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Gayana (Concepc.) v.68 n.2 supl.TIProc Concepción 2004

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Gayana 68(2) supl. t.I. Proc. : 54-59, 2004 ISSN 0717-652X

AN ALTERNATIVE PROPOSAL: ACOUSTIC TECHNIQUES TO ASSES DETECTION AND MONITORING OF TOXIC ALGAL BLOOMS Silvia Blanc† , Patricia Mosto ‡ , Marta E. de Milou† & Carlos Benítez† † Naval Service of Research and Development (SENID), 327 th Libertador Ave., 1638, Vicente López, Argentina.

[email protected], [email protected], [email protected] ‡ Dept. of Biological Sciences of the Rowan University of New Jersey. NJ 08028, USA. [email protected]

ABSTRACT An alternative methodology consisting in the application of acoustic techniques to asses detection and monitoring of toxic algal blooms is presented here. Successive steps in this direction led to already reported results [Blanc et al., 2000] achieved under the frame of a multidisciplinary research programme conducted in the Argentinean Naval Service of Research and Development. First efforts were focused in the examination of the acoustical scattering behaviour of single-species cultures of Skeletonema costatum. For this purpose a pulse-echo electronic equipment was developed, including home-made piezoelectric transducers with an operating frequency centred at 2.6 MHz. A slight modified Johnson's fluid-sphere model was used. Numerical Abundance estimates from at-lab acoustic determinations of Volume Backscattering Strengths were obtained. When they were compared with predicted values computed through the adopted model and traditional optical counting, satisfactory results were obtained. More recently, single cultures of dinoflagellates, namely, Gymnodinium sp., were selected to enable a further stage of acoustic laboratory measurements. Controlled observations of its optimum growth conditions along a six-months period have been performed. The effects of different patterns of light and temperature have been analysed. Simultaneous work on modifying existing models of Backscattering Cross-Sections to provide a realistic description of dinoflagellates physical properties is being performed.

INTRODUCTION Consciousness about the remarkable importance of studying microscopic cells of drifting phytoplankton has significantly improved along the last two decades within the scientific community even among researchers not directly involved in marine biology, phycology and ecology disciplines. In particular, many exhaustive and rigorous studies about the two well known main types of microscopic marine algae, diatoms and dinoflagellates, have been reported in the literature. Since these oceanic groups have a relevant first-step position in the whole food web and they account for more than half of the photosynthesis on earth, providing oxygen to the atmosphere and regulating the carbon dioxide that controls the earth's climate, investment of time and scientific efforts in their investigation from a wide spectrum of approaches seems a task well-worthy to be encouraged. On the other hand, current increasingly occurrence of the so-called micro-algae "blooms" in coastal waters has become a natural phenomenon that cannot be neglected, mainly in the cases of some species producing endo- or exotoxins that may harmfully affect aquatic life leading even to produce direct health effects such as the well known "red tides". Traditionally, determinations of Numerical Abundance of marine micro-algae, are usually held because of their role as biological indicators of ecosystem integrity and their rapid response to water quality changes. They are routinely obtained by counting subsamples of specimens caught with planktonic nets. This process, usually tedious and time consuming, provides detailed descriptions, as well as quantitative estimates of species with a time-lag after samples are taken. This article deals with an alternative methodology consisting in the simultaneous application of sound scattering theoretical modelling (Greenlaw, 1979; Kristensen et al., 1986) and electro-acoustic measurements (Stanton et al., 1998) to asses detection and monitoring of toxic algae blooms through estimates of Numerical Abundance and consequent Biomass of phytoplanktonic algae. For this purpose, a multidisciplinary research programme has being conducted in the Argentinean Naval Service of Research and Development . Successive steps in this direction led to already reported results [Blanc et al., 2000] which can be summarised as an acoustical exploration of phytoplanktonic micro-algae response to ultrasound sonorization. Regarding the followed experimental approach, a pulse-echo electronic equipment was designed and constructed, including home-made piezoelectric transducers with an operating frequency centred at 2.6 MHz, in order to accomplish at-lab acoustical controlled essays of scattering of sound from single-species cultures of diatoms, namely, Skeletonema costatum (Fig. 1). Fig . 2 schematically shows the experimental set-up. Concerning the theoretical approach, a fluid-sphere model, the so-called high-pass model due to Johnson (Johnson, 1977) has been slightly modified to include the effect of the siliceous elastic-shelled cell wall of diatoms on their acoustic behaviour. Comparisons between theoretical values of Volume Scattering Strengths [Urick, 1975], SV, compatible with the scattering model adopted and traditional optical micro-algae counting, showed a reasonable good agreement with electro-acoustic measurements. That first stage of encouraging results justified further work on improving the method to conduct at-sea measurements in the future. Previously, analogue at-lab essays with single-species cultures of dinoflagellates, namely, Gymnodinium sp., were planned. From the biological point of view, the results of controlled observations of their optimum growth conditions along a six-months period are presented here. From the acoustic point of view, first results on an attempt of modelling Backscattering cross-sections, SIGMA (m2 ), of the selected species with a realistic description of their physical properties are reported.

