In this study, fillers (gravel and brick) of different surface areas were installed on both banks of two test channels so as to have the same capacity, and effects of differences in surface area and porosity of the streamside on aquatic biota and activity of the attached biofilm were investigated at downstream distance. The results showed that an increase in surface area and porosity causes change in the composition of microbial community, increasing the number of total bacteria and nitrifying bacteria and improving oxygen uptake activity. Although there was no evident difference found in the biofilm microfauna between the two test channels, a result was obtained that oxygen uptake rate by protozoa and metazoa evaluated by laboratory batch experiments quadruples farther downstream in the gravel-filled channel. Furthermore, it was clear, from the result that there observed many Physidae and Ephemeroptera that prey on deposits such as detritus precipitated in the interstices, that the amount and diversity of macrobenthos increased in the gravel-filled channel. These results suggest that surface area and porosity are extremely important factors in the detritus food chain constructed by settling and accumulation of particulate organic matter, and it is considered that controlling them can create a diverse aquatic biota
- 105 - INFLUENCE OF STREAMSIDE SURFACE AREA ON AQUATIC BIOTA AND BIOFILM ACTIVITY J. Hiratsuka*, J. H. Kim**, H. Tanaka**, H. Sasaki* and R. Sudo** * Department of Resources and Environmental Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan ** Center for Environmental Science in Saitama, 914 Kamitanadare, Kisai-machi, Saitama 347-0015, Japan ABSTRACT In this study, fillers (gravel and brick) of different surface areas were installed on both banks of two test channels so as to have the same capacity, and effects of differences in surface area and porosity of the streamside on aquatic biota and activity of the attached biofilm were investigated at downstream distance. The results showed that an increase in surface area and porosity causes change in the composition of microbial community, increasing the number of total bacteria and nitrifying bacteria and improving oxygen uptake activity. Although there was no evident difference found in the biofilm microfauna between the two test channels, a result was obtained that oxygen uptake rate by protozoa and metazoa evaluated by laboratory batch experiments quadruples farther downstream in the gravel-filled channel. Furthermore, it was clear, from the result that there observed many Physidae and Ephemeroptera that prey on deposits such as detritus precipitated in the interstices, that the amount and diversity of macrobenthos increased in the gravel-filled channel. These results suggest that surface area and porosity are extremely important factors in the detritus food chain constructed by settling and accumulation of particulate organic matter, and it is considered that controlling them can create a diverse aquatic biota. KEYWORDS Aquatic biota; biofilm; detritus; macrobenthos; river restoration; surface area INTRODUCTION In Japan, river modification and the organization of agricultural water for controlling and utilizing water channels have been rapidly accomplished after the war. Such conversion to a linear and monotonous river structure results in the loss of habitat for a diverse species of life (Shimatani and Kayaba, 1997). On the other hand, although the environmental standard achievement rate (BOD standard) of Japanese rivers seems to show an improvement of their water quality along with popularization of sewerage, to 81.0 % in 1998, many problems remain, such as the enrichment of rivers due to inorganic nutrients, the diversification of pollution load sources such as trace toxic chemicals, and the decrease in discharge. Especially in urban rivers, these problems are concentrated