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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 03 nov 1995 to 14 feb 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 202 (1984) "Daphnia sp., Acute Immobilisation test and Reproduction test
Deviations:
not specified
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Vanillin purity: 99.9%
CAS: 121-33-5
Identity confirmed by IR spectrum
Loss on drying < 0.1%
Date received: 27/10/95
Storage conditions: room temperature
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
See the endpoint study summary "Aquatic toxicity" for the read-across justification and properties of the analogue material.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for analysis were taken at all test concentrations three times during the test.

Test solutions

Vehicle:
no
Details on test solutions:
Stock solutions (5g/L) were prepared in distilled water. The stock solutions were further diluted in the test medium to concentrations 10, 18, 32, 56 and 100 mg/L.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Strain A
- Source: University of Cardiff, UK
- Stock culture: cultured in Elendt M7 medium with continuous feeding with green algae (selenastrum capricornutum)
- Age of parental stock (mean and range, SD): Adult Daphnia magna (age 19 days) were isolated 24 hours prior to initiation of the test. Young daphnids produced overnight (less than 24 hours) were used in the test.
- Feeding during test: yes
- Food type/Amount/Frequency: fed daily with Selenastrum capricornutum (13-25*10E6 cells/animal/day) corresponding to 100-185 µg C/animal/day.

ACCLIMATION: none, daphnids were bred with the same manner as for the test.

QUARANTINE (wild caught): not applicable

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
The adult Daphnia were transferred to fresh media in clean vessels with a Pasteur pipette with smoothed opening. Young daphnids were counted using a stereo microscope and then discarded.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d

Test conditions

Hardness:
no data
Test temperature:
20 ± 1°C
pH:
pH new medium: 7.7-7.9
pH old medium: 7.5-9
Dissolved oxygen:
7.4 to 13 mg/L
Nominal and measured concentrations:
Nominal: 0, 10, 18, 32, 56 and 100 mg/L
Initial measured concentrations ranged from 99 to 121% of the nominal concentrations.
Calculated weighted mean concentrations on the 21 days of exposure were 5,9, 10, 24,1, 42,2 and 95,2 mg/l respectively.
Details on test conditions:
TEST SYSTEM
- Test vessel: flasks
- Type (delete if not applicable): open / closed: no data
- Material, size, headspace, fill volume: glass/-/-/50 ml
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 3 times per week (Days 3, 5, 7, 10, 12, 14, 17, 19)
- No. of organisms per vessel: 1 (10 per concentration)
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7 with corresponding composition
No more data
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- Adjustment of pH: only the dilution medium was adjusted to pH 7.8 ± 0.2 by addition of HCl or NaOH during its preparation
- Photoperiod: 16h light/8h dark
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- pH: it was measured before and after each renewal of test solutions.
- Dissolved oxygen: it was measured before renewal after 3, 7, 12 and 21 days.
- Animals: the animals were observed daily and any immobilized animals were recorded. The number of live offspring was counted and aborted eggs/neonates noted.
Young daphnids were considered to be dead if no sign of movement was apparent during examination under a stereo microscope. Adult Daphnia which were unable to swim within 15 seconds after gentle agitation were considered immobilised.

VEHICLE CONTROL PERFORMED: no vehicle

RANGE-FINDING STUDY: none
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95%CL: 21-27 mg/L
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
16 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% CL: 13-19 mg/L
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
5.9 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
63 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
other: geometric mean of TWA measured conc.
Details on results:
- Mortality of parent animals:
Immobilisation of adults was only observed at the highest test concentration (100 mg/L). Immobilisation first occured after 3 days. 50% were immobilised after 9 days and all were immobilised (dead) after 13 days. Since no immobilisation was observed at lower concentrations, the EC50 for immobilisation can only be estimated as the geometric mean of the two concentrations causing 0 and 100% immobilisation.

- No. of offspring produced per female (average):
Control: 112
10 mg/L: 109
18 mg/L: 71
32 mg/L: 40
56 mg/L: 0.7
100 mg/L: 0

- Type and number of morphological abnormalities/ behavioural abnormalities: No significant effect of Vanillin on the size or feeding rate of the daphnids were noted.

- Time to first brood release (day nr.):
Control: 8
10 mg/L: 8
18 mg/L: 9
32 mg/L: 9
56 mg/L: 14 (aborted eggs were observed from day 9)
100 mg/L: /

No more data
Reported statistics and error estimates:
The LOEC (reproduction, 21 days) was calculated by ANOVA using Dunnett's method.

Any other information on results incl. tables

Measurement of Vanillin concentrations showed that removal of Vanillin from the solution occured between the replacements of solutions. The rate of removal was higher in the second and third week of the test than in the first week. The most probable mechanism of removal is microbial degradation. Adapted bacteria may have developed in the solution and were transfered with the parent animals at replacement. TheBOD5/COD ratio (0.72) confirms that Vanillin is readily biodegradable. The contribution of accumulation of Vanillin in Daphnia is supposed to be of minor importance for the removal between replacement.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Control mortality <=20%: 0%; Dissolved oxygen >= 3 mg/l: >= 7.2 mg/L; Cumulative young per animal (control group) >=60: 100-135; pH variation fresh-replacement<= 1.5 units: <= 1.1 units.
Conclusions:
LOEC (reproduction) 21 days: 18 mg/L (nominal); 10 mg/L (meas.)
NOEC (reproduction) 21 days: 10 mg/L (nominal); 5.9 mg/L (meas.)
EC50 (reproduction) 21 days: 24 mg/L (nominal); 16 mg/L (meas.)
Executive summary:

Vanillin has been tested on the reproduction of Daphnia magna for 21 days according to the OECD202 and the GLP (NIVA, 1996). Test solutions were renewed 3 times per week. Vanillin concentration of 100 mg/L resulted in a lethal effect starting at day 3. No lethality was observed at 56 mg/L and lower concentrations during 21 days exposure. Sublethal effects of Vanillin included delayed occurence of the first brood, aborted broods and reduced number of live offspring. The EC50 for effect of reproduction (21 days) was estimated at 24 mg/L, LOEC was 18 mg/L and NOECat 10mg/L (all nominal concentartions). Since exposure concentrations were fluctuating, weighed mean concentrations were calculated and led to measured EC50 for effect on reproduction was 16 mg/L, NOEC at 5.9 mg/L and LOEC at 10mg/L.