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EC number: 221-070-0 | CAS number: 2997-92-4
- 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
Additional ecotoxological information
Administrative data
- Endpoint:
- additional ecotoxicological information
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 8 January to 11 April 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Cross-referenceopen allclose all
- Reason / purpose for cross-reference:
- reference to other study
Reference
- Endpoint:
- short-term toxicity to aquatic invertebrates
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- Batch number: 2018/019/92
Purity: 98.5% both by UV and autotitrator
Expiry date: 21 January 2020 - Analytical monitoring:
- yes
- Details on sampling:
- At the beginning and at the end of the test, the content of the test item in the test solutions was determined using HPLC-UV. The measured concentrations at the start of the test were between 105 % and 107 % of the nominal concentration and between 82 % and 91 % of the nominal concentration at the end of the test. The concentrations 10 mg/L, 22 mg/L and 46 mg/L were diluted before measurement to lie within the calibrated range (10 mg/L dilution factor 4, 22 mg/L and 46 mg/L dilution factor 10). The recovery rate from Daphnia test medium of 108.6 % was also taken into account.
- Vehicle:
- no
- Details on test solutions:
- A stock solution containing 100.4 mg/L test item in dilution water was prepared. The concentrations to be tested were prepared by dilution of this stock solution with dilution water.
- Test organisms (species):
- Daphnia magna
- Details on test organisms:
- Specification
Species Daphnia magna
Authority STRAUS Strain Berlin
Sex female
Age between 0 and 24 hours
Origin Umweltbundesamt Berlin In-house breeding since 27. September 2007
Selection of the test system was made following the proposal of the guidelines.
Animal Husbandry
Daphnia magna is bred in the LAUS GmbH throughout the year. The animals are kept for the use in toxicity tests. They multiply by parthenogenesis, thus being genetically identical. The keeping is performed similar to the method described in the OECD guideline.
Vessels preserving glasses, nominal volume 2 L
Medium M4-Medium (recipe of ELENDT)
Food green algae (Desmodesmus subspicatus)
Medium renewal twice a week
Photo period 16/8 hours, using neon tubes
Temperature 20 2 °C - Test type:
- static
- Water media type:
- other: Elendt M4
- Limit test:
- no
- Total exposure duration:
- 48 h
- Test temperature:
- 19.6 - 21.5 oC
- pH:
- 7.5 - 7.7
- Dissolved oxygen:
- 8.0 - 9.0 mg O2 / L
- Nominal and measured concentrations:
- Time 0: 90.6 - 107 % Nominal
48h: 79.4 - 91% Nominal
See Table below - Details on test conditions:
- Selection of Daphnia
23 hours before the start of the test, the adult animals were separated from the young. 0.5 hours before test start, the adults were caught with the help of a glass tube, and the newborn daphnia (age < 24h) were sieved from the medium and immediately placed into a beaker containing dilution water. After the settling-in period, animals which showed no apparent damage were used for the test. Switching from M4-medium (husbandry) to Dilution water (test) has been shown not to cause any detrimental effects for test daphnia, therefore no acclimation period of 48 h was performed. This is a deviation from OECD Guideline No. 202 and EU-Method C.2, which can be considered uncritical.
Study Performance
Using a glass tube, the daphnia were caught and lifted from the beaker. They were put on a small sieve, and the medium surrounding the animals was sucked off using absorbent paper. Immediately after that, the animals were put into the respective test solution. The test vessels were left to stand for 48 hours. After 24 and 48 hours, the immobilised daphnia were counted. Daphnia are considered immobilised when they do not perform any movements or are only able to move their antennae when the beaker is gently agitated. Daphnia which are trapped at the surface of the test solution are also considered immobilised. The pH, the concentration of dissolved oxygen and the content of the test item in the test vessels were measured at the beginning and at the end of the test. - Reference substance (positive control):
- yes
- Remarks:
- Potassium dichromate
- Key result
- Duration:
- 24 h
- Dose descriptor:
- EC50
- Effect conc.:
- 22 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mobility
- Key result
- Duration:
- 48 h
- Dose descriptor:
- EC50
- Effect conc.:
- 10.39 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mobility
- Key result
- Duration:
- 48 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 4.6 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mobility
- Key result
- Duration:
- 48 h
- Dose descriptor:
- LOEC
- Effect conc.:
- 10 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mobility
- Results with reference substance (positive control):
- Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study (201902R201). The 24h-EC50 value was determined as 1.98 mg/L. The value was within the requested range for the 24h-EC50 between 0.6 - 2.1 mg/L of potassium dichromate K2Cr2O7 as specified in the OECD guideline.
