Juniper, Juniperus virginiana, ext.

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Dec 2017 - 23 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

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

Version / remarks:

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 to confirm WAF procedure

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, a sample was taken from the highest test concentration after 24 hours of exposure.

Frequency at t=0 h, t=24 h and at t=96 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 Fraction procedure was applied.

Details on test solutions:

The batch of Cedarwood Texas oil crude tested was a 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 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. No undissolved material was observed in any of the solutions when inspected under a microscope. All test solutions were clear and colorless at the end of the preparation procedure. Any residual volumes were discarded.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
Species: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
Mean length*: Range-finding test: 2.6 ± 0.3 cm, Final test: 2.2 ± 0.2 cm
Mean weight*: Range-finding test: 0.21 ± 0.10 g, Final test: 0.15 ± 0.07 g
Characteristics: F1 from a single parent-pair bred in UV-treated water.
Reason for selection: This system has been selected as an internationally accepted species.
Total fish used: 61

* Ten fish of the batch used for the test, were weighed and measured prior to the start of the test.

ACCLIMATION
Quarantine/Acclimatisation: At least 12 days after delivery
Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel- Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
Measurements: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, pH and temperature were measured before transferring the fish to the test system.
Water quality: parameters Were kept within the optimum limits for the respective fish species.
Feeding: Daily with pelleted fish food (Essence (300-500 um), Coppens International bv, Helmond, The Netherlands)
Validity of batch: In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.

FEEDING DURING TEST
No feeding from 24 hours prior to the test and during the total test period

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

180 mg CaCO3 per litre

Test temperature:

20-24°C

pH:

Start of test (0 hr): 7.9- 8.0
End of test (96hr): 7.2 - 7.6

Dissolved oxygen:

Start of test (0 hr): 8.4 - 8.5
End of test (96hr): 5.9 -7.6

Nominal and measured concentrations:

Nominal: WAFs prepared at loading rates of 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/L.

Measured TOC concentrations corrected for the control treatment
Loading rate (mg/L): control, 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/L.
TOC (t=0hr): n.a, 0.54, 0.29, 0.68, 0.77, 2.9 and 5.3 mg/L
TOC (t=96hr): n.a, 0.19, 1.2, 0.9, 1.4, n.a and n.a mg/L

n.a. – not applicable

Details on test conditions:

TEST SYSTEM
- Test duration: 96 hours
- Test type: Static
- Test vessels: 6.5 litres, all-glass, containing 6.2 litres of test solution. Since the test item was volatile the study was performed in air-tight vessels and the media were not aerated.
- Number of fish: 7 per concentration
- Loading: 0.17 g fish/Litre, i.e. 7 fish per 6.2 litres of test medium
- Aeration: The test media were not aerated during the test.
- Introduction of fish: Within 27 minutes after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 1.0 unit and 1.0°C.

TEST MEDIUM / WATER PARAMETERS
- Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
- Intervals of water quality measurement: dissolved oxygen content, pH and temperature were recorded at the start and at the end of the test in all vessels with surviving fish. Additionally, after 24 hours of exposure, at the highest test concentration with surviving fish.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Illumination: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality and other effects: At 3, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 to observe for any dead or severely distressed fish. Dead fish were removed when observed.

TERMINAL PROCEDURES
Euthanasia: At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study : was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
- Three fish per concentration were exposed to WAFs prepared at loading rates of 0.46, 1.0, 10 and 100 mg/L.
- Dissolved oxygen concentrations, pH and temperature were only measured in the lowest and the highest test concentration.
- Samples for determination of TOC concentration were taken also at t=72h.

- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

pentachlorophenol (PCP)

Key result

Duration:

96 h

Dose descriptor:

LL50

Remarks:

24, 48, 72 and 96h-LL50

Effect conc.:

6.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%-confidence interval (4.6 -10 mg/L)

Duration:

3 h

Dose descriptor:

LL50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%-confidence interval (10-22 mg/L)

Details on results:

Measured Concentrations:
At the start of the test, the measured TOC concentrations increased with increasing loading rates. Between 20 and 37% of the nominal TOC concentrations were observed to be dissolved in test medium, with the exception of the lowest test concentration, where 140% was found. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared.

Mortality and Other Effects:
No effects were observed at the four lowest test concentrations and the control throughout the test. All fish exposed to the highest concentration were found dead after the first 3 hours of exposure, while none of fish exposed to WAF of 10 mg/L survived the first 24 hours of exposure. Clinical effects were observed at the WAF prepared at a loading rate of 4.6 mg/L

Acceptability of the Test
1. No mortality was observed in the control at the end of the test.
2. Test conditions were maintained constant throughout the test.
3. The dissolved oxygen concentration has been at least 60% of the air saturation value throughout the test (>5 mg/L at 22°C).

