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Feasibility study of effect of ultrasound on water chestnuts
Ultrasound in Med. & Biol., Vol. 32, No. 4, pp. 595– 601, 2006 Copyright © 2006 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/06/$–see front matter
doi:10.1016/j.ultrasmedbio.2005.12.013
● Original Contribution FEASIBILITY STUDY OF EFFECT OF ULTRASOUND ON WATER CHESTNUTS JUNRU WU* and MEIYIN WU† *Department of Physics, University of Vermont, Burlington, VT, USA; and †Center for Earth and Environmental Science, Plattsburgh State University, Plattsburgh, NY, USA (Received 19 September 2005, revised 16 December 2005, in final form 22 December 2005)
Abstract—Water chestnut (Trapa natans L.), an annual aquatic plant with floating leaves was first introduced into North America in 1874. Since then, wild populations have quickly become established in many locations within Northeastern USA. Due to its detrimental effects on the overall health of aquatic ecosystems, millions of dollars have been spent to control the water chestnut infestations in the North America through mechanical harvesting and manual removal, with limited success. The potential for continued expansion of the infestations demonstrates an urgent need for an effective control method. This study examined the potential of ultrasound application as an alternative control strategy for water chestnut management. Various frequencies and amplitudes of ultrasound generated by submerged transducers were applied directly to water chestnuts harvested from Lake Champlain. Substantial damages on water chestnut cells as well as penetrated petitoles were observed at the following tested frequencies of ultrasound, 20 kHz, 187 kHz, 469 kHz, 519 kHz and 2.34 MHz. Among them, 20 kHz ultrasound of 1.9 MPa acoustic pressure amplitude demonstrated the most significant damages within 10 s of ultrasound exposure. The treated plants started to die within 72 h and the mortality rate of water chestnut plants treated with the ultrasound application was 100%. (E-mail: [email protected]) © 2006 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound, Bioeffects, Plant management.
confirmed in Connecticut, Delaware, Maryland, Massachusetts, New Hampshire, New Jersey, New York, Pennsylvania, Vermont, Virginia and in Canada. Moreover, water chestnut has a potential further to spread into warmer regions of the USA, since it is native to tropical and subtropical climates. As a result, this species is now listed on “State Noxious Weed Lists” by 35 States in the USA (Pemberton 2002). In Lake Champlain, water chestnut management programs currently rely on the continuation of an inefficient, expensive and labor-intensive harvesting program. Since 1982, over $5.3 million has been spent to control the advance of water chestnut and to prevent the lake-wide spread of water chestnut, with limited success. The potential for continued northward advance of the infestation in Lake Champlain, as well as other water bodies in the North America, demonstrates an urgent need for a more effective control approach. Studies have been conducted to identify alternative control methods. Unfortunately, to date, no cost-effective methods are available to control water chestnut infestation.
INTRODUCTION Water chestnut (Trapa natans L.) is an annual aquatic plant with floating leaves around a central stem and feathery, adventitious submerged structures (Fig. 1). It is native to Southern Europe, tropical Africa and Asia (Agrawal and Ram 1995) and was first introduced into North America in 1874 (Countryman 1977) as a collection for a botanical garden of Harvard University. The gardener released this plant into local waters. Since then, wild populations have quickly become established in many locations within Northeastern USA. This species now occupies significant areas of southern Lake Champlain, six Lake Champlain tributaries and at least six other lakes and ponds in New York and Vermont (Wibbe 1886; Crow and Hellquist 2000). Water chestnut (T. natans) occurs from the northeast, west to the Great Lakes and south to Washington, D.C.; it has also been
Address correspondence to: Prof. Junru Wu, Cook Physical Science Building, Department of Physics, University of Vermont, Burlington, VT 05405-0125. E-mail: [email protected]
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Fig. 1. Illustration of water chestnut.
