2. Faecal contamination and hygiene aspect concerning the usage of treated waste water and channel water for irrigation of potatoes (Solanum tuberosum)

SAFIR Safe and High Quality Food Production using Low Qua lity Waters and Improved Irrigation Systems and Management

Contract-No. 023168

A Specific Targeted Research Project

under the Thematic Priority ' Food Quality and Safe ty '

Work Package 4 • Impact on Food and Soil Properties

Deliverable D4_4 Survival in soil of residual pathogens in treated low quality water and their contamination of food products (report). Due date: Actual submission date: Start date of project: 01-10-05 Duration: 48 months Deliverable Lead contractor: UoC (KVL) Participant(s) (Partner short names) CAAS, CAU, CER, NAGREF, KVL, SSICA,

UB Author(s) in alphabetic order: Andersen, M. N., Battilani, A., Dalsgaard, A.,

Ensink, J. H. J., Fletcher, T., Forslund, A., Gola, S., Jovanovic, Z., Kljujev, I., Plauborg, F., Psarras, G., Raicevic, V., Stikic, R., Sandei, L.

Contact for queries: A. Forslund

Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Groennegaardsvej 15, DK-1870 Frederiksberg C, Denmark Tel. +45 3533 2725 Fax +45 3533 2757 E-Mail [email protected]

Dissemination Level: (PUblic, Restricted to other Programmes Participants, REstricted to a group specified by the consortium, COnfidential only for members of the consortium)

PU

Deliverable Status: Revision 1.0 Project co-funded by the European Commission within the Sixth Framework Programme (2002-2006)

Deliverable 4_4 2 / 35

Contents 1. Transport of human pathogens in leachate in sub-surface drip irrigated soil....................4

1.1 Abstract......................................................................................................................4

1.2 Introduction ................................................................................................................4

1.3 Materials and methods...............................................................................................5

1.3.1 Study site and climatic conditions.......................................................................5

1.3.2 Facility and experimental lay-out ........................................................................6

1.3.3 Soil types and irrigation system ..........................................................................7

1.3.4 Microorganisms ..................................................................................................7

1.3.5 Preparation and enumeration of bacteriological and phage parameters ............8

1.3.6 Irrigation strategy and frequency ........................................................................9

1.4 Results .....................................................................................................................10

1.5 Discussion and conclusion.......................................................................................11

1.6 Acknowledgement....................................................................................................13

1.7 References...............................................................................................................13

2. Faecal contamination and hygiene aspect concerning the usage of treated waste water and channel water for irrigation of potatoes (Solanum tuberosum)........................................18

2.1 Abstract....................................................................................................................18

2.2 Introduction ..............................................................................................................18

2.3 Methodology ............................................................................................................19

2.3.1 Study sites ........................................................................................................19

2.3.2 Irrigation practices ............................................................................................20

2.3.3 Sample collection..............................................................................................21

Water ...........................................................................................................................21

Soil...............................................................................................................................21

Potato ..........................................................................................................................21

2.3.4 Sample analysis................................................................................................21

Bacteriology.................................................................................................................21

Parasitology.................................................................................................................22

2.4 Results .....................................................................................................................22

2.4.1 Italy ...................................................................................................................22

2.4.2 Serbia ...............................................................................................................23

2.5 Conclusion ...............................................................................................................24


2.7 References...............................................................................................................25

3. Faecal contamination and hygiene aspect of soil and on tomatoes (Solanum Lycopersicum) irrigated with partially treated waste water.....................................................28


3.1 Abstract....................................................................................................................28

3.2 Introduction ..............................................................................................................28

3.3 Methodology ............................................................................................................29

3.3.1 Study sites ........................................................................................................29

3.3.2 Irrigation practices ............................................................................................30

3.3.3 Sample collection..............................................................................................31

Water ...........................................................................................................................31

Soil...............................................................................................................................31

Tomato ........................................................................................................................31

3.3.4 Sample analysis................................................................................................31

Bacteriology.................................................................................................................31

Parasitology.................................................................................................................31

3.4 Results .....................................................................................................................32

3.4.1 Italy ...................................................................................................................32

3.4.2 Crete ................................................................................................................33

3.5 Conclusion ...............................................................................................................34


3.7 References...............................................................................................................34


1. Transport of human pathogens in leachate in subsurface drip irrigated soil

A. Forslund, F. Plauborg, M. N. Andersen and A. Dalsgaard

1.1 Abstract

The risk for contamination of groundwater and potatoes through sub-surface drip irrigation with low quality water was explored in large-scale lysimeters containing compact coarse sand and sandy loam. The human pathogens, Salmonella Senftenberg, Campylobacter jejuni and E. coli O157:H7, and the virus indicator Salmonella Typhimurium bacteriophage 28B, were added weekly through irrigation tubes during one month followed by irrigation with groundwater containing no pathogens for additional six month. Two weeks after irrigation was started, phage 28B was detected at low concentrations (2 pfu/mL) in leachate from both sandy loam and coarse sand lysimeters. After 27 days, phages continued to be present in similar concentrations in leachate from lysimeters containing coarse sand, while no phages were found in lysimeters with sandy loam. None of the three added bacterial pathogens were found in any leachate samples during the entire study period. All bacterial pathogens and phage 28B were found on potato samples harvested just after the application of test organisms was terminated. Under conditions with compact soil, limited macropores and low water velocity, human bacterial pathogens seems to be retained in the soil matrix and die-off before leaching to groundwater. However, vira may leachate to groundwater and represent a health risk as only few virus particles are needed to cause human disease. The findings of bacterial pathogens and phage 28 on all potato samples suggest that a main risk associated with sub-surface drip irrigation with low quality water is faecal contamination of root crops, in particular those consumed raw.

1.2 Introduction

Clean freshwater is a limited resource and its use for crop irrigation is in fierce competition with the demand for household and industrial consumption, but also nature will need its water, as stated by the European Water Framework Directive (2000/60/EC). On top of this, the problem will be amplified by changes in climate and precipitation patterns reducing groundwater deposit as a consequence of decreased precipitation across Europe (IPCC 2007; EEA 2003). These limitations in access to clean freshwater already force agriculture, especially in Central Europe and the Mediterranean area, to search for alternative water sources and irrigation strategies to sustain food production. Even in humid areas irrigated agriculture may foresee reduction in water availability as climate change scenarios forecast a decrease in summer precipitation (IPCC 2007). Hence, low quality water, e.g. treated/untreated wastewater or surface water run-off, will increasingly be used for irrigation in agriculture. Already today, low quality waters are used to irrigate food crops in Australia, the Mediterranean and elsewhere, e.g. Israel has for decades used treated wastewater in irrigated agriculture (Lazarova et al. 2000). The water scarcity calls for different measures to save water and increase productivity in irrigated agriculture. The efficiency of crops to take up water is significantly increased by the use of sub-surface drip irrigation, mainly due to reduced soil evaporation, but also because water requirements of the plants can be more precisely can be met (Ayars et al. 1999; Shahnazari 2007). However, the sub-surface soil application of treated wastewater, which often still contains pathogenic microorganisms, may potentially increase pathogen survival by preventing their exposure to the harmful effects of UV-light and desiccation. Pathogens in protected soil environments may subsequently be


transferred to crop roots and could therefore pose a risk for consumers, in particular when such produce are consumed raw, e.g. carrots.

Irrigation of agricultural land with low quality water, in particular sub-surface irrigation, may potentially also lead to contamination of groundwater as the irrigation water may contain high numbers of faecal microorganisms, and occasionally disease-causing pathogens like Salmonella, Campylobacter, Shigella, enteric viruses, and protozoan parasites (Calci et al. 1998; Nwachuku and Gerba 2008; U.S. EPA 1992). Waterborne illness associated with consumption of contaminated groundwater is common in United States and Europe, but often these outbreaks are related to faecal contamination taking place in the distribution system or from surface run-off, e.g. into wells (Abbaszadegan et al. 2003; Craun 1991; Craun et al. 2006; Kramer et al. 2001). It is unknown to what extent groundwater aquifers are contaminated due to irrigation with faecal contaminated water and subsequent transport of pathogens through soil to groundwater.

The processes determining the vertical movement of pathogens through soil are complex and depend on type of soil and its properties; climatic conditions; the initial concentration of pathogens; as well as the type of vegetation (Chu et al. 2003; Entry et al. 2000; McMurry et al. 1998). Preferential water movement, e.g. through roots and earthworm channels and naturally occurring cracks, is probably the primary route by which microorganisms move through the soil (Abu-Ashour et al. 1998; Artz et al. 2005; McMurry et al. 1998). The survival of pathogens in soil depends on many parameters such as temperature, moisture content, pH, soil composition and inhibitory competition from indigenous mikroflora (Abu-Ashour et al. 1994; Mawdsley et al. 1995).

The objective of the current study was to determine the occurrence and survival of human pathogens in leachate following sub-surface irrigation using artificially contaminated water. The experiment was carried out in a semi-field environment where potatoes in lysimeters with clay and sandy soils were irrigated with water spiked with Salmonella, Campylobacter, pathogenic E. coli and a virus indicator (bacterial phage).

1.3 Materials and methods

1.3.1 Study site and climatic conditions

The experiments were carried out from August 2007 to March 2008 in Mid Jutland, Denmark at the Research Center Foulum (56o30´N, 9o35´E). The climate is temperate with an annual average rainfall of about 800 mm. Weather data were collected at the local climate station located 500 m from the study site. Figure 1 shows some important weather characteristics during the study period. Total precipitation was 372 mm with temperatures around two degrees higher than normal in January and February.


-5

0

5

10

15

20

25

01/08/07

31/08/07

30/09/07

30/10/07

29/11/07

29/12/07

28/01/08

27/02/08

Date

°C

-5

0

5

10

15

20

25

mm

/day

Prec

Temp

ET0

Figure 1 : Daily average temperature (Temp), reference evapotranspiration (ET0) and precipitation (Prec) during the study period.

1.3.2 Facility and experimental lay-out

The experiment was carried out at a large semi-field facility, consisting of concrete lysimeters measuring 2.70 m × 1.60 m with a soil depth of 1.40 m totalling 6.05 m3. These are accessible from underground basement corridors where percolation water is collected (Figure 2).

Figure 2: The cross-section of the lysimeters shows the 4 rows which each consist of 25 lysimeters filled with the same soil type. Leachate samples were collected through the drainage pipe in the basement corridor.

