Remain up-to-date with changing legislation and technological advances. Recent technological advances within analytical instrumentation have meant significant enhancements can now be made in the methods of analysis within the Environmental testing market.
For example, the introduction of Rapid resolution liquid chromatography instrumentation and improvements within the hyponated Mass spectrometry area have led to a generation of instruments offering high throughput and robust detection within environmental samples. These improvements offer possibilities of reductions in limits of detection for parameters of interest.
With the introduction of technological advances, increasing demands for new parameters and ever more tighter Environmental Quality Standards, laboratory providers are reviewing their service to identify areas for development. Over the last 12 months, STL have reviewed a number of key analytical areas with the aim of incorporating new technologies available within the analytical sector to deliver a range of benefits:
- Reducing (where required) Limits of detection
- Increased robustness of methodologies
- Meeting legislative requirements
- Increased throughput
- Reduced sample volume and solvent usage
Potable water analysis
Monitoring of potable water for organic compounds such as pesticides, herbicides, fungicides and insecticides has been well established within the UK water industry for many years. This has led to customers having suites of organic compounds that are required for testing within the laboratories.
The laboratory approach to these testing requirements has been to group the compounds into classes such as organochlorine pesticides or organophosphorous pesticides. These groups are assessed for their chemical and physical nature and then suitable extraction and instrumentation techniques applied for analysis. This has traditionally led to lengthy extraction procedures with high solvent usage and multiple instrumental analyses.
With the advances in analytical detection techniques it has now been possible to re-visit the requirements of customers and rationalise groups of pesticides, fungicides, herbicides and insecticides into hydrophilic and hydrophobic compounds.
Hydrophilic compounds, which have a strong affinity to water, have traditionally been more difficult to analyse than hydrophobic compounds due to their higher aqueous solubility. This has meant liquid chromatographic techniques have often been the analytical technique of choice. However due to the previous limitations of this technique it has often been preferred that an involved extraction procedure be implemented followed by gas chromatographic detection.
With the recent introduction of rapid resolution liquid chromatography and triple quadruple mass spectrometric detection these limitations have significantly reduced and it is now possible to successfully analyse compounds including suburea and triazine herbicides, organophosphorous and organonitrogen pesticides within rapid analytical suites and meet all regulatory quality requirements.
The sample preparation stage can also be minimised by the use of new technologies, reducing time between sampling to analysis at the laboratory and significantly reducing solvent usage and preparation times. It also minimises possible losses of compounds of interest in an extraction phase and hence improves precision of the technique.
The detection system works on the principle of initial selection of a precursor ion from the original mass spectrum (Q1). This then passes through a secondary collision cell, which further breaks this ion down into product ions of which those representative of the compound will pass through the third quadrupole (Q3) for detection and quantitation purposes.
This technique leads to a high degree of selectivity, which reduces background noise hence increasing sensitivity and reducing effects from varying sample matrices.
STL have successfully been able to apply this technique within several water types and meet the standards as required for potable analysis within England and Wales.
The gains that this technique has given in the potable water analysis area are as summarised below:
- Reduction in sample volumes required for analysis, of benefit when less sample is available
- Increased speed of analysis from point of sampling, leading to less chance of deterioration of the sample
- Increased robustness of analytical data obtained, and therefore higher quality of results
- Increased selectivity within sample types and more accurate analysis
- Increased sample throughput and therefore greater lab capacity and the potential for reduced turnaround times
With changes within the environment and its use, new challenges for potable water analysis continue to occur. The compounds identified as potential pollutants are often amenable to the novel technology approach.
Figure 1: Structure of a) PFOS/PFOSA and b) PFOA
Perfluorooctane sulfonate (PFOS) and related 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). With the continued use of such materials, it is expected that the anion (Figure 1) will be present in the environment, probably associated with metal cations for some time.
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, the main manufacturer of PFOS voluntarily phased out manufacture of the substance in 2001. However, some companies still produce it 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.
Analytical methods are therefore required to determine PFOS and related compounds in surface waters and also in potable waters due to concerns for wildlife and potential effects on human health.
Analysis of PFOS and related compounds have been reported in human maternal and cord blood samples by extraction using solid phase extraction cartridges and analysis using liquid-chromatography mass spectrometry (LCMS) with electrospray ionisation (Inoue et. al 2004) and in seawater using LCMS/MS (Yamashita et. al 2004).
