Advancements in Wastewater Analysis: new EU directives and subsequent regulation on environmental protection continue to place technical demands on analytical laboratories.
The requirement to meet low limits of detection for emerging compounds in complex matrices represents a considerable challenge for traditional methods and therefore new procedures utilising advanced techniques such as high resolution or tandem mass spectrometry have become common in environmental testing laboratories.
The use of Gas Chromatography Mass Spectrometry (GCMS) and Liquid Chromatography Mass Spectrometry (LCMS) is playing an increasingly important function in many modern laboratories. This article reviews analytical methodology used to determine some traditional contaminants as well as some emerging contaminants, which may require routine monitoring in future.
Wastewater is a term used to describe spent or used water discharged from residential homes, industry, farms, hospitals and other sources of anthropogenic origin. Wastewater contains dissolved or suspended solids and is composed of a complex mixture of chemical contaminants as well as microorganisims, paper fibres, plant material, proteins, pharmaceuticals, hair colorants, emulsified oils and hundreds of industrial chemicals.
Analysis of contaminants in wastewater, often required at trace or ultra trace concentrations is difficult due to the complex composition of wastewater. Therefore analytical techniques used to determine trace contaminants in wastewater need to be specific and allow analytes to be detected without interference from other compounds present in the sample (often at concentrations considerably higher than the compounds of interest).
Mass spectrometry coupled to gas chromatography (GC) and liquid chromatography (LC) are ideal techniques for the determination of organic contaminants in wastewater due to the separation capability of GC and LC coupled to the selectivity and sensitivity of mass spectrometry. This coupling of chromatography with mass selective detection allows the routine determination of many contaminants in parts-per-trillion (ppt) or lower concentrations.
In recent years developments in chromatography such as the introduction of Ultra High Performance Liquid Chromatography (UPLC or UHPLC), which utilises the same separation methodology as conventional HPLC, but uses columns packed with smaller particles (generally about 2 µm particle size or less) has further enhanced the applicability of LC in wastewater analysis. The smaller particles used in UHPLC increase column efficiency, which results in higher chromatographic resolution, and speed of analysis.
Higher resolution and speed is particularly suitable for complex matrices where separation of analytes from other compounds is desirable, sometimes essential to obtain good quantitative results.
Mass spectrometry has also developed considerably over the past decade with the introduction of bench-top high resolution mass spectrometry including time of flight (TOF) and orbitrap MS instruments and tandem mass spectrometry (MSMS) instrumentation such as triple quadrupole (QQQ), quadrupole-trap (QQT) and quadrupole-time-of-flight (Q-TOF) mass spectrometers.
Such instrumentation allows Analytical Chemists to determine contaminants in environmental and wastewater samples that would have been difficult to detect before. These advancements also enable Analytical Chemists to meet new legislative requirements, to analyse emerging compounds and identify unknowns in samples.
Substances of concern
The environmental analysis industry in the UK is heavily influenced by European Community/UK legislative developments. Laboratories performing environmental and wastewater analysis need to be aware of the changes in a wide range of sectors including pollution control and prevention, remediation and sustainable development, in order to meet the requirements of customers and other stakeholders.
In the UK, water companies are responsible for the safe discharge of treated effluent into the aquatic environment and need to ensure that such discharges meet statutory requirements in terms of the quality of their discharge and ensuring the prevention of adverse environmental impacts from discharge to receiving waters, which invariably includes protecting the environment and wildlife from damage and the safeguarding of human health.
The EU has some of the highest environment standards in the world, which have been developed over a number of decades, which aim to address a wide range of issues, including protecting the environment from harmful substances and reducing health problems caused by pollution. A range of substances that have high acute toxicity, are carcinogenic or persistent in the environment have been regulated by the EC. A list of substances considered particularly harmful to the environment has been classified by the EC as Priority Substances or as Priority Hazardous Substances, which are considered as extremely harmful (Table 1).
Compliance with priority substance standards will be used by regulators to define ‘good chemical status’ for the Water Framework Directive (WFD). The European Commission will undertake periodic reviews of this list of priority substances and update the requirements for monitoring and compliance on a regular basis.
