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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 October 2009 to 6 November 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Version / remarks:
March 2006
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
Oxygen conditions:
other: Sealed vessels
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Totnes Sewage Treatment Works, Devon, UK. This works treats sewage of predominantly domestic origin. At the laboratory, the activated sludge was kept aerated at room temperature and the pH maintained at 7.0 ± 1.0 until initiation of the test on the same day.

- On the exposure start day the activated sludge was centrifuged, washed and resuspended in the test medium and the solids concentration determined. This sludge was then diluted in medium to a final sludge solids concentration of 4 mg l-1, and added to the test bottles.
Duration of test (contact time):
21 d
Initial conc.:
684 mg/L
Based on:
other: equivalent to a nominal 400 mg l-1 as carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: 85 mg of KH2PO4, 217.5 mg of K2HPO4, 334 mg of Na2HPO4.2H2O, 5 mg of NH4Cl, 22.5 mg of MgSO4.7H2O, 36.4 mg of CaCl2.2H2O, 0.25 mg of FeCl3.6H2O.
- Test temperature: 22 ± 2°C.
- pH: pH of the test medium prior to use was measured and adjusted as necessary to 7.4 ± 0.2.
- Other: Sufficient replicates were prepared for five replicates to be analysed for inorganic carbon at the end of the study, together with one replicate for pH analysis, and triplicate analysis on each other sample occasion.

TEST SYSTEM
- Culturing apparatus: nominal 125 ml serum bottles, sealed with butyl rubber septa and aluminium crimp caps.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Inoculum blanks contained no test or reference substance, in order to demonstrate there was no other carbon source in the medium.
- Toxicity control: Toxicity controls contained the test and reference substances, both at nominal 20 mg l-1, as carbon, and were used if the test substance failed to degrade during the test period, to show if the failure had been due to inhibition of the inoculum by the test substance.
- Other: Positive controls contained sodium benzoate at nominal 20 mg l-1, as carbon, to demonstrate the viability of the inoculum.

Total Organic Carbon (TOC) DETERMINATION
- The TOC of the acetonitrile and sodium benzoate stock solutions was determined using tube kits provided by Hach Lange Ltd using a DR2800 spectrophotometer for evaluation. In a two stage process, inorganic carbon is expelled from the sample, then the remaining organic carbon is oxidised to carbon dioxide. The carbon dioxide passes through a membrane into an indicator cuvette, where a colour change occurs, which is evaluated spectrophotometrically.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
Not applicable
Test performance:
At the end of the 28 day test period, the pH values were 7.4 in the inoculum blank bottles, 6.9 in the sodium benzoate bottles, 7.1 in the acetonitrile bottles and 6.5 in the toxicity control bottles.
The incubator temperature remained within the range 22 ± 2°C of its set-point from day 1 of the study onwards (but was just below 20ºC during the first 24 hours of the study).
Parameter:
% degradation (CO2 evolution)
Value:
70
Sampling time:
21 d
Remarks on result:
other: 62% degradation in the 10 day window from day 4 to day 14
Details on results:
The measured TOC of the acetonitrile stock solution was 313 mg C 1^-1, lower than the nominal concentration of 400 mg C 1^-1. It was considered possible that the low result was due to loss of acetonitrile by evaporation from the test solution, either before the analysis was done, or during removal of inorganic carbon. Therefore, a fresh acetonitrile stock solution was prepared and analysed with and without the inorganic carbon removal step. The organic carbon measured in this stock was 291 and 337 mg C 1^-1, with and without IC removal, respectively. This indicates that the low result was probably not due to loss of acetonitrile by evaporation. It was decided to calculate the biodegradation results based on both nominal and measured TOC results, for completeness. More than 60% degradation was achieved within the 10 day window as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms.
Results with reference substance:
The measured TOC of the sodium benzoate stock solution was 400 mg C 1^-1, equal to the nominal concentration of 400 mg C 1^-1. On day 28 sodium benzoate (the reference substance) attained a mean level of biodegradation (based on the IC:TOC ratio) of 91%.

