Concrete of Salvia sclarea L. (Lamiaceae) obtained from leaves, flowers and twigs by apolar hydrocarbon solvent extraction with or without purification by molecular distillation

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:

2017-05-29 to 2017-08-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline No. 201. All validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

signed on January 10, 2017

Specific details on test material used for the study:

Not applicable

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from the control and all test concentrations (from a replicate of each test concentration without algae dedicated exclusively to chemical analyses) at the start and the end of the test. 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 AND APPLICATION OF TEST SOLUTION
- Method: The study was carried out using WAFs (Water Accommodated Fractions). The WAFs were prepared under closed conditions and by slow-stirring. The mixing vessels were 1 L cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. A magnetic stirring bar was placed in each mixing vessel completely filled with test water (with a minimum of 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 screw caps after weighing. Each glass flask was placed in a water bath for 10 minutes at 50°C, followed by sonication for approx. 10 minutes. Based on experience on similar substances, these steps (heating and sonication) were essential to remove the paste fragments stuck to the glass of the flasks and to extract the soluble fraction of the test item as much 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 23 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 removed via the drain port. Then the WAFs were directly added into test flasks containing a fixed amount of inoculum (5.103 cells.mL-1 per vessel) that were immediately sealed after filling with a minimum of headspace. At the start of the test, the solution was observed to be clear and colourless.

- Controls: Test water without test substance but treated in the same way as the test substance solutions (WAFs).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 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.
- Stock culture: 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.
- Pre-culture: 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 x 10^4 cells/mL. 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

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

No data

Test temperature:

22.6-23.0 °C throughout the test (average value: 22.9 °C)

pH:

Start(t=0 h): 8.39 - 8.42
End (t=72 h): 8.16 - 10.13

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

no data

Nominal and measured concentrations:

Nominal concentrations: 10.0; 17.7; 31.6; 56.2 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace.
Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Initial cells density: An initial cell density of 5.10^3 cells.mL-1 using the exponentially growing pre-culture.
- Replicates: 6 controls and 3 replicates of each loading rate for counting. Moreover, a replicate of each treatment without algae was prepared for chemical analyses.

GROWTH MEDIUM
- Standard medium used: Yes; Original medium of OECD TG 201. Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water (for all treatments and inoculum suspension): 7 mL of NaHCO3 was added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg.L-1, according to the study plan.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: Continuous illumination
- Light intensity and quality: Continuous illumination with a light intensity of 4,440-8,880 lux and did not vary more than ± 15% from the average light intensity over the incubation area.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.
- Other:
pH: At the start and the end of the test in one vessel per concentration and the control (same vessel at t=0h and t=72h).
Temperature of Medium: Measured continuously in the growth chamber, over the study period.
Light Intensity: Light intensity was measured once (t=0 h) during the test at 5 positions distributed over the experimental area at the surface of the test media.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.78
- Results used to determine the conditions for the definitive study: Based on the results of a range-finding test, test solutions used in the definitive test were prepared to obtain the following loading rates: 10.0; 17.7; 31.6; 56.2 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

37.528 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: nd - 46.403 mg/L

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

41.195 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. limits: nd - nd

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

42.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: nd - 49.927 mg/L

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

43.513 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. limits: nd - nd

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

54.55 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 17.065 - 71.424 mg/L

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

48.318 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. limits: nd - nd

Remarks:

This value should be taken with caution

Results with reference substance (positive control):

On February 14, 2017 (most recent test), the 72h-EC50 was 1.438 mg.L-1 for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (expected 72h-EC50: 0.92 mg.L-1 to 1.46 mg.L-1).

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.

Table 6.1.5/1: pH-values during the final test

	Nominal concentration(mg test item.L^-1)*
	Control	10.0	17.7	31.6	56.2	100.0
Start t=0h	8.41	8.39	8.42	8.40	8.42	8.42
End t=72h	9.91	10.13	10.01	10.07	9.06	8.16

 * WAF prepared at the given loading rate.

Table 6.1.5/2: Algal cell densities during the final test (expressed as density of algal cells/mL x 10⁴)

	Replicate	Nominal concentration  (mg test item.L^-1)*
		Control	10.0	17.7	31.6	56.2	100.0
t=24h	1	3.6	7.2	5.2	5.6	3.6	1.6
	2	4.0	4.0	4.8	4.8	4.8	1.2
	3	4.4	2.0	3.2	3.2	2.4	1.2
	4	2.8
	5	3.2
	6	4.8
	Mean	3.8	4.4	4.4	4.5	3.6	1.3
	Std. Dev.	0.75	2.62	1.06	1.22	1.20	0.23
t=48h	1	16.0	20.8	21.6	16.8	3.6	0.8
	2	15.2	28.8	16.8	16.4	4.0	1.2
	3	16.4	20.4	19.6	16.8	1.6	1.2
	4	17.2
	5	13.6
	6	24.8
	Mean	17.2	23.3	19.3	16.7	3.1	1.1
	Std. Dev.	3.92	4.74	2.41	0.23	1.29	0.23
t=72h	1	44.0	120.0	80.0	87.5	15.2	0.8
	2	58.8	137.5	62.5	87.5	6.8	0.4
	3	62.8	100.0	110.0	77.5	0.8	0.8
	4	66.8
	5	60.0
	6	90.0
	Mean	63.7	119.2	84.2	84.2	7.6	0.7
	Std. Dev.	15.02	18.76	24.02	5.77	7.23	0.23

* WAF prepared at the given loading rate.

