AACL Bioflux, Volume 5(2) June 30, 2012

Contents

Dewiyanti, I., Karina, S., 2012. Diversity of Gastropods and Bivalves in mangrove ecosystem rehabilitation areas in Aceh Besar and Banda Aceh districts, Indonesia. Aquaculture, Aquarium, Conservation & Legislation 5(2):55-59.

Sinitean, A., Petrovici, M., 2012. Usage of biotic indices in evaluating the impact of the urban centres on the quality of the water in rivers. Aquaculture, Aquarium, Conservation & Legislation 5(2):60-63.

Corre, Jr, V., Faisan, Jr, J., Carton-Kawagoshi, R. J., Elle, B. J., Traifalgar, R. F., Caipang, C. M., 2012. Evidence of WSSV transmission from the rotifer (Brachionus plicatilis) to the black tiger shrimp (Penaeus monodon) postlarvae and means to control rotifer resting eggs using industrial disinfectants. Aquaculture, Aquarium, Conservation & Legislation 5(2):64-68.

Gheorghievici, L. M., Pompei, I., Gheorghievici, G., Tanase, I., 2012. The influence of abiotic factors on suppliers of organic matter in the peloidogenesis process from Lake Techirghiol, Romania. Aquaculture, Aquarium, Conservation & Legislation 5(2):69-78.

Musuka, C. G., Musonda, F. F., 2012. Current and future prospects of commercial fish farming in Zambia. Aquaculture, Aquarium, Conservation & Legislation 5(2):79-87.

Mohammadi, F., Mousavi, S. M., Rezaie, A., 2012. Histopathological study of parasitic infestation of skin and gill on Oscar (Astronotus ocellatus) and Discus (Symphysodon discus). Aquaculture, Aquarium, Conservation & Legislation 5(2):88-93.

Sezen, S., Olmez, M., 2012. Production of Aphanius mento (Heckel, 1843) under controlled conditions. Aquaculture, Aquarium, Conservation & Legislation 5(2):94-98.

Rhyne, A. L., Tlusty, M. F., 2012. Trends in the marine aquarium trade: the influence of global economics and technology. Aquaculture, Aquarium, Conservation & Legislation 5(2):99-102.

International Standard Serial Number Online ISSN 1844–9166; Printed ISSN 1844-8143

Published by Bioflux – five issues/year

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.

International Editorial Board

40 Countries (5 continents): Romania, USA, Guyana, Canada, Republic of Moldavia, Belgium, Italy, Spain, Peru, Turkey, Venezuela, Poland, Croatia, Germany, Finland, Slovakia, India, Pakistan, Israel, Switzerland, Hungary, Bangladesh, Palestinian Authority, Nigeria, South Korea, Greece, England, China, Japan, France, Thailand, Malaysia, Indonesia, Iran, Philippines, Norway, Republic of Armenia, Brazil, Portugal and Slovenia.

Editorial Board Expanded

Editor-in-Chief Rhyne Andrew: Roger Williams University; New England Aquarium, Boston (USA) Gavriloaie Ionel-Claudiu (reserve): Technical College "INFOEL" Bistrita (Romania).

Editors Arockiaraj A. Jesu: Cheju National University (South Korea) Bavaru Adrian: Ovidius University, Constanta (Romania) Caipang Cristopher M.A.: Univ. Nordland, Bodoe (Norway); Indep. res. (Philippines) Codreanu Mario: USAMV Bucuresti, Bucharest (Romania) Dimaggio Matthew A.: University of Florida (USA) Mehrad Bahareh: Gorgan University of Agricultural Sciences and Nat. Res. (Iran) 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) Petrescu-Mag I. Valentin: USAMV Iasi, Iasi; USAMV Cluj, Cluj-Napoca (Romania) Parvulescu Lucian: West University of Timisoara (Romania) Petrescu-Mag Ruxandra Malina: UBB Cluj-Napoca, Bioflux SRL (Romania) Petrovici Milca: West University of Timisoara (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)

Journal Secretary Nowak Michal: University of Agriculture in Krakow (Poland)

