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M. Iwamoto, T. Ayers, B.E. Mahon, D.L. Swerdlow (2010)Epidemiology of seafood associated infections in the United States
Bérenger Colsoul (2021)Review for "Stocking density effect on the growth and mortality of juvenile European flat oyster ( Ostrea edulis Linnaeus, 1758)"
J. Bilbao, O. Muñiz, J. Rodríguez, M. Revilla, Aitor Laza‐Martinez, S. Seoane (2020)Assessment of a sheltered euhaline area of the southeastern Bay of Biscay to sustain bivalve production in terms of phytoplankton community composition
T. Humphrey, K. Martin (1993)Bacteriophage as models for virus removal from Pacific oysters (Crassostrea gigas) during re-laying
Epidemiology and Infection, 111
S. Chinnadurai, K. Elavarasan, V. Geethalakshmi, V. Kripa, Kolliyil Mohamed (2022)Development of a depuration protocol for commercially important edible bivalve molluscs of India: Ensuring microbiological safety.
Food microbiology, 110
A. Mudadu, C. Spanu, J. Pantoja, M. Santos, C. Oliveira, S. Salza, G. Piras, M. Uda, S. Virgilio, L. Giagnoni, J.G. Pereira, T. Tedde (2022)Association between Escherichia coli and Salmonella spp. food safety criteria in live bivalve molluscs from wholesale and retail markets
K. Azpeitia, L. Ferrer, M. Revilla, J. Pagaldai, D. Mendiola (2016)Growth, biochemical profile, and fatty acid composition of mussel (Mytilus galloprovincialis Lmk.) cultured in the open ocean of the Bay of Biscay (northern Spain)
E. Suffredini, L. Lanni, G. Arcangeli, T. Pepe, R. Mazzette, G. Ciccaglioni, L. Croci (2014)Qualitative and quantitative assessment of viral contamination in bivalve molluscs harvested in Italy.
International journal of food microbiology, 184
O. Muñiz, M. Revilla, J. Rodríguez, Aitor Laza‐Martinez, A. Fontán (2019)Annual cycle of phytoplankton community through the water column: Study applied to the implementation of bivalve offshore aquaculture in the southeastern Bay of Biscay
D.W. Cook, R.D. Ellender (1986)Relaying to decrease the concentration of oyster‐associated pathogens
S. Malham, P. Rajko-Nenow, E. Howlett, Karen Tuson, T. Perkins, D. Pallett, Hui Wang, C. Jago, Davey Jones, J. McDonald (2014)The interaction of human microbial pathogens, particulate material and nutrients in estuarine environments and their impacts on recreational and shellfish waters.
Environmental science. Processes & impacts, 16 9
E. Fernández, X. Álvarez‐Salgado, R. Beiras, A. Ovejero, G. Méndez (2016)Coexistence of urban uses and shellfish production in an upwelling-driven, highly productive marine environment: The case of the Ría de Vigo (Galicia, Spain)
Regional Studies in Marine Science, 8
INTRODUCTIONMost bivalve mollusc production in Europe takes place in estuaries and Rías, many of which are subject to varying degrees of microbiological contamination (Fernández et al., 2016). According to Regulation (EU) 2019/627, shellfish production areas are classified into three different quality levels (A, B and C) based on the level of Escherichia coli in the flesh and intravalvular liquid. Class A areas are those from which live bivalve molluscs may be collected for direct human consumption, Class B areas are those from which live bivalve molluscs may be collected and placed on the market for human consumption only after treatment in a purification centre or after relaying, and Class C areas are those from which live bivalve molluscs may be collected and placed on the market only after relaying over a long period to reduce polluting substances to an acceptable level for human consumption. Most live bivalve molluscs intended for direct consumption are treated in purification centres (e.g., Mudadu et al., 2022; Romalde et al., 2002; Suffredini et al., 2014) because relaying is not practised in many countries due to unavailability of clean waters in many areas (Richards, 1988), making it very difficult to market live bivalves from C areas.Additionally, Regulation (EC) No 853/2004 establishes a relaying period of at least 2 months, which is not attractive for mollusc producers from an economic point of view. However, this relaying time, which depends, among other factors, on the mollusc species, water quality and environmental conditions (Chinnadurai et al., 2023; Cook & Ellender, 1986; WHO and FAO, 2018; Richards, 1988; Son & Fleet, 1980), can be shortened by the competent authority based on the food business operator's risk analysis. A priori, the most rapid relaying can be achieved at low shellfish density and clean waters with high salinity (Lee et al., 2008; Richards, 1988). However, most of the relaying studies have been carried out in estuaries or inlets (e.g., Lees et al., 2010), and, as far as we know, there is no experimental information on the relaying time required in open waters, at least in southern Europe. Therefore, the aim of this study was to determine the minimum time required for relaying mussels and