Santalum austro-caledonicum, ext.

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:

weight of evidence

Study period:

2017-02-21 to 2017-11-03

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: This study present vulnerabilities and a Klimisch 4 was assigned.

Remarks:

As an UVCB susbtance, the study was performed under WAF conditions according to OECD 23. Study was then performed at nominal loading rates. TOC measurement is performed to control the stability of the substance during the study. The vulnerability is from the 0.57mg/L loading rates. Indeed at 48h the recovery is highre than the 20% allowed by the OECD 202 guideline. Regarding the nature of the substance, such behaviour is not exceptionnal. However, as it happens at one of the lowest concentration, the statistical regression is highly influenced by this deviation. Therefore, even if the whole report cannot be invalidated, the study is considered as Klimish 4 and other studies are performed to validate the result.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

OECD Series on testing and assessment, No. 23, “Guidance document on aquatic toxicity
testing of difficult substances and mixtures”, December 15, 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

10 January 2017

Specific details on test material used for the study:

Not applicable

Analytical monitoring:

yes

Details on sampling:

- Chemical analyses:
Single samples for analysis were taken from the control and all test solutions at the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h). Concentration of dissolved organic material was checked by analysis of total organic carbon (TOC) in the control medium and the WAFs. TOC analysis was not performed in compliance with the OECD GLP principles but was adapted to fit the specific parameters of the test item, in accordance with ISO 17025.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for fresh media at t=0h and t=24h) were prepared under closed conditions and by slow-stirring to avoid production of a dispersion.
The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 5 L, except for the preparation of the lowest loading rates where a 10 L glass bottle were used. A magnetic stirring bar was placed in each mixing vessel and 5.0 to 5.5 L of test water (depending on the brim capacity of the bottles; 10.0 to 10.5 L for the lowest loading rates) were added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. Then the mixing vessels were closed immediately. The mixing was carried out at a speed that was slow enough not to cause dispersion or emulsification of the undissolved fraction of the test item. Mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 ± 2 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for 1 hour before use. The first 100 mL were removed via the drain port. Then the WAFs were directly added into test vessels that were immediately sealed with screw caps after introduction of daphnids. No small bubble was observed in the test vessels. The test solution in test vessels was observed to be clear and colourless at all loading rates. The test was carried out without adjustment of the pH.

- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna (Straus), clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Reason for selection: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at study initiation: < 24 h
- Breeding Conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water was changed three times a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

Total water hardness of the test water was approximately 250 mg/L (as CaCO3).

Test temperature:

between 20.6 and 20.8 °C throughout the test (average value: 20.8°C), and complied with the requirements (20°C ± 2°C, constant within 1°C).

pH:

8.08 - 8.58
Guideline requirements: pH between 6.0-9.0, and not varying by more than 1.5 units

Dissolved oxygen:

8.27 - 9.25 mg O2/L
oxygen: ≥ 60% of the air-saturation value at the end of the test in controls and test vessels.

Salinity:

None

Conductivity:

No data

Nominal and measured concentrations:

Nominal : control, 0.32; 0.57; 1.01; 1.80; 3.21 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screw caps. Each test vessel will be uniquely identified with study code, replicate number, date of experimentation and concentration.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Renewal rate of test solution: A semi-static test was performed with renewal of test solutions after 24 h (because of the properties of the test item)
- No. of daphnids: 20 per treatment group (including controls), divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace.
- Introduction of Daphnids: Daphnids were introduced into the test vessels each completely filled with test solution and without headspace immediately after filling the test vessels with test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as prescribed by the OECD Guideline 202

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED:
- Immobility: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Immobile animals were eliminated from the vessels as soon as they were discovered. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.
- pH and dissolved O2: At start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h) from all test concentrations and control.
- Temperature of Medium: Measured continuously in a temperature controlled vessel next to the test vessels, over the entire study period, beginning at the start of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.78
- Range finding study: Ten daphnids per concentration (5 per vessel, in duplicate) were exposed to the nominal loading rates 0.32, 1.00, 3.20, 10.00 and 32.00 mg.L-1 and to a control.
- Results used to determine the conditions for the definitive study: The per cent immobility was 0, 0, 30; 100 and 100% (24 h) and 0, 0, 40; 100 and 100% (48 h) at 0.0, 0.32; 1.00; 3.20; 10.00 and 32.00 mg.L-1, respectively. Based on the results of a range-finding test, test solutions used in the definitive test were prepared to obtain the following loading rates (spaced by a factor of approximately 1.78): 0.32, 0.57, 1.01, 1.80 and 3.21 mg.L-1.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

