Aquacult Int

DOI 10.1007/s10499-012-9498-4

Environmental conditions in a lagoon and their possible

effects on shellfish contamination by Giardia
and Cryptosporidium

Matteo Francavilla • Pasquale Trotta • Marianna Marangi •

Paolo Breber • Annunziata Giangaspero

Received: 24 June 2011 / Accepted: 9 January 2012

Ó Springer Science+Business Media B.V. 2012

Abstract Over a 1-year period, we carried out an analysis for the presence of Giardia
and Cryptosporidium (oo)cysts in 934 specimens taken from four species of shellfish
farmed in the Varano Lagoon. We also studied the chemical, physical and meteo-climatic
parameters in the same environment, to evaluate the possible effects of environmental
conditions on protozoan contamination in farmed shellfish. Using both IF and molecular
tests, all 38 pools of shellfish gave negative values for Giardia and Cryptosporidium
oo/cysts, thus confirming previous investigations. The environmental factors considered
having a negative influence on the survival of Giardia and Cryptosporidium are high water
temperatures (8–29°C) and high salinity levels (23–32 ppt). However, the small amount of
freshwater discharged into the lagoon (up to 0.0091 m3 s-1), the long-lasting solar irra-
diation (mean of 896 W m-2) and the shallowness of the lagoon (mean 2.5 m) may also
have a synergic effect in limiting the presence of Giardia and Cryptosporidium in the
lagoon.

Keywords Giardia  Cryptosporidium  Shellfish  Chemical–physical parameters 

Meteo-climatic parameters  Lagoon

Introduction

Shellfish farming has expanded rapidly in recent decades (FAO 2010) due to the worldwide
economic importance of molluscs as food. Shellfish farming is a socially and economically
important industry in Italy; shellfish are the most farmed aquatic species (about 150,000

M. Francavilla  P. Trotta  P. Breber

Istituto di Scienze Marine, Consiglio Nazionale delle Ricerche (CNR), Via Pola 4, 71010 Lesina, Italy

M. Marangi  A. Giangaspero (&)

Dipartimento di Scienze delle Produzioni, dell’Ingegneria e della Meccanica e dell’Economia
Applicate ai Sistemi Agro-Zootecnici, University of Foggia, Via Napoli 25, 71100 Foggia, Italy
e-mail: a.giangaspero@unifg.it

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 Aquacult Int

tons annually) and account for over 10% (about 70,000 tons) of fishery products (Mattei
and Pellizzato 1997). Several species of shellfish are farmed along the Italian coast, and the
most important production area involves clams (Ruditapes (Tapes) philippinarum,
Chamelea gallina, Ruditapes (Tapes) decussatus) and intensive farming of mussels Mytilus
galloprovincialis (Prioli 2001; Lucchetti 2003) along the Adriatic coast. Lagoons are
considered to be very suitable for shellfish farming, and the most farmed lagoons in Italy
are the Venice, Marano and Sacca di Goro lagoons along the northern Adriatic coast, the
Orbetello lagoon on the Tyrrhenian coast and the Varano Lagoon in southern Italy (Rossi
and Paesanti 1992). Lagoon environments have high inputs of nutrients of continental
origin, which can sustain high rates of primary productivity (Castel et al. 1996). Lagoons
are prime locations for farming filter-feeding molluscs because of the ready availability of
particulate nutrients in lagoon environments, together with generally shallow water and
easy access to land-based facilities. However, geo-morphological features such as con-
finement and shallowness mean that coastal lagoons are highly productive systems, but that
they are also highly vulnerable to eutrophication, harmful algal blooms, oxygen depletion
and dystrophic outbreaks (Kjerfve 1994; Castel et al. 1996; De Wit et al. 2001; Viaroli
et al. 2006); this is a consequence of their position between the land and open sea and of
their consequent exposure to anthropogenic pressures from urban, industrial and tourism
development and from nutrients and pollutants from heavily exploited watersheds (Gönenç
and Wolfin 2005). These ecosystems can also be contaminated with waterborne pathogens
from diffuse or point-source discharges of human sewage or agricultural waste within their
catchment areas. Molluscs can filter large volumes of water and can also retain micro-
organisms and must be considered a potential vehicle of infection transmission if eaten raw
or very lightly cooked (Robertson 2007).
Giardia and Cryptosporidium spp. are important enteric protozoan pathogens for
humans and animals and have been found in contaminated water and edible shellfish all
over the world, including Italy (Robertson 2007; Giangaspero et al. 2009). In a previous
study, Giangaspero et al. (2009) studied the presence and species/genotypes of Giardia
and Cryptosporidium in water flowing into the Varano Lagoon (Apulia Region, southern
Italy), and in shellfish (R. decussatus, R. philippinarum and M. galloprovincialis) farmed
in the lagoon throughout a 1-year period (February 2006 to January 2007). High con-
centrations of Giardia cysts and Cryptosporidium oocysts were detected in almost all
samples of water flowing into the lagoon. However, no contamination was detected in the
shellfish sampled, and this absence of contamination is assumed to be related to Varano
Lagoon’s physical and ecological factors. Several studies have evaluated the persistence
of Giardia and Cryptosporidium (oo)cyst pathogens in water as a function of both sur-
vival and transport. Different pathogens persist for different periods of time, and the most
important mode of inactivation or mortality may vary significantly. Factors that have been
shown to influence the viability of Giardia and Cryptosporidium protozoans include
temperature, salinity, exposure time of pathogens, hydrodynamics, water discharge, solar
radiation (visible and UV) and ammonia concentrations (Brookes et al. 2004; Fayer et al.
2004).
In view of these findings, we tested shellfish farmed in the Varano Lagoon for the
presence of Giardia and Cryptosporidium (oo)cyst over a 1-year period and also studied
the lagoon’s chemical, physical and meteo-climatic parameters in order to evaluate the
possible effects of environmental conditions on protozoan contamination in farmed
shellfish.

