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

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:

07 April 2017 - 25 October 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: Terpenes 2 (Thujopsene), Cedarwood Texas oil distilled
Appearance: Almost colourless, very pale yellow liquid
Batch: B-64543
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions: 25 January 2019 (expiry date)

Analytical monitoring:

yes

Remarks:

TOC analysis for 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 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.

Vehicle:

no

Remarks:

Water Accomodated Fractions (WAFs) were used

Details on test solutions:

Preparation of test solutions started with loading rates individually prepared ranging between 0.46 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. In the second full test, the lowest concentrations were prepared as dilutions of the WAF prepared at a loading rate of 0.46 mg/L in medium.

All test solutions were clear and colorless at the end of the preparation procedure. No undissolved material was observed in any of the WAFs when inspected under a microscope. Any residual volumes were discarded.

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).

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

pH:

First full test:
pH t=0h : 7.9 - 8.0
pH t=48h: 7.7 - 7.8

Second full test:
pH t=0h : 7.8 - 7.9
pH t=48h: 7.8 - 7.9

Dissolved oxygen:

First full test:
O2 t=0h : 8.8 - 9.2
O2 t=48h: 8.6 -9.1

Second full test:
O2 t=0h : 9.2-9.4
O2 t=48h: 8.2 - 8.7

Nominal and measured concentrations:

Nominal loading rates:
First full test: WAFs prepared at loading rates of 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/L.
Second full test: prepared at 0.46, 1.0, 2.2, 4.6 and 10 mg/L and solutions containing 22 and 46% of the WAF prepared at a loading rate of 0.46 mg/L

First full test:
TOC measurements corrected for controls:
Loading rates (mg/L): Controls, 0.46,1.0, 2.2, 4.6,10 and 22 mg/L
WAFs: t=0h: n.a, n.q., n.q, n.q, n.q, 0.26,0.71 mg TOC/L
WAFs: t=48h n.a, 0.070, 0.091, 0.19, n.q, 0.32,0.86 mg TOC/L

Second full test:
TOC measurements corrected for controls:
Loading rates (mg/L): Control, 0.46, 1.0, 2.2, 4.6, and 10 mg/L
WAFs: t=0h: n.a, 3.0, 2.4, 0.90, 0.78, 1.1 mg TOC/L
WAFs: t=48h * n.a, 2.3, 0.85, 0.47, 1.1, 0.43 mg TOC/L

n.q. – not quantifiable, n.a. – not applicable * no correction was applied as no TOC concentration was detected in the control

Details on test conditions:

Test Concentrations:
First full test: Daphnids were exposed to WAFs prepared at loading rates of 0.46,1.0, 2.2, 4.6,10 and 22 mg/L.
Second full test: WAFs prepared at 0.46, 1.0, 2.2, 4.6 and 10 mg/L and solutions containing 22 and 46% of the WAF prepared at a loading rate of 0.46 mg/L
Controls: Test medium without test item or other additives

Test Procedure and 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 23 minutes after preparation of the test solutions.

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 second full 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.
- Samples for analysis were collected in 50 mL vials.

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

1.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% cl (0.41-2.9)

Remarks:

effect parameters based on both full tests and were expressed in terms of nominal loading rates

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% cl ( 8.3-2585 mg/L)

Remarks:

effect parameters based on both full tests and were expressed in terms of nominal loading rates

Duration:

48 h

Dose descriptor:

EL0

Effect conc.:

0.22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (46% of WAF prepared at 0.46 mg/L)

Remarks:

effect parameters based on both full tests and were expressed in terms of nominal loading rates

Details on results:

First full test:
Very low concentrations of the test item were measured. Only at the two highest concentrations a slight increase of the TOC concentrations was observed. No immobility or other effects were observed in the control treatment. Although the TOC values before correction indicate an increase of concentrations, the biological responses show no clear dose related effects. Especially, the responses recorded in WAF of 4.6 mg/L show strong deviation to what was observed in other groups. As at least 76% immobilisation was observed at the end of the exposure and the observed effects cannot be related either to the applied dose or to the measured TOC concentrations, no EL50 value could be determined based on this test and a second full test was performed. These test conditions remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: ≥3 mg/L at the end of the test). The temperature continuously measured in a temperature control vessel varied at 20°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).

