Authors: 
Dr. Sharon Swales
Managing Director
Harrogate, UK


Sean McLaughlin
Research Director, Environmental Fate and Metabolism
Wareham, MA


Active ingredients in plant protection products (PPP), pharmaceuticals, veterinary medicines, and other general chemicals are introduced into the environment, either deliberately (e.g., PPP), through excretion from humans/animals, or during general use (e.g., disinfectants). Since the environment consists of many different and diverse biological systems and the route of entry into it depends on the product type, an essential part of environmental protection involves understanding the environmental fate (fate) of these active ingredients. It is imperative to know where the compound will end up following its point of entry (distribution) and how fast it dissipates or degrades within an environmental compartment (soil, sediment, water), as this can directly impact the biological organisms within that compartment.

Non-radiolabeled or radiolabeled material can be used to determine transformation rates or mobility in the various environmental compartments. However, radiolabeled material is required to determine the transformation pathway (route) and establish mass balance. The use of radiolabeled material to determine the distribution and fate of compounds has been a standard practice in the regulated chemical industry for over 30 years. Many of the standard test guidelines directly reference the use of radiolabeled material, but also stipulate that non-radiolabeled material may be used but only for determination of the rate of degradation.
This article is designed to provide an understanding of the advantages and/or disadvantages of conducting environmental fate studies with radiolabeled versus non-radiolabeled material.

Fate Pathways
The types of study required to determine the fate of a compound depend on the end use of, and risk assessment for, the product containing the active ingredient. While some core requirements are in place for pharmaceuticals and veterinary medicines and the requirements for other more general chemical products are driven by exposure scenarios based on usage volumes, the most extensive requirements are for PPPs that are applied directly to the environment.

Studies are designed to determine the rate and/or route of degradation (biodegradation studies), mobility in soil, and the ultimate destination of the compound (e.g., in groundwater, as residues irreversibly bound to soil/sediment, or by mineralization to carbon dioxide). Transformation routes may be hydrolytic, biotic, or photolytic and the effect of these can be investigated in individual studies: aerobic mineralization in surface water, aerobic/anaerobic transformation in soil or water-sediment, or photolysis in soil or water. To determine whether a compound is likely to leach through soil into groundwater, adsorption/desorption studies can be used.

Studies to Determine the Route and Rate of Transformation
To thoroughly determine the route of transformation, full accountability of the compound, i.e., “mass balance,” should be ensured throughout the duration of the test. This makes the use of radiolabeled compound essential for determination of mass balance and degradation products that occur throughout the duration of the study, including potentially transient intermediates. The use of radiolabeled compound also enables a distinction between a compound degrading in the test system and one binding irreversibly to soil or sediment. Loss of a compound following application to a test system does not necessarily imply that it is degrading, other mechanisms, e.g., sorption or volatilization that cannot be quantified without the use of radiolabeled compound could be occurring. However, if the only result required is the rate of degradation, full accountability is not required and only the compound under test needs to be monitored.

While it is acceptable to use non-radiolabeled compound for determination of the rate of degradation, it is important to understand the challenges and potential limitations of not using a radiolabel:

1. Without a radiolabel for tracing, the loss processes may be unknown, and the perceived degradation rate could be attributed to reversible or irreversible binding to soil, particularly at later sampling intervals. Extraction solvents that remove all applied compound immediately after application of the test compound may be less effective for aged samples, leading to a perception of the compound degrading rather than adsorbing to the soil. Developing an extraction method for effective analyte recovery throughout the duration of the study can be problematic.
2. Being able to determine the full mass balance with radiolabeling, even for a rate of degradation study, ensures the study data is accurate. When radioactivity is not present in the analyzed extract, it can be characterized as unextracted residue or volatile material, e.g., CO₂.
3. Test systems for these studies are natural systems, collected from fields, ponds, or rivers. While the pesticide history may be known, it does not mean that these systems are free of environmental toxicants, which can lead to high levels of matrix interference in an analytical method developed for a non-radiolabeled compound. For a compound that is a small molecule, matrix interference may be the result of genuine contamination of a similarly small molecule, an issue that is exacerbated when monitoring common metabolites to chemical classes.
4. Alternately, use of a radiolabel ensures that there is no matrix interference: when radio-HPLC is used in tandem with LC-MS, it is quick and easy to demonstrate that the peak being measured can be attributed only to the applied compound of interest.
5. For a non-radiolabeled fate study, the analytical method needs a greater degree of stringency for accuracy and precision than a typical residue method to suitably verify the T0 concentration and to provide consistent rate kinetics. Additional time to develop and validate will may be needed to develop an appropriate method of analysis.
6. LOD and LOQ tend to be lower for radiolabeled detection techniques, so even when concentrations are low the analytical method allows the compound to be quantified.

