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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Remarks:
Due to the nature of the test material (UVCB), measured concentrations were not necessarily representative of the whole substance. Therefore, statistical analysis and end points were expressed terms of nominal concentrations only.
Vehicle:
no
Details on test solutions:
- All test solutions were prepared individually and without the use of any solubilising agent. Nominal concentrations of the test material were prepared by adding weighed amounts of the test material into a 1 L glass jar and labelled appropriately. Due to the adhesive nature of the test material, the weighed amounts were transferred onto a pre-weighed square glass slide. The glass slide containing the test material was added directly to the test vessel to avoid any loss of the test material during transfer.
- Test mixtures prepared in the absence of ATU and identified as: FT1, FT2, FT3, FT4, and FT5, corresponded to nominal test material concentrations of 10, 100, and 3 replicates of 1000 mg/L, respectively. Similarly test mixtures prepared in the presence of ATU included FN1, FN2, FN3, FN4, and FN5, corresponded to nominal test material concentrations of 10, 100, and 3 replicates of 1000, mg/L, respectively. All treatments were aerated continuously at a rate of 500 to 1000 mL/minute during the 3 hour test period.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Guelph Wastewater Treatment Plant, Guelph, Ontario
- The aerobic sludge was continuously aerated from the time of collection until use in preparation of the inoculum. Upon receipt in the lab, the sludge was adjusted to the test temperature of 20 ± 2°C, washed three times with laboratory chlorine-free well water (groundwater, water hardness ~ 350 mg/L as CaCO3; pH 8.0 – 8.5), and then analysed for solids content (i.e., mean dry weight as g/L).
- The mean dry weight of the sludge (5.1 g/L) was measured by drying 1.0 mL portions (n = 3) of the washed sludge on pre-weighed foil boats in a drying oven at 100°C for approximately 2 hours.
- The volume of inoculum required for the range-finding test was 6 L. The inoculum was prepared by adding 3,550 mL of washed activated sludge (dry wt = 5.1 g/L), 2,150 mL of RO water and 300 mL of synthetic sewage feed. The inoculum was aerated overnight at 20 ± 2°C.
- On the day of test initiation, the prepared inoculum was continuously aerated throughout the day. Prior to test initiation, the pH of the inoculum was determined to be 6.7 in the range test and pH adjustment was not required (acceptable range: pH 6 to 8). The inoculum was mixed well prior to sub-sampling for use in preparation of the test solutions.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2°C
pH:
6.7
Nominal and measured concentrations:
Nominal: 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 1 L glass
- Fill volume: 500 mL
- Aeration: 0.5-1.0 L/min
- No. of vessels per concentration: single replicates of 10 and 100 mg/L; triplicate exposures of 1000 mg/L with and without ATU
- No. of vessels per control: 2 inoculum controls each with and without ATU and 3 reference toxicant concentrations
- No. of vessels per abiotic control: 1
- Sludge concentration: Approximately 1.5 g of suspended solids per litre
- Nutrients provided for bacteria: The synthetic sewage feed which contains nutrients, peptone, meat extract and urea was prepared on the day prior to test initiation. This was used as a nutrient solution to provide sustenance to the bacteria contained in the activated sludge which allows normal aerobic microbial respiration to take place. The synthetic sewage feed for the range-finding test was prepared the day prior to test initiation.
- Nitrification inhibitor used: N-allylthiourea. A 2320 mg/L stock solution of ATU was prepared by adding 0.5803 g into a 250 mL volumetric flask and making this up to volume with distilled water. The addition of 2.5 mL of this stock solution to the incubation mixture volume of 500 mL was to achieve a final concentration of 11.6 mg/L ATU as recommended.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse Osmosis (RO) water

