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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
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on test solutions:
The test substance and 3,5-dichlorophenol were added to the incubation vessels using stock solutions of 0.25 g/L and 0.5 g/L, respectively. The stock solution of 3,5-dichlorophenol was prepared by dissolving 0.05g substance in 1 mL of 1M NaOH, then diluting this to approximately 30 mL with deionised water, adding under stirring 0.5M H2SO4 to the point off incipient precipitation and finally diluting the mixture to 100mL with deionised water.
The test substance stock was prepared by weighing 0.05g in a measuring flask and diluting it to 100mL. The concentration of the active ingredient in the stock solution was 0.25g/L
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:
static
Water media type:
freshwater
Total exposure duration:
30 min
Test temperature:
20°C
pH:
7.1 - 7.3
Nominal and measured concentrations:
Not specified
Details on test conditions:
In the test, the respiration rate of activated sludge fed with a standard amount of synthetic sewage was measured. The respiration rate of the same activated sludge in the presence of various concentrations of the test substance under otherwise 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 if the cintrols. Somce aspects of the test are listed below:
- the test compound and the reference compound concentrations were spaced by a factor of two
- the contact time was 30 minutes

The respiration rates of the activated sludge were measured un a Biological Oxygen Monitor (BOM). The dry weight of the inoculum was determined by filtrating 100mL of the activated sludge over a preweighed 12 µm Schleicher adn Schull filter. This filter was dried for 1.5 hours at 104 deg C and weighed after cooling. Dry weight was calculated by subtracting the weighed filters.
Reference substance (positive control):
yes
Remarks:
3,5 dichlorophenol
Key result
Duration:
30 min
Dose descriptor:
EC50
Remarks:
calculated
Effect conc.:
ca. 11 mg/L
Nominal / measured:
not specified
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% confidence interval: 3-27 mg/L
Key result
Duration:
30 min
Dose descriptor:
other: EC20
Remarks:
calculated
Effect conc.:
ca. 5 mg/L
Nominal / measured:
not specified
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
30 min
Dose descriptor:
other: EC80
Remarks:
calculated
Effect conc.:
ca. 24 mg/L
Nominal / measured:
not specified
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
30 min
Dose descriptor:
EC10
Remarks:
calculated
Effect conc.:
ca. 4 mg/L
Nominal / measured:
not specified
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, C12-16 ADBAC (49.9% active in water) 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
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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. by dissolving 0.1258g of 3,5-dichlorophenol in 5mL of 2M NaOH solution, adjusting its pH to 7.9 with 2M HCl solution and then making it up to 250 mL wiht ultrapure water.

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. The suspension was aerated vigorously and kept at approximately 20 deg C in the dark.
- 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:
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.:
ca. 15.5 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% confidence limits of 12.7 and 19 mg/L
Remarks:
equivalent to 7.75 mg a.i./L (95% confidence limits of 6.35 and 9.5 mg a.i./L)
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
ca. 3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: calculated - equivalent to 1.6 mg a.i./L
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 3 h EC20, EC50 and EC80 were determined to be 6.8, 15.5 and 35.5 mg/L respectively (equivalent to 3.4, 7.75 and 17.8 mg a.i./L, respectively). The 3 h NOEC was established at 3.2 mg/L (equivalent to 1.6 mg a.i./L).
Executive summary:

A study was conducted to determine the toxicity to microorganisms of the test substance, C12 -16 ADBAC (49 -51% active) 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). No analytical dose determination was performed. 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. Under the study conditions, the 3 h EC20, EC50 and EC80 were determined to be 6.8, 15.5 and 35.5 mg/L respectively (equivalent to 3.4, 7.75 and 17.8 mg a.i./L, respectively). The 3 h NOEC was established at 3.2 mg/L (equivalent to 1.6 mg a.i./L) (Mayer, 2001).

Description of key information

Based on the study results, the 3 h EC50 and NOEC values of the test substance for toxicity to micro-organisms were determined to be 7.75 and 1.6 mg a.i./L (measured) respectively; these values have been considered further for hazard/risk assessment.

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, C12-16 ADBAC (49.9% active in water) 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, C12 -16 ADBAC (49 -51% active) 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). No analytical dose determination was performed. 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. Under the study conditions, the 3 h EC20, EC50 and EC80 were determined to be 6.8, 15.5 and 35.5 mg/L respectively (equivalent to 3.4, 7.75 and 17.8 mg a.i./L, respectively). The 3 h NOEC was established at 3.2 mg/L (equivalent to 1.6 mg a.i./L) (Mayer, 2001).

Based on the above studies, the same effect levels were concluded in the biocide assessment report on C12-16 ADBAC by RMS Italy. Both the studies were judged reliable, but the results of the test conducted for a longer period (3h) was considered to be more appropriate as it also provides the lowest endpoints, hence the US ISC data (Mayer, 2001) was selected as the key value (ECHA biocides assessment report, 2015).Therefore, in line with the biocides assessment report on C12-16 ADBAC by RMS Italy (ECHA biocides assessment report, 2015), 3 h EC50 and NOEC values of 7.75 and 1.6 mg a.i./L (measured) respectively has been considered further for hazard/risk assessment.