Vanillin

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 30 jan 2007 to 30 oct 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Preliminary test
A sample of 5 mL was taken from the 100 mg/L nominal solution container at the beginning of the test and kept at -20°C.
As the test item was found to be toxic at this limit test concentration after 24 hours, sampling was cancelled and samples already taken were not
analyzed.

- Definitive test
Samples of 5 mL were taken from each test solution container (except for the control) at the beginning of the test and kept at -20°C until analysis.
Samples totaling 5 mL were also taken at T24 and T48 hours at each concentration (except for the control) and kept at -20°C until analysis.

Vehicle:

no

Details on test solutions:

The stock solutions, for the preliminary and definitive tests, were prepared by dissolving the test item directly in M4 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 M4 reconstituted water to provide a geometric series of concentrations:
• 0, 0.1, 1, 10 and 100 mg/L for the preliminary test,
• 0, 5.62, 10.0, 17.8, 31.6, 56.2 and 100 mg/L for the EC50 test.

The pH range considered as appropriate for a normal life of test organisms is circa 6 to 9.
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 incorporation of the animals.
Test vessels containing daphnids were filled directly from the test solution containers immediately after preparation and test solutions remained
unchanged.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna STRAUS-clone 5, an invertebrate crustacean (water flea).
Reason for this choice: species commonly used in Europe for aquatic toxicity testing and recommended in OECD and EEC guidelines.
Breeder: CIT.
Origin: Centre Technique du Bois et de l'Ameublement, Paris, France.
Life stage: at the beginning of the test, first instar daphnids between 6 and 24 hours old and produced from parthenogenically reproducing brood
female population.

- Feeding during test
Culture animals are fed with a diet of between 0.1 and 0.2 mg of carbon per Daphnia per day, in the form of the algal strain Scenedesmus subspicatus which is cultured at CIT, during breeding, but animals were not fed during the test.

Acclimation: animals are held in water of the same quality as the test water from birth until use in the test.
Allocation of the animals: animals were randomly placed first into one beaker containing water of the same quality and conditions as the culture
water and then 6 hours later, the remaining daphnids were randomly assigned to the test item solutions using a pipette.
Number of animals: 20 animals in all test item solutions and in the control, with five animals per vessel.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

238 - 289 mg/L CaCO3

Test temperature:

18 - 22°C

pH:

between 6 and 9 and did not vary by more than 1 unit in the control and in each test solution during the test.

Dissolved oxygen:

> 3 mg/L

Nominal and measured concentrations:

Nominal: 0, 5.62, 10, 17.8, 31.6, 56.2 and 100 mg/L.
Geometric mean 0-48h (measured): Not determined, 2.23, 3.61, 12.9, 26.8, 50.5 and 90.2 mg/L.

Details on test conditions:

Test solutions were not changed over the 48-hour test period.

The other test conditions were as follows:
- Temperature in water: between 20°C and 21.6°C in the preliminary test and between 20.2°C and 20.8°C in the definitive test.
- Light/dark cycle: 16/8 hours.
- Duration of test: 48 hours.
- Loading: test vessels were loaded at one daphnid for 10 mL of solution.
- Forced aeration: was not used during the test. Dilution water was aerated prior to the addition of the test item.

The pH and dissolved oxygen values of the control and the highest concentration in the preliminary test and of all definitive test concentrations were measured at the beginning and the end of the tests.
Temperature was measured at the beginning and the end of the test, and the maximum and minimum temperatures during the test were recorded.
Water hardness was checked at the beginning and the end of the test in the control solution.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

36.79 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 26.8 - 50.5

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

26.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Details on results:

All validity criteria of the study were respected.

Measured concentrations were within ± 20% of the corresponding nominals at the 24 hour sampling time, then dropped below 80% at concentrations of 17.8 mg/L and higher, and were below the limit of quantification (< 1 mg/L) at concentrations of 5.62 and 10 mg/L at the 48 hours sampling time.
The study results are therefore based on geometric means of measured concentrations in test solutions during the corresponding exposure periods, i.e. 24 or 48 hours.