1.034 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

95% Confidence Limit (mg/L) = [0.877 - 1.220]

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

0.998 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 0.840 - 1.187 mg/L

Details on results:

After 24 hours of exposure, immobilisation rates were 0% at 0.32 and 0.57 mg.L-1 (loading), 65% at 1.01 mg.L-1 (loading), 85% at 1.80 mg.L-1 (loading) and 100% at 3.21 mg.L-1 (loading). After 48 hours of exposure, immobilisation rates were 0% at 0.32 (loading), 5% at 0.57 mg.L-1 (loading), 65% at 1.01 mg.L-1 (loading), 85% at 1.80 mg.L-1 (loading) and 100% at 3.21 mg.L-1 (loading).

Results with reference substance (positive control):

On January 10, 2017 (most recent test), the 24h-EC50 was 1.22 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.

Reported statistics and error estimates:

The 24 and 48-hour EL50 including the 95% confidence interval using Probit-analysis were determined by the computer program ToxRat.
The evaluation of the effects was based on the nominal WAFs concentrations (nominal loading values).

Analytical results

The analytical results of this test (see table 6.1.3/1 below) showed that WAFs concentrations were overall stable between the start and the end of each exposure period (t=0h-t24h_Old and t=24h_Fresh - t=48h),within or the ± 20% of the initial TOC concentrations values. It should be noted that a WAF is by definition a complex mixture for which the individual concentration of each constituent differs due to its properties (e.g. solubility, adsorption, volatilisation, bioaccumulation…). Due to these differences, interactions between certain constituents of the mixture may occur and affect the behaviour of a given constituent which consequently would not react in the same way that if it was alone in the mixture. Therefore, results are provided as Effective Loading Rate 50, according to the OECD No. 23.

Table 6.1.3/1. Concentrations of the test item in test water - Results of the determination of TOC analysis (mg/L) - Final test.

Nominal concentration* (mg test item.L^-1)	Start (t=0h)	t=24h Old	Relative loss to initial value (t=0h - t=24hold)(%)	t=24h Fresh	End (t=48h)	Relative loss to initial value (t=24hfresh- t=48h)(%)	Geometric mean TOC concentrations (mg.L^-1)
Control	0.91	0.83	9	0.80	0.99	-24	0.88
0.32	1.04	0.91	13	1.15	1.03	10	1.03
0.57	1.16	0.96	17	0.99	1.17	-18	1.07
1.01	1.40	1.26	10	1.21	1.29	-7	1.29
1.80	1.65	1.38	16	1.72	1.65	4	1.59
3.21	2.33	2.02	13				2.17

* WAF prepared at the given loading rate.

  Grey boxes: no further analysis was performed for concentrations in which all daphnids were immobilised at t=24h (see biological section).

Table 6.1.3/2: Acute immobilisation of daphnids after 24 and 48 h in the final test

Nominal Concentration*(mg test item.L^-1)	Replicate	Number of daphnids exposed	Response at 24h		Response at 48h
			Number	Total %	Number	Total %
Control	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.32	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.57	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 1 0	5
1.01	1 2 3 4	5 5 5 5	3 5 3 2	65	3 5 3 2	65
1.80	1 2 3 4	5 5 5 5	5 4 3 5	85	5 4 3 5	85
3.21	1 2 3 4	5 5 5 5	5 5 5 5	100	5 5 5 5	100

 

* WAF prepared at the given loading rate

 

Water quality parameter values throughout the test:

The results of measurement of pH and oxygen concentrations remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen: ≥ 60% of the air-saturation valueat the end of the test), suggesting that pH and oxygen concentrations were not influenced by the test item.

Furthermore, the temperature of the test medium was situated between 20.6 and 20.8 °C throughout the test (average value: 20.8 °C), and complied with the requirements (20°C ± 2°C, constant within 1°C).

 

Validity criteria of the study:

Controls: In the control, no daphnids became immobilized nor trapped at the surface of the water or showed signs of stress.

Dissolved [O2]: Dissolved oxygen concentration at the end of the test was ≥ 60 % of the air-saturation value in controls and test vessels.

Thus the validity criteria have been fulfilled in the present study.

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value estimated was 0.998 mg test item/L.

Executive summary:

A study was performed according to the OECD Guideline 202 and with GLP compliance to assess the acute toxicity of the test item SANDALWOOD AUSTROCALEDONIEN OIL to the freshwater invertebrate Daphnia magna. It was investigated in a 48h closed semi-static test using Water Accommodated Fractions (WAFs). The criterion measured was the EL₅₀ (Median Effective Loading rate), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.  