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Materials and methods

Study area: the Varano Lagoon

The Varano Lagoon lies on the northern coast of the Gargano Promontory (southern
Adriatic Sea) (41.88°N; 15.75°E) (Fig. 1). It has a surface area of about 65 km2 and
resembles a lake because of its trapezoidal shape and its shores sloping straight down to the
water. The water depth is fairly uniform along the shoreline. The minimum and maximum
water depths are 0.5 m near the shoreline and 5.8 m in the centre of the lagoon, respec-
tively (Brambati 1988).
The lagoon is partially isolated from the southern Adriatic Sea by a coastal barrier, with
two channels: Capoiale (5.22 m deep) at its western end and Varano (4.31 m deep) at its
eastern end. These channels allow the lagoon to communicate with the sea through the
hydrodynamic balance created by tidal level, wind strength and direction, and anthropo-
genic action.
The lagoon’s main freshwater sources are the San Nicola springs (underwater springs
discharging groundwater into the south-western area of the lagoon) and the Bagno spring in
the southern area. Other freshwater sources bringing rainfall run-off into the lagoon along
with sewage from the municipal wastewater treatment plants are the Sant’Antonino

Adriatic Sea
Capoiale Outlet
Varano Outlet

41.92

St7
St3
LATITUDE (WGS84 - Decimal Degrees)

St1 Tr5
galloprovincialis
decussatus

41.9
Tapes
Oestrea

Mytilus
gigas

41.88 St4

Tr1 St2
Tapes
philippinarum

41.86 St6
St5 Tr4

Tr3
41.84 Tr2

41.82

15.64 15.66 15.68 15.7 15.72 15.74 15.76 15.78 15.8 15.82 15.84 15.86

LONGITUDE (WGS84 - Decimal Degrees)

Fig. 1 The Varano Lagoon. Sampling sites in lagoon: St1, St2, St3, St4, St5, St6, St7. Freshwater
tributaries: Tr1 (San Nicola springs), Tr2 (Bagno spring), Tr3 (San Francesco channel), Tr4
(Sant’Antonino channel), Tr5 (Idrovora Muschiaturo channel). Farms of Ruditapes decussatus and Mytilus
galloprovincialis (41°530 N–15°440 E). Artificial bench of Crassostrea gigas (41°54.150 N–15°43.510 E).
Natural bench of Ruditapes philippinarum (41°51.750 N–15°48.230 E)

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channel from Cagnano Varano and the San Francesco channel from Carpino. Sant’An-
tonino discharges chlorine-treated wastewater produced by the approximately 9,000
inhabitants of Cagnano Varano, and San Francesco discharges chlorine-plus UV-treated
wastewater produced by the approximately 5,000 inhabitants of Carpino. Both channels
flow into the lagoon in the south-eastern area. The Idrovora Muschiaturo is another
freshwater tributary draining water from agricultural lands, which discharges into the
eastern area of the Varano Lagoon.
In the Varano Lagoon, there are two shellfish farms (R. decussatus and M. gallopro-
vincialis) with a total area of 750 9 1,200 m, in the area around coordinates 41°530 N–
15°440 E of the lagoon, and there is also an artificial bench of Crassostrea gigas of
30 9 15 m, around coordinates 41°54.150 N–15°43.510 E (Fig. 1). In addition, there is a
natural bench of R. philippinarum, of about 200 m2, located at the inlet of the Sant’An-
tonino sewage channel (41°51.750 N–15°48.230 E).

Shellfish sampling and processing

From April 2007 to February 2008, we collected by hand 600 specimens of M. gallo-
provincialis, 129 specimens of R. decussatus, 129 specimens of R. philippinarum and 125
specimens of C. gigas, in the Varano Lagoon. The specimens were kept at 0–5°C until they
reached the laboratory, where they were measured, weighed (live weight) and pooled
(2 pools of 30 molluscs month/site) for convenience (Table 1).

Haemolymph concentration and direct immunofluorescence test

Following procedures adopted from previous work (Graczyk et al. 1999, 2001; Molini
et al. 2007), haemolymph was aspirated from each mollusc (approximately 100 lL/clam)
and pooled according to the site of collection and to the number of molluscs. Each pool
was concentrated in a 1 M sucrose solution and centrifuged at 4009g for 15 min. The
interface was then aspirated, re-suspended in 4 mL of saline (0.9% NaCl) and centrifuged
at 6009g for 10 min. The supernatant was finally removed by aspiration, leaving 1.5 mL
of concentrated sample volume including the pellet. The haemolymph (50 lL from each
pool) was dried on glass microscope slides and examined by fluorescence microscopy after
staining with FITC-labelled antibodies as described by the manufacturer (MERIFLUORÒ
Cryptosporidium/Giardia; Meridian Diagnostic, Cincinnati, Ohio, USA).

