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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 12, 2012 - Dec 14, 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Sampling method: Pooled samples of the test media at the beginning and end of each renewal period (0 and 24 h; 24 and 48 h) were analysed
- Sample storage conditions before analysis: The samples were analysed immediately after sampling by liquid chromatography (HPLC)
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia magna
- Strain: Straus
- Source: The stock culture was obtained from the German Federal Environment Agency, Institute for Water, Soil, and Air Hygiene (Berlin).
- Age at study initiation (mean and range, SD): aged less than 24 hours at the start of the test
- Method of breeding: Specimens used in the test were bred in the laboratory at Fraunhofer IME.
- Feeding during test: no
- Food type: fed daily with an algal suspension (Desmodesmus subspicatus containing Artemio Fluid JBL).
- Frequency: daily

- Acclimation period: To maintain the stock culture, batches of 30 - 50 adult daphnids were held at room temperature in approximately 1.8 L of dilution water for one week
- Acclimation conditions (same as test or not):
- Type and amount of food:
- Feeding frequency:
- Health during acclimation (any mortality observed):
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
1.2 mmol/L
Test temperature:
19.7± 1°C
at start: 8.39-8.53
at end: 8.20-8.35
Dissolved oxygen:
at start: 8.2 - 8.6 mg/L
at end: 7.7-8.6 mg/L
not stated
Nominal and measured concentrations:
Range finder test: nominal concentrations: control, 1, 10, and 100 mg/L (corresponding to 0.36, 3.6, and 35.8 mg a.i./L).
Definitive test: the nominal concentrations of the test item used were 6.25, 12.5, 25.0, 50.0, and 100 mg/L (corresponding to 2.2, 4.5, 9.0, 17.9, and 35.8 mg a.i./L). The concentrations were spaced by a factor of 2 and agreed upon with the monitor.
Details on test conditions:
- Test vessel: 60 mL glass beakers
- No. of organisms per vessel: 4
- No. of vessels per concentration (replicates): 4

- Source/preparation of dilution water: Schmallenberg district water production plants, mostly fed by small springs and percolation. The purification process occurs on site at Fraunhofer IME and includes filtration with activated charcoal, passage through a lime-stone column, and aeration to the point of oxygen saturation

- Photoperiod: The daphnids were maintained under a light/dark cycle of 16/8 hours.
- Light intensity:light intensity did not exceed 1125 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality and clinical signs (sub-lethal and abnormal behavior) were recorded at 3 h, 24 h, 48 h, 72 h and 96 h after fish addition.

- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: The test included one control tank and five tanks with nominal test concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg test item/L. Analysis of test media revealed concentrations of test item between 96 and 106 % of nominal for fresh, and between 96 and 108 % of nominal for the aged test media. Since one measurement was found to be > 120 % of nominal, the geometric mean was used for the final determination of effect data. The geometric mean measured concentrations were calculated to be 0.663, 1.32, 2.60, 5.08 and 9.59 mg test item/L.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
48 h
Dose descriptor:
Effect conc.:
19.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
Results with reference substance (positive control):
EC50: The latest 24h determined EC50 was 1.03 mg/L (April 2012).
Reported statistics and error estimates:
The percent immobilisation (test endpoint) of each test concentration will be compared with a control consisting of dilution water only. If the analyses demonstrate that the deviation from the nominal concentration is above 20 %, all effect data will be based on the measured concentrations. If possible, a NOEC/LOEC is calculated by using ANOVA followed by Dunnett’s or Williams’ tests or an appropriate non-parametric test. When the test results show a concentration-response relationship, the data is analysed by regression to determine the EC50/20/10 including the 95 % confidence interval using Probit-analysis assuming log-normal distribution of the values. The computer software ToxRat® Professional v.210 will be used for statistical evaluations.
Validity criteria fulfilled:
At 24 h, 5% immobilisation effect was observed in the 100 mg/L test concentration. After 48 h, the concentration dependent effect was more apparent. Immobilisation was 20 and 100% in the 50.0 and 100 mg/L test concentrations resulting in a NOEC of 50.0 mg/L corresponding to 17.9 mg a.s./L. The EC50 was 55.5 mg/L, corresponding to 19.9 mg a.i./L.

No immobility was observed in the control. There were no signs of disease or stress, e.g. discolouration or abnormal behaviour, observed in any replicate. All surviving specimens appeared normal.

Description of key information

Key study [Schlechtriem, (2013)]: OECD guideline 202 and EU guideline C.2. The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-hour EC50 value of 19.9 mg a.i./L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
19.9 mg/L

Additional information

Three studies were available for this endpoint on the test substance. A new study was selected as key study [(Schlechtriem, 2013)]. This study was run according to the OECD guideline 202 and EU guideline C.2. The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-hour EC50 value of 19.9 mg a.i./L.

Two other studies are available which gave a 48 -Hour EC50 of 16 and 0.96 mg a.i./L respectively [Kamp (1997) and Wetton (2000)]. The study of Wetton (2000) was rated Klimisch 2 and considered a supporting study. The study of Kamp was rated Klimisch 1. Although the obtained EC50 value was clearly lower than the EC50 value obtained in the study from Schlechtriem (2013), this value was considered an outlier and therefore not used for the PNEC derivation.