Cuprate(6-), [2-[[[[3-[[4-chloro-6-[[4-[[4-chloro-6-[(3-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,5-disulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulfophenyl]azo]phenylmethyl]azo]-5-sulfobenzoato(8-)]-, pentasodium hydrogen, (SP-4-3)-

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-10 to 2017-05-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All tested concentration levels and the control were analytically verified via photometric analysis in fresh media at the start of exposure and at water renewal (0 and 24 hours) and in 24-hours old media at renewal and at the end of the exposure (24 and 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 and at renewal (0 and 24 hours), samples of the fresh media were taken after preparation of all concentration levels and analyzed. At renewal and at the end of the exposure (24 and 48 hours), samples of the 24-hours old media were taken directly from the test vessels and analyzed.

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

Vehicle:

no

Details on test solutions:

Stock solution
A stock solution (100 mg/L of the test item were weighed out) was freshly prepared with dilution water (see Table 2) prior to the start of the exposure (0 hours) and the water renewal (24 hours). The stock solution was mixed thoroughly by manual agitation.

Test concentrations
5 test item concentrations in a geometric series with a separation factor of 2, prepared by diluting the stock solution of 100 mg/L with dilution water, were tested as follows: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L.
The test 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 method
Based on the results of a static preliminary range finding test, the study was performed under semi-static conditions with a renewal of the test solutions after 24 hours, since the measured concentrations did not remain stable over a period of 48 hours under static test conditions.

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), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.

Culture feeding
The culture 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.

Origin of the food algae
Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

see any other information on material and methods incl. tables

Test temperature:

see any other information on material and methods incl. tables

pH:

see any other information on material and methods incl. tables

Dissolved oxygen:

see any other information on material and methods incl. tables

Conductivity:

see any other information on material and methods incl. tables

Nominal and measured concentrations:

Nominal: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L

Details on test conditions:

Test method
Based on the results of a static preliminary range finding test, the study was performed with a semi-static test design with a renewal of the test solutions after 24 hours, since the measured concentrations did not remain stable over a period of 48 hours under static test 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 in each test replicate (see ‘Application’)

Dilution water
Same as culture medium

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

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for at the start of the exposure 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 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 (start of the exposure) or test solution (water renewal) by 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

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

42.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 34.0 - 47.6

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

15.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 12.0 - 20.4

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 2017-05-04 to 2017-05-05.

EC50-Value (with 95% confidents limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 1.60 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000)
0.6 - 2.1 mg/L, acc. to OECD 202 (2004)
95% confidence limits 1.43 - 1.79 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.
All effect concentrations (EC10 / 50 / 100) given are based on the nominal test item concentrations, since the measured test item concentrations were within ± 20% of the nominal concentrations. The concentration-effect relationships of the test item after 24 and 48 hours are shown graphically.

EC-values and statistical analyses
The EC10- and the EC50-values (after 24 and 48 hours) were calculated by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism. The EC50-value for the reference item and its 95% confidence limits were calculated accordingly.
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

The daphnids counted immobile were trapped in the meniscus of the test solutions. They were put under the water surface by dripping with the test solution by a pipette and counted immobile, if they were not able to swim within the water body.Other adverse effects did not appear.

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

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

ReactiveBlue 160 Nominal test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	100^1)	100^1)	100^1)	100^1)	100	100^1)	100^1)	100^1)	100^1)	100
50.0	60^1)	40^1)	100^1)	80^1)	70	100^1)	100^1)	80^1)	80^1)	90
25.0	0	0	0	20^1)	5	40^1)	100^1)	60^1)	60^1)	65
12.5	0	0	0	0	0	20^1)	60^1)	20^1)	60^1)	40
6.25	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

¹⁾= daphnids trapped in the meniscus of the test solutions

Absolute Numbers of immobileDaphnids after 24 and 48 hours ofExposure in the Definitive Test

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

ReactiveBlue 160 Nominal test item concentration [mg/L]	Number of immobile Daphnids / Total number of Daphnids
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
50.0	3 / 5	2 / 5	5 / 5	4 / 5	14 / 20	5 / 5	5 / 5	4 / 5	4 / 5	18 / 20
25.0	0 / 5	0 / 5	0 / 5	1 / 5	1 / 20	2 / 5	5 / 5	3 / 5	3 / 5	13 / 20
12.5	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	1 / 5	3 / 5	1 / 5	3 / 5	8 / 20
6.25	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
Control	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

Additional Observations during the Definitive Test

All tested concentration levels were visually clear and concentration-related coloured from dark blue (100 mg/L) to light blue (6.25 mg/L) throughout the exposure period.

