Antonio Lonigro

Giardia cysts and Cryptosporidium oocysts in membrane-filtered municipal wastewater used for irrigation

ABSTRACT A wastewater tertiary treatment system based on membrane ultrafiltration and fed with secondary-treated municipal wastewater was evaluated for its Giardia cyst and Cryptosporidium oocyst removal efficiency. Giardia duodenalis (assemblages A and B) and Cryptosporidium parvum were identified in feed water but were found in filtered water only during occasional failure of the filtration system.

Giardia and Cryptosporidium in inflowing water and harvested shellfish in a lagoon in Southern Italy.

Giardia and Cryptosporidium spp. are important enteric protozoan pathogens for humans and animals, and have been found to contaminate water as well as edible shellfish all over the world. This is the first study to simultaneously investigate the presence of Giardia and Cryptosporidium in inflowing water and harvested shellfish in a geographically closed environment (Varano Lagoon, Southern Italy). Samples of treated wastewater were collected each month - at the outlet from the treatment plant, and downstream at the inlet into the lagoon - from the channels flowing into the Lagoon, together with specimens of Ruditapes decussatus and Mytilus galloprovincialis from shellfish-farms on the same lagoon. Giardia cysts were found by immunofluorescence (IF) microscopy in 16 out of 21 samples of treated wastewater and in 7 out of 21 samples from downstream water channels, and viable cysts were also detected by a beta-giardin RT-PCR. G. duodenalis Assemblages A and B were identified by small ribosomal subunit (18S-rDNA) and triosephosphate isomerase (tpi)-PCR, followed by sequencing. Cryptosporidium oocysts were found by IF in 5 out of 21 wastewater samples, and in 8 out of 21 samples from water channels. Molecular analysis identified the zoonotic species Cryptosporidium parvum by oocyst wall protein (COWP)-PCR and sequencing. Higher concentrations of Giardia cysts than Cryptosporidium oocysts were registered in almost all wastewater and water samples. IF and molecular testing of shellfish gave negative results for both protozoa. Wastewaters carrying Giardia and Cryptosporidium (oo)cysts are discharged into the Lagoon; however, the shellfish harvested in the same environment were found to be unaffected, thus suggesting that physical, ecological and climatic conditions may prevent contamination of harvested shellfish.

Membrane filtration of municipal wastewater effluents for implementing agricultural reuse in southern Italy.

Membrane filtration was investigated at field scale in order to assess its effectiveness for reusing municipal effluents in agriculture. The study was started on April 2002 and ended on September 2007, as part of a national R&D project (AQUATEC). Preliminary results, which we already reported elsewhere, concerned the first two project years while this paper refers to the subsequent period. Three different crops (processing tomato, fennel and lettuce) were grown in rotation at a test field located in Apulia (Southern Italy) and irrigated with membrane filtered municipal secondary effluents. The quality of the reclaimed water was monitored chemically and microbiologically, and compared with conventional water pumped from a local well. Both water sources were used in parallel for irrigating two plots of the test field. The results showed that the microbiological quality of the treated wastewater was comparable to or even higher than that of the conventional source. Protozoan (oo)cysts were experimentally identified as effective indicators of possible failures of the filtration system. Moreover, long term heavy metals accumulation trends were monitored in soil and crops, showing that despite some lead and copper accumulation in the soil, no measurable increase of these metals was observed in the edible parts of the crops.

Molecular detection of Cyclospora in water, soil, vegetables and humans in southern Italy signals a need for improved monitoring by health authorities.

To date, in Europe, there is scant information on the occurrence of Cyclospora in water from treatment plants and in humans, and no data are available on soil or fresh plant products. Here, we undertook the first molecular survey of Cyclospora in multiple biological matrices collected from the Apulia region of southern Italy. Samples of irrigation water from four municipal treatment plants, eight different types of vegetables or fruit (cucumber, lettuce, fennel, celery, tomato, melon, endive and chicory) and soil from the same farms on which these plants were grown, as well as faecal samples from humans living in the same region were tested by qPCR-coupled single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. Cyclospora was detected in 15.5% of all 213 samples tested. Specifically, this protist was detected in (i) treated water (21.3% of 94 samples), well water (6.2% of 16), but not drinking water (0% of 3); (ii) soil (11.8% of 51 samples) and vegetables (12.2% of 49), with the highest prevalence (18.7%) on fennel; and (iii) human stools (27.5% of 40 samples). In environmental and food samples, Cyclospora was detected mainly in autumn and was significantly more prevalent in the faeces from humans of 40-50 years of age. This is the first comprehensive molecular survey of Cyclospora in environmental, food and human faecal samples in Europe. These data suggest that irrigation water, soil and vegetables might be contaminated by Cyclospora cayetanensis, which might represent a source of infection to humans in the study area and calls for monitoring by health authorities.

