Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 627-132-7 | CAS number: 1227096-04-9
- 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
Biodegradation in water: screening tests
Administrative data
Link to relevant study record(s)
- Endpoint:
- biodegradation in water: ready biodegradability
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 30-Nov-2022 until 09-Jan-2023
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Guideline:
- OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
- Version / remarks:
- 1992
- Deviations:
- yes
- Remarks:
- One minor deviation from the guidelines of the Closed Bottle test (OECD TG 301D) was introduced: Ammonium chloride was omitted from the medium more details see principles other than guideline
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
- Version / remarks:
- 2009
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)
- Version / remarks:
- 1994
- Deviations:
- no
- Principles of method if other than guideline:
- A minor deviation from the guidelines of the Closed Bottle test (OECD TG 301D) was introduced.
Ammonium chloride present in the mineral salt medium was omitted because it may be converted to nitrate which leads to an oxygen consumption not related to the biodegradation of the test substance. The omission of ammonium does not affect the biodegradability because ammonium is not limiting in the Closed Bottle test as was demonstrated by the biodegradation of the reference substance. The omission of ammonium does make the quantification of the biodegradation of the test substance more accurate.
Justfiication of the omission of ammonium chloride from the mineral salts medium of the OECD 301D ready biodegradation test
The ready biodegradation test medium
Ammonium chloride is added to the standard ready biodegradation test (RBT) medium as macro-nutrient for the growth of the microorganisms that are responsible for the biodegradation of the test substance. Ammonium chloride is added in RBT tests under the assumption that there is a nitrogen limitation in RBTs. The OECD 301D test uses however the lowest test substance concentration of all RBTs and the omission of ammonium chloride does not result in nitrogen limitation as shown by the biodegradation of the reference compound in these tests.
Ammonium chloride added with the medium in the OECD 301D test results in an excess of ammonium in the test which will be oxidized to nitrate. Nitrification is performed by a small group of autotrophic bacteria which are not involved in the mineralization of the test substance. The test substance will be mineralized by heterotrophic bacteria. Adding ammonium chloride to the standard ready biodegradation test medium, not needed for growth of the heterotrophic bacteria, results only in growth of the nitrifying autotrophic bacteria.
Stringency of RBTs
A positive RBT result informs about three aspects of biodegradation: i) the presence and pervasiveness of competent microorganisms in the environment; ii) the ultimate biodegradation of the test substance; and iii) rates of biodegradation in the environment. RBTs are regarded as the most stringent biodegradation tests because biodegradation needs to be achieved within a certain time frame using a relatively small inoculum and a relative high concentration of one test substance.
Meaning, in the relatively small RBT inoculum already enough competent microorganisms should be present to achieve the mineralization of the test substance in the required time frame. The inoculum concentration required for ready biodegradation test is described in the OECD guidelines and is measured at the start of an RBT. Hence, the stringency of RBTs is confirmed (checked) at the start of the RBT.
Organic substances are biodegraded in ready biodegradability tests by heterotrophic micro-organisms capable of utilizing the substance as carbon and energy source. The ammonium present in the standard RBT medium is oxidized by nitrifying bacteria. These nitrifying bacteria utilizing ammonium as energy source and carbon dioxide as carbon source (autotrophic growth) and are not involved in the biodegradation of organic substances. Hence, the numbers of nitrifying bacteria in the inoculum of RBTs do not affect/influence the stringency of the tests.
Accuracy of RBTs
In RBTs the respiration of the inoculum blank (the endogenous respiration*) is used as the value for the background respiration. On top of this background respiration, it should be possible to accurately measure the respiration by the test substance. In the OECD 301D test there is only a maximum amount of oxygen available at the start of the OECD 301D test (~9 mg O2/L at 20°C, saturation of oxygen in water). Oxygen concentrations in the test should stay aerobic (≥ 0.5 mg O2/L) and a maximum endogenous respiration of 1.5 mg O2/L is allowed. This means that there is ~7 mg O2/L left for the biodegradation assessment of the test substance. If the endogenous respiration would use more oxygen there is less oxygen available to assess the biodegradation of the test substance resulting in a less accurate biodegradation assessment. The validity criteria of the inoculum blank therefore ensures the accuracy of the measured oxygen consumption by the test substance.
* Endogenous respiration is defined as: a situation in which living organisms oxidize some of their own cellular mass instead of oxidizing substrates they take from the environmental matrix.
