Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2016-11-21 to 2016-12-23, with the definitive exposure phase from 2016-12-20 to 2016-12-23 at the test facility.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

Determination of the test Item
No analytical monitoring was carried out. Due to the low solubility, a specific analytical method is not available and the concentrations are too low for unspecific analysis as the determination of the total organic carbon content.

Vehicle:

no

Details on test solutions:

Preparation of the Saturated solution
A saturated solution with a nominal loading of 5.00 mg/L was prepared once 24 ± 1 hour 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) 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 will be 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 will always be 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 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 (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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Not specified

Test temperature:

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

pH:

Nominal test item loading pH-values
[mg/L] Start; 0 hours End; 72 hours
5.00 7.75 8.38
2.50 7.78 8.61
1.25 7.80 8.93
0.625 8.04 9.13
0.313 8.14 9.34
Control 8.07 9.26

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

No analytical monitoring

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 6305 cells/mL
- Control end cells density: Mean 2579163 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:
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 0.064
Na2EDTA 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: 5298 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 definitive test at the saturated solution.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

1.86 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI

Remarks:

1.66 - 2.03

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

2.27 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI

Remarks:

2.16 - 2.34

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

3.08 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI

Remarks:

2.92 - 3.36

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

< 0.313 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

0.313 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.836 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI

Remarks:

< 0.313 - 1.29

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

1.34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI

Remarks:

1.15 - 1.60

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

1.88 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI

Remarks:

1.67 - 2.06

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 were clear throughout the exposure phase. Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. All effect v alues are given based on the nominal test item loadings.
- 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 2016-10-11 to 2016-10-14. 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 0.435 0.764 ± 0.513
95% confidence interval 0.414 – 0.458
Yield inhibition
EyC50 0.230 0.402 ± 0.293
95% confidence interval 0.179 – 0.322
SD = Standard deviation

Reported statistics and error estimates:

ELx-values and statistical Analyses
ELx-values with confidence intervals of growth rate inhibition and for yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEL, LOEL and statistical analyses
The NOEL and LOEL were determined by calculation of statistically significant differences of growth rates and yield.
The following statistical tests were conducted:

Shapiro-Wilk’s test on normal distribution was calculated with a significance level 0.01.

Levene’s test on variance homogeneity was calculated with a significance level 0.01.

Monotonicity of Concentration/Response was calculated by trend analysis by contrasts (significance level 0.05).

SD Jonckheere-Terpsta test procedure rate was done for growth with a significance level 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 Version 3.2.1, ToxRat Solutions GmbH

Cell Densities

Nominal test item loading			Replicate		Cell density [cells/mL]
[mg/L]		[% of the saturated solution]		No.		0 hours		24 hours		48 hours		72 hours
5.00		100		1		6305		4650		3509		3317
			2		6305		5496		6426		16332
			3		6305		3900		2756		< LOQcell density
			Mean		6305		4682		4230		n.a.
2.50		50.0		1		6305		16535		128052		515332
			2		6305		6939		23907		489002
			3		6305		6897		12208		471874
			Mean		6305		10124		54722		492069
1.25		25.0		1		6305		30687		354576		2334737
			2		6305		28417		403429		2288675
			3		6305		18819		258014		1803694
			Mean		6305		25974		338673		2142369
0.625		12.5		1		6305		27526		312674		2338909
			2		6305		33012		402453		2555803
			3		6305		30765		366145		2305137
			Mean		6305		30434		360424		2399950
0.313		6.25		1		6305		29677		352151		2344603
			2		6305		26159		342871		2338740
			3		6305		29218		311637		2195140
			Mean		6305		28351		335553		2292828
Control			1		6305		35919		418020		2578693
			2		6305		33892		418866		2682714
			3		6305		33531		367284		2751667
			4		6305		30441		372702		2526316
			5		6305		31459		363388		2358473
			6		6305		31989		367955		2577115
			Mean		6305		32872		384703		2579163

