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
12. Procedure
12.1 For each blank, test material and reference being tested, prepare a 1 % inoculum by the following dilution:
12.1.1 Add 900 mL of water to each of the 2-L Erlenmeyer flasks.

12.1.2 To each 2-L Erlenmeyer flask, add 10 mL of the phosphate buffer stock solution; 1 mL each of the magnesium sulfate, calcium chloride, ammonium sulfate, and trace elements stock solutions; 4 mL of the ferric chloride stock solution; and 10 mL of the sludge inoculum or a sufficient volume of the inoculum to give 30 mg/L suspended solids. The Erlenmeyer flasks now contain at least 928 mL of solution.

12.1.3 In the case in which a pre-adapted combined inoculum is used and in which the test materials are structurally related, use the same composite inoculum (see 8.3.6) for the blank, reference, and test flasks.

12.2 Aerate the aqueous mixture in the Erlenmeyer flasks with CO2-free air for at least 1 h to purge the system of CO2. Carbon dioxide-free air can be bubbled into the aqueous medium with the aid of a long glass tube (or equivalent). A sodium hydroxide scrubbing apparatus used to provide CO2-free air is shown in Fig. 1.

12.3 Measure the pH in each Erlenmeyer flask by Test Methods D1293 or an equivalent method. Using dilute HCl or NaOH, adjust the pH to 7 +/- 0.5 before adding the test material or reference material.

12.4 The carbon concentration of the test material or reference material in the test medium shall be 10 to 20 mg C/L. Calculate the weight of the test or reference material needed to produce 10 to 20 mg of carbon per litre based upon the carbon content of the material as determined previously (see 9.2).

12.5 Addition of the Test Material or Reference Material:
12.5.1 Add the test material or reference material gravimetrically to the replicate Erlenmeyer flasks. If in order to accomplish this, the material is weighed into or onto a small object, then both the material and the object shall be added to the flask.

NOTE 3 - An example of a small object might be a glass fiber filter. The test or reference material is added to the respective shake flasks as a measured weight adsorbed onto the surface of the filter. This enables an accurate weight to be dosed into each flask and increases the surface area of the hydrophobic test or reference material. A blank glass fiber filter should also be added to each blank shake flask.

12.5.2 Sonication of the test material or reference material in 5 mL of water while still in/on a small object is allowed as a means of obtaining a better dispersion of insoluble materials in the test medium. If sonication is performed, the object shall also be added to the flask. In addition, if sonication is performed on the test material, the reference material shall also be sonicated in an identical manner prior to its addition to the test medium.

12.6 Along with the flasks containing test materials or reference materials, additional replicate flasks shall contain the test medium and the inoculum with no additional carbon source added. These flasks shall be blanks.

12.7 Add sufficient volume of water to achieve a final volume of 1000 mL in each flask.

12.8 Add 10 mL of the 0.1M Ba(OH)2 solution to the conical alkaline trap unit.

12.9 Place the charged Ba(OH)2 trap and stopper securely on top of the Erlenmeyer flask. Connect the tops of the inlet and outlet vent tubes with a single piece of non-permeable tubing and ensure that each assembled Gledhill shake flask unit is a closed system. The stopper should fit tightly on the Erlenmeyer flask and the tubing connecting the inlet and outlet vent tubes should be secured tightly in place to ensure that the test system is closed and to prevent any leaks (for example, the loss of evolved CO2 or the entry of ambient CO2 into the flask).

12.10 Start the test by agitating the Erlenmeyer flasks in a shaker table or with a magnetic stirrer set at a moderate speed (for example, 150 to 200 rpm). Ensure that each flask is agitated at about the same speed.

12.11 Run the test at 22 +/-2°C, and record the test temperature throughout the test period.

12.12 At the time when the temperature is taken, it is also a convenient time to inspect the shake flasks to ensure that the stopper and connecting vent tubing are tightly secured in place.

12.13 Maintain the Erlenmeyer flasks in darkness to prevent photodegradation of the test material and growth of photosynthetic bacteria and algae.

12.14 Carbon Dioxide Evolution and Analysis:
12.14.1 The CO2 produced in each Erlenmeyer flask reacts with Ba(OH)2 in the conical trap and is precipitated as barium carbonate (BaCO3).

12.14.2 When significant BaCO3 precipitate is evident, the Ba(OH)2 solution is removed from the conical trap for analysis. The trap is rinsed with 10 mL of CO2-free distilled water and this water rinse combined with the collected Ba(OH)2 solution.
12.14.2.1 Analysis - The amount of CO2 produced is determined by titrating the Ba(OH)2 with standard hydrochloric acid. Titrate the combined Ba(OH)2-water rinse mixture immediately after removing and collecting it from each Erlenmeyer flask. Exercise care to minimize exposure to air to avoid absorbing ambient CO2. Titrate with standard hydrochloric acid to a colorless phenolphthalein end point using a graduated burette. If an automatic titrator is used, titrate to a potentiometric end point of pH 8.3 (that is, phenophthalein equivalent end point).

12.14.3 The trap is refilled with 10 mL of fresh 0.1M Ba(OH)2 solution. The inlet and outlet tubes are opened, and the aqueous solution in the flask is then sparged with CO2-free air through the inlet tube. Flasks should be sparged for approximately the same length of time (for example, a few minutes) for the blank and for the reference and test materials. The outlet and inlet tubes are resealed tightly and the flasks are placed back on the shaker or stirrer, and agitation resumed.

12.14.4 Sampling of the Ba(OH)2 traps and titration may be required every one to three days for the first ten days and then every five to seven days until a plateau of CO2 evolution is reached. A plateau is reached after evidence of biodegradation has occurred and the production of CO2 is either no longer detectable or is equal to the CO2 produced by the blanks for two consecutive sampling times.

12.14.5 Because the results of the titration are added to produce the final test result, the uncertainty in the final result increases as the number of titrations increases. Consideration of this point is advisable when establishing the frequency of titrations. Titrations of trap samples shall be carried out at the same frequency for the blank, and reference and test flasks, and the method of analysis shall follow the procedure described in 12.14.2.1.

12.14.6 The test shall continue for at least 28 days or, if longer, until the CO2 evolution has reached a plateau. If on day 28, a plateau has not been achieved, that is, CO2 production is still being detected in the conical trap (signifiant BaCO3 precipitation), perform a titration on the Ba(OH)2 solution in the trap and continue to monitor CO2 evolution until a plateau has been reached.

12.14.7 Once the CO2 evolution has reached a plateau, measure the final pH of the Erlenmeyer flask contents before adding concentrated acid to terminate the biodegradation study. A minimal volume of the aqueous solution can be removed for pH determination. Then add 1 mL of concentrated hydrochloric acid or concentrated sulfuric acid to each of the Erlenmeyer flasks to decompose inorganic carbonate and to release the trapped CO2. Restopper the Erlenmeyer flasks and continue agitation overnight to collect the released CO2 in the barium hydroxide traps. Remove the trap sample on the next day and perform the final titration on the barium hydroxide solution as described in 12.14.2 and 12.14.2.1.