 Day  Percentage Biodegradation
   Acetonitrile (20 mg C l-1)  Reference substance (20 mg C l-1)
 11  69
 7  52  81
 14  62  81
 21  70  91
Validity criteria fulfilled:
yes
Remarks:
Reference substance results were within the expected range.
Interpretation of results:
readily biodegradable
Conclusions:
More than 60% degradation was achieved within the 10-day window, so acetonitrile can be classified as readily biodegradable
Executive summary:

In a guideline (OECD 310) and GLP study by Brixham Environmental Laboratories (2010) acetonitrile was determined to be readily biodegradable by non-acclimated activated sludge (> 60% degradation within 10 -days; 70% degradation after 21 days).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 March 1998 - 14 May 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Ten different sources in Japan, such as sewage-treatment plants, rivers, lakes and seas.
- Concentration of sludge: 30 mg/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
other: BOD
Parameter followed for biodegradation estimation:
TOC removal
Parameter followed for biodegradation estimation:
test mat. analysis
Remarks:
: GC
Details on study design:
TEST CONDITIONS
- Test temperature: 25±1˚C
- pH: 7.0
- pH adjusted: No


TEST SYSTEM
- Number of culture flasks/concentration:
3 bottles (test substance + sludge)
1 bottle (test substance + water)
1 bottle (reference substance + sludge)
- Measuring equipment: continuous BOD meter


SAMPLING
- Sampling frequency: continuous for BOD; After 28 days for TOC and GC


CONTROL AND BLANK SYSTEM
- Inoculum blank: 1 bottle was set
Reference substance:
aniline
Remarks:
: 100mg/L
Parameter:
other: BOD
Value:
65
Sampling time:
28 d
Remarks on result:
other: Mean value
Parameter:
% degradation (TOC removal)
Value:
84
Sampling time:
28 d
Remarks on result:
other: Mean value
Parameter:
other: GC
Value:
88
Sampling time:
28 d
Remarks on result:
other: Mean value
Details on results:
- Biodegradabily by BOD after 28 days: 72 %, 67 % and 56 % (mean: 65 %)
- Biodegradabily by TOC after 28 days: 94 %, 89 % and 70 % (mean: 84 %)
- Biodegradabily by GC after 28 days: 97 %, 92 % and 73 % (mean: 88%)

- Kinetics of test substance(BOD):
24, 23, 20 % (7th day)
32, 30, 26 % (14th day)
53, 52, 40 % (21st day)
72, 67, 56 % (28th day)

- Degradation products: No
Results with reference substance:
- Kinetics of control substance (BOD):
60 % (7th day), 70 % (14th day)
Interpretation of results:
readily biodegradable
Executive summary:

The CITI of Japan (1998) reported a biodegration study of acetonitrile according to OECD guideline 301C (modified MITI test I) and under GLP conditions. Biodegradation results after 28 days were:

by BOD - 65%

by TOC - 84%

by GC - 88%

Although the test substance was not completely biodegradated in this study, the BOD-curve was still on the upward trend after 28 days. Therefore, additional test was performed at a lower concentration of test substance (30 mg/L), and the degradation of the test substance measured by TOC and GC reached 100% after 14 days.

Based on these results, acetonitrile is considered to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Basic data given. Design does not meet guidelines.
Guideline:
other: American Public Health Association (1980) Standard Methods
Principles of method if other than guideline:
The method used was that of American Public Health Association (1980) Standard Methods for Examination of Water and Wastewater. American Public Health Association. Washington D.C.
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Initial conc.:
3.2 mg/L
Based on:
test mat.
Parameter:
other: BOD5
Value:
> 57.5
Sampling time:
5 d
Details on results:
The 5 day BOD was 1.15 +/- 0.17 mmol/mmol acetonitrile. Since the concentrations of acetonitrile tested are expressed as a range, it is not possible to express BOD as mg O2/l.

Mixed microbial cultures, acclimated to 45 model chemicals (including acetonitrile), were incubated for up to 20 days with 0.4-3.2 mg acetonitrile/l, at 21 ± 3 deg C. Dissolved oxygen concentrations were measured using a probe. Biodegradation was assessed based on the 5 day BOD/theoretical oxygen demand.

