WO2014027346A1

WO2014027346A1 - Device and methods for determination of health of corals and aquatic plants

Info

Publication number: WO2014027346A1
Application number: PCT/IL2013/050684
Authority: WO
Inventors: Zvy DUBINSKY; Yulia PINCHASOV GRINBLAT
Original assignee: Bar Ilan University
Priority date: 2012-08-15
Filing date: 2013-08-12
Publication date: 2014-02-20

Abstract

There are provided devices and methods for real time detection of health of aquatic environment, by utilizing the photoacoustic effect. The devices and methods are used for a non-invasive, non-destructive real time detection and identification of health of corals, by determining the photosynthesis efficiency by utilizing the photoacoustic effect.

Description

DEVICE AND METHODS FOR DETERMINATION OF HEALTH OF CORALS

AND AQUATIC PLANTS

FIELD OF THE INVENTION

The present invention relates to device and methods for determination in real-time of health of aquatic environment and in particular of corals and aquatic plants, by determining photosynthesis efficiency utilizing the photoacoustic effect.

BACKGROUND OF THE INVENTION

The photoacoustic effect is the conversion between light and acoustic waves due to absorption and localized thermal excitation. In other words, photoacoustic effect is the formation of sound waves, following light absorption in a material sample. To obtain a photoacoustic effect, intensity of light must vary, either periodically (modulated) or as a single flash (pulsed). The photoacoustic effect may be quantified by measuring the formed sound (pressure changes) with appropriate detectors, such as hydrophone (for example, microphones or piezoelectric sensors). The electric output (current or voltage) from these detectors and its time variation represents the photoacoustic signal. Various uses, which utilize the photoacoustic effect, are known, such as, for example: photoacoustic spectroscopy, in which the photoacoustic signal may be used in spectroscopic studies of either opaque or transparent objects; study of energy in chemical reactions where light participates, such as, for example, in the study of photosynthesis. Several different mechanisms that produce the photoacoustic effect have been described: One mechanism is photo thermal, and it is based on the heating effect of the light and the consequent expansion of the light-absorbing material, which includes the following stages: conversion of the absorbed pulsed or modulated radiation into heat energy; temporal changes of the temperatures at the site where radiation is absorbed; and expansion and contraction following these temperature changes, which are "translated" to pressure changes. The changes in pressure, which occur in the region where light was absorbed, propagate within the sample body and can be sensed by an adequate sensor. An additional mechanisms are related to photophysical processes and photochemical reactions following light absorption, such as, for example, change in the material balance of the sample and/or the gaseous phase around the sample; change in the molecular organization, which results in molecular volume changes.

The photoacoustic effect has been used in various settings. For example, Pinchasov Y., et.al. (2005) discloses photo acoustics as a diagnostic tool for probing the physiological status of phytoplankton.

For example, Pinchasov Y. et.al. (2006) discloses the effect of lead on photosynthesis, as determined by photo acoustics in synechococcus leopoliensis (cyaobacteria). For example, Pinchasov Y. et.al. (2007) discloses the use of photo acoustics as a novel tool for the determination of photo synthetic energy storage efficiency in phytoplankton.

For example, Pinchasov Y. et.al. (2008) discloses photo synthetic efficiency as function of nutrient status in phytoplankton from irrigation and drinking water reservoirs, determined by photoacoustics.

For example, Pinchasov-Grinblat Y. et.al. (2010) discloses comparison of two methods for estimating energy storage efficiency in phytoplankton photosynthesis.

For example, Pinchasov-Grinblat Y. et.al. (2011) discloses the effect of photoacclimation on photosynthetic energy storage efficiency, determined by photoacoustics.

For example, US patent application No. US 2009/0103083 is directed to acoustic and optical illumination technique for underwater characterization of objects/environment.

Algae are the main primary producers in all water bodies, marine, freshwater, natural and man made, and as such they provide the energy basis for all aquatic ecosystems. The biomass and vitality of the algal population responds rapidly to seasonal changes in environmental factors such as temperature, light, vertical mixing, eutrophication, pollution and nutrient limitation. Typical processes resulting from human activities such as non-point sources of agrochemicals, sewage outflows, and effluents from food, animal and other chemical processing industries as well as from urban and rural runoff, contribute considerable amounts of nitrogen and phosphorous, in many cases in addition to heavy metals, to natural and artificial water bodies. Corals are among the most productive ecosystems on Earth, because they host symbiotic algae (for example, dinoflagellates (zooxanthellae)) which provide them with large amount of photo synthates for their energetic requirements. Coral reefs face unprecedented pressures on local, regional, and global scales as a consequence of climate change and anthropogenic disturbances. The responses to such stress are often a decrease in the photo synthetic efficiency of the symbiotic dinoflagellates, as well as bleaching, which involves the mass expulsion of these symbionts, or loss of their pigments.

The anthropogenic increase in atmospheric CO₂ causes two global climate change related processes in the marine domain, seawater warming and ocean acidification. Both of these have significant adverse effects on reef building corals. Sea warming affects corals by increasing both the frequency and severity of coral bleaching events whereas the decrease in seawater pH interferes with the skeletal calcification. The well being of reef building corals depends on their mutualistic symbiosis with the endocelllar symbionts, the zooxanthellae which supply most of the energy required by host and symbiont. Hence, it is of great interest and importance to be able to detect any incipient decline in the efficiency of the photo synthetic light utilization efficiency of the zooxanthellae.

Thus, there is an unmet need in the art for device and methods that utilize the photoacoustic effect to provide real time, non-invasive and non-destructive detection of health of aquatic environment, and in particular, of health of corals, for the early identification of environmental changes. SUMMARY OF THE INVENTION

The present invention, in embodiments thereof, provides novel device and methods that utilize photoacoustic effect, for the real-time detection and/or identification of the condition of aquatic environments and changes thereto. In particular, the device and methods of the present invention are configured to provide non-destructive, non-invasive detection and/or identification of the health condition of corals and aquatic plants, such as macroalgea, in real time, and thereby provide indication as to the condition of the aquatic environment. In some embodiments, the device and methods of the present invention can be used in-situ, i.e. within the aquatic environment itself, without the need to remove habitats (such as corals) from the environment. The health condition of corals and/or aquatic environment may be affected by various environmental factors, such as, for example, warming, acidification, pollution, and the like. In some embodiments, the device and methods disclosed herein may therefore be used to provide real-time warning of any detrimental changes in various aquatic habitats. In some embodiments, the device of the invention may be a portable, submersible device configured to provide real time, noninvasive, non-destructive measurements of the aquatic environment. Furthermore, the device and methods of the present invention provide an additional advantage as they can be used not only on homogeneous algal cultures or assemblages, but also within the aquatic environment on the complex geometry of coral colonies, their rough and uneven surface, which pose a serious hurdle on measurements thereof by other means.

It has further been found by the inventors that changes (such as decrease) in plant and coral health in aquatic environment may be detectable as changes (such as increase) in thermal dissipation of energy, which in turn can advantageously be detected/measured externally, non-invasively, in the habitat itself, in real-time, by utilizing the device and methods of the present invention. It has been shown by the inventors of the present invention that the photoacoustic effect can be applied beyond homogeneous algal suspensions, and can surprisingly be used in real time for detection of health of corals hosting symbiotic algae, zooxanthellae. It has further been shown that the photosynthetic energy storage efficiency of zooxanthellate corals, which can be readily determined by the device and methods of the present invention, is a rapid and reliable reporter of the status of corals since it declines readily under any stress or disease.

In some embodiments, the device and methods of the present invention can be used to provide real-time warning of detrimental developments in water supply reservoirs due to, for example, algal proliferation resulting from eutrophication, or conversely, due to pollution which may cause the collapse of healthy algal assemblages. In some embodiments, the device and methods of the present invention utilize the photoacoustic effect, which is a conversion between light and acoustic waves due to absorption and localized thermal excitation.

According to some embodiments, there is provided a device for identification in real time of the condition of aquatic environment, the device comprising: a) a first light source generating light pulses b); a second light source generating a continuous saturating light; c) an hydrophone sensor configured to sense transient pressure changes generated by heat dissipation of light fraction not utilized by the aquatic environment; and d) a processor configured to process the pressure changes sensed by the hydrophone to produce a signal indicative of photosynthesis activity in the aquatic environment.

According to some embodiments, there is provided a device for determination of health of coral, the device comprising: a) a first light source capable of generating light pulses; b) a second light source capable of generating a continuous light; c) a sensing unit configured to sense transient pressure changes generated by heat dissipation of light fraction not absorbed by the coral, wherein the light is produced by the first light source or the second light source; and d) a processing logic configured to produce a signal indicative of photosynthesis efficiency of the coral, based on the pressure changes detected by the sensing subunit.

According to some embodiments, the device may further include an imaging subunit configured to acquire an image of the coral. According to further embodiments the device may further include a display, such as an LCD display. According to additional embodiments the device may further include a memory storage subunit, such as, for example, a flash memory. In further embodiments, the device may further include a power source, such as a rechargeable battery. According to additional embodiments, the device may further include an input subunit, such as a keyboard, configured to control the device and/or to input/retrieve data from the device.

In some embodiments, the device is substantially water proof. In some embodiments, the device is portable. In some embodiments, the first light source generates light pulses of less than about 1 microsecond. In some embodiments, the first light source comprises an array of light emitting diodes (LED) generating red light at a wavelength in the range of about 620-740 nm. In some embodiments, the second light source comprises an array of light emitting diodes (LED) generating blue light at a wavelength in the range of about 450-490nm. In some embodiments, the blue light is saturating light.

According to some embodiments, the sensing subunit may include an hydrophone selected from, but not limited to : a microphone, a piezoelectric element, a ceramic sensor or combinations thereof.

According to some embodiments, there is provided a method for determining in real time, the health condition of a coral in an aquatic environment, the method comprising: a) detecting transient pressure changes generated by heat dissipation of light fraction not utilized/absorbed by the coral, wherein the light is produced by a first light and a second light, b) producing a signal indicative of photosynthesis efficiency of the coral, based on the pressure changes detected by a sensor; and c) determining the health condition of the coral based on changes in the photo synthetic efficiency of the coral over time.

According to some embodiments, the method further comprises obtaining an image of the coral. In some embodiments, the method further comprises transmitting the signal indicative of photosynthesis efficiency to a remote location.

According to some embodiments, the method may further be used to alert of changes in the aquatic environment in which the coral is residing.

Further embodiments, features, advantages and the full scope of applicability of the present invention will become apparent from the detailed description and drawings given hereinafter. However, it should be understood that the detailed description, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1A. Schematic illustration of a block diagram of a device, according to some embodiments;

Fig. IB. Schematic illustration of a front view of the front end of the device, according to some embodiments;

Fig. 1C. Schematic illustration of a front view of the rear end of the device, according to some embodiments; Fig. 2. Schematic illustration of a benchtop photoacoustic measurement system. Abbreviations shown in the scheme: L - Laser source; S - beam-shaping slits; BS - beam splitter; PAC - photoacoustic cell with suspension of algae; D - stainless- steel photoacoustic detector; P - low-noise preamplifier; A - low noise amplifier; PD - photodiode; TR - trigger signal; B - background light source; O - oscilloscope; and C - computer;

