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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 27th May 2015 and 29th June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Int. Trade & Industry's Chemical Substances Control Law (Law Clause #117, 1973), following methodology outlined in MITI Gazette, 19 July 1974,para. 7-1: Method for Testing the Biodegradability of Chemical Substances by micro-organisms
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
A mixed population of active sewage sludge micro-organisms were obtained between 2 March 2015 and 5 March 2015 from ten different sampling sites around the UK. The sampling sites were as follows:

a) Domestic sewage plants (x3)
i) Liverpool
ii) Loughborough, Leicestershire
iii) Gloucester

b) Industrial sewage plant (x1)
i) Derby

c) Freshwater samples (x3)
i) Leeds and Liverpool Canal, Liverpool
ii) River Derwent, Belper, Derbyshire
iii) River Severn, Gloucester

d) Lake Water (x1)
i) Allestree Lake, Derby

e) Sea Water Samples (x2)
i) Huttoft, Eastern Coast
ii) Hightown, North Western Coast


The sample types and volumes sampled from each site were as follows:

a) Sewage Plants : 1 liter of return sludge at each sewage disposal plant
b) Freshwater, lake and sea: 1 liter of surface water and 1 liter of surface soil on the bank/beach which is in contact with the atmosphere.

The samples obtained from the sampling sites were mixed thoroughly and the mixture allowed to settle. The floating foreign matter was removed and the supernatant filtered through a coarse filter paper. The filtrate (2 liters) was then mixed with 2 liters of supernatant removed from a previously established culture and 2 liters of synthetic sewage and transferred to a culture vessel. The pH of the culture mixture was 7.2 and constantly aerated via a narrow bore glass pipette at a temperature of approximately 25 °C.

The culture was allowed to settle 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 re-started again. Synthetic sewage was prepared by dissolving glucose, peptone and monopotassium phosphate in deionized 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.:
100.6 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
DOC removal
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
Procedure
Preparation of Seeded Dilution Water
Determination of the suspended solids level of the active sludge culture was carried out according to the method given in the Japanese Industrial Standard JISK 0102-1981. The suspended solids (ss) concentration of the culture was determined to be 4300 mg ss/L. Seeded dilution water was prepared by addition of 209.3 mL of the active sludge culture to a final volume of 600 mL of mineral medium to give a suspended solids level of 1500 mg ss/L, so that on subsequent dilution in the preparation of the test series, a suspended solids level of 30 mg ss/L was obtained.

Experimental Preparation
Test Item
The test item was dissolved directly in mineral medium.

An amount of test item (167.7 mg) was dissolved in mineral medium with the aid of high shear mixing at approximately 7500 rpm, for 30 minutes and the volume adjusted to 1 liter to give a 167.7 mg/L stock solution. To ensure sufficient volume for dosing, this stock solution was prepared in triplicate prior to the three stock solutions being pooled to give one stock solution of 167.7 mg/L. An aliquot (300 mL) of this stock solution was diluted with mineral medium (190 mL) and inoculum (10 mL) to give the final test concentration of 100.6 mg/L. The volumetric flask containing the stock solution were inverted several times to ensure homogeneity.

A further amount of test item (167.7 mg) was dissolved in deionized water with the aid of high shear mixing at approximately 7500 rpm, for 30 minutes and the volume adjusted to 1 liter to give a 167.7 mg/L stock solution. To ensure sufficient volume for dosing, this stock solution was prepared in duplicate prior to the two stock solutions being pooled to give one stock solution of 167.7 mg/L. Additional vessels were prepared as the inoculated test item vessels using deionized water (500 mL) and mineral medium (500 mL) without the addition of inoculum.

A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guideline, however in error a test concentration of 100.6 mg/L was employed.

Reference Item
A reference item, aniline (C6H5NH2), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium with the aid of ultrasonication for approximately 10 minutes. An aliquot (50 mL) of this stock solution was diluted with mineral medium (440 mL) and the inoculum (10 mL), to give the test concentration of 100 mg/L. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity.

Preparation of Test System
The following test preparations were prepared and inoculated in 500 mL bottles:

a) Four replicate bottles containing inoculated mineral medium to act as the inoculum control.
b) Three replicate bottles containing inoculated mineral medium and the reference item, aniline, at a concentration of 100 mg/L to act as the procedure control.
c) Eight replicate bottles containing inoculated mineral medium and the test item at a concentration of 100.6 mg/L.
d) Five replicate bottles containing the test item in deionized water alone at a concentration of 100.6 mg/L.
e) One bottle containing the test item in mineral medium alone at a concentration of 100.6 mg/L.

