Food Control 47 (2015) 436e443

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Food Control

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Labelling accuracy in Tasmanian : An investigation using DNA barcoding

* Richard Lamendin a, , Karen Miller b, Robert D. Ward c a Institute for Marine and Antarctic Studies, University of Tasmania, Cnr Alexander Street & Grosvenor Street, Sandy Bay, Tasmania 7000, Australia b Australian Institute of Marine Science, University of Western Australia Oceans Institute (M096), 35 Stirling Highway, Crawley, Western Australia 6009, Australia c Commonwealth Scientific Research and Industrial Research Organisation, Marine and Atmospheric Research, GPO Box 1538, Hobart, Tasmania 7001, Australia article info abstract

Article history: Mislabelling of seafood products has been documented in numerous countries for over three-quarters of Received 12 May 2014 a century. With a trend towards increased consumption of seafood, the informed consumer demands Received in revised form accurately labelled products that provide full disclosure of composition. DNA barcoding can be used to 8 July 2014 accurately identify a seafood product to species based on its genetic signature, and so provides a means Accepted 20 July 2014 to test the authenticity and accuracy of seafood labelling. This can be especially useful for products such Available online 29 July 2014 as fillets which have few or no unambiguous identifying characters, and can easily be mislabelled. We investigated labelling accuracy in seafood retailers in Tasmania, Australia. Thirty-eight seafood products Keywords: Barcoding were obtained from seafood retailers, sequenced for the barcoding gene region cytochrome oxidase fi CO1 subunit 1(CO1), and subsequently identi ed to species level by querying GenBank and Barcode of Life Seafood Data Systems (BOLD) DNA sequence records. Results were compared with standard fish names (SFN) Mislabelling prescribed under the Australian Fish Names Standard (AFNS) and FishBase. Of the 38 samples, none were Species identification deemed to have been mislabelled under Australian regulation, although in some cases naming dis- Standard fish names crepancies and ambiguity may cause confusion for some consumers. Our work, while reflecting high standards in Tasmanian seafood, highlights the need for mandatory standard labelling across all seafood products so as to eliminate any possible misrepresentation. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction countries, is a prime example (Stoyke, Hamann, Rdeck & Gowick, 2013). Such reports highlight an issue fundamental to the prob- Numerous studies have shown that the mislabelling of products lem of mislabelling: the inability of the consumer to accurately for human consumption is widespread and has been occurring for detect what it is they are about to eat. The ramifications are clear: at some time (e.g. Hanner, Becker, Ivanova, & Steinke, 2011; Jacquet & best mislabelling is the product of dishonest behaviour; at worst, it Pauly, 2008; Wong & Hanner, 2008). Meat products of high-market is fraud and may have serious financial, legal and health conse- value are known targets for species substitution and adulteration. quences (Jacquet & Pauly, 2008). One study revealed that 68% of meat products sampled contained In the case of seafood, the absence of external morphological species not declared on the product's labelling (Cawthorn, features, particularly in processed products such as fish fillets, Steinman & Hoffman, 2013). The ‘horse meat scandal’ of 2013, hinders a person's ability to distinguish certain products or species involving the adulteration of beef burgers in multiple European from one another (Barbuto, Galimberti, Ferri, Labra & Malandra et al., 2010; Wong & Hanner, 2008). Various cooking and presen- tation methods add to this effect by masking the character, or Abbreviations: FSANZ, Food Standards Australia New Zealand; ANZFSC, Australia edible qualities, of that which is being eaten. Seafood retailers may New Zealand Food Standards Code; SSA, Seafood Service Australia; AFNS, Australian take advantage of the subtlety in fish fillet characteristics and Fish Names Standard; SFN, Standard Fish Name; BOLD, Barcode of Life Data Sys- contribute to what is now perceived as a growing form of economic tems; ABARES, Australian Bureau of Agricultural and Resource Economics and adulteration (Hanner et al., 2011). Financial incentives are thought Sciences. fi e * Corresponding author. to provide the strongest motivation to mislabel sh either with E-mail address: [email protected] (R. Lamendin). more appetising titles, or as higher-priced species (Jacquet & Pauly, http://dx.doi.org/10.1016/j.foodcont.2014.07.039 0956-7135/© 2014 Elsevier Ltd. All rights reserved. R. Lamendin et al. / Food Control 47 (2015) 436e443 437

