ASTM D6731 Standard Test Method for Determining the Aerobic, Aquatic Biodegradability of Lubricants or Lubricant Components in a Closed Respirometer
6. Apparatus
6.1 Closed Respirometer:
6.1.1 The principle of a closed respirometer is given in Fig. 2. When testing volatile compounds, the apparatus used shall be appropriate or adapted to this particular purpose in accordance with the manufacturer's instructions. Exercise care that the closed respirometer apparatus is well sealed to prevent any loss (for example, leakage) of volatile compounds from the system or of oxygen into the system.

6.1.2 The test mixture is stirred by a magnetic stirrer in the test flask, which is filled with sufficient volume to minimize headspace and prevent delay of O2 and CO2 diffusion through the air-water phases. This volume is dependent on the selected flask size, and is normally specified by the manufacturer of the respirometer. If biodegradation takes place, the microorganisms consume oxygen and produce carbon dioxide. Oxygen from the headspace is then dissolved in the liquid to reestablish chemical equilibrium. The carbon dioxide produced by the microorganisms diffuses into the headspace where it is trapped in an absorbent solution or material and the total pressure in the flask then decreases.

6.1.3 This pressure drop is detected by a manometer, which produces a signal that results in the electrolytic generation of oxygen. When the original pressure is re-established, the signal is stopped and the quantity of electricity used is measured. The amount of electricity used is proportional to the amount of consumed oxygen. This is indicated on a plotter or a printer, or the data are collected using an appropriate software program.

6.2 Water-Bath or Constant Temperature Room, to comply with 11.2.

6.3 Centrifuge.

6.4 pH-meter.

6.5 Analytical Balance, capable of weighing to appropriate precision and accuracy (for example, +/- 0.0001 g).

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 decreasing 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 Calcium Chloride Solution - Dissolve 27.5 g of anhydrous calcium chloride (CaCl2) or 36.4 g of calcium chloride dihydrate (CaCl2·2H2O) in water and dilute to 1 L.

7.3.2 Ferric Chloride Solution - Dissolve 0.25 g ofiron (III) chloride hexahydrate (FeCl3·6H2O) in water and dilute to 1 L. Prepare this solution just before use or add a drop of concentrated hydrochloric acid (HCl) or 0.4 g/L of ethylenediaminetetraacetic acid (EDTA).

7.3.3 Magnesium Sulfate Solution - Dissolve 22.5 g of magnesium sulfate heptahydrate (MgSO4·7H2O) in water and dilute to 1 L.

7.3.4 Phosphate Buffer Solution - Dissolve 8.5 g of anhydrous potassium dihydrogen phosphate (KH2PO4), 21.75 g anhydrous potassium monohydrogen phosphate (K2HPO4), 33.4 g disodium hydrogen phosphate dihydrate (Na2HPO4·2H2O), and 0.5 g ammonium chloride (NH4Cl) in water and dilute to 1 L. Alternatively, 50.3 g of disodium hydrogen phosphate, heptahydrate (Na2HPO4·7H2O) may be used in place of Na2HPO4·2H2O. The pH of this solution shall be about 7.4.

8. Inoculum Test Organisms
8.1 Sources of the Inoculum - Activated sewage-sludge from a sewage-treatment plant that treats principally domestic waste may be considered as an aerobic inoculum. An inoculum derived from soil or natural surface waters, or any combination of the three sources, may also be used in this test method. Allowance for various and multiple inoculum sources provides access to a greater diversity of biochemical competency and potentially represents more accurately the capacity for biodegradation. 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 h 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 minimal or no effluent from industry.
8.1.1.1 Using CO2 -free air, aerate sludge in the laboratory for 4 h. Depending on the number of test systems, sufficient volume of the mixed liquor is sampled and homogenized for 2 min at medium speed using a high-sheer/high-speed blender. Withdraw a sample for the determination of the dry weight of the suspended solids (8.2.2). Keep the inoculum continuously well mixed until all sample preparation is completed to avoid solids settling.

8.1.1.2 Calculate the volume of homogenized mixed liquor necessary to achieve a final sludge dry-weight concentration in the test medium of 30 mg/L (suspended solids, 8.2.2). The inoculum prepared from the homogenized mixed liquor may be used to prepare a composite inoculum (8.1.5), pre-adapted to the test material (8.3.1), or added directly to the test systems (12.2).

8.1.1.3 Alternatively, settle the homogenized sludge for 30 min or longer (if required) and decant the liquid supernatant for use as inoculum. The inoculum prepared from the supernatant may be used to prepare a composite inoculum (8.1.5), pre-adapted to the test material (8.3.2), or added directly to the test systems (12.2).

8.1.1.4 It is optional to pre-condition the inoculum. Preconditioning consists of aerating the activated sludge for up to seven days. Sometimes pre-conditioning improves the precision of the test method by reducing the amount of oxygen consumption in the blank controls.

NOTE 1 - Exercise care in pre-conditioning because of the sensitivity of inocula to prolonged aeration and starvation conditions. Pre-conditioning should be applied in situations where it is known that the inoculum source consistently shows a high internal respiration rate.

