Reaction mass of [3R-(3α,3aβ,6α,7β,8aα)]-octahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-6-ol and [3R-(3α,3aβ,6α,7β,8aα)]-octahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl acetate

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

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

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2000)

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: June 2013; signature: February 2016

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The saturated solution and four dilution levels out of the saturated solution was tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution. The concentrations were based on the results of a preliminary range finding test (non-GLP, Erlenmeyer flasks, with headspace in OECD medium). All concentration levels and the control were analytically verifiedthe test item via GC-MS at the start (0 hours) with algae and at the end of the exposure (72 hours) with and without algae. This resulted in analytically measured geometric mean measured test item concentrations of the sum of two components of 0.177, 0.347, 0.796, 1.69 and 3.29 mg/L, respectively.- Sampling method: 6 mL each of all test item concentrations and the control were filled into a 15 mL centrifuge tube containing approximately 1 g sodium chloride. 1 mL cyclohexane was added and the samples were shaken properly for 1 min. The cyclohexane phase was removed with a pipette and transferred into a 4 mL vial containing a hint of sodium sulphate for drying the samples. The samples were diluted with cyclohexane including 50µL gamma-decalactone (20 mg/L in cyclohexane) as internal standard prior to analysis. The full dilution steps for samples is documented in the full study report.- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C before preparation, if necessary. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM- Common name: Pseudokirchneriella subcapitata- Strain: HINDÁK, SAG 61.81- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to to 2567 – 5130 lux for 24 hours per day.ACCLIMATION- Acclimation period: No. However, a three days old preculture, prepared in dilution water, was used as inoculum.- Culturing media and conditions (same as test or not): No. Culture Medium: Nutrient medium Z according to LÜTTGE et al. (1994) Botanica Acta, Journal of the German Botanical Society, No. 3 Volume 107 page 111-186 (June 1994), THIEME-VERLAG.Dilution water: OECD medium was used: (mg/L) - NH4Cl: 15 ; MgCl2.6 H2O: 12 ; CaCl2.2 H2O: 18 ; MgSO4.7H2O: 15 ; KH2PO4: 1.6 ; FeCl3.6H2O: 0.064 ; Na2EDTA.2H2O: 0.1 ; H3BO3: 0.185 ; MnCl2.4H2O: 0.415 ; ZnCl2: 3x10-3 ; Na2MoO4.2H2O: 7x10-3 ; CoCl2.6H2O: 1.5x10-3 ; CuCl2.2H2O: 1x10-5 ; NaHCO3: 50 ; pH 8.1 +/- 0.2. This medium has a nominal hardness of 0.24 mmol Ca+Mg/L.- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Ca+Mg: 0.24 mmol/L

Test temperature:

Nominal range: 21 - 24 °C, controlled at ± 2°C - measured room temperature values were min: 21.5 ; max 22.5 and mean 22.0 °C

pH:

0 hours: pH 7.7 ± 0.2 and pH 8.11 (controls); 72 hours: pH 8.33 - 9.50 (definitive test concentrations) and pH 9.42 (controls). pH did not vary more than 1.5 units in the control.

Nominal and measured concentrations:

Preliminary test: 0 (control), 1.0, 10.0 and 100.0 % as a nominal loading of saturated solutionFinal test: Solutions containing: 0 (control), 6.25, 12.5, 25.0, 50.0, 100.0 % as a nominal loading of a saturated solution (dilution factor 2; nominal)See table 2 for nominal and measured concentrations during the course of the test.

Details on test conditions:

TEST SYSTEM- Test vessel: Glass. Sterile Erlenmeyer flasks- Type: Closed - Static.- Material, size, headspace, fill volume: volume: 250 mL, sealed with cotton wool plugs. Test volume 100 mL- Aeration: Vessel shaken continuously. Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm- Initial cells density: nominal: 5 x 10^3 - 10^4 and current: 6305 cells/mL- Control end cells density: Mean (of replicates after 72 hours) 2186223 cells/mL (or ca. x 347 increase in cell density)- No. of vessels per concentration (replicates): 3 replicates of each test concentration.- No. of vessels per control (replicates): 6 replicates of the control- No. of vessels per vehicle control (replicates): Not applicable.GROWTH MEDIUM- Standard medium used: Yes. OECD Medium as indicated in OECD TG 201.TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: Prepared according to guidelines (see 'Details on test organisms' field for more details on composition).- Culture medium different from test medium: Yes. (see 'Details on test organisms' field)- Intervals of water quality measurement: Start and end of the test period.OTHER TEST CONDITIONS- Sterile test conditions: No.- Adjustment of pH: No.- Photoperiod: 24 hours ; continuous - Light intensity and quality: Fluorescent tubes, OSRAM L 36 W/865, cool daylight; approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1- Salinity (for marine algae): Not applicable.EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :- Determination of cell concentrations: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found in the preliminary range finding test at the saturated solution.- Other: Initial cell density: Microscopic evaluation of the cells was carried out daily. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation of algae cells.TEST CONCENTRATIONS- Spacing factor for test concentrations: 2 (nominal) In definitive test justified from the results of the range finding study.- Justification for using less concentrations than requested by guideline: Not applicable.- Range finding study: Yes.- Test concentrations: Two replicates per concentration and four for the control were exposed to dilutions representing 0 (control), 1.0, 10.0, 100 % in a saturated solution as a nominal loading in a non-GLP preliminary test.- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Geometric mean measured test item concentration of the sum of the two components

