Essential oil of Cistus ladaniferus L (Cistaceae) obtained from stems and leaves by distillation

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:

2016-11-22 to 2016-12-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to the OECD 202 guideline. All validity criteria were successful.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

14 January 2016

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

In order to verify the initial concentrations and maintenance of the exposure concentrations of the test item during the ecotoxicological testing, chemical analyses in daphnids test solutions were performed:
- In fresh solutions at the beginning of the test (T = 0h) and after renewal (T=24h)
- In old solutions (T = 24h and T= 48h)
- for the control (i.e. daphnids test medium filtered) and at the lowest, the highest concentrations and a concentration around the expected EC50 concentration
The test tube were pooled (two by two) for chemical analyses, corresponding to one replicate.

Schedule analysis at initial time:
- T0h Abiotic Control Fresh: 1 replicate
- T0h Abiotic 5.3 mg/L Fresh: 2 replicates
- T0h Abiotic 30.8 mg/L Fresh: 2 replicates
- T0h Abiotic 100.0 mg/L Fresh: 2 replicates
All the “Fresh” solutions were analysed taken directly from the starting solutions without daphnids.

Schedule analysis at 24h:
- T24h Abiotic Control Old: 1 replicate
- T24h Abiotic 5.3 mg/L Old: 2 replicates
- T24h Abiotic 30.8 mg/L Old: 2 replicates
- T24h Abiotic 100.0 mg/L Old: 2 replicates

- T24h Biotic Control Old: 1 replicate
- T24h Biotic 5.3 mg/L Old: 2 replicates
- T24h Biotic 30.8 mg/L Old: 2 replicates
- T24h Biotic 100.0 mg/L Old: 2 replicates

- T24h Abiotic Control Fresh: 1 replicate
- T24h Abiotic 5.3 mg/L Fresh: 2 replicates
- T24h Abiotic 30.8 mg/L Fresh: 2 replicates
- T24h Abiotic 100.0 mg/L Fresh: 2 replicates

Schedule analysis at final time:
- T48h Abiotic Control Old: 1 replicate
- T48h Abiotic 5.3 mg/L Old: 2 replicates
- T48h Abiotic 30.8 mg/L Old: 2 replicates
- T48h Abiotic 100.0 mg/L Old: 2 replicates

- T48h biotic Control Old: 1 replicate
- T48h biotic 5.3 mg/L Old: 2 replicates
- T48h biotic 30.8 mg/L Old: 2 replicates
- T48h biotic 100.0 mg/L Old: 2 replicates

The measured concentrations of the “CISTUS ORGANIC OIL” should not vary more than ± 20 % of nominal or initial measured concentration during the test.

Vehicle:

no

Details on test solutions:

TEST ITEM PREPARATION
According to the study monitor, the test item was not soluble in the test medium (see composition in § 9.2.2), a special solubilisation protocol was carried out before the experimentation, according to the OECD n°23 guidance document on aquatic toxicity testing of difficult substances and mixtures.
This method consisted in stirring each test item nominal concentrations in test medium during 24 hours, in the dark and 20 ± 2°C.
All the test solutions (six concentrations) were then filtered (cellulose acetate with a mesh size of 0.45 µm) to remove suspended particles: solutions obtained were named WAF (Water Accommodated Fraction).
The test was carried out under semi static conditions, which means that the WAF solutions were renewed at 24h.

TEST SOLUTIONS
According to the results of the preliminary test, this study was conducted at six concentration levels: 5.3 - 9.5 – 17.1 – 30.8 – 55.6 – 100.0 mg/L.
The test will contain a minimum of 44 glass test tubes as follow:
Control: 8 replicates (biotic), (including 4 test tubes for chemical analysis)
Concentration 5.3 mg/L: 8 replicates (biotic) (including 4 test tubes for chemical analysis)
Concentration 9.5 mg/L: 4 replicates (biotic)
Concentration 17.1 mg/L: 4 replicates (biotic)
Concentration 30.8 mg/L: 8 replicates (biotic) (including 4 test tubes for chemical analysis)
Concentration 55.6 mg/L: 4 replicates (biotic)
Concentration 100.0 mg/L: 8 replicates (biotic) (including 4 test tubes for chemical analysis)

Each glass test tube will contain 10 mL of test solution and 5 daphnids (biotic); they were covered in order to avoid evaporation and dust deposition. The test tube were pooled (two by two) for chemical analyses. Some abiotic solutions (without daphnids) were stored in the same conditions (test conditions) for analyses.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Daphnia magna Straus, clone number 5, from at least the third generation (i.e. the third brood) obtained with a non-cyclic parthenogenesis reproduction.
The daphnids breeding was carried out in closed bottles placed in a climatic chamber free from any toxic vapour and in medium M4.
Daphnids used in this experimentation were previously filtered in order to obtain animals from a size between 560 µm and 800 µm (corresponding to daphnids less than 24 hours old).
- Feeding during test: None

