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The term emerging substances often invokes thoughts of novel or new compounds entering the environment. Many of these “emerging” compounds, however, have in fact been used for decades and as such they have been present as environmental contamination for many years. The types of compounds are those familiar to most of us; pharmaceuticals and personal care products, industrial chemicals and detergents and disinfectants.
The reason they are emerging is that we are only just beginning to understand, by virtue of improved analytical techniques and environmental studies, the impact these compounds are having our ecosystems.
Clearly the number of compounds in use within the UK would preclude their target analysis – let alone legislation – to take place; however, that is exactly what is required as set out under the Environmental Quality Substances (EQS) directive (2013) and the Water Frame Work (WFD) directive (2000).
Fortunately the EQS and WFD directives do allow some degree of pragmatism in that a priority list or watch list of compounds need be agreed and monitored.
The watch list is a small group of compounds, initially 10, but rising to 14, which are anticipated to pose a threat to human health or the environment but for which insufficient evidence is available. Each member state is responsible for the monitoring of these compounds over a period of typically two to four years by which time the compounds are either deselected, or if evidence suggests, highlighted as potential priority substances or priority hazardous substances in the EQS directive.
Current watch list compounds
Three compounds have already been listed; the non-steroidal anti-inflammatory drug (NSAID) diclofenac, plus the hormones 17-beta-estradiol (E2), and 17-alpha-ethinylestradiol (EE2).
Of the two hormones, 17-alpha-ethinylestradiol (EE2), a synthetic estradiol derivative, is used almost exclusively in contraceptive pills whereas 17-beta-estradiol (E2) is a naturally occurring hormone responsible for the secondary sex characteristics in women.
The proposed monitoring levels for these compounds vary from 10 ng/L for diclofenac down to 0.035 ng/L for EE2.
Future watch list compounds
The European Commission’s Joint Research Centre (JRC) published a comprehensive report entitled “Development of the first Watch List under the Environmental Quality Standards Directive” in the late summer of 2015 that detailed 25 potential candidates for inclusion. Of the list two were inorganic compounds (chromium trioxide and free cyanide), 14 were pesticides (herbicides, insecticides and biocides), four were industrial chemicals and intermediates and four were pharmaceuticals (fluoroquinolone and macrolide antibiotics).
Of the list of 25 candidates, a total of 10 substances or groups of substances were recommended for inclusion on the first watch list based on their PNEC and PEC values.
The Predicted No Effect Concentration (PNEC) is the threshold of any given compound at which below this concentration no adverse effects of exposure in the environment are measured. They are conservative as often they are based on single worst case exposure studies and extrapolated out to the wider environment. The predicted environmental concentration, or PEC, is an estimate of the concentration of any given compound based on exposure models such as European Union System for the Evaluation of Substances (EUSES). The table below shows the proposed list, thier uses plus their PNEC and Predicted Environmental Concentration (PEC) values.
The values in the table marked with an asterisk (*), with the exception of 2-Ethylhexyl 4-methoxycinnamate (EHMC), are based on the PNEC in water. Given EHMC’s propensity for adsorption to sediment, the PNEC and PEC values above are based on sediment concentrations corrected for dry weight.
Pharmaceuticals – a special case?
Although making up only a fraction of the 25 potential candidates, the inclusion of three more pharmaceutical compounds, specifically antibiotics, into the final list is interesting. Although in this case the compounds were chosen for their direct toxicity to the aquatic environment there is a growing concern that the increase in antibiotic resistance may provide some grounds for further testing on these classes of compound.
Most European or global legislation that focuses on organic compounds does so because of a proven or probable biologically adverse effect of that compound on the environment or on human health. Legislation like the Stockholm convention – the so called list of ‘Dirty Dozen’ compounds – brought into effect in European law in 2004, looks to control and reduce the compounds that are known to be persistent in the environment and bio-accumulate.
Covering compounds such as the pesticide DDT, the fluorosurfactant and flame retardant PFOS, as well as unintentionally produced by-products such as dioxins and furans, the convention aims to halt the spread of these compounds by eliminating their production.
Similarly more recent legislation looking at endocrine disruptors (WFD 2000, REACH, Plant Protection Products Regulation (EC) 1107/2009 and the Biocidal Products Regulation (EU) 528/2012) is looking to restrict the discharge of a range of compounds known to mimic hormone behaviour in aquatic fauna.
With the obvious exception of pesticides however, the compounds under investigation were never designed to have these biological effects. Compare this to the pharmaceuticals currently being discharged into the environment via wastewater treatment works (WWTW) and there are clearly some valid reasons for further study.
It is true that the persistency of some of the major pharmaceuticals are significantly less than that of those present in the Stockholm convention (PFOS has an environmental half-life of approx. 40 years compared with ibuprofen with one of several weeks), but what is different for the pharmaceuticals is that they are being constantly replenished via WWTWs in to Europe’s surface water ways.
