Report on the Equivalence of EU and US Legislation for the Sanitary Production of Live Bivalve Molluscs for Human Consumption

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1 Report on the Equivalence of EU and US Legislation for the Sanitary Production of Live Bivalve Molluscs for Human Consumption EU Scientific Veterinary Committee Working Group on Faecal Coliforms in Shellfish, August BACKGROUND Filter-feeding bivalve molluscan shellfish accumulate micro-organisms, including human pathogenic bacteria and viruses, when grown in sewage-polluted waters and can present a significant health risk when consumed raw or lightly cooked. Most countries have developed sanitary controls for the production and marketing of bivalve molluscs to minimise these public health risks. In the European Union they are controlled by EU Directive 91/492. In the United States by interstate trading agreements set out in the FDA National Shellfish Sanitation Program Manual of Operations. These regulations cover similar ground on the requirements for clean growing areas, the controls and processing requirements for more contaminated areas, the hygiene conditions for processing and dispatch establishments, requirements for marketing documentation etc. Third country imports into the EU and US have to be produced to the same standard as domestic products. Major exporting nations, such as New Zealand, have therefore developed programs for compliance with the regulations of their target export markets. A mutual recognition of EU and US controls for bivalve molluscs is still in discussion in the framework of the Sanitary and Phytosanitary Agreement of the World Trade Organisation. Comparison of US and EU legislative requirements shows that they share a similar philosophy in many respects and are likely to deliver similar end results. However, in one important respect they take a different approach which makes direct comparison difficult. Sanitary controls under both systems are underpinned by a classification of harvesting areas according to the degree of pollution as judged by faecal indicators. The purpose of this classification is to ensure that, a; water quality is adequate in areas from which molluscs are harvested for direct human consumption, b; where mollusc processing procedures are required to render products fit for consumption that contamination levels do not exceed safe limits, c; where pollution levels are excessive that harvesting for human consumption is prohibited. For these classifications or grading of mollusc harvest areas EU Directive 91/492 relies on microbiological analysis of shellfish flesh whereas the US FDA sanitation program relies on microbiological analysis of growing waters. Unfortunately bacterial counts in shellfish do not directly correlate with bacterial counts in seawater and therefore the equivalency of these approaches for delivering the same level of public health protection is not immediately apparent. This has hindered negotiation on recognition of equivalency for free trade purposes. Recognising this principally as a technical difficulty the EU Commission has requested expert guidance from the Scientific Veterinary Committee. This reports details the conclusions of an expert working group set up to consider this issue and make recommendations on equivalency to facilitate further negotiations. 2. BIVALVE MOLLUSCS Bivalve molluscs are a type of shellfish that have two shell halves which hinge together. Species commonly commercially exploited in Europe include the native or flat oyster (Ostrea edulis), pacific oyster (Crassostrea gigas), common blue mussel (Mytilus edulis) and Mediterranean blue mussel (Mytilus galloprovincialis), cockles (Cerastoderma edule), king scallops (Pecten maximus) and queen scallops (Chlamys opercularis), and various clams including the native clam or palourde (Tapes

2 descussatus), the hard shell clam (Mercenaria mercenaria), the manila clam (Tapes philippinarum), and the razor shell clam (Ensis spp.). With the exception of scallops these are normally static animals that attach themselves to, or bury themselves in, the sea bed or other submerged surface. They feed by filtering small particles such as algae from the surrounding water. Many of the commercial species are common in inshore estuaries or similar shallow or drying areas where nutrient levels are high and the waters are sheltered. Dense beds of the animals can occur in productive areas and have been an important source of food since prehistoric times. Indigenous species such as cockles, mussels and the native oyster continue to be harvested from natural populations, however, the characteristics of bivalve molluscs also make them suitable for cultivation. Nowadays the cultivation of indigenous species such as mussels and oysters is supplemented by breeding and farming introduced species such as pacific oysters and manila clams. It is important to recognise that bivalve mollusc species vary greatly in their characteristics and habitats. Those adapted to drying conditions close their shells tightly when out of the water to retain a marine environment around their fleshy internal parts. Such species (oysters, mussels and clams) can survive for extended periods out of the water and can be traded as live animals. Other species such as cockles are less hardy and are normally processed soon after harvest, however, they may also be traded as live animals if carefully handled. Scallops and other species not adapted to drying conditions soon die out of water and are normal handled as chilled or processed fishery product. Species adapted to drying conditions can be cultivated in the intertidal range which facilitates handling and harvest. Oysters are frequently grown in bags or similar containers raised off the foreshore on trestles, however, they may also be cultivated broadcast directly on the sea bed if conditions are suitable. Manila clams can be cultivated in containers in a similar way. Oysters, clams and scallops can also be cultivated suspended in the water column in lantern nets. Mussels can be cultivated loose on the sea bed in the intertidal region or in deeper waters but are also commonly grown attached to ropes for ease of mechanical harvesting. Such ropes can be suspended from floating structures in deeper waters or hung from poles in the intertidal range. Other indigenous species (cockles and various clams) bury themselves in the substratum and feed through siphon tubes. Such species grow in both the intertidal range and continually submerged as do indigenous populations of native oyster and mussel. Stocks may be harvested whilst submerged using various forms of dredge towed from a boat or may be raked or gathered when exposed. Harvesting methods vary from sophisticated and expensive high capacity mechanical devices to small scale local hand racking and gathering. Scallops are the only mobile bivalve species commonly harvested. Although there is a small scallop farming industry most are harvested from wild stocks by trawl, or diver, in deeper off-shore waters. In addition to their varying habitats bivalve molluscs vary greatly in such physiological attributes as their filtration, growth and activity rates, and in their response to environmental stress. This variability has important ramifications for various aspects of health monitoring and control. 