The Water Framework Directive
The Water Framework Directive 2000/60/EC is a European Union directive focusing on ecology and the protection of the aquatic environment. It is the single most important piece of water legislation in the EU.
The Water Framework Directive (WFD) requires that EU surface water bodies, including rivers, certain lakes, estuaries and coastal waters achieve good status by 2015. The WFD looks at the ecological health of surface water bodies as well as defining traditional chemical standards. The WFD sets out a number of objectives required to prevent deterioration of the status of surface water and groundwater bodies. The status of a water body is judged using separate ‘ecological and chemical classification’ systems. To achieve ‘good status’ overall, a water body must achieve both good ecological and good chemical status.
The chemical classification system for surface waters, used for the most polluting substances, has only two classes (good or failing to achieve good), which are assessed according to whether water meets Environmental Quality Standards (EQS) for substances listed in the Dangerous Substances Directive and associated daughter directives, and in the WFD Priority List Substances. The directive will help deal with diffuse pollution as well as point source discharges and is designed to:
• Protect and enhance the status and prevent further deterioration of ecosystems and wetlands which depend on good water quality • Promote the sustainable use of water to protect water resources • Protect and improve the water environments through the reduction and followed by the cessation of the discharge of ‘priority’ substances • Reduce groundwater pollution
The implementation of the WFD depends on the availability of analytical methods for the priority substances specified in the directive. The WFD requires the monitoring of water quality to be performed by methods that conform to CEN/ISO quality standards to ensure data generated within member state countries is of comparable and equivalent scientific quality throughout the EU.
Emerging Pollutants and Endocrine Disrupting Compounds
Advances in Environmental Analytical Chemistry have allowed Analytical Chemists to detect a wide range of contaminants in environmental and wastewaters. Many of these substances are of anthropogenic origin, originating from chemicals used in industrial process and from consumer use e.g. surfactants, drugs and pharmaceuticals and compounds excreted by the human body e.g. hormones.
Contaminants that have recently been discovered in the environment include substances such as pharmaceuticals and personal care products, endocrine disrupting compounds and metabolites of pesticides and other persistent organic pollutants. Although historically compounds that were toxic, persistent or bioaccumulated in tissue were of particular interest, emerging compounds including pharmaceuticals and personal care products are of increasing concern because they can have adverse biological affects on wildlife due to their biological activity, they are continually introduced into the environment, so even if they degrade more are being continually added.
The presence of trace quantities of endocrine disrupting chemicals (EDC) in the aquatic environment has become an issue of considerable concern. EDCs encompass a large range of compounds, many of which have been detected in wastewater and in the aquatic environment.
The list of suspected endocrine disrupters includes: alkylphenol ethoxylates, alkylphenols, alkylphenol carboxylates and other degradation products as well as a wide range of other organic, inorganic, and organometallic substances. EDCs can be divided into two categories: natural compounds e.g. hormones found in the bodies of humans and animals; phytoestrogen, which are substances found in some plants and man-made or industrial substances, which include a wide range of compounds – for example compounds that have been manufactured specifically to have endocrine effects e.g. ethynyloestradiol used in the oral contraceptive pill.
Some endocrine disrupting compounds found in the environment are breakdown products of other manufactured chemicals. Endocrine disrupting chemicals can alter or disrupt natural hormone function of wildlife. There is considerable evidence that aquatic organisms downstream of some sewage treatment works show endocrine disruption as a result of exposure to substances in the effluent.
Research has indicated that the natural oestrogens, oestrone (E1) and 17-β-oestradiol (E2) and the synthetic oestrogen ethynyloestradiol (EE2) are responsible for most of the oestrogenic activity found in sewage effluents. The Environment Agency of England and Wales has proposed predicted no effect concentrations (PNEC) values for all three free steroids expressed as annual averages of 0.1 ng/L (EE2), 1 ng/L (E2) and 3 ng/L (E1). Therefore in order to test for compliance to these targets, very sensitive analytical methods are needed.
Typically, wastewater samples require extraction, concentration, cleanup and analysis using advanced analytical instrumentation. Samples are usually derivatised to produce non-polar derivatives prior to GCMSMS analysis but when utilising LCMS the free steroids can be analysed directly after sample extraction and cleanup without the need for derivatisation. Various LCMS techniques have been successfully applied to the analysis of steroid oestrogens including LC-QQQ, LCTOF and LC-Orbitrap MS.
