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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 August to 25 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 209 with GLP statement. All validity criteria were fulfilled.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
No additional information
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: No correction was made for the purity/composition of the test item. 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. The test substance was added by pipetting the required amount just below the surface of the test medium. These mixtures were briefly stirred (ca. 5 minutes) in closed dark brown bottles. 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. Optimal contact between the test item and test organisms was ensured applying continuous aeration and stirring.
- Controls: Test medium without test item and treated in the same way as the test item solutions.
Test organisms (species):
other: Micro-organisms in activated sludge
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of the sludge: 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 pH was 7.6 on the day of testing. 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.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
18-21 °C
pH:
pH in all test vessels, before addition of sludge was between 7.8 and 7.9. After the 3 hour exposure period the pH was between 7.5 and 8.3.
Dissolved oxygen:
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.
27 and 30 mg oxygen per one gram of activated sludge, for the combined limit/range-finding and final test respectively.
Salinity:
None
Nominal and measured concentrations:
Five loading rates: 46, 100, 220, 460 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass open bottles/vessels
- 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.
- Air supply: Clean, oil-free air
- No. of vessels per concentration (replicates): 5 replicates per test group
- No. of vessels per control (replicates): 6 replicates for the control

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO / Milli-RO water (Tap-water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA))
- Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition: CaCl2.2H2O: 211.5 mg/L; MgSO4.7H2O: 88.8 mg/L; NaHCO3: 46.7 mg/L; KCl: 4.2 mg/L
- Synthetic medium (=sewage feed): 16 g peptone; 11 g meat extract; 3 g urea; 0.7 g NaCl; 0.4 g CaCl2.2H2O; 0.2 g MgSO4.7H2O; 2.8 g K2HPO4; Dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

PERFORMANCE OF THE TEST
- 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). The pH was determined. Thereafter 250 mL activated sludge was added. This was the start of the test. 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.
- The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls.
- The medium temperature was recorded continuously in a temperature control vessel(s). The temperature control vessel(s) was/were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.
- Oxygen recording: Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study: In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/L were tested. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. In addition, a blank control (6 replicates) and a nitrification control (2 replicates) were included. Furthermore, an abiotic control (1 replicate) and the highest loading rate with a nitrification inhibitor (3 replicates) were tested.
Test procedure and conditions were similar to those applied in the final test with the following exceptions:
- Inhibitor of nitrification: A 2.32 g/L solution of N-allylthiourea (ATU, Merck Schuchardt OHG, Hohenbrunn, Germany) was prepared. 2.5 mL of this solution was added to 500 mL final test medium (final ATU concentration: 11.6 mg/L).
- Results used to determine the conditions for the definitive study:
The combined limit/range-finding test showed no inhibition, 9 and an average of 37% inhibition of the respiration rate at a loading rate of 10, 100 and 1000 mg/L, respectively.
There was no oxygen uptake from abiotic processes and the result at 1000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was 46%. Based on the results of the total and heterotrophic respiration, nitrification was calculated not to be inhibited. Therefore, N-allylthiourea was not used in the final test. The temperature continuously measured in the temperature control vessels ranged between 19 and 22 °C during the test, and complied with the requirements as laid down in the study plan (20 ± 2 °C). The pH in all test vessels, before addition of sludge was between 7.5 and 7.6. After the 3 hour exposure period the pH was between 7.4 and 8.3.
Based on the results, the final test was performed with the loading rates of 46, 100, 220, 460 and 1000 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
other: NOEL
Effect conc.:
46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EL10
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% CI 0.31-75 mg/L
Key result
Duration:
3 h
Dose descriptor:
other: EL50
Effect conc.:
960 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 46 mg per litre. At higher loading rates the inhibitory effect of test substance on aerobic waste water (activated sludge) bacteria increased with increased loading rate, ranging from 22% inhibition at 100 mg/L to 47% at 1000 mg/L.
Results with reference substance (positive control):
The batch of activated sludge was tested for sensitivity with the reference substance 3,5-dichlorophenol, and showed normal sensitivity.
The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (6.0 and 5.5 mg/L, for the combined limit/range-finding and final test respectively).
Reported statistics and error estimates:
ECx and ELx: For the reference item calculations calculation of EC50 values was based on probit analysis using linear maximum likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.
Calculation of ELx values were based on probit analysis using linear maximum likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding loading rates of the test item.
NOEL determination: An effect was considered to be significant if statistical analysis of the data obtained for the test loading rates compared with those obtained in the blank control revealed significant inhibition of the respiration rate (Welch-t test for Inhomogeneous Variances with Bonferroni-Holm Adjustment, α=0.05, one-sided, smaller).
The calculations were performed with ToxRat Professional v. 3.0.0 (ToxRat Solutions® GmbH, Germany).

