Occurrence of parasites in waters used for crops irrigation and vegetables from the Savannah of Bogotá, Colombia

The World Health Organization (WHO) has established as a criterion of parasitological quality for irrigation water, ≤ 1 helminth egg/liter, which guarantees the safety in agricultural products. In this study, the presence of parasites in surface water used for irrigation of crops (n = 96) and vegetables (celery and lettuce) (n = 120), from the Former La Ramada irrigation district, was evaluated using conventional and molecular parasitological methods. Our findings showed contamination of irrigation systems in the study area with domestic wastewater, demonstrated by the presence of Ancylostomatidae eggs, Ascaris spp., Hymenolepis spp., Trichuris spp., Capillaria spp., Giardia spp. cysts, and oocysts of Toxoplasma gondii and Cryptosporidium spp. A prevalence of 33% and 23.3% was calculated for helminths and protozoa, respectively in vegetables, representing a possible risk to human and animal health in relation to these parasites. These findings show the need for continuous monitoring of the water quality used for crop irrigation, as well as the safety of food, taking into account the values established in national and international regulations.


Introduction
Water is an essential input for agricultural production and plays a fundamental role in food security (Mabhaudhi et al. 2016).According to The Food and Agriculture Organization (FAO 2017), agriculture uses 70% of freshwater withdrawals worldwide, with the use of urban effluents for crop irrigation being a very common practice (Almuktar et al. 2018;Pedrero et al. 2010).The quality of irrigation water differs by region, country and location, depending on local sanitation conditions.In some countries, crop irrigation is carried out with treated wastewater, while in others, where sanitation conditions and treatment systems are poor or nonexistent, contaminated water is used without any control (Jiménez et al. 2016).
In Colombia, less than 40% of wastewater is treated, then raw wastewater or wastewater diluted with surface water is used for irrigation, generating possible risks to public health, especially when used for the production of food for direct consumption (Ofori et al. 2021).The regulations and guidelines around the world regarding agricultural water reuse are principally human-health focused, unsatisfactory concerning some of the possible hazardous contaminants such as emerging pollutants (including the parasites), and with large discrepancies when compared with each other (Shoushtarian and Negahban-Azar 2020).
Several studies have reported the risk faced by consumers of agricultural products that have been irrigated with wastewater or contaminated sources (Dickin et al. 2016, Amoah et al. 2018a, Khan et al. 2022), and it has even been estimated that about 70% of diarrhea cases are caused by the consumption of food contaminated with chemical and/ or biological agents, including parasites (Kirk et al. 2017;Gizaw 2019).Foodborne parasites constitute one of the most representative and relevant etiological groups in public health, mainly in developing countries (Food and Agriculture Organization of the United Nations 2014; Rodriguez-Morales et al. 2016).This is generated not only by the use of effluents contaminated with wastewater for irrigation crops, but also by the inappropriate handling of fruits and vegetables during their production chain (cultivation, distribution, processing, packaging and sale) (Briñez et al. 2012;Olea et al. 2012).
This study evaluates the occurrence of parasites in waters used for crop irrigation and vegetables from the Savannah of Bogotá, Colombia.We assess the parasitological water quality coming from the middle basin of the Bogotá River, in the area of the irrigation district of La Ramada, where due to the economic growth of the region, raw domestic and industrial wastewater is also discharged directly into the irrigation channels (Argüello and Bustos 2018).Vegetables such as lettuce and celery were analyzed, using parasitological and PCR assay methods, for contamination with helminth and protozoan parasites endemic in Colombia and important in the Savannah of Bogotá, Colombia.

