Shale oils

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5th August - 20th September 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
- Sampling method:
Just before the start of the test: - duplicate samples from each test medium (without daphnids)
- duplicate samples from the control (without daphnids)
After 48 hours (stability samples): - duplicate samples from each test medium
- duplicate samples from the control

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Four days before the start of the test, four individual mixtures with loading rates of 3.2, 10, 32, and 100 mg/L were prepared. Test item amounts of 7.3, 20.4, 73.7, and 230.5 mg were mixed into 2280, 2040, 2300, and 2300 mL of test water, respectively, to obtain the loading rates mentioned above. 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 (Schleicher & Schuell, Type NC45, pore size 0.45 μm) just before the start of the test. 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 daphnids as WAFs. For practical reasons, the test medium with the loading rate of 1.0 mg/L had to be prepared by diluting the WAF with the loading rate of 3.2 mg/L with test water. In addition to the WAFs, a control (test water without test item) was tested in parallel.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater water flea
- Strain: Daphnia Magna straus
- Source: A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield/UK in 1992. Since that time, the clone is bred in the laboratories of RCC.
- Age at study initiation (mean and range, SD): 6 -24 hours
- Weight at study initiation (mean and range, SD): NDA
- Length at study initiation (length definition, mean, range and SD): NDA
- Valve height at study initiation, for shell deposition study (mean and range, SD):NDA
- Peripheral shell growth removed prior to test initiation: NDA
- Method of breeding: bred in the laboratories of RCC
- Feeding during test: None

ACCLIMATION
Not applicable as the daphnids were bred in the laboratory where tsting was conducted.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

N/A

Hardness:

250 mg/L as CaCO3

Test temperature:

21 ºC

pH:

7.9

Dissolved oxygen:

6.8 - 8.6 mg/L

Salinity:

NDA

Nominal and measured concentrations:

1.0, 3.2, 10, 32 and 100 mg/L as WAF loading rates.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL test tubes with glass stoppers
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: filled to 50 mL
- Aeration: aerated prior to the start of the study until oxygen saturation was reached
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: loading rate was less than one daphnia per 5 mL of test solution.

TEST MEDIUM / WATER PARAMETERS
Reconstituted test water: analytical grade salts were dissolved in purified 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)
Na : K = 10 : 1 (based on molarity)
The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period the test water was not aerated.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light, 8 hours dark
- Light intensity: 570 - 740 lux

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

TEST CONCENTRATIONS
- 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):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL (3.2 to 10)

Details on results:

The reported biological results are based on the loading rates of the test item Shale Oil since water accommodated fractions (WAFs) were tested. The biological results are listed in Table 1.

During the test period of 24 hours, no immobility of the test organisms was observed in the control and at the loading rates of 1.0 and 3.2 mg/L. At the next higher loading rate 10 mg/L, the immobilization rate was 90% after 24 hours. At the two highest loading rates of 32 and 100 mg/L, all daphnids were immobile.

The 24-hour EC50 of the test item was calculated to be 6.3 mg/L loading rate (95% confidence limits could not be calculated). The 24-hour EC0 was 3.2 mg/L loading rate and the 24-hour EC100 amounted to 32 mg/L loading rate.

After 48 hours of exposure, no immobility was observed at the loading rates of 1.0 and 3.2 mg/L. At the higher loading rates of 10 mg/L and above, all daphnids were immobile after 48 hours test duration.

The 48-hour EC50 was calculated to be 5.7 mg/L loading rate with 95% confidence limits of 3.2 and 10 mg/L. The 48-hour EC0 and the 48-hour NOEC (highest concentration tested without toxic effects after 48 hours) of Shale Oil were 3.2 mg/L loading rate since no immobilization of test organisms was observed up to and including this loading rate. The 48-hour EC100 amounted to 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 entire test duration. The test media with the loading rates of 32 and 100 mg/L were colored by the test item.

Results with reference substance (positive control):

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control at least once a year. The latest result of the positive control test in February 2005 (48-hour EC50: 0.53 mg/L, RCC Study No. 859199) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 1996 to 2005: 48-hour EC50: 0.53 - 1.1 mg/L).

Reported statistics and error estimates:

The 24 hour EC50 was calculated using the moving average method.
The 48-hour EC50 could not be calculated by Probit Analysis or Moving Average Interpolation due to the steep concentration-effect relationship. Instead, the 48-hour EC50 was determined as the geometric mean value of the two consecutive test concentrations with 0% and 100% immobility, and the 95% confidence limits for the EC50 as the test concentrations with 0% and 100% immobility.

Table 1: Effect of Shale Oil on the Mobility of Daphnia Magna

Loading rate	Number of daphnids tested	Immobilized daphnids after 24 hours		Immobilized daphnids after 48 hours
		Number	%	Number	%
Control	20	0	0	0	0
1.0	20	0	0	0	0
3.2	20	0	0	0	0
10	20	18	90	20	100
32	20	20	100	20	100
100	20	20	100	20	100

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour EC50 to Daphnia magna was determined as 5.7 mg/L with 95 % CL of 3.2 - 10 mg/L.

Executive summary:

The short term toxicity of the test material to freshwater invertebrates was investigated in a study which was conducted in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions.

During this study, daphnia magna straus were exposed to shale oils middle fraction at WAF loading rates of 1.0, 3.2, 10, 32 and 100 mg/L for a period of 48 hours.

The 48-hour EC50 to Daphnia magna was determined as 5.7 mg/L with 95 % CL of 3.2 - 10 mg/L.

Description of key information

In an acute toxicity to aquatic organisms test, daphnia magna straus were exposed to shale oils middle fraction at WAF loading rates of 1.0, 3.2, 10, 32 and 100 mg/L, in accordance with OECD 202, EU method C.2, and performed to GLP standard
The 48-hour EC50 to Daphnia magna was determined as 5.7 mg/L with 95 % CL of 3.2 - 10 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

5.7 mg/L

Additional information

The short term toxicity of the test material to freshwater invertebrates was investigated in a study which was conducted in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During this study, daphnia magna straus were exposed to shale oils middle fraction at WAF loading rates of 1.0, 3.2, 10, 32 and 100 mg/L for a period of 48 hours.

The 48-hour EC50 to Daphnia magna was determined as 5.7 mg/L with 95 % CL of 3.2 - 10 mg/L.