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Nitrogen uptake and growth rate of kelp (Laminaria saccharina) grown in an outdoor culture system using… Subandar, Awal 1991

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NITROGEN UPTAKE AND GROWTH RATE OF KELP (Laminaria saccharina) GROWN IN A N OUTDOOR CULTURE SYSTEM USING SALMON CULTURE EFFLUENT  By A W A L SUBANDAR Sarjana, Bogor Agricultural University, Indonesia, 1985  A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T O F T H E REQUIREMENTS FOR T H E D E G R E E O F MASTER O F SCIENCE  in T H E F A C U L T Y O F G R A D U A T E STUDIES (BIO-RESOURCE ENGINEERING DEPARTMENT)  We accept this thesis as conforming to the required standard  T H E UNIVERSITY O F BRITISH C O L U M B I A April 1991 © Awal Subandar, 1991  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make  it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ABSTRACT  Salmon netpen culture operation is going to grow in the next 5 years in British Columbia, and this culture generates nutrient loading. Kelp could utilize this nutrient to grow. To investigate the feasibility of an integrated culture of kelp and salmon, 3 consecutive experiments (each lasted 9 days) using kelp (Laminaria saccharina) (length 15 cm) were conducted at Marine Ecosystem Programme (West Vancouver) during April-September, 1990. The objectives were to test the effect of flow and kelp density on nitrogen removal, nitrogen uptake, growth, biomass and dissolved oxygen production of kelp. The flow treatment consisted of 3 levels (8.1-24, 25-44.1, 65.2-83.5 1 h" ), and 1  the density treatment consisted of 4 levels (0, 10, 15, 20 plants/tank or 0, 370, 555, 740 kelp/m , respectively).  The kelp were grown in 12 Plexiglas tanks (45x45x20 cm" ).  3  Water samples of N H  3  + 4  and N0 " (morning, afternoon), growth measurement and 3  biomass harvesting were conducted every 3 days. Luxurious uptake of nitrogen was not evident because C/N ratio (10-11) was stable in all experiments. The treatments with kelp demonstrated higher total nitrogen removal than the controls (no kelp). The removal rate ranged 32.9-339 /xmol l" h" (301  1  40%). The total nitrogen uptake rate ranged 6.1-22.5 /xmol g" dry mass h" . The high1  1  flow, low-density tank had the highest total nitrogen uptake. The mean uptake based on 3 days growth for all flow-density combinations were similar. The range of mean uptake based on 3 days growth was 5.4-8.3 /xmol g" dry mass h" . The kelp utilized 1  ii  1  NH  + 4  and N0 " equally. No differences of total nitrogen ( N H 3  + 4  and N0 "), N H 3  + 4  and  N0 " uptake values were evident between morning and afternoon sampling time for all 3  flow-density combinations. The growth ranged between 6.5-9 % d" . The biomass production ranged from 1  1.3-2.1 g per sampling. The highest growth rate and biomass production was performed by kelp in the high-flow, low-density tank. The biomass production appeared to increase as nitrogen uptake increased. Intraspecific competition to light appeared to occur in the medium and high kelp density tanks. Tanks with kelp contained higher dissolved 0 than control (no kelp) tanks. No 2  difference of dissolved 0 was evident in the morning and afternoon periods in all flow2  density combinations. The range of dissolved 0 in the kelp tanks was 7.1-9.5 mg 1-1, 2  and the highest value was found in the low-flow, high-density tank. The low-density and high-flow combination would be applicable in an integrated culture of kelp and salmon. Depending on the current and the desired N removal efficiency, the suggested kelp raft will consist of a number of kelp units, which are between 120 to 480 m long and 0.15 m in cross sectional area. 2  The result of high growth and biomass production would serve the purpose of maximum nitrogen purification of salmon culture effluent. In addition, the cultured kelp would provide oxygen through photosynthetic activity. A model for predicting total nitrogen uptake was presented.  iii  TABLE OF CONTENTS  Abstract  ii  List of Tables  v  List of Figures  vi  Acknowledgements  vii  I. Introduction  1  II. Objectives  4  III. Literature Reviews 3.1. Nutrient Loading 3.2. Kelp Studies 3.3. Integrated Culture of Seaweed and Marine Animals IV. Material and Methods  6 8 12 13  V. Results 5.1. Physical Parameter Profiles 5.2. Nitrogen Removal and Uptake 5.3. Growth 5.4. Dissolved Oxygen 5.5. Uptake Prediction  18 18 34 35 37  VI. Discussion 6.1. Nitrogen Removal and Uptake 6.2. Growth 6.3. Dissolved Oxygen  39 48 51  VII. Conclusions and Suggestions  52  References  56  iv  LIST OF TABLES  Table  Title  Page  1. Carbon-Nitrogen Ratio of Kelp (L. saccharina) Grown in Different Flow-Density Combinations.  28  2. Total Nitrogen (Ammonium and Nitrate) Removal Rates (/xmol l" h") .... 29 by Control (No Kelp) and Kelp (L. saccharina) Grown in Different Flow-Density Combinations. 1  1  3. Total Nitrogen (Ammonium and Nitrate) Uptake Rates Measured Twice Daily and Every 3 Days (/im g" dry mass h") of Kelp (L. saccharina) Grown in Different Flow-Density Combinations.  32  4. Ammonium, Nitrate Uptake Rates (/im g" dry mass h") and Percentage of Contribution to Total Nitrogen Uptake Rate of Kelp (L. saccharina) Grown in Different Flow-Density Combinations.  33  5. Removal Rates of Ammonium and Total Nitrogen (Ammonium and Nitrate) (%) of Kelp (L. saccharina) Grown in Different Flow-Density Combinations.  34  6. The Specific Growth Rates (% d") and Production (g) Level of Kelp (L. saccharina) Grown in Different Flow-Density Combinations.  36  7. Dissolved Oxygen Concentrations in Control (No Kelp) and Kelp (L. saccharina) Tanks (mg l") in Different Flow-Density Combinations in the Morning and Afternoon.  38  8. Nitrogen Uptake Rates of Kelp in Different Culture System and Nitrogen Supply as Reported in Literatures.  47  9. Specific Growth Rates of Different Species of Kelp Grown in Different Conditions as Reported in Literatures.  50  1  1  1  1  1  1  v  LIST OF FIGURES  Figure  Title  Page  1. Description of Experimental Layout. Includes Salmon Culture Unit, Head Tank, 3 Control Tanks, 9 Kelp Tanks.  14  2.a. Irradiance Levels During Experiment I (July 31-August 9, 1990) Data were Recorded Every Half Hour.  19  2.b. Irradiance Levels During Experiment II (August 20-29, 1990) Data were Recorded Every Half Hour.  20  2.c. Irradiance Levels During Experiment III (September 10-19, 1990) Data were Recorded Every Half Hour.  21  3.a. Temperature Fluctuation in the Head Tank, a Kelp Tank (High Flow, ... 22 Low Density) and Air During Experiment I (July 31-August 8, 1990). Data were Recorded Every Half an Hour. 3.b. Temperature Fluctuation in the Head Tank, a Kelp Tank (Low Flow, .... 23 High Density) and Air During Experiment II (August 20-29, 1990). Data were Recorded Every Half an Hour. 3.c. Temperature Fluctuation in the Head Tank, a Kelp Tank (Low Flow, .... 24 Medium Density) and Air During Experiment III (September 10-19, 1990). Data were Recorded Every Half an Hour. 4.a. Nitrogen Concentration in the Effluent from the Salmon Tank During Experiment I (July 31-August 8, 1990).  25  4.b. Nitrogen Concentration in the Effluent from the Salmon Tank During Experiment II (August 20-29, 1990).  26  4.c. Nitrogen Concentration in the Effluent from the Salmon Tank During Experiment III (September 10-19, 1990).  27  vi  ACKNOWLEDGEMENTS  First of all, I am very thankful to the Overseas Fellowship Program Board, Ministry of Research and Technology (Indonesia) for giving me an opportunity to pursue my master degree in Canada. To finish the research, I certainly have received a lot of help from some people. First of all, I am deeply grateful to my supervisor, Dr. Royann Petrell, for her guidance, support and encouraging advice which led to the successful completion of this work. I am also very thankful to Dr. Kwang Victor Lo for his financial support. My committee members, Drs. Paul Richard and Paul Harrison, offered a lot of practical and theoretical suggestions which were very crucial for the progress of the work. I am also thankful to Dr. Louis Druehl for giving me many suggestions and useful references in the early stage of the research. I cannot forget Scott Mattice for his invaluable help in assembling the experimental structure; Colin Savage who assembled the data acquisition system, and without whose technical knowledge a very large amount of data would have been lost at one stage of the experiment. I am also deeply thankful to Trev Neufeld for his assistance in providing fish feed during the experiment; to Niel Jackson, Jurghen Pehlke and Moez Bouraoui for giving their hands in the construction of the tanks. I am indebted to Kedong Yin and Maureen Soon and Dr. Steve Calvert for their assistance in the chemical analyses, to Adeleine Chen and Dr. Ping Liao for allowing me to use their laboratory facilities, and to Dr. George Kruzynski for his encouraging advice and for placing some equipment at my disposal. My thanks to Ping Ma, Soenarto and Kaan Katircioglu for their guidance in dealing with statistical analysis, and to Broer Titayanto Pieter for his support in finishing this report. I wish to thank David Holtzman for his invaluable help in obtaining some research fund. I am indebted to Kang Hendra Jitno for his advice and help in computer work. Finally, my special appreciation is given to my parents, brothers and Haryanti for their support and patience during my long study abroad.  