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The role of algae in a deep wastewater self-regeneration pond
Wat. Res. Vol. 34, No. 14, pp. 3666±3674, 2000 7 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter
PII: S0043-1354(00)00107-X www.elsevier.com/locate/watres
THE ROLE OF ALGAE IN A DEEP WASTEWATER SELFREGENERATION POND M. ARAUZO*, M. F. COLMENAREJO, E. MARTIÂNEZ and M. G. GARCIÂA Centro de Ciencias Medioambientales, C.S.I.C. Serrano 115 dpdo., 28006, Madrid, Spain (First received 18 November 1998) AbstractÐThe present investigation was designed to gain information on the role of algae in a deep wastewater self-regeneration pond. The experimental pond was continuously fed with secondary euent from a municipal wastewater treatment plant. Biological, physical and chemical pro®les were recorded throughout the water column during the period July 1996 to December 1997. The removal eciencies of several chemical and sanitary indicators were calculated. The time course of species biomass was used to evaluate phytoplankton dynamics and its relationship with the performance of the deep pond. The eciency of the system seemed to be conditioned by temperature and phytoplankton biomass. Improved performance was observed during periods of mixing. Chlorococcales and Volvocales grew extensively during strati®cation periods. Euglenophyceae dominated during the mixing. When the total phytoplankton biomass exceeded 15,000 mgC mÿ3, the removal eciencies of the chemical oxygen demand (COD) and the suspended solids acquired negative values. These phytoplankton blooms were associated with an intense increase in pH and, consequently, in NH3 levels, in the epilimnion, during the strati®cation. When such a bloom occurred, considerable decreases in zooplankton and bacterial biomasses were observed, which temporarily destabilized the trophic structure of the pond. 7 2000 Elsevier Science Ltd. All rights reserved Key wordsÐurban wastewater, deep pond, algae, removal eciency, strati®cation, mixing
INTRODUCTION
An experimental deep self-regeneration pond was designed with the aim of improving the quality of wastewater euent for its reuse. The pond was supplied with secondary euent produced at a conventional treatment plant in which urban wastewater is treated. It was constructed at Plataforma Experimental de la Finca La Poveda (Arganda del Rey, Comunidad de Madrid, Spain). This facility is managed by Centro de Ciencias Medioambientales (C.S.I.C. ). Deep treatment ponds oer theoretical advantages over conventional stabilization ponds, which have greater surfaces and are shallower. They occupy less surface area and, in arid and semi-arid areas, summer evaporation losses are reduced. Deep ponds could become an inexpensive, simple way to improve the sanitary and chemical quality of water and, at the same time, be used as reservoirs for agricultural irrigation. Nevertheless, deep treatment ponds are infrequently used and their properties have not been extensively investigated (Moreno et al., 1984a, b, 1988; Berna et al., 1986; Torres et al., 1997). Previous studies have generally been directed
towards hydraulic, design and sanitary aspects. Despite being the key to the optimization of performance, to date, the biological communityÐthe engine of wastewater self-regenerationÐhas been the focus of very little research in deep treatment ponds (Soler et al., 1991). It is known that, in pond systems, algae stimulate bacteria, and bacteria stimulate algae (Palmer, 1980). In this way, the chemical units that comprised the organic waste eventually become incorporated into the algae as stable organic components of living cells. The death and decomposition of large numbers of algae lead to undesirable conditions similar to those caused by the original wastewater in the pond. Consequently, this needs to be avoided to keep the trophic structure well balanced for optimum performance. The present investigation involves the in-depth examination of the role of algae in a deep urban wastewater self-regeneration pond. Evaluation was made of the phytoplankton community, the dynamics of its populations and its relationship with the performance and the trophic structure of the pond. MATERIALS AND METHODS
*Author to whom all correspondence should be addressed. Fax: +34-1-564-0800; e-mail: [email protected]
The experimental pond was 4.75 m deep, had a volume of 2161 m3 and was continuously fed with secondary eu-
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Algae in a deep self-regeneration pond ent from a municipal wastewater treatment plant. The pond had the shape of an inverted truncated square pyramid, measuring 10 10 m at the bottom and 31 31 m at the top. The lining material was geotextile felt, over which a layer of high-density polyethylene was laid in order to prevent leakage. The in¯uent port was ®xed close to the bottom (4.5 m) during the course of the study. Euent ports were at depths of 1.5, 2.5 and 3.75 m from the surface layer, and all three were kept open throughout the experimental period. The ¯ow was regulated through a control valve and a magnetic ¯ow-meter. The pond was continuously fed with a ¯ow of 11 2 3 m3 hÿ1 from July 1996 to December 1997. This provided a mean hydraulic retention time of 924 days. The experimental design encompassed three time scales: . On a fortnight basis: physical, chemical and biological parameters were monitored at dierent depths of the water column (meter by meter) in the center of the pond. . On a twice per week basis: physical, chemical, biological and microbiological parameters on the in¯uent and the three euents were monitored in order to evaluate the performance of the pond. . On a daily basis: the input ¯ow and the functioning of the system and maintenance were checked. Samples were taken between 9:00 and 10:00 in the morning. Given the great variability of in¯uent composition depending on the time of day, composite samples were made collecting aliquots every hour over each 24 h period. These aliquots were linked to the rate of ¯ow. Sample taking was carried out with an automatic sampler equipped with a refrigeration system (model Streamline 800 SL). For the collection of samples at dierent depths from the center of the pond, an immersible, peristaltic pump was used. Temperature and oxygen pro®les were recorded using a YSI-57 dissolved oxygen ®eld meter. The following parameters were evaluated: . Physical and chemical parameters of in¯uent, euents and deep pro®les: temperature, dissolved oxygen levels, pH, conductivity, chemical oxygen demand (COD), total phosphorus, soluble reactive phosphorus (PO3ÿ 4 ), nitrate, nitrite and ammonia (NH+ 4 \ NH3) levels, Kjeldhal nitrogen content, total solids, total dissolved solids and total suspended solids (®xed and volatile). . Biological parameters in deep pro®les: bacterial community biomass, phytoplankton community biomass (primary producers) and zooplankton community biomass (secondary producers). . Microbiological analysis in in¯uent and euents: total coliforms (as a sanitary indicator). Physical, chemical and microbiological analyses were performed according to APHA (1989). Total nitrogen was taken as the sum (expressed in mgN lÿ1) of nitrate, nitrite and Kjeldhal nitrogen. NH3 was calculated from the quantity of its ionized form, taking into account pH and temperature (Alabaster and Lloyd, 1980). The euphotic zone was de®ned as the depth reached by only 1% of surface solar radiation. This was determined from measurements made by a Secchi disk, previously calibrated with an LI-COR 192 S photometer, and assuming that the extinction of light follows Lambert's law (Hutchinson, 1957). Removal eciencies were calculated as ``(In¯uent±Euent) 100/In¯uent''. Given the great variability of in¯uent quality throughout the study, monthly mean values of removal eciency have been used. Total bacteria were estimated by optical microscopy, bacterial density by the extension method of Breed (Harrigan and McCane, 1979) and biomass according to Norland (1993). Phytoplankton and protozoan densities (by species) were estimated by optical inverted microscopy
