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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 August 2016 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
CAS RN: 15337-18-5
Description: yellow viscous liquid
Purity: 96.6%
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Nominal amounts of test item (5, 50 and 500 mg) were each separately smeared onto GF/A filter paper (70 mm) prior to being 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 ºC and 21 ºC, to maximize the dissolved test item concentration.

Single test vessel replicates were prepared for the two lower concentrations and three replicates were prepared for the highest 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 of the 1000 mg/L test concentration).

The pH of the test item dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 and 8.0, as necessary.

A filter paper (GF/A filter paper, 70 mm) was added to each control vessel to ensure consistency between the test and control vessels. The control group was maintained under identical conditions, but was not exposed to the test item.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on 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 7.8 measured using a Hach HQ40d Flexi 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 two consecutive dry weights within 4% were attained. The suspended solids concentration was equal to 3.0 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
The temperature recorded in the first and last control vessels were measured to be 22 ºC.
pH:
Adjusted to between pH 7.0 and 8.0.
Dissolved oxygen:
Ranged from 5.9 to 8.5 mg O2/L.
Nominal and measured concentrations:
10, 100 and 1000 mg/L
Details on test conditions:
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.
Finally two further control vessels were prepared. The addition of the inoculum to the reaction mixture was considered the start of the 3-hour exposure period for each vessel.

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 a final volume of 1 liter of deionized reverse osmosis water with the aid of ultrasonication. The pH of the synthetic sewage stock was 7.4.

Observations
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. The temperature was recorded in one of the control vessels at the start and end of the incubation.

pH Measurements
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.

Oxygen Concentration
The oxygen concentrations in all vessels were measured after 30 minutes contact time.

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 was measured as dissolved oxygen. 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 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
Key result
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
Key result
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
Details on results:
No statistically significant toxic effects were shown at any of the test concentrations employed (P≥0.05). It was therefore considered justifiable not to perform a definitive test. It was considered unnecessary to test at concentrations in excess of 1000 mg/L.
Results with reference substance (positive control):
The EC50 value for the reference item satisfied the validation criterion.

Validation Criteria

The coefficient of variation of oxygen uptake in the control vessels was 5.23% and the specific respiration rate of the controls was 27.97 mg oxygen per gram dry weight of sludge per hour.

Results

Inhibition of Respiration Rate

 

Test Item

3,5-dichlorophenol

ECx(3 Hours)
(mg/L)

95% Confidence Limits (mg/L)

ECx(3 Hours)
(mg/L)

95% Confidence Limits (mg/L)

EC10

>1000

-

1.5

-

EC20

>1000

-

2.2

-

EC50

>1000

-

7.2

5.5 - 9.4

EC80

>1000

-

23

-

NOEC

1000

-

-

-

 

Validity criteria fulfilled:
yes
Conclusions:
No effects of the test item were observed on the respiration of activated sewage sludge following a 3-hour exposure, resulting in an EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) following the 3-hour exposure was 1000 mg/L.
Executive summary:

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010), No. 209, "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a measured temperature of approximately 22 ºC with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

No effects of the test item were observed on the respiration of activated sewage sludge following a 3-hour exposure, resulting in an EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) following the 3-hour exposure was 1000 mg/L.

It was considered unnecessary to test at concentrations in excess of 1000 mg/L.

The 3-hour EC50 value for the reference item, 3,5-dichlorophenol, was determined to be 7.2 mg/L, with 95% confidence limits of 5.5 to 9.4 mg/L.

Description of key information

No effects of the test item were observed on the respiration of activated sewage sludge following a 3-hour exposure, resulting in an EC50 value of greater than 1000 mg/L.  The No Observed Effect Concentration (NOEC) following the 3-hour exposure was 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information