3. HEALTH EFFECTS Human health problems arising from the consumption of bivalve molluscs are well recognised internationally and have been recorded since medieval times. The association of shellfish-transmitted infectious disease with sewage pollution became well documented in the late 19th and early 20th century with numerous outbreaks of typhoid fever in several European countries, the US and elsewhere. These hazards have been documented as a cause of concern by various international agencies. The United Nations in their comprehensive report on the marine environment in 1990 stated that "the present state of knowledge indicates that the most clearly identified health risk associated with coastal pollution by urban waste water is the transmission of disease by the consumption of shellfish harvested in contaminated areas" (United Nations Environment Programme, 1990). Disease outbreaks can occur on an epidemic scale as graphically illustrated by an outbreak of Hepatitis A in Shanghai, China in Almost 300,000 cases were traced to the consumption of contaminated clams (Halliday et al., 1991). Disease outbreaks have been reported in many countries, including Europe and the US, and have been extensively reviewed (Rippey 1994). In the developed world outbreaks with a known aetiology are

3 predominantly caused by small round structured viruses (SRSV s) of the Norwalk or Norwalk-like family which cause gastroenteritis. A smaller proportion of cases are caused by Hepatitis A virus and from bacterial agents of infection such as Salmonella and various vibrio species. The majority of cases continue to have an undefined aetiology, however, the clinical symptoms are mostly consistent with viral gastroenteritis. The available evidence therefore suggests that in the developed world viral infections are the predominant cause of infectious disease following shellfish consumption. These hazards occur predominantly because of the filter-feeding method of bivalve molluscs. These animals concentrate and retain human pathogens derived from sewage contamination of their shallow, in-shore, growing waters. They can also accumulate toxic algae and naturally occurring pathogenic bacteria in the same way. These hazards are compounded by the traditional consumption of bivalve shellfish raw, or only lightly cooked, and by the consumption of the whole animal including the viscera. Scallops which are both harvested from less polluted off-shore waters, and where the viscera is not consumed, do not present the same infectious disease hazard as oysters, mussels, cockles and clams. Univalve molluscs (eg winkles and whelks) are not filter feeders and do not present the same infectious disease hazards as the bivalves. It should be remembered that these circumstances are largely unique to bivalve molluscs and that they therefore require specific and targeted control measures to contain the risk. 4. MOLLUSC PROCESSING Conventionally, two different forms of commercial process are available for reducing the disease hazard from shellfish subject to pollution. For shellfish sold as a processed product heat treatment (cooking) may be used. Various heat treatment processes have been described varying from pasteurisation through to sterilisation by canning. Research in the UK has shown that hepatitis A virus can be inactivated by raising the temperature of shellfish meats to 90 C and holding that temperature for 90 seconds (Millard et al., 1987). Commercial heat treatments providing adequate health safeguards are laid down by Commission Decisions 93/25/EEC and 96/77/EC. Heat processing is not however applicable for shellfish sold live which constitute the bulk of the infectious disease hazard. Here, selfpurification, either in tanks (depuration) or in the natural environment (relaying), can be used. For depuration, harvested animals are transferred to tanks of clean seawater where they continue to filter feed for a period during which time sewage contaminants are purged out by the normal physiological processes. Depuration periods commonly vary from 1 to 7 days. Depuration systems also vary and include processes where water is static or changed in batches through to systems where seawater is flushed through continually or recycled through a steriliser. Water sterilisation processes include ozone, chlorination, UV irradiation and iodophores. Depuration is widely used in many countries including Australia, the UK, France, Italy, Spain and elsewhere, it is less widely used in the US. Depuration is also often used to add value to shellfish recognised as fit for direct consumption by harvesting area criteria. Relaying involves the transfer of harvested animals to cleaner estuaries or inlets for self-purification in the natural environment. This process can be used as an alternative to depuration for lightly polluted shellfish. Shellfish can only be held for relatively short periods in depuration tanks but can obviously be maintained for much longer periods in the natural environment. This makes relaying also suitable for treating more heavily polluted shellfish where longer periods (minimum two months in EU Directive 91/492) are required to remove contaminants such as enteric viruses causing gastroenteritis. The main disadvantages of relaying are the limited availability of suitable unutilised clean coastal areas and of obtaining ownership rights to those areas, the difficulty of controlling water quality and other water parameters and the susceptibility of stock to poaching. Combinations of these processes may also be used, for instance in France traditional practices include holding molluscs in claires (man-made tidally submersible ponds) for several months and then in degorgeoirs (wet storage ponds) for two days.

