Concrete of Cistus ladaniferus (Cistaceae) obtained from stems and leaves by organic solvents extraction

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2015-06-29 to 2016-02-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This substance is a natural extract. Natural extract can be gum, resinoid, concrete or absolute. This natural extract belongs to the group of Natural Complex Substances (NCS): UVCB sub-type 3, where the source is biological, and the process is refinement (ECHA Guidance on Identification and naming of substances under REACH, version 2.1 – May 2017, Section 4.3.1 and EFEO/IFRA Guidelines on substance identification and sameness of natural complex substances (NCS) under REACH and CLP, version of August 5, 2015).
The source substance (labdanum gum) and the registered substance are obtained from the same botanical source: Cistus ladaniferus (Cistaceae). The rawest extract is a resin obtained from shrubs Cistus ladanifer. The name of that first extract is Labdanum gum. Labdanum gum contains all the constituents available from the extraction of Cistus ladaniferus (Cistaceae). The other Cistus ladaniferus (Cistaceae) extracts are obtained from the source substance Labdanum gum. Therefore, the compositions, and then the expected behaviour are covered by Labdanum gum data. Therefore, we consider as reliable the read across from Labdanum gum dossier to this dossier.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

referenced as Method C.3 of Commission Regulation No. 761/2009

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Remarks:

2015-03-05

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

PREPARATION OF WAFs AND APPLICATION OF TEST SOLUTION
The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the WAFs. The volume of the mixing vessel was approximately 1L. A magnetic stirring bar was placed in each mixing vessel completely filled with test water (without headspace). The loading rates of the test item were weighed in glass flasks (approximate volume: 100 mL) filled with minimum headspace with test water (from the mixing vessel) and were immediately sealed with screwcaps after weighing. Each glass flask was placed in a water bath for 10-15 minutes at 50°C (following recommendations of the sponsor), followed by sonication for 10 minutes. This step was essential to remove the gum pellets stuck to the glass of the flasks and to extract a maximum of soluble fraction of the test item as possible. Then the mixing vessels were carefully filled with the contents of the glass flasks and thereafter were closed immediately. The 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 at room temperature, the WAFs were allowed to stand for 1 hour before use and were extracted via the drain port. The first 100 mL were discarded. Then the WAFs were directly added into flasks containing a fixed amount of inoculum to obtain a cell density of 5.103 cells.mL-1 per vessel. At the start of the test, the solution was observed to be clear and colourless to slightly yellow at the two tested loading rates. The test was carried out without adjustment of the pH.
Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 100 and 1000 mg test item.L-1 and to a control.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata, CCAP 278/4
- Source (laboratory, culture collection): Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 -75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions according to the test guidelines.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (=test water) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.

ACCLIMATION
2 to 4 days before the start of the test, cells from the algal stock culture were inoculated in test water at a cell density of 1.104 cells.mL-1. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Remarks:

Original medium of OECD TG 201

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

No data.

Test temperature:

23°C ± 2°C

pH:

Not varying by more than 1.5 units at the end of the test in the control : 8.11-8.41 (start) - 10-26-10.73(end)

Dissolved oxygen:

No data.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

100 and 1000 mg.L-1 (nominal loading rates).

Details on test conditions:

TEST SYSTEM
Test vessel:
100 mL, all-glass closed flasks with ground glass stopper, filled with 50 mL of test medium.
Each test vessel was uniquely identified with study code, replicate number, date of experimentation and concentration.

Replicates:
6 replicates for the control and per loading rates. Chemical analyses were taken from additional replicates (with algae): - one additional series for sampling for analysis of test concentrations at t=0h - and another for sampling for analysis of test concentrations at t=72h. Volume of 50 mL of algal suspension per replicate

Initial cells density: 5 000 cells.mL-1 per vessel

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
The stock solutions 1 and 3 were sterilised by autoclaving (120 °C, 15 min), then stored in the dark at 4°C. The stock solutions 2 and 4 were sterilised by membrane filtration (mean pore diameter 0.2 μm). The growth medium was prepared by adding an appropriate volume of the stock solutions 1-4 to water.
To 500 mL of sterilised water were added: - 10 mL of stock solution 1
- 1 mL of stock solution 2
- 1 mL of stock solution 3
- 1 mL of stock solution 4
Then, the mixture was made up to 1000 mL with sterilised water. The pH of this solution was approximately in the range of 8.1 ± 0.2.
Stock solution 1: macronutrients
NH4Cl 1.5 g.L-1
MgCl2·6H2O 1.2 g.L-1
CaCl2·2H2O 1.8 g.L-1
MgSO4·7H2O 1.5 g.L-1
KH2PO4 0.16 g.L-1
Stock solution 2: iron
FeCl3·6H2O 64 mg.L-1
Na2EDTA·2H2O 100 mg.L-1
Stock solution 3: trace elements
H3BO3 185 mg.L-1
MnCl2·4H2O 415 mg.L-1
ZnCl2 3 mg.L-1
CoCl2·6H2O 1.5 mg.L-1
CuCl2·2H2O 0.01 mg.L-1
Na2MoO4·2H2O 7 mg.L-1
Stock solution 4: bicarbonate
NaHCO3 50 g.L-1


OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity and quality:4 440-8 880 lux


EFFECT PARAMETERS MEASURED:
Cell numbers were counted daily by microscope using a counting chamber.

