[2-[ethyl[3-methyl-4-[(3-phenyl-1,2,4-thiadiazol-5-yl)azo]phenyl]amino]ethyl]trimethylammonium methyl sulphate

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:

from 2018-05-14 to 2018-05-17, with the definitive exposure phase from 2018-05-15- to 2018-05-17

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[2-[ethyl[3-methyl-4-[(3-phenyl-1,2,4-thiadiazol-5-yl)azo]phenyl]amino]ethyl]trimethylammonium methyl sulphate

EC Number:

248-957-5

EC Name:

[2-[ethyl[3-methyl-4-[(3-phenyl-1,2,4-thiadiazol-5-yl)azo]phenyl]amino]ethyl]trimethylammonium methyl sulphate

Cas Number:

28313-51-1

Molecular formula:

C22H29N6S.CH3O4S

IUPAC Name:

{2-[ethyl({3-methyl-4-[(E)-2-(3-phenyl-1,2,4-thiadiazol-5-yl)diazen-1-yl]phenyl})amino]ethyl}trimethylazanium methyl sulfate

Test material form:

solid: particulate/powder

Details on test material:

Basic Red 23 MeSO4

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 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:

Stock solution
A stock solution of 100 mg test item/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 test item solution was stirred for 24 ± 1 hours (1100 rpm, room temperature) 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. The following filtrate, i.e. the test item solution, was used as a test item solution in the test. During filtration, the filter was always kept covered.
The test item solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.

Test concentrations
5 test item concentrations in a geometric series with a separation factor of 2.2, prepared by dilution of the stock solution 100 mg/L with dilution water, were tested as follows:
0.640 - 1.41 - 3.10 - 6.82 - 15.0 mg/L, corresponding to the geometric mean measured concentrations 0.470 – 1.13 – 2.45 – 5.92 – 13.3 mg/L.
The test item concentrations were selected 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 (Clone 5).

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

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.

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 +/- 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. The algae were cultured at the test facility.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution water
0 hours:Total hardness [mg CaCO3/L]: 248

Test temperature:

18 - 22°C, constant within ± 1°C
20 °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
test item concentration
[mg/L] pH-value Dissolved O2 concentration [mg/L]
15.0 8.11 8.92
6.82 8.18 8.91
3.10 8.19 8.95
1.41 8.30 8.93
0.640 8.33 8.94
Control 8.35 8.97


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
test item concentration
[mg/L] pH-value Dissolved O2 concentration [mg/L] Replicate number
15.0 7.69 8.42 1
6.82 7.72 8.34 2
3.10 7.73 8.58 1
1.41 7.80 8.33 1
0.640 7.82 8.37 1
Control 7.90 8.33 1

Water Quality Parameters of the Dilution Water at the Start of the Exposure (0 hours)
Dilution water dated: pH-value Dissolved O2 concentration [mg/L] Temperature [°C] Conductivity [µS/cm] Total hardness [mg CaCO3/L]
15.05.2018 8.21 9.06 20.2 615 248

Dissolved oxygen:

see above

Conductivity:

See above

Nominal and measured concentrations:

nominal concentrations: 0.640 - 1.41 - 3.10 - 6.82 - 15.0 mg/L
geometric mean measured concentrations: 0.470 – 1.13 – 2.45 – 5.92 – 13.3 mg/L.

Details on test conditions:

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 for the limit concentration 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 out 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

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

2.18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: <0.640 - 4.96

Effect concentrations 2

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

15.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: extrapolated value is given, because the calculated value is near to the concentration range (0.640 – 15.0 mg/L nominal)

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

2.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 1.57 - 3.08

Effect concentrations 5

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 5.41 - 7.28

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

Test of the Reference Item

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 05-03 to 2018-05-04.

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


 

Reported statistics and error estimates:

Methods of evaluation
The EC100-values after 24 and 48 hours were empirically derived from the observation data. 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 concentrations.

EC-values and statistical analyses
The EC10- and EC50-values (after 24 and 48 hours 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 concentration-effect relationships are shown graphically.
An EC50 value for the reference item was calculated accordingly. The respective 95% 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 95% confidence limits, since the calculated values were classified as ambiguous by the software.
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.
- SigmaPlot, SPSS INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

 Measured Exposure Concentrations during the Definitive Test

 

The concentrations of the test item Basic Red 23 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 90 to 97% of the nominal values. At the end of the exposure period (48 hours), the measured concentrations were within the range of 56 to 83% of the nominal values.

