2-[2-[4-[(2-cyanoethyl)ethylamino]-o-tolyl]vinyl]-1,3,3-trimethyl-3H-indolium dihydrogen phosphate

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:

2018-06-25 to 2018-07-05, with the definitive exposure phase from 2018 07 03 to 2018-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[2-[4-[(2-cyanoethyl)ethylamino]-o-tolyl]vinyl]-1,3,3-trimethyl-3H-indolium dihydrogen phosphate

EC Number:

276-470-8

EC Name:

2-[2-[4-[(2-cyanoethyl)ethylamino]-o-tolyl]vinyl]-1,3,3-trimethyl-3H-indolium dihydrogen phosphate

Cas Number:

72208-20-9

Molecular formula:

C25H30N3.H2O4P C25H32N3O4P

IUPAC Name:

2-[2-[4-[(2-cyanoethyl)ethylamino]-o-tolyl]vinyl]-1,3,3-trimethyl-3H-indolium dihydrogen phosphate

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analytically verified via HPLC-DAD in the fresh media at the start of exposure (0 hours) and in the old media at the end of the exposure (48 hours). The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000)

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples were taken from the freshly prepared concentration levels and the control for analysis.
At the end of the exposure (48 hours), samples for the analyses of the old media were taken from the test replicates containing daphnids.


Criteria for the analytical monitoring (target)
Recoveries of the test item should be within ± 20% of the nominal or initially measured concentrations.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the stock solution
A stock solution (100 mg/L test item were weighed out) was prepared with dilution water one day before the start of the exposure (at -24 hours). The test item was mixed with the dilution water by constant stirring with a magnetic stirrer at approximately 1100 rpm for 24 hours at room temperature. After completion of stirring, the stock solution was checked via laser beam (Tyndall effect) for undissolved test item. Since no Tyndall effect was observed in the stock solution 100 mg/L, the stock solution was used un-filtrated for the preparation of further dilution levels by dilution with dilution water.

Test concentrations
5 test item concentrations in a geometric series with a separation factor of 2, prepared by dilution of the stock solution 100 mg/L with dilution water, were tested as follows: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L
The test item concentrations were based on the results of a non-GLP preliminary range finding test.

Control
Dilution water without test item incubated under the same conditions as the test groups

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system: Daphnia magna STRAUS, obtained from continuous laboratory cultures.

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline.

Origin Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination;
light intensity of max. 1500 lx

Culture medium Elendt M4, according to OECD 202, Annex 3 (2004), is used. The composition of the culture medium is presented in the table below.

 

Composition of the Culture Medium Elendt M4
according to OECD 202, Annex 3 (2004)

Component Concentration [mg/L]
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 x 5 H2O 7.47
NaNO3 0.274
KH2PO4 0.143
K2HPO4 0.184
Na2EDTA x 2 H2O 2.50
FeSO4 x 7 H2O 0.996
H3BO3 2.86
MnCl2 x 4 H2O 0.361
LiCl 0.306
SrCl2 x 6 H2O 0.152
RbCl 0.0710
NaBr 0.0160
Na2MoO4 x 2 H2O 0.0615
CuCl x 2 H2O 0.0168
ZnCl2 0.0130
CoCl2 x 6 H2O 0.0100
KI 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamin 0.0010
Biotin 0.00075
pH 8.2 to 0.8

Feeding of the culture stocks
The daphnids were fed 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. The algae were cultured at the test facility.