a) Skeletonema costatum (small cells linked firmly with long external tubes of the marginal strutted. processes). Source: UTEX Bank of Data, LB 2308. Date: 5/17/94.

b) Image of Skeletonema costatum that was sonorized at 2.6 MHz during at-lab essays.

c) Skeletonema costatum. Illustrative details. Source:

d) Skeletonema costatum. Cells unions. Source: Skeletonema_costatum.htm

Fig. 1: Diatoms: Illustrative details of single-species cultures of Skeletonema costatum.

Fig. 2: Schematic diagram of acoustic measurements.

MATERIAL AND METHODS Biological aspects. In order to enable the exploration of the acoustic response of single-species cultures of micro-phytoplankton algae through controlled systematic at-lab sonorization, some practical procedures associated to biological aspects of the essayed samples could not be neglected. An optimum system to ensure their growth and maintenance had to be set for each selected specimen. First efforts were focused in the examination of the acoustical scattering behaviour of a diatom, namely, Skeletonema costatum, mostly due to the quite easy conditions required by traditional culture methodologies (Zaijic, 1970). When an attempt to use equivalent recommendations from the literature with the selected dinoflagellate, Gymnodinium sp., several practical problems could not be easily overcome (Fig. 3). Evaluations of its growth and maintenance were performed along six months at the Dept. of Biological Sciences of the Rowan University of New Jersey through two successive three-months duration series of at-lab essays. The 1 rst. Serial was devoted to analyse the effect of simultaneous variations in temperature and light:darkness regimens while the 2 nd. Serial attempted to analyse the influence of fluorescent light intensity. Each serial consisted of two experiments and each of these experiments was performed with an unique culture along nearly successive periods of time (approximately 10 weeks-duration). While cultures were submitted to three different external conditions during both experiments of 1 rst. Serial, other five different external conditions were set for the other two experiments of the 2 nd. Serial. Every time that optical counting of number of cells was done, a measurement duplication was also held with the objective of avoiding rough measuring uncertainties. Table 1 summarises external conditions to which the cultures were submitted during both experiments within each serial. Prime numbers only indicate the above mentioned measurements duplications. In all cases, manual shaking was about 2 or 3 times/day during approximately 20 min. in all cases. Gymnodinium sp. single-cultures were obtained from the Algae Cultures Collection of Texas University (UTEX LB 1654). The used culture medium was a slight modification of Erdschreiber's medium [Provasolli et al., 1957]. It contained (a) 1000 ml of filtered sea water (using filters of the type Whatman No 1) from coastal areas with low pollution levels; (b) 1 ml of a mixture of salts composed by NaNO3 (2g/100ml), Na2 HPO4 .12H2 O (0,3g/100ml) and B 12 vitamin (150mg/100ml); and (c) 50 ml of

overlying soil (mixture consisting of garden-soil with distilled water in a 1:4 soil-water ratio, where the overlying fluid has been filtered). Garden soil with low silt-clay content, moderate humus content and lack of commercial fertilisers was used. The components indicated in (c) and (b) were added to component set in (a), before being submitted to autoclave effects. At that precise moment, a solid precipitate usually appeared in the mixture. It had to be eliminated before inoculating the culture medium with Gymnodinium sp. Each culture was performed in a Erlenmeyer whose volume of 250 ml was filled with 150 ml of culture medium and inoculated with 2 ml of the original culture of Gymnodinium sp. Traditional optical counting of Numerical Abundance (Number of cells/ml) was done at the beginning of each experiment. Subsequent counting at regular time intervals along 10 or 11 weeks were carried out with a Sedgwift Rafter camera and a phase-contrast Leitz Laborlux microscope.

a) Gymnodinium sp. Illustrative details. Source:

b) Image of Gymnodinium sp. that will be sonorized at 3 MHz during at-lab essays.

c) Photo from a single-species culture of Gymnodinium

d) Photo from a single-species culture of Gymnodinium

sp. observed along 1 rst. Serial. Dept. Biological Sciences. Rowan University of New Jersey.

sp. observed along 1 rst. Serial. Dept. Biological Sciences. Rowan Univ.of New Jersey.

