AACL Bioflux, Volume 8(1) February 28, 2015
Contents
Sarong M. A., Jihan C., Muchlisin Z. A., Fadli N., Sugianto S., 2015 Cadmium, lead and zinc contamination on the oyster Crassostrea gigas muscle harvested from the estuary of Lamnyong River, Banda Aceh City, Indonesia. AACL Bioflux 8(1):1-6.
Pojas R. G., Tabugo S. R. M., 2015 Fluctuating asymmetry of parasite infested and non- infested Sardinella sp. from Misamis Oriental, Philippines. AACL Bioflux 8(1):7-14.
Bobiles R. U., Soliman V. S., Yamaoka K., 2015 Changes in otolith structure of seagrass siganid Siganus canaliculatus during settlement. AACL Bioflux 8(1):15-25.
Hontiveros G. J. S., Serrano Jr. A. E., 2015 Nutritional value of water hyacinth (Eichhornia crassipes) leaf protein concentrate for aquafeeds. AACL Bioflux 8(1):26-33.
Bahrevar R., Faghani-Langroudi H., 2015 Effect of fish meal replacement by blood meal in fingerling rainbow trout (Oncorhynchus mykiss) on growth and body/fillet quality traits. AACL Bioflux 8(1):34-39.
Muchlisin Z. A., Rinaldi F., Fadli N., Adlim M., Siti-Azizah M. N., 2015 Food preference and diet overlap of two endemic and threatened freshwater fishes, depik (Rasbora tawarensis) and kawan (Poropuntius tawarensis) in Lake Laut Tawar, Indonesia. AACL Bioflux 8(1):40-49.
Musman M., Rahmad A., Dewiyanti I., Sofia C., Sulistiono H., 2015 A comparative study on the efficacy of mixed tannins, hydrolysable tannins, and condensed tannins of Avicennia marina as anti-ectoparasite against Trichodina sp. AACL Bioflux 8(1):50-56.
Mehrad B., Shabanpour B., Jafari S. M., Pourashouri P., 2015 Characterization of dried fish oil from Menhaden encapsulated by spray drying. AACL Bioflux 8(1):57-69.
Panchai K., Hanjavanit C., Rujinanont N., Wada S., Kurata O., Hatai K., 2015 Experimental pathogenity of Achlya species from cultured Nile tilapia to Nile tilapia fry in Thailand. AACL Bioflux 8(1):70-81.
Mimeche F., Biche M., 2015 Length-weight relationships of four non-native cyprinid from the semiarid region in North-East of Algeria. AACL Bioflux 8(1):82-88.
Palm H. W., Nievel M., Knaus U., 2015 Significant factors affecting the economic sustainability of closed aquaponic systems. Part III: plant units. AACL Bioflux 8(1):89- 106.
Francisco F. C., Simora R. M. C., Nunal S. N., 2015 Deproteination and demineralization of shrimp waste using lactic acid bacteria for the production of crude chitin and chitosan. AACL Bioflux 8(1):107-115.
Esanu V. O., Gavriloaie C., Oroian I. G., Burny P., 2015 Some considerations concerning the artificially colored aquarium fish trade. AACL Bioflux 8(1):116-121. International Standard Serial Number Online ISSN 1844–9166; Print ISSN 1844-8143
Published by Bioflux – six issues/year (bimonthly); in cooperation with New England Aquarium (USA) and Natural Sciences Museum Complex (Constanta, Romania)
The journal includes original papers, short communications, and reviews on Aquaculture (Biology, Technology, Economy, Marketing), Fish Genetics and Improvement, Aquarium Sciences, Fisheries, Ichtiology, Aquatic Ecology, Conservation of Aquatic Resources and Legislation (in connection with aquatic issues) from wide world.
Editor-in-Chief: Petrescu-Mag I. Valentin: USAMV Cluj, Cluj-Napoca; University of Oradea (Romania) Gavriloaie Ionel-Claudiu (reserve): SC Bioflux SRL, Cluj-Napoca (Romania)
Journal Secretary: Nowak Michal: University of Agriculture in Krakow (Poland)
Editors: Adascalitei Oana: Maritime University of Constanta (Romania) Arockiaraj A. Jesu: Cheju National University (South Korea) Bavaru Adrian: Ovidius University, Constanta (Romania) Botha Miklos: Bioflux SRL, Cluj-Napoca (Romania) Breden Felix: Simon Fraser University (Canada) Brezeanu Gheorghe: Romanian Academy of Science, Bucharest (Romania) Bud Ioan: USAMV Cluj (Romania) Burny Philippe: Universite de Liege (Belgium) Caipang Cristopher M.A.: Temasek Polytechnic (Singapore) Codreanu Mario: USAMV Bucuresti, Bucharest (Romania) Cosma Dorin: West University of Timisoara (Romania) Creanga Steofil: USAMV Iasi, Iasi (Romania) Cristea Victor: Dunarea de Jos University of Galati (Romania) Dimaggio Matthew A.: University of Florida (USA) Dutu Mircea: The Ecological University of Bucharest (Romania) Gaina Viorel: University of Craiova (Romania) Gal Denes: Research Inst. for Fisheries, Aquaculture & Irrigation, Szarvas (Hungary) Georgescu Bogdan: USAMV Cluj, Cluj-Napoca (Romania) Grozea Adrian: USAMVB Timisoara (Romania) Hermosilla Joeppette J., Tokyo Univ.of Marine Sci.& Tech., Minato, Tokyo (Japan) Mehrad Bahareh: Gorgan University of Agricultural Sciences and Nat. Res. (Iran) Miclaus Viorel: USAMV Cluj, Cluj-Napoca (Romania) Molnar Kalman: Hungarian Academy of Sciences, Budapest (Hungary) Muchlisin Zainal Abidin: Universiti Sains (Malaysia), Syiah Kuala University (Indonesia) Murariu Dumitru: Grigore Antipa Museum of Natural History, Bucharest (Romania) Muscalu Radu: Sterlet SRL, Timisoara (Romania) Olivotto Ike: Universita Politecnica delle Marche, Ancona (Italy) Oroian Ioan: USAMV Cluj, Cluj-Napoca (Romania) Papadopol Nicolae: Natural Sciences Museum Complex, Constanta (Romania) Parvulescu Lucian: West University of Timisoara (Romania) Pasarin Benone: USAMV Iasi, Iasi (Romania) Petrescu-Mag Ruxandra Malina: Babes-Bolyai University, Cluj-Napoca (Romania) Petrovici Milca: West University of Timisoara (Romania) Rhyne Andrew: Roger Williams University; New England Aquarium, Boston (USA) Sima Nicusor Flaviu: USAMV Cluj, Cluj-Napoca (Romania) Tlusty Michael F.: New England Aquarium, Boston (USA) Vesa Stefan Cristian: Iuliu Hatieganu UMF, Cluj-Napoca (Romania) Wittenrich Matthew L.: University of Florida (USA)
Editorial councellor: Velter Victor: UEFISCDI, Bucharest (Romania)
Scientific Reviewers: Abdullah Khalid: Agricultural Research Institute Ratta Kulachi (Pakistan) Adewolu Morenike A.: Lagos State University, Ojo (Nigeria) Aftabuddin Sheikh: University of Chittagong (Bangladesh) Al-Qutob Mutaz: Al-Quds University (Palestinian Authority) Alves-Vianna Rafael: Federal University of Vicosa (Brazil) Antone Veronica: Natural Sciences Museum Complex, Constanta (Romania) Banaduc Doru Stelian: Lucian Blaga University of Sibiu (Romania) Banatean-Dunea Ioan: USAMVB Timisoara (Romania) Berkesy Corina: ICPE Bistrita (Romania)
ii Boaru Anca: USAMV Cluj, Cluj-Napoca (Romania) Brunio Erwin O.: Tokyo University of Marine Science & Technology (Japan) Calado Ricardo J. G.: University of Aveiro, Aveiro (Portugal) Celik Meryem Yesim: University of Sinop (Turkey) Chiorean Adriana: Natural Sciences Museum Complex, Constanta (Romania) Cocan Daniel: USAMV Cluj, Cluj-Napoca (Romania) Cosier Viorica: USAMV Cluj, Cluj-Napoca (Romania) Costa Daniel Ribeiro: Federal Rural University of Amazon, Belem (Brazil) Costea Ramona Ioana: "Stefan cel Mare" University of Suceava, (Romania) Cotutiu Mihaela Ioana: Technical College INFOEL, Bistrita (Romania) Covaciu-Marcov Severus D.: University of Oradea (Romania) Covrig Ilie: USAMV Cluj, Cluj-Napoca (Romania) Csep Laszlo: Bioflux, Cluj-Napoca (Romania) Curlisca Angelica: Natural Sciences Museum Complex, Constanta (Romania) Dediu Lorena: Dunarea de Jos University of Galati (Romania) Docan Angela: Dunarea de Jos University of Galati (Romania) Dragan Alina-Aida - Technical University, Cluj-Napoca (Romania) Galca Valerica: USAMV Iasi, Iasi (Romania) Gavrilovic Ana: University of Dubrovnik, Dubrovnik (Croatia) Gorgan Lucian Dragos: UAIC Iasi, Iasi (Romania) Hajirezaee Saeed: University of Teheran, Karaj (Iran) Hoha Gabriel: USAMV Iasi, Iasi (Romania) Kabir Milad: Gorgan University of Agricultural Sciences and Nat. Res. (Iran) Karayucel Ismihan: University of Sinop, Sinop (Turkey) Kosco Jan: Presov University, Presov (Slovakia) Luangpirom Ampa: Khon Kaen University, Khon Kaen (Thailand) Malos Cristian: Babes-Bolyai University, Cluj-Napoca (Romania) Manko Peter: Presov University, Presov (Slovakia) Monwar Mohammad M.: University of Chittagong (Bangladesh) Muntean Octavian Liviu: UBB Cluj-Napoca (Romania) Munteanu Florin: Natural Sciences Museum Complex, Constanta (Romania) Murray Joanna: CEFAS, Lowestoft (England) Musuka Confred G.: The Copperbelt University, Kitwe (Zambia) Ndimele Prince Emeka: Lagos State University, Ojo, Lagos (Nigeria) Nita Victor: Nat. Inst. Marine Research & Dev. Gr. Antipa, Constanta (Romania) Odagiu Antonia: USAMV Cluj, Cluj-Napoca (Romania) Oprea Lucian: Dunarea de Jos University, Galati (Romania) Oroian Teofil: USAMV Cluj, Cluj-Napoca (Romania) Pacioglu Octavian: Roehampton University, London (England) Palm Harry: University of Rostock, Rostock (Germany) Parsaeimehr Ali: National Academy of Science, Yerevan (Republic of Armenia) Perdikaris Costas: Technological Educational Institute of Epirus (Greece) Peteiro Cesar: Spanish Institute of Oceanography (IEO), Santander (Spain) Petrescu Dacinia Crina: Babes-Bolyai University, Cluj-Napoca (Romania) Popescu Irinel Eugen: UAIC Iasi, Iasi (Romania) Rahman Mohammed Mahabubur: Kochi University, Kochi (Japan) Rahmati-holasoo Hooman: University of Tehran, Tehran (Iran) Rosioru Corina: UBB Cluj-Napoca (Romania) Rus Vasile: USAMV Cluj, Cluj-Napoca (Romania) Sakkaravarthi Karuppiah: Annamalai University, Tamil Nadu (India) Sas Istvan: University of Oradea, Oradea (Romania) Suteu Mihai: Independent researcher, Cluj-Napoca (Romania) Taati M. Mehdi: Gorgan University of Agricultural Sciences and Nat. Res. (Iran) Talu Stefan: Technical University of Cluj (Romania) Turcu Mihaela Claudia: University of Turku, Turku (Finland) Udoh James P.: University of Uyo, Uyo (Nigeria) Vintila Iuliana: "Dunarea de Jos" University Galati (Romania) Zamfirescu Stefan: UAIC Iasi, Iasi (Romania)
Contact Publisher SC Bioflux SRL, 54 Ceahlău Street, Cluj-Napoca, 400488, Romania, European Union. Ioan Valentin Petrescu-Mag, e-mail: [email protected] Alternate Journal Contact: Ionel-Claudiu Gavriloaie, e-mail: [email protected]
Journal Secretary: Michał Nowak, Department of Ichthyobiology and Fisheries, University of Agriculture in Kraków, ul. T. Spiczakowa 6, 30-199 Kraków, Poland, European Union, e-mail: [email protected]
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vi AACL BIOFLUX Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society
Cadmium, lead and zinc contamination on the oyster Crassostrea gigas muscle harvested from the estuary of Lamnyong River, Banda Aceh City, Indonesia 1Muhammad A. Sarong, 1Cut Jihan, 2Zainal A. Muchlisin, 3Nur Fadli, 4Sugianto Sugianto
1 Department of Biology, Faculty of Teaching Training and Education, Syiah Kuala University, Banda Aceh, Indonesia; 2 Department of Aquaculture, Faculty of Marine and Fisheries, Syiah Kuala University, Indonesia; 3 Department of Marine Sciences, Faculty of Marine and Fisheries, Syiah Kuala University, Indonesia; 4 Department of Soil Science, Faculty of Agriculture, Syiah Kuala University, Indonesia. Corresponding author: M. A. Sarong, [email protected]
Abstract. The aim of the present study was to examine the heavy metals lead (Pb), cadmium (Cd), and zinc (Zn) in the oysters (Crassoscrea sp.) muscle harvested from the estuary of Lamnyong River, Banda Aceh City, Indonesia. The samplings were conducted two times (in May 2013 and March 2014) at four sampling locations. The samples were analyzed for Atomic Absorption Spectrometry (AAS). The study revealed that Pb, Cd and Zn were detected in the oysters. The concentration of Zn increased from 3.778 ppm to 11.567 ppm on May 2013 and March 2014, respectively; the concentration of Pb was undetected to 0.017 ppm on May 2013 and March 2014, respectively; while the concentration of Cd was decreased from 0.152 ppm to 0.015 ppm in the same period. The concentration of Pb was highest in station IV (0.029 ppm), Cd in station I (0.093 ppm), and Zn in stations II and III (8.069 ppm and 8.030, respectively). It was concluded that the concentrations of lead, cadmium and zinc have exceeded the maximum limit for aquatic organism and therefore the oysters from this estuary area were not safe to be consumed. Key Words: heavy metals, mollusk, domestic disposal, water quality, fertilizer, pesticide.
Introduction. Lamnyong river is situated in the sub district of Syiah Kuala, Banda Aceh, Indonesia. This river has an important role for the local people by providing water resources for domestic needs and livelihoods. Field observations indicate that there is a variety of anthropogenic activities along the river of Lamnyong, for example agriculture, fishing port, traditional market and workshops. These anthropogenic activities are alleged to produce waste that can contaminate and give a negative impact on the aquatic organisms especially on the mollusks settling at the bottom of the river. One of the possible pollutants produced from anthropogenic activities is heavy metals. Heavy metals accumulate in aquatic organisms, especially in a filter feeders organism like oysters and then contaminate humans through the food chain (Amriani et al 2011). Oysters (Crassostrea gigas) are one of the groups of mollusk which have high economic values. These aquatic animals live by attaching themselves to mangrove roots, rocks, dock poles and other objects in waters. Oysters are very susceptible to be contaminated by heavy metals due to their filter feeder habits (Bouilly et al 2006). Accordingly, this study examined three types of heavy metals, namely: lead (Pb), cadmium (Cd) and zinc (Zn) in oysters (C. gigas) harvested from the estuary of Lamnyong river. It was presumed that these heavy metals have a higher potential for polluting the Lamnyong river due to the agricultural activities, fisheries and workshops located along the watershed.
AACL Bioflux, 2015, Volume 8, Issue 1. 1 http://www.bioflux.com.ro/aacl Lead and cadmium are non-essential metals; these heavy metals are toxic for human even at low concentration, while zinc is an essential heavy metal needed by living organisms at low concentration, thus is known toxic at high level concentration for animals and humans as well (Darmono 1999). Lead causes anemia, impaired renal function, nervous system, and brain and skin disorders (Palar 2008). Furthermore, cadmium is risky for blood vessel pressure. When cadmium enters the body, it is then most likely to accumulate in the kidneys, liver and partially released through the digestive tract (Mifbakhuddin et al 2010), while zinc will cause health problems, particularly diarrhea at a particular level (Effendi 2003). Studies on the heavy metal contaminations on the aquatic organisms in Aceh Province, Indonesia, are very crucial because of its rapid growth of population and industries that threaten the environment. However, unfortunately, very limited information on heavy metal contamination of the aquatic organisms is available, except a study by Sarong et al (2013) on cadmium contamination on fish at Keureto river. To date, no study has been conducted on the heavy metal contaminations especially of lead, cadmium and zinc on the oyster tissue harvested from the estuary of Lamnyong river. Cadmium, lead and zinc are often used as an indicator of heavy metal pollution in waters caused by human activities (Ravanelli et al 1997; Simbolon et al 2014). This study is crucial because most of the oysters traded in the local market come from this area. Hence, the objective of the present study was to examine the lead, cadmium and zinc concentration on the oysters harvested from several locations within the estuary area of Lamnyong river, Banda Aceh, Indonesia.
Material and Method
Site and time. The study was conducted at four sampling sites within the estuary area of Lamnyong river, Banda Aceh, Indonesia. The characteristics of every sampling site are as follow: station I is a tributary of Lamnyong river near Simpang Mesra (95o35’6” E, 5o57’2” N) with residential areas, fish markets and residential sewages; station II is the Lamnyong river watershed close to Krueng Cut bridge (95o35’6” E, 5o57’9” N); station III is situated at Tibang village, which has mangrove green belts and fish pond areas (95o35’0” E, 5o58’7” N); station IV represents an area of the Lamnyong river mouth (Alue Naga) and fishing ports (95o34’8” E, 5o60’5” N) (Figure 1).
Figure 1. The map of Syiah Kuala sub district, Banda Aceh city, showing sampling sites.
AACL Bioflux, 2015, Volume 8, Issue 1. 2 http://www.bioflux.com.ro/aacl Sampling and sampling preparation. Samplings were done two times, the first one in May 2013 and the second one in March 2014. Three plots were determined randomly at left-right and the center of the river. The plot size was 1m x 1m and only the oysters with size larger than 2 cm were collected at the first sampling in May 2013, and 3 cm minimum oysters were collected at the second sampling in March 2014. The tissues of oysters were removed from the shell, preserved in the crushed ice (4oC) and then transported to the laboratory in the Faculty of Sciences, Syiah Kuala University for an analysis of lead, cadmium and zinc. The samples were prepared based on a standard manual for Spectrophotometry analysis proposed by AOAC (1999) and Indonesian National Standard (SNI 06-6989.7). The main physical (temperature, water current and depth) and chemical (pH, dissolved oxygen, salinity) water quality parameters were measured when the oysters were sampled.
Heavy metal analysis. The oyster muscle was used for lead, cadmium and zinc analysis by using a standard procedure of the Atomic Absorption Spectrophotometry (AOAC 1999; Petrovici & Pacioglu 2010; Grd et al 2012).
Results and Discussion. The study revealed that cadmium and zinc were found in May 2013, but lead was not detected in the oyster mussel at that time. However, these heavy metals (cadmium, zinc and lead) were detected in March 2014. The results showed that lead and zinc levels on the oyster muscle increased during the last two years of 2013 and 2014. For example, lead has increased from 0 ppm to 0.019 ppm, while zinc increased from 3.778 ppm to 11.567 pm in respective years, but cadmium decreased from 0.152 ppm in 2013 to 0.015 ppm in 2014 (Table 1). Based on the sampling locations, lead was highest at station IV, cadmium at station I and zinc at stations II and III (Table 2). It indicates that all locations have been polluted by these heavy metals higher than the limit levels.
Table 1 Average concentration of lead, cadmium and zinc in the oysters (Crassostrea gigas) harvested from the estuary of Lamnyong river, Banda Aceh, Indonesia, in May 2013 and March 2014
Heavy metal May 2013 March 2014 Lead (ppm) 0.000 0.019 Cadmium (ppm) 0.152 0.015 Zinc (ppm) 3.778 11.567
Table 2 The average levels and standard deviation (SD) of lead, cadmium and zinc on oyster muscle (Crassostrea gigas) from two sampling times in May 2013 and March 2014 according to the sampling sites
Heavy metals (ppm) Sampling station Lead Cadmium Zinc I 0.004±0.009 0.093±0.059 7.122±1.577 II 0.003±0.006 0.081±0.099 8.069±5.744 III 0.004±0.009 0.074±0.073 8.030±4.877 IV 0.029±0.039 0.086±0.083 7.470±5.099
Heavy metal contaminants often occur in open waters due to industrial activities, agricultural, horticultural and domestic wastes (Rahman et al 2010; Petrescu-Mag et al 2010; Al-Baggou et al 2011; Mohammed et al 2011). Therefore, we speculate that the heavy metals of lead, cadmium and zinc sources mostly came from workshops (vehicles and welding), agricultural activities (pesticide and fertilizer) and household waste disposals located along the Lamnyong river. This is in agreement to Liu et al (2014) that lead is commonly used in paint and canning industries, gildings and pesticides. While
AACL Bioflux, 2015, Volume 8, Issue 1. 3 http://www.bioflux.com.ro/aacl cadmium probably came from domestic disposals and oil spills from fishermen boats (Hidayat & Novita 2012). According to Huamain et al (1999), fertilizers and pesticides contribute significantly to soil fertility but are also major sources of pollution to aquatic organisms. Similarly, Cyrille et al (2012) reported that the source of cadmium mostly comes from industrial and domestic wastes, fertilizers and pesticides used in plantations. According to Amin (2002), the source of zinc in the waters comes from settlement disposals which entered the waters without being treated. Besides domestic pollutants, the intensive agricultural activities were also a potential source of zinc. The concentration of heavy metals in the waters probably changes by seasons, where the high level would occur during the dry season (i.e. March 2014 was in the dry season in Banda Aceh). Similarly, Fisher et al (2000) reported that the heavy metals concentration on Crassostrea virginica muscles in Tampa Bay, Florida was higher during dried seasons because the concentration of heavy metals on the water was higher by inspissation due to the decline of water volume. In addition, Edward (2014) reported that the concentration of heavy metals was higher in the sediment compared to in the waters as recorded in Wawobatu Bay Waters, Southeast of Sulawesi. In general, the concentration of the heavy metals, in particular lead and cadmium, was higher in Station IV. This location is the mouth of Lamnyong river which connects directly to the sea. A similar finding was reported by Lauenstein et al (2002) that heavy metals accumulation in the oyster (C. virginica) occurred highest in an estuary area of a polluted river in the waters of Carolina, USA. Besides toxic to human, the heavy metals also give a negative impact to aquatic biodiversity. For example, Samman et al (2014) found that there was a strong relationship between mercury concentration and the density of popaco snail (Telescopium telescopium) in Kao Bay, North Halmahera, where the density of snail was lower when the concentration of mercury was higher. According to the Indonesian Government Law No. 82 Year 2011, the limit of cadmium for fisheries purposes is 0.01 ppm, while based on the Indonesian Ministry of Environment Year 2004, the maximum standard of lead, cadmium and zinc on aquatic organisms is 0.008 ppm, 0.001 ppm and 0.05 ppm, respectively. Therefore, the oysters in the estuary of Lamnyong River have been contaminated by the heavy metals of lead, cadmium and zinc in which the concentration of these heavy metals have exceeded the permitted limit. Therefore, the oysters in the estuary of Lamnyong River are not safe for human consumption.
Conclusions. The concentration of lead, cadmium and zinc on the oysters (Crassostrea gigas) harvested from the estuary of Lamnyong river increased from May 2013 to March 2014 in which the concentration of these heavy metals has exceeded the maximum limit. Therefore, it is concluded that the oysters from this estuary have been contaminated by lead, cadmium and zinc and are not safe to be consumed by humans.
Acknowledgements. We express appreciate to Dr. Yunisrina Qismullah Yusuf for proof reading of the manuscript. The technical assistance by Badan Percepatan Publikasi Internasional, Universitas Syiah Kuala and all members of this task force are also acknowledged.
References
Al-Baggou B. K., Naser A. S., Mohammad F. K., 2011 Hydrogen peroxide potentiates organophosphate toxicosis in chicks. HVM Bioflux 3(2):142-149. Amin B., 2002 [Distribution of heavy metals of Pb, Cu and Zn on the sediment of Telaga Tujuh Karimun waters, Riau Isle]. Jurnal Natur Indonesia 5(1):9-16 [in Indonesian]. Amriani, Hendrarto B., Hadiyarto A., 2011 [Bio-accumulation of heavy metals of lead (Pb) and zinc (Zn) on the blood mussel, Anadara granosa L. and mangrove mussel, Polymesoda bengalensis L. at Kendari Bay]. Jurnal Ilmu Lingkungan 9(2):45-50 [in Indonesian].
AACL Bioflux, 2015, Volume 8, Issue 1. 4 http://www.bioflux.com.ro/aacl AOAC., 1999 AOAC official methods 199.10. Association of Official Analytical Chemists. Maryland, USA, pp. 40-50. Bouilly K., Gagnaire B., Bonnard M., Thomas-Guyon H., Renault T., Miramand P., Lapegue S., 2006 Effect of cadmium on aneuploidy and hemocyte parameter in the Pacific oyster, Crassostrea gigas. Aquatic Toxicology 78:149-156. Cyrille Y. D. A., Victor K., Sanogo T. A., Boukary S., Joseph W., 2012 Cadmium accumulation in tissue of Sarotherodon melanotheron (Ruppel, 1852) from the Aby Lagoon system in Cote d’Ivoire. International Journal of Environmental Research and Public Health 9:821-830. Darmono, 1999 [Cadmium (Cd) in the environment and its effect on the animal health and productivity]. Wartazoa 8(1):28-32 [in Indonesian]. Edward, 2014 [Heavy metals content in sediment in Wawobatu Bay Waters, Kendari, Southeast of Sulawesi]. Depik 3(2):157-165 [in Indonesian]. Effendi H., 2003 [Study on the water quality for water resource and environmental management]. Yogyakarta: Kanisius, 258 pp. [in Indonesian]. Fisher W. S, Oliver L. M, Winstead J. T., Long E. R., 2000 A survey of oyster Crassostrea virginica from Tampa Bay, Florida: associations of internal defense measurement with contaminant burdens. Aquatic Toxicology 51:115-138. Grd D., Dobsa J., Simunic-Meznaric V., Tompic T., 2012 Analysis of heavy metals concentration in wastewater along highways in Croatia. Journal of Computing and Information Technology 20(3):209–215. Hidayat D., Novita N. P. I., 2012 [Distribution of heavy metal Cd in sediment of Way Kuala Estuaria Bandar Lampung]. Molekul 7(1):82-88 [in Indonesian]. Huamain C., Zhang C., Tu C., Zhu Y., 1999 Heavy metal pollution in soils in China: status and countermeasures. Ambio 28(2):130-134. Indonesian Government Law No. 82, Year 2011 Indonesian national standard for heavy metals of lead (Pb), cadmium (Cd) for fisheries product. SNI 2354.5:2011. Badan Standardisasi Nasional: Jakarta. Indonesian Ministry of Environment Law, 2004 Indonesian national standard for heavy metals of zinc (Zn) for fisheries product. SNI 06-6989.7. Badan Standardisasi Nasional: Jakarta. Lauenstein G. G., Cantillo A. Y., O’Conner T. P., 2002 The status and trends of trace element and organic contaminant in oysters, Crassostrea virginica, in the waters of the Carolina, USA. The Science of the Total Environmental 285:79-87. Liu G., Yu Y., Hou J., Xue W., Liu X., Liu Y., Wang W., Alsaedi A., Hayat T., Liu Z., 2014 An ecological risk assessment of heavy metal pollution of the agricultural ecosystem near a lead-acid battery factory. Ecological Indicator 47:210-218. Mifbakhuddin, Astuti R., Awaludin A., 2010 [Effect of vinegar submersion on the cadmium concentration on muscle of green mussel, Perna viridis]. Jurnal Kesehatan 3(1):14- 20 [in Indonesian]. Mohammed E. H., Wang G., Xu Z., Liu Z., Wu L., 2011 Physiological response of the intertidal copepod Tigriopus japonicus experimentally exposed to cadmium. AACL Bioflux 4(1):99-107. Palar H., 2008 [Contamination and toxicology of heavy metals]. Rineka Cipta, Jakarta, 152 pp. [in Indonesian]. Petrescu-Mag I. V., Păsărin B., Todoran C. F., 2010 Metallurgical, agricultural and other industrial related chemical pollutants: biomonitoring and best model organisms used. Metalurgia International 15:38-48. Petrovici M., Pacioglu O., 2010 Heavy metal concentrations in two species of fish from the Crişul Negru River, Romania. AACL Bioflux 3(1):51-60. Rahman M. M., Rahman M. M., Chongling Y., Islam K. S., 2010 Changes in growth and antioxidant enzymes activities during cadmium stress in the mangrove plant Kandelia candel (L.) Druce. AES Bioflux 2(1):15-24. Ravanelli M., Tubertini O., Valcher S., Martinotti W., 1997 Heavy metal distribution in sediment cores from western Ross sea (Antactica). Water, Air and Soil Pollution 99:697-704.
AACL Bioflux, 2015, Volume 8, Issue 1. 5 http://www.bioflux.com.ro/aacl Samman A., Lumbanbatu D. T. F., Setyobudiandi I., 2014 [Mercury concentrations and its relationship to the density index of popaco snail (Telecopium telescopium) in Kao Bay, North Halmahera]. Depik 3(2):128-136 [in Indonesian]. Sarong M. A., Mawardi A. L., Adlim M., Muchlisin Z. A., 2013 Cadmium concentration in three species of freshwater fishes from Keuretoe River, Northern Aceh, Indonesia. AACL Bioflux 6(5):486-491. Simbolon A. R., Riani E., Wardiatno Y., 2014 Status pollution and heavy metal content on scallop (Placuna placenta) in Tangerang coastal waters. Depik 3(2):91-98 [in Indonesian].
Received: 22 November 2014. Accepted: 27 December 2014. Published online: 02 January 2015. Authors: Muhammad Ali Sarong, Department of Biology, Faculty of Teaching Training and Education, Syiah Kuala University, Banda Aceh 23111, Indonesia, e-mail: [email protected] Cut Jihan, Department of Biology, Faculty of Teaching Training and Education, Syiah Kuala University, Banda Aceh 23111, Indonesia, e-mail: [email protected] Zainal A. Muchlisin, Department of Aquaculture, Faculty of Marine and Fisheries, Syiah Kuala University, Banda Aceh 23111, Indonesia, e-mail: [email protected]. Nur Fadli, Department of Marine Sciences, Faculty of Marine and Fisheries, Syiah Kuala University, Banda Aceh 23111, Indonesia, e-mail: [email protected] Sugianto Sugianto, Department of Soil Science, Faculty of Agriculture, Syiah Kuala University, Banda Aceh 23111, Indonesia, e-mail: [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. How to cite this article: Sarong M. A., Jihan C., Muchlisin Z. A., Fadli N., Sugianto S., 2015 Cadmium, lead and zinc contamination on the oyster Crassostrea gigas muscle harvested from the estuary of Lamnyong River, Banda Aceh City, Indonesia. AACL Bioflux 8(1):1-6.
AACL Bioflux, 2015, Volume 8, Issue 1. 6 http://www.bioflux.com.ro/aacl AACL BIOFLUX Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society
Fluctuating asymmetry of parasite infested and non-infested Sardinella sp. from Misamis Oriental, Philippines Rowina G. Pojas, Sharon R. M. Tabugo
Department of Biological Sciences, Mindanao State University – Iligan Institute of Technology, Iligan City, Philippines. Corresponding author: S. R. M. Tabugo, [email protected]
Abstract. Fluctuating asymmetry (FA) is defined as subtle, random deviation from perfect symmetry and is the most commonly used estimate of developmental stability. It has been found to increase with the presence of stressors including parasite infestation. In this study, potential relationships were investigated between the presence of parasite and its effect to FA as a widely employed measure of developmental stability on Sardinella sp. FA levels as asserted in many studies increases with developmental instability. Sardinella sp. from Misamis Oriental were collected and assessed for the presence of parasites in the gills. Fluctuating asymmetry in the traits were analyzed using landmark method for shape asymmetry, via Symmetry and Asymmetry in Geometric Data (SAGE) program. A total of twelve landmark points were used and a total of 200 fishes were evaluated. Procrustes ANOVA showed insignificant levels of FA for Sardinella sp. found without parasite while those found with parasites showed significant levels of FA. Thus, in consistency with other studies, that the presence of parasites may affect the developmental stability of an organism as seen in the asymmetry measurement of the left and right sides of the organism, which implies the extent to which the organism is able to buffer any disturbance. Hypothesis assumes that parasites can cause an increased level of FA due to the stress it induced in the development of the organism. An increase level of FA has implications on species fitness and adaptation. Presence of parasites has negative impact on host fitness. Key Words: Adaptation, development of the organism, species fitness, developmental stability.
Introduction. Developmental stability is defined as the ability of an organism to buffer its development against genetic or environmental disturbances encountered during development to produce the genetically predetermined phenotype (Waddington 1942) and, as such, it is a fundamental characteristic of development. The most commonly used estimate of developmental stability has been the fluctuating asymmetry (FA). The underlying assumption of this measure is that the development of both sides of a bilaterally symmetric organism is influenced by identical genes, and thus non-directional differences between sides must be environmental in origin and reflect accidents occurring during development (Waddington 1942; Moller & Swaddle 1997). FA refers to a pattern of bilateral variation in a sample of individuals, where the mean of right minus left values of a trait is zero and the variation is normally distributed about that mean (Palmer 1994). Typically, one or more indexes are calculated that express FA as a variance, or an average absolute value, of the difference between the right and left elements of a bilateral pair for a sample of individuals (Palmer & Strobeck 1986). A highly significant FA (high FA value) indicates a lower developmental stability. It is usually assumed that elevated levels of FA are the result of environmental and/or genetic stress experienced by the organism during ontogeny which perturbs the normal developmental program (Valen 1962; Palmer & Strobeck 1986; Palmer 1994; Markow 1994). FA variance of populations and absolute FA values of individuals has been found to increase with stress (Leung & Forbes 1996; Graham et al 1993). Both genomic and environmental changes can increase FA which represents a possible deterioration in developmental homeostasis apparent in adult morphology. Noteworthy, is that genetic perturbations include intense directional selection and certain specific genes. While,
AACL Bioflux, 2015, Volume 8, Issue 1. 7 http://www.bioflux.com.ro/aacl environmental perturbations leading to elevated levels of FA have been associated with environmental variables such as temperature extremes, protein deprivation, audiogenic stress, pollution, parasites etc. (Palmer & Strobeck 1986; Mpho et al 2000). As such, it has been proposed as a general tool for biomonitoring stress within populations regardless of the nature of stress. FA has been used as a bioindicator in a broad array of applications. Recent studies on FA have focused on naturally selected traits and how asymmetry is associated with environmental stress (Siikamaki & Lammi 1998; Imasheva et al 1999; Reimchen & Nosil 2001). For example, fish from polluted or otherwise altered waters exhibit higher levels of asymmetry in gill rakers and fin rays than those inhabiting nonpolluted or natural habitats (Ostbye et al 1997; Campbell et al 1998), logging and consequent habitat alteration leads to increased mandibular asymmetry in shrews (Badyaev et al 2000), and food deprivation in the laboratory is associated with higher levels of asymmetry in feather growth in some birds (Swaddle & Witter 1994). Moreover, another potential source of environmental stress is parasitism by macroparasites and pathogens. Several studies have demonstrated a positive correlation between levels of parasitism and extent of asymmetry in various traits (Moller 1996), and these studies are consistent with the widespread assumption drawn from other work that parasitism does affect FA levels and can be detrimental to host individuals (Martin & Hosken 2009). In this study, potential relationships were investigated between the presence of parasite and its effect to FA as a widely employed measure of developmental stability on Sardinella sp. FA levels as asserted in many studies increases with developmental instability. Sardinella sp. was considered of economic value because of its high nutritional content as they are rich in vitamins and minerals. They are also a natural source of marine omega-3 fatty acids, which reduce the occurrence of cardiovascular disease, and good source of vitamin D, calcium, vitamin B12, and protein. This species is commercially harvested as an important food source in Iligan City, Misamis Oriental and nearby localities (Mahrus et al 2012). Also much interest has been devoted to the determination and examination of FA as an indicator of individual quality and fitness. Here, a hypothesis assumes that parasites can cause an increased level of FA in the organism due to the stress it induced in the development of the organism (Graham et al 1993; Martin & Hosken 2009). Hence, this study generally aims to test if the presence of parasites (e.g. in the gills) may affect developmental stability of Sardinella sp. through the use of fluctuating asymmetry as an estimate of developmental stability. Specifically, check the presence of parasites in Sardinella sp. samples from Misamis Oriental and investigate if there is any significant difference in FA measurement between infested and non-infested samples of Sardinella sp.
Material and Method. Samples of Sardinella sp. come from Misamis Oriental. Two- dimensional images of the sample were taken. The samples were then thoroughly checked in their gills for the presence of any parasite. Gills of the samples were scraped and examined using conventional microscopy. A total of 200 fishes were evaluated. A total of 12 landmarks were digitized using the tpsDig software. The location of the landmarks and the anatomical descriptions of each are presented in Figure 1 and Table 1. Overall and localized fluctuating asymmetries were determined by subjecting the paired landmark coordinates to Procrustes ANOVA following the method of Klingenberg et al 1998 and using Symmetry and Asymmetry in Geometric Data (SAGE) software.
Figure 1. Location of the 12 landmark points on Sardinella sp.
AACL Bioflux, 2015, Volume 8, Issue 1. 8 http://www.bioflux.com.ro/aacl Table 1 Position of the twelve landmarks selected in Sardinella sp.
Position Landmark # Anatomical landmarks 1 Lower anterior of the premaxilla 2 Lower posterior of the premaxilla 3 Upper posterior of the premaxilla 4 Upper anterior of the premaxilla 5 Center of eye 6 Anterior margin through the midline of the eye 7 Posterior margin through the midline of the eye 8 Superior margin of the eye 9 Inferior margin through the midline of the eye 10 Dorso-lateral angle of the operculum 11 Posterior margin of the operculum 12 Isthmus
FA levels were assessed using the “Symmetry and Asymmetry in Geometric Data” (SAGE) program, version 1.0. The software analyzed the x and y coordinates of the landmarks per individual, using a configuration protocol for both lateral sides of the head of Sardinella sp. Procrustes superimposition analysis was performed with the original and mirrored configurations of the right and left lateral sides simultaneously. The least squares Procrustes consensus of set of landmark configurations and their relabelled mirror images is a perfectly symmetrical shape, while FA is the deviation from perfect bilateral symmetry (Marquez 2006; Klingenberg et al 1998). The squared average of Procrustes distances for all specimens is the individual contribution to the FA component of variation within a sample. To detect the components of variances and deviations, a Procrustes ANOVA was used. Sides (directional asymmetry; DA), Individual x sides (fluctuating asymmetry; FA), and their respective error were included as effects. The ANOVA used most frequently for fluctuating asymmetry is a two-way, mixed-model ANOVA with replication. The main fixed effect is sides (S), which has two levels (left and right). The block effect is individuals (I), which is a random sample of individuals from a population. The sides by individuals interaction (S x I) is a mixed effect. Finally, an error term (m) represents measurement error (replications within sides by individuals). The effect called sides is the variation between the two sides; it is a measure of directional asymmetry. The effect called individuals is the variation among individual genotypes; the individuals mean square is a measure of total phenotypic variation and it is random. Meanwhile, the individual by sides interaction is the failure of the effect of individuals to be the same from side to side. It is a measure of fluctuating asymmetry and antisymmetry thus, a mixed effect. The error term is the measurement, and is a random effect. Only Individual x Sides interaction denotes fluctuating asymmetry (FA) (Samuels et al 1991; Palmer & Strobeck 1986, 2003; Carpentero & Tabugo 2014). Moreover, to detect the components of variances and deviations, Principal Component Analysis (PCA) of the covariance matrix associated with the component of FA variation were also performed for the samples to carry out an interpolation based on a thin-plate spline and then visualize shape changes as landmark displacement in the deformation grid (Albarran-Lara et al 2010; Marquez 2006).
Results and Discussion. An underlying hypothesis of FA analysis is that bilaterally symmetrical traits should be in principle identical on either side of the body since they are said to be governed by the same genes thus, non-directional differences between the sides must be environmental in origin and reflect accidents occurring during development (Palmer 1994; Valen 1962; Gangestad & Thornhill 1999; Martin & Hosken 2009). However, deviations from perfect asymmetry were described as common and thought to convey information about developmental stability of individuals and populations.
AACL Bioflux, 2015, Volume 8, Issue 1. 9 http://www.bioflux.com.ro/aacl Developmental stability is defined as the ability of a genotype to resist developmental perturbations and its common estimate is through FA although, other types of symmetry may also convey to some extent information about developmental stability. As such, there is a direct relationship between FA and developmental instability and FA was thought to reflect an organism’s ability to cope with genetic and environmental stress during development and considered to reflect a population’s average state of adaptation, coadaptation, fitness and individual quality (Graham et al 2010; Parsons 1990). Moreover, it is thought to increase under both environmental and genetic stress (Graham et al 2010). A potential source of environmental stress is parasitism by macroparasites and pathogens and this have been demonstrated through several studies which showed a positive correlation between levels of parasitism and extent of asymmetry in various traits (Moller 1996). Such studies were consistent with the widespread assumption that parasitism does affect FA levels and can be detrimental to host individuals (Martin & Hosken 2009). FA of the right and left lateral sides of the head of Sardinella sp. were assessed through Procrustes method using SAGE software. It was investigated whether there was any significant difference in FA measurement between infested and non- infested samples of Sardinella sp. Index of FA using the coordinates was determined and final result of the Procrustes ANOVA is shown in Table 2. It was noted that the individual by sides interaction is the failure of the effect of individuals to be the same from side to side. It is a measure of fluctuating asymmetry and antisymmetry thus, a mixed effect. The error term is the measurement, and is a random effect. Only Individual x Sides interaction denotes fluctuating asymmetry (FA) (Palmer & Strobeck 1986; Galbo & Tabugo 2014). Hereby, the interaction of ‘Side x Individuals’ showed a high value of mean square and a low value of mean square measurement error. Thus, the F value suggested highly significant FA for all parasite infested samples of Sardinella sp. from Misamis Oriental where *P<0.001. The results of the Procrustes ANOVA indicated a random variation (FA) between the right and left lateral sides of the head of Sardinella sp. rather than non- random differences among sides. Results implied that presence of parasite affects fluctuating asymmetry in host species. This is consistent with other studies where parasitism has significant effect on the degree of fluctuating asymmetry on the host (Moller 1992; Polak 1993). The mechanisms by which parasites cause increased character asymmetry in general and for Sardinella sp. in particular are unknown and still subject to further investigations though there were underlying pre-conceived notions. But most likely, parasite-induced nutritional deprivation of various forms destabilizes host development and elevates levels of fluctuating asymmetry (Polak 1993, 1997). Parasitism can limit host nutrient availability by reducing host food intake, digestion, absorption and nutrient assimilation (Whitefield 1979; Thompson 1983; Polak 1997). Parasites may compete with their hosts for resources, and therefore impinge upon host metabolism, growth and development (Schall et al 1982; Goater et al 1993). Some experimental studies have shown that nutritional stress directly leads to elevated asymmetry (Sciulli et al 1979; Swaddle et al 1994; Imasheva et al 1999), although others have shown no such effect (Hovorka & Robertson 2000; Bjorksten et al 2000). In this case, there is high parasitic load in Sardinella sp. samples examined such that more likely parasitism lead to nutritional deprivation which in turn caused increased fluctuating asymmetry. Most of the commonly encountered fish parasites were protozoans (e.g. ciliates, flagellates, myxozoans, microsporidians, and coccidians). The costs of parasitism could result in increased physiological stress during development and thus, directly induce high levels of asymmetry. Supporting this idea were some experimental manipulations of parasite loads which provide evidence for a direct causal link between FA and parasites. However, FA and parasites may be associated for several other reasons (Polak 1997; Thomas et al 1998). The higher the FA (significant FA) it is more developmentally unstable the organism (Palmer 1994). Thus, FA can be used as an indicator of individual quality and adaptation thereby, also demonstrating the potential for FA as a biomarker of stress and developmental instability of populations. Moreover, according to Mpho et al (2000), the possible causes of developmental instability were well studied and include a wide range of
AACL Bioflux, 2015, Volume 8, Issue 1. 10 http://www.bioflux.com.ro/aacl environmental factors (e.g. deviant climatic conditions, food deficiency, parasitism, pesticides) and genetic factors (e.g. inbreeding, hybridization, novel mutants). Such factors may also increase stress to populations.
Table 2 Procrustes ANOVA results for samples Sardinella sp. with- and without parasites from Misamis Oriental
Effect SS DF MS F P Significance Sardinella sp. withouth parasites Sides 0 20 0 0 1 ns Individuals x 1.1604e-031 1780 6.5192e-035 9.5074e-032 1 ns Sides Measurement 2.4685 3600 0.0006857 - - - error Sardinella sp. with parasites Sides 0.013025 32 0.00040703 3.1328 2.018e-008 ****** Individuals x 0.12057 928 0.00012993 1.6483 0 ****** Sides Measurement 0.15234 1920 7.8825e-005 - - - error Side = directional asymmetry; individual x sides interaction = fluctuating asymmetry; * P<0.001 significant, ns – statistically insignificant (P>0.05); significance was tested with 99 permutations.
Another way to examine the variability of landmark points in tangent space is to run a principal component analysis (PCA) on the tangent coordinates derived from Procrustes analysis. First principal component depicts vectors at landmarks that show the magnitude and direction in which that landmark is displaced relative to the others. The second depicts the difference via the thin plate splines, an interpolation function that models change between landmarks from the data of changes in coordinates of landmarks. Here, the red dots represent the morphological landmarks used in the study while the blue arrows indicate the direction as well as the magnitude of the fluctuation. The percentage values of PCA represent the level of variability in the data (Marquez 2006). Based on the percentage of overall variation exhibited by PC1 and PC2, parasite infested samples exhibits higher variation compared to non infested samples. Thus, higher FA was also exhibited by parasite infested samples. Hence, the presence of parasites may contribute or increase variability (Table 3 & Figure 3).
Table 3 Variance explained by first two principal components between Sardinella sp. with- and without parasites from Misamis Oriental
Parasite +/- PC 1 (%) PC 2 (%) + 10.7 89.3 - 52.0 23.2
AACL Bioflux, 2015, Volume 8, Issue 1. 11 http://www.bioflux.com.ro/aacl
Figure 3. PCA implied deformation for individual x side interaction of fluctuating asymmetry of Sardinella sp. with- and without parasites.
