Widespread microbiological groundwater contamination in the South-eastern Salento (Puglia-Italy)

Abstract

In the Salento peninsula (Puglia Region, South-East Italy), underground waters are a fundamental resource for the population because they constitute the principal reservoir for drinking water and irrigation. They are, however, affected by overexploitation. The risk factors in the Salento arise mainly from anthropic activities, especially tourism and agriculture (leaking wells, sewage and inadequate waste disposal procedures). The Southern Salento is recognized to be at high risk of pathologies characterised by oral–faecal transmission. From 2001 to 2009 the incidence of typhoid fever in the Salento was 12.11/100 000 inhabitants as against 2.91 in Italy. Enteritis caused by rotaviruses is an important cause of hospitalization of paediatric-aged children in the Salento, with high social costs. An effective monitoring system for the conservation and management of water bodies and the protection of public health is therefore fundamental. The present study sought to determine the microbiological and chemical–physical quality of groundwater in the Salento and to analyse the factors associated with contamination. The results indicated widespread pollution from salt and microbial contamination. Contamination from faecal microorganisms posed a significant risk of human infection in 100% of samples. Furthermore, the water was unsuitable even for irrigation in a high percentage of cases (31.8%), which is of considerable significance given that agriculture is one of the most important economic activities in the area under study. The high salt concentration was probably due to excessive extraction of water for intensive irrigation, especially in summer. Under these circumstances, some of mitigation activity is necessary. Furthermore, it would be advisable to decrease the pollution load from anthropic activities in the territory and to reduce water consumption in order to conserve groundwater resources especially.

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Environmental impact

Throughout the world there is evidence of contaminated groundwater leading to outbreaks of disease and contributing to endemic background disease in situations where groundwater used for drinking water or irrigation has become contaminated. In the studied area (Salento Peninsula, Apulia, Italy) groundwater is the principal reservoir for drinking water and irrigation. The risk factors in the Salento arise mainly from anthropic activities such as agriculture (leaking wells, sewage and inadequate waste disposal procedures). Moreover the Southern Salento is recognized to be at high risk of pathologies characterised by oral–faecal transmission (incidence of typhoid fever was 12.11/100 000 inhabitants) The aim of this study was to estimate the microbial and chemical–physical quality of groundwater in the South-eastern Salento (Province of Lecce, Italy), with the purpose of formulating mitigation strategies. For the reasons expounded above the authors believe the contribution of this research study warrants its publications in the Journal of Environmental Monitoring.

1 Introduction

With the world population steadily increasing, there is rising demand for water.

The use of groundwater as a source of drinking water is often preferred because of its generally good microbial quality in its natural state. Nevertheless, it is easily contaminated and outbreaks of disease from contaminated groundwater sources have been reported by countries at all levels of economic development.^1,2

The microbiological contamination of groundwater has profound implications for public health, particularly in small communities and developing countries where groundwater is the only source of drinking and irrigation water.^3,4

However, understanding the impact of groundwater on public health is often difficult and the interpretation of health data is a complex task. It is made even more difficult by the fact that many water supply systems that use groundwater are small and both outbreaks and background levels of disease are thus unlikely to be detected, especially in countries with limited health surveillance.⁵

The global incidence of waterborne disease is significant, though it can only be estimated, since reliable data are not sufficiently available for direct assessment.⁶ The contribution of groundwater to the global incidence of waterborne disease cannot be assessed easily, as there are many competing transmission routes. Confusion of socioeconomic and behavioural factors is frequent, definitions of outcome vary and, exposure–risk relationships are often unclear.⁷ Many outbreaks of waterborne disease could have been prevented by better understanding and management of groundwater in terms of public health. Pathogenic contamination has often been associated with simple deficiencies in sanitation but also with inadequate understanding of how to attenuate disease agents in surface waters.⁸

Throughout the world there is evidence of contaminated groundwater leading to outbreaks of disease and contributing to endemic background disease in situations where groundwater used for drinking water or irrigation has become contaminated.⁹ However, diarrhoeal disease transmission is also commonly due to poor excreta disposal practices.¹⁰ Furthermore, water that is of good quality at its source may be re-contaminated during extraction, transport and household storage.^11,12

The most comprehensive reports of waterborne disease outbreaks come from two countries, the USA and the UK, and some indications of the role of groundwater in the spread of infectious diarrhoeal disease can be estimated in these countries. Indeed, outbreaks of waterborne disease in these countries due to the consumption of untreated groundwater have been described by many authors.^7,13–16

In developing countries, evidence of the role of groundwater in causing disease outbreaks is more limited, although there have been numerous studies of the impact of drinking water, sanitation and hygiene on diarrhoeal disease. In a review of diarrhoeal disease in Nepal in relation to water and sanitation, Pokhrel and Viraraghavan¹⁷cite examples from South Asia where contamination of groundwater supplies has led to outbreaks of disease. A study of the local population in Uganda recorded an overall incidence rate of waterborne disease of 80.1 per 1000.¹⁸ Few studies of this issue have been carried out in Italy. After a major gastroenteritis outbreak, which was reported in a vacation resort in Central Italy in 2003,¹⁹ environmental investigations revealed serious faecal contamination of the groundwater and the presence of Noroviruses in the seawater near the resort. In many cases the groundwater, responsible for secondary contamination of drinking water, had probably been contaminated by illegal household sewage disposal.¹⁹

In the Salento, groundwater is a fundamental resource for the population, because it constitutes the principal reservoir for drinking water and irrigation. It is, however, affected by overexploitation. The risk factors in the Salento arise mainly from anthropic activities such as agriculture (leaking wells, sewage and inadequate waste disposal procedures).

