Disinfestant Chemicals to Control Waterborne Pathogens Are Deactivated by Peat Particles in Irrigation Water
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1 Refereed Manuscript Proc. Fla. State Hort. Soc. 124: Disinfestant Chemicals to Control Waterborne Pathogens Are Deactivated by Peat Particles in Irrigation Water Jinsheng Huang*, Dustin P. Meador, Paul R. Fisher, Danilo B. Decio, and W.E. Horner University of Florida, Environmental Horticulture Department, P.O. Box 1167, Gainesville FL Additional index words. chlorine, bleach, activated peroxide, peroxyacetic acid, quaternary ammonium chloride, disinfestation, disinfection, oxidation reduction potential, sanitation, water treatment Recycling of irrigation water increases water use efficiency, but can also increase the risk of disease transmission to crops. Disinfestant chemicals applied to control pathogens may also react with unfiltered peat and other organic particles present in recycled water. The objective was to quantify the persistence of sodium hypochlorite (using Clorox Regular-Bleach), activated peroxide (using ZeroTol ), and quaternary ammonium chloride (QAC, using GreenShield ) in water to which peat-based substrates were added. Free chlorine concentration dropped rapidly (within 3 mins.) from 2 to mg L 1 following addition of.2 grams (dry weight) of a 6% peat/4% perlite (v/v) substrate to 1L of chlorinated water, and total chlorine dropped from 2 to.3 mg L 1. Concentrations of hydrogen dioxide (H 2 O 2 ) and peroxyacetic acid (PAA) in the activated peroxide solution and QAC in the Greenshield solution also decreased in the presence of a 1% peat substrate, but were less sensitive than chlorine to peat substrate. At mg L 1 H 2 O 2 and 2 mg L 1 PAA, residual concentration decreased to 217 and 3 mg L 1 at for H 2 O 2 and PAA, respectively, following addition of 1 gram (dry weight, equivalent to 1 ml L 1 ) of a 1% peat substrate to 1 L of activated peroxide solution. QAC concentration dropped from 615 to 25 mg L 1 within one day following 1 gram of the dry peat substrate in 1 L of QAC solution. Results emphasize that disinfestant efficacy decreases with increasing organic load, particularly for chlorine solutions, and the need for both filtration and real time monitoring of sanitizing chemical concentration. Water disinfestation is a treatment to reduce the risk of introducing disease via irrigation water and to control growth of bacterial, fungal, and viral organisms in the system. Irrigation water can be disinfested by chemical treatments using either oxidizing agents or other modes of action such as quaternary ammonium products. Chemical disinfestation treatments include technologies such as chlorine, hydrogen peroxide, activated peroxygen (also termed peroxyacetic acid) and copper (Cu 2+ ) ionization. Sodium hypochlorite, the active ingredient in common household bleach, is widely used to control waterborne pathogens and algae in irrigation water. Once added to water, sodium hypochlorite is converted to the hypochlorite ion (OCl ) and hypochlorous acid (HOCl), which along with dissolved chlorine gas are collectively termed free chlorine. The balance between these two chemicals is determined by the of the water, whereby hypochlorous acid predominates at solution below 7.5 and hypochlorite ions are favored at above 7.5 (Fig. 1). The concentration of hypochlorous and hypochlorite ions influence the sanitizing strength of the solution, because hypochlorous acid has a reported germicidal effectiveness about 1 times greater than hypochlorite (White, 1968). Chlorine oxidizes living tissue and organic compounds, resulting in damage to membranes, regulatory enzymatic functions and nucleic acids of microorganisms (Stewart and Olson, 1996). We thank the industry partners of the Young Plant Research Center (floriculturealliance.org) for supporting this research. The use of trade names in