Conclusions. Fluctuating asymmetry is defined as deviations from symmetry which may be caused by environmental stresses, developmental instability and genetic problems during development. It is thought that the more perfectly symmetrical an organism is, the better it has been able to handle developmental stress and has more developmental stability. Significant values for FA were observed in parasite infested Sardinella sp. suggesting deviations in its bilateral symmetry. Meanwhile, Sardinella sp. without parasite had insignificant value for FA. Hence, this study provides experimental evidence that parasites can affect fluctuating asymmetry in Sardinella sp. The results of the study demonstrates the potential of FA as a biomarker of stress and its efficacy in measuring developmental stability in Sardinella sp. Findings confirmed and correspond to a number of studies suggesting that fluctuating asymmetry is thus a useful tool in determining developmental stability.
Acknowledgements. The authors would like to thank the faculty of BRTCM, MSU-IIT, to their families for the unending moral and financial support.
References
Albarran-Lara A. L., Mendoza-Cuenca L., Valencia-Avalos S., Gonzalez-Rodriguez A., Oyama K., 2010 Leaf fluctuating asymmetry increases with hybridization and introgression between Quercus magnoliifolia and Quercus resinosa (Fagaceae) through an altitudinal gradient in Mexico. Int J Plant Sci 171:310-322. Badyaev A. V., Foresman K. R., Fernandes M. V., 2000 Stress and developmental stability: vegetation removal causes increased fluctuating asymmetry in shrews. Ecology 81:336–345. Bjorksten T., David P., Pomiankowski A., Fowler K., 2000 Fluctuating asymmetry of sexual and nonsexual traits in stalk-eyed flies: a poor indicator of developmental stress and genetic quality. J Evol Biol 13:89–97.
AACL Bioflux, 2015, Volume 8, Issue 1. 12 http://www.bioflux.com.ro/aacl Campbell W. B., Emlen J. M., Hershberger W. K., 1998 Thermally induced chronic developmental stress in coho salmon: integrating measures of mortality, early growth, and developmental instability. Oikos 81:398–410. Carpentero E. R., Tabugo S. R. M., 2014 Determining developmental instability via fluctuating asymmetry in the shell shape of Arctica islándica Linn. 1767 (ocean quahog). European Journal of Zoological Research 3(3):1-7. Galbo K. R., Tabugo S. R. M., 2014 Fluctuating asymmetry in the wings of Culex quinquefasciatus (Say) (Diptera: Culicidae) from selected barangays in Iligan City, Philippines. AACL Bioflux 7(5):357-364. Gangestad S. W., Thornhill R., 1999 Individual differences in developmental precision and fluctuating asymmetry: a model and its implications. J Evol Biol 12:402–416. Goater C. P., Raymond R. D., Bernasconi M. V., 1993 Effects of body size and parasite infection on the locomotory performance of juvenile toads. Bufo bufo. Oikos 66:129-136. Graham J. H., Raz S., Hagit H., Nevo E., 2010 Fluctuating Asymmetry: methods, theory and applications. Symmetry 2:466-495. Graham J. H., Freeman D. C., Emlen J. M., 1993 Developmental stability: A sensitive indicator of populations under stress. In: Environmental toxicology and risk assessment, ASTM STP 1179. Landis W. G., Hughes J. S., Lewis M. A. (eds), American Society for Testing Materials, Philadelphia, PA. Hovorka M. D., Robertson R. J., 2000 Food stress, nestling growth, and fluctuating asymmetry. Can J Zool 78:28–35. Imasheva A. G., Bosenko D. V., Bubli O. A., 1999 Variation in morphological traits of Drosophila melanogaster (fruit fly) under nutritional stress. Heredity 82:187–192. Klingenberg C. P., McIntyre G. S., Zaklan S. D., 1998 Left-right asymmetry of fly wings and the evolution of body axes. Proceedings of the Royal Society of London B, Biological Sciences 265:1255–1259. Leung B., Forbes M. R., 1996 Fluctuating asymmetry in relation to stress and fitness: effects of trait type as revealed by meta-analysis. Ecoscience 3:400–413. Mahrus, Sumitro S. B., Widodo N., Sartimbul A., 2012 The Association between Genetic Variations and Omega-3 Production on Sardinella lemuru in Lombok Strait. IOSR Journal of Agriculture and Veterinary Science 1(6):12-16. Markow T. A., (Ed) 1994 Developmental Instability: Its origins and evolutionary implications. Dordrecht, The Netherlands: Kluwer. Marquez E., 2006 Sage: symmetry and asymmetry in geometric data. Ver 1.0. http://www.personal.umich.edu/~emarquez/morph/ Martin O. Y., Hosken D. J., 2009 Longevity and Development Stability in the Dung Fly Sepsis cynipsea, as affected by the ectoparasite mite, Pediculoides mesembrinae. J Insect Sci 9(66):1-9. Moller A. P., 1996 Parasitism and developmental instability of hosts: a review. Oikos 77:189–196. Moller A. P., 1992 Parasites differentially increase the degree of fluctuating asymmetry in secondary sexual characters. J Evol Biol 5:691–699. Moller A. P., Swaddle J. P., 1997 Asymmetry, Developmental Stability, and Evolution. Oxford University Press, Oxford. Mpho M., Holloway G. J., Callaghan A., 2000 The effect of larval density on life history and wing asymmetry in the mosquito Culex pipiens. Bull Entomol Res 90:279-283. Ostbye K., Oxnevad S. A., Vollestad L. A., 1997 Developmental stability in perch (Perca fluviatilis) in acidic aluminum-rich lakes. Can J Zool 75:919–928. Palmer A. R., 1994 Fluctuating asymmetry analysis: a primer. In: Developmental Instability: Its Origins and Evolutionary Implications. Markow T. A. (ed), London: Kluwer Academic. Palmer A. R., Strobeck C., 2003 Fluctuating asymmetry analyses revisited. In: Developmental Instability: causes and consequences. Polak M. (ed), New York, USA: Oxford University Press. Palmer A. R., Strobeck C., 1986 Fluctuating asymmetry - measurement, analysis, patterns. Annu Rev Ecol Syst 17:391-421.
AACL Bioflux, 2015, Volume 8, Issue 1. 13 http://www.bioflux.com.ro/aacl Parsons P. A., 1990 Fluctuating asymmetry: an epigenetic measure of stress. Biol Rev Camb Philos Soc 65(2):131–145. Polak M., 1997 Parasites, fluctuating asymmetry, and sexual selection. In: Parasites and Pathogens: effects on host hormones and behavior. Beckage N. E. (ed), pp. 246– 276, New York: Chapman & Hall. Polak M., 1993 Parasites increase fluctuating asymmetry of male Drosophila nigrospiracula: implications for sexual selection. Genetica 89:255–265. Reimchen T. E., Nosil P., 2001 Lateral plate asymmetry, diet and parasitism in threespine stickleback. J Evol Biol 14:632–645. Samuels M. L., Casella G., McCabe G. P., 1991 Interpreting blocks and random factors: rejoiner. J Am Stat Assoc 86:798-808. Schall J. J., Bennet A. F., Putman P. W., 1982 Lizards infected with malaria: physiological and behavioral consequences. Science 217:1057-1059. Sciulli P. W., Doyle W. J., Kelley C., Siegel P., Siegel M. I., 1979 The interaction of stressors in the induction of increased levels of fluctuating asymmetry in the laboratory rat. Am J Phys Anthropol 50:279–284. Siikamaki P., Lammi A., 1998 Fluctuating asymmetry in central and marginal populations of Lychnis viscaria in relation to genetic and environmental factors. Evolution 52:1285–1292. Swaddle J. P., Witter M. S., 1994 Food, feathers and fluctuating asymmetries. Proc R Soc Lond B Biol Sci 255:147–152. Swaddle J. P., Witter M. S., Cuthill I. C., 1994 The analysis of fluctuating asymmetry. Anim Behav 48:986–989. Thomas F., Ward D. F., Poulin R., 1998 Fluctuating asymmetry in an insect host: a big role for big parasites? Ecol Lett 1:112-117. Thompson S. N., 1983 Biochemical and physiological effects of metazoan endoparasites on their host species. Comp Biochem Physiol B 74(2):183–211. Valen V., 1962 A study of fluctuating asymmetry. Evolution 16:125-142. Waddington C. H., 1942 Canalization of development and the inheritance of acquired characters. Nature 150:563-565. Whitfield P. J., 1979 The biology of parasitism: an introduction to the study of associating organisms. Baltimore, MD: University Park Press.
Received: 01 December 2014. Accepted: 03 January 2015. Published online: 24 January 2015. Authors: Rowina Gomez Pojas, MSU-Iligan Institute of Technology, College of Science and Mathematics, Department of Biological Sciences, Philippines, Lanao del Norte, Iligan City, Tibanga, Andres Bonifacio Avenue, 9200, e-mail: [email protected]. Sharon Rose Malanum Tabugo, MSU-Iligan Institute of Technology, College of Science and Mathematics, Department of Biological Sciences, Philippines, Lanao del Norte, Iligan City, Tibanga, Andres Bonifacio Avenue, 9200, e-mail: 9200, [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. How to cite this article: Pojas R. G., Tabugo S. R. M., 2015 Fluctuating asymmetry of parasite infested and non-infested Sardinella sp. from Misamis Oriental, Philippines. AACL Bioflux 8(1):7-14.
AACL Bioflux, 2015, Volume 8, Issue 1. 14 http://www.bioflux.com.ro/aacl AACL BIOFLUX Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society
Changes in otolith structure of seagrass siganid Siganus canaliculatus during settlement 1Renan U. Bobiles, 1Victor S. Soliman, 2Kosaku Yamaoka
1 Coastal Resources Management Unit, Bicol University Tabaco Campus, Tabaco City, Philippines; 2 Graduate School of Kuroshio Science, Kochi University, Kochi, Japan. Corresponding author: R. U. Bobiles, [email protected]
Abstract. Coral reef fishes upon settlement undergo morphological changes. Their otoliths or ear bones that provide auditory and balance functions reflect integral changes associated with the transition from pelagic larval to demersal juvenile stages. In the seagrass siganid Siganus canaliculatus, otolith sulcus area grew at a slower rate with sagittal area and body size in both stages. Sulcus area apparently contracted or remained constant during this transition. The one-day difference between sampling of pre- and post-settled fish did not produce significant difference in sulcus area. Sulcus area-to-sagittal area ratio was higher at pre- than at post-settled fish. Sagittal area increased and it grew at faster rate with body size which indicates a shift to improved food availability and less harsher environment for the young fish. Sagittal growth was higher at post- than at pre-settlement. Body size and otolith area were several magnitude larger than sulcus area, thus, they dimensionally outgrew the sulcus. The faster growth of sulcus before settlement suggests the fish depends more on it during this phase than after settlement. Key Words: Siganus canaliculatus, otolith area, settlement, sulcus, growth.
Introduction. Settlement is the transition of organism from pelagic to benthic environment which coincides with metamorphosis (Wellington & Victor 1992). During metamorphosis there are various changes occurring in the external and internal characteristics as well as the behavior of the fish (Fukahara 1988). Sagittal otoliths of newly metamorphosed and settled flatfishes display distinct secondary growth centres (accessory primordia) at the circumference of the larval otolith (Karakiri et al 1989; Sogard 1991). Alhossaini et al (1989) and Karakiri et al (1989) showed that settlement of North Sea plaice (Pleuronectes platessa) is synchronous during the formation of accessory primordia, a secondary growth center in the otolith structure associated during metamorphosis. The otolith of Siganus fuscescens and Siganus guttatus during settlement is associated with rapid reduction of otolith increment width and increase in Strontium and Calcium ratio 4–5 days before the event (Yamada & Shibuno 2007; Yamada & Baba 2009). Otolith microstructural changes shown in the transition zone in the daily increment pattern are believed to occur during settlement (Sponaugle & Cowen 1994). This transition zone is characterized by rapid reduction of otolith increment width and lessening of increment opacity in S. guttatus (Soliman et al 2009). Otolith growth, morphometrics and shape vary even at intra-specific level due to difference in growth of the fish (Campana & Casselman 1993) and life history (Ballagh et al 2012). Variability in growth during the larval stage is typically high and has been attributed to three causes: water temperature during the larval life stages (McCormick & Molony 1995), food availability (McCormick & Molony 1993) and delays in settlement once a stage of physiological competence has been reached (Cowen 1991). Differences in otolith structure depend not only on differential growth rates but also on the consistency of the environmental conditions encountered during the life history (Pothin et al 2006) and otolith can be completely reshaped by environmental conditions during ontogeny (Vignon 2012). The difference in metabolic rates linked to environmental factors may
AACL Bioflux, 2015, Volume 8, Issue 1. 15 http://www.bioflux.com.ro/aacl influence the shape and growth rate of the otoliths (Wilson 1985). Siganids are highly lunar periodic spawners which occurs on or a day about new moon, and restricted settlement period of one to three days with a short planktonic larval duration (Soliman et al 2010). In growth evaluation of S. fuscescens before, during and after settlement, it showed that during settlement there is sudden increase in the growth rate with influence of spatial difference (Mellin et al 2009). The Siganus canaliculatus commonly known as seagrass siganids characterized by a “V” shaped sulcus (Figure 1). The sulcus is a depression on the medial surface of the sagittal otolith (Secor et al 1991) that forms where the maculus or sensory tissue comes into contact with the otolith (Aguirre & Lombarte 1999). The sulcus and the growth of the macula may reflect their function as organs of equilibrium and hearing (Gauldie 1988; Lombarte 1992; Lombarte & Popper 1994; Arellano et al 1995). Gauldie (1988) modeled otolith function in which the sagittal otolith acts as a mechanism of levers through which sound waves are transformed into shearing forces in the plane of the hair cell in the macula. The change in the macula parallels growth of the sulcus, which has something to do with the fish auditory responses. Moreover, the shape of the sensory macula also varies through ontogenetic development of teleost fishes (Platt & Popper 1981) due to difference in auditory feedback on fish hearing (Aguirre & Lombarte 1999; Lombarte 1992; Arellano et al 1995). Changes in sulcus acusticus shape could occur in relation to fish growth and greater complexity in the crystalline structure of the sulcus acusticus (Montanini 2015). Importance in the relationship of sagitta area and sulcus is related on their function as a mechanical receptor that processes acoustic and postural information has well been established (Schuijf 1981; Fay 1984; Gauldie 1988). Similarly, the variation in the sensory macula and the shape of the sagittal otolith could be a form of adaptation in variable environments.
Figure 1. Characteristic of S. canaliculatus otolith (Sagitta) with prominent horn shaped rostrum and anti-rostrum and its “V” shaped sulcus indicated by gray dotted line.
The sagittal size is associated with environmental characteristics as depth and low-light of the environment (Paxton 2000); Ekau (1991) also reported a relationship between the shape of the fish, its ecotype and otolith size. The relation of environmental condition and otolith morphology could provide a way for explaining characteristic of biological significance. The objectives of the study were to compare the relationships of otolith area, sulcus area and fish body size associated during the settlement of Siganus canaliculatus. Variation in otolith shape and growth in reef fishes within and among
AACL Bioflux, 2015, Volume 8, Issue 1. 16 http://www.bioflux.com.ro/aacl species were affected by changes in the environmental condition along the fish life history (Pothin et al 2006; Wilson 1985).
Material and Method
Field sampling. Samples were collected on the month of May 2010 during the siganid “fry run” or recruitment that usually occur in the east coast of San Miguel Island in the region; pre-settled juveniles were caught by bagnet that operated offshore and post- settled juveniles were caught by encircling net that operated in the seagrass areas. The island is in Lagonoy Gulf (Bicol Region), northeastern Philippines (Figure 2). The gulf is endowed with a wide array of habitats with varying productivity. Garces & Valmonte- Santos (1995) characterized the gulf into three habitat namely coral reef/seagrass and seaweeds area, estuary and deeper area as to productivity. Productivity estimate using zooplankton biomass, chlorophyll a, and using the light and dark bottle consistently reveal that estuary is the most productive followed by coral reef/seagrass and seaweeds area, and the less productive is the deeper area of the gulf. The period of sampling of post-settled juveniles was done a day after the pre-settled individuals collection. The 30 individuals were collected for each stage (pre-settlement and post-settlement) sorted from other species of siganids, then preserved in 95% ethanol.
Figure 2. Location where S. canaliculatus samples were obtained (shaded red) from the east coast of San Miguel Island in Lagonoy Gulf, Philippines (original drawing).
Laboratory procedures. Fish body lengths were measured to the nearest mm at 0.01 mm accuracy using vernier caliper. Total length (TL) was measured from the tip of the upper jaw to the tip of the tail and standard length (LS) was measured from the tip of the upper jaw to the caudal peduncle, body depth (BD) vertical distance between the dorsal and ventral margins of the body measured at the base of the pectoral fin where it joins the body. Right sagittal otolith was used in the analysis. Sagittae were used in the study because of its high degree of variability in shape compared to lapilli and asteriscii. The otolith non-linear morphometrics were obtained using the image analysis software Digimizer (Version 3.7.0.0). The assumption of pre-settled and post-settled juvenile was verified and resolved through microstructure analysis of the sagittae. The presence or absence of settlement mark was the basis for classifying between post-settled and pre-settled fish. The
AACL Bioflux, 2015, Volume 8, Issue 1. 17 http://www.bioflux.com.ro/aacl transition from pelagic larval (pre-settled) to demersal stage (post-settled) corresponds to a marked reduction in opacity of otolith increment and abrupt width reduction relative to preceding increments where the reduced increment is the settlement mark (Soliman et al 2010). The otolith images were obtained using Olympus CX41 microscope with a digital image system (CMOS Camera) attached. Digital images of the otolith were analyzed using Digimizer to determine the parameters otolith area (Oa) and sulcus area (Sa) for the two settlement stages (Figure 3).
Sulcus Area
Length
Otolith Area
Width
Figure 3. Schematic representation of sagittal otolith (Secor et al 1999), the shaded part represent the sulcus area (Sa) and the non-shaded part is the otolith area (Oa).
Data analysis. Test for normality of size distribution was performed using Shappiro Wilks. Statistical differences in the fish morphometrics between the two stages were done using T-test. Functional relationships of the fish standard length, Oa and Sa were examined though least squares linear regression. Growths of the sagittal area and sulcus area relative to fish length were determined by the slope of the regression equation. The slope coefficients (b) for the growth relationship between the area of the Oa and LS, the area of the Sa and LS and the area of the Sa and Oa, were determined by fitting a power equation y = axb to the data and tested for significant deviations from isometry. In the equation the value of the dependent variable (y), what is being predicted or explained, Alpha (a) constant; equals the value of Y when the value of X = 0 sometimes referred to as the intercept, X is the value of the Independent variable and Beta (b) the coefficient of X; the slope of the regression line. Negative allometric growth was exhibited by sulcus area with respect to fish LS and Oa except for the Oa relative to fish LS which showed positive allometry for the two settlement stages of S. canaliculatus. Gould (1966) as cited by Arellano et al (1995) showed that slope (b) of sagittal area and sulcus area regression equal to a value of 2 indicates isometry. A slope differing from 2 shows either positive allometry (> 2) or negative allometry (< 2) with respect to length. Growth of sulcus relative to otolith area is isometric if the slope equal to 1, otherwise growth is allometric. Comparisons in the slope were tested to determine significant difference during the transition stage. The test statistic is Student’s t, computed as the difference between the two slopes divided by the standard error of the difference between the slopes (Cohen et al 2003).
AACL Bioflux, 2015, Volume 8, Issue 1. 18 http://www.bioflux.com.ro/aacl Results
Population structure. Sixty S. canaliculatus specimens (larvae and juveniles) were obtained from samples caught by bagnet and seine net (Table 1). The pre-settled fish from bagnet ranged from 22.68 to 27.84 mm LS while the post-settled fish from seine net 6 ranged from 25.28 to 28.88 mm LS. Otolith area of pre-settled fish was 1.82 x 10 – 2.78 x 106 µm2 and for post-settled fish it was 2.04 x 106 to 2.92 x 106 µm2. Area of sulcus in pre-settled group was 5.21 x 105 – 7.58 x 105 µm2. Post-settled group had a range of 5.96 x 105 - 7.61 x 105 µm2 in sulcus area. The ratio of sulcus area to otolith area in pre- settled group was 3.61–4.01 and it was 3.26-3.83 in post-settled group. Pairwise comparison using Student t-test showed that body size (Ls, TL and BD), and Oa as well as sulcus to otolith/sulcus area ratio were significantly different between the two settlement stages. Sulcus areas between the stages were not significantly different.
Table 1 Fish and otolith morphometric measurements of S. canaliculatus specimens
Morphometric variable Pre-settled (Mean ± S.D.) Post-settled (Mean ± S.D.) Standard length (mm)* 25.58 ± 1.270 26.95 ± 0.884 Total length (mm)* 29.62 ± 1.589 31.772 ± 0.939 Body depth (mm)* 7.86 ± 0.498 8.254 ± 0.438 Otolith area (sqµm)* 222556.08 ± 25273.459 234088.53 ± 21064.296 Sulcus acusticus area (sqµm)ns 65416.61 ± 5808.67 63308.61 ± 3158.65 Otolith area/Sulcus area (Oa:Sa)* 3.798 ± 0.106 3.470 ± 0.116 *Significantly different at α = 5%; ns – not significant.
The Shappiro-Wilks test for normality showed that the size distribution of S. canaliculatus during the settlement stages (pre-settled p = 0.4871, post-settled p = 0.8412) came from normally distributed population as shown in Figure 4. Fish body length in the two settlement stages had both unimodal distribution.
Figure 4. Length-frequency distribution of S. canaliculatus during the two settlement stages.
Growth of the otolith area (Oa) and the sulcus area (Sa). The linear regressions between Oa, Sa and LS are presented in Figures 5a-c and Table 2. Significant relationship 2 was exhibited between Oa and LS both in pre-settled (r = 0.91, n = 30) and post settled 2 (r = 0.89, n = 30) stages. The Sa versus LS also showed high relationship in pre-settled (r2 = 0.95, n = 30) and post-settled (r2 = 0.77, n = 30) fish. Similarly, growth of Sa relative to Oa showed functional relationship in both pre-settled (r2 = 0.95, n = 30) and post-settled (r2 = 0.80, n = 30) stages.
AACL Bioflux, 2015, Volume 8, Issue 1. 19 http://www.bioflux.com.ro/aacl
A
B
C
Figure 2. Regression relationships of standard length (Ls), otolith area, and sulcus area during the transition from pre-settled to post-settled stage of S. canaliculatus.
Table 2 Regression equations and parameters of standard length, otolith and sulcus area
Degrees of freedom Stages Model (Equation) r2 a b (df) b LS v. Oa (Oa = a x LS ) 0.91 5.26 2.32 29 b Pre-settled LS v. Sa (Sa = a x LS ) 0.95 5.02 1.86 29 Oa v. Sa (Sa = a + b x Oa) 0.95 11161.2 0.33 29 b LS v. Oa (Oa = a x LS ) 0.89 4.16 2.49 29 b Post-settled LS v. Sa (Sa = a x LS ) 0.77 6.67 1.35 29 Oa v. Sa (Sa = a + b x Oa) 0.80 33219.7 0.15 29
AACL Bioflux, 2015, Volume 8, Issue 1. 20 http://www.bioflux.com.ro/aacl The pairwise comparison of the slope (b) using t-test was made to test the difference between the growth in settlement stages of S. canaliculatus (Table 3). Relative to LS, significant difference was observed in Oa (t = -8.10, p < 0.05) and Sa (t = -2.11, p < 0.05). Sulcus was significantly different (t = -8.30, p < 0.05) with respect to otolith area. Growth of sulcus with respect to fish length and otolith area is relatively higher in pre- settled higher than in post-settled. Otolith areas are significantly different between the stages.
Table 3 Pairwise comparison of slope of otolith and sulcus area using Student t-test
Slope of line Regression t-stat. df n Pre-settled Post-settled
Ls vs otolith area* 2.176 2.491 -8.100 56 60 Ls vs sulcus area* 1.862 1.350 -2.116 56 60 Otolith area vs sulcus area* 0.335 0.146 -8.304 56 60 * Significantly different at α = 5%.
Discussion. The significant difference of change in body size of the settling S. canaliculatus juveniles indicated a physiologic activity involving growth (Leis & McCormick 2002). Change in body size was associated with change in growth (slope) of sulcus and otolith area during metamorphosis. Early life ecology of several siganids species such as S. canaliculatus includes pelagic larvae (Woodland 2001). This transition from pre-settled to post-settled stage implies abundance of food towards the settlement area (McCormick & Molony 1992; Soliman et al 2010), and it could be that the conditions obtaining stimulated in direct and subtle ways a differential increase in growth (positive allometry) of otolith area relative to fish length. An increase of one variable (fish body size) was concomitant with the increase of the other (otolith area) at a relatively faster rate of growth. Improved situation of settling area enhanced the growth of fish that contributed to the otolith size difference. The general shape of the sagittal change in relation to fish size (Gonzalez Naya et al 2012), fast growing fish tend to form long and thin otolith in contrast with slow growing fish that posses compact and short ones (Campana & Thorrold 2001). Similarly, food variation, somatic growth and otolith growth are significantly correlated (Aguilera et al 2009). Habitat characteristic such as refuge availability (Adams & Ebersole 2004) and food availability (McCormick & Malony 1992) affect post-settlement juvenile abundance and number of recruits (McCormick & Malony 1993). Sulcus as part of otolith associated with the auditory function (Platt & Popper 1981) showed different pattern of growth (negative allometry) relative to fish length. Growth was most likely attributed to diminishing reliance to auditory sense during ontogeny. This is supported in Gauldie (1988) and Lombarte (1992) who reported that pelagic fishes are more sensitive to sound frequencies than demersal fishes. The higher average Sa:Oa ratio of pre-settled fish is probably linked with pelagic nature of the juvenile. Pelagic migration would create intense physical contact between the sensory hairs of the macula and the sulcus due to environmental perturbation like strong current. This physical contact provides a fulcrum for the rotation of the otolith that generates the shearing forces to activate the kinocilia of the macula (Schuijf 1981). The comparative study of otolith morphology and its relation to environmental parameters provides a method for elucidating characteristics of biological importance (Aguirre & Lombarte 1999). Significant difference in the growth (slope) of otolith area relative to fish length was manifested during the ontogenetic shift (from pre-settled to post-settled) of S. canaliculatus. Higher growth of otolith area with respect to body size in post-settled fish could be attributed to increased metabolic processes involved, thus, the incorporation of calcium carbonate could have been faster. It seems that during the transition or shift in the habitat, ecological factor greatly influenced and masked the expressions of the genetic factor in the formation of otolith by indirect influence on the growth rate (Campana & Casselman 1993). Primarily, change in growth during the
AACL Bioflux, 2015, Volume 8, Issue 1. 21 http://www.bioflux.com.ro/aacl transition from pre-settled to post-settled stages coincides with metamorphosis and associated morphological changes in some reef fishes (Leis & McCormick 2002), which could explain the pronounced difference in the otolith growth in the siganid juveniles. Sulcus growth with respect to otolith area and body size in pre-settled fish was higher than in post-settled fish. Pelagic migration creates intense physical contact between the sensory hairs of the macula or its approximation the sulcus, because of environmental and biological perturbations such as strong current and predator avoidance and where visual impediment was experienced due to murky environment (Schuijf 1981). This may indicate that individuals with relatively larger sulcus area are more sensitive to sound frequency. Rogers & Cox (1988) also found that fishes are most sensitive to sound where the ambient noise is high such as in reef areas. Furthermore, Aguirre & Lombarte (1999) reported that in hakes, Mullus surmuletus which are associated with rocky reefs and M. barbatus which inhabit muddy bottoms, the slope of sulcus is increasing with body size. Both species of Mullus feed on benthic species mainly using sound perception, with little mobility and are either cryptic or hidden in the substratum whereby visual recognition is difficult. For the pre-settled S. canaliculatus, higher growth (slope) of sulcus could be expected as it performs its function as mechanoreceptors (auditory), in contrast to post-settled juveniles where environmental condition was relatively less turbulent and auditory function was very much compensated with visual senses in a clear environment of the seagrass beds where they settled. Changes in the growth of sulcus could have been associated with the reduction in auditory dependence and increase visual reliance in the seagrass area.
Conclusions. Pelagic to demersal transition of S. canaliculatus imposed a physiological change in the fish. These changes were evident in the otolith structure between pre- settled and post-settled stage. In the transition, only the growth of otolith area in post- settled fish relative to body length was relatively higher than in pre-settled. During ontogeny, the pattern of growth in otolith area and sulcus area was allometric. The rate of growth in otolith area was characterized by positive allometry which means there was increased rate of growth as the fish settled. The growth rate of sulcus exhibited negative allometry indicating a decrement in rate of growth during its transition from pre-settled to post-settled stage. This variation can be highly likely due to differences in food abundance and spatial niches habitat characteristic as a means of adaptation.
Acknowledgements. This research was undertaken in part with the support of the Japan Society for the Promotion of Science Exchange Program for Southeast Asia Young Researchers to R. U. B. The otolith microstructure study was undertaken at the Fish Ecology and Population Dynamics Laboratory of the Bicol University Tabaco Campus, Tabaco City, Philippines and at the Laboratory of Marine Bioresource Production in Kochi University, Kochi, Japan. The assistance by Dr. Yohei Nakamura and Dr. Yoshinori Morooka during the stay of R.U.B. in Kochi University is sincerely acknowledged. The research was implemented under the auspices of the Siganid Recruitment Ecology Program that has been funded by Bicol University.
References
Adams A. J., Ebersole J. P., 2004 Processes influencing recruitment inferred from distributions of coral reef fishes. Bulletin of Marine Science 75:153-174. Aguilera B., Catalán I. A., Palomera I., Olivar M. P., 2009 Otolith growth of European sea bass (Dicentrarchus labrax L.) larvae fed with constant or varying food levels. Scientia Marina 73(1):173-182. Aguirre H., Lombarte A., 1999 Ecomorphological comparisons of sagittae in Mullus barbatus and M. surmuletus. Journal of Fish Biology 55:105–114. Alhossaini M., Liu Q., Pitcher T. J., 1989 Otolith microstructures indicating growth and mortality among plaice, Pleuronectes platessa L., post-larval sub-cohorts. Journal of Fish Biology 35(supplement A):81–90.
AACL Bioflux, 2015, Volume 8, Issue 1. 22 http://www.bioflux.com.ro/aacl Arellano R. V., Hamerlynck O., Vinex M., Mees J., Hostens K. Gijselinck W., 1995 Changes in the ratio of the sulcus acusticus area to the sagitta area of Pomatoschistus minutus and P. lozanoi (Pisces, Gobiidae). Marine Biology 122:355– 360. Ballagh A. C., Welch D. J., Newman S. J., Allsop Q., Stapley J. M., 2012 Stock structure of the blue threadfin (Eleutheronema tetradactylum) across northern Australia derived from life-history characteristic. Fisheries Research 121-122:63-72. Campana S. E., Casselman J. M., 1993 Stock discrimination using otolith shape analysis. Canadian Journal of Fisheries and Aquatic Sciences 50:1062-1083. Campana S. E., Thorrold S. R., 2001 Otoliths, increments and elements: keys to a comprehensive understanding of fish populations?. Canadian Journal of Fisheries and Aquatic Sciences 58:30-38. Cohen J., Cohen P., West S. G., Aiken L. S., 2003 Applied multiple regression/correlation analysis for the behavioral sciences. 3rd edition, Mahwah N. J.: Lawrence Earlbaum Associates. Cowen R. K., 1991 Variation in the planktonic larval duration of the temperate wrasse Semicossyphus pulcher. Marine Ecology Progress Series 69:9-15. Ekau W., 1991 Morphological adaptations and mode of life in high Antarctic fish. In: Biology of Antarctic fishes. di Prisco G., Maresca B., Tota B. (eds), Springer-Verlag, Berlin, pp. 23–39. Fay R. R., 1984 The goldfish ear codes the axis of acoustic particle motion in three dimensions. Science 225:951–954. Fukahara O., 1988 Morphological and functional development of larval and juvenile Limanda yokohamae (Pisces: Pleuronectidae) reared in the laboratory. Marine Biology 99:271-281. Gauldie R. W., 1988 Function, form and time-keeping properties of fish otoliths. Comparative Biochemistry and Physiology 91A:395–402. Garces L. R., Valmonte-Santos R. A., 1995 Assessment of water quality in Lagonoy Gulf: Part IV. Species composition and biomass of plankton communities. In: Resource and ecological assessment of Lagonoy gulf, Philippines. Volume 2: Technical Monograph. Silvestre G., Luna C., Soliman V., Garces L. (eds), ICLARM Technical Report. Gonzalez Naya M. J., Tombari A., Volpedo A., Gomez S. E., 2012 Size related changes in sagittal otolith of Australoheros facetus (Pisces; Cichlidae) from South America. Journal of Applied Ichthyology 28:752–755. Karakiri M., Berghahn R., von Westernhagen H., 1989 Growth differences in 0-group plaice Pleuronectes platessa as revealed by otolith microstructure analysis. Marine Ecology Progress Series 55:15–22. Leis J. M., McCormick M. I., 2002 The Biology, behavior, and ecology of the pelagic, larval stage of coral reef fishes. In: Coral reef fishes - dynamics and diversity in a complex ecosystem. Sale P. (ed), Academic Press, San Diego, pp. 171-199. Lombarte A., 1992 Changes in otolith area: sensory area ratio with body size and depth. Environmental Biology of Fishes 33:405-410. Lombarte A., Popper A. N., 1994 Quantitative analyses of postembryonic hair cell addition in the otolithic endorgans of the inner ear of the European hake, Merluccius merluccius (Gadiformes, Teleostei). Journal of Comparative Neurology 345:419– 428. McCormick M. I., Molony B. W., 1992 Effects of feeding history on the growth characteristics of a reef fish at settlement. Marine Biology 114:165-173. McCormick M. I., Molony B. W., 1993 Quality of the reef fish Upeneus tragula (Mullidae) at settlement: is size a good indicator of condition? Marine Ecology Progress Series 98:45-54. McCormick M. I., Molony B. W., 1995 Influence of water temperature during the larval stage on size, age and body condition of a tropical reef fish at settlement. Marine Ecology Progress Series 118:59-68.
AACL Bioflux, 2015, Volume 8, Issue 1. 23 http://www.bioflux.com.ro/aacl Mellin C., Galzin R., Ponton D., Vigliola L., 2009 Back-calculated larval and juvenile growth trajectories of coral reef fish: how to untangle fast growth and selection for fast growth? Aquatic Biology 6:31-39. Montanini S., Stagioni M., Valdre G., Tommasini S., Vallisneri M., 2015 Intra-specific and inter-specific variability of sulcus acusticus of the sagittal otoliths in two gurnard species (Scorpaeniformes, Triglidae). Fisheries Research 161:93-101. Paxton J. R., 2000 Fish otoliths: do sizes correlate with taxonomic group, habitat and/or luminescence? Philos Trans R Soc Lond B Biol Sci 355:1299–1303. Platt C., Popper A. N., 1981 Fine structure and function of the ear. In: Hearing and sound communication in fishes. Tavolga W. N., Popper A. N., Fay R. R. (eds), New York: Springer Verlag, pp. 3–38. Pothin K., Gonzales-Salas C., Chabanet P., Lecomte-Finiger R., 2006 Distinction between Mulloidichthys flavolineatus juveniles from Reunion Island and Mauritius Island (South-west Indian Ocean) based on otolith morphometrics. Journal of Fish Biology 69:38-53. Rogers P. H., Cox M., 1988 Underwater sound as a biological stimulus. In: Sensory biology of aquatic animals. Atema J., Fay R. R., Popper A. N., Tavolga W. N. (eds), New York: Springer Verlag, pp. 131–149. Schuijf A., 1981 Models of acoustic localization. In: Hearing and sound communication in fishes. Tavolga W. N., Popper A. N., Fay R. R. (eds), New York: Springer Verlag, pp. 267–310. Secor D. H., Dean J. M., Laban E. H., 1991 Manual for otolith removal and preparation for microstructural examination: a user manual. Electric Power Research Institute and the Belle W. Baruch Institute for Marine Biology and Coastal Research, 85 pp. Sogard S. M., 1991 Interpretation of otolith microstructure in juvenile winter flounder (Pseudopleuronectes americanus): ontogenetic development, daily increment validation and somatic growth relationships. Canadian Journal of Fisheries and Aquatic Sciences 48:1862–1871. Soliman V. S., Yamada H., Yamaoka K., 2009 Validation of daily sagittal increments in the golden rabbitfish Siganus guttatus (Bloch) using known-age larvae and juveniles. Journal of Applied Ichthyology 25(4):438-441. Soliman V. S., Yamaoka K., Yamada H., 2010 Early life-history of the spiny siganid Siganus spinus (Linnaeus 1758) inferred from otolith microstructure. Journal of Applied Ichthyology 26:540–545. Sponaugle S., Cowen R. K., 1994 Larval durations and recruitment patterns of two Caribbean gobies (Gobiidae): contrasting early life histories of demersal spawners. Marine Biology 120:133-143. Vignon M., 2012 Ontogenetic trajectories of otolith shape during shift in habitat use: interaction between otolith growth and environment. Journal of Experimental Marine Biology and Ecology 420-421:26-32. Wellington G. M., Victor B. C., 1992 Regional differences in duration of the planktonic larval stage of reef fishes in the Eastern Pacific Ocean. Marine Biology 113:491-498. Wilson R. R., 1985 Depth-related changes in sagitta morphology in six macrourid fishes of the Pacific and Atlantic Oceans. Copeia 1985:1011–1017. Woodland D. J., 2001 Siganidae – Rabbitfishes (Spinefoots). In: FAO species identification guide for fishery purposes. The living marine resources of the Western Central Pacific, Vol. 6, Bony Fishes, Part 4 (Labridae to Latimeriidae), estuarine crocodiles, sea turtles, sea snakes and marine mammals. Carpenter K. E., Niem V. H. (eds), FAO, Rome, pp. 3627–3650. Yamada, H., Baba, K., 2009 Ontogenetic changes of trace elements in otoliths and their spatial variations in rabbitfish, Siganus fuscescens. Bulletin of the Japanese Society of Fisheries Oceanography 73, 8-15. Yamada H., Shibuno T., 2007 Changes in otolith microstructure and microchemistry of two Siganid fishes during settlement period. Nippon Suisan Gakkaishi 73:859–866.
AACL Bioflux, 2015, Volume 8, Issue 1. 24 http://www.bioflux.com.ro/aacl Received: 28 November 2014. Accepted: 22 January 2015. Published online: 24 January 2015. Authors: Renan U. Bobiles, Coastal Resources Management Unit, Bicol University Tabaco Campus, M. H. Del Pilar Street, Tayhi, Tabaco City 4511, Albay Philippines, Philippines, e-mail: [email protected] Victor S. Soliman, Coastal Resources Management Unit, Bicol University Tabaco Campus, M. H. Del Pilar Street, Tayhi, Tabaco City 4511, Albay Philippines, Philippines, e-mail: [email protected] Kosaku Yamaoka, Graduate School of Kuroshio Science, Kochi University, 200 Otsu, Monobe, Nankoku city, Kochi, Japan, e-mail: [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. How to cite this article: Bobiles R. U., Soliman V. S., Yamaoka K., 2015 Changes in otolith structure of seagrass siganid Siganus canaliculatus during settlement. AACL Bioflux 8(1):15-25.
AACL Bioflux, 2015, Volume 8, Issue 1. 25 http://www.bioflux.com.ro/aacl AACL BIOFLUX Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society
Nutritional value of water hyacinth (Eichhornia crassipes) leaf protein concentrate for aquafeeds 1,3Gaily Jubie S. Hontiveros, 2,3Augusto E. Serrano Jr.
1 College of Fisheries, Mindanao State University, General Santos City, Philippines; 2 National Institute of Molecular Biology and Biotechnology, University of the Philippines Visayas, Miagao, Iloilo, Philippines; 3 Institute of Aquaculture, College of Fisheries and Ocean Sciences, University of the Philippines Visayas, Miagao, Iloilo, Philippines. Corresponding author: A. E. Serrano Jr., [email protected]
Abstract. The present study evaluated the nutritive value of water hyacinth leaf protein concentrate (WHLPC) as a potential feed ingredient for aquafeeds in general and measured the Apparent Digestibility of the Ingredient (ADI) for dry matter in Oreochromis niloticus adults. Concentrating the water hyacinth meal resulted in a 248% increase in crude protein. The apparent dry matter digestibility (ADMD) of WHLPC was relatively high at 76.4%. Cadmium, copper, and lead increased after protein concentration but were still considered much lower than the allowable limits set by the European Union for animal feedstuffs. The most limiting amino acid was methionine followed by lysine. The chemical score of the WHLPC was estimated to be 38.9% while the Essential Amino Acid Index (EAAI) was 0.88; the latter index indicated that the WHLPC was a useful protein source and with amino acid supplementation or protein complementation could convert it to a good quality protein source for aquafeeds. Key Words: Eichhornia crassipes, leaf concentrate, apparent digestibility of ingredient, chemical score, EAAI.
Introduction. Given the current level of per capita consumption of aquatic foods, it is projected that the world will require an additional 23 million tons by 2020 (FAO 2012). Considering that most of the world’s major fish stocks are overfished and catches are either static or declining, aquaculture could be the only option to increase total fish production. However, aquaculture largely depends on quality of feeds, the sustainability of which is affected by the supply of animal and plant proteins, oils and carbohydrates. The key aquafeed ingredients such as fish meal, soybean meal, and various oilseed cakes are are in direct competition with terrestrial animal husbandry (Sumagaysay-Chavoso 2007). In Asia, these ingredients are produced in much lesser quantities than what is being consumed for feed manufacture (De Silva & Hasan 2007). The perennial shortage of this key raw material is highly problematic for feed manufacturers and can seriously affect aquaculture sustainability and profitability. Increased awareness of the likelihood of fish meal scarcity has led to efforts in reducing dependence on this ingredient. One of the solutions is incorporating plant-based raw materials to satisfy the protein requirement of cultured species. Attention has been focused on replacing fish meal with soybean meal. A large volume of soybean meal used in aquafeed production is imported especially in the Philippines. One of the untapped resources are aquatic macrophytes which include water hyacinth (Eichhornia crassipes), water lettuce (Pistia stratiotes), duckweed (Lemna spp.) among others. These aquatic plants are considered pests and pose many negative impacts on bodies of water. Water hyacinth is the most noxious among these and can be one of the bigger problems in managing water systems including the Philippines (Valencia 2012). Many water quality problems such as low dissolved oxygen, high carbon dioxide, and toxic nitrogenous compounds are caused by aquatic vegetation. Water-level manipulation, through clogging drains and intakes, in ponds and raceways can also be complicated by aquatic vegetation (Masser 2000). The use of water hyacinth in fish feed could reduce the present
AACL Bioflux, 2015, Volume 8, Issue 1. 26 http://www.bioflux.com.ro/aacl dependence on other competitive agricultural crops included in compounding feeds and could put a nuisance aquatic plant into beneficial use. The objective of the present study is to evaluate the nutritive value of water hyacinth leaf protein concentrate (WHLPC) as a potential feed ingredient for aquafeeds.
Material and Method. The present study was conducted in May to July, 2013 at the Multispecies Hatchery of the University of the Philippines Visayas in Miagao, Iloilo, Philippines.
Production of water hyacinth leaf protein concentrate. Water hyacinth was collected from a freshwater pond in Culasi, Antique, Philippines. Leaves were dried until about 10% moisture and were ground using a hammer mill. WHLPC was produced using methods described by Virabalin et al (1993). Briefly, hyacinth leaf powder was soaked in water at 1:3 (w/v) ratio for 30 min and homogenized in a blender for 5 min. Sodium hydroxide (NaOH) solution was used to adjust the pH to 9.0 and allowed to stand. The resulting slurry was filtered through a cheese cloth, the filtrate collected, acidified with hydrochloric acid (HCl) to pH 2.0 and flocculate allowed to settle. Further flocculation was enhanced by heating in a water bath between 60 and 80oC for 10 min the precipitate of which was collected, oven-dried at 60oC, and ground to fine powder. Proximate composition of the raw water hyacinth leaf powder and WHLPC were analyzed. Moisture was measured using a thermo-balance (Mettler Toledo HB43 halogen moisture analyzer). Ash content was determined after incineration in a muffle furnace at 550oC for 12 h. Crude protein was measured after block digestion and steam distillation using Foss TecatorTM digestion system and Foss KjeltecTM 8200 auto-distillation unit. Crude fat was extracted using Foss SoxtecTM 2050 automatic system and fiber was determined using Foss FibertecTM 2010 system. Amino acid composition of WHLPC was analyzed by a service laboratory (SGS Taiwan) using liquid chromatography with tandem mass spectrometry (LC-MSMS) following standard methods (AOAC 2003).
Preparation of the test diets. A reference and test diets were formulated with chromic -1 oxide (Cr2O3) used as an inert marker at a concentration of 10 g kg in the reference diet (Table 1). Ingredients were purchased from the Southeast Asian Fisheries Development Center-Aquaculture Department (SEAFDEC/AQD). The test diet was prepared as a 70:30 mixture of the reference diet to the test ingredient. Dry ingredients were thoroughly mixed first before the liquid component (i.e. oil and distilled water). The resulting dough was pelleted and subsequently oven-dried at 60oC for 12 h. Feeds were then cut and sieved to appropriate size (2 mm) pellets and stored at -20oC until use.
Table 1 Composition of the reference and test diets in g kg-1
Ingredient Ref. diet Test diet Danish meal 255.8 Squid meal 81.0 Defatted soybean meal 260.0 Copra meal 152.5 Cod liver oil 10.0 Soybean oil 10.0 Corn starch 177.7 Vitamin mixa 13.0 Mineral mixb 30.0 c Chromic oxide (Cr2O3) 10.0 Reference diet 700.0 Water hyacinth leaf protein concentrate (test ingredient) 300.0 a Vitamin premix (kg-1 of diet): vitamin A - 15600 IU; vitamin D3 - 2600 IU; vitamin E - 260 IU; vitamin B1 - 104 mg; vitamin B2 - 104 mg; vitamin B6 - 65 mg; vitamin B12 - 26 µg; niacin - 520 mg; calcium pantothenate - 260 mg; biotin - 0.52 mg; folic acid - 23.4 mg; ethoxyquin - 6.5 mg; b Mineral premix (kg-1 of diet): iron - 1200 mg; manganese - 300 mg; zinc - 1200 mg; copper - 120 mg; iodine - 54 mg; cobalt - 600 µg; selenium - 6 mg; c Sigma-Aldrich chromic (III) oxide.
AACL Bioflux, 2015, Volume 8, Issue 1. 27 http://www.bioflux.com.ro/aacl Digestibility set up and fecal collection. Male Nile tilapia (Oreochromis niloticus) were obtained from the University of the Philippines Visayas Freshwater Aquaculture Station, Miagao, Iloilo and transported to the University hatchery. Prior to the digestibility trial, fish were acclimatized to the laboratory conditions and fed a commercial diet (27% crude protein, 6% crude lipid) at 4% body weight for 7 days. Four tanks (67 L) in a static system were randomly assigned to each diet. Sixteen adult fish (46.17 ± 6.34 g) were divided into 8 experimental tanks (Figure 1) and allowed to acclimate for 3 days. Fish were fed with the reference or test diet to apparent satiation twice daily (08:00 and 14:00 h). Tanks were cleaned before the first feeding; about 70% water was replaced daily with previously chlorine-treated freshwater (0 ppt). Continuous aeration was provided and water quality parameters were monitored regularly. Water temperature and pH were measured twice daily (08:00 and 14:00 h). Levels of dissolved oxygen were determined by titration, and total ammonia nitrogen and nitrite were analyzed by colorimetry using test kits (Advance Pharma Co., Ltd., Bangkok, Thailand).