The influence of the nitrification of the ammonium nitrogen supplemented with the mineral salts medium on the accuracy validity criteria was compared for the different respirometric RBTs. The biodegradation assessment in the OECD 301B test is based on the measurement of evolved CO2 and the accuracy of the test is therefore not affected by the nitrification of the ammonium nitrogen in the mineral salt medium. The OECD 301C, 301D and 301F are respirometric tests based on measurement of the oxygen consumption. Nitrification in the control bottles will in these tests be mistaken for endogenous respiration. Assuming a complete oxidation of the mineral salts ammonium nitrogen results in an additional oxygen consumption of 0.6 mg/L in the OECD 301D test and of 6 mg/L in the OECD 301C and 301F test. The contribution of this oxygen consumption is 40% of the allowed endogenous respiration in the OECD 301D test and is only 10% of the allowed endogenous respiration in the OECD 301C and 301F test. For the OECD 301D test it is known (see first paragraph above) that nitrogen is not limited in the test and therefore the supplemented ammonium nitrogen is an excess that will be oxidized to nitrate. In the OECD 301C and 301F a higher test substance concentration is used and therefore the nitrogen is expected to be limited to biodegrade all the test substance. In these tests part of the dosed mineral salts ammonium nitrogen will be incorporated in new biomass and the actual oxygen consumption by the nitrification is therefore expected to be < 6 mg/L. The contribution of the oxygen consumption of the allowed endogenous respiration in the OECD 301C and 301F will be <10%.
In conclusion: The back-ground respiration of the other RBTs is not or only slightly influenced by the addition of ammonium in the mineral salts medium compared to the influence it has on the back-ground respiration in the OECD 301D test.
High level of variation in RBT results.
The amount and rate of oxygen consumption by the nitrification of the ammonium chloride dosed with the mineral salts medium will mainly depend on the initial numbers of nitrifying bacteria present in the inoculum. These numbers will vary throughout the year because of the seasonal changes. Nitrifying bacteria are sensitive and relative slow growing bacteria. Low bacteria numbers at the start of the tests or an initial delay in growth by toxic effects is therefore not easily to overcome over a 28 days test period. A test substance that is (slightly) toxic to nitrifying bacteria will delay or stop the growth of nitrifying bacteria in the test bottles. In such a case the inoculum blank (with no hampering of the growth of nitrifying bacteria) will overestimate the background respiration in the test bottles resulting in lower (false) biodegradation values. Analysis of formed nitrate and nitrite in the OECD 301D test and control bottles allow a correction for the additional oxygen consumption. These analyses will however also introduce analytical inaccuracy and hence an increased variation (inaccuracy) of the final calculated test substance biodegradation.
The endogenous respiration in the OECD 301D test medium without addition of ammonium chloride and using bacterial densities (cells/L) in the prescribed range of the test guideline varies in general in the range of 1 ± 0.2 mg/L. The addition of ammonium nitrogen in the mineral salts medium would result in max 0.6 mg/L addition oxygen consumption and could therefore result in failing the endogenous respiration validity criteria. The result of adding ammonium chloride in the OECD 301D will therefore result in a higher chance of invalidating test results.
In summary:
An accurate and stringent assessment of the biodegradation potential of a test substance in the OECD 301D test is possible by omitting the ammonium chloride from the RBT test medium. The omission of ammonium chloride from the OECD 301D medium is justified because:
• There is no nitrogen limitation for the growth of heterotrophic micro-organisms in the OECD 301D test and therefore no additional nitrogen source needs to be added with the medium;
• The omission of ammonium chloride does not affect the stringency of the test because the initial bacterial density at the start of the test is demonstrated to be in the prescribed range;
• The omission of ammonium chloride improves the accuracy of the measured oxygen consumption by the test substance.
• It is much more difficult in the OECD 301D test compared to the other RBTs to fulfill the accuracy criteria when ammonium chloride is added (“biased” effect of ammonium between RBTs).
• Supplementing ammonium chloride in the mineral salts medium of the OECD 301D test will introduce a higher variability and more invalidated test results caused by nitrifying bacteria which are not involved in the biodegradation of the test substances.
Based on the above the omission of ammonium chloride from the OECD 301D test medium should be accepted. In addition, test results from OECD 301D tests where ammonium chloride was omitted from the medium are also accepted in other regulations (a.o. biocidal product directive, ecolabeling, etc…).
Justification for the use of river water as test medium:
According to OECD TG 301 it is clearly stated that an alternative source for the inoculum like surface water (e.g. river water) can be used for the test. Furthermore, also the
REACH guidance on Information Requirements and Chemical Safety Assessment (2017) mentions and justifies that micro-organisms (~10^5 cells/mL) in surface waters can be used as inoculum for the closed bottle test.
