2-isopropyl-N-hydroxyethyl 1,3-oxazolidine;methyl carbonato-N-ethyl-2-isopropyl-1,3-oxazolidine;reaction mass of: carbonato-bis-N-ethyl-2-isopropyl-1,3-oxazolidine

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 1998-10-27 to 1999-01-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is regarded as reliable without restrictions because it was conducted in compliance with GLP regulation and guideline.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

cited as: Directive 92/69/EEC

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

At approximately 24 h intervals after the start of incubation, samples were taken for cell counting from all flasks other than those used for determining water quality.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 100 mg/L stock solution was prepared by addition of Incozol LV to algal nutrient medium, the stock solution was stirred using a magnetic stirrer for approximately 2 h. After stirring, the test media were prepared by dilution of the stock solution, and after filling, the test vessels were sealed. A control treatment was prepared containing algal nutrient medium only.
At the start and at the end of the exposure period all test media were clear and colourless.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: S. capricornutum,
- Strain: CCAP 278/4
- Source: culture derived from the Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, Ambleside, Cumbria, UK.
- Method of cultivation:
To prepare the algal culture, medium stock solutions containing the various nutrients were prepared with deionised water. The stock solutions were then added to deionised water in the appropriate quantities. The algal culture medium was autoclaved before the addition of NaHCO3. The NaHCO3 was added to the algal culture medium after filter sterilisation (0.2 µm filter).

ACCLIMATION
- Acclimation period: no
- Culturing media and conditions: same as test conditions
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.2 - 22.8 °C

pH:

7.9 - 8.7

Nominal and measured concentrations:

nominal concentrations: 100, 50, 25,12.5, 6.3, 3.2 mg/L.
measured concentrations: 111, 60, 35, 27, 16, 13 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL (nominal volume)
- Material, size, headspace, fill volume:
- Aeration: no
- Type: closed
- Type of flow-through: static
- Initial cells density: 1E+03 cells/mL
- Control end cells density: 1E+03 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
To prepare the algal culture, medium stock solutions containing the various nutrients were prepared with deionised water. The stock solutions were then added to deionised water in the appropriate quantities. The algal culture medium was autoclaved before the addition of NaHCO3. The NaHCO3 was added to the algal culture medium after filter sterilisation (0.2 µm filter).

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 16h light
- Light intensity and quality: 6580 – 7460 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations:
Cell counts were carried out using a Z1 Coulter Counter (Coulter Electronics Ltd, Luton, UK). Corrections for cell counts were made by subtraction of background counts from a vessel incubated with the test vessels but containing no algae. Initial cell concentration of the starter culture was performed using a Coulter Counter. The acceptability of electronic counting was carried out by comparison of cell concentrations using a haemocytometer and light microscope.

TEST CONCENTRATIONS
- nominal concentrations : 100, 50, 25,12.5, 6.3 and 3.2 mg/L.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CI: 11.9 to 47.6 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

93.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 74.2 to 126 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

During the definitive study anomalous high background counts were obtained from the additional test vessels, these high background counts were not related to concentration of test material present. Therefore on each sampling occasion a mean value was obtained from all of the background counts, the mean was used in the calculation of the results. In addition counts at 24 h from two of the three flasks inoculated at 6.3 mg/L were high, both these and one flask from the 50 mg/L treatment were excluded from the result calculations.

Confirmatory test
As a result of the problems encountered with the background counts and the exclusion of three flasks from the calculations a confirmatory test was performed. The confirmatory test was conducted using manual counting with a haemacytometer and a light microscope. It was not possible in the definitive test to perform manual counts due to dilution of the samples for electronic counting. Due to the level of cell inoculation required for a sealed test system cell counts were made at 72 h only and only a result based on the average specific growth rate could be calculated. However this single value is sufficient to serve the purpose of the confirmatory study.