Table 6.1.7/3: Final test – Overview of the results

 

Flask

 

Loading rate

(mg/L)

pH

 

Mean respiration rate

 

% Inhibition of the respiration rate relative to the blank control

(mean value)

Start

End

(mg O2/l h)

 

(mg O2/g h)1

 

Blank control

 

-

7.8

7.5-8.0

44

30

-

T1

46

7.8

7.7-7.9

41

27

7

T2

100

7.8

7.7-7.8

35

23

22*

T3

220

7.8

7.5-7.9

27

18

39*

T4

460

7.8

7.5-7.9

27

18

40*

T5

1000

7.8

7.5-7.7

24

16

47*

¹)The amount of suspended solids in the final test mixture was 1.5 g/L

* Statistically significantly different compared to control

 

Acceptability of the test

 

The mean blank control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (27 and 30 mg oxygen per one gram of activated sludge, for the combined limit/range-finding and final test respectively).

The coefficient of variation of oxygen uptake in blank control replicates did not exceed 30% at the end of the definitive test (12-17%).

The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (6.0 and 5.5 mg/L, for the combined limit/range-finding and final test respectively).

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, test substance was not toxic to waste water bacteria (activated sludge) at a loading rate of 46 mg/L (NOEL). The EL50 was estimated to be 960 mg/L.
Executive summary:

The effect of test substance on the respiration rate of activated sludge was investigated after a contact time of 3 h, according to OECD Guideline 209 with GLP statement.

The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, the test item – Milli-RO water mixtures were briefly stirred (ca. 5 minutes). Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test item and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

 

Following a combined limit/range-finding test, final test was performed with five loading rates, 46, 100, 220, 460 and 1000 mg/L, increasing with a factor 2.2 were tested. Five replicates per loading rate and six replicates for an untreated control group were tested.

 

No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 46 mg/L. At higher loading rates the inhibitory effect of test substance on aerobic waste water (activated sludge) bacteria increased with increased loading rate, ranging from 22% inhibition at 100 mg/L to 47% at 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.

 

Under the test conditions, test substance was not toxic to waste water bacteria (activated sludge) at a loading rate of 46 mg/L (NOEL). The EL10 was determined at a loading rate of 25 mg/L but is not taken into account considering the high 95% confidence interval (0.31 - 75 mg/L). The EL50 was estimated to be 960 mg/L.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, (eco)toxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances are structurally related, in that both are 1-[(x,x)-dimethyl-1-cyclohexen-1-yl]-pent-4-en-1-one, which can exist as alpha (1-[(5,5)-dimethyl...) or beta (1-[(3,3)-dimethyl...) forms, meaning the position of the double bond in the hexane cycle differs.
The target substance is the isomer alpha (1-(5,5-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one).
The source substance is a mixture of isomer alpha (1-(5,5-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one), present as the major constituent between 60 and 75% in the mixture, and corresponding to the target (mono-constituent) substance; and isomer beta (1-(3,3-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one), present between 25 and 35%.

3. ANALOGUE APPROACH JUSTIFICATION
The similarity of the composition, physicochemical and environmental properties and ecotoxicological profile between the target and source substances is pronounced. Therefore, isomerism is not expected to significantly influence the toxicity to microorganisms between the target and source substances.

