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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 January to 24 January 2019
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 (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1812101500R supplied by Sponsor
- Expiration date of the lot/batch: 10 December 2020
- Purity test date: 99.9116% w/w

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature under nitrogen in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: 10.4 µg/L
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
- Preliminary purification step (if any): None
- Final dilution of a dissolved solid, stock liquid or gel: added directly to water
- Final preparation of a solid: N/a

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
- other information:
Analytical monitoring:
no
Remarks:
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the ho mogeneity, concentration or stability of the test item formulation. This exception with regard to GLP has been reflected in the GLP compliance statement.
Vehicle:
yes
Remarks:
Deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Range-Finding Test
Test Item Preparation The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dispersed directly in water.
Nominal amounts of test item (5, 50 and 500 mg (500 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates). The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld
meter and adjusted to between pH 7.0 and 8.0 if necessary.

Definitive Test
In the Range Finding Test the dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.
No statistically significant toxic effects were shown at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture:
- Name and location of sewage treatment plant where inoculum was collected: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK
- Method of cultivation: A synthetic sewage of a specified composition, was added to each test vessel to act as a respiratory substrate:
The pH of the synthetic sewage stock used to feed the activated sewage sludge and used in tests to measure total and heterotrophic respiration was between pH 7.3 and 7.4.
- Preparation of inoculum for exposure:
- Pretreatment:
- Initial biomass concentration:A mixed population of activated sewage sludge micro-organisms was obtained on 16 January 2019 for the test to measure total respiration and on 23 January 2019 for the test to measure heterotrophic respiration from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
Preparation of Inoculum
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the
day of collection the activated sewage sludge (9.5 liters) was fed synthetic sewage (475 mL). The pH of the sample on the day of the test was 7.2 measured using a Hach HQ40d Flexi handheld meter.
Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
ca. 20 °C
pH:
7.2 - 7.8
Dissolved oxygen:
6.14 - 8.76 mg O2/L
Nominal and measured concentrations:
Nominal concentrations
Details on test conditions:
Range-Finding Test - Test Item Preparation
In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L (three replicates for the test concentration of 1000 mg/L). The test item was dispersed directly in water.
Nominal amounts of test item (5, 50 and 500 mg (500 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).
The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld meter (see Table 1) and adjusted to between pH 7.0 and 8.0 if necessary.
The exposure conditions for each flask were as described below.
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
The control group was maintained under identical conditions but not exposed to the test item.
Reference Item Preparation
A reference item, 3,5-Dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 20 minutes. The pH of this stock solution was measured to be pH 6.3 and was adjusted to pH
7.4 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.
The exposure conditions for each flask were as described below.

Specific Inhibitor of Nitrification Preparation
For the purpose of the test a specific inhibitor of nitrification, allylthiourea (ATU) was used. A stock solution of 1.16 g/L was prepared by dissolving a nominal amount of the reference item (290 mg) directly in water (250 mL) with the aid of ultrasonication for approximately 20 minutes. The pH of this stock solution was measured to be pH 6.7 and adjusted to pH 7.2 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution. An aliquot (5 mL) of the stock solution was removed and dispersed in the test vessels as described in Sections 3.5.2.1 and 3.5.2.2 to give the final concentration of 11.6 mg/L.

Preparation of Test System
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added to a final volume of 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. Two additional control vessels were then prepared prior to the test item vessels as described above. Finally, two further control vessels were prepared.
The test was conducted under normal laboratory lighting in a temperature controlled room at a measured temperature of approximately 20 °C.
Reference substance (positive control):
yes
Remarks:
A reference item, 3,5-Dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum.
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:
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:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
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:
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:
NOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
15 mg/L
Nominal / measured:
nominal
Conc. based on:
other: 3,5-dichlorophenol
Basis for effect:
inhibition of heterotrophic respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
1.4 mg/L
Nominal / measured:
nominal
Conc. based on:
other: 3,5-dichlorophenol
Basis for effect:
inhibition of nitrification rate
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
6 mg/L
Nominal / measured:
nominal
Conc. based on:
other: 3,5-dichlorophenol
Basis for effect:
inhibition of total respiration

The following Tables from the report are attached below in the background material section:

Table 1: pH Values of the Test Item Preparations after Stirring and Prior to the Addition of Inoculum in the Range-Finding Test to Measure Total Respiration

Table 2: Dissolved Oxygen Concentrations and Saturation of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test to Measure Total Respiration

Table 3: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test to Measure Total Respiration

Table 4: pH values of the test preparationsat the Start and End of the Exposure Period in the Range-Finding Test to Measure Total Respiration

Table 5: Observations on the Test Preparations throughout the Test Period in the Range-Finding Test to Measure Total Respiration

Table 6: pH Values of the Test Item Preparations after Stirring and Prior to the Addition of Inoculum in the Range-Finding Test to Measure Heterotrophic Respiration

Table 7: Dissolved Oxygen Concentrations and Saturation of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test to Measure Heterotrophic Respiration

Table 8: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test to Measure Heterotrophic Respiration

Table 9: pH Values of the Test Preparations at the Start and End of the Exposure Period in the Range-Finding Test to Measure Heterotrophic Respiration

Table 10: Observations on the Test Preparations throughout the Test Period in the Range-Finding Test to Measure Heterotrophic Respiration

Table 11: Percentage Calculated Inhibition Values after 3 Hours Contact Time in the Range-finding Test to Measure Nitrification Respiration

Figure 1: Concentration-Response Curve: 3,5-Dichlorophenol for Total Respiration – 3 Hour Contact Time

Figure 2: Concentration-Response Curve: 3,5-Dichlorophenol for Heterotrophic Respiration – 3 Hour Contact Time

Figure 3: Concentration-Response Curve: 3,5-Dichlorophenol for Nitrification Respiration – 3 Hour Contact Time

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the total, heterotrophic and nitrification respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure for the total, heterotrophic and nitrification respiration of activated sewage sludge micro-organisms was 1000 mg/L.
Executive summary:

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Methods

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (three replicates of the 1000 mg/L test concentration) with and without the presence of allylthiourea (ATU) for a period of 3 hours at measured temperatures of between 19 °C and 20 °C with the addition of a synthetic sewage as a respiratory substrate.

The rates of respiration (total respiration, heterotrophic respiration and nitrification respiration) were determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

Results

The effect of the test item on the total, heterotrophic and nitrification respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure for the total, heterotrophic and nitrification respiration of activated sewage sludge micro-organisms was 1000 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Description of key information

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (three replicates of the 1000 mg/L test concentration) with and without the presence of allylthiourea (ATU) for a period of 3 hours at measured temperatures of between 19 °C and 20 °C with the addition of a synthetic sewage as a respiratory substrate.

The rates of respiration (total respiration, heterotrophic respiration and nitrification respiration) were determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

The effect of the test item on the total, heterotrophic and nitrification respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure for the total, heterotrophic and nitrification respiration of activated sewage sludge micro-organisms was 1000 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Key value for chemical safety assessment

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

Additional information