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
7. Test Procedure
7.1 Test solutions should not be aerated during the test unless absolutely necessary to maintain adequate dissolved oxygen levels.
7.2 The exposure temperature, organism mass per test solution volume, and test organism size should be appropriate and standard for the species and experimental design as defined in Guide E 729 or applicable regulatory guidelines.
7.3 Test organisms should be placed in the test solutions within 60 min after preparation at study initiation. For the mechanical dispersion technique, test organisms should be added to the test vessels within 60 min prior to test material addition to minimize physical coating of the organisms. The test exposure period begins upon addition of test organisms (or upon addition of test material if organisms are added first). Standard laboratory procedures appropriate to the toxicity test being conducted should be followed during the study. The exposure loads used in the toxicity test are based on the loading rate of test material into the test system (for mechanical dispersion) or into the preparation vessels (for WAF or WSF). The exposure loads will be used to calculate the LLXX, ILXX, or ELXX for the selected test species (9).
7.4 The test solutions may be renewed either to maintain adequate loading levels of the test material (loss may be due to test material instability) or to maintain adequate levels of dissolved oxygen. Renewal may not be common with the mechanical dispersion technique, but is sometimes prompted by dissolved oxygen or material stability requirements. The loading rate may change if significant material remains adhering to the cylinder and test vessel when using this technique. Acute and chronic studies may be renewed at 24 or 48 h intervals with subsequent and separate WAF/WSF preparations or solution addition (mechanical dispersion technique). Renewals should take place as rapidly as possible after test solution preparation. Some understanding of material composition is necessary to determine the appropriate frequency of renewal. A minimum of 75 % of the test solution should be replaced with each renewal.
8. Characterization
8.1 The exposure matrix may be analyzed either to demonstrate the qualitative maintenance of exposure conditions or to measure the quantitative concentrations of individual constituents. The analytical procedure used should be appropriate for the test material.
8.1.1 If the test material is uniformly dispersed throughout the test vessel, samples for analytical characterization of the exposure matrix are taken from the midpoint of the vessel.
8.1.2 If the test material is not uniformly distributed throughout the test vessel as may occur during the WAF or mechanical dispersion procedures, multiple samples or larger volumes (greater than 100 mL) may be necessary for analytical characterization.
8.1.3 If the exposure matrix samples cannot be analyzed immediately, they should be minimally handled, extracted or preserved, or both, as appropriate for stabilizing the test material constituents, and stored in the dark under reduced temperature (4°C) to minimize loss of test material by microbial degradation, hydrolysis, photolysis, reduction, sorption, and volatilization. If the exposure matrix will be stored at reduced temperature, the entire volume, including vessel walls, should be extracted to minimize the effects of partitioning.
8.1.4 The precision and bias of the analytical method used to measure test material constituents should be determined in dilution water or a substance comparable to the dilution water.
