Removal of nitrogen, phosphorus and COD from waste water using sand filtration system with Phragmites Australis

War. Res. Vol. 21, No. 10, pp. 1217-1224, 1987 Printed in Great Britain. All rights reserved

0043-1354/87 $3.00+0.00 Copyright © 1987 Pergamon Journals Ltd

REMOVAL OF NITROGEN, PHOSPHORUS A N D COD FROM WASTE WATER USING SAND FILTRATION SYSTEM WITH P H R A G M I T E S A U S T R A L I S ARIYAWATHIE G. WATHUGALA*, TAKAO SUZUKI and YASUSHI KURIHARA'~ Biological Institute, Faculty of Science, Tohoku University, Sendal 980, Japan

(Received August 1986) Abstract--The removal efficiencies of nitrogen, phosphorus and COD from waste water were examined using sand filtration systems with Phragmites australis (Cav.) Trin. ex. Steudel. The quality of effluent waters from the system with plant were far better than those from the one without plant, implying Phragmites could incorporate nitrogen and phosphorus into its tissues and promote phosphorus absorption onto the sand by the release of oxygen from the roots. The P-pot provided with the infiuent containing 198 mgl -~ of total nitrogen and 21 mgl -~ of total phosphorus had the highest biomass of Phragmites. Harvestable above-ground biomass accounted for about 3.5 kg m-2 and removable nitrogen and phosphorus accounted for 69 and 6 g m -2 respectively. The removal rates of total nitrogen and phosphorus in the system with Phragmites receiving variable amounts of COD were almost at the same level and also much better than those of the systems without plant, implying that the different COD concentrations in the influent media do not impair the removal efficiencies of nitrogen and phosphorus. Also Phragmites was found to resist COD concentration as high as 128 m g l - ' , and signs of dogging were not detected in this system throughout the experiment.

Key words--nitrogen, phosphorus, COD, sand filtration system, Phragmites, removal etticiency, distribution of N and P in the pot

INTRODUCTION The use of vascular aquatic plants for the removal o f nitrogen, phosphorus and other nutrients from the domestic and agricultural wastes is in increasing demand (Toth, 1972; Tourbier and Pierson, 1976; O'Briem, 1981; Wolverton, 1982). As the aquatic macrophyte, Phragmites australis has been successfully employed to purify waste water, using reed ponds (de Jong, 1976) and natural marsh (Toth, 1972). As these systems require a large area, it is hoped that a system with emerged plant will be elaborated to reduce the area of land needed for treatment (de Jong, 1976). While a few studies have been reported on sand or gravel filtration system with Phragmites (Wolverton, 1982; Butijn and Greiner 1985; Gersberg et al., 1986), the ability o f plants in nutrient removal from waste water has not yet been fully examined. In our preliminary studies (Suzuki et al., 1985) on the effects of Phragmites australis on the removal of nitrogen, phosphorus and C O D in sand filtration pot systems, we obtained results wherein no nitrogen and phosphorus were detected in the effluents obtained by percolating an influent medium containing 10 mg 1of nitrogen and 1.0 mg 1- ~ of phosphorus.

*Present address: Department of Botany, University of Colombo, Colombo 3, Sri Lanka. tTo whom correspondence should be addressed.

The present experiments were therefore designed and conducted (A) to find out the level to which the concentration of nitrogen and phosphorus in the infiuent can be raised without impairing with the effective removal of nutrients by the sand filtration system using Phragmites, and to clarify the quantitative role of plants and sand in nutrient removal, and (B) to study the removal efficiency of C O D at C O D different levels in the influent. MATERIALS AND M E T H O D S

Experiment (A ) Eight polyethylene pots (dia: 68 cm, height: 71 cm, capacity: 2001., Osaka Suiko Co. Lid) were filled to depth of 35 cm with fiver sand previously washed to remove all litter and clay particles (particle size: 0.25-2.0mm). Young Phragmites australis shoots were transplanted at the rate of about 150 shoots m -2 in four pots (P-pots), and another four pots without plants were used as the controls (C-pots). The pots were placed in the field under a roof of Panasola (Mitsui Toatsu Chemical Co. Ltd) to prevent rain water entering them. The concentrations of total nitrogen and phosphorus in the infiuent media were made 4, 8, 16 and 32 times the original concentrations (TN = 11.9, TP = 1.3 mg 1- i) by adding (NH 4)3PO4 3H20 and NH4NO 3 into the 15,000 times diluted Corn Steep Liquor (Oji Corn Starch Co. Ltd). In this case, the COD concentration was left constant (12.6mgl-L). (Hereafter, the Ppots provided with 4, 8, 16 and 32 times concentrated influent media are referred as 4P, 8P, 16P and 32P respectively. Similarly, the respective C-pots are referred as 4C, 8C, 16C and 32C.) The resultant infiuent media concentrations are given in Table 1. Infiuent was continuously dosed from the top of the pot and the effluent discharged from the bottom after percolating through the sand (Fig. 1).