Considering the extent to which pollution can affect water supply sources and groundwater in particular, the system of controls introduced thanks to EU directive 2000/60/EC²⁰ may be considered the first step towards safer water distribution. This needs to be integrated with further and more complete prevention measures that take account of the risk level specific to local conditions.^21,22

The aim of this study was to estimate the microbial and chemical–physical quality of groundwater in the South-eastern Salento (Province of Lecce, Italy), with the purpose of formulating mitigation strategies.

Bacterial indicators (Total coliforms, Faecal coliforms, Escherichia coli and Enterococci), viral indicators (somatic coliphages) and pathogenic viruses (Rotaviruses) were all measured in order to assess the sanitary risks. This approach was adopted because currently applied biological indicators, such as coliforms, do not provide information on the risks posed to public health by enteric viruses in water sources.²³ Waterborne disease outbreaks have occurred in systems that tested negative for coliforms, and positive coliform results do not necessarily correlate with viral risk. It is widely recognized that bacterial indicators do not co-occur exclusively with infectious viruses, nor do they respond in the same manner to environmental or engineered stressors.

Somatic coliphages have been proposed as model viruses in water quality control,²⁴ and feasible standardised methods for their enumeration are available.²⁵ They are analogous in structure, size, composition and morphology to pathogenic human enteric viruses but unlike these viruses, they are easily detected by simple, fast and inexpensive methods. They are excreted in high quantities (10–10⁸ PFU g⁻¹ of stool), following the same environmental dispersion dynamics as enteric viruses. For these reasons somatic coliphages could be used as indicators of viral contamination.²⁶

Group A rotaviruses are the greatest cause of acute gastroenteritis in children below 2 years of age.²⁷ They are widely distributed in nature and are excreted in large numbers in the faeces of infected individuals.^28–30 There are many cases of waterborne epidemics due to rotaviruses,³¹ which have been isolated in sewage, river water, groundwater and drinking water.³² Furthermore Rotaviruses have been proposed as indicators of faecal viral contamination of water by the United States Environmental Protection Agency.³³

2 Materials and methods

2.1 The case study region

The studied area is located in South-eastern Italy (Fig. 1). It has a total area of 140.8 km² and a demographic density of 163 inhabitants per km², with a total population of 22 889 inhabitants.³⁴

Fig. 1 Distribution of wells in the studied area.

Most of the land in the area is used for agriculture, which accounts for 31% of the economy. Livestock rearing accounts for only 2.33% of the land in the area under study.³⁴

In addition, tourism is constantly increasing; it is concentrated above all in the summer, and is centred on in the city of Otranto, which contains more than 96% of tourist buildings. The official wells are evenly distributed throughout the territory and nearly all of them are used for irrigation.³⁴

2.2 Sampling strategy and water analysis

The study was carried out in the period October 2007 to September 2008. Bimonthly sampling was performed at 22 wells, all drawing from the same aquifer, located in five villages in the South-eastern Salento: Otranto, Uggiano la Chiesa, Giurdignano, Muro Leccese and Poggiardo (Fig. 1).

Of the 22 examined wells, 10 were affected by risk factors located within 200 metres (the limit indicated as a buffer by the Water Framework Directive 2000/60/CE²⁰). Six of the wells are used both for drinking water and irrigation, and 16 are used only for irrigation (Table 1).

Table 1 Characteristics of studied wells

	Well ID	GPS coordinates		Depth below surface/m	Use	Risk from
		E	N
Otranto	0	281740.76	4448940.53	76	Irrigation	Tourism
	1	2813990.5	4458558.9	94	Irrigation	Agriculture–tourism
	2	2813195.27	4454785.42	82	Irrigation–drinking water	Agriculture
	3	2814035.2	4455224.7	65	Irrigation	Agriculture–tourism
	4	2814778.17	4452634.3	90	Irrigation–drinking water	Agriculture
	5	2814995.5	4452471.1	75	Irrigation	Agriculture
	6	2816415.9	4450836.5	70	Irrigation	Agriculture–oil mill–wastewater treatment plant
	7	2817236.9	4447532.8	83	Irrigation–drinking water	Agriculture–tourism–livestock
Giurdignano	8	2812850.0	4449063.0	63	Irrigation	Agriculture
	9	2812562.94	445894.0	98	Irrigation	Agriculture
	10	2810715.9	4448635.9	62	Irrigation	Agriculture
Uggiano la Chiesa	11	2813983.7	4447339.0	52	Irrigation	Agriculture
	12	2813711.4	4445115.6	68	Irrigation	Agriculture
	13	2815945.61	4444074.71	65	Irrigation–drinking water	Agriculture
Muro leccese	14	2804543.99	4444413.32	70	Irrigation	Agriculture
	15	2803366.78	4443291.78	72	Irrigation–drinking water	Agriculture
	16	2806914.4	4446267.1	65	Irrigation	Agriculture
	17	2805437.6	4447400.9	80	Irrigation–drinking water	Agriculture
Poggiardo	18	2807750.26	4437984.61	84	Irrigation	Agriculture–rubbish dump
	19	2808255.99	443999.61	87	Irrigation	Agriculture–rubbish dump
	20	2808836.55	4438720.56	81	Irrigation	Agriculture–rubbish dump
	21	2809785.52	4439710.54	80	Irrigation	Agriculture–rubbish dump

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Each sampling was performed between 09:00 and 15:00 in calm weather conditions; for each well two samples were collected (A and B) in 1000 ml sterile bottles (Abba type). In order to prevent any external contamination, water was run through the tap for 10 minutes in order to clean the pipe before collection. Moreover the tap was sterilized with a Bunsen burner before sampling. The samples were then taken to a laboratory in a refrigerator (+4 °C) and were examined within 4–5 hours.

