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EC number: 930-776-4 | CAS number: -
- Life Cycle description
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Toxicity to microorganisms
Administrative data
- Endpoint:
- activated sludge respiration inhibition testing
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The study was conducted between 11 December 2014 and 12 December 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 015
- Report date:
- 2015
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Silicomanganese manufacturing fumes dedusting solid residues
- EC Number:
- 930-776-4
- IUPAC Name:
- Silicomanganese manufacturing fumes dedusting solid residues
- Test material form:
- other: granular solid
- Details on test material:
- Identification: SiMn fumes
Physical state/Appearance: dark brown granular solid
Batch: 04042012
Purity: not supplied
Expiry Date: not supplied
Storage Conditions: room temperature in the dark
Constituent 1
Sampling and analysis
- Analytical monitoring:
- not required
Test solutions
- Vehicle:
- no
- Details on test solutions:
- Test Water:
The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).
Synthetic Sewage:
A synthetic sewage of the following composition was added to each test vessel to act as a respiratory substrate:
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
dissolved in 1 liter of water with the aid of ultrasonication.
The pH of the synthetic sewage stock was 7.1. The pH value was measured using a Hach 160 handheld meter.
Range-Finding Test:
Test Item Preperation:
In the range-finding test activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dispersed directly in water.
Nominal amounts of test item (5, 50 and 500 mg (500 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at temperatures of between 20 °C and 22 °C, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).
The pH of the test item dispersions was measured after stirring using a Hach 160 handheld meter and adjusted to between pH 7.0 and 8.0.
The exposure conditions for each flask are described in 'details on test conditions' section.
The control group was maintained under identical conditions but not exposed to the test item.
Reference Item Preparation:
A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 15 minutes. The pH of this stock solution was measured to be pH 5.7 and was adjusted to pH 7.7 using 1.0 M NaOH. The pH values were measured using a Hach 160 handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to give the final concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.
The exposure conditions for each flask are described in 'details on test conditions' section.
Test organisms
- Test organisms (species):
- activated sludge of a predominantly domestic sewage
- Details on inoculum:
- A mixed population of activated sewage sludge micro-organisms was obtained on 12 December 2014 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
Prepearation of Inoculum:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and was used on the day of collection. The pH of the sample was 7.3 measured using a Hach 160 handheld meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L.
Study design
- Test type:
- static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 3 h
- Post exposure observation period:
- Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge.
Test conditions
- Test temperature:
- The test was conducted under normal laboratory lighting in a temperature controlled room between 20 and 22 ºC.
- pH:
- The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a Hach HQ40d Flexi handheld meter.
See results section for further details. - Dissolved oxygen:
- The oxygen concentrations in all vessels were measured after 30 minutes contact time.
See results section for further details. - Nominal and measured concentrations:
- Nominal test concentrations: 10, 100 and 1000 mg/L.
- Details on test conditions:
- Preparation of Test System:
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. The test item vessels were prepared as described in 'details on test solutions' section. Finally two further control vessels were prepared.
The test was conducted under normal laboratory lighting in a temperature controlled room between 20 and 22 ºC.
Evaluations:
Measurement of the Respiration Rates:
As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period. - Reference substance (positive control):
- yes
- Remarks:
- 3,5-dichlorophenol
Results and discussion
Effect concentrationsopen allclose all
- Duration:
- 3 h
- Dose descriptor:
- EC50
- Effect conc.:
- > 1 000 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- inhibition of total respiration
- Remarks:
- respiration rate
- Duration:
- 3 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 1 000 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- inhibition of total respiration
- Remarks:
- respiration rate
- Details on results:
- Range-finding test:
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L .
No statistically significant toxic effects were shown at all of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
The coefficient of variation of oxygen uptake in the control vessels was 4.1% and the specific respiration rate of the controls was 23.86 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.
The validation criterion for the reference item EC50 value was also satisfied.
