Benzyl propionate

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:

from 2016-03-21 to 2016-11-24

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)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100, 50, 25, 12.5, 6.25 mg/L and control
- 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: A stock solution of 100 mg/L was freshly prepared with dilution water. The stock solution was stirred for 15 minutes at 1100 rpm and room temperature until the test item was completely dissolved.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: Pseudokirchneriella subcapitata
- Age of inoculum: three days old
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE x m^-2 ^x s-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:

21.5 - 22.0 °C

pH:

8.07 - 8.13 (0 hours), 7.71 - 9.39 (72 hours)

Salinity:

NA

Nominal and measured concentrations:

nominal concentrations: 100, 50.0, 25.0, 12.5 and 6.25 mg/L
Geometric mean measured / geometric mean measured exposure test item concentration: 93.7, 43.5, 17.1, 4.43, 2.17 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile headspace flasks
- Headspace, fill volume: glass flaks without headspace was used to reduce losses of test item, fill volume: 59 mL
- Initial cells density: 6305 cells/mL
- Control end cells density: 522898 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Medium used: Nutrient medium Z according to Lüttge et al. (1994)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to OECD Guideline 201

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: 24 hours per day
- Light intensity and quality: 4466 - 5469 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter (determined at 0, 24, 48 and 72 hours)
- Other: Microscopic evaluation of the cells at the start and the end of the
incubation period was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

- Range finding study
- Test concentrations: 100, 10, 1, 0.1 mg/L
- Results used to determine the conditions for the definitive study: Growth rate inhibition was determined to be 100%, 2%, -2% and 0% at 100, 10.0, 1.0 and 0.1 mg/L, respectively.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16.7 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.:

9.98 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

11.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no
- Effect concentrations exceeding solubility of substance in test medium: The test media were clear throughout the exposure phase.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 0.460 mg/L (95% CL: 0.443 - 0.481) based on growth rate (with headspace); 1.15 mg/L (95% CL: 0.922 - 1.32) based on growth rate (without headspace);
- Other: The toxicity of potassium dichromate to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2016-04-26 to 2016-04-29 (with headspace) and 2015- 11-10 to 2015-11-13 (without headspace), respectively.

Reported statistics and error estimates:

EC10-, EC20- and ECso-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. The following statistical tests were conducted:
- Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01.
- Levene’s test on variance homogeneity was done with a significance level 0.01.
- Monotonicity was done by trend analysis by contrasts (significance level 0.05).
- William’s multiple sequential t-test was done with a significance level 0.05.

Table 1: 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 / geometric mean measured exposure test item concentration	Replicate	Growth rate	Inhibition of growth rate	Yield	Inhibition of yield
[mg/L]	No.	[d^-1]	[%]	[cells/mL]	[%]
93.7	1 2 3	-0.231 -0.182 -0.257	100 100 100	-3154 -2658 -3386	100 100 100
	Mean	(+) -0.223	100	(+) -3066	100
43.5	1 2 3	0.032 0.074 0.057	98 95 96	631 1573 1181	100 100 100
	Mean	(+) 0.054	96	(+) 1128	100
17.1	1 2 3	0.754 0.638 0.709	49 57 52	54170 36452 46561	90 93 91
	Mean	(+) 0.700	52	(+) 45728	91
4.43	1 2 3	1.54 1.58 1.50	-4 -7 -2	627633 713895 552761	-21 -38 -7
	Mean	(-) 1.54	-4	(-) 631430	-22
2.17	1 2 3	1.48 1.52 1.48	0 -3 0	520652 600515 522626	-1 -16 -1
	Mean	(-) 1.49	-1	(-) 547931	-6
Control	1 2 3 4 5 6	1.47 1.49 1.50 1.46 1.44 1.48		518003 548758 559526 489643 461707 521921
	Mean	1.47		516593

