Carbonic acid, ethyl methyl ester

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:

Feb 12 - Feb 15, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed in compliance with the Good Laboratory Practice (GLP) regulations (revised in 1997, ENV/MC/CHEM(98)17). The method followed the recommendations of the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

433-480-9

EC Name:

-

Cas Number:

623-53-0

Molecular formula:

C4H8O3

IUPAC Name:

ethyl methyl carbonate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: A volume of test sample was diluted with acetone to give a final theoretical concentration of 20 mg/l.
- Sample storage conditions before analysis: n.a.

Test solutions

Vehicle:

yes

Details on test solutions:

The test medium (reconstituted water as vehivle and test material) was freshly prepared.

NaNO3 25.5 mg/1
MgC12.6H20 12.164 mg/1
CaC12.2H20 4.41 mg/I
MgSO4.7H20 14.7 mg/1
K2HPO4 1.044 mg/1
NaHCO3 15.0 mg/1
H3BO3 0.1855 mg/1
MnC12.4H20 0.415 mg/1
ZnC12 0.00327 mg/1
FeC13.6H20 0.159 mg/1

CoC12.6H20 0.00143 mg/I
Na2MoO4.2H20 0.00726 mg/1
CuCI2.2H20 0.000012 mg/1
Na2EDTA.2H20 0.30 mg/I
Na2SeO3.5H20 0.000010 mg/I

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCI. The prepared media was sterilised by 0.2 µm membrane filtration and stored in darkness.

Test organisms

Test organisms (species):

Scenedesmus sp.

Details on test organisms:

The test was carried out using Scenedesmus subspicatus strain CCAP 276/20. Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, The Ferry House, Far Sawrey, Ambleside, Cumbria. 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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

250 mg CaCO3

Test temperature:

24+-1°C

pH:

8

Dissolved oxygen:

80 %

Salinity:

for details see composition of reconstituted water "details on test conditions"

Nominal and measured concentrations:

Range finding study: 1, 10, 100 mg/l
Main study: 100 mg/l

Details on test conditions:

The test concentration to be used in the definitive study was determined by preliminary range-finding studies. The initial range-finding study was conducted by exposing Scenedesmus subspicatus cells to a series of nominal test concentrations of 1.0, 10 and 100 mg/I for a period of 72 hours.

Due to the volatile nature of the test material the study was conducted in 250 tnl glass conical flasks sealed with ground glass stoppers to reduce evaporation/volatilisation of the test material. Two replicate flasks were prepared for each control and test concentration. The test material was dissolved directly in culture medium.

An amount of test material (100 mg) was dissolved in culture medium and the volume adjusted to 500 ml to give a 200 mg/l stock solution from which a series of dilutions were made to give further stock solutions of 20 and 2.0 mg/I. An aliquot (100 ml) of each of the stock solutions was separately mixed with algal suspension (100 ml) to give the required test concentrations of 1.0, 10 and 100 mg/l.

The results of the initial range-finding study indicated that at higher test concentrations, algal growth was stimulated. Therefore in order to confirm these findings a second range-finding study was conducted at concentrations of 1.0, 10 and 100 mgll.

An amount of test material (100mg) was dissolved in culture medium with the aid of ultrasonication (for approximately 10 minutes) and the volume adjusted to 500 ml to give a 200 mg/1 stock solution. A series of dilutions were made from this stock solution to give further stock solutions of 20 and 2.0 mg/I. An aliquot (100 ml) of each of the stock solutions was separately mixed with algal suspension (100 ml) to give the required test concentrations of 1.0, 10 and 100 mg/I.

In each range-finding study the stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The control group was maintained under identical conditions but not exposed to the test material.

At the start of the range-finding studies a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer II Particle Counter. The flasks were then sealed with ground glass stoppers and incubated (Gallenkamp INR - 401-010W) at 24± 1°C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 100 rpm for 72 hours.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer lI Particle Counter.


Based on the result of the range-finding studies a "limit test" was conducted for the definitive study at a test concentration of 100 mg/L confirm that at the maximum test concentration given in the OECD/EEC Test Guidelines, no adverse effect on algal growth was observed.

Exposure conditions
As in the range-finding study 250 ml glass conical flasks were used. Six flasks each containing 100 ml of solution were prepared for the treatment group and three flasks each containing 100 ml were prepared for the control.
The control group was maintained under identical conditions but not exposed to the test material.

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.35 x 10^6 cells per ml. This suspension was diluted to a cell density of 2.32 x 10^4 cells per ml prior to use. At initiation of the study the culture contained a nominal cell density of 10^4 cells per ml.

Due to the volatile nature of the test material, the flasks were sealed with ground glass stoppers and incubated (Gallenkamp INR-401-010W) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 100 rpm for 72 hours.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter° Multisizer II Particle Counter.

The pH of each control and test flask was determined at initiation of the study and after 72 hours exposure. The temperature within the incubator was recorded daily.

Water samples were taken from the control (replicates R1 - R3 pooled) and the 100 mg/i test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at each occasion and stored frozen (approximately -20°C) for further analysis if necessary.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 62 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 62 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 62 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Range-finding Studies
The results of both range-finding studies showed no adverse effect on growth at the test concentrations of 1.0, 10 and 100 mg/l. Indeed algal growth increased relative to the control cultures at the higher test concentrations.

