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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 29 September 2008 and 15 October 2008
Reliability:
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.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
A study to determine the General Physico-Chemical properties of the test material (Project Number: 2551/0013) determined the water solubility of the test material to be 0.224 mg/l. Pre-study solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. A test concentration of 0.20 mg/l (by visual inspection) was obtained using a preliminary solution in dimethylformamide. At higher test concentrations precipitation of the test material was observed on addition of the test material solvent stock solution to water.

Based on this information the test material was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.


The test concentration to be used in the definitive test was determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.00070, 0.0070 and 0.070 mg/l. The test material was prepared using a preliminary solution in dimethylformamide.

An amount of test material (50 mg) was dissolved in dimethylformamide and the volume adjusted to 25 ml to give a 50 mg/25 ml solvent stock solution. An aliquot (50 microl) of this solvent stock solution was dispersed in 500 ml of reconstituted water prior to removal of any undissolved test material by centrifugation at 10000 g for 30 minutes to give the 0.070 mg/l test concentration from which serial dilutions were prepared in reconstituted water to give the remainder of the test concentrations of 0.0070 and 0.00070 mg/l.

Each solvent stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The test material concentration in the test samples was determined by high performance liquid chromatography (HPLC) using an external standard. The test material gave a chromatographic profile consisting of two peaks. The results have been calculated using the total peak area associated with the test material.

The reference substance was stored at room temperature prior to the study.

The method was developed by the Department of Analytical Services, Harlan Laboratories Ltd.

A volume of test sample was extracted with dichloromethane (3 x 50 ml). The extracts were filtered through anhydrous sodium sulphate. The combined extracts were evaporated to dryness and the residue re-dissolved in acetonitrile to give a final theoretical concentration of 7.0 mg/l.

Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 10 mg/l for the pre-study media preparation trial and 7.0 mg/l for all other analyses.

The standards and samples were analysed by HPLC using the following conditions:
HPLC System : Agilent Technologies 1050 incorporating autosampler and workstation
Column : Synergi Polar RP, (150 x 4.6 mm id)
Column temperature : 30ºC


Mobile phase : acetonitrile:water* (65:35 v/v)
Flow rate : 1 ml/min
UV/Vis detector wavelength : 210 nm
Injection volume : 10 µl
Retention time : approximately 6 minutes

An amount of test material (550 mg) was dispersed, in duplicate, in 11 litres of test medium to give initial test material dispersions of 100 mg/l. These were stirred using a propeller stirrer at approximately 1500 rpm at approximately 21ºC for periods of 24 and 48 hours.

Samples were taken for analysis following removal of any undissolved test material by centrifugation at 10000 or 40000 g for 30 minutes or following filtration through 0.2 microm Gelman Acrocap filters with the first 100 or 500 ml being discarded.

* Prepared by ELGA Purelab Option R-15 water purification
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Method: Based on this information the test material was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.

Eluate: n/a
Differential loading: n/a
Controls: A positive control (Safepharm Laboratories Project No: 0039/0977) conducted approximately every six months used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.

An amount of reference material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (50 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/l stock solution. Aliquots (16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.

Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Exposure conditions for the positive control were similar to those used in the definitive test.

The temperature was maintained at 21°C.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide

- Concentration of vehicle in test medium (stock solution and final test solution): An amount of test material (50 mg) was dissolved in dimethylformamide and the volume adjusted to 25 ml to give a 50 mg/25 ml solvent stock solution.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): None.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a suspension of algae (Chlorella sp.). Culture conditions ensured that reproduction was by parthenogenesis.

Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.



ACCLIMATION
- Acclimation period: 24 hours
- Acclimation conditions (same as test or not):
- Type and amount of food: a suspension of algae (Chlorella sp).
- Feeding frequency: fed daily
- Health during acclimation (any mortality observed): No mortality observed.


QUARANTINE (wild caught)
- Duration:
- Health/mortality
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
n/a
Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test temperature:
Temperature was maintained at approximately 21 deg C to 22 deg C throughout the test.

Some of the temperatures were observed to be slightly in excess of the range given in the protocol of 20 ± 1°C. This deviation was considered not to have affected the outcome or the validity of the test given that no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperature range was within guideline specification.
pH:
Please refer to the table in Attachment 3.
Dissolved oxygen:
The dissolved oxygen concentration was approximately air-saturated value.

Please refer to the table in Attachment 3
Salinity:
Not applicable due to the use of freshwater throughout the study.
Nominal and measured concentrations:
Please refer to the table in Attachment 2.
Time-weighted mean measured concentration: 0.070 mg/l
Details on test conditions:
For the purpose of the definitive test the test material was prepared using a preliminary solution in dimethylformamide.

An amount of test material (50 mg) was dissolved in dimethylformamide and the volume adjusted to 25 ml to give a 50 mg/25 ml solvent stock solution. An aliquot (50 microl) of this solvent stock solution was dispersed in 500 ml of reconstituted water prior to removal of any undissolved test material by centrifugation at 10000 g for 30 minutes to give the 0.070 mg/l test concentration.

