4-methyl-4-phenylpentan-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:

17 Mar - 03 Mar 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted 2006, corrected 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Dec 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office of Public Health, Consumer protection directorate, Notification authority for chemicals, Bern, Switzerland (16 Dec 2015)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations except 0.32 mg/L and the control at the start of the test (without algae) and at the end of the test (with algae) were analysed. At the end of the test, the treatment replicates were pooled for sampling. The samples from the nominal test concentration of 0.32 mg/L were not analyzed because this concentration was below the determined NOEC.
- Sampling method: Duplicate samples were taken from all test media.
- Sample storage conditions before analysis: Frozen at -20 ± 5 °C immediately after sampling. Pre-experiments found that the test item was stable under these storage conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 100 mg/L was prepared by dissolving 50.23 mg test item in 500 mL test water by intense stirring during 15 min at room temperature. The lower test item concentrations were prepared in a series of dilution steps just before the start of the test.
- Controls: yes, test medium control
- Evidence of undissolved material: All test media were clear solutions throughout the test period.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: unicellular green algae
- Strain: SAG 61.81
- Source: supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Göttingen, Germany)
- Age of inoculum: An inoculum was prepared 3 d before the start of the exposure.
- Method of cultivation: The algae were cultivated at the test facility under standard conditions according to the guidelines. The algae were cultivated under test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

15 mg/L CaCO3 (AAP medium)

Test temperature:

22 °C

pH:

0 h: 7.2 (control and treatments)
72 h: 7.9 (control), 7.4 - 8.2 (treatments)

Nominal and measured concentrations:

control, 0.32, 1.0, 3.2, 10, 32, and 100 mg/L (nominal)
< LOQ, < LOQ, 0.978, 3.15, 10.1, 32.5, and 101 mg/L (measured at 0 h)
< LOQ, < LOQ, 0.922, 3.02, 8.81, 29.1, and 93.8 mg/L (measured 72 h)

Details on test conditions:

TEST SYSTEM
- Test vessel: 75 mL Erlenmeyer flasks filled with 30 mL test medium
- Type: Covered with a glass lid
- Initial cell density: 1.11 * 10E+04 relative fluorescent units (5000 cells/mL)
- Control end cells density: 139.1 * 10E+04 relative fluorescent units
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes (AAP medium)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Culture medium same as test medium (AAP medium).
- Intervals of water quality measurement: The light intensity was measured at the start of the test. The temperature in the incubator was monitored and recorded continuously. The pH was measured and recorded in each treatment at the start and end of the test. The appearance of the test media was visually controlled and recorded daily during the exposure period.

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination with LED light
- Light intensity and quality: Mean value of 68 µE*s^-1*m^-2 (65 - 70 µE*s^-1*m^-2)
- Other: Test flasks were incubated in a temperature controlled orbital shaker (Multitron-Pro, Infors HT) and were positioned randomly and repositioned daily.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Daily, by fluoresence measurement (SpectraMax I3x, Molecular Devices Ltd); Excitation: 440 nm; Emission: 680 nm
- Other: For the measurement of biomass, 100 µL algal suspension was withdrawn daily from each test flask and was not replaced.
- Other: The appearance of the algal cells (shape and size) in the control and the 32 mg/L treatment was visually inspected by microscope at the end of the test. Algal cell density at the highest nominal concentration of 100 mg/L was too low for a reliable examination.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: about 3.2
- 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: Average inhibition of growth after 72 h was 4.4% and 92% in the 10 mg/L and 100 mg/L treatments, respectively.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 45 - 54 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 27 - 32 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval: 28 - 31 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval: 8.7 - 11 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: No
- Any observations that might cause a difference between measured and nominal values: No remarkable observations. All test media were clear solutions throughout the test period.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the sensitivity of the test system was within the range recommended by the guideline (ErC50 (72 h): 0.9 - 1.5 mg/L)
- ErC50 (72 h): 1.0 mg/L
- Other: The positive control is tested twice a year. The most recent test dates from May 2017.

Reported statistics and error estimates:

The EC10/20/50 values for the inhibition of average growth rate and yield and the 95% confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression. For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test and Welch t-test with Bonferroni-Holm-adjustment, where appropriate. Statistical analysis was performed using ToxRat Professional.

