INTRODUCTION

Urea in natural waters is a significant compound being one of the major nitrogen sources for phytoplankton as well as a key organic compound in the biogeochemical cycle. There is now an appreciable amount of data on the distribution of urea concentrations in reservoirs (Mitamura et al., 1990, 1994, 1995, 1997). The uptake of urea nitrogen by phytoplankton in artificial reservoirs was measured with the ¹⁵N tracer technique (Mitamura et al., 1993, 1997). Urea carbon decomposition by reservoir phytoplankton has been demonstrated using ¹⁴C- 1abelled urea (Mitamura et al., 1989, 1990, 1995). Results indicated that urea played a significant role as a nitrogen source for phytoplankton populations in reservoirs, and was decomposed more effectively by phytoplankton than by bacteria, at least, under natural conditions.

¹⁴C- and ¹⁵N-labelled urea have been used to measure the rate of urea carbon decomposition and urea nitrogen uptake. The rates determined using these two isotopes, however, were not always equal. Harrison et al. (1985) and Price and Harrison (1988) reported that the urea carbon decomposition rates using ¹⁴C-labelled urea were higher than those using ¹⁵N-labelled urea by marine phytoplankton. On the other hand, Mitamura and Saijo (1986) measured equivalent values of both rates by freshwater phytoplankton in Lake Biwa. Horrigan and McCarthy (1982) also found that both rates had similar values using Thalassiosira pseudonana and Skeletonema costatum. To obtain further information on urea cycling in freshwater, the relationship between rates of both urea carbon decomposition and urea nitrogen uptake by reservoir phytoplankton was examined.

MATERIALS AND METHODS

Field investigations of urea carbon decomposition and urea nitrogen uptake were made at a station near the dam of Lakes Paro, Soyang, Chuncheon and Uiam located in the upper reaches of the North Han River, Lake Chungju in the South Han River, and Lake Paldang where the North and South Han Rivers converge at the upper area of the reservoir (Fig. 1).

To measure the rate of urea carbon decomposition, sample waters were collected from various depths of the upper euphotic zone (0~3m depth) with a Van Dorn plastic sampler. Twenty-ml water samples taken from each layer were poured into three series of clear transparent glass bottles. Crystalline ¹⁴C-1abelled urea (Amersham; sp act, 1.85 GBq/mmol) was dissolved in sterile, deionized distilled water, and then the stock ¹⁴C-1abelled urea solution was stored at -20°C. After adding 0.5 ml of the diluted ¹⁴C- 1abell-ed urea solution (containing 2 nmol 12Curea and 3.7 kBq ¹⁴C-urea) to each bottle, 0.2 ml of concentrated formaldehyde solution was immediately added to a series of control bottles. The second series of bottles was wrapped in a black sheet for a determination of the urea carbon decomposition rate in the dark. To measure the in situ rate, the transparent and dark bottles were suspended from a buoy at the respective depths from which the water samples were collected. After leaving the bottles from noon to sunset, biological activity was stopped by adding formaldehyde solution to each bottle. To measure the rates under continuous light intensity in the laboratory, on the other hand, the transparent (light) and dark bottles were incubated in a water tank under 140 μEinst m^-2 sec^-1 using daylight type fluorescent lamps at temperatures which were similar to the surface water temperature of the investigated reservoirs. After four hours of incubation, the urea carbon decomposing activity was terminated by adding formal- dehyde solution. Sample water in each glass bottle of the field and laboratory experiments was then filtered through a Millipore HA type filter. The filter was put in a scintillation vial, and 10 ml of Bray scintillation fluid was added. The radioactivity was then measured with a 1iquid scintillation spectrometer (ALOKA model LSC-651) to determine the rate of urea carbon incorporation into particulate organic matter. The filtrate of each sample was poured into a separate 50-ml glass bottle with a screw cap, and a CO₂ absorption tube containing 0.5 ml of n-ethanolamine was inserted into each bottle to absorb the ¹⁴CO₂ liberated from the sample solution by acidification. After adding 1 ml of 1M sulfuric acid solution to each filtrate, the bottles were sealed tightly and left for four days at room temperature. After adding the scintillation fluid to n-ethanolamine containing ¹⁴CO₂ liberated from sample water in the glass bottle, the radioactivity was determined as described above. The rate of urea carbon decomposition was calculated as the sum of the carbon incorporation rate into the particulate matter and the CO₂ liberation rate into the water from the urea.

