© 2011 libby rohrer rens · libby rohrer rens december 2011 chair: danielle d treadwell major:...
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CHLORINE DIOXIDE AS A SANITIZING AGENT IN RECIRCULATING IRRIGATION FOR GREENHOUSE HYDROPONIC BELL PEPPERS
By
LIBBY ROHRER RENS
A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2011
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© 2011 Libby Rohrer Rens
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To my family
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ACKNOWLEDGMENTS
I would like to thank Danielle Treadwell for her guidance and support throughout
the process of my master degree. Additionally I would like to thank the other members
of my graduate committee, Daniel Cantliffe and Jerry Bartz, for their expertise. I want to
thank Michael Alligood for his assistance with the implementation of the field
components of my research. I would finally like to thank the Horticultural Sciences
Department at the University of Florida for the funding of my graduate degree.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 7
LIST OF FIGURES .......................................................................................................... 8
ABSTRACT ..................................................................................................................... 9
CHAPTER
1 INTRODUCTION .................................................................................................... 11
Closed-loop Production Systems for Greenhouse Vegetables ............................... 11 Sanitizing the Nutrient Solution ............................................................................... 12
Properties of Chlorine Dioxide ................................................................................ 14 In Summary ............................................................................................................ 16
2 CHLORINE DIOXIDE AS AN IRRIGATION SANITIZING AGENT REDUCES HYDROPONIC BELL PEPPER GROWTH ............................................................. 17
Materials and Methods............................................................................................ 17
Objectives ......................................................................................................... 17 Experimental Design ........................................................................................ 17
Transplant Production ...................................................................................... 18 Irrigation System .............................................................................................. 18 Fertilization ....................................................................................................... 19
Chlorine Dioxide Preparation. ........................................................................... 19 Data Collection ................................................................................................. 19
Statistical Analysis ............................................................................................ 20 Results and Discussion........................................................................................... 21
Fall 2009 ........................................................................................................... 22
Spring 2010 ...................................................................................................... 23
3 RESIDUAL CHLORINE DIOXIDE CONCENTRATION CHANGES OVER TIME IN RECIRCULATING HYDROPONIC IRRIGATION SOLUTIONS ......................... 35
Materials and Methods............................................................................................ 35
Objectives ......................................................................................................... 35 Experimental Design ........................................................................................ 35 Water Sources and Sampling ........................................................................... 35 Experimental Procedure ................................................................................... 36 Statistical Analysis ............................................................................................ 37
Results and Discussion........................................................................................... 37 Residual Chlorine Dioxide ................................................................................ 37
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Chlorine Dioxide Demand................................................................................. 39
4 HYDROPONIC BELL PEPPER GROWTH REDUCES DUE TO METHODS OF CHLORINE DIOXIDE IRRIGATION APPLICATION ............................................... 46
Materials and Methods............................................................................................ 46 Objectives ......................................................................................................... 46 Experimental Design ........................................................................................ 46 Transplant Production ...................................................................................... 46 Irrigation System .............................................................................................. 47
Chlorine Dioxide Production ............................................................................. 47 Chlorine Dioxide Application ............................................................................. 48 Data Collection ................................................................................................. 48 Statistical Analysis ............................................................................................ 49
Results and Discussion........................................................................................... 49 Residual Chlorine Dioxide Content ................................................................... 49
Bell Pepper Growth .......................................................................................... 50
5 CONCLUSIONS ..................................................................................................... 56
APPENDIX ADDITIONAL FIGURES ............................................................................. 58
LIST OF REFERENCES ............................................................................................... 64
BIOGRAPHICAL SKETCH ............................................................................................ 70
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LIST OF TABLES
Table page 2-1 Concentration of fertilizers in the nutrient solution used to produce
greenhouse bell peppers in Fall 2009 and Spring 2010 in Citra, FL. .................. 25
2-2 Fall 2009 comparison of greenhouse bell pepper growth in perlite and pine bark media in Citra, FL. ...................................................................................... 26
2-3 Pepper plant growth response to ClO2 concentration in perlite and pine bark media in Citra, FL. .............................................................................................. 27
3-1 Concentration of fertilizers in the nutrient solution used to produce greenhouse bell peppers Spring 2011 in Citra, FL. ............................................ 41
3-2 Residual chlorine dioxide. ................................................................................... 42
3-3 Chlorine dioxide demand. ................................................................................... 43
4-1 Concentration of fertilizers in the nutrient solution used to produce greenhouse bell peppers in Spring 2011 in Citra, FL. ......................................... 53
4-2 Pepper plant growth responses to two application strategies of ClO2 and two soilless medias in Citra, FL. ................................................................................ 54
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LIST OF FIGURES
Figure page 2-1 Design of greenhouse bell pepper production system in Citra, FL showing a
single plot composed of ten plants. .................................................................... 28
2-2 Fall 2009 bell pepper whole-plant dry weight in response to increasing ClO2 concentration in Citra, FL. .................................................................................. 29
2-3 Fall 2009 bell pepper plant height in response to increasing ClO2 concentration in Citra, FL. .................................................................................. 30
2-4 Fall 2009 bell pepper leaf area in response to increasing ClO2 concentration in Citra, FL. ......................................................................................................... 31
2-5 Spring 2010 bell pepper whole-plant dry weight in response to increasing ClO2 concentration in Citra, FL. .......................................................................... 32
2-6 Spring 2010 bell pepper plant height in response to increasing ClO2 concentration in Citra, FL. .................................................................................. 33
2-7 Spring 2010 bell pepper leaf area in response to increasing ClO2 concentration in Citra, FL. .................................................................................. 34
3-1 Chlorine dioxide residual after10 mg L-1 treatment. ............................................ 44
3-2 Chlorine dioxide residual after 20 mg L-1 treatment. ........................................... 45
4-1 Residual chlorine dioxide in the nutrient solution used to produce greenhouse bell peppers in Citra, FL. .................................................................................... 55
A-1 Fall 2009 root systems of pepper plants grown in perlite media irrigated with 0 to 40 mg L-1 chlorine dioxide. ........................................................................... 58
A-2 Fall 2009 root systems of pepper plants grown in pine bark media irrigated with 0 to 40 mg L-1 chlorine dioxide. ................................................................... 59
A-3 Spring 2010 root systems of pepper plants grown in perlite media irrigated with 0 to 10 mg L-1 chlorine dioxide. ................................................................... 60
A-4 Spring 2010 root systems of bell pepper plants grown in pine bark media irrigated with 0 to 10 mg L-1 chlorine dioxide. ..................................................... 61
A-5 Spring 2010 bell pepper plants. .......................................................................... 62
A-6 Water samples used in chlorine dioxide demand experiments. .......................... 63
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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science
CHLORINE DIOXIDE AS A SANITIZING AGENT IN RECIRCULATING IRRIGATION
FOR GREENHOUSE HYDROPONIC BELL PEPPERS
By
Libby Rohrer Rens
December 2011
Chair: Danielle D Treadwell Major: Horticultural Sciences
Sanitation of greenhouse irrigation systems with chlorine dioxide was investigated
for its use in hydroponic bell pepper (Capsicum annum, L. ‘Legionnaire’) production.
The goal of this project was to evaluate the plant response to chlorine dioxide
concentrations recommended for pathogen control in applied hydroponic systems and
was broken down into three objectives. The first objective was to determine the
response of bell pepper growth when exposed to a range of concentrations of chlorine
dioxide within the nutrient solution. The second objective was to determine the ClO2
demand of irrigation solutions used in recirculating hydroponic systems. The final
objective was to determine the impact of ClO2 application strategy and potting media on
greenhouse bell pepper growth. Together, these objectives help to optimize a
recommendation for chlorine dioxide application in commercial greenhouse systems.
In the first greenhouse experiments, plant growth, including plant height, fresh
weight and dry weight, decreased quadratically in response to increasing concentrations
of chlorine dioxide up to 40 mg L-1. Plants grown in pine bark media were less impacted
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by chlorine dioxide than plants grown in perlite, likely due to the greater organic matter
content in the pine bark media leading to reduction of chlorine dioxide before coming
into contact with plant roots.
The chlorine dioxide demands of hydroponic nutrient solution, nutrient solution
leachate from pine bark media, and nutrient solution leachate from perlite media were
determined over a period of four hours in lab experiments. Chlorine dioxide demand
was dependant on both water source and initial application concentration over time. All
hydroponic solutions had a greater chlorine dioxide demand than deionized and well
water treatments, with pine bark leachate having the greatest demand. These results
indicate that higher concentrations of chlorine dioxide are needed to meet the demand
of irrigation water, and initial treatment doses should be tested at a range of
concentrations to determine the minimum treatment that will create an optimal
sanitizing residual.