Honorary Editors Appelbaum Samuel: University of the Negev (Israel) Bourne Godfrey Roderick: Univ. Missouri-St Louis (USA), CEIBA (Guyana) Breden Felix: Simon Fraser University (Canada) Brezeanu Gheorghe: Romanian Academy of Science, Bucharest (Romania) Bud Ioan: USAMV Cluj (Romania) Bura Marian: USAMVB Timisoara (Romania) Burny Philippe: Universite de Liege (Belgium) Creanga Steofil: USAMV Iasi, Iasi (Romania) Cristea Victor: Dunarea de Jos University of Galati (Romania) Grozea Adrian: USAMVB Timisoara (Romania) Miclaus Viorel: USAMV Cluj (Romania) Molnar Kalman: Hungarian Academy of Sciences, Budapest (Hungary) Naghiu Alexandru: USAMV Cj(Romania), Nat.Acad.Agric.Sci. (Italy), Univ.Leon (Spain) Oroian Ioan: USAMV Cluj, Cluj-Napoca (Romania) Papadopol Nicolae: Natural Sciences Museum Complex, Constanta (Romania) Pasarin Benone: USAMV Iasi, Iasi (Romania) Usturoi Marius Giorgi: USAMV Iasi, Iasi (Romania)

Editorial Councellor Velter Victor: UEFISCDI, Bucharest (Romania)

Scientific Reviewers Abdullah Khalid: Agricultural Research Institute Ratta Kulachi (Pakistan) Abraham T. Jawahar: West Bengal Univ. of Animal & Fishery Sciences (India) Adewolu Morenike A.: Lagos State University, Ojo (Nigeria)

ii 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) Blas Raul Humberto Sevillano: Univ. Nacional Agraria La Molina (Peru) Boaru Anca: USAMV Cluj, 3M AGC SRL, Cluj-Napoca (Romania) Brunner Andreas: Swiss Federal Institute of Technology (Switzerland) 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 (Romania) Costa Daniel Ribeiro: Federal Rural University of Amazon, Belem (Brazil) Costea Ramona Ioana: USAMV Cluj (Romania) Cotutiu Mihaela Ioana: Technical College INFOEL, Bistrita (Romania) Covaciu-Marcov Severus D.: University of Oradea (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) Gal Denes: Research Inst. for Fisheries, Aquaculture & Irrigation, Szarvas (Hungary) Galca Valerica: USAMV Iasi, Iasi (Romania) Galca Ioan: USAMV Iasi, Iasi (Romania) Gavrilovic Ana: University of Dubrovnik, Dubrovnik (Croatia) Gospic Daniel: G2O d.o.o. (Slovenia) Gorgan Lucian Dragos: UAIC Iasi (Romania) Hajirezaee Saeed: University of Teheran, Karaj (Iran) Hoha Gabriel: USAMV Iasi, Iasi (Romania) Iorga Eugen: Institutul de Tehnologii Alimentare (Rep. Moldavia) Islam Kazi Shakila: Xiamen University, Xiamen (China) Jakab Endre: UBB Cluj-Napoca (Romania) Kabir Milad: Gorgan University of Agricultural Sciences and Nat. Res. (Iran) Karayucel Ismihan: University of Sinop (Turkey) Kempkes Michael: Deutschen Guppyförderation (Germany) Kosco Jan: Presov University, Presov (Slovakia) Luangpirom Ampa: Khon Kaen University, Khon Kaen (Thailand) Malos Cristian: UBB Cluj-Napoca (Romania) Manko Peter: Presov University, Presov (Slovakia) Montana Carmen: UNELLEZ, Barinas (Venezuela), University of Texas (USA) Muntean Octavian Liviu: UBB Cluj-Napoca (Romania) Munteanu Florin: Natural Sciences Museum Complex, Constanta (Romania) Murray Joanna: CEFAS, Lowestoft (England) Muscalu-Nagy Cristina: Sterlet SRL, Timisoara (Romania) 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) Parsaeimehr Ali: National Academy of Science, Yerevan (Republic of Armenia) Perdikaris Costas: Technological Educational Institute of Epirus (Greece) Perkovic Anica: University of Josip Juraj Strossmayer, Osijek (Croatia) Petrescu Dacinia Crina: UBB Cluj-Napoca (Romania) Popescu Irinel Eugen: UAIC Iasi, Iasi (Romania) Raghavan Rajeev: St. Albert’s College, Kochi (India) 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) Sahlean Tiberiu: University of Bucharest (Romania) Sas Istvan: University of Oradea, Oradea (Romania) Savin Anatol: ASM Institute of Zoology (Rep. Moldavia) Taati M. Mehdi: Gorgan University of Agricultural Sciences and Nat. Res. (Iran) Talu Stefan: Technical University of Cluj (Romania) Tugui Olimpia Ramona: Univ. Lyon II Lumiere (France) Turcu Mihaela Claudia: University of Turku, Turku (Finland) Udoh James P.: University of Uyo, Uyo (Nigeria)

iii Vintila Iuliana: "Dunarea de Jos" University Galati (Romania) Zamfirescu Stefan: UAIC Iasi, Iasi (Romania).

