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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
Described in "Principles of method if other than guideline"
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
Described in "Principles of method if other than guideline"
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test item in cases where the test item is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (50 mg/l) of test item in reconstituted water for a period of 24 hours prior to removing any undissolved test item present by filtration (0.2 µm Sartorius Sartopore, first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution of the test item.
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Farnesene
IUPAC Name:
Farnesene
Constituent 2
Reference substance name:
(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
EC Number:
242-582-0
EC Name:
(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Cas Number:
18794-84-8
IUPAC Name:
7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Test material form:
other: liquid
Details on test material:
Namel (as cited in report): Farnesene
Description : clear, colourless liquid
Chemical name: trans-β-Farnesene;
trans-7,11-dimethyl-3-methylene-1,6,10-dodecatriene
(6E)-7,11-dimethyl-3-methylidenedodeca-1,6,10-triene
CAS number : 18794-84-8
Empirical fomula: C15H24
Smiles notation: C(C=C)(CCC=C(CCC=C(C)C)C)=C
Molecular weight:204.35
substance type: organic

Expiry date : 21 January 2012
Storage conditions: approximately 4°C, in the dark



Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES of Farnesene (surrogate)
- Melting point: 253K
- Boiling point: 250 - 260 C
- Vapour pressure:
- Water solubility (under test conditions):<0.0001 g/L at 20 C
- log Pow: >6.5
-Density: 0.83
Molecular weight = 204
Molecular Formula C15H24

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The concentration of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours (see attached Appendix 6).

Test solutions

Vehicle:
no
Details on test solutions:
Pre-study media preparation trial
Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication. An estimate of the water solubility for the test item using EPIWIN (Version 3.20) indicated that the solubility was approximately 0.0090 mg/l.

Based on this information the test item 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 item under test conditions (see Appendix 5 in any other information on materials including methods section).

Range-finding test
The results obtained from the pre-study media preparation trial conducted indicated that a saturated solution method of preparation followed by the removal of any undissolved test item by filtration was most appropriate for this test item.
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 0.10, 1.0, 10 and 100% v/v saturated solution.
An amount of test item (550 mg) was added to the surface of 11 litres of reconstituted water and stirred using a propeller stirrer at approximately 1500 rpm for 24 hours. After stirring any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give the 100% v/v saturated solution test concentration. Serial dilutions were then prepared in reconstituted water to give the remainder of the test concentrations.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21C to 22°C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. 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. Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
The control group was maintained under identical conditions but not exposed to the test item.
A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored at approximately -20°C prior to analysis.

Definitive test
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration, no immobilisation or adverse reactions to exposure were observed.

Experimental preparation
An amount of test item (550 mg) was added to the surface of 11 litres of reconstituted water and stirred using a propeller stirrer at approximately 1500 rpm for 24 hours. After stirring any undissolved test item was removed by filtration through a 0.2µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give the 100% v/v saturated solution test concentration.
The concentration of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours (see attached Appendix 6).

Physico-chemical measurements
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi hand held meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
Evaluation of data

An estimate of the EC50 values was given by inspection of the immobilisation data.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Test Species
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in 150 ml glass beakers containing Elendt M7 medium (see attached Appendix 2) in a temperature controlled room at approximately 20 DegC. 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.

Please see attached appendix 2 for details on Reconstituted Water – Elendt M7 Medium

Study design

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

Test conditions

Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test temperature:
Water temperature was recorded daily throughout the test
Temperature was maintained at 21°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.
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
pH:
pH values were recorded at the start and termination of the test (see attached Appendix 7). The pH values were measured using a Hach HQ30d Flexi hand held meter.
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl
There were no treatment related differences for pH.
Dissolved oxygen:
The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentrations were recorded at the start and termination of the test (see attached Appendix 7). The dissolved oxygen concentration was measured using a Hach HQ30d Flexi hand held meter.
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:
NA - Freshwater used
Nominal and measured concentrations:
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration, no immobilisation or adverse reactions to exposure were observed.
Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.0015 mg/l. This does not infer that no test item was in solution, just that which was so was at a concentration of less that the LOQ.
Details on test conditions:
Exposure conditions
In the definitive 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 21degC to 22degC 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.