Results with reference substance (positive control):

Under the conditions of the present test, all carp exposed to a PCP concentration of 0.46 mg/L died within 24 hours, while no mortality occurred at 0.10 and 0.22 mg/L. The 96h-LC50 for carp exposed to PCP was 0.32 mg/L (95 % confidence interval between 0.22 and 0.46 mg/L). This effect was already reached within 24 hours of exposure.

The range of the 96h-LC50 for carp is generally between 0.10 and 0.46 mg/L based on historical data of reference tests performed approximately every 3 months from April 1988 until the end of 2000, and annually since then. Hence, the sensitivity of carp originating from the present.

Reported statistics and error estimates:

The LL50 could not be determined using the maximum likelihood estimation method. This was because there was no concentration between the highest concentration (A) at which 0% mortality and the lowest concentration (B) at which 100% mortality occurred. Instead, the LL50 was calculated as (AB)½, with A and B being limits of the 95% confidence interval.

Sublethal observations / clinical signs:

Incidence of Mortality and Total Mortality During the Final Test

Cedarwood Texas oil crude Loading rate (mg/L)	Initial number of fish	Cumulativemortality					Total Mortality (%)
		3h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
0.46	7	0	0	0	0	0	0
1.0	7	0	0	0	0	0	0
2.2	7	0	0	0	0	0	0
4.6	7	0	0	0	0	0	0
10	7	0	7	7	7	7	100
22	7	7	7	7	7	7	100

 

Clinical Effects Observed During the Final Test

 

Cedarwood Texas oil crude Loading rate (mg/L)	Time of recording (hours)	Specification of effects	Relative number
4.6	24	Immobile	7/7
	48	Immobile Loss of equilibrium	2/7 5/7
	72 - 96	Hypoactive swimming anddarker color	7/7
10	3	Discolored and loss of equilibrium Immobile	2/7 5/7

Validity criteria fulfilled:

yes

Remarks:

see details on results

Conclusions:

The 96-h LL50 for Cyprinus carpio exposed to Cedarwood Texas oil crude was 6.8 mg/L (95% confidence interval between 4.6 and 10 mg/L), and was already reached after 24 hours of exposure.

Executive summary:

An evaluation of acute toxic effects of Cedarwood Texas oil crude in Cyprinus carpio was investigated in accordance to GLP and OECD 203 guidelines. Based on the results of a preceding range-finding test, Water Accommodated Fractions (WAFs) of Cedarwood Texas oil crude were individually prepared at loading rates ranging between 0.46 and 100 mg/L and used as test concentrations. Seven fish per concentration were exposed to a control and to WAFs prepared at loading rates of 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/L under static conditions. The total exposure period was 96 hours and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of the test. No effects were observed in the four lowest WAFs throughout the test.  No fish survived exposure in the two highest WAFs. The measured TOC concentrations increased with increasing loading rates 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. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 96h-LL50 was 6.8 mg/L (95% confidence interval between 4.6 and 10 mg/L) and was already reached after 24 hours of exposure.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The result derived from read across is sufficiently reliable because all Annex XI criteria are met.

Justification for type of information:

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

According to Annex VIII, 9.1.3 of the REACh Regulation (EC) No 1907/2006, Short-term toxicity on fish (Fish acute toxicity) is standard information required for the registration of substances manufactured or imported in quantities of ten tonnes 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 “Fish acute toxicity” for the substance Cedarwood Virginia oil [CW Virginia] because a 96-hour toxicity study, according to OECD test guideline 203, is available for the substance Cedarwood Texas oil crude [CW TX crude], which composition is very similar to the target substance, with some variations only in the concentrations of the constituents.

The read-across is based on the comparison of the composition of two UVCBs that are very close one another, the raw material being the very similar, with the same process except for an additional distillation step for the target substance.

This read-across is based on the argument that the target and the source substances have similar toxicological properties and mode of action due to the structural similarities in their constituents.

This read-across prediction intends to provide acute fish toxicity information as a basic element to be used for the classification for aquatic environmental hazards according to CLP Regulation EC/1272/2008.

By means of this read-across approach, vertebrate animal testing is avoided.


This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the chemical compositions of both UVCB substances, and the absence of significant difference between the concentrations of the constituents; or when there are variations in concentrations, the source UVCB substance is the at least comparable or worst-case regarding acute aquatic toxicity.
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, which are summarised hereafter.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

96 h

Dose descriptor:

LL50

Remarks:

24, 48, 72 and 96h-LL50

Effect conc.:

6.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%-confidence interval (4.6 -10 mg/L)

Duration:

3 h

Dose descriptor:

LL50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%-confidence interval (10-22 mg/L)

Details on results:

Measured Concentrations:
At the start of the test, the measured TOC concentrations increased with increasing loading rates. Between 20 and 37% of the nominal TOC concentrations were observed to be dissolved in test medium, with the exception of the lowest test concentration, where 140% was found. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared.