3667
(UtermoÈhl, 1958) and biomass according to Rott (1981). The densities and biomass of Rotifera and Crustacea (by species) were estimated following the method of Dumont et al. (1975). Zooplankton biomass was de®ned as the sum of the biomasses (expressed in mgC mÿ3) of Protozoa, Rotifera and Crustacea. Dierences in pond performance between the mixing and the strati®cation were estimated by the Student's ttest. One-way analyses of variance have been performed to compare variables recorded during the mixing and during the strati®cation, both in the epilimnion and the metalimnion (BMDP7D; Dixon et al., 1992). In the case of homogeneity of variances, a parametric test was applied to data. A non-parametric method was used when there was heterogeneity of variances (Brown-Forsythe; Dixon et al., 1992). A Levene test was used to previously determine the homogeneity of variances (Dixon et al., 1992). RESULTS
Stable, thermal strati®cations were produced during the summer (until August in 1996 and from April to September in 1997). During strati®cation, the mixed upper level (epilimnion) was observed to extend from the surface to a depth of 2 m. A lower strati®cation level of decreasing temperature gradient (metalimnion) was observed from 2 to 4.75 m. It was not possible to de®ne a stable hypolimnion. During the remaining periods the pond was mixed throughout its depth. The phytoplankton species observed during the study period are shown in Table 1. The mean values of the physical, chemical, bioTable 1. Phytoplankton species in the deep pond from July 1996 to December 1997 Bacillariophyceae Nitzschia graciliformis Lange-Bertalot and Simonsen 1978a Nitzschia palea (KuÈtzing) W. Smith 1856a Chlorophyceae Chlamydomonas sp. Ehrb. 1833a Chlamydomonas reinhardtii Dang. 1888 Chlorococcum infusionum (Schrank) Menegh. 1842 Coelastrum microporum NaÈg. in A. Br. 1855 Eudorina elegan Ehr. 1833 Micractinium pusillum Fresenius 1858 Monoraphidium komarkovae Nyg. 1979 Monoraphidium minutum (Naeg.) Kom-Legn. Oocystis solitaria Writt. in Writt. and Nordst. 1879 Pandorina morum (O. F. MuÈller) Bory 1824 Scenedesmus acumuminatus (Lagerh.) Chod. 1902 Scenedesmus arcuatus (Lemm.) Lemm. 1899 Scenedesmus quadricauda (Turp.) BreÂb. Sensu Chod. 1913, 1926 Euglenophyceae Euglena hemichromata Skuja 1948 Euglena oxyuris Schmarde 1946a Euglena proxima Dangeard 1901 Euglena viridis Ehrenberg 1830 Lepocinclis ovum (Ehr.) Lemm. 1901 Cyanophyceae Oscillatoria agardhii Gomont 1892a Oscillatoria chlorina KuÈtz ex Gomont 1892a Oscillatoria tennuis Agardha a
Species showing low occurrence and biomass (no more than two occurrences and biomass lower than 0.5% of the total phytoplankton biomass).
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logical and sanitary variables, recorded in the deep pond during mixing and strati®cation periods (both in the epilimnion and metalimnion layers), are shown in Table 2. A signi®cant dierence was observed between both periods with respect to the water temperature. A signi®cant reduction in the euphotic zone and increases in phytoplankton biomass, pH, NH3, total suspended solids and chlorophyll ``a'' were observed in the epilimnion during strati®cation. The lowest zooplankton level was in accordance with scarce dissolved oxygen in the metalimnion. Signi®cantly higher levels of total solids, total nitrogen and total coliforms were observed during strati®cation (both in the epilimnion and in the metalimnion) than during mixing. The time course of some of the removal eciency variables is shown in Fig. 1. The best performance (in terms of greatest reductions) was observed for nitrate, nitrite and total coliforms. The total coliforms, COD and suspended solid removal eciencies were greater during the mixing than during the strati®cation, showing t-values of 7.06, 4.28 and 4.99, respectively ( p < 0.0001, 15 degrees-of-freedom). However, nitrite removal was more ecient during the strati®cation, with a t-value of ÿ2.89 ( p < 0.02, 15 degrees-of-freedom). The remaining indicators of performance showed no signi®cant dierences between the periods of mixing and strati®cation. Water temperature was highly related to phytoplankton biomass (r = 0.61; p < 0.001). How both these variables seem to directly or indirectly control
the eciency of the system is shown in Table 3. The phytoplankton community time course throughout the water column is shown in Figs 2±4. Chlorococcales and Volvocales grew extensively during strati®cation, although species such as Chlamydomonas reinhardtii, Eudorina elegans, Pandorina morum, Micractinium pusillum, Chlorococcum infusionum, Coelastrum microporum, Monoraphidium komarkovae and Scenedesmus acuminatus were also present during mixing, but in a smaller proportion. Euglenophyceae dominated during mixing when the pond showed best performance (Figs 1 and 2). A high concentration of euglenoid ¯agellates was observed in the deepest layers, which received no light. Cyanophyceae and Bacillariophyceae were weakly represented and in no case surpassed 0.5% of the total phytoplankton biomass. Five signi®cant blooms, with biomasses higher than 15,000 mgC mÿ3, were observed as follows: at the end of the ®rst strati®cation period (dominated by Oocystis solitaria, Eudorina elegans and Pandorina morum ), at the end of the ®rst spring mixing period (Euglena viridis and Lepocinclis ovum ), in the middle (Chlamydomonas reinhardtii ) and at the end of the second strati®cation period (Oocystis solitaria, Coelastrum microporum and Chlorococcum infusionum ) and, ®nally, at the beginning of the second autumn mixing period (Lepocinclis ovum). Whenever total phytoplankton biomass exceeded 15,000 mgC m3, the removal eciencies of COD and suspended solids acquired negative values (Figs 1 and 2). Positive correlation was shown by NH3 level
Table 2. One-way analysis of variance for several variables in the deep ponda Variables
Euphotic zone (m) Temperature (8C) Dissolved oxygen (mgO2 lÿ1) pH Conductivity (mS cmÿ1) COD (mgO2 lÿ1) Total phosphorous (mgP lÿ1) ÿ1 PO3ÿ 4 (mgP l ) ÿ1 NOÿ 3 (mg l ) ÿ1 NOÿ 2 (mg l ) ÿ1 NH+ 4 (mg l ) NH3 (mg lÿ1) Total nitrogen (mgN lÿ1) Total solids (mg lÿ1) Total dissolved solids (mg lÿ1) Total suspended solids (mg lÿ1) Fixed suspended solids (mg lÿ1) Volatile suspended solids (mg lÿ1) Chlorophyll ``a'' (mg lÿ1) Phytoplankton biomass (mgC mÿ3) Bacterial biomass (mgC mÿ3) Zooplankton biomass (mgC mÿ3) Total coliforms (CFU 100 mlÿ1) a