In the final greenhouse experiment, bell pepper plants grown in pine bark media
were not impacted by 20 mg L-1 ClO2 application, whereas plants grown in perlite had a
significant growth reduction compared to the 0 mg L-1 control. Chlorine dioxide
application as a single-dose versus slow-release treatment was not as important as
media on plant growth.
Overall, pepper plants grown in pine bark media were less sensitive to chlorine
dioxide treatments as compared to perlite media, and this shows potential for use in
combination with concentrations of up to 20 mg L-1 chlorine dioxide.
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CHAPTER 1 INTRODUCTION
Closed-loop Production Systems for Greenhouse Vegetables
Agricultural practices consume 128 billion gallons of water per day, accounting
for one third of the total freshwater withdrawn annually throughout the US (Kenny et al.
2009). The Federal Clean Water Act (FCWA), defined water quality load allocations to
reduce pollution of surface and ground waters. In 2005, Florida’s Department of
Agriculture and Consumer Services (FDACS) initiated a Best Management Practices
(BMPs) program for farmers and ranchers that includes a suite of recommended
practices designed to reduce risk to water quality and increase water efficiency (FDACS
2005;2006). Many of these practices are easily applied in greenhouse vegetable
production systems.
The United States produces 1,636 acres of greenhouse vegetables, with 622
acres in California, followed by Pennsylvania (68), New York (59), and Florida (47)
(USDA-NASS, 2009). Greenhouse vegetable production has added advantages over
field production including controlled atmosphere (carbon dioxide, humidity, temperature,
and light), exclusion of pests and inclusion of beneficial insects, controlled fertilization
and irrigation schedules, and higher planting densities which leads to its increase in
yield compared to field grown vegetables. While irrigation volume per acre can be
increased in greenhouse systems, the yield is often 3 to 10 times greater than field
production (Cantliffe and Vansickle 2009.; Cook et al. 2005; Jovicich et al. 2007;
Rouphael et al. 2004) meaning water use efficiency of greenhouse vegetable production
is higher than in field production. In previous studies, water use efficiency (grams of
water per kilogram of fruit) in the greenhouse was greater than in the field by 33% in
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cucumbers (Jovicich et al. 2007), and 54% in zucchini (Rouphael et al. 2005). To further
maximize greenhouse water use efficiency, irrigation can be conserved by utilizing a
closed-loop irrigation system where excess irrigation is collected and reapplied to the
crop. Recirculating irrigation solution has the immediate production benefits of reducing
greenhouse inputs of water up to 30% and fertilizer up to 50% (Ruijs and Van Os 1991;
Ruijs 1993; Van Os 1999; Van Os et al. 1991) thereby decreasing demand for fresh
water and reducing risk to water quality. In the Netherlands greenhouse crops are
required to be produced in closed loop systems (Van Os, 1999). As the salt content of
the nutrient solution (measured as electrical conductivity (EC) in mmol dm-3) increases
after leaching from the plants it must be dispensed out of the system once it reaches a
toxic crop-dependant threshold (Van Os, 1999; Shannon, 1998).
Sanitizing the Nutrient Solution
Recycled hydroponic nutrient solution can act as a primary and secondary source
of pathogens. A wide variety of fungal, bacterial, and viral waterborne pathogens have
been reported in recirculating hydroponic systems (Amsing 1995; Atmatjidou et al.
1991; Berkelmann et al. 1995; Buttner et al. 1995; Hong and Moorman 2005; Jenkins Jr
and Averre 1983; Menzies et al. 1996; Stanghellini and Rasmussen 1994; Stewart-
Wade 2011; Werres et al. 2007). The risk of pathogen transmission is reduced when the
nutrient solution is sanitized before recirculation. The most common methods of
sanitizing irrigation water include mechanical filtration; heat and UV treatments; and the
additions of oxidants such as chlorine, chlorine dioxide, and ozone (Ehret et al. 2001;
US-EPA 1999; Van Os 1999). Each method has its own set of benefits and drawbacks
for use in hydroponics. Slow-sand filtration is highly effective at removing fungi and
bacteria from water, however it may be insufficient against some organisms such as
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viruses and nematodes (Van Os et al. 1999). Filtration is commonly a pre-treatment
used in conjunction with another method of sanitizing. Heating, or pasteurizing, the
nutrient solution to 95°C for 30 seconds is the most frequently used method in the
Netherlands and has been well-documented to effectively remove pathogens from the
irrigation solution when combined with filtration (Beardsell et al. 2010; Ehret et al. 2001;
Van Os 1999). The use of Ultraviolet (UV) lamps is another common method and has
been proven to be effective for sanitizing recirculating systems (Buyanovsky et al. 1981;
Ehret et al. 2001; Mebalds et al. 1996; Stanghelini et al. 1984). An important advantage
to the use of non-chemical means of water sanitation is the lack of harmful disinfection
byproducts (DBPs) that are created by many chemical disinfectants. However, in many
cases a greenhouse grower aims to treat components within the hydroponic system
such as holding tanks, irrigation lines, or emitters which harbor algae, biofilm, and
pathogen propagules (Konjoian, 2011) which can be accomplished by using a residual
chemical sanitizer.
A benefit to using a chemical sanitizer is that in most cases a residual can be
maintained in the nutrient solution, providing longer term control and disinfection of
irrigation components throughout the hydroponic system (Beardsell et al. 2010; Gagnon
et al. 2005; Ehret et al. 2001; Van Os 1999). Ozone is a strong oxidizing agent used to
successfully sanitize irrigation without the formation of toxic DBPs unless in the
presence of bromine. While ozone has a high oxidation potential and quick reaction
time, it is unstable and breaks down quickly in water, leaving no measurable residual
(Beardsell et al. 1996; 2010; Sorlini and Collivignarelli 2005; US-EPA 1999). Chlorine,
injected as sodium hypochlorite, is the most commonly used sanitizer used for
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hazardous disinfection byproducts in water, a limited pH range, and at some rates has a
phytotoxic effect on plants (Beardsell et al. 2010; Narkis and Kott 1992; Premuzic et al.
2007; Saha 2009; 2011; US-EPA 1999; Sorlini and Collivignarelli 2005). Chlorine
dioxide (ClO2) is an oxidant emerging as an alternative to chlorine. Compared to
chlorine, ClO2 does not form hazardous disinfection byproducts in water, is functional at
pH 4 through pH 10, and is active against chlorine-resistant pathogens. (Fisher et al.
2009; Gagnon et al. 2005; Huang et al. 1997; Narkis and Kott 1992; Sorlini and
Collivignarelli 2005; Stevens 1982; US-EPA 1999). Chlorine dioxide has only rarely
been tested in hydroponic systems for phytotoxic effects on plants (Carrillo, Puente, &
Bashan, 1996).
Properties of Chlorine Dioxide
Chlorine dioxide is currently used commercially for paper pulp bleaching,
municipal water treatment, and postharvest surface sanitizing of fruits and vegetables
(Gagnon et al. 2005; Gomez-Lopez et al. 2009; Narkis and Kott 1992; Olsen et al. 2003;
Roberts and Reymond 1994; Shin et al. 2011). It is highly explosive when compressed
and cannot be liquefied; therefore it must be produced on site, most commonly by an
acidification of sodium chlorite. Chlorine dioxide is applied as a gas or a gas in water
solution with concentrations up to 3000 mg L-1 at 25°C. It oxidizes by electron transfer
and has two and a half times the oxidative capacity of chlorine, without risk of toxic
halogenated DBP formation. Upon oxidation, 50% to 70% of ClO2 is converted to
chlorite (ClO2-), and 30% is converted to chlorate (ClO3
-) and chloride (Cl-) (Gomez-
Lopez et al. 2009; Stevens 1982; Sorlini and Collivignarelli 2005;US-EPA 1999;
Veschetti et al. 2005). Important oxidation reactions and end products include: [ClO2 +
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e- ClO2- ], [ClO2
- + OCl- = ClO3 - + Cl-], [2ClO2 + 2OH ClO2
- + ClO3- + H2O], [ClO2
-
+ 2H2O + 4e- Cl- + 4OH] (Stevens 1982; US-EPA 1999; Veschetti et al. 2005)
Chlorine dioxide has been well-documented for its effectiveness as a sanitizer on
a wide variety of plant pathogens including fungi (Beardsell et al. 1996; Chastagner and
Riley 2002; 2004; 2005; Copes et al. 2004; Mebalds et al. 1996; Roberts and Reymond
1994), fungal-like microbes (Beardsell et al. 1996; Hong and Moorman 2005; Mebalds
et al. 1996), bacteria (Gomez-Lopez et al. 2009; Hong and Moorman 2005; Yao et al.
