Registration Dossier

Ecotoxicological information

Short-term toxicity to fish

Currently viewing:

Administrative data

Link to relevant study record(s)

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:
1st August - 20th September 2005
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Study conducted on middle weight fraction of shale oils.
according to
OECD Guideline 203 (Fish, Acute Toxicity Test)
according to
EU Method C.1 (Acute Toxicity for Fish)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
Details on sampling:
- Concentrations: all
- Sampling method:
Just before test start (Day 0) and on the last day of test medium preparation (Day 3):
- duplicate samples from each test medium
- duplicate samples from the control
At the end of the first test medium renewal period (Day 1) and at the end of the last renewal period (Day 4):
- duplicate samples from each test medium
- duplicate samples from the control
Since all test fish were dead at the loading rates of 32 and 100 mg/L after 24 hours of exposure, these test media were not prepared anymore after the first test medium renewal and, thus, no samples were taken at the last test medium renewal period.

All samples were taken from the approximate center of the test vessels without mixing the test media and were immediately analyzed without prior storage.
The concentration of DOC was measured in all duplicate test medium and control samples.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Four days before the start of the test and prior to each test medium renewal, five individual mixtures with the loading rates mentioned above were prepared as follows:

Test item amounts of 5.3-5.5 mg, 17.0-17.3 mg, 53-55 mg, 170-173 mg, and 530-550 mg of the test item were mixed into 5300-5500 mL of test water to obtain the loading rates mentioned above. The deviation of the effective weights to the target amounts of the test item was below 1% for each weighing. The test item was mixed into the test water as homogeneously as possible by intense stirring on magnetic stirrers. No auxiliary solvent or emulsifier was used.

The dispersions were stirred at room temperature in the dark for 96 hours in completely filled and tightly closed Erlenmeyer flasks to dissolve maximum concentrations of the different compounds of the test item in the test water.

The long stirring period of 96 hours was chosen to ensure that the equilibrium in WAF preparation was attained. In a pre-experiment (without GLP), approximately the same concentration of dissolved organic carbon was measured in filtrates after stirring for 3, 24 and 96 hours indicating that the equilibrium was attained before 96 hours stirring for the main compounds in the WAFs.

After the stirring period of 96 hours, the equilibrated dispersions were filtered through membrane filters (pore size 0.45 μm) just before the start of the test and prior to each test medium renewal. The suction pressure of the filtration unit was reduced as much as possible to avoid losses of the volatile compounds of the test item during filtration. The filtrates of the dispersions with different loading rates of the test item were tested on the fish as WAFs. Additionally, a control (test water without test item) was tested in parallel.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Common name: Zebra fish
- Strain: Brachydanio rerio
- Source: H. Eggimann, CH-4133 Pratteln, Switzerland
- Age at study initiation (mean and range, SD): NDA
- Length at study initiation (length definition, mean, range and SD): 2.8 ± 0.1cm
- Weight at study initiation (mean and range, SD): 0.16 ± 0.03g
- Method of breeding: NDA
- Feeding during test: Not fed

- Acclimation period: In accordance with the test guidelines, the fish were held in the laboratories of RCC for more than two weeks without any medication. Prior to the test start, they were acclimated for one week to the test water and temperature.
- Acclimation conditions (same as test or not): yes.
- Type and amount of food: commercial fish diet.
- Feeding frequency: During holding and acclimatization until one day before the start of the test, the fish were fed.
- Health during acclimation (any mortality observed): No fish died in the test fish batch and all fish were healthy.
Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
Post exposure observation period:
250 mg/L as CaCO3
Test temperature:
21 - 23 ºC
7.1 - 7.8
Dissolved oxygen:
6.1 - 7.7 mg O2/L
Nominal and measured concentrations:
Nominal WAF concentrations: 1.0, 3.2, 10, 32, and 100 mg/L

In the water accommodated fractions (WAF’s) with the loading rates of 1.0, 3.2 and 10 mg/L, the concentration of DOC measured in the samples were approximately in the same range as the DOC concentrations measured in the control. Thus, the quantification of the DOC originating from the test item was not possible due to the background values measured in the control.
In the WAF’s with the loading rates of 32 and 100 mg/L, concentrations of DOC at the start of the test medium renewal period were 6.7 and 17.7 mg/L, respectively. Over the test medium renewal period of 24 hours, the concentration of DOC decreased to 61 and 48% of the initially measured values. The decrease of the DOC concentration in these test media was considered to be due to adsorption of test item compounds on the test fish and on the glass surfaces of the test vessels.
Details on test conditions:
- Test vessel: 5L Erlenmeyer flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: filled to the top.
- Aeration: yes
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): every 24 hours
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: 0.22 g/L

