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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 October 2006 to 1 February 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
-Description: amber coloured paste
- Storage: room temperature, in the dark, under nitrogen
Analytical monitoring:
yes
Details on sampling:
Prior to analysis, samples were extracted with dichloromethane three times following the addition of sodium chloride. The extracts were filtered through anhydrous sodium sulphate, combined, evaporated to dryness and redissolved in 1% (v/v) phosphoric acid in propan-1-ol to achieve concentrations with the validated calibration range for quantitation.
Vehicle:
yes
Remarks:
(acetonitrile)
Details on test solutions:
Due to the very low water solubility of the test material, it was not possible to prepare aqueous stock solutions and test solutions were therefore prepared using an auxiliary solvent (acetonitrile) at nominal concentrations of 0.0020, 0.0063, 0.020, 0.063 and 0.20 mg/L. Prepared stock solutions and test solutions were inverted several times to ensure adequate mixing and homogeneity.

Pre-test dosing trials confirmed that the test material could not be quantified in test medium in untreated or centrifuged test solutions samples, so it was considered unnecessary to analyze test solution samples during the test. Samples of the solvent stock solution used to prepare the media were analyzed and measured concentrations were in excess of 80% of nominal confirming correct dosing of the test system. The effect concentrations were based on nominal test concentrations.

Solutions were observed to be clear, colorless at test initiation and green tinged by test termination due to the presence of algal feed.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Source: in-house laboratory cultures
- Age of parental stock: first instar; parent generation used on study was less than 24 hours old
- Feeding during test: yes; daily
- Food type: suspension of unicellular algal culture (Chlorella sp.)
- Feeding amount: 0.1 – 0.2 mg C/daphnid/day, dependent on age and size of daphnids; equal amounts given to each daphnid (5 – 10 µL).

ACCLIMATION
Acclimation conditions were the same as those used in the definitive test.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Culture conditions ensure that reproduction was by parthenogenesis. Gravid adults were isolated the day before test initiation, such that the young daphnids produced overnight were less than 24 hours old.
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
21 d
Hardness:
124 - 162 mg CaCO3/L in the solvent control and highest surviving treatment group
Test temperature:
20 – 22˚C
pH:
7.7 – 8.5
Dissolved oxygen:
6.9 - 8.9 mg/L (>78% air saturate value, ASV)
Nominal and measured concentrations:
Nominal concentrations: control (0), solvent control (100 µL/L ACN), 0.0020, 0.0063, 0.020, 0.063 and 0.20 mg/L
Details on test conditions:
- Test vessel: 150 mL glass flasks, covered with plastic lid
- Fill volume: 100 mL
- Aeration: None
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution: Flow through conditions were attempted in preliminary trials, but it was not possible to maintain concentrations in the test material in the solvent stock solution. Test solutions were therefore renewed daily.
- No. of vessels per solvent control or test concentration (replicates): 10 vessels each containing an individual parent daphnid
- Source/preparation of dilution water: dechlorinated, softened tap water
- Photoperiod: 16 h light / 8 h darkness, with 20 min transition period
- Light intensity: 403 to 510 lux
Reference substance (positive control):
not specified
Key result
Duration:
21 d
Dose descriptor:
EC50
Remarks:
(95% confidence limits, 0.038 – 0.077 mg/L)
Effect conc.:
0.054 mg/L
Nominal / measured:
nominal
Basis for effect:
mortality
Remarks:
immobilization
Remarks on result:
other: (95% confidence limits, 0.038 – 0.077 mg/L)
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.084 mg/L
Nominal / measured:
nominal
Basis for effect:
reproduction
Remarks on result:
other: (95% confidence limits, 0.056 – 0.13 mg/L)
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.063 mg/L
Nominal / measured:
nominal
Basis for effect:
mortality
Remarks:
immobilization
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.2 mg/L
Nominal / measured:
nominal
Basis for effect:
reproduction
Remarks:
mortality (immobiization)
Reported statistics and error estimates:
The EC50 (immobilization) values and associated confidence limits at 24 hours, and at 7, 14 and 21 days and EC50 (reproduction) value with associated confidence limit at 21 days were calculated by the trimmed Spearman-Karber method using the ToxCalc software package (ToxCalc 1999). Parent mortalities/immobilization were compared to the solvent control group using the corrected chi-squared statistic. The LOEC and NOEC for numbers of live young produced per adult over 21 days and parently growth (length at test termination) were compared with the control and solvent control groups using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance and Dunnett’s multiple comparison procedure using the SAS computer software package. The 0.2 mg/L test group data were not included in the statistical analysis due to 100% immobilization prior to test termination.
Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test material resulted in significant mortalities at the test concentrations of 0.063 and 0.20 mg/L resulting in 70% and 100% mortalities (immobilization) by Days 21 and 2, respectively. The 14- and 21-day EC50 values for immobilization were determined for the parental Daphnia (P1) to be 0.058 and 0.054 mg/L (95% confidence limits, 0.038 – 0.089 mg/l and 0.038 – 0.077 mg/L, respectively).

