4'-methoxyacetophenone

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:

2017-04-28 to 2017-06-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

ANALYSIS OF TEST CONCENTRATIONS
- Sampling method: duplicate samples were taken from the test media of all test concentrations and the control at the start of the test (withoutalgae) and at the end of the test (containing algae; test medium of the treatment replicates pooled)
- Sample storage conditions before analysis: frozen at -20 ± 5 °C until sample preparation and at room temperature until the start of the analysis

DETERMINATION OF ALGAL BIOMASS
- 100 μL per sampling of the algal suspension was withdrawn daily from each test flask (not replaced)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solution of 100 mg/L (highest test solution) prepared by completely dissolving 50.05 mg of the test item in 500 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature
- test media prepared just before the start of the test
- Controls: Six replicates (without test item) were exposed under the same conditions as the test concentrations. Potassium dichromate was tested as a reference item
- Replicates per test concentration: 3
- Replicates of the control: 6
- Evidence of undissolved material: No

Test organisms (species):

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

Details on test organisms:

- Strain: No. 61.81 SAG
- Sorce: Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen, Germany)
- Cultivation: at IES Laboratories under standardized conditions according to the test guidelines, cultivated under the test conditions and kept in the exponential growth phase until
inoculation of the test solutions
- Inoculum culture: set up three days before the start of the exposure
- Evaluation of the algal quality and experimental conditions: tested with potassium dichromate (May 2017, IES Study Number 20170077)
- Nominal algal cell density in test solutions: 5000 cells/mL (determined using an electronic particle counter)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

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

Test temperature:

24 °C

pH:

Control: 7.3-7.9
Test item: 7.3-7.9

Dissolved oxygen:

Not measured

Salinity:

Not measured

Conductivity:

Not measured

Nominal and measured concentrations:

Nominal: control, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
Measured (0 h fresh): < LOQ, n.a., 0.974, 3.08, 10.0, 32.4 and 104 mg/L
Measured (72 h aged): < LOQ, n.a., 0.853, 2.85, 9.72, 32.2 and 103 mg/L
n.a. = not analyzed, since this concentration was below the NOEC determined in this test and thus not relevant for the interpretation of the biological results

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks, volume: 75 mL
- Type: Covered with a glass dish
- Fill volume: 30 mL
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Agitation: Test containers were placed on an orbital shaker (test flasks were positioned randomly and repositioned daily)

GROWTH MEDIUM
- Standard medium used: Yes, AAP Medium (see table in section "Any other information on materials and methods incl. tables)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Ultrapure water prepared in-house using an ELGA water purification system

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: 24 h/day
- Light intensity and quality: 74 μE s-1 m-2 (range: 72 to 76 μE s-1 m-2, measured at nine places in the experimental area); Light homogeneity was within ± 15 % over incubation area.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

28 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 26-30 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 51-56 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:

EC10

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 11-14 mg/L

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% CI: 28-31 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: Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities.
- Any stimulation of growth found in any treatment: No
- Any observations that might cause a difference between measured and nominal values: No
- Measured concentrations: The measured concentrations of the test item during the exposure interval were in the range between 96 and 104 % of the nominal values at the start of the test and 85 and 103 % at the end of the test
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid: yes
- 72-h EC50 Growth rate inhibition: 1.0 mg/L
- Conclusion: Sensitivity of the test system was within the range recommended by the guideline (72-hour EC50 for the growth rate 0.9-1.5 mg/L)

Reported statistics and error estimates:

The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 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®.

Table 1: Biomass of Algae

Nominal Test Item Concentration [mg/L]	Repl. No.	Biomass of Algae*
		24 Hours	48 Hours	72 Hours
Control	1	6.3	39.4	205.9
	2	6.0	39.6	212.8
	3	4.3	35.7	194.3
	4	5.0	35.9	188.5
	5	5.6	35.0	196.2
	6	5.5	37.2	201.6
	Mean	5.4	37.1	199.9
	SD	0.7	2.0	8.7
0.32	1	4.2	40.5	212.1
	2	4.9	35.8	198.1
	3	5.0	38.7	202.4
	Mean	4.7	38.3	204.2
	SD 0.4	2.4	7.2
1.0	1	4.8	39.2	210.3
	2	4.5	38.3	210.6
	3	5.1	36.5	194.8
	Mean	4.8	38.0	205.3
	SD 0.3	1.4	9.0
3.2	1	5.5	36.2	183.6
	2	4.6	34.6	197.4
	3	4.6	34.2	187.5
	Mean	4.9	35.0	189.5
	SD	0.5	1.1	7.1
10	1	5.2	38.7	183.7
	2	5.6	34.2	185.8
	3	5.2	34.3	191.6
	Mean	5.3 35.8 187.0
	SD	0.3	2.6	4.1
32	1	4.9	22.7	90.0
	2	4.8	24.0	89.2
	3	4.3	23.6	97.9
	Mean	4.7	23.4	92.4
	SD	0.3	0.7	4.8
100	1	3.0	3.2	2.5
	2	3.9	2.9	1.7
	3	3.4	3.2	2.9
	Mean	3.4	3.1	2.4
	SD	0.5	0.2	0.6

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (mean of duplicate measurements per replicate) and is given as relative fluorescence units (x 104). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 1.36 x 10⁴ relative fluorescence units.

