4,8-Cyclododecadien-1-one

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:

03-07-2014 to 03-02-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: June 2013; signature: November 2013

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Definitive test: 0.00 (control), 1.00, 3.16, 10.0, 31.6 and 100 mg/L. All concentration levels and the control were analytically verified via GC-MS at the start (0 hours) and at the end of the exposure (72 hours).
- Sampling method: Analytical evaluation of the concentrations of the test item were carried out via GC-MS from freshly prepared media after 0 hours (without algae) and old test media after 72 hours (with algae) of exposure. Separate replicates for the test item analysis at the beginning of the exposure were prepared without algae. For the test item analysis after 72 hours, separate replicates were prepared with algae at the beginning of the exposure and incubated under test conditions. The method was validated prior to the study according to SANCO 3029/99 rev.4 (2000).
- Sample storage conditions before analysis: Original samples were stored at 6 ± 2 °C before preparation, if necessary. All prepared samples were stored at room temperature (in autosampler) before start of analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution (100 mg/L of the test item were weighed out) was freshly prepared with dilution water before the start of exposure. The stock solution was stirred with a magnetic stirrer at approximately 1100 rpm for 1 h at room temperature. 5 test item concentrations in a geometric series with a separation factor of 3.16 (or factor √10) were tested as follows: 0 (control) - 1.00 – 3.16 – 10.00 – 31.60 – 100.00 mg/L. The definitive test concentrations are based on the results of a non GLP preliminary range finding test.
- Eluate: No
- Controls: Six replicates of dilution water without test item tested under the same conditions as the test groups.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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
- Age of inoculum (at test initiation): A four day old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE·m-2·s-1 for 24 hours per day.
- Culture medium: Nutrient medium Z according to LOTTGE et al. (1994).

ACCLIMATION
- Acclimation period: No. However, a four 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: (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 ; NaHCO3* : 250 ; MES monohydrate*: 2665.6 ; pH 8.1 +/- 0.2. This medium has a nominal hardness of 0.24 mmol Ca+Mg/L.
* additional compounds were added to enable sufficient growth under conditions without headspace.
- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

no

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: 23 ; max 24.0 and mean 23.5 °C

pH:

0 hours: pH 8.01 ± 0.2 (and 8.07 in control); 72 hours: pH 8.42-9.25 (definitive test concentrations) and pH 9.04 (controls). pH did not vary more than 1.5 units.

Nominal and measured concentrations:

Preliminary test: 0 (control), 1.0, 10 and 100 mg/L as a nominal test item concentration (non-GLP range finding test)
Definitive Test: 0 (control), 1.00, 3.16, 10.0, 31.6, 100 mg/L as a nominal test item concentration
Analytically measured concentrations: The measured concentrations of the test item at the start of the exposure (0 hours) were in the range of 92 to 101 % of the nominal values. The measured concentrations of the test item at the end of the exposure (72 hours) were in the range of 87 to 101 % of the nominal value. See Table 3 for nominal and measured concentrations at initial exposure and during the course of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass.
- Type: Closed - Static. Sterile headspace flasks
- Material, size, headspace, fill volume: , volume: 119 mL, with aluminium tops with PFTE seals. Minimum headspace.
- Aeration: No. Vessel shaken continuously. Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: Nominal: approximately 5 x 10^3 – 10^4 cells/ml and current: 7085 cells/mL
- Control end cells density: Mean (of replicates after 72 hours) 430972 cells/ml (or ca. x60.8 increase in cell density)
- No. of vessels per concentration (replicates): 3 replicates of each test concentration; 1 extra replicate of each test group for sampling purposes
- 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. adjusted-Medium M2.