No statistically significant differences were observed in the growth of the surviving parent organisms.

The 21-day EC50 for reproduction was determined to be 0.084 mg/L (95% confidence limits, 0.056 – 0.13 mg/L).

The Lowest Observed Effect Concentration was considered to be 0.063 mg/L based on significant mortalities observed in the parental generation, although there was no significant effect on reproduction at this concentration. The No Observed Effect Concentration was considered to be 0.20 mg/L for immobilization and reproduction.
Executive summary:

The study was conducted to assess the effect of the test material on the reproduction of Daphnia magna over a 21-day period according to OECD Guideline No. 211 (1998). Due to the very low water solubility of the test material, it was not possible to prepare aqueous stock solutions and, following extensive solubility trials with several solvents, the test solutions were prepared using acetonitrile (ACN) at nominal concentrations of 0.0020, 0.0063, 0.020, 0.063 and 0.20 mg/L. A negative and solvent control (100 µL/L ACN) groups were tested concurrently. Flow-through conditions were attempted in preliminary trials, but it was not possible to maintain concentrations in the test material in the solvent stock solution. Test solutions were therefore renewed daily. The Daphnia were fed daily with an algal suspension. The numbers of adult and young Daphnia (live and dead) and Daphnia with eggs or young in the brood pouch were determined daily. Length of each surviving parent animal was determined at test termination.

Pre-test dosing trials confirmed that the test material could not be quantified in test medium in untreated or centrifuged test solutions samples, so it was considered unnecessary to analyze test solution samples during the test. Samples of the solvent stock solution used to prepare the media were analyzed and measured concentrations were in excess of 80% of nominal confirming correct dosing of the test system. The effect concentrations were based on nominal test concentrations.

Exposure of Daphnia magna to the test material resulted in significant mortalities at the test concentrations of 0.063 and 0.20 mg/L resulting in 70% and 100% mortalities (immobilization) by Days 21 and 2, respectively. The 14- and 21-day EC50 values for immobilization were determined for the parental Daphnia (P1) to be 0.058 and 0.054 mg/L (95% confidence limits, 0.038 – 0.089 mg/l and 0.038 – 0.077 mg/L, respectively). No statistically significant differences were observed in the growth of the surviving parent organisms. The 21-day EC50 for reproduction was determined to be 0.084 mg/L (95% confidence limits, 0.056 – 0.13 mg/L). The Lowest Observed Effect Concentration was considered to be 0.063 mg/L based on significant mortalities observed in the parental generation, although there was no significant effect on reproduction at this concentration. The No Observed Effect Concentration was considered to be 0.20 mg/L for immobilization and reproduction.

Description of key information

In an OECD Guideline 211 study, conducted according to GLP, theeffect of the test material on the survival, growth and reproduction of Daphnia magna was determined following exposure to the test material for 21 days ( Safepharm Laboratories, Ltd, 2008). The 21-day EC50 values for immobilization and reproduction were determined for the parental Daphnia (P1) to be 0 0.054 mg/L (95% confidence limits, 0.038 – 0.077 mg/L) and 0.084 mg/L (95% confidence limits, 0.056 – 0.13 mg/L), respectively. No statistically significant differences were observed in the growth of the surviving parent organisms. The Lowest Observed Effect Concentration (LOEC) was considered to be 0.063 mg/L based on significant mortalities observed in the parental generation, although there was no significant effect on reproduction at this concentration. The No Observed Effect Concentration (NOEC) was considered to be 0.20 mg/L for immobilization and reproduction.

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