vii  I. INTRODUCTION Aquaculture is a growing industry in British Columbia. The total predicted production of 17,000 tonnes in 1991 is 3 times greater than it was 4 years ago. Sixtythree percent of the total production value is salmon. Salmon farming is going to grow for the next 5 - 10 years (Kenney, 1990). The growth will be favoured by the relaxation of tight provincial government restriction (Crutchfield, 1989) and availability of 700 potential sites (Anonymous, 1986). The above production generates nutrient loading into marine environment. Ackefors and Enell (1990) postulated that nitrogen loading rate was a function of the amount of fish produced and feed used, and the nitrogen content of feed and fish. Based on the current production level and feed composition used by farmers (7.2% nitrogen, Deacom, Pers. Comm.), the nutrient loading to B.C. coastal waters is approximately 78 kg N/ton fish produced or 1,326 tonnes in 1991. Some studies proved that nutrient loading could decrease water quality in and around netpens, such as decreased dissolved oxygen level in and around netpens (Kadowaki and Hirata, 1984; Phillips et al., 1985), decreased water transparency around netpens (Enell, 1987), and increased sedimentation beneath netpens (Kennedy et al., 1976; Weston, 1986). To minimize the effects of nutrient loading, some efforts have been applied, such as regular rotation of netpens, the use of submersible mixer or food/faecal trap (Beveridge, 1987). Weston (as interviewed by Orians, 1989) estimated that food/faecal trap would not be effective because the device only trapped 10% of total nitrogen 1  loading, and the trapped matter could not be used for anything due to high salt content. Another potential option to lower the amount of nutrient released into marine environment is to try integrated culture of salmon and macroalgae. The growth of marine macroalgae in coastal waters is limited by low nitrogen availability (Topinka and Robbins, 1976). Excessive supply of nitrogen from netpens can be cleaned by growing commercial kelp, while oxygen essential for cultured fish in netpens is generated by the kelp. The assumption made in this study was that phosphorous would not be a limiting factor to growth. Atkinson and Smith (1983) show that the C:N:P atomic ratio of benthic marine macroalgae is around 550:30:1. The level of dissolved phosphorous loading around netpens is around 2.2-4 kg P/ton fish produced (Ackefors and Enell, 1990; Enell, 1987). The ratio of N/P is, therefore, between 18.75-37.5. The focus of this study is on nitrogen and not phosphorous, because based on the above calculation the ratio in salmon culture effluent and plants are very similar. To a lesser extent, integrated culture of kelp and salmon provides some other advantages. Kelp is available in coastal waters around B.C., and its culture techniques has been proven (Druehl, 1987). Kelp has economic value for its chemical content, mainly iodine, alginate, laminaran and mannitol (Druehl, 1988; Hoppe, 1979) and use as a food source (Saito, 1976). Recently, the use of seaweed for marine sanitation has received more attention and demonstrates promising results.  Markovtsev and Krupnova (1988) claimed that  culture of Laminaria algae provided a reliable biological filter for purification of fish  3 processing effluent.  Levin and McNeil (1989) found that cultured macroalgae could  purify effluent from a land based salmon culture. In this study, investigation on kelp (Laminaria saccharina) grown in salmon waste water effluent was conducted to estimate the technical feasibility of an integrated salmonkelp culture.  H. OBJECTIVES  To study an integrated culture of kelp and salmon, an experiment was conducted. The objectives were to estimate the amount of nitrogen removed from the system, to estimate nitrogen uptake by kelp (L. saccharina) grown in different combinations of flow and density, to estimate the growth and biomass production of the kelp, and to measure dissolved oxygen generated by the kelp. Nitrogen removal was defined as the difference between inorganic nitrogen concentration of inflow and outflow; nitrogen uptake was the difference between inorganic concentration of inflow and outflow per unit mass of kelp. The hypotheses tested in the experiment were: 1. Carbon-nitrogen ratio fluctuates following nitrogen supply from the fish tank. 2. Nitrogen removal of the treatments with kelp is greater than the control (without kelp). 3. Nitrogen removal depends on kelp density as well as flow rate. 4. Nitrogen uptake depends on kelp density as well as flow rate. 5. Nitrogen uptake in the morning is lower than in the afternoon following photosynthetic activity of kelp. 6. Nitrogen uptake by kelp in a continuous culture system is higher than nitrogen uptake by kelp in a batch culture system. 7. Growth of kelp depends on kelp density as well as flow rate. 8. Kelp biomass production depends on kelp density as well as flow rate. 9. The tanks with kelp should contain higher dissolved oxygen concentration than the control (without kelp) tanks. 4  5 10. The amount of dissolved oxygen generated depends on kelp density as well as flow rate. 11. Dissolved oxygen concentration in the morning is lower than dissolved oxygen concentration in the afternoon following the level of photosynthetic activity of kelp.  HI. LITERATURE REVIEWS  3.1. Nutrient Loading The waste produced from intensive cage salmonid culture consists mainly of uneaten food, excreta, and, to a lesser extent, other materials such as fish fatalities, scales, mucus (Phillips et al., 1985). Most of nitrogen(57-86%) is usually found in dissolved form, and phosphorus (5478%) is usually found in particulate form (Enell, 1987; Gowen and Bradbury, 1987; Ackefors and Enell, 1990). The dissolved nitrogen forms are primarily ammonium and urea (Weston, 1986; Enell, 1987). The magnitude of nutrient loading depends generally on the size of venture, site hydrographical characteristics, farming practice (Phillips et al., 1985; Gowen and Bradbury, 1987), feeding conversion ratio and nitrogen/phosphorous content in feed (Gowen and Bradbury, 1987; Ackefors and Enell, 1990). To calculate nutrient loading, Ackefors and Enell (1990) propose this simple equations: kg nitrogen  = ( A x C ) - (B x C J dn  kg phosphorous = (A x C ) - (B x C ) dp  fp  Where A = wet mass of dry pellets used per year, B = wet mass of fish produced per year, C = nitrogen (C ) and phosphorous (C ) content of dry pellets (% of wet mass), d  dn  dp  C = nitrogen (C ) and phosphorous (C ) content of fish (% of wet mass); this variable f  to  fp  is rather constant for salmon and rainbow trout ( C = 3%, Cfp = 0.4%). fe  Besides this source of nutrient loading, other sources exist: decomposition of 6  7 material deposited beneath netpens and excretory products of fouling organisms attached to netpens structure, which encompass phosphate, ammonia, amino nitrogen and urea (Beveridge, 1987). Almost 30% of nitrogen in feed will end up on the bottom in form of particulate material (Phillips et al., 1985). The nutrient deposited could be released back to water column in form of ammonium, phosphate and silicate. The rate of releasing back of ammonium, by desorption and biological process, was between 10-30% (Ackefors and Enell, 1990).  A 50 tonnes salmon farm required approximately 500 kg feed/day,  containing 40 kg nitrogen, would generate 1.2-3.6 kg of N H released back from 3  sediment (Gowen and Bradbury, 1987). Some researchers have found increasing nitrogen around salmon netpens, but the extent of the impact varies from place to place (Beveridge, 1987). Weston (1986) measured ammonia concentration inside netpens holding 27,000 kg salmon was 0.020 mg/1, whereas the N H concentration at 30 m upcurrent was 0.007 mg/1 and it was 0.012 3  mg/1 at 30 m downcurrent. Korman (1989) observed the concentration of N H at the 3  surface within salmon netpens at Sechelt Inlet, B.C. was 4 nm, and the N H  3  concentration at the downstream of the netpens was much less than the concentration inside the netpens.  Weston as interviewed by Orians (1989) stated that the higher  concentration area typically extended about 15-20 m from the site, and in some area reached 50 m.  8 3.2. Kelp Studies Kelp common to B.C. are Macrocystis integrifolia, Laminaria saccharina, L. groenlandica, Nereocystis leutkana (Scagel, 1967).  Due to incompleteness of culture  technology for N. leutkana (Foreman, Pers. Comm.), the former species has more potential to be grown alongside salmon netpens. In this study, the Laminaria has been chosen because this kelp grows around Vancouver (Druehl, Pers. Comm.), and the technology for its extensive culture has been proven in B.C. (Druehl et al., 1988). The production levels of L. saccharina and L. groenlandica are 3.0-8.0 and 9.6-20.5 kg wet mass/m rope used in culture, respectively (26.4 and 33.0 mt/ha). Besides that, kelp contents iodine, alginate, laminaran and mannitol in significant amount (Druehl, 1988; Hoppe, 1979), and kelp is a nutritious food stuff (Saito, 1976). Furthermore, kelp culture could be economical because its fertilizer will come from salmon waste. In some Asian countries, such as Japan and China, cultured kelp was fertilized using either the porous container method or spraying method (Lobban and Wynee, 1981; Druehl, 1988). To successfully culture kelp in particular, or macroalgae in general, an understanding of its biology is essential.  