Lavender, Lavandula angustifolia, ext.

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-26 to 2018-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 203 and EU Method C.1 with GLP compliance. All validity criteria were fulfilled but a minor deviation was observed (concerning temperature values; see 3.2.). This deviation did not affect the integrity of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

minor deviation. See "Dissolved oxygen" section in the Endpoint Study Record.

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

yes

Remarks:

minor deviation. See "Dissolved oxygen" section in the Endpoint Study Record.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2017-01-10

Specific details on test material used for the study:

No additional information

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) and every 24 hours thereafter in old and new solutions. 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 OF STOCK AND TEST SOLUTIONS:
- Method: The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for fresh media at t=0h, t=24h, t=48h and t=72h) 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 5.5 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 after filling and introduction of fish with a minimum of headspace. The test solutions in test vessels were observed to be clear and colourless. 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):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST SYSTEM
- Common name: Zebra fish
- Source: Elevage de la Grande Rivière - B. et C. Romano - La Fond Garel – 69490 SAINT FORGEUX
- Sex: Male and female
- Reason for selection: The system has been selected as an internationally accepted species.

HOLDING
- Quarantine: At least 12 days after delivery under similar temperature and light conditions as used in the test.
- Holding and test water: Reconstituted water, as prescribed by the OECD Guideline 203.
Stock solutions:
a) CaCl2.2H2O: 11.76 g.L-1
b) MgSO4.7H2O: 4.93 g.L-1
c) NaHCO3: 2.59 g.L-1
d) KCl: 0.23 g.L-1
An aliquot (25 mL) of each solution (a to d) was added to each litre (final volume) of deionised water (conductivity < 10 µS.cm-1).
The pH of this solution was in the range of 6 to 8.5 and the water hardness was between 10 and 250 mg.L-1 (as CaCO3).

- Measurements: pH, nitrate and nitrite concentration, ammonia concentration, total water hardness and dissolved oxygen: once a week. Temperature: every day.
- Feeding: Daily with flaked food or live food (such as brine shrimp nauplii).
- Validity of batch: In the batch of fish which was used for the test, no mortality during the seven days prior to the start of the test was observed. Moreover, the average length and weight of the fish used during the final test was 2.98 cm and 0.253 g, respectively. Hence, this batch was considered to be valid.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

The water hardness was between 10 and 250 mg.L-1 (as CaCO3).

Test temperature:

23°C ± 2°C, constant within 2°C.

pH:

6.0-8.5, constant within 1 unit

Dissolved oxygen:

> 60% of air saturation

Salinity:

Not applicable

Conductivity:

Not mentionned

Nominal and measured concentrations:

Nominal concentrations : 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 1.80): 6, 10, 19, 33 and 60 mg.L-1.

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass aquaria, of 3.5 L capacity filled with minimum headspace (approximately 1 cm) and closed by placing a glass plate on the aquarium and sealed with Vaseline. Each test vessel was uniquely identified with study code, date of experimentation and treatment group.
- Aeration: No aeration of the test solutions occurred throughout the test.
- test type: A semi-static test was performed, with daily renewal of the test solutions and the control in closed test vessels, in order to maintain the dissolved oxygen concentration higher than 60 % of the air-saturation value throughout the test, and to prevent the dissipation of the test item from test solutions because of its volatility and the expected degradation of some constituents.
- No. of organisms per vessel: 7 fish per vessel filled with minimum headspace
- Biomass loading rate: loading of fish not exceeding 1.0 g.L-1

OTHER TEST CONDITIONS
- Adjustment of pH: The test was carried out without adjustment of the pH.
- Photoperiod: 16h light : 8 h dark
- Light intensity: no data

EFFECT PARAMETERS MEASURED:
- Mortality and other effects: Fish were inspected at 24, 48, 72 and 96 hours following the start of exposure. Dead fish recorded were removed when observed.
- pH and dissolved O2: Measured daily in all vessels with surviving fish.
- Temperature of medium: Measured daily in all vessels with surviving fish; temperature was measured continuously in the control vessel and in the highest loading rate with surviving fish.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.80
- Range finding study: yes
- Test concentrations: 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 1.80): 6, 10, 19, 33 and 60 mg.L-1.
- Results used to determine the conditions for the definitive study: The range-finding test was carried out using WAFs (Water Accommodated Fractions) of the test item over a range of nominal loading rate of 1, 10, 32 and 100 mg.L-1 and to a control. After 72h, the total mortality was: 0, 0, 0, 100 and 100% at 0 (control), 1, 10, 32 and 100 mg/L respectively.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

29.173 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL: 21.063 - 40.881 mg/L

Duration:

72 h

Dose descriptor:

LL50

Effect conc.:

31.435 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL: 22.652 - 44.480 mg/L

Duration:

48 h

Dose descriptor:

LL50

Effect conc.:

33.88 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL: 24.387 - 48.615 mg/L

Duration:

24 h

Dose descriptor:

LL50

Effect conc.:

44.399 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

mortality (fish)

Details on results:

Biological results:
After the 96 hours of exposure, mortality was 0% at 6 and 10 mg.L-1, 14.3% at 19 mg.L-1, 57.1% at 33 mg.L-1 and 100% at 60 mg test item.L-1. Therefore, the highest loading rate without effect was 10 mg.L-1 and the lowest loading rate with 100% mortality was 60 mg.L-1.
The 24, 48, 72 and 96-hour LL50s were calculated by the computer program ToxRat using Probit-analysis.

