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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start : 29 March 2012 Completion : 16 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study without significant deficiencies
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
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 25 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 6.4.1 and 6.6). Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The sludge was coarsely sieved, 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 7.8 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.
16 ml synthetic medium, 250 ml sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates.
Test type:
static
Water media type:
freshwater
Total exposure duration:
3 h
Post exposure observation period:
none
Hardness:
CaCl2.2H2O 211.5 mg/l
MgSO4.7H2O 88.8 mg/l
NaHCO3 46.7 mg/l
KCl 4.2 mg/l
Test temperature:
The temperatures measured in the various vessels ranged between 17.3 and 20.8°C and were except for the deviation of max. 0.7°C as described in paragraph 6.9.1, within the ranges prescribed by the protocol.
pH:
The pH in all test series, before addition of sludge was between 4.3 and 7.5. After the 3 hour exposure period the pH was between 4.3 and 8.4.
Dissolved oxygen:
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)
Salinity:
NA
Nominal and measured concentrations:
Nominal loading rates (mg/l) were 0, 100, 180, 320, 560 and 1000.
Details on test conditions:
Contact time: 3 hours, during which aeration and stirring took place.

Vessels: All glass bottles/vessels.

Milli-RO / Milli-Q 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).

Synthetic medium (=sewage feed)
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-Q water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

Air supply: Clean, oil-free air.

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.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
560 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:

No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 100 mg di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) / litre (Bonferroni t-Test: α=0.05 Toxstat). At higher loading rates the inhibitory effect of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 26% inhibition at a loading rate of 180 mg/l to 65% at loading rate of 1000 mg/l.
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/l for total respiration (5.3 and 4.9 mg/l).
Reported statistics and error estimates:
The EC10 was at a loading rate of 110 mg/l (95% confidence interval: 45 - 280 mg/l).
The EC20 was at a loading rate of 170 mg/l (95% confidence interval: 72 - 400 mg/l).
The EC50 was at a loading rate of 560 mg/l (95% confidence interval: 270 - 1190 mg/l).
The EC80 was above a loading rate of 1000 mg/l.

Final Test Results

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

19.4

7.5

8.0

40

27

 

C 2

0

19.7

7.5

8.0

39

26

 

C 3

0

19.5

7.4

7.9

44

29

 

C 4

0

18.2

7.4

8.0

39

26

 

C 5

0

20.2

7.4

7.9

51

34

 

C 6

0

19.7

7.4

7.9

44

29

 

Mean

0

 

 

 

43

29

 

SD

0

 

 

 

5

3

 

CV (%)

0

 

 

 

12

12

 

 

 

 

 

 

 

 

 

R 1

5.0

19.4

7.5

8.2

25

17

42

R 2

12

19.5

7.5

8.2

13

9

70

R 3

30

18.5

7.5

8.3

5

3

88

 

 

 

 

 

 

 

 

T 1a

100

19.4

7.1

8.0

39

26

9

T 1b

100

18.9

7.2

8.1

34

23

21

T 1c

100

19.2

7.2

8.0

36

24

16

T 1d

100

19.0

7.1

8.0

35

23

18

T 1e

100

20.0

7.2

8.0

42

28

2

 

 

 

 

 

 

 

 

T 2a

180

19.4

6.9

8.0

34

23

21*

T 2b

180

19.5

6.9

7.9

33

22

23*

T 2c

180

18.2

6.9

8.0

28

19

35*

T 2d

180

18.4

6.9

8.0

29

19

32*

T 2e

180

19.7

6.9

7.9

35

23

18*

 

 

 

 

 

 

 

 

T 3a

320

19.2

6.6

7.9

32

21

25*

T 3b

320

19.9

6.5

7.9

31

21

28*

T 3c

320

20.8

6.6

7.8

36

24

16*

T 3d

320

20.0

6.5

7.8

32

21

25*

T 3e

320

18.2

6.5

8.0

22

15

49*

T 4a

560

18.5

5.6

8.0

23

15

46*

T 4b

560

17.9

5.4

8.1

19

13

56*

T 4c

560

18.4

5.5

8.0

21

14

51*

T 4d

560

18.1

5.5

8.0

20

13

53*

T 4e

560

18.0

5.6

8.0

20

13

53*

 

 

 

 

 

 

 

 

T 5a

1000

19.3

4.3

8.0

16

11

63*

T 5b

1000

17.7

4.3

8.0

15

10

65*

T 5c

1000

17.3

4.3

8.0

12

8

72*

T 5d

1000

18.6

4.3

7.9

15

10

65*

T 5e

1000

18.8

4.3

7.9

16

11

63*

C:           Control

R:           Reference substance, 3,5-dichlorophenol

T:           Test substance, di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA)

CV:         Coefficient of variation

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

*             Statistically significant (Bonferroni t-Test:α=0.05 Toxstat

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 100 mg/l (NOEC).



Executive summary:

The influence of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) 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.

 

DNNSA is a UVCB substance (purity 100%). The test material was a brown highly viscous liquid. 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 with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 25 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. 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 9 to 14 minutes.

 

The final test was performed based on the result of a preceding combined limit/range-finding test. Five loading rates, ranging from 100 to 1000 mg/l, increasing with a factor 1.8 were tested. Five replicates per loading rate and sixreplicates for an untreated control group were tested.

No statistically significant inhibition of the respiration rate of the sludge was recordedat or below a loading rate of 100 mg di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) /litre(Bonferroni t-Test:α=0.05 Toxstat). At higher loading rates theinhibitory effect of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 26% inhibition at a loading rate of 180 mg/l to 65% at loading rate of 1000 mg/l.

 

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.

 

In conclusion, di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 100 mg/l. The effect parameters for the inhibition of the respiration rate are presented below.

 

Parameter

di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA)

Loading rate (mg/l)

NOEC

100

EC10

110 (45 - 280 mg/l)

EC20

170 (72 - 400 mg/l)

EC50

560 (270 - 1190 mg/l)

EC80

>1000

(  ) Between brackets the 95% confidence intervals are given.

 

Description of key information

di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 100 mg/l (NOEC). 

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information