Study area and sample collection
The study was carried out in the Former La Ramada irrigation and drainage district, located in the municipalities of Mosquera and Funza (Cundinamarca), Colombia.This district is responsible for providing permanent irrigation and drainage services for water from the middle basin of the Bogotá River through hydraulic infrastructure works (Corporación Autónoma Regional de Cundinamarca (CAR) 2010; Corporación Autónoma Regional de Cundinamarca (CAR) 2011), which extend through the municipalities of Mosquera, Tenjo, Funza, Cota, Madrid and Bojacá.
Water sampling was taken in six sampling sites from irrigation canals of the Former La Ramada district (Fig. 1): Fig. 1 Geographical distribution of the sampled sites of water used for irrigation and vegetables (celery and lettuce), from the Former La Ramada irrigation district site 1: longitude: − 74.240309, Latitude: 4.634444;site 2: longitude: − 74.196457, Latitude: 4.698042;site 3: longitude: − 74.218966, Latitude: 4.683125;site 4: longitude: − 74.197369, longitude: 4.690720, site 5: longitude: − 74.196457, Latitude: 4.698042 and site 6: longitude: − 74.190544, Latitude: 4.692900.Sites one, two, and three were selected because of their predominant agricultural activity, along with animal husbandry, primarily cattle, pigs, and poultry (Fig. 1 Agriculture Zone).Sites four to six had a high influence of industrial, agricultural and residential activities.At these sites, a total of 96 water spot samples were collected during the months of June to November 2019; 60 samples in the dry season during the months of July, August, and September to 2019, when irrigation water is used all the time for cultivation, and 36 samples in the rainy season during May, June, October, and November of 2019, when irrigation water was used less.
Each sample, comprising a minimum of 2 L of water was collected, transported in a clean plastic container, washed previously with Tween 80 to avoid the adhesion of parasitic structures and stored at 4 °C.The maximum processing time was 96 hours after collection.
A total of 120 samples of fresh vegetables (lettuce (n = 108) and celery (n = 12)) were collected, during harvest at Site 1, Site 3 and Site 4, because they were irrigated with the irrigation canal waters.Those vegetables are eaten raw, they have a high consumption by the local population, large volume of trade and consumption in Bogota city and availability at all times of the year.A completely randomized design was used, selecting 3 farms in each chosen site, Three plants were taken in each farm for a total of 9 plants per site, and the sampling unit was made up by quartering.A total of eight samplings were conducted during the years 2019 and 2021 in both the rainy and dry season.

Detection and enumeration of parasitic forms in irrigation water samples
For the detection and enumeration of helminth eggs in the water, the samples were processed using the method of Bailenger (1979) modified by Ayres and Mara (1997).Once the concentration of each sample was completed, it was observed using brightfield microscopy with a McMaster Chamber.

Molecular detection of protozoa in irrigation water
The direct centrifugation methodology described in the Environmental Protection Agency (EPA) method 1693 (EPA 2014) was used.The sediment obtained was subjected to DNA extraction using the Fast DNA Spin Kit for soil Cat: 116560200 MP Biomedicals, with the aid of the Super FastPrep-2MT instrument (MP Biomedicals), following the manufacturer's instructions.The recovered DNA was quantified using NanoDrop™ One-ThermoScientific and stored at − 20 °C until analysis.
Molecular detection (Table 1) was performed individually for each protozoan species using qPCR with different sets of specific primers and probes reported by Mejía et al.  2020), using the Applied biosystems 7500.For the identification of T. gondii oocysts, the primers described by Opsteegh et al. (2010) were used, slightly modified for amplification detection using the Sybr select master mix thermo kit, for which 10 μL of the mix, 1 μL of each primer (final concentration 0.5 μM) and 3 μL of the sample were used, for a final volume of 15 μL.DNA extracted from cat feces donated by Dr. João Luis Garcia (Universidad de Londrina (UEL) Paraná, Brazil) was used as a positive control for this parasite.For Cryptosporidium spp. the methodology described by Burnet et al. (2013) was used.DNA from C. cayetanensis and Cryptosporidium spp.donated by Dr. Ana Luz Galván from the Universidad de Antioquia, (Medellín-Colombia) were used as controls.In the case of Giardia spp.DNA was obtained from culture in TYS-33 medium (Keister 1983).

Physicochemical and microbiological characteristics of irrigation water samples
Physicochemical (pH, dissolved oxygen (DO), conductivity and temperature) and microbiological (most probable number (MPN) of total coliforms and Escherichia coli) parameters were measured in water samples used for crop irrigation.
In each sample, a multiparameter DO / pH / Conductivity (HACH HQ40D) was used.Following the manufacturer's instructions, the measurement of the physicochemical parameters was carried out in situ.
For the identification of microbiological parameters, the methodology described in the Standard Methods for the Examination of Water and Wastewater (9221) was followed (APHA et al. 2017).The procedure was performed by calculating the MPN of coliforms and E. coli.Both physicochemical and microbiological results were analyzed with GraphPad prism v5.0.

