4-ethenylphenyl acetate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-15 to 2018-02-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

434-600-2

EC Name:

-

Cas Number:

2628-16-2

Molecular formula:

CH3CO2C6H4CH=CH2

IUPAC Name:

4-ethenylphenyl acetate

Test material form:

liquid

Specific details on test material used for the study:

Purity analysed 98.4 % w/w

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- analytical samples of all test item concentrations and control were taken at test start (0 hours fresh), at 24 hours (aged), 48 hours (aged) and at test end (72 hours aged).
- three replicates per treatment group containing algae were set up for the analytical sampling
- Storage before analysis: All samples were stored deep frozen until they were transferred to the analytical laboratory.

Test solutions

Vehicle:

no

Details on test solutions:

AAP-Medium (according to Annex 3 of OECD 201)

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: Hindak
- Source (laboratory, culture collection):
- Age of inoculum (at test initiation): purchased from a commercial supplier MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany.
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state.
- culture conditions are as follows:
-- illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 - 120 μEm-2s-1)
-- temperature: 21 - 24 °C
-- culture flasks: 100 mL Erlenmeyer flasks
-- CO2 supply by shaking on a rotating shaker, approximately 105 rpm


ACCLIMATION
3 to 4 days before start of the test, test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth.

Study design

Test type:

static

Water media type:

freshwater

Remarks:

synthetic growth medium

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

At 24, 48 and 72 hours from the test initiation, the number of cells in each replicate was determined

Post exposure observation period:

not applicable

Test conditions

Hardness:

not specified

Test temperature:

22.2 – 23.3 °C

pH:

7.59 – 9.18 (control)

Dissolved oxygen:

not specified

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

0.5 x 10e4 cells/mL (targeted initial cell density)

Details on test conditions:

TEST SYSTEM
- Test vessel: serum bottles with crimp caps
- Type : closed with crimp caps
- Material, size, headspace, fill volume: 120 mL, minimal headspace above test solution to reduce volatilisation due to he high vapour pressure of the substance.
- Aeration: agitator added to each serum bottle
- CO2 supply: NaHCO3 was added
- Renewal rate of test solution (frequency/flow rate): no renewal, static test. dens
- Initial cells density: 0.5 × 10^4 cells/mL.
- Average control end cells density: 68.77 × 10^4 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP medium
- Detailed composition if non-standard medium was used: according to Annex 3 of OECD 201


OTHER TEST CONDITIONS
- Sterile test conditions: yes/n
- Adjustment of pH: to pH 7.5
- Photoperiod: Continuously
- Light intensity and quality: 88.8 μEm-2s-1 (mean)


EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: fluorescence detection every 24h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 4, a larger spacing factor was selected due to the flat dose response curve in the non-GLP range finding study
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: GLP range-finding test with nominal test item concentrations of 100,10.0, 1.00, 0.100, 0.0100 mg/L and control, was used to decide upon the test concentrations
- Test concentrations: 100, 25.0, 6.25, 1.56, 0.391 mg/L and control

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is tested in a separate study twice a year at the test facility

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.715 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.246 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.463 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Flocculation: only singular cells

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 0.703 mg/L

Reported statistics and error estimates:

The statistical evaluation for the 72 hours period was performed for growth rate and yield using SAS® (2002–2010).
A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Levene Test. The NOEC and LOEC were determined by using a multiple comparison method (Dunnetts-ttest, left sided, for growth rate and yield). The EC10, 20, 50-values for were determined by probit analysis following the normal (growth rate nominal and actual; yield actual) and logistic (yield nominal) distribution, respectively. Due to statistical reasons inhibition-values above 100 % were set to 100 and values below 0 % were set to zero. Only concentrations within a clear concentration response relation were used for calculations.

Any other information on results incl. tables

Biological results:

Average cell numbers:

Conc.	Average cell numbers [10^4/mL]
[mg/L]	0 h	24 h	48 h	72 h
Control	0.6	2.39	16.55	68.77
0.391	0.6	2.36	15.68	68.81
1.56	0.6	2.31	17.03	72.69
6.25	0.6	2.17	13.11	52.7
25	0.6	1.59	2.92	4.45
100	0.6	0.72	0.56	0.41

Percentage inhibition of growth rate and yield:

Conc.	% Inhibition of growth rate	% Inhibition of yield
[mg/L]	0 h – 72 h	0 h – 72 h
Control	0	0
0.391	0.2	-0.1
1.56	-1	-5.8
6.25	5.8	23.6
25	57.9	94.4
100	108	100.3

Analytical results:

Test item, nominal concentration (mg/L)	sampling	measured concentration (mg/L)	% of nominal	geometric mean (mg/L)
Control	0h fresh	< LOQ	-	-
	24h aged	< LOQ	-
	48h aged	< LOQ	-
	72h aged	< LOQ	-
0.391	0h fresh	0.422	108	0,117
	24h aged	0,176	45
	48h aged	< LOQ	-
	72h aged	< LOQ	-
1.56	0h fresh	1.5	96,2	0.246
	24h aged	0.974	62.4
	48h aged	< LOQ	-
	72h aged	< LOQ	-
6.25	0h fresh	6.11	97.8	0.463
	24h aged	3	48
	48h aged	< LOQ	-
	72h aged	< LOQ	-
25	0h fresh	20.8	83.2	3.05
	24h aged	23.7	94.8
	48h aged	3.47	13.9
	72h aged	< LOQ	-
100	0h fresh	101	101	44.1
	24h aged	92.3	92.3
	48h aged	76.3	76.3
	72h aged	5.31	5.31

