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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03-03-2017 - 12-07-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Identification: Cedarwood Oil Virginia
Appearance: Pale yellow to yellow liquid
Batch: 1002960562
Purity/Composition: 100.0% (UVCB)
Test item storage: At room temperature
Stable under storage conditions until: 31 August 2017 (expiry date)

Test facility test item number: 207801/A
Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Essential oil of Junipers Virginiana L. (Cupressaceae) obtained from the wood by steam distillation
CAS Number 85085-41-2
Molecular structrue: UVCB
Molecular formula: UVCB
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Volatile: Not indicated
Solubility in water: Not available
Stability in water: Not available
Analytical monitoring:
yes
Remarks:
TOC analysis
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 h and t=48 h
Volume: 40 mL
Storage: Samples were stored in a refrigerator (2 - 8°C) until analysis.
At the end of the exposure period, the replicates were not pooled at each concentration before sampling. Instead samples were taken from one vessel of each group.
Additionally, reserve samples of 40 mL were taken for possible analysis. If not used, these samples were stored in a refrigerator (2 - 8°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Analysis set-up: No. of repeats: at least 3

Vehicle:
no
Remarks:
Water Accomodated Fractions (WAFs) were used
Details on test solutions:
The batch of Cedarwood Oil Virginia tested was a pale yellow to yellow liquid. The test item was a UVCB substance and not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item. Preparation of test solutions started with loading rates individually prepared ranging between 1.0 and 100 mg/L. A 2-day period of magnetic stirring in closed vessels with minimal headspace and in the dark was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle overnight. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. All WAFs were microscopically inspected for the presence of undissolved test material. All test solutions were clear and colorless at the end of the preparation procedure with the exception of the highest test concentration of the first full test, in which particles of undissolved materials were observed under the microscope. This solution was also observed to be slightly hazy.
Any residual volumes were discarded.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: For the test, young daphnids with an age of < 24 hours were selected from parental daphnids older than two weeks.

Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
180 mg/L expressed as CaCO3
Test temperature:
First and second test: The temperature continuously measured in a temperature control vessel varied between 20 and 21°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).
pH:
First test:
pH t=0h : 8.0
pH t=48h: 7.8 - 8.0

Second test:
pH t=0h : 7.7 - 7.8
pH t=48h: 7.9 - 8.0
Dissolved oxygen:
First test:
O2 t=0h: 8.7 - 9.2 mg/L
O2 t=48h: 8.5 - 9.0 mg/L

Second test:
O2 t=0h: 8.8 - 9.0 mg/L
O2 t=48h: 8.0 - 8.3 mg/L
Nominal and measured concentrations:
First test
Nominal: WAFs prepared at loading rates 0, 1.0, 2.2, 4.6, 10, 22 mg/L.
TOC measurements corrected for controls:
WAFs: t=0h: n.a., 0.18, 0.70, 1.0, 2.2, 6.4 mg TOC/L
WAFs: t=48h n.a. 0.02, 0.47, 1.0, 1.9, 5.6 mg TOC/L

Second test:
Nominal: WAFs prepared at loading rates 0, 1.0, 2.2, 4.6, 10, 22 mg/L.
TOC measurements corrected for controls:
WAFs: t=0h: n.a., 0.021, 0.21, 0.56, 1.76, 3.83 mg TOC/L
WAFs: t=48h n.a. 0.10, 0.44, 0.69, 1.94, 3.72 mg TOC/L

TOC measurement test substance: 82.79% TOC
Details on test conditions:
Test duration: 48 hours
Test type: Static
Test vessels: 60 mL, all-glass, closed with no headspace
Medium: Adjusted ISO medium
Number of daphnids: 20 per concentration
Loading: 5 per vessel containing 60 mL of test solution
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids: Within 40 minutes after preparation of the test solutions.

Immobility (including mortality): At 24 hours and at 48 hours.
pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

The project started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to a control and a WAF prepared at a loading rate of 100 mg/L in the limit test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to WAFs prepared at loading rate of 1.0 and 10 mg/L in the combined range-finding test.
• Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration.

A full test was performed with test procedure and conditions were similar to those applied in the second full test with the following exceptions:
• 40 mL samples were stored in 50 mL vials prior to analysis.
Reference substance (positive control):
yes
Remarks:
K2Cr2O7
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
1.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval between 1.1 and 1.5 mg/L
Remarks:
EL50 value based on 2 full tests
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval between 3.5 and 11 mg/L
Remarks:
EL50 value based on 2 full tests
Details on results:
- First Full Test:
It should be noted that the samples taken in this test were, inadvertently, stored with a small headspace that could cause loss of test item from the solutions. As the extent of loss could not be assessed, the full test was repeated. The measured TOC concentrations increased with the loading rate at the start of the test indicating proper preparation of WAFs. In general 22-35% of the test item was found to be dissolved in test medium at the start of the test. Note, that values below 1.0 mg TOC/L should be considered indicative.