this publication does not imply endorsement of the products named or criticism of similar ones not mentioned. *Corresponding author: phone: (352) ; huangj@ufl.edu % Total Chlorine 1% 9% 8% 7% 6% 5% 4% 3% 2% 1% % Hypochlorous acid HOCl (strong sanitizer) HOCl OCl Fungi, bacteria, algae spores and viruses may be deactivated with adequate concentration and contact time. A 2 mg L 1 concentration of free chlorine has low risk of phytotoxicity, while providing adequate sanitizing strength of many (but not all) pathogen species and life stages (Cayanan et al., 8, 9; Hong et al., 3). Hydrogen dioxide (H 2 O 2 ) degrades in water, producing oxygen (O 2 ) molecule and water (H 2 O). Activated peroxygen products [such as ZeroTol (27% hydrogen dioxide; BioSafe Systems Hypochlorite OCl - (weak sanitizer) Fig.1. Effect of on the forms of free chlorine at 2 C. Calculated from pka value of 7.58 determined by Morris, Proc. Fla. State Hort. Soc. 124:
2 LLC, East Hartford, CT) and X3 (6.9% hydrogen dioxide, Phyton Corp., New Hope, MN )] stabilize H 2 O 2 with acetic acid to form the strong sanitizer peracetic or peroxyacetic acid (CH 3 OOOH), also called PAA. Label rates are application and product-specific, ranging from 1 to 5 mg L 1 H 2 O 2 and 1 to mg L 1 CH 3 OOOH (peroxyacetic acid) for activated peroxygen products. High rates are used for surface sanitation, and lower rates for continuous water treatment. Quaternary ammonium chloride (QAC) functions as a cationic surface active agent with antimicrobial activity due to cellular disruption of the cell membrane (Isquith et al., 1972). Since QAC compounds are positively charged, the cationic surface active agents are attracted to negatively charged materials such as bacterial proteins, including structural components of the cell and bacterial enzymes. Disorganization and denaturation of essential proteins and cellular constituents are the main mode of antibacterial activity of QAC compounds. (Fredell, 1994). Some QAC products are commercially available for greenhouse application [such as Greenshield (1% QAC, Whitmire Micro-Gen, Research Libraries, Inc., St. Louis., MO)]. The reaction of chlorine and other disinfestants in water is a dynamic chemical process, and their effectiveness is influenced by a number of factors. Proper disinfestation requires frequent monitoring of the solution and a thorough understanding of the factors involved. These factors include the of the solution, residual concentration of oxidizing agents (such as chlorine concentration), water temperature, amount of organic matter present, exposure time, the growth stage of the pathogens present, and fertilizer (N and micronutrient, such as Fe 2+ and Cu 2+ ). Hypochlorous, hypochlorite ions and other sanitizing agents such as H 2 O 2, PAA and QAC may react with peat, plant material, and micronutrient chelates carried in the water (Percival et al., ). The residual level of active ingredient concentration is reduced during sanitizing because the chemical structure of the disinfestant becomes modified. Residual concentration of sanitizing chemicals is very important for effective sanitation. However, there are limited research data on the persistence of sanitizing chemicals in recycled water system with the presence of organic matter. The objective of this study was to quantify the persistence of sodium hypochlorite (using Clorox Regular-Bleach), activated peroxide (using ZeroTol ), and quaternary ammonium chloride (QAC, using GreenShield ) in water containing peat-based substrates. Materials and Methods Three experiments were conducted to quantify the changes of residual concentration of three sanitizing chemicals (chlorine, activated peroxides, and QAC) in peat-based substrates. Tested chemicals included Clorox Regular Bleach (6.15% sodium hypochlorite), ZeroTol and GreenShield. Experiments involved titration of sanitizing chemicals with increasing