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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-09-10
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 and no deviations were observed.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on 03-05 June 2013 / signed on 05 November 2013
Specific details on test material used for the study:
No additional information
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
See table 6.1.7/1: Dilution table for the test item

- Control: Test medium without test or reference item
Test organisms (species):
activated sludge
Details on inoculum:
- Source: Municipal sewage treatment plant of 31137 Hildesheim, Germany.
- Pretreatment: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 2.94 g/L corresponding to 1.47 g/L in the test vessel
Dry solids concentration of sludge: 2.94 g/L; Dry solids concentration in test solution: 1.47 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
none
Post exposure observation period:
None
Hardness:
None
Test temperature:
Nominal: 18-22 °C; actual: 21.3 °C
pH:
- pH value of the activated sludge: 7.71
- pH value of the synthetic waste water: 7.41
Dissolved oxygen:
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flatbed recorder.
Salinity:
No data
Nominal and measured concentrations:
10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL Erlenmeyer flasks, DIN 12380
- Aeration: Shaking of flasks at 150 rpm to keep the dissolved oxygen concentration above 60-70% saturation and to maintain the sludge flasks in suspension.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6 (Triplicates at the beginning of the test + triplicates at the end of the test)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic waste water according to OECD Guideline 209
- Volume of the test medium: 500 mL

OTHER TEST CONDITIONS
- Adjustment of pH: Adjustment to 7.5 ± 0.5 was not necessary.

EFFECT PARAMETERS MEASURED
- The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates.

TEST CONCENTRATIONS
- Preliminary test
- Test concentrations: 8.98, 89.8 and 898 mg/L
- Results used to determine the conditions for the definitive study: No indication for the requirement to measure the inhibition of nitrification in the definitive study was observed.
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
93.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 81.5-108 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
275 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 214-339 mg/L
Details on results:
The mean inhibition of respiration for the test item replicates were -3, 2, 12, 53 and 58% at 10, 32, 100, 320 and 1000 mg/L, respectively.
Results with reference substance (positive control):
EC50 value for the reference item was 101 mg/L (95% CI: 95.3-107 mg/L). This is in the recommended range of validity of 53-155 mg/L.
Reported statistics and error estimates:
The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) and Dunnett's test was used for NOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The α-value (acceptable probability of incorrectly concluding that there is a difference) is α=0.05.

The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression and linear regression, respectively, using software GraphPadPrism, Calculations of the confidence intervals for the EC-values were carried out using standard procedures.

Table 6.1.7/3: Inhibitions in the Preliminary test

Nominal test item concentration (mg/L)

 

pH (test item in demin. water)

pH (adjusted to)

pH (test item in medium)

Inhibition (%)

Preliminary test – without pH adjustment

8.98

6.16

-

7.37

24

6.20

-

7.38

-3

89.8

6.19

-

7.39

10

7.20

-

7.41

1

898

7.20

-

7.39

52

6.18

-

7.40

52

898 stirring overnight

6.14

-

7.39

52

5.98

-

7.40

54

 

Additional replicates were prepared to determine the inhibition of nitrification. Two replicates 898 mg/L were measured with addition of Allylthiourea (ATU final concentration 11.6 mg/L). No indication for the requirement to measure the inhibition of nitrification in the definitive study was observed.

Table 6.1.7/4: Inhibitions in the Preliminary test (with / without ATU)

 

Nominal test item concentration (mg/L)

Inhibition of total respiration (%)

Inhibition of heterotrophic respiration (%)

Inhibition of nitrification (%)

898

52

33

64

52

64

45

 

 

Table 6.1.7/5: Oxygen uptake rates and inhibition of the respiration of the control and test item concentrations

 

Test concentration (mg/L)

Replicates

Oxygen uptake rate R (mg O2/L.h)

Inhibition (%)

Mean Inhibition (%)

