Source: UNIVERSITY OF ILLINOIS submitted to
NIFA-BARD COLLABORATIVE: EXPOSURE RISKS OF PATHOGENS AND DISINFECTION BYPRODUCTS FROM ON-SITE TREATED RAINWATER AND DRAINAGE WATER FOR IRRIGATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1011892
Grant No.
2017-68007-26307
Project No.
ILLU-000-626
Proposal No.
2016-10258
Multistate No.
(N/A)
Program Code
A8103
Project Start Date
Mar 15, 2017
Project End Date
Mar 14, 2021
Grant Year
2017
Project Director
Nguyen, T. H.
Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
Civil and Env Eng
Non Technical Summary
Rising prices of vegetables and fruits motivate the development of community gardens and commercial greenhouses in cities throughout the US. At the same time, soilless substrate-based or hydroponic greenhouses for the production of flowers, fruits, and vegetables have been developed in Israel for years. However, in both countries, these farms face significant challenges including the cost of freshwater used for irrigation. The presence of microbial contaminants in the harvested rainwater (RW) and the drainage water (DW) from soilless farms prevent the use or recycling of these waters to substitute for freshwater. Although disinfection is likely to inactivate plant and human pathogens in these waters, toxic disinfection byproducts (DBPs) may be formed. In general, the extent of human pathogen survival and of formation of DBPs in disinfected RW and DW, as well as the possible residual toxicity of these waters, are not well known. To address these knowledge gaps, we propose the following objectives: 1) characterize the microbial community, including pathogens in RW and DW collected in both Illinois and Israel; 2) determine the disinfection efficacy and mechanisms of well-characterized strains of rotavirus (RV) and coxsackievirus (CX) in these water samples by chlorination alone and by chlorination preceded by ozonation or solar irradiation; 3) identify which disinfection method generates the lowest levels of mammalian cell cytotoxicity and genotoxicity in the disinfected RW and DW; 4) determine the characteristics of organic matter in RW and DW that may be linked to the formation of reactive oxygen species that inactivate RV and CX upon solar irradiation, the formation of selected representative toxic DBPs typically present under various disinfection treatments, the total organic halogens (TOX), and the toxicity; and 5) transfer this knowledge to growers and consumers in the US and Israel through Extension activities. This comprehensive study will determine the relative potential human health impacts of irrigation water used in urban and small farms in both dry climates (Israel and the American South and Southwest) and temperate climates (the American North and Midwest).
Animal Health Component
0%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1110210115040%
1110210110140%
1110210202020%
Knowledge Area
111 - Conservation and Efficient Use of Water;

Subject Of Investigation
0210 - Water resources;

Field Of Science
2020 - Engineering; 1101 - Virology; 1150 - Toxicology;
Goals / Objectives
The primary goal of this project is to generate science-based information to allow the growers to safely choose an application of the irrigation water based on parameters that encompass efficient economics, sustainability, and reduce exposure risk of pathogens and toxic chemicals generated through disinfection to humans. Specific goals are:1) Identify the microbial constituents of rainwater (RW) and drainage water (DW) longitudinally and temporally to determine (1) the link between bacterial microbiomes with chemical compositionof RW and DW, and (2) the risk of pathogen exposure by growers and consumers of crops grown in the studied community-based garden in Illinois (IL) and soilless hydroponic farms in Israel (IS).2) Determinethe efficacy of virus disinfection, together with the mechanisms controlling virus inactivation by SoChlor, OzChlor, and Chlor obtained by a comprehensive set of virus strainsto develop recommendations on disinfection for the safe recycle of RW and DW.3) Determinecytotoxicity, genotoxicity, thiol reactivity, and up regulation of antioxidant response element in human cells. These findingswill be linked to fluorescence characterization of the same water samples. 4) Characterize the chemical composition of the studied RW and DW. Specifically, thefluorescence of organic matter components will be related to (a) appearance of disinfection byproductsduring chlorination, (b) change of the dissolved organic matter composition during pretreatment via sunlight and ozone, (c) toxicity of disinfected waters, and (d) virus inactivation by reactive oxygen species upon sunlight irradiation.5) Transfer this knowledge to growers and consumers in the US and Israel through Extension activities. This comprehensive study will determine the relative potential human health impacts of irrigation water used in urban and small farms in both dry climates (Israel and the American South and Southwest) and temperate climates (the American North and Midwest).
