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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
Study period:
January 25th, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
(2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
(2007)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 10 g/L was prepared by adding 5.0 g test substance to 500 ml of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Magnetic stirring for 18 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear and colourless stock solution corresponding to the test concentration were then added to the test media.

- Controls: Blank-control, nitrification control, abiotic control and toxicity control
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 sewage.
- Preparation of inoculum for exposure: 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 (3.4 g/L of sludge). 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.
- Performance of the test: The synthetic medium (16 ml) and an appropriate amount of the test substance stock 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19 to 22°C
pH:
- Before addition of sludge was between 7.5 and 7.8.
- After the 3 hour exposure period the pH was between 7.5 and 8.0.
Dissolved oxygen:
- At the start of the test the dissolved oxygen concentration was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C)
Nominal and measured concentrations:
- Nominal: Blank-control, 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All-glass bottles
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: All-glass, 1 L capacity, filled with 500 ml of test solution
- Aeration: Yes. 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 vessels per concentration (replicates): 3 replicates for the 1000 mg/L test concentration and 1 replicate for the other test concentrations
- No. of vessels per control (replicates): Blank-control (6 replicates), nitrification control, abiotic control and toxicity control
- Concentration of suspended solids in test solution: 1.7 g/L of sludge

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)
- Intervals of water quality measurement
pH: At the beginning and at the end of the test period.
Temperature: Continuously during the test period.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): After the 3-hour contact time the oxygen consumption was recorded for a period of 11-20 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x10
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
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
Details on results:
- The combined limit/range-finding test showed no inhibition of the respiration rate at 10 mg/L. At 100 and 1000 mg/L an inhibition of 21% and an average of 29% inhibition were observed, respectively. The inhibition observed at 1000 mg/L was statistically significant but below 50%. Thus, the EC50 was above the highest concentration tested (1000 mg/L). No NOEC could be determined since it was observed a statistically significant difference in inhibition at 1000 mg/L in relation to the control group, and only one replicate was used for the lower test concentrations (i.e. 10 and 100 mg/L). Therefore, the statistical significance could not be determined at test concentrations of 10 and 100 mg/L.

- There was no significant oxygen uptake from abiotic processes. The results at 1000 mg/L with a nitrification inhibitor showed no heterotrophic inhibition of the respiration rate. The reviewer calculated that at 1000 mg/L, the heterotrophic respiration was inhibited 4% and the nitrification respiration was inhibited 92%.

- Acceptability of the test:
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 10 mg/L for total respiration (accepted range of 2 to 25 mg/L)
Results with reference substance (positive control):
The EC50 was 9.9 mg 3,5-Dichlorophenol/L.

Study results Flask

Conc. (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

20.4

7.6

8.0

37

22

 

C 2

0

20.7

7.6

7.8

34

20

 

C 3

0

19.4

7.6

7.9

44

26

 

C 4

0

20.7

7.8

7.7

31

18

 

C 5

0

19.8

7.7

7.7

35

21

 

C 6

0

21.8

7.7

7.6

46

27

 

 

Mean: 38

22

 

SD: 6

3

 

CV (%): 16

16

CN

0

20.8

7.5

8.0

26

15

31

R 1

5.0

20.6

7.6

7.9

28

16

26

R 2

12

21.0

7.6

7.8

15

9

60

R 3

30

20.8

7.6

7.8

6

4

84

T1

10

20.8

7.6

7.8

42

25

-11

T2

100

20.8

7.5

7.7

30

18

21

T3a

1000

20.2

7.5

7.7

28

16

26

T3b

1000

21.1

7.5

7.7

26

15

31

T3c

1000

21.3

7.5

7.6

27

16

29

Mean

 

27

 

16

 

 

29

TA

1000

21.1

7.7

7.5

1

1

97

TN

1000

21.2

7.5

7.6

26

15

0

C: Blank Control

CN: Nitrification control

R: Reference substance, 3,5-dichlorophenol

T: Test substance, Accelerator (PT 25E or PT 25E/2)

CV: Coefficient of variation

SD: Standard deviation

TA: Abiotic control of test substance.

TN: Test substance with N-allylthiourea.

¹ The amount of suspended solids in the final test mixture was 1.7 g/l.

2 Relative to CN

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test Accelerator (PT 25E or PT 25E/2) was slightly toxic to waste water (activated sludge) bacteria at or below a 1000 mg/L.
Executive summary:

Toxic effects of Accelerator (PT 25E or PT 25E/2) to waste water (activated sludge) bacteria were investigated according to OECD guideline 209 and GLP principles. Nominal test concentrations were the following: 0 (blank control), 10, 100 and 1000 mg/L.

After 3h of incubation Accelerator (PT 25E or PT 25E/2) did not cause an inhibition of the respiration rate at 10 mg/L. At 100 and 1000 mg/L an inhibition of 21% and an average of 29% inhibition were observed, respectively. The inhibition at 1000 mg/L was statistically significant but below 50%. The EC50 was estimated to be >1000 mg/L related to the respiration rate. No NOEC could be determined since it was observed a statistically significant difference in inhibition at 1000 mg/L in relation to the control group, and only one replicate was used for the lower test concentrations (i.e. 10 and 100 mg/L). Therefore, the statistical significance could not be determined at test concentrations of 10 and 100 mg/L. There was no significant oxygen uptake from abiotic processes. The results at 1000 mg/L with a nitrification inhibitor showed no heterotrophic inhibition of the respiration rate. The reviewer calculated that at 1000 mg/L, the heterotrophic respiration was inhibited 4% and the nitrification respiration was inhibited 92%. The study is considered to be reliable without restrictions.

Description of key information

Toxic effects of Accelerator (PT 25E or PT 25E/2) to waste water (activated sludge) bacteria were investigated

according to OECD guideline 209 and GLP principles. The 3 h EC50 was estimated to be >1000 mg/L related to

the respiration rate. The study is considered to be reliable without restrictions.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

Toxic effects of Accelerator (PT 25E or PT 25E/2) to waste water (activated sludge) bacteria were investigated according to OECD guideline 209 and GLP principles. Nominal test concentrations were the following: 0 (blank control), 10, 100 and 1000 mg/L.

After 3h of incubation Accelerator (PT 25E or PT 25E/2) did not cause an inhibition of the respiration rate at 10 mg/L. At 100 and 1000 mg/L an inhibition of 21% and an average of 29% inhibition were observed, respectively. The inhibition at 1000 mg/L was statistically significant but below 50%. The EC50 was estimated to be >1000 mg/L related to the respiration rate.No NOEC could be determined since it was observed a statistically significant difference in inhibition at 1000 mg/L in relation to the control group, and only one replicate was used for the lower test concentrations (i.e. 10 and 100 mg/L). Therefore, the statistical significance could not be determined at test concentrations of 10 and 100 mg/L.There was no significant oxygen uptake from abiotic processes. The results at 1000 mg/L with a nitrification inhibitor showed no heterotrophic inhibition of the respiration rate. The reviewer calculated that at 1000 mg/L, the heterotrophic respiration was inhibited 4% and the nitrification respiration was inhibited 92%. The study is considered to be reliable without restrictions.