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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 April - 24 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Vetiveria zizanioides, ext.
EC Number:
282-490-8
EC Name:
Vetiveria zizanioides, ext.
Cas Number:
84238-29-9
Molecular formula:
Not available due to the UVCB nature of the substance
IUPAC Name:
Essential oil of Vetiveria zizanioides (Poaceaes)
Constituent 2
Reference substance name:
Vetiveria zizanioides, ext.
IUPAC Name:
Vetiveria zizanioides, ext.
Details on test material:
- Name of test material (as cited in study report): Vetiveria zizanioides, ext.
- CAS Number: 84238-29-9
- Substance type: Clear brown liquid
- Physical state: liquid
- Analytical purity: Not indicated by the sponsor; treated as 100% pure
- Lot/batch No.: ADL 53 901
- Expiration date of the lot/batch: 04 January 2011
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature in the dark

- Stability in water: Unknown
- Solubility in water: 10-20 mg/mL (estimated)

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Source:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Treatment:
The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 4.3 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (45 minutes) and the liquid was decanted for use as inoculum at the amount of 10 ml/l of mineral medium.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
2.7 other: mg/107 mL
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Composition of medium:
Stock solutions of mineral components
A) 8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli-Q water and made up to 1 litre,
pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-Q water
and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-Q water and
made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-Q water and
made up to 1 litre.

Mineral medium
1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water.

- Test temperature: 19.2-21.2ºC
- pH: 8.0 at study start
- Sludge, suspended solids concentration: 4.3 g/L. Before use, the sludge was allowed to settle (45 minutes) and the liquid was decanted for use as inoculum at the amount of 10 ml/l of mineral medium.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus:
- Test bottles: Glass serum bottles, sealed with butyl rubber stoppers and crimp-on aluminium seals. In general, bottles were filled to give a headspace to liquid ratio of 1:2 (107 ml to 160 ml-capacity bottle).
- Incubation: In the dark on an orbital shaker, with a shaking rate sufficient to keep the bottle well mixed and in suspension (shaking rate: 150 to 200 rpm).
- Number of culture flasks/concentration:
Per series, five test bottles for analysis at the end of the test and triplicate test bottles for other time intervals.
- Measuring equipment:
Shimadzu TOC-VCPH total organic carbon analyzer combined with a Shimadzu ASI-V autosampler.
- Test performed in closed vessels due to significant volatility of test substance: yes

CONTROL AND BLANK SYSTEM
Test set-up:
Inoculum blank: containing inoculated medium
Procedure control: containing inoculated medium and reference substance.
Test substance: containing inoculated medium and test substance.
Toxicity control: containing inoculated medium, reference substance and test substance.

STATISTICAL METHODS:
ThIC = TOC
TIC = (mg C in the liquid + mg C in the headspace)
TIC = (VL * CL) + (VH *CH)
Since the negligible amount of IC in the gaseous phase will be ignored (VH * CH =0): TIC = VL *CL

Where:
VL = volume of liquid in the bottle (litre)
CL = concentration of IC in the liquid (mg/l as carbon)
VH = volume of the headspace (litre)
CH = concentration of IC in the headspace (mg/l as carbon)

Percentage degradation = (TICt-TICb)/TOC * 100

Where:
TICt = mg TIC in test bottle at time t;
TICb = mean mg TIC in blank bottles at time t;
TOC = mg TOC added initially to the test bottles.

The results, expressed as percentage biodegradation against incubation time, were presented graphically.
A figure of more than 10% degradation was considered significant.

PREPARATION OF TEST SUBSTANCE SOLUTIONS
Vetiveria zizanioides, ext. was a clear brown liquid. Vetiveria zizanioides, ext. was treated as 100% pure. Since the organic carbon content of Vetiveria zizanioides, ext. could not be calculated, a sample of the pure test substance was taken for determination of the Total Organic Carbon (TOC) content. TOC analysis was performed using a Shimadzu TOC-VCPH total organic carbon analyzer combined with a Shimadzu SSM-5000A (Solid Sample module for Total Organic Carbon Analyzer) (Shimadzu, Kyoto, Japan). Calibration standards were Glucose (C6H12O6, 99.5%, Sigma, Steinheim, Germany) as Total Carbon (TC) standard and Sodium carbonate (Na2CO3, p.a., Merck, Darmstadt, Germany) as Inorganic Carbon (IC) standard. The TOC content of Vetiveria zizanioides, ext. was determined to be 79.72%.

Since Vetiveria zizanioides, ext. was poorly soluble in water, 2.7 mg test substance, corresponding to a final organic carbon concentration of 20 mg C/l, was added directly to the test medium (final volume bottle 107 ml). The substances were incubated in a buffer-mineral salts medium inoculated with a mixed population of micro-organisms. Note that the test substance was added immediately before closing the bottles. The test was performed in sealed bottles with a headspace of air.