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v News !

In April, 12, 2012

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and

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were merged into one journal, namely:

AACL Bioflux (1980).

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We thank: Natural Sciences Museum Complex (Constantza, Romania) and Bioflux publishing house (Cluj-Napoca, Romania) for this collaborative effort !

vi Aquaculture, Aquarium, Conservation & Legislation OPEN ACCESS International Journal of the Bioflux Society Research Article

Diversity of Gastropods and Bivalves in mangrove ecosystem rehabilitation areas in Aceh Besar and Banda Aceh districts, Indonesia

Dewiyanti Irma, Karina Sofyatuddin

Department of Marine Sciences, Faculty of Marine and Fisheries, Syiah Kuala University, Darussalam, Banda Aceh, Indonesia.

Abstract. This study was conducted in mangrove rehabilitation area on May to November 2011. The objective of the present study was to evaluate the diversity of Gastropods and Bivalves based on the different level of vegetation age of mangrove. Sampling were conducted on 1 x 1 m2 of square transects. We recorded 14 species of Gastropods and 5 species of Bivalves with abundance of 371 individuals/m2 and 28 individuals/m2, respectively. The highest abundance of Gastropod was found on 4 years vegetation age on Cerithidae cingulata (150 individuals/ m2), while the highest abundance of Bivalves was found on 3 years vegetation age on Pedalion isognomum (8 individuals/m2). In conclusion, the condition of community structure post tsunami was unstable.

Key Words: mangrove, rehabilitation, community structure, Gastropods, Bivalves.

Copyright: 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.

Corresponding Author: Dewiyanti Irma, [email protected]

The changes of mangrove forest from the stable vegetation pre Introduction tsunami to the newly vegetations covered by sapling and seedling certainly caused a serious impact to the biotic life in changing Mangrove forests are a typical forest ecosystem growing along of community structures in relation to abundance and diversity. the coast or estuaries and they are affected by the tide and flow Most of Bivalves, not only were consumed by the local people, with a wide variety of environments throughout the tropical but also were used as a commercial alternative livelihood of lo- and sub-tropical regions (Nybakken 1992; Aksornkoae 1993). cal people to increase their income. Gastropods and Bivalves Mangrove plays an important role in the lifecycle of various are generally benthos organism and they are regularly used as aquatic organisms such as fishes, shrimps and mollusks espe- bio-indicators of aquatic healthy. Gastropods and Bivalves can cially for spawning, nursery and feeding grounds (Pramudji produce a billion of larvae in the form of meroplankton that sus- 2001). Mangrove as a feeding ground contributes to the habi- tains the biotic population and they have a role in food chain. tat complexity and diversity of the macrofauna associated in The observation of Gastropods and Bivalves populations in mangrove ecosystem, while crustaceans and mollusks are the mangrove ecosystem is important to evaluate their condition dominant macrofauna in this ecosystem (Hutchings & Saenger in the post tsunami rehabilitation program in Aceh where the 1987; Sasekumar 1974). The macrofauna diversity and abun- new communities have begun to form. Hence, the objective dance may reflect biological indicators of changing habitat in of the present study was to assess the species composition of mangrove ecosystem (Macintosh et al 2002). Gastropods and Bivalves in mangrove rehabilitation area post The past tsunami in late 2004 has destroyed and reduced the tsunami based on the different level of vegetation age. mangrove ecosystem in Aceh coastal zone and it resulted in a negative impact on mangrove biota for example Gastropods and Bivalves. According to Dewiyanti (2005) Rhizopora api- Material and Methods culata, R. stylosa and Avicennia marina were the dominant The study was conducted in the area of mangrove rehabilita- species of mangrove pre tsunami in Ulee Lheue coastal, Banda tion in Banda Aceh and Aceh Besar districts, Indonesia. The Aceh city. Furthermore, Dewiyanti (2005) reported that there study was conducted for six months from May to November were 22 species of Gastropods and 17 species of Bivalves oc- 2011. The mangrove vegetation in the study areas are the new- curred before tsunami in mangrove area of Ulee-Lheue where ly formed vegetation post mangrove rehabilitation program. the most dominant families were Cerithidae and Isognomonidae Five sampling sites (10 m x 10 m) were selected by stratified for Gastropods and Bivalves, respectively.