4 5. CONTROL MEASURES AND LEGISLATIVE STANDARDS Control measures are aimed at limiting the microbiological burden, and hence the infectious hazard risk, of bivalve molluscs entering the human food chain. Quantification of risk generally relies on traditional bacterial indicators of faecal contamination such as the faecal coliforms or E.coli. These can be measured in the shellfish themselves (EU approach) or in the shellfish growing waters (US FDA approach). It is generally accepted that the most effective and reliable approach to control is to harvest shellfish from areas with good water quality. Removing contamination by mollusc processing is a less effective option although still widely used in many countries for areas where coastal populations cause water quality deterioration. Most agencies recognise that harvested shellfish which meet the microbiological standard of less than 230 E.coli, or 300 faecal coliforms, in 100g of shellfish flesh are fit for direct human consumption. This, together with standards for specific pathogens (such as salmonella), chemicals and algal biotoxins, is the end-product standard set out in EU Directive 9l/492. Both EU and US legislative approaches employ a grading or classification approach with standards set for categories varying from waters considered safe to those prohibited for harvesting. Intermediate standards are set for areas of lower quality from which shellfish must be processed to reduce the disease hazard prior to consumption. Point source pollution inputs are generally identified through a shore line survey, or similar identification, and quality monitoring programs designed accordingly. US FDA Approved and EU Category A standards describe the cleanest growing areas from which shellfish can be taken for direct human consumption. All shellfish from EU Category A areas must contain less than 230 E.coli, or 300 faecal coliforms in 100g of shellfish flesh. The US FDA program gives a choice of using either total or faecal coliforms to establish a classification. It further expresses standards in two components, a geometric mean (GM) count of results and an upper standard which not more than 10% of results can exceed. Approved areas must comply with a total coliform GM of 70 per 100ml water with not more than 10% of samples exceeding 230 per 100ml. Alternatively they can comply with a faecal coliform GM of 14 per 100ml water with not more than 10% of samples exceeding 43 per 100ml. Both the EU and the US systems base standards on a 5-tube 3-dilution most probable number (MPN) test. The US FDA program refines this standard with the concept of Conditionally Approved areas. Such areas meet the prescribed standards only for certain periods because of predictable pollution events. They may be harvested during periods when they meet the standards subject to a management plan. Shellfish cannot be harvested for direct consumption from shellfish growing areas exceeding the above levels of contamination, they may however be taken for depuration or relaying. Unfortunately, these processes are not completely effective particularly for the removal of the human pathogenic viruses responsible for the bulk of shellfish associated infections. A number of instances of viral gastroenteritis have been documented following consumption of purified shellfish which meet specified safe bacteriological limits. For this reason an upper threshold is generally placed on the degree of contamination beyond which it is not sensible to employ short-term purification procedures. EU Category B and US FDA Restricted classifications describe upper thresholds for such areas. EU Category B areas must contain less than 4600 E.coli, or 6000 faecal coliforms per 100g of shellfish flesh in 90% of samples. US FDA Restricted areas must comply with a total coliform GM of 700 per 100ml water with not more than 10% of samples exceeding 2,300 per 100ml. Alternatively they can comply with a faecal coliform GM of 88 per 100ml water with not more than 10% of samples exceeding 260 per 100ml. In a similar way to approved areas restricted areas can also be Conditionally Restricted. Protracted relaying should effectively remove viruses from more highly contaminated shellfish and is incorporated as an option in EU legislation. Shellfish contaminated up to the level of Category C must be relaid for a minimum period of 2 months to reduce contaminants to acceptable levels before they can be placed on the market. Shellfish from Category C areas must contain less than 60,000 faecal coliforms per 100g of shellfish flesh. This treatment may be combined with depuration if relaying alone is not sufficient to meet the microbiological end-product standard. US FDA controls do not incorporate an equivalent to EU category C. Shellfish growing areas exceeding these prescribed levels of

