Diethyl maleate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 May 2005 to 29 September 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes

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:

- Name of test material (as cited in study report): DIETHYLMALEINATE
- Physical state: Colourless liquid.
- Batch No.: ETLM 201.
- Expiration date of the lot/batch: 30 June 2005.
- Stability under test conditions: Stability in aqueous solution: stable
- Storage condition of test material: Room temperature, in the dark, may be used under light.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal 7, 14, 28, 55 and 110 mg test substance / L
- Sampling method: Samples of all test media for determination of the actual test substance concentrations were taken immediately before introduction of the daphnia and after 48 hours.
- Sample storage conditions before analysis: The samples were analysed on the day of sampling.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Immediately before the beginning of the test the highest concentration was prepared by dissolving 27.6 mg test substance in 250 mL reconstituted water. The other test substance concentrations used were then obtained by taking 6.3, 12.5, 25 and 50 mL of the highest concentration and adding up to 100 mL each with reconstituted water. The resulting nominal test substance concentrations were: 7, 14, 28, 55 and 110 mg test substance / L
- Controls: For the negative control group only reconstituted water according to ISO 6341 was used.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
Neonates of Daphnia magna (Crustacea, Cladocera), less than 24 hours old, were obtained by hatching ephippia, supplied in the microbiotest "Daphtoxkit F TM "(Daphnia magna; MicroBioTests Inc., Industriezone "De Prijkels", Venecoweg 19 , 9810 Nazareth, Belgium). The ephippia were hatched in the laboratory in a petri dish containing reconstituted water in a temperature controlled room at approx. 20-22 °C under continuous illumination of
approximately 6 000 Lux. According to MicroBioTests Inc. the largest hatching occurs between 72 and 80 hours and the daphnia must be collected at the latest 90 hours after initiation of hatching. Approximately 2 hours before the start of the test the neonates were fed with a suspension of Spirulina powder (MicroBioTests Inc.), as also in traditional cultures the neonates have the opportunity to take up some feed particles. The neonates were collected for this test at 86 hours after initiation of hatching.
- Feeding during test: no


ACCLIMATION
- Acclimation period: < 24h
- Acclimation conditions (same as test or not): same
- Type and amount of food: Approximately 2 hours before the start of the test the neonates were fed with a suspension of Spirulina powder, as also in traditional Daphnia cultures the neonates have the opportunity to take up some feed particles.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Test conditions

Hardness:

250 mg CaCO3/L

Test temperature:

20°C

pH:

Between 7.6 and 7.9 in the test substance solutions and between 7.7 and 7.8 in the control media.

Dissolved oxygen:

At least 7.4 mg/L.

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal concentrations: 7, 14, 28, 55 and 110 mg test substance / L.
Measured concentrations at 0 hours: 6, 12, 24, 48 and 95 mg test substance / L
Measured concentrations at 48 hours: 6, 11, 23, 47 and 92 mg test substance / L
For test substance concentrations see also the attached background material: Key. Fenzl. Acute Toxicity Daphnia_ Tables and figures.pdf

Details on test conditions:

TEST SYSTEM
- Test vessel: Disposable multiwell test plates with 30 test wells with a transparent lid, provided with the testkit. Each plate has four 10 mL wells each (replicates A,B,C,D) for the control and for 5 concentrations. Additionally, for each concentration the plates are provided with "rinsing wells" to prevent dilution of the test substance during the transfer of the daphnia from the hatching petri dish to the test wells (see below).
- Material, size, headspace, fill volume: 10 mL policarbonate wells (according to Microbiotest), filled with 10 mL medium
- Aeration: No extra aeration, but slight circulation of air is still possible when the wells are closed with the transparent lid.
- No. of organisms per vessel: 5.
- No. of vessels per concentration (replicates): 4.
- No. of vessels per control (replicates): 4.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The stock solutions for preparation of the reconstituted water according to ISO 6341 were obtained by MicroBioTests Inc. (Batch No. ISO D300902). The water was aerated and the pH, the dissolved oxygen concentration and the water hardness were checked before using.
- Culture medium different from test medium: no.
- Intervals of water quality measurement: Temperature daily. Oxygen and pH at the start and the end of the test. Total hardness before the start.

OTHER TEST CONDITIONS
- Adjustment of pH: no.
- Photoperiod: 4 a.m. to 8 p.m.
- Light intensity: /



EFFECT PARAMETERS MEASURED: 24 and 48 hours after start of exposure to the test substance the neonates were observed for their swimming behaviour. Animals not able to swim within approximately 15 s after gentle agitation of the test container are evaluated as immobilised. Additionally immobilised animals trapped at the surface were counted to evaluate a possible influence of the test substance to the water surface tension.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: nominal 2.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

95 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence limit: upper: 135; lower:67; slope: 3

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

53 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence limit: upper: 62; lower: 46; slope: 3

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 95 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

95 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

24 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

24 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 7

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

24 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 8

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

24 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: Some immobilised test animals were trapped at the medium surface. This may indicate a possible influence of the test substance to the surface tension. For details please see also the attached background material: Key. Fenzl. Acute Toxicity Daphnia_ Tables and figures.pdf
- Mortality of control: no mortality and no immobilisation in the negative control group.
- Other adverse effects control: no.
- Abnormal responses: 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: not applicable.

Results with reference substance (positive control):

The batch of ephippia is provided together with a specification sheet with batch number and date of expiry. Additionally for confirmation of the sensitivity of the test animals of the used batch the values of a reference test with potassium dichromate are provided. Batch No. of the ephippia in the test: DM310105. 24h EC 50 of potassium dichromate: 1.09 mg/L. EC50 24h acceptability range according OECD guideline 202 is 0.6 to 2.1 mg/L.

Reported statistics and error estimates:

EC50-values and confidence limits were determined by the method of W. Thompson, modified by C.S. Weil, Drug and Chemical Toxicology, 6 (6), 595-603 (1983).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour EC50 in Daphnia magna was 53 mg / L in a aqueous solution of Diethylmaleinate, based on immobilisation of the test animals. The
NOEC is estimated to be 24 mg/L. The results are based on the measured concentrations at the start of the test.

Executive summary:

The acute toxicity of "DIETHYLMALEINATE" to Daphnia magna was determined in a 48 hours static test. The following guideline was applied: Annex 5 (92/69/EEC) to Commission Directive 92/32/EEC, Part C; Methods for determination of ecotoxicity, publication No: L383, 29 December 1992: C.2. Acute Toxicity for Daphnia.

Neonates of Daphnia magna, not more than 24 hours old, were exposed to five concentrations of the test substance (nominal: 7, 14, 28, 55 and 110 mg test substance / L; measured initial: 6, 12, 24, 48 and 95 mg test substance / L) in reconstituted water according ISO 6341.

The 48-hour EC50 in Daphnia magna was 53 mg / L in a aqueous solution of Diethylmaleinate, based on immobilisation of the test animals. The NOEC is estimated to be 24 mg/L. The results are based on the measured concentrations at the start of the test.