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Ecotoxicological information

Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
GLP compliance:
yes
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:
yes
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in the samples was determined by means of gas chromatography with flame ionization detection, Samples (ca. 20 mL) were withdrawn from the control, lowest, middle and highest concentration. Samples from the other concentrations were also withdrawn and stored for additional analyses, when needed. The concentration of the test substance in the solution withdrawn directly from the stock solution was also determined.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 will be taken in single-fold and analyzed in duplicate. Samples were taken at 0, 24, 48, 72 and 96 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 The initial samples for analysis (0 hours) were withdrawn from the vessels after addition of the test organisms and stabilization of the test system.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.
Vehicle:
no
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 (ref.nr. 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):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
The test fish (Zebra-fish, Brachydanio rerio) were obtained from an established commercial hatchery (Bio International, Horn, The Netherlands), and registered in the in-house purchase register as batch : 020298. Quarantine and transportation procedures were in accordance with the Standard Operating Procedure A-324 [4.6]. All fish were held in reconstituted water (DSW) of an approximately 16-hour daylight photoperiod and observed for at least 12 days prior to testing. During the holding period the fish were fed daily with a standard commercial fish food, and this was stopped approximately 24 hours before testing. The batch had a mortality of 3 % and was accepted because less than 5% mortality was observed within 7 days of holding. The dissolved oxygen concentration was more than 80% of the air-saturation value throughout the holding period. Records were held on the quarantine procedures. The wet weight of the fish used in the test, based on the weight of ten fish of the same batch, was 0.77 gram. The mean length of the fish was 2.4 cm. The biomass loading did not exceed the maximum loading of 1 gram wet weight ll.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
22.3 - 24.7°C
pH:
8.1 - 8.4
Dissolved oxygen:
92 - 107%
Nominal and measured concentrations:
Nominal: 0.462, 0.578, 0.721, 0.902 and 1.127 mg/L
Measured (geometric mean): 0.061, 0.065, 0.068, 0.071, 0.083 mg/L
Details on test conditions:
- Glassware: The test was performed in test vessels containing 1000 mL of DSW. The test vessels were closed with plastic stoppers. All other equipment was made of glass.
- 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 litre: 100 mg NaHCO3, 20 mg KHCO3, 200 mg CaCl2 2H2O and 180 mg MgSO4 7H2O.
- Apparatus and test conditions: The test was carried out in a temperature controlled area with a temperature of 23 ± 2°C. The photoperiod was 16 hours of daylight supplied by fluorescent tubes (8 ± 2 µmol/m2/s). The test vessels were placed in a random manner. Aeration was achieved using a Pasteur pipette connected to a compressor with purified air.
- Determination of test parameters: The pH and oxygen of all test substance samples and controls were measured daily. The pHs were determined with the aid of a pH meter (pH 196, WTW, Weilheim, Germany), and the oxygen concentration was determined using a oxygen monitor (Oxi 196, WTW, Weilheim, Germany) The temperature was measured continuously using a temperature sensor coupled to a data logger (Elbanton, Kerkdriel, The Netherlands). The light intensity in the test chamber 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 on the toxicity of the test substance to fish. In this investigation 100% survival was found at a concentration of 20% of the stock solution and at a concentration of 100% of the stock solution adverse behaviour was observed. The definitive test was performed as a static test using 10 fish per test concentration and control. The concentration range was made using a stock solution. The test was performed in single-fold. The control consisted of DSW alone. The fish were allocated randomly in the test vessels. The test vessels were aerated gently during the test. The pH was not adjusted during the test. The fish were not fed during the performance of the test. Mortalities and other abnormalities were recorded within the first 6 hours of testing and subsequently every 24 hours. Those fish which were not able to breathe and which did not react to a gentle mechanical touch were considered to be dead. Dead individuals were removed at each observation and a record was maintained of mortality and abnormal behaviour for each concentration tested.
- Statistical evaluation: The nominal and measured concentrations of the test substance were used for the calculation of the LC50 values, and to plot the dose-response curve. The LC50 values were calculated using the Binomial method using the computer program ToxCalc of Tidepool Scientific Software. Confidence limits (95%) were also calculated. 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, where possible. 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 96 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. If values were below the detection limit, the detection limit values were used for calculation.
- Validity criteria: The test should be valid if no more than 10% of the control test fish die or exhibit abnormalities during the test period (OECD, EEC). The dissolved oxygen concentration should be > 60% of the air-saturation throughout the test (OECD, EEC). The pH should not vary more than one unit during the test (EEC).
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
not determinable
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 tests with fish: The concentration of the test substance in test media from the 96 hour fish toxicity study were between 0 and 0.95 at the beginning of the test. Already after 48 hours the test substance concentration in all test media had decreased below the detection limit of 0.04 mg/L. This rapid decrease may be due to absorption onto the fish or the test vessel wall. The test substance may also be metabolized by the fish. Based on the observed behavioural effects at the highest dose it is likely that some material remained in solution.

Test environment
- The light intensity during the test in the test chamber was between 9.73 and 7.35 µmol/m2/s

Validity criteria
- The test was valid as shown by the oxygen and pH values and by the absence of mortality in the control after 96 hours of testing.

Test results
- During the test partial mortalities were observed only at the highest concentration tested (1.127 mg/L nominal). 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. Only moderate effects could be determined, and therefore no LC50 value could be calculated. The nominal No Observed Effect Concentration (NOEC) found for the test substance is 0.902 mg/L and based on the measured concentrations the NOEC is 0.071 mg/L. During the test period changes in the behaviour and mortalities were only observed at the highest concentration tested. This observation is in agreement with the results of the chemical analyses which revealed that after 24 hours the test substance was only detected in the medium with the highest dose.
Sublethal observations / clinical signs:

Analysis of test substance in test media

 

0 hr

24 hr

48 hr

72 hr

96 hr

1.127 mg/L

0.950

0.063

nd

nd

nd

0.721 mg/L

0.650

nd

nd

nd

nd

0.462 mg/L

0.362

nd

nd

nd

nd

0 mg/L

nd

nd

nd

nd

nd

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

Validity criteria fulfilled:
yes

Description of key information

The LD50 could not be determined.

Key value for chemical safety assessment

Additional information

The short-term toxicity to fish of the test substance was determined in an acute toxicity test which is described in the OECD Guideline 203 and was performed in accordance with the principles of Good Laboratory Practice (GLP). Ten fish per test concentration (0.462, 0.578, 0.721, 0.902, and 1.127 mg/L) were exposed to the test substance for 96 hours in a static test. During the test the pH varied no more than 0.3 units, and the oxygen concentrations were all above 60% of the saturation values. The temperature varied between 22.3 and 24.7°C throughout the test. The test is valid as shown by the absence of mortality in the control. The measured concentration of the test substance in test media were between 0 and 0.95 mg/L at the beginning of the test. Already after 48 hours the test substance concentration in all test media had decreased below the detection limit of 0.04 mg/L. The LC50 value could not be calculated due to the low solubility and the low toxicity at the tested concentrations. The No Observed Effect Concentration (NOEC) derived from nominal concentrations is 0.902 mg/L and 0.071 mg/L when derived from the measured concentrations. The NOEC based on measured values is questionable since a small toxic effect at the highest dose was observed and all concentrations, except the first measurement, are below the detection limit.