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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Concentrations were adjusted for the purity of the test material.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Since the test substance was insoluble in water at the concentrations to be used in this study, a primary stock solution was not prepared. Test vessels were prepared individually by weighing out the appropriate amount of test material and adding it directly to the respective vessels.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Wareham Waste Water Treatment Facility, Wareham, Massachusetts.
- Preparation of inoculum for exposure: Before use, the sludge solution was passed through a 2-mm stainless steel sieve, concentrated by centrifuging for 10 minutes at 1 000 rpm, and the resulting supernatant poured off. The sludge was washed with approximately 500 mL of laboratory well water and centrifuged at 1 000 rpm for 10 minutes, for a total of four times, with the supernatant being discarded each time.
Based on the percent moisture of 95.12 %, 245.90 g of the sludge was added to 3554.1 mL of laboratory well water. Two packets of synthetic sewage feed were dissolved in 500 mL of purified reagent water prior to its addition to the inoculum. The inoculum received 200 mL (50 mL per litre of inoculum) of an undiluted synthetic sewage feed and was aerated overnight at a temperature of 20 ± 2 °C. The batch of inoculum was prepared 1 day before test initiation.
- Composition of synthetic sewage sludge feed: Tryptone (46.93 %), beef extract powder (32.26 %), urea (8.798%), NaCl (2.053 %), CaCl2.2H2O (1.173 %), MgSO4.7H2O (0.5865 %), K2HPO4 (8.212 %), purified reagent water (brought to a total volume of 500 mL).
- Initial biomass concentration: Following an inoculum adjustment of 1.16 L of laboratory well water, resulting in a final volume of 5.16 L for the inoculum, the solids content of the batch was determined to be 3.00 g/L.
Test type:
static
Water media type:
freshwater
Remarks:
Mixture of unadulterated water from a 100-metre bedrock well and dechlorinated Town of Wareham well water.
Limit test:
no
Total exposure duration:
3 h
Test temperature:
The temperature in the chamber where aeration was performed ranged from 20.5 to 21.1 °C. The temperature of the respirator water bath ranged from 20.0 to 20.1 °C.
pH:
The measured pH value in the abiotic control was 7.56. The pH values of the other vessels ranged from 7.91 to 7.99.
Nominal and measured concentrations:
0, 10, 100, 1000 mg/L (nominal).
Details on test conditions:
TEST SYSTEM
- Test vessel: 1 L glass beaker
- Fill volume: 16 mL synthetic sewage feed, 250 mL inoculum (except for the abiotic control). Total volume after water added was 500 mL.
- Aeration: Yes. Solutions were aerated using a Welch air pump set to deliver room air at a rate of 0.5 to 1 L/minute.
- No. of vessels per concentration: One replicate (10, 100 mg/L); triplicate (1000 mg/L)
- No. of vessels per control: Duplicate
- No. of vessels per abiotic control: One replicate

PREPARATION OF TEST SOLUTIONS
At time zero, 16 mL of synthetic sewage feed was added to a 1-L beaker. An aliquot (250 mL) of the microbial inoculum was added and the solution was brought to a final volume of 500 mL with laboratory well water. This mixture was identified as Control 1. An abiotic control was prepared with test material, synthetic sewage feed, and laboratory well water with no microbial inoculum. The positive control test systems were prepared by adding appropriate volumes of the 3,5-dichlorophenol stock solution to the sludge mixture. Another control, identified as Control 2, was prepared in the same manner as Control 1, 15 minutes after the last reference substance test system was prepared. Lastly, test material systems were prepared at each test concentration by adding appropriate amounts of the test material to the sludge mixture.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water, laboratory well water, was a mixture of unadulterated water from a 100-metre bedrock well and dechlorinated Town of Wareham well water.
- Water quality measurements: Total hardness, total alkalinity, pH, and conductivity were measured weekly. Representative samples of the town water were analysed biannually for the presence of pesticides, PCBs, and toxic metals. None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analysed.

OTHER TEST CONDITIONS
- Adjustment of pH: The batch of inoculum had a pH value of 7.25, which was adjusted to a final pH of 7.40 on the day of dosing with 1 N sulfuric acid.

EFFECT PARAMETERS MEASURED
- Following 3 hours of aeration, a 23-mL aliquot of each sludge mixture was transferred to a 50-mL cylinder placed inside a 20 ± 1 °C water bath placed over a stir plate. The cylinders received a stir bar and were fitted with a dissolved oxygen probe after receiving the sample. The probes were attached to a Strathkelvin Strathtox Respirometer and a computer, which ran the Strathkelvin Strathtox software version 2.0. The Strathkelvin system was used to monitor the depletion of oxygen of the samples (six at a time). The sludge mixtures were stirred continuously while the oxygen measurements were taken. The oxygen measurements were terminated after 10 minutes. The pH of the sludge mixture was measured immediately after the respiration measurement.
- The temperature of the environmental chamber in which the test was conducted was monitored using a minimum/maximum thermometer during the 3-hour contact time. Solution pH was measured with a pH meter. The Strathtox Respirometer was used for determination of O2 consumption. The oxygen depletion rate was measured in every sample from each test system.

