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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 October 2007 to 26 October 2007
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
Commission Directive 92/69/EEC
GLP compliance:
yes (incl. QA statement)
(Date of inspection: 21 August 2007 Date of Signature: 15 October 2007)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
trimethyl[2-({2-[(2-methylprop-2-enoyl)oxy]ethyl phosphonato}oxy)ethyl]azanium
Details on test material:
- Name of test material (as cited in study report): MPC
- Substance type: White powder
- Physical state: Solid
- Lot/batch No.: 270103
- Storage condition of test material: Room temperature in the dark.

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: Control and 100 mg/l test group.

- Sampling method: Water samples were taken from the control and each test group (4 replicates pooled) at 0 hours and 48 hours for quantitative analysis.

- Sample storage conditions before analysis: Duplicate samples were taken and stored frozen at approximately -20ºC for further analysis if necessary.

Test solutions

Details on test solutions:
An amount of test material (200 mg) was dissolved in reconstituted water and the volume adjusted to 2 litres to give the test concentration of 100 mg/l. The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Water flea.
- Source: In-house laboratory cultures.
- Age at study initiation (mean and range, SD): Less than 24 hours old (1st instar).
- Method of breeding: Parthenogenesis.
- Feeding during test: Adult Daphnia was maintained in polypropylene vessels containing approximately 2 litres of reconstituted water. Each culture was fed daily with a suspension of algae (Chlorella sp.). Gravid adults were isolated the day before initiation of the test. The young were removed from the cultures and used for testing.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:
Not applicable.

Test conditions

250 mg CaCO3/L (reconstituted water)
Test temperature:
21 - 22ºC. Some of the temperatures were measured to be slightly in excess of the 20 ± 1ºC but this was considered not to affect the resu;ts of the test.
(0-h) 7.9
(48-h) 8.0 - 8.1
Dissolved oxygen:
8.4 - 8.8 mg O2/l (94 - 99% of air saturation value).
Not reported.
Nominal and measured concentrations:
100 mg/l (nominal concentration)
Details on test conditions:
- Test vessel:
- Type (delete if not applicable): closed.
- Material, size, headspace, fill volume: 250 ml glass jars with approximately 200 ml of test preparation.
- Aeration: No.
- Renewal rate of test solution: Not renewed.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

- Source/preparation of dilution water: Reconstituted Water.
Stock Solutions
a) CaCl2.2H2O : 11.76 g/l
b) MgSO4.7H2O: 4.93 g/l
c) NaHCO3: 2.59 g/l
d) KCl: 0.23 g/l
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water. The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.

- Conductivity: <5 µS cm-1 (deionised water).

- Adjustment of pH: pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl (deionised water).
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/l (nominal concentration)

- Results used to determine the conditions for the definitive study: No immobilisations up to 1.0 mg/l after 48 hours exposure. A single immobilised daphnid was observed in each of the 10 and 100 mg/l test concentrations after 48 hours exposure. This immobilisation was considered to be due to natural causes rather than a true toxic effect given that no immobilisation or sub-lethal effects of exposure were observed in the remaining daphnids.Based on this result, a limited test was conducted as a main test with the concentration of 100 mg/l.
Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
48 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
48 h
Dose descriptor:
Effect conc.:
100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
There was no imobilisation in 20 daphnids exposed to a test concentration of 100 mg/l for a period of 48 hours.

- Mortality of control: Not reported.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At o hurs and throughout the test period, all control and test vessels were observed to contain clear colourless solutions.

Results with reference substance (positive control):
- Results with reference substance valid?: yes

- EC50:
3-h EC50: > 3.2 mg/l
24-h EC50: 1.2 mg/l, 95%CL of 1.1 - 1.4
48-h EC50: 0.75 mg/l, 95%CL of 0.56 - 1.0 (the mean of 0.81 mg/l, sd of 0.21)).

Reported statistics and error estimates:
Not reported.

Any other information on results incl. tables

Table Analytical results


Nominal Concentration (mg/l)

Concentration Found (mg/l)

Expressed as a percent of the nominal concentration (%)

0 hours





100 R1 - R2




100 R3 - R4







48 hours





100 R1 - R2




100 R3 - R4



LOQ = Limit of quantitation

R1 - R4 = Replicates 1 to 4

Applicant's summary and conclusion

Validity criteria fulfilled:
The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.
Executive summary:

Introduction. A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).


Methods. Following a preliminary range-finding tests, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a concentration of 100 mg/l for 48 hours at a temperature of 21 to 22°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of 21°C to 22°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.


Results. The 48-Hour EC50 for the test material to Daphnia magna based on nominal test concentrations was greater than 100 mg/l and correspondingly the No Observed Effect Concentration was 100 mg/l.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal and so the results are based on nominal concentrations only.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.

The 48-Hour EC50 for the reference material to Daphnia magna based on nominal test concentrations was 0.75 mg/l with 95% confidence limits of 0.56 – 1.0 mg/l. The No Observed Effect Concentration was 0.56 mg/l.