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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 September 2012 - 12 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test water used was deionised reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
INOCULUM
Type: a mixed population of activated sewage sludge micro-organisms.
Source: the aeration stage of the Severn Trent Water PLC sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
Date of collection: 11 September 2012.
Preparation: 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 (10 litres) was fed synthetic sewage sludge (500 mL).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
Not reported.
Test temperature:
20 ± 2 °C
pH:
7.5 - 8.3
Dissolved oxygen:
4.5 - 7.6 mg O2/L
Nominal and measured concentrations:
The nominal concentrations used in the test were 10, 100 and 1000 mg/L. Three replicates were employed at the 1000 mg/L concentration level.
Details on test conditions:
SYNTHETIC SEWAGE
A synthetic sewage was added to each test vessel to act as a respiratory substrate, containing the following components dissolved in 1 litre of water with the aid of ultrasonication:
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

The pH of the synthetic sewage stock used to feed the activated sewage stock was 7.2 and the pH of the synthetic sewage sludge used in the test was 7.1. The pH values were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

RANGE-FINDING TEST
Amounts of test material (5, 50 and 500 mg) were each separately dispersed in approximately 200 mL of deionised reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours in order to maximise the dissolved test material 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.
The pH of the test material dispersions was measured after stirring and adjusted to between pH 7.0 and 8.0 if necessary.
The control group was maintained under identical conditions but not exposed to the test material.

PREPARATION OF INOCULUM
The pH of the sample on the day of the test was 7.5. 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 (which had been rinsed three times with 20 mL deionised reverse osmosis water prior to drying in an oven) using a Buchner funnel which was then rinsed 3 times with 10 mL of deionised 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 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 prior to use.

PREPARATION OF TEST SYSTEM
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 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference material vessels and the test material vessels. Finally two further control vessels were prepared.

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 7.0 and 2.0 mg O2/L). In the case of 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.

Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge. The pH of the control, reference and test material preparations was measured at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time. The oxygen concentrations in all vessels were measured after 30 minutes contact time.
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:
RANGE-FINDING TEST
Oxygen consumption rates and percentage inhibition values for the control, test and reference materials are given in Table 1. The pH values of the test preparations at the start and end of the exposure period are given in Table 2.

No statistically significant toxic effects were shown at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.

The following results were derived:
EC20: >1000 mg/L
EC50: >1000 mg/L
EC80: >1000 mg/L
NOEC: 1000 mg/L
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

The dissolved oxygen concentrations after 30 minutes contact time were less than 60 to 70 % of the dissolved oxygen saturation level of 8.9 mg O2/L with the exception of the control replicate R1 and reference material vessels. This deviation was considered to have had no adverse effect on the study given that all oxygen consumption values were measured/calculated over the linear portion of the traces.

The coefficient of variation of oxygen uptake in the control vessels was 6.3 % and the specific respiration rate of the controls was 26.2 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7.0 mg O2/L and 2.0 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:
EC20: 2.7 mg/L
EC50: 9.0 mg/L (95 % confidence limits 7.2 - 11 mg/L)
EC80: 30.4 mg/L

The validation criteria for the reference material were satisfied.

Table 1 Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)

Initial O2 Reading (mg O2/L)

Measurement Period (minutes)

Final O2 Reading (mg O2/L)

O2 Consumption Rates (mg O2/L/hour)

% Inhibition

 

Control

R1

4.7

4

1.9

42.00

-

R2

5.5

5

2.2

39.60

-

R3

5.1

5

1.8

39.60

-

R4

5.9

7

1.7

36.00

-

 

Test

Material

10

5.3

5

1.9

40.80

[4]

100

4.8

4

2.1

40.50

[3]

1000 R1

4.5

4

1.8

40.50

[3]

1000 R2

5.4

5

1.9

42.00

[7]

1000 R3

5.5

5

2.1

40.80

[4]

Reference

Material

3.2

6.8

10

1.8

30.00

24

10

7.0

10

3.9

18.60

53

32

7.6

9

6.5

7.33

81

[ ] = increase in respiration rate as compared to the controls

R1 - 4 = Replicates 1 - 4

 

Table 2 pH Values of the Test Preparations at the Start and End of the Exposure Period in the Range-Finding Test

Nominal Concentration (mg/L)

pH

0 Hours

3 Hours

 

Control

R1

7.6

8.2

R2

7.5

8.2

R3

7.7

8.1

R4

7.5

8.2

 

Test

Material

10

7.6

8.1

100

7.7

8.1

1000 R1

7.7

8.1

1000 R2

7.5

8.1

1000 R3

7.6

8.0

Reference

Material

3.2

7.6

8.3

10

7.5

8.3

32

7.6

8.3

Validity criteria fulfilled:
yes
Conclusions:
The 3 hour EC50 was >1000 mg/L. The 3 hour NOEC was 1000 mg/L.
Executive summary:

The potential of the test material to cause toxic effects to microorganisms was investigated in accordance with the standardised guideline OECD 209.

In a 3 hour toxicity study, activated sewage sludge from a plant treating predominantly domestic sewage was exposed to the test material. In a range-finding study, the sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 100 and 1000 mg/L (3 replicates at the highest concentration) 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 a blank control and reference material.

The effect of the test material on the respiration of activated sewage sludge microorganisms gave a 3 hour EC50 > 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; no toxic effects were seen at any of the test concentrations employed and it was therefore considered justifiable not to perform a definitive test.

Description of key information

The 3 hour EC50 > 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours 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

The potential of the test material to cause toxic effects to microorganisms was investigated in accordance with the standardised guideline OECD 209. The study was conducted in line with GLP and was well reported. It was therefore awarded a reliability score of 1 in accordance with the criteria of Klimisch (1997).

In a 3 hour toxicity study, activated sewage sludge from a plant treating predominantly domestic sewage was exposed to the test material. In a range-finding study, the sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 100 and 1000 mg/L (3 replicates at the highest concentration) 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 a blank control and reference material.

The effect of the test material on the respiration of activated sewage sludge microorganisms gave a 3 hour EC50 > 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; no toxic effects were seen at any of the test concentrations employed and it was therefore considered justifiable not to perform a definitive test.