2008). In doing so the fish benefits from an improved public image 2010). Marko et al., (2004) suggest that deliberate mislabelling not while the vendor benefits from increased sales and revenue. Ample only defrauds consumers but also distorts the status of fish stocks evidence attests to the widespread nature of mislabelling in the as perceived by customers. This may be the case in Ireland, where a seafood industry throughout the world (Table 1). false perception of high availability of (a depleted species) A lack of regulation and enforcement is also responsible for suggests healthy stocks (Miller & Mariani, 2010). seafood mislabelling (Hanner et al., 2011; Miller & Mariani, 2010). As a means to authenticate animal meat products and bypass In Ireland, where rates of mislabelling have been found to be high, the inherent problems of morphology-based identification such issues are dealt with by at least three government agencies. In methods, a number of molecular diagnostic techniques have been this case, an excess of bureaucracy and conflicting interests impede developed (Wong & Hanner, 2008; Yamashita et al., 2008). Among successful enforcement of seafood labelling and traceability stan- them is DNA barcoding (Galimberti et al., 2013). Hebert, Cywinska, dards (Miller & Mariani, 2010). Of course, not all mislabelling may Ball, and deWaard (2003) established the idea that the DNA be deliberate. The often confusing repertoire of names applied to sequence of a specific mitochondrial gene could serve as the core of the same fish, reflecting the cultural, ethnic and geographic di- a system to unambiguously identify all animals across the globe. versity of the peoples who caught and consume it (for example The target gene, cytochrome c oxidase unit 1 (CO1), approximately indigenous fishermen, European settlers, later immigrants etc.), 650 base pairs in length, is responsible for encoding a portion of the may contribute to product misrepresentation. In Australia, the terminal enzyme of the respiratory chain of mitochondria, and popular angling and food fish Argyrosomus hololepidotus is importantly, is known to possess low within-species compared to commonly known as either ‘Jewfish’, ‘Mulloway’, ‘Butterfish’ or between-species variation (Ward, Hanner, & Hebert, 2009). Bar- ‘Kingfish’, depending on the state in which the animal is caught coding takes advantage of this gene to identify animals to the (NSW/Qld, Vic, SA, WA respectively): its Standard Fish Name for species level. Barcodes of unidentified specimens may be queried trade purposes Australia-wide is Jewfish (Australian Fish Names against reference sequences (linked to formally identified voucher Standard AS SSA 5300). specimens held in public collections) housed in the publicly- The consequences of mislabelling go beyond the economic. accessible Barcode of Life Data systems (BOLD) (Ratnasingham & Should distributors, retailers or restaurants buy fish of lesser value Hebert, 2007; Wong & Hanner, 2008). and trade them as higher value substitutes, the consumer forfeits The barcoding process has proven successful in the identifica- the price difference and vendors in turn secure increased profits tion of fish species including (Holmes, Steinke, & Ward, (Jacquet & Pauly, 2008). Such practices serve to undermine con- 2009; Ward, Holmes, & Yearsley, 2008; Ward, Zemlak, Innes, Last, sumer confidence in fish and seafood products (Hanner et al., 2011; & Hebert, 2005). Importantly, it can be easily applied to seafood. Yearsley, Last, & Ward, 1999) and place honest suppliers or retailers Whole fish, fillets, fins, fragments, juveniles, larvae, eggs or any at a disadvantage in a market where others may undercut them. On other properly preserved tissue may be used for identification a broader scale, such acts nullify actions taken by the consumer to (Becker, Hanner, & Steinke, 2011) and the technique can be applied uphold conservation prohibitions (Hanner et al., 2011). The intro- to raw, smoked or cooked specimens (Hanner et al., 2011). duction of seafood awareness campaigns, certification programs Although DNA barcoding is well established in Australia (see (e.g. Marine Stewardship Council) and ecolabels (Jacquet & Pauly, Ward et al., 2009; Ward, Holmes, White, Last, 2008; Ward et al., 2007; Roheim & Sutinen, 2006) aim to persuade seafood con- 2005), its application for the detection of mislabelling of seafood sumers to select more sustainable or environmentally-friendly has rarely been attempted. Here, we use DNA barcoding to inves- products (Von der Heyden, Barendse, Seebregts, & Matthee, tigate the incidence of mislabelling specifically in Tasmanian sea- 2010). Without accurate labels however, consumers are pre- food products. The state of Tasmania represents one of Australia's vented from making such choices (Logan, Alter, Haupt, Tomalty, & most valuable fisheries. In the 2009e10 period, Tasmanian fish Palumbi, 2008), and regardless of any good intentions, ‘the represented a combined beach/farm gate value of $563.8 million “wrong” choice will inadvertently be made’ (Von der Heyden et al., and 25% of the country's total fisheries production. Tasmanian fish products are considered, locally and internationally, to be of high quality and this is linked to advanced technology and farming Table 1 practices, combined with independent assessments of sustain- Examples of reported cases of mislabelling by country. ability (TSIC 2013). The success of Tasmanian seafood is also Country Notes Source attributed to the strong focus placed on marketing to domestic consumers and the use of certified and easily recognised labels that Australia 23.2% of seafood products Anonymous. Food Standards ‘ ’ ‘ ’ mislabelled Australia New Zealand attest to the industry's clean and green credentials. Labelling Canada 41.2% of seafood products Hanner et al., 2011 accuracy on the