Study design

Test type:

static

Water media type:

freshwater

Remarks:

Elendt M4, according to OECD 202, Annex 3 (2004)

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution water
0 hours: Total hardness [mg CaCO3/L]: 293

Test temperature:

target: 18 - 22°C, constant within ± 1°C
measured: 18.5 - 19 °C

pH:

Water Quality Parameters in fresh Media at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) per concentration level and control)

Nominal pH-value Dissolved O2 concentration
test item [mg/L]
concentration
[mg/L]
10.0 7.81 9.01
5.00 7.87 9.02
2.50 7.93 9.02
1.25 7.93 8.99
0.625 7.94 8.88
Control 8.40 9.04


Water Quality Parameters in old Media at the End of the Exposure (48 hours)
(measured in one replicate (containing daphnids) per concentration level and control)

Nominal pH-value Dissolved O2 concentration Replicate number
test item [mg/L]
concentration
[mg/L]
10.0 7.56 7.84 2
5.00 7.54 7.77 2
2.50 7.45 8.24 2
1.25 7.54 8.12 2
0.625 7.59 8.02 2
Control 7.62 8.40 2


Water Quality Parameters of the Dilution Water at the Start of the Exposure (0 hours)
Dilution water pH-value Dissolved Temperature Conductivity Total hardness
dated: O2 concentration [mg/L] [°C] [µS/cm] [mg CaCO3/L]
2018-07-03 8.40 9.04 19.4 608 293

Dissolved oxygen:

see above

Salinity:

Not applicable, freshwater

Conductivity:

Dilution water day 0: 608 [µS/cm]

Nominal and measured concentrations:

Nominal test item concentrations: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L

Details on test conditions:

Test method The study was performed under static conditions.

Test duration 48 hours

Test vessels Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Test volume 20 mL

Dilution water Same composition as the culture medium

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used per concentration level and control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization
Acclimatization of the daphnids was not necessary, because the composition of the dilution water was equivalent to the culture medium.

Application
20 g test solution per replicate was weighed into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by a pipette.

Test temperature (target) 18 - 22 °C, constant within ± 1 °C

Illumination (target) Diffuse light, light intensity of max. 1500 lx

Photoperiod (target) 16/8 hours light/dark cycle

Feeding The daphnids were not fed during the study.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

4.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: < 0.625 - 9.10 mg/L

Remarks:

confidence limits

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

2.68 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Cl: 2.50 - 5.00 mg/L

Remarks:

Confidence limits

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

4.91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 2.52 - > 10.0 mg/L

Remarks:

Confidence limits

Effect concentrations 4

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.54 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Cl: 2.50 - 5.00 mg/L

Remarks:

Confidence limits

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2018 06-20 to 2018-06-21. For results of the most recent of the monthly performed reference tests, see the table below.

EC50-Value (with 95% Confidence Limits) of the Reference Item Potassium dichromate
based on nominal concentrations [mg/L], (0 - 24 hours)

Current Study Valid Range
EC50 2.03 mg/L 0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
95% confidence limits 1.00 – 4.00 mg/L 0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A

Reported statistics and error estimates:

Methods of evaluation
The EC100-values after 24 and 48 h were empirically derived from the observation data (lowest concentration level resulting in 100% immobilization). The effect levels (EC10 / 50 / 100) are given based on the nominal concentrations, since the measured test item concentrations were within ± 20% of the nominal values

EC-values and statistical analyses
The EC10- and EC50-values (after 24 h of exposure) were calculated by sigmoidal dose-response regression with the software GraphPad Prism. The respective 95% confidence limits were calculated from the standard error and the t distribution. In case that the confidence limits could not be calculated by the software, the respective confidence limits were empirically derived from the observation data.
The EC10- and EC50-values (after 48 h of exposure) were calculated by straight line regression with the software GraphPad Prism. Only the highest concentration level without any effect (EC0) and the lowest concentration level causing 100% immobilization (EC100) were taken into account, since only one partial effect occurred. Both concentration levels (EC0 and EC100) were used as confidence limits.
An EC50 value for the reference item was calculated by sigmoidal dose-response regression with the software GraphPad Prism. The respective confidence limits were empirically derived from the observation data as follows: The highest concentration level without any effect (EC0) and the lowest concentration level causing 100% immobilization (EC100) were used as confidence limits.
All calculations were carried out from the best-fit values with the software GraphPad Prism.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