Validity criteria fulfilled:

yes

Conclusions:

Based on the areas under the growth curves 72-h E(b)C50 of Incozol LV to Pseudokirchneriella subcapitata was observed to be 21.6 mg/L (with 95 % confidence limits of 11.9 to 47.6 mg/L). Based on the average specific growth rate the 72-h E(r)C50 of Incozol LV to P. subcapitata was observed to be 93.1 mg/L (with 95 % confidence limits of 74.2 to 126 mg/L). Based on the biomass the NOEC of Incozol LV was observed to be 3.2 mg/L. Based on the growth rate the NOEC of Incozol LV was observed to be 6.3 mg/L.

Executive summary:

A study was conducted according to OECD TG 201 and Directive 92/69/EEC to determine the effects of Incozol LV on the alga Pseudokirchneriella subcapitata. A 72-hour sealed study was performed. Therefore, 100 mg/L stock solution was prepared by direct addition of Incozol LV to the algal nutrient medium and stirred for approximately 2 h. Aliquots of the stock solution were used and further diluted with algal nutrient medium to achieve the desired concentration series. A control treatment was prepared containing algal nutrient medium only. The test was conducted with 6 concentrations as follows: 100, 50, 25, 12.5, 6.3 and 3.2 mg/L. Two tests were performed, the definitive test using electronic particle counting and a confirmatory test which was conducted using a light microscope and a haemacytometer. Due to low initial cell inoculum it was not possible to perform counts using the light microscope and haemacytometer at 24 and 48 h, therefore, it was only possible to calculate a 72 h E(b)C50 based on the areas under the growth curves. Based on the areas under the growth curves the 72-h E(b)C50 of Incozol LV to Pseudokirchneriella subcapitata was observed to be 21.6 mg/L (with 95 % confidence limits of 11.9 to 47.6 mg/L). Based on the average specific growth rate the 72-h E(r)C50 of Incozol LV to P. subcapitata was observed to be 93.1 mg/L (with 95 % confidence limits of 74.2 to 126 mg/L). Based on the biomass the NOEC of Incozol LV was observed to be 3.2 mg/L. Based on the growth rate the NOEC of Incozol LV was observed to be 6.3 mg/L.

Description of key information

Based on the areas under the growth curves 72-h E(b)C50 of Incozol LV to Pseudokirchneriella subcapitata was observed to be 21.6 mg/L (with 95 % confidence limits of 11.9 to 47.6 mg/L). Based on the average specific growth rate the 72-h E(r)C50 of Incozol LV to P. subcapitata was observed to be 93.1 mg/L (with 95 % confidence limits of 74.2 to 126 mg/L). Based on the biomass the NOEC of Incozol LV was observed to be 3.2 mg/L. Based on the growth rate the NOEC of Incozol LV was observed to be 6.3 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

93.1 mg/L

EC10 or NOEC for freshwater algae:

6.3 mg/L

Additional information

A study was conducted according to OECD TG 201 and Directive 92/69/EEC to determine the effects of Incozol LV on the alga Pseudokirchneriella subcapitata. A 72-hour sealed study was performed. Therefore, 100 mg/L stock solution was prepared by direct addition of Incozol LV to the algal nutrient medium and stirred for approximately 2 h. Aliquots of the stock solution were used and further diluted with algal nutrient medium to achieve the desired concentration series. A control treatment was prepared containing algal nutrient medium only. The test was conducted with 6 concentrations as follows: 100, 50, 25, 12.5, 6.3 and 3.2 mg/L. Two tests were performed, the definitive test using electronic particle counting and a confirmatory test which was conducted using a light microscope and a haemacytometer. Due to low initial cell inoculum it was not possible to perform counts using the light microscope and haemacytometer at 24 and 48 h, therefore, it was only possible to calculate a 72 h E(b)C50 based on the areas under the growth curves. Based on the areas under the growth curves the 72-h E(b)C50 of Incozol LV to Pseudokirchneriella subcapitata was observed to be 21.6 mg/L (with 95 % confidence limits of 11.9 to 47.6 mg/L). Based on the average specific growth rate the 72-h E(r)C50 of Incozol LV to P. subcapitata was observed to be 93.1 mg/L (with 95 % confidence limits of 74.2 to 126 mg/L). Based on the biomass the NOEC of Incozol LV was observed to be 3.2 mg/L. Based on the growth rate the NOEC of Incozol LV was observed to be 6.3 mg/L.