phenyl pent-4-enoate

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected June 2013; signature: November 2013

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All relevant concentration levels and the control were analytically verified via HPLC-DAD at the start (0 hours), 24 hours and at the end of the exposure (72 hours).
- Sampling method: Analytical evaluation of the concentrations of the test item were carried out via HPLC-DAD from freshly prepared media after 0 hours (with algae), 24 hours (with algae) and old test media after 72 hours (with algae) of exposure. Three replicates per concentration and six for the control (without test item) were prepared. Separate replicates for each measuring time were prepared. All samples for the test item analysis were taken from additionally prepared replicates with algae. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).
- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C if necessary. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDÁK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE·m-2·s-1 for 24 hours per day.

ACCLIMATION
- Acclimation period: No. However, a three days old preculture, prepared in dilution water, was used as inoculum.
- Culturing media and conditions (same as test or not): No. Culture Medium: Nutrient medium Z according to LÜTTGE et al. (1994) Botanica Acta, Journal of the German Botanical Society, No. 3 Volume 107 page 111-186 (June 1994), THIEME-VERLAG.
Dilution water: (mg/L) - 3x AAP dilution water:
NaNO3 76.5, MgCI2.6H2O 36.492, CaCI2.2H2O 13.23, MgSO4.7H20 44.1, K2HPO4 3.132, NaHC03 45, H3BO3 0.5565, MnCI2.4H2O 1.246, ZnCI2 9.81 x 10^-3, CoCI2.6H2O 4.29 x10^-3, CuCI2.2H2O 0.036 x10^-3, NaMoO4.2H2O 21.78 x10^-3, FeCl3.6H2O 0.4794, Na2-EDTA 0.9, MES monohydrate* 2665.6, NaHCO3* 250. pH 7.5 +/- 0.1. * additional compounds were added to enable sufficient growth under conditions without headspace.
- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

Nominal range: 21 - 24 °C, controlled at ± 2°C - measured room temperature values were min: 21.5 ; max 23.0 and mean 22.3 °C

pH:

0 hours: pH 7.42 ± 0.1 (and 7.48 in control); 72 hours: pH 7.47-7.69 (definitive test concentrations) and pH 8.36 (controls). pH did not vary more than 1.5 units.

Nominal and measured concentrations:

Preliminary test: 0 (control), 1.0, 10, 50 and 100 mg/L as a nominal test item concentration (non-GLP range finding test)
Final test: Solutions containing: 0 (control), 7.90, 11.9, 17.8, 26.7 and 40.00 mg/L
See table 3 for nominal and geometric mean measured concentrations at initial exposure and during the course of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass.
- Type: Closed - Static. Sterile headspace flasks
- Material, size, headspace, fill volume: , volume: 59 mL, with aluminium tops with PFTE seals. Minimum headspace.
- Aeration: Vessel shaken continuously. Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: nominal: 5 x 10^3 - 10^4 and current: 6153 cells/ml
- Control end cells density: Mean (of replicates after 72 hours) 642046 cells/ml (or ca. x104 increase in cell density)
- No. of vessels per concentration (replicates): 3 replicates of each test concentration; 1 extra replicate of each test group for sampling purposes
- No. of vessels per control (replicates): 6 replicates of the control
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. x3 AAP medium used (see guideline OECD TG 201)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Prepared according to guidelines (see 'Details on test organisms' field for more details on composition).
- Culture medium different from test medium: Yes. (see 'Details on test organisms' field)
- Intervals of water quality measurement: Start and end of the test period.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 60 - 120 µE/m2/s ; within ± 15 % over incubation area
- Salinity (for marine algae): Not applicable.

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. Dilution water was used as background signal. No self-fluorescence was found at the concentration level of 40.00 mg/L in the range finding test.
- Other: Initial cell density: Microscopic evaluation of the cells was carried out at the start and end of the exposure. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0. In definitive test justified from the results of the range finding study.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: Three replicates per concentration were exposed to dilutions representing 1.0, 10, 50 and 100 mg/L based on nominal test item concentration in a preliminary test.
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: 9.87 - 12.9 mg/L ; based on geometric mean measured concentrations

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 4.35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: not available ; based on geometric mean measured concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 4.35 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.:

< 4.35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% C.I.: not available ; based on geometric mean measured concentrations

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 4.35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% C.I.: not available ; based on geometric mean measured concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 4.35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% C.I.: < 7.90 - 8.59 mg/L ; based on nominal concentrations

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 7.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: not available ; based on nominal concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 7.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on nominal concentrations

Details on results:

- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start of the incubation period and at test end revealed no morphological abnormalities in the test item concentration or control.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels:
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: No low dose stimulation was observed in the definitive test. Within the preliminary test: Yes. At low concentrations 1.00 mg/L nominal test item concentration a growth rate and yield inhibition was negative at 72h. This was low dose stimulation and was taken into account during the study.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate reduction (ERC50: 0-72h) was 1.15 mg/L with a 95% confidence interval ranging from 0.922 to 1.32 mg/L without headspace. The EC50 for yield inhibition (EYC50: 0-72h) was 0.509 mg/L with a 95% confidence interval ranging from 0.443 to 0.574 mg/L without headspace.
The results with headspace and without headspace were within the test facility SOPs (historic values).
- Other: The sensitivity of the test system was in agreement with the historical data.

