**MARINE ECOLOGY PROGRESS SERIES Mar Ecol Prog Ser**  **V ol. 229: 1–10, 2002**  **Published March 20**

**INTRODUCTION**

Coastal power stations generally use seawater as cooling water and thus release seawater with elevated temperature into nearshore environments. In addition, antifouling chemicals are injected into intake seawaterto prevent the growth of fouling organisms on the surface of the cooling systems. Chlorination of seawater ismostly employed by coastal power stations and thereby causes formation of chlorination by-products which might potentially inhibit microbes (Langford 1990). During the passage through cooling systems, intake seawater containing natural microbes is thus exposed to both high temperature (rarely exceeding 40°C) and antifouling chemicals for a short time (e.g. about 10 to 30 min). At the outfalls, thermal effluents become mixed with receiving seawater, and elevated tempera-

© Inter-Research 2002 · www.int-res.com *Corresponding author. E-mail: bccho@plaza.snu.ac.kr 

# Effects of thermal effluents from a power station **on bacteria and heterotrophic nanoflagellates**
**in coastal waters**

**Dong H. Choi 1**  

## , Jong S. Park **1** 

## , Chung Y. Hwang **1** 

## , Sung H. Huh **2**



## , **Byung C. Cho 1,**  

## *

**1 School of Earth and Environmental Sciences and Research Institute of Oceanography, Seoul National University,**  **Seoul 151-742, Korea**

**2 Department of Oceanography, Pukyong National University, Pusan 608-737, Korea** 

ABSTRACT: To investigate effects of thermal effluents from a coastal power station on bacteria and heterotrophic nanoflagellates (HNF) in adjacent coastal waters, surface distributions of bacteria and
HNF and interrelations between microbial and environmental variables were studied in November1998 and July, August and November 1999 near Hadong Power Station, Korea. In addition, manipulation experiments with treatments of high temperature (40°C; temperature in the cooling system of the power station) and additions of hypochlorite and dilution experiments of thermal effluents with intake seawaters were carried out. Water temperature in the discharge-channel was always 5 to 10°C higher than the ambient temperature, but bacterial production, HNF abundance and grazing rates on bacteria, as well as chlorophyll a concentrations, were always lower there and increased with distance from the power station. Manipulation experiments showed that addition of hypochlorite had much more deleterious effects on bacteria and HNF (95 to 98% inhibition of bacterial production and 25 to 45% decrease in HNF abundance at 0.13 ppm of residual chlorine) than high temperature (9 to 39% inhibition of bacterial production but no inhibition of HNF abundance at 40°C). Dilutions of thermal effluents from the outlet and condenser tube with intake seawater would bring <0.03 ppm of

r esidual chlorine, but inhibitory effects of thermal effluents on bacterial production and HNF grazing activity (23 to 69% inhibition of bacterial production and 31 to 36% inhibition of HNF grazing) were shown, indicating inhibitory potential of chlorination by-products in the discharged water on coastal microbes. Analyses of horizontal distributions of microbial variables, manipulation and dilution experiments gave consistently similar results of inhibitory effects of thermal effluents on bacteria and HNF. Our work suggests that a microbial ecological approach is useful in estimating influences of thermal pollution on microbes in aquatic environments.

KEY WORDS: Thermal effluents · Chlorination · Bacteria · Heterotrophic nanoflagellates

Resale or republication not permitted without written consent of the publisherMar Ecol Prog Ser 229: 1–10, 2002

ture and chlorination by-products are always observed near the discharge area (Jenner et al. 1997, Allonier et al. 1999). Therefore, thermal discharges from a coastal power station have 2 main components of pollution to coastal waters, i.e. high temperature and chemicals formed during antifouling procedures.