- Reported statistics and error estimates:
- The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.3.0. No error estimates were provided due to mathematical reasons.
- Validity criteria fulfilled:
- yes
- Conclusions:
- The 48h EC50 value was determined to be 10.39 mg/L.
- Executive summary:
The 48h EC50 value was determined to be 10.39 mg/L.
Although the substancei s known to hydrolyse under environmental conditions, analysis showed sufficient stability in the 48 hours of this test under the laboratory conditions used.
The instablity in water is thought to be partly to do with interaction with ions in hard water under alkaline conditions.
Immobility (cumulative)
Nominal conc. (mg/L) | 24h (%) | 48h (%) |
Control | 0 | 0 |
2.2 | 0 | 0 |
4.6 | 0 | 0 |
10 | 0 | 40 |
22 | 50 | 100 |
46 | 100 | 100 |
Mean measured Test Item Concentrations
Nominal conc. (mg/L) | Time 0 (mg/L) | 48h (mg/L) | % Nominal (Time 0) | % Nominal (48h) |
Control | 0 | 0 | 0 | 0 |
2.2 | 2.31 | 1.96 | 105 | 89 |
4.6 | 4.82 | 3.93 | 105 | 85 |
10 | 10.62 | 9.12 | 106 | 91 |
22 | 23.60 | 18.07 | 107 | 82 |
46 | 48.63 | 41.88 | 106 | 91 |
- Reason / purpose for cross-reference:
- reference to other study
Reference
- Endpoint:
- toxicity to aquatic algae and cyanobacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20 May - 31 May 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Alga, Growth Inhibition Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- Batch : 2018/019/92
Purity: 98.5% by UV and titrator
Expiry: 21 January 2020 - Analytical monitoring:
- yes
- Details on sampling:
- 0h and 72h
- Vehicle:
- no
- Details on test solutions:
- A stock solution containing 100 mg/L test item in algal medium (demineralised water enriched with minerals but without algae) was prepared. The concentrations to be tested were prepared by dilution of this stock solution with algal medium.
- Test organisms (species):
- Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
- Details on test organisms:
- Unicellular freshwater green alga.
Genus Desmodesmus
Species subspicatus
SAG Strain Number 86.81
Taxonomic position Chlorophyta - Chlorophyceae
Origin and Culture
The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared. - Test type:
- static
- Water media type:
- other: OECD TG 201 medium
- Limit test:
- no
- Total exposure duration:
- 72 h
- Test temperature:
- 22.4-24.3oC
- pH:
- 8.3 - 8.5
- Details on test conditions:
- 4440-8880 Lux
- Reference substance (positive control):
- yes
- Remarks:
- Potassium dichromate 0.28 and 0.65 mg/L
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.16 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- LOEC
- Effect conc.:
- 0.53 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- 1.3 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.16 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- biomass
- Key result
- Duration:
- 72 h
- Dose descriptor:
- LOEC
- Effect conc.:
- 0.53 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- biomass
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- 0.29 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- biomass
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 1.95 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- biomass
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- > 18.62 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Details on results:
- No negative impact on microscopic cell evaluation at all test concentrations.
- Results with reference substance (positive control):
- 72h ErC50 0.65 mg/L
72h EyC50 0.28 mg/L - Reported statistics and error estimates:
- Calculation of results was performed with the help of validated software (Microsoft Ex-cel®). The estimation of the biological data was accomplished using the software Tox-Rat® Professional, version 3.3.0. The details of calculation are stated in Annex 4: Statisti-cal calculation using ToxRat® Professional 3.3.0.
- Validity criteria fulfilled:
- yes
- Conclusions:
- ErC50 > 18.62 mg/L
- Executive summary:
It is likely that the test material, or degradation products, interfered with the mineral content of the test media causing inhibition of growth due to removal of key nutrients from the small test vessels. This is something that would not be seen in the wider environment with moving water systems that allow for minerals to be replenished.