as a result of promoting river modifications from the viewpoint of land utilization and disaster prevention (Environment Agency, 2000). Recently, while ecological engineering is drawing attention in that utilizes the self-design function of an ecosystem for recovering original aquatic ecosystems (Mitsch and Jørgensen, 1989; Shield Jr. et al., 1995), river restorations of diverse scales are being performed in Japan as well (Seki and Yabe, 1993). River restoration applied to small rivers is often performed by placing natural materials such as stones or artificial vegetation instead of concrete bank or by giving variety to the river cross sections with the objective of improving the diversity of the river ecosystem or contributing to water quality improvement. In this case, one of environmental factors brought to the aquatic ecosystem by river restoration is an increase in the surface area and porosity of riverbanks and riverbed. An increase in the surface area and porosity of riverbanks and riverbed creates new supermicrohabitats, which is considered to greatly influence the microbial community that occupies the lower trophic level of the aquatic ecosystem and to cause a change to circulate materials such as carbon, nitrogen, and phosphorus. It is an objective of this study to clarify the influence of the surface area and porosity of the streamside on aquatic biota, where an experimental investigation has been attempted using test channels. The contents of - 106 - the investigation include, (1) the influence of an increase in surface area and porosity of streamside on the amount and succession along with the downstream distance of aquatic biota in each trophic level, and (2) a separate evaluation of the activity of each microbial community using the oxygen production rate and uptake rate of the biofilm attached to the streamside. MATERIALS AND METHODS Description of test channel As shown in Figure 1, installed on both banks of test channels are bricks 6×9.5×20 cm in size and gravels which is 4-8 cm in diameter so that they have approximately the same packing rate. The flow velocity of the flow center and channel cross sectional area are set approximately the same between the two channels, and only in the surface area ratio the gravel-filled channel has a value 5.5 times higher than the other. Listed in Table 1 is the operating condition of the test channels. In the experiment, artificial wastewater was added to pumped-up pond water, which was poured in as sample water. The composition of the artificial wastewater was fish extract 40 g･l -1 , NH 4 Cl 8 g･l -1 , and KH 2 PO 4 4 g･l -1 (TOC, T-N, and T-P are 16, 7.4, and 2.6 g･l -1 , respectively), and a feed pump was used for continuous addition at a rate of 5 l･d -1 . The experiment was performed from July to October in 2000. At 3, 10, 20, and 30 m points of the gravel-filled channel, stainless-steel gabions of a cubic shape of 20 cm in size were preinstalled for serving each activity experiment and biomass investigation. A sheet was put on the bottom of the gabion in order not to leak any sediment. Analytic method of water quality Collecting and analyzing sample water were performed at each point in the daytime and nighttime at a frequency of once a week from August, one month since the experiment started. Water temperature, pH (D-25, HORIBA), DO (DO-14P, TOA), SS, dissolved organic carbon (DOC; TOC-5000, Shimadzu), dissolved total nitrogen (DTN), NH 4 -N, NO 2 -N, NO 3 -N, and PO 4 -P were determined by Standard Methods (APHA, 1998). Filtration was performed using the 0.45 µm membrane filter. Observation of aquatic biota Investigation of the biota was performed at each point of the both channels in October 2000. Preprocessing of each sample was performed by extracting a 20 cm section of bricks and gravels in a gabion, peeling all attached deposits, and fixing the capacities of brick-filled channel