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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:
04 October 2012 to 14 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test material was dispersed directly in water. Due to its light sensitive nature the test material was weighed out under non-actinic lighting and all test vessels were shielded from light with foil.
Amounts of test material (5, 50 and 500 mg (in triplicate)) 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.
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: 13 November 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 (450 mL).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C
pH:
7.2 - 7.9
Dissolved oxygen:
4.6 - 6.7 mg O2/L
Nominal and measured concentrations:
- Nominal concentrations: 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL conical flask
- Material, size, headspace, fill volume: 500 mL; 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added
- Aeration: Yes; the mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1.0 litre per minute
- No. of vessels per concentration (replicates): 1 replicate at the 10 and 100 mg/L nominal concentration; 3 replicates at the 1000 mg/L nominal concentration
- No. of vessels per control (replicates): 4
- Suspended solids concentration of activated sludge: 3.0 g/L prior to use.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used was deionised reverse osmosis water
- Total organic carbon: Less than 1 mg/L Dissolved Organic Carbon (DOC).
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of the control, reference material and test material preparations was measured at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours of contact time. The oxygen concentrations in all vessels were measured after 30 minutes of contact time.

OTHER 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 and 2.8 g K2HPO4.
- Adjustment of pH: Yes. The pH of the test material dispersions was measured after stirring and adjusted to between pH 7.0 and 8.0 if necessary.
The pH of the synthetic sewage stock used to feed the activated sewage sludge was 7.1 and the pH of the synthetic sewage stock used for the test was pH 6.9 and adjusted to 7.2 using 1.0 M NaOH.
- Photoperiod: The test vessels were shielded from light with foil due to the light sensitive nature of the test material.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
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 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.
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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: Yes; the study was carried out as a range-finding test. Based on the results it was considered to be unnecessary to carry out a definitive test.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
>= 100 - <= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Oxygen consumption rates and percentage inhibition values for the control, test and reference materials are given in Table 1.
The dissolved oxygen concentrations after 30 minutes contact time in some of the vessels were below 60 % of the dissolved oxygen saturation level of 8.9 mg O2/L. 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.
No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L, however statistically significant toxic effects were shown at the test concentration of 1000 mg/L. Based on these results, it was considered unnecessary to perform a definitive test in order to obtain an exact NOEC and an EC50 value for the test material.

The following results were derived:
EC20: 100 - 1000 mg/L
EC50: 100 - 1000 mg/L
EC80: >100 mg/L
NOEC: 100 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 31.00 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 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.

There was a relatively large increase in respiration rate observed in the 10 and 100 mg/L test vessels and this was considered to be due to the possible hormetic response of the activated sewage sludge micro-organisms to the test material, i.e. the stimulation of biological activity due to the presence of test material at concentrations below its toxic level.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: The EC50 value was determined to be 9.7 mg/L with 95 % confidence limits of 8.0 - 12 mg/L
- Other: The reference material was included at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. Aliquots of a 0.5 g/L stock solution (3.2, 10 and 32 mL) were 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 material was inverted several times to ensure homogeneity of the solution.
Reported statistics and error estimates:
The percentage inhibition values were plotted against concentration for the reference material only, a line fitted using the Xlfit software package (IDBS) and the EC20, EC50 and EC80 values determined from the equation for the fitted line.
The EC20, EC50 and EC80 values for the test material were determined by inspection of the inhibition of respiration rate data.
95 % confidence limits were calculated for the reference material EC50 values using the method of Litchfield and Wilcoxon (1949).
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Table 1: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours of Contact Time

Nominal Concentration (mg/L)

Replicate

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

4.0

3

1.8

44.00

-

R2

4.6

3

2.3

46.00

-

R3

4.2

3

1.8

48.00

-

R4

3.0

1

2.2

48.00

-

10

-

3.7

2

2.0

51.00

[10]

100

-

4.0

2

2.0

60.00

[29]

1000

R1

7.8

10

6.8

6.00

87

R2

7.7

10

6.9

4.80

90

R3

8.1

10

7.2

5.40

88

3,5-DCP 3.2

-

5.2

5

2.0

38.40

17

3,5-DCP 10

-

6.8

10

3.2

21.60

54

3,5-DCP 32

-

6.9

10

5.5

8.40

82

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

R1 - 4 = Replicates 1 - 4

3,5-DCP = 3,5-Dichlorophenol

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the effect of the test material on the respiration of activated sewage sludge gave a 3-hour EC50 value of 100 to 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours of exposure was 100 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 under GLP conditions.

Activated sewage sludge was exposed to an aqueous dispersion of the test material in the dark at concentrations of 10, 100 and 1000 mg/L (3 replicates at 1000 mg/L) 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 of contact time and compared to data for the control and a reference material (3,5-dichlorophenol).

No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L, however statistically significant toxic effects were shown at the test concentration of 1000 mg/L. Based on these results, it was considered unnecessary to perform a definitive test in order to obtain an exact EC50 value for the test material. The validity criteria for the test were fulfilled.

Under the conditions of this study, the effect of the test material on the respiration of activated sewage sludge gave a 3-hour EC50 value of 100 to 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours of exposure was 100 mg/L.

Description of key information

3-hour EC50 value of 100 to 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours of exposure was 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 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 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria of Klimisch (1997).

Activated sewage sludge was exposed to an aqueous dispersion of the test material in the dark at concentrations of 10, 100 and 1000 mg/L (3 replicates at 1000 mg/L) 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 of contact time and compared to data for the control and a reference material (3,5-dichlorophenol).

No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L, however statistically significant toxic effects were shown at the test concentration of 1000 mg/L. Based on these results, it was considered unnecessary to perform a definitive test in order to obtain an exact EC50 value for the test material. The validity criteria for the test were fulfilled.

Under the conditions of this study, the effect of the test material on the respiration of activated sewage sludge gave a 3-hour EC50 value of 100 to 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours of exposure was 100 mg/L.