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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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:

All loading levels and the control were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (48 hours).
At the start of the exposure (0 hours), sampling was carried out after preparation of the test concentrations.
At the end of the exposure (48 hours), samples were taken directly from the test vessels. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Recoveries of the test item should be within ± 20 % of the nominal or initially measured concentrations.
The effect levels were based on the geometric mean measured concentrations.

Test solutions

Vehicle:

no

Details on test solutions:

The saturated solution (20.0 mg/L test item were weighed out) was prepared with dilution water (as specified below) one day before the start of the exposure (at -24 hours). The test item was dispersed in the dilution water by stirring with a magnetic stirrer at approximately 1100 rpm for 24 hours at room temperature. After completion of stirring, the test item dispersion was filtered with a membrane filter (0.45 µm, RC, MACHEREY-NAGEL) to achieve the saturated solution.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Clone 5
- Source:
Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
Breeder: DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Age at study initiation (mean and range, SD): 2 to 24 h old daphnids from a healthy stock were used for the study
- Method of breeding:
Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 +- 2°C, in an incubator, 16 h illumination; light intensity
of max. 20 µE x m-2 x s-1
Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
Cultur feeding: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and
Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: At least 2 h in dilution water.
- Acclimation conditions (same as test or not): Same as test conditions.
- Health during acclimation (any mortality observed): No mortality observed.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

265 mgCaCO3/L

Test temperature:

18 - 22°C, constant within ± 1°C
At start of exposure: 19.8

pH:

pH at the Start of the Exposure (0 hours)
(measured in one additional replicate per concentration level and control)

Geometric mean
measured test item
concentration
[mg/L] pH-value

16.7 7.36
8.08 7.33
3.63 7.30
1.78 7.28
0.837 7.23
Control 7.63


pH at the End of the Exposure (48 hours)
(measured in all replicates)

Geometric mean
measured test item
concentration
[mg/L] pH-values
Replicates
1 2 3 4
16.7 7.35 7.39 7.41 7.41
8.08 7.35 7.36 7.36 7.37
3.63 7.32 7.33 7.34 7.35
1.78 7.29 7.29 7.31 7.31
0.837 7.22 7.24 7.26 7.26
Control 7.04 7.16 7.18 7.20

Dissolved oxygen:

O2-concentration at the Start of the Exposure (0 hours)
(measured in one additional replicate per concentration level and control)

Geometric mean
measured test item
concentrations
[mg/L]
Dissolved
O2-concentration
[mg/L]
16.7 4.53
8.08 7.38
3.63 8.14
1.78 8.59
0.837 8.68
Control 9.03



O2-concentration at the End of the Exposure (48 hours)
(measured in all replicates)

Geometric mean
measured test item
concentrations
[mg/L] Dissolved O2 -concentration [mg/L]
Replicates
1 2 3 4
16.7 8.67 8.83 8.87 8.95
8.08 8.37 8.26 8.31 8.22
3.63 8.65 8.68 8.65 8.58
1.78 8.70 8.69 8.71 8.76
0.837 8.70 8.70 8.57 8.68
Control 8.64 8.60 8.68 8.75

Nominal and measured concentrations:

Nominal: 1.25 - 2.50 - 5.00 - 10.0 - 20.0 mg/L (factor 2)
Geometric mean measured concentrations:
0.837 - 1.78 - 3.63 - 8.08 - 16.7 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (4 cm ID x 7 cm H), 50 mL capacity, loosely covered with watch glasses
- Test volume: 20 mL
- Aeration: None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water, according to OECD 202, Annex 3
Component Concentration [mg/L]
KCl 5.76
NaHCO3 64.8
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123

- Water Quality Parameters of the Dilution Water prior to the Start of the Exposure (0 h):
pH-value DissolvedO2-concentration[mg/L] Temperature[°C] Conductivity[µS/cm] Total hardness [mg CaCO3/L]
7.63 9.03 19.8 642 265

- Culture medium different from test medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: test start (0h) and test end (48h)

OTHER TEST CONDITIONS
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 20 µE x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobilisation, 24 h interval

