Phenethyl 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 2017-03-13 to 2017-03-17

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: 6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution, and a control
- Sampling method: The samples for the test item analysis after 0, 24, 48 and 72 hours were prepared with algae and incubated under test conditions. Analytical monitoring did also comprise the degradation product.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal loading of 1 mg test item/L was prepared with demineralized water one day prior to the start of the exposure. This dispersion was stirred for 24 hour with 1100 rpm at room temperature. After stirring, the saturated solution was taken from the homogenous liquid phase. The components of the dilution water were added to the saturated solution. Test concentrations were acquired by further dilution of the saturated solution.
- Test concentrations: 6.25 - 12.5 - 25 - 50 - 100 % of the saturated solution.
- Control: Six replicates (without test item) were incubated under the same conditions as the test concentrations

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

ACCLIMATION
- Acclimation period: four days
- Culturing media and conditions (same as test or not): Nutrient medium Z, same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Nominal: 0.24 mmol Ca+Mg/L

Test temperature:

22.0 – 23.5 °C, mean 22.8°C

pH:

7.79 to 8.25 at test start and 9.07 to 9.59 at test end

Nominal and measured concentrations:

Nominal concentrations: 6.25 - 12.5 - 25 - 50 - 100 % of the saturated solution
Measured initial concentration: 11.7 - 25.3 - 50.8 - 109 - 224 µg/L
TWA: 4.57 - 11.6 - 21.2 - 45.3 - 96.3 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks
- Type: Open
- Material, size, headspace, fill volume: volume: 250 mL, sealed with cotton wool plugs, fill volume: 100 mL
- Initial cells density: 6839 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: range of the measured values: 4757 to 5808 lux, mean value: 5429 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Chlorophyll a-fluorescence
- Other: Microscopic evaluation: at the start and the end of the incubation period; Physico-chemical Properties: The pH-values at the start of exposure were measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-values were measured from pooled samples of each test item concentration and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 1, 10 and 100% saturated solution (nominal loading level of 1.00 mg/L)
- Results used to determine the conditions for the definitive study: The saturated solution and the dilution levels of 1 and 10% of the saturated solution were visually clear throughout the exposure period. Growth rate inhibitions were 1%, 1%, 15% for 1, 10 and 100% saturated solution, respectively.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 96.3 µg/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.:

62.3 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 96.3 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 3.63 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

21.2 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield

Details on results:

- Microscopic evaluation: revealed no morphological abnormalities.
- Physicochemical Data: The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The test item solutions were visually clear throughout the exposure phase.
- Chemical analysis of test item concentrations: The measured concentrations of the test item after 24 hours ranged from 50% to 62% of the initially measured values at the start of the exposure (0 hours). After 48 hours the measured concentrations of the test item were between 20% and 23% and at the end of the exposure between

Results with reference substance (positive control):

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

Cell Densities:

Time weighted average test item concentration [µg/L]	Replicate No.	Cell density [cells/mL]
		0 hours	24 hours	48 hours	72 hours
96.3	1	6839	23848	190557	1330199
	2	6839	20530	163005	1381345
	3	6839	20284	159541	924128
	Mean	6839	21554	171034	1211891
45.3	1	6839	24334	219068	1557022
	2	6839	34057	293458	1714733
	3	6839	19541	169595	1126549
	Mean	6839	25977	227374	1466101
21.2	1	6839	32532	327669	1979302
	2	6839	32298	292311	1973392
	3	6839	29401	280397	1813398
	Mean	6839	31410	300126	1922031
11.6	1	6839	35827	261776	2255283
	2	6839	31321	330501	1883154
	3	6839	31540	231884	1604891
	Mean	6839	32896	274720	1914443
3.63	1	6839	39312	337301	2034019
	2	6839	30762	376405	2360092
	3	6839	34902	279378	1553452
	Mean	6839	34992	331028	1982521
Control	1	6839	39792	371547	2289107
	2	6839	36207	409597	2356581
	3	6839	37682	362453	2265933
	4	6839	35081	331169	2167035
	5	6839	34630	369511	2080249
	6	6839	31827	337465	1964029
	Mean	6839	35870	363624	2187156

