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Ecotoxicological information

Toxicity to microorganisms

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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 August to 23 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: In compliance with GLP, according to the OECD 209 .

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
yes
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Principles of method if other than guideline:
Doses: 0, 10, 100, 1000
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): MK-0954 Free Acid
- Molecular formula (if other than submission substance):C22H23ClN6O
- Molecular weight (if other than submission substance):422.91
- Physical state:white powder
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Purity test date: not provided
- Lot/batch No.: not available
- Expiration date of the lot/batch: 24 April 2013
- Stability under test conditions:
- Storage condition of test material: At room temperature in the dark
-Stability under storage conditions: stable
-water solubility: insoluble
-hygroscopic: no

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 ml of test substance 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, 250 ml sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates (see also paragraph 6.3 and 6.5). Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- source : Municipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewageMunicipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage

- Preparation of the sludge: The sludge was coarsely sieved (1 mm), washed and diluted with ISO-medium. 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 7.3 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

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
not applicable

Test conditions

Hardness:
no data
Test temperature:
Except for the deviations (R1 (reference substance) 17.5°C and TN (test substance with N-allylthiourea 17.0°C), the temperatures measured in the various vessels ranged between 18.2 and 19.7°C and were within the ranges prescribed by the protocol.
pH:
The pH in all test series, before addition of sludge was between 7.2 and 7.5. After the 3 hour exposure period the pH was between 7.0 and 8.0.

Dissolved oxygen:
dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/l at 20°C)
Nominal and measured concentrations:
10, 100, and 1000 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: glass open bottles/vessels
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 1L
- 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 organisms per vessel: Number of micro-organisms was determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium.
- No. of vessels per concentration (replicates):one replicate for 10 and 100 mg/L, three replicate for 1000 mg/L
- No. of vessels per control (replicates):two replicates for blank control and one nitricifation control of the sludge , an abiotic control and the highest concentration with a nitrification inhibitor
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Synthetic sewage: in accordance to TG 209

OTHER TEST CONDITIONS
- Adjustment of pH: in all test series, before addition of sludge was between 7.2 and 7.5. After the 3 hour exposure period the pH was between 7.0 and 8.0.
- Other: The temperature ranges between 18.2 and 19.7°C except for two test vessels was below 18°C; Reference1:17.5°C and test substance with N-allylthiourea

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The oxygen consumption was recorded for a period of 10 to 15 minutes. The pH and temperature were determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations/loading rates and controls. Determination of oxygen was performed with oxygen probes connected to a BlueBox, a multichannel measuring and controlling system.

TEST CONCENTRATIONS

- 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 (2 replicates) was included. Furthermore, an abiotic control and the highest loading rate with a nitrification inhibitor were tested.

- Test concentrations: 10, 100 and 1000 mg/l
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
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
Remarks:
respiration rate
Details on results:
In the combined limit/range-finding test no statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg MK-0954 Free Acid per litre (Two Sample t-Test : α=0.05 Toxstat).

There was no oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was slightly higher than the total inhibition.
Results with reference substance (positive control):
- Results with reference substance valid? yes
1.The mean control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.
The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test.
2.The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/l for total respiration (6.5 mg/l)
Reported statistics and error estimates:
95% confidence limits were calculated for the reference item EC50 value.
Chi-square test for normality
F-test for equality of two variances
Analysis of variance (Anova table)
2 Sample t-Test

Any other information on results incl. tables

Flask

Conc./

loading

rate

(mg/l)

Temp.

(°C)

pH before addition of sludge

pH after

3 h contact time

Respiration rate

(mg O2/l h)

Respiration rate

(mg O2/g h)¹

% Inhibition respiration relative to the control (mean value)

C 1

0

19.3

7.5

7.8

30

20

C 2

0

18.1

7.5

7.8

28

19

Mean

0

29

19

SD

0

1

1

CV (%)

0

5

5

CN

0

18.6

7.5

8.0

20

13

R 1

5.0

17.5

7.5

7.9

16

11

45

R 2

12

19.2

7.5

7.9

11

7

62

R 3

30

19.5

7.4

7.9

5

3

83

T1

10

19.7

7.4

7.8

35

23

-21

T2

100

19.2

7.4

7.7

33

22

-14

T3a

1000

18.3

7.2

7.5

27

18

7

T3b

1000

18.2

7.3

7.6

26

17

10

T3c

1000

18.7

7.4

7.5

25

17

14

Mean

T 3

26

17

10

TA

1000

19.4

7.2

7.0

0

0

100

TN

1000

17.0

7.3

7.7

15

10

25²

 

 

C:          Blank Control

CN:       Nitrification control

R:          Reference substance, 3,5-dichlorophenol

T:          Test substance, MK-0954 Free Acid

SD:         Standard deviation

CV:         Coefficient of variation

TA:         Abiotic control of test substance.

TN:         Test substance withN-allylthiourea.

¹            The amount of suspended solids in the final test mixture was 1.5 g/l.

2             Relative to CN

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, MK-0954 Free Acid was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).

The EC50 exceeded a loading rate of 1000 mg/l.
Executive summary:

The influence of MK-0954 Free Acid on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The study procedures described in this report were based onthe OECD guideline No. 209, 2010.In addition, the procedures were designed to meet the test methods of theCouncilRegulation (EC) No. 440/2008 of, Publication No. L142, Part C11 and ISO Standard 8192 (2007).

 

The batch of MK-0954 Free Acid tested was a white powderand tested as received.The test substance was not completely soluble in test medium at the loading rates initially prepared.Therefore, the test substance – Milli-RO water mixtures were magnetically stirred for a period of approximately 24 hours. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for 10-15 minutes.

 

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 concentrations consisted of one replicate. In addition, blank-controls and a nitrification control of the sludge were included. Furthermore, an abiotic control and the highest concentration with a nitrification inhibitor were tested.

 

In thecombined limit/range-finding test no statistically significant inhibition of the respiration rate of the sludge was recordedat a loading rate of 1000 mg MK-0954 Free Acid per litre(Two Sample t-Test : α=0.05 Toxstat).

There was no oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition.

The batch of activated sludge was checked for sensitivity by testing the reference substance

3,5-dichlorophenol, which showed normal sensitivity.

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