1217

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ARIYAWATHIE G, WATHUGALAet al.

1218

Table l. Chemical compositionsof the influent media (mgl ]) Pot series

4P, 4C

8P, 8C

16P, 16C 32P, 32C

Total N NH:N NO2 + NO~ N

47.6 25.3 19.2

95.2 52.7 39.5

198.4 107.3 80.0

380.8 216.6 161.0

Total P PO+P COD

5.2 4.2 12.6

10,4 9.4 12.6

20.8 19.8 12.6

41.6 40.6 12.6

The depth of the overlying water above the sand was maintained at 20 cm and the retention time was adjusted at 2 weeks. The experiment was conducted from 25 June to 2 November. The effluent waters of the pots were sampled once in 2 4 weeks for chemical analysis, and those for the analysis of dissolved chemical components were filtered through Whatman GF/C filter paper immediately after the sampling. The loss of water from the system due to evapotranspiration was measured as the difference between the volumes of influent and effluent. After the overlying water was taken throughly for the chemical analysis at the end of the experiment, all the Phragmites shoots were harvested and the sedimented and wall-attached materials from the pots taken for the estimation of biomass, nitrogen and phosphorus. The interstitial waters of the pots were sampled by drawing them from the bottom of the pots, and the sand was then sampled from the top in a stratified manner in 5, I0, 10, and 10cm depth intervals. Rhizome and root samples were taken for the estimation of biomass and nitrogen and phosphorus content. Total kjeldahl nitrogen, ammonia and orthophosphate were estimated according to Ukita et al. (1979). Nitrite and nitrate were estimated according to Strickland and Parsons (1972). Total phosphorus was determined by the persulphate method described by Harwood et al. (1969). The nitrogen contents of plant, sand, and sedimented and wallattached materials were estimated using a CN analyzer (Yanaco Model MT-500, Yanagimoto MFG. Co. Ltd). The estimation of total phosphorus in those samples was carried out according to Andersen (1976).

Four influent media containing 21, 42, 85 and 128 mg 1of COD were prepared by diluting Corn Steep Liquor. (NH4)3PO4 . 3H20 and (NH4)2SO4 were then dissolved appropriately in those media to obtain final concentrations of about 190 mg 1-l of total nitrogen and 21 mg 1-t of total phosphorus. The experiment's operating conditions were similar to those previously described and the flow was started in mid June and continued until mid November. (Hereafter the W-pots provided with inttuent media containing 21, 42, 85 and 128mgl -~ of COD are referred as 1P', 2P', 4P' and 6P' respectively. Similarly the respective C'-pots are referred as 1C', 2C', 4C' and 6C'.) The effluent waters were sampled once in 2-4 weeks and the concentrations of total nitrogen and phosphorus estimated as described and the COD estimated by the potassium permanganate oxidation method. At the end of the experiment, plant height and biomass were estimated.

RESULTS

(A ) Removals o f Nitrogen and Phosphorus from the Influent Media and Distribution o f Total Nitrogen and Phosphorus in the Pot System Chemical analysis o f effluent waters The changes in the a m m o n i a and nitrite + nitrate concentrations in the effluent waters are shown in Figs 2(a) and (b). All the effluents were free o f a m m o n i a in the early stage o f the experiment, with a m m o n i a first appearing in 32C after about 60 days o f operation. This concentration increased very rapidly from there on and reached a very high level. In contrast, 32P was effective in removing a m m o n i a for a longer period, but here also, a rapid increase in

Experiment (B ) Eight pots (dia: 50cm, height: 80cm, capacity: 1301.) were prepared as described. Young Phragmites australis shoots were then transplanted (178 shoots m -z) in four of the eight pots (F-pots) with the remaining four pots without plants used as the controls (C'-pots).