2010), and viruses (Gomez-Lopez et al. 2009; Hong and Moorman 2005). The current
recommended residual concentration of ClO2 for eliminating pathogens in hydroponic
nutrient solution varies based on the targeted pathogen (Copes et al. 2004). Maintaining
a concentration of 3 mg L-1 for 8 to12 minutes is one practice recommended to
greenhouse growers; however some pathogens required maintaining the concentration
of ClO2 from 0.25 to 20 mg L-1 for up to 20 minutes for adequate pathogen control
(Beardsell et al. 1996; 2010; Fisher et al. 2009; James et al. 1996; Mebalds et al. 1996).
The appropriate initial dose of ClO2 required to sufficiently sanitize water is
increased by the oxidant demand of the systems water. Recirculating nutrient solution
will contain contamination from root exudates, media, and fertilizers and therefore will
have a higher organic load and ClO2 demand compared to fresh water. In different
surface water samples the concentration of ClO2 decreased by 1 to 3 mg L-1 within an
hour (Beardsell et al. 1996; DeMers and Renner 1992; James et al. 1996) . Waste water
samples may have demands near 7 mg L-1 (Narkis and Kott 1992; Veschetti et al.
2005). Limited research has been performed on the ClO2 demand of recirculated
greenhouse hydroponic nutrient solutions intended for recirculation.
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While previous studies have demonstrated the potential for ClO2 to provide
excellent control of plant pathogens, but only a few studies describe ClO2’s effects on
plant growth. Foliar applications of ClO2 up to 20 mg L-1 on herbaceous bedding plant
species and woody shrub species showed no adverse effects (Copes et al. 2003).
Daffodil bulbs had no growth effect when surface sanitized by submersion in a 10 mg L-
1 solution of ClO2 for 4 hours to control Fusarium (Chastagner and Riley 2002). One
study investigated the use of ClO2-sanitized municipal water as irrigation on radish and
lettuce seedlings and found that dilute solutions of ClO2 in the nutrient solution in the
form of one to five applications had no long term effects on plant growth, while more
concentrated solutions yielded growth reductions and leaf chlorosis, however ClO2
concentrations were unreported (Carrillo et al. 1996). A tomato, pepper and cucumber
grower in California reported utilizing a ClO2 residual of up to 0.5 mg L-1 to eliminate
biofilm from tubing in irrigation systems without observing adverse phytotoxic effects
(Konjoian, 2011). Additional studies on ClO2’s effect on plant growth are needed before
a recommendation can be made for its use as an irrigation sanitizer (Beardsell et al.
2010; Mebalds et al. 1996).
In Summary
Chlorine dioxide is well-documented to effectively eliminate many viral, bacterial,
and fungal pathogens that present issues in municipal and agricultural systems. Despite
the apparent chemical and sanitizing benefits of ClO2 there are limited studies reporting
potential phytotoxic effects of ClO2 when used as an irrigation sanitizing agent for
greenhouse plants. The few reports that are available indicate that plant damage may
occur, but the potential benefits of its use warrant further investigation.
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CHAPTER 2 CHLORINE DIOXIDE AS AN IRRIGATION SANITIZING AGENT REDUCES
HYDROPONIC BELL PEPPER GROWTH
Materials and Methods
Objectives
Chlorine dioxide is well-documented to effectively eliminate many viral, bacterial,
and fungal pathogens that present issues in municipal and agricultural systems;
however there is limited research on the effects of ClO2 as an irrigation disinfectant on
greenhouse-grown vegetables. The objectives of this research were to determine the
response of bell pepper growth when exposed to a range of concentrations of ClO2
within the nutrient solution.
Experimental Design
Two experiments were designed to evaluate a range of ClO2 concentrations on
bell pepper growth and development. Treatments for both experiments included two
types of potting media (medium grade perlite and composted pine bark) and four
concentrations of ClO2. Treatments were arranged in a randomized complete block
design and replicated three times. Each plot consisted of 10 potted pepper plants. In the
first experiment (Fall 2009), the concentrations of ClO2 tested were 0, 10, 20, and 40
mg L-1. The greatest concentration of ClO2 was selected based on previously published
journal articles and personal communication with the manufacturer. In Fall 2009 both 20
mg L-1 and 40 mg L-1 had a significant negative impact on pepper growth and
development, and therefore were replaced by lower concentrations of ClO2 in the
spring. In Spring 2010 the experiment was repeated using the same methods, except
the concentrations of ClO2 tested were 0, 2.5, 5, and 10 mg L-1 to evaluate the lower
range of ClO2 concentrations. A 10 mg L-1 concentration was maintained but this time
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as the highest concentration and treatments were compared to the same control of 0
mg L-1 as in Fall 2009.
Transplant Production
Bell pepper [Capsicum annum, L. ‘Legionnaire’ (Siegers Seed Co, Holland, MI)]
transplants were seeded in 72-cell plastic trays into MetroMix 200 potting media (Sun
Gro, Vancouver, Canada) on 16 Sept. 2009 and 28 Jan. 2010 in a fan and pad
polyethylene greenhouse on the University of Florida (UF) campus in Gainesville.
Seedlings were fertilized weekly with 20N–8.8P–16.6K (4 ml L-1, Spectrum Group, St.
Louis, MO) once the first true leaves appeared. Pepper seedlings were transplanted into
a one-half-acre, passively-ventilated, saw-tooth style greenhouse (Top Greenhouses
Ltd., Barkan, Israel) at the UF Plant Sciences Research and Education Unit in Citra, Fl
on 5 Nov. 2009 for the fall trial and 15 March 2010 for the spring trial. Transplanting
dates were typical for vegetable producers in Florida.
Irrigation System
Each plot was established with an independent irrigation system. For each plot,
nutrient solution was stored in a 100-gallon plastic reservoir, injected into drip lines by a
pony pump (Little Giant PP1-S, Oklahoma City, OK) through 0.75 inch polyethylene
pipe set to 10 psi pressure, and supplied to plants through pressure compensating
emitters (Flow: 2 L hr-1, Netafim, Tel-Aviv, Israel). Individual peppers were planted in
12.1 L Bato-bucket pots (General Hydroponics, Sebastopol, Ca) containing a 2.5 cm
irrigation reservoir on the bottom of each pot. Two emitters supplied nutrient solution to
each potted pepper. Irrigation was initially set to run 30 seconds every 45 minutes from
7:00 AM – 5:00 PM, and subsequently maintained at a frequency to produce 10% to
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20% leachate as recommended for greenhouse vegetables. Leachate from each plot
(composite of ten pots) was collected in a three-gallon reservoir.
Fertilization
Fertilizers used in the nutrient solution were a combination of 6N-5.28P-23.24K
containing trace elements, and calcium nitrate (Verti-gro Inc., Summerfield,FL) and
were supplied at the labeled rate for hydroponic vegetables in the Fall 2009 trial. The
concentrations applied in mg L-1 were 252N-63P-278K were higher than the UF-IFAS
recommended rate for greenhouse bell pepper, and therefore were reduced to
recommended rates of 131N-48P-214K for the Spring 2010 trial (Table 2-1).
Chlorine Dioxide Preparation.
Gaseous ClO2 (Z-SeriesTM Sachets, ICA TriNova, Newnan, GA) was produced on
site by combining sulfuric acid (H2SO4) impregnated in zeolite particles and sodium
chlorite (NaClO2) in a sachet with one gas permeable side. Sachets were placed in the
irrigation supply tank with the gas-permeable side down, allowing gaseous ClO2 to
dissolve into the nutrient solution. Sachets were designed for the diffusion of ClO2 gas
into the water over a five to seven day period. Sachets were applied eleven days after
transplanting on 16 Nov., 2009 and 26 March, 2010.
Data Collection
The experiment was terminated before fruit set, six weeks after transplanting on
15 Dec 2009, and 22 April, 2010. Bell pepper growth was evaluated using
measurements of plant height, fresh weight, dry weight and leaf area four weeks after
ClO2 application. Plant height (cm) was measured from the cotyledons to the tip of the
highest meristem and was recorded and averaged on the center eight plants per plot.
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Destructive samples were collected and averaged on the center five plants per plot and
included root and shoot fresh and dry weights as well as leaf area.
All growth parameters are reported as an average per plant. The bell pepper
plants destructively sampled for weight were cut at the media level and shoots were
weighed immediately. Leaves, with petioles removed, were then collected and scanned
for whole-plant leaf area (cm2) (LI-3100 LI-COR, Lincoln, NE). Roots were placed in
plastic storage bags at 13°C and washed with water within 24 hours until free of media.
