2-[[4-(diethylamino)-2-methylphenyl]azo]-5-nitrobenzene-1,3-dicarbonitrile

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:

from 2017-10-16 to 2017-10-20, with the definitive exposure phase from 2017-10-17 to 2017-10-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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 (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-[2-[(2,6-dicyano-4-nitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

EC Number:

255-473-8

EC Name:

N-[2-[(2,6-dicyano-4-nitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

Cas Number:

41642-51-7

Molecular formula:

C20H19N7O3

IUPAC Name:

N-{2-[(2,6-dicyano-4-nitrophenyl)diazenyl]-5-(diethylamino)phenyl}acetamide

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The test concentration of Disperse Blue 165 was analytically verified via HPLC-DAD in the saturated solution, the dilutions and the control at the start of the exposure (0 hours). At the start of the exposure (0 hours), sampling was carried out after preparation of the loading levels. At test start the measured test item concentrations were below the LOD in all loading levels. At the end of the exposure (72 hours), no analytical monitoring was carried out, as all test loadings can be considered to be below the LOD in the aged media too.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the Saturated solution
A saturated solution with a nominal loading of 100 mg test item/L was prepared once 24 hours prior to the start of the exposure. An appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted into a glass bottle with an appropriate amount of dilution water. The saturated solution was stirred for 24 hours (1100 rpm, at 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 of the filtrate were discarded. The filtration was interrupted for 10 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. During filtration, the filter was always kept covered.
The solution was checked via laser beam (Tyndall effect), which was positive.

Test loading
All terms related to concentration level are given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations. The saturated solution and four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution. The test loadings are based on the results of a non-GLP preliminary range finding test.

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

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 23.0 mean value: 22.5

pH:

Nominal test item loading pH-values
[% of the saturated solution] Start; 0 hours End; 72 hours
100 7.76 8.11
50.0 7.80 8.18
25.0 7.85 8.62
12.5 7.90 8.92
6.25 7.94 8.98
Control 7.98 8.91

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 6839 cells/mL
- Control end cells density: Mean 1936653 cells/mL
- No. of vessels per concentration (replicates): 3
- 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 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064a2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 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: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 5911 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test at the saturated solution.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

12.5 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate und yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

25 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

40.8 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 38.3 - 43.2

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

24.7 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 20.8 - 29.9

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: The test media clear throughout the exposure period.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2017-10-10 to 2017-10-13. 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 0.535 0.745 ± 0.524
95 % confidence interval 0.523 – 0.547
Yield inhibition
EyC50 0.320 0.398 ± 0.291
95 % confidence interval 0.249 – 0.339
SD = Standard deviation

Reported statistics and error estimates:

EL-values and statistical analyses
EL-values of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEC, LOEC and analyses
The NOEC / LOEC was determined by calculation of statistical significance of growth rate and yield.
The following statistical tests were conducted:
Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01.
Levene’s test on variance homogeneity was done with a significance level of 0.01.
Monotonicity of Concentration/Response was assessed by trend analysis by contrasts (significance level: 0.05).
William’s Multiple Sequential t-test on significant differences was done with a significance level of 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.
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- ToxRat Professional, Version 3.2, TOXRAT SOLUTIONS GMBH

Any other information on results incl. tables

Cell Densities

Nominal test item loading		Replicate		Cell density [cells/mL]
[% of the saturated solution]		No.		0 hours		24 hours		48 hours		72 hours
100		1		6839		2810		LOQcell density		LOQcell density
		2		6839		3445		LOQcell density		2892
		3		6839		3041		LOQcell density		LOQcell density
		Mean		6839		3099		n.a.		n.a.
50.0		1		6839		5856		7094		40333
		2		6839		5651		5543		25741
		3		6839		4952		8905		48780
		Mean		6839		5486		7181		38285
25.0		1		6839		19898		140581		1179112
		2		6839		11287		67009		650032
		3		6839		19418		155129		1392534
		Mean		6839		16868		120906		1073893
12.5		1		6839		27599		228384		1765716
		2		6839		24764		190639		1582080
		3		6839		31730		273924		1924364
		Mean		6839		28031		230982		1757387
6.25		1		6839		30955		273526		2138371
		2		6839		35350		303945		2476324
		3		6839		32155		326440		2273787
		Mean		6839		32820		301304		2296161
Control		1		6839		32213		276359		1520693
		2		6839		31804		293353		2191859
		3		6839		34519		266960		1761912
		4		6839		34115		314631		2026130
		5		6839		34425		338004		1889252
		6		6839		33135		331707		2230072
		Mean		6839		33369		303502		1936653

