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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:
25 Feb - 26 Feb 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
GLP compliance:
yes (incl. certificate)
Remarks:
With the exception that no analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Amounts of test item (5, 16, 50, 160 and 500 mg) were each separately dispersed in approximately 250 mL of water and subjected to ultrasonication for approximately 15 min followed by prolonged mixing for 24 h in order to maximise the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (200 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 32, 100, 320 and 1000 mg/L.
- Differential loading: yes
- Controls: The control group was maintained under identical conditions but not exposed to the test item.
- Reference substance: Two stock solutions of 50 and 160 mg/L were prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 15 minutes. Aliquots (10 and 100 mL) of the 160 mg/L stock solution were removed and dispersed with activates sewage sludge, synthetic sewage and water to give the final concentrations of 3.2 and 32 mg/L. Similarly, a 100 mL aliquot of the 50 mg/L stock solution was used to prepare the 10 mg/L concentration. The volumetric flasks containing the reference item were inverted several times to ensure homogeneity of the solutions.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum: Obtained on 26 Feb 2010 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Pretreatment: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and was used on the day of collection. The pH of the sample was 7.4 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 GF/A filter paper using a Buchner tunnel which was then rinsed 3 times with 10 ml of deionised reverse osmosis water and filtration continued tor 3 minutes. The filter paper was then dried in an oven at approximately 105°C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.8 g/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
21 +/- 1°C
pH:
7.3 - 7.9 (at the beginning of the test)
7.8 - 8.4 (after 3 hours)
Nominal and measured concentrations:
Nominal: control, 10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL darkened glass Biological Oxygen Demand (BOD) bottle)
- Aeration: clean, oil-free compressed air at a rate of approx. 0.5 - 1 L/min
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water a total hardness of approx. 140 mg/L as CaCO3.

OTHER TEST CONDITIONS
- Light intensity: normal laboratory lighting


EFFECT PARAMETERS MEASURED: oxygen consumption
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (Sigma-Aldrich Batch No. 05910HJ)
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
15 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:
EC50
Effect conc.:
27 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 22 - 33 mg/L
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
83 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.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50: 8.1 mg/L (95% CL: 6.4 - 10 mg/L)
Reported statistics and error estimates:
The percentage inhibition values were plotted against concentration, a line fitted using the XIfit software package (IDBS) and the EC15, EC20, EC50 and EC80 values determined from the equation for the fitted line. 95% CL were calculated for the EC50 values using the method of Litchfield and Wilcoxon.

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of 27 mg/L, 95% confidence limits 22 - 33 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 13 mg/L.

Table 1: Oxygen consumption rates and percentage inhibition values after 3 hours contact time

Nominal concentration [mg/L]

Initial O2 reading

[mg O2/L]

Measurement period [min]

Final O2 reading

[mg O2/L]

O2 Consumption rates

[mg O2/L/min]

% inhibition

Control

R1

6.5

8

2.8

0.46

-

 

R2

6.1

8

2.9

0.40

-

Test item

10

6.0

8

2.5

0.44

[2]

 

32

7.2

10

5.3

0.19

56

 

100

8.2

10

7.5

0.07

84

 

320

8.2

10

7.7

0.05

88

 

1000

8.6

10

8.4

0.02

95

3,5-dichlorophenol

3.2

7.1

10

4.0

0.31

28

 

10

7.8

10

5.9

0.19

56

 

32

8.3

10

7.5

0.08

81

[] Increase in respiration rate as compared to controls

Observations made at 0 hours, 30 minutes and 3 hours showed that the control vessels contained a dark brown dispersion and the reference item vessels contained a dark brown dispersion with no undissolved reference item visible.

Observations made of the test item vessels at 0 hours prior to the addition of activated sewage sludge and synthetic sewage showed that the test concentration of 10 mg/L contained a clear colourless solution and the test concentration of 32 mg/L contained a very slightly cloudy dispersion with no undissolved test item visible. The test concentration of 100 mg/L contained a slightly cloudy dispersion with no undissolved test item visible and the test concentrations of 320 and 1000 mg/L contained cloudy dispersions with no undissolved test item visible. Observations made of the test item vessels after 30 minutes and 3 hours contact time showed that the test concentration of 10 mg/L contained a dark brown dispersion with no undissolved test item visible and the test concentrations of 32 and 100 mg/L contained dark brown dispersions with foam on the surface and no undissolved test item visible. The test concentrations of 320 and 1000 mg/L contained dark brown/grey dispersions with foam on the surface and no undissolved test item visible.

Description of key information

NOEC (3 h): 13 mg/L for activated sludge microorganisms (OECD 209)

Key value for chemical safety assessment

EC50 for microorganisms:
27 mg/L
EC10 or NOEC for microorganisms:
13 mg/L

Additional information

One study investigated the effects of the test substance Atmer 163 (CAS No. 97925-95-6) to activated sludge microorganisms (Clarke, 2010). The test was performed according to the OECD Guideline 209 (GLP). Activated sewage sludge from a domestic sewage treatment plant was used as inoculum and exposed to nominal concentrations of 10, 32, 100, 320 and 1000 mg/L. The study resulted in an EC50 (3 h) of 27 mg/L (95% CL: 22 - 33 mg/L) based on the respiration of microorganisms in activated sewage sludge and in a NOEC (3 h) of 13 mg/L. It can be assumed that the test substance has toxic effects to microorganisms in activated sewage sludge.