5-(benzoylamino)-3-[[5-[[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]methyl]-1-sulpho-2-naphthyl]azo]-4-hydroxynaphthalene-2,7-disulphonic acid, lithium 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:

2018-03-05 to 2018-03-07

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:

4-(benzoylamino)-6-[[5-[[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]methyl]-1-sulpho-2-naphthyl]azo]-5-hydroxynaphthalene-1,7-disulphonic acid, lithium sodium salt

EC Number:

280-426-3

EC Name:

4-(benzoylamino)-6-[[5-[[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]methyl]-1-sulpho-2-naphthyl]azo]-5-hydroxynaphthalene-1,7-disulphonic acid, lithium sodium salt

Cas Number:

83400-11-7

Molecular formula:

C32 H21 Cl F2 N6 O11 S3 . x Li . x Na C32H(21-x-y)ClF2Li(x)N6Na(y)O11S3

IUPAC Name:

lithium sodium 4-benzamido-6-[2-(5-{[(5-chloro-2,6-difluoropyrimidin-4-yl)amino]methyl}-1-sulfonatonaphthalen-2-yl)diazen-1-yl]-5-hydroxynaphthalene-1,7-disulfonate

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 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 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 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 (100 mg/L of the test item was weighed out) was freshly prepared with dilution water (see Table 2) 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 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 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 GmbH & Co. KG, 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]: 241

Test temperature:

18 - 22°C, constant within ± 1°C
19.5 - 20.5 °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]
100 7.45 9.18
50 7.42 9.18
25 7.38 9.19
12.5 7.36 9.17
6.25 7.33 9.11
Control 7.43 9.16


Water Quality Parameters in old Media at the End of the Exposure (48 hours)
(measured in one replicate (containing daphnids) per concentration and control)
Nominal
test item concentration
[mg/L] 48 hours
pH-value Dissolved
O2 concentration
[mg/L] Replicate number
100 7.40 7.90 2
50 7.25 8.36 2
25 7.45 8.36 2
12.5 7.44 8.37 2
6.25 7.49 7.58 2
Control 7.53 8.12 2

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]
2018-03-05 7.44 9.21 20.8 617 241

Dissolved oxygen:

see above

Conductivity:

Dilution water day 0: 617 [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 per concentration level 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 other: mg test item/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 100 other: mg test item/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: mg test item/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: mg test item/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 100 other: mg test item/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 other: mg test item/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

A reference test was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium (acc. to OECD 202 (2004), Annex 3) under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility.
Reference item Potassium dichromate p.a. (SIGMA)
Purity 99.0%
Batch No. MKBV0900V
Storage stability 2021-11-25
Test concentrations 1.00 – 2.00 – 4.00 mg/L
Ranges of validity EC50 (24 hours): 0.6 - 2.4 mg/L, according to AQS P 9/2 (clone 5)
EC50 (24 hours): 0.6 - 2.1 mg/L, according to OECD 202 (clone A)
Test duration 2018-03-13 to 2018-03-14

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 1.66 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)

Reported statistics and error estimates:

Methods of evaluation
No calculations were made because no effects on Daphnia magna were observed.
All effect concentrations (EC10 / 50 / 100) given were based on the nominal concentrations 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 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.

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

Method Validation

Requirements of the method validation

According to SANCO 3029/99 rev.4 (2000) using the following criteria. 

Parameter, Acceptance Criteria and Results of the Method Validation

Parameter	Acceptance criteria	Result
Linearity	5 standard concentrations, r^2≥ 0.992	0.4 to 4.0 mg test item/L (n = 6), r^2≥ 0.992	x
Lowest calibration standard	S/N≥9	0.4 mg test item/L, S/N =373	x
Limit of Detection (LOD)	S/N of≥3	Not necessary if the S/N of thelowest calibration standard >30	-
Limit of Quantification (LOQ)	At least 20% above lowest calibration level after sample preparation	3.00 mg test item/L (1 x LOQ) 100 mg test item/L (33.3 x LOQ)	x
Accuracy^1) (Fortified samples)	Mean recovery rate of 70-110% (ideally 80-100%) per fortification level (2 levels)	1 x LOQ: 101% (n = 5) 33.3 x LOQ: 101% (n = 4)	x
Precision^1)	Relative standard deviation≤20% per fortification level	1 x LOQ: 1.1% 33.3 x LOQ: 0.46%	x
Specificity (HPLC-DAD)	Comparison of spectra of sample peaks against spectra obtained from standard peaks	Spectra compared and test item verified	x
	Blank values < 30% of LOQ	Blank values < 30% of LOQ

x               = Criterion fulfilled

-                 = not determined

1)               = For details, see below

 

Preparation of Fortified Samples

LOQ Level		Control		1		33.3
Stock solution [mg test item/L]		-		100
Medium		-		Daphniadilution water
Spiking solution		-		Stock solution
Replicates		2		5		5
Concentration of the LOQ [mg test item/L]		-		3.00		100
Medium for preparation		Daphniadilution water
Volume of spiking solution [mL]		-		0.12		-
Volume of medium [mL]		-		3.88		-
Dilution factor		-		-		50
Dilution medium		-		-		Daphniadilution medium
Sample volume [mL]		Approx. 1		Approx. 1		0.1
Finale volume [mL]		Approx. 1		Approx. 1		5.0

 

 

 MeasuredConcentrations and Percent of Nominal Concentrations of the Fortified Samples ofReactiveRed 158

 Fortified concentrations*: 3.10 mg/L (1 x LOQ) and 103 mg/L (33.3 x LOQ) of the test item

Replicate	ReactiveRed158
	Daphniadilution water
	1 x LOQ		33.3 x LOQ
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
1	3.06	99	105	101
2	3.14	101	104	101
3	3.12	101	105	101
4	3.14	101	104	100
5	3.14	101	84.7^1)	82^1)
Mean	3.12	101	104	101
SD ±	0.04		0.5
CV [%]	1.1		0.46

Meas. conc.    = measured concentration of the test item, dilution factor taken into account

%                     = percent concentration of the fortified sample

*                       = weighing factor taken into account

¹⁾                      = outlier due to Grubb’s test

SD                   = Standard deviation

CV                   = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

No effects on Daphnia magna were observed. Based on the nominal concentrations of the test item Reactive Red 158, the 48 hours-EC10 / 50 / 100 for Daphnia magna was > 100 mg/L.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test itemReactive Red 158 were determined at the test facility according to OECD 202 (2004) from2018-03-05 to 2018-03-07.

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 ofapprox. 55 g/L (20 °C, OECD 105). The test item concentrations showed a concentration related slightly red colour and were visually clearly dissolved throughout the exposure period.

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

The concentrationsof the test itemwereanalytically verified viaHPLC-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 98 to 104 % of the nominal values. At the end of the exposure period (48 hours), the measured test itemconcentrations in the old mediawere in the range of 96 to 101 % of the nominal values. Since the measured concentrations of the test itemReactiveRed 158remainedstable 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 158.

The validity criteria of the test guideline were fulfilled.

 

EC₁₀-, EC₅₀-and EC₁₀₀-Values

(based on the nominal concentration of the test item)

ReactiveRed158
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