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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:
2017-08-14 to 2017-08-17
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:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Determination of the test item
All test solutions and the control were analytically verified via HPLC-
DAD in fresh media at the start of the exposure (0 hours). Since all recoveries in all test solutions were Details of the analytical method are presented in section 14. Analytical results are presented in section 7.2.3.

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples of the fresh media were
taken after preparation of the test solutions and analyzed.
Since all recoveries in all test solutions were
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:

Preparation of the saturatedt solution
The 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 was 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.

Test item loadings
5 loading levels of the test item in a geometric series with a separation factor of 2, prepared by diluting 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 section 7.1.

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

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 dated:

0 hours: 2017-08-05 Total hardness [mg CaCO3/L]: 173
Test temperature:
18 - 22°C, constant within ± 1°C
2017-08-05 21.0 °C


pH:

Water Quality Parameters in fresh Media at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) of all loading levels and the control)
Dilution level of saturated solution
[%] Disperse Orange 32
Nominal test item loadings
[mg/L] pH-value Dissolved
O2 concentration [mg/L]
100 100 8.12 8.10
50.0 50.0 8.15 8.09
25.0 25.0 8.20 8.14
12.5 12.5 8.29 8.27
6.25 6.25 8.38 8.66
Control Control 8.37 8.75


Table 9: Water Quality Parameters in old Media at the End of the Exposure (48 hours)
(measured in all replicates (containing daphnids) of all loading levels and the control)
Dilution level of saturated solution
[%] Disperse Orange 32
Nominal test item loadings
[mg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
100 100 7.77 7.75 7.79 7.79 7.98 8.17 8.23 7.82
50.0 50.0 7.79 7.60 7.73 7.73 7.94 8.15 7.84 8.00
25.0 25.0 7.81 7.78 7.77 7.78 7.73 8.07 8.38 8.22
12.5 12.5 7.86 7.79 7.79 7.80 8.18 8.49 8.45 8.50
6.25 6.25 7.89 7.69 7.74 7.76 8.30 8.46 8.55 8.54
Control Control 7.97 7.70 7.69 7.67 8.20 8.06 8.25 8.42

Dissolved oxygen:
Water Quality Parameters in the fresh Media at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) WAF and control)
Nominal loading level of
the test item in the water
accommodated fraction
[mg/L] pH-value
Dissolved
O2 concentration

[mg/L]
100 8.45 8.37
50 8.55 8.34
25 8.52 8.37
12.5 8.52 8.40
6.25 8.31 8.50
Control 8.60 8.39


Table 10: Water Quality Parameters in the old Media at the End of the Exposure (48 hours)
(measured in all replicates containing daphnids)
Nominal loading level of
the test item in the water
accommodated fraction
[mg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
100 7.71 - - - 8.35 - - -
50 7.72 - - - 8.35 - - -
25 7.73 - - - 8.53 - - -
12.5 7.72 - - - 8.48 - - -
6.25 7.74 - - - 8.29 - - -
Control 7.80 - - - 8.68 - - -

Water Quality Parameters in fresh Media at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) of all loading levels and the control)
Dilution level of saturated solution
[%] Disperse Orange 32
Nominal test item loadings
[mg/L] pH-value Dissolved
O2 concentration [mg/L]
100 100 8.12 8.10
50.0 50.0 8.15 8.09
25.0 25.0 8.20 8.14
12.5 12.5 8.29 8.27
6.25 6.25 8.38 8.66
Control Control 8.37 8.75


Table 9: Water Quality Parameters in old Media at the End of the Exposure (48 hours)
(measured in all replicates (containing daphnids) of all loading levels and the control)
Dilution level of saturated solution
[%] Disperse Orange 32
Nominal test item loadings
[mg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
100 100 7.77 7.75 7.79 7.79 7.98 8.17 8.23 7.82
50.0 50.0 7.79 7.60 7.73 7.73 7.94 8.15 7.84 8.00
25.0 25.0 7.81 7.78 7.77 7.78 7.73 8.07 8.38 8.22
12.5 12.5 7.86 7.79 7.79 7.80 8.18 8.49 8.45 8.50
6.25 6.25 7.89 7.69 7.74 7.76 8.30 8.46 8.55 8.54
Control Control 7.97 7.70 7.69 7.67 8.20 8.06 8.25 8.42


Conductivity:

Table 10: 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]
2017-08-15 8.37 8.75 21.0 472 173

Nominal and measured concentrations:

All test solutions of the test item Disperse Orange 32 and the control were analytically verified via HPLC-DAD in fresh media at the start of the exposure (0 hours). Details of the analytical method are presented in section 14.
The measured concentrations in the samples were < LOD at the start of the exposure (0 hours). Therefore, analytical motoring at the end of the exposure was waived and the test item concentrations were considerd to be below the LOD in the 48-hours media, too. For results, see Table 6.

Table 6: Measured Concentrations of the Test Item at the Start of Exposure
Dilution level of saturated solution
[%] Sampling: 0 hours
Nominal loading
of the test item
[mg/L] Disperse Orange 32
Meas. conc.
[mg/L]
100 100 < LOD
50.0 50.0 < LOD
25.0 25.0 < LOD
12.5 12.5 < LOD
6.25 6.25 < LOD
Control Control < LOD
Meas. conc. = measured concentration of the test item, mean value of 2 injections
LOD = limit of detection (0.02 mg/L of the test item)

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 concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
other: EL 10,50,100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
76.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Results with reference substance (positive control):
A reference test was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium (Table 2) under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility. The results of the most recent test are presented in section 7.3.

