Trisodium 4-[[4-[[(2,3-dichloro-6-quinoxalinyl)carbonyl]amino]-2-sulphonatophenyl]azo]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate

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:

2017-03-13 to 2017-03-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

p-[4,5-dihydro-4-[[2-methoxy-5-methyl-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonic acid, sodium salt

EC Number:

263-417-9

EC Name:

p-[4,5-dihydro-4-[[2-methoxy-5-methyl-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonic acid, sodium salt

Cas Number:

62121-75-9

Molecular formula:

C20H22N4O11S3.xNa C20H(22-x)N4NaxO11S3; x<=2

IUPAC Name:

sodium 4-[4-(2-{2-methoxy-5-methyl-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl]benzene-1-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 fresh media at the start of the exposure (0 hours) and in old media at the end of the exposure (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 (0 hours), samples of the fresh media were taken after preparation of the limit concentration and analyzed.
At the end of the exposure (48 hours), samples of the old media were taken for analysis from additional replicates, which were prepared with test media, but without daphnids. These additional replicates were incubated under test conditions until sampling.

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

Test solutions

Vehicle:

no

Details on test solutions:

Limit concentration A nominal concentration of 100 mg/L of the test item was tested in a limit test.

Preparation of the saturated solution

The limit concentration (100 mg/L test item were weighed out) was prepared with dilution water one day before the start of the exposure (at -24 hours). The test item was mixed with the dilution water by constant stirring with a magnetic stirrer at approximately 1100 rpm for 24 hours at room temperature. After completion of stirring, undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used as a limit concentration in the test. During filtration, the filter was always kept covered.

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), 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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

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:

A nominal concentration of 100 mg/L of the test item was tested in a limit test.

Details on test conditions:

Test method The study was performed under static 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 composition as the culture medium (see Table 2)

Number of daphnids 20 daphnids, divided into 4 replicates, each with 5 daphnids, were
and replicates used for the limit concentration 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 forthe 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

Results and discussion

Effect concentrations

Effect concentrations 1

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 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 43.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

recovery lower due to hydrolysis activity

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 2017-03-09 to 2017-03-10.

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 2.05 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 2.00 - 3.71 mg/L


 

Reported statistics and error estimates:

Methods of evaluation
The effects of the limit concentration were deduced directly from the observed immobilization rates.
The effect levels (EC10 / 50 / 100) given are based on the geometric mean measured concentration.

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 Prism, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

The percentage of immobility, determined in the limit concentration and in the control after 24 and 48 hours, is given below. The absolute numbers of immobile daphnids are presented below. Other adverse effects did not appear.

The study was performed under static conditions.

In thelimit concentrationof 100 mg/L of the test itemReactive Yellow 15, which corresponds to a geometric mean measured concentration of 43.8 mg/L, no effects on Daphnia magna were observed.

Immobilization Ratesafter 24 and 48 hours ofExposure in the Definitive Test

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

ReactiveYellow 15 Geometric mean measured test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
43.8	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)

ReactiveYellow 15 Geometric mean measured 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
43.8	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

 

The limit concentration was yellow and visually clear during the exposure period.

 

Measured Exposure Concentrations during the Definitive Test

 

The concentration of the test itemReactiveYellow 15 was 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) in the limit concentration and in the control.

The measured concentration in the limit concentration was 52.7 mg/L at the start of the exposure (0 hours) and 36.5 mg/L at the end of the exposure (48 hours), representing 69% of time 0 measured test item concentration.The geometric mean measured concentration is 43.8 mg/L.

 Measured Concentrations of the Test Itemduring the Definitive Test

Sampling date:	2017-03-14 Start of the exposure, 0 hours	2017-03-16 End of the exposure, 48 hours		Geometric mean measured test item concentration [mg/L]
Start of analysis:	2017-03-14	2017-03-16
Nominal test item concentration [mg/L]	Reactive Yellow 15
	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%
100	52.7	36.5	69	43.8
Control	< LOQ	< LOQ

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

%                     = percent of the initially measured concentration of the test item

LOQ       = limit of quantification of the analytical method (0.500 mg test item/L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In the limit concentration of 100 mg/L of the test item Reactive Yellow 15, which corresponds to a geometric mean measured concentration of 43.8 mg/L, no effects on Daphnia magna were observed.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effectsof a limit concentration of 100 mg/Lof the test item Reactive Yellow 15 were determined at the test facility according to OECD 202 (2004) from 2017-03-13 to 2017-03-16, with the definitive exposure phase from 2017‑03‑14 to 2017-03-16.

The limit test was conducted under static conditions over a period of 48 hours.The limit concentration was yellow and visually clear during the exposure period.Twenty daphnids, divided into 4 replicates with 5 daphnids each, were exposed to the limit concentration and the control.

The concentration of the test item ReactiveYellow 15 was 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) in the limit concentration and in the control.

The measured concentration in the limit concentration was 52.7 mg/L at the start of the exposure (0 hours) and 36.5 mg/L at the end of the exposure (48 hours), representing 69% of time 0 measured test item concentration.The geometric mean measured concentration is 43.8 mg/L.

The effect concentrations given are based on the geometric mean measured concentration and the nominal concentration of the limit concentration of the test item ReactiveYellow 15.

The validity criteria of the test guideline were fulfilled.

 

EC_{10 / 50 / 100}-Values

(based on the geometric mean measured concentration of the test item)

Effect concentrations	Test duration [hours]	Reactive Yellow15
		Geometric mean measured test item concentration [mg/L]	Nominal test item concentration [mg/L]
EC10 / 50 / 100	24	> 43.8	> 100 mg/L
	48	> 43.8	> 100 mg/L

  

 

In the limit concentration of 100.0 mg/L of the test itemReactiveYellow 15,
which corresponds to a geometric mean measured concentration of 43.8 mg/L,

no effects onDaphnia magnawere observed.