At test start 5000 algal cells/mL were incubated; 6 replicates of the controls and 3 replicates of each test loading rate.

Std. Dev.: standard deviation.

 

Validity criteria of the study

- Cell density in Controls: 128-fold increase within 72 hours. The specific growth rate was determined at 1.61 day^-1, so greater than 0.92 day^-1.

- Coefficient of Variation:

1. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 24.5%.

2. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 4.8%.

Thus the validity criteria were respected in this study.

 

Analytical results

Concentration of dissolved organic material in the control and the WAFs was checked by TOC analysis at the start and at the end of the test.

Due to the complex nature of the substance, it was difficult to well solubilize the substance in the water medium(heating, sonication, mixingwithout headspace…).

Even if the TOCanalysisindicated that organic compounds were found at low value in the loading rates, analytical resultsshowed thatWAFs concentrations were overall stablebetween the start and the end of the exposure period. Itshould be noted that aWAF 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, and since the test item was a UVCB substance, the results were based on thenominaltest loading rates.

 

Table 6.1.5/3: Concentrations of the test item in test water - Results of the determination of TOC analysis (mg.L^-1) - Final test.

Nominal concentration* (mg test item.L^-1)	Start (t=0h)	End (t=72h)
Control	0.37	0.99
10.0	1.53	2.58
17.7	1.78	3.05
31.6	4.22	5.86
56.2	7.79	9.74
100.0	12.9	16.4

* WAF prepared at the given loading rate.

Physico-chemical parameter values throughout the test

The results of measurement of pH are presented in Table 6.1.5/1. The temperature in the incubator was situated between 22.6 and 23.0°C throughout the test (average value: 22.9°C). The mean light intensity was 4827 lux (range: 4580-5110 lux). Thus, all test conditions remained within the ranges prescribed by the study plan (pH: not varying by more than 1.5 units at the end of the test in the control; temperature: 23°C ± 2°C, constant within 2°C; light intensity: 4440-8880 lux and shall not vary more than ± 15% from the average light intensity over the incubation area).

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on nominal concentrations, the 72-hour EL50 for the parameter growth rate was determined to be 54.550 mg.L-1. The 72-hour EL10 for growth rate was 37.528 mg L-1.

Executive summary:

A study was performed according to OECD Guideline 201 added by WAF protocol according to OECD 23 on complex substances, to assess the test item, CLARY SAGE CONCRETE for its ability to generate toxic effects in Pseudokirchneriella subcapitata, under static conditions. 

Following a preliminary range-finding test, algal cells were exposed toWater Accommodated Fractions (WAFs)of the test itemover a range of nominal loading values of10.0, 17.7, 31.6, 56.2 and 100.0mg.L^-1and to a control. The 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 control and the WAFs was checked by TOC analysisat the startand the end of the test.

 

Overall, the analytical results of this test showed that WAFs concentrations were stable throughout the experiment. 

 

Under the experimental conditions and based on nominal concentrations, the 72-hour EL50 for the parameter growth rate was determined to be 54.550 mg.L-1. The 72-hour EL10 for growth rate was 37.528 mg L-1.

Description of key information

Under the experimental conditions and based on nominal concentrations, the 72-hour EL50 for the parameter growth rate was determined to be 54.550 mg.L-1. The 72-hour EL10 for growth rate was 37.528 mg L-1.

Based on the result of this study, the substance CLARY SAGE CONCRETE would not be acutely toxic to aquatic organisms in accordance with the classification of the CLP.

Key value for chemical safety assessment

EC50 for freshwater algae:

54.55 mg/L

EC10 or NOEC for freshwater algae:

37.528 mg/L

Additional information

A reliable study was available to assess the test item, CLARY SAGE CONCRETE for its ability to generate toxic effects in Pseudokirchneriella subcapitata, under static conditions. 

Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the test itemover a range of nominal loading values of 10.0, 17.7, 31.6, 56.2 and 100.0mg.L^-1and to a control. The 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 control and the WAFs was checked by TOC analysis at the start and the end of the test.

Overall, the analytical results of this test showed that WAFs concentrations were stable throughout the experiment. 

This toxicity study was considered acceptable for that endpoint.