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed toWater Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of0.32, 0.57, 1.01, 1.80 and 3.21 mg/L and to a control.The immobility of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours.Concentration of dissolved organic material in the control and the WAFs was checked by TOC analysis at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

The analytical results of this test showed that WAFs concentrations were overall stablebetween the start and the end of each exposure period (t=0h-t24h_Oldand t=24h_Fresh-t=48h), within the ± 20% of the initial TOC concentrations values. After 48 hours of exposure, immobilisation rates were 0% at 0.32 mg/L (loading), 5% at 0.57 mg/L (loading), 65% at 1.01 mg/L (loading), 85% at 1.80 mg:L (loading) and 100% at 3.21 mg/L (loading).

The EL₅₀ at each of the observation times was as follows:

Time (h)	EL50(mg test item.L^-1)	95% confidence limits (mg test item.L^-1)
24	1.034	0.877 – 1.220
48	0.998	0.840 – 1.187

The 24 and 48-hour EL₅₀including the 95% confidence interval were calculated by the computer program ToxRat.

Therefore, under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value estimated was 0.998 mg test item/L.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-02-21 to 2017-11-03

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other:

Remarks:

As an UVCB susbtance, the study was performed under WAF conditions according to OECD 23. Study was then performed at nominal loading rates. TOC measurement is performed to control the stability of the substance during the study. The vulnerability is from the 0.32 mg/L and 0.57mg/L loading rates. Indeed at 48h the recovery is higher than the 20% allowed by the OECD 202 guideline. Regarding the nature of the substance, such behaviour is not exceptionnal. However, as it happens at one of the lowest concentration, the statistical regression is highly influenced by this deviation. Therefore, even if the whole report cannot be invalidated, the study is considered as Klimish 4 and other studies are performed to validate the result.

Justification for type of information:

This study present vulnerabilities and a Klimisch 4 was assigned. Full justification can be found in the endpoint summary.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

OECD Series on testing and assessment, No. 23, “Guidance document on aquatic toxicity
testing of difficult substances and mixtures”, December 15, 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

10 January 2017

Specific details on test material used for the study:

Not applicable

Analytical monitoring:

yes

Details on sampling:

- Chemical analyses:
Single samples for analysis were taken from the control and all test solutions at the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h). Concentration of dissolved organic material was checked by analysis of total organic carbon (TOC) in the control medium and the WAFs. TOC analysis was not performed in compliance with the OECD GLP principles but was adapted to fit the specific parameters of the test item, in accordance with ISO 17025.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for fresh media at t=0h and t=24h) were prepared under closed conditions and by slow-stirring to avoid production of a dispersion.
The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 5 L, except for the preparation of the lowest loading rates where a 10 L glass bottle were used. A magnetic stirring bar was placed in each mixing vessel and 5.0 to 5.2 L of test water (depending on the brim capacity of the bottles; 11.0 L for the lowest loading rates) were added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. Then the mixing vessels were closed immediately. The mixing was carried out at a speed that was slow enough not to cause dispersion or emulsification of the undissolved fraction of the test item. Mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 ± 2 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for 1 hour before use. The first 100 mL were removed via the drain port. Then the WAFs were directly added into test vessels that were immediately sealed with screw caps after introduction of daphnids. No small bubble was observed in the test vessels. The test solution in test vessels was observed to be clear and colourless at all loading rates. The test was carried out without adjustment of the pH.

- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna (Straus), clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Reason for selection: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at study initiation: < 24 h
- Breeding Conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water was changed three times a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

Total water hardness of the test water was approximately 250 mg/L (as CaCO3).

Test temperature:

between 20.8 and 20.9 °C throughout the test (average value: 20.9°C), and complied with the requirements (20°C ± 2°C, constant within 1°C).

pH:

7.56 - 8.65
Guideline requirements: pH between 6.0-9.0, and not varying by more than 1.5 units

Dissolved oxygen:

7.81 - 8.45 mg O2/L
oxygen: ≥ 60% of the air-saturation value at the end of the test in controls and test vessels.