Fine roots were captured using a series of mesh screens and combined with the intact
root systems to obtain fresh and dry weights. Fresh tissues were dried in a forced air
oven at 60°C until a constant weight.
Statistical Analysis
Proc GLM was used to determine main and interaction effects (SAS v9.2, Cary,
NC). Due to significant interactions between data parameters and year, data were
analyzed and are presented by year. Regression analyses were conducted for leaf
area, dry mass, and plant height in response to increasing ClO2 concentration, and
those relationships were best described by quadratic curves. Proc GLM was used to
determine the impact that media had on leaf area, dry mass, and plant height in
instances where there were no ClO2 concentration-media interaction. Averages were
computed by media using the least square means (LSmeans) option and compared
among media types using Fisher’s Protected Least Significant Difference Test (LSD)
and treatment concentrations were compared using Tukey’s Honestly Significantly
Difference (HSD) test.
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Results and Discussion
Commercial greenhouse bell pepper production in Florida commonly includes two
annual growing seasons in fall and spring. In the spring season, peppers are
transplanted into the greenhouse in February, and fruit are harvested in May to July
before rising greenhouse temperatures damage the fruit. In the fall, peppers are
transplanted into the greenhouse in August and fruit are harvested in January. In the
fall, pepper plants develop as the seasonal weather cools and days shorten, slowing
plant growth and producing smaller yields as compared to the spring season (Prieto et
al. 2007). This difference in plant growth was observed in this study between Fall 2009
and Spring 2010 seasons but was not considered to interfere with observations in
response to treatments. The objective of this study was to evaluate pepper plant growth
response to ClO2 concentrations over the short term to quickly identify problems
associated with excessive concentrations of the sanitizer, therefore yield was not
evaluated.
In both seasons, plants grown in pine bark were inherently larger and were less
impacted by ClO2 than plants grown in perlite (Table 2-3). Chlorine dioxide reacts with
organic compounds in pine bark media such as amines, aldehydes, and phenols and is
chemically reduced minimally active byproducts before reaching the plant roots
(Gagnon et al. 2005; Stevens, 1982; US-EPA, 1999). Perlite is created from heated and
expanded obsidian, and as an inorganic media it does not contain chemicals that react
with ClO2, allowing it to directly contact plant roots. This inherent difference between the
growing media is likely to be responsible for the increased impact from ClO2 observed
on plants growing in perlite media.
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Fall 2009
In Fall 2009 there were significant main effects from media and ClO2 concentration
on all plant growth parameters (Table 2-2). Quadratic trend lines were best fit to
describe the influence that increasing ClO2 concentrations have on plant dry weight,
plant height, and leaf area. Pepper plant growth was greater among plants grown in
pine bark than in perlite, hence the intercept, which is at 0 mg L-1 ClO2, was significantly
higher than that for perlite. Growth of pepper plants in both media types quickly reduced
in response to increasing ClO2 concentrations. While the magnitude of growth reduction
in response to increasing ClO2 concentrations was the same for plants in perlite and
pine bark, the difference in y-intercepts indicates that the maximum percent growth
reduction was lower for bell pepper plants grown in pine bark.
Plant dry weight decreased following a quadratic trend and was predicted to reach
4.9 g with treatment of 31.4 mg L-1 ClO2 (Fig. 2-2). At this concentration the percent
decrease in whole plant dry weight for plants grown in pine bark was 67% while the
decrease for plants grown in perlite was 96% compared to a control of no ClO2.
A quadratic line was fit to describe the response of plant height to increasing
concentrations of ClO2 (Fig 2-3). The decrease in plant height was 8.1 after treatment
with 40 mg L-1 ClO2. At this concentration the percent decrease in plant height for plants
grown in pine bark was 37% while the decrease for plants grown in perlite was 43% at
40 mg L-1 of ClO2 compared to a control of 0 mg L-1 ClO2.
The curves describing leaf area in response to increasing ClO2 concentration
followed a quadratic trend until they reduced by 773.2 cm2 at a concentration of 29.2
mg L-1 ClO2 (Fig 2-4). For plants grown in perlite, the estimated leaf area at this
concentration was a negative value. Therefore plants grown in perlite were considered
23
to have a minimum leaf area of 0 cm2. The maximum percent decrease in leaf area for
plants grown in pine bark was 67% while the decrease for plants grown in perlite was
100% at 29.2 mg L-1 ClO2 compared to a control of no ClO2.
Spring 2010
In 2010 there was a significant interaction between ClO2 concentration and media
in addition to their main effects for all plant growth parameters and were best fit to
quadratic trend lines. Plant growth as measured by several parameters was greater
among plants grown in pine bark than in perlite. Hence the intercept, which is at 0 mg L-
1 ClO2, was significantly higher than that for perlite. Growth of pepper plants in both
medias quickly reduced in response to increasing ClO2 concentration, however the
plants grown in pine bark did not respond as negatively as plants grown in perlite.
The response of dry weight to increasing ClO2 concentration was fit to a quadratic
line (Fig 2-5). The curves reach minimums at 9.0 mg L-1 and 7.4 mg L-1 for perlite and
pine bark; respectively which corresponds to a dry weight decrease of 15.2 g (75%) for
perlite and 10.1 g (36%) for pine bark compared to a control of no ClO2.
The response of plant height to increasing ClO2 concentration was best fit to a
quadratic line for each media tested (Fig 2.6). The minimum height response was
observed at 10.0 mg L-1 and 7.9 mg L-1 for perlite and pine bark; respectively which
corresponds to a plant height decrease of 9.7 cm (35%) for perlite and 6.0 cm (19%) for
pine bark compared to a control of 0 mg L-1.
The curves describing plant leaf area in response to increasing ClO2 concentration
follow a quadratic trend (Fig. 2.7) until they reach minimums at a ClO2 concentration of
8.2mg L-1 and 6.3 mg L-1 for perlite and pine bark; respectively which corresponds to a
leaf area decrease of 1517 cm2 (84%) for perlite and 899 cm2 (32%) for pine bark.
24
This study is the first of its kind to investigate the phytotoxic response of plants to
ClO2 applied as an irrigation sanitizer. By design of the ClO2 sachets used for
treatments, ClO2 up to 40 mg L-1 was gradually dosed into the nutrient solution reservoir
and used for all irrigation events throughout the experiment. This study highlights that
the use of high concentrations of ClO2 in the nutrient solution has adverse effects on
plant growth and are exacerbated in growing media with lower ClO2 demand. The plant
response is reduced when lower concentrations are used and when an organic media
type is used. The use of fewer applications and/or reduced concentrations of ClO2, as
well as the economic benefit of plant protection are worthy of future investigation.
25
Table 2-1. Concentration of fertilizers in the nutrient solution used to produce
greenhouse bell peppers in Fall 2009 and Spring 2010 in Citra, FL.
* EDTA: Ethylenediaminetetraacetic acid
Nutrient
Potassium nitrate KNO3 K 177 134
N 63 48
Magnesium sulfate MgSO4 Mg 36 28
S 48 38
Ammonium phosphate (NH4)3PO4 N 9 7
P 7 5
Potassium phosphate KH2PO4, K2HPO4, K 101 79
K3PO4 P 56 41
Sodium borate Na2B4O7 B 0.36 0.86
Copper EDTA* Cu 0.36 0.26
Iron EDTA* Fe 2.40 1.80
Manganese EDTA* Mn 0.36 0.26
Zinc EDTA* Zn 0.36 0.26
Sodium molybdate Na2MoO4 Mo 0.06 0.05
Calcium nitrate Ca(NO3)2 Ca 254 109
N 180 77
Manganese sulfate MnSO4 Mn -- 0.7
S -- 0.42
2010
mg*L-1
Chemical name Molecular Formula
2009
mg*L-1
26
Table 2-2. Fall 2009 comparison of greenhouse bell pepper growth in perlite and pine bark media in Citra, FL.
X Fisher’s LSD at α = 0.05
Leaf Area
(cm2)
Fresh Shoot
Weight (g)
Fresh Root
Weight (g)
Total
Fresh
Weight (g)
Dry Shoot
Weight (g)
Dry Root
Weight (g)
Total Dry
Weight (g)
Height
(cm)
Perlite 253.02 15.1 6.4 21.4 1.80 0.54 2.34 13.7
PineBark 644.61 34.5 15.7 49.8 3.48 0.86 4.34 17.8
LSDx
135.37 5.76 1.83 7.12 0.61 0.13 0.73 1.39
p-value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
27
Table 2-3. Pepper plant growth response to ClO2 concentration in perlite and pine bark media in Citra, FL.