Project Methods
We propose a comprehensive study to evaluate the potential for exposure of growers and consumers to pathogens and DBPs present in disinfected harvested RW and recirculating DW from soilless farms that may have adverse health impact. In Task 1, samples of RW and DW from gardens and farms in both IL and IS will be subjected to microbial community characterization using both next-generation (NextGen) sequencing and microfluidic quantitative polymerase chain reaction (MFqPCR). Specifically, in IL, rainwater will be collected by rain barrels at El Paseo Community Garden in Chicago, IL, and a green roof at the University of Illinois at Urbana-Champaign. In IS, rainwater will be collected on the Volcani center campus. In all locations, samples from the storage tank will be taken freshly after the rain and over time to determine the changes in microbiome during the storage time. DW will be collected from Patkin Farm (IL), a soilless farm of strawberries, Yesha (IS), and Fine Hydroponics farm, Bnei Netzarim (IS), and also from greenhouse experiments at Ramat-Negev Research and Development (IS), where tomatoes grow on soilless media including tuff, coconut coir, and ground stone. This field experiment is situated in Ramat a-Negev and under the supervision by Dr. Asher Bar-Tal, the Volcani Center, Agricultural Research Organization (ARO).In Task 2, inactivation of RV and CX viruses will be studied in RW collected in IL and in solutions with dissolved freeze-dried HS and diluted organic extracts from RW and DW collected in IS. The laboratory-cultivated viruses will be added to these solutions, allowing a comparison at a consistent solution condition for these three schemes of disinfection: chlorination alone (Chlor), solar disinfection followed by chlorination (SoChlor), and ozonation followed by chlorination (OzChlor). In Task 3, the organic extracts containing DBPs from the RW and DW samples before and after chlorination, SoChlor and OzChlor, will be sent from IS to IL. For this Task we will employ a battery of four in vitro quantitative bioassays targeting relevant biological endpoints: (1) Cytotoxicity: with the reduction in cell growth and viability compared to controls, cytotoxicity captures a wide range of toxic insults; (2) Genotoxicity: by damaging genomic DNA, genotoxic agents are associated with mutagenicity, carcinogenicity, and the induction of developmental defects; (3) Thiol Reactivity: cysteine thiols are the predominant soft nucleophiles in biological systems and serve to protect the cell from reactive toxicants; and (4) Up regulation of the antioxidant response element in human cells: cellular damage by reactive oxygen species (ROS) will be measured. In Task 4, RW and DW samples collected in IS before and after disinfection and the HS extract from IL samples before and after disinfection will be characterized for common water quality parameters and by excitation-emission matrices (EEMs) of fluorescence, using the protocol developed by Dr. Borisover's group at the Volcani Center at both Volcani and the University of Illinois. In Task 5, educational materials will be developed and delivered to growers in Chicago and IS through the Illinois Extension office and the Galilee Society in IS, respectively. Dr Sabbah of Braude College is also an investigator at Galilee Society.

Progress 03/15/17 to 03/14/21

Outputs
Target Audience:Members of the target audience included researchers, growers, consumers, and Extension educators. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We trained one postdoctoral fellow (Bhuvaneshwari) in Israel, one postdoctoral fellow (Dong) and a graduate student (Fuzawa) at Illinois on this project. In addition, we are working closely with our colleagues in the Extension office of Cook County in Chicago to disseminate the results to growers. How have the results been disseminated to communities of interest?We presented the study findings to hydroponic and aquaponic growers in Chicago. The workshop has been recorded and posted on the Extension YouTube Channel under Local Foods. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1 (Illinois Team): Out of twelve rainwater samples selected for analysis, sixsamples were collected in October 2016 and 6 samples were collected in June-July 2017.These water samples were coagulated and filtered through 1.6 µm and 0.22 µm pore size filters to collect bacteria and viruses, respectively. DNA from the collected microbes was extracted and subjected to quantitative polymerase chain reaction (qPCR) to quantify the common bacterial pathogens. Shiga Toxin producing E. coli (STEC) gene stx1 and stx2 was detected in 19.4% and 22.2% of the samples, respectively, Enterococcus spp. was detected in 19.4% of the samples, ftsz gene of E. coli and udiA gene of E. coli was found in 13.9% of the samples and Campylobacter jujeni was found in 11.1% of the samples. Out of the samples analyzed, samples collected in 2017 showed higher concentration of detected bacterial pathogen. Objective 2 (Illinois Team): We determined the disinfection efficacy and inactivation mechanisms of peracetic acid-based sanitizer (PAA) under pHs relevant for vegetable sanitation for rotavirus (RV) and Tulane virus (TV; a human norovirus surrogate). RV was significantly more susceptible than TV to PAA disinfection. Our results also suggest that PAA caused both genome damage and loss of binding functions to inactivate TV and RV. We also determined if the effectiveness of peracetic acid (PAA), a sanitizer used in the vegetable industry, is affected by the location of viruses (virus-contaminating produce surfaces or inside the tissues) in microgreen arugula. For both internalized TV and RV, the disinfection efficacy was less than 2-log10 in PFU using all the tested PAA concentrations and exposure times examined here.Thus, both the type and location of virus in fresh vegetables may influence the virus disinfection of postharvest vegetables. Therefore the optimization of sanitation for postharvest fresh vegetables is needed to reduce foodborne viral infection risks.The findings presented here potentially contribute to the design of a robust sanitation strategy for RV and TV using PAA to prevent foodborne disease. Objective 3 (Illinois Team):We evaluated the impact of Ct values and different ways to achieve the same Ct value by ozonation or chlorination on cytotoxicity of disinfected wastewaters from four agricultural sources. The overall results indicate that the cytotoxicity of aquaponics and hydroponics water may increase when higher Ct values are applied to inactivate recalcitrant pathogens, and to counteract the potential increase in cytotoxicity at high Ct values, for both ozonation and chlorination, lower disinfectant dose