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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 September 2009
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
Version / remarks:
April 1984
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Lot No.: 9052S 13209211
Appearance: Colourless liquid
Purity: 100% (confirmed via GC analysis)
Storage: Sample was stored at ambient temperature, in the containerin which it was received, until required for testing
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A nominal 5000 mg l-1 stock solution of acetonitrile was prepared in reverse osmosis (RO) water. This stock was observed to be a clear colourless solution and the pH was measured as 5.8, which was adjusted to 6.4 using 2M sodium hydroxide solution. Test solutions were prepared by the addition of a known volume of this stock solution to a total volume of 500 ml RO water, synthetic sewage and activated sludge as described in the experimental design.

PREPARATION OF REFERENCE SUBSTANCE
A nominal 500 mg l-1 stock solution of the reference substance, 3,5-dichlorophenol, was prepared in RO water. This stock solution was observed to be clear and colourless and its pH was measured as 6.8 on the day of the test.
Test organisms (species):
activated sludge
Details on inoculum:
- Laboratory culture: Totnes Sewage Works, Devon, UK. This works treats sewage of predominantly domestic origin.
- Preparation of inoculum for exposure: The activated sludge was settled and the concentrate washed with tap water. The washed settled sludge was fed with 50 ml of OECD synthetic sewage feed per litre of sludge per day and aerated at room temperature, until it was used in the test. The total filte able solids concentration was determined on the day of the test and was found to be 4763 mg l-1. The pH was measured as 5.4, and was subsequently adjusted to 7.2 with 2 M sodium hydroxide prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Remarks on exposure duration:
3 hours for 3,5-DCP
Post exposure observation period:
Not applicable
Hardness:
Not reported
Test temperature:
20 ± 2°C
pH:
Control (30 mins): 7.2 to 7.7
Control (3 hrs): 6.4 to 7.7
3,5-DCP: 7.5 to 8.1
Acetonitrile: 7.1 to 7.5
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal 10, 32, 100, 320 and 1000 mg l-1 concentrations of acetonitrile were prepared together with two control culture flasks. Five flasks containing the reference substance, 3,5-dichlorophenol, at nominal concentrations of 3.2, 10, 32 and 100 mg l-1, together with an abiotic flask at 100 mg l-1 were also prepared.
Details on test conditions:
This test measures the respiration rate of an activated sludge thirty minutes, or three hours, after feeding an excess, but standard amount, of a synthetic sewage and compares this with the respiration rate of the same activated sludge in the presence of the test chemical. 3,5-dichlorophenol is used as a reference substance as it has known inhibitory effects on respiration and ensures that the batch of sludge used in the test shows a normal level of sensitivity.

Each flask contained an excess of the synthetic sewage, sufficient activated sludge to give final solids concentrations of 1600 mg l-1, an appropriate quantity of either acetonitrile, or 3,5-dichlorophenol stock solution, and RO water to give a final flask contents volume of 500 ml.

Flasks were set up in batches of six and aerated at 20 ± 2°C for thirty minutes for the acetonitrile vessels and three hours for the 3,5-dichlorophenol vessels. The aeration time was shorter for the acetonitrile flasks, because of the volatility of the test substance (as permitted by the guideline, Ref 1). Each batch included a control flask and five test or reference substance flasks. The temperatures of the flask contents were measured at the end of the aeration period using a mercury-in-glass thermometer.

The respiration rate of each culture was measured after thirty minutes, or three hours, as applicable, and compared with the mean respiration rate of the two control cultures, which were measured at both thirty minutes and three hours. The rate of oxygen uptake was measured in glass sample tubes into which microcathode oxygen electrodes were inserted. The electrodes were connected to an interface unit, which converted the current produced by the electrodes into dissolved oxygen readings. These were in turn transferred to a computer, which calculated the respiration rate in each flask over the linear part of the curve and compared it to the mean of the two control cultures. The rates of oxygen uptake were expressed as mg O2 l-1 h-1.

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (97%)
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
30 min
Dose descriptor:
other: EC20
Effect conc.:
320 - 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
30 min
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
30 min
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
In test flasks dosed with acetonitrile, 32% inhibition was observed in the 1000 mg l-1 test concentration, 10% in the 320 mg l-1 test concentration, and less than 10% in all the other tested concentrations.

The 10% effect concentration (EC10) is defined as the concentration, estimated from the data obtained, resulting in a 10% reduction in the respiration rate of activated sludge within the period of the test. Percentage inhibition less than or equal to 10% was within the expected experimental variability of the test and was considered not to be an effect of acetonitrile. The no observed effect concentration (NOEC) is therefore equivalent to the EC10 in this test.
Results with reference substance (positive control):
The reference substance, 3,5-dichlorophenol, caused substantial inhibition of the respiration rate of the activated sludge. From the results obtained the three hour EC50 value was estimated to be 6.2 mg l-1. This is within the expected normal range of 5 to 30 mg l-1 (Ref 1) indicating the sludge was responding normally and confirming the viability of the sludge organisms. The respiration rates in the two control flasks were within 15% of each other. Therefore, the mean control respiration rate was used in calculation of the percentage inhibition.
Reported statistics and error estimates:
The EC50 value was estimated by computer software.