Toxicological endpoints for the test item:

	Test item[mg/L]
	nominal	actual /95 % confidence limits
ErC10(Growth rate)^1)	8.37	0.715
95 % confidential limits	6.48 - 10.1	0.525 – 0.906
ErC20^1)	11.4	1.09
95 % confidential limits	9.27 - 13.4	0.855 – 1.34
ErC50^1)	20.4	2.45
95 % confidential limits	17.6 - 23.6	2.01 – 3.07
EyC10(Yield)^2)	4.53	0.360^1)
95 % confidential limits	3.62 - 5.35	0.0792 – 0.654
EyC20^2)	5.93	0.493^1)
95 % confidential limits	4.98 - 6.84	0.167 – 0.932
EyC50^2)	9.40	0.900^1)
95 % confidential limits	8.20 - 10.9	0.477 – 2.67
NOEC^3)	1.56	0.246
LOEC^3)	6.25	0.463

¹⁾Probit analysis following normal distribution

²⁾Probit analysis following logistic distribution

³⁾Following Dunnetts-t-test (left-sided, p<0.05) for growth rate and for yield

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Significant inhibitory effects were determined for growth rate and yield at test item concentrations of 6.25 mg/L (nominal) and 0.463 mg/L (actual) and above. The EC50 values for growth rate and yield inhibition were found to be 2.45 mg/L and 0.9 mg/L respectively (measured, geometric mean). The EC10 values for growth rate and yield inhibition were found to be 0.715 mg/L and 0.360 mg/L respectively (measured, geometric mean).

Executive summary:

The objective of this study was to determine the inhibitory effects of the test item on the growth of the single cell green alga Pseudokirchneriella subcapitata. The study has been performed according to OECD TG 201 and in compliance to GLP.

Based on the results of a GLP range-finding test with nominal test item concentrations of 100, 10.0, 1.00, 0.100, 0.0100 mg/L and control, the following nominal test item concentrations were tested in the main test (spaced by a factor of 4.0): 100, 25.0, 6.25, 1.56, 0.391 mg/L and control. Six replicates were employed for the control and three for each test item concentration. Three additional replicates per treatment group containing algae were set up for the analytical sampling.

Analytical samples were taken and analysed in the main test from control and all test item concentrations at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and 72 hours from aged test solutions. Since the test concentration decreased over time, the effect concentrations have been expressed as measured, geometric mean values.

All test validity criteria were fulfilled.

Significant inhibitory effects were determined for growth rate and yield at test item concentrations of 6.25 mg/L (nominal) and 0.463 mg/L (measured, geometric mean) and above. The overall LOEC was therefore determined to be 6.25 mg/L (nominal) and 0.463 mg/L (measured, geometric mean), the corresponding NOEC was set at 1.56 mg/L (nominal) and 0.246 mg/L(measured, geometric mean).

The EC10-value for growth rate (ErC10) was determined to be 8.37 mg/L (nominal) and 0.715 mg/L

The objective of this study was to determine the inhibitory effects of the test item on the growth of the single cell green alga Pseudokirchneriella subcapitata. The study has been performed according to OECD TG 201 and in compliance to GLP.

Based on the results of a GLP range-finding test with nominal test item concentrations of 100, 10.0, 1.00, 0.100, 0.0100 mg/L and control, the following nominal test item concentrations were tested in the main test (spaced by a factor of 4.0): 100, 25.0, 6.25, 1.56, 0.391 mg/L and control. Six replicates were employed for the control and three for each test item concentration. Three additional replicates per treatment group containing algae were set up for the analytical sampling.

Analytical samples were taken and analysed in the main test from control and all test item concentrations at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and 72 hours from aged test solutions. Since the test concentration decreased over time, the effect concentrations have been expressed as measured, geometric mean values.

All test validity criteria were fulfilled.

Significant inhibitory effects were determined for growth rate and yield at test item concentrations of 6.25 mg/L (nominal) and 0.463 mg/L (measured, geometric mean) and above. The overall LOEC was therefore determined to be 6.25 mg/L (nominal) and 0.463 mg/L

(measured, geometric mean), the corresponding NOEC was set at 1.56 mg/L (nominal) and 0.246 mg/L

(measured, geometric mean)

The EC10-value for growth rate (ErC10) was determined to be 8.37 mg/L (nominal) and 0.715 mg/L (measured, geometric mean). The EC10-value for yield (EyC10) was 4.53 mg/L (nominal) and 0.360 mg/L (measured geometric mean).

The EC50-value for growth rate (ErC50) was determined to be 20.4 mg/L (nominal) and 2.45 mg/L (measured, geometric mean). The EC50-value for yield (EyC50) was 9.40 mg/L (nominal) and 0.900 mg/L (measured, geometric mean).