Reaction mass of (3E)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-1-en-1-yl)butan-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)butan-2-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:

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:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000)

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: February 2014; signature: April 2014

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All loading levels and the control were analytically verified via GC-MS at the start (0 and 72 hours) and at the end of the exposure. Test substance loadings were prepared as Water Accommodated Fraction (WAF).
- Sampling method: The analysis described within this report was the analysis of one of the constituents measured at a specific wavelength.
The test concentrations of tracer constituent were measured using gas chromatography with mass spectrometric detection system (GC-MS). At the start of the definitive test, two samples (50 mL) were taken from the freshly-prepared control and test media. After 72 hours, the contents of the replicate flasks for each group were pooled and further samples taken for analysis. This was to assess the stability of the WAF.
- Sample storage conditions before analysis: All original samples were stored at room temperature before preparation and a separate sample stored frozen if required.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: Strain No. CCAP 278/4.
- Source (laboratory, culture collection): the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd.
- Age of inoculum (at test initiation): 3 days (in pre-culture under the same conditions as the test).
- Method of cultivation: Primary liquid cultures (100 mL) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 24°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth, characterised by a cell density of 8.7 x 10^5 cells/mL

ACCLIMATION
- Acclimation period: None.
- Culturing media and conditions (same as test or not): Yes.
Stock Culture, Pre-Culture and definitive test OECD Test Medium: NH4Cl 15 mg/L; MgCl2.6H2O 12 mg/L; CaCl2.2H2O 18 mg/L; MgSO4.7H2O 15 mg/L; KH2PO 1.6 mg/L; FeCl3.6H2O 0.064 mg/L; Na2EDTA.2H2O 0.1 mg/L; H3BO3 186 µg/L; MnCl2.4H2O 416 µg/L; ZnCl2 1.5 µg/L; CoCl2.6HO 0.75 µg/L; CuCl2.2H2O 0.1 µg/L; Na2MoO4.2H2O 7 µg/L; NaHCO3 50 mg/L; pH ca. 8
- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Not reported. See 'Details on test organism' field for details on test medium. Initial dilution water resistivity: 18 Megohm/cm.

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 21 and 23°C. Temperature remained within the limits prescribed by the guideline (21-24°C, constant within 2°C).

pH:

0 hours: pH 7.9 ± 0.15 ; 72 hours: pH 7.84-8.25 (definitive test concentrations) and pH 8.5 (controls). pH did not vary more than 1.5 units.

Nominal and measured concentrations:

Preliminary test: 0 (control), 0.1, 1.0, 10 and 100 mg/L as a nominal loading of a WAF
Final test: Solutions containing: 0 (control), 2.56, 6.4, 16.0, 40.0 and 100 mg/L prepared as a nominal loading rate of WAF

Details on test conditions:

TEST SYSTEM
- Test vessel: Flask (Glass)
- Type (delete if not applicable): Closed - Static (loosely stoppered and with aluminium foil).
- Material, size, headspace, fill volume: Glass, 250 ml, filled to 100 ml
- Aeration: Vessel shaken continuously.
- Initial cells density: 1 x 10^4 cells/ml.
- Control end cells density: Mean (of replicates after 72 hours) ca. 175.0 x10^4 cells/ml
- 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; 1 extra replicate of test group for sampling purposes at 24h; 1 replicates of intermediate concentration without algae.
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. adjusted-Medium M2.
- Detailed composition if non-standard medium was used: Stock Culture, Pre-Culture and definitive test OECD Test Medium: NH4Cl 15 mg/L; MgCl2.6H2O 12 mg/L; CaCl2.2H2O 18 mg/L; MgSO4.7H2O 15 mg/L; KH2PO 1.6 mg/L; FeCl3.6H2O 0.064 mg/L; Na2EDTA.2H2O 0.1 mg/L; H3BO3 186 µg/L; MnCl2.4H2O 416 µg/L; ZnCl2 1.5 µg/L; CoCl2.6HO 0.75 µg/L; CuCl2.2H2O 0.1 µg/L; Na2MoO4.2H2O 7 µg/L; NaHCO3 50 mg/L; pH ca. 8

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Filtered, dechlorinated tap water which had been softened and treated by reverse osmosis, before microfiltration and purification (resistivity of 18 Megohm/cm). Stock solutions were sterilised by autoclaving or by membrane filtration before being stored at 4°C in the dark. This was used to generate the dilution medium described above.
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Not reported.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 4440 to 8880 lux (nominal)
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell density using Coulter Z Series Particle Count and Size Analyser. Algal medium used as blank.
- Other: Initial cell density: Cells were counted using a microscope and a counting chamber.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5. 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: Three replicates per concentration were exposed to dilutions representing 0.1, 1.0, 10 and 100 mg/L (filtered and unfiltered) prepared as a WAF in the range-finding test. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval. In the definitive test 5 concentrations were used at 2.56, 6.4, 16.0, 40.0 and 100 mg/L prepared as WAF.
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

56.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 56.5 - 61.4 mg/L; nominal based on WAF

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

24.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 19.5 - 30.9 mg/L; nominal based on WAF

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

17.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL:14.0 - 20.4 mg/L; nominal based on WAF

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

12.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 7.27 - 20.8 mg/L; nominal based on WAF

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

6.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): With the exception of some swollen cells observed at nominal loading rates of 40 and 100 mg/L at 24 hours, no other microscopic abnormalities of the cells were detected. Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: No.
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: Yes. At low concentrations 2.56 - 40 mg/L WAF yield inhibition was negative between 24-48h. This was low dose stimulation and was limited to < 15%. It was not observed other than at 6.40 mg/L WAF at 48-72 hours and was taken into account in effect level calculation.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Final test: During the exposure period the measured concentrations (from the measured tracer constituent) decreased in the definitive test from nominal values. No observations were made to account for the difference.
- Effect concentrations exceeding solubility of substance in test medium: No.