part of vendors is therefore central to the continued mislabelled success of Tasmanian fisheries, and given the high economic value & Ireland 25% of cod and haddock Miller Mariani, 2010 of the industry, even conservative rates of mislabelling and sub- products mislabelled; Ireland 28.2% of cod mislabelled Miller et al., 2012 stitution would compromise the environmentally-aware efforts Italy 77% of 'palombo' Barbuto et al., 2010 made by both retailer and consumer. products substituted; Italy 32% seafood products Filonzi, Chiesa, Vaghi, & 2. Methods mislabelled Marzano, 2010 Italy 79% jellyfish products Armani et al., 2013 mislabelled 2.1. Sample collection and DNA extraction South Africa 50% of seafood products Von der Heyden et al., 2010 mislabelled Fifty-one seafood products (fresh, uncooked and unprocessed fi South Africa 31% sh seafood Cawthorn et al., 2012 fillets) were acquired anonymously from 15 fishmongers, markets mislabelled Spain 20% of hake sampled Machado-Schiaffino, or supermarkets across the Hobart (Tasmania) metropolitan area. found to be mislabelled Martinez, & From these products a small section of muscle tissue was removed Garcia-Vazquez, 2008 and stored in plastic vials at 20 C until processed. DNA was United States 77% of red snapper Marko et al., 2004 extracted from a 25 mg sub-sample of each fillet using the Qiagen mislabelled DNeasy Blood and Tissue Kit (Qiagen, USA) according to 438 R. Lamendin et al. / Food Control 47 (2015) 436e443 manufacturer protocols, but with minor adjustments to improve even at the low 80% and 84% respectively did align with the label at DNA yields. Specifically, the final elution buffer (AE) was warmed to point of sale. H54 matched with Nemadactylus bergi at 93% in 56 C before being applied to the sample, and the final incubation GenBank, but with Nemadactylus valenciennesi in BOLD at 100%; period in this buffer at room temperature was extended to 10 min. there is currently no COI barcode sequence for N. valenciennesi in Final DNA concentration was then measured using a NanoDrop GenBank. H43 matched with Lutjanus erythropterus/Lutjanus 8000 spectrophotometer. malabaricus at 98% in GenBank but at 100% in BOLD. In a few instances, individual sample barcodes matched to 2.2. PCR amplification and DNA multiple congeneric species. Barcode sample H38 matched to three species of the Mustelus genus when performing a BLAST search of A fragment of the CO1 gene was PCR amplified using primers GenBank: Merluccius antarcticus, Merluccius lenticulatus and Mer- FishF1 and FishR1 (Ward et al., 2005). Each PCR reaction mixture luccius palumbes (Table 2). The BOLD search however provided a consisted of 6.25 ml GoTaq Green Master mix (Promega), 0.1 mMof match to M. antarcticus at 99.65%. H43 as already mentioned each primer, and 2e46 ng of template DNA in a total reaction vol- matched two species of Lutjanid snapper (L. malabaricus and ume of 12.5 ml. PCR amplification reactions were conducted on Bio- L. erythropterus) with both search engines. This could reflect Rad C1000 thermal cyclers and consisted of a hot start of 95 C for possible misidentifications in the BOLD database between these 2 min, followed by 35 cycles of 94 C for 30s, 51 C for 30s and 72 C two very similar species, or perhaps hybridization or haplotype for 1 min, an extension stage of 72 C for 10 min and a final holding sharing. Likewise sample H59 matched to two species from the temperature of 15 C. PCR products were visualised on 1% agarose genus Seriolella: S. punctata and S. porosa. In this instance, the gels stained with Midori Green DNA stain (GeneWorks). PCR sam- sample is likely to be of the local species S. punctata rather than the ples with all but the very faintest bands were purified in accordance South American species S. porosa. with protocols outlined in a Qiagen QIAquick PCR purification kit (Qiagen, USA). DNA concentration of the purified PCR product was 3.1. Assessing fish names measured by spectrophotometer (NanoDrop 8000) and the product sequenced at the Australian Genome Research Facility in Brisbane, Of the 38 seafood products analysed for this study, none was Queensland, on the AB 3730xl platform. Sequencing was performed deemed to have been mislabelled or substituted (Table 2). However, in both forward and reverse directions. there were several cases where names considered obsolete, too broad, or potentially misleading were being used by retailers. For 2.3. Data evaluation example, we obtained several samples labelled as Gummy flake or deep-sea flake, boarfish or ocean (Table 2) even though these Forward and reverse sequences of each sample were aligned names are considered obsolete and may refer to multiple species of and analysed using MEGA version 5.2 (Tamura et al., 2011). fish. Sequencing errors were scanned for by eye and either removed or corrected with reference to electropherogram trace files. In this 4. Discussion way a contiguous sequence file was created for each sample. Identification of DNA sequences to species was achieved by per- DNA barcoding has shown that seafood retailers in Tasmania forming a BLAST search of GenBank (blast.ncbi.nlm.nih.gov) and have high labelling standards and that there is very little evidence using the BOLD identification engine to search species-level bar- that mislabelling occurs within the state. This is in stark contrast