0 7.5 a 21.0 a 1213.3 a
10 4.3 b 18.7 a 631.3 b
20 3.0 b 17.2 ab 394.3 b
40 2.6 b 14.4 b 339.6 b
p-value
0 27.8 a 30.5 a 2793.5 a
2.5 21.7 b 27.8 ab 2262.5 b
5 19.9 b 27.3 ab 2210.4 b
10 18.7 b 24.9 b 1995.8 b
p-value
0 5.2 a 18.9 a 618.6 a
10 2.1 b 14.9 ab 203.2 b
20 1.5 b 11.2 bc 133.6 b
40 0.6 b 9.9 c 56.8 b
p-value
0 20.8 a 28.3 a 1868.4 a
2.5 11.8 b 22.7 b 915.6 b
5 8.0 bc 19.3 bc 553.1 c
10 5.2 c 18.4 c 372.7 c
p-value
-- Perlite Media --
0.0006
0.00180.00450.0054
0.0029
<0.00010.0002<0.0001
0.00050.00140.0023
0.0039
Dry
Weight (g)
Plant Height
(cm)
Leaf Area
(cm2)
ClO2
mg L-1
-- Pine Bark Media--
28
Figure 2-1. Design of greenhouse bell pepper production system in Citra, FL showing a single plot composed of ten plants.
29
Figure 2-2. Fall 2009 bell pepper whole-plant dry weight in response to increasing ClO2 concentration in Citra, FL. Main effects of media (p<0.0001) and mg L-1 (p<0.0001) were significant, however there was no significant interaction between the two (p=0.6950). y=dry mass and x=chlorine dioxide concentration. (R2=0.90).
0
1
2
3
4
5
6
7
8
9
0 10 20 30 40
Dry
We
igh
t (g
)
mg*L-1
Pine Bark y = 0.005x² - 0.31x + 7.3
Perlite y = 0.005x² - 0.31x + 5.1
30
Figure 2-3.Fall 2009 bell pepper plant height in response to increasing ClO2 concentration in Citra, FL. Main effects of media (p<0.0001) and mg L-1 (p<0.0001) were significant, however there was no significant interaction between the two (p=0.1928). y=plant height in cm and x=chlorine dioxide concentration. (R2=0.88).
0.0
5.0
10.0
15.0
20.0
25.0
0 10 20 30 40
Heig
ht
(cm
)
mg*L-1
Pine Bark y = 0.0043x² - 0.37x + 21.5
Perlite y = 0.0043x² - 0.37x + 18.5
31
Figure 2-4. Fall 2009 bell pepper leaf area in response to increasing ClO2 concentration in Citra, FL. Main effects of media (p<0.0001) and mg L-1 (p<0.0001) were significant, however there was no significant interaction between the two (p=0.1648). y=leaf area and x=chlorine dioxide concentration. (R2=0.87).
-200
0
200
400
600
800
1000
1200
1400
0 10 20 30 40
Lea
f A
rea
(cm
2)
mg*L-1
Pine Bark y = 0.904x²- 52.9x + 1150.4
Perlite y = 0.904x² - 52.9x + 648.6
32
Figure 2-5. Spring 2010 bell pepper whole-plant dry weight in response to increasing ClO2 concentration in Citra, FL. Main effects of media (p<0.0001) and mg L-1 (p<0.001) were significant, as well as the interaction between the two (p=0.0125). y = dry mass and x = chlorine dioxide concentration. (R2=0.95).
0
5
10
15
20
25
30
35
0 2.5 5 7.5 10
Dry
We
igh
t (g
)
mg*L-1
Pine Bark y = 0.186x² - 2.7x + 27.9 Perlite y = 0.186x² - 3.4x + 20.1
33
Figure 2-6. Spring 2010 bell pepper plant height in response to increasing ClO2 concentration in Citra, FL. Main effects of media (p<0.0001) and mg L-1 (p<0.001) were significant, as well as the interaction between the two (p=0.0264). y=plant height in cm and x=chlorine dioxide concentration. (R2=0.91).
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
0 2.5 5 7.5 10
Heig
ht
(cm
)
mg*L-1
Pine Bark y = 0.0968x² - 1.52x + 31.1
Perlite y = 0.0968x² - 1.94x + 27.5
34
Figure 2-7. Spring 2010 bell pepper leaf area in response to increasing ClO2 concentration in Citra, FL. Main effects of media (p<0.0001) and mg L-1 (p<0.001) were significant, as well as the interaction between the two (p=0.0001). y=leaf area and x=chlorine dioxide concentration. (R2=0.98).
0
500
1000
1500
2000
2500
3000
3500
0 2.5 5 7.5 10
Lea
f A
rea
(cm
2)
mg*L-1
Pine Bark y = 22.6x² - 285.2x + 2821 Perlite y = 22.6x² - 370.6x + 1806
35
CHAPTER 3 RESIDUAL CHLORINE DIOXIDE CONCENTRATION CHANGES OVER TIME IN
RECIRCULATING HYDROPONIC IRRIGATION SOLUTIONS
Materials and Methods
Objectives
Limited research has been performed on the ClO2 demand of greenhouse
hydroponic nutrient solutions intended for recirculation. The objective of this research is
to determine the ClO2 demand of water used in recirculating irrigation systems and to
further characterize the ClO2 sequestration of two common water sources and three
hydroponic irrigation solutions.
Experimental Design
Chlorine dioxide (Z-SeriesTM Sachets, ICA Trinova, Newnan, GA) was added to
two water sources and three hydroponic irrigation solutions and subsequently sampled
for residual ClO2 concentration over a period of four hours. Treatments included two
concentrations of ClO2 (10 mg L-1 and 20 mg L-1) and five water samples (deionized
water (DI), well water only, nutrient solution in well water, well water-nutrient solution
leachate from pine bark media, and well water-nutrient solution leachate from perlite
media), each measured at five time points (0.25, 0.5, 1, 2, and 4 hours). The experiment
was repeated four times over two days with two concurrent replications per day and was
conducted in the Plant Mineral Nutrition Laboratory at the University of Florida’s (UF)
main campus in Gainesville, FL.
Water Sources and Sampling
Water samples were collected from a hydroponic bell pepper production system
located at the UF Institute of Food and Agricultural Sciences (IFAS) Plant Sciences
Research and Education Unit (PSREU) greenhouses in Citra, FL. Hydroponic nutrient
36
solution was prepared using UF-IFAS recommendations for greenhouse bell pepper
production (Table 3-1) and prepared using PSREU well water. Well water was sampled
before the experiments were performed and tested low for chloride and other potential
contaminants. Leachate samples were collected from the bell pepper production system
and prepared as follows. Two benches were established with independent irrigation
systems and each supplied nutrient solution to ten pepper plants (Capsicum annum, L.
‘Legionnaire,’ Siegers Seed Co, Holland, MI). One soilless media type (perlite or pine
bark) was used at each bench and individual plants were potted into 12.1 L plastic pots
with drainage holes (Bato-buckets, General Hydroponics, Sebastopol, CA). At each
bench, nutrient solution was stored in a 55-gallon reservoir, injected by a pony pump
(Little Giant PP1-S, Oklahoma City, OK) through 0.75 inch polyethylene pipe set to 10
psi pressure, and supplied to plants through pressure compensating emitters (2L hr-1,
Netafim, Tel-Aviv, Israel). Irrigation events occurred between 7:00 am and 5:00 pm, and
were maintained at a frequency to produce 20% to 30% leachate, as recommended for
greenhouse vegetables with recirculating irrigation. Leachate from each bench
(composite of ten pots) was collected in a 5-gallon reservoir. Nutrient solution was not
recirculated for the purpose of data collection. Eight weeks after transplanting, leachate
from perlite and pine bark plots was accumulated for one week and used in laboratory
experiments.
Experimental Procedure
Water samples were pH adjusted to 6.0 with commercially available hydroponic
pH buffers (General Hydroponics, Sebastopol, CA). Chlorine dioxide was added to 300
mL of the sampled water at a concentration of 10 mg L-1 or 20 mg L-1 dependant on the
treatment and the solutions were tested for residual ClO2 at 0.25, 0.5, 1, 2, and 4 hours
37
after the ClO2 addition. The experiment was terminated four hours after ClO2 addition as
previous studies report suppression of many pathogens occurs well within this range
(Beardsell et al. 1996; 2010; Fisher et al. 2009; James et al. 1996; Mebalds et al. 1996).
Residual ClO2 concentration was measured using a titration procedure adapted from
Mahovic et al, 2009. Samples were combined with potassium iodide in pH 7.0
phosphate buffer and titrated using sodium thiosulfate to a colorless endpoint. Two-
normal sulfuric acid was then added to achieve a pH of 2.0 which produced a color
change, and a second titration with sodium thiosulfate was performed to a colorless
endpoint. The concentration of ClO2 was calculated from this procedure.