OTHER TEST CONDITIONS
- Adjustment of pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

- Photoperiod:
a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.

- Light intensity:
Not recorded

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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.

TEST CONCENTRATIONS

- Spacing factor for test concentrations:
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration, no immobilisation or adverse reactions to exposure were observed.

- Justification for using less concentrations than requested by guideline:
Not applicable

- Range finding study

- Test concentrations:

0.10, 1.0, 10 and 100% v/v saturated solution
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilization
Remarks on result:
other: 95% CL not stated
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilization
Remarks on result:
other: 95% CL not stated
Details on results:
Range-finding Test
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1, which is in "Overall Remarks" attachments.
No immobilisation was observed at the test concentrations of 10 and 100% v/v saturated solution.
A single immobilised daphnid was observed at 0.10% v/v saturated solution after 48 hours exposure. This was considered to be due to natural causes rather than a toxic effect given that no immobilisation was observed at the higher test concentrations of 10 and 100% v/v saturated solution. All the daphnids at 1.0% v/v saturated solution were observed to be immobilised after 48 hours exposure. These were considered to be due to contamination from an unknown source given that no immobilisation was observed at the higher test concentrations of 10 and 100% v/v saturated solution.
Based on this information, a single test concentration of four replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the highest attainable test concentration, no immobilisation or adverse reactions to exposure were observed.
Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.0015 mg/l. This does not infer that no test item was in solution, just that which was so was at a concentration of less that the LOQ.

Definitive Test
Immobilisation data
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.

There was no immobilisation in 20 daphnids exposed to a test concentration of 100% v/v saturated solution for a period of 48 hours. Inspection of the immobilisation data gave the following results:


Time (h) EC50 (% v/v Saturated Solution)
24 >100
48 >100


The No Observed Effect Concentration after 24 and 48 hours exposure was 100% v/v saturated solution. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
Observations on test item solubility
The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

Physico-chemical measurements
The results of the physico-chemical measurements are given in the attached Appendix 7. Temperature was maintained at 21°C to 22°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
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.

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.
Verification of test concentrations
Analysis of the test preparations at 0 and 48 hours (see attached Appendix 6) showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.0015 mg/l. This does not infer that no test item was in solution, just that which was, was at a concentration of less than 0.0015 mg/l.
Results with reference substance (positive control):
A positive control (Harlan Laboratories Ltd., Project Number: 41102080) used potassium dichromate as the reference item. Details of the positive control are given in the attached Appendix 3. The positive control was conducted between 7 June 2011 and 9 June 2011.
Reported statistics and error estimates:
Not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna is considered to
have a 48-Hour EC50 of greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution.The test showed no toxic effects at saturation.

Executive summary:

The freshwater invertebrate Daphnia magna is considered to have a 48-Hour EC50 of greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution. The test showed no toxic effects at saturation.

Experimental results:

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 OECD 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.

Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication. An estimate of the water solubility for the test item using EPIWIN (Version 3.20) indicated that the solubility was approximately 0.0090 mg/l. A pre-study media preparation trial indicated that the use of a saturated solution method of preparation followed by the removal of any undissolved test item by filtration was most appropriate for this test item.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100% v/v saturated solution for 48 hours at a temperature of 21°C to 22°C under static test conditions. The test item solutions were prepared by stirring an excess (50 mg/l) of test item in reconstituted water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results and Conclusion.

The 48-Hour EC50 for the test item to Daphnia magna based on nominal test concentrations was greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.0015 mg/l. This does not infer that no test item was in solution, just that which was, was at a concentration of less than 0.0015 mg/l. The test showed no toxic effects at saturation.