Reported statistics and error estimates:

All 95% confidence intervals for EC50 were calculated using the likelihood ratio method (Donaldson and Schnabel, 1985). The EC10 (with 95% confidence interval) were also estimated by reparameterising the above formulae.
For yield and growth rate, Williams’ test (1971, 1972) was also used to compare each treated group with control unless there was evidence of a non-monotonic dose-response relationship, in which case Dunnett’s test (1955, 1964) was used.

Validity criteria fulfilled:

yes

Conclusions:

The test substance 72h-EL50 (growth rate reduction) was 56.5 (C.I. 52.1 - 61.4) mg/L based on nominal loading rate concentrations. The NOELR was 6.40 mg/L.

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 under GLP. Triplicate algal cultures, with an initial cell density of ca. 1 x 10^4/mL, were exposed to water accommodated fractions (WAF) of test substance prepared from aqueous mixtures with nominal loading rates of 2.56, 6.40, 16.0, 40.0 and 100 mg/L. Aliquots of the test substance were added to OECD medium; these mixtures were stirred for approximately 24 hours in the dark and then left to stand for approximately 24 hours in the dark before the WAFs were removed and filtered (0.2 μm pore size), before being used directly as test media. The cultures were incubated in an orbital incubator under continuous illumination with the algal cultures at temperatures ranging from 21.4 to 22.3°C for 72 hours. The test substance comprised several constituents. One constituent was analysed as a tracer to assess the stability of the WAF system. The analysis described within this report was the analysis of one of the constituents measured at a specific wavelength using the gas chromatography with mass spectrometric detection system (GC-MS) method. The concentrations of the tracer constituent decreased from 70 to 54% of the initially measured concentrations (at the start of the test), showing some decline during the exposure. Since the test substance is a complex mixture, its toxicity cannot be attributed to a single constituent or a mixture of constituents, but to the test substance as a whole. Therefore the nominal loading rates of the test substance were used in the determination of study end-points, as it is recommended in the OECD Guidance Document No 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD, 2000). Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the growth rate and yield. The following study end-points were derived from the data: 72 -hours EL50 (yield): 24.3 (C.I. 19.5 – 30.9). Whereas the 72 -hours EL50 (growth rate): 56.5 (C.I. 52.1 – 61.4) mg/L; EL10: 17.1 (C.I. 14.0 – 20.4) mg/L respectively. The No Observed Effect Loading Rate (NOELR) was determined to be 6.4 mg/L.

Description of key information

ErL50 (algae; growth rate) = 56.5 (C.I. 52.1 - 61.4) mg/L ; WAF loading 72hour-freshwater, OECD TG 201, 2015

ErL10 (algae; growth rate)= 17.1 (C.I. 14.0 – 20.4) mg/L; WAF loading 72 hour-freshwater, OECD TG 201, 2015

NOELR (algae; growth rate) = 6.40 mg/L ; WAF loading, 72 hour-freshwater, OECD TG 201, 2015

Key value for chemical safety assessment

EC50 for freshwater algae:

56.5 mg/L

EC10 or NOEC for freshwater algae:

6.4 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 under GLP. Triplicate algal cultures, with an initial cell density of ca. 1 x 10^4/mL, were exposed to water accommodated fractions (WAF) of test substance prepared from aqueous mixtures with nominal loading rates of 2.56, 6.40, 16.0, 40.0 and 100 mg/L. Aliquots of the test substance were added to OECD medium; these mixtures were stirred for approximately 24 hours in the dark and then left to stand for approximately 24 hours in the dark before the WAFs were removed and filtered (0.2 μm pore size), before being used directly as test media. The cultures were incubated in an orbital incubator under continuous illumination with the algal cultures at temperatures ranging from 21.4 to 22.3°C for 72 hours. The test substance comprised several constituents. One constituent was analysed as a tracer to assess the stability of the WAF system. The analysis described within this report was the analysis of one of the constituents measured at a specific wavelength using the gas chromatography with mass spectrometric detection system (GC-MS) method. The concentrations of the tracer constituent decreased from 70 to 54% of the initially measured concentrations (at the start of the test), showing some decline during the exposure. Since the test substance is a complex mixture, its toxicity cannot be attributed to a single constituent or a mixture of constituents, but to the test substance as a whole. Therefore the nominal loading rates of the test substance were used in the determination of study end-points, as it is recommended in the OECD Guidance Document No 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD, 2000). Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the growth rate and yield. The following study end-points were derived from the data: 72 -hours EL50 (yield): 24.3 (C.I. 19.5 – 30.9). Whereas the 72 -hours EL50 (growth rate): 56.5 (C.I. 52.1 – 61.4) mg/L; EL10: 17.1 (C.I. 14.0 – 20.4) mg/L respectively. The No Observed Effect Loading Rate (NOELR) was determined to be 6.4 mg/L.