Dosing Methodologies for Environmental Toxicity Testing: A Look at Challenges & Potential Solutions

June 14, 2017

There are several methods that are available for successfully dosing environmental toxicity (ecotox) exposures. While there are a variety of dosing methodologies available, not all test substances are appropriate for them - especially in the case of aquatic exposures.

An understanding of the test substance itself is critical to the decision on an appropriate dosing method. It is vital to understand the water solubility of the test substance but it is also important to understand the functional limit of solubility under testing conditions. Water solubility, which we tend to treat as a stand-alone concept, can differ greatly among dilution matrices (based on factors such as water characteristics and pH) as well as differing testing temperature. It is possible for the limit of solubility under testing conditions to differ among test types. In order to understand the best way to maintain consistent concentrations over the course of an exposure, behaviors like adsorption and degradation also need to be well understood. And of course there are practical considerations that are associated with the needs of specific exposure designs and durations such as volume restrictions on stock solutions or test substance availability. In cases where a flow through system is required and testing needs to be performed at the limit of solubility of a test substance, stock volumes that exceed 500 L per day become unmanageable.

A test substance might exhibit solubility in a specific delivery method but may show insolubility when a dilution water interface is reached. For example, a material may be soluble in solvent at a high concentration but once that solvent stock is delivered into water, additional energy maybe required to reach water solubility.  The impact of this can be extreme in flow-through systems which are either continuously dosing or have a short residence time for the high concentration to mix. The effects of poorly soluble test substances, particularly undissolved material, can be detrimental to the exposure with issues such as inhomogeneous solutions, variable analytical recoveries, unintended oral exposure and potential physical toxicity to test organisms. Mechanisms like filtration and centrifugation may be employed for some study designs when undissolved material is observed, but both may be impacted by the potential of a material to adsorb and are limiting factors depending on the volume of the solution – these procedures may be effective for small volumes (i.e. 1-2L) but they become unreasonable for larger volume stocks.

So what options are there for dosing? Probably the most favorable type of dosing solution is a direct addition water stock. This dosing method eliminates the need for additional controls (i.e. solvent controls) which is especially advantageous when testing with vertebrates. It also eliminates the potential that the solvent negatively impacts the exposure. Remember that solubility in water does not imply stability and so the stability of a dosing stock and therefore the preparation schedule needs to be well understood. As mentioned previously, the volume required can become a restriction and, thus, needs to be considered. Mixing energy also needs to be carefully controlled in order to maintain consistency when more than one stock needs to be prepared during the course of an exposure.  In the case that a test substance is stable, extended mixing time may be employed to solubilize the material but there might be limitations for this procedure when a flow through system is utilized.
Sonication can be a powerful method to enhance solubility of a solid test substance. This method is highly restricted by volume, although small volumes can be sonicated prior to a final large dilution. Sonication can be used during flow through testing to enhance solubility at the interface of a solvent solution and the dilution water. In all cases where sonication is used, care should be given to ensure that the temperature of the solution is not raised to temperatures that would impact the test substance.
In some cases, pH adjustment can provide a concentrated aqueous solution. This procedure is molecule-specific and can depend on the buffering capacity of some dilution matrices. If the final pH of the stock solution is outside of the prescribed testing range, the amount that can be delivered to the exposure system is limited.

A methodology that is often employed for oil-based products, mixtures and multi-constituent products is the Water Associated Fraction (WAF). The mixing of these WAFs needs to be managed extremely carefully as factors such as mixing speed, vortex height and settling time can have a dramatic effect on the aqueous fraction. While this method can provide the most conservative mechanism to evaluate toxicity, the WAF may have different composition than the parent product due to differences in solubility amongst the components, with the emphasis on components with lower molecular weights. The analytical confirmation of these solutions can be very challenging, since solubility may differ among the components of the parent material and can be present at very low levels. TOC can be used as a rough estimate of concentration when specific analytes of interest cannot be confirmed.
The use of co-solvents can be a valuable tool in ecotox testing to facilitate solubility in water. Several organic solvents are allowable for use in aquatic toxicology testing and this can be especially useful in acute testing where the control and solvent control are not used for statistical comparison or data interpretation, but are used as a mechanism to evaluate performance. The use of solvent does provide a nutrient source for microbial growth and can have a dramatic impact on the water quality of a system if this is not carefully managed. In addition, test substances which are impacted by microbial degradation can be much more challenging to keep at consistent levels, especially in a chronic exposure. Any time a solvent is used, the risk exists that a statistical difference in performance of the control and solvent control groups could impact the interpretation of the results, potentially resulting in rejected studies and the need for repeat work.  

To avoid the use of co-solvent for dosing, saturator columns (or generator columns) have become a routine dosing methodology at Smithers Viscient. This method is especially robust for materials with a tendency to adsorb. Glass columns are packed with an inert substance (glass beads or glass wool) and then the test substance is applied to the substrate using a concentrated solvent stock. Once the solvent is evaporated, dilution water is passed through the column. The resulting effluent is the functional limit of solubility. This a non-solvent delivery and the issues associated with the use of solvent are eliminated. Columns can sometimes be used for several weeks before they are considered to be exhausted. Columns generally require a large amount of test substance, making them less attractive for substances available in small quantities (including radiolabeled materials) and they require analytical work up front to understand the column profile and longevity.

Selecting the appropriate method for dosing an ecotox study is one of the most critical steps in assuring a consistent and stable exposure for the duration of the study. The proper dosing technique requires the necessary knowledge and experience to evaluate the  many physical, chemical and biological factors. However, when done correctly, provides reliable and reproducible results and in many cases, can eliminate the confounding influence of co-solvents.


About The Author | Lee E. Sayers

Lee E. Sayers is a Senior Research Biologist at Smithers Viscient and has been part of the Ecotox team since 2000. Prior to joining our team she was a microbiologist specializing in bioassays for the detection of viral contaminates for BioReliance, a CRO located in Rockville, MD. Lee holds dual BA degrees in biology and classical Greek from Colgate University. Lee spent several years directing the aquatic acute programs as well as the chronic testing with daphnids. She has been instrumental in developing novel approaches to assessing chronic toxicity to daphnia in closed systems. She currently oversees the chronic testing with mysid and fish, including fish full life cycle testing. During the EPA Tier I testing, Lee was involved in the conduct of 16 Fish Short Term Reproduction Assays (FSTRA) and 13 Amphibian Metamorphosis Assays (AMA). Lee has been involved in studies for known hormone-type APIs or materials suspected to have endocrine activity, ranging from early life stage testing, fish sexual development testing, to modified life cycle and multi-generational testing focusing on endocrine endpoints. Throughout her tenure at Smithers Viscient Lee has been very active in developing programs to assess biomarker endpoints (e.g. vitellogenin and acetylcholinesterase activity) in various species of fish and birds. Lee is well versed in the challenges of dosing aquatic exposure systems with difficult test substances and had spent several years supporting the development of non-solvent techniques for dosing.

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