Three series of 200-ml transparent glass bottles were provided for the measurement of the urea nitrogen uptake rate. After adding five concentrations of ¹⁵N-1abelled urea solution to each bottle, considering the ambient concentrations in the surface water, these bottles were incubated simultaneously with the urea carbon decomposition measurements. After the biological activity of phytoplankton was stopped by adding formaldehyde solution to each bottle, sample water in each one was filtered through a glass fiber filter (Whatman GF/C) which was free of organic matter by ignition at 420°C. The ratio of ¹⁵N to ¹⁴N in each filter sample was determined by optical emission spectrometry with a ¹⁵N analyzer (JASCO type NIA-1) after dry combusting with calcium oxide and copper oxide in a capillary by the micro-Dumas’ method. The nitrogen uptake rates of urea were extrapolated using a linear transformation of the following Michaelis-Menten equation, assuming that the rate was related to the ambient concentration of urea: V=Vmax∙S/(Ks+S), where V is the nitrogen uptake rate of urea, S is the urea concentration, Vmax is the nitrogen uptake rate at saturating levels of S, and Ks is the half-saturation constant at which V=Vmax/2.

Photosynthetic activity was measured by the ¹⁴C technique of Steemann Nielsen (1952), simultaneously during both experiments for the urea carbon decomposition and the urea nitrogen uptake measurements. The total CO₂ in the sample water was determined with an infrared CO₂ analyzer, as described by Satake et al. (1972).

To determine the ambient concentration of nitrogenous nutrients and particulate matter, the collected sample waters were immediately filtered through glass fiber filters (Whatman GF/C) treated by ignition at 420°C. The filters and filtrates were then stored at -20°C in a deep freezer until chemical analysis in the laboratory. The concentration of urea was determined by the method of Newell et al. (1967). Ammonia was determined by the method of Sagi (1966), nitrite after Bendschneider and Robinson (1952), and nitrate by the method of Wood et al. (1967). Particulate nitrogen (PN) was determined with a CHN Corder (YANACO type MT-3). Chlorophyll -a concentration was determined by the method of SCOR/Unesco (1966).

RESULTS AND DISCUSSION

1. Distribution of urea

Distributions of urea concentration in the upper euphotic zone of investigated reservoirs were listed in Table 1. The concentration of urea ranged from 0.1 to 0.6 μmol/L in the six reservoirs. No appreciable change in the vertical distribution of urea concentration was observed. The urea nitrogen levels in Lakes Paro and Chuncheon were comparable to the ammonia concentrations, but had much lower values in Lakes Chungju and Paldang. The contribution of urea nitrogen to the total nitrogenous nutrients (the sum of urea, ammonia, nitrite, nitrate nitrogen) was considerably low, ranging from 0.1 to 5.3%, due to the high concentration of nitrate nitrogen.

2. Urea carbon decomposition

Table 2 shows the urea carbon decomposition rate in transparent and dark bottles measured by the in situ and laboratory experiments in all six reservoirs.

The urea carbon decomposition rates in the upper euphotic zone were 2 to 70μg ureaCm^-3 h^-1 in the light and 1 to 16 μg ureaC m^-3 h^-1 in the dark. The decomposition rates of urea in Lakes Paro and Soyang showed lower values than those observed in the other four reservoirs. The highest rates were obtained at the surface layer of Lakes Chuncheon and Paldang.

The chlorophyll-a specific rate of the urea carbon decomposition rate, calculated as the average value in each reservoir, was 5 to 12 μg ureaC mgchl.a^-1 h^-1 in the light and 2 to 5 μg ureaC mgchl.a^-1 h^-1 in the dark in the six reservoirs (Fig. 2). On the other hand, ratios of the urea carbon decomposition rate to the photosynthetic carbon uptake rate were 3 to 8 μg ureaC/ mgphoto.C. The urea carbon decomposition rate seems to be related to the standing crop of reservoir phytoplankton and their photosynthetic activity. These findings indicate that a considerable part of urea carbon decomposition is associated with the photosynthesis of phytoplankton.

3. Urea nitrogen uptake

As shown in Table 2, the urea nitrogen uptake rates, measured simultaneously with the urea carbon decomposition, were 7 to 170 μg ureaNm^-3 h^-1 in the light and 3 to 120 μg ureaN m^-3 h^-1 in the dark in all six reservoirs, respectively. The uptake rates generally decreased with depth. The rates in Lakes Chuncheon and Paldang were considerably higher compared with those in the other four reservoirs. The rates showed higher values in the light than in the dark bottles. The distribution patterns of urea nitrogen uptake showed a similar tendency, to those of urea carbon decomposition.

