Reaction mass of disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate and disodium 4-amino-3-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

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 2018-04-24 to 2018-04-27, with the definitive exposure phase from 2018-04-25 to 2018-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to OECD and GLP guidelines

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

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4-amino-3-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

EC Number:

270-629-5

EC Name:

Disodium 4-amino-3-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Cas Number:

68460-07-1

Molecular formula:

C28H22N8O8S2.2Na

IUPAC Name:

disodium 4-amino-3-({4-[(2-amino-4-hydroxyphenyl)diazenyl]phenyl}diazenyl)-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

Constituent 2

Reference substance name:

Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

EC Number:

272-559-0

EC Name:

Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Cas Number:

68877-33-8

Molecular formula:

C28H23N9O7S2.2Na

IUPAC Name:

disodium 4-amino-3-({4-[(2,4-diaminophenyl)diazenyl]phenyl}diazenyl)-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

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 exposure and at renewal of the test solutions (0 and 24 hours) and in 24-hours old media at renewal and at the end of the exposure (24 and 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 exposure and at renewal of the test solutions (0 and 24 hours), sampling was carried out after preparation of the test item concentrations.
At renewal and at the end of exposure (24 and 48 hours), samples of the 24-hours old media were taken from the test vessels.

Criteria for the analytical monitoring
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
The stock solution (150 mg test item/L were weighed out) was freshly prepared with demineralized water one day prior to the start of the exposure (day -1) and prior to the renewal of the test solutions (day 0 hours). The solution was stirred for 24 hours at approx. 1100 rpm at room temperature. Prior to testing, the nutrient salts for the medium were added to obtain the final stock solution.

Test concentrations
5 test item concentrations in a geometric series with a separation factor of 2, prepared by dilution of the stock solution 150 mg test item/L with dilution water (see Table 1), were tested as follows: 9.38 - 18.8 - 37.5 - 75.0 - 150 mg test item/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 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, 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:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution water
0 hours:Total hardness [mg CaCO3/L]: 249
24 hours: Total hardness [mg CaCO3/L]: 231

Test temperature:

18 - 22°C, constant within ± 1°C
20 °C

pH:

Water Quality Parameters in fresh Media at the Start of the Exposure and at Renewal
(0 and 24 hours)
(measured in one additional replicate (without daphnids) per concentration level and control)
Nominal test item loading
[mg/L] 0 hours 24 hours
pH-value Dissolved
O2 concentration
[mg/L] pH-value Dissolved
O2 concentration [mg/L]
150 7.79 8.41 - -
75.0 7.89 8.61 7.78 8.82
37.5 7.92 8.72 7.72 8.96
18.8 7.97 8.69 7.78 8.99
9.38 8.00 8.79 7.82 9.02
Control 8.10 8.85 7.81 9.05
- = not determined due to 100% immobilization after 24 h

Water Quality Parameters in the 24-hours old Media at Renewal and at the End of the Exposure (24 and 48 hours)
(measured in one appropriate replicate (containing daphnids) per concentration level and control)
Nominal test item loading
[mg/L] 24 hours 48 hours
pH-value Dissolved
O2 concentration
[mg/L]
Replicate
number pH-value Dissolved
O2 concentration
[mg/L] Replicate number
150 7.93 8.09 1 - - -
75.0 7.73 8.14 3 7.63 8.26 2
37.5 7.74 7.97 4 7.62 8.24 2
18.8 7.77 7.61 2 7.63 8.22 2
9.38 7.86 7.94 2 7.63 8.31 2
Control 7.84 7.96 2 7.71 8.99 2
- = not determined due to 100% immobilization after 24 h


Water Quality Parameters of the Dilution Water at the Start of the Exposure and at Renewal
(0 and 24 hours)
Dilution water
dated: pH-Value

Dissolved
O2 concentration
[mg/L] Temperature

[°C] Conductivity

[µS/cm] Total hardness

[mg CaCO3/L]
0 hours: 2018-04-25 7.75 8.84 20.2 619 249
24 hours: 2018-04-26 7.80 9.08 19.5 613 231

Dissolved oxygen:

see above

Conductivity:

Dilution water day 0: 619 [µS/cm]
Dilution water day 1: 613 [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.:

23.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI (< 9.38 - 33.5)

Effect concentrations 2

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 33.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI (21.7 - 40.4)

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

150 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

11.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI (< 9.38 - 17.0)

Effect concentrations 5

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

29.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI (21.6 - 39.4)

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

37.5 mg/L

Nominal / measured:

nominal

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 2018 04-10 to 2018-04-11. For results of the most recent of the monthly performed reference tests, see table below.

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 1.89 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 EC100-values (after 24 and 48 hours) were empirically derived from the observation data.
All effect concentrations are given as nominal concentrations since the test item remained within ± 20% of the nominal values.

EC-values and statistical anaylses
The EC10- and the EC50-values (after 24 and 48 hours) were calculated by sigmoidal dose-response regression or straight line regression with the software GraphPad Prism. The respective 95% confidence limits were calculated from the standard error and the t distribution. If the confidence limit exceeded the actual tested concentration range, the limit was given as below the lowest or above the highest tested concentration. All calculations were carried out from the best-fit values with the software GraphPadPrism. The concentration-effect relationships after 24 and 48 hours are shown graphically.
An EC50 value was calculated for the reference item by sigmoidal dose-response regression from the best-fit values with the software GraphPad Prism. 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.
 
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:

Based on the nominal concentrations of the test item, the 48 hours-EC50 for Daphnia magna was 29.8 mg/L.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined at the test facility according to OECD 202 (2004) from 2018-04-24 to 2018-04-27, with the definitive exposure phase from 2018-04-25 to 2018-04-27.

The study was conducted under semi-static conditions over a period of 48 hours with five dilution levels (nominal: 9.38 to 150 mg/L) prepared from the stock solution in a geometric series with a separation factor of 2.

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

The concentrations of the test item were determined at the start of the exposure intervals (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours) in all test media and the control via HPLC-DAD.The measured concentrations of the test item in fresh media were in the range of 84 to 103% and in old mediain the range of 82 to 100% of the nominal values. Since the measured concentrations of the test item remained stable within ± 20% of the nominal concentrations throughout the exposure period, the nominal concentrations were used for evaluation.

All effect concentrations given in are based on the nominal concentration of the test item.

The validity criteria of the test guideline were fulfilled.

 

EC₁₀-, EC₅₀- (with Confidence Limits) and EC₁₀₀-Values

                     (based on the nominal concentration of the test item)

test item
Effect concentrations	Test duration [hours]	Nominal test item concentration [mg/L]
EC10	24	23.6	(Cl: < 9.38 – 33.5)
	48	11.8	(Cl:< 9.38 – 17.0)
EC50	24	33.2	(Cl: 21.7 – 40.4)
	48	29.8	(Cl: 21.6 – 39.4)
EC100	24	150
	48	37.5