3-Oxazolidineethanol, 2-(1-methylethyl)-

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:

from 2008-04-08 to 2008-04-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.0; 3.2; 10; 32; 100 mg/L
- Sampling method: directly in culture medium
- Sample storage conditions before analysis: No, samples were taken at 0, 22, 46 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Amounts of test material (200 and 64 mg) were each separately dissolved in culture medium and the volume adjusted to 2 litres to give 100 and 32 mg/L stock solutions, respectively. A series of dilutions was made from these stock solutions to give further stock solutions of 10, 3.2 and 1.0 mg/L. An aliquot (1500 mL) of each of the stock solutions was separately inoculated with algal suspension (10.8 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test material degradation products in the test solutions were verified by chemical analysis at 0 and 72 hours.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CCAP 276/20
- Source: Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Method of cultivation: The cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

ACCLIMATION
- Acclimation period: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 1E+03 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 1E+04 - 1E+05 cells/mL.
- Culturing media and conditions (same as test):
NaNO3: 25.5 mg/L; MgCl2.6H2O: 12.164 mg/L; CaCl2.2H2O: 4.41 mg/L; MgSO4.7H2O: 14.7 mg/L; K2HPO4: 1.044 mg/L; NaHCO3: 15.0 mg/L; H3BO3: 0.1855 mg/L; MnCl2.4H2O: 0.415 mg/L; ZnCl2: 0.00327 mg/L; FeCl3.6H2O: 0.159 mg/L; CoCl2.6H2O: 0.00143 mg/L; Na2MoO4.2H2O: 0.00726 mg/L; CuCl2.2H2O: 0.000012 mg/L; Na2EDTA.2H2O: 0.30 mg/L, Na2SeO3.5H2O: 0.000010 mg/L.
The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl. For the purposes of the range-finding and definitive test, additional sodium bicarbonate (500 mg/L) was added to the prepared culture medium prior to use.
The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

7.6 - 7.7

Nominal and measured concentrations:

The test material was known to degrade rapidly in water to give isobutyraldehyde and diethanolamine. The relative amounts of each degradant were calculated to be 45.3 % isobutyraldehyde and 66 % diethanolamine. The test concentrations were monitored by analysis of the test samples for the degradants only. For the calculation of test sample results, the nominal concentration for each degradant was adjusted to account for their relative percentages.
Analysis of the test preparations at 0 hours showed measured test concentrations in the range of 92 % to 116 % of nominal for diethanolamine and 81 % to 87 % of nominal for isobutyraldehyde.
Analysis of the test preparations at 72 hours showed measured test concentrations in the range of 88 % to 101 % of nominal for diethanolamine and less than the limit of quantitation (LOQ) of the analytical method employed to 71 % of nominal for isobutyraldehyde.
Due to the volatile nature of the degradation product isobutyraldehyde, an additional test replicate was prepared at 0 hours and incubated alongside the test to provide samples for unopened vessel analysis at 72 hours. Analysis of the additional test replicates showed measured test concentrations in the range of 82 % to 96 % of nominal for diethanolamine and less than the LOQ to 77 % of nominal for isobutyraldehyde were obtained. Given that these measured concentrations were similar to those obtained from the test vessels which had been opened, it was considered that the decline in measured concentrations of isobutyraldehyde seen in the 72-Hour test samples was due to possible adsorption of the test material to the algal cells present rather than losses due to volatility. Whilst the preliminary recovery analyses conducted in the presence of algal cells indicated that no immediate adsorption occurred this does not preclude long-term adsorption over the test period. Adsorption was not a factor in the preliminary stability analyses conducted as no algal cells were present.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed
- Material, size, headspace, fill volume: 250 mL
- Initial cells density: 4.29E+03 cells/mL
- Control end cells density: 2.56E+05 cells/mL
- No. of organisms per vessel: 4.29E+03 cells/mL
- No. of vessels per concentration (replicates): 3 flasks
- No. of vessels per control (replicates): 6 flasks

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: continous illumination
- Light intensity and quality: intensity approximately 7000 lux provided by warm white lighting (380 - 730 nm)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Coulter® Multisizer Particle Counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: max. 3.125
- Range finding study: yes
- Test concentrations in the range finding study: 100, 10, 1.0 and 0.10 mg/L
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L. Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

85 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: yes
- Colour differences: At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, 1.0, 3.2, 10 and 32 mg/L test cultures were observed to be green dispersions whilst the 100 mg/L test cultures were observed to be pale green dispersions.
- Flocculation: no
- Adherence to test vessels: no
- pH- value: The pH values of the control cultures were observed to increase from pH 7.6 - 7.7 at 0 hours to pH 9.5 - 9.6 at 72 hours. The increase in pH after 72 hours was in excess of that recommended in the Test 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.
- Any observations that might cause a difference between measured and calculated values of the degradants isobutyraldehyde and diethanolamine : Absorption of isobutyraldehyde to algal cells is assumed due to low recovery of the substance after 72 hours in unopened vessels.

