I ANNALES • Ser. hist. nal. • 13 • 2003 • 1 • Supplement short scientific article UDK 639.32:504.064(262.3-18) received: 2003-09-19 THE INFLUENCE OF FISH CAGE AQUACULTURE ON BACTERIOPLANKTON IN THE BAY OF PiRAN (GULF OF TRIESTE, ADRIATIC SEA) Valentina TURK & Alenka MALE! National Institute of Biology, Marine Biology Station Piran. St-6330 Piran, Fornace 4Î E-mail: turk@mbss.org ABSTRACT The impact of fish cages on bacterioplankton was examined in enclosures containing seawater from cage and non-cage sites and natura! plankton from the Bay of Piran (northern Adriatic). Nutrient enriched seawater stimulates bacteria! production and abundance. This observation was further investigated in situ, where for 3 months submerged bio-filter with Schizobrachiella sanguínea dominating fouling community was enclosed in mesocosm. Based on the results of bacterial production and abundance, we assumed thai filter feeding could remove significant amount of bacteria attached on suspended panicles and thus depleting bacterioplankton in the water column. Key words: aquaculture, environmental impact, bacterioplankton, cyanobacteria, Adriatic Sea INFLUENZA DELLA PISCICOLTURA IN GABBÍE SUL BATTERIOPLANCTON NELLA BA1A DI PIRANO (GOLFO DI TRIESTE, MARE ADRIATICO) SíNTESI L'impatto di un allevamento di pesci in gabbie su! batterioplancion é stato esaminato in contenitori da laboratorio contenenti acqua marina proveniente dali'allevamento,. da un sito prossimo all'allevamento e da una stazione di conirollo con plancton naturaie della baia di Pirano (Adriático settenlrionale). L'acqua proveniente dali'allevamento di pesci, arricchita in nutrienti, stimola la produzione e i'abbondanza batterica. Ta/e osservazione é stata successi-vamente investígala in situ con l'immersione per tre mes i di bio-filtrí con una comunita di fouling con predominante Schizobrachiella sanguínea inclusa nel mesocosmo. In base ai risultati di produzione ed abbondanza batterica, gli autori concludono che l'alimentazione di filtri pub rimuovere quantitá signiticanti di bailen legati al particellato sospeso e quindi diminuiré il batterioplancton nella colorína d'acqua. Parole chiave: acquacoltura, imparto ambiéntale, batíerioplancton, cianobatteri, mare Adriático 41 ANNALES • Set. hist. nat. • 13 • 2003 • 1 • Supplement Valentin* TURK A Alerk» MAUfc THE INrUHNCE Oi I I5H CAGF AQUACULTURC ON BACTERlOPLANKTON IN Hit" 6AV OF PiRAN .. .I7-4J INTRODUCTION Bacteria play a central role in all major nutrient cycles in the marine environment (Azam, 1998). They are also most important organisms involved in different systems designed to treat domestic wastewaters. During the last few years, a series of papers was published addressing the impact of fish farming on water column chemistry and the effect on plankton distribution (Pitta et at., 1999; Alortgi et a!., 2002). However, the role of bacteria in the environment impacted by the caged fish culture has received little attention. Bacterioplankton community and abundance in pelagic system can be regulated by bottom-up and top down regulating forces. The fish farming activity may have direct and indirect effects on the components of the microbial food web either by changing nutrient status of the environment or by altering prey and predator community composition. The present study is part of the EU funded project BlOFAQs (Bio-fiitration and Aquaculture: an Evaluation of Hard Substrate Deployment Performance Within Mariculture Developments) (Angel, 2001; Black et a/., 2001), The main objective was to assess the effectiveness of deployment of artificial substrates (bio-filters) in the water column in reducing the environmental impacts of cage fish culture. By providitig surface area for sessile biota and microbial colonization, bio-filters would facilitate uptake of organic and inorganic matter released by farmed fish. With in this project the microbial dynamics was followed in the enrichment experiment, using seawater from fish cage as nutrient source for enclosed plankton population, and seawater collected at unimpacted area. Additionally, microbial abundance and production was measured in mesocosm with enclosed bio-filters together with surrounding water during 2 diel cycles. MATERIAL AND METHODS Enrichment experiment was undertaken in July 2001 and lasted for 5 days (from 30tirnc*.YtnUil KV n for chemical / ANALYSES S \ \ \; k^vm "-mwf i K-RSPEX.