 

LOQ_{cell density}  = Limit of quantification of the cell density (2736 cells/mL).             n.a.       = not applicable

 

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 loading	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[% of the saturated solution]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		n.a.	100		n.a.	100
	2		-0.287	100		-3947	100
	3		n.a.	100		n.a.	100
	Mean	(+)	n.a.	100	(+)	n.a.	100
50.0	1		0.592	69		33494	98
	2		0.442	76		18902	99
	3		0.655	65		41941	98
	Mean	(+)	0.563	70	(+)	31446	98
25.0	1		1.72	9		1172273	39
	2		1.52	19		643193	67
	3		1.77	6		1385695	28
	Mean	(+)	1.67	11	(+)	1067054	45
12.5	1		1.85	1		1758877	9
	2		1.82	3		1575241	18
	3		1.88	0		1917525	1
	Mean	(-)	1.85	2	(-)	1750548	9
6.25	1		1.92	-2		2131532	-10
	2		1.96	-5		2469485	-28
	3		1.94	-3		2266948	-17
	Mean	(-)	1.94	-3	(-)	2289322	-19
Control	1		1.80			1513854
	2		1.92			2185020
	3		1.85			1755073
	4		1.90			2019291
	5		1.87			1882413
	6		1.93			2223233
	Mean		1.88			1929814

n.a. = not applicable

 

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

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.55	2.15	1.71	1.80	0.311	17.3	16.9
	2	1.54	2.22	2.01	1.92	0.351	18.2
	3	1.62	2.05	1.89	1.85	0.216	11.7
	4	1.61	2.22	1.86	1.90	0.309	16.3
	5	1.62	2.28	1.72	1.87	0.359	19.2
	6	1.58	2.30	1.91	1.93	0.363	18.8
				Mean	1.88
				SD ±	0.05
				CV [%]	2.57

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Disperse Blue 165 was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item loadings): The EL50-values with 95% confidence intervals for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 40.8 (38.3 – 43.2) and 24.7 (20.8 – 29.9)% of the saturated solution, respectively. The NOEL-values for both inhibition of growth rate and yield after 72 hours were 12.5% of the saturated solution, respectively. All effect levels are far above the water solubility limit of the substance. The substance is not considered to be harmful to freshwater algae.

Executive summary:

The toxicity of Disperse Blue 165 to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016 C.3 from 2017-10-16 to 2017-10-20, with the definitive exposure phase from 2017-10-17 to 2017-10-20 at the test facility. The aim of the study was the determination of NOEL, LOEL, EL₁₀, EL₂₀- and EL₅₀-values of growth rate and yield over a period of 72 hours.

 

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A saturated solution with a nominal loading of 100 mg test item/L was prepared with dilution water 24±1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out. The saturated solution was stirred for 24 ±1hour (1100 rpm, at room temperature) with a magnetic stirrer. Undissolved particles were removed by membrane filtration. The following filtrate, i.e. the saturated solution was used in the test. The solution was checked via laser beam (Tyndall effect), which was positive.

 

The saturated solution and four dilution levels out of the saturated solution were tested in the definitive test in a geometrical series with a dilution factor of 2:6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution. Three replicates were tested for each test item concentration and six replicates for the control. The test media were clear throughout the test period. The environmental conditionswere within the acceptable limits.

The measured concentrations of the test item were all < LOD (0.00625 mg/L)at the start of the exposure (0 hours), therefore no analysis at the end of the exposure was necessary.

 

All effect values given are based on the nominal test item loadings of DisperseBlue 165.

NOEL, LOEL, EL_x-values and 95% Confidence Intervals ofDisperseBlue 165(0 - 72 hours)

              based on the nominal test item loadings

	Growth Rate Inhibition nominal test item loadings [% saturated solution]
NOEL	12.5
LOEL	25.0
ErL10	24.1 (21.3 – 27.4)
ErL20	29.1 (26.4 – 32.0)
ErL50	40.8 (38.3 – 43.2)
	Yield Inhibition nominal test item loadings [% saturated solution]
NOEL	12.5
LOEL	25.0
EyL10	14.1 (10.9 – 17.6)
EyL20	16.5 (13.1 - 20.0)
EyL50	24.7 (20.8 – 29.9)