TEST CONCENTRATIONS
Limit test at 100 and 1000 mg/L (nominal loading rates) according to results obtained during range-finding test (No significant inhibition of the growth rate was recorded for all test concentrations; 1, 10, 100, 1000 mg/L).

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rates of test item

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rates of test item

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rates of test item

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rates of test item

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rates of test item

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rates of test item

Basis for effect:

other: Yield

Details on results:

Based on the results of a range-finding test, a limit test was performed to demonstrate that the test item had no effect on Pseudokirchneriella subcapitata under the experimental conditions at the loading rates of 100 and 1000 mg test item.L-1.
After 24, 48 and 72 hours of exposure no inhibition of algal growth relative to control values was observed at nominal WAFs concentrations of 100 and 1000 mg.L-1 (identical growth curves). Thus, ErLx and EyLx values could not be determined due to the absence of effect. The 72-hour NOELR for the parameters growth rate and yield was determined to be at least 1000 mg test item.L-1.

Results with reference substance (positive control):

No data.

Reported statistics and error estimates:

None

Range-finding test results:

In the range-finding test, algal cells were exposed to the loading rates of 1, 10, 100 and 1000 mg.L^-1 (loading) and to a control. The test system was maintained in a temperature controlled cabinet (23°C ± 2°C) with continuous illumination for a period of 72 hours.

Algal cell densities during the range-finding test (expressed as density of algal cells.mL-1x10⁴).

Replicate		Nominal concentration (mg test item.L^-1 )*
		Control	1	10	100	1000
t=24h	1	2.4	3.2	2.8	2.8	4.4
	2	3.2	3.2	4.4	3.2	2.8
	3	5.2	2.8	3.2	4.8	4.4
	Mean	3.6	3.1	3.5	3.6	3.9
t=48h	1	17.2	19.6	20.8	20.8	18.8
	2	16.8	16.4	20	18.4	16.8
	3	19.6	20.4	19.2	18.8	17.2
	Mean	17.9	18.8	20	19.3	17.6
t=72h	1	50.4	49.2	48	42.8	42.8
	2	47.6	43.2	40.8	47.2	44
	3	45.2	48	43.6	42.4	43.6
	Mean	47.7	46.8	44.1	44.1	43.5

Final test:

pH-values during the Final Test

	Nominal concentration (mg test item.L^-1)*
	Control	100	1000
Start t=0h	8.41	8.13	8.11
End t=72h	10.26	10.73	10.57

Algal cell densities during the final test (expressed as density of algal cells.mL^-1x10⁴)

	Nominal concentration  (mg test item.L^-1)*
	Control	100	1000
t=24h	1	4.8	3.6	4.8
	2	4.4	4.8	3.2
	3	2.8	4.8	3.6
	4	3.2	3.6	2.8
	5	4.4	3.2	4.4
	6	2.8	2.8	4
	Mean	3.7	3.8	3.8
	Std. Dev.	0.9	0.83	0.75
t=48h	1	28.8	27.6	26.4
	2	26	31.6	26.4
	3	25.6	23.2	30
	4	24.4	27.6	27.2
	5	25.2	24	27.6
	6	25.6	26	23.2
	Mean	25.9	26.7	26.8
	Std. Dev.	1.51	3.02	2.21
t=72h	1	64.8	66	66.8
	2	63.6	68.4	67.2
	3	65.6	64	67.6
	4	67.6	67.6	65.6
	5	64.8	65.6	66.4
	6	67.6	66.4	66.8
	Mean	65.7	66.3	66.7
	Std. Dev.	1.63	1.55	0.69

Validity criteria of the study

Cell density in Controls:

131-fold increase within 72 hours

Coefficient of Variation:

-The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 10%.

- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 0.5%.

Thus the validity criteria were respected in this study.

