Ethyl pyruvate

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:

24 November 2017 - 31 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl pyruvate

EC Number:

210-511-2

EC Name:

Ethyl pyruvate

Cas Number:

617-35-6

Molecular formula:

C5H8O3

IUPAC Name:

ethyl 2-oxopropanoate

Test material form:

liquid

Details on test material:

Identification: EC#210-511-2
CAS No.: 89800-10-2
Chemical name: ethyl pyruvate
Appearance/physical state: very pale yellow liquid
Batch: 61112
Purity: 98.54%
Expiry date: 30 September 2018
Storage conditions: room temperature, in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1, 3.2, 10, 32, 100 mg/L
- Sampling method: Samples taken from controls and test groups at 0, 24, 48, and 72 hours for immediate quantitative analysis
- Sample storage conditions before analysis: frozen

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was dissolved directly in culture medium.
- Controls: biological control (no test item), and positive control (potassium dichromate)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not specified

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): not specified
- Method of cultivation: not specified

ACCLIMATION
- Acclimation: Maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: no

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Not specified

Test temperature:

24 ±1 °C

pH:

Control: 7.7
0.15 mg/L: 7.5 - 8.3
0.22 mg/L: 7.5 - 8.2
0.35 mg/L: 7.5 - 8.2
1.5 mg/L: 7.1 - 8.0
31 mg/L: 6.6 - 7.6

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal - 1.0, 3.2, 10, 32, 100 mg/L
Measured - 0.15, 0.22, 0.35, 1.5, 31 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed (plugged with polyurethane foam bungs)
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 5.00 x 10e3 cells per mL
- Control end cells density: 8.97 x 10e5 cells per mL
- No. of organisms per vessel: 5.00 x 10e3 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: n/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionized water
- Culture medium different from test medium: no
- Intervals of water quality measurement: Initiation of the test and after 72 hours exposure

OTHER TEST CONDITIONS
- Sterile test conditions: not specified
- Adjustment of pH: Adjusted to 7.5 with 0.1N NaOH or HCl
- Photoperiod: continuous illumination
- Light intensity and quality: 7000 lux, provided by warm white lighting (380 to 730 nm)
- Salinity (for marine algae): not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Coulter® Multisizer Particle Counter
- Chlorophyll measurement: not specified
- Other: not specified

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not specified
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study: 0.10, 1.0, 10 and 100 mg/L
- Test concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 2.5 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:

NOEC

Effect conc.:

ca. 0.35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 1.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 2.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 1.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no, however misshapen cells were observed to be present in the test cultures at 1.5 and 31 mg/L.
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: not specified
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Full results in Annex 1 and Table 2.

Results with reference substance (positive control):

- Results with reference substance valid? yes

ErC50 (0 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L

No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955).

Any other information on results incl. tables

Annex 1.

Inhibition of Growth Rate

ErC10 (0 to 72 hour) : 3.6 mg/L

ErC20 (0 to 72 hour) : 7.3 mg/L

ErC50 (0 to 72 hour) : 25 mg/L

Inhibition of Yield

EyC10 (0 to 72 hour) : 0.79 mg/L

EyC20 (0 to 72 hour) : 1.0 mg/L

EyC50 (0 to 72 hour) : 2.3 mg/L; 95% confidence limits 1.6 to 3.3 mg/L

Table 1. Results for Spiked Recovery Samples

Nominal Concentration of Test Item cnom	Fortified Concentration of Test Item in the Spiked Sample cfort	Sample Preparation Factor F	Determined Concentration of Test Item in the Spiked Sample c	Mean Analytical Recovery Rate R	Precision (Relative Standard Deviation of Recovery)
[mg/L]	[mg/L]		[mg/L]	[%]	[%]
1.00	1.03	1 1 1 1 1	0.557	43	14.6
			0.404
			0.431
			0.413
			0.409
10.0	10.6	1 1 1 1 1	8.36	78	2.6
			8.40
			8.34
			7.87
			8.24
100	103	1 1 1 1 1	90.5	88	1.2
			91.8
			91.1
			92.4
			89.5
Acceptance Target				80-120	<10

Table 2. Inhibition of Growth Rate and Yield in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.074	-	1.04E+06	-
	R2	0.071		8.38E+05
	R3	0.073		9.21E+05
	R4	0.073		9.55E+05
	R5	0.072		8.67E+05
	R6	0.069		7.31E+05
	Mean	0.072		8.92E+05
	SD	0.002		1.05E+05
0.15	R1	0.078	[8]	1.36E+06
	R2	0.080	[11]	1.61E+06
	R3	0.077	[7]	1.26E+06
	Mean	0.078	[9]	1.41E+06	[58]
	SD	0.002		1.80E+05
0.22	R1	0.075	[4]	1.14E+06
	R2	0.079	[10]	1.51E+06
	R3	0.078	[8]	1.32E+06
	Mean	0.077	[7]	1.32E+06	[48]
	SD	0.002		1.88E+05
0.35	R1	0.078	[8]	1.35E+06
	R2	0.073	[1]	9.85E+05
	R3	0.073	[1]	9.58E+05
	Mean	0.075	[3]	1.10E+06	[23]
	SD	0.003		2.21E+05
1.5	R1	0.068	6	6.45E+05
	R2	0.066	8	5.58E+05
	R3	0.065	10	5.33E+05
	Mean	0.066	8	5.79E+05	35
	SD	0.002		5.90E+04
31	R1	0.034	53	5.41E+04
	R2	0.029	60	3.46E+04
	R3	0.032	56	4.43E+04
	Mean	0.032	56	4.43E+04	95
	SD	0.003		9.75E+03

*       In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R   = Replicate

-    = Not applicable

SD   =Standard Deviation

[ ] = Increase in growth as compared to the control

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:

Yield:
EC50: 2.3mg/l
NOEC: 0.35 mg/l
LOEC: 1.5 mg/l

Growth Rate:
EC50: 25 mg/l
NOEC: 0.35 mg/l
LOEC: 1.5 mg/l

Executive summary:

 Introduction

A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriellasubcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods

Following a preliminary range-finding test and stability analyses,Pseudokirchneriella subcapitatawas exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Results.

As expected for an ester, it was noted that the parent test item (ethyl pyruvate) hydrolyzed readily when added to test media, reverting to the parent carboxylic acid and alcohol in solution. Given this, samples were provided for immediate chemical analysis at 0, 24, 48 and 72 hours in bottles spiked with 1% v/v formic acid to effectively halt the hydrolysis reaction and attain the reaction equilibrium. 

 

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.46 to 93 mg/L (46% to 93% of the nominal test concentrations). It was considered that failure to attain near nominal concentrations in all test preparations was due to hydrolysis of the parent test item occurring during the time taken to prepare all serial dilutions. A decline in measured test concentrations was observed after each 24-Hour period in the range of less than the limit of quantification (LOQ), determined to be 0.21 mg/L, to 57 mg/L at 24 hours, less than the LOQ to 6.2 mg/L at 48 hours, and to less than the LOQ in all test preparations at 72 hours. 

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The geometric mean measured test concentrations were determined to be 0.15, 0.22, 0.35, 1.5 and 31 mg/L.

Exposure of Pseudokirchneriella subcapitatato the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	25		*		0.35	1.5
Yield	2.3	1.6	-	3.3	0.35	1.5

*It was not possible to calculate 95% confidence limits for the E_rC₅₀value as the data generated did not fit the models available for the calculation of confidence limits.