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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 July 2019 to 28 January 2020
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
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
REAGENTS
- All reagents used were ACS reagent grade or higher quality.
- The solutions were prepared using GenPure deionised water matching the requirements of Type 1 water of ASTM D1193.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
TEST SYSTEM
- The biological test system contained a mixture of microorganisms common for the activated sludge wastewater treatment process. The organisms responsible for the decomposition of organic materials are principally aerobic and facultative aerobic bacteria.
- The test system was chosen because it is representative of a treatment process that may receive the test substance.

TEST INOCULUM
- Activated sludge collected from the Easton Wastewater Treatment Facility, Easton, Maryland, USA was used to prepare inoculum for the test. The Easton facility receives wastewater predominately from domestic sources.
- The sludge was sieved using a 2 mm screen and was kept under aeration in a temperature controlled room.
- Synthetic sewage (50 mL per litre of sludge) was added at the end of the day the sludge was collected.
- Prior to use, the sludge was allowed to settle for 30 minutes and the supernatant above the settled solids was removed. Total suspended solids (TSS) concentration of the sludge was determined.
- The inoculum was prepared by diluting an aliquot of the settled sludge with an aliquot of deionised (reverse osmosis) water to achieve target TSS concentration of approximately 3000 mg/L.
- The inoculum was maintained at an average temperature of 20 ± 2 °C, continuously aerated, and used for testing the same day as prepared.
- The pH and TSS concentration of the inoculum were determined prior to use on the day of the study.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Not applicable
Hardness:
Not reported
Test temperature:
20.3 to 21.9 °C
pH:
7.82 to 7.91 at start of test (see Table 1, attached)
Dissolved oxygen:
See Table 2 (attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal concentrations of 10, 100 mg/L and 1000 mg/L
Details on test conditions:
OBJECTIVE
- The objective of this study was to assess the effects of the test item on activated sludge microorganisms by determining influence of this compound on activated sludge respiration rate.

EXPERIMENTAL DESIGN
- The test contained control, reference, and treatment groups.
- The control replicates were used to determine the background respiration rate of the sludge and were not exposed to the test or reference substances.
- The reference group was dosed with 3, 5-dichlorophenol, a known inhibitor of respiration, at concentrations of 3, 15, and 50 mg/L. The test substance was tested at nominal dose levels of 10, 100, and 1000 mg/L. The 1000 mg/L treatment was tested in triplicate.
- An abiotic control was treated with the test item at a dose level of 1000 mg/L to examine the potential for abiotic reactions of the test substance to consume or release oxygen under the tested conditions.

REFERENCE SUBSTANCE STOCK SOLUTION
- A 1000 mg/L stock solution of 3,5-dichlorophenol was prepared by dissolving 1.0003 g of material in 20 mL of 1N NaOH and then adding 40 mL of deionised (GenPure) water. While stirring, approximately 16 mL of 1N H2SO4 was added to reach the point of incipient precipitation.
- The solution of 3,5-dichlorophenol was then diluted to the final volume of 1 L with GenPure water.
- The pH of the solution was adjusted by adding 1N H2SO4 and the flask was filled up to the volume with GenPure water. The final pH of the reference substance stock solution was 7.55.
- The reference substance solution was administered to the reference group by volumetric addition.
- Volumetric addition is the most appropriate route of administration of aqueous solutions.

TEST CONDITIONS AND APPARATUS
- Control, reference, and treatment test mixtures were incubated at 20 ± 2 ºC and aerated for three hours at a rate sufficient to provide aerobic conditions and maintain solids in suspension.
- The mixtures were prepared and aerated in 1000 mL Erlenmeyer flasks and then transferred to fill 300 mL biochemical oxygen demand (BOD) bottles to conduct dissolved oxygen (DO) measurements.
- Each test flask was identified by project number, test substance identification, test concentration, and a flask number.

SYNTHETIC SEWAGE
- Synthetic sewage was prepared according to the procedure presented in Appendix 4 (attached).
- The pH of synthetic sewage was 7.40.

PRE-TEST pH EVALUATION
- Effect of the test substance on the pH of the inoculum/water mixtures was assessed prior to performing of the test. A pH pre-test evaluation of the test substance treated mixtures was performed using test mixtures prepared by placing 16 mL of synthetic sewage and adding appropriate amount of the test substance according to the table below.
- Deionized (reverse osmosis, RO) water (200 mL) was then added, the mixture was stirred with a magnetic stir bar, and the pH was measured and adjusted, if necessary, to become close to 7.5 by adding aliquots of 1.0 M NaHCO3. After this, an appropriate amount of deionized water was added to bring total volume of water plus 1.0 M NaHCO3 to 234 mL. Then, a 250 mL aliquot of inoculum was added providing total volume of 500 mL and final pH was measured.