~ 100 .,.+ Z

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australis _

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JuI. Aug. Sep. Oct. Nov.

Fig. 1. Experimental set-up.

Fig. 2. The concentrations of ammonia(a), nitrite+ nitrate(b) and orthophosphate(c) in the effluent waters of the P and C-pots.

Removal of N, P and COD by

concentration was observed toward the end of the experiment. Even though traces of ammonia were detected in 16P effluent at the final sampling, the .concentration was much lower than that of 16C, which was even higher than those of 32P at the sampling days except November. 8P, 4P and 4C effluents were free of ammonia throughout the experiment, implying a very high removal rate. On the other hand, nitrite + nitrate were presented in all effluents, except 4P, even from the very beginning of the experiment. The concentrations in all the P-pots except 32P, were lower than those of the respective C-pots. Concentrations in 32P increased very rapidly to reach a very high level even higher than that of the influent toward the end of the experiment, implying that Phragmites play an important role in ammonia removal through nitrification. The 4P effluent was free of nitrite + nitrate throughout the experiment. The total nitrogen were almost equal to the sum of inorganic nitrogen components in the respective effluents. The results obtained for the determinations of effluent water orthophosphate are presented in Fig. 2(c). The orthophosphate appearance pattern was very similar to that of ammonia in Fig. 2(a). Here also, all the effluents were free of orthophosphate in the early stage of the experiment and was first detected in 32C. The effluent concentrations of 32C, 16C and 8C even reached those of their respective influents by the end of the experiment suggesting, that the sand in those pots had reached their saturation levels in absorbing phosphorus. 32P was effective in removing orthophosphate for a longer period and a rapid increase was observed after about 120 days, indicating that its sand had also reached the

Phragmites

1219

saturation level. Even though the effluents of 16P and 4C contained a little orthophosphate toward the end of the experiment, these two pots together with 8P and 4P, which were free of orthophosphate throughout the experiment, can be considered as very effective in removing orthophosphate. The changes of total phosphorus were quite similar in pattern and concentration levels to those of orthophosphate.

Evapo transpirat ion The measurements of loss of water from the pot systems due to evapotranspiration are listed in Table 2. The rates of evapotranspiration of the P-pots were much higher than those of the C-pots. Among the P-pots, 16P had the highest rate, which may be attributed to the highest biomass of Phragmites (Table 3).

Percentage removals of total nitrogen and phosphorus Using the cumulative values of the total inputs and outputs, the total nitrogen and phosphorus removals as percentages of their respective inputs were calculated. The results are presented in Figs 3(a) and (b) respectively. All the P-pots showed higher removal rates of both total nitrogen and phosphorus than the respective C-pots. It was very clear, especially in the C-pots, that the removal rates decreased with time, and also that the decrease started earliest at the highest loading level. The total nitrogen and phosphorus removal rates at the end of the experiment were found to decrease with the increasing loading in both P and C-pot series. Even so, all the P-pots, except 32P regarding total nitrogen, showed very high removal rates of both total nitrogen and phosphorus.

Biomass of Phragmites Table 2. The loss of water from the pot systems due to evapotranspiration Evapotranspiration Pot series

ml m -2 d -~

% of influent

4P 8P 16P 32P

8393 7693 10,227 9303

46 42 56 51

4C 8C 16C 32C

3173 3077 2478 2245

14 13 II l0

The shoot numbers and the biomass of different

Phragmites tissues at the end of the experiment are presented in Table 3. 16P had the highest stem and leaf, hence the highest total biomass. Even though 32P had the highest numbers of shoots, its aboveground and also its total biomass was the lowest.

Amounts of nitrogen and phosphorus incorporated in the plants The nitrogen and phosphorus concentrations in the stems, leaves, rhizomes and roots of Phragmites

Table 3. Shoot numbers and biomass of different tissues of experiment

Phragmites australis at end of

Biomass Pot series 4P 8P 16P 32P

Shoot number No. p o t 175 187 174 234

g dry w t p o t -t Stem

Leaf

Rhizome

Root

Total

601 519 746 507

260 280 353 231

194 311 275 222

175 128 121 78

1230 1238 1495 1038

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ARIYAWATHIEG. WATHUGALAet al.