In principle, organic chemicals should be introduced in all vessles (bottles) of OECD TG 301 and OECD TG 310 irrespective of the inoculum used. The biodegradable organic carbon introduced should in all cases be limited in order to guarantee a low respiration by the microorganisms introduced. Endogenous respiration (oxidation of storage material and protein) by the micro-organisms introduced with inocula is the major controbutor and should be <= 1.5 mg/L at day 28 in the closed bottle test (validity criterion). The endogenous respiration in the blank control bottles (river water) was 1.2 mg/L at day 28. When this validity criterion is met, it is shown that the test substance is the major source of carbon for energy and growth in the test. - GLP compliance:
- yes (incl. QA statement)
- Oxygen conditions:
- aerobic
- Inoculum or test system:
- natural water: freshwater
- Remarks:
- River water was sampled from the Rhine near Heveadorp, The Netherlands (28-Nov-2022). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream.
- Details on inoculum:
- River water was sampled from the Rhine near Heveadorp, The Netherlands (28-Nov-2022).
The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km
upstream. The river water was aerated for 7 days before use to reduce the endogenous
respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum.
The particles were removed by sedimentation after 1 day while moderately aerating.
The Colony forming units (CFU) in the particle free and preconditioned river water was
determined by a colony count method based on the ISO 6222 (1999) guideline. The
preconditioned river water used in the closed bottles was diluted 10x and 100x in a sterile
peptone solution (1 g/L). Subsequently 1 ml of the peptone dilutions was transferred on a
sterile petri dish and yeast extract agar was added. The yeast extract agar contained per liter
of water 6 g tryptone, 3 g yeast extract and 15 g agar. Yeast extract agar plates were
incubated for 68 hours at a temperature of 22.7 °C. Only CFU counts between 30 and 300
were regarded as accurate and accepted for calculation of the CFU content. The inoculum
concentration in the BOD bottles determined by colony count was 4.4E+05 CFU/L. - Duration of test (contact time):
- 28 d
- Initial conc.:
- 2 mg/L
- Based on:
- test mat.
- Details on study design:
- Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.
Nutrients and stocks
The river water used in the Closed Bottle test contained per liter of water 8.50 mg KH2PO4, 21.75 mg K2HPO4, 33.41 mg Na2HPO4·2H2O, 22.51 mg MgSO4·7H2O, 27.51 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification that is not related to the biodegradation of the test substance.
The test substance was added to the bottles from a stirred aqueous stock emulsion of 1 g/L with a pH of 5.7. This homogenous and stable emulsion of the test substance was prepared by acidifying the test substance stock first to pH 3.0 using a 2 M HCl solution. The acidified mixture was subsequently homogenized by stirring. Next, the pH of the homogenous emulsion was adjusted to pH 5.6 by using a 1 M NaOH solution and the final volume was adjusted with demineralized water to obtain a 1 g/L test substance concentration. The stock emulsion was stirred at ambient temperature until use. The reference substance sodium acetate was added to the bottles using an aqueous stock solution of 1.0 g/L.
Test procedures
The Closed Bottle test (OECD TG 301D) was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only river water incubated with a regular stirring of the content, 10 bottles containing river water and the test substance incubated with a regular stirring of the content, 6 bottles containing river water and sodium acetate and 10 bottles containing only river water. The content of the bottles was stirred 10 times per day for 5 minutes to improve the bioavailability of the test substance in the bottles. The regular stirring of the content in the bottles was achieved by using a stirrer bar and stirrer plate together with a time switch to start and stop the stirrer plate.
Volumes of 0.6 mL of the test substance stock emulsion were added directly to the bottles already filled with nutrient spiked river water and subsequently closed. The concentrations of the test substance and sodium acetate in the bottles were 2.0 mg/L and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28. - Reference substance:
- acetic acid, sodium salt
- Key result
- Parameter:
- % degradation (O2 consumption)
- Remarks:
- based on ThODNO3
- Value:
- 66
- Sampling time:
- 28 d
- Parameter:
- % degradation (O2 consumption)
- Remarks:
- based on ThODNH3
- Value:
- 69
- Sampling time:
- 28 d
- Validity criteria fulfilled:
- yes
- Interpretation of results:
- readily biodegradable
- Remarks:
- Assuming complete nitrification as a worst-case of the test substance organic nitrogen, and calculating the biodegradation based on the ThODNO3 the test substance was biodegraded by 66% at day 28 in the Closed Bottle test. Based on ThODNH3 the test substance was biodegraded by 69%.
- Conclusions:
- Under the study conditions, the test substance was determined to be classified as readily biodegradable based on ≥60% biodegradation reached at day 28 (66% biodegradation based on ThODNO3).