Detection of parasites in vegetable samples
Thirty grams (30 g) of each type of directly consumed vegetable (celery or lettuce) were taken, and the methodology described by Matosinhos and collaborators (Matosinhos et al. 2016) was followed.Due to the large amount of organic detritus, an additional step was performed, to allow the visualization of protozoan cysts and oocysts using the Formol-Ether Concentration Method.Following the technique described by Triviño-Valencia et al. (2016), this sample was observed using light microscopy to characterize helminth eggs and larvae, along with protozoa (Entamoeba spp., Endolimax spp., and others).Furthermore, detection of Giardia spp.cysts and Cryptosporidium spp.oocysts was performed by immunofluorescence, using the A100FLK Aqua-Glo™ G/C Direct Comprehensive Kit (Waterborne Inc.®) following the manufacturer's instructions.The criteria for positivity are based on the parameters defined in EPA method 1623.1 (EPA 2014; Palacios 2017).

Detection of helminth eggs and larvae in irrigation water by microscopy
It was found that 68% (65/96) of the samples were positive for helminth eggs.Ancylostomatidae was found in 42/96 (43.75%) of the samples and Ascaris spp. was found in 33/96 (34.37%) of the samples were the predominant parasites with averages of 1.53 and 0.81 eggs per liter (eggs/L) respectively.Followed by Hymenolepis spp. was found in 11/96 of the samples (11.45%),Trichuris spp.were observed in 5/96 of the samples (5.20%) and Capillaria spp. was found in 2/96 of the samples (2.08%) with averages of 0.22, 0.02 and 0.008 eggs/L respectively.Nematode larvae were observed (52.08%) with an average of 1.9 nematode larvae per liter of water.
Due to resource limitations and sample volume, a specific number of samples were selectively evaluated for Cryptosporidium spp.and C. cayetanensis.Of the 96 samples available, 62 water samples were evaluated for Cryptosporidium and 88 samples were evaluated for C. cayetanensis.
Considering the season of sample collection, the presence of parasites was higher in the rainy season with 68.3%, with a higher prevalence of Cryptosporidium spp.followed by T. gondii and Giardia spp.(Table 2).

Identification of physicochemical and microbiological parameters in irrigation water samples
Table 3 shows the average values of physicochemical and microbiological data for each of the 6 sampling points.The data show higher MPN values for total coliforms and E. coli in the area with purely agricultural activity.Likewise, higher average pH and temperature values are observed in this same zone.In contrast, it was found that the zone with mixed activity (agricultural, urban-industrial), obtained a higher average value of dissolved oxygen (DO) and conductivity.

Detection of parasites (protozoa and helminths) in vegetables
The detection of parasites in vegetables grown in the Former La Ramada area showed that 49.16% (59/120) of the samples (lettuce and celery) were positive for at least one helminth egg, cyst or protozoan oocyst (Table 4).The prevalence of helminths and protozoa detected in the 120 vegetable samples showed that 33.33% (40/120) of the vegetables were positive for helminth eggs, with Ascaris spp.being the most prevalent parasite, with an average of 0.43 eggs/30 g of sample analyzed, followed by Ancylostomatidae, Hymenolepis spp., Trichuris spp.and Capillaria spp.(Fig. 2).Of the samples, 23.33% (28/120) presented some protozoa, with Entamoeba spp.being the most representative, detected with an average of 0.45 cysts/30 g, followed by Giardia spp., Cryptosporidium spp.and Endolimax spp.(Table 4).Entamoeba spp.were detected in the three points evaluated, with a sample of lettuce in which 17 cysts were reported (site 3).Site 1 (n = 93) had the highest diversity of parasites; predominantly Ascaris spp., Entamoeba spp.and Giardia spp.The data also showed that 7.5% (9/120) of the vegetables were contaminated with both helminths and protozoa; 5.83% (7/120) of these samples were collected at site 1; the sample with the highest diversity of parasites had 4 Ancylostomatidae eggs, 1 Capillaria spp.egg and 3 Cryptosporidium spp.oocysts (Table 5).
Nematode larvae were observed in 39.16% (47/120) of the samples, with an average of 1.68 larvae/30 g of sample analyzed, with site 1 showing the highest values of these parasitic stages (Fig. 3A).Immunofluorescence assays detected the presence of 4 samples positive for Cryptosporidium spp.oocysts and 7 for Giardia spp.cysts (Fig. 3B).

