N-(4-chlorophenyl)-2-[(4-methyl-2-nitrophenyl)azo]-3-oxobutyramide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed OECD study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-(4-chlorophenyl)-2-[(4-methyl-2-nitrophenyl)azo]-3-oxobutyramide

EC Number:

260-618-3

EC Name:

N-(4-chlorophenyl)-2-[(4-methyl-2-nitrophenyl)azo]-3-oxobutyramide

Cas Number:

57206-89-0

Molecular formula:

C17H15ClN4O4

IUPAC Name:

N-(4-chlorophenyl)-2-[(4-methyl-2-nitrophenyl)diazenyl]-3-oxobutanamide

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
Analytical evaluation of the saturated solution of N-(4-Chlorophenyl)-2-((4-methyl-2-nitrophenyl)azo)-3-oxobutyramide and the control was carried out via HPLC-DAD from freshly prepared media after 0 hours. Separate replicates for the test item analysis at the beginning of exposure were prepared without algae. The recovery rates of the test item at the start was below the SysQL. Therefore, no analytical evaluation after 72 h was carried out.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution
The saturated solution with a nominal loading of 1 mg/L (test item was weighed out) was prepared with demineralized water two days prior to the start of the exposure (0 h). The dispersion 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). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL filtrate were discarded. The filtration was interrupted for ca. 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. After the separation treatment the components of the dilution water were added to the saturated solution. The following filtrate, i.e. the saturated solution, was used in the test. During filtration the filter was always kept covered. 

Test concentration
The saturated solution was tested in a limit test.

Control
Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Test organism
Desmodesmus subspicatus CHODAT SAG 86.81
(formerly Scenedesmus subspicatus CHODAT)

Reason for the selection of the test system
According to the guideline Desmodesmus subspicatus CHODAT SAG 86.81 is suitable for this type of study.

Origin
Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen

Cultivation at test facility
Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE•m-2•s-1 for 24 h per day.

Culture medium
Nutrient medium Z according to LÜTTGE et al. (1994).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

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

pH:

pH-value
Start; 0 hours End; 72 hours
Saturated solution with
a nominal loading
of 1 mg/L 7.51 8.01
Control 7.93 8.11

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Saturated solution with a nominal loading of 1 mg/L

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 4847 cells/mL
- Control end cells density: Mean 834269 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: 74.0



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.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: 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):

The toxicity of potassium dichromate (SIGMA, batch number MKBQ9179V, purity 100.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Desmodesmus subspicatus was determined over a period of 72 hours from 2015-10-19 to 2015-10-22. For results see Table 8. The reference item toxicity is in the valid range following test facility SOPS.

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 0.685 0.671 ± 0.401
95 % confidence interval 0.603 - 0.794
Yield Inhibition
EyC50 0.362 0.320 ± 0.139
95 % confidence interval 0.328 - 0.398
SD = Standard deviation

Reported statistics and error estimates:

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

NOEC and LOEC values
NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As standard a t-test was used for NOEC/LOEC calculations. When running a t-test 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 tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

 
Software
The data for the tables in this report were computer generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
• Excel, MICROSOFT CORPORATION
• SigmaPlot, SPSS INC.

Any other information on results incl. tables

Cell Densities

 

	Replicate	Cell density [cells/mL]
	No.	0 hours	24 hours	48 hours	72 hours
Saturated solution with a nominal loading of 1 mg/L	1	4847	27514	155406	758864
	2	4847	27471	182441	819548
	3	4847	30304	156421	901303
	4	4847	29645	160899	780367
	5	4847	29212	183990	813752
	6	4847	25349	175870	796386
	Mean	4847	28249	169171	811703
Control	1	4847	25912	136202	760163
	2	4847	27206	161655	786428
	3	4847	28033	161914	917370
	4	4847	23117	141244	910202
	5	4847	25176	168808	826355
	6	4847	23021	137179	805093
	Mean	4847	25411	151167	834269

         

 

 

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		Inhibition of growth rate	Yield		Inhibition of yield
	No.	[d^-1]		[%]	[cells/mL]		[%]
Saturated solution with a nominal loading of 1 mg/L	1		1.68	2		754017	9
	2		1.71	0		814701	2
	3		1.74	-2		896456	-8
	4		1.69	1		775520	7
	5		1.71	0		808905	2
	6		1.70	1		791539	5
	Mean	(-)	1.71	1	(-)	806856	3
Control	1		1.69			755316
	2		1.70			781581
	3		1.75			912523
	4		1.75			905355
	5		1.71			821508
	6		1.70			800246
	Mean		1.72			829422

Section-by-Section and Average Specific Growth Ratesof 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	1.66	1.72	1.69	0.031	1.83	5.83
	2	1.73	1.78	1.58	1.70	0.103	6.07
	3	1.76	1.75	1.73	1.75	0.012	0.66
	4	1.56	1.81	1.86	1.75	0.161	9.20
	5	1.65	1.90	1.59	1.71	0.167	9.76
	6	1.56	1.79	1.77	1.70	0.127	7.44
				Mean	1.72
				SD ±	0.03
				CV [%]	1.51

CV= Coefficient of variation

SD= Standard deviation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study N-(4-Chlorophenyl)-2-((4-methyl-2-nitrophenyl)azo)-3-oxobutyramide was found not to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-value of the test item for both inhibition of growth rate and inhibition of yield after 72 hours was 1.00 mg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were both > 1.00 mg/L, respectively. All effect levels are given based on the saturated solution prepared with a nominal loading of 1 mg/L of N-(4-Chlorophenyl)-2-((4-methyl-2-nitrophenyl)azo)-3-oxobutyramide .

Executive summary:

The toxicity of N-(4-Chlorophenyl)-2-((4-methyl-2-nitrophenyl)azo)-3-oxobutyramide  to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) and Council Regulation (EC) No.761/2009/Method C.3 (2009) . 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 4847 cells/mL. A saturated solution with a nominal concentration of 1.00 mg/L was tested. Sixr eplicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of the test item N-(4-Chlorophenyl)-2-((4-methyl-2-nitrophenyl)azo)-3-oxobutyramide in the saturated solution and the control were analytically verified via HPLC-DAD at the start of exposure. The concentration of the saturated solution was < 0.1 mg/L (i.e. below the lowest calibration standard of the analytical method). F

All effect values of N-(4-Chlorophenyl)-2-((4-methyl-2-nitrophenyl)azo)-3-oxobutyramide are given based on the nominal concentration.

 

NOEC, LOEC, EC - values ofN-(4-Chlorophenyl)-2-((4-methyl-2-nitrophenyl)azo)-3-oxobutyramide (0-72 hours)

based on the saturated solution, prepared with 1 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