Multiplex PCR for the detection and quantification of zoonotic taxa of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels

Giardia duodenalis, Cryptosporidium parvum and Toxoplasma gondii are important parasitic protists linked to water- and food-borne diseases. The accurate detection of these pathogens is central to the diagnosis, tracking, monitoring and surveillance of these protists in humans, animals and the environment. In this study, we established a multiplex real-time PCR (qPCR), coupled to high resolution melting (HRM) analysis, for the specific detection and quantification of each G. duodenalis (assemblage A), C. parvum and T. gondii (Type I). Once optimised, this assay was applied to the testing of samples (n = 232) of treated wastewater and mussels (Mytilus galloprovincialis). Of 119 water samples, 28.6% were test-positive for G. duodenalis, C. parvum and/or both pathogens; of 113 mussel samples, 66.6% were test-positive for G. duodenalis, C. parvum and/or both pathogens, and 13.2% were test-positive for only T. gondii. The specificity of all amplicons produced was verified by direct sequencing. The oo/cysts numbers (per 5 μl of DNA sample) ranged from 10 to 64. The present multiplex assay achieved an efficiency of 100% and a R(2) value of >0.99. Current evidence indicates that this assay provides a promising tool for the simultaneous detection and quantitation of three key protist taxa.

Nutrient recovery and crop yield enhancement in irrigation with reclaimed wastewater: a case study

Abstract The paper reports results of treated wastewater reuse field experiments carried out in Apulia (Southern Italy). Fennel and lettuce were irrigated with four different water sources: three reclaimed wastewater streams, obtained by applying different treatment schemes to the same municipal wastewater, and a conventional source (well water). Differences between the three effluents were significant in terms of suspended solids and faecal indicators. Both lettuce and fennel yields were enhanced by the high content of nutrients in the effluent of one of the treatment plants, which had been operated for partial nitrogen removal. In particular, fennel productivity was enhanced by replacing chemical fertirrigation with the supply of nutrients contained in the irrigation water.

Vegetable crop irrigation with tertiary filtered municipal wastewater

Abstract The use of tertiary membrane-filtered municipal wastewater for irrigation as an alternative to natural freshwater sources was evaluated. Membrane filtration was considered as a viable technology to reclaim wastewater for irrigation, and the microbial and heavy metal impact on crops and soil was studied. The results of 2 years of research (2003 – 2004), carried out in Cerignola in the South of Italy, are reported. Tertiary treatment was carried out using a membrane filtration pilot plant with a hollow fibre submerged system. The water produced was used for drip irrigation of three vegetable crops in succession – processing tomato, fennel and lettuce – and compared with conventional water. Microbiological analyses were performed on the water used for irrigation, on soil samples and on marketable crops. Results show that the microbial content of soil and crops did not show relevant differences in relation to the two types of water. The measured values of heavy metals concentration in crops never exceeded toxic values. The filtered wastewater never caused an increase of bacterial concentration in the soil nor on the edible part of crops. Therefore, tertiary filtered municipal wastewater can be considered a valid alternative source of water for vegetable crop irrigation.

Development and validation of an analytical method based on liquid chromatography–tandem mass spectrometry detection for the simultaneous determination of 13 relevant wastewater-derived contaminants in lettuce

AbstractWe present an analytical method developed and validated to study the potential uptake of 13 selected drugs (ten pharmaceuticals, one illicit drug, and two transformation products) into lettuce plants from contaminated water and soil. Some of the selected drugs (i.e., cocaine, methadone, cis-diltiazem, valsartan, and valsartan acid), which are commonly present in treated wastewater, were investigated for the first time in plant tissues. The method is based on ultrasonic solvent extraction with acetonitrile–methanol (1:1, v/v) and subsequent automated extract cleanup and analysis by means of online solid-phase extraction–liquid chromatography–tandem mass spectrometry. Optimum extraction conditions were selected after evaluation of analyte recoveries with four different extraction techniques (ultrasonic solvent extraction, solid–liquid extraction, pressurized liquid extraction, and a “quick, easy, cheap, effective, rugged, and safe” based method) and six different solvent mixtures. Furthermore, two different solid-phase extraction cleanup sorbents were evaluated. The method developed has high sensitivity (with limits of detection between 0.1 and 12.6 ng per gram dry weight and limits of quantification between 0.5 and 42.0 ng per gram dry weight), satisfactory accuracy (with analyte relative recoveries above 80% for all analytes but acridone and oxcarbazepine), and good repeatability (with relative standard deviations below 9% for all analytes). As part of the validation procedure, the analytical method was applied to the analysis of lettuce plants irrigated with water fortified with the selected compounds for the entire growing period. The results obtained evidenced the transfer of all the investigated drugs into lettuce leaves. Graphical Abstractᅟ

Effects of residual disinfectant on soil and lettuce crop irrigated with chlorinated water

The accidental or continuous release of residual chlorine in water reclaimed for irrigational purposes could compromise the crop yield and increase the load of toxic organo-halogenated compounds, posing additional risks for environment and human health. This study was aimed at assessing the consequences of using chlorinated water for irrigating lettuce crops grown in pots with two different types of soil. The results show that the accumulation of extractable organo-halogenated compounds (EOX) in soil, roots and leaves is directly related to the chlorine concentration in the irrigation water. The accumulation of EOX in sandy soils is not significant, while it reached up to 300% of the control in the silty-clay soil, demonstrating that the phenomenon is linked to the organic matter content in the soil. The accumulation of EOX in the soil appears to play a significant role in subsequent bioaccumulation in cultures irrigated with tap water (long term memory effect). Chloramines also demonstrated to have similar impacts as the free chlorine from hypochlorite. The consistent bioaccumulation of 400-700μgClkg-1 of EOX in the leaves of crops irrigated with just 0.2mgClL-1 of residual chlorine, as compared to levels below the detection limit of 75μgClkg-1 in the control crops, evidences the potential impact on food chain and human health.