Mixing throughout its depth, n = 19 (mean2SD)
1.0820.34 12.923.3 1.221.3 7.620.3 18992360 107227 2.320.7 2.020.6 1.020.6 0.320.4 37.126.8 0.520.5 34.525.2 10132184 9882181 2429 423 1928 1052120 460525260 9512372 10562986 5.5E+0526.4E+05
Strati®cation Epilimnion, n = 15 (mean2SD)
Metalimnion, n = 15 (mean2SD)
0.7120.15$ 19.822.6$ 2.323.2 7.920.4$ 19712209 121216$ 2.020.5 1.520.5 0.920.4 0.320.5 38.429.1 2.022.1$ 36.725.9$ 11292101$ 1095296$ 35211$ 422 31211$ 1802105$ 12,585210,997$ 9152260 83621842 1.1E+0627.6E+05$
± 18.722.3$ 0.220.1$ 7.620.2 20472211 130218$ 2.320.8 1.920.7 0.820.4 0.120.0 41.228.9 0.620.3 40.325.6$ 11482104$ 11202107$ 3229$ 524 2828$ 1252101 518224116 12462351 1682331$ 1.2E+0621.0E+06$
Three groups were considered: mixing, strati®cation-epilimnion, strati®cation-metalimnion. Means and standard deviations during the mixing (throughout its depth) and during the strati®cation (both in the epilimnion and metalimnion) are shown. The given variables, with the same symbols after two values, indicates a non-signi®cant dierence (at the level p < 0.05) between the groups corresponding to those two values; n: number of data.
Algae in a deep self-regeneration pond
and log10 phytoplankton biomass (r = 0.51, p < 0.05) during strati®cation in the epilimnion, while NH3 was negatively correlated with log10 zooplankton biomass (r=ÿ0.49, p < 0.10) and log10 bacterial biomass (r=ÿ0.48, p < 0.10). However, during the periods of maximum phytoplankton biomass the epilimnion remained oxygenated, even during the night (unpublished data). DISCUSSION
The algae observed in the deep pond were of a
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wide distribution and common to organic polluted ecosystems (Palmer, 1980). These included Euglena viridis, Chlamydomonas reinhardtii, Eudorina elegans, Pandorina morum, Coelastrum microporum, Micractinium pusillum and Oscillatoria chlorina. Phytoplankton biomasses recorded were in accordance with those reported by Soler et al. (1991) in a similar deep pond in Murcia (southern Spain), but with no continuous water supply. However, in contrast to the ®ndings of these authors, higher biomasses were recorded during the strati®cation period than during the mixing, as usually happens
Fig. 1. Time course of the removal eciency [(in¯uent±euent) 100/in¯uent] of several variables in the deep pond from July 1996 to December 1997.
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in highly productive natural ecosystems (Arauzo et al., 1996), as well as in conventional treatment ponds. Opportunistic species were dominant during the strati®cation (Chlorophyceae, mainly Chlorococcales ). Motile green algae (Volvocales ) were also well represented during strati®cations, which is infrequent in conventional treatment ponds during the warm season (Dinges, 1982). The absence of Cyanophyceae was probably due to the high ratio of ``dissolved inorganic nitrogen/soluble reactive phosphorus'' over the entire study period (which was never less than 15). These conditions do not favor the development of nitrogen-®xing species (Pearsall, 1930). However, it should be noted that Soler et al. (1991) observed extensive growth of Cyanophyceae in their deep pond during the major part of strati®cation. Likewise, Dinges (1982) reported the prevalence of Cyanophyceae in conventional treatment ponds during the warm season. As in the present investigation, Soler et al. (1991) reported the domination of motile algae during the mixing (Euglenophyceae in our pond, Chlamydomonas in theirs). All-green euglenoid ¯agellates and Chlamydomonas are known to be photoauxotrophic (Leedale, 1967; Harris, 1989). Furthermore, some green euglenoids are facultatively heterotrophic (Harris, 1989). This was illustrated by the fact that the highest concentrations of Euglena viridis and Lepocinclis ovum were observed close to the sediment in the pond (Fig. 2), far from the reach of solar radiation. These species may have a double function in the deep pond: providing oxygen by means of photosynthesis in the euphotic zone and mineralizing organic matter at night, or in the aphotic zone. However, it must not be overlooked that certain strains of Euglena and Chlamydomonas have the unusual ability to use acetate in photoassimilation. Consequently, these organisms might produce little or no oxygen and might be inecient in stimulating aerobic bacteria to act on wastewater (Palmer, 1980). High levels of dissolved oxygen have been observed throughout the column during
the Euglena viridis bloom, while levels lower than 1 mg lÿ1 were registered during the Lepocinclis ovum bloom. The increase in NH+ 4 levels at the expense of NO3 during the mixing phase, which may favor the development of Euglenophyceae (Margalef, 1983), may be another factor related to Euglenophycean domain during the mixing period. During the strati®cation the system has been observed to collapse at times. When total phytoplankton biomass exceeded 15,000 mgC mÿ3, the removal eciencies of COD and suspended solids acquired negative values. During such bloom periods, the photosynthetic activity of the high concentration of algae causes the pH to rise above 8 in the epilimnion. This is associated with an intense increase in NH3, reaching levels considered toxic for aquatic organisms (Alabaster and Lloyd, 1980). These processes may lead to large reductions in zooplankton and bacterial biomass in the epilimnion and may temporarily destabilize the trophic structure of the system during such phytoplankton blooms. It could be argued that it was the possible anoxia in the epilimnion, not the NH3 toxicity, which was responsible for such zooplankton mortality, but this hypothesis must be ruled out: epilimnion did remain oxygenated, even during the night. In the light of these results, it appears to be of the utmost importance for the improvement in performance to maintain the balance of the trophic structure (bacteria, algae and zooplankton). It is felt that further knowledge on the eect of NH3 on the biological community may permit to obtain better stabilization of the biological community and, therefore, better performance. On the other hand, suspended solids, COD or total phosphorus are frequently used as indicators of performance in treatment plants. However, since in pond systems the stabilization of wastewater as living forms (mainly as phytoplankton biomass) is favored, the use of such indicators in wastewater regeneration ponds may be less justi®ed. This was con®rmed by the negative correlation between phy-
Table 3. Pearson correlation between removal eciency of some variables and phytoplankton biomass and water temperaturea Removal eciency
Variables in the deep pond
Total coliforms COD Suspended solids Fixed suspended solids Volatile suspended solids Total nitrogen Nitrate Nitrite Ammonia Organic nitrogen Total phosphorus SRP a
Monthly mean values were used (n = 17). at any of the three preceding levels.