^ CHAMBER t:^,,. !:• Mil Stl Fig. 1: Scheme of the mesocosm in situ enclosure experiment. SI. 1: Shema mezokozemskega ekspcrimenia v naravnem okolju. In situ enclosure (mesocosm) (Fig. 1) experiments were ¡performed on the 3,d and 5,h months after biofilters immersion into natural environment (Plate I: Fig. 1) at the station near fish cages (station SL), and at the station 200 rn from the cages (station CL) (Plate II; f ig. 14). The mesocosm represented an in situ enclosure of selected bio-filter in natural environment. Each selected bio-filter was enclosed within a clear acrylic plastic octagon box, with a volume of about 110 litres (Fig. 1). A fine scale profiler with sensors (Sea Bird and Sea Tech) was connected to the chamber to measure, each hour, temperature, conductivity, dissolved oxygen and fluorescence. Divers collected samples for chemical and biological parameters from the chamber five tunes over 24 hours, from 26 to 27 September and 21 to 22 November at the station CL and at the station SI from 27 to 28 September 2001 and from 23 to 24 November 2001. At the same time intervals water column characteristics were performed using fine-scale profiler (CTD - University of Australia). The seawater for chemical and hio- ANNALL-S • Ser. hist. nat. • 13 • 2003 ■ 1 • Supplement Valentin» TURK & Alenki MAI iT THE INFLUt'MCF Of FISHCAGf AQUACUI. liiRf ON 8ACTFRIOFIANKTON IN I'Ht BAY OF PIRAN ..., 37-42 logical parameters was collected at three different depths (5 m, 8 m, 11 m) using a membrane pump (flow rate 20 I per minute). Heterotrophic bacteria were counted according to the Porter and Feig protocol (Porter & Feig, 1980) and the biovoiume of bacteria was converted into carbon biomass using 20 fg C cell'1 as the conversion factor (lee & Fuhrman, 1987). Cyanobacteria were counted in green excitation using an epifluorescence microscope U'akahashi ei at., 1985), Bacterial production (BP) was measured by 3H-leucine incorporations according to procedures of Smith & Azam (1992). Each time, 1,7 ml of seawater was incubated with L-[4,5-3H] leucine (20 nM final, Amersham) for 1 hour at in situ temperature. All samples were done in triplicate. Bacterial production was calculated as in Simon & Azam (1989). At. the same time, ^H-thymidine incorporation method was used in parallel samples (Fuhrman & Azam, 1982). Triplicates of eac h sample were incubated for one hour at in situ temperature with 250pCi }H-thymidine I"1 (sp. act. 80 Ci minor1, Amersham). Moles of thymidine incorporated were converted to cells produced by the conversion factor 2 x 10,s cells mole"1. RESULTS AND DISCUSSION An enrichment experiment was set up to examine the impact of fish farming on nutrients and microplankton distribution. During the five days of incubation, natural bacterioplankton community showed significant enhancement on production rates and biomass accumulation in the enclosures with the water from the fish cage (treatment C) and nearby station (treatment B), compared to the seawater from the non-impacted area (treatment A). Abundance of bacteria increased from 0.5 to 4.3 x 10s cells I'1 in the treatment B, from 0.6 to 3.8 x 10s ceils !"' in the treatment C three days after the inoculation (Fig. 2). The highest bacterial production was measured on the second day with the vaiue of 2.02 pg C I'1 h"' in the treatment B, compared to the production of I.01 pg C I'1 h*1 in the treatment C (Fig. 2). Cyanobacteria were more abundant in the treatment with seawater froin fish farm (treatment C) and increased