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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:
February 06 to February 08, 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
Adopted: April 13, 2004
GLP compliance:
yes (incl. QA statement)
Certificate from May 31, 2016
Analytical monitoring:
Details on sampling:
The concentrations of the test item in the water phase was measured by chemical analysis. Samples were taken from fresh and aged test solutions and the control prior to distribution to the test vessels. At the end of the test, samples were taken directly from two representative replicates of the test concentration and control. Analysis of all samples was performed at test end.
Oxygen concentration, pH value, and temperature of the test solutions were checked at test start and end. The pH value should not vary by more than 1.5 units.
Details on test solutions:
To prepare the test item concentrations, 101.7 mg of the test item, corresponding to a nominal test concentration of 100 mg active substance (a.s.)/L were stirred in 1 L dilution water for 48 hours. After stirring, the stock solution was filtered through a 0.22 µm PES filter. For the non-GLP range-finder test, three nominal concentrations were prepared thereof through serial dilution (i.e. 1.0, 10.0 and 100 mg a.s./L). For the definitive test (i.e. limit test), no further preparation was required.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: water flee
- Source: German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene, bred in the laboratory of the Fraunhofer IME.
- Age: 4 - 24 hours old at test start
- Feeding during test: no
- pre-treatment: Adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving. Batches of 30 to 50 animals were held at room temperature in ca. 1.8 L dilution water for one week. During this week the daphnids were fed daily with an algal suspension (Desmodesmus subspicatus) and ArtemioFluid (JBL). Algae growing in the log-phase were centrifuged and the pellet was re-suspended in a few mL of medium. 30 mL of this suspension was given to 1 L Daphnia medium. The water was changed once per week. Newborn D. magna were separated by sieving, the first generation was discarded. Individuals applied in the test were transferred with a bore Pasteur pipette a few hours after sieving to ensure applying only healthy specimens.
Test type:
Water media type:
Limit test:
Alcalinity: 1.6 mmol/l
Total hardness: 1.1 mmol/l
Ca-hardness: 0.8 mmol/l
Mg-hardness: 0.3 mmol/l
Test temperature:
18 – 22 °C (with a variation of less than 1 °C) throughout the test
mean temperature of 18.8 °C throughout the test
pH 8.03 – 8.57 throughout the test
Dissolved oxygen:
8.8 - 9.4 mg diss. oxygen/L throughout the test
not reported
270 µS/cm
Nominal and measured concentrations:
Nominal: 0 and 100 mg test item/L
initial, measured: 0 and 45.38 µg test item/L
final, measured: 0 and 46.86 µg test item/L
geometric mean: 0 and 46.11 µg test item/L
Details on test conditions:
- Test vessel: glass beakers
- Type: covered with glass panes to prevent from evaporation
- Fill volume: 50 mL
- Aeration: no
- Type of flow-through: static exposure
- organisms per vessel: 5
- Rreplicates: 6 per control and treatment (i.e. 100 mg/L)

- Dilution water: Purified (filtration with activated charcoal, passage through a limestone column, and aeration) tap water, sourced from the Schmallenberg district water production plants, mostly fed by small springs and percolation. To avoid copper contamination, plastic water pipes are used for the testing facilities. Water chemistry is regularly recorded. The pH values of the tap water (i.e. 7.1 – 8.4) are within the recommended range and although the hardness (i.e. 140 - 250 mg/L as CaCO3) is lower than OECD recommendations, historical sensitivity data indicates suitable test conditions.