Kelp growth is determined by physical  (irradiance, temperature, current), biological (species, type of tissue, nutritional history) and chemical factors (ambient concentration, chemical species, internal nitrogen) (Lobban et al., 1985). Irradiance is the most important factor in determining the growth of a plant, due to the role of irradiance in photosynthetic activity. Growth of L. longicruris followed the seasonal pattern of irradiance when nitrogen was abundant year round (Anderson et al.,  9 1981; Gagne et al., 1982). Boden (1979) showed that irradiance was a controlling factor for L. saccharina. The maximum growth rate (1.15 cm/day) occurred at a depth of 9 m. Growth rates at 1 and 3 m were 40% of the maximum growth rate, and the growth rate at 17 m was much reduced. In general, irradiance saturation level of Laminariales and other macrophytes is between 30-100 /iE m s (Harrison and Druehl, 1982). 2  1  As cold water macroalgae, the optimum temperature of kelp is slightly above 10°C (Bolton and Luning, 1982), and L. saccharina has the broadest range of optimum temperature (10-15°C) among the member of genus Laminaria (Bolton and Luning, 1982; Druehl, 1967). Other macroalgae, such as L. longicruris and L. digitata have temperature optimas just at 10°C. The temperature optima of L. hyperborea is at 15°C. Increasing or decreasing temperature could reduce specific growth rate of all species. An increase in temperature up to 20°C reduced the specific growth rate of L . saccharina by 30-60%, and a decrease in temperature to 5  oC  reduced the specific growth  rate of L. saccharina by 17-40% (Bolton and Luning, 1982). Gerard and Mann (1979) reported that nutrient uptake of L. longicruris was enhanced by increasing water movement.  Whitford and Schumacher (1961, 1964)  suggested that current produced a steep diffusion gradient, which could increase exchange of materials between macroalgae and surrounding waters.  Lapointe and  Ryther (1979) argued that nitrogen availability was a function of both concentration and • flow rate near thallus of macroalgae.  In nature, seaweed populations were often  described as more luxuriant in turbulent waters than in stagnant waters (Conover, 1968).  10 In study with M.pyrifera, Gerard (1982 ) calculated a minimum current of 4 cm s" was b  1  necessary for the growth of the kelp, whereas 7.5 cm s" for Ulva lactuca (Parker, 1981). 1  The winter growth of L. longicruris correlated significantly with an increase in ambient dissolved N0 " (Chapman and Craigie, 1977; Asare and Harlin, 1983). Wheeler 3  and North (1981) reported the same tendency for M.pyrifera. Fertilization to increase dissolved N0 " could increase summer growth of the kelp (Chapman and Craigie, 1977). 3  In B.C., production of L. groenlandica was positively correlated with nutrient concentration, and negatively correlated with temperature (Druehl et al., 1988). Penniman (1988) reported that the growth of lamina of juvenile of L. longicruris increased from 0.187 doubling day" (at 9 /xmol N) to 0.205 doubling day" (at 20 and 36 1  1  /xmol N) at temperature 15°C, and the growth rate at 5°C was slightly lower than at 15°C at the same nitrogen concentration. In laboratory scale, Chapman et al. (1978) observed a linear relationship between the growth of L. saccharina and N0 " concentration up to 10 /xmol, and a luxury 3  consumption of N0 " for N0 " concentration above 10 /xmol. 3  3  Besides that, the  chlorophyll content and photosynthetic capacity of the macroalgae also increased with increasing N0 " in the surrounding medium. 3  Generally increasing the nitrogen concentration accelerates growth of various macroalgae (De Boer, 1979; Lapointe and Ryther, 1979; Amat and Braud, 1990; Rui et al., 1990; Fredriksen and Rueness, 1989; Indergaard and Knutsen, 1990); however, ammonium at more than 100 fimol is considered toxic ( Harrison, Pers. Comm.). Nutrient species could affect uptake rate and growth rate of algae. The maximum  11 N0 " uptake rate of L . longicruris was 7-10 /imol h" g" dry mass, and this rate was not 1  1  3  affected by the presence of N H .  In other case, N0 " uptake was inhibited in  +  4  2  proportion to the concentration of N0 " in the medium (Harlin and Craigie, 1978). 3  Laminaria groenlandica simultaneously took up N0 " and N H , and the uptake rates +  3  were identical and equal when only N0 " or N H 3  et al., 1986).  + 4  4  was present in the medium (Harrison  Haines and Wheeler (1978) observed that NOy and N H  + 4  were  simultaneously absorbed by M.pyrifera, and the NOy uptake by Hypnea musciformis was reduced 50% in the presence of N H , while N H +  4  + 4  uptake was not affected by the  presence of N 0 \ 3  Some studies indicated that kelp could store nitrogen during winter when ambient nitrogen was high (Chapman and Craigie, 1977; Wheeler and North, 1981; Asare and Harlin, 1983; Rosell and Srivastava, 1985). Laminaria longicruris stored reserve N0 " up 3  to 150 /xmol g" fresh mass, or approximately 28,000 times than ambient concentration 1  (Chapman and Craigie, 1977). However, Asare and Harlin (1983) found the storage level of L. longicruris was only 560 times the ambient concentration, possibly because of high nitrogen concentration in the surrounding marine environment. Adult and juvenile sporophyte of M. pyrifera contained nitrogen reserve up to 10,000 times larger than ambient nitrogen concentration (North as cited by Gerard, 1982 ). . a  An increase in total nitrogen tissue could lead to a decrease in nutrient uptake (Rui et al., 1990). Amat and Braud (1990) showed that every 1% dry mass of total nitrogen tissue decreased nitrogen uptake of Chondrus crispus as much as 1.5 ng N g"  1  dry mass min" .' 1  12 3.3. Integrated Culture of Seaweed and Marine Animals In Asia, integrated culture of seaweed and marine animals is a common practice. The culture of Gracilaria with shrimp (Penaeus monodori) and crab (Scylla serrata) or milkfish {Chanos chanos) in ponds are widely applied in Japan, China, Korea, Vietnam, Taiwan, Philippines and India (Gomez and Corrales, 1988). In Japan, luxurious growth of Hypnea and Gracilaria has been noted in netpens of yellowtail (Seriolla quinqueradiata) (Gomez and Corrales, 1988). Although an integrated culture of seaweed and marine animals is widely practised in Asia, its use for purification purpose has not been extensively used. Markovtsev and Krupnova (1988) reported that the culture of Laminaria around discharge of a fishprocessing plant at southern Maritime Kray area (Soviet Union) could purify the effluent, which contained up to 120 mg total N l" . 1  In an experimental culture oiPalmaria palmata, receiving waste water from landbased salmon culture, Levin and McNeil (1989) claimed that the red macroalgae removed virtually all of the inorganic nitrogen present in the effluent. Fujita et al. (1989) did an experiment on integrated culture of various species of macroalgae with (land based) salmon. Despite a very low stocking density (1 g l") and a high flow rate (40 1  volume changes d' ) used in 11-liter tanks, the cultured macroalgae removed 30-50% of 1  N H (initial concentration 12.8 /xmol) in the salmon culture effluent. Besides that, the 4  specific growth rate of the cultured macroalgae increased.  The growth rates of P.  palmata, Gigartina exasperata, Gracilaria lemaneiformis, Farlovia mollis, and Iridaea cordata were 15.4, 2.8, 9.0, 4.6, 3.0% d" , respectively. 1  IV. MATERIALS AND METHODS  Three consecutive experiments (each 9 days long) on nitrogen uptake of L. saccharina receiving waste water from a fish tank were conducted at an outdoor facility of the Marine Ecosystem Programme, West Vancouver (April 25-September 19, 1990). The treatments tested were combinations of densities and flow rates.  The density  treatment consisted of 4 levels (0, 10, 15, 20 kelp/tank or 0, 370, 555, 740 kelp/m ) and 3  the flow rate treatment consisted of 3 levels (8.1-24, 25-44.1, 65.2-83.5 1 h" ). 1  The kelp used were collected at Brockton Point, Stanley Park, Vancouver, B.C., on April 25 and May 22, 1990. The kelp were kept in styrofoam coolers and covered with seawater. The kelp were brought directly to the laboratory. Before placing them in kelp tanks, the kelp were detached from their holdfasts, and they then were reattached to marked rocks. The kelp were trimmed to 15 cm measured from stipe junction. The kelp were conditioned to salmon culture effluent for 2-3 months before the experiments were initiated (Figure 1). They were placed in 12 Plexiglas tanks (45x45x20 cm ). The tank inlet and outlet were positioned in such a way as to increase mixing. 