Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability rating was 1 because study followed OECD 209 test guidelines and was carried out under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MCP 2484
- Physical state: liquid, amber-color
- Analytical purity: 100%

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A sample of activated sludge was obtained the day before the start of the test from Worlingworth Sewage Treatment Works (Suffolk, UK), which treats predominantly domestic waste. In the laboratory, the sample was maintained under aerobic conditions until required. The concentration of suspended solids in a homogenised sample was determined on the day of collection and immediately before the start of the test.
On the day of collection, an aliquot (10 mL) of the activated sludge was filtered through a dried and preweighed Whatman GF/C filter paper, which was then dried again at approximately 105 C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added to the stock of activated sludge and this was aerated overnight. On the day of the test, the MLSS content of the sludge was determined (in triplicate) and adjusted to 4 g/L by the addition of tap water. The pH and temperature of the sludge were also measured. Aliquots (200 mL) were then added to each mixture to give a final MLSS concentration of 1.6 g/L.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Test temperature:
Temperature was between 19.5 to 20 C
pH:
pH reported in range 7.3-7.8 for blank controls, 7.2-8.0 for positive controls (3.5-DCP) and 7.3-7.8 for test material (MCP 2484)
Dissolved oxygen:
Initial Dissolved Oxygen ranged from 7.0-7.7 in the definitive limit test
Nominal and measured concentrations:
Nominal loading concentrations: 10, 30, 100, 300, 1000 mg/L Test material MCP 2484

Positive control (3,5-dichlorophenol) (3,5-DCP) was tested at 3, 10, 32 mg/L
Details on test conditions:
Nominal loadings of the test substance were added to the test vessels, by adding appropriate quantity of test material in one-litre test beakers and RO water (284 ml) was added. The pH of the mixtures was then determined and no adjustment was necessary. Additions of synthetic sewage (16 mL) and microbial inoculum (200 ml) were made at fifteen minute-intervals to give a final volume of 500 mL for each blank control, positive control and test vessels at the specified exposure concentrations /loadings.

The prepared mixtures were aerated for three hours in a thermostatically-controlled water bath (19.5-20.0 C), using an aerator connected to a laboratory supply of oil-free compressed air (one litre/minute). Following the exposure period, a well-mixed sample of each mixture was transferred to a biochemical oxygen demand (BOD) bottle. The rate of oxygen consumption was measured, over a period of approximately 10 minutes or until the dissolved oxygen concentration fell below 2 mgO2/L using a Yellow Springs Instrument (YSI) dissolved oxygen meter with a self stirring bottle probe connected to a chart recorder. The pH and temperature of the samples were measured at the start and end of the test.

The prepared mixtures were aerated for three hours in a thermostatically-controlled water bath (19.5-20.0 C), using an aerator connected to a laboratory supply of oil-free compressed air (one litre/minute). Following the exposure period, a well-mixed sample of each mixture was transferred to a biochemical oxygen demand (BOD) bottle. The rate of oxygen consumption was measured, over a period of approximately 10 minutes or until the dissolved oxygen concentration fell below 2 mgO2/L using a Yellow Springs Instrument (YSI) dissolved oxygen meter with a self stirring bottle probe connected to a chart recorder. The pH and temperature of the samples were measured at the start and end of the test.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
other: EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: nominal loadings
Duration:
3 h
Dose descriptor:
other: NOEL
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: nominal loadings
Details on results:
The test substance, MCP2484, did not show any biologically significant inhibitory effect on the respiration rate of activated sludge at any of the loading concentrations employed in the test. The 3 hr EL50 of test substance could not be calculated but it must be greater than 1000 mg/L, the highest nominal loading level tested. The 3 hr NOEL was estimated also to be > 1000 mg/L nominal loading. The test substance is not expected to cause acute toxicity to microbial organisms in activated sludge inoculum at its maximum water solubility limits.
 
Results with reference substance (positive control):
For the positive reference substance, 3,5-DCP, a 3-hour EC50 = 8.5 mg/L value was determined and this fulfilled the validity criterion relating to sensitivity to inhibition (acceptable EC50 range 5 to 30 mg/L). The validity criterion relating to the respiration rates in the control (variation not greater than 15%) was also satisfied.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test substance was not inhibitory to activated sludge microbial respiration at the highest loading of 1000 mg/L nominal exposure. Therefore, the test material is not expected to adversely impact microbial activity in a wastewater treatment plant.
Executive summary:

The test substance did not inhibit microbial respiration in a sludge sample at any of the exposure levels tested including the high loading of 1000 mg/L, all loading which exceeds its maximum water solubility (<0.001 mg/L). The 3 hour EL50 was > 1000 mg/L (nominal loading) and the 3 hour NOEL was >1000 mg/L (nominal loading). Therefore, the test substance is not expected to adversely impact microbial activity in a wastewater treatment plant.