Biol Trace Elem Res (2010) 134:342–351

DOI 10.1007/s12011-009-8477-8

Analysis of Bacteria, Parasites, and Heavy Metals

in Lettuce (Lactuca sativa) and Rocket Salad
(Eruca sativa L.) Irrigated with Treated Effluent
from a Biological Wastewater Treatment Plant

Meire Nikaido & Karina A. A. Tonani & Fabiana C. Julião & Tânia M. B. Trevilato &
Angela M. M. Takayanagui & Sérgio M. Sanches & José L. Domingo &
Susana I. Segura-Muñoz

Received: 30 June 2009 / Accepted: 17 July 2009 /

Published online: 29 July 2009
# Humana Press Inc. 2009

Abstract This study aimed to evaluate the viability of using treated residuary water from
the Biological Wastewater Treatment Plant of Ribeirão Preto to grow vegetables, through
the characterization and quantification of parasites, coliforms, and heavy metals. Three
equal cultivation areas were prepared. The first was irrigated with treated/chlorinated
(0.2 mg L−1) wastewater, the second one with treated wastewater without chlorination, and
the third site with potable water, which was the control group. The presence of
Hymenolepis nana, Enterobius vermicularis, nematode larvae, and Entamoeba coli was
verified in lettuce (Lactuca sativa) samples. Although nematode larvae were observed in
rocket salad (Eruca sativa L.), no significant differences were found between the number of
parasites and type of irrigation water used. No significant differences were found between
the number of fecal coliforms in vegetables and the different types of irrigation. However,
the vegetables irrigated with treated effluent without chlorination showed higher levels of
fecal coliforms. The risk of pathogens is reduced with bleach addition to the treated effluent

M. Nikaido : K. A. A. Tonani : F. C. Julião : S. I. Segura-Muñoz (*)

Laboratory of Ecotoxicology and Environmental Parasitology,
Maternal Infant Nursing and Public Health Department, University of São Paulo, College of Nursing,
WHO Collaborating Centre for Nursing Research Development, Avenida Bandeirantes,
3900, Campus Universitário, Ribeirão Preto, São Paulo, Brazil
e-mail: [email protected]

T. M. B. Trevilato
Laboratory of Pediatrics, Metals Division, Hospital das Clínicas, Medical School,
University of São Paulo, Ribeirão Preto, São Paulo, Brazil

A. M. M. Takayanagui : S. M. Sanches
Laboratory of Environmental Health, Maternal Infant Nursing and Public Health Department,
University of São Paulo, College of Nursing, WHO Collaborating Centre for Nursing Research
Development, Ribeirão Preto, São Paulo, Brazil

J. L. Domingo
Laboratory of Toxicology and Environmental Health, School of Medicine, IISPV,
“Rovira i Virgili” University, Reus, Catalonia, Spain
Parasite Analysis in Vegetables Irrigated with Treated Wastewater 343

at 0.2 mg/L. Concentration of heavy metals in vegetables does not mean significant risks to
human health, according with the parameters recommended by the World Health
Organization.

Keywords Wastewater reuse . Vegetables . Coliforms . Enteroparasites . Heavy metals

Introduction

In agriculture, the planned use of reclaimed wastewater is an alternative to control pollution

of water bodies, provision of water for crops, nutrient cycling, and increased agricultural
production [1]. Risks to public health arising from the use of wastewater are related to the
presence of chemicals, organic and inorganic toxic substances, and potentially pathogenic
microorganisms, which above certain concentrations may be harmful to humans [1–3].
Human infection by parasites occurs mainly by fecal-oral route. Parasites can be
transmitted by ingestion of untreated or inadequately treated water and contaminated food
(raw vegetables and poorly washed fruits), which in turn can be also contaminated by cysts
carried by insects, from person to person, through contaminated hands, or places of human
settlement such as kindergartens, orphanages, and others. Samples of lettuce have been
used to detect intestinal parasites [4, 5], as these can cause severe health damage, meaning a
serious problem still persisting in developing countries.
Food is the main source of human contamination by heavy metals. Such contamination
can occur during the use of contaminated water in irrigation or from polluted soils. Metals
such as lead, mercury, and cadmium are highly toxic not only for plants but also for humans
and animals. They are pollutants of water, soils, and culture, causing diseases including
cancer and degeneration of multiple tissues, being even lethal to consumers of highly
contaminated crops [6].
Taking the above into account, in this study, we evaluated the viability of using treated
residuary water from the Biological Wastewater Treatment Plant of Ribeirão Preto (RP-
BWTP), Brazil, to grow lettuce (Lactuca sativa) and rocket salad (Eruca sativa L.), through
the characterization and quantification of parasites, coliforms, and heavy metals.

