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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-02-16 to 2011-01-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
(2004)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
Commission Regulation (EC) No. 440/2008
Deviations:
no
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 (1 00 mg/1) of test item in reconstituted water for a period of 24 hours prior to removal of the aqueous phase by mid-depth siphon to give a saturated solution of the test item.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
1,4-Benzenedicarboxylic acid, 1,4-diisononyl ester
EC Number:
700-453-0
Cas Number:
59802-05-0
Molecular formula:
C26H42O4
IUPAC Name:
1,4-Benzenedicarboxylic acid, 1,4-diisononyl ester
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 1.0, 10 and 100% v/v saturated solution. In the definitive test a nominal concentration of 100% v/v saturated solution was tested.
- Sampling method: Samples of the test preparations were taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. Water samples were taken from the control (replicates R1 - R4 pooled) and the 100% v/v saturated solution test group (replicates R1 - R2 and R3 - R4 pooled) at 0 and 48 hours for quantitative analysis. Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Pre-study solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. 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. Therefore, an amount of test item (200 mg) was dispensed in duplicate, on to the surface of 2 litres of reconstituted water with the aid of magnetic stirring at a rate such that a dimple was formed at the water surface for periods of 24 or 48 hours. After stirring the aqueous phase was removed from the bottom of the mixing vessel and samples taken for chemical analysis.
For the definitive test an amount of test item (500 mg) was dispensed on to the surface of 5 litres of reconstituted water prior to stirring slowly via magnetic stirrer for 24 hours. After 24 hours the stirring was stopped and the aqueous phase removed by mid-depth siphoning to give a 100% v/v saturated solution of test item.
- Controls: test medium without test substance and positive control. The control group was maintained under identical conditions but not exposed to the test substance.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: in-house laboratory cultures
- Feeding during test: no feeding during exposure

ACCLIMATION
- Acclimation period: 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.
- Acclimation conditions: Culture conditions ensured that reproduction was by parthenogenesis.
- Type and amount of food: Each culture was fed daily with a suspension of algae (Chlorella sp.).
- The reconstituted water was used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
- The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures. 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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaC03.
Test temperature:
20 °C
pH:
7.8 ± 0.2
Dissolved oxygen:
air saturation value (ASV) in excess of 100%.
Conductivity:
<5 µS cm-1
Nominal and measured concentrations:
Nominal concentration of 100 % saturated solution
Analysis of the test preparations at 0 and 48 hours showed measured concentrations to be less than the limit of quantitation (LOQ), which was assessed to be 0.00027 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass jars containing approximately 250 ml of test preparation
- Type (delete if not applicable): covered to reduce evaporation
- Aeration: no aeration
- 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: reconstituted water as recommended in guideline
- Culture medium different from test medium: same

OTHER TEST CONDITIONS
- Adjustment of pH: The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary). No pH-Adjustment during exposure.
- The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 21 °C with a photoperiod of 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. Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 1.0, 10 and 100% v/v saturated solution.
- Results used to determine the conditions for the definitive study: No immobilisation was observed at the test concentrations of 1.0, 10 and 100% v/v saturated solution. Chemical analysis of the 100% v/v saturated solution test samples at 0 and 48 hours showed measured test concentrations of less that the limit of quantitation (LOQ) of the analytical method employed were obtained. 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.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
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:
mobility
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:
mobility
Details on results:
Analysis of the test preparations at 0 and 48 hours showed measured concentrations to be less than the limit of quantitation (LOQ), which was assessed to be 0.00027 mg/L and so the results are based on nominal test concentrations only.
There was no immobilisation in 20 daphnids exposed to a test concentration of 100% v/v saturated solution for a period of 48 hours.

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.
Results with reference substance (positive control):
- Results with reference substance valid? The results from the positive control with potassium dichromate were within the normal range for this reference item. The mean 48-Hour EC50 value calculated from all positive controls was 0. 77 mg/1 (sd = 0.19).
- Relevant effect levels: The 48-Hour EC50 for the reference item to Daphnia magna based on nominal concentrations was 0.75 mg/L with 95% confidence limits of 0.65 - 0.86 mg/1. The No Observed Effect Concentration was 0.32 mg/L.
- Dose-response test: concentrations of 0.32 mg/L, 0.56 mg/L, 1.0 mg/L, 1.8 mg/L and 3.2 mg/L were tested

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 100% v/v saturated solution. Correspondingly the No Observed Effect Concentration was 100% v/v saturated solution. Analysis of the test preparations at 0 and 48 hours showed measured concentrations to be less than the limit of quantitation (LOQ), which was assessed to be 0.00027 mg/L and so the results are based on nominal test concentrations only. This study showed that there were no toxic effects at saturation.