Silybum marianum, ext.

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:

05 - 08 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item, controls and vehicle.
Name: Extract of Chardon marie without support
Batch/Lot Number: VDO06205A
Description: Light yellow brown cloudy oil
Purity: Considered as 100%. No correction for purity of the Test Item was applied because this is an UVCB Test Item.
Expiry date: 13 July 2019
Storage condition: Controlled room temperature (15-25 °C, ≤ 70 RH%)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety.

Analytical monitoring:

yes

Details on sampling:

NPOC analysis
Alle glassware (sample tubes, beakers, flasks) were caregully washed with chromosulphuric acid and then by ultrapure water to remove organic contaminants.

Fore measurement of non-purgable organic organic content, the samples were acidified with 2% (v/v) amount from 1 M HCL. The CO2 released by acidification was removed by purging the solutions for 90 seconds with synthetic air (Sparge gas flow 80 ml/min).

50 ul of these acidified samples were injected for NPOC analysis. At least three injections were made from each solution.

Vehicle:

yes

Remarks:

OECD medium

Details on test solutions:

Formulation
Because the test item is poorly soluble in water, test solutions were prepared using a saturated solution method (water accommodated fraction, WAF) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
Saturated test item solutions (nominal loading rates of 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L) were individually prepared by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the study. These solutions were shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give the appropriate WAF solutions.
The test solutions were prepared and distributed into test vessels prior to introduction of algae.

Untreated Control:
Algal growth medium was inoculated with algal cells (without test item) and was examined in parallel to the test item concentrations.

Reference Control:
For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 19/009-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 22 – 25 January 2019.
The 72h ErC 50: 0.87 mg/L, (95 % confidence limits: 0.80 – 0.95 mg/L)
The 72h EbC 50: 0.62 mg/L, (95 % confidence limits: 0.57 – 0.68 mg/L)
The 72h EyC 50: 0.52 mg/L, (95 % confidence limits: 0.48 – 0.57 mg/L)

These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Dilution Water:
Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Culture conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

other: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 104 algal cells per mL test medium.

Post exposure observation period:

Not specified

Hardness:

Not specified

Test temperature:

Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.5 - 22.6 °C measured in the flask and between 21.3 and 23.4 °C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.36 –8.98 during the experiment.

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

The following nominal concentrations were tested: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rates WAFs.

Details on test conditions:

The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 104 algal cells per mL test medium.

The test was performed with three replicates per test concentration and six replicates in the control group. Volumes of 100 mL algal suspension per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.

Preliminary Range Finding Test
A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.

Test item concentrations in the Definitive Test
Because slight inhibition was observed at the highest concentration level (100 mg/L WAF) during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 2.0 and one control were tested in the main experiment.
The following nominal concentrations were tested: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rates WAFs.
The test item concentrations were analytically determined at the start and at the end of the experiment. Concentrations (with the exception of the lowest concentration of 6.25 mg/L nominal loading rate WAF at the start of the test and at the highest level of 100.0 mg/L nominal loading rate WAF at the end of the test) could not be measured during the experiment due to limitation of the analytical method used (see details in Appendix 4). Therefore, biological results are expressed in nominal loading rates WAFs (water accommodated fraction) based on OECD TG NO. 23 on poorly soluble and multi-constituent substances.

Observations
The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

ANALYTICAL MEASUREMENTS
Analytical measurement was performed at the applied test concentration levels and from the control at the beginning and at the end of the experiment.
The samples were analysed by NPOC method. The test item is a plant-extract UVCB, the water soluble fraction causing biological effects is unknown hence it is not possible to perform a more appropriate analytical method. For a UVCB, it is usually most appropriate to use the Loading Rate to express the concentration data.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

99.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAFs

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. limits 75.74 – 130.61 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAFs

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAFs

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

29.08 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAFs

Basis for effect:

other: yield

Remarks on result:

other: 95 % conf. limits 25.91 – 32.64 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAFs

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAFs

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

47.21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAFs

Basis for effect:

biomass

Remarks on result:

other: 95 % conf. limits 40.89 – 54.50 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAFs

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAFs

Basis for effect:

biomass

Details on results:

VALIDITY
The cell density in the control cultures increased by the factor of 73.50 within three days.
The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 13.78 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.01 %.
All validity criteria were met; therefore, the study can be considered as valid.

