Disodium 1-amino-4-[[3-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-2-methyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

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 2018-01-31 to 2018-02-02

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:

Disodium 1-amino-4-[[3-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-2-methyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

EC Number:

276-374-6

EC Name:

Disodium 1-amino-4-[[3-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-2-methyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Cas Number:

72139-17-4

Molecular formula:

C25H16ClF2N5O8S2.2Na

IUPAC Name:

disodium 1-amino-4-({3-[(5-chloro-2,6-difluoropyrimidin-4-yl)amino]-2-methyl-5-sulfophenyl}amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The limit concentration and the control 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).

Determination of the test item
The limit concentration level 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 (48 hours).


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 Recoveries of the test item should be within ± 20% of the initially
monitoring (target) measured concentrations.

Test solutions

Vehicle:

no

Details on test solutions:

Limit concentration
A limit concentration of 100 mg/L of the test item Reactive Blue 114 was tested. The limit concentration was based on the results of a non-GLP preliminary range finding test.

Preparation of the limit concentration
The limit concentration (100 mg/L of the test item were weighed out) was freshly prepared with dilution water (Table 2) prior to the start of the exposure (at 0 hours). The limit concentration was mixed thoroughly by manual agitation.

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 Dr. Nowak, Mayenbrook 1, 28870 Ottersberg, 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, 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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution water
0 hours:Total hardness [mg CaCO3/L]: 208

Test temperature:

18 - 22°C, constant within ± 1°C
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 limit concentration and control)
Nominal test item concentration
[mg/L] pH-value Dissolved
O2 concentration [mg/L]
100 7.64 9.08
Control 7.68 9.10


Water Quality Parameters in old Media at the End of the Exposure (48 hours)
(measured in one replicate (containing daphnids) of the limit concentration and the control)
Nominal test item concentration
[mg/L] 48 hours (old media)
pH-value Dissolved
O2 concentration [mg/L]
100 7.49 8.15
Control 7.87 8.17


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]
2018-01-31 7.74 9.19 20.8 640 208

Dissolved oxygen:

see above

Conductivity:

Dilution water day 0: 640 [µS/cm]

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.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 100 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.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 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.:

> 100 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 2018-01-30 to 2018-01-31. 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 1.47 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

Reported statistics and error estimates:

Methods of evaluation The EC10 / 50 / 100-values after 24 and 48 hours were empirically derived from the observation data.

EC-values and statistical analyses for reference item
The EC50-value for the reference item and its confidence limits 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 concentration-effect relationships of the test item are shown graphically.

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

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal limit concentration of the test item Reactive Blue 114, the 48-hour EC50 for Daphnia magna was > 100 mg/L. No effects on Daphnia magna STRAUS were observed.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Reactive Blue 114 were determined at the test facility according to OECD 202 (2004) from 2018-01-31 to 2018-02-02.

The study was conducted under static conditions over a period of 48 hours with a test item solution with the nominal limit concentration of 100 mg/Lof the test item Reactive Blue 114.

The test item concentration was blue, visually clear throughout the exposure.

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

The concentrations of the test item Reactive Blue 114 were analytically verified via HPLC-DAD in fresh media at the start (0 hours) and in old media at the end of the exposure (48 hours) in the limit concentration and in the control.

The measured concentration of the test item at test start was 107% of the nominal test item concentration. At 48 hours, the measured concentration was 100% of the nominal test item concentration.

The measured test item concentrations remained stable within±20% of the nominal limit concentration of the test item over the test period. The effect concentrations given in the table below are based on the nominal limit concentration of the test item Reactive Blue 114.

The validity criteria of the test guideline were fulfilled.

 

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

(based on the nominal concentration of the test item)

Effect levels		Test duration [hours]		Reactive Blue 114 Nominal test item concentration [mg/L]
EC10 (with 95% confidence limits)		24		> 100
		48		> 100
EC50 (with 95% confidence limits)		24		> 100
		48		> 100
EC100		24		> 100
		48		> 100