3  The tanks and kelp used were randomly reallocated before each experiment. The tanks were placed into 2 raceways containing flowing seawater in order to minimize temperature increases. All the tanks received effluent water from a salmon tank through a head tank (Figure 1). The salmon stocking density was approximately 8 kg m-3, and the fish were fed until satiation once a day at approximately 9:00 a.m. The head and  13  Fr  HEAD TANK  SALMON TANK J - : DRAIN  6  : PUMP  CONTROL AND KELP TANKS  Figure 1. Description of Experimental Layout. Includes Salmon Culture Unit, Head Tank, 3 Control Tanks, 9 Kelp Tanks.  15 kelp tanks were cleaned every 2 days in order to prevent periphyton from growing, which could block irradiance and interfere with nitrogen uptake. Regular water sampling was conducted every three days in the morning (before the fish were fed) and again in the afternoon (approximately 5 hours after the fish were fed). The plant length and biomass were measured on 60% of the kelp density every third day. Water samples were collected in the head tank twice every day: once in the morning and once in the afternoon. Salinity (YSI model 33), pH (Hanna Instrument model HI 8521) and flow rate were measured daily. Row rate was calculating after measuring the amount of time to fill a given volume of water. Dissolved oxygen (YSI model 57) was measured twice every day: once in the morning and once in the afternoon. Irradiance (Licor Li 185B) was measured in a high kelp density tank, and water temperature was measured at 3 kelp tanks (copper constant thermocouple connected to YSI model 33). Irradiance and water temperature were recorded every half hour using an automatic data logger especially constructed for use in saltwater experiments (Petrell and Savage, 1990). In order to maintain a steady biomass, kelp were trimmed when lamina length was more than 15 cm. The wet mass harvested was used to estimate production. The amount of kelp harvested was kept in plastic bags and it was kept frozen until the C/N ratio analysis was performed. Water samples were collected from the outlet of kelp tanks. Each sample was taken with 60 ml syringe, and then it was injected into a 30 ml Nalgene wide mouth bottle through a 934 A H Whatman filter held by Swinnex 25 mm Millipore filter holder.  16 The first 10 ml was always discarded, and the next 10 ml was used for rinsing the bottle. The required sample was 25 ml, so the remaining 15 ml was not used. All the bottles and apparatus were washed in HC1 10% v/v before sampling. All the water samples were frozen until chemical tests were done. Ammonium and nitrate analyses were done using a Technicon Auto Analyzer II, and the C/N test was done using Carlo Erba Analyzer N2A1500 at Oceanography Department, U.B.C. Each sample of both water and C/N consisted of two replicates. Nitrogen removal was calculated using the following formula: V = f(S S) r  Where V = nutrient uptake (jumol N g" dry mass h" ), f = flow rate (1 h-1), S = inflow 1  inorganic nitrogen concentrations  1  (/nmol N), S =  residual inorganic nitrogen  concentrations (/xmol N). Nitrogen uptake was calculated according to the following formula (Rosenberg et al., 1984): V = {f(S S)}/w r  The explanation of each variable was identical with the variables of the nitrogen removal formula, and W = seaweed biomass ( g dry mass). The punched hole method (Parker, 1947 as quoted by Chapman, 1973) was applied to estimate growth rate.  Five mm holes were made on the first day of  experiment, and hole movements were measured from the stipe junction every 3 days. The growth was calculated using the growth rate formula (Brinkhuis, 1985) as follows: U = {ln(W -W)}/t t  17 Where U = specific growth rate (% d" ), W = distance from stipe junction at time t, 1  t  W = initial distance, t = interval time. To estimate the mean uptake rate based on 3 days growth (between 2 samplings), the following approach was used: V  = Nmean  Where V  m e a n  xU  tissue  = mean uptake rate between 2 sampling periods (3 days interval) (jimol  g" dry mass), N 1  = total nitrogen tissue (/xmol g" dry mass), U = specific growth rate 1  t i s s u e  (% d" ). 1  A four-way analysis of covariance (ancova) was done on the pooled data (Sokal & Rohlf, 1981) of uptake and growth using the SYSTAT package. The approach was to model uptake and growth as a linear function of independent variables (irradiance, temperature, pH, salinity, nitrogen supply, wet mass). Experiment number, day, flow and density in nominal data were included in the model as the main effects as well as interaction among the different main effects. The modelling process was progressive, where initially all the main effects and independent variables, were tried and subsequently, model components which did not significantly contribute in the model (p>0.10) were removed (Friedlander et al., 1990). To obtain a quantitative model, multiple regression analysis (SYSTAT package) was run on the uptake data.  V. RESULTS  5.1. Physical Parameter Profiles The range of irradiance was 0-1874 ixE m" s' . 2  1  The mean day irradiances of  experiment I, II, III were 106, 99, 179 ixE m" s", respectively. High irradiance levels 2  1  occurred during the first and last experiments (Figure 2). The range of irradiance during experiment I was 0-1212 /xE rn" s' , and during experiment III was 0-1875 ixE m' s". 2  1  2  1  The range of temperature was 10.2-22.9°C. The mean temperatures of experiment I, II and III were 15.6, 13.6 and 14.6°C, respectively (Figure 3). Salinity was fairly constant throughout the experiments period (27-29°/^) in all flow-density combinations. The range of pH was 6.92-7.85, and the pH of the control tanks was usually lower than the kelp tanks.  5.2. Nitrogen Removal and Uptake Supply of total nitrogen ( N H  + 4  and N0 ") from the fish tank fluctuated every day 3  and usually was lower in the morning before the salmon were fed (Figure 4). Nitrogen concentration was usually higher every third day. The head tank was cleaned every second day, and cleaning probably removed beneficial nitrogen utilizing organisms. Nitrogen was not limiting for the kelp, because the C-N ratios were stable throughout the experiments (Table 1). The ranges of N H , N0 " and total nitrogen +  4  3  were 6.2-25.4, 12.9-40.0, 19.7-52.7 /xmol, respectively. Treatments with kelp had higher total nitrogen removal than control (no kelp) (/xmol l' h" ). This trend occurred for all levels of flow and density (p<0.05, Table 2). 1  1  18  2  Figure 2.a. Irradiance Levels During Experiment I (July 31-August 8,1990) Data were Recorded Every Half an Hour.  2  A 8 5 6 7 DAYS Figure 2.b. Irradiance Levels During Experiment II (August 20-29,1990) Data were Recorded Every Half an Hour. 1  2  3  4  O  2  ^  1.5  CO CM  E  _  a. S 8 31  -  c o ca sz.  1 *=!  0.5 -  1  V 4  5 8 DAYS Figure 2.c. Irradiance Levels During Experiment III (Septemberl 0-19,1990) Data were Recorded Every Half an Hour.  1  2  3 4 A. Head Tank  5 6 7 8 B. Kelp Tank C. Air  9  10  Figure 3.a. Temperature Fluctuation in the Head Tank, a Kelp Tank (Low Flow, Medium Density) and Air During Experiment I (July 31-August 8,1990). Data were Recorded Every Half an Hour.  30  10  1  1  1  2  1  1  3 4 A. Head Tank  1  1  5 6 7 B. Kelp Tank  1  8 C. Air  1 9  1  1' 10  Figure 3.b. Temperature Fluctuation in the Head Tank, a Kelp Tank (Low Flow, High Density) and Air During Experiment II (August 20-29,1990). Data were Recorded Every Half an Hour.  30  25 P LU DC 3 OC UJ CL  20  2  LU  15 -  10 1  3 4 A. Head Tank  5 6 B. Kelp Tank  7  8  10  C. Air  Figure 3.c. Temperature Fluctuation in the Head Tank, a Kelp Tank (Low Flow, Medium Density) and Air During Experiment III (September 10-19,1990). Data were Recorded Every Half an Hour.  I  I  I  I  4  I  DAYS  I  I  6  L  8  A. NH (Morning)  B . NH, (Afternoon)  C. NO, (Morning)  D. NQ (Afternoon)  E. NH and N0 (Morning)  F. NH and NQ (Afternoon)  4  4  3  10  4  Figure 4.a. Nitrogen Concentration in the Effluent from the Salmon Tank During Experiment I (July 31-August 8,1990).  60  i  i  i  2  i  4  i  DAYS  1  i  6  1  8  1  A. NH (Morning)  B. NH, (Afternoon)  C. NQ, (Morning) E. NH and N0 (Moming)  D. NQ (Afternoon) F. NH and NQ (Afternoon)  4  4  3  1—  10  4  Figure 4.b. Nitrogen Concentration in the Effluent from the Salmon Tank During Experiment II (August 20-29,1990).  60  i  i  i  i  i  4  2  i  i  6  i  i  8  i  10  DAYS A. NH (Morning)  B. NH (Afternoon)  C. NO, (Morning) E. NH and N0 (Morning)  D. NO, (Afternoon) F. NH and NO, (Afternoon)  4  4  3  4  4  Figure 4.c. Nitrogen Concentration in the Effluent from the Salmon Tank During Experiment III (September 10-19,1990).  K>  28 Table 1. Carbon-Nitrogen Ratios of Kelp (L. saccharina) Grown In Different Flow-Density Combinations.  Flow-Density  Experiment I  Experiment II  Experiment III  Mean  High-Low  9.9  10.1  10.4  10.2  High-Medium  11.2  10.4  10.3  10.6  High-High  11.2  10.3  9.6  10.4  Medium-Low  10.3  9.9  10.0  10.1  MediumMedium  10.7  10.3  10.0  10.3  Medium-High  10.5  10.8  10.5  10.6  Low-Low  10.9  10.4  10.5  10.7  Low-Medium  10.9  10.6  10.3  10.6  Low-High  10.9  10.6  10.3  10.6  A model of removal (r = 0.746) obtained from a four-way analysis of covariance 2  (ancova) was found. Y  ijkl  = M+a +^ +7 +5 +ai8 +Q S +/3a + 5 +T+N i  j  k  1  ij  !  