ANALYTICAL RESULTS

The measured concentrations in the test concentrations ranging from 1.0 – 100 mg/L were 98 – 102% of the nominal values at the start of the test and 88 – 95% at the end of the test (Table 1). Therefore, the test item was correctly dosed and was stable in the test media for the duration of the test of 72 h and the biological results were related to the nominal concentrations.

 

Table 1. Analytical results

Sampling day/sample age[d/h]	Nominal [mg/L]	Measured [mg/L]	Sample preparation factor	Determined concentration of test item [mg/L]	% if nominal concentration
0/0 (fresh)	Control	n.d.	2	< LOQ	n.a.
	1.0	0.489	2	0.978	98
	3.2	1.57	2	3.15	98
	10	5.03	2	10.1	101
	32	16.3	2	32.5	102
	100	50.4	2	101	101
3/72 (aged)	Control	n.d.	2	< LOQ	n.a.
	1.0	0.461	2	0.922	92
	3.2	1.51	2	3.02	95
	10	4.41	2	8.81	88
	32	14.6	2	29.1	91
	100	46.9	2	93.8	94

n.d. = no test item detected

n.a. = not applicable

LOQ = 0.6 mg/L

 

BIOLOGICAL RESULTS

Table 2. Average growth rates.

Nominal test item concentration [mg/L]	Average growth rate µ and inhibition of µ (Ir)
	0 – 24 h		0 – 48 h		0 – 72 h
	µ [day^-1]	Ir [%]	µ [day^-1]	Ir [%]	µ [day^-1]	Ir [%]
Control	1.470	0.0	1.613	0.0	1.610	0.0
0.32	1.464	0.4	1.649	-2.2	1.628	-1.1
1.0	1.414	3.8	1.631	-1.1	1.617	-0.5
3.2	1.328#	9.6#	1.604	0.6	1.593	1.1
10	1.178#	19.9#	1.540	4.5	1.557*	3.3*
32	0.927#	36.9#	1.339*	17.0*	1.382*	14.1*
100	0.851#	42.1#	0.284*	82.4*	-0.003*	100.2*

*: Mean value statistically significantly lower than in the control (according to a Welch t-test after Bonferroni-Holm correction, one-sided smaller, α = 0.05)

#: Mean value statistically significantly lower than in the control (according to a Williams t-test, one-sided smaller, α = 0.05)

Note: Percentage inhibition values in excess of 100% are obtained when the biomass at the end of the interval is lower than at the start of the interval.

 

VALIDITY CRITERIA

Table 3: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	The biomass in the control increased by a factor of 125 over 72 h.	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	The mean coefficient of variation of the daily growth rates in the control (section-by section growth rates) during 72 h was 9.2%.	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus.	The coefficient of variation of the average specific growth rates in the replicates of the control after 72 h was 0.8%.	Yes

 

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Conclusions:

The study was performed according to OECD 201 and GLP. The ErC50 (72 h) was calculated to be at 48 mg/L and the ErC10 (72 h) at 30 mg/L based on nominal concentrations.

Description of key information

ErC50 (72 h) = 48 mg/L (Pseudokirchneriella subcapitata, OECD 201, nominal)

ErC10 (72 h) = 30 mg/L (Pseudokirchneriella subcapitata, OECD 201, nominal)

Key value for chemical safety assessment

EC50 for freshwater algae:

48 mg/L

EC10 or NOEC for freshwater algae:

30 mg/L

Additional information

One experimental study is available investigating the effects of the substance to freshwater algae. The study was performed according to OECD 201 (GLP) under static conditions using Pseudokirchneriella subcapitata as test organism. Nominal test concentrations of 0.32, 1.0, 3.2, 10, 32, and 100 mg/L (nominal) and a control were tested. Since the measured test item concentrations were within 80 and 120% of nominal at test start and test end, all endpoints were evaluated using the nominal concentrations. The ErC10 (72 h) was determined to be at 30 mg/L (95% confidence intervals 27 - 32 mg/L) and the ErC50 (72 h) at 48 mg/L (95% confidence intervals 45 - 54 mg/L).