Test substance

Nominal test concentration (mg l-1)

pH

 Respiration rate (mg O2l-1h-1)

Percentage inhibitionb

 

Start

Enda

Control (30 mins)

Control

7.7

7.2

41.3

-

 

Control (3 hrs)

Control

7.7

6.4

22.7

-

 

3,5-DCPc

 100 (abiotic)

7.8

7.6

0

-

 

3,5-DCP

100

7.5

8.1

1.7

93

 

3,5-DCP

32

7.5

8.1

2.2

91

 

3,5-DCP

10

7.5

8.0

8.8

64

 

3,5-DCP

3.2

7.5

7.9

16.7

31

 

Control (30 mins)

Control

7.5

7.1

46.7

-

 

Control (3 hrs)

Control

7.5

6.6

25.7

-

 

Acetonitrile

1000

7.5

7.5

30.0

32

 

Acetonitrile

320

7.5

7.2

39.4

10

 

Acetonitrile

100

7.5

7.2

53.4

<10

 

Acetonitrile

32

7.5

7.2

51.2

<10

 

Acetonitrile

10

7.5

7.1

46.7

<10

 

Validity criteria fulfilled:
yes
Remarks:
Reference substance data were within the expected range.
Conclusions:
The resulting 30 minute EC50 and NOEC values of acetonitrile, based on inhibition of respiration rate of activated sewage sludge, were determined to be >1000 mg/L and 320 mg/L, respectively.
Executive summary:

In a guideline (OECD 209) and GLP study by Brixham Environmental Laboratory (2009) the 30 -min NOEC for acetonitrile in an Activated Sludge Respiration Inhibition Test was determined to be 320 mg/L.

Endpoint:
toxicity to microorganisms, other
Remarks:
Inhibition of cell multiplication
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No information available
GLP compliance:
not specified
Analytical monitoring:
not specified
Test organisms (species):
Entosiphon sulcatum
Test type:
not specified
Water media type:
freshwater
Test temperature:
25 degree C.
pH:
7
Details on test conditions:
Type: aquatic
Duration:
72 h
Dose descriptor:
other: TT
Effect conc.:
1 810 mg/L
Executive summary:

Bringmann and Kuhn (1980) reported a 72 -hour toxicity threshold (TT), based on inhibition of cell multiplication, of 1810 mg/L for acetonitrile in (Entosiphon sulcatum).

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
according to guideline
Guideline:
other: Owen et al (1979)
Principles of method if other than guideline:
An anaerobic toxicity assay conducted according to the method of Owen et al., 1979.
GLP compliance:
not specified
Analytical monitoring:
not specified
Test organisms (species):
anaerobic bacteria
Test type:
not specified
Water media type:
freshwater
Test temperature:
35 degree C.
pH:
7
Details on test conditions:
Type: other
Duration:
48 h
Dose descriptor:
IC50
Effect conc.:
28 000 mg/L
Executive summary:

Blum and Speece (1991) reported a 48 -hour IC50 value, based on inhibition of gas production, of 28000 mg/L for acetonitrile in anaerobic bacteria.

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No information available
GLP compliance:
not specified
Analytical monitoring:
not specified
Test organisms (species):
aerobic microorganisms
Test type:
not specified
Water media type:
freshwater
Test temperature:
25 - 35 degree C.

Details on test conditions:
Type: soil
Duration:
15 h
Dose descriptor:
IC50
Effect conc.:
7 500 mg/L
Executive summary:

Blum and Speece (1991) reported a 15 -hour IC50 value, based on inhibition of oxygen uptake, of 7500 mg/L for acetonitrile in aerobic bacteria.

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No information available.
GLP compliance:
not specified
Analytical monitoring:
not specified
Test organisms (species):
Nitrosomonas sp.
Test type:
not specified
Water media type:
freshwater
Test temperature:
25 degree C.
pH:
6.5 - 8
Details on test conditions:
Type: other
Duration:
24 h
Dose descriptor:
IC50
Effect conc.:
73 mg/L
Executive summary:

Blum and Speece (1991) reported a 24 -hour IC50 value, based on inhibition of ammonia consumption, of 73 mg/L for acetonitrile in (Nitrosomonas sp).

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No information available
GLP compliance:
not specified
Analytical monitoring:
not specified
Test organisms (species):
Pseudomonas putida
Test type:
not specified
Water media type:
freshwater
Test temperature:
25 degree C.
pH:
7
Details on test conditions:
Type: aquatic
Duration:
16 h
Dose descriptor:
other: TT
Effect conc.:
680 mg/L
Executive summary:

Bringmann and Kuhn (1980) reported a 16 -hour toxicity threshold (TT), based on inhibition of cell multiplication, of 680 mg/L for acetonitrile in (Pseudomonas putida).