As can be seen in Fig 2, the activity of urea nitrogen uptake by reservoir phytoplankton, calculated from the nitrogen uptake rate by unit amount of chlorophyll-a, was 5 to 46 μg ureaN mgchl.a^-1 h^-1 in the light and 2 to 21 μg ureaN mgchl.a^-1 h^-1 in the dark. A similar distribution pattern was observed for PN specific rates for urea nitrogen uptake. The ratios of the urea nitrogen uptake rate to the photosynthetic carbon uptake rate were 7 to 41 μg ureaN/mgphoto. C. The urea nitrogen uptake rates were closely related to the photosynthetic activity as well as to the urea carbon decomposition. The variations in the chlorophyll-a specific rate of urea carbon decomposition and urea nitrogen uptake might be due to water temperature in the field and laboratory incubation, differences in the utilizable activity of phytoplankton species, and the effect of other nitrogenous nutrients on urea carbon decomposition, as suggested by Mitamura and Saijo (1986).

4. Cycling of urea

The daily rates of urea carbon decomposition were estimated from the light and dark values during in situ or laboratory incubation. The daily nitrogen uptake rate was also estimated in the same manner. The daily rates of urea carbon decomposition and urea nitrogen uptake were 0.1 to 1.0 mgC m^-3 day^-1 and 0.1 to 3.8 mgN m^-3 day^-1 in the upper euphotic zone of the six reservoirs (Table 2). These daily rates in Lake Paro were low compared with those in the other five reservoirs. Both rates showed a similar change and resembled the values of primary productivity.

The turnover time of urea in steady state can by expressed as the time necessary to utilize an amount of urea equivalent to the ambient concentration. As shown in Fig. 3, the range in turnover times of the upper euphotic zone was 1 to 94 days calculated from the urea carbon decomposition rates and 3 to 107 days from the urea nitrogen uptake rates in six reservoirs. The times from both carbon and nitrogen calculations showed almost the same value. The turnover times tended to increase with depth. Much shorter turnover times were obtained in Lake Chungju. On the other hand, the times in Lake Paro required much longer periods. The present turnover times resemble the values obtained by previous investigators (McCarthy et al., 1982, Mitamura and Saijo, 1986). Brief turnover times indicate that urea, as reflected in the term regenerated production, are associated with rapidly recycled forms, as reported by Dugdale and Goering (1967).

In the euphotic zone of reservoirs located on the North and South Han River System, urea in the regenerated form of a nitrogenous compound makes a major contribution as a nitrogen source to the growth of phytoplankton populations, and plays a significant role in the biogeochemical nitrogen cycle.

5. Relationship between urea carbon decomposition and urea nitrogen uptake

As can be seen from the results of both experiments using ¹⁴C- and ¹⁵N-labelled urea, the distributions of urea carbon decomposition showed a similar pattern to those of urea nitrogen uptake. Carpenter et al. (1972) and Mitamura and Saijo (1986) suggested that the urea decomposing activity has a close relationship with the utilization of urea nitrogen by phytoplankton. Data serving to clarify the relationship between urea carbon decomposition and urea nitrogen uptake, however, are still insufficient. Using coastal marine diatoms, Price and Harrison (1988) reported that both rates, determined using these two isotopes, showed significant differences from the expected 1 : 1 ratio (as molar unit). Mitamura and Saijo (1986) found that the ratio of urea carbon decomposition to urea nitrogen uptake, since values were calculated as unit mole of urea, averaged 0.98 in Lake Biwa.

To clarify the relationship between the urea carbon decomposition rate measured with ¹⁴C- 1abelled urea and the urea nitrogen uptake rate measured with ¹⁵N-1abelled urea, all the values obtained in the upper euphotic zone of the six reservoirs were plotted in Fig. 4. A close linear relation between the two methods was observed. Based on the linear regression analysis for the urea carbon decomposition rate against the urea nitrogen uptake rate, the following equation was derived: UC (rate of urea carbon decomposition; μMurea m^-3 h^-1)=0.70UN (rate of urea nitrogen uptake; μMurea m^-3 h^-1)+0.08 (r=0.844, p< 0.001). Weight ratios of the urea carbon decomposition rate to the urea nitrogen uptake rate were 0.2 to 0.5 in the light and 0.1 to 0.5 in the dark, respectively. No significant differences were observed in the ratio under a variety of environmental conditions such as water temperature, irradiance, urea and other nutrient concentrations in water, standing crop of phytoplankton, or species structure of phytoplankton.

The present slope (0.70; molar rate) and the ratio of urea carbon decomposition to urea nitrogen uptake (0.37; average weight ratio) indicate that practically equivalent amounts of urea nitrogen and urea carbon decomposition (although the urea nitrogen uptake rate was somewhat higher than that of urea carbon decomposition) are simultaneously taken up as a nitrogen source for phytoplankton. Although this seems to be a significant process in the utilization of urea by phytoplankton populations in reservoirs, the detail physiological pathway remains obscure.