Coarse

sand

Coarse sand

SandyLoam Loamy

sand Sandy loam

Coarse sand

Coarse sand


During the experiment a mobile roof covered the facility to prevent rain reaching the soil and irrigation of the individual lysimeters was controlled by a computer. The lysimeters were irrigated with groundwater by sub-surface drippers to reduce soil evaporation. Potato plants of the cultivar Folva were planted in the spring and the tops of potato plants had just been removed when the study was initiated to prevent further evaporation and transpiration from the crop. Each lysimeter contained 20 potato plants distributed equally with tubers harvested on August 28, 2007. The experiment included 30 lysimeters of which 15 contained coarse sand and 15 sandy loam. For each soil type, 5 different irrigation applications were applied in triplicates, including control lysimeters. Volumes of irrigation water and collected leachate were measured, and sub-samples were taken for microbiological analyses.

1.3.3 Soil types and irrigation system

Two different soil types, coarse sand and sandy loam, typical for Danish agriculture and climatic conditions were studied (Table 1). Before placement in the lysimeter which took place in 1993, the soils had been used for agriculture purposes for many years. During the collection, each soil type was separated into three diagnostic horizons, which were homogenized and vibrated back to their original dry bulk density (Nielsen and Møberg 1985). Table 1: Physical properties of soil types in the lysimeters

Soil type Horizon Depth

Organic

matter

Clay

<2 µm

Silt

2-20 µm

Fine sand

20-200 µm

Coarse sand

200-2000 µm

Bulk

density Total

porosity

(cm)

(g/100 g)

(g/cm3) Vol. %

Ap 0-30 1.9 5.8 2.1 17.8 72.3 1.41 47.0

Bhs 30-70 0.7 5.9 0.5 14.1 78.6 1.46 45.0

Coarse sand

C 70-

140 0.2 5.2 0.7 19.0 74.9 1.50 44.0

Ap 0-30 2.3 17.6 12.9 48.0 19.2 1.44 47.0

EB 30-70 0.5 21.6 13.4 43.9 20.6 1.53 44.0 Sandy loam

Cg 70-140

0.3 21.7 15.8 40.2 22.0 1.55 43.0

The tubers were in the spring ridged with 15 cm of soil and the drip lines placed on the soil with the emitters located midway between seed potatoes, i.e., 15 cm from each potato plant. Emitters in the drip lines (NETAFIM, Tel Aviv, Israel) could deliver 1 L/h (hours) and were located 30 cm apart.

1.3.4 Microorganisms

The human pathogenic bacteria Campylobacter jejuni (NCTC 11168), Escherichia coli serotype O157:H7 (ATCC 43888) and a Nalidixic acid-resistant Salmonella Senftenberg 775W were used in the experiment. Salmonella Typhimurium phage 28B (Lilleengen 1948) was used as an indicator for virus transport. Phage 28B has neither been shown to occur in environmental samples nor in faeces and has previously been used to model groundwater flow (Carlander et al. 2000; Johansson et al. 1998).


1.3.5 Preparation and enumeration of bacteriological and phage parameters

For preparation of test solution to be added to the irrigation water, E. coli O157:H7, C. jejuni and S. Senftenberg 775W resistant to Nalidixic acid were grown over night at 37 °C in Brain Heart Infusion broth (BHI; Oxoid, Basingstoke, England). For growth of Salmonella, 40 µg/mL Nalidixic acid were added. C. jejuni was incubated microaerofilic. Concentrations of the solutions with bacterial test organisms were calibrated by OD600. Preparation and enumeration of phage 28B by lysing the Salmonella Typhimurium type 5 host strain was done as described previously (Adams 1959; Höglund et al. 2002). During each of the 4 irrigations, the total number of the individual human pathogens and phage 28B contained in the water applied to each lysimeter were 3 × 108 cfu (colony forming units) and 6 × 1010 pfu (plaque forming units), respectively.

E. coli O157:H7, S. Senftenberg and C. jejuni in leachate were enumerated by direct plating on agar plates with a detection limit of 1 cfu/mL. Leachate from the individual lysimeters was collected in sterile 30-L bottles, which were weighed before a sub-sample of 300 mL was taken. The sub-sample was stored at 5 °C and analys is initiated within a maximum of 12 h. Soil collected for analysis was pooled samples of six 30-50 g representative soil samples collected from each lysimeter. Initially, soil samples from all lysimeters were analyzed before irrigation with water spiked with human pathogens and phage 28B. At harvest, soil from lysimeters that had received 4 applications of pathogens was collected on the ridge near the drip emitter. Samples of 10 g of soil was added to 90 mL Maximum Recovery Diluent (MRD, Oxoid) and treated in ultra sound bath for 30 s to remove microorganisms from the surface of the soil particles. The detection limit for soil analyses was 10 cfu/g.

The surface of the potato, including any soil attached to the potato, was analyzed for pathogens and phage 28B. Approximately 600 g of potatoes was collected in a sterile plastic bag and weighed and 200 mL of MRD dilution solution was added to the bag. Any microorganisms present on the surface of the potatoes were removed by gently hand massage for 30 s, and the solution subsequently serially diluted before enumeration. Potato samples were only collected from lysimeters that received 4 applications of water containing pathogens and the phage as well as controls irrigated with water containing no pathogens. In each lysimeter, potatoes were collected from two different potato plants and analyzed for the added microorganisms. Concentration of pathogens and phages on potato surfaces was express as units per surface area.

E. coli O157:H7, S. Senftenberg and C. jejuni were counted on Sorbitol MacConkey Agar (Merck, Darmstadt, Germany), MacConkey Agar (Merck) incl. Nalidixic acid and Campylobacter Blood-free Selective Agar (Oxoid), respectively, according to the manufactures directions. Suspected colonies of E. coli O157 and Salmonella were confirmed by agglutination in antiserum (SSI, Copenhagen, Denmark) and Campylobacter by microscopy as typically curved motile bacteria.

Initial analyses of the soils and leachate showed that E. coli O157, Campylobacter, Salmonella or lysing phages were not present. It was further shown that phage 28B did not lyse S. Senftenberg 775W.


1.3.6 Irrigation strategy and frequency

All lysimeters were irrigated during the potato-growing season with groundwater by sub-surface drip irrigation following different strategies. Before the start of the experiment all lysimeters were rewetted to field capacity, and hereafter irrigated as described in the following and shown in Figure 3. From August 1, water was applied to create a slow drainage reflecting natural conditions. Then initiated on November 28, 6 mm water was applied daily to create a steady state water flow and leaching at a soil water status close to saturated water content. In this period, deviation from the strategy was due to frost in the period December 16 to January 5.

0

100

200

300

400

500

01/08/07

31/08/07

30/09/07

30/10/07

29/11/07

29/12/07

28/01/08

27/02/08

Date

Acc

um

ula

ted

wa

ter

(mm

)

0

100

200

300

400

500Drainage sandy loam

Drainage coarse sand

Irrigation

Figure 3: Volume of accumulated irrigation water and leachate colleted during the study period.

Lysimeters received in triplicate between 1-4 applications with water containing added microorganisms, i.e. all 24 lysimeters received one application three weeks before harvest; 18 lysimeters received another application two weeks before harvest; 12 lysimeters received a third application one week before harvest; and 6 lysimeters received a fourth application 1 day before harvest. The 6 control lysimeters were irrigated with groundwater not added any microorganisms.

The four applications with added microorganisms were done weekly with a total of 30 L applied to each lysimeter over a 24-h period. Between these applications, each lysimeter was irrigated daily totalling 1 mm per week. This strategy was chosen to reflect average volumes of rain expected during winter seasons. After the last application of microorganisms the potatoes were harvested on August 28 and irrigation with 3 mm per week continued for eleven weeks. Since only a few leachate samples contained low concentrations of the phage, the frequency of irrigation was increased to 6 mm daily for a 3-month period. This strategy was chosen to have almost the entire pore volume exchanged in the lysimeters during the study period. During this last irrigation period, water samples colleted every second and fourth week were analyzed.


1.4 Results

The occurrence and survival of pathogens and phage 28B in leachate from lysimeters were studied during 212 days through the cold season (August 1, 2007 to February 28, 2008). During the first 28 days, irrigation water containing pathogens were applied once a week. For the remaining study period, only groundwater was used for irrigation. With the chosen irrigation strategy, the distribution of pathogens and the phage would cover a depth of approximately 8 mm if macropore flow did not occur. Drainage water was collected and analyzed weekly during the one-month period where pathogens and the phage were added.

Figure 3 shows the accumulated volumes of irrigation water and collected leachate. From November 28, leaching occurred at an approximately constant rate (steady state). In total, the amounts of irrigation water leached and irrigated were similar around 450 mm corresponding to 17 % more than normal precipitation for this location (Figure 1) and hence, somehow hydraulic conditions were created similar to natural conditions in some years.

Low concentrations of the phage, i.e. 2 pfu/mL, were detected in the drainage water 15 days after the initial application of test organisms. The phage was detected in 2 out of 12 lysimeters with sandy loam and in coarse sand, respectively. Irrespective of soil type, one of these lysimeters had received water containing the phage ones and the other lysimeter received such water twice. After 27 days, phages were detected in three lysimeters containing coarse sand at a concentration of 2 pfu/mL, while no phages were found in sandy loam lysimeters. Although identical concentrations of the phage were found, these three lysimeters were originally irrigated ones, twice and three times, respectively, with water containing the phage. Due to the limited number of lysimeters that leached phages and the low concentration of phages found, it was not possible to estimate a difference in the leachate pattern of the phage between the two soil types and frequency of irrigation. After the four weekly applications of water containing pathogens and the phage, phages were not detected during the remaining 185 days of the study period. During the entire study period there were not isolated any bacterial pathogens in the drainage water, even that around 0.75 of the total pore volume had been exchanged.