to 0.6 l and the gravel-filled channel to 2 l. This was filtered with a 1 mm mesh sieve, and macrobenthoses of the remaining were directly counted. The portion that passed through the sieve was dispersed by a homogenizer to make a uniform suspended sample, which was used for counting and measurement of total bacteria (epifluorescence microscopic method), nitrifying bacteria (ELISA kits, “Kenshutsu-kun”, Yakult Honsya), chlorophyll-a and pheophytin-a (Lorenzen, 1967). Protozoa and metazoa were counted at 100-400 magnifications under an optical microscope after the suspended sample was further diluted by 5-10 times. SS and VSS of the suspended sample were also measured. Measurement of oxygen production/uptake rate of biofilm An appropriate amount (that will make the VSS after measurement 35 ± 10 mg) of the suspended sample was injected to a 200 ml incubation bottle where diluted BOD solution was put, a DO electrode (DO-25A, TOA) with a stirrer was inserted airtight, and the DO concentration change was measured at an illumination of 5000 lux in a water bath of 20°C. When measuring DO, Glucose 5 mg, 1 mg as N of each of NH 4 Cl, Table 1. Operation conditions in test channels Filler Discharge Velocity Water level Cross sectional area Surface area (m 3 ･d -1 ) (m･s -1 ) (m) (m 2 ) (m 2 ･m-flow direction -1 ) brick 130 0.125 0.118 0.0252 0.55 gravel 101 0.127 0.122 0.0257 3 - 107 - NaNO 2 , and KNO 3 were added, and the maximum activity was measured. In this study inhibitors, each of which works only for one of the microbial, were used respectively in order to separate and quantify DO production/uptake rates by algae, heterotrophic bacteria, nitrifying bacteria, protozoa and metazoa in the biofilm. Amounts of added inhibitors were DCMU (an inhibitor of photosynthesis of phytoplankton) 1 mg, ATU (an inhibitor of respiration of nitrifying bacteria) 4 mg, and nystatin (an inhibitor of cell membrane functions of protozoa and metazoa) 4 mg. Nystatin was stirred for 30 minutes after its addition, and measurements were taken after the reaction. All the DO measurements were performed for 30 minutes in each inhibition stage, in two series at each point of the both channels. Oxygen production rate (O P ) and oxygen uptake rate (O R ) of each microbial community were calculated by linearly approximating change of DO concentration. RESULTS AND DISCUSSION The average water quality of both test channels at individual points are listed in Table 2 (average water temperature in daytime and nighttime were 27.4°C, 25.8°C, respectively). From the fact that DO increases more in the brick-filled channel during the daytime and decreased greatly in the gravel-filled channel during the nighttime, it was clear that biofilm having such different microbial community were formed and that the primary production was prominent in the brick-filled channel and that the biodegradation was prominent in the gravel-filled channel. The percentage SS removal was 22.7 % and 32.9 % in the brick-filled channel and the gravel-filled channel, respectively, where the gravel-filled channel showed higher SS removal effect. From the results that NH 4 -N decreased greatly in the daytime when uptake of algae was active and that it decreased modestly also in the nighttime, it is evident that nitrification proceeded in both channels. Biotas of both channels are listed in Table 3. Although total number of bacteria per surface area virtually does not change at the inflow section, while it decreased downstream in the brick-filled channel, it increased in the gravel-filled channel, and the trend was especially significant for nitrifying bacteria. As seen from the DOC change in Table 2, biodegradable organic matter are almost all removed while flowing down to the 10-20 m point. Therefore, it can be seen that bacterial community utilizing biodegradable dissolved organic matter as the substrate is predominant