Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 November 1993 to 16 December 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP 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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

other: mixed population of activated sludge microorganisms from domestic (x3), industrial (x1), river( x1), lake (x1) and seawater (x2)

Details on inoculum:

Ten different sampling sites as follows:
a) City domestic sewage plants (x3)
i ) Li verpool
ii) Derby
iii) Bristol
b) Industrial sewage plant (x1)
i) Belper
c) River samples (x3)
i) River Mersey
ii) River Trent
iii) River Severn
d) Lake Water (x1)
i) Allestree Lake, Derby
e) Sea Water Samples (x2)
i) Skegness
ii) Southport

The sludge samples obtained from the sampling sites were mixed thoroughly and the mixture allowed to stand. The floating foreign matter was removed and the supernatant filtered through Whatman No. 2 filter paper. The filtrate was then mixed with approximately 2 litres of supernatant from an existing culture and transferred to a 10 litre culture vessel. The pH of the culture mixture was adjusted to pH 7.0 ± 1.0 with sodium hydroxide or phosphoric acid and constantly aerated via a narrow bore glass pipette at a temperature of 24 ± 1°C. The culture was settled daily for approximately 30 minutes and approximately 1/3 of the volume of the supernatant removed. An equal volume of 0.1% synthetic sewage was added and the aeration started again. Synthetic sewage was prepared by dissolving glucose, peptone and monopotassium phosphate in deionised water at a concentration of 0.1% (w/v).
The pH of the synthetic sewage and culture was adjusted daily to within the range pH 7.0 ± 1.0 with sodium hydroxide or phosphoric acid. The sludge was found to form a clear supernatant on settling and to have an active microflora including a variety of protozoa, including ciliates, flagellates and a large population of motile bacteria. The sludge formed cloudy flocs when on aeration.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

Culture Medium

Deionised water purified by reverse osmosis (Elgastat Spectrum R01) was aerated and allowed to stand for about 20 hours at approximately 21°C to give a dissolved oxygen concentration of approximately 9 mg O2/l.

To 1 litre of water was added 3 ml of solutions 1 - 4.

Solution 1: K2HPO4 21.75 g/l
KH2PO4 8.50 g/l
Na2HPO4·12H20 44.60 g/l
NH4Cl 1.70 g/l

Solution 2: MgSO4·7H20 22.50 g/l

Solution 3: CaCl2 27.50 g/l

Solution 4: FeCl3·6H20 0.25 g/l

Method

Vessels: 500 ml amber glass bottles
Apparatus: Camlab BOD apparatus. A closed system oxygen consumption measuring apparatus using a direct reading manometer scale. Carbon dioxide produced was adsorbed using potassium hydroxide (45% w/v). Agitation of the test solution was brought about by rotary stirring using a magnetic stirrer.
Preparation of seeded dilution water: Determination of the suspended solids level of the activated sludge culture was carried out according to the method given in the Japanese Industrial Standard JISK 0102-1981. The suspended solids concentration of the culture was determined to be 4000 mg ss/l. Seeded dilution water was prepared by addition of 83.45 ml of the activated sludge culture to 10 litres of culture medium to give a suspended solids level of approximately 33.38 mg ss/l so that on dilution with an aliquot of standard material stock solution, a suspended solids loading of 30 mg ss/l was obtained. For the preparation of the control and test material with inoculum vessels, the seeded dilution water was further diluted by the addition of deionised water to give a suspended solids value of 30 mg ss/l.
Test Concentrations:
a) Culture medium with inoculum.
b) Culture medium with inoculum and "standard material" aniline: concentration 100 mg/l.
c) Culture medium with inoculum and test material: concentration 100 mg/l.
d) Deionised water with test material only: concentration 100 mg/l.
Triplicate bottles of each series were prepared.
Preparation of Test Concentrations:
a) Three replicates consisting of 428 ml of the diluted seeded dilution water to give a suspended solids value of 30 mg ss/l.
b) 900 ml of seeded dilution water plus 100 ml of a 1000 mg/l stock solution of aniline to give a test concentration of 100 mg/l and a suspended solids value of 30 mg ss/l and dispensed to 3 replicates of 157 ml.
c) Three replicates consisting of 15.7 mg of test material washed into each exposure vessel using 157 ml of the diluted seeded dilution water to give a test concentration of 100 mg ss/l and a suspended solids value of 30 mg/l.
d) Three replicates consisting of 15.7 mg of test material washed into each exposure vessel using 157 ml of deionised water to give a test concentration of 100 mg/l.
Test volumes: The test volumes used for the control, test and standard material series were assigned following the calculation of the Theoretical Oxygen Demand (ThOD).
Observations: The appearance of the test material dispersions was recorded on days 0 and 28. Direct readings were taken daily from the manometer scales of the Camlab BOD Apparatus. These readings were multiplied by the corresponding conversion factor for the test volume employed in the study to give the BOD value. Temperatures were recorded daily and pH values of the control, test and standard material solutions were recorded on days 0 and 28.