Figure 1. Digestibility set up used in the study.
The digestibility trial lasted for 8 weeks from May to July 2013. Fecal collection started 3 days after feeding adult Nile tilapia with the reference or test diet to allow evacuation of all previously ingested material (Koprucu & Ozdemir 2005) and repeatedly done daily until the required fecal amount in the analysis had been collected. Fish were netted and feces were collected by manual stripping following the procedure of Stone et al (2008). Gentle pressure was applied to the abdomen using a moderate squeezing motion with the thumb and forefinger. Three times weekly, manual stripping was done and fish were promptly returned to the experimental tank. Fecal matter was pooled by treatment, weighed, and stored at -20oC until analysis. Triplicate samples of the diets and fecal matter were homogenized and analyzed for moisture and ash contents of the samples according to standard methods (AOAC 1995). Moisture was measured by drying to a constant weight in an oven at 105oC. Ash content was determined after incineration in a muffle furnace at 550oC for 12 h. Samples were then acid-digested and chromium (III) was estimated using Varian SpectrAA 55 double beam flame atomic absorption spectrometer.
Analytical methods and calculations. The apparent digestibility coefficients (ADC) of the diets and test ingredient were calculated according to the equations described by Cho et al (1985): ADC of reference or test diet = 1- (F/D x Dcr/Fcr) where F = percent of nutrient in feces; D = percent of nutrient in diet; Dcr = percent of
Cr2O3 in diet, and Fcr = percent of Cr2O3 in feces.
ADC of test ingredient, ADCI = ADCT + [((1 - s) DR)/s DI] (ADCT − ADCR)
AACL Bioflux, 2015, Volume 8, Issue 1. 28 http://www.bioflux.com.ro/aacl where ADCI = ADC of test ingredient; ADCT = ADC of test diet; ADCR = ADC of reference diet; DR = percent dry matter in the reference diet; DI = percent dry matter in the test ingredient; s = proportion of test ingredient in test diet (i.e. 0.3 in this study), and 1 – s = proportion of reference diet in test diet (i.e. 0.7 in this study). Amino acid results were expressed as grams per 100 g determined amino acid for protein. The essential amino acid (A/E) ratio (Arai 1981) of each essential amino acid (EAA) was calculated as the percentage of the total EAA. The chemical score of the WHLPC was determined by the following formula:
Chemical score = % limiting Essential Amino Acid (EAA) in WHLPC/ % corresponding EAA in chicken egg
The essential amino acid index (EAAI) of the two diets was determined from the formula:
n Essential Amino Acid Index = √[aa1/AA1Xaa2/AA2…aa10/AA10] where aa1 is the A/E ratio in the feed [(EAA/total EAA)×100], AA1 is the A/E ratio in the chicken egg [(EAA/total EAA)×100]. Chicken egg protein is considered by nutritionists to be the most complete protein and therefore use it as the standard dietary protein in evaluating other protein sources for a generalized animal diet. EAAI is the geometrical mean of the ratio of all essential amino acids in the evaluated protein relative to their content in a highly nutritive reference protein, viz., whole egg (Oser 1959).
Results. Proximate composition of the WHLPC is shown in Table 2. Concentrating protein by the combined acidification and heating resulted in an increased protein content from about 9% (unpubl. data) to about 22.4%. However, crude fiber content remained very high at about 55%. Since aquatic macrophytes could play as adsorbent of heavy metals and thus could pose risk when accumulated in food organisms such as culture fish and shellfish, three heavy metals were analyzed and is presented in Table 3. Increases in cadmium, copper, and lead were noted after protein concentration but were still considered much lower than the allowable limits set by the European Union (EC 2002). The amino acid profile of the WHLPC is shown in Table 4 and indicated a very high Essential Amino Acid Index but a considerable low chemical score when compared to amino acid profile of the whole chicken egg. The most and second limiting essential amino acids were methionine and lysine (about 39% and 72% of the corresponding EAA in chicken egg), respectively. The in vivo ADC of the reference and test diets were not significantly different from each other (Table 5). The Apparent Digestibility Coefficient of Ingredient (ADCI) for the dry matter of WHLPC was determined to be about 76% in tilapia.
Table 2 Nutrient composition (dry basis) of water hyacinth leaf protein concentrate
Component (g kg-1) Dry matter 919.0 Crude protein 223.5 Crude lipid 70.7 Crude fiber 54.8 NFE 511.2 Ash 139.7
AACL Bioflux, 2015, Volume 8, Issue 1. 29 http://www.bioflux.com.ro/aacl Table 3 Heavy metal content (dry basis) of water hyacinth leaf meal and leaf protein concentrate
Detected level (mg kg-1) Metal Water hyacinth leaf meal Water hyacinth leaf protein concentrate Cadmium 0.20 ± 0.02 0.31 ± 0.02 Copper 5.25 ± 0.14 22.09 ± 0.88 Lead 0.39 ± 0.03 0.44 ± 0.06 Values reported are means ± S.E.M. of three replicates.
Table 4 Amino acid composition (% of protein), A/E ratio*1, Essential Amino Acid Index (EAAI) and chemical score of the water hyacinth leaf protein concentrate
A/E WHLPC (% CP) Chemical score WHLPC Chicken Egg*2 Essential amino acids Arginine 6.58 13.07 10.6 107.68 Histidine 2.22 4.41 4.2 91.06 Isoleucine 5.47 10.86 11.0 86.78 Leucine 9.56 18.98 15.4 108.18 Lysine 5.06 10.05 12.2 72.36 Phenylalanine 6.01 11.93 10.0 104.67 Methionine 1.31 2.60 5.9 38.89 Threonine 5.27 10.46 8.9 102.68 Tryptophan 1.42 2.82 2.6 95.20 Valine 7.46 14.81 11.9 108.68 Non-essential amino acids Alanine 6.49 Aspartic acid 10.21 Cystine 0.38 Glycine 6.51 Glutamic acid 7.31 Proline 5.62 Tyrosine 2.92 Serine 10.21 EAAI 0.88 Chemical score of the WHLPC 38.89 *1 - the essential amino acid (A/E) ratio of each essential amino acid (EAA) was calculated as the percentage of the total EAA (10 EAAs); *2 - from FAO (1981).
Table 5 Apparent dry matter digestibility (ADMD) coefficients of diets and test ingredient evaluated in the digestibility study
Apparent digestibility coefficient (%)
Diet Ingredient (ADCI) Reference diet 98.8 ± 0.2 76.4 ± 11.2 Test diet 92.1 ± 3.3 76.4 ± 11.2 Values reported are means ± S.E.M. of three replicates.
Discussion. Results of the present study indicated that water hyacinth leaf meal concentrate exhibited a considerable potential as an aquafeed ingredient. As far as the authors are concerned, among the process of increasing the protein content of water hyacinth, the method used in the present study was similar with the method used by Chavez et al (2014) which exhibited the highest increase in protein. Saha & Ray (2011) have fermented the water hyacinth with Bacillus megaterium and B. subtilis and obtained a maximum increase of only 126% (from 13.37% to 16.88%). In the present study, the increase was about 248% (from about 9% to 22.35%). Furthermore, animal nutritionists
AACL Bioflux, 2015, Volume 8, Issue 1. 30 http://www.bioflux.com.ro/aacl consider an ingredient as a protein supplement if it contains at least 20% crude protein, thus the WHLPC in the present study was prepared to be a protein supplement as traditionally defined. Despite increases in the three heavy metal cadmium, copper and lead were observed (0.31, 22.1 and 0.44 mg kg-1), these values were considerably lower than the allowable limits set by the European Union for animal feed ingredients of 1, 25 and 5 mg kg-1, respectively (EC 2002). However, this data was true at least from the samples taken from Culasi, Antique, Philippines; it is recommended that macrophytes should be sourced from known unpolluted sources. The water hyacinth meal was moderately digestible in the Nile tilapia (76.4%) as an ingredient in the present study. This value was near the apparent protein digestibility values of the diets which included raw water hyacinth leaf meal of 80.1% 82.7% for meal and fermented leaf meal fed to Labeo rohita juveniles, respectively (Saha & Ray 2011). A-Rahman Tibin et al (2012) measured the ADCs of diets containing increasing proportion of water hyacinth meal (10-25%) to have an average value of 62.6%. But it should be noted that these values are ADCs of the diets and not of the ingredient; the present study measured the latter. At any rate, the water hyacinth leaf meal without any treatment process like fermentation (Saha & Ray 2011) or acid/heat treatment (in the present study) could lead to lower ADC values presumably because of the antinutritional factors it contained. Tannin and phytic acid contents in water hyacinth leaf meal were estimated to be 0.98% and 0.42%, respectively (Saha & Ray 2011). Tannins inhibit protease activity and possibly of activities of other digestive enzymes or by forming indigestible complexes with dietary protein (Krogdahl 1989). Phytic acid binds protein and minerals which could cause poor bioavailability of both nutrients (Cain & Garling 1995; Hossain & Jauncey 1989, 1993; Spinelli et al 1983). Similar to other plant proteins, it was expected that there would be an imbalance in the amino acid content of water hyacinth concentrated meal. The most limiting amino acid observed was methionine with an A/E ratio to that of the chicken egg to be 38.9% which was also the chemical score; the second limiting amino acid was lysine (A/E ratio of 72.4%). The observed pattern was similar with the observations from various cereal grains and legume seeds showing either lysine or methionine to be the limiting amino acid (Lieder 1965). It could probably be the result of the acid- and heat-treatment of the water hyacinth leaf meal. Chemical score is based on the assumption that whole egg protein is of the highest biological value and thus the most suitable for growth which could be limited by the EAA in the diet whose ratio to its content in the whole egg protein is the lowest (Hepher 1988). Although chemical score is important in determining the relative value of dietary protein, the other essential amino acid could also have an effect on the nutritive value of the dietary protein as reflected in the essential amino acid index. The EAAI index of the WHLPC was estimated to be 0.88. Oser (1959) developed a criteria for protein quality of feedstuff which was later used by Peñaflorida (1989). The criteria classified good-quality protein sources to have an EAAI greater than or equal to 0.90, useful protein sources to have a value of 0.80, and inadequate when the value was below 0.70. Thus, WHLPC in the present study could be considered a useful protein source but perhaps when supplemented with the most limiting AA or combined with a complimentary protein source could be classified as good quality protein. In general, chemical scores and EAAI are very good indicators of biological value of dietary protein especially when use as sole source of protein. However, animal nutritionist almost always recommend varied sources of protein for reasons that each source could be complimentary with another source which ultimately could balance the essential amino acids. Knowing the EAA profile of a nonconventional protein source is critical in the formulation of a well balanced diet down to its EAA profile.
Conclusions. The present study showed that WHLPC could be a protein source for aquafeeds having a crude protein of more than 20%; it is relatively digestible in the Nile tilapia with ADC for dry matter of 76.4%. When sourced from an unpolluted source, concentrating the leaf meal by acid and heat-treatment resulted in slightly higher levels of heavy metals such as cadmium, copper and lead but were still much lower than the
AACL Bioflux, 2015, Volume 8, Issue 1. 31 http://www.bioflux.com.ro/aacl allowable limit for animal feedstuff set by the European Union Council. Although the chemical score was low (38.9%) due to the most limiting amino acid methionine, its Essential Amino Acid Index of 0.88 resulted in the classification of the WHLPC as a useful protein and was very near the index of 0.90 for good quality protein. Amino acid supplementation or dietary protein complementation could easily make the WHLPC an excellent dietary protein source for aquafeeds.
Acknowledgements. The authors wish to express their gratitude to the Philippine Department of Science and Technology (DOST), Philippine Council for Agriculture, Aquatic and Natural Resources Research and Development (PCAARRD) for the funding and DOST- Accelerated Science and Technology Human Resource Development Program (ASTHRDP) for the scholarship provided to Ms. Hontiveros. They also wish to thank the UPV Office of the Research and Extension for (OVCRE) for the additional funding and publication support.
References
A-Rahman Tibin M. E., Abol-Munafi A. B., Amiza A. M., Khoda Bakhsh H., Adam Sulieman H. M., 2012 Apparent digestibilty coefficient of pelleted fish feed incorporated with water hyacinth (Eichhornia crassipes). Online Journal of Animal and Feed Research 2:30-33. AOAC International, 2003 Official methods of analysis of AOAC International. 17th Edition, 2nd Revision. Gaithersburg, MD, USA, Association of Analytical Communities. Method No. 994.12. Arai S., 1981 A purified test diet for coho salmon Oncorhynchus kisutch fry. Bulletin of the Japanese Society of Scientific Fisheries 47:547-550. Cain K. D., Garling D. L., 1995 Pretreatment of soybean meal with phytase for salmonid diets to reduce phosphorous concentration in hatchery effluents. The Progressive Fish-Culturist 57:114-119. Chavez N. N., Traifalgar R. F., Gonzaga J. V., Corre V. L. Jr., 2014 Growth performance of Penaeus monodon fed diets containing water hyacinth leaf protein concentrate. ABAH Bioflux 6:195-201. Cho C. Y., Cowey C. B., Watanabe T., 1985 Finfish nutrition in Asia: methodological approaches to research and development. Ottawa, Ontario, IDCR-233e, IDRC, Canada, 154 pp. De Silva S. S., Hasan M. R., 2007 Feeds and fertilizers: the key to long-term sustainability of Asian aquaculture. In: Study and analysis of feeds and fertilizers for sustainable aquaculture development. Hasan M. R., Hecht T., De Silva S. S., Tacon A. G. J. (eds), FAO Fisheries Technical Paper, No. 497, Rome, FAO, pp. 19–47. EC, 2002 OJ L 14, 30.5.2002 on undesirable substances in animal feed. Pages 10. Directive 2002L0032: The European Parliament and The Council of the European Union. FAO, 1981 Amino acid contents of food and biological data on protein. Rome, Italy: Food Policy and Food Science Service, Nutrition Division, FAO. Available at: http://www.fao.org/docrep/005/AC854T/AC854T46.htm#chI.I.8. Accessed February 1, 2015. FAO, 2012 The State of World Fisheries and Aquaculture. Rome: FAO. Available at: http://www.fao.org/docrep/016/i2727e/i2727e00.htm. Accessed February 1, 2015. Hepher B., 1988 Nutrition of pond fishes. Cambridge University Press, Cambridge, U.K., 388 pp. Hossain M. A., Jauncey K., 1989 Nutritional evaluation of some Bangladeshi oilseed meals as partial substitutes for fish meal in the diet of common carp, Cyprinus carpio L. Aquaculture Research 20:255-268. Hossain M. A., Jauncey K., 1993 The effects of varying dietary phytic acid, calcium and magnesium levels on the nutrition of common carp, Cyprinus carpio. In: Fish nutrition in practice. Kaushik J., Luquet P. (eds), Paris: INRA Editions, pp. 705-715.
AACL Bioflux, 2015, Volume 8, Issue 1. 32 http://www.bioflux.com.ro/aacl Koprucu K., Ozdemir Y., 2005 Apparent digestibility of selected feed ingredients for Nile tilapia (Oreochromis niloticus). Aquaculture 250:308–316. Krogdahl A., 1989 Alternative protein sources from plants containing anti-nutrients affecting digestion in salmonids. In: Proceedings of the Third International Symposium on Feeding and Nutrition in Fish. Takeda M., Watanabe T. (eds), Toba, Japan, pp. 253-261. Lieder U., 1965 Das Eiweiss der Nahrung der Karpfen. Dtsch Fisch Ztg 12:16-26. Masser M. P., 2000 Aquatic vegetation control. In: Encyclopedia of aquaculture. Stickney R. R. (ed), pp. 51-61. Oser B. L., 1959 An integrated essential amino acid index for predicting the biological value of proteins. In: Protein and amino acid nutrition. Albanese A. A. (ed), Academic Press, New York, USA, pp. 281-295. Peñaflorida V. D., 1989 An evaluation of indigenous protein sources as potential component in the diet formulation for tiger prawn, Penaeus monodon, using essential amino acid index (EAAI). Aquaculture 83:319-330. Saha S., Ray A. K., 2011 Evaluation of nutritive value of water hyacinth (Eichhornia crassipes) leaf meal in compound diets for rohu, Labeo rohita (Hamilton, 1822) fingerlings after fermentation with two bacterial strains isolated from fish gut. Turkish Journal of Fisheries and Aquatic Sciences 11:199-207. Spinelli J., Houle C. R., Wekell J. C., 1983 The effect of phytates on the growth of rainbow trout (Salmo gairdneri) fed purified diets containing varying quantities of calcium and magnesium. Aquaculture 30:71-83. Stone D. A. J., Gaylord T. G., Johansen K. A., Overturf K., Sealey W. M., Hardy R. W., 2008 Evaluation of the effects of repeated fecal collection by manual stripping on the plasma cortisol levels, TNF-α gene expression, and digestibility and availability of nutrients from hydrolyzed poultry and egg meal by rainbow trout, Oncorhynchus mykiss (Walbaum). Aquaculture 275:250-259. Sumagaysay-Chavoso N. S., 2007 Analysis of feeds and fertilizers for sustainable aquaculture development in the Philippines. In: Study and analysis of feeds and fertilizers for sustainable aquaculture development. Hasan M. R., Hecht T., De Silva S. S., Tacon A. G. J. (eds), FAO Fisheries Technical Paper, No. 497, FAO, Rome, pp. 269-308. Valencia G. R., 2012 DOST launches solution to the water hyacinth problem. Available at: http://www.dost.gov.ph/index.php?option=com_content&view=article&id=1104:dos t-launches-solution-to-the-water-hyacinth-problem&catid=1:latest&Itemid=150. Virabalin R., Kositsup B., Punnapayak H., 1993 Leaf protein concentrate from water hyacinth. Journal of Aquatic Plant Management 31:207-209.
Received: 09 January 2015. Accepted: 02 February 2015. Published online: 03 February 2015. Authors: Gaily Jubie S. Hontiveros, College of Fisheries, Mindanao State University, General Santos City, Philippines, e-mail: [email protected] Augusto Erum Serrano Jr., University of the Philippines Visayas, National Institute of Molecular Biology and Biotechnology, Philippines, Miagao, Iloilo 5023, e-mail: [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. How to cite this article: Hontiveros G. J. S., Serrano Jr. A. E., 2015 Nutritional value of water hyacinth (Eichhornia crassipes) leaf protein concentrate for aquafeeds. AACL Bioflux 8(1):26-33.
AACL Bioflux, 2015, Volume 8, Issue 1. 33 http://www.bioflux.com.ro/aacl AACL BIOFLUX Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society
Effect of fish meal replacement by blood meal in fingerling rainbow trout (Oncorhynchus mykiss) on growth and body/fillet quality traits Roghayeh Bahrevar, Hamid Faghani-Langroudi
Department of Fisheries, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran. Corresponding author: H. Faghani-Langroudi, [email protected]
Abstract. A feeding trial was conducted to evaluate the potential of replacing fish meal by processed blood meal, in practical diets for fingerling rainbow trout Oncorhynchus mykiss. Fish meal was replaced by 0%, 10%, 20% and 30% of blood meal. The diet with 0% blood meal was used as control. Fingerling rainbow trout were reared in 12 ponds. Each dietary treatment was tested in groups of 30 fish per pond arranged in a completely randomized design. Fish were fed by the diets for 8 weeks. Percentage weight gain, specific growth rate, survival rate, feed conversion ratio and body composition of fish were estimated. There were significant differences (p < 0.05) in growth performance among fish fed by diets (0–30% fish meal replacement) with those fed diet control as feed. No significant difference was observed in survival rate among fish fed by the experimental diets. These results showed that blood meal is not a suitable protein source as fish meal replacement for fingerlings rainbow trout. Growth, nutrient utilization and body composition were either not improved or were significantly influenced by gradually replacing fish meal by blood meal. Key Words: FCR, SGR, diet, protein source, carcass analyze.
Introduction. In recent years, many researchers have tried different kind of animal meal (bone meal, blood meal, hemoglobin meal, poultry meal, and meat meal) to substitute fish meal in diets and the percentage of substitution has varied according to species, fish size and feeding habits (Zhou et al 2004; Barnes et al 2012; Dedeke et al 2013). Initially, the alternative raw materials were selected because they were less expensive and more available than fish meal and fish oil, but it is currently also necessary to consider raw materials with an adequate balance in amino acid profile, good digestibility, high level protein content, and a suitable palatability be considered good protein sources in fish diets (Lunger et al 2007; Antolović et al 2012; Bayraktar & Bayır 2012). Fish meal is a major protein source in aqua feeds, especially for carnivorous fish species because it is an excellent source of essential nutrients such as indispensable amino acids, essential fatty acids, vitamins, minerals, attractants and unknown growth factors (Zhou et al 2004; Sheen et al 2014). However, increasing demand, unstable supply and high price of the fish meal with the expansion of aquaculture made it necessary to search for alternative protein sources (Lunger et al 2007; FAO 2010). Blood meal is one of the alternative protein sources for fish meal in diets for many fish species, due to its high protein content, reasonable price and steady supply (El- Sayed 1999). Blood meal, a lysine-rich ingredient (6–8% lysine), is produced using a wide variety of processing techniques (Bureau et al 1999). Some researchers have tried a mixture of vegetable and animal meals, e.g., Martínez-Llorens et al (2008), who replaced 10% of fish meal by blood or hemoglobin meal, with a constant quantity of poultry meal and soybean meal, and obtained better results with a 5% blood meal inclusion. In most studies, commercial fish weight is not reached, so the effect of protein source dietary inclusion on sensorial changes in fish muscle cannot be evaluated. Some authors reported this effect on fillet quality traits (De Francesco et al 2007; Martínez-Llorens et al 2009). Estimates of lysine availability in blood meals appear to be quite highly variable across species and/or studies, even for the
AACL Bioflux, 2015, Volume 8, Issue 1. 34 http://www.bioflux.com.ro/aacl same type of drying technique. The objective of the present study is evaluating effects of fish-meal replacement by blood-meal on growth performance and carcass analyze, in diets for fingerling rainbow trout (Oncorhynchus mykiss).
Material and Method. A feeding trial was conducted at the reproduction and breeding center of rainbow trout located in Tonekabon, north of Iran, using fingerling rainbow trout, O. mykiss. The study was done from February to April 2014. In total 360 fingerlings were acclimatized to the rearing conditions for a 1-week period prior to the feeding trial. For the eight week feeding trial, homogenous groups of 30 rainbow trout with an average initial weight of 10±1g (Mean ± SD) were randomly distributed among twelve ponds. Each pond was supplied with a water flow of 1.5-2 L S-1. During the experiment period, the quality and quantity of water including oxygen, pH and temperature, were measured and controlled. Biometry was performed at the end of each week. Formulation of diets according to then nutritional requirements of fish was determined by the UFFDA software (Table 1). Amount of energy and protein in diets were considered identical.
Table 1 Composition of the experimental diets on a dry matter basis in 100 g dry diet
Diets (replacement percentage) Ingredients 1 (0%) 2 (10%) 3 (20%) 4 (30%) Fish meal 50.00 45.00 40.00 35.00 Blood meal 0.00 5.00 10.00 15.00 Meat meal 13.86 16.60 20.21 23.20 Wheat meal 2.59 2.29 2.20 2.72 Maize meal 27.09 24.65 20.13 16.62 Maize oil 4.00 4.00 5.00 5.00 Pellet binder 1.00 1.00 1.00 1.00 Mineral mix 0.50 0.50 0.50 0.50 Vitamin mix 0.50 0.50 0.50 0.50 Collin chloride 0.36 0.36 0.36 0.36 Antioxidant 0.10 0.10 0.10 0.10
At the end of the feeding trials, 8 specimens of each treatment were used for carcass analyze. Analyses of crude protein, moisture and ash in the diets were performed by standard methods (AOAC 2000). Dietary lipid was determined by the method of Kinsella et al (1977). Growth, biometric and economic indexes considered were as follows (Xue et al 2006): body weight increase (BWI) = 100 (W1 – W0) (W0)-1; growth rate (GR) = (W1–W0) (t)−1; feed conversion ratio (FCR) = [feed offered (g)] [weight gain (g)]-1; specific growth rate (SGR) = 100 (lnW1–lnW0) (t)−1; condition factor (CF) = 100 (W1) (FL³)-1; survival rate (SR) = 100 (N1) (N0)-1; final body weight (FBW) = W1–W0. Where W0 and W1 were wet body weight of fish at the start and end of the experiment, respectively; FL and t were fork length and duration of experiment (8 weeks), respectively; N0 and N1 were number of fish at the start and end of the experiment, respectively. Statistical analyses of data were carried out with SPSS 16.0 software package (SPSS; Chicago, IL). Normality was tested using Kolmogorov-Smirnoff test. Homogeneity was checked using the absolute residuals according to Levene’s test. Effect of treatment was carried out using one-way ANOVA followed by a post hoc Duncan's multiple range test. In all statistical tests used, p < 0.05 was considered statistically different.
Results. The fish readily accepted all diets. At the end of 8 weeks of the feeding trial, negative effects on growth performance were observed when 10% of fish meal protein was replaced by blood meal protein. Also FCR was increased during the feeding trial. Significant differences were found in weight gain, FCR, SGR, GR, CF and FBW when the replacement level for fish meal protein was increased from 0 to 10% (Table 2). The percentages of BWI were significantly decreased from 19.32±4.06 to 14.13±2.30 after 8
AACL Bioflux, 2015, Volume 8, Issue 1. 35 http://www.bioflux.com.ro/aacl weeks, when the replacement level for fish meal protein was increased from 0 to 30% (Table 2). Among the treatments, numerically the higher growth performance was found in fish fed fish meal free diet (blood meal 0%). Similarly, SGR, CF, and PR were also higher in blood meal 0% group and these parameters were significantly different between the treatments. No difference was also found in SR among different treatments. Survival ranged from 100% to 99.72% in fish fed the test diets and there were no differences among treatments at the end of the feeding trial.
Table 2 Growth performance of fingerlings rainbow trout fed by different levels of fish and blood meals in experimental diets for 8 experimental weeks
Diet group (replacement percentage) Growth factor 1 (0%) 2 (10%) 3 (20%) 4 (30%) BWI 19.32±4.06c 17.64±3.58b,c 16.02±3.14a,b 14.13±2.30a SGR 1.09±0.21c 1.00±0.18a,b 0.92±0.17b 0.82±0.12a FCR 1.06±0.05a 1.18±0.06b 1.31±0.06c 1.52±0.07d SR 100.00±0.00a 100.00±0.00a 99.86±0.68a 99.72±0.96a GR 0.55±0.14d 0.47±0.11c 0.41±0.08b 0.33±0.07a CF 1.65±0.04d 1.43±0.05c 1.32±0.05b 1.04±0.14a FBW 40.94±1.32d 36.59±1.27c 32.75±1.40b 27.81±0.71a Different letters indicate significant differences (p ≤ 0.05).
The whole body proximate composition of fish at the start and end of the feeding trial are shown in Table 3. All the fish showed a change in the analyzed parameters compared to those of the initial values. There was significant difference (p < 0.05) in the final whole body proximate composition among the groups of fish fed the different experimental diets (Table 3). Table 3 Proximate composition of fingerlings rainbow trout fed by different levels of fish and blood meals in experimental diets for 8 experimental weeks
Diet group (replacement percentage) Carcass analyze 1 (0%) 2 (10%) 3 (20%) 4 (30%) Ash 2.20±0.50a 2.77±0.22b 1.74±0.12a 1.69±0.02a Moisture 72.99±1.00a 74.44±1.29a,b 75.08±1.01a,b 75.51±1.00b Lipid 5.69±0.01c 5.63±0.01b 5.62±0.01a,b 5.61±0.01a Protein 17.24±0.01a 17.55±0.00b 17.78±0.00c 17.77±0.00c Different letters indicate significant differences (p ≤ 0.05).
Discussion. Fishery products, either in the form of low-value trash fish or rendered as fish meal, are presently the major sources of protein in the grow-out culture of most fish species and constitutes up to 70% by weight of their diet (Tacon 1995). As the demand for fish meal and marine fishery products for aquaculture increases while their availability decreases, the cost is expected to rise (Tacon 1995; Fasakin et al 2005). Reduction of the fish meal dependency is becoming more important for the sustainability and profitability of commercial fish farming. Individual rendered animal protein meals, such as blood meal often have deficiencies or excesses in essential amino acids that may affect the overall productivity of cultured fish (Fasakin et al 2005). Moreover, these diets are not always representative of what is commonly used in the industry and the use of rendered animal protein ingredients needs to be evaluated in more practical diets. This study has demonstrated that replacement of fish meal protein by processed animal by- product meals for example blood meal allowed growth rates reduce than those exhibited by the control groups. Obtained results indicated the fingerling rainbow trout readily accepted the diets at all levels of fish meal replacement by blood meals as shown by the high feed conversion ratios of 1.06–1.52. This conforms to another study where diets with inclusion levels of meat meal ranging from 30 to 70% as substitute for fish meal have been readily accepted by rainbow trout (Watanabe et al 1993). Possible reasons for the reduced
AACL Bioflux, 2015, Volume 8, Issue 1. 36 http://www.bioflux.com.ro/aacl growth of fingerling rainbow trout at total replacement by blood meal may be due to deficiencies in essential nutrients such as essential amino acids. The dietary essential amino acid requirements of rainbow trout have not been defined. In the absence of amino acid requirement data, the amino acid profile of the whole body tissue of the animal has been used as an index of the essential amino acid requirements (Wilson & Poe 1985). Fish meals, in general, have been reported to have good essential amino acid profiles for fish (Cuzon & Guillaume 1991). On the other hand, the animal by-product meals were lower in some essential amino acids (methionine, lysine, and isoleucine). Deficiencies in essential amino acids may explain the decline in growth performance of fingerlings rainbow trout particularly at full replacement levels of fish meal by blood meal. Other possible explanation for the low performance at increasing levels of fish meal substitution by blood meal may be the resulting effect on diet digestibility. Several authors have mentioned that poor growth and feed utilization of fish fed feeds containing alternatives of fish meal e.g., spray-dried blood meal, may be due to low protein digestibility and essential amino acid deficiency (mainly to deficient processing of rendered meals) (Bureau et al 1999; De Francesco et al 2007; Martínez-Llorens et al 2008, 2009). Millamena (2002) has shown that apparent digestibility values of blood meal were generally lower than for fish meals for Epinephelus coioides. From the economic standpoint, replacement of fish meal by cheaper animal by- product meals in a practical diet for rainbow trout can alleviate the problem of low fish meal availability and high cost. Fish survival was not affected by experimental diets and similar results were obtained by Millamena (2002). The results of this study clearly demonstrated that growth performances of fish in the dietary treatments in the present experimental condition were not satisfactory. The growth rate was comparable to that observed in other studies (Sevgili & Erturk 2004; Azevedo et al 2004; Kikuchi et al 2007). In this study, the whole body proximate composition of fish at the start and end of the feeding trial showed a change in the analyzed parameters compared to those of the initial values. There was significant difference in the final whole body proximate composition among the groups of fish fed the different experimental diets. The increasing in moisture content from 72.99% to 75.51% and the decreasing in ash content from 2.20% to 1.69% with increasing levels of blood meal were reflected in the proximate analysis of the diets. This increase of moisture level after 8 weeks revealed decreasing levels of lipid.
Conclusions. Based on overall performances of fish, blood meal is not a suitable protein source as fish meal replacement for fingerling rainbow trout. Growth, nutrient utilization and body composition were either not improved or were significantly influenced by gradually replacing fish meal with blood meal. Further studies to determine the long-term effect on the performance of fish fed the fish meal replacement diet under on-farm conditions is suggested.
References
Antolović N., Kožul V., Antolović M., Bolotin J., 2012 Effects of partial replacement of fish meal by soybean meal on growth of juvenile saddled bream (Sparidae). Turkish Journal of Fisheries and Aquatic Sciences 12:247-252. AOAC (Association of Official Analytical Chemists), 2000 Association of official analytical Chemists. 17th edition, AOAC, Washington, DC, pp. 21-447. Azevedo P. A., Lesson S., Cho C. Y., Bureau D. P., 2004 Growth, nitrogen and energy utilization of juvenile from four salmonid species: diet, species and size effects. Aquaculture 234:393-414. Barnes M. E., Brown M. L., Rosentrater K. A., Sewell J. R., 2012 An initial investigation replacing fish meal with a commercial fermented soybean meal product in the diets of juvenile rainbow trout. Open Journal of Animal Sciences 2:234-243.
AACL Bioflux, 2015, Volume 8, Issue 1. 37 http://www.bioflux.com.ro/aacl Bayraktar K., Bayır A., 2012 The effect of the replacement of fish oil with animal fats on the growth performance, survival and fatty acid profile of rainbow trout juveniles, Oncorhynchus mykiss. Turkish Journal of Fisheries and Aquatic Sciences 12:661- 666. Bureau D. P., Harris A. M., Cho C. Y., 1999 Apparent digestibility of rendered animal protein ingredients for rainbow trout (Oncorhynchus mykiss). Aquaculture 180:345- 358. Cuzon G., Guillaume J., 1991 Recommendations for practical feed formulation. In: The Crustacean Nutrition Newsletter. Castell J. D., Corpron K. E. (eds), The International Working Group on Crustacean Nutrition, Halifax, Nova Scotia, Canada B3J 2S7, pp. 52–60. Dedeke G. A., Owa S. O., Olurin K. B., Akinfe A. O., Awotedu O. O., 2013 Partial replacement of fish meal by earthworm meal (Libyodrilus violaceus) in diets for African catfish, Clarias gariepinus. International Journal of Fisheries and Aquaculture 5(9):229-233. De Francesco M., Parisi G., Pérez-Sánchez J., Gomez-Requeni F., Medale F., Kaushik S. J., Mecatti M., Poli B. M., 2007 Effect of high-level fish meal replacement by plant proteins in gilthead sea bream (Sparus aurata) on growth and body/fillet quality traits. Aquaculture Nutrition 13:361–372. El-Sayed A. F. M., 1999 Alternative dietary protein sources for farmed tilapia, Oreochromis spp. Aquaculture 179:149-168. FAO, 2010 The state of the world fisheries and aquaculture. FAO Fisheries and Aquaculture Department. Food and Agriculture Organization of the United Nation. Rome, Italy, 218 pp. Fasakin E. A., Serwata R. D., Davies S. J., 2005 Comparative utilization of rendered animal derived products with or without composite mixture of soybean meal in hybrid tilapia (Oreochromis niloticus × Oreochromis mossambicus) diets. Aquaculture 249:329-338. Kikuchi K., Furuta T., Ishizuka H., Yanagawa T., 2007 Growth of tiger puffer, Takifugu rubripes, at different salinities. Journal of the World Aquaculture Society 38:427– 434. Kinsella J. E., Shimp J. L., Mai J., Weihrauch J., 1977 Fatty acid content and composition of freshwater finfish. Journal of the American Oil Chemists' Society 54:424-429. Lunger A. N., McLean E., Craig S. R., 2007 The effects of organic protein supplementation upon growth, feed conversion and texture quality parameters in juvenile cobia (Rachycentron canadum). Aquaculture 264:342–352. Martínez-Llorens S., Tomás Vidal A., Garcia I. J., Torres M. P., Cerda M. J., 2009 Optimum dietary soybean meal level for maximizing growth and nutrient utilization of on-growing gilthead sea bream (Sparus aurata). Aquaculture Nutrition 15:320- 328. Martínez-Llorens S., Tomás Vidal A., Moñino A. V., Ader J. G., Torres M. P., Cerda M. J., 2008 Blood and haemoglobin meal as protein sources in diets for gilthead sea bream (Sparus aurata): effects on growth, nutritive efficiency and fillet sensory differences. Aquaculture Research 39:1028–1037. Millamena O. M., 2002 Replacement of fish meal by animal by-product meals in a practical diet for grow-out culture of grouper Epinephelus coioides. Aquaculture 204:75-84. Sevgili H., Erturk M. M., 2004 Effects of replacement of fish meal with poultry by-product meal on growth performance in practical diets for rainbow trout, Oncorhynchus mykiss. Akdeniz Universitesi Ziraat Fakultesi Dergisi 17(2):161-167. Sheen S. S., Chen C. T., Ridwanudin A., 2014 The effect of partial replacement of fish meal protein by dietary hydrolyzed fish protein concentrate on the growth performance of orange-spotted grouper Epinephelus coioides. Journal of Aquaculture and Marine Biology 1(2):00006. DOI: 10.15406/jamb.2014.01.00006. Tacon A. G. J., 1995 The potential for fish meal substitution in aquafeeds. Infofish International 3:29–34.
AACL Bioflux, 2015, Volume 8, Issue 1. 38 http://www.bioflux.com.ro/aacl Watanabe T., Pongmaneerat J., Sato S., Takeuchi T., 1993 Replacement of fish meal by alternative protein sources in rainbow trout diets. Nippon Suisan Gakkaishi 59:1573–1579. Wilson R. P., Poe W. E., 1985 Relationships of whole body and egg essential amino acid pattern to amino acid requirement patterns in channel catfish, Ictalurus punctatus. Comparative Biochemistry and Physiology 80B:385–388. Xue M., Luo L., Wu X., Ren Z., Gao P., Yu Y., Pearl G., 2006 Effects of six alternative lipid sources on growth and tissue fatty acid composition in Japanese sea bass (Lateolabrax japonicus). Aquaculture 260:206-214. Zhou Q. C., Tan B. P., Mai K. S., Liu Y. J., 2004 Apparent digestibility of selected feeding ingredients for juvenile cobia Rachycentron canadum. Aquaculture 241:441–451.
Received: 03 January 2015. Accepted: 27 January 2015. Published online: 01 February 2015. Authors: Roghayeh Bahrevar, Department of Fisheries, Tonekabon Branch, Islamic Azad University, P. O. Box: Tonekabon 46815-559, Tonekabon, Iran, e-mail: [email protected] Hamid Faghani-Langroudi, Department of Fisheries, Tonekabon Branch, Islamic Azad University, P. O. Box: Tonekabon 46815-559, Tonekabon, Iran, e-mail: [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. How to cite this article: Bahrevar R., Faghani-Langroudi H., 2015 Effect of fish meal replacement by blood meal in fingerling rainbow trout (Oncorhynchus mykiss) on growth and body/fillet quality traits. AACL Bioflux 8(1):34-39.
AACL Bioflux, 2015, Volume 8, Issue 1. 39 http://www.bioflux.com.ro/aacl AACL BIOFLUX Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society
Food preference and diet overlap of two endemic and threatened freshwater fishes, depik (Rasbora tawarensis) and kawan (Poropuntius tawarensis) in Lake Laut Tawar, Indonesia 1Zainal A. Muchlisin, 1Fakhrurrazi Rinaldi, 2Nur Fadli, 3Muhammad Adlim, 4,5Mohd N. Siti-Azizah
1 Department of Aquaculture, Faculty of Marine and Fisheries, Syiah Kuala University, Banda Aceh 23111, Indonesia; 2 Department of Marine Sciences, Faculty of Marine and Fisheries, Syiah Kuala University, Banda Aceh 23111, Indonesia; 3 Department of Chemistry, Faculty of Teaching Training and Education, Syiah Kuala University, Banda Aceh 23111, Indonesia; 4 School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia; 5 Centre for Marine and Coastal Studies, Universiti Sains Malaysia, Penang, Malaysia. Corresponding author: Z. A. Muchlisin, [email protected]
Abstract. Depik (Rasbora tawarensis) and kawan (Poropuntius tawarensis) are endemic and threatened fishes occurring in Lake Laut Tawar, Indonesia. The populations of both depik and kawan have been declining sharply over the last few decades. Several biological aspects of both species have been previously reported. However, there is no information on the feeding biology of depik and kawan, hence here we present the evaluation of feeding biology of these fishes. Sampling was conducted in July 2013 using gillnets. A total of 42 depik and 45 kawan fishes were examined. The food occurrence, dietary shift, diet similarity and diet overlap were evaluated in this study. Phytoplankton (especially algae) and zooplankton were the primary and secondary food sources, respectively. However, their favorite food were Closteriopsis longissima and Arcella vulgaris. Higher food similarity was observed between length classes of 55-70 mm and 71-85 mm for depik; and between 71-85 mm and 101-115 mm for kawan. It was concluded that the depik and kawan are plankton feeder (planktonphagous). Based on diet overlap index, there is a moderate degree of overlapping of food preference between depik and kawan. Key Words: plankton feeder, food similarity, diet overlap, stomach content, Closteriopsis longissima, Arcella vulgaris.
Introduction. At least 11 species of freshwater fishes have been recorded in Lake Laut Tawar where two of them are endemic to this lake namely depik - Rasbora tawarensis, and kawan - Poropuntius tawarensis (Muchlisin & Siti-Azizah 2009). Laut Tawar is the biggest lake in the Aceh Province originating from an old volcanic caldera. The lake is a focal point of the local people providing water resources for domestic needs, agricultural, and aquaculture activities as well as industries. Economic and conservation evaluations have shown that both species are economically and ecologically important fishes in Aceh waters, Indonesia (Muchlisin 2013). However, both fishes are listed in the IUCN red list as vulnerable (IUCN 1990) and updated by CBSG as critically endangered (CBSG 2003) due to ecological perturbation, pollution, introduced species and unfriendly fishing practices in Lake Laut Tawar. The depik population has been decreasing sharply during the last two decades (Muchlisin et al 2011b) and according to local fishermen; kawan is also becoming very rare and difficult to catch in recent times. Depik and kawan have the potential to be cultured as food and ornamental fishes, hence information on the bio-ecology of these fishes is crucial in the food and aquarium fishery industries. The growth pattern and reproductive biology of these species have been extensively investigated (Muchlisin et al 2010a; 2010b; 2011a; 2011b). However, there is no report on their feeding habits. In the aquaculture point of view, information
AACL Bioflux, 2015, Volume 8, Issue 1. 40 http://www.bioflux.com.ro/aacl on the feeding habit is crucially needed when intiating a systematic programme for the development of feeding and breeding technologies starting from wild populations (Vine 1998). Feeding provides the source of nutrition and energy for growth, reproduction and other physiological activities in most living organisms including fish. Nyunja et al (2002) reported that besides influence on growth, feed also plays an essensial role in fish abundance, migration and distribution, and therefore information on the feeding habit and feeding interaction among species are crucially important in relation to strategize a better conservation program (Balik et al 2003). In fish as in many other organisms, feeding habits show ontogenetic variation (Weliange & Amarasinghe 2003) and seasonal changes in food availability and ontogenetic dietary shifts can affect both predator–prey and competitive interactions amongst size-structured fish communities (Winemiller 1989). Diet analysis is also necessary to demonstrate the trophic overlap among species within a community (Bascinar & Saglam 2009). This information is essential in determining the intensity of the interspecific interaction in fish community (Morte et al 2001). The knowledge of feeding biology is useful to select such species of fish for developing culture program and produce an optimum yield by utilizing all the available potential food sources in the water bodies without any competition or predation (Manon & Hossain 2011) and also useful in determining of policy on the introducing fish species into a new environment for fishing or recreational purposes (Muchlisin 2011c, 2012). The study of the food and feeding habits of fish is a subject of continuous research because it constitutes the basis for the development of a successful fisheries management program on fish capture and culture (Oronsaye & Nakpodia 2005). The feeding biology of several tropical fishes have been well documented, for example: Sardinella gibbosa (Nyunja et al 2002), Sargochromis codringtonii (Moyo 2004), Chrysichthys nigrodigitatus and Brycinus nurse (Oronsaye & Nakpodia 2005), Oreochromis niloticus and Sarotherodon galilaeus (Oso 2006), Garra cambodgiensis and Mystacoleucus marginatus (Mazlan et al 2007), Loricaria lentiginosa (Salvador et al 2009). Here, we report preliminary observations of feeding habits of the two endemic fishes (depik and kawan) in Lake Laut Tawar, Indonesia.
Material and Method
Sampling. Fish samples were collected on July 2013 using a series of mesh size of experimental gillnets (1-3 cm) from several sites in the lake at 04°36′43″N 096°55′25″E (Figure 1).
Figure 1. The map of northern part of Lake Laut Tawar showing the sampling location.
AACL Bioflux, 2015, Volume 8, Issue 1. 41 http://www.bioflux.com.ro/aacl A total of 32 depik and 35 kawan fishes were caught during the sampling. Collected fishes were counted, rinsed and anesthetized in a solution of Tricaine Methanesulfonate (MS222), then after preserved in 10% formalin. The sampled fishes were transported to laboratory for further analysis.
Stomach content analysis. All fish samples were analyzed for stomach contents in Laboratory of Ichthyology of Syiah Kuala University, Indonesia. The total lengths of fish samples were measured and weighed to the nearest 0.1 mm and 0.01 g, respectively. The specimens were abdominally dissected by using a pair of surgical scissors, and then their stomachs were taken and weighed to the nearest to 0.01 g and then dried at room temperature for two hours. The stomachs were measured for the length and then dissected, and the contents emptied into a petri-dish. The stomach contents were added with 10 mL sterile tap water and thoroughly mixed. One drop of stomach content solution was transferred into a sedge wick rafter cell using a teat pipette. The food items were then enumerated under a compound microscope. Each food item was identified to the lowest possible taxonomic level. The identification of food item (plankton) was based on Prescott (1978), Verlencar & Desai (2004), and Bellinger & Sigee (2010).
Food occurrence. The food items were isolated and grouped based on their types and enumerated using a microscope. The occurrence of each food item was scored and then converted to a percentage of the ratio of the number of times an item occurred to the total number of guts analyzed. The percentage abundance of each food item was also computed from the ratio of the number of a particular item in the stomach to the total number of items in the stomach.
Dietary shifts, diet overlap and food similarity. To evaluate the ontogenic shift in dietary preference, the sampled fishes were divided into four length classes i.e. (a) 55-70 mm, (b) 71-85 mm, (c) 86-100 mm, and (c) 101-115 mm. The food compositions of each class were evaluated and compared. Diet overlap between depik and kawan was determined using Schoener’s diet overlap index (Schoener 1970):
Cxy = 100–0.5 (Σlpxi-pyil), th where Cxy is the degree of overlap, pxi is the proportion of the i resource (in this case, th prey type) used by species x, and pyi is the proportion of the i prey type used by species y, and the vertical bars indicate absolute positive values of the difference. Index values range from 0 to 100; with a value of 0 indicating no overlap and a value of 100 indicating complete overlap. Diet overlap values above 60 were considered biologically significant overlap (Wallace 1981). The food similarity between length classes of every species were examined using Plymouth Routines in Multivariate Ecological Research package (PRIMER- E) software (Clarke & Gorley 2006).