Potatoes were collected from lysimeters that received contaminated irrigation water four times with the last application done one day before harvest. Potato samples were stored at 5 °C and analyzed within 3 weeks for bacterial pathog ens and phages present on the surface of the potatoes, including those present in soil adhering to the potato. Phage 28B was found on potatoes in 5 out of 6 samples from lysimeters containing coarse sand at a concentration of 6.5 × 103 ± 1.2 × 104 pfu/cm2 potato surface. However, the phage was not detected on potatoes grown in sandy loam. S. Senftenberg was found in 4 out of 6 lysimeters independent of soil type at a concentration of 34 ± 66 cfu/cm2. E. coli O157:H7 was isolated from all potato samples from coarse sand (28 ± 21 cfu/cm2), while 2 out of 6 sandy loam samples contained this pathogen (7.4 ± 21 cfu/cm2). C. jejuni was recovered in 1 out of 6 samples for both soil types. Concentration of C. jejuni in coarse sand was 4 cfu/cm2 and in sandy loam the concentration was approximately 20 times higher. All four pathogens were detected in only one potato sample.

Following four irrigations with water containing pathogens and the phage, soil samples were collected during potato harvest and stored for 11 weeks at 5 °C. All sandy loam and 83% of coarse sand samples, respectively, contained the phage at a concentration ranging from 2 to 6 × 104 pfu/g. Bacterial pathogens could not be isolated from any soil sample.


1.5 Discussion and conclusion

Phage 28B was detected at low concentrations (2 pfu/mL) in drainage water from both sandy loam and coarse sand lysimeters 15 days after the irrigation with water containing bacterial pathogens and the phage was initiated. After 27 days, phages continued to be present in a similar concentration in leachate from three lysimeters containing coarse sand, while no phages were found in sandy loam lysimeters. However, none of the three added bacterial pathogens were found in the leachate during the weekly samplings. It should be noted that the concentration of human enteric viruses typically range from 100-700 pfu/L in domestic sewage (Oron et al. 1995; Snowdon et al. 1989) and that Salmonella occurs in wastewater ranging from a few cells to 8000 cfu/100 mL (Bitton 2005). These concentrations of pathogens are 3-5 Log10 units lower than those applied in our experiments.

The fate of the microorganisms in soil depends mainly of inactivation and transport processes involving advection, dispersion, adsorption and decay (Yates and Yates 1990). In our study, the phage was transported faster than the average velocity of the irrigation water since only 0.75 of the total soil pore volume was exchanged at the end of the experiment. Microorganisms may be excluded from the smaller pores in soil and forced to travel only through larger pores, e. g. fractures and cracks, where their average velocity are greater than that of the soil medium taken as a whole. Carlander et al. (2000) also studied transport of phage 28B and detected the phage in leachate 2-24 hours following application after passage of clay soil lysimeters with a dept of 1.2 m. This fast transport was explained by rapid flow of soil water in macropores. This suggests that even though the soil in the lysimeters used in our study were established 14 years ago, limited numbers of macropores were present to facilitate a fast transport of microorganisms. It is therefore likely that matrix flow of irrigated water was predominant in the used lysimeters. Other studies have reported a much faster breakthrough of phages, e.g. when 6-120 times larger volumes of irrigation water was applied (Carlander et al. 2000; McLeod et al. 2001; McLeod et al. 2003; Pang et al. 2008), but also during natural rain conditions (Nicosia et al. 2001), as a consequence of macropore flow. The irrigation rate in the present study was adjusted to simulate natural rain events of approximately 1 mm/day when pathogens and the phage were initially applied.

The recovery of phage 28B, but not any of the bacterial pathogens may be explained by the filtration of the larger sized bacterial cells (0.5-5 µm) compared to the smaller size of phages (20-200 nanometre). Other transport studies have shown a faster transport of bacteriophages through soil compared to E. coli (Hijnen et al. 2005; Sinton et al. 1997), since bacteria easier become trapped in soil as a consequence of the larger cell size. But also small particles with similar sizes as vira can be retained in the fine pores of the soil matrix (Cumbie and McKay 1999). Our findings of phage 28B in leachate 27 days after it was applied in lysimeters containing coarse sand, but not in any lysimeters with sandy loam, are supported by a general enhanced trapping of microorganisms in clay soil compared to larger sand particle (Winfield and Groisman 2003). Clay minerals further provide better adsorption sites for microorganisms than sand (Gerba et al. 1984). Another parameter that influences the recovery of bacteria in the leachate could be the bulk density of the soil. Artz et al. (2005) reported the leaching rate of E. coli O157:H7 to decrease with increased bulk density and with a bulk density of 1.15 g/cm3 less than 0.4 % of added bacterial cells could be recovered. The bulk density in our study ranged from 1.41 to 1.55 g/cm3 and a low leaching rate would therefore be expected.

The long duration of the experiment totalling 212 days was a consequence of the irrigation strategy to apply water volumes at a frequency similar to normal precipitation during winter time together with the large volume of the lysimeters. Pathogen survival time in soil has been


shown to vary from 4 to 160 days (Abu-Ashour et al. 1994; Sjogren 1994) and Salmonella may survive in coarse sand up to 64 days in unsaturated conditions (Parker and Mee 1982). With the water volume and frequency of irrigation applied in our study, it is likely that the survival time of pathogens was exceeded due to their die-off because of detrimental impacts of various factors in the soil. The exposure to the soil environment may also have induced a so-called VBNC (Viable-But-Not-Culturable) stage of bacterial cells. VBNC cells can not be cultured by traditionally cultured-based methods, but only by direct detection, including DNA-based methods. Since there are diverting findings and opinions regarding the possibility of VBNC cells to resuscitate to an infective stage (Koenraad et al. 1997; Winfield and Groisman 2003), only bacterial pathogens that could be cultured were enumerated in the present study. Thus, we do not know if a VBNC stage of the added bacterial pathogens were present in the collected leachate.

In the present study, human bacterial pathogens were studied instead of faecal indicators like thermotolerant coliforms and E. coli. This was done as differences in cell surface, but also other properties of microorganisms may affect their transport through and survival in soil. Bolster et al. (2006) observed a faster transport of C. jejuni compared to E. coli. Even though E. coli is used as an indicator for the presence of pathogenic bacteria, Salmonella has shown better survival in the soil environment compared to E. coli (Winfield and Groisman 2003). Phage 28B was chosen as a virus indicator as its detection and enumeration is fast and simple. Also, it is problematic to use pathogenic viruses for experiments in the external environment. Bacteriophages are commonly used as surrogate for human enteric viruses, like adenovirus and rotavirus (Leclerc et al. 2000), and phage 28B have been applied in many other transport and survival studies (McLeod et al. 2003; Ottoson et al. 2008; Vinnerås et al. 2008).

The use of sub-surface drip irrigation protects pathogens from the lethal exposure of ultraviolet light and desiccation at the soil surface (Beard 1940). Sub-surface irrigation minimizes the contact between irrigation water and crops like fruits and plants with edible parts located above the soil (Oron et al. 1991; Oron et al. 2001); however, root crops are clearly at risk of faecal contamination by this irrigation method. In our study, all the three added bacterial pathogens were isolated from the surface of potatoes, including adhering soil particles, grown in the lysimeters irrigated with contaminated water. Drip emitters were placed in the middle of two potato plants and the frequency of pathogen detection varied between the soil types. No phages were detected on potato samples grown in sandy loam indicating possible adsorption to clay minerals. Straub et al. (1992) observed an increase in sorption of virus with increases in clay content. Phages were found in both types of soil when soil samples were taken close to the drip emitter. This corresponds with the finding of Assadian et al. (2005) and Enríquez et al. (2003) that observed the highest concentration of bacteriophages in the proximity of the drip emitter.

Our study suggests that in compact soils with a low water velocity, human bacterial pathogens will be retained in the soil matrix and be inactivated before leaching to groundwater. The leaching of small amounts of phages used as model for viruses is problematic, since only few viruses are needed to cause disease in humans. A main risk associated with sub-surface drip irrigation using low quality water seems to be contamination of root crops, in particular those eaten raw.


1.6 Acknowledgement

We would like to thank Annika Holmqvist for the provision of Salmonella Senftenberg 775W and the phage host strain S. Typhimurium type 5 and Jacob Ottoson for providing us with the Salmonella Typhimurium phage 28B. The study was supported by the “Safe and High Quality Food Production using Low Quality Waters and Improved Irrigation Systems and Management” project (SAFIR, EU, FOOD-CT-2005-023168) funded by the European Commission.

1.7 References

Abbaszadegan, M., M. LeChevallier, and C.P. Gerba. 2003. Occurrence of viruses in U.S. groundwaters. Journal of American Water Works Association 95: 107-120.

Abu-Ashour, J., D.M. Joy, H. Lee, H.R. Whiteley, S. Zelin. 1994. Transport of microorganisms through soil. Water, Air and Soil Pollution 75: 141-157.

Abu-Ashour, J., D.M. Joy, H. Lee, H.R. Whiteley, S. Zelin. 1998. Movement of bacteria in unsaturated soil columns with macropores. Transactions of the Asae 41, no. 4: 1043-1050.

Adams, M.H. 1959. Bacteriophages. New York: Interscience Publishers, Inc.

Artz, R.R.E., J. Townend, K. Brown, W. Towers, and K. Killham. 2005. Soli macropores and compaction control the leaching potential of Escherichia coli O157:H7. Environmental Microbiology 7, no. 2: 241-248.

Assadian, N.W., G.D. Di Giovanni, J. Enciso, J. Iglesias, W Lindemann. 2005. The transport of waterborne solutes and bacteriophage in soil subirrigated with a wastewater blend. Agriculture, Ecosystems and Environment 111: 279-291.

Ayars, J.E., C.J. Phene, R.B. Hutmacher, K.R. Davis, R.A. Schoneman, S.S. Vail, and R.M. Mead. 1999. Subsurface drip irrigation of row crops: a review of 15 years of research at the Water Management Research Laboratory. Agricultural Water Management 42: 1-27.

Beard P.J. 1940. Longevity of Eberthella Typhosus in various soils. American Journal of Public Health 30: 1077.

Bitton, G. 2005. Pathogens and parasites in domestic wastewater. In: Wastewater microbiology, 109-151. New York: John Wiley & Sons.

Bolster, C.H., S.L. Walker, and K.L. Cook. 2006. Comparison of Escherichia coli and Campylobacter jejuni transport in saturated porous media. Journal of Environmental Quality 35, no. 4: 1018-1025.


Calci, K.R., W. Burkhardt III, W.D. Watkins, and S.R. Rippey. 1998. Occurrence of male-specific bacteriophage in feral and domestic animal wastes, human feces, and human-associated wastewaters. Applied and Environmental Microbiology 64, no. 12: 5027-5029.