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Fig. 3. Removal rates of total nitrogen(a) and total phosphorus(b) as percentages of total inputs in the P and C-pots.

grown at different nutrient levels are presented in Fig. 4. The concentrations of both nitrogen and phosphorus in various tissues were of different levels and, within a tissue, an increase was seen with the increasing concentrations in the influent media. Using the biomass of different tissues and the concentrations of nitrogen and phosphorus in those tissues, the total amounts of nitrogen and phosphorus incorporated in the above and under-ground parts of Phragmites in each pot were calculated (Fig. 5). All the values in the above-ground parts were higher than those of the underground parts. Even though an increase in nitrogen and phosphorus concentrations in the tissues were observed with the increasing concentrations in the influent media (Fig.

rl4P

~iSP

116P

132P

4), 16P had the highest amount of both nitrogen and phosphorus, attributed to the highest biomass. Amounts o f nitrogen and phosphorus retained in the sand

The vertical distribution of nitrogen and phosphorus in the sand of the P and C-pots are shown in Fig. 6. The concentrations of both nitrogen and phosphorus in respective layers of sand increased with increasing concentrations in the influent media. Concentration gradients of nitrogen and phosphorus were observed in the P-pot, these concentrations being decreased with the depth. In the C-pots, however, such gradients were observed only for nitrogen at lower loadings. After subtracting the amounts already present in the sand prior to the experiment, the total amounts of nitrogen and phosphorus retained in the sand were calculated, and the results will be incorporated in Figs 7(a) and (b). Distribution of total nitrogen and phosphorus in the pot systems

0 ~ O

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L

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.~0.2

Stem

Leaf

Rhizome

Root

Fig. 4. The concentrations of nitrogen(a) and phosphorus(b) in different tissues of Phragmites austra/is.

In order to compare the distributions of nitrogen and phosphorus in different fractions of the P and C-pots within a series with each other, the percentage values of their respective inputs were shown in Figs 7(a) and (b). In the P-pots, the plants and sand were responsible for higher fractions of nitrogen and phosphorus retained in the pot. The percentages of nitrogen and phosphorus incorporated in the plant decreased with the increasing loading, and the values in the sand increased with the increasing loading, but only up to 16P. In the C-pot series, even though the amounts associated with sand increased with the increasing concentrations in the influent, the per-

Removal of N, P and COD by Phragmites (b)

(a)

(c)

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,° I

E

J¢ Ul Ul ¢0

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/ 41~ t~F ~bv' 32P

4P

8 P 16P 32P

4P

8 P 16P 32P

Fig. 5. The biomass(a) and the amounts of nitrogen(b) and phosphorus(c) in the above-ground (white bar) and underground (black bar) parts of Phragmites australis. TN (mglg) 0.2 0.4

0 I

E .0-5 5 -15 I t~ 25-35 ~-zs

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TN(mglg) TP(mglg) 0.2 0.4 0.2 04 0

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Fig. 6. Vertical distributions of the concentrations of total nitrogen and phosphorus in the sand of the P and C-pots. Broken lines indicate the original concentrations.

a

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[] Plant DSand moutput [] Balance ~ ] O v e r l y i n g a n d interstitial w a t e r s ,. s e d i m e n t e d a n d a t t a c h e d materials

&O

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8C

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32C

16C

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b)

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16P

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4C

8C

Fig. 7. The distributions of total nitrogen(a) and total phosphorus(b) in the P and C-pots as percentages of their total inputs.

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ARIYAWATHIEG.

WATHUGALA

et al.

phorus balance, except for 32P, all the pots showed very small values. (B) COD Removals COD Oi-

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Jul

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1 Sep

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I Nov

Fig. 8. The concentrations of COD in the effluent waters of the F-pot(a) and C'-pot(b).

