Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data is from peer reviewed journal
Justification for type of information:
Data is from peer reviewed journal
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
Biodegradation study was conducted for 21 days for evaluating the percentage biodegradability of test substance (2R,5S)-5-methyl-2-(propan-2-yl)cyclohexan-1-one by using Pseudomonas citronellolis DSM 50332 as an inoculum.
GLP compliance:
not specified
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of test material (IUPAC name): (2R,5S)-5-methyl-2-(propan-2-yl)cyclohexan-1-one- Common name: Menthone - Molecular formula: C10H18O- Molecular weight: 154.2512 g/mol- Smiles notation: C1([C@@H](CC[C@@H](C1)C)C(C)C)=O- InChl: 1S/C10H18O/c1-7(2)9-5-4-8(3)6-10(9)11/h7-9H,4-6H2,1-3H3- Substance type: Organic- Physical state: Liquid- Other: Test chemical was obtained from Fluka, Neu-Ulm, Germany.
Oxygen conditions:
anaerobic
Inoculum or test system:
other: Pseudomonas citronellolis DSM 50332 (Microorganisms)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Test inoculum Pseudomonas citronellolis DSM 50332 was obtained from the Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany.
Duration of test (contact time):
21 d
Initial conc.:
308.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
TEST CONDITIONS- Composition of medium: Anoxic media was used for the study. The medium contained (per liter of distilled water) 1 g of NaCl, 0.1 g of MgCl2.7H2O, 0.04 g of CaCl2, 0.5 g of KCl, 0.125 g of NH4Cl, 0.2 g of Na2SO4, 0.4 g of KH2PO4, 1.2 g of K2HPO4, and 0.85 g of NaNO3. After autoclaving, 2 ml of a non-chelated trace element mixture, 2 ml of a selenite-tungstate solution, vitamins, and 20 ml of a 1 M NaHCO3 solution were added, and the pH was adjusted to 7.0.- Additional substrate: 10 mM nitrate, 0.5 ml 2,2,4,4,6,8,8-heptamethylnonane (HMN), and 2 mM menthone (test chemical) was added as an additional substrate. - pH: 7.0- pH adjusted: YesTEST SYSTEM- Culturing apparatus: Glass tubes (21 ml) were used as a test vessel for the study. CONTROL AND BLANK SYSTEM- Other: Pasteurized samples and inoculated preparations without nitrate or test chemical were used as controls.
Parameter:
% degradation (test mat. analysis)
Value:
1.13
Sampling time:
21 d
Remarks on result:
other: Other details not known
Details on results:
Test substance undergoes 1.13% degradation by Test mat. analysis parameter in 21 days.

Microbial growth on test chemical menthone was observed within 10 days to 3 weeks. The disappearance of test chemical in mud-free enrichment cultures was quantified.

 

Table: Amount of test chemical consumed by nitrate reducing enrichment cultures.

 

Substrate

Amt. of monoterpene consumed (µmol)

Amt. of nitrate consumed (µmol)

Bacterial growth

Amt. of gas formed (ml)

No. of electrons released from complete substrate oxidation to CO2 per no. of electrons consumed by nitrate reduction (%)

Menthone

22.6

150

+0.254

ND

167

 

 

Where,

ND = not determined

Validity criteria fulfilled:
not specified
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The percentage degradation of test substance (2R,5S)-5-methyl-2-(propan-2-yl)cyclohexan-1-one was determined to be 1.13% degradation by Test mat. analysis parameter in 21 days. Thus, based on percentage degradation, (2R,5S)-5-methyl-2-(propan-2-yl)cyclohexan-1-one is considered to be not readily biodegradable in water.
Executive summary:

Biodegradation study was conducted for 21 days for evaluating the percentage biodegradability of test substance (2R,5S)-5-methyl-2-(propan-2-yl)cyclohexan-1-one by using Pseudomonas citronellolis DSM 50332 as an inoculum.Test inoculum Pseudomonas citronellolis DSM 50332 was obtained from the Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany. Initial test substance conc. used for the study was 308.5 mg/l (2 mM). Anoxic media was used for the study.The medium contained (per liter of distilled water) 1 g of NaCl, 0.1 g of MgCl2.7H2O, 0.04 g of CaCl2, 0.5 g of KCl, 0.125 g of NH4Cl, 0.2 g of Na2SO4, 0.4 g of KH2PO4, 1.2 g of K2HPO4, and 0.85 g of NaNO3. After autoclaving, 2 ml of a non-chelated trace element mixture, 2 ml of a selenite-tungstate solution, vitamins, and 20 ml of a 1 M NaHCO3 solution were added, and the pH was adjusted to 7.0.Enrichment cultures were inoculated with activated sludge obtained from a local wastewater plant (Lintel, Osterholz-Scharmbeck, Germany). Enrichment cultures were maintained and test chemical consumption was measured in 21-ml glass tubes containing 15 ml of anoxic medium (10 mM nitrate, 0.5 ml2,2,4,4,6,8,8-heptamethylnonane (HMN), and 2 mM menthone (test chemical)). The overpressure due to gas formation was measured with a syringe, and 10 mM nitrate was added when the electron acceptor was depleted. The gas formed was analyzed by gas chromatography. Bacterial growth was observed daily and required between 3 - 22 days. Monoterpene contents were determined with a gas chromatograph equipped with flame ionization detectors and connected to a digital data-analyzing system. The amounts of monoterpene dissimilated were calculated from the differences in monoterpene contents between the pasteurized controls and the grown enrichment cultures. A model MAT ITS 40 ion trap system was used for the gas chromatography-mass spectrometry analysis.Microbial growth on test chemical menthone was observed within 10 days to 3 weeks. The disappearance of test chemical in mud-free enrichment cultures was quantified.The percentage degradation of test substance (2R,5S)-5 -methyl-2 -(propan-2 -yl)cyclohexan-1 -one was determined to be 1.13% degradation by Test mat. analysis parameter in 21 days. Thus, based on percentage degradation, (2R,5S)-5 -methyl-2 -(propan-2 -yl)cyclohexan-1 -one is considered to be not readily biodegradable in nature.

Description of key information

Biodegradation study was conducted for 21 days for evaluating the percentage biodegradability of test substance (2R,5S)-5-methyl-2-(propan-2-yl)cyclohexan-1-one by usingPseudomonas citronellolis DSM 50332 as an inoculum (Jens Harder and Christina Probian, 1995).Test inoculum Pseudomonas citronellolis DSM 50332 was obtained from the Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany. Initial test substance conc. used for the study was 308.5 mg/l (2 mM). Anoxic media was used for the study.The medium contained (per liter of distilled water) 1 g of NaCl, 0.1 g of MgCl2.7H2O, 0.04 g of CaCl2, 0.5 g of KCl, 0.125 g of NH4Cl, 0.2 g of Na2SO4, 0.4 g of KH2PO4, 1.2 g of K2HPO4, and 0.85 g of NaNO3. After autoclaving, 2 ml of a non-chelated trace element mixture, 2 ml of a selenite-tungstate solution, vitamins, and 20 ml of a 1 M NaHCO3 solution were added, and the pH was adjusted to 7.0.Enrichment cultures were inoculated with activated sludge obtained from a local wastewater plant (Lintel, Osterholz-Scharmbeck, Germany). Enrichment cultures were maintained and test chemical consumption was measured in 21-ml glass tubes containing 15 ml of anoxic medium (10 mM nitrate, 0.5 ml2,2,4,4,6,8,8-heptamethylnonane (HMN), and 2 mM menthone (test chemical)). The overpressure due to gas formation was measured with a syringe, and 10 mM nitrate was added when the electron acceptor was depleted. The gas formed was analyzed by gas chromatography. Bacterial growth was observed daily and required between 3 - 22 days. Monoterpene contents were determined with a gas chromatograph equipped with flame ionization detectors and connected to a digital data-analyzing system. The amounts of monoterpene dissimilated were calculated from the differences in monoterpene contents between the pasteurized controls and the grown enrichment cultures. A model MAT ITS 40 ion trap system was used for the gas chromatography-mass spectrometry analysis.Microbial growth on test chemical menthone was observed within 10 days to 3 weeks. The disappearance of test chemical in mud-free enrichment cultures was quantified.The percentage degradation of test substance (2R,5S)-5 -methyl-2 -(propan-2 -yl)cyclohexan-1 -one was determined to be 1.13% degradation by Test mat. analysis parameter in 21 days. Thus, based on percentage degradation, (2R,5S)-5 -methyl-2 -(propan-2 -yl)cyclohexan-1 -one is considered to be not readily biodegradable in nature.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Experimental study and predicted data for the target compound (2R,5S)-5-methyl-2-(propan-2-yl)cyclohexan-1-one (CAS No. 89-80-5) and varioussupporting studiesfor its structurally similar read across substance were reviewed for the biodegradation end point which are summarized as below:

 