Second full test:
Unexpectedly high concentrations were measured in the two lowest WAFs. These concentrations were higher than expected based on the original loading rates. The reason for such high values is not known and these concentrations were considered not reliable. The concentrations measured at the two highest WAFs could be considered dose-related. It should be noted that a set of samples was analysed several times (until exhaustion of the available liquid) due to a mistake in a program. Because the internal Total Carbon control deviated from the standard after each run, the results were rejected. Nevertheless, a significant deviation in measured concentrations was observed in this analysis. This indicates a possible issue with homogeneity of test solutions. Visually samples contained no undissolved material but formation of micro emulsions cannot be excluded. No immobility or other effects were observed in the control and the four lowest concentrations during the exposure period. After the first 24 hours of exposure, immobilisation was only observed in the highest WAF (15%), while at the end of the test a dose-response was observed at the three highest concentrations tested despite the fact that no dose-response was observed for the measured TOC concentrations.These test conditions remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: ≥3 mg/L at the end of the 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).


Acceptability of the Test:
1. In the control, no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.
2. 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 0.95 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-values were calculated from the weibits (Weibull analysis) of the percentages of affected daphnids and the logarithms of the corresponding test item loading rate using the maximum likelihood estimation method. It should be noted that the data from WAF prepared at 4.6 mg/L in the first full test was excluded from the analysis. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Number of Introduced Daphnids and Incidence of Immobility in the First Full Test

Time (h)	Replicate	Terpenes 2 (Thujopsene), Cedarwood Texas oil distilled Loading rate (mg/L)
		Control	0.46	1.0	2.2	4.6	10	22
0	A	5	5	5	5	5	5	5
	B	5	5	6*	5	5	5	5
	C	5	5	5	5	5	5	5
	D	5	5	5	5	5	5	5
	Total introduced	20	20	21	20	20	20	20
24	A	0	0 (5)	0 (1)	0 (1)	0	2 (2)	5 (5)
	B	0	0	0	0	0	0	2 (5)
	C	0	2 (5)	0	3 (5)	0	5 (5)	4
	D	0	2	0	0 (5)	0	1 (5)	2
	Total immobilised	0	4	0	3	0	8	13
	Effect %	0	20	0	15	0	40	65
48	A	0	4 (3)	5 (1)	5 (4)	1	3 (2)	5 (1)
	B	0	5	4 (1)	5 (2)	0	3	5 (4)
	C	0	4	5	5 (1)	0	5 (2)	5 (2)
	D	0	5^#	2	5 (3)	1	5 (3)	4
	Total immobilised	0	18	16	20	2	16	19
	Effect %	0	90	76	100	10	80	95

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility,^#Microscopic observation revealed no test item attached to the daphnids * First full test: Vessel B of the WAF prepared at 1.0 mg/L contained 6 instead of 5 daphnids. Evaluation: This was taken into account during calculations of effects.

Number of Introduced Daphnids and Incidence of Immobility in the Second Full Test

Time (h)	Replicate	Terpenes 2 (Thujopsene), Cedarwood Texas oil distilled; Loading rate (mg/L)
		% of WAF prep. at a loading rate of 0.46 mg/L			WAF prep. at loading rate (mg/L)
		Control	0.10	0.22	0.46	1.0	2.2	4.6	10
0	A	5	5	5	5	5	5	5	5
	B	5	5	5	5	5	5	5	5
	C	5	5	5	5	5	5	5	5
	D	5	5	5	5	5	5	5	5
Total introduced		20	20	20	20	20	20	20	20
24	A	0	0	0	0	0	0	0	0 (2)
	B	0	0	0	0	0	0	0 (1)	1
	C	0	0	0	0	0	0	0 (1)	1 (5)
	D	0	0	0	0	0	0	0 (1)	1 (4)
Total immobilised		0	0	0	0	0	0	0	3
	Effect %	0	0	0	0	0	0	0	15
48	A	0	0	0	0	0	3^#	4 (4)	5 (1)
	B	0	0	0	0	0	3 (1)	5 (2)	3
	C	0	0	0	0	0	1	4 (3)	5 (1)
	D	0	0	0	0	0	3	3 (3)	4
Total immobilised		0	0	0	0	0	10	16	17
	Effect %	0	0	0	0	0	50	80	85

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility,^#Microscopic observation revealed no test item attached to the daphnids

Validity criteria fulfilled:

yes

Remarks:

see details on results

Conclusions:

The 48h- EL50 for Daphnia magna exposed to Terpenes 2 (Thujopsene), Cedarwood Texas oil distilled was 1.4 mg/L based on nominal loading rates (95% confidence interval between 0.41 and 2.9 mg/L).