STL have developed a sensitive analytical method to determine PFOS and PFOA in raw and potable waters. Samples are analysed using solid phase extraction after the addition of internal standards e.g. 13C4-PFOS and 13C4-PFOA. Analysis of the extract is carried out using Liquid chromatography tandem mass spectrometry (LCMS/MS) using negative ion electrospray in the multiple reaction monitoring (MRM) mode.
Validation of the analytical procedure shows that PFOS and PFOA can be analysed in raw and treated water with limits of detection below 0.025 µg/L.
Landfill leachate and discharges to groundwaters
With the increasing diversity of sample matrix types within the area of landfill leachate and discharges from landfill to groundwater the complexity of analysis within the laboratory has become more evident especially within the area of organics analysis.
As part of the risk assessment of landfills for discharges to groundwater and subsequent setting of groundwater control and trigger levels, the Environment Agency have issued the following guidance document: “Hydrogeological Risk Assessments for Landfills”
The requirements ensure compliance with both the EC Groundwater Directive (80/68/EEC) and Landfill Directive (99/31/EC).
Within this document Appendix 7 sets out typical minimum reporting values for selected List1 substances in clean groundwater. These values will probably be used in assessing whether a predicted discharge of the List1 substance to groundwater is discernible, and consequently whether the discharge complies with the Groundwater Directive. Below is a selection of pesticides included in this table:
|Endosulphan||0.005||Endosulphan a and b, each to 0.005µg/l|
These minimum reporting values are extremely challenging using conventional laboratory techniques. With the added complexity that sample matrices are varied, the introduction of novel technology has now been implemented at STL.
The challenges in lowering limits of detection whilst maintaining selectivity within the sample matrix, meant that direct aqueous injection onto liquid chromatographic systems was not a feasible option at this stage. However, advances within the gas chromatographic mass spectrometric techniques and their affordability within the environmental sector has enabled STL to successfully introduce improved detection to analyse a wide variety of pesticides, insecticides herbicides and organo-nitrogen compounds within the varied leachate, effluent and groundwater sample matrix type.
To ensure effective sample introduction onto the chromatographic system a two-stage extraction process has been instigated. This enables effective simultaneous extraction of both hydrophilic and hydrophobic compounds. Detection of a large number of compounds is made possible by utilising multiple reaction monitoring (MRM) where several product ions can be monitored simultaneously.
Below are examples of two of the compounds showing the detection at their low concentration range and linearity across the range of analysis.
The above technique has enabled the laboratory to improve limits of detection whilst increasing the scope of organic compounds available. The methodology is robust and can accommodate a variety of different sample matrix types.
With the introduction of raw surface water monitoring in line with the Water Framework Directive during 2008, it is anticipated that this technique will encompass many of the requirements for analysis of identified priority hazardous substances within this directive and translations into English law.
Rapid detection of Legionella by Polymerase Chain Reaction (PCR)
Legionella are commonly occurring bacteria, found in both natural and artificial water systems. In favourable conditions, the bacteria are able to rapidly multiply, potentially reaching hazardous levels. Favourable conditions for Legionella bacterial growth are water temperatures of 20-45°C and the presence of nutrients in the form of sediment, scale, sludge and biofilms, which often occur in man-made water systems.
Legionella are fastidious, strictly aerobic, non-spore forming gram- negative bacilli. The organisms are typically 0.3-0.7 µm wide and 2-3 µm long. They do not typically grow on conventional media and require specific media and growth promoters for isolation. The media of choice is GVPC – BCYE that promotes the selective isolation of Legionella and suppresses non-target micro-organisms. Legionella require cysteine for growth however there are exceptions e.g. Legionella oakridgensis.
Legionella was first discovered in 1976 following the outbreak in the Bellevue Stratford Hotel in Philadelphia where an American Legionnaires convention had taken place. In this outbreak 182 veterans developed an acute respiratory illness, which resulted in 29 deaths. Following an investigation by the Centres for Disease Control in Atlanta, Legionella pneumophila was isolated in 1977.
Retrospective analysis has shown isolation as far back as 1947. A number of serotypes have since been identified with up to 30 different species. It has been documented that the mode of infection is that via inhalation of contaminated droplets of water (aerosols) containing Legionella directly into the lungs of susceptible individuals and there has been some literature suggesting a link with drinking or ingestion of contaminated water.