Carlander, A, P. Aronsson, G. Allestam, T.A. Stenstrom, K. Perttu. 2000. Transport and Retention of Bacteriophages in two types of Willow-Cropped Lysimeters. Journal of Environmental Science and Health Part A 35, no. 8: 1477.

Chu, Y., Y. Jin, T. Baumann, and M.V. Yates. 2003. Effect of soil properties on saturated and unsaturated virus transport through columns. Journal of Environmental Quality 32: 2017-2025.

Craun, G.F. 1991. Causes of waterborne outbreaks in the United States. Water Sciences and Technology 24: 17–20.

Craun, M.F., G.F. Craun, R.L. Calderon, and M.J. Beach. 2006. Waterborne outbreaks reported in the United States. Journal of Water and Health 4, no. 2: 19-30.

Cumbie, D.H., L.D. McKay. 1999. Influence of diameter on particle transport in a fractured shale saprolite. Journal of Contaminant Hydrology 37: 139-157.

EEA. 2003. Europe’s environment: The third Environmental assessments report No 10.

Enríquez, C.E., A. Absar, E.M. Suarez-Rey, C.Y. Choi, G. Oron, and C.P. Gerba. 2003. Survival of bacteriophages MS-2 and PRD-1 in turfgrass irrigated by sursurface drip irrigation. Journal of Environmental Engineering 129, no. 9: 852-857.

Entry, M.A., R.K. Hubbard, J.E. Thies, and J.J. Fuhrmann. 2000. The influence of vegetation in riparian filterstrips on coliform bacteria: I. Movement and survival in water. Journal of Environmental Quality 29, no. 4: 1206-1214.

European Water Framework Directive (2000/60/EC)

http://ec.europa.eu/comm/environment/water/water-framework/index_en.html

Accessed November 12, 2009

Gerba, C.P., and G. Bitton. 1984. Microbial pollutants: Their survival and transport pattern to groundwater. In Groundwater pollution microbiology, eds. G. Bitton and C.P. Gerba, 65-88. New York: John Wiley & Sons.

Hijnen, W.A.M., A.J. Brouwer-Hanzens, K.J. Charles, and G.J. Medema. 2005. Transport of MS2 phage, Escherichia coli, Clostridium perfringens, Cryptosporidium parvum, and Giardia intestinalis in a gravel and a sandy soil. Environmental Science and Technology 39, no. 20: 7860-7868.


Höglund, C., N. Ashbolt, T.A. Stenström, L. Svensson. 2002. Viral persistence in source-separated humane urine. Advances in Environmental Research 6: 265-275.

IPCC. 2007. Climate Change 2007: Synthesis Report. Contribution of Working Groups I, II and III to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. IPCC, Geneva, Switzerland.

Johansson, P.-O., B. Espeby, B. Nilsson, G. Allestam. 1998. Artificial groundwater recharge in Stockholm – II Column test design and tracer tests. In: Artificial Recharge of Groundwater, ed. Peters, J.H. et al., 383-385. Netherland, Rotterdam: Balkema AA.

Koenraad, P.M.F.J., F.M. Rombouts, S.H.W. Notermans. 1997. Epidemiological aspects of thermophilic Campylobacter in water-related environments: A review. Water Environment Research 69, no. 1: 52 -63.

Kramer, M.H., G. Quade, P. Hartemann, and M. Exner. 2001. Waterborne diseases in Europe 1986-1996. Journal of American Water Works Association 93: 48-53.

Lazarova, V., G. Cirelli, P. Jeffrey, M. Salgot, N. Icekson, and F. Brissaud. 2000. Enhancement of integrated water management and water reuse in Europe and the Middle East. Water Science and Technology 42, no.1-2: 193-202.

Leclerc, H., S. Edberg, V. Pierzo, and J.M. Delattre. 2000. Bacteriophages as indicators of enteric viruses and public health risk in groundwaters. Journal of Applied Microbiology 88: 5-21.

Lilleengen, K. 1948. Typing of Salmonella Typhimurium by means of a bacteriophage. Ph.D. thesis. The bacteriological and hygienical Department of the Royal Veterinary College, Stockholm, Sweden.

Mawdsley, J.L., R.D. Bardgett, R.J. Merry, B.F. Pain, M.K. Theodorou. 1995. Pathogens in livestock waste, their potential for movement through soil and environmental pollution. Applied Soil Ecology 2: 1-15.

McLeod, M., J. Aislabie, J. Smith, R. Fraser, A. Roberts, and M. Taylor. 2001. Viral and chemical tracer movement through contrasting soils. Journal of Environmental Quality 30: 2134-2140.

McLeod, M., J. Aislabie, J. Ryburn, A. McGill, and M. Taylor. 2003. Microbial and chemical tracer movement through two Southland soils, New Zealand. Australian Journal of Soil Research 41: 1163-1169.


McMurry, S.W., M.S. Coyne, and E. Perfect. 1998. Fecal coliform transport through intact soil blocks amended with poultry manure. Journal of Environmental Quality 27: 86-92.

Nicosia, L.A., J.B. Rose, L. Stark, and M.T. Stewart. 2001. A field study of virus removal in septic tank drainfields. Journal of Environmental Quality 30: 1933-1939.

Nielsen, J.D., and J.P. Møberg. 1985. Classification of soil profiles at Danish

Research stations. Danish Journal of Plant Soil Science 89: 157–167.

Nwachuku, N., and C.P. Gerba. 2008. Occurrence and persistence of Escherichia coli O157:H7 in water. Reviews in Environmental Science and Biotechnology 7: 267-273.

Oron, G., Y. DeMalach, Z. Hoffman, Y. Keren, H. Hartman, and N. Plazner. 1991. Wastewater disposal by sub-surface trickle irrigation. Water Science and Technology 23, no. 10-12: 2149-2158.

Oron, G., M. Goemans, Y. Manor, and J. Feyen. 1995. Poliovirus distribution in the soil-plant system under reuse of secondary wastewater. Water Research 29, no. 4: 1069-1078.

Oron, G., R. Armon, R. Mandelbaum, Y. Manor, C. Campos, L. Gillerman, M. Salgot, C. Gerba, I. Klein, and C. Enriquez. 2001. Secondary wastewater disposal for crop irrigation with minimal risks. Water Science and Technology 43, no. 10: 139-146.

Ottoson, J.R., A. Schnürer, and B. Vinnerås. 2008. In situ ammonia production as a sanitation agent during anaerobic digestion at mesophilic temperature. Letters in Applied Microbiology 46: 325-330.

Pang, L., M. McLeod, J. Aislabie, J. Šimunek, M. Close, and R. Hector. 2008. Modeling transport of microbes in ten undisturbed soils under effluent irrigation. Vadose Zone Journal 7: 97-111.

Parker, W.F., and B.J. Mee. 1982. Survival of Salmonella adelaide and fecal coliforms in coarse sands of the Swan Coastal Plain, Western Australia. Applied and Environmental Microbiology 43, no. 5:981-986.

Shahnazari, A., F. Fulai, M.N. Andersen, S-E Jacobsen, and C.R. Jensen. 2007. Effects of partial root-zone drying on yield, tuber size and water use efficiency in potato under field conditions. Field Crops Research 100: 117-124.

Sinton, L.W., R.K. Finlay, L. Pang, and D.M. Scott. 1997. Transport of bacteria and bacteriophages in irrigated effluent into and through an alluvial gravel aquifer. Water, Air and Soil Pollution 98: 17-42.


Sjogren, R.E. 1994. Prolonged survival of an environmental Escherichia coli in laboratory soil microcosms. Water, Air and Soil Pollution 75: 389-403.

Snowdon, J.A., D.O. Cliver, and J.C. Converse. 1989. Land disposal of mixed human and animal wastes: A review. Waste Management and Research 7: 121-134.

Straub, T.M., I.L. Pepper, and C.P. Gerba. 1992. Persistence of viruses in desert soils amended with anaerobically digested sewage sludge. Applied and Environmental Microbiology 58, no. 2:636-641.

U.S. EPA (1992) Guidelines for water reuse. United States Environmental Protection Agency, Washington.

Vinnerås, B., A. Nordin, C. Niwagaba, K. Nyberg. 2008. Inactivation of bacteria and viruses in human urine depending on temperature and dilution rate. Water Research 42: 4067-4074.

Winfield, M.D., and E.A. Groisman. 2003. Role of nonhost environments in the lifestyle of Salmonella and Escherichia coli. Applied and Environmental Microbiology 69, no. 7: 3687-3694.

Yates, M.V., and S.R. Yates. 1990. Modelling microbial transport in soil and groundwater. AMS News 56, No. 6: 324-327.


2. Faecal contamination and hygiene aspect concerni ng the usage of treated waste water and channel water for irrigation of potatoes (Solanum tuberosum)

A. Forslund, J. H. J. Ensink, A. Battilani, I. Kljujev, S. Gola, V. Raicevic, Z. Jovanovic, R.

Stikic, T. Fletcher and A. Dalsgaard

2.1 Abstract

Clean water has become one of the limiting factors to achieve increased food production in Europe and some southern member states of the European Union now face seasonal water shortages. To overcome water shortages the European Water Framework Directive promotes and encourages the use of treated urban wastewater in agriculture. However, the use of poor quality water in agriculture poses potential health risks. The application of wastewater through subsurface drip irrigation lines could overcome public health concerns by minimizing contact with wastewater but will the subsurface application minimize the risk of contamination of vegetables grown in soil?

To test this hypothesis, the quality of soil and potatoes irrigated by sprinkler irrigation, furrow and subsurface drip irrigation using treated urban wastewater and untreated channel water were compared at experimental sites near Belgrade, Serbia and in Bologna, Italy. Water, soil and potato samples were collected during growing season and analysed for the presence of the faecal indicator organisms E. coli. In addition water and potatoes in Italy were analysed for the occurrence of helminth eggs.

The study found elevated levels of E. coli in irrigation water (Italy mean: 1.7 colony forming units (cfu)/ml and Serbia 11 cfu/ml), but low concentrations of E. coli in soil (Italy mean: 1.0 cfu/g and Serbia 1.1 cfu/g) and detected even lower concentration of E. coli on potatoes (Italy mean: 1.0 cfu/g and Serbia 0.0 cfu/g). The vast majority (>84%) of collected samples were free of E. coli. No helminth eggs were found in irrigation water or on the surface of potatoes.