Geometric Mean of Measured Concentrations
Nominal |
Geometric Mean |
mg/L |
mg/L |
0.32 |
0.16 |
1 |
0.53 |
3.2 |
1.79 |
10 |
5.46 |
32 |
18.62 |
From the cell numbers, the Growth Rate µ and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table:
Table9.6‑a Growth Rate µ, Yield
Nominal Concentration in mg/L |
Parameter |
Growth Rate (0-72 h) [day-1] |
Yield (0-72 h) |
Blank control |
Mean |
1.80 |
529007 |
SD |
0.02 |
33641 |
|
0.1 |
Mean |
1.79 |
514240 |
SD |
0.01 |
12341 |
|
0.32 |
Mean |
1.79 |
519620 |
SD |
0.01 |
8559 |
|
1 |
Mean |
1.76 |
464660 |
SD |
0.01 |
19577 |
|
3.2 |
Mean |
1.50 |
215007 |
SD |
0.03 |
19027 |
|
10 |
Mean |
1.43 |
172080 |
SD |
0.00 |
1273 |
|
32 |
Mean |
1.43 |
170927 |
SD |
0.00 |
248 |
SD = Standard Deviation
The following mean inhibition values were calculated for the Growth Rate µ and the Yield.
A clear dose-response relationship could be observed. However, the inhibition in the two highest concentrations was nearly identical. The highest concentration was not used for evaluation so that the graphical evaluation is not falsified.
Inhibition Values
Nominal concentration in mg/L |
% Inhibition |
|
Growth Rate (0-72 h) |
Yield (0-72 h) |
|
Blank control |
0 |
0 |
0.1 |
0.50 |
2.79 |
0.32 |
0.30 |
1.77 |
1 |
2.37 |
12.16 |
3.2 |
16.57 |
59.36 |
10 |
20.60 |
67.47 |
32 |
20.72 |
67.69 |
All validity criteria were met
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- other: SANCO 3029/99 revision 4
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- 2,2'-azobis[2-methylpropionamidine] dihydrochloride
- EC Number:
- 221-070-0
- EC Name:
- 2,2'-azobis[2-methylpropionamidine] dihydrochloride
- Cas Number:
- 2997-92-4
- Molecular formula:
- C8H18N6.2ClH
- IUPAC Name:
- 2,2'-diazene-1,2-diylbis(2-methylpropanimidamide) dihydrochloride
- Test material form:
- solid
Constituent 1
- Specific details on test material used for the study:
- Batch; 2018/19/59
Purity: 98.5%
Expiry Date: 7 August 2019
Results and discussion
Any other information on results incl. tables
Specificity
The condition “peak areas in blank < 30 % of the peak areas at the proposed LOQ” was fulfilled.
Calibration
A linear calibration function was calculated. Correlation coefficient => 0.99 This condition is fulfilled with correlation coefficient 0.99998, the working range 0.05 – 5 mg/L can be used.
Accuracy & Precision
Alagae, Low: Accuracy 103.5%, RSD 4.5%
Algae, High: Accuracy 104.8%, RSD 0.7%
Daphnia, Low: Accuracy 106.8%, RSD 0.9%
Daphnia, High: Accuracy 110.5%, RSD 0.7%
All accuracy values lay in the demanded range of 70 – 110 %, with the exception of the test item solution 46 mg/L in daphnia test medium. However, the deviation of 0.5% is so small that it can be neglected and the repeatability of the measurements of five independent samples is sufficient with an RSD of 0.7 %. As the values lay outside the range 100 ± 5 %, the mean recovery rates will be taken into account in the calculation of the test item concentrations in the subsequent ecotoxicological studies. The condition for precision “RSD ≤ 20 % per level” was fulfilled for both media and levels.
Stability
The test item was considered unstable in algal test medium under the test conditions as the residual concentration was < 80 % of the initial concentration at the end of the incubation period. Nevertheless, an analytical monitoring of the Algal Growth Inhibition Test is possible. In daphnia test medium under the test conditions was the test item considered stable as the residual concentration was > 80 % of the initial concentration at the end of the incubation period
Applicant's summary and conclusion
- Conclusions:
- This analytical method is considered valid for the analysis of 2, 2'-azobis[2-methylpropionamidine] dihydrochloride in the matrices Algal and Daphnia test medium.
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