to code records within BOLD (www.boldsystems.org). Top species other parts of the world, where similar studies have shown that matches obtained from these searches were compared to relevant mislabelling can occur in as much as 77% of seafood products species' names corresponding to their common/market names as (Table 1), and indicates that consumers can have confidence that derived from FishBase and the Australian Fish Names Standard AS the Australian regulatory system is providing an adequate frame- SSA 5300 (SSA 2007). In cases where common names listed in work for the consistent and accurate naming of fish products by either were inconsistent with one another, a search of synonymous retailers. common, market or vernacular names was performed. In this way, a strict literal interpretation of fish names was avoided, allowing a 4.1. Limitations of sampling processed fish measure of flexibility (in favour of the vendor) in determining whether there had been mislabelling. This approach was required Although previous studies have shown that DNA barcoding can given the lack of definitive Australian laws regulating the naming of be used successfully on all manner of processed fish samples fish seafood products (see Discussion). including those that have been smoked, dried and/or frozen (Becker et al., 2011), we did experience difficulty obtaining se- 3. Results quences from some of our samples, despite the fact that these were sampled as fresh fillets from fishmongers. Thirteen samples could Of 51 seafood samples collected from commercial outlets across not be sequenced despite multiple efforts of altering the extraction Hobart, 35 yielded sequences of sufficient quality to create process and PCR conditions. The observed failure rate of 25.49% is contiguous sequences 530 bp enabling accurate identification to high when compared to other barcoding studies: for example, species using GenBank and/or BOLD reference databases. Addi- Hanner et al. (2011) observed a failure rate of only 6.7%. The failure tionally, three samples (H27, H43, H74) yielded sequences from just of some of our samples to amplify and sequence may be related to one primer of high-enough quality to be used in the final analysis. transport, handling and storage, whereby fish may have suffered Thus the total number of seafood products analysed was 38. under a freeze/thaw process that resulted in the degradation of The majority of samples were accurately matched their muscle tissue DNA, either before or after sample collection (or (similarity 99%) to species in both GenBank and BOLD (Table 2). during both). The nature of sample preservation and storage can Four samples (H27, H54, H43 and H74) matched to species with less certainly influence DNA quality and ultimately affect the success of confidence within the GenBank database (80%, 93%, 98% and 84%, processes such as barcoding (Evans, 2007; Miller, Williams, respectively). H27 and H74 could not be assigned to any species Rowden, Knowles, & Dunshea, 2010). Nevertheless, we believe using the BOLD search engine, although GenBank identifications the failure of a number of samples to amplify, from what is a small Table 2 List of all barcoded samples with advertised names matched to identification results.

Sample Advertised name Description a Sequence Expected species GenBank Match BOLD Match SSA standard Mislabelled? ID length (bp) identification similarity identification similarity fish name H3 Yellowfin SFN 592 Thunnus albacares Thunnus albacares 100% Thunnus albacares; obesus 100.00% Yellowfin Tuna No H27 Hake SFN 768 Merluccius spp. Merluccius spp. 80% Unable to match 0% Hake Unable to determine H28 Saddletail Snapper SFN 579 Lutjanus malabaricus Lutjanus erythropterus; 99% Lutjanus malabaricus 99.83% Saddletail No malabaricus Snapper H29 Smoked Cod Questionable 574 Multiple species Merluccius paradoxus 99% Merluccius paradoxus 100.00% Hake Possibly H31 Gummy Flake Obsolete 563 Mustelus spp. Mustelus antarcticus 100% Mustelus antarcticus 100.00% Gummy Shark No H33 Gummy Flake Obsolete 596 Mustelus spp. Mustelus antarcticus 99% Mustelus antarcticus 99.83% Gummy Shark No H34 Pink Ling SFN 571 Genypterus blacodes Genypterus blacodes; 99% Genypterus blacodes 100.00% Pink Ling No tigerinus H36 Blue Grenadier SFN 550 Macruronus novaezelandiae Macruronus 99% Macruronus magellanicus; 99.64% Blue Grenadier No novaezelandiae novaezelandiae H37 Silver SFN 621 Seriolella punctata Seriolella punctata 99% Seriolella punctata; porosa 100.00% Silver Warehou No Warehou/Trevally H38 Deep-Sea Flake Obsolete 577 Multiple species Mustelus antarcticus; 99% Mustelus antarcticus 99.65% Gummy Shark No lenticulatus; palumbes 436 (2015) 47 Control Food / al. et Lamendin R. H41 Pink Ling SFN 597 Genypterus blacodes Genypterus blacodes 99% Genypterus blacodes 100.00% Pink Ling No H42 Barramundi SFN 592 Lates calcarifer Lates calcarifer 99% Lates calcarifer 100.00%100.00% Barramundi Saddletail No No H43 Saddletail Snapper SFN 249 Lutjanus malabaricus Lutjanus erythropterus; 98% Lutjanusmalabaricus erythropterus; Snapper H44 Yellowfin Tuna SFN 618 Thunnus albacaresmalabaricus Thunnus albacares 100% Thunnus albacares 100.00% Yellowfin Tuna No H45 Pink Ling SFN 618 Genypterus blacodes Genypterus blacodes 99% Genypterus blacodes 100.00% Pink Ling No H46 Boarfish Obsolete 613 Multiple species Paristiopterus