The toxicity of chlorine to a common vascular aquatic plant

Water Res Vol. 18. No. 8. pp. 1037-1043. 1984 Printed in Great Britain. All rights reser',ed

0043-1354 8453.00+0.00 Cop.~right ~ 1984 Pergamon Press Ltd

THE TOXICITY OF CHLORINE TO A COMMON VASCULAR AQUATIC PLANT CHARLES H. WATKINS and RICHARD S. HAMMERSCHLAG EcologicaI Services Laboratory, National Capital Region, National Park Service, Washington, DC 20242, U.S.A. (Received December 1983) Abstract--Myriophyllum spicatum was exposed to various chlorine concentrations on a continuous and intermittent basis in 96-h toxicity studies utilizing a proportional diluter. Continuous exposure to chlorine concentrations as low as 0.05 mg 1-~ total residual chlorine (TRC) depressed shoot and total plant dry weights approx. 30~, relative to controls. Shoot length was depressed approx. 16°;oat this concentration. Chlorophyll a was depressed 25~o at 0.1 mg 1-t TRC. However, intermittent exposure of plants to chlorine for three 2-h periods daily for 96 h indicated an insensitivity to repeated short term chlorine exposure at all concentrations but 1.0 mg 1- ~TRC. These results indicate that high level chlorine discharges from waste water facilities and electric generating plants could be a contributing factor impacting nearby submerged aquatic vegetation. INTRODUCTION The biotic impacts associated with the use of chlorine as a disinfectant and biocide have been the subject of considerable scientific investigation in the last decade (Brungs, 1973, 1976; Jolley, 1978; Jolley et al., 1978; 1980). Acute and sublethal toxic effects have been observed at various chlorine concentrations to a variety of marine and freshwater organisms. Concern over chlorine's impact on primary productivity has extended research to the phytoplankton community where mortality, reduction in cell growth, as well as depression of photosynthesis, respiration and nutrient uptake have been documented. One area that has not received sufficient attention is the toxicity of chlorine to vascular aquatic macrophytes. Chlorine toxicity to emergent aquatic plants has only been implicated in studies of chlorinated sewage effluent application to freshwater tidal wetlands (Whigham and Simpson, 1978). Reduction in above-ground biomass, change in species composition and elimination of species was attributed to chlorine by those authors although no chlorine measurements were made. In 1976 National Park Service studies were begun to investigate the basis for the disappearance (since the 1930s) of submerged and emergent aquatic vegetation in the upper Potomac estuary near Washington, DC. Field studies involved placing wild rice (Zyzania aquatica L.) and a number of species of submerged aquatic plants in selected areas of the upper Potomac and Anacostia estuaries. Inhibitions in plant growth were observed, seemingly correlated with proximity to electric generating and waste treatment plant outfalls (Wester and Rawles, 1979). Differences in turbidity, suspended solids, depth and flow rate, etc., in these field tests made meaningful comparisons of plant growth data difficult. Nonetheless, the combination of observed depression of

test plant growth near the mentioned facilities, the known discharge of chlorine by these facilities and well described biocidal activity of chlorine suggested chlorine be investigated for its potential role. The purpose of this study was to develop, under controlled laboratory conditions, quantitative data on chlorine toxicity to a c o m m o n submerged aquatic plant. MATERIALS AND METHODS Eurasian watermilfoil (Myrioph.vllm spicatum) was exposed to chlorine concentrations of 0.0, 0.02, 0.05, 0.1, 0.3, 0.5 and 1.0 mg 1-t measured as total residual chlorine (TRC) on a continuous and intermittent basis in 96-h toxicity studies. Chlorine's instability necessitated the use of a proportional diluter to sustain toxicant concentrations. Municipal water aged and filtered through activated carbon to remove chlorine, NH4+ and other materials served as the water source. The toxicant was obtained by bubbling commercially available CI., gas into distilled water. Diluter system Though similar to a Mount and Brungs type apparatus (Mount and Brungs, 1967), the diluter (Fig. 1) incorporated a number of modifications designed to accommodate the wide chlorine concentration range studied. The lower cells of the Mount system were replaced with six plastic centrifuge tubes (after Chandler et al., 1974) for retaining different volumes of partially diluted chlorine. Every 6.5 min, 500-ml flows from water cells 4 to 9 emptied these centrifuge tubes, delivering an appropriate quantity of chlorine for final dilution and delivery to the six 11, toxicant test chambers. The flow from water cell 10 triggered the vacuum system and delivered chlorine free water to the control test chamber. Flows from water cells 2 and 3 simultaneously diluted chlorine from the Marriotte bottle chemical metering device (McAllister et al., 1972) and the secondary toxicant reservoir for subsequent refill of their respective centrifuge tube series 1-3 and 4-6. Volumes retained in the secondary toxicant reservoir and the centrifuge tubes were determined by the height of the drain in each, the excess flowing to the next container. Fine adjustments were achieved using glass beads which served to displace the toxicant solution. Toxicant solution draining from tubes 3 and 6 was discarded. Retention box couples R~-R., and R3-R4 served as mixing