SAMPLING
- Sampling frequency:
Inoculum blank and test substance: day 1, 7, 14, 21 and 28
Procedure control: day 1, 14 and 28
Toxicity control: day 1 and 28.

- Sampling method:
1 ml 7 M NaOH was injected through the septum of each bottle and the bottles were shaken for 1 h at the test temperature.
Thereafter, the bottles were removed from the shaker and allowed to settle. The supernatant was used for IC analysis.

Results and discussion

Test performance:
IC DETERMINATIONS

IC CONCENTRATION IN BLANK BOTTLES (MEAN VALUES)
Nominal day: - IC (mg/l)
1 - 1.815
7 - 2.050
14 - 2.200
21 - 2.475
28 - 2.722

IC CONCENTRATION IN REFERENCE SUBSTANCE (MEAN VALUES)
Nominal day: IC (mg/l)
1 - 1.934
14 - 18.36
28 - 16.68

IC CONCENTRATION IN VETIVERIA ZIZANOIDES, EXT. BOTTLES (MEAN VALUES)
Nominal day: IC (mg/l)
1 - 1.870
7 - 2.059
14 - 3.153
21 - 6.164
28 - 7.001

IC CONCENTRATION IN BOTTLES OF THE TOXICITY CONTROL (MEAN VALUES)
Nominal day: IC (mg/l)
1 - 2.025
28 - 19.44


% Degradationopen allclose all
Parameter:
% degradation (inorg. C analysis)
Value:
0.3
Sampling time:
1 d
Parameter:
% degradation (inorg. C analysis)
Value:
0
Sampling time:
7 d
Parameter:
% degradation (inorg. C analysis)
Value:
4.8
Sampling time:
14 d
Parameter:
% degradation (inorg. C analysis)
Value:
18.4
Sampling time:
21 d
Parameter:
% degradation (inorg. C analysis)
Value:
21.4
Sampling time:
28 d
Details on results:
Reference substance: 0.6, 80.8 and 69.8% degraded after 1, 14 and 28 days, respectively.
Toxicity control (ref. substance and test substance): 0.5 and 41.8% degraded after 1 and 28 days, respectively.

BOD5 / COD results

Results with reference substance:
The mean percentage degradation of the reference substance has reached the level for ready biodegradability (81%) by 14 days.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
other: Relative biodegradation values revealed 21% biodegradation of Vetiveria zizanioides, ext. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.
Conclusions:
Vetiveria zizanioides, ext. was not readily biodegradable under the conditions of the head space test presently performed.
Executive summary:

The study procedures were based on the OECD guideline No. 310, 2006.In addition, the procedures were designed to meet the test methods of the ISO International Standard 14593, 1999.

Vetiveria zizanioides, ext. was a clear brown liquid. Vetiveria zizanioides, ext. was treated as 100% pure. Since the organic carbon content of Vetiveria zizanioides, ext. could not be calculated, a sample of the pure test substance was taken for determination of the Total Organic Carbon (TOC) content. The TOC content of Vetiveria zizanioides, ext. was determined to be 79.72%.

 

Since Vetiveria zizanioides, ext. was poorly soluble in water, 2.7 mg test substance, corresponding to a final organic carbon concentration of 20 mg C/l, was added directly to the test medium (final volume bottle 107 ml).The substances were incubated in a buffer-mineral salts medium inoculated with a mixed population of micro-organisms. Note that the test substance was added immediately before closing the bottles. The test was performed in sealed bottles with a headspace of air.

The test consisted of four groups:

1.        Blank control: bottles containing inoculated medium;

2.        Procedure control: bottles each containing inoculated medium and 1-Octanol at             20 mg C/l;

3.        Test substance: bottles each containing inoculated medium and Vetiveria zizanioides, ext. at 20 mg C/l;

4.        Toxicity control: bottles each containing inoculated medium and Vetiveria zizanioides, ext. at 20 mg C/l and 1-Octanol at 20 mg C/l.

 

The CO2evolution resulting from the aerobic biodegradation of the test substance was determined by measuring the inorganic carbon (IC) produced in the test bottles in excess of that produced in blank vessels containing inoculated medium only. The extent of biodegradation was expressed as a percentage of the theoretical maximum IC production, based on the quantity of test substance (as organic carbon) added initially.

 

The relative biodegradation values calculated from the measurements performed during the test period revealed 21% biodegradation of Vetiveria zizanioides, ext. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control,Vetiveria zizanioides, ext.was found not to inhibit microbial activity.

 

Since all criteria for acceptability of the test were met, this study was considered to be valid.

 

In conclusion, Vetiveria zizanioides, ext. is designated as not readily biodegradable.