and longer contact time should be adopted. In summary, for both aquaponics and hydroponics, ozonation led to lower cytotoxicity than chlorination. Disinfection by ozone is preferred. To ensure pathogen inactivation and minimize toxicity, low concentration of ozone or chlorine over long exposure time is preferable. Objective 3 (Israel Team):We studied the toxicity of chlorinated effluents using genetically modified bioluminescent bacterial strains underwent plasmid fusion with different promotors responding to genotoxicity, cytotoxicity, and reactive oxygen species (ROS) mediated stress. Three types of secondary wastewater effluents were collected from treatment plants and chlorinated using oneand ten mg L-1 of sodium hypochlorite at various contact times lasting from 0.5 to 72 hours. Induction of bioluminescence of bacterial strains in response to the DBP production upon water chlorination was considered as the toxicity pattern. Disinfection timeand chlorine concentration dependent development of genotoxicityand cytotoxicity followed with decline at a more extended period of interaction signify the production and disappearance of DBPs. Induction in the bioluminescence of bacteria suggests cellular metabolism-altering and DNA-damaging properties of disinfected effluents. In the case of ROS mediated stress, there were no significant changes in the observed induction pattern between controls and chlorinated effluents. The mechanism-specific induction pattern varied depending on the wastewater effluent nature. Objective 3 (Israel Team): We have compared genetically modified bioluminescent (GMB) bacteria receive plasmid fusion with promotor's response to specific reporter genes lux CDABE, and Chinese hamster ovary (CHO) cells in characterizing the toxicity of water samples collected from a wastewater treatment plant, lakes and streams, hydroponics and aquaponics farms situated in northern Israel. In addition to the source water toxicity, the GMB bacteria were also used to test the toxicity of the XAD extracts i.e., concentrating the source water samples using XAD resin, and diluted to the concentrations causing 50% growth inhibition of the CHO cell lines. The GMB bacteria-based toxicity of the above-mentioned diluted XAD extracts was found variable among different water samples thus indicating that both the monitoring tools act distinctively. It was also demonstrated that sample pre-concentration based on adsorption by XAD resins with the following elution by organic solvents was not able to represent the material responsible for the major toxicity of the whole water samples towards the GMB bacteria. Therefore, the use of XAD resin extracts may lead to major underestimating of the water sample toxicity. Thus, the GMB bacteria assay provided a mechanism-specific biomonitoring tool to probe the toxicity of water samples without a need for the pre-concentration step. Objective 4 (Israel Team):We have studied the strengths of correlations with formation of chlorination disinfection byproducts. The statistical results that the water source, DBP predictors are the most significant parameter followed by disinfection agents, whereas DBPs type is less parameter for DBP formation. Among the water types, potentially showed the correlations of DBP predictors with DBP formation. UV absorbance and OC concentrations over SUVA and DBP predictors served as better predictors, which suggests that non-fluorescent OM present in waters may have a high reactivity towards chlorination reactions. When considering, RI and PARAFAC showed stronger correlations with DBP concentrations as compared to PP. Humic-like substances showed a correlation with DBP formation than proteinaceous substances of OM. Objective 5 (Illinois and Israel Teams):We organized a workshop in Chicago for aquaponic and hydroponic growers. We presented the project findings. We also completed a workshop evaluation. The evaluation showed that the participants' knowledge level increased after the workshop and the participants also planned to implement knowledge learned from the workshop in their practice. Dr. Massalha has presented the project for science teachers in high schools north of Israel. We have planned a workshop for growers and farmers in Israel. The workshop had intended to include presentation of the involved researcher fromIsrael (Dr. Borisover, Dr. Massalha and Prof. Sabbah) as well as from the United States(Prof. Nguyen). In addition, the plan was supposed to include a tour and demonstration of technical aspects (i.e. safety measures if RW and DW used for irrigation, practices of soilless farming, vegetables and food in diets in Israel). This workshop was planned for April-May 2020, however, the COVID-19 pandemic stopped us from the planned workshop. We hope to conduct the workshop after the pandemic is under control in Israel and in the United States.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Fuzawa, M., E. Araud, J. Li, J.L. Shisler and T.H. Nguyen. 2019. Free Chlorine Disinfection Mechanisms of Rotaviruses and Human Norovirus Surrogate Tulane Virus Attached to Fresh Produce Surfaces. Environmental Science & Technology.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Fuzawa, M., H. Bai, J.L. Shisler and T.H. Nguyen. 2020. The Basis of Peracetic Acid Inactivation Mechanisms for Rotavirus and Tulane Virus Under Conditions Relevant for Vegetable Sanitation, Applied and Environmental Microbiology. 86(19): p. e01095-20.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Fuzawa, M., R.L. Smith, K.-M. Ku, J.L. Shisler, H. Feng, J.A. Juvik and T.H. Nguyen. 2020. Roles of Vegetable Surface Properties and Sanitizer Type on Annual Disease Burden of Rotavirus Illness by Consumption of Rotavirus-Contaminated Fresh Vegetables: A Quantitative Microbial Risk Assessment, Risk Analysis. 40(4): p. 741-757.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Bhuvaneshwari, M., Eltzov, E., Veltman, B., Shapiro, O., Sadhasivam, G. and Borisover, M. 2019. Toxicity of Chlorinated and Ozonated Wastewater Effluents Probed by Genetically Modified Bioluminescent Bacteria and Cyanobacteria Spirulina sp. Water Research 164, 114910.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Bhuvaneshwari, M. and Borisover, M. 2020. Strengths of Correlations With Formation of Chlorination Disinfection Byproducts: Effects of Predictor Type and Other Factors. Environmental Science and Pollution Research, 27, 53375352.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Bhuvaneshwari, M., Massalha, N., Eltzov, E., Nguyen, T.N., Sabbah, I. and Borisover, M. 2020. Water Toxicity Evaluations: Comparing Genetically Modified Bioluminescent Bacteria and CHO Cells as Biomonitoring Tools. Ecotoxicology and Environmental Safety, 203, 110984.
  • Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Fuzawa, M., Duan, J., Shisler, J.L. and Nguyen, T.H. 2021. Peracetic Acid Sanitation on Arugula Microgreens Contaminated With Surface-Attached and Internalized Tulane Virus and Rotavirus. Food and Environmental Virology.


Progress 03/15/19 to 03/14/20

Outputs
Target Audience:Members of the target audience included researchers, growers, and consumers. Changes/Problems:We have received a one-year no-cost extension to finish the following tasks: 1.Determine efficacy and mechanisms of virus inactivation in hydroponics water and in plants grown hydroponically; 2.Compare genetically-modified bioluminescent bacteria and CHO cell lines for water toxicity evaluation; and 3.We will organize a workshop in Israel to facilitate communication between researchers and those in the hydroponics industry. What opportunities for training and professional development has the project provided?We trained one postdoctoral fellow (Bhuvaneshwari) in Israel and a graduate student (Fuzawa) at Illinois on this project. How have the results been disseminated to communities of interest?In addition to peer-reviewed publications and conference presentations, we are working closely with our colleagues in the Extension office of Cook County in Chicago to disseminate the results to the growers. What do you plan to do during the next reporting period to accomplish the goals?Determine efficacy and mechanisms of virus inactivation in hydroponics water and in plants grown hydroponically. Compare genetically modified bioluminescent bacteria and CHO cell lines for water toxicity evaluation We will organize a workshop in Israel to facilitate communication between researchers and those in the hydroponics industry.

Impacts
What was accomplished under these goals? For the third year of the project, the Illinois team focused on Objective 2 and the Israel team focused on Objectives 3 and 4 of the project. Objective 2 (Illinois Team): We determined the disinfection efficacy and inactivation mechanisms of peracetic acid-based sanitizer (PAA) under pHs relevant for vegetable sanitation for rotavirus (RV) and Tulane virus (TV; a human norovirus surrogate). RV was significantly more susceptible than TV to PAA disinfection. For a 2-log10 reduction of virus titer, RV required a 1 mg/L concentration of PAA for 3.5 minutes, while TV required a 10 mg/L PAA concentration for 30 minutes of exposure. The higher resistance of TV can be explained, in part, by significantly more aggregation of TV in PAA solutions. The PAA mechanism of virus inactivation was then explored by quantifying (i) viral genome integrity and replication using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and (ii) virus-host cell interactions using a cell-free binding assay with porcine gastric mucin conjugated with magnetic beads (PGM-MBs). Under conditions described above, only 10% of PAA-treated TV still bound to PGM-MBs, and only 60% of TV NSP genes remained intact. For RV, 80% of the RV lost their ability to bind to sialic acid-specific binding sites on the PGM-MBs. Linear correlations between the reduction of RV titers and RV genome damage suggest that genome damage was responsible for about 30% of RV inactivation. Our results suggest that PAA caused both genome damage and loss of binding functions to inactivate TV and RV. The findings presented here potentially contribute to the design of a robust sanitation strategy for RV and TV using PAA to prevent foodborne disease. Objectives 3 and 4 (Israel Team). We examined the toxicity effects of chlorinated and ozonated wastewater effluents on three genetically modified bioluminescent bacteria, in comparison to the naturally isolated cyanobacteria, Spirulina strains as test systems. Three different secondary wastewater effluents were collected from treatment plants, chlorinated using sodium hypochlorite (at 1 and 10 mg L-1 of chlorine) or treated using 3-4 mg L-1 of ozone at different contact times. As compared to a cyanobacterial Spirulina sp., the genetically modified bacteria enhancing bioluminescence at the presence of stress agents demonstrated greater sensitivity to the toxicity induction and have also provided mechanism-specific responses associated with genotoxicity, cytotoxicity, and reactive oxygen species (ROS) generation in wastewater effluents. Effects of effluent chlorination time and chlorine concentration revealed by means of bioluminescent bacteria suggest the formation of genotoxic and cytotoxic DBPs followed with their possible disappearance at longer times. Ozonation could degrade genotoxic compounds in some effluents, but the cytotoxic potential of wastewater effluents may certainly increase with ozonation time. No induction of ROS-related toxicity was detected in either chlorinated or ozonated wastewater effluents. UV absorbance- and fluorescence emission-based spectroscopic characteristics may be variously correlated with changes in genotoxicity in ozonated effluents, however, no associations were obtained in chlorinated wastewater effluents. The bacterial response to the developed mechanism-specific toxicity differs among wastewater effluents, reflecting variability in effluent compositions. We also used 574 linear associations collected from 70 experimental research papers published over the period of 