il  jl  7  kl  Where Yijkl = removal rate at the i-th experiment, the j-th day, the k-th flow and the 1-th density, /x = constant, a = experiment, /3 = day, 7 = flow, 5 = density, i = index of experiment (1,2,3), j = index of day (1,2), k = index of flow (1,2,3), 1 = index of density (1,2,3,4), T = temperature, N = supply of total nitrogen ( N H  + 4  and N0 "). 3  29 Table 2. Total Nitrogen (Ammonium and Nitrate) Removal Rates (/imol l" h") by Control (No Kelp) and Kelp (L. saccharina) Grown in Different Flow-Density Combinations. 1  Flow-Density  Removal Rate  High-Control  54.4 + 22.3  High-Low  263.3 + 170.2  High-Medium  303.6 + 266.3  High-High  339.0 + 234.4  Medium-Control  47.6 + 33.0  Medium-Low  201.6 + 83.1  Medium-Medium  233.6 + 143.3  Medium-High  224.5 + 125.5  Low-Control  32.9 + 12.2  Low-Low  169.8 + 14.9  Low-Medium  192.9 + 88.5  Low-High  203.6 + 74.6  1  As main effects, flow and density affected significantly removal rate (jumol l" h") 1  1  (p<0.05) (Table 2). The significant physical parameters were water temperature and incoming nitrogen (p<0.05, respectively). No significant differences of uptake values in the morning and afternoon for every flow-density combination were evident. As a result, the morning and afternoon data of each flow-density combination were pooled.  30 After pooling the data, a Tukey test was done to determine which treatment affected uptake. No significant differences of uptake were seen among all flow-density combinations unless the first sampling of each experiment was included. At the high and medium flow levels, the low density kelp tanks demonstrated higher total nitrogen uptake than the medium and high density kelp tanks (p<0.05, Table 3); however, at the low flow level, the different kelp density tanks did not indicate any significant differences in uptake. At the low and medium kelp densities, the high flow tanks had higher uptake values than the medium and low flow tanks (p<0.05). At the high density, the uptake value of the high flow tank was not significantly different from the uptake value of the medium flow tank, but it was significantly different from the uptake value of the low flow level tank (p<0.006). A high level of irradiance occurred at the first and the last experiment, and this level possibly affected the kelp. Three kelp died in the last experiment as they lost their basal (meristematic portion of the blade). In terms of mean uptake values based on growth, measured every 3 days (interval between 2 samplings), no significant differences were evident among all flow-density combinations (Table 3). Ancova test (four-ways) on the pooled daily uptake data yielded the following model (r = 0.723). 2  Yijkl = M+«i+^j+7 +S +a/S +a5 + 5 +a 5 +T+N k  1  ij  iI  7  kl  !7  ikl  Where Yijkl = uptake rate at the i-th experiment, the j-th day, the k-th flow and the 1-th density, JJL = constant, a = experiment, /3 = day,  7  == flow, 5 = density, i = index of  31 experiment (1,2,3), j = index of day (1,2), k = index of flow (1,2,3), 1 = index of density (1,2,3,4), T = temperature, N = supply of total nitrogen ( N H  + 4  + N0 "). 3  As main effects, flow and density treatments significantly affected daily uptake (p<0.05). The important physical parameters were temperature and incoming nitrogen. In terms of nitrogen form, N H  and NO " contributed equally to total nitrogen  + 4  a  uptake (Table 4). No significant difference was found between morning and afternoon uptake of N H  + 4  or N0 ", so the data were pooled according to each nitrogen form. The 3  uptake pattern of N H  + 4  was similar to the uptake pattern of total nitrogen. At the high  flow level, a significant difference of N H  + 4  uptake was evident between the low and high  kelp density tanks (p<0.05). At the medium and low densities, the high flow tanks had greater N H  + 4  uptakes than the medium and low flow tanks (p<0.05). No significant  differences of N0 " uptake were found for all flow-density combinations. In control 3  tanks, total nitrogen removal consisted of 60% N0 " removal and 40% N H 3  + 4  removal  (p<0.1). The range of removal rate of total nitrogen was 30-40% and the range of removal rate of N H  + 4  was 30-45%. At the low and high kelp densities, significant differences of  removal rate were found between the high and low flow tanks (p< 0.075 and p<0.03, respectively) (Table 5).  32 Table 3. Total Nitrogen (Ammonium and Nitrate) Uptake Rates Measured Twice Daily and Every 3 Days (nmo\ g" dry mass h") of Kelp (L. saccharina) Grown in Different Flow-Density Combinations. 1  FlowDensity  Mean of Morning Measurement  1  Mean of Afternoon Measurement  Mean of Morning and Afternoon Measurements  Mean of Uptake of 3 Days Period (Based on Growth Rates)  20.9+10.2 (9)  23.6+10.6 (12)  22.5 + 10.3 (21)  8.3+2.9 (9)  HighMedium  17.9+7.9 (10)  16.3+7.6 (12)  17.0+7.6 (22)  6.9+2.5 (9)  HighHigh  13.9+6.5 (9)  12.9+9.3 (12)  13.3+8.1 (21)  6.7+2.5 (9)  MediumLow  14.5+5.9 (10)  15.1+5.8 (12)  14.8+5.7 (22)  6.9+1.3 (9)  MediumMedium  8.9+5.4 (10)  6.7+2.4 (12)  7.7+3.9 (22)  6.5 + 1.9 (9)  MediumHigh  9.4+3.5 (9)  7.7+5.4 (12)  8.5+4.6 (21)  5.5 + 1.9 (9)  Low-Low  9.5+5.1 (10)  8.9+6.7 (12)  9.2+5.7 (22)  6.9+2.9 (9)  LowMedium  7.7+4.2 (10)  8.5+4.2 (11)  8.1+4.1 (21)  6.7+2.2 (9)  LowHigh  6.4+3.8  5.9+2.8 (12)  6.1+3.2 (21)  5.4+1.9 (9)  HighLow  (9)  33 Table 4. Ammonium, Nitrate Uptake Rates (/umol g dry mass" h") and Percentage of Contribution to Total Nitrogen Uptake Rates of Kelp (L. saccharina) Grown in Different FlowDensity Combinations. 1  Ammonium Uptake  %  Nitrate Uptake  %  12.1+7.3  53.7  10.4+9.3  46.3  High-Medium  8.4+5.6  49.3  8.6+9.8  50.7  High-High  6.5+4.5  48.7  6.8+7.5  51.3  Medium-Low  7.5+4.1  50.9  7.3+4.4  49.1  MediumMedium  3.9+2.8  50.6  3.8+5.1  49.4  MediumHigh  4.2+2.2  49.6  4.3+4.2  50.4  Low-Low  4.6+3.4  50.1  4.6+4.5  49.9  Low-Medium  4.3+4.1  52.3  3.3+3.7  47.7  Low-High  2.9+1.8  47.7  3.2+2.4  52.3  High-Control*)  21.3+9.3  39.2  33.1 + 11.8  60.8  MediumControl*)  17.8+8.5  37.4  29.8+15.6  62.6  Low-Control*)  12.5+5.1  37.9  20.4+7.3  62.1  FlowDensity  High-Low  ;  1  The results were obtained without dividing with biomass. The unit: /xmol l" h . 1  1  34 Table 5. Removal Rates of Ammonium and Total Nitrogen (Ammonium and Nitrate) (%) of Kelp (L. saccharina) Grown in Different Flow-Density Combinations.  Flow-Density  NH  + 4  Removal Rate  Total Nitrogen Removal Rate  High-Low  29.6+11.5  25.8+6.1  High-Medium  31.0+12.3  27.7+7.9  High-High  31.4+12.6  28.6+8.3  Medium-Low  34.9+11.9  30.0+6.7  Medium-Medium  39.8+14.7  34.4+11.2  Medium-High  36.9+12.4  33.0+9.5  Low-Low  38.6+14.8  34.4+13.6  Low-Medium  40.1+11.8  34.5+9.5  Low-High  43.9+15.9  39.9+13.8  5.3. Growth In terms of lamina length increment, the range of growth varied between 6.5-9 % d" . The growth of kelp in the low density kelp tanks was significantly higher than the 1  growth of kelp in the high density kelp tanks for all flow levels (p<0.05); however, it was not significantly higher than the growth of kelp in the medium density kelp tanks. Growth recorded at different flow levels was not significantly different for all levels of kelp density (Table 6). An ancova model of growth developed was found (r = 0.502): 2  35 Y  = /i+ai+7 +P+T+N  i k  Where Y  k  i k  = growth rate at the i-th experiment and k-th density level, JU = constant, a.  = experiment, 7 = density, i = index of experiment (1,2,3), k = index of density (1,2,3,4), P = pH, T = temperature, N = concentration of total nitrogen supply ( N H  + 4  and N0 "). 3  Density affected growth significantly (p<0.01), and the important physical parameters were temperature, pH and concentration of total nitrogen supply. The biomass production of the low density kelp tank was significantly greater than the biomass production of the high density kelp tank at medium and low flow levels (p<0.10). At high density kelp, biomass production in the high flow tank was only significantly different from the production in the low flow tank (p< 0.025).  5.4. Dissolved Oxygen Dissolved oxygen concentration in the kelp tanks was always higher than dissolved oxygen concentration in the control (without kelp) tanks in both morning and afternoon sampling periods.  However, the morning and afternoon data of dissolved oxygen  concentration in each kelp tank was not significantly different. As the result, these data were pooled. In pooled data, flow appeared to affect dissolved oxygen concentration more than density in this experiment. In all flow levels, no significant difference of dissolved oxygen concentration was found among the high, medium and low kelp density tanks. In all density levels, a significant difference of dissolved oxygen concentration occurred only  36 between the high and low flow tanks in both morning and afternoon sampling periods (p<0.05, Table 7). Supersaturation level was often found in tanks with very low flow rates.  Table 6. The Specific Growth Rates (% d") and Production Level (g) of Kelp (L. saccharina) Grown in Different Flow-Density Combinations. 