amounts of peat-based substrates added to a deionized water sample. Measured variables for all experiments included ORP (oxidation reduction potential, mv),, concentration of active ingredients from the tested sanitizing chemicals [free chlorine and total chlorine, or hydrogen peroxide (H 2 O 2 ) and peracetic acid (PAA), or quaternary ammonium chloride (QAC)]. The concentration of free chorine represented the concentration of hypochlorous acid and hypochlorite in the solution. The concentration of total chlorine represented both free chlorine and other chlorine species. Free and total chlorine concentrations were measured using a Thermo Scientific Orion AQUAfast IV AQ colorimeter utilizing the Orion AQUAfast IV Free Chlorine DPD Powder Pack and Total Chlorine DPD Powder Pack. Solution was measured using a Corning 43 meter. ORP was measured using an Oakton WD double-junction ORP electrode connected to Corning 43 meter. Hydrogen peroxide and PAA were measured using a CHEMets hydrogen peroxide Kit model K-551 and Peracetic Acid Kit model K-795, respectively. QAC concentration was measured using Taylor K-965 Quaternary Ammonium Compounds and Polyquat test kit. Experiment I: Titration of a chlorine solution with peat perlite A 6% Canadian sphagnum peat: 4% perlite substrate by volume containing preplant fertilizer, lime, and wetting agent (Conrad Fafard, Inc., Agawam, MA) was dried at 15 C for 24 h, with a dry density of 116 g L 1. Solution, ORP, and free and total chlorine residual concentrations were measured in a 2 mg L 1 free chlorine solution. The dried peat perlite was then added at a rate of,.2,.4,.6,.8, 1., 2., 3., 4., 5., 6., and 7. g per liter of solution to 12 aliquots of ml of the 2 mg L 1 free chlorine solution with three replicates per aliquot (1 g of peat perlite mix contained approximately.55 g peat and.45 g perlite). Each treatment was stirred constantly for 3 min and, ORP, free and total chlorine residual concentrations were then measured. Experiment II: Titration of an activated peroxide solution with a peat substrate A 1% peat substrate by volume containing pre-plant fertilizer, limestone, and wetting agent was dried at 15 C for 24 hours, with a dry bulk density of 11.6 g L 1 (Pindstrup Mosebrug A/S Pindstrup DK-855 Ryomgaard, Denmark). Activated peroxide solution was made from ZeroTol at a rate of 1 ml L 1, with the initial measurements of H 2 O 2 and PAA residual concentrations, at and 22 mg L 1, respectively. The dried peat was then added at rate of,.2,.6, 1., 2., 3., 4., 6., 8., and 1. g per liter of solution to 15 aliquots of 2L of ZeroTol solutions with three replicates per aliquot. Each treatment was stirred constantly for 3 min initially and solutions were stored in the dark at room temperature for 7 d. Solution, ORP, H 2 O 2 and PAA residual concentrations were measured after 4 h, and at, 2, and 7. Experiment III: Titration of a quaternary ammonium chloride (QAC) solution with a peat substrate Quaternary ammonium chloride (QAC) solution was prepared by adding 4 ml GreenShield solution to 1 L of deionized water, resulting in the concentration of QAC averaged at 615 mg L 1. A peat substrate (dry density of 11.6 g L 1 ) containing preplant fertilizer, lime, and wetting agent (Pindstrup Mosebrug A/S Pindstrup DK-855 Ryomgaard, Denmark) was added to Greenshield solution at rate of, 1.7, 5, 1, 2, 3, 4, 5, and 75 ml per liter of solution to 9 aliquots of 3L of QAC solution with three replicates per aliquot.. Solutions were stored in room temperature at dark for 7 d and solution, ORP, and QAC concentrations were measured at, 2, 3 and 7. Statistical analysis Experiments in all cases were analyzed as complete randomized designs with three replicates. Data were analyzed using ANOVA with SAS PRO GLM. Means were separated using Tukey HSD test and 95% confidence interval (CI) was calculated. 29 Proc. Fla. State Hort. Soc. 124: 211.