No significant immobilisation was observed in the control and the lowest concentration during the exposure period. It should be noted that an effect of ≤ 10% is allowed for the control treatment and therefore, considered not significant. A number of daphnids were observed to be trapped at the surface of test solutions after 24 hour of exposure. It was impossible to reimmerse them to assess mobility without opening the vessels. Therefore, all daphnids trapped at the surface after the first 24 hours of exposure were assumed to be immobilised (worst-case-scenario). Daphnids observed to be trapped at the surface at the end of the test were reimmersed before assessment of immobility. A dose related increase of effects was observed at both time points. All daphnids exposed to the highest concentration were immobilised already after 24 hours of exposure.

- Second full test:
Similiar to the first full test, the measured TOC concentrations increased with the loading rate at the start of the test indicating proper preparation of WAFs. Despite the fact that samples were stored without a headspace in this test, the measured concentrations were slightly lower than in the first test. A maximum of 21% of the test substance was found to be dissolved in test medium at the start of the test. It should be noted, that values below 1.0 mg TOC/L should be considered indicative. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared.

Despite the lower concentrations measured in this test, the immobilisation rate was slightly but not significantly higher. No immobilisation was observed in the control treatment during the exposure period, while 40% of daphnids exposed to WAF prepared at 1.0 mg/L were immobilised at the end of the exposure. A number of daphnids were trapped at the surface of test solutions at both time points. This time it was possible to submerge all the floating daphnids before assessment of immobility without opening the vessels.

Validity and EL50 calculation:
Both full tests met their validity criteria and showed similar results; i.e. the effects were below 50% at the lowest loading rate and ≥ 90% of daphnids were immobilised at the higher concentrations. In both tests the same loading rates were prepared and therefore, it is justified to calculate the EL50 values based on both tests.
Results with reference substance (positive control):
In conclusion, the actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at Charles River Den Bosch.
The 24h-EC50 was 0.95 mg/L with a 95% confidence interval between 0.85 and 1.1 mg/L.
The 48h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.52 and 0.66 mg/L.

Reported statistics and error estimates:
Calculation of EL50: The 24 and 48h-EL50-value was calculated from the logits (Logit analysis) of the percentages of affected daphnids and logarithms of the corresponding test substance loading rates using the maximum likelihood estimation method. The analysis was performed on the combined results of both full tests.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

 Summary of the effects at 24 and 48 h (combined tests, 40 Daphnids per concentration)

CW OIL VIRGINIA

Loading rate (mg/L)

Immobility %

t=24h

Immobility %

t=48h

Control

0

0

1.0

0

23

2.2

15

95

4.6

38

98

10

65

100

22

100

100

 

Validity criteria fulfilled:
yes
Remarks:
In the control, no daphnids became immobilised or showed other signs of disease or stress. The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.
Conclusions:
In conclusion, the 48h- EL50 for Daphnia magna exposed to Cedarwood Virginia Oil was 1.3 mg/L based on nominal loading rates (95% confidence interval between 1.1 and 1.5 mg/L) based on the data of two independent tests.
Executive summary:

A full OECDTG 202 GLP test was performed twice with Daphnia magna, based on the results of a preceding range-finding test. Water Accommodated Fractions (WAFs) of Cedarwood Virginia oil were prepared and used as test concentrations.Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs individually prepared at loading rates of 1.0, 2.2, 4.6, 10 and 22 mg/L under static conditions. The total exposure period was 48 hours and samples for Total Organic Carbon (TOC) analyses were taken at the start and at the end of the test. The exposure was performed in airtight closed vessels with headspace reduced to minimum. The measured TOC concentrations increased with the loading rate at the start of both tests indicating proper preparation of WAFs. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. In conclusion, the 48h- EL50 for Daphnia magna exposed to Cedarwood Oil Virginia was 1.3 mg/L based on nominal loading rates (95% confidence interval between 1.1 and 1.5 mg/L). The EL50value was calculated based on results of two independently conducted tests.

 

Description of key information

A full OECDTG 202 GLP test was performed twice with Daphnia magna, based on the results of a preceding range-finding test. Water Accommodated Fractions (WAFs) of Cedarwood Virginia oil were prepared and used as test concentrations.Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs individually prepared at loading rates of 1.0, 2.2, 4.6, 10 and 22 mg/L under static conditions. The total exposure period was 48 hours and samples for Total Organic Carbon (TOC) analyses were taken at the start and at the end of the test. The exposure was performed in airtight closed vessels with headspace reduced to minimum. The measured TOC concentrations increased with the loading rate at the start of both tests indicating proper preparation of WAFs. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. In conclusion, the 48h- EL50for Daphnia magna exposed to Cedarwood Oil Virginia was 1.3 mg/L based on nominal loading rates (95% confidence interval between 1.1 and 1.5 mg/L). The EL50value was calculated based on results of two independently conducted tests.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.3 mg/L

Additional information