Materials and Methods

Preparation of Nursery Seedlings

Three nursery seedlings of lettuce (L. sativa) and rocket salad (E. sativa L.), 2×3 m each
(about 120 plants were planted in each nursery), were demarcated and replicated at the RP-
BWTP. Soil was previously prepared with organic fertilizer. The first of the three sites was
irrigated with treated effluent chlorinated at 0.2 mg L−1, the second one with treated effluent
without addition of chlorine, and the third site (control) was irrigated with drinking water
(Fig. 1).

Parasitological and Bacteriological Analysis

Irrigation water was collected in sterile containers of 1 L and kept under refrigeration at 4°C
until the time of analysis, which did not exceed 24 h. Vegetables were collected in sterile
bags, individually defoliated and carefully washed, leaf-by-leaf, with 700 mL of distilled
344 Nikaido et al.

Fig. 1 Beds of vegetables (from

left to right, the first site irrigated
with treated/chlorinated effluent
to 0.2 mg L−1, the second site
with treated effluent without the
addition of chlorine, and the third
one with drinking water)

water [7]. Part of this water was used for the P/A test (Colilert®), while the remaining was
filtered in a drum sieve with mesh gauze, being then transferred to a conical bottom cup to
settle for 1 h before parasitological analysis.
The spontaneous sedimentation technique was used for the analysis of parasites. Reading
was performed with the Sedgwick-Rafter counting cell (Pyse-SGI Limited, Kent, UK). For
bacteriological analysis, the P/A Colilert® test was used, being the reading performed by
the multiple-tube technique.

Metal Analysis

Samples were collected in polyethylene containers previously cleaned with nitric acid
(Suprapur, E. Merck, Darmstadt, Germany) solution (30% v/v) for removal of interferences.
For the analysis of soil samples, the “Aqua Regia Metal Extraction Method” (HNO3:HCl,
3:1) was used in Teflon pumps. Approximately 1 g of each sample was weighed, and
10 mL of “Aqua Regia” were added [8]. For analysis of vegetables, about 2 g of vegetable
in nature were weighed and 10 mL of nitric acid (Suprapur) added. The Teflon pumps were
resting for 8 h at room temperature. Afterwards, they were put into an oven at 80°C for
eight additional hours. Solutions were cooled, filtered with a 12.5-cm paper filter, and
diluted to 25 mL with deionized water [8].
Water for irrigation was fixed adding high purity HNO3 and kept at −18°C until time of
analysis. Samples were centrifuged, and the supernatant separated for further reading for
heavy metals analysis according to standard methods for the examination of water and
wastewater. The concentrations of cadmium (Cd), copper (Cu), chromium (Cr), lead (Pb),
and zinc (Zn) were determined by inductively coupled plasma spectroscopy (Perkin Elmer,
Optima 3000 DV) at the Chemical Analysis Center of the São Carlos Institute of Chemistry,
University of São Paulo, Brazil. Mercury (Hg) levels were determined by atomic absorption
spectrometry with hydride generation in a Varian spectrophotometer model AA-200 at the
Laboratory of Pediatrics, Metals Division at Clinics Hospital of the University of São Paulo
at Ribeirão Preto Medical School.

Statistics

The Kruskal–Wallis non-parametric statistical test was performed through the Graph Pad
Prism Statistical Program (Version 3.02 for Windows, Graph Pad Software, San Diego, CA,
Parasite Analysis in Vegetables Irrigated with Treated Wastewater 345

USA). Multiple comparisons test was performed to check for differences between the types
of irrigation. The significance level was set at p<0.05.

Results and Discussion

Soil

The current mean concentrations of Cd, Cu, Cr, Pb, and Zn in soil were within the
maximum established by the Report of Established Guiding Values for Soil and
Groundwater in the State of São Paulo [9].