Results with reference substance (positive control):

- Results with reference substance valid: yes
- EC50: 0.31 mg/L; 95 % confidence limits 0.28 - 0.35 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) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and all test concentrations 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).

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave an ErC50 (0 - 72 h) of 110 mg/L, an EyC50 (0 - 72 h) of 85 mg/L and an EbC50 (0 - 72 h) of 90 mg/L. The Lowest Observed Effect Concentration based on growth rate, yield and biomass integral was 100 mg/L, and the No Observed Effect Concentration was 32 mg/L.

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, 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 test, Desmodesmus subspicatus was exposed to an aqueous solution of the test material at concentrations of 1.0, 3.2, 10, 32 and 100 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.

A degradation product of the test material, isobutyraldehyde, was known to be volatile and hence testing was conducted in completely filled, stoppered test vessels in order to minimise possible losses due to volatilisation. Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatus was exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 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.

 

In terms of growth rate, exposure of Desmodesmus subspicatus to the test material gave an ErC50 (0 - 72 h) value of 110 mg/L. The Lowest Observed Effect Concentration based on inhibition of growth rate was 100 mg/L and the No Observed Effect Concentration was 32 mg/L.

In terms of yield, exposure of Desmodesmus subspicatus to the test material gave an EyC50 (0 - 72 h) value of 85 mg/L. The Lowest Observed Effect Concentration based on yield was 100 mg/L and the No Observed Effect Concentration was 32 mg/L.

In terms of biomass integral (area under growth curve), exposure of Desmodesmus subspicatus to the test material gave an EDC50 (0 - 72 h) value of 90 mg/L. The Lowest Observed Effect Concentration based on inhibition of biomass integral was 100 mg/L and the No Observed Effect Concentration was 32 mg/L.

 

The test material was known to degrade rapidly in water to give isobutyraldehyde and diethanolamine. The relative amounts of each degradant were calculated to be 45.3 % isobutyraldehyde and 66 % diethanolamine. The test concentrations were monitored by analysis of the test samples for the degradants only. For the calculation of test sample results, the nominal concentration for each degradant was adjusted to account for their relative percentages.

Analysis of the test preparations at 0 hours showed measured test concentrations in the range of 92 % to 116 % of nominal for diethanolamine and 81 % to 87 % of nominal for isobutyraldehyde.

Analysis of the test preparations at 72 hours showed measured test concentrations in the range of 88 % to 101 %) of nominal for diethanolamine and less than the limit of quantitation (LOQ) of the analytical method employed to 71 % of nominal for isobutyraldehyde.

Due to the volatile nature of the degradation product isobutyraldehyde, an additional test replicate was prepared at 0 hours and incubated alongside the test to provide samples for unopened vessel analysis at 72 hours. Analysis of the additional test replicates showed measured test concentrations in the range of 82 % to 96 % of nominal for diethanolamine and less than the LOQ to 77 % of nominal for isobutyraldehyde were obtained. Given that these measured concentrations were similar to those obtained from the test vessels which had been opened, it was considered that the decline in measured concentrations of isobutyraldehyde seen in the 72 -hour test samples was due to possible adsorption of the test material to the algal cells present rather than losses due to volatility. Whilst the preliminary recovery analyses conducted in the presence of algal cells indicated that no immediate adsorption occurred this does not preclude long-term adsorption over the test period. Adsorption was not a factor in the preliminary stability analyses conducted as no algal cells were present.

Given that the test preparations were a mixture of two degradants, the results are based on nominal test concentrations only.

 

Exposure of Desmodesmus subspicatus to the reference material, potassium dichromate, gave an ErC50 (0 - 72 h) of 0.57 mg/L; 95 % confidence limits 0.48 - 0.66 mg/L, an EyC50 (0 - 72 h) of 0.32 mg/L; 95 % confidence limits 0.29 - 0.35 mg/L, and an EbC50 (0 - 72 h) of 0.31 mg/L; 95 % confidence limits 0.28 - 0.35 mg/L. The Lowest Observed Effect Concentrations based on inhibition of growth rate, yield and biomass integral were 0.50, 0.125 and 0.125 mg/L, respectively, and the No Observed Effect Concentrations were 0.25, 0.0625 and 0.0625 mg/L, respectively.