Polybrominated diphenyl ethers
Polybrominated diphenyl ethers (PBDE) are used as flame retardants in foam padding, plastics, fabrics, computer plastics, upholstered furniture, textiles, televisions and other products. PBDEs have been found to be persistent in the environment; they are bioaccumulative and have endocrine disrupting properties, particularly lower brominated molecules (Br 1-5), which can affect hormone levels in the thyroid gland. The commercial mixture of pentabromodiphenyl ether, predominantly contains the penta-BDE congeners (50-62%), however the mixture also contains tetra-BDEs (24-38%) and hexa-BDEs (4-8%), as well as traces of the tri-BDE (0-1%).
Two of the three commercial PBDE products have recently been banned in the EU and parts of the USA but nevertheless they are ubiquitous in the environment. The EU Environmental Quality Standards for the sum of all PBDEs is 0.5 ng/L. For reliable measurement of PBDEs at EQS levels, sensitive analytical methods are required which have a limit of quantification (LOQ) equal to or lower than 30% of EQS.
PBDEs in wastewater and environmental samples are analysed following sample extraction and concentration and can be determined by GCMS or GCMS/MS using electron ionisation or chemical ionisation. GCMS/MS provides greater selectivity and sensitivity compared with GCMS. Gas chromatography combined with inductively coupled plasma mass spectrometry (ICP-MS) has also been successfully applied to the analysis of PBDEs. ICP-MS possesses several advantages:
• High sensitivity to PBDE congeners • Absolute selectivity between halogens, suffering no interference from fluorine, chlorine or iodine containing compounds e.g. PCBs • No suppression of response from co-eluting compounds • Compound Independent Calibration (CIC), useful for the quantification of congeners for which standards are expensive or unavailable
Organotin compounds are man made compounds containing tin. The most used organotin compounds to date have been Tributyltin (TBT) compounds which are substances containing the tributyltin moiety in their chemical structure. TBT uses include wood preservation, as antifouling pesticides in marine paints (to protect from algal and barnacle growth), antifungal compounds in textiles and industrial water systems, such as cooling tower and refrigeration water systems. Tributyltin oxide is the most widely used compound in TBT–containing commercial products.
TBT compounds can be toxic to wildlife, especially fish, molluscs and other aquatic organisms. TBT compounds are released into the environment from their use as marine antifouling paints on ships and from their use in wood preservatives. TBT is included in the UK Surface Waters (Dangerous Substances) (Classification) Regulations and the European Union Water Framework Directive (WFD) Priority list substances.
Organotin compounds can have adverse effects on marine organisms even at sub parts-per-trillion (ng/L) levels, which is reflected in the very low environmental quality standard for TBT and its compounds of 0.2 ng/L. These low limits are a challenge even for the most sensitive analytical techniques such as GCMS or GC-ICPMS, therefore an extraction and pre-concentration step is invariably involved in their analysis.
Organotin compounds with less than four alkyl groups e.g. TBT compounds are also too polar to analyse directly by GC and must be derivatised to form non-polar alkyltin compounds prior to analysis. In the past, most methods were based on extraction with troplone (a complexing agent) and n-hexane followed by derivatisation using Grignards reagent. Most standard methods in current use utilise in-situ derivatisation of samples using tetraethylborate to produce ethyl organotin derivatives. The ethylated derivatives are extracted into hexane, the extract is concentrated and analysed. Using GCMS/MS and GC-ICP-MS limits of detection lower than 0.2 ng/L TBT can be achieved.
Perfluorinated organic compounds
Fluorinated organic compounds, such as perfluorooctane sulfonate (PFOS) and related compounds including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonamide (PFOSA) belong to a class of compounds known as perfluoroalkyl substances (PFAS). The term PFOS-related substances refers to any substance which contains the PFOS moiety (C8F17SO2).
PFOS and related substances are contained in cleaning products, fire fighting foams, carpets, textiles, paper and packaging, coating additives, leather and photographic materials. These substances have been shown to be persistent and bioaccumulative in the environment. They are highly toxic to certain species of wildlife e.g. honey bees. With regard to human health, the OECD has concluded that there is a significant association between exposure to PFOS and bladder cancer and increased risks of neoplasams of the male reproductive system and gastrointestestinal tract.
