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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
Specific details on test material used for the study:
- Appearance: Off-white powder
- Storage conditions: At room temperature in the dark
- Stability: Stable for at least 12 months under storage conditions
Analytical monitoring:
Details on sampling:
The concentration of the test substance in the samples was determined by means of gas chromatography with flame ionization detection. Samples (ca. 20 mL) were withdrawn from the control, lowest, middle and highest concentration. Samples from the other concentrations were also withdrawn and stored for additional analyses, if needed. The concentration of the test substance in the solution withdrawn directly from the stock solution was also determined. The samples were stored in the refrigerator in the dark until analysis. All samples were taken in single-fold and analyzed in duplicate. Samples were taken at 0, 24 and 48 hours of testing. The initial samples for analysis (0 hours) were withdrawn from the vessels after addition of the test organisms and stabilization of the test system. The samples were stored for a maximum period of 4 weeks.
Details on test solutions:
A test substance stock solution was prepared by weighing out the test substance (0.50091 g) using an analytical balance (Sartorius, 160P, Breukelen, The Netherlands). This was transferred to a separatory funnel filled with 5 liter DSW. This mixture was ultrasonically treated for approximately 20 minutes and subsequently mixed for approximately 20 hours at room temperature using a magnetic stirrer. Thereafter, the liquid was filtered using a glass fibre filter (Schleicher& Schull, GF 92 Glasfaser filter ( 421057 X 27358), 1 µm Ø) to remove all particles. The clear aqueous filtrate was used as stock solution for preparation of the desired concentration range.
Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna (Straus, clone IV, RIZA,. The Netherlands) was obtained from the laboratory parthenogenetic culture. This culture was kept as a synchronous brood stock at the GLP cluster of Aquasense Lab. The Daphnids were maintained in the synthetic culture medium (Elendt M-4), which is a reconstituted water. During culturing the Daphnids were fed with algae (Scenedesmus). The neonates used for test performance were between 0 and 24 hours old. The quality of the Daphnids is checked every 6 months and compared to inter-laboratory test data.
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Test temperature:
22.3 - 24.7°C
8.1 - 8.4
Dissolved oxygen:
92 - 107
Nominal and measured concentrations:
Nominal: 0.223, 0.335, 0.502, 0.752, and 1.127 mg/L
Measured (geometric mean): 0.168, 0.270, 0.472, 0.610, and 0.858 mg/L
Details on test conditions:
- Glassware: The test were performed in small vessels of approximately 70 ml total volume. The test vessels for each test were placed in separate racks, and were kept open to the air.
- Dilution medium: The dilution water used for testing and dilutions was Dutch Standard Water (DSW), having a pH of 7.8 ± 0.5. The DSW was prepared using deionized water, which has a conductivity of less than 0.5 µS/mm. The dilution water was aerated before being used in the test. The air was purified by active coal, cotton filter and water. The deionized water was prepared using a water purification system (Milli-Q, Millipore, Breda, The Netherlands). The water contained the following minerals per lite: 100 mg NaHCO3, 20 mg KHCO3, 200 mg CaCl2 H2O and 180 mg MgS04 7H2O.
- Apparatus and test conditions: The test were performed in glass vessels containing 50 mL of medium. The fluids were not aerated during the test period, and the Daphnids were not fed. The temperature in the incubator was kept constant between 18 and 22°C ± 1°C with a light regime of 16 hours of artificial light per day (8 ± 2 µmol/m2/s). The test was static with the fluids not being renewed during the test. The Daphnids were randomly placed In the test vessels.
- Determination of test parameters: The pH and oxygen of all test substance samples and controls were measured at the beginning (in pooled samples of each concentration) and end of the test (in all test concentrations). The pH values were determined with the aid of a pH meter (pH 196, VVTVV, Weilheim, Germany), and the oxygen concentration were determined using anoxygen monitor (Oxi 196, WTW, Weilheim, Germany). The temperature were measured continuously using a temperature sensor coupled to a data logger (Elbanton, Kerkdriel, The Netherlands). The light intensity was measured at the beginning and end of the test.
- Test method: The test concentration range used in this study was derived from the results of preliminary investigations. In this investigation 100% immobility was found at a concentration of 100% of the stock solution and no immobility at concentrations of 20% or lower. The definitive test was performed as a static test using 20 Daphnids per test concentration and control. The concentration range was made using the stock solution. The test were initiated by exposing 20 Daphnids divided into 4 groups of five organisms. The control consisted of DSW alone. The Daphnids were observed every 24 hours for immobilisation and other adverse behavioral effects. Daphnids which were not able to swim for 15 seconds after gentle agitation of the test vessel were considered Immobile.
- Statistical evaluation: The nominal concentrations of the test substance were used to plot the dose-response curve. The EC50 values were calculated using the Maximum Likelihood Probit method using the computer program ToxCalc of Tidepool Scientific Software. Confidence limits (95%) were calculated for each EC50 value. The program was validated with a known dataset before use. The NOEC was calculated using the threshold values of Williams test and the 100% mortality was derived from the results as presented. Calculation procedures are based on both nominal values (calculated from the measured concentration of the stock solution), and geometric means of measured values of the concentration range (whenever the recovery was < 80% after 48 hours of testing as described in the OECD 202 Guideline and EEC 67/548 C2 Directive). The geometric means of concentrations which were not measured by chemical analysis, were estimated on the basis of the average values of the preceeding and following measured concentration at each sampling time.
- Validity criteria: For the test to be valid, the number of floating, and /or immobile Daphnids in the control group must not exceed 10% at the end of the test, and the Daphnids should not be trapped at the surface (OECD/EEC). The dissolved oxygen concentration should be > 60% of the air-saturation throughout the test (OECD/EEC). The pH of the test medium should not vary more than one unit during the test (EEC).
Reference substance (positive control):
Key result
48 h
Dose descriptor:
Effect conc.:
0.331 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
48 h
Dose descriptor:
Effect conc.:
0.396 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
Analysis results
- General: GC analysis of pure extraction solvent (dibutylether) and a test substance standard solution showed that the test substance appears as a single peak which is free from interferences from the solvent and solvent impurities. Analysed samples were test media samples containing high, medium, low and zero test substance dose concentrations. The samples were taken at the beginning of the test (t=0 hr), at the end of the test and between times after every 24 hours. After shaking and sonication, none of the samples contained visible particles or aggregates so none of the samples was centrifuged.
- Samples from toxicity test with Daphnia magna: The concentration of the test substance in the test media from the Daphnia magna tests at t=0 hours were between 0 and 1.14 mg/L. The current chemical analyses do not give a clue for the cause of the test substance losses. Some of these values are above 100%, which was probably due to variations during preparation of the test substance test series. During the test the concentrations dropped gradually to approximately 50% of the initial values. This decrease could not be fully caused by photodegradation or adsorption onto the test vessel wall, based on an investigation of the photodegradation of the test substance under light conditons. The results of this investigation indicated that the decrease in the concentration was almost identical in samples incubated in the light and dark. Additionally, it was found that only 4.5% of the total amount of the test substance was adsorbed onto the test vessel wall during an incubation period of 72 hours.

