1. Project Aims

The twinkling artifact (TA) is a rapid color-shift that selectively highlights hard objects such as kidney stones in color-Doppler ultrasound images; however, its inconsistent appearance has limited its clinical use. Our objective is to develop an ultrasound imaging protocol to enhance kidney stone detection in space, addressing ExMC Gap 4.13. In the 3rd year renewal proposal, the aims are: AIM 1: Develop, refine, and test in space analogs improved ultrasound imaging protocols to enhance kidney stone detection. AIM 2: Determine the effect of breathing gas composition on twinkling in swine. AIM 3: Determine the role of bacteria in twinkling.

2. Key Findings

Twinkling was reduced when 9 swine were exposed to elevated carbon dioxide (CO2) levels (similar to what is found on the International Space Station (ISS)) with the degree of reduction in twinkling correlating with the concentration of CO2; twinkling increased when swine were exposed to 100% oxygen. When 4 swine were exposed to elevated CO2 levels versus normal air, there was an unrecoverable decrease in twinkling over the course of the study. Concentrations of gases in the blood and urine suggest both O2 and CO2 contribute to twinkling. Imaged 7 human subjects in the hyperbaric chamber and found a statistically significant increase in twinkling when subjects breathe 100% O2 at 1.6 ATA (the decompression stop) compared to initial twinkling levels. Growing several different species of bacteria on sterilized stones did not induce or increase twinkling; bacteria grown on agar plates also did not twinkle. Found evidence suggesting the contribution of internal micro-cracks to twinkling in addition to the surface crevice bubbles. Low frequency ultrasound was found to enhance twinkling if already present on stones, but did not cause non-twinkling stones to twinkle. Published 2 papers in peer-reviewed scientific journals; 4 additional papers are in preparation. Presented at 5 scientific conferences including an invited talk at the Acoustical Society of America Meeting in Honolulu, HI. Mentored a Pacific Science Center summer high school student and a summer undergraduate student. Organized a Wide World of Sound Booth at Engineering Discovery Days as Chair of the Outreach Committee, Cascadia Regional Chapter, Acoustical Society of America. Was interviewed for 2 National Space Biomedical Research Institute (NSBRI)/University of Washington promotional videos and a book on women in science. Accepted a tenure-track assistant professor position in the Graduate Program in Acoustics, Department of Aerospace Engineering at the Pennsylvania State University.

3. Impact

We have discovered that breathing CO2 significantly reduces twinkling in swine and have shown that breathing normal air (0.04% CO2) is insufficient to restore twinkling. The results also suggest that breathing elevated O2 may restore or enhance twinkling, which is supported in our human hyperbaric study. Bacteria grown on sterile stones or agar plates was found to be insufficient to induce or enhance twinkling; bacteria may need to be present in the stone formation process to contribute to twinkling. While increasing the energy delivered to the stone or lowering the transmitted frequency has been found to enhance twinkling, there remains some stones that do not twinkle. Further investigation into the stone formation process and/or the presence or absence of internal micro-cracks, which have been shown to play a role in twinkling in addition to the surface crevice bubbles, may help elucidate why some stones are resistant to twinkling so new techniques can be developed.

4. Proposed research

While this project is ended with NSBRI, we plan to finish recruiting and imaging the last subject for the human hyperbaric study and plan to submit 3 more journal publications. We are also looking to investigate in humans the effect of either an elevated CO2 or O2 environment on kidney stone twinkling.

Improving ultrasound for kidney stone detection would allow emergency rooms to diagnose kidney stones immediately rather than sending the patient to radiology for an x-ray or CT. Ultrasound could also be used for more routine monitoring of kidney stones so that steps could be taken to avoid emergency surgery. While we have not found a way for ultrasound to predict stone composition, significant improvements have been made in kidney stone imaging and sizing. We have shown that surface crevice bubbles and internal micro-cracks contribute to the ultrasound twinkling artifact and we continue to increase our understanding of kidney stone formation. Furthermore, we have found that breathing elevated levels of oxygen may enhance twinkling, which could help make kidney stones even easier to diagnose, particularly for non-expert sonographers. In space, ultrasound is one of the few imaging technologies that can be safely flown, and our improved kidney stone detection protocols will make ultrasound a more robust tool for early stone detection, which is critical for minimizing mission disruption and reducing the risk of an unpredictable and life-threatening renal stone incident.