gallipavo 100% Paristiopterus gallipavo 100.00% Yellowspotted No Boarfish H47 Ocean Trout Obsolete 624 Oncorhynchus mykiss Oncorhynchus mykiss 100% Oncorhynchus mykiss 100.00% Rainbow Trout No H48 Gummy Flake Obsolete 563 Mustelus spp. Mustelus antarcticus 99% Mustelus antarcticus 99.82% Gummy Shark No H49 Blue-eye Trevalla SFN 586 Hyperoglyphe antarctica Hyperoglyphe antarctica 100% Hyperoglyphe antarctica 100.00% Blue-eye Trevalla No H51 Ling Obsolete 597 Genypterus spp. Genypterus blacodes 99% Genypterus blacodes 99.83% Pink Ling No H52 Obsolete 596 Oncorhynchus & Salmo spp. Salmo salar 100% Salmo salar 100.00% No H53 Morwong SFN 579 Multiple species Nemadactylus bergi 99% Nemadactylus sp. king 100.00% Morwong No H54 Queen Snapper Obsolete 564 Nemadactylus valenciennesi Nemadactylus bergi 93% Nemadactylus valenciennesi 100.00% Blue Morwong No e

H56 Yellowfin Tuna SFN 586 Thunnus albacares Thunnus albacares 100% Thunnus albacares 100.00% Yellowfin Tuna No 443 H57 Pink Ling SFN 588 Genypterus blacodes Genypterus blacodes 99% Genypterus blacodes 99.32% Pink Ling No H58 Boarfish Obsolete 600 Multiple species Paristiopterus gallipavo 100% Paristiopterus gallipavo 100.00% Yellowspotted Possibly Boarfish Seriolella punctata; S. porosa 100.00% Silver Warehou No H59 Spotted Trevally Obsolete 530 Seriolella punctata Seriolella punctata; 100% S. porosa H60 Snapper SFN 631 Multiple species Lutjanus malabaricus 100% Lutjanus malabaricus 100.00% Saddletail Possibly Snapper H63 Gummy Shark SFN 580 Mustelus antarcticus Mustelus antarcticus 100% Mustelus antarcticus 100.00% Gummy Shark No H64 Alfonsino SFN 546 Beryx decadactylus; splendens Beryx decadactylus 100% Beryx decadactylus 100.00% Imperador Possibly H65 SFN 606 Hoplosethus atlanticus Hoplostethus atlanticus 100% Hoplostethus atlanticus 100.00% Orange Roughy No H67 Pink Ling SFN 579 Genypterus blacodes Genypterus blacodes 99% Genypterus blacodes 100.00% Pink Ling No H70 Salmon Obsolete 595 Oncorhynchus & Salmo spp. Salmo salar 100% Salmo salar 100.00% Atlantic Salmon No H71 Ocean SFN 564 Multiple species Helicolenus percoides 99% Helicolenus percoides 99.64% Ocean Perch No H72 Ocean Trout Obsolete 604 Oncorhynchus mykiss Oncorhynchus mykiss 100% Oncorhynchus mykiss 100.00% Rainbow Trout No H73 Silver SFN 569 Seriolella punctata Seriolella punctata; 100% Seriolella punctata; S. porosa 100.00% Silver Warehou No Warehou/Trevally S. porosa H74 Ocean Trout Obsolete 1157 Oncorhynchus mykiss Oncorhynchus mykiss 84% Unable to match 0% Rainbow Trout Unable to determine H76 Ocean Trout Obsolete 685 Oncorhynchus mykiss Oncorhynchus mykiss 99% Oncorhynchus mykiss 99.84% Rainbow Trout No a ‘Description’ refers to whether the advertised name is a standard fish name (SFN) or is obsolete. Sequence length is measured in base pairs (bp). ‘Expected Species’ is the expected scientific name based on the advertised name, determined using FishBase and the Australian Fish Names Standard AS SSA 5300. Identifications listed here were obtained by querying GenBank and BOLD barcode records. The ‘SSA Standard Fish Name’ is the name 439 prescribed under the AFNS. 440 R. Lamendin et al. / Food Control 47 (2015) 436e443 sample size, does not warrant questioning of the integrity of Tas- The Australia and New Zealand Food Standards Code (ANZFSC) manian seafood markets and the quality of their product. has been adopted as the required standards for food produced, sold There was no evidence that samples which did not amplify or or imported in the States, Territories and Commonwealth of sequence successfully belonged to a certain species, nor were they Australia and in New Zealand. It falls under, but is not restricted to, acquired from a single vendor (indeed, they appeared to fail at Tasmania's Food Act of 2003 (Standard 1.1.1). Standard 1.2.2 of the random). In most cases, where one sample of a certain species ANZFSC requires that the label on a package of food must include a failed, that same species was successfully barcoded using another name or description sufficient to indicate the true nature of the sample suggesting there is unlikely to be any other methodological food. In the case of fish or fish products sold from retail cabinets limitation such as the use of PCR primers that are not suited to however, where such items are kept and displayed on ice and then certain species. While the primers used in this study are not truly packaged in the presence of the purchaser (i.e.: at a fishmonger), universal, they have been used across a wide and diverse range of certain information must be presented. A label on, or in connection fish species (see Ward et al., 2009; Ward, Holmes, & Yearsley, 2008; with, display of the food must identify: the prescribed name of the Ward, Holmes, White, et al., 2008; Ward et al., 2005) adding further food and, in any other case, a name or description of the food weight to our conclusion that failure to amplify relates to the sufficient to indicate the true nature of the food (Standard 1.2.2). sample rather than the method. Despite a relatively high failure Issues of misleading or deceptive conduct relating to the sale of rate, DNA barcoding proved to be an excellent method for testing food are treated in the Food Act (2003) under Part II, sections 15, 18 labelling accuracy in Tasmanian fish products. and 22: in effect such conduct is prohibited.

