ASTM D6081 Standard Practice for Aquatic Toxicity Testing of Lubricants
ASTM D6081 Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation
6. Preparation of Exposure Matrix
6.1 Experimental design considerations for fish and invertebrates will determine the test species, number of loading rates tested, and number of organisms per treatment. Experimental design considerations for algae will determine the test species, number of loading rates tested, and appropriate initial biomass per treatment.
6.2 An acute or chronic test intended to allow calculation of an LLXX, ILXX, or ELXX will typically consist of a dilution water or test medium control and a series of at least five loading rates of test material.
6.3 Test material should be added directly to the dilution water to prepare the exposure matrix. Use of a solvent or chemical dispersant is not recommended because these may alter the nature of the exposure to the test material and lead to non-representative ecotoxicity relationships.
6.4 Preparation of Water-Accommodated Fraction (WAF):
6.4.1 Components dissolved in the water phase or entrained as stable droplets in the water phase are evaluated in a WAF study (6). Individual WAFs must be generated for each test exposure loading. Serial dilutions of a single WAF are not appropriate due to differential solubility of constituents at low exposure loads (6,7). A schematic of the WAF setup is provided in Fig. 1.
6.4.2 Vessels for WAF preparation should be of suitable shape and size for the experimental design. The vessels should be of sufficient size to maintain the vortex intensity and optimize surface area contact between the test material and the water. Height to diameter ratio for the WAF preparation at initiation should be between 1:1 and 2:1, and solutions should not exceed 20 L per individual preparation. If larger volumes of test solution are required for experimental design, separate WAF preparations of the same test load may be combined. Aspirator bottles or other vessels with outlet ports at the bottom are preferable for WAF collection, if the test material is less dense than water. If a glass siphon is used to remove the WAF, it should be in place prior to the introduction of the test material. WAF solutions from test materials which are denser than water should be decanted from the top of the vessel. For these dense fluids, a propeller-driven system which stirs the test solution from the top of the vessel may be preferable to a stir plate which stirs the test solution from the bottom of the vessel. In all cases, care should be taken to ensure that non-dispersed test material is not collected with the WAF.
6.4.3 Vessels for WAF preparation should be filled with the appropriate volume of dilution water, and be prepared for stirring as described in 6.4.2. Test material should be slowly added to the top of each vessel after dilution water addition. The vessel should be capped with foil or a non-reactive covering and stirred at the test temperature. For test temperatures different than room temperature, the WAF preparation vessels may need to be housed in water baths or incubators to maintain the desired temperature. If photodecomposition of the test material is likely, the stirring phase should be conducted in the dark. Preparation vessels should be inspected occasionally to ensure an appropriate vortex is maintained.
6.4.4 The amount of test material added to each vessel should be determined by the desired nominal exposure load. Nominal exposure loads may be based on direct weight of material added per volume solution (wt/vol) or calculated on the basis of test material density and added on a vol/vol basis.
6.4.5 The stir plates should be run at a sufficient speed to ensure a vortex depth of 10 to 35 % of the test solution height in the WAF preparation vessel. The vortex in all WAF preparation vessels for any one experiment should be the same. Exercise care to ensure that test material is not pulled down to the bottom of the vessel, and that the rate of stirring is not so vigorous as to promote emulsification.
6.4.6 The duration of WAF preparation may depend on the results of an optional equilibrium/stability study (see 6.7). In the absence of this study, a standard 20 to 24-h mixing and 1 to 4-h settling period should be used (6,8).
6.4.7 Upon completion of the WAF mixing and settling period, the aqueous solution should be drained or decanted from below the surface of each preparation vessel. The first 5 to 10 mL of solution decanted from the vessel should be discarded. The remaining solution should be used undiluted in the exposure vessels.
6.5 Preparation of Water-Soluble Fraction (WSF):
6.5.1 Only components dissolved in the water phase are evaluated in a WSF study (6). WSFs are prepared by filtration of WAFs through a 0.45 µm nominal cellulose-acetate filter or by centrifugation to remove undissolved material. Preliminary testing may be necessary to ensure that the filter disc will not remove test material constituents which are dissolved in the water phase. Individual WSFs must be generated for each test exposure load. Serial dilutions of a single WSF are not appropriate due to differential solubility of constituents at low exposure loads. A schematic of the WSF setup is provided in Fig. 2.
