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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
June 13th - 7th 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 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
N/A
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Amounts of the test item of 5.1, 16.0, 50.1, 160.2 and 499.5 mg were weighed onto glass slides and were transferred to the respective stirring vessel. Then, 260 mL of tap water was added to each vessel. By using the volume of 260 mL of tap water, the stirring vessels were almost completely filled. Then, the stirring vessels were completely sealed using glass stoppers.
The test item was mixed into the tap water using ultrasonic treatment over 15 minutes and intense stirring. The dispersions were stirred over 24 hours at room temperature in the dark to dissolve a maximum amount of the test item and/or to disperse it as homogeneously as possible. No emulsifiers or solvents were used.
After the stirring period, the content of the stirring vessels were transferred to the test vessels. Then, the stirring vessels were rinsed with 24 mL tap water and 16 mL of synthetic wastewater which were also transferred to the test vessels. Finally, 200 mL of the activated sludge inoculum was added

Control:
The reference item 3,5-dichlorophenol was tested in parallel under otherwise identical conditions of the test, and functioned as a positive control. The nominal concentrations tested were of 5, 16 and 50 mg/L. A stock solution of 3,5-dichlorophenol was prepared according to the test guidelines: 0.5 g of 3,5-dichlorophenol was dissolved in 10 mL 1 M NaOH and diluted to about 30 mL with purified water. Excess of NaOH was neutralized with 0.5 mol/L H2SO4 to the point of incipient precipitation. Thereafter, the mixture was made up to one liter with purified water. The pH was determined to be 7.5. The final concentration of 3,5-dichlorophenol amounted to 500 mg/L. Aliquots of this 3,5-dichlorophenol stock solution were mixed with synthetic wastewater and tap water in the respective test flasks to obtain the desired concentrations.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: ARA Ergolz II, Füllinsdorf, Switzerland

- Method of cultivation: Incubated in a laboratory wastewater treatment plant (Husmann unit) for one week. The Husmann unit consisted of an aeration vessel (3.3 L) and a settling vessel (5 L). The aeration vessel was continuously aerated with compressed air to maintain aerobic conditions and to keep sludge flocs in suspension. An airlift pump was used to recycle the activated sludge from the settling vessel intermittently back to the aeration vessel.

- Preparation of inoculum for exposure: The water flow through the unit was approximately 0.55 liter per hour resulting in a hydraulic retention time of approximately 6 hours. The sludge was fed with synthetic wastewater which was continuously dosed to the aeration tank at a concentration of dissolved organic carbon (DOC) of approximately 110 to 160 mg/L in the influent.
One day before the performance of the test, the volume of three liters activated sludge were removed from the activated sludge unit. Then, the sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. Then, the sludge was resuspended in three liters of tap water and fed with 150 mL of synthetic wastewater and was kept at room temperature under continuous aeration until use.
Before use, an aliquot of the sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, the sludge was diluted with tap water to obtain a concentration equivalent to 4 g dry material per liter. The pH of the activated sludge inoculum was adjusted from 8.2 to 7.4 with a diluted sulfuric acid solution.

- Pretreatment: Over two weeks prior to the test start, the test item was continuously dosed to the aeration tank by means of an organic stock solution in acetone. The concentration of the test item in the water inflow was 15 mg/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
N/A
Hardness:
NDA
Test temperature:
20 ºC
pH:
7.5 - 8.3
Dissolved oxygen:
7.9 - 8.5 mg O2/L
Salinity:
NDA
Nominal and measured concentrations:
Nominal: 10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 2000 mL glass beakers containing 500 mL of test media.
- Aeration: yes
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): No renewal
- No. of organisms per vessel: not reported.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: 1.6g/l dry weight sludge

TEST MEDIUM / WATER PARAMETERS
Tap water and synthetic waste water

Synthetic wastewater:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2 · 2H2O
0.2 g MgSO4 · 7H2O
2.8 g K2HPO4
filled up to a final volume of 1 litre with deionized water

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the activated sludge inoculum was adjusted from 8.2 to 7.4 with a diluted sulfuric acid solution.
- Photoperiod: NDA
- Light intensity: NDA

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : respiration rate

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study: NDA
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
154 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL of 108 – 222 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
47 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL of 26 – 71 mg/L
Details on results:
Up to and including the nominal concentration of 32 mg/L, the test item Shale Oil had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours. At the higher test item concentrations of 100, 320 and 1000 mg/L, the respiration rate was inhibited by 37, 68 and 97%, respectively.

The calculated 3-hour EC-values and their 95% confidence limits were as follows:

NOEC (determined as the calculated 3-hour EC15): 47 mg/L 95% confidence interval: 26 – 71 mg/L
EC20: 59 mg/L 95% confidence interval: 34 – 86 mg/L
EC50: 154 mg/L 95% confidence interval: 108 – 222 mg/L
EC80: 402 mg/L 95% confidence interval: 272 – 718 mg/L

The oxygen consumption rates of the two controls (run at the start or at the end of the test) differed by 7.2% (guideline-recommended maximum variation: 15%).
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 11 mg/L with 95% confidence limits of 3.8 and 23 mg/L. The 3-hour EC50 is within the guideline-recommended range of 5-30 mg/L, confirming the suitability of the activated sludge used.
Reported statistics and error estimates:
The 3-hour EC-values for the test item and reference item and their 95%-confidence limits were calculated by Probit Analysis. All test results were based on the nominal concentrations of the test item and the reference item.

Table 1: Influence of Shale Oil on oxygen consumption of activated sludge

Flask

Test Chemical

Nominal Concentration of test chemical (mg/L)

Oxygen consumption rate (mg O2/L min-1)

Inhibition (%)

1

Control

0

1.015

-

10

Control

0

1.087

-

2

Shale Oil

10

1.054

-0.3

3

Shale Oil

32

0.940

10.6

4

Shale Oil

100

0.659

37.3

5

Shale Oil

320

0.338

67.8

6

Shale Oil

1000

0.029

97.3

7

3,5-dichlorophenol

5

0.841

19.9

8

3,5-dichlorophenol

16

0.353

66.5

9

3,5-dichlorophenol

50

0.074

93.0

Mean Oxygen consumption rate for control = 1.051 mg O2/L min-1(standard deviation = 7.2)

Validity criteria fulfilled:
yes
Conclusions:
The EC50 to activated sludge of shale oil, middle fraction was determined as 154 mg/L, with 95 % CL of 108 – 222 mg/L.
Executive summary:

The inhibitory effect of the test item Shale Oil on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the EU Commission Directive 88/302/EEC, Part C.11 (1988) and the OECD Guideline for Testing of Chemicals, No. 209 (1984).The activated sludge inoculum was pre-adapted to the test item at the nominal concentration of 15 mg/L for two weeks prior to the start of the test. The following nominal concentrations of the test item were tested: 10, 32, 100, 320 and 1000 mg/L.

The EC50 to activated sludge of shale oil, middle fraction was determined as 154 mg/L, with 95 % CL of 108 – 222 mg/L.

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

Description of key information

The EC50 to activated sludge of shale oil, middle fraction was determined as 154 mg/L, with 95 % CL of 108 – 222 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
154 mg/L
EC10 or NOEC for microorganisms:
47 mg/L

Additional information

Similar values for short-term toxicity to daphnia were observed between the middle fraction and heavy 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 toxicity to microorganisms. Hence, the study conducted on the middle fraction of shale oil, is considered robust enough to be the key study for the heavy fraction.