Vanillin

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: screening test, other

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles.

Principles of method if other than guideline:

Anaerobic degradation using Hungate technique with acclimation phase prior degradation assessment

GLP compliance:

no

Oxygen conditions:

anaerobic

Inoculum or test system:

anaerobic sludge

Details on inoculum:

- Source of inoculum/activated sludge: Inoculum from a laboratory  anaerobic digester fed primary settled sewage sludge on a 15 days  detention 
time.
- Preparation of inoculum for exposure: No details
- Concentration of sludge: 10% (vol/vol) of the anaerobic bacteria culture
- Initial cell/biomass concentration: No details

Duration of test (contact time):

16 d

Initial conc.:

300 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CH4 evolution

Parameter followed for biodegradation estimation:

CO2 evolution

Parameter followed for biodegradation estimation:

other: test substance monitoring

Details on study design:

TEST CONDITIONS
- Composition of medium: Prereduced medium containing various  oligoelements and 1% of vitamin (vol/vol), 0.001 g resazurin  (oxidation-
reduction indicator, and C/N/P ratio was 100/15/1 as described by Wolin et al. (1963).
- Test temperature: 35 deg.C
- pH: 7.0
- pH adjusted: yes
- Aeration of dilution water: Bubbling with oxygen-free air (30% CO2, 70%  N2).
- Suspended solids concentration: No details
- Test substance concentration: 300 mg/l
- Continuous darkness: no 

TEST SYSTEM
- Culturing apparatus: 250 ml sealed serum bottles
- Number of culture flasks/concentration: 10
- Method used to create anaerobic conditions: The medium was boiled,  followed by addition of sodium sulphide (reducing agent). Moreover,  serum-bottles were flushed (500 ml/min) with oxygen-free gas for 20 min  before inoculation.
- Measuring equipment: Gas composition (CH4, CO2) was checked with gas  partitioner (Fisher-Hamilton, model 29).

SAMPLING
- Sampling frequency: daily
- Sampling method: For gas: with 1ml gas-tight glass syringe. 
Test  medium: plastic disposal syringe (volume not specified).

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes

Value:

72

Sampling time:

16 d

Details on results:

Parameters followed for biodegradation estimation: CH4 and CO2 evolution, test substance monitoring


Raw data for anaerobic degradation of vanillin (n=10)

Acclimation lag necessary to an optimum anaerobic degradation: 12 +/- 1.2 days
Period of gas production: 16 +/- 1.1 days
Degradation of substrate carbon to gas: 72 +/- 1.4%


Degradation products: not measured

Validity criteria fulfilled:

not specified

Interpretation of results:

other: biodegradation observed

Conclusions:

72% of degradation after 16 days.

Reference 2

Endpoint:

biodegradation in water: screening test, other

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Method: Degradation of effluent with an isolated fungal species in an aerated batch reactor

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

other: Aspergillus terreus

Details on inoculum:

- Source of inoculum/activated sludge: The experiment was performed with an isolated fungal species: Asperigullus terreus provided by the
Microbiology Department of the Faculty of the University of Granada, Spain.
- Laboratory culture: Yes
- Method of cultivation: Growth medium: malt-extract-agar
- Preparation of inoculum for exposure: 1.9x10E+8 viable spores/ml were suspended in saline solution. For each effluent concentration, 2 15ml
tube ware inoculated with 1.5 ml of suspension, incubated at 28 deg. C for 72h with constant shaking. Each tube was then diluted with Minimum
Maintenance Medium (MMM), defined by Jachekar et al. (1981) to a volume of 150ml and incubated in two 500 ml flasks (28 deg. C, 72h)

DETAILS ON EFFLUENT
- Effluent samples were drawn from the final centrifugation step of "Pieralisi" process, from olive oil-extraction industry, into sterile flasks.
- Physical and chemical characteristics of the effluent is as follows:
pH: 5.26 COD: 212.25 g/l BOD: 179.45 g/l Total solids: 55.59 g/l Volatile solids: 44.46 g/l Settled solids: 7.45 g/l
Suspended solids: 2.44 g/l Dissolved solids: 45.4 g/l Total phenolic compounds: 0.7%
- The samples were diluted to concentrations between 20 and 80% using MMM.

TEST SAMPLE PEPARATION
- After the last step of the inoculum treatment, the two 500 ml flasks containing fugal cultures ware combined and added to effluent samples to
a volume of 3000 ml in a 5 litre batch reactor

Duration of test (contact time):

6 d

Initial conc.:

5.6 mg/L

Based on:

test mat.