Microbial and chemico-physical analyses were performed for every considered parameter on six samples for each of the 22 wells, making 132 analyses in total.

The temperature, salinity and pH of the well water were measured in situ with a multiparametric probe (WTW MultiLine P4).

The results are given as the average for each well of the values recorded at all samplings. The indicators of faecal contamination were compared with the limits established by Italian law D. lgs 31/2001³⁵ was passed in response to European Union directive 98/83/EC (drinking water), the Italian law on irrigation waters³⁶ and the Ministerial Decree of 23 March 2000³⁷ concerning the quality of irrigation water in terms of salinity and electrical conductivity.

Total coliforms, E. coli, Enterococci, somatic coliphages and Rotaviruses were evaluated for each sample.

For the estimation of Total coliforms (TF), 100 ml sub-samples were filtered. The filter membranes were placed on m-Endo agar plates which were then incubated for 24 hours at a temperature of 37 °C; the count was performed by considering dark red colonies with a metallic reflex, and the results were expressed as CFU per 100 ml (colony-forming units).³⁸

E. coli were identified using TTC agar; the yellow colonies were enumerated after 24 h at 37 °C.³⁹

Faecal Enterococci (FE) were identified using m-Enterococcus agar. After filtration and incubation of the plates at 37 °C for 48 hours, the pink-red colonies were considered as positive.³⁸

The results were expressed as CFU (colony-forming units) per 100 ml, taking the mean of 2 determinations.

To detect Rotaviruses and somatic coliphages, 100 litres of water from each well were concentrated by the tangential flow ultrafiltration technique, using polypropylene membranes with a 10 kDa molecular weight cut-off. These filters have been used in previous studies to concentrate viruses present in the water, particularly enteroviruses and bacteriophages, and have been shown to have a high capacity for recovery of both the former and the latter.^40,41,42

In order to eliminate particulate matter, the samples were pre-filtered with 12 µm polypropylene membranes. To recover the viral particles that could have remained attached to the membrane, the pre-filter was washed with 3% Beef Extract (B.E.) at pH 9.⁴⁰

Before the viral concentration phase the filtering membrane was conditioned with 3% B.E., pH 7.⁴¹

The concentration of the water samples was performed in two consecutive steps using firstly a MAXIFLEX 25 ECO filtration system (Schleicher & Schuell, Dassel, Germany) equipped with a polysulfone membrane with a surface area of 0.1 m² and secondly an ULTRAN-MINIFLEX filtration system (Schleicher & Schuell, Dassel, Germany) equipped with a membrane with a smaller surface area (0.0024 m²).

The concentration process (in both steps) was completed by washing the filters with 3% B.E. at pH 9, in order to remove any viral particle that could have adhered to them.⁴¹ A volume of about 300 ml was obtained after the first concentration and about 40 ml after the second.

The concentrated samples were neutralized with HCl 1 N, decontaminated with chloroform (1 : 10) and processed according to the following procedures.

For rotavirus identification, viral RNA was extracted using the “QIAamp Viral RNA mini Kit” (QIAGEN AG, Basel, Switzerland), following the manufacturer's instructions. RT-Nested PCR for human rotaviruses was performed following the protocol described by Gilgen et al.,⁴³ in which two pairs of primers are used (RV1/RV2 and RV3/RV4), both specific to the amplification of two fragments of the VP7 gene. In the first amplification a genomic fragment of 1059 bp was obtained, while in the second amplification a G-type-specific fragment of 346 bp was obtained (nucleotides 50 to 395). A positive control (rotavirus RNA) and negative control (RNase-Free water) were included for each PCR assay. PCR products were resolved on 2% agarose.

The G-genotype was determined by aligning the nucleotide sequences obtained with the reference sequences available in the GenBank database using the BLAST programme (NCBI).⁴⁴

The enumeration of somatic coliphages was performed using the double-layer agar technique.⁴⁵E. coliWG5, a strain isolated by the Department of Microbiology at the University of Barcelona, Spain, was used as the host strain. The reference phage for positive controls was ΦX 174.

The results were expressed as PFU (plaque-forming units) per 100 ml, taking the mean of 3 measurements.

2.3 Data analysis

Statistical analysis was carried out using StatSoftStatistica8®. Descriptive statistical analysis was performed on each parameter in order to describe the basic features of the data.

The spatial–temporal distribution was analyzed for each parameter and two-way ANOVA (α 0.05) was performed on the standardized data in order to identify statistically significant variation.

3 Results

3.1 Physical parameters

The mean values of the chemical–physical parameters (temperature, pH and electrical conductivity) are shown in Table 2. The temperature values were homogeneous (P > 0.05), except for a small increase near the coast. The pH ranged from 7.3 to 7.7. The electrical conductivity values were high in all sampling sites, indicating saltwater intrusion into the groundwater, especially where extraction rates were higher due to extensive irrigation. In 45.5% of samples the conductivity ranged from 0.25 to 0.75 mS cm⁻¹. In the remaining 54.5% of samples, mean electrical conductivity was over 0.75 mS cm⁻¹. This is the threshold value for high salinity water, above which water is not suitable for irrigation (Table 3). No significant variation (P > 0.05) in conductivity between sites related to the distance from the sea was recorded.

Table 2 Mean values of physical parameters at each sampling site

	ID wells	Temperature/°C			pH			Electrical conductivity/mS cm^−1
		Mean ± sd	Min	Max	Mean ± sd	Min	Max	Mean ± sd	Min	Max
Otranto	0	18.4 ± 0.7	17.9	19.9	7.3 ± 0.2	7.1	7.5	1.1 ± 0.7	1.1	1.2
	1	18.4 ± 0.3	18.0	18.7	7.3 ± 0.1	7.1	7.4	1.7 ± 0.6	1.6	1.8
	2	18.0 ± 0.6	17.0	19.0	7.6 ± 0.2	7.4	7.8	0.5 ± 0.6	0.5	0.6
	3	18.2 ± 1.1	16.5	19.2	7.4 ± 0.2	7.1	7.6	0.6 ± 0.1	0.5	0.7
	4	18.3 ± 5.3	10.1	26.0	7.5 ± 0.2	7.3	7.7	0.9 ± 0.03	0.8	0.9
	5	18.0 ± 0.8	17.2	19.5	7.4 ± 0.2	7.1	7.7	1.1 ± 0.01	1	1.1
	6	19.6 ± 0.6	18.3	19.8	7.4 ± 0.3	7.1	7.8	1.1 ± 0.2	0.6	1.1
	7	17.9 ± 0.4	17.4	18.3	7.6 ± 0.2	7.3	7.8	0.6 ± 0.02	0.5	0.6
Giurdignano	8	17.4 ± 0.6	16.6	18.4	7.4 ± 0.2	7.2	7.7	1.5 ± 0.03	1.5	1.6
	9	17.9 ± 0.3	17.5	18.2	7.5 ± 0.2	7.3	7.8	1.2 ± 0.4	0.6	1.4
	10	17.3 ± 0.5	16.4	17.9	7.3 ± 0.2	7.0	7.6	0.6 ± 0.02	0.6	0.7
Uggiano la chiesa	11	18.0 ± 0.6	17.2	19.0	7.6 ± 0.1	7.5	7.9	0.6 ± 0.02	0.6	0.7
	12	18.0 ± 0.4	17.4	18.4	7.4 ± 0.2	7.2	7.6	0.9 ± 0.01	0.92	0.95
	13	18.5 ± 0.5	17.5	19.1	7.5 ± 0.1	7.3	7.7	0.7 ± 0.02	0.7	0.8
Muro Leccese	14	16.5 ± 1.3	14.6	17.9	7.5 ± 0.2	7.4	7.8	0.8 ± 0.02	0.78	0.84
	15	16.6 ± 1.1	17.4	18.0	7.4 ± 0.1	7.2	7.5	0.6 ± 0.03	0.5	0.6
	16	17.8 ± 0.6	18.8	18.4	7.5 ± 0.3	7.1	7.9	0.7 ± 0.04	0.6	0.8
	17	17.6 ± 0.6	16.8	18.2	7.4 ± 0.1	7.2	7.6	0.8 ± 0.02	0.79	0.85
Poggiardo	18	18.1 ± 0.5	17.5	18.7	7.5 ± 0.3	7.2	7.9	0.4 ± 0.2	0.8	0.9
	19	18.5 ± 0.7	17.4	19.3	7.4 ± 0.02	7.3	7.5	0.8 ± 0.02	0.8	0.9
	20	14.6 ± 1.6	14.2	18.4	7.6 ± 0.2	7.3	7.8	0.8 ± 0.01	0.8	0.9
	21	17.7 ± 1.0	15.7	18.7	7.4 ± 0.4	7.1	8.0	0.7 ± 0.01	0.5	0.8

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Table 3 Suitability for agriculture based on conductivity

Salinity	mS cm^−1	No. of samples	Evaluation	% of samples analyzed
Low	<0.25	0	Suitable for irrigation in every kind of soil	0
Medium	0.25–0.75	10	Suitable for irrigation of salt-tolerant plants and well-drained soil	45.5
High	0.75–2.25	12	Not suitable for soil with poor drainage	54.5
Very high	>2.25	0	Not suitable in general, for any kind of soil	0

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3.2 Microbial parameters

The results (see Table 4) show widespread contamination of faecal origin.

Table 4 Mean values of microbial indicators recorded in each well during the monitoring period^35–37