Results and Discussion. The results showed that depik and kawan fed primarily on planktons; both phytoplankton and zooplankton, therefore these fishes can be categorized as plankton feeders or planktonphagous. A total of 21 species of planktons were identified in the stomachs of kawan where algae were predominant, while 16 species of planktons were recorded in the depik stomach. Similarly, algae were also the predominant food items for depik. The food occurrence analysis in depik showed that Closteriopsis longissima was the most frequently found (50% of fish samples), followed by Arcella vulgaris (28.6% of fish samples). C. longissima, Cocconeis sp. and A. vulgaris were the major food items in the stomachs of kawan with occurrence of 48.57%, 45.71% and 40.0%, respectively (Table 1). The predominant food item in the length class of 55-70 mm and 71-85 mm of depik was C. longissima. However, the food preference was for Trachelomonas curta at the length class of 86-100 mm and A. vulgaris at the length class of 101-115 mm. C. longissima was found at all length classes, moreover Alona guttata was also recorded at all length classes except at 101-115 mm length class. In addition, Dinophysis sp. was found at length class of 86-100 mm and 101-115 mm, while Aphanothece nidulans was only found at 101-115 mm length class (Table 2). In the kawan fish, A. vulgaris, C.
AACL Bioflux, 2015, Volume 8, Issue 1. 42 http://www.bioflux.com.ro/aacl longissima and Cocconeis sp. were predominant food items at length class of 71-85 mm, 86-100 mm and 101-115 mm. Several food items were specific to certain length classes; Didymosphenia geminata was only found at 71-85 mm length class, Biddulphia sp. and Canthocamptus sp. were only found at 86-100 mm, while Anuraeopsis fissa was found at length classes of 86-100 mm and 101-115 mm (Table 2). The higher similarity of food items in the depik was found between length class of 55-70 mm and 71-85 mm (54.86%; Table 3). In the kawan, the highest food similarity was found between 71-85 mm and 101-115 mm (59.11%), followed by 86-100 mm and 101-115 mm (57.03%; Table 4). Diet overlap index between depik and kawan was 62.15 indicating a moderate degree of competition in food preference of both endemic species. Detailed information of diet overlap is presented in Table 5. Observation on the gill structures of depik and kawan revealed that both species have high densities of gill rakers; probably the gill rakers function to filter planktons from the water which enters through the mouth and out through the gills. The favorite food of depik and kawan were C. longissima and A. vulgaris. Cocconeis sp. was also preferred by kawan fish, in contrast to the depik where this food item was not found in its stomach. This indicates that depik and kawan prefer C. longissima and A. vulgaris as their primary food sources. We postulate that these planktons were the most abundant in Lake Laut Tawar as had been reported by Nurfadillah et al (2012).
Table 1 Occurence of food items (planktons) in the depik and kawan stomachs
Depik (n = 32) Kawan (n = 35) Food items (plankton) Occurence % Occurence % Alona guttata 4 12.50 - - Amphora normani 1 3.13 - - Amphorellopsis sp. - - 1 2.86 Anuraeopsis fissa - - 3 8.57 Aphanothece nidulans 1 3.13 - - Arcella vulgaris 9 28.13 14 40.00 Biddulphia sp. - - 1 2.86 Brachionus sp. 2 6.25 - - Canthocamptus sp. - - 1 2.86 Chalotrix sp. 1 3.13 1 2.86 Closterium gracile 1 3.13 - - Closteriopsis longissima 16 50.00 17 48.57 Cocconeis sp. - - 16 45.71 Cyclotella sp. - - 1 2.86 Cymatopleura sp. - - 1 2.86 Dictyosphaerium sp. - - 1 2.86 Didymosphenia geminata - - 1 2.86 Dinophysis sp. 2 6.25 1 2.86 Fragilaria sp. - - 1 2.86 Melosira italica 1 3.13 - - Microcystis aeruginosa 1 3.13 2 5.71 Netrium oblongum 1 3.13 - - Oscillatoria sp. - - 1 2.86 Paramecium caudatum 2 6.25 - - Peridinium sp. - - 1 2.86 Phacus curvicauda 2 6.25 1 2.86 Pleurotaenium trabecula 1 3.13 - - Stephanodiscus sp. - - 10 28.56 Tetmemorus laevis - - 1 2.86 Trachelomonas curta 3 9.38 3 8.57
AACL Bioflux, 2015, Volume 8, Issue 1. 43 http://www.bioflux.com.ro/aacl Table 2 The stomach content of depik and kawan according to length classes
Length Food items of depik Food items of kawan classes 50-70 mm Alona guttata, n/a Amphora normani, Arcella vulgaris, Chalotrix sp., Closterium gracile, Closteriopsis longissima, Netrium oblongum, Paramecium caudatum, Pleurotaenium trabecula 71-85 mm Alona guttata, Arcella vulgaris, Arcella vulgaris, Didymosphenia geminata, Closteriopsis longissima, Closteriopsis longissima, Melosira italica Cocconeis sp., Stephanodiscus sp., Trachelomonas curta 86-100 mm Alona guttata, Amphorellopsis sp., Closteriopsis longissima, Anuraeopsis fissa, Dinophysis sp., Arcella vulgaris, Microcystis aeruginosa, Biddulphia sp., Phacus curvicauda, Canthocamptus sp., Trachelomonas curta Closteriopsis longissima, Cocconeis sp., Cyclotella sp., Dictyosphaerium sp., Dinophysis sp., Microcystis aeruginosa, Peridinium sp., Phocus curvicauda, Stephanodiscus sp., Tetmemorus laevis, Trachelomonas curta 101-115 mm Aphanothece nidulans, Anuraeopsis fissa, Arcella vulgaris, Arcella vulgaris, Closteriopsis longissima, Chalotrix sp., Dinophysis sp., Closteriopsis longissima, Phocus curvicauda, Cocconeis sp., Trachelomonas curta Dictyosphaerium sp., Fragilaria sp., Microcystis aeruginosa, Oscillatoria sp., Stephanodiscus sp., Trachelomonas curta
Table 3 The similarity of food items (%) of depik fish among length classes
Length classes (mm) 55-70 71-85 86-100 101-115 55-70 71-85 54.86 86-100 18.1 29.44 101-115 34.64 44.92 46.47
AACL Bioflux, 2015, Volume 8, Issue 1. 44 http://www.bioflux.com.ro/aacl Table 4 The similarity of food items (%) of kawan fish among length classes
Classes length (mm) 71-85 86-100 101-115 71-85 86-100 51.67 101-115 59.11 57.03
Table 5 th Diet overlap between depik and kawan fish in Lake Laut Tawar. Pxi is the proportion of the i th prey type fed by kawan fish, pyi is the proportion of the i prey type fed by depik fish
Food items Pxi (kawan) Pyi (depik) Alona guttata - 12.50 Amphora normani - 3.13 Amphorellopsis sp. 2.86 - Anuraeopsis fissa 8.57 - Aphanothece nidulans - 3.13 Arcella vulgaris 40.00 28.13 Biddulphia sp. 2.86 - Brachionus sp. - 6.25 Canthocamptus sp. 2.86 - Chalotrix sp. 2.86 3.13 Closterium gracile - 3.13 Closteriopsis longissima 48.57 50.00 Cocconeis sp. 45.71 - Cyclotella sp. 2.86 - Cymatopleura sp. 2.86 - Dictyosphaerium sp. 2.86 - Didymosphenia geminata 2.86 - Dinophysis sp. 2.86 6.25 Fragilaria sp. 2.86 - Melosira italica - 3.13 Microcystis aeruginosa 5.71 3.13 Netrium oblongum - 3.13 Oscillatoria sp. 2.86 - Paramecium caudatum - 6.25 Peridinium sp. 2.86 - Phacus curvicauda 2.86 6.25 Pleurotaenium trabecula - 3.13 Stephanodiscus sp. 28.57 - Tetmemorus laevis 2.86 - Trachelomonas curta 8.57 9.38 Total 225.74 150.05 Cxy = 100 – 0.5 ( Pxi – Pyi ) Cxy = 100 – 0.5 (75.69) Cxy = 100 – 37.85 Cxy = 62.15
Comparison of total body length to alimentary tract length of depik fish was 1.2 (i.e. the alimentary tract length was 1.2 times of total body length), while in the kawan fish was 1.5, indicating an omnivorous feeding habit pattern where both kawan and depik consume zooplankton and phytoplankton, while algae were predominant in stomachs of the fish samples. According to Huet (1971), omnivorous fishes have alimentary tract slightly longer than their body length as observed in this study. A similar study on bada fish - Rasbora argyrotaenia from Musi River, South Sumatera was conducted by Arsyad & Syaefudin (2010). They reported that bada fish fed on algae especially Ulothrix sp. as its primary food item. This report corresponds to our study in that both depik and kawan fed on algae but the preferred food item were different; C. longissima and A. vulgaris were
AACL Bioflux, 2015, Volume 8, Issue 1. 45 http://www.bioflux.com.ro/aacl the foremost food items for depik and kawan, probably due to these food items being the most abundant in Lake Laut Tawar as mentioned earlier. While the feeding habit of fishes are species dependent, the availability of food in the environment, also plays a major role. However, some species show selectivity on one or two types of food. According to Nikolsky (1963) food availability determines the population size, population dynamic, growth and condition of fish. Based on food item occurrence of food, it is shown that depik and kawan fed on algae more frequently than on other food items. Therefore, algae is the primary food for depik and kawan. The highest food similarity in depik was found between length class 55-70 mm and 71-85 mm (54.86%), while the food similarity of kawan was highest between 71-85 mm and 101- 115 mm (59.11%) length classes. According to Cohen et al (2002), food similarity was affected by fish size, food availability and its abundance. However, some fishes are selective in their food preferences, meaning that although available in low quantities in the waters, these foods are often found in the alimentary tract of the fish. Winemiller (1989), stated that when fish reached a bigger size it would change its feeding habit. However, no significant changes in feeding habit were detected in the depik and kawan at all length classes. A similar finding was found in the zooplanktivorous Hemirhamphus limbatus in Sri Lanka reservoirs where the feeding habit of this fish did not vary with body size (Weliange & Amarasinghe 2003). In contrast, significant changes in feeding habit were detected between adult and juvenile of the European anchovy Engraulis encrasicolus and the European pilchard Sardina pilchardus in the Gulf of Lions (Costalago et al 2012), Diapterus rhombeus and Micropogonias furnieri (Pessanha & Araujo 2014). Furthermore, while food preference may also change seasonally (Manon & Hossain 2011) in several fishes such as Moenkhausia icae and Leporinus fasciatus (Ropke et al 2014), other species have a constant feeding habit as recorded in Oreochromis mossambicus, O. niloticus and Amblypharyngodon melettinus (Weliange & Amarasinghe 2003). However this was not evaluated in our study due to limitation of sampling duration. Based on the diet overlap analysis, A. vulgaris, Chalotrix sp., C. longissima, Cocconeis sp., Dinophysis sp., Microcystis aeruginosa, Phacus curvicauda, T. curta were the foremost food items for both depik and kawan (Table 5). Wootton (1998) stated that utilisation of the same types of foods illustrates the overlap of the food resources by two or more species of fish in the same habitat. The diet overlap index of depik and kawan was 62.15 indicating a moderate degree of overlapping. Hilderbrand & Kershner (2004) reported that the degree of diet overlapping was related to food availability, type of habitat, drift timing and the ability of fish to utilize the food available in the waters. In addition, the diet overlap would be higher when the fish fed a similar type of food items in the same relative amounts. Furthermore, Krebs (1978) stated that when higher degree of diet overlapping occurred, it would eliminate one competing species. If the food sources in the waters become a limiting factor it will affect the growth of fish and only fish that can compete and utilize the available food can grow well.
Conclusions. The depik and kawan fishes are plankton feeders (planktonphagous), feeding on phytoplankton and zooplankton, where the favorite food items were C. longissima and A. vulgaris. Hence, algae was the primary food item for depik and kawan. The highest food similarity in depik was found between class length of 55-70 mm and 71- 85 mm; and between 71-85 mm and 101-115 mm for kawan. The diet overlap index showed that there was a moderate degree of overlapping of food resources between depik and kawan.
References
Arsyad M. N., Syaefudin A., 2010 Food and feeding habit of rasbora (Rasbora argyrotaenia, Blkr) in the down stream of Musi River. The 2nd Proceedings of International Conference on Indonesian Inland Waters. Research Institute for Inland Fisheries, Palembang, pp. 217-224.
AACL Bioflux, 2015, Volume 8, Issue 1. 46 http://www.bioflux.com.ro/aacl Balik I., Karasahin B., Ozkok R., Cubuk H., Uysal R., 2003 Diet of silver crucian carp Carassius gibelio in Lake Egirdir. Turkish Journal of Fisheries and Aquatic Sciences 3:87-91. Bascinar N. S., Saglam H., 2009 Feeding habits of black scorpionfish Scorpaena porcus, in the South Eastern Black Sea. Turkish Journal of Fisheries and Aquatic Sciences 9:99-103. Bellinger E. G., Sigee D. C., 2010 Freshwater algae: identification and use as bioindicators. John Wiley & Sons, Ltd, New Jersey, 254 pp. CBSG, 2003 Conservation assessment and management plan for Sumatran threatened species. IUCN-SSC Conservation Breeding Specialist Group, Apple Valley, USA, 117 pp. Clarke K. R., Gorley R. N., 2006 Primer V6: User manual and tutorial. Playmouth Marine Laboratory, UK, 192 pp. Cohen J. E., Jonsson T., Carpenter S. R., 2002 Ecological community description using the food web, species abundance, and body size. Proceedings of National Academy of Sciences USA 100:1781-1786. Costalago D., Navarro J., Álvarez-Calleja I., Palomera I., 2012 Ontogenetic and seasonal changes in the feeding habits and trophic levels of two small pelagic fish species. Marine Ecology Progress Series 460:169-181. Hilderbrand R. H., Kershner J. L., 2004 Influence of habitat type on food supply, selectivity and diet overlap of bonneville cutthroat trout and non native brook trout in Beaver Creek, Idaho. North American Journal of Fisheries Management 24:33-40. Huet M., 1971 Textbook of fish culture: breeding and cultivation of fish. Fishing News (Book) Ltd, London, 436 pp. IUCN, 1990 IUCN red list of threatened animal. IUCN, Gland and Cambrige.http://www.iucnredlist.org/. Krebs C. J., 1978 The experimental analysis of distribution and abundance. 2nd edit.,
H arper and Row Publisher, New York, US, 678 pp. Manon M. R., Hossain M. D., 2011 Food and feeding habit of Cyprinus carpio var. specularis. Journal of Science Foundation 9:163-169. Mazlan A. G., Samat A., Amirrudin., Anita T., 2007 Aspects on the biology of Garra cambodgiensis and Mystacoleucus marginatus (Cyprinidae) from Ulu Dungun, Terengganu. Malaysian Applied Biology 36:67-72. Morte S., Redon M. J., Sanz-Brau A., 2001 Diet of Scorpaena porcus and Scorpeana notata (Pisces: Scorpaenidae) in the Western Mediteranean. Cahiers de Biologie Marine 42:333-344. Moyo N. A. G., 2004 The feeding habits of Sargochromis codringtonii in Lake Kariba, Zimbabwe. Hydrobiologia 522:321-327. Muchlisin Z. A., 2013 Study on potency of freshwater fishes in Aceh waters as a basis for aquaculture and conservation development programs. Jurnal Iktiologi Indonesia 13:91-96. Muhclisin Z. A., Siti-Azizah M. N., 2009 Diversity and distribution of freshwater fishes in Aceh waters, northern Sumatra Indonesia. International Journal of Zoological Research 5:62-79. Muchlisin Z. A., Musman M., Siti-Azizah M. N., 2010a Spawning seasons of Rasbora tawarensis (Pisces: Cyprinidae) in Lake Laut Tawar, Aceh Province, Indonesia. Reproductive Biology and Endocrinology 8:49. Muchlisin Z. A., Musman M., Siti-Azizah M. N., 2010b Length-weight relationships and condition factors of two threatened fishes, Rasbora tawarensis and Poropuntius tawarensis, endemic to Lake Laut Tawar, Aceh Province, Indonesia. Journal of Applied Ichthyology 26:949-953. Muchlisin Z. A., Musman M., Fadli N., Siti-Azizah M. N., 2011a Fecundity and spawning frequency of Rasbora tawarensis (Pisces: Cyprinidae) an endemic species from Lake Laut Tawar, Aceh, Indonesia. AACL Bioflux 4:273-279. Muchlisin Z. A., Fadli N., Rudi E., Mendo T., Siti-Azizah M. N., 2011b Estimation of production trend of the depik, Rasbora tawarensis (Teleostei,Cyprinidae), in Lake Laut Tawar, Indonesia. AACL Bioflux 4:590-597.
AACL Bioflux, 2015, Volume 8, Issue 1. 47 http://www.bioflux.com.ro/aacl Muchlisin Z. A., 2011c [Policy analysis of introducing alien species of fish in inland waters of Aceh Province]. Jurnal Kebijakan & Riset Sosek Kelautan dan Perikanan 1(1):79- 89 [in Indonesian]. Muchlisin Z. A., 2012 First report on introduced freshwater fishes in the waters of Aceh, Indonesia. Archives of Polish Fisheries 20(2):129-135. Nikolsky G. V., 1963 The ecology of fishes. Academic Press, London, 352 pp. Nurfadillah, Damar A., Adiwilaga E. M., 2012 [Community of phytoplankton in Lake Laut Tawar, Aceh Tengah District, Aceh Province]. Depik 1:93-98 [in Indonesian]. Nyunja J. A., Mavuti K. M., Wakwabi E. O., 2002 Tropic ecology of Sardinella gibbosa (Pisces: Clupeidae) and Atherinomorous lacunosus (Pisces: Atherinidae) in Mtwapa Creek and Wasini Channel, Kenya. Western Indian Ocean Journal of Marine Science 1:181-189. Oronsaye C. G., Nakpodia F. A., 2005 A comparative study of the food and feeding habits of Chrysichthys nigrodigitatus and Brycinus nurse in a tropical river. Pakistan Journal of Scientific and Industrial Research 48:118-121. Oso J. A., Ayodele I. A., Fagbuaro O., 2006 Food and feeding habits of Oreochromis niloticus (L.) and Sarotherodon galilaeus (L.) in a tropical reservoir. World Journal of Zoology 1:118-121. Pessanha A. L. M., Araujo F. G., 2014 Shifts of the feeding niche along the size dimension of three juvenile fish species in a tidal mudflat in southeastern Brazil. Marine Biology 161:543-550. Prescott D. W., 1979 How to know the freshwater algae. M.W.C. Brown Company Publishers, Iowa, 348 pp. Ropke C. P., Ferreira E., Zuanon J., 2014 Seasonal changes in the use of feeding resources by fish in stands of aquatic macrophytes in an Amazonian floodplain, Brazil. Environmental Biology of Fishes 97:401-414. Salvador Jr. L. F., Salvador G. N., Santos G. B., 2009 Morphology of the digestive tract and feeding habits of Loricaria lentiginosa Isbrucker, 1979 in a Brazilian reservoir. Acta Zoologica 90:101-109. Schoener T. W., 1970 Non-synchronous spatial overlap of lizards in patchy habitats. Ecology 51:408-418. Verlencar X. N., Desai S., 2004 Phytoplankton identification manual. National Institute of Oceanography, Dhargalkar V. K., Ingole B. S. (eds), Dona Paula, Goa, 40 pp. Vine N. G., 1998 Aspects of the biology of the doublesash butterflyfish Chaetodon marleyi (Pisces: Chaetodontidae). Master Thesis, Rhodes University, USA, 198 pp. Wallace R. K. Jr., 1981 An assessment of diet-overlap indexes. Transactions of the American Fisheries Society 110:72-76. Weliange W. S., Amarasinghe U. S., 2003 Seasonality in dietary shifts in size-structured freshwater fish assemblages in three reservoirs of Sri Lanka. Environmental Biology of Fishes 68:269-282. Winemiller K. O., 1989 Ontogenetic diet shifts and resource partitioning among piscivorous fishes in the Venezuelan llanos. Environmental Biology of Fishes 26:177- 199. Wootton R. J., 1998 Fish ecology. Blackie and Sons Ltd., Glasgow, 212 pp.
AACL Bioflux, 2015, Volume 8, Issue 1. 48 http://www.bioflux.com.ro/aacl Received: 11 January 2015. Accepted: 01 February 2015. Published online: 04 February 2015. Authors: Zainal A. Muchlisin, Department of Aquaculture, Faculty of Marine and Fisheries, Syiah Kuala University, Banda Aceh 23111, Indonesia, e-mail: [email protected]. Fakhrurrazi Rinaldi, Department of Aquaculture, Faculty of Marine and Fisheries, Syiah Kuala University, Banda Aceh 23111, Indonesia, e-mail: [email protected] Nur Fadli, Department of Marine Sciences, Faculty of Marine and Fisheries, Syiah Kuala University, Banda Aceh 23111, Indonesia, e-mail: [email protected] Muhammad Adlim, Department of Chemistry, Faculty of Teaching Training and Education, Syiah Kuala University, Banda Aceh 23111, Indonesia, e-mail: [email protected] M. Nor Siti-Azizah, School of Biological Sciences Universiti Sains Malaysia Penang 11800, Malaysia, and Centre for Marine and Coastal Studies Universiti Sains Malaysia, e-mail: [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. How to cite this article: Muchlisin Z. A., Rinaldi F., Fadli N., Adlim M., Siti-Azizah M. N., 2015 Food preference and diet overlap of two endemic and threatened freshwater fishes, depik (Rasbora tawarensis) and kawan (Poropuntius tawarensis) in Lake Laut Tawar, Indonesia. AACL Bioflux 8(1):40-49.
AACL Bioflux, 2015, Volume 8, Issue 1. 49 http://www.bioflux.com.ro/aacl AACL BIOFLUX Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society
A comparative study on the efficacy of mixed tannins, hydrolysable tannins, and condensed tannins of Avicennia marina as anti-ectoparasite against Trichodina sp. 1Musri Musman, 1Aulia Rahmad, 1Irma Dewiyanti, 2Chairin Sofia, 2Hendro Sulistiono
1 Faculty of Marine and Fishery, Syiah Kuala University, Darussalam-Banda Aceh, Indonesia; 2 Brackish Water Aquaculture Centre, Ujung Batee-Aceh Besar, Indonesia. Corresponding author: M. Musman, [email protected]
Abstract. The research objective was to compare the efficacy of mixed tannins, hydrolysable tannins and condensed tannins from Avicennia marina leaves against protozoan ectoparasite Trichodina sp. Mixed tannins obtained from the leaves of A. marina was further separated into hydrolysable tannins and
condensed tannins. The condensed tannins were to be highly toxic (LC50 = 19.82 ppm) to Trichodina sp. compared to both hydrolysable tannins (LC50 = 61.76 ppm) and mixed tannins (LC50 = 64.81 ppm) displayed also toxic to the ectoparasite. The results addressed that condensed tannins have a good prospect to be used as an agent for controlling the ectoparasitic diseases in fish.
Key Words: Grey mangrove, white mangrove, LC50, ciliate protists, protozoa, ectoparasite control.
Introduction. Tilapia (Oreochromis niloticus) has been cultivated in several countries, including Indonesia. Tilapia can be cultured in a variety of habitats (in fresh, brackish, and marine water) due to its euryhaline nature. Hence, it can be a subsistence level to meet local protein needs and move the mainstream seafood markets (FAO 2014). An inhibiting factor in tilapia fish farming is a trichodina disease that cause many deaths addressed by parasitic protozoa groups, especially the type of Trichodina sp. Symptoms of infected fish among others are a white spots on their skin and gills, gasp for air at the surface, flashing, scraping, and weight loss. The fish are showing signs of damage to the skin are often accompanied by secondary infections (Hendrick 1998). The ectoparasite Trichodina sp. has shape likes a plate or a hat that covered his body cilia tip section. Usually Trichodina sp. attack to the outer part of the body organs such as the skin, fins, and gills through denticle organs that functions as suction on the host infestation. Trichodina sp. uses cilia to migrate from host to host where the parasite lives and foraging (Lom & Dykova 1992). Protozoan parasite control can be done by breaking the cycle of life. To break the cycle of life, the fish after treatment was quarantined for 3 days at a temperature of about 25-27°C, then transferred it to the place of cultivation. The use of synthetic chemical-based antiparasitics against ectoparasites infection it is already clear that is not recommended, because the synthetic antibiotics can cause resistance to the pathogens. Therefore, a natural antibiotic substance to control this disease should be sought. A number of experts are eager to study natural substances derived from the mangrove to lead to the discovery of bioactive compounds which can be used for pharmaceuticals, antibiotic substances, and feed. The studies shown constituents of Avicennia marina leaf, which contain alkaloids, terpenoids, saponins,
AACL Bioflux, 2015, Volume 8, Issue 1. 50 http://www.bioflux.com.ro/aacl glycosides, flavonoids, and tannins (Poompozhil & Kumarasamy 2014; Mouafi et al 2014). Plant tannins based on the building block of their chemical moieties are grouped into two main classes, i. e. hydrolysable tannins, and condensed tannins (Ribereau- Gayon 1972; Sarkar & Howarth 1976; Hahn et al 1984; Khanbabee & Ree 2001). Hydrolysable tannins are hexahydroxydiphenic acid esters of glucose or other polyols whereas condensed tannins are flavonoid polymers (Haslam 1979). The most common modes of tannins action are interference with the cell membrane and cell wall, interference with nucleic acids and enzyme interactions (Mukhopadhyay & Peterson 2006; Hugo & Russell 1982; Tenover 2006). Tannins have ability to bind with protein (Bate-Smith & Swain 1962; Hahn et al 1984) and preserve animal hides (White 1957; Maxson & Rooney 1972). Tannins have shown antibacterial (Banso & Adeyemo 2007), anticarcinogenic, antimutagenic, antimicrobial (Cos et al 2004; Awika et al 2006), antioxidant and antiradical (Amarowicz et al 2004; Alasalvar et al 2006) properties. Research on an efficacy of mixed, hydrolysable, and condensed tannins of A. marina against ectoparasites Trichodina sp., as far as authors knows based on the literatures searching has not been done. Therefore, study to control ectoparasite using the tannins extracts of A. marina against Trichodina sp. needed.
Material and Method
Identification of organism. The host of ectoparasite Trichodina sp. was tilapia (O. niloticus) captured using nets at the Kuta village (coordinate 5° 57’ 41” N 95° 33’ 08” E), Lamprit Subdistrict, Banda Aceh. The fishes taken for this study were the size of 3-7 cm, amounting to 240 fishes was attacked by the ectoparasites. Ectoparasite Trichodina sp. found on the outside of the body organs of fish was identified (Noga 2010; Purwanti et al 2012). Scraping mucus from skin surface of infected fish on both sides of the fish with a cover glass, and then placed it on an object glass and spilled some water, and observed using a Nikon Eclipse E200 microscope. After soaking 48 hours, the fish was put in a container with clean water so that the fish fresh back. Trichodina sp. found in fish body was observed to approve its death due to exposure the treated extracts.
Plant material. 1.4 kg of A. marina fresh leaves collected at Gano village (coordinate 5° 58’ 67” N 95°32’ 94” E) of Syiah Kuala Subdistrict, Banda Aceh were dried under the sunbeam for 5 days to give 750 g dried material. The amount of moisture removed was calculated and the sample was then stored in a herb room at 10°C with a relative humidity of less than 50% until used.
Isolation of the A. marina-leaf extract. Isolation of the dried material was run at the chemical laboratory of Teacher Training and Education Faculty of Syiah Kuala University, Banda Aceh. 750 g of A. marina dried leaves were ground in a blender to get the powder in 40 meshes. 250 g of the powder was transferred to dark-colored flasks, poured 1500 mL of 96% (v/v) m e t h a n o l on the powder and then let to rest for 24 hours. Afterwards, the slurry was filtered through Whatman #1 filter paper. The filtrate was evaporated to dryness under vacuum at 65°C. 13.4 g of the crude extract was obtained as the mixed tannins (MTs) and stored at 4°C until further analyses.
Separation of a mixed tannins using Sephadex LH-20. Method to separate the mixed tannins was based on Hagerman (2002) method. 4 g of the mixed tannins were suspended in 5 mL of 95% (v/v) ethanol and then applied to a chromatographic column (45x180 mm) packed with Sephadex LH-20 that had been equilibrated with 95% (v/v) ethanol. The column was rinsed with 2500 mL of ethanol 95% (v/v) to get hydrolysable tannins, and condensed tannins were eluted from the column using 1500 mL of 65% (v/v) acetone. Test for tannins and phenolic compounds. About 2 mg of each of the extract was boiled with 10 mL of water for 5 minutes, then cooled and filtered. Lead acetate test. To each of the 1 mL aliquot extracts, 5 drops of 1% lead acetate
AACL Bioflux, 2015, Volume 8, Issue 1. 51 http://www.bioflux.com.ro/aacl solution was added. The formation of white precipitate indicated the presence of tannins (Kokate 1997). Ferric chloride test. To 1 mL aliquot of each of the extracts, 5 drops of 5% ferric chloride solution was added. Hydrolysable tannins forms bluish-black precipitate whereas condensed tannins forms greenish-brown precipitate (Jain et al 2013). Gelatin test. To 1 mL aliquot of each of the extracts, 5 drops of 1% gelatin solution containing sodium chloride was added. Formation of white precipitate indicated the presence of tannins (Tiwari et al 2011).
Toxicity tests. Bioassays were conducted in the laboratory of Brackish Water Aquaculture Center (BBAP) Ujong Batee, Aceh Besar District in four replications for each experimental unit in a completely randomized design using tannins extracts of A. marina against ectoparasite Trichodina sp. The tannins were dissolved in water to make concentration 60-100 ppm (mixed tannins experimental unit), 50-90 ppm (hydrolysable tannins experimental unit), 10-50 ppm (condensed tannins experimental unit) on the basis of preliminary testing. Ten individuals Trichodina sp. were introduced into a 0.5 L vessel containing 400 mL of treated extract. Mortalities were recorded 48 hours later. To verify that Trichodina sp. lives normally, a control was prepared and used in the same condition. Mortality was recorded 48 h after treatment and the mortality was defined as the body structure of Trichodina sp. stunted (Ajizah 2004). The main parameters observed in this study were Trichodina sp. attacked to the body of the fish by looking under a microscope and the extracts. The physico-chemical water parameters, i. e. pH, temperature, dissolved oxygen were also monitored.
Statistical analysis. Data analyses were performed with SPSS version 18.0. Prior to analysis all data were transformed using arcsin square root transformation in order to reach the assumptions of the analysis of variance (ANOVA). The results of the acute toxicity experiments were analyzed for each experimental unit separately, using a one- way ANOVA followed by Duncan’s test at 5% of significance. For each experimental unit, the four replicates used for each extracts concentration yielded a mortality percentage. The data obtained in the form of dead parasites due to exposure the tannins of A. marina then analyzed using Trimmed Spearman-Karber (TSK) program version 1.5 to calculate LC50 with confidence intervals at 5% level. Means are given ± SE.
Results and Discussion. The results shown in Table 1 revealed that the tannins of A. marina have significant effect on mortality of protozoan ectoparasite Trichodina sp. attacking tilapia (Oreochromis niloticus) where p = 0.05. Mortality of ectoparasites Trichodina sp. increased with increasing concentration of the given extract on each experimental unit. Mortality data of Trichodina sp. after exposure the tannins of A. marina was incorporated into the program TSK version 1.5 to obtain results LC50. A one-way between treatments ANOVA was conducted to compare the effect of tannins of A. marina on mortality of ectoparasite Trichodina sp. There was a significant effect of concentration of mixed tannins [F(5, 18) = 126.9, p = 0.0], hydrolysable tannins [F(5, 18), 119.1, p =0.0], and condensed tannins [F(5, 18) = 322.8, p = 0.0] on mortality of ectoparasite Trichodina sp. at the p = 0.05 level for the six conditions of each experimental unit. Post hoc comparisons using the Duncan’s test indicated that the mean for the control condition was significantly different than the other concentrations for each experimental unit. Mortality data of Trichodina sp. were analyzed using the TSK program version 1.5 in order to get LC50 (median lethal concentration) values of tannins of A. marina against ectoparasite Trichodina sp. on tilapia. The LC50 values of mixed, hydrolysable, and condensed tannins of A. marina to Trichodina sp. on tilapia were 64.81, 61.76, and 19.82 ppm, respectively. The values indicated that the condensed tannins were highly toxic to Trichodina sp. than hydrolysable tannins and mixed tannins. The highly toxic condensed tannins to Trichodina sp. were due to condensed tannins most ready precipitate proteins at pH values near the isoelectric point of the proline-rich proteins which have a very high affinity for tannins (Hagerman 1989; Hagerman & Butler 1980). However, the loss of
AACL Bioflux, 2015, Volume 8, Issue 1. 52 http://www.bioflux.com.ro/aacl conformational mobility of intramolecular biphenyl linkages in mixed tannins and hydrolysable tannins reduce capacity to bind to protein (Haslam & Lilley 1985). The death of Trichodina sp. in the treatment was due to the presence of tannin-toxic substances of A. marina. Therefore, the tannins of A. marina can act as an anti-ectopasite against Trichodina sp.
Table 1 Percent mortality and LC50 of Trichodina sp. due to introduce mixed, hydrolysable, and condensed tannins of Avicennia marina in 48 hours exposure time
Extract % Mortality Median lethal A one-way ANOVA result concentration (mean±SE*, n = 40) concentration value
MTs (ppm)
There was a significant LC [confidence Control 0a 50 effect of MTs on mortality interval, 60 4.3±0.3b of Trichodina sp. at the p=0.05](ppm)/48h = 70 5.8±0.6c p = 0.05 for the six 64.81 [54.74-76.73] 80 8.0±0.4d treatments [F(5,18) 90 9.8±0.3e =126.9, p = 0.0] 100 10e sig
HTs (ppm)
There was a significant LC (confidence Control 0a 50 effect of HTs on mortality interval, 50 2.5±0.3b of Trichodina sp. at the p=0.05](ppm)/48h = 60 4.5±0.3c p = 0.05 for the six 61.76 [50.98-74.82] 70 5.5±0.3c treatments [F(5,18) 80 6.8±0.3d =119.1, p = 0.0] 90 7.5±0.3d sig
CTs (ppm)
There was a significant LC (confidence Control 0a 50 effect of CTs on mortality interval, 10 3.3±0.3b of Trichodina sp. at the p=0.05](ppm)/48h = 20 4.8±0.3c p = 0.05 for the six 19.82 [11.46-34.26] 30 6.3±0.3d treatments [F(5,18) 40 8.8±0.3e =322.8, p = 0.0] 50 10f sig *Means followed by same letter are not significantly different at 5% level (Duncan’s test following ANOVA). MTs - mixed tannins, Hts - hydrolysable tannins, CTs - condensed tannins.
Observation of the behavior of infected tilapia Trichodina sp. showed that at 15 minutes after contact to tannins tilapia look more active, where the movement was faster and openings the cover of the operculum was also faster than usual. This is caused by the reaction caused by exposure to toxic substances such as tannins in the test container. Compared with controls, tilapia was seen moving calm with normal operculum openings. The value of physico-chemical parameters of the water in this study, i.e. temperature was in range 28°-30°C, dissolved oxygen 5.1-7.8 mg/L, and pH 6.8-8.9. The value range is not much different from the control, i.e. temperature was in range of 29°-30°C, dissolved oxygen 6.2-8.5 mg/L, and pH was in range of 7.5-9.0 in which the Trichodina sp. found as much as 100% live. These clues can be stated that the death of Trichodina sp. due solely to tannins of A. marina. Observation under the microscope showed the differences of the body structure of Trichodina sp. in the living and the dead form as shown in Figure 1. The body structure of dead Trichodina sp. showed the organs were not intact and the body was more faded (B) than the alive (A). Tannins have the ability to interact with and precipitate proteins via
AACL Bioflux, 2015, Volume 8, Issue 1. 53 http://www.bioflux.com.ro/aacl the formation of cross links between collagen fibers in animal skins (Gupta & Haslam 1980). The deformations due to tannins action to disorder and to damage of cell membrane (Akiyama et al 2001) disrupted the permeability of the cell itself.
Figure 1. Trichodina sp. in alive form (A) and in a dead form (B) after exposure of the tannins extracts.
Conclusions. The condensed tannins been proved to be highly toxic against ectoparasite Trichodina sp. than the hydrolysable and mixed tannins.
Acknowledgements. The authors are thankful to Dr. Saida Rasnovi, Botanist of Syiah Kuala University, for support to identification of the plant material.
References
Ajizah A., 2004 [Sensitivity of Salmonella typhimurium to Psidium guajava L. leaf extracts]. Bioscientiae 1:31-8. [In Indonesian]. Akiyama H., Fuji K., Yamasaki O., Oono T., Iwatsuki T., 2001 Antibacterial action of several tannins against Staphylococcus aureus. J Antimicrob Chemother 48:487-91. Alasalvar C., Karamac M., Amarowicz R., Shahidi F., 2006 Antioxidant and antiradical activities in extracts of hazelnut kernel (Corylus avellana L.) and hazelnut green leafy cover. J Agric Food Chem 54(13):4826-4832. Amarowicz R., Troszynska A., Barylko-Pikielna N., Shahidi F., 2004 Polyphenolics extracts from legume seeds: correlations between total antioxidant activity, total phenolics content, tannins content and astringency. Journal of Food Lipids 11(4):278-286. Awika J. M., McDonough L. W., Rooney L. W., 2006 Decorticating sorghum to concentrate healthy phytochemicals. J Agric Food Chem 53:6230–6234. Banso A., Adeyemo S. O., 2007 Evaluation of antibacterial properties of tannins isolated from Dichrostachys cinerea. Afr J Biotechnol 6(15):1785-1787. Bate-Smith E. C., Swain T., 1962 Comparative biochemistry. Vol. III., Mason H. S., Florkin M. (eds), Academic Press, London and New York. Cos T., De Bruyne N., Hermans S., Apers D. V., Berghe A., Vlietinck J., 2004 Proanthocyanidins in health care: Current and new trends. Curr Med Chem 11:1345- 1359. FAO, 2014 Market Reports: Tilapia - December 2014, http://[email protected]. Gupta R. K., Haslam E., 1980 Vegetable tannins-structure and biosynthesis. In: Proceedings of the International Symposium held during the 36th Annual Meeting of the Institute of Food Technologists 10-13 June 1976, St. Louis MO. IDRC-145e, Ottawa, Ont. Hagerman A. E., 1989 Chemistry of tannin-protein complexation. In: Chemistry and significance of condensed tannins. Hemingway R. W., Karchesy J. J. (eds), Plenum Press, New York. Hagerman A. E., 2002 The tannin handbook. Miami University Oxford, OH 45056 www.users.muohio.edu/hagermae/ Hagerman A. E., Butler L. G., 1980 Condensed tannin purification and characterization of tannin-associated proteins. J Agric Food Chem 28:947
AACL Bioflux, 2015, Volume 8, Issue 1. 54 http://www.bioflux.com.ro/aacl Hagerman A. E., Butler L. G., 1981 The specificity of proanthocyanidin-protein interactions. J Biol Chem 256:4494-4497. Hahn D. H., Rooney L. W., Earp C. F., 1984 Tannins and phenols of sorghum. Cereal Foods World 29(12):776. Haslam E., 1979 Shikimic acid metabolites, in comprehensive organic chemistry. Vol. 5, Haslam E. (ed), Pergamon, Oxford and London. Haslam E., Lilley T. H., 1985 New polyphenols for old tannins. In: The biochemistry of plant phenolics. Van Sumere C. F., Lea P. J. (eds), 25:237-256, Clarendon Press, Oxford. Hendrick R. P., 1998 Relationship of the host, pathogen and environment: Implication for diseases of cultured and wild fish population. J Aquat Anim Health 10:107–111. Hugo W. B., Russell A. D., 1982 Types of antimicrobial agents. In: Principles and practice of disinfection, preservation and sterilization. Russell A. D., Hugo W. B., Ayliffe G. A. J. (eds), pp. 8-106, Blackwell Science Ltd. Jain P., Jain S., Pareek A., Sharma S., 2013 A comprehensive study on the natural plant phenols: perception to current scenario. Bull Pharm Res 3(2):90-106. Khanbabee K., Ree T. V., 2001 Tannins: classification and definition. Society of Chemistry 18:641-649. Kokate C. K., 1997 Practical pharmacognosy. 4th ed., New Delhi, Vallabh Prakashan. Lom J., Dykova I., 1992 Protozoan parasites of fishes. Developments in Aquaculture and Fisheries Science, 26. Elsevier: Amsterdam, 315 pp. Maxson E. D., Rooney L. W., 1972 Two methods of tannin analysis for Sorghum bicolor (L.), Moench grain. 12:253. Mouafi F. E., Abdel-Aziz S. M., Bashir A. A., Fyiad A. A., 2014 Phythochemical analysis and antimicrobial activity of Mangrove leaves (Avicennia marina and Rhizophora stylosa) against some pathogens. World Appl Sci J 29(4):547-554. Mukhopadhyay A., Peterson R. T., 2006 Fishing for new antimicrobials. Curr Opin Chem Biol 10:327-333. Noga E. J., 2010 Fish disease diagnosis and treatment. 2nd edition, Wiley-Blackwell, USA, 538 pp. Poompozhil S., Kumarasamy D., 2014 Studies on phythochemical constituents of some selected mangroves. Journal of Academia and Industrial Research 2(10):590-592. Purwanti R., Susanti R., Tri Martuti N. K., 2012 [Effect of ginger extract to the decline in the number of protozoan ectoparasites on tiger grouper juvenile]. Journal of Life Science 1(2):71-77. [In Indonesian]. Ribereau-Gayon P., 1972 Plant phenolics. Oliver and Boyd, Edinburgh and London. Sarkar J. K., Howarth R. E., 1976 Specificity of the vanillin test for flavonols. J Agric Food Chem 24(2):317-320. Tenover F. C., 2006 Mechanisms of antimicrobial resistance in bacteria. Am J Med 119:S3-S10. Tiwari P., Kumar B., Kaur M., Kaur G., Kaur H., 2011 Phytochemical screening and extraction: A review. Internationale Pharmaceutica Sciencia 1(1):98-106. White T., 1957 Tannins-their occurrence and significance. J Sci Food Agric 8:377.
AACL Bioflux, 2015, Volume 8, Issue 1. 55 http://www.bioflux.com.ro/aacl Received: 10 December 2014. Accepted: 25 January 2015. Published online: 04 February 2015. Authors: Musri Musman, Syiah Kuala University, Faculty of Marine and Fishery, Indonesia, Darussalam-Banda Aceh 23111, e-mail: [email protected] Aulia Rahmad, Syiah Kuala University, Faculty of Marine and Fishery, Indonesia, Darussalam-Banda Aceh 23111, e-mail: [email protected] Irma Dewiyanti, Syiah Kuala University, Faculty of Marine and Fishery, Indonesia, Darussalam-Banda Aceh 23111 e-mail: [email protected] Chairin Sofia, Brackish Water Aquaculture Centre, Indonesia, Ujung Batee-Aceh Besar 23381, e-mail: [email protected] Hendro Sulistiono, Brackish Water Aquaculture Centre, Indonesia, Ujung Batee-Aceh Besar 23381, e-mail: [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. How to cite this article: Musman M., Rahmad A., Dewiyanti I., Sofia C., Sulistiono H., 2015 A comparative study on the efficacy of mixed tannins, hydrolysable tannins, and condensed tannins of Avicennia marina as anti-ectoparasite against Trichodina sp. AACL Bioflux 8(1):50-56.
AACL Bioflux, 2015, Volume 8, Issue 1. 56 http://www.bioflux.com.ro/aacl AACL BIOFLUX Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society
Characterization of dried fish oil from Menhaden encapsulated by spray drying 1Bahareh Mehrad, 1Bahareh Shabanpour, 2Seyed M. Jafari, 1Parastoo Pourashouri
1 Faculty of Seafood Technology, Gorgan University of Agriculture Science and Natural Resources, Gorgan, Iran; 2 Faculty of Food Science, Gorgan University of Agriculture Science and Natural Resourses, Gorgan, Iran. Corresponding author: B. Mehrad, [email protected]
Abstract. This study aimed to evaluating the phisycochemical characterization of encapsulated fish oil from menhaden and potential of maltodextrin in combination with different wall materials in its microencapsulation process by spray drying, in order to maximize encapsulation efficiency. Maltodextrin (MD) mixed with fish gelatin (FG), κ carrageenan (κc) and both of them. The feed emulsions used for particle production were characterized for stability. The best encapsulation efficiency was obtained for MD:FG followed by the MD:FG+κc combination, while the lowest encapsulation efficiency was obtained for MD:κc, which also showed poorer emulsion stability. Particles were hollow, with the active material embedded in the wall material matrix, and had no apparent cracks or fissures. Key Words: fish oil, menhaden, spray drying, κ Carrageenan, fish gelatin.
Introduction. Functional food including n-3 lipids is one of the fastest growing food product groups in the US and Europe (Frost and Sullivan Research Service 2005). Mainly, marine lipids have received growing notice for the past decade because of their useful health properties of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on diseases like cardiovascular diseases (Yaqoob 2004), rheumatoid arthritis (Kremer 2000) and Crohn's disease (Belluzzi et al 2000). The best way to secure an increased intake of these healthy fatty acids in the people is through increase dietary fish consumption or partakes of fish oil capsule. An alternative way of increasing the intake of EPA and DHA could be via incorporation of fish oil into food products by substituting vegetable or animal fat into products such as salad dressing, milk and yogurt (Let et al 2005, 2007). Due to their unsaturated character, n-3 polyunsaturated fatty acids (PUFA) are highly prone to accelerated oxidative rancidity. Lipid oxidation causing formation of undesirable off-flavours (e.g. fishy and rancid off-flavour) and unhealthy compounds such as free radicals and reactive aldehydes (Let et al 2003). Therefore, for successful development of PUFA enriched food, preventing the happening of the lipid oxidation is important. Microencapsulating the oil with different biopolymers could be a very good way to overcome these adverse features (Cho et al 2003; Díaz-Rojas et al 2004). This structure could serve as: (a) vehicle for carrying the useful component to the preferred place action; (b) protection of the functional component from chemical or biological degradation (e.g. oxidation); (c) masking of the undesired properties of active component (e.g. odour and taste); and (d) controlling the release of the functional ingredient (Weiss et al 2006). This technology has been extensively used in pharmaceutical as well as in food industry. Choosing the best wall materials and encapsulation technique are important steps in food encapsulation. Previous researches have emphasized that the best way to emulsify fish oil is to use a kind of wall materials, which could function, as a carrier matrix and as an emulsifier (Sheu & Rosenberg 1998). Even the best mixture of
AACL Bioflux, 2015, Volume 8, Issue 1. 57 http://www.bioflux.com.ro/aacl biopolymers for encapsulating fish oil used with different encapsulating methods can create both stable and unstable powders. Hence, in this research, three biopolymers (maltodextrin (MD), Gelatin from cold- water fish skin (FG) and κ-carrageenan (κc)) were evaluated in combination as wall materials for fish oil (fish oil from menhaden - which was bought from Sigma-Aldrich). Maltodextrin is a filler matrix (Rosenberg et al 1993), which is cheap, extremely soluble in water and able to produce stable emulsion (Anandaraman & Reineccius 1986). Gelatin from marine sources (warm-water and cold-water fish skins, bones, and fins) is a potential substitute for bovine gelatin (Kim & Mendis 2006; Rustad 2003). One of the most important benefits of gelatin from marine sources is that they do not have the risk of occurrence of Bovine Spongiform Encephalopathy. Fish gelatin is acceptable for Islam, and can be used with least limitations in Judaism and Hinduism. Carrageenan is linear water-soluble sulfated polysaccharides that extracted from red seaweeds. Because of their biocompatibility and ability to produce thermoreversible hydrogels, carrageenan has been widely used as gelling agent in food and pharmaceutical industries (Stephen et al 1995). Within the carrageenan family, κ carrageenan originates the strongest gels and, hence, in the last decade this biopolymer has been studied a lot as a carrier for controlled drug release (Daniel-da-Silva et al 2011; Keppeler et al 2009; Leong et al 2011; Santo et al 2009). Spray drying (SD) is one of many standard methods to encapsulate food ingredients. SD is a common method used for encapsulation of food components (Desobry et al 1997). It is an easy and low-cost technique in which either proteins or polysaccharides or a mixture of both can be used to create the wall for microcapsule. However, SD has some disadvantages, first disadvantage is high temperature for drying (sometimes higher than 200°C) and second one is only appropriate for matrices that are extremely soluble in water (Gharsallaoui et al 2007). Spray drying an emulsion that includes sensitive components such as fish oil is risky. Due to the high drying temperature used in spray drying, deterioration of sensitive components because of oxidation has been reported (Hogan et al 2003; Kolanowski et al 2006). The main objective of this study was to investigate the effect of wall materials types (fish gelatin, κ carrageenan, maltodextrin and the combination of fish gelatin and κ carrageenan) on microencapsulation of fish oil using spray drying. The outcomes are compared based on encapsulation efficiency and powders physicochemical properties by related examinations.
