North East File Collection - NEFC

• Pyridine was implicated as a cause in the mass mortalities events (MMEs) that occurred during the autumn 2021 in the NE coast of England involving mainly crabs and lobsters. This was inferred from high pyridine levels reported in some crabs from the affected area, following analysis by the Environment Agency (EA).
• The analytical screening method used by the EA (although accredited for water samples), was neither quantitative nor validated for biota (or for sediment) samples but was used within this incident to identify lines for potential follow up.
• Due to continuing external concerns over pyridine, Defra commissioned this work at Cefas to develop and validate a robust quantitative method for pyridine in these environmental matrices.
• This method was used to re-analyse stored samples collected during the MMEs.
• Cefas analysts developed and validated a method, using a head space injection gas chromatography coupled to a mass spectrometer, HS-GC-MS technique. The limit of detection (LOD) and the limit of quantification (LOQ) of the method was 0.006mg/kg and 0.02mg/kg wet weight (ww) for the shellfish matrix and 0.002mg/kg and 0.008mg/kg ww for the sediment matrix, respectively.
• The analytical method developed in this study, demonstrated fit-for-purpose performance criteria for biota matrices, including a recovery range of 89-101% and an associated within batch coefficient of variation (relative standard deviation) of 2-3% across three concentration levels (5, 25 and 500mg/kg).
• Reanalysis of crustacean samples that had originally returned high indicative pyridine levels with the EA method (3-429mg/kg) demonstrated very low concentrations of the chemical (<0.22-0.077mg/kg, over 3 orders of magnitude lower). Analysis of additional crab samples, unrelated to the events, also demonstrated the presence of pyridine at very low levels (<0.02-0.139mg/kg).
• A single mussel sample returned a value of 2.36mg/kg.
• Pyridine levels in sediments collected in November 2021 all returned values between the LOD and LOQ. Three sediment samples collected in January 2022 returned values above the LOQ (0.014, 0.008, 0.009mg/kg ww), although below 2x LOQ.
• Both sediment and biota samples returned levels of pyridine within an expected range based on the low environmental persistence, and high biodegradation rate of the chemical.
• It is therefore considered very unlikely that pyridine, as a single chemical entity, was the cause of the crab and lobster mortalities during autumn 2021.

In the autumn of 2021, a mass mortality event (MME) concerning primarily, marine decapods (crabs and lobsters), occurred along the NE coast of the UK. As first responders for marine mortality incidents, the Environment Agency (EA), launched a major pollution investigation, which involved, amongst other things, collecting samples (water, sediment, and biological material (referred to hereafter as biota) to analyse for chemical pollutants to determine possible causes. Screening for several hundreds of contaminants1 did not reveal the presence or levels of toxic substances(s) which could definitively explain the event. Pyridine was detected in tissues of some of the affected crabs using the screening qualitative method of analysis which initiated further investigation. The United Kingdom Accreditation Service (UKAS) accredited ISO/IEC 17025 GCMS screening method for chemicals in waters 2. The EA method include pyridine in the target database but is not validated (or intended for routine use) in either biota or sediment matrices. Application of this method to the tissues of some affected and unaffected crab tissues indicated the presence of unexpectedly high levels of pyridine. It was acknowledged from the outset that the method could only provide a qualitative data. The reasons for this were (1) the sample matrix, in this case water compared to biota, can have a substantial effect upon the reliability of results in non-target matrices, (2) the analytical approach, qualitative versus fully quantitative, was not designed to determine precise numerical values. Thus, the accuracy of pyridine levels indicated by these exploratory screening tests was highly uncertain. Confirmation was required to verify the potential significance of these findings in the context of the MME.

In December 2021 Defra commissioned the Centre for Environment, Fisheries and Aquaculture Science (Cefas) to develop and validate a fully quantitative method for pyridine in biota and sediments. This report describes the development, validation, and performance characteristics of the method, and presents quantitative analyses of archived samples, collected, and analysed originally during the initial investigation.