Initial conc.:

1.4 mg/L

Based on:

test mat.

Details on study design:

TEST CONDITIONS
- Composition of medium: The experimental medium used is Minimum Maintenance Medium (MMM)
- Test temperature: 28 deg. C
- pH: No details
- Test duration: 6 days
- Aeration of experimental medium: with compressed air at a rate of 720 ml of filtered air per min.
- Test substance concentration: Effluent samples ware diluted between 20 and 80% with MMM and diluted again with Aspergillus terreus suspension
at a rate of 10%. Vanillin concentration ranged between 1.4 and 5.6 mg/l (with total phenolic content: 0.7%, Vanillin content in total phenolic load: 0.52% and COD: 212.25 g/l).
- Control substance concentration: No

TEST SYSTEM
- Culturing apparatus: Aerated, thermostated and stirred batch reactor.
- Number of culture flasks/concentration: 1
- Method used to create aerobic conditions: Bubbling (720 ml of filtered air/min.

SAMPLING
- Sampling frequency: No details ("Samples were taken at regular intervals")

CONTROL AND BLANK SYSTEM: No

Reference substance:

not required

Value:

62.5

Sampling time:

6 d

Details on results:

Vanillin degradation rate by A. terreus after 6 days in an aerated batch reactor, contained in olive oil-extraction effluent, is 62.52%.

Degradation products: not measured

Interpretation of results:

other: biodegradation observed

Conclusions:

Vanillin is biodegradable after 6 days in an aerated batch reactor, contained in olive oil-extraction effluent.

Reference 3

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: Experimental data obtained at Chemical Inspection and Testing Institute, Japan, according to OECD guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Deviations:

not specified

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Preparation of activated sludge: A mix of old and fresh activated sludge is performed every three months.
Fresh activated sludge is a mixture of ten samples from diferent city and industrial sewage plants, soils and rivers in Japan. This mixture was then cultured at pH=7+/-1 under aeration
Concentration: 30 mg/L
No other information on the inoculum available.

Duration of test (contact time):

14 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

TOC removal

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS: no data
TEST SYSTEM
- Volume of test solution: 300 mL
- Number of culture flasks/concentration: 3
- magnetic stirrer
- Test temperature: 25°C +/- 1°C
- Culture medium: according to Japanese Industrial Standards JIS K 0102-1986-21 adjusted to pH = 7
No other data

SAMPLING: No data

CONTROL AND BLANK SYSTEM
- Inoculum blank: 1 test vessel with 300mL of basal culture medium (sludge)
- water + test substance at 100 mg/L
- reference substance: sludge + aniline at 100 mg/L
- Concentration of activated sludge (as suspended solid concentration): 30mg/l
Volume of test solution: 300ml
Cultivated temperature: 25deg.C
Cultivation duration: 14days

Parameter:

% degradation (TOC removal)

Value:

97 - 100

Sampling time:

14 d

Details on results:

The results published on the CERI website are as follow:

Parameter percentage average%)
biodegradation(%)
BOD 96 98 96 97
TOC 97 100 100 99
HPLC 100 100 100 100


The conclusion reported in the Official Bulletin of Economy, Trade and Industry of Japan, published Dec. 12th 1993, was: "Chemical substance determinated to be ready biodegradable".

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Conclusions:

Vanillin is readily biodegradable.

Executive summary:

In this study, the biodegradation of Vanilline (initial concentration of 100 mg/L) was studied during 14 days according to OECD guideline 301C. Inoculum used in the test was an activated sludge at a concentration of 30 mg/L obtained by mixing 10 different samples from different city and industrial sewage plants, soils and rivers in Japan. The percentage of biodegradation was estimated by following 3 parameters: the Biological Oxygen Demand (BOD), the Total Organic Carbon (TOC) and the test substance analysis (estimated by HPLC). The corresponding percentages of biodegradation obtained after 14 days were 97 %, 99 % and 100% respectively. Under the test conditions Vanillin is therefore considered as readily biodegradable.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 10 nov 1995 to 14 feb 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study following recognized methods

Qualifier:

according to guideline

Guideline:

ISO 5815 (Water quality - Determination of Biochemical Oxygen Demand after 5 Days (BOD5) - Dilution and Seeding Method)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Norvegian Standard NS4748

Deviations:

not specified

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): microorganisms in effluent from an activated sludge simulation unit (Husman) - cultivated on synthetic sewage (OECD 303a) and domestic sewage was used in seed in the test.

