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Diss Factsheets

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 19 October 2009 and 13 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Remarks:
The study plan was adapted in accordance with OECD 23; Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Remarks:
The study plan was adapted in accordance with OECD 23; Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Details on test material:
- Substance type: UVCB
- Physical state: liquid
- Analytical purity: ~90%
- Lot/batch No.: CM71217681
- Expiration date of the lot/batch: 27-Apr-2010
- Stability under test conditions: Unknown; is excluded from the statement of compliance.
- Storage condition of test material: At room temperature (range of 20 ± 5 °C), light protected.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal Concentration control, 10, 18, 32, 56, 100 (% v/v Saturated Solution)
- Sampling method: 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 methanol.
- Sample storage conditions before analysis: no data available.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: saturated solution
- Eluate: not applicable.
- Differential loading: In each of six ground glass stoppered conical flasks an amount of test material (100 mg) was dispersed in 500 ml of reconstituted water to give initial test material dispersions of 200 mg/l. These were mixed with the aid of shaking (INFORS Aerotron) at 300 rpm at a temperature of 30°C for 24 hours. After the shaking period the contents of the replicate flasks were pooled and then allowed to cool to approximately 20C over a period of approximately 24 hours. The undissolved test material was then removed by centrifugation at 10000 g for 30 minutes to give a 100% v/v saturated solution.
Aliquots (200 and 360 ml) of the 100% v/v saturated solution were each separately dispersed in a final volume of 2 litres of reconstituted water to give the 10 and 18% v/v saturated solution test concentrations respectively. Further aliquots (320 and 560 ml) of the 100% v/v saturated solution were each separately dispersed in a final volume of 1 litre of reconstituted water to give the 32 and 56% v/v saturated solution test concentrations respectively.

- Controls: The control group was maintained under identical conditions but not exposed to the test material.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none.
- Concentration of vehicle in test medium (stock solution and final test solution): none.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic observations of the 10 to 100% v/v saturated solution test concentrations showed no micro particles or micro-dispersions to be present.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water Flea (Daphnia)
- Strain: Daphnia magna
- Source: In house laboratory cultures
- Age at study initiation (mean and range, SD): 1st instar
- Weight at study initiation (mean and range, SD): NDA
- Length at study initiation (length definition, mean, range and SD): NDA
- Method of breeding: parthenogenises
- Feeding during test: The daphnids received no food during exposure

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test temperature:
The temperature was maintained at ADD°C throughout the duration of the test.
pH:
7.8
Dissolved oxygen:
The dissolved oxygen concentration was approximately air-saturation value
Salinity:
Not applicable.
Nominal and measured concentrations:
concentrations of 10, 18, 32, 56 and 100% v/v saturated solution (0.0073 mg/l based on geometric mean measured concentration of the 100% v/v saturated solution)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml covered jar
- Material: glass
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): not applicable
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): not applicable.
- Biomass loading rate:NDA

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water
- Total organic carbon: NDA
- Particulate matter: NDA
- Metals:NDA
- Pesticides:NDA
- Chlorine:NDA
- Alkalinity:NDA
- Ca/mg ratio:NDA
- Conductivity:NDA
- Culture medium different from test medium: No difference
- Intervals of water quality measurement: N/A


OTHER TEST CONDITIONS
- Adjustment of pH: Using NaOH or HCl were necessary
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods
- Light intensity: NDA


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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: ~1.8
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution (0.00021, 0.0021, 0.021 and 0.21 mg/l based on chemical analysis of the 100% v/v saturated solution).
- Results used to determine the conditions for the definitive study: No immobilisation was observed at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution (equivalent to 0.00021, 0.0021 and 0.021 mg/l based on chemical analysis of the 100% v/v saturated solution). However, immobilisation was observed at 100% v/v saturated solution (equivalent to 0.21 mg/l based on chemical analysis). Based on this information test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution were selected for the initial tests.

Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks:
immobilisation
Remarks on result:
other: 95% Cl = 0.45-0.56 mg/l
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks:
immobilisation
Details on results:
- Behavioral abnormalities: no effect.
- Observations on body length and weight: No data.
- Other biological observations: No data.
- Mortality of control: 0
- Other adverse effects control: no effect.
- Abnormal responses: none.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none.
- Effect concentrations exceeding solubility of substance in test medium: N/A
Results with reference substance (positive control):
- Results with reference substance valid? Yes
The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.5 mg/l (95% confidence limits of 0.45 – 0.56 mg/L).

The No Observed Effect Concentration after 48 hours was 0.0073 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

Time (h): 48 h
EC50 (mg/l): 0.0073
95% Confidence limits (mg/l): no data.