``log10 phytoplankton biomass''
Water temperature
ÿ0.48 ÿ0.82 ÿ0.71 ns ÿ0.43+ ns ns 0.82 ns ÿ0.44+ ÿ0.41+ ns
ÿ0.82 ÿ0.77 ÿ0.67 ns ÿ0.47 ns ns 0.71 ns ÿ0.48 ÿ0.45+ ns
: p < 0.01 signi®cance; : p < 0.05 signi®cance; +: p < 0.10 signi®cance; ns: non-signi®cance
Algae in a deep self-regeneration pond
toplankton and the removal eciencies of these indicators. Depending on their future application, euents could be treated with sand ®lters to eliminate phytoplankton. Alternatively, the eventual balancing of the trophic structure of the pond will prevent an excess in phytoplankton biomass.
3671 CONCLUSIONS
. The role of algae in a deep wastewater self-regeneration pond has been examined. To a great extent, the processes observed are common to other types of treatment ponds; however, its
Fig. 2. Contour plots for phytoplankton biomass (total and according to species) in the deep pond from July 1996 to December 1997.
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depth, which causes a succession of mixing and strati®cation over the annual cycle, has an important in¯uence on phytoplankton dynamics, as well as the system's performance. . Phytoplankton biomass was related to water temperature. Both variables seem to control the eciency of the deep treatment pond. . The best performance (greatest reductions) was shown by nitrate, nitrite and total coliforms. The removal eciency of total coliforms, COD and suspended solids was better during the mixing
period than during strati®cation. In contrast, greater nitrate removal eciency was recorded during the latter period. . Chlorococcales and Volvocales grew extensively during strati®cation. Euglenophyceae dominated during mixing, when the pond showed best performance. Flagellate algae seem to have an important role in the functioning of a deep pond, mainly during mixing, but also during strati®cation. This may be a distinctive feature of deep ponds as opposed to shallower treatment ponds.
Fig. 3. Contour plots for phytoplankton species biomass in the deep pond from July 1996 to December 1997.
Algae in a deep self-regeneration pond
. The absence of Cyanophyceae was probably due to the high ratio of ``dissolved inorganic nitrogen/soluble reactive phosphorus'' over the study period. . During the strati®cation the system has been observed to collapse at times. When total phytoplankton biomass rose to beyond 15,000 mgC mÿ3, the removal eciencies of COD and suspended solids acquired negative values. During these bloom periods, intense photosynthetic ac-
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tivity due to high concentrations of algae caused the pH to rise above 8, leading to an intense increase in NH3 to levels toxic for aquatic organisms. These processes may lead to large reductions in zooplankton and bacterial biomass and may temporarily destabilize the trophic structure. Nevertheless, such processes are not exclusive to deep ponds, but may also occur in any kind of organic polluted ponds. . It seems to be highly important to keep the
Fig. 4. Contour plots for phytoplankton species biomass in the deep pond from July 1996 to December 1997.
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trophic structure (bacteria, algae and zooplankton) well balanced for optimum pond performance. Further knowledge on the NH3 toxicity observed may permit the future stabilization of the biological community during the warm season.
AcknowledgementsÐThe authors wish to acknowledge the ®nancial support of Comunidad de Madrid (COR 0010/94 CAM ). A. Rubio, F. Abileo and A. Arellano performed the chemical analyses.