Reconstituted water: analytical grade salts were dissolved in deionized water to obtain the following nominal concentrations:
CaCl2 × 2H2O : 2.0 mmol/L (= 294 mg/L)
MgSO4 × 7H2O : 0.5 mmol/L (= 123 mg/L)
NaHCO3 : 0.75 mmol/L (= 65 mg/L)
KCl : 0.075 mmol/L (= 5.8 mg/L)
Water Hardness : 2.5 mmol/L (= 250 mg/L as CaCO3)
Alkalinity : 0.8 mmol/L
Ratio of Ca : Mg = 4 : 1 (based on molarity)
Ratio of Na : K = 10 : 1 (based on molarity)
The test water was aerated prior to the preparation of the test media until oxygen saturation was reached.

- Adjustment of pH: NDA
- Photoperiod: 16 hours light / 8 hours dark.
- Light intensity: 60 - 350 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality

- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study: Conducted
Reference substance (positive control):
96 h
Dose descriptor:
Effect conc.:
5.7 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95 % CL of 3.2 - 10
96 h
Dose descriptor:
Effect conc.:
1 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
The biological results (observed visible abnormalities, mortalities and the LC50-values) are listed in Table 1.

In the control and at the loading rate of 1.0 mg/L, all fish survived until the end of the test and no visible abnormalities were observed. At the next higher loading rate of 3.2 mg/L, some test fish showed visible abnormalities during the test. However, all test fish survived and no symptoms of toxicity were observed anymore at the end of the test after 96 hours. At the next higher loading rate of 10 mg/L, all fish showed strong symptoms of toxicity and died during the test period. At the two highest loading rates of 32 and 100 mg/L, all fish died within 3 hours of exposure.

The 96-hour NOEC (highest loading rate tested without observable toxic effects after the exposure period of 96 hours) of Shale Oil to zebra fish amounted to 1.0 mg/L loading rate. The 96-hour LOEC (lowest loading rate tested with observable toxic effects) was 3.2 mg/L loading rate.

The 96-hour LC50 of Shale Oil was calculated to be 5.7 mg/L loading rate with a 95% confidence interval of 3.2 to 10 mg/L. The 96-hour LC0 was 3.2 mg/L loading rate and the 96-hour LC100 was 10 mg/L loading rate.

No remarkable observations were made concerning the appearance of the test media with the loading rates of 1.0 to 10 mg/L. These test media were clear solutions throughout the test medium renewal periods. The test media with the loading rates of 32 and 100 mg/L were colored by the test item. The test medium with the highest loading rate of 100 mg/L was slightly turbid at the end of the test medium renewal period of 24 hours.
Results with reference substance (positive control):
Reported statistics and error estimates:
The test fish were observed after approximately 3, 24, 48, 72 and 96 hours test duration for mortality and visible abnormalities. Dead fish were removed at least once daily and discarded.
The LC50 and the 95% confidence interval at the observation dates after 24, 48, and 72 hours test duration were calculated by Moving Average Interpolation (Ref. 2, 3). The NOEC, LOEC, LC0 and LC100 were determined directly from the raw data.
However, the LC50 at the observation intervals after 3 and 96 hours could not be calculated by Probit Analysis or Moving Average Interpolation due to the steep concentration-effect relationship. Instead, the LC50-values were determined as the geometric mean values of the two consecutive test concentrations with 0% and 100% mortality, and the 95% confidence limits for the LC50 values as the test concentrations with 0% and 100% mortality.
All calculations are based on the loading rates of the test item.
Validity criteria fulfilled:
The LC50 of shale oils, middle fraction to zebra fish was 5.7 mg/L
Executive summary:

In an acute aquatic toxicity study according to OECD 203 and performed to GLP, zebra fish were exposed to shale oil, middle fraction at nominal WAF loading rate concentrations of 1.0, 3.2, 10, 32, and 100 mg/L.

The LC50 of shale oils, middle fraction to Brachydanio rerio was 5.7 mg/L.

Based on the rationale for read-across, it is considered acceptable to use this study to address the same endpoint for the light fraction of shale oil.

Description of key information

The LC50 of shale oils, middle fraction to Brachydanio rerio was 5.7 mg/L loading rate.

Key value for chemical safety assessment

LC50 for freshwater fish:
5.7 mg/L

Additional information

Similar values for short-term toxicity to daphnia were observed between the middle fraction and light fraction of shale oil. This, combined with analytical data which showed the two fractions to be compositionally similar, support the validity of a read-across approach to address the short-term toxicity to fish endpoint. Hence, the study conducted on the middle fraction of shale oil, is considered robust enough to be the key study for the light fraction.