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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 November 2007 to 20 November 2007
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:
1984
Deviations:
yes
Remarks:
Copper(II)sulphate pentahydrate was used as reference substance. As per updated guideline (2010), this reference substance can also be used.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
No pretreatment was used. The dry sludge concentration was 3.92 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
not reported
Test temperature:
21.6 °C
pH:
7.04
Nominal and measured concentrations:
Nominal concentrations for pre-test: 10, 100, 1000 mg/L; main study: 1000 mg/L (nominal)
Details on test conditions:
The test substance was stirred in water for 24 hours before testing to yield a suspension.
Reference substance (positive control):
yes
Remarks:
Copper(II)sulphate pentahydrate
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 total respiration
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: foam with test substance flakes was observed when test vessels were aerated;
- Effect concentrations exceeding solubility of substance in test medium: solubility was exceeded;
- Adsorption (e.g. of test material to the walls of the test container): none reported
- Blank controls oxygen uptake rate: 33.6 and 32.0 mg O2/L.h
- Coefficient of variation of oxygen uptake rate in control replicates: not reported


 
Results with reference substance (positive control):
Results with the reference substance were valid. The EC50 for copper(II)sulphate pentahydrate was determiined to be 107 mg/L (acceptable range: 53-157 mg/L)
Reported statistics and error estimates:
The EC50 of the reference substance was calculated with probit analysis using the software SigmaPlot.

Percentage inhibition (%) in the range-finding test:

10 mg/L: -10%

100 mg/L: 6%

1000 mg/L: -3%

Percentage inhibition (%) in main study:

1000 mg/L: 4%

 

Validity criteria fulfilled:
yes
Conclusions:
There was a 4% inhibition at 1000 mg/L. The NOEC (3h) was determined to be 1000 mg/L, and the EC50 (3h) was > 1000 mg/L.
Executive summary:

The study was conducted according to OECD test guideline 209 (1984). The test substance did show only minimal inhibition at 1000 mg/L. The 3 hour EC50 of the test substance was calculated as >1000 mg/L. The NOEC was determined to be 1000 mg/L using direct observation.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 December 1999 to 19 January 2000
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:
1984
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
1987
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Version / remarks:
Draft guideline
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
- Concentrations: 32, 100, 320, 1000 and 3200 mg/L
- Sampling method: not reported;
- Sample storage conditions before analysis: not reported.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preliminary investigational work conducted for the validation of mixing period indicated that the concentration of dissolved organic carbon increased over time until 144 hours where a plateau was reached. It was therefore considered appropriate to stir the test item/water combination for a total preparation period of 144 hours prior to exposing test organisms in order to allow equilibration between the test item and aqueous phase to occur and to ensure that the test organisms were exposed to the maximum dissolved test material/leachate concentration.
- Controls: yes, maintained under identical conditions but not exposed to test item;
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): the test item was prepared by a direct dispersion in water. Amounts of test material (16, 50, 160, 500 and 1600 mg) were each separately dispersed in approximately 250 mL of water with the aid of stirring by magnetic stirrer (ca. 100% vortex depth) for a total preparation period of 144 hours. Synthetic sewage (16 mL), activated sewage sludge (200 mL) and water were added to a final volume of 500 mL to give the required concentrations of 32, 1 00, 320, 1000 and 3200 mg/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): yes, from 100 mg/L onwards;
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Severn Trent Water PIc sewage treatment plant at Belper, Derbyshire, United Kingdom;
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of 21 °C and was used on the day of collection;
- Pretreatment: no
- Initial biomass concentration: suspended solids were equal to 4.0 g/L prior to use;
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
no
Hardness:
100 mg/L as CaCO3
Test temperature:
21 °C
pH:
7.9-8.0 (controls 7.8-7.9)
Dissolved oxygen:
7.0-8.0 mg/L (controls 7.5-8.1)
Salinity:
not appplicable
Conductivity:
249-533 µS/cm
Nominal and measured concentrations:
32, 100, 320, 1000 and 3200 mg/lL (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL conical flasks;
- Aeration: with compressed air via narrow bore glass tubes at a rate of approximately 0.5 -1 litre per minute;
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per abiotic control (replicates): -
TEST WATER PARAMETERS
- Source/preparation of dilution water: The test water used for both the range-finding and definitive studies was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 12480 Duplex Water Softener) giving water with a total hardness of approximately 100 mg/L as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
SYNTHETIC SEWAGE
- as per guideline;
OTHER TEST CONDITIONS
- Adjustment of pH: not reported;
- Photoperiod: not reported;
EFFECT PARAMETERS MEASURED: oxygen consumption after 30 minutes and 3 hours;
RANGE FINDING STUDY
- Test concentrations: 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
3 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: Value obtained by extrapolation of the concentration response curve as no concentration tested resulted in 50% inhibition of respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
4 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: Value obtained by extrapolation of the concentration response curve as no concentration tested resulted in 50% inhibition of respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
UNDISSOLVED TEST ITEM
Observations made throughout the study period showed that at the test concentrations of 100, 320, 1000 and 3200 mg/L grey dispersions were formed with small particles of test material dispersed throughout the test preparation and floating on the surface. This is due to the insoluble nature of the test material in water. At the test concentration of 32 mg/L, a dark grey dispersion was formed, however no undissolved test material was visible.
- Blank controls oxygen uptake rate: within normal ranges;
- Coefficient of variation of oxygen uptake rate in control replicates: +/- 0% (30 min), +/- 1% (3 hours)
RANGE-FINDING STUDY:
Oxygen consumption rates and percentage inhibition values were determined in two range-finding studies for the control, test item and reference material.
The results of the initial range-finding study indicated a flat response of the activated sewage sludge micro-organisms to the test item, with inhibition values of 3%, 11 % and 16% being obtained for the 10, 100 and 1000 mg/L test concentrations, respectively.
The results of the second range-finding study showed that no significant inhibition of respiration occurred at 1000 mg/L, however at the test concentration of 2000 mg/lL 29% inhibition occurred. The difference in inhibition values obtained for the 1000 mg/L test concentration in the two rangefinding studies performed is considered to be a result of the relatively flat response of the activated sewage sludge to the test material. Based on these results a test concentration range of 32, 100, 320, 1000 and 3200 mg/L was selectedf or the definitive study in ordert o ensuret hat a No ObservedE ffect Concentration was obtained and to possibly obtain an EC50 value.
Results with reference substance (positive control):
Results with reference substance valid. yes
- 3,5-dichlorophenol, EC50 (30 minutes): 16 mg/L
- 3,5-dichlorophenol, EC50 (3 hours): 15 mg/L;
Reported statistics and error estimates:
none
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge resulted in a 3 -hour EC50 of 4500 mg/L (extrapolated value from the concentration response curve as no concentration tested resulted in 50% inhibition of respiration. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 320 mg/L.
Executive summary:

A GLP study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", EEC Commission Directive 87/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800. Following preliminary range-finding studies, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 32, 100, 320, 1000 and 3200 mg/L for a period of 3 hours at 21°C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 30 minutes and 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol. The effect of the test item on the respiration of activated sewage sludge resulted in a 3 -hour EC50 of 4500 mg/L (extrapolated value from the concentration response curve as no concentration tested resulted in 50% inhibition of respiration.

The No Observed Effect Concentration (NOEC) after 3 hours exposure was 320 mg/L.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 May 2019 to 22 May 2019 (experimental phase)
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
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was pipetted directly into the test vessels;
- Controls: yes, maintained under identical conditions but not exposed to test item.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Pretreatment: Upon arrival in the test facility, the sludge was filtrated, washed with tap water 3 times and re-suspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed /L
- The dry matter was determined as 3.1 g suspended solids/L, giving a concentration of 1.55 g suspended solids/L in the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
no
Hardness:
0.88 mM
Test temperature:
18.5 – 20.9 °C
pH:
8.2-8.3 (controls 8.3)
Salinity:
not appplicable
Conductivity:
210 µS/cm
Nominal and measured concentrations:
2.5, 25, 250 and 2500 mg/L (nominal, corresponding to 1, 10, 100 and 1000 mg/L SiO2 in the test item)
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (800 – 1000 mL) were used as test vessels.Narrow-neck glass bottles with flat bottoms (250 mL) were used for O2 measurements.
- Aeration: purified air, using Pasteur pipettes;
- No. of vessels per concentration (replicates): 1 (lower concentrations), 5 (highest concentration)
- No. of vessels per control (replicates): 1 (positive control), 2 before and 2 after measuring positive control and test item, respectively.
- No. of vessels per abiotic control (replicates): -
TEST WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water, CoA available;
SYNTHETIC SEWAGE
- as per guideline;
EFFECT PARAMETERS MEASURED: oxygen consumption after 3 hours;
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 2 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: value corresponds to >1000 mg SiO2/L
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
2 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: value corresponds to 1000 mg SiO2/L
Details on results:
Validity criteria were fulfilled:
- The coefficient of variation of oxygen uptake rate in control replicates was +/- 6.9% (i.e. less than 30%)
- The mean specific respiration rate of the blank controls was 27.61 mg oxygen per gram dry weight of sludge per hour (i.e. was > 20 mg oxygen per gram dry weight of sludge per hour).
- The reference substance results were valid, the EC50 for 3,5-dichlorophenol was within the expected range: 2 to 25 mg/L.
Results with reference substance (positive control):
Results with reference substance valid: yes
- 3,5-dichlorophenol, EC50 (3 hours): 10 mg/L (95% CI 5.4-15 mg/L);
Reported statistics and error estimates:
For the treatments with the test item concentration 2500 mg/L (corresponding to 1000 mg SiO2/L), it was tested whether the differences between treatment and blank control were significant. Based on equality of variance, the t-test was used (level of significance: 95%).
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge resulted in a 3 -hour EC50 of > 2500 mg/L (corresponding to 1000 mg SiO2/L). The No Observed Effect Concentration (NOEC) after 3 hours exposure was 2500 mg/L (corresponding to 1000 mg SiO2/L)


Executive summary:

A GLP study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals No 209, adopted in 2010 and Commission Regulation (EU) No. 2016/266 amending Regulation (EC) No. 440/2008, EU-Method C.11., adopted in 2015. Activated sewage sludge from a mainly domestic sewage treatment plant was exposed to the test item at concentrations of 2.5, 25, 250, and 2500 mg/L (corresponding to 1, 10, 100 and 1000 mg SiO2/L) at a temperature of 20 +/- 2 °C for a period of 3 hours with the addition of synthetic sewage as respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol. The effect of the test item on the respiration of activated sewage sludge resulted in a 3 -hour EC50 of > 2500 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 2500 mg/L (corresponding to 1000 mg SiO2/L).

Description of key information

In GLP studies in accordance with OECD testing guideline 209 and EU methods, no effects were noted with colloidal and surface-treated (hydrophobic) synthetic amorphous silica (SAS) up to and including the highest tested concentration of 1000 mg/L (tested as dispersions). Effects on the respiration of activated sewage sludge were noted with another surface-treated SAS (tested as dispersion; after stirring for 144 hours), resulting in a No Observed Effect Concentration (NOEC) after 3 hours exposure of 320 mg/L. The 3-hour EC50 was 4500 mg/L (extrapolated value from the concentration response curve as no concentration tested resulted in 50% inhibition of respiration).

Key value for chemical safety assessment

Additional information