from 3.3 to 7.8 x 10' cells I1 in treatment B, and from 3.1 up to II.0 x 10' ceils i"1 in treatment C within four days of incubation, and only up to 2.8 x 10' cells l'! in the control bottle. Natural population of heterotrophic bacteria quickly responded to the nutrient enriched seawater, preceding autotrophic organisms, which was also reported in previous studies (Pitta et at., 1996; Malej eta/v 2003). Fig. 2: The bacterial abundance (—-) and production (□} in different nutrient treatments during the enrichment experiment lasting from 30"' fut y to 5"'August 2001. SI. 2: Gostota bakterij (■—) in produkcija (□) v različnih hranilnih razmerah v obogatitvenem poizkusu v času od 30. julija do 5. avgusta 2001. f 1.9 «J Z 1 x.. J a *0C» MÛC »joči» ot. -s#»>i. av o 2 % l ¡7 00 »A) Fig. 3: Bacterial abundance and bacterial production in the water column during 24-hour cycle at the station near the fish cages (station SL) and the station 200m away from the cages (station CL) in September 2001. Si. 3: Gostota bakterij in bakterijska produkcija v vodnem stolpcu (5 m, 8 m, 11 m) na postaji blizu rihjih kletk (postaja SL) in na postaji, oddaljeni 200m od ribjih kletk (postaja CL) 24-mesečnem ciklu v septembru 2001. 39 ANNALE5 • Ser. hist. nat. • 13 • 2003 • I • Supplement —- wiprthM .i I", f. ■■■ «• .o \VUj THEI-.lli.TNCf 01 MSH CA~: AQOACTiTlURf ON f A- = i :¡AKM "I ¡n I Hi BAY Of PISA-»- .. . 3- ■>.' Tab. 1: Average bacterial abundance, bacterial production (BP) and P/B ratio in the water column at the station near the fish cages (station SL) and the station located 200 m away (station CL) during 24-hour measurement from 26 to 28 September and from 27 to 29 November 2001. Tab. J: Povprečne gostote bakterij, bakterijske produkcije (BP) in P/B razmerja v vodnem stolpcu na postaji blizu ribjih kletk (postaja SL) in na postaji, oddaljeni 200 m (postaja CL) v 24-urnih meritvah od 26. do 28. septembra in od 27. do 29. novembra 2001. Station CL SL SL/CL ! Date No. Average ±SD No. Average ± SD % 26/28 September 2001 Abundance (cells I"') 1 1 1.57 x 10s 3.75 x 10s 11 1.49 x 10* 4.57 x 10" 95 I BP (ÏH-Thy) (MfiCI 'd-') 15 3.78 1.63 15 3.97 1.86 105 BP ('H-Leu) (PRC I"ri1) 15 4.80 2.27 15 5.84 3.09 122 P/B id') 11 0.178 11 0.247 27/29 November 2001 Abundance (cells I"') 8 1.15 x 10s 1.29 x 10d 8 1.10 x 10'J 7.64 x Î 0' 96 BP pH-Leu) (pgC r'd"') 15 3.55 0.66 15 3.92 1.43 110 P/B (cl1) 8 0.154 8 0.175 Based on laboratory results, we decided to examine the possible effect of fish farm on bacterioplankton dynamic in the field. During the study of diurnal dynamics, five samples at three different depths (5 m, 8 m and 11 m) were analysed for each station (fig. 3). Vertical distribution of heterotrophic bacteria was similar at all sampled depths, and the abundance varied from 1.41 x 109 cells I"' to 2.3 x 105 cells I"1. The bacterial production measured as 'H-leucine incorporation varied from 0.2 to 1.9 pg C fV at the station CL and from 0.2 to U pg C I'V at the station SL, with the highest values at 5m depth over the 24 hour experiment (l:ig. 3). Comparison between both sampling locations is presented in Table 1, based on the results of bacterial abundance and production rates during the 24-hour measurements in September and November. The average number of bacteria was 1.49 x 10' cells"' (±3.8x 10 , n=H) at the station near the fish cages, compared to the average number of 1.57 x 109 cells ' (±4.6x 10s, n=11) at the station located 200 m away. Bacterial production measured as 1H-leucine incorporation was 5.84 pg C T'd"' (±3.1 pg C 11 d"', n=15) and 4.8 pg C I 'd1 (±2.3 pg C I"' d~\ n-15} at the station SL and CL, respectively. Similar were results of the bacterial production measured as ' H-thymidine incorporation (Tab. 1). No difference between both stations was recorded in November. The average number of bacteria was 1.1S x 109 ceils I"' at the station CL and 1.10 x I05 cells l'f at the station SL. The bacterial production