TESTMETHODS

Biodegradation is "the destruction of chemical compounds by the biological action of living organisms" (3). It can be divoded into primary and ultimate biodegradation. Primary degradation is the maximum extent of degradation needed to change the identity of the compound. Ultimate biodegradation or mineralization involves the complete conversion of a compound to C02, H20 and other inorganic compounds. OECD - 301 B (modified sturm test) is based on the ultimate biodegradation of surfactant.

Biodegradability can be affected in the presence of other compunds. It was found that there is retardation of biodegradation of LAS by sublethal concentrations of mercuric chloride (4). Microbial organisms adapted in actived sludge can degrade phenol, and can utilize phenol as a sole source of carbon for their metabolism. However, degradation is affected by the presence of phenol above 360 mg/1 (5). This is substrate inhibition.

Methods Available

A variety of tests are used to assess the biodegradability surfactants. They include screening tests to indicate ready biodegradability, tests of inherent biodegradation and simulation tests to assess removal by waste treatment processes.

Tests can be conducted using radiolabelled surfactants also. There are other tests, like BOD mesurement, CO2 evolution, shake culture tests, and enrichment cultures.

OECD Method

OECD divides biodegradation tests into three tyes: screening, inherent and simulation (6).

Screening tests generally employ a simple aqueous medium containing mineral salts and a small number of unacclimatised microogranisms to which the test compound is added. The purpose of screeing tests is to provide unequivocal evidence that the test compound will biodegrade in the environment.


Inherent tests employ a higher concentration of micro-organisms and may last for several months. Compounds passing the test for inherent biodegradability are also tested using simulation methods, such as continuous activated sludge, tricking filter systems, etc. to assess their bahaviour during wastewater treatment.

Degree of biodegradation for a specific compound varies with test method and that the chemical, physical and biological characteristics of the compound must be considered when choosing the test (7) (8).

BOD screening test - can be employed to estimate the extent of biodegradation in an isolated system of known composition. Interpretation of the results is rather difficult, due to the complexity of the metabolic processes involved, the variety of metabolic by-products formed, and the multiple pathways of possible oxygen utilisation by the microorganisms.

C02 evolution test - is used to assess ultimate biodegradation. This method is not quantitative because the C02 evolved usually falls short of 100% of the theoretical value. Some of the carbon is converted to new cells, i.e. used for biomass production. Some carbon is converted to C02 and third possible short term fate of substrate carbon is the formation of intermediate metabolites, which may accumulate in the test medium.

Therefore, OECD (1981) concluded that results of atleast 60% of the theoretical oxygen demand or of theoretical C02 production indicates that the chemical should be classified as readily biodegradable and is hence readily removed in the environment.

OECD - 301 B (modified sturm test) (4)

Principle

Aerobic micro-organisms, utilising an organic substrate as carbon and energy source, convert the molecules into new cells, C02 and wastes. By measuring the amount of C02 provided and comparing the quantity with the theoretical yield, calculated from the substrate carbon content, a measure of ultimate biodegradability can be made. The test duration is limited to 28 days and it has been found that materials degrading extensively in this period are readily degraded. Hence, a result regarded as positive by this test procedure indicates that the substance tested is readily and ultimately biodegradable.

Test procedure and performance

Assembly is prepared as shown in Fig. (2). First four slacks are to get the C02 - free air is passed through four 4-four litre flasks. Flasks A and B are for test material. Flask C is standard sodium acetate (220 ppm) for checking the performance of test as "control". Sodium acetate should give 100% biodegradation at the end of 28 days. Flask D is inoculum blank - Inoculum used is the activated sludge (1%). The inoculum should normally contain 106 to 20 x 106 colony forming units per ml.

Bubbles of C02-free air are passed through the solution at the rate of 50-100 ml / min. per flask, i.e. approximately 1-2 bubbles per second.

Carbon dioxide produced in the flask is then absorbed in 0.025 N Barium hydroxide, and precipitated as barium carbonate. The amount of C02 produced is determined by titrating the unreacted barium hydroxide with standard 0.05 NHC1. Periodically, every 2-3 days, the two absorbers (4-1 flasks) are removed and filled with 100 ml fresh barium hydroxide solution is placed at the far end of series.

The test is run at 25 + 2°C temperature.