Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 16 May 2016 and 17 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
not required

Test solutions

Vehicle:
no
Details on test solutions:
N/A

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 17 May 2016 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and was used on the day of collection. The pH of the sample was 7.7 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 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.

Study design

Test type:
static
Water media type:
freshwater
Remarks:
The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Hardness:
Not reported
Test temperature:
20”C
pH:
6.9 - 7.6
Dissolved oxygen:
5.5 - 7.3 mg O2/L
Salinity:
Not reported
Nominal and measured concentrations:
5, 50 and 500 mg(nominal)
Details on test conditions:
Range-Finding Test
est 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. The test item was dispersed directly in water.
Nominal amounts of test item (5, 50 and 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 temperatures of between 20 C and 21 C, 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 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 6.2 and was adjusted to pH 7.2 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

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 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 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. The test item vessels were prepared as describedabove. 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 20 ºC.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrations
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Range-Finding Test

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L.
No statistically significant toxic effects were shown at the test concentration of 10 mg/L, however statistically significant toxic effects were shown at the test concentrations of 100 and 1000 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
Based on these results (the EC50 value for the test item was greater than 100 mg/L) and after discussion with the Sponsor, it was considered unnecessary to perform a definitive test in order to obtain a NOEC and/or an exact EC50 value for the test item

Any other information on results incl. tables

Validation Criteria

The coefficient of variation of oxygen uptake in the control vessels was 1.6% and the specific respiration rate of the controls was 20.86 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied. The validation criterion for the reference item EC50 value was also satisfied.

Inhibition of Respiration Rate

The following results were derived:

   X-19574 Amines, C16-18 and C18-unsatd. alkyl, O,O-di-Bu phosphorothioates, CASRN 97808-07-6      3,5-dichlorophenol    
    ECx (3 Hours) (mg/L)  95% Confidence Limits (mg/L)  ECx (3 Hours) (mg/L) 95% Confidence Limits (mg/L) 
 EC10  10 -100  -  1.7  -
 EC20  10 -100  -  2.5  -
 EC50  100 -1000  -  7.5  5.9 -9.6
 EC80  100 -1000  -  23  -

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 100 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 (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a measured temperature of 20 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.

Results

The effect of the test item on the respiration of activated sewage sludge gave a 3 Hour EC50 value of greater than 100 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L