No correlation was found between irrigation water, soil and produce quality, nor was a significant difference detected between sprinkler, furrow or subsurface drip irrigated soils and produce, indicating that soil was a very effective barrier in protecting food safety and consumer.

2.2 Introduction

Potatoes, the world most important vegetable crop, are grown in soil and irrigation of potato plants has a positive productivity effect on the yield of potatoes (Walker et al., 1999). The demand and need of water for irrigation by agriculture in European has increased during the last decades to meet food crop demand and due to altered precipitation pattern. Decline in groundwater deposit can be a consequence of decreased precipitation across Europe leading to scarcity of clean water (EEA 2003; Falloon and Betts, 2009; IPCC 2007). Treated waste water has been used to irrigate food crops in Australia, the Mediterranean and Israel (Lazarova et al. 2000). The use of low quality water for irrigation of agricultural food crop production as a substitute for groundwater would be an alternative if documentation of microbiological safety for farm workers and consumers can be ensured. There is a growing awareness that food crops can be contaminated with human pathogenic microorganisms via irrigation or post harvest washing with contaminated water, contact with soil or manure, or during the food distribution chain to markets (Beuchat, 1996; Beuchat and Ryu, 1997).


Incidence and outbreaks of disease associated with the consumption of raw fruit and vegetable are becoming numerous (Beuchat, 2002; Sivapalasingam et al, 2004) and the source of pathogens involved in fresh produce contamination may originate from faeces or contaminated water (FDA, 1998).

Low quality water can contain high numbers of faecal microorganisms including occasionally disease-causing pathogens like Salmonella, Campylobacter, Shigella, enteric viruses, protozoan parasites and helminth pathogens (Calci et al. 1998; Nwachuku and Gerba 2008; Steele and Odumeru, 2004; U.S. EPA 1992). This implies a potential risk of introducing faecal pathogens to crops through waste water irrigation or manure fertigation (FDA, 2001). In guidelines for microbial quality of irrigation water, E. coli and helminth eggs are used as indicators of faecal pollution (Barrell et al, 2000; Edberg et al, 2000; WHO, 2006) with the latter also being pathogenic to humans.

Pathogen survival can be affected by waste water application methods. Surface irrigation methods like furrow and sprinkler irrigation expose the microbial pathogens to high temperature, desiccation and UV-light leading to a faster die-off on the surface of the soil (Hutchison et al, 2004). By the subsurface irrigation technique the pathogens are protected from these factors in the soil and their survival depends on parameters such as temperature, moisture content, pH, soil composition and inhibitory competition from indigenous mikroflora (Abu-Ashour et al. 1994; Gerba et al, 1978; Mawdsley et al. 1995).

The research findings presented here were part of the SAFIR project, which was funded under an EU FP6 grant. The overall aim of the project was to develop irrigation management strategies for the production of high quality and safe vegetable crops using treated wastewater. Experimental plots in Bologna, Italy, were irrigated with treated (domestic) wastewater using conventional irrigation techniques (sprinkler irrigation) and subsurface drip irrigation. In Serbia, experimental plots were irrigated with channel water using furrow irrigation and subsurface drip irrigation. Potatoes were grown in the experimental plots. The objective was to assess whether the soil could act as barrier and so guarantee food safety. For this assessment irrigation water, soil and produce samples were collected during 2 cropping seasons in the period from March 2007 to September 2008 and analyzed for the presence of E. coli and helminth ova.

2.3 Methodology

2.3.1 Study sites

The Italian experimental site was located outside Bologna in the Po valley, Northern Italy (44°34´ N, 11°32´ E). The area is predominantly rur al. The experimental field was part of a commercial farm and had not been used for research purposes before. Rainfall during the cropping season was 263.3 mm in 2007 and 301.8 in 2008. The average temperature during growing season in 2007 and 2008 was 17.5°C, respect ively. A total of 18 plots (2 different irrigation application types, 3 different water qualities and 3 replicates), each comprising of 105 m2 were cultivated with potatoes (Agata variety). A total of 462 potato plants were grown per plot. Potatoes were grown on a silty-clay soil (sand 24%, silt 41%, clay 35%), with a bulk density of 1.234 t m-3, a field capacity of 0.345 m3m-3, and a wilting point of 0.214 m3m-3. The shallow groundwater table was located at 0.8 m depth from April to early June.


The experimental site in Serbia was situated in a vegetable commercial farm (“Salate Centre”), located 10 km north of the Serbian capital, Belgrade. The average annual rain fall here was 273.4 mm 187.3 mm in 2007 and 2008, respectively. Average temperatures in the two seasons were 20.6°C. The soil of the field was silty-clay and it was developed on alluvial deposit. The topsoil (0-0.4 m) contained 4% coarse sand, 23% fine sand, 42% silt, 29% clay and 2% of organic matter. The subsoil (0.4-0.8 m) contained 6% coarse sand, 28% fine sand, 39% silt, 26% clay and 1% organic matter. The bulk density was 1530 t cm-3 for both soil depths. Field capacity was 0.32 m3m-3 and a wilting point of 0.20 m3m-3. During the last decade the land had been used for production of lettuce, tomato and cabbage. Field area was divided into 18 plots in a randomized block design and full irrigation strategy was added to 9 of the plots. The potato variety Liseta was used for the study. Plot area was 11.70m x 6 m, with 8 rows and 39 potato plants per row.

2.3.2 Irrigation practices

At the Italian site three different water qualities were used for irrigation: i) tap water, ii) primary treated wastewater (PTWW) and iii) secondary treated wastewater (STWW) (Table 1). Tap water was provided through the municipal water supply system and was groundwater without any additional treatment. PTWW and STWW were obtained from a small wastewater treatment plant serving the nearby village (population <2000 inhabitants). At the study site PTWW underwent further treatment by MBR (Membrane Bio Reactor) technology (Grundfos, Bjerringbro, Denmark), while STWW was further treated with sand filter. MBR treated PTWW (MBR-water) and sand filtrated STWW were stored on site and connected to the field site by two separate, 600 meter long, pipelines. Storage time could reach up to 72 hours before use in agriculture.

Plots were irrigated by mini sprinkler (Netafim Ltd, Tel Aviv, Israel??) or by subsurface drip lines (Netafim Ltd). Drip lines were placed at 0.75 meter distance and subsurface lines were buried at 10 cm depth. The distance between drip emitters was 30 cm and each emitter was able to supply 1.6 L/h. The drip line emitters were located exactly in the middle between two plants. Irrigation frequency was fixed at every second day to obtain optimal soil water dynamic. Crops received water every day.

Table 1: Water types and irrigation type for Italian and Serbian field site

Location Water type Irrigation type

Italy Tap water Sprinkler

Subsurface drip

MBR watera Sprinkler

Subsurface drip

STWWb with sand filtration (SF) Sprinkler

Subsurface drip

Serbia Channel water Furrow

Channel water with sand filtration Subsurface drip

Channel water with sand filtration (SF) and

heavy metal removal devise (HM)

Subsurface drip

a MBR water: Primary treated waste water treated by the MBR technology, b STWW: Secondary treated waste water


In Serbia, channel water of different qualities were used for irrigation: i) Channel water used in furrow irrigation directly without treatment, ii) channel water treated through a sand filter for subsurface drip irrigation and iii) channel water treated by sand filtration and heavy metal removal devise (HM)(Netafim Ltd.) for subsurface drip irrigation (Table 1). Water for irrigation was supplied from the channel which was located 100 m away from the experimental field.

Plots were irrigated by furrow irrigation or by subsurface drip lines (Netafim Ltd). Water pipelines reached the head of each furrow and a valve and flow meter were installed to control flow rate. Drip lines were placed at 0.75 meter distance and were buried at 10 cm depth. The distance between drip emitters (Netafim Ltd) was 30 cm and each emitter was able to supply 1 L/h. The drip line emitters were located exactly in the middle between two plants. Crops received water daily.

2.3.3 Sample collection

Water

Water samples were collected between 7 to 15 times during the cropping season. For bacterial analysis of water quality, a composite sample consisting of three individual 1 litre samples was collected over a 4 hours period. Samples were collected in 1-litre sterile glass bottles. For the helminth egg analysis a 10-litre composite sample was required. Samples were collected and kept in clean plastic containers until further processing.

Samples were stored in a cool box and transported to the local laboratory for further analysis. Analysis of water samples for E. coli was commenced on the day of collection. Samples for helminth egg analysis were stored at 4 – 5 °C until further processing. During the seasons 2007 and 2008 a total of 45 and 21 water samples were analysed at the Italian field site, respectively. At the Serbian site 21 water samples were analysed each year through the two seasons.

Soil

Soil samples were collected before planting of potatoes, during the growing season and at the time of harvest. Before treated waste water application there were from each plot collected a composite sample consisting of 3 sub-samples at two depths: 0-30 cm and 31 – 60 cm. During the irrigation period a composite sample of 8 sub-samples for each depth was collected. The soil from the sub-samples was collected in 1-litre sterile plastic bag and mixed well. Soil samples were collected with an 8 cm auger and within a 25 cm radius of a drip emitter. The sub-samples were taken random in each plot. During the seasons 2007 and 2008 a total of 108 and 180 soil samples were analysed at the Italian field site. At the Serbian site 18 and 36 soil samples, respectively, were analysed through the two seasons.

Potato

Within each plot 3 potato plants were randomly selected at some distance apart. Potato plants were harvested and a total of 4-6 potatoes were selected for the bacteriological analysis. The potatoes were picked with a sterile plastic bag. For the helminth egg analysis a composite sample containing 1 potato from each of the same 3 potato plants chosen for bacteriology analysis was analysed for the presence of helminth eggs. During both 2007 and 2008 18 potato samples were analysed each year at the Italian field site for E. coli and helminth eggs. At the Serbian site 9 potato samples were analysed each year at harvest time for E. coli.

2.3.4 Sample analysis

Bacteriology

Water, soil and potato sample analysis was carried out using a chromogenic medium (CM1046) (Oxoid, Hampshire, UK), selective for E. coli. Samples were analysed according to the pour plate method. Briefly water samples were diluted 10-fold and one ml of sample


dilutions was transferred to an empty Petri-dish and mixed with 15-20 ml of medium with a temperature of 45 ± 1 °C. Plates were incubated at 37 °C for 24 hours. Dark purple to indigo blue colonies were counted as E. coli.