Extensive research has been conducted on interactions between ultrasound and organism cells/tissues, particularly on bioeffects on plant leaves, seeds and roots. Mechanisms of bioeffects of ultrasound are known to include “thermal” and “mechanical effects” in general (NCRP 2002). When ultrasound is absorbed by a medium such as plant cells, energy associated with ultrasound would be converted into heat and resulted in an increase in cell temperature, which is known as the thermal effect. When ultrasound passes through an aqueous medium and plants, bubble activities may be generated, which is called acoustic cavitation. Acoustic cavitation causes changes in plant cells including microstreaming of a cell’s internal structure and a mass disruption of the cell wall (Coakley and Nyborg 1978). Acoustic cavitation, the dominant mechanism in the ultrasound application, is especially evident on aquatic plants, due to the presence of gas in the interconnected gas chambers inside plant petioles (Fig. 2). Documented effects of ultrasound on plant cells include chromosomal anomalies, cell death, damage to or destruction of cellular structures, reduced growth rates, changes in the osmotic potential of cells and chemical changes within the liquid being cavitated (Miller 1979; Soar 1985; Newroth and Soar 1986). Harvey and Loomis (1928) documented the effects of ultrasound on cells of common water weed, Elodea canadensis Michx, under a microscope, including changes in fluid flow, stirring of intracellular contents, rotation of organelles and cell disruption during exposure to ultrasound at 400 kHz. The cell disruption of Elodea was later documented to proceed in two stages upon exposure of ultrasound
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(Miller 1983). At the first stage, the vacuolar membrane was disrupted by mixing of cytoplasm with the vacuolar contents. At the second stage, the plasma membrane was found to be broken permanently and the leaf lost its viability (Miller 1983). In another study, ultrasound was documented effectively to damage plant cells and tissues of Eurasian watermilfoil, Myriophyllum spicatum L., with a single exposure for only several seconds (Newroth and Soar 1985; Soar 1985). Immediate damage consisted of rupturing or flooding of Eurasian watermilfoil’s aerechyma, deterioration of plant tissues and biomass reduction. It was reported that 100% mortality of Eurasian watermilfoil after two exposures of ultrasound (Newroth and Soar 1985). Newroth and Soar (1985) also compared the effectiveness of ultrasound with other common control measures of Eurasian watermilfoil, including mechanical harvesting, water-level drawdown, biologic control, hydraulic dredging, rototilling, herbicides, bottom barrier application and diver-operated dredging. They concluded that ultrasound was “one of the most promising approaches” and had “advantages for management and high levels of effectiveness in treatment of shoot and root tissues” of Eurasian watermilfoil. The objective of this study is to examine the feasibility of ultrasound application for water chestnut control using commercially available transducers. Ultrasound of various frequencies and amplitudes generated by submerged transducers was applied directly to water chestnut plants. Damages and mortality of water chestnuts caused by ultrasound applications were observed under a controlled laboratory environment to determine the effectiveness of future ultrasound applications for water chestnut management in Lake Champlain. EXPERIMENTAL METHOD Ultrasonic sources Ultrasound source transducers used included: 1) ½-inch (12 mm) diameter planar nonfocusing transduc-
Fig. 2. The gas chambers inside a petiole of water chestnut.
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ers (Panametrics, Waltham, MA, USA) of the following resonance frequencies: 200 kHz, 1 MHz and 2 MHz; 2) a focusing 2.5-inch (63.5 mm) diameter transducer (model H-104, Sonic Concepts, Inc., Bothell, WA, USA) of 2-inch (50.8 mm) focal length was used for 500-kHz sound source; and 3) a sonicator (model S-3000, Misonix Inc., Farmingdale, NY, USA), which had a cylindrical ultrasonic horn (12 mm diameter and 14 cm length) and produced 20-kHz nonfocusing sound waves. When the Misonix sonicator was used, the Misonix S-3000 power supply was the source of the electrical power; no other power supply was needed. Optimizing acoustic parameters A laboratory study was first performed to determine the optimal frequency, acoustic pressure amplitude and the minimum ultrasound exposure duration successfully to destroy gas chambers inside of water chestnut petioles. A computer-controlled measurement system (NTR Systems, Seattle, Washington, USA) that included three linear position manipulators and a digital oscilloscope (model 9310, LeCroy, Chestnut, NY, USA) used as a digitizer was used to measure an in situ 2-D cross-axis sound field. A calibrated pvdf membrane hydrophone with a 0.2 mm diameter electrode (Sonic Consulting, Inc. Wyndmoor, PA, USA) was used as a sound-wave sensor for all megahertz frequencies and a calibrated pvdf hydrophone of 6 mm diameter hydrophone (model 8103, Brüel & Kjær, Nærum, Denmark), a charge amplifier (model 2635, Brüel & Kjær, Nærum, Denmark) were used for 20 kHz and submegahertz sound fields. The 3-D position of a hydrophone was controlled by a computer via three linear manipulators (NTR Systems, Seattle, Washington, USA). A transducer was electronically connected to HP 3314A function generator (Hewlett Packard, CA, USA) and an ENI A-300 RF power amplifier (ENI, Rochester, NY, USA). A block diagram is included in Figure 3. When a 20-kHz sound field was used, it was generated by a 20-kHz horn driven by a power supply of a sonicator (model S-3000, Misonix Inc. Farmingdale, NY, USA); it is not shown in the block diagram. When a nonfocusing transducer was used, a hydrophone was scanned at a plane that is perpendicular to the acoustic axis of the sound field and 1 cm distant from the surface of the transducer. When a focusing transducer was used, a hydrophone sensing element was scanned at the focal plane of the sound field. The in situ spatial-peak pulse-average intensity, ISPPA (NCRP 1983), was also calculated postmeasurements. After the sound field mapping, a portable single ultrasound transducer of known resonance frequency described above was then submerged in a water tank. A water chestnut leaf with its petiole dissected from a plant
Fig. 3. Block diagram and illustration of insonification.