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
Thin cells were observed at the highest examined concentration of 100.0 mg/L nominal loading rate WAF 48 and 72 hours after start of the experiment.

AVERAGE SPECIFIC GROWTH RATES
The results of the statistical evaluation (based on Bonferroni t-Test; a=0.05) show that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value at the concentration range of 25.0 – 100.0 mg/L nominal loading rate WAF, accordingly the No Observed Effect Loading Rate (NOELR) was determined as 12.5 mg/L nominal loading rate WAF.
The 72 h ErL50 value was determined [by Probit analysis (TOXSTAT software)] as 99.46 mg/L (95 % confidence limits: 75.74 – 130.61 mg/L) nominal loading rate WAF.

AREAS UNDER THE GROWTH CURVES
The results of the statistical evaluation (based on Bonferroni t-Test; a=0.05) show that the 0-72 h areas were statistically significantly different from the untreated control value at the concentration range of 25.0 – 100.0 mg/L nominal loading rate WAF, accordingly the No Observed Effect Loading Rate (NOELR) was determined as 12.5 mg/L nominal loading rate WAF.
The 72 h EbL50 value was determined [by Probit analysis (TOXSTAT software)] as 47.21 mg/L (95 % confidence limits: 40.89 – 54.50 mg/L) nominal loading rate WAF.

YIELD
The results of the statistical evaluation (based on Bonferroni t-Test; a=0.05) show that the 0-72 h yield was statistically significantly different from the untreated control value at the tested concentration range of 25.0 – 100.0 mg/L nominal loading rate WAF, accordingly the No Observed Effect Loading Rate (NOELR) was determined as 12.5 mg/L nominal loading rate WAF.
The 72 h EyL50 value was determined [by Probit analysis (TOXSTAT software)] as 29.08 mg/L (95 % confidence limits: 25.91 – 32.64 mg/L) nominal loading rate WAF.

Results with reference substance (positive control):

The 72h ErC 50: 0.87 mg/L, (95 % confidence limits: 0.80 – 0.95 mg/L)
The 72h EbC 50: 0.62 mg/L, (95 % confidence limits: 0.57 – 0.68 mg/L)
The 72h EyC 50: 0.52 mg/L, (95 % confidence limits: 0.48 – 0.57 mg/L)

Reported statistics and error estimates:

Not specified

Growth Rates (m) and Percentage Inhibition ofmduring the Test Period

NominalConcentration [mg/L nominal loading rate WAF]		Growth rate (m) and % inhibition ofm
		0–24 h			0–48 h			0–72 h
		m		%		m		%		m		%
Control		0.0569		0.0		0.0596		0.0		0.0597		0.0
6.25		0.0569		0.0		0.0606^+		-1.7		0.0592		0.8
12.5		0.0609		-7.0		0.0598		-0.3		0.0592		0.8
25.0		0.0538		5.5		0.0578		3.0		0.0461*		22.8
50.0		0.0569		0.0		0.0544*		8.6		0.0379*		36.5
100.0		0.0538		5.5		0.0512*		14.1		0.0349*		41.5

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

⁺ : at this value the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

NominalConcentration [mg/L nominal loading rate WAF]	Area under the Growth Curves (A) and Percentage Inhibition of A
	0–24 h		0–48 h		0–72 h
	A	%	A	%	A	%
Control	36.0	0.0	270.0	0.0	1338.0	0.0
6.25	36.0	0.0	280.0	-3.7	1328.0	0.7
12.5	40.0	-11.1	280.0	-3.7	1320.0	1.3
25.0	32.0	11.1	244.0	9.6	744.0*	44.4
50.0	36.0	0.0	224.0	17.0	548.0*	59.0
100.0	32.0	11.1	192.0*	28.9	456.0*	65.9