Executive summary:

A full OECDTG 202 GLP tests was performed twice with Daphnia magna, and based on the results of a combined limit/range-finding test. Water Accommodated Fractions (WAFs) were prepared at individual loading rates 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 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/L, in the first full test. In the second full tests daphnids were exposed to WAFs prepared at 0.46, 1.0, 2.2, 4.6 and 10 mg/L and solutions containing 22 and 46% of the WAF prepared at 0.46 mg/L.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 a minimum. The measured TOC concentrations showed that the test item was poorly soluble in test medium and the results were inconclusive. Moreover, there was an indication of inhomogeneity of test solutions. For these reasons and because the TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. At the end of the first full test, 76-100% immobility was observed in all WAFs beside 4.6 mg/L where only 10% daphnids were immobilised. In the second full test, a dose-related immobilisation was observed at the end of the test. Based on the observed differences in both tests, despite the same preparation procedure, it was assumed that additional testing would not improve understanding of the behaviour of the test item in an aquatic environment. Therefore, it was decided to combine results of both tests for the calculation of the EL50 values. 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 Terpenes 2 (Thujopsene), Cedarwood Texas oil distilled was 1.4 mg/L based on nominal loading rates (95% confidence interval between 0.41 and 2.9 mg/L).

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 May 2017 - 13 Sep 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: Cedarwood Texas oil crude
Appearance: Yellow liquid (determined by Charles River Den Bosch)
Batch: LS160616
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions: until 15 June 2018 (expiry date)

Analytical monitoring:

yes

Remarks:

TOC analysis for 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 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.

Vehicle:

no

Remarks:

Water Accomodated Fractions (WAFs) were used

Details on test solutions:

Preparation of test solutions started with loading rates individually prepared ranging between 0.46 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. In the second full test, the lowest concentrations were prepared as dilutions of the WAF prepared at a loading rate of 0.46 mg/L in medium. No undissolved material was observed in any of the solutions when inspected under a microscope besides for the WAF of 100 mg/L in the range-finding test which contained micro-particles.

All test solutions were clear and colorless at the end of the preparation procedure with the exception of the highest test concentration of the combined limit/range-finding test which was observed to be slightly hazy. Any residual volumes were discarded.

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).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

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:

First full test:
pH t=0h : 8.0
pH t=48h: 7.8-8.0

Second full test:
pH t=0h : 7.8 - 8.6
pH t=48h: 7.7

Dissolved oxygen:

First full test:
O2 t=0h : 8.8 - 9.1
O2 t=48h: 7.0 -8.2

Second full test:
O2 t=0h : 8.2 - 8.7
O2 t=48h: 6.9-7.3

Nominal and measured concentrations:

Nominal loading rates:
First full test: WAFs prepared at loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L.
Second full test: WAFs prepared at 0.46, 1.0, 2.2, 4.6 and 10 mg/L and solutions containing 22 and 46% of the WAF prepared at a loading rate of 0.46 mg/L

First full test:
TOC measurements corrected for controls:
Loading rates (mg/L): Controls, 1.0, 1.8, 3.2, 5.6 and 10mg/L
WAFs: t=0h: n.a, n.q., n.q, 0.29, 0.49, 1.6 mg TOC/L
WAFs: t=48h n.a, n.q, n.q, n.q, 0.41,1.4 mg TOC/L

Second full test:
TOC measurements corrected for controls:
Loading rates (mg/L): Control, 0.46, 1.0, 2.2, 4.6, and 10 mg/L
WAFs: t=0h: n.a, n.q., n.q, n.q, 0.27, 1.5 mg TOC/L
WAFs: t=48h n.a, 0.17, 0.25, 0.5, 1.2, 1.9 mg TOC/L

n.q. – not quantifiable, n.a. – notapplicable

Details on test conditions:

Test Concentrations:
First full test: Daphnids were exposed to WAFs prepared at loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L.
Second full test: WAFs prepared at 0.46, 1.0, 2.2, 4.6 and 10 mg/L and solutions containing 22 and 46% of the WAF prepared at a loading rate of 0.46 mg/L
Controls: Test medium without test item or other additives

Test Procedure and 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 34 minutes after preparation of the test solutions.