Micro-organisms are numerically abundant in coastal waters and carry out many ecologically important

r oles in coastal ecosystems. Changes in microbialactivities caused by changes in environmental conditions will thus confer significant impacts on functionsof coastal ecosystems. In fact, marine bacterial responses to additions of inhibitory substances and organic pollutants have been extensively studied (Azam et al. 1977, Siebers et al. 1981, Hudak & Fuhrman 1988, Riemann & Lindgaard-Jørgensen 1990). Responses of marine bacteria and heterotrophic nanoflagellates (HNF) to temperature changes have also been studied in detail (Marrasé et al. 1992, Shiah & Ducklow 1994). Thus, if thermal effluents have deleterious effects onnatural marine microbes, they should be easily detected. Some studies have examined effects of chlorination on marine natural phytoplankton (Carpenter et al. 1972, Eppley et al. 1976). However, studies on
effects of thermal effluents on both natural marine bacteria and HNF using ecologically quantitative approaches are rare. To compare bacterial activities, earlier studies either used the heterotrophic potential approach to calculate maximum uptake velocity (V max ) (Davis & Coughlan 1978) or measured uptake rates of

3 H-glucose added as a tracer (Larrick et al. 1981). However, these approaches do not quantitatively assess the bacterial utilization of organic matter or in

situ growth rates of bacteria in the sea (Azam & Fuhrman 1984). Further, V max has been shown toincrease gradually as the range of added glucose concentration is increased (Azam & Hodson 1981). Plate
counts have also been used to determine the effect of thermal effluents on bacteria (Solski 1974, Guthrie et al. 1978). It is now known that plate count approachesgrossly underestimate total bacteria, total viable bacteria or bacterial diversity in samples (Ferguson et al. 1984, Giovannoni & Rappé 2000). Thus, global effects of thermal effluents on marine bacteria occurring naturally might not have been accurately measured. Based on earlier studies, however, Langford (1990) concluded that short-term temperature exposure had little effect on bacteria over the range normally found in thermal discharges and that the total effects of athermal discharge would mainly depend on the concentrations of chlorine residuals present.

The goals of the present study were to assess the effects of thermal discharge on coastal bacteria and HNF using well-established, quantitative, microbial ecological techniques and to confirm the conclusions of Langford (1990). To analyze effects of the thermal effluent on coastal bacteria and HNF, we measured bacterial abundance and production, HNF abundanceand grazing rates on bacteria along with environmental parameters in coastal waters around a coastal power station. We also carried out manipulation and dilution experiments to evaluate the effects of thermal
effluents on coastal bacteria and HNF.

**MATERIALS AND METHODS**

**A coastal power station at Hadong.** Hadong PowerStation is located between Stn I (intake) and D (discharge) in a coastal area of Kwang-yang Bay, Korea (Fig. 1). The inlet for intake of seawater is located at 10 m depth, and thermal effluents are discharged to the receiving seawater at the outlet. At present,

4 power plants are operating, and a direct cooling system is employed. The time taken for the coolingwater to travel between the dosing point and the outfall is ca 20 min. To prevent growth of biofilms on thesurface of condenser tubes, sodium hypochlorite produced from electrolysing of seawater is applied continuously. The initial dose of sodium hypochlorite is ca 0.35 ppm chlorine. However, the residual chlorine
concentration of seawater in the undiluted effluent stream is kept at ca 0.05 to 0.10 ppm. No significant difference was found in pH between intake and outlet areas (Huh 2000).

**Study area and sample collection.** During 4 investigations in November 1998 and July, August and November 1999 in the Hadong area (Fig. 1), seawater samples were collected with 5 l Niskin bottles from 0, 3 and 5 m depth. Stn D was located at the center of the outlet, which was connected to an adjacent coastal area. At this station, surface seawater samples were collected with a clean 1 l polycarbonate bottle. Each sample was used for determining chlorophyll a (chl a)

, HNF abundance, HNF grazing rates on bacteria and bacterial abundance and production.