Description of key information

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-hour period and gave an ErC50 (0 - 72 h) of 110 mg/L, an EyC50 (0 - 72 h) of 85 mg/L and an EbC50 (0 - 72 h) of 90 mg/L. The Lowest Observed Effect Concentration (LOEC) based on growth rate, yield and biomass integral was 100 mg/L, and the No Observed Effect Concentration (NOEC) was 32 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

110 mg/L

EC10 or NOEC for freshwater algae:

32 mg/L

Additional information

A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, 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 test, Desmodesmus subspicatus was exposed to an aqueous solution of the test material at concentrations of 1.0, 3.2, 10, 32 and 100 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.

A degradation product of the test material, isobutyraldehyde, was known to be volatile and hence testing was conducted in completely filled, stoppered test vessels in order to minimise possible losses due to volatilisation. Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatus was exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 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.

 

In terms of growth rate, exposure of Desmodesmus subspicatus to the test material gave an ErC50 (0 - 72 h) value of 110 mg/L. The Lowest Observed Effect Concentration based on inhibition of growth rate was 100 mg/L and the No Observed Effect Concentration was 32 mg/L.

In terms of yield, exposure of Desmodesmus subspicatus to the test material gave an EyC50 (0 - 72 h) value of 85 mg/L. The Lowest Observed Effect Concentration based on yield was 100 mg/L and the No Observed Effect Concentration was 32 mg/L.

In terms of biomass integral (area under growth curve), exposure of Desmodesmus subspicatus to the test material gave an EDC50 (0 - 72 h) value of 90 mg/L. The Lowest Observed Effect Concentration based on inhibition of biomass integral was 100 mg/L and the No Observed Effect Concentration was 32 mg/L.

 

The test material was known to degrade rapidly in water to give isobutyraldehyde and diethanolamine. The relative amounts of each degradant were calculated to be 45.3 % isobutyraldehyde and 66 % diethanolamine. The test concentrations were monitored by analysis of the test samples for the degradants only. For the calculation of test sample results, the nominal concentration for each degradant was adjusted to account for their relative percentages.

Analysis of the test preparations at 0 hours showed measured test concentrations in the range of 92 % to 116 % of nominal for diethanolamine and 81 % to 87 % of nominal for isobutyraldehyde.

Analysis of the test preparations at 72 hours showed measured test concentrations in the range of 88 % to 101 %) of nominal for diethanolamine and less than the limit of quantitation (LOQ) of the analytical method employed to 71 % of nominal for isobutyraldehyde.

Due to the volatile nature of the degradation product isobutyraldehyde, an additional test replicate was prepared at 0 hours and incubated alongside the test to provide samples for unopened vessel analysis at 72 hours. Analysis of the additional test replicates showed measured test concentrations in the range of 82 % to 96 % of nominal for diethanolamine and less than the LOQ to 77 % of nominal for isobutyraldehyde were obtained. Given that these measured concentrations were similar to those obtained from the test vessels which had been opened, it was considered that the decline in measured concentrations of isobutyraldehyde seen in the 72 -hour test samples was due to possible adsorption of the test material to the algal cells present rather than losses due to volatility. Whilst the preliminary recovery analyses conducted in the presence of algal cells indicated that no immediate adsorption occurred this does not preclude long-term adsorption over the test period. Adsorption was not a factor in the preliminary stability analyses conducted as no algal cells were present.

Given that the test preparations were a mixture of two degradants, the results are based on nominal test concentrations only.

 

Exposure of Desmodesmus subspicatus to the reference material, potassium dichromate, gave an ErC50 (0 - 72 h) of 0.57 mg/L; 95 % confidence limits 0.48 - 0.66 mg/L, an EyC50 (0 - 72 h) of 0.32 mg/L; 95 % confidence limits 0.29 - 0.35 mg/L, and an EbC50 (0 - 72 h) of 0.31 mg/L; 95 % confidence limits 0.28 - 0.35 mg/L. The Lowest Observed Effect Concentrations based on inhibition of growth rate, yield and biomass integral were 0.50, 0.125 and 0.125 mg/L, respectively, and the No Observed Effect Concentrations were 0.25, 0.0625 and 0.0625 mg/L, respectively.