In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace. - Results with reference substance (positive control):
- The following results were derived for 3,5-dichlorophenol:
3 hour EC10: 1.6 mg/L
3 hour EC 20: 2.4 mg/L
3 hour EC 50: 7.5 mg/L (95% Confidence Limits; 5.9 - 9.6 mg/L)
3 hour EC80: 23 mg/L
Any other information on results incl. tables
The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in the table below:
Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test
Nominal |
Dissolved Oxygen Concentration |
Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mg O2/L |
|
Control |
R1 |
6.8 |
76 |
|
R2 |
6.2 |
70 |
|
R3 |
6.6 |
74 |
|
R4 |
6.2 |
70 |
Test Item |
10 |
6.3 |
71 |
|
100 |
6.0 |
67 |
|
1000 R1 |
6.4 |
72 |
|
1000 R2 |
7.4 |
83 |
|
1000 R3 |
6.3 |
71 |
3,5-dichlorophenol |
3.2 |
7.0 |
79 |
|
10 |
7.6 |
85 |
|
32 |
8.2 |
92 |
R1– R4= Replicates 1 to 4
Oxygen consumption rates and percentage inhibition values for the control, test and reference items after 3 hours contact time are given in the table below:
Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Timein the Range-Finding Test
Nominal |
Initial O2 |
Measurement Period |
Final O2Reading |
O2Consumption Rates |
% |
|
Control |
R1 |
5.6 |
6 |
1.8 |
38.00 |
- |
|
R2 |
5.7 |
7 |
1.6 |
35.14 |
- |
|
R3 |
5.6 |
6 |
2.1 |
35.00 |
- |
|
R4 |
5.5 |
6 |
2.0 |
35.00 |
- |
Test Item |
10 |
4.9 |
5 |
1.8 |
37.20 |
[4] |
|
100 |
4.5 |
4 |
2.1 |
36.00 |
[1] |
|
1000 R1 |
5.4 |
6 |
1.8 |
36.00 |
[1] |
|
1000 R2 |
6.9 |
9 |
2.2 |
31.33 |
12 |
|
1000 R3 |
5.9 |
7 |
1.9 |
34.29 |
4 |
3,5-dichlorophenol |
3.2 |
6.6 |
10 |
2.2 |
26.40 |
26 |
|
10 |
7.5 |
10 |
5.2 |
13.80 |
61 |
|
32 |
7.8 |
10 |
7.0 |
4.80 |
87 |
[Increase in respiration rate as compared to controls]
R1– R4= Replicates 1 to 4
The pH values of the test preparations at the start and end of the exposure period are given in the table below:
pH Values of the Test Preparations at the Start and End of the Exposure Period in the Range-finding Test
Nominal |
pH |
||
0 Hours |
3 Hours |
||
Control |
R1 |
7.6 |
7.5 |
|
R2 |
7.6 |
7.6 |
|
R3 |
7.5 |
7.6 |
|
R4 |
7.5 |
7.6 |
Test Item |
10 |
7.6 |
7.5 |
|
100 |
7.5 |
7.6 |
|
1000 R1 |
7.5 |
7.6 |
|
1000 R2 |
7.5 |
7.8 |
|
1000 R3 |
7.5 |
7.6 |
3,5-dichlorophenol |
3.2 |
7.5 |
7.9 |
|
10 |
7.6 |
8.1 |
|
32 |
7.6 |
8.0 |
R1– R4= Replicates 1 to 4
Observations made on the test preparations throughout the range-finding test are given in the table below:
Observations on the Test Preparations throughout the Test Periodin the Range-Finding Test
Nominal Concentration (mg/L) |
Observations on Test Preparations |
|||
0 Hours* |
30 Minutes Contact Time |
3 Hours Contact Time |
||
Control |
R1 |
Pale yellow/brown dispersion |
Dark brown dispersion |
Dark brown dispersion |
|
R2 |
Pale yellow/brown dispersion |
Dark brown dispersion |
Dark brown dispersion |
|
R3 |
Pale yellow/brown dispersion |
Dark brown dispersion |
Dark brown dispersion |
|
R4 |
Pale yellow/brown dispersion |
Dark brown dispersion |
Dark brown dispersion |
Test Item |
10 |
Pale yellow/brown dispersion, test item visible settled on bottom of flask |
Dark brown dispersion, no test item visible |
Dark brown dispersion, no test item visible |
|
100 |
Pale orange/brown dispersion, test item visible settled on bottom of flask |
Dark brown dispersion, test item visible settled on bottom of flask |
Dark brown dispersion, test item visible settled on bottom of flask |
|
1000 R1 |
Brown dispersion, test item visible settled on bottom of flask |
Dark brown dispersion, test item visible settled on bottom of flask |
Dark brown dispersion, test item visible settled on bottom of flask |
|
1000 R2 |
Brown dispersion, test item visible settled on bottom of flask |
Dark brown dispersion, test item visible settled on bottom of flask |
Dark brown dispersion, test item visible settled on bottom of flask |
|
1000 R3 |
Brown dispersion, test item visible settled on bottom of flask |
Dark brown dispersion, test item visible settled on bottom of flask |
Dark brown dispersion, test item visible settled on bottom of flask |
3,5-dichlorophenol |
3.2 |
Pale yellow/brown dispersion, no undissolved reference item visible |
Dark brown dispersion, no undissolved reference item visible |
Dark brown dispersion, no undissolved reference item visible |
|
10 |
Pale yellow/brown dispersion, no undissolved reference item visible |
Dark brown dispersion, no undissolved reference item visible |
Dark brown dispersion, no undissolved reference item visible |
|
32 |
Pale yellow/brown dispersion, no undissolved reference item visible |
Dark brown dispersion, no undissolved reference item visible |
Dark brown dispersion, no undissolved reference item visible |
*Observations made prior to the addition of activated sewage sludge
R1– R4= Replicates 1 to 4
Applicant's summary and conclusion
- Validity criteria fulfilled:
- yes
- Conclusions:
- The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.
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