ASTM D6139 Aerobic Aquatic Biodegradation of Lubricants or Their Components
ASTM D6139 Standard Test Method for Determining the Aerobic Aquatic Biodegradation of Lubricants or Their Components Using the Gledhill Shake Flask
8. Inoculum Test Organisms
8.1 Sources of the Inoculum - The following provides several options for where and how to obtain an appropriate inoculum:
8.1.1 Inoculum from Activated Sludge - Activated sludge freshly sampled (that is, less than 24 hs old) from a well-operated predominantly domestic sewage treatment plant (that is, one with no recent upsets and operating within its design parameters) may be used. This sewage treatment plant should receive no more than 25 % of its influent from industrial source(s).
8.1.1.1 Using CO2-free air, aerate sludge in the laboratory for 4 h. Five hundred millilitres of the mixed liquor is sampled and homogenized for 2 min at medium speed in a blender10 or equivalent high -speed mixer. Allow to settle for 30 min.
8.1.1.2 If the supernatant still contains high levels of suspended solids at the end of 30 min, allow to settle for another 30 to 40 min.
8.1.1.3 Decant sufficient volume of the supernatant to provide either a 1 % (by volume) inoculum or 30 mg/L of suspended solids for each test Erlenmeyer flask. Avoid carry-over of sludge solids which might interfere with the measurement of CO2 production.
8.1.1.4 It is optional to pre-condition the inoculum. Preconditioning consists of aerating the activated sludge in mineral medium solution for up to seven days. Sometimes pre conditioning improves the precision of the test method by reducing blank values.
NOTE 2 - Exercise care in pre-conditioning because of the sensitivity of inocula to prolonged aeration and starvation conditions. Pre-conditioning should be applied mainly in situations where it is known that the inoculum source consistently shows a high internal respiration rate.
8.1.2 Inoculum From Soil:
8.1.2.1 Suspend 100 g of soil in 1000 mL of water.
8.1.2.2 Allow the suspension to settle for 30 min.
8.1.2.3 Filter the supernatant through a coarse filter paper or glass wool plug, and discard the first 200 mL. The filtrate is aerated immediately and continuously until used.
8.1.3 Inoculum from Surface Water:
8.1.3.1 Filter surface water through a coarse filter paper or glass wool plug, discarding the first 200 mL.
8.1.3.2 Aerate the filtrate until used.
8.1.4 Composite Inoculum - The three inoculum sources may be combined in any proportion and mixed well.
8.2 Enumeration of Microorganisms:
8.2.1 APHA Test Method 9215, or equivalent, shall be used to enumerate the microorganisms in the inoculum. The inoculum shall contain 10(6) to 10(8) colony-forming units (CFU) per millilitre. It is optional to measure the total bacterial count of the inoculum using the dip slide technique with a commercially available diagnostic kit.
8.2.2 Alternatively, APHA Test Method 2540B shall be used to determine the sludge dry-weight per unit volume. Calculate the volume of mixed liquor necessary to achieve a final sludge dry-weight concentration in the test medium of 30 mg/L (suspended solids).
8.3 Pre-adaptation of the inoculum is allowed and can be accomplished as follows:
8.3.1 Supplement inoculum with 25 mg/L vitamin-free casamino acids and 25 mg/L of yeast extract.
8.3.2 The test medium solution shall be prepared as follows: each litre of the test medium is prepared by measuring out the following volumes of the six stock solutions listed below, combining them, mixing, and diluting to 1 L with water. Multiples of this test medium solution can be prepared at one time (scale volumes proportionally).
8.3.2.1 Ammonium Sulfate Solution, 1 mL,
8.3.2.2 Calcium Chloride Solution, 1 mL,
8.3.2.3 Ferric Chloride Solution, 4 mL,
8.3.2.4 Magnesium Sulfate Solution, 1 mL,
8.3.2.5 Phosphate Buffer Solution, 10 mL,
8.3.2.6 Trace Elements Solution, 1 mL.
8.3.3 Add 100 mL of supplemented inoculum and 900 mL test medium to a 2-L Erlenmeyer flask
8.3.4 Add test materials incrementally during the acclimation period at concentrations equivalent to 4, 8, and 8 mg carbon/L on days 0, 7, and 11, respectively.
8.3.5 The inoculum flask(s) will be maintained at a temperature of 22 +/-2°C in the dark and will be agitated on a shaker table or with a magnetic stirrer at a moderate speed (for example, 150 to 200 rpm).
8.3.6 On day 14, homogenize the culture in a blender for at least 1 min and refilter the medium through glass wool prior to use as the inoculum for the test. If pre-adaptation is conducted for a series of functionally or structurally related test materials (may include reference material), media from the separately prepared inoculum may be combined before final filtration. The enumeration of microorganisms in the final pre-adapted inoculum shall be carried out using the method described in 8.2.
9. Test Material and Reference Material
9.1 This section addresses specific requirements pertaining to the carbon concentrations of the test material and reference material as well as the appropriate choice of reference materials.
9.2 The carbon content of a test material shall be measured by Test Methods D5291 or an equivalent procedure.
9.3 The test material shall be added to provide 10 to 20 mg carbon per litre (mg C/L) in the test medium. This will ensure that sufficient carbon is present to yield CO2 which can be adequately measured by the trapping procedure described in this test method should the test material be biodegradable.
9.4 Reference - A material known to be biodegradable shall be tested simultaneously with the test material.
9.4.1 For water-insoluble test materials, the suggested reference is a low erucic acid rapeseed oil, also called LEAR, such as canola oil. The fatty acid profile of low erucic acid rapeseed oil shall contain a maximum of 2 % erucic acid by weight.
9.4.2 Sodium benzoate or aniline is suggested as a reference material if the test material is water-soluble.
9.4.3 The reference will be added in the same manner as the test material to provide a carbon concentration of 10 to 20 mg C/L in the flask.
9.4.4 The results from flasks containing the reference verify the viability of the inoculum.
9.5 The test method will be performed in a minimum of two replicates on all test and reference materials although triplicates are recommended.
9.6 Exercise care to obtain representative samples from test and reference materials.
10. Hazards
10.1 This test method includes the use of hazardous chemicals. Avoid contact with chemicals and follow the manufacturers' instructions and Material Safety Data Sheets (MSDS).
10.2 This test method includes the use of potentially harmful microorganisms. As such, execution of this test method must be carried out under the guidance of qualified personnel who understand the safety and health aspects of working with microorganisms. Minimally, review the test method with an industrial hygienist before initiating any activity. Avoid contact with the microorganisms by using gloves and other appropriate protective equipment and sterile procedures. Use good personal hygiene.
10.3 Sterilize materials and supplies contaminated with biologically active cultures before discarding or reusing them.
10.4 Chemicals should be disposed of as described in Guide D4447 or as prescribed by current regulations.
11. Preparation of Apparatus
11.1 Cleaning - The following is a suggested method for cleaning glassware and equipment to avoid organic contamination which may affect test results. The glassware and equipment used to prepare and store stock solutions and test solutions should be cleaned before use. Items should be washed with detergent and rinsed with water, a water-miscible organic solvent, water, acid (such as 10 % concentrated hydrochloric acid), and at least twice more with distilled, deionized water. Some organic solvents may leave a film that is insoluble in water. The presence of this film is not acceptable and may lead to false positive results. At the end of every test, all items that are to be used again should be immediately (a) emptied, (b) rinsed with water, and (c) cleaned as stated previously.
