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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 August 2013 to 19 December 2013
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Infineum UK, E00031-633
- Rest date: 30 January 2014
- Purity: 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature, in the dark
Analytical monitoring:
no
Details on sampling:
The test was conducted at nominal EXP0700332 concentrations of 10, 100 and 1000 mg/L.
The reference item, 3,5-DCP, was tested at nominal concentrations of: 5, 15 and 30 mg/L.
Appropriate weights of EXP0700332 were weighed directly onto glass cover slips and these added to the appropriate test vessel (500 mL glass conical flask) to give the required test concentrations. The flasks had the appropriate amount of deionised water (234 mL) and synthetic sewage feed (16 mL) added. At time ‘0’ the microbial inoculum (250 mL) was added to each vessel to give a final concentration of 500 mL. This process was repeated at 15 minute intervals for each test concentration, reference substance and control.
Vehicle:
no
Details on test solutions:
A 1 g/L reference item stock solution was prepared by adding 3,5-DCP (0.09997 g) to a volumetric flask (100 mL) and bringing to volume with deionised water. The solution was ultra-sonicated for approximately 30 min to aid dissolution. Aliquots were then added to the appropriate test vessel to give the required reference item test concentrations.
Prior to initiation of the test, both control vessels and reference item vessels were prepared by adding 16 mL of synthetic sewage feed and an appropriate volume of deionised water. An appropriate volume of the reference item was added to the reference item vessels and the weighed test item then added to the appropriate test item vessels. At time ‘0’ microbial inoculum (250 mL) was added to control vessel C1 to give a final volume of 500 mL. This process was repeated at 15 min intervals for each concentration of test and reference substance.
Test organisms (species):
activated sludge
Details on inoculum:
A sample (ca.2 x 5L, Batch A and Batch B) of activated sewage sludge was obtained from Haddington Municipal Sewage Works (a local sewage processing plant, which handles predominantly domestic sewage) on 04 September 2013. The solid content of the Batch A sludge was determined by shaking the bottle of sludge to homogenise thoroughly. Duplicate sub-samples (5 mL) of the homogenised sludge were dried in an oven at ca.105¿C and the suspended solids content determined as 2.218 g/L. Following the solid content determination both batches of the sludge were constantly aerated. Sewage sludge ca. 1000 mL was removed from Batch A sludge, leaving ca. 3.750 L remaining. Synthetic sewage feed was then added to this sludge at a rate of 50 mL/L of sludge and the sludge left aerating overnight. The solid content of Batch A sludge was then re-determined and found to be 3.942 g/L, which was too high for use. To reduce the solid content of Batch A sludge, Batch B sludge was allowed to settle for ca 30 min and ca. 1.5 L of supernatant removed. An aliquot (1.1 L) of the supernatant was added to Batch A sludge and the solid content re-determined as 3.250 g/L, which was accepted for use on the test.
Test type:
static
Water media type:
other: Deionised Water
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
After the 3 hour contact time, the dissolved oxygen content was recorded at 30 second intervals for a period of 10 minutes, or until 2 consecutive readings of 0.00 mg O2/L were recorded.
Test temperature:
20.2-20.9 degrees Celsius
pH:
pH 7.5 +/- 0.5
Nominal and measured concentrations:
EXP0700332 Nominal concentrations of 10 mg/L, 100 mg/L and 1000 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL conical flask
- Aeration: yes
- No. of vessels per concentration (replicates): 1 vessel per concentration: 5 EXP0700332 concentrations (10, 100, 1000, 1000, 1000 mg/L), 3 refernce substance concentrations (3 vessels and 2 control vessels
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 3.250 g/L
- Weight of dry solids per volume of reaction mixture per unit of time: 3.250 g/L
- Nutrients provided for bacteria: Synthetic sewage feed
- Nitrification inhibitor used (delete if not applicable): none
Reference substance (positive control):
yes
Remarks:
3, 5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
No inhibition of respiration was noted at 10, 100 and 1000 mg EXP0700332/L. As there was no inhibition of respiration at any EXP0700332 concentration, the 3 h EC10/ELr10, EC20/ELr20, EC50/ELr50 and EC80/ELr80 for EXP0700332 to activated sludge respiration was estimated as >1000 mg/L, under test conditions.
Results with reference substance (positive control):
The 3 h EC50 (respiration inhibition) for 3,5-dichlorophenol to activated sludge was estimated as 10.8 mg/L, with 95 % confidence limits of 9.29 and 12.6 mg/L, under test conditions.
Reported statistics and error estimates:
The respiration rate for each vessel was calculated from the recorded oxygen concentrations and expressed as mg O2/L/h for the period between approximately 7.0 and 2.0 mg O2/L. For the control vessels, the recorded oxygen concentrations for the period between 6.55 and 2.08 mg O2/L (Replicate 1) were used to calculate the respiration rate, with the recorded oxygen concentrations for the period between 6.20 and 2.43 mg O2/L (Replicate 2) being used. For the 10 mg EXP0700332/L vessel, the recorded oxygen concentrations for the period between 6.03 and 2.30 mg O2/L were used to calculate the respiration rate and for the 100 mg EXP0700332/L vessel, the recorded oxygen concentrations for the period between 5.58 and 2.47 mg O2/L were used. For the three replicate 1000 mg EXP0700332/L vessels the recorded oxygen concentrations for the period between 5.63 and 2.24 mg O2/L, 4.28 and 2.49 mg O2/L and 5.15 and 2.39 mg O2/L for replicates I, II and III, respectively, were used. For the 5 mg/L reference item vessel, the recorded oxygen concentrations for the period between 6.57 and 2.34 mg O2/L were used. Where respiration rate was low (15 and 30 mg/L reference item) calculations were conducted over the full linear range. The inhibition effect of EXP0700332 and 3,5-DCP is calculated from the respiration rate expressed as a percentage of the mean of the two control respiration rates.
Validity criteria fulfilled:
yes
Conclusions:
No inhibition of respiration was noted at 10, 100 and 1000 mg EXP0700332/L. As there was no inhibition of respiration at any EXP0700332 concentration, the 3 h EC10/ELr10, EC20/ELr20, EC50/ELr50 and EC80/ELr80 for EXP0700332 to activated sludge respiration was estimated as >1000 mg/L, under test conditions.
The 3 h EC50 (respiration inhibition) for 3,5-dichlorophenol to activated sludge was estimated as 10.8 mg/L, with 95 % confidence limits of 9.29 and 12.6 mg/L, under test conditions.
The coefficient of variation of oxygen uptake rate in control replicates was 12%. The oxygen uptake rate in the controls was 33.0 and 27.8 mg oxygen/g of activated sludge/h, for control replicates 1 and 2, respectively.
The test met guideline validity criteria as the coefficient of variation in control replicates was less than 30%, the EC50 (3 h) for the reference item was in the range 2 to 25 mg/L and the oxygen uptake in the controls was greater than 20 mg oxygen/g of activated sludge/h.
Executive summary:

This study was conducted to determine the effects of EXP0700332 on sewage microorganisms by measuring the respiration rate under defined conditions. The study was designed to be appropriate for submission to Regulatory Authorities using procedures outlined in OECD Guideline 209 (July 2010).

The test was conducted at the following nominal concentrations of EXP0700332: 10, 100 and 1000 mg/L. Single vessels were tested for test concentrations of 10 and 100 mg/L, triplicate vessels were tested at 1000 mg/L. A reference item (3,5-dichlorophenol) was tested as a positive control at the following nominal concentrations: 5, 15 and 30 mg/L. Duplicate control vessels were also tested.

The prepared vessels were aerated for 3 h, after which dissolved oxygen concentrations were measured at 30 sec intervals for up to a maximum period of 10 min. The respiration rate (mg O2/L/h) was calculated over the linear phase of oxygen consumption for each vessel (ca 7.0 – 2.0 mg O2/L). Where respiration rate was low, the calculation was conducted over the full linear range. The respiration inhibition (%) was compared to the mean control vessel respiration rate, for both the reference and test items.

The coefficient of variation of oxygen uptake rate in control replicates was 12%.

No inhibition of respiration was noted at 10, 100 and 1000 mg EXP0700332/L. As there was no inhibition of respiration at any EXP0700332 concentration, the 3 h EC10/ELr10, EC20/ELr20, EC50/ELr50 and EC80/ELr80 for EXP0700332 to activated sludge respiration was estimated as >1000 mg/L, under test conditions.

The 3 h EC50 (respiration inhibition) for 3,5-dichlorophenol to activated sludge was estimated as 10.8 mg/L, with 95 % confidence limits of 9.29 and 12.6 mg/L, under test conditions.

The test met guideline validity criteria as the coefficient of variation in control replicates was less than 30%, the EC50 (3 h) for the reference item was in the range 2 to 25 mg/L and the oxygen uptake in the controls was greater than 20 mg oxygen/g of activated sludge/h.

Description of key information

The study was conducted to GLP and designed to be appropriate for submission to Regulatory Authorities using procedures outlined in OECD Guideline 209 (July 2010). The test was conducted at the following nominal concentrations of EXP0700332: 10, 100, 1000 mg/L. A reference item (3,5 - dichlorophenol) was tested as a positive control at 5, 15 and 30 mg/L. Duplicate control vessels were also tested.

No inhibition of respiration was noted at 10, 100 and 1000 mg EXP0700332/L. As there was no inhibition of respiration at any EXP0700332 concentration, the 3 h EC10/ELr10, EC20/ELr20, EC50/ELr50 and EC80/ELr80 for EXP0700332 to activated sludge respiration was estimated as >1000 mg/L, under test conditions.

The 3 h EC50 (respiration inhibition) for 3,5-dichlorophenol to activated sludge was estimated as 10.8 mg/L, with 95 % confidence limits of 9.29 and 12.6 mg/L, under test conditions.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information