1  Flow-Density  Growth  Production  High-low  9.0 + 2.8 (54)  2.1 + 1.0 (54)  High-medium  8.7 + 2.9 (80)  2.0 + 1.0 (79)  High-high  7.7 + 2.4 (108)  1.9 + 0.9 (108)  Medium-low  8.6 + 2.9 (54)  2.1 + 0.8 (54)  Medium-medium  7.8 + 2.4 (80)  1.8 + 0.9  Medium-high  7.1 + 2.8 (106)  1.6 + 0.9 (108)  Low-low  8.1 + 2.4 (54)  1.8 + 0.9  7.7 + 2.8 (80)  1.8 + 0.9  6.5 + 2.4 (107)  1.3 + 0.8 (108)  Low-medium Low-high  (81)  (54) (81)  37 5.5. Uptake Prediction A multiple regression for predicting uptake was developed. The important factors of the model (r = 0.64) were flow rate, plant density, irradiance level and plant biomass. 2  V = 14.924 + 0.141F -1.207D + 0.0081 + 0.069B Where V = N uptake (/xmol g" dry mass h" ), F = flow rate (1 h" ), D = kelp density 1  1  1  (number of kelps/tank), I = irradiance level (/xE m" s"), B = plant wet biomass (g). 2  1  This model was based on the flow ranged 8.1-83.5 1, the density ranged 0-20 kelp/tank (0-740 kelp m" ), the irradiance level ranged 80-1874 /xE m" s", and the plant 3  2  biomass ranged 2.5-8.2 g l"  1  1  38 Table 7. Dissolved Oxygen Concentrations in Control (No Kelp) and Kelp (L. saccharina) Tanks (mg 1 in Different Flow-Density Combinations in the Morning and Afternoon. 4)  Flow-Density  Morning  Afternoon  High-Control  5.2+0.7 (23)  5.4+0.7 (19)  5.3+0.7 (42)  High-Low  7.0+0.9 (23)  7.1+1.1 (19)  7.1+1.0 (42)  High-Medium  7.1 + 1.1 (23)  7.2+1.1 (19)  7.1+1.1 (42)  High-High  7.2+1.0 (22)  7.3+1.0 (20)  7.2+1.0 (42)  MediumControl  5.4+0.7 (23)  5.3+0.7 (19)  5.4+0.7 (42)  Medium-Low  7.9+1.5 (23)  7.7+1.1 (19)  7.9+1.3 (42)  Medium-Medium  9.2+2.5 (22)  8.3+0.9 (19)  8.8+1.9 (41)  Medium-High  8.4+1.6 (23)  8.5+1.4 (20)  8.4+1.5 (43)  Low-Control  5.7+0.8 (23)  5.8+1.1 (20)  5.7+0.8 (43)  Low-Low  8.6+2.0 (22)  8.4+1.4 (18)  8.5+1.7 (40)  Low-Medium  8.8+1.7 (23)  8.9+1.6 (19)  8.9+1.7 (42)  Low-High  9.9+2.9 (23)  8.9+1.9 (17)  9.5+2.5 (40)  Mean  VI. DISCUSSION  6.1. Nutrient Removal and Uptake Although concentration of nitrogen supply fluctuated every day, nitrogen was not limiting (as revealed by stable C/N ratio) and ammonium was far below the toxic level (100 jiimol, Harrison, Pers. Comm.). A variable C/N ratio has been associated with plant response to nitrogen storage and indicate possible nitrogen limitation to specific growth rate (Atkinson and Smith, 1983). A stable C/N ratio (10-11) for every flow-density combination in every experiment indicated that nitrogen was not limiting. Similar results were reported by Chapman et al. (1978), Lapointe and Ryther (1979). Niell (1976); Hanisak (1979); Fredriksen and Rueness (1989) reported that C/N of macroalgae varied between 5-40, and the critical ratios were 10-15. At the critical ratio nitrogen was not limiting and no storage of nitrogen was evident. The general consensus is ratio higher than the upper threshold indicates that nitrogen is a limiting factor for growth, and a ratio lower than the lower threshold indicates that macroalgae stores nitrogen. The long acclimation (2-3 months) in the experimental culture system allowed the kelp to adapt to the nitrogenous rich environment of fish waste. During the acclimation period the plants did not appear to store nitrogen, because the nutritional history of the kelp involved nutrient-rich environment. Kelp in this study were collectedfroman nitrogenous rich area (tidal area at Brockton Point, Stanley Park) and, then, they were grown in salmon culture effluent, so the logical conclusion was that the plants did not store nitrogen. This conclusion was supported by a constant C/N ratio. Espinoza and Chapman (1983) showed that L. 39  40 longicruris from nitrogenous rich environment did not store nitrogen in laboratory culture, whereas population from poor environment did store. Anderson et al. (1981) reported that because of high nitrogen availability year round at Bic Island, Quebec, photosynthates could be used for immediate growth and no large buildup of carbohydrate reserves was found. Control (without kelp) demonstrated lower nitrogen removal than treatment with kelp.  This was not surprising due to high flushing rate (1-3 times h") and regular 1  cleaning, which prevented bacteria, phytoplankton and periphyton from utilizing available nitrogen in control tanks. Removal rate in the control tanks ranged between 32.9-54.4 umo\ l h . 1  1  No significant differences of total nitrogen uptake were found among treatments with kelp due to large variances in the data. Some factors possibly contributed to the large variances of total nitrogen uptake. Because no kelp trimming was applied until water samplings were finished in both the morning and afternoon periods, each treatment with kelp had a very high biomass in the first sampling of every experiment. The biomass before trimming was roughly 60-80% greater than the biomass after trimming in each treatment with kelp. The high biomass condition in the first sampling of every experiment led to high uptake values, which were usually 2-3 times higher than the uptake values of the following samplings. Besides that, Oliger and Santelices (1981) noted that large differences of physiological responses, such as nutrient uptake and growth, could occur if diverse individuals from field-collected samples were used in experiments. Plants could vary according to age, reproductive stage, nutrient status and  41 collection site.  Gerard et al. (1987) believed that differential growth and nutrient  assimilation rates of two different origin populations strongly suggested genetic differentiation, although further information about plants grown from spores under experimental condition was still required. None of the previous factors were within the scope of this thesis. High irradiance level and high temperature appeared to act synergistically to affect the fluctuative uptake capability of kelp. Irradiance, which ranged between 0-1875 /xE m" s" during the experiment, was often very high and caused photoinhibition 2  1  (photobleaching). The symptom was a white, fragile lamina due to pigment bleaching as plant's response to high irradiance level (Ramus et al, 1976; Dring, 1981; Ramus and Van der Meer, 1983). Lapointe and Tenore (1981) found that uptake N0 " of U.fasciata 3  was inversely proportional to irradiance level. Some studies, such as the studies by Lapointe and Tenore (1981) and Lapointe (1981), demonstrated that seaweed grown under a low irradiance level had more pigment than plants grown under high irradiance level. Yoneshigue and Oliveira (1987) reported that irradiance level (450 /xE m" s") 2  1  accelerated the death of transplanted kelp which required cold water temperature and low irradiance level for optimum growth. The water temperature during the experiment ranged from 10.2-22.9°C. The optimum temperature range of L. saccharina is 10-15°C (Bolton and Luning, 1982). At 20°C the kelps are extremely fragile and lose pigment at the tip of the lamina. The plants disintegrated at 23°C. Gerard and Du Bois (1988) observed that growth rate of two different population of L. saccharina declined as water temperature increased to  42 exceed 20°C, and as the experiment progressed, deleterious effects became evident in increases mortality rate. In brief, the cultured kelp clearly suffered photoinhibition, and, then it was further affected by higher than optimal range of temperature. Photoinhibition started during the first experiment, in which the duration of irradiance level higher than 100 /xE m" s" 2  1  ranged from 7-10 hours (irradiance range was 0-1200 /xE m" s-1, and irradiance mean 2  was 106 juE m" s"). The irradiance level in the second experiment was lower than the 2  1  first experiment, and the cultured kelp appeared to recover from photobleaching's effect. The duration of irradiance level higher than 100 / i E m" s" in the second experiment 2  1  ranged from 7-9 hours. The irradiance level in this experiment ranged 0-670 /xE m" s' , 2  1  and the irradiance mean was 98 ixE m" s". In the last experiment, the kelp endured a 2  high irradiance level again.  1  Three kelp died as they lost their basal section.  The  duration of irradiance level higher than 100 /xE m" s" ranged from 7-9.5 hours. The 2  1  range of irradiance level in the last experiment was 0-1875 juE m" s", and the irradiance 2  1  mean was 179 jxE m" s". Yarish et al. (1986) found that three species of red algae 2  1  survived long term exposure to high temperature (25°C) at low irradiance level (10-20 fxE m" s"), but not when the irradiance level was doubled. Yoneshigue and Oliveira 2  1  (1987) reported that temperature of approximately 20°C acted synergistically with high irradiance to perpetuate gradual erosion of lamina and eventually death of cultured kelp. In nature, Laminaria faces pigment bleaching (due to nitrogen starvation during summer) which causes lamina break up and this is aggravated by high temperature (Chapman and Craigie, 1978).  