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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 24 September 2015 to 18 July 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The temperature was below the range prescribed by the study plan (20 ± 2°C) during the exposure phase, the temperature was 20-23°C. The validity criteria for the controls and reference item were met and therefore this deviation is considered not to have influenced the sludge.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
GLP compliance:
yes
Remarks:
The certificate copy was not included in the study report.
Test material information:
Composition 1
Specific details on test material used for the study:
APPEARANCE
Reddish solid below 25°C / liquid above 25°C

SOURCE OF TEST MATERIAL
- Source and batch/lot No.of test material: Solvay / MR92143651 / 206887/A
- Expiration date of the batch: 01 January 2017
- Purity/Composition: 86.4%
- Purity/composition correction factor: No correction factor required
- Purity test date: 13 April 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature.
- Stability under test conditions: until 01 January 2017 (expiry date)
- Solubility of the test substance in the vehicle (20°C): Insoluble in Water
- Stability of the test substance in the vehicle (20°C): Not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Test substance handling: Must be heated to 40 - 50°C until complete melting of the crystallized part before being used in liquid form.
- No correction was made for the purity/composition of the test item.
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, 1-Litre test bottles were filled with 200 mL of test item mixtures in Milli- RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 24 hours. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rates. To ensure a reliable weighing of the test item, minimum reliable amount 1 mg, all volumes/amounts used for the lowest concentration in the final test (1.0 mg/L) were multiplied by a factor 2. Optimal contact between the test item and test organisms was ensured applying continuous aeration and stirring.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Test system: Micro-organisms in activated sludge.
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's- Hertogenbosch, The Netherlands.
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use. The initial pH was 5.8 on the day of testing. The sludge was buffered to a pH of 7.1 using sodium bicarbonate solution.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
Hardness:
Unspecified
Test temperature:
The temperature continuously measured in the temperature control vessels ranged between 20 and 23°C during the test, and was slightly outside the range prescribed by the study plan (20± 2°C).
pH:
The pH in all test vessels, before addition of sludge was between 7.4 and 7.7. After the 3 hour exposure period the pH was between 7.1 and 8.1.
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Conductivity:
Unspecified
Nominal and measured concentrations:
Seven loading rates tested: 1.0, 3.2, 10, 32, 100, 320 and 1000 mg/L.
Details on test conditions:
TEST SYSTEM
- Contact time: 3 hours, during which aeration and stirring took place.
- Test vessel: All glass open bottles/vessels.
- Air supply: Clean, oil-free air.
- Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration: 5 replicates.
- No. of vessels per control: 6 replicates.
- Blank / Nitrification controls: Test medium without test item and treated in the same way as the test item solutions. In the test series for heterotrophic respiration ATU was added.
(See table I in the "Any other information on materials and methods incl. tables" tab.)
- Nutrients provided for bacteria: Synthetic medium [16g peptone, 11g meat extract, 3g urea, 0.7g NaCl, 0.4g CaCl2.2H2O, 0.2g MgSO4.7H2O & 2.8g K2HPO4 dissolved in Milli-RO water, made up to 1 litre and filtered]. The pH was within 7.5 ± 0.5.
- Nitrification inhibitor used: N–allylthiourea, (ATU)

TEST MEDIUM / WATER PARAMETERS
- The synthetic medium (16 mL) made up to 50 mL with Milli-RO and 200 mL test item solution were mixed (total volume 250 mL). To ensure a reliable weighing of the test item, minimum reliable amount 1 mg, all volumes/amounts used for the lowest concentration in the final test (1.0 mg/L) were multiplied by a factor 2. The pH was determined. Thereafter 250 mL activated sludge was added. This was the start of the
test.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- The final test was performed based on the result of a preceding combined limit/range-finding test loading rates, ranging from 1.0 to 1000 mg/L, increasing with a factor 3.2.
- Test concentrations: Seven loading rates: 1.0, 3.2, 10, 32, 100, 320 and 1000 mg/L.
Reference substance (positive control):
yes
Remarks:
The reference item was 3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
other: NOELR
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: ELR50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: NOELR
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Duration:
3 h
Dose descriptor:
other: NOELR
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Details on results:
- Blank controls oxygen uptake rate: 20 mg oxygen per one gram of activated sludge.
- Coefficient of variation of oxygen uptake rate in control replicates: 16%
Results with reference substance (positive control):
- Results with reference substance valid? Yes, the EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration.
- Relevant effect levels: EC50 = 4.0 mg/L (95% confidence limits: 1.7 - 9.1)
Reported statistics and error estimates:
Normal-distribution and variance-homogeneity of data were tested using the Shapiro Wilk`s and Levene´s tests. No departures from normality or variance homogeneity were detected. Normal-distribution and variance-homogeneity requirements are fulfilled.
Inhibition of the respiration rate were then statistically analyzed comparing the data obtained for the test loading rates compared with those obtained in the blank control using the Williams Multiple Sequential t-test Procedure.

A summarized overview of the study results is presented in Table II and Table III.

