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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes
Analytical monitoring:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The synthetic sewage feed (16 ml) and an adequate amount of the test substance loading were mixed and made up to 250 ml with Milli-RO water in a 1 litre bottle. The pH was determined. Thereafter 250 ml activated sludge was added. This was the start of the test. The mixture was then aerated during the contact time, using a pipette as an aeration device.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Preparation of inoculum for exposure: Coarsely sieved then washed and diluted with ISO medium.

- Amount suspended solids: 3.0 g/l of sludge, as used for the test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Oxygen consumption measured and recorded for approximately 10 minutes.After the 3-hour contact time the oxygen consumption was recorded for a period approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
Determination of oxygen was performed with oxygen probes connected to a BlueBox, a multichannel measuring and controlling system.
Test temperature:
19.6 - 21.1°C
pH:
The pH in all test series, before addition of sludge remained within the range of 7.5 ± 0.5 (7.3-7.5). After the 3 hour exposure period the pH increased to a maximum of 8.3.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: All glass bottles/vessels.

- Aeration: Clean, oil-free air.

- No. of vessels per concentration (replicates): Loading rates of 10, 100 and 1000 mg/l. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate.

- No. of vessels per control (replicates): Blank control: three replicates. Also, abiotic control and the highest loading rate with a nitrification inhibitor were tested.

- No. of vessels per reference (replicates): Three concentrations, each one replicate.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).



Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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:
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 in general comparable to the total inhibition.
Results with reference substance (positive control):
- Results with reference substance valid? Yes. The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/l for total respiration. The controls 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.

Flask

Conc. / loading

Rate

(mg/l)

Temp.

(°C)

pH before addition of sludge

pH after 3 h contact time

Oxygen conc. at the start (~ mg O2/l)

Respi-

ration rate

(mg O2/l h)

Respi-

ration rate (mg O2/g h)¹

% Inhibition respiration relative to the control (mean value)

C 1

0

21.1

7.8

7.9

8.6

38

25

C 2

0

20.3

7.8

7.9

8.6

39

26

C 3

0

20.5

7.8

7.9

8.0

36

24

C 4

0

20.4

7.8

8.0

8.0

33

22

C 5

0

20.6

7.8

8.0

8.0

34

23

C 6

0

20.6

7.8

8.0

8.0

37

25

Mean

 

 

 

 

 

36

24

SD

 

 

 

 

 

2

2

CV

(%)

 

 

 

 

 

6

6

 

 

 

 

 

 

 

 

 

CN

0

20.9

7.8

8.2

8.8

21

14

 

 

 

 

 

 

 

 

 

R 1

5.0

21.1

7.8

8.0

8.8

23

15

36

R 2

12

20.9

7.8

8.1

8.6

13

9

64

R 3

30

20.3

7.7

8.2

8.8

7

5

81

T 1

10

20.4

7.7

7.9

8.2

40

27

-11

T 2

100

19.6

7.7

8.0

8.1

33

22

9

T3a

1000

20.2

7.7

7.9

8.0

47

31

-30

T3b

1000

20.6

7.7

7.9

7.8

43

29

-19

T3c

1000

20.8

7.7

7.9

8.1

44

29

-22

T3 mean

45

30

-24

TA

1000

20.8

7.7

7.4

8.8

0

0

100

TN

1000

20.4

7.7

8.3

8.0

26

17

-242

C:          Blank Control

CN:        Nitrification control

R:          Reference substance, 3,5-dichlorophenol

T:           Test substance, (Chloromethyl)triethoxysilane

CV:         Coefficient of variation

ATU        N-allylthiourea

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

2              Relative to CN

TA:         Abiotic control of test substance.

TN:         Test substance with ATU.

                         

Validity criteria fulfilled:
yes
Conclusions:
An EC50 of >1000 mg/l and a NOEC of ≥1000 mg/l for toxicity to microorganisms were determined in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP.

Description of key information

3-hr EC50of > 1000 mg/l and a NOEC of ≥ 1000 mg/l (OECD 209), RL1 

Key value for chemical safety assessment

Additional information

One OECD 209 study is available for the registered substance. The study was conducted to standard guideline (OECD 209), GLP and fulfilled the validity criteria (NOTOX, 2011). There was no observed effect of respiration rate of tested microorganisms at the test concentration after 3 hours, and so the EC50 and NOEC values were assessed to be >1000 mg/l and ≥1000 mg/l.