Molecular study

All haemolymph samples were also subjected to molecular tests to confirm the direct
immunofluorescence (IF) testing. DNA was extracted using QIAamp DNA Mini Kit (Qiagen)
for PCR and stored at -20°C. Two loci were used for Giardia spp. genotyping: a fragment of
about 250 bp within the gene encoding for the triosephosphate isomerase (TPI) and a frag-
ment of about 130 bp within the gene encoding for the small ribosomal subunit (18S-rDNA).
A semi-nested PCR protocol was performed for amplification of the TPI gene. The
primary PCR used the forward primer AL3544 and the reverse primer AL3545, while the
second step used the reverse primer TPR1 (Sulaiman et al. 2003).
For amplification of 18S-rDNA, primary PCR was performed using the primers RH11
and RH4 (Hopkins et al. 1997). The Giar-F and Giar-R internal primers (Read et al. 2002)
were used for the nested PCR.

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Table 1 Species of shellfish

Species of shellfish Month of No. of No. of
farmed in the Varano Lagoon and
collection specimens pools
number of specimens and pools
subjected to immunofluorescence
test (IF) and PCR tests for the Crassostrea gigas April 07 13 1
detection of Giardia and May 07 23 2
Cryptosporidium July 07 11 1
August 07 18 1
October 07 20 1
November 07 23 1
January 08 17 1
Subtotal 125 8
Tapes decussatus May 07 50 2
July 07 30 1
August 07 25 1
October 07 24 1
Subtotal 129 5
Tapes philippinarum May 07 50 2
July 07 30 1
August 07 27 1
October 07 22 1
Subtotal 129 5
Mytilus galloprovincialis
April 07 60 2
May 07 60 2
June 07 60 2
July 07 60 2
August 07 60 2
September 07 60 2
October 07 60 2
November 07 60 2
December 07 60 2
January 08 60 2
February 08 60 2
Subtotal 600 20
Total 983 38

All DNA samples were also subjected to a semi-nested PCR to amplify a 400-bp
fragment of the N-terminal domain of the gene encoding for the Cryptosporidium spp.
outer protein wall (COWP). Two sets of degenerated primers were used: CRY15D and
CRY9D in the first step and CRYINT2D and CRY9D in the second step (Traversa et al.
2008).
All specific Giardia spp. and Cryptosporidium spp. PCR products were purified using a
Montage PCR Millipore kit and sequenced using the Dye Deoxy Terminator Cycle
Sequencing Kit (Applied Biosystems) on an ABI-PRISM 377 sequencer. Multiple align-
ment of the nucleotide sequences was performed using Clustal X and compared with
those available in GenBank for Giardia and Cryptosporidium species and genotype
identification.

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Water sampling and chemical–physical parameters monitoring

From April 2007 to February 2008, water samples were taken monthly and on the basis of
weather conditions, in seven geo-referenced stations (St1, St2, St3, St4, St5, St6 and St7)
in the Varano Lagoon and at the inlet into the lagoon of its five main freshwater sources:
San Nicola (Tr1), Bagno (Tr2), San Francesco (Tr3), Sant’Antonino (Tr4) and Idrovora
Muschiaturo (Tr5) (Fig. 1). The sample stations were chosen in order to achieve the best
sample grid describing the lagoon according to the marine, groundwater and discharge
canal influence and according to results of previous monitoring programs (Spagnoli et al.
2002; Specchiulli et al. 2008).
Temperature (°C), salinity (psu) and pH were measured in situ using a ISY QS600
multiprobe. These chemical–physical parameters were measured in surface water at all
sites and in the water column in the lagoon. Water samples were taken in triplicate at each
site and at a depth of 50 cm in the lagoon. The samples were preserved in refrigerated
dark bottles and then transferred to the Marine Science Institute laboratory for ammonia
(N–NH3) analysis. Water samples were filtered (Wathman GF/F) and ammonia nitrogen
(N–NH3) were assayed (three replicates) using an automated multiparameter autoanalyzer
(EasyChem Plus, Systea srl) according to Grasshoff et al. (1999).

Meteo-climatic parameters

Atmospheric temperature (°C), rainfall (mm), wind direction (N degrees), wind speed
(m s-1) and global solar radiation (W m-2) data were collected using an automatic
weather station (LSI Lastem, BABUC-ABC) on the Marine Science Institute building not
far from the Varano Lagoon. Meteo-climatic data were automatically registered and saved
at 5 min intervals and then downloaded to a computer using Info ABC 4.01 Software and
processed.

