Dibutyl itaconate

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 14 November 2015 to 08 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

(2011)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2009)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23

Version / remarks:

(2000)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Not completely soluble in test medium at 100 mg/L

Analytical monitoring:

yes

Details on sampling:

- Concentrations sampled: all test concentrations (and the control), at t=0, t=24h and t=72h
- Volume: 2.0 mL
- At the end of the exposure period, the replicates were pooled at each concentration before sampling
- Sample storage conditions before analysis: in a freezer

Vehicle:

no

Details on test solutions:

- Preparation of test solutions started with a loading rate of 100 mg/L applying a 2-day period of magnetic stirring to ensure maximum dissolving of the test item in test medium. After a 1-hour period for settlement, the aqueous Water Soluble Fraction was siphoned off and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. All final test solutions were 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 10^4 cells/mL.

Test organisms (species):

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

Details on test organisms:

- Species: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2) at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

21 - 23°C

pH:

7.6 - 8.3

Nominal and measured concentrations:

- 0.32, 1.0, 3.2, 10, 32 and 100% of a WSF, prepared at a loading rate of 100 mg/L
- Time Weight Average (TWA): 0.14, 0.47, 1.6, 6.1, 19 and 63 mg/L (measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 105 x 10^4 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Also 1 replicate without algae, at 10% of a WSF, prepared at a loading rate of 100 mg/L

TEST MEDIUM: M2; according to OECD 201, formulated using Milli-RO water

OTHER TEST CONDITIONS
- Illumination: Continuously using TLD-lamps with a light intensity within the range of 84 to 85 μE.m^-2.s^-1
- Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. A floating layer and precipitation were observed in the highest test concentration after 24 hours, which remained for the rest of the study duration. Consequently, algal densities were determined by use of a microscope and a counting chamber throughout the test.
- Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

COMBINED LIMIT/RANGE-FINDING TEST
- Test concentrations: 0.1, 1.0, 10 and 100% of a WSF, prepared at a loading rate of 100 mg/L
- Results used to determine the conditions for the definitive study:
-- At 100%, there was 93.4% inhibition of growth rate
-- At 10%, there was 38.2% inhibition of growth rate
-- At 1.0%, there was 2.0% inhibition of growth rate
-- Samples taken from 1.0 and 100% of the WSF prepared at a loading rate of 100 mg/L were analysed. The initial test item concentrations were 0.91 and 99 mg/L, respectively. Both concentrations decreased during the test period, i.e. to 51 and 62% of initial after 72 hours of exposure (to 0.466 and 61.6 mg/L)

DETERMINATION OF THE EXPOSURE CONCENTRATIONS
The Time Weight Average (TWA) of the concentrations of the test item measured in the samples taken at the start (t=0), after 24 hours (t=24h) and at the end of the test (t=72h).

CALCULATION OF THE ECx values
Calculation of ECx values was based on probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition versus the logarithms of the corresponding TWA concentrations of the test item. The calculations were performed with ToxRat Professional v. 3.0.0. (ToxRat Solutions® GmbH, Germany).

Reference substance (positive control):

yes

Remarks:

(sensitivity check using potassium dichromate)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.9 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 6.4 - 7.4 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 1.1 - 1.5 mg/L

Details on results:

- Measured test substance concentrations: At the start of the test, the measured test concentrations were 0.23, 0.67, 2.3, 7.7, 25 and 76 mg/L in 0.32, 1.0, 3.2, 10, 32 and 100% of the WSF prepared at a loading rate of 100 mg/L, respectively. All concentrations decreased during the test period, i.e. were 77-91% of initial after 24 hours and 29-64% of initial after 72 hours of exposure. The measured concentration in the sample taken from the solution without algae decreased similarly. Given this result, the Time Weight Average exposure concentrations were calculated (see the table below) and used to determine the effect parameters.
- Growth rates were in the range of the control at the lowest concentration during the 72-hour test period, whereas the growth rates of algae exposed to TWA concentrations from 0.47 mg/L and higher were increasingly inhibited, resulting in 96% inhibition at a TWA exposure concentration of 63 mg/L. Statistically significant inhibition of growth rate was found at test concentrations of 0.47 mg/L (TWA) and higher. However, biologically relevant inhibition (>10% inhibition of growth rate) was observed only at the four highest test concentrations and the NOEC was therefore set at a TWA concentration of 0.47 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):

In the latest sensitivity check, performed in the period 14 - 17 December 2015, an 72h-EC50 of 1.5 mg/L for growth rate inhibition was determined. The sensitivity of the algal culture was within the range determined with the historical data collected at the testing laboratory (0.82 - 2.3 mg/L).