- Culture and test medium: dilution water

- Adjustment of pH: no
- Photoperiod: light/dark cycle of 16/8
- Light intensity: 910 – 980 lx (corresponding to 12.1 – 13.1 µE/(m² * s))
- Test concentrations: Range-finder: 1.0, 10.0 and 100.0 mg a.s./L, Definitive test: limit test (100.0 mg a.s./L)
- vehicle used: no
Reference substance (positive control):
potassium dichromate
48 h
Dose descriptor:
Effect conc.:
> 46.11 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Lumilux® Yellow CD397
Basis for effect:
Details on results:
The test item up to and including its solubility limit in the test medium is not acutely toxic to Daphnia magna under the chosen test conditions. No effect of the test item was observed on immobility of daphnids during the test. With ≤ 10 % control mortality and a dissolved oxygen concentration > 3 mg/L in control and test vessels at the end of the 48h-exoposure period, the test is considered valid. Details are given in the tables below "any other information on results including tables".
Results with reference substance (positive control):
According to the guideline no reference item is required. However, the sensitivity of the test clone was checked twice a year in non-GLP tests by using potassium dichromate (K2Cr2O7) as reference substance. The latest 24h-EC50 (November 2017) was 1.435 mg/L (95 % confidence limits: 1.233 – 1.745 mg/L). K2Cr2O7 is a recommended reference substance for acute effects on daphnids according to the OECD guideline 202. The EC50 was within the expected range as noted in the OECD guideline of 0.6-2.1 mg/L.
Reported statistics and error estimates:
In this report, numerical values are frequently rounded to a smaller degree of precision (number of digits) than used in the actual calculation. Minor differences in results obtained from calculations with such rounded values in comparison to those obtained with higher precision values are possible. They are, however, well within the limits of the experimental accuracy and thus of no practical concern. Statistical calculations were not performed since no effects on the immobilization on the introduced daphnids occurred.

Table: Mobile and immobile daphnids after 48 h and percent immobilization compared to the controls. Concentrations given as geometric mean measured concentrations.

Geometric mean concentration [µg/L]

Total Introduced



% Immobility











Table: Dilution water, chemical properties



Conductivity (µS/cm)


Alcalinity (mmol/l)


Tot. Hardness (mmol/l)


Ca-hardness (mmol/l)


Mg-hardness (°d)


NPOC (mg/L)


NO3 (mg/L)


NO2 (mg/L)

< 0.005

NH4 (mg/L)

< 0.01

PO4 (mg/L)


Cl (mg/L)

< 0.02

Cd (µg/L)

<0.04(LOD) - <0.11 (LOQ)

Cr (µg/L)

< 0.43 (LOD)

Cu (µg/L)

< 0.77 (LOQ) – 0.86

Fe (µg/L)

0.67 – 1.81

Mn (µg/L)

< 0.15 (LOQ) – 0.17

Ni (µg/L)

< 0.60 (LOQ)

Pb (µg/L)

< 2.01 (LOD)

Zn (µg/L)

1.45 – 1.99

Validity criteria fulfilled:
≤ 10 % control mortality; diss. oxygen concentration > 3 mg/L in control and test vessels at the end of the 48h-exoposure period
The potential immobilising effect of the test item Lumilux® Yellow CD397 (N-[2-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]naphthalene-2-sulphonamide) on freshwater cladocere Daphnia magna was investigated in a static exposure system for 48 h according to OECD 202 (2004). Nominal concentrations of 0 and 100 mg test item/L have been prepared in dilution water and were chemically analysed using LC-MS (LOQ = 0.05 µg a.s./L) at the start and the end of the test. Based on the water accommodated fraction (WAF) of the test item and its mean measured concentration of 46.1 µg test item/L, the 48h-ErC50 and EyC50 were determined to be > 46.1 µg test item/L (corresponding to a maximum solubility of the test item in test solution). The NOEC >= 46.1 µg/L. All validity criteria of the test were met.

Description of key information

In a GLP- and guideline-conform (OECD 202, 2004) acute immobilization test with freshwater cladocere Daphnia magna and test item Lumilux® Yellow CD397, the 48h-EC50 was determined to be > 46.1 µg test item/L (WAF, geometric mean, corresponding to the maximum solubility of he test item in test solution). The NOEC is >= 46.1 µg test item/L. Hence, the test item is not acutely toxic to Daphnia magna up to and including its solubility limit in the test medium under the chosen test conditions.

Key value for chemical safety assessment

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