Analytical results:
Concentration of dissolved organic material in the control and the WAFs was checked by TOC analysis at the start (t=0h) and every 24 hours thereafter in old and new solutions. 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. Therefore, according to the results of the TOC analysis, and since the test item was a UVCB substance, the results were based on nominal test loading rates.

Water quality parameters and environmental conditions throughout the test:
The results of measurement of pH and oxygen concentrations (mg.L-1) remained within the ranges prescribed by the study plan (pH: 6.0-8.5, constant within approx. 1 unit; oxygen > 60% of air saturation). The temperature values measured during the study in test vessels were situated between 23 °C ± 2 °C, but were sometimes higher than the requirements, and more particularly in the controls with 2.7°C difference between the lowest and the highest temperature value. This minor deviation was considered not to affect the results of the test as no impact was observed on the controls throughout the duration of the test.

Results with reference substance (positive control):

On December 20, 2017 (most recent test), the 24h-LC50 was 195.554 mg.L-1. Hence, the sensitivity of this batch of Danio rerio was in agreement with internal historical data.

Sublethal observations / clinical signs:

Table 6.1.1/1: pH-values during the final test:

 

		Nominal concentration(mg test item.L^-1)*
		Control	6	10	19	33	60
Start t=0h		7.28	7.59	7.39	7.57	7.43	7.31
t=24h	Old	7.27	7.24	7.21	7.22	7.08	7.19
	Fresh	7.80	7.59	7.54	7.45	7.26
t=48h	Old	7.21	7.32	7.41	7.32	7.28
	Fresh	7.01	7.70	7.56	7.54	7.38
t=72h	Old	7.31	7.27	7.32	7.20	7.10
	Fresh	7.70	7.77	7.67	7.53	7.47
End t=96h		7.38	7.15	7.25	7.11	7.17

* WAF prepared at the given loading rate.

Grey boxes: no further analysis was performed for concentrations in which all fish were dead.

Table 6.1.1/2: Dissolved oxygen concentrations (mg.L^-1) during the final test:

		Nominal concentration(mg test item.L^-1)*
		Control	6	10	19	33	60
Start t=0h		8.83	8.71	8.64	8.66	8.65	8.70
t=24h	Old	7.53	7.10	7.09	6.42	5.85	7.48
	Fresh	8.47	8.45	8.50	8.49	8.39
t=48h	Old	7.87	7.38	7.51	7.24	7.10
	Fresh	8.53	8.43	8.49	8.24	8.26
t=72h	Old	7.80	7.38	7.32	7.05	6.72
	Fresh	8.39	8.59	8.48	8.53	8.56
End t=96h		8.27	7.92	7.42	7.24	7.44

* WAF prepared at the given loading rate.

Grey boxes: no further analysis was performed for concentrations in which all fish were dead.

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

Table 6.1.1/3. Temperatures (°C) measured during the final test:

 

		Nominal concentration(mg test item.L^-1)*
		Control	6	10	19	33	60
Start t=0h		23.3	23.6	24.0	23.2	22.7	22.4
t=24h	Old	24.6 Min 24.5 Max 24.8	24.6	24.7	24.3	23.9	24.4 Min 23.7 Max 24.8
	Fresh	22.1	23.1	23.8	22.9	22.7
t=48h	Old	23.0 Min 22.3 Max 23.6	24.6	24.9	24.5	24.4 Min 23.1 Max 24.6
	Fresh	22.4	23.8	24.0	23.7	23.6
t=72h	Old	23.2 Min 22.3 Max 23.7	24.4	24.7	24.5	24.5 Min 23.1 Max 24.6
	Fresh	22.5	23.2	23.5	23.0	22.4
End t=96h		23.8 Min 22.2 Max 24.1	24.8	25.0	24.8	24.9 Min 22.4 Max 25.0

* WAF prepared at the given loading rate.

Grey boxes: no further analysis was performed for concentrations in which all fish were dead.

Temperature was measured continuously in the control and in the highest loading rate with surviving fish in order to obtain the values of temperature fluctuations throughout the test; Min: lowest temperature recorded; Max: highest temperature recorded.