Figs. 3A-B. Graphs showing changes in photosynthetic and chlorophyll in the macroalgea P. pavonica, as measured by the photoacoustic method, under various experimental conditions (C=control, +N=addition of nitrogen, +P=addition of phosphorous, +NP=addition of nitrogen and phosphorous). Fig. 3A shows changes of photosynthetic efficiency (%) over time (Hours) under the various experimental conditions. Fig. 3B shows changes of photoacoustic signal (arbitrary units) over time (Hours) under the various experimental conditions;

Figs. 4A-B. Graphs showing changes in photosynthetic and chlorophyll in the macroalgea U. Rigida, as measured by the photoacoustic method, under various experimental conditions (C=control, +N=addition of nitrogen, +P=addition of phosphorous, +NP=addition of nitrogen and phosphorous). Fig. 4A shows changes of photosynthetic efficiency (%) over time (Hours) under the various experimental conditions. Fig. 4B shows changes of photoacoustic signal (arbitrary units) over time (Hours) under the various experimental conditions;

Figs. 5A-B. Graphs showing changes in photosynthetic and chlorophyll in the macroalgea H. musciformis, as measured by the photoacoustic method, under various experimental conditions (C=control, +N=addition of nitrogen, +P=addition of phosphorous, +NP=addition of nitrogen and phosphorous). Fig. 5A shows changes of photosynthetic efficiency (%) over time (Hours) under the various experimental conditions. Fig. 5B shows changes of photoacoustic signal (arbitrary units) over time (Hours) under the various experimental conditions; Fig. 6. Schematic illustration of a benchtop photoacoustic measurement system. Abbreviations shown in the scheme: L - Laser; PAC - photoacoustic cell with coral fragment; D - stainless-steel photoacoustic detector; P - low-noise preamplifier; A - low noise amplifier; TR - trigger signal; BL - background light source; BC - heating bath circulator; O - oscilloscope; and C - computer; Figs. 7A-C. Graphs showing photoacoustic signal (PAS) over time (μβ) from 3 different coral fragments (Fig. 7A, Fig. 7B and Fig. 7C, respectively). PAS in "dark" is the photoacoustic signal generated by a laser pulse in the dark and PAS in "light" is a signal obtained under saturating continuous light;

Fig. 8. A graph showing the effect of temperature increase (°C) on photosynthetic efficiency (%) of Styllophora Pistillata.

DETAILED DESCRIPTION OF THE INVENTION

According to some embodiments, the present invention provides devices and methods for the determination and identification of the condition of aquatic environment, and changes thereto, by providing real-time assessment of the condition of the environment by utilizing the photoacoustic effect. In some embodiments, the device and methods of the invention utilize the photoacoustic effect to determine, in real time, within the aquatic environment, the photosynthetic efficiency, which is indicative of the health condition of the aquatic environment. In some embodiments, the device and methods are used to determine the health condition of corals or other aquatic plant, by determining the photosynthetic activity under various light conditions/regimes, such as, for example, red light pulses and continuous blue light. In some embodiments, the device is portable, configured to be carried by a user and provide real time assessment of the health of the aquatic environment.

In some embodiments, and without wishing to be bound to any theory or mechanism, the device and methods of the present invention utilize the photoacoustic effect, whereby heat dissipation of the fraction of light energy not utilized by photosynthesis in the aquatic environment under various light conditions/regimes is compared. Heat dissipated from the photosynthetic system generates a pressure transient that may be sensed and processed by the device. Thus, by comparing the signal produced under the various light conditions (for example, light pulses in the dark as compared to saturating light), the efficiency of light storage by the photosynthetic system can be computed and be indicative as to the condition of the aquatic environment. For example, reduction in the health condition of the aquatic environment may be evident as in increase in the photoacoustic signal (i.e. increase in heat dissipation), since a smaller portion of light energy is stored by the photosynthetic system and larger portion is converted to heat. When the energy of the photons absorbed in a sample is degraded to heat the thermal expansion of the material causes a volume increase:

AV= aAH÷Cpp (1) where a is the thermal expansivity, a= 1÷V (5V÷oT)p, p, the density, Cp, the heat capacity of the medium and ΔΗ is the heat liberated or enthalpy change.

The photoacoustic method allows the direct determination of the energy storage efficiency of photosynthesis by relating the energy stored by photosynthesis to the total light energy absorbed by the plant material. Depending on the efficiency of the photosynthetic system, a variable fraction of the absorbed light energy is stored, thereby affecting the heat evolved and the resulting photoacoustic signal. The higher the photosynthetic efficiency, the larger will be the difference between the stored energy with and without ongoing photosynthesis. By exposing the plant cells to continuous, saturating, background light, no storage of any of the pulse energy can take place, whereas in the absence of such light, a maximal fraction of the pulse energy is stored by photosynthesis. As referred to herein, the terms "photosynthetic storage efficiency", "Φ", "maximal photosynthetic storage efficiency" or "PS max" may interchangeably be used and are determined as the complement of the ratios of the photoacoustic signal generated by a weak pulse of light in the dark (PAdark), to that obtained under strong continuous illumination (PAlight or PAsat), according to equation no. 2:

Φ = (PAught - PAdark)) ÷ PAlight = 1 - PAdark÷P Alight (2)

As referred to herein, the term "real time" is directed to include an operation that is performed at a spatial and/or temporal location as with the condition which is being measured/determined by the operation.

As referred to herein, the terms "aquatic environment" and "aquatic habitat" may interchangeably be used. The terms are directed to include any aquatic surrounding, such as, deep water, sea, ocean, pond, lake, sweet water source, and the like, as well as any habitats residing in the environment, such as, for example, corals, aquatic plants, algae, plankton, and the like.

As referred to herein, the terms "condition" and/or "health" of an aquatic environment or coral are meant to include any parameter that is indicative of the status of the aquatic environment and/or habitats thereof. The condition/health may be affected by various environmental conditions, such as, for example, but not limited to: ocean warming, acidification, water pollution, eutrophication, and the like.

According to some embodiments, there is provided a device for real time identification of health of aquatic environment, the device comprising: a) a first light source generating light pulses b); a second light source generating a continuous saturating light; c) an hydrophone configured to sense transient pressure changes generated by heat dissipation caused by light fraction not utilized in the aquatic environment; and d) a processor configured to process the pressure changes sensed by the hydrophone to produce a signal indicative of photosynthesis activity in the environment. In some embodiments, the device may further include one or more imaging subunits, configured to acquire an image and/or a series of images. The imaging subunit may include, for example, but not limited to: a CCD, a digital camera, a camcorder, and the like, or any combination thereof. In some embodiments, the device may further include a memory subunit configured to store data measured/produced by the device. The memory subunit may include any type of memory storing subunit, such as, for example, flash memory, ROM, and the like. The data stored on the memory may include, for example, measurements, images, calculations, and the like. In some embodiments, the memory subunit is integrated with the processor (processing logic) unit.

In some embodiments, images acquired by the imaging subunit may be transmitted to a remote location, where they may be inspected/viewed in real-time. In some embodiments, the transmission of the acquired images may be by any route, such as, for example, wires, wireless, or both. The transmission may be performed by the transmission subunit, such as, for example, a Wi-Fi subunit.

In some embodiments, the device may further include a power source, such as, for example, a rechargeable battery and a charger.

In some embodiments, the device may further include one or more additional subunits configured to detect/measure environmental parameters, such as, for example, water temperature, ambient light intensity, pressure, and the like.

In additional embodiments, the device may further include a display, such as an LCD screen or and LCD touch screen, configured to display images and/or data to the operator of the device (user). In addition, the device may further include an input subunit, such as a keyboard, configured to allow the user to control the device (such as, for example, control light intensity, light duration, and the like) and/or input data into the device. In some embodiments, the device may further include any one or more additional functional units, configured to detect, identify and/or analyze the photoacoustic effect resulting from the photosynthesis in the aquatic environment.

In some embodiments, the device is configured to detect photosynthesis of symbiotic algae residing in/on coral cells. In some embodiments, the device is configured to detect photosynthesis of seaweed and seagrasses.

In some embodiments, the device has a housing having a cylindrical shape. In some embodiments, the device has a housing having a cubical shape. In some embodiments, the device is configured to be submersible, i.e. placed in the aquatic environment In some embodiments, the device can be placed/carried at various depths, such as, for example, in the range of about 0.1 to about 50 meters under water level. In some embodiments, the device is essentially or substantially water proof. In some embodiments the device is portable. In some embodiments, the device is configured to be carried by a user (operator), for example, by one or more handles. In some embodiments, the user is a diver, which carries and operates the device within the aquatic environment.

Reference is now made to Figs. 1A-C, which show schematic illustrations of various views of a device, according to some embodiments. As shown in Fig. 1A, which shows a block diagram of a device, according to some embodiments, device (2) has a front panel (6) facing the front end (4) (i.e. the end which is proximal to the object being detected/measured (for example, a coral); and a rear panel (10), facing the rear end (8 (i.e. the end which is distal to the detected/measured object and is proximal to the user). The device further includes a first light source (shown as Led Array 12 in Fig. 1A) and a second light source (shown as White Led 14 in Fig. 1A), located at the front panel and facing the front end. The device front panel further includes an hydrophone (shown as microphone 16 in Fig. 1A). Further located at the front panel is an imaging subunit (shown as camera 18 in Fig. 1A). The device may further include a processor (processing logic) subunit (shown as Main CPU 20 in Fig. 1 A) and a memory subunit (shown as Memory 22 in Fig. 1A). In addition, the device further includes a power source (shown as Battery and Charger 24, in Fig. 1A). Additional subunits, configured to detect environmental parameters are located in the device. The environmental parameters are shown as Pressure sensor 26 and Light sensor 28 in Fig. 1A. A communication subunit (shown as Wi-Fi subunit 30 in Fig. 1 A) is further located in the device. In addition, at the rear panel, facing the rear end a display and input subunits are located. The display subunit (shown as LCD touch display 32 in Fig. 1A) is optionally connected to the imagine subunit (20) and is configured to display images acquired by the imaging subunit to a user. Further located on the rear panel is a connector (shown as connector 34) configured to connect the device to various other devices/sources, such as, for example, external computer, external power source, and the like.

As shown in Fig. IB, which shows a schematic front view of the front panel of a device, according to some embodiments, on the front panel a first light source (shown as Led array 52) and a second light source (54), are located. Further located on the front panel is hydrophone (56) and an imaging subunit (58). As shown in Fig. 1C, which shows a front view of the rear panel of a device, according to some embodiments, a display subunit (60) is located. Further located on the rear panel a connector (62) configured to connect the device to power source and/or to any other device (such as a remote controller, remote computer, and the like).

In some embodiments, the first and second light source may be identical or different. In some embodiments, the first and/or second light sources may include an array of light sources that may be identical or different. In some embodiments, the light source may be selected from, but not limited to: a light emitting diode (LED), laser, halogen light, flash light, lamp, fiber optics, and the like. Each possibility is a separate embodiment.

In some exemplary embodiments, the first light source may include a first array of light emitting diode(s) (LEDs) that may include any number of LEDs, such as, in the range of about 1 -50 LEDs. For example, the array may include 2-40 LEDs. For example, the array may include 5-25 LEDs. For example, the array may include 10-15 LEDs. For example, the array may include 9-18 LEDs.