5 contamination, or areas for which harvesting area survey and classification has not been conducted, are prohibited for harvesting for human consumption in both US and EU legislation. They also both contain clauses to suspend harvesting from classified areas following a pollution emergency. 6. COMPARISON OF EU AND US LEGISLATIVE APPROACH In addition to criteria for harvesting area classification both EU and US legislation set out requirements for other aspects such as bivalve transport, wet storage, depuration, relaying, analytical methods and movement documentation. In many respects EU and US requirements for these aspects are either similar or differences do not have a trade or public health implication. These other aspects are not therefore considered further here. The main difference in legislation hindering negotiation on recognition of equivalency for free trade purposes is the approach taken to classification of harvesting areas. EU Directive 91/492 relies on microbiological analysis of shellfish flesh whereas the US FDA sanitation program relies on microbiological analysis of growing waters. Unfortunately bacterial counts in shellfish do not directly correlate with bacterial counts in water and therefore the equivalency of these approaches for delivering the same level of public health protection is not immediately apparent. This report therefore concentrates on a detailed analysis of the comparability of approach and standards for the classification of shellfish harvesting areas and whether, or not, equivalent levels of public health protection are provided. The US FDA legislation allows the use of either the total or faecal coliform standard. However, it is generally accepted that faecal coliforms are a more specific indicator of faecal pollution. Indeed, reference is made to possible limitations of the total coliform approach in the specifications for approved areas. EU Directive 91/492 specifies only faecal coliforms, or the even more specific, E.coli. It is therefore only possible to compare the faecal coliform, or nearly comparable E.coli, standards when evaluating the equivalency of these legislative approaches. Table 1 summarises the relevant standards. Table 1. Synopsis of standards Shellfish treatment required US FDA Classification Microbiological standard per 100ml water non required Approved GM < 14 FC s and 90% < 43 FC s EU Classification Category A Microbiological standard per 100g shellfish all samples <230 E. coli or all samples <300 FC s purification or relaying Restricted GM < 88 FC s and 90% < 260 FC s Category B 90% < 4600 E. coli or 90% < 6000 FC s protected relaying (>2 months) - - Category C all samples <60,000 FC s note: FC s = faecal coliforms, GM = geometric mean, 90% = 90%-ile compliance The US FDA National Shellfish Sanitation Program Manual of Operations supplements the above legislative standards for harvesting area classification with various other detailed requirements and guidance. Some important additional factors include: requirements for a detailed growing area sanitary survey (which includes identification of pollution sources) prior to classification and updated periodically thereafter; the establishment of a prohibited zone adjacent to each sewage treatment plant outfall; the requirement to draw up maps showing the boundaries and classifications of each growing area; the stipulation that a minimum number of samples from each station shall be used to assess compliance (15

6 for approved areas); and the requirement to site sampling stations next to actual or potential sources of pollution and to time sampling to reflect adverse pollution conditions. EU Directive 91/492 does not set out in this way such detailed requirements for harvesting area classification. However, it should be remembered that EU Directives are framework documents used by EU member states to frame their own implementing legislation. Further details and guidance of this nature, needed to implement the basic criteria set out in the Directive, will be found in member states domestic legislation (Decree , Journal Republique Francaise) or in guidance documentation produced by competent authorities in each member state (MAFF, 1992). It is not possible, within this document, to address the various detailed procedures used by different member states to achieve the basic criteria set out in the Directive. For the purposes of this report therefore comparison of equivalency has to be based on the legislative standards set out in table RELATIONSHIP BETWEEN BACTERIAL COUNTS IN SHELLFISH AND IN WATER EU Directive 91/492 stipulates bacteriological criteria for shellfish flesh but these standards are not translated into an equivalent water quality standard. US FDA standards are based on bacterial counts in water with no equivalent flesh counts. The first issue is therefore to determine whether a relationship can be established between bacterial counts in shellfish and their overlying growing waters. Scrutiny of the scientific literature reveals little information on this topic. Only a limited number of laboratory and environmental studies have been reported and certainly no consensus relationship has yet emerged. However, discussion among experts revealed that competent authorities in several member states had on occasion monitored, for various purposes, bacteriological levels in both shellfish and seawater. Matched pairs of data were available for growing areas in the UK, Ireland, Italy, Holland and France. In addition data was available from a study conducted in New Zealand and submitted to the EU Commission as a basis for third country equivalency. Additional general observations were available from a report on the Canadian shellfish sanitation program. Furthermore the UK data set had already been subject to statistical analysis. This study was therefore taken as a starting point for addressing the issue of comparability. Initially UK data was analysed to establish the relationship, if any, between counts in flesh and water. Subsequently other data sets were compared with the UK data set to determine whether the UK data set was representative and whether data could be pooled to provide a European data set. Finally the relationship between E.coli counts in flesh and water derived from this pooled data set was modelled to provide estimates of seawater quality equivalent to the legislative standards set down in EU Directive 