1997-2019 to compare the strengths of the correlations of Abs, Fluor, and OC with DBPs concentrations. The correlations were expressed as approximately normally distributed Z-scores using Fisher variance-stabilizing transformation. The effects of specific prediction method, chlorination agent, water source, and DBPs type, with consideration of possible effects due to the presence of bromide, are examined against Z-scores by ANOVA, testing main effects and some variables interactions. The performed analysis is a first attempt to expose differences and patterns in correlation strengths associated with DBPs formation, based on systematically covered broad existing literature. Abs and OC concentration of water samples tend to demonstrate the strongest correlations with DBPs formation as compared with specific UV absorbance (SUVA) or intensity of fluorescence emission. Correlations of DBPs formation during chloramination demonstrated weaker strengths as compared with other chlorination agents, suggesting more caution in predicting DBPs concentrations, based on simple descriptors such as Abs, OC, and Fluor. In a series of different water types, the correlations with DBPs formation are expected to be enhanced, when wastewater is chlorinated. Non-fluorescent matter may be an important contributor to DBPs formation during water chlorination. When fluorescence intensity is considered as a predicting tool, choosing humic-like rather than proteinaceous fluorescence may enhance the strengths of the correlations with DBPs formation. Different performances of Abs, OC and Fluor in correlating with DBPs formation may be beneficial for their concurrent use helping to optimize removal of different DBPs precursors.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Fuzawa, M., Araud, E., Li, J., Shisler, J.L. and Nguyen, T.H. 2019. Free chlorine disinfection mechanisms of rotaviruses and human norovirus surrogate tulane virus attached to fresh produce surfaces. Environmental Science & Technology 53(20), 11999-12006, doi: 10.1021/acs.est.9b03461.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Bhuvaneshwari, M., Eltzov, E., Veltman, B., Shapiro, O., Sadhasivam, G. and Borisover, M. 2019. Toxicity of chlorinated and ozonated wastewater effluents probed by genetically modified bioluminescent bacteria and cyanobacteria Spirulina sp. Water Research 164, 114910. doi: 10.1016/j.watres.2019.114910.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Fuzawa, M., R.L. Smith, K.-M. Ku, J.L. Shisler, H. Feng, J.A. Juvik and T.H. Nguyen. 2020. Roles of vegetable surface properties and sanitizer type on annual disease burden of rotavirus illness by consumption of rotavirus-contaminated fresh vegetables: A quantitative microbial risk assessment. Risk Analysis, 2020, doi:10.1111/risa.13426.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Manivannan, B. and Borisover, M. 2020. Strengths of correlations with formation of chlorination disinfection byproducts: Effects of predictor type and other factors. Environmental Science and Pollution Research. doi:10.1007/s11356-019-06976-0.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: M. Bhuvaneshwari and Mikhail Borisover. 2019. Disinfected (chlorinated) water: What affects the correlations between the formation of by-products and organic matter descriptors? 13th Dahlia Greidinger International Symposium 2019, 5th March, Technion Israel Institute of Technology, Israel, Poster presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: M. Bhuvaneshwari and Mikhail Borisover. 2019. Predicting formation of chlorinated disinfection byproducts with aquatic organic matter descriptors: The influencing factor. The 12th student's conference for environmental, Earth, and planetary science, EPScon 2019, 14th March, Weizmann Institute, Israel, Poster presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: M. Bhuvaneshwari and Mikhail Borisover. 2019. Aquatic organic matter descriptors correlated with the formation of chlorination disinfection byproducts: What works better and which factors are important? EGU 2019 General Assembly, 8th April, Vienna, Austria, Poster presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: M. Bhuvaneshwari, Evgeni, E., Boris, V., Orr, S. and Mikhail Borisover. 2019. Differential toxicity of chlorinated wastewater effluents on genetically modified bacteria and naturally occurring cyanobacteria. 17th International Conference on Chemistry and Environment (ICCE), 16-20th June 2019, Thessaloniki, Greece, Oral presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: M. Bhuvaneshwari and Mikhail Borisover. 2019. Formation of chlorinated disinfection byproducts (DBPs) in water: The strengths of correlations between DBPs and organic matter-based predictors. 17th International Conference on Chemistry and Environment (ICCE), 16-20th June 2019, Thessaloniki, Greece, Poster presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: M. Bhuvaneshwari and Mikhail Borisover. 2019. Genetically modified bioluminescent bacteria as a toxicity monitoring tool for chlorinated and ozonated wastewater effluents. The student's conference held in Institute of Soil, Water and Environmental Sciences, Agricultural Research Organization, 6th August 2019, The Volcani Center, Israel, Oral presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: M. Bhuvaneshwari, Eltzov, E., Shapiro, O. and Mikhail Borisover. 