1037

t038

CHARLES H. VCaTKINS and RICHARD S. HAMMERSCHLAG WATER INFLUENT

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chambers while delaying refill of the centrifuge tubes until flows from cells 4 to 9 had passed. Approximate chlorine concentrations in the diluter were as follows: Marriotte bottle, 2000 mg 1-~; secondary toxicant reservoir and centrifuge tubes 4 to 6, 14 mg 1-'; centrifuge tubes 1 to 4, 7 mg 1- J. Chlorine concentrations in the test chambers var/ed no more than 10°/~ from the intended value during diluter cycling. In the intermittent chlorination studies an automatic timer was used in conjunction with a solenoid operated drain valve in retention box R,. The normally open drain valve was closed for three l-h intervals (7-8 a.m., 3--4 p.m., 11-12 p.m.) each 24-h period permRting toxicant delivery. Chlorine concentrations in the test chambers rose gradually (Fig. 2) reaching 90% of the target concentration after live diluter cycles or 32 rain. At the end of the chlorination period concentrations fell off more rapidly due to the natural dissipation of the toxicant. It should be noted that there was considerable overlap among the test concentrations as chlorine levels approached and receded from the target concentration. Further, target concentrations were achieved ['or only a fraction of the 2-h period plants were

exposed to chlorine. Mean chlorine levels during this period were thus substantially lower than those reported. The water supply system (Fig. 3) consisted of a series or live 190-1. polyethylene tasks, four of which were connected by three 7.7 x 35-cm filters each containing 815cm 3 activated carbon. When tank T s emptied, float switch S, activated a relay system which turned off pump P: and turned on pump P~ and the two nutrient supply pumps. Refill o f T s took 5 min at which time float switch Si shut off Pj and the nutrient pumps and restored the flow to the diluter. This process occurred every 4.5 h. Since inlet pipes to 1"2, T~ and "I"4were 5-cm lower than the water level set in T~ by the float valve, water gradually flowed ~through the filters, refilling "1"4prior to the start of the next pumping cycle. The supply system contributed to dechlorination by aging the municipal water approx. 16 h through a hatching effect. Total residual chlorine was measured with an amperometric titrator (Fisher model No. 393) modified by the addition of a digital extended range voltmeter. Titrations were performed repeatedly throughout the 96-h test on