The potato surfaces were washed in 200 ml sterile Peptone Saline Diluent (CM0733, Oxoid) and the washing water was analysed similarly to water samples. Ten gram of soil was stomached for 30 seconds following addition of 90 ml of distilled water. A 10-fold dilution of the mixed solution was then analysed by the same method used for water samples.

Parasitology

Water samples and surface of potatoes were analysed for the occurrence of helminth eggs according to the modified Bailenger method (Ayres and Mara, 1996) with zinc sulphate solution replaced by saturated NaCl-glucose solution.

2.4 Results

From the Italian field site irrigation water and potatoes surface were analysed for the presence and the concentration of E. coli and helminth eggs, respectively. In addition soil samples were analysed for E. coli. In Serbia, the presence and concentration of E. coli were investigated in irrigation water, soil and on potato surfaces.

2.4.1 Italy

A limited number of samples contained E.coli and no helminth eggs were detected. The irrigation water was positive for E. coli in 2007 and 2008 in 31 and 33% of the samples, respectively. E. coli was not detected in any tap water samples. Secondary treated waste water treated through sand filter contained E. coli in 13% of the water samples while 80% of the water samples treated by the MBR system were positive for E. coli. The mean concentration of E. coli was higher in MBR-water compared to STWW treated with sand filtration during the first season while the opposite was observed in the second year (Table 2).

Table 2: Presence of E. coli and helminth eggs in tap water, STWW and MBR-water in Italy.

Mean concentration of E. coli (cfu/ml) and helminth eggs (eggs/L) [maximum value]

Season

Total no. of

samples Organisms

No. of positive samples Tap water MBR-water STWW and SF

2008 21 E. coli 7 ND 1.00 [1] 5.37 [400]

12 Helminth eggs 0 ND ND ND

2007 45 E. coli 14 ND 4.06 [20] 1.28 [7]

15 Helminth eggs 0 ND ND ND aSF: Sand filtration, bUV: UV treatment, ND: Not detected

Initial soil samples taken before irrigation did not contain E. coli. During irrigation and at harvest time only one soil sample (1%) contained E. coli in 2007. This soil sample had been irrigated with MBR-water by sprinkler irrigation and was collected from the lower soil depth. E. coli was not found in soil irrigated with tap water or sand filtrated STWW.


Table 4: Presence of E. coli in soil in Italy.

Mean concentration of E. coli (cfu/g) [maximum value]

Season Time of

sampling

Total no. of

samples

No. of positive samples Tap water MBR-water

STWW and SF

2008 Before irrigation 6 0 ND ND ND

During irrigation 180 2 ND 1.04 [10] 1.05 [20]

2007 Before irrigation 6 0 ND ND ND

During irrigation 108 1 ND 1.19 [500] ND

ND: Not detected

In 2008 E. coli was detected in 1% of the soil samples (2 samples out of a total of 180 soil samples). The first soil sample had been irrigated with MBR-water by sprinkler irrigation and was collected from the top soil layer. The other soil sample had received sand filtrated STWW by subsurface drip irrigation and here the deeper soil layer sample contained E. coli. Both soil samples contained low concentrations of E. coli (Table 3). Potato samples were all negative for E. coli in 2007, while one sample obtained in 2008 contained E. coli with a concentration similar to what was found in soil samples (Table 4). Helminth eggs were not found in any type of irrigation water nor on potatoes (Table 3 + 4).

Table 6: Concentration of E. coli and helminth eggs on potatoes in Italian field site.

Mean concentration of E. coli (cfu/g) and helminth eggs (eggs/g)

[maximum value]

Season

Total no. of

samples Organisms

No. of positive samples Tap water MBR-water STWW and SF

2008 18 E. coli 1 ND ND 1.06 [3]


2007 18 E. coli 0 ND ND ND


ND: Not detected

2.4.2 Serbia

In 2007, E. coli were found in 86% of the water samples. All channel water samples both before and after sand filtration contained E. coli. After the heavy metal remover devise E. coli was detected in 66% of irrigation water samples with a mean concentration of 2.7 cfu/ml (Table 5). In 2008, the prevalence of E. coli in the channel water was 90% and the distribution between the different channel water types was similar to findings in 2007. No E. coli was found on the surface of potato samples during the two seasons (Table 6).

Seventeen per cent of the soil samples analysed in 2008 prior to irrigation with channel water was initiated contained E. coli while all soil samples collected the first year before irrigation had E. coli (Table 7).


Table 5: Presence of E. coli in channel water and treated channel water in Serbia.

aSF: Sand filtration, bHM: Heavy metal remover device

Table 6: Concentration of E. coli on potatoes in the Serbian field site.

aSF: Sand filtration, bHM: Heavy metal remover device, ND: Not detected

Table 7: Presence of E. coli in soil in Serbia.

aSF: Sand filtration, bHM: Heavy metal remover device, ND: Not detected

The mean concentration of E. coli found in these initial soil samples in 2007 was up to 380 times higher compared to the E. coli concentration in the channel water. In 2007, E. coli was detected in 17% of the soil samples collected during the irrigation period and all of them had been irrigated with sand filtrated STWW through subsurface drip irrigation. Only the lower soil fraction was positive for E. coli. Soil samples collected during the 2008 season were all negative for E. coli.

2.5 Conclusion

Low levels of E. coli were found in irrigation water from Italy and Serbia. Only one potato sample was positive for E. coli during the 2 years of study. Soil samples taken before irrigation with treated waste water or channel water, respectively, contained higher concentration of E. coli then found in soil during the irrigation period. This suggests other possible faecal contamination sources than the irrigation water e.g. environmental sources like wild animals, birds, etc.

Mean concentration of E. coli (cfu/ml) [maximum value]

Season

Total no.

of

samples

Organisms No. of

positive samples Channel

water Channel water

and SFa Channel water, SF a and HMb

2008 21 E. coli 19 25.6 [100] 21.2 [90] 9.99[110]

2007 21 E. coli 18 13.3 [30] 8.88 [10] 2.68 [10]


Season

Total no.

of

samples

Organisms No. of

positive samples Channel

water Channel water

and SFa Channel water, SF a and HMb




Season Time of sampling

Total no.

of

samples

No. of positive samples Channel

water Channel

water and SFa Channel water, SF a and HMb

2008 Before irrigation 18 3 ND 117 [37000] ND

During irrigation 36 0 ND ND ND

2007 Before irrigation 18 18 619 [3150] 2809 [5000] 1031 [4100]

During irrigation 18 3 ND 3.98 [40] ND


2.6 Acknowledgement

This study was supported by EU Commission (FP6 projects SAFIR and CROPWAT) and Serbian Ministry of Science (TR 20025). Also, the authors are grateful to Netafim company (A.C.S. Ltd. Netafim, Israel) and Avi Schweitzer for donating the irrigation system.

2.7 References

Abu-Ashour, J., D.M. Joy, H. Lee, H.R. Whiteley, S. Zelin. 1994. Transport of microorganisms through soil. Water, Air and Soil Pollution 75, 141-157.

Ayres, R. M. and D. D. Mara. 1996. Analysis of Wastewater for Use in Agriculture – A Laboratory Manual of Parasitological and Bacteriological Techniques. WHO, Geneva, Switzerland.

Barrell, R. A. E., P. R. Hunter and G. Nichols. 2000. Microbiological standards for water and their relationship to risk. Communicable Disease and Public Health 3, No. 1, 8-13.

Beuchat. L. R. 1996. Pathogenic Microorganisms Associated with fresh Produce. Journal of Food Protection 59, No. 2, 204-216.

Beuchat, L. R. 2002. Ecological factors influencing survival and growth of human pathogens on raw fruit and vegetables. Microbes and Infection 4, 413-423.

Beuchat, L. R., and J. H. Ryu. 1997. Produce handling and processing practices. Emerging Infectious Diseases 3, no. 4, 459-465.

Calci, K.R., W. Burkhardt III, W.D. Watkins, and S.R. Rippey. 1998. Occurrence of male-specific bacteriophage in feral and domestic animal wastes, human feces, and human-associated wastewaters. Applied and Environmental Microbiology 64, no. 12, 5027-5029.

Edberg, S. C., E. W. Rice, R. J. Karlin, M. JU. Allen. 2000. Escherichia coli: the best biological drinking water indicator for public health protection. Symposium Series Society for Applied Microbiology 29, 106S-116S.

EEA. 2003. Europe’s environment: The third Environmental assessments report No 10.

Falloon, P., and R. Betts. 2009. Climate impacts on European agriculture and water management in the context of adaptation and mitigation – The importance of an integrated approach. Science of the Total Environment, Article in press.

FDA (U.S. Food and Drug Administration). 1998. Guidance for Industry: Guide to Minimize Microbial Food Safety Hazards for Fresh Fruits and Vegetables. Available at: http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/ProduceandPlanProducts/ucm064574.htm



FDA (U.S. Food and Drug Administration). 2001. Analysis and Evaluation of Preventive Control Measures for the Control and Reduction/Elimination of Microbial Hazards on Fresh and Fresh-Cut Produce. Available at: http://www.fda.gov/Food/ScienceResearch/ResearchAreas/SafePracticesforFoodProcesses/ucm090977.htm Accessed November 12, 2009

Gerba, C. P., J. L. Melnick and C. Wallis. 1978. Fate of Wastewater Bacteria and Viruses in Soil. Journal of the Irrigation and Drainage Division 101, No. 3, 157-174.

Hutchison, M. L., L. D. Walters, A. Moore, K. M. Crookes, and S. M. Avery. 2004. Length of Time before Incorporation on Survival of Pathogenic Bacteria Present in Livestock Wastes Applied to Agricultural Soil. Applied and Environmental Microbiology 70, No. 9, 5111-5118.

IPCC. 2007. Climate Change 2007: Synthesis Report. Contribution of Working Groups I, II and III to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. IPCC, Geneva, Switzerland.

Lazarova, V., G. Cirelli, P. Jeffrey, M. Salgot, N. Icekson, and F. Brissaud. 2000. Enhancement of integrated water management and water reuse in Europe and the Middle East. Water Science and Technology 42, no.1-2, 193-202.

Mawdsley, J.L., R.D. Bardgett, R.J. Merry, B.F. Pain, M.K. Theodorou. 1995. Pathogens in livestock waste, their potential for movement through soil and environmental pollution. Applied Soil Ecology 2, 1-15.