Control

1

24.8

-

-

2

24.0

3

22.4

4

18.4

5

20.0

6

19.2

Test item

10

1

21.2

1

-3

2

21.2

1

3

22.4

-4

4

23.2

-8

5

22.4

-4

32

1

20.0

7

2

2

23.6

-10

3

21.6

0

4

19.6

9

5

20.4

5

100

1

18.0

16

12

2

19.2

11

3

19.2

11

4

19.2

11

5

19.6

9

320

1

10.4

52

53

2

10.0

53

3

10.4

52

4

9.6

55

5

10.0

53

1000

1

9.2

57

58

2

9.2

57

3

9.2

57

4

8.8

59

5

8.8

59

 

 

Coefficient of variation of oxygen uptake rates of the control: 12.3%

Suspended solids in the test vessel: 1.47 g/L

Table 6.1.7/6: Specific respiration rates of the control replicates

 

Control

Replicates

Specific respiration rate Rs(mg O2/g.h)

1

17

2

16

3

15

4

13

5

14

6

13

Mean value

15

 

Suspended solids in the test vessel: 1.47 g/L Validity criteria:

- The mean specific oxygen uptake rate of the control replicates was 15 mg O2/g.h and therefore not meeting the criterion (validity criterion: should be ≥ 20 mg 02/g.h). The oxygen uptake of activated sludge from municipal treatment plants is influenced by seasonal fluctuations and fluctuations in sewage flow etc. The lower oxygen uptake rate reflects the range of common uptake rates of activated sludge from municipal treatment plants and was considered to have no impact on quality and integrity of the study. Despite the lower respiration, the EC50-value of the reference item was in the required range. Therefore, no impact on the results of the test item can be expected through lower respiration.

- The coefficient of variation of the oxygen uptake rates in the control replicates was 12.3 % (validity criterion: < 30 %).

- The EC50 of the reference item was in the range of 53 - 155 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, the NOEC of test item is 32 mg/L. The test item is not toxic up to the concentration of 32 mg/L to activated sludge of a municipal sewage treatment plant. The EC10 is 93.9 mg/L, the EC20 is 125 mg/L and the EC50 is 275 mg/L of the test item. The EC80 is > 1000 mg/L.
Executive summary:

The effect of test item on the respiration rate of activated sludge was assessed according to OECD Guideline 209 with GLP statement.

The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations 10, 32, 100, 320 and 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates.

 

The mean inhibition of respiration for the test item replicates were -3, 2, 12, 53 and 58% at 10, 32, 100, 320 and 1000 mg/L, respectively.

 

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC50-value for the reference item was 101 mg/L.

 

Under the test conditions, the NOEC of test item is 32 mg/L. The test item is not toxic up to the concentration of 32 mg/L to activated sludge of a municipal sewage treatment plant. The EC10 is 93.9 mg/L (95% CI: 81.5 -108 mg/L), the EC20 is 125 mg/L (95% CI: 112 -144 mg/L)

and the EC50 is 275 mg/L (95% CI: 214 -339 mg/L) of the test item. The EC80 is > 1000 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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[further information is included as attachment to Iuclid section 13]

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)
Both the target and the source substances are mono-constituents. They are structurally related, with the same 2,6,6-trimethylcyclohexyl ring branched with 2-buten-1-one chain at the C-1 position. They differ by the number and location of the endocyclic double bonds: the target has a single one in cyclohex-2-enyl, while the source substance has two, in 1,3-cyclohexadienyl.
The target and source substances are typically pure. Some impurities were reported above 1.0% but none of these impurities may contribute to the classification of the substances. Therefore, it is concluded that the impurities will not affect the validity of the read-across.

3. ANALOGUE APPROACH JUSTIFICATION
Based on structural similarity, comparing physicochemical and environmental fate properties and ecotoxicological profile, both substances are not expected to have significantly difference on the activated sludge respiration inhibition.
The Activated Sludge Respiration Inhibition test (ASRIT), performed on the source substance, was conducted according to OECD Test Guideline 209 and the test material was clearly identified. The result of the study is adequate for the purpose of prediction.
Therefore, based on the considerations above, it can be concluded that the result of the ASRIT study conducted with the source substance is likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 9.1.4 of the REACH regulation.