Toxicity of chlorine

1039

CONSTANTHEAD CYLINDER

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samples withdrawn from each test chamber, using commercially available buffer, potassium iodide and phenylarsine oxide solutions. The phenylarsine oxide was diluted 50% with distilled water permitting a more controlled approach to the end-point. A 15-h photoperiod was provided by four Durotest Vitalite 122-cm fluorescent tubes suspended l l-cm above the test chambers and acclimation tank, yielding 164 microeinstein units m -z s -) (8800 Ix) at water surface. Nutrients The nutrient regimen was provided by a modified Hoagland's solution of inorganic micro- and macronutrients (Table 1). A single stock solution containing all chemicals except magnesium sulfate was metered by a peristaltic pump at the rate of 0.63 ml 1-) to water supply tank T~ during each filling cycle. Magnesium sulfate was prepared separately to prevent precipitation and metered to T~ at the rate of 0.1 ml 1-~. These rates yielded approx. 0.025 Hoagland's concentration when fully diluted in the water supply and provided for healthy plant growth yet minimized algal growth during the tests. Plant specimens M. spicatum was collected from the headwaters of Nanjemoy Creek, Maryland, a tributary of the Potomac estuary 55 miles downriver from Washington, DC. Stems were cut approx. 10cm from the apical meristem, rooted below the apical meristem in mud obtained from a local pond and maintained in concrete holding tanks in a greenhouse or an indoor 378-1. polyethylene tank containing aged municipal and Potomac river water. At the beginning of each experiment, 40 plant stems, each containing an apical growing point, were retrieved and rinsed in aged municipal water to remove surface debris. Stems were cut to 5-cm lengths and inserted to depth of 3-cm in 30-ml plastic cups containing mud covered by a l-cm layer of sand. (In order to encourage growth of a single terminal shoot any budding branches were removed prior to rooting in the cups, at the end of the acclimation period and at the end of day 2 of the test.) The plants were placed in a 35-1. aquarium for a 5-day acclimation period. Fresh nutrient/water solution was introduced throughout the acclimation period at a rate of 5 1 h-L Water supply, nutrient concentration, photoperiod and temperature were identical to those existing during the actual test. At the end of the acclimation period, 24 plants were selected and loosely sorted into three similar sized groups of 8 plants each. Seven plants from each group were randomly assigned to the test chambers; 1 plant to the control chamber and 1 to each of the six test concentration chambers. Before placing in the test chambers, stem length was

WR 18,8-~H

measured from soil surface to apical meristem. The number of nodes occurring between soil surface and apical meristem of each plant were counted. The three remaining plants (one from each group) were set aside and called representative plants, dried in an oven at 3 9 C for 2 days and then weighed. Growth measurement At the end of the 96-h test period, stem length was measured and node counts taken. The plants were then removed from the test chambers and cups. Roots were washed of any mud and organic debris and then separated from the shoot. One of the four leaves from each of the third, fourth and fifth node of each plant was randomly selected for chlorophyll extraction. Dry weights were obtained separately for the remaining shoot material and roots of each plant. Dry weights of the cell material obtained in the chlorophyll extraction process were subsequently added to the dry weight of the shoot from each plant. Data for increase in shoot dry weight was obtained in each test by subtracting an average value of the representative plants' dry weights from the final dry weight of the shoots in each treatment. Roots were incipient at the beginning of each test and their initial dry weight was considered to be zero. Total plant dry weight equalled corrected shoot dry weight plus root dry weight. Chlorophyll extractions were performed using a procedure adapted from the spectrophotometric determination of chlorophyll a in algae (APHA, 1975). Immediately after separation from the shoot, the three leaves were combined in individual beakers containing 0.2-ml MgCO 3 suspension diluted with 5-ml distilled water. After approx. 30 rain, leaf material was macerated in 5-ml 90)
1040

CHARLES H . WATKINS a l l d RICHARD S. HA.'.IMERSCHLAG

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Continuous chlorination Continuous 96-h exposure of M. spicatum t o a chlorine concentration of 0.05 mg 1-~ TRC resulted in a statistically significant 16.2% reduction m shoot length relative to control plants (Table 2). Higher chlorine concentrations depressed shoot growth further, reaching an 88.2% reduction at 1.0 mgl -~. A reduction of 5.8% at 0.02 mg 1- x was not statistically significant. Depression of shoot growth appeared to result from the inhibition of internodal stem elongation, as plants typically gained two to five nodes over the course of each test regardless of chlorine concentration. Dry weight data indicated statistically significant reductions of 30.9 and 28.5% for the shoot and total plant dry weight parameters at 0 . 0 5 m g l -~ TRC. Higher chlorine concentrations depressed biomass increase severely, reaching an almost total suppression at 0.5 mg 1-~. Shoot biomass was actually reduced relative to representative plants at 1.0 mg I Root dry weights were not statistically different from control plants at 0.02 and 0.05 mg 1- ~, but suffered a significant 34.8~ reduction at 0. l mg t-' and a 7 0 ~ reduction at 1.0 mg I-'. The higher chlorine concentrations did not affect roots as severely as shoots,

