N-[2-[(2,6-dicyano-p-tolyl)azo]-5-(diethylamino)phenyl]methanesulphonamide

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 2017-11-12 to 2017-11-16, with the definitive exposure phase from 2017-11-13 to 2017-11-16

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-p-tolyl)azo]-5-(diethylamino)phenyl]methanesulphonamide

EC Number:

269-887-1

EC Name:

N-[2-[(2,6-dicyano-p-tolyl)azo]-5-(diethylamino)phenyl]methanesulphonamide

Cas Number:

68385-96-6

Molecular formula:

C20H22N6O2S

IUPAC Name:

N-[2-[(2,6-dicyano-p-tolyl)azo]-5-(diethylamino)phenyl]methanesulphonamide

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 Red 343 (Ethyl) 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 (with algae). At test start the measured test item concentrations were below the LCL in all loading levels. Therefore, no analytical monitoring was carried out, at the end of the exposure (72 hours).

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the Saturated solution
A saturated solution with a nominal loading of 60 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 negative.

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 √10: 0.600 - 1.90 - 6.00 - 19.0 - 60.0% of the saturated solution.
These loadings were 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): 3 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
60.0 8.13 8.10
19.0 8.14 8.13
6.00 8.15 8.26
1.90 8.15 8.41
0.600 8.18 8.53
Control 8.17 8.48

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 777327 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: 6265 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.:

19 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.:

60 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.:

47.5 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 40.4 - 56.5

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

29.5 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 15.5 - 51.0

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.

NOEL, LOEL and analyses
The NOEL and LOEL were determined by calculation of statistically significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEL/LOEL calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. 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. The Normality test failed for growth rate. Therefore, the data were transformed (Y= Y squared).

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.

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
60.0		1		6839		11035		25317		71100
		2		6839		5455		8135		11541
		3		6839		11743		26525		43080
		Mean		6839		9411		19992		41907
19.0		1		6839		32477		79538		572019
		2		6839		37752		115277		668636
		3		6839		24813		65716		443217
		Mean		6839		31681		86844		561291
6.00		1		6839		38068		113989		778829
		2		6839		36000		168980		1240318
		3		6839		34920		108646		806509
		Mean		6839		36329		130538		941885
1.90		1		6839		34665		181831		1179632
		2		6839		34867		149441		1257698
		3		6839		34683		101091		761975
		Mean		6839		34738		144121		1066435
0.600		1		6839		38147		127103		793283
		2		6839		36649		187297		1489730
		3		6839		39915		116886		1367365
		Mean		6839		38237		143762		1216793
Control		1		6839		33583		106551		604264
		2		6839		35204		100272		656698
		3		6839		28998		57991		637679
		4		6839		37700		209810		887735
		5		6839		38665		249194		907222
		6		6839		27245		63053		970365
		Mean		6839		33566		131145		777327

 

 

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]		[%]
60.0	1		0.780	50		64261	92
	2		0.174	89		4702	99
	3		0.613	61		36241	95
	Mean	(+)	0.523	67	(+)	35068	95
19.0	1		1.48	6		565180	27
	2		1.53	3		661797	14
	3		1.39	12		436378	43
	Mean	(-)	1.47	7	(-)	554452	28
6.00	1		1.58	0		771990	0
	2		1.73	-10		1233479	-60
	3		1.59	-1		799670	-4
	Mean	(-)	1.63	-4	(-)	935046	-21
1.90	1		1.72	-9		1172793	-52
	2		1.74	-11		1250859	-62
	3		1.57	0		755136	2
	Mean	(-)	1.68	-7	(-)	1059596	-38
0.600	1		1.59	-1		786444	-2
	2		1.80	-14		1482891	-92
	3		1.77	-12		1360526	-77
	Mean	(-)	1.72	-9	(-)	1209954	-57
Control	1		1.49			597425
	2		1.52			649859
	3		1.51			630840
	4		1.62			880896
	5		1.63			900383
	6		1.65			963526
	Mean		1.57			770488

 

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	1.59	1.16	1.74	1.49	0.302	20.2	32.0
	2	1.64	1.05	1.88	1.52	0.429	28.2
	3	1.45	0.69	2.40	1.51	0.854	56.5
	4	1.71	1.72	1.44	1.62	0.156	9.6
	5	1.73	1.86	1.29	1.63	0.299	18.4
	6	1.38	0.84	2.73	1.65	0.976	59.1
				Mean	1.57
				SD ±	0.07
				CV [%]	4.45

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Disperse Red 343 (Ethyl) 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 47.5 (40.4 – 56.5) and 29.5 (15.5 – 51.0)% of the saturated solution, respectively. The NOEL-values for both inhibition of growth rate and yield after 72 hours were 19.0% of the saturated solution. All effect levels are far above the water solubility limit.

Executive summary:

The toxicity of DisperseRed 343 (Ethyl) to the unicellular freshwater green algaPseudokirchneriella subcapitatawas determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 266/2016 C.3from2017-11-12 to2017-11-16, with the definitive exposure phase from 2017-11-13 to 2017-11-16at 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 60 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 negative.

 

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 √10:0.600 - 1.90 - 6.00 - 19.0 - 60.0% 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 < lowest calibration level 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 Disperse Red 343 (Ethyl).

 

NOEL, LOEL, EL_x-values and 95% Confidence Intervals ofDisperseRed 343 (Ethyl)(0 - 72 hours)

              based on the nominal test item loadings

	Growth Rate Inhibition nominal test item loadings [% saturated solution]
NOEL	19.0
LOEL	60.0
ErL10	19.9 (12.5 – 27.1)
ErL20	26.5 (19.1 – 33.4)
ErL50	47.5 (40.4 – 56.5)
	Yield Inhibition nominal test item loadings [% saturated solution]
NOEL	19.0
LOEL	60.0
EyL10	13.2 (5.23 – 31.6)
EyL20	16.5 (7.88 – 36.4)
EyL50	29.5 (15.5 – 51.0)