Water discharge of freshwater tributaries

The water discharge of the Varano Lagoon tributaries (Tr1, Tr2, Tr3, Tr4, Tr5) was
estimated during one month (December) using measurements of depth and current velocity
along the channel cross-section, according to the formula:
Q¼Sv
where Q was the discharge (m3 s-1), S was the cross-section area of the portion of the
channel occupied by the flow (m2), and v was the flow speed (m s-1) measured at a depth
(d) of 0.6  d (m) (where d value is from 0.1 to 1.1 m). Flow speed was measured with a
Van Wheel FA flow meter (Höntzsch Instruments, Germany).

Results

Giardia and Cryptosporidium (oo)cysts from shellfish

A total of 983 specimens of shellfish and 38 pools of shellfish haemolymph (5 of

R. decussatus, 5 of R. philippinarum, 8 of C. gigas and 20 of M. galloprovincialis) were
sampled and analysed. Both IF and molecular tests gave negative results for Giardia and
Cryptosporidium oo/cysts in all pools for the four shellfish species.

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(a) 35 St1 St2 St3 St4 St5 St6 St7

30

25

T (°C)
20

15

10

0
7

07

08

8
-0

-0

l-0

-0

-0

-0

-0

-0

-0
n-

n-
pr

ay

ug

ct

ov

ec

b
Ju
Ju

Se

Ja

Fe
O
A

D
M

N
(b) 34 St1 St2 St3 St4 St5 St6 St7
32

30
Salinity (psu)

28

26

24

22

20
7

07

07

08

08
-0

-0

l-0

-0

-0

-0

-0
n-

p-

n-

b-
pr

ay

ug

ct

ov

ec
Ju
Ju

Se

Ja

Fe
O
A

D
M

(c) 9.0 St1 St2 St3 St4 St5 St6 St7

8.8

8.6

8.4
pH

8.2

8.0

7.8

7.6
7

08
07

7
07

08
7

7
7

7
7

l-0

-0
-0

-0
-0

-0
-0

n-
n-

p-

b-
ct
pr

ec
ug

ov
ay

Ju

Ja
Ju

Se

Fe
O
A

D
M

Fig. 2 Physical–chemical parameters at seven stations (St1, St2, St3, St4, St5, St6 and St7) in the Varano
Lagoon

Chemical–physical parameters

Figure 2 shows variations in temperature, salinity and pH at the seven Varano Lagoon
monitoring stations (St1, St2, St3, St4, St5, St6 and St7).
Surface water temperature was lowest in winter (8.59°C in January) and highest (29°C)
in summer (Fig. 2a). The surface water salinity was lowest in winter (23.65 psu in January)
and highest (32.65 psu) in summer (Fig. 2b). Moreover, water salinity and temperature
were found to be strictly correlated (r = 0.699, p \ 0.001). The pH ranged from a min-
imum of 7.79 in January to a maximum of 8.80 in November (Fig. 2c).

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Fig. 3 Physical–chemical Salinity (psu)

parameters measured along the
(a)
22 24 26 28 30 32 34
water column in the central 0.0
station (St4) of the Varano -0.5 Apr-07
Lagoon May-07
-1.0
-1.5 Jul-07

Depth (m)
-2.0 Aug-07

-2.5 Oct-07
Nov-07
-3.0
Jan-08
-3.5
Feb-08
-4.0
-4.5

Temperature (°C)
(b)
7 12 17 22 27 32
0.0
-0.5 Apr-07
-1.0 May-07
-1.5 Jul-07
Depth (m)

-2.0 Aug-07
-2.5 Oct-07
-3.0 Nov-07
-3.5 Jan-08
-4.0 Feb-08
-4.5
-5.0

Water column analysis in peripheral monitoring stations (St1, St2, St3, St5, St6, St7)
showed no variations in temperature and salinity from the surface to the bottom (average
depth of 2.5 m). However, at the central station (St4) in the deepest area of the lagoon
(average depth 5 m), we observed stratification in the water column in the late winter-
early spring, corresponding to the highest gradients of salinity and temperature between
the surface and the bottom (Fig. 3). In particular, we found a DSalinity of 6.87 psu in
April (23.77 psu at the water surface and 30.62 psu at the bottom) (Fig. 3a) and a
DTemperature of 2.33°C in February (9.57°C at the water surface and 11.90°C at the
bottom) (Fig. 3b). The water column was well mixed in the warmer month’s of late
summer-early autumn.
Variations in salinity and pH measured in the main freshwater tributaries of the Varano
Lagoon (Tr1, Tr2, Tr3, Tr4, Tr5) are shown in Fig. 4. In Tr5 and Tr4 a great variability
were found in salinity, ranging from 8 psu in winter to 24–32 psu in autumn (Fig. 4a).
Water salinity in Tr1, Tr2 and Tr3 was quite constant and ranged from 2 to 5 psu
(Fig. 4a). The pH showed a peak value in Tr5 (8.33 in April) and in Tr3 (8.39 in July),
whereas the lowest pH (7.37) was found in Tr1 in February (Fig. 4b).
Ammonia (N–NH3) concentrations in the lagoon (Fig. 5a) ranged from 24.13 to
1.62 lM, with a mean value of 22 lM in summer–autumn and 5 lM in winter–spring.
In freshwater tributaries Tr4 and Tr3, ammonia concentrations ranged from 482.95 to
0.70 lM with the highest concentration (482.95 and 310.47 lM, respectively) in late
spring. Tr1 and Tr2 had constantly low concentrations of ammonia (\1 lM), while Tr5
showed intermediate concentrations ranging from a minimum of 5.84 lM in spring to a
maximum of 86.03 lM in winter.

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(a) 35
Tr5 Tr4 Tr3 Tr2 Tr1
30

25

Salinity (psu)
20

15

10

0
7

07

07

08

08
-0

-0

l-0

-0

-0

-0

-0
n-

p-

n-

b-
pr

ay

ug

ct

ov

ec
Ju
Ju

Se

Ja

Fe
O
A

D
M

N
(b)
8.5 Tr5 Tr4 Tr3 Tr2 Tr1

8.0
pH

7.5

7.0
7

07

07

08

08
-0

-0

l-0

-0

-0

-0

-0
n-

p-

n-

b-
pr

ay

ug

ct

ov

ec
Ju
Ju

Se

Ja

Fe
O
A

D
M

Fig. 4 Salinity (a) and pH (b) measured in the main freshwater tributaries (Tr1, Tr2, Tr3, Tr4, Tr5) of the
Varano Lagoon

Meteo-climatic parameters

In the study area, the average daily atmospheric temperature ranged from 0.3°C in winter
to 34.75°C in summer (Fig. 6a). Rainfall was more frequent in autumn and winter,
although there were some rainstorms in late spring and early summer. The daily rainfall
pattern is shown in Fig. 6a; the range was 0–16.8 mm, and total rainfall in the period
studied was about 241 mm.
Wind speed ranged from 0.31 to 11.23 m s-1 with a mean value of 3.5 m s-1. When we
analysed wind direction distribution, we observed that the prevailing directions were N
(12.97%), NNW (10.62%) and SSW (10.90%), and SW (8.28%) (Fig. 6b).
Global Solar Radiation was measured hourly, and mean distribution is shown in Fig. 6c.
The general pattern was of relatively low levels during winter (mean value at noon
411 W m-2) and high levels from spring throughout late summer, with a maximum global
solar radiation of 1,005 W m-2 (mean value at noon 896 W m-2), and intermediate levels
in the remaining periods.

Water discharge of freshwater tributaries

Water discharge of the main freshwater sources of the Varano Lagoon is shown in Table 2:
Tr1 and Tr2 discharged the greatest amounts of water (0.2 and 0.11 m3 s-1, respectively);
Tr5 discharged an intermediate amount (0.04 m3 s-1); and Tr3 and Tr4 discharged the
smallest amounts (0.0091 and 0.0083 m3 s-1, respectively).

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(a) 30
St1 St2 St3 St4 St5 St6 St7
25

N-NH3 (microM)
20

15

10

0
7

08

08
7

07

07
-0

l-0

-0

-0

-0

-0
-0

n-

b-
n-

p-
ay

ug

ct

ov

ec
pr

Ju

Ja

Fe
O
Ju

Se

D
M

N
A

(b) 600
Tr5 Tr4 Tr3 Tr2 Tr1

500
N-NH3 (microM)

400

300

200

100

0
7

08

08
7

07

07
-0

l-0

-0

-0

-0

-0
-0

n-

b-
n-

p-
ay

ug

ct

ov

ec
pr

Ju

Ja

Fe
O
Ju

Se

D
M

N
A

Fig. 5 Ammonia (N–NH3) concentrations: a at seven stations (St1, St2, St3, St4, St5, St6 and St7); and
b in the main freshwater tributaries (Tr1, Tr2, Tr3, Tr4, Tr5) of the Varano Lagoon

Discussion

Encysted protozoans (Giardia and Cryptosporidium) from human and animal faeces are
transported directly or by rainfall-initiated run-off from agricultural, suburban and urban
land, wastewater discharges and other sources into rivers, streams, estuaries and coastal
waters, thus contaminating the sea and its fauna. Accumulation of these pathogens by
marine, lake and/or lagoon shellfish—farmed for human consumption—is a potential risk
for human health, especially if they are eaten raw (Robertson 2007). Giardia and Cryp-
tosporidium (oo)cysts have been detected in molluscs in several areas, such as the UK
(Freire-Santos et al. 2000), Egypt (Negm 2003), the USA’s Atlantic coast (Fayer et al.
2003), Spain’s Galician coast (Gómez-Couso et al. 2006a, b), the Netherlands (Schets et al.
2007), France (Touron et al. 2007) and Ireland (Lucy et al. 2008). In Italy, Giardia and
Cryptosporidium (oo)cysts have been found in edible shellfish (Chamelea gallina) from the
Adriatic coast (Giangaspero et al. 2004, 2005) and in Manila clams (R. philippinarum)
from the Venice Lagoon (Molini et al. 2007).
The Varano Lagoon is one of the most important areas for shellfish farming in Italy,
and it has been previously observed that a high number of (oo)cysts of Giardia and
Cryptosporidium were discharged into the lagoon by sewage channels (Tr3 and Tr4)
(Giangaspero et al. 2009). However, interestingly, shellfish (R. decussatus and
M. galloprovincialis) farmed in the same environment were found by both IF and PCR