91/492. These estimates could then be compared to those set down in the US FDA sanitation manual to determine the degree of equivalency of health protection. This was only attempted for EU categories A and B as there is no direct equivalent in the US regulations for EU category C. The UK data set resulted from a joint project between the Ministry of Agriculture and Local Authorities and was based on monitoring already in place for the purpose of compliance with Directive 91/492. The study aimed to establish the relationship between bacterial levels in seawater and in shellfish and to extend this to the calculation of bacteriological water quality standards equivalent to those set out in the Directive for shellfish. Six geographically separate Local Authorities took seawater samples simultaneously with shellfish taken for monitoring purposes. All samples were tested by the appropriate local Public Health Laboratory Service (PHLS) laboratory as routine monitoring samples. Shellfish were tested for E.coli by the standard approved MPN protocol in the UK (MAFF et al., 1992). Water samples were tested for E.coli by the standard method in use in the PHLS laboratory which was either membrane filtration or MPN. The survey extended over a period of 32 months between 1991 and 1994 and generated 602 matched results spanning 3 shellfish species (mussels, Ostrea edulis (native flat oysters) and Crassostrea gigas (Pacific oysters), 6 different harvesting areas with 46 different sampling sites, 4 different PHLS laboratories and covering all seasons of the year. This wide range of parameters was felt to be sufficient to minimise bias from any particular variable. The use of a number of different laboratories should help ensure that results are representative and applicable to the routine monitoring scenario.

7 As a preliminary assessment of these data, figure 1 shows for all sites the individual numbers of E. coli in shellfish (ns) plotted against the corresponding number of E. coli in seawater (nw). Superimposed is a line joining the geometric means of E. coli in shellfish (gms) to the corresponding geometric means in seawater (gmw). To allow for zeroes in the data, numbers of E. coli have been increased by 1 throughout. Even at this gross level of aggregation (all sites, all species) there is some tendency for ns to increase with nw. This is made clearer in figure 2 which shows a bubble plot (bubble size proportional to sample size) of the site specific geometric means for shellfish (gms) plotted against the site geometric means for seawater (gmw) on a logarithmic scale, together with a weighted linear regression line [log10(gms) = log10(gmw) ] and its 95% confidence limits. This represents the relationship between sites in their geometric mean levels of E. coli in shellfish and water, and there is a clear indication that sites with generally high levels of E. coli in seawater have generally high levels in shellfish, and vice versa. This analysis pools all shellfish species. However, scrutiny of site geometric means revealed consistent differences in the relationship according to species. Figure 3 shows geometric mean levels of E. coli in shellfish and water for each site plotted according to species tested. Also shown are the weighted linear regression lines for each species. It was particularly noticeable that there was no significant difference between the calculated slopes for each individual species. This analysis shows that a consistent and statistically significant relationship exists between E.coli levels in shellfish and in water when data is aggregated at the geometric mean level and examined on a site specific basis. However, the actual bioaccumulation ratio will depend on the shellfish species tested and the seawater geometric mean. 8. COMPARISON OF UK DATA SET WITH DATA FROM OTHER EU MEMBERS STATES Additional data sets were available from Ireland, Italy, Holland, France and New Zealand. The number of matched pairs are summarised by species in table 2. The mussel species for the French and Italian data sets were M. galloprovincialis, for the UK, Holland and Ireland M. edulis, and for New Zealand Greenshell mussels. Significant data sets were only available for mussels, C.gigas and O.edulis. Table 2. Summary of data sets and sampling stations Country Mussels C.gigas O.edulis Cockles Palourde Venus Callistr France 30 (3) 360 (18) Holland 306 (2) Ireland 1462 (1) 258 (1) 1478 (1) Italy 87 (5) 8 (1) 47 (1) 9 (1) 2 (1) New Zealand 143 (23) UK 313 (23) 111 (6) 178 (17) 1 (1) Again, as a preliminary assessment, figure 4 shows the individual numbers of E. coli in shellfish (ns) plotted for all the above data against the corresponding number of E. coli in seawater (nw) with a line superimposed joining the geometric means in shellfish (gms) and in seawater (gmw). In comparison to figure 1 this gross data aggregation (all sites, all species) again shows a tendency for ns to increase with nw. The analysis for the UK data set in section 7 indicates that a consistent and statistically significant relationship exists between E.coli levels in shellfish and in water when data is aggregated at the geometric mean level and examined on a site specific basis. Information on the number of sampling stations for the data sets is indicated in brackets in table 2. Sampling site information was not available for the Irish data set which therefore had to be aggregated as a single station and not available for all records in the Italian data set. These are indicated by italics which denotes limited applicability of statistical approach based on analysis of data by sampling station.

