Sage, Salvia officinalis, 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:

key study

Study period:

From 2018-01-23 to 2018-03-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

signed on 2017-01-10

Specific details on test material used for the study:

Not applicable.

Analytical monitoring:

yes

Details on sampling:

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:

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.
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. A magnetic stirring bar was placed in each mixing vessel and test water was added. The loading rates of the test item were weighed on weighing boats that afterwards were placed above each mixing vessel and rinsed with test water. The mixing vessels were then carefully filled with the remaining volume of test water to obtain 5.0 to 5.7 L of test medium (depending on the brim capacity of the bottles) in order to use a maximum volume and to minimise headspace. 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 at least 1 hour before use. The first 100 mL were discarded 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. The test was carried out without adjustment of the pH.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Daphnia magna (Straus), clone 5
- Sex: Female
- Origin: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.
- Validity of batch: Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at test start: < 24 hours old
- 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:

The total water hardness was approximately 250 mg.L-1 (as CaCO3).

Test temperature:

The temperature of the test medium was situated between 20.7 and 21.0°C throughout the test (average value: 20.9°C).

pH:

7.74 - 8.45
pH requirements: 6.0-9.0, not varying by more than 1.5 units

Dissolved oxygen:

8.01 - 8.62 mg O2/L
oxygen: ≥ 60% of the air-saturation value at the end of the test in controls and test vessels.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Loading rates: 3.2, 5.7, 10.1, 18.0 and 32.0 mg.L-1.

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screw caps. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- 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
- Reconstituted water (Elendt M4 medium), as prescribed by 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 treatment group.
- 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: 5 daphnids per vessel, in duplicate were exposed to the nominal loading rates 1, 10, 32 and 100 mg.L-1 and to a control.
- Results used to determine the conditions for the definitive study: after 24 hours of exposure, immobilisation rates were 90% at 32 mg.L-1 100% at 100 mg.L-1; after 48 hours of exposure, immobilisation rate was 10% at 10 mg.L-1 and 100% at 32 mg.L-1. Based on the results of a range-finding test, the following nominal concentrations: .3.2, 5.7, 10.1, 18.0 and 32.0 mg.L-1 (loading rates) were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

10.996 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%CL: ND

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

30.824 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%CL: 24.876-48.242

Details on results:

The evaluation was based on the nominal WAFs concentrations.
After 48 hours of exposure, immobilisation rates were 0% at 3.2 and 5.7 mg.L-1, 20% at 10.1 mg.L-1 and 100% at 18.0 and 32.0 mg.L-1.
The 48-hour EL50 value was determined by the computer program ToxRat.

Results with reference substance (positive control):

On August 17, 2017 (most recent test), the 24h-EC50 was 0.92 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 evaluation of the effects was based on the nominal WAFs concentrations (nominal loading values). The software ToxRat® Professional was used for the determination of the effective concentrations.

Biological results

Acute immobilisation of daphnids after 24 and 48 hours 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
3.2	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
5.7	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
10.1	1 2 3 4	5 5 5 5	0 0 0 0	0	0 2 1 1	20
18.0	1 2 3 4	5 5 5 5	1 2 0 1	20	5 5 5 5	100
32.0	1 2 3 4	5 5 5 5	3 1 3 3	50	5 5 5 5	100

* WAF prepared at the given loading rate.

Analytical results

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 stable between the start and the end of the test, within the ± 20% of the initial TOC concentrations values, except the first day at 3.2mg.L^-1and the 2^ndday at 5.7, 18.0 and 32.0mg.L^-1 where important gains were observed. This probable overestimation constituted a deviation from the test guideline (higher than ± 20% of the initial concentrations values) but 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. Besides, biological results were in line with those of the range-finding test, where analytical results were consistent.

Moreover, due to the complex nature of the WAF and since the test item was a UVCB substance, the results were based on the nominal test loading rates and asEffective Loading Rate 50, according to the OECD No. 23.

Concentrations of the test item in test water - Results of the determination of TOC analysis (mg.L^-1) - 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)(%)
Control	0.97	0.87	10	0.96	1.14	-19
3.2	1.56	2.20	-41	1.61	1.63	-1
5.7	2.25	2.00	11	1.94	4.39	-126
10.1	2.92	2.58	12	2.76	2.70	2
18.0	4.12	4.04	2	4.05	5.98	-48
32.0	6.41	5.76	10	5.92	8.99	-52

* WAF prepared at the given loading rate.

Water quality parameters and environmental conditions throughout the test:

The results of measurement of pH and oxygen concentrations (mg.L^-1) are presented below. 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 20.7 and 21.0°C throughout the test (average value: 20.9°C), and complied with the requirements as laid down in the study plan (20°C ± 2°C, constant within 1°C).

 

pH-values during the final test

		Nominal concentration(mg test item.L^-1)*
		Control	3.2	5.7	10.1	18.0	32.0
Start t=0h		8.45	8.12	8.05	8.20	8.18	8.10
t=24h	Old	7.74	7.94	8.00	8.04	8.02	8.06
	Fresh	8.17	8.16	8.20	8.08	8.06	8.13
End t=48h		8.09	8.12	8.12	8.12	8.06	8.15

* WAF prepared at the given loading rate.