4.2. Confidence in species assignment using barcoding 4.4. Compliance to naming standards

In the majority of cases, no discrepancies were found between Standard 2.2.3 of the ANZFSC does not, however, define specific species assigned to each barcode by GenBank and BOLD (Table 2). In names for fish. Instead, it suggests the Australian Fish Names one case however, GenBank appeared unable to adequately resolve Standard (AFNS) AS SSA 5300 for guidance on standard fish names differences between congenerics of the Nemadactylus genus. Sam- to be used in Australia. Because the AFNS has not been incorporated ples H53 and H54 were matched to the species N. bergi with sim- into the ANZFSC (and thus not included in Commonwealth legis- ilarity values of 93 and 99%. The same search using BOLD matched lation), compliance to it cannot be subject to monitoring or these two barcodes to N. sp. king and Nemadactylus valencienessi enforcement by any appropriate state body e in this case the Tas- with similarity values of 100%, suggesting the current absence of manian Local Government's Department of Health and Human these species barcodes from the GenBank library, and thus greater Services Food Unit (Food Enforcement Contacts, FSANZ). In view of resolving power in the BOLD database. Attempts to resolve such this, a business may choose to advertise their seafood products inconsistencies by examining specimen vouchers revealed that the with any name they see fit, so long as they do not breach the matches provided by BOLD were to specimens forming part of on- subsections outlined above (sections 15, 18 and 22). The number of going research projects. Notwithstanding this fact, they have been obsolete fish names observed at retailers and recorded here included in Table 2. Note that the BOLD reference library in- (Table 2) is testament that this approach persists in the industry. corporates all GenBank records as well as those submitted to BOLD. Our results have shown that in certain cases, retailers have not The greater number of BOLD records reflects the fact that many are complied with the AFNS while simultaneously adhering to Tas- part of on-going research projects that will eventually be published manian State law. The Australian Fish Names Standard AS SSA 5300 in GenBank (Wong & Hanner, 2008). It is worth noting here that prescribes a standard fish name (SFN) for species produced or BOLD is characterised by its requirement for a persistent linkage traded in Australia. It specifies that fish sold to consumers (e.g. between a barcode sequence and its source, and a secure envi- retail sales and restaurants) must be identified by their standard ronment for its storage, organisation and query. Formal guidelines fish name. Again, this specification only holds if the business elects govern barcode designation while sequence records are required to to comply with the Standard, and doing so is not mandatory. Ac- meet quality standards. Importantly, the specimens from which cording to the Seafood Services Australia (SSA) website (https:// they derive are subject to expert taxonomic review (Ratnasingham seafood.net.au/page/?pid¼684) only three Tasmanian businesses & Hebert, 2007). GenBank, by contrast, does not meet this standard, are licensed by SSA for compliance with approved fish names in and so may contain barcodes with associated taxonomic problems accordance with the Standard. Interestingly, one of these was tar- and other erroneous information (though this is likely to be less of a geted for sampling in Hobart, and of three seafood products ac- concern where the species investigated are commercial and in quired from it one was advertised and sold under an obsolete general easily identified to species). market name: sample H58 e a portion of ‘Boarfish’. When queried The identities of samples H27 and H74 cannot be said to have in GenBank and BOLD the sample barcode returned matches with been truly determined: the percentage scores for match similarity the species Paristiopterus gallipavo e the Yellowspotted Boarfish (80 and 84% respectively) do not meet the threshold for true species (Table 2). In this case a broad, all-encompassing, yet obsolete name identification, despite the GenBank match aligning with the label at has been used to identify a fish that should otherwise be labelled by the point of sale. its Standard Fish Name. While this case is not strictly one of mis- labelling, it is a reflection of lack of compliance with the Standard. 4.3. Australian regulation and the Tasmanian story Another fourteen seafood product samples were advertised and sold under a name deemed obsolete by SSA1 (Table 2). While the Genetic identification of fish fillets via DNA barcoding revealed no mislabelling in retailers in the greater Hobart area. Approxi- mately 75% of all outlets in the Hobart metropolitan area were 1 On 22 July 2013, Seafood Services Australia announced that it would cease sampled and our results attest to the honesty and accuracy of these trading. Reductions in the availability of funding to primary production industries retailers. A future study should capture a wider range of seafood are reported to have played a significant role in the decision to close the company's doors (https://seafood.net.au/news/news.item.php?pid¼329). Despite this, re- outlets (to include restaurants, for example), and more Australian sponsibility for the project to develop and promote the Australian Fish Names cities, but it is probable that the Australian regulatory framework is Standard is to be transferred to another organisation. For this reason, the SFN list encouraging positive attributes in the industry. continues to be referred to in this discussion. R. Lamendin et al. / Food Control 47 (2015) 436e443 441 usage of obsolete names may well be common, it can be argued that species belonging to this family). Presumably sample H60, with its such terms meet the expectations of the general public, and are vague title, fits within this taxonomic boundary somewhere. therefore acceptable in the eyes of the Australian consumer. For this Indeed it does: once barcoded, the sample returned matches to L. reason, it is also argued that the clauses of Tasmania's Food Act malabaricus, the Saddletail Snapper (CAAB code 37 346007). The (2003) outlined above have not been breached. AFN list however, matches the term ‘Snapper’ to a single species: Chrysophrys auratus. While in essence both L. malabaricus and C. auratus are snappers, the AFNS dictates that only the latter may 4.5. Confusion and convolution in seafood labelling be described by the common name ‘Snapper’. A similar story can be told for the term ‘Morwong’: when searched the SFN list provides Sample H64 highlights the fact that some inconsistency lies links to 15 species formerly identified by this name; FishBase within the two sources of reference information used in this study provides matches to 8 separate species. In contrast to ‘Snapper’, to distinguish species names. Advertised and sold as ‘Alfonsino’ (a ‘Morwong’ may be used to represent a ‘group’ of species under the standard fish name), the sample was barcoded and matched to AFNS e one comprised of Nemadactylus macropterus and Nem- Beryx decadactylus. FishBase confirms this pairing of common and adactylus sp. The species matches to N. bergi obtained by searching scientific names, while the SSA Standard Fish Names List attributes GenBank and BOLD subsequently fall within this group. the species to the name Imperador. According to the SFN list These two cases highlight an important point: in both we cannot Alfonsino is the name attributed to Beryx splendens. (Meanwhile, be sure whether the vendors intended to use the broad (and FishBase recognises both Imperador and Alfonsino as common therefore misleading) name or the species-specific name (which in names of B. decadactylus, and to make things more confusing this case would imply mislabelling). The issue however remains identifies B. splendens as the Splendid Alfonsino.) academic. The retailers from which these samples were acquired In other cases certain seafood products have been advertised are not ones electing to comply with the AFNS. While they may and sold under names too broad to accurately describe which have used names potentially too vague to accurately describe the species they might be (Table 3). As described earlier, sample H58, species being advertised, they cannot be accused of mislabelling the ‘Boarfish’ is one example. Another is sample H60, and usage of their seafood. the term ‘Snapper’. The Codes for Australian Aquatic Biota (CAAB) There is one case of a seafood product whose label, though not database, maintained by the CSIRO Division of Marine and Atmo- strictly incorrect, is potentially misleading. Sample H29 was spheric Research Australia (CMAR), identifies 77 listings under advertised and sold as ‘Smoked Cod’, but when barcoded yielded a Lutjanidae e the Snappers (FishBase identifies 17 genera and 110 sequence matched to Merluccius paradoxus e a species more commonly known as Hake. While Hake may be alternatively Table 3 known, and in this case advertised, as Cod, one might argue that the A selection of seafood products available for purchase in Australia, with advertised use of ‘Smoked Cod’ is misrepresentative (There are numerous (market) and alternative common names. examples of fish with common and standard names featuring the Advertised name Common (obsolete) namesa word ‘cod’).Interestingly, supplementary nutritional information

Yellowfin Tuna ahi, Allison tuna, 'fin, yellowfin, yellowfinned albacore provided by the retailer's online shopping service lists as one of the Hake Cape hake, Pacific hake, South Atlantic hake ingredients ‘Hake fillets’. This information was not present on the Saddletail Snapper saddletail seaperch, large mouth nannygai, label for the seafood product; it could only be found online. large-mouthed nannygai, malabar blood snapper, Regardless of the information available online, a label with ‘Smoked nannygai Hake’ might in this case be more appropriate. Gummy Flake Australian smooth hound, flake, grey gummy shark, gummy, shark, smooth dog-shark, sweet William Miller and Mariani (2010) examined Irish seafood and what Smoked Cod eucla, bearded rock, red, forkbeard, violet, slender, might be considered the ‘real’ cod. Of 131 samples of Atlantic cod luminous cod (Gadus morhua) purchased across Dublin, 28% were found to be Pink Ling kingclip, rock ling, ling mislabelled. Cod is acknowledged to be a popular seafood histori- Blue Grenadier blue hake, grenadier, hoki, New Zealand whiptail, New Zealand whip-tail, whiptail cally, and despite falling stocks is in high demand. Satisfying con- Silver snotty nose trevally, snottynose trevally, spotted sumers in the Irish market has meant substituting Cod for imported Warehou/Trevally trevalla, spotted trevally, spotted warehou, trevally whitefish of lesser value: at once ‘creating a false perception of Barramundi barra, giant perch, palmer, silver barramundi market availability’ and cod stock health (Miller & Mariani, 2010). Boarfish sharpsnout deepsea, malayan deepsea, yellowspotted, In Italy significant commercial fraud (species substitutions c. 80%) is giant, bigspine, blackspot ‘ ’ Ocean Trout rainbow trout reported for shark seafood products, or palombo (Barbuto et al., Blue-eye Trevalla big eye, bigeye, blue eye, blue eye cod, blue-eye, 2010). This mislabelling took place despite European Commission blue-eye cod, blue-eye trevalla, blue-eyed trevalla, regulations relating to the labelling and traceability of seafood bluenose being in effect (EC, 2000, 2001). Under Article 2 of the directive, any Salmon chinook, chinook salmon, coho, king, king salmon, “ Pacific salmon, pink, quinnat salmon, rainbow labelling used must not be such as could mislead the purchaser to trout, silver a material degree, particularly: (i) as to the characteristics of the Morwong