2019. Examining the toxicity of chlorinated wastewater effluents: Genetically modified bioluminescent bacteria vs. cyanobacteria Spirulina sp. The International Conference on Environmental Indicators (ICEI), 9-12th September 2019, Technion, Israel, Oral presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: M. Bhuvaneshwari and Mikhail Borisover. 2019. Strengths of correlation between chlorinated disinfection byproducts (DBPs) formation with aquatic organic matter descriptors - the critical parameters. The International Conference on Environmental Indicators (ICEI), 9-12 September 2019, Technion, Israel, Oral presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: M. Bhuvaneshwari, Evgeni Eltzov, Boris Veltman and Mikhail Borisover. 2019. Biomonitoring the toxicity of chlorinated and ozonated wastewater effluents using genetically modified bioluminescent bacteria. The student's conference held in Institute of Soil, Water and Environmental Science, Agricultural Research Organization, 26th December 2019, The Volcani Center, Israel, Oral presentation.


Progress 03/15/18 to 03/14/19

Outputs
Target Audience:Our target audience included growers, researchers, and Extension educators. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We trained one postdoctoral fellow and a graduate student on this project. In addition, we are working closely with our colleagues in the Extension office of Cook county in Chicago to disseminate the results to the growers. How have the results been disseminated to communities of interest?We organized a workshop in March 2018 in Chicago to present the findings of the projects to a group of growers. The workshop was recorded and posted on the youtube channel of the Illinois Extension local food group (https://www.youtube.com/watch?v=QqIqcQpDb4Qand https://www.youtube.com/watch?v=m5dOZu53gOg). We also submitted an impact story to NIFA and a video on our project to the NIFA international office (https://nifa.usda.gov/international-partnership-videosUSDA/NIFAInternationalPartnerships Video webpage andhttps://www.youtube.com/channel/UCfdDfqYSXZ5daqILCmX7-VA). The findings of the review on pathogens in aquaponics were presented at CRWAD (Conference of Research Workers in Animal Diseases) in Chicago this past December. A second presentation of this material was given to the Controlled Environment Ag Chicago group on 2/12/2019, which is a network of aquaponics farmers and researchers that meets to collaborate on a regular basis. What do you plan to do during the next reporting period to accomplish the goals?We will plan to do the following: 1. Determine efficacy and mechanisms of virus inactivation in hydroponics water and in plants grown hydroponically; 2.Collaborate with a hydroponics facility in West Virginia to model and predict basil downy mildew outbreaks. Additionally, we will conduct a risk assessment for one of the aquaponics systems at Loyola University in Chicago. The risk of pathogen introduction into the system will be examined, possibly along with other factors; and 3.We will continue to work with the Controlled Environment Ag Chicago group to facilitate communication between researchers and those in the aquaponics industry.

Impacts
What was accomplished under these goals? For the second year of the project, the Illinois team focused on Objectives 2 and 5 and the Israel team focused on Objectives 3 and 4 of the project. Objective 1 (Illinois team): Out of 12 rainwater samples selected for analysis, 6 samples were collected in October 2016 and 6 samples were collected in June-July 2017.These water samples were coagulated and filtered through 1.6 µm and 0.22 µm pore size filters to collect bacteria and viruses, respectively. DNA from the collected microbes was extracted and subjected to quantitative polymerase chain reaction (qPCR) to quantify the following bacteria: Enterococcus, general E. coli, Enteropathogenic E. coli, Shiga-toxin producing E. coli, Shigella spp, S. flexneri, Campylobacter jejuni, C. lari, Salmonella, Clostridium prefringes, Pseudomonas, Legionella pneumophila, Mycobacteria spp, Listeria monocytogenes, Vibrio cholerae, Giardia lambia and Cryptosporidium parvum. The following bacteria were detected at least once: Campylobacter jejuni (cadF), Enteropathogenic E. coli (EHEC), general E. coli (ftsZ and uidA), and Shiga-toxin producing E. coli (stx2 and stx1). Microfluidic quantitate polymerase chain reaction (mfqPCR) was performed for different rainwater samples collected in different conditions. Shiga Toxin producing E. coli (STEC) gene stx1 and stx2 was detected in 19.4% and 22.2% samples, respectively, Enterococcus spp. was detected in 19.4% of the samples, ftsz gene of E. coli and udiA gene of E. coli was found in 13.9% of the samples and Campylobacter jujeni was found in 11.1% of the samples. Out of the samples analyzed, samples collected in 2017 showed higher concentration of detected bacterial pathogen. Objective 2 (Illinois team): We determined free chlorine disinfection efficacy and mechanisms for RVs (strain OSU and Wa) and HuNoV surrogate Tulane virus (TV) attached to surfaces of either greenhouse- or field-grown 'Red Russian' kale and southern giant curly mustard. We found that microbial density and disinfection efficacies