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Toxicity to microorganisms

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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 20 June 1995 to 23 June 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
This acute microbial toxicity study was not performed according to international guideline but according to internal standard protocol, with GLP statement.
Qualifier:
no guideline followed
Principles of method if other than guideline:
This study was performed according to internal standard protocol.
This test uses the measurement of dissolved oxygen utilization as an indicator of microbial oxidation in a closed test system. If the test substance is inhibitory, oxygen utilization will decrease at higher concentrations of test substance.
The procedure described in this test method is based on ASTM's "Microbiological Inhibition Testing Procedure,'" and is a modification of the standard five-day BOD analysis. The test measures the threshold inhibition concentration of the test substance to a mixed microbial inoculum by measuring the oxidation change over various concentrations of the test substance and comparing this to the oxidation change of an easily degradable substance such as D-glucose. The toxicity threshold concentration is defined as the lowest concentration of the test substance that produces a reduction in biochemical oxidation.
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test substance was added to the test bottles by direct weight. The test substance was weighed onto slides which were dropped into the appropriate bottles.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge mixed liquor was collected on 19 June 1995 from the DRWPCC which receives predominately domestic waste. Upon arrival at WESTON, the sludge was screened through a 2 mm sieve and the total suspended solids (TSS) were determined. The sludge was distributed into a semi-continuous activated sludge (SCAS) unit at a TSS concentration of approximately 2500 mg/L. The unit was aerated at a rate adequate to maintain the solids in suspension.
On 20 June 1995, 25 mL of sludge was collected from the SCAS unit and diluted to a TSS concentration of approximately 250 mg/L with BOD dilution water. The sludge was placed on a stir plate and aerated until used.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 d
Remarks on exposure duration:
none
Post exposure observation period:
None
Hardness:
No data
Test temperature:
20 +/- 0.2°C
pH:
No data
Dissolved oxygen:
See table 6.1.7/2 in "Any other information on results incl. tables".
Salinity:
Not applicable
Nominal and measured concentrations:
- Nominal concentrations: 0.7, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0 and 100 mg active/L.
- Measured concentrations: not measured.
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Biochemical oxygen demand (BOD) bottles with ground glass stoppers and plastic sealing caps.
- Type (delete if not applicable): closed
See table 6.1.7/1 in "Any other information on materials and methods incl. tables" for details on test procedure.

TEST MEDIUM / WATER PARAMETERS
The test medium was biochemical oxygen demand dilution water containing the following standard reagent solutions per 19 liters of water:
19 mL magnesium sulfate solution, 2.25% (w/v);
19 mL calcium chloride solution, 2.75% (W/V);
19 mL phosphate buffer, pH 7.2;
19 mL ferric chloride solution, 0.025% (W/V).
The dilution water was aerated with compressed air and a gas dispersion tube to bring the dissolved oxygen of the water to near saturation (8.0 mg O2/L at 27°C to 9.2 mg O2/L at 20°C).

OTHER TEST CONDITIONS
no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Initial and final (after 3 days) dissolved oxygen (DO).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.4-2.5
Reference substance (positive control):
no
Key result
Duration:
3 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
See table 6.1.7/2 in "Any other information on results incl. tables" and figure in "Illustration".
The residual DO concentrations of the test substance were plotted against the nominal testing concentrations. Horizontal lines representing the average initial DO of the dilution water (8.3 mg O2/L) and the average residual DO of D-glucose (4.8 mg O2/L) were drawn on the graph.
The toxicity threshold concentration is the lowest concentration of test substance producing a reduction in biochemical oxidation compared to the D-glucose control. Graphically, this is the point where the test substance line crosses the D-glucose line and continues to increase with concentration.
The toxicity threshold concentration of the test substance was > 100 mg active/L, the highest concentration tested.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
None

Table 6.1.7/2: Initial and final dissolved oxygen (DO) measurements

Test treatment

Nominal testing concentration (mg active/L)

Replicates

Initial DO

(mg O2/L)

Final DO

(mg O2/L)

