1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-nortall-oil alkyl derivs.

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 17 August 2011 and 20 April 2012.

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:

- Concentrations: Standard solutions of test item were prepared in acetonitrile at nominal concentrations of 0.010, 0.050, 0.10, 0.20, 0.50, 1.0, 5.0 and 10 mg/1.

Sample Preparation:
The test samples (200 ml) plus 40 g of sodium chloride were 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 redissolved in acetonitrile to give a final theoretical concentration of 1.8 to 5.0 mg/l.

- Sample storage conditions before analysis:
Samples were stored at approximately -20°C prior to analysis. Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Vehicle:

yes

Details on test solutions:

Range finding test:
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.025, 0.25, 2.5 and 25 mg/l. The test item was dissolved directly in reconstituted water. An amount of test item (50 mg) was dissolved in 2 litres of reconstituted water with the
aid of ultrasonication for 10 minutes followed by vigorous stirring via a magnetic stirrer for 10 minutes to give the 25 mg/I test concentration from which serial dilutions were prepared in reconstituted water to give the remainder of the test series. Each prepared concentration was inverted several times to ensure adequate mixing and
homogeneity.

The reconstituted water was prepared as follows:
a) CaCI2.2H20 - 11.76 g/l
b) MgS04.7H20 - 4.93 g/l
c) NaHC03 - 2.59 g/l
d) KCI - 0.23 g/l

An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCI and was aerated until the dissolved oxygen concentration was approximately air-saturation value. The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaC03.

For the purpose of the definitive test the test item was dissolved directly in reconstituted water.
An amount of test item (50 mg) was dissolved in 2 litres of reconstituted water with the aid of ultrasonication for 10 minutes followed by vigorous stirring via a magnetic stirrer for 10 minutes to give a 25 mg/l stock solution. An aliquot (500 ml) of the 25 mg/l stock solution was diluted in a final volume of 5 litres of reconstituted water to give the 2.5 mg/l test concentration. Aliquots (10, 18, 32, 56, 100, 180, 320 and 560 ml) of the 2.5 mg/l test concentration were each separately diluted in a final volume of 1 litre of reconstituted water to give the remainder of the test concentrations of 0.025, 0.045, 0.080, 0.14, 0.25, 0.45, 0.80 and 1.4 mg/l.

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

Test organisms (species):

Daphnia magna

Details on test organisms:

Adult Daphnia were maintained in 150 ml glass beakers containing Elendt M7 medium (see Appendix 2) 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 mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. 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.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaC03.

Test temperature:

Temperature was maintained at 20°C to 22°C throughout the test.

Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCI

Dissolved oxygen:

The dissolved Oxygen concentration ranged between 8.6 and 8.8 mg O2/l after 48 hours.

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed in the control 'group.

Salinity:

Not applicable as this was a fresh water study

Nominal and measured concentrations:

Based on the results of the range-finding test the following nominal test concentrations were assigned to the definitive test: 0.025, 0.045, 0.080, 0.14, 0.25, 0.45, 0.80, 1.4 and 2.5 mg/I.

Details on test conditions:

As in the range-finding test, 250 ml glass jars containing approximately 250 ml of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 20°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 group was maintained under identical conditions but not exposed to the test item.

The test preparations were not renewed during the exposure period. 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.:

0.098 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% confidence limits of 0.056 - 0.17 mg/l

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.056 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Details on results:

Range finding study:
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 (please see tables in the attachment section).

No immobilisation was observed at the nominal test concentration of 0.025 mg/l. However, immobilisation was observed at 0.25, 2.5 and 25 mg/l.
Based on this information test concentrations of 0.025, 0.045, 0.080, 0.14, 0.25, 0.45, 0.80, 1.4 and 2.5 mg/I were selected for the definitive test.

Chemical analysis of the test preparations showed measured concentrations to range from 34% to 55% of nominal at 0 hours and from 26% to 60% of nominal at 48 hours. Although a decline in measured concentration was observed at the lower test concentrations over the 48-Hour test period it was considered that the test item was generally stable under test conditions.