in the brick-filled channel. This is also supported from the result that the decrease trend of the total number of bacteria and the decrease trend of DOC in the brick-filled channel mostly correspond to each other. On the other hand, in the gravel-filled channel as there are reports that the biodegradation is promoted by the existence of interstitial voids (Gantzer et al., 1988; Leu et al., 1996), it is considered that the total number of bacteria increased because a group of bacteria which utilize particulate organic matter trapped in the gravel interstices increased. Therefore, it is indicated that an accumulation of particulate organic matter by multilayer structure in the gravel-filled channel has a large impact on the microbial community and causes to construct the detritus food chain. Figure 2 shows nitrogen mineralization rate, nitrification rate, and denitrification rate from batch experiments performed by dividing into three layers in the depth direction of the gravel-filled channel. For the batch experiments, gabions at 10 m and 20 m points were collected, 5-8 pieces of gravel in each layer were used without peeling off. Measurements of NH 4 -N, NO 2 -N, and NO 3 -N were done at every 12 hours for 48 hours, and nitrogen mineralization, nitrification, and denitrification rate were calculated from these concentration changes. All the experiments including the contrast experiment were performed at 20 ± 2°C in the dark condition. Table 2. Average water quality in daytime and nighttime during August to October 2000 Parameter brick-filled channel gravel-filled channel (mg/l except pH) 3m 10m 20m 30m 3m 10m 20m 30m day 7.74 7.78 7.82 7.83 7.74 7.75 7.76 7.82 pH night 7.54 7.59 7.55 7.54 7.59 7.54 7.48 7.46 day 8.00 8.13 8.35 8.62 7.80 7.89 8.04 8.31 DO night 8.48 8.40 8.27 8.16 8.52 8.21 7.84 7.61 day 8.08 7.61 6.73 6.53 8.87 6.46 6.02 4.93 SS night 9.52 8.61 7.84 7.06 10.06 8.93 8.49 7.76 day 3.82 2.94 2.91 2.95 3.83 3.12 2.93 2.97 DOC night 3.58 3.03 3.07 3.07 3.48 3.14 3.08 3.01 day 0.314 0.312 0.305 0.293 0.395 0.377 0.363 0.381 DTN night 0.346 0.359 0.349 0.356 0.331 0.340 0.331 0.336 day 0.131 0.126 0.118 0.112 0.194 0.179 0.155 0.150 NH 4 -N night 0.165 0.157 0.150 0.160 0.188 0.170 0.169 0.170 day 0.047 0.052 0.053 0.059 0.049 0.058 0.078 0.069 NO 2+3 -N night 0.035 0.033 0.041 0.043 0.036 0.047 0.054 0.070 day 0.049 0.044 0.041 0.046 0.067 0.062 0.069 0.064 PO 4 -P night 0.044 0.050 0.054 0.057 0.072 0.067 0.068 0.067 - 108 - From the VSS distribution, it is indicated that removed VSS precipitates downward and that many detritus are accumulated in the bottom layer. Therefore, each measurement result of the bottom layer was all underestimated. However, because the detritus supply is performed more sufficiently in the lower part, the activity of bacteria, protozoa, and metazoa that mineralize particulate organic matter and its degradation by-products was higher in the middle layer than in the upper layer. The result that nitrification rate was lower in the upper layer than that in the middle layer is considered to be caused by the inhibition of sunlight and competition with algae on the surface (Lipschultz et al., 1985; Azov and Tregubova, 1995). Although the DO condition in the batch experiment is different from the test channels, the profile of denitrification rate suggests that there exist much heterotrophic bacteria in the upper layer. As a result of performing similar activity experiments of the brick-filled channel, mineralization rate was 1.2 mgN･gVSS -1 ･d -1 , which lower than those in the both upper and middle layer of the gravel-filled channel. Also, nitrification rate and denitrification rate were 3.4 and 6.8 mgN･gVSS -1 ･d -1 , respectively, which showed almost the same rate with those in the middle layer of the gravel-filled channel. Although many flagellated protozoa occurred in the gravel-filled channel, there was no