132852
		Mean		4910		27705	190963	1050586
Control		1		4910		26117	161764	935439
		2		4910		22278	159597	905211
		3		4910		25477	191728	1191041
		4		4910		23781	157519	880365
		5		4910		28462	175087	1018981
		6		4910		25138	152915	945086
		Mean		4910		25209	166435	979354

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 100 mg test item/L	1		1.80	-2.10		1083904	-11.2
	2		1.81	-2.83		1126656	-15.6
	3		1.81	-2.85		1128112	-15.8
	4		1.75	0.57		940176	3.52
	5		1.73	2.09		867264	11.0
	6		1.81	-2.85		1127942	-15.8
	Mean	(-)	1.79	-1.33	(-)	1045676	-7.31
Control	1		1.75			930529
	2		1.74			900301
	3		1.83			1186131
	4		1.73			875455
	5		1.78			1014071
	6		1.75			940176
	Mean		1.76			974444

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	1.67	1.82	1.76	1.75	0.0762	4.36	7.42
	2	1.51	1.97	1.74	1.74	0.228	13.1
	3	1.65	2.02	1.83	1.83	0.186	10.2
	4	1.58	1.89	1.72	1.73	0.157	9.06
	5	1.76	1.82	1.76	1.78	0.0332	1.87
	6	1.63	1.81	1.82	1.75	0.104	5.96
				Mean	1.76
				SD ±	0.04
				CV [%]	2.08

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 100 mg/L. All effect levels are given based on the saturated solution prepared with nominal 100 mg test item/L. As the test item is highly insoluble in water, no suitable method for determination of the test item could be established.

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 4910 cells/mL. A saturated solution with a nominal concentration of 100 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 the test item is highly insoluble in water and no suitable method for determination of the test item could be established. All effect levels are given based on the saturated solution prepared with nominal 100 mg test item/L.

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

                   based on the saturated solution prepared with 100 mg test item/L

                               

	Rate-related Inhibition
NOEC	100
LOEC	> 100
ErC10	> 100
ErC20	> 100
ErC50	> 100
	Inhibition of Yield
NOEC	100
LOEC	> 100
EyC10	> 100
EyC20	> 100
EyC50	> 100

Description of key information

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 100 mg/L. All effect levels are given based on the saturated solution prepared with nominal 100 mg test item/L. As the test item is highly insoluble in water, no suitable method for determination of the test item could be established.