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

Yield (Y) and Percentage Inhibition of Y during the Test Period

NominalConcentration [mg/L nominal loading rate WAF]	Yield (Y) and % inhibition of Y
	0–72 h
	Y	%
Control	72.5	0.0
6.25	70.0	3.4
12.5	70.0	3.4
25.0	26.7*	63.2
50.0	14.3*	80.2
100.0	11.3*	84.4

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

TEST CONDITIONS

pH-Values in the Test Media at the Start and End of the Test

NominalConcentration [mg/L nominal loading rate WAF]	pH-values
	Start	End
Control	7.56	8.83
		8.90
		8.95
		8.88
		8.79
		8.98
6.25	7.64	8.84
		8.90
		8.94
12.5	7.38	8.90
		8.73
		8.60
25.0	7.41	8.06
		8.06
		8.05
50.0	7.36	8.04
		8.03
		8.04
100.0	7.36	8.12
		7.98
		7.97

       

Temperature in the Climate Chamber and in the Test Media during the Test

Parameter		Exposure Time
		Day 0	Day 1	Day 2	Day 3
temperature (ºC) measured in flask		22.5	22.6	22.6	22.6
min / max temperature (ºC) of the climate chamber	max	23.4	23.3	23.4	23.3
	min	21.3	21.5	21.4	21.3

 

 CELL NUMBER

Cell Number (x 10⁴cell/mL) determined in the Main Experiment

 

NominalConcentration [mg/L nominal loading rate WAF]		Number of cells
		0 h	24 h	48 h	72 h
Control		1	4	18	75
		1	5	19	76
		1	5	16	75
		1	3	19	76
		1	4	16	69
		1	3	17	70
	Mean	1.00	4.00	17.50	73.50
	SD	0.0	0.9	1.4	3.1
6.25		1	4	19	70
		1	5	19	74
		1	3	17	69
	Mean	1.00	4.00	18.33	71.00
	SD	0.0	1.0	1.2	2.6
12.5		1	4	16	75
		1	5	18	70
		1	4	19	68
	Mean	1.00	4.33	17.67	71.00
	SD	0.0	0.6	1.5	3.6
25.0		1	3	16	28
		1	4	16	30
		1	4	16	25
	Mean	1.00	3.67	16.00	27.67
	SD	0.0	0.6	0.0	2.5
50.0		1	4	13	15
		1	5	13	15
		1	3	15	16
	Mean	1.00	4.00	13.67	15.33
	SD	0.0	1.0	1.2	0.6
100.0		1	3	12*	13*
		1	4	12*	12*
		1	4	11*	12*
	Mean	1.00	3.67	11.67	12.33
	SD	0.0	0.6	0.6	0.6

*   : Thin, deformed cells

  

Measured carbon contents and the calculated test item concentrations of the test with 98% confidence interval

Nominal loading rate WAF mg/L	Measured carbon content at the start (corrected by control	Calculated test item content at the start*	Measured carbon content at the end (corrected by control	Calculated test item content at the end*
6.25	BQL	-	1.20 +/- 0.9574	1.55 +/- 1.231
12.5	BQL	-	BQL	-
25	BQL	-	BQL	-
50	BQL	-	BQL	-
100	1.35 +/- 0.1076	1.73 +/- 0.1384	BQL	-

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

The effect of Extract of Chardon marie without support test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours.

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 25.0 – 100.0 mg/L nominal loading rate WAFs, therefore the overall NOEC was determined as 12.5 mg/L nominal loading rate WAF and LOEC was determined as 25.0 mg/L nominal loading rate WAF.

Executive summary:

The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours. Since slight inhibition was observed at the highest concentration level (100 mg/L nominal loading rate WAF) during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control were tested in the main experiment.

