ANALYSIS METHODS
Monitoring checks on bathing water are carried out every year from 1st April to 30th September. Samples are taken every fortnight for a total of 12 routine samples every year.
When the analyses carried out on a sample are unsatisfactory, even if just for one single established parameter, as well as trying to find and eliminate the possible causes of pollution, 5 supplementary water samples are taken on different days, in the same point. Furthermore, samples are also taken from the areas close by in order to be able to define the boundaries of the polluted area, which may have to be subjected to temporary bathing restrictions. Usually, additional samples are taken 50 and 100 metres to the north and south of the point where the unsatisfactory results were registered as well as, of course, in the point itself.
MONITORING METHODS
 The methods used for taking samples are defined in attachment 2 of DPR 470/82 (Presidential Decree) and the Ministry of Health Circular Letter dated 19/02/91, entitled "The Quality of Bathing Waters - Code of Conduct".
Samples are taken in the stretch of sea normally used by bathers and, more precisely, where seawater is between 80 and 120 centimetres deep. Along the coast of Emilia Romagna, this depth is usually registered only a few metres from the shoreline although it does vary depending on the tides.
In accordance with established regulations, samples are taken between 9.00 and 15.00, normally from a boat.
At the monitoring site, by law, the following are measured and recorded:
- approximate distance from the shore
- date and time
- water and air temperature, taken using a thermometer
- state of the sea (motion of the waves)
- direction and intensity of surface currents (visual assessment)
- direction and intensity of the wind (visual assessment)
- the presence of oils, phenols and surface-active agents (visual and olfactory assessment)
- anomalous changes in colour (visual assessment)
- transparency (Secchi's disc)
- dissolved oxygen and pH (using electrometric instruments)
Monitoring for the assessment of microbiological parameters (coliforms, streptococci and salmonella) is carried out using a sterile bottle that holds at least 500 ml of water. Using a special set of pincers, the open bottle is lowered to a depth of 30 centimetres. Once it has been extracted, the bottle is closed and placed into a refrigerated container awaiting analysis, which must be carried out within 24 hours.
Other samples may be collected on the surface, after visual corroboration, in order to check for the presence of oils, phenols and surface-active agents under laboratory conditions.
ANALYTIC METHODS
Microbiological assessment
The technical departments (analytic areas) of the provincial section of ARPA (Regional Agency for the Protection of the Environment) carry out all microbiological assessments.
Once they have been delivered to the laboratory, samples are promptly prepared for examination, which must be carried out the same day.
Analysis checks are carried out in accordance with existing laws (DPR 470/82).
Since 1995, the four regional laboratories qualified to carry out analyses on bathing water have used the membrane filter technique to assess for total coliforms, faecal coliforms and faecal streptococci. Before 1995, the laboratories of Ravenna and Rimini used the MPN technique.
Appropriate quantities of water to be examined, which may have been diluted depending on the probable level of pollution of the sample, are filtered through a sterile membrane (0.45 µ porosity) of cellulose nitrate using special filters connected to an air pump. The filters are then washed with sterile distilled water and the membrane is transferred onto agar in a Petri dish. This is then incubated in a thermostat at the appropriate temperature and for the pre-set length of time, as described in chart 4.
Chart 4 - Microbiological Assessment
| Parameters | Agar | Incubation temperature | Incubation time | Appearance of the colonies |
| Total coliforms | m-Endo Agar Les or m-Endo Agar * | 37 °C | 24 hours | Red colonies with metallic reflections |
| Faecal coliforms | m-FC Agar | 44 °C | 24 hours | Blue colonies |
| Faecal streptococci | m-Enterococcus Agar or Slanetz-Bartley | 37 °C | 48 hours | Pink and red colonies |
*in connection with laboratory experience
The information acquired from laboratory practices has allowed us to make the following observations:
Filtering of appropriate quantities of sea water
On the basis of experience and historical data recordings from the monitoring sites, it is clear that it is necessary to use appropriate quantities of water. In particular, with regards to total coliforms, it is preferable to filter either 50 ml or 10 ml in order to obtain a sufficient but not excessive number (30-80) of typical colonies (red with metallic reflections) on the dish, thus avoiding possible masking by other forms of flora that may confuse the analyses.
Use of substrata that are always the same in terms of composition and quality
With regards to the preparation of the m-Endo Agar substratum, it is best to adopt the following techniques:
- only prepare the agar at the time of use;
- only dissolve the powder for a few minutes;
- keep the dishes that are ready out of sunlight;
- carry out periodical test checks with wild-type strain.
Interpretation of the results.
 When examining water, in particular seawater, m-Endo Agar substrata allow for the growth of interference microorganisms that do not belong to the coliform group. Generally, these are microorganisms from the Vibrionacea and Pseudomonadacea families that form colonies that can easily be confused with coliforms. Red colonies with metallic reflections covering either the entire colony or just part of it are without doubt total coliforms. Red colonies without metallic reflections must be subjected to further confirmation checks. The number of non-typical colonies that need to be tested should indicatively be equal to the square root of the total numbered colonies or in any case, at least 5 per dish. These colonies should then be transferred onto selective agar dishes and the following checks carried out:
- Cytochrome oxidase (with ready to use sticks or using a specific reagent prepared at the time): this assessment makes it possible to distinguish Enterobacteria, which include coliforms, from Vibrionacee (Vibrio spp., Aeromonas spp.);
- Lactose fermentation, with the production of gas, for colonies that proved negative to cytochrome oxidase checks;
- Possible identification using miniaturized systems.
The faecal coliform and faecal streptococci parameters do not give rise to particular problems of interpretation and the reference analytical techniques are those described in DPR 470/82 and in Ministry of Health Decree dated 12/08/85.
Chemical and physical parameters
Transparency: is assessed using Secchi's disc and is expressed in metres. The measurement is read by sinking a white disc, with a diameter of 30 cm, into the water tied to a graduated rope. The distance of the disc's disappearance from sight is then assessed. From an operational point of view, the measurement is taken on the shaded side of the boat and the observer must maintain a constant distance from the water.
Colour: water colour is assessed visually and a record must be taken of any anomalous colour changes.
pH: this is usually assessed at the monitoring site using an electrometric instrument.
Mineral oils, surface-active agents and phenols: the assessment of the presence of these substances can either be carried out at the monitoring site by means of a visual (oils and surface-active agents) and olfactory (oils and phenols) inspection or it can be assessed in a laboratory. In the latter case, special samples should be collected.
Dissolved oxygen: assessment can be carried out at the monitoring site using electrometric instruments or it can be carried out in a laboratory, using chemical assessment methods. Portable gaugers indicate the concentration of oxygen in terms of mg/l. As DPR 470/82 states that the dissolved oxygen parameter be expressed in saturation percentage, it is necessary to also determine the salinity and temperature of the water and to then apply the formula that transforms the milligrams measured into the saturation percentage. In fact, the solubility of oxygen in water is influenced both by temperature and salinity. If this parameter is assessed in a laboratory, then a water sample needs to be collected, taking particular care to avoid air particles getting into the bottle and altering the real value of dissolved oxygen present. In order to get around this problem, bottles with a two-way tap are used. The oxygen is then fixed, for subsequent titration in laboratory, by adding magnesium solphate and iodide-sodium azide. Another sample is also taken in order to assess chlorides and therefore, salinity in the laboratory.
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