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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 27 July 2016 to 02 August 2016
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
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected on 03-05 June 2013 / signed on 01 February 2016
Specific details on test material used for the study:
- Water solubility: not miscible
Analytical monitoring:
no
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 of a predominantly domestic sewage
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: 2.71 g/L corresponding to 1.36 g/L in the test vessel
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, actually measured 19.1°C
pH:
- pH value of the activated sludge: 7.40
- pH value of the synthetic waste water: 7.27
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 5 minutes on a flat bed recorder
Salinity:
No data
Nominal and measured concentrations:
1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL Erlenmeyer flasks, DIN 12380
- Aeration: Shaking aeration, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension. The flasks were closed with an active carbon trap that allows air exchange.
- No. of vessels per concentration (replicates): 3
- 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
A preliminary test (non GLP) was carried out with the following test item concentrations: 10 - 100 - 1000 mg/L with two replicates each and additionally two replicates with 1000 mg/L to study the inhibition of the nitrification using Allylthiourea ATU. The pH was not adjusted. The predicted Henrys Law Constant (25°C) [HENRYWIN v3.20] of 6.27E-005 atm-m3/mole (Bond Method) and 1.44E-004 atm-m3/mole (Group Method ), respectively, indicate a volatility that can be considered as close to non-volatile in practice. Nevertheless, the precautions mentioned in the guidelines (shaking aeration, direct pipetting) were taken. Based on the results of the preliminary test it can be concluded that the heterotrophic respiration as well as the nitrification are not inhibited. Therefore, the definitive study was carried out with the concentration of 1000 mg/L in 3 replicates (limit test to determine the NOEC).
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The mean inhibition of respiration for the test item replicates was 4% at 1000 mg/L.
The NOEC of the test substance is >= 1000 mg/L. The test substance caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L. No nitrification inhibition was observed at 1000 mg/L in the preliminary test and this endpoint was therefore not evaluated in the definitive test.
Results with reference substance (positive control):
EC50 value for the reference item was 102 mg/L (95% CI: 98.4-106 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) was used for NOEC calculation.When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-value for both tests was 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

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, direct aeration

10

-

-

-

1

-

-

-

-1

100

-

-

-

6

-

-

-

-1

1000

-

-

7.57

4

-

-

-

3

Table 6.1.7/4: Inhibition of the Nitrification in the Preliminary test (with / without ATU)

 

Nominal test item concentration (mg/L)

Inhibition of total respiration (%)

Inhibition of heterotrophic respiration + ATU* (%)

Inhibition of nitrification (%)

1000

4

19

-8

3

7

-2

  * ATU = N-Allylthiourea

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

 

Test and reference item concentration (mg/L)

Repl.

Oxygen uptake rate (mg O2/L.h)

Inhibition (%)

Mean inhibition (%)

Control

1

2

3

4

5

6

24.4

24.0

24.0

22.4

22.8

22.8

-

-

Test item

1000

1

2

3

22.4

22.4

22.4

4

4

4

4

Reference item

58

1

2

3

17.6

18.0

18.0

25

23

23

24

Reference item

100

1

2

3

12.4

12.8

12.4

47

45

47

46

Reference item

180

1

2

3

4.8

4.8

4.8

79

79

79

79

 

Repl. = replicate

Coefficient of variation of oxygen uptake rates of the control: 3.5%

Suspended solids in the test vessel: 1.36 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

17.9

2

17.6

3

17.6

4

16.5

5

16.8

6

16.8

Mean value

17.2

 

Repl. = replicate

Suspended solids in the test vessel: 1.36 g/L

Validity criteria:

- The mean specific oxygen uptake rate of the control replicates was 17.2 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 3.5 % (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 3h-NOEC of the test substance is > 1000 mg/L. The test substance caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 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 limit test item concentration 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 was 4% at 1000 mg/L.

 

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 102 mg/L.

 

Under the test conditions, the 3h-NOEC of the test substance is > 1000 mg/L. The test substance caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 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 structural similarity between the source and the target substances (stereoisomers) and comparable properties related to the target endpoints.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Refer to the Test material section of the source and target records.

3. ANALOGUE APPROACH JUSTIFICATION
See attached document in Iuclid section 13

4. DATA MATRIX
See attached document in Iuclid section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Remarks:
Read-Across justification document
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Validity criteria fulfilled:
not applicable
Conclusions:
According to the experimental study performed on the source substance, the 3h-NOEC of the target substance is determined to be greater than 1000 mg/L.
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 (Reaction Mass of 1-[(1R*,6S*)-2,2,6-Trimethylcyclohexyl]-3-hexanol and 1-[(1S*,6S*)-2,2,6-Trimethylcyclohexyl]-3-hexanol (EC#947-716-8)) have been read-across for this endpoint. The target and source substances are structurally related, in that both are reaction mass of stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol. They differ by the number of constituents. The source substance is a reaction-mass of both cis (major) and trans isomers, with both 3R and 3S hydroxyl group (i.e. 4 diastereoisomers), whereas the target substance is a reaction-mass of two trans 1R,6S diastereoisomers, with both 3R and 3S hydroxyl group. The cis form (unknown absolute configuration, i.e. 1 or 2 possible isomers) being considered as an impurity. Thus, two isomers are common between the source and target substances, the other two being diastereoisomers. 


 


 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 limit test item concentration 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 was 4% at 1000 mg/L. 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 102 mg/L. Under the test conditions, the 3h-NOEC of the test substance is > 1000 mg/L. The test substance caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 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 > 1000 mg/L

Key value for chemical safety assessment

Additional information

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


This valid study was performed according to OECD Guideline 209 (ASRIT) in compliance with GLP on the source substance, Reaction Mass of 1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and 1-[(1S*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol (EC 947-716-8). The target and source substances are structurally related, in that both are reaction mass of stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol. They differ by the number of constituents. The source substance is a reaction-mass of both cis (major) and trans isomers, with both 3R and 3S hydroxyl group (i.e. 4 diastereoisomers), whereas the target substance is a reaction-mass of two trans 1R,6S diastereoisomers, with both 3R and 3S hydroxyl group. The cis form (unknown absolute configuration, i.e. 1 or 2 possible isomers) being considered as an impurity. Thus, two isomers are common between the source and target substances, the other two being diastereoisomers. 


 


To assess the effect of the substance on the respiration rate of activated sludge, the test was carried out under static conditions with the limit test item concentrations 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 was 4% at 1000 mg/L. The EC50-value for the reference item was 102 mg/L. Under the test conditions, the 3h-NOEC of the test substance is > 1000 mg/L. The test substance caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L.


 


This source substance is considered adequate for read-across purposes based on structural similarity (stereoisomers), on similar basic physico-chemical properties and similar environmental profile between the registered substance and the analogue material used (see IUCLID section 13 for justification).