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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:
27 April 2012 to 06 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
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 in water (tap water purified by reverse osmosis). Magnetic stirring for 59 minutes in the combined limit/range finding test or for 25 minutes in the additional range finding test and final test was applied to accelerate dissolution and to ensure homogeneity. Stock solutions corresponding to the test concentration were then added to the test media.
- Controls: The reference substance was used as a positive control, tested at 5.0, 12 and 30 mg/L. Untreated test medium controls were run concurrently.
- Evidence of undissolved material: There was no evidence of undissolved test material all stock solutions were clear and colourless.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- 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 approximately 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.7 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
NDA
Test temperature:
Ranged from 17.8 to 20.7 ºC
pH:
Before he addition of the sludge ranged from 7.2 to 7.6.
pH after exposure ranged from 7.8 to 8.4.
Dissolved oxygen:
Dissolved oxygen was above 60-70% saturation (60% of ari saturation is > 5 mg/L at 20 ºC) at the start of the test.
Salinity:
NDA
Nominal and measured concentrations:
Nominal test concentrations: 0.10, 0.32, 1.0, 3.2, 10, 32, 100, 320 and 1000 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass bottles
- Type: Open
- Aeration: Continuous aeration was achieved using a pipette as an aeration device, delivering clean oil free air. Aeration was sufficient to maintain the dissolved oxygen concentration and to maintain suspension of the sludge.
- No. of organisms per vessel: 250 mL of activated sludge.
- No. of vessels per concentration (replicates): 5 replicates per group.
- No. of vessels per control (replicates): 6 replicates.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis and subsequently passed over activated carbon and ion-exchange cartridges.

SYNTHETIC MEDIUM
- Dissolved in dilution water, made up to 1L and filtered. The pH was within 7.5 ± 0.5.
- Volume added to each sample: 16 mL
- Composition:
> 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

OTHER TEST CONDITIONS
- Adjustment of pH: No.

EFFECT PARAMETERS MEASURED:
pH: Was determined immediately before initiation of the test. Initiation of the test was signified by addition of the activated sludge. The pH was also determined post exposure.
Temperature: Determined in the reaction mixture post exposure.
Oxygen consumption: Was determined over a period of 9 to 14 minutes after the 3 hour exposure duration. Measurements were performed with an oxygen probe connected to a multichannel measuring and controlling system.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Initially a combined limit/range finding study was performed followed by an additional range finding study. The experimental set up for both can be seen in Table 1.
Combined limit/range finding study:
- Test concentrations: 10, 100 and 1000 mg/L
- Test conditions: Similar to the main test with the following exceptions; sludge pH on test day was 7.6 and a 2.32 g/L solution of N-allythiourea was used as a nitrification inhibitor.
Additional range finding study:
- Test concentrations: 0.01, 0.1, 1.0, 10, 100 and 1000 mg/L
- Test conditions: Similar to the main test.
- Results used to determine the conditions for the definitive study:
The combined limit/range-finding test showed 43% inhibition of the respiration rate at 10 mg/L, 47% at 100 mg/L and 58% (mean value) at 1000 mg/L.
Since not enough information was obtained to perform a final test, an additional range-finding test was performed at 0.01, 0.1, 1.0, 10 100 and 1000 mg/L.
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 lower than the total inhibition. N-allylthiourea was not used in the additional range-finding test and the final test.
The temperatures measured in the various vessels ranged between 18.8 and 20.6°C and were within the ranges prescribed by the protocol. The pH in all test series, before addition of sludge was 7.4. After the 3 hour exposure period the pH was between 7.2 and 8.2.
The additional range-finding test showed no inhibition of the respiration rate at and below 0.1 mg/L and 3% inhibition of the respiration rate at 1.0 mg/L, 13% at 10 mg/L, 23% at 100 mg/L and 43% at 1000 mg/L. Therefore, the NOEC in the additional range-finding test was at approximately 1.0 mg/L and the expected EC50 was above the highest concentration tested.
The temperatures measured in the various vessels ranged between 18.1 and 19.6°C and were within the ranges prescribed by the protocol. The pH in all test series, before addition of sludge was between 7.3 and 7.4. After the 3 hour exposure period the pH was between 7.9 and 8.1.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (99.8%)
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
1
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL 0.1 - 7.9 mg/L
Key result
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
2.1
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL 4.4 - 110 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL 4.4 - 110 mg/L
Details on results:
No biologically and/or statistically significant inhibition of the respiration rate of the sludge was recorded at and below 1.0 mg/L. Inhibition was observed at 0.32 mg/L, however this response was not concentration related. There was no increase in % inhibition of the respiration rate with increasing concentration at this level, and therefore considered as not significant for the test.

