Physical Chemistry-Getting the Basics Right

January 23, 2019

The importance of study design, with particular reference to UVCBs

Physical chemistry studies give one an opportunity to understand the behaviour of the test item and
therefore underpin the design and consideration of studies required in further sections of a regulatory
dossier. The timeframes of regulatory studies regularly overlap with this battery of tests and also require
knowledge of the physical-chemical properties of the item.
The success and prompt completion of a regulatory package of studies is intimately linked to the base set of physical chemistry tests. Commercial pressures increasingly require condensed phasing of studies, giving less tolerance for unanticipated challenges and delays.
It is therefore important that physical chemistry studies are designed thoughtfully, with excellent
communication between the study directors and the various groups reliant on this data. In these instances, real-time communication informs decisions on dosing strategies and test design, not only from the value generated by the physical chemistry group but also how that value was generated.
With substances that are difficult to test, it is imperative that physical-chemical tests are approached and
constructed in a way that gives complete and reliable information to the data produced. Quality through
design of these tests can support correct decision-making in associated regulatory studies. The time taken to consider such designs carefully expedites the entire regulatory process.
Smithers Viscient believes a congruent approach between physical chemistry and ecotoxicology study
designs is of the utmost importance, especially when testing low solubility, volatile, surface-active, unstable, persistent and/or unknown or variable composition, complex reaction products and biological (UVCB) compounds. Of particular importance are the implications of water solubility, partition coefficient, surface tension and vapour pressure.

UVCB example
As an example, imagine a UVCB compound which contains some surface-active components and other suspected persistent, bioaccumulative and toxic (PBT) components of low water-solubility. We will consider the implications for water solubility testing and subsequently the dosing regimens in aquatic toxicology studies. For this endpoint, it is important to get the basics right.
During the development of analytical methodology, we seek to assign markers representative of the total hydrocarbon structures present in the water-soluble fraction. These markers need to be based on more than just what we can simply analyse: they are chosen to inform the behaviour of the whole test item and represent the range of functional groups present in the product.
The analytical methodology developed for water-solubility measurements for UVCB test item must be
approached in unison with the one used to demonstrate test item concentrations in aquatic ecotoxicology media. Also, due to the presence of surface-active components, we need to ensure any sensitive analytical end-points are measuring true solubility and not a fine dispersion or emulsion of test item in the water column.
Critical micelle concentration (CMC) and turbidity measurements are useful confirmatory experiments that should be conducted in tandem with other quantitative analytical techniques. We would expect a series of solubilities to be present. Some components may be fully soluble and not reach a ‘saturation concentration’, while other extremely insoluble components may still affect the apparent solubility of further components within the water-soluble fraction.

Design considerations
In terms of design consideration, we would employ an adaption of the OECD 105 methodology for
shake-flask. For a complex liquid product the traditional column elution method is not appropriate.
However, reducing kinetic energy to the system of a shake-flask design is a key element; shaking or higher stirring rates can lead to excess kinetic energy that increase the potential for emulsions to form and disrupt the boundary layer with air.
For this reason, a slow-stirring methodology, with a stirring rate of <100 rpm, and only a dimple present on the surface, is most appropriate. The reduced energy to the system necessitates a longer equilibration time than the typical 72 hours of the shake-flask method. Equilibrium solubility in distilled water would be assessed at a controlled temperature (20°C) for between two and four weeks.
It is critical that the ionic strength of the aqueous media is controlled and understood if pH regulation is
required through the use of buffers. Likewise, potential interactions between the buffers and the test item components need to be assessed fully.
This becomes very important when seeking to understand solubility interactions in ecotoxicology media. If the liquid test substance is more dense than the water column, we would consider application to a glass slide suspended just beneath the water surface. If the volatility of the test substance is a concern, this testing would be conducted in near-zero headspace conditions.

