Diethyl maleate

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:

12 April 2010 to ..........

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP compliant study; no chemical analyses of actual test substance concentrations performed

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl maleate

EC Number:

205-451-9

EC Name:

Diethyl maleate

Cas Number:

141-05-9

Molecular formula:

C8H12O4

IUPAC Name:

diethyl (Z)-but-2-enedioate

Details on test material:

Test Item: Diethylmaleate
Common Name: DEM
CAS No.: 141-05-9
Chemical Name (IUPAC): Maleicaciddiethylester
Physical Appearance: Liquid
Purity as per Certificate of Analysis: 99%
Batch No.: LEDM1A9002
Manufactured Date: Jan. 2010
Expiry Date: Jan. 2011
Retest Date: no retest before Jan. 2011
Recommended Storage Condition: Ambient (+18 to +36ºC)

Date of receipt of test item at test facility: 29 March 2010
Test Item code by test facility: D011-03

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Test solutions of all the five test concentrations were analysed along with the negative control for the active ingredient concentration at the beginning and end of the test using an in-house validated analytical method.

Test solutions

Vehicle:

no

Details on test solutions:

A stock solution of 10 mg/mL was prepared by mixing 250 mg of the test item in test medium and making up the volume up to 25 mL in a volumetric flask, with test medium.
Stock solution was freshly prepared and used within 10 minutes of preparation.

Five concentrations of the test item, a negative control and a positive control were prepared as follows:

G1 (Negative control): 10 mL of algal cell suspension were made up to the mark in a 1 L volumetric flask using the test medium. From this, 150 mL each were dispensed into 6 test containers.

G2 (2 mg/L): 10 mL of algal cell suspension and 0.2 ml of stock were made up to the mark in a 1 L volumetric flask using test medium. From this, 150 mL were dispensed into 3 test containers.

G3 (5 mg/L): 10 mL of algal cell suspension and 0.5 mL of stock were made up to the mark in a 1 L volumetric flask using test medium. From this, 150 mL were dispensed into 3 test containers.

G4 (12.5 mg/L): 10 mL of algal cell suspension and 1.25 mL of stock were made up to the mark in a 1 L volumetric flask using test medium. From this, 150 mL were dispensed into 3 test containers.

G5 (31.25 mg/L): 10 mL of algal cell suspension and 3.125 mL of stock were made up to the mark in a 1 L volumetric flask using test medium. From this, 150 mL were dispensed into 3 test containers.

G6 (78.13 mg/L): 10 mL of algal cell suspension and 7.813 mL of stock were made up to the mark in a 1 L volumetric flask using test medium. From this, 150 mL were dispensed into 3 test containers.

G7 (Positive control) (0.9 mg/L of K2Cr2O7): 5 mL of algal cell suspension and 0.45 mL of 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
- Source (laboratory, culture collection): Norwegian Institute for Water Research, P.O. Box 173, Kjelsas, N-0411, Oslo, NORWAY
- Age of inoculum (at test initiation): To adapt and to ensure that the algae are in the exponential growth phase, inoculum culture was prepared by inoculating Pseudokirchneriella subcapitata of continuous 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.
- 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: 3 days
- Culturing media and conditions: same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

no data

Test temperature:

Temperature inside the algal growth chamber was recorded once daily.
The temperature of the algal growth chamber ranged between 22.7 and 23.4 °C.

pH:

The pH of the the test solutions was checked at the beginning and at the end of the test.
The pH of the negative control ranged between 7.92 and 8.47. The pH of test item solutions ranged between 7.92 and 8.49 during the test period. The pH of the positive control ranged between 7.96 and 8.57 during the test period

Dissolved oxygen:

no data

Salinity:

not appropriate

Nominal and measured concentrations:

nominal concentrations: 2, 5, 12.5, 31.25 and 78.13 mg/L
measured concentrations:

Details on test conditions:

The test was conducted using 250 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 the negative control group and 3 replicates were maintained for each of the test concentration and positive control groups. The initial cell biomass was approximately 1 x 104 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 One hundred milligrams (100 mg) of Potassium dichromate (K2Cr2O7) were mixed with 10 mL of test medium (10 mg/mL).
This stock solution was further diluted in test medium to get the required concentration of Potassium dichromate per mL of the test medium.
Dilution: 1 mL of stock + 9 mL of test medium = 1 mg/mL

The algal biomass in each flask was determined at 24, 48 and 72 hours after the start of the test using a haemocytometer.
The measured cell concentrations in the test cultures and controls were tabulated together with the concentrations of the test item and the times of measurement.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

201.97 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.:

32.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

44.47 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

10.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

20.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.51 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

see Attachment

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 inhibition of growth was determined by calculating the ErC and EyC (the index “r” refers to growth rate and the index “y” refers to yield) by Probit analysis of Finney (1971) using in-house developed and validated computer programme.
The equation of growth rate and 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

see Attachment for Tables

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 2, 5, 12.5, 31.25 and 78.13 mg/L (factor of 2.5) resulted in 0, 2.32, 6.71, 15.95 and 100 % inhibition of average growth rate and 0, 9.46, 24.98, 49.66 and 100 % inhibition of yield, respectively. There was 38.51 % and 81.47 % inhibition of average growth rate and yield, respectively in the positive control.

Executive summary:

The effect of Diethylmaleate 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 2, 5, 12.5, 31.25 and 78.13 mg/L (factor of 2.5) 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, 2.32, 6.71, 15.95 and 100 % and the inhibition of yield was 0, 9.46, 24.98, 49.66 and 100 % at the tested concentrations of 5, 12.5, 31.25 and 78.13 mg/L, respectively when compared to the negative control. There was 38.51 % and 81.47 % inhibition of average growth rate and yield of alga, respectively, in the positive control.

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

At 72 hours	EC values (mg/L)
ErC50	201.97
ErC20	44.47
ErC10	20.16
EyC50	32.63
EyC20	10.15
EyC10	5.51