4-methyloxazole-5-carbonitrile

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:

08 June 2010-11 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-methyloxazole-5-carbonitrile

EC Number:

213-709-7

EC Name:

4-methyloxazole-5-carbonitrile

Cas Number:

1003-52-7

Molecular formula:

C5H4N2O

IUPAC Name:

4-methyl-1,3-oxazole-5-carbonitrile

Details on test material:

Name of test material (as cited in study report): CMO
- Substance type: Clear colourless liquid
- Physical state: Liquid
- Analytical purity: 99.9%
- Lot/batch No.: 485
- Expiration date of the lot/batch: 04 December 2010
- Stability under storage conditions: Stable
- Storage condition of test material: In refrigerator (2-8°C) in the dark under nitrogen
- Stability in water: Unknown
-Solubility in water: Approximately 4.5 g/L

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below
Frequency: at t=0 h, t=24 h and t=72 h
Volume: 3 ml
Storage: Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of CMO tested was a clear colour liquid with a purity of 99.9% and the substance was completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with a loading rate of 100 mg/l applying 1 day of magnetic stirring. The lower test concentrations were prepared by subsequent dilutions of the 100 mg/l concentration in test medium. The final test solutions were all clear and colourless

Test organisms

Test organisms (species):

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

Details on test organisms:

- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1.
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E4 cells/ml.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
in a climate room at a temperature of 21-24°C.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/l

Test temperature:

between 22.8 and 23.7°C.

pH:

Between 8.1 and 8.2

Nominal and measured concentrations:

Nominal concentrations: Control and 0.10, 1.0, 10 and 100 mg/l
Measured concentrations:At the start of the test, the actual test concentration was 76 mg/l at the expected nominal concentration of 100 mg/l. During the test period this concentration fluctuated but remained in agreement with initial (83-115%). However, the actual test concentration in the vessel without algae decreased during the test period, i.e. to 42% of initial at the end of the test period, indicating that CMO adsorbs to algal cells. Given these results, the effect parameters were based on the initial test concentration.

Details on test conditions:

TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: 100 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 1920000 cells/ml
Replicates:
6 replicates of both the control and the highest test concentration;
3 replicates of each lower test concentration;
1 extra replicate of the control and each test concentration for sampling purposes;
1 or 2 extra replicates of each test concentration without algae.


GROWTH MEDIUM
- Standard medium used: yes
OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps of the type Cool-white of 30 Watt, with a light intensity within the range of 84 to 91 µE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h NOEyC, 72 h ErC50, 72 h EyC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations: control and 0.1, 1.0, 10 and 100 mg/l

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 76 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 76 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

76 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 76 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Inhibition of yield increased with increasing concentration of CMO from 10 mg/l upwards resulting in 21% inhibition at 76 mg/l.
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Results with reference substance (positive control):

- EC50:The EC50 for growth rate reduction (ERC50: 0-72h) was 1.1 mg/l with a 95% confidence interval ranging from 1.0 to 1.3 mg/l.
The EC50 for yield inhibition (EYC50: 0-72h) was 0.59 mg/l with a 95% confidence interval ranging from 0.51 to 0.68 mg/l.

Reported statistics and error estimates:

Statistical analysis of the data for growth rate reduction was not needed as the effects recorded were not significant (<10%).
Statistically significant inhibition of yield was found at test concentrations of 76 mg/l and higher (Bonferroni t and Tukey test, a = 0.05).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, no reduction of growth rate was recorded at any of the concentrations of CMO tested. CMO inhibited the yield of this fresh water algae species significantly by 21% at 76 mg/l.

The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EYC50: 0-72h) was >76 mg/l.

The NOEC for growth rate reduction and yield inhibition was 76 and < 76 mg/l, respectively.