AIM 2: Determine the effect of breathing gas composition on twinkling in swine. A total of 13 swine have been exposed to elevated levels of carbon dioxide (CO2) in air at 0.8%, 0.54%, and 0.27% (approximately 6, 4, and 2 mm Hg, respectively). The 9 swine initially breathing 100% oxygen (O2) show a statistically significant reduction in twinkling when the swine were exposed to air with elevated CO2 with the degree of decrease in twinkling proportional to the concentration of CO2 (i.e., higher CO2 levels causes more reduction in twinkling: 0.8% > 0.54% > 0.27%). A second set of 4 swine were oscillated between normal air (0.04% CO2) and elevated CO2 at 0.54%. These swine showed a significant decrease in twinkling over the course of the study with only minor increases in twinkling when the exposure was shifted from the elevated CO2 to normal air. The slight increases in twinkling from exposure to normal air were not sufficient to recover from the loss in twinkling from exposure to the elevated CO2. Results from measuring the concentrations of gases in the blood and urine suggest both O2 and CO2 contribute to twinkling as fluctuations from oscillating the respiratory gas and overall trends are observed for both O2 and CO2. The results in swine are supported by the human hyperbaric study where a statistically significant increase in twinkling was observed when subjects breathe 100% O2 at 1.6 ATA (the decompression stop) compared to initial twinkling levels on ambient air.

AIM 3: Determine the role of bacteria in twinkling. Preliminary studies where twinkling was evaluated in fresh kidney stones before sections of the stone and surrounding solution were analyzed for bacteria suggest that bacteria may play a role in twinkling. In the lab, growing two different types of bacteria on sterilized human kidney stones did not induce or increase twinkling. Further testing showed bacteria grown on agar plates did not twinkle. It remains possible that bacteria present in the stone formation process contribute to twinkling, but bacteria, alone or in a biofilm, do not show twinkling with the current ultrasound equipment.

2016 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 8-11, 2016. , Feb-2016

Engineering and Urology Society 31st Annual Meeting, American Urological Association, San Diego, CA, May 7, 2016. Abstract Book, p. 70. http://engineering-urology.org/am/31EUS_2016.pdf ; accessed 3/24/17. , May-2016

Engineering and Urology Society 31st Annual Meeting, American Urological Association, San Diego, CA, May 7, 2016. Abstract Book, p. 35. http://engineering-urology.org/am/31EUS_2016.pdf ; accessed 3/24/17. , May-2016

2017 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 23-26, 2017. , Jan-2017

Journal of the Acoustical Society of America. 2016 Oct;40(4):3030. http://doi.org/10.1121/1.4969401 , Oct-2016

Aerospace Medicine and Human Performance. 2016 Mar;87(3). , Mar-2016

2017 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 23-26, 2017. , Jan-2017

45th Annual Ultrasound in Industry Association Symposium, Seattle, WA, April 4-6, 2016. , Apr-2016

Aerospace Medical Association, Abstract Book, April - May, 2017. In press as of March 2017. , Mar-2017

Journal of the Acoustical Society of America. 2016 Oct;140(4):2983. http://doi.org/10.1121/1.4969238 , Oct-2016

2016 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 8-11, 2016. , Feb-2016

Engineering and Urology Society 31st Annual Meeting, American Urological Association, San Diego, CA, May 7, 2016. Abstract Book, p. 33. http://engineering-urology.org/am/31EUS_2016.pdf ; accessed 3/24/17. , May-2016

Journal of the Acoustical Society of America. 2017 May;141(5)3955. Meeting abstract issue, http://dx.doi.org/10.1121/1.4988989 , May-2017

1. Project Aims

The twinkling artifact (TA) is a rapid color-shift that selectively highlights hard objects such as kidney stones in color-Doppler ultrasound images; however, its inconsistent appearance has limited its clinical use. Our objective is to develop an ultrasound imaging protocol to enhance kidney stone detection in space, addressing ExMC Gap 4.13.

AIM 1: Develop ultrasound imaging protocols to enhance kidney stone detection in space.

AIM 2: Manipulate existing elastic wave and bubble dynamic models to aid in refining kidney stone detection protocols.

