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Diss Factsheets

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:
24 Sep - 22 Oct 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Qualifier:
according to guideline
Guideline:
ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test))
GLP compliance:
yes (incl. QA statement)
Remarks:
Department of Health of the Government of the United Kingdom

Test material

Constituent 1
Chemical structure
Reference substance name:
Vinyl benzoate
EC Number:
212-214-3
EC Name:
Vinyl benzoate
Cas Number:
769-78-8
Molecular formula:
C9H8O2
IUPAC Name:
vinyl benzoate

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK (obtained on 24 Sep 2008)
- Laboratory culture: no
- Pretreatment: The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded). In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2-free air for approximately 1 h whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.6 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately 1 h prior to removal of an aliquot (2 L) of the supernatant for use in the test. The supernatant was maintained on aeration using CO2-free air until use.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
20 mg/L
Based on:
TOC
Initial conc.:
27.4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: according to guideline
- Test temperature: 20 ± 1 °C
- pH: 7.6
- pH adjusted: yes; 7 M sodium hydroxide
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 125 mL glass Wheaton bottles (total volume when full 160 mL) each containing 107 mL of solution.
- Number of culture flasks/concentration: 33
- Method used to create aerobic conditions: constantly shaken
- Measuring equipment:
DOC measurement: On days 0 and 28 samples were removed from the control and standard material vessels prepared for DOC analysis and filtered through Gelman 0.45 µm Acrocap filters (approximately 5 mL discarded) prior to DOC analysis. DOC analysis of the test material and toxicity control vessels was not possible due to the insoluble nature of the test material in water. The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser or a Shimadzu TOC-VCSH TOC analyser. Samples (27, 13 or 50 µL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.
IC measurement: Triplicate control, standard material and test material vessels were sacrificed on days 0, 2, 5, 8, 12, 14, 16 and 21 for IC analysis. On day 28, five replicate vessels were sacrificed for IC analysis. Triplicate toxicity control vessels were sacrificed on days 0, 8 and 14 for IC analysis. An aliquot (1.0 mL) of concentrated orthophosphoric acid was injected through the septum of each vessel taken for analysis in order to lower the pH of the medium to < 3. The vessels were then shaken at approximately 150 rpm (INFORS TR-225 orbital platform shaker) for 1 h at 20 ± 1 °C prior to samples being withdrawn from the headspace and analysed for IC. The principle of this method is that after acidification to a pH value of < 3 and equilibration at 20 ± 1 °C, the equilibrium constant for the distribution of CO2 between the liquid and gaseous phases in the test vessels is 1.0 and hence only the CO2 concentration in the headspace needs to be determined. The samples were analysed using a Shimadzu TOC-Vcsh TOC analyser. Samples (50 µL) taken from the headspace were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.
- Test performed in closed vessels: All vessels were sealed using Teflon lined silicon septa and aluminium crimp caps

SAMPLING
- Sampling frequency: on days 0, 2, 5, 8, 12, 14, 16 and 21 for IC analysis
- Sampling method: One vessel was removed and analysed. On day 28, five replicate vessels were sacrificed for IC analysis. Triplicate toxicity control vessels were sacrificed on days 0, 8 and 14 for IC analysis.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 33 replicates
- Abiotic sterile control: yes, 33 replicates
- Toxicity control: yes, 9 replicates
- Other: positive control: yes, 33 replicates
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
94
Sampling time:
28 d

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 100% degradation after 14 and 28 d thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

The test material attained 94% degradation after 28 days and satisfied the 10-day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD 310.

Table 1: Percentage biodegradation

Day

% Degradation

Sodium benzoate

Test material

Toxicity control

0

-1

0

0

2

74

63

-

5

89

72

-

8

94

83

83

12

95

90

-

14

100

94

99

16

95

93

-

21

100

92

-

28

100

94

-

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable