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Toxicity to microorganisms

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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 24, 2003 to December 04, 2003
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)
Specific details on test material used for the study:
- Purity: 49.9% of test substance in water.
Analytical monitoring:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Nature: secondary activated sludge
- Source: Nieuwgraaf in Duiven, The Netherlands, a sewage treatment plant treating predominantly domestic sewage
- Sampling site: Sewage treatment plant
- Laboratory culture: No, the activated sludge was directly used after getting it from the sewage treatment plant.
- Preparation of inoculum for exposure: Prior to use it was homogenised with a syringe
- Pretreatment: No
- Initial cell concentration: The dry weight of the activated sludge in the incubation vessel was 1.5 g/L
Test type:
other: biodegradation
Water media type:
freshwater
Total exposure duration:
28 d
Test temperature:
20°C
pH:
7.1 - 7.3
Nominal and measured concentrations:
Not specified
Details on test conditions:
- Organic substrate: The soil was amended with powdered alfalfa meal (Medicago sativa), 5 g of alfalfa meal per kg of soil (dry weight).
- Aeration of dilution water: No
- Suspended solids concentration: The dry weight of the activated sludge in the incubation vessels was 1.5 g/L
- Method of soil incubation: individual subsamples
- Aeration: No
Reference substance (positive control):
no
Key result
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
11 mg/L
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: calculated
Duration:
30 min
Dose descriptor:
other: EC20
Effect conc.:
5 mg/L
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: calculated
Duration:
30 min
Dose descriptor:
other: EC80
Effect conc.:
24 mg/L
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: calculated
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
4 mg/L
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The EC50 of the test substance for activated sludge was 11 mg a.i./L with 95% confidence limits of 3 and 27 mg a.i./L. The EC10, EC20 and EC80 were 4, 5 and 24 mg a.i./L, respectively.
Results with reference substance (positive control):
The EC50 of the reference compound 3,5-dichlorophenol (5.2 mg/L) was within the prescribed range of 5 to 30 mg/L.
Reported statistics and error estimates:
The EC values were computed from the best fitted line (least square method) through the points given by the probit of the percentage inhibition and the logarithm of the concentration of the test substance. The EC10, 25, 50 values were computed from the best fitted line (least-squares method) through the points given by the probit of the percentage inhibition and the logarithm of the concentration of the test substance. Confidence limits were computed on the basis of Fieller's theorem. All computations were performed using the TOXCALC version 5.0 program.
Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the 3h EC50 of the test substance for activated sludge was 11 mg a.i./L (with 95% confidence limits of 3 and 27 mg a.i./L). The EC10, EC20 and EC80 were 4, 5 and 24 mg a.i./L, respectively. The test substance was therefore considered harmful to activated sludge.
Executive summary:

A study was conducted to determine the toxicity to microorganisms of the test substance according to OECD Guideline 209, in compliance with GLP. The respiration inhibition test was performed on activated sludge fed with a standard amount of synthetic sewage. The respiration rate of the same activated sludge in the presence of various concentrations of the test substance under identical conditions was also measured. The inhibitory effect of the test substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. The validity of the test was shown by two criteria. First, the control respiration rates were within 15% of each other and secondly, the EC50 of the reference compound 3, 5-dichlorophenol (5.2 mg/L) was within the prescribed range of 5 to 30 mg/L. No analytical dose determination was performed. Under the study conditions, the 3 h EC50 of the test substance for activated sludge was 11 mg a.i./L (with 95% confidence limits of 3 and 27 mg a.i./L). The 3 h EC10, EC20 and EC80 were 4, 5 and 24 mg a.i./L, respectively (Geerts, 2004).

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 12, 2000 to June 20, 2000
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)
Specific details on test material used for the study:
- Physical state: Pale yellow soloured liquid
- Analytical purity: 49 to 51%
- Lot/batch No.: K 9225906
- Storage condition of test material: Room temperature
Analytical monitoring:
no
Details on test solutions:
A stock solution of the test was prepared by dissolving 2.0008 g of the test substance in 1,000 mL of BOD dilution water.
A stock solution of the reference substance was prepared according to OECD guideline no 209.

The pH of the sludge suspension was found to be 7.1 before use. Control mixtures were prepared by combining 16 mL of synthetic sewage feed with the appropriate volumes of the test substance stock solution and dilution water to a volume of 300 mL in one litre glass beaker.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: Municipal oxidation ditch, which is used to treat the domestic sewage (Hazerwoude Dorp, the Netherlands)
- Preparation of inoculum for exposure: Approximately 20 litres of activated sludge was taken from the municipal oxidation ditch, which is used to treat the domestic sewage. The sample was centrifuged and the supernatant discarded. The activated sludge was washed thrice by centrifugation and re-suspension in tap water twice and once in dilution water. The dry weight of the sludge suspension was determined. The mixed liquor suspended solids content was found to be 7.1 g/L.
- Initial biomass concentration: The dry weight was checked prior to use in the test and was found at a mixed liquor suspended solids level of 3.7 g/L.
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
3 h
Test temperature:
21.9 - 23°C
pH:
7.6 - 7.8
Nominal and measured concentrations:
Nominal concentrations: 0, 3.2, 10, 32, 100, 320 and 1,000 mg/L
Details on test conditions:
A stock solution of the test was prepared by dissolving 2.0008 g of the test substance in 1,000 mL of BOD dilution water.
A stock solution of the reference substance was prepared according to OECD guideline no 209.

The pH of the sludge suspension was found to be 7.1 before use. Control mixtures were prepared by combining 16 mL of synthetic sewage feed with the appropriate volumes of the test substance stock solution and dilution water to a volume of 300 mL in one litre glass beaker.

The test was started by adding 200 mL of sludge suspension to the mixtures with and without test substance. The incubation of mixtures was started at the intervals of 12 min and the first and last sample were controls without the test substance. The mixtures were aerated vigorously and incubated at the temperature between 21.9 and 23°C for 3h. After 3h, a sample of each mixture was poured into a BOD bottle of and stirred vigorously. An oxygen electrode (connected to a WTW OXI 2000 meter) was inserted into the sample and oxygen concentration was recorded every minute during a period of about 10 min.

Simultaneously, three concentrations (5, 12 and 30 mg/L) of the reference substance 3,5-dichlorophenol were tested. Each concentration of reference was diluted with dilution water to 284 mL, mixed with 16 mL of synthetic sewage feed and 200 mL sludge suspension.

The BOD dilution water was prepared according to NEN Standard No. 6634.
The synthetic sewage feed was prepared according to OECD guideline no. 209.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
7.75 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% confidence limits of 6.35 and 9.5 mg a.i./L
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1.6 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: calculated
Details on results:
The EC50 of the test substance for activated sludge was 7.75 mg a.i./L with 95% confidence limits of 6.35 and 9.5 mg a.i./L. The EC20 and EC80 were 3.4 and 17.8 mg a.i./L, respectively. The NOEC of the test substance was found to be 1.6 mg a.i./L.
Results with reference substance (positive control):
- Results with reference substance valid: yes
- Relevant effect levels: The 3-h EC50 of reference substance was found to be 5 mg/L, which is in the range prescribed by OECD guideline (range of 5-30 mg/L).

The respiration rates of two controls of the test were found to be 13.69 and 14.75 mg O2/g/h, respectively. The difference between the two values amounted to 3.8% being within the validity criterion (15%) of OECD guideline. Further, the 3-h EC50 of reference substance was found to be 5 mg/L, which is in the range prescribed by OECD guideline (range of 5-30 mg/L).

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the EC50 of the test substance for activated sludge was 7.75 mg a.i./L. The EC20 and EC80 were 3.4 and 17.8 mg a.i./L, respectively. The NOEC of the test substance was found to be 1.6 mg a.i./L.
Executive summary:

A study was conducted to determine the toxicity to microorganisms of the test substance (49 -51% purity) according to OECD Guideline 209, in compliance with GLP. A mixture of activated sludge, synthetic sewage feed and a range of concentrations of the test or reference substance were prepared in 1 L glass beakers. The mixtures were aerated and, after an incubation period of 3 h at a temperature between 21.9 and 23°C, the decrease in the oxygen concentration in the mixtures was recorded during a period of 10 min. The inhibitory effect of the test substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. The substance was tested at nominal concentrations of 0, 1, 3.2, 10, 32, 100, 320 and 1000 mg/L (equivalent to ca. 0, 0.5, 1.6, 5.0, 16, 50, 160 and 500 mg a.i./L) . A control test with reference substance 3,5-dichlorophenol yielded an EC50 value of 5 mg/L, which is within the range of prescribed by guideline. The validity criteria of the guideline were fulfilled. No analytical dose determination was performed. Under the study conditions, the 3 h EC50 was 7.75 mg a.i./L. The 3 h EC20 and EC80 were 3.4 and 17.8 mg a.i./L, respectively. The 3 h NOEC was found to be 1.6 mg a.i./L (Mayer, 2011).

Description of key information

Based on inhibition of total respiration, the 3 h EC50 and NOEC values due to the test substance were determined to be 7.75 and 1.6 mg a.i./L (nominal) respectively

Key value for chemical safety assessment

EC50 for microorganisms:
7.75 mg/L
EC10 or NOEC for microorganisms:
1.6 mg/L

Additional information

Study 1. A study was conducted to determine the toxicity to microorganisms of the test substance according to OECD Guideline 209, in compliance with GLP. The respiration inhibition test was performed on activated sludge fed with a standard amount of synthetic sewage. The respiration rate of the same activated sludge in the presence of various concentrations of the test substance under identical conditions was also measured. The inhibitory effect of the test substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. The validity of the test was shown by two criteria. First, the control respiration rates were within 15% of each other and secondly, the EC50 of the reference compound 3, 5-dichlorophenol (5.2 mg/L) was within the prescribed range of 5 to 30 mg/L. No analytical dose determination was performed. Under the study conditions, the 3 h EC50 of the test substance for activated sludge was 11 mg a.i./L (with 95% confidence limits of 3 and 27 mg a.i./L). The 3 h EC10, EC20 and EC80 were 4, 5 and 24 mg a.i./L, respectively (Geerts, 2004).

Study 2. A study was conducted to determine the toxicity to microorganisms of the test substance (49 -51% purity) according to OECD Guideline 209, in compliance with GLP. A mixture of activated sludge, synthetic sewage feed and a range of concentrations of the test or reference substance were prepared in 1 L glass beakers. The mixtures were aerated and, after an incubation period of 3 h at a temperature between 21.9 and 23°C, the decrease in the oxygen concentration in the mixtures was recorded during a period of 10 min. The inhibitory effect of the test substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. The substance was tested at nominal concentrations of 0, 1, 3.2, 10, 32, 100, 320 and 1000 mg/L (equivalent to ca. 0, 0.5, 1.6, 5.0, 16, 50, 160 and 500 mg a.i./L) . A control test with reference substance 3,5-dichlorophenol yielded an EC50 value of 5 mg/L, which is within the range of prescribed by guideline. The validity criteria of the guideline were fulfilled. No analytical dose determination was performed. Under the study conditions, the 3 h EC50 was 7.75 mg a.i./L. The 3 h EC20 and EC80 were 3.4 and 17.8 mg a.i./L, respectively. The 3 h NOEC was found to be 1.6 mg a.i./L (Mayer, 2011).