Reaction mass of [(2S,3R,4S,5S,6R)-6-[[(2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxymethyl]-3,4,5-trihydroxyoxan-2-yl] (1S,2R,4aS,6aR,6aR,6bR,8R,8aR,9R,10R,11R,12aR,14bS)-8,10,11-trihydroxy-9-(hydroxymethyl)-1,2,6a,6b,9,12a-hexamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylate and [(2S,3R,4S,5S,6R)-6-[[(2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxymethyl]-3,4,5-trihydroxyoxan-2-yl] (4aS,6aR,6aS,6bR,8R,8aR,9R,10R,11R,12aR,14bS)-8,10,11-trihydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylate

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:

Experimental start date 18 November 2020

Experimental completion date 24 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Reaction mass of Madecassoside and Terminoloside
IUPAC Name: Reaction mass of [(2S,3R,4S,5S,6R)-6-[[(2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxymethyl]-3,4,5-trihydroxyoxan-2-yl] (1S,2R,4aS,6aR,6aR,6bR,8R,8aR,9R,10R,11R,12aR,14bS)-8,10,11-trihydroxy-9-(hydroxymethyl)-1,2,6a,6b,9,12a-hexamtheyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylate and {(2S,3R,4S,5S,6R)-6-[[(2R,3R,4R,5S,6R)-3,4-dihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxymethyl]-3,4,5-trihydroxyoxan-2-yl] (4aS,6aR,6aS,6bR,8R,8aR,9R,10R,11R,12aR,14bS)-8,10,11-trihydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a,hexamethyl-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylate
Batch: 200409041286
Purity: 96%
Physical State/Appearance: White crystalline powder at 20 °C and at 1013hPa
Expiry Date: 12 May 2023
Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Verification of Test Concentrations
Samples were taken from the control and the 100 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours and stored frozen prior to analysis. A set of duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Range-Finding Test
A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions

Definitive Test
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1.0 L to give a 100 mg/L stock solution

Test organisms (species):

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

Details on test organisms:

Raphidocelis subcapitata is a freshwater unicellular alga, representative of primary producers found in natural waters and can therefore be considered as an important non-target organism in freshwater ecosystems.

The test was carried out using Raphidocelis subcapitata strain CCAP 278/4. Liquid cultures of Raphidocelis subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
Approximately 3 to 4 days before the start of the test, inoculum cultures of algae was set up at an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 C until the algal cell density was approximately 105 to 106 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

All test and control cultures were inspected microscopically at 72 hours

Test temperature:

Temperature was maintained at 24 ±1 ºC throughout the test.

pH:

The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.

The pH value of the control cultures was observed to increase from pH 7.8 at 0 hours to pH 8.6 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Salinity:

Not applicable as freshwater study

Nominal and measured concentrations:

Range-Finding Test
0.10, 1.0, 10 and 100 mg/L

3.4.2 Definitive Test
Based on the result of the range-finding test a "limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no effect on algal growth was observed.

Details on test conditions:

Experimental Design and Study Conduct
Range-Finding Test
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Raphidocelis subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.

The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration.

A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of the stock solutions was separately inoculated with algal suspension (2.7 mL) to give an initial nominal cell density of 5.00 x 103 cells/mL.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron incubator) at 24 ±1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were analyzed on the day of sampling. Duplicate samples were taken and stored frozen for further analysis if required.

Definitive Test
Based on the result of the range-finding test a "limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no effect on algal growth was observed.

Experimental Preparation
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1.0 L to give a 100 mg/L stock solution. An aliquot (1.0 L) of each of the stock solutions was inoculated with algal suspension (4.8 mL) to give an initial nominal cell density of 5.00 x 103 cells/mL.

The stock solution and the prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours (see Annex 4).

Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and 100 mg/L treatment groups.
The control group was maintained under identical conditions but not exposed to the test item.
Pre culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.04 x 106 cells per mL. Inoculation of 1.0 L of test medium with 4.8 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron incubator) at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Reference substance (positive control):

yes

Remarks:

A positive control test using potassium dichromate as the reference item was performed twice in a 12 month period to demonstrate satisfactory conditions of the test.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-finding Test
The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100 mg/L.
Based on this information a single test concentration of six replicates, of 100 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines, no effect on growth was observed.

Chemical analysis of the test preparations at 0 hours (see Annex 4) showed measured test concentrations to range from 0.11 to 100 mg/L (95% to 107% of nominal). Chemical analysis of the 72 hours showed measured test concentrations to range from 0.11 to 104 mg/L (103% to 108% of nominal) indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparation at 0 hours showed a measured test concentration of 91 mg/L. There was no significant change in the measured concentration at 72 hours and so the results are based on nominal test concentrations only.

Growth Data
From the data given in Table 2 and Table 4, it is clear that the growth rate (r) and yield (y) of Raphidocelis subcapitata (CCAP 278/4) were not affected by the presence of the test item at a nominal test concentration of 100 mg/L over the 72 Hour exposure period.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.
Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErC10 (0 to 72 hour) : >100 mg/L
ErC20 (0 to 72 hour) : >100 mg/L
ErC50 (0 to 72 hour) : >100 mg/L*

where ErCx is the test concentration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100 mg/L test group using a Student-t test incorporating Levene’s test on Variance Homogeneity and Shapiro-Wilk’s test on Normal Distribution. There were no statistically significant differences (P0.05), between the control and 100 mg/L test group and therefore the No Observed Effect Concentration based on growth rate was 100 mg/L. Correspondingly the Lowest Observed Effect Concentration (LOEC) based on growth rate could not be determined.