The measured test item concentrations were not within the range of ± 20% of the nominal concentrations. Therefore, the geometric mean measured concentrations were calculated.

 Measured Concentrations and Percent of the Nominal Concentration of the Test ItemBasic Red23during the Definitive Test

Sampling date	Start of the exposure, fresh media, 0 hours		End of the Exposure, old media, 48 hours
Nominal test item concentration [mg/L]	Basic Red23
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Geometric mean measured concentration [mg/L]
15.0	14.2	95	12.5	83	13.3
6.82	6.57	96	5.33	78	5.92
3.10	2.78	90	2.15	69	2.45
1.41	1.32	93	0.978	69	1.13
0.640	0.619	97	0.357	56	0.470
Control	< LOQ		< LOQ

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

%                     = percentage of the nominal concentration of the test item

LOQ                = limit of quantification (0.300 mg test item/L)

 Preliminary Range Finding Test (non-GLP)

 

A non-GLP preliminary range finding test was conducted at the test facility under static conditions over a period of 48 hours with three nominal test item concentrations of 1, 10 and 100 mg/L prepared with dilution water. The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle).

The test item solution was prepared in the same way as in the definitive test. The Tyndall effect was negative for the saturated solution after filtration of undissolved material. The test item solutions were visually clear and concentration related pink to red coloured.

In the range finding test, two replicates per concentration level and control, each with five daphnids, were tested.

Immobilization Rates in the non-GLP Preliminary Range Finding Test

(n = 10, divided into 2 replicates with 5 daphnids each)

Nominal test item concentration [mg/L]		IMMOBILIZATION [%]
		24 hours				48 hours
		Replicates				Replicates
		1		2		MV		1		2		MV
100		100		100		100		100		100		100
10		40		60		50		100		100		100
1		0		0		0		0		0		0
Control		0		0		0		0		0		0

 

 Measured Concentrations of Basic Red 23 during the non-GLPPreliminary Range Finding Test

Analytical system: HPLC-DAD

Sampling	Start of the exposure, 0 hours		End of the Exposure, 48 hours
Nominal test item concentration [mg/L]	BasicRed 23
	Measured concentration [mg/L]	%	Measured concentration [mg/L]	%
100	97.4	97	99.2	99
10	8.80	88	7.75	78
1	0.822	82	0.600	60
Control	< LCL		< LCL

%                     = percent of the nominal concentration of the test item

LCL                  = lowest calibration Level (50µg/L of the test item)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the geometric mean measured concentrations of the test item Basic Red 23, the 48-hours EC50 for Daphnia magna was 5.33 (95% confidence limits: 4.58 – 6.34 mg/L).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item BasicRed 23 were determined at the test facility according to OECD 202 (2004) from 2018-05-14 to 2018-05-17, with the definitive exposure phase from 2018-05-15- to 2018-05-17.

The study was conducted under static conditions over a period of 48 hours with five nominal test item concentrations in a geometric series with a separation factor of 2.2.All tested concentration levels were concentration-depended pink coloured throughout the exposure period.

 

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

The concentrations of the test item Basic Red 23 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 90 to 97% of the nominal values. At the end of the exposure period (48 hours), the measured concentrations were within the range of 56 to 83% of the nominal values.

The initially measured concentrations of the test item were within ± 20% of the nominal concentrations. Until the end of the exposure period, the test item recoveries decreased to a minimum of 56% of the nominal values, which indicates that the test item is not stable under test conditions. Therefore, all effect concentrations given are based on the nominal concentrations and the geometric mean measured concentrations of the test item Basic Red 23.

The validity criteria of the test guideline were fulfilled.

 

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

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

Effect concentrations		Test duration [hours]		BasicRed 23
			Nominal test item concentration [mg/L]		Geometric mean measured test item concentration [mg/L]
EC10 (with 95% confidence limits)		24		2.18 (<0.640 - 4.96)		1.73 (<0.470 – 4.28)
		48		2.25 (1.57 – 3.08)		1.76 (1.19 – 2.47)
EC50 (with 95% confidence limits)		24		15.4 *		13.7*
		48		6.21 (5.41 – 7.28)		5.33 (4.58 – 6.34)
EC100		24		> 15.0		> 13.3
		48		15.0		13.3

* = extrapolated value is given, because the calculated value is near to the concentration range (0.640 – 15.0 mg/L nominal)