 

 

Table 6.1.1/4. Incidence of mortality and total mortality during the final test:

 

	Nominal concentration(mg test item.L^-1)*
Nominal Cumulative Mortality	Control M     S	6 M     S	10 M     S	19 M     S	33 M     S	60 M     S
t=24h	0      0	0      0	0      0	0      1	0      2	7^a     0
24h Total Mortality (%)	0	0	0	0	0	100
t=48h	0      0	0      0	0      0	1      0	2      1	7    0
48h Total Mortality (%)	0	0	0	14.3	28.6	100
t=72h	0      0	0      0	0      0	1      0	3      1	7    0
72h Total Mortality (%)	0	0	0	14.3	42.9	100
t=96h	0      0	0      0	0      0	1      6^b	4      3^c	7    0
96h Total Mortality (%)	0	0	0	14.3	57.1	100

* WAF prepared at the given loading rate.

M: Number of dead fish; S: Number of surviving fish but with sublethal effects

^aAt t=0h, sublethal effects were rapidly observed (less than 1 hour) after the fish were introduced into test vessels. All fish were dead at t=4h and removed from the test vessel.

^bFish with pronounced sublethal effects (weakening, slow swimming, some with loss of colour).

^cThe surviving fish were very weak.

The initial number of fish in each test vessel was 7.

Validity criteria of the study:

- Controls: No mortality was observed in the control at the end of the test.

- Dissolved [0₂]: The dissolved oxygen concentration was at least 60% of the air saturation value throughout the test.

Thus the present study can be considered valid.

Validity criteria fulfilled:

yes

Conclusions:

The toxic effect of test item Huile essentielle de lavande fine to the zebrafish (Danio rerio) was investigated in a closed semi-static test. Under the experimental conditions and based on nominal loading rates, the 96-hour LL50 value estimated was 29.173 mg.L-1.

Executive summary:

This study was performed to assess the acute toxicity of the test item Huile essentielle de lavande fine to the zebrafish Danio rerio. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 203, "Fish Acute Toxicity Test", referenced as Method C.1 of Commission Regulation No. 440/2008 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23). The criterion measured was the LL₅₀ (Median Lethal Loading rate), a statistically derived loading rate which is expected to cause death in 50% of test animals within a period of 96 hours.

Following a preliminary range-finding test, groups of seven fish were exposed toWater Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 6, 10, 19, 33 and 60 mg/L in aquaria closed over the study period with a minimum of headspace. The total test period was 96 hours and test solutions were renewed on a daily basis. The mortality of the fish was determined by visual observation after 24, 48, 72 and 96 hours.Concentration of dissolved organic material in the control and the WAFs was checked by analysis of Total Organic Carbon (TOC) at the start of the test and every24 hours thereafter in old and new solutions.

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. Since the test item was a UVCB substance, the results were based on nominal test loading rates. After the 96 hours of exposure, mortality was 0% at 6 and 10 mg:L, 14.3% at 19mg:L, 57.1% at 33mg/L and 100% at 60 mg test item/L.

The LL₅₀ values at each of the observation times were as follows:

Time (h)	LL50(mg test item.L^-1)	95% Confidence limits (mg test item.L^-1)
24	44.399	n.d.
48	33.880	24.387 – 48.615
72	31.435	22.652 – 44.480
96	29.173	21.063 – 40.881

n.d.: not determined due to mathematical reasons or inappropriate data

Statistical analyses were performed by the computer program ToxRat.

Under the experimental conditions andbased on nominal loading rates, the 96-hour LL₅₀ value estimated was 29.173 mg/L.

Description of key information

The toxic effect of test item Huile essentielle de lavande fine to the zebrafish (Danio rerio) was investigated in a closed semi-static test. Under the experimental conditions and based on nominal loading rates, the 96-hour LL50 value estimated was 29.173 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

29.173 mg/L

Additional information

For that endpoint, an experimental study was available on the registered substance. This study was performed to assess the acute toxicity of the test item Huile essentielle de lavande fineto the zebrafish Danio rerio. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 203, "Fish Acute Toxicity Test", referenced as Method C.1 of Commission Regulation No. 440/2008 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Following a preliminary range-finding test, groups of seven fish were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of6, 10, 19, 33 and 60 mg/Lin aquaria closed over the study period with a minimum of headspace. The total test period was 96 hours and test solutions were renewed on a daily basis. The mortality of the fish was determined by visual observation after 24, 48, 72 and 96 hours. Concentration of dissolved organic material in the control and the WAFs was checked by analysis of Total Organic Carbon (TOC) at the start of the test and every 24 hours thereafter in old and new solutions.

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 initialTOCconcentrations values. Since the test item was a UVCB substance, the results were based onnominal test loading rates. After the 96 hours of exposure, mortality was 0% at 6 and 10 mg/L, 14.3% at 19mg/L, 57.1% at 33 mg/Land 100% at 60 mg test item/L.

Besides one minor deviation that was considered not to affect the integrity of the study, all validity criteria were fulfilled. It can be considered that the requirements of the test guidelines were respected and this study was therefore acceptable for that endpoint.