Discussion
Foodborne parasites have long been a "neglected" group of pathogens, attributed to vulnerable or low-resource populations (Robertson 2018).However, in both developed and developing countries, there have been numerous cases of outbreaks associated with the consumption of fresh produce, which have increased considerably (Kopper et al. 2009;Ryan and Cacciò 2013;Carstens et al. 2019).The variety and types of microorganisms found in these foods (especially vegetables) vary substantially, depending on several factors  such as the type of crop, region of origin, growth conditions, handling and transport circumstances, among others.In Colombia, the Ministry of Health reported in 2021, 603 foodborne disease (FBD) outbreaks, with an estimated 6883 affected people (Instituto Nacional de Salud 2022).Although those reports broadly classify the etiologic agents that can cause FBDs, it is evident from those reports that the Entamoeba genus is the gastrointestinal protozoa most commonly associated with outbreaks and food contamination, after viruses and bacteria (Varela et al. 2016;INS 2018;Montañez et al. 2020).The real impact of these infections and the true number of outbreaks caused by these parasites are unknown and underestimated in Colombia, due to the lack of mandatory reporting and the absence of an active surveillance system at the national level (INS 2018).
In this study, different parasitological and molecular techniques were used to evaluate the quality of water used for agriculture and some vegetables (lettuce and celery) crop in this zone.These methodologies allowed the detection of protozoa and helminths in food crops and water samples.
The results showed that the water sources being used for crop irrigation in this area of the country do not comply with the guidelines established by the World Health Organization regarding their parasitological quality (should have less than 1 egg/L to guarantee human health safety).For example, in sampling site 6, the average for Ascaris spp.Was 2.6 eggs/L and for Hymenolepis spp.1.23 eggs/L and for site 1 the average for Ancylostomatidae was 5.54 eggs/L.The presence of helminth and protozoan has also been found in the food (lettuce and celery) grown in this area.33.33% (40/120) of the vegetables were positive for helminth eggs, with Ascaris spp.being the most prevalent parasite, with an average of 0.43 eggs/30 g of sample analyzed, followed by Ancylostomatidae, Hymenolepis spp., Trichuris spp.and Capillaria spp.
In the review of the literature from Colombia, there are no studies with these same characteristics that have simultaneously evaluated the parasitological quality of water used for agriculture and food irrigated with this water.According to the physicochemical and microbiological 1.40% 0.01 0-1 3 6.12% 0.12 0-3 characteristics of the water used for irrigation analyzed in this study (Table 3), the results of positivity for Giardia cysts (10.41%),Cryptosporidium oocysts (51.62%) and T. gondii (18.75%) obtained by qPCR and the occurrence of different helminths are not surprising, since fecal contamination of these effluents is evident.
As can be seen in Table 3, the sites considered in the agricultural zone have anoxic conditions and high electrical conductivity values.In the mixed zone conductivity remains high despite higher DO values, which is a clear indicator of the discharge of wastewater of both industrial and domestic origin, affecting the development of both biological activity and degradation processes (Abella and Martínez 2014).
In line with the findings of this current study, in Morocco, Amahmid et al. (2022) found crops contaminated with various levels of parasite eggs and cysts due to irrigation with raw sewage.Lettuce samples were positive (27.7%) for one or more helminth eggs including the pathogens Ascaris and Trichuris with an average of 4.7 eggs/kg.These values differ from the current study where 33.33% (40/120) of the vegetables were positive for helminth eggs, with Ascaris spp.being the most prevalent parasite, and an average of 0.43 eggs/30 g, followed by Ancylostomatidae, Hymenolepis spp., Trichuris spp.and Capillaria spp.The dissimilarities observed in these studies may be a result of the level of environmental sanitation in different countries.Moreover, in our research, in zones where the hygienic settings of the irrigation water canal structure were not suitable, the occurrence of eggs, cysts and larvae parasites in vegetables was apparent.
In Mexico, Chaidez et al. (2005) evaluated the presence of Giardia spp.and Cryptosporidium spp. in 58 irrigation water samples and found that 48.2% and 50% of the samples were positive, respectively.Results coincide with our study where the characteristics of the agricultural areas were similar.
It is worth noting the importance of this study's finding on T. gondii prevalence (18.75%), which is one of the most prevalent protozoa in the water samples studied and, according to published reports, is the main cause of outbreaks in Latin America (Rosado-García et al. 2017;Minuzzi et al. 2021).
The presence of Cryptosporidium spp.oocysts in 51.62% (32/62) of the water samples evaluated is not surprising, since these results are consistent with documented studies around the world, where the presence of this parasite is found in water used for crop irrigation, as documented in Greece and United Kingdom (Spanakos et al. 2015;Bodley-Tickell et al. 2002).
The research developed by Palacios (2017) determined the presence of Cryptosporidium spp. in calf feces and water samples (Ecuador).The author showed that in calves there is a prevalence of 93.3% of Cryptosporidium spp.(112 positive cases out of 120), while in water, a concentration of 5 oocysts/100 mL was determined.Based on these findings, the researcher concluded that the high prevalence of Cryptosporidium spp. in calves may be the cause of the presence of this parasite in water.
Colombia is a tropical country with dry and rainy seasons.The contamination of irrigation channels may be related to runoff in rainy seasons, and to the discharge of untreated domestic water during the dry season, among other causes (Steele and Odumeru 2004;Almuktar et al. 2018).
Referring to the evaluation of the presence of helminth eggs, it was found that Ancylostomatidae (43.75%) and Ascaris spp.(34.37%) were the most prevalent genera in irrigation water samples.These results are in agreement with the findings of Amoah et al. (2018b) in South Africa, Hajjami et al. (2012) in Morocco, Ortiz and López (2012) in Colombia and Trang do et al. (2006) in Vietnam.
Regarding the concentration of eggs, previous studies have reported total values of 0.1 to 3 helminth eggs per liter of untreated water, and 0.1 to 1 viable helminth egg per liter of treated water in the Savannah of Bogotá (Campos-Pinilla et al. 2008;Campos et al. 2018).Our results reported average numbers ranging from 0 to 5.54 eggs/L and an average of 1.9 nematode larvae per L.These results suggest that these samples are not in compliance with WHO quality criteria guidelines (Campos et al. 2018).