Evaluation of growht rate and yield inhibition after 72 hours:

Time weighted average test item concentration [µg/L]	Replicate No.	Growth rate [d^-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
96.3	1	1.76	9	1323360	39
	2	1.77	8	1374506	37
	3	1.64	15	917289	58
	Mean	(+) 1.72	10	(+) 1205052	45
45.3	1	1.81	6	1550183	29
	2	1.84	4	1707894	22
	3	1.70	1	1119710	49
	Mean	(+) 1.78	7	(+) 1459262	33
21.2	1	1.89	2	1972463	10
	2	1.89	2	1966553	10
	3	1.86	3	1806559	17
	Mean	(-) 1.88	2	(-) 1915192	12
11.6	1	1.93	-1	2248444	-3
	2	1.87	3	1876315	14
	3	1.82	5	1598052	27
	Mean	(-) 1.88	2	(-) 1907604	13
3.63	1	1.90	1	2027180	7
	2	1.95	-1	2353253	-8
	3	1.81	6	1546613	29
	Mean	(-) 1.88	2	(-) 1975682	9
Control	1	1.94		2282268
	2	1.95		2349742
	3	1.93		2259094
	4	1.92		2160196
	5	1.91		2073410
	6	1.89		1957190
	Mean	1.92		2180317

Measured concentrations and percentage of the nominal concentration of test item in fresh medium (0 hours) to old medium (72 hours):

Sample time	0 h	24 h		48 h		72 h
Dilution Level of the Saturated Solution [%]	Meas. conc. [µg/L]	Meas. conc. [µg/L]	[%]	Meas. conc. [µg/L]	[%]	Meas. conc. [µg/L]	[%]
100	224	118	53	50.2	22	6.46	2.9
50.0	109	54.9	50	22.5	21	3.05	2.8
25.0	50.8	28.3	56	11.8	23	0.94	1.9
12.5	25.3	14.9	59	5.69	23	0.546	2.2
6.25	11.7	7.27	62	2.29	20	< LOQ
Control	< LOQ	< LOQ		< LOQ		< LOQ

Meas. Conc. = Measured concentration of the test item (enrichment and dilution factor taken into account)

%= Percent of the initially measured concentration of the test item

LOQ= Limit of quantification (0.5 µg test item/L)

Measured concentrations and percentage of the nominal concentration of metabolite phenylethyl alcohol in fresh medium (0 hours) to old medium (72 hours):

Sample time	0 h	24 h		48 h		72 h
Dilution Level of the Saturated Solution [%]	Meas. conc. [µg/L]	Meas. conc. [µg/L]		Meas. conc. [µg/L]		Meas. conc. [µg/L]
		1)	2)	1)	2)	1)	2)
100	< LOQ	2.3	4.75	12.5	25.8	16.8	34.8
50.0	< LOQ	1.03	2.12	5.11	10.6	9.31	19.2
25.0	< LOQ	0.591	1.22	2.48	5.12	3.00	6.20
12.5	< LOQ	< LOQ		1.39	2.87	2.60	5.37
6.25	< LOQ	< LOQ		0.750	1.55	< LOQ
Control	< LOQ	< LOQ		< LOQ		0.784	1.62

Meas. Conc. =Measured concentration of the test item (enrichment and dilution factor taken into account)

1) = Measured concentration of the metabolite

2) = Measured concentration of the metabolite calculated with molar ratio to test item. (molar ratio = M(test item) / M(metabolite)

LOQ= Limit of quantification (0.5 µg metabolite/L)

Mean calculated concentrations (µg/L) of the test item from measured concentrations of test item and metabolite phenylethyl alcohol:

Dilution level of saturated solution (%)	0h	24h	48h
100	224	81.6	50.7
50	109	38.0	22.1
25	50.8	19.7	11.3
12.5	25.3	14.9	5.7
6.25	11.7	7.3	2.6
Control	< LOQ	< LOQ	< LOQ

Validity criteria fulfilled:

yes

Conclusions:

In this study, 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 time weighted average test item concentration): The EC50-values for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 96.3 µg/L (saturated solution), respectively. The EC10-values, ErC10 and EyC10 were 62.3 µg/L and <3.63 µg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 21.2 µg/L.