The changes in the COD concentrations in the effluents are shown in Fig. 8. The concentrations of the F - p o t s were lower than those of the respective C'-pots in the early stage of the experiment and the concentrations in the respective P' and C'-pots became almost equal toward the end. The concentrations in all the effluents seemed to be dependent on that of the influent. In spite of the even higher concentration such as 128 mgl ~ in the influent, 6P" effluent seemed to have even lower concentrations than that of 4C'. Removal rates o f total nitrogen, total phosphorus and COD

centage values of nitrogen in the sand were more or less at the same level. On the phosphorus, however, a decrease was seen with the increasing loading. At higher loadings, the amounts in sand in the P-pots (16P and 32P) were higher than those in the C-pots. In both P and C-pot series, the output increased with the increasing loading of both nitrogen and phosphorus. Outputs of the P-pots being always lower than those of the respective C-pots. The fractions presented as "overlying and interstitial waters + sedimented and attached materials" were more or less at the same level, implying that they had increased with the increasing concentrations of influent. This increase is due to the increase in concentrations in the interstitial and mainly in the overlying waters, and not due to the amounts in the algal biomass. The differences between inputs and all other estimated amounts are presented as the balance. The amounts of nitrogen calculated as balance may be due to denitrification (Kickuth, 1976), and these amounts were higher in the P-pots than those in their respective C-pots. However, on the phos-

Making use of the total nitrogen, total phosphorus and COD concentrations in the influent and effluent waters, and the loss of water due to evapotranspiration, the total inputs, outputs, and then the total removals of the nutrients at the end of the experiment were calculated (Table 4). The removals of total nitrogen, total phosphorus and COD in the W-pots were higher than those of the respective C'-pots. As expected, phosphorus removal in the W-pots was 100%, and that of the C'-pots, much lower. The removals and the removal rates of COD increased with the increasing loading along the P' and C'-pot series. Plant growth The Phragmites biomass and also the average height of the plants at the end of the experiment showed very little change from one pot to the other. The plants in 4P' were the tallest, and those in the 2P' had the highest biomass. In general, the plants in all four pots appeared healthy throughout the experiment.

Table 4. The total inputs, outputs and removals of total nitrogen, phosphorus and COD in the P' and C'-pots at the end of the experiment F-pots Pot series

1P'

2P"

C-pots 4P'

6P'

IC'

2C'

4C'

6C'

Total nitrogen Input gpot ~ Output g pot ~ Removal g pot t % Removal

136 27 109 80

136 17 119 88

134 26 108 81

134 21 113 84

136 50 86 63

136 48 88 65

134 48 86 64

134 45 89 66

14.8 0.0 14.8 100

14,8 0.0 14.8 100

14.7 0.0 14.7 100

14.7 0,0 14.7 100

14.8 3.6 11.2 76

14.8 2.1 12.7 86

14.7 2.2 12,5 85

14.7 2.4 12.3 84

15.7 1,2 14.5 92

30.6 0,8 29,8 97

59.6 1.8 57.8 97

89,0 2.0 87.0 98

15.7 2.3 13.4 85

30.6 3.5 27, I 89

59,6 5.2 54.4 91

89.0 5.2 83,8 94

Total phosphorus Input gpot ~ Output g pot ~ Removal gpot ~ % Removal

COD Input g pot ~ Output gpot ~ Removal g pot ~ % Rerqoval

Removal of N, P and COD by Phragmites DISCUSSION

At all nutrient concentration levels in the influent media studied, the P-pots showed better removal of ammonia, nitrite + nitrate (except 32P), total nitrogen, orthophosphate and total phosphorus than their respective C-pots, implying that the presence of Phragmites is effective in removing nutrients from the waste water. The removal rates of total nitrogen and phosphorus in the P'-pots which received variable amounts of COD were almost at the same level and also much better than those of the respective C - p o t s (Table 4), implying that the different concentrations of COD in the influent media do not impair with the removal efficiencies of nitrogen and phosphorus and that Phragmites was found to resist COD concentration as high as 128 mg 1-1, and signs of clogging were not detected in this pot throughout the experiment. In some cases, the nitrite + nitrate concentration in the effluents were higher than those of the respective influents [Fig. 2(b)], probably due to the ability of emerged macrophytes to transport oxygen down to the roots, thereby stimulating the growth of nitrifying bacteria (Gersberg et al., 1986; Armstrong, 1964). The nitrate can then percolate through to the oxygenpoor zone where it will be removed from the system by denitrification (Gersberg et al., 1983). The fact that the balance values of nitrogen in the pot with Phragmites were higher than those without Phragmites (Fig. 7) may be explained by the promoting activity of Phragmites for denitrification. It may therefore be pointed out that the presence of emerged macrophyte was significant in ammonia removal and denitrification. Gersberg et al. (1986) showed that bulrush and reed were superior in removing ammonia, when the influent ammonia-nitrogen concentration was 24.7mgl -1. Wolverton (1982) also showed a high ammonia removal rate by a rock-reed filter at 12.4 mg 1-l initial ammonia concentration. These inflow ammonia-nitrogen concentration levels are similar to or lower than that of the influent of 4P in this experiment, and probably may be too low to evaluate the potential of Phragmites in nutrient removal. In this study, the removal rates of total nitrogen and phosphorus of 16P, which received 198mgl -~ of total nitrogen and 21 mg1-1 of total phosphorus indicated 88 and 99% respectively at the end of the experiment. 16P also produced the highest Phragmites biomass. Large quantities of nitrogen and phosphorus incorporated into the plant tissues could be removed from the system by harvesting. The harvestable aboveground biomass of 16P accounted for about 3.5 kg m -2 (dry wt basis), and is higher than most of the values reported by Dykyjova (1978) and Kvet (1973a) for Phragmites. The amounts of nitrogen and phosphorus that can be removed with above-ground biomass of 16P ac-

