Shale oils, light

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

29th July - 20th September 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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 read across from shale oil, middle fraction

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Since no substance specific analysis of the different compounds of the test item could be performed, the concentration of dissolved organic carbon (DOC) was measured in the test media as sum parameter of the different organic compounds. For this, the following samples were taken:

Just before the start of the test: - duplicate samples from each test medium (without algae)
- duplicate samples from the control (without algae)
After 72 hours: - duplicate samples from each test medium (without algae) (stability samples)
- duplicate samples from the control (without algae)

For the 72-hour stability samples, additional flasks with adequate volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test but without algae. All samples were analyzed immediately after sampling without prior storage.

The concentration of DOC was measured in all duplicate test medium and control samples.

Vehicle:

no

Details on test solutions:

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, 10.8, 33.9, and 108.0 mg were mixed into 2280, 1080, 1060, and 1080 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 algae 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):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Scenedesmus subspicatus
- Source (laboratory, culture collection): SAG, Institute for Plant Physiology, University of Göttingen, D-37073 Göttingen, Germany
- Age of inoculum (at test initiation): four days
- Method of cultivation: laboratory culture.

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: NDA

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

N/A

Hardness:

NDA

Test temperature:

22 - 23ºC

pH:

7.9 - 9.4

Dissolved oxygen:

NDA

Salinity:

NDA

Nominal and measured concentrations:

1.0, 3.2, 10, 32, and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml erlenmeyer flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: completely filled (approx 60 mL)
- Aeration: NDA
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): No
- Initial cells density: 10,000 cells/mL
- Control end cells density: 638,800 cells/ml
- No. of organisms per vessel: 600,000 cells per vessel
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: same as test medium

TEST MEDIUM / WATER PARAMETERS
The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines, but modified according to the International Standard ISO 14442: the concentration of NaHCO3 was increased by 200 mg/L to 250 mg/L (as carbon source for the algal growth), and 6 mmol/L HEPES-buffer was added to keep the pH in the test media as constant as possible. Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations:

Macro-nutrients:
NaHCO3 250.0 mg/L
CaCl2 × 2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
MgSO4 × 7 H2O 15.0 mg/L
MgCl2 × 6 H2O 12.0 mg/L
KH2PO4 1.6 mg/L
HEPES-buffer 1430.0 mg/L

Trace elements:
Na2EDTA × 2 H2O 100.0 μg/L
FeCl3 × 6 H2O 80.0 μg/L
MnCl2 × 4 H2O 415.0 μg/L
H3BO3 185.0 μg/L
Na2MoO4 × 2 H2O 7.0 μg/L
ZnCl2 3.0 μg/L
CoCl2 × 6 H2O 1.5 μg/L
CuCl2 × 2 H2O 0.01 μg/L

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, HEPES-buffer added
- Photoperiod: continuously illuminated
- Light intensity and quality: measured light intensity of about 7800 Lux (mean value), range: 6630 to 8670 Lux
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement: NDA
- Other: The shape of the algal cells was examined microscopically in these samples.

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:

72 h

Dose descriptor:

EC50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL of 9.3 - 44

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL of 23-44

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no differences observed microscopically.
- Unusual cell shape: No effects
- Colour differences: change in colour of the test media at highst concentration
- Flocculation: NDA
- Adherence to test vessels: NDA
- Aggregation of algal cells: NDA
- Other: NDA
- Any stimulation of growth found in any treatment: Not significantly
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The latest result of the positive control test in 2004 (72-h EC50 for the growth rate: 0.65 mg/L, RCC Study No. 852335) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 1996 to 2004: 72-h EC50: 0.44–1.16 mg/L).

Reported statistics and error estimates:

See any other remarks on materials and methods.

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

The influence of the test item Shale Oil on the growth of Scenedesmus subspicatus is shown in Table 1–5.

The test item had a statistically significant inhibitory effect on the growth (biomass and growth rate) of Scenedesmus subspicatus after the exposure period of 72 hours at the loading rates of 32 and 100 mg/L (results of Dunnett-tests, one-sided, α = 0.05, see Tables 4 and 5). Thus, the loading rate of 32 mg/L was determined as the 72-hour LOEC (lowest concentration tested with toxic effects).

The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours) was determined to be the loading rate of 10 mg/L since up to and including this loading rate both the mean biomass and the mean growth rate of the algae were not statistically significantly lower than in the control (Tables 4 and 5).

The EC-values as well as the NOEC and LOEC were calculated for both parameters, the algal biomass (b) and the growth rate (r), after 72 hours test duration:

Parameter (0-72h)	Biomass b (mg/l loading rate)	Growth rate r (mg/l loading rate)
EC50 95% confidence limits	20 9.3-44	32 23-44
EC10 95% confidence limits	9.4 0.7-16	11.3 5.8-17
EC90 95% confidence limits	43 25-562	88 60-177
NOEC	10	10
LOEC	32	32

The microscopic examination of the algal cells after 72 hours test period showed no difference between the algae growing at the loading rate of 100 mg/L and the algal cells in the control. The shape and size of the algal cells growing in the test media containing the test item at up to this test concentration were obviously not affected.

In the control the cell density has increased from a nominal N = 1 × 10⁴ cells/mL at the start of the test (0 hours) to N = 64 × 10⁴ cells/mL (mean value) after 72 hours (Table 1). Thus, the algal growth in the control was sufficiently high under the conditions of the test. The validity criterion of increase of cell density by at least a factor of 16 over the duration of the study was

fulfilled.

No remarkable observations were made concerning the appearance of test media with the loading rates of 1.0 to 32 mg/L. These test media were clear solutions throughout the entire test duration. The test medium with the loading rate of 100 mg/L was colored by the test item.

At the start of the test, the pH values in the test medium and the control ranged from 7.9 to 8.0. At the end of the test, pH values of 7.9 and 9.4 were measured. The increase of the pH during the test was obviously caused by the CO₂-consumption of the algae due to their rapid growth or their high densities in the closed test system (although the test media were buffered). The water temperature ranged from 22 to 23 °C.

Validity criteria fulfilled:

yes

Remarks:

The validity criterion of increase of cell density by at least a factor of 16 over the duration of the study was fulfilled.

Conclusions:

The biomass EC50 of shale oils, middle fraction to Scenedesmus subspicatus was 20 mg/L.
The growth rate EC50 of shale oils, middle fraction to Scenedesmus subspicatus was 32 mg/L.

Executive summary:

In an acute aquatic toxicity study according to OECD 201 and performed to GLP, Scenedesmus subspicatus 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 biomass EC50 of shale oils, middle fraction to Scenedesmus subspicatus was 20 mg/L.

The growth rate EC50 of shale oils, middle fraction to Scenedesmus subspicatus was 32 mg/L.

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

Description of key information

The biomass EC50 of shale oils, middle fraction to Scenedesmus subspicatus was 20 mg/L loading rate.
The growth rate EC50 of shale oils, middle fraction to Scenedesmus subspicatus was 32 mg/L loading rate.

Key value for chemical safety assessment

EC50 for freshwater algae:

32 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 long-term toxicity to aquatic invertebrates 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.