**Bacterial abundance and production.** Samples for
measurements of bacterial abundance were fixed with 0.2 μm filtered, borate-buffered formalin (final conc. of

2 %). Bacteria, stained with DAPI (4’,6’-diamidino-2-phenylindole) and collected on black 0.2 μm polycarbonate filters, were counted under UV excitation usingan epifluorescence microscope (Porter & Feig 1980). Bacterial production was measured by the 3 H-thymidine and

14 C-leucine incorporation method (Ducklow et al. 1992).

Te n milliliter samples, in triplicate, were filled into sterile polypropylene tubes, and both 3 H-thymidine (specific activity = 84 Ci mmol–1 ) and 14 C-leucine (specific activity
= 315 mCi mmol–1 ) were added to the tubes, each at 10 nM (final conc.). All the tubes were incubated in the 2Choi et al.: Effects of thermal effluents on coastal microorganisms

dark for 0.5 to 1 h at in situ water temperature. Formalin-killed samples (finalconc. of 2%) served as blanks. After incubation, 3 H-thymidine and 14 C-leucine incorporation was stopped by adding 2% formalin. The samples were filtered onto 0.2 μm cellulose nitrate filters (Whatman) and rinsed according to Ducklow et al. (1992). The filters were radio-assayed.The incorporated radioactivity was converted to cell number produced using

a conversion factor of 2.65 × 10 18 cells
mol–1 thymidine incorporated and 0.18 ×
10 18 cells mol–1 leucine incorporated (Ducklow et al. 1992).

**HNF abundance and grazing rates**

**of HNF.** Samples for measurements of
HNF abundance were fixed immediately with alkaline Lugol solution (finalconc. of 0.5%) and borate-buffered formalin (final conc. of 3%, Sherr et al. 1989). Primulin-stained HNF collected on 0.4 μm polycarbonate filters (25 mmdiameter) were enumerated with UV excitation using an epifluorescence microscope (Caron 1983). HNF grazing rates on bacteria were measured by using fluorescently labeled bacteria(FLB) prepared according to Sherr et al. (1987). For detailed descriptions, see Cho et al. (2000). Briefly, FLB were prepared by staining natural bacterial samplescollected from coastal waters with 5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF). The added concentration of FLB was from 4 to 14% of the bacterial abundance. Grazing experiments were conducted in
the dark at in situ temperature. Subsamples of 30 ml were collected at 0, 10, 20 and 30 min, fixed and kept

r efrigerated until microscopic examinations. Subsamples were primulin-stained and filtered, and at least 150 microscopic fields were counted. Bacterivory rates were measured over the linear portion of the uptake curve. The ranges for the coefficient of variation (i.e.

100 × SD over the mean) of the slopes from duplicate bottles were from 2 to 40% (mean ± SD: 13.7 ± 9.2%). Grazing rates were calculated by multiplying the ratio of bacterial abundance to FLB by the mean values of ingested FLB (l–1 h –1 ).

**Manipulation and dilution experiments.** We carried out manipulation and dilution experiments to assess
the short-term toxic effects of thermal effluents on bacteria and HNF in coastal waters. In the first manipulation experiment, using intake seawater, we assessedinfluences of temperature and chlorine (added as sodium hypochlorite) on coastal microbes: (1) 0.5 ppm (final conc.) of chlorine was added to 2 polycarbonate bottles, which were incubated at in situ temperature; (2) addition of 0.5 ppm (final conc.) of chlorine and increase of water temperature to 40 ± 1°C were applied in 2 other bottles; (3) water temperature was increased to 40 ± 1°C in the remaining 2 bottles. Another 2 bottles were employed as controls and incubated at in situ temperature. Before the treatments, 4 bottles were pre-incubated for ca 40 min at 40°C, the temperature inside the cooling system. Incubations of samples lasted 2 h, and subsampling was carried out every hour. In a second series of experiments, we only focused on influences of varying concentrations ofchlorine on coastal bacteria and HNF. We added varying concentrations (0.13, 0.25 and 0.5 ppm chlorine equivalent) of sodium hypochlorite to coastal seawater samples and incubated samples for 2 h at ambient temperature. Subsamples were collected every hourfor measurements of bacterial abundance and production and HNF abundance. In dilution experiments, the
effluents were cooled to near in situ coastal water temperature and then diluted with coastal seawater 2- (or 5-), 10-, and 100-fold. After 30 min incubations of coastal waters, the effluents and variously diluted effluents, samples were collected for measurements of
microbial variables.