Definitive study:

Chemical analysis of the 0.25 to 2.5 mg/I nominal test concentrations (corresponding to the No Observed Effect Concentration and above) showed measured concentrations to range from 0.0846 to 1.611 mg/I at 0 hours and from 0.0267 to 1.472 mg/I at 48 hours. Given that these results were below the nominal test concentrations and, in general, no significant decline in measured concentration was observed over the 48-Hour test period, it was considered justifiable to base the results on the mean measured test concentrations. The mean measured test concentrations were determined to be:
Nominal Test Concentration (mg/l) Mean measured test concentartion (mg/l) Expressed as a percentage of nominal
0.25 0.056 22
0.45 0.17 38
0.80 0.37 46
1.4 0.73 52
2.5 1.5 60

Immobilisation data
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2 (please see tables in the attachments section).
Analysis of the immobilisation data by the probit method (Finney 1971) at 24 hours and the geometric mean method at 48 hours based on the mean measured test concentrations gave the following results:

Time (h) EC50 (mg/l) 95% Confidence limits (mg/l)
24 0.17 0.13 - 0.21
48 0.098 0.056 - 0.17*

* Concentrations resulting in 0% and 100% immobilisation respectively.

The No Observed Effect Concentration after 24 and 48 hours exposure was 0.056 mg/l.
The No Observed Effect Concentration is based on the immobilisation at this concentration.
The slope and its standard error of the response curve at 24 hours was 5.4 (SE - 1.2). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of the response curve at 38 hours.

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd., Project No: 41104946) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l. Exposure conditions for the positive control were similar to those in the definitive test with the exception of the temperatures during testing which ranged from 21°C to 22°C.

Analysis of the immobilisation data by the geometric mean method at 24 hours and the trimmed Spearman-Karber method (Hamilton et al 1977*) 48 hours based on the nominal test concentrations gave the following results:
Time EC50 (mg/l) 95% confidence limits
24 1.3 1.0 - 1.8
48 1.1 1.0 - 1.3

The No Observed Effect Concentrations after 24 and 48 hours were 1.0 and 0.56 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration. The results from the positive control with potassium dichromate were within the normal range for this reference item.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and, based on mean measured test concentrations, gave a 48-Hour EC50 value of 0.098 mg/l with 950/0 confidence limits of 0.056 - 0.17 mg/l. The No Observed Effect Concentration at 48 hours was 0.056 mg/l.

Executive summary:

Introduction:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the GECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods:

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test item at nominal test concentrations of 0.025, 0.045, 0.080, 0.14, 0.25, 0.45, 0.80, 1.4 and 2.5 mg/l for 48 hours at a temperature of 20°C to 22°C under static test conditions. The number of immobilised Daphnia were recorded after 24 and 48 hours.

Results:

Chemical analysis of the 0.25 to 2.5 mg/l nominal test concentrations (corresponding to the No Observed Effect Concentration and above) showed measured concentrations to range from 0.0846 to 1.611 mg/l at 0 hours and from 0.0267 to 1.472 mg/l at 48 hours. Given that these results were below the nominal test concentrations and, in general, no significant decline in measured concentration was observed over the 48-Hour test period, it was considered justifiable to base the results on the mean measured test concentrations. These were calculated to be 0.056, 0.17, 0.37, 0.73 and 1.5 mg/l.

The 48-Hour EC50 for the test item to Daphnia magna based on mean measured test concentrations was 0.098 mg/l with 950/0 confidence limits of 0.056 - 0.17 mg/l. The No Observed Effect Concentration was 0.056 mg/l.

Description of key information

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and, based on mean measured test concentrations, gave a 48-Hour EC50 value of 0.098 mg/l with 950/0 confidence limits of 0.056 - 0.17 mg/l. The No Observed Effect Concentration at 48 hours was 0.056 mg/l.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.098 mg/L

Additional information

Introduction:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the GECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods:

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test item at nominal test concentrations of 0.025, 0.045, 0.080, 0.14, 0.25, 0.45, 0.80, 1.4 and 2.5 mg/l for 48 hours at a temperature of 20°C to 22°C under static test conditions. The number of immobilised Daphnia were recorded after 24 and 48 hours.

Results:

Chemical analysis of the 0.25 to 2.5 mg/l nominal test concentrations (corresponding to the No Observed Effect Concentration and above) showed measured concentrations to range from 0.0846 to 1.611 mg/l at 0 hours and from 0.0267 to 1.472 mg/l at 48 hours. Given that these results were below the nominal test concentrations and, in general, no significant decline in measured concentration was observed over the 48-Hour test period, it was considered justifiable to base the results on the mean measured test concentrations. These were calculated to be 0.056, 0.17, 0.37, 0.73 and 1.5 mg/l.

The 48-Hour EC50 for the test item to Daphnia magna based on mean measured test concentrations was 0.098 mg/l with 95% confidence limits of 0.056 - 0.17 mg/l. The No Observed Effect Concentration was 0.056 mg/l.