Publication Abstract

Project ME4117 – Development and Improvement of Analytical Methods for Marine Monitoring. 1.1 – Refinement of the methods of extraction and analysis for the determination of polycyclic aromatic hydrocarbons in marine sediments and biota

Project ME4117 – Development and Improvement of Analytical Methods for Marine Monitoring. 1.1 – Refinement of the methods of extraction and analysis for the determination of polycyclic aromatic hydrocarbons in marine sediments and biota

H. S. Rumney, K. Potter, A. Papachlimitzou, P. K. Mellor, A. Sypniewska-Huk

Polycyclic aromatic hydrocarbons (PAH) are important and ubiquitous contaminants in UK waters. They are present in fossil fuels (particularly crude oil and its refined products) and are also formed by the incomplete combustion of organic material. PAHs enter the marine environment from domestic sewage, industrial effluents, atmospheric transport following emissions to air and following the spillage or disposal of oil and petroleum products. As there are many sources of PAHs and they are readily absorbed onto sedimentary particles, this has lead to high concentrations being recorded in marine sediments, particularly in historically industrialized estuaries.   Hydrocarbon analyses are carried out at the Cefas Lowestoft Laboratory for three main reasons.

• To investigate gradients of concentration around point sources (oil spills, oil production platform discharges, etc.);
• To investigate the distribution of these contaminants over a wider geographical area (baseline/benchmark studies, particularly within the UK Clean Seas Environment Monitoring Programme);
• In support of laboratory-based experiments. PAHs can exert both acute and chronic toxic effects on marine biota. Generally it is the low molecular weight PAH (e.g. 2- and 3-ring compounds) which exert acute toxicity, and some of the higher molecular weight PAH (with 5-rings or more), which have carcinogenic potential and so can exert chronic toxicity.

The main objective of this project is to develop and validate new analytical techniques for the analysis of existing priority analytes, thereby reducing the analytical effort required, or offering improved method performance, spatial or temporal resolution. To achieve this, a review of contemporary literature was initially undertaken to assess the latest developments in PAHs analysis. Focus was placed on published literature, reporting , methodological steps in which improvements had been identified; these included sample extraction, extract concentration and clean up, and instrumental (GC-MS) analysis. Subsamples of sediment and biota, depending on availability time, were to be analysed to compare the suitability of a pressurised liquid extraction (PLE) technique and compared to the current alkaline saponification extraction technique. This was based upon the fact that PLE allows the sample batch to be extracted with more automation, thus saving operator time. For the concentration of sample extracts, it was decided to compare the current method of rotary evaporation with a more automated system involving the Horizon DryVap^TM . This has the capability to evaporate six samples at a time with little operator intervention. As the extraction techniques to be employed will not use an in-line clean up procedure, it will still be necessary to remove any co-extracted interferences, elemental sulphur residual lipids and pigments from crude extracts. Our current clean up method is already efficient, but we have decided to investigate the use of commercially available, solid phase extraction cartridges containing neutral alumina to compare with our current deactivated alumina glass column chromatography approach. The advantages here include savings in staff time and costs involved with column preparation and a reduction in health and safety hazards associated with the preparation of the columns.   As GC-MS is the most commonly employed technique for the analysis of volatile, organic contaminants and is one that Cefas currently applies, it was decided to investigate ways to improve analysis by using a more selective GC column to improve analyte separation. In addition, the application of GC with triple quadrupole MS (i.e., MS/MS) detection was explored with the aim of improving analyte selectivity and detection sensitivity.   The programme of method development, refinement and validation reported herein has highlighted the complexities of analysing marine matrices for PAHs. Optimised sample preparation techniques need to consider the properties of the particular sample matrix under investigation. These important properties include the nature of the sediment or biota sample, their lipid and water content and, for analysis by gas chromatography with mass spectrometric detection, the volatility of the analytes. In terms of extraction, the automated SpeedExtractor™ provided efficiency (consumables and operator time) gains for processing dry sediments and, once optimised, it was capable of handling wet materials, which are more typically the samples that Cefas is required to analyse.   Sequential extraction using a non-polar solvent (pentane) followed by a more polar solvent (dichloromethane) produced an optimised extraction procedure. However, there was still evidence of low recoveries of both low and high molecular weight PAHs. The elimination of co-extracted water from extracts using various drying agents also highlighted their impact on the more volatile PAHs. Until further investigations have been undertaken to resolve this problem, Cefas will continue to use their current method of extraction involving alkaline saponification and liquid/liquid solvent extraction followed by the use of anhydrous sodium sulphate for removing the water.   Application of the automated DryVap™ extract concentrator demonstrated significant efficiency gains in this aspect of extract preparation. The automated approach compared well, in terms of performance, with our conventional method of rotary evaporation, whilst concentrating three times as many samples in the same time period. Also, more control is available to the analyst performing this task, since solvent sensors prevent complete evaporation taking place, thus minimising the potential loss of volatile analytes. However, the recovery of the surrogate standard chrysene d-12 was still only approximately 50%. Since it was apparent that the DryVap process was suitable for extracts from both dry and wet sediments and from biota (mussel) tissue, Cefas intends to adopt this procedure into their routine standard operating procedures of sample preparation.   Investigations of the application of neutral alumina, solid phase extraction (Bond Elut Al-N; SPE) for extract clean up demonstrated no adverse effect on the quality of the PAH concentration data compared to our current clean up method involving glass column chromatography and deactivated alumina. Where an increase in cost in terms of acquiring commercially available SPE cartridges is incurred, this is off-set by the efficiency gains of reduced staff time and reduced solvent consumption. On this basis, as well as the promising performance of SPE, Cefas intends to integrate this approach into their routine, standard operating procedures.   Regarding gas chromatographic (GC) separation of target PAHs, the application of a PAH specific GC column (PAH Select™) has clearly highlighted the gains which can be made in sample turnaround times. The quality of chromatography has been proved and the separation of problematic partially and/or co-eluting PAHs has also been shown. This has reduced the uncertainty associated with our conventional GC method and so improved the accuracy of PAH determinations. However, further GC method refinements are required to improve resolution and responses of some of the earlier-eluting as well as later-eluting compounds. Once these are complete, Cefas intends to utilise this column for the separation of target PAHs in routine marine sample analysis.  

With the recent investment made by Cefas in a triple quadrupole (MS/MS) mass spectrometer for the detection of PAHs, improvements in terms of their unambiguous identification were immediately recognised. The limited work conducted on the newly acquired instrument showed its successful application to PAHs extracted from sediments. Further investigations and validation exercises are required before GC-MS/MS can be applied to marine biota. Again, it is Cefas intention to introduce this tool of detection and quantitation into their routine monitoring and surveillance programmes, as well as apply it in the future, to new areas of PAH research.

H. S. Rumney, K. Potter, A. Papachlimitzou, P. K. Mellor, A. Sypniewska-Huk

Cefas Technical Report