Material and Method. The experiments were conducted between November 2013 to September 2014 in Gorgan University of Agricultural Science and Natural Resources labs. The materials were obtained from the following sources: fish oil (from menhaden), κ- carrageenan, gelatin (from cold water fish skin), acetic acid and sodium hydroxide purchased from Sigma-Aldrich Inc., St Louis, MO, USA. n-hexane (95%), isooctane, methanol and sulfuric acid were purchased from Dr. Mojallali Inc., Tehran, Iran. All chemicals used in this study were of analytical grade. Purified water was used for the preparation of all solution. All experiments and analysis were carried out in triplicate.
Emulsion preparation. Wall materials ratio including FG and κc, maltodextrin as filler matrix and soy lecithin as emulsifier are summarized in Table 1. Coating materials were dissolved in distilled water, followed by gentle stirring with a magnetic stirrer (for 30 min) to achieve a homogenous shell solution. The solution was allowed to hydrate for 24 h before emulsion preparation to ensure a full dissolution of materials, followed by cooling down to room temperature. The wall material concentration was 20% (Fernandes et al 2013a, b) and the amount of fish oil used was 25% of the mass of the wall materials (Jafari et al 2008). Coarse emulsions were prepared using an Ultraturrax IKA T25 homogeniser (Germany) at 12,500 rpm for 5 min. There was no addition of antioxidant during preparation of emulsion.
AACL Bioflux, 2015, Volume 8, Issue 1. 58 http://www.bioflux.com.ro/aacl Table 1 Composition of the wall materials for the each treatment used as a feed solution for the spray-drying process
Wall material (g 100 g−1 of solution) Coar material - fish oil # FG Κc Maltodextrin Soy lecithin (g 100 g−1 of solution) 1 7.5 - 32.5 - 10 2 - 2.5 37 0.5 10 3 7.5 2.5 29.5 0.5 10
Emulsion characterization
Creaming stability measurement. Ten grams of emulsion were transferred into a test tube (internal diameter 15 mm, height 125 mm) and then stored for 1 month at room temperature. After storage, some emulsions separated into an optically opaque “cream” layer at the top and a transparent (or turbid) “serum” layer at the bottom. We defined the serum layer as the sum of any turbid and transparent layers. The total height of the emulsions (HE) and the height of the serum layer (HS) were measured. The extent of creaming was characterized as % serum = 100 (HS/HE) (Surh et al 2007).
Emulsion color. Color of emulsions was measured using a tintometer (Lovibond CAM- System 500, UK). L* is the lightness, a* and b* represent the colors where –a* is greenness, +a* is redness, −b* is blueness, and +b* is yellowness.
Spray drying of emulsions. The emulsions were prepared in the same way as described before and reserved in glass beaker. Directly after the emulsification, the spray drying of emulsion took place. A spray-dryer (model MSD 1.0; Labmaq do Brasil, Ribeirão Preto, Brazil) equipped with a two-fluid nozzle atomiser was used to convert the liquid emulsions into solid powders. Emulsions were fed into the spray dryer chamber, drying time was very short. The inlet and outlet temperature of spray dryer were set ±180°C and ±80°C respectively. Microcapsules were collected in a glass container. The produced microcapsules were transferred instantly into a glass container and immersed in ice- water bath (Anwar & Kunz 2011).
Powders analysis
1. Moisture content. The moisture content of the microcapsules was determined gravimetrically by drying them in oven at 105°C for 24 h. The moisture content (%) was recorded for each sample after stable weight was obtained.
2. Color of microcapsules. Color of microcapsules was measured using a tintometer (Lovibond CAM-System 500, UK). L* is the lightness, a* and b* represent the colors where –a* is greenness, +a* is redness, −b* is blueness, and +b* is yellowness.
3. Free surface oil of powders. Fifteen mL n-hexane was added to 2.5 g microcapsule sample. The resulting solution was mixed with a vortex mixer (Chiltern International, Slough, UK, operating at the speed of 4) for 2 min and then centrifuged (Centrifuge 5702, Eppendorf, Hamburg, Germany) at 8,000 rpm for 20 min. The supernatant was filtered with the whatman filter paper and filter paper then washed twice with n-hexane (Baik et al 2004; Hardas et al 2000). After that, n-hexane was evaporated in a rotary evaporator (RE 111 Rotavapor, Type KRvr TD 65/45, BUCHI, Switzerland) at 70°C, and the solvent-free extract was dried in oven at 105°C. The amount of free surface fish oil was determined gravimetrically.
4. Total oil. Two mL of acetate buffer (pH 3.0) was added to 0.5 g microcapsule sample and vortexed for 2 min. The resulting solution was then extracted with 25 mL n- hexane/isopropanol (3:1 v/v). The tubes were then centrifuged for 15 min at 1600 rpm
AACL Bioflux, 2015, Volume 8, Issue 1. 59 http://www.bioflux.com.ro/aacl (Centrifuge 5702, Eppendorf, Hamburg, Germany). The clear organic phase then collected and aqueous phase was re-extracted with the solvent mixture (Baik et al 2004;
Hardas et al 2000). After filtration through anhydrous Na2So4 the solvent was evaporated in a rotary evaporator (RE 111 Rotavapor, Type KRvr TD 65/45, BUCHI, Switzerland) at 70°C, and the solvent-free extract was dried at 105°C. The amount of total oil was determined gravimetrically.
5. Calculation of encapsulation efficiency. From the quantitative determinations above detailed, the encapsulation efficiency (EE) was calculated as follows:
EE = encapsulated oil (g/100 g powder) × 100/total oil (g/100 g powder) encapsulated oil = total oil-surface free oil
6. Water solubility of microcapsules. Powders samples (0.1 g) were placed in centrifuge tubes containing 5 mL distilled water and incubated at 37°C for 5 h. The supernatants were obtained from centrifugation (KUBOTA, Japan) at 2500 ×g for 15 min and top up to 10 mL. The concentration of the soluble protein in the aqueous phase was determined using Biuret method.
7. Controlled release and core retention. Controlled-release method was performed according to the modified method of Gan et al (2008). Encapsulates (0.1 g) were placed in a glass test tube containing 2 mL of pepsin solution (2 mg mL-1 citric acid, pH 2.0). The suspension was incubated at 37°C in a water bath for 5 h. Samples from each test tube were drawn at intervals of 1 h until 5 h of incubation and placed at a Whatman filter paper to adsorb the released oil. The filter papers were then dried and the released oil content measured. The controlled-release of the fish oil from the encapsulated powder was expressed as proportion (%) of fish oil released from the encapsulates to fish oil retained in the dry encapsulates.
8. Particle size analysis. The particle size of the particles were determined by mixing 0.01 g of each samples with 0.5 mL water at room temperature (25±2°C) at a constant stirring rate of 400 rpm using a magnetic stirrer. The droplet size distributions of the resulting dispersions were determined by the Nano Zetasizer (Malvern Instruments Ltd., Worcestershire, UK). Triplicate experiments were performed.
Statistical analysis. In all cases, samples were analyzed in triplicate (n = 3). Significance of results was tested by an analysis of variance (ANOVA) and Duncan's Multiple-Range Test. Significance of differences was defined at p < 0.05.
Results and Discussion. This research was conducted to evaluate the effects of different wall materials on phisycochemical characteristics of spray-dried microcapsules. Fish oil was emulsified with three combinations of matrices as listed in Table 1, and dried by spray dryer to produce fish oil powders.
Emulsion characterization. The percentage of separation observed in the emulsions produced with different types of wall materials are shown in Table 2. The stability assessment showed that MD:FG emulsion was stable, but κc and MD:κc+FG emulsions were unstable to sum extent, which demonstrated the formation of a small separation layer and a foam phase, 24 h after their homogenization. This was unexpected, since lecithin and FG are well known by their good emulsifying ability. In line with Dickinson & Matsumura (1991) the unfolding of the protein molecules at the droplets surface, which would increase protein–protein interaction leading to flocculation during emulsification and as a result reducing the emulsion stability, may have caused this effect. The unfolding of protein molecules of the oil–water interface may lead to changes in secondary and tertiary structure, and consequently exposure of their residues which would be linked (–S–S– linkages or disulphide linkages) within the native globular structure leading to the formation of intermolecular interaction at the oil–water interface
AACL Bioflux, 2015, Volume 8, Issue 1. 60 http://www.bioflux.com.ro/aacl and flocculating. Another hypothesis that can be considered to explain this unusual behaviour is that the stability of protein-stabilized emulsions is a function of pH and other parameters.
Table 2 Characterization of emulsions prepared with different types of wall materials
Formulation % Separation L* a* b* MD:FG - 90.32a 2.54bc 0.27b MD:κc 13.2±0.09 88.17a 2.27c 0.63a MD:FG + κc 1.4±0.01 90.03a 3.64ab 0.59a L* is the lightness, a* and b* represent the colors where –a* is greenness, +a* is redness, −b* is blueness, and +b* is yellowness. Comparison within the rows was shown in the table with the data written as mean (n = 3). Means within the same column not followed by the same letter are significantly different at p < 0.05 level of significance, according to Duncan's Multiple-Range Test.
Therefore, depending on the emulsions’ pH, the emulsifying capacity of FG may have been lower than usual (Huynh et al 2008), affecting the emulsion stability. Color data of the microcapsules has shown that MD:κc emulsion had the lowest values in lightness (L*) and higher values in blueness (b*). This is mainly due to using lecithin as emulsifier in this treatment.
Moisture content. Moisture content is an important attribute of products since is directly related to stability. Dried products are more stable than liquid formulations, on the subject of the physicochemical aspects and microbiological spoilage. The moisture is mainly related to the drying conditions, however the composition of the formulations also plays an important role because drying could promote changes in water binding and dissociation that will affect the properties the dried product. High moisture content affects the shelf life of encapsulated fish oil (Baik et al 2004; Drusch et al 2006). This is probably because of a decrease in the glass transition temperature to values below the storage temperature, leading to relatively higher mobilities of molecules and reaction rates. The generated microcapsules were analyzed according to the method explained previously and results are reported in Figure 1. Moisture content of microcapsules varied from a minimum value of 3.05% (MD:FG) to a maximum value of 3.46% (MD:FG+κc). There was no significant difference in the moisture content of microcapsules as affected by wall materials. It could be related to the same amount of water applied for aqueous phase preparation or smaller difference in particle size of finished microcapsules, which is an index of crust formation time. The crust keeps water within the particle, so that the interior moisture cannot be simply evaporate. Results showed that moisture content of microcapsule coated with MD:FG was lower than two other matrices. FG has lower molecular weight, and therefore, water diffusivities are greater for solutions of MD:FG than for that of MD:κc (El-Sayed et al 1990). Proteins could therefore, migrate quickly to the surface of droplet and form a continuous glass phase at droplet surface earlier than does κc. The formed case on droplet surface is consequently transformed to a tough leather-like skin and avoids the moisture evaporation. Crust acts as a barrier against moisture evaporation and thus powders with higher moisture content were produced. Rosenberg & Sheu (1996) reported a similar observation for the effect of lactose on crust formation during microencapsulation of volatiles in whey protein-based wall systems. In addition, the lactose glass phase reduces the diffusion of solvent through the wall by enhancing the hydrophilic character of the wall matrix (Moreau & Rosenberg 1996). Rahman & Labuza (1999) suggest that the diffusion of oxygen may vary with different water content and water activity. The rate of diffusivity depends on the porosity of matrices and adsorbed water may form a protective layer against oxidation. Thus, the physical, chemical, and microbial stability of food depends highly on the water content (Rahman & Labuza 1999). According to the mentioned theory, MD:κc powders which had the highest moisture contents showed better stabilities than other samples. Therefore,
AACL Bioflux, 2015, Volume 8, Issue 1. 61 http://www.bioflux.com.ro/aacl this study suggests that higher water content in a sample could correlate with better stability against oxidation. In addition, the moisture content is critical for formed microcapsules. High moisture will induce high viscosity and stickiness of microcapsules, resulting in the formation of inter-particle bridges that lead to caking and particle collapse and the release/oxidation of the core material (Beristain et al 2002; Drusch et al 2006, 2007; Le Meste et al 2002; Partanen et al 2005).
Figure 1. Moisture content of microcapsules (%). FG = microcapsules with fish oil as core and fish gelatin+Maltodextrin as wall material, κc = microcapsules with fish oil as core and κ carrageenan+Maltodextrin as wall material, FG + κc = microcapsules with fish oil as core and fish gelatin and κ carrageenan+Maltodextrin as wall materials.
Color of microcapsules. L* value is a measure of the lightness and a* and b* values are the measurement of redness and yellowness color of microcapsules respectively. Color data of the microcapsules is shown in Table 3. MD:κc had the lowest values in lightness (L*) and higher values in redness (a*). Comparing the effects of wall materials on color of microcapsules show that b* is higher in MD:κc, which is related to using lecithin as emulsifier in this treatment, or maybe because of the high amount of free surface oil in this treatment (Drusch et al 2006).
Table 3 Color measurements of microcapsules with different wall materials
Formulation L* a* b* MD:FG 92.54a 2.82b 0.92b MD:κc 91.35b 3.43a 1.87a MD:FG + κc 92.39a 3.11ab 0.67b L* is the lightness, a* and b* represent the colors where –a* is greenness, +a* is redness, −b* is blueness, and +b* is yellowness. Comparison within the rows was shown in the table with the data written as mean (n = 3). Means within the same column not followed by the same letter are significantly different at p < 0.05 level of significance, according to Duncan's Multiple-Range Test.
Surface oil, total oil and encapsulation efficiency. The surface oil expresses the amount of oil that is nonencapsulated and it is an important parameter determining the product quality because the non-encapsulated oil is prone to oxidize thus may lead to the development of off-flavors and affect the acceptability of the product (Drusch & Berg 2008). The presence of large amounts of oil on the surface of powders is undesirable, since the surface oil not only deteriorates quickly causing off-flavor but also affects the wettability and dispersability of powders (Drusch & Mannino 2009). Previous researches
AACL Bioflux, 2015, Volume 8, Issue 1. 62 http://www.bioflux.com.ro/aacl showed that the amount of surface oil increased with the increasing emulsion droplet size. The possible explanation for the higher remaining oil on the surface of particles in spray drying method was the breakdown of the large emulsion droplets during atomization. This problem led to lower stability of the particles during storage, since there was no protection against oxidation, and the hydro peroxides were easily decomposed and formed off-flavor products (Soottitantawat et al 2003). The result of surface oil is shown in Figure 2. MD:κc powders shown the higher amount of free surface oil than other treatments.
Figure 2. Free surface oil content of microcapsules (%). FG = microcapsules with fish oil as core and fish gelatin+Maltodextrin as wall materials, κc = microcapsules with fish oil as core and κ carrageenan+Maltodextrin as wall materials, FG + κc = microcapsules with fish oil as core and fish gelatin and κ carrageenan+Maltodextrin as wall materials.
The total oil content indicates the total extractable oil of microcapsules which includes both surface oil and encapsulated oil. Total oil of microcapsules is shown in Figure 3 where the MD:FG microcapsules had the highest amount of total oil.
Figure 3. Total oil content of microcapsules (%). FG = microcapsules with fish oil as core and fish gelatin+Maltodextrin as wall materials, κc = microcapsules with fish oil as core and κ carrageenan+Maltodextrin as wall materials, FG + κc = microcapsules with fish oil as core and fish gelatin and κ carrageenan+Maltodextrin as wall materials.
Encapsulation efficiency (EE) reflects the real amount of fish oil that is encapsulated inside the matrix (Figure 4). Among the three formulas, MD:κc exhibited the lowest EE value. Encapsulation efficiency (EE) obtained with any wall material is statistically (p < 0.05) different from one to another. According to Figure 4, encapsulation efficiency varied from 71.77 to 87.65% and was significantly influenced by wall material.
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Figure 4. Oil recovery and encapsulation efficiency of microcapsules with fish oil and three matrices. FG = microcapsules with fish oil as core and fish gelatin+Maltodextrin as wall materials, κc = microcapsules with fish oil as core and κ carrageenan+Maltodextrin as wall materials, FG + κc = microcapsules with fish oil as core and fish gelatin and κ carrageenan+Maltodextrin as wall materials.
In the last years, special attention has been given to the studies aiming at improving the encapsulation efficiency during drying of food flavors and oils, by minimizing the amount of unencapsulated oil present at the surface of powder particles and thus preventing lipid oxidation and volatile losses, and extending product's shelf life (Desai & Park 2005). According to Jafari et al (2008), the main factors that affect encapsulation efficiency of microencapsulated oils and flavors are the type of wall material, the properties of the core materials (concentration, volatility), the characteristics of the emulsion (total solids, viscosity, droplets size) and the conditions of the drying process. Thus, it is important to optimize the drying process, in order to obtain the minimal surface oil in the powder particles. The EE for MD:FG treatment was higher than other treatments which indicated that most of the oil was encapsulated and less oil was on the surface of the microcapsules. Both wall material selection and emulsion properties (stability, viscosity and droplet size) can affect the process efficiency and the microencapsulated product stability. A successful microencapsulation must result in a powder with minimum surface oil and maximum retention of the active material.
Water solubility and controlled release. Water solubility of microcapsules for controlled core release in an aqueous environment is very importance for the functionality of microcapsules. Microcapsule design requires limited or delayed water solubility (Lee & Rosenberg 2000). The water solubility of encapsulated powders is presented in Figure 5. This principle may be used to control the release of the core material. It has long been recognized that controlled release technologies are of an interest not only for pharmaceutical industries but also for food industries. Encapsulation is always a key for putting a controlled release function in a food product (Lakkis 2007). However, in most cases, it is the encapsulant (i.e. shell material for reservoir-type, or outer matrix for matrix-type microcapsule) that has a critical effect on controlled release kinetics. Different biopolymers has proven to be a useful strategy to form shell walls with maximal protection and tailored controlled release properties for core substances (Islan et al 2012; Piculell et al 1995). In all cases in this study, core release was time dependent. The efficiency of protection or controlled release mainly depends on the composition and structure of wall material (Young et al 1993).
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Figure 5. Water solubility of encapsulated powders. FG = microcapsules with fish oil as core and fish gelatin+Maltodextrin as wall materials and FG + κc = microcapsules with fish oil as core and fish gelatin and κ carrageenan+Maltodextrin as wall materials.
Controlled release of oil through different types of microcapsules is shown in Figure 6.
Figure 6. Cumulative release of fish oil from microcapsules during incubation at 37°C. FG = microcapsules with fish oil as core and fish gelatin+Maltodextrin as wall materials, κc = microcapsules with fish oil as core and κ carrageenan+Maltodextrin as wall materials, FG + κc = microcapsules with fish oil as core and fish gelatin and κ carrageenan+Maltodextrin as wall materials.
From Figure 6 it can be seen that fish oil release from powders is illustrated by two different phases. Initially (0-3 hr) a quick release of fish oil is observed (“burst effect”) followed by a period during which the release becomes steady (after 3 hr) representing a constant release (“lag time”). The burst effect has been observed by other researchers in different polymeric matrices and may be because of the volume increase of polymer when immersed in liquid media. In aqueous liquid media, hydrophilic polymers start to hydrate causing relaxation of the polymer chain. In the present case, it is assumed that this effect is the main responsible for the initial release of fish oil from matrix; we believe that it is the fish oil that is not physically or chemically linked to matrix that will be released at this point of the process. In contrast, the subsequent almost zero order fish oil release phase results from the overlapping of several effects, such as the increase in the diffusion pathways with time, which is at least partially compensated by the increase in device porosity (polymer degradation), and the maintenance of approximately linear fish oil concentration gradients over prolonged periods of time within the microparticles
AACL Bioflux, 2015, Volume 8, Issue 1. 65 http://www.bioflux.com.ro/aacl (Faisant et al 2003). The release characteristics of microcapsules are strongly dependent on their permeability (which was affected by the polarity, density, porosity, homogeneity and thickness of shell wall materials) (Donhowe & Fennema 1993). None of the microcapsules used achieved complete core release after the incubation period showing that a certain amount of the fish oil was trapped within the network and was not released into the pepsin solution.
Conclusions. In this work it was possible to evaluate the performance of different wall materials combinations in the fish oil microencapsulation by spray drying. The MD:FG combination showed the best encapsulation efficiency result. This study clearly indicates the usefulness of fish gelatin as wall material in effective oil encapsulation in conjunction with Maltodextrin. Encapsulation of fish oil from menhaden in those matrices conducted also to differentiated releasing profiles that are explained by the dissimilar properties of the wall. Those different patterns may be very useful in finding the most suited formulation and processing condition for a specific end use of encapsulates.
Acknowledgements. The authors thank Gorgan University of Agricultural Science and Natural Resources for the financial support.
References
Anandaraman S., Reineccius G. A., 1986 Stability of encapsulated orange peel oil. Food Technology 40(11):88–93. Anwar S. H., Kunz B., 2011 The influence of drying methods on the stabilization of fish oil microcapsules: comparison of spray granulation, spray drying, and freeze drying. Journal of Food Engineering 105:367–378. Baik M. Y., Suhendro E. L., Nawar W. W., McClements D. J., Decker E. A., Chinachoti P., 2004 Effects of antioxidants and humidity on the oxidative stability of microencapsulated fish oil. Journal of the American Oil Chemists Society 81(4):355– 360. Belluzzi A., Boschi S., Brignola C., Munarini A., Cariani G., Miglio F., 2000 Polyunsaturated fatty acids and inflammatory bowel disease. American Journal of Clinical Nutrition 71:339S-342S. Beristain C. I., Azuara E., Vernon-Carter E. J., 2002 Effect of water activity on the stability to oxidation of spray-dried encapsulated orange peel oil using mesquite gum (Prosopis juliflora) as wall material. Journal of Food Science 67:206–211. Cho Y. H., Shim H. K., Park J., 2003 Encapsulation of fish oil by an enzymatic gelation process using transglutaminase cross-linked proteins. Journal of Food Science 68:2717-2723. Daniel-da-Silva A. L., Ferreira L., Gil A. M., Trindade T., 2011 Synthesis and swelling behavior of temperature responsive k-carrageenan nanogels. Journal of Colloid and Interface Science 355:512–517. Desai K. G. H., Park H. J., 2005 Recent developments in microencapsulation of food ingredients. Drying Technology 23(7):1361-1394. Desobry S. A., Netto F. M., Labuza T. P., 1997 Comparison of spray-drying, drum-drying and freeze-drying for beta-carotene encapsulation and preservation. Journal of Food Science 62(6):1158-1162. Díaz-Rojas E. I., Pacheco-Aguilar R., Lizardi J., Argüelles-Monal W., Valdez M. A., Rinaudo M., Goycoolea F. M., 2004 Linseed pectin: gelling properties and performance as an encapsulation matrix for shark liver oil. Food Hydrocolloids 18:293-304. Dickinson E., Matsumura Y., 1991 Time-dependent polymerization of β-lactoglobulin through disulphide bonds at the oil–water interface in emulsions. International Journal of Biological Macromolecules 13(1):26–30. Donhowe I. G., Fennema O., 1993 Water vapor and oxygen permeability of wax films. Journal of the American Oil Chemists Society 70:867–873.
AACL Bioflux, 2015, Volume 8, Issue 1. 66 http://www.bioflux.com.ro/aacl Drusch S., Berg S., 2008 Extractable oil in microcapsules prepared by spray-drying: localisation, determination and impact on oxidative stability. Food Chemistry 109(1):17–24. Drusch S., Mannino S., 2009 Patent-based review on industrial approaches for the microencapsulation of oils rich in polyunsaturated fatty acid. Trends in Food Science & Technology 20(6–7):237–244. Drusch S., Serfert Y., van den Heuvel A., Schwarz K., 2006 Physicochemical characterization and oxidative stability of fish oil encapsulated in an amorphous matrix containing trehalose. Food Research International 39:807–815. Drusch S., Serfert Y., Scampicchio M., Schmidt-Hansberg B., Schwarz K., 2007 Impact of physicochemical characteristics on the oxidative stability of fish oil microencapsulated by spray-drying. Journal of Agricultural and Food Chemistry 55(26):11044–11051. El-Sayed T. M., Wallack D. A., King C. J., 1990 Changes in particle morphology during drying of drops of carbohydrate solutions and food liquids. 1. Effects of composition and drying conditions. Industrial and Engineering Chemistry Research 29(12):2346– 2354. Faisant N., Siepmann J., Richard J., Benoit J. P., 2003 Mathematical modeling of drug release from bioerodible microparticles: effect of gamma-irradiation. European Journal of Pharmaceutics and Biopharmaceutics 56:271–279. Fernandes R. V. B., Borges S. V., Botrel D. A., 2013a Influence of spray drying operating conditions on microencapsulated rosemary essential oil properties. Ciência e Tecnologia de Alimentos 33:171–178. Fernandes R. V. B., Borges S. V., Botrel D. A., Silva E. K., da Costa J. M. G., Queiroz F., 2013b Microencapsulation of rosemary essential oil: characterization of particles. Drying Technology 31:1245–1254. Frost and Sullivan Research Service. End-user analysis of the global omega-3 PUFA markets. London: Frost and Sullivan Research Service. 2005. Gan C. Y., Cheng L. H., Easa A. M., 2008 Evaluation of microbial transglutaminase and ribose cross-linked soy protein isolate-based microcapsules containing fish oil. Innovative Food Science and Emerging Technologies 9:563–569. Gharsallaoui A., Roudaut G., Chambin O., Voilley A., Saurel R., 2007 Applications of spray-drying in microencapsulation of food ingredients: an overview. Food Research International 40(9):1107–1121. Hardas N., Danviriyakul S., Foley J. L., Nawar W. W., Chinachoti P., 2000 Accelerated stability studies of microencapsulated anhydrous milk fat. Lebensmittel- Wissenschaft & Technologie 33:506-513. Hogan S. A., O’riordan E. D., O’Sullivan M., 2003 Microencapsulation and oxidative stability of spray-dried fish oil emulsions. Journal of Microencapsulation 20(5):675– 688. Huynh T. V., Caffin N., Dykes G. A., Bhandari B., 2008 Optimization of the microencapsulation of lemon myrtle oil using response surface methodology. Drying Technology 26(3):357–368. Islan G. A., de Verti I. P., Marchetti S. G., Castro G. R., 2012 Studies of ciprofloxacin encapsulation on alginate/pectin matrixes and its relationship with biodisponibility. Applied Biochemistry and Biotechnology 167:1408–1420. Jafari S. M., Assadpoor E., He Y., Bhandari B., 2008 Encapsulation efficiency of food flavours and oils during spray drying. Drying Technology 26(7):816-835. Keppeler S., Ellis A., Jacquier J. C., 2009 Cross-linked carrageenan beads for controlled release delivery systems. Carbohydrate Polymers 78:973–977. Kim S., Mendis E., 2006 Bioactive compounds from marine processing byproducts – a review. Food Research International 39:383–393. Kolanowski W., Ziolkowski M., Weissbrodt J., Kunz B., Laufenberg G., 2006 Microencapsulation of fish oil by spray drying-impact on oxidative stability. Part 1. European Food Research and Technology 222(3–4):336–342. Kremer J. M., 2000 n-3 Fatty acids supplement in rheumatoid arthritis. American Journal of Clinical Nutrition 71:349S-351S.
AACL Bioflux, 2015, Volume 8, Issue 1. 67 http://www.bioflux.com.ro/aacl Lakkis J. M., 2007 Encapsulation and controlled release technologies in food systems. Blackwell Publishing Ltd., 256 pp. Le Meste M., Champion D., Roudaut G., Blond G., Simatos D., 2002 Glass transition and food technology: a critical appraisal. Journal of Food Science 67:2444–2458. Lee S. J., Rosenberg M., 2000 Preparation and some properties of water-insoluble, whey protein-based microcapsules. Journal of Microencapsulation 17:29–44. Leong K. H., Chung L. Y., Noordin M. I., Mohamad K., Nishikawa M., Onuki Y., Morishitab M., Takayamabet K., 2011 Carboxymethylation of kappa-carrageenan for intestinal- targeted delivery of bioactive macromolecules. Carbohydrate Polymers 83:1507– 1515. Let M. B., Jacobsen C., Meyer A. S., 2005 Sensory stability and oxidation of fish oil enriched milk is affect by milk storage temperature and oil quality. International Dairy Journal 15:173-182. Let M. B., Jacobsen C., Meyer A. S., 2007 Ascorbyl palmitate, gamma-tocopherol, and EDTA affect lipid oxidation in fish oil enriched salad dressing differently. Journal of Agricultural and Food Chemistry 55:2369-2375. Let M. B., Jacobsen C., Frankel E. N., Meyer A. S., 2003 Oxidative flavour deterioration of fish oil enriched milk: effects of oil type and ethylenediaminetetraacetate (EDTA). European Journal of Lipid Science and Technology 105:518-528. Moreau D. L., Rosenberg M., 1996 Oxidative stability of anhydrous milkfat microencapsulated in whey proteins. Journal of Food Science 61:39–43. Partanen R., Hakala M., Sjövall O., Kallio H., Forssell P., 2005 Effect of relative humidity on the oxidative stability of microencapsulated sea buckthorn seed oil. Journal of Food Science 70:37–43. Piculell L., Bergfeldt K., Nilsson S., 1995 Factors determining phase behaviour of multi- component polymer systems. In: Biopolymer mixtures. Harding S. E., Hill S. E., Mitchell J. R. (eds), Nottingham, UK: NottinghUniversity Press, pp. 13–35. Rahman M. S., Labuza T. P., 1999 Water activity and food preservation. In: Handbook of food preservation. Rahman M. S. (ed), Marcel Dekker, New York, pp. 339–382. Rosenberg M., Sheu T. Y., 1996 Microencapsulation of volatiles by spray-drying in whey protein-based wall systems. International Dairy Journal 6(3):273–284. Rosenberg M., Young S. L., Brooker B. E., Colombo V. E., 1993 Whey proteins as microencapsulating agents – microencapsulation of anhydrous milkfat – structure evaluation. Food Structure 12(1):31–41. Rustad T., 2003 Utilisation of marine by-products. Electronic Journal of Environmental, Agricultural and Food Chemistry 2:458–463. Santo V. E., Frias A. M., Carida M., Cancedda R., Gomes M. E., Mano J. F., Reis R. L., 2009 Carrageenan-based hydrogels for the controlled delivery of PDGF-BB in bone tissue engineering applications. Biomacromolecules 10:1392–1401. Sheu T. Y., Rosenberg M., 1998 Microstructure of microcapsules consisting of whey proteins and carbohydrates. Journal of Food Science 63(3):491–494. Soottitantawat A., Yoshii H., Furuta T., Ohkawara M., Linko P., 2003 Microencapsulation by spray drying: influence of emulsion size on the retention of volatile compounds. Journal of Food Science 68(7):2256–2262. Stephen A. M., Philips G. O., Williams P. A., 1995 Food polysaccharides and their applications. New York: Marcel Dekker, pp. 205–217. Surh J., Vladisavljevi Cacute G. T., Mun S., McClements D. J., 2007 Preparation and characterization of water/oil and water/oil/water emulsions containing biopolymer- gelled water droplets. Journal of Agricultural and Food Chemistry 55:175-184. Weiss J., Takhistov P., McClements J. D., 2006 Functional materials in food nanotechnology. Journal of Food Science 71:R107-R116. Yaqoob P., 2004 Fatty acids and the immune system: from basic science to clinical applications. Proceedings of Nutrition Society 63:89-104. Young S. L., Salda X., Rosenberg M., 1993 Microencapsulating properties of whey proteins. 1. Microencapsulation of anhydrous milk fat. Journal of Dairy Science 76(10):2868–2877.
AACL Bioflux, 2015, Volume 8, Issue 1. 68 http://www.bioflux.com.ro/aacl Received: 15 January 2015. Accepted: 09 February 2015. Published online: 11 February 2015. Authors: Bahareh Mehrad, Faculty of Seafood Technology, Gorgan University of Agriculture Science and Natural Resources, Basij Square, Gorgan, Iran, e-mail: [email protected] Bahareh Shabanpour, Faculty of Seafood Technology, Gorgan University of Agriculture Science and Natural Resources, Basij Square, Gorgan, Iran, e-mail: [email protected] Seyed Mahdi Jafari, Faculty of Food Science, Gorgan University of Agriculture Science and Natural Resourses, Basij Square, Gorgan, Iran, e-mail: [email protected] Parastoo Pourashouri, Faculty of Seafood Technology, Gorgan University of Agriculture Science and Natural Resources, Basij Square, Gorgan, Iran, e-mail: [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. How to cite this article: Mehrad B., Shabanpour B., Jafari S. M., Pourashouri P., 2015 Characterization of dried fish oil from Menhaden encapsulated by spray drying. AACL Bioflux 8(1):57-69.
AACL Bioflux, 2015, Volume 8, Issue 1. 69 http://www.bioflux.com.ro/aacl AACL BIOFLUX Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society
Experimental pathogenicity of Achlya species from cultured Nile tilapia to Nile tilapia fry in Thailand 1Kwanprasert Panchai, 1Chutima Hanjavanit, 2Nilubon Rujinanont, 3Shinpei Wada, 3Osamu Kurata, 4Kishio Hatai
1 Applied Taxonomic Research Center, Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand; 2 Department of Fisheries, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand; 3 Laboratory of Aquatic Medicine, School of Veterinary Medicine, Nippon Veterinary and Life Science University, Tokyo 180–8602, Japan; 4 Microbiology and Fish Disease Laboratory, Borneo Marine Research Institute, Universiti Malaysia Sabah, 88400, Kota Kinabalu, Malaysia. Corresponding author: C. Hanjavanit, [email protected]
Abstract. Experimental infection of Nile tilapia (Oreochromis niloticus) fry using 6 Achlya isolates from cultured Nile tilapia with water mold infections was attempted. The experimental fish were exposed to 1.0 x 102 and 1.0 x 104 zoospores mL-1 of each Achlya isolate after ami-momi treatment. The cumulative mortality rates of fish exposed to 1.0 x 104 zoospores mL-1 of A. klebsiana BKKU1003, and A. diffusa BKKU1012 were 88.8 and 77.7%, respectively. A. klebsiana BKKU1003 was more pathogenic than the other isolates. Histopathological examination of the skin of Nile tilapia fry exposed to 1.0 x 102 zoospores mL-1 of A. klebsiana BKKU1003 showed numerous hyphae grew on the skin surface and some areas of skin were sloughed. The fish exposed to 1.0 x 104 zoospores mL-1 of A. klebsiana BKKU1003 showed massive accumulated hyphae on skin lesions with necrosis of the epidermal cells and the hyphae penetrated from the epidermis to the musculature without granulomatous response surrounding the hyphae. We found that it is possible to infect tilapia fry by exposing them to zoospores of Achlya after the ami-momi treatment. Key Words: oomycete, ami-momi treatment, Oreochromis niloticus, histopathology.
Introduction. Water mold infection has usually found in Nile tilapia (Oreochromis niloticus) in Thailand’ hatcheries under natural condition. It is caused by a member in Family Saprolegniaceae included genera Achlya, Aphanomyces and Saprolegnia (Yuasa et al 2000). This infection has been a serious problem and continued to occur in intensive culture during the cool season (Willoughby & Lilley 1992; Chinabut et al 1995; Chukanhom & Hatai 2004; Hanjavanit et al 2012). Water mold infection could occur throughout the year, especially when water temperatures fluctuate, handling stress occurs, fish density is extremely high, or skin parasites exist (Noga 1993). Saprolegniasis has been reported from cultured economic fish and eggs in Thailand (Willoughby & Lilley 1992; Chinabut et al 1995; Lawhavinit et al 2002; Chukanhom & Hatai 2004), and Achlya spp. were isolated from dead fish (Willoughby & Lilley 1992) and from eggs of common carp (Cyprinus carpio) (Chukanhom & Hatai 2004), eggs of Nile tilapia (Panchai et al 2007), eggs of Mekong giant catfish (Pangasianodon gigas) (Abking et al 2012) and eggs of African catfish (Clarias gariepinus) (Hanjavanit et al 2012). They were also isolated from the diseased Nile tilapia (Panchai et al 2014). Some species of Saprolegniaceae was mentioned as primary pathogen (Yuasa & Hatai 1995; Stueland et al 2005), whereas most species were mentioned as secondary pathogens (Lilley & Roberts 1997; Sosa et al 2007). Therefore, it is important to determine whether the isolated strains are able to infect fish under laboratory condition. An ami-momi (AM) treatment is a notable method to enhance the sensitivity of fish to water mold for artificial infection, which produces the
AACL Bioflux, 2015, Volume 8, Issue 1. http://www.bioflux.com.ro/aacl 70 physical stress and scarification of fish skin (Hatai & Hoshiai 1993; Yuasa & Hatai 1995; Grandes et al 2001; Hussein & Hatai 2002; Kiryu et al 2002; Stueland et al 2005; Hanjavanit et al 2010; Hussein et al 2013) by shaken fish in a scoop net in the air (Hatai & Hoshiai 1993). Objectives of this study were to induce Achlya spp. infection to Nile tilapia fry under the AM treatment and examine the histopathological characters of the infected fish.
Material and Method
Source of experimental fish. Approximately three hundred healthy Nile tilapia fry (average 0.7–0.8 g in body weight and 3–4 cm in total length) were provided by Khon Kaen Inland Fisheries Research and Development Center and were used for the experimental infection. The fish were acclimatized at 25°C for one week. The fish were fed with commercial formula food (GF Feed, Krungthai Feedmill Public Co., Ltd., Bangkok, Thailand) daily and starved for a few days before the experiment. The experiment was carried out at Department of Biology, Faculty of Science, Khon Kaen University, Thailand during November to December 2014.
Induction of tilapia skin lesion. Two sets of fish were prepared and designed for induction of skin lesion. Group I, three fish were random selected and then shaken in a fan-shaped scoop net (10 cm in diameter) in the air for 0, 1, 2, 3, 4 and 5 minutes, respectively. This shaking process is the AM treatment (Hatai & Hoshiai 1993). The fish were then rinsed with sterilized tap water (STW) to eliminate excess mucus and then placed into each 500 mL of STW for 7 days. The experiment was carried out in a plastic tank (8 cm wide x 15 cm long x 11 cm high with water depth 4.5 cm), which placed in an experimental pond at constant temperature of 25°C. Aeration was supplied during the experiment. Cumulative mortality rates were also noted each day during the test period. Group II, the fish after the AM treatment as described above were immediately fixed in 10% phosphate buffered formalin (PBF) solution and decalcified with ethylene diamine tetra-acetic acid (EDTA) solution for histological examination. Three parts including head, trunk and caudal regions (0.5 x 0.5 cm) of the fixed fish were dissected (Figure 1) and processed into paraffin method. After that, the samples were sectioned at 5 μm with a sliding microtome. All of the section slides were stained with hematoxylin and eosin (H & E). The thickness of epidermis, which was defined as distance between apical surface of the uppermost nucleated epidermal cell and basement membrane (Wisenden & Smith 1997) and was measured by an ocular micrometer at 40x magnification (n = 5). The epidermal thickness was statistically analyzed using two-way ANOVA with Fisher’s least significant difference (LSD) multiple comparison test to determine significant differences between region and treatment (Zar 2010). Three replicates of each treatment were conducted. Nine fish were used for each treatment.
Figure 1. The positions of skin section: head (A), trunk (B), caudal regions (C) of Nile tilapia fry.
AACL Bioflux, 2015, Volume 8, Issue 1. http://www.bioflux.com.ro/aacl 71 Achlya isolates used in this study. Achlya isolates (A. klebsiana BKKU1003, A. bisexualis BKKU1007, A. diffusa BKKU1012, Achlya sp. BKKU1117, A. prolifera BKKU1125 and Achlya sp. BKKU1127) were obtained from net cage-cultured Nile tilapia with water mold infections on the Nam Phong River in Khon Kaen Province, northeastern Thailand from September 2010 to August 2011 (Table 1) (Panchai et al 2014). The identified to species was examined by morphological structures of reproductive organs on hemp seeds (Cannabis sativa) cultures according to key references (Kitancharoen et al 1995; Johnson et al 2002; Chukanhom & Hatai 2004) and molecular identification (Lilley et al 2003; Phadee et al 2004; Muraosa et al 2009, 2012). They were routinely maintained on glucose yeast extract (GY) agar (Hatai & Egusa 1979) at 25°C and transferred to fresh GY agar every month and were deposited at the Department of Biology, Faculty of Science, Khon Kaen University, Thailand.
Table 1 Achlya spp. used in this study were isolated from Nile tilapia with water mold infections in cage culture in the Nam Phong River, Khon Kaen Province
Species Remark Location Date Achlya klebsiana BKKU1003 Farm A, Ban Hua Sua–tent, 23 September 2010 Nam Phong district A. bisexualis BKKU1007 Farm B, Ban Hua Sua–tent, 23 September 2010 Nam Phong district A. diffusa BKKU1012 Farm C, Ban Hua Sua–tent, 23 September 2010 Nam Phong district Achlya sp. BKKU1117 Farm B, Ban Hua Sua–tent, 19 January 2011 Nam Phong district A. prolifera BKKU1125 Farm C, Ban Hua Sua–tent, 19 January 2011 Nam Phong district Achlya sp. BKKU1127 Farm C, Ban Hua Sua–tent, 20 February 2011 Nam Phong district
Preparation of zoospores. Zoospore suspensions from each isolate were prepared as follows: a small piece of the 3 days growing edge agar (5 x 5 cm) with hyphae (agar block) was cut off. Three agar blocks were placed in a plastic Petri dish containing GY broth for 3 days at 25°C. After that, only mycelia were cut and washed in successive baths of sterilized tap water (STW) and continuous incubation for 16–18 h at 25°C to get zoospores. The number of zoospores was counted using a hemocytometer (Neubauer counting chamber, Erma®) and adjusted to approximately 1.0 x 102 and 1.0 x 104 zoospores mL-1 for experimental infection.
Experimental design of artificial infection. Fish were randomly taken from holding tank and subdivided into four groups. Group A: AM group, fish (n = 3) were shaken in a fan-shaped scoop net in the air for 2 minutes and then rinsed with STW to eliminate excess mucus. Group B: non-AM group, both groups A and B were exposed to 1.0 x 102 and 1.0 x 104 zoospores mL-1. Group C: AM group, and group D: non-AM group were control of groups A and B, respectively, which exposed to STW. The experimental fish were maintained in the same condition as described in Experiment induction of tilapia skin lesion. Numbers of fish exposed to each isolate of each concentration and the control group were nine. Both infected and controlled groups were kept observation for 7 days by naked eyes. These experiments were performed after the experimental protocol had been approved by the Institutional Animal Ethics Committee, Khon Kaen University, Thailand (Reference NO. 0514.1.12.2/33). To confirm the presence of the Achlya isolates, triplicate samples of the infected hyphae from infected moribund fish were repeatedly placed onto GY agar for identification. Infection and cumulative mortality rates were also daily recorded during the assay and tested statistically using one-way ANOVA with Fisher’s least significant
AACL Bioflux, 2015, Volume 8, Issue 1. http://www.bioflux.com.ro/aacl 72 difference (LSD) multiple comparison test to determine significant differences among treatment (Zar 2010).
Histopathological examination. Gross pathological changes and lesions were observed and photographed. After removal of some hyphae as described above, the fish were routinely necropsied. The histopathology of hyphal infection was verified. All fish were fixed in 10% PBF solution and skin lesions were removed for preparing permanent slides by paraffin method, the same procedure as in Experiment induction of tilapia skin lesion. All slides were stained with H & E. Some selected slides were also stained with periodic acid-Schiff (PAS) reagent, Giemsa, Gram stain and Uvitex 2B (Wada et al 2003) to observe water mold hyphae in the tissues.
Results
Induction of tilapia skin lesion. Gross observation of the control and the treatment groups appeared normal (Figure 2). It was found that fish of the control and treatment groups did not die after the AM treatment for 1 and 2 minutes. Whereas, the cumulative mortality was occurred at day 1 after the AM treatment for 3 to 5 minutes (Table 2).
Figure 2. Gross morphology of Nile tilapia fry after net shaking. A: the control group; B: the treatment group shaking for 2 minutes.
Table 2 Cumulative mortality of Nile tilapia fry/total fish examined after the ami-momi treatment (D = day)
Durations No. of dead fish / No. fish examined Total (minute) 1D 2D 3D 4D 5D 6D 7D Control 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 3 1/9 1/9 0 0 0 0 0 2/9 4 2/9 0 0 0 0 0 0 2/9 5 5/9 0 0 0 0 0 0 5/9
Histological examination of induction of tilapia skin lesion. From the microscopic examination of the skin on the head, trunk and caudal regions of the control and AM groups consisted of epidermis, dermis and hypodermis covering muscular layer. The epidermis of the control group consisted of stratified squamous epithelium with scattered mucous cells (Figure 3A) and more mucous cells were found at the head region than the trunk and caudal regions (Figure 3B-C). The epidermis at the head region was thicker than those of the trunk and caudal regions with significant difference (p < 0.05, Table 3). The epidermis of the AM groups after 1-2 minutes treatment was composed of 3-4 layers of epidermal cells and no mucous cells appeared (Figure 3D-F). The AM group after 3 minutes treatment, the most outer cells of epidermis was necrosis and consisted of 2-3
AACL Bioflux, 2015, Volume 8, Issue 1. http://www.bioflux.com.ro/aacl 73 epidermal cells layer. Whereas, the most outer cells of epidermis of the AM groups after 4 and 5 treatment were also necrosis and only 1-2 epidermal cells layer occurred (Figure 3G-I). It was also found that the epidermal thickness of all AM groups was thinner than that of the control group with significant difference (p < 0.05, Table 3) except the AM group after 1-2 minutes treatment showed non-significant difference from the control group (p > 0.05, Table 3).