Pacific oysters in Basque open waters to comply with the health standards for live bivalve molluscs set out in the Commission Regulation (EC) No 2073/2005, amended by Commission Regulation (EU) 2015/2285 on microbiological food safety criteria: (i) absence of Salmonella spp. in 25 g of flesh and intravalvular fluid and (ii) low presence of E. coli (20% of the samples may contain E. coli between 230 and 700 most probable number (MPN)/100 g of flesh and intravalvular fluid, while the remaining 80% must contain < 230 MPN/100 g).MATERIAL AND METHODSPrior to the relaying experiment, mussels (Mytilus galloprovincialis) and Pacific oysters (Magallana gigas) from the Basque coast (SE Bay of Biscay) were deployed for 6 weeks in the Pasaia port (43° 20′ N, 01° 56′ W; Figure 1) close to a stream discharging untreated wastewaters, a possible contamination source of faecally derived enteric pathogens (e.g., Iwamoto et al., 2010; Malham et al., 2014). The relaying experiment was carried out in an offshore aquaculture experimental site (43° 21.39′ N, 2° 26.90′ W; (Figure 1) located 2 miles off the coast at Mendexa. It consists of a 100‐m long longline installed at 35–50 m depth that can support high currents and waves up to 9 m high (Azpeitia et al., 2016). The area presents high salinities and relatively low chlorophyll ‘a’ and nutrient levels (Muñiz et al., 2019). Previous studies have found that it meets the requirements to be classified as zone A (unpublished data). Contaminated mussels and Pacific oysters were deployed for relaying for 21 days (20 June 2022–11 July 2022). For each species, 25 bags of 1.5 kg each were interspersed every 0.5 m along the longline at 5 m depth. At each sampling time, five bags of each species were removed at 0, 2, 8, 14 and 21 days of depuration for Salmonella spp. and E. coli determination. Additionally, seawater temperature was recorded every 10 min using an AQUATEC sensor installed at 5 m depth, and rainfall data were obtained from the Basque Meteorological Agency (Euskalmet) from a nearby station at Mendexa. The samples were shipped refrigerated to the laboratory in insulated boxes and were submitted for microbiological analysis within 24 h of collection. Each replicate was processed for E. coli enumeration and Salmonella spp. detection according to Regulation (EC) 2285/2015 using ISO/TS 166649‐3 and ISO 6579 methods, respectively.1FIGUREMap of the study area showing the locations where shellfish were contaminated for 6 weeks (Pasaia port) and the relaying site located in the offshore aquaculture experimental site (Mendexa) on the Basque coast (SE Bay of Biscay)Statistics were computed with Statgraphics (StatPoint Technologies). As data did not follow a normal distribution, the geometric mean was calculated at each sampling time for each species. The non‐parametric Kruskal–Wallis one‐way test followed by the Mann–Whitney U post hoc test was applied for comparing the median values of E. coli with relaying days for each species. Regressions between the geometric mean of E. coli levels and relaying days were carried out, and when values were below the quantification limit, half of the value was used. All analyses were set at α = 0.05.RESULTS AND DISCUSSIONThe mean daily seawater temperature fluctuated from 19.5 to 21.8°C (Figure 2). On the other hand, except on the second day when rainfall exceeded 10 L m−2 in 24 h, precipitation was low throughout the experiment (Figure 2). The environmental variables seemed favourable for bacteria elimination (Humphrey & Martin, 1993) as in summertime high temperatures and low rainfall are usual (Azpeitia et al., 2016; Bilbao et al., 2021; Zorita et al., 2021).2FIGUREDaily mean seawater temperature measured every 10 min at 5 m depth at the relaying site in waters off the Basque coast (line), and daily rainfall data obtained from the Basque Meteorological Agency (Euskalmet) from a nearby station (bars)At the beginning of the experiment, mussels and Pacific oysters presented geometric mean values of E. coli of 13,068 and 14,662 MPN/100 g, respectively (Figure 3; Supporting Information Table S1). These values meet the requirements of a class C area after Regulation (EU) 2019/627 (i.e., none of the replicates exceeded 46,000 MPN/100 g). After relaying, a significant decrease in E. coli concentration was observed in mussels (geometric mean values of 202, 57, 88 and 31 MPN/100 g after 2, 8, 14 and 21 days, respectively) and in Pacific oysters (geometric mean values of 271, 32, 32 and 31 MPN/100 g after 2, 8, 14 and 21 days, respectively; Figure 3). Furthermore, the significant regression found between the geometric mean of E. coli levels and relaying days in mussels (R2 = 0.88, n = 5, p < 0.05) and Pacific oysters (R2 = 0.92, n = 5, p < 0.01) indicated that bacteria load decreased as relaying progressed (Figure 4). On the other hand, Salmonella spp. was detected in all mussel and