Cypress, Cupressus funebris, ext.

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Cypress, Cupressus funebris, ext.

EC Number:

285-360-9

EC Name:

Cypress, Cupressus funebris, ext.

Cas Number:

85085-29-6

Molecular formula:

Not applicable due to UVCB nature of the substance

IUPAC Name:

Essential oil of Cedarwood obtained from the wood of Cupressus funebris (Cupressaceae) by steam distillation

Test material form:

other: Pale yellow to yellow liquid

Remarks:

could crystallize

Details on test material:

- Name of test material (as cited in study report): Cedarwood Oil China
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: no data
- Lot/batch No.: confidential information

Specific details on test material used for the study:

Identification: Cedarwood Oil China
Appearance: Pale yellow to yellow liquid
Batch: 1002515317
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: 207805/A
Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Essential oil of Chamaecyparis funebris (Cupressaceae) obtained from the wood by steam distillation
Molecular formula: UVCB
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Solubility water: Not available
Stability in water: Not available

Sampling and analysis

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 50 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 50 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.

No. of repeats: at least 3

Test solutions

Vehicle:

no

Remarks:

Water Accomodated Fractions (WAFs) were used

Details on test solutions:

The batch of Cedarwood Oil China tested was a pale yellow to yellow liquid. The item was a UVCB substance 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 in the combined limit/range-finding test and between 0.22 and 22 mg/L in the final test. 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 test solutions were inspected under the microscope for the presence of undissolved test material.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system:
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.

Breeding
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).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/L expressed as CaCO3

Test temperature:

The temperature continuously measured in a temperature control vessel varied between 20 and 21°C during the test

pH:

pH t=0h : 7.9-8.0
pH t=48h: 8.0-8.1

Dissolved oxygen:

O2 t=0h: 8.6-9.0 mg/L
O2 t=48h: 8.3-9.0mg/L

Nominal and measured concentrations:

Nominal loading rates 0.22, 0.46, 1.0, 2.2, 4.6, 10, 22 mg/L
TOC measurements corrected for controls:
WAFs: t=0h: 0.41, -0.12, 0.14, 0.28, 0.61, 1.9, 2.9 mg TOC/L
WAFs: t=48h 0.21, 0.02, 0.21, 0.24, 0.41, 1.5, 2.5 mg TOC/L

TOC measurement test substance: 82.7% TOC

Details on test conditions:

Test duration : 48 hours
Test type: Static
Test vessels: 60 mL, all-glass, closed airtight with headspace reduced to minimum
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 20 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. 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 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.

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

2.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval between 2.6 and 3.3 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EL0

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

8.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 4.9 - 15

Details on results:

No immobility was observed in the control and at the three lowest WAFs tested throughout the entire exposure period. After 24 hours of exposure, 95 and 85% of daphnids were immobile at the two highest WAFs tested, respectively while no effects were observed at WAFs prepared at loading rate of 2.2 and 4.6 mg/L. At the end of the exposure period, 10% of daphnids were immobile in the WAF prepared at 2.2 mg/L, while effects were completed at the three highest WAFs tested. Also in the full test, a number of daphnids were observed to be trapped at the surface of test solutions. Microscopic observation performed at the end of the test revealed that no test substance was attached to the daphnids exposed to the WAF prepared at a loading rate of 4.6 mg/L.

The measured TOC concentrations increased with the loading rate at the start of the test indicating proper preparation of WAFs. In the highest concentration 2.9 mg TOC/L was measured at the start of the test (this corresponds to 16% of the original loading rate) . It should be noted, that values below 1.0 mg TOC/L should be considered indicative.

Results with reference substance (positive control):

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 0.64 mg/L with a 95% confidence interval between 0.56 and 0.73 mg/L.
The 48h-EC50 was 0.48 mg/L with a 95% confidence interval between 0.43 and 0.53 mg/L.

Reported statistics and error estimates:

The 24 and 48h-EL50-value was calculated from the weibits (Weibull analysis) of the percentages of affected daphnids and the logarithms of the corresponding test item loading rates using the maximum likelihood estimation method.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

 

CW oil China Loading rate (mg/L)	Immobility % t=24h	Immobility % t=48h
Control	0	0
0.22	0	0
0.46	0	0
1.0	0	0
2.2	0	10
4.6	0	100
10	95	100
22	85	100

Applicant's summary and conclusion

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:

The 48h-EL50 for Daphnia magna exposed to Cedarwood oil Chinese was 2.9 mg/L (95% confidence interval between 2.6 and 3.3 mg/L).

Executive summary:

A full OECDTG 202 GLP test was performed with daphnia magna, based on the results of a preceding range-finding test. Water Accommodated Fractions (WAFs) of Cedarwood oil Chinese w

ere 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 0.22, 0.46, 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 the test 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 Chinese was 2.9 mg/L (95% confidence interval between 2.6 and 3.3 mg/L).