Table 2: Average Growth Rates (μ)

Nominal Test Item Concentration [mg/L]	Average Growth Rateμ(day-1) and Inhibition ofμ(Ir)
	0-24 h		0-48 h		0-72 h
	μ	Ir [%]	μ	Ir [%]	μ	Ir [%]
Control	1.378	0.0	1.653	0.0	1.663	0.0
0.32	1.236	10.3	1.668	-0.9	1.670	-0.4
1.0	1.262	8.4	1.665	-0.7	1.672	-0.5
3.2	1.273	7.6	1.624	1.8	1.646	1.1
10	1.363	1.1	1.634	1.2	1.641#	1.3
32	1.230*	10.8	1.423*	13.9	1.406#	15.5
100	0.918*	33.4	0.414*	74.9	0.180#	89.2

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

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

Table 3: Yield (Y)

Nominal Test Item Concentration [mg/L]	Yield Y (x 104) and Inhibition of Y (Iy)
	0-24 h		0-48 h		0-72 h
	Y	Iy [%]	Y	Iy [%]	Y	Iy [%]
Control	4.08	0.0	35.76	0.0	198.52	0.0
0.32	3.33	18.3	36.94	-3.3	202.84	-2.2
1.0	3.45	15.4	36.63	-2.4	203.89	-2.7
3.2	3.51	13.8	33.64	5.9	188.12*	5.2
10	3.96	3.0	34.39	3.8	185.68*	6.5
32	3.30*	19.1	22.06#	38.3	91.01*	54.2
100	2.07*	49.3	1.76#	95.1	1.03*	99.5

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

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

Table 4: Analytical Results for Test Samples

Sampling Day / Sample Age [d/h]	Nominal Concentration of Test Item Cnom [mg/L]	Measured Concentration of Test Item x [mg/L]	Sample Preparation Factor F	Determined Concentration of Test Item c [mg/L]	% of Nominal Concentration
0/0 (fresh)	Control	n.d.	1.2	< LOQ	n.a.
	1.0	0.812	1.2	0.974	97
	3.2	2.57	1.2	3.08	96
	10	8.35	1.2	10	100
	32	27.0	1.2	32.4	101
	100	86.6	1.2	104	104
3/72 (aged)	Control	n.d.	1.2	< LOQ	n.a.
	1.0	0.711	1.2	0.853	85
	3.2	2.37	1.2	2.85	89
	10	8.10	1.2	9.72	97
	32	26.8	1.2	32.2	101
	100	86.1	1.2	103	103

n.d. = no test item detected

n.a. = not applicable

LOQ: 0.23 mg/L

Remark: The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

Validity criteria fulfilled:

yes

Conclusions:

The test item had an inhibitory effect on the growth of the freshwater green algae Pseudokirchneriella subcapitata.
The 72-hour ErC50 value for inhibition of growth rate was 53 mg(L (95% CI: 51-56 mg/L). The 72-hour ErC10 for inhibition of growth rate resulted in 28 mg/L.

Executive summary:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No. 2016/266, C3. The nominal test item concentrations tested were 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel. The measured concentrations of the test item in the test media of the test concentrations of 1.0 to 100 mg/L were between 96 and 104 % of the nominal values at the start of the test and 85 and 103 % at the end of the test. Thus, the test item was stable in the test media over the test period of 72 hours. The biological results were related to the nominal concentrations of the test item. The 72-hour ErC50 value for inhibition of growth rate was 53 mg(L (95% CI: 51 - 56). The 72-hour ErC10 for inhibition of growth rate resulted in 28 mg/L (95% CI: 26 - 30).

Description of key information

The toxicity of the test item to the freshwater green algae Pseudokirchneriella subcapitata was determined in a test according to OECD guideline 201 and EU Method C.3.

The test determined an 72-hour ErC50 of 53.0 mg/L and an 72-hour ErC10 of 28 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

53 mg/L

EC10 or NOEC for freshwater algae:

28 mg/L

Additional information

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No. 2016/266, C3. The nominal test item concentrations tested were 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel. The measured concentrations of the test item in the test media of the test concentrations of 1.0 to 100 mg/L were between 96 and 104 % of the nominal values at the start of the test and 85 and 103 % at the end of the test. Thus, the test item was stable in the test media over the test period of 72 hours. The biological results were related to the nominal concentrations of the test item. The 72-hour ErC50 value for inhibition of growth rate was 53 mg(L (95% CI: 51 - 56). The 72-hour ErC10 for inhibition of growth rate resulted in 28 mg/L (95% CI: 26 - 30).