Because of these concerns, 3M, the main manufacturer of PFOS voluntarily phased out manufacture of the substance in 2001. However, some companies still produce PFOS and it is also used in some industrial processes. Hazard assessments carried out by several OECD countries have concluded that PFOS and related substances are a concern in the environment and for human health.
In the UK, the Environment Agency of England and Wales has undertaken a risk assessment and have given a proposed no effect concentration for freshwater of 25 µg/L. Analysis of PFOS and related compounds can be undertaken by extraction and concentration of water samples using solid phase extraction cartridges and analysis using LCMS/MS using negative ion electrospray in the multiple reaction monitoring (MRM) mode, allowing limits of detection in the low ng/L to be achieved.
Heavy metals A number of heavy metals are included in various environmental regulations in the UK, such as the elements lead, copper, nickel, cadmium, platinum, zinc and mercury. Unlike many organic pollutants, which over time eventually degrade to carbon dioxide and water, heavy metals will accumulate in the environment as elements. Regulation of metals is due to concerns regarding bioaccumulation and toxicity as well as other factors, although the scientific basis for regulation of heavy metals is widely debated, especially with respect to bioavailability.
Currently used EQSs for freshwaters in the UK are based on acute and chronic laboratory toxicity data. In some countries, a single concentration, usually of dissolved metal, is used. In other countries, including England and Wales, the beneficial (protective) effect of water hardness is taken into account, so that harder waters have higher EQSs. The concentration of metals in bioavailable form are not necessarily proportional to the total concentration of the metal.
Laboratory analysis of heavy metals in water samples is performed after dissolution of samples via the action of microwaves in the presence of nitric acid. The digestion ensures that any suspended or colloidal forms are converted to soluble forms. Filtered (or dissolved or soluble) metals may be determined, by filtration of samples through a 0.45µm membrane filter prior to analysis.
Analysis can be undertaken using a number of techniques including Atomic Absorption (AA) spectroscopy, Inductively Coupled Plasma (ICP) – Atomic Emission Spectrometry (AES) or Mass Spectrometry (MS).
ICP-MS first introduced into laboratories in the 1980s has become the technique of choice for the determination of low-level metals. Detection limits using ICP-MS are equal to or better than those attainable using graphite furnace atomic spectroscopy. However, ICP-MS instruments equipped with nominal resolution quadrupole mass analysers are influenced by a number of argon-containing polyatomic species, which can interfere with the detection of elements, particularly in the mass range m/z 40-80.
A collision cell operating as a ‘Kinetic Energy Discriminator’ can be used to reduce the background and remove interferences, but this often also results in a reduction of instrument sensitivity. Alternatively, interferences can be removed using a dynamic reaction cell (DRC) which uses reaction gases such as ammonia, methane, hydrogen, and helium, which are selected based upon their ability to undergo a gas phase chemical reaction with polyatomic or other interfering species. ICP-DRC-MS instruments allow limits of detection in the ppt range to be achieved in wastewater samples.
The introduction of stringent environmental quality standards and the requirement to determine compounds of emerging concern in environmental and wastewater samples at ultra low concentrations, requiring limits of detection in the low parts per billion and, for some biologically active substances, at low parts per trillion (or lower) concentrations, provide significant analytical challenges.
Recent advances and availability of advanced analytical instrumentation allow Analytical Chemists to detect a vast range of substances in environmental and wastewater samples. Many of the advances are related to the introduction of high resolution chromatography instrumentation, coupled to high resolution or tandem mass spectrometry instrumentation into environmental testing laboratories which, when combined with innovative sample preparation and analysis techniques, allows Analytical Chemists to analyse and report substances at lower concentrations than ever done before. In addition, new substances including metabolites, wastewater degradation products and disinfection by-products are continually being identified and reported.
Dr Rakesh Kanda, Principal Research Scientist, Severn Trent Services, Analytical Services, Britten Road, Reading, Berkshire, RG2 OAU
Email: [email protected]
Published: 10th Dec 2010 in AWE International