Any other information on results incl. tables

Test Substance:

Verification of test concentrations

Analysis of the 10% to 32% v/v saturated solution test concentrations at 0 and 48 hours showed measured concentrations to be less than the limit of quantitation (LOQ) for the analytical method which was assessed as 0.0024 mg/l.

 

Analysis of the 56% and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations to be 0.00531 and 0.00918 mg/l respectively.

 

Analysis of the 56% and 100% v/v saturated solution test preparations at 48 hours showed measured test concentrations to be 0.00423 and 0.00587 mg/l respectively.

 

The geometric mean measured test concentration of the 100% v/v saturated solution was calculated to be 0.0073 mg/l.

 

Observations on test material solubility

The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

           

Immobilisation data

Cumulative immobilisation data from the exposure ofDaphnia magnato the test material during the definitive test are given in the following Table

 

Time (h)

EC50 (mg/l)

24

>0.0073

48

>0.0073

 

Positive Control

The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in the following Table.

 

Time (h)

EC50 (mg/l)

95% Confidence limits (mg/l)

24

0.95

0.81- 1.1

48

0.50

0.45-0.56

 

The No Observed Effect Concentrations after 24 and 48 hours was 0.32 mg/l. 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 material.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
No immobilisation or adverse effects were observed at the geometric mean measured test concentration of 0.0073 mg/l. Consequently, the 48-Hour EC50 for the test material to Daphnia magna based on the geometric mean measured test concentration was greater then 0.0073 mg/l and correspondingly the No Observed Effect Concentration was 0.0073 mg/l.
This study showed that there were no toxic effects at saturation.
Executive summary:

SUMMARY

Introduction.A study was performed to assess the acute toxicity of the test material toDaphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphniasp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods.A determination of the General Physico-Chemical Properties study conducted on the test material (Harlan Laboratories Ltd. Project Number: 0525/0967) gave a water solubility value for the test material of less than or equal to 0.138 mg/l at 20.0 ± 0.5°C. Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A further media preparation trial indicated that a dissolved test material concentration of approximately 0.0442 mg/l was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test and initial tests, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at concentrations of 10, 18, 32, 56 and 100% v/v saturated solution (0.0073 mg/l based on geometric mean measured concentration of the 100% v/v saturated solution) for 48 hours at a temperature of approximately 20C under static test conditions. The test material solution was prepared by shaking an excess (200 mg/l) of test material in reconstituted water in sealed vessels at approximately 300 rpm at a temperature of 30°C for 24 hours (INFORS Aerotron). After the shaking period the contents of the vessels were pooled and allowed to cool to approximately 20°C for approximately 24 hours prior to the removal of any undissolved test material by centrifugation at 10000gfor 30 minutes to produce a saturated solution with a 0-Hour measured concentration of 0.00918 mg/l. The difference observed between the measured concentration obtained from the pre-study media preparation trial and definitive test was considered to be due to slight variation when removing the supernatant from a centrifuged test sample, the low water solubility of the test material and the problems associated with the preparation of saturated solutions. The number of immobilisedDaphniawere recorded after 24 and 48 hours.

A positive control conducted alongside the definitive test used potassium dichromate as the reference material.Daphnia magnawas 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 approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results.Analysis of the 10% to 32% v/v saturated solution test concentrations at 0 and 48 hours showed measured concentrations to be less than the limit of quantitation (LOQ) for the analytical method which was assessed as 0.0024 mg/l.

Analysis of the 56% and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations to be 0.00531 and 0.00918 mg/l respectively.

Analysis of the 56% and 100% v/v saturated solution test preparations at 48 hours showed measured test concentrations to be 0.00423 and 0.00587 mg/l respectively. This slight decline in measured concentration over the 48-Hour test period was contrary to the stability analysis which showed the test material to be stable over the test period. Results from a Study to Determine the General Physico-Chemical Properties of the test material (Harlan Laboratories Ltd Project Number: 0525/0967) indicated a high potential for the test material to adsorb to organic matter. Therefore the slight loss of the test material over the test period was considered to be due to possible adsorption of the test material to the test organisms and/or their organic waste products.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentration of the 100% v/v saturated solution, as no effects were observed at this concentration, in order to give a "worst case" analysis of the data. This approach is recommended by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). The geometric mean measured concentration was calculated to be 0.0073 mg/l.

No immobilisation or adverse effects were observed at the geometric mean measured test concentration of 0.0073 mg/l. Consequently, the 48-Hour EC50 for the test material toDaphnia magnabased on the geometric mean measured test concentration was greater then 0.0073 mg/l and correspondingly the No Observed Effect Concentration was 0.0073 mg/l.

This study showed that there were no toxic effects at saturation.

The 48-Hour EC50 for the reference material toDaphnia magnabased on nominal concentrations was 0.50 mg/l with 95% confidence limits of 0.45 – 0.56 mg/l. The No Observed Effect Concentration was 0.32 mg/l.