4-methylcyclohexanone

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:

2015-11-25 to 2016-04-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP-compliant study, performed according to guideline, fully adequate for assessment

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)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and 100 mg/L
- Sampling method: Duplicate samples were taken from the bulk test preparation at 0 hours and from the pooled replicates at 96 hours
- Sample storage conditions before analysis: Samples were stored frozen immediately after sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (200 mg) was dissolved in culture medium and the volume adjusted to 2 litres to give a 100 mg/L stock solution. The test media was prepared just before inoculation with algal suspension. The entire volume of the 100 mg/L test concentration was inoculated with 14.8 mL of algal suspension to give a nominal cell density of approximately 5.00 × 10^3 cells/mL.
- Controls: Blank control
- Evidence of undissolved material: None reported

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Method of cultivation: The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C

ACCLIMATION
- Acclimation period: Not reported
- Culturing media and conditions: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 - 10^5 cells/mL.
- Any deformed or abnormal cells observed: Not reported

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

0.15 mmol/L (= 15 mg/L as CaCO3) (calculated)

Test temperature:

24 ± 1°C

pH:

Start of test: 7.5 - 7.6
End of test: 10.3 - 10.4

Dissolved oxygen:

Not reported

Nominal and measured concentrations:

Control and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks sealed with ground glass stoppers
- Type: closed
- Material, size, headspace, fill volume: 250 mL glass flasks. Due to the potentially volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization.
- Aeration: None. In order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.
- Initial cells density: 5000 cells/mL
- Control end cells density: 2.94 x 10^5 cells/mL at 72 hours
- No. of vessels per concentration: 6
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: Yes, reconstituted test water (ASTM medium) prepared according to OECD Test Guideline No 201

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: The culture medium was the same as the test medium
- Intervals of water quality measurement: Temperature was recorded daily and pH was determined at the start and end of the test

OTHER TEST CONDITIONS
- Sterile test conditions: Not reported
- Adjustment of pH: The pH of the culture medium was adjusted to 7.5
- Photoperiod: Continuous illumination
- Light intensity and quality: Mean measured light intensity of 6982 Lux (range: 6790 to 7180 Lux) by warm white lighting (380 to 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell densities were determined at 24, 48, 72 and 96 hours using an electronic particle counter
- Chlorophyll measurement: None
- Other: The shape and size of the algal cells was inspected microscopically at the end of the test

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: Control, 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No dose-response relationship up to 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Duration:

96 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Details on results:

- Exponential growth in the control: Not reported
- Observation of abnormalities: After 96 hours there were no abnormalities detected in the control or test cultures
- Colour differences: At the start of the test all control and test cultures were observed to be clear, colourless solutions. After the 96-hour test period all control and test cultures were observed to be bright green dispersions.
- Other: The increase of pH from the start to the end of the test was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth. CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. This increase was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Analysis of the 100 mg/L test concentration at 0 and 96 hours showed measured test concentrations of 87 and 84 % of nominal respectively were obtained

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50: 1.5 mg/L

Reported statistics and error estimates:

Statistical analysis of the growth rate, yield and biomass integral data was carried out for the control and 100 mg/L test concentration using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Table 1. Inhibition of growth rate, yield and biomass integral in the definitive test (0 to 72 hours)

Nominal concentration (mg/L)	Growth rate (cells/mL/hour)	Growth rate % inhibition	Yield (cells/mL)	Yield % inhibition	Biomass integral (cells/mL)	Biomass integral % inhibition
Control	0.057	-	2.89E+05	-	5.02E+06	-
100	0.058	[1]	3.14E+05	[8]	5.92E+06	[18]

Values in square brackets indicate an increase in growth as compared to controls

Table 2. Inhibition of growth rate, yield and biomass integral in the definitive test (0 to 96 hours)

Nominal concentration (mg/L)	Growth rate (cells/mL/hour)	Growth rate % inhibition	Yield (cells/mL)	Yield % inhibition	Biomass integral (cells/mL)	Biomass integral % inhibition
Control	0.053	-	7.74E+05	-	1.78E+07	-
100	0.053	0	7.85E+05	[1]	1.91E+07	[8]

Values in square brackets indicate an increase in growth as compared to controls

Validity criteria fulfilled:

yes

Remarks:

For the control cultures after 72 hours, the cell concentration increased by a factor of 59, the mean coefficient of variation for section by section specific growth rate was 30 %, and the coefficient of variation for average specific growth rate was 2 %.

Conclusions:

No statistically significant effects to algal growth rate, yield and biomass integral was observed at 100 mg/L after 72 and 96 hours. The 72- and 96-h NOEC was determined to be 100 mg/L.

Executive summary:

The toxicity of the test item to algae (Pseudokirchneriella subcapitata) was assessed according to EU Method C.3 and OECD Guideline 201. The study is reliable without restrictions, as it is GLP-compliant and was conducted according to standard guidelines. Following a preliminary range-finding test, algae were exposed to an aqueous solution of the test item at a single nominal concentration of 100 mg/L (six replicate flasks per treatment) for 96 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. No statistically significant effects to algal growth rate, yield and biomass integral was observed between the control and 100 mg/L test item concentration after 72 and 96 hours. The 72- and 96-h NOEC was determined to be 100 mg/L.

Description of key information

The toxicity of the test item to algae (Pseudokirchneriella subcapitata) was assessed according to EU Method C.3 and OECD Guideline 201. The study is reliable without restrictions, as it is GLP-compliant and was conducted according to standard guidelines. Following a preliminary range-finding test, algae were exposed to an aqueous solution of the test item at a single nominal concentration of 100 mg/L (six replicate flasks per treatment) for 96 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. No statistically significant effects to algal growth rate, yield and biomass integral was observed between the control and 100 mg/L test item concentration after 72 and 96 hours. The 72- and 96-h NOEC was determined to be 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L