7-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-4-hydroxy-3-[(4-methoxy-2-sulphophenyl)azo]naphthalene-2-sulphonic acid, sodium salt

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:

2018-06-05 to 2018-06-07

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:

7-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-4-hydroxy-3-[(4-methoxy-2-sulphophenyl)azo]naphthalene-2-sulphonic acid, sodium salt

EC Number:

286-839-5

EC Name:

7-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-4-hydroxy-3-[(4-methoxy-2-sulphophenyl)azo]naphthalene-2-sulphonic acid, sodium salt

Cas Number:

85391-83-9

Molecular formula:

C21H14ClF2N5O8S2.xNa

IUPAC Name:

7-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-4-hydroxy-3-[(4-methoxy-2-sulphophenyl)azo]naphthalene-2-sulphonic acid, sodium salt

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 the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure period (48 hours) as specified below. The analytical 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 of the fresh media (control and limit concentration) were taken after preparation of the limit concentration and analyzed.
At the end of the exposure (48 hours), samples of the old media (control and limit concentration) were taken directly from the test vessels.

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 of the test item was weighed out) was freshly prepared with dilution water prior to the start of the exposure (at 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 (see Table 2), were tested as follows:
6.25 - 12.5 - 25.0 - 50.0 - 100 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 group.

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:

see any other information on materials and methods incl. tables

Test temperature:

see any other information on materials and methods incl. tables

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:

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:

other: EC10/50/100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

other: EC10/50/100

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 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 EC10 / 50 / 100-values after 24 and 48 hours for the test item were empirically derived from the observation data. Since no effects on Daphnia magna were observed in the limit concentration of the test item, no calculations were made. All effect concentrations (EC10 / 50 / 100) given were based on the nominal concentration of the test item.

EC-values and statistical analyses for reference item
An EC50-value was calculated for the reference item by sigmoidal dose-response regression. The respective 95 % confidence limits for the EC50 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 calculation was not possible 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

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 four nominal test item concentrations of 0.1, 1, 10 and 100 mg/L prepared with dilution water. The test item solutions were visually clear and concentration dependent red to orange coloured.

The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle). 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		0		20		10		0		20		10
10		0		0		0		0		0		0
1		0		0		0		0		0		0
0.1		0		0		0		0		0		0
Control		0		0		0		0		0		0

 

MeasuredConcentrations ofReactiveRed 123during 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]	ReactiveRed 123
	Measured concentration [mg/L]	%	Measured concentration [mg/L]	%
100	96.4	96	98.6	99
10	8.94	89	9.51	95
1	0.996	100	0.965	97
0.1	0.121	121	0.0855	85
Control	< LCL		< LCL

%                     = percent of the nominal concentration of the test item

LCL                  = Lowest Calibration Level (0.05 mg/L of the test item)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In all tested concentrations of the test item Reactive Red 123, no effects on Daphnia magna were observed.
Based on the nominal concentrations of the test item Reactive Red 123, the 48 hours-EC10 / 50 / 100 for Daphnia magna were > 100 mg/L.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Reactive Red 123 were determined at the test facility according to OECD 202 (2004) from 2018-06-05 to 2018-06-07.

The study was conducted under static conditions over a period of 48 hours with five concentration levels prepared by dilution from the stock solution of the test item (nominal concentration 100 mg/L). The tested concentration levels were 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L.

The test itemis a red powder with awater solubility of 50 g/L (25 °C). The test item concentrations showed aconcentration-dependent red to orange colouringand were visually clear throughout the exposure period. The control was colourless and visually clear as well.

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

The concentrationsof the test item 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) 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 96 to 99% of the nominal values. At the end of the exposure period (48 hours), the measured test item concentrations in the old media were in the range of 95 to 98% of the nominal values. Since the measured concentrations of the test item Reactive Red 123 remained stable within ± 20% of the nominal concentrations throughout the exposure period, the nominal concentrations were used for evaluation.

All effect concentrations (EC_{10 / 50 / 100}) are based on the nominal concentrations of the test item Reactive Red 123.

The validity criteria of the test guideline were fulfilled.

 

EC₁₀-, EC₅₀-and EC₁₀₀-Values

(based on the nominal concentration of the test item)

Reactive Red 123
Effect concentrations	Test duration [hours]	Nominal test item concentration [mg test item/L]
EC10	24	> 100
	48	> 100
EC50	24	> 100
	48	> 100
EC100	24	> 100
	48	> 100