**Other analyses.** W ater temperature was measured with a digital thermometer (APPA Technology Co.). Salinity was measured with a temperature-salinity bridge (YSI 30). Samples for chl a measurements were filtered on GF/F (Whatman) filters. The filters werekept at –20°C and brought to the laboratory. Concen3

Fig. 1. Study area and sampling stations in Kwang-yang Bay, KoreaMar Ecol Prog Ser 229: 1–10, 2002

trations of chl a were measured spectrophotometrically according to Parsons et al. (1984). Concentrations of

r esidual free-chlorine were measured directly at the sampling points using a portable photometer (YSI 9600) and the recommended tablets (Palintest Ltd, Catalogue No. YAP 021 containing DPD (diethyl-p-phenylene diamine ) ). The tablets were added to seawater samples,and a pink color developed as a result of a reaction. Linear regression and t-tests were performed using SPSS for Windows (SPSS Inc. Version 8.0).

**RESULTS**

**Surface distributions of temperature,**

**chl** a **, bacterial and HNF variables,**

**and residual chlorine**

Only data from surface waters were presented here, because data from the surfacemore clearly showed the patterns of influences of thermal discharges than those
from subsurface waters. Surface water temperature at the outlet was between 27.8 and
29.9°C in summer and between 23.2 and 27.2°C in November, and always from 5 to 10°C higher at the outlet (i.e. Stn D) than at any other station (Fig. 2A). Surface water temperature was, however, almost uniform within distances of 1 to 5 km from the power station throughout the investigations. Chl a concentrations were always low at Stn D,but increased with distance (Fig. 2B), except in August 1999. Bacterial abundance
was also lower at Stn D than at other stations, except in August 1999 (Fig. 2C). Bacterial production was always lower at Stn D, and increased with distance (Fig. 2D). Both HNF abundance and HNF grazing rates on bacteria were always lower at Stn D than at
other stations and increased with distance(Fig. 2E,F). The increase of bacterial production, HNF abundance and HNF grazing rates with distance from the power station was more pronounced in summer than inwinter (Fig. 2D–F). Concentrations of residual chlorine were below 0.03 ppm in coastal waters including Stn D during this study (data not shown), with the exception of 1 sample that had 0.05 ppm (in August 1999 at the surface at Stn C2).

**Microbial ecological interrelationships**

In a temperature vs salinity diagram (Fig. 3A), it was obvious that data fromStn D distributed separately from the remaining data, due to the high temperature there. When data from Stn D were 4

Fig. 2. (A) Temperature, (B) chlorophyll a (chl a ), and (C, D) bacterial and (E, F) heterotrophic nanoflagellate (HNF) variables vs distance from thepower station (BP-TDR, bacterial production based on thymidine incorporation). Mean ± 1 SD of data are shown for stations at the same distance.When error bars are not shown, 1 SD is less than the size of the symbol. Different symbols represent different investigation dates: ( j ) November 1998, ( d ) July, ( m ) August, and ( ✚ ) November 1999Choi et al.: Effects of thermal effluents on coastal microorganisms

excluded, some interrelationships improved very significantly (Fig. 3B–D), illustrating the negative effectsof the effluents on the corresponding microbial variables (i.e. low bacterial abundance and production and
HNF abun dance). The remaining interrelationships (Fig. 3E–H) showed trends typical of coastal waters,

r egardless of inclusion of Stn D data; bacterial cell production increased with
increases in chl a (Fig. 3E), and bacterial protein synthesis rates were tightly coupled with DNA synthesis rates (Fig. 3F). HNF abundance and grazing
rates increased with increases in bacterial cell production (Fig. 3G,H).

