Ethyl 3-phenyloxirane-2-carboxylate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 November 2012 - 26 February 2012

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:

2006

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)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group at t=0, t=72 and t=96 hours for quantative analysis. Duplicate samples were taken at each occasion and stored at approximately -20°C for further analysis if necessary. Samples without algal cells were taken at the start of the test, incubated alongside the test and taken for analysis at t=72 and t=96 hours.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: an amount of test item (1100 mg) was dissolved in culture medium with the aid of prolonged stirring at approximately 1500 rpm for 24 hours. After stirring, as a precautionary measure any undissolved material was removed by filtration through a 0.2 µm Sartorius Sartopore filter to give a stock solution with a 0-hour measured concentration of 84 mg/L. A series of dilutions was made from this stock solution to give further stock solutions of 41, 20, 9.3 and 4.7 mg/L. An aliquot (1500 mL) of each of the stock solutions was seperately inoculated with algal suspension (42 mL). The stock solutions and each of the prepared concentrations were inverted several times to ensure mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 21 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

ACCLIMATION
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyrethane foam stoppers an kept under constant agitation by an orbital shaker (100-150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4-10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

Temperature was maintained at 24 ± 1 °C throughout the test.

pH:

The pH of each control and test flask was determined at initiation of the test and after 96 hours exposure. The pH was maintained within 7.8 - 10.6

Nominal and measured concentrations:

Based on the results of the range-finding test the following test concentrations were assigned to the definitive test:
Nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg/L
Measured concentrations: 4.69, 9.31, 19.7, 40.7 and 83.9 mg/L at t=0; 0.655, 1.48, 13.8, 33.1, 68.2 mg/L at t=72 for nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L respectively
Measured concentrations for the test solutions without algae: 3.90, 7.75, 15.1, 34.4 and 70.9 mg/L at t=72 for nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L respectively

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks were used. Six flasks each completely filles with test preparation were used for the control group and three flasks each completely filled were used for each treatment group. The flasks were sealed with ground glass stoppers to reduce losses due to volatilisation. The control group was maintained under identical conditions but not exposed to the test material.

- Initial cells density:
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 3.54E+05 cells per mL. Inoculation of 1500 mL of test medium with 42 mL of this algal suspension gave an initial cell density of 1E+04 cells per mL and had no significant dilution effect.

- No. of vessels per concentration (replicates):
3

- No. of vessels per control (replicates):
6

- No. of vessels per vehicle control (replicates):
None

GROWTH MEDIUM
- Detailed composition of medium:
NaNO3 25.5 mg/L
MgCl2.6H2O 12.164 mg/L
CaCl2.2H2O 4.41 mg/L
MgS04.7H20 14.7 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 1855 mg/L
MnCl2.4H20 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.159 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H20 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionised water (EIga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with O.1N NaOH or HCl. The prepared media was sterilised by 0.2 µm membrane filtration and stored in darkness. The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture with the addition of 500 mg/L of sodium bicarbonate to provide a sufficient supply of CO2 and to counteract in the increase in pH due to algal growth in an enclosed system.

- Photoperiod:
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous illumination and constantly shaken at approximately 150 rpm for 96 hours.

- Light intensity and quality:
intensity approximately 7000 lux

- Salinity (for marine algae):
Not applicable

- Determination of cell concentrations:
Samples were taken at 0, 24, 48, 72 and 96 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

- Results used to determine the conditions for the definitive study:
The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L. Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

zinc chloride

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

36 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- It was not possible to calculate the 95% confidence limits for the EC50 value as the data generated did not fit the models available for the calculation.
- Effect concentrations were based on initial measured test concentrations because results from the analysis of samples without algal cells showed that test substance likely adsorbed to the algae. According to OECD 201 it can be assumed that test substance adsorbed to algae still contributes to the toxicity of the test substance.

Results with reference substance (positive control):

72h-EC50 for growth rate: 0.23 (95%-CI: 0.20-0.28)
72h-LOEC for growth rate: 0.032
72h-NOEC for growth rate: 0.010

Compared to historical data for the reference item, the current study with the reference item gave lower effect concentrations. This is due to the fact that test vessels were sealed to reduce losses of test item through volatility. Therefore it can be stated that the results obtained from the definitive test gave a worst case result for the test item.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the growth rate, yield and biomass integral data after 72 hour for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001). Results showed there were no statistically differences between the control, 4.7 and 9.3 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different from the control (P<0.05).

