p-methoxybenzyl acetate

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:

2016-02-29 to 2016-03-03

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:

2011

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: The control and the samples 0.253 mg/L to 2.53 mg/L were analysed undiluted. The samples 8.00 mg/L, 25.3 mg/L and 80 mg/L were diluted with mobile phase, prior to analysis.
- Sampling method: All concentration levels and the control were analytically verified via HPLC-DAD after 0 hours (start of exposure) and 72 hours (end of exposure). Separate replicates for the test item analysis at the beginning of exposure were prepared with algae. For the test item analysis after 72 hours samples were taken from the test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solution of 80.0 mg/L was freshly prepared with dilution water and stirred for 20 min at room temperature and 1100 rpm.
- Controls: Six replicates (without test item) were exposed under the same conditions as the test concentrations. Potassium dichromate was tested as a reference item.
- Evidence of undissolved material: No

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDAK, SAG 61.81
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE * m*exp-2 * s*exp-1 for 24 hours per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

21 - 24 °C, controlled at ± 2 °C

pH:

8.1 ± 0.2

Dissolved oxygen:

No data was provided.

Salinity:

Freshwater was used.

Conductivity:

No data was provided.

Nominal and measured concentrations:

Nominal: 0.253 - 0.800 - 2.53 - 8.00 - 25.3 - 80.0 mg/L
Measured: 0.198 - 0.621 - 1.99 - 6.99 - 23.1 - 76.7 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks, volume: 250 mL
- Type: sealed with cotton wool plugs
- Fill volume: 100 mL
- Initial cells density: Nominal: approximately 5 x 10*exp3 - 10*exp4 cells/mL Current: 6305 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to guideline

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 h/day
- Light intensity and quality: 60 - 120 µE * m*exp-2 * s*exp-1; Light homogeneitywas within ± 15 % over incubation area.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: yes, Chlorophyll-a-fluorescence daily with excitation at 436 nm, emission at 685 nm

TEST CONCENTRATIONS
- Spacing factor for test concentrations: dilution factor of √10 (~ 3.16)
- Range finding study: yes
- Test concentrations: nominal, 100, 10, 1.0 and 0.1 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.99 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

23.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

28.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 24.6-69.1

Remarks:

95 % Confidence Intervals

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

39.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 28.8-70.5

Remarks:

95 % Confidence Intervals

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

59.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 37.2-73.9

Remarks:

95 % Confidence Intervals

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.99 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

23.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.83 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Remarks on result:

other: 4.07-12.1

Remarks:

95 % Confidence Intervals

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

15.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Remarks on result:

other: 10.9-19.6

Remarks:

95 % Confidence Intervals

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Remarks on result:

other: 30.4-49.4

Remarks:

95 % Confidence Intervals

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 (e.g. precipitation) 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 98 and 104 % (0 hours) and 59 to 94 % (72 hours) of the nominal value.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid: yes
- EC50: Growth rate inhibition: 0.564 mg/L (CI: 0.546-0.582 mg/L, within the valid range of 0.797+/- 0.428 mg/L), Yield inhibition: 0.322 mg/L (CI: 0.239-0.342 mg/L, within the valid range of 0.415+/- 0.275 mg/L)

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 and LOEC were determined by calculation of statistically significant differences of growth rates and yield. 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 a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Geometric Mean measured test item concentration [mg/L]	Replicate No.	Growth rate [d*exp-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
76.7	1	0.342	82	11290	99
	2	0.427	78	16376	99
	3	0.341	82	11211	99
	Mean	(+) 0.37	81	(+) 12959	99
23.1	1	1.81	6	1449338	28
	2	1.81	6	1414777	29
	3	1.8	6	1368546	32
	Mean	(+) 1.81	6	(+) 1410887	29
6.99	1	1.87	3	1719342	14
	2	1.9	1	1889722	6
	3	1.9	1	1855500	7
	Mean	(-) 1.89	2	(-) 1821521	9
1.99	1	1.87	3	1707953	15
	2	1.91	1	1933473	3
	3	1.92	0	1997746	0
	Mean	(-) 1.9	1	(-) 1879724	6
0.621	1	1.2	0	2013758	0
	2	1.93	0	2043189	-2
	3	1.91	1	1906102	5
	Mean	(-) 1.92	0	(-) 1988350	1
0.198	1	1.91	1	1907595	5
	2	1.94	-1	2098497	-5
	3	1.96	-2	2243676	-12
	Mean	(-) 1.93	-1	(-) 2083256	-4
Control	1	1.93		2057847
	2	1.94		2115581
	3	1.94		2144785
	4	1.86		1667360
	5	1.9		1883239
	6	1.94		2130465
	Mean	1.92		1999880