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(a) 18 Rainfall Atmospheric Temperature 40

16 35
14
30
Rainfall (mm) 12
25

T (°C)
10
20
8
15
6
4 10
2 5
0 0

08
07

7
07

08
7

7
7

7
7

l-0

-0
-0

-0
-0

-0
-0

n-
n-

p-

b-
ct
pr

ec
ug

ov
ay

Ju

Ja
Ju

Se

Fe
O
A

D
M

N
Date

N
(b) NNW 16 NNE % Prev
NW 12 NE

8
WNW ENE
4
W 0 E

WSW ESE

SW SE

SSW SSE
S

(c) 1200
Solar Radiation (W/sq.m)

1000

800

600

400

200

0
7

07

08

08
-0

-0

-0

l-0

-0

-0

-0

-0
p-

n-

b-
pr

ay

ug

ct

ov

ec
Ju
Ju

Se

Ja

Fe
O
A

D
M

Date

Fig. 6 Meteo-climatic parameters in the area of the Varano Lagoon: a rainfall and atmospheric
temperature; b wind prevalence; c global solar radiation

tests to be uncontaminated (Giangaspero et al. 2009). The present study confirmed these
results.
These findings further support the hypothesis that the presence of one or more envi-
ronmental factors may prevent contaminated water reaching the shellfish farm area and
contaminating molluscs (Giangaspero et al. 2009).

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Table 2 Water discharge

Tributaries Water discharge
of the main freshwater tributaries
(m3 s-1)
of the Varano Lagoon
Tr1 0.2
Tr2 0.11
Tr3 0.0091
Tr4 0.0083
Tr5 0.04

Temperature and salinity

Temperature and salinity are considered the most critical factors determining the fate of
Cryptosporidium and Giardia oocysts in the environment (DeRegnier et al. 1989; Johnson
et al. 1997; Lim et al. 1999; Olson et al. 1999; Nasser et al. 2003, Reinoso et al. 2008). In
fact, the ability of Cryptosporidium oocysts to initiate infection has been linked to finite
carbohydrate energy reserves in the form of amylopectin, consumed in direct response to
ambient temperatures (Fayer et al. 1998b).
Several authors have studied the effects of temperature on Cryptosporidium and Giardia
(oo)cyst viability and/or infectiousness in different aquatic environments, that is, deionized
water (Fayer et al. 1996a, 1998b; Olson et al. 1999; Jenkins et al. 2003; King et al. 2005;
McGuigan et al. 2006; Peng et al. 2008), river water (DeRegnier et al. 1989; Robertson
et al. 1992; Pokorny et al. 2002) and seawater.
Significant reductions in oocyst viability have been identified in seawater trials: salinity
of 20 psu and higher proved to have a significant effect on Cryptosporidium infection
ability (Fayer et al. 1998a). In particular, oocysts held at 20°C at salinities of 0 and 10 psu
survived for 12 weeks, at 20 psu for 4 weeks and at 30 psu for only 2 weeks. Freire-Santos
et al. (1999) registered an inactivation percentage of 81.5% in artificial seawater (35 psu)
at 18°C and after a storage time of 40 days. Robertson et al. (1992) found a mortality of
43% of oocysts immersed in laboratory-simulated seawater (37 psu) at 4°C for a storage
period of 35 day in the dark. Oocysts were also found to maintain infection ability for at
least 1 year in simulated seawater (33 psu) at 6–8°C, in the dark (Tamburrini and Pozio
1999). Johnson et al. (1997) reported a very short inactivation time (4 days) of C. parvum
oocycts stored in artificial seawater (28–35 psu) in the dark and at temperatures that ranged
from 22 to 26°C. They also found that sunlight under the same conditions of salinity and
temperature significantly reduced the inactivation time (from 4 to 2 days, respectively).
Interestingly, the same authors also found a very short inactivation time of Giardia cysts in
artificial seawater; an inactivation percentage of 90% was registered after 64.4 h at a
salinity of 28 psu, at temperatures ranging from 22 to 26, and in the dark. Increased salinity
(from 28 to 35 psu) was found to reduce the inactivation time (to 53.7 h). Moreover,
similar to findings for Cryptosporidium oocysts, they observed that sunlight significantly
reduced the inactivation time of Giardia cysts from 64.4 to 1.5 h.
Temperature and salinity in the Varano Lagoon are well correlated and show a seasonal
variation. Therefore, it is probable that at those temperatures and salinity levels (8–29°C
and 23–32 psu), (oo)cysts discharged into Varano Lagoon cannot survive longer than a few
weeks in winter and a few days in summer.