Pacific oyster replicates at the beginning of the experiment (Table S1). However, after 2 days of relaying, Salmonella spp. was no longer found, suggesting that the risk from these enteric pathogens was reduced rapidly. This is consistent with previous research on different species of oysters (e.g., Cook & Ellender, 1986; Son & Fleet, 1980). As for the health standards for live bivalve molluscs set out in the Commission Regulation (EC) No 2073/2005, amended by the Commission Regulation (EU) 2015/2285, 80% of the mussel samples contained E. coli below 230 MPN/100 g after 8 and 21 days but not after 14 days (only 60% of the samples; Figure 5). In contrast, 100% of the Pacific oysters achieved the health standards after 8 days (Figure 5).3FIGUREBoxplot of Escherichia coli levels in mussels and Pacific oysters deployed at 5 m depth for 21 days at the relaying site in waters off the Basque coast. Boxplots represent the median (line), 25%–75% percentiles (box) and min–max (whisker). Significant differences in median values (p < 0.05) between pairs of treatments (days) are identified by different letters. MPN: most probable number4FIGURERegression between geometric mean of E. coli levels and relaying days in mussels and Pacific oysters. Note: Values reported as < 30 MPN/100 g were assigned a value of 15 MPN/100 g5FIGUREPercentage of replicates showing different E. coli levels at each sampling day. Satisfactory E. coli levels (≤230 MPN/100 g; light grey), marginal (between 230 MPN/100 g and 700 MPN/100 g; dark grey) and unsatisfactory (700 MPN/100 g; black)Thus, under the studied conditions, the relaying time in open waters could be less than the 2 months established in the regulations. However, it should be noted that favourable conditions have been used for the elimination of bacteria: low density of individuals, low rainfall (i.e., high salinity) and high temperatures. In less favourable conditions, longer times are to be expected. Although this research cannot be considered a food business operator's risk analysis, it provides basic information on the minimum time required for offshore relaying of bivalves from C zones so as to meet the microbiological standards for human consumption. However, it must be taken into account that after the relaying period and before being placed on the market, molluscs must also comply with contaminants and biotoxin health standards to ensure food safety (Lee et al., 2008).CONCLUSIONAt the beginning of the relaying experiment, mussels and Pacific oysters accumulated Salmonella spp. and E. coli concentrations corresponding to class C areas, and after 2 days of relaying, Salmonella spp. was no longer detected in both bivalve species. Pacific oysters needed 8 days to show E. coli values below the acceptable threshold for human consumption, while mussels needed 21 days. Under the studied conditions, the mollusc relaying period in open waters could be reduced while ensuring food safety. Finally, this work provided basic data for the shellfish relaying in open waters.AUTHOR CONTRIBUTIONSIzaskun Zorita: Conceptualisation; formal analysis; funding acquisition; supervision; writing ‐ original draft. Oihana Solaun: Conceptualisation; investigation; writing‐review & and editing. José Germán Rodríguez: Conceptualisation; formal analysis; methodology; writing‐review & editing.ACKNOWLEDGEMENTSThis work was supported by the Fisheries and Aquaculture Directorate, Department of Economic Development, Sustainability and Environment of the Basque Government through BIOTOX Project (European Maritime and Fisheries Funds 00002‐INA2021‐33). We wish to thank AZTI staff for their valuable assistance. This is contribution number 1144 from AZTI, Marine Research, Basque Research and Technology Alliance (BRTA).CONFLICT OF INTERESTThe authors declare that there is no conflict of interest.DATA AVAILABILITY STATEMENTData are available in the article's Supporting Information.ETHICS STATEMENTI declare that all individuals listed as authors qualify as authors and have approved the submitted version; that the work is original and that is not under consideration by any other journal.PEER REVIEWThe peer review history for this article is available at: https://publons.com/publon/10.1002/aff2.97REFERENCESAzpeitia, K., Ferrer, K., Revilla, M., Pagaldai, J. & Mendiola, D. (2016) Growth, biochemical profile, and fatty acid composition of mussel (Mytilus galloprovincialis Lmk.) cultured in the open ocean of the Bay of Biscay (northern Spain). Aquaculture, 454, 95–108. https://doi.org/10.1016/j.aquaculture.2015.12.022Bilbao, J., Muñiz, O., Rodríguez, J.G., Revilla, M., Laza‐Martínez, A. & Seoane, S. 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Aquaculture Fish and Fisheries – Wiley
Published: Apr 1, 2023
Keywords: Bay of Biscay; bivalves; Escherichia coli; longline; relaying time; Salmonella
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