3 Results and Discussion Experiment I: Titration of a chlorine solution with peat perlite ORP,, free and total chlorine concentration rapidly decreased as peat perlite concentration per liter of solution increased. The concentration of free chlorine was almost undetectable when.2 g of growing substrate (equivalent to 1.7 ml in volume) or more was added to a liter of chlorinated water (Fig.2A). Total chlorine also dropped quickly when.2 g of growing substrate was added and remained around.3 mg L 1. Initial solution was around 8.3 and slightly decreased with increasing organic load and leveled off at 7.. Initial ORP was around 73 mv, and its value decreased with increasing peat based organic load (Fig. 2B). The organic load from the substrate therefore rapidly consumed a 2 mg L 1 free chlorine in the solution to almost undetectable levels. A reduction in the residual free chlorine concentration corresponds to low sanitation strength, as measured by ORP, which dropped below 45 mv as the concentration of substrate per liter increased. Results emphasize the need for filtration to reduce organic load, in order to maintain ORP and free chlorine at levels required for pathogen control. Chlorine (mg L -1 ) A B Total Chlorine Free Chlorine Grams of Growing Medium per L of Solution Fig. 2. Residual free chlorine, total chlorine concentration (A),, and ORP (B) measurements as increasing amounts of growing medium (Fafard 2P) were added to the sodium hypochlorite-treated water. Contact time was 3 min. Error bars represent 95% CI. ORP Grams of Growing Medium per L of Solution ORP (mv) Experiment II: Titration of activated peroxide solution with peat substrate When ZeroTol was mixed in deionized water at a rate of 1 ml per liter, the initial concentration of H 2 O 2 and PAA was and 22 mg L 1, respectively. Residual concentration of H 2 O 2 gradually decreased as peat concentration per L of solution increased (Fig. 3A). The loss of H 2 O 2 was measured at 4 h,, and was 33%, 53%, and 64%, respectively, for the peat concentration of 1 g per liter. The H 2 O 2 was undetectable at when the amount of peat was over 6 g per liter of solution. Residual concentration of PAA in peat-zerotol solution decreased rapidly as increasing the amount of peat substrate (Fig. 3B). Over 5% of PAA was consumed after 4 h when the concentration of peat reached above 4 g per L of solution, with the maximum of consumption 73% at 1 g per L of ZeroTol solution. PAA continued to degrade over time with over 9% consumed at for peat concentration above 6 g per L of solution. PAA was undetectable at when peat concentration was over 4 g per L of solution. Solution slightly increased from initial 3.8 up to 4.6 as increasing concentration of peat (Fig. 3C). Solution did not change significantly over time, except at for treatments contained over 4 g per L of substrate. Solution ORP decreased slightly from initial 44 mv to around 38 mv as increasing the amounts of peat in ZeroTol solution (Fig. 3D). ORP values decreased significantly from 38 mv to up to mv at for treatments with more than 4 g peat in 1 L of ZeroTol solution. This corresponded with the PAA changes as indicated in Figure 3B that there were no PAA present in the solution for treatments with over 4 g peat per liter of solution. Experiment III: Titration of a QAC solution with peat substrate Residual QAC concentration decreased as the amount of peat substrate in the GreenShield solution increased (Fig. 4). At day 1, QAC dropped to 25 mg L 1 from initial 563 mg L 1 when the volume of peat was 1 g (or 1 ml) per liter, with approximately 56% sanitizing strength loss. More than 9% QAC was lost when over 4 g (or 4 ml) peat was added to 1L of GreenShield solution. QAC was almost undetectable when more than 5 g peat was added into 1L of solution. At, up to 34% QAC remained for the solution mixed with less than 2 g peat per liter of solution. ORP values in all solution were the same, with an average of 348 ±15 mv over the course of experiment. Solution did not change significantly, with the mean of 4.8 ±.3 (standard deviation). When the three sanitizing chemicals were applied at the label rate, the sensitivity of the active ingredients to peat contamination could be rated in the order of from very sensitive to less sensitive as chlorine, QAC, PAA and H 2 O 2 ( Fig. 5). In situations with high organic load, activated peroxide and QAC products may be more