43 Because photoinhibiton in this experiment occurred at the apex of lamina, nitrogen uptake could have been affected. Davison and Stewart (1984) postulated that most nitrate uptake occurred in mature lamina rather than meristematic area. In nature, kelp canopy (mature section) has been determined to be the most productive site in the entire kelp system (Wheeler, 1980 ). b  Besides the temperature, nitrogen supply was another important covariate mentioned in both model. This could be explained by Michaelis-Menten expression in which the higher the nutrient concentration, the higher the uptake rate, and vice versa. Since the nitrogen supply in this experiment fluctuated with fish activity, the uptake rate of nitrogen, in turn, probably fluctuated. Even though Michaelis-Menten described also an asymptotic uptake, in which above a certain nutrient concentration the uptake could not increase, this did not occur during the experiment because the nitrogen supply from the fish tank was not saturating. Harrison et al. (1986) observed that nitrogen uptake of L. groenlandica still linearly increased up to N H  + 4  or N 0 - concentration 60 uM. 3  Amat and Braud (1990) reported that an increasing supply of nitrogen enhanced uptake rate of Chondrus crispus. Lapointe and Tenore (1981) found that N0 " uptake of U. 3  fasciata depended mostly on daily nitrogen supply of that nutrient. A pattern of daily uptake was distinguished if high values from the first sampling in every experiment were not included in the analysis. The high uptake values of the first sampling in every experiment were usually 2-3 times higher than the following samplings. Those high uptake values were caused by a high biomass since no trimming was applied in the first samplings. By neglecting extreme values of the first sampling of  44 every experiment, a distinct pattern of uptake was distinguished.  At the high and  medium flow levels, daily uptake rate of the low density kelp was higher than the medium and high densities kelp. This could be related to a canopy (self shading) effect. The low density kelp ranged 2.5-4.0 g l" , which was below the suggested level of 5 1  g 1-1 (Harrison and Druehl, 1982). The medium density kelp ranged between 4.5-5.9 g l" , and the high kelp density ranged between 6.25-8.16 g l" . Amat and Braud (1990) 1  1  found out that an increase in density of C. crispus from 1.48 to 4.28 g dry mass l"  1  decreased uptake from 26.8 to 17.2 /ig N g dry mass" min" . 1  1  At low flow level, however, no difference was found among all kelp density levels. Debris (mainly fish faeces) accumulated and covered kelp lamina at low flow level, and this would affect uptake which occurred through all plant surface. At the low and medium kelp densities, the uptake in the high flow tanks was higher than the uptake in the medium and low flow tanks. At the high kelp density, the uptake in the high flow tank was only higher than the uptake in the low flow tank. This pattern could be explained on the basis of an increased nitrogen availability in the high and medium flow culture conditions. Turbulent enhancement of gaseous and nutrient diffusion through the nutrient depleted boundary layer adjacent to plant thallus could have increased nutrient uptake (Whitford and Schumacher, 1961, 1964; Conover, 1968; Doty, 1971). Lapointe and Ryther (1979) reported that nitrogen availability was a function of both nitrogen concentration and flow level rate near plant thallus. Wheeler (1977) showed that the uptake of NO " by the kelp M.pyrifera was dependent on water s  velocity (flow level). Current slightly enhanced N H  + 4  uptake rates by Ulva lactuca discs  45 except at the highest N H  + 4  concentration (115-145 /umol) Parker (1981) and Gracilaria  tikvahiae (Parker, 1982). Irregardless of kelp density, uptake values in the medium and low flow tanks were not significantly different due possibly to the slight difference in flow rate. The flow range in the medium flow tanks (25-44.11 h") was too close to the flow range in the low 1  flow tanks (8.1-24 1 h ). 1  The level of N H  + 4  uptake by the kelp in this experiment was similar to NO " a  uptake. This tendency occurred for all treatments with kelp. The similar results were also reported by Harrison et al. (1986), Harlin and Craigie (1978). Ammonium ( N H ) +  4  and N0 " were utilized simultaneously by L. groenlandica and L. longicruris (Harrison 3  et al., 1986; Harlin and Craigie, 1978), and the uptake rates were equal to uptake rates when only N H  + 4  or N0 " was present in medium. Meanwhile, in all control tanks, 3  contribution of N0 " (60%) was higher than N H 3  (40%), and this indicated the  + 4  presence of nitrifying bacteria in the tanks. Uptake values obtained in this experiment (total nitrogen, ammonium, nitrate) were generally higher than results reported elsewhere (Table 8). For instance, Harrison et al. (1986) reported that total nitrogen, N H  + 4  and N0 " uptake of L. groenlandica were 3  16.8, 9.75 and 7.05 ttm g" dry mass h" , respectively. The results of total nitrogen, N H 1  1  + 4  and N0 " uptake of the high flow-low density combination in this experiment were 3  22.476, 12.075 and 10.401 itm g" dry mass h'\ respectively. The higher results in this 1  experiments can be explained in three ways. First, most of other investigations were involved with a batch culture system, whereas this experiment used a continuous culture  46 system. Second, nitrogen supply in this experiment was higher than concentrations used in other reported experiments (Table 8). Third, the cultured kelp possibly consisted mostly of young individuals collected at spring time, which could perform higher nitrogen uptake than older kelp. Harrison et al. (1986) observed that N H  + 4  and N0 " uptake rate 3  of young (first year) L. groenlandica was three times higher than older (second and third year) kelp. Since both form of nitrogen ( N H  and N0 ") were present in the system,  + 4  no general conclusion could be made if only N H  3  + 4  was present as it occurred in netpens  operation. The mean uptake based on 3 days growth (between 2 samplings) was usually lower than mean daily uptake of each flow-density combination. The low results seemed to be acceptable, because uptake rate at night time was always lower than uptake rate at day time (Chapman, 1987). Harrison et al. (1986) reported that N0 " uptake rate of 3  first year L. groenlandica at night time was half of N0 " uptake rate at day time, while 3  NH  + 4  uptake rate of the same kelp at night time was one-third of N H  + 4  uptake rate at  day time. The low mean uptake rate of 3 days period could be explained also in the following manner. Most of nitrogen uptake occurred in the mature section of kelp's lamina (Davison and Stewart, 1984), from which the sample of total nitrogen tissue was taken, and the kelp suffered photoinhibition mostly at the apex (mature section) of the lamina. The photoinhibition could reduce the level of total nitrogen tissue. In terms of removal rate, the removal rate of N H  + 4  and total nitrogen ranged 30-  45% and 30-40%, respectively. The similar results were obtained by Fujita et al. (1989), in which they found the removal rate of N H  + 4  was 30-50%.  47  Table 8. Nitrogen Uptake Rates of Kelp in Different Culture System and Nitrogen Supply (as Reported in Literature).  Species L. digitata  Uptake (/iM/g/h)  Concentration, Culture System  Source  3 M M NOy  6.9 + 0.2  Indergaard and Jansen (1976)  batch L. longicruris L. groenlandica  7 - 10  batch  Harlin and Craigie (1978)  7.05 + 3.45  N0 " (morning)  Harrison et al.  3  (1986)  NH  9.75 + 2.40  + 4  total  16.80  3.6 + 0.9 2.1 + 0.6 5.7  (morning)  - "  N0 " (evening) 3  NH  + 4  (evening) total  20 /imol N0 " 3  20 /imol N H  + 4  batch L. longicruris  6.7 + 1.8  Chapman (1987)  11.7 /iM N0 " 3  8.3 + 1.8  14.9 /xM N0 3 "  8.2 + 0.6  17.2 /xM N 0 3  batch Red algae  3.8 - 6.4  12.8 jumol N H  + 4  Fujita et al. (1989)  continuous  48 The highest removal rate of N H  + 4  (44%) and total nitrogen (40%) were in the  low flow-high density tank, whereas the lowest removal rate of N H  + 4  (30%) and total  nitrogen (26%) were in the high flow-low density tank. The low flow-high density tank had significantly higher removal rate than the high flow-low density tank, because the former had more biomass than the latter and longer residence time. tendency occurred for uptake.  The opposite  In summary, for nitrogen removal purpose the  combination of 740 kelp/m" in flow of 15 1 h" should be used. For nitrogen uptake by 3  1  kelp the combination of 370 kelp/m" in flow of 70 1 h" should be used. 3  1  6.2. Growth In term of lamina increment, the growth rate in the low density kelp treatment was significantly higher than the growth in the high density kelp treatment at all flow levels, but it was not different from the growth rate in the medium density kelp treatment. Canopy (self-shading) effect appeared to perpetuate this pattern. High kelp density (6.25-8.16 g l") was higher than the suggested density of 5 g l" (Harrison and 1  1  Druehl, 1982). Lapointe and Tenore (1981) conceived that specific growth rate of U. fasciata decreased from 0.36 to 0.002 doubling d" as density increased from 0.2 to 3.6 1  kg wet mass m" . 