Figure 3: Dinoflagellates: Illustrative details of single-species cultures of Gymnodinium sp.

Modelling acoustic Backscattering cross-sections. Whenever a signal is emitted within the sea from an acoustic source, it meets a great variety of inhomogeneities which implies discontinuities in the physical properties of the medium such as density and compressibility, that means an acoustic impedance contrast with the fluid medium. These inhomogeneities intercept and reradiate a fraction of the incident acoustical energy producing "sound volume scattering" at the receiver terminals. Acoustic scattering effects due to the presence of single-species cultures of Skeletonema costatum could be already detected [Blanc et al., 2000] and consequently, their Volume Backscattering Strengths, SV, were computed from echo voltages measurements. When those experimental values were compared with theoretical predictions, a slightly modified Johnson's model [Johnson, 1977] for Backscattering cross-sections was used. Its main modification consisted in the inclusion of silica shell cellwall effects that characterise Skeletonema costatum. It is pointed out here that these organisms have a basic pill-box structure (FIG. 1) that may provide a geometrically complex cross-section to incident acoustic waves. An analogous Backscattering cross-sections model for single-species cultures of Gymnodinium sp. will be needed in further stages of this research project. For this purpose, a first attempt to use the same model has been performed. However, these organisms main physical properties, significantly different from those of Skeletonema costatum, are taken into account, such as the ones derived from their typical theca (armour of cellulose arranged to protect the interior of the cell) that give them a streamlined shape.

RESULTS AND DISCUSSION Optimum growth conditions for Gymnodinium sp. FIG. 4 graphically shows the results obtained for three different environmental conditions, as indicated in TABLE I, in both experiments of the 1 rst. Serial. From observation of its plotting _ Number of cells/ml vs. Weeks _, it derives that the so-called growth Conditions N° 1-1' and N° 3-3' produced the major number of cells. In particular, it is observed that under Conditions N° 1-1' algae population achieves its maximum during the time interval 22 nd -28 th days after the culture initiation, whereas the maximum occurs in the period 43 rd -48 th days under Conditions N° 3-3'. For Conditions N° 2-2', the cultures suffer a gradual declination till algae death is completed in the period 15 th -20 th days. Meanwhile, in the 2 nd Serial, some significant differences with theoretical predictions reported in the specialised literature are found [Carefoot, 1968; Zaijic, 1970]. FIG. 5 shows the results for five environmental conditions, as indicated in TABLE I. Plotting observations show that the maximum growth corresponds to Conditions N° 2-2'. Likewise, algae growth turned out to be relatively good for Conditions N° 3-3'. On the other hand, at low light intensities no algae growth took place. Modelling acoustic Backscattering cross-sections of Gymnodinium sp. FIGS. 6 and 7 show 3D plotting Backscattering crosssections, SIGMA, (in logarithmic scale) vs. equivalent sphere radius and frequency, for the respective diatoms and dinoflagellates cultures here examined. Different estimated density and sound speed contrasts values (that is, ratios of the assumed spherical scatterer to the medium properties) were used in each case. Therefore, the obtained two-variable function, SIGMA reaches a maximum predictable value of 2.2 x 10 -13 m2 in Skeletonema costatum but a maximum nearly two orders of magnitude less, 5.8 x 10 -15 m2 , in Gymnodinium sp., for 1 MHz - 4 MHz frequencies range and 0.1 mm 15 mm equivalent sphere radius range. In spite of their different properties, both types of phytoplankton have been modelled following a well known successfully proved assumption, namely, acoustically small non-spherical bodies whose dimensions are less than the wave-length have a sound scattering behaviour equivalent to actual spheres of the same volume and same average physical properties. At this stage of the research programme conducted in the Argentinean Naval Service of Research and Development presented here, efforts have been focused on investigating the best conditions for the quick growth of cultures of Gymnodinium sp. Results lead to conclude that the best conditions are obtained at 27°C, under a 16:8 hs light:darkness cycle and at 70 cm of light source-culture. In addition to this, simultaneous efforts have been invested in theoretical modelling of an appropriate first-approximation description of their Backscattering cross-section destined to go on with further steps of this project.

