2. Introduction
2.1. Biodegradability testing
2.1.1. Tests on ready biodegradability
Table 1: Applicability of test methods
Test Analytical method Suitability for compounds which are:
Poorly soluble volatile adsorbing
DOC Die-Away Dissolved organic carbon - - +/-
CO2 Evolution Respirometry: CO2
evolution + - +
MITI (I) Respirometry: oxygen
consumption + +/- +
Closed Bottle Respirometry: dissolved
oxygen +/- + +
Modified OECD
Screening Dissolved organic carbon - - +/-
Manometric
Respirometry Oxygen consumption + +/- +
i) DOC Die-Away Test (OECD 301A, EC C.4.A, ISO 7827)
This test is used for non-volatile substance which has solubility in water of at least 100 mg/l. The carbon content and the major components purity or relative proportions are preferred to be known. It’s similar to the Modified OECD Screening test (301 E) but allows using much higher microbial cell densities.
9 A measured volume of inoculated mineral medium with a known concentration of the test substance (10-40 mg DOC/l), is aerated in the dark or diffuse light at 22 ± 2oC. DOC analysis at frequent intervals over a 28-day period is used to follow the degradation. The biodegradation degree is calculated by expressing the concentration of removed DOC as a percentage of the concentration initially present.
The percentage degradation (Dt): [
] where:
Dt = % degradation at time t,
Co = the initial concentration of DOC in the inoculated culture medium containing the test substance (mg DOC/l),
Ct = the concentration of DOC in the inoculated culture medium containing test substance at time t (mg DOC/l),
Cbl(o) = the initial concentration of DOC in blank inoculated mineral medium (mg DOC/l), Cbl(t) = concentration of DOC blank inoculated mineral medium at time t (mg DOC/l).
When abiotic sterile control is used, the percentage abiotic degradation is calculated using:
% abiotic degradation =
where:
Cs(o) = DOC concentration in sterile control at day 0, Cs(t) = DOC concentration in sterile control at day t.
ii) CO2 Evolution (Modified Sturm Test) (OECD 301B, EC C.4.C, ISO 9439)
This test is also used for non-volatile substance. The carbon content and the major components purity or relative proportions are preferred to be known.
A known concentration of the test substance (10-20 mg DOC or TOC/l) in a measured volume of inoculated mineral medium is aerated by controlled rate-passage of carbon dioxide-free air in dark or in diffuse light. Degradation is followed over 28 days by determining the produced CO2 which is expressed as a percentage of ThCO2. The
10 absorbed CO2 in barium or sodium hydroxide, in the absorption bottle, is measured by titration of the remaining hydroxide or as inorganic carbon.
When 0.0125 M Ba(OH)2 is used as the absorbent, the amount remaining is assessed by titrating with 0.05 M HCl. (Thus 50 ml HCl would be needed to titrate 100 ml Ba(OH)2).
Since 1 mmol of CO2 is produced for every mmol of Ba(OH)2 reacted to BaCl2 and 2 mmol of HCl are needed for the titration of the remaining Ba(OH)2, and given that the molecular weight of CO2 is 44 g, the weight of CO2 produced (mg) is calculated by:
The weight of CO2 produced from the test substance is the difference between the weights of CO2 produced from the inoculum plus test substance and from the inoculum alone.
The biodegradation percentage is calculated from:
% degradation = CO CO2
2
% degradation = CO2
Where, 3.67 is the conversion factor (44/12) for carbon to CO2. The percentage of ThCO2
values calculated for each of the days, up to that time, on which it was measured.
When NaOH is used as the absorbent, calculate the amount of CO2 produced after any time interval from the concentration of inorganic carbon and the volume of absorbent used. Calculate the percentage degradation from:
% of ThCO2 =
Display the course of degradation graphically and calculate the percentage removal achieved at the plateau at the end of the test.
When an abiotic control is used, the percentage abiotic degradation is calculated by:
% abiotic degradation = CO2
CO2
iii) Modified MITI Test (I) (OECD 301C, EC C.4.F)
In this test, insoluble and volatile substances can be measured with certain precautions.
The insoluble substances should be well dispersed physically not with chemical means.
11 While for the volatile substances, the volume of "dead" gas space in the automatic respirometer should be kept to a minimum. The ThOD is calculated.
The oxygen uptake by an inoculated solution of the test substance in a mineral medium is measured automatically in a darkened, enclosed respirometer at 25 + 1°C over a period of 28 days. Evolved CO2 is absorbed by soda lime. Biodegradation is expressed as the percentage oxygen uptake of the theoretical uptake (ThOD). By using additional specific chemical analysis made at the beginning and end of incubation, the primary biodegradation percentage can be calculated, and the ultimate biodegradation can be calculated by DOC analysis.
BOD is expressed as mg oxygen/mg test substance can be calculated by dividing the oxygen uptake (mg) by the test substance (mg) after a given time, corrected for that taken up by the blank inoculum control after the same time, by the weight of the test substance used.