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 second full 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.
- Samples for analysis were collected in 50 mL vials.

Range-Finding Test
A range-finding test was performed with procedure and conditions were similar to those applied in the full tests with the following exceptions:
- Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to WAFs prepared at 0.46, 1.0 and 10 mg/L and to a control.
- Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration.
- Samples for analysis were collected in 50 mL vials.

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

0.76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% cl (0.59-0.98 mg/L)

Remarks:

effect parameters based on both full tests and were expressed in terms of nominal loading rates

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

4.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% cl ( 4.0-5.5 mg/L)

Remarks:

effect parameters based on both full tests and were expressed in terms of nominal loading rates

Duration:

48 h

Dose descriptor:

EL0

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (22% of WAF prepared at 0.46 mg/L)

Remarks:

effect parameters based on both full tests and were expressed in terms of nominal loading rates

Details on results:

First full test:
The measured TOC concentrations increased with the loading rate at the start of the test at the three highest WAFs. In general 8.7-19% of the test item was found to be dissolved in test medium at the start of the test.No immobility or other effects were observed in the control treatment. At 24 hour of exposure a dose-response ranging from 10% in the lowest to 100% immobility at the highest WAF was observed. At the end of the test immobility of at least 95% was observed in all WAFs. Therefore, no EL50 value could be determined based on this test and a second full test was performed. These test conditions remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: >= 3 mg/L at the end of the 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).

Second full test:
The measured TOC concentrations increased with the loading rate at the start of the test at the three highest WAFs. In general 7.0-18% of the test item was found to be dissolved in test medium at the start of the test. The concentrations measured in the WAF prepared at a loading rate of 10 mg/L were comparable to these measured in the first full test. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared.No immobility or other effects were observed in the control and the lowest concentration during the exposure period. After the first 24 hours of exposure, immobilisation was only observed in the highest WAF. At the end of the test a scattered dose-response was observed.These test conditions remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: >= 3 mg/L at the end of the 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).

Acceptability of the Test:
1. In the control, no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.
2. 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 0.95 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-values were calculated with the Spearman-Karber procedure without trim. This procedure was chosen as some of the concentrations deviated from the doseresponse. The analysis was performed on the combined results of both full tests. The 48h-EL0 – was determined as the highest loading rate without effect. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Number of Introduced Daphnids and Incidence of Immobility in the First Full Test

Time (h)	Replicate	Cedarwood Texas oil crude; Loading rate (mg/L)
		Control	1.0	1.8	3.2	5.6	10
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	1	3 (5)	3 (4)	4 (4)	5 (2)
	B	0	0	1 (5)	2 (2)	4	5 (5)
	C	0	1 (4)	1 (5)	1 (4)	4 (2)	5 (4)
	D	0	0 (5)	1 (3)	2 (2)	2 (5)	5
	Total immobilised	0	2	6	8	14	20
	Effect %	0	10	30	40	70	100
48	A	0	4	5	5	5	5
	B	0	5^#	5	5	5	5
	C	0	5 (1)	5	5	5	5
	D	0	5 (1)	5 (1)	5	5	5
	Total immobilised	0	19	20	20	20	20
	Effect %	0	95	100	100	100	100

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility,^#Microscopic observation revealed no test item attached to the daphnids

Number of Introduced Daphnids and Incidence of Immobility in the Second Full Test

Time (h)	Replicate	Cedarwood Texas oil crude; Loading rate (mg/L)
		% of WAF prep. at a loading rate of 0.46 mg/L			WAF prep. at loading rate (mg/L)
		Control	22	46	0.46	1.0	2.2	4.6	10
0	A	5	5	5	5	5	5	5	5
	B	5	5	5	5	5	5	5	5
	C	5	5	5	5	5	5	5	5
	D	5	5	5	5	5	5	5	5
Total introduced		20	20	20	20	20	20	20	20
24	A	0	0	0	0	0 (1)	0 (4)	0 (5)	5 (2)
	B	0	0	0	0	0	0 (5)	0 (2)	4
	C	0	0	0	0	0 (1)	0	0	3 (4)
	D	0	0	0	0 (2)	0	0	0	3 (1)
Total immobilised		0	0	0	0	0	0	0	15
	Effect %	0	0	0	0	0	0	0	75
48	A	0	0	0	2	2 (1)	4 (4)	5 (1)	5
	B	0	0	0	1	1 (1)	5 (5)	2	5
	C	0	0	0	4	0	2	1	5
	D	0	0	2 (4)	4 (2)^#	1 (1)	1	1	5
Total immobilised		0	0	2	11	4	12	9	20
	Effect %	0	0	10	55	20	60	45	100

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility,^#Microscopic observation revealed no test item attached to the daphnids,

Validity criteria fulfilled:

yes

Remarks:

see details on results

Conclusions:

The 48h- EL50 for Daphnia magna exposed to Cedarwood Texas oil crude was 0.76 mg/L based on nominal loading rates (95% confidence interval between 0.59 and
0.98 mg/L).