AIM 3: Determine how hypobaric and hyperbaric conditions alter the TA.

AIM 4: Determine how urine pH and stone type affect the TA.

AIM 5: Determine how exposure to gas concentrations unique to space travel vehicles alters the TA.

2. Key Findings

Exposing swine to 6 mm Hg carbon dioxide in air, the upper end of what is found on the International Space Station (ISS), significantly reduces or eliminates the TA. In ex vivo kidney stones of all major stone types, we found that increasing the acoustic energy delivered to the stone enhances the TA. A damped, shock wave lithotripter pulse can be used to expand bubbles on the stone surface and introduce transient twinkling. We proposed a modification to the current crevice bubble hypothesis of twinkling to include internal microcracks. A variety of bacterial species have been found on 6/6 fresh human kidney stones. Results on twinkling in these stones suggest a relationship between bacteria type and bubbles (or the TA). Published 2 papers in peer-reviewed scientific journals; 2 additional publications are currently in draft form. Presented at 5 scientific conferences, including an invited talk at an International Medical Physics conference. Represented the National Space Biomedical Research Institute (NSBRI) with an ultrasound and kidney stone demo at the 2015 SpaceCOM Expo. Mentored a summer high school student who finished with a demo at the Pacific Science Center. Mentored a high school student senior project. Organized a booth at the University of Washington Engineering Discovery Days. Was approved to begin the hyperbaric human subjects research study to verify bubbles exist on in situ human kidney stones. Grew calcium oxalate crystals in the lab with and without protein and found that they differed significantly from human kidney stones.

3. Impact

We have discovered that breathing CO2 significantly diminishes or eliminates twinkling, which has important implications for detecting kidney stones in flight and suggests the need for an effective countermeasure. We also found that bacteria may form the bubbles that cause stones to twinkle. As bacteria behave differently in space, it is important to determine the relationship between bacteria and bubbles. While increasing the energy delivered to the stone has been found to enhance twinkling, there remains some stones that do not show the TA. We are focusing on new ways to enhance twinkling such as low frequency or dual frequency ultrasound. We also are investigating whether we can predict stone composition based on the reflected Doppler ultrasound signal. Finally, we propose a caveat to the crevice bubble hypothesis of twinkling to include the contribution of internal microcracks, increasing our understanding of the fundamental physics of the TA.

4. Proposed research

We will continue our efforts to recruit subjects for the human hyperbaric experiment. In swine, we will test the influence of CO2 levels on kidney stone twinkling and whether oxygen is a potential countermeasure to restore twinkling. We plan to collect more fresh kidney stones for bacterial analysis, in addition to growing calcium oxalate crystals in the presence of bacteria in the lab to see if we can mimic human kidney stone formation. We will determine whether low frequency or dual frequency ultrasound can make all kidney stones twinkle. We will also determine whether the twinkling signal can be used to predict stone composition.

The Integrated Medical Model team defines two renal stone scenarios; the best case scenario (i.e., where stones pass safely and spontaneously) is predicted to occur in 68% of cases where stones are small (<5 mm diameter). However, as stones increase to 5-10 mm in diameter, stones are predicted to pass safely and spontaneously in less than 50% of cases. These data show the need for a diagnostic tool that allows for routine monitoring of people at risk for developing kidney stones both on Earth and in space. Currently, kidney stones are detected with x-ray or computed tomography (CT), both of which expose the patients to ionizing radiation. Our technology will make ultrasound a more robust tool to detect small kidney stones, thereby reducing patient exposure to ionizing radiation and reducing the cost associated with kidney stones. This technology would allow emergency rooms to diagnose kidney stones immediately, rather than sending the patient to radiology for a CT. In addition, more than 50% of stone-formers have a repeat stone incident within 5 years. Our technology would allow for more routine monitoring so steps could be taken to avoid emergency surgery. Should we find that ultrasound can be used to predict stone type, doctors can help direct treatment to those that are known to be successful for that stone type. Further, if bacteria are found to play a significant role in stone formation or detection, it may help in the development of medication to prevent stone formation in the first place! In space, ultrasound is one of the few imaging technologies that can be safely flown, and our improved kidney stone detection protocols will make ultrasound a more robust tool for early stone detection, which is critical for minimizing mission disruption and reducing the risk of an unpredictable and life-threatening renal stone incident.