BOD = mg O2
= mg O2 / mg test substance The percentage biodegradation is then obtained from:
% degradation = % ThOD = O2. /
The primary biodegradation percentage can be calculated from the loss of specific (parent) chemical:
Dt = where:
Dt = % primary degradation at time t, normally 28 days;
Sa = residual amount of test chemical in inoculated medium at end of the test (mg);
Sb = residual amount of test chemical in the abiotic control at the end of the test (mg).
iv) Closed Bottle test (OECD 301D, EC C.4.E, ISO 10707)
In this test, volatile and insoluble substances can be measured with taking proper precautions such as shaking the bottles periodically during the incubation in the insoluble substances cases.
12 The test substance solution, usually at 2-5 mg/l, is inoculated with a relatively small amount of mixed population micro-organisms and kept in completely closed bottles at constant temperature in the dark. By analyzing the dissolved oxygen over a 28-d period, the degradation can be followed. The percentage of ThOD is the amount of biological oxygen uptake during the test substance biodegradation, corrected by the blank inoculum uptake run in parallel, or, less satisfactorily COD.
To calculate the ThOD, the substance formula and its purity, or major components relative proportions, should be known. The COD should be measured if the ThOD can’t be calculated, but sometimes, false high values of biodegradation percentage may be obtained if the test substance is not oxidized completely in the COD test.
BOD = mg O2/l uptake by test substance - mg O2/l uptake by blank
mg test substance/l in vessel = mg O2/mg test substance The percentage biodegradation is then obtained from:
% degradation = O2. /
% degradation = O2. /
v) Modified OECD Screening test (OECD 301E, EC C.4.B)
This test is used for non-volatile test substances with solubility in water of at least 100 mg/l. The carbon content and the purity of major components are preferred to be known.
A relatively low concentration of micro-organisms is used.
A measured volume of the medium containing a known test substance concentration of (10-40 mg DOC/l) is inoculated with 0.5 ml effluent per liter of medium. The mixture is aerated at 22 + 2°C in dark or diffused light. DOC analysis at frequent intervals over a 28 d period is used to follow the degradation. The biodegradation degree is calculated by expressing the concentration of removed DOC as a percentage of the concentration initially present. Primary biodegradation can be calculated from additional chemical analysis for a specific compound at the beginning and end of the test.
[
] where:
13 Dt = % degradation at time t,
Co = the initial concentration of DOC in the inoculated culture medium containing the test substance (mg DOC/l),
Ct = the concentration of DOC in the inoculated culture medium containing test substance at time t (mg DOC/l),
Cbl(o) = the initial concentration of DOC in blank inoculated mineral medium (mg DOC/l), Cbl(t) = concentration of DOC blank inoculated mineral medium at time t (mg DOC/l).
When abiotic sterile control is used calculate the percentage abiotic degradation using:
% abiotic degradation =
where,
Cs(o) = DOC concentration in sterile control at day 0, Cs(t) = DOC concentration in sterile control at day t.
vi) Manometric Respirometry (OECD 301F, EC C.4.D ISO 9408)
In this test, volatile and insoluble substances can be measured with taking proper precautions.
To calculate the ThOD, the substance formula and its purity, or major components relative proportions, should be known. The COD should be measured if the ThOD can’t be calculated, but sometimes, false high values of biodegradation percentage may be obtained if the test substance is not oxidized completely in the COD test.
A known test substance concentration (100 mg test substance/l giving at least 50-100 mg ThOD/l) in a measured volume of inoculated mineral medium is stirred in a closed flask for up to 28 days at a constant temperature (+1°C or closer). The oxygen consumption is evaluated either by assessing the quantity of oxygen (produced electrolytically) necessary needed to keep constant gas volume in the respirometer flask, or from volume or pressure change (or a combination of the two) in the apparatus. KOH solution or another suitable absorbent is used to absorb the evolved CO2. The amount of microbial oxygen uptake during the test substance biodegradation (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD or, less acceptably, COD.
14 Primary biodegradation can be calculated from additional chemical analysis for a specific compound at the beginning and end of the test, and ultimate biodegradation by DOC analysis.
BOD = mg O2/l uptake by test substance - mg O2/l uptake by blank
mg test substance/l in vessel = mg O2/mg test substance The percentage biodegradation is then obtained from:
% degradation = O2. /
% degradation = O2. /
vii) Biochemical Oxygen Demand (EC C.5)
This test is applied to water-soluble organic compounds; however, it may be applied for volatile and low water soluble compounds by taking special measures and precautions.
A measured amount of the substance is dissolved or dispersed in a suitable medium, inoculated with micro-organisms, well-aerated, and incubated in the dark at a constant defined temperature.
The BOD is measured by determining the difference in dissolved oxygen content at the beginning and at the end of the test. The duration of the test must be at least five days and not more than 28 days.
A blank must be measured in parallel with the test sample.