Negative inhibition = growth stimulation

 

Measured Concentrations and Percentage of the Nominal Concentration of in Fresh Medium (0 hours) and Old Medium (72 hours)

Sampling date	2016-02-29 0 h fresh medium		2016-03-03 72 h old medium
Nominal test item concentration [mg/L]	Test substance
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Geometric Mean measured test item concentration [mg/L]
80	78.3	98	75	94	76.7
25.3	25.9	102	20.7	82	23.1
8	8.16	102	6	75	6.99
2.53	2.56	101	1.55	61	1.99
0.8	0.811	101	0.475	59	0.621
0.253	0.262	104	0.15	59	0.198
Control	< LOQ		< LOQ

Meas. Conc.= Measured concentration of the test item (dilution factor taken into account), mean value of two injections

% = Percentage of the nominal concentration of the test item

LOQ = Limit of quantification (0.13 mg test item /L)

Validity criteria fulfilled:

yes

Conclusions:

The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 59.9 (37.2 - 73.9) mg/L and 38.0 (30.4 - 49.4) mg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 28.8 (24.6 - 69.1) mg/L and 7.83 (4.07 - 12.1) mg/L, respectively. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 6.99 mg/L, respectively.

Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 C.3. The study was conducted under static conditions with an initial cell density of 6305 cells/mL. Six concentrations were tested in a geometrical series with a dilution factor of 3.16: 0.253 - 0.800 - 2.53 - 8.00 - 25.3 - 80.0 mg/L corresponding to geometric mean measured test item concentrations of 0.198 - 0.621 - 1.99 - 6.99 - 23.1 - 76.7 mg/L. The concentrations of the test item in all test concentrations and the control were analytically verified by HPLC-DAD at the start and the end of exposure. The measured concentrations of the test substance in the fresh media (0 h) were between 98 and 104 % of the nominal values. The measured concentrations in the old media (72 h) containing algae were in the range of 59 to 94 % of the nominal values. The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 59.9 (37.2 - 73.9) mg/L and 38.0 (30.4 - 49.4) mg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 28.8 (24.6 - 69.1) mg/L and 7.83 (4.07 - 12.1) mg/L, respectively. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 6.99 mg/L, respectively.

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. The test determined an EC50 value of 59.9 mg/L, an EC10 value of 28.8 mg/L and a NOEC of 6.99 mg/L. The values are based on the growth rate.

Key value for chemical safety assessment

EC50 for freshwater algae:

59.9 mg/L

EC10 or NOEC for freshwater algae:

28.8 mg/L

Additional information

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 C.3. The study was conducted under static conditions with an initial cell density of 6305 cells/mL. Six concentrations were tested in a geometrical series with a dilution factor of 3.16: 0.253 - 0.800 - 2.53 - 8.00 - 25.3 - 80.0 mg/L corresponding to geometric mean measured test item concentrations of 0.198 - 0.621 - 1.99 - 6.99 - 23.1 - 76.7 mg/L. The concentrations of the test item in all test concentrations and the control were analytically verified by HPLC-DAD at the start and the end of exposure. The measured concentrations of the test substance in the fresh media (0 h) were between 98 and 104 % of the nominal values. The measured concentrations in the old media (72 h) containing algae were in the range of 59 to 94 % of the nominal values. The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 59.9 (37.2 - 73.9) mg/L and 38.0 (30.4 - 49.4) mg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 28.8 (24.6 - 69.1) mg/L and 7.83 (4.07 - 12.1) mg/L, respectively. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 6.99 mg/L, respectively.