Effluents and Water Used for Irrigation

The results revealed the presence of parasites in the treated/chlorinated effluent. The
chlorination of treated effluent decreased the number of parasites. However, no significant
differences in the number of parasites were observed when treated/chlorinated effluent was
compared to the treated effluent without chlorination. No parasitic structures were found in
samples of drinking water (Table 1).
Various recent studies have shown a high incidence of parasitic structures in wastewater
[10–12]. For example, an average concentration of 27.4 helminth eggs per liter was recently
found in Cambodia [13]. In a study also carried out in Ribeirão Preto, Brazil, during 2006
and 2007, wastewater with average concentration of 27 Ascaris lumbricoides eggs per liter,

Table 1 Parasitic Frequency in

the Water Used for Irrigation of Parasites Mean ± SD
Lettuce and Rocket Salad
(Number of Eggs/Liter) Treated/chlorinated effluent
Larvae of nematode 9.9±8.1
Entamoeba coli 0.1±0.3
Enterobius vermicularis 0.1±0.3
Hymenolepis nana 5.5±4.1
Ascaris lumbricoides 0.2±0.4
Schistosoma mansoni 0.1±0.3
Treated effluent
Larvae of nematode 11.1±9.5
Entamoeba coli 0.4±0.8
Enterobius vermicularis 0.4±0.6
Hymenolepis nana 8.1±10.7
Ascaris lumbricoides 1±1.1
Schistosoma mansoni 0±0
Drinking water
Larvae of nematode 0±0
Entamoeba coli 0±0
Enterobius vermicularis 0±0
Hymenolepis nana 0±0
Ascaris lumbricoides 0±0
Schistosoma mansoni 0±0
346 Nikaido et al.

162 larvae of nematode per liter, and ten Giardia lamblia cists per liter was found [12].
Parasitological structure concentrations in the effluents of RP-BWTP decreased compared
with the concentrations obtained in 2006/2007 (Table 1). These results highlight the need
for judicious measures on the use of the effluent for irrigation, due to the presence of
pathogenic microorganisms that can endanger human health.
The results of fecal coliforms in the treated/chlorinated effluent and drinking water were,
in general terms, within the maximum limit allowed by Centro de Vigilância Sanitária Rule
No. 21/1991 of Center of Health Surveillance of São Paulo State [14] (Fig. 2). It is
established that irrigation water should not contain concentrations of fecal coliforms higher
than 1,000 per 100 mL of sample. Concentrations of fecal coliforms between 1.5×103 and
1.8×104 most probable number (MPN)/100 mL were found in Viçosa, MG, Brazil [11]. In
Paraíba, also in Brazil, Barros et al. [15] observed that the water used for irrigation of
vegetables in five gardens presented concentrations of fecal coliforms well above the
maximum allowed. Those results exceed the values found in the treated/chlorinated effluent
used for irrigation of lettuce and rocket salad, at the present study. Therefore, the treated/
chlorinated effluent shows better conditions of use for irrigation of crops than many other
watercourses in the country, which receive frequently illegal discharges of domestic and
industrial sewage.
The average pH obtained in the analysis of the treated/chlorinated effluent and treated
effluent was 6.9, which is within the maximum allowed by Resolution Conselho Nacional
de Meio Ambiente 357/2005 [16]. The pH value should be considered regarding the reuse
of wastewater in agriculture. It must be taken into account that when the water used for
irrigation presents very acidic or basic pH, serious problems of nutrition and toxicity to
plants, as well as problems of corrosion and incrustation in irrigation systems, can occur
[17]. Therefore, the pH of irrigation water and wastewater did not affect the nutrition and
development of lettuce and rocket salad. Chlorination of treated effluent was also effective
to reduce biochemical oxygen demand from 7.47 to 4.57 mg L−1. The recorded temperature
of the treated/chlorinated effluent, treated effluent, and drinking water was within levels
expected for tropical countries like Brazil [17].
The concentrations of Cd, Cu, Hg, Pb, and Zn were analyzed in the treated/chlorinated
effluent, treated effluent, and drinking water. According to the US Environmental
Protection Agency [18], all metals, except Hg for which no maximum level has been
specified, were within the maximum values allowed. With the exception of Pb, metal
concentrations found in the three types of water used to irrigate the vegetables were within
the parameters established by Canada, a country with rigorous environmental controls.