Statistical Analysis
The data were analyzed as ClO2 residual (the concentration remaining in the water
sample), and ClO2 demand (the treatment concentration minus the residual). Proc
Glimmix was used to perform repeated measures analysis (SAS V9.2, Cary, NC).
Chlorine dioxide concentrations were compared among all treatments to determine
differences between water samples and treatment concentration over four hours.
Averages were computed using the least squared means (LSMeans) option and
compared among treatments using Tukey’s Honestly Significant Difference Test (HSD).
Results and Discussion
Residual Chlorine Dioxide
In these experiments residual ClO2 concentrations were influenced by water
source, initial application concentration of ClO2 and time in significant three-way
interactions (p<0.0001; concentration-water-hour interaction p=0.0025). The residual
concentration of ClO2 throughout the four hour duration of the experiment was
38
significantly different between 10 and 20 mg L-1 treatment concentrations for each of the
water sources and time points tested (Table 3-2).
At the 15 minute time point the ClO2 residual in the DI and well water samples
remained relatively constant, whereas the residual in the leachates and nutrient solution
decreased by up to 80%. After four hours the residual concentration was reduced from
the treatment concentration in all water types (p<0.0001). Chlorine dioxide residuals in
DI water, well water, and nutrient solution treated with both initial concentrations, and
pine bark leachate treated with 20 mg L-1, gradually decreased throughout the four
hours. Only pine bark leachate receiving 10 mg L-1 ClO2 dropped to a minimum by 15
minutes and remained at approximately 2.5 mg L-1 throughout the 4 hours. In all other
treatments the residual ClO2 concentration changed considerably throughout the
treatment time. The response of ClO2 in perlite leachate observed in this experiment
was unexpected, as the residual concentration initially decreased by 65%, increased
over the next two hours, and then decreased again by the fourth hour. The researchers
believe that this response is an interference caused by the interaction between minerals
in the perlite media and chemicals used in the ClO2 titration method rather than a
generation of ClO2 within the leachate solution.
A ClO2 concentration of 3 mg L-1 for 8-12 minutes is one practice recommended to
greenhouse growers for control of waterborne pathogens (Fisher et al. 2009; Mebalds et
al. 1996). In this study, the use of 10 mg L-1 was more than a sufficient dose to attain
the recommended concentration-time in all water samples except for the pine bark
leachate where a higher initial dose is recommended.
39
Chlorine Dioxide Demand
Chlorine dioxide demand of the water samples was influenced by water source,
initial application concentration of ClO2 and time in significant three-way interactions
(p<0.0001; mg L-1-water-hour interaction p=0.0025) (Table 3-3). The magnitude of ClO2
demand would be same for both treatment concentrations if water quality a primary
factor in the determination of ClO2 demand, however not all water samples followed this
trend. Deionized water, well water and nutrient solution had similar demands of ClO2
while neither of the leachate samples had the same magnitude of loss between
treatment concentrations at any time point.
Chlorine dioxide demand of fresh well water was similar to the demand of DI water
over the 4 hour period at both the 10 and 20 mg L-1 initial concentrations and was less
than solutions containing fertilizers, root exudates, or leachate from pine bark. In the
nutrient solution, oxidizers such as ClO2 will oxidize ferrous iron (2+) ions to the ferric
iron (3+) and manganous manganese (2+) to (4+). These oxidized ions react with water
to form insoluble precipitates (US-EPA, 1999). These reactions may partially account
for the higher demand of ClO2 in the nutrient and leachate solutions. The precipitation of
iron and manganese will also impact the fertilizer concentrations needed to grow
greenhouse vegetables, however the use of chelated forms of these fertilizers will
maintain the concentration of iron and manganese in the desired form. The nutrient
solution accumulates more organic matter after flowing through the irrigation system,
the matrix of potting media and plant roots, and being dispensed as leachate. Leachate
from pine bark media also contains organic compounds such as amines, aldehydes,
and phenols which reduce ClO2 (Gagnon et al. 2005; Stevens, 1982; US-EPA, 1999).
These organic compounds account for the higher ClO2 demand of pine bark leachate as
40
compared to the nutrient solution. Other studies have found similar demands when
treating waste water samples including organic suspended solids with ClO2 (Narkis and
Kott 1992; Veschetti et al. 2005).
Concentrations of chlorine dioxide needed to adequately sanitize recirculated
greenhouse water will need to be high enough to meet the demand of the water as well
as to provide a sufficient residual to control pathogens over time. The demand will vary
by system, water quality, fertilizers used, and amount of recycled water in the solution.
These results indicate that ClO2 demand should be examined over a range of
concentrations to determine the minimum treatment dose that will create an optimal
ClO2 residual long enough to sanitize plant pathogens in hydroponic leachate intended
for recirculation without negatively impacting plant growth. In addition, as the ClO2
treated nutrient solution passes through the hydroponic system, it is anticipated that the
ClO2 concentration will decline further when ClO2 comes into contact with biofilm and
organic matter within the system (Gagnon et al. 2005). To compensate for this, a higher
treatment concentration is required in order to maintain a sufficient ClO2 residual while
the nutrient solution continues to recirculate throughout the irrigation system.
41
Table 3-1. Concentration of fertilizers in the nutrient solution used to produce greenhouse bell peppers Spring 2011 in Citra, FL.
Ca 160
N 102
Phosphoric acid H3PO4 P 51
Potassium chloride KCl K 157
Mg 48
S 67
Copper sulfate CuSO4 Cu 0.32
Iron EDTA* Fe 3.2
Manganese sulfate MnSO4 Mn 1.03
Sodium borate Na2B4O7 B 0.749
Sodium molybdate Na2MoO4 Mo 0.07
Zinc sulfate ZnSO4 Zn 0.34
Nutrient mg*L-1
Calcium nitrate
Magnesium sulfate
Ca(NO3)2
MgSO4
Molecular
FormulaChemical name
42
Table 3-2. Residual chlorine dioxide.
xMean separation in columns by Tukey's HSD test at α =0.05 (lowercase letters). yMean separation in rows by Tukey's HSD test at α =0.05 (uppercase letters).
PPM Water Sample
10 DI water 10.0 cx
Ay
9.7 b A 8.7 c B 7.6 c C 5.8 d D
Well Water 9.3 c A 9.3 b A 8.6 c A 7.4 c B 5.8 de C
Nutrient Solution 5.7 e A 4.8 c AB 4.3 de AB 3.8 d BC 2.6 f C
Pine Bark Leachate 2.2 f A 2.6 c A 2.9 e A 2.7 d A 3.1 ef A
Perlite Leachate 2.9 f C 4.0 c BC 7.1 cd A 7.9 c A 5.7 de AB
20 DI water 19.6 a A 18.6 a B 16.9 a C 15.0 a D 11.6 ab E
Well Water 18.9 a A 16.8 a AB 17.1 a AB 15.4 a BC 12.4 ab C
Nutrient Solution 16 a A 15.9 a A 14.9 ab AB 13.5 b B 9.9 bc C
Pine Bark Leachate 9.6 c A 8.8 b AB 8.8 c B 8.5 c B 7.5 cd C
Perlite Leachate 7.5 d C 9.7 b BC 13.5 b AB 14.9 a A 12.6 a AB
4 Hrs0.25 Hr 0.5 Hr 1 Hr 2 Hrs
mg*L-1
43
Table 3-3. Chlorine dioxide demand.
xMean separation in columns by Tukey's HSD test at α =0.05.
*Nonsignificant from zero at α=0.05.
Water Sample PPM
DI water 10 0.0* ex
0.3* g 1.4 d 2.3 e 4.2 e
20 0.4* e 1.4 efg 3.2 cd 5.0 d 8.4 bc
Well Water 10 0.8* e 0.8* fg 1.4 d 2.6 e 4.2 de
20 1.1* e 3.2 def 3.0 cd 4.6 d 7.6 bc
Nutrient Solution 10 4.3 d 5.1 bcd 5.7 bc 6.2 bc 7.4 c
20 3.9 d 4.1 cde 5.1 bc 6.5 b 10.1 ab
Pine Bark Leachate 10 8.8 c 7.4 bcd 7.1 b 7.3 b 6.9 cd
20 10.4 b 11.2 a 11.3 a 11.5 a 12.5 a
Perlite Leachate 10 7.1 c 6.0 bc 2.9 cd 2.1 e 4.3 de
20 12.5 a 10.3 a 6.5 b 5.1 cd 7.4 c
(mg*L-1
)
0.25 Hr 4 Hrs2 Hrs1 Hr0.5 Hr
44
Figure 3-1. Chlorine dioxide residual after10 mg L-1 treatment. Error bars represent standard error of the mean.
