Vanillin

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 19 feb 2007 to 30 oct 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Test solutions analysis
- Preliminary test
At T0 hour, a sample was taken from the limit test solution (100 mg/L nominal) before addition of algae and stored at -20°C until analysis.
At T24 and T48 hours, samples were taken from each limit test solution replicate, pooled (2 x 5 mL in total) and stored at -20°C until analysis.
Further samples were taken at T24 and T48 hours from the limit test solution which contained no algae and had been run alongside the test to
determine the influence of adsorption (at the surface of algae cells) and/or bioaccumulation on the possible decrease in test item concentration
throughout the test.

As the test item was found to be toxic at the limit test concentration after 48 hours, no sampling was taken at T72 hours and samples already taken were not analyzed.

- Definitive tests
For each definitive test, at T0 hour, samples were taken from each test solution before addition of algae, except for the control, and stored at -20°C
until analysis.
At T24, T48 and T72 hours, samples were taken from all test replicates at each concentration (except for the control), pooled by concentration
(2 x 5 in total) and stored at -20°C until analysis. Before analysis, these samples containing algae were centrifuged and the supernatant was analyzed separately to verify the test item concentration in solution throughout the test.
Further samples were taken at T24, T48 and T72 hours from the solutions which contained no algae and had been run alongside the test to
determine the influence of adsorption (at the surface of algae cells) and/or bioaccumulation on the possible decrease in test item concentration
throughout the test.

Following a suspicion of bacteria contamination in the first definitive tests, the samples in the first and second definitive tests were not analyzed.

Vehicle:

no

Details on test solutions:

The stock solutions, for the preliminary and definitive tests, were prepared by dissolving the test item directly in OECD reconstituted water
After agitation, the stock solutions were immediately used to prepare the test solutions.

Test solutions were prepared by further dilution of the stock solution with OECD reconstituted water to provide a geometric series of concentrations:
• 0.1, 1, 10 and 100 mg/L for the preliminary test,
• 2.20, 4.85, 10.7, 23.5, 51.7, 114 and 250 mg/L for the EC50 tests.

In the second definitive test, all preparations (stock solution and test solutions) were prepared under sterile conditions (laminar flow hood). The
stock solution and the reconstituted water (OECD medium) were sterilized by membrane filtration (diameter 0.22 µm).

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Pseudokirchneriella subcapitata.
Strain No.: CCAP 278/4.
Reason for this choice: species commonly used in Europe for aquatic toxicity testing and recommended in OECD and EEC guidelines.
Cultured at: CIT.
Origin: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN, Argyll PA37
1QA, Scotland.
Culture method: the algae are cultured under sterile conditions and maintained at exponential growth rate.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

34 +/- 17 mg/L CaCO3

Test temperature:

Controlled daily in a flask containing test water but no algae, run alongside the test. Between 21°C and 25°C and did not vary by more than ± 2°C
during the test.

pH:

The pH values of the control and of all the test concentrations (except for those of the range-finding concentrations) were measured at the
beginning and the end of the tests. For the control, these values did not deviate by more than 1.5 units throughout the tests.
The pH of all test solutions remained within the range 6.0 +/- 0.2 to 9.0 +/- 0.2 after preparation and there was therefore no adjustment of pH before addition of the algae.

Nominal and measured concentrations:

definitive test:
Nominal: 0, 2.2, 4.85, 10.7, 23.5, 51.7, 114 and 250 mg/L.
Measured geometric mean 0-72h: Not determined, 1.71, 4.22, 9.79, 21.1, 47.0, 111 and 259 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: open
- Aeration: yes
Glass test vessels were filled directly from the test solution containers immediately after preparation and algae were then added to these vessels.
Test solutions remained unchanged throughout the study.

OTHER TEST CONDITIONS
- Illumination: continuous. A set of 15 and 30 W Osram Fluora® fluorescent tubes between "white" and "daylight" type (spectral range 400 to 700 nm) were set approximately 40 cm above the cultures. These emit light measured using a lux meter equipped with a spherical collector at a level
corresponding to half the height of the cultures in their conical flasks (corresponding to an irradiance of 252 mW/m2 for 15 W and 476 mW/m2 for 30 W tubes x no. of bulbs = 3724 mW/m2). The light intensity at each position to be occupied by a culture flask is measured regularly
(approximately 7000 lux). The color temperature of the fluorescent lamps is between 4400 K and 5000 K.
- Duration of test: 72 hours.
- Transfer: the number of cells in the algal pre-culture was counted and the quantity of pre-culture to be added to the test solutions was calculated
to give a concentration of 1 x 10E4 cells/mL. The algal pre-culture was then added where appropriate to the 100 mL test solutions and all the 250 mL conical flasks stoppered with sterile cotton wool wrapped with lint and transferred to the agitator.
- Culture homogeneity: the solutions were agitated throughout the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2 for definitive test.
- Range finding study: 0.1, 1, 10 and 100 mg/L
- Test concentrations (nominal): 0, 2.2, 4.85, 10.7, 23.5, 51.7, 114 and 250 mg/L in the definitive test
- Results used to determine the conditions for the definitive study: result in preliminary study

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

120 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 110-130 mg/L

Results with reference substance (positive control):

- Results with reference substance valid: yes
- 72-hour ErC50: 1.18 and 1.04 mg/L

Validity criteria fulfilled:

yes

Conclusions:

Harmful for aquatic organisms

Executive summary:

In a 72 hours algae study (CIT, 2008), the acute toxicity of the test item Rhovanil Extra Pure (Vanilline) was evaluated in the algal strain Pseudokirchneriella supcapitata using a 72-hour static test according to current EC and OECD guidelines, and GLP.