freshly harvested from Lake Champlain was mounted on a plastic holder. When a nonfocusing transducer was used, the sample/sample holder was positioned at 1 cm from the transducer. Consequently, the petiole was exposed to a near-field ultrasound field generated by the transducer. When a focusing transducer was used, the leaf/holder was located at its focal region. The ultrasound exposure tank’s dimensions were 40 ⫻ 45 ⫻ 75 cm3 and it was filled with water of 22° C. After ultrasound exposure, the petiole was dissected perpendicularly, as shown in Figure 4, using a brand new sharp razor blade. As many as seven cross-sections were obtained, and each cross-section was thinner than 1 mm. A dissected cross-section of the petiole then was examined using an optical microscope. Particular attention was paid to the integrity of the gas chambers. When it was determined that a 20-kHz sound field was most effective to destroy petioles of the plant, three water chestnut leaves with their petioles undissected from three plants freshly harvested from Lake Champlain were exposed to the near-field of the 20-kHz horn for 10 s by aiming the horn directly at the petioles. Three similar water chestnut leaves that were not exposed to any sound field were used as control. Both insonified and uninsonicated leaves were kept in a water tank at room temperature (22° C) for observation. EXPERIMENTAL RESULTS AND DISCUSSION Characteristics of sound field of transducers of 20 kHz, 200 kHz, 500 kHz, 1 MHz and 2 MHz used in this
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Table 1. Summary of characteristics of sound sources
Fig. 4. Illustration of the dissection of a petiole.
study are summarized in Table 1. From the Table, it is evident that the 500-kHz focusing transducer has the smallest – 6-dB beamwidth and the highest acoustic pressure amplitude and the 20-kHz transducer has the second highest acoustic pressure amplitude. Figures 5 and 6, are 2-D mappings of a nonfocused 20-kHz and focused 500-kHz sound field, respectively. A picture (Fig. 7) of a cross-section of a partially damaged petiole after exposure to ultrasound of 2 MHz for 1 min is compared with that of another cross-section of a petiole that was not exposed to sound field (control). The similar petiole damages due to other submegahertz and megahertz frequencies listed in Table 1 were also observed. To generate such damages, a long exposure (⬎30 s) is needed. Other common phenomenon for petioles after exposure of the submegahertz and megahertz frequencies was that they all appeared to be unbroken from outside immediately following the treatment. That is to say that, although the microscopic structure was comprised, the macroscopic integrality was still kept. Figure 8 is a picture of petioles after they were exposed to 20-kHz ultrasound for various times. After 15-s exposure, the surface of the petiole seemed to be indented. It is clear that, for 40-s and 45-s exposure cases, the petioles were severely damaged; differing from the submegahertz and megahertz frequencies exposure cases, the plant integrity now was lost. Figure 9 contains two pictures; the top one is for a petiole after 1 min exposure of 20 kHz ultrasound and the bottom one shows its broken gas chamber. It is evident that, after 1 min exposure, the definite gas chamber division did not exist any more. Three water chestnut leaves with their petioles undissected from three plants freshly harvested from Lake Champlain and exposed to the 20-kHz horn for 10 s all died in 72 h. Other three similar water chestnut leaves
Frequency (Hz)
–6 dB beam diameter (mm)
Highest acoustic pressure amplitude (MPa)
ISPPA (W/cm2)
F(Pr,f )
20 k 200 k 500 k 1M 2M
12 12 3 12 12
1.9 1.2 2.8 1.3 1.3
860 340 1.9 400 400
13.4 2.7 4.0 1.3 0.9
that were not exposed to any sound field and were used as control were healthy after 72 h. When the structure of the plant was damaged, internal transport/exchange of energy, nutrients, water and gases were interrupted. Leaves will then turned yellow/brown and dead. The plants were determined dead once all the leaves were detached from the stem and no new leaf was produced. Figure 10 shows that air bubbles existed inside a petiole after 1-MHz ultrasound exposure for 1 min; a similar phenomenon occurred at other frequencies. It suggested acoustic cavitation (bubble activities under ultrasound) may have played a primary role in damaging plants. Usually speaking, since the acoustic attenuation of plants was relatively low (Fukuhara 2002) in the frequency-range used and particularly when exposure time was limited to 10 s, the thermal effect should be minimal. A mechanical index (MI) was developed as an indicator of the potential for nonthermal damage caused by acoustic cavitation (Apfel and Holand 1991; NCRP 2002) for diagnostic ultrasound. A quantity F(Pr, f ) related to MI can be expressed as follows:
Fig. 5. A 2-D mapping of a nonfocused 20-kHz sound field generated by a horn of 6-mm radius at a cross-axial plane that is 1 cm from the sound source (near-field). It can be seen that the acoustic pressure is near uniform (⬃1.9 MPa) within an area |x| ⱕ 6mm and |y| ⱕ 6mm, a characteristic of the near-field of a nonfocusing sound field.