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: The pH-value at the start and the end of the exposure was measured from pooled replicates per concentration level and control after measurement of the cell density. The room temperature was measured continuously. Light intensity was measured prior to the start of the exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No.
- Photoperiod: 24 hour day/light; continuous
- Light intensity and quality: 92.1 - 109.6 µE/m2/s ; within ± 15 % over incubation area

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 at the concentration level of 100 mg/L in the range finding test.
- Other: Initial cell density: Microscopic evaluation of the cells was carried out at the start and end of the exposure. 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: Approximately, 3.16. 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
- Test concentrations: 0 (control), 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, 3% growth inhibition at 0.1 mg/L; 3% growth inhibition at 1.0 mg/L; 19% growth inhibition at 10 mg/L; 100% growth inhibition at 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

28.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: analytically confirmed nominal test item concentrations; 95% CL 27.1 - 29.9

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.97 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: analytically confirmed nominal test item concentrations; 95% CL 8.66 - 11.5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: analytically confirmed nominal test item concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

12.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: analytically confirmed nominal test item concentrations; 95% CL 10.6 - 16.1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: analytically confirmed nominal test item concentrations

Details on results:

- Exponential growth in the control (for algal test): Yes.
- 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:
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: Yes. At low concentrations: 1.00 and 3.16 mg/L nominal test item concentration a growth rate and yield inhibition was negative at 72h. This was low dose stimulation and was limited and was taken into account in the data analysis and effect level calculations.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not applicable. The measured concentrations of the test item at the start of exposure were in the range of 92- 101%. At the end of exposure (72 h) the measured concentrations were in the range of 87- 101 % of the nominal values of all concentrations. Therefore, the nominal concentration was used for calculations.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate reduction (ERC50: 0-72h) was 0.661 mg/L with a 95% confidence interval ranging from 0.638 to 0.685 mg/L. The EC50 for yield inhibition (EYC50: 0-72h) was 0.341 mg/L with a 95% confidence interval ranging from 0.286 to 0.377 mg/L.
The results 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:

EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The NOEC/LOEC were determined by calculation of statistically significant differences of growth rate and yield inhibition. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05.

Test for Normality
- Results Growth rate: The statistical program states significant differences for the analytically confirmed nominal test item concentrations 10.0 to 31.6 mg/L compared to the control. No statistically significant difference was found at 3.16 mg/L concentration. Therefore, the NOEC was determined to be 3.16 mg/L.
- Results for Yield: The statistical program states significant differences for the analytically confirmed nominal test item concentrations 10.0 to 31.6 mg/L compared to the control. Therefore, the NOEC was determined to be 3.16 mg/L.

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

Nominal test item concentration [mg/L]	Growth rate inhibition [%]	Yield inhibition [%]
100.0	100	100
10.0	19	57
1.0	3	14
0.1	3	17

Negative values = growth stimulation

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 [mg/L]	Replicate No.	Growth rate [d^-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
100	1	n.a.	100	n.a.	100
	2	n.a.	100	n.a.	100
	3	n.a.	100	n.a.	100
	Mean	n.a.	100	n.a.	100
31.6	1	0.595	57	35099	92
	2	0.586	57	33977	92
	3	0.637	53	40849	90
	Mean	(+) 0.606	56	(+) 283528	91
10.0	1	1.25	8	297538	30
	2	1.22	11	265832	7
	3	1.24	9	287213	32
	Mean	(+) 1.24	10	(+) 283528	33
3.16	1	1.35	1	400591	6
	2	1.34	2	383228	10
	3	1.31	4	350492	17
	Mean	(-) 1.33	3	(-) 378104	11
1.00	1	1.43	-4	509886	-20
	2	1.40	-2	469083	-11
	3	1.37	0	418519	1
	Mean	(-) 1.40	-2	(-) 465829	-10
Control	1	1.40		464534
	2	1.33		379210
	3	1.37		424263
	4	1.34		392856
	5	1.37		428811
	6	1.39		453645
	Mean	1.37		423887

negative inhibitions = increase of growth.

n.a. = not applicable.