Figure 3. Cross section of skin from the head (A, D, G), trunk (B, E, H) and caudal (C, F, I) regions of Nile tilapia fry after the ami-momi treatment showing epidermis (E), scale (s), dermis (D), hypodermis (H), muscle (M) (H & E). A-C: the control group; D-F: the AM group for 2 minutes; G-I: the AM group for 5 minutes (arrow heads = mucous cells).
AACL Bioflux, 2015, Volume 8, Issue 1. http://www.bioflux.com.ro/aacl 74 From the results of mortality and histological characteristics of skin of the AM groups, the 2 minutes AM treatment was selected as a representative duration time for experimental infection because no fish died and it was possible to induce the scarification of skin.
Table 3 Mean standard deviation (SD) of epidermal thickness of Nile tilapia fry (n = 5) of both control and treatment groups after net shaking
Epidermal thickness (Mean SD) (µm) Durations (minute) Head Trunk Caudal Control 211.0a 7.1 149.0b 2.1 113.0c 12.5 1 192.0a,d 5.5 138.0b,g 2.1 108.0c,j 5.5 2 179.0a, d 12.9 138.0b,g 2.1 111.0c, j 3.6 3 157.0d 21.7 140.0g 8.9 96.4j 3.6 4 132.0e 9.5 121.0h 6.2 77.4k 2.1 5 21.4f 6.2 13.1i 4.1 8.3l 2.1 Different letters within a column indicate statistically significant differences between treatments (p < 0.05).
Experimental design of artificial infection. All the Achlya isolates used in group A (AM group) were able to infect the experimental fish. The infection was clearly visible at the site of injured areas of experimental fish and mortality were occurred at days 3 and 2 when exposed to 1.0 x 102 and 1.0 x 104 zoospores mL-1 by naked eyes, respectively. The moribund fish revealed serious oomycete infections, characteristically with hemorrhage comprising of cotton like tuft on the skin surface of forehead, dorsal fin and caudal peduncle (Figures 4A, 5A). An analysis of infection rates and cumulative mortality is summarized in Table 4. Based on the infection rate and cumulative mortalities when the fish exposed to 1.0 x 104 zoospores mL-1 of each isolate, it was divided into 3 groups. Group I, high cumulative mortality 77.7–88.8% included A. klebsiana BKKU1003 and A. diffusa BKKU1012. Group II, moderately cumulative mortality 55.5–66.6% included Achlya sp. BKKU1117, A. prolifera BKKU1125 and Achlya sp. BKKU1127. Group III, low cumulative mortality 33.3% was A. bisexualis BKKU1007. Neither sign of oomycete infection (0% infection) nor mortality (0% mortality) was recorded in group B (non-AM group) as shown in Table 4. It was also found that all fish in groups B (non-AM group), C (control of AM group) and D (control of non-AM group) were able to survive throughout the experimental period (7 days). During the experiment, the Achlya spp. used in the experiments were successfully re-isolated from the lesions on the skin or muscle of some moribund fish. The morphology and growth patterns of the re-isolated Achlya were identical to those of the artificial isolates (Kitancharoen et al 1995; Johnson et al 2002; Chukanhom & Hatai 2004), fulfilling Koch’s postulates.
Histopathological examination of experimental artificial infection. From microscopic examination of infected skin lesions with all isolates, showed hyphae covering the surface of the epidermis, the upper epidermal cell layers of skin lesions were disorganized and completely sloughed. Epidermal cells affected with hyphae were severely necrotized (Figure 4B, C). Whereas, isolate of A. klebsiana BKKU1003 in 1.0 x 104 zoospores mL-1 caused highest infection (9/9 moribund fish, 100%). Three out of 9 moribund fish (33.3%) had massive accumulations of hyphae on the skin lesions and they penetrated the epidermis, passing through the dermis into the musculature of the trunk region (Figure 5B, C). Scattered capillaries and inflammatory cells were observed in the dermal layer. No granulomas were found surrounding the hyphae. The hyphae were not easily visible with H&E stain, but they were clearly observed with PAS and Uvitex 2B. Giemsa and Gram stains failed to reveal any bacteria within the lesions.
AACL Bioflux, 2015, Volume 8, Issue 1. http://www.bioflux.com.ro/aacl 75
Figure 4. A: Nile tilapia fry exposed to 1.0 x 102 zoospores mL-1 of A. klebsiana BKKU1003 for 3 days. Note the hyphae on the head, dorsal and ventral fins and caudal peduncle. B: Histopathology of skin of moribund tilapia fry showing hyphae with pink color (arrows) attached on epidermis (PAS). C: Serial section of B showing positive Uvitex 2B-H&E stain of hyphae with blue-white fluorescence (arrows).
Figure 5. A: Nile tilapia fry exposed to 1.0 x 104 zoospores mL-1 of A. klebsiana BKKU1003 for 4 days. Note the hyphae on the head, dorsal fin and caudal peduncle. B: Histopathology of skin of moribund tilapia fry showing aseptate hyphae with pink color (arrows) invaded musculature (PAS). C: Serial section of B showing positive Uvitex 2B-H&E stain of hyphae with blue-white fluorescence (arrows).
AACL Bioflux, 2015, Volume 8, Issue 1. http://www.bioflux.com.ro/aacl 76 Table 4 Infection (no. fish with infection/no. fish examined) and mortality (no. fish with mortality/no. fish examined) of Nile tilapia fry experimentally exposed to zoospores of each Achlya isolate for 7 days
Exposure level Ami-momi treatment Non-ami-momi treatment 1.0x102 zoospores mL-1 1.0x104 zoospores mL-1 1.0x102 zoospores mL-1 1.0x104 zoospores mL-1 No. fish No. fish No. fish No. fish No. fish No. fish No. fish No. fish Species with with with with with with with with infection/ mortality/ infection/ mortality/ infection/ mortality/ infection/ mortality/ No. fish No. fish No. fish No. fish No. fish No. fish No. fish No. fish examined examined examined examined examined examined examined examined (%) (%) (%) (%) (%) (%) (%) (%) Control 0/9 (0)a* 0/9 (0)a 0/9 (0)a 0/9 (0)a 0/9 (0)a 0/9 (0)a 0/9 (0)a 0/9 (0)a A. klebsiana BKKU1003 3/9 (33.3)b 3/9 (33.3)b 9/9 (100)c 8/9 (88.8)d 0/9 (0)a 0/9 (0)a 0/9 (0)a 0/9 (0)a A. bisexualis BKKU1007 1/9 (11.1)a,b 0/9 (0)a 4/9 (44.4)b 3/9 (33.3)b 0/9 (0)a 0/9 (0)a 0/9 (0)a 0/9 (0)a A. diffusa BKKU1012 3/9 (33.3)b 2/9 (22.2)a,b 8/9 (88.8)c 7/9 (77.7)c,d 0/9 (0)a 0/9 (0)a 0/9 (0)a 0/9 (0)a Achlya sp. BKKU1117 2/9 (22.2)a,b 1/9 (11.1)a,b 7/9 (77.7)c 5/9 (55.5)c 0/9 (0)a 0/9 (0)a 0/9 (0)a 0/9 (0)a A. prolifera BKKU1125 1/9 (11.1)a,b 0/9 (0)a 7/9 (77.7)c 6/9 (66.6)c,d 0/9 (0)a 0/9 (0)a 0/9 (0)a 0/9 (0)a Achlya sp. BKKU1127 1/9 (11.1)a,b 1/9 (11.1)a,b 8/9 (88.8)c 6/9 (66.6)c,d 0/9 (0)a 0/9 (0)a 0/9 (0)a 0/9 (0)a *Different letters within a column indicate statistically significant differences between treatments (p < 0.05).
AACL Bioflux, 2015, Volume 8, Issue 1. http://www.bioflux.com.ro/aacl 77 Discussion. Water mold infection is a common problem in aquaculture and it is widespread in all stages of the fish life cycle (Bruno et al 2011). Experimental infections to demonstrate the pathogenicity of Saprolegnia or Aphanomyces have been attempted by some methods, such as zoospore injection into the muscle or exposure to zoospore suspension (Howe et al 1998; Khan et al 1998; Johnson et al 2004). These experiments could not reproduce the clinical signs of water mold infection, especially in salmonid fish. However, Hatai & Hoshiai (1993) succeeded in reproducing the clinical signs of saprolegniasis using the AM treatment, which enhanced the susceptibility of fish to Saprolegnia infection and the AM treatment predisposes fish infection because it causes stress and a decrease in epidermal thickness and in the number of mucous cells. In the present study, we selected 2 minutes of AM treatment for induced the lesion of skin of Nile tilapia fry due to there were no fish died during the experiment. This result was supported by other researches who used 2 minutes for the AM treatment in juvenile of rainbow trout (Oncorhynchus mykiss) (Yuasa & Hatai 1995; Grandes et al 2001), fingerlings of masu salmon (O. masou), sockeye salmon (O. nerka), brown trout (Salmo trutta), Japanese char (Salvelinus leucomaenis) (Hussein & Hatai 2002), adult of platyfish (Xiphophorus maculates) (Hanjavanit et al 2010) and sub-adult Nile tilapia (Hussein et al 2013). The pathogenicity tests using 6 isolates of the genus Achlya from infected Nile tilapia were carried out using Nile tilapia fry. We succeeded in producing experimental infection using the AM treatment. All Achlya isolates demonstrated their pathogenicity toward the test fish. The levels of infection and mortality were classified into 3 groups, which were high infection (77.7–88.8% cumulative mortality), moderately infection (55.6–66.6% cumulative mortality), and low infection (33.3% cumulative mortality) to Nile tilapia fry. From this study, it may be stated that 6 isolated Achlya spp. from cultured Nile tilapia have pathogenicity to Nile tilapia fry. The fish exposed to A. klebsiana BKKU1003 showed a high infection ratio and cumulative mortality than those exposed to others, and the differences were statistically significant (p < 0.05). Similar results using A. klebsiana were reported by Chukanhom & Hatai (2004) who demonstrated pathogenicity against platy (Xiphophorus maculates), which showed 100% infection rate of injured fish when exposed to 1.0 x 104 zoospores mL-1. In the present study, the isolate of A. bisexualis BKKU1007 showed the lowest pathogenicity, which was similar to the pathogenicity test against guppy (Poecilia reticulata) reported by Lawhavinit et al (2002), which showed 33% infection rate of injured fish when exposed to 1.0 x 102 zoospores mL-1. The pathogenicity differences among Achlya species may be due to the water mold species or the number of zoospores (Howe et al 1998). In the current study, the histopathology of Nile tilapia fry exposed to zoospores of Achlya after the AM treatment showed numerous hyphae covering the surface of the epidermis and invading into the muscle. Whereas, Hussein et al (2013) reported the experimental infection of A. proliferoides in sub-adult tilapia and found mycelial mats attached to the surface of the epidermis, with some penetrating only in the dermal layer. The difference of these pathological findings may be caused by the size or stage of fish. In addition, this may be related to the differences in pathogenicity of the isolates or host response (Sosa et al 2007). No granulomas were found around the hyphae in the affected musculature, which may be due to the rapid growth of the hyphae, as mentioned by Hatai & Hoshiai (1992). The AM treatment is a kind of physical trauma, which could conduct neuroendocrine stress leading to immunosuppression in fish (Hatai & Hoshiai 1994). Moreover, the treatment decreased in epidermal thickness and in the number of mucous cells, which has been proposed to increase risk of fungal infection (Pickering 1977; Richards & Pickering 1979). Noga (1993) stated that the importance of mucous and skin as a physical barrier against external pathogens. In addition, the direct cause of death in the infected fish is most likely related to massive osmoregulatory system problems and a lethal dilution of body fluids (Grandes et al 2001; Fontenot & Neiffer 2004). The healthy fish directly exposed to a number of zoospores (1.0 x 104 zoospores mL-1), it was found that no water mold infection appeared. This pointed out that the AM treatment is needed as a primary inducer for the water mold infection and these Achlya spp. might be
AACL Bioflux, 2015, Volume 8, Issue 1. http://www.bioflux.com.ro/aacl 78 opportunistic pathogens which was supported by Lilley & Roberts (1997) and Sosa et al (2007), who stated that species of the genus Achlya are usually known as saprophytic or secondary, opportunistic invaders. Environmental factor such as temperature may play an important role, as the water mold infection usually occurs in the cool season (Willoughby & Lilley 1992; Chinabut et al 1995; Chukanhom & Hatai 2004; Hanjavanit et al 2012). The present study maintained an experimental temperature at 25°C for artificial infection because this temperature is in a range that caused the water mold infection in nature.
Conclusions. This is the first report to induce Achlya spp. infection in Nile tilapia fry under the AM treatment in Thailand. It seems likely that the AM treatment has the same effect as handling or crowded conditions for the fish, inducing stress and disturbing their defense mechanisms. This treatment may also induce external mycotic zoospores to make contact with the skin, which serves as a highly supportive nutritional source. Our comparative study showed that 1.0 x 104 zoospores mL-1 of A. klebsiana BKKU 1003 had the highest pathogenicity. These results demonstrate the first success using the AM treatment to induce Achlya spp. experimental infection using the same host.
Acknowledgement. This work was strongly supported by the National Research University Program, Khon Kaen University, Thailand and grant No. W-2553-Ph.d-02, which is gratefully acknowledged. The authors wish to express their sincere thanks to the Graduate School of Khon Kaen University for supporting the first author for oversea research of this project in Japan.
References
Abking N., Fuangsawat W., Lawhavinit O., 2012 Achlya spp. isolated from eggs of the Mekong giant catfish (Pangasianodon gigas, Chevey). Proceedings of 47th Kasetsart University Annual Conference, Veterinary medicine, Bangkok, pp. 187-196. Bruno D. W., West P. V., Beakes G. W., 2011 Saprolegnia and other Oomycetes. In: Fish diseases and disorders. Volume 3, viral, bacterial and fungal infections. Woo P. T. K., Bruno D. W. (eds), CABI Publishing, Wallingford, Oxfordshire, pp. 669–720. Chinabut S., Robert R. J., Willoughby G. R., Pearson M. D., 1995 Histopathology of snakehead, Channa striatus (Bloch), experimentally infected with the specific Aphanomyces fungus associated with epizootic ulcerative syndrome (EUS) at different temperatures. Journal of Fish Diseases 18:41–47. Chukanhom K., Hatai K., 2004 Freshwater oomycete isolated from eggs of the common carp (Cyprinus carpio) in Thailand. Mycoscience 45:42–48. Fontenot D. K. Neiffer D. L., 2004 Wound management in teleost fish: biology of the healing process, evaluation and treatment. Veterinary Clinics of North America: Exotic Animal Practice 7:57–86. Grandes J. M. F., Diez M. F., Gancedo J. M. A., 2001 Experimental pathogenicity in rainbow trout Onchorhynchus mykiss (Walbaum), of two distinct morphotypes of long-spine Saprolegnia isolates obtained from wild brown trout Salmo trutta L., and river water. Journal of Fish Disease 24:351–359. Hanjavanit C., Panchai K., Wada S., Kurata O., Hatai K., 2010 Artificial infection by water molds following net-shake treatment in the platyfish (Xiphophorus maculates). Aquaculture Science 58:533–537. Hanjavanit C., Rakmanee C., Kitancharoen K., Hatai K., 2012 Freshwater oomycete isolates from African catfish Clarias gariepinus eggs in Thailand. Aquaculture Science 60:269–276. Hatai K., Egusa S., 1979 Studies on the pathogenic oomycete of mycotic granulomatosis III. Development of the medium for egg–oomycete. Fish Pathology 13:147–152. Hatai K., Hoshiai G., 1992 Mass mortality in cultured coho salmon (Oncorhynchus kisutch) due to Saprolegnia parasitica Coker. Journal of Wildlife Diseases 28:532– 536.
AACL Bioflux, 2015, Volume 8, Issue 1. http://www.bioflux.com.ro/aacl 79 Hatai K., Hoshiai G., 1993 Characteristics of two Saprolegnia species isolated from coho salmon with saprolegnisis. Journal of Aquatic Animal Health 5:115–118. Hatai K., Hoshiai G., 1994 Pathogenicity of Saprolegnia parasitica Coker. In: Salmon Saprolegniasis. Maeller G. J. (ed), U.S. Department of Energy, Portland, Oregon, pp. 87–98. Howe G. E., Rach J. J., Olson J. J., 1998 Method for inducing saprolegniosis in channel catfish. Journal of Aquatic Animal Health 10:62–68. Hussein M. A., Hatai K., 2002 Pathogenicity of Saprolegnia species associated with outbreaks of salmonid saprolegniosis in Japan. Fisheries Science 68:1067–1072. Hussein M. A., Hassan W. H., Mahmoud M. A., 2013 Pathogenicity of Achlya proliferoides and Saprolegnia diclina (Saprolegniaceae) associated with Saprolegniosis outbreaks in cultured Nile tilapia (Oreochromis niloticus). World Journal of Fish and Marine Sciences 5:188–193. Johnson R. A., Seymour R. L., Padgett D. E., 2002 Biology and the systematics of the Saprolegniaceae. Available at: http://dl.uncw.edu/digilib/biology/oomycete/ taxonomy and systematics/padgett book/ (Accessed July 21, 2010). Johnson R. A., Zabrecky J., Kiryu Y., Shields J. D., 2004 Infection experiments with Aphanomyces invadans in four species of estuarine fish. Journal of Fish Disease 27:287–295. Khan M. H., Marshall L., Thomson K. D., Campbell R. E., Lilley J. H., 1998 Susceptibility of five fish species (Nile tilapia, rosy barb, rainbow trout, stickleback and roach) to intramuscular injection with the oomycete fish pathogen, Aphanomyces invadans. Bulletin of the European Association of Fish Pathologists 18:192–197. Kiryu Y., Shields J. D., Vogelbein W. K., Zwerner D., Kator H., 2002 Induction of skin ulcers in Atlantic menhaden by injection and aqueous exposure to the zoospores of Aphanomyces invadans. Journal of Aquatic Animal Health 14:11–24. Kitancharoen N., Hatai K., Ogihara R., Aye D. N. N., 1995 A new record of Achlya klebsiana from snakehead, Channa striatus, with fungal infection in Myanmar. Mycoscience 36:235–238. Lawhavinit O., Chukanhom K., Hatai K., 2002 Effect of Tetrahymena on the occurrence of achlyosis in the guppy. Mycoscience 43:27–31. Lilley J. H., Roberts R. J., 1997 Pathogenicity and culture studies comparing the Aphanomyces involved in epizootic ulcerative syndrome (EUS) with other similar fungi. Journal of Fish Diseases 20:135–144. Lilley J. H., Hart D., Panyawachira V., Kanchanakhan S., Chinabut S., Sӧderhäll K., Cerenius L., 2003 Molecular characterization of the fish-pathogenic fungus Aphanomyces invadans. Journal of Fish Diseases 26:263–275. Muraosa Y., Sano A., Hatai K., 2012 Molecular identification of marine crustacean- pathogenic Peronosporomycetes using DNA sequences of ITS1 and their pathogenicity for nauplii of brine shrimps. Fish Pathology 47:41–48. Muraosa Y., Morimoto K., Sano A., Nishimura K., Hatai K., 2009 A new Peronosporomycete, Halioticida noduliformans gen. et sp. nov., isolated from white nodules in the abalone Haliotis spp. from Japan. Mycoscience 50:106–115. Noga E. J., 1993 Water mould infections of freshwater fish: recent advances. Annual Review of Fish Diseases 3:291–304. Panchai K., Hanjavanit C., Kitacharoen N., 2007 Characteristics of Achlya bisexualis isolated from eggs of Nile tilapia (Oreochromis niloticus). KKU Research Journal 12:195–202. Panchai K., Hanjavanit C., Rujinanont N., Wada S., Kurata O., Hatai K., 2014 Freshwater oomycete isolated from net cage cultures of Oreochromis niloticus with water mold infection in the Nam Phong River, Khon Kaen Province, Thailand. AACL Bioflux 7:529-542. Phadee P., Kurata O., Hatai K., Hirono I., Aoki T., 2004 Detection and identification of fish-pathogenic Aphanomyces piscicida using polymerase chain reaction (PCR) with species–specific primers. Journal of Aquatic Animal Health 16:220–230. Pickering A. D., 1977 Seasonal changes in the epidermis of the brown trout, Salmo trutta (L.). Journal of Fish Biology 10:561–566.
AACL Bioflux, 2015, Volume 8, Issue 1. http://www.bioflux.com.ro/aacl 80 Richards R. H., Pickering A. D., 1979 Changes in serum parameters of Saprolegnia- infected brown trout, Salmo trutta L. Journal of Fish Diseases 2:197–206. Sosa E. R., Landsberg J. H., Kiryu Y., Stephenson C. M., Cody T. T., Dukeman A. K., Wolfe H. P., Vandersea M. W., Litaker R. W., 2007 Pathogenicity studies with the fungi Aphanomyces invadans, Achlya bisexaulis, and Phialemonium dimorphosporum: induction of skin ulcers in striped mullet. Journal of Aquatic Animal Health 19:41–48. Stueland S., Hatai K., Skaar I., 2005 Morphological and physiological characteristics of Saprolegnia spp. strains pathogenic to Atlantic salmon, Salmo salar L. Journal of Fish Disease 28:445–453. Wada S., Yorisada Y., Kurata O., Hatai K., 2003 Histological detection of aquatic fungi by Uvitex 2B, a fluorescent dye. Fish Pathology 38:49–52. Willoughby L. G., Lilley J. H., 1992 The ecology of aquatic fungi in Thailand and the fish diseases relationship. The Aquatic Animal Health Research Institute Newsletter Article 1:5–6. Wisenden B. D., Smith R. J. F., 1997 The effect of physical condition and shoal-mate familiarity on proliferation of alarm substance cells in the epidermis of fathead minnows. Journal of Fish Biology 50:799-808. Yuasa K., Hatai K., 1995 Relationship between pathogenicity of Saprolegnia spp. isolates to rainbow trout and their biological characteristics. Fish Pathology 30:101–106. Yuasa N., Chaloakpantharat P., Teabsree S., 2000 Fungal diseases in economically important fishes and disease prevention (in Thai with English Abstract). KKU fund for research reports, Khon Kaen University, 25 pp. Zar J. H., 2010 Biostatistical analysis. Prentice–Hall International Inc., New Jersey, 944 pp.
Received: 09 January 2015. Accepted: 12 February 2015. Published online: 14 February 2015. Authors: Kwanprasert Panchai, Applied Taxonomic Research Center, Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand, e-mail: [email protected] Chutima Hanjavanit, Applied Taxonomic Research Center, Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand, e-mail: [email protected] Nilubon Rujinanont, Department of Fisheries, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand, e-mail: [email protected] Shinpei Wada, Laboratory of Aquatic Medicine, School of Veterinary Medicine, Nippon Veterinary and Life Science University, Tokyo 180–8602, Japan, e-mail: [email protected] Osamu Kurata, Laboratory of Aquatic Medicine, School of Veterinary Medicine, Nippon Veterinary and Life Science University, Tokyo 180–8602, Japan, e-mail: [email protected] Kishio Hatai, Microbiology and Fish Disease Laboratory, Borneo Marine Research Institute, Universiti Malaysia Sabah, 88400, Kota Kinabalu, Malaysia, e-mail: [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. How to cite this article: Panchai K., Hanjavanit C., Rujinanont N., Wada S., Kurata O., Hatai K., 2015 Experimental pathogenicity of Achlya species from cultured Nile tilapia to Nile tilapia fry in Thailand. AACL BIOFLUX 8(1):70-81.
AACL Bioflux, 2015, Volume 8, Issue 1. http://www.bioflux.com.ro/aacl 81 AACL BIOFLUX Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society
Length–weight relationships of four non-native cyprinid from the semiarid region in North-East of Algeria 1Fateh Mimeche, 2Mohamed Biche
1 Department of Agricultural Sciences, University of M’Sila, M’Sila, Algeria; 2 Department of Zoology and Forestry, National Agronomic Institute, El Harrach, Algeria. Corresponding author: F. Mimeche, [email protected]
Abstract. Length-weight (L-W) relationships for 4 non-native cyprinid species collected in the Ain Zada reservoir (North-East of Algeria): Cyprinus carpio (Linnaeus, 1758), Hypophtalmichthys molitrix (Valenciennes, 1844), Hypophtalmichthys nobilis (Richardson, 1845) and Carassius auratus gibelio (Bloch, 1782). The values of the exponent b are in the L-W relationships ranged from 2.43 to 3.37. This is the first L-W parameters reported for four non-native freshwater fish in Algeria. Key words: Stock biomass, fish condition indicators, population dynamics, stock assessment.
Introduction. Cyprinid fishes have received much attention from evolutionary biologists, as they show a wide distribution around the world and occur in almost every freshwater environment (Szlachciak & Strakowska 2010). Cyprinidae family includes the greatest number of species used by humans of any family of fishes in the North Africa (Mimeche et al 2013). In Algeria, about 27 non-native fish species was introduced and at least 303 introduction events, either intentional or accidental, were recorded in the literature (Kara 2011). The Asian carp is introduced in Algeria between 1858 and 1931 (Kara 2011; Dieuzeide & Roland 1951; Kottelat 1997). In 1986, the authorities introduced for the first time the alevins, spawner phytophagous and carnivorous in the Ain Zada reservoir, for control and aquaculture. These introductions reflect prevailing attitudes and values by the public authorities in which the primary concern is a socioeconomic benefit (Kara 2011). In the aim to transform the growth-in-length equations to growth-in-weight, the length–weight relationships (LWR) have been used for estimate stock biomass from limited sample sizes, as indicators of fish condition and used for stock assessment models, also to compare the life histories of some species among regions and other aspects of fish population dynamics (Kohler et al 1995; Petrakis & Stergiou 1995; Gonçalves et al 1997; Moutopoulos & Stergiou 2002, Andreu-Soler et al 2006) and is an important tool in fish biology, physiology, ecology and fisheries assessment (Oscoz et al 2005). This paper tries to apply the recommendations given by Froese (2006). The present paper is the first published information on length–weight relationships of 4 non-native fish species in the reservoirs of semiarid regions of Algeria.
Material and Method. This study was conducted in the Ain Zada Reservoir (05'' 80' 36° N, 40'' 18' 05° E), a big dam located in a semiarid region in the Bou Sellam basin (Figure 1), northeastern Algeria (Bordj Bou Arreridj City). The area has a semiarid Mediterranean climate, characterized by a relatively temperate winter also hot and dry summer, the rainy season runs among September and May, announcing the end of the wet season. The Bou Sellam basin is characterized too by an interannual variability of rainfall,
AACL Bioflux, 2015, Volume 8, Issue 1. http://www.bioflux.com.ro/aacl 82 where we note that much of the rain falls in this region were during a few weeks in the form of downpour, then great droughts ahead after these downpours. The average annual rain falls vary between 300 and 700 mm. The Ain Zada reservoir is fed by Oued Bou Sellam, the reservoir stored a volume of water about 125 hm³ and regulating a volume of 50 hm3 per year to ensure the drinking and industrial water needs for the rapidly growing populations of Bordj Bou Arreridj and Setif cities.
N Water Vegetation Urban area Oued Express way El Mahdia National road
20 Km
Ain Taghrout
ALGERIA AFRICA
Figure 1. Location of the study area.
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Fish samples were collected monthly from January 2012 until June 2012 using multi- mesh gill nets. In this reservoir, Luciobarbus callensis is the only native species and coexists with other non-native fish. The captured specimens were preserved in neutralized formaldehyde solution (7%) and transported into the laboratory for identification to the lowest taxonomic level according to Kottelat & Freyhof (2007). Total length (TL; ±0.1 mm) and weight (W; ±0.1 g) were recorded. The length–weight relationships were calculated using the formula (Le Cren 1951): W = a TLb
were estimated by linear regression after logarithmic transformation of the data (Froese 2006): log W = log a + b log TL
where: W - total weight; TL - total length; a - the intercept; b - the regression slope.
Statistical analyses were performed with SPSS (SPSS, Chicago, IL, USA) software package.
Results and Discussion. In this study, four species from Cyprinidae family were sampled/recorded in the reservoir: Cyprinus carpio (Linnaeus, 1758), Hypophtalmichthys molitrix (Valenciennes, 1844), Hypophtalmichthys nobilis (Richardson, 1845) and Carassius auratus gibelio (Bloch, 1782). Other species at exist in this reservoir but not captured in this study; Perca fluviatilis (Linnaeus, 1758), Abramis brama (Linnaeus, 1758), and Aspius aspius (Linnaeus, 1758) cited by Kara (2011). Ctenopharyngodon idella (Valenciennes, 1844) and Squalius cephalus (Linnaeus, 1758) catched by the professional fisherman; this is the first record of S. cephalus in this area. Mimeche et al (2013) reported his presence in K’sbo reservoir (M’Sila City) in the south of this basin. Length and weight mean are summarized in Table 1. According to fishbase data (http://www.fishbase.org version 01⁄ 2015), we report new maximum total lengths for 2 species: 112 cm in H. molitrix, 37.5 cm in C. auratus gibelio.
Table 1 Mean length and weight values of fishes collected in Ain Zada reservoir
Length (cm) Weight (g) Species N Mean ± SD Min-Max Mean ± SD Min-Max C. carpio carpio 114 29.00 ± 0.66 19.9-48.6 344.00 ± 23.52 122.0-1360.7 H. molitrix 95 24.17 ± 1.70 13.2-112 9596.87 ± 445.27 14.2-20000.0 H. nobilis 91 23.15 ± 0.90 83-132 19774.67 ± 5666.52 6500-36000 C. auratus gibelio 93 23.66 ± 0.26 24.6-37.5 453.00 ± 13.58 281.9-870.3 N - individuals, SD - Standard deviation.
This research provides the first reference on length–weight relationships for four non- native freshwater fish in Algeria (Table 2). All length–weight relationships were highly significant (P < 0.001), with r² values being greater than 0.87. Slopes (b values) of the length–weight relationships ranged from 2.43 ± 0.05 for C. carpio to 3.37 ± 0.14 for H. nobilis (Table 2).
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Table 2 Length–weight relationship (LWR) parameters and values (a and b) and condition factor for five fish species caught in Ain Zada reservoir
SE SE Species N a CI 95% (a) b CI 95% (b) r² (a) (b) C. carpio carpio 114 -1.04 0.07 1.18-0.89 2.43 0.05 2.33-2.53 0.95 H. molitrix 95 -2.43 0.12 2.67-2.19 3.26 0.06 3.14-3.39 0.96 H. nobilis 91 -2.58 0.28 3.14-2.02 3.37 0.14 3.09-3.64 0.87 C. auratus gibelio 93 -1.79 0.11 2.02-1.56 3.04 0.07 2.88-3.19 0.94 N- individuals, SE - the standard error of the slope (a, b), CI - confidence interval, r² - the coefficient of determination.
Parameter b values remained mostly within the expected range of 2.5–3.5 (Froese 2006), but can vary between 2 and 4 (Bagenal & Tesch 1978) for most fishes. The mean value of 3.025 (SE = 0.21). The median of b was 3.150, whereas 75% of the values ranged between 3.04 and 3.37 (Figure 2).
Figure 2. Box-Whiskers plots of the exponent b of the length-weight relationships (W = aLb) for the four fish species in Ain Zada Reservoir. The central box covers 75% of data values, the horizontal line indicates the median, and the vertical line represents the range of the values.
The length–weight relationship in fishes can be affected by habitat, season, gonad maturity, sex, health, preservation techniques, even time of day (because of changes in stomach fullness); and differences in the observed length ranges of the specimen caught (Oliva-Paterna et al 2009). However, the population of C. carpio is outside of the expected range (2.436 ± 0.051). We can explain the low b-values by the effects of environmental seasonality, the reproductive cycle of the species and to the transfer of energy to the gonads (Mimeche et al 2013). The b-values calculated for C. carpio and H. nobilis, H. molitrix and C. auratus gibelio from the Ain Zada reservoir in north east of Algeria when compared with those obtained by other authors in different area are presented in Table 3.
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Table 3 Parameters b and length obtained from different areas for four fish species
Total Length (cm) Species Locality (Area) b Reference Mean Min- Max Kleanthidis et Lake Volvi Greece - 7.8-18.1 2.67 al (1999) Lake Niushan Ye et al 46.05 12.4-82.3 2.966 China (2007) all regions of Treer et al - 15.20-73.50 2.895 C. carpio Croatia (2008) Segura river basin Andreu-Soler (southeast-ern - 10.0-23.8 3.68 et al (2006) Spain) Iberian Peninsula Miranda et al - 7.1-59 3.070 (Spain) (2006) Lake Niushan in Ye et al 39.70 15.2-64.1 3.162 China (2007) H. molitrix Wanner & Missouri River USA - 23.1-88.0 3.13-3.70 Klumb (2009) Lake Niushan Ye et al 45.45 24.2-73.4 3.167 China (2007) H. nobilis Wanner & Missouri River USA - 32.2-120.0 2.75-2.96 Klumb (2009) Kleanthidis et Lake Volvi Greece 8.2 25.2 3.11 al (1999) C. auratus Lake Niushan Ye et al 14.84 5.5-31.8 3.100 gibelio China (2007) All regions of Treer et al - 5.10-29.20 2.976 Croatia (2008)
Conclusions. C. carpio and H. nobilis, H. molitrix and C. auratus gibelio present a large tolerance range overlooked the variable environmental conditions in semiarid reservoir and establish new viable populations disperse widely and incorporated in large numbers in the ecosystem. Tarkan et al (2012) showing the growth of the fishes introduced into artificial water bodies is faster than that observed in populations of natural lakes and flowing waters. Thus, certain characteristics of the species as a strong physiological tolerance or dispersal limitation (Lauzeral et al 2010; Segurado et al 2011), a high reproductive rate and reproductive strategies (Ruesink 2005; Tarkan et al 2012), aggressive behavior and competitiveness (Conrad et al 2011; Moyle & Marchetti 2006). Environmental conditions similar to those of the native zone (Moyle & Marchetti 2006).
Acknowledgements. This study was funded by the University of M’Sila. We greatly acknowledge to M. Nouijem Yacine Lecturer in the University of M’Sila, administrative staff and the fisherman of the Ain Zada Reservoir. We dedicate this work to the memory of Belmaloufi Elkhier contributing in this study. We sincerely thank Radia Mimeche for her help in English language.
References
Andreu-Soler A., Oliva-Paterna F. J., Torralva M., 2006 A review of length–weight relationships of fish from the Segura River basin (SE Iberian Peninsula). J Appl Ichthyol 22:295–296.
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Bagenal T. B., Tesch F. W., 1978 Age and growth. In: Methods for assessment of fish production in fresh waters. Pp. 101–136, IBP Handbook No 3, Blackwell Scientific Publications, Oxford. Conrad J. L., Weinersmith K. L., Brodin T., Saltz J. B., Sih A., 2011 Behavioural syndromes in fishes: a review with implications for ecology and fisheries management. J Fish Biol 78:395-435. Dieuzeide R., Rolland J., 1951 Le laboratoire d'hydrobiologie et de pisciculture d'eau douce du Mazafran. Bull Stat Aquic Pêche Castiglione 3:190–207. Froese R., 2006 Cube law, condition factor and weight–length relationships: history, meta-analysis and recommendations. J Appl Ichthyol 25:241–253. Gonçalves J. M. S., Bentes L., Lino P. G., Ribeiro J., Canario A. V. M., Erzini K., 1997 Weight–length relationships for selected fish species of the small-scale demersal fisheries of the southwest coast of Portugal. Fish Res 30:253–256. Kara H. M., 2011 Freshwater fish diversity in Algeria with emphasis on alien species. Eur J Wildl Res 58:243–253. Kleanthidis P. K., Sinis A. I., Stergiou K. I., 1999 Length-weight relationships of freshwater fishes in Greece. Naga, The ICLARM Quarterly 22(4):37-41. Kohler E. N., Casey J., Turner P., 1995 Length–weight relationships for 13 species of sharks from the western North Atlantic. Fish Bull 93:412–418. Kottelat M., 1997 European freshwater fishes. A heuristic checklist of the freshwater fishes of Europe (exclusive of former USSR), with an introduction for non- systematics and comments on nomenclature and conservation. Biologia 52:1-271. Kottelat M., Freyhof J., 2007 Handbook of European freshwater fishes. Publications Kottelat, Cornol, Switzerland, 646 pp. Lauzeral C., Leprieur F., Beauchard O., Duron Q., Oberdorff T., Sébastien Brosse S., 2010 Identifying climatic niche shifts using coarse-grained occurrence data: a test with non-native freshwater. Glob Ecol Biogeogr 20:407–414. Le Cren E. D., 1951 The length–weight relationship and seasonal cycle in gonad weight and condition in the perch Perca fluviatilis. J Anim Ecol 20:210-219. Miranda R., Oscoz J., Leunda P. M., Escala M. C., 2006 Weight–length relationships of cyprinid fishes of the Iberian Peninsula. J Appl Ichthyol 22:297–298. Mimeche F., Biche M., Ruiz-Navarro A., Oliva–Paterna F. J., 2013 Population structure, age and growth of Luciobarbus callensis (Cyprinidae) in a man-made lake from Maghreb (NE, Algeria). Limnetica 2:391-404. Moutopoulos D. K., Stergiou K. I., 2002 Length-weight and length-length relationship of fish species from the Aegean Sea (Greece). J Appl Ichthyol 18:200–203. Moyle P. B., Marchetti M. P., 2006 Predicting invasion success: Freshwater fishes in California as a model. Bioscience 56:515-524. Oliva-Paterna F. J., Torralva M., Carvalho E. D., 2009 Length-weight relationships for 20 species collected in the Jurumirim reservoir (Paranapanema Basin, Brazil). J Appl Ichthyol 25:360-361. Oscoz J., Campos F., Escala M. C., 2005 Weight–length relationships of some fish species of the Iberian Peninsula. J Appl Ichthyol 21:73–74. Petrakis G., Stergiou K. I., 1995 Weight–length relationships for 33 fish species in Greek waters. Fish Res 21:465–469. Ruesink J. L., 2005 Global analysis of factors affecting the outcome of freshwater fish introductions. Conserv Biol 19:1883-1893. Segurado P., Santosa J. M., Pontb D., Melcher A. H., Jalond D. G., Hughes R. M., Ferreira M. T., 2011 Estimating species tolerance to human perturbation: Expert judgment versus empirical approaches. Ecol Indic 11:1623–1635. Szlachciak J., Strakowska E., 2010 Morphological characteristics and variation of rudd Scardinius erytrophthalmus (L.) from the Łuknajno Lake, Poland. AACL Bioflux 3(2):91-101. Tarkan A. S., Copp G. H., Top N., Özdemir N., Önsoy B., Bilge G., Filiz H., 2012 Are introduced gibel carp Carassius gibelio in Turkey more invasive in artificial than in natural waters? Fish Manag Ecol 19:178–87.
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Treer T., Sprem N., Torcu-Koc H., Sun Y., Piria M., 2008 Length–weight relationships of freshwater fishes of Croatia. J Appl Ichthyol 24:626–628. Wanner G. A., Klumb R. A., 2009 Length-weight relationships for three Asian Carp species in the Missouri River. J Freshw Ecol 24:489-495. Ye S., Li Z., Feng G., Cao W., 2007 Length-weight relationships for thirty fish species in Lake Niushan, a shallow macrophytic Yangtze Lake in China. Asian Fish Sci 20:217- 226. *** http://www.fishbase.org version 01⁄2015
Received: 08 December 2014. Accepted: 26 January 2015. Published online: 17 February 2015. Authors: Fateh Mimeche, University of M’Sila, Faculty of Science, Department of Agricultural Sciences, Algeria, M’Sila, 28000, e-mail: [email protected] Mohamed Biche, National Agronomic Institute, Department of Zoology and Forestry, Algeria, Avenue Hassan Badi - El Harrach - Algiers, 16200, e-mail: [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. How to cite this article: Mimeche F., Biche M., 2015 Length–weight relationships of four non-native cyprinid from the semiarid region in North-East of Algeria. AACL Bioflux 8(1):82-88.
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AACL BIOFLUX Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society
Significant factors affecting the economic sustainability of closed aquaponic systems. Part III: plant units Harry W. Palm, Madeline Nievel, Ulrich Knaus
University of Rostock, Faculty of Agricultural and Environmental Sciences, Aquaculture and Sea-Ranching, Justus-von-Liebig-Weg 6, 18059 Rostock, Germany. Corresponding author: H. W. Palm, [email protected]
Abstract. The hydroponic unit of two identical closed ebb-flow substrate aquaponic systems for warm- water fish were tested for water parameter differences of dissolved oxygen (DO) [mg L-1], temperature [°C], pH and phosphorus [mg L-1] under Nile tilapia (Oreochromis niloticus) cultivation. Each system contained 3.7 m3 water, and the relationship of the water volume in the aquaculture tank to the settling basin (sedimenter, clarifier), the biofilter and the hydroponic units was 2.25:1:0.075:0.6 (fish tank:hydroponic unit = 3.75). The hydroponics were built as gravel bed aggregate systems with a single nutrient enriched water inlet for each of four plant units, and a horizontal sub-irrigation towards the outlet. Related to an increasing feed input into the fish tanks, DO levels in the hydroponic units were lowest inside the system (1-4.7 mg L-1). This is a favourable place for oxygen level monitoring to indicate a stable performance. Declining DO trends were observed in both cycles, with significant differences (p < 0.05) within cycle I and II on 4 consecutive days. Oxygen means of cycle I and II were significant only on day I. Inside the 2 m2 plant boxes, slightly decreasing trends in DO distribution towards the outlet were observed. Time series of all four experimental days (ANOVA, p < 0.05) showed varying values of phosphorus with highest levels on sampling day III in both cycles. Following an increase in feed input, a sluggish phosphorus accumulation inside the plant boxes was observed. Parallel arrangement of the 4 plant boxes in each cycle had no influence onto water parameters within each plant box. Within each cycle no trend was observed, total parameter values differed only slightly, influenced by the system design of the hydroponic units. Plant growth was different in cycle I and II. Best growth was recorded close to the nutrient enriched water inlet into the plant boxes, assessing the chosen central nutrient water irrigation system as sub-optimal for plant growth. Cucumber and zucchini showed better biomass gain (sum 7.60 kg) in plant box II of cycle II than in other plant boxes, suggesting variable conditions inside the plant boxes of the tested aquaponic system. Key Words: aquaponics, hydroponics, sub-irrigation, system design, ebb and flow system, fish and plant combination, Tilapia.
Zusammenfassung. Die Hydroponikeinheit zweier identischer Ebbe-und Flut Substrat- Aquaponiksysteme, mit Haltung von Nil Tilapien (Oreochromis niloticus), wurden hinsichtlich der physikalisch-chemischen Wasserparameter Sauerstoff [mg L-1], Temperatur [°C], pH und Phosphor [mg L-1] verglichen. Die Aquaponiksysteme wurden im geschlossen Süßwasserkreislauf betrieben. Jedes System besaß ein Wasservolumen von 3,7 m³, mit einem Verhältnis des Aquakulturbehälters zum Sedimenter, dem Biofilter und der Hydroponikeinheit von 2,25:1:0,075:0,6 (Fischbehälter:Hydroponik Einheit = 3,75). Die Hydroponikeinheit des Aquaponiksystems bestand aus Kiessubstrat mit einem zentralen Einlauf der Nährstofflösung am Anfang jeder Pflanzenkiste und einer horizontal linearen Wasserverteilung über dem Substrat zum Auslauf hin. In Abhängigkeit eines kontinuierlich erhöhten Futtermitteleintrages in den Fischbehältern zeigte der Sauerstoffgehalt in der Hydroponik die geringsten Werte (1-4,7 mg L-1). An dieser Stelle lässt sich der Sauerstoffgehalt zur Überprüfung der Systemstabilität am besten beobachten. Abnehmende Sauerstoffverhältnisse wurden in beiden Kreisläufen beobachtet, mit signifikanten Unterschieden (p < 0,05) innerhalb Kreislauf I und II an allen 4 Datenaufnahmetagen. Die Mittelwerte von Sauerstoff zwischen Kreislauf I und II waren nur an Tag I signifikant im Vergleich zu den anderen Tagen. Innerhalb der 2 m² Pflanzenkisten wurden teilweise gering abnehmende Sauerstoffverteilungen vom Einlauf zum Ablauf beobachtet. Der Phosphorgehalt aller vier Tage (ANOVA, p < 0,05) zeigte variierende Werte mit höchsten Anteilen an Tag III in beiden Kreisläufen. Durch erhöhte Futtermittelvergaben kam es zu einer zeitlich versetzten Akkumulation von Phosphor in den Pflanzenkisten (Trägheitseffekt). Die parallele Anordnung der Pflanzenkisten zeigte keinen Einfluss auf die Wasserparameter. Innerhalb eines Kreislaufes wurden keine Trends beobachtet, die Werte zeigten nur geringe Differenzen, beeinflusst durch den Aufbau der Hydroponikeinheiten. Das Pflanzenwachstum war zwischen Kreislauf I und II verschieden. Das beste Pflanzenwachstum wurde nahe dem Einlauf am Anfang der Pflanzenkisten beobachtet, wodurch ein zentraler Nährstoffeinfluss als
AACL Bioflux, 2015, Volume 8, Issue 1. 89 http://www.bioflux.com.ro/aacl suboptimal für das Pflanzenwachstum bewertet werden kann. Gurken und Zucchini zeigten besseres Wachstum (Summe 7,60 kg) in Pflanzenkiste II von Kreislauf II als in den anderen Pflanzenkisten. Schlüsselworte: Aquaponik, Hydroponik, Bewässerung, System Design, Ebbe- und Flut System, Fisch und Pflanze Kombination, Tilapia.