Mortality and Other Effects:
No effects were observed at the four lowest test concentrations and the control throughout the test. All fish exposed to the highest concentration were found dead after the first 3 hours of exposure, while none of fish exposed to WAF of 10 mg/L survived the first 24 hours of exposure. Clinical effects were observed at the WAF prepared at a loading rate of 4.6 mg/L

Acceptability of the Test
1. No mortality was observed in the control at the end of the test.
2. Test conditions were maintained constant throughout the test.
3. The dissolved oxygen concentration has been at least 60% of the air saturation value throughout the test (>5 mg/L at 22°C).

Results with reference substance (positive control):

Under the conditions of the present test, all carp exposed to a PCP concentration of 0.46 mg/L died within 24 hours, while no mortality occurred at 0.10 and 0.22 mg/L. The 96h-LC50 for carp exposed to PCP was 0.32 mg/L (95 % confidence interval between 0.22 and 0.46 mg/L). This effect was already reached within 24 hours of exposure.

The range of the 96h-LC50 for carp is generally between 0.10 and 0.46 mg/L based on historical data of reference tests performed approximately every 3 months from April 1988 until the end of 2000, and annually since then. Hence, the sensitivity of carp originating from the present.

Reported statistics and error estimates:

The LL50 could not be determined using the maximum likelihood estimation method. This was because there was no concentration between the highest concentration (A) at which 0% mortality and the lowest concentration (B) at which 100% mortality occurred. Instead, the LL50 was calculated as (AB)½, with A and B being limits of the 95% confidence interval.

Sublethal observations / clinical signs:

Incidence of Mortality and Total Mortality During the Final Test

Cedarwood Texas oil crude Loading rate (mg/L)	Initial number of fish	Cumulativemortality					Total Mortality (%)
		3h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
0.46	7	0	0	0	0	0	0
1.0	7	0	0	0	0	0	0
2.2	7	0	0	0	0	0	0
4.6	7	0	0	0	0	0	0
10	7	0	7	7	7	7	100
22	7	7	7	7	7	7	100

 

Clinical Effects Observed During the Final Test

 

Cedarwood Texas oil crude Loading rate (mg/L)	Time of recording (hours)	Specification of effects	Relative number
4.6	24	Immobile	7/7
	48	Immobile Loss of equilibrium	2/7 5/7
	72 - 96	Hypoactive swimming anddarker color	7/7
10	3	Discolored and loss of equilibrium Immobile	2/7 5/7

Validity criteria fulfilled:

yes

Remarks:

see details on results

Conclusions:

Based on the read across result from Cedarwood Texas oil crude, Cedarwood Virginia Oil is concluded to have a 96-h LL50 of 6.8 mg/L (95% confidence interval between 4.6 and 10 mg/L).

Executive summary:

Based on the read across result from Cedarwood Texas oil crude, Cedarwood Virginia Oil is concluded to have a 96-h LL50 of 6.8 mg/L (95% confidence interval between 4.6 and 10 mg/L). An evaluation of acute toxic effects of Cedarwood Texas oil crude in Cyprinus carpio was investigated in accordance to GLP and OECD 203 guidelines. Based on the results of a preceding range-finding test, Water Accommodated Fractions (WAFs) of Cedarwood Texas oil crude were individually prepared at loading rates ranging between 0.46 and 100 mg/L and used as test concentrations. Seven fish per concentration were exposed to a control and to WAFs prepared at loading rates of 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/L under static conditions. The total exposure period was 96 hours and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of the test. No effects were observed in the four lowest WAFs throughout the test.  No fish survived exposure in the two highest WAFs. The measured TOC concentrations increased with increasing loading rates 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. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 96h-LL50 was 6.8 mg/L (95% confidence interval between 4.6 and 10 mg/L) and was already reached after 24 hours of exposure.

Description of key information

Based on the read across result from Cedarwood Texas oil crude, Cedarwood Virginia Oil is concluded to have a 96-h LL50 of 6.8 mg/L (95% confidence interval between 4.6 and 10 mg/L). An evaluation of acute toxic effects of Cedarwood Texas oil crude in Cyprinus carpio was investigated in accordance to GLP and OECD 203 guidelines. Based on the results of a preceding range-finding test, Water Accommodated Fractions (WAFs) of Cedarwood Texas oil crude were individually prepared at loading rates ranging between 0.46 and 100 mg/L and used as test concentrations. Seven fish per concentration were exposed to a control and to WAFs prepared at loading rates of 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/L under static conditions. The total exposure period was 96 hours and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of the test. No effects were observed in the four lowest WAFs throughout the test.  No fish survived exposure in the two highest WAFs. The measured TOC concentrations increased with increasing loading rates 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. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 96h-LL50 was 6.8 mg/L (95% confidence interval between 4.6 and 10 mg/L) and was already reached after 24 hours of exposure.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

6.8 mg/L