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
[mg/L]	[% of the saturated solution]	No.	[d^-1]		[%]	[cells/mL]		[%]
5.00	100	1		-0.214	100		-2988	100
		2		0.317	84		10027	100
		3		n.a.	100		n.a.	100
		Mean	(+)	n.a.	95	(+)	n.a.	100
2.50	50.0	1		1.47	27		509027	80
		2		1.45	28		482697	81
		3		1.44	28		465569	82
		Mean	(+)	1.45	28	(+)	485764	81
1.25	25.0	1		1.97	2		2328432	10
		2		1.96	2		2282370	11
		3		1.89	6		1797389	30
		Mean	(-)	1.94	3	(+)	2136064	17
0.625	12.5	1		1.97	2		2332604	9
		2		2.00	0		2549498	1
		3		1.97	2		2298832	11
		Mean	(-)	1.98	1	(+)	2393645	7
0.313	6.25	1		1.97	2		2338298	9
		2		1.97	2		2332435	9
		3		1.95	3		2188835	15
		Mean	(-)	1.97	2	(+)	2286523	11
Control		1		2.00			2572388
		2		2.02			2676409
		3		2.03			2745362
		4		2.00			2520011
		5		1.97			2352168
		6		2.00			2570810
		Mean		2.00			2572858

 

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	1.74	2.45	1.82	2.00	0.391	19.5	20.8
	2	1.68	2.51	1.86	2.02	0.439	21.8
	3	1.67	2.39	2.01	2.03	0.361	17.8
	4	1.57	2.50	1.91	2.00	0.471	23.6
	5	1.61	2.45	1.87	1.97	0.429	21.7
	6	1.62	2.44	1.95	2.00	0.412	20.6
				Mean	2.00
				SD ±	0.02
				CV [%]	0.89

 

SD = Standard deviation CV = Coefficient of variation

Validity criteria fulfilled:

yes

Conclusions:

In this study, Vat Black 25 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) after 72 hours were 3.08 (2.92 – 3.36) mg/L, corresponding to 61.6 (58.5 – 67.2) % of the saturated solution, respectively. The EL50-values with 95 % confidence intervals for inhibition of yield (EyL50) after 72 hours were 1.88 (1.67 – 2.06) mg/L, corresponding to 37.5 (33.4 – 41.1) % of the saturated solution, respectively. The NOEL-values for inhibition of growth rate and yield after 72 hours were 1.25 mg/L (corresponding to 25.0 % of the saturated solution) and < 0.313 mg/L (corresponding to < 6.25 % of the saturated solution), respectively.

Executive summary:

The toxicity of Vat Black 25 (Batch no.:20151007) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 C.3 from 2016-11-21 to 2016-12-23, with the definitive exposure phase from 2016-12-20 to 2016-12-23 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 6305 cells/mL. A saturated solution with a nominal loading of 5.00 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), 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 2:6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution, corresponding to the nominal test item loadings of 0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L. 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.

 

All effect values given are based on the nominal test item loadings of Vat Black 25.

 

 NOEL, LOEL, EL_x-values and 95 % Confidence Intervals ofVat Black 25 (0 - 72 hours)

 based on the nominal test item loadings

	Growth Rate Inhibition nominal test item loadings
	[mg/L]	[% saturated solution]
NOEL	1.25	25.0
LOEL	2.50	50.0
ErL10	1.86 (1.66 – 2.03)	37.1 (33.2 – 40.5)
ErL20	2.27 (2.16 – 2.34)	45.4 (43.2 – 46.7)
ErL50	3.08 (2.92 – 3.36)	61.6 (58.5 – 67.2)
	Yield Inhibition nominal test item loadings
	[mg/L]	[% saturated solution]
NOEL	< 0.313	< 6.25
LOEL	0.313	6.25
EyL10	0.836 (< 0.313 – 1.29)	16.7 (< 6.25 – 25.7)
EyL20	1.34 (1.15 – 1.60)	26.8 (23.0 – 32.1)
EyL50	1.88 (1.67 – 2.06)	37.5 (33.4 – 41.1)

Description of key information

Key value determined in a GLP accredited laboratory study performed in accordance with OECD Guideline 201 (Alga, Growth Inhibition Test).

According to this test, the test item does have toxic effects on alga.

Key value for chemical safety assessment

EC50 for freshwater algae:

3.08 mg/L

EC10 or NOEC for freshwater algae:

1.25 mg/L

Additional information

In a GLP study conducted in accordance with OECD Guideline 201, Vat Black 25 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) after 72 hours were 3.08 (2.92 – 3.36) mg/L, corresponding to 61.6 (58.5 – 67.2) % of the saturated solution, respectively. The EL50-values with 95 % confidence intervals for inhibition of yield (EyL50) after 72 hours were 1.88 (1.67 – 2.06) mg/L, corresponding to 37.5 (33.4 – 41.1) % of the saturated solution, respectively. The NOEL-values for inhibition of growth rate and yield after 72 hours were 1.25 mg/L (corresponding to 25.0 % of the saturated solution) and < 0.313 mg/L (corresponding to < 6.25 % of the saturated solution), respectively.