Immobilization Rates after 24 and 48 hours of Exposure in the Definitive Test

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

Nominal test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
10.0	80	40	80	80	70	100	100	100	100	100
5.00	60	40	40	100	60	100	100	100	100	100
2.50	0	0	0	0	0	0	0	0	0	0
1.25	0	0	0	0	0	0	0	0	0	0
0.625	0	100	0	0	25	0	100	0	0	25
Control	0	0	0	0	0	0	0	0	0	0

 

Absolute Numbers of immobile daphnids after 24 and 48 hours of Exposure in the Definitive Test

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

Nominal test item concentration [mg/L]	Number of immobile Daphnids / Total number of Daphnids
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	Sum	1	2	3	4	Sum
10.0	4 / 5	2 / 5	4 / 5	4 / 5	14 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
5.00	3 / 5	2 / 5	2 / 5	5 / 5	12 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
2.50	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
1.25	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
0.625	0 / 5	5 / 5	0 / 5	0 / 5	5 / 20	0 / 5	5 / 5	0 / 5	0 / 5	5 / 20
Control	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

 

All tested concentration levels were visually clear and concentration-depended rose to pink coloured throughout theexposure period.

The concentrations of the test item Basic Red 15 phosphate liquid were analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure (48 hours) in all tested concentration levels and in the control.

The measured concentrations of the test item at the start of the exposure (0 hours) were in the range of 98 to 109% of the nominal values. At the end of the exposure period (48 hours), the measured concentrations were within the range of 91 to 98% of the nominal values.

The measured concentrations of the test item were within ± 20% of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test.

Measured Concentrations and Percent of the Nominal Concentration of the Test Item Basic Red15phosphate liquid during the Definitive Test

Sampling date	Fresh media, 0 hours		Old media, 48 hours
Nominal test item concentration [mg/L]	Basic Red15 phosphate liquid
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
10.0	9.79	98	9.14	91
5.00	4.98	100	4.69	94
2.50	2.46	98	2.34	94
1.25	1.33	107	1.21	97
0.625	0.684	109	0.612	98
Control	< LOQ		< LOQ

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

%                  = percent of the nominal concentration of the test item

LOQ             = limit of quantification (0.3 mg/L of the test item)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item Basic Red 15 phosphate, the 48-hours EC50 for Daphnia magna was 3.54 mg/L (95% confidence limits: 2.50 – 5.00 mg/L).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Basic Red 15 phosphate were determined at the test facility according to OECD 202 (2004) from 2018-06-25 to 2018-07-05, with the definitive exposure phase from 2018‑07‑03 to 2018-07-05.

The study was conducted under static conditions over a period of 48 hours with five nominal test item concentrations in the range of0.625 to 10.0 mg/L, prepared with dilution water in a geometric series with a separation factor of 2. All tested concentration levels were visually clear and concentration-depended rose to pink coloured throughout the exposure period.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control.

The concentrations of the test item Basic Red 15 phosphate liquid were analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure (48 hours) in all tested concentration levels and in the control.

The measured concentrations of the test item at the start of the exposure (0 hours) were in the range of 98 to 109% of the nominal values. At the end of the exposure period (48 hours), the measured concentrations were within the range of 91 to 98% of the nominal values.

The measured concentrations of the test item were within ± 20% of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test. Therefore, all effect concentrations given are based on the nominal concentrations of the test item Basic Red 15 phosphate liquid.

The validity criteria of the test guideline were fulfilled.

 

EC₁₀-, EC₅₀- (with 95% Confidence Limits) and EC₁₀₀-Values

(based on the nominal concentrations of the test item)

Effect concentrations		Test duration [hours]		Basic Red 15 phosphate liquid Nominal test item concentration [mg/L]
EC10 (with 95% confidence limits)		24		4.48		(95% Cl: < 0.625 – 9.10)
		48		2.68		(95% Cl: 2.50 – 5.00)
EC50 (with 95% confidence limits)		24		4.91		(95% Cl: 2.52 – > 10.0)
		48		3.54		(95% Cl: 2.50 – 5.00)
EC100		24		> 10.0
		48		5.00