TEST PROCEDURE
- Test mixtures were prepared at 15-minute intervals starting with the first control.
- Each control vessel contained 16.0 mL of synthetic sewage, 234 mL of deionized water, and 250 mL of inoculum for a total volume of 500 mL.
- Reference substance dosed biotic mixtures were prepared by combining 16.0 mL of synthetic sewage, appropriate volume of the reference substance stock solution, and deionised water to achieve total volume of 250 mL.
- Test substance biotic mixtures were prepared by placing in the test vessel 16.0 mL of synthetic sewage, appropriate amount of test substance and adding sufficient amount of 1 M NaHCO3 (1.0 mL for 1000 mg/L) to achieve pH within the range of 7.5 ±0.5.
- After this, appropriate volume of deionized water was added to bring total volume the mixture to 250 mL according to the attached table.
- After the addition of deionized water, 250 mL of inoculum was added into each vessel except abiotic control to bring the total volume to 500 mL and the vessels were placed under aeration for three hours prior to start DO measurements.

SAMPLE ANALYSIS
- After 3 hours of aeration, the contents of the first control vessel was transferred into a BOD bottle and dissolved oxygen concentration was measured every 10 seconds over a 10 minute period or until the dissolved oxygen measurement dropped below 2.0 mg O2/L using a YSI Model 5000 Dissolved Oxygen Meter.
- The respiration rate in all other vessels was determined in an identical manner.
- The contact time for each concentration of reference or test substance with the inoculum was three hours.

CALCULATIONS
- A respiration rate was calculated for each test mixture and expressed in mg O2/L/hour. The rate was calculated using dissolved oxygen (DO) values from the linear portion of the graph between approximately 7.0 mg O2/L and 2.0 mg O2/L, or over a 10-minute period if DO did not reach approximately 2.0 mg O2/L. However, values outside this range could be used provided their gradients did not change as they passed through the 7.0 mg O2/L and 2.0 mg O2/L points.
- The respiration rate was calculated using the equation Respiration Rate = [(initial DO – final DO)/(final time – initial time)] × 3600 s/h
- Percent inhibition was calculated using the equation Percent inhibition = (1 – (2Rs / (RC1 + RC2) * 100 % where Rs = oxygen consumption rate at a given concentration of the test or reference substance; RC1 = oxygen consumption rate in Control 1; RC2 = oxygen consumption rate in Control 2.
- Percent inhibition of the test substance at 1000 mg/L was also calculated adjusted by oxygen consumption in abiotic control using the equation Percent inhibition = (1- [2(Rs – R4)] / (RC1 + RC2) * 100 % where RA = oxygen consumption rate in abiotic control at test substance concentration 1000 mg/L.

QUALITY CONTROL
- The test was considered valid if the three following criteria were met:
(i) The two control respiration rates should not be less than 20 mg oxygen per one gram dry weight of activated sludge in an hour.
(ii) The two control oxygen uptake rates should have a coefficient of variation of 30 % or less.
(iii) The EC50 in the three-hour span of 3,5-dichlorophenol should be in the range of 2 to 25 mg/L.