In some exemplary embodiments, the first light source may include an array of LEDs, emitting red light at a wave length in the range of about 620-740 nm (Frequency of about 480-400THz) at various intensities (brightness). For example, the array of LEDs may include one or more LEDs emitting red light at a wave length of about 625nm. For example, the array of LEDs may include one or more LEDs emitting red light at a wave length of about 630 nm. For example, the array of LEDs may include one or more LEDs emitting red light at a wave length of about 650 nm. For example, the array of LEDs may include one or more LEDs emitting red light at a wave length of about 660 nm. In some embodiments, the light beam may have a diameter of about 1-30 mm. In some embodiments, the light beam may have a diameter of about 5-20 mm.

In some embodiments, the first light source may produce pulses of light. The light pulses may be at a duration of about 0.01-5000 microseconds, with intervals in the range of about 0.01 -5000 microseconds. For example, the light pulses may have a duration of about 0.1-5 microseconds, with intervals in the range of about 0.1-5 microseconds. For example, the light pulses may have a duration of about 0.2-2 microseconds. For example, the light pulses may have a duration of about 0.5-1.5 microseconds. For example, the light pulses may have a duration of about 0.75-1 microseconds. For example, the light pulses may be at intervals of about 0.2-2 microseconds. For example, the light pulses may be at intervals of about 0.5-1.5 microseconds. For example, the light pulses may be at intervals of about 0.75-1 microseconds.

In some embodiments, the second light source may include a second array of light emitting diode(s) (LEDs) that may include any number of LEDs, such as, in the range of about 1-50 LEDs. For example, the second array may include 2-40 LEDs. For example, the second array may include 5-25 LEDs. For example, the second array may include 10- 15 LEDs.

In some embodiments, the second array of LEDs, may include LEDs emitting blue light at a wave length in the range of about 450-490 nm (Frequency of about 670-610THz) at various intensities (brightness). For example, the second array of LEDs may include one or more LEDs emitting blue light at a wave length of about 450nm. For example, the second array of LEDs may include one or more LEDs emitting blue light at a wave length of about 470 nm. For example, the second array of LEDs may include one or more LEDs emitting blue light at a wave length of about 490 nm. In some embodiments, the second light source may provide continuous light. In some embodiments, the light beam may have a diameter of about 1-30 mm. In some embodiments, the light beam may have a diameter of about 5-20 mm.

In some exemplary embodiments, the first array of LEDs includes 5 LEDs, each emitting light at a wavelength of about 660 nm, at light pulses of 1 microsecond, with intervals of microsecond between the pulses.

In some exemplary embodiments, the second array of LEDs, include 5 LEDs, each emitting continuous light at a wavelength of about 450 nm.

According to some embodiments, the hydrophone may include any type of hydroponic sensor capable of detecting/sensing pressure changes. For example, the hydrophone may be selected from, but not limited to: microphone, piezoelectric element, ceramic sensor, and the like.

According to some embodiments, the processor of the device is any type of processing logic configured to store, calculate, analyze and/or display data. In some embodiments, the processor is a Central Processing Unit (CPU).

According to some embodiments, there are provided methods for real-time determination or identification of the condition of an aquatic environment, utilizing the photoacoustic effect. In some embodiments, the method may include one or more of the following steps: a) detection in real time of the photosynthetic efficiency in the aquatic environment under continuous, saturating light by detecting heat dissipation of the fraction of saturating light not utilized by photosynthesis; b) detection in real time of the photosynthetic efficiency in the aquatic environment under light pulses (i.e. in the dark), by detecting heat dissipation of the fraction of saturating light not utilized by photosynthesis; c) comparing the photosynthetic efficiency determined in step a) with the photosynthetic efficiency determined in step b), to determine the overall photosynthetic efficiency in the aquatic environment; d) identification of changes over time of the overall photosynthetic efficiency in the aquatic environment to determine the condition of the aquatic environment.

In some embodiments, the changes in the aquatic environment, as can be identified in step d) above herein, may include increase in the overall photosynthetic efficiency or reduction in photosynthetic efficiency, each may be indicative of detrimental developments in the aquatic environment. For example, increase in the overall photosynthetic efficiency may be indicative of increase in bio mass of habitats of the aquatic environment. Such a condition may result, for example, due to algal proliferation because of eutrophication, which in turn, can induce negative environmental effects, such as hypoxia, which may cause reductions in specific fish and other animal populations in the aquatic environment. For example, decrease in the overall photosynthetic efficiency may be indicative of an event leading to reduction in number of habitats in the aquatic environmental or reduction in habitats well being. Such an event may be, for example, pollution, warming, acidification, and the like. The habitats may be, for example, algal assemblages, symbiotic algae (zooxanthnthellae) that symbiotically reside in coral cells, aquatic plants such as macroalgae, seaweeds, seagrass, and the like. In some embodiments, the habitats do not include phytoplankton.

In some embodiments, the step(s) of determining the photosynthetic efficiency (such as in steps a) and b)) are performed by utilizing the photoacoustic effect, whereby the fraction of light energy not utilized/absorbed by the photosynthetic system (i.e. in the process of photosynthesis occurring in the aquatic environment, by habitats thereof) dissipate as heat, which generates a pressure transient that can be sensed, by a sensor, such as, an hydrophone, and further processed, for example, by a processing logic.

According to some embodiments, any of the calculation or computing steps of the method may further include a step of determining ambient light at different depths and/or hours (time) in which the measurements are being performed to take this into account in the various calculations. For example, the ambient light at different depths and hours can be used to generate P vs. I curves.

According to some embodiments, the method for real-time determination or identification of the condition of an aquatic environment may be specifically used for the detection of health of various aquatic habitats, such as, for example, corals. Determination of the photosynthetic efficiency, by the methods of the present invention of algal assemblages of symbiotic algae (zooxanthnthellae) that symbiotically reside in coral cells is indicative of the coral health. For example, reduction in the photosynthetic efficiency of these symbiotic algae is indicative of reduction of health of the coral itself, which may, in turn be indicative of detrimental changes in the aquatic environment, such as, for example, pollution, water warming, acidification, and the like.