	ID wells	Bacteria (CFU per 100 ml)									Viruses
		Total coliforms^a			Escherichia coli ^b			Enterococci^c			Rotavirus	Somatic coliphages (PFU per 100 ml)
		Mean ± sd	Min	Max	Mean ± sd	Min	Max	Mean ± sd	Min	Max		Mean ± sd	Min	Max
a Limits for drinking water 0 CFU per 100 ml. b Limits for drinking water 0 CFU per 100 ml. Limits for irrigation water 100 CFU per 100 ml. c Limits for drinking water 0 CFU per 100 ml.
Otranto	0	71.2 ± 89.6	6	250	15.2 ± 22.7	0	55	2.2 ± 4.3	0	10	—	11.6 ± 5.2	0	14
	1	58.8 ± 78.2	0	200	23.2 ± 38.3	0	100	1.4 ± 1.1	0	2	—	4.8 ± 8.1	2	24
	2	7.2 ± 12.0	0	30	1.8 ± 3.6	0	8	0.0	0	0	—	2.8 ± 2.1	0	6
	3	36.2 ± 36.1	1	100	10.2 ± 16.1	0	40	34.3 ± 81.0	0	200	—	4.2 ± 3.6	0	9
	4	95.0 ± 80.2	3	250	64.2 ± 91.5	10	250	0.7 ± 1.6	0	4	—	18.3 ± 7.8	10	32
	5	100.8 ± 92.5	5	200	73.2 ± 80.7	3	200	2.8 ± 5.5	0	14	—	25.4 ± 16.6	3	43
	6	68.7 ± 50.0	0	100	11.5 ± 22.7	0	60	1.5 ± 3.2	0	8	—	12.4 ± 3.7	6	16
	7	98.7 ± 81.0	17	200	42.2 ± 27.4	6	80	0.5 ± 1.2	0	3	—	18.2 ± 5.4	12	26
Giurdignano	8	35.3 ± 56.3	10	150	3.0 ± 2.7	0	6	0.0	0	0	—	7.8 ± 4.2	0	12
	9	163.8 ± 178.0	30	500	130.0 ± 188.4	2	500	0.2 ± 0.4	0	1	—	24.7 ± 15.8	0	46
	10	34.2 ± 81.3	0	200	16.7 ± 40.2	0	100	0.0	0	0	—	5.8 ± 13.2	0	35
Uggiano la chiesa	11	21.3 ± 20.3	0	50	17.5 ± 22.3	0	46	0.0	0	0	—	8.6 ± 4.7	0	12
	12	19.7 ± 26.7	0	65	4.5 ± 4.2	0	11	0.0	0	0	—	9.4 ± 10.1	0	21
	13	70.2 ± 113.4	4	300	69.3 ± 114.5	3	300	2.5 ± 3.9	0	10	—	17.4 ± 11.4	0	35
Muro Leccese	14	17.7 ± 30.9	0	80	1.8 ± 4.2	0	10	0.7 ± 1.0	0	2	—	3.4 ± 3.7	0	8
	15	78.2 ± 950.	0	200	52.8 ± 77.6	0	200	2.3 ± 4.2	0	10	—	16.9 ± 14.7	3	33
	16	10.3 ± 14.2	0	35	1.5 ± 3.7	0	9	0.0	0	0	—	1.7 ± 4.7	0	12
	17	0.2 ± 0.4	0	1	0.0	0	0	0.0	0	0	—	0.0	0	0
Poggiardo	18	9.2 ± 16.3	0	40	3.2 ± 7.3	0	18	1.2 ± 2.4	0	6	—	<1	0	0
	19	23.3 ± 39.2	0	100	1.0 ± 2.5	0	6	0.0	0	0	—	<1	0	0
	20	68.3 ± 51.5	20	150	7.0 ± 8.4	0	20	1.5 ± 2.4	0	6	—	5.8 ± 2.4	0	12
	21	37.5 ± 79.0	0	200	1.5 ± 2.4	0	5	5.8 ± 14.3	0	35	—	2.4 ± 3.2	0	9

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For each parameter the Anova analysis showed high spatial and temporal variability (P < 0.05).

Total coliforms (TC) had values above zero in 79.9% of the 132 analyzed samples, with a mean concentration of 51.15 CFU per 100 ml. The maximum value was 500 CFU per 100 ml. TC had values above zero in all 22 wells that were examined. Spatial distribution analysis (Table 4) highlighted a higher concentration of TC at well 9 (164 CFU per 100 ml) located in the urban area of Giurdignano, wells 4 and 5 (95 and 101 CFU per 100 ml respectively) located in the centre of Otranto, and wells 0 (71 CFU per 100 ml) and 7 (99 CFU per 100 ml) located in south-east of Otranto. The lowest TC values were recorded in Muro Leccese and Poggiardo.

Concentrations of E. coli were high in 61.4% of samples (81 cases); the mean value was 25 CFU per 100 ml and the maximum was 500 CFU per 100 ml. Wells affected by this contamination were 9 (130 CFU per 100 ml) in Giurdignano, 4 (64 CFU per 100 ml) and 5 (73 CFU per 100 ml) in the centre of Otranto and 13 (70 CFU per 100 ml) in Uggiano La Chiesa. The lowest contamination from E. coli was again recorded in Muro Leccese and Poggiardo (Table 4). Contamination from Enterococci (Table 4) was lower than the other faecal indicators. The mean concentration was 2.57 CFU per 100 ml and the maximum was 200 CFU per 100 ml. In 31.82% of the studied wells Enterococci were always absent. The highest contamination from Enterococci was recorded in well 3 (34 CFU per 100 ml) located in the centre of Otranto.

The mean values for somatic coliphages were between 0 and 25 PFU per 100 ml, with a maximum of 46 PFU per 100 ml recorded in Uggiano la Chiesa in January (Table 4).

Rotaviruses were always absent (Table 4).

Comparing the results with the legal limits for drinking water and irrigation water, it was found that 95.4% of wells were not suitable for drinking water, while 31.8% were not suitable for agriculture (Table 5).

Table 5 Number of wells with water not suitable for drinking or irrigation

Use	No. of wells	% of wells
Water not suitable for drinking	21	95.4
Irrigation water with infection risk (E. coli > 100 CFU per 100 ml)	7	31.8

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4 Discussion

In the Salento groundwater is a fundamental resource for the population because it is the principal reservoir for drinking water and irrigation.