Results with reference substance (positive control):

- Results with reference substance valid?: yes
24-hour EC50: 1.11 mg/L

Reported statistics and error estimates:

Immobilization was determined at 0, 24 and 48 hours.
Determination of the EC50:
When for at least two concentrations, immobilization is > 0% and < 100%, the EC50 is calculated according to Probit analysis (i.e. Finney's method, published by E. Weber, combined with Bliss's method). The confidence interval limits are calculated statistically according to Fieller's method.

When at only one concentration, immobilization is > 0% and < 100%, the EC50 is also calculated by Probit analysis. In this case, the highest concentration causing no immobilization and the lowest concentration producing 100% immobilization are used as confidence limits.

If at all concentrations, immobilization is 0% or 100%, the EC50 corresponds to the geometric mean of the highest concentration causing no immobilization and the lowest concentration producing 100% immobilization. In this case, the highest concentration causing no immobilization and the lowest concentration producing 100% immobilization can also be used as confidence limits.

The immobilization of daphnids (% relative to the control) are summarized in the table below:

Group	Control	RHOVANIL Extra-Pure
	Nominal concentration (mg/L)
	0	5.62	10.0	17.8	31.6	56.2	100
	Means of measured concentration (mg/L)
0 hour	ND	5.12	10.3	18.5	33.2	59.6	106
Geometric mean 0-24 h	ND	4.7	9.69	18.0	32.0	59.7	104
Geometric mean 0-48 h	ND	2.23	3.61	12.9	26.8	50.5	90.2
	Immobilization (%)
24 hours	0	0	0	0	0	70	100
48 hours	0	0	0	0	0	100	100

ND: not determined

The toxicity parameters, including the EC50 and EC0 (i.e. NOEC), are summarized in the table below:

	Endpoints (mg/L) (1)
	24 hours	48 hours
EC100	104	50.5
EC50	49.1 (32.0-104)	36.79  (26.8-50.5)
EC0	32	26.8

(1) EC100: lowest concentration with 100% immobilization; EC50: Median (50%) Effective Concentration (numbers in brackets correspond to the highest concentration causing no immobilization and the lowest concentration producing 100% immobilization); EC0: highest concentration without immobilization

Validity criteria fulfilled:

yes

Conclusions:

Harmful for aquatic organisms.

Executive summary:

The 48 hours acute toxicity of Vanilline to Daphnia magna was studied under static conditions. Daphnids were exposed to test chemical at nominal concentrations of 0, 5.62, 10, 17.8, 31.6, 56.2 and 100 mg/L for 48 hours. Immobilization and was observed . 

The 48 hours EC₅₀ was 36.79 mg/L.

The 48 hours NOEC based on immobilization was 26.8 mg/L.

Based on the results of this study, Vanilline is considered as harmful to Daphnia magna.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

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.

Principles of method if other than guideline:

Method: static method

GLP compliance:

not specified

Analytical monitoring:

not specified

Details on test solutions:

no data

Test organisms (species):

Daphnia magna

Details on test organisms:

10 animals per concentrations

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Details on test conditions:

3 replicates per concentration.
No more data on test conditions.

Reference substance (positive control):

not specified

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

180 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

log (EC50) = -3.499 mol/L

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: No test substance information, no guideline, secondary literature.

Principles of method if other than guideline:

no data

GLP compliance:

not specified

Analytical monitoring:

not specified

Test organisms (species):

Daphnia magna

Test type:

not specified

Water media type:

freshwater

Total exposure duration:

48 h

Details on test conditions:

no data

Reference substance (positive control):

not specified

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

48.1 mg/L

Description of key information

CIT report (2009) was chosen as key study, since conducted according to OECD guideline and GLP.
Daphnids were exposed to test chemical at nominal concentrations of 0, 5.62, 10, 17.8, 31.6, 56.2 and 100 mg/L for 48 hours in static conditions. Immobilization was observed . The 48 hours EC50 was 36.79 mg/L, and the 48 hours NOEC was 26.8 mg/L. 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

36.79 mg/L

Additional information

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

Based on the results of this study, Vanilline is considered to be harmful to Daphnia magna.

Another study on Natural Vanillin was chosen as supporting study.

The 48-hours acute toxicity of Natural Vanillin (purity 98%) to Daphnia magna was studied under static conditions. The 48-hours EC50 was higher than 10 mg/L but less than 100 mg/L (based on nominal concentration). 

Based on the results of this study, Natural Vanilline is considered to be harmful to Daphnia magna.