The Defra report “Wastewater treatment in the United Kingdom – 2012” estimates that over 10 billion litres of wastewater are collected and shipped through the UK’s sewage system. While the removal rate for more traditional contaminants such as polyaromatic hydrocarbons (PAHs) is very high, often 90% or more, for certain pharmaceuticals this removal rate can be as low as 30%.
Another compelling reason for further study is the cocktail effect that all of the different classes and subclasses of compounds being released have on aquatic environment. It has been estimated that over 3,000 individual pharmaceutical compounds have been licensed for use in the UK, but little if any have been studied for their environmental effect and certainly not as part of a combined effect study.
Targeted versus non-targeted analysis
A key driver in the inclusion of compounds into the watch list is the ability of analytical laboratories to analyse the candidate compounds at a suitable level – typically a level less than the Predicted No Effect Concentration (PNEC). The current list has a range of PNEC concentrations ranging from the ng/L level to the mg/L level meaning that in some cases at least advanced level chromatographic tandem mass spectrometric systems are required, e.g. gas chromatography tandem mass spectrometry (GC-MS/MS) or liquid chromatography tandem mass spectrometry (LC-MS/MS).
Contract analytical laboratories, almost regardless of the industry or sector they serve, are based around targeted analysis. Clients or sponsors will provide a list of compounds of interest and the laboratory will devise, develop and implement an analytical solution to meet that requirement. The principle is true from contaminated soil labs running thousands of samples a day looking at gross level hydrocarbon contamination right down to drinking water labs looking at trace level pesticides.
The most sensitive analytical tools used in the laboratory are very much designed for this purpose – instrumentation like GC-MS/MS or LC-MS/MS can see down to sub ng/L levels with relative ease, but this sensitivity comes at the cost of the ability to determine a wider range of compounds outside of the small target list that the instrument method was designed for.
While true that more common laboratory analytical technique such as flame ionisation or electron capture detectors (e.g. GC-FID or GC-ECD) are more comprehensive in the range of compounds they detect, they often lack the sensitivity or confirmatory ability of an MS based detector to be used in studies of this sort.
Whether one looks at GC-MS/MS or LC-MS/MS, the fundamental principles are the same:
- A suitable sample extract is introduced into the instrument and the chromatographic part of the instrument begins to separate out the constituent parts. This separated sample stream is introduced into the tandem mass spectrometer.
- The sample enters the mass spectrometer and is ionised.
- The ions enter the first quad (Q1) and are filtered based on their mass to charge (m/z) ratios.
- Only those ions exhibiting the correct m/z ratio proceed to the next stage, the collision cell (Q2).
- The ions are bombarded with gas to physically fragment them in to smaller fragments.
- The smaller fragments are further filtered (Q3), again based on their mass to charge (m/z) ratios.
- Only those ions exhibiting the correct m/z ratio proceed to the detector.
This desire for high sensitivity at the cost of limited analytical scope has meant that often compounds are not being detected because they are not being looked for. It could be said that the same scenario applies to legislation – how can you regulate something that has no supporting evidence, but the lack of regulation means that no-one has the drive to monitor for it?
This limitation has been the subject of more recent technological advancement – the ultimate goal for the laboratory is to be able to take a sample and effectively ask the question; “what pollutants are in this?” and it is the advent of quadrupole time of flight (TOF) and high mass accuracy GC and LC systems that has begun the journey towards being able to answer this question.
Unlike their highly sensitive tandem MS cousins, they have the ability to obtain continuous scanning data from the entire analytical run. While the continuous scanning capability has been around for some years now, recent improvements have seen the sensitivity of these instruments approach that of the tandem MS systems.
With no filtration of ions or selective fragmentations, the TOF is able to obtain full screen information on molecular ions and isotope patterns from every ion that is produced. Being able to be combined to LC or GC, the chromatographic separation for TOF is the same as for other forms of MS detector; the key difference between TOF and MS/MS lies in the mechanism of determining ions.
In TOF, the ion’s m/z ratio is determined by a time measurement. The ion is formed and then accelerated along a flight tube of known length where the time taken to cover this distance is measured. From this measurement a highly accurate determination of mass can be made and a substance’s molecular formula determined. This information can then direct further experimentation to enable a structural identification, and therefore a chemical identify, to be made.
The future
This advancement in analytical technology is bringing instrumentation and analytical tools that were once the reserve of research labs in to some of the more advanced contract laboratories. As the technology evolves further, and uptake of the technology becomes more widespread, the unit price will invariably drop meaning that an even greater number of laboratories will be able to adopt this technology.
While the advancement in the electronics and hardware design of the high mass accuracy instrumentation has been a major step in improving the commercial viability of the product. It has only been the improvement of the accompanying software, enabling sample data to be reviewed and interrogated, that a major missing part of the puzzle has been found.
Modern software can automatically and quickly analyse datafiles so that molecular formula generation, database or library searches, deconvolution, and isotope pattern matching can be undertaken for confident compound identification.
The ability to interrogate huge volumes of data quickly and efficiently – data often comprising several thousand megabytes for every sample – is a key step in moving away from targeted lists and heading towards a more holistic approach to environmental regulation.
