Acetonitrile

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

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.

Reference 2

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).

Reference 3

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.

Reference 4

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.

Reference 5

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).

Reference 6

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).

Reference 7

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).

Reference 8

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