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Environmental fate & pathways

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 17 July 2014 and 22 August 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
A mixed population of sewage treatment micro-organisms was obtained on 17 July 2014 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum
The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at 21 ± 1 ºC prior to use.
Duration of test (contact time):
28 d
Details on study design:
Experimental Preparation
Test Item
At the request of the Sponsor and following the recommendations of the International Standards Organisation (ISO, 1995) the test item was dispensed onto a filter paper* using a gas tight syringe. Using this method enables relatively small amounts of test item to be added accurately to the test vessels.

An amount of test item (66 µL, equivalent to 50 mg of test item determined by preliminary weighings) was dispensed onto filter paper* which was resting on a piece of foil and added immediately to mineral medium (495 mL) and inoculum (5 mL) to give the test concentration of 100 mg/L. Due to the volatile nature of the test item each vessel was placed immediately on the respirometer after the addition of the inoculum.

A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guideline.
Inoculum control vessels were prepared containing mineral medium (495 mL) and inoculum (5 mL).

A filter paper* resting on a piece of foil was added to each inoculum control vessel in order to maintain consistency between the test and inoculum control vessels.

Abiotic Test
A nominal amount of sodium azide (10.00 g) was dissolved in mineral medium and the volume adjusted to 500 mL to give a 20 g/L stock solution.

An amount of test item (66 µL, equivalent to 50 mg of test item determined by preliminary weighings) was dispensed onto a filter paper* which was resting on a piece of foil and added immediately mineral medium (445 mL) with an aliquot (50 mL) of the 20 g/L sodium azide stock solution to give the test concentration of 100 mg test item/L and 2000 mg sodium azide/L. Due to the volatile nature of the test item each vessel was immediately placed on the respirometer after the addition of the inoculum.


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

a) Five replicate bottles containing inoculated mineral medium to act as the inoculum control plus a filter paper*.
b) Two replicate bottles containing inoculated mineral medium plus a filter paper* and the reference item, aniline, at a concentration of 100 mg/L.
c) Five replicate bottles containing inoculated mineral medium and the test item on a filter paper* at a concentration of 100 mg/L.
d) Two replicate bottles containing inoculated mineral medium, the reference item, aniline, at a concentration of 100 mg/L and the test item on a filter paper* at a concentration of 100 mg/L to act as toxicity control vessels.
e) Four replicate bottles containing inoculated mineral medium, the test item on a filter paper* at a concentration of 100 mg/L and sodium azide at a concentration of 2000 mg/L to act as abiotic test vessels.

A filter paper was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.

Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.

All vessels were inoculated with the prepared inoculum at a rate of 1% v/v.

On Day 0 the reference item and sodium azide was added (where appropriate) and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter prior to the addition of test item (where appropriate). If necessary the pH values were adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the addition of the inoculum and test item.

Two of the five inoculum control and test item vessels and two of the four abiotic test vessels were sacrificed for immediate chemical analysis. All remaining inoculum control, test item, procedure control, toxicity control and abiotic test 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 test with a magnetically coupled stirrer.

As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine solution (50% v/v) causing a net reduction in gas pressure within the sample flask. 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 computer.

The test was conducted in diffuse light at temperatures of approximately 24 ºC.

On Day 28 an assessment of the biological oxygen demand data was made and the most consistent vessels (two inoculum control, one procedure control, two test item, one toxicity control and two abiotic test vessels) chosen for calculating and reporting purposes. The remaining vessels were discarded and are not reported.

The remaining vessels which were not sampled were discarded and are not reported. Additional replicate vessels were prepared and incubated in order that in the event of a leak in the test system a replicate vessel could be discarded without jeopardizing the integrity of the test.

Evaluations
Physico-chemical Measurements
The temperature of the water bath was recorded daily.


pH Measurements
On Day 0 the pH of each of the test vessels was determined prior to the addition of the inoculum and, where appropriate, the addition of the test item using a Hach HQ40d Flexi handheld meter. The pH values were adjusted where necessary to pH 7.4 ± 0.2 using diluted hydrochloric acid. The required quantity of inoculum and test item, where appropriate, was then added to each vessel.

On Day 28 the pH of the inoculum control and procedure control vessels was determined. Due to the volatile and oily nature of the test item it was considered inappropriate to determine the pH of the vessels containing test item.


Total Viable Counts
In order to confirm that abiotic conditions were present in the abiotic test vessels at the end of the test, total viable counts were performed. An aliquot (100 µL) of sample was dispensed onto a Tryptone Soya Agar (TSA) plate and spread over the plate prior to incubation at approximately 25 ºC for approximately 2 days. After the incubation period, the number of colony forming units (cfu) were determined by direct counting of the colonies on each agar plate.


Compound Specific Analyses
On Day 0, two inoculum control, two test item and two abiotic test vessels were sacrificed for compound specific analysis. On Day 28 chemical analysis of the two inoculum control, test item and abiotic test vessels from which the oxygen consumption values were taken was

Data Evaluation
Calculation of Theoretical Oxygen Demand
The Theoretical Oxygen Demand (ThOD) for a compound CcHhClclNnPpSsOoNana was calculated by:

ThOD (NO3)(mgO2/mg) = (16(2c + ½ (h-cl) + 5/2n +5/2p + 3s + ½ na – o) / molecular weight

Percentage Biodegradation
The percentage biodegradation in terms of oxygen consumption was calculated as follows:

% degradation = ((BOD-B) / ThOD) x 100

Where:
BOD = Biological Oxygen Demand of the test item or reference item (mgO2/L)
B = Oxygen consumption in basal mineral medium to which inoculum is added (control) (mgO2/L)
ThOD = Theoretical oxygen demand to completely oxidize the reference and/or test item (mgO2/L)
Reference substance:
aniline
Key result
Parameter:
% degradation (O2 consumption)
Value:
73
Sampling time:
28 d
Details on results:
Total Viable Counts
The total viable counts from the abiotic test vessels confirmed that abiotic conditions had been present as the number of total viable counts were very low.