Analytical results

Concentration of dissolved organic material in the WAFs was checked by analysis of the Chemical Oxygen Demand (ST-COD) in the control medium and the WAFs at the start and the end of the test. Although every effort was made to extract and solubilize the soluble fraction of the test item in test water (heating, sonication, mixing without headspace …), analytical results of this test indicate that few organic compounds were found in WAFs. However, these results showed that WAFs concentrations were overall stable throughout the experiment (by subtracting the control value from the one of the WAFs). It should be noted that increases in COD values at t=72h were due to algal development.

Summary of Analytical Results:

COD (mg O2.L^-1)
Nominal concentration* (mg test item.L^-1)	Start (t=0h)	End (t=72h)
Control	<LOQ	33
100	18	48
1000	22	48

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on nominal loading rates of test item, the 72-hour NOELR for the parameters growth rate and yield was determined to be at least 1000 mg test item.L-1. The 72h-LOELR and 72h-EL50 were found to be higher than 1000 mg test item.L-1, for both parameters (growth rate and yield).

Executive summary:

A study was performed to assess the test item LABDANUM GUM for its ability to generate toxic effects in Pseudokirchneriella subcapitata. The method followed was designed to be

compliant with the OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 761/2009 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 100 and 1000 mg test item.L^-1 and to a control. The potential inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Concentration of dissolved organic material in the WAFs was checked by analysis of the Chemical Oxygen Demand (COD) in the control medium and the WAFs at the start and at the end of the test. These chemical analyses were performed only to provide an indication of the concentration of dissolved organic material in the WAFs and their stability.

Analytical results showed that few organic compounds were found in WAFs, although every effort was made to extract and solubilise the soluble fraction of the test item in test water (heating, sonication, mixing without headspace …).

After 24, 48 and 72 hours of exposure, no inhibition of algal growth relative to control values was observed in any of the test loading rates. Thus, ErLx and EyLx values could not be determined due to the absence of effect at the tested loading rates. Therefore, under the experimental conditions and based on nominal loading rates of test item, the 72-hour NOELR for the parameters growth rate and yield was determined to be at least 1000 mg test item.L-1. The 72h-LOELR and 72h-EL50 were found to be higher than 1000 mg test item.L-1 under the test conditions, for both parameters (growth rate and yield).

Besides one minor deviation (concerning some pH values) that did not affect the integrity of the study, this study respected the requirements of the guideline and the validity criteria have been fulfilled. Therefore, the present study is considered acceptable for that endpoint.

Results synopsis:

72-hour NOELR > = 1000 mg test item.L^-1 (based on growth rate and yield)

72-hour EL50 > 1000 mg test item.L^-1 (based on growth rate and yield)

72-hour LOELR > 1000 mg test item.L^-1 (based on growth rate and yield)

Description of key information

Based on a read-across from an experimental GLP study performed on the analogue substance Labdanum gum according to the OECD 201 guideline, the following results have been extrapolated to the registered substance:

-       72h-EL50 > 1000 mg/L (based on growth rate)

-       72h-NOELR >= 1000 mg/L (based on growth rate)

Key value for chemical safety assessment

EC50 for freshwater algae:

1 000 mg/L

EC10 or NOEC for freshwater algae:

1 000 mg/L

Additional information

For that endpoint, a study on the registered substance was not available. Therefore, in order to assess the toxicity of the registered substance on freshwater algae, the results from an experimental OECD 201 study performed on the analogue substance Labdanum gum have been used.

In this study, the toxic effect of the test item LABDANUM GUM to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a static limit test using Water Accommodated Fractions. The method followed was designed to be compliant with the OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 761/2009 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 100 and 1000 mg test item/L and to a control. The potential inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Concentration of dissolved organic material in the WAFs was checked by analysis of the Chemical Oxygen Demand (COD) in the control medium and the WAFs at the start and at the end of the test. These chemical analyses were performed only to provide an indication of the concentration of dissolved organic material in the WAFs and their stability.

Analytical results showed that few organic compounds were found in WAFs, although every effort was made to extract and solubilise the soluble fraction of the test item in test water (heating, sonication, mixing without headspace …).

After 24, 48 and 72 hours of exposure, no inhibition of algal growth relative to control values was observed in any of the test loading rates. Thus, ErLx and EyLx values could not be determined due to the absence of effect at the tested loading rates. Therefore, under the test conditions and based on nominal loading rates of test item, the 72-hour NOELR for the parameters growth rate and yield was determined to be at least 1000 mg test item.L-1. The 72h-LOELR and 72h-EL50 were found to be higher than 1000 mg test item.L-1 under the test conditions, for both parameters (growth rate and yield).

Besides one minor deviation (concerning some pH values) that did not affect the integrity of the study, the validity criteria were fulfilled and the study respected the requirements of the guideline. This study was therefore considered acceptable for that endpoint and the read-across justification is provided in the iuclid study record.