Dissolved oxygen concentrations (mg.L^-1) during the final test

		Nominal concentration(mg test item.L^-1)*
		Control	3.2	5.7	10.1	18.0	32.0
Start t=0h		8.60	8.58	8.62	8.56	8.61	8.55
t=24h	Old	8.07	8.14	8.15	8.17	8.17	8.18
	Fresh	8.32	8.42	8.43	8.50	8.46	8.42
End t=48h		8.01	8.06	8.12	8.22	8.16	8.13

* WAF prepared at the given loading rate.

All these dissolved-oxygen concentrations correspond to values > 60% of the air-saturation value.

 

 

Validity criteria of the study:

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

Dissolved [0₂]: 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:

The toxic effect of test item Sage dalmatian oil to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test using Water Accommodated Fractions. Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value estimated was 10.996 mg.L-1.

Executive summary:

A study was performed to assess the acute toxicity of test item Sage dalmatian oil to Daphnia magna. The followed method was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphniasp., Acute Immobilisation Test” (1), referenced as Method C.2 of Commission Regulation No. 440/2008 (amended byCommission Regulation (EU) 2016/266)(2) and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23) (7). 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 of3.2, 5.7, 10.1, 18.0 and 32.0mg.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 analysis of Total Organic Carbon (TOC) 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 stable between the start and the end of the test except the first day at 3.2mg.L^-1andthe 2^ndday at 5.7, 18.0 and 32.0mg.L^-1where important gains were observed. This probable overestimation constituted a deviation from the test guideline (higher than ± 20% of the initial concentrations values) but 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. Besides, biological results were in line with those of the range-finding test, where analytical results were consistent.

Since the test item was a UVCB substance, the results were based on the nominal test loading rates.After 48 hours of exposure, immobilisation rates were 0% at 3.2 and 5.7mg.L^-1, 20% at 10.1mg.L^-1and 100% at 18.0 and 32.0mg.L^-1.

Results synopsis:

Test: Toxic effect of SAGE DALMATIAN OIL to Daphnia magna according to OECD guidelines No. 202 and 23.

48h-EL50: 10.996 mg/L

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

key study

Study period:

From 2017-11-03 to 2017-11-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: method is used; calculation method applicable for that endpoint.

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 acute toxicity to daphnia was determined using a validated QSAR for the Mode of Action in question. 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 already known from literature or calculated using the iSafeRat QSAR model. 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:

not specified

Specific details on test material used for the study:

Not applicable.

Analytical monitoring:

not required

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

Not applicable.

Test organisms (species):

Daphnia magna

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.:

9.6 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: result based on the typical composition given by the supplier of the substance

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)
constituent 1	2.4
constituent 2	1.7
constituent 3	0.91
constituent 4	0.64
constituent 5	0.59A
constituent 6	0.0027
constituent 7	0.25
constituent 8	0.0041
constituent 9	0.37
constituent 10	0.26
constituent 11	0.2
constituent 12	0.14
constituent 13	0.054
constituent 14	0.144
constituent 15	0.067
constituent 16	0.048
constituent 17	0.048
constituent 18	0.038
constituent 19	0.019

Validity criteria fulfilled:

yes

Conclusions:

Calculated 48h-EL50 of Sage Dalmatian for the typical composition is of 9.6 mg test item/L.

Executive summary:

Sage Dalmatian oil is a Natural Complex Substance (UVCB) with a well-defined composition (>98%). 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). The typical composition of the substance provided by the supplier of the 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 are already known from literature or predicted using the iSafeRat QSAR model. Each value has been included as a supporting study 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 48h-EL50 was 9.6 mg test item/L for the typical composition of Sage Dalmatian Oil.

This toxicity study is acceptable and can be used for that endpoint.

 

Results Synopsis

Test Type: Calculation method

48h-EL50: 9.6 mg test material/L based on the typical composition

Description of key information

Two reliable studies were available to assess the acute toxicity of SAGE DALMATIAN OIL to aquatic invertebrates:

- one test performed according to OECD 202 guideline and under GLP compliance that gave a 48h-EL50 of 10.996 mg/L

-a calculation method that replaces an OECD 202 study and guideline for Testing of Chemicals No. 23 (i.e. WAF conditions) that gave a 48h-EL50 of 9.6 mg/L.

Since these two results are reliable, the geometric mean between these two results was used for that endpoint and gave a 48h-EL50 of 10.27 mg/L.

Based on the results of these two studies, Sage dalmatian oil would not be classified as acute 1 to aquatic organisms in accordance with the classification of the CLP.

Synopsis of the acute toxicity to aquatic invertebrates:

Test item: Sage dalmatian oil

48h-EL50 = 10.27 mg/L (geometric mean, key value)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

10.27 mg/L

Additional information

Sage Dalmatian Oil is a Natural Complex Substance (UVCB) with a well-defined composition. The WAF experimental study and the calculation method are based on typical compositions of the substance.

it appears that the calculation method gives an accurate prediction (error factor below 2 in comparison to the experimental study) although it is slightly more conservative than the experimental result.

Therefore, this calculation method can be considered as valid to be used alone for other ecotoxicological endpoints.