1 Evidence gathered under the Defra-group investigation into Crustacean Mortality of Autumn 2021 - data.gov.uk

2 0754Testing Multiple (ukas.com)

There are several methods that can be used for the analysis of pyridine in environmental samples (including biota and sediments) (IARC 2000). Following internal literature review, method development in this study focused upon analysis using headspace injection gas chromatography (HS-GC) coupled to a mass spectrometer (MS) used in tandem mode (MS/MS). The choice of HS-GC-MS over the more traditional injection techniques that use gas chromatography (GC), was informed by the need to minimise loss of pyridine during initial sample processing. Standard GC methods involve injection of a liquid sample or sample extract onto the front end of a GC column, whilst the head space technique injects part of the sample that has been volatilized whilst contained within a sealed vial. This means that volatile chemicals can be “extracted” from the sample directly without the need for additional steps (such as Soxhlet and further clean-up of the extracts). HS-GC-MS approaches therefore bypass the risk of loss of target chemicals during sample preparation steps and reduces interferences.

The instrument used for sample injection was a Shimadzu HS-20 headspace system. Separation of pyridine was performed with a Shimadzu GC-2010Plus (Shimadzu, Japan) using a Rxi-5Sil MS column 60m x 0.25mm id x 1.0µm (Restek, Bellefonte, PA, USA). This very long column with relatively thick stationary phase was needed to separate pyridine from the solvent front and from the deuterated form. The analyser was a Shimadzu TQ8050 triple quadrupole MS used in electron ionization (EI) mode. Instrumental parameters are summarized in Table 1.

Identity of native pyridine (not labelled) and deuterated pyridine (d5 labelled pyridine) were confirmed by their spectrum, obtained running the MS in full scan (Figure S1, Annex I). To improve sensitivity and selectivity, quantification of pyridine was carried out in multiplereaction monitoring mode (MRM) using the parameters summarized in Table 2.

Table 1: Instrumental parameters used for the analysis of pyridine by HS-GC-MS.

Table 2: MS/MS parameters (transitions and collision energies) used for the analysis of native pyridine and deuterated pyridine.

The stability of pyridine during laboratory storage is not known, so fresh standards for native pyridine (stock standards) were prepared weekly to improve accuracy of both reference and sample quantitation. To account and correct for any losses of pyridine during the sample preparation and analysis, d5-pyridine was added to samples, blanks, and standards as an internal standard at the beginning of the process and was used as an injection standard for quantification of the native pyridine, thereafter, referred to as pyridine. The d5 standard was prepared <14 days before use (since providing the standards, samples, blanks, and reference materials were run in the same batch and were spiked simultaneously, the d5 standard concentration is not a critical quantification parameter).

The volatility of chemicals in head space usually varies depending on the composition/type of matrix (interfering or enhancing the signal of the pyridine). To account for this expected variation, the calibration curves used in this study were carried out using a matrix matched calibration approach. In brief, both clean biota (lobster, Homarus gammarus, shellfish matrix) collected from the Isle of Man in 2022) and sediment (previously collected, characterised as clean and stored sediment from Shoebury East beach, Essex, (sediment matrix)) were utilised as blank matrixes. These blank samples were spiked at different levels to create both the calibration curves (9 levels) and the in-house reference materials (3 levels). During samples analysis, only the lowest 5 points of the calibration curve were used for quantification due to all samples registering pyridine levels close to the bottom of the calibration range.

Note: The number of samples from the mortality event required that analyses extended over several days. To ensure consistency of test results, samples were divided in batches. For each batch samples, calibration standards and reference materials were all prepared together (same day) and they were all analysed in the instrument together.

Validation was carried out to evaluate the performance characteristics of the quantitative pyridine method in biota (using lobster as shellfish matrix). Whilst previous analyses undertaken at the EA were considered only a qualitative screen, pyridine values from crustacean tissues were recorded within the range 3mg/kg (low) to 439mg/kg (high). Therefore, it was considered important to ensure that the quantitative method was reliable over an equivalent linear concentration range, and that validation was carried out over that range. Consequently, the blank matrix (lobster muscle tissue) was spiked at 3 concentration levels: 5mg/kg (low), 25mg/kg (medium) and 500mg/kg (high). Six replicates were used at each concentration level. Results for the validation are given in Table 3. Recoveries for all levels ranged between 89% and 101% and the associated within batch Relative Standard Deviation (RSD) between 2% and 3%. Consequently, performance characteristics were well within the accepted range of target values as per Cefas analytical quality requirements (Annex II, Table S1). Method standardised uncertainty was 8.11% and the expanded uncertainty 16%, both calculated following the equations in Annex II. These criteria were based on those of Eurachem (2014).

These results confirmed the suitability of the method used for the quantitation of pyridine in biota samples.

Table 3: Concentrations and method performance characteristics for the validation tests using lobster as blank shellfish matrix spiked with native pyridine at 5, 25 and 500mg/kg (wet weight).

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