REFERENCES
Alabaster J. S. and Lloyd R. (1980) Water Quality Criteria for Freshwater Fish. Butterworths, London. APHA (1989) Standard methods for the examination of water and wastewater. American Public Health Association. Washington DC, 1134 pp. Arauzo M., Alvarez Cobelas M., Vicioso J. and Verdugo M. (1996) The phytoplankton of some gravel-pit lakes in Spain. Hydrobiologia 333, 19±27. Berna L. M., Torrella F., Soler A., SaÂez J., Llorens M. and Martõ nez I. (1986) Microbiological, physical and chemical study of water self-autodepuration by means of deep lagooning (Estudio de la autodepuracioÂn microbioloÂgica y fõ sico-quõ mica de aguas residuales por lagunaje profundo). Anales de BiologõÂa 10(2), 45±59. Dinges R. (1982) Natural Systems for Water Pollution Control. Van Nostrand Reinhold Co, New York, 252 pp. Dixon W. J., Brown M. D., Engelman L., Frane J. W., Hill M. A., Jennrich R. I. and Toporek J. D. (1992) BMDP Statistical Software Manual, Vols I and II, University of California Press Berkeley, 1500 pp. Dumont H. J., van del Velde I. and Dumont S. (1975) The dry weight estimate of biomass in a selection of Cladocera, Copepoda and Rotifera from the plankton, periphyton and benthos of continental waters. Oecologia 19(1), 75±97. Harrigan W. F. and McCane M. E. (1979) Laboratory
Methods in Food and Dairy Microbiology. Academic Press, London, New York. Harris H. H. (1989) The Chlamydomonas Sourcebook. Academic Press, San Diego, 780 pp. Hutchinson G. E. (1957) A Treatise on Limnology, Vol. I, Wiley New York, 1015 pp. Leedale G. F. (1967) Euglenoid Flagellates. Prentice-Hall, USA, 242 pp. Margalef R. (1983) Limnology (LimnologõÂa). Omega, Barcelona, 1010 pp. Moreno M. D., Soler A., SaÂez J. and Romera P. (1984a) Study on the hydrodynamic behaviour of a deep waste stabilisation pond. Tribune du Cebedeau 37(489490), 323±328. Moreno M. D., Soler A., Moreno J., SaÂez J. and Moreno J. (1984b) Thermal simulation of deep stabilisation ponds. Tribune du Cebedeau 37(491), 403±410. Moreno M. D., Medina M. A., Moreno J., Soler A. and SaÂez J. (1988) Modelling the performance of deep waste stabilisation ponds. Wat. Res. Bull. 24(2), 377±387. Norland S. (1993) The relationship between biomass and volume of bacteria. In Handbook of Methods in Aquatic Microbial Ecology, eds P. F. Kemp, B. F. Sherr, E. B. Sherr and J. J. Cole, pp. 303±308. Lewis Publishers, Boca Raton 394 pp. Palmer C. M. (1980) The identi®cation, signi®cance, and control of algae in water supplies and in polluted water. In Algae and Water Pollution. Castle House Publications Ltd, UK, 123 pp. Pearsall W. H. (1930) Phytoplankton of English lakes, I. The proportion in the water of some dissolved substances of biological importance. J. Ecol. 18(2), 306± 320. Rott E. A. (1981) Some results of phytoplankton counting intercalibrations. Schweiz. Z. Hydrol. 43(1), 34±62. Soler A., SaÂez J., LloreÂns M., Martõ nez I., Torrella F. and Berna L. M. (1991) Changes in physico-chemical parameters and photosynthetic microorganisms in a deep wastewater self-depuration lagoon. Wat. Res. 25(6), 689±695. Torres J. J., Soler A., SaÂez J. and OrtunÄo F. (1997) Hydraulic performance of a deep stabilisation pond. Wat. Res. 31(4), 679±688. UtermoÈhl H. (1958) Zur Vervollkommnung der quantitativen Phytoplankton-Methodik. Mitt. int. Verein. theor. angew. Limnol. 9, 38.
PII: S0043-1354(00)00107-X www.elsevier.com/locate/watres
THE ROLE OF ALGAE IN A DEEP WASTEWATER SELFREGENERATION POND M. ARAUZO*, M. F. COLMENAREJO, E. MARTIÂNEZ and M. G. GARCIÂA Centro de Ciencias Medioambientales, C.S.I.C. Serrano 115 dpdo., 28006, Madrid, Spain (First received 18 November 1998) AbstractÐThe present investigation was designed to gain information on the role of algae in a deep wastewater self-regeneration pond. The experimental pond was continuously fed with secondary euent from a municipal wastewater treatment plant. Biological, physical and chemical pro®les were recorded throughout the water column during the period July 1996 to December 1997. The removal eciencies of several chemical and sanitary indicators were calculated. The time course of species biomass was used to evaluate phytoplankton dynamics and its relationship with the performance of the deep pond. The eciency of the system seemed to be conditioned by temperature and phytoplankton biomass. Improved performance was observed during periods of mixing. Chlorococcales and Volvocales grew extensively during strati®cation periods. Euglenophyceae dominated during the mixing. When the total phytoplankton biomass exceeded 15,000 mgC mÿ3, the removal eciencies of the chemical oxygen demand (COD) and the suspended solids acquired negative values. These phytoplankton blooms were associated with an intense increase in pH and, consequently, in NH3 levels, in the epilimnion, during the strati®cation. When such a bloom occurred, considerable decreases in zooplankton and bacterial biomasses were observed, which temporarily destabilized the trophic structure of the pond. 7 2000 Elsevier Science Ltd. All rights reserved Key wordsÐurban wastewater, deep pond, algae, removal eciency, strati®cation, mixing
INTRODUCTION
An experimental deep self-regeneration pond was designed with the aim of improving the quality of wastewater euent for its reuse. The pond was supplied with secondary euent produced at a conventional treatment plant in which urban wastewater is treated. It was constructed at Plataforma Experimental de la Finca La Poveda (Arganda del Rey, Comunidad de Madrid, Spain). This facility is managed by Centro de Ciencias Medioambientales (C.S.I.C. ). Deep treatment ponds oer theoretical advantages over conventional stabilization ponds, which have greater surfaces and are shallower. They occupy less surface area and, in arid and semi-arid areas, summer evaporation losses are reduced. Deep ponds could become an inexpensive, simple way to improve the sanitary and chemical quality of water and, at the same time, be used as reservoirs for agricultural irrigation. Nevertheless, deep treatment ponds are infrequently used and their properties have not been extensively investigated (Moreno et al., 1984a, b, 1988; Berna et al., 1986; Torres et al., 1997). Previous studies have generally been directed
towards hydraulic, design and sanitary aspects. Despite being the key to the optimization of performance, to date, the biological communityÐthe engine of wastewater self-regenerationÐhas been the focus of very little research in deep treatment ponds (Soler et al., 1991). It is known that, in pond systems, algae stimulate bacteria, and bacteria stimulate algae (Palmer, 1980). In this way, the chemical units that comprised the organic waste eventually become incorporated into the algae as stable organic components of living cells. The death and decomposition of large numbers of algae lead to undesirable conditions similar to those caused by the original wastewater in the pond. Consequently, this needs to be avoided to keep the trophic structure well balanced for optimum performance. The present investigation involves the in-depth examination of the role of algae in a deep urban wastewater self-regeneration pond. Evaluation was made of the phytoplankton community, the dynamics of its populations and its relationship with the performance and the trophic structure of the pond. MATERIALS AND METHODS