Table II: Final test – Overview of the results: Total and heterotrophic respiration
Replicate Loading
rate
(mg/L)
pH Mean respiration rate % Inhibition
of the
respiration
rate
(mean value)
Start End (mg O2/L h) (mg O2/g h)¹
Blank control   7.6 7.1 - 7.7 29.38 19.58  
T1 1.0 7.6 7.1 - 7.3  44.97 29.98 -53
T2 3.2 7.6 7.5 28.11 18.74 4
T3 10 7.6 7.5 27.57 18.38 6
T4 32 7.6 7.3 - 7.5 28.22 18.81 4
T5 100 7.6 7.3 25.62 17.08 13
T6 320 7.6 7.3 27.12 18.08 8
T7 1000 7.4 - 7.5 7.1 - 7.2 25.02 16.68 15*
Nitrification control   7.6 - 7.7 7.9 - 8.1 18.62 12.41  
T1 + ATU 1.0 7.6 7.8 - 7.9 26.44 17.63 -42
T2 + ATU 3.2 7.6 7.9 20.65 13.77 -11
T3 + ATU 10 7.6 7.9 21.99 14.66 -18
T4 + ATU 32 7.6 7.9 23.43 15.62 -26
T5 + ATU 100 7.6 7.9 19.47 12.98 -5
T6 + ATU 320 7.6 7.8 18.30 12.20 2
T7 + ATU 1000 7.4 – 7.5 7.8 16.23 10.82 13
¹) The amount of suspended solids in the final test mixture was 1.5 g/L.
* Statistically significantly different compared to control

Table III: Final test – Overview of the calculations for nitrification respiration
Replicate Loading
rate
(mg/L)
Total
Respiration
rate
(mg O2/l.h)
RT
Heterotrophic
Respiration
rate
(mg O2/l.h)
RH
Nitrification
Respiration
rate
(mg O2/l.h)
RN
% Inhibition of the
respiration
rate
(mean value)
Blank 0 29.38 18.62 10.76 -
1 1.0 44.97 26.44 18.53 -72
2 3.2 28.11 20.65 7.46 31
3 10 27.57 21.99 5.58 48
4 32 28.22 23.43 4.79 56
5 100 25.62 19.47 6.15 43
6 320 27.12 18.30 8.82 18
7 1 000 25.02 16.23 8.79 18

No statistically significant inhibition of the total respiration rate of the sludge was recorded at or below a loading rate of 320 mg 1 -Phenyldecane-1,3 -dione per litre.

No statistically significant inhibition of the heterotrophic respiration rate and nitrification was observed at any of the tested loading rates. Since the heterotrophic respiration was stimulated at or below the concentration of 320 mg/L and the nitrification is calculated using both total and heterotrophic respiration the inhibition of nitrification could be overestimated for these concentration. The effect on nitrification inhibition at all concentrations was not statistically significant different from the blank control.

For total, heterotrophic and nitrification inhibition the ELR50 was beyond the range tested (>1000 mg/L).

Validity criteria fulfilled:
yes
Remarks:
OECD validity criteria, see field "overall remarks"
Conclusions:
Under the conditions of this present test 1-Phenyldecane-1,3-dione was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 320 mg/L (NOELR total respiration).
For total, heterotrophic and nitrification inhibition the ELR50 was beyond the range tested (>1000 mg/L).
Executive summary:

The influence of 1-Phenyldecane-1,3-dione on the activity of activated sludge was evaluated by measuring the [total, heterotrophic and nitrification] respiration rate under defined conditions, according to EU Method C.11 and OECD 209 Guidelines.

The final test was performed based on the result of a preceding combined limit/range-finding test loading rates, ranging from 1.0 to 1000 mg/L, increasing with a factor 3.2 were tested. Five replicates per loading rate and six replicates for an untreated control group were tested. The final test was performed with and without a nitrification inhibitor (N–allylthiourea, (ATU)).

No statistically significant inhibition of the total respiration rate of the sludge was recorded at or below a loading rate of 320 mg of test item /L. No statistically significant inhibition of the heterotrophic respiration rate and nitrification was observed at any of the tested loading rates. Since the heterotrophic respiration was stimulated at or below the concentration of 320 mg/L and the nitrification is calculated using both total and heterotrophic respiration the inhibition of nitrification could be overestimated for these concentration. The effect on nitrification inhibition at all concentrations was not statistically significant different from the blank control. For total, heterotrophic and nitrification inhibition the ELR50 was beyond the range tested (>1000 mg/L).

The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Description of key information

One study allowing to measure the influence of 1-Phenyldecane-1,3-dione on the activity of activated sludge according to EU Method C.11 and OECD 209 Guidelines is available. That key study was carried out without major deviation to the guideline and according to GLP.

That test was performed based on the result of a preceding combined limit/range-finding test loading rates, ranging from 1.0 to 1000 mg/L, increasing with a factor 3.2 were tested. Five replicates per loading rate and six replicates for an untreated control group were tested. The final test was performed with and without a nitrification inhibitor (N–allylthiourea, (ATU)).

No statistically significant inhibition of the total respiration rate of the sludge was recorded at or below a loading rate of 320 mg of test item /L. No statistically significant inhibition of the heterotrophic respiration rate and nitrification was observed at any of the tested loading rates. Since the heterotrophic respiration was stimulated at or below the concentration of 320 mg/L and the nitrification is calculated using both total and heterotrophic respiration the inhibition of nitrification could be overestimated for these concentration.

The effect on nitrification inhibition at all concentrations was not statistically significant different from the blank control. For total, heterotrophic and nitrification inhibition the ELR50 was beyond the range tested (>1000 mg/L). 

Key value for chemical safety assessment

EC10, LC10 or NOEC for microorganisms:
320 mg/L

Additional information