Tetrasodium [3-[[8-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-1-hydroxy-3,6-disulpho-2-naphthyl]azo]-4-hydroxynaphthalene-1,5-disulphonato(6-)]cuprate(4-)

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 2017-12-11 to 2017-12-13.

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:

Tetrasodium [3-[[8-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-1-hydroxy-3,6-disulpho-2-naphthyl]azo]-4-hydroxynaphthalene-1,5-disulphonato(6-)]cuprate(4-)

EC Number:

238-739-8

EC Name:

Tetrasodium [3-[[8-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-1-hydroxy-3,6-disulpho-2-naphthyl]azo]-4-hydroxynaphthalene-1,5-disulphonato(6-)]cuprate(4-)

Cas Number:

14692-76-3

Molecular formula:

C23H10ClCuN7Na4O14S4

IUPAC Name:

tetrasodium [3-[[8-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-1-hydroxy-3,6-disulpho-2-naphthyl]azo]-4-hydroxynaphthalene-1,5-disulphonato(6-)]cuprate(4-)

Test material form:

solid: particulate/powder

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 fresh media at the start of the exposure (0 hours) and in old media at the end of the exposure period (48 hours) as specified below.
The samples were analysed with a HPLC-DAD method, implemented under non-GLP and documented finally in the GLP raw data. The method was validated.

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples were taken from all 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 directly from the test vessels.

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

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution
The stock solution (100 mg/L of the test item were weighed out) was freshly prepared with dilution water before the start of the exposure (0 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 dilution of the stock solution 100 mg/L with dilution water, were tested as follows: 6.25 - 12.5 - 25 - 50 - 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 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 ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, was 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


Composition of the Culture Medium according to ELENDT (1990)
Component Concentration [mg/L]
CaCl2 x 2 H2O 147
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 4.30
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18
Na2EDTA x 2 H2O 5.00
FeSO4 x 7 H2O 1.99
H3BO3 0.29
MnCl2 x 4 H2O 0.36
LiCl 0.30
SrCl2 x 6 H2O 0.15
RbCl 0.071
NaBr 0.016
Na2MoO4 x 2 H2O 0.063
CuCl x 2 H2O 0.017
ZnCl2 0.013
CoCl2 x 6 H2O 0.010
KJ 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamine 0.0010
Biotin 0.00075
pH 8.2  0.8

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]: 176

Test temperature:

18 - 22°C, constant within ± 1°C
19.0 °C

pH:

see any other information on materials and methods incl. tables

Dissolved oxygen:

see any other information on materials and methods incl. tables

Conductivity:

see any other information on materials and methods incl. tables

Details on test conditions:

Number of daphnids 20 daphnids, divided into 4 replicates, each with 5 daphnids, were
and replicates 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 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 were 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:

EC50

Effect conc.:

74.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: CI (54.0 - 94.7)

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

21.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: CI (16.4 - 28.0)

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 2017-12-06 to 2017-12-07. For results of the most recent of the monthly performed reference tests, see the tebale 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 1.64 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 concentrations (EC10 / 50 / 100) were based on the nominal concentrations of the test item.

EC-values and statistical analyses
The EC10- and the 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. All calculations were carried out from the best-fit values with the software GraphPad Prism. The concentration-effect relationships are shown graphically. The EC50-value for the reference item with 95% confidence limits was 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 Prism5, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

Immobilization Ratesafter 24 and 48 hours ofExposure 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
100	100	100	60	80	85	100	100	100	100	100
50.0	40	0	0	40	20	100	80	80	100	90
25.0	0	40	20	20	20	40	80	40	40	50
12.5	0	0	0	0	0	60	20	20	20	30
6.25	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

Absolute Numbers of immobileDaphnids after 24 and 48 hours ofExposure 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 h					48 h
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	5 / 5	5 / 5	3 / 5	4 / 5	17 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
50.0	2 / 5	0 / 5	0 / 5	2 / 5	4 / 20	5 / 5	4 / 5	4 / 5	5 / 5	18 / 20
25.0	0 / 5	2 / 5	1 / 5	1 / 5	4 / 20	2 / 5	4 / 5	2 / 5	2 / 5	10 / 20
12.5	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	3 / 5	1 / 5	1 / 5	1 / 5	6 / 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 test item concentrations were visually clear throughout the exposure period. The concentration levels showed a concentration-related purple color.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item Reactive Blue 13 (Na), the 48 hours-EC50 for Daphnia magna was 21.6 mg/L (95% confidence limits: 16.4 – 28.0 mg/L).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Reactive Blue 13 (Na) were determined at the test facility according to OECD 202 (2004) from 2017-12-11 to 2017-12-13.

The study was conducted under static conditions over a period of 48 hours with five nominal concentrations of the test item ranging from 6.25 to 100 mg/Lin a geometric series with a separation factor of 2.

The test item is a dark brown solid. The water solubility is approx. 238 g/L. All test item concentrations were visually clear throughout the exposure period. The concentration levels showed a concentration-related purple color.

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

The concentrationsof the test itemwereanalytically verified via HPLC-DAD in fresh media at the start of the exposure (0 hours) and in old media at the end of the exposure period (48 hours) in all tested concentration levels and in the control.

The measured concentrations of the test item in fresh media at the start of the exposure (0 hours) were in the range of 82 to 105% of the nominal values in all test item concentrations. At the end of the exposure period (48 hours), the measured test itemconcentrations in the old mediawere in the range of 88 to 106% of the nominal values of all test concentrations.The measured concentrations of the test item Reactive Blue 13 (Na) remained stable within ± 20% of the nominal concentrations throughout the exposure period, which indicates that the test item concentrations were successfully maintained for the duration of the test.

All effect concentrations (EC_{10 / 50 / 100}) given in the table below are based on the nominal concentrations of the test item Reactive Blue 13 (Na). 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)

Reactive Blue 13 (Na)
Effect concentrations	Test duration [hours]	Nominal concentrations of the test item [mg/L]
EC10	24	27.8 (< 6.25 – 81.1)
	48	8.30 (< 6.25 – 11.9)
EC50	24	74.5 (54.0 – 94.7)
	48	21.6 (16.4 – 28.0)
EC100	24	> 100
	48	100