8 As a first approach the station specific geometric mean E.coli concentrations in shellfish were calculated for each species (gms) and plotted against the corresponding geometric mean concentration in seawater (gmw). All of the data except the Irish data were used to construct a weighted regression line of gms on gmw, shown plotted in figure 5a with its 95% confidence limits. Figure 5b shows the same information with points represented by a bubble whose size is proportional to the number of records at a station. Figures 6a,b are similar to Figures 5a,b except that only UK data have been used to construct the reference regression line. It can be seen by inspection that the regression lines for all data pooled are similar to that for the UK data alone. The statistical equations of the overall weighted regression lines are: All data (except Irish) log 10 gms = log 10 gmw UK data log 10 gms = log 10 gmw Section 7 shows that with the UK data set species specific effects are significant therefore this analysis was extended to comparison of species by country where the data allowed. Figures 7a,b 8a,b and 9a,b show the individual weighted regression lines for each country, separately for mussels, C. gigas and O. edulis respectively. Because of the short ranges of E. coli concentrations in seawater, the separate regression lines for each species have been fitted with a common regression slope. The regression coefficients for each country are given in table 3. There were statistically significant differences between countries for mussels. However, inspection of figure 7b and table 3 shows that mussel results for France, Holland and the UK form a cluster of similar results with Italy and New Zealand being outliers. This comparability may reflect similar geographical and environmental conditions prevailing in these European countries. Differences in the Italian data set may be influenced by the partial lack of station information. Figure 8 and table 3 shows that the French and UK data for C.gigas are very similar and, statistically, not significantly different. Absence of station information for the Irish data set precluded its inclusion in the analysis however single geometric means for the entire data set are plotted by species in figures 7 to 9 for reference. For mussels and C. gigas inspection of figures 7 and 8 shows these mean points to be broadly consistent with the regression lines for other European countries showing that this data set is likely to be comparable to other European data sets for these species. For O.edulis the Irish data set contained the only data for comparison with the UK data set. Figure 9 shows good agreement between the mean value of the Irish data set for O.edulis and the regression slope generated from the UK data set. This shows that the UK data set is likely to be representative for this species. Table 3. Summary of regression coefficients by country and species. Country Mussels C. gigas O. edulis France Holland 1.34 Ireland Italy 0.90 New Zealand 1.95 UK Pooled slope s.e

9 To facilitate further analysis data was pooled for France, Holland and the UK into a European data set. Table 4 shows the revised intercepts when the model is re-fitted with countries grouped accordingly for the three major species. Grouped data were fitted with a common regression slope of In brackets are the corresponding predicted E. coli concentrations in shellfish (per 100g) at a seawater concentration of 100 per ml. These figures show that for the pooled data set a seawater geometric mean of 100 will give a bioconcentration factor in shellfish of 5.9 for mussels, 2.6 for C.gigas and 6.9 for O.edulis. Figures for the Italian and New Zealand data set are also given for comparison. Table 4. Summary of intercepts for grouped data with predicated E.coli concentrations in shellfish Country Mussels C. gigas O. edulis Pooled (Holland + France + UK) 1.38 (594) 1.02 (261) 1.45 (694) Italy 0.85 (176) New Zealand 1.89 (1921) 9. RELATIONSHIP BETWEEN CATEGORY A AND B COMPLIANCE AND E.COLI IN SEAWATER. The above analysis establishes a relationship between counts in shellfish flesh and in seawater at the geometric mean level. However, this is not directly useful for establishing compliance with the categories laid down in the EU Directive. Category B requires 90% of shellfish samples to comply with a limit value of 4600 E. coli per 100g flesh. Category A requires all samples to comply with a limit value of 230 E. coli per 100g flesh. Category B compliance was approached by using the pooled data set to model the proportion of shellfish values below 4600, i.e. the Class B compliance rate, which is assumed to depend on the average density of E. coli at a site, which we will estimate by gmw. Let Ni be the number of animals sampled at the i th station and ri the number of animals for which the corresponding numbers of E. coli fall below Then, if the level in one animal is unaffected by those in another, ri can be treated as the outcome of a Binomial trial of Ni samples with probability p of compliance and 1-p of non-compliance, where p depends on gmw. The relationship between p and gmw is assumed to be the logistic equation, i.e. α+ e p = 1 + e β log gmw α+ β log gmw which was found to be reasonable. Figure 10 shows a bubble plot for the pooled data set of the observed values of ri/ni plotted against the geometric mean number of E. coli in seawater, with bubble size proportional to Ni. Superimposed is the fitted logistic regression for all of the data with its 95% confidence limits. The fitted model gives a = 5.53 and b = The gmw value corresponding to a 90% compliance is obtained by solving 0 log. 9 log 01. = α+ β gm w for gmw, giving gmw = 111.7, with 95% confidence limits of 81 and 154. Roughly then, we can say that to achieve a 90% compliance of Category B at a station, the average number of E. coli in seawater should be no greater than a geometric mean of 110, with 95% confidence limits for this estimate of approximately The model described was fitted to the pooled data regardless of shellfish species. However, the analyses in sections 7 and 8 show that species specific effects may be significant. When the model is extended to