Introduction. Economic sustainability of aquaponics, the combination of aquaculture and hydroponics, depends on a variety of factors, including system and feed design, animal welfare or parasite and pathogen control (Palm et al 2014a). Rakocy et al (2006) described the essential components of aquaponic systems as the fish rearing tanks, components for settable and suspended solids removal, biofilter, sump and hydroponic units for plant production. However, size, arrangement and the management of fish and plant production determine their functionality. Palm et al (2014a) referred to the component ratios (fish-to-plant-units, water volume, clarifier volume or biofilter volume), water exchange rates, aeration, waste removal and different cultivation methodologies such as batch, staggered, intercropping or polyculture. Successful aquaponic production must consider both optimal fish and plant species selection (Palm et al 2014b) as well as the best suitable cultivation technologies. Soilless cultivation in a nutrient solution (hydroponics, Raviv & Lieth 2008) includes the use of different aggregates (organic or inorganic substrates) and techniques for the nutrient supply such as floating raft aquaponics (Rakocy 1989; Rakocy et al 2004; Al-Hafedh et al 2008), nutrient film technique (NFT, Kloas et al 2011), deep-flow technique (DFT) or aeroponics. In the latter, the plant roots are sprayed directly with a nutrient enriched solution with the help of water nozzles (Farran & Mingo-Castel 2006). Most common are aggregate systems using inert substrates like stone wool or gravel for plant root fixation and water preservation (Raviv & Lieth 2008). Rakocy et al (2006) suggested economical savings with the use of substrates for biofiltration in hydroponics at the same time. The oxidation of ammonia to nitrite and nitrate by bacteria (e.g. Nitrosomonas, Nitrobacter) settling on a substrate can replace additional biofilters commonly used in recirculation aquaculture and closed non-aggregate aquaponic systems. Sand as a substrate was studied by McMurtry et al (1997) in a system with hybrid tilapia (Oreochromis mossambicus X Oreochromis niloticus) and tomatoes (Solanum lycopersicum). Graber & Junge (2009) tested a light-expanded clay aggregate (LECATM) as a substrate for the cultivation of aubergine (Solanum melongena), tomato and cucumber (Cucumis sativus) with Nile tilapia (O. niloticus) and Eurasian perch (Perca fluviatilis), resulting in highest nutrient removal rates under tomato culture. Lewis et al (1978) examined in outdoor hydroponic tanks with gravel the production of three varieties of tomatoes and channel catfish (Ictalurus punctatus), revealing excellent water quality parameters. Also Palm et al (2014b) studied the effect of a gravel based aquaponic system (ebb-and flow) on Nile tilapia, African catfish (Clarias gariepinus) and different plants (lettuce - Lactuca sativa, tomato, cucumber and basil - Ocimum basilicum), demonstrating a significantly better plant growth with the Nile tilapia. The substrate selection has direct consequences for biofilter activity and nutrient availability for the cultivated plants within the tested system. Graber & Junge (2009) compared the LECATM system (aquaponics), a stand alone fertilized hydroponics and a soil cultivation under irrigation with fish tank water. The LECATM substrate functioned as a biofilter with positive effects on nitrification and micro- and macronutrient uptake by the plants. Fish growth of Nile tilapia and Eurasian perch was identical to traditional aquaculture production, and tomatoes showed highest nutrient recycling capability, followed by aubergine and cucumber. Comparison of the optimal fertilized standalone hydroponic system with aquaponics demonstrated less nitrogen (factor 3) and phosphorus (factor 10) in the aquaponic system, with equal yields of tomatoes in aquaponics, hydroponics, and soil cultivation. However, the fish tank water showed a 45 times lower concentration of potassium in comparison to the hydroponic system, negatively affecting the tomato fruit quality from aquaponic production. The experiment demonstrated an adequate use of the LECATM trickling filter system for plant
AACL Bioflux, 2015, Volume 8, Issue 1. 90 http://www.bioflux.com.ro/aacl production, an almost identical tomato yield in both systems (hydroponic, aquaponic) and no fishy taste of the fruits. The aquaponic system in the present study was built as a low-tech warmwater closed recirculating gravel ebb-and flow system for the production of Nile tilapia in freshwater (Palm et al 2014a). Different plants were cultivated with a single central nutrient water inflow at one side of each of 4 plant boxes in each cycle, followed by water irrigation towards a single plant box outlet. We herewith report the physical and chemical water parameters inside each plant box during 4 experimental days with different feed input. Possible effects of the water distribution within the plant boxes onto oxygen and phosphorus levels, with relevance for the observed plant growth, are discussed.
Material and Method
Experimental design and data collection. Two closed aquaponic recirculation units (cycle I, cycle II) were built for freshwater and warmwater fish and plant cultivation (Palm et al 2014a, b). The water volume was 3.7 m3 in each recirculation system, with a relationship of the water volume in the aquaculture unit (1,800 L), the sedimenter (800 L), the biofilter (60 L volume biocarrier), and the hydroponic unit (480 L) of 2.25:1:0.075:0.6 (fish tank:hydroponic unit = 3.75). The plant boxes (1.00 x 2.00 x 0.30 m) were laid out with polyethylene foil (3 mm) and filled with gravel (2,000 kg each cycle) as a substrate, with a maximum water level of 20 cm (120.00 L). The plant boxes were equipped with a water siphon (bell pipe) that allowed one maximum water level per hour (ebb and flow system, 24 times per day). The filtered amount of water through the plant boxes was set for 11,520 L per day, passing through the hydroponic unit 3.1 times in 24 hours. Each plant box in both units was equipped with 9 polyvinyl chloride (pvc) test tubes, with a height of 15 cm and 1.2 cm in diameter, and an arrangement of three tubes in one row (Figure 1).
Figure 1. Schematic overview of one 2 m2 plant box with inflow, plant cultivation area, test tubes, gravel substrate, water flow, outflow and sump.
The nutrient water inflow was set at the centre of one side of each box, with the water flowing towards the outlet. Physical water parameters of temperature [°C], oxygen [mg L-1] and pH were taken from the test tubes using a HQ40D multimeter (Hach Lange GmbH, Germany). Additional probes were filtered with a Whatman GF6 glass filter, and the phosphorus [mg L-1] content was analysed by using a spectral photometer DR-3900 (Hach Lange GmbH, Germany). Samples were taken on four days (09.07.2012 (day I), 16.07.2012 (day II), 23.07.2012 (day III) 30.07.2012 (day IV)) under daily feed input
AACL Bioflux, 2015, Volume 8, Issue 1. 91 http://www.bioflux.com.ro/aacl levels of 243.00 g, 327.5 g, 175.00 g, and 215.00 g respectively, during the steady phase of sub-experiment III (SE III) as described by Palm et al (2014a).
Fish and plant species. The aquaculture units of cycle I and II were stocked on the 21.03.2012 with 398 Nile tilapia postlarvae of 0.50 g (Palm et al 2014a). The total experimental time of 160 days was divided into three sub-experiments (SE I, SE II and SE III), with the present sampling describing the water parameters in the plant boxes of four sampling days (duration) within SE III, recorded as day I (equals day 111 of Palm et al 2014a), day II (day 118), day III (day 125) and day IV (day 132). Fish weight on day 112 in cycle I was 25.94 g (±9.73) and 22.64 g (±9.17) in cycle II, whereas fish weight on day 132 in cycle I was 46.83 g (±23.10) and in cycle II 41.55 g (±16.04). Fish were fed with E-2P Stella (Skretting) with 47% crude protein, 14% crude lipid, 2.60% crude fibre and 6.50% crude ash, one time daily by hand. Plant cultivation in each cycle was different. Seeds originated from N. L. Chrestensen Erfurter Samen- und Pflanzenzucht GmbH (Erfurt, Germany). Cycle I was planted with 60 tomato type Moneymaker, 11 butterhead lettuce type Mona, 11 lettuce type Lollo rosso, and 11 spinach (Spinacia oleracea) type Matador. Plant box I was planted with tomato (n = 15) and lettuce (n = 8), plant box II with tomato (n = 15) and spinach (n = 8), plant box III with tomato (n = 15) and lettuce (Lollo rosso, n = 8) and plant box IV with tomato (n = 15), lettuce (n = 3), spinach (n = 3) and lettuce (Lollo rosso, n = 3). A wider range of plant species was cultivated in cycle II: 5 tomato type Moneymaker, 10 paprika (Capsicum annum) type Yolo Wonder, 5 broccoli (Brassica oleracea var. italica) type Calabrese natalino, 8 butterhead lettuce type Mona, 8 zucchini (Cucurbita pepo) type Diamant F1 hybrid, 7 cucumber type Montea, 8 spinach type Matador, 5 aubergine type Early Long Purple 3, 8 lettuce type Lollo rosso, 3 peppermint (hybrid Mentha × piperita), 3 basil, 3 chives (Allium schoenoprasum) type Polyvit and 3 rosemary (Rosmarinus officinalis). Plant box I was planted with tomato (n = 5), broccoli (n = 5), paprika (n = 5) and lettuce (n = 8). Plant box II was planted with zucchini (n = 8), cucumber (n = 7) and spinach (n = 8). In plant box III was grafted paprika (n = 5), aubergine (n = 5) and lettuce (Lollo rosso, n = 8). Plant box IV was planted with rosemary (n = 3), peppermint (n = 3), chives (n = 3) and basil (n = 3).
Statistical analyses. Tests were performed in order to identify possible differences of physical and chemical water parameters inside the plant boxes, caused by the plant box system design (water inflow, ebb-and flow interval) and different plant species cultivation, as compared between cycle I and cycle II and inside the boxes of each cycle. Within the hydroponic unit two groups (means, comparison between cycle I and II and between each box of cycle I and cycle II) were compared and tested using the Shapiro- Wilk test, followed by the t-test and Levene statistics, in the case of normal distribution. Otherwise, the Mann-and-Whitney test was performed in order to determine significant differences at the p < 0.05 level. Analyse of variance (ANOVA, p < 0.05) was used to determine significant differences between more than two groups of water parameters with Levene test and post hoc Tukey-HSD test at variance homogeneity. Otherwise, the Dunnett-T3 test for variance inhomogeneity was used. All data were analysed by Microsoft Excel (2010) and the SPSS 20.0 statistical software package (IBM).
Results. Dissolved oxygen (DO) [mg L-1] values as determined from the 9 test tubes in each plant box decreased for cycle I and II from day I until IV (Figure 2, Table 1). Insignificant differences were found in cycle I on day II and III, and in cycle II on day III and IV. In cycle I the linear regression was y = -1.1245x+5.0598 (R² = 0.7735), and the absolute DO levels of cycle II were nearly the same, with the regression curve y = -1.0338x+4.8139 (R² = 0.7734). Temperature decreased significantly in both cycles, from 25.39°C (±0.52, day I) to 22.29°C (±0.14, day IV) in cycle I and from 25.68°C (±0.60, day I) to 22.49°C (±0.23, day IV) in cycle II (Table 1). The observed pH values differed only slightly during the experiment, with values around 7.6. Cycle I showed two homogenous groups of pH with 7.64 (day I, day IV) and 7.62 (day II, day III, Table 1) whereas the pH values of
AACL Bioflux, 2015, Volume 8, Issue 1. 92 http://www.bioflux.com.ro/aacl cycle II were not significant on day I (7.65 ±0.04), day II (7.63 ±0.02) and day III (7.66 ±0.08) but differed on day II (7.63 ±0.02) and day IV (7.65 ±0.03). Levels of phosphorus were highest in cycle I and day III with 2.14 mg L-1 (±0.62), followed by day IV with 1.72 mg L-1 (±0.27), day I with 1.19 mg L-1 (±0.34) and day II with the lowest value of 0.94 mg L-1 (±0.09). The same trend was observed in cycle II, with highest phosphorus values on day III with 1.47 mg L-1 (±0.40), followed by day IV with 1.34 mg L-1 (±0.27), and non-significant values of day I with 0.96 mg L-1 (±0.25) and day II with 0.89 mg L-1 (±0.06, Table 1).
Figure 2. Oxygen [mg L-1] gradient on sampling days I, II, III, IV and in cycles I & II. Lowercase showing significant groups (p < 0.05, t-test) of cycle I & II on the same experimental day, capital letter showing significant groups (p < 0.05, ANOVA) of the same cycle (cycle I = bold, cycle II = light) on all days (time series).
Plant growth differed between cycle I and II. In cycle II, growth of cucumber and zucchini (plant box II) showed better biomass weight gain (sum 7.60 kg) than all other plant species. In general, a better biomass weight gain of the plants was observed close to the nutrient water inflow in each of sampled plant boxes (Figure 3). The comparison of water parameters between each plant box of cycle I & II showed no trends due to the parallel arrangement. Means of water parameters of plant boxes were combined and tested independently in cycle I and II on the different sampling days (Tables 2, 3). In cycle I, oxygen [mg L-1] was the solidest parameter. Non- significant differences were found in groups of oxygen [mg L-1], temperature [°C] and pH on day II (Table 2) and day III (Table 3). In general, more significant differences of tested parameters were found than non-significant (p < 0.05). In contrast, cycle II showed the same amount of significant and non-significant differences between means of water parameters. Also, oxygen [mg L-1] was the most stable parameter in cycle II. DO levels, temperature and pH were non-significant. Phosphorus [mg L-1] showed significant differences between plant boxes of both cycles with moderate variations on all experimental days and partly higher levels on the fourth day. The comparison of water parameters at the different positions inside each box showed partly decreasing trends. Each plant box (n = 4) in cycle I and cycle II was tested for water parameter significance (ANOVA, p < 0.05) on the respective sampling day. Three groups of test tubes (n = 9) were named as “front” (near the nutrient water inflow), “middle” and “back” (near the outflow, Tables 4, 5, 6, 7). In both cycles, pH was the most stable parameter with no significant differences on all days and positions,
AACL Bioflux, 2015, Volume 8, Issue 1. 93 http://www.bioflux.com.ro/aacl followed by oxygen levels with 5 significant groups in total. Slightly decreasing trends of oxygen were observed on day III in cycle I box II (1.60 mg L-1 front tubes, 1.52 mg L-1 middle position, 1.16 mg L-1 back position) and cycle II on day IV in box I with 1.15 mg L-1 (front), 0.97 mg L-1 (middle) and 0.85 mg L-1 (back position). Day II showed best results with no significant differences of all parameters in both cycles and positions.
Figure 3. Better growth of cucumber and lettuce near the centred plant box inflow at the top of the box.
Discussion. The present study describes the water parameters within two identical ebb- and flow gravel bed hydroponic units under closed recirculating aquaponic conditions. Palm et al (2014a, b) identified an optimal feed input level of 200 g per day for the tested Nile tilapia aquaponics, reaching a steady state phase where DO levels and conductivity in the fish tank appeared relatively stable though working under very low water removal rates of 1.37 %. The sedimenter, a separate biofilter and the gravel bed system, beside the cultivated fish, were the most oxygen consuming units. We herewith describe the chemo-physical characteristics within the plant boxes during the end of sub- experiment II of Palm et al (2014a), with a feed input above the carrying capacity of 243.00 g (day 111 in Palm et al 2014a) and 327.5 g (day 118) as well as under subsequently reduced feed input during the steady phase of 175.00 g (day 125) and 215.00 g (day 132). The observed DO levels inside the plant boxes were low, following a negative linear regression, and reaching its lowest level of nearly 1 mg L-1 at the final day of sampling. This result is corresponding to Lennard & Leonard (2004), comparing closed constant flow and reciprocating (flood and drain) aquaponic systems under production of Murray cod (Maccullochella peelii peelii) and Green oak lettuce (Lactuca sativa) in a gravel bed hydroponics. Decreasing oxygen conditions were found in both aquaponic systems (more in the flood and drain system design) at an incrementally feeding rate of 1.0 and 1.5% d-1. The daily feed ratio of 2.5% d-1 in the present study was much higher, consequently negatively affecting the DO levels. While the DO level on day I with nearly 5 mg L-1 was still relatively high, caused by the constantly increasing feed input during subexperiment III (see Palm et al 2014a), this values dropped significantly to between 1.01 and 1.19 mg L-1 (cycle I & II) within the next three weeks, demonstrating overfeeding of the aquaponic system. During these three samplings, the hydroponic units had lower oxygen values in comparison to the global mean of both systems in DO (data from Palm et al 2014a). The values differed on day II (difference of -1.7 mg L-1 in cycle I and -1.57 mg L-1 in cycle II inside the hydroponics compared with the global cycle mean of DO with 3.47 mg L-1) day III (-3.43 mg L-1 in cycle I and -3.59 mg L-1 in cycle II
AACL Bioflux, 2015, Volume 8, Issue 1. 94 http://www.bioflux.com.ro/aacl compared with 4.96 mg L-1) and day IV (-2.74 mg L-1 in cycle I and -2.56 mg L-1 in cycle II compared with 3.75 mg L-1). Palm et al (2014a) suggested the oxygen level as an adequate indicator for system stability during aquaponic fish cultivation. However, under application of substrate hydroponics, the oxygen levels inside the plant boxes indicated the overfeeding of the system more rapid compared with other parts of the system, owed to the fact that overfeeding results in an accumulation of oxygen demanding faeces and feed reside inside the different filters, especially the plant box substrate. Temperature of the hydroponic units showed direct seasonal influences with the plant cultivation in summer (July). A change of natural light illumination, influenced by cloud intensity, resulted in different water temperatures with highest values on day II near 30°C and lowest on the latter two sampling days close to 22°C (Table 1). For plant growth, Rakocy et al (2006) recommended temperatures close to 24°C (75°F), but some common garden and winter crops could growth at temperatures at 18.3°C (65°F). For cucumber water temperatures were partly in its optimal range with 23-25°C (Göhler et al 2002). Other cultivated plants like herbs might have had more problems with suboptimal water temperatures at late summer conditions. The most stable water parameter inside the plant boxes of each cycle (front, middle and back position) was pH with insignificant values from 7.61-7.81 on all sample days (Tables 4, 5, 6, 7). Optimum pH range for nitrification is found to be between 7.0 and 9.0, whereas nutrient solubility is best for plant growth from 5.5-6.5 for hydroponic systems (Rakocy et al 2006). The gravel substrate hydroponic unit of the present system was more optimized for nitrification processes of root bacteria with possible suboptimal effects on plant growth. Generally, a compromise of nutrient solubility and nitrification in aquaponics is found by pH close to 7.0 (Rakocy et al 2006). Otherwise cultivated plants can affect pH depending on the species. Marschner et al (1995) cited in Raviv & Lieth (2008) described acidification of the root surroundings by chickpea (Cicer arietinum) and an increase of pH by corn (Zea mays). In our study the selection of different plants like vegetables and herbs could have influenced the root environment in case of pH, reflecting the mixture of plants that were cultivated inside the boxes (pH = 7.61-7.81). Also, insignificant pH values were found on the sampling days between cycle I and II (Table 1) except on day IV with very small differences. This indicates a homogenous water pH relation in the two gravel hydroponic units (cycle I and II) with the same plant growing conditions. Some plants like cucumber and zucchini showed better weight gain with possible better adaptation to higher pH levels and an indication for the importance of plant species selection for the present aquaponic system. The sampled hydroponic units were planned with a single water inflow at the top of the plant boxes, sub-irrigation, and a central drain close to the end of the plant boxes. This caused consequences for the oxygen distribution and consequently plant growth. We observed a better plant growth close to the central water inlet as illustrated in Figures 1 & 3. Similarly, the oxygen level was slightly decreasing on day III in cycle I box II and on day IV in cycle II box I from the inlet towards the outlet. This result corresponds to a marked oxygen depletion reported by Gislerød & Kempton (1983) in nutrient film technique (NFT) hydroponics, from the inlet (5-7 mg L-1) to the lower end of gullies (1-5 mg L-1) under cultivation of cucumber. Cucumber plants close to the inlet showed a better height (15 cm) than plants close to the outlet. For a uniform plant growth in a gravel substrate hydroponic unit and by using longer than wide plant systems a non- central irrigation system (e.g. overhead or drip) should be recommended. The oxygen level inside the plant boxes has direct effects onto plant growth. According to Gislerød & Kempton (1983), cucumber plants could be stressed if the oxygen levels fall below 3 mg L-1. In our study, the plant growth in all boxes was moderate because of generally lower oxygen values below 3 mg L-1 during the third sub-experiment (except on day I in cycle II all boxes). The phosphorus levels (P) inside the hydroponic units appeared low with a sigmoid curve expression (Figure 4). In aquaponic systems phosphorus is originating directly from the fish feed input and, according to fish species, digestion. Sigmoid phosphorus curves reflect an accumulation of the fish feed overload the days before. Highest feed input was observed on sampling day II with 327.50 g d-1, resulting in higher P quantities on day III
AACL Bioflux, 2015, Volume 8, Issue 1. 95 http://www.bioflux.com.ro/aacl and IV (Figure 4). This demonstrates a general sluggish phosphorus accumulation in gravel plant box systems. We can characterize our present low tech aquaponics as a phosphorus undersaturated system under the given feed input level between 175 g (day III) and 327.5 g (day II). Maximum observed P values were 2.68 mg L-1 (±0.39 box IV cycle I day III), and do not meet the requirements e.g. of cucumber with 40 mg L-1 in rock wool substrate hydroponics or 25 mg L-1 in NFT hydroponic systems (Göhler et al 2002). In closed aquaponic systems Lennard & Leonard (2004) reported also insignificantly low P levels of 3.87-4.04 mg L-1 (constant flow vs. flood and drain, Mann– Whitney-test). Palm et al (2014b) reported better growth of different plants (lettuce, tomato, cucumber and basil) with a higher phosphorus load close to 5 mg L-1 and the cultivation of Nile tilapia. Even lower levels of phosphorus were recorded by Danaher et al (2013) with insignificant values of 0.7-1.1 mg L-1 in the aquaculture water in comparison with an ordinary clarifier and swirl separator (t-test). For the cultivation of water spinach (Ipomoea aquatica), the authors suggested P concentrations of 0.8 % (overall) in plant tissue with no signs of nutrient deficiencies at a daily feeding rate of 103 g m-2 of hydroponic plant growing area d-1, much lower than in the present study.
Figure 4. Phosphorus [mg L-1] gradient on sampling days I, II, III, IV between cycle I & II and on all days of cycle I & II (time series). Lowercase showing significant groups (p < 0.05, t-test) of cycle I & II on the same experimental day, capital letter showing significant groups (p < 0.05, ANOVA) of the same cycle (cycle I = bold, cycle II = light) on all days (time series).
AACL Bioflux, 2015, Volume 8, Issue 1. 96 http://www.bioflux.com.ro/aacl Table 1 Daily feed input [g] and duration [day] (Palm et al 2014a) and physico-chemical parameters of cycle I and II (O. niloticus) between day I, II, III, IV and in comparison of cycle I and II on the specific day
Parameter Day I Day II Day III Day IV Mean ± SD Mean ± SD Mean ± SD Mean ± SD Feed input [g]1 243.00 327.50 175.00 215.00 Duration [day]1 111 118 125 132 Cycle I Oxygen [mg L-1] 4.68a, m±2.91 1.77b, m±0.80 1.53b, m±0.45 1.01c, m±0.20 Temperature [°C] 25.39a, m±0.52 30.46b, m±0.07 22.00c, m±0.15 22.29d, m±0.14 pH 7.64a, m±0.02 7.62b, m±0.02 7.62b, m±0.12 7.64a, m±0.02 Phosphorus [mg L-1] 1.19a, m±0.34 0.94b, m±0.09 2.14c, m±0.62 1.72d, m±0.27 Cycle II Oxygen [mg L-1] 4.46a, z±0.89 1.90b, m±0.66 1.37c, m±0.52 1.19c, m±0.39 Temperature [°C] 25.68a, z±0.60 30.41b, m±0.12 21.99c, m±0.47 22.49d, z±0.23 pH 7.65ab, m±0.04 7.63a, m±0.02 7.66ab, m±0.08 7.65b, z±0.03 Phosphorus [mg L-1] 0.96a, z±0.25 0.89a, z±0.06 1.47b, z±0.40 1.34c, z±0.27 Means (± SD), different letters in groups (a, b, c, d) showing significant differences (ANOVA, p < 0.05) between values of one independent cycle (time series, along all days); different letters (m, z) showing significant differences (t-test, p < 0.05) in comparison of cycle I and II on the specific experimental day (I, II, III, IV). 1Feed input [g] and duration [day] data adopted from Palm et al (2014a).
AACL Bioflux, 2015, Volume 8, Issue 1. 97 http://www.bioflux.com.ro/aacl Table 2 Physico-chemical parameters between plant boxes (I, II, III, IV) within cycle I and cycle II on day I and day II
Box I Box II Box III Box IV Parameter Cycles Mean ± SD Mean ± SD Mean ± SD Mean ± SD Day I Oxygen [mg L-1] Cycle I 2.31a±0.51 2.66a±0.53 5.02a±2.65 8.72b±0.06 Cycle II 4.42a±0.34 4.56a±1.65 4.15a±0.55 4.71a±0.33 T [°C] Cycle I 25.38a±0.37 25.73ab±0.17 25.82b±0.19 24.64c±0.09 Cycle II 25.69a±0.67 26.10a±0.39 25.54a±0.56 25.39a±0.57 pH Cycle I 7.66a±0.01 7.62b±0.02 7.65ac±0.03 7.62bc±0.02 Cycle II 7.67a±0.08 7.62a±0.02 7.65a±0.01 7.64a±0.02 Phosphorus [mg L-1] Cycle I 1.18a±0.30 1.45b±0.03 1.42b±0.74 0.70c±0.06 Cycle II 0.97ab±0.14 0.93ab±0.14 0.86b±0.42 1.07a±0.11 Day II Oxygen [mg L-1] Cycle I 2.07a±0.94 1.89a±0.52 1.93a±1.04 1.20a±0.25 Cycle II 1.43a±0.35 2.00a±0.64 1.98a±0.18 2.19a±1.00 T [°C] Cycle I 30.50a±0.07 30.46a±0.05 30.42a±0.04 30.46a±0.09 Cycle II 30.32a±0.18 30.41a±0.09 30.48a±0.07 30.44a±0.05 pH Cycle I 7.62a±0.00 7.62a±0.00 7.63a±0.03 7.61a±0.02 Cycle II 7.64a±0.02 7.62b±0.01 7.62ab±0.00 7.62ab±0.01 Phosphorus [mg L-1] Cycle I 0.88a±0.03 0.91b±0.03 0.89ab±0.03 1.08c±0.07 Cycle II 0.85a±0.03 0.95b±0.05 0.92b±0.07 0.86a±0.03 Means (± SD), different letters in groups showing significant differences (ANOVA, p < 0.05).
AACL Bioflux, 2015, Volume 8, Issue 1. 98 http://www.bioflux.com.ro/aacl Table 3 Physico-chemical parameters between plant boxes (box I, II, III, IV) within cycle I and cycle II on day III and day IV
Box I Box II Box III Box IV Parameter Cycles Mean ± SD Mean ± SD Mean ± SD Mean ± SD Day III Oxygen [mg L-1] Cycle I 1.41a±0.49 1.43a±0.25 1.73a±0.48 1.56a±0.51 Cycle II 1.36a±0.58 1.03a±0.30 1.42a±0.61 1.67a±0.41 T [°C] Cycle I 21.97a±0.17 21.96a±0.12 22.03a±0.12 22.04a±0.18 Cycle II 21.92a±0.80 22.31b±0.26 22.03b±0.11 21.68b±0.10 pH Cycle I 7.62a±0.01 7.62a±0.01 7.62a±0.01 7.62a±0.02 Cycle II 7.78a±0.03 7.61b±0.01 7.62b±0.01 7.62b±0.01 Phosphorus [mg L-1] Cycle I 1.24a±0.12 2.19b±0.14 2.44c±0.38 2.68c±0.39 Cycle II 1.77a±0.55 1.30b±0.32 1.28b±0.21 1.51a±0.18 Day IV Oxygen [mg L-1] Cycle I 1.07a±0.16 1.01a±0.18 0.98a±0.19 0.98a±0.27 Cycle II 0.99a±0.15 0.97a±0.17 1.38a±0.35 1.43a±0.53 T [°C] Cycle I 22.32ab±0.11 22.34a±0.10 22.18b±0.16 22.30ab±0.13 Cycle II 22.81a±0.15 22.53b±0.09 22.30c±0.10 22.33c±0.05 pH Cycle I 7.62a±0.01 7.65b±0.02 7.65bc±0.01 7.66c±0.01 Cycle II 7.66a±0.06 7.65a±0.01 7.66a±0.01 7.65a±0.01 Phosphorus [mg L-1] Cycle I 1.32a±0.03 1.73b±0.12 1.92c±0.19 1.91c±0.05 Cycle II 1.57a±0.08 1.06b±0.03 1.08b±0.04 1.62c±0.04 Means (± SD), different letters in groups showing significant differences (ANOVA, p < 0.05).
AACL Bioflux, 2015, Volume 8, Issue 1. 99 http://www.bioflux.com.ro/aacl Table 4 Physico-chemical parameters within the plant boxes at the specific positions (front, middle, back) of cycle I on day I and II
Cycle I Cycle I Parameter Box Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD front middle back front middle back Day I Day II Oxygen [mg L-1] I 2.63a±0.52 2.29a±0.60 2.01a±0.33 2.04a±1.10 2.08a±1.09 2.09a±1.07 II 2.21a±0.35 2.52ab±0.41 3.24b±0.07 1.78a±0.30 2.05a±0.84 1.85a±0.50 III 2.81a±0.48 3.84a±0.35 8.43b±0.94 1.92a±1.27 1.88a±1.16 1.98a±1.18 IV 8.79a±0.01 8.67b±0.05 8.70b±0.03 1.23a±0.28 1.20a±0.27 1.18a±0.30 Temperature [°C] I 25.27a±0.31 25.80b±0.10 25.07a±0.12 30.53a±0.06 30.53a±0.06 30.43a±0.06 II 25.77ab±0.12 25.90a±0.00 25.53b±0.06 30.47a±0.06 30.47a±0.06 30.43a±0.06 III 25.87a±0.15 25.87a±0.12 25.73a±0.29 30.43a±0.06 30.43a±0.06 30.40a±0.00 IV 24.67a±0.15 24.67a±0.06 24.60a±0.00 30.50a±0.10 30.43a±0.06 30.43a±0.12 pH I 7.65a±0.01 7.66a±0.01 7.66a±0.01 7.62a±0.00 7.63a±0.01 7.62a±0.00 II 7.62a±0.01 7.63a±0.03 7.61a±0.01 7.62a±0.00 7.62a±0.00 7.63a±0.01 III 7.64a±0.03 7.63a±0.04 7.66a±0.00 7.63a±0.04 7.62a±0.04 7.62a±0.04 IV 7.62a±0.02 7.63a±0.03 7.62a±0.00 7.61a±0.02 7.61a±0.02 7.62a±0.02 Phosphorus [mg L-1] I 1.42a±0.45 1.05a±0.03 1.06a±0.03 0.87a±0.04 0.89a±0.02 0.88a±0.02 II 1.44a±0.04 1.45a±0.02 1.45a±0.02 0.91a±0.02 0.91a±0.02 0.92a±0.04 III 1.42a±0.06 1.43a±0.03 1.40a±0.12 0.89a±0.05 0.88a±0.03 0.90a±0.01 IV 0.68a±0.05 0.73a±0.06 0.70a±0.05 1.10a±0.08 1.07a±0.07 1.07a±0.07 Means (± SD), different letters in groups showing significant differences (ANOVA, p < 0.05).
AACL Bioflux, 2015, Volume 8, Issue 1. 100 http://www.bioflux.com.ro/aacl Table 5 Physico-chemical parameters within the plant boxes at the specific positions (front, middle, back) of cycle I on day III and IV
Cycle I Cycle I Parameter Box Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD front middle back front middle back Day III Day IV Oxygen [mg L-1] I 1.77a±0.64 1.44a±0.34 1.02a±0.03 1.09a±0.13 1.05a±0.14 1.08a±0.25 II 1.60a±0.25 1.52ab±0.15 1.16b±0.04 1.03a±0.26 1.01a±0.15 1.00a±0.21 III 1.96a±0.67 1.68a±0.41 1.54a±0.42 0.95a±0.17 1.01a±0.26 0.97a±0.20 IV 1.65a±0.59 1.44a±0.52 1.59a±0.63 0.99a±0.29 0.99a±0.33 0.95a±0.33 Temperature [°C] I 21.77a±0.12 22.03b±0.06 22.10b±0.10 22.30ab±0.10 22.43a±0.06 22.23b±0.06 II 22.03a±0.12 21.83a±0.06 22.00a±0.10 22.37a±0.12 22.27a±0.06 22.40a±0.10 III 22.03ab±0.06 22.17a±0.06 21.90b±0.00 22.00a±0.17 22.23a±0.06 22.30a±0.00 IV 22.07a±0.06 22.23b±0.06 21.83c±0.06 22.43a±0.12 22.30a±0.00 22.17a±0.06 pH I 7.63a±0.01 7.62a±0.00 7.61a±0.01 7.62a±0.01 7.62a±0.00 7.63a±0.01 II 7.61a±0.01 7.62a±0.01 7.62a±0.01 7.64a±0.02 7.64a±0.02 7.65a±0.01 III 7.62a±0.01 7.61a±0.01 7.62a±0.01 7.65a±0.01 7.64a±0.02 7.66a±0.01 IV 7.62a±0.01 7.63a±0.03 7.61a±0.02 7.66a±0.01 7.66a±0.01 7.66a±0.01 Phosphorus [mg L-1] I 1.24a±0.08 1.21a±0.16 1.26a±0.13 1.30a±0.02 1.34b±0.02 1.32ab±0.02 II 2.18a±0.16 2.15a±0.15 2.23a±0.11 1.70a±0.05 1.72a±0.03 1.76a±0.20 III 2.76a±0.13 2.34b±0.38 2.22b±0.35 1.83a±0.13 1.88ab±0.19 2.05b±0.19 IV 2.77a±0.22 2.79a±0.43 2.48a±0.44 1.90a±0.03 1.92a±0.06 1.92a±0.05 Means (± SD), different letters in groups showing significant differences (ANOVA, p < 0.05).
AACL Bioflux, 2015, Volume 8, Issue 1. 101 http://www.bioflux.com.ro/aacl Table 6 Physico-chemical parameters within the plant boxes at the specific positions (front, middle, back) of cycle II on day I and II
Cycle II Cycle II Parameter Box Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD front middle back front middle back Day I Day II Oxygen [mg L-1] I 4.26a±0.30 4.30a±0.14 4.71a±0.40 1.26a±0.16 1.39a±0.32 1.63a±0.51 II 5.38a±2.94 3.97a±0.48 4.33a±0.63 1.84a±0.81 2.09a±0.68 2.06a±0.69 III 4.36a±0.37 3.81a±0.57 4.29a±0.70 2.01a±0.20 1.97a±0.19 1.95a±0.23 IV 4.92a±0.41 4.76a±0.11 4.45a±0.30 2.15a±1.10 2.19a1.20 2.22a±1.16 Temperature [°C] I 26.00a±0.10 26.23a±0.31 24.83b±0.12 30.17a±0.21 30.37a±0.06 30.43a±0.15 II 26.07ab±0.15 26.50a±0.20 25.73b±0.32 30.47a±0.12 30.40a±0.10 30.37a±0.06 III 25.97a±0.15 25.83a±0.21 24.83b±0.15 30.47a±0.06 30.53a±0.06 30.43a±0.06 IV 25.87a±0.15 25.53ab±0.55 24.77b±0.15 30.47a±0.06 30.40a±0.00 30.47a±0.06 pH I 7.67a±0.09 7.67a±0.09 7.67a±0.09 7.65a±0.03 7.64a±0.03 7.64a±0.03 II 7.61a±0.02 7.62a±0.02 7.62a±0.02 7.61a±0.01 7.62a±0.01 7.62a±0.00 III 7.65a±0.01 7.65a±0.01 7.66a±0.00 7.62a±0.00 7.62a±0.01 7.62a±0.01 IV 7.64a±0.02 7.64a±0.02 7.63a±0.02 7.62a±0.01 7.62a±0.01 7.62a±0.01 Phosphorus [mg L-1] I 0.90a±0.03 1.14b±0.06 0.89a±0.13 0.86a±0.02 0.86a±0.04 0.84a±0.03 II 0.97a±0.13 0.89a±0.14 0.92a±0.15 0.93a±0.05 0.96a±0.49 0.95a±0.04 III 1.04a±0.69 0.70a±0.15 0.85a±0.06 0.95a±0.07 0.90a±0.06 0.91a±0.07 IV 1.03a±0.16 1.13a±0.07 1.07a±0.06 0.85a±0.02 0.87a±0.01 0.86a±0.04 Means (± SD), different letters in groups showing significant differences (ANOVA, p < 0.05).
AACL Bioflux, 2015, Volume 8, Issue 1. 102 http://www.bioflux.com.ro/aacl Table 7 Physico-chemical parameters within the plant boxes at the specific positions (front, middle, back) of cycle II on day III and IV
Cycle II Cycle II Parameter Box Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD front middle back front middle back Day III Day IV Oxygen [mg L-1] I 1.43a±0.83 1.46a±0.57 1.19a±0.49 1.15ab±0.15 0.97a±0.03 0.85b±0.01 II 1.31a±0.41 0.94a±0.12 0.84a±0.01 1.09a±0.27 0.94a±0.06 0.88a±0.08 III 1.24a±0.71 1.59a±0.76 1.42a±0.58 1.38a±0.35 1.39a±0.45 1.36a±0.42 IV 1.96a±0.59 1.61a±0.32 1.45a±0.12 1.56a±0.70 1.42a±0.52 1.31a±0.57 Temperature [°C] I 21.63a±1.01 21.50a±0.62 22.63a±0.15 22.87a±0.15 22.87a±0.06 22.70a±0.20 II 22.40a±0.44 22.23a±0.23 22.30a±0.10 22.47a±0.06 22.60a±0.10 22.53a±0.06 III 22.17a±0.06 21.97b±0.06 21.97b±0.06 22.33a±0.06 22.33a±0.15 22.23a±0.06 IV 21.77a±0.06 21.67a±0.06 21.60a±0.10 22.33a±0.06 22.30a±0.00 22.37a±0.06 pH I 7.81a±0.06 7.78a±0.01 7.77a±0.01 7.71a±0.09 7.62a±0.02 7.65a±0.01 II 7.61a±0.01 7.61a±0.01 7.62a±0.00 7.64a±0.01 7.65a±0.02 7.66a±0.01 III 7.62a±0.01 7.61a±0.01 7.61a±0.01 7.66a±0.01 7.66a±0.00 7.65a±0.01 IV 7.61a±0.01 7.61a±0.01 7.62a±0.01 7.66a±0.01 7.64a±0.00 7.66a±0.01 Phosphorus [mg L-1] I 1.72a±0.23 2.01a±0.87 1.59a±0.26 1.45a±0.10 1.59a±0.06 1.60a±0.06 II 1.03a±0.10 1.38b±0.19 1.49b±0.38 1.04a±0.04 1.06a±0.03 1.09b±0.01 III 1.16a±0.24 1.42a±0.19 1.27a±0.10 1.05a±0.03 1.10b±0.01 1.10ab±0.05 IV 1.46a±0.23 1.60a±0.16 1.48a±0.12 1.60a±0.03 1.62a±0.03 1.65a±0.55 Means (± SD), different letters in groups showing significant differences (ANOVA, p < 0.05).
AACL Bioflux, 2015, Volume 8, Issue 1. 103 http://www.bioflux.com.ro/aacl Different phosphorus levels were found inside the hydroponic units in comparison to the whole (or global) system values on a specific day (see Palm et al 2014a). Highest P values were observed on day III with 138.06 % of P in cycle I and 94.84 % P in cycle II (Table 1) in comparison of 1.55 mg L-1 P (±0.16 = 100 %) as mean of both systems. All other values [mg L-1 or %] of P inside the plant boxes were lower compared with the mean of both cycles, on day I with 83.80 % in cycle I and 67.60 % in cycle II (100 %= 1.42 mg L-1 ±0.08), day II with 37.00 % in cycle I and 35.04 % in cycle II (100 %= 2.54 mg L-1 ±0.05) and on day IV with 67.45 % in cycle I and 52.55 % in cycle II (100 %= 2.55 mg L-1 ±0.00). Phosphorus levels below 100 % (as difference from global P mean) inside the plant boxes demonstrate either plant growth or P accumulation inside the gravel substrate. In soil, the most important form of phosphorus is orthophosphate 3 (PO4 , Röber & Schacht 2008), which is highly reactive. Raviv & Lieth (2008) described the decline of P concentration inside nutrient solutions following a fertilizer application being a widespread phenomenon of two mechanisms. The first very fast decrease is based on electrostatic reactions and a high affinity of the ion to charged surfaces within seconds or minutes. This is followed by a slow formation (hours, days) of new solid metal-P-compounds. The latter is found under acidic pH’s with AL, Fe and Mn or neutral or basic pH’s with Ca and Mg. Also the characteristics of the gravel substrate surface might have had a direct influence on the phosphorus accumulation. Göhler et al (2002) could demonstrate the best results of phosphate accumulation (Ca[H2PO4]2) in the laboratory with expanded clay “Lecaton” (grain size 8-16 mm) after 4 h in contrast to smaller grain sizes. The amount of phosphorus aggregation was decreasing after 8, 24 and partly 48 hours. The applied gravel substrate in the present study had a given grain size between 16-32 mm (coarse gravel, pebble), with a good ability of particle and phosphorus accumulation as well as root bacteria development. Consequently, the applied gravel combined with the surface affinity and formation of compounds might have influenced the measured phosphorus levels inside the plant boxes, combined with the different feed input levels (Figure 4) as also reported by Raviv & Lieth (2008). Nevertheless, our system had a low phosphorus level, not favourable for the cultivation of all plant species. We could not observe any distinct pattern in the tested water parameters inside the plant boxes, though some variability occurred. The parallel arrangement of the gravel filled plant boxes had no adverse effects onto the water parameter distribution inside each cycle (Tables 2, 3). Each plant box was connected to the nutrient enriched water with a distance of 1.5 m to the next plant box inlet. No clear trend was observed, differences between the four tested plant boxes of each cycle were small. However, on each sampling day, varying means of significant groups were observed between the plant boxes of one cycle, partly changing on another day and plant box. Most insignificant parameters were found on day II (in total five groups), followed by day III (four groups in total, Tables 2, 3). This result demonstrates that the tested system design in general enables similar conditions for the cultivated plant species in each box. However, still some variability inside the different plant boxes occurred, and the phosphorus values were generally different between the boxes of the identically built cycles I and II, most probably resulting from different needs and nutrient uptake of the cultivated plants. This makes predictions of water parameters and growth factors inside the hydroponic units difficult, challenging future system up-scaling of plant box numbers in order to increase the yield of plant cultivation.
Conclusions. The present study describes the water parameter differences of dissolved oxygen (DO), temperature, pH and phosphorus inside a gravel hydroponic unit of a warmwater ebb- and flow aquaponic system. In both cycles, linear decreasing oxygen levels on the four experimental days were strongly influenced by the daily feed input and the system functioning, suggesting that oxygen measurements inside the gravel bed might be a more sensible earlier indicator for system functioning of a closed substrate aquaponics. The observed pH is a result of the selected fish and plant species combination, with compromising effects of acidifying and non-acidifying root effects. Phosphorus distribution was sigmoid between the four days and showed sluggish
AACL Bioflux, 2015, Volume 8, Issue 1. 104 http://www.bioflux.com.ro/aacl accumulation as a consequence of higher feed inputs the days before. In general, phosphorus accumulation was low, characterising the present system as unsaturated in phosphorus. Water parameter of the hydroponic unit itself had lower values in comparison to the combined system parameters. Comparison of each plant box (I, II, III, IV) inside each cycle showed no trends of water parameters in means. Therefore, the parallel plant box installation had no adverse effect onto water parameters, enabling an easy system up-scaling of more than four plant boxes. Inside some plant boxes, a slightly decreasing trend of oxygen was found as a result of suboptimal subsurface irrigation from the central inflow towards the outlet. Better plant growth was observed closer to the central inlets. A de-central irrigation system in gravel substrate hydroponic units can be recommended in order to enhance oxygen levels, nutrient supply and subsequent plant growth in order to achieve economical sustainability.
Acknowledgements. We thank the Ministry of Agriculture, Environment and Consumer Protection of Mecklenburg Western Pomerania for supporting research in aquaponic fish and plant production. This project was partially funded through the pilot project “FishGlassHouse: Innovationsinitiative zur ressourceneffizienten Nahrungsmittel- produktion in MV” (European Fisheries Found-EFF) and INNOLIFE (Institut für Innovationsdesign), Wezlar, Germany, for material support.
References
Al-Hafedh Y. S., Alam A., Beltagi M. S., 2008 Food production and water conservation in a recirculating aquaponic system in Saudi Arabia at different ratios of fish feed to plants. Journal of the World Aquaculture Society 39:510-520. Danaher J. J., Shultz R. C., Rakocy J. E., Bailey D. S., 2013 Alternative solids removal for warm water recirculating raft aquaponic systems. Journal of the World Aquaculture Society 44(3):374-383. Farran I., Mingo-Castel A. M., 2006 Potato minituber production using aeroponics: effect of plant density and harvesting intervals. American Journal of Potato Research 83(1):47-53. Gislerød H. R., Kempton R. J., 1983 The oxygen content of flowing nutrient solutions used for cucumber and tomato culture. Scientia Horticulturae 20:23-33. Göhler F., Molitor H. D., Roth K., Wohanka W., 2002 [Soilless culture in horticulture]. Eugen Ulmer GmbH & Co. Stuttgart, 268 pp. [in German]. Graber A., Junge R., 2009 Aquaponic systems: nutrient recycling from fish wastewater by vegetable production. Desalination 246:147-156. Kloas W., Rennert B., Van Ballegooy C., Drews M., 2011 Aquaponic system for vegetable and fish production. United States Patent Application Publication. Pub. No.: US 2011/0131880 A1, 7 pp. Lennard W. A., Leonard B. V., 2004 A comparison of reciprocating flow versus constant flow in an integrated, gravel bed, aquaponic test system. Aquaculture International 12(6):539-553. Lewis W. M., Yopp J. H., Schramm H. L. Jr., Brandeburg A. M., 1978 Use of hydroponics to maintain quality of recirculated water in a fish culture system. Transactions of the American Fisheries Society 107(1):92-99. Marschner H., 1995 Mineral nutrition of higher plants. 2nd edition. London: Academic Press, 889 pp. McMurtry M. R., Sanders D. C., Cure J. D., Hodson R. G., Haning B. C., Amand E. C. S., 1997 Efficiency of water use of an integrated fish/vegetable co-culture system. Journal of the World Aquaculture Society 28(4):420-428. Palm H. W., Seidemann R., Wehofsky S., Knaus U., 2014a Significant factors influencing the economic sustainability of closed aquaponic systems. Part I: system design, chemo-physical parameters and general aspects. AACL Bioflux 7(1):20-32. Palm H. W., Bissa K., Knaus U., 2014b Significant factors affecting the economic sustainability of closed aquaponic systems. Part II: fish and plant growth. AACL Bioflux 7(3):162-175.
AACL Bioflux, 2015, Volume 8, Issue 1. 105 http://www.bioflux.com.ro/aacl Rakocy J., 1989 Hydroponic lettuce production in a recirculating fish culture system. In: Island perspectives. Vol. 3. Agricultural Experiment Station, University of the Virgin Islands, pp. 5-10. Rakocy J., Masser M., Losordo T., 2006 Recirculating aquaculture tank production systems: aquaponics - integrating fish and plant culture. Southern Regional Aquaculture Center (SRAC), Publication No. 454, 16 pp. Rakocy J. E., Bailey D. S., Shultz R. C., Thoman E. S., 2004 Update on tilapia and vegetable production in the UVI aquaponic system. Proceedings of the Sixth International Symposium on Tilapia in Aquaculture, Manila, Philippines. Bolivar R. B., Mair G. C., Fitzsimmons K. (eds), pp. 676-690. Raviv M., Lieth J. H., 2008 Soilless culture: theory and practice. Elsevier, 608 pp. Röber R., Schacht H., 2008 [Plant nutrition in horticulture]. E. Ulmer, 444 pp. [in German]. *** Microsoft, 2010 Microsoft Excel [computer software]. Redmond, Washington: Microsoft. *** SPSS Inc., 2013 Statistic Software Package, Chicago, IL, USA.
