Enteric bacterial contamination and survival on produce during irrigation with dairy wastewater in the fi eld

The goals of this study were to quantify enteric bacterial contamination and survival on several different types of produce during irrigation with wastewater from a dairy operation. Dairy wastewater was used to irrigate three different types of vegetable crops: lettuce, carrot, and bell pepper. This study was conducted over two consecutive growing seasons. Irrigation water and vegetable samples were examined for Escherichia coli and Clostridium perfringens. In the dairy wastewater, E. coli and C. perfringens concentrations averaged 8.2 × 10MPN/100 mL and 5.0 × 10 CFU per 100 mL, respectively. Analysis of variance test results indicated that E. coli and C. perfringens concentrations detected on the three crops after irrigation were statistically different (p< 0.0001). The greatest contamination occurred on the carrots followed by lettuce and bell peppers. E. coli and C. perfringens were recovered from the carrots, bell peppers, and soil 49 days after wastewater irrigation of the plots had ceased. Moisture content of the soil was statistically significantly related to survival of the organisms in the soil. doi: 10.2166/wrd.2013.161 om https://iwaponline.com/jwrd/article-pdf/3/2/102/377827/102.pdf er 2018 Faezeh D. Manshadi 1540 Southwest Expressway, Apt. 245 San Jose, CA 95126, USA Martin Karpiscak Research Scientist, Office of Arid Lands Studies, University of Arizona, Tucson, Arizona, USA 85719 Charles P. Gerba (corresponding author) Department of Soil, Water, and Environmental Science, University of Arizona, Bldg 90, Room 409, Tucson, Arizona, USA 85721 E-mail: gerba@ag.arizona.edu


INTRODUCTION
It is estimated that approximately 10% of the irrigated crops in the world are irrigated with wastewater or sewage polluted surface waters ( Jimenez et al. ). In some countries, 80% of the vegetable production is irrigated with wastewater (Pedrero et al. ). Plants might become contaminated in the field through the use of contaminated irrigation water (Beuchat &  The goal of this research was to obtain data on transfer of two naturally occurring bacteria found in dairy wastewater, E. coli and Clostridium perfringens, onto three different vegetables whose edible parts were located in the soil, on the soil surface and above the soil surface. Dairy wastewater is currently used in Arizona to irrigate crops for animal consumption (Karpiscak et al. ). An additional purpose was to obtain data on survival of these organisms on plants growing in the field and in the soil. E. coli was selected because of its common use as an indicator for enteric bacterial pathogens and C. perfringens because of its longer survival in the environment. The wastewater passed through a solid separator as the only treatment system. Wastewater was transported by a tanker truck to the field plots and then diluted 1:1 in a second tanker truck containing non-disinfected well water (producing diluted dairy wastewater) before application to the field. Water was applied by furrow irrigation. Dairy wastewater, diluted dairy wastewater, and well water samples were taken weekly during the irrigation of the vegetables and tested for E. coli and C.

METHODS
perfringens. Samples were transported in ice-packed coolers to the laboratory. Three vegetable varieties were chosen for this project: lettuce (Lactuca sativa), carrot (Daucus carota), and bell pepper (Capsicum annuum).
This study was conducted over 2 consecutive years. In the first year growing season, diluted dairy wastewater was applied to vegetable plots weekly from May through July. Two harvests were made from each vegetable plot during this time, 2 weeks apart from late May to early July depending upon the crop. In the second year the growing season was extended an additional 5 weeks to assess survival of the bacteria on the crops and soil after irrigation with wastewater was terminated. During this part of the study, the fields were irrigated only with well water. Samples of produce and soil were placed in plastic bags and transported to the laboratory in ice-packed coolers. A total of 12 vegetable (four vegetables selected from each location) and soil (two soil samples selected from each location) samples were randomly collected from the plots for each harvest period from areas not previously sampled.

Irrigation water analysis
Dairy wastewater, diluted dairy wastewater, and well water were analyzed for the presence of E. coli and C. perfringens.
Wastewater was serially diluted in Tris-buffered saline The efficiency of recovery of E. coli and C. perfringens was determined in laboratory experiments by adding known concentrations of the organisms to the different vegetables and processing as described in the previous paragraph.