Negative inhibition = growth stimulation

Table 2: Measured Concentrations and Percentage of the Nominal Concentration of test item in Fresh Medium (0 hours) and Old Medium (72 hours with and without algae)

Sampling date	2016-08-01 0 h fresh medium		2016-08-04 72 h old medium without algae		2016-08-04 72 h old medium with algae
Start of analysis	2016-08-01		2016-08-04		2016-08-04
Nominal concentration of the test item [mg/L]	BENZYL PROPIONATE
	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Geometric mean measured concentration^1)[mg/L]
100	101	101	82.1	82	86.9	87	93.7
50.0	50.1	100	31.2	62	33.4	67	40.9
25.0	25.1	100	7.81	31	5.26	21	11.5
12.5	12.5	100	< LOQ		< LOQ		3.06
6.25	6.27	100	< LOQ		< LOQ		2.17
Control	< LOQ		< 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 (1.5 mg test item /L)

¹⁾               =  Values < LOQ were taken with ½ LOQ (0.75 mg/L) into account. Data were taken from old medium with algae.

Table 3: Measured Concentrations and Percentage of the Nominal Concentration of test item in Old Medium (24 hours and 48 hours)

Sampling date	2016-08-02 24 h (with algae)		2016-08-03 48 h (with algae)
Start of analysis	2016-08-05		2016-08-05
Nominal concentration of the test item [mg/L]	BENZYL PROPIONATE
	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]
50.0	47.6	95^1)	42.2	84^1)
25.0	24.5	98^1)	17.8	71
12.5	12.2	98	2.33	19

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 (1.5 mg test item /L)

¹⁾                =  Reanalysed on 2016-08-08

Validity criteria fulfilled:

yes

Conclusions:

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured and geometric mean measured exposure test item concentrations): The EC50 and EC10 value was determined to be 16.7 mg/L and 9.98 mg/L based on growth rate.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3. The study was conducted under static conditions with an initial cell density of 6305 cells/mL for 72 hours. Five concentrations were tested in a geometrical series with a dilution factor of 2 (nominal): 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L. The measured concentrations of the test item in the fresh media (0 h) were in the range of 100 to 101% of the nominal value. The measured concentrations in the old media (72 h) were in the range of < LOQ to 87% of the nominal values of all concentrations. Therefore, all effect values given are based on the geometric mean measured (nominal 6.25 and 100 mg/L) and the time weighted average mean measured exposure (nominal 12.5, 25.0 and 50.0 mg/L) test item concentrations. In this study, the test item was found to inhibit the growth of the freshwater green alga after 72 hours with the following effect values (based on geometric mean measured and geometric mean measured exposure test item concentrations): The EC50 and EC10 value was determined to be 16.7 mg/L and 9.98 mg/L based on growth rate.

Description of key information

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50 value and EC10 value was determined to be 16.7 mg/L and 9.98 mg/L based on growth rate.

Key value for chemical safety assessment

EC50 for freshwater algae:

16.7 mg/L

EC10 or NOEC for freshwater algae:

9.98 mg/L

Additional information

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3. The study was conducted under static conditions with an initial cell density of 6305 cells/mL for 72 hours. Five concentrations were tested in a geometrical series with a dilution factor of 2 (nominal): 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L. The measured concentrations of the test item in the fresh media (0 h) were in the range of 100 to 101% of the nominal value. The measured concentrations in the old media (72 h) were in the range of < LOQ to 87% of the nominal values of all concentrations. Therefore, all effect values given are based on the geometric mean measured (nominal 6.25 and 100 mg/L) and the time weighted average mean measured exposure (nominal 12.5, 25.0 and 50.0 mg/L) test item concentrations. In this study, the test item was found to inhibit the growth of the freshwater green alga after 72 hours with the following effect values (based on geometric mean measured and geometric mean measured exposure test item concentrations): The EC50 and EC10 value was determined to be 16.7 mg/L and 9.98 mg/L based on growth rate.