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Diss Factsheets

Ecotoxicological information

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 2018-01-30 to 2018-02-02, with the definitive exposure phase from 2018-01-31 to 2018-02-02
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

Constituent 1
Chemical structure
Reference substance name:
Sodium bis[4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-3-hydroxy-N,N-dimethylbenzene-1-sulphonamidato(2-)]chromate(1-)
EC Number:
278-137-2
EC Name:
Sodium bis[4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-3-hydroxy-N,N-dimethylbenzene-1-sulphonamidato(2-)]chromate(1-)
Cas Number:
75214-69-6
Molecular formula:
C36H34CrN10NaO8S2
IUPAC Name:
hydroxylamine
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 Recoveries of the test item should be within ± 20% of the nominal or
monitoring (target) initially measured concentrations.



Test solutions

Vehicle:
no
Details on test solutions:
Preparation of the saturated solution
A saturated solution (100 mg/L test item were 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 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 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. Tyndall effect was positive.

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, were tested as follows: 0.625 - 1.25 - 2.50 - 5.00 - 10.0% of the saturated solution.
The test concentrations were 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 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:
no
Total exposure duration:
48 h

Test conditions

Hardness:
Dilution water
0 hours:Total hardness [mg CaCO3/L]: 208
Test temperature:
18 - 22°C, constant within ± 1°C
18.5 - 19.0 °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 20 daphnids, divided into 4 replicates, each with 5 daphnids, were
and replicates used for each concentration level 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 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 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 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 concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 339 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
160 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: CI (140 - 191)
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 2018-01-30 to 2018-01-31. For results of the most recent of the monthly performed reference tests, see the 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 1.47 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 effect levels (EC10/50/100) were based on the geometric mean measured concentrations of the test item.

EC-values and statistical analyses
The EC10/50/100 after 24 hours and the EC100 after 48 hours were empirically derived from the observed immobilization rates.
The EC10/50-values after 48 hours were calculated 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 Prism5. The concentration effect relationships are shown graphically.
The EC50-value for the reference item was also calculated 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 Prism5.

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

Immobilization Ratesafter 24 and 48 hours of Exposure in the Definitive Test

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

Geometric mean measured

test item

concentration

[µg/L]

IMMOBILIZATION [%]

24 hours

48 hours

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

339

0

0

0

20  

5

100

100

100

100

100

134

0

0

0

0

0

40

40

20

20

30

  70.3

0

20  

0

0

5

20

20

20

20

20

  32.0

20  

0

0

0

5

20

0

0

0

5

  15.8

0

0

0

0

0

0

0

0

0

0

Control

0

0

0

0

0

0

0

0

0

0

Absolute Numbers of immobile daphnids after 24 and 48 hours of Exposure in the Definitive Test

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

Geometric mean measured

test item

concentration

[µg/L]

Number of immobile Daphnids / Total number of Daphnids

24 h

48 h

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

339

0 / 5

0 / 5

0 / 5

1 / 5

1 / 20

5 / 5

5 / 5

5 / 5

5 / 5

20 / 20

134

0 / 5

0 / 5

0 / 5

0 / 5

0 / 20

2 / 5

2 / 5

1 / 5

1 / 5

6 / 20

  70.3

0 / 5

1 / 5

0 / 5

0 / 5

1 / 20

1 / 5

1 / 5

1 / 5

1 / 5

4 / 20

  32.0

1 / 5

0 / 5

0 / 5

0 / 5

1 / 20

1 / 5

0 / 5

0 / 5

0 / 5

1 / 20

  15.8

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 test item loadings of 2.5 to 10 % showed concentration related slight reddish color and were visually clearly dissolved throughout the exposure period. Other loading levels and the control were colorless and visually clearly dissolved throughout the exposure period.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Based on the geometric mean measured concentrations of the test item Acid Red 279, the 48 hour-EC50 for Daphnia magna was 160 (140 – 191) µg test item/L.
Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Acid Red 279 were determined at the test facility according to OECD 202 (2004) from 2018-01-30 to 2018-02-02, with the definitive exposure phase from 2018-01-31 to 2018-02-02.

The study was conducted under static conditions over a period of 48 hours with five loading levels prepared by dilution from the saturated solution of the test item (nominal loading 100 mg/L). The tested loadings levels were0.625 - 1.25 - 2.50 - 5.00 - 10.0 % of the saturated solution (separation factor 2).

The test itemis a red solid with awater solubility ofapprox. 3.8 g/L (20°C, OECD 105). The test item loadings of 2.5 to 10 % showed concentration related slight reddish color and were visually clearly dissolved throughout the exposure period. Other loading levels and the control were colorless and 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 item 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) in all tested loading 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 25 to 403 µg/L. At the end of the exposure period (48 hours), the measured test itemconcentrations in the old media were in the range of 19.3 to 286 µg/L. Since the measured concentrations of the test item Acid Red 279 did not remainstable within ± 20% of the initial measured concentrations throughout the exposure period, the geometric mean measured concentrations were calculated as follows 15.8 – 32.0 – 70.3 – 134 – 339 µg/L.

All effect concentrations (EC10 / 50 / 100) given in the table below are based on the geometric mean measured concentrations of the test item Acid Red 279.

The validity criteria of the test guideline were fulfilled.

 

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

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

Acid Red 279

Effect concentrations

 

Test duration

[hours]

Geometric mean measured test item concentrations

[µg test item/L]

EC10

24

> 339

48

80.3 (62.5 – 102)

EC50

24

> 339

48

160 (140 – 191)

EC100

24

> 339

48

  339