Our study showed that faecal contamination of the groundwater was significant and widespread in the 5 studied municipalities of the South-eastern Salento. Bacterial contamination of groundwater is a common problem in many rural areas and it may be related to manure spreading or livestock rearing.⁴⁶ In our study, the high concentration of faecal indicators (mainly E. coli) suggests that human waste was the source of this contamination; inadequate management of liquid waste is the strongest anthropogenic impact in the region, including its tourist areas, especially in the summer. The possible sources of human waste in this area include two wastewater disposal systems that discharge waste directly into the groundwater of Otranto and Uggiano la Chiesa. The high concentrations of bacteria and coliphages recorded in the groundwater monitored suggest that disinfection of the sewage was insufficient. Moreover, the high number of holiday homes in the studied area generates strong pressure due to their sub-standard septic tanks, which discharge wastewater into the ground.

The high contamination from faecal microorganisms poses a serious risk of infection and thus compromises the quality of drinking water in almost 100% of samples. Moreover, in a high percentage of cases the water was not suitable for irrigational use either, and this is a serious problem given that agriculture is one of the most important sectors of the economy in the studied area. The hygiene of agricultural product (mainly vegetables to be eaten raw),^47,48 however, also represents a health risk,⁴⁹ given the possible spread of oral–faecal diseases (e.g. typhoid, hepatitis A, salmonella, diarrhoea, etc.) and considering the high consumption of this type of product. This takes on even greater importance if you consider that the studied area and the southern Salento in general are recognized as being at high risk of pathologies of oral–faecal transmission. During the last few years the incidence of Hepatitis A has fallen, thanks partly to vaccination in the Puglia Region. The incidence of typhoid fever on the other hand is still high. The data recorded in the period 2001–2009 show an incidence of typhoid fever in the Salento of 12.11/100 000 inhabitants as against 10.94 in Puglia and 2.91 in Italy. It is reasonable to suppose that such data are fairly homogenous throughout the Salento and therefore also in the 5 municipalities considered.⁴⁹

The presence of Somatic coliphages in the analyzed water shows real faecal contamination. They were detected even in samples without any bacterial contamination (Total coliforms and E. coli absent). These findings suggest that quantification of somatic coliphages should be combined with the faecal indicators normally used to evaluate the quality of water for drinking or irrigation, in order to provide a more detailed analysis of the hygienic and sanitary quality of these waters.

Rotaviruses were always absent, although epidemiological data show that infections caused by rotaviruses are a major cause of hospitalisation of paediatric-aged children in the Salento peninsula, with high social costs.^50–53 Contrary to the EPA's indications, their absence in microbiologically contaminated samples suggests that they are not suitable as indicators of viral contamination.

The high salinity values are presumably caused by excessive extraction of water, especially in summer when the demand for water is higher both for drinking and irrigation. This was observed not only near the coast (where marine waters tend to invade the fresh water-bearing stratum which lies on a deeper stratum of saltwater) but throughout the studied area.

5 Conclusions

Our study has shown a situation of widespread pollution from salt and microbial contamination.

These circumstances require the timely implementation of a routine monitoring system for water bodies, in accordance with the European water directives, which establishes groundwater quality standards. These standards need to take account of local and regional conditions (e.g. hydrogeology, nature of topsoil, interactions with associated aquatic and terrestrial ecosystems, types of pressures, etc.). Microbial contamination of groundwater should therefore receive special attention in any groundwater monitoring programme, given the high incidence of waterborne disease in the studied area.⁴⁹

Furthermore, it would be advisable to reduce the pollution load from anthropic activities in the area and to promote sustainable use of water, reducing consumption through the reuse of treated wastewater in agriculture.

Acknowledgements

This study was supported and funded by the CUIS- Interprovincial Consortium of the University of Salento. Furthermore, we acknowledge the excellent cooperation of the municipal administrations of the Otranto, Uggiano la Chiesa, Muro Leccese Giurdignano and Poggiardo.