*Author to whom all correspondence should be addressed. Fax: +34-1-564-0800; e-mail: [email protected]
The experimental pond was 4.75 m deep, had a volume of 2161 m3 and was continuously fed with secondary eu-
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Algae in a deep self-regeneration pond ent from a municipal wastewater treatment plant. The pond had the shape of an inverted truncated square pyramid, measuring 10 10 m at the bottom and 31 31 m at the top. The lining material was geotextile felt, over which a layer of high-density polyethylene was laid in order to prevent leakage. The in¯uent port was ®xed close to the bottom (4.5 m) during the course of the study. Euent ports were at depths of 1.5, 2.5 and 3.75 m from the surface layer, and all three were kept open throughout the experimental period. The ¯ow was regulated through a control valve and a magnetic ¯ow-meter. The pond was continuously fed with a ¯ow of 11 2 3 m3 hÿ1 from July 1996 to December 1997. This provided a mean hydraulic retention time of 924 days. The experimental design encompassed three time scales: . On a fortnight basis: physical, chemical and biological parameters were monitored at dierent depths of the water column (meter by meter) in the center of the pond. . On a twice per week basis: physical, chemical, biological and microbiological parameters on the in¯uent and the three euents were monitored in order to evaluate the performance of the pond. . On a daily basis: the input ¯ow and the functioning of the system and maintenance were checked. Samples were taken between 9:00 and 10:00 in the morning. Given the great variability of in¯uent composition depending on the time of day, composite samples were made collecting aliquots every hour over each 24 h period. These aliquots were linked to the rate of ¯ow. Sample taking was carried out with an automatic sampler equipped with a refrigeration system (model Streamline 800 SL). For the collection of samples at dierent depths from the center of the pond, an immersible, peristaltic pump was used. Temperature and oxygen pro®les were recorded using a YSI-57 dissolved oxygen ®eld meter. The following parameters were evaluated: . Physical and chemical parameters of in¯uent, euents and deep pro®les: temperature, dissolved oxygen levels, pH, conductivity, chemical oxygen demand (COD), total phosphorus, soluble reactive phosphorus (PO3ÿ 4 ), nitrate, nitrite and ammonia (NH+ 4 \ NH3) levels, Kjeldhal nitrogen content, total solids, total dissolved solids and total suspended solids (®xed and volatile). . Biological parameters in deep pro®les: bacterial community biomass, phytoplankton community biomass (primary producers) and zooplankton community biomass (secondary producers). . Microbiological analysis in in¯uent and euents: total coliforms (as a sanitary indicator). Physical, chemical and microbiological analyses were performed according to APHA (1989). Total nitrogen was taken as the sum (expressed in mgN lÿ1) of nitrate, nitrite and Kjeldhal nitrogen. NH3 was calculated from the quantity of its ionized form, taking into account pH and temperature (Alabaster and Lloyd, 1980). The euphotic zone was de®ned as the depth reached by only 1% of surface solar radiation. This was determined from measurements made by a Secchi disk, previously calibrated with an LI-COR 192 S photometer, and assuming that the extinction of light follows Lambert's law (Hutchinson, 1957). Removal eciencies were calculated as ``(In¯uent±Euent) 100/In¯uent''. Given the great variability of in¯uent quality throughout the study, monthly mean values of removal eciency have been used. Total bacteria were estimated by optical microscopy, bacterial density by the extension method of Breed (Harrigan and McCane, 1979) and biomass according to Norland (1993). Phytoplankton and protozoan densities (by species) were estimated by optical inverted microscopy
3667
(UtermoÈhl, 1958) and biomass according to Rott (1981). The densities and biomass of Rotifera and Crustacea (by species) were estimated following the method of Dumont et al. (1975). Zooplankton biomass was de®ned as the sum of the biomasses (expressed in mgC mÿ3) of Protozoa, Rotifera and Crustacea. Dierences in pond performance between the mixing and the strati®cation were estimated by the Student's ttest. One-way analyses of variance have been performed to compare variables recorded during the mixing and during the strati®cation, both in the epilimnion and the metalimnion (BMDP7D; Dixon et al., 1992). In the case of homogeneity of variances, a parametric test was applied to data. A non-parametric method was used when there was heterogeneity of variances (Brown-Forsythe; Dixon et al., 1992). A Levene test was used to previously determine the homogeneity of variances (Dixon et al., 1992). RESULTS
Stable, thermal strati®cations were produced during the summer (until August in 1996 and from April to September in 1997). During strati®cation, the mixed upper level (epilimnion) was observed to extend from the surface to a depth of 2 m. A lower strati®cation level of decreasing temperature gradient (metalimnion) was observed from 2 to 4.75 m. It was not possible to de®ne a stable hypolimnion. During the remaining periods the pond was mixed throughout its depth. The phytoplankton species observed during the study period are shown in Table 1. The mean values of the physical, chemical, bioTable 1. Phytoplankton species in the deep pond from July 1996 to December 1997 Bacillariophyceae Nitzschia graciliformis Lange-Bertalot and Simonsen 1978a Nitzschia palea (KuÈtzing) W. Smith 1856a Chlorophyceae Chlamydomonas sp. Ehrb. 1833a Chlamydomonas reinhardtii Dang. 1888 Chlorococcum infusionum (Schrank) Menegh. 1842 Coelastrum microporum NaÈg. in A. Br. 1855 Eudorina elegan Ehr. 1833 Micractinium pusillum Fresenius 1858 Monoraphidium komarkovae Nyg. 1979 Monoraphidium minutum (Naeg.) Kom-Legn. Oocystis solitaria Writt. in Writt. and Nordst. 1879 Pandorina morum (O. F. MuÈller) Bory 1824 Scenedesmus acumuminatus (Lagerh.) Chod. 1902 Scenedesmus arcuatus (Lemm.) Lemm. 1899 Scenedesmus quadricauda (Turp.) BreÂb. Sensu Chod. 1913, 1926 Euglenophyceae Euglena hemichromata Skuja 1948 Euglena oxyuris Schmarde 1946a Euglena proxima Dangeard 1901 Euglena viridis Ehrenberg 1830 Lepocinclis ovum (Ehr.) Lemm. 1901 Cyanophyceae Oscillatoria agardhii Gomont 1892a Oscillatoria chlorina KuÈtz ex Gomont 1892a Oscillatoria tennuis Agardha a
Species showing low occurrence and biomass (no more than two occurrences and biomass lower than 0.5% of the total phytoplankton biomass).
