Benzyl cinnamate

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 2017-04-25 to 2017-04-28

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:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.00632 - 0.0200 - 0.0632 - 0.200 - 0.632 - 2.00 mg/L
- Sampling method: All concentration levels and the control were analytically verified via GC-MS after 0, 24, 48 and 72 hours (end of the exposure).
- 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 (on an autosampler).

Vehicle:

yes

Remarks:

DMSO

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The stock solutions were prepared in measuring flasks (10 or 20 mL capacity) by weighing out appropriate amounts of the test item and transferring it into the measuring glass flasks. Thereafter, the flasks were filled up with DMSO to 10 and 20 mL. The test concentrations were prepared by adding 0.1 mL/L of the appropriate stock solutions to the dilution water.
- Test concentrations: 0.00632 - 0.0200 - 0.0632 - 0.200 - 0.632 -2.00 mg/L
- Control: Test water was used without addition of the test item.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: HINDAK, SAG 61.81
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 hours per day.
- Culture medium: Nutrient medium Z according to Lüttge et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

22 – 23°C

pH:

7.97-9.41

Nominal and measured concentrations:

Nominal concentrations: 0.00632 - 0.0200 - 0.0632 - 0.200 - 0.632 - 2.00 mg/L
Measured concentrations (time weighted average test item concentration): 0.00435 - 0.0200 - 0.0486 - 0.156 - 0.411 - 1.72 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks
- Type: closed
- Material, size, headspace, fill volume: 59 mL, with aluminum tops with PTFE seals
- Initial cells density: 8548 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, As recommended in OECD TG 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24 hours/day light
- Light intensity and quality: Mean light intensity: 5719 lux (range: 5030 to 6284 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm.
- Microscopic evaluation: 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.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.386 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.122 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.049 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.156 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.158 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.043 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yiield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.004 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Microscopic Examination: no morphological abnormalities. There was no statistically significant difference between the control and solvent control regarding growth rate and yield.
- Physico-chemical data: The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The test media were clear throughout the exposure phase.
- Analytical data: The measured concentrations of the test item at the start of the exposure (0 hours) ranged from 99% to 191% of the nominal test item concentrations. After 24 hours the measured concentrations of the test item were between 77% and 187%, after 48 hours between 38% and 80% and at the end of the exposure between

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: most recent test: yield 0.230 mg/L (95% CI: 0.179-0.322), growth rate 0.435 mg/L (95% CI: 0.414-0.458)

Mean cell densities (3 replicates):

TWA concentration (mg/L)	cell densities (cells/mL)
	0h	24h	48h	72h
1.72000	8548	5222	3171	< LOQ
0.41100	8548	18686	30081	74777
0.15600	8548	31444	81801	378219
0.04860	8548	41915	150738	629554
0.02000	8548	40900	189068	659731
0.00435	8548	43748	187871	715559
solvent control	8548	41210	199883	721426
control	8548	47684	224605	739592

Evaluation after 72h

TWA concentration (mg/L)	growth rate (d-1)	% inhibition	yield (cells/mL)	% inhibition
1.72000	n.a.	100	n.a.	100
0.41100	0.691	53	66229	91
0.15600	1.26	15	369671	48
0.04860	1.43	3	621006	13
0.02000	1.45	2	651183	9
0.00435	1.48	0	707011	1
solvent control	1.48		712878
control	1.49		731044

Measured concentrations

Nominal Test item Concentration (mg/L)	Time weighted average Test item Concentration (mg/L)
2.00	1.72
0.632	0.411
0.200	0.156
0.0632	0.0486
0.0200	0.0200
0.00632	0.00435

Validity criteria fulfilled:

yes

Conclusions:

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchnerielia subcapitata after 72 hours with the following effect values (based on time weighted average test item concentration): The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.386 (0.349 - 0.434) mg/L and 0.158 (0.139 - 0.178) mg/L, respectively. The EC10-values with 95% confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 0.122 (0.0906 - 0.162) mg/L and 0.0435 (0.0227 - 0.0640) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 0.0486 and 0.00435 mg/L, respectively.

Description of key information

ErC50 (72 h) = 0.386 mg/L (TWA;Pseudokirchneriella subcapitata; OECD 201)

ErC10 (72 h) = 0.0.122 mg/L (TWA; Pseudokirchneriella subcapitata; OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

0.386 mg/L

EC10 or NOEC for freshwater algae:

0.122 mg/L

Additional information

For this endpoint one test is available. The influence of test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a study conducted according to OECD Guideline 201 and GLP.

The study was performed under static conditions. For preparing the sock solutions a slow stirring procedure was not appropriate since the test item hydrolysis fastly. Therefore, to achieve the maximum solubility of the test item DMSO was used as a solvent. Six test item concentrations were tested in a geometrical series with a dilution factor of 3.16: 0.00632 - 0.0200 - 0.0632 - 0.200 - 0.632 - 2.00 mg/L.

All concentration levels and the (solvent) control were analytically verified via GC-MS after 0 (start of exposure), 24, 48 and 72 hours (end of the exposure). Analytical monitoring also comprised the degradation product Benzylalcohol.

Using the sum of the measured concentrations of the test item and the calculated amount resulted from the metabolite (molecular ratio was taken into account) the time weighted average (TWA) concentrations were calculated (0.00435 – 0.02 – 0.0486 – 0.156 – 0.411 – 1.72 mg/L).

The EC50-values with for inhibition of growth rate and yield after 72 hours were 0.386 mg/L and 0.158 mg/L, respectively. The EC10-values for inhibition of growth rate and yield after 72 hours were 0.122 mg/L and 0.0435 mg/L, respectively.