Lavender, Lavandula angustifolia, ext.

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 January 2017 to 08 February 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)

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

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 the Chemical Oxygen Demand (ST-COD) in the control medium and the WAFs. Regarding properties of the test item, COD 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. 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 5.5 L of test water (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 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).
The water hardness (286 mg.L-1 as CaCO3) of the batch of test water used to prepare the WAFs was slightly above the recommended value of 250 mg.L-1 (as CaCO3) given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control throughout the duration of the test.

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:

7.49 - 8.33
Guideline requirements: pH between 6.0-9.0, and not varying by more than 1.5 units

Dissolved oxygen:

7.24 - 9.19 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, 10; 18; 32; 56 and 100 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 1.0, 3.2; 10.0; 32.0; 100.0 mg.L-1 and to a control.
- Results used to determine the conditions for the definitive study: The per cent immobility was 0, 0, 0, 0, 10 and 100 (24 h) and 0, 0, 0, 0, 50 and 100 (48 h) at 0.0, 1.0, 3.2; 10.0; 32.0 and 100.0 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): 10, 18, 32, 56 and 100 mg.L-1.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

36.172 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

95% Confidence Limit (mg/L) = [30.648 - 42.666]

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

21.995 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 19.973 - 24.222 mg/L

Details on results:

After 24 hours of exposure, immobilisation rates were 0% at 10 and 18 mg.L-1 (loading), 50% at 32 mg.L-1 (loading), 80% at 56 mg.L-1 (loading) and 100% at 100 mg.L-1 (loading). After 48 hours of exposure, immobilisation rates were 5% at 10 mg.L-1 (loading), 10% at 18 mg.L-1 (loading) and 100% at 32, 56 and 100 mg.L-1 (loading).
The 24-hour EL50 including the 95% confidence interval using probit analysis was determined by the computer program ToxRat. At t=48h, an EL50 value was also determined by the software but due to lack of concentration/response data this value (21.286 mg.L-1) could not be valid. Indeed, the probit model assumes that the probability of immobilisation at any levels follows an integrated normal distribution, which was not the case in the present study. Bross (1950) showed that with small sample sizes, the Spearman-Karber point estimator gave a more accurate and precise calculation of median effective dose values than maximum likelihood even when assuming an underlying logistic distribution. Therefore, since the probit model was not appropriate in this study at t=48h, it was decided to use a non-parametric test (the trimmed Spearman-Karber method) for estimation of the EL50.

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 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. The 24-hour EL50 including the 95% confidence interval was determined using probit analysis. At t=48h, an EL50 value was also determined by the software ToxRat but due to lack of concentration/response data the value could not be considered as valid. Indeed, the probit model assumes that the probability of immobilisation at any levels follows an integrated normal distribution, which was not the case in the present study. Bross (1950) showed that with small sample sizes the Spearman-Karber point estimator gave a more accurate and precise calculation of median effective dose values than maximum likelihood even when assuming an underlying logistic distribution. Therefore, since the probit model was not appropriate in this study at t=48h, it was decided to use a non-parametric test (the trimmed Spearman-Karber method) for estimation of the EL50.

Analytical results

Concentration of dissolved organic material in the control and the WAFs was checked by analysis of the Chemical Oxygen Demand (ST-COD) 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 each exposure period (t=0h-t24h_Old and t=24h_Fresh -t=48h), within the ± 20% of the initial COD concentration values (in mg O₂.L^-1). 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: 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
10^a	1 2 3 4	5 5 5 5	0 0 0 0	0	1 0 0 0	5
18^a	1 2 3 4	5 5 5 5	0 0 0 0	0	0 1 1 0	10
32	1 2 3 4	5 5 5 5	3 5 1 1	50	5 5 5 5	100
56	1 2 3 4	5 5 5 5	4 4 4 4	80	5 5 5 5	100
100	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

^aAt t=48h, the swimming speed of mobile daphnids was slower than the one of the controls.

 

Water quality parameter values throughout the test:

The results of measurement of pH and oxygen concentrations remained within the limits required (pH: 6.0 to 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. However, it can be noted that the water hardness (286 mg.L^-1as CaCO₃) of the batch of test water used to prepare the WAFs was slightly above the recommended value of 250 mg.L^-1(as CaCO₃) given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control throughout the duration of the test.

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 as laid down in the study plan (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 concentrations, the 48-hour EL50 value was estimated to be 21.995 mg test item.L-1 (loading).

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 LAVENDER OIL POPULATION 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 preliminary range-finding test, 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 10, 18, 32, 56and 100mg.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 the Chemical Oxygen Demand (ST-COD) 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 CODconcentration values(in mg O₂.L^-1).After 48 hours of exposure, immobilisation rates were 5% at 10mg.L^-1(loading), 10% at 18mg.L^-1(loading) and 100% at 32, 56 and 100 mg.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	36.172	30.648 – 42.666
48	21.995*	19.973 - 24.222*

Statistical analyses were performed by the computer program ToxRat (8).

* Since the probit model was not appropriate at t=48h, the trimmed Spearman-Karber method was used for estimation of the EL₅₀includingthe 95% confidence interval.

Therefore, under the experimental conditions and based on nominal concentrations, the 48-hour EL50 value was estimated to be 21.995 mg test item.L-1 (loading).

Description of key information

A reliable study was available to assess the acute toxicity of the registered substance to aquatic invertebrates:

It is one test performed according to the OECD 202 guideline and under GLP compliance that gives a 48h-EL50 of 21.995 mg/L (loading)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

21.995 mg/L

Additional information

A study was performed according to the OECD Guideline 202 and with GLP compliance to assess the acute toxicity of the test item LAVENDER 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. Different qualities of the registered substance exist, and the test has been realised on one of the quality. The quality named "POPULATION" (also known as "FINE") has been selected because it is the most representative in Europe.

Following a preliminary range-finding test, the daphnids were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 10, 18, 32, 56 and 100 mg.L^-1 and to a control. The analytical results of this test showed that WAFs concentrations were overall stable during the test.

After 48 hours of exposure, immobilisation rates were 5% at 10mg.L^-1(loading), 10% at 18mg.L^-1(loading) and 100% at 32, 56 and 100mg.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	36.172	30.648 – 42.666
48	21.995*	19.973 - 24.222*

Statistical analyses were performed by the computer program ToxRat (8).

* Since the probit model was not appropriate at t=48h, the trimmed Spearman-Karber method was used for estimation of the EL₅₀includingthe 95% confidence interval.

This study comply with the requirements of the guideline. It is therefore considered as acceptable for that endpoint.