According to some embodiments, the method may further include a step of providing an image of the aquatic environment, such as, for example, an image of a coral being examined for photosynthesis efficiency. The image may be provided by a CCD, a digital camera, a camcorder, and the like.

According to some embodiments, the method may further include a step of storing data on a digital media, such as, for example, flash memory, hard disk, and the like.

According to some embodiments, the method may further include a step of transmitting, by any communication route, the data produced by the method to a remote location. The data may include, for example, but not limited to: data regarding the photoacoustic measurements, data regarding the photosynthetic efficiency calculations, images, and the like.

According to some embodiments, each of the steps of the methods disclosed herein may be performed separately, or continuously. The various determinations and calculations may be performed by the device of the present invention and the results may be provided by any communication route to a remote location, where the results may be inspected/analyzed in real time to provide warning of changes in the aquatic environment. According to some embodiments, the device and methods of the present invention may be used to scan/measure a portion of a coral or even the entire coral. This may be achieved, by acquiring data from various regions of the coral, to produce a data set indicative of the coral condition/health. In some embodiments, statistical analysis may be performed on data obtained from various portions of the coral to produce a data set indicative of the condition of the entire coral, or portions thereof. The data set may include one or parameters that may be measured/calculated by the device and methods of the present inventions, such as, for example, but not limited to: images of the coral or any portion thereof; photosynthetic efficiency of the coral or any portion thereof, chlorophyll concentration or any portion thereof.

EXAMPLES

Example 1- determination of effect of nutrient enrichment on macroalgae by the photoacoustic method Seaweeds are algae that live in the sea. These marine plants are often called

"benthic algae" which means that they are attached to substratum in the sea by their holdfast that anchors them to the substrate. Most of the seaweeds are macroalgae which are distributed in three phyla: Chlorophyta which are often called green algae (-1200 species), Rhodophyta, the red algae (-6000 species) and Heterokontophyta or brown algae (-1750 species). Seaweeds grow in the photic zone which is in the range between the surface water levels (in the intertidal zone) and down to -200 meters deep in the open ocean. Viva rigida C. Agardh, Hypnea musciformis (Wulfen in Jacquin) J.V. Lamouroux and Padina pavonica (Linnaeus), Thivy Agardh are green, red and brown algae, respectively.

The photoacoustic method was used to determine the effect of nutrient changes in the aquatic environment on various species of macroalgae from three different phyla that have different types of thallus morphologies and diverse pigment assortments. Viva rigida has green flat sheet-like thallus with toothed margins. This thallus is composed of two cell layers. Hypnea musciformis has red-green narrow, cylindrical, branched thallus morphology. Its branch apices are slightly upcurved, flattened hooks. Padina pavonica is characterized by flat calcified "ear-like" blades which has circinnately inrolled apical margins.

The photo synthetic storage efficiency, Φ, was calculated according to equation (2).

Φ = (P Ai_ight - P Ada_rk)) ÷P Ai_ight = 1 -P A_dark÷PAii_ght (2)

Detection of the pressure wave in an aqueous suspension of algae was performed over 10-20 microseconds after the laser pulse.

Experimental procedure:

Device and method: The experimental setup is schematically shown in Figure 2. The overall procedure is similar to that described in Pinchasov et al. (2005). The second harmonic of a Continuum Minilite Q-Switched Nd-Yag laser at 532 nm was used. The signal was processed with a Tektronix TDS 430A oscilloscope. The submersible, stainless steel enclosed hydrophone detector contained a 10 mm diameter resonating ceramic disc (BM 500, Sensor, Ontario, Canada). The sample was placed in a 16 mm quartz glass cell (PAC). The laser (L) pulse, after passing through a pair of 1 mm wide slits (S) is incident upon the suspension of algae whose pigments absorb part of the laser light. Depending on the experimental conditions, a variable fraction of the absorbed light pulse is stored in the products of photosynthesis. The remainder of the absorbed light is converted to heat producing an acoustic wave. This is intercepted by a detector (D), containing the above ceramic disc. A small portion of the laser pulse is deflected by a beam splitter (BS) and used to trigger the Tektronix TDS 430A oscilloscope, where the amplified (Amptek A-250 Preamp and Stanford Research A 560 Amp) photoacoustic signal is recorded. The signal contains a noisy background and later reflections from the walls of the vessel as well as from impedance mismatch within the detector. Signal to noise was improved by averaging over 128 pulses, and by taking root mean square (RMS) values over the time of the recorded signal (-10 μβ). Weak (-20 μ3 cm-2), 5ns pulses at 532 nm wavelength, were used as a probe for ongoing photosynthesis. The source of the background light (B), was a quartz -halogen illuminator (Cole-Parmer 4971). The intensity of the background light was adjusted to the desired level by neutral density filters and measured with a LiCor light meter equipped with a cosine quantum sensor. Algae samples: The samples of three common macro algae species: Ulva rigida, Hypnea musciformis and Padina pavonica were collected from the intertidal abrasion platforms at Bat Yam (located in the middle of the Israeli Mediterranean). All samples were kept at 22 ± 0.1 °C in 100 mL Erlenmeyer during 192 hours under continuous irradiance at -200 ± 5.0 μΐ m-2 s-1. Chlorophyll was determined by photoacoustic method, based on the proportionality of the photoacoustic signal to the amount of pigment.

The samples were exposed to 3 treatments: nitrogen (was added as NaN03, at concentration of 50 ml/L from the stock of 75.0 g/L), phosphorus (was added as NaH2P04, at concentration of 5 ml from the stock of 5.0 g/L), and nitrogen and phosphorus together. Controls were kept in seawater alone.