Measured concentrations:

%WSF	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72h
0.32	0.229	0.188	0.0667	0.14
1.0	0.671	0.514	0.332	0.47
3.2	2.27	1.80	1.19	1.6
10	7.74	6.65	4.67	6.1
10 (without algae)	7.61	6.08	4.41	5.7
32	24.8	18.9	15.9	19
100	76.4	69.8	47.1	63

Inhibition of growth rate (total test period):

- 63 mg/L: 96.0%

- 19 mg/L: 82.9%

- 6.1 mg/L: 40.3%

- 1.6 mg/L: 16.8%

- 0.47 mg/L: 7.1%

- 0.14 mg/L: 1.1%

Validity criteria fulfilled:

yes

Conclusions:

In a study performed in accordance with OECD 201 (2011) and according to GLP principles, an 72h-EC50 of 6.9 mg/L for growth rate was determined for the substance, based on TWA concentrations. The 72h-EC10 for growth rate was determined to be 1.3 mg/L.

Executive summary:

In a study performed in accordance with OECD 201 (2011) and according to GLP principles, the toxicity of the substance to the freshwater algae Pseudokirchneriella subcapitata was investigated. As the substance was not completely soluble in test medium at 100 mg/L, preparation of test solutions started with a loading rate of 100 mg/L applying a 2-day period of magnetic stirring and a 1-hour period for settlement before the aqueous Water Soluble Fraction (WSF) was siphoned off and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to a control, whereas three replicates per group were exposed to 0.32, 1.0, 3.2, 10, 32 and 100% of a WSF prepared at a loading rate of 100 mg/L. Samples for analytical confirmation of exposure concentrations were taken at the start and after 24 and 72 hours of exposure. All concentrations decreased during the test period, i.e. were 77-91% of initial after 24 hours and 29-64% of initial after 72 hours of exposure. Given this result, the Time Weight Average exposure concentrations were calculated to be 0.14, 0.47, 1.6, 6.1, 19 and 63 mg/L. Under the conditions of the present study, the 72h-EC50 and 72h-EC10 for growth rate were 6.9 and 1.3 mg/L, respectively.

Description of key information

In a study performed in accordance with OECD 201 (2011) and according to GLP principles, an 72h-EC50 of 6.9 mg/L for growth rate was determined for the substance, based on TWA concentrations. The 72h-EC10 for growth rate was determined to be 1.3 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

6.9 mg/L

EC10 or NOEC for freshwater algae:

1.3 mg/L

Additional information

In a study performed in accordance with OECD 201 (2011) and according to GLP principles, the toxicity of the substance to the freshwater algae Pseudokirchneriella subcapitata was investigated. As the substance was not completely soluble in test medium at 100 mg/L, preparation of test solutions started with a loading rate of 100 mg/L applying a 2-day period of magnetic stirring and a 1-hour period for settlement before the aqueous Water Soluble Fraction (WSF) was siphoned off and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to a control, whereas three replicates per group were exposed to 0.32, 1.0, 3.2, 10, 32 and 100% of a WSF prepared at a loading rate of 100 mg/L. Samples for analytical confirmation of exposure concentrations were taken at the start and after 24 and 72 hours of exposure. All concentrations decreased during the test period, i.e. were 77-91% of initial after 24 hours and 29-64% of initial after 72 hours of exposure. Given this result, the Time Weight Average exposure concentrations were calculated to be 0.14, 0.47, 1.6, 6.1, 19 and 63 mg/L. Under the conditions of the present study, the 72h-EC50 and 72h-EC10 for growth rate were 6.9 and 1.3 mg/L, respectively.