suitable for disinfestation than chlorine. However, the efficacy of all these disinfectants decreased as increasing organic load. To provide adequate sanitation, filtration of non-target organic matter and surface cleaning as a pretreatment are needed before the application of these sanitizing chemicals. The protocols in this study could be adjusted to evaluate the persistence of other chemical disinfectants in solution such as chlorine dioxide, ozone, copper, etc. Measurements of sanitizing ingredient concentrations are crucial to monitor the potential residual activity of sanitizing agents and biological load which influences sanitizing agent consumption or chemical demand. Oxidation reduction potential (ORP) can be used to evaluate Proc. Fla. State Hort. Soc. 124:
4 8 A H2O2 (ppm) B PAA (ppm) C D ORP (mv) Fig. 3. Hydrogen peroxide (H 2 O 2 ) and peracetic acid (PAA) concentration (A B), and ORP (C D) measured in ZeroTol solution as increasing amounts of peat substrate (Pindstrup peat, medium particle, with adjusted to 5.) were added to the solution. ZeroTol was applied at the label rate for mist at 1 ml per liter. Measurements were taken after 4 h and at, 2, and 7. Dry bulk density of the peat was 11.6 g/l. Error bars represent 95% confidence interval (95%CIs). QAC (ppm) Day 1 Day 2 Day Fig. 4. Quaternary ammonium chloride (QAC) concentration changes as increasing amounts of peat substrate (Pindstrup peat, medium particle, with adjusted to 5.) were added to the GreenShield solution. GreenShield was applied at the label rate for floor mats at 4 ml per liter (equivalent to 1 tbsp per gal). QAC was measured at, 2, 3, and 7. Dry bulk density of the peat was 11.6 g/l. Error bars represent 95% confidence interval (95% CIs). Fig. 5. Percentage of residual active ingredient concentration in the peat based substrate solutions after either 3 min for sodium hypochlorite (free chlorine), or after 1 d for activated peroxide (hydrogen peroxide and PAA) and quaternary ammonium chloride (QAC). 292 Proc. Fla. State Hort. Soc. 124: 211.
5 sanitizing strength of chlorine products and other oxidizers such as chlorine dioxide and ozone (Suslow, 4). ORP, however, could not be used for monitoring the residual activity of non-oxidizer chemicals such as QAC products. Because the organic load that accumulates during irrigation recycling would vary in commercial plant production, depending on biofilm and organic matter from surfaces and pots, the most efficient system to maintain adequate sanitizing power would require real time monitoring of active ingredient concentration in the recirculated irrigation solution. Literature Cited Cayanan, D.F., Y. Zheng, P. Zhang, T. Graham, M. Dixon, C. Chong, and J. Llewellyn. 8. Sensitivity of five container-grown nursery species to chlorine in overhead irrigation water. HortScience 43: Cayanan, D.F., P. Zhang, W. Liu, M. Dixon, and Y. Zheng. 9. Efficacy of chlorine in controlling five common plant pathogens. HortScience 44: Cayanan, D.F., M. Dixon, Y. Zheng, and J. Llewellyn. 9. Response of container-grown nursery plants to chlorine used to disinfest irrigation water. HortScience 44: Fredell, D.L Biological properties and applications of cationic surfactants. In: J. Cross and E.J. Singer (eds.). Cationic surfactants: Analytical and biological evaluation. Marcel Dekker, New York. Hong, C.X., P.A. Richardson, P. Kong, and E.A. Bush. 3. Efficacy of chlorine on multiple species of Phytophthora in recycled nursery irrigation water. Plant Dis. 87: Isquith, A.J., E.A. Abbott, and P.A. Waters Surface-bonded antimicrobial activity of an organosilicone quaternary ammonium chloride. Appl. Microbiol. 24: Morris, J.C The acid ionization constant of HOCl from 5 to 35 ºC. J. Phys. Chem. 7: Percival, S.L., J.T. Walker, and P.R. Hunter.. Microbiological aspects of biofilms and drinking water. CRC Press. p White, G.C Chlorination and dechlorination: A scientific and practical approach. J. Amer. Water Works Assoc. 6:54. Stewart, M.H. and B.H. Olson Bacterial resistance to potable water disinfectants. Modeling disease transmission and its prevention by disinfection. Cambridge University Press, Cambridge, UK. p Suslow, T.V. 4. Oxidation-reduction potential (ORP) for water disinfection monitoring, control, and documentation. ANR Catalog Publ. No Univ. of California, Davis. Proc. Fla. State Hort. Soc. 124:
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