TEST CONDITIONS
- The temperatures in the temperature controlled room during the test ranged from 20.3 to 21.9 °C.
- The results of pre-test pH evaluations (see Table 1, attached) indicated that the after addition of the test substance and titration with 1 M NaHCO3 the pH of the inoculum/synthetic sewage/water mixture remained within the pH range of 7.5 ± 0.5.
- Total suspended solids (TSS) concentration of the activated sludge after removal of the
supernatant layer was 6027 mg/L (average of three replicates). The inoculum was prepared by mixing 2960 mL of activated sludge with 3040 mL of deionized water. The resulting TSS concentration in the inoculum was 3053 mg/L (average of three replicates). The initial pH of inoculum (pH = 8.67) was adjusted to 7.86 by adding 4 mL 2 N HCL.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (Sigma Aldrich; CAS 591-35-5; batch MKBS0425V; purity 99.7 %; expiry date assigned by test house 31 October 2019; storage conditions not stated)
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
Details on results:
RESULTS
- Dissolved oxygen concentrations in the test mixtures are presented in Table 2 (attached) and calculated respiration rates and percent inhibition are shown in Table 3 (attached).
- The respiration rates in the two controls were 32.5 and 50.0 mg O2/L/h corresponding to 21.3 mg O2/g sludge solids/h and 32.8 mg O2/g sludge solids/h, respectively (see Table 4, attached), based on the concentration of the sludge solids in the test mixtures of 1.53 g/L.
- The average respiration rate for the two controls (27.0 mg O2/g sludge solids/h) was greater than 20 mg O2/g sludge solids/h, which is considered as the low limit of validity of the test.
- The coefficient of variation of respiration rates in the two control vessels was 30.0 % (see Table 4, attached). This value was within the 30 % limit established for the test.
- The respiration rate in biotic test mixtures dosed with the test substance at concentrations of 10 and 100 mg/L was 2336 and 2362 mg O2/L/h, respectively (see Table 3, attached). Compared to the untreated control test mixtures, the values of respiration inhibition of the test substance treated test vessels at concentrations of 10 and 100 mg/L were -5564 % and -5625 %, respectively. Negative values of respiration inhibition are frequently observed for some compounds at relatively low concentrations but biochemical mechanism of enhancement of respiration by the test item is not known.
- The average (of 3 replicates) respiration rate in biotic test mixture dosed with the test substance at 1000 mg/L was 606 mg O2/L/h (Table 3), which corresponds to an average respiration inhibition of -1369 %. Negative value of respiration inhibition at all tested concentrations indicated that EC50 of the test item is greater than the highest concentration tested based on the available experimental data.
- The abiotic treatment mixture dosed with 1000 mg/L of the test substance provided a respiration rate of 0.3 mg O2/L/h indicating that there was a minimal uptake of oxygen resulting from abiotic reactions of the test substance.
- Graphs of percent inhibition versus concentration of the reference substance and test substance are presented in Figures 1 and 2, respectively (attached).
Results with reference substance (positive control):
- The validity of the test was supported by the results for the 3,5-dichlorophenol reference group.
- Calculations with binomial test indicated that confidence level of the EC50 being within limits from 3 mg/L to 50 mg/L was above 95 %.
- Calculations with probit method provided EC50 value of 14.0 mg/L with 11.5 mg/L and 17.1 mg/L 95% confidence limits. This EC50 value was within the 2 to 25 mg/L range considered acceptable for the test.
Reported statistics and error estimates:
- When the dose response pattern (percent inhibition versus test substance concentration) allowed calculation of an EC50 value, the data were analysed using the computer program designed by C.E. Stephan.
- The program was designed to calculate the EC50 value and the 95% confidence interval by probit analysis, the moving average, or binomial probability with nonlinear interpolation.
- Confidence limits (95%) for the EC50 and the EC50 value itself (when applicable) were determined using binomial probability with nonlinear interpolation only.
Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, of the test item did not inhibit activated sludge respiration at dose levels of 10, 100 mg/L, and 1000 mg/L. The EC50 of the test item was determined to be greater than the highest concentration tested based on the available experimental data and 1000 mg/L can be taken as No Observed Effect Concentration (NOEC).
Executive summary:

GUIDELINE

The study was performed based on procedures in the OECD Guideline for the Testing of Chemicals, Guideline 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation).

 

METHODS

The test contained control, reference, and treatment groups. The control replicates were used to determine the background respiration rate of the sludge and were not exposed to the test or reference substances. The reference group was dosed with 3, 5-dichlorophenol, a known inhibitor of respiration, at concentrations of 3, 15, and 50 mg/L. The test substance was tested at nominal dose levels of 10, 100, and 1000 mg/L. The 1000 mg/L treatment was tested in triplicate. An abiotic control was treated with the test item at a dose level of 1000 mg/L to examine the potential for abiotic reactions of the test substance to consume or release oxygen under the tested conditions.

 

RESULTS

Under the test conditions, of the test item did not inhibit activated sludge respiration at dose levels of 10, 100 mg/L, and 1000 mg/L. The EC50 of the test item was determined to be greater than the highest concentration tested based on the available experimental data and 1000 mg/L can be taken as No Observed Effect Concentration (NOEC).

Description of key information

The EC50 of the test item was determined to be greater than the highest concentration tested based on the available experimental data and 1000 mg/L can be taken as No Observed Effect Concentration (NOEC) (OECD 209).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

GUIDELINE

The study was performed based on procedures in the OECD Guideline for the Testing of Chemicals, Guideline 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation).

 

METHODS

The test contained control, reference, and treatment groups. The control replicates were used to determine the background respiration rate of the sludge and were not exposed to the test or reference substances. The reference group was dosed with 3, 5-dichlorophenol, a known inhibitor of respiration, at concentrations of 3, 15, and 50 mg/L. The test substance was tested at nominal dose levels of 10, 100, and 1000 mg/L. The 1000 mg/L treatment was tested in triplicate. An abiotic control was treated with the test item at a dose level of 1000 mg/L to examine the potential for abiotic reactions of the test substance to consume or release oxygen under the tested conditions.

 

RESULTS

Under the test conditions, of the test item did not inhibit activated sludge respiration at dose levels of 10, 100 mg/L, and 1000 mg/L. The EC50 of the test item was determined to be greater than the highest concentration tested based on the available experimental data and 1000 mg/L can be taken as No Observed Effect Concentration (NOEC).