Interpretation of results:
readily biodegradable
Executive summary:

Babeu and Vaishnav (1987) reported 57.5% aerobic degradation (BOD/ThOD) in 5 days using mixed microbial cultures acclimated to several chemicals including acetonitrile.

Endpoint:
biodegradation in water: screening tests
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
Model cited in R.7 guidance
Justification for type of information:
QSAR prediction
Guideline:
other: US EPA EPIWIN Suite v3.20
Principles of method if other than guideline:
BIOWIN v 4.10
Remarks on result:
other: Predicts that acetonitrile is readily biodegradable.
Executive summary:

US EPA BIOWIN software (v4.10) predicts that acetonitrile is ready biodegradable.

Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Limited information available. Not widely recognized data sources.
Principles of method if other than guideline:
Not described.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Initial conc.:
490 mg/L
Based on:
test mat.
Parameter:
other: oxygen demands
Value:
17
Sampling time:
1 d

In a bench-scale fill and draw study, maintained at 20 degree C, with 1 day acclimation, 17% removal was observed, based on the difference between the oxygen demands of the influent and effluent. There was no theoretical oxidation based on the ratio of observed and calculated oxygen demand.

Interpretation of results:
other: not readily biodegradable in 1 day
Executive summary:

Plack and Ruchhoft (1947) reported 17% removal of acetonitrile in 1 day (based on oxygen demands) in a bench-scale fill and draw study with non-acclimated (< 1 day) activated sludge. There was no theoretical oxidation based on the ratio of observed and calculated oxygen demand.

Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Limited information available. Not widely recognized data source.
Principles of method if other than guideline:
The method described follows closely that of Directive 84/449/EEC.C7, and is described in one citation (Envirofate, 1994) as being the 'MITI' method.
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
other: activated sludge
Value:
30
Sampling time:
14 d

The ratio of concentrations of activated sludge: test substance was 100:30 (ppm). The test was conducted at 25 degree C and pH 7. Biodegradation was assessed from BOD/ThOD, and also from direct analysis of the test material. No data were presented but acetonitrile was described as 'well biodegradable'.

Interpretation of results:
other: described by authors as well-biodegradable
Executive summary:

Sasaki (1978) reported acetonitrile as "well-biodegradable" in an aerobic test with activated sludge. The data are not presented but the classification must be understood as more than 30% degradation (BOD/ThOD) in 14 days.

Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Limited information available
Principles of method if other than guideline:
Not described. Handbook source.
GLP compliance:
not specified
Oxygen conditions:
anaerobic
Remarks on result:
other: The half-life for aqueous anaerobic degradation has been estimated to be 4-16 weeks.

The half-life for aqueous anaerobic degradation has been estimated to be 4-16 weeks.

Executive summary:

Howard (1991) estimated the half-life for aqueous anaerobic degradation of acetonitrile to be 4-16 weeks.

Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1960
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Limited information available. Not a widely recognized data source.
Principles of method if other than guideline:
No data available.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: acclimated river water
Initial conc.:
10 g/L
Based on:
test mat.
Parameter:
other: removal
Value:
100
Sampling time:
4 d
Parameter:
other: theoretical oxidation
Value:
> 60
Sampling time:
4 d

Ludzack F.J. et al. Biochemical oxidation of some commercially important organic cyanides. In; Proc. 13th Indust. Waste Conf. Eng. Bull. Purdue Univ. Eng. Ext. Ser. 297 -312, 1958 (cited in Ludzack and Ettinger, 1960).

Interpretation of results:
readily biodegradable
Executive summary:

Ludzack (1960) reported 100% removal of acetonitrile (60% theoretical oxidation) after 4 days by acclimated river water.

Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1961
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Limited information available. Not a widely recognized data source.
Principles of method if other than guideline:
No information available.
GLP compliance:
no
Oxygen conditions:
anaerobic
Inoculum or test system:
other: digester sludge
Initial conc.:
40 mg/L
Based on:
test mat.
Parameter:
not specified
Sampling time:
1 mo
Remarks on result:
other: acetonitrile was apparently not digested

Acetonitrile neither inhibited nor aided anaerobic digestion. Acetonitrile was apparently not digested. Mean gas production was slightly reduced compared to that for the reference feed.