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Ammonia concentration

Free ammonia (NH3) is another influential factor in the inactivation of C. parvum oocysts.
It occurs naturally in the environment as a product of urea hydrolysis and of microbio-
logical degradation of proteins and other nitrogen-containing compounds (Jenkins et al.
1998). Important sources of ammonia include fertilizers, human and animal waste. Free
ammonia (NH3) and ionized ammonia (NH4?) represent two forms of reduced inorganic
nitrogen which exist in equilibrium depending upon the pH of the waters in which they are
found. Ammonium (NH4?) is the prevalent form at pH values below 7, while at pH values
over 9, most ammonium (NH4?) is converted to ammonia (NH3). NH3 is particularly
harmful to aquatic organisms (Arauzo and Valladolid 2003) and potentially toxic to
(oo)cysts. Fayer et al. (1996b) reported that oocysts were totally inactivated when exposed
to an atmosphere of pure ammonia at 21 and 23°C for 24 h. Jenkins et al. (1998) showed
that 24-h exposure to a concentration of 1,020 mg NH3 per litre inactivated 64.5% of
C. parvum oocysts. Reinoso et al. (2008) reported that C. parvum oocyst viability fol-
lowing 4 days’ exposure to 5 and 50 mg NH3 per litre was 41.5 and 14.8%, respectively.
Increasing the ammonia concentration and exposure time increased the inactivation of
C. parvum oocysts.
The maximum N–NH3 concentration in the Varano Lagoon was 24.13 lM (corre-
sponding to 410.21 lg NH3 per litre), which is very low compared to the inactivating
concentrations reported above. Thus, the impact of free ammonia on (oo)cyst viability in
the body of Varano Lagoon can be considered negligible. On the other hand, although
ammonia concentrations in sewage channels Tr3 and Tr4 sometimes reached values
(482.95 and 310.47 lM corresponding to 8.2 and 5.3 mg NH3, respectively) within the
minimum values able to inactivate pathogens (5–50 mg NH3 per litre), pH values (\9)
registered in all monitored stations (lagoon and sources) demonstrated that the real free
ammonia concentration was even lower than that measured in the laboratory (where we
measured the sum of N–NH3 and N–NH4?), because it was in equilibrium with its ionized
form (NH4?). Thus, in general, ammonia concentration cannot be considered an important
parameter to justify (oo)cyst inactivation.

Hydrodynamic pattern and water discharge

According to Brookes et al. (2004), another important factor to be considered for studying
the pathogens’ fate in a water body is the hydrodynamic pattern.
Pathogen transport is predominantly driven by inflows and basin-scale circulation
patterns including wind-driven currents and internal waves. Although wind-driven currents
only influence the surface layer, inflows can occur at any depth in a stratified reservoir, and
internal waves can generate significant internal currents that can act in different directions
at different depths (Deen et al. 2000). The Varano Lagoon’s hydrodynamic pattern is
completely wind-dominated, and the tidal variability and freshwater inflows only play a
minor role (Villani et al. 2000). Although we found that the prevailing winds in this area
come from N to NW and SSW to SW, the effect of southerly winds was very low because
the lagoon is sheltered by the Gargano Promontory close to its southern shore. Therefore,
the principal winds were only those from N to NW, which induced currents in the same
direction. This in turn made the south-eastern area of the lagoon, where (oo)cysts were
found to be discharged by Tr3 and Tr4 channels (Giangaspero et al. 2009), a very confined
area, according also to the results achieved by Spagnoli et al. (2002) and Specchiulli et al.
(2008). Salinity values found in Tr4 (Fig. 4a), which are constantly higher than at other

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sources, confirm the prevalence in that area of N-NW currents, causing an upstream input
of lagoon water into the mouth of Tr4. We did not observe the same phenomenon at Tr3
because the mouth of the channel was higher than the lagoon water level.
Therefore, pathogens brought and discharged by Tr3 and Tr4 into the lagoon remained
confined in that area and exposed to possible inactivating environmental factors for a long
time.
The speed at which an inflow carrying pathogens arrives into a water body and the
resulting dilution of its characteristics are all of critical importance in determining the
hydrodynamic distribution of pathogens in lakes and lagoons (Brookes et al. 2004).
Considerable water discharge can cause a decrease in salinity, in temperature and also in
residence time in the area where the inlet discharges into the water body. However,
reductions in all these parameters can prolong the survival of (oo)cysts and their infection
ability. This might explain why Giangaspero et al. (2005) found clams infected by
Cryptosporidium along the Adriatic coast (Abruzzo-Italy). In fact, the authors sampled
shellfish at 500 m from the mouths of four rivers (Tordino, Tronto, Vibrata and Vomano)
which discharged large volumes of water into the sea. In particular, the mean water
discharge of the Tordino, Tronto, Vibrata and Vomano rivers was 6, 17, 0.53 and
15 m3 s-1, respectively (ARTA Abruzzo 2005). These volumes of water discharge could
be enough to reduce salinity, temperature and the residence time of water in the sampling
area, prolonging the survival of pathogens and their infectivity.
In the Varano Lagoon, we found that the water volume discharged into the lagoon by
Tr3 and Tr4—reported in previous findings as carrying pathogens (Giangaspero et al.
2009)—was very low (0.0091 and 0.0083 m3 s-1, respectively) and was not enough to set
a gradient of salinity and/or temperature or to disperse (oo)cysts by horizontal transport.
Consequently, when pathogens arrive in the Varano Lagoon, it may be hypothesized that
they move rapidly from the chemical–physical conditions of the source into those of the
lagoon, which are not favourable for their long-term survival.
In this study, although we measured water discharge only once, this was done during the
rainiest period of the year; thus, we may assume that the values represent the maximum
water discharge for each tributary during the study period.