Reference item Potassium dichromate p.a. (SIGMA)

Purity 99.0%

Batch number MKBV0900V

Expiry date 2021-11-25

Test concentrations 1.00 – 2.00 – 4.00 mg/L

Ranges of validity EC50 (24 hours): 0.6 - 2.4 mg/L, according to AQS P 9/2 (clone 5),
EC50 (24 hours): 0.6 - 2.1 mg/L, according to OECD 202 (clone A)

Exposure phase 2017-08-03 to 2017-08-04

Reported statistics and error estimates:
Methods of evaluation The EL10- and the EL50-values after 24 hours and the EL100-values after 24 and 48 hours were empirically derived from the observed immobilization rates.
All effect levels (EL10 / 50 / 100) given are based on the nominal loading levels of the test item.

EL-/EC-values and The EL10- and the EL50-values with 95% confidence limits after
statistical analyses 48 hours were calculated by sigmoidal dose-response regression. The loading-effect relationship is shown graphically.
The EC50-value with 95% confidence limits for the reference item was calculated accordingly 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


Any other information on results incl. tables

Method of determination          Analytical evaluation of the test itemDisperseOrange 32wascarried out via HPLC-DAD on a reversed phase column using external standard calibration. Analysis was performedin gradient modewith a Diode Array Detector.

 

Equipment                             Autosampler:                      G1329A,Agilent 1100

                                              Autosampler-thermostat:     G1330B,Agilent1100

                                              Vacuum degasser:              G1322A,Agilent1100

                                              Column oven:                     G1316A,Agilent1100

                                              HPLC-pump:                       G1312A, Agilent1100

                                              Detector:                            G1315B Diode Array Detector (DAD),
                                          Agilent1100

                                              Software:                           Empower 2 (Build No: 2154),Waters

 

Additional Equipment              Piston stroke pipettes,Thermo Scientific

                                              Positive-displacement pipettes,Gilson Medical

                                              

Reagents                                HPLC water, for HPLC,VWR

Acetonitrile, super gradient,VWR

N,N-Dimethylformamide, ≥ 99.8% Emsure

 

Analytical standard                  The test item was used for preparation of the external standards.

 

Conditions of analysis

 

Column                                  Nucleodur C18 ec, Gravity, 3 µm, 125 x 3 mm, batch: 37400033,Macherey-Nagel

Column temperature                 25 °C

Mobile phase (gradient)           A: HPLC water

                                              B:Acetonitrile

 

Table11:    Gradient Table

Time [min]

A [%]

B [%]

 0.00

50

50

 1.00

50

50

 4.00

10

90

 6.00

10

90

 6.50

50

50

 9.00

50

50

 

Flow rate                                 1.0 mL/min

Run time                                 9 min

Injection volume                      100 µL

Wavelength                             430 nm


 

Preparation of the standards    A stock solution of 100 mg test item/L in dimethylformamid was diluted to 4 mg/L with acetonitrile : HPLC water (50 : 50) and this solution was further diluted to 7 concentrations with acetonitrile : HPLC water (50 : 50). The calibration range was 0.05 to 3.20 mg/L. The signal-to-noise ratio of the lowest calibration standard was 12 and the signal-to-noise ratio of the limit of detection (0.02 mg/L) was 3. For the calibration curve and chromatograms, seeFigure2toFigure5.

 

Preparation of the samples      The control and the samples were analyzed without dilution.

 

Sample storage                       All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

 

Evaluation                               Quantification of the standards was calculated by peak area based on the external standard.

 


 

1.1      Method Validation

 

Requirements of the               Recoveries at 0 hours were < lowest calibration level, therefore

method validation                    method validation was not necessary.

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Based on the nominal loadings of the test item, the 48 hour-EL50 for Daphnia magna was 76.5 mg/L (95% confidence limits: 68.2 – 83.8 mg/L).

Executive summary:

In the acute immobilization test toDaphnia magna(STRAUS), the effects of the test itemDisperseOrange 32 were determined at the test facility according to OECD 202 (2004) from2017-08-14 to 2017-08-17, with the definitive exposure phase from 2017-08-15 to 2017-08-17.

The study was conducted under static conditions over a period of 48 hours with a saturated solution with a nominal loading of100 mg/Lof the test itemDisperse Orange 32and further four dilution levels in a geometric series with a separation factor of2(6.25 to 50% of the saturated solution, corresponding to nominal loading levels of6.25 to 50.0 mg/Lof the test item).All tested concentration levels were visually clear throughout the exposure period. The two highest loading levels (50 and 100% of the saturated solution) were slightly yellow during of the exposure,while lower loading levels were colorless.

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

The concentrations of the test itemDisperseOrange 32were analytically verified via HPLC-DAD in fresh media at the start of the exposure (0 hours) in all test sollutions and in the control. Details of the analytical method are presented in section 14.

The measured concentrations in all test solutions were < 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 concentrations were considered to be below the LOD in the 48-hours old media, too.

Exposures are expressed in terms of the original concentration of the test itemDisperseOrange 32in water at the start of the mixing period (loading rate). The effect values are presented inTable 1.

 

The validity criteria of the test guideline were fulfilled.

 

Table1:   EL10-, EL50- EL100-Values (with 95% Confidence Limits)

(based on the nominal loading of the test item)

Effect loading levels

Test

duration

[hours]

Disperse Orange32

Nominal loading of the test item

[mg/L]

EL10

24

> 100 mg/L

48

                                  50.0 (CI: 42.2 – 60.8)

EL50

24

> 100 mg/L

48

                                   76.5 (CI: 68.2 – 83.8)

EL100

24

> 100 mg/L

48

                             > 100 mg/L