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Ecotoxicological information

Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 May 2003 to 17 December 2003
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
July 1992
according to guideline
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
Official Journal of the European Communities, L 383, 172 - 178, 29 December 1992
GLP compliance:
Analytical monitoring:
Details on sampling:
Concentrations: duplicate samples were taken from the blank-control and the 100 mg/l concentration for analysis

Frequency: at t= 0 h and at t= 72 h from freshly prepared test solutions; at t= 24 h and t= 96 h from 24-hour old solutions

Sampling method: 10 ml volume from the approximate centre of the test vessels;

Sample storage before analysis: stored in a freezer until transportation to TNO Nutrition and Food Research for analysis

Additionally, singular reserve samples of 10 ml were taken. These samples were stored in a freezer for possible analysis for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

After receipt on 17 July 2003 (range finding study) and on 18 August 2003 (main study) the samples were stored at < -18°C until analysis on 24 July 2003 and 21 August 2003, respectively. At the request of the study director on 21 August 2003 sample 373657-5 (a sample from the rangefinding study) was reanalysed.
Details on test solutions:
Preparation of stock solutions was performed by weighing 500 mg of test substance and adding it to 5 litre of test medium in a silanised volumetric flask. Preparation was conducted under dimmed light conditions. After magnetical stirring for 12 minutes under dark conditions, the resulting solutions were clear and colourless. Lower test concentrations were prepared by subsequent dilution of the stock. Since the pyrophosphate pmt of the test substance causes a drop in the pH of the test medium, pH of the test medium was adjusted with ortho-Phosphoric acid (85 %) to 6.0 in the final test.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Species: Zebra-fish (Danio rerio, Teleostei Cyprinidae)
- Source: Atlanta, Hellevoetsluis, The Netherlands
- Mean length: Range-finding test: 2.5 ± 0.2 cm; Final test: 2.3 ± 0.2 cm
- Mean weight: Range-finding test: 0.25 ± 0.08 g; Final test: 0.29 ± 0.11 g
- Characteristics: F1 from a single parent-pair bred in UV-treated water
- Total fish used: 26

- Quarantine/ Acclimatisation: at least 12 days after delivery
- Feeding: Daily with Trouvit
- Medium: ISO-medium [ISO International standard 734]
- Measurements: pH, nitrate and nitrite concentration and ammonia concentration: once a week; temperature: every day
- Health during acclimation (any mortality observed): mortality during the seven days prior to the start of the test was less than 5%
Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
250 mg CaCO3/l
Test temperature:
21.0 and 21.6 °C
5.6 - 6.4
The pH in the 100 mg/l concentration was just below 6.0 (the lower limit
prescribed by the protocol) throughout the test. Also the pH in the blank-control
was just below 6.0 in the freshly prepared medium at t= 72 h. Since the given pH
range in the protocol describes the optimum pH range for Zebra-fish (6.0 - 8.5) this
was not considered to have affected the study. For details on the pH values see attachment "T-1063FM - results acute toxicity to zebrafish.pdf".
Dissolved oxygen:
6.9 - 9.1 mg/l
Nominal and measured concentrations:
Nominal test concentration: 100 mg/L
Measured concentrations: Analysis of the samples taken during the test showed that measured concentrations were in agreement with nominal during both the first and the last 24 hours of the test (100-93% and 108-93% of nominal, respectively). Therefore, results were based on nominal concentrations.
For details on the chemical analysis (measured concentrations of T-1063FM) see attachment “T-1063FM - results acute toxicity to zebrafish.pdf”).

Details on test conditions:
- Test type: Semi-static, with renewal of test solutions after each 24-hour test period
- Test vessel: 6.5 litres, all-glass with perspex cover lids
- Aeration: no
- Feeding: No feeding from 48 hours prior to the test and during the total test period.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.41 g fish/litre, i.e. 7 fish per 5 litres of test medium
- Introduction of fish: within 10 minutes after preparation of the test media
- Euthanasia: at the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2 % ethylene glycol monophenylether in water

Test medium: ISO-medium [ISO International Standard 7346], formulated using RO-water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA), aerated until the dissolved oxygen concentration had reached saturation and the pH had stabilised. After aeration the hardness was 250 mg CaC03 per litre and the pH was 7.9, after adjustment it was 6.0-6.1.
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen content, pH and temperature: Daily in all vessels, beginning at the start of the test (day 0).

- Adjustment of pH: yes, see test medium
- Illumination: 16 hours photoperiod daily, light was dimmed to 3 lux.

- Mortality and other effects: At 3, 24, 48, 72 and 96 hours following the start of exposure. Dead fish were removed when observed.

- Range finding study:
Test procedure and conditions were similar to those applied in the final test with the following exceptions: Three fish per concentration were exposed to a range of 0.1 to 100 mg/l increasing by a factor of 10. Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration. Samples for analyses were taken from the 10 mg/l concentration.
After 96 hours of exposure no mortality or clinical effects were observed at any of the test concentrations. The analytical results showed that at a concentration of 10 mg/l the measured concentration was in agreement with nominal during both the first and the last 24 hours of the test (77 - 91 % and 96 - 92 % of nominal, respectively). The LC50 was expected to be above the nominal concentration of 100 mg/l.
- Limit test: 100 mg/l (regulatory limit concentration)
Reference substance (positive control):
Pentachlorphenol (PCP)
Key result
96 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
Basis for effect:
mortality (fish)
Details on results:
In the control group, no fish showed abnormal behaviour (including mortality).
Mortality and clinical effects: One fish died at the 100 mg/l test concentration. No clinical effects were observed. Based on the results of the range finding test, the death of one fish was not considered to be test substance related. However, no NOEC could be determined.
Observations on the exposure media: After 3 hours of exposure the 100 mg/l test concentration was observed to be hazy with a test substance floating layer. During the rest of the test period the 100 mg/l test concentration was observed to be hazy with both a floating layer and test substance precipitate. The blank-control was clear and colourless.

For details on the mortality, the dissolved oxygen concentrations and the temperature, see attachment "T-1063FM - results acute toxicity to zebrafish.pdf".
Results with reference substance (positive control):
Under the conditions of the present test Pentachlorphenol induced no lethal effects in zebra-fish at or below 0.15 mg/l.
The 24h-LC50 was 0.29 mg/l with 0 % mortality at 0.22 mg/l and 80 % mortality at 0.32 mg/l.
The 96h-LC50 was 0.24 mg/l with a 95 % confidence interval between 0.21 and 0.30 mg/l. This effect was already reached within 48 hours of exposure.
The range of the 96h-LC50 for zebra-fish is generally between 0.18 mg/l and 0.30 mg/l (with a 95% confidence interval between 0.28 and 0.39 mg/l) based on historical data of reference tests performed by NOTOX. The response observed in zebra-fish originating from the present batch falls within this range. The validity criteria were fullfilled. For details see attachment "T-1063FM - results acute toxicity to zebrafish.pdf".
Validity criteria fulfilled:
no mortality of control animals; concentration of dissolved oxygen in all test vessels >60 % of the air saturation; test conditions were maintained constant throughout the test
Under the conditions of the present test T-1063FM induced 14% mortality in zebra-fish at 100 mg/l. Therefore the 96h-LC50 is above 100 mg/l.

Description of key information

The LC50 for zebra-fish is above 100 mg/l (EU Method C.1, OECD guideline 203).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
Effect concentration:
> 100 mg/L

Additional information