- Preparation of inoculum for exposure: The seeding water was withdrawn from the sedimentation tank and prepared by filtration through a coarse filter.

Duration of test (contact time):

5 d

Initial conc.:

2 mg/L

Based on:

test mat.

Initial conc.:

4 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

other: BOD and COD

Details on study design:

TEST CONDITIONS
- Composition of medium:
*Stock solutions for mineral medium:
(a) Potassium dihydrogen orthophosphate, KH2PO4 : 8.50 g
Dipotassium hydrogen orthophosphate, K2HPO4 : 21.75 g
Disodium hydrogen orthophosphate dihydrate, Na2HPO4.2H2O : 33.40 g
Ammonium chloride, NH4Cl : 0.50 g

Dissolve in water and make up to 1 litre. The pH of the solution should be 7.4.

(b) Calcium chloride, anhydrous, CaCl2 : 27.50 g
or
Calcium chloride dihydrate, CaCl2.2H20 : 36.40 g

Dissolve in water and make up to 1 litre.

(c) Magnesium sulphate heptahydrate, MgS04.7H20: 22.50 g

Dissolve in water and make up to 1 litre.
(d) Iron (III) chloride hexahydrate, FeCl3.6H20 : 0.25 g

Dissolve in water and make up to 1 litre.
Note: one drop of concentrated HCl or 0.4 g EDTA disodium salt will be added per liter so these solutions do not need to be made up right before they are used.

*Preparation of mineral medium:
Mix 1 ml of solution (a) with 800 ml water, then add 1 ml of solutions (b), (c) and (d) and make up to one liter.

- Test temperature: 19.6 -20.2°C
- pH: not measured
- inoculum concentration: 5 ml of inoculum in 1000 ml of test medium. This gives a plate count of approx. 10 exp3 CFU per ml in the test medium.
- Aeration of dilution water: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: bottles
No more data

SAMPLING: no data

CONTROL AND BLANK SYSTEM
- Inoculum blank: no
- Abiotic sterile control: no
- Toxicity control: yes


STATISTICAL METHODS: none

Reference substance:

other: solution of glucose (150 mg/L) and glutamic acid (150 mg/L) for BOD method

Reference substance:

other: Potassium hydrogenphtalate (0.42 g/L in distilled water) for COD method

Parameter:

BOD5

Value:

1 260 mg O2/g test mat.

Parameter:

COD

Value:

1 760 mg O2/g test mat.

Parameter:

BOD5*100/COD

Value:

72

Results with reference substance:

The BOD5 of the reference solution (150 mg/L glucose and 150 mg/L glutamic acid) was 190 mg/L. This value falls within the acceptable BOD5 range, 180-230 mg/L.
The COD of Potassium hydrogen phtalate (0.42 g/L) was 487 mg/L. The ThOD was 500 mg/L.

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Conclusions:

Vanillin is readily biodegradable when measured as BOD5/COD.

Executive summary:

In a study (NIVA, 1996), the potential for aerobic biodegradation of Vanillin was investigated by determination of the biochemical oxygen demand (BOD5) and the chemical oxygen demand (COD). The tests were performed in accordance with ISO 5815 method.

The BOD5/COD after 5 days was 0.72.

In these conditions, Vanillin is considered as readily biodegradable.

Reference 5

Endpoint:

biodegradation in water: screening test, other

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Study well conducted, but performed with isolated bacterial strain from soil, and colorimetric and semi-quantitative analytical methods.

Qualifier:

no guideline followed

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

other: other bacteria: pure culture of vanillin-destroying bacteria extracted from soil

Details on inoculum:

- Source of inoculum: Pure bacterial strain isolated from Alabama (US) soil
- Concentration of inoculum: Not indicated

Exposure conditions
- The exposition occurred in a 2-liter flask with 250 ml of a mineral nutriment solution (in g/l : 0.4 NaNO3, 0.4 K2HPO3, 0.2 MgSO4, 0.4 NH4Cl,
0.2 KCl) containing vanillin (0.4 g/l). The Flask was sterilized before bacterial inoculation and incubated at 25-30°C.

Duration of test (contact time):

8 d

Initial conc.:

0.4 g/L

Based on:

test mat.