Test environment
- The pH values during the 48 hours of testing showed a maximum deviation of 0.2 units. The oxygen values exceeded the 60% of the saturation values. The temperature during the test varied between 19.8 and 20.6°C with a mean value of 20.3°C. The light intensity measured at the beginning and end of the test in the test chamber was between 8.30 and 8.90 µmol/m2/s, which is in agreement with the light intensity of 8 ± 2 µmol/m2/s as recommended in the OECD/EEC guidelines.

Test results
- All Daphnids survived after 24 hours of testing at the concentrations 0.223 and 0.335 mg/L. The highest two concentrations showed 100% mortality after 48 hours of testing, and partial immobilities were found at all other concentrations after 48 hours of testing. Because the test concentrations dropped below 80% of the initial concentration, the calculation of the effects were also performed using the geometric means of the measured concentrations The EC50 value calculated after 48 hours of testing based on the nominal values is 0.396 mg/L (0.345 - 0.453 95% confidence limits) and the NOEC derived from the results is 0.223 mg/L. Based on the measured concentrations the EC50 is 0.331 mg/L (0.280 - 0.383 95% confidence limits), and the NOEC is 0.168 mg/L. No other deviations, such as changes in the behaviour or other effects have been observed.

Validity of test
- The test is valid as shown by the oxygen and pH values and by the absence of immobility in the controls after 48 hours of testing, which is less than the maximum acceptable value of 10% as described in the OECD/EEC guidelines.
Results with reference substance (positive control):
The results of the quality control test with potassiumdichromate (24-Feb-1998: 24Hr-EC50, 2.0 mg/L) are in agreement with the criteria mentioned in the Study Protocol, indicating that the sensitivity of the Daphnids has not changed significantly.

 Analysis of the test substance in test media


0 hr

24 hr

48 hr

1.127 mg/L




0.502 mg/L




0.223 mg/L




0 mg/L




nd: Not determinable (below detection limit of 0.039 mg/L)

Validity criteria fulfilled:

Description of key information

The 48h-EC50 is 0.331 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.331 mg/L

Additional information

The short-term toxicity to daphnia of the test substance was determined in an acute toxicity test which is described in the OECD Guideline 202 and was performed in accordance with the principles of Good Laboratory Practice (GLP). Twenty Daphnids per test concentration (0.223, 0.335, 0.502, 0.752, and 1.127 mg/L were exposed to the test substance for 48 hours in a static test. During the test the pH varied no more than 0.2 units, and the oxygen concentrations were all above 60% of the saturation values. The temperature varied between 19.8 and 20.6°C throughout the test. Therefore the test meets the requirements and is valid as shown by the absence of immobility in the controls. The EC50 value calculated after 48 hours of testing based on the nominal values is 0.396 mg/L (0.345 - 0.453 95% confidence limits) and the NOEC derived from the results is 0.223 mg/L. Based on the measured concentrations the EC50 is 0.331 mg/L (0.280 - 0.383 95% confidence limits), and the NOEC is 0.168 mg/L. Chemical analyses showed that the initial concentrations were substantially achieved, as shown by the percentage recovery at t=0, but the concentration gradually decreased during the 48 hours of testing. This decrease is not an inherent property of the test substance and may be attributed, to some extent, to adsorption onto the test vessel wall or onto the Daphnids.