In an experimental key study from peer reviewed journal (1995), biodegradation experiment was conducted for 21 days for evaluating the percentage biodegradability of test substance (2R,5S)-5-methyl-2-(propan-2-yl)cyclohexan-1-one (CAS no. 89-80-5) by using Pseudomonas citronellolis DSM 50332 as an inoculum. Test inoculum Pseudomonas citronellolis DSM 50332 was obtained from the Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany. Initial test substance conc. used for the study was 308.5 mg/l (2 mM). Anoxic media was used for the study.The medium contained (per liter of distilled water) 1 g of NaCl, 0.1 g of MgCl2.7H2O, 0.04 g of CaCl2, 0.5 g of KCl, 0.125 g of NH4Cl, 0.2 g of Na2SO4, 0.4 g of KH2PO4, 1.2 g of K2HPO4, and 0.85 g of NaNO3. After autoclaving, 2 ml of a non-chelated trace element mixture, 2 ml of a selenite-tungstate solution, vitamins, and 20 ml of a 1 M NaHCO3 solution were added, and the pH was adjusted to 7.0.Enrichment cultures were inoculated with activated sludge obtained from a local wastewater plant (Lintel, Osterholz-Scharmbeck, Germany). Enrichment cultures were maintained and test chemical consumption was measured in 21-ml glass tubes containing 15 ml of anoxic medium (10 mM nitrate, 0.5 ml2,2,4,4,6,8,8-heptamethylnonane (HMN), and 2 mM menthone (test chemical)). The overpressure due to gas formation was measured with a syringe, and 10 mM nitrate was added when the electron acceptor was depleted. The gas formed was analyzed by gas chromatography. Bacterial growth was observed daily and required between 3 - 22 days. Monoterpene contents were determined with a gas chromatograph equipped with flame ionization detectors and connected to a digital data-analyzing system. The amounts of monoterpene dissimilated were calculated from the differences in monoterpene contents between the pasteurized controls and the grown enrichment cultures. A model MAT ITS 40 ion trap system was used for the gas chromatography-mass spectrometry analysis. Microbial growth on test chemical menthone was observed within 10 days to 3 weeks. The disappearance of test chemical in mud-free enrichment cultures was quantified. The percentage degradation of test substance (2R,5S)-5 -methyl-2 -(propan-2 -yl)cyclohexan-1 -one was determined to be 1.13% degradation by Test mat. analysis parameter in 21 days. Thus, based on percentage degradation, (2R,5S)-5 -methyl-2 -(propan-2 -yl)cyclohexan-1 -one is considered to be not readily biodegradable in nature.

 

In a prediction using the Estimation Programs Interface Suite (EPI suite, 2017), the biodegradation potential of the test compound (2R,5S)-5 -methyl-2 -(propan-2 -yl)cyclohexan-1 -one (CAS No. 89 -80 -5) in the presence of mixed populations of environmental microorganisms was estimated.The biodegradability of the substance was calculated using seven different models such as Linear Model, Non-Linear Model, Ultimate Biodegradation Timeframe, Primary Biodegradation Timeframe, MITI Linear Model, MITI Non-Linear Model and Anaerobic Model (called as Biowin 1-7, respectively) of the BIOWIN v4.10 software. The results indicate that chemical (2R,5S)-5-methyl-2-(propan-2-yl)cyclohexan-1-one is expected to be not readily biodegradable.

 

Next study was experimental study done from from authoritative database (HSDB, 2017), in this study the biodegradation study was conducted for 28 days for evaluating the percentage biodegradability of test chemical. Activated sludge was used as a test inoculum for the study. The percentage degradation of test chemical was determined to be 0% by BOD parameter in 28 days. Thus, based on percentage degradation, test chemical is considered to be not readily biodegradable in nature.

 

In a supporting study from authoritative database (J-CHECK and HSDB, 2017) biodegradation experiment was conducted for 28 days for evaluating the percentage biodegradability of test chemical. The study was performed according to OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I) under aerobic conditions. Activated sludge was used as a test inoculums for the study. Concentration of inoculum i.e, sludge used was 30 mg/l and initial test substance conc. used in the study was 100 mg/l, respectively. The percentage degradation of test substance was determined to be 0% and 5% by BOD, TOC removal and GC parameter in 28 days. Thus, based on percentage degradation, test chemical is considered to be not readily biodegradable in nature.

 

On the basis of above results for target chemical (2R,5S)-5-methyl-2-(propan-2-yl)cyclohexan-1-one (from peer reviewed journal and EPI suite, 2017) and for its read across substance (from authoritative database J-CHECK and HSDB, 2017), it can be concluded that the test substance (2R,5S)-5-methyl-2-(propan-2-yl)cyclohexan-1-one can be expected to be not readily biodegradable in nature.