AACL Bioflux Volume 5 | Issue 2 Page 55 http://www.bioflux.com.ro/aacl Dewiyanti et al 2012 random sampling method with dividing the study areas into Gastropods are more active compared to Bivalves; they can several layers or strata based on characteristics of the different move up and down follow the tides. According to Tee (1982) planting age. According to field assessment, there were five lev- almost all Gastropods are categorized as a tree climber, mov- els of mangrove vegetation age found at study area i.e. from 1 ing up and down to follow the tide, for example they go down year old to 5 years old. Every site was a representative of man- to the ground that are not flooded during low tide. grove age level where site one was representative of one year We found that total abundance of Gastropods and Bivalves was old and situated in Deah Glumpang village (05°33’42.3” N, 371 individual with average density of 74 ind/m2 and 28 indi- 95°17’35.7” E), site two was representative of two years old and viduals with average density of 6 ind/m2, respectively (Table situated in Lampageu village (05°33’08.2” N, 95°14’13.9”E), 1 and Table 2). site three was representative of three years old and located in Deah Glumpang village (05°33’34.3” N, 95°17’21.0” E), site Table 1. Average of Gastropods abundance in study sites (ind/m2) four was representative of four years old and located in Lamnga Families/Species Study sites Total village (05°37’01.6” N, 95°23’46.6” E), and site five was rep- 1 2 3 4 5 resentative of five years old and occurred in Lamnga village (05°36’35.8” N, 95°19’25.6” E) (Figure 1). Buccinidae Biological observation includes the retrieval of mangrove data Cantharus fumosus 0 0 0 1 0 1 and mollusks (Gastropods and Bivalves). Samples were collect- Cerithiidae ed using the square transects of 1 x 1 (m) into 10 x 10 (m) as a plot determined randomly in very sampling site and every plot Cerithium patulum 7 12 9 0 0 28 Clypeomorus granosum 5 5 5 5 2 22 Clypeomorus moniliferum 0 0 4 0 0 4 Conidae Phasmaconus radiatus 1 0 1 0 0 2 Littorinidae Littorina scabra 6 4 3 3 2 18 Littorina undulata 0 1 1 0 0 2 Nassariidae Nassarius olivaceus 1 1 0 1 0 3 Nassarius distortus 1 2 7 4 0 14 Figure 1. The map of study sites located at Banda Aceh and Aceh Neritidae Besar districts. Nerita undata 1 1 1 0 0 3 had three square transects. Mollusk samples were collected in Nerita planospira 2 1 1 1 0 5 two ways: by collecting for epifauna and taking the substrate to a depth of 15 cm for infauna. Mollusk samples were sorted and Nerita. sp 1 0 0 3 0 4 preserved in bottle sample containing 70% alcohol. Each bot- Potamididae tle sample was labeled with the site, date and other important Cerithidea cingulata 0 0 39 150 74 263 information. The samples were then transported to the labora- Terebralia palustris 0 0 0 1 0 1 tory and identified based on Dharma (1988) and Roberts et al (1982) and FAO (1998). The C-organic contents were analyzed Total Individual 25 27 71 169 79 371 based on Schumacher (2002) and the compositions of substrate Number of Species 9 8 10 8 3 14 were examined based on standard pipette method (Sudjadi et al 1971; Van Der Pol 1983). Furhermore, texture of substrate was gained by using textural triangle according to percentage of sand, The higher abundance of Gastropods was obtained in study site 2 silt, and clay fraction (Sudjadi et al 1971; Van Der Pol 1983). 4 (169 ind/m ) which was dominated by Potamididae (151 ind/ 2 m ). These results are in agreement with Dharma (1988) who reported that mangrove forest is the most preferred habitat for Results and Discussion families Potamididae. Moreover, Budiman (1988) explained Species composition and abundance that Potamididae had a wide geographical distribution and also There were 14 species of Gastropods and 5 species of Bivalves found in high abundance in mangrove ecosystem. In addition, belonging to seven and four families, respectively. These results the higher abundance of Bivalves was found in study site 3 with were lower compared to Macintosh et al (2002) who recoded 33 density of 14 ind/m2 dominated by Isognomonidae (8 ind/m2). species of mollusks in mangrove rehabilitation area in Thailand. Overall, Ostreidae had high abundance in present study (13 ind/ Gastropods and Bivalves obtained during the observation gen- m2). According to Plaziat (1984), Isognomonidae and Ostreidae erally live on the surface of the substrate, attached to the roots, are abundant in mangrove area because they have higher adap- stems and leaves of mangrove trees. tation ability in changing environmental factors such as drought The results of observation showed that Gastropods had high abun- due to low tide and salinity. dance and distribution probably due to their mobile characteristic.