TEST CONCENTRATIONS
- Test concentrations:
Nominal: 1.25 - 2.50 - 5.00 - 10.0 - 20.0 mg/L (spacing factor 2 )
Geometric mean measured concentraion: 0.837 - 1.78 - 3.63 - 8.08 - 16.7 mg/L

- Range finding study:
A non GLP preliminary range finding test under static conditions over a period of 48 hours was conducted at the test facility with a saturated solution of the test item with a nominal loading rate of 20.0 mg/L and a further dilution level with a nominal loading rate of 5.00 mg/L.
The test item dispersion of 20.0 mg/L was stirred with approximately 1100 rpm for 24 hours at room temperature. The test item dispersion was filtrated with a membrane filter (0.45 µm, RC, MACHEREY-NAGEL) to achieve the saturated solution. A further dilution level with a nominal loading rate of 5.00 mg/L was prepared with the dilution water by diluting the saturated solution.
The saturated solution was dark violet and clear throughout the exposure phase. The loading level of 5.00 mg/L was violet and clear throughout the exposure phase. The carapaces of the daphnids were violet discoloured.
In the range finding test, two replicates per treatment group and control, each with ten daphnids, were tested.
The biological results are presented and the analytical results showed stable recoveries during the range finding test are presented in the tables below.

Immobilisation Rates in the non GLP Preliminary Range Finding Test
(n = 20, 2 replicates with 10 daphnids each)

Nominal test item concentration
[mg/L] IMMOBILISATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
20.0 40* 20* 30 100* 100* 100
5.00 0* 0* 0 0* 0* 0
Control 0 0 0 0 0 0

MV = mean value
* = carapaces of the daphnids violet discoloured

Measured Concentrations of Duasyn Blue T 48 gefriergetrocknet during the non GLP Range Finding Test

Sampling date 2014-11-18
0 h
Start of the exposure
Nominal
test item
concentration
[mg/L] Duasyn Blue T 48 gefriergetrocknet
Meas. conc.
[mg/L] %
20.0 15.0 75
5.00 3.69 74
Control < 0.0500
Meas. conc. = measured concentration of the test item, mean value of 2 injections, dilution factors taken into account
% = percent of the nominal concentration of the test item

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

16.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 8.14 - 16.7

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

5.98 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 2.73 - 9.55

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 16.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: Not applicable

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

15.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 11.7 - > 16.7

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 16.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 16.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: not applicable

Details on results:

See below

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 hours. The EC50-value with 95 % confidence limits (CI) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 1.51 mg/L (CI 1.08 - 2.10 mg/L)
The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Any other information on results incl. tables

Biological Data

The percentage immobility, determined in all test and control groups after 24 and 48 h under static conditions, is given in the table below.

 

Immobilisation Rates after 24 and 48 h of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Duasyn BlueT 48 gefriergetrocknet		IMMOBILISATION [%]
Nominal loading level of the test item   [mg/L]	Geometric mean measured test item concentration [mg/L]	24 hours					48 hours
		Replicates					Replicates
		1	2	3	4	MV	1	2	3	4	MV
20.0	16.7	0	40	0	20	15	20	80	40	80	55
10.0	8.08	0	0	0	0	0	40	0	0	40	20
5.00	3.63	0	0	0	0	0	0	0	0	0	0
2.50	1.78	0	0	0	0	0	0	0	0	0	0
1.25	0.837	0	0	0	0	0	0	0	0	0	0
Control		0	0	0	0	0	0	0	0	0	0

MV = mean value

 

The tested solutions showed a concentration related violetcolour and were visually clear throughout the exposure period. The saturated solution was dark violet coloured.

Measured Exposure Concentrations during the Definitive Test

The concentrations of the test item were analytically verified via LC-MS/MS at the start of the exposure (0 hours) and at the end of the exposure (48 hours) in all loading levels and the control.

The measured concentrations of the test item at the start of the exposure (0 hours) were in the range of 75 to 81 % of the nominal values. At the end of the exposure (48 hours), the measured concentrations of the test item were in the range of 60 to 87 % of the nominal values.

The measured test item concentrations did not remain within ±20 % of the nominal loadings or the initially measured concentrations. The geometric mean measured concentrations of the test item were calculated to be: 0.837 – 1.78 – 3.63 – 8.08 – 16.7 mg/L.