Dilution water controls

-

1

2

8.3

8.2

8.1

8.1

D-glucose controls

6

1

2

8.3

8.3

4.8

4.7

ST 02 C 95

0.7

1

2

-

8.2

4.7

-

1.0

1

2

-

8.2

4.5

-

2.0

1

2

-

8.2

4.8

-

5.0

1

2

-

8.2

4.6

-

10.0

1

2

-

8.2

4.3

-

20.0

1

2

-

8.2

4.4

-

50.0

1

2

-

8.3

4.1

-

100.0

1

2

-

8.3

4.3

-

Validity criteria fulfilled:
not applicable
Conclusions:
The lowest concentration of test substance producing a reduction in biochemical oxidation was >100 mg active/L, the highest concentration tested.
Executive summary:

This acute microbial toxicity study was performed to determine the concentration of the test substance that is acutely toxic to aerobic aquatic microorganisms. The procedure described in this test method is based on ASTM's "Microbiological Inhibition Testing Procedure", and is a modification of the standard five-day BOD analysis, with GLP statement.

The microbial inoculum was activated sludge. Nominal testing concentrations for the test substance were 0.7, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, and 100.0 mg active/L. The reference substance D-glucose was tested at a nominal concentration of 6 mg active/L. Dilution water controls were also included in the test design. The test was set up in duplicate. One set of bottles was designated for measuring initial dissolved oxygen (DO) concentrations only and the other set of bottles was designated for measuring final DO concentrations only.

A series of 300-mL BOD bottles was filled with a mixture of microbial inoculum, dilution water, D-glucose, and varying concentrations of test substance. Initial DO readings were taken on one set of bottles. Afterwards the test substance bottles were discarded, while the

dilution water and D-glucose controls plus the second set of bottles were sealed and incubated at 20 ± 0.2 °C. Final DO concentrations were measured three days later.

The residual DO concentrations of the test substance were plotted against the testing concentrations and compared to the D-glucose control. The toxicity threshold concentration of the test substance (corresponding to the lowest concentration of test substance producing a reduction in biochemical oxidation compared to the D-glucose control) was >100 mg active/L, the highest concentration tested.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
(Q)SAR
Remarks:
QSAR result on the C6 isomers of the transformation product of the registered substance
Adequacy of study:
supporting study
Study period:
From 2016-01-05 to 2016-01-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
QSAR value. The substance, the C6 isomers of the transformation product of the registered substance, falls into the applicability domain of the model QSAR
Justification for type of information:
1. SOFTWARE
iSafeRat® HA-QSAR to predict the toxicity (30-180 min EC50) in the activated sludge, respiration inhibition test: Toxicity to microorganisms (Activated Sludge, Respiration Inhibition)

2. MODEL (incl. version number)
iSafeRat® ASRIT HA-QSAR v1.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
C6 isomers of the transformation product of the registered substance: C1C(C)(C)CCCC1C(C)OC(C)(C)COH
Water solubility = 35.0 mg/L (KREATIS internal study, 2015), derived using the log Kow experimental value at 4.33