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1041

probably due to the protection from the chlorine afforded by the rooting medium. Chlorophyll a measurements indicated statistically insignificant reductions of approx. 10°~oat 0.02 and 0.05mg1-~ TRC. Higher concentrations progressively affected this parameter and were statistically significant, reaching a 71.4~/o reduction at 1.0 mg 1- t. A gradual blackening of leaf tissue was noted at 0.1, 0.3, 0.5 and 1.0 mg 1-~, being consistently more pronounced in the basal leaves. The severity and rapidity of this necrotic discoloration was concentration dependent, noticeable at 1.0 mg 1-' within 6 h. With the exception of meristematic tissue, leaves exposed to 1.0 mg 1-~ were subsequently bleached and by day 3 were prone to detachment. Growing points and newly generated lateral buds at all concentrations retained their color, and remained viable though were observed to be progressively reduced in size and general vigor with increasing chlorine concentrations. Gas bubbles, possibly indicating oxygen production, were observed adhering to leaf surfaces by day 2 of each test. Bubble density seemed to vary inversely with chlorine concentration and was noticeably reduced on 0.02 mg l -t plants compared with controls. Further reductions were evident at higher concentrations with 0.5 and 1.0mg 1-~ plants having virtually no attached bubbles. One test was run in which plants were exposed to continuous chlorination for 5 days followed by a 5-day chlorine-free recovery period. Heavy algal growth precluded chlorophyll a and dry weight measurement, however shoot length data indicated a depressed ability to resume normal growth immediately following release from chlorine exposure at 0 . 1 m g l -t and above. While upper leaves at 0.1 mg 1-~ were able to recover normal color, lower leaves at the higher concentrations were not and they eventually died. New growth was progressively less vigorous at 0.3 and 0.5 mg 1-t with 1.0 mg 1-~ plants spindly and stunted. Stem length measurements taken during the recovery period indicated evidence of a relative acceleration in shoot growth for all concentrations in the latter half of the recovery period. Although not reflected clearly in the mean data presented in Table 2, superior growth under low chlorine conditions was noted in a number of the continuous chlorination tests. Using an average value of the three plants in each treatment, a chlorine concentration of 0.02 mg 1-~ resulted in an increase (greater than 15~ relative to controls) in two out of six tests in the parameters length and chlorophyll a, three out of six in the parameters shoot and total plant dry weights and four out of six tests in root dry weight. Length, chlorophyll a and root dry weight were increased in two out of six tests at 0.05 mgl -t. Intermittent chlorination

Under intermittent chlorination conditions (Table 3), results were highly variable and for many parameters plants grown in the three lowest target concen-

1042

CHARLES H. WATKL','Sand RICHARDS. HAMMERSCHLAG

trations did significantly better than the controls. Chlorine at 0.02 mg I- resulted in substantial increases in length and shoot, root and total plant dry weights. Shoot and total plant dry weights were similarly increased at 0.05 and 0.1 mg l ~ TRC. Data for all parameters were not significantly different t¥om controls at 0.3 and 0.5 mg 1-'. A target chlorine concentration of 1.0mg 1- resulted in significant depressions of only length, root dry weight and chlorophyll a. Epiphytic growth proved to be a recurrent problem in these studies. In the continuous chlorination tests epiphyton were frequently evident on chlorine-free control plant leaf surfaces and test chamber wails by day 3. Plants and test chambers exposed to chlorine remained free of algal growth. In the intermittent tests, epiphyton was not limited to the controls. appearing with progresswe delay on plants and chamber walls exposed to 0.02 and 0.05 mg 1-~ TRC.

DISCUSSION Results from this study demonstrated chlorine toxicity to the vascular aquatic plant, M . spicatum, at relatively high chlorine concentrations compared to most phytoplankton toxicity data, While Brook and Baker (1972) reported a 50~/; reduction in photosynthesis and respiration rates at 0.34 mg I -T TRC, more recent research indicates a much greater sensitivity. In 2- to 4-h incubation studies Eppley et al. (1976), documented a 5 0 ~ inhibition in ~4C-uptake rates at 0.1 mg I-~. Similar reductions were noted at 0.01 mg I-~ with a 24-h incubation period. Toetz et al. (1977), documented a 50~. reduction in NO3 uptake rates with 0.028mgl -~. Nitrate uptake was completely blocked at 0 . l i n g I -~ Research by Brooks and Liptak (1979) indicated measurable reductions in ~4C-uptake rates following a 30min exposure to chlorine concentrations as low is 0.003 mg 1-~, although good recovery at all concentrations below 0.1 m g l -~ was noted in a subsequent 24-h observation period. Chlorophyll a was observed to decline only slightly at concentrations below 0.1 mg 1-~. in apparent agreement with the present work despite differences in exposure time. In this study, concentrations required to achieve a 50~ reduction in growth under continuous chlorination conditions occurred in the 0.1-0.4mgl -~ range (from Table 2) depending on the parameter measured. Inhibitions of plant growth were nevertheless substantiated at fairly low chlorine levels, 0.05 mgl-~. Results from the intermittent chlorination tests indicated an insensitivity of growth reduction to the repeated short term exposures at all target concentrations but 1.0 mg 1-~ TRC during the 96-h test period used in this study. While the 50~ inhibition level is a useful basis of comparison, it should be applied cautiously when comparing results from studies employing dissimilar