Salinity:

None

Conductivity:

No data

Nominal and measured concentrations:

Nominal : control, 0.32; 0.57; 1.01; 1.80; 3.21 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screw caps. Each test vessel will be uniquely identified with study code, replicate number, date of experimentation and concentration.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Renewal rate of test solution: A semi-static test was performed with renewal of test solutions after 24 h (because of the properties of the test item)
- No. of daphnids: 20 per treatment group (including controls), divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace.
- Introduction of Daphnids: Daphnids were introduced into the test vessels each completely filled with test solution and without headspace immediately after filling the test vessels with test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as prescribed by the OECD Guideline 202

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED:
- Immobility: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Immobile animals were eliminated from the vessels as soon as they were discovered. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.
- pH and dissolved O2: At start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h) from all test concentrations and control.
- Temperature of Medium: Measured continuously in a temperature controlled vessel next to the test vessels, over the entire study period, beginning at the start of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.78
- Range finding study: Ten daphnids per concentration (5 per vessel, in duplicate) were exposed to the nominal loading rates 0.32, 1.00, 3.20, 10.00 and 32.00 mg.L-1 and to a control.
- Results used to determine the conditions for the definitive study: The per cent immobility was 0, 0, 30; 100 and 100% (24 h) and 0, 0, 40; 100 and 100% (48 h) at 0.0, 0.32; 1.00; 3.20; 10.00 and 32.00 mg.L-1, respectively. Based on the results of a range-finding test, test solutions used in the definitive test were prepared to obtain the following loading rates (spaced by a factor of approximately 1.78): 0.32, 0.57, 1.01, 1.80 and 3.21 mg.L-1.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

0.92 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

95% Confidence Limit (mg/L) = [0.781 - 1.085]

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

0.835 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 0.708 - 0.986 mg/L

Details on results:

After 24 hours of exposure, immobilisation rates were 0% at 0.32 mg.L-1 (loading), 5% at 0.57 mg.L-1 (loading), 75% at 1.01 mg.L-1 (loading), 90% at 1.80 mg.L-1 (loading) and 100% at 3.21 mg.L-1 (loading). After 48 hours of exposure, immobilisation rates were 0% at 0.32 (loading), 15% at 0.57 mg.L-1 (loading), 75% at 1.01 mg.L-1 (loading), 95% at 1.80 mg.L-1 (loading) and 100% at 3.21 mg.L-1 (loading).

Results with reference substance (positive control):

On January 10, 2017 (most recent test), the 24h-EC50 was 1.22 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.

Reported statistics and error estimates:

The 24 and 48-hour EL50 including the 95% confidence interval using Probit-analysis were determined by the computer program ToxRat.
The evaluation of the effects was based on the nominal WAFs concentrations (nominal loading values).

Analytical results

The analytical results of this test (see table 6.1.3/1 below) showed that WAFs were overall stable throughout the test period. Indeed, TOC content in WAFs was maintained around ± 20-30% during the first part of the test (t=0h-t24h_Old), and was satisfactorily preserved within ± 20% of the initial concentration during the second exposure period (t=24h_Fresh-t=48h), except at 0.32mg/L with a gain of 35%. It would be difficult to determine the specific cause of this last observation, as well as gains observed in controls. However, this was considered not to affect the results and the integrity of the study regarding properties of the test item and the non-specific analytical method.

It should be noted that a WAF is by definition a complex mixture for which the individual concentration of each constituent differs due to its properties (e.g. solubility, adsorption, volatilisation, bioaccumulation…). Due to these differences, interactions between certain constituents of the mixture may occur and affect the behaviour of a given constituent which consequently would not react in the same way that if it was alone in the mixture.

Therefore, results are provided as Effective Loading Rate 50, according to the OECD No. 23.

Table 6.1.3/1: Concentrations of the test item in test water - Results of the determination of TOC analysis (mg/L) - Final test.

Nominal concentration* (mg test item.L^-1)	Start (t=0h)	t=24h Old	Relative loss to initial value (t=0h - t=24hold)(%)	t=24h Fresh	End (t=48h)	Relative loss to initial value (t=24hfresh- t=48h)(%)	Geometric mean TOC concentrations (mg.L^-1)
Control	0.86	1.60	-86	1.09	1.52	-39	1.23
0.32	1.51	1.12	26	1.14	1.54	-35	1.31
0.57	1.18	1.54	-31	1.47	1.76	-20	1.47
1.01	1.39	1.77	-27	1.56	1.52	3	1.55
1.80	2.14	1.98	7	1.93	1.82	6	1.96
3.21	2.34	2.66	-14				2.49

* WAF prepared at the given loading rate.

Grey boxes: no further analysis was performed for concentrations in which all daphnids were immobilised at t=24h (see biological section).