1223

count for 69 and 6 g m -2 respectively. These values are higher than those reported by Kvet (1973b), de Jong (1976) and Dykyjova (1978) on Phragmites, and our estimate for nitrogen is in the upper range of the values reported for the nitrogen removal potential of the above-ground biomass of reed (Wolverton, 1982). Wathugala et al. (1985) reported that the amount of phosphorus incorporated in the above-ground biomass of Zizania latifolia accounted for 7.9 g m -2. This value is a little higher than that obtained from Phragmites (6.0gm -2) in this experiment. It is known however, that Phragmites distributed over a wide area can tolerate extreme environmental conditions concerning pH, salinity, oxygen depletion and pollution (Ranwell et al., 1964; Haslam, 1972; Yamasaki, 1984), and these should be an advantage for the application of Phragmites in wastewater treatment. Working with artificial marsh systems with emerged vegetation (bulrush) for the treatment of municipal waste waters, Spangler et al. (1976) found that the harvested shoots contained only 14% of the total phosphorus, and the remainder were in nonbiotic materials associated with the gravel. In the present study, the percentage of phosphorus absorbed onto the sand of the P-pots were higher than those of the respective C-pots [Fig. 7(b)]. In the P-pots, gradients of nitrogen and phosphorus concentrations in the sand were observed (Fig. 6), the upper most layer being the most concentrated. The upper 15 cm layer contained more than 50% of the total nitrogen and phosphorus retained in the sand. The aerobic conditions created by the release of oxygen into the substratum through the root systems of Phragmites (Armstrong, 1964; Teal and Kanwisher, 1966) should be responsible for this additional absorption of phosphorus onto the sand in the P-pots. Spangler et al. (1976) Showed that aerobic conditions were maintained in the rhizosphere and the amount of phosphorus in the gravel zone of the pond with emerged vegetation was higher than that of the control. Therefore, emerged macrophyte was found to make indirect contributions in nutrient removal by stimulating the nutrient absorption onto the sand and by promoting the denitrification activity as mentioned, in addition to the direct nutrient uptake. The concentrations of nitrogen, phosphorus and COD in the influent media in 16P which exhibited the highest removal of nitrogen and phosphorus, may correspond to the secondary effluent of waste treatment facilities for domestic animals in Japan (Sano et al., 1980), it woud therefore be suggested that such effluents can be polished by using a sand filtration system with Phragmites. In the application of this kind of a system, porous tubes can be embedded at the bottom of the sand bed to collect the clean water. Paddy fields where cultivation has been stopped through government policy, can be suggested as an area available for this purpose. In continuous operation of this system for many years, the upper most

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ARIYAWATHIE G. WATHUGALAet al.

layers of sand should be c o n c e n t r a t e d with phosphorus. Therefore, the u p p e r sand layer must be replaced with a new layer, a n d the removed sand can be used to improve n u t r i e n t p o o r soils for cultivation purposes, otherwise the sand bed can be operated every o t h e r year. The Phragmites shoots harvested at the end of the growing season can be used in m a k i n g traditional crafts, thatching, fences a n d wind breakers etc., a n d can also be converted into c o m p o s t a n d used as a soil additive ( K u r i h a r a et al., 1986). Acknowledgement--This study was supported in part by a grant in aid for scientific research from Ministry of Education, Culture and Science, Japan (No. 58030003).

REFERENCES

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