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logical and sanitary variables, recorded in the deep pond during mixing and strati®cation periods (both in the epilimnion and metalimnion layers), are shown in Table 2. A signi®cant dierence was observed between both periods with respect to the water temperature. A signi®cant reduction in the euphotic zone and increases in phytoplankton biomass, pH, NH3, total suspended solids and chlorophyll ``a'' were observed in the epilimnion during strati®cation. The lowest zooplankton level was in accordance with scarce dissolved oxygen in the metalimnion. Signi®cantly higher levels of total solids, total nitrogen and total coliforms were observed during strati®cation (both in the epilimnion and in the metalimnion) than during mixing. The time course of some of the removal eciency variables is shown in Fig. 1. The best performance (in terms of greatest reductions) was observed for nitrate, nitrite and total coliforms. The total coliforms, COD and suspended solid removal eciencies were greater during the mixing than during the strati®cation, showing t-values of 7.06, 4.28 and 4.99, respectively ( p < 0.0001, 15 degrees-of-freedom). However, nitrite removal was more ecient during the strati®cation, with a t-value of ÿ2.89 ( p < 0.02, 15 degrees-of-freedom). The remaining indicators of performance showed no signi®cant dierences between the periods of mixing and strati®cation. Water temperature was highly related to phytoplankton biomass (r = 0.61; p < 0.001). How both these variables seem to directly or indirectly control
the eciency of the system is shown in Table 3. The phytoplankton community time course throughout the water column is shown in Figs 2±4. Chlorococcales and Volvocales grew extensively during strati®cation, although species such as Chlamydomonas reinhardtii, Eudorina elegans, Pandorina morum, Micractinium pusillum, Chlorococcum infusionum, Coelastrum microporum, Monoraphidium komarkovae and Scenedesmus acuminatus were also present during mixing, but in a smaller proportion. Euglenophyceae dominated during mixing when the pond showed best performance (Figs 1 and 2). A high concentration of euglenoid ¯agellates was observed in the deepest layers, which received no light. Cyanophyceae and Bacillariophyceae were weakly represented and in no case surpassed 0.5% of the total phytoplankton biomass. Five signi®cant blooms, with biomasses higher than 15,000 mgC mÿ3, were observed as follows: at the end of the ®rst strati®cation period (dominated by Oocystis solitaria, Eudorina elegans and Pandorina morum ), at the end of the ®rst spring mixing period (Euglena viridis and Lepocinclis ovum ), in the middle (Chlamydomonas reinhardtii ) and at the end of the second strati®cation period (Oocystis solitaria, Coelastrum microporum and Chlorococcum infusionum ) and, ®nally, at the beginning of the second autumn mixing period (Lepocinclis ovum). Whenever total phytoplankton biomass exceeded 15,000 mgC m3, the removal eciencies of COD and suspended solids acquired negative values (Figs 1 and 2). Positive correlation was shown by NH3 level
Table 2. One-way analysis of variance for several variables in the deep ponda Variables
Euphotic zone (m) Temperature (8C) Dissolved oxygen (mgO2 lÿ1) pH Conductivity (mS cmÿ1) COD (mgO2 lÿ1) Total phosphorous (mgP lÿ1) ÿ1 PO3ÿ 4 (mgP l ) ÿ1 NOÿ 3 (mg l ) ÿ1 NOÿ 2 (mg l ) ÿ1 NH+ 4 (mg l ) NH3 (mg lÿ1) Total nitrogen (mgN lÿ1) Total solids (mg lÿ1) Total dissolved solids (mg lÿ1) Total suspended solids (mg lÿ1) Fixed suspended solids (mg lÿ1) Volatile suspended solids (mg lÿ1) Chlorophyll ``a'' (mg lÿ1) Phytoplankton biomass (mgC mÿ3) Bacterial biomass (mgC mÿ3) Zooplankton biomass (mgC mÿ3) Total coliforms (CFU 100 mlÿ1) a
Mixing throughout its depth, n = 19 (mean2SD)
1.0820.34 12.923.3 1.221.3 7.620.3 18992360 107227 2.320.7 2.020.6 1.020.6 0.320.4 37.126.8 0.520.5 34.525.2 10132184 9882181 2429 423 1928 1052120 460525260 9512372 10562986 5.5E+0526.4E+05
Strati®cation Epilimnion, n = 15 (mean2SD)
Metalimnion, n = 15 (mean2SD)
0.7120.15$ 19.822.6$ 2.323.2 7.920.4$ 19712209 121216$ 2.020.5 1.520.5 0.920.4 0.320.5 38.429.1 2.022.1$ 36.725.9$ 11292101$ 1095296$ 35211$ 422 31211$ 1802105$ 12,585210,997$ 9152260 83621842 1.1E+0627.6E+05$
± 18.722.3$ 0.220.1$ 7.620.2 20472211 130218$ 2.320.8 1.920.7 0.820.4 0.120.0 41.228.9 0.620.3 40.325.6$ 11482104$ 11202107$ 3229$ 524 2828$ 1252101 518224116 12462351 1682331$ 1.2E+0621.0E+06$
Three groups were considered: mixing, strati®cation-epilimnion, strati®cation-metalimnion. Means and standard deviations during the mixing (throughout its depth) and during the strati®cation (both in the epilimnion and metalimnion) are shown. The given variables, with the same symbols after two values, indicates a non-signi®cant dierence (at the level p < 0.05) between the groups corresponding to those two values; n: number of data.
Algae in a deep self-regeneration pond
and log10 phytoplankton biomass (r = 0.51, p < 0.05) during strati®cation in the epilimnion, while NH3 was negatively correlated with log10 zooplankton biomass (r=ÿ0.49, p < 0.10) and log10 bacterial biomass (r=ÿ0.48, p < 0.10). However, during the periods of maximum phytoplankton biomass the epilimnion remained oxygenated, even during the night (unpublished data). DISCUSSION
The algae observed in the deep pond were of a
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wide distribution and common to organic polluted ecosystems (Palmer, 1980). These included Euglena viridis, Chlamydomonas reinhardtii, Eudorina elegans, Pandorina morum, Coelastrum microporum, Micractinium pusillum and Oscillatoria chlorina. Phytoplankton biomasses recorded were in accordance with those reported by Soler et al. (1991) in a similar deep pond in Murcia (southern Spain), but with no continuous water supply. However, in contrast to the ®ndings of these authors, higher biomasses were recorded during the strati®cation period than during the mixing, as usually happens
Fig. 1. Time course of the removal eciency [(in¯uent±euent) 100/in¯uent] of several variables in the deep pond from July 1996 to December 1997.