Table 1 Concentrations of test material in test media samples determined using HPLC analysis

Sample time (h)	Nominal concentration (mg/L)	Measured concentration (mg/L)	Relative to initial (%)
0	Control	<LOQ	-
	6.25	4.69	-
	12.5	9.31	-
	25	19.7	-
	50	40.7	-
	100	83.9	-
72	6.25	0.655	14
	12.5	1.48	16
	25	13.8	70
	50	33.1	81
	100	68.2	81
72 without algae	6.25	3.90	83
	12.5	7.75	83
	25	15.1	77
	50	34.4	85
	100	70.9	85
96	Control	<LOQ	-
	6.25	<LOQ	-
	12.5	<LOQ	-
	25	4.44	23
	50	29.7	73
	100	64.1	76
96 without algae	6.25	3.68	78
	12.5	7.32	79
	25	15.6	79
	50	32.8	81
	100	67.6	81

Table 2 Mean inhibition of growth rate, yield an biomass in the definitive test

Measured test concentration (t=0)	Growth rate (cells/mL/h)		Yield cells/mL		Biomass integral (cells/mL/h)
	0-72 h	% inhibition	0-72 h	% inhibition	0-72 h	% inhibition
Control	0.092	-	7.51E+05	-	1.64E+07	-
4.7	0.091	1	7.00E+05	7	1.61E+07	2
9.3	0.090	2	6.47E+05	14	1.27E+07	22
20	0.065	29	9.90E+04	87	3.30E+06	80
41	0.033	64	1.90E+03	100	8.48E+05	95
84	0.028	70	-2.78E+03	100	6.47E+05	96

Validity criteria fulfilled:

yes

Remarks:

Cell concentration of the control cultures increased 77.1 fold, the mean CV of the section by section growth rate in the controls was 22%%, the CV of the average specific growth rate in controls was 3%, the CV of the cell density values in control was 12%

Conclusions:

The 72h-ErC50 and 72h-NOEC were 36 and 9.3 mg/L, respectively.

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The study was performed according to OECD TG No 201 and GLP. Algal cultures were exposed to solutions of the test material at nominal concentrations of 0, 6.25, 12.5, 25, 50 and 100 mg/L for 96 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Test concentrations were verified by analysis at t=0, t=72 and t=96. Results showed a significant decrease in test concentrations especially at the low concentrations. However, results from analysis of samples without algae at t=72 and at t=96 showed that test solutions remained 77 -85% and 78 -81% of initial measured test concentrations, respectively. By including analysis of media without algae, the authors demonstrated that the test substance adsorbed to the algae. According to OECD TG 201 it can be assumed that test substance that adsorbs to algae still contributes to the toxicity and the initial concentrations can be used. In addition, even if the geometric mean concentration would be used EC50 would be still > 10 mg/l and the NOEC > 1 mg/l. Therefore, EC50 and NOEC are based on initial measured concentrations. 72h-ErC50 and 72h-NOEC were determined to be 36 and 9.3 mg/L respectively. All validity criteria were fulfilled and the study was considered valid without restrictions.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2016-2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across information

Justification for type of information:

The algae 72h-ErC50 and NOEC of Ethylphenylglycidateis are derived from read across from Ethyl 3methylphenylglycidate. The documentation of this read across can be found in the Aquatic endpoint summary, the accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

36 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Validity criteria fulfilled:

yes

Remarks:

Adequate and reliable documentation of the read across is presented.

Conclusions:

The 72h-ErC50 and 72h-NOEC algae toxicity of Ethylphenylglycidate are 36 and 9.3 mg/L, respectively (based on read across from Ethyl 3methylphenylglycidate.

Description of key information

The 72h-ErC50 and NrOEC of algae for Ethylphenylglycidate are 36 and 9.3 mg/l, respectively. These values are based on read across from Ethyl-3methylphenylglycidate tested according to OECD TG 201.

Key value for chemical safety assessment

EC50 for freshwater algae:

36 mg/L

EC10 or NOEC for freshwater algae:

9.3 mg/L

Additional information

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The study was performed according to OECD TG No 201 and GLP. Algal cultures were exposed to solutions of the test material at nominal concentrations of 0, 6.25, 12.5, 25, 50 and 100 mg/L for 96 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Test concentrations were verified by analysis at t=0, t=72 and t=96. Results showed a significant decrease in test concentrations at t=72 and at t=96. However, results from analysis of samples without algae at t=72 and at t=96 showed that test solutions remained 77 -85% and 78 -81% of initial measured test concentrations, respectively. By including analysis of media without algae, the authors demonstrated that the test substance adsorbed to the algae. According to OECD201 it can be assumed that test substance that adsorbs to algae still contributes to the toxicity and the initial concentrations can be used. Therefore, EC50 and NOEC are based on initial measured concentrations. 72h-ErC50 and 72h-NOEC were determined to be 36 and 9.3 mg/L respectively. All validity criteria were fulfilled and the study was considered valid without restrictions.