**Manipulation and dilution experiments** 

One of the typical results is shown(Fig. 4A), in which increased temperature (40°C) and/or hypochlorite addition were applied to nearshore waters. Increased temperature had somewhatinhibitory effects on bacterial abundance and bacterial production. After

1 h of exposure to 40°C, bacterial abundance decreased substantially (21%

r eduction) compared to the control and
was still lower after 2 h of incubation. Bacterial protein synthesis rate, after

2 h of exposure to 40°C, was 61% of the control value. Additions of hypochlorite (giving 0.5 ppm chlorine equivalent) caused almost complete inhibition of bacterial production. Addition of 0.5 ppm chlorine as hypochlorite also
caused a ca 50% decrease in bacterial abundance. But, an addition of 0.13 ppm chlorine as hypochlorite didnot cause any change in bacterial abundance (see Fig. 4B). Double treatments of high temperature and chlorine (0.5 ppm) caused similar decreases in bacterial abundance and production as chlorine (0.5 ppm). Thus, the results
indicate that effects of low levels of
chlorine are dominant over the effectsof high temperature (40°C). Interestingly, high temperature had no inhibitory effect on HNF abundance. After

2 h of incubation, HNF abundance, in contrast, increased significantly (24% increase compared to control). After 2 hof hypochlorite additions, HNF abundance significantly decreased to 31% of the control. Combined effects of high temperature and chlorine (0.5 ppm) were statistically similar (p > 0.05) to thechlorine effect on HNF abundance during the 2 h of incubation, suggesting no synergistic effect of high temperature and chlorine on HNF. Overall, these results 5

Fig. 3. Interrelationships between temperature and (A) salinity, (B) bacterial abundance (BA), (C) bacterial production based on thymidine incorporation (BP-TDR) and (D) heterotrophic nanoflagellate (HNF) abundance; between chlorophyll a (chl a ) and BP-TDR (E); between bacterial production based on leucine incorporation (BP-LEU) and BP-TDR (F); between BP-TDR and HNF abundance (G); and between BP-TDR and HNF grazing rates (GR) (H). D: data from discharge-channel. Solid and dashed lines: regression lines for all data and data without outlet, respectively (the statistics and equations for solid and dashed lines are shown at the top and bottom, respectively, on the right side of each panel)Mar Ecol Prog Ser 229: 1–10, 2002

suggest that the effect of chlorine would be more dominant during a short-term period than that of high temperature to coastal bacteria and HNF in the study area.

In other experiments, various concentrations of chlorine were added to seawater (Fig. 4B). It was clearly shown that a chlorine concentration as low as 0.13 ppm almost completely inhibited bacterial cell productionand protein production activities. Decreases in bacterial and HNF abundances were detected at concentrations >0.13 ppm chlorine after 1 h of incubation.

We further tested the effects of thermal effluents by diluting discharged waters with nearshore waters (Fig. 5). In one experiment (Fig. 5A) in which outletwater was used, bacterial production showed somewhat lower values than those expected from mixing
of the nearshore waters and effluents. In another experiment, in which condenser water was used (Fig. 5B), the effluent showed no inhibitory effect on bacterial abundance at 10- and 100-fold dilutions,similar to the first experiment, but there were significant inhibitory effects on bacterial production, HNF grazing rates and HNF abundance. The inhibitory effects were the greatest at the lowest dilution. Although drastic decreases in bacterial production were observed in both outlet and condenser waters, substantial amounts (4 to 21% of bacterial production found in inlet seawater) of bacterial production were observed (Fig. 5). 6

Date Dilution Bacterial production- Bacterial production- HNF HNF factor TdR Leu abundance grazing rates at outlet ( × 10 7 cells l–1 h –1 ) ( × 10 7 cells l–1 h –1 ) ( × 10 6 HNF l–1 ) ( × 10 7 cells l–1 h –1 )

