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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-04-10 to 2013-04-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
- Concentrations: a series of nominal test concentrations of 10, 100 and 1000 mg ai/L, a negative control and a positive control
- Sampling method: As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe.
- Sample storage conditions before analysis: no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 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 ai/L. The test item was dissolved directly in water. All test concentrations were corrected for a test item content of 70 %.
- Controls: Negative control and 3,5-dichlorophenol as positive control
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
activated sludge
Details on inoculum:
- Laboratory culture: No, a mixed population of activated sewage sludge micro-organisms was obtained on 10 April 2013 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- Method of cultivation: 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.
- Preparation of inoculum for exposure: On the day of collection the activated sewage sludge (10 liters) was fed synthetic sewage sludge (500 mL). The pH of the sample on the day of the test was 7.7 measured using a WTW pH/Oxi 340I pH and dissolved oxygen 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 one 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.
- Pretreatment: At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 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 approximately 0.5 – 1 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. Then, the test item vessels were prepared. Finally two further control vessels were prepared.
- Initial biomass concentration: 16 mL of synthetic sewage (34.1 g/L) was diluted to 250 mL with water and 250 mL of inoculum (3.0 g/L) added in a 500 mL conical flask. This results to 9.275 g/500 mL (after recalculation).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC)
Test temperature:
20 ± 2 °C
pH:
control at 0 hours: 7.4-7.6 mg/L, after 3 hours: 8.1-8.3 (n=4)
test item: at 0 hours 7.2-7.5, after 3 hours 7.9-8.2
positive control: at 0 hours 7.6, after 3 hours 8.3-8.5
Dissolved oxygen:
control after 30 minutes: 5.8-6.7 mg/L (n=4)
test item: 6.0 mg/L for 10 mg/L, 6.1 mg/L for 100 mg/L, 5.1-7.1 mg/L for 1000 mg/L (n=3)
positive control: 6.2 mg/L for 3.2 mg/L, 7.1 mg/L for 10 mg/L, 6.7 mg/L for 32 m mg/L
Salinity:
not applicable
Nominal and measured concentrations:
test item concentrations of 10, 100 and 1000 mg ai/L
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: no information on material, 500 mL sie, 500 ml fill volume
- Aeration: yes, aeration with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 – 1 liter per minute.
- No. of organisms per vessel: 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum
- No. of vessels per concentration (replicates): 1 for 10 and 100 mg/L, 3 for 1000 mg/L
- No. of vessels per control (replicates): 4
- Biomass loading rate: 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC)
- Total organic carbon: ess than 1 mg/L Dissolved Organic Carbon (DOC)

OTHER TEST CONDITIONS
- Adjustment of pH: pH values of the test item stock solutions prior to the addition of inoculum were adjusted (prior to adjustment pH 1.6-3.5, after adjustment pH 7.2-7.5)
- Photoperiod: normal laboratory lighting
- Light intensity: normal laboratory lighting

EFFECT PARAMETERS MEASURED: respiration rate measured as change of oxygen concentration after 3 hours of exposure

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: yes
- Test concentrations: the same as for the definitive test
- Results used to determine the conditions for the definitive study: the results of the range finding test are used for the definitive test
Reference substance (positive control):
yes
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
corrected by purity
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
corrected by purity
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: The validation criterion for the reference item EC50 value was also satisfied.
Reported statistics and error estimates:
The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC20, EC50 and EC80 values determined from the equation for the fitted line. 95 % confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).
The results of the study are considered valid if (i) the EC50 (3-Hour contact time) for 3,5-dichlorophenol lies within the range 2 to 25 mg/L (ii) the specific respiration rate of the blank controls should not be less than 20 mg oxygen per gram dry weight of sludge per hour (iii) the coefficient of variation of oxygen uptake rate in control replicates should not be more than 30 % at the end of the test.

Table 1 Summary

  Methyl phosphonic acid 70% 3,5-dichlorophenol
ECx (3 Hours) (mg ai/L) 95% Confidence Limits (mg ai/L) ECx (3 Hours) (mg/L) 95% Confidence Limits (mg/L)
EC10 - - 2.3 -
EC20 - - 3.3 -
EC50 >1000 - 10 8.2 - 12
EC80 - - 32 -
NOEC 100 - - -

Table 2 Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test

Nominal Concentration (mg ai/L) Initial O2 Reading (mg O2/L) Measurement Period (minutes) Final O2 Reading (mg O2/L) O2 Consumption Rates (mg O2/L/hour) % Inhibition
Control R1 4.3 3 2.2 42 -
Control R2 5 4 2.2 42 -
Control R3 4.7 3 2.3 48 -
Control R4 6.2 7 1.9 36.86 -
Test Item 10 4.3 4 1.7 39 8
Test Item 100 4.7 5 2.1 31.2 26
Test Item 1000 R1 5.8 7 1.9 33.43 21
Test Item 1000 R2 7 10 2.8 25.2 40
Test Item 1000 R3 7.2 10 3.4 22.8 46
3,5-dichlorophenol 3.2 5.8 7 1.8 34.29 19
3,5-dichlorophenol 10 7.5 10 4 21 50
3,5-dichlorophenol 32 8.1 10 6.7 8.4 80
Validity criteria fulfilled:
yes
Conclusions:
Methyl phosphonic acid 70 % was determined to have a 3-Hour EC50 value of greater than 1000 mg ai/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg ai/L.
Executive summary:

A valid, reliable and conclusive study was performed to assess the effect of the test item Methyl phosphonic acid 70 % 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 active ingredient (ai)/L (3 replicates of the 1000 mg ai/L test concentration) for a period of 3 hours at a temperature of 20±2 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was 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 respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg ai/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg ai/L.

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

The reference item gave a 3-Hour EC50 value of 10 mg/L.

Description of key information

The effect of the test item Methyl phosphonic acid 70 % on the respiration of activated sewage sludge micro-organisms gave a 3-hour EC50 value of greater than 1000 mg ai/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg ai/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

In a valid, reliable and conclusive study according to OECD TG 209 (2010), the effect of the test item Methyl phosphonic acid 70 % on the respiration of activated sewage sludge micro-organisms gave a 3-hour EC50 value of greater than 1000 mg ai/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg ai/L, which is considered as the key value for risk assessment.