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Dr Bruce Petrie and Dr Barbara Kasprzyk-Hordern look at pharmacologically active compounds in waste water.
Pharmacologically active compounds (PACs), such as pharmaceutical drugs used by the human population, can be found in the aquatic environment; for example, in rivers. Some of the most commonly used PACs are sold in hundreds of tonnes a year in the UK alone. Usage of PACs is likely to increase in the future due to an ageing population in Western countries and an increase in consumption levels in the developing world.
The presence of PACs in the environment is mainly attributed to the often poor metabolism of these compounds in humans and their incomplete removal during sewage treatment. They were first reported in the effluent of sewage treatment plants (STPs) in the 1970s (Hignite and Azarnoff, 1977). Figure 1 shows daily loads of pharmaceuticals and other emerging pollutants in a typical waste water treatment plant. Due to insufficient treatment, high levels of PACs are released into receiving waters.
Daily contaminant loads
A major concern regarding the release of pharmaceuticals and personal care products (PPCPs) into the environment is their possible impact on aquatic organisms. A landmark case in 1998 by Jobling et al reported a high incidence of intersexuality in wild populations of roach fish throughout the UK.
The possible impact of PPCPs, however, is not only limited to the aquatic environment. A drop of around 95 percent in India’s vulture population was attributed to diclofenac, an anti-inflammatory drug (BirdLife International, 2014). It was found that vultures feeding on carcasses of livestock that were previously treated with the drug died because it caused the vultures to suffer renal failure.
Proposed legislation
Growing understanding of the possible ecological impact of PACs in the environment has seen the proposal of new environmental regulation in Europe. In 2012, the hormones 17β-estradiol and 17α-ethinylestradiol and the non steroidal anti-inflammatory drug, diclofenac, were proposed as priority hazardous chemicals by the European Commission. Such is the level of concern posed by this chemical type that the proposed quality standards were 0.4, 0.035 and 100 ng l-1, respectively. A much larger number of PACs are known to be present, but their impact within the aquatic environment either individually or synergistically is unknown.
There is also very little understanding of impacts from metabolites of PACs and their transformation by-products. To date, around 200 PACs have been observed in STP effluents and river waters globally. Despite a great depth of ongoing research into the occurrence and fate of these chemicals in STPs and the environment, several areas need further study.
Misconceptions and understudied areas
The following sections outline some of the understudied areas and common misconceptions concerning PACs in the environment.
Sampling and analytical protocols
Sampling is the first and arguably the most important step for the determination of PACs in sewage and river waters. Current sampling protocols, however, are not suitable to adequately monitor PACs and a number of uncertainties exist.
There are two main sampling options available: grab sampling, which involves the collection of a sample at a given point in time, and composite sampling, which tends to be carried out over a 24 hour period and can be collected in a time or flow proportional manner.
Grab sampling is the most common approach used as it is user friendly. As PAC concentration can vary throughout the day, the major flaw of this strategy is that it can only give a snapshot of PAC levels.
Alternatively, composite sampling can collect a sample representative for a system over a 24 hour period. The stability of PACs in such samples is an uncertainty and this needs to be addressed with the validation of a suitable preservation technique.
Cooling to 4˚ C has been found insufficient to stop biodegradation for some PACs across a 12 hour time period (Baker and Kasprzyk-Hordern, 2013). Consequently, currently applied sampling protocols have uncertainties and can introduce considerable bias into findings, prior to any analytical measurement. More comprehensive sampling campaigns are also needed to better understand spatial and temporal trends of PAC occurrence in the environment. Prescription cost analysis information shows that some PACs have seasonal trends; for example, the quantity of antihistamines and sunscreen agents prescribed are 100 percent higher in summer months than winter months (National Health Service, 2012). The PACs pseudoephedrine and pholcodine, used in used in nasal decongestants and cough suppressants, respectively, showed greater quantities prescribed in winter months (Figure 3).
Currently employed analytical methods tend to focus on a limited number of compounds and do not include metabolites or transformation by-products. The determination of such compounds, however, is essential to assess PAC fate and possible ecological impact, as loss of the parent compound does not necessarily mean complete mineralisation or indeed a reduction in toxicity. Transformation by-products formed by biologically mediated reactions within the STP or the environment can be more persistent and potent than the parent compound. The limiting factor here is the application of low resolution mass spectrometers for PAC determinations. Although these are excellent for quantitative analysis of known compounds, they do not facilitate the identification of unknown compounds such as transformation by-products. This emphasises how little is actually known regarding PAC fate and behaviour in the environment.
Furthermore, a lack of analysis is undertaken on solids, such as suspended particulate matter, sewage sludge, sediments and soil. This is because in most analytical laboratories, good analytical approaches are lacking. Nevertheless, analysis here is essential to determine fate for compounds, especially those with a high susceptibility to partition to solid organic matter, such as antibiotics.
Chirality within the environment
A concept often overlooked is that PACs are chiral and exist in the form of two or more enantiomers (Kasprzyk-Hordern, 2010). These molecules usually have a chiral centre and enantiomers are non-superimposable on one another, as seen in figure 4. The concern posed is that different enantiomers of the same compound can behave very differently when exposed to a biologically mediated environment. This can result in the enrichment of one specific enantiomer. Enantiomers can have varying degrees of toxicity; for example, the S enantiomer of the antidepressant fluoxetine, or Prozac, is ten times more toxic to the fat head minnow than the R enantiomer (De Andrés et al, 2009).
