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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The RSS is based on the draft report
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
2008
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance: stable for up to 72 hours in test water
Analytical monitoring:
yes
Details on sampling:
Duplicate samples were taken from the freshly prepared test media (without food) of all test concentrations and the control at one treatment period of the first, second and last week of the test (Day 0, 7 and 16).
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). Samples were taken from the actual test by combining the contents of all replicate test beakers after the end of the test medium renewal periods (with food) as well as from samples incubated during the renewal periods under the test conditions, however without food and daphnids.
All samples were frozen (at about –20 °C) immediately after sampling and stored until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Although the water solubility of the test material is 1.85 g/L some precipitation was observed in test water. Thus, at the start of the test and before each test medium renewal a preparation with nominal 100 mg test material/L was prepared by dispersing 200 mg of the test material (dosing range: 200.0 to 200.1 mg) in 2000 mL of test water. After ultrasonic treatment (15 minutes) and intense stirring (three hours at room temperature in the dark), preparations were filtered through a membrane filter (Whatman, Type NC45, pore size 0.45 µm). As a precaution, filters were pre-conditioned with 200 mL filtrate to avoid possible losses of dissolved test material due to adsorption on the filter material.
The undiluted filtrate was diluted with adequate volumes of test water to prepare the test media with nominal 32, 10, 3.2, 1.0, and 0.32 mg/L (dilutions 1:3.2, 1:10, 1:32, 1:100, and 1:320)
- Controls: test water only
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flee
- Strain/clone: STRAUSS
- Source: in-house culture originally obtained from the Daphnia Collection of the University of Basel/Switzerland in 2015
- Feeding during test
- Food type: suspension of freshly grown green algae (Desmodesmus subspicatus) and a commercial fish diet (TETRA MIN Hauptfutter, TETRA-Werke, 49304 Melle / Germany)
- Amount: 0.20 mg total organic carbon/animal/day
- Frequency: daily

ACCLIMATION
- Acclimation period: culture medium, temperature and light conditions were identical to those in the test.
- Type and amount of food: green algae (Desmodesmus subspicatus) or a suspension of freshly grown green algae (Desmodesmus subspicatus) and a commercial fish diet (TETRA MIN Hauptfutter, TETRA-Werke, 49304 Melle / Germany), three times a week
- Health during acclimation (any mortality observed): no mortality or signs of stress were observed and the brood stock was healthy.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Stock animals were maintained separately in a 100 mL glass beaker filled with about 80 mL culture medium and were transferred twice a week to fresh medium. The condition of the stock animals was frequently checked.
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, by removing the offspring <24 hours before start of the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
2.5 mmol/L (250 mg CaCO3/L)
Test temperature:
19-21°C
pH:
7.6-8.4
Dissolved oxygen:
8.0-9.6 mg O2/L
Nominal and measured concentrations:
See "Attached background material" - Table 1 Test Material Concentrations.pdf
As the test item was stable during the test medium renewal periods of 48 and 72 hours, the mean measured test item concentrations over the test period were calculated for each test concentration as the arithmetic mean over all measurements (in the freshly prepared and aged test media) of the three analyzed renewal periods. For the aged test media, the analytical measurements from samples with food were taken into account.
Reference substance (positive control):
not required
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival, growth and reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
9.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival, growth and reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
6.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 9.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks on result:
other: the highest test concentration with surviving parental daphnids.
Details on results:
- Behavioural abnormalities: none
- Other biological observations:none
- Other adverse effects control: none
- Abnormal responses: none

The detailed biological results obtained for reproduction and parental mortality and body length are presented in "Attached background material" - "Tables & Figures_long-term toxicity to daphnia.pdf".
Reported statistics and error estimates:
Statistical analysis was performed using ToxRat Professional®, Version 3.2.1.
The NOEC for the mean reproduction rates of the daphnids at the test concentrations was determined by a Bonferroni-Holm Test, 'a' = 0.05, one-sided smaller.
The NOEC for the mean body length of the daphnids at the different test concentrations was determined by a Williams t test, 'a' = 0.05, one-sided smaller.
The NOECmortality was determined directly from the raw data.
Additionally, the EC10 value for inhibition of reproduction rate after 21 days was calculated by Weibull Analysis using linear maximum likelihood regression based on the mean values per concentration level.
The EC10 of the body length after 21 days could not be calculated due to the low toxic effect of the test item on the body length up to the highest test concentration with surviving daphnids.
Validity criteria fulfilled:
yes
Remarks:
mortality of parent animals in the blank control: ≤ 20%; mean surviving offspring in the blank control: ≥ 60;

Description of key information

overall 21-d NOEC for Daphnia magna 3.2 mg/L (OECD TG 211)

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
3.2 mg/L

Additional information