Inhibition of Yield
EyC10 (0 to 72 hour) : >100 mg/L
EyC20 (0 to 72 hour) : >100 mg/L
EyC50 (0 to 72 hour) : >100 mg/L*

Where EyCx is the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as in Section 6.2.3. There were no statistically significant differences (P0.05), between the control and 100 mg/L test group and therefore the NOEC based on yield was 100 mg/L. Correspondingly the LOEC based on yield could not be determined.

Results with reference substance (positive control):

A positive control (Covance study number YY61NS) used potassium dichromate as the reference item at concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L. The positive control was conducted between 20 January 2020 and 06 March 2020.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.2 mg/L; 95% confidence limits 1.1 to 1.3 mg/L
EyC50 (0 to 72 hour) : 0.51 mg/L; 95% confidence limits 0.45 to 0.58 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.125 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.25 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item*.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 195 after 72 hours.  This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Nominal cell density of control at 0 hours                 :           5.00 x 10³ cells per mL

Mean cell density of control at 72 hours                    :           9.73 x 10⁵ cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 5% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Test Item Solubility

At the start of the test, all control and test cultures were observed to be clear colorless solutions.  After the 72‑Hour test period all control and test cultures were observed to be green dispersions.

Cell Densities and Percentage Inhibition of Growth from the Range‑finding Test

Nominal Concentration (mg/L)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	5.49E+03	1.55E+06	-	-
	R2	5.51E+03	2.16E+06
	Mean	5.50E+03	1.85E+06
0.10	R1	5.72E+03	1.68E+06	0	0
	R2	5.10E+03	2.02E+06
	Mean	5.41E+03	1.85E+06
1.0	R1	6.19E+03	1.87E+06	4	11
	R2	6.10E+03	1.44E+06
	Mean	6.15E+03	1.66E+06
10	R1	4.84E+03	1.62E+06	4	12
	R2	6.95E+03	1.64E+06
	Mean	5.90E+03	1.63E+06
100	R1	6.25E+03	1.78E+06	4	10
	R2	5.75E+03	1.56E+06
	Mean	6.00E+03	1.67E+06

 

 

Table 2             Cell Densities and pH Values in the Definitive Test

Nominal Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 Hours	24 Hours	48 Hours	72 Hours	72 Hours
Control	R1	7.8	2.77E+04	1.51E+05	9.20E+05	8.6
	R2		2.98E+04	1.45E+05	9.62E+05
	R3		3.07E+04	1.69E+05	1.00E+06
	R4		2.80E+04	1.49E+05	9.06E+05
	R5		3.07E+04	1.63E+05	9.71E+05
	R6		3.33E+04	1.89E+05	1.07E+06
	Mean		3.00E+04	1.61E+05	9.73E+05
100	R1	7.7	3.29E+04	1.71E+05	1.16E+06	8.7
	R2		5.63E+04	1.70E+05	1.06E+06
	R3		4.03E+04	1.55E+05	1.03E+06
	R4		2.81E+04	1.70E+05	1.37E+06
	R5		3.09E+04	1.61E+05	1.07E+06
	R6		3.19E+04	1.78E+05	1.07E+06
	Mean		3.67E+04	1.68E+05	1.13E+06

 

Table 3             Daily Specific Growth Rates for the Control Cultures in the Definitive Test

Treatment		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 to 1	Day 1 to 2	Day 2 to 3
Control	R1	0.071	0.070	0.075
	R2	0.074	0.066	0.079
	R3	0.076	0.071	0.074
	R4	0.072	0.070	0.075
	R5	0.076	0.069	0.074
	R6	0.079	0.072	0.072
	Mean	0.075	0.070	0.075

 

Table 4             Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.072	-	9.15E+05	-
	R2	0.073		9.57E+05
	R3	0.074		9.98E+05
	R4	0.072		9.01E+05
	R5	0.073		9.66E+05
	R6	0.075		1.07E+06
	Mean	0.073		9.68E+05
	SD	0.001		6.10E+04
100	R1	0.076	[4]	1.16E+06
	R2	0.074	[1]	1.06E+06
	R3	0.074	[1]	1.02E+06
	R4	0.078	[7]	1.37E+06
	R5	0.075	[3]	1.07E+06
	R6	0.075	[3]	1.07E+06
	Mean	0.075	[3]	1.12E+06	[16]
	SD	0.002		1.27E+05

 

*          Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts                                 from 3 counts for each of the replicate flasks

R       =  Replicate

-        =  Not applicable

*            Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from                           3 counts for each of the replicate flasks

R    = Replicate

R    = Replicate

*          In accordance with the OECD test guideline only the mean value for yield for each test concentration is                              calculated

R       = Replicate

SD    =  Standard Deviation

[...]    =Increase growth compared to the controls

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated and based on the nominal test concentration gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) was 100 mg/L.

Executive summary:

1.1                   Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata).  The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) No 761/2009.

1.2                   Methods

Following a preliminary range-finding test, Raphidocelis subcapitata was exposed to an aqueous solution of the test item at a nominal concentration of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^® Multisizer Particle Counter.

1.3                   Results.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal (91% and 89% of nominal) and so the results are based on nominal test concentrations only.

Exposure of Raphidocelis subcapitata to the test item gave EC₅₀ values based on the nominal test concentrations of greater than 100 mg/L.  The No Observed Effect Concentration (NOEC) was 100 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Description of key information

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated and based on the nominal test concentration gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) was 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L