Meas Est Meas Meas Est Meas Meas Est Meas Meas Est Meas Est Est Est Est

Nov 1998 3.20 0.66 1.26 0.52 0.34 1.44 0.23 0.08 0.23 0.33 – – –
Jul 1999 3.82 nd 12.09 – nd 14.63 – 0.38 1.30 0.29 0.20 0.74 0.27 Aug 1999 3.20 0.20 2.10 0.10 0.02 2.92 0.01 0.10 0.36 0.28 0.18 0.16 1.09
Nov 1999 2.45 0.29 0.73 0.39 0.09 0.65 0.13 0.08 0.12 0.68 0.06 0.07 0.78 T able 1. Comparisons of measured (Meas) and estimated (Est) values of bacterial production, HNF (heterotrophic nanoflagellate) abundance and grazing rate at the outlet. Dilution factor of condenser water (F) with receiving seawater at the outlet was calculated on the basis of water temperatures at the outlet, coastal waters (within 2 km from the outlet), and condenser tubes. Esti

mated value equals (average value for the coastal waters) × [ (F –1)/ F ], assuming that microbial variables in condenser water were zero. nd: not detected; –: not determined Fig. 4. (A) Effects of increased temperature (40°C) and hypochlorite on microbial variables. Different symbols represent differ ent treatments: ( s ) control, ( ✚ ) addition of 0.5 ppm chlorine as hypochlorite, ( d ) increased temperature, ( j ) increased temperature and addition of 0.5 ppm chlorine. (B) Effects of different concentrations of chlorine added as hypochlorite on microbial variables. Different symbols represent different concentrations of chlorine: ( s ) control, ( j ) 0.13, ( ✚ ) 0.25, and ( r ) 0.5 ppm chlorine. Experiments were done in August 1999. Error bars represent 1 SD. When error bars are not shown, 1 SD is less than the size of the symbolChoi et al.: Effects of thermal effluents on coastal microorganisms

**DISCUSSION**

Analyses of horizontal distributions ofmicrobiological variables, interrelationships between microbiological variables and temperature, and manipulation and dilution experiments gave consistently similar results: thermal effluents seemed to have inhibitory effects on bacterial production and on HNF grazing rates and abundance.

Tr eatments of full-load chlorination and high temperature in the cooling systemseemed to be highly effective in reducing activities and abundances of natural marine bacteria and HNF. Further,our results suggest that inhibitory effects are overwhelmingly due to chlorination and not to thermal loading of the
effluents.

A noteworthy observation of thisstudy was that bacterial and HNF activities were significantly inhibited ondiluting the effluents with intake seawater 10- to 100-fold. Since such dilutions of the effluents with intake seawater (<0.03 ppm chlorine) will lower
the chlorine concentration below 0.03 ppm, which is the natural background level (Eppley et al. 1976), no inhibitory effects on micro-organisms would occur.Thus, the inhibitory effects of the effluents on bacteria and HNF at such dilutions strongly suggest the presence of inhibitory substances (e.g. chlorinationby-products) formed during the chlorination process in the effluents.

The presence of inhibitory chlorination by-products in the thermal effluents also helps explain the strikingly low values of bacterial production and HNF grazing and abundance in the outlet, where the residual chlorine concentration was already at natural background levels. Mixing of the thermal effluent with receiving seawater at the outlet is calculated to bring a 3.2 ± 0.6-fold dilution ofthe effluent, based on temperature data from the outlet, coastal seawater and condenser tubes. If chlorination treatments were assumed to have a completely
lethal effect on marine microbes in the effluents and dilution of the effluents with receiving water did not cause any effect on micro-organisms, ca 2/3 of the values for microbial variables of the coastal waters wereexpected to be found at the outlet (this might be a conservative estimate). If some bacteria and HNF survivedduring the chlorination treatment (see Fig. 5), the values would be somewhat higher. In reality, measured values were much lower than the expected ones(Table 1): bacterial cell production and protein production was on average 34 ± 22% and 12 ± 11% of the expected values, respectively (Table 1). HNF abundance and grazing rate was, on average, 41 ± 23% and 71 ±41% of the expected values, respectively. If the positive effects on metabollism of temperature increases at the outlet are considered, the estimated values would be much higher than those in Table 1. Therefore, the
ratios of measured to estimated values could be much lower than those in Table 1. This implies that inhibitory substances (i.e. presumably chlorination by-products) formed during the chlorination process were present in the thermal discharges and further inhibited bacterialand HNF activities in the outlet. Interestingly, the in7

