Registration Dossier

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
There were slight deviations with initial and final dissolved oxygen concentrations. Please see 'Principles of method if other than guideline' for further details.
Principles of method if other than guideline:
There were slight deviations with initial and final dissolved oxygen concentrations, some preparations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L, respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.The dissolved oxygen concentrations after 30 minutes contact time in some of the vessels was below 60% of the dissolved oxygen saturation level of 8.9 mg O2/L. This deviation was considered to have had no adverse effect on the study given that all oxygen consumption values were determined over the linear portion of the traces.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
An amount of test item (50 mg) was dissolved in water with the aid of ultrasonication for approximately 30 minutes and the volume adjusted to 500 mL to give a nominal 100 mg/L stock solution from which dilutions were made to give 1.0, 3.2, 10 and 32 mg/L stock solutions. An aliquot (50 mL) of the 1.0 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 0.10 mg/L. Similarly, aliquots (50 mL) of the 3.2, 10, 32 and 100 mg/L stock solutions were used to prepare the nominal test concentrations of 0.32, 1.0, 3.2 and 10 mg/L. Five replicates were prepared per concentration. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C overnight prior to use in the test. On the day of collection the activated sewage sludge (15 liters) was fed synthetic sewage (750 mL). The pH of the sample on the day of the definitive test was 7.8. Determination of the mixed liquor suspended solids (MLSS) level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed three times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 °C for at least one hour and allowed to cool before weighing. This process was repeated until two successive dry weights within 5% was attained. The suspended solids concentration was equal to 3.0 g dry weight/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
Approximately 21°C.
Nominal and measured concentrations:
0.1, 0.32, 1.0, 3.2 and 10 mg/L nominal.
Details on test conditions:
At the start of the test (time "0"), 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical glass flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels (in ascending concentration order) with appropriate amounts of the reference item being added. Two additional control vessels were then prepared prior to the test item vessels being prepared (in ascending concentration order). Finally two further control vessels were prepared. The addition of the inoculum to the reaction mixture was considered the start of the 3-hour exposure period for each vessel.

A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g Peptone
, 11 g Meat extract
, 3 g Urea
, 0.7 g NaCl
, 0.4 g CaCl, 2.2H2O
, 0.2 g MgSO, 4.7g H2O,
2.8 g K2HPO4, then
dissolved in 1 liter of water with the aid of ultrasonication.

The pH of the test item stock solutions was measured using Hach 160 handheld meter and adjusted, where as necessary, to between pH 7.0 and 8.0 with 1.0M NaOH

.
Observations
Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge. The temperature was recorded in one of the control vessels at the start and end of the incubation period. The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour exposure period. The oxygen concentrations in all vessels were measured after 30 minutes contact time.

Measurement of the Respiration Rates

As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured continuously over the linear portion of the oxygen consumption trace (where possible, between approximately 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over a period of up to 10 minutes.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
3.1 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 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L, respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

The dissolved oxygen concentrations after 30 minutes contact time in some of the vessels was below 60% of the dissolved oxygen saturation level of 8.9 mg O2/L. This deviation was considered to have had no adverse effect on the study given that all oxygen consumption values were determined over the linear portion of the traces.
Results with reference substance (positive control):
The EC50 value for the reference item (11 mg/L) also satisfied the validation criterion.

Range Finding Study:

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.

Statistically significant respiration inhibition was observed at all of the test concentrations employed (p = 0.05). Based on these results and after discussion with the Study Monitor for the Sponsors, it was considered necessary to perform a definitive test to obtain a NOEC and, if possible, an EC10 value for the test item. Based on this information test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were selected for the definitive test.