Results:

The results are shown in Figs 3A-B, 4A-B and 5A-B. In general, in all tested samples, the photo synthetic efficiency (Fig 3A, Fig. 4A and Fig. 5A) and chlorophyll concentration (Fig. 3B, Fig. 4B and Fig. 5A), that were determined by photoacoustic method decreased with time. Macroalgae rapidly exhausted nutrients in the water, and within 190 hours, the controls declined to some 70 % in P. pavonica (Figs. 3A-B) and H. musciformis (Figs. 5A-B), and about 50 % in U. rigida (Figs. 4A-B), of the initial values. The addition of nutrients (such as, nitrogen (N) and/or phosphorous (P) slowed down, but did not prevent, such decline (~ 20 % in U. rigida (Figs. 4A-B) and ~ 50 % and 30 % in P. pavonica and H. musciformis, respectively, (Figs. 3A-B and Figs. 5A-B).

Altogether, these results, which are in agreement with results obtained by other methods, indicate that the photoacoustic method can surprisingly be used for the ecophysiological studies, detection and identification of macroalgae.

Example 2- determination of photosynthetic energy storage efficiency of zooxanthellate corals by the photoacoustic method

Experimental Procedure - The second harmonic of a Continuum Minilite Q- S witched Nd-Yag laser at 532 nm was used. The signal was processed with a Tektronix TDS 430A oscilloscope. The submersible, stainless steel enclosed detector contained a 10 mm diameter resonating ceramic disc (BM 500, Sensor, Ontario, Canada)

Device and method: The experimental setup is shown schematically in Fig. 6. The samples used were 2-5 cm long branches "nubbins" of three zooxanthellae containing reef builders, the corals Stylophora pistillata (Genus Styiophora, smooth cauliflower coral) and Acropora (Genus Acropora, small polyp stony coral in the Phylum Cnidaria) and Millepora dichotoma (Genus Millepora, net fire coral). A sample was placed in a 16mm square glass cell (PA), perpendicular to the laser beam (light source). The laser (L) pulse, (S) is incident upon the sample where the pigments of the symbiotic algae, the zooxanthellae, absorb part of the laser light. Depending on the experimental conditions (as detailed below), a variable fraction of the absorbed light pulse is stored in the products of photosynthesis. The remainder of the absorbed light is converted to heat producing an acoustic wave. This is intercepted by the hydrophone (D), containing the ceramic disc, and amplified (Amptek A-250 Preamp and Stanford Research A). The photoacoustic signal is recorded on the Tektronix TDS 430A oscilloscope that is triggered by the laser pulse generator. Series of 32- 128 pulses in which the sample was in the dark, were alternated by an identical series of light signals, when the sample branch is concomitantly illuminated by saturating continuous light. The source of the background light E, was a quartz -halogen illuminator (Cole-Parmer 4971). The RMS values over the time of the recorded signal (-10 μβ), after each laser pulse were used.

In order to obtain a photosynthesis versus energy relationship, the intensity of the background light was adjusted to the desired level by neutral density filters and measured with a LiCor light meter equipped with a cosine quantum sensor.

The efficiency of photosynthesis under different ambient irradiance levels was determined. By increasing the continuous background light intensity (E) from zero to saturation of photosynthesis, an increasing fraction of the photo synthetic reaction centers of the tested plant is closed at any time, and a decreasing fraction of the probe laser pulse energy is stored in the photosynthetic reaction centers. A corresponding increase in the fraction of the pulse energy is converted to heat, which is sensed by the photoacoustic detector (PA_E). From the detector responses, the photosynthetic energy-storage versus background light-intensity relationship was obtained. PSE is the fraction of maximal photosynthetic energy storage efficiency at a given continuous background light intensity E, as shown in Equation 3:

PSE = (PA_E - PAdark)/ (PA_sat - PA_dark) (3) where "sat" and "dark" are directed to with and without saturating background illumination, respectively.

Coral samples: The coral samples, collected a from the Gulf of Eilat (Aqaba) were kept in the lab, at 24°C, under white fluorescent light at -220 μΐ m^"2 s^"1 PAR. The efficiency percentages were normalized to areal chlorophyll and to zooxanthellae densities harboring the coral samples. To obtain these values, following measurements, the tissue from the entire nubbin was stripped by air brush and algal cell counts in the resulting slurry were performed under the microscope in a Neubauer cytometer. The area of the nubbin was determined by the aluminum foil method. Chlorophyll a was determined spectrophotometrically following overnight extraction with dimethyl formamide of centrifuged cells at room temperature. From these values, the areal chlorophyll concentrations ^g chlorophyll cm^"2) and zooxanthellae densities (cells cm^"2) were calculated.

Results:

Areal chlorophyll concentration: The laser beam was aimed at the coral nubbin to cover the area of 1 cm^" , thereby averaging the small scale differences in zooxanthellae distributions in the different polyp parts and the coenosarc areas between these small polyp species. These measurements were conducted with the saturating background light on, hence the thermal signal depends only on the light absorbed by chlorophyll, regardless of any energy that would have been stored had we made these measurements in the dark. The results were calibrated against extracted chlorophyll. A good correlation was obtained, regardless of the species-specific differences in polyp size and skeletal architecture, indicating that the photoacoustic method provide a powerful- non destructive method for following the effects of partial through complete bleaching, photo acclimation and of eutrophication.

Photosynthetic light utilization efficiency determinations: By alternating the photoacoustic signals obtained from series of laser pulses in the dark with identical series while the nubbins were exposed to saturating continuous light, the efficiency of photosynthetic energy storage was determined for two corals and one zooxanthellate hydrozoan. Comparing the efficiencies measured herein with efficiencies determined by other methods, such as oxygen evolution studies indicates that the values derived by photoacoustics from healthy organisms are likely to present maximal values and are higher than most reported for various aquatic phototrophs. Hence, the results presented herein demonstrate that the photoacoustic method is superior (i.e. more accurate and specific) to other detection methods. Additionally, the Photoacoustic based method allows the nondestructive comparison of various states of energy utilization by aquatic phototrophs such as zooxanthellate corals and hydro zoans, seaweeds and seagrasses in response to photoacclimation, eutrophication and pollution.

