Elsevier

Talanta

Volume 116, 15 November 2013, Pages 251-258
Talanta

Full method validation for the determination of hexachlorobenzene and hexachlorobutadiene in fish tissue by GC–IDMS

https://doi.org/10.1016/j.talanta.2013.04.080Get rights and content

Highlights

  • A method for simultaneous determination of HCB and HCBD in fish tissue is proposed.

  • The separation and quantification are carried out by GC–IDMS.

  • Full and detailed validation of the method is presented.

  • Measurement uncertainty is deeply investigated and estimated at EQS levels.

  • The method was successfully tested on Silurus glanis from River Ebro (Spain).

Abstract

This paper summarizes the validation strategy and the results obtained for the simultaneous determination of hexachlorobenzene (HCB) and hexachlorobutadiene (HCBD) in fish tissue with a maximum of about 10% m/m fat content using a GC–IDMS technique. The method is applicable for the determination of HCB and HCBD at trace levels in different kinds of fish tissue samples in accordance with the requirements of the EU Directive 2008/105/EC establishing Environmental Quality Standard (EQS) levels for biota in aquatic ecosystems (10 ng/g for HCB and 55 ng/g for HCBD).

The method validation aimed to assess performance parameters such as linearity, limit of detection/limit of quantification (LOD/LOQ), trueness, selectivity, intermediate precision, repeatability, stability of the extracts and robustness. The validation experiments have been performed by using uncontaminated fish tissue. Trueness was evaluated by using a certified reference material (NIST SRM 1947) (where applicable) and by the standard addition method. Very good linear signal-concentration curves were obtained for both analytes over the whole range of calibration. The repeatability and the intermediate precision of the method, expressed as relative standard deviation (RSD) and calculated at the EQS level, were estimated to be below 3% both for HCB and HCBD. The limits of quantification were 3.7 ng/g for HCB and 15.7 ng/g for HCBD in the fish.

An uncertainty budget for the measurement of both HCB and HCBD in fish at about the EQS levels, applying the described method, has been established in the order of 10%.

The analytical method and its performance characteristics take into account the requirements of EU Directive 2009/90/EC regarding the establishment of minimum performance criteria for the methods of analysis to be used in the water monitoring activity of the Water Framework Directive. Finally, the validated method was successfully tested on contaminated Silurus glanis from Ebro River (Spain). The method will be used in the homogeneity, stability and interlaboratory comparison studies for the characterization of a new candidate certified reference material.

Introduction

Hexachlorobenzene (HCB) and hexachlorobutadiene (HCBD) are considered as harmful for the environment and especially for aquatic ecosystems. HCB was first used in agriculture as fungicide on several seeds of crops as well as a wood preserving agent. Furthermore, it was applied as a porosity-control agent in the manufacturing of graphite anodes, as a fluxing agent in the production of aluminum, as a peptizing agent in the manufacturing of nitroso and styrene rubber for tires, and as a chemical intermediate in dye manufacturing [1]. HCBD has been used in a wide range of industrial applications [2], for instance, as a hydraulic fluid, a heat transfer liquid, by-product in the synthesis of chlorinated hydrocarbons, used in the production of aluminum and graphite rods, as solvent for polymers as well as a pesticide and herbicide in agricultural activities.

The EU Water Framework Directive (WFD) [3] lays down a strategy against pollution of all EU waters (rivers, lakes, ground and coastal waters) and requires Member States to implement specific measures for pollution control and prevention. In this respect one of the “daughter” Directives [4] lays down the Environmental Quality Standards (EQS) for 33 priority substances and other 8 pollutants with the aim of achieving a good chemical status of surface water before 2015. As foreseen in Article 3 of this Directive, EU Member States that opt to apply EQS for sediment and biota, shall apply an EQS of 10 μg/kg for HCB and 55 μg/kg for HCBD, respectively. These EQSs are established for prey tissue (i.e. wet weight) choosing the most appropriate indicator among fish, mollusks, crustaceans and/or other biota. Although the level of these contaminants has decreased over the last decades [5], [6], both HCB and HCBD are substances of major concerns for affecting not only the environment but also human and animal health. Therefore, their contamination levels should be monitored on a regular basis.