Executive summary:

An OECDTG 202 GLP test was performed with Daphnia magna, based on the results of a combined limit/range-finding test and a range-finding test. Water Accommodated Fractions (WAFs) of Cedarwood Texas oil crude were prepared at individual loading rates 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 1.0, 1.8, 3.2, 5.6 and 10 mg/L, in the first full test. In a second full test daphnids were exposed to WAFs prepared at 0.46, 1.0, 2.2, 4.6 and 10 mg/L and solutions containing 22 and 46% of the WAF prepared at 0.46 mg/L.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 a 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. At the end of the first full test, 95% or more daphnids were immobilised at the end of the exposure in all concentrations. In the second full test, a dose-response was observed but it was scattered. Therefore, it was decided to combine results of both tests for the calculation of the EL50 values. 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 Cedarwood Texas oil crude was 0.76 mg/L based on nominal loading rates (95% confidence interval between 0.59 and 0.98 mg/L).

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

The full read across justification report is attached under "Attached justification".

22 December 2017 READ-ACROSS STUDY / CW TX T1 / DAPHNIA ACUTE TOX I&B9W8768R001F1.0

According to Annex VII, 9.1.1 of the REACh Regulation (EC) No 1907/2006, “Aquatic toxicity - Short-term toxicity testing on invertebrate (preferred species Daphnia)” is standard information required for the registration of substances manufactured or imported in quantities of one tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).

A read-across approach appears appropriate to predict the endpoint “Daphnia acute toxicity” for the substance Cedarwood Texas Terpenes 1 (Cedrene) [CW TX T1] because:

A Daphnia acute toxicity study, according to OECD test guideline 202 is available for the substance Cedarwood Texas oil - Crude [CW TX Crude], which composition is very similar to the target substance because Source substance is produced from it;

The comparison of Daphnia acute toxicity in different Cedarwood oils shows that the 48h-EL50 is very equivalent in this group, and that CW TX Crude, can be considered as the worst-case based on its lowest acute ecotoxicity value.

The screening by a known constituents’ approach indicates the constituents that may drive the acute ecotoxicity, and a comparison with other test results shows that Cedarwood Texas Terpenes 2 (Thujopsene) [CW TX T2] would be the more relevant source regarding its composition by using the maximum possible weight percentages, but would not put in the read-across prediction as much precaution as by using CW TX Crude.

This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the compositions, the understanding of which constituents are driving the ecotoxicity, and the comparison of the acute daphnia toxicity test data in the group of Cedarwood oils;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, are summarised.

Reason / purpose for cross-reference:

read-across source

Test organisms (species):

Daphnia magna

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

0.76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% cl (0.59-0.98 mg/L)

Remarks:

effect parameters based on both full tests and were expressed in terms of nominal loading rates

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

4.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% cl ( 4.0-5.5 mg/L)

Remarks:

effect parameters based on both full tests and were expressed in terms of nominal loading rates

Duration:

48 h

Dose descriptor:

EL0

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (22% of WAF prepared at 0.46 mg/L)

Remarks:

effect parameters based on both full tests and were expressed in terms of nominal loading rates

Details on results:

First full test:
The measured TOC concentrations increased with the loading rate at the start of the test at the three highest WAFs. In general 8.7-19% of the test item was found to be dissolved in test medium at the start of the test.No immobility or other effects were observed in the control treatment. At 24 hour of exposure a dose-response ranging from 10% in the lowest to 100% immobility at the highest WAF was observed. At the end of the test immobility of at least 95% was observed in all WAFs. Therefore, no EL50 value could be determined based on this test and a second full test was performed. These test conditions remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: >= 3 mg/L at the end of the 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).