Conclusions
In conclusion, this study establishes that there is a potential risk of transmission of parasites of public health interest from the consumption of vegetables grown in the Savannah of Bogotá, Colombia.Molecular detection showed that the most relevant parasite was Cryptosporidium spp.and in terms of geographic region, the industrial and urban region had the highest concentration of Giardia spp.The detection of DNA of T. gondii, Giardia spp.and Cryptosporidium spp. in addition to the observation of different helminth eggs in water samples used for irrigation and vegetables, shows that fecal contamination of effluents (with animal and human feces) is evident.Therefore, it is necessary to inform stakeholders -farmers, consumers, regulatory entities-to carry out a risk analysis of the production chain of food grown in this area of the country, based on Good Agricultural Practices (GAPs), to implement appropriate preventive measures to reduce the risk of contamination and possible transmission of these parasites.
It is recommended to carry out viability tests to obtain more robust data on the risk that these parasites represent for the community.
To our knowledge, this is the first investigation describing T. gondii detection in irrigation water samples collected from the territory of Savannah of Bogotá, Colombia using sensitive molecular tools.

Fig. 3
Fig. 3 Detection of intestinal parasites in vegetables irrigated with water from the farmer La Ramada irrigation district.Panel a: Nematode larvae detected in vegetable samples, bright field microscopy observation.Panel b: Giardia spp.cysts (b) and Cryptosporidium

Table 2
Molecular detection by qPCR of Giardia spp., T. gondii, Cryptosporidium spp.and C. cayetanensis in irrigation water from the Former La Ramada district Data represent the percentage of intestinal parasites in irrigation water collected on both A (Agricultural) and I (urban-industrial) land.* n = 62 **n = 88

Table 4
Prevalence of intestinal parasites in vegetable samples (Lettuce and Celery)

Table 5
Prevalence of parasites detected at three sampling sites in the former La Ramada irrigation and drainage district Pos (Positive samples); Prev.(Prevalence); X̄ (average value of helminth eggs, cysts or oocysts of protozoa in 30 g of plant material analyzed); Min-Max (Minimum and maximum values of parasites detected in vegetables)