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Fig. 6. A 2-D mapping of a focused 500-kHz sound field at its focal plane that is 2 in (50.8 mm) from the sound source.
F共Pr, f 兲 ⫽
Pr共MPa兲
兹 f 共MHz兲
(1)
where Pr is the negative acoustic pressure amplitude
Fig. 7. Comparison of a cross-section of an insonified (2 MHz, 2 min) petiole (top) with that of an uninsonified petiole (bottom).
expressed in MPa and f is the central frequency of ultrasound in MHz. In this study, the low frequencies used were much below the 1-MHz limit of diagnostic imaging applications. Nevertheless, F(Pr, f ), that is related to MI, may still be a good indicator for the plant destruction due to acoustic cavitation. As shown in Table 1, the 20-kHz sound source certainly has the highest F(Pr, f ). It is agreeable with our observation that the 20-kHz sound source caused the severest damage to the plant. Although the 500-kHz focused sound field has the
Fig. 8. Cross-sections of petioles after various time exposures to the 20-kHz sound field.
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highest acoustic pressure amplitude at its focal region as shown in Table 1, its F(Pr, f ) is still lower that that of 20-kHz case, as its frequency is much higher. Another disadvantage of the focused sound field is that it is critical to place the plant at its focus to get maximum acoustic pressure amplitude. Since the focal zone is relatively small, it is time-consuming and impractical to use a focused sound field in the future field applications. The results of the feasibility study performed by us strongly suggest that a 20-kHz nonfocused sound field may provide us with an effective alternative technique to manage water chestnuts in Lake Champlain. In addition to its effectiveness described above, ultrasound technique also has the following advantages compared with other management techniques: 1) no foreign substances need to be added during treatment; 2) it does not adversely affect drinking or irrigation water quality; 3) treatment may start as soon as ice breaks, before a large biomass develops; 4) it may avoid collection and disposal of plant materials; 5) results can be observed im-
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Fig. 10. Air bubbles that can be observed by an optical microscope existed inside of petiole after 1 min 1-MHz ultrasound exposure.
mediately; and 6) ultrasound electronic equipment has a long life expectancy. The authors want to emphasize that our study is preliminary and only of feasibility in nature. The commercially available single transducers were used in this study. In field applications, a multiple-transducer-array controlled by a computer may be used to increase the efficiency and reduce the cost. To achieve this goal, more research and technical development are needed. REFERENCES
Fig. 9. Top panel: a picture of a petiole after 1 min exposure to 20-kHz sound field. Bottom panel: a picture of a cross-section of a petiole after 1 min exposure to 20-kHz sound field. The gas chambers were totally broken and definite divisions no longer existed.
Agrawal A, Ram HYM. In vitro germination and micropropagation of water chestnut (Trapa sp.). Aquatic Botany 1995;15:135–146. Apfel RE, Holland CK. Gauging the likelihood of cavitation from short-pulse, low-duty cycle diagnostic ultrasound. Ultrasound Med Biol 1991;17:179 –185. Coakley WT, Nyborg WT. Cavitation: Dynamics of gas bubbles; applications. In: Fry FJ, editor. Ultrasound: Its applications in medicine and biology. New York: Elsevier, 1978. Countryman WD. Water chestnut (Trapa natans L.) in Lake Champlain. Proc., Lake Champlain Basin Environ. Conf. Miner Institute for Man and Environment, Chazy, NY, 1977. Crow EC, Hellquist CB. Aquatic and wetland plants of Northeastern North America, vol. 1. Madison, WI: University of Wisconsin Press, 2000. Fukuhara M. Acoustic characteristics of botanical leaves using ultrasonic transmission waves. Plant Sci 2002;162:521–528. Harvey EN, Loomis AL. Further observations on the effect of high frequency sound waves on living matter. Biol Bull 1928;55:459 – 469. Miller DL. A cylindrical-bubble model for the response of plant-tissue gas bodies to ultrasound. J Acoust Soc Am 1979;65:1313–1321. Miller DL. Further examination of the effects of ultrasonic activation of gas bodies in Elodea leaves. Environ Exp Botany 1983;23:393– 405. NCRP. Biological effects of ultrasound: mechanisms and clinical implications. Bethesda, MD: National Council on Radiation Protection and Measurements, 1983.
