9-[bis(2-hydroxyethyl)amino]-5-[(3-methylphenyl)amino]-7-phenylbenzo[a]phenazin-7-ium chloride

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:

2015

Report date:

2015

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:

8-[bis(2-hydroxyethyl)amino]-12-[(3-methylphenyl)amino]-10-phenyl-5,10λ⁵-diazatetraphen-10-ylium chloride

EC Number:

941-946-2

Molecular formula:

C33H31ClN4O2

IUPAC Name:

8-[bis(2-hydroxyethyl)amino]-12-[(3-methylphenyl)amino]-10-phenyl-5,10λ⁵-diazatetraphen-10-ylium chloride

Details on test material:

purity; 89.7 % (w/w) (main component)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The test item contents of all test item concentration and control solutions were analytically verified at the beginning and the end of exposure. Separate replicates for the test item analysis at the beginning of the exposure were prepared without algae. For the test item analysis after 72 hours, samples were taken out of the test replicates.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10.0 mg/L was freshly prepared with dilution water. The stock solution was stirred for 24 hours (1000 rpm) with a magnetic stirrer. No filtration was necessary.

- Eluate: Dilution water
- Differential loading: 5 concentrations were tested in a geometrical series with a dilution factor of 3.16:
0.100 - 0.316 - 1.00 - 3.16 - 10.0 mg/L.
The concentrations are based on the results of a preliminary range-finding test (non-GLP).

- Controls: Six replicates (without test item) were tested under the same test conditions as the test vessels.
- pH-Controls: None

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x 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.: 21.0 max.: 23.5, mean value: 22.3 °C

pH:

Geometric mean measured pH-value
test item concentration
[mg/L] Start; 0 hours End; 72 hours
5.87 7.58 7.82
0.468 7.64 7.86
0.105 7.69 7.99
0.0394 7.81 8.06
0.0340 7.92 8.11
Control 8.09 8.21

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 25 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 4998 cells/mL
- Control end cells density: Mean 1223669 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 0.064
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: 80.5 - 98.2 µE x m^-2 x s^-1, mean value: 88.9 µE x m^-2 x s^-1



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.:

0.105 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.468 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.119 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: < 0.0340 - 0.135

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.139 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.121 - 0.156

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.219 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.197 - 0.244

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.105 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition oy Yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.468 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.112 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: 0.0437 - 0.377

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: 0.0460 - 0.385

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.142 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: 0.106 - 0.415

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):

- Results with reference substance valid? Yes

Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.586
0.678 ± 0.414
95 % confidence interval 0.522 - 0.818 0.678 ± 0.414
Yield Inhibition
EyC50 0.401 0.319 ± 0.141
95 % confidence interval 0.285 - 0.469 0.319 ± 0.141

SD = Standard deviation

Reported statistics and error estimates:

EC-values of the growth rate and yield inhibition were calculated by non-linear sigmoidal dose-response regression.
The values for NOEC/LOEC were determined by calculation of statistical significance of inhibition of growth rate and yield. As standard methods, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC 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. Equal Variance test failed for both growth rate and yield.

Any other information on results incl. tables

Cell Densities

Geometric mean measured test item concentration	Replicate		Cell density [cells/mL]
[mg/L]	No.		0 hours	24 hours		48 hours		72 hours
5.87	1		4998	<LOQ		2885		<LOQ
	2		4998	2564		2352		<LOQ
	3		4998	5315		5514		<LOQ
	Mean		4998	n.a.		3584		n.a.
0.468	1		4998	2668		<LOQ		<LOQ
	2		4998	4011		<LOQ		<LOQ
	3		4998	11569		3456		<LOQ
	Mean		4998	6083		n.a.		n.a.
0.105	1		4998	22147		138577		663863
	2		4998	31576		310685		1688003
	3		4998	34665		267266		1235448
	Mean		4998	29463		238843		1195771
0.0394	1		4998	24555		298466		1226278
	2		4998	44129		340490		1670213
	3		4998	39746		329679		1541892
	Mean		4998	36143		322878		1479461
0.0340	1		4998	31135		296430		1361468
	2		4998	38143		302070		1589565
	3		4998	34375		275284		1411410
	Mean		4998	34551		291261		1454148
Control		1	4998		29762		238284		1122326
		2	4998		34673		310577		1454162
		3	4998		21155		247909		1114096
		4	4998		18998		264862		1308364
		5	4998		26283		301871		1289955
		6	4998		40725		247502		1053109
		Mean	4998		28599		268501		1223669