All vessels were inoculated with the prepared inoculum at a rate of 30 mg suspended solids (ss)/L.

On Day 0, one inoculated mineral medium, three test item in inoculated mineral medium, and one test item in deionized water vessel were sampled for compound specific analysis. Additionally, one inoculated mineral medium, one procedure control, one test item in inoculated mineral medium, and one test item in deionized water vessel were sacrificed for pH/DOC determination.

All remaining inoculum control, procedure control and test item vessels were placed in a CES Multi-Channel Aerobic Respirometer.

The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the study with a magnetically coupled stirrer.

As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed in the ethanolamine (50% v/v) CO2 trap causing a net reduction in gas pressure within the sample flask (see Figure 1). The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer's own battery backed memory was collected on the hard disk drive of a non-dedicated P.C.

The test was conducted in diffuse lighting at a temperature of 25 ± 0.2 °C.

On Day 28, one inoculated mineral medium, one procedure control, three test item in inoculated mineral medium and one test item in deionized water vessel were selected for pH determination compound specific analysis and DOC analysis.

The remaining test item vessels which were not sampled were stored frozen to allow for identification of degradation products if required.
Reference substance:
aniline
Test performance:
The BOD of the inoculated mineral medium (control) was 49.02 mg O2/L at the end of the test and therefore satisfied the validation criterion given in the OECD Guidelines. Although the control BOD was in excess of 30 mg O2/L at the end of the test, this was considered not to affect the integrity of the study given that the upper level of 60 mg O2/L given in the OECD Guidelines was not exceeded and that all other validation criteria were satisfied.

The pH of the inoculated test item vessels at the end of the study ranged between 7.5 and 7.7 and therefore satisfied the validation criterion given in the OECD Guidelines.

The difference between the extremes of replicate BOD values at the end of the test was 12% and therefore satisfied the validation criterion given in the OECD Guidelines.
Key result
Parameter:
% degradation (O2 consumption)
Value:
63
Sampling time:
28 d
Parameter:
% degradation (DOC removal)
Value:
60
Sampling time:
28 d
Parameter:
% degradation (test mat. analysis)
Value:
100
Sampling time:
28 d
Details on results:
The test item attained 71%, 60% and 59% biodegradation with a mean of 63% biodegradation calculated from oxygen consumption values after 28 days.
The biodegradation rates calculated from the results of the DOC analyses were 60%, 60% and 59% with a mean of 60% biodegradation after 28 days.
The results of the compound specific analyses showed that the test item vessels attained 100% biodegradation after 28 days.

Aniline attained 77% biodegradation after 28 days calculated from oxygen consumption values, and 93% biodegradation calculated from the results of the DOC analyses. The biodegradation rates calculated from the DOC analyses were higher than those calculated from oxygen consumption values. This was considered to be due to incorporation of aniline into the microbial biomass prior to biodegradation, and hence oxygen consumption occurring.

Chemical analysis of the inoculated test item preparations on Day 0 gave mean measured concentrations of 101%, 106% and 103% of nominal. The test item in deionized water vessel on Day 0 gave a result of 104% of nominal. A decline in measured test concentration in the inoculated test item vessels was observed on Day 28 to less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.083 mg/L. The test item in deionized water also showed a decline in measured test concentration on Day 28 to 32% of nominal.

 

An additional peak was shown on the Day 28 chromatography for all the test item and control vessels at approximately 1.7 minutes. In order to attempt to identify this peak, additional analysis was conducted on the stored inoculated control, inoculated test item and test item in deionized water vessels from Day 28 which indicated that the mass spectra of this peak had only two significant ions at 77 and 45 atomic mass unit (amu). A good match to the National Institute of Standards Technology (NIST) library could not be made, however matches suggested it was dimethyl-silanediol. This was considered not to be test item related but rater due to column bleed.

 

The NIST library match for the peak at 7.3 minutes in the additional analysis was disagreed with due to the presence of an ion at 224 amu implying that the compound eluting at this time point was the test item.

Based on the results of the chemical analysis there was calculated to be a 100% loss of parent test item in the inoculated test item vessels and 68% loss of test item in the deionized water vessels over the study period. Therefore, the difference in the loss of test item between the inoculated test item vessels and the test item in deionized water vessels on Day 28 was considered to be due to degradation of the test item which was observed from the oxygen consumption values. The differences in degradation values of the test item obtained by oxygen consumption (mean of 60%) and compound specific analysis (mean of 100%) were considered to be due to the test item hydrolyzing thereby reducing the amount of parent test item detected on Day 28.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item can be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301C.
Executive summary:

Introduction

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No. 301C "Ready Biodegradability; Modified MITI Test", US EPA Draft Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (n)) and the requirements of the Japanese Ministry of International Trade and Industry's Chemical Substances Control Law (Law Clause No 117, 1973). The study followed the methodology outlined in the MITI Gazette, 19 July 1974 under paragraph 7-1: Method for Testing the Biodegradability of Chemical Substances by Micro-organisms.  