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:

250 mg/L of CaCO3

Test temperature:

19.7 - 20.1°C

pH:

7.7 - 8.0

Dissolved oxygen:

8.6 - 9.2 mg/L

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentrations: Control; 5.3; 9.5; 17.1; 30.8; 55.6 and 100 mg/L

Details on test conditions:

TEST DETAILS
- Test vessel: glass test tube
- Type: closed
- Each glass test tube contained 10 mL of test solution and 5 daphnids (biotic); they were covered in order to avoid evaporation and dust deposition. The test tubes were pooled (two by two) for chemical analyses.
- Renewal rate of test solution: Renewal of test solutions at 24h
- No. of organisms per vessel: 5
- Test medium: The medium M4 ELENDT was prepared from concentrated stock solutions in ultrapure water as described in the guideline.
- Light intensity: Darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Observations of the daphnid’s immobility and behaviour were done after 24 and 48 hours in control and exposed groups.
- Any sign of stress observed on the daphnids after 48 hours of exposure to the test item were reported.
- pH and dissolved oxygen were measured before and after renewal of solutions (including the controls).

RANGE-FINDING STUDY
- Test concentrations: control, 5, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: 0; 0; 5 and 100% of immobilized daphnids at 48h at 0; 5; 10 and 100 mg/L respectively. Based on the results of the preliminary test, it has been proposed to use the following nominal concentrations for the study: 0; 5.3; 9.5; 17.1; 30.8; 55.6 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

94.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

63.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No sign of stress was observed on the mobile daphnids after 48 hours of exposure to the highest concentration.
The EC50 24h and 48h was calculated with the scientific software Minitab 17, with probits analysis. Nethertheless, the raw data could not permit to calculate confidence limits (p=0.95).
The results obtained for the test item “CISTUS ORGANIC OIL” on the toxicological endpoint “immobilisation” based on nominal test item concentration give:
- EC50-24h = 94.2 mg/L
- EC50-48h = 63.2 mg/L
As the confidence interval can not be given and according to the OECD 202 guideline and the §26, we can approximate the EC50 48h with the geometric mean between the highest concentration causing no immobility (= 30.8 mg/L) and the lowest concentration producing 100 per cent immobility (=100.0 mg/L):
--> EC50-48h = 55.5 mg/L

Results with reference substance (positive control):

The last toxic response obtained on the reference item K2Cr2O7 (November 11, 2016) gives an EC50-24h of 1.16 mg/L (1.06 – 1.28), indicating that the daphnids sensitivity is correct (within the accepted interval of 0.6 – 2.1 mg/L fixed by the International Standard ISO 6341 December 2012 as mentioned in the OECD 202 Guideline) and in accordance with historical data obtained by the laboratory.

Reported statistics and error estimates:

See "details on results"

Table 6.1.3/1: Daphnids immobilisation

Raw data (daphnids immobilisation in cumulative number for each observation time 24 and 48 hours) are presented hereafter:

Nominal test item concentrations  in mg/L	Number of daphnids exposed at the start	Immobilised daphnids (%)
		at 24h	at 48h
Control	40	0 (0%)	0 (0%)
5.3	40	0 (0%)	0 (0%)
9.5	20	0 (0%)	0 (0%)
17.1	20	0 (0%)	0 (0%)
30.8	41	0 (0%)	0 (0%)
55.6	20	0 (0%)	1 (5%)
100.0	43	32 (74%)	43 (100%)

Table 6.1.3/2: Chemical analysis of daphnids solutions

The table below summarizes the results achieved and the variation between initial measurement and after 24 hours of exposure.

 

Nominal test item concentration	At initial time (T0h) (FRESH)	After 24 hours of exposure (OLD)	Variation (%)
	At 24h after renewal (FRESH)	At 48h final time (OLD)
	mg NPOC/L
Test item Abiotic 5.3 mg/L	3.4	3.3	-2.9
	< 2	< 2	NA
Test item Abiotic 30.8 mg/L	5.7	6.8	+19.3
	7.5	7.2	-4.0
Test item Abiotic100 mg/L	16.3	18.9	+16.0
	18.4	16.1	-12.5

NA: non applicable

 

Nominal test item concentration	At initial time (T0h) (FRESH)	After 24 hours of exposure (OLD)	Variation (%)
	At 24h after renewal (FRESH)	At 48h final time (OLD)
	mg NPOC/L
Test item Biotic 5.3 mg/L	3.4	3.3	-2.9
	< 2	< 2	NA
Test item Biotic 30.8 mg/L	5.7	6.7	+17.5
	7.5	6.9	-8.0
Test item Biotic100 mg/L	16.3	17.1	+4.9
	18.4	14.8	-19.6

NA: non applicable

 

At the nominal concentration of 5.3 mg/L, the test item « CISTUS ORGANIC OIL» was considered stable under test conditions because the measured concentrations of the test item in Biotic or Abiotic solutions did not vary more than ± 20 % of initial measured or nominal concentration during the first renewal, but the variation was not calculable during the second renewal (T24h to T48h) because the results were lower than the LOQ (2 mg/L).