was 3.92 pg C f'd"' at the station SL and 3.55 pg C I'V at the station SL (Tab. 1). The P/B ratios tor the heterotrophic bacteria were from 0.070 to 0,342 for the station CL and from 0.071 to 0.642 at the station SL. Although the average biomass of bacteria was higher at the station located 200 m away from the fish farm, bacterial production was higher (5-22%) at the station near the fish cages. However, the abundance and pro- duction rates were in the range reported for the Gulf of Trieste (Turk el al., 2001). Similar results were reported from other areas in the Mediterranean (Pitta ef a!., 1999), when the plankton community structures and abundance were more dependent on seasonal environmental characteristics and locations than by the presence of fish farming. In contrast to the resujts in the water column that did not show a significant difference, the results obtained in the enclosures were different, thus eliminating currents and tides. In situ mesocosm experiments were performed in order to relate microbial dynamics to sessile biota on bio-filter. The experiment was performed in Fig. 4: Results of bacterial abundance and bacterial production in the mesocosm experiment at the station near the fish cages (station SL) and at the station location above 200 m away (station CL) during 24-hour cycle in September 2001. SI. 4: Gostota bakterij in bakterijska produkcija v mezokozemskem poizkusu na postaji blizu ribjih kletk (postaja SL) in na postaji, oddaljeni 200 m (postaja CL) v 24-ttrnem ciklu, septembra 2001. 54 I ANNALES • Ser. hist. nal. • 13 • 2003 • 1 • Supplement y.i!ct!iir>.p TUCK A AknU \lAlt!- THÏ WfLOCNtf OF FISH C ,u,r AQViACOLTURf ~ fSAf iTSIOW.AWrON IN ThtBAY Ol PIRAfÎ September using the bio-filter, submerged for 3 months near the fish cages at a depth of 5 m. Dominating fouling community on the enclosed bio-filter was bryozoan (Schizobrachiella sanguined) (Frumen ef at., this volume). Over a die! cycle, bacterial numbers constantly decreased throughout the experiment from 2.2 x \(f cells I"1 to 1.1 x 10' cells t"' at the station SL (Fig. 4), Contrary to bacterial abundance, production showed an increase from midnight and early morning up to 4.5 pg C I ' h'1 m the enclosure with bio-filter at the station near the fish farm (station SL). According to high bacterial production and constant decrease in number during the night, it i?. assumed that the majority of the bacteria produced were consumed, presumably due to grazing of sessile organisms on bio-filters. Studies on bryozoans feeding indicate that small particles are principal food source (Hudges, 2001}. Our preliminary study did not reveal any large-scale eutroph¡cation or significant differences between stations near ihe fish farm cages and open water stations. However, a pronounced response of bacterial community was observed after addition of enriched water from the fish farm in laboratory experiment and for in situ mesocosm experiment. Results from mesocosm experiment showed that filter feeding could remove significant amount of attached bacteria on suspended particles and thus deplete bacteriopiankton in the water column. However, bacteria within the peiagic ecosystem recycle the excreted nutrients and additional investigations should be considered in the future. VPLIV MARIKULTURE NA BAKTERIOPLANKTON V PIRANSKEM ZALIVU Valentina TURK & Alenka MALE! Nacionalni inštitut za biologijo, Morska bioioška postaja, SI <>330 Piran, fornače 4 J E-mail-. turk@mbi.5.or£ POVZETEK Avtorici sta preučevali vpliv gojenja rib v kletkah na bakterioplankton v morski vodi z naravnim plnaktonom, vzeti z lokacij s kletkami in 200 m od njih v Piranskem zalivu. S hranilnimi snovmi obogatena morska voda spodbuja produketjo in gostoto bakterij. Raziskave sta opravili tudi v naravnem okolju, v katerem je bil za 3 mesece potopljeni biof/lter s prevladujočo vrsto Schizobrachiella sanguinea obdan z mezokozmom. 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