Table 3: Concentrations and Percent of the Nominal Concentration of Test Substance (CBI) in Fresh Medium (0 h) and Old Medium (72 h)

Sampling	0 h fresh medium		72 h old medium
Nominal test item concentration [mg/L]	Test Item
	Calculated conc. [mg/L]	[%]	Calculated conc. [mg/L]	[%]
100	95.7	96	97.6	98
31.6	31.2	99	31.4	99
10.0	10.1	101	8.72	87
3.16	3.05	97	3.11	98
1.0	0.920	92	1.01	101
Control	< SysQL		< SysQL

Calculated Conc. = Calculated concentration of the test item, based on the measurement (sum of peak area) of three isomers, single determinations, dilution factor taken into account.

% = Percent of the nominal concentration of the test item.

SysQL = System quantification limit (5 ug/L test item).

Validity criteria fulfilled:

yes

Conclusions:

The test item 72h-EC50 (growth rate reduction) was 28.5 (C.I. 27.1 – 29.9) mg/L. The corresponding EC10 was 9.97 (C.I. 8.66 – 11.5) mg/L and the NOEC was 3.16 mg/L based on analytically confirmed nominal concentrations.

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 GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 7085 cells/mL. With regard to the volatility of the test item, glass flasks without headspace were used to reduce losses of the test item. Five test item solutions with a geometric series of 3.16 (nominal) and concentrations of 0 (control), 1.00, 3.16, 10.00, 31.60 and 100.00 mg/L were freshly prepared from a stock solution of 100 mg/L in dilution water stirred for 1 hour at 1100 rpm at room temperature. Three replicates were tested for each test item concentration and six replicates in the control. The environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via GC-MS at test start (0 hours) and at the end (72 hours) of the exposure. The recovery rates of the test item at the start were in the range of 92 - 101%. At the end of the exposure test item concentrations were in the range of 87 - 101 % of the nominal test item concentrations. All effect values given are based on the analytically confirmed nominal concentrations of test item. The validity criteria of the test guideline were fulfilled. The EC50 for growth rate reduction (72h-ErC50) was 28.5 (C.I. 27.1 – 29.9) mg/L. The corresponding EC10 was 9.97 (C.I. 8.7 – 11.5) mg/L and the NOEC was 3.16 mg/L.

Description of key information

ErC50 (algae; growth rate) = 28.5 (C.I. 27.1 - 29.9) mg/L ; analytically confirmed nominal concentration, 72hour-freshwater, OECD TG 201, 2015

ErC10 (algae; growth rate)= 9.97 (C.I. 8.66 - 11.5 ) mg/L ; analytically confirmed nominal concentration, 72hour-freshwater, OECD TG 201, 2015

NOEC (algae; growth rate) = 3.16 mg/L ; analytically confirmed nominal concentration, 72hour-freshwater, OECD TG 201, 2015

Key value for chemical safety assessment

EC50 for freshwater algae:

28.5 mg/L

EC10 or NOEC for freshwater algae:

3.16 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 GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 7085 cells/mL. With regard to the volatility of the test item, glass flasks without headspace were used to reduce losses of the test item. Five test item solutions with a geometric series of 3.16 (nominal) and concentrations of 0 (control), 1.00, 3.16, 10.00, 31.60 and 100.00 mg/L were freshly prepared from a stock solution of 100 mg/L in dilution water stirred for 1 hour at 1100 rpm at room temperature. Three replicates were tested for each test item concentration and six replicates in the control. The environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via GC-MS at test start (0 hours) and at the end (72 hours) of the exposure. The recovery rates of the test item at the start were in the range of 92 - 101%. At the end of the exposure test item concentrations were in the range of 87 - 101 % of the nominal test item concentrations. All effect values given are based on the analytically confirmed nominal concentrations of test item. The validity criteria of the test guideline were fulfilled. The EC50 for growth rate reduction (72h-ErC50) was 28.5 (C.I. 27.1 – 29.9) mg/L. The corresponding EC10 was 9.97 (C.I. 8.66 – 11.5) mg/L and the NOEC was 3.16 mg/L.