Essential oil of Cedarwood Texas obtained from the wood of Juniperus mexicana (Cupressaceae) by distillation - Cedrol

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Apr 2017 - 29 Jun 2017

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)

Version / remarks:

Adopted Apr 13, 2004.

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.

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Cedrol, Cedarwood Texas oil distilled
Appearance: Light yellow, solid mass
Batch: B-64530
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 25 January 2019 (expiry date)
Test Facility test item number: 207804/A
Purity/Composition correction factor: No correction factor required
Test item handling: Test item should be equilibrated to 100ºC for several minutes until completely liquefied to obtain a homogeneous sample.
Chemical name (IUPAC, synonym or trade name: Cedarwood Texas oil distilled, Cedrol
Molecular structure: UVCB
Solubility in water: Not available, excepted limited solubility
Stability in water: Not available

Analytical monitoring:

yes

Remarks:

TOC analysis for confirmation WAF preparation

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.

Vehicle:

no

Remarks:

Water Accomodated Fractions were applied

Details on test solutions:

The batch of Cedrol, Cedarwood Texas oil distilled tested was a light yellow solid mass UVCB substance. The test item was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.

In order to obtain a homogeneous sample, the test item was heated for approximately 1¼ hour at 95ºC prior to the preparation of test solutions. The container was closed during warming. After warming, the appearance of the test item changed from a light yellow solid mass to a light yellow liquid.
Preparation of test solutions started with loading 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 test solutions were clear and colorless at the end of the preparation procedure. Microscopic examination of all WAFs revealed no undissolved test item present in these solutions. 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 selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.

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).

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Hardness:

180 mg/L ( expressed as CaCO3)

Test temperature:

The temperature continuously measured in a temperature control vessel was 20°C during the test

pH:

Start of the experiment (t=0h): 7.9
End of the experiment (t=48h) 7.7-7.9

Dissolved oxygen:

Start of the experiment (t=0h): 8.6-9.2
End of the experiment (t=48h) 8.5-9.1

Nominal and measured concentrations:

Nominal WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L
TOC measurements corrected for controls:
WAFs: t=0h: n.a., 2.6, 7.1, 10, 9.3, 7.6 mg TOC/L
WAFs: t=48h n.a. 2.4, 6.7, 9.6, 9.1, 7.5 mg TOC/L

TOC measurement test substance: 80% 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 36 minutes after preparation of the test solutions.

Test medium:
Composition of medium M7:
Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
Macro salts: CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCl: 4.2 mg/L

Medium M7: trace elements, macronutrients and vitamins are added to freshly prepared ISO medium to reach the following concentrations:
Trace elements:
B 0.125 mg/L
Fe 0.05 mg/L
Mn 0.025 mg/L
Li, Rb and Sr 0.0125 mg/L
Mo 0.0063 mg/L
Br 0.0025 mg/L
Cu 0.0016 mg/L
Zn 0.0063 mg/L
Co and I 0.0025 mg/L
Se 0.0010 mg/L
V 0.0003 mg/L
Na2EDTA.2H2O 2.5 mg/L
Macro nutrients: Na2SiO3.9H2O 10.0 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L
Vitamins: Thiamine 75.0 μg/L
B12 1.0 μg/L
Biotin 0.75 μg/L

Measurements and Recordings:
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.

Combined Limit/Range-Finding 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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL (8.9-14 mg/L)

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL (8.5-21 mg/L)

Details on results:

A preliminary combined range finding/limit test showed EL50 to be between 10 and 100 mg/L.
In the full test, no immobility was observed in the control treatment during the entire exposure period. At the lowest loading rate tested, the immobility increased from 10% after the first 24 hours of exposure to 30% at the end of the test. All daphnids exposed to higher loading rates were immobilised already after 24 hours of exposure.

The measured TOC concentrations increased with the loading rate at the three lowest loading rates the start indicating proper preparation of WAFs. In general 9.4-50% of the test substance was found to be dissolved in test medium. It should be noted, that values below 1.0 mg TOC/L should be considered indicative. At the higher concentrations a steady-state was reached indicating that the maximum solubility of the test substance in medium approximated 10 mg TOC/L. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared.

All validity criterias were met. In the control, no daphnids became immobilised or showed signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium. In addition, the oxygen concentration at the end of the test was ≥ 3 mg/L in control and test vessels.

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.

 Summary of the effects at 24 and 48 h (20 Daphnids per concentration)

Cedrol CW Texas Oil Distilled WAF Loading rate (mg/L)	Immobility % t=24h	Immobility % t=48h
Control	0	0
10	10	30
18	100	100
32	100	100
56	100	100
100	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 Cedrol, Cedarwood Texas oil distilled was 11 mg/L (95% confidence interval between 8.9 and 14 mg/L).

Executive summary:

This study was performed to assess the acute toxicity of Cedrol, Cedarwood Texas oil distilled on Daphnia magna, and was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202. A full test was performed based on the results of a preceding range-finding test. Water Accommodated Fractions (WAFs) of Cedrol, Cedarwood Texas oil distilled were prepared and used as test concentrations. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs prepared at loading rates of 10, 18, 32, 56 and 100 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. Due to the potential volatile nature of the test item, the exposure was performed in airtight closed vessels with headspace reduced to minimum. The measured TOC concentrations increased with the loading rate at the three lowest loading rates the start. At the higher concentrations a steady-state was reached indicating that the maximum solubility of the test substance in medium approximated 10 mg TOC/L. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 48h-EL50 for Daphnia magna exposed to Cedrol, Cedarwood Texas oil distilled was 11 mg/L (95% confidence interval between 8.9 and 14 mg/L).

Description of key information

This study was performed to assess the acute toxicity of Cedrol, Cedarwood Texas oil distilled on Daphnia magna, and was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202. A full test was performed based on the results of a preceding range-finding test. Water Accommodated Fractions (WAFs) of Cedrol, Cedarwood Texas oil distilled were prepared and used as test concentrations. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs prepared at loading rates of 10, 18, 32, 56 and 100 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. Due to the potential volatile nature of the test item, the exposure was performed in airtight closed vessels with headspace reduced to minimum. The measured TOC concentrations increased with the loading rate at the three lowest loading rates the start. At the higher concentrations a steady-state was reached indicating that the maximum solubility of the test substance in medium approximated 10 mg TOC/L. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 48h-EL50 for Daphnia magna exposed to Cedrol, Cedarwood Texas oil distilled was 11 mg/L (95% confidence interval between 8.9 and 14 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

11 mg/L

Additional information

Based on loading rate.