EXPERIMENTAL DESIGN
- Blank Controls: Blank controls containing only sewage feed, sludge and RO water in the absence and presence of ATU were prepared for the test. These controls were used to determine baseline respiration in the test system. Blank controls, with (FNB1 and FNB2) and without (FB1 and FB2), ATU were established, one at the beginning and one at the end of the test period.
- Abiotic Control: One abiotic control (FA) was established containing only sewage feed, RO water and the highest concentration of test material. This control was used to evaluate the abiotic oxygen uptake by the test material, if any.
- Test Treatments: All test solutions were prepared individually and without the use of any solubilising agent. Nominal concentrations of the test material were prepared by adding weighed amounts of the test material into a 1 L glass jar and labelled appropriately. Due to the adhesive nature of the test material, the weighed amounts were transferred onto a pre-weighed square glass slide. The glass slide containing the test material was added directly to the test vessel to avoid any loss of the test material during transfer. Test mixtures prepared in the absence of ATU were identified as FT1, FT2, FT3, FT4, and FT5, corresponded to nominal test material concentrations of 10, 100, and 3 replicates of 1000 mg/L, respectively. Similarly test mixtures prepared in the presence of ATU included FN1, FN2, FN3, FN4, and FN5, corresponded to nominal test material concentrations of 10, 100, and 3 replicates of 1000, mg/L, respectively. All treatments were aerated continuously at a rate of 500 to 1000 mL/minute during the 3 hour test period. After a 3 hour aerated exposure in the test, the respiration rate was determined in each container from the measurement of oxygen depletion in units of mg/L/h using a DO meter. DO measurements were taken every 30 seconds over the course of 10 minutes for each vessel in the test.
- Reference Toxicant Series: A reference toxicant 3,5-dichlorophenol was tested with a known 3 hour EC50, serving as a positive control for the test system. Five hundred (500) mL of a 1000 mg/L stock solution of the reference material were prepared by weighing 0.1253 g and adding this to a 250 mL volumetric flask. The stock solution was prepared on the day prior to test initiation. The reference solutions were prepared from lowest to highest concentration. The following nominal concentrations of the reference toxicant were tested: 3.25, 10, and 32.5 mg/L corresponding to treatments FR1, FR2 and FR3, respectively. The reference toxicant vessels were prepared by adding 3.25 mL, 10 mL and 32.5 mL of a 500 mg/L stock solution of 3,5-dichlorophenol.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Details on results:
RANGE-FINDING TEST
- Inoculum Controls: The average respiration rates of the Inoculum Controls in the absence and presence of ATU were 76.9 and 46.1 mg O2/L/h, respectively. The CV of respiration rates between the controls was not more than 30% at the end of the test and the oxygen uptake rate of the controls was ≥ 20 mg of oxygen per gram of activated sludge in an hour. According to the test acceptability criteria, this test was acceptable for the determination of activated sludge respiration inhibition.
- Abiotic Control: There was no significant oxygen uptake in the abiotic control in the range-finding test. At 1000 mg/L test material concentration the abiotic oxygen uptake rate was -0.7 mg O2/L/h (the negative value indicates stimulation relative to the control).
- Test Material: The 3 hour EC50s for total, heterotrophic and nitrification inhibition were greater than 1000 mg/L. The 3 hour NOECs for total, heterotrophic and nitrification inhibition were all reported to be 1000 mg/L.
- Based on these results and in consultation with the Study Sponsor, further testing was not needed.
Results with reference substance (positive control):
The reference toxicant (3,5-dichlorophenol) was tested at concentrations of 3.25, 10 and 32.5 mg/L. The reference toxicant achieved a 3-hour EC50 of 5.1 mg/L (95% confidence limits: 2.52 – 10.20 mg/L), indicating that the test system was acceptable for the determination of activated sludge respiration inhibition.
Reported statistics and error estimates:
The 3 hour EC50 and NOEC were determined using Equal Variance t Two-Sample Test.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the 3-hour EC50s for total, heterotrophic and nitrification inhibition were greater than 1000 mg/L (nominal concentration). The 3 hour NOECs for total, heterotrophic and nitrification inhibition were reported to be 1000 mg/L (nominal concentration).
Executive summary:

The toxicity of the test material to aquatic microorganisms was investigated in accordance with the standardised guideline OECD 209, under GLP conditions in an activated sludge respiration inhibition test.

Due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results are expressed here in terms of nominal concentrations only.

A range-finding test which included single replicates of 10 and 100 mg/L of the test material and three replicates of 1000 mg/L was performed. All of the criteria for test validity were met.

Under the conditions of this study, the 3 hour EC50s for total, heterotrophic and nitrification inhibition were greater than 1000 mg/L (nominal concentration). The 3 hour NOECs for total, heterotrophic and nitrification inhibition were reported to be 1000 mg/L (nominal concentration).

Description of key information

Under the conditions of this study, the 3 hour EC50s for total, heterotrophic and nitrification inhibition were greater than 1000 mg/L (nominal concentration). The 3 hour NOECs for total, heterotrophic and nitrification inhibition were reported to be 1000 mg/L (nominal concentration).

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The toxicity of the test material to aquatic microorganisms was investigated in accordance with the standardised guideline OECD 209, under GLP conditions in an activated sludge respiration inhibition test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results are expressed here in terms of nominal concentrations only.

A range-finding test which included single replicates of 10 and 100 mg/L of the test material and three replicates of 1000 mg/L was performed. All of the criteria for test validity were met.

Under the conditions of this study, the 3 hour EC50s for total, heterotrophic and nitrification inhibition were greater than 1000 mg/L (nominal concentration). The 3 hour NOECs for total, heterotrophic and nitrification inhibition were reported to be 1000 mg/L (nominal concentration).