§ 796.3100 Aerobic aquatic biodegradation.
(a) Introduction—(1) Purpose. (i) This Guideline is designed to develop data on the rate and extent of aerobic biodegradation that might occur when chemical substances are released to aquatic environments. A high biodegradability result in this test provides evidence that the test substance will be biodegradable in natural aerobic freshwater environments.
(ii) On the contrary, a low biodegradation result may have other causes than poor biodegradability of the test substance. Inhibition of the microbial inoculum by the test substance at the test concentration may be observed. In such cases, further work is needed to assess the aerobic aquatic biodegradability and to determine the concentrations at which toxic effects are evident. An estimate of the expected environmental concentration will help to put toxic effects into perspective.
(2) Definitions. (i) “Adaptation” is the process by which a substance induces the synthesis of any degradative enzymes necessary to catalyze the transformation of that substance.
(ii) “Ultimate Biodegradability” is the breakdown of an organic compound to CO2, water, the oxides or mineral salts of other elements and/or to products associated with normal metabolic processes of microorganisms.
(iii) “Ready Biodegradability” is an expression used to describe those substances which, in certain biodegradation test procedures, produce positive results that are unequivocal and which lead to the reasonable assumption that the substance will undergo rapid and ultimate biodegradation in aerobic aquatic environments.
(3) Principle of the test method. This Guideline method is based on the method described by William Gledhill (1975) under paragraph (d)(1) of this section. The method consists of a 2-week inoculum buildup period during which soil and sewage microorganisms are provided the opportunity to adapt to the test compound. This inoculum is added to a specially equipped Erlenmeyer flask containing a defined medium with test substance. A reservoir holding barium hydroxide solution is suspended in the test flask. After inoculation, the test flasks are sparged with CO2-free air, sealed, and incubated, with shaking in the dark. Periodically, samples of the test mixture containing water-soluble test substances are analyzed for dissolved organic carbon (DOC) and the Ba(OH)2 from the reservoirs is titrated to measure the amount of CO2 evolved. Differences in the extent of DOC disappearance and CO2 evolution between control flasks containing no test substance, and flasks containing test substance are used to estimate the degree of ultimate biodegradation.
(4) Prerequisites. The total organic carbon (TOC) content of the test substance shall be calculated or, if this is not possible, analyzed, to enable the percent of theoretical yield of carbon dioxide and percent of DOC loss to be calculated.
(5) Guideline information. (i) Information on the relative proportions of the major components of the test substance will be useful in interpreting the results obtained, particularly in those cases where the result lies close to a “pass level.”
(ii) Information on the toxicity of the chemical may be useful in the interpretation of low results and in the selection of appropriate test concentrations.
(6) Reference substances. Where investigating a chemical substance, reference compounds may be useful and an inventory of suitable reference compounds needs to be identified. In order to check the activity of the inoculum the use of a reference compound is desirable. Aniline, sodium citrate, dextrose, phthalic acid and trimellitic acid will exhibit ultimate biodegradation under the conditions of this Test Guideline method. These reference substances must yield 60 percent of theoretical maximum CO2 and show a removal of 70 percent DOC within 28 days. Otherwise the test is regarded as invalid and shall be repeated using an inoculum from a different source.