10 allow the a terms to be different for each species, there is a significant improvement in the model, with no evidence of lack of fit. The fitted model for category B compliance in shellfish with a different a term for each species are summarised in table 5 with the combined figure also given for reference. There were statistically significant differences between species. The calculated E.coli geometric means in 100ml seawater corresponding to a 90% shellfish compliance rate for category B for each species are also given. Table 5. Summary of Class B compliance by species for the pooled data set. Species Slope Intercept GM in water for 90% compliance Mussels C. gigas O. edulis combined Category A requires all samples to comply with a limit value of 230 E. coli per 100g flesh. However, there is no guidance in the Directive covering the time period this applies to or whether single isolated discrepant results should disqualify areas otherwise well within the standard limits. In practice most implementing authorities have had to make some allowance for such results. For the purpose of this report experts took the pragmatic view that 95% compliance with the criteria was a reasonable reflection of sample compliance for category A within the EU generally. 95% compliance with the limit value of 230 E. coli was therefore taken as the numerical criteria for compliance with category A for the purposes of this analysis. The above analytical approach was used to model the E.coli seawater geometric mean equivalent to 95% compliance with category A in shellfish. The pooled data set was analysed to provide figures for all species combined and for each individual species. The results are shown in table 6 and show that the all species combined geometric mean in seawater corresponding to a 95% compliance with category A in shellfish was 1.1 E.coli per 100ml. The species specific geometric means are also given and vary between 0.8 and 3 E.coli per 100ml. Table 6. Summary of Class A compliance by species for the pooled data set. Species Slope Intercept GM in water for 95% compliance Mussels C. gigas O. edulis combined COMPARISON OF EU AND US LEGISLATIVE STANDARDS The analysis of the UK and European data sets in sections 7 and 8 clearly shows that a relationship exists between E.coli counts in shellfish and in seawater. Analysis showed widely variable values for shellfish and seawater which determined poor predictive value for individual observations. However, data aggregation at the geometric mean level for each sampling station showed that a statistically significant relationship existed between E.coli levels in shellfish and in seawater. This relationship was modelled in section 9 to establish targets for geometric mean levels of E.coli in seawater compatible with category A and B compliance in shellfish. Emerging from the study were significant species specific effects suggesting that the three shellfish species represented in the pooled data set

11 bioaccumulated E.coli from seawater at different rates and to different concentrations. This species dependant effect obviously complicates the comparison of shellfish standards with those of the US FDA based on global water quality standards. However, comparison of geometric mean water quality equivalence values for category A of between 0.8 and 3.0 E.coli per 100ml shows that EU category A is clearly more stringent for all species studied than the US FDA geometric mean for approved areas of 14 per 100ml. The all data combined geometric mean water quality equivalence value for category B of 110 E.coli per 100ml is about equivalent to the US FDA geometric mean for restricted areas of 88 per 100ml. However here species specific effects are more apparent with seawater geometric means for shellfish compliant with category B ranging from 50 for mussels and 128 for O. edulis, to 586 for C. gigas. From these figures it is clear that for mussels the provisions of EU category B are more stringent than US FDA restricted, for O. edulis the provisions of both sets of standards are much the same, whereas for C. gigas the provisions of US FDA restricted are more stringent than those of EU category B. For the purpose of this report it is important to establish the scale of these differences in terms of compliance with the US and EU standards. Table 7 summarises the figures given above and compares, for the pooled European data set, predicted compliance rates with the EU standards when the US FDA geometric mean water quality criteria values are used. Table 7. Comparison of predicted compliance rates for EU categories using US standards Category Species GM in water Predicted compliance rate for 95% compliance for FDA approved (GM=14) A Mussels % A C. gigas % A O. edulis % A combined % Category Species GM in water Predicted compliance rate for 90% compliance for FDA restricted (GM=88) B Mussels % B C. gigas % B O. edulis % B combined % We see that application of the US FDA approved area criteria would give an overall compliance with EU category A of only 70% for the pooled European data set. Species specific compliance with category A using US criteria would range from 82% for C. gigas to around 60% for both O. edulis and mussels. Clearly therefore, on the basis of this analysis legislative standards for EU category A compliance are more stringent than those for US FDA approved areas for all species studied. Application of the US FDA restricted area criteria gives an overall compliance with EU category B of 91.4%. This is remarkably similar to the stipulated compliance regime of 90%. We can therefore say that if data for all species in the pooled European data set is combined to obtain an approximate average water value for EU category B that this would give almost identical results to the US FDA criteria for restricted areas. However, individual species would vary in their compliance rates for EU category B. Species specific compliance rates for EU category B using US FDA restricted area criteria would be 86% for mussels, 92% for O. edulis and 97% for C. gigas. On the basis of this analysis, for mussels legislative standards for EU category B compliance are more stringent than those for US FDA restricted areas, for native oysters (O. edulis) standards are nearly equivalent, and for pacific oysters (C. gigas) US FDA standards are more stringent than EU standards. However these differences are not large in practical terms.