< LOQ     = below the limit of quantification

n.a.                        = data not determinable

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Geometric mean measured test item concentration [mg/L]	Replicate	Growth rate		Growth rate inhibition	Yield		Inhibition of yield
	No.	[d^-1]		[%]	[cells/mL]		[%]
5.87	1		<LOQ	100		<LOQ	100
	2		<LOQ	100		<LOQ	100
	3		<LOQ	100		<LOQ	100
	Mean	(+)*	n.a.	100	(+)*	n.a.	100
0.468	1		<LOQ	100		<LOQ	100
	2		<LOQ	100		<LOQ	100
	3		<LOQ	100		<LOQ	100
	Mean	(+)*	n.a.	100	(+)*	n.a.	100
0.105	1		1.63	11		658865	46
	2		1.94	-6		1683005	-38
	3		1.84	0		1230450	-1
	Mean	(-)	1.80	2	(-)	1190773	2
0.0394	1		1.83	0		1221280	0
	2		1.94	-6		1665215	-37
	3		1.91	-4		1536894	-26
	Mean	(-)	1.89	-3	(-)	1474463	-21
0.0340	1		1.87	-2		1356470	-11
	2		1.92	-5		1584567	-30
	3		1.88	-3		1406412	-15
	Mean	(-)	1.89	-3	(-)	1449150	-19
Control	1		1.80			1117328
	2		1.89			1449164
	3		1.80			1109098
	4		1.86			1303366
	5		1.85			1284957
	6		1.78			1048111
	Mean		1.83			1218671

negative inhibitions = increase of growth

* = not included in statistical analyses due to values < LOQ

Section-by-Section and Average Specific Growth Rates of 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	1.78	2.08	1.55	1.80	0.266	14.7	24.5
	2	1.94	2.19	1.54	1.89	0.327	17.3
	3	1.44	2.46	1.50	1.80	0.571	31.7
	4	1.34	2.63	1.60	1.86	0.687	37.0
	5	1.66	2.44	1.45	1.85	0.521	28.2
	6	2.10	1.80	1.45	1.78	0.325	18.2
				Mean	1.83
				SD ±	0.04
				CV [%]	2.24

CV= Coefficient of variation

SD= Standard deviation

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study,the test item was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values: The NOEC-value of the test item for both inhibition of growth rate and inhibition of yield after 72 hours was 0.105 mg/L, the LOEC for both parameters was 0.468 mg/L. The EC50-values with 95 % confidence intervals of the test item for inhibition for growth rate (ErC50) and yield (EyC50) after 72 hours were determined to be 0.219 (0.197 – 0.244) mg/L and 0.142 (0.106 – 0.415) mg/L.
All effect levels are given based on the geometric mean measured concentrations .

Executive summary:

The toxicity of the test irem to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) and Council Regulation (EC) 440/2008/Method C.3. 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 4998 cells/mL. A stock solution with a nominal concentration of 10.0 mg/L was tested.Five concentrations were tested in a geometrical series with a dilution factor of 3.16:0.100 - 0.316 - 1.00 - 3.16 - 10.0 mg/L.Three replicates were tested for the test item concentrations and six for the control. Environmental conditions were determined to be within the acceptable limits.

The test item concentration in the test and control solutions were analysed by LC-MS/MS at the start and the end of exposure. The recovery rate of the test item was in the range of 80 – 96 % of the nominal concentration at the test start and between 1 and 37 % at the test end.
All effect value sof Duasyn BlueT 48 gefriergetrocknet are given based on the geometric mean measured test item concentrations.

 

NOEC, LOEC, EC-values and Confidence Intervals of thetest item (0-72 hours)

               based on geometric mean measured test item concentrations [mg/L]

                               

	Growth Rate Inhibition Geometric mean measuredtest item concentration [mg/L]
NOEC	0.105
LOEC	0.468
ErC10	0.119 (CI: < 0.0340 – 0.135)
ErC20	0.139 (CI: 0.121 – 0.156)
ErC50	0.219 (CI: 0.197 – 0.244)
	Inhibition of Yield Geometric mean measuredtest item concentration [mg/L]
NOEC	0.105
LOEC	0.468
EyC10	0.112 (CI: 0.0437 – 0.377)
EyC20	0.120 (CI: 0.0460 – 0.385)
EyC50	0.142 (CI: 0.106 – 0.415)