evident difference recognized in microfauna between the two channels. It was shown that algae grew greatly at 11.2 µgChl.a･ cm -2 in the inflow section of the brick-filled channel compared with the gravel-filled channel, but Chl.a Table 3. The succession of biotas according to downstream distance in test channels in October 2000 brick-filled channel gravel-filled channel Biota 3 m 10 m 20 m 30 m 3 m 10 m 20 m 30 m Bacteria (10 6 cells・cm -2 ) 113.2 88.8 74.3 57.7 97.5 110.3 229.0 134.7 Nitrifying bacteria 27.7 4.3 7.1 17.3 27.4 20.6 54.8 66.2 ammonia oxidizing 26.8 4.2 6.7 16.7 25.6 20.0 51.2 64.0 nitrite oxidizing 0.9 0.1 0.4 0.6 1.8 0.6 3.6 2.2 Protozoa (N per cm 2 ) 1360 845 2086 1228 2463 1440 3520 3253 Mastigophora 858 726 1821 1144 2087 1347 2987 2933 Sarcodina 21 21 28 21 33 40 40 93 Ciliata 481 98 237 63 343 53 493 227 crawling 321 42 70 7 127 13 13 80 stalked 7 3 40 free swimming 153 56 167 56 213 40 480 107 Metazoa (N per cm 2 ) 279 105 98 91 110 53 100 120 Rotatoria 147 84 70 63 60 40 60 27 Gastrotricha 7 7 7 7 13 27 Nematoda 105 7 14 21 33 7 27 Oligochaeta 14 7 7 67 Ostracoda 7 13 7 Copepoda 3 Tardigrada 7 7 Chlorophyll-a (µg・cm -2 ) 11.2 3.6 2.9 2.9 4.0 3.7 6.3 3.1 Macrobenthos (N per m 2 ) 326 465 442 558 1493 1946 1787 1879 Diptera 279 465 442 465 1400 1800 1267 1713 Gastropoda 93 73 133 487 153 Ephemeroptera 20 13 33 13 Hirudinea 47 VSS (mg・cm -2 ) 4.3 1.6 1.8 2.5 3.5 4.1 8.4 4.2 024681012 Fig. 2. Vertical distribution of mineralization ( ), nitrification ( ) and denitrification ( ) rate in the gravel-filled channel . upper layer (0-4cm) middle layer (4-8cm) VSS mg cm -2 bottom layer (8-12cm) 0123456 mgN gVSS -1 d -1 - 109 - concentration decreased afterwards. Van Zanten and van Dijk (1994) reported that rotifers and phytoplankton have a positive correlation, and this reported result coincides with that seen in the brick-filled channel. While macrobenthos, Diptera such as Chironomus sp. occurs many, are of about 330 - 560N･m -2 in the brick-filled channel, those are of about 1500-1950 N･m -2 and the occurrence of Ephemeroptera and Gastropoda were observed in the gravel-filled channel. As shown in Figure 3 and Figure 4, abundance of Ephemeroptera that live hidden in accumulated deposits show a similar trend to the abundance of algae which are their food source, and it is recognized that omnivorous Gastropoda (Physidae) which prefer a dark condition have strong positive correlation with gravel-attached VSS. On the other hand, from the result that macrobenthos were fewer at the 3 m point of the brick-filled channel where Chl.a and VSS increased greatly, it is clear that shading effect and accumulation of particulate organic matter by the multilayer structure play an important role in creating microhabitat of macrobenthos. Table 4 shows the results of measuring oxygen production (O P ) and uptake rate (O R ) in biofilm. The method of grasping riverbed activity from oxygen uptake rate is well known as a highly repeatable and easy to measure method for estimating biofilm activity (Hanes and Irvine, 1968). Moreover, it is possible to separate a specific microbial activity and quantify it by adding an inhibitor (Surmacz-Gorska et al., 1996). While O P /O R was 0.69 in average for the case of brick-filled channel, it turned out to be 0.10 in average for the gravel-filled channel, which means that oxygen uptake accompanying degradation is remarkable compared with the primary production in the gravel-filled channel. Then, as shown in Table 5, there was about eight times difference in oxygen production rate per Chl.a between both channels, and abundance ratio per Chl.a of Pheo.a, which inactive algae have, in the gravel-filled channel was more than twice as high. Table 4. Comparison of oxygen production and uptake rate of biofilm between brick-filled channel and gravel-filled channel in October 2000 O P , O R gO 2 ･m -2 ･d -1 ( % ) Filler 3m 10m 