Methods

The test item was prepared as an aqueous solution at a concentration of 100.6 mg/L, inoculated with micro-organisms from a laboratory culture originating from 10 different sites throughout the UK and incubated in the dark at 25±0.2 °C for 28 days. The biodegradation of the test item was assessed by the measurements of daily oxygen consumption, DOC analyses on Days 0 and 28 and compound specific analyses on Days 0 and 28. Control solutions with inoculum and the reference item, aniline, were used for validation purposes.

Results

The test item attained 71%, 60% and 59% with a mean of 63% biodegradation calculated from oxygen consumption values after 28 days. The biodegradation rates from the DOC analyses were 60%, 60% and 59% with a mean value of 60%.   

The results of the compound specific analyses showed that the test item vessels attained 100% biodegradation after 28 days.  

Chemical analysis of the inoculated test item preparations on Day 0 gave mean measured concentrations of 101%, 106% and 103% of nominal. The test item in deionized water on Day 0 gave a result of 104% of nominal. A decline in measured test concentration in the inoculated test item vessels was observed on Day 28 to less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.083 mg/L. The test item in deionized water also showed a decline in measured test concentration on Day 28 to 32% of nominal.  

An additional peak was shown on the Day 28 chromatography for all the test item and control vessels at approximately 1.7 minutes. In order to attempt to identify this peak, additional analysis was conducted on the stored inoculated control, inoculated test item and test item in deionized water vessels from Day 28 which indicated that the mass spectra of this peak had only two significant ions at 77 and 45 atomic mass unit (amu). 

A good match to the National Institute of Standards Technology (NIST) library could not be made, however matches suggested it was dimethyl-silanediol. This was considered not to be test item related but rater due to column bleed. The NIST library match for the peak at 7.3 minutes in the additional analysis was disagreed with due to the presence of an ion at 224 amu implying that the compound eluting at this time point was the test item. Therefore the difference in the results from chemical analysis compared to the results obtained from oxygen consumption and dissolved organic carbon analysis on Day 28 was considered to be due to hydrolysis of the test item over the study period.

Based on the results of the chemical analysis there was calculated to be a 100% loss of parent test item in the inoculated test item vessels and 68% loss of test item in the deionized water vessels over the study period. Therefore, the difference in the loss of test item between the inoculated test item vessels and the test item in deionized water vessels on Day 28 was considered to be due to degradation of the test item which was observed from the oxygen consumption values. 

The differences in degradation values of the test item obtained by oxygen consumption (mean of 60%) and compound specific analysis (mean of 100%) were considered to be due to the test item hydrolyzing thereby reducing the amount of parent test item detected on Day 28.   

The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301C.  

The following table summarizes the results from Days 7, 14, 21 and 28, calculated from oxygen consumption values,analyses and the compound specific test item analyses in a Modified MITI Biodegradability test with SAKURA Salicylate: 

Identification

Biodegradation (%)

Oxygen Consumption

Day 28

Day 7

Day 14

Day 21

Day 28

DOC Analyses

Compound Specific Analyses

Test Item (100.6 mg/L) Plus Inoculum

R1

55

65

69

71

60

100

R2

51

58

59

60

60

100

R3

50

57

59

59

59

100

Mean

52

60

62

63

60

100

R1– R3= Replicates 1 to 3

Description of key information

The substance is readily biodegradable under the conditions of a test according to OECD TG 301C (MITI test).

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The potential for biodegradation of the substance was determined in a screening study according to OECD TG 301C (MITI test) and in compliance with GLP criteria. In this study, 100 mg/L test substance was inoculated with active sewage sludge from predominantly domestic sewage (obtained from 10 different sampling sites around the UK) for a period of 28 days under aerobic conditions in the dark. During the incubation period the biological oxygen demand (BOD) was measured and biodegradation expressed as percentage of the theoretical uptake (ThOD). In addition, the biodegradation of the test item was assessed by measurements of dissolved organic carbon (DOC) and compound specific analyses on Days 0 and 28.

Based on oxygen consumption the test item was biodegraded 63% at day 28. Biodegradation calculated from the results of the DOC analyses reached 60% after 28 days whereas the results of the compound specific analyses showed that the test substance was no longer present in the test solution after 28 days. This difference was considered to be due to hydrolysis of the substance during the test. Based on these findings the substance is qualified as readily biodegradable.