Soil analysis
Two randomly selected soil samples from each plot were collected before and after the application of wastewater coli. C. perfringens was detected by the membrane filter method as previously described.

Survival experiments
Experiments were conducted to determine the persistence of the studied enteric bacteria on the produce and the soil after wastewater irrigation was terminated in the second year. Three carrot, bell pepper, and soil samples were taken from each treatment plot weekly from July 7 through August 20 during which time the plots were irrigated with well water. Samples were analyzed for the presence of E. coli and C. perfringens. Samples were processed and analyzed as previously described.

Efficiency of methods
The efficiency of recovery of the studied organisms is shown in  suggests that depending on where the edible part of the crops is situated (i.e., in the soil or above the soil) has a statistically significant effect on the degree of contamination on the surface of the vegetable crops.

Soil
Two soil samples were collected from each plot before and after application of dairy wastewater to the plots. Another set of samples was taken at the second harvest of each  At the end of second year, E. coli and C. perfringens concentrations of 25 MPN/g and 133 CFU/g were detected in the experimental soil plots, respectively. There were positive correlations (p < 0.05) between E. coli and C. perfringens density and soil moisture content.

Survival study
To assess how long the bacteria would survive on the produce and soil in the second year wastewater irrigation was terminated after the second harvest and subsequent irrigation was with well water only. A total of eight bell pepper and carrot samples were collected weekly from experimental plots after wastewater irrigation had ceased. Lettuce was not studied as it was beyond the growing season in southern Arizona. E. coli and C. perfringens were recovered from all samples of vegetables and soil 49 days after wastewater flooding of the plots had ceased (Tables 5 and 6).
E. coli concentrations ranged from 0.2 to 124 MPN/g and 1 to 172 MPN/g on bell peppers and carrots, respectively (Table 5). Concentration of C. perfringens on bell peppers and carrot samples ranged from 0.02 to 0.8 CFU/g, and 1 to 11 CFU/g, respectively (Table 5)   Wachtel et al. ). The problem with laboratory-grown bacteria is that they were grown under nutrient-rich conditions and they need to adapt to low-nutrient conditions in the new environment which may cause them to die off faster than expected or their behavior may be different from indigenous microorganisms.
As might be expected, the more contact the crop had with the soil, the greater the contamination of the crop.
Washing dirt off crops or in the case of lettuce removing the outermost leaves of the lettuce heads will reduce the microbial load but not necessarily eliminated it (Maxcy ; Fascioio et al. ). We have previously tested laboratory-grown E. coli seeded into irrigation water (nonwastewater) used for furrow irrigation of lettuce and bell pepper (Stine et al. ). In these studies, the transfer of E. coli to lettuce was found to be very low (0.00007%), and no transfer could be detected to the bell peppers.
Only one irrigation event was conducted in those studies.
Our current study also suggests low transfer, even through the produce was irrigated multiple times with wastewater during the growing season. However, E. coli were routinely detected at low levels on the bell pepper, which did not occur in our previous study with laboratory-grown E. coli.  shown that even crops whose edible portion is grown above the ground may become contaminated and that pro- Additional studies would be needed to assess low level survival of pathogens in the field. Our studies suggest some caution in using only laboratory data to judge the survival of enteric bacteria on produce in the field.

CONCLUSIONS
The potential for contamination of three types of vegetables, lettuce, carrots, and bell peppers, was assessed during surface irrigation with the diluted dairy wastewater. The findings of this study can be summarized as follows: • The concentrations of both E. coli and C. perfringens on three crops were significantly different (p < 0.0001) which reflected the distance the edible portion grew above the ground.
• The positive correlations (p < 0.05) between E. coli and C. perfringens density and soil moisture content indicates that the soil moisture has an important effect in occurrence and survival of these enteric microorganisms in soil.
• E. coli and C. perfringens survived on the vegetables and in soil for 49 days after wastewater application ceased.