4. DATA MATRIX
See attached document, included in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Key result
Duration:
3 h
Dose descriptor:
other: NOEL
Effect conc.:
46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EL10
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% CI 0.31-75 mg/L
Key result
Duration:
3 h
Dose descriptor:
other: EL50
Effect conc.:
960 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 46 mg per litre. At higher loading rates the inhibitory effect of test substance on aerobic waste water (activated sludge) bacteria increased with increased loading rate, ranging from 22% inhibition at 100 mg/L to 47% at 1000 mg/L.
Validity criteria fulfilled:
not applicable
Conclusions:
Under the test conditions, it can be concluded that the test substance and the target substance are not toxic to waste water bacteria (activated sludge) at a loading rate of 46 mg/L (NOEL). The EL50 was estimated to be 960 mg/L.
Executive summary:

No study was performed on the target substance to assess the toxicity to microorganisms. However, a study was performed on a source substance, to assess the effect of the test substance on the respiration rate of activated sludge after a contact time of 3 hours, according to OECD Guideline 209 with GLP statement.

The source substance is a mixture of two isomers: 1-((5,5)-Dimethyl-1-cyclohexen-1-yl)pent-4-en-1-one (present at ca. ~67% in the mixture, corresponding to the target substance) and 1-((3,3)-Dimethyl-1-cyclohexen-1-yl)pent-4-en-1-one (present at ca. ~30% in the mixture).

 

Following a combined limit/range-finding test, the final test was performed with five loading rates, 46, 100, 220, 460 and 1000 mg/L, increasing with a factor 2.2 were tested. Five replicates per loading rate and six replicates for an untreated control group were tested.

No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 46 mg/L. At higher loading rates the inhibitory effect of test substance on aerobic waste water (activated sludge) bacteria increased with increased loading rate, ranging from 22% inhibition at 100 mg/L to 47% at 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.

 

Under the test conditions, it can be concluded that the test substance and the target substance are not toxic to waste water bacteria (activated sludge) at a loading rate of 46 mg/L (NOEL).

The EL10 was determined at a loading rate of 25 mg/L but is not taken into account considering the high 95% confidence interval (0.31 - 75 mg/L). The EL50 was estimated to be 960 mg/L.

Description of key information

Read-across, OECD Guideline 209, GLP, key study, validity 1:
3h-NOEL (activated sludge) = 46 mg/L;

3h-EL50 (activated sludge) = estimated 960 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
960 mg/L
EC10 or NOEC for microorganisms:
46 mg/L

Additional information

No study was performed on the registered substance to assess the toxicity to microorganisms. However, a study was performed on a read-across substance, to assess the effect of the test substance on the respiration rate of activated sludge after a contact time of 3 hours, according to OECD Guideline 209 with GLP statement.

This source substance is a mixture of two isomers: 1-(5,5-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one, present as the major constituent at ca. 66% in the mixture, and corresponding to the registered/target (mono-constituent) substance, and 1-(3,3-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one, present at ca. 30% as second constituent.

Following a combined limit/range-finding test, the final test was performed with five loading rates, 46, 100, 220, 460 and 1000 mg/L. Five replicates per loading rate and six replicates for an untreated control group were tested. No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 46 mg/L. At higher loading rates the inhibitory effect of test substance on aerobic waste water (activated sludge) bacteria increased with increased loading rate, ranging from 22% inhibition at 100 mg/L to 47% at 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.

Based on structural/composition similarity, comparing physicochemical and environmental properties and ecotoxicological profile, both substances (the source and the target substances) are not expected to be significantly different in terms of their effects on the activated sludge respiration inhibition. Therefore, under the test conditions, the registered substance also is considered not toxic to waste water bacteria (activated sludge) at a loading rate of 46 mg/L (NOEL). An EL10 value was determined at a loading rate of 25 mg/L but is not taken into account considering the high 95% confidence interval (0.31 - 75 mg/L). The EL50 was estimated to be 960 mg/L.