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached QPRF

6. ADEQUACY OF THE RESULT
See attached QPRF
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
not applicable
Remarks:
(QSAR model)
Qualifier:
equivalent or similar to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
not applicable
Remarks:
(QSAR model)
Principles of method if other than guideline:
The purpose of this QSAR model is to accurately predict the toxicity to microorganisms as would be expected in a laboratory experiment following OECD Guideline 209 and EC method C.11 for specific, named modes of action to provide a value that can effectively replace a 30 to 180 minutes EC50 value from an experimental study. The regression based method used to achieve this has been fully validated following the OECD (2004) recommendations (refer to the QMRF with KREATiS QMRF identifier: KTS/QMRF/ASR/01 for further details).
GLP compliance:
no
Specific details on test material used for the study:
- Mode of action: non-polar narcotic (MOA 1 or MechoA 1.1)
- Log Kow = 4.33 (from experimental study)
- Water solubility = 35.0 mg/L (KREATIS internal study, 2015)
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
Not applicable
Test organisms (species):
activated sludge
Details on inoculum:
This study is not performed on a single or known species as such. Results were acquired from studies where the following test system was used:
Activated sludge collected and treated according to the method outlined in OECD Guideline 209.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
180 min
Remarks on exposure duration:
Results from a test duration of 30 to 180 min were used for this algorithm. The data were separated into 2 groups (30 and 180 min) but the variability between results was not considered important enough to merit the production of two separate models.
Post exposure observation period:
None
Hardness:
The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.
Test temperature:
The temperatures varied from approximately 20 to 25 °C depending on the studies used to construct the algorithm. This small difference is not expected to contribute to the variability of the EC50 values found in experimental data.
pH:
Test results were taken from studies with measured pHs between 6 - 9.
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
This study is typically performed using nominal concentrations. In the case where the test substance was highly volatile this could have caused bias and attempts were made to use measured values when these were available.
Details on test conditions:
Following OECD Guideline 209, all studies used a static test design. For suspected volatile substances only tests performed in closed vessels were accepted unless accompanying analytical monitoring proved such a design was not necessary.
Reference substance (positive control):
no
Remarks:
(QSAR model)
Key result
Duration:
180 min
Dose descriptor:
EC50
Effect conc.:
> 35 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: The 30-180min EC50 to activated sludge was predicted as greater than the water solubility value within the exposure period of the test.
Details on results:
The water solubility value given as input to the iSafeRat® ASRIT HA-QSAR model falls within the descriptor domain of the model between log water solubility (in log (mol/L)) of -2.74 to 1.01. Below this value the algorithm is no longer applicable but the toxicity of the substance can be considered as greater than the solubility limit. Moreover the test substance is attributed to the class of non-polar narcotic compounds.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
QSAR statistical parameters are given in the QMRF and the QPRF

No additional information

Validity criteria fulfilled:
yes
Remarks:
The substance falls into the applicability domain of the QSAR model.
Conclusions:
The 30-180min EC50 of the C6 isomers of the transformation product of the registered substance to activated sludge was predicted as greater than the water solubility value within the exposure period of the test.
Executive summary:

A QSAR model was used to calculate the inhibition of respiration to activated sludge of the test item. This QSAR model has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 209 and EU Method C.11. The criterion predicted was the 30 to 180 minutes EC50 (Median Effective Concentration for specific respiration rate), a statistically derived concentration which is expected to cause 50% inhibition of intrinsic rate of respiration of the test system within a period of 30 to 180 minutes as an effect on the

organic carbon oxidation process. The results from this study do not include the supplementary test results that may be required to determine the effect on nitrification. It should be noted that a specific test duration time could not be determined for this model as 30 and 180 minutes data were available but not in sufficient quantity to separate them into two separate models.

The respiration inhibition of activated sludge was determined using a validated QSAR model for the Mode of Action in question (MOA 1 or MechoA 1.1, non-polar narcosis). The QSAR model is based on validated data for a training set of 12 chemicals derived from 30-180 minute EC50 for which the concentrations of the test item had been determined by chemical analyses over the test period. Further valid data were available but the values were all above the water solubility of the substance and were therefore not included as part of the regression. On the other hand, these data

could be used to determine the point at which the regression no longer holds true and results from experimental studies on this MOA are expected to be greater than the subcooled liquid water solubility limit (i.e. test substance concentrations up to 100 mg/L will result in no effect on activated sludge respiration). The water solubility value given as input to the iSafeRat® ASRIT HA-QSAR model do not fall within the descriptor domain of the model between log water solubility (in log (mol/L)) of -2.72 to 1.01 where the EC50 can be calculated. However it falls within the applicability domain of the model between log water solubility (in log (mol/L)) of -4.64 to -3.01 where the EC50 is considered as greater than the subcooled liquid water solubility value within the exposure period of the test. The water solubility of the substance was predicted by the iSafeRat® solubility module using the experimental log Kow value of the substance, measured at 4.33.