Second full test:
The measured TOC concentrations increased with the loading rate at the start of the test at the three highest WAFs. In general 7.0-18% of the test item was found to be dissolved in test medium at the start of the test. The concentrations measured in the WAF prepared at a loading rate of 10 mg/L were comparable to these measured in the first full test. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared.No immobility or other effects were observed in the control and the lowest concentration during the exposure period. After the first 24 hours of exposure, immobilisation was only observed in the highest WAF. At the end of the test a scattered dose-response was observed.These test conditions remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: >= 3 mg/L at the end of the 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).

Acceptability of the Test:
1. In the control, no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.
2. 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 Texas distilled Terpenes 1 (Cedrene) was 0.76 mg/L based on nominal loading rates (95% confidence interval between 0.59 and 0.98 mg/L).

Executive summary:

For CW Texas distilled Terpenes 1 (Cedrene) read across was applied from CW Texas Crude oil. An OECDTG 202 GLP test was performed with Daphnia magna, based on the results of a combined limit/range-finding test and a range-finding test. Water Accommodated Fractions (WAFs) of Cedarwood Texas oil Crude were prepared at individual loading rates 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 1.0, 1.8, 3.2, 5.6 and 10 mg/L, in the first full test. In a second full test daphnids were exposed to WAFs prepared at 0.46, 1.0, 2.2, 4.6 and 10 mg/L and solutions containing 22 and 46% of the WAF prepared at 0.46 mg/L.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 a 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. At the end of the first full test, 95% or more daphnids were immobilised at the end of the exposure in all concentrations. In the second full test, a dose-response was observed but it was scattered. Therefore, it was decided to combine results of both tests for the calculation of the EL50 values. 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 Cedarwood Texas oil crude was 0.76 mg/L based on nominal loading rates (95% confidence interval between 0.59 and 0.98 mg/L).

Description of key information

Cedarwood Texas distilled Terpenes 1 (Cedrene)

For CW Texas distilled Terpenes 1 (Cedrene) read across was applied from CW Texas Crude oil. An OECDTG 202 GLP test was performed with Daphnia magna, based on the results of a combined limit/range-finding test and a range-finding test. Water Accommodated Fractions (WAFs) of Cedarwood Texas oil Crude were prepared at individual loading rates 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 1.0, 1.8, 3.2, 5.6 and 10 mg/L, in the first full test. In a second full test daphnids were exposed to WAFs prepared at 0.46, 1.0, 2.2, 4.6 and 10 mg/L and solutions containing 22 and 46% of the WAF prepared at 0.46 mg/L.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 a 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. At the end of the first full test, 95% or more daphnids were immobilised at the end of the exposure in all concentrations. In the second full test, a dose-response was observed but it was scattered. Therefore, it was decided to combine results of both tests for the calculation of the EL50 values. 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 Cedarwood Texas oil crude was 0.76 mg/L based on nominal loading rates (95% confidence interval between 0.59 and 0.98 mg/L).

Cedarwood Texas distilled Terpenes 2 (Thujopsene)

A full OECDTG 202 GLP tests was performed twice with Daphnia magna, and based on the results of a combined limit/range-finding test. Water Accommodated Fractions (WAFs) were prepared at individual loading rates 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 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/L, in the first full test. In the second full tests daphnids were exposed to WAFs prepared at 0.46, 1.0, 2.2, 4.6 and 10 mg/L and solutions containing 22 and 46% of the WAF prepared at 0.46 mg/L.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 a minimum. The measured TOC concentrations showed that the test item was poorly soluble in test medium and the results were inconclusive. Moreover, there was an indication of inhomogeneity of test solutions. For these reasons and because the TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. At the end of the first full test, 76-100% immobility was observed in all WAFs beside 4.6 mg/L where only 10% daphnids were immobilised. In the second full test, a dose-related immobilisation was observed at the end of the test. Based on the observed differences in both tests, despite the same preparation procedure, it was assumed that additional testing would not improve understanding of the behaviour of the test item in an aquatic environment. Therefore, it was decided to combine results of both tests for the calculation of the EL50 values. 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 Terpenes 2 (Thujopsene), Cedarwood Texas oil distilled was 1.4 mg/L based on nominal loading rates (95% confidence interval between 0.41 and 2.9 mg/L).

Additional information

Key EL50 values are :

CW Tx distilled Terpenes 1 (Cedrene): EL50 = 0.76 mg/L

CW Tx distilled Terpenes 2 (Thujopsene): EL50 = 1.4 mg/L