Effects of ultrasound on water chestnuts ● J. WU NCRP. Exposure criteria for medical diagnostic ultrasound: II. Criterial based on all known mechanisms. Bethesda, MD: National Council on Radiation Protection and Measurements, 2002. Newroth PR, Soar RJ. Eurasian watermilfoil management using newly developed technologies. Proceedings of the fifth annual conference and international symposium on applied lake and watershed management, Nov. 13–16, 1985, Lake Geneva, Wisconsin.
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Pemberton RW. Water chestnut. In: Van Driesche R, editor. Biological control of invasive plants in the Eastern United States. USDA Forest Service Publication FHTET-2002– 04, 2002. Soar RJ. Laboratory investigation on ultrasonic control of Eurasian water milfoil. 1st International Symposium on Watermilfoil and Related Haloragaceae Species, July 23–24, 1985, Vancouver, Canada. Wibbe JH. Notes from Schenectady. Bulletin of the Torrey Botanical Club 1886;13:39.
doi:10.1016/j.ultrasmedbio.2005.12.013
● Original Contribution FEASIBILITY STUDY OF EFFECT OF ULTRASOUND ON WATER CHESTNUTS JUNRU WU* and MEIYIN WU† *Department of Physics, University of Vermont, Burlington, VT, USA; and †Center for Earth and Environmental Science, Plattsburgh State University, Plattsburgh, NY, USA (Received 19 September 2005, revised 16 December 2005, in final form 22 December 2005)
Abstract—Water chestnut (Trapa natans L.), an annual aquatic plant with floating leaves was first introduced into North America in 1874. Since then, wild populations have quickly become established in many locations within Northeastern USA. Due to its detrimental effects on the overall health of aquatic ecosystems, millions of dollars have been spent to control the water chestnut infestations in the North America through mechanical harvesting and manual removal, with limited success. The potential for continued expansion of the infestations demonstrates an urgent need for an effective control method. This study examined the potential of ultrasound application as an alternative control strategy for water chestnut management. Various frequencies and amplitudes of ultrasound generated by submerged transducers were applied directly to water chestnuts harvested from Lake Champlain. Substantial damages on water chestnut cells as well as penetrated petitoles were observed at the following tested frequencies of ultrasound, 20 kHz, 187 kHz, 469 kHz, 519 kHz and 2.34 MHz. Among them, 20 kHz ultrasound of 1.9 MPa acoustic pressure amplitude demonstrated the most significant damages within 10 s of ultrasound exposure. The treated plants started to die within 72 h and the mortality rate of water chestnut plants treated with the ultrasound application was 100%. (E-mail: [email protected]) © 2006 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound, Bioeffects, Plant management.
confirmed in Connecticut, Delaware, Maryland, Massachusetts, New Hampshire, New Jersey, New York, Pennsylvania, Vermont, Virginia and in Canada. Moreover, water chestnut has a potential further to spread into warmer regions of the USA, since it is native to tropical and subtropical climates. As a result, this species is now listed on “State Noxious Weed Lists” by 35 States in the USA (Pemberton 2002). In Lake Champlain, water chestnut management programs currently rely on the continuation of an inefficient, expensive and labor-intensive harvesting program. Since 1982, over $5.3 million has been spent to control the advance of water chestnut and to prevent the lake-wide spread of water chestnut, with limited success. The potential for continued northward advance of the infestation in Lake Champlain, as well as other water bodies in the North America, demonstrates an urgent need for a more effective control approach. Studies have been conducted to identify alternative control methods. Unfortunately, to date, no cost-effective methods are available to control water chestnut infestation.
INTRODUCTION Water chestnut (Trapa natans L.) is an annual aquatic plant with floating leaves around a central stem and feathery, adventitious submerged structures (Fig. 1). It is native to Southern Europe, tropical Africa and Asia (Agrawal and Ram 1995) and was first introduced into North America in 1874 (Countryman 1977) as a collection for a botanical garden of Harvard University. The gardener released this plant into local waters. Since then, wild populations have quickly become established in many locations within Northeastern USA. This species now occupies significant areas of southern Lake Champlain, six Lake Champlain tributaries and at least six other lakes and ponds in New York and Vermont (Wibbe 1886; Crow and Hellquist 2000). Water chestnut (T. natans) occurs from the northeast, west to the Great Lakes and south to Washington, D.C.; it has also been
Address correspondence to: Prof. Junru Wu, Cook Physical Science Building, Department of Physics, University of Vermont, Burlington, VT 05405-0125. E-mail: [email protected]
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Fig. 1. Illustration of water chestnut.