Received: 05 January 2015. Accepted: 22 February 2015. Published online: 27 February 2015. Authors: H. W. Palm, University of Rostock, Faculty of Agricultural and Environmental Sciences, Aquaculture and Sea- Ranching, Justus-von-Liebig-Weg 6, D-18059 Rostock, Germany, e-mail: [email protected] Madeline Nievel, University of Rostock, Faculty of Agricultural and Environmental Sciences, Aquaculture and Sea-Ranching, Justus-von-Liebig-Weg 6, D-18059 Rostock, Germany, e-mail: [email protected] Ulrich Knaus, University of Rostock, Faculty of Agricultural and Environmental Sciences, Aquaculture and Sea- Ranching, Justus-von-Liebig-Weg 6, D-18059 Rostock, Germany, e-mail: [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. How to cite this article: Palm H. W., Nievel M., Knaus U., 2015 Significant factors affecting the economic sustainability of closed aquaponic systems. Part III: plant units. AACL Bioflux 8(1):89-106.
AACL Bioflux, 2015, Volume 8, Issue 1. 106 http://www.bioflux.com.ro/aacl AACL BIOFLUX Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society
Deproteination and demineralization of shrimp waste using lactic acid bacteria for the production of crude chitin and chitosan Farramae C. Francisco, Rhoda Mae C. Simora, Sharon N. Nuñal
Institute of Fish Processing Technology, College of Fisheries and Ocean Sciences, University of the Philippines Visayas, Miagao 5023, Iloilo, Philippines. Corresponding author: R. M. C. Simora, [email protected]
Abstract. Deproteination and demineralization efficiencies of shrimp waste using two Lactobacillus species treated with different carbohydrate sources for chitin production, its chemical conversion to chitosan and the quality of chitin and chitosan produced were determined. Using 5% glucose and 5% cassava starch as carbohydrate sources, pH slightly increased from the initial pH of 6.0 to 6.8 and 7.2, respectively after 24 h and maintained their pH at 6.7 to 7.3 throughout the treatment period. Demineralization (%) in 5% glucose and 5% cassava was highest during the first day of treatment which was 82% and 83%, respectively. Deproteination (%) was highest in 5% cassava starch on the 3rd day of treatment at 84.4%. The obtained chitin from 5% cassava and 5% glucose had a residual ash and protein below 1% and solubility of 59% and 44.3%, respectively. Chitosan produced from 5% cassava and 5% glucose had protein content below 0.05%; residual ash was 1.1% and 0.8%, respectively. Chitosan solubility and degree of deacetylation were 56% and 33% in 5% glucose and 48% and 29% in 5% cassava, respectively. The advantage this alternative technology offers over that of chemical extraction is large reduction in chemicals needed thus less effluent production and generation of a protein-rich liquor, although the demineralization process should be improved to achieve greater degree of deacetylation. Key Words: waste utilization, alternative carbon source, bioprocessing, lactic acid bacteria.
Introduction. In seafood industries, shellfish waste management is a huge problem especially the crustacean sector which lacks cost-effective outlets for their waste (Raja et al 2012). About 45% of processed seafood consists of shrimp, the waste of which is composed of exoskeleton and cephalothorax (Gortari & Hours 2013). This waste represents 50-70% of the weight of the raw material, and it contains valuable components such as chitin, protein, and pigments, their amounts depending on the processing conditions, the species, the body parts, the seasonal variations, etc. (Xu et al 2008). Crustacean shells are the most important chitin source for commercial use due to their high content and ready availability (Subasinghe 1995). Chitin is a linear water- insoluble polymer consisting of β-(14) linked units of 2-acetamido-2-deoxy-D- glucopyranose (N-acetylglucosamine; GlcNAc; A-unit) (Heggset 2012). Chitosan, on the other hand, is a co-polymer of glucosamine and N-acetyloglucosamine which is partially deacetylated chitin (Yen et al 2009). Due to their biocompatibility, non-toxicity, biodegradability and film forming characteristics, chitin and chitosan are widely applied in agriculture (Khorrami et al 2012), biomedicine and food industries (Shirai et al 2001). The conventional methods for chitin extraction from crustaceans are chemical processes which involve the use of strong acid for demineralization and strong base for deproteination. A final bleaching step leads to a colorless chitin (Xu et al 2008). The chemical deacetylation of chitin into chitosan also requires strong chemical conditions (Stevens et al 1998). It has been reported that chemical chitin purification is extremely hazardous, energy consuming and damaging to the environment owing to the high mineral acid and base involved (Healy et al 2003).
AACL Bioflux, 2015, Volume 8, Issue 1. 107 http://www.bioflux.com.ro/aacl An alternative treatment of crustacean waste with lactic acid bacteria (LAB) for the production of chitin has been studied and reported (Rao et al 2000). However, few studies have reported the use of co-culture cultivation of proteolytic LAB strains and the use of cheap carbon source such as cassava (Manihot esculenta) flour. Thus, this study aimed to produce crude chitin from shrimp waste through lactic acid bacteria treatment coupled with mild chemical post treatment for chitin conversion into chitosan and compare in terms of solubility and proximate composition.
Material and Method
Microorganism. Lactic acid bacteria used in this study were isolated from ‘burong bangus’, a traditionally low salt fermented cooked rice and milkfish (Chanos chanos) mixture, and raw tuna (Katsuwonus sp.). L137 strain was isolated from ‘burong bangus’ and identified in a previous study by Olympia et al (1986) as Lactobacillus plantarum, a starch-hydrolyzing LAB. T1 strain was isolated from raw tuna (Katsuwonus sp.) and identified to genus-level according to Bergey’s Manual for Determinative Bacteriology (1957) as Lactococcus sp. These LAB strains were chosen for the study because they were found to be heterofermentative and proteolytic when tested in skim milk agar, which are important properties of LAB for deproteination and demineralization purposes. However, L137 strain, after prolonged storage, was found to be negative for amylolytic activity when tested on starch agar. These strains were stored at 4oC on MRS agar (Pronadisa) as the maintenance medium at the University of the Philippines Visayas Microbiology Laboratory, Miagao, Iloilo. Tests were conducted between February and August 2014.
Preparation of inoculum. The inoculum used was a co-culture of T1 and L137 strains. Optical Density (OD) at 540 nm and corresponding CFU m L-1 of individual cultures of T1 and L137 in 100 mL sterile MRS broth incubated at 37°C for 48 hours were determined. To prepare the inoculum for the treatment, 200 mL sterile MRS broth in E-flasks were inoculated with 1 mL aliquot of each 48h-culture of T1 and L137 strains to serve as mother culture and incubated at 37°C for 24 hours. Prior to inoculation, OD at 540 nm and CFU m L-1 of the mother culture were determined.
Protease assay. Cell-free supernatants were obtained after centrifugation of the 24h co- culture of T1 and L137 strains at 1398 x g at 4°C for 40 min. The filtrate was assayed for proteolytic activity (Akinkugbe & Onilude 2013). Protease activity was conducted using the method of Sigma-Aldrich using casein as substrate. Protease activity was determined in terms of Units (U) defined as the amount (µmoles) of enzyme that catalyzes the reaction of 1 µmole of casein per minute. All assays were carried out in triplicate.
Shrimp wastes. Fifteen (15) kilograms of frozen cultured white shrimp (Penaeus merguiensis) (80-100 mm) were procured from a local market in Roxas City, Capiz. Frozen whole shrimp were kept in ice in a sealed polystyrene container during transport and immediately stored overnight at -20°C upon arrival at the laboratory. Whole shrimp was thawed in running tap water before use. Shrimp heads and shells (with tails) were removed and separated from the meat, washed several times to eliminate any adhering meat with running tap water and dried overnight in an oven at 105°C. The dried shrimp heads and shells were milled into flakes using a Hammer mill (Culatti).
Microbial extraction of chitin. Twenty four (24) hour co-culture of T1 and L137 strains in MRS broth were used as inoculum (10% v/w). Glacial acetic acid (approx. 5 mL) was added to bring down the pH of shrimp waste to 6.0 (Rao & Stevens 2005). Treatment of 200 g shrimp waste inoculated with 20 mL of inoculum (10%) and 5% (w/v) carbon source (Treatment A with glucose (Gibco); Treatment B with cassava flour dissolved in distilled water; and Treatment C with no added carbohydrate source as control) was conducted in duplicate 300 mL E-flasks, covered with aluminum foil, at 37°C in a controlled temperature incubator shaker for 7 days. The slurry was filtered through a
AACL Bioflux, 2015, Volume 8, Issue 1. 108 http://www.bioflux.com.ro/aacl cheese cloth to separate the solid materials. This crude chitin was washed with distilled water, oven dried, weighed, analyzed and further converted into chitosan. All experiments were carried out in triplicate.
Chitosan preparation. The prepared dried crude chitin was placed into a flask with 55% NaOH solution with chitin to NaOH solution ratio of 1:25 (g mL-1), in a water bath at 95°C for 4 hours (Khorrami et al 2012). The produced chitosan was washed with distilled water and dried at 105°C overnight. Fourier Transform Infrared Spectroscopy (FTIR) spectrum of chitosan samples were measured and compared among treatments. All experiments were carried out in triplicate.
Proximate analysis. Moisture content was determined using the AOAC method (1990). Ash content was determined by burning the samples in a crucible at 600°C in a furnace for 2 hours (AOAC 1990). The pH value was monitored every 24 hours using a hand held pH meter (Milwaukee pH600). Growth trends in terms of CFU mL-1 developed on MRS agar plates was monitored during 7-day treatment period (24 hour interval). Protein content was measured using the standard biuret protein assay in samples before and after treatment. Lowry assay was used to determine protein content in chitin and chitosan, where protein concentrations are very low. Deproteination (%DP) and demineralization (%DM) were calculated using the equations by Rao & Stevens (2005). Chitin recovery (%CR) was determined as chitin derived (g) in reference to the original amount of chitin present in shrimp heads or shells. Chitin yield (%CY) was calculated as chitin derived (dry based, g) in reference to the original wet sample quantity of heads or shells (Rao & Stevens 2005). Lipid content was measured according to the standard methods by AOAC (1990).
Characterization of crude chitin and chitosan
Solubility of crude chitin and chitosan. Solubility of chitin was determined by dissolving 1 3 g of dried chitin in 100 cm of dimethyl acetamide/lithium chloride (DMA/LiCl) solution for 12 h and subsequently centrifuged to determine the percentage of insoluble chitin. The DMA/LiCl solution was prepared by dissolving 8 g of anhydrous lithium chloride overnight in 100 cm3 of DMA. The solubility of chitosan was determined by dissolving 1% (w/v) chitosan in a solution of 1% glacial acetic acid for 24 h under continuous stirring (Rao & Stevens 2005). The equation was used below:
% Solubility =
Degree of deacetylation of chitosan. The DD of chitosan was determined using a FTIR (AVATAR 330 FTIR ThermoNicolet) instrument with frequency of 4000-400 cm-1. The DD of the chitosan was calculated using the baseline by Khan et al (2002).
Statistical analysis. Data were analyzed by one-way analysis of variance (ANOVA). All data sets were tested for normality. If significant differences were indicated, individual groups were compared using the Tukey’s Range Test.
Results and Discussion
Shrimp waste composition. Shrimp waste form the major fraction (53%) by weight of the whole shrimp as shown in Table 1. The moisture, ash, and protein content in the shrimp biowaste were 77.26, 7.49, and 7.39% respectively. Mass balance was calculated based on minerals, protein, and lipid data with an error < 10%, balance is reasonably accurate (Rao & Stevens 2005). The quality of chitin and chitosan produced from crustacean shells is partially dependent on the type of raw material used. As observed in this study, the protein and ash values of shrimp shell wastes were generally low as
AACL Bioflux, 2015, Volume 8, Issue 1. 109 http://www.bioflux.com.ro/aacl compared to previous studies by Rao & Stevens (2005), Aytekin & Elibol (2010), and Jung et al (2007) possibly due to the small size of the shrimp samples used and the preparation method of the shell wastes in which all adhering meat was removed and washed. Furthermore, proximate composition of shrimps, crustaceans and other aquatic organisms has found to be varied due to the seasonal factors, climatic factors, geographic factors, habitat, developmental stage, sex, and sexual maturation (Pillay & Nair 1971).
Table 1 Composition of shrimp waste
Shrimp fraction Shrimp waste Meat Whole shrimp waste Moisture content Ash Protein Total lipid (%) (%) (% wb) 46 53 77.26±1.0 7.49±0.42 7.39±0.33 0.50±0.05
Microbial extraction of crude chitin. The pH slightly increased for the first 24 hours of treatment and decreased after 48 hours. Treatment C (control) showed pronounced increment in pH over the 7-day treatment. Treatment A (with 5% glucose) showed little change in pH reaching its lowest pH of 6.7 on days 2 and 7. Treatment B (with 5% cassava flour) also displayed minimal change in pH reaching its lowest pH of 6.6 on day 6. There was no significant difference in pH among treatments during the 7-day treatment period. Medium pH likely depends on the content of the energy source such as glucose and sucrose (Jung et al 2005). In this study, 5% carbohydrate source and 10% inoculum levels were applied, similar to the ratio applied by Rao & Stevens (2005). However, medium pH was maintained by 5% glucose and 5% cassava flour treatments possibly due to the inadequate source of carbohydrates in the substrate and the complex structure of cassava starch. Franco et al (1998) stated that starches that naturally present a porous surface, such as corn (Zea mays) starch, are degraded easier than those with a smooth surface such as cassava starch. Moreover, high proportions of amylose and amylopectin, 18 to 25% and 80% in cassava flour, respectively, are more resistant to enzymes (Rocha et al 2010) and generally, lactic acid bacteria are deficient in amylolytic characters especially for the highly branched starch (Boontawan 2010). In addition, the low acid production of all treatments may be caused by the heterofermentative and proteolytic properties of two LAB strains used. During heterofermentative lactic acid biosynthesis, carbon dioxide and ethanol are present, thus rendering a lactic acid yield production (Serna-Cock & Rodríguez-de Stouvenel 2005). Faithong et al (2010) reported that degradation of small fish and shrimp by proteases yields short chain peptides and free amino acids. Furthermore, lactic acid bacteria are generally recognized as non-toxicogenic, although some species isolated from fish and its products can produce biogenic amines. Biogenic amines are physiologically degraded by oxidative deamination process catalyzed by amine oxidase with the production of aldehydes, ammonia and hydrogen peroxide (Zaman et al 2009) which are basic nitrogenous compounds. Demineralization (%) was highest during the first day of treatment with 82.4%, 82.9%, and 86.6% for treatments A, B, and C, respectively (Figure 1A) and declined thereafter. This could be due to the low carbohydrate source added (5%) which also led to low acid production during the 7-day treatment. On the other hand, Jung et al (2006) reported a high demineralization efficiency of 97.2% after 7 days of co-fermentation by Lactobacillus paracasei subsp. tolerans KCTC-3074 and Serratia marcescens FS-3 of red crab (Chionoecetes japonicus) shell waste due to higher carbohydrate source added (10%) which yielded high lactic acid. Consequently, cell growth gradually decreased as energy sources were used up. Ghorbel-Bellaaj et al (2012) added that the addition of carbohydrate source to shrimp waste medium has no significant effect on deproteination but its effect is more important on demineralization. Deproteination (%) was highest in treatment B (84.4%) followed by A (44.8%) and C (12.6%) during the 3rd day of treatment (Figure 1B). Owing to the efficient
AACL Bioflux, 2015, Volume 8, Issue 1. 110 http://www.bioflux.com.ro/aacl deproteination, especially in treatment B with cassava as carbon source, higher value was reported in this study than the previous studies by Jung et al (2006) and Shirai et al (1998). Sumantha et al (2005) stated that the progressive decrease in proteolytic activity with increasing fermentation time could possibly be due to ending of production, as enzymes are primary metabolites and it could also be due to enzyme inactivation. Towards the end of treatment period, the control, devoid of carbon source, showed comparable values while treatment A (5% glucose) displayed lower values. According to Ghorbel-Bellaaj et al (2012), this is mainly owing to the induction of the repressive effect of glucose on protease production or catabolic repression.
Figure 1. Changes in demineralization (A) and deproteination (B) of shrimp waste with LAB over the 7-day treatment period.
Chitin recovery (CR) was significantly highest in shrimp waste treated with 5% glucose followed by 5% cassava and control, respectively (Table 2). Chitin yield (CY) was generally low among treatments. Significantly highest CY was noted in treatment A, but showed no significant difference from treatment B. Rao & Stevens (2005) reported similar low results, wherein 50–60% of solid material, assumed to be chitin, is lost. This could be due to chitin, occurring partially in aggregates of small particle size in the shrimp heads or shells, were lost during filtration or sequential washing. In the standard procedures, chitin particles were supposed to be retained by cloth filtration, but small chitin particles might be lost during cloth filtration (Rao & Stevens 2005).
Table 2 Chitin recovery and yield (%) of shrimp waste
Sample Protein (g) Ash (g) Chitin recovery (%) Chitin yield (%) Treatment A (5% glucose) Original 0.22 17.20 - - Residue 0.059 6.49 48.3±4.51a 6.5±0.5a Treatment B (5% cassava) Original 0.24 17.73 - - Residue 0.059 5.1 37.3±4.04b 6.0±1.0ab Treatment C (control) Original 0.29 19.78 - - Residue 0.026 4.95 27.0±3.00c 4.5±0.5b Superscripts in column denote significant difference (α = 0.05) based on one way ANOVA analysis.
Quality of crude chitin and chitosan. There were no significant (p>0.05) differences in protein contents among the three prepared chitins (Table 3). Protein contents of S. marcescens FS-3 alone and L. paracasei subsp. tolerans KCTC-3074 plus S. marcescens FS-3 cofermentation of red crab shell waste were 3.62% and 10.62%, respectively, after
AACL Bioflux, 2015, Volume 8, Issue 1. 111 http://www.bioflux.com.ro/aacl days of fermentation (Jung et al 2006) which were higher than the protein contents in the present study. This indicated the efficiency of deproteination by co-cultures of two LAB in this study. However, the repressive effect of glucose on protease production led to high residual protein content in treatment A (0.094%). Significant difference in ash content was observed between treatments where highest residual ash content was noted in the control. The result was higher than reported by Rao & Stevens (2005). This could be due to low acid production which led to low demineralization efficiencies during treatment leaving high residual ash content. Chitin solubility (%), among the three treatments, was highest in treatment B. However, there was no significant difference in solubility between prepared chitins. Solubility was low due to the high residual ash content in the produced chitins. All treatments were brownish in color; this was probably due to the low lactic acid produced during treatment which was inefficient in removing the pigments in the shrimp waste. The protein (%) content of produced chitosan was significantly highest (p ≤ 0.05) in the control (Table 3). According to Rao & Stevens (2005), the use of 50% NaOH nullified possible effects that might be caused by differences in the protein content of chitin. Ash content showed significant difference between treatments; the highest residual ash was observed in the control, the reason being that; alkaline deacetylation involved only the removal of acetyl groups from the molecular chain of chitin, leaving the chitin backbone intact and producing a compound (chitosan) with a high degree chemical reactive amino group (-NH2). Treatment A has significantly highest solubility (56%) among the prepared chitosans. Solubility is influenced by residual ash content; the lower the ash content, the higher the solubility, as observed in treatment A. Treatment A showed the highest degree of deacetylation (DD%) among all treatments (Table 3) however, there was no significant difference in DD% between three treatments. The study of Nessa et al (2010) on the process for the preparation of chitin and chitosan from prawn (Penaeus indicus) shell waste demonstrated that duration of deacetylation affected mostly the degree of deacetylation and solubility of the product. In general, prepared chitosan showed low solubility and DD (%) owing to the short duration of deacetylation process of chitin to chitosan which was four hours. Moreover, No & Meyers (1995) reported that it is estimated that deacetylation must be at least 85% complete in order to achieve the desired solubility. The color of produced chitosans was light yellow; this could be due to the quality of chitin used as raw material for chitosan conversion since characteristics of chitosan were affected by the conditions of chitin extraction.
Table 3 Quality of crude chitin and chitosan
Synthesized Property A B C Chitin Protein (%) 0.094a 0.085a 0.081a Ash (%) 0.76a 0.9b 1.3c Solubility (%) 44.3a 59.0ab 39.0a Chitosan Protein (%) 0.032a 0.033a 0.039b Ash (%) 0.8a 1.1b 1.3c Solubility (%) 56.0a 48.0b 41.0c Degree of deacetylation (%) 33.0a 29.0a 26.0a Superscripts in row denote significant difference (α = 0.05) based on one way ANOVA analysis.
Conclusions. The results of the present study showed that chitin for its conversion to chitosan can be produced through microbial treatment of lactic acid bacteria. However, the alternative process for chitin production is still less efficient than conventional chemical treatment. A ratio of 10% (v/w) inoculum and 5% (w/w) glucose and cassava starch were insufficient to produce desirable acid concentration in the demineralization
AACL Bioflux, 2015, Volume 8, Issue 1. 112 http://www.bioflux.com.ro/aacl process due to inadequate energy source. However, the co-cultures of T1 and L137 showed efficient deproteination in treatment B (with added 5% cassava) despite low demineralization and that glucose (treatment A) exhibited repressive effect on protease production. Prepared chitin and chitosan showed high residual ash content; however, protein content was relatively low. Produced chitin and chitosan showed low solubility (%) due to high ash content and low degree of deacetylation (%), respectively. The produced chitin and chitosan from shrimp shell waste could be used in a variety of applications especially in food, biomedical and pharmacological industries. The advantagethis technology offers over that of chemical extraction is large reduction in chemicals needed thus less effluent production and generation of a protein-rich liquor fraction.
Acknowledgements. The authors are grateful to the Philippine Department of Science and Technology (DOST) – Accelerated Science and Technology Human Resource Development Program (ASTHRDP) for granting the scholarship, to Ms. Francisco and to the Office of the Vice Chancellor for Research and Extension of the University of the Philippines Visayas for the supplementary research funding and publication support.
References
Akinkugbe A. O., Onilude A. A., 2013 Selective comparability and physiological studies of lactic acid bacteria protease and Calotropis procera (linn) extracts. Peer J PrePrints 1:e4v1 http://dx.doi.org/10.7287/peerj.preprints.4v1. AOAC, 1990 Official Methods of Analysis, 15th ed.; Association of Official Analytical Chemists: Washington, DC, 246 pp. Aytekin O., Elibol M., 2010 Cocultivation of Lactococcus lactis and Teredinobacter turnirae for biological chitin extraction from prawn waste. Bioprocess Biosyst Eng 33:393- 399. Bergey D. H., Breed R., American Society for Microbiology, 1957 Bergey’s Manual for Determinative Bacteriology. 7th edition. Williams and Wilkins Co., Baltimore, USA, pp. 1-8. Boontawan P., 2010 Development of lactic acid production process from cassava by using lactic acid bacteria. Unpublished doctoral dissertation. Suranaree University of Technology, 194 pp. Faithong N., Benjakul S., Phatcharat S., Binsan W., 2010 Chemical composition and antioxidative activity of Thai traditional fermented shrimp and krill products. Food Chemistry 119:133-140. Franco C. M. L., Ciacco C. F., Tavares D. Q., 1998 The structure of waxy corn starch: effect of granule size. Starch 50:193-198. Ghorbel-Bellaaj O., Younes I., Maâlej H., Hajji S., Nasri M., 2012 Chitin extraction from shrimp shell waste using Bacillus bacteria. International Journal of Biological Macromolecules 51:1196-1201. Gortari M. C., Hours R. A., 2013 Biotechnological processes for chitin recovery out of crustacean waste: a mini-review. Electronic Journal of Biotechnology 16:14-14. Healy M., Green A., Healy A., 2003 Bioprocessing of marine crustacean shell waste. Acta Biotechnologica 23:151-160. Heggset E. B., 2012 Enzymatic degradation of chitosans – a study of the mode of action of selected chitinases and chitosanases. PhD Thesis summary, Norwegian University of Science and Technology, pp. 1-2. Jung W. J., Jo G. H., Kuk J. H., Kim K. Y., Park R. D., 2005 Demineralization of crab shells by chemical and biological treatments. Biotechnology and Bioprocess Engineering 10:67-72. Jung W. J., Jo G. H., Kuk J. H., Kim K. Y., Park R. D., 2006 Extraction of chitin from red crab shell waste by cofermentation with Lactobacillus paracasei subsp. tolerans KCTC-3074 and Serratia marcescens FS-3. Applied Microbiology and Biotechnology 71:234-237.
AACL Bioflux, 2015, Volume 8, Issue 1. 113 http://www.bioflux.com.ro/aacl Jung W. J., Jo G. H., Kuk J. H., Kim Y. J., Oh K. T., Park R. D., 2007 Production of chitin from red crab shell waste by successive fermentation with Lactobacillus paracasei KCTC-3074 and Serratia marcescens FS-3. Carbohydrate Polymers 68:746-750. Khan T. A., Peh K. K., Ch'ng H. S., 2002 Reporting degree of deacetylation values of chitosan: the influence of analytical methods. J Pharm Pharmaceut Sci 5:205-212. Khorrami M., Najafpour G. D., Younesi H., Hosseinpour M. N., 2012 Production of chitin and chitosan from shrimp shell in batch culture of Lactobacillus plantarum. Chemical and Biochemical Engineering 26:217-23. Nessa F., Masum S. M., Asaduzzaman M., Roy S. K., Hossain M. M., Jahan M. S., 2010 A process for the preparation of chitin and chitosan from prawn shell waste. Bangladesh Journal of Scientific and Industrial Research 45:323-330. No H. K., Meyers S. P., 1995 Preparation and characterization of chitin and chitosan – a review. Journal of Aquatic Food Product Technology 4:27-52. Olympia M., Valenzuela A., Takano M., 1986 Isolation of an amylolytic lactic acid bacteria in burong bangus. Paper presented at the 7th World Food Congress, September 26- October 2, Singapore. Pillay K. K., Nair N. B., 1971 The annual reproductive cycles of Uca annulipes, Portunus pelagicus and Metapenaeus affinis (Decapoda: Crustacea) from the south-west coast of India. Marine Biology 11:152-166. Raja R., Chellaram C., John A. A., 2012 Antibacterial properties of chitin from shell wastes. Indian Journal of Innovations and Developments 1:7-10. Rao M. S., Stevens W. F., 2005 Chitin production by Lactobacillus fermentation of shrimp biowaste in a drum reactor and its chemical conversion to chitosan. Journal of Chemical Technology and Biotechnology 80:1080-1087. Rao M. S., Munoz J., Stevens W. F., 2000 Critical factors in chitin production by fermentation of shrimp biowaste. Applied Microbiology and Biotechnology 54:808- 813. Rocha T. S., Carneiro A. P. A., Franco C. M. L., 2010 Effect of enzymatic hydrolysis on some physicochemical properties of root and tuber granular starches. Ciencia e Tecnologia de Alimentos 30:544-551. Serna-Cock L., Rodríguez-de Stouvenel A., 2005 Producción biotecnológica de ácido láctico: estado del arte. Ciencia e Tecnologia de Alimentos 5:54-65. Shirai K., Palella D., Castro Y., Guerrero-Legarreta I., Saucedo-Castaneda G., Huerta- Ochoa S., Hall G. M., 1998 Characterization of chitins from lactic acid fermentation of prawn wastes. Advance in Chitin Science 3:103-110. Shirai K., Guerrero I., Huerta S., Saucedo G., Castillo A., Gonzales R. O., Hall M. G., 2001 Effect of initial glucose concentration and inoculation level of lactic acid bacteria in shrimp waste ensilation. Enzyme Microb Technol 28:446-452. Stevens W. F., Cheypratub P., Haiqing S., Lertsutthiwong P., How N. C., Chandrkrachang S., 1998 Alternatives in shrimp biowaste processing. In: Advances in shrimp biotechnology. Flegel T. W. (ed), National Center for Genetic Engineering and Biotechnology, Bangkok, pp. 11-23. Subasinghe S., 1995 The development of crustacean and mollusk industries for chitin and chitosan resources. In: Chitin and chitosan the versatile environmentally friendly modern materials. Zakaria M. B., Muda W. M. W., Abdullah M. P. (eds), Universiti Kebangsaan Malaysia, pp. 27-34. Sumantha A., Sandhya C., Szakacs G., Soccol C. R., Pandey A., 2005 Production and partial purification of a neutral metalloprotease by fungal mixed substrate fermentation. Food Technology and Biotechnology 43:313-319. Xu Y., Gallert C., Winter J., 2008 Chitin purification from shrimp wastes by microbial deproteination and decalcification. Applied Microbiology and Biotechnology 79:687- 97. Yen M. T., Yang J. H., Mau J. L., 2009 Physicochemical characterization of chitin and chitosan from crab shells. Carbohydrate Polymers 75:15-21. Zaman M. Z., Abdulamir A. S., Abu Bakar F., Selamat J., Bakar J., 2009 A review: microbiological, physicochemical and health impact of high level of biogenic amines in fish sauce. American Journal of Applied Sciences 6:1199-1211.
AACL Bioflux, 2015, Volume 8, Issue 1. 114 http://www.bioflux.com.ro/aacl Received: 07 January 2015. Accepted: 11 February 2015. Published online: 27 February 2015. Authors: Farramae C. Francisco, Institute of Fish Processing Technology, College of Fisheries and Ocean Sciences, University of the Philippines Visayas, Miagao 5023 Iloilo, Philippines, e-mail: [email protected] Rhoda Mae C. Simora, Institute of Fish Processing Technology, College of Fisheries and Ocean Sciences, University of the Philippines Visayas, Miagao 5023 Iloilo, Philippines, e-mail: [email protected]. Sharon N. Nuñal, Institute of Fish Processing Technology, College of Fisheries and Ocean Sciences, University of the Philippines Visayas, Miagao 5023 Iloilo, Philippines, e-mail: [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. How to cite this article: Francisco F. C., Simora R. M. C., Nuñal S. N., 2015 Deproteination and demineralization of shrimp waste using lactic acid bacteria for the production of crude chitin and chitosan. AACL Bioflux 8(1):107-115.
AACL Bioflux, 2015, Volume 8, Issue 1. 115 http://www.bioflux.com.ro/aacl AACL BIOFLUX Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society
Some considerations concerning the artificially colored aquarium fish trade 1Valentin O. Eşanu, 2Claudiu Gavriloaie, 3Ioan G. Oroian, 1,4Philippe Burny
1 University of Liege, Agro-Bio Tech Gembloux, Belgium; 2 SC Bioflux SRL, Cluj-Napoca, Romania; 3 University of Agricultural Sciences and Veterinary Medicine (USAMV), Faculty of Agriculture, Cluj-Napoca, Romania; 4 Walloon Center for Agricultural Research, Belgium. Corresponding author: V. O. Eşanu, [email protected]
Abstract. Humans kept ornamental fish in their homes from ancient times. During time, the ornamental fish industry became a profitable enterprise. Color, size and shape are important traits when we describe a fish as a phenotypically uniform line, strain or breed; a standardized color, shape and size describe better a fish population and thus it is promoted better on the market. Several varieties of fish are being artificially colored to improve marketability. Painted, dyed or tattooed fish are terms for a new variety of pet fish whose scales have been transformed from monochrome silver into vivid pigmentations using artificial colors or lasers. These practices produce many health problems for fish. We should not to put money above the life and welfare of any living thing. Education of the customers is a very important thing, because artificially colored fish continue to exist because of demand. Key Words: tattooed fish, dyed fish, petshop, aquarium fish.
Introduction. The creations of nature have long been kept captive in human care, in simulated environments; concerning the fish, humans were interested in keeping ornamental fish in their homes from ancient times (Kaszony 1970). Gradually the freshwater aquarium became a popular and educational activity for humans of all ages (Petrescu-Mag et al 2013a). The beneficial effects of aquarium on human health and well being were demonstrated long time ago (Petrescu-Mag 2007). Currently, aquariums are a very useful and effective tool in some psychologically disorders treatment both in old people and orphan children (Petrescu-Mag et al 2013a). Aquarium fish trade has also a negative impacts on biodiversity (Burlacu et al 2009a, b; Păsărin & Petrescu-Mag 2011). The aquarium industry became a profitable enterprise for many companies from all over the world and mainly for Southeast Asian companies of aquaculture (Ng & Tan 1997; Petrescu-Mag 2007). Color, size and shape are important traits when we describe a fish as a phenotypically uniform line, strain or breed; a standardized color, shape and size describe better a fish population and thus it is promoted better on the market (Petrescu-Mag et al 2013b). Several varieties of fish are being artificially colored to improve marketability. Painted, dyed or tattooed fish are terms for a new variety of pet fish whose scales have been transformed from monochrome silver into vivid pigmentations using artificial colors or lasers (Broome 2013). A comprehensive list of artificially colored fish species available on the market can be found on http://www.deathbydyeing.org/colormedead.htm. In the present paper we will discuss about the main procedures used in artificially fish coloring and also about the negative effects of these unnatural processes upon the fish.
Methods of coloration. There are a number of methods for introducing artificial colour to fish, although specific information on methodology is sometimes scarce. The results of the coloring process varies depending on the type of fish and the method used. The most used varieties for coloration are the albino strains of various species.
AACL Bioflux, 2015, Volume 8, Issue 1. 116 http://www.bioflux.com.ro/aacl Colour-enhancing foods. Many varieties of "colour-enhancing" foods for aquarium fishes are available to the consumer. Generally, these foods contain natural dyes, such as beta carotene, and are not harmful to fish, but the effect is temporary, because once they are no longer fed the treated food, fish eventually lose their color (Sharp 2006). There are some claims that colored food also contains unnatural dyes, which can negatively affect fish’s growth and development (Hirte-Runtsch 2008).
Genetic modification. Aquarium fish genetically modified to fluoresce in different bright colours under white or ultraviolet light are now available commercially worldwide, under the trade name GloFish. Initially only zebrafish (Brachydanio rerio) was involved (Gavriloaie 2007), but some other species as Puntigrus tetrazona and Gymnocorymbus ternetzi followed (Curry 2012; Roberts 2013) (Figure 1). The technology was developed originally to produce a zebrafish capable of detecting environmental pollution, especially heavy metals (Gong et al 2003; Mag & Petrescu 2006). These fish are just as hardy and healthy as the regular varieties.
Figure 1. Some breeds of GloFish (Source: http://www.glofish.com/about/glofish-display/).
The use of hormones. Usually fish are kept in water containing large amounts of artificial hormones, for several reasons. Most often this is done to make the fish breed, especially for species which are difficult to breed normally. Another reason is to make the young fish grow and mature more quickly. Finally, fish are „juiced” with hormones in order to obtain more intensive colours. This might also be used for making females of certain fish species look more colorful (Wiegert 2012). These aspects makes fish more tempting to buy. Unfortunately, the treatment will also make the fish sterile, the intense colour only lasts for a few weeks and then it fades to a very pale fish, and at worst it will greatly reduce the normal life span of the fish (http://www.thekrib.com/Fish/steroids.html thekrib.com).
Injection of dyes. The practice of dying live fish started in the late 1970s in Asian fish farms, with glasfish - Parambassis ranga (McMahon & Burgess 1998). These fish are injected with stripes of bright colored pigments along their dorsal and ventral lines (Figure 2). For this, needles are used to inject dye under the skin by many punctures in order to achieve the desired effect. The same needle is used to inject several fish. The size of the needle compared to the size of the fish means that there will be a significant tissue damage at the injection site (http://www.fishtanksandponds.co.uk/ethics/fish-keepings- hall-of-shame.html). After the glassfish, other species followed. One of the most frequents is parrot fish, a hybrid cichlid species of still unknown origin. In some cases, this creates an all- over color change, and in others, it creates little pockets of dye (Hirte-Runtsch 2008).
AACL Bioflux, 2015, Volume 8, Issue 1. 117 http://www.bioflux.com.ro/aacl The dyeing process cause a high mortality in fish, and the few surviving fish, most will die within the two months following the trauma (Sharp 2006). Anyway, the dye eventually fades within six to ten months (Wiegert 2013). Only about 10% of the fish that survive for sale will keep their coloration for any length of time (Sharp 2006). The practice of painting these fish has nearly eliminated the availability of the unpainted variety in the pet industry (http://www.firsttankguide.net/painted.php).
Figure 2. Dyed glassfish - Parambassis ranga (Source: https://fishcompetstore.files.wordpress.com/2013/11/dsc_6695.jpg).
Dipping. This process involves the fish being dipped in a caustic solution which removes the fishes mucus coat. Then the fish is dipped in a solution with the dye and finally it is placed in a solution which contains an irritating substance which causes the fish to regenerate its mucus coat (http://www.fishtanksandponds.co.uk/ethics/fish-keepings- hall-of-shame.html). This method is also very stressful to the fish, and has a high mortality rate (Sharp 2006).
Tattooing. There is a recent trend in the aquarium market for tattooed fish believed to bring luck and prosperity to their owners. Fish tattooing has spread to parrot fish (Figure 3), livebearers (Figures 4 and 5), goldfish and others (Adams 2011). The fish are tattooed with dye using a special "low intensity laser" which leaves a permanent mark (Clarke 2006).
Figure 3. Tattooed parrot fish: with Chinese characters - left, with flowers - right (Source: for the left image - http://www.odditycentral.com/pics/tattooed-fish-sold-as-lucky-charms-in-china.html; for the right image - http://www.practicalfishkeeping.co.uk/content.php?sid=829).
AACL Bioflux, 2015, Volume 8, Issue 1. 118 http://www.bioflux.com.ro/aacl
Figure 4. Tattooed livebeares (Source: http://www.underwatertimes.com/news.php?article_id=01769543281).
Figure 5. Tattooed mollies with numbers (Source: http://news.asiaone.com/News/AsiaOne+News/Singapore/Story/A1Story20080117-45461.html).
These fish are being laser-tattooed with intricate patterns like numbers, flags, hearts, flowers (http://www.aquariumlife.net/articles/ethics/dyed-fish/256.asp) or Chinese characters like “luck”, “happiness”, or “May your business boom” (Daub 2009). Using this technique, the words "I love you" are written on fish around Valentine's day (http://www.fishtanksandponds.co.uk/ethics/fish-keepings-hall-of-shame.html).
Health hazards to artificially colored fish. First of all, dyeing, dipping and laser tattooing cause a great pain to fish. Yes, fish do feel pain, they respond to tissue- damaging stimuli similar to stressed mammals (Weis 2011). In the case of dyeing, using the same needle for a large number of fish greatly increases the risk of disease transmission (Hirte-Runtsch 2008). A fish’s immune system is seriously compromised through injection. Evidence can be found in the ratio of painted glassfish exhibiting lymphocystis to those left unpainted (Donston & Lass 2012). Dyed fish can be very lethargic and unhealthy, unlike their normal counterparts. Many suffer from kidney failure (http://www.theaquariumwiki.com/Dyed_fish; MacMahon & Burgess 1998), skin disease or they may be vulnerable to attacks in the tank because of their unfamiliar look (http://hongkong.coconuts.co). The dipping fish in dye solutions, though not as invasive as tattooing or injection, removes the fishes' first line of protection whcih is the mucus layer, affecting the gills as well, which affects then the respiration (Hirte-Runtsch 2008). Tattooing seems to have a much lower mortality rate than injection, but it is still elevated above the unmodified fish rates, as is the frequency of disease (Wiegert 2012).
Conclusions and Recommendations. Fish are naturally beautiful the way they are, that is why they do not need to be painted or tattooed. We should not to put money above the life and welfare of any living thing. Education of the customers is a very important thing, because artificially colored fish continue to exist because of demand. A
AACL Bioflux, 2015, Volume 8, Issue 1. 119 http://www.bioflux.com.ro/aacl good fish store should either label fish as dyed or tattooed. Since we live in a world of supply and demand, the consumers have the power to stop the trade with these unnatural colored fish. Every time someone buys a painted, dyed, or tattooed fish, contributes to the continuation of this practice. So, next time you visit a pet shop, think twice about buying unusually colored fish.
References
Adams J., 2011 Live fish keyring, tattooed fish and preserved fish ‘art’ are really, really tacky. Available at: http://reefbuilders.com/2011/04/08/live-fish-keyring-tattooed- preserved/. Accessed January 26, 2015. Broome H., 2013 Pet shop owners dunk fish craze. Available at: http://www.northernstar.com.au/news/pet-shop-owners-dunk-fish-craze/1935172/. Accessed February 02, 2015. Burlacu L., Radu C. F., Crăciun N., Sahlean T., Gavriloaie I. C., Bucur C., 2009a Body mass-related modifications involved in starvation at pumpkinseed Lepomis gibbosus (Linnaeus, 1758) (Teleostei, Centrarchidae). AACL Bioflux 2(1):57-61. Burlacu L., Radu C. F., Gavriloaie I. C., Sahlean T., Crăciun N., Bucur C., 2009b Variation of growth-related values within age categories and sexes in a pumpkinseed – Lepomis gibbosus (Linnaeus 1758), (Teleostei, Centrarchidae) - population. AACL Bioflux 2(1):63-70. Clarke M., 2006 Company offers custom fish tattoos with laser. Available at: http://www.practicalfishkeeping.co.uk/content.php?sid=829. Accessed February 01, 2015. Curry C., 2012 Genetically modified neon ‘GloFish’ could threaten natural species: report. Available at: http://abcnews.go.com/blogs/technology/2012/09/neon-genetically- modified-glofish-could-threaten-natural-species/. Accessed February 02, 2015. Daub E., 2009 Novelty vs. cruelty: the ethics of dyed or tattooed aquarium fish. Available at: http://blogs.thatpetplace.com/thatfishblog/2009/08/14/novelty-vs-cruelty-the- ethics-of-dyed-or-tattooed-aquarium-fish/#.VOZN0yxiBmk. Accessed December 27, 2014. Donston P., Lass D., 2012 Painted, tattooed and dyed fish: pros & cons. Available at: http://www.petproductnews.com/ppn-editorial-blog/fish-absolutely/painted- tattooed-and-dyed-fish.aspx. Accessed January 29, 2015. Gavriloaie I. C., 2007 [Fishes as bioindicators]. Ecoterra 14:16-17 [in Romanian]. Gong Z., Wan H., Tay T. L., Wang H., Chen M., Yan T., 2003 Development of transgenic fish for ornamental and bioreactor by strong expression of fluorescent proteins in the skeletal muscle. Biochemical and Biophysical Research Communications 308(1):58-63. Hirte-Runtsch S., 2008 Dyed fish. Online Aquarium Fish Magazine. Available at: http://www.fishlore.com/aquariummagazine/may08/dyedfish.htm. Accessed November 30, 2014. Kászoni Z., 1970 [Acvaristica]. Scientific Publishing House, Bucharest, 282 pp. [in Romanian]. Mag I. V., Petrescu R. M., 2006 [Fish as bioindicator of water quality]. Environment and Progress 8:215-218 [in Romanian]. MacMahon S., Burgess P., 1998 Why it’s cruel to dye. Practical Fishkeeping. March 1998, pp. 114-115. Ng P. K. L., Tan H. H., 1997 Freshwater fishes of Southeast Asia: potential for the aquarium fish trade and conservation issues. Aquarium Science and Conservation 1(2):79-90. Păsărin B., Petrescu-Mag I. V., 2011 What we expect from Poeciliids for the future in terms of evolution. Poec Res 1(1):24-26. Petrescu-Mag I. V., 2007 [Sex control in guppyculture]. Academicpres, Cluj-Napoca, ISBN-978-973-744-094-5 [in Romanian].
AACL Bioflux, 2015, Volume 8, Issue 1. 120 http://www.bioflux.com.ro/aacl Petrescu-Mag R. M., Păsărin B., Şonea C. G., Petrescu-Mag I. V., 2013a Customer preferences and trends for aquarium fish in Transylvania (Romania). North-Western Journal of Zoology 9(1):166-171. Petrescu-Mag R. M., Creanga S., Petrescu-Mag I. V., 2013b Mendelian laws in aquaculture and cuniculture: simple and efficient. AACL Bioflux 6(2):111-114. Roberts D., 2013 Electric green barb GloFish: an introduction. Available at: http://blog.petsolutions.com/live-fish/electric-green-barb-glofish-an-introduction/. Accessed December 14, 2014. Sharp S., 2006 Artificially colored aquarium fish. Death by dyeing. Available at: http://freshaquarium.about.com/cs/beginnerinfo/a/paintedfish.htm. Accessed February 03, 2015. Weis J. S., 2011 Do fish sleep? Fascinating answers to questions about fish. Rutgers University Press, 217 pp. Wiegert J., 2012 Avoid dyed or painted fish. Available at: http://www.fishchannel.com/fish-exclusives/fama/conservation-corner/avoid-dyed- or-painted-fish.aspx. Accessed February 02, 2015. Wiegert J., 2013 Parrot fish: good or bad for the hobby? Available at: http://www.tfhmagazine.com/details/articles/parrotfish-good-or-bad-for-the-hobby- full-article.htm. Accessed February 02, 2015. *** http://www.deathbydyeing.org/colormedead.htm. ***http://hongkong.coconuts.co/2015/02/17/tai-po-aquarium-slammed-selling-get-rich- tattooed-fish-chinese-new-year. Accessed February 17, 2015. *** http://www.thekrib.com/Fish/steroids.html thekrib.com. *** http://www.theaquariumwiki.com/Dyed_fish. *** http://www.firsttankguide.net/painted.php. *** http://www.glofish.com/about/glofish-display/. *** https://fishcompetstore.files.wordpress.com/2013/11/dsc_6695.jpg. ***http://www.odditycentral.com/pics/tattooed-fish-sold-as-lucky-charms-in-china.html. *** http://www.practicalfishkeeping.co.uk/content.php?sid=829. *** http://www.underwatertimes.com/news.php?article_id=01769543281. *** http://news.asiaone.com/News/AsiaOne+News/Singapore/Story/A1Story20080117- 45461.html. *** http://www.fishtanksandponds.co.uk/ethics/fish-keepings-hall-of-shame.html.
Received: 17 February 2015. Accepted: 27 February 2015. Published online: 28 February 2015. Authors: Valentin O. Eşanu, University of Liege, Agro-Bio Tech Gembloux, Belgium, e-mail: [email protected] Claudiu Gavriloaie, SC Bioflux SRL, Cluj-Napoca, Romania, 54 Ceahlau Street, Cluj-Napoca 400488, Romania, e-mail: [email protected] Ioan Gheorghe Oroian, Department of Environment and Plant Protection, Faculty of Agriculture, University of Agricultural Sciences and Veterinary Medicine, 3-5 Calea Mănăştur Street, 400372Cluj-Napoca, Romania, e-mail: [email protected] Philippe Burny, Walloon Center for Agricultural Research, Belgium, e-mail: [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. How to cite this article: Eşanu V. O., Gavriloaie C., Oroian I. G., Burny P., 2015 Some considerations concerning the artificially colored aquarium fish trade. AACL Bioflux 8(1):116-121.
AACL Bioflux, 2015, Volume 8, Issue 1. 121 http://www.bioflux.com.ro/aacl