TEST CONCENTRATIONS
A range-finding test was conducted using test concentrations of 10, 100 and 1000 mg/L. A definitive test was not conducted since the NOEC was > 1000 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (purity: 97.6 %)
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
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:
- At test termination, the respiration rate for the two control vessels was determined to be 45.3 and 46.5 mg O2/L/hr, which met the acceptability criterion (i.e., within 30 % of each other during the test). The average oxygen uptake rates of the two control vessels was determined to be 30.6 mg O2/g of sludge/hr, which met the acceptability criterion (i.e., > 20 mg O2/g of sludge/hr). The respiration rate in the test material vessels (10, 100, 1000, 1000, and 1000 mg/L) was 42.9, 42.0, 42.9, 42.0, and 42.1 mg O2/L/hr, respectively. Compared to the controls (mean of two values), respiratory inhibition by the test material in test vessels (10, 100, 1000, 1000, and 1000 mg/L) was 6.5, 8.5, 6.5, 8.5, and 8.3 %, respectively.
- Based on these results, the EC50 and EC10 values were both empirically estimated to be > 1000 mg/L. The NOEC was empirically estimated to be > 1 000 mg/L, since there was no statistical significance between the 1000 mg/L treatments and the controls.
- In order to confirm that no statistically significant effect was observed between the controls and the highest test substance treatment group (1000 mg/L), a one-tailed t-Test was conducted. The t-Test resulted in a value of 0.06 when the respiration rates of the controls were compared to the respiration rates of the highest test substance treatment group (1000 mg/L). Since the result of this t-Test was greater than 0.05, there is not a statistically significant difference between the respiration rates of the controls and the respiration rates of the highest test substance treatment group (1000 mg/L).
- The respiration rate of the abiotic control was 1.3 mg/h, which was considered insignificant since the oxygen concentration was near saturation at the end of the measurement.
Results with reference substance (positive control):
Respiration rate in the 3,5-dichlorophenol test vessels (1.0, 3.0, 10, and 30 mg/L) was 39.6, 40.3, 28.8, and 3.3 mg O2/L/hr, respectively. Compared to the controls (mean of two values), respiratory inhibition by 3,5-dichlorophenol in test vessels was 13.7, 12.2, 37.3, and 92.8 %, respectively. Based on these results, the EC50 value for 3,5-dichlorophenol was calculated to be 8.8 mg/L, which was within the acceptable limits as specified in the OECD 209 Guideline (i.e., 2 to 25 mg/L).
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the EC50 and EC10 values were both empirically estimated to be > 1000 mg/L. The NOEC was empirically estimated to be > 1000 mg/L, since there was no statistical significance between the 1000 mg/L treatments and the controls.
Executive summary:

The toxicity of the test material to microorganisms was investigated in a study which was conducted in accordance with the standardised guideline OECD 209, under GLP conditions.

During the study activated sewage sludge was exposed to test material at concentrations of 10, 100 and 1000 mg/L for a period of 3 hours at a temperature of approximately 20 ± 2 °C with the addition of activated sewage sludge as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and the positive control, 3,5-dichlorophenol.

At test termination, the respiration rate for the two control vessels was determined to be 45.3 and 46.5 mg O2/L/hr, which met the acceptability criterion (i.e., within 30 % of each other during the test). The average oxygen uptake rates of the two control vessels was determined to be 30.6 mg O2/g of sludge/hr, which met the acceptability criterion (i.e., > 20 mg O2/g of sludge/hr). The respiration rate in the test material vessels (10, 100, 1 000, 1 000, and 1000 mg/L) was 42.9, 42.0, 42.9, 42.0, and 42.1 mg O2/L/hr, respectively. Compared to the controls (mean of two values), respiratory inhibition by the test material in test vessels (10, 100, 1000, 1000, and 1000 mg/L) was 6.5, 8.5, 6.5, 8.5, and 8.3 %, respectively.

Under the conditions of the study, the EC50 and EC10 values were both empirically estimated to be > 1000 mg/L. The NOEC was empirically estimated to be > 1000 mg/L, since there was no statistical significance between the 1000 mg/L treatments and the controls.

Description of key information

The EC50 and EC10 values were both empirically estimated to be > 1 000 mg/L. The NOEC was empirically estimated to be > 1 000 mg/L, since there was no statistical significance between the 1 000 mg/L treatments and the controls.

Key value for chemical safety assessment

Additional information

The toxicity of the test material to microorganisms was investigated in a study which was conducted in accordance with the standardised guideline OECD 209, under GLP conditions.

The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Duringthe study activated sewage sludge was exposed to test material at concentrations of 10, 100 and 1 000 mg/L for a period of 3 hours at a temperature of approximately 20 ± 2 °C with the addition of activated sewage sludge as a respiratory substrate. The rate of respiration was determined after 3 hours contacttime andcompared to data for the control and the positive control, 3,5-dichlorophenol.

At test termination, the respiration rate for the two control vessels was determined to be 45.3 and 46.5 mg O2/L/hr, which met the acceptability criterion (i.e., within 30 % of each other during the test). The average oxygen uptake rates of the two control vessels was determined to be 30.6 mg O2/g of sludge/hr, which met the acceptability criterion (i.e., > 20 mg O2/g of sludge/hr). The respiration rate in the test material vessels (10, 100, 1 000, 1 000, and 1 000 mg/L) was 42.9, 42.0, 42.9, 42.0, and 42.1 mg O2/L/hr, respectively. Compared to the controls (mean of two values), respiratory inhibition by the test material in test vessels (10, 100, 1 000, 1 000, and 1 000 mg/L) was 6.5, 8.5, 6.5, 8.5, and 8.3 %, respectively.

Under the conditions of the study, the EC50 and EC10 values were both empirically estimated to be > 1 000 mg/L. The NOEC was empirically estimated to be > 1 000 mg/L, since there was no statistical significance between the 1 000 mg/L treatments and the controls.