Extensive research has been conducted on interactions between ultrasound and organism cells/tissues, particularly on bioeffects on plant leaves, seeds and roots. Mechanisms of bioeffects of ultrasound are known to include “thermal” and “mechanical effects” in general (NCRP 2002). When ultrasound is absorbed by a medium such as plant cells, energy associated with ultrasound would be converted into heat and resulted in an increase in cell temperature, which is known as the thermal effect. When ultrasound passes through an aqueous medium and plants, bubble activities may be generated, which is called acoustic cavitation. Acoustic cavitation causes changes in plant cells including microstreaming of a cell’s internal structure and a mass disruption of the cell wall (Coakley and Nyborg 1978). Acoustic cavitation, the dominant mechanism in the ultrasound application, is especially evident on aquatic plants, due to the presence of gas in the interconnected gas chambers inside plant petioles (Fig. 2). Documented effects of ultrasound on plant cells include chromosomal anomalies, cell death, damage to or destruction of cellular structures, reduced growth rates, changes in the osmotic potential of cells and chemical changes within the liquid being cavitated (Miller 1979; Soar 1985; Newroth and Soar 1986). Harvey and Loomis (1928) documented the effects of ultrasound on cells of common water weed, Elodea canadensis Michx, under a microscope, including changes in fluid flow, stirring of intracellular contents, rotation of organelles and cell disruption during exposure to ultrasound at 400 kHz. The cell disruption of Elodea was later documented to proceed in two stages upon exposure of ultrasound
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(Miller 1983). At the first stage, the vacuolar membrane was disrupted by mixing of cytoplasm with the vacuolar contents. At the second stage, the plasma membrane was found to be broken permanently and the leaf lost its viability (Miller 1983). In another study, ultrasound was documented effectively to damage plant cells and tissues of Eurasian watermilfoil, Myriophyllum spicatum L., with a single exposure for only several seconds (Newroth and Soar 1985; Soar 1985). Immediate damage consisted of rupturing or flooding of Eurasian watermilfoil’s aerechyma, deterioration of plant tissues and biomass reduction. It was reported that 100% mortality of Eurasian watermilfoil after two exposures of ultrasound (Newroth and Soar 1985). Newroth and Soar (1985) also compared the effectiveness of ultrasound with other common control measures of Eurasian watermilfoil, including mechanical harvesting, water-level drawdown, biologic control, hydraulic dredging, rototilling, herbicides, bottom barrier application and diver-operated dredging. They concluded that ultrasound was “one of the most promising approaches” and had “advantages for management and high levels of effectiveness in treatment of shoot and root tissues” of Eurasian watermilfoil. The objective of this study is to examine the feasibility of ultrasound application for water chestnut control using commercially available transducers. Ultrasound of various frequencies and amplitudes generated by submerged transducers was applied directly to water chestnut plants. Damages and mortality of water chestnuts caused by ultrasound applications were observed under a controlled laboratory environment to determine the effectiveness of future ultrasound applications for water chestnut management in Lake Champlain. EXPERIMENTAL METHOD Ultrasonic sources Ultrasound source transducers used included: 1) ½-inch (12 mm) diameter planar nonfocusing transduc-
Fig. 2. The gas chambers inside a petiole of water chestnut.