The nominal concentrations oftest item used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and100.0 mg/L nominal loading rate WAFs.

The test item concentrations were analytically determined at the start and at the end of the experiment. Concentrations (with the exception of the lowest concentration of 6.25 mg/L nominal loading rate WAF at the start of the test and at the highest level of 100.0 mg/L nominal loading rate WAF at the end of the test) could not be measured during the experiment due to limitation of the analytical method used. Therefore,biological results areexpressed innominal loading rates WAFs (water accommodated fraction) based on OECD TG NO. 23 on poorly soluble and multi-constituent substances.

The test design included three replicates at each test concentration and six replicates for the untreated controls.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (a= 0.05) by TOXSTAT software.

The E_rL₅₀, E_bL₅₀and E_yL₅₀values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software.

 

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 25.0 – 100.0 mg/L nominal loading rate WAFs,therefore the overall NOEC was determined as 12.5 mg/L nominal loading rate WAF and LOEC was determined as 25.0 mg/L nominal loading rate WAF. The biological results are summarised in the following table:

Influence of Extract of Chardon marie without support on the Growth of Pseudokirchneriella subcapitata

Parameter (0-72 h)	Growth rate (r) [mg/L]		Yield (y) [mg/L]	Biomass (b) [mg/L]
		Calculation based on the nominal concentrations (nominal loading rate WAFs)
EL50	99.46		29.08	47.21
95 % conf. limits	75.74 – 130.61		25.91 – 32.64	40.89 – 54.50
NOELR	12.5		12.5	12.5
LOELR	25.0		25.0	25.0

Description of key information

The effect of Extract of Chardon marie without support test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours.

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 25.0 – 100.0 mg/L nominal loading rate WAFs, therefore the overall NOEC was determined as 12.5 mg/L nominal loading rate WAF and LOEC was determined as 25.0 mg/L nominal loading rate WAF.

Key value for chemical safety assessment

EC50 for freshwater algae:

29.08 mg/L

EC10 or NOEC for freshwater algae:

12.5 mg/L

Additional information

The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours. Since slight inhibition was observed at the highest concentration level (100 mg/L nominal loading rate WAF) during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control were tested in the main experiment.

The nominal concentrations oftest item used in the main experimentwere: 6.25, 12.5, 25.0, 50.0 and100.0 mg/L nominal loading rate WAFs.

The test item concentrations were analytically determined at the start and at the end of the experiment. Concentrations (with the exception of the lowest concentration of 6.25 mg/L nominal loading rate WAF at the start of the test and at the highest level of 100.0 mg/L nominal loading rate WAF at the end of the test) could not be measured during the experiment due to limitation of the analytical method used. Therefore,biological results areexpressed innominal loading rates WAFs (water accommodated fraction) based on OECD TG NO. 23 on poorly soluble and multi-constituent substances.

The test design included three replicates at each test concentration and six replicates for the untreated controls.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (a= 0.05) by TOXSTAT software.

The E_rL₅₀, E_bL₅₀and E_yL₅₀values of the test item and their confidence limits were calculated using Probit analysisby TOXSTAT software.

 

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 25.0 – 100.0 mg/L nominal loading rate WAFs,therefore the overallNOEC was determined as 12.5 mg/L nominal loading rate WAF and LOEC was determined as 25.0 mg/L nominal loading rate WAF. The biological results are summarised in the following table:

Influence of Extract of Chardon marie without supporton the Growth of Pseudokirchneriella subcapitata

Parameter (0-72 h)	Growth rate (r) [mg/L]		Yield (y) [mg/L]	Biomass (b) [mg/L]
		Calculation based on the nominal concentrations (nominal loading rate WAFs)
EL50	99.46		29.08	47.21
95 % conf. limits	75.74 – 130.61		25.91 – 32.64	40.89 – 54.50
NOELR	12.5		12.5	12.5
LOELR	25.0		25.0	25.0