The 30-180min EC50 of the C6 isomers of the transformation product of the registered substance to activated sludge was predicted as greater than the water solubility value within the exposure period of the test.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
(Q)SAR
Remarks:
QSAR result on the C7 isomer of the transformation product of the registered substance
Adequacy of study:
supporting study
Study period:
From 2016-03-14 to 2016-03-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
QSAR value. The substance, the C7 isomer of the transformation product of the registered substance, falls into the applicability domain of the model QSAR
Justification for type of information:
1. SOFTWARE
iSafeRat® HA-QSAR to predict the toxicity (30-180 min EC50) in the activated sludge, respiration inhibition test: Toxicity to microorganisms (Activated Sludge, Respiration Inhibition)

2. MODEL (incl. version number)
iSafeRat® ASRIT HA-QSAR v1.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
C7 isomer of the transformation product of the registered substance: OCC(C)(C)OC1CC(C)(C)CCCC1C
Water solubility = 29.1 mg/L (KREATiS – Internal Study (2016))

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached QPRF

6. ADEQUACY OF THE RESULT
See attached QPRF
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
not applicable
Remarks:
(QSAR model)
Qualifier:
equivalent or similar to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
not applicable
Remarks:
(QSAR model)
Principles of method if other than guideline:
The purpose of this QSAR model is to accurately predict the toxicity to microorganisms as would be expected in a laboratory experiment following OECD Guideline 209 and EC method C.11 for specific, named modes of action to provide a value that can effectively replace a 30 to 180 minutes EC50 value from an experimental study. The regression based method used to achieve this has been fully validated following the OECD (2004) recommendations (refer to the QMRF with KREATiS QMRF identifier: KTS/QMRF/ASR/01 for further details).
GLP compliance:
no
Specific details on test material used for the study:
- Mode of action: non-polar narcotic (MOA 1 or MechoA 1.1)
- log Kow : 4.40 (KREATIS, 2016)
- Water solubility = 29.1 mg/L (KREATIS internal study, 2016)
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
Not applicable
Test organisms (species):
activated sludge
Details on inoculum:
This study is not performed on a single or known species as such. Results were acquired from studies where the following test system was used:
Activated sludge collected and treated according to the method outlined in OECD Guideline 209.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
180 min
Remarks on exposure duration:
Results from a test duration of 30 to 180 min were used for this algorithm. The data were separated into 2 groups (30 and 180 min) but the variability between results was not considered important enough to merit the production of two separate models.
Post exposure observation period:
None
Hardness:
The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.
Test temperature:
The temperatures varied from approximately 20 to 25 °C depending on the studies used to construct the algorithm. This small difference is not expected to contribute to the variability of the EC50 values found in experimental data.
pH:
Test results were taken from studies with measured pHs between 6 - 9.
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
This study is typically performed using nominal concentrations. In the case where the test substance was highly volatile this could have caused bias and attempts were made to use measured values when these were available.
Details on test conditions:
Following OECD Guideline 209, all studies used a static test design. For suspected volatile substances only tests performed in closed vessels were accepted unless accompanying analytical monitoring proved such a design was not necessary.
Reference substance (positive control):
no
Remarks:
(QSAR model)
Key result
Duration:
180 min
Dose descriptor:
EC50
Effect conc.:
> 29.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: The 30-180min EC50 to activated sludge was predicted as greater than the water solubility value within the exposure period of the test.
Details on results:
The water solubility value given as input to the iSafeRat® ASRIT HA-QSAR model falls within the descriptor domain of the model between log water solubility (in log (mol/L)) of -2.74 to 1.01. Below this value the algorithm is no longer applicable but the toxicity of the substance can be considered as greater than the solubility limit. Moreover the test substance is attributed to the class of non-polar narcotic compounds.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
QSAR statistical parameters are given in the QMRF and the QPRF

No additional information

Validity criteria fulfilled:
yes
Remarks:
The substance falls into the applicability domain of the QSAR model.
Conclusions:
The 30-180min EC50 of the C7 isomer of the transformation product of the registered substance to activated sludge was predicted as greater than the water solubility value within the exposure period of the test.
Executive summary:

A QSAR model was used to calculate the inhibition of respiration to activated sludge of the test item. This QSAR model has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 209 and EU Method C.11. The criterion predicted was the 30 to 180 minutes EC50 (Median Effective Concentration for specific respiration rate), a statistically derived concentration which is expected to cause 50% inhibition of intrinsic rate of respiration of the test system within a period of 30 to 180 minutes as an effect on the

organic carbon oxidation process. The results from this study do not include the supplementary test results that may be required to determine the effect on nitrification. It should be noted that a specific test duration time could not be determined for this model as 30 and 180 minutes data were available but not in sufficient quantity to separate them into two separate models.