References

1. C. O'Reilly, Epi-Aid #2004-076 Final Trip Report: Outbreak of Gastroenteritis with Multiple Etiologies among Resort Island Visitors and Residents—Ohio, 2004, Centers for Disease Control and Prevention, Atlanta, GA, 2005.
2. S. Fetouani, M. Sbaa, M. Vanclooster and B. Bendra, Assessing ground water quality in the irrigated plain of Triffa (north-east Morocco), Agric. Water Manage., 2008, 95, 133–142.
3. C. E. O'Reilly, A. B. Bowen, N. E. Perez, J. P. Sarisky, C. A. Shepherd and M. D. Miller, et al., A waterborne outbreak of gastroenteritis with multiple etiologies among resort island visitors and residents: Ohio, 2004, Clin. Infect. Dis., 2007, 44, 506–512.
4. G. K. Holz, Seasonal variation in groundwater levels and quality under intensively drained and grazed pastures in the Montagu catchment, NW, Tasmania, Agric. Water Manage., 2009, 96, 255–266.
5. A. Prüss and A. Havelaar, The Global Burden of Disease Study and Applications in Water, Sanitation and Hygiene, in Water Quality: Guidelines, Standards and Health, ed. L. Fewtrell and J. Bartram, IWA Publishing, London, 2001, pp. 43–59.
6. A. Prüss-Üstün, D. Kay, L. Fewtell and J. Bartram, Unsafe Water, Sanitation and Hygiene, in Comparative Quantification of Health Risks: Global and Regional Burden of Disease Attributable to Selected Major Risk Factors, ed. M. Ezzati, A. D. Lopez, A. Rodgers and C. J. L. Murray, World Health Organization, Geneva, 2004, vol. 2, pp. 1321–1352.
7. P. Payment and P. R. Hunter, Endemic and Epidemic Infectious Intestinal Disease and its Relationship to Drinking Water, in Water Quality: Guidelines, Standards and Health, ed. L. Fewtrell and J. Bartram, IWA Publishing, London, 2001, pp. 61–88.
8. R. Jamieson, R. Gordon, D. Joy and H. Lee, Assessing microbial pollution of rural surface waters: a review of current watershed scale modelling approaches, Agric. Water Manage., 2009, 70, 1–17.
9. A. Rahman and H. K. Lee, Domestic water contamination in rapidly growing megacities of Asia: case of Karachi, Pakistan, Environ. Monit. Assess., 1997, 44, 1–3.
10. V. Curtis, S. Cairncross and R. Yonli, Domestic hygiene and diarrhoea—pinpointing the problem, Trop. Med. Int. Health, 2000, 5(1), 22–32.
11. S. Toze, Reuse of effluent water—benefits and risks, Agric. Water Manage., 2006, 80, 147–159.
12. M. D. Sobsey, Managing Water in the Home: Accelerated Health Gains from Improved Water Supply, World Health Organization, Geneva, 2002.
13. S. H. Lee, D. A. Levy, G. F. Craun, M. J. Beach and R. L. Calderon, Surveillance for Waterborne-Disease Outbreaks—United States, 1999–2000, Morbidity and Mortality Weekly Report, CDC, 2002, vol. 51, pp. 55–58.
14. G. F. Craun, R. L. Calderon and N. Nwachuku, Causes of Waterborne Outbreaks Reported in the United States, 1991–1998, in Drinking Water and Infectious Disease, Establishing the Links, ed. P. Hunter, M. Waite and E. Rochi, CRC Press and IWA Publishing, Boca Raton, Florida, 2003, pp. 105–117.
15. G. F. Craun, R. L. Calderon and M. F. Craun, Waterborne Outbreaks Caused by Zoonotic Pathogens in the United States, in Waterborne Zoonoses: Identification, Causes and Control, ed. J. A. Cotruvo, A. Dufour, G. Rees, J. Bartram, R. Carr, D. O. Cliver, G. F. Craun, R. Fayer and V. P. J. Gannon, IWA Publishing, London, 2004, pp. 120–135, WHO.
16. T. T. Fong, L. S. Mansfield, D. L. Wilson, D. J. Schwab, S. L. Molloy and J. B. Rose, Massive microbiological groundwater contamination associated with a waterborne outbreak in Lake Erie, south Bass island, Ohio, Environ. Health Perspect., 2007, 115(6), 856–864.
17. D. Pokhrel and T. Viraraghavan, Diarrhoeal disease in Nepal vis-à-vis water supply and sanitation status, J. Water Health, 2004, 2(2), 71–81.
18. G. W. Nasinyama, S. A. McEwen, J. B. Wilson, D. Waltner-Towers, C. L. Gyles and J. Opuda-Asibo, Risk factors for acute diarrhoea among inhabitants of Kampala District, Uganda, SA, Med. J., 2000, 90(90), 891–898.
19. G. Migliorati, V. Prencipe, A. Ripani, C. Di Francesco, C. Casaccia and S. Crudeli, et al., Gastroenteritis outbreak at holiday resort, Central Italy, Emerging Infect. Dis., 2008, 14(3), 474–478.
20. Council Directive 2000/60/EC of the European Parliament and of the Council of the 23 October 2000 establishing a framework for Community action in the field of water policy, Official Journal No L 327, 22 Dec. 2000.
21. R. J. Lieberman, L. C. Shadix, B. S. Newport, C. P. Frebis, M. W. N. Moyer, R. S. Safferman, et al., American Water Works Association Research Foundation, Microbial Monitoring of Vulnerable Public Groundwater Supplies, report 90914, 2002.
22. A. Locas, C. Barthe, A. B. Margolin and P. Payment, Groundwater microbiological quality in Canadian drinking water municipal wells, Can. J. Microbiol., 2008, 54(6), 472–478.
23. F. Lucena, A. E. Duran, A. Moron, E. Calderon, C. Campos, C. Gantzer, S. Skraber and J. Jofre, Reduction of bacterial indicators and bacteriophages infecting faecal bacteria in primary and secondary wastewater treatments, J. Appl. Microbiol., 2004, 97, 1069–1076.
24. G. A. Palmateer, B. J. Dutka, E. M. Janzen, S. M. Meissner and M. Sakellaris, Coliphages and bacteriophages as indicators of recreational water quality, Water Res., 1991, 25, 355–357.
25. ISO 10705-2, Water quality: detection and enumeration of bacteriophages, Part 2: enumeration of somatic coliphages, 2000.