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ers (Panametrics, Waltham, MA, USA) of the following resonance frequencies: 200 kHz, 1 MHz and 2 MHz; 2) a focusing 2.5-inch (63.5 mm) diameter transducer (model H-104, Sonic Concepts, Inc., Bothell, WA, USA) of 2-inch (50.8 mm) focal length was used for 500-kHz sound source; and 3) a sonicator (model S-3000, Misonix Inc., Farmingdale, NY, USA), which had a cylindrical ultrasonic horn (12 mm diameter and 14 cm length) and produced 20-kHz nonfocusing sound waves. When the Misonix sonicator was used, the Misonix S-3000 power supply was the source of the electrical power; no other power supply was needed. Optimizing acoustic parameters A laboratory study was first performed to determine the optimal frequency, acoustic pressure amplitude and the minimum ultrasound exposure duration successfully to destroy gas chambers inside of water chestnut petioles. A computer-controlled measurement system (NTR Systems, Seattle, Washington, USA) that included three linear position manipulators and a digital oscilloscope (model 9310, LeCroy, Chestnut, NY, USA) used as a digitizer was used to measure an in situ 2-D cross-axis sound field. A calibrated pvdf membrane hydrophone with a 0.2 mm diameter electrode (Sonic Consulting, Inc. Wyndmoor, PA, USA) was used as a sound-wave sensor for all megahertz frequencies and a calibrated pvdf hydrophone of 6 mm diameter hydrophone (model 8103, Brüel & Kjær, Nærum, Denmark), a charge amplifier (model 2635, Brüel & Kjær, Nærum, Denmark) were used for 20 kHz and submegahertz sound fields. The 3-D position of a hydrophone was controlled by a computer via three linear manipulators (NTR Systems, Seattle, Washington, USA). A transducer was electronically connected to HP 3314A function generator (Hewlett Packard, CA, USA) and an ENI A-300 RF power amplifier (ENI, Rochester, NY, USA). A block diagram is included in Figure 3. When a 20-kHz sound field was used, it was generated by a 20-kHz horn driven by a power supply of a sonicator (model S-3000, Misonix Inc. Farmingdale, NY, USA); it is not shown in the block diagram. When a nonfocusing transducer was used, a hydrophone was scanned at a plane that is perpendicular to the acoustic axis of the sound field and 1 cm distant from the surface of the transducer. When a focusing transducer was used, a hydrophone sensing element was scanned at the focal plane of the sound field. The in situ spatial-peak pulse-average intensity, ISPPA (NCRP 1983), was also calculated postmeasurements. After the sound field mapping, a portable single ultrasound transducer of known resonance frequency described above was then submerged in a water tank. A water chestnut leaf with its petiole dissected from a plant
Fig. 3. Block diagram and illustration of insonification.
freshly harvested from Lake Champlain was mounted on a plastic holder. When a nonfocusing transducer was used, the sample/sample holder was positioned at 1 cm from the transducer. Consequently, the petiole was exposed to a near-field ultrasound field generated by the transducer. When a focusing transducer was used, the leaf/holder was located at its focal region. The ultrasound exposure tank’s dimensions were 40 ⫻ 45 ⫻ 75 cm3 and it was filled with water of 22° C. After ultrasound exposure, the petiole was dissected perpendicularly, as shown in Figure 4, using a brand new sharp razor blade. As many as seven cross-sections were obtained, and each cross-section was thinner than 1 mm. A dissected cross-section of the petiole then was examined using an optical microscope. Particular attention was paid to the integrity of the gas chambers. When it was determined that a 20-kHz sound field was most effective to destroy petioles of the plant, three water chestnut leaves with their petioles undissected from three plants freshly harvested from Lake Champlain were exposed to the near-field of the 20-kHz horn for 10 s by aiming the horn directly at the petioles. Three similar water chestnut leaves that were not exposed to any sound field were used as control. Both insonified and uninsonicated leaves were kept in a water tank at room temperature (22° C) for observation. EXPERIMENTAL RESULTS AND DISCUSSION Characteristics of sound field of transducers of 20 kHz, 200 kHz, 500 kHz, 1 MHz and 2 MHz used in this
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Table 1. Summary of characteristics of sound sources
Fig. 4. Illustration of the dissection of a petiole.
study are summarized in Table 1. From the Table, it is evident that the 500-kHz focusing transducer has the smallest – 6-dB beamwidth and the highest acoustic pressure amplitude and the 20-kHz transducer has the second highest acoustic pressure amplitude. Figures 5 and 6, are 2-D mappings of a nonfocused 20-kHz and focused 500-kHz sound field, respectively. A picture (Fig. 7) of a cross-section of a partially damaged petiole after exposure to ultrasound of 2 MHz for 1 min is compared with that of another cross-section of a petiole that was not exposed to sound field (control). The similar petiole damages due to other submegahertz and megahertz frequencies listed in Table 1 were also observed. To generate such damages, a long exposure (⬎30 s) is needed. Other common phenomenon for petioles after exposure of the submegahertz and megahertz frequencies was that they all appeared to be unbroken from outside immediately following the treatment. That is to say that, although the microscopic structure was comprised, the macroscopic integrality was still kept. Figure 8 is a picture of petioles after they were exposed to 20-kHz ultrasound for various times. After 15-s exposure, the surface of the petiole seemed to be indented. It is clear that, for 40-s and 45-s exposure cases, the petioles were severely damaged; differing from the submegahertz and megahertz frequencies exposure cases, the plant integrity now was lost. Figure 9 contains two pictures; the top one is for a petiole after 1 min exposure of 20 kHz ultrasound and the bottom one shows its broken gas chamber. It is evident that, after 1 min exposure, the definite gas chamber division did not exist any more. Three water chestnut leaves with their petioles undissected from three plants freshly harvested from Lake Champlain and exposed to the 20-kHz horn for 10 s all died in 72 h. Other three similar water chestnut leaves
