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
6. Apparatus
6.1 Carbon Dioxide Scrubbing Apparatus (see Fig. 1):
6.1.1 The following are required to produce a stream of CO2-free air for aeration and for sparging aqueous solutions and mixtures (for example, test medium, sewage inoculum):
6.1.1.1 Erlenmeyer flask, One 1-L with side arm containing 500 mL of 10 M sodium hydroxide (NaOH), and fitted with a rubber stopper and an inlet tube that extends below the level of the NaOH solution or an equivalent apparatus or system.

6.1.1.2 Erlenmeyer flask, One 1-L with side arm containing 500 mL of distilled water and fitted with a stopper and inlet tube, or an equivalent apparatus or system.

6.1.1.3 It is optional to add an empty 1-L Erlenmeyer flask in series with the flasks to prevent liquid carryover.

6.1.1.4 It is optional to add a 1-L Erlenmeyer flask containing 500 mL of 0.1M barium hydroxide [Ba(OH)2] solution to monitor for possible breakthrough CO2.

6.1.2 Connect the flasks in series as shown in Fig. 1, using vinyl or other suitable non -gas-permeable tubing, to a pressurized air system and purge air through the scrubbing solution.

6.1.3 The CO2 scrubbing apparatus upstream of the Erlenmeyer flask containing the Ba(OH)2 may be substituted with an alternative system which effectively and consistently produces CO2-free air (that is, containing <1 ppm CO2).

6.2 Incubation/Biodegradation Apparatus - Gledhill-type Shake Flask Units8 (see Fig. 2) - Each test material, reference, or blank control requires the following:
6.2.1 Erlenmeyer Flasks, 2-L - 2-L Erlenmeyer flasks are used to hold the 1 L of total final aqueous volume but larger volume Erlenmeyer flasks (as large as 3 to 4-L) may be used if 2 to 3-L final aqueous volumes are required. The amounts described here are for 1-L final aqueous volumes carried out in 2-L Erlenmeyer flasks; scale procedure accordingly if larger final aqueous volumes and larger Erlenmeyer flasks are necessary.

6.2.2 Stoppers - Each stopper is fitted with a conical alkaline trap, an outlet and an inlet vent tube (see Fig. 2). Ensure that the stopper fits tightly in the Erlenmeyer flask to prevent any leaks.

6.2.3 Conical Alkaline Trap Tube or Unit - Glass, 40- mL conical tube (borosilicate glass, No. 8120 centrifuge tube or equivalent) welded to a glass support rod, or an equivalent apparatus, will be used to hold the Ba(OH)2 solution for trapping the evolved CO2 from aerobic biodegradation. The opening in the alkaline trap tube is large enough to permit CO2 diffusion into the barium hydroxide solution. The support rod of the conical trap shall fit tightly in the stopper.

6.2.4 Inlet and Outlet Vent Tubes - The inlet vent tube attached to the stopper extends down into the flask so that it will be immersed below the surface of the aqueous medium and will be used for sparging. The outlet vent tube will be situated significantly above the level of the aqueous medium and will be used for venting. The two vent tubes shall fit tightly in the stopper.

6.2.5 Flexible tubing which is non-permeable to CO2 will be used to connect the tops of inlet and outlet vent tubes to form a closed system.

6.2.6 Agitators - Incubator-shaker table unit or equivalent, or stirrers may be used to agitate the aqueous mixture in the Erlenmeyer flasks.

6.3 Analytical Balance, to weigh out test material or reference material to be added to the test flask (capable of weighing to appropriate precision and accuracy, for example, +/-0.0001 g)

6.4 Titration Apparatus for Measuring the Production of CO2:
6.4.1 Appropriate graduated burette filled with standard HCl solution.

6.4.2 Alternatively, an automatic titration apparatus in which the burette dispenser is filled with standard HCl solution. Automatic titrations are carried out to a potentiometric end point of pH 8.3 (that is, phenolphthalein end point equivalent)

6.5 Glass Wool, for filtering the inoculum.

7. Reagents and Materials
7.1 Purity of Reagents - Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all reagents conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society where such specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity to permit its use without lessening the accuracy of the determination.

7.2 Purity of Water - Unless otherwise indicated, references to water shall be understood to mean reagent water as defined by Type II of Specification D1193.

7.3 Prepare the following stock solutions:
7.3.1 Ammonium Sulfate Solution (40 g/L) - Dissolve 40.0 g ammonium sulfate [(NH4)2SO4] in water and dilute to 1 L.

7.3.2 Calcium Chloride Solution (27.5 g/L) - Dissolve 27.5 g anhydrous calcium chloride (CaCl2) in water and dilute to 1 L.

7.3.3 Ferric Chloride Solution (0.25 g/L) - Dissolve 0.25 g ferric chloride hexahydrate (FeCl3•6 H2O) in water and dilute to 1 L.

7.3.4 Magnesium Sulfate Solution (22.5 g/L) - Dissolve 22.5 g magnesium sulfate heptahydrate (MgSO4•7 H2O) in water and dilute to 1 L.

7.3.5 Phosphate Buffer - Dissolve 8.5 g potassium dihydrogen phosphate (KH2PO4), 21.7 g potassium monohydrogen phosphate (K2HPO4), 50.3 g sodium monohydrogen phosphate heptahydrate (Na2HPO4•7 H2O) [or alternatively, 33.4 g of sodium monohydrogen phosphate dihydrate (Na2HPO4•2H2O), the dihydrate equivalent form] and 1.7 g ammonium chloride (NH4Cl) in water and dilute to 1 L.

7.3.6 Trace Elements Solution - Dissolve 0.035 g manganous chloride tetrahydrate (MnCl2•4 H2O), 0.057 g boric acid (H3BO3), 0.043 g zinc sulfate heptahydrate (ZnSO4•7 H2O) and 0.037 g ammonium molybdate tetrahydrate [(NH4)6Mo7O24•4 H2O] in water and dilute to 1 L.

7.4 Barium Hydroxide Solution, 0.1 M, is prepared by dissolving 32.0 g barium hydroxide octahydrate [Ba(OH)2•8H2O] in distilled water and diluting to 1 L. Filter free of solid material, confirm molarity by titration with standard acid, and store under nitrogen sealed as a clear solution to prevent absorption of CO2 from the air. It is recommended that 2 L be prepared at a time when running a series of tests.

7.5 Vitamin-free Casamino Acids.

7.6 Yeast Extract.

7.7 Phenolphthalein.

7.8 Standardized Hydrochloric Acid (0.190 - 0.210 M).