did not differ between cultivars (kale and mustard) and growth locations (greenhouse and field). Free chlorine disinfection efficacies for RV strain OSU and Wa were higher when they were attached to mustard than to kale, although similar disinfection efficacies were observed for TV attached to mustard and kale. Linear correlation between the log10 reduction in infectivity and the percentage reduction in genome damage after free chlorine disinfection was observed for TV and RV strain OSU, suggesting that viral inactivation was partially caused by genome damage. We also developed a binding assay to determine whether free chlorine compromises the virus ability to bind to the host cell receptors and causes permeability of the viral capsids. Based on the binding assay results, we found that exposure to chlorine at 1.7 mg/L over 1 minute caused the binding protein VP4 of RV strain OSU to lose its ability to bind to the host cell receptors but did not cause viral capsid permeability. TV lost binding ability and became permeable after being exposed to free chlorine at 29 mg/L over 1 minute. These findings suggest that enteric viruses have different susceptibility to free chlorine treatments and produce surface properties are important to consider in food sanitation strategy to reduce foodborne viral infection risks. Objective 3 and 4 (Israel team). Disinfection byproducts (DBPs) formed during chlorination of any water sources are becoming emerging contaminants, and a potent toxicant can cause health risks to humans. We evaluated the toxicity of whole chlorinated effluents on three genetically modified bioluminescent bacteria and one naturally isolated cyanobacteria spirulina strains. Three different wastewater effluents (W1, W2, and W3) were collected from treatment plants located in Raanana, Kibbutz Yagur and Shafdan, then chlorinated using 1 and 10 mg L-1 of sodium hypochlorite (NaOCl) at different contact times such that finally the residual chlorine was quenched using sodium thiosulfate. Development of toxicity of chlorinated effluents towards test strains was unique between them. Chlorination affects wastewater cytotoxicity and genotoxicity potentials determined in comparison to non-chlorinated effluents, such that these effects, both negative and positive, depend on chlorine concentration and disinfection time. Chlorination did not induce significant ROS-associated toxicity. In respect to chlorination time, initial increasing trend may appear in developments of both genotoxicity and cytotoxicity potentials, followed by a further significant decrease, indicating the competition between the formation of toxic DBPs and their disappearance. The differences in cytotoxicity and genotoxicity indices between three effluents are indicative of differences in effluent quality and composition which may have a greater significance than chlorine concentration and disinfection time. Toxicity of chlorinated water on cyanobacteria was comparable to that of non-chlorinated effluents, which suggests that the production of DBPs was not strong enough to inhibit the growth of algae. The bioluminescent bacteria were found to be more susceptible to changes in toxicity of wastewater than cyanobacteria, and overall, effluents W1 and W3 showed higher toxicity effects compare to effluent W2 on both the test systems. Objective 5 (both teams). We organized a workshop in Chicago in March 2018 to disseminate the results. The workshop was recorded and available through the Illinois Extension youtube channel. In Feb. 2019, we also presented at another meeting of the Controlled Environment Ag Chicago group.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Dong, S., Massalha, N., Plewa, M.J. and Nguyen, T.H. 2018. The impact of disinfection Ct values on cytotoxicity of agricultural wastewaters: Ozonation vs. chlorination. Water Research 144, 482-490.
  • Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Manivannan Bhuvaneshwari and Mikhail Borisover. 2019. Absorbance-, fluorescence- and organic carbon-based correlations with the formation of chlorination disinfection byproducts: Roles of the prediction method, water source, chlorination agent and type of byproduct. Submitted to Water Research.
  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2019 Citation: Bhuvaneshwari Manivannan and Mikhail Borisover. 2019. A poster on "Aquatic organic matter descriptors correlated with formation of chlorination disinfection byproducts: What works better and which factors are important?" is to be presented in EGU 2019 general assembly, 8th April, Vienna, Austria.
  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2019 Citation: Bhuvaneshwari Manivannan and Mikhail Borisover. 2019. A poster on "Disinfected (chlorinated) water: What affects the correlations between the formation of byproducts and organic matter descriptors?" is to be presented at the 13th Dahlia Greidinger International Symposium 2019, 5th March, Technion Israel Institute of Technology, Israel.
  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2019 Citation: Bhuvaneshwari Manivannan and Mikhail Borisover. 2019. A poster on "Predicting formation of chlorinated disinfection byproducts with aquatic organic matter descriptors: The influencing factors" is to be presented in EPScon 2019, 14th March, Weizmann Institute, Israel.