Solubility challenges
With UVCB test items, solubility dynamics do not function in the same classical manner as single-constituent compounds. Interactions between the solvating water molecules and the solute components are affected by the presence of interactions within the component mixture. It is therefore important that more than one loading rate is tested over at least one order of magnitude difference.
As the loading rate is decreased, we can often see there is the potential for significant decreases in the ratio of soluble to less soluble components in the water-soluble fraction. Therefore, at lower loading rates, the low-solubility PBT components can have a much higher influence within this fraction.
REACH guidance has suggested loading rates of 1,000 and 100 mg/L, which correlates with the highest
concentration for testing dispersions in the OECD guidance document on aquatic toxicity testing on difficult
substances and mixtures. Data on the water-soluble fractions, as provided by a correctly designed
experiment, would therefore generate information on the bioavailable portion of test item (in conjunction
with dispersibility limits and CMCs), so as not to underestimate observed toxicity concentrations.
Each of the decisions made in the water-solubility testing impacts the test item dosing regimen for aquatic toxicology studies. Media preparation techniques such as water-accommodated fractions (WAFs) or low-energy stirring are directly analogous to the slow-stirring methodology. Information gathered, and the design of the water-solubility experiment, therefore inform the decision-making process for the exposure regime.

Key considerations 
In our example above, consideration given to the design of the ecotoxicology studies is educated by the
strategy and results of the water solubility test. These include:
● the very low rate of stirring test item media to decrease kinetic energy to the system and the ensuing
risk of micro-emulsions;
● the consequent equilibration time that can extend into weeks when preparing media;
● the predicted solubility effect from ionic strength (or lack) of the aqueous media including the
identity of salts;
● the need for non-solvent delivery of the UVCB test item;
● the avoidance of separation techniques such as filtration or centrifugation when preparing a surface
active test item in media;
● the understanding of the test item components’ solubilities at lower temperatures of media
preparation beyond simple Arrhenius predictions;
● the selection of the correct marker components in alignment with the water solubility testing to give
evidence to solubility, stability and a concentration effect level; and
● the variance in toxicological effects from the preparation of separate WAFs for each dosing rate
supported by water-solubility results at additional loading rates.
In this case, if we had not paid careful consideration to the design of the physical chemistry and reliant
aquatic toxicity tests it is entirely possible that the wrong outcomes could have been determined. For
example, incorrect selection of a marker (highly soluble surface-active component) would suggest complete, homogenous dissolution of our test item and a higher water solubility with bioavailable fraction for the aquatic toxicology tests.
This lack of rigour may have precluded the use of a WAF or the expectation for micro-emulsions to form, in turn underestimating the toxicity of test item components. This, of course, would be incorrect, clearly showing the importance of getting the basics right. This principle extends to our other physical chemistry tests, not simply attributing a value to a measurement with accuracy and precision, but conscientiously preparing the design of that measurement to accurately understand the value.
Is our surface tension result pH-dependent or does it consider the need for equilibrium solubility of a
complex test item before measurement? Is our volatility assessment with vapour pressure designed to assess the impact of individual volatile components or do we see large deviations from the Clapeyron-Clausius equation from a skewed weight loss at low temperatures?
How do we assess whether degradation has impacted the vapour pressure calculation of a UVCB? And what of partition coefficient where surface activity, multiple components and low aqueous solubility make this test a complex trade-off between a blend of testing strategies?

It is important to ensure the values generated and reported for the physical-chemical tests fully describe the range of the test items characteristics. In each of these examples the implications consequences of not understanding the behaviour of the test item, with reference to underpinning the design and consideration of studies required in further sections of the regulatory dossier, are too great, to settle for a number in favour of understanding the behaviour of the test item and therefore underpinning the design and consideration of studies required in further sections of the regulatory dossier. Getting the basics right, starting with quality through design of the physical chemistry tests is paramount to the expeditious success of the regulatory


About The Author | Alasdair Clipston

Alasdair Clipston is the Director of Chemistry at Smithers Viscient, EU. Alasdair has spent 15 years within contract research and testing organisations. His experience includes operational leadership and study direction; managing residue analysis, ecotox support, physical chemistry and batch analysis teams in support of regulatory testing for plant protection products, biocides, pharmaceuticals and general industrial chemicals.

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