AIM 2: Manipulate existing elastic wave and bubble dynamic models to aid in refining kidney stone detection protocols. The linear elastic wave model was successfully coupled to the bubble dynamics model and then used to guide the development of stone imaging protocols.

AIM 3: Determine how hypobaric and hyperbaric conditions alter the TA. Recruitment is currently ongoing for the human hyperbaric research study to verify bubbles are present on in situ stones. In the lab, twinkling on macroscopically rough-surfaced stones has always decreased as expected upon exposure to hyperbaric pressure and increased when exposed to hypobaric pressure. Macroscopically smooth-surfaced stones do not always respond as expected to hypo- or hyperbaric pressure, which has led to the addition of internal microcracks to the crevice bubble hypothesis of twinkling.

AIM 4: Determine how urine pH and stone type affect the TA. Imaging ex vivo stones across physiologically-relevant pHs indicated that the TA is only minimally influenced by pH. Yet stone composition was found to significantly influence twinkling through surface roughness. We discovered large differences in composition and architecture in stones classified as the same type, which was reflected in the amplitude and stability of the twinkling signal. This prompted the creation of a calcium oxalate stone farm, where essentially an artificial kidney was fed solutions supersaturated with calcium and oxalate. The grown crystals were much more fragile and less dense than real stones, suggesting that urine salt supersaturation alone is insufficient to form kidney stones.

AIM 5: Determine how exposure to gas concentrations unique to space travel vehicles alters the TA. When 4 swine were exposed to increased carbon dioxide at levels 20x that found on Earth (8000 ppm, 6 mm Hg), the TA was significantly diminished or eliminated in 10-15 minutes. Twinkling remained at the low to non-existent level until the pig was returned to oxygen, where twinkling reappeared within 15 minutes. The same pattern in the TA repeated when the pigs were again exposed to carbon dioxide and then oxygen. While the blood showed large changes in carbon dioxide and oxygen concentrations, only small changes were found in the urinalysis. Based on how quickly twinkling responded to the change in breathing gas, we expect that gas exchange is the mechanism by which twinkling is reduced.

2015 Northwest Urological Society Annual Meeting, Seattle, WA, December 4-5, 2015. , Dec-2015

Journal of the Acoustical Society of America. 2015 Sep;138(3):1747. http://dx.doi.org/10.1121/1.4933515 , Sep-2015

33rd World Congress of Endourology and Shock Wave Lithotripsy, October 2015. Abstract Book p. A201-A202. , Oct-2015

2015 NASA Human Research Program Investigators' Workshop, Galveston, TX, January 13-15, 2015. Abstract #0418. , Jan-2015

2015 NASA Human Research Program Investigators' Workshop, Galveston, TX, January 13-15, 2015. Abstract #0136. , Jan-2015

Journal of the Acoustical Society of America. 2015 Sep;138(3):1746. http://dx.doi.org/10.1121/1.4933510 , Sep-2015

Aerospace Medicine and Human Performance. 2015 Mar;86(3):221. , Mar-2015

2015 NASA Human Research Program Investigators' Workshop, Galveston, TX, January 13-15, 2015. Abstract #0130. , Jan-2015

130th Engineering and Urology Society Annual Meeting, New Orleans, LA, May 16, 2015. Abstract Book, p. 35. , May-2015

Omics International Conference on Medical Physics, Birmingham, UK, August 3-5, 2015. , Aug-2015

2016 NASA Human Research Program Investigators' Workshop, Galveston, TX, February 8-11, 2016. , Feb-2016

Omics International Conference on Medical Physics, Birmingham, UK, August 3-5, 2015. , Aug-2015

1. Specific Aims/Objectives

The twinkling artifact (TA), a rapid color-shift that selectively highlights hard objects in color-Doppler ultrasound images, has the potential to improve kidney stone detection; however, its inconsistent appearance has limited its use. Recently, it was hypothesized that crevice bubbles on the surface of stones cause twinkling, and bubbles are going to be very sensitive to the changes in gravity and pressure that occur during space travel. Our objective is to develop an ultrasound imaging protocol to enhance kidney stone detection in space, addressing ExMC Gap 4.13.

