Concrete of Rosa Damascena (Rosaceae) obtained from fresh flowers by extraction with an apolar solvent

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:

2017-02-07 to 2017-02-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Read-across from an analogue substance.
The analogue substance Turkish Rose Absolute and the registered substance Turkish Rose Concrete have the same botanical origin: Rosa Damascena (Rosaceae). They are both obtained from fresh rose flowers. They follow the same production pattern. They both have the same main constituents and in similar concentrations, regarding their respective SIP. Based on these similarities, both substances are not expected to show different ecotoxicological behaviour. Thereby, results from the experimental OECD 202 study assessing the toxicity of the analogue substance Turkish Rose Absolute on the aquatic invertebrate Daphnia magna can be used as a read accross in the present dossier.

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 10.0 mg/L Fresh: 2 replicates
- T0h Abiotic 41.0 mg/L Fresh: 2 replicates
- T0h Abiotic 105.0 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 10.0 mg/L Old: 2 replicates
- T24h Abiotic 41.0 mg/L Old: 2 replicates
- T24h Abiotic 105.0 mg/L Old: 2 replicates

- T24h Biotic Control Old: 1 replicate
- T24h Biotic 10.0 mg/L Old: 2 replicates
- T24h Biotic 41.0 mg/L Old: 2 replicates
- T24h Biotic 105.0 mg/L Old: 2 replicates

- T24h Abiotic Control Fresh: 1 replicate
- T24h Abiotic 10.0 mg/L Fresh: 2 replicates
- T24h Abiotic 41.0 mg/L Fresh: 2 replicates
- T24h Abiotic 105.0 mg/L Fresh: 2 replicates

Schedule analysis at final time:
- T48h Abiotic Control Old: 1 replicate
- T48h Abiotic 10.0 mg/L Old: 2 replicates
- T48h Abiotic 41.0 mg/L Old: 2 replicates
- T48h Abiotic 105.0 mg/L Old: 2 replicates

- T48h biotic Control Old: 1 replicate
- T48h biotic 10.0 mg/L Old: 2 replicates
- T48h biotic 41.0 mg/L Old: 2 replicates
- T48h biotic 105.0 mg/L Old: 2 replicates

The measured concentrations of the “ROSE TURKISH ABSOLUTE” should not vary more than ± 20 % of nominal or initial measured concentration during the test.

Vehicle:

no

Details on test solutions:

TEST ITEM PREAPARATION
The test item was not soluble in the test medium, 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
This study was conducted on six concentration levels: 10.0 – 16.0 – 25.6 – 41.0 – 65.6 – 105.0 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 10.0 mg/L: 8 replicates (biotic) (including 4 test tubes for chemical analysis)
Concentration 16.0 mg/L: 4 replicates (biotic)
Concentration 25.6 mg/L: 4 replicates (biotic)
Concentration 41.0 mg/L: 8 replicates (biotic) (including 4 test tubes for chemical analysis)
Concentration 65.6 mg/L: 4 replicates (biotic)
Concentration 105.0 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:

260 mg/L of CaCO3

Test temperature:

19.9 - 20.1°C

pH:

7.0 - 7.5

Dissolved oxygen:

9.0 - 9.1 mg/L

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentrations: Control; 10.0, 16.0, 25.6, 41.0, 65.6 and 105.0 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
- 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; 0 and 60% of immobilized daphnids and 0; 0; 5 and 100 mg/L respectively at 24 and 48h.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

48.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 43.8 - 54.8 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

29.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 25.6 - 32.5 mg/L

Details on results:

See "any other information on results incl. tables"
The EC50 24h and 48h was calculated with the scientific software Minitab 17, with probits analysis.

Results with reference substance (positive control):

The last toxic response obtained on the reference item K2Cr2O7 (February 16, 2017) gives an EC50-24h of 1.11 mg/L (1.01 – 1.22), 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:

None

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%)
10.0	39	0 (0%)	0 (0%)
16.0	20	0 (0%)	0 (0%)
25.6	20	1 (5%)	9 (45%)
41.0	40	12 (30%)	33 (83%)
65.6	20	15 (75%)	20 (100%)
105.0	40	40 (100%)	40 (100%)

No sign of stress was observed on the mobile daphnids after 48 hours of exposure to the highest concentration.

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 10.0 mg/L	4.5	4.5	0.0
	4.0	4.3	+ 7.5
Test item Abiotic 41.0 mg/L	16.0	15.9	- 0.6
	17.3	17.8	+ 2.9
Test item Abiotic105.0 mg/L	39.8	39.5	- 0.8

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 10.0 mg/L	4.5	4.5	0.0
	4.0	4.1	+ 2.5
Test item Biotic 41.0 mg/L	16.0	15.9	- 0.6
	17.3	16.5	- 4.6
Test item Biotic105.0 mg/L

NA: non applicable

 

For all concentrations, the test item « ROSE TURKISH ABSOLUTE» 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 and the second renewal. 

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 (8.7 mg/L)

All the validity criteria were successful.

Validity criteria fulfilled:

yes

Conclusions:

The results obtained for the test item “ROSE TURKISH ABSOLUTE” on the toxicological endpoint “immobilisation” give an EC50-48h = 29.1 mg/L, based on nominal test item concentration.

Executive summary:

The aim of this study was to assess the effects of the test item “ROSE TURKISH ABSOLUTE”on the daphnids Daphnia magna, according to the OECD 202 guideline, in semi-static conditions. The study was performed under 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, 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).

According to chemical analyses, the test item 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 therefore based on nominal concentrations.

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 (8.7 mg/L)

 

All the validity criteria were successful.

Daphnids immobilisation results:

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%)
10.0	39	0 (0%)	0 (0%)
16.0	20	0 (0%)	0 (0%)
25.6	20	1 (5%)	9 (45%)
41.0	40	12 (30%)	33 (83%)
65.6	20	15 (75%)	20 (100%)
105.0	40	40 (100%)	40 (100%)

The results obtained for the test item “ROSE TURKISH ABSOLUTE” on the toxicological endpoint “immobilisation” give an EC50-48h = 29.1 mg/L, based on nominal test item concentration.

Description of key information

Based on a read-across from an experimental GLP study performed on the analogue substance Turkish rose absolute according to the OECD 202 guideline, the following result has been extrapolated to the registered substance:

-       48h-EC50 = 29.1 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

29.1 mg/L

Additional information

For that endpoint, data on the registered substance was not available. Therefore, in order to assess the acute toxicity of the registered substance to aquatic invertebrates, the results from an experimental OECD 202 study performed on the analogue substance Turkish rose absolute have been used.

In this study, the toxic effect of the test item Turkish rose absolute to the freshwater invertebrate Daphnia magna was investigated in a 48h-closed semi-static test using Water Accommodated Fractions. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23). The effect was based on mobility.

The results obtained for the test item “ROSE TURKISH ABSOLUTE” on the toxicological endpoint “immobilisation” gave an EC50-48h = 29.1 mg/L, based on nominal test item concentration.

The study respected the requirements of the guideline and the validity criteria were successful. This study was therefore considered acceptable for that endpoint and the read-across justification is provided in the iuclid study record.