4',4'''-azobis[N-(9,10-dihydro-9,10-dioxo-1-anthryl)[1,1'-biphenyl]-4-carboxamide]

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 2017-09-25 to 2017-11-24, with the definitive exposure phase from 2017-11-21 to 2017-11-24

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:

4',4'''-azobis[N-(9,10-dihydro-9,10-dioxo-1-anthryl)[1,1'-biphenyl]-4-carboxamide]

EC Number:

235-436-2

EC Name:

4',4'''-azobis[N-(9,10-dihydro-9,10-dioxo-1-anthryl)[1,1'-biphenyl]-4-carboxamide]

Cas Number:

12227-50-8

Molecular formula:

C54H32N4O6

IUPAC Name:

4',4'''-azobis[N-(9,10-dihydro-9,10-dioxo-1-anthryl)[1,1'-biphenyl]-4-carboxamide]

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

no

Remarks:

No analytical monitoring was carried out due to the low solubility of the test item. No analytical monitoring was carried out, since no suitable method for the determination of the test item could be established.

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 homogenized before weighing (pounded in a mortar). 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) 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 15 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 will always be kept covered.
The solution was checked via laser beam (Tyndall effect). No Tyndall effect was observed.

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.

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

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 7.96 8.07
50.0 8.00 8.61
25.0 8.03 8.99
12.5 8.05 9.13
6.25 8.06 9.28
Control 8.11 9.31

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 2368726 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: 5956 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. Self-fluorescence was observed in the saturated solution. A separate replicate of each test item concentration were prepared without algae and treated like the other test replicates. The self-fluorescence of each measuring point was taken into account in the evaluation.

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.:

25 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.:

50 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.:

69.3 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 62.2 - 80.9

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

40.7 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 37.0 - 44.1

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.

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
100		1		6839		5317		4753		5446
		2		6839		5891		6146		7102
		3		6839		5288		6339		8232
		Mean		6839		5499		5746		6927
50.0		1		6839		12615		76829		613554
		2		6839		14265		83129		963082
		3		6839		12278		56008		562431
		Mean		6839		13053		71989		713022
25.0		1		6839		26215		246610		2092366
		2		6839		25252		266109		1909432
		3		6839		31236		256014		2216545
		Mean		6839		27568		256244		2072781
12.5		1		6839		27842		304674		2188254
		2		6839		31069		320345		2454578
		3		6839		30332		296235		2195218
		Mean		6839		29748		307085		2279350
6.25		1		6839		31017		340002		2411016
		2		6839		27798		319333		2301935
		3		6839		29995		328813		2499557
		Mean		6839		29603		329383		2404169
Control		1		6839		29890		355700		2208771
		2		6839		28603		339970		2644745
		3		6839		32471		270752		2407037
		4		6839		26569		307129		2208244
		5		6839		30194		334153		2233115
		6		6839		30285		337992		2510442
		Mean		6839		29669		324283		2368726

 

 

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		-0.0759	100		-1393	100
	2		0.0126	99		263	100
	3		0.0618	97		1393	100
	Mean	(+)	-0.0005	99	(-)	88	100
50.0	1		1.50	23		606715	74
	2		1.65	15		956243	60
	3		1.47	25		555592	76
	Mean	(+)	1.54	21	(+)	706183	70
25.0	1		1.91	2		2085527	12
	2		1.88	4		1902593	19
	3		1.93	1		2209706	6
	Mean	(-)	1.90	2	(-)	2065942	13
12.5	1		1.92	1		2181415	8
	2		1.96	-1		2447739	-4
	3		1.92	1		2188379	7
	Mean	(-)	1.94	1	(-)	2272511	4
6.25	1		1.96	0		2404177	-2
	2		1.94	0		2295096	3
	3		1.97	-1		2492718	-6
	Mean	(-)	1.95	0	(-)	2397330	-2
Control	1		1.93			2201932
	2		1.99			2637906
	3		1.96			2400198
	4		1.93			2201405
	5		1.93			2226276
	6		1.97			2503603
	Mean		1.95			2361887

Negative inhibition = increase of growth

 

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	1.48	2.48	1.83	1.93	0.508	26.4	24.4
	2	1.43	2.48	2.05	1.99	0.525	26.5
	3	1.56	2.12	2.19	1.96	0.345	17.7
	4	1.36	2.45	1.97	1.93	0.547	28.4
	5	1.49	2.40	1.90	1.93	0.460	23.9
	6	1.49	2.41	2.01	1.97	0.463	23.5
				Mean	1.95
				SD ±	0.03
				CV [%]	1.30

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Vat Yellow 33 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 69.3 (62.2 – 80.9) and 40.7 (37.0 – 44.1)% of the saturated solution, respectively. The NOEL-values for both inhibition of growth rate and yield after 72 hours were 25.0% of the saturated solution, respectively. The effect levels are far above the water solubility limit. Vat dyes are practically insoluble in water and organic solvents.

Executive summary:

The toxicity of Vat Yellow 33 to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016 Method C.3 from2017-09-25 to 2017-11-24, with the definitive exposure phase from 2017-11-21 to 2017-11-24 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) 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). No Tyndall effect was observed.

 

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 conditions were within the acceptable limits.

No analytical monitoring was carried out due to the low solubility ofthe test item. No suitable method for the determination of the test item could be established.

 

All effect values given are based on the nominal test item loadings of Vat Yellow 33.

 

NOEL, LOEL, EL_x-values and 95% Confidence Intervals of Vat Yellow 33 (0 - 72 hours)

              based on the nominal test item loadings

	Growth Rate Inhibition nominal test item loadings [% saturated solution]
NOEL	25.0
LOEL	50.0
ErL10	39.2 (32.5 – 43.5)
ErL20	49.2 (46.5 – 52.3)
ErL50	69.3 (62.2 – 80.9)
	Yield Inhibition nominal test item loadings [% saturated solution]
NOEL	25.0
LOEL	50.0
EyL10	23.2 (19.0 – 28.2)
EyL20	28.6 (24.8 - 32.8)
EyL50	40.7 (37.0 – 44.1)