AIM 1: Develop ultrasound imaging protocols to enhance kidney stone detection in space.

AIM 2: Manipulate existing elastic wave and bubble dynamic models to aid in refining kidney stone detection protocols.

AIM 3: Determine how hypobaric and hyperbaric conditions alter the TA.

AIM 4: Determine how urine pH and stone type affect the TA.

AIM 5: Determine how exposure to gas concentrations unique to the space travel vehicles alters the TA.

2. Key Findings

In ex vivo kidney stones of all major stone types, we found that increasing the acoustic energy delivered to the stone enhances the TA. Hypobaric conditions were found to enhance the TA and hyperbaric conditions were found to diminish the TA. High-magnification, high-speed imaging has found a crevice bubble on the stone surface that oscillated when exposed to ultrasound. The linear elastic stone model has been successfully coupled with a bubble dynamics model. A new hypothesis to describe the origin of the bubbles in twinkling - namely bacteria – was generated.

Published 4 papers in scientific journals, including, "Focused ultrasound to displace renal calculi: threshold for tissue injury," "Preclinical safety and effectiveness studies of ultrasonic propulsion of kidney stones," "Ultrasound-guided tissue fractionation by high intensity focused ultrasound in an in vivo porcine liver model," and "Pulsed focused ultrasound treatment of muscle mitigates paralysis-induced bone loss in the adjacent bone: A study in a mouse model."

Submitted 2 papers that are in review to J. Fluid Mech. and Ultrasound Med. Biol. Presented at 4 scientific conferences. Represented the National Space Biomedical Research Institute (NSBRI) at the 2014 congressional demonstration. Mentored a summer high-school student who finished with a demo at the Pacific Science Center.

3. Impact

We have discovered methods to enhance the TA that can be programmed into NASA's flexible ultrasound system; some of these enhancements can also be implemented on commercial ultrasound machines. All of our experimental results support the original hypothesis that crevice bubbles on the kidney stone surface cause twinkling and, for the first time, we have observed a bubble on the stone surface. We have also hypothesized that bacteria cause these bubbles to form, which enhances our understanding of the etiology of the TA and stone disease. Furthermore, we have shown that stone composition and environment influence twinkling, which could be used to enhance stone detection or potentially be used to predict stone composition.

4. Proposed Research

We will test the influence of breathing increased carbon dioxide levels on twinkling in a pig model. We will utilize modeling to determine what parameters most strongly influence twinkling. We plan to image more bubbles on the stone surface with higher magnification, high-speed imaging to look for correlations between the twinkling amplitude and number of bubbles. We will leverage the ex vivo hyperbaric experimental results in an IRB to test whether bubbles are present on in situ kidney stones by recruiting human subjects for tests in a hyperbaric chamber. We will grow calcium oxalate crystals in the lab to determine whether pure crystals twinkle. We plan to determine the bacteria load on fresh stones, comparing stones that twinkle with those that weakly twinkle.

The Integrated Medical Model team defines two renal stone scenarios: the best case scenario (i.e., where stones pass safely and spontaneously) is predicted to occur 68% of cases where stones are small (< 5 mm diameter). However, as stones increase to 5-10 mm in diameter, stones are predicted to pass safely and spontaneously in less than 50% of cases. These data show the need for a diagnostic tool that allows for routine monitoring of people at risk for developing kidney stones, both on Earth and in space. Currently, kidney stones are detected with x-ray or CT, both of which expose the patients to ionizing radiation. Our technology will make ultrasound a more robust tool to detect small kidney stones, thereby reducing patient exposure to ionizing radiation and reducing the cost associated with kidney stones. This technology would allow emergency rooms to diagnose kidney stones immediately, rather than sending the patient to radiology for a CT. In addition, more than 50% of stone-formers have a repeat stone incident within 5 years. Our technology would allow for more routine monitoring so steps could be taken to avoid emergency surgery. In space, ultrasound is one of the few imaging technologies that can be flown, and our improved kidney stone detection protocols will make ultrasound a robust tool for early stone detection, which is critical for minimizing mission disruption and reducing the risk of an unpredictable and life-threatening renal stone incident.