6.5.2 The procedures described in 6.4.3-6.4.7 for the WAF preparation are the initial steps of the WSF procedure.
6.5.3 Upon completion of the WAF mixing and settling period, the aqueous solution should be decanted from below the surface of each preparation vessel. The first 5 to 10 mL of solution decanted from the vessel should be discarded, with the remaining solution used to prepare the WSF. Filtration through a 0.45 µm filter or low speed centrifugation (to remove droplets but not to break an emulsion) is performed on the solution to remove undissolved material. The filtrate or centrifugation supernatant is the WSF exposure matrix and should be used undiluted in the exposure vessels.
6.6 Preparation of a Mechanical Dispersion:
6.6.1 In the mechanical dispersion technique, a motor-driven propeller assembly is used to continually move test material and dilution water in the test vessel during the study (6,7,10). This procedure should not be used for small-sized, slowly, or poorly-swimming organisms. It is most appropriate for juvenile fish evaluated in acute studies. A schematic of the mechanical dispersion setup is provided in Fig. 3.
6.6.2 Each test vessel is equipped with a central cylinder equipped with a three-bladed propeller. The cylinder should be of appropriate configuration to allow water and test material to spill into the top of the cylinder and be released from small apertures at the bottom of the cylinder. The pitch of the propeller should be between 30 to 45 % and should be the same among test vessels. Screening with a non-reactive material should be used at the top and bottom of the cylinder, as necessary, to prevent organism movement into the cylinder.
6.6.3 Each test vessel must be filled with a sufficient volume of dilution water to enable operation of the mechanical dispersion apparatus. The stirring speed in each vessel should be adjusted from 1000 to 1500 rpm, creating a vortex within the cylinder of 12 to 20 mm (5 to 10 % of cylinder height). Motor speed should be checked daily and evaporative losses should be replaced with dilution water, as needed, to ensure consistent motor speed.
6.6.3.1 The dimensions and configuration of the test vessels have not been specified. Both circular and rectangular vessels have been used with this technique. The test vessels should be the same for all test treat rates during a study and should be appropriate for the organism being evaluated.
6.6.4 The amount of test material added to each vessel should be determined by the desired nominal exposure load. Nominal exposure loads may be based on direct weight of material added per volume solution (wt/vol) or calculated on the basis of test material density and added on a vol/vol basis.
6.6.5 Test material should be slowly added to the top of the test vessel. The test material may be added directly into the cylinder, but this is not mandatory. For viscous or semi-solid lubricants or their constituents, application of test material to petri dishes, glass plates or TFE-fluorocarbon pads before addition to the test vessels is preferable to direct addition to the water, to ensure maximum surface area for test material exposure and reproducible test results.
6.7 Equilibrium/Stability Study (Optional):
6.7.1 The stirring and settling periods used to generate individual WAFs or WSFs are material-sensitive and can be optimized (11). Analytical characterization studies can be done to determine the mixing time which results in maximum test material concentration in the matrix and the duration of matrix stability.
6.7.2 To conduct an equilibrium/stability study, prepare at least one WAF or WSF exposure matrix at the highest anticipated loading rate to be used in the toxicity study for each time interval to be evaluated. The same dilution water and vessel configuration which will be used during the toxicity study should be used during the equilibrium/stability study. Individual WAF or WSF exposure matrices should be sampled at different stirring intervals (that is, 3 h, 6 h, 12 h, 24 h, 36 h) to evaluate test material loading and stability in the exposure matrix. For preparation vessels such as aspirator bottles or other vessels with an outlet port, the matrix can be collected from the port during the equilibrium/stability study. For preparation vessels without an outlet port, the matrix should be sampled near the bottom with a glass siphon. Analyze the matrix solution using an analytical procedure appropriate for the test material (for example, gas chromatography (GC), high pressure liquid chromatography (HPLC), infrared spectrophotometry (IR)).
6.7.3 Based on the analytical characterization results, determine the appropriate stirring and settling periods for WAF or WSF preparation.