Fig. 5. Dilution experiments. (A) Discharged waters from outlet or (B) freshly discharged waters from condenser tubes were diluted in different ratios, withintake seawater. Shaded bars: the difference between the measured (bars without shading) and predicted values (total height of bars) when 2 types of waters were mixed with given ratios. BA: bacterial abundance; BP-TDR and BP-LEU(hatched): bacterial production measured by thymidine and leucine incorporation, respectively; HNF: heterotrophic nanoflagellate; GR: HNF grazing rate. Experiments A and B were done in August and November 1999, respectively. Error bars represent 1 SD. When error bars are not shown, 1 SD is less than the size of the symbolMar Ecol Prog Ser 229: 1–10, 2002

hibitory effects were apparently more pronounced in summer than in winter (Table 1), although our data were limited. Consistently, formations of halogenated compound in the discharge area are known to be higher in summer than in winter (Allonier et al. 1999), probably related to the nature of the organic matter in
the seawater.

In seawater, humic substances, constituting substantial fractions of dissolved organic matter (Thurman1985), could serve as precursors of toxic organo-halogenated compounds produced during chlorination in seawater. Chlorination by-products are detected from the thermal effluents of coastal nuclear power stations in which low-level chlorination was routinely used.Bromoform, dibromoacetic acid and dibromoacetonitrile are the main compounds (Allonier et al. 1999). Further, the bromoform concentration in the plume of a coastal power plant was measurable (0.26 μg l–1 ) at a
distance of 1.5 to 15 km from the outlet of thermal effluents, although concentration of residual oxidants was below 0.03 ppm in the field (Jenner et al. 1997).
Chlorinated humic substances from diverse sources are known to genetically mutate a strain of Salmonella typhimurium (Meier et al. 1987, Watt et al. 1996). As Allonier et al. (1999) suggested the possible toxicity of such compounds towards aquatic organisms, diverse halogenated compounds formed during chlorination of seawater might have induced toxic effects on bacteria and HNF or even have lethal effects in our samples.

As Langford (1990) concluded, in the cooling water system, chlorination seems to be the overwhelmingly

r esponsible reason for the inhibitory effects. Combined treatments of chlorination and high temperature gave similar effects as chlorination treatment (Fig. 4). All the treatments of seawater with chlorination (0.13 to0.5 ppm) caused drastic inhibition of bacterial production and significant reduction of HNF and bacterial abundances, indicating some lethal effects on bacteria and HNF (Fig. 4). Similarly, 14 C-glucose uptake by bacteria was reported to be null when chlorination was in progress (Davis & Coughlan 1978), indicating total suppression of bacterial activity by chlorination. Wood (1979, cited in Langford 1990) also reported that 0.1 mg l–1 r esidual chlorine caused at least 90% reductions of culturable bacterial counts. In this respect,observations of detectable bacterial production in condenser waters are interesting, suggesting the presence of chlorination-resistant bacteria, presumably derived from biofilms on walls of the cooling systems. Overall,
it seems that chlorination is lethal for a fraction of the exposed natural marine bacteria and HNF in cooling water and induces injury in a large proportion of the

r emaining cells.

In contrast, the effects of high temperature were notas pronounced as those of chlorination. In an experiment where water temperature increased to 40°C for1 to 2 h (Fig. 4A), bacterial cell production and abundance were only from 9 to 22% and 10 to 21% lower,

r espectively, than the control incubated at in situ temperature. High temperature seems to have no negative effect on HNF abundance at all: HNF abundance, incontrast, increased ca 24% after 2 h incubation compared to control. Thus, short-term (<30 min) exposure
of coastal bacteria and HNF to 40°C would not seem to cause detrimental damage to bacteria (or probablyHNF either) during passage through the cooling system. At the outlet, water temperature was 7 to 10°Cand up to 5°C higher than ambient seawater temperature in November and in summer, respectively. Bacteria and HNF in the discharge area could be released from high-temperature (e.g. 40°C) stress, but still be exposed to temperatures elevated above ambient coastal seawater. Although we did not test the effects of temperatures elevated 5 to 10°C above the in situtemperature, such temperatures may not have inhibitory effects on bacteria and HNF (Marrasé et al. 1992, Shiah & Duckow 1994). Rather, they might have stimulating effects on microbial activities (Massana et al. 1994, Shiah & Duckow 1994, Vaqué et al. 1994).