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No information available
GLP compliance:
not specified
Analytical monitoring:
not specified
Test organisms (species):
Uronema parduzci
Test type:
not specified
Water media type:
freshwater
pH:
6.9
Details on test conditions:
Type: aquatic
Duration:
20 h
Dose descriptor:
other: TT
Effect conc.:
5 825 mg/L
Executive summary:

Bringmann and Kuhn (1980b) reported a 20 -hour toxicity threshold (TT), based on inhibition of cell multiplication, of 5825 mg/L for acetonitrile in (Uronema parduzci).

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No information available
GLP compliance:
not specified
Analytical monitoring:
not specified
Test organisms (species):
Chilomonas paramaecium
Test type:
not specified
Water media type:
freshwater
Test temperature:
20 degree C.
pH:
6.9
Details on test conditions:
Type: aquatic
Duration:
48 h
Dose descriptor:
other: TT
Effect conc.:
942 mg/L
Executive summary:

Bringmann and Kuhn (1981) reported a 48 -hour toxicity threshold (TT), based on inhibition of cell multiplication, of 942 mg/L for acetonitrile in (Chilomonas paramaecium).

Description of key information

In a guideline (OECD 209) and GLP study by Brixham Environmental Laboratory (2009) the 30 -min EC50 for acetonitrile in an Activated Sludge Respiration Inhibition Test was determined to be >1000 mg/L and the NOEC was 320 mg/L.  Previous data on the toxicity of acetonitrile to aquatic microorganisms was evaluated in the EU RAR.  Reported toxicity values ranged from 73 mg/L to 28,000 mg/L.  The lowest value by an order of magnitude was reported by Blum and Speece (1991), a 24-hour IC50 of 73 mg/L in Nitrosomas sp., based on inhibition of ammonia consumption.  

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
320 mg/L

Additional information

In a guideline (OECD 209) and GLP study by Brixham Environmental Laboratory (2009) the 30 -min EC50 for acetonitrile in an Activated Sludge Respiration Inhibition Test was determined to be >1000 mg/L, and the NOEC was 320 mg/L.

Previous data on the toxicity of acetonitrile to microorganisms was reviewed in the EU Risk Assessment Report (2000). A summary of these data is shown in the table below. Blum and Speece (1991) present data on three groups of environmental bacteria that can be relevant when considering the microbial activity of domestic WWTPs. The IC50 values (the concentrations of acetoni­trile causing 50% bacterial inhibition compared to controls) are considered valid for this assessment and were summarized as follows:

           1. Aerobic heterotrophs.- These bacteria predominate in activated sludge systems and in natural aerobic environments where they convert organic material to carbon dioxide and water. The inhibition in oxygen uptake by the bacteria was monitored after 15 hours exposure and used to calculate and IC50 of 7500 mg/L.

           2.Nitrosomas sp.- These bacteria convert ammonia nitrogen to nitrite as the first most sensitive step in the biologi­cal oxidation of inorganic nitrogen. The inhibition in ammonia consumption by the bacteria was monitored after 24 hours exposure and used to calculate an IC50 of 73 mg/L.

           3. Methanogens.- Methanogens form part of the consortium of bacteria that convert organic matter to carbon dioxide and methane under anaerobic conditions. The inhibition of gas production by the bacteria was monitored after 48 hours exposure and used to calculate an IC50 of 28000 mg/L.

Additional data on (Pseudomona putida),and several protozoa species have been published by Brigmann and Kühn (1980ª, 1980b, 1981). The lowest toxicity threshold is 680 mg/l reported for (P. putida), while TT values for protozoa species are in the range of g/l.

 

Toxicity of acetonitrile to aquatic microorganisms (EU Risk Assessment Report, 2000)

SPECIES

TEST TYPE

COMMENTS

DURATION

(hour)

TOXICITY

END POINT

(mg/l)

REFERENCE

Chillmonas paramecium(Protozoa)

Inhibition of cell multiplication

Valid as additional information

48

TT = 942

Bringmann and Kühn (1981)

 

Entosiphon sulcatum(Protozoa)

Inhibition of cell multiplication

Valid as additional information

72

TT = 1810

Bringmann and Kühn (1980a)

 

Pseudomona putida(Bacteria)

Inhibition of cell multiplication

Valid as additional information

16

TT = 680

Bringmann and Kühn (1980a)

 

Uronema parduzci(Protozoa)

Inhibition of cell multiplication

Valid as additional information

20

TT = 5825

Bringmann and Kühn (1980b)

 

Nitrosomas

(Bacteria)

Inhibition of ammonia consumption

Valid for the assessment

24

IC50= 73

Blum and Speece (1991)

Aerobic micro-organisms

Inhibition of oxygen uptaque AFNOR and ETAD

Valid for the assessment

15

IC50= 7500

Blum and Speece (1991)

Metanogenic bacteria

Inhibition of gas production

Valid for the assessment

48

IC50= 28000

Blum and Speece (1991)

 TT = toxicity threshold