26. W. O. K. Grabow, Bacteriophages: update on application as models for viruses in water, Water SA, 2001, 27, 251–268.
27. U. D. Parashar, C. J. Gibson, J. S. Bresee and R. I. Glass, Rotavirus and severe childhood diarrhea, Emerging Infect. Dis., 2006, 12, 304–306.
28. A. de Donno, T. Grassi, A. Idolo, G. Gabutti and M. Guido, The epidemiology of rotavirus infection and the distribution of G-types in Salento (Italy) during 2004–2005, Int. J. Humanist. Stud., 2008, 1, 26–29.
29. T. Grassi, A. de Donno, M. Guido and G. Gabutti, G-Genotyping of rotaviruses in stool samples in Salento, Italy, J. Prev. Med. Hyg., 2006, 47(4), 138–141.
30. T. Grassi, A. de Donno, M. Guido and G. Gabutti, The epidemiology and disease burden of rotavirus infection in the Salento peninsula, Italy, Turk. J. Pediatr., 2008, 50, 132–136.
31. T. Grassi, F. Bagordo, A. Idolo, F. Lugoli, G. Gabutti and A. de Donno, Rotavirus detection in environmental water samples by tangential flow ultrafiltration and RT-nested PCR, Environ. Monit. Assess., 2010, 164, 199–205.
32. B. Gratacap-Cavallier, O. Genoulaz, K. Brengel-Pesce, H. Soule, P. Innocenti-Francillard and M. Bost, et al., Detection of human and animal rotavirus sequences in drinking water, Appl. Environ. Microbiol., 2000, 66, 2690–2692.
33. EPA Environmental Protection Agency, Regulatory impact analysis for the proposed ground water rule, Fed. Regist., April 5, 2000.
34. Province of Lecce, Online Statistics Office, 2006, http://www.provincia.le.it/ssi/statistica/default.html.
35. Decreto Legislativo 2 febbraio 2001, n. 31 “Attuazione della direttiva 98/83/CE relativa alla qualità delle acque destinate al consumo umano” Gazzetta Ufficiale n. 52 del 3 marzo 2001—Supplemento Ordinario n. 41.
36. Decreto 12 giugno 2003, n. 185, Ministero dell'Ambiente e della Tutela del Territorio, Regolamento recante norme tecniche per il riutilizzo delle acque reflue in attuazione dell'articolo 26, comma 2, del decreto legislativo 11 maggio 1999, n. 152. GU n. 169 del 23-7-2003.
37. Decreto Ministero delle Politiche Agricole e Forestali 23 Marzo 2000, Approvazione dei metodi ufficiali di analisi delle acque per uso agricolo e zootecnico, GURI del 13 Aprile 2000 n. 87 (Suppl Ord).
38. American Public Health Association, Standard Methods for the Examination of Water and Wastewater, American Public Health Association, 1015 Fifteenth Street, NW, Washington, DC, 20th edn, part 9000, 1998.
39. EN ISO 9308-1, Water quality—Detection and enumeration of Escherichia coli and coliform bacteria—Part 1: membrane filtration method, 2000.
40. A. Carducci, L. Cantiani and M. A. Ruschi, A contribution to the standardization of methods for the concentration of enterovirus from seawater, Ig. Mod., 1994, 45, 707–721.
41. K. H. Oshima, Efficient and Predictable Recovery of Viruses and Cryptosporium parvum Oocysts from Water by Ultrafiltration System, New Mexico: Water Resources Research Institute, Technical Completion Report, 316 ( 2001) 1–55.
42. W. O. K. Grabow, Bacteriophages: update on application as models for viruses in water, Water SA, 2001, 27, 251–268.
43. M. Gilgen, D. Germann, J. Luthy and P. Hubner, Three-step isolation method for sensitive detection of enterovirus, rotavirus, hepatitis a virus, and small round structured viruses in water samples, Int. J. Food Microbiol., 1997, 37, 189–199.
44. http://blast.ncbi.nlm.nih.gov/Blast.cgi .
45. EN ISO 10705–2, Water quality: detection and enumeration of bacteriophages, Part 2: enumeration of somatic coliphages, 2000.
46. E. Dominguez-Mariani, A. Carrillo-Chàvez, A. Ortega and M. T. Orozco-Esquivel, Wastewater reuse in Valsequillo agricultural area, Mexico: environmental impact on groundwater, Water, Air, Soil Pollut., 2004, 155, 251–267.
47. C. I. Gallimore, C. Pipkin, H. Shrimpton, A. D. Green, Y. Pickford, C. McCartney, G. Sutherland, D. W. Brown and J. J. Gray, Detection of multiple enteric virus strains within a foodborne outbreak of gastroenteritis: an indication of the source of contamination, Epidemiol. Infect., 2005, 133(1), 41–47.
48. S. Cheong, C. Lee, S. W. Song, W. C. Choi, C. H. Lee and S. J. Kim, Enteric viruses in raw vegetables and groundwater used for irrigation in South Korea, Appl. Environ. Microbiol., 2009, 75(24), 7745–7751.
49. Epidemiology of Infection diseases, http://www.simi.iss.it/bancaDati.aspx.
50. A. de Donno, T. Grassi, F. Bagordo, A. Idolo, A. Cavallaro and G. Gabutti, Collaborative group for the surveillance of rotavirus infection. Emergence of unusual human rotavirus strains in Salento, Italy, during 2006–2007, BMC Infect. Dis., 2009, 15(9), 43.
51. T. Grassi, D. Donia, A. de Donno, A. Idolo, C. Alfio, C. Alessandri and M. Divizia, Detection and molecular characterization of human rotaviruses isolated in Italy and Albania, J. Med. Virol., 2010, 82(3), 510–518.
52. D. Martinelli, R. Prato, M. Chironna, A. Sallustio, G. Caputi, M. Conversano, M. Ciofi Degli Atti, F. P. D'Ancona, C. A. Germinario and M. Quarto, Large outbreak of viral gastroenteritis caused by contaminated drinking water in Apulia, Italy, May–October 2006, Euro Surveill., 2007, 12(4), E070419.1.
53. C. Scarcella, S. Carasi, F. Cadoria, L. Macchi, A. Pavan, M. Salamana, G. L. Alborali, M. M. Losio, P. Boni, A. Lavazza and T. Seyler, An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009, Euro Surveill., 2009, 14(29), 19274.

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