This means that the traditional approach of monitoring concentration without considering enantiomeric distribution could actually overestimate, or indeed underestimate, the risk posed to the environment. Despite this information, there is a lack of knowledge on the enantiomeric distribution of such chemicals in the environment due to the difficulties associated with their analysis.
As an example, methods of detection used for quantitation, such as tandem mass spectrometry for environmental analysis, are unable to distinguish between enantiomers of the same compound. They therefore need to be separated from one another chromatographically, which in itself is difficult because enantiomers have identical physicochemical properties. Chiral stationary phases are needed, such as enzyme based stationary phases, as they can successfully separate enantiomers for their quantification individually. At present, however, these are specific for only a few groups of PACs. The ongoing development of improved analytical stationary phases for the successful enantiomeric separation of a broader range of PACs will aid understanding of their fate and behaviour in the environment.
Ecotoxicity
Enantiomers of a chiral PAC can exhibit very different potencies, but this phenomenon has only been studied for a few compounds and for a limited number of aquatic indicator species. A greater understanding of ecotoxicity at the enantiomeric level to a range of indicator species is needed.
Currently, the majority of ecotoxicity testing is done on single PACs in controlled laboratory conditions, at relatively high concentrations compared with those encountered in the environment. Although these tests are very useful in assessing the possible impact of individual PACs to aquatic organisms, they do not represent conditions that are experienced within the environment. An example of this is that aquatic organisms present within the environment are not exposed to a single PAC, but to a diverse mixture. The impact of mixtures is unknown due to the complex nature of determining their impact synergistically.
PACs are continuously discharged into the environment and therefore present throughout the lifetime of the organism, yet there is a lack of information on the chronic impact of PACs to exposed aquatic organisms. Studies are required to assess multigenerational impact of PAC mixtures while at environmentally relevant concentrations. On the other hand, environmental waters contain dissolved organic matter which could potentially reduce PAC bioavailability. It is clear that there are several areas of ecotoxicological research that need to be addressed in order to better understand the impact of PACs in the environment.
Future vision and recommendations
The following recommendations for future improvements address analytical approaches, environmental monitoring and the treatment of waste water.
Analytical approaches
Improved approaches to sampling waste waters and river waters are anticipated. A more standardised sampling strategy will help ensure reliable and representative data is attained. This is likely to involve flow proportional composite sampling, with the use of a suitable preservation technique.
In terms of analytical methods, growing understanding of the importance of chirality and improved chiral stationary phases will result in more analysis undertaken at the enantiomeric level. Routine monitoring will also likely incorporate more analysis of solids for better fate understanding. This will help developing understanding of the role of physicochemical properties of PACs to their removal by adsorption onto biomass during sewage treatment.
The greatest breakthrough in analytical instrumentation is the ongoing development of high resolution mass spectrometers capable of high accuracy measurements. They enable quantitative targeted screening and qualitative non-targeted screening to be undertaken simultaneously. The identification of unknown compounds is essential, as they can be used to find previously undetermined PACs of interest for further investigation and reveal pathways of biotransformation. Findings from chemical analysis need to be supported with bioanalytical methods. Chemical analysis needs to drive the design of ecotoxicological studies to ensure typical environmentally relevant conditions are used. This will enable better understanding of the ecological impact of typically observed PAC concentrations in the environment.
Waste water treatment
The ever increasing demand placed on water resources by a growing population has seen increased focus on possible options for re-use of water. The extraction of surface waters for re-use demands an improved understanding of PAC fate and removal during waste water treatment. Operation of STPs could then be modified to specifically target improved removals of PACs. Studies have shown that this could be achievable for processes such as activated sludge, where process operation impacted removal (Clara et al, 2005). This will help reduce the release of PACs into surface waters. There is also a growing focus on reducing the carbon footprint of the sewage treatment process and recovering valuable resources during processing. This is likely to result in the incorporation of more novel process types into the sewage treatment flow sheet.
Processes such as high rate anaerobic reactors or algae systems for effluent polishing are promising, as they can generate energy through the production of biogas. Due to their use not being widespread, however, at present there are a very limited number of studies which have assessed their potential to remove PACs. Also, processes or operational conditions that reduce PAC content in digested sludge are beneficial as they provide an alternative pathway for release into the environment.
Environmental monitoring
The focus of research in this area has been on the receiving aqueous environment, but due to the diverse range of physicochemical properties exhibited by PACs, some have a tendency to partition into sludge during sewage treatment. Despite undergoing anaerobic digestion, notable concentrations of some PACs are still reported in treated sludge or biosolids (Stasinakis et al, 2008; Guerra et al, 2014). The majority of these biosolids are then applied directly to agricultural farmland as a fertiliser. It is anticipated that environmental monitoring will be widened to also include the terrestrial environment. Monitoring and analysis strategies should essentially be the same for both the aqueous and terrestrial environments. Both targeted and non targeted analytical screening approaches are recommended for amended soils, as well as for measuring concentration at the enantiomeric level. In addition to undertaking chemical determinations, toxicological based assessments are also required here.
Due to limited research on the occurrence of PACs in amended soils, there has been no driver to measure biological toxicity. Indirect methods such as soil respiration rate can help establish the possible impact of biosolid application to soils, but at present there are no standard methods of measuring toxicity to exposed organisms in amended soils.
Published: 27th May 2014 in AWE International