Global solar radiation

Solar radiation is a genotoxic agent because UV radiation is the most damaging and
mutagenic component of the electromagnetic spectrum, and its detrimental effects on
bacteria, fungi, plants and animals are known. Solar radiation is considered to be another
influential factor causing Giardia and Cryptosporidium (oo)cyst inactivation. Johnson et al.
(1997) investigated the effect of natural solar inactivation of Cryptosporidium and Giardia
in marine waters and identified a 90% reduction in viability (measured using excystation)
after an exposure period of 4/2 days and 1.5 h, respectively. Batch process solar disin-
fection (SODIS) under simulated solar conditions (830 W m-2) has identified even more
rapid reductions (4–10 h) in both (oo)cyst excystation and infection ability (McGuigan
et al. 2006). Recently, King et al. (2008) demonstrated that solar UV can dramatically
affect C. parvum oocyst infectivity in environmental waters of varying water quality and
may be a major driving factor behind oocyst inactivation in the environment.
UV radiation represents approximately 3–4% of incoming solar (\2,800 nm) radiation
(Kirk 1994; Ziegler and Benner 2000). Brookes et al. (2004) reported that for summer
conditions at mid-latitudes, a typical measurement for average hourly incoming shortwave
radiation around midday is 1,000 W m-2, which corresponds to approximately 30 W m-2

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of UV light. For a 1-h exposure, this corresponds to a cumulative UV light dose of

10.800 mJ cm-2. This is extremely high compared to water treatment dosages (Craik et al.
2001), which are typically in the range 20–120 mJ cm-2, and so it is highly likely that UV
radiation under environmental conditions in lakes and lagoons has the potential to inac-
tivate Giardia and Cryptosporidium, at least in summer.
In the Varano Lagoon, solar radiation levels higher than 830 W m-2 (radiation used for
SODIS process)—corresponding to approximately 25 W m-2 of UV light—were regis-
tered continuously for 6 months (from spring to the end of the late summer). This UV light
dose may have a synergic effect with other environmental factors (i.e. temperature) in
inactivating (oo)cysts in the lagoon and in the sewage channels. However, the vertical
distribution of UV light in a water body is closely related to depth and is attenuated by
dissolved organic carbon (DOC) (Kirk 1994; Morris et al. 1995; King et al. 2008). In this
study, we did not measure DOC concentration in the lagoon, but it should be noted that the
area where pathogens were discharged (Tr3 and Tr4 mouths) was very shallow (maximum
depth 1.5 m).
Besides the environmental conditions studied, it is known that in water bodies receiving
pathogens, including Giardia and Cryptosporidium, the exposure time to environmental
inactivating factors is strongly correlated with the residence time of water. A removal rate
of 55.8% of Giardia and 78.1% of Cryptosporidium oocysts has been found to occur in a
water body with a retention time of about 18 days (Bertolucci et al. 1998).
In the Varano Lagoon, the residence time of water was found to be 1,032 days
(Giordani et al. 2005). According to the literature, this period can be considered as long
enough to inactivate (oo)cysts by means of temperature and salinity. This could, therefore,
justify findings of infected shellfish in estuaries and/or lagoons with temperature and
salinity levels similar to those of the Varano Lagoon, but with a shorter residence time of
water. Some examples are the Venice Lagoon (10–14 days) (Giordani et al. 2005),
Chesapeake Bay (68–30 days) (Hagy et al. 2000), estuaries on the Galician coast of NW
Spain (3–8 days) (Dale et al. 2004) and the Seine estuary in France (27.5–14 days)
(Akopian et al. 2001).
In conclusion, this study demonstrated that environmental factors, that is, chemical–
physical and meteo-climatic parameters—as also shown by Ortega et al. (2009)—may
influence the absence of some protozoan parasites such as Giardia and Cryptosporidium.
In the Varano Lagoon, the main factors that can be considered as negatively influencing
the survival of these protozoans are temperature and salinity, which are strictly correlated.
But the low volumes of freshwater discharged into the lagoon, the long-lasting solar
irradiation and the shallowness of the lagoon may also have a synergic effect in reducing
the life of the investigated pathogens in this body of water.
The environmental ecology of the Varano Lagoon is very peculiar and cannot change
much from year to year; conditions can be modified only if a huge amount of water is
discharged into the lagoon, causing salinity to fall and thus favouring the diffusion and
survival of pathogens. In view of this, public health institutions must monitor the sanitary
conditions of the lagoon via strict screening of the amount of water flowing into the lagoon,
particularly wastewater (i.e. urban and/or livestock run-off)—which may be also illegally
discharged—and by surveying harvested shellfish, whose role as bioindicators of water
contamination is widely recognized (Graczyk et al. 2003; Molini et al. 2007).

Acknowledgments This work was supported by Research Grant: Progetto Esplorativo PE_100 (CIP
PE_087) (2006–2008) from Regione Puglia, Italy. Thanks also to Sarah Jane Christopher for her patient
English revision of the manuscript.

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 Aquacult Int

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