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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:
2016-06-10 to 2016-08-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
(2012)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Deviations:
no
Principles of method if other than guideline:
Due to the poorly water soluble test item, a modification of the standard method for the preparation of aqueous media was performed according to the guidance document on aquatic toxicity testing of difficult substances and mixtures (OECD 2000) and ECETOC Monograph No. 26 (1996).
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a solution of the test item at a nominal concentration of 0.080 mg/L for a period of 21 days. The test concentration of 0.080 mg/L was considered to be the limit of solubility of the test item.
- Sampling method: Water samples were taken from the control and each surviving test group for quantitative analysis. Samples of the fresh test preparations were taken from the bulk test preparations on Days 0, 6, 13 and 20 and of the expired test preparations (replicates pooled) on Days 1, 7, 14 and 21.
- Sample storage conditions before analysis: Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on the results of the preliminary range-finding test, a "limit test" was conducted at a concentration of 0.080 mg/L to confirm that at highest attainable test concentration no reduction of reproduction or adverse reactions to exposure were observed. A nominal amount of test item (20 mg) was dissolved in dimethylformamide and the volume adjusted to 5 mL to give a 4.0 mg/mL stock solution. A dilution was made from this stock solution to give a further stock solution of 0.80 mg/mL. An aliquot (200 µl) of the 0.80 mg/mL stock solution was dispersed in 2 liters of test water and stirred with a magnetic stirrer for 30 minutes to give the test concentration of 0.080 mg/L. The test preparations were renewed daily throughout the test.
- Controls: dilution water and solvent control
- Chemical name of vehicle: dimethylformamide
- Concentration of vehicle in test medium: 100 µL/L in the test substance group and in the solvent control
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea Daphnia magna
- Strain/clone: Daphnia magna Straus Clone 5
- Source: in-house laboratory culture
- Feeding during test
- Food type: mixture of algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension
- Amount: approx. 0.1 to 0.2 mg/carbon/daphnid/day
- Frequency: daily

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium 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. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
252 to 274 mg/L as CaCO3
Test temperature:
20 to 22 °C
pH:
7.5 to 8.2
Dissolved oxygen:
>= 7.5 mg O2/L
Nominal and measured concentrations:
nominal concentration: 0.080 mg/L; measured initial test substance concentration: 0.0491 to 0.0588 mg/L, measured test substance concentration (old media):
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass vessels
- Type (delete if not applicable): covered with a plastic lid
- Material, size, headspace, fill volume: 150 mL glass vessels with 100 mL test preparation
- Renewal rate of test solution (frequency/flow rate): daily renewal
- No. of organisms per vessel: single daphnid pre vessel
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10
- Biomass loading rate: 1 daphnia / 100 ml

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (Elendt M7 medium)
- Ca/mg ratio: 252 to 274 mg/L as CaCO3
- Culture medium different from test medium: same medium
- Intervals of water quality measurement: Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal in the control and the test concentration group

OTHER TEST CONDITIONS
- Photoperiod: 16 h light, 8 h darkness
- Light intensity: 416 to 649 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F1) daphnia and the number of discarded unhatched eggs were counted daily. An assessment was also made of the general condition and size of the parental daphnia as compared with the controls. At the end of the test, the length of each surviving parent animal was determined.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by 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 (ECETOC, 1996 and OECD, 2000). Based on the results of a preliminary range-finding test, a "limit test" was conducted at a concentration of 0.080 mg/L to confirm that at highest attainable test concentration no reduction of reproduction or adverse reactions to exposure were observed. The test concentration of 0.080 mg/L was considered to be the limit of solubility of the test item.
- Range finding study
- Test concentrations: nominal concentrations of 0.0080 and 0.080 mg/L.
- Results used to determine the conditions for the definitive study: Given that there was no immobilization or effect on the number of live young in the first brood at the highest attainable test concentration, a "limit test" at a concentration of 0.080 mg/L was selected for the definitive test.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
> 0.019 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other:
Remarks:
no significant effects on parental daphnia generation mortalities
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 0.019 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.019 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
> 0.019 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other:
Remarks:
no significant impairment of reproduction
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 0.019 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.019 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
This study showed that there were no significant effects on parental daphnia generation mortalities, no impairment of reproduction and no reduction of parental daphnia generation body length at the highest attainable test concentration under experimental conditions. The NOEC was 0.019 mg/L as there were no significant mortalities (immobilization) observed in the parental generation (P1) and there were no significant differences (P>=0.05) in terms of the number of live young produced per adult when compared to the control after 21 days. As there were no effects at the highest attainable test concentration, a LOEC was not determined. At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Analysis of the fresh test preparations on Days 0, 6, 13 and 20 showed measured test concentrations to range from 0.0491 to 0.0588 mg/L. A decline in measured test concentration of the old test preparations was observed on Days 1, 7, 14 and 21. The decline resulted in measured concentrations of less than the limit of quantification (LOQ determined to be 0.0052 mg/L) to 0.0135 mg/L. Given the decline in the measured concentration of the old test preparations it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data. The geometric mean measured test concentration calculated using all the measured concentrations was determined to be 0.019 mg/L.

Reported statistics and error estimates:
Two-sample t-test incorporating Shapiro-Wilk's test on normal distribution and Levene’s test on variance homogeneity.
The "Lowest Observed Effect Concentration" (LOEC) was not determined as the test was conducted as a limit test and no effects were observed at the highest attainable test concentration.

Summary of Findings Following the Exposure of Daphnia magna for 21 Days

Nominal Concentration
(mg/L)

Geometric Mean Measured Concentration (mg/L)

Percentage Survival of Parental (P1) Generation

Total Number of Live Young

Number of Live Offspring Per Parent Surviving to the End of the Test

Number of Live Offspring at the End of the Test*

Control

80

1321

150

150

Solvent Control

100

1602

160

160

0.080

0.019

90

1528

167

167

*Per Parent at the Start of the Test Excluding Parental Accidental or Inadvertent Mortalities

Validity criteria fulfilled:
yes
Remarks:
All control validity criteria were successfully fulfilled, therefore the results of the study are considered to be valid.
Conclusions:
Exposure of Daphnia magna to the test item resulted in no significant mortalities. The 21-Day EC50 (immobilization) value, based on geometric mean measured concentrations, for the parental daphnia generation (P1) was estimated to be greater than 0.019 mg/L. No significant impairment of reproduction was observed at the concentration employed during the test. The 21-Day EC50 (reproduction) based on geometric mean measured concentrations was greater than 0.019 mg/L. The NOEC based on geometric mean measured concentration was 0.019 mg/L. This study showed that there were no effects on parental immobilization or reproduction at the highest attainable test concentration under experimental conditions.

Description of key information

A chronic toxicity study with Daphnia magna showed no significant effects on parental daphnia generation mortalities, no impairment of reproduction and no reduction of parental daphnia generation body length at the highest attainable test concentration under experimental conditions.

Key value for chemical safety assessment

Additional information

A chronic toxicity test with Daphnia magna has been performed in GLP compliance according to the OECD guideline TG 211. Based on the results of a preliminary test, a limit test with a test concentration of 0.08 mg/L has been performed, which is represents the limit of water solubility. This study showed that there were no significant effects on parental daphnia generation mortalities, no impairment of reproduction and no reduction of parental daphnia generation body length at the highest attainable test concentration under experimental conditions. The geometric mean measured test concentration calculated using all the measured concentrations has been determined to be 0.019 mg/L. The EC50 (21 days) for immobilization of the parental daphnia and the 21-day EC50 for reproduction and the corresponding NOEC have been estimated to be greater than 0.019 mg/L based on the geometric mean.