Executive summary:

The toxicity of test item to the unicellular freshwater green alga Pseudokirchnerieila subcapitata was determined according to the principles of OECD 201. The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with a factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution, corresponding to the time weighted average test item concentrations 3.63 - 11.6 - 21.2 - 45.3 - 96.3 µg/L.

The concentrations of test item were determined at the start of the exposure (0 hours) and at the end of the exposure (72 hours) of all tested dilutions of the saturated solution and the control via GC-MS. Additional replicates were analysed after 24 hours and 48 hours of the exposure due to instability of the test item. The concentrations of one of the metabolites (Phenylethyl alcohol) of test item were determined at the start and after 24 hours, 48 hours and 72 hours of the exposure.

The measured concentrations of the test item after 24 hours ranged from 50% to 62% of the initially measured values at the start of the exposure (0 hours). After 48 hours the measured concentrations of the test item were between 20% and 23% and at the end of the exposure between <LOQ and 2.9% of the initially measured values. Therefore one metabolite was determined in all samples and was calculated with the molar ratio to test item. Using the sum of the measured concentrations of the test item and the calculated amount resulted from the metabolite the time weighted average was calculated. No significant adsorption to glass or algae was observed. At the end of the exposure, the concentration of the 100% saturated solution was in the additional replicate without algae 34.7 µg/L (15% of the initially measured value). Thus, even without the presence of algae the concentration of the test item degrades due to abiotic factors.

The test item (including its metabolite) was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on time weighted average test item concentration): The EC50-values for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 96.3 µg/L, respectively. The EC10-values, ErC10 and EyC10  were 62.3 µg/L and <3.63 µg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 21.2 µg/L.

Description of key information

For this endpoint, there is one study available in which the toxicity to algae was assessed according to OECD 201. The ErC50 after 72 hours was > 96.3 µg/L (saturated solution, TWA) and the ErC10 after 72 h was 62.3 µg/L (TWA) (Pseudokirchneriella subcapitata).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

62.3 µg/L

Additional information

The toxicity of test item to the unicellular freshwater green alga Pseudokirchnerieila subcapitata was determined according to OECD 201. The study was conducted under static conditions. A saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with a factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution, corresponding to the time weighted average test item concentrations 3.63 - 11.6 - 21.2 - 45.3 - 96.3 µg/L.

The concentrations of test item were determined at the start of the exposure (0 hours) and at the end of the exposure (72 hours) of all tested dilutions of the saturated solution and the control via GC-MS. Additional replicates were analysed after 24 hours and 48 hours of the exposure due to instability of the test item. The concentrations of one of the metabolites of test item were determined at the start and after 24 hours, 48 hours and 72 hours of the exposure.

The measured concentrations of the test item after 24 hours ranged from 50% to 62% of the initially measured values at the start of the exposure (0 hours). After 48 hours the measured concentrations of the test item were between 20% and 23% and at the end of the exposure between <LOQ and 2.9% of the initially measured values. Therefore one metabolite was determined in all samples and was calculated with the molar ratio to test item. Using the sum of the measured concentrations of the test item and the calculated amount resulted from the metabolite the time weighted average was calculated. No significant adsorption to glass or algae was observed. At the end of the exposure, the concentration of the 100% saturated solution was in the additional replicate without algae 34.7 µg/L (15% of the initially measured value). Thus, even without the presence of algae the concentration of the test item degrades due to abiotic factors.

The test item (including its metabolite) was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on time weighted average test item concentration): The EC50-values for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 96.3 µg/L, respectively. The EC10-values, ErC10 and EyC10  were 62.3 µg/L and <3.63 µg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 21.2 µg/L.