Fig. 2 Concentration of fecal 1.0×10

08

coliforms in water used for

Fecal coliform per 100 mL

irrigation of vegetables
06
1.0×10

1.0×1004

02
1.0×10

00
1.0×10
water used for irrigation
treated effluent drinking water
treated/chlorinated effluent
Parasite Analysis in Vegetables Irrigated with Treated Wastewater 347

Canada sets maximum levels of Cd, Cu, Pb, and Zn of 0.01, 0.2, 0.01, and 2 mg/L,
respectively [19].

Vegetables

The presence of Hymenolepis nana, Enterobius vermicularis, nematode larvae, and

Entamoeba coli was verified in lettuce (L. sativa) samples. Although nematode larvae
were observed in rocket salad (E. sativa L.), no significant differences were found between
the number of parasites and type of irrigation water used. Therefore, the number of parasitic
structures in the vegetables irrigated with treated/chlorinated effluent did not differ from the
results corresponding to vegetables irrigated with fresh water (Table 2). It can be assumed
that contamination of vegetables might have occurred through the soil, wind, and insects in
the environment, as the drinking water used for irrigation of crops did not present any
parasitic structure.
Takayanagui et al. [4] found contamination by intestinal parasites in Ribeirão Preto in
33% of lettuce samples acquired in local markets. Lettuce and rocket salad, highly
contaminated with larvae of nematode, E. coli, E. vermicularis, H. nana, and other

Table 2 Parasitic Frequency in

Lettuce and Rocket Salad (Num- Parasites Vegetables Mean ± SD
ber of Eggs/Liter)
Treated/chlorinated effluent
Larvae of nematode Lettuce 2.5±2.5
Rocket salad 1.7±2.6
Entamoeba coli Lettuce 0.1±0.3
Rocket salad 0.1±0.2
Enterobius vermicularis Lettuce 0.1±0.2
Rocket salad 0±0
Hymenolepis nana Lettuce 0±0
Rocket salad 0.1±0.2
Treated effluent
Larvae of nematode Lettuce 2.3±1.7
Rocket salad 1.3±1.2
Entamoeba coli Lettuce 0.1±0.2
Rocket salad 0.1±0.2
Enterobius vermicularis Lettuce 0±0
Rocket salad 0±0
Hymenolepis nana Lettuce 0.1±0.2
Rocket salad 0.1±0.3
Drinking water
Larvae of nematode Lettuce 5.1±6.3
Rocket salad 2±2.6
Entamoeba coli Lettuce 0.1±0.5
Rocket salad 0±0
Enterobius vermicularis Lettuce 0±0
Rocket salad 0±0
Hymenolepis nana Lettuce 0±0
Rocket salad 0±0
348 Nikaido et al.

parasites, were found in the retail market of Florianópolis, SC, Brazil. In a study performed
in Florianópolis, Brazil, the percentage of contamination was 60% for lettuce and 56% for
rocket salad. Many cultivars sometimes use water from poor sources and, without
appropriate surveillance of marketed products, expose consumers to diseases transmitted by
contaminated water and food [20].
In the current study, no significant differences were found between the number of fecal
coliforms in vegetables and the different types of irrigation. However, the vegetables
irrigated with treated effluent without chlorination showed higher values of fecal coliforms
(Fig. 3). The risk of pathogens is reduced with bleach addition to the treated effluent at a
concentration of 0.2 mg/L.
In a recent study performed in Ghana, levels of fecal coliforms higher than 1.5×105
MPN/100 mL were found in lettuce samples irrigated with untreated effluent [21]. In turn, a
high level of contamination by fecal coliforms was found in lettuce sold by greengrocers,
markets, and supermarkets, 3.8×105, 8.6×105, and 3.2×105 MPN/100 mL, respectively, in
Lavras, Brazil [5]. These values exceeded the current levels found in lettuce irrigated with