Each solvent stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Preliminary stability analysis showed a decline in measured test concentrations over 48 hours. Therefore, in an attempt to minimise losses of test material the test media were freshly prepared at 0 and 24 hours prior to dosing using a semi-static test regime.

The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media) (see attachment 2).

In the definitive test 250 ml glass jars containing approximately 250 ml of test preparation were used. At the start of the study 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test, control and solvent control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21°C to 22°C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control and solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 µl/l of dimethylformamide.

Semi-static test conditions were employed in the test in an effort to maintain near nominal test concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.

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.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.07 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: The test concentration of 0.070 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and having due regard to the amount of auxiliary solvent permitted in the study.
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.07 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: The NOEC is based upon zero immobilisation at this concentration.
Details on results:
The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

Temperature was maintained at approximately 21ºC to 22ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Results with reference substance (positive control):
- Mortality: Yes
- EC50/LC50: 0.7 mg/l
- Other: n/a

The results from the positive control with potassium dichromate were within the normal range for this reference material.
Reported statistics and error estimates:
None.

Definitive Test:

Immobilisation data

There was no immobilisation in 20 daphnids exposed to a time-weighted mean concentration of 0.070 mg/l for a period of 48 hours.  Inspection of the immobilisation data gave the following results:

Time (h)

EC50 (mg/l)

95% Confidence limits
(mg/l)

24

>0.070

-

48

>0.070

-

The No Observed Effect Concentration after 24 and 48 hours exposure was 0.070 mg/l.  The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The test concentration of 0.070 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guidelines.  Other various recognised auxiliary solvents were used during preliminary solubility work, however, dimethylformamide was found to give the best testable dispersion of the test material in water.  

Positive Control

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al 1977) at 24 hours and the probit method (Finney 1971) at 48 hours based on the nominal test concentrations gave the following result



Time (h)

EC50 (mg/l)

95% Confidence limits
(mg/l)

3

> 3.2*

-

24

0.97

0.85

-

1.1

48

0.70

0.62

-

0.80

The No Observed Effect Concentration after 24 and 48 hours were 0.56 and 0.32 mg/l respectively.  The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

* A 3-Hour EC50 value was not calculated as less than 50% immobilisation occurred at this tipoint.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and, based on the time-weighted mean measured test concentration, gave a 48-Hour EC50 of greater than 0.070 mg/l. Correspondingly the No Observed Effect Concentration was 0.070 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.

Methods.A Study to Determine the General Physico-Chemical Properties of the test material gave a water solubility value for the test material of 0.224 mg/l. Based on this information the test material was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore media preparation trials were conducted in order to determine the solubility of the test material under test conditions. 

Pre-study media preparation trials indicated that the use of preliminary solution in auxiliary solvent to spike the test medium followed by centrifugation at 10000 g to remove any undissolved test material was the most suitable method of preparation. 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a time-weighted mean measured concentration of 0.070 mg/l for 48 hours at a temperature of 21°C to 22°C under semi-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 under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.

Chemical analysis of the freshly prepared media at 0 and 24 hours showed measured test concentrations to range from 0.0424 to 0.123 mg/l. A decline in measured test concentrations was observed in the old media at 24 and 48 hours in the range of 0.0324 to 0.0780 mg/l. This decline was inline with the preliminary stability analyses conducted which indicated that the test material was unstable in culture medium.

Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations in order to give a "worst case" analysis of the data.

The 48-Hour EC50 based on the time-weighted mean measured test concentrations was greater than 0.070 mg/l and correspondingly the No Observed Effect Concentration was 0.070 mg/l.

The 48-Hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.70 mg/l with 95% confidence limits of 0.62 – 0.80 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

 

Description of key information

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.

Methods.A Study to Determine the General Physico-Chemical Properties of the test material gave a water solubility value for the test material of 0.224 mg/l. Based on this information the test material was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore media preparation trials were conducted in order to determine the solubility of the test material under test conditions. 

Pre-study media preparation trials indicated that the use of preliminary solution in auxiliary solvent to spike the test medium followed by centrifugation at 10000gto remove any undissolved test material was the most suitable method of preparation. 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a time-weighted mean measured concentration of 0.070 mg/l for 48 hours at a temperature of 21°C to 22°C under semi-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 under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.

Chemical analysis of the freshly prepared media at 0 and 24 hours showed measured test concentrations to range from 0.0424 to 0.123 mg/l. A decline in measured test concentrations was observed in the old media at 24 and 48 hours in the range of 0.0324 to 0.0780 mg/l. This decline was inline with the preliminary stability analyses conducted which indicated that the test material was unstable in culture medium.

Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations in order to give a "worst case" analysis of the data.

The 48-Hour EC50 based on the time-weighted mean measured test concentrations was greater than 0.070 mg/l and correspondingly the No Observed Effect Concentration was 0.070 mg/l.

The 48-Hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.70 mg/l with 95% confidence limits of 0.62 – 0.80 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.07 mg/L

Additional information

The test concentration of 0.070 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guidelines.