 

At the nominal concentration of 30.8 and 100 mg/L, the test item « CISTUS ORGANIC OIL» was considered stable under test conditions because the measured concentrations of the test item in Biotic or Abiotic solutions did not vary more than ± 20 % of initial measured or nominal concentration during the test.

Table 6.1.3/3: Validity criteria conformity

	Values for OECD 202	Validity criteria for the test
Immobilised daphnids in the control	=< 10 %	Ok (0%)
Dissolved Oxygen concentration at the end of the test	>= 3 mg/L	Ok (8.2 mg/L)

All the validity criteria were successful.

Validity criteria fulfilled:

yes

Conclusions:

The results obtained for the test item “CISTUS ORGANIC OIL” on the toxicological endpoint “immobilisation” give an EC50-48h = 63.2 mg/L, based on nominal test item concentration.

Executive summary:

The aim of this study was to assess the effects of the test item “CISTUS ORGANIC OIL”on the daphnids Daphnia magna, according to the OECD 202 guideline, in semi-static condition during 48 hours and with GLP compliance. The concentrations which immobilise 50% of the daphnids exposed to the test item after 24 and 48 hours, reported as EC₅₀-24h and EC₅₀-48h were evaluated, relative to the tested concentrations.

As the test item was not soluble in the test medium, according to the sponsor, a special solubilisation protocol was carried out before the experimentation. This method consisted in stirring each test item nominal concentrations in test medium during 24 hours. The test solution was then filtered through a membrane filter to remove suspended particles: solutions obtained are named WAF (Water Accommodated Fraction).

The test item « CISTUS ORGANIC OIL» was considered stable under test conditions because the measured concentrations of the test item in Biotic or Abiotic solutions did not vary more than ± 20 % of initial measured or nominal concentration during the test.

The values are based on nominal test item“CISTUS ORGANIC OIL”concentration.

Validity criteria conformity:

	Values for OECD 202	Validity criteria for the test
Immobilised daphnids in control	<10%	Yes (0%)
Dissolved Oxygen concentration	>3 mg/L	Yes (8.2 mg/L)

 

All the validity criteria were successful.

Daphnids immobilisation

Nominal test item concentrations in mg/L	Number of daphnids exposed at the start	Immobilised daphnids (%)
		at 24h	at 48h
Control	40	0 (0%)	0 (0%)
5.3	40	0 (0%)	0 (0%)
9.5	20	0 (0%)	0 (0%)
17.1	20	0 (0%)	0 (0%)
30.8	41	0 (0%)	0 (0%)
55.6	20	0 (0%)	1 (5%)
100.0	43	32 (74%)	43 (100%)

The results with geometric mean will not be retained because the result is more dependent of the conditions of the study (concentration range) than Probits analysis.

The results obtained for the test item “CISTUS ORGANIC OIL” on the toxicological endpoint “immobilisation” based on nominal test item concentration give an EC50-48h = 63.2 mg/L.

Description of key information

The experimental GLP study performed according to the OECD 202 guideline in order to determine the toxic effect of the registered substance to the aquatic invertebrate Daphnia magna gives a 48h-EC50 of 63.2 mg/L (nominal).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

63.2 mg/L

Additional information

For that endpoint, an experimental study performed on the registered substance Cistud Organic Oil was available. This study intended to assess the effects of the test item “CISTUS ORGANIC OIL” on Daphnia magna, according to the OECD 202 guideline, in semi-static condition during 48 hours and with GLP compliance.

The concentrations which immobilise 50% of the daphnids exposed to the test item after 24 and 48 hours, reported as EC₅₀-24h and EC₅₀-48h were evaluated, relative to the tested concentrations.

As the test item was not soluble in the test medium, according to the sponsor, a special solubilisation protocol was carried out before the experimentation. This method consisted in stirring each test item nominal concentrations in test medium during 24 hours. The test solution was then filtered through a membrane filter to remove suspended particles: solutions obtained are named WAF (Water Accommodated Fraction). Based on chemical analyses, the results are expressed in nominal concentrations.

All the validity criteria were successful. This study is considered acceptable for that endpoint.