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 2.40 - 2.66 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.07 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Geometric mean measured test item concentration of the sum of the two components

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 0.984 - 1.16 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Geometric mean measured test item concentration of the sum of the two components

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.886 - 1.11 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 0.179 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

Geometric mean measured test item concentration of the sum of the two components

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.347 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Geometric mean measured test item concentration of the sum of the two components

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): Yes. (> or = 16-fold increase was seen: actual: 347-fold)- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start of the incubation period and at test end revealed no morphological abnormalities in the test item concentration or control.- Unusual cell shape: No.- Colour differences: None.- Flocculation: Not reported.- Adherence to test vessels: None reported.- Aggregation of algal cells: No.- Other:- Any stimulation of growth found in any treatment: No.- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Final test: None reported.- Effect concentrations exceeding solubility of substance in test medium: Not reported. Effect concentrations based on loading rates (WAFs).

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.- EC50: The EC50 for growth rate reduction (ERC50: 0-72h) was (with head-space) 0.460 mg/L with a 95% confidence interval ranging from 0.443 to 0.481 mg/L. The equivalent EC50 for yield inhibition (EYC50: 0-72h) was 0.317 mg/L with a 95% confidence interval ranging from 0.208 to 0.347 mg/L. The results with headspace and without headspace were within the test facility SOPs (historic values).- Other: The sensitivity of the test system was in agreement with the historical data.

Reported statistics and error estimates:

The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. The following statistical tests were conducted:1. Growth rate and yield: Shapiro-Wilk's test on normal distribution was done with a significance level 0.05.Growth rate and Yield: Normality check was passed (p > 0.01).2. Growth rate and yield: equal variance test was done with a significance level 0.05.Variance homogeneity check was passed (p > 0.01).3. Dunnet's Test: Growth rate: and Yield: Full details of the statistical analysis is tabulated and presented in the full study report.The P values for Dunnett's and Duncan's tests are currently unavailable except for reporting that the P's are greater or less than the critical values of .05 and .0Growth Rate: P<0.050 for all exposures versus control except 62.5 mg/L and 125 mg/L ("Do Not Test" result; according to the program a result of Do Not Test should be treated as if there is no significant difference between the means. even though one may appear to exist). The program states significant differences for the nominal test item concentrations 253, 506 and 1010 mg/L compared to the control. Therefore, the NOEC is 126 mg/L.Yield: P<0.050for all exposures versus control except 0.126 g/L. The program states significant differences for the nominal test item concentrations 62.5, 253, 0.506 and 1010 g/L compared to the control. A mean inhibition of yield < 13% is considered to be not biologically significant. Therefore, the NOEC is considered to be 126 mg/L.Note: all effect levels are based on measured concentrations during the course of the study. Specifically, geometric mean measured concentrations of the sum of two constituents.

Table 1. Results of the Preliminary Range Finding Test (non GLP, 0 - 72 hours)

Saturated Solution [%]	Growth Rate Inhibition [%]	Yield Inhibition [%]
100	100	100
10	4	20
1	3	18

Measured concentrations for constituent 2 were determined during the course of the RF study and are reported in the full study report.

 

Table 2. Percentage reduction in growth rate and inhibition of yield in the definitive test

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-)

Nominal test item concentration [% sat. sol.]	Geometric mean measured test item concentration of the sum of the two components [mg/L]	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
		No.	[d-1]		[%]	[cells/mL]		[%]
100	3.29	1		0.63	68		35263	98
		2		0.67	66		40424	98
		3		0.59	70		31099	99
		Mean	(+)	0.63	68	(+)	35595	98
50.0	1.69	1		1.41	28		426058	80
		2		1.41	27		430625	80
		3		1.47	24		516492	76
		Mean	(+)	1.43	27	(+)	457725	79
25.0	0.796	1		1.80	7		1410199	35
		2		1.85	5		1627206	25
		3		1.79	8		1348012	38
		Mean	(+)	1.82	7	(-)	1461806	33
12.5	0.347	1		1.91	2		1943046	11
		2		1.93	1		2028293	7
		3		1.91	2		1941863	11
		Mean	(-)	1.92	2	(-)	1971067	10
6.25	0.177	1		1.90	2		1905328	13
		2		1.92	2		1978510	9
		3		1.89	3		1797304	18
		Mean	(-)	1.90	2	(+)	1893714	13
Control		1		1.92			1996777
		2		1.97			2308671
		3		1.95			2206849
		4		1.92			1992886
		5		1.99			2460051
		6		1.94			2114273
		Mean		1.95			2179918