AACL Bioflux Volume 5 | Issue 2 Page 56 http://www.bioflux.com.ro/aacl Dewiyanti et al 2012

Table 2.Average of Bivalves abundance in study sites (ind/m2) was a contribution of litter, especially leaves, from the man- Families/Species Study sites Total grove vegetation. We found that the age of vegetation did not affect the abun- 1 2 3 4 5 dance of Bivalves. However, the higher abundance of Bivalves Isognomonidae was found at study site 3 with 3 years old of mangrove. This Pedalion isognomum 1 2 8 0 0 11 is probably due to the higher density of mangrove (3 ind/m2) Lucinidae at this site, as it had a lot of root branching used for Bivalves living on. Mangrove roots represent the area of hard substrate Lucina sp. 1 1 0 0 0 2 colonized by massive sponge, oyster (Ostreidae), and barnacle Ostreidae (Cannicci et al 2008). Moreover, Guerreiro et al (1996) ex- Crassostrea cucullata 0 1 3 0 0 4 pressed that sediment textures, tidal elevation and forest type affect the distribution of macrofauna in mangrove ecosystem. Saccostrea echinata 0 0 2 5 2 9 Cerithidea cingulata was the dominant species in the study Veneridae area. We assumed that this species had a good adaptation on Gafrarium dispar 0 1 1 0 0 2 environmental condition at these sites and therefore it won the Total 2 5 14 5 2 28 competition for food and habitat. In addition, we found three species of native mollusks in mangrove ecosystem i.e. C. cin- Number of species 2 4 4 1 1 5 gulata, Terebralia palustris and Nerita planospira; three faculta- In general, the abundance of Gastropods has increased with tive mollusks i.e. Littorina scabra, C. patulum, and Crassostrea vegetation age increasing, for example study site 4, the vegeta- cucullata and two migrant mollusks i.e. Nerita undata and tion of four years old had the highest abundance of Gastropods Clypeomorus moniliferum. compared to other sites. We recorded that the site 4 had higher The species richness and abundance of Gastropods and Bivalves C-organic content (0.9%) compared to other sites, therefore we has decreased in post tsunami. For example, at the site 1, before assumed that the higher abundance of Gastropods at site 4 prob- tsunami, the species richness of Gastropod consisted in 11 species with density of 52 ind/m2 and they decreased to only 9 spe- ably due to higher in C-organic content. Contribution of organic 2 matter was influenced by the litters falling in the surrounding cies with density of 25 ind/m after tsunami. Before tsunami the vegetation area and would be the primarily contribution in the Cerithiidae had high abundance; however the species composi- substrate. In addition, we also recorded that the percentage of tion was dominated by Potamididae after tsunami. Macintosh silt and sand were higher at site 4, i.e. 15% and 83% respec- et al (2002) reported that Potamididae was one of Gastropods tively, but with lower percentage of clay (2%). According to families which was more representative of the younger plan- Rangan (1996) the substrate condition influences the develop- tation, assumed as they graze the young mangrove leaves like ment of biotic communities, where muddy with a little clay is Littorinidae and therefore, Macintosh et al (2002) suggested using a desirable substrate for Gastropods. this family as bio-indicator of ecological changes. Nevertheless, The value of C-organic content in the substrate was 0.8%, 0.8%, Asthon et al (2003) stated that young sapling leaves probably 0.7%, 0.9% and 0.6% for sites 1, 2, 3, 4 and 5, respectively. The was a better food source for mangrove Gastropods evidenced C-organic content recorded in the present study (post tsunami) by his observation that Gastropods was positively correlated was lower than the previous study (pre tsunami). This condition with sapling species number. exists probably because tsunami brought out the sands from oth- Decreasing in species richness and abundance post tsunami er sites into mangrove area. According to Plaziat (1984) types probably due to changing in community structure of mangroves, of substrates had strong relationship with nutrient in sediment. where the density of mangrove community in pre tsunami was Sukardjo (1994) found that high quantities of C-organic matter high than in post tsunami. The tsunami catastrophe destroyed in Tanah Grogot, East Kalimantan was from high density of trees mangrove vegetation and also disrupted community structure because the fallen litter of mangrove leaves contributes signifi- of biotic that lives in it. In general, the C-organic content be- cantly to the higher organic matter in the soils. Furthermore, fore tsunami was also higher than after tsunami at site 1; the there was a connection between the size of substrate with car- C-organic content before tsunami was 1.06% and it decreased bon content in the substrate and also oxygen dissolved. Sand to 0.8% after tsunami. soil enables good oxidation and it makes high oxygen content Mangrove rehabilitation program is one of the solutions to restore and less organic matter compared with refine soil. the mangrove ecosystem damaged due to the tsunami and later The sediment examination showed that the percentage of sand, on mangrove biota including Gastropods and Bivalves will re- silt, and clay fractions have ranged from 82.5 to 91.0%, 5.0 to turn back to this ecosystem. Ashton et al (2003) and Aksornkoae 15.0%, and 2.0 to 5.0, respectively. Percentage of sand was (1993) mentioned that anthropogenic and natural disturbance high probably because sampling location directly receives ma- often result in diversity loss to natural ecosystem and loss of terial brought from the sea by the current despite in mangrove diversity have a significant effect on ecosystem functioning. area during tsunami in late December 2004. Nybakken (1992) mentioned that particle sedimentation depends on the size of Biological indices the particle and current. The bigger and heavier particles will The diversity index (Shannon index) has varied from 0.51 to settle faster than the smaller particles and weak current will de- 2.95 at site 5 and site 1, respectively, with average of 2.02 in- posit material longer than the strong current. Silt fraction was dicating a low value. The evenness index (E) has varied from higher than clay; this condition can be explained because there 0.26 to 0.85 with an average of 0.58 (Figure 2). These average