Definitive Study
From the data, it is clear that neither the growth (r) or the biomass (b) of Scenedesmus subspicatus (CCAP 276/20) was affected by the presence of the test material over the 72-hour exposure period.

The results obtained for the test material vessels in the definitive study confirmed the findings of the two range-finding studies in which a significant increase in algal growth was observed al the test concentration of 100 mg/l. This was considered to be due to a possible hormetic effect whereby biological processes, such as growth, are stimulated by the presence of a substance at concentrations that are below the toxic level of the substance to the test organism.

The EC50 value with respect to biomass, EbCsn (72h), was determined by inspection of the area under the growth curve data after 72 hours.

The EC50 value with respect to growth rate, ErC50 (0 - 72 h), was determined by inspection of the growth rates for the period 0 - 72 hours.

Accordingly the following results were determined from the data:
EbC5a (72 h) : >100 mg/I
ErC50 (0 - 72 h): >100 mg/1

Observations on cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control cultures, however, some cell debris was observed to be present in the test cultures.

Observations on test material solubility
At 0 hours the control and test cultures were all observed to be clear colourless solutions. After the 72 hour study duration the control cultures were observed to be pale green dispersions and the 100 mg/I test cultures were observed to be bright green dispersions. This was due to the presence of algal cells.

The pH values of the control cultures were observed to increase from pH 7.5 at 0 hours to pH 10.1 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation C02 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. This effect was increased by the requirement to use test vessels sealed with ground glass stoppers due to the volatile nature of the test material and hence gaseous exchange between the test vessels and the atmosphere was prevented. The increase in pH after 72 hours was in excess of that recommended in the EEC Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

Chemical analysis of the test solutions at 0 hours showed measured test concentrations ranging from 103% to 104% of nominal. Analysis of the test solutions at 72 hours showed a marked decline in measured test concentrations to 33% of nominal. Given that the pre-study stability analysis conducted showed the test material to be stable in culture medium over the 72-hour study period this decline was considered to be due to one or a combination of the following:

I. Volatile nature of the test material - despite efforts to minimise losses of test material, i.e the use of sealed vessels, it was not possible to completely seal the test system due to the requirement to open the test vessels after each 24 hour period in order to take samples for the determination of cell density. This was not apparent in the pre-study stability analyses as sealed vessels with a minimum head space were used.

2. Adsorption to algal cells - the initial recovery analyses conducted showed that the test material did not immediately adsorb to algal cells, however this does not preclude long term adsorption to algal cells over the study period. Adsorption was not a factor in the pre-study stability analyses as no algal cells were present.

Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations in order to give a "worst case" analysis of the data. The time-weighted mean measured test concentrations were determined to be:

-----------------------------------------------------------------------------------------------------------------------
Nominal Concentration (mg/I) Time-Weighted Mean Measured Test Expressed as a % of the Nominal Test
Concentration (mg/l) Concentration
-----------------------------------------------------------------------------------------------------------------------
100 (R1-R3) 62 62
100 (R4-R6) 61 61
-----------------------------------------------------------------------------------------------------------------------
The following results are based on the time-weighted mean measured test concentrations:

EbC50 (72 h) > 62 mg/I (meas. TWA) or > 100 mg/L (nominal)
ErC50 (0-72 h) > 62 mg/I (meas. TWA) or > 100 mg/L (nominal)
No Observed Effect Concentration (NOEC) > 62 mg/I (meas. TWA) or > 100 mg/L (nominal)

The use of the time-weighted mean measured test concentrations (TWA) in the calculation of the EC50 and NOEC values had a significant effect on the results of the study. This was considered to be due to the volatile nature of the test material under the study conditions and/or adsorption of the test material to algal cells

Any other information on results incl. tables

see attached table

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

Purpose
The purpose of this assay was to identify the aquatic toxicity potential of the test material in algea to provide a rational basis for hazard estimation for the test item in aquatic environments.

Study Design
The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).
Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to an aqueous solution of the test material at a concentration of 100 mg/1 (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 daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer II Particle Counter.


Results
Exposure of Scenedesfnus subspicatus to the test material gave EC50 values of greater than 100 mg/l and correspondingly the No Observed Effect Concentration was greater than 100 mg/l.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/I.
Chemical analysis of the test solutions at 0 hours showed measured test concentrations ranging from 103% to 104% of nominal. Analysis of the test solutions at 72 hours showed a marked decline in measured test concentrations to 33% of nominal. Given that the pre-study stability analysis conducted showed the test material to be stable in culture medium over the 72 hour study period this decline was considered to be due to the volatile nature of the test material and/or adsorption of the test material to algal cells.
Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations in order to give a "worst case" analysis of the data. The EC50 values based on the time-weighted mean measured test concentrations were greater than 62 mg/1 and correspondingly the No Observed Effect Concentration was greater than 62 mg/l.

Conclusion
The effect of the test material on the growth of Scenedesmus subspicatus has been investigated and gave EC50 values of greater than nominal 100 mg/1. Correspondingly the No Observed Effect Concentration was greater than 100 mg/1.
Based on the time-weighted mean measured test concentrations the EC50 values were estimated to be greater than 62 mg/l. Correspondingly the No Observed Effect Concentration was greater than 62 mg/1.