Reported statistics and error estimates:

EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The NOEC / LOEC was determined by calculation of statistically significant differences of growth rate and yield using Williams Sequential t-test Procedure. A Shapiro-Wilk's Normality test and a Levene's Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The a-value
(acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.01
Test for Normality
- Results Growth rate: Shapiro-Wilk's Normality check was passed (P> 0.01). Levene's test indicated non-homogeneous variances. Therefore Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm indicated that the NOEC was lower than 7.900 mg/L. The LOEC was indicated to be 4.35 mg/L.
- Results for Yield: Shapiro-Wilk's Normality check was passed (P> 0.01). The Levene test indicates variance homogeneity (P > 0.010). Trend analysis by Contrasts (Monotonicity of Concentration/Response) revealed a linear trend. thus the selected Williams test was performed. The Williams Multiple Sequential t-test Procedure indicated that the NOEC was lower than 7.900 mg/L. The LOEC was indicated to be 4.35 mg/L.

Table 1. Results of the Preliminary Range Finding Test (non GLP, 0 - 72 hours)

Nominal Test Item Concentration [mg/L]	Growth Rate Inhibition [%]	Yield Inhibition [%]
100	100	100
50.0	100	100
10.0	0	1
1.00	-3	-13

Negative values = growth stimulation

 

Table 2. Percentage reduction in growth rate and inhibition of yield in the definitive test (at 72 hours)

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-)

Nominal test item concentration	Geometric mean measured test item concentration*	Replicate		Growth rate	Growth rate inhibition		Yield	Yield Inhibition
[mg/L]	[mg/L]	No.		[d-1]	[%]		[Cells/mL]	[%]
40.0	36.6	1		n.a.	100		n.a.	100
		2		n.a.	100		n.a.	100
		3		n.a.	100		n.a.	100
		Mean	(+)	n.a.	100	(+)	n.a.	100
26.7	18.0	1		-0.09	100		-1480	100
		2		-0.04	100		-753	100
		3		0.12	93		2556	100
		Mean	(+)	-0.01	100	(+)	108	100
17.8	16.4	1		0.86	45		74624	88
		2		0.70	55		44437	93
		3		0.92	40		92223	86
		Mean	(+)	0.83	47	(+)	70428	89
11.9	11.2	1		1.00	36		115781	82
		2		0.92	41		90129	86
		3		1.02	34		126525	80
		Mean	(+)	0.98	37	(+)	110812	83
7.90	4.35	1		1.36	12		354280	44
		2		1.31	15		306864	52
		3		1.37	11		371591	42
		Mean	(+)	1.35	13	(+)	344245	46
Control		1		1.54			611683
		2		1.57			670859
		3		1.55			630875
		4		1.52			575525
		5		1.55			643576
		6		1.57			682839
		Mean		1.55			635893

n.a. = not applicable, cell density < limit of quantification (2736 cells/mL)

* = excluding the values < LOQ

 

Table 3. Measured Concentrations and Percentage of the nominal Concentration of the Test Item

Sampling			24 h		48 h		72 h
Nominal test item concentration [mg/L]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Geometric Mean measured concentration after 72 h [mg/L]	Percentage to Nominal test item concentration [%]
40.0	38.1	95	37.5	94	35.9	90	34.8	87	36.6	91.5
26.7	24.6	92	24.3	91	9.68	36	< LOQ*		18.0	67.4
17.8	16.4	92	16.5	93	< LOQ*		< LOQ*		16.4	92.1
11.9	11.2	94	11.2	94	< LOQ*		< LOQ*		11.2	94.1
7.90	7.53	95	7.28	92	6.35	80	1.03	13	4.35	55.1
Control	< LOQ		< LOQ		< LOQ		< LOQ		< LOQ	< LOQ

Meas. conc. = Measured concentration of the test item, dilution factor taken into account

% = Percentage of nominal concentration of the test item

LOQ = Limit of quantification (0.8 mg/L)

*= Not taken into account for evaluation of Geometric mean

Validity criteria fulfilled:

yes

Conclusions:

The EC50 for growth rate reduction (72h-ErC50) based on geometric mean measured concentrations, but excluding measured values lower than the Limit of Quantification was 11.5 (C.I. 9.87 - 12.9) mg/L. The corresponding EC10 was < 4.35 mg/L and the NOEC was < 4.35 mg/L.

Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6153 cells/mL. The definitive test was repeated due to varying 0 hour analytical recoveries of the test item concentration. After a change in test media preparation the analytical recoveries at 0 hours was in the range of 80 – 120 % nominal test item concentration. The definitive test as reported was conducted with regard to the volatility of the test item, glass flasks without headspace were used to reduce losses of the test item. Five concentrations were freshly prepared with dilution water (adjusted to pH 6 ± 0.1 with 1M NaOH or HCI). The solutions were stirred for 24 hours (1100 rpm, 30°C ± 2°C) with a magnetic stirrer. Six test item solutions were employed with a dilution factor of 1.5 (nominal) and concentrations of 0 (control), 7.90, 11.9, 17.8, 26.7 and 40.0 mg/L. This corresponded to a geometric mean measured test item concentrations after 72 hours (excluding the values lower than the LOQ) of 0 (control), 4.35, 11.2, 16.4, 18.0, 36.6 mg/L, respectively. Three replicates were tested for each test item concentration and six replicates for the control. The test item solutions were clear (negative Tyndall effect) and the test item was dissolved throughout the test period. The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were fulfilled. The concentrations of the test item were analytically verified via HPLC-DAD at test start (0 hours), after 24 hours and at the end (72 hours) of the exposure. The recovery rates of the test item at the start were in the range of 92 - 95 %. After 24 and 48 hours, the recovery ranges were 91 - 94 % and < Limit of Quantification (LOQ), respectively. At the end of the test, concentrations were in the range of < LOQ to 87%. Based on the decline of the analytical recoveries and the nature of the test item (chemical class ester), the observed effects may be related to the test item or possible degradation products. All effect values were based on the nominal test item concentration after 72 hours and the geometric mean measured test item concentrations after 72 hours, excluding the values lower than the LOQ. This approach was used since analytical recoveries varied between the concentrations in a non-concentration related manner. The EC50 for growth rate reduction (72h-ErC50) based on nominal concentrations was 8.14 (C.I. < 7.90 – 8.59) mg/L. The corresponding EC10 was < 4.35 mg/L and the NOEC was < 4.35 mg/L. The EC50 for growth rate reduction (72h-ErC50) based on geometric mean measured concentrations, but excluding measured values lower than the Limit of Quantification was 11.5 (C.I. 9.87 - 12.9) mg/L. The corresponding EC10 was < 4.35 mg/L and the NOEC was < 4.35 mg/L.

Description of key information

ErC50 (algae; growth rate) = 11.5 (C.I. 9.87 - 12.9) mg/L GMM concentration, 72-hour, freshwater, OECD TG 201, 2017

Key value for chemical safety assessment

EC50 for freshwater algae:

11.5 mg/L

Additional information

Key study: OECD TG 202, 2017 : The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6153 cells/mL. The definitive test was conducted with regard to the volatility of the test item, glass flasks without headspace were used to reduce losses of the test item. Five concentrations were freshly prepared with dilution water (adjusted to pH 6 ± 0.1 with 1M NaOH or HCI). The solutions were stirred for 24 hours (1100 rpm, 30°C ± 2°C) with a magnetic stirrer. Six test item solutions were employed with a dilution factor of 1.5 (nominal) and concentrations of 0 (control), 7.90, 11.9, 17.8, 26.7 and 40.0 mg/L. This corresponded to a geometric mean measured test item concentrations after 72 hours (excluding the values lower than the LOQ) of 0 (control), 4.35, 11.2, 16.4, 18.0, 36.6 mg/L, respectively. Three replicates were tested for each test item concentration and six replicates for the control. The test item solutions were clear (negative Tyndall effect) and the test item was dissolved throughout the test period. The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were fulfilled. The concentrations of the test item were analytically verified via HPLC-DAD at test start (0 hours), after 24 hours and at the end (72 hours) of the exposure. The recovery rates of the test item at the start were in the range of 92 - 95 %. After 24 and 48 hours, the recovery ranges were 91 - 94 % and < Limit of Quantification (LOQ), respectively. At the end of the test, concentrations were in the range of < LOQ to 87%. Based on the decline of the analytical recoveries and the nature of the test item (chemical class ester), the observed effects may be related to the test item or possible degradation products. All effect values were based on the nominal test item concentration after 72 hours and the geometric mean measured test item concentrations after 72 hours, excluding the values lower than the LOQ. This approach was used since analytical recoveries varied between the concentrations in a non-concentration related manner. The EC50 for growth rate reduction (72h-ErC50) based on nominal concentrations was 8.14 (C.I. < 7.90 – 8.59) mg/L. The corresponding EC10 was < 4.35 mg/L and the NOEC was < 4.35 mg/L. The EC50 for growth rate reduction (72h-ErC50) based on geometric mean measured concentrations, but excluding measured values lower than the Limit of Quantification was 11.5 (C.I. 9.87 - 12.9) mg/L. The corresponding EC10 was < 4.35 mg/L and the NOEC was < 4.35 mg/L.