Validity criteria fulfilled:

yes

Conclusions:

The test substance 72h-EC50 (growth rate reduction) was 2.53 (C.I. 2.40 - 2.66) mg/L based on geometric measured concentrations of two constituents. The NOEC was 0.347 mg/L based on geometric measured concentrations of two constituents.

Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines in accordance with GLP. The aim of the study was the determination of NOEC, LOEC, EC10, EC20- and EC50-values of growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6305 cells/mL. A saturated solution with a nominal loading 1 mL test item/L was prepared once with demineralized water four days prior to the start of the exposure. Five concentrations were tested in a geometrical series with a dilution factor of 2: 6.25, 12.5, 25.0, 50.0, 100% of the saturated solution which corresponds to nominal test item concentrations (based on mean relative density of the mixture of 1.01) of 0.0632, 0.126, 0.253, 0.506, 1.01 g/L. The corresponding geometric mean measured concentration of the sum of the two components of the test item was 0.179, 0.347, 0.796, 1.69, 3.29 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. No Tyndall effect was observed in the saturated solution. The measured concentrations of the test item at the start were in the range of 0.273 – 3.97 mg/L. At test end, the test item concentrations were in the range of 42 - 69% of the initial values of all concentrations. Since the test item contains two constituents and the biological effects cannot be attributed to one of these constituents, all effect values given are based on the nominal test item concentrations and the geometric mean measured concentrations of the sum of the two components. The EC50 for growth rate reduction (72h-ErC50) based on geometric mean measured concentration of two constituents was 2.53 (C.I. 2.40-2.66) mg/L. The ErC10 was 1.00 (C.I. 0.886 – 1.11) mg/L. The EC50 for inhibition of yield (72h-EyC50) based geometric mean measured concentration of two constituents was 1.07 (C.I. 0.984-1.16) mg/L. The EyC10 was < 0.179 mg/L. The corresponding NOEC was 0.347 mg/L, respectively.

Description of key information

ErC50 (algae; growth rate) = 2.53 (C.I. 2.40 - 2.66) mg/L, GMM of two constituents, 72hour-freshwater, OECD TG 201, 2016
ErC10 (algae; growth rate)= 1.00 mg/L (C.I. 0.886 - 1.11), GMM of two constituents; 72 hour-freshwater, OECD TG 201, 2016
NOEC (algae; growth rate) = 0.347 mg/L, GMM of two constituents; 72 hour-freshwater, OECD TG 201, 2016

Key value for chemical safety assessment

EC50 for freshwater algae:

2.53 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L

Additional information

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines in accordance with GLP. The aim of the study was the determination of NOEC, LOEC, EC10, EC20- and EC50-values of growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6305 cells/mL. A saturated solution with a nominal loading 1 mL test item/L was prepared once with demineralized water four days prior to the start of the exposure. Five concentrations were tested in a geometrical series with a dilution factor of 2: 6.25, 12.5, 25.0, 50.0, 100% of the saturated solution which corresponds to nominal test item concentrations (based on mean relative density of the mixture of 1.01) of 0.0632, 0.126, 0.253, 0.506, 1.01 g/L. The corresponding geometric mean measured concentration of the sum of the two components of the test item was 0.179, 0.347, 0.796, 1.69, 3.29 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. No Tyndall effect was observed in the saturated solution. The measured concentrations of the test item at the start were in the range of 0.273 – 3.97 mg/L. At test end, the test item concentrations were in the range of 42 - 69% of the initial values of all concentrations. Since the test item contains two constituents and the biological effects cannot be attributed to one of these constituents, all effect values given are based on the nominal test item concentrations and the geometric mean measured concentrations of the sum of the two components. The EC50 for growth rate reduction (72h-ErC50) based on geometric mean measured concentration of two constituents was 2.53 (C.I. 2.40-2.66) mg/L. The ErC10 was 1.00 (C.I. 0.886 – 1.11) mg/L. The EC50 for inhibition of yield (72h-EyC50) based geometric mean measured concentration of two constituents was 1.07 (C.I. 0.984-1.16) mg/L. The EyC10 was < 0.179 mg/L. The corresponding NOEC was 0.347 mg/L, respectively.