N-[2-[(2,6-dicyano-p-tolyl)azo]-5-(diethylamino)phenyl]methanesulphonamide

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:

2018-02-19 to 2018-03-15, with the definitive exposure phase from 2018-03-13 to 2018-03-15

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)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-[2-[(2,6-dicyano-p-tolyl)azo]-5-(diethylamino)phenyl]methanesulphonamide

EC Number:

269-887-1

EC Name:

N-[2-[(2,6-dicyano-p-tolyl)azo]-5-(diethylamino)phenyl]methanesulphonamide

Cas Number:

68385-96-6

Molecular formula:

C20H22N6O2S

IUPAC Name:

N-[2-[(2,6-dicyano-p-tolyl)azo]-5-(diethylamino)phenyl]methanesulphonamide

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) Since the measured test item concentration was even below the LOD in fresh media at the start of the exposure period, the analytical monitoring at the end of the exposure was waived and the test item concentration was considered to be below the LOD in the 48 hours old media, too.

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.

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 saturated solution
A saturated solution (100 mg/L test item was weighed out) was prepared with dilution water (see Table 2) 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 the highest concentration level and as a stock solution for the preparation of further dilution levels by dilution with dilution water. During filtration, the filter was always kept covered.
The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was observed to be negative.

Test item loadings
5 loading levels of the test item in a geometric series with a separation factor of 2, prepared by dilution of the saturated solution with dilution water (see Table 2) were tested as follows: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution.
The test concentrations are based on the results of a non-GLP preliminary range finding test. For results, see Annex II.

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. The composition of the culture medium is presented in Table 2.
 

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]: 246

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)
Dilution Level of the Saturated solution
[%] pH-value Dissolved
O2 concentration [mg/L]
100 7.66 9.60
50.0 7.60 9.45
25.0 7.63 9.41
12.5 7.67 9.47
6.25 7.64 9.38
Control 7.63 9.42


Water Quality Parameters in old Media at the End of the Exposure (48 hours)
(measured in the replicate with the highest immobilization rate (containing daphnids) per concentration and control)
Dilution Level of the Saturated solution
[%] 48 hours
pH-value Dissolved
O2 concentration
[mg/L] Replicate number
100 7.61 8.96 3
50.0 7.61 8.22 2
25.0 7.60 8.39 1
12.5 7.66 8.20 1
6.25 7.62 8.06 1
Control 7.62 8.49 1


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]
2018-03-13 7.61 9.90 20.2 642 246

Dissolved oxygen:

see above

Conductivity:

Dilution water day 0: 642 [µS/cm]

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

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 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: % saturated solution

Nominal / measured:

nominal

Conc. based on:

other: saturated solution

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

other: saturated solution

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

other: saturated solution

Basis for effect:

mobility

Effect concentrations 4

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

other: saturated solution

Basis for effect:

mobility

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

other: saturated solution

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

other: saturated solution

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 2018 02-27 to 2018-02-28.

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 1.24 mg/L (CI: <1.00 – 2.00)
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

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48 hour-EC50 for Daphnia magna was >100 mg test item/L.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item DisperseRed 343 (Ethyl) (batch no.:2016-5-2) were determined at the test facility according to OECD 202 (2004) from 2018-02-19 to 2018-03-15, with the definitive exposure phase from 2018-03-13 to 2018-03-15.

The study was conducted under static conditions over a period of 48 hours with five concentration levels prepared by dilution from a saturated solution of the test item (nominal loading 100 mg/L). The tested concentration levels were 0.625 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution, corresponding to mg test item/L.

The test item is a red solid with awater solubility of < 0.024 mg/L (OECD 105). The test item concentrations were colourless and visually clear throughout the exposure period.

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

The concentrations of the test item were analytically verified via HPLC-DAD in fresh media at the start of the exposure (0 hours) in the saturated solution and in the control.

The measured concentration in the saturated solution was < LOD (LOD = 0.02 mg/L with a S/N ratio of 3) at the start of the exposure (0 hours).Therefore, analytical monitoring at the end of the exposure was waived and the test item concentration was considered to be below the LOD in the 48-hours old media, too.

 

 EC₁₀-, EC₅₀-and EC₁₀₀-Valuesand 95 % Confidence Limits

(based on the dilution level of the saturated solution of the test item)

Disperse Red 343 (Ethyl)
Effect concentrations	Test duration [hours]	Dilution Level of the Saturated Solution [%]
EC10	24	>100
	48	>100
EC50	24	>100
	48	>100
EC100	24	>100
	48	>100