Fig. 3 Concentration of fecal 05

1.0×10
coliforms in lettuce and rocket
salad samples
Fecal coliform per 100 mL

04
1.0×10

03
1.0×10

02
1.0×10
Lettuce
treated effluent drinking water
treated/chlorinated effluent

05
1.0×10
Fecal coliform per 100 mL

04
1.0×10

03
1.0×10

02
1.0×10

01
1.0×10
Rocketsalad
treated effluent drinking water
treated/chlorinated effluent
Parasite Analysis in Vegetables Irrigated with Treated Wastewater 349

treated/chlorinated effluent, whose maximum value obtained was <2×103 MPN/100 mL.
The presence of E. coli in irrigation water and vegetables might indicate the potential
presence of pathogens [22]. Studies have suggested that sewage treatment, coupled with the
chlorination of the effluent, decrease the concentration of fecal coliforms in water,
minimizing risks associated with the potential presence of other microorganisms [11].
Monitoring of crops should not be based only on the type of irrigation used. Other
factors that might lead to contamination from the plantation to the consumer’s table, as
shown in several studies [4, 23], should be also taken into account.
The concentrations of Cd, Cu, Hg, Pb, and Zn (Table 3) found in lettuce irrigated with
treated/chlorinated effluent have been compared with the results reported for Bolivia: 0.091,
1.36, 0.110, 0.810, and 10.4 mg kg−1 for Cd, Cu, Hg, Pb, and Zn, respectively [19]. All these
metals, except Cd, presented levels below those observed in the lettuce grown in Bolivia.
The World Health Organization [24] established guidelines for the daily intake of Cu and
Zn of 2–4 and 15 mg/day, respectively, and limits the intake of Cd and Pb to 0.07 and
0.43 mg/day, respectively. For the vegetables analyzed in the present study, the daily
consumption of lettuce and rocket salad should not increase the intake of these metals until
harmful concentrations.

Conclusions

Scarcity of water, not only absence of water but mainly leak of good quality water, is a
problem in a number of zones around the world, requiring an immediate action and
intervention in the preservation and conscious use of available water resources. During
extreme conditions of scarcity of water, the reuse of non-drinking water in agriculture may
be necessary.
The results of the present study show that wastewater chlorination at 0.2 mg L−1 after
biological treatment at the RP-BWTP, allowed the use of effluent for irrigation of raw-eaten
vegetables, like lettuce (L. sativa) and rocket salad (E. sativa L.), getting vegetables with
concentration of parasites structures and coliform bacteria and heavy metal concentrations,
whose consumption should not pose significant additional risks to human health. Reuse of
water requires ongoing monitoring of both the effluent used and the vegetable in question.
Supervision of these cultivars might facilitate a potential use of effluent in the future and
minimize risks of contamination by parasites, coliforms, and heavy metals.

Table 3 Concentrations of Heavy Metals in Vegetables (Milligrams/Kilogram)

Cd Cu Hg Pb Zn

Irrigated with drinking water

Mean ± SD Lettuce 0.030±0.500 0.740±0.700 0.000±0.000 0.150±0.230 1.740±0.490
Rocket salad 0.000±0.000 1,466±1.610 0.001±0.005 0.026±0.044 1.501±0.289
Irrigated with treated/chlorinated effluent
Mean ± SD Lettuce 0.110±0.190 1,550±1.470 0.010±0.020 0.030±0.040 2.350±3.840
Rocket salad 0.001±0.003 0.175±0.164 0.002±0.004 0.124±0.154 1.440±0.285
Irrigated with treated effluent
Mean ± SD Lettuce 0.050±0.060 0.290±0.190 0.000±0.000 0.170±0.160 1.470±0.420
Rocket salad 0.000±0.000 0.838±0.836 0.005±0.009 0.071±0.124 1.911±1.960
350 Nikaido et al.

The consumption of raw vegetables irrigated with treated and chlorinated wastewater is
recommended only if systematic programs are adopted to assure the sanitary qualification
of the crops, including future risk assessments of enteric viruses and others pathogenic
bacteria such as Salmonella and Shigella.

Acknowledgments This study was funded by the State of São Paulo Research Foundation (FAPESP)
process no. 06/59920-8 and no. 06/55788-8 and obtained support and approval from the AMBIENT,
Environmental Services of Ribeirão Preto SA. The authors thank Carlos Roberto Ferreira, Karina Carregari,
Félix A. de Moura, Aparecido M. Gouveia, and Edivaldo R. Oliveira from RP-BWTP, for the assistance
during nursery seedlings preparation and crops plantation.

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