45
Figure 3-2. Chlorine dioxide residual after 20 mg L-1 treatment. Error bars represent standard error of the mean.
46
CHAPTER 4 HYDROPONIC BELL PEPPER GROWTH REDUCES DUE TO METHODS OF
CHLORINE DIOXIDE IRRIGATION APPLICATION
Materials and Methods
Objectives
Despite the apparent chemical and sanitizing benefits of ClO2 there are limited
studies optimizing the application strategies of ClO2 for sanitizing greenhouse irrigation
and the effects of those strategies on plant growth. The objective of this research is to
determine the impact of ClO2 application strategy and potting media on greenhouse bell
pepper growth.
Experimental Design
Treatments included two types of media (medium grade perlite and composted
pine bark), two concentrations of ClO2 (0 and 20 mg L-1) and two methods of ClO2
application (single-dose and slow-release). Treatments were arranged in a randomized
complete block design and replicated three times.
Transplant Production
Bell pepper [Capsicum annum, L. ‘Legionnaire’ (Siegers Seed Co, Holland, MI)]
transplants were seeded in 72-cell plastic trays using MetroMix 200 potting media (Sun
Gro, Vancouver, Canada) on 7 January, 2011 and were grown in a controlled
environment chamber (Conviron, Controlled Environments Limited, Winnepeg,
Manitoba, Canada) on the University of Florida (UF) campus in Gainesville. Seedlings
were fertilized weekly with 20N–8.8P–16.6K (4 ml L-1, Spectrum Group, St. Louis, MO)
once the first true leaves appeared. Pepper seedlings were transplanted on February
47
14, 2011 at the UF Plant Sciences Research and Education Unit (PSREU) in Citra, Fl in
a one-half-acre, passively-ventilated, saw-tooth style greenhouse (Top Greenhouses
Ltd., Barkan, Israel).
Irrigation System
Each plot was established with an independent irrigation system. For each plot,
nutrient solution was stored in a 55 gallon plastic reservoir, injected into drip lines by a
pony pump (Little Giant PP1-S, Oklahoma City, OK) through 0.75 inch polyethylene
pipe set to 10 psi pressure, and supplied to plants through pressure compensating
emitters (Flow: 2L hr-1, Netafim, Tel-Aviv, Israel). Individual peppers were planted in
12.1 L Bato-bucket pots (General Hydroponics, Sebastopol, Ca) containing a 2.5 cm
irrigation reservoir on the bottom of each pot. Two emitters supplied nutrient solution to
each potted pepper. Irrigation was initially set to run 30 seconds every 45 minutes from
7:00 AM – 5:00 PM, and subsequently maintained at a frequency to produce 20% to
30% leachate as recommended for closed-loop irrigation of greenhouse vegetables.
Leachate from each plot (composite of ten pots) was collected in a three-gallon
reservoir for each plot. The nutrient solution was prepared using UF-IFAS
recommendations for greenhouse bell pepper production and prepared using PSREU
well water, detailed in Table 4-1 (Jovicich, Cantliffe, & P. J. Stoffella, 2004).
Chlorine Dioxide Production
Chlorine dioxide was produced on site using proprietary sachets designed for the
diffusion of ClO2 gas into the water over a 7-day period (Z-SeriesTM Sachets, ICA
TriNova, Newnan, GA). Gaseous ClO2 was produced by combining sulfuric acid
(H2SO4) impregnated in zeolite particles and sodium chlorite (NaClO2) into a sachet with
48
one gas permeable side. Sachets were placed into water with the gas-permeable side
down allowing gaseous ClO2 to diffuse into the water.
Chlorine Dioxide Application
Chlorine dioxide applications were made 10 weeks after transplant on 24 April,
2011. Chlorine dioxide was applied to the irrigation supply tanks using two methods,
single-dose and slow-release. For the single-dose method, a ClO2 producing sachet
was first placed in well water to produce a concentrated ClO2 stock solution. Well water
was selected because this was the source of irrigation for crops in the greenhouse.
Concentrated ClO2 solution was then added to the 200-liter irrigation supply reservoir at
a volume equivalent of 4000 mg of ClO2 or 20 mg ClO2 per liter of water. For the slow-
release method, ClO2 producing sachets were placed directly into the irrigation
reservoir, which led to a calculated production of 4000 mg ClO2 per 200 liters of water
over a period of 7 days.
Data Collection
The experiment was terminated 11 weeks after transplanting on 5 May, 2011. Bell
pepper growth was evaluated using measurements of plant height, fresh weight, dry
weight, and fruit set eleven days after ClO2 application. Plant height (cm) was measured
from the cotyledons to the tip of the highest meristem and was recorded and averaged
on the center eight plants per plot. Destructive samples were collected and averaged on
the center three plants per plot and included root and shoot fresh and dry weights as
well as fruit number and fresh weight. Residual ClO2 concentration was measured using
a titration procedure adapted from Mahovic et al, 2009. Samples were combined with
potassium iodide in pH 7.0 phosphate buffer and titrated using sodium thiosulfate to a
colorless endpoint. Two-normal sulfuric acid was then added to achieve a pH of 2.0
49
which produced a color change, and a second titration with sodium thiosulfate was
performed to a colorless endpoint. The concentration of ClO2 was calculated from this
procedure.
Each of the three bell pepper plants destructively sampled was cut at the media
level. Pepper fruits were removed and undamaged fruit number was recorded and
weighed separately from shoots. Roots were stored in plastic storage bags at 13°C and
washed within 48 hours with water until free of media. Fine roots were captured using a
series of mesh screens and combined with the intact root systems to obtain fresh and
dry weights. Shoots and roots were dried in a forced air oven at 60°C until a constant
weight.
Statistical Analysis
Proc Mixed was used to determine main and interaction effects (SAS v9.2, Cary,
NC). Interaction effects were not significant (a=0.05) therefore main effects are
reported. Averages were computed for main effects using the least squared means
(LSMeans) option and compared among treatments using Tukey’s Honestly
Significantly Different Test (HSD). Proc Glimmix was used to perform repeated
measures analysis of residual ClO2 in the nutrient solution and compared between
application methods.
Results and Discussion
Residual Chlorine Dioxide Content
Residual ClO2 concentration in the irrigation solution was different between
application methods throughout the experiment (Fig. 4-1). The concentration of ClO2 in
the slow-release treatment continued to increase over the first 3 days after treatment
onset, reaching a maximum of 14.7 mg L-1 and then gradually decreased over the next
50
7 days. Residual ClO2 in the single-dose treatment quickly decreased to 10.1 mg L-1
after one day and continued to steadily decrease through day ten. Following the first
day after ClO2 addition, the residual concentration in the slow-release treatment was
consistently greater than the residual in the single-dose treatment. There were no
significant differences in any of the plant growth parameters between application
methods despite the differences in residual ClO2 concentrations between treatments.
Under the conditions of this experiment the ClO2 remaining in the nutrient solution
was at a concentration for a duration previously found to sufficiently sanitize many plant
pathogens found in hydroponic irrigation (Beardsell et al. 1996; Chastagner and Riley
2002; 2004; 2005; Copes et al. 2004; Mebalds et al. 1996; Roberts and Reymond
1994; Hong and Moorman 2005; Gomez-Lopez et al. 2009; Yao et al. 2010). As the
sanitized solution flows through the hydroponic system it comes into contact with
biofilm, algae and pathogens harbored in irrigation lines and emitters. Maintaining a
high residual in the nutrient solution allows for disinfestation of irrigation components in
addition to the water itself (Coosemans 1995; Gagnon et al. 2005; Huang et al. 1997).
As control of a variety of pathogens has been reported at lower residual concentrations
than those used in this experiment, studies investigating the effect of lower treatment
doses and application methods of ClO2 on plants grown in inorganic media, such as
perlite, are warranted.
Bell Pepper Growth
Pepper plants from the control treatments consistently yielded healthier, more
vigorous plants in terms of fresh weight, dry weight, plant water content, and change in
plant height compared with plants in the ClO2 treatments (Table 4-2). Plants irrigated
with ClO2 had 8.6% less biomass and accumulated less than half of the height over the
51
10-day treatment interval. The ClO2 application method did not significantly impact any
plant growth parameters, although the slow release treatment was associated with
lower fresh weight, dry weight, and fruit weight. Plants grown in pine bark had a greater
fresh weight, percent water content and an increase in height compared to plants grown
in perlite. The growth of plants grown in pine bark was not impacted by either ClO2
treatment. Pepper plants grown in perlite were observed to be severely wilted with the
application of ClO2, and had lower fresh weight, percent water content, fruit weight, and
height change in response to ClO2 application.
