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:
01 May - 25 June 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study conducted in compliance with OECD Guideline 202 with minor deviations: static test system; temperature in control vessels was sometimes outside the recommended range; oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100 %
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
static test system; temperature in control vessels was sometimes outside the recommended range; oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100 %
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Physical state: Clear, colourless viscous liquid (after melting)
- Lot/batch No.: 11GP0077
- Expiration date of the lot/batch: 1 January 2014
- Storage condition of test material: Room temperature in the dark
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No data

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L LR WAF
- Sampling method: Samples were taken for analysis at 0 (fresh media) and 48 h (old media) from the control and each loading rate WAF test group in the definitive test and from the control and 100 mg/L loading rate WAF in the additional experiment.
- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was heated at approximately 60 °C to liquefy prior to use. Amounts of test item (20, 36 and 64 mg) were each separately added to glass slides and suspended in the water columns of 20 L of reconstituted water to give the 1.0, 1.8 and 3.2 mg/L loading rates respectively. Further amounts of test item (112, 200, 360, 320, 560 and 250 mg) were each separately added to the surface of 20, 20, 20, 10, 10 and 2.5 L of reconstituted water to give the 5.6, 10, 18, 32, 56 and 100 mg/L loading rates respectively. After the addition of the test item, 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 h and the mixtures allowed to stand for 1 h. 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. Microscopic inspection of the WAFs showed no microscopic particles of test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L loading rate WAFs.
- Controls: The control group was maintained under identical conditions but not exposed to the test item.

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: 1st instar (< 24 h old)
- Method of breeding: Parthenogenesis
- Feeding during test: No

ACCLIMATION
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: A mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
- Feeding frequency: Daily

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
No data

Test conditions

Hardness:
250 mg/L as CaCO3
Test temperature:
20-23 °C
pH:
8.0-8.1
Dissolved oxygen:
8.5-8.8 mg O2/L
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L LR WAF
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass jars containing approximately 200 mL of test preparation
- Aeration: No
- No. of organisms per vessel: Five
- No. of vessels per concentration (replicates): 4 replicates
- No. of vessels per blank control (replicates): 4 replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water- Elendt M7 Medium
- Conductivity: < 5 µS/cm
- Intervals of water quality measurement: Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test.

EQUIPMENTS:
- The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter.
- The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

OTHER TEST CONDITIONS
- Photoperiod: 16 h of continuous artificial light and 8 h continuous darkness with 20 min dawn and dusk transition periods

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Any immobilization or adverse reactions to exposure were recorded at 24 and 48 h 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 sec after gentle agitation.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1.0, 10 and 100 mg/L (nominal loading rates)
- Results used to determine the conditions for the definitive study: In the range-finding test, no significant immobilization was observed at the 1.0 mg/L loading rate WAF. However, immobilization was observed at 10 and 100 mg/L loading rate WAF. Based on this information loading rates of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L, using a stirring period of 23 h followed by a 1-h standing period, were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (non-concurrent): 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
mobility
Details on results:
- Immobilization: A single immobilized daphnid was observed at 56 mg/L loading rate WAF and three immobilized daphnids were observed at 100 mg/L loading rate WAF after 48 h exposure. Given the results from the additional experiment, which showed no immobilization at 100 mg/L loading rate WAF, it was considered that the observed immobilization was possibly due to natural causes rather than a toxic effect of the test item.
Results with reference substance (positive control):
- Results with reference substance valid: Yes
- EC50 (24 h): 1.3 mg/L (1.0-1.8 mg/L)
- EC50 (48 h): 1.1 mg/L (1.0-1.3 mg/L)
- NOEC after 24 and 48 h were 1.0 and 0.56 mg/L, respectively.
Reported statistics and error estimates:
No data

Any other information on results incl. tables

- Validation of Mixing Period: Pre-study investigational work indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 h. Therefore, for the purpose of testing the test item was prepared using a stirring period of 23 h followed by a 1 h settlement period.

- Chemical analysis results: Analysis of the test preparations at 0 h showed measured concentrations of carbon to range from below the limit of quantitation (LOQ) (assessed as 1.0 mg C/L) to 8.90 mg/L. Analysis of the test preparations at 48 h showed measured concentrations of carbon to range from less than the LOQ to 5.83 mg/L. The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
mortality in control < 10 % (no immobilisation observed); dissolved oxygen concentration > 3 mg O2/L
Conclusions:
Under the test conditions, the 48 h EL50 (Effective Loading rate) for 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 100 mg/L loading rate WAF.
Executive summary:

An acute aquatic toxicity study was performed in accordance with GLP and OECD Guideline 202. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item, over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L for 48 h at a temperature of 20-23 °C under static test conditions in the dark. An additional experiment was conducted following the completion of the definitive test to support the results from the definitive test. The number of immobilized Daphnia and any adverse reactions to exposure were recorded after 24 and 48 h.

A single immobilized daphnid was observed at 56 mg/L loading rate WAF and three immobilized daphnids were observed at 100 mg/L loading rate WAF after 48 h exposure. Given the results from the additional experiment, which showed no immobilization at 100 mg/L loading rate WAF, it was considered that the observed immobilization was possibly due to natural causes rather than a toxic effect of the test item.

Total Organic Carbon (TOC) analysis of the test preparations at 0 h showed measured concentrations of carbon to range from below the limit of quantitation (LOQ) (assessed as 1.0 mg C/L) to 8.90 mg/L. Analysis of the test preparations at 48 h showed measured concentrations of carbon to range from less than the LOQ to 5.83 mg/L. The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Under the test conditions, the 48 h EL50 (Effective Loading rate) for 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 100 mg/L loading rate WAF.