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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 25 September 2012. Experimental completion date: 26 September 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Amounts of test item (in triplicate) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 min followed by magnetic stirring for 24 h 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).
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 °C overnight prior to use in the test. On the day of collection the activated sewage sludge (5 L) was fed synthetic sewage sludge (250 mL). The pH of the sample on the day of the test was 7.9 measured using a WTW pH/Oxi 3401 pH and dissolved oxygen 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 GFIA 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 °C for at least 1 h 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 prior to use.

*rinsed 3 times with 20 mL deionized reverse osmosis water prior to drying in the oven
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C.
pH:
7.3 - 84

The pH of the test item dispersions was measured after stirring using a WTW pH/Oxi 3401 pH and dissolved oxygen meter and adjusted to between pH 7.0 and 8.0 if necessary.
Dissolved oxygen:
5.6 - 7.3 mg O2/L
Nominal and measured concentrations:
Nomical test concentrations: 10, 100 and 1000 mg/L
Amount of test item: 5, 50 and 500 mg
Details on test conditions:
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 approximately 0.5 - 1 L per min. Thereafter, at 15 min 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.

As each vessel reached 3 h 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 7.0 mg 02/L and 2.0 mg 02/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 min period.

The test was conducted under normal laboratory lighting in a temperature controlled room.

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 pH of the control, reference item and test item preparations was measured at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.


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 CaC12.2H20
0.2 g MgS04.7H20
2.8 g K2HP04
dissolved in 1 L of water with the aid of ultrasonication.
The pH of the synthetic sewage stock used to feed the activated sewage sludge was pH 7.0 and the pH of the synthetic sewage stock used for the test was pH 7.0. The pH values were measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
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:
The dissolved oxygen concentrations after 30 min contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg 02/L.

The coefficient of variation of oxygen uptake in the control vessels was 3.8% and the specific respiration rate of the controls was 29.17 mg oxygen per gram dry weight of sludge per hour. The validation criteria was therefore been satisfied.

The validation criterion for the reference item ECS0 value was also satisfied.

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7.0 mg 02/L and 2.0 mg 02/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.
Reported statistics and error estimates:
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.

The following results were derived:

 

Test item

Reference substance

ECx (3 Hours)

(mg/L)

95 % confidence limits (mg/L)

ECx (3 Hours)

(mg/L)

95 % confidence limits (mg/L)

EC20

>1000

-

3.4

-

EC50

>1000

-

11

8.5 - 14

EC80

>1000

-

33

-

NOEC

1000

-

-

-

 

It was considered unnecesary and unrealistic to test at concentrations in excess of 1000 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge micro-organisms was assessed according to OECD Guideline 209, The 3-Hour EC50 value was determined to be > 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 h exposure was 1000 mg/L.

Description of key information

The effect of the test item on the respiration of activated sewage sludge micro-organisms was assessed according to OECD Guideline 209, The 3-Hour EC50 value was determined to be > 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 h exposure was 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

Introduction

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) ".

Methods

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 temperature of 20 ± 2 °C 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.

Results

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

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

The reference item gave a 3-Hour EC50value of 11 mg/L, 95% confidence limits 8.5 - 14 mg/L.