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M. Arauzo et al.
in highly productive natural ecosystems (Arauzo et al., 1996), as well as in conventional treatment ponds. Opportunistic species were dominant during the strati®cation (Chlorophyceae, mainly Chlorococcales ). Motile green algae (Volvocales ) were also well represented during strati®cations, which is infrequent in conventional treatment ponds during the warm season (Dinges, 1982). The absence of Cyanophyceae was probably due to the high ratio of ``dissolved inorganic nitrogen/soluble reactive phosphorus'' over the entire study period (which was never less than 15). These conditions do not favor the development of nitrogen-®xing species (Pearsall, 1930). However, it should be noted that Soler et al. (1991) observed extensive growth of Cyanophyceae in their deep pond during the major part of strati®cation. Likewise, Dinges (1982) reported the prevalence of Cyanophyceae in conventional treatment ponds during the warm season. As in the present investigation, Soler et al. (1991) reported the domination of motile algae during the mixing (Euglenophyceae in our pond, Chlamydomonas in theirs). All-green euglenoid ¯agellates and Chlamydomonas are known to be photoauxotrophic (Leedale, 1967; Harris, 1989). Furthermore, some green euglenoids are facultatively heterotrophic (Harris, 1989). This was illustrated by the fact that the highest concentrations of Euglena viridis and Lepocinclis ovum were observed close to the sediment in the pond (Fig. 2), far from the reach of solar radiation. These species may have a double function in the deep pond: providing oxygen by means of photosynthesis in the euphotic zone and mineralizing organic matter at night, or in the aphotic zone. However, it must not be overlooked that certain strains of Euglena and Chlamydomonas have the unusual ability to use acetate in photoassimilation. Consequently, these organisms might produce little or no oxygen and might be inecient in stimulating aerobic bacteria to act on wastewater (Palmer, 1980). High levels of dissolved oxygen have been observed throughout the column during
the Euglena viridis bloom, while levels lower than 1 mg lÿ1 were registered during the Lepocinclis ovum bloom. The increase in NH+ 4 levels at the expense of NO3 during the mixing phase, which may favor the development of Euglenophyceae (Margalef, 1983), may be another factor related to Euglenophycean domain during the mixing period. During the strati®cation the system has been observed to collapse at times. When total phytoplankton biomass exceeded 15,000 mgC mÿ3, the removal eciencies of COD and suspended solids acquired negative values. During such bloom periods, the photosynthetic activity of the high concentration of algae causes the pH to rise above 8 in the epilimnion. This is associated with an intense increase in NH3, reaching levels considered toxic for aquatic organisms (Alabaster and Lloyd, 1980). These processes may lead to large reductions in zooplankton and bacterial biomass in the epilimnion and may temporarily destabilize the trophic structure of the system during such phytoplankton blooms. It could be argued that it was the possible anoxia in the epilimnion, not the NH3 toxicity, which was responsible for such zooplankton mortality, but this hypothesis must be ruled out: epilimnion did remain oxygenated, even during the night. In the light of these results, it appears to be of the utmost importance for the improvement in performance to maintain the balance of the trophic structure (bacteria, algae and zooplankton). It is felt that further knowledge on the eect of NH3 on the biological community may permit to obtain better stabilization of the biological community and, therefore, better performance. On the other hand, suspended solids, COD or total phosphorus are frequently used as indicators of performance in treatment plants. However, since in pond systems the stabilization of wastewater as living forms (mainly as phytoplankton biomass) is favored, the use of such indicators in wastewater regeneration ponds may be less justi®ed. This was con®rmed by the negative correlation between phy-
Table 3. Pearson correlation between removal eciency of some variables and phytoplankton biomass and water temperaturea Removal eciency
Variables in the deep pond
Total coliforms COD Suspended solids Fixed suspended solids Volatile suspended solids Total nitrogen Nitrate Nitrite Ammonia Organic nitrogen Total phosphorus SRP a
Monthly mean values were used (n = 17). at any of the three preceding levels.
``log10 phytoplankton biomass''
Water temperature
ÿ0.48 ÿ0.82 ÿ0.71 ns ÿ0.43+ ns ns 0.82 ns ÿ0.44+ ÿ0.41+ ns
ÿ0.82 ÿ0.77 ÿ0.67 ns ÿ0.47 ns ns 0.71 ns ÿ0.48 ÿ0.45+ ns
: p < 0.01 signi®cance; : p < 0.05 signi®cance; +: p < 0.10 signi®cance; ns: non-signi®cance
Algae in a deep self-regeneration pond
toplankton and the removal eciencies of these indicators. Depending on their future application, euents could be treated with sand ®lters to eliminate phytoplankton. Alternatively, the eventual balancing of the trophic structure of the pond will prevent an excess in phytoplankton biomass.
3671 CONCLUSIONS
. The role of algae in a deep wastewater self-regeneration pond has been examined. To a great extent, the processes observed are common to other types of treatment ponds; however, its
Fig. 2. Contour plots for phytoplankton biomass (total and according to species) in the deep pond from July 1996 to December 1997.
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depth, which causes a succession of mixing and strati®cation over the annual cycle, has an important in¯uence on phytoplankton dynamics, as well as the system's performance. . Phytoplankton biomass was related to water temperature. Both variables seem to control the eciency of the deep treatment pond. . The best performance (greatest reductions) was shown by nitrate, nitrite and total coliforms. The removal eciency of total coliforms, COD and suspended solids was better during the mixing
period than during strati®cation. In contrast, greater nitrate removal eciency was recorded during the latter period. . Chlorococcales and Volvocales grew extensively during strati®cation. Euglenophyceae dominated during mixing, when the pond showed best performance. Flagellate algae seem to have an important role in the functioning of a deep pond, mainly during mixing, but also during strati®cation. This may be a distinctive feature of deep ponds as opposed to shallower treatment ponds.
Fig. 3. Contour plots for phytoplankton species biomass in the deep pond from July 1996 to December 1997.
Algae in a deep self-regeneration pond
. The absence of Cyanophyceae was probably due to the high ratio of ``dissolved inorganic nitrogen/soluble reactive phosphorus'' over the study period. . During the strati®cation the system has been observed to collapse at times. When total phytoplankton biomass rose to beyond 15,000 mgC mÿ3, the removal eciencies of COD and suspended solids acquired negative values. During these bloom periods, intense photosynthetic ac-
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tivity due to high concentrations of algae caused the pH to rise above 8, leading to an intense increase in NH3 to levels toxic for aquatic organisms. These processes may lead to large reductions in zooplankton and bacterial biomass and may temporarily destabilize the trophic structure. Nevertheless, such processes are not exclusive to deep ponds, but may also occur in any kind of organic polluted ponds. . It seems to be highly important to keep the
Fig. 4. Contour plots for phytoplankton species biomass in the deep pond from July 1996 to December 1997.
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trophic structure (bacteria, algae and zooplankton) well balanced for optimum pond performance. Further knowledge on the NH3 toxicity observed may permit the future stabilization of the biological community during the warm season.
AcknowledgementsÐThe authors wish to acknowledge the ®nancial support of Comunidad de Madrid (COR 0010/94 CAM ). A. Rubio, F. Abileo and A. Arellano performed the chemical analyses.
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