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Long-term toxicity to aquatic invertebrates

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long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Feb 2013 to 10 Apr 2013
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
For determination of the test item concentrations, duplicate samples were taken from all test concentrations and from the control at one treatment period of the first, second and last week of the test (Day 0, 7 and 16, respectively).

To determine the maintenance of the test item concentration in the test media, stability samples were taken at the end of two test medium renewal periods of 48 hours (Days 2 and 9) and at the end of one renewal period of 72 hours (Day 19). The following stability samples were taken in duplicate:
- Samples taken from the actual test by combining the contents of all replicate test beakers after the end of the test medium renewal period.
- Samples incubated during the renewal periods under the test conditions, however without food and daphnids.

Immediately after the samples were taken, an appropriate amount of methanol was added to each sample to stabilize the test item during the storage period (1 mL methanol per mL sample volume). Then the samples were stored deep-frozen (at about –20 °C) till date of analysis. According to pre-experiments to the storage stability the test item is sufficiently stable in test media samples under these storage conditions.
Details on test solutions:
- Method: Prior to the start of the test and prior to each test medium renewal, a stock solution of nominal 15 mg/L was freshly prepared by completely dissolving 45 mg of the test item (dosed in the range of 45.0 to 45.3 mg) in 3000 mL of test water using ultrasonic treatment for 15 minutes and stirring for 15 minutes at room temperature. This intensively stirred stock solution was used in a series of dilutions to prepare the test media of all test item concentrations.
Test organisms (species):
Daphnia magna
Details on test organisms:
The study was performed with females of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield/UK in 1992. Since this date, the clone is successfully bred at Harlan Laboratories in culture medium identical to the medium used for the test. The temperature and light conditions were identical to those of the test.
During breeding, daphnids are generally fed three times a week with an algal suspension of the green algae Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany) and cultivated at Harlan Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany). Each stock animal was maintained separately in a 100 mL glass beaker filled with about 80 mL culture medium and was transferred twice a week to fresh medium. The condition of the stock animals was frequently checked. No signs of stress were observed and the brood stock was healthy. The daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first brood progeny. At the start of the test, the test animals were less than 24 hours old.
Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
Test temperature:
19 - 21 °C
7.5 - 8.0
Dissolved oxygen:
> 7.8 mg/L
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 0.015, 0.047, 0.15, 0.47 and 1.5 mg/L
Details on test conditions:
- Test vessel: glass beakers
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- Renewal rate of test solution: In this semi-static test, the test media of all treatments were renewed on Days 2, 5, 7, 9, 12, 14, 16 and 19 of the test period (every Monday, Wednesday, and Friday). At these dates, the surviving test animals were carefully transferred by means of glass tubes from the old test vessels into the freshly prepared test medium.
- Feeding: The test animals were fed daily with a food mixture containing a suspension of green algae of the species Scenedesmus subspicatus (freshly grown at Harlan Laboratories) and a fish food suspension. The fish food suspension was prepared by dispersing 10 g of powdered commercial fish diet (TETRA MIN Hauptfutter, obtained from TETRA-Werke, 49304 Melle / Germany) in 500 mL of test water. The suspension was allowed to stand for 4 hours. Then, 400 mL of the supernatant were taken, diluted 1:1 with test water and boiled. The suspension was stored deep frozen in small quantities until use. The carbon contents of the algal and fish food suspensions were determined using a Shimadzu TOC 5000A Analyzer. The food amount (based on the measured concentrations of the total organic carbon (TOC) in the food suspensions) was 0.20 mg TOC per Daphnia and day.

- Photoperiod: A 16-hour light to 8-hour dark cycle with a 30 minute transition period was used. Light intensity during the light period was between approximately 390 and 560 Lux.
Key result
21 d
Dose descriptor:
Effect conc.:
>= 1.5 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
21 d
Dose descriptor:
Effect conc.:
> 1.5 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
- Mortality: In the control and in all concentrations up to and including 1.46 mg/L, the survival of the test animals at the end of the test was at least 90% or higher. Mortality up to 20% is regarded as natural and tolerated by the test guideline. Thus, the survival of Daphnia magna over 21 days was not affected by the test item up to and including the highest mean measured concentration of 1.5 mg/L.
- First brood: The first young offspring released from their parent animals were recorded in the control and at all test concentrations at observation on Day 9. No delay of the first brood was observed. Thus, the time of the first brood was not affected by the test item up to and including the highest mean measured concentration of 1.46 mg/L.
- Reproduction: The mean reproduction rate of the daphnids in the control was 106 ± 7.8 living offspring per surviving adult (mean ± standard deviation). No significant inhibitory effect of the test item on the mean reproduction rate was determined up to and including the highest mean measured concentration of 1.46 mg/L .
- Abnormalities: No visible abnormalities were observed in the test animals during the test.
Reported statistics and error estimates:
The Dunnett t test (one-sided smaller, a = 0.05) was used to used to calculate the mean reproduction rate.
Validity criteria fulfilled:

Description of key information

The 21-d NOEC value based on reproduction is >1.5 mg/L in aquatic invertebrates (Daphnia magna).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
1.5 mg/L

Additional information

The long-term toxicity to aquatic invertebrates was determined in a study according to OECD TG 211 and in compliance with GLP criteria (Harlan Laboratories, 2013). In this study, daphnids (Daphnia magna, 10 per concentration) were exposed to nominal concentrations of 0 (control), 0.015, 0.047, 0.15, 0.47 and 1.5 mg/L for 21 days under semi-static conditions. In this semi-static test, the test media of all treatments were renewed on days 2, 5, 7, 9, 12, 14, 16 and 19 of the test period. Analytical confirmation of nominal test concentrations (LC-MS analysis of the main component) showed that all test concentrations remained within ± 20% of nominal concentrations throughout the test. Therefore, effect concentrations are expressed as nominal. Mortality, first brood, reproduction rate and abnormalities of the test organisms were recorded throughout the test. Survival of the test organisms was at least 90 % or higher at the end of the test. Furthermore, the first brood and reproduction rate were not affected by the test item and no abnormalities were observed throughout the test. Based on these findings, the 21-d NOEC was determined to be ≥ 1.5 mg/L, with a LOEC of > 1.5 mg/L. The 21-d EC50 for the inhibition of the reproduction rate was > 1.5 mg/L.