Nwachuku, N., and C.P. Gerba. 2008. Occurrence and persistence of Escherichia coli O157:H7 in water. Reviews in Environmental Science and Biotechnology 7, 267-273.

Sivapalasingam, S., C. R. Friedman, L. Cohen, and R. V. Tauxe. 2004. Fresh Produce: A Growing Cause of Outbreaks of Foodborne Illness in the United States, 1973 through 1997. Journal of Food Protection 67, No. 10, 2342-2353.

Steele, M., and J. Odumeru. 2004. Irrigation Water as Source of Foodborne Pathogens on Fruit and Vegetables. Journal of Food Protection 67, No. 12, 2839-2849.

U.S. EPA. 1992. Guidelines for water reuse. United States Environmental Protection Agency, Washington.

Walker, T. S., P. E. Schmiediche and R. J. Hijmans. 1999. World trends and patterns in the potato crop: An economic and geographic survey. Potato Research 42, 241-264.


WHO. 2006. Guidelines for the safe use of wastewater, excreta and greywater, Volume 2: Wastewater use in agriculture. World Health Organization, Geneva, Switzerland.


3. Faecal contamination and hygiene aspect of soil and on tomatoes (Solanum Lycopersicum) irrigated with part ially treated waste water.

A. Forslund, J. H. J. Ensink, A. Battilani, S. Gola, L. Sandei, G. Psarras, T. Fletcher and A.

Dalsgaard

3.1 Abstract

The demand for fresh produce by European consumers has increased dramatically over the last century as a result of changing food habits and rapid population growth. Water has become one of the limiting factors to achieve increased production and some areas within the European Union now face seasonal water shortages. To overcome water shortages the European Water Framework Directive promotes and encourages the use of treated urban wastewater in agriculture. However, the use of poor quality water in agriculture poses potential health risks. The application of wastewater through subsurface drip irrigation lines could overcome public health concerns by minimizing contact with wastewater as well as minimizing contamination of vegetables grown on plants.

To test this hypothesis, the quality of soil and tomatoes irrigated by sprinkler irrigation, surface and subsurface drip irrigation using treated urban wastewater were compared at experimental sites in Chania, Crete and Bologna, Italy. Water, soil and tomato samples were collected at regular intervals and analysed for the presence of the faecal indicator organisms E. coli and helminth eggs.

The study found elevated levels of E. coli in irrigation water (Italy mean: 2.5 colony forming units (cfu)/ml and Crete 1.1 cfu/ml), but low concentrations of E. coli in soil (Italy mean: 1.8 cfu/g and Crete 2.2 cfu/g) and found even lower concentrations on tomatoes (Italy mean: 0.0 cfu/g and Crete 1.4 cfu/g). The vast majority (>85%) of collected samples were free of E. coli. No helminth eggs were found in the irrigation water (Crete and Italy) or on the tomatoes (Crete) during the 2 seasons. In Italy, 11 % of the tomato samples contained helminth eggs in 2007 while none were detected in 2008.

No correlation was found between irrigation water, soil and produce quality, nor was a significant difference detected between sprinkler, surface drip or subsurface drip irrigated soils and produce, indicating that soil was a very effective barrier in protecting food safety and consumer.

3.2 Introduction Since the beginning of the 20th century the demand for water by European agriculture has, as a result of rapid population growth and urbanization, more than doubled (Lavalle et al, 2009). Imbalances in water supply and availability are experienced by many European Union (EU) member countries around the Mediterranean, particularly in the summer months mainly as a result of low precipitation and peak demands for irrigation water by agriculture and the tourist industry. The use of urban wastewater in agriculture has often been propagated as a way to overcome water scarcity and to protect aquatic ecosystems from contamination. In several countries around the Mediterranean treated urban wastewater as been incorporated as a resource into integrated water resource management programmes, with Israel currently at the forefront. The European Water Framework Directive (91/271/EEC), advocated a similar approach and specifies that treated wastewater should be used in agriculture where and whenever appropriate (EU, 1991). However, human pathogens, organic and inorganic constituents, found in urban wastewater are a matter of concern for both farmer health and


food safety (FDA, 2001). To overcome these public health concerns water quality guidelines for the safe use of wastewater in agriculture were developed by the United States Environmental Protection Agency (USEPA) and the World Health Organization (WHO). The USEPA guideline advocates a no risk approach, which means that water used for irrigation purposes should effectively be free of pathogens and that all wastewater should undergo tertiary treatment processes before it is allowed to be used in agriculture (USEPA, 2004). This is in contrast to the WHO guidelines which use health based targets and the assumption that no additional cases of disease should occur as a result of exposure to wastewater or wastewater irrigated produce (WHO, 2006). For this the WHO guidelines use a Quantitative Microbial Risk Assessment (QMRA) model which calculates, based on a permissible annual disease risk, a required reduction in pathogens concentrations. The WHO guidelines advocate a multiple barrier approach and the required reduction in pathogen concentration is not expected to be only met through wastewater treatment technology, but will depend on the type of crop grown (crop consumed uncooked vs. crops consumed cooked and industrial crops), the degree of mechanization (labour intensive vs. mechanized) and how irrigation water is applied (basin vs. bed and furrow irrigation and sprinkler vs. surface or subsurface drip irrigation) (WHO, 2006). Neither the USEPA nor the WHO guidelines have been adopted by the EU, though some countries and regions have adopted guidelines along the lines of the WHO. However the lack of centrally set EU water quality standards, combined with the introduction of the 'fork to farm' principle and product standards, means that vegetable farmers are expected to control product quality – like primary producers in other industries- and as a result the use of treated wastewater in agriculture remains uncommon within EU member states. The research findings here presented were part of the SAFIR project and were funded under an EU FP6 grant. The primary objective of the overall project was: to develop irrigation management strategies for the production of high quality and safe vegetable crops using treated wastewater. At experimental plots in Chania, Crete and Bologna, Italy, tomatoes were irrigated at experimental plots with treated (domestic) wastewater using conventional irrigation techniques (sprinkler and surface drip irrigation) and subsurface drip irrigation in order to assess whether the soil could act as barrier and so protect farmer health and guarantee food safety. For this assessment irrigation water, soil and produce samples were collected during 2 cropping seasons in the period from March 2007 to September 2008 and analysed for the presence of E. coli and helminth ova.

3.3 Methodology

3.3.1 Study sites

The Italian experimental site was located outside Bologna in the Po valley, Northern Italy (44°34´ N, 11°32´ E). The area is predominantly agr icultural. The experimental field is part of a commercial farm and had not been used for research purposes before. Rainfall during the cropping season was 178.5 mm in 2007 and 230.8 mm in 2008. The average temperature during growing season 2007 and 2008 was 22.8°C and 23.5°C, respectively. A total of 18 plots (2 different irrigation application types, 3 different water qualities and 3 replicates), each comprising of 105 m2 were cultivated with processing tomatoes (Perfect Peel variety). A total of 350 tomato plants were grown per plot. Tomatoes were grown on a silty-clay soil with a shallow groundwater table, at 0.8 m depth from April to early June.

The Greek experimental plots were located on the Island of Crete in the peri-urban areas of Chania. Vegetable cultivation is common in this part of the island, with cultivation occurring more or less continuous throughout the year. The average annual rain and temperature in 2007 was 50.6 mm and 22.7°C. In 2008 there were 34. 8 mm rain and the average


temperature was 21.9°C during the cropping season. Here 12 plots (2 different irrigation application types, 2 different water qualities and 3 replicates), were selected, each comprising an area of 50 m2 with of total of 100 tomato (Verdoun variety) plants per plot.

3.3.2 Irrigation practices At the Italian site three different water qualities were used for irrigation: i) tap water, ii) primary treated wastewater (PTWW) and iii) secondary treated wastewater (STWW) (Table1). Tap water was provided through the municipal water supply system and was groundwater without any additional treatment. PTWW and STWW were obtained from a small wastewater treatment plant serving the nearby village (population <2000 inhabitants). At the study site PTWW underwent further treatment by MBR (Membrane Bio Reactor) technology (Grundfos, Bjerringbro, Denmark), while STWW was further treated with sand filter. PTWW and STWW were stored on site and connected to the field site by two separate, 600 meter long, pipelines. Storage time could reach up to 72 hours before use in agriculture.

Plots were irrigated by mini sprinkler (Netafim Ltd, Tel Aviv, Israel) or by subsurface drip lines (Netafim Ltd) Drip lines were placed at 1.5 meter distance and subsurface lines were buried at 10 cm depth. The distance between emitters was 40 cm and these were positioned exactly in the middle between two plants. Their discharge rate was 1.6 L/h. Crops received water every day. Processing tomatoes received 251 mm of irrigation water in 2007.

Table 1: Water and irrigation types in Italy and on Crete

Location Water type Irrigation type

Italy Tap water Sprinkler

Subsurface drip

MBR watera Sprinkler

Subsurface drip

STWWb with sand filtration Sprinkler

Subsurface drip

Crete Tap water Surface drip

Subsurface drip

STWWb with sand filtration and UV-treatment Surface drip

Subsurface drip a MBR water: Primary treated waste water treated by the MBR technology, b STWW: Secondary treated waste water

On Crete two different water qualities were used for irrigation: i) Tap water and ii) secondary treated wastewater (STWW). STWW was obtained from the Chania wastewater treatment plant and transported to the experimental site, where it was stored in four 5m3 tanks. Tomatoes were after planting for a period of 5 weeks irrigated with tap water, after which secondary effluent was used for irrigation of the assigned plots. Plots were irrigated by surface drip lines (Netafim Ltd, Tel Aviv, Israel) or by subsurface drip lines (Netafim Ltd) (Table 1). Drip lines were placed at 1 meter distance and subsurface lines were buried at 15 cm depth. The distance between emitters was 50 cm with the dripper located next to each plant. Drip emitter was able to supply 1.6 L/h. Crops received water every day.


3.3.3 Sample collection

Water

Water samples were collected fortnight during the cropping season. For bacterial analysis of water quality, a composite sample consisting of three individual 1-L samples was collected over a 4-hrs period and subsequent pooled. Samples were collected in 1-L sterile glass bottles. For the helminth egg analysis, a 10-L composite sample was required. Samples were collected and kept in clean plastic containers until further processing.

