Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July to August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD, EPA, etc)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): XP-7688
- Substance type: white powder
- Physical state: Solid
- Analytical purity: 99.5 wt% obtained by proton NMR
- Purity test date: 99.5 wt% (16 May 2011)
- Lot/batch No.: 10188-1 JM
- Expiration date of the lot/batch: 28 February 2015
- Storage condition of test material: Room temperature in the dark
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
For details on properties of the test item, refer to Chapter 4. Physical and chemical properties

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- the test item was dispersed directly in deionised reverse osmosis water. Amounts of test item were dispersed in deionised reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours in order to maximise the dissolved test item concentration.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Culture source: A mixed population of activated sewage sludge micro-organisms obtained on 21 July 2011 from the aeration stage of the Severn Trent Water Pic sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- Method of cultivation: Maintained on continuous aeration in the laboratory at a temperature of approximately 21°C.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Hardness:
< 1 mg/L Dissolved Organic Carbon (DOC) = content in the deionised reverse osmosis water.
Test temperature:
20 +/- 2°C
pH:
- Control vessels :
* Start of the exposure period: 7.5 (mean, SD=0.08, n=4)
* End of the exposure period: 7.6 (mean, SD=0.05, n=4)
- Test item:
* Start of the exposure period: 7.5 (mean, SD=0.05, n=5)
* End of the exposure period: 7.6 (mean, SD=0.045, n=5)
- Reference substance:
* Start of the exposure period: 7.4 (mean, SD=0.05, n=3)
* End of the exposure period: 7.9 (mean, SD=0.06, n=3)
Dissolved oxygen:
Control vessels :
* Start of the exposure period: 5.275 mg O2/L (mean, SD=0.22, n=4)
- Test item:
* Start of the exposure period: 5.22 mgO2/L (mean, SD=0.15, n=5)
- Reference substance:
* Start of the exposure period: 7.1 mgO2/L (mean, SD=0.8, n=3)
Nominal and measured concentrations:
- Nominal concentration of the test item: 10, 100, 1000 mg/L
- Nominal concentration of the reference item : 3.2, 10, 32 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL conical flask
- Type (delete if not applicable): closed
- Aeration: 0.5 to 1 litre per minute.
- No. of vessels per concentration (replicates): 1 replicate for the concentration of 10 and 100 mg/L nominal concentration and 3 replicates for the concentration of 1000 mg/L; 1 replicates for each of the 3 concentrations used for the reference substance.
- No. of vessels per control (replicates): 4 replicates
- Biomass loading rate: Suspended solid concentration: 2.8 g/L - 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 deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105°C for at least 1-Hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.

TEST MEDIUM / WATER PARAMETERS
- Preparation of the synthetic sewage:
A synthetic sewage was added to each test vessel to act as a respiratory substrate:
* Peptone: 16g
* Meat extract: 11g
* Urea: 3g
* NaCI: 0.7 g
* CaCl2.2H20: 0.4g
* MgS04.7H20: 0.2 g
* K2HP04: 2.8 g
Dissolved in 1 litre of water with the aid of ultrasonication. The pH of the synthetic sewage stock was 6.9 and adjusted to 7.1 using 1.0 M NaOH. The pH values were measured using a WTW pH320 pH meter.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- The Biological Oxygen Demand (BOD)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Factor of 10
- Range finding study: The current test description was a range finding study and no definitive test has been conducted as it has been considered not justifiable.
- Test concentrations: 10, 100 and 1000 mg/L nominal concentration of the test item
Reference substance (positive control):
yes
Remarks:
3.5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
other: EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: -
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other:
Remarks on result:
other: -
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: -
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: -
Results with reference substance (positive control):
The coefficient of variation of oxygen uptake in the control vessels was 3% and the specific respiration rate of the controls was 22.2 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 --> refer to the below table for further details.
Reported statistics and error estimates:
Calculation of the respiration rate (R, mg 02/1/h):
Linear part of the recorded oxygen decrease graph according to the following equation:
R = (Q1 - Q2)/delta t x 60
Where:
Q1 = oxygen concentration at the beginning of the selected section of the linear phase (mg/l);
Q2 = oxygen concentration at the end of the selected section of the linear phase (mg/l);
Delta t = time interval between these two measurements (min)

The specific respiration rate, Rs, expressed as the amount of oxygen consumed per gram dry weight of sludge per hour (mg/gh) was deduced according to the following equation:
Rs = R / SS
Where:
SS = concentration of suspended solids in the test mixture (g/l).

The percentage inhibition was calculated according to the equation:
% inhibition = [1 - (R/Rbc)] x 100
Where:
Rbc is the mean respiration rate of the blank controls.

The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (lOBS) and the EC15, EC20, EC50 and EC80 values determined from the equation for the fitted line. The EC15, EC20 , EC50 and EC80 values for the test item were determined by inspection of the inhibition of respiration rate data. 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).

Any other information on results incl. tables

* Oxygen consumption rates and percentage inhibition values after 3 hours contact time

 Substance  Nominal concentration (mg/L)  Initial O2 reading (mg O2/L) Measurement period (min)   Final O2 Reading (mg O2/L) O2 consumption rates (mg O2/L/hour)  % inhibition 
 Control  R1 5.2 5 2.5 32.4  -
   R2 5.0  2.5  30.0 
   R3 5.4  2.3  31.0 
   R4 5.5  2.4  31.0 
 Test item 10  5.2  2.5  32.4   
   100 5.4  2.4  30.0   
   1000 R1 5.2  2.5  32.4   
   1000 R2 5.3  2.6  32.4   
   1000 R3 5.0  2.7  34.5   
3.5 -dichlorophenol   3.2 6.3  10  2.4  23.4  25 
   10 7.1  10  5.1 12.0  61 
   32 7.9  10 7.3  3.6  88 

* Dissolved oxygen concentrations of the test preparations after 30 minutes contact time

Substance    Nominal concentration (mg/L) Dissolved oxygen concentration (mg O2/L) 
 Control  R1  6.2
   R2  5.8
   R3  6.1
   R4  6.2
 Test item  10  6.4
   100  6.1
   1000 R1  5.7
   1000 R2  5.9
   1000 R3  6.1
 3.5 -dichlorophenol  3.2  6.4
   10  6.8
   32  7.0

EC values for the reference susbtance: 3.5 -dichlorophenol :

 

3.5 -dichlorophenol

 
   ECx (3 hours) (mg/L)  95% Confidence limits (mg/L)
 EC20  2.5  -
 EC50  7.5  5.9 -9.5
 EC80  23  -
 NOEC  2.1  -

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of 6H-Dibenz [c,e] [1,2] oxaphosphorin 6,6'( 1,2-ethanediyl) bis-6,6'-dioxide on the 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 was 1000 mg/l.
Executive summary:

Introduction. A study was performed to assess the effect of 6H-Dibenz [c,e] [1,2] oxaphosphorin 6,6'( 1,2-ethanediyl) bis-6,6'-dioxide on the respiration of activated sewage sludge. The method followed was 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) 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.

Results. The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l. The reference item gave a 3-Hour EC50 value of 7.5 mgtl, 95% confidence limits 5.9 - 9.5 mg/l.