- Executive summary:
test was performed, which allows the biodegradability to be measured in an aerobic aqueous
medium. The ready biodegradability was determined in the Closed Bottle test performed
according to slightly modified OECD, EU and ISO Test Guidelines.
The test substance (2.0 mg/L) was exposed to river water, which was spiked to a mineral
nutrient solution, dosed in closed bottles, incubated in the dark at 22.6 to 22.7°C and the
content in the bottles was regularly stirred (10 times per day for 5-minute periods) for 28 days.
The degradation of the test item was assessed by the measurement of oxygen consumption.
According to the results of this study, the test item did not cause a reduction in the endogenous
respiration at day 7. The test substance is therefore considered to be non-inhibitory to the
inoculum.
Assuming complete nitrification of the test substance organic nitrogen, and calculating the
biodegradation based on the ThODNO3 the test substance was biodegraded by 66% at day 28 in
the Closed Bottle test. Over 60% biodegradation was not achieved within a period of 10 days
(14 days for the Closed Bottle test) immediately following the attainment of 10%
biodegradation. The time window criterion should however not be applied because amines, di-
C16-C18-alkyl methyl is a mixture of constituents (UVCB). The biodegradation of the test
substance is therefore an addition of different biodegradation curves, and the time window
should not be used as a pass/fail criterion. Amines, di-C16-C18-alkyl methyl is classified as
readily biodegradable based on the ≥60% biodegradation reached at day 28.
The test is valid as shown by an endogenous respiration of 1.30 mg/L determined in the control
bottles that were incubated with a regular stirring of the content and by the total mineralization
of the reference compound, sodium acetate. Sodium acetate was degraded by 84% of its
theoretical oxygen demand after 14 days. Furthermore, the differences of extremes of the
duplicate values at day 28 were less than 20%. Finally, the most important criterion was met
by oxygen concentrations >0.5 mg/L in all bottles during the test period.
Reference
Table I Dissolved oxygen concentrations (mg/L) in the closed bottles.
Time (days) | Oxygen concentration (mg/L) | |||
| Mcs | Mts | Mc | Ma |
0 | 9.0 | 9.0 | 9.0 | 9.0 |
| 9.0 | 9.0 | 9.0 | 9.0 |
Mean (M) | 9.00 | 9.00 | 9.00 | 9.00 |
7 | 8.1 | 5.8 | 8.5 | 4.5 |
| 8.1 | 5.6 | 8.4 | 4.5 |
Mean (M) | 8.10 | 5.7 | 8.45 | 4.50 |
14 | 8.1 | 4.3 | 8.2 | 3.8 |
| 8.0 | 3.9 | 8.3 | 3.9 |
Mean (M) | 8.05 | 4.10 | 8.25 | 3.85 |
21 | 7.7 | 2.5 | 7.9 | - |
| 7.8 | 2.9 | 8.0 | - |
Mean (M) | 7.75 | 2.70 | 7.95 | - |
28 | 7.7 | 3.1 | 7.8 | - |
| 7.7 | 3.2 | 7.8 | - |
Mean (M) | 7.70 | 3.15 | 7.80 | - |
Mcs River water with nutrients, bottle content regularly stirred
Mts River water with nutrients and test substance (2.0 mg/L), bottle content regularly stirred
Mc River water with nutrients
Ma River water with nutrients and sodium acetate (6.7 mg/L)
Table II Oxygen consumption (mg/L) and the calculated percentages biodegradation (BOD/ThOD) of the sodium acetate and the test substance in the Closed Bottle test. Biodegradation of the test substance is calculated both without nitrification (BOD/ThODNH3) and with nitrification (BOD/ThODNO3).
Time (days) | Oxygen consumption (mg/L) | Biodegradation (%) | |||
Test substance | Acetate | Test substance | Acetate | ||
ThODNH3 | ThODNO3 | ||||
0 | 0.00 | 0.00 | 0 | 0 | 0 |
7 | 2.40 | 3.95 | 36 | 35 | 76 |
14 | 3.95 | 4.40 | 60 | 57 | 84 |
21 | 5.05 | - | 76 | 73 | - |
28 | 4.55 | - | 69 | 66 | - |
Description of key information
Key value for chemical safety assessment
- Biodegradation in water:
- readily biodegradable
- Type of water:
- freshwater
Additional information
Ready biodegradability of Amines, bis(hydrogenated tallow alkyl) methyl has been investigated through OECD Guideline 301 D, modified according to the recommendations of ECETOC (1985) or Blok et al. using emulsifier.
Activated sludge were cultured for 28 days in presence of Amines, bis(hydrogenated tallow alkyl) methyl.
More than 86% biodegradation of test substance occured after 28 days. The substance was considered to be readily biodegradable.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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.