Samples were stored in a cool box and transported to the local laboratory for further analysis. Analysis of water samples for E. coli on the day of collection. Samples for helminth egg analysis were stored at 4 – 5 °C until further processing. During the seasons 2007 and 2008 a total of 57 and 33 water samples were analysed at the Italian field site, respectively. At the Greek site 42 and 20 water samples were analysed through 2007 and 2008, respectively.

Soil

Soil samples were collected before planting of tomato plants, during the growing season at the time of harvest. Before irrigation there were from each plot collected a composite sample consisting of 3 sub-samples at two depths: 0-20 cm and 21 – 40 cm, while during the irrigation period a composite sample of 8 sub-samples for each depth was collected. The soil from the sub-samples was collected in 1 L sterile plastic bag and mixed well. Soil samples were collected with a 2 cm auger and within a 25 cm radius of a drip emitter. The sub-samples were taken random in each plot. During the seasons 2007 and 2008, a total of 114 and 222 soil samples were analysed at the Italian field site, respectively. At the Greek site 28 and 76 soil samples were analysed through the two seasons.

Tomato

Within each plot 3 plants were randomly selected at some distance apart and 1 - 6 tomatoes were picked from each plant for the bacteriological analysis. The tomatoes were picked with a sterile plastic bag and tomatoes from the lower part of the plants were chosen. For the helminth egg analysis, a composite sample containing 1 tomato from each of the same 3 tomato plants chosen for bacteriology analysis were analysed for the presence of helminth eggs. In Italy, 18 tomato samples were analysed for the presence of E. coli and helminth eggs on the tomato surface in both 2007 and 2008. On Crete there were analysed 24 tomato samples for E. coli and 12 tomato samples for helminth eggs in 2007. The same number of samples were analysed the following year.

3.3.4 Sample analysis

Bacteriology

Water, soil and tomato sample analysis was done on a chromogenic medium (CM1046) (Oxoid, Hampshire, UK), selective for E. coli. Samples were analysed according to the pour plate method. Briefly water samples were diluted 10-fold and one ml of sample dilutions was transferred to an empty Petri-dish and mixed with 15-20 ml of medium with a temperature of 45 ± 1 °C. Plates were incubated at 37 °C for 24 hours. Dar k purple to indigo blue colonies were counted as E. coli colonies.

The surface of the tomatoes were washed in 200 ml sterile Peptone Saline Diluent (CM0733, Oxoid) and the washing water were analysed similarly to water samples. Ten gram of soil added 90 ml of distilled water were stomached for 30 seconds, thereafter 10-fold diluted and analysed likewise the water samples.

Parasitology

Water samples and surface of tomatoes were analysed for the occurrence of helminth eggs according to the modified Bailenger method (Ayres and Mara, 1996) with zinc sulphate solution replaced by saturated NaCl-glucose solution.


3.4 Results

Irrigation water and the surface of tomatoes were analysed for the presence and concentration of E. coli and helminth eggs, respectively. Soil were analysed for E. coli.

3.4.1 Italy

In Bologna, a limited number of samples contained E.coli and even less contained helminth eggs. Both in 2007 and 2008, 36% of the irrigation water samples were positive for E. coli. E. coli was not detected in tap water. Secondary treated waste water treated through sand filter contained E. coli in 37% of the water samples while 73% of the MBR-water samples were positive for E. coli. The concentration and maximum value of E. coli found in water samples are listed in Table 2.

Table 2: Presence of E. coli and helminth eggs in tap water, STWW and MBR-water

Concentration of E. coli (cfu/ml) and helminth eggs (eggs/L) [maximum value]

Location Season Total no.

of samples

Organisms No. of

positive samples

Tap water

MBR-water

STWW and SFa

STWW, SFa and UVb

Italy 2008 33 E. coli 12 ND 1.21 [4] 14.5 [1000] -

12 Helminth eggs 0 ND ND ND -

2007 57 E. coli 21 ND 3.76 [20] 3.39 [3000] -


Crete 2008 20 E. coli 0 ND - - ND

2 Helminth eggs 0 ND - - ND

2007 42 E. coli 6 1.14 [8] - - 1.40 [79]

24 Helminth eggs 0 ND - - ND aSF: Sand filtration, bUV: UV treatment, ND: Not detected

Initial soil samples taken in 2007 before irrigation did not contain E. coli while 6% of soil samples collected the following spring contained E. coli before irrigation. Soil samples taken during irrigation and at harvest time were positive for E. coli in 26% of the soil samples in 2007. Of these soil samples 18% had been irrigated with tap water, 36% received STWW inclusive sand filtration and the rest (46%) of the soil samples had been irrigated with MBR-water. In tap water irrigated plots, 80% of the positive samples had received subsurface drip irrigation while 20% had been sprinkler irrigated. For STWW the distribution between subsurface drip irrigation and sprinkler were identical. In MBR-water irrigated soil samples the majority of soil samples positive for E. coli had been sprinkler irrigated (77%). Soil samples were furthermore divided in soil samples taken from the top 20 cm and soil samples taken 20 to 40 cm below the surface. 67% of the E. coli positive soil samples were found in the top layer. In 2008 it was only possible to detect E. coli in 1% of the soil samples arising from plots irrigated with sand filtrated STWW by subsurface drip irrigation.

Table 3 illustrates the concentration and maximum value of E. coli detected in soil distributed on water types. Tomato samples were all negative for E. coli in the two years samples were analysed (Table 4).


Table 3: Presence of E. coli in soil

Concentration of E. coli (cfu/g) [maximum value]

Location Year Time of sampling Total no. of

samples

No. of positive samples Tap water MBR

water STWW with

SFa STWW with SFa and UVb

Italy 2008 Before irrigation 6 1 2.08 [80] ND ND -

During irrigation 216 2 ND ND 1.19 [23000] -

2007 Before irrigation 6 0 ND ND ND -

During irrigation 108 28 1.58 [50] 7.11 [480000]

2.50 [1200] -

Crete 2008 Before irrigation 4 0 ND - - ND

During irrigation 72 6 1.30 [60] - - 1.78 [9400]

2007 Before irrigation 4 3 18.8 [360] - - 7.45 [12]

During irrigation 24 14 2.98 [71] - - 3.29 [52] aSF: Sand filtration, bUV: UV treatment, ND: Not detected

Helminth eggs were not found in any water samples during the two years. Only in 2007, helminth eggs were found on the surface of two tomato samples. One of the samples originated from a plot irrigated with tap water while the other had received secondary treated waste water. Species of helminth eggs found was in both cases Strongoloides.

3.4.2 Crete

No helminth eggs were found in water samples and on the surface of tomatoes during 2007 and 2008, respectively. In 2007, E. coli were found in water samples in 14% of water samples with two-thirds of these were found in STWW treated by UV-light with a mean concentration of 1.4 cfu/ml (Table 2). In 2008, no E. coli were detected in irrigation water or on tomato surfaces. In 2007, only two tomato samples contained E. coli with a concentration of 13,157 cfu/g (surface drip irrigation) and 554 cfu/g (subsurface drip irrigation). Both samples had been irrigated with UV-treated STWW (Table 4).

Initial soil samples taken in 2008 before irrigation did not contain E. coli, while 75% of soil samples collected before irrigation the first year contained E. coli. In 2008, it was possible to detect E. coli in 8% of the soil samples collected during the irrigation period and half of them had been irrigated with tap water and half with UV-treated STWW. About 30% of the E. coli positive soil samples were irrigated with surface drip irrigation while the rest received sursurface drip irrigation. Soil samples taken in 2007 were positive for E. coli in 58% of the soil samples. Of these soil samples, 57% had been irrigated with tap water and 43% received STWW inclusive sand filtration and UV-treatment. For both tap water and UV-treated STWW, 50% of the E. coli positive soil samples had received subsurface drip irrigation while 50% had been surface drip irrigated. Soil samples were furthermore divided in soil samples taken from the top 20 cm and soil samples taken 20 to 40 cm below the surface. A total of 60% of the E. coli positive soil samples were found in the top layer. Table 3 illustrates the concentration and maximum value of E. coli detected in soil distributed on water types.


Table 4: Concentration of E. coli and helminth eggs on tomatoes

Concentration of E. coli (cfu/g) and helminth eggs (eggs/g)

[maximum value] Location Year Organisms No. of

samples Tap water MBR-water STWW and

SFa STWW, SFa

and UVb

Italy 2008 E. coli 18 ND ND ND -

Helminth eggs 18 ND ND ND -

2007 E. coli 18 ND ND ND -

Helminth eggs 18 1.02 [1.13] ND 1.02 [1.13] -

Crete 2008 E. coli 24 ND - - ND

Helminth eggs 12 ND - - ND

2007 E. coli 24 ND - - 4.21 [13157]

Helminth eggs 12 ND - - ND aSF: Sand filtration, bUV: UV treatment, ND: Not detected

3.5 Conclusion

The results of the study indicate that there does not seem to be any correlation between the level of faecal pollution in the water used for irrigation and on the irrigated crops. Further studies of E. coli isolated from irrigation water and soil may in light if a connection can be established

3.6 Acknowledgement

This study was supported by EU Commission (FP6 projects SAFIR). The authors thanks Grundfos and Netafim company for guidance.

3.7 References

Ayres, R. M. and D. D. Mara. 1996. Analysis of Wastewater for Use in Agriculture – A Laboratory Manual of Parasitological and Bacteriological Techniques. WHO, Geneva, Switzerland.

European Water Framework Directive (2000/60/EC)

http://ec.europa.eu/comm/environment/water/water-framework/index_en.html


FDA (U.S. Food and Drug Administration). 2001. Analysis and Evaluation of Preventive Control Measures for the Control and Reduction/Elimination of Microbial Hazards on Fresh and Fresh-Cut Produce. Available at: http://www.fda.gov/Food/ScienceResearch/ResearchAreas/SafePracticesforFoodProcesses/ucm090977.htm Accessed November 12, 2009


Lavalle, C., F. Micale, T. D. Houston, A. Camia, R. Hiederer, C. Lazar, C. Conte, G. Amatulli and G. Genovese. 2009. Climate change in Europe. 3. Impact on agriculture and forestry. A review. Agronomy for Sustainable Development 29. No. 3, 433-446.

USEPA, 2004, Guidelines for water reuse. US Environmental Protection Agency, Washington DC, US.

WHO. 2006. Guidelines for the safe use of wastewater in agriculture. World Health Organization, Geneva, Switzerland.