 

Table 6.1.3/2: Acute immobilisation of daphnids after 24 and 48 h in the final test

Nominal concentration*(mg test item.L^-1)	Replicate	Number of daphnids exposed	Response at 24h		Response at 48h
			Number	Total %	Number	Total %
Control	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.32	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.57	1 2 3 4	5 5 5 5	0 0 0 1	5	2 0 0 1	15
1.01	1 2 3 4	5 5 5 5	4 4 2 5	75	4 4 2 5	75
1.80	1 2 3 4	5 5 5 5	5 4 4 5	90	5 5 4 5	95
3.21	1 2 3 4	5 5 5 5	5 5 5 5	100	5 5 5 5	100

* WAF prepared at the given loading rate

 

Water quality parameter values throughout the test:

The results of measurement of pH and oxygen concentrations (mg/L) are presented in Tables 2 and 3. These parameters remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen:≥ 60% of the air-saturation valueat the end of the test), suggesting that pH and oxygen concentrations were not influenced by the test item. Furthermore, the temperature of the test medium was situated between 20.8 and 20.9 °C throughout the test (average value: 20.9 °C), and complied with the requirements (20°C ± 2°C, constant within 1°C).

 

Validity criteria of the study:

Controls: In the control, no daphnids became immobilized nor trapped at the surface of the water or showed signs of stress.

Dissolved [O2]: Dissolved oxygen concentration at the end of the test was ≥ 60 % of the air-saturation value in controls and test vessels.

Thus the validity criteria have been fulfilled in the present study.

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value estimated was 0.835 mg.L-1.

Executive summary:

A study was performed according to the OECD Guideline 202 and with GLP compliance to assess the acute toxicity of the test item SANDALWOOD AUSTROCALEDONIEN OIL to the freshwater invertebrate Daphnia magna. It was investigated in a 48h closed semi-static test using Water Accommodated Fractions. The criterion measured was the EL₅₀(Median Effective Loading rate), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.  

Following a previous test, where a 48-hour EL₅₀ of 0.998 mg/L was determined, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 0.32, 0.57, 1.01, 1.80 and 3.21mg.L^-1and to a control.The immobility of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours.Concentration of dissolved organic material in the control and the WAFs was checked by TOC analysis at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

The analytical results of this test showed that WAFs concentrations were overall stablebetween the start and the end of each exposure period.After 48 hours of exposure, immobilisation rates were 0% at 0.32 (loading), 15% at 0.57mg.L^-1(loading), 75% at 1.01mg.L^-1(loading), 95% at 1.80mg.L^-1(loading) and 100% at 3.21mg.L^-1(loading).

The EL₅₀at each of the observation times was as follows:

Time (h)	EL50(mg test item.L^-1)	95% confidence limits (mg test item.L^-1)
24	0.920	0.781 – 1.085
48	0.835	0.708 – 0.986

The 24 and 48-hour EL₅₀including the 95% confidence interval were calculated by the computer program ToxRat.

Therefore, under the experimental conditions and based on nominal concentrations, the 48-hour EL50 value estimated was 0.835 mg.L-1 (loading).

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

calculation (if not (Q)SAR)

Remarks:

Estimated by calculation

Adequacy of study:

weight of evidence

Study period:

2017-09-25 to 2017-09-29

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, and documentation / justification is limited

Remarks:

Result is a calculation based on constituents own ecotoxicity. Each constituent ecotoxicity is produced by a validated QSAR model. Unfortunately, one of the constituent is out of the Applicability Domain of the model. That constituent represents less than 3% of the total composition. Therefore it is complex to invalid the study as long as disregard it completely. That's the reason the study is Klimish 4.

Justification for type of information:

The registered substance is a Natural Complex Substance (UVCB) with a well-defined composition for which the relative percentage of each constituent is known.
Therefore, it has been decided that the ecotoxicity of the substance will be derived from knowledge of the constituents. The calculation is based on QSARs on consttiuents. QMRF and QPRF are attached in the part "attached background material".
However, this study present vulnerabilities and a Klimisch 4 was assigned. Full justification can be found in the endpoint summary.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

calculation method

Principles of method if other than guideline:

The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analyzable fraction of a WAF study.
Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents. In the calculation the second step is to remove this non-bioavailable fraction.
The final step is to determine the truly bioavailable fraction of the WAF per constituent. The EC50s of each constituent are predicted using the iSafeRat QSAR model. Each value and QMRF/QPRF has been provided in the IUCLID. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Effective Loading rate of the WAF.
The method has been validated using data derived from 48-hour EC50 tests on aquatic invertebrates, for which the concentrations of the test item had been determined by chemical analyses over the test period. Further to this the effective loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the non-bioavailable fraction.