ACKNOWLEDGMENTS The authors acknowledge the valuable collaboration provided by Mr. Michael Furey during at-lab biological measurements and data analysis.

REFERENCES Blanc, S.; Benitez, C.; Milou, M.; Mosto, P.; Lascalea, G. & Juárez, R. 2000. "Acoustical behaviour of phytoplanktonic algae". Acoustics Letters, 23, (9). [ [ Links ]1] Blanc, S.; Benitez, C. E.; Milou, M.; Mosto, P.; Lascalea, G. 2000. "Acoustical estimation of diatoms' biomass". Proceedings of the Fifth European Conference on Underwater Acoustics, ECUA 2000, Lyon, France, 1479-1484. [ [ Links ]2] Blanc, S.; Mosto, P.; Benitez, C.; Juárez, R.; Milou, M. & Lascalea, G. 1998, "Acoustic response of phytoplanktonic volume scatterers at ultrasonic frequencies as an indicator of pollution in sea waters" in Environmental Coastal Regions, edited by C. A. Brebbia, WITPress Computational Mechanics Publications, Southampton, UK, 231-241. [ [ Links ]3] Blanc, S.; Milou, M.; Benitez, C.; Mosto, P.; Juárez, R. & Lascalea, G. 1998. "Indirect Analysis of Coastal Seas Pollution: Ultrasonic Volume Scattering Cross Sections of Phytoplankton." Proceedings of Coastal and Marginal Seas Meeting. UNESCO, Paris, France, in Oceanography 11, 2, 44. [ [ Links ]4] Carefoot, R. 1968. Culture of freshwater dinoflagellate Peridinium cinstum. Journal of Phycology. 4: 129-131. [ [ Links ]5] Clay, C. C. & Medwin, H. 1977. Acoustical oceanography: Principles and Applications. Ed. by John Wiley & Sons, pp. 24. [ [ Links ]6] Foote, K. 1990. "Speed of sound in Euphausia superba". Journal of the Acoustical Society of America, 87, 1405-1408. [ [ Links ]7] Greenlaw, Ch. F. 1979. "Acoustical estimation of zooplankton populations". Limnology Oceanography, 24 (2), 226-242. [ [ Links ]8] Holliday, D. V. 1980. "Volume scattering strengths and zooplankton distributions at acoustic frequencies between 0.5 and 3 MHz". Journal of the Acoustical Society of America 67 (1), 135-146. [ [ Links ]9] Johnson, R. 1977. "Sound scattering from a fluid sphere revisited". Journal of the Acoustical Society of America , 61, 375-377. [ [ Links ]10] Kristensen, A. & Dalen, J. 1986. "Acoustic estimation of size distribution and abundance of zooplankton". Journal of the Acoustical Society of America , 80 (2), 601-611. [ [ Links ]11] Lee, R. 1995, Phycology. University of Cambridge Pub., pp. 645. [ [ Links ]12] Provasolli, L.; Mc Laughin, J. & Droop, M. 1957. "The development of acquisition media for marine algae". Archives Mikrobiology, 25, 392-428. [ [ Links ]13] Stanton, T. K.; Chu, D.; Wiebe, P.; Martin, L. V. & Eastwood, R. 1998. "Sound scattering by several zooplankton groups. I. Experimental determination of dominant scattering mechanisms". Journal of the Acoustical Society of America, 103 (1), 225-235. [ [ Links ]14] Stanton, T. K. & Chu, D. 1998. "Sound scattering by several zooplankton groups. II. Scattering models". Journal of the Acoustical Society of America 103 (1), 236-253. [ [ Links ]15] Urick, R. 1975. Principles of Underwater Sound, edited by Mc Graw-Hill Inc., 216. [ [ Links ]16] Zaijic, J. E. 1970. Properties of algae. New York-London. Plenum Press.367 pp. [ [ Links ]17]

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