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Administrative data

short-term toxicity to fish
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
199-01-25 to 1999-01-29
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes (incl. QA statement)

Test material

Details on test material:
- Name of test material (as cited in study report): Tri (hexyl, octyl, decyl) citrate
- Substance type: pure active substance
- Physical state: liquid
- Stability under test conditions: in water for at least 2 hours
- Storage condition of test material: in original container, at room temperature, in the dark
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
- Melting point: -29°C (freezing point: -35.5°C)
- Boiling point: test substance showed decomposition above 260°C
- Vapour pressure: 8.5 x 10E-3 p at 25°C
- Water solubility (under test conditions): < 1 mg/L
- Solubility in organic solvents: soluble
- log Pow: > 6
- pKa: not applicable
- Base or acid catalysis of test material: no data
- UV absorption: no data
- Stability of test material at room temperature: stable
- pH dependance on stability: no data

OTHER PROPERTIES (if relevant for this endpoint)
- Adsorption characteristics: no data

Sampling and analysis

Analytical monitoring:
Details on sampling:
In a pre-experiment (without GLP) the dissolved organic carbon contents (DOC) of the test substance solution (same preparation as in the test) was determined. In the solution the same amount of DOC was determined as in the blank control. Thus, no dissolved test substance could be verified in test water. Therefore, no analytical dose verification in the test media of the acute toxicity test with fish could be done.

Test solutions

Details on test solutions:
- Method: A supersaturated stock suspension of the test substance with a nominal concentration of 100 mg/L was prepared by weighing 503 mg of the test substance into 5 L test water, following ultrasonic treatment for 10 minutes and intense stirring. The supersaturated stock suspension was stirred on a magnetic stirrer at room temperauter in the dark over 3 days . Thereafter the suspension was filtered through a cellulose-nitrate filter with a defined pore size of 0.2 µm just before the start of the test.
- Controls: reconstituted water
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no, suspension was filtered

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Common name: Zebra fish
- Strain: Brachydanio rerio
- Source: City Zoo, Darmstadt, Germany
- Age at study initiation (mean and range, SD): juveniles (no further details mentioned)
- Length at study initiation (length definition, mean, range and SD): 3.11 ± 0.16 cm (mean of 10 fish from the test fish batch)
- Weight at study initiation (mean and range, SD): 0.27 ± 0.06 g (mean of 10 fish from the test fish batch)
- Method of breeding: not mentioned
- Feeding during test: none

- Acclimation period: 7 weeks
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: TETRA MIN Hauptfutter, TETRA-Werke, Melle, Germany (commercial fish diet)
- Feeding frequency: not mentioned
- Health during acclimation (any mortality observed): no fish died and all fish were healthy

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
Post exposure observation period:

Test conditions

2.5 mmol/L (= 250.0 mg/L) as CaCO3
Test temperature:
21 - 23 °C
7.6 - 7.9
Dissolved oxygen:
8.1 - 8.9 mg/L
Nominal and measured concentrations:
filtrated WAF
Details on test conditions:
- Test vessel:
- Material, size, headspace, fill volume: 9 L glass aquaria with 5 L test medium
- Aeration: slightly aerated during test
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.378 g/L

- Source/preparation of dilution water: according to guideline
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4:1 (based on molarity)
- Conductivity: not mentioned
- Culture medium different from test medium: no
- Intervals of water quality measurement: not mentioned

- Adjustment of pH: no
- Photoperiod: 16 h light
- Light intensity: 712 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The test fish were observed after approx. 2, 24, 48, 72 and 96 hours test duration for symptoms of intoxication and mortality. The water temperature, pH-values and the dissolved oxygen concentrations were determined daily in test concentration and control. The behaviour of the test substance in test water was determined once every day during the test.
Reference substance (positive control):

Results and discussion

Details on results:
- Behavioural abnormalities: not observed
- Observations on body length and weight: not made
- Other biological observations: no signs of intoxication were observed
- Mortality of control: no mortality occured
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations were made concerning the behaviour of the test substance in the test medium
- Effect concentrations exceeding solubility of substance in test medium: not applicable, filtrated test solution
Reported statistics and error estimates:
The NOEC and the LC0 were determined directly from the raw data. The LOEC, the LC50 and the LC100 could not be quantified due to the absence of toxicity of the test substance.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table: Mortality and symptoms of intoxication

Treatment   Number of fish with intoxication symptoms and intoxication symtpoms / number of dead fish / number of fish tested after            
  2 h   24 h  48 h  72 h  96 h
 control  0 / 0 / 7  0 / 0 / 7  0 / 0 / 7  0 / 0 / 7  0 / 0 / 7
 filtrate of test substance  0 / 0 / 7  0 / 0 / 7  0 / 0 / 7  0 / 0 / 7  0 / 0 / 7

Possible intoxication symptoms with abbreviations: strongly extended gills (AK), apathy (AP), distended abdomen (BA), fins frayed out at the border (FV), Exopthalmus (GA), convulsions (KR), fish mainly at the water surface (OB), mucus secretion (SA), fish lying on side of back (SR), strong ventilation (SV), tumbling during swimming (TS)

Applicant's summary and conclusion

Validity criteria fulfilled:
no fish died in the control and oxygen saturation was always > 60%
Due to low water solubility limit of the test substance a filtrate of a supersaturated stock suspension of nominal 100 mg/L was tested. Thus no concentrations above the solubility limit of the test substance in the used test water were tested. Due to the low water solubility limit no analytical dose verfication could be done in the present test. Therefore, the biological results could not be related to a specific concentration but to the solubility limit of Tri (hexyl, octyl, decyl) citrate in the test medium.
In the cotnrol and in the undiluted filtrate of the supersaturated stock suspension all fishes survived until the end of the test and no signs of intoxication were observed. Therefore, the 96-hour NOEC and the 96-hour LC0 of Tri (hexyl, octyl, decyl) citrate to Zebra fish were determined to be at least up to the solubility limit. The NOEC and the LC0 might even be higher, but concentrations in excess of the solubility limit had not been tested. The 96-hour LOEC, the 96-hour LC50 and the 96-hour LC100 were clearly higher than the solubility limit of the test substance in the used test water.