Frequency (Hz)
–6 dB beam diameter (mm)
Highest acoustic pressure amplitude (MPa)
ISPPA (W/cm2)
F(Pr,f )
20 k 200 k 500 k 1M 2M
12 12 3 12 12
1.9 1.2 2.8 1.3 1.3
860 340 1.9 400 400
13.4 2.7 4.0 1.3 0.9
that were not exposed to any sound field and were used as control were healthy after 72 h. When the structure of the plant was damaged, internal transport/exchange of energy, nutrients, water and gases were interrupted. Leaves will then turned yellow/brown and dead. The plants were determined dead once all the leaves were detached from the stem and no new leaf was produced. Figure 10 shows that air bubbles existed inside a petiole after 1-MHz ultrasound exposure for 1 min; a similar phenomenon occurred at other frequencies. It suggested acoustic cavitation (bubble activities under ultrasound) may have played a primary role in damaging plants. Usually speaking, since the acoustic attenuation of plants was relatively low (Fukuhara 2002) in the frequency-range used and particularly when exposure time was limited to 10 s, the thermal effect should be minimal. A mechanical index (MI) was developed as an indicator of the potential for nonthermal damage caused by acoustic cavitation (Apfel and Holand 1991; NCRP 2002) for diagnostic ultrasound. A quantity F(Pr, f ) related to MI can be expressed as follows:
Fig. 5. A 2-D mapping of a nonfocused 20-kHz sound field generated by a horn of 6-mm radius at a cross-axial plane that is 1 cm from the sound source (near-field). It can be seen that the acoustic pressure is near uniform (⬃1.9 MPa) within an area |x| ⱕ 6mm and |y| ⱕ 6mm, a characteristic of the near-field of a nonfocusing sound field.
Effects of ultrasound on water chestnuts ● J. WU
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Fig. 6. A 2-D mapping of a focused 500-kHz sound field at its focal plane that is 2 in (50.8 mm) from the sound source.
F共Pr, f 兲 ⫽
Pr共MPa兲
兹 f 共MHz兲
(1)
where Pr is the negative acoustic pressure amplitude
Fig. 7. Comparison of a cross-section of an insonified (2 MHz, 2 min) petiole (top) with that of an uninsonified petiole (bottom).
expressed in MPa and f is the central frequency of ultrasound in MHz. In this study, the low frequencies used were much below the 1-MHz limit of diagnostic imaging applications. Nevertheless, F(Pr, f ), that is related to MI, may still be a good indicator for the plant destruction due to acoustic cavitation. As shown in Table 1, the 20-kHz sound source certainly has the highest F(Pr, f ). It is agreeable with our observation that the 20-kHz sound source caused the severest damage to the plant. Although the 500-kHz focused sound field has the
Fig. 8. Cross-sections of petioles after various time exposures to the 20-kHz sound field.
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highest acoustic pressure amplitude at its focal region as shown in Table 1, its F(Pr, f ) is still lower that that of 20-kHz case, as its frequency is much higher. Another disadvantage of the focused sound field is that it is critical to place the plant at its focus to get maximum acoustic pressure amplitude. Since the focal zone is relatively small, it is time-consuming and impractical to use a focused sound field in the future field applications. The results of the feasibility study performed by us strongly suggest that a 20-kHz nonfocused sound field may provide us with an effective alternative technique to manage water chestnuts in Lake Champlain. In addition to its effectiveness described above, ultrasound technique also has the following advantages compared with other management techniques: 1) no foreign substances need to be added during treatment; 2) it does not adversely affect drinking or irrigation water quality; 3) treatment may start as soon as ice breaks, before a large biomass develops; 4) it may avoid collection and disposal of plant materials; 5) results can be observed im-
Volume 32, Number 4, 2006
Fig. 10. Air bubbles that can be observed by an optical microscope existed inside of petiole after 1 min 1-MHz ultrasound exposure.
mediately; and 6) ultrasound electronic equipment has a long life expectancy. The authors want to emphasize that our study is preliminary and only of feasibility in nature. The commercially available single transducers were used in this study. In field applications, a multiple-transducer-array controlled by a computer may be used to increase the efficiency and reduce the cost. To achieve this goal, more research and technical development are needed. REFERENCES
Fig. 9. Top panel: a picture of a petiole after 1 min exposure to 20-kHz sound field. Bottom panel: a picture of a cross-section of a petiole after 1 min exposure to 20-kHz sound field. The gas chambers were totally broken and definite divisions no longer existed.
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