Ethyl 2-methyl-1,3-dioxolane-2-acetate

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:

8 April 2013 to 25 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and the 100 mg/L test group (replicates R1 - R6 pooled) at 0 and 72 hours for quantitative analysis. All 0-hour samples were stored at approximately -20 °C prior to analysis, whilst the 72 hour samples were analyzed on the day of receipt. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20 °C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
100 mg of test material was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/L stock solution. This stock solution was inoculated with algal suspension (6.5 mL) to give the required test concentration of 100 mg/L.
The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4

CULTURE MEDIA AND CONDITIONS
Master cultures were maintained by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

7.3 - 7.4 (0 hours); 8.1 - 8.4 (72 hours)

Nominal and measured concentrations:

0, 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed (the test vessels were plugged with polyurethane foam bungs)
- Fill volume: 100 mL
- Aeration: the flasks were constantly shaken at approximately 150 rpm for 72 hours
- Initial cells density: 5 x 10^3 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition of medium used: 25.5 mg/L NaNO3, 12.16 mg/L MgCl2.6H2O, 4.41 mg/L CaCl2.2H2O, 14.6 mg/L MgSO4.7H2O, 1.044 mg/L K2HPO4, 15.0 mg/L NaHCO3, 0.186 mg/L H3BO3, 0.415 mg/L MnCl2.4H2O, 0.00327 mg/L ZnCl2, 0.160 mg/L FeCl3.6H2O, 0.00143 mg/L CoCl2.6H2O, 0.00726 mg/L NaMoO4.2H2O, 0.000012 mg/L CuCl2.2H2O, 0.30 mg/L Na2EDTA.2H2O
The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH of HCl.

TEST MEDIUM
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux, provided by warm white lighting (380 - 730 nm)

EFFECT PARAMETERS MEASURED
Samples were taken at 0, 23, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter

WATER QUALITY MEASUREMENT
The pH of the control and 100 mg/L treatment group was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

TEST CONCENTRATIONS
- Range finding study: The range-finding study was conducted under the same conditions as were used in the definitive study except only 2 replicate flasks were included for each control and test concentration.
- Test concentrations: 0, 0.10, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (0.25, 0.50, 1.0, 2.0, 4.0 mg/L)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

RANGE-FINDING TEST
The results of the range-finding test showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100 mg/L. Based on this information a single test concentration of six replicates, of 100 mg/L was selected for the definitive test.
Chemical analysis of the 100 mg/L test preparation at 0 and 72 hours showed measured test concentrations to range from 134% to 137% of nominal indicating that the test material was stable over the test duration. Whilst the measured concentrations obtained were in excess of 120% of nominal this was considered to have had no adverse effect on the outcome of the test given that no effect on growth was observed.

DEFINITIVE TEST
Neither the growth rate (r) nor the yield (y) of the algae tested was affected by the presence of the test material at a nominal concentration of 100 mg/L over the 72 hour exposure period. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.
- Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.
- Observations on Test Material Solubility
At the start of the test all control and test cultures were observed to be clear and colourless solutions. After the 72 hour test period all control and test cultures were observed to be green dispersions.
- Physico-Chemical Measurements
The pH value of the control cultures was observed to increase from pH 7.4 a 0 hours to pH 8.1 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the test guidelines.
- Verification of Test Concentrations
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 122% to 126% of nominal and so the results are based on nominal test concentrations only. Whilst the measured concentrations obtained were in excess of 120% of nominal this was considered to have had no adverse effect on the outcome of the test given that no effect on growth was observed.

Results with reference substance (positive control):

Exposure of Pseudokirchneriella subcapitata to the reference item gave the following results:
ErC50 (0 – 72 h) : 1.2 mg/L; 95% confidence limits 1.0 – 1.3 mg/L
EyC50 (0 – 72 h) : 0.52 mg/L; 95% confidence limits 0.45 – 0.62 mg/L

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

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/L test concentration 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).

Table 1: Definitive Test Results

Nominal conc. (mg/L)		Cell densities (cells/mL)				Growth rate (cells/mL/hour)		Yield (cells/mL)
		0 h	23 h	48 h	72 h	0 - 72 h	% inhibition	0 - 72 h	% inhibition*
Control	R1	5.73^3	2.61^4	1.54^5	7.62^5	0.070	-	7.56^5	-
	R2	7.50^3	2.68^4	1.32^5	6.72^5	0.068		6.64^5
	R3	6.24^3	2.59^4	1.68^5	8.71^5	0.072		8.65^5
	R4	6.18^3	2.40^4	1.59^5	8.93^5	0.072		8.87^5
	R5	7.62^3	2.61^4	1.18^5	7.09^5	0.069		7.02^5
	R6	7.04^3	2.42^4	1.02^5	6.03^5	0.067		5.96^5
	Mean	6.72^3	2.55^4	1.39^5	7.52^5	0.070		7.45^5
100	R1	6.91^3	2.63^4	1.64^5	9.12^5	0.072	[3]	9.05^5
	R2	5.94^3	2.55^4	1.62^5	7.96^5	0.070	0	7.90^5
	R3	6.61^3	2.74^4	1.49^5	7.24^5	0.069	1	7.18^5
	R4	5.81^3	3.93^4	1.47^5	7.91^5	0.070	0	7.85^5
	R5	7.89^3	3.11^4	1.67^5	8.04^5	0.071	[1]	7.96^5
	R6	5.71^3	2.65^4	1.46^5	7.14^5	0.069	1	7.09^5
	Mean	6.48^3	2.94^4	1.56^5	7.90^5	0.070	[0]	7.84^5	[5]

 * In accordance with the OECD test guideline only the mean value for yield was calculated

R1 - R6 = replicates 1 to 6

[increase in growth as compared to controls]

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 72-hour EC50 was determined to be in excess of 100 mg/L, based on growth; the 72-hour NOEC was determined to be 100 mg/L.

Executive summary:

The effect of the test material on the growth of Pseudokirchneriella subcapitata was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 201 and EU Method C.3.

Following a preliminary range-finding test, the algae were exposed to an aqueous solution of the test material at a concentration of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Samples of the algal populations were removed and cell concentrations determined for each control and treatment group.

Exposure of the algae to the test material gave an EC50 value of greater than 100 mg/L and a No Observed Effect Concentration of 100 mg/L, based on both growth rate and yield. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 122% to 126% of nominal and so the results are based on nominal test concentrations only.

Description of key information

72-hour EC50 > 100 mg/L, 72-hour NOEC = 100 mg/L, based on growth rate (Pseudokirchneriella subcapitata), OECD 201, EU Method C.3, Vryenhoef (2013).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The effect of the test material on the growth of Pseudokirchneriella subcapitata was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 201 and EU Method C.3.

Following a preliminary range-finding test, the algae were exposed to an aqueous solution of the test material at a concentration of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Samples of the algal populations were removed and cell concentrations determined for each control and treatment group.

Exposure of the algae to the test material gave an EC50 value of greater than 100 mg/L and a No Observed Effect Concentration of 100 mg/L, based on both growth rate and yield. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 122% to 126% of nominal and so the results are based on nominal test concentrations only.

Since the study was conducted under GLP conditions and in accordance with standardised guidelines it was assigned a reliability score of 1 in line with the criteria of Klimisch (1997).