Two types of illness have been documented Legionnaires disease and Pontiac fever. Legionnaires disease is the more serious illness and results in a fatality rate of approximately 12%. Legionella pneumophila serogroup 1 is predominantly the organism implicated with Legionnaires disease. However, infection has also been documented with non Legionella pneumophila bacteria, these have been due to Legionella species particularly Legionella micdadei, Legionella bozemanii, Legionella dumoffii, Legionella oakridgensis, Legionella longbeachae, Legionella anisa, Legionella feeleii and Legionella wadsworthii.
Employers and premise owners are required to monitor water systems for the presence of Legionella bacteria. Requirements for statutory checks are stipulated in the HSE Approved Code of Practice and Guidance document, L8 Legionnaires’ Disease – The control of Legionella Bacteria in Water Systems, which sets the requirements for testing and details the different circumstances under which water must be sampled and analysed. It also provides comprehensive guidance on the following topics:
- Identification and assessment of the risk from Legionella bacteria
- Prevention and Control of Risk from Exposure to Legionella bacteria
- Guidance on the control of Legionella in water systems including cooling towers, cooling systems, hot and cold water systems
- Treatment and control programmes
- Monitoring for the effectiveness of treatment for chemical and microbiological parameters
STL’s dedicated Microbiology facility has recently introduced a second category of Legionella testing to support monitoring programmes. The Polymerase Chain Reaction (PCR) method may be used in conjunction with the standard methodology or as an investigational tool to monitor water treatment following a positive result using the standard method, particularly if early indication is required on the effectiveness of treatment. The results can be available in 24 hrs or within 5 hours for emergency analysis.
The method relies on the capture of the target organism via membrane filtration followed by extraction of DNA from intact bacterial/ protozoan/ algal cells. Then a PCR run is carried out of the purified DNA to amplify the target DNA to a threshold level in order for it to be detected by the instrument. The target DNA is specific to the organisms being isolated i.e. Legionella pneumophila or Legionella species. The typical genes targeted are the MIP gene and the 16S r RNA genes respectively. The PCR reaction involves a number of stages:
- • Denaturation – the double stranded DNA is separated into two single strands by heating it to a temperature of 95°C
- • Annealing – The mixture is then cooled and primers added to enable them to anneal to the target sequence on the DNA
- • When DNA polymerase is added the primers enable the DNA polymerase to synthesise the new strands of target DNA
- • The above 3 stages are then repeated 20 to 40 times to amplify the target DNA effectively doubling the target DNA during each new PCR cycle
The results are displayed graphically as real time curves on the VDU display of the instrument if a positive or negative result, giving the quantified target DNA result. TaqMan technology is used to provide a quantitative measurement of the DNA. The TaqMan probe carries two markers at its ends a fluorophore and a quencher. The probe is stable and emits no fluorescence as long as the two remain in close proximity. During the PCR reaction, the DNA polymerase splits the probe and liberates the fluorophore allowing the fluorescent signal intensity to be measured. The PCR result will be presence, absence, inhibition or Enumeration. If Legionella are present above a certain threshold, the quantification is expressed as Genomic Units/Litre. Therefore the result is an indication of target DNA content, not an expression of colony forming units as determined by the standard method.
The ability of this method to rapidly assess contamination levels is particularly advantageous in a number of applications;
- • Response times are considerably reduced – essential when monitoring high-risk water systems, the application of effective treatment regimes or the effectiveness of remedial actions
- • Timely day-to-day monitoring of treatment performance and ‘fine- tuning’ of chemical applications
- • The method enables a rapid assessment of the cleanliness of a water system, for example, during pre-commissioning investigations or where water systems have previously failed L8 guidelines
- • Real Time results demonstrating the Legionella status of the water system at the time of sampling
It can be seen that although challenges within the Environmental testing market are ever increasing with both more varied sample matrices and more stringent environmental quality standards, laboratories are able to meet this demand by investment in the latest technologies available and development of new or improved methodologies. It can be seen that in order for laboratories to design analytical packages that meet the needs of their customers, it is more important than ever that they remain up-to-date with both changing legislation and technological advances.
Published: 10th Jan 2007 in AWE International