Ethyl 2-chloroacetoacetate

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:

14 June 2010 - 23 July 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

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult sunstances and mixtures, OECD series on testing and assesment number 23, December 14, 2000.

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2-chloroacetoacetate

EC Number:

210-180-4

EC Name:

Ethyl 2-chloroacetoacetate

Cas Number:

609-15-4

Molecular formula:

C6H9ClO3

IUPAC Name:

ethyl 2-chloro-3-oxobutanoate

Constituent 2

Reference substance name:

Chloracetessigester

IUPAC Name:

Chloracetessigester

Details on test material:

- Name of test material (as cited in study report): Chloracetessigester
- Substance type: Clear slightly yellowish liquid
- Physical state: Liquid
- Analytical purity: 93.9%
- Lot/batch No.: 001008
- Expiration date of the lot/batch: 26 July 2010
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature in the dark
-Stability in water: Unknown
-Solubility in water: Yes, 17 g/L
- Density: 1.18 g/cm3 (20°C)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

During the final test duplicate 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 4 ml
Storage Not applicable, samples were analysed on the day of sampling.

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

Test solutions

Vehicle:

no

Details on test solutions:

The batch of Chloracetessigester tested was a clear slightly yellowish liquid with a purity of 93.9% and the substance appeared to be completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with the highest test concentration of 100 mg/l applying a 15-minute period of magnetic stirring. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. During the final test dilutions were prepared from a stirring solution and all test vessels used were incubated for 1 day with the respective test solutions as to prevent any possible adsorption to glass ware. After the pre-incubation period vessels were filled with freshly prepared solutions. All final test solutions were visually clear and colourless.

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1.
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation):3 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:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol(24 mg CaCO3/L)

Test temperature:

between 21.5 and 23.7°C

pH:

between 5.2 and 8.3

Nominal and measured concentrations:

Nominal concentrations: control and 0.32, 1.0, 3.2, 10, 32 and 100 mg/l
Measured concentrations: At the start of the test, the mean measured concentrations were 0.11, 0.37 and 4.3 mg/l at nominal concentrations of 10, 32 and 100 mg/l, respectively. Measured concentrations could not be quantified (<0.1 mg/l) at any of the lower test concentrations. Concentrations after 24 and 72 hours of exposure could only be measured in samples taken from nominally 100 mg/l, being respectively 0.38 and 0.11 mg/l. All lower concentrations were below quantification. It was not possible to determine time weighted average concentrations for the range tested. Consequently, results were based on both nominal and initial measured exposure concentrations.

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: 1235000 cells/ml
Replicates:
3 replicates of each test concentration.
6 replicates of the control.
2 replicates of the highest concentration without algae.
1 extra replicate of each test concentration for sampling purposes.

GROWTH MEDIUM
- Standard medium used: yes
OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: The pH of thesolution of the loading rate of 100 mg/l was adjusted from 5.9 to 6.6 using sodium hydroxide (1N, Merck, Darmstadt, Germany)
- 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 68 to 78 µE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h NOEyC, 72 h ErC10, 72 h EyC10, 72 h ErC50, 72 h EyC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: control and 0.32, 1.0, 3.2, 10, 32 and 100 mg/l

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7,)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 0.06 - 0.43 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.83 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 0.31 - 2.2 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.23 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CI 0.07 - 0.71 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 4.1 - 45 mg/L

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CI 1.3 - 7.0 mg/L

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 13 - 131 mg/L

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CI 7.0 - 34 mg/L

Effect concentrations 10

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 11

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Microscopic observations at the end of the test of the algae exposed to nominal 3.2 and 10 mg/l revealed a normal and healthy appearance of the exposed cells when compared to the control.

Results with reference substance (positive control):

Potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/l and higher.
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:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (ANOVA, Tukey test, Bonferroni t-test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding nominal and initial measured concentrations of the test substance.
Statistically significant reduction of growth rate was found at nominal test concentrations of 10 mg/l and higher (Bonferroni t test, a = 0.05).
Statistically significant inhibition of yield was found at test concentrations of 10 mg/l and higher (Bonferroni t test, a = 0.05).

Any other information on results incl. tables

-

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, Chloracetessigester reduced growth rate and inhibited the yield of this fresh water algae species significantly at nominally 10 mg/l and higher. This corresponded to an initial measured concentration of 0.11 mg/l.

The EC50 for growth rate reduction (ERC50: 0-72h) based on nominal concentrations was 41 mg/l with a 95% confidence interval ranging from 13 to 131 mg/l. When based on initial measured concentrations this corresponded to 0.83 mg/l with a 95% confidence interval ranging from 0.31 to 2.2 mg/l.

The EC50 for yield inhibition (EYC50: 0-72h) based on nominal concentrations was 15 mg/l with a 95% confidence interval ranging from 7.0 to 34 mg/l. When based on initial measured concentrations this corresponded to 0.23 mg/l with a 95% confidence interval ranging from 0.07 to 0.71 mg/l.

The NOEC for both growth rate reduction and yield inhibition based on nominal concentrations was 3.2 mg/l with a corresponding initial measured exposure concentration below 0.1 mg/l.

Executive summary:

-