Sodium 2-[[[2,5-dichloro-4-[(2-methyl-1H-indol-3-yl)azo]phenyl]sulphonyl]amino]ethanesulphonate

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 10 16 to 2017-10-19, with the definitive exposure phase from 2017 10 17 to 2017-10-19

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:

Sodium 2-[[[2,5-dichloro-4-[(2-methyl-1H-indol-3-yl)azo]phenyl]sulphonyl]amino]ethanesulphonate

EC Number:

273-402-9

EC Name:

Sodium 2-[[[2,5-dichloro-4-[(2-methyl-1H-indol-3-yl)azo]phenyl]sulphonyl]amino]ethanesulphonate

Cas Number:

68959-19-3

Molecular formula:

C17H16Cl2N4O5S2.Na

IUPAC Name:

sodium 2-[[[2,5-dichloro-4-[(2-methyl-1H-indol-3-yl)azo]phenyl]sulphonyl]amino]ethanesulphonate

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The saturated solution 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).

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples of the fresh media (control and saturated solution) were taken after preparation of the saturated solution and analyzed. At the end of the exposure (48 hours), samples of the old media (control and saturated solution) 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 concentration.

Test solutions

Vehicle:

no

Details on test solutions:

Limit concentration
A saturated solution with a nominal loading of 100 mg/L of the test item was tested in a limit test. The limit loading level was selected based on the results of a non-GLP preliminary range finding test.

Preparation of the Saturated solution
The saturated solution with a nominal loading of 100 mg test item/L was prepared 24 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out and transferred with an appropriate amount of dilution water. The saturated solution was stirred for 24 hour (1100 rpm) with a magnetic stirrer at room temperature. 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 10 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. 20 µEm-2  s-1

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:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution water
0 hours:Total hardness [mg CaCO3/L]: 169

Test temperature:

18 - 22°C, constant within ± 1°C
20.8 °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 and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids were
used for each loading level and the control.

Age of the daphnidsat 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 was not necessary, because 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 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.:

> 26.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

other: EC10/50/100

Effect conc.:

> 26.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

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 10-05 to 2017-10-06.

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 1.92 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 1.11 - 2.00 mg/L

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 geometric mean measured 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 Prism, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

Immobilization Rates after 24 and 48 hours ofExposure in the Definitive Test

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

Acid Yellow 230 Nominal loading of the test item [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100*	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

* = saturated solution

 

 

 Absolute Numbers of immobileDaphnids after 24 and 48 hours ofExposure in the Definitive Test

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

Acid Yellow 230 Nominal loading of the test item [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
100*	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

* = saturated solution

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In the saturated solution prepared with a nominal loading of 100 mg/L of the test item Acid Yellow 230, no effects on Daphnia magna were observed.
Based on the geometric mean measured concentrations of the test item Acid Yellow 230, the 48 hours-EC10 / 50 / 100 for Daphnia magna was > 26.2 mg/L.

No effects were found at the maximum water solubility level in Elendt M4 medium.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of a saturated solution with a nominal loading of 100 mg/L of the test item AcidYellow 230 were determined at the test facility according to OECD 202 (2004) from 2017‑10‑16 to 2017-10-19, with the definitive exposure phase from 2017‑10‑17 to 2017-10-19.

The limit test was conducted under static conditions over a period of 48 hours.The saturated solution was yellow and visually clear at the start of the exposure phase. After 24 and 48 hours of exposure, yellow flakes were on the bottom of the test vessels.Twenty daphnids, divided into 4 replicates with 5 daphnids each, were exposed to the saturated solution and the control.

The concentration of the test item AcidYellow 230 was 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) in the saturated solution and in the control.

The measured concentration in the saturated solution was 29.6 mg/L at the start of the exposure (0 hours) and 23.2 mg/L at the end of the exposure (48 hours).The geometric mean measured concentration is 26.2 mg/L.

The effect concentrations are based on the geometric mean measured concentration of the saturated solution of the test item Acid Yellow 230.

No effects were found at the maximum water solubility level in Elendt M4 medium.

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]	Acid Yellow230 Geometric mean measured concentration of the test item [mg/L]
EC10 / 50 / 100	24	> 26.2
	48	> 26.2*

* Maximum water solubility level in Elendt M4 medium