The main criterion measured is the EC50, a statistically derived concentration resulting in 50% reduction of the average specific growth rate (ErC50) or the yield (EyC50) relative to the control.

The 72 -hour ErC50 (geometric mean) was 120 mg/L.

The 72 -hour EyC50 (geometric mean) was 78.6 mg/L.

The NOEC was 47 mg/L.

The validity criteria were fulfilled.

In this study, Vanilline is considered as harmful to algae.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Only one concentration tested and visual inspection of growth compared to a control flask.

Principles of method if other than guideline:

Visual inspection of growth compared to a control flask.

GLP compliance:

no

Analytical monitoring:

no

Test organisms (species):

other: different species, see results

Details on test organisms:

no data

Test type:

not specified

Water media type:

not specified

Limit test:

yes

Total exposure duration:

21 d

Test temperature:

22°C

Nominal and measured concentrations:

One concentration tested: 2 mg/L.

Details on test conditions:

Tests were carried out in 25 ml Erlen flasks incubated at a constant temperature of 22°C in an illuminated culture room. The number of algal cells in the test medium at the beginning of the test averaged approximately 125000 per ml. During the period of incubation, the amount of visible algal growth was recorded at 3, 7, 14 and 21 days. Growth was then compared to control.

Reference substance (positive control):

not specified

Details on results:

The results were recorded as Toxic, where no growth occurred in the presence of the test chemical, but did occur in the control flask; Partially toxic, where growth occurred but the amount was not as great as that in the control; Non-toxic, where growth was similar in amount to that in the control.

For vanillin, these results were reported:
- Microcystis aeruginosa and Cylindrospermum licheniform (Algae, blue, cyanobacteria): population growth similar to control up to 21 days exposure at 2 mg/L.
- Scenedesmus obliquus and Chlorella variegata (green algae) : Biomass lower than in the control after 3 days exposure (visual estimation) of 2 mg/L. No more difference observed with the control between 7 and 21 days exposure.
- Nitzschia palea (diatom): Biomass lower than in the control after 7 days exposure (visual estimation) at 2 mg/L. No more difference observed with the control after 14 and 21 days exposure.
- Gomphonema parvulum (diatom): no growth occurred after 3 days exposure 2 mg/L. However, some growth was detectable at 7, 14 and 21 mg/L

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Test conditions and methods poorly described. Only one concentration tested

Principles of method if other than guideline:

no data

GLP compliance:

no

Analytical monitoring:

yes

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

no data

Test type:

not specified

Water media type:

not specified

Total exposure duration:

80 h

Test temperature:

26°C

Details on test conditions:

no data

Duration:

80 h

Dose descriptor:

EC50

Effect conc.:

ca. 1 mmol/L

Basis for effect:

biomass

Details on results:

growth inhibition:
after 80h:  50% at 1 mmole/l       
after 160h: 30% at 1 mmole/l

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 is poorly described and not in accordance with guidelines.

Principles of method if other than guideline:

no data

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

not specified

Details on test solutions:

no data

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

no data

Test type:

not specified

Water media type:

freshwater

Total exposure duration:

5 d

Post exposure observation period:

no data

Details on test conditions:

no data

Reference substance (positive control):

not specified

Duration:

5 d

Dose descriptor:

LOEC

Effect conc.:

>= 152.1 mg/L

Basis for effect:

other: no data

Duration:

5 d

Dose descriptor:

other: LCIC

Effect conc.:

> 152.1 mg/L

Basis for effect:

other: no data

Details on results:

LCIC > 1000 µM (LCIC = Lowest Complete Inhibition Concentration)
LOEC = 1000 µM

other alga: Pediastrum simplex
LCIC > 1000 µM
LOEC > 1000 µM

Results with reference substance (positive control):

no data

Reported statistics and error estimates:

no data

Description of key information

CIT report (2009) was selected as key study, since conducted according to OECD guideline and GLP.
The algae Pseudokirchneriella supcapitata was exposed to vanillin in a 72-hour static test.
The 72 -hour ErC50 (geometric mean) was 120 mg/L.
The 72 -hour EyC50 (geometric mean) was 78.6 mg/L
The NOEC was 47 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

120 mg/L

EC10 or NOEC for freshwater algae:

47 mg/L

Additional information

Only one study (CIT, 2009) was selected as key study, because it was conducted according to OECD guideline and GLP.

In this study, Vanilline is considered to be harmful to algae.

Other studies were not well described and/or unreliable.