Biodegradation
The test item attained 73% biodegradation after 28 days based on oxygen consumption values. The test item failed to meet the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. However, in accordance with the Revised Introduction to the OECD Guidelines for Testing of Chemicals (OECD, 2006), if testing on a complex mixture is performed, and it is anticipated that a sequential biodegradation of the individual structures takes place, then the 10-Day window should not be applied to interpret the results of the test.

The toxicity control attained 62% biodegradation after 14 days and 70% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Chemical Analysis
Chemical analysis of the 100 mg/L test preparation at 0 hours showed a mean measured concentration of 102% of nominal was obtained. A decline in the mean measured test concentration was observed on Day 28 to 7% of nominal (93% loss over the test duration assuming 100% recovery on Day 0).

The losses observed by chemical analysis were higher than those observed by oxygen consumption. This was considered to be due to a combination of volatility of the test item and incorporation of the test item, or degradation products of the test item, into the microbial biomass. In such cases the micro-organisms present utilize carbon originating from the test item to increase their biomass by incorporation the carbon into new cells. This effectively removes the test item from the aqueous phase and hence reduces the apparent biodegradation of the test item as measured by oxygen consumption.

Chemical analysis of the abiotic test vessels on Day 0 showed a mean measured concentration of 108% of nominal was obtained. Analysis on Day 28 showed a mean measured concentration of 70% of nominal was obtained. Given the low total viable counts from the abiotic test vessels on Day 28 and the low biological oxygen consumption values from these vessels it was considered that this 35% loss of test item measured by chemical analysis on Day 28 occurred during sampling and analysis given the volatile nature of the test item.
Results with reference substance:
Aniline (procedure control) attained 69% biodegradation after 14 days and 75% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Validation Criteria

The mean BOD of the inoculated mineral medium (control) was 24.36 mg O2/L after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The difference between extremes of replicate BOD values at the end of the test was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.

Theoretical Oxygen Demand Values

Calculation of Theoretical Oxygen Demand (ThOD) for the test and reference items.

Information supplied by the Sponsor indicated that the ThOD value for the test item was
3.40 mg O2/mg.

Therefore for a test concentration of 100 mg/L, the ThOD will be 340 mg O2/L.

 

Reference Item (Procedure Control): Aniline C6H5NH2       mol wt = 93.13

 

ThOD (NO3) = ((16[12 + 3.5 + 2.5]) / 93.13) = 3.09 mg O2/mg

Therefore, for a test concentration of 100 mg/L, the ThOD will be 309 mg O2/L.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
In an OECD 301F manometric respirometry test conducted in compliance with GLP, Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics attained 73% degradation in 28 days. The 10-day window criterion is not applicable for complex substances where sequential degradation of the constituents takes place (OECD, 2006). The substance was therefore concluded to be readily biodegradable.
Executive summary:

Introduction

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301F, “Ready Biodegradability; Manometric Respirometry Test” referenced as method C.4-D of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (q)).

 

Methods…….

The test item at a concentration of 100 mg/L was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of approximately
24 ºC for 28 days. 

 

At the request of the Sponsor and following the recommendations of the International Standards Organisation (ISO, 1995), the test item was adsorbed onto a filter paper prior to subsequent dispersal in test media Using this method enables relatively small amounts of test item to be added accurately to the test vessels.

 

The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values on Days 0 to 28 and by compound specific analysis on Days 0 and 28. Control solutions with inoculum and the reference item, aniline, together with a toxicity control were used for validation purposes.

 

Results…….

The test item attained 73% biodegradation after 28 days and therefore can be considered to be readily biodegradable.

 

Chemical analysis of the 100 mg/L test preparation at 0 hours showed a mean measured concentration of 102% of nominal was obtained. A decline in measured test concentration was observed on Day 28 to approximately 7% of nominal (93% loss over the test duration). 

 

The losses observed by chemical analysis were higher than those observed by oxygen consumption. This was considered to be due to a combination of volatility of the test item and incorporation of the test item, or degradation products of the test item, into the microbial biomass. In such cases the micro-organisms present utilize carbon originating from the test item to increase their biomass by incorporation the carbon into new cells. This effectively removes the test item from the aqueous phase and hence reduces the apparent biodegradation of the test item as measured by oxygen consumption.

 

Chemical analysis of the abiotic test vessels on Day 0 showed a mean measured concentration of 108% of nominal was obtained. Analysis on Day 28 showed a mean measured concentration of 70% of nominal was obtained. Given the low total viable counts from the abiotic test vessels on Day 28 and the low biological oxygen consumption values from these vessels it was considered that this 35% loss of test item measured by chemical analysis on Day 28 occurred during sampling and analysis given the volatile nature of the test item.  

Description of key information

Biodegradation in water: readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Hydrocarbons, C15-C19, n-alkanes, isoalkanes <2% aromatics has been tested in an OECD 301F (manometric respirometry) test conducted in compliance with GLP (Best, 2014). The test substance attained 73% biodegradation in 28 days and was therefore considered to be readily biodegradable. The result is considered to be reliable and selected as key study.