2  Most of the growth of L. saccharina, which is phototaxis positive, occurs in the first 20 cm basal and very little occurs above that section (Brinkhuis, unpub. data in Brinkhuis, 1984). Lapointe et al. (1976) reported an increased pigment levels in nitrogen enriched Gracilaria, and the enhanced pigment appeared to increase the photon  49 gathering 'antennae' allowing these plants to achieve some of the highest growth (yields) and solar conversion efficiencies reported for outdoor algal cultures or fast growing food crops such as sugar and rice. The ancova model developed indicated that pH, temperature and nitrogen supply were important factors that affected kelp's growth. Penniman (1988) reported that the growth of L . longicruris increased from 0.187 doubling d" (at 9 limol) to 0.025 doubling 1  d" (at 20 and 36 /imol) at temperature 15°C. Druehl et al. (1988) reported that the 1  production of L. groenlandica in British Columbia was positively correlated with N0 " 3  concentration in the marine environment. The results calculated using the punched-hole method could underestimate the . growth rate of the cultured kelp in this experiment because the method estimated only one dimensional growth (length) (Harrison, Pers. Comm.) The specific growth rate of the cultured kelps ranged from 6.5-9% d" . Those 1  results were generally higher than unfertilized kelps reported in the literatures (Table 9). For example, Gerard et al. (1987) reported that the growth rate of L. saccharina in nature (ambient nitrogen was 0.1-16 /xmol) was 1.6-6% d" . The growth rate of L. 1  saccharina in the high flow-low density combination was 9 % d-1. The high specific growth rate of L. saccharina (5-19% d") reported by Bolton and Luning (1982) was 1  obtained in optimum conditions of enriched seawater and temperature (range 10-15°C), whereas in this experiment the kelps endured photoinhibition and unfavourable temperature range during the experiment.  50  Table 9. Specific Growth Rates of Different Species of Kelp In Different Condition as Reported in Literatures.  Species L. saccharina  SGR (% d )  Notes  1  1.6 - 6.0  L. saccharina  1.5 - 18  L. longicruris  19  L. digitata  Source  nature  Gerard et al.  0.1-16 M M  (1987)  batch culture enriched seawater optimum temp.  Bolton and Luning ( 1 9 8 2 )  batch culture 3 uM NOy  Indergaard and Jensen ( 1 9 7 6 )  7 - 9  L. hyperborea  6.4  L. digitata  0.2 - 2.3  M. pyrifera  3.6  nature  Gerard  M. pyrifera  5 - 8  nature  Wheeler and North ( 1 9 8 1 ) Druehl  M. pyrifera  2.8 +  1.6  wet weight  4.3 +  2.4  frond elongation  (1982 ) B  (1984)  batch culture  The biomass production was similar in all treatments, except for production in the low and high density kelp tanks at both the medium and low flow levels. The biomass production ranged between  1.3-2.1  g. Two reasons can be given to explain this.  First, the short duration ( 9 days) of the experiment did not allow the kelp to double its biomass. De Boer et al.  (1978)  suggested  21  days for biomass doubling time. Second,  the cultured kelp probably consisted most of young individuals (collected at spring time),  51 and young plants grew more in length than mass mode (Mann and Mann, 1981). Druehl (1984) found no discernable difference between fertilized and control plants (M.pyrifera) on wet mass basis, but frond (lamina) elongation were significantly greater for fertilized plants.  6.3. Dissolved Oxygen Treatment with kelp demonstrated higher 0  2  than control (without kelp) in  morning and afternoon sampling periods. However, dissolved oxygen concentration in the morning and afternoon of all flow-density combinations were not significantly different from each other.  Photosynthetic activity possibly reached saturating level  before afternoon time. Irradiance level achieved saturating level around 9-10 a.m. The sampling interval was actually too large to detect hourly trends of photosynthetic activity. Even though nitrogen supply usually increased in the afternoon (after fish feeding), this increase was not reflected in an increase in dissolved oxygen production. This finding supported the work of Chapman et al. (1978) in which photosynthetic activity of L. saccharina (measured in term of 0 production) was similar at 10 and 500 2  /xmol external N 0 . Furthermore, Brinkhuis et al. (1976) revealed that many seaweeds 3  reached photosynthetic saturation at 10-50% of full incident light. Treatments of the low flow level in all kelp density levels significantly demonstrated greater dissolved oxygen concentration than treatment of the high flow level. At the low flow level, the 0  2  produced stayed longer in tanks, while at high flow  level it would be flushed away promptly.  VH. CONCLUSIONS AND SUGGESTIONS The purpose of this study was to assess the feasibility of an integrated culture of salmon and kelp. The culture of kelp within 3 meters to a netpen farm would purify soluble ammonium generated through nutrient loading effect. The combination of the high flow and low density in this study was shown to be effective to clean ammonium from salmon culture effluent, and this would be applicable in netpens culture. The kelp density suggested for rope culture around a netpen is 110 kelp/m' or 370 kelp/m" . 2  3  Ammonium free fertilizer discharging from netpen culture could enhance the growth of kelp.  The biomass production appeared to increase as nitrogen uptake  increased. Through photosynthetic activity, the kelp generated dissolved oxygen, which is a critical factor in netpens culture. More available dissolved oxygen would be valuable for the purpose of increasing the productivity of salmon netpens culture. More specially the following conclusion can be made from the experimental results. 1. The treatments with kelp demonstrated higher total nitrogen removal than the controls (without kelp). 2. Removal rate of total nitrogen was primarily controlled by kelp density. 3. Kelp density and flow rate were significant factors in affecting nitrogen uptake of L. saccharina in this experiment. The combination of high flow and low density demonstrated the highest total nitrogen uptake compared to the other 52  53 flow-density combinations. Light and temperature acted synergistically to perpetuate photoinhibition. 4. The ancova (analysis of covariance) model developed suggested the importance of incoming nitrogen and temperature as an important factor affecting nitrogen uptake. 5. This experiment demonstrated that continuous system could give greater uptake and growth rates than a batch system. 6. Growth of L . saccharina in this experiment was affected by kelp density. 7. Biomass production was affected by kelp density and appeared to increase as nitrogen uptake increased. 8. Daily dissolved oxygen concentration of the treatments with kelp were higher than the controls (without kelp). 9. Dissolved oxygen concentration was significantly less pronounced in the high flow level than in the low flow level. 10.  A model developed for predicting nitrogen uptake was found to be adequate under the condition of the experiment.  Suggestions for Further Work 1. It is recommended to use the density level 110 kelp/m" or 370 kelp/m' and 2  3  high flow rate in further experiments. Constant biomass should be maintained throughout the experiments, including the first sampling. 2. Kelp utilizes nitrogen (ammonium, nitrate) in the light time as well as dark  54 time. To obtain more succinct nitrogen uptake pattern, it is suggested to conduct a 24 hour sampling. 3. To avoid significant oxygen depletion due to kelp consumption during the dark period, an investigation of a 24 hour oxygen fluctuation should be conducted. 4. Since only ammonium is found in and around netpens culture operation, a study focused on ammonium uptake should be done. 5. Another experiment to investigate the effect of light and temperature should be conducted. 6. Other factors, such as nutrient status, reproductive stage, age, genetics, could contribute to variances of uptake, and these factors could be the basis for another study. Uptakes, flow rates and residence time obtained or used in these work can be used to determine the number and size of the kelp line needed to remove a given amount of N from a salmon netpen farm. The size of one unit is calculated as follows: its length is obtained by multiplying the residence time by the current velocity at the netpen site. Its cross-sectional area, on the other hand, is obtained through the division of flow rate by the current velocity at the netpen site, or it is the market-sized kelp dimensions (which ever is largest). The number of units is calculated.by the following formula: Number of units = (Culture Volume, m /unit)/(Uptake Rate, ixmol/h/kg kelp, db)x 3  (Desired N Removal Efficiency, /imol/h)x(Kelp Stocking Density, kg/m ). 3  55 Depending on the current and the desired N removal efficiency, the suggested kelp raft will consist of a number of units, which are between 120 to 480 m long and 0.15 m in cross-sectional area. If the current velocity 0.1 m/s and 1.1 tons of N is released 2  per year, 5 units are needed to remove 75% of the nutrients.  REFERENCES Anonymous. 1986. Industrial Organization of the B.C. Salmon Aquaculture Industry (Final Report). The DPA Group Inc. Vancouver, B.C. Ackefors, H and M . Enell. 1990. Discharge of Nutrients from Swedish Fish Farming to Adjacent Sea Areas. Ambio 19(1): 28-35. Amat, M.A. and J.P. Braud. 1990. Ammonium Uptake by Chondrus crispus Stackhouse (Gigartinales, Rhodophyta) in Culture. Hydrobiologia 204/205: 467-471. Anderson, R.M.; A Cardinal and J. 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