Report No SR 97 (07) F

Investigation of the relationship between indicator bacteria in mussel flesh and intervalvular fluid and surrounding waters Phase 3

D P Milne, J E Higgins and I J Brodie

SR 97 (07) F

January 1998


  1. The EC Directive on the Quality Required of Shellfish Waters, commonly known as the EC Shellfish Waters Directive (79/923/EEC) stipulates a microbiological requirement of 300 faecal coliforms per 100ml of shellfish flesh and intervalvular fluid (FIV). After two years of sampling, it emerged from results collated from all over the UK, that mussels collected from sites remote from any notable sewage input were consistently exceeding this standard. Archives revealed that there was a great dearth of research in this area; therefore it was decided, in 1992, to fund a preliminary investigation (report SR3553) into the relationship between Faecal Coliform (FC) levels in mussel FIV and their surrounding waters. It soon became clear that there was no empirical relationship and a further study (report SR[97]03F) revealed a complex relationship, which would probably take years to develop. This fueled a search for an alternative indicator bacterium. Faecal streptococci (FS) was found to be a promising candidate and evidence from a new investigation (report SR[97]04F) strengthened this case. In addition, the above report exposed the 24 hour analysis delay phenomenon and described the development of a pour plate method, as an alternative to the Most Probable Number (MPN) method, for enumerating mussel FS FIV.
  2. The objectives of this study SR[97]05F which follows on from report SR[97]04F were:
    1. To monitor FC and FS levels in mussels which have been relayed from highly polluted waters to non-polluted waters and from moderately polluted waters to highly polluted waters.
    2. To monitor FC and FS in mussels and their adjacent waters, with the express purpose of further developing the empirical relationship.
    3. To further develop the FS pour plate method for the quantitative analysis of mussel FIV.
    4. To investigate the apparent 24 hour analysis delay phenomenon exhibited by FC and FS in mussel flesh.

  3. There is a significant linear and polynomial relationship between FS in mussel FIV and FS in their surrounding waters, when the FIV was analysed by the MPN method.
  4. There is a very strong significant linear and polynomial relationship between FS in mussel FIV and FS in their surrounding waters, when the FIV was analysed by the project pour plate method.
  5. The pour plate method displays a higher correlation coefficient than the MPN method. Therefore, not only is the pour plate method quicker and cheaper, but it is also more reproducible.
  6. The case to replace FC FIV levels with FS FIV levels, as the major bacteriological parameter in Directive 79/923/EEC, is becoming more powerful. A preliminary approach to the European Environment Agency, to discuss the topic, should be made. The case should be strengthened by further studies.
  7. The experiment in which mussels collected from a polluted site, were relayed to an apparent non-polluted site, will have to be declared void. FC and FS water levels recorded in the adjacent waters ranged from 30-180/l00ml and 200-2000/l00ml respectively. Therefore, the site could not be classified as nonpolluted as the standards required were set at <l0/l00ml for both indicators.
  8. The experiment in which mussels collected from a moderately polluted site were relayed to a highly polluted site provided further evidence that FIV FS levels are a suitable indicator of faecal pollution in mussels. After the relaying period was completed, the levels in both the tidal and sub-tidal zones increased by an order of magnitude. These experiments should be continued.
  9. The time delay work carried out for this report has demonstrated that FIV FC and FS levels appear to differ significantly 24 hours after sampling, compared to 6 hours or less after sampling. As it is common practice all over the UK for procedures relating to mussel sampling and analysis to include a 24 hour or greater delay, these findings could have profound and lasting repercussions. Therefore, this phenomenon should be studied in greater detail.

Key Words

Mussels, Mytilus edulis, faecal coliforrns, faecal streptococci, Most Probable Number, Pour Plate Method, 24 hour analysis delay phenomenon, EC Shellfish Hygiene Directive, EC Shellfish Waters Directive.

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