AIM 2: Manipulate existing elastic wave and bubble dynamic models to aid in refining kidney stone detection protocols. The existing elastic wave and bubble dynamic model has been successfully coupled. We have also been able to observe one instance of a bubble oscillating on the stone surface with high-magnification, high-speed imaging when exposed to color Doppler ultrasound.

AIM 3: Determine how hypobaric and hyperbaric conditions alter the twinkling artifact (TA).

Task 3.1: To test the effect of increased and decreased ambient pressure on the TA in ex vivo human kidney stones. Increasing the ambient pressure has been found to diminish twinkling, with the exact pressure threshold to eliminate twinkling ranging from 3 atm (absolute) to greater than 8 atm, depending on the exact location and stability of twinkling on the individual stone, the gas content of the liquid and stone, and the amplitude and number of cycles in the Doppler pulse. On the other hand, hypobaric conditions have been found to enhance twinkling, which could be utilized in the first aim to enhance kidney stone detection in space.

Task 3.2: To test the effect of increased ambient pressure on the TA in humans. We have reopened discussions with the Virginia Mason Hyperbaric Center and have successfully shown that twinkling can disappear in their chamber at physiologically safe pressure levels. We are pursuing an IRB to be able to complete this task.

AIM 4: Determine how urine pH and stone type affect the TA. Preliminary results in solutions with acidic pH suggest that urine pH affects twinkling. Preliminary results monitoring twinkling in all major stone types indicate that twinkling occurs, though the strength and stability of twinkling is lower for some stone types, such as uric acid.

AIM 5: Determine how exposure to gas concentrations unique to the space travel vehicles alters the TA. The concentration of gases in the solution surrounding the kidney stone influences the twinkling artifact. Preliminary results monitoring twinkling in solutions with increased carbon dioxide concentrations suggest that gas composition also affects the TA.

2014 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 12-13, 2014. Abstract 3129. http://www.hou.usra.edu/meetings/hrp2014/pdf/3129.pdf , Feb-2014

Journal of the Acoustical Society of America. 2014 Oct;136(4):2301. http://dx.doi.org/10.1121/1.4900325 , Oct-2014

14th International Symposium on Therapeutic Ultrasound, Las Vegas, NV, April 2-5, 2014. Abstract Book, April 2014: 283. , Apr-2014

Journal of the Acoustical Society of America. 2014 Oct;136(4):2249. http://dx.doi.org/10.1121/1.4900116 , Oct-2014

2014 IEEE International Ultrasonics Symposium, Chicago, IL, September 3-6, 2014. Abstract Book, p. 626. http://ewh.ieee.org/conf/ius_2014/press/IUS%202014%20Abstract%20Book.pdf ; accessed 1/22/2015. , Sep-2014

2014 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 12-13, 2014. Abstract 3184. http://www.hou.usra.edu/meetings/hrp2014/pdf/3184.pdf , Feb-2014

14th International Symposium on Therapeutic Ultrasound, Las Vegas, NV, April 2-5, 2014. Abstract Book, April 2014: 252. , Apr-2014

Journal of the Acoustical Society of America. 2014 Apr;135(4):2344-5. http://dx.doi.org/10.1121/1.4877703 , Apr-2014

Astronauts are at an increased risk of forming kidney stones because of dehydration and altered bone metabolism. A stone, while innocuous in the kidney, will often pass spontaneously causing debilitating pain that will affect mission operations. Even worse, large stones can become obstructing when they attempt to pass, resulting in a serious infection or even death without surgical intervention. The goal of this proposal is to develop an ultrasound imaging protocol to detect stones before they become dangerous. Early detection will allow for planned intervention through the administration of stone-dissolving medications, scheduled transport to Earth, or an ultrasound-based stone pushing technique in development at the University of Washington. The twinkling artifact is a rapid color change that can highlight hard objects, such as kidney stones, on a grey-scale ultrasound image; however, twinkling currently appears inconsistently on clinical ultrasound machines. Our team recently showed that twinkling is caused by bubbles on the stone surface and bubbles will be very sensitive to the changes in gravity and pressure that occur in space. In this proposal, we will use our knowledge of bubbles and ultrasound to increase twinkling. Using modeling and experimentation in environments that mimic space, we will develop and test imaging protocols to demonstrate their ability to detect stones in astronauts before they grow large enough to become dangerous.