Neither immobility or any adverse effects were observed at the concentration level 6.25 mg/L and in the control.

 

Measured Exposure Concentrations during the Definitive Test

 

The concentrations of the test item Reactive Blue 160 were analytically verified via photometric analysis at a wavelength of 618 nm in fresh media of all concentration levels and the control at the start of the exposure and at renewal (0 and 24 hours) and in 24-hours old media at renewal and at the end of the test (24 and 48 hours).

The measured concentrations of the test item in fresh media at the start of the exposure and at renewal (0 and 24 hours) were in the range of 98 to 105% of the nominal values and 90 to 105% in 24-hours old media at renewal and at the end of the test (24 and 48 hours).

The measured test item concentrations were all 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 Nominal Concentration of the Test ItemReactiveBlue 160during the Definitive Test

Sampling date	2017-05-10 Start of the  exposure interval 0 hours		2017-05-11 End of the  exposure interval 24 hours		2017-05-11 Start of the  exposure interval 24 hours		2017-05-12 End of the  exposure interval 48 hours
Start of analysis	2017-05-10		2017-05-11		2017-05-11		2017-05-12
Nominal test item concentration [mg/L]	ReactiveBlue 160
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
100	105	105	101	101	105	105	92.9	93
50.0	51.7	103	51.7	103	51.7	103	52.5	105
25.0	25.9	103	25.0	100	25.9	103	25.0	100
12.5	12.9	103	12.3	98	12.6	101	12.0	96
6.25	6.14	98	5.66	90	6.30	101	5.98	96
Control	< LOQ		< LOQ		< LOQ		< LOQ

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

%                  = percent of the nominal concentration of the test item

LOQ       = limit of quantification of the analytical method (0.750 mg/L of the test item)

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item Reactive Blue 160, the 48-hours EC50 for Daphnia magna STRAUS was 15.9 mg/L (95% confidence limits: 12.0 - 20.4 mg/L).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Reactive Blue 160 were determined at the test facility according to OECD 202 (2004) from 2017-05-10 to 2017-05-12.

The study was conducted under semi-static conditions over a period of 48 hours with 5 concentrations of the test item Reactive Blue 160 in the range of 6.25 - 100 mg/L, prepared in a geometric series with a separation factor of 2.

All tested concentration levels were visually clear and concentration-related coloured from dark blue (100 mg/L) to light blue (6.25 mg/L) 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 Reactive Blue 160 were analytically verified via photometric analysis at a wavelength of 618 nm in fresh media of all concentration levels and the control at the start of the exposure and at renewal (0 and 24 hours) and in 24-hours old media at renewal and at the end of the test (24 and 48 hours).

The measured concentrations of the test item in fresh media at the start of the exposure and at renewal (0 and 24 hours) were in the range of 98 to 105% of the nominal values and 90 to 105% in 24-hours old media at renewal and at the end of the test (24 and 48 hours).

The measured test item concentrations were all 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 in the table below are based on the nominal concentrations of the test item Reactive Blue 160.

The validity criteria of the test guideline were fulfilled.

 

EC₁₀-, EC₅₀-(with 95% Confidence Limits), EC₁₀₀-Valuesand NOEC

(based on the nominal concentrations of the test item)

ReactiveBlue 160
Effect concentrations	Test duration [hours]	Nominal test item concentrations [mg/L]
EC10 (with 95% confidence limits)	24	28.7	(Cl: 21.1 – 38.8)
	48	7.56	(Cl:≤6.25 – 9.99)
EC50 (with 95% confidence limits)	24	42.9	(Cl: 34.0 – 47.6)
	48	15.9	(Cl: 12.0 – 20.4)
EC100	24	100
	48	100

Description of key information

Based on the nominal concentrations of the test item Reactive Blue 160, the 48-hours EC50 for Daphnia magna STRAUS was 15.9 mg/L (95% confidence limits: 12.0 - 20.4 mg/L) in an OECD 202 guideline study..

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

15.9 mg/L