The respiration inhibition of activated sludge was determined using a validated QSAR model for the Mode of Action in question (MOA 1 or MechoA 1.1, non-polar narcosis). The QSAR model is based on validated data for a training set of 12 chemicals derived from 30-180 minute EC50 for which the concentrations of the test item had been determined by chemical analyses over the test period. Further valid data were available but the values were all above the water solubility of the substance and were therefore not included as part of the regression. On the other hand, these data

could be used to determine the point at which the regression no longer holds true and results from experimental studies on this MOA are expected to be greater than the subcooled liquid water solubility limit (i.e. test substance concentrations up to 100 mg/L will result in no effect on activated sludge respiration). The water solubility value given as input to the iSafeRat® ASRIT HA-QSAR model do not fall within the descriptor domain of the model between log water solubility (in log (mol/L)) of -2.72 to 1.01 where the EC50 can be calculated. However it falls within the applicability domain of the model between log water solubility (in log (mol/L)) of -4.64 to -3.01 where the EC50 is considered as greater than the subcooled liquid water solubility value within the exposure period of the test. The water solubility of the substance was predicted by the iSafeRat® solubility module.

The 30-180min EC50 of the C7 isomer of the transformation product of the registered substance to activated sludge was predicted as greater than the water solubility value within the exposure period of the test.

Description of key information

Internal standard procedure, GLP, key study, validity 2:

The toxicity threshold concentration of the test substance, corresponding to the lowest concentration of test substance producing a reduction in biochemical oxidation compared to the D-glucose control, was >100 mg active/L, the highest concentration tested.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

One valid key study is available to assess the acute microbial toxicity of the registered substance to aerobic aquatic microorganisms. This study was not performed according to international guidelines but according to internal standard protocol, with GLP statement. The microbial inoculum was activated sludge. Nominal testing concentrations for the test substance were 0.7, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, and 100.0 mg active/L. The reference substance D-glucose was tested at a nominal concentration of 6 mg active/L. Dilution water controls were also included in the test design. The test was set up in duplicate. One set of bottles was designated for measuring initial dissolved oxygen (DO) concentrations only and the other set of bottles was designated for measuring final DO concentrations only. A series of 300-mL BOD bottles was filled with a mixture of microbial inoculum, dilution water, D-glucose, and varying concentrations of test substance. Initial DO readings were taken on one set of bottles. Afterwards the test substance bottles were discarded, while the dilution water and D-glucose controls plus the second set of bottles were sealed and incubated at 20 ± 0.2 °C. Final DO concentrations were measured three days later. The residual DO concentrations of the test substance were plotted against the testing concentrations and compared to the D-glucose control. The toxicity threshold concentration of the test substance, corresponding to the lowest concentration of test substance producing a reduction in biochemical oxidation compared to the D-glucose control, was >100 mg active/L, the highest concentration tested.

In addition, the toxicity of the transformation product (C6 and C7 isomers) of the registered substance to microorganisms was evaluated.

The first piece of evidence is the toxicity information presented in the screening non-GLP biodegradation study (OECD 301F; Firmenich, 2013) performed on the transformation product of the registered substance (mixture of C6 and C7 isomers). No toxicity was observed in this study, at 105 mg/L of test substance. According to the biodegradation table presented in the corresponding Endpoint Study Record, the biodegradation in the toxicity test was greater than 25% within 14 days which according to the guideline confirms that the test substance is not inhibitory.

The second piece of evidence is the predicted toxicity effect of the transformation product (C6 and C7 isomers) of the registered substance in activated sludge with iSafeRat® High Accuracy QSAR v1.1. This QSAR result was considered reliable as the substances (C6 and C7 isomers) fall within the applicability domain of the model. The 30-180min EC50 value of C6 and C7 isomers to activated sludge was predicted as greater than the water solubility value (predicted at 35 mg/L for C6 and 29.1 mg/L for C7) within the exposure period of the test. Therefore, no toxicity is predicted on sludge dwelling microorganisms for the transformation product of the registered substance.