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in the european union , official controls on the production and distribution stages of live bivalve molluscs for human consumption include a sampling plan of production areas based on the study of contamination sources ( sanitary survey ) , critical for the classification .
the aim of the sanitary survey is the classification of areas in three classes , a , b or c. bivalves harvested from class a areas may be placed on the market for direct human consumption ; bivalves from class b areas must undergo purification ; the ones from class c areas must be relayed over a long period ( european commission , 2004 ) .
natural beds of clams ( chamelea gallina ) in the district of san benedetto del tronto ( south marche ) , hardly exploited for harvesting , have been recently reclassified ( marche region , 2013 ) and three of the seven areas previously classified as class a have been reclassified as class b areas .
furthermore , in the marche region , class a areas have been reduced from 39 to 16 .
some authors focused on the intermittent faecal contamination sources that may cause a seasonal trend of e. coli contamination ( campos et al . ,
2013 ) , depending mostly on the weather ( ritter et al . , 2002 ) , but also on the death rate of e. coli in sea water ( fujioka et al . , 1981 ;
kim and hur , 2010 ; rozen and belkin , 2001 ; troussellier et al . , 1998 ) .
recently , in the same district of san benedetto del tronto , a statistically significant relationship between rainfall and e. coli values exceeding the food safety criteria for direct human consumption [ > 230 most probable number ( mpn)/100 g ] has been demonstrated , particularly in areas with the highest density of inhabitants ( ciccarelli et al . , 2012 ) .
the aim of this study was to verify , by means of a statistical test , the seasonal trend of e. coli contamination monitored in the natural beds of clams in the district above mentioned .
we analysed the results of the monitoring of e. coli levels in the classified areas of the district of san benedetto del tronto ( figure 1 ) from 2005 to 2012 , provided by algaeadria database , a monitoring network for the whole adriatic area in order to collect and process homogeneous information on biotoxins , microbiological and phytoplankton ( http://algaeadria.org ) .
results are referred to two established sampling sites in each classified area : the first site ( i ) was 0.6 - 0.7 km offshore , 3 - 6 m deep ; and the second site ( ii ) was 1.2 - 1.3 km offshore , 6 - 9 m deep . in general , samples were collected quarterly , but during early years they were collected 4-monthly and later , bimonthly .
microbial analyses were performed by the veterinary public health laboratory of marche and umbria , fermo office , with the in force mpn method . by graphical data analysis tools (
helsel and hirsch , 2002 ) , two classes of results were recognised : compliant , if values were 230 mpn/100 g , and non - compliant , if values were > 230 mpn/100 g , whose rates and monthly distribution were evaluated . focusing on that ( figure 2 ) ,
data were related at three different periods : january - june and july - december ( combination a : 6 months and 6 months ) ;
march - september and october - february ( combination b : 7 months and 5 months ) ; march - june and july - february ( combination c : 4 months and 8 months ) .
the non - compliant rates were evaluated at each site and each seasonal combination as mentioned above ( figure 3 ) .
furthermore , non - parametric fisher s exact test was applied to data from each sampling site : the columns of the 2x2 contingency tables were the compliant and non - compliant values ; the lines were values in the first period of time and in the second one for each combination ( table 1 ) .
despite an irregular trend during the year , the non - compliant values of e. coli appeared to increase in the early half of the year , as shown in figure 2 .
indeed , the rates of non - compliant values were higher in the first period of speculated combinations than those in the second period , as in figure 3 . as regards the results of fisher s exact test , shown in table 1 , only for the site 19.02-i ( figure 1 )
p values seemingly confirmed that the rates of non - compliant values from march to june ( combination c ) were significantly higher than those from july to february ( p=0.043 ) .
nevertheless , if the bonferroni correction method for multiple tests is applied , the comparisonwise rate gets p0.016 ( p/k with k=3 ) , so none of p values rejected the null hypothesis .
the graphical analysis revealed , first of all , that e. coli had an irregular trend during the year in general , with spikes in early months .
this could be due to the local distribution of precipitations ( ciccarelli et al . , 2012 ) . in effect , by the comparison of the rates of non - compliant results in different seasonal combinations , we found that they significantly increased , in particular , for combination b , from march to september , and , for combination c , from march to june . this may allow a seasonal reclassification of the clam harvesting areas .
however , only for one sampling site ( 19.02-i ) and for combination c , the different rates appeared to have statistical significance , when the bonferroni correction is not applied .
this evidence could scientifically support a seasonal classification of production areas and could allow a more detailed classification due to the identification of the periods of time , during the year , with the lowest microbial levels .
statistical tools , such as non - parametric fisher s exact test , help to point out the significance of seasonal trends of e. coli contamination in shellfish . in effect , by the use of these methods , our study verified the seasonality of non - compliant values with a statistical significance . finally , it is intended to give a new scientific based support to competent authorities for seasonal classifications of bivalve mollusc harvesting areas and place on the market shellfish products meeting the requirements for direct human consumption .
in the european union , the classification of shellfish harvesting areas depends on levels of escherichia coli checked in shellfish flesh and determines whether post - harvest treatment required before shellfish can be sold for human consumption . 
 nevertheless , intermittent sources of contamination , such as rainfall and runoff from agricultural and urban lands , may give rise to seasonal variations of e. coli concentration , hence an annual classification could not be correct . in this study 
 , we investigated the microbial trend in clams ( chamelea gallina ) harvested from the district of san benedetto del tronto , italy . 
 the algaeadria database , a monitoring network for the whole adriatic area , provided results from 2005 to 2012 . 
 e. coli values compliant and non - compliant with food safety criteria were evaluated by graphical data analysis tools and one - tailed fisher s exact test . 
 the results showed a clear general seasonal trend and , in one of the considered areas , the non - compliant values from july to february were significantly lower than those from march to june ( p<0.05 ) . 
 these findings may scientifically support a seasonal classification .