 
Adsorption/Desorption Studies
Adsorption/desorption studies determine how much of the compound will adsorb to a stationary phase, e.g., typically soil, but also sewage sludge or sediment, and how much will stay in the aqueous phase. The pre-requisite for these studies is for the compound to remain stable throughout the duration of the test, with >95% accountability for the applied compound. Therefore, this type of study could be conducted with or without radiolabeled compound.
It is important to understand the challenges and potential limitations of not using a radiolabel for adsorption/desorption studies:

1. A non-radiolabeled compound requires a validated analytical method for soil and calcium chloride, with LOD and LOQ values that must be several orders of magnitude below what can be a very low water solubility. The guideline requires use of five concentrations in the test, with the highest concentrations limited by the water solubility.
2. Matrix interference can be much more significant in these studies than in the corresponding rate of degradation studies due to the low concentrations of test compound being used. This issue can be eliminated by using a radiolabel.
3. These studies require preliminary steps, including solubility and adsorption to test vessels. Any losses at this stage are easier to track with a radiolabel and make the next steps easier to navigate.
4. It much easier to verify the extraction of >95% of the compound by the presence of a radiolabel, with no ambiguity between poor extraction and degradation of compound. For a non-labeled analytical method to meet the OECD 106 study requirements, the mass balance requirement can significantly impact development time.
5. In a radiolabel study, if a compound is demonstrably stable after the preliminary stages, conducting the definitive isotherms test using the indirect method (LSC of aqueous phase and determination of soil concentration by difference) saves both time and cost. With the new EFSA checklist for OECD 106 studies, non-radiolabeled compounds will need to be conducted using the direct method of analysis, which is time consuming and significantly more expensive.

   
Challenges with Radiolabeled Compounds
The downside of using a radiolabel is that synthesis of a ¹⁴C-compound is expensive, and for some compounds that could be cost prohibitive. A lengthy synthesis could lead to significant delays for a registration package. There could be challenges with the position of the radiolabel in the molecule: sometimes the most stable one is the most expensive and choosing the wrong radiolabel site, e.g., a labile side chain or terminal methyl group, could be problematic.
The accuracy of the data generated depends on the specific activity of the starting compound. While ¹⁴C is the label of choice, it has a low specific activity, which could lead to problems in a large molecule. Compounds with a tritium (³H) label can be produced at a higher specific activity. However, because tritium exchange is a common problem in aqueous systems it is not an ideal label for environmental testing.

Conclusion
While acceptable from a guideline perspective, environmental fate testing with non-radiolabeled compounds limit the amount of information that can be obtained and make it more difficult to track the fate of a chemical once mechanisms such as adsorption, degradation, and/or volatilization start to occur. Once the compound can no longer be extracted easily from a solid matrix or is present in an aqueous phase, assessing the mechanism responsible for the disappearance becomes difficult. For these reasons, even testing with mixtures (UVCBs) or large complex molecules may benefit from targeted radiolabeled positions on certain parts of a representative molecule in a mixture. The higher upfront costs and time needed for radio-synthesis may seem excessive when data is needed for a registration package, but in the long term they provide more robust results and often the studies can be completed in a shorter timescale because the analytics are less challenging.