4. DATA MATRIX
See attached document in Iuclid section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Remarks:
Read-across justification document
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
93.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 81.5-108 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
275 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 214-339 mg/L
Validity criteria fulfilled:
not applicable
Conclusions:
According ot the experimental study performed on the source substance, the 3h-NOEC of the target substance can be determined at 32 mg/L. The target substance is considered not toxic up to the concentration of 32 mg/L to activated sludge of a municipal sewage treatment plant. The 3h-EC10 and EC50 values are determined at 93.9 mg/L and 275 mg/L, respectively.
Executive summary:

No experimental study is available on the target substance to assess the toxicity of the registered substance to microorganisms. Therefore, good quality data for a related source substance have been read-across for this endpoint. Both the target and the source substances are mono-constituents. They are structurally related, with the same 2,6,6-trimethylcyclohexyl ring branched with 2-buten-1-one chain at the C-1 position. They differ by the number and location of the endocyclic double bonds: the target has a single one in cyclohex-2-enyl, while the source substance has two, in 1,3-cyclohexadienyl.

Based on structural similarity, comparing physicochemical properties, environmental fate properties and ecotoxicological profile, both substances are not expected to have significantly difference on the activated sludge respiration inhibition.

The Activated Sludge Respiration Inhibition test (ASRIT), performed on the source substance, was conducted according to OECD Test Guideline 209 with GLP statement. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations 10, 32, 100, 320 and 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates.

The mean inhibition of respiration for the test item replicates were -3, 2, 12, 53 and 58% at 10, 32, 100, 320 and 1000 mg/L, respectively.

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC50-value for the reference item was 101 mg/L.

Under the test conditions, the NOEC of test item is 32 mg/L. The test item is not toxic up to the concentration of 32 mg/L to activated sludge of a municipal sewage treatment plant. The EC10 is 93.9 mg/L (95% CI: 81.5 -108 mg/L), the EC20 is 125 mg/L (95% CI: 112 -144 mg/L)

and the EC50 is 275 mg/L (95% CI: 214 -339 mg/L) of the test item. The EC80 is > 1000 mg/L.

Therefore, based on the considerations above, it can be concluded that the result of the ASRIT study conducted with the source substance is likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 9.1.4 of the REACH regulation.

Description of key information

Read-across, OECD Guideline 209, GLP, key study, validity 1:

3h-NOEC = 32 mg/L;

3h-EC50 = 275 mg/L (95% CI: 214 -339 mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:
275 mg/L
EC10 or NOEC for microorganisms:
32 mg/L

Additional information

To assess the toxicity of the registered (target) substance to microorganisms, one experimental study is available on a source substance.

Both the target and the source substances are mono-constituents. They are structurally related, with the same 2,6,6-trimethylcyclohexyl ring branched with 2-buten-1-one chain at the C-1 position. They differ by the number and location of the endocyclic double bonds: the target has a single one in cyclohex-2-enyl, while the source substance has two, in 1,3-cyclohexadienyl.

Based on structural similarity, comparing physicochemical properties, environmental fate properties and ecotoxicological profile, both substances are not expected to have significantly difference on the activated sludge respiration inhibition.

This valid study was performed according to OECD Guideline 209 (ASRIT) in compliance with GLP on the source substance, (2E)-1-(2,6,6-trimethyl-1,3-cyclohexadien-1-yl)-2-buten-1-one (CAS 23726 -93 -4). To assess the effect of the substance on the respiration rate of activated sludge, the test was carried out under static conditions with the test item concentrations 10, 32, 100, 320 and 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item (copper (II) sulphate pentahydrate) were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates were -3, 2, 12, 53 and 58% at 10, 32, 100, 320 and 1000 mg/L, respectively. The EC50-value for the reference item was 101 mg/L. Under the test conditions, the NOEC of test item is 32 mg/L. The test item is not toxic up to the concentration of 32 mg/L to activated sludge of a municipal sewage treatment plant. The EC10 is 93.9 mg/L (95% CI: 81.5 -108 mg/L) and the EC50 is 275 mg/L (95% CI: 214 -339 mg/L) of the test item.

Based on the considerations above, it can be concluded that the result of the ASRIT study conducted with the source substance is likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 9.1.4 of the REACH regulation.