The results are shown in Figs. 7A - C, which show graphs of the photoacoustic signal (PAS) with and without saturating background light from three different coral species. The photosynthetic efficiency, Φ, was calculated according to equation (2), and was determined in Stylophora pistillata (Fig. 7A) to be 28.32 %; in Acropora (Fig. 7B) to be 32.49 % and in Millepora dichotoma (Fig. 7C) to be 33.17 %.

Example 3 - Effect of temperature increase on photosynthetic efficiency of a zooxanthellate coral as determined by the photoacoustic method The experimental setup in as described in Example 2.

A fragment of Styllophora Pistillata coral, was placed into the photoaocustic cell and incubated at initial temperature of 22 °C, and increased gradually up to 30 °C. The coral fragment was incubated for 18 hours in the cell, and tested again. The photosynthetic efficiency, Φ, was calculated according to equation (2). As shown in Fig. 8, the photosynthetic efficiency, Φ, was almost unchanged over 4 hours, and slowly decreased during the next 20 hours. Initial values of photosynthetic efficiency were between 26 - 28 %, and after 24 hours the photosynthetic efficiency decreased to about 18.27 %.

References

Dubinsky, Z.,. Feitelson, J. and Mauzerall D.C. (1998). Listening to Phytoplankton.

Measuring Biomass and Photosynthesis by Photoacoustics. Journal of Phycology, 34, 888- 892.

Pinchasov Y., Kotliarevsky D., Dunisky Z., Mauzerall D.C. and Feitelson J. (2005). Photoacoustics as a diagnostic tool for probing the physiological status of phytoplankton. Israel Journal of Plant Sciences, 53, 1-10.

Pinchasov Y., Berner T., Dubinsky Z. (2006). The effect of lead on photosynthesis, as determined by photoacoustics in synechococcus leopoliensis. Water, Air and Soil Pollution, 175, 117-125.

Picnhasov Y., Porat R, Zur B and Dubinsky Z (2007). Photoacoustics: a novel tool for the determination of photosynthetic energy storage efficiency in phytoplankton. Hydrobiologia, 579, 251-256.

Pinchasov Y. Port R., Zur B., Singer L, and Dubinsky Z (2008). Photosynthetic efficiency as function of nutrient status in phytoplankton from irrigation and drinking water reservoirs, determined by photoacoustics. Israel Journal of Plant Sciences, 56, 69-74.

Pinchasov-Grinblat Y., Iluz D., Alster A., Perelman A., and Dubinsky Z. (2010). Comparison of two methods for estimating energy storage efficiency in phytoplankton photosynthesis. Journal of Oceanography and Marine Science 15, 86-92.

Pinchasov-Grinblat Y.,Hofman R. and Dubinsky Z. (2011). The effect of photoaclimation on photosynthetic energy storage efficiency, determined by photoacoustics. Open Journal of Marine Science, 1, 43-49.

Claims

1. A portable device for determination of health of coral in real time, the device comprising: a) a first light source capable of generating light pulses; b) a second light source capable of generating a continuous light; c) a sensing unit configured to sense transient pressure changes generated by heat dissipation of light fraction not absorbed by the coral, wherein the light is produced by the first light source or the second light source; and d) a processing logic configured to produce a signal indicative of photosynthesis efficiency of the coral, based on the pressure changes detected by the sensing subunit; said portable device is configured for use in an aquatic environment.

2. The device of claim 1 further comprising an imaging subunit configured to acquire an image of the coral.

3. The device of claim 1 , further comprising a display.

4. The device of claim 1 further comprising memory subunit.

5. The device of claim 1 further comprising a power source.

6. The device of claim 1 further comprising an input subunit.

7. The device of claim 1 , being substantially water proof.

8. The device of claim 1 , further comprising a transmitter configured to transmit data acquired by the device to a remote location.

9. The device of claim 1 , wherein the first light source generates light pulses of less than about 1 microsecond.

10. The device of claim 1 , wherein the first light source comprises an array of light emitting diodes (LED) generating red light at a wavelength in the range of about 620-740 nm.

11. The device of claim 1, wherein the second light source comprises an array of light emitting diodes (LED) generating blue light at a wavelength in the range of about 450-490nm.

12. The device of claim 11, wherein the blue light is saturating light.

13. The device of claim 1, wherein the sensing unit comprises an hydrophone selected from a piezoelectric element, a microphone, ceramic plate, or combinations thereof.

14. A method for determining in real time, the health condition of a coral in an aquatic environment, the method comprising: a) detecting transient pressure changes generated by heat dissipation of light fraction not utilized/absorbed by the coral, wherein the light is produced by a first light and a second light, b) producing a signal indicative of photosynthesis efficiency of the coral, based on the pressure changes detected by a sensor; c) determining the health condition of the coral based on changes in the photo synthetic efficiency of the coral over time.

15. The method of claim 14, wherein the method further comprises obtaining an image of the coral.

16. The method of claim 14, wherein the first light source generates light pulses of less than about 1 microsecond.

17. The method of claim 14, wherein the first light source comprises an array of light emitting diodes (LED) generating red light at a wavelength in the range of about 620-740 nm.

18. The method of claim 14, wherein the second light source comprises an array of light emitting diodes (LED) generating blue light at a wavelength in the range of about 450-490nm.

19. The method of claim 18, wherein the blue light is saturating light.

20. The method of claim 14, wherein the sensor is a hydrophone.

21. The method of claim 14 further comprising transmitting the signal indicative of photosynthesis efficiency to a remote location.