Exposure Conditions

Duration: 28 days
Temperature: 25 ± 1°C
Light regime: The cultures were shielded from light
Agitation: By magnetic stirrers

Reference substance:

aniline

Parameter:

% degradation (O2 consumption)

Value:

4

Sampling time:

28 d

Details on results:

Aniline attained a 96% degradation from calculation of the results of the DOC analysis and 100% from the COD analyses. Results of the compound sepecific analysis showed that Nigrosine Base EX attained -2% degredation after 28 days.

Parameter:

COD

Value:

100

Results with reference substance:

Aniline attained 51% degradation, calculated from the oxygen consumption values after 7 days and 66% degradation after 14 days, confirming the suitability of the inoculum and culture conditions.
Aniline was degraded to 96% from calculation of the results of the DOC analysis and 100% from the COD analysis after 28 days of incubation indicating that the inoculum was viable and exerting normal biodegradative activity. In the presence of the test material, degradation of Nigrosine Base EX ranged from1% to 4% which indicates that the test material was not inhibitory to the bacterial inoculum. The observed variation in the degradation of aniline in the presence of Nigrosine is not considered to be significant.

Calculation of Theoretical Oxygen Demand (ThOD) for the test and standard material.

By dividing the % composition of each element by its atomic weight an empirical formula was obtained.

Calculated ThOD (N03) = 2.91 mg O2/mg

Therefore from a test concentration of 100 mg/l, the ThOD will be 291 mg O2/L.

Standard Material: Aniline, molecular weight of 93.13, for a test concentration of 100 mg/l, the ThOD will be 309 mg O2/L.

Infrared Spectroscopy

The infrared spectrum of the test material was shown to be similar to that supplied by the Sponsor, confirming the identity.

The infrared spectrum of the test material at initiation and termination of the study showed the test material to be stable during storage.

Validation of Method of Analysis

The Method of Analysis has been validated to be suitable for the purposes of this study.

Validation Criteria

Aniline attained 51% degradation, calculated from the oxygen consumption values, after 7 days and 66% degradation after 14 days, confirming the suitability of the inoculum and culture conditions.

Percentage Degradation Values

Nigrosine Base EX attained 4% degradation calculated from the oxygen consumption values after 28 days.

Aniline attained 96% degradation from calculation of the results of the DOC analyses and 100% from the COD analyses.

Results of the compound specific analyses showed that Nigrosine Base EX attained -2% degradation after 28 days.

Validity criteria fulfilled:

yes

Interpretation of results:

other: not readily biodegradable

Conclusions:

The test material cannot be considered to be readily biodegradable under the strict terms and conditions of the OECD Guideline No. 301C.

Executive summary:

In an assessment of ready biodegradability study, modified MITI test (370/135), the test material was determined to not be readily biodegradable according to OECD Guideline 301C, having attained 4% degradation after 28 days.

Description of key information

The test material cannot be considered to be readily biodegradable under the strict terms and conditions of the OECD Guideline No. 301C.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

In an assessment of ready biodegradability study, modified MITI test, conducted to GLP and with a Klimisch score of 1, the test material was determined to not be readily biodegradable, having attained 4 % degradation after 28 days. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).