20m 30m brick 2.77 1.47 0.80 1.12 Oxygen production rate (O P ) gravel 0.35 0.23 0.06 0.06 brick 3.40 1.60 1.73 1.94 Oxygen uptake rate (O R ) gravel 1.88 1.55 2.88 1.67 brick 0.09 ( 2.6) 0.08 ( 5.2) 0.00 ( 0.0) 0.03 ( 1.5) nitrifying bacteria gravel 0.25 (13.3) 0.03 ( 2.1) 0.47 (16.2) 0.18 (10.6) brick 0.04 ( 2.3) 0.03 ( 1.7) 0.06 ( 2.9) protozoa and metazoa gravel 0.05 ( 3.5) 0.08 ( 2.9) 0.23 (13.7) brick 3.32 (97.4) 1.48 (92.5) 1.71 (98.3) 1.85 (95.6) heterotrophic bacteria and algae gravel 1.63 (86.7) 1.47 (94.4) 2.33 (80.9) 1.27 (75.8) brick 0.81 0.92 0.46 0.58 O P / O R gravel 0.19 0.15 0.02 0.04 Table 5. Average value of algal activity in 3, 10, 20, and 30 m point Filler of streamside O P / Chl.a (mgO 2 ･mgChl.a ‐ 1 ・d -1 ) Pheo.a / Chl.a (-) brick 33.0 ± 8.1 0.83 ± 0.28 gravel 4.2 ± 3.9 1.98 ± 0.27 0 2 4 6 8 0 10 20 30 40 50 0 5 10 15 20 25 30 Fig. 3. Relationship between ephemeroptera and Chl.a in the gravel-filled chennel Chl.a Ephemeroptera Chl.a µg cm -2 Ephemeroptera N m -2 DISTANCE m . . 0 2 4 6 8 10 0 100 200 300 400 500 600 700 0 5 10 15 20 25 30 Fig. 4. Relationship between gastropoda and VSS in the gravel-filled channel VSS Gastropoda VSS mg cm -2 Gastropoda N m -2 DISTANCE m ･ ･ - 110 - As shown in Table 4, O R of protozoa and metazoa increased downstream in the gravel-filled channel, and maximumized in the outflow section, being 0.23 gO 2 ･m -2 ･d -1 which is four times higher than that at the 10 m point, and all of points showed higher oxygen uptake than in the brick-filled channel. It is well known that predation effect of protozoa and metazoa are influenced by the activity of bacteria (Berk and Botts, 1984). From this, it is considered that both were activated synergistically downstream. Oxygen uptake rate by nitrifying bacteria in the gravel-filled channel is maintained high at an average of 10 % of the total O R , shown a similar change to the number of nitrifying bacteria. On the other hand, oxygen uptake rate by these in the brick-filled channel is minor, which means that heterotrophic bacteria and epilithic algae are dominant. In this way, it is possible to evaluate the activity of microbial community in each trophic level by the oxygen production/uptake experiments using various inhibitors. CONCLUSIONS Through test channel experiments an investigation was performed on the influence of differences in the surface area of the streamside in a flow water system on succession of aquatic biotas and microbial activities. As the result, the gravel-filled multilayer structure promotes removal of SS, and by that heterotrophic bacteria and macrobenthos that actively utilize detritus increased. Furthermore, in the internal space of the gravel-filled channel, nitrifying bacteria increase and become activated. Although there was no large difference seen between species compositions of protozoa and metazoa, their density per surface area was high in the gravel-filled channel, and oxygen uptake rate downstream also became higher. Based on the above, it suggests that surface area and porosity of streamside are important controlling factors to promote the detritus food chain to high trophic levels, and to diversify aquatic biota including microorganisms. REFERENCES Azov Y. and Tregubova T. (1995). Nitrification processes in stabilisation reservoirs. Wat. Sci. Tech., 31(12), 313-319. Berk S. G. and Botts J. A. (1984). Indirect effects of chlorinated wastewater on bacteriovorous protozoa. Environ. Pollut. Ser. 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Tech., 29(3), 49-51. . 0.356 0.3 31 0.340 0.3 31 0.336 day 0 .13 1 0 .12 6 0 .11 8 0 .11 2 0 .19 4 0 .17 9 0 .15 5 0 .15 0 NH 4 -N night 0 .16 5 0 .15 7 0 .15 0 0 .16 0 0 .18 8 0 .17 0 0 .16 9 0 .17 0 day 0.047. Mastigophora 858 726 18 21 114 4 2087 13 47 2987 2933 Sarcodina 21 21 28 21 33 40 40 93 Ciliata 4 81 98 237 63 343 53 493 227 crawling 3 21 42 70 7 12 7 13 13 80 stalked