Several studies have been carried out to determine HCB in different matrices such as sediment [7], [8], [9], [10], biota [11], [12], [13], [14], [15], [16], [17], [18], [19] and environmental waters [20], [21], [22]. In many cases, not only HCB but also HCBD was detected in sediment [8], [9], [10], biota [16], [17] and water [20], [22]. In the context of the monitoring activities of the priority substances, Article 6 of the Directive 2009/90/EC [23] prescribes that the laboratories appointed as responsible for the water monitoring shall demonstrate their competence by analysis of available reference materials.

The long-term goal of this project is to support the implementation of the Water Framework Directive through provision of a tailored certified reference material (CRM) [24]. In order to properly characterize the CRM during its development and production, a validated analytical method has to be available. This is a prerequisite for working according to ISO Guide 34 and ISO/IEC 17025 [25] which contain the general requirements for the competence of reference material producers and for the competence of testing and calibration laboratories, respectively.

This paper summarizes the method validation of a ‘fit-for-purpose’ analytical procedure for the determination of HCB and HCBD in a fish matrix according to Directive 2009/90/EC. The method is based on accelerated solvent extraction (ASE) followed by column clean-up and GC-IDMS (gas chromatography isotopic dilution mass spectrometry) separation and detection [26], [27]. Utmost care was placed on the full method validation assessing linearity, limit of detection (LOD), limit of quantification (LOQ), repeatability and reproducibility [20] but also recovery [28], [29], [30] and trueness (using a CRM) [31] as well as robustness and stability as also recommended by the European Commission [32]. In addition, estimations of the individual uncertainty contributions of each parameter as well as of the final expanded uncertainties have been performed.

Section snippets

Reagents and certified reference material used

Acetone was provided by VWR International (Fontenay-sous-bois, France), while n-hexane Emsure® grade, iso-octane Suprasolv® grade, anhydrous Na2SO4 and Florisil® for column chromatography (0.150–0.250 mm) were obtained from Merck (Darmstadt, Germany). Diatomaceous earth Celite® 545 AW – reagent grade was purchased from Supelco (Bellafonte, PA, USA).

SRM 1947 – Lake Michigan Fish Tissue was provided by the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA).

Calibration standards

Isotopically

Method validation

Before method validation, several steps of the analytical procedure have been optimized such as the extraction step using various ASE parameters, different types and amounts of solid phase adsorbent, to set the best fat/fat retainer ratio for the clean-up. In addition, several types and volumes of the elution solvents in the clean-up step were tested until the optimal conditions were found. The GC–MS parameters have also been studied in detail and optimized.

The in-house validation procedure was

Conclusion

To our knowledge this paper presents the first fully validated method that allows the simultaneous quantification of HCB and HCBD in fish tissue at EQS levels. The obtained results fulfill the requirements of Article 4 of EU Directive 2009/90/EC with regard to the uncertainty of the measurement of 50% or below estimated at the level of relevant EQS. Regarding the limit of quantification equal or below a 30% of the relevant EQS, the minimum criteria is fulfilled for HCBD, while is sufficiently

Acknowledgment

The authors would like to thank the staff of EC-JRC-IRMM, RM Unit for valuable discussions and Silvia Lacorte from IIQAB-CSIC, Spain for supplying the fish samples. Dr. M. Schantz and Dr. S. Wise from NIST are acknowledged for providing SRM 1947.

The opportunity for Ms. Burcu Binici to contribute to this publication came via a one year traineeship at EC-JRC-IRMM, funded by the European Union's IPA TR0802.09 (EMIT) project.

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    National Food Chain Safety Office, Pesticide Analytical Laboratory, Ország út 23, 2481 Velence, Hungary.

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