measures of toxicity. The o~erall growth measures used in this study would be expected to be tar tess sensitive than the biochemical indicators used m phytoplankton research. Further. in an experimental design of this nature percent growth relatwe ~o control is directly related to the time period of the study. Given a longer test period, greater reductions in growth would normally be expected. Epiphyton growth on test plants was a complicating factor in this study. Relying principally on the gross measures length and biomass to detect toxicity. individual tests were mtended to last 10 days or more. However. m preliminary continuous chlorination tests using this longer protocol, 0.02 and 0.05 mg 1 plants rapidly outgrew the controls after day 4. presumably as epiphyton density on the latter increased. Depression of host photosynthesis has been correlated with the presence of epiphyton which act as a barrier to carbon uptake and reduce light penetration to leaf surfaces (Sand-Jensen. 1977). An inhibitory effect is further supported by plan! growth patterns in the intermittent tests. The propensity for superior growth under low chlorine conditions was shifted to the 0.05 and 0. I mg 1-' plants coincident with the appearance of epiphyton on 0,02 and 0.05 mg I -~ plants. In order to reduce the influence of epiphyton, tests were limited to 4 days, a time frame which impaired the ability of length and biomass measures to evidence small changes in plant growth rates. Furthermore, variation in the epiphyton populations which frequently developed within even this time frame may explain the relatively superior growth of lower dose chlorinated plants observed m some intermittent and continuous chlorination tests. Differences in gas bubble density on leaf surfaces in this study are suggestive of a chlorine induced inhibition of photosynthesis similar to that documented with phytoplankton. Further research is clearly needed in this area. Future studies employing the more instantaneous and sensitive measures of radioactive labelled carbon and nutrient uptake would avoid the complications encountered in this bioassay and may document inhibition of important biochemical processes at chlorine concentrations consistent with phytoplankton toxicity data. The relative toxicities of free and combined chlorine should also be investigated in recogninon of the predominance and persistence of the combined form in aquatic systems. Chlorine's effect on reproduction and plants' sensitivity to chlorine when weakened by other physical and chemical stresses are other areas needing study. Research documenting recovery in carbon uptake following short term exposure to chlorine (Brooks and Liptak, 1979), and the ability of rapidly regenerating phytoplankton communities to compensate for dead cells (Goldman and Davidson. 1977) suggests that intermittent chlorination may have little overall impact on primary productivity of phytoplankton. The same may not hold true for rooted aquatic plants which lack phytoplankton's intrinsic

Toxicity of chlorine regeneration rate and have comparatively complex life cycles (see, for example Sculthorpe, 1967), rendering them potentially less adaptive to stress. Repeated short-term exposures from power plants alone or persistent chlorine residuals formed in conjunction with chlorinated sewage effluent may have long term impacts on these populations. While chlorine at low concentrations is clearly not lethal to M. spicatum in the short-term, this study does demonstrate growth impairment at such chlorine levels. Thus if conditions are ever appropriate for sustaining these chlorine levels in the upper Potomac estuary or elsewhere, chlorine would be a contributing factor to stress and decline in sensitive aquatic plant populations. Further, growth reduction apparent only over the course of a growing season at chlorine concentrations currently below the level of detectability is a possibility which should not be dismissed. The impact of chlorine on vascular aquatic plant populations would, in any case, appear to be subtle and would likely occur only in conjunction with other environmental stresses. For example Phillips et al. (1978), in discussing early growth of submerged aquatic vegetation, stresses the necessity of plants reaching the photic zone where net photosynthesis can occur, before stored energy is depleted. Under highly turbid conditions, such as exist in the Potomac estuary, inhibition of stem growth from chlorine might restrict plant populations to a shallower margin. Reduced success would be exacerbated by a developed predator population leading to overall vegetation decline. Acknowledgements--The authors wish to express their thanks to Horace V. Wester who should be credited with initiating and conducting wide ranging investigations into chlorine's impact on aquatic macrophytes in the upper Potomac estuary. Thanks also to William W. Dix of the American University who was responsible for the design concept of the diluter used in this study. This work was supported by the Ecological Services Laboratory of the National Park Service, National Capital Region, Washington, DC. Disclaimer: Mention of a product or manufacturer does not constitute endorsement or approval by the U.S. Government or its agencies. REFERENCES

