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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.