A reduction in plant growth from the slow-release treatment was expected as it is
supported by previous research (Davies et al. 2010a; 2010b). Despite the differences in
residual ClO2 concentration in the nutrient solution, this experiment highlights that at 20
mg L-1, the application strategy was not as important as media on plant growth. Plant
growth in pine bark consistently outperformed plant growth in perlite media. The
addition of ClO2 to the nutrient solution further reduced plant growth in the perlite media,
causing severe wilt and biomass reduction; however there was no significant effect on
plants grown in pine bark.
This study demonstrated that under the conditions of this experiment the use of 20
mg L-1 ClO2 as an irrigation sanitizing agent had no impact on pepper plants grown in
pine bark. Under both ClO2 application methods the ClO2 residual was maintained at a
concentration-time well above that previously recommended for pathogen control.
Therefore, reduced concentrations for use on plants grown in inorganic media types,
such as perlite and sand, could be investigated. Furthermore, after optimization of non-
52
phytotoxic concentrations, ClO2 should be tested against pathogens in applied, in-situ
hydroponic systems.
53
Table 4-1. Concentration of fertilizers in the nutrient solution used to produce greenhouse bell peppers in Spring 2011 in Citra, FL.
Ca 160
N 102
Phosphoric acid H3PO4 P 51
Potassium chloride KCl K 157
Mg 48
S 67
Copper sulfate CuSO4 Cu 0.32
Iron EDTA* Fe 3.2
Manganese sulfate MnSO4 Mn 1.03
Sodium borate Na2B4O7 B 0.749
Sodium molybdate Na2MoO4 Mo 0.07
Zinc sulfate ZnSO4 Zn 0.34
Nutrient mg*L-1
Calcium nitrate
Magnesium sulfate
Ca(NO3)2
MgSO4
Molecular
FormulaChemical name
54
Table 4-2 Pepper plant growth responses to two application strategies of ClO2 and two soilless medias in Citra, FL.
a Change in height over 10 day treatment interval
b Fruit were harvested before reaching marketable size and are reported as total fruit weight per plant.
x Value not significantly different from 0, α =0.05.
Media
Pine Bark 3.1 608.3 135.4 77.5 71.4 87.5 659
Perlite 1.2 525.3 125.6 75.8 68.1 89.8 689.8
p-value 0.0063 0.0192 0.0843 0.0051 0.0045 <0.0001 0.6882
ClO2 (mg L-1
)
0 2.9 612.6 136.4 77.5 71.5 88.6 731.4
20 1.3 520.9 124.6 75.7 68 88.6 617.3
p-value 0.0189 0.0111 0.0422 0.0036 0.0036 0.9866 0.1507
Full Strength Solution2.2 568.3 131.0 76.6 69.9 88.6 705.5
Extended Diffusion2.1 565.2 130.0 76.6 69.6 88.7 643.3
p-value 0.8286 0.9210 0.8529 0.9813 0.8104 0.7773 0.4207
Pine Bark
0 mg L-1
3.7 637.0 139.7 77.8 71.9 87.6 659.5
20 mg L-1
2.5 576.0 130.2 77.2 71 87.4 658.5
p-value 0.2952 0.2705 0.2623 0.4832 0.6102 0.8535 0.9942
Perlite
0 mg L-1
2.1 588.2 133.2 77.3 71.1 89.7 803.3
20 mg L-1
0.2x
462.3 118 74.3 65.1 89.8 576.2
p-value 0.0428 0.0116 0.0634 0.0028 0.0012 0.598 0.0146
Treatment
--- Treatment main effects ---
--- Growth response from ClO 2 concentration by media ---
Strategy
Root %
Water
Fruit
Weighty
(g)
Height
changez
(cm)
Plant
Fresh
Weight (g)
Plant Dry
Weight
(g)
Plant %
Water
Shoot %
Water
55
Figure 4-1 Residual chlorine dioxide in the nutrient solution used to produce greenhouse bell peppers in Citra, FL. Bars represent standard error of the mean. Both treatments were applied with the same amount of ClO2 by weight.
56
CHAPTER 5 CONCLUSIONS
Chlorine dioxide is well-documented to effectively eliminate many viral, bacterial,
and fungal pathogens that present issues in municipal and agricultural systems. Despite
the apparent chemical and sanitizing benefits of ClO2 there are limited studies reporting
potential phytotoxic effects of ClO2 when used as an irrigation sanitizing agent for
greenhouse plants. The few reports that are available indicate that plant damage may
occur, but the potential benefits of its use warrant further investigation. This research
was split into three objectives. First, to determine the response of bell pepper growth
when exposed to a range of concentrations of ClO2 within the nutrient solution, and to
identify the concentration associated with minimal negative effects on plant growth.
Secondly, to determine the ClO2 demand of water used in recirculating irrigation
systems, and to further characterize the ClO2 sequestration of two common water
sources and three hydroponic irrigation solutions. And finally, to determine the impact of
ClO2 application strategy and potting media on greenhouse bell pepper growth.
Pepper plants grown in pine bark were less impacted by ClO2 than plants grown in
perlite for all experiments. Chlorine dioxide reacts with organic compounds in pine bark
media such as amines, aldehydes, and phenols and is chemically reduced minimally
active byproducts before reaching the plant roots (Gagnon et al. 2005; Stevens, 1982;
US-EPA, 1999). Perlite is created from heated and expanded obsidian, and as an
inorganic media it does not contain chemicals that react with ClO2, allowing it to directly
contact plant roots. This inherent difference between the growing media is likely to be
57
responsible for the increased impact from ClO2 observed on plants growing in perlite
media.
As the sanitized solution flows through the hydroponic system it comes into
contact with biofilm, algae and pathogens harbored in irrigation lines and emitters.
Maintaining a high residual in the nutrient solution allows for disinfestation of irrigation
components in addition to the water itself (Coosemans 1995; Gagnon et al. 2005;
Huang et al. 1997). As control of a variety of pathogens has been reported at lower
residual concentrations than those used in this experiment, studies investigating the
effect of lower treatment doses and application methods of ClO2 on plants grown in
inorganic media, such as perlite, are warranted.
The initial concentration of chlorine dioxide initially dosed into the system needs to
be applied at a sufficiently high dose in order to meet the demand of irrigation water and
to attain the desired ClO2 residual to treat pathogens. The demand will vary by system,
water quality, fertilizers used, and amount of recycled water in the solution. In addition,
as the ClO2-treated nutrient solution passes through the hydroponic system, it is
anticipated that the ClO2 concentration will decline further when ClO2 comes into
contact with biofilm and organic matter within the system (Gagnon et al. 2005). To
compensate for this, a higher treatment concentration is required in order to maintain a
sufficient ClO2 residual while the nutrient solution continues to recirculate throughout the
irrigation system. ClO2 demand should be examined over a range of concentrations to
determine the minimum treatment dose that will create an optimal ClO2 residual long
enough to sanitize plant pathogens in hydroponic leachate intended for recirculation
without negatively impacting plant growth.
58
APPENDIX ADDITIONAL FIGURES
Figure A-1. Fall 2009 root systems of pepper plants grown in perlite media irrigated with 0 to 40 mg L-1 chlorine dioxide.
59
Figure A-2. Fall 2009 root systems of pepper plants grown in pine bark media irrigated
with 0 to 40 mg L-1 chlorine dioxide.
60
Figure A-3. Spring 2010 root systems of pepper plants grown in perlite media irrigated
with 0 to 10 mg L-1 chlorine dioxide.
61
Figure A-4. Spring 2010 root systems of bell pepper plants grown in pine bark media
irrigated with 0 to 10 mg L-1 chlorine dioxide.
62
Figure A-5. Spring 2010 bell pepper plants. From left to right: Plants grown in perlite
media irrigated with 0, 2.5, 5, and 10 mg L-1 chlorine dioxide. Plants grown in pine bark media irrigated with 0, 2.5, 5, and 10 mg L-1 chlorine dioxide.
63
Figure A-6. Water samples used in chlorine dioxide demand experiments. From left to
right: deionized water, well water only, well water plus nutrient solution, well water- nutrient solution leachate from pine bark media, and well water-nutrient solution leachate from perlite media.
64
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BIOGRAPHICAL SKETCH
Libby Rohrer Rens was born in 1985 in Waupun, Wisconsin. She received her
Bachelor of Science degree in Horticultural Sciences and Plant Pathology from the
University of Wisconsin-Madison in December 2008. During pursuing her degree she
had the opportunity to work closely with orchard growers on the development of
Integrated Pest Management programs on their farms. In August 2009 she began her
graduate studies in the Department of Horticultural Sciences at the University of Florida
in Gainesville working on irrigation sanitation of greenhouse grown bell pepper.