8.1.2 Inoculum from Secondary Effluent - Alternatively, the inoculum can be derived from the secondary effluent of a treatment plant or laboratory-scale unit receiving domestic sewage.
8.1.2.1 Allow the secondary effluent to settle for 1 h and collect the supernatant or filter the effluent through a coarse filter paper. After supernatant collection or effluent filtration, aerate the sample using CO2 -free air in the laboratory for 4 h. The inoculum may be used to prepare a composite inoculum (8.1.5), pre-adapted to the test material (8.3.2), or added at this point to the test systems (12.2). Up to 100 mL of this type of inoculum may be used per litre of medium.

8.1.3 Inoculum from Surface Water - A further source for the inoculum is surface water. In this case, collect a sample of an appropriate surface water (for example, river or lake) and keep aerobic until required.
8.1.3.1 Filter the surface water through a coarse filter paper or glass wool plug, and discard the first 200 mL. Aerate the remaining filtered sample using CO2 -free air in the laboratory for 4 h. The inoculum may be used to prepare a composite inoculum (8.1.5), pre-adapted to the test material (8.3.1), or added directly to the test systems (12.2). Up to 100 mL of this type of inoculum may be used per litre of medium.

8.1.4 Inoculum from Soil:
8.1.4.1 Suspend 100 g of soil in 1000 mL of water.

8.1.4.2 Allow the suspension to settle for 30 min.

8.1.4.3 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. The soil inoculum may be used to prepare a composite inoculum (8.1.5), pre-adapted to the test material (8.3.1), or added directly to the test systems (12.2). Up to 100 mL of this type of inoculum may be used per litre of medium.

8.1.5 Composite Inoculum - The four inoculum sources may be combined in any proportion and mixed well.

8.2 Determination 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(7) colony-forming units 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 for inoculum from activated sludge, 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 any inoculum type to a test material is allowed. A sufficient volume of pre-adapted inoculum in test medium shall be incubated for 14 days to yield a minimum of 100 mL of inoculated medium for each respirometer test system; that is, 100 mL for each blank, test material, and positive control material replicate. When developing preadapted inoculum for more than one test material, individual cultures will be prepared separately for each test material. Pre-adaptation can be accomplished as follows:
8.3.1 Pre-adaptation of Homogenized Mixed Liquor Inoculum - Supplement the calculated volume of homogenized mixed liquor inoculum necessary to achieve a suspended solids concentration of 30 mg/L (8.1.1.2) with 25 mg/L of vitamin-free casamino acids and 25 mg/L of yeast extract.
8.3.1.1 Add the supplemented inoculum to a 2-L Erlenmeyer flask. Add 10 mL of phosphate buffer solution, 1 mL of magnesium sulfate solution, 1mL of ferric chloride solution, and 1mL of calcium chloride solution to the 2-L Erlenmeyer flask. Add sufficient volume of water to the 2-L Erlenmeyer flask to achieve a total volume of 1000 mL. Prepare separate inoculated test medium for each test material requiring preadaptation.

8.3.2 Pre-adaptation of inoculum prepared from one of the following sources: activated sludge supernatant, 8.1.1.3; secondary effluent, 8.1.2.1; surface water, 8.1.3.1; soil, 8.1.4.3; or composite, 8.1.5. Supplement inoculum with 25 mg/L of vitamin-free casamino acids and 25 mg/L of yeast extract.
8.3.2.1 Add 100 mLofthe supplemented inoculum prepared in 8.3.2 to a 2-L Erlenmeyer flask. Add 10 mL of phosphate buffer solution, 1mL of magnesium sulfate solution, 1mL of ferric chloride solution, and 1mL of calcium chloride solution to the 2-L Erlenmeyer flask. Add sufficient volume of water to the 2-L Erlenmeyer flask to achieve a total volume of 1000 mL. Prepare separate inoculated test medium for each test material requiring pre-adaptation.

8.3.3 The inoculum flasks are maintained at a temperature of 22°C (+/- 2°C) in the dark and are agitated on a shaker or shaker table or with magnetic stirrers at a moderate speed (for example, 150 to 200 rpm). 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, to ensure the use of a consistent amount of test material.

8.3.4 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 materials, media from the separately prepared flasks may be combined before final filtration.

9. Test Material and Reference Material
9.1 This section addresses specific requirements pertaining to the theoretical oxygen demand (ThO2) of the test material and reference material as well as the appropriate choice of the reference material.

9.2 The theoretical oxygen demand (ThO2) shall be determined based on results of elemental analysis and calculations in 13.1.

9.3 The test material shall be added to the appropriate respirometer test systems to obtain a loading of 50 to 100 mg/L and a test material ThO2 requirement of 50 to 200 mg O2/L in the test medium.

9.4 Reference - A material known to be biodegradable shall be tested simultaneously with the test material.
9.4.1 For water soluble test materials, suggested reference materials are sodium benzoate and aniline.

9.4.2 For water insoluble test materials, the suggested reference material is low erucic acid rapeseed oil, also called LEAR or canola oil. The low erucic acid rapeseed oil shall contain a maximum of 2 % by weight erucic acid.

9.4.3 The reference material will be added to the appropriate respirometer test systems to obtain a loading of 50 to 100 mg/L, in order to require 50 to 200 mg O2/L as reference material ThO2 in the test medium in the same manner as the test material.

9.4.4 The results from flasks containing the reference material verify the viability of the inoculum.

9.5 The test method shall be performed in a minimum of two replicate test systems on all test and reference materials, but triplicates are preferred.