Dimethyl acetylsuccinate

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:

13 April 2010 to XXXXXXXXXXX

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl acetylsuccinate

EC Number:

233-897-4

EC Name:

Dimethyl acetylsuccinate

Cas Number:

10420-33-4

Molecular formula:

C8H12O5

IUPAC Name:

dimethyl 2-acetylsuccinate

Details on test material:

- Batch manufactured and supplied by: DSM Fine Chemicals, Austria Nfg GmbH & Co KG, St.-Peter-Straße 25, P.O. Box 933, 4021 Linz / Austria
- Test Item: Dimethylacetylsuccinate
- Common Name: DMAS
- CAS No.: 10420-33-4
- Chemical Name (IUPAC): (+/-)Acetylsuccinicaciddimethylester
- Physical Appearance: Solid
- Purity as per Certificate of Analysis: 97.8%
- Batch No.: LEDM5A1003
- Manufactured Date: Dec. 2009
- Expiry Date: not applicable
- Retest Date: Dec. 2011
- Recommended Storage Condition: Ambient (+18 to +36ºC)
- Note: Date of receipt of test item at test facility: 29.03.2010
- Test Item code by test facility: D011-05

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal 1.2, 3.7, 11, 33 and 100 mg/L
- Sampling method: no data
- Sample storage conditions before analysis: no data
The test solutions of all the five test concentrations were analysed along with the negative control for the active ingredient concentration at the start and at the end of treatment using an in-house validated analytical method.

Test solutions

Vehicle:

not specified

Details on test solutions:

A stock solution of 10 mg/mL was prepared by dissolving 250 mg of the test item in the test medium and making up the volume up to 25 mL in a volumetric flask, with test medium.

This stock was further diluted in the test medium to get the required concentrations per mL of the test medium.
Dilution: 1 mL of stock + 9 mL of test medium = 1 mg/mL
Stock solution and dilution were freshly prepared and used within 30 minutes of preparation.

Five concentrations of the test item, a negative control and a positive control were prepared as follows:
G1 (Negative control): 20 mL algal cell suspension were made up to 2000 mL using test medium. From this, 150 mL each was dispensed into 6 Nos. 250 mL test containers.
G2 (1.2 mg/L): 20 mL algal cell suspension and 2.4 mL dilution of test item stock were made up to 2000 mL using test medium in duplicate and pooled. From this, 150 mL each was dispensed into 3 Nos. 250 mL test containers and 300 mL each into 4 Nos. 500 mL test containers.
G3 (3.7 mg/L): 20 mL algal cell suspension and 0.74 mL test item stock were made up to 2000 mL using test medium. From this, 150 mL each was dispensed into 6 Nos. 250 mL test containers.
G4 (11 mg/L): 20 mL algal cell suspension and 2.2 mL test item stock solution were made up to 2000 mL using test medium. From this, 150 mL each was dispensed into 6 Nos. 250 mL test containers.
G5 (33 mg/L): 10 mL algal cell suspension and 3.3 mL test item stock solution were made up to 1000 mL using test medium. From this, 150 mL each was dispensed into 3 Nos. 250 mL test containers.
G6 (100 mg/L): 10 mL algal cell suspension and 10 mL test item stock solution were made up to 1000 mL using test medium. From this, 150 mL each was dispensed into 3 Nos. 250 mL test containers.
G7 (Positive control)(0.9 mg/L of K2Cr2O7): 5 mL algal cell suspension and 0.45 mL dilution of the positive control were mixed with 500 mL of test medium. From this, 150 mL each were dispensed into 3 test containers.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) NIVA-CHL 1.
- Source: Norwegian Institute for Water Research, P.O. Box 173, Kjelsas, N-0411, Oslo, NORWAY
- Age of inoculum: 3 days
- Method of cultivation: Algal cells were maintained at the test facility in conical flasks by incubating inside an algal growth chamber under a continuous illumination of 8000 ± 1600 Lux and a temperature between 21 and 25°C (±2°C). Once in 5 to 10 days, sub-culturing was done from the continuous culture into fresh medium.

ACCLIMATION
- Acclimation period: To adapt and to ensure that the algae are in the exponential growth phase, inoculum culture was prepared by inoculating Pseudokirchneriella subcapitata of continuous/mother culture to test medium 3 days before the start of the test. The flasks were incubated inside the algal growth chamber under the test conditions.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Test conditions

Hardness:

not applicable, the OECD medium was used

Test temperature:

22.8 to 23.6°C
For more information please see: Key.Bopanna 2010. DMAS, Alga, Growth Inhibition Test Tables and Figures.pdf

pH:

Between 7.94 and 8.61 in the relevant test media
For more information please see: Key.Bopanna 2010. DMAS, Alga, Growth Inhibition Test Tables and Figures.pdf

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

Nominal: 1.2, 3.7, 11, 33 and 100 mg/L
Measured 0h: 0.695, 2.918, 8.810, 28.186 and 92.117 mg/L
Measured 72h: 0.012, 0.019, 0.055, 0.839 and 34.766 mg/L
or more information please see: Key.Bopanna 2010. DMAS, Alga, Growth Inhibition Test Tables and Figures.pdf

Details on test conditions:

The test was conducted using 250 mL and 500 mL conical flasks (test containers) with seven groups representing, one negative control, five concentrations of the test item and one positive control. Six replicates were maintained for the negative control group and 3 replicates were maintained for each of the test concentration and positive control groups for observation. The extra replicates prepared were used for analysis of active ingredient concentration. The initial cell biomass was approximately 1 x 10 to the power of 4 cells/mL of test medium. The test containers were incubated at 21 to 24ºC with a continuous light intensity of 4440 to 8880 Lux. Algal cells were counted at every 24 hours interval up to the end of the treatment period.