Grey, jackass, blue, dusky, banded, crested, red foodstuff and, in particular, as to its nature, identity, properties, morwong, silver perch, seabream, butterfish composition, quantity, durability, origin or provenance, method of Queen Snapper blue morwong, queens fish, sea , southern manufacture or production[…]”. Such prohibitions or restrictions blue morwong, queen morwong Spotted Trevally silver warehou also apply to the presentation of foodstuffs, their packaging and Snapper goldband snapper, brownstripe snapper, crimson advertising (EC, 2000). They are in effect quite similar to the reg- snapper, fiveline snapper, saddletail snapper ulations outlined in Tasmania's Food Act (2003) (see above) hence Alfonsino imperador, splendid alfonsino, alfonsin the stark difference in labelling accuracy between Europe and Orange Roughy deepsea perch, red roughy, reef fillets, sea perch Ocean Perch coral cod, coral perch, jock stewart, ocean perch, Australia (or at least Tasmania) is interesting. red gurnard perch, red rock perch, scarpee, sea perch Mislabelling of cod in the UK was found to be less severe than in Ireland according to Miller, Jessel, and Mariani (2012), and their a Some fish have over time acquired numerous common or vernacular names. Note: the advertised name may not necessarily be the appropriate Standard Name, experience may well explain our results for Tasmania. Heightened refer to Table 2 for these. levels of consumer awareness in the UK, likely driven by the 442 R. Lamendin et al. / Food Control 47 (2015) 436e443 presence and influence of environmental non-governmental or- Australia New Zealand Food Standards Code, Standard 1.1.1. Preliminary provisions e ’ ganisations (ENGOs), has potentially helped to create a demand for Application, interpretation and general prohibitions . Australia New Zealand Food Standards Code, Standard 1.2.2. Food Identification detailed information on product labels and sustainable choices Requirements. within the market place, thus eliminating the economic and cul- Australian Fish Names Standard AS SSA 5300. (2007). Seafood Services Australia. tural environment required for mislabelling and substitution to Barbuto, M., Galimberti, A., Ferri, E., Labra, M., Malandra, R., Galli, P., et al. (2010). DNA barcoding reveals fraudulent substitution s in shark seafood products: the occur (Miller et al., 2012). Informed consumers may therefore be Italian case of “palombo” (Mustelus s). Food Research International, 43,376e381. crucial to reducing commercial fraud in food industries. Becker, S., Hanner, R., & Steinke, D. (2011). Five years of FISH-BOL: brief status The absence of mislabelling observed in the Tasmanian seafood report. Mitochondrial DNA, 22,3e9. fl Cawthorn, D. M., Steinman, H. A., & Witthuhn, R. C. (2013). A high incidence of industry is possibly, as in the UK, a re ection of high consumer species substitution and mislabelling detected in meat products sold in South awareness in the marketplace. Hobart comprises a cosmopolitan Africa. Food Research International, 32,440e449. population with strong links to the fishing and marine scientific European Commission (EC). (2000). Directive 2000/13/EC of the European Parlia- ment and of the Council of 20 March 2000 on the approximation of the laws of communities: factors which may contribute to a strong desire for the member states relating to the labelling, presentation and advertising of informative and accurate seafood labelling. Furthermore, Tasmania foodstuffs. Official Journal of European Communities, LI09,29e42. is an island state surrounded by managed fisheries, and with a European Commission (EC). (2001). Commission Regulation (EC) No 2065/2001 of relatively small population has little or no need for imported, and 22 October 2001 laying down detailed rules for the application of Council Regulation (EC) No 104/2000 as regards informing consumers about fishery and potentially mislabelled, seafood. A pilot survey using DNA finger- products. Official Journal of European Communities, L278,6e8. printing conducted to identify fish species sold throughout Evans, T. C. (2007). DNA damage e The major cause of missing pieces from the DNA Australian retail outlets revealed mislabelling approximating 23%; puzzle. New England Biolabs, Inc.. from NEB expressions, 2.1. Filonzi, L., Chiesa, S., Vaghi, M., & Marzano, F. M. (2010). Molecular barcoding reveals (http://www.foodstandards.gov.au/publications/pages/ mislabelling of commercial fish products in Italy. Food Research International, 43, pilotsurveyontheidentityoffish/Default.aspx Barramundi and Red 1383e1388. Emperor were the focus of these sampling efforts due to the fact Galimberti, A., De Mattia, F., Losa, A., Bruni, I., Federici, S., Casiraghi, M., et al. (2013). DNA barcoding as a new tool for food traceability. Food Research International, that no ambiguity in marketing names purportedly exists for either 50,55e63. species. The one Barramundi sample acquired and successfully Hanner, R., Becker, S., Ivanova, N. V., & Steinke, D. (2011). FISH-BOL and seafood barcoded in our study (H42) was unequivocally matched to Lates identification: geographically dispersed case studies reveal systemic market substitution across Canada. Mitochondrial DNA, 22,106e122. calcarifer, the accepted pairing under the AFNS. Further sampling Hebert, P. D. N., Cywinska, A., Ball, S. L., & deWaard, J. R. (2003). Biological would be required to determine if there are any substitutions identifications through DNA barcodes. Proceedings of the Royal Society, 270, involving the imported Nile Perch (Lates niloticus), a congeneric of 313e321. Holmes, B. H., Steinke, D., & Ward, R. D. (2009). Identification of shark and ray fins the Australian Barramundi (Ward et al., 2008). using DNA barcoding. Fisheries Research, 95, 280e288. Jacquet, J. L., & Pauly, D. (2007). The rise of seafood awareness campaigns in an era of collapsing fisheries. 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