Acetonitrile was classified as partially biodegradable under anaerobic conditions based on a calculated integrated assessment index (IAI) of 0.7286 in a study conducted in Beijing, China (Ludzack, 1961; cited in HSDB, 2009). The IAI relates to data collected from a model consisting of an initial acetonitrile concentration of 100 mg/L in an inorganic medium and anaerobic sludge from wastewater treatment digestor tanks in Beijing (Hongwei, Y. et al. Ecotox Environ Safety 63: 299 -305, 2006).

Executive summary:

Ludzack (1961) reported that acetonitrile was apparently not digested in a 1 -month study with digestion bottles containing sludge from a sewage treatment works.

Description of key information

Acetonitrile has been determined to be readily biodegadable in guideline studies (OECD 310; 301C) conducted under GLP conditions (Brixham, 2010; CITI of Japan, 1998), and by other supporting information.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

In a guideline (OECD 310) and GLP study by Brixham Environmental Laboratories (2010) acetonitrile was determined to be readily biodegradable by non-acclimated activated sludge (> 60% degradation within 10 -days; 70% degradation after 21 days). The CITI of Japan (1998) reported a biodegration study of acetonitrile according to OECD guideline 301C (modified MITI test I) and under GLP conditions. Biodegradation results at a concentration of 100 mg/L after 28 days were 65%, 84% and 88% as measured by BOD, TOC, and GC, respectively. The BOD-curve was still on the upward trend after 28 days, therefore, additional testing was performed at a lower concentration of test substance (30 mg/L), and the degradation of the test substance measured by TOC and GC reached 100% after 14 days. Based on these results, acetonitrile is considered to be readily biodegradable.

Evidence for the biodegradation of acetonitrile by non-adapted bacterial populations has been reported by a number of other investigators. In a review, Sasaki (1978) characterized acetonitrile as “well- biodegradable”, which based on classification is understood as more than 30% of degradation (BOD/ThOD) in 14 days. In early work, Plack and Ruchhoft (1947) indicated 17% removal of acetonitrile in 1 day (based on oxygen demands) in a bench-scale fill and draw study with non-acclimated (< 1 day) activated sludge. US EPA BIOWIN software (v4.10) predicts that acetonitrile is ready biodegradable.

There is evidence of the rapid degradation of acetronitrile by adapted microbial populations. Ludzack and Ettinger (1960) reported previous works from Ludzack et al. indicating 100% removal (60% theoretical oxidation) after 4 days in acclimated river water, and more than 98% removal (more than 70% theoretical oxidation) in acclimated activated sludge in 28 days. In addition, Babeu and Vaishnav (1987) reported 57.5% of degradation (BOD/ThOD) in 5 days using mixed microbial cultures acclimated to several chemicals including acetonitrile.

The anaerobic degradation information available for acetonitrile suggest no digestion of acetonitrile in a 1-month study with digestion bottles (Ludzack, 1961) and an estimated half-life of 4-16 weeks under anaerobic conditions (Howard, 1991).

Biodegradation test results for acetonitrile

Type

Source of microorganisms

Method

Result

Detection

Ref


Aerobic

activated sludge

<1 day acclimation

Bench-scale fill and draw study

17%, 1day

O2 demand

Plack, 1947

Aerobic

river water, acclimated (19 days) population

Other

100%, 4day

O2 demand

Ludzack, 1960

Aerobic

Other

Other

98%, 28day

O2 demand

Ludzack. 1960

Aerobic

Acclimated mixed microbial cultures

Othera

>58%, 5day

BOD/ThOD

--

Babeu, 1987

Aerobic

Active sludge

Japanese methodb

30%, 14day

BOD/ThOD

--

Saski, 1978

 Aerobic  Activated sludgenon-adapted  OECD 301C  65%, 28dayBOD84%, 28dayTOC88%, 28dayGC --  CITI of Japan, 1998 
 Aerobic  Activated sludgenon-adapted  OECD 310  70%, 21dayCO2 evolution  --  Daniel, 2010

 

aAmerican Public Health Association 1980

bDirective 84/449/EEC.C7