APHA (1975) Standard Methods for the Examination of Water and Wastewater, 14th Edition. American Public Health Association, Washington, DC.

1043

Brook A. J. and Baker A. L. (1972) Chlorination at power plants: impact on phytoplankton productivity. Science 176, 1414-1415. Brooks A. S. and Liptak N. E. (1979) The effect of intermittent chlorination on freshwater ph~toplankton. Water Res. 13, 49-52. Brungs W. A. (1973) Effects of residual chlorine on aquatic life. J. Wat. Pollut. Control Fed. 45, 2180-2193. Brungs W. A. (1976) Effects of wastewater and cooling water chlorination on aquatic life. U.S. Environmental Protection Agency, Ecological Research Series. EPA600/3-76-098. Chandler J. H., Sanders H. O. and Walsh D. F. (1974) An improved chemical delivery apparatus for use in intermittent-flow bioassays. Bull. encir, contain. Toxic. 12, 123-128. Eppley R. W., Renger E. H. and Williams P. M. (1976) Chlorine reactions with seawater constituents and the inhibition of photosynthesis of natural marine phytoplankton. Estuarine coast. Mar. Sci. 4, 147-161. Goldman J. C. and Davidson J, A. (1977) Physical model of marine phytoplankton chlorination at coastal power plants. Enrir. Sci. Technol. 11, 908-913. Jolley R. L. (Ed.) (1978) Water Chlorination: Em'ironmental Impact and Health Effects, Vol. 1. Ann Arbor Science, Ann Arbor, MI. Jolley R. L., Brungs W. A. and Cumming R. B. (Eds) (1980) Water Chlorination: Environmental Impact and Health Effects, Vol. 3. Ann Arbor Science, Ann Arbor, MI. Jolley R. L., Gorchev H. and Hamilton D. H. (Eds) (1978) Water Chlorination: Enrironmental Impact and Health Effects, Vol. 2. Ann Arbor Science, Ann Arbor, MI. McAllister W. A., Mauck W. L. and Mayer F. k. (1972) A simplified device for metering chemicals in intermittentflow bioassays. Trans. Am. Fish. Soc. 3, 55-57. Mount D. I. and Brungs W. A. (1967) A simplified dosing apparatus for fish toxicology studies. Water Res. I, 21-29. Phillips G. L., Emison D. and Moss B. (1978) A mechanism to account for macrophyte decline in progressively eutrophicated freshwaters. Aquat. Bot. 4, [03-126. Sand-Jensen K. (1977) Effect of epiphytes on eelgrass photosynthesis. Aquat. Bot. 3, 55-63. Sculthorpe C. D. (1967) The Biology of Vascular Aquatic Plants. Ballantyne, London. Toetz D., Varga L. and Pierce M. (1977) Effects of chlorine and chloramine on uptake of inorganic nitrogen by phytoplankton. Water Res. II, 253-258. Wester H. V. and Raw[es S. D. (1979) Impact of Chlorine Pollution in the Upper Potomac and Anacostia Estuaries (abs). First Conference on Scientific Research in the National Parks, New Orleans. Whigham D. F. and Simpson R. L. (1978) Nitrogen and phosphorus movement in a freshwater lidal wetland receiving sewage effluent. Coastal Zone "78," Proceedings of the Symposium on Technical, Environmental, Socioeconomic and Regulatory Aspects of Coastal Zone Management, ASCE/San Francisco, pp. 2189-2203.