We re the low values of bacterial production at the
outlet due to the effects of low-level chlorination on 3 Hthymidine and 14 C-leucine? In other words, chlorination might cause chemical transformation of the tracers so that incorporation rates of the tracers are reduced. Thus, bacterial production could be underestimated due
to the lowered concentrations of tracers at the outlet and in condenser samples. We checked this possibility by adding stock solution of tracers to double-distilled water in which hyperchlorite was contained (final conc. of 0.03 and 0.1 ppm chlorine equivalent). After 30 min of incubations with hyperchlorite, the stocks wereadded (vol. of 140 μl) to 10 ml seawater to measure bacterial production. There were no significant differences between hyperchlorite-treated and control tracer stocks for bacterial production rate measurements (data not shown). Thus, low values of bacterial production were due to inhibitory effects of thermal effluents, not due to chemical alterations of tracers by chlorination.

So far, we have presumed that toxic effects arise mainly from chlorination and chlorination by-products. However, any inhibitory substance might yield similar

r esults. Other possible toxic substances could be heavy metals, antifouling paint, or ash discharges (Larrick et al. 1981, Langford 1990). Distributions of total heavy metals of various elements (Fe, Zn, Cu, Cr, Pb and Cd)were not different among stations including the discharge channel (Huh 2000, S.-R. Yang pers. comm.).Further, Hadong Power Station does not treat the cooling system with antifouling paint or other coatings. Also, no ash effluents are discharged near the thermal 8Choi et al.: Effects of thermal effluents on coastal microorganisms

discharges in the study area. Thus, it seems that the observed inhibitory effects of thermal effluents on coastal microbes were actually due to chlorination and chlorination by-products.

Finally, our results suggested that the range in which thermal effluents affect bacteria and HNF in receiving coastal waters might be proportional to 100 times the volume of the flux of condenser water. However, thiscould be a maximal estimate because of potential chlorine decay due to volatilization of chlorine, progressive
chemical reactions of chlorine with seawater constituents, and the photochemical destruction of chlorine during mixing in the receiving water (Eppley et al. 1976). Depressed activities of bacteria and HNF in the affected region would slow down regeneration of nutrients and flow of energy and material through the microbial food web. Efforts in developing techniques
to reduce the toxic effects of thermal effluents are increasing. Recently, with the aid of ‘mussel monitor’, ‘pulse-chlorination’ technology using short (i.e. a few minutes), intermittent chlorination has reduced the amount of chlorine by 50% compared to continuous chlorination (Jenner & Polman 1998). As chlorination is still the most widely applied method for antifouling, pulse-chlorination would be preferable with fewer subsequent environmental effects.

In conclusion, thermal effluents had inhibitory effects on bacterial production and on HNF grazing rates on bacteria. The inhibitory effects seemed to be mostlydue to chlorination of seawater and chlorination byproducts in the effluents. Our results suggest that natural marine micro-organisms seem to be sensitive to thermal pollution, and microbial ecological approaches are useful in assessing the magnitude of influences of
thermal effluents on coastal microbes.

Acknowledgements. We thank Korea Electric Power Corporation (KEPCO) for their cooperation during the study. Wethank Dr Jed Fuhrman for careful reading of an earlier version of our manuscript. We thank 4 anonymous reviewers for helpful comments. The present study was supported (in part) by the BK 21 Project of the Korean Government and a KEPCO project.

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Editorial responsibility: Otto Kinne (Editor), Oldendorf/Luhe, Germany Submitted: December 12, 2000; Accepted: August 16, 2001 Proofs received from author(s): March 4, 2002