12 11. DISCUSSION AND RECOMMENDATIONS On the basis of analysis of the pooled European data set, legislative standards for EU category A compliance are more stringent than those for US FDA approved areas. Additional confirmatory information comes from Canadian experiences with their shellfish sanitation program modelled on the US FDA system. They report that sampling of shellstock in areas classified approved, and complying with, the FDA criteria occasionally fail the bacteriological flesh standards. These failures are mainly in intertidal clam species more susceptible to contaminants carried on the freshwater interface than continuously immersed Canadian cultured oysters, mussels and scallops. A policy of closure on bacteriological counts in clam shellstock has overcome this problem but it illustrates that under certain circumstances EU criteria can be more demanding than US FDA criteria. These various factors would seem to suggest that shellstock sampling, when properly performed, can reveal potential contamination problems in shellfish which water sampling alone might miss. It should be remembered that within the EU many different bivalve species are harvested from a variety of diverse habitats and aquaculture systems (see section 2). In addition great variation in hydrographical and environmental parameters will occur between, for instance, warmer southern states and colder northern states. The universally applicable nature of the shellstock approach to assessing shellfish quality has benefits for the very diverse nature of bivalve harvesting within the EU. However, when comparing the implementation of US and EU requirements it should be remembered that the US FDA manual of operations is more prescriptive than the EU Directive regarding the precise details for harvesting area monitoring and compliance (see section 6). This may tend to make the US FDA requirements more uniformly applied than the EU requirements. It is difficult to quantify such effects however it may counterbalance, to some extent, the more stringent nature of the EU standards for category A areas. In conclusion statistical analysis shows that EU legislative standards for category A shellfish harvesting areas are more stringent than US FDA standards for approved areas for all shellfish species studied. This may be counter balanced to some extent by the more detailed and prescriptive monitoring and classification requirements specified in the US FDA regulations. Many of the above factors also apply to consideration of the equivalency between EU category B and US FDA restricted. Analysis of the pooled European data set showed that if all species are combined to give an approximate average water value for EU category B that this would give almost identical results to the US FDA criteria for restricted areas. This shows that, in general terms, both sets of legislation are putting a similar upper threshold contamination limit on shellfish destined for commercial depuration. However individual species specific effects were noted which suggested that, for mussels, EU legislative standards for category B compliance are more stringent than those for US FDA restricted areas, standards were nearly equivalent for native oysters (O. edulis), and US FDA standards were more stringent for pacific oysters (C. gigas). It is very difficult to quantify what, if any, public health implication would arise from these differences. From the trading viewpoint for both sets of standards greater stringency for one species is balanced with less stringency against another. On balance the recommendation of the working group is that as the differences seen are not large, in percentage compliance rate terms (see table 7), it is recommended that EU category B and US FDA restricted are recognised as offering equivalent levels of public health protection for the purposes of negotiation on mutual recognition of trading standards.

13 REFERENCES Council Directive of 15 July 1991 laying down the health conditions for the production and the placing on the market of live bivalve molluscs (91/492/EEC). Official Journal of the European Communities 1991; L 268: Halliday ML, Kang L, Zhou T, Hu M, Pan Q, Fu T, Huang Y, Hu S. An epidemic of hepatitis a attributable to the ingestion of raw clams in shanghai china. Journal of Infectious Disease 1991; 164: MAFF, DoH and PHLS Working Group. Bacteriological examination of shellfish. PHLS Microbiology Digest 1992; 9: National Shellfish Sanitation Program, Manual of Operations Part 1, 1993 Revision. US Department of Health and Human Services, Public Health Service, Food and Drug Administration. United Nations Environment Programme. Joint Group of Experts on the Scientific Aspects of Marine Pollution: The State of the Marine Environment. UNEP Regional Seas Reports and Studies 1990; No Decree No du 28 avril 1994 relatif aux conditions sanitaires de production et de mise sur le marche des coguillages vivants. Journal Officiel de la Republique Francaise, 30th Avril: Millard JH, Appleton H, Parry JV. Studies on heat inactivation of hepatitis A virus with special reference to shellfish. Epidemiology and Infection 1987; 98: Rippey SR. Infectious diseases associated with molluscan shellfish consumption. Clinical Microbiology Reviews 1994; 7:

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