Details on study design:

no data

Reference substance:

not required

Value:

100

Sampling time:

4 d

Details on results:

Raws results are given in the table below. The mean degradation time is around 2.5 days (estimated by log-normal regression)

Table: Kinetic of vanillin degradation (mg/l)

Degradation Vanillin quantified by
time (days) Estes reagent (mg/l)
0 249
1 257
2 177
3 56
4 23
5 22
6 26
7 10
8 0


Degradation products: yes
Degradation products (CAS No./EC No./EINECS Name): 121-34-6 204-466-8 vanillic acid

Kinetic of testsubst.	:	2 day(s) = 71   %
		4 day(s) = 9   %
		6 day(s) = 10   %

Interpretation of results:

readily biodegradable

Reference 6

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD Guideline, but on raw effluent without any chemical analysis.

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Deviations:

not specified

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

DETAILS ON INOCULUM:
- Source of inoculum/activated sludge: Activated sludge from municipal sewage treatment plant, Saint Fons, France.
- Preparation of inoculum for exposure: Sludge was washed 2 times and transferred immediately in a carbon-free growth medium, containing also
the industrial effluent.
- Concentration of sludge: 1 g/l dry matter

DETAILS ON TEST SAMPLE:
Test sample was made by pooling 3 effluent samples collected every day during 3 days (October, 23, 24 & 25th 1986).

Duration of test (contact time):

28 d

Initial conc.:

250 mg/L

Based on:

DOC

Details on study design:

TEST CONDITIONS
- Composition of medium: carbon-free growth medium
- Test temperature: no data
- pH: no data
- Suspended solids concentration: 1g/l
- Test substance concentration: 250 mg DOC/l
- Control substance concentration: No control substance

TEST SYSTEM
- Number of culture flasks/concentration: 2
- Measuring equipment: Carbona anlyzer (Beckman)

SAMPLING
- Sampling frequency: 0, 0.75, 2, 4, 6, 8, 14, 22, 28 days

CONTROL AND BLANK SYSTEM
- Inoculum blank: No inoculum blank system
- Abiotic sterile control: 2 replicates sterilised with HgCl2 (100mg/l)
- Toxicity control: No toxicity control system

STATISTICAL METHODS:
No details

Reference substance:

not required

Value:

37.2

Sampling time:

18 h

Value:

78.5

Sampling time:

2 d

Value:

81

Sampling time:

4 d

Value:

81.3

Sampling time:

6 d

Value:

83.3

Sampling time:

28 d

Details on results:

The degradation curve reaches a plateau after 2 days of incubation; the degradation rate is therefore 78.5 %. The maximum
degradation value was obtained after day 4 and was constant during the 24 last days of the test. This effluent was not degradable by abiotic
mechanisms.

The industrial effluent collected downstream a vanillin plant is inherently and rapidly biodegradableby activated sludge from municipal STP.

The main problem with that study is that no analytical measure was provided. The effluent composition in organic compound was not determined
and the real degradation rate of vanillin can not be assessed.

Degradation products: not measured

Validity criteria fulfilled:

not specified

Interpretation of results:

other: The raw effluent produced by a vanillin factory is inherently and rapidly biodegradable

Conclusions:

The raw effluent produced by a vanillin factory is inherently and rapidly biodegradable

Reference 7

Endpoint:

biodegradation in water: screening test, other

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: No enough data for the assessment

GLP compliance:

no

Oxygen conditions:

anaerobic

Inoculum or test system:

other: benthic microorganisms of an eutrophic lake

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): from samples of bottom deposites at 2 
sites of the eutrophic  lake Jeziorak (Poland), 26 % of microorganisms were  able to utilize Vanillin as sole source of carbon.

Duration of test (contact time):

6 d

Initial conc.:

100 mg/L

Based on:

test mat.

Sampling time:

6 d

Description of key information

The MITI study  (1993) is selected as key study. Although GLP were not mentioned, this study was performed according to OECD 301C test guideline and was generated by the Japanese Competent Authorities. The vanillin biodegradation rate reached 97% (relative to biochemical oxygen demand) after 14 days.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

Other studies were selected as supporting studies: NIVA study confirms the ready biodegradation potential of vanillin. Martinez (1993), Healy (1979) and RP (1987) demonstrated the potential of Vanillin for biodegradation in various conditions (anaerobic, effluents. . .).