Registration Dossier

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:
2009-10-07 to 2010-10-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conform study under GLP.

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
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Test material form:
solid
Details on test material:
Sponsor's identification: LCE08089
Product code: LCE08044
Description: white solid
Batch number: 0901600021
Date received: 31 July 2009
Storage conditions: room temperature in the dark
Specific details on test material used for the study:
Sponsor's identification: LCE08089
Product code: LCE08044
Description: white solid
Batch number: 0901600021
Date received: 31 July 2009
Storage conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal loading rates of fresh media 0, 10, 18, 32, 56, and 100 mg/l
- Sampling method: Samples of the control and each loading rate WAF test group were taken for analysis at 0 (fresh media), 24 (fresh and old media) and 48 hours (old media).
- Sample storage conditions before 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:
Due to the low aqueous solubility and complex nature of the test material for the purposes of the definitive test the test material was prepared as a Water Accommodated Fraction (WAF).
Amounts of test material (200, 360, 640, 1120 and 2000 mg) were each separately added to the surface of 20 litres of reconstituted water to give the 10, 18, 32, 56 and 100 mg/l loading rates respectively. After the addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 500 ml discarded) to give the 10, 18, 32, 56 and 100 mg/l loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed there to be no particles or micro-dispersions of test material present.
Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media).
- Differential loading: Loading rates of 10, 18, 32, 56 and 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.
- Controls: negative control: test medium without test item; positive control: test medium plus potassium dichromate
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic observations made on the 100 mg/l loading rate WAF indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length).

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain: not specified
- Source: in-house laboratory cultures.
- Age at study initiation (mean and range, SD): Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.
- Weight at study initiation (mean and range, SD): not applicable
- Length at study initiation (length definition, mean, range and SD): no data
- Valve height at study initiation, for shell deposition study (mean and range, SD): not applicable
- Peripheral shell growth removed prior to test initiation: not applicable
- Method of breeding: 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.
- Feeding during test: no

ACCLIMATION
- Acclimation period: not applicable

QUARANTINE (wild caught)
not applicable

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
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:
21°C to 22°C
pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl
Dissolved oxygen:
The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Salinity:
Not reported as such, however detailed mineral compostion of the reconstituted water is given in the original study report.
Nominal and measured concentrations:
see attached table on nominal and measured concentrations
Details on test conditions:
TEST SYSTEM
- Test vessel: glass jars
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 250 ml, 50 ml, 200 ml
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- 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: no data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: resconstituted water was used, based on mineral stock solution and deionised water
- Total organic carbon: below LOQ
- Particulate matter: not applicable
- Metals: not applicable
- Pesticides: not applicable
- Chlorine: not applicable
- Alkalinity: Not reported as such, however detailed mineral compostion of the reconstituted water is given in the original study report.
- Ca/Mg ratio: 1:2 (mol:mol)
- Conductivity: Not reported as such, however detailed mineral compostion of the reconstituted water is given in the original study report.
- Culture medium different from test medium: no
- Intervals of water quality measurement: 0, 24, and 48 h

OTHER TEST CONDITIONS
- Adjustment of pH: if necessary, with NaOH or HCl
- Photoperiod: 16 h
- Light intensity: no data

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.75
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: yes
- Test concentrations: loading rate 1.0, 10 and 100 mg/l
- Results used to determine the conditions for the definitive study: No significant immobilisation was observed at 1.0 and 10 mg/l loading rate WAF. However, immobilisation was observed at 100 mg/l loading rate WAF. Based on this information loading rates of 10, 18, 32, 56 and 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: effective loading rate
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
56 mg/L
Nominal / measured:
nominal
Conc. based on:
other: efective loading rate
Basis for effect:
mortality
Remarks on result:
other: reported as "No Observed Effect Loading Rate"
Details on results:
- Behavioural abnormalities: none reported
- Observations on body length and weight: none reported
- Other biological observations: none reported
- Mortality of control: no
- Other adverse effects control: none reported
- Abnormal responses: none reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: see "details on analytical methods" and "details on test solutions"
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50/LC50: EC50 = 0.78 at 48 h
- Other:
Reported statistics and error estimates:
no statistics applied

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour Effective Loading Rate 50 value of greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate at 48 hours was 56 mg/l loading rate WAF.
Executive summary:

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 Regulation (EC) No. 440/2008.

Information provided by the Sponsor indicated that the test material was a mixture of components. Given this and also the poor solubility of the test material in water it was considered that the most suitable method of preparation for this material was as a Water Accommodated Fraction (WAF).

Based on analytical work conducted during the study, it was considered appropriate to determine the dissolved test material concentration present in the test samples by Total Organic Carbon (TOC) analysis.

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/l for 48 hours under semi-static test conditions at a temperature of 21°C to 22ºC. The number of immobilised Daphnia were recorded after 24 and 48 hours.

The 48-Hour Effective Loading Rate 50 value for the test material to Daphnia magna based on nominal loading rates was greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 56 mg/l loading rate WAF.

Total Organic Carbon (TOC) analysis of the test preparations was performed on the fresh test media at 0 and 24 hours and on the old/expired test media at 24 and 48 hours. The results obtained indicated that measured concentrations of around the limit of quantitation (LOQ) were obtained from both the fresh and old test media samples at 0, 24 and 48 hours.

Given that toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, and that the dissolved test material was around the quantifiable limit of the analytical method, the results were based on nominal loading rates only.