At higher concentrations the inhibitory effect of the test material on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from an average of 25% inhibition at 3.2 mg/L to 51% at 32 mg/L. Starting at 32 mg/L the inhibition of the respiration rate levelled off at approximately 50% inhibition.
Results with reference substance (positive control):
- Results with reference substance were valid.
- Relevant effect levels: Inhibition of respiration was observed between 52 and 86% at concentrations ranging from 5.0 to 30 mg/L, see Table 3.

Table 3. Study Results

Flask

Concentration (mg/L)

Respiration Rate (mg O₂/L.h)

Respiration Rate (mg O₂/L.h)1

% Inhibition Respiration3

C1

0

32

21

 

C2

0

43

29

 

C3

0

34

23

 

C4

0

35

23

 

C5

0

37

25

 

C6

0

32

21

 

 Mean

0

36

24

 

SD

0

4

3

 

CV (%)

0

12

12

 

 

R1

5.0

17

11

52

R2

12

10

7

72

R3

30

5

3

86

 

T1 a

0.10

36

24

-1

T1 b

0.10

35

23

1

T1 c

0.10

32

21

10

T1 d

0.10

28

19

21

T1 e

0.10

37

25

-4

 

T2 a

0.32

32

21

10*²

T2 b

0.32

35

23

1*²

T2 c

0.32

28

19

21*²

T2 d

0.32

30

20

15*²

T2 e

0.32

24

16

32*²

 

T3 a

1.0

32

21

10

T3 b

1.0

34

23

4

T3 c

1.0

37

25

-4

T3 d

1.0

34

23

4

T3 e

1.0

25

17

30

 

T4 a

3.2

27

18

24

T4 b

3.2

27

18

24*

T4 c

3.2

28

19

21*

T4 d

3.2

24

16

32*

T4 e

3.2

27

18

24*

 

T5 a

10

22

15

38*

T5 b

10

20

13

44*

T5 c

10

18

12

49*

T5 d

10

17

11

52*

T5 e

10

19

13

46*

 

T6 a

32

16

11

55*

T6 b

32

19

13

46*

T6 c

32

18

12

49*

T6 d

32

16

11

55*

T6e

32

18

12

49*

 

T7 a

100

16

11

55*

T7 b

100

19

13

46*

T7 c

100

22

15

38*

T7 d

100

18

12

49*

T7 e

100

18

12

49*

T8 a

320

20

13

44*

T8 b

320

18

12

49*

T8 c

320

16

11

55*

T8 d

320

16

11

55*

T8 e

320

15

10

58*

T9 a

1000

18

12

49*

T9 b

1000

17

11

52*

T9 c

1000

18

12

49*

T9 d

1000

11

7

69*

T9 e

1000

17

11

52*

C: Control,

R: Reference substance,

T: Test material,

SD: standard deviation,

CV: Coefficient of variation,

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

1: The amount of suspended solids in the test mixture was 1.5 g/L,

2: This response was not concentration related,

3: Percentage inhibition respiration relative to the control (mean value).

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, the test material was not toxic to waste water (activated sludge) bacteria at or below a 1.0 mg/l (NOEC). The EC50 was determined to be 22 mg/L (95% confidence interval 4.4 to 110 mg/L). Since all criteria for acceptability of the test were met, this study was considered to be valid.
Executive summary:

The toxicity of the test material to microorganisms was determined in a activated sludge respiration inhibition study, performed in line with GLP and to the standardised guidelines OECD 209 and EU method C.11.

Samples of activated sludge were exposed to the test material under freshwater static conditions for 3 hours at nine nominal concentrations ranging between 0.10 and 1000 mg/L, increasing with a spacing factor of 3.2. 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. Blank controls were run concurrently for comparison. The batch of activated sludge was checked for sensitivity by testing the reference substance (3,5- dichlorophenol), which showed normal sensitivity. All acceptability criteria were met and thus the study was considered to be valid.

Under the conditions of the test, the test material was not toxic to waste water (activated sludge) bacteria at or below a 1.0 mg/l (NOEC). The EC₅₀ was determined to be 22 mg/L (95% confidence interval 4.4 to 110 mg/L).

Description of key information

The toxicity of 3,5-Dimethylpyrazole to microorganisms was determined in a key study (Desmares-Koopmans, 2012) which was performed to GLP and the standardised guidelines OECD 209 and EU Method C 11. The study determined to the NOEC to be 1.0 mg/L and the EC50 22 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
22 mg/L
EC10 or NOEC for microorganisms:
1 mg/L

Additional information

The toxicity to microorganisms of the test material was determined in a GLP compliant study (Desmares-Koopmans, 2012) performed in accordance with standardised guidelines, with a sufficient level of detail to assess the quality of the presented data. The study was performed to a good standard and was assigned a reliability score of 1 in accordance with Klimisch (1997).

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