AACL Bioflux Volume 5 | Issue 2 Page 57 http://www.bioflux.com.ro/aacl Dewiyanti et al 2012 values indicate that the community condition was unstable. Acknowledgements However, the mollusk community at site 1 was relatively sta- We would like to express our gratitude to the Directorate General ble compared to other sites. of Higher Education through Syiah Kuala University for providing research grant. Our deepest thanks to the head of Deah Glumpang, Deah Baroe and Lamnga villages for their supporting and very good cooperation. Thanks to Roby, Sarah, and Marli for their field assistance during the research study. We express appreciation to Dr. Z. A. Muchlisin for his suggestions during manuscript preparation.

References Aksornkoae, S., 1993. Ecology and management of mangroves. IUCN Wetlands Programe. IUCN, pp. 1-6 Bangkok, Thailand. Ashton, E. C., Donald, J. M., Peter, J. H., 2003. A baseline study of diversity and community ecology of crab and molluscan macrofauna in the Sematan mangrove forest, Serawakm Malaysia. Journal of Tropical Ecology 19:127-142. Budiman, A., 1988. Some aspects on the ecology of mangrove whelk Figure 2. The diversity index (H’), evenness index (E) and Telecospium Telecospium (Linne, 1758) (Mollusca, Gastropoda: Dominance index (D) according to study sites. Potamididae). Treubia 29(4):237-245. Cannicci, S., Damien, B., Sara, F., Thomas, J. S., Joachim, O., Farid, In addition, the dominance index (D) has ranged between 0.19 D. G., 2008. Faunal impact on vegetation structure and ecosystem at site 1 to 0.84 at site 5 with average of 0.46 indicating a mod- function in mangrove forests: a review. Aquatic Botani 89:186-200. erate value. The dominance index showed the degree of pre- Dewiyanti, I., 2005. Structure of mollusca community (Gastropods and dominance of one or a few species in an ecological community. Bivalves) with its associates in mangrove ecosystem at Ulee-Lheue, Legendre & Legendre (1983) explained that if the dominance Banda Aceh. Nad [Struktur Komunitas Moluska (Gastropoda dan index values obtained are between of 0.4 to 0.6 it can be catego- Bivalvia) Serta Asosianya Pada Ekosistem Mangrove Di Kawasan rized as moderate, above 0.6 is higher and below 0.4 is lower. Pantai Ulee-Lheue Banda Aceh NAD]. Jurnal Natural 5(2):12-16. The result showed that C. cingulata was a dominant species at Dharma, B., 1988. Indonesian shells [Siput dan Kerang Indonesia]. 111 sites 4 and 5, where the dominance index was higher than 0.6, pp., Sarana Graha, Jakarta. indicating the dominance index at both stations were fallen FAO species identification guide for fishery purposes, 1998. The liv- into higher category. While at sites 1, 2, and 3, the dominance ing marine resources of the Western Central Pasific: Volume 1 indices were lower than 0.4 and it was categorized into lower Seaweeds, corals, bivalves and gastropods. Carpenter K. E., Niem category. Overall, we found that the low diversity index, the V. H (eds), pp. 1-686. Food and Agriculture Organization of the unevenly distributed individuals within species and the mod- United Nations, Rome, Italy. erate evenness and dominance showed an unstable community Guerreiro, J. S., Freitas, P., Pareira, J., Paula, J., Macia, A., 1996. Sediment structure at the study sites post tsunami. Macintosh et al (2002) macrobenthos of mangrove flats at Inhaca Island, Mozambique. Cah Bio Mar 37:309-327. explained that high dominance of one species probably indicates a stressful environment while a higher diversity indicates Hutchings, P., Saenger, P., 1987. Ecology of mangroves. Heatwole H (ed), 388 pp., University of Queensland Press, St. Lucia, Australia. a stable condition of ecosystem. For comparison, the stability of Gastropods and Bivalves com- Legendre, L., Legendre, P., 1983. Numerical Ecology. 337 pp., Elsevier munity structures in pre tsunami was in moderate category Scientific, Publishing Company, New York. (Dewiyanti 2005). Meanwhile, the condition of community Macintosh, D. J., Ashton, E. C., Havanon, S., 2002. Mangrove rehabili- structure generally has been changed become unstable in post tation and intertidal biodiversity: a study in the Ranong mangrove ecosystem Thailand. Estuarine, Coastal and shelf Science 55:331-345. tsunami period. Nybakken, J. W., 1992. Marine Biology: an ecological approach [Biologi Laut, Suatu Pendekatan Ekologis]. Translated by: M. Eidman, D. Conclusion G. Bengen, Koesoebiono, M. Hutomo dan Sukristijono, pp. 363- Species richness of Gastropods was higher compared to that of 375, PT Gramedia, Jakarta. Bivalves and the values decreased after tsunami, however their Plaziat, J. C., 1984. Mollusk distribution in the Mangal. Hydrobiology species compositions were similar between pre and post tsuna- of the Mangal, pp. 111-143, Dr W. Junk Publishers, The Hague. mi. Gastropods were dominated by Potamididae while Bivalves Pramudji, P., 2001. The role of mangrove forest ecosystems as habitat were dominated by Isognomonidae and Ostreidae. In general, for marine organisms [Ekosistem Hutan Mangrove dan Peranannya diversity of Gastropods and Bivalves in the study areas was in Sebagai Habitat Berbagai Fauna Akuatik]. Oseana 26(4):13-23. lower category. The abundance of Gastropods increased with Rangan, J., 1996. Community structure and typology of Gastropods increasing vegetation age, but the age of vegetation did not af- on mangrove forest zone, Kulu district, Minahasa, North Sulawes fect the abundance of Bivalves, and the community structure [Struktur dan Tipologi Komunitas Gastropoda pada Zona Hutan at the present study was unstable. Mangrove Perairan Kulu, Kabupaten Minahasa, Sulawesi Utara]. Program Pasca Sarjana, IPB, Bogor, Indonesia.

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Roberts, D., Soemodihardjo, S., Kastoro, W., 1982. Shallow water Van Der Pol, F., 1983. Analytical methods of the service laboratory for marine molluscs of North-West Java, pp. 143, P3O – LIPI, Jakarta. soil, plant and water analysis: part 1 methods for soil analysis. Soil Sasekumar