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Fish Product Mislabelling: Failings of Traceability in the Production Chain and Implications for Illegal, Unreported and Unregulated (IUU) Fishing

Fish Product Mislabelling: Failings of Traceability in the Production Chain and Implications for... Increasing consumer demand for seafood, combined with concern over the health of our oceans, has led to many initiatives aimed at tackling destructive fishing practices and promoting the sustainability of fisheries. An important global threat to sustainable fisheries is Illegal, Unreported and Unregulated (IUU) fishing, and there is now an increased emphasis on the use of trade measures to prevent IUU-sourced fish and fish products from entering the international market. Initiatives encompass new legislation in the European Union requiring the inclusion of species names on catch labels throughout the distribution chain. Such certification measures do not, however, guarantee accuracy of species designation. Using two DNA- based methods to compare species descriptions with molecular ID, we examined 386 samples of white fish, or products labelled as primarily containing white fish, from major UK supermarket chains. Species specific real-time PCR probes were used for cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) to provide a highly sensitive and species-specific test for the major species of white fish sold in the UK. Additionally, fish-specific primers were used to sequence the forensically validated barcoding gene, mitochondrial cytochrome oxidase I (COI). Overall levels of congruence between product label and genetic species identification were high, with 94.34% of samples correctly labelled, though a significant proportion in terms of potential volume, were mislabelled. Substitution was usually for a cheaper alternative and, in one case, extended to a tropical species. To our knowledge, this is the first published study encompassing a large-scale assessment of UK retailers, and if representative, indicates a potentially significant incidence of incorrect product designation. Citation: Helyar SJ, Lloyd HaD, de Bruyn M, Leake J, Bennett N, et al. (2014) Fish Product Mislabelling: Failings of Traceability in the Production Chain and Implications for Illegal, Unreported and Unregulated (IUU) Fishing. PLoS ONE 9(6): e98691. doi:10.1371/journal.pone.0098691 Editor: Konstantinos I. Stergiou, Aristotle University of Thessaloniki, Greece Received November 22, 2013; Accepted May 7, 2014; Published June 12, 2014 Copyright:  2014 Helyar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was jointly funded by Greenpeace and The Sunday Times. The funder Greenpeace provided support in the form of salary for author NB, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funder The Sunday Times provided support in the form of salary for author JL, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing Interests: The authors have the following interests: This study was jointly funded by Greenpeace and The Sunday Times. Co-author Jonathan Leake is employed by The Sunday Times. Co-author Niall Bennett is employed by Greenpeace. There are no patents, products in development or marketed productsto declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: sarah.helyar@matis.is ¤ Current address: Food Safety, Environment & Genetics, Matı ´s, Reykjavı ´k, Iceland of the Johannesburg Plan of Implementation to restore them to a Introduction level that can produce maximum sustainable yield by 2015 [3]. In recent years, concerns about the health of the oceans and the A major threat for the sustainable management of these effects of over-exploitation of fisheries have increased. Consumer valuable resources is Illegal, Unreported and Unregulated (IUU) demand for seafood is growing with the contribution of fish to the fishing. Current estimates suggest that globally up to 25% of average annual diet reaching a record of 18.8 kg per person per fisheries catches fall within IUU practices [4–6], identifying it as year in 2011 [1], as compared to 17.1 Kg in 2008 [2]. This is the single largest threat to achieving sustainability. Both the FAO partly due to an increase in the range of species consumed, and an [7] and the European Union [8] have placed increasing emphasis increase in aquaculture. Fish products were worth a record $217.5 on the use of trade measures to prevent IUU-sourced fish and fish billion in 2010, up over 9% from 2009, and these trends are products from entering international trade. One component of this expected to continue. The increasing demand for fish highlights increased regulation has required the inclusion of binomial species the need for the sustainable management of aquatic resources; nomenclature on catch labels throughout the distribution chain 87.3% of world fish stocks are classed as overexploited, depleted or [9]. recovering: a number which continues to increase [1], with 29.9% In addition to top down pressure for improved labelling and of stocks classed as overexploited and unlikely to meet the targets traceability of fish products, many consumers are increasingly aware of nutritional and environmental issues regarding fisheries, PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e98691 Fish Mislabelling and IUU leading to shifts in attitude regarding acceptable species, catch into numbered tubes filled with 96% ethanol. Sample details location and catch methods [10]. In parallel, due to globalization including the place and date of purchase, species designation, and of the industry, consumers are encountering an increasing number eco-labelling were entered into a database linked to photographs of the packaging. Sample identities were not disclosed until of fish species and/or an escalation in common names applied to the same species. Such drivers have led to a greater demand for completion of molecular genetic analyses, when molecular and sample IDs were cross-referenced. informative labelling, including the use of ‘eco-labelling’. Although labelling to provide additional ecological information about a product is often voluntary, the FAO recognised that it could Molecular methods contribute to improved fisheries management and convened a DNA was extracted with the E-Z 96 Tissue DNA kit (Omega- Technical Consultation in 1998, which resulted in their Guidelines biotek), then quantified with a Nanodrop 1000 (Thermo for the Eco-labelling of Fish and Fishery Products from Marine Scientific), and standardised to either 5 ng/mL or 2 ng/mL Capture Fisheries [11]. Informative labelling is particularly depending on original concentration. Real-time PCR assays were important for processed items because any recognizable external carried out on all samples on an Applied Biosystems 7700 real- morphological features are typically removed, leaving consumers time sequence detection system. The 25 mL reactions contained reliant on product labelling for content information. However, it 200 nM of each of the two species specific probes (see Table 1), has been argued that any such labelling scheme, whether 300 nM of the GAD-F and GAD-R primers (Taylor et al. 2002), voluntary or legislated, requires policing in order to prevent 9.163 ml 2X Taqman Universal PCR Master Mix (UNG+ROX misuse and fraud [12].The mislabelling of a fish product may be and passive reference) (Applied Biosystems), 15 ng of DNA, and unintentional if, for example, species that are morphologically (depending on DNA concentration) either 10.417 or 6.917 mL similar are caught together, such as in many tropical or coral reef PCR grade H O (Sigma). Reactions were run in optical 96-well fisheries [13–17]. Alternatively, mislabelling may not be acciden- reaction plates using optical adhesive covers (Applied Biosystems). tal, such as where product substitutions are from species that do Plates were analysed under real-time conditions on two dye layers not occur in the same ocean [18–20], or for lesser value species with eight ‘no template controls’ (NTCs) per 96-well plate, and 2 [21,22]. However, whether intentional or not, the outcome can be positive controls for each of the two target species. The assay was serious for management and sustainability targets. In addition to run using the default cycling conditions [25]. the direct impacts of depletion from IUU fishing, substitutions and In addition to the real-time PCR, all samples were sequenced misidentification that occur before fish are landed will inflate the for approximately 655 bp from the 59 region of the COI gene inaccuracies in catch and forecast statistics. from mitochondrial DNA using primers developed by Ward [27]. Several recent studies of mislabelling have been undertaken in Tests were run with all combinations of the four available primers, Europe [19,22,23], yielding rates of mislabelling of up to 32% but the combination of FishF1/FishR2 produced consistently good [19]. Most mislabelled products have originated from small-scale PCR products in the species tested, and was therefore used retailers and convenience food outlets (e.g. fish and chip shops) but throughout (see Table 1). PCRs were carried out in 30 mL the major supermarkets have not hitherto been thoroughly reactions containing 15 mL of 2 x PCR Mastermix (containing investigated. Supermarket chains account for 72% of the total 0.75 U of Taq polymerase (buffered at pH 8.5), 400 mM each fish retail market in the UK (excluding canned products) [24]. If dNTP, 3 mM MgCl (Promega)), 9 mL PCR grade H O (Sigma), 2 2 comparable rates of mislabelling occur in supermarket products it 15 pmol each primer, and 3.0 mL of DNA template. The PCRs consisted of a denaturation step of 2 min at 95uC followed by 35 is thereby likely to have a substantial impact on efforts to manage the respective fisheries sustainably. It is therefore necessary to cycles of 30 seconds at 94uC, 30 seconds at 54uC, and 1 min at 72uC, followed by a final extension of 10 min at 72uC and then establish to what extent mislabelling of fish products occurs in the major retailers of the fish food supply chain, which is addressed in held at 4uC. PCR products were visualized on 1.2% agarose gels. If a single clear band was produced, PCR products were sent to this study. GATC (Germany, http://www.gatc-biotech.com) for sequencing. The current study uses two DNA-based methods to identify the DNA from 48 samples was re-extracted as independent replicates species of origin for 386 samples collected from major supermarket of real-time PCR and sequencing, including all samples where chains around the UK. Species-specific real-time PCR probes [25] molecular data contradicted species designations, and an addi- for cod (Gadus morhua), and haddock (Melanogrammus aeglefinus) were tional randomly chosen 33 samples to test repeatability of DNA- used to provide a highly sensitive test for the major species of white based species ID. fish sold in British supermarkets. Additionally, DNA barcoding [26] using fish-specific COI primers [27] was employed. The COI mitochondrial gene has been validated for forensic species Species identification identification [28] to determine its reproducibility and limitations Real-time PCR. The results were analysed using the by testing its ability to provide accurate results under a variety of Sequence Detection Software version 1.71 (Applied Biosystems). conditions. To our knowledge, the current findings represent the The DRn values for each cycle and dye layer were then exported first large-scale assessment of fish product authentication across to MS Excel and additional manual processing was carried out. major UK supermarket retailers. First, the mean and standard deviation of the endpoint (PCR cycle 40) DRn values of the NTCs were calculated for each dye layer. z*M-values (z*M = M+(3.89xSD)+C) were then calculated where Materials and Methods M = mean of the NTC DRn, SD is the standard deviation of the Sample collection NTC DRn and 3.89 is the one tailed Z-value for the 99.999% 386 samples of processed white fish, ranging from fillets to fish confidence interval, C is a constant (0.3) introduced to overcome fingers and fish cakes, were collected from six leading supermarket the slight increase in fluorescence of samples above the NTC chains, at multiple locations across England, Scotland and Wales fluorescence due to spectral bleeding between dye layers. Samples (Table S1in File S1). Approximately 20 mg of tissue was taken which had DRn values larger than the value of z*M were from the centre of each product to ensure minimal DNA damage considered to have a fluorescence significantly greater than the from production, processing, or contamination. These were placed NTCs, and therefore to be positive reactions. PLOS ONE | www.plosone.org 2 June 2014 | Volume 9 | Issue 6 | e98691 Fish Mislabelling and IUU Table 1. list of all primers used. Sequence 59-39 Reporter Quencher COD P CTTTTTACCTCTAAATGTGGGAGG - - HAD P CTTTCTTCCTTTAAACGTTGGAGG - - GAD-F GCAATCGAGTYGTATCYCTWCAAGGAT FAM Non-fluorescent GAD-R CACAAATGRGCYCCTCTWCTTGC TET Non-fluorescent FishF1 TCAACCAACCACAAAGACATTGGCAC - - FishR2 ACTTCAGGGTGACCGAAGAATCAGAA - - COD P, HAD P, GAD-F, and GAD-R were used in the real-time-PCR, and FishF1 and FISHR2 were used for the sequencing PCRs. doi:10.1371/journal.pone.0098691.t001 COI sequencing. Successfully sequenced COI amplicons Of 179 samples labelled as cod, 57 were specified as Atlantic were manually checked and edited to remove ambiguous base cod (Gadus morhua) and 20 as Pacific cod (Gadus macrocephalus), while calling in BioEdit (Ibis Biosciences). Sequences were tested against for the remainder (102) there was no specification for either the the Barcode of Life database (BOLD) [29]. In addition, reference species or catch area. In total, 9 (5.03%) of these cod samples were sequences for all species genetically identified and all species not verified as cod by DNA data, including 1 (0.56%) identified as indicated on sample packaging, were downloaded from BOLD Melanogrammus aeglefinus (haddock), and 2 (1.11%) highly processed and aligned with the sample sequences in Clustal X [30], the samples that were found to have a mixed species composition (see Neighbour-joining tree was constructed in MEGA5 [31] with Table 2: #1892; G. morhua/G. chalcogramma and #1886; G. morhua/ 1000 bootstrap replicates. M. aeglefinus). From the 57 samples labelled specifically as Atlantic cod, 51 had congruent label and DNA-based designations, while 6 Results (10.5%) were genetically identified as Pacific cod (G. macrocephalus). 155 samples were labelled as haddock (M. aeglefinus). Of these, For consistency, all samples are referred to by the labelled 146 generated a molecular ID in agreement with labelling (5.81% species unless otherwise stated. Of 386 samples, 371 (97.4%) mislabelled), with 6 (3.87%) identified as G. morhua (Atlantic cod), 1 produced DNA of sufficient quality for further analysis. Label (0.65%) as G. macrocephalus (Pacific cod) and 2 (1.29%) exhibited a designations indicated primarily cod (179), haddock (155) and mixed species composition (see Table 2: #1452 and #1847; G. pollock (32). morhua/M. aeglefinus). Real-time-PCR. All samples labelled as hake or Alaskan In addition, one of the four hake (labelled as Merluccius capensis) pollack showed negative results for probes designed to identify cod samples was identified as Merluccius paradoxus (cape hake), one and haddock. The sample labelled as whiting was positive for cod. whiting (Merlangius merlangus) sample was identified as Micromesistius For the samples labelled as haddock (155), the haddock probe was poutassou (blue whiting), and one Alaskan Pollack was also found to positive in 134 samples (86.5%), while the cod probe gave a contain the Vietnamese catfish Pangasius hypophthalmus. Overall, our positive result for 6 samples, both probes were amplified in 7 survey indicated a rate of mislabelling of 5.66%. All samples and samples (inconclusive result) and neither were amplified in 8 results are presented in Tables S1 and S2 in File S1, with detailed samples (negative). All cod labelled as originating from the Pacific results of the mislabelled samples in Table 2 and Table S3 in File were negative for both the cod and haddock probes. Out of the S1. Sequence similarity with all reference samples is demonstrated Atlantic cod samples (57), the cod specific probe amplified in 47 in Figure 1, and the details of the reference sequences used are in samples (82.5%), both probes were positive in 3 samples Table S4 in File S1. (inconclusive result) and neither in 7 samples (negative). For the cod samples which did not indicate a catch location (102), the cod Discussion specific probe was positive in 80 samples, the haddock specific probe was positive in 2 samples, both amplified in 8 samples Our study represents, to our knowledge, the largest published (inconclusive result) and neither in 12 samples (negative). Real- survey to date of mislabelling within the fish products sold by UK time-PCR results are presented in Table 2. supermarkets. Samples were taken of products from leading COI sequencing. All sequence data has been submitted to brands and supermarket ‘‘own brands’’ from 6 major supermarket NCBI, under accession numbers KJ614671 to KJ615069 (Table chains across the UK. Previous studies have examined the food S2 in File S1). 48 samples have two sequences listed as these retail sector and found high rates of mislabelling, particularly in samples were re-extracted as independent replicates to ensure the restaurants and fast-food outlets [22,32]. Within our study of repeatability of the methods. supermarket-sourced samples the overall inconsistency between The majority of sequences were identified with a sequence product label and genetic species identification was 5.66%. This is identity greater than 99.5% in the BOLD database, with considerably lower than observed in other sectors: 25% within sequences from two samples falling below this threshold. mixed sectors [22]; 25% within markets and restaurants [32]; 32% Additionally, two samples could not be matched unambiguously within fishmongers [19]. Nevertheless, if our data are represen- due to 100% sequence identity at COI at the taxon-pairs involved. tative of overall trends, with over 4 billion fish products consumed The sequence data matched with Gadus chalcogramma/G. finnmarch- (C. Roberts, unpublished data) the incidence of mislabelling could ica (Alaskan and Norwegian Pollock respectively; previously exceed 200 million products annually in the UK alone. This level Theragra sp.), or Gadus macrocephalus and Gadus ogac (Pacific and of misinformation raises considerable concern in terms of Greenland cod respectively): these are both instances where the consumer information and protection. It also presents substantial (sub-) species designation is debatable (see Discussion). PLOS ONE | www.plosone.org 3 June 2014 | Volume 9 | Issue 6 | e98691 Fish Mislabelling and IUU Table 2. Summary of all mislabelled samples. Identification Code Species reported (type) Area of Catch real-time PCR First sequence identity Second sequence identity 1415 Cod (breaded fillet) Atlantic Negative Gadus macrocephalus Gadus macrocephalus 1426 Cod (breaded fillet) Atlantic Negative Gadus macrocephalus Gadus macrocephalus 1446 Cod (breaded fillet) Atlantic Negative Gadus macrocephalus Gadus macrocephalus 1747 Cod (precooked meal) Atlantic Negative Gadus macrocephalus Gadus macrocephalus 1889 Cod (precooked meal) Atlantic Negative Gadus macrocephalus Gadus macrocephalus 1975 Cod (breaded fillet) Atlantic Negative Gadus macrocephalus Gadus macrocephalus 1886 Cod (fish cakes) NA Inconclusive Gadus morhua Melanogrammus aeglefinus 1765 Cod (fish cakes) NA Melanogrammus aeglefinus Melanogrammus aeglefinus Melanogrammus aeglefinus 1892 Cod (fish fingers) NA Gadus morhua Gadus chalcogrammus Gadus morhua 1470 Haddock (precooked meal) Atlantic Gadus morhua Gadus morhua Gadus morhua 1812 Haddock (fish cakes) Atlantic Gadus morhua Gadus morhua Gadus morhua 1888 Haddock (precooked meal) Atlantic Gadus morhua Gadus morhua Gadus morhua 1977 Haddock (breaded fillet) Atlantic Gadus morhua Gadus morhua Gadus morhua 1989 Haddock (precooked meal) Atlantic Gadus morhua Gadus morhua Gadus morhua 1868 Haddock (precooked meal) Atlantic Gadus morhua Gadus morhua Gadus morhua 1851 Haddock (precooked meal) Atlantic Negative Gadus macrocephalus Gadus macrocephalus 1452 Haddock (fish cakes) Atlantic Inconclusive Gadus morhua Melanogrammus aeglefinus 1847 Haddock (fish cakes) Atlantic Inconclusive Gadus morhua Melanogrammus aeglefinus 1763 Alaskan Pollack (fish cakes) Pacific Negative Pangasius hypophthalamus Gadus chalcogrammus 1813 Hake (M. capensis) (breaded fillet) NA Negative Merluccius paradoxus Merluccius paradoxus 1848 Whiting (precooked meal) NA Inconclusive Micromesistius poutassou Micromesistius poutassou NA: Not available from packaging. Negative: neither of the real-time PCR probes amplified. Inconclusive: both real-time PCR probes amplified. First and second sequence identities are the result of independent DNA extractions and sequencing (see methods for details). doi:10.1371/journal.pone.0098691.t002 challenges for the sustainable management of the respective thought to be extremely low, and currently only of local fisheries. importance. The total reported catch for this stock from 2009– Genetic identification of products was carried out with species 2011 was 586 metric tons [35], while for the same three years, the total reported catch for G. macrocephalus was 1,165,420 metric tons. specific real-time PCR, and by matching sample COI sequences with those of known species in the BOLD database with high ($ Greenland cod is also no longer considered a separate species, but is now classed as a subspecies of Pacific cod, G. macrocephalus 99.5%) sequence identity [33]. Such independent testing yields a high degree of certainty to the identifications, as more than 98% of [36,37]. In the case of the Pollack species, G. finnmarchica was species pairs have shown greater than 2% COI sequence identified from a few samples from the northern tip of Norway divergence [34]. The BOLD database was used in preference to [38] and recent molecular evidence has shown it to be indistinct the nucleotide sequence database in GenBank (www.ncbi.nlm.nih. from the Alaskan Pollock (G. chalcogramma) [39–41]. gov/), to ensure that the queried sequences were matched to From all samples labelled as Atlantic cod, the majority of those taxonomically-validated specimens. Of all the sequences submit- found to be mislabelled were genetically identified as Pacific cod. ted, only two returned a match with less than 99.5% identity. Both This category of mislabelling could not originate at the pre-landing of these were from highly processed samples (one labelled as cod, stage; as is evident from their common names; these species are the other as haddock), and also returned inconclusive results for harvested from different oceans. The implication, therefore, is that the real-time-PCR (both cod and haddock probes amplified). Both intentional mislabelling has occurred at a later stage in the supply sequences were genetically identified as M. aeglefinus (haddock), chain. The incentive could be to supply products that mirror the although with relatively low sequence similarity (99.49% and preferences of the buying public, and so presumably fetch a higher 98.6%). For both of these sequences, the next closest match was G. price. This class of mislabelling may have little direct impact on morhua, rather than the next closest relative of haddock, Merlangius the Atlantic cod stocks but it may influence efforts to sustainably merlangus (see Figure 1), supporting the conclusion that the DNA manage stocks of Pacific cod. More importantly perhaps for this amplified was a mix of more than one species, and therefore that particular case of mislabelling is the issue of consumer misinfor- these products had a mixed species composition. mation and protection as it indicates that at some point in the Ambiguous results occurred when a sample matched with both supply chain there appears to be either negligence or a wilfully fraudulent attempt to provide inaccurate product information. Alaskan and Norwegian pollock (Gadus ( = Theragra) chalcogramma and G. finnmarchica, respectively), or with Pacific and Greenland Such instances erode consumer confidence and can undermine cod (Gadus macrocephalus and G. ogac, respectively), because trust in product labelling, including any associated eco-labels. congeners have 100% sequence identity at COI. However, in Samples labelled as M. aeglefinus (haddock) show a different the case of Pacific and Greenland cod, catches of G. ogac are pattern of mislabelling. The majority of mislabelled products were PLOS ONE | www.plosone.org 4 June 2014 | Volume 9 | Issue 6 | e98691 Fish Mislabelling and IUU Figure 1. Neighbour-joining tree showing all mislabelled samples together with representative reference sequences taken from BOLD. Reference sequences are colour coded according to species and samples tested are colour coded according to the species stated on the packaging. Samples that have two sequences are labelled a and b. doi:10.1371/journal.pone.0098691.g001 identified as G. morhua (Atlantic cod). Haddock and Atlantic cod In addition to the mislabelling of cod and haddock presented are frequently caught together in a mixed fishery and have similar here, other mislabelling instances were found. One highly market values, with cod slightly more valuable on average. As a processed (fish cake) sample labelled as containing Alaskan Pollack result there is minimal direct benefit to intermediaries in the (G. chalcogramma) was found to also contain Pangasius hypophthalmus. production chain to encourage such mislabelling. Alternatively, it P. hypophthalmus, or Vietnamese catfish, is a freshwater species from has been suggested that such mislabelling may arise by an Southeast Asia, legally described in the UK as Basa, Panga(s), accidental consequence of the mixed fishery [23]. However, while Pangasius, River cobbler or any of these combined with ‘catfish’ we accept such possibility, mislabelling undeniably benefits the [42]. Without performing a quantitative test for the presence of P. primary producer. Mislabelling G. morhua (Atlantic cod) as M. hypophthalmus, we were unable to estimate the relative quantities of aeglefinus (haddock) enables fishermen to land undersized or over the 2 species in this product (made of minced fish). It was therefore quota Atlantic cod and so profit from fish that should currently be not possible to determine whether this reflected inadvertent discarded. Irrespective of the underlying cause, if the mislabelling contamination through inadequate cleaning of the production line occurs before the fish are landed (for example, if filleted and frozen between products, or deliberate substitution of a cheaper product. at sea), such IUU activities will likely exceed catch quotas (TAQ) In either case it is unlikely to significantly affect catch data or to for a major North Atlantic fishery. The rate of mislabelling contribute to IUU. However, this accidental or fraudulent (3.87%) is comparatively low compared to other recent studies behaviour is a serious issue for consumer misinformation and [22]. However, if we extrapolate such incidence to the TAQ for trust, given the concerns over potentially increased contaminant 2011, it represents an additional 2188 tonnes of Atlantic cod (or an levels in Pangasius species (such as mercury) [43], which may result excess 2.9% of the Atlantic cod TAQ for 2011) being landed and in avoidance by some consumer groups. recorded as haddock. PLOS ONE | www.plosone.org 5 June 2014 | Volume 9 | Issue 6 | e98691 Fish Mislabelling and IUU Four hake samples were tested and one, labelled as M. capensis, as increasing media attention, the importance of consumer was identified as M. paradoxus (25% mislabelled, although the low confidence in the fisheries sector and revised EU legislation sample size requires caution). Historically, hake has been assessed [51,52] will collectively highlight and tackle mislabelling practices. as a single species, as separation of catches has not always been Nevertheless, only genetic testing across the supply chain can possible [44,45]. However species-specific assessments are now assess the scale and likely key stages of highest risk. It also appears being conducted. The shallow water M. capensis stock is above increasingly likely that such practices are more frequent at the sustainable levels, with catches below maximum sustainable levels more highly processed end of the market, where opportunities for and is certified by the Marine Stewardship Council (MSC). The detection and/or levels of discrimination are reduced. As deep-water M. paradoxus stock is below precautionary levels, and a witnessed recently in the wake of the horsemeat scandal across rebuilding plan is in place [46]. The mislabelling of this species, Europe, the complexities of the modern food production chain whether intentional or not, at a rate even well below that observed demand close scrutiny at all stages to ensure authenticity and here is a cause for serious concern, as such a practice would compliance. A forensic framework of genetic testing using compromise restoration of M. paradoxus to sustainable levels. validated reference databases [47,53–55] is expected to provide One noteworthy pattern to emerge is the variation in amount of an increasingly effective approach for detection, prosecution and mislabelling found among the different levels of processing: within ultimate deterrence of illegal activity. Such actions are likely not the fresh/frozen fillets (n = 84) no mislabelling was identified; in only to protect policy compliant end-users and the wider fishing battered/breaded fillets (n = 84), fish fingers (n = 31), and pre- industry but importantly also enhance prospects for achieving cooked meals (n = 128), the respective mislabelling rates were sustainability of exploited marine resources. 7.14%, 6.45% and 5.47% respectively. In fishcakes (n = 44), which are composed of minced fish, mislabelling rates of 13.6% were Supporting Information identified. However, these data are insufficient to identify where in this production chain, pre- or post-landing, yield higher rates of File S1 Supporting Information. Table S1. Summary of illegal activity. Targeted sampling at discrete stages across the sampling effort for samples which produced a DNA of sufficient supply chain is required: from on-board during sample catch to quality for testing. Samples are recorded by reported species and final retailer outlet. Alternatively it may be an inadvertent are split by supermarket (own brand/other brand items). Table consequence of the particular processing activity, such as S2. Genetic analyses for all samples. The sample identification inadequate cleaning of processing machinery. Huxley-Jones et al. number, product labelling (reported species and catch location), [23] found lower levels of mislabelling in processed products, such processing level* and results from the real time PCR and COI as fish fingers, than filleted products, and suggested that this may sequencing are given for each sample. Second sequence identity is be due to greater economic gains associated with the mislabelling the result of new DNA extraction and sequencing for mislabelled, of fillets. In contrast, our study included more diverse forms of ambiguous, and control samples. Genbank accession numbers are processing (from fresh fillets through fish fingers and precooked provided for each sequence. * classification of processing level (1: meals to fish cakes consisting of minced fish), and has demon- fresh or frozen fillets, 2: battered or breaded fillets, 3: fish fingers, strated a clear pattern of mislabelling, from zero in unprocessed 4: pre-cooked meals, 5: fishcakes). Table S3. Additional data for fish fillets to the highest levels of mislabelling in the most highly- the mislabelled samples. Query sample details, including species processed category. labelled on packaging, COI sequence and the Genbank accession The main trends highlighted here have been the substitution of number are given. Reference species is the closest sequence match G. morhua (Atlantic cod) with G. macrocephalus (Pacific cod) in in the BOLD database, together with the catch location, BOLD primarily filleted products, and the substitution of M. aeglefinus ID number and Genbank accession number of the reference (haddock) with G. morhua (Atlantic cod) in precooked meals and fish sample. Sequence similarity (% identity) between sample and cakes. Both aspects of mislabelling have a detrimental effect on G. reference is shown (* indicates sequence matches lower than morhua: substitution with G. microcephalus creates an erroneous 99.5%). Sequence similarity to the next closest match is also impression of the abundance of the former, undermining work shown. Table S4. BOLD and Genbank identifiers for the carried out by seafood awareness campaigns such as Seafood reference sequences used in Figure 1. Watch and the Marine Stewardship Council, to educate consum- (XLSX) ers and provide tools for informed purchasing decisions. However, cod is one of the species for which there are now sufficient genomic Acknowledgments resources to move beyond species identification and allow traceability to population level [47]. Testing by regulatory and Help with sampling was provided by Amy Sherborne, Willie McGee, and certifying bodies would improve consumer confidence in products Charlotte Hunt-Grubbe. Martin Taylor, Delphine Lallias and Wendy that are proven to fulfil claims of having been sourced from Grail all provided technical advice. We would also like to thank the two sustainably harvested stocks. In addition, as suggested here, if the reviewers for their insightful comments, which have greatly improved the clarity. substitution of M. aeglefinus with G. morhua is occurring at sea, the implications of such IUU activity would compromise the recovery of these heavily exploited species. Author Contributions Previous studies have reported relatively high rates of mislabel- Conceived and designed the experiments: SJH GRC JL. Performed the ling of seafood products globally [13,48], in Europe [19,22,49], experiments: HapDL MdB. Analyzed the data: SJH HapDL. Wrote the and South Africa [21,50]. However, many studies have focused on paper: SJH HapDL GRC MdB JL NB. smaller convenience food outlets and/or restaurants. Actions such References 1. FAO (2012) The State of the World’s Fisheries and Aquaculture. Rome: Food 2. FAO (2011) The State of the World’s Fisheries and Aquaculture. Rome: Food and Agriculture Organization of the United Nations. and Agriculture Organization of the United Nations. PLOS ONE | www.plosone.org 6 June 2014 | Volume 9 | Issue 6 | e98691 Fish Mislabelling and IUU 3. WSSD (2002) United Nations Plan of Implementation of the World Summit on 31. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) Sustainable Development A/CONF.199/20. 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Ursvik A, Breines R, Christiansen JS, Fevolden SE, Coucheron DH, et al. (2007) Mislabelling of a depleted reef fish. Nature 430: 309–310. A mitogenomic approach to the taxonomy of pollocks: Theragra chalcogramma and 14. Ardura A, Pola IG, Ginuino I, Gomes V, Garcia-Vazquez E (2010) Application T. finnmarchica represent one single species. BMC Evolutionary Biology 7 1): 86. of barcoding to Amazonian commercial fish labelling. Food Research 40. Byrkjedal I, Rees DJ, Christiansen JS, Fevolden SE (2008) The taxonomic status International 43: 1549–1552. of Theragra finnmarchica Koefoed, 1956 (Teleostei: Gadidae): perspectives from 15. Crego-Prieto V, Campo D, Perez J, Garcia-Vazquez E (2010) Mislabelling in morphological and molecular data. Journal of Fish Biology 73 5): 1183–1200. megrims: implications for conservation. In:Tools for Identifying Biodiversity 41. 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Filonzi L, Chiesa S, Vaghi M, Marzano FN (2010) Molecular barcoding reveals African Journal of Marine Science 34(1): 43–53. mislabelling of commercial fish products in Italy. Food Research International 46. Rademeyer RA, Butterworth DS, Plaganyi EE (2008) Assessment of the South 43: 1383–1388. African hake resource taking its two-species nature into account. African Journal 20. Armani A, Castigliego L, Tinacci L, Gianfaldoni D, Guidi A (2011) Molecular of Marine Science 30(2): 263–290. characterization of icefish, (Salangidae family), using direct sequencing of 47. Nielsen E, Cariani A, Mac Aoidh E, Maes G, Milano I, et al. (2012) Gene- mitochondrial cytochrome b gene. Food Control 22: 888–895. associated markers provide tools for tackling illegal fishing and false eco- 21. von der Heyden S, Barendse J, Seebregts AJ, Matthee CA (2010) Misleading the certification. Nature Communications 3:851. masses: detection of mislabelled and substituted frozen fish products in South 48. 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Available: ftp://ftp. African market. Food Research International 46(1): 30–40. fao.org/FI/DOCUMENT/fcp/en/FI_CP_UK.pdf. Accessed: April 2012. 51. EC (European Commission) (2000) European Council Regulation No 104/2000 25. Taylor MI, Fox C, Rico I, Rico C (2002) Species-specific TaqMan probes for of 17 December 1999 on the common organization of the markets in fishery and simultaneous identification of (Gadus morhua L.), haddock (Melanogrammus aeglefinus aqua-culture products. Official Journal of the European Communities, L17, 22– L.) and whiting (Merlangius merlangus L.). Molecular Ecology Notes 2(4): 599–601. 52. 26. Hebert PDN, Ratnasingham S, DeWaard JR (2003) Barcoding animal life: 52. EC (European Commission) (2001) Commission Regulation (EC) No 2065/2001 cytochrome c oxidase subunit 1 divergences among closely related species. of 22 October 2001 laying down detailed rules for the application of Council Proceedings of the Royal Society B: Biological Sciences 270: S96–9. Regulation (EC) No 104/2000 as regards informing consumers about fishery 27. Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PDN (2005) DNA barcoding and aquaculture products. Official Journal of the European Communities, L278, Australia’s fish species. Philosophical Transactions of the Royal Society B 360: 6–8. 1847–57. 53. Glover KA (2010) Forensic identification of fish farm escapees: the Norwegian 28. Dawnay N, Ogden R, McEwing R, Carvalho GR, Thorpe RS (2007) Validation experience. Aquaculture Environment Interactions 1: 1–10. of the barcoding gene COI for use in forensic genetic species identification. 54. Glover KA, Haug T, Oien N, Walloe L, Lindblom L, et al. (2012) The Forensic Science International 173: 1–6. Norwegian minke whale DNA register: a data base monitoring commercial 29. Ratnasingham S, Hebert PDN (2007) BOLD: The Barcode of Life Data System harvest and trade of whale products. Fish and Fisheries 13: 313–332. (www.barcodinglife.org) Molecular Ecology Notes 7(3): 355–364. 55. Ogden R (2008) Fisheries forensics: the use of DNA tools for improving 30. Higgins DG, Sharp PM (1988). CLUSTAL: a package for performing multiple compliance, traceability and enforcement in the fishing industry. Fish and sequence alignment on a microcomputer. Gene 73: 237–244. Fisheries 9(SI): 462–472. PLOS ONE | www.plosone.org 7 June 2014 | Volume 9 | Issue 6 | e98691 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS ONE Pubmed Central

Fish Product Mislabelling: Failings of Traceability in the Production Chain and Implications for Illegal, Unreported and Unregulated (IUU) Fishing

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© 2014 Helyar et al
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1932-6203
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Abstract

Increasing consumer demand for seafood, combined with concern over the health of our oceans, has led to many initiatives aimed at tackling destructive fishing practices and promoting the sustainability of fisheries. An important global threat to sustainable fisheries is Illegal, Unreported and Unregulated (IUU) fishing, and there is now an increased emphasis on the use of trade measures to prevent IUU-sourced fish and fish products from entering the international market. Initiatives encompass new legislation in the European Union requiring the inclusion of species names on catch labels throughout the distribution chain. Such certification measures do not, however, guarantee accuracy of species designation. Using two DNA- based methods to compare species descriptions with molecular ID, we examined 386 samples of white fish, or products labelled as primarily containing white fish, from major UK supermarket chains. Species specific real-time PCR probes were used for cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) to provide a highly sensitive and species-specific test for the major species of white fish sold in the UK. Additionally, fish-specific primers were used to sequence the forensically validated barcoding gene, mitochondrial cytochrome oxidase I (COI). Overall levels of congruence between product label and genetic species identification were high, with 94.34% of samples correctly labelled, though a significant proportion in terms of potential volume, were mislabelled. Substitution was usually for a cheaper alternative and, in one case, extended to a tropical species. To our knowledge, this is the first published study encompassing a large-scale assessment of UK retailers, and if representative, indicates a potentially significant incidence of incorrect product designation. Citation: Helyar SJ, Lloyd HaD, de Bruyn M, Leake J, Bennett N, et al. (2014) Fish Product Mislabelling: Failings of Traceability in the Production Chain and Implications for Illegal, Unreported and Unregulated (IUU) Fishing. PLoS ONE 9(6): e98691. doi:10.1371/journal.pone.0098691 Editor: Konstantinos I. Stergiou, Aristotle University of Thessaloniki, Greece Received November 22, 2013; Accepted May 7, 2014; Published June 12, 2014 Copyright:  2014 Helyar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was jointly funded by Greenpeace and The Sunday Times. The funder Greenpeace provided support in the form of salary for author NB, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funder The Sunday Times provided support in the form of salary for author JL, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing Interests: The authors have the following interests: This study was jointly funded by Greenpeace and The Sunday Times. Co-author Jonathan Leake is employed by The Sunday Times. Co-author Niall Bennett is employed by Greenpeace. There are no patents, products in development or marketed productsto declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: sarah.helyar@matis.is ¤ Current address: Food Safety, Environment & Genetics, Matı ´s, Reykjavı ´k, Iceland of the Johannesburg Plan of Implementation to restore them to a Introduction level that can produce maximum sustainable yield by 2015 [3]. In recent years, concerns about the health of the oceans and the A major threat for the sustainable management of these effects of over-exploitation of fisheries have increased. Consumer valuable resources is Illegal, Unreported and Unregulated (IUU) demand for seafood is growing with the contribution of fish to the fishing. Current estimates suggest that globally up to 25% of average annual diet reaching a record of 18.8 kg per person per fisheries catches fall within IUU practices [4–6], identifying it as year in 2011 [1], as compared to 17.1 Kg in 2008 [2]. This is the single largest threat to achieving sustainability. Both the FAO partly due to an increase in the range of species consumed, and an [7] and the European Union [8] have placed increasing emphasis increase in aquaculture. Fish products were worth a record $217.5 on the use of trade measures to prevent IUU-sourced fish and fish billion in 2010, up over 9% from 2009, and these trends are products from entering international trade. One component of this expected to continue. The increasing demand for fish highlights increased regulation has required the inclusion of binomial species the need for the sustainable management of aquatic resources; nomenclature on catch labels throughout the distribution chain 87.3% of world fish stocks are classed as overexploited, depleted or [9]. recovering: a number which continues to increase [1], with 29.9% In addition to top down pressure for improved labelling and of stocks classed as overexploited and unlikely to meet the targets traceability of fish products, many consumers are increasingly aware of nutritional and environmental issues regarding fisheries, PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e98691 Fish Mislabelling and IUU leading to shifts in attitude regarding acceptable species, catch into numbered tubes filled with 96% ethanol. Sample details location and catch methods [10]. In parallel, due to globalization including the place and date of purchase, species designation, and of the industry, consumers are encountering an increasing number eco-labelling were entered into a database linked to photographs of the packaging. Sample identities were not disclosed until of fish species and/or an escalation in common names applied to the same species. Such drivers have led to a greater demand for completion of molecular genetic analyses, when molecular and sample IDs were cross-referenced. informative labelling, including the use of ‘eco-labelling’. Although labelling to provide additional ecological information about a product is often voluntary, the FAO recognised that it could Molecular methods contribute to improved fisheries management and convened a DNA was extracted with the E-Z 96 Tissue DNA kit (Omega- Technical Consultation in 1998, which resulted in their Guidelines biotek), then quantified with a Nanodrop 1000 (Thermo for the Eco-labelling of Fish and Fishery Products from Marine Scientific), and standardised to either 5 ng/mL or 2 ng/mL Capture Fisheries [11]. Informative labelling is particularly depending on original concentration. Real-time PCR assays were important for processed items because any recognizable external carried out on all samples on an Applied Biosystems 7700 real- morphological features are typically removed, leaving consumers time sequence detection system. The 25 mL reactions contained reliant on product labelling for content information. However, it 200 nM of each of the two species specific probes (see Table 1), has been argued that any such labelling scheme, whether 300 nM of the GAD-F and GAD-R primers (Taylor et al. 2002), voluntary or legislated, requires policing in order to prevent 9.163 ml 2X Taqman Universal PCR Master Mix (UNG+ROX misuse and fraud [12].The mislabelling of a fish product may be and passive reference) (Applied Biosystems), 15 ng of DNA, and unintentional if, for example, species that are morphologically (depending on DNA concentration) either 10.417 or 6.917 mL similar are caught together, such as in many tropical or coral reef PCR grade H O (Sigma). Reactions were run in optical 96-well fisheries [13–17]. Alternatively, mislabelling may not be acciden- reaction plates using optical adhesive covers (Applied Biosystems). tal, such as where product substitutions are from species that do Plates were analysed under real-time conditions on two dye layers not occur in the same ocean [18–20], or for lesser value species with eight ‘no template controls’ (NTCs) per 96-well plate, and 2 [21,22]. However, whether intentional or not, the outcome can be positive controls for each of the two target species. The assay was serious for management and sustainability targets. In addition to run using the default cycling conditions [25]. the direct impacts of depletion from IUU fishing, substitutions and In addition to the real-time PCR, all samples were sequenced misidentification that occur before fish are landed will inflate the for approximately 655 bp from the 59 region of the COI gene inaccuracies in catch and forecast statistics. from mitochondrial DNA using primers developed by Ward [27]. Several recent studies of mislabelling have been undertaken in Tests were run with all combinations of the four available primers, Europe [19,22,23], yielding rates of mislabelling of up to 32% but the combination of FishF1/FishR2 produced consistently good [19]. Most mislabelled products have originated from small-scale PCR products in the species tested, and was therefore used retailers and convenience food outlets (e.g. fish and chip shops) but throughout (see Table 1). PCRs were carried out in 30 mL the major supermarkets have not hitherto been thoroughly reactions containing 15 mL of 2 x PCR Mastermix (containing investigated. Supermarket chains account for 72% of the total 0.75 U of Taq polymerase (buffered at pH 8.5), 400 mM each fish retail market in the UK (excluding canned products) [24]. If dNTP, 3 mM MgCl (Promega)), 9 mL PCR grade H O (Sigma), 2 2 comparable rates of mislabelling occur in supermarket products it 15 pmol each primer, and 3.0 mL of DNA template. The PCRs consisted of a denaturation step of 2 min at 95uC followed by 35 is thereby likely to have a substantial impact on efforts to manage the respective fisheries sustainably. It is therefore necessary to cycles of 30 seconds at 94uC, 30 seconds at 54uC, and 1 min at 72uC, followed by a final extension of 10 min at 72uC and then establish to what extent mislabelling of fish products occurs in the major retailers of the fish food supply chain, which is addressed in held at 4uC. PCR products were visualized on 1.2% agarose gels. If a single clear band was produced, PCR products were sent to this study. GATC (Germany, http://www.gatc-biotech.com) for sequencing. The current study uses two DNA-based methods to identify the DNA from 48 samples was re-extracted as independent replicates species of origin for 386 samples collected from major supermarket of real-time PCR and sequencing, including all samples where chains around the UK. Species-specific real-time PCR probes [25] molecular data contradicted species designations, and an addi- for cod (Gadus morhua), and haddock (Melanogrammus aeglefinus) were tional randomly chosen 33 samples to test repeatability of DNA- used to provide a highly sensitive test for the major species of white based species ID. fish sold in British supermarkets. Additionally, DNA barcoding [26] using fish-specific COI primers [27] was employed. The COI mitochondrial gene has been validated for forensic species Species identification identification [28] to determine its reproducibility and limitations Real-time PCR. The results were analysed using the by testing its ability to provide accurate results under a variety of Sequence Detection Software version 1.71 (Applied Biosystems). conditions. To our knowledge, the current findings represent the The DRn values for each cycle and dye layer were then exported first large-scale assessment of fish product authentication across to MS Excel and additional manual processing was carried out. major UK supermarket retailers. First, the mean and standard deviation of the endpoint (PCR cycle 40) DRn values of the NTCs were calculated for each dye layer. z*M-values (z*M = M+(3.89xSD)+C) were then calculated where Materials and Methods M = mean of the NTC DRn, SD is the standard deviation of the Sample collection NTC DRn and 3.89 is the one tailed Z-value for the 99.999% 386 samples of processed white fish, ranging from fillets to fish confidence interval, C is a constant (0.3) introduced to overcome fingers and fish cakes, were collected from six leading supermarket the slight increase in fluorescence of samples above the NTC chains, at multiple locations across England, Scotland and Wales fluorescence due to spectral bleeding between dye layers. Samples (Table S1in File S1). Approximately 20 mg of tissue was taken which had DRn values larger than the value of z*M were from the centre of each product to ensure minimal DNA damage considered to have a fluorescence significantly greater than the from production, processing, or contamination. These were placed NTCs, and therefore to be positive reactions. PLOS ONE | www.plosone.org 2 June 2014 | Volume 9 | Issue 6 | e98691 Fish Mislabelling and IUU Table 1. list of all primers used. Sequence 59-39 Reporter Quencher COD P CTTTTTACCTCTAAATGTGGGAGG - - HAD P CTTTCTTCCTTTAAACGTTGGAGG - - GAD-F GCAATCGAGTYGTATCYCTWCAAGGAT FAM Non-fluorescent GAD-R CACAAATGRGCYCCTCTWCTTGC TET Non-fluorescent FishF1 TCAACCAACCACAAAGACATTGGCAC - - FishR2 ACTTCAGGGTGACCGAAGAATCAGAA - - COD P, HAD P, GAD-F, and GAD-R were used in the real-time-PCR, and FishF1 and FISHR2 were used for the sequencing PCRs. doi:10.1371/journal.pone.0098691.t001 COI sequencing. Successfully sequenced COI amplicons Of 179 samples labelled as cod, 57 were specified as Atlantic were manually checked and edited to remove ambiguous base cod (Gadus morhua) and 20 as Pacific cod (Gadus macrocephalus), while calling in BioEdit (Ibis Biosciences). Sequences were tested against for the remainder (102) there was no specification for either the the Barcode of Life database (BOLD) [29]. In addition, reference species or catch area. In total, 9 (5.03%) of these cod samples were sequences for all species genetically identified and all species not verified as cod by DNA data, including 1 (0.56%) identified as indicated on sample packaging, were downloaded from BOLD Melanogrammus aeglefinus (haddock), and 2 (1.11%) highly processed and aligned with the sample sequences in Clustal X [30], the samples that were found to have a mixed species composition (see Neighbour-joining tree was constructed in MEGA5 [31] with Table 2: #1892; G. morhua/G. chalcogramma and #1886; G. morhua/ 1000 bootstrap replicates. M. aeglefinus). From the 57 samples labelled specifically as Atlantic cod, 51 had congruent label and DNA-based designations, while 6 Results (10.5%) were genetically identified as Pacific cod (G. macrocephalus). 155 samples were labelled as haddock (M. aeglefinus). Of these, For consistency, all samples are referred to by the labelled 146 generated a molecular ID in agreement with labelling (5.81% species unless otherwise stated. Of 386 samples, 371 (97.4%) mislabelled), with 6 (3.87%) identified as G. morhua (Atlantic cod), 1 produced DNA of sufficient quality for further analysis. Label (0.65%) as G. macrocephalus (Pacific cod) and 2 (1.29%) exhibited a designations indicated primarily cod (179), haddock (155) and mixed species composition (see Table 2: #1452 and #1847; G. pollock (32). morhua/M. aeglefinus). Real-time-PCR. All samples labelled as hake or Alaskan In addition, one of the four hake (labelled as Merluccius capensis) pollack showed negative results for probes designed to identify cod samples was identified as Merluccius paradoxus (cape hake), one and haddock. The sample labelled as whiting was positive for cod. whiting (Merlangius merlangus) sample was identified as Micromesistius For the samples labelled as haddock (155), the haddock probe was poutassou (blue whiting), and one Alaskan Pollack was also found to positive in 134 samples (86.5%), while the cod probe gave a contain the Vietnamese catfish Pangasius hypophthalmus. Overall, our positive result for 6 samples, both probes were amplified in 7 survey indicated a rate of mislabelling of 5.66%. All samples and samples (inconclusive result) and neither were amplified in 8 results are presented in Tables S1 and S2 in File S1, with detailed samples (negative). All cod labelled as originating from the Pacific results of the mislabelled samples in Table 2 and Table S3 in File were negative for both the cod and haddock probes. Out of the S1. Sequence similarity with all reference samples is demonstrated Atlantic cod samples (57), the cod specific probe amplified in 47 in Figure 1, and the details of the reference sequences used are in samples (82.5%), both probes were positive in 3 samples Table S4 in File S1. (inconclusive result) and neither in 7 samples (negative). For the cod samples which did not indicate a catch location (102), the cod Discussion specific probe was positive in 80 samples, the haddock specific probe was positive in 2 samples, both amplified in 8 samples Our study represents, to our knowledge, the largest published (inconclusive result) and neither in 12 samples (negative). Real- survey to date of mislabelling within the fish products sold by UK time-PCR results are presented in Table 2. supermarkets. Samples were taken of products from leading COI sequencing. All sequence data has been submitted to brands and supermarket ‘‘own brands’’ from 6 major supermarket NCBI, under accession numbers KJ614671 to KJ615069 (Table chains across the UK. Previous studies have examined the food S2 in File S1). 48 samples have two sequences listed as these retail sector and found high rates of mislabelling, particularly in samples were re-extracted as independent replicates to ensure the restaurants and fast-food outlets [22,32]. Within our study of repeatability of the methods. supermarket-sourced samples the overall inconsistency between The majority of sequences were identified with a sequence product label and genetic species identification was 5.66%. This is identity greater than 99.5% in the BOLD database, with considerably lower than observed in other sectors: 25% within sequences from two samples falling below this threshold. mixed sectors [22]; 25% within markets and restaurants [32]; 32% Additionally, two samples could not be matched unambiguously within fishmongers [19]. Nevertheless, if our data are represen- due to 100% sequence identity at COI at the taxon-pairs involved. tative of overall trends, with over 4 billion fish products consumed The sequence data matched with Gadus chalcogramma/G. finnmarch- (C. Roberts, unpublished data) the incidence of mislabelling could ica (Alaskan and Norwegian Pollock respectively; previously exceed 200 million products annually in the UK alone. This level Theragra sp.), or Gadus macrocephalus and Gadus ogac (Pacific and of misinformation raises considerable concern in terms of Greenland cod respectively): these are both instances where the consumer information and protection. It also presents substantial (sub-) species designation is debatable (see Discussion). PLOS ONE | www.plosone.org 3 June 2014 | Volume 9 | Issue 6 | e98691 Fish Mislabelling and IUU Table 2. Summary of all mislabelled samples. Identification Code Species reported (type) Area of Catch real-time PCR First sequence identity Second sequence identity 1415 Cod (breaded fillet) Atlantic Negative Gadus macrocephalus Gadus macrocephalus 1426 Cod (breaded fillet) Atlantic Negative Gadus macrocephalus Gadus macrocephalus 1446 Cod (breaded fillet) Atlantic Negative Gadus macrocephalus Gadus macrocephalus 1747 Cod (precooked meal) Atlantic Negative Gadus macrocephalus Gadus macrocephalus 1889 Cod (precooked meal) Atlantic Negative Gadus macrocephalus Gadus macrocephalus 1975 Cod (breaded fillet) Atlantic Negative Gadus macrocephalus Gadus macrocephalus 1886 Cod (fish cakes) NA Inconclusive Gadus morhua Melanogrammus aeglefinus 1765 Cod (fish cakes) NA Melanogrammus aeglefinus Melanogrammus aeglefinus Melanogrammus aeglefinus 1892 Cod (fish fingers) NA Gadus morhua Gadus chalcogrammus Gadus morhua 1470 Haddock (precooked meal) Atlantic Gadus morhua Gadus morhua Gadus morhua 1812 Haddock (fish cakes) Atlantic Gadus morhua Gadus morhua Gadus morhua 1888 Haddock (precooked meal) Atlantic Gadus morhua Gadus morhua Gadus morhua 1977 Haddock (breaded fillet) Atlantic Gadus morhua Gadus morhua Gadus morhua 1989 Haddock (precooked meal) Atlantic Gadus morhua Gadus morhua Gadus morhua 1868 Haddock (precooked meal) Atlantic Gadus morhua Gadus morhua Gadus morhua 1851 Haddock (precooked meal) Atlantic Negative Gadus macrocephalus Gadus macrocephalus 1452 Haddock (fish cakes) Atlantic Inconclusive Gadus morhua Melanogrammus aeglefinus 1847 Haddock (fish cakes) Atlantic Inconclusive Gadus morhua Melanogrammus aeglefinus 1763 Alaskan Pollack (fish cakes) Pacific Negative Pangasius hypophthalamus Gadus chalcogrammus 1813 Hake (M. capensis) (breaded fillet) NA Negative Merluccius paradoxus Merluccius paradoxus 1848 Whiting (precooked meal) NA Inconclusive Micromesistius poutassou Micromesistius poutassou NA: Not available from packaging. Negative: neither of the real-time PCR probes amplified. Inconclusive: both real-time PCR probes amplified. First and second sequence identities are the result of independent DNA extractions and sequencing (see methods for details). doi:10.1371/journal.pone.0098691.t002 challenges for the sustainable management of the respective thought to be extremely low, and currently only of local fisheries. importance. The total reported catch for this stock from 2009– Genetic identification of products was carried out with species 2011 was 586 metric tons [35], while for the same three years, the total reported catch for G. macrocephalus was 1,165,420 metric tons. specific real-time PCR, and by matching sample COI sequences with those of known species in the BOLD database with high ($ Greenland cod is also no longer considered a separate species, but is now classed as a subspecies of Pacific cod, G. macrocephalus 99.5%) sequence identity [33]. Such independent testing yields a high degree of certainty to the identifications, as more than 98% of [36,37]. In the case of the Pollack species, G. finnmarchica was species pairs have shown greater than 2% COI sequence identified from a few samples from the northern tip of Norway divergence [34]. The BOLD database was used in preference to [38] and recent molecular evidence has shown it to be indistinct the nucleotide sequence database in GenBank (www.ncbi.nlm.nih. from the Alaskan Pollock (G. chalcogramma) [39–41]. gov/), to ensure that the queried sequences were matched to From all samples labelled as Atlantic cod, the majority of those taxonomically-validated specimens. Of all the sequences submit- found to be mislabelled were genetically identified as Pacific cod. ted, only two returned a match with less than 99.5% identity. Both This category of mislabelling could not originate at the pre-landing of these were from highly processed samples (one labelled as cod, stage; as is evident from their common names; these species are the other as haddock), and also returned inconclusive results for harvested from different oceans. The implication, therefore, is that the real-time-PCR (both cod and haddock probes amplified). Both intentional mislabelling has occurred at a later stage in the supply sequences were genetically identified as M. aeglefinus (haddock), chain. The incentive could be to supply products that mirror the although with relatively low sequence similarity (99.49% and preferences of the buying public, and so presumably fetch a higher 98.6%). For both of these sequences, the next closest match was G. price. This class of mislabelling may have little direct impact on morhua, rather than the next closest relative of haddock, Merlangius the Atlantic cod stocks but it may influence efforts to sustainably merlangus (see Figure 1), supporting the conclusion that the DNA manage stocks of Pacific cod. More importantly perhaps for this amplified was a mix of more than one species, and therefore that particular case of mislabelling is the issue of consumer misinfor- these products had a mixed species composition. mation and protection as it indicates that at some point in the Ambiguous results occurred when a sample matched with both supply chain there appears to be either negligence or a wilfully fraudulent attempt to provide inaccurate product information. Alaskan and Norwegian pollock (Gadus ( = Theragra) chalcogramma and G. finnmarchica, respectively), or with Pacific and Greenland Such instances erode consumer confidence and can undermine cod (Gadus macrocephalus and G. ogac, respectively), because trust in product labelling, including any associated eco-labels. congeners have 100% sequence identity at COI. However, in Samples labelled as M. aeglefinus (haddock) show a different the case of Pacific and Greenland cod, catches of G. ogac are pattern of mislabelling. The majority of mislabelled products were PLOS ONE | www.plosone.org 4 June 2014 | Volume 9 | Issue 6 | e98691 Fish Mislabelling and IUU Figure 1. Neighbour-joining tree showing all mislabelled samples together with representative reference sequences taken from BOLD. Reference sequences are colour coded according to species and samples tested are colour coded according to the species stated on the packaging. Samples that have two sequences are labelled a and b. doi:10.1371/journal.pone.0098691.g001 identified as G. morhua (Atlantic cod). Haddock and Atlantic cod In addition to the mislabelling of cod and haddock presented are frequently caught together in a mixed fishery and have similar here, other mislabelling instances were found. One highly market values, with cod slightly more valuable on average. As a processed (fish cake) sample labelled as containing Alaskan Pollack result there is minimal direct benefit to intermediaries in the (G. chalcogramma) was found to also contain Pangasius hypophthalmus. production chain to encourage such mislabelling. Alternatively, it P. hypophthalmus, or Vietnamese catfish, is a freshwater species from has been suggested that such mislabelling may arise by an Southeast Asia, legally described in the UK as Basa, Panga(s), accidental consequence of the mixed fishery [23]. However, while Pangasius, River cobbler or any of these combined with ‘catfish’ we accept such possibility, mislabelling undeniably benefits the [42]. Without performing a quantitative test for the presence of P. primary producer. Mislabelling G. morhua (Atlantic cod) as M. hypophthalmus, we were unable to estimate the relative quantities of aeglefinus (haddock) enables fishermen to land undersized or over the 2 species in this product (made of minced fish). It was therefore quota Atlantic cod and so profit from fish that should currently be not possible to determine whether this reflected inadvertent discarded. Irrespective of the underlying cause, if the mislabelling contamination through inadequate cleaning of the production line occurs before the fish are landed (for example, if filleted and frozen between products, or deliberate substitution of a cheaper product. at sea), such IUU activities will likely exceed catch quotas (TAQ) In either case it is unlikely to significantly affect catch data or to for a major North Atlantic fishery. The rate of mislabelling contribute to IUU. However, this accidental or fraudulent (3.87%) is comparatively low compared to other recent studies behaviour is a serious issue for consumer misinformation and [22]. However, if we extrapolate such incidence to the TAQ for trust, given the concerns over potentially increased contaminant 2011, it represents an additional 2188 tonnes of Atlantic cod (or an levels in Pangasius species (such as mercury) [43], which may result excess 2.9% of the Atlantic cod TAQ for 2011) being landed and in avoidance by some consumer groups. recorded as haddock. PLOS ONE | www.plosone.org 5 June 2014 | Volume 9 | Issue 6 | e98691 Fish Mislabelling and IUU Four hake samples were tested and one, labelled as M. capensis, as increasing media attention, the importance of consumer was identified as M. paradoxus (25% mislabelled, although the low confidence in the fisheries sector and revised EU legislation sample size requires caution). Historically, hake has been assessed [51,52] will collectively highlight and tackle mislabelling practices. as a single species, as separation of catches has not always been Nevertheless, only genetic testing across the supply chain can possible [44,45]. However species-specific assessments are now assess the scale and likely key stages of highest risk. It also appears being conducted. The shallow water M. capensis stock is above increasingly likely that such practices are more frequent at the sustainable levels, with catches below maximum sustainable levels more highly processed end of the market, where opportunities for and is certified by the Marine Stewardship Council (MSC). The detection and/or levels of discrimination are reduced. As deep-water M. paradoxus stock is below precautionary levels, and a witnessed recently in the wake of the horsemeat scandal across rebuilding plan is in place [46]. The mislabelling of this species, Europe, the complexities of the modern food production chain whether intentional or not, at a rate even well below that observed demand close scrutiny at all stages to ensure authenticity and here is a cause for serious concern, as such a practice would compliance. A forensic framework of genetic testing using compromise restoration of M. paradoxus to sustainable levels. validated reference databases [47,53–55] is expected to provide One noteworthy pattern to emerge is the variation in amount of an increasingly effective approach for detection, prosecution and mislabelling found among the different levels of processing: within ultimate deterrence of illegal activity. Such actions are likely not the fresh/frozen fillets (n = 84) no mislabelling was identified; in only to protect policy compliant end-users and the wider fishing battered/breaded fillets (n = 84), fish fingers (n = 31), and pre- industry but importantly also enhance prospects for achieving cooked meals (n = 128), the respective mislabelling rates were sustainability of exploited marine resources. 7.14%, 6.45% and 5.47% respectively. In fishcakes (n = 44), which are composed of minced fish, mislabelling rates of 13.6% were Supporting Information identified. However, these data are insufficient to identify where in this production chain, pre- or post-landing, yield higher rates of File S1 Supporting Information. Table S1. Summary of illegal activity. Targeted sampling at discrete stages across the sampling effort for samples which produced a DNA of sufficient supply chain is required: from on-board during sample catch to quality for testing. Samples are recorded by reported species and final retailer outlet. Alternatively it may be an inadvertent are split by supermarket (own brand/other brand items). Table consequence of the particular processing activity, such as S2. Genetic analyses for all samples. The sample identification inadequate cleaning of processing machinery. Huxley-Jones et al. number, product labelling (reported species and catch location), [23] found lower levels of mislabelling in processed products, such processing level* and results from the real time PCR and COI as fish fingers, than filleted products, and suggested that this may sequencing are given for each sample. Second sequence identity is be due to greater economic gains associated with the mislabelling the result of new DNA extraction and sequencing for mislabelled, of fillets. In contrast, our study included more diverse forms of ambiguous, and control samples. Genbank accession numbers are processing (from fresh fillets through fish fingers and precooked provided for each sequence. * classification of processing level (1: meals to fish cakes consisting of minced fish), and has demon- fresh or frozen fillets, 2: battered or breaded fillets, 3: fish fingers, strated a clear pattern of mislabelling, from zero in unprocessed 4: pre-cooked meals, 5: fishcakes). Table S3. Additional data for fish fillets to the highest levels of mislabelling in the most highly- the mislabelled samples. Query sample details, including species processed category. labelled on packaging, COI sequence and the Genbank accession The main trends highlighted here have been the substitution of number are given. Reference species is the closest sequence match G. morhua (Atlantic cod) with G. macrocephalus (Pacific cod) in in the BOLD database, together with the catch location, BOLD primarily filleted products, and the substitution of M. aeglefinus ID number and Genbank accession number of the reference (haddock) with G. morhua (Atlantic cod) in precooked meals and fish sample. Sequence similarity (% identity) between sample and cakes. Both aspects of mislabelling have a detrimental effect on G. reference is shown (* indicates sequence matches lower than morhua: substitution with G. microcephalus creates an erroneous 99.5%). Sequence similarity to the next closest match is also impression of the abundance of the former, undermining work shown. Table S4. BOLD and Genbank identifiers for the carried out by seafood awareness campaigns such as Seafood reference sequences used in Figure 1. Watch and the Marine Stewardship Council, to educate consum- (XLSX) ers and provide tools for informed purchasing decisions. However, cod is one of the species for which there are now sufficient genomic Acknowledgments resources to move beyond species identification and allow traceability to population level [47]. Testing by regulatory and Help with sampling was provided by Amy Sherborne, Willie McGee, and certifying bodies would improve consumer confidence in products Charlotte Hunt-Grubbe. Martin Taylor, Delphine Lallias and Wendy that are proven to fulfil claims of having been sourced from Grail all provided technical advice. We would also like to thank the two sustainably harvested stocks. In addition, as suggested here, if the reviewers for their insightful comments, which have greatly improved the clarity. substitution of M. aeglefinus with G. morhua is occurring at sea, the implications of such IUU activity would compromise the recovery of these heavily exploited species. 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