Progress 03/15/17 to 03/14/18

Outputs
Target Audience:Members of the target audience included agricultural researchers, growers (especially of small and urban farm), and consumers. We organized a workshop in Chicago on April 6th to present the project findings to growers in this area. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We trained one postdoctoral fellow and a graduate student on this project. In addition, we are working closely with our colleagues in the Extension office of Cook county in Chicago to disseminate the results. How have the results been disseminated to communities of interest?We have published one peer-reviewed paper in Environmental Science & Technology. In addition, we have organized a workshop for urban growers in Chicago on April 6. We are working on posting the recording of the workshop online. What do you plan to do during the next reporting period to accomplish the goals?We plan to do the following: 1.Determine efficacy and mechanisms of virus inactivation in hydroponics water and in plants grown hydroponically; and 2.Determine and quantify human and plant pathogens of aquaponics and hydroponics water samples.

Impacts
What was accomplished under these goals? For the first year of the project, we focused on objectives 1, 3, 4, and 5. 1) We collected six water samples from aquaponics and hydroponics farms in the Chicago area. We used 16S sequencing to screen the presence of bacterial pathogens. We found no foodborne pathogens such as E. coli and Salmonella in these water samples. We found environmental opportunistic pathogens, Pseudomonas, Legionnella, and Mycobacteria in most of the samples. The presence of these bacteria are not likely to cause harm for consumers. However, long term exposure of farm workers to these bacteria may be a concern. In year two of the project, we will further quantify and identify these bacteria. We also did not detect Pythium spp (plant pathogens) in these samples. Because of the presence of opportunistic pathogens in these water samples, we further investigated how to disinfect these waters with chlorination and ozonation with the goal with minimal formation of toxic disinfection byproducts. 2) To ensure safe treatment of the aquaponics and hydroponics water, it is vital to determine a disinfection strategy with minimal formation of toxicity. The cumulative disinfectant concentration over time (Ct concept) is important for pathogen control during reuse water disinfection. We evaluated the impact of Ct values and different ways to achieve the same Ct value by ozonation or chlorination on cytotoxicity of disinfected wastewaters from four agricultural sources. N-Acetylcysteine (NAC) thiol reactivity was used to determine cytotoxicity mechanism. Results showed that for two sources and for both ozonation and chlorination, in general higher Ct values led to higher cytotoxicity. The ozonated waters were at least 10% less toxic and as much as 22.4 times less toxic than either the non-disinfected controls or the chlorinated waters. Chlorination consistently produced higher cytotoxicity than ozonation, by at least 2.2 folds and as much as 22.4 folds. Given the same Ct values, the combination of higher average disinfection concentration and shorter contact time produced finished waters with higher toxicitythan the combination of lower average disinfection concentration and longer contact time. NAC thiol reactivity was positively and significantly correlated with cytotoxicity, and agreed with 80% of the cytotoxicity ranking orders, indicating that the mechanism of cytotoxicity at least in part stemmed from the thiol-specific reagents that acted as thiol pool quenchers. The overall results indicate that the cytotoxicity of aquaponics and hydroponics water may increase when higher Ct values are applied to inactivate recalcitrant pathogens, and to counteract the potential increase in cytotoxicity at high Ct values, for both ozonation and chlorination, lower disinfectant dose and longer contact time should be adopted. In summary, for both aquaponics and hydroponics, ozonation led to lower cytotoxicity than chlorination. Disinfection by ozone is preferred. To ensure pathogen inactivation and minimize toxicity, low concentration of ozone or chlorine over long exposure time is preferable. 3) We also explored partially treated municipal wastewateras an unconventional water source for agriculture. We investigated chronic mammalian cell cytotoxicity of wastewaters from four sources and their optical spectroscopic properties with or without chlorination or ozonation. Samples from effluents of activated sludge, nitrification tower, facultative waste stabilization pond, and maturation waste stabilization pond were either chlorinated or ozonated. The wastewater samples were analyzed for fluorescence excitation emission matrix, specific fluorescence index (SFI), and specific UV absorbance at 254 nm (SUVA). Before and after disinfection the wastewater samples were quantitatively analyzed for in vitro mammalian cell cytotoxicity. We found that the organic extracts from the ozonated samples induced lower cytotoxicity responses than those from the chlorinated or the nondisinfected samples. To develop correlations between SFI, SUVA, and cytotoxicity, we analyzed 21 independent samples. Significant linear correlations found among these samples suggest that under the tested conditions, cytotoxicity was preferentially influenced by the fluorescence and SUVA of their composite organic agents. These two spectroscopic parameters may be used as indicators for the potential cytotoxicity of nondisinfected, ozonated, or chlorinated municipal wastewaters, allowing on-line monitoring of potential to form toxic disinfection byproducts and subsequent modification of treatment.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Massalha, N., Dong, S., Plewa, M.J., Borisover, M. and Nguyen, T.H. 2018. Spectroscopic indicators for cytotoxicity of chlorinated and ozonated effluents from wastewater stabilization ponds and activated sludge. Environmental Science & Technology, 2018, 52 (5), 3167-3174, DOI: 10.1021/acs.est.7b05510.