Measured Concentrations and Percent of the Nominal Loading Rates of the Test Item  during the Definitive Test

Sampling date	2015-03-03 Start of the exposure, 0 hours		2015-03-05 End of the exposure, 48 hours
Start of analysis	2015-03-03		2015-03-05
Nominal loading level of the test item [mg/L]	Test item					Geometric mean measured test item concentration [mg/L]
	Meas. conc. [mg/L]	%		Meas. conc. [mg/L]	%
20.0	16.1	81		17.3	87	16.7
10.0	8.03	80		8.13	81	8.08
5.00	4.07^1)	81		3.24^3)	65	3.63
2.50	2.00^1)	80		1.58^3)	63	1.78
1.25	0.938^2)	75		0.746^3)	60	0.837
Control	< LOQ			< LOQ

Meas. conc.    = measured concentration of the test item, mean value of 2 injections, dilution factor taken into account

%                     = percent of the nominal loading level of the test item

LOQ                = limit of quantification (5.00 µg test item/L)

¹⁾                      = reanalysis on 2015-03-05, mean value of 2 replicates with 2 injections each, dilution factor taken into account

²⁾                      = reanalysis on 2015-03-05, mean value of 3 replicates with 2 injections each, dilution factor taken into account

³⁾             = reanalysis on 2015-03-07, mean value of 3 replicates with 2 injections each, dilution factor taken into account

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the geometric mean measured concentrations of the test item, the 48 hour-EC50 for Daphnia magna was 15.2 mg/L (95 % confidence limits: 11.7 – > 16.7 mg/L). The EC10 after 48 hours was calculated to be 5.98 mg/L (95 % confidence limits: 2.73 – 9.55 mg/L).

Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the test item were determined according to OECD 202 (2004).

The study was conducted under static conditions over a period of 48 hourswith a saturated solution with a nominal loading of 20.0 mg/L of the test item and further dilution levels prepared with dilution water in a geometric series with a separation factor of 2(nominal loading rates of 1.25 to 10.0 mg/L).

The test item has a water solubility of 0.8 g/L (20 °C).The saturated solution with a nominal loading of 20.0 mg/L of the test item was prepared with dilution water one day before the start of the exposure (at -24 hours) and was used for testing and as stock solution for the preparation of all further tested dilution levels. A test item dispersion of 20.0 mg/L was stirred with approximately 1100 rpm for 24 hours at room temperature. After completion of stirring, the test item dispersion was filtered with a membrane filter(0.45 µm, RC,Macherey-Nagel) to achieve the saturated solution. Further dilution levels were prepared by diluting the saturated solution with dilution water. The tested solutions showed a concentration related violet colour and were visually clear throughout the exposure period. The saturated solution was dark violet coloured.

Twenty daphnids were exposed to each loading level and the control.

The concentrations of the test item were analytically verified via LC-MS/MS at the start of the exposure (0 hours) and at the end of the exposure (48 hours) in all loading levels and the control.

The measured concentrations of the test item at the start of the exposure (0 hours) were in the range of 75 to 81 % of the nominal values. At the end of the exposure (48 hours), the measured concentrations of the test item were in the range of 60 to 87 % of the nominal values.

Since the measured test item concentrations did not remain within ± 20 % of the nominal loadings or the initially measured concentrations, the effect values given are based on the geometric mean measured concentrations of the test item calculated to be: 0.837 – 1.78 – 3.63 – 8.08 – 16.7 mg/L.

The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 hours) and at the end of the exposure (48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

  

 

EC₁₀-, EC₅₀- (with Confidence Interval), EC₁₀₀-Values

(based on the geometric mean measured concentrations of the test item)

Test item
Effect levels	Test duration [h]	Toxicity endpoint based on the geometric mean measured test item concentrations [mg/L]
EC10 (with 95 % confidence limits)	24	16.4	(Cl: 8.14 – 16.7)
	48	5.98	(Cl: 2.73 – 9.55)
EC50 (with 95 % confidence limits)	24	> 16.7	(Cl: Not applicable)
	48	15.2	(Cl: 11.7 – > 16.7)
EC100	24	> 16.7
	48	> 16.7