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
9. Interferences/Complexities
9.1 Limitations to the methods described in this practice might arise and thereby influence aquatic toxicity test results and complicate data interpretation. The following factors should be considered when testing poorly water-soluble lubricants or lubricant constituents.
9.1.1 Test material may be altered by storage beyond the acceptable shelf life or under inappropriate environmental conditions.

9.1.2 Volatility of one or more of the components in the neat test material can occur at various stages in the course of the study.
9.1.2.1 Improper storage of materials with volatile components can lead to a change in the composition of the test material.

9.1.2.2 Preparation of the exposure matrix for a material with a volatile component in an open vessel or a vessel with a large headspace can lead to a change in the matrix composition over time.

9.1.2.3 Organism exposure to the exposure matrix of a material with a volatile component in an open vessel or a vessel with a large headspace can lead to a change in the matrix composition over time.

9.1.2.4 Improper sampling or storage, or both, of matrix samples which contain a volatile component prior to characterization or analysis can lead to a change in the matrix composition over time.

9.1.3 Test materials with chemically different components may show compartmentalization of some of the components.
9.1.3.1 Materials/components that have low water solubility may compartmentalize out of the water phase (for example, floating on surface, clumping on the vessel walls).

9.1.3.2 Materials/components that are ionic may bind at active sites on glass and plastic vessels and so be removed from the water phase.

9.1.3.3 Materials/components with large log Kow (octanol-water partition coefficients) values may adsorb onto non-glass vessels.

9.1.3.4 Materials/components may be lost by absorption on the filter disc during preparation of a WSF exposure matrix.

9.1.4 Physical characteristics of the test material may make the addition of the material to the test system difficult.
9.1.4.1 Quantification of highly viscous materials during material addition is often difficult due to retention of some of the material on the measuring/transport container.

9.1.5 The ion composition of the dilution water used for aquatic toxicity testing may influence the integrity of the test material composition.
9.1.5.1 Materials/components may react/chelate with ions in the dilution water with a resulting change in characteristics (for example, precipitation of components, altered solubility, increased bioavailability).

9.1.5.2 The ionic strength of the aqueous phase may affect the partitioning (or lack thereof) of non-aqueous hydrophobic components into that phase.

9.1.6 Any alterations in the mixing energy or mixing vessel shape can influence the form of the material in the exposure matrix.
9.1.6.1 Mixing energy that is too great may create an emulsion, while mixing energy that is too low may not optimize the distribution of the test material in the exposure matrix.

9.1.6.2 Using mixing vessels of different shapes or volumes to prepare the exposure matrices for the same test can produce different mixing levels and subsequently different exposure loads for some test materials.

9.1.6.3 Vessels used for preparing the exposure matrix should provide maximum surface area for contact between the test material and dilution water. Vessels that are narrow at the opening (for example, volumetric flasks, Erlenmeyer flasks) should not be used if the contact zone is in the restricted portion of the vessel.

9.1.7 Exposure matrix samples that are collected for characterization can vary in composition during the preparation phase and the exposure phase of the test.
9.1.7.1 Samples collected at different times from the same mixing vessel may not be comparable.

9.1.7.2 The physical characteristics of the test material may make some sampling methods inappropriate.

9.1.7.3 The physical characteristics of the test material may make some preservation methods inappropriate.

9.1.8 Test materials evaluated using the exposure methods recommended in this practice may affect the bioassay test organisms independently of their inherent toxicity.
9.1.8.1 Materials/components that move to the surface of the exposure vessel may reduce oxygen transfer and cause a reduction in the dissolved oxygen.

9.1.8.2 Suspended test material may adhere to the test organisms and cause injury independent of the inherent toxicity of the test material (for example, fish suffocating due to test material covering on gills, organisms adhering to the sides of the test vessel, and so forth).

9.1.9 The characteristics of the exposure matrix (for example, suspended test material) or the design of the exposure vessel (for example, no head space) can make the observation and enumeration of test organisms difficult, particularly when using the mechanical dispersion technique or toxicity test vessels with lids.

10. Calculation or Interpretation of Results
10.1 The LLXX, ILXX, or ELXX and its 95 % confidence limits shall be calculated on the basis of nominal loading rates.

10.2 Standard statistical programs can be used to calculate the LLXX, ILXX, or ELXX. The method used should appropriately take into account the number of test vessels per treatment and the number of test organisms per vessel.

11. Keywords
11.1 aquatic toxicity; ecotoxicity; effect load; incident load; lethal load; lubricant; mechanical dispersion; water accommodated fraction (WAF); water soluble fraction (WSF)