GLP compliance:

no

Specific details on test material used for the study:

None

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

not applicable

Test organisms (species):

Daphnia sp.

Details on test organisms:

not applicable

Test type:

other: calculation method

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

48h-EL50 (effective loading rate of WAF)

Post exposure observation period:

not applicable

Hardness:

Hardness is not a necessary component of the WAF calculation

Test temperature:

The Temperature is not a necessary component of the WAF calculation.

pH:

The pH is not a necessary component of the WAF calculation

Dissolved oxygen:

The oxygen concentration is not a necessary component of the WAF calculation

Salinity:

Salinity is not a necessary component of the WAF calculation.

Nominal and measured concentrations:

The calculation determines measured concentrations

Details on test conditions:

calculation method

Reference substance (positive control):

not required

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

9.1 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Based on a typical composition

Details on results:

not applicable

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

At this 48-hour EL50 the expected concentrations of each constituent in the mixture (based on thermodynamic calculation) are as follows

constituents	concentration in the WAF (mg.L^-1)
(Z)-α-santalol	3.7
(Z)-β-santalol	1.5
(Z)-trans-α-bergamotol	0.72
(Z)-lanceol	0.62
(Z)-epi-β-santalol	0.28
α-santalene	0.00098
(Z)-γ-curcumen-12-ol	0.13
trans-β-santalol	0.11
β-santalene	0.0018
epi-β-santalene	0.0018
(Z)-α-santalal	0.024

Validity criteria fulfilled:

not applicable

Conclusions:

The 48h-EL50 is 9.1 mg test item/L, based on typical composition.

Executive summary:

The registered substance SANDALWOOD AUSTROCALEDONIEN OIL is a Natural Complex Substance (UVCB) with a well-defined composition. Its acute toxicity to aquatic invertebrates has been investigated using an in-house calculation method that replaces an OECD 202 study and guideline for Testing of Chemicals No. 23 (i.e. WAF conditions).

A typical composition has been investigated.

 

The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analysable fraction of a WAF study. In the calculation the second step is to remove this non-bioavailable fraction. Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents.These two reasons explain why ecotoxicity values from WAF studies are always higher for non-polar narcotic mixtures than the calculated values from CLP additivity calculation.

The final step is to determine the truly bioavailable fraction of the WAF per constituent. The EC50s of each constituent were predicted using the iSafeRat QSAR model (and QPRF as well as the QMRF are provided in the IUCLID). An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Effective Loading rate of the WAF.

The 48-h EL50 was 9.1 mg test material/L.

Based on the results of this study, the substance would not be classified as acute 1 to aquatic organisms in accordance with the classification of the CLP.

Results Synopsis

Test Type: Calculation method replacing an OECD 202 test

48h-EL50: 9.1 mg test material/L based on the typical composition

Description of key information

In a weight of evidence approach, the registered substance exhibits a 48h-EL50 = 1.96 mg/L (geometric mean of three 48h-EL50 values).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.96 mg/L

Additional information

For that endpoint, three studies assessing the acute toxicity of the registered substance on aquatic invertebrates, were available:

- Two experimental studies performed under GLP conditions according to the OECD 202 guideline. They were performed in 48h closed semi-static tests using Water Accommodated Fractions (WAFs).

- One calculation method based on validated QSAR approachthat replaces an OECD 202 study and guideline for Testing of Chemicals No. 23 (i.e. WAF conditions).

 

None of these three studies were regarded as key study. They were all considered as Klimisch 4.

 

In one hand, both experimental studies suffer deviations in analytical recovery, indeed gain of matters were observed at the lowest concentrations. That deviation was observed at the same concentration and lead to the same conclusion. This deviation does not disqualify the studies but surely influence the value of the EL50s as a worst-case approach. Therefore, studies were considered as Klimish 4 and used as Weight of Evidence.  

In the other hand, to increase the evidence, we performed calculation based on constituent’s approach. That approach is based on QSAR calculations for every constituent performed by iSafeRat® holistic HA-QSAR v1. One of the constituent is unfortunately out of the applicability domain of the model. But that constituent represents less than 3% of the total composition. Therefore, a Klimish 4 was considered relevant for that study.

 

As a conclusion, the results of these three studies were used in a weight of evidence approach and the key ecotoxicity value for that endpoint was the geometric mean between the three EL50.