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EC number: 258-094-6 | CAS number: 52673-15-1
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2 017
- Report date:
- 2017
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
- Reference substance name:
- 2-(3-methoxypropyl)-9(or 10)-methylbenzimidazo[2,1-b]benzo[lmn][3,8]phenanthroline-1,3,6(2H)-trione
- EC Number:
- 258-094-6
- EC Name:
- 2-(3-methoxypropyl)-9(or 10)-methylbenzimidazo[2,1-b]benzo[lmn][3,8]phenanthroline-1,3,6(2H)-trione
- Cas Number:
- 52673-15-1
- Molecular formula:
- C25H19N3O4
- IUPAC Name:
- 17-(3-methoxypropyl)-6(or7)-methyl-3,10,17-triazahexacyclo[13.6.2.0²,¹.0,.0¹²,²².0¹,²³]tricosa-1(21),2,4(9),5,7,12(22),13,15(23),19-nonaene-11,16,18-trione
Constituent 1
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 wereDetails 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 µEm-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
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Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.