Negative Control: The test medium itself was used as the negative control.
Positive Control: 0.9 mg/L of K2Cr2O7

The algal biomass in each flask was determined at 24, 48 and 72 hours after the start of the test using a haemocytometer.

GROWTH MEDIUM
- Standard medium used: yes OECD medium


For more information please see: Key.Bopanna 2010. DMAS, Alga, Growth Inhibition Test Tables and Figures.pdf

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

40.95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: fiducial limits: 15.83 and 403.95 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

8.93 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: fiducial limits: 3.45 and 88.14 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.03 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: fiducial limits: 1.56 and 39.77 mg/L

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: not reported
- Unusual cell shape: not reported
- Colour differences: not reported
- Flocculation: not reported
- Adherence to test vessels: not reported
- Aggregation of algal cells: not reported
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

Potassium dichromate (K2Cr2O7); 0.9 mg/L; There was 41.19 % and 83.53 % inhibition of average growth rate and yield, respectively, in the positive control.

Reported statistics and error estimates:

The concentration/effect relationship was determined using the following two approaches (growth rate and yield).

Average growth rate:
The average growth rate (µ) for exponentially growing culture was calculated using Equation 1.

Equation 1: µ = (ln Nn-ln N1) / tn-t1)
Where,
N1 = nominal number of cells/mL at time t1
Nn = measured number of cells/mL at time tn
t1 = time of the start of the treatment
tn = time of nth measurement after beginning of treatment

The percent inhibition of growth rate (%Ir) for each treatment replicate was calculated using Equation 2.

Equation 2: % Ir = ((µc-µT) / µc) * 100
Where,
% Ir = percent inhibition in average specific growth rate
µc = mean value for average specific growth rate (µ) in the control group
µT = mean value for average specific growth rate in the treatment group

The per cent inhibition is plotted against the logarithm of the test item concentration.


Yield:
The yield was calculated by finding out the difference between the cell numbers at the beginning and end of the test for each replicate of the controls and the treatment groups. For each group, the mean value for yield along with variance was estimated. The per cent inhibition in yield (%Iy) was calculated for each treatment replicate using the Equation 3.

Equation 3: % Iy = ((Yc-Yt) / Yc) * 100
Where,
Yc : Mean value for yield in the negative control group
Yt : Mean value for yield in the treatment group


The EC10, EC20 and EC50 were calculated for the definitive test.

The EC values were not calculated for inhibition of growth rate as the percent growth inhibition were not more than 50 % in any of the tested concentration. The inhibition of yield was determined by calculating the EyC (the index the index y refers to yield) by Probit analysis of Finney (1971) using in-house developed and validated computer programme.

The equation of yield was Y = a + bX, where X is the log concentration and Y is the probit value with a and b as constants.

Any other information on results incl. tables

For more information please see: Key.Bopanna 2010. DMAS, Alga, Growth Inhibition Test Tables and Figures.pdf

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of the alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) to nominal concentrations of 1.2, 3.7, 11, 33 and 100 mg/L (factor of approximately 3) resulted in 0.34, 0.99, 10.44, 14.17 and 24.28 % inhibition of average growth rate and 1.46, 4.12, 36.04, 45.54 and 64.91 % inhibition of yield at the tested concentration of 1.2, 3.7, 11, 33 and 100 mg/L, respectively. There was 41.19 % and
83.53 % inhibition of average growth rate and yield, respectively in the positive control.

The EC values calculated for yield on the basis of the nominal concentration of the test item are presented below:

At 72 hours EC values (mg/L)
EyC50 40.95
EyC20 8.93
EyC10 4.03

Executive summary:

The effect of Dimethylacetylsuccinate was tested on the growth of fresh water single cell green alga Pseudokirchneriella subcapitata.

The alga was exposed to the test item at the nominal concentrations of 1.2, 3.7, 11, 33 and 100 mg/L (factor of approximately 3) along with a negative control and a positive control. The cell growth was measured at 24, 48 and 72 hours after the initiation of the test.

The concentration / effect relationship was determined using two factors, namely, growth rate and yield. The inhibition of growth rate was 0.34, 0.99, 10.44, 14.17 and 24.28 % and the inhibition of yield was 1.46, 4.12, 36.04, 45.54 and 64.91 % at the tested concentrations of 1.2, 3.7, 11, 33 and 100 mg/L, respectively, when compared to the negative control. There was 41.19 % and 83.53 % inhibition of average growth rate and yield of alga, respectively, in the positive control.

The EC values were not calculated for inhibition of growth rate as the percent growth inhibition were not more than 50 % in any of the tested concentrations and hence E_rC₅₀value is considered more than 100 mg/L.

The EC values calculated for yield on the basis of the nominal concentration of the test item are presented below:

 

At 72 hours	EC values (mg/L)
EyC50	40.95
EyC20	8.93
EyC10	4.03