Oxygen consumption rates and percentage inhibition values for the control, test and reference items are tabulated as follows. The temperatures recorded in the first and last control vessels were 21 °C and 22 °C, respectively. The coefficient of variation of oxygen uptake in the control vessels was 2.42% and the specific respiration rate of the controls was 27.27 mg oxygen per gram dry weight of sludge per hour. 

pH Values of the Test Preparations at the Start and End of the Exposure Period in the Range-finding Test

Nominal
Concentration
(mg/L)

pH

0 Hours

3 Hours

Control

R1

7.6

7.7

 

R2

7.6

7.8

 

R3

7.7

7.8

 

R4

7.7

7.8

Test Item

10

7.6

7.9

 

100

7.6

7.8

 

1000 R1

7.4

7.9

 

1000 R2

7.5

7.9

 

1000 R3

7.6

7.9

3,5-Dichlorophenol

3.2

7.6

8.0

 

10

7.6

8.2

 

32

7.7

8.3

 


R1– R4= Replicates 1 to 4

Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Definitive Test

Nominal

Concentration

 (mg/L)

Initial O2

 
Reading

 (mg O2/L)

Measurement Period

(minutes)

Final O2Reading

 (mg O2/L)

O2Consumption Rates

(mg O2/L/hour)

% Inhibition

Control

R1

4.8

4

2.1

40.50

-

 

R2

4.6

4

1.9

40.50

-

 

R3

3.5

2

2.1

42.00

-

 

R4

4.3

3

2.2

42.00

-

 

R5

6.3

6

2.2

41.00

-

 

R6

6.5

7

1.9

39.43

-

Test Item

0.10 R1

3.8

3

1.8

40.00

2

 

0.10 R2

3.4

2

2.0

42.00

[3]

 

0.10 R3

4.5

4

1.9

39.00

5

 

0.10 R4

4.3

3

2.3

40.00

2

 

0.10 R5

4.3

4

1.8

37.50

8

 

0.32 R1

4.3

4

1.7

39.00

5

 

0.32 R2

4.4

3

2.3

42.00

[3]

 

0.32 R3

3.3

2

2.0

39.00

5

 

0.32 R4

4.2

4

1.6

39.00

5

 

0.32 R5

4.2

4

1.5

40.50

1

Test Item

1.0 R1

4.1

3

2.1

40.00

2

 

1.0 R2

4.3

4

1.7

39.00

5

 

1.0 R3

4.3

3

2.2

42.00

[3]

 

1.0 R4

3.2

2

1.9

39.00

5

 

1.0 R5

4.2

3

2.2

40.00

2

 

3.2 R1

4.2

4

1.8

36.00

12*

 

3.2 R2

4.4

4

2.0

36.00

12*

 

3.2 R3

4.8

5

1.8

36.00

12*

 

3.2 R4

5.2

5

2.3

34.80

15*

 

3.2 R5

5.7

6

2.1

36.00

12*

 

10 R1

5.9

7

2.2

31.71

22*

 

10 R2

6.3

8

2.0

32.25

21*

 

10 R3

6.7

8

2.5

31.50

23*

 

10 R4

6.1

8

2.0

30.75

25*

 

10 R5

6.1

8

2.0

30.75

25*

3,5-Dichlorophenol

3.2

5.1

5

2.3

33.60

18

 

10

5.9

10

2.4

21.00

49

 

32

7.5

10

5.9

9.60

77

  R1– R5= Replicates 1 to 5
[Increase in respiration rate as compared to controls]
* Statistically significant respiration inhibition compared to the control vessels (p = 0.05).

Validity criteria fulfilled:
yes
Conclusions:
The effect of 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione on the respiration of activated sewage sludge resulted in a 3-hour EC10 value of 3.1 mg/L. The No Observed Effect Concentration (NOEC) following the 3-hour exposure was 1.0 mg/L.
Executive summary:

In an OECD 209 study, conducted according to GLP, the 3 hour respiration inhibition EC10 to activated sewage sludge micro-organism of 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione is 3.1 mg/L, and the NOEC is 1.0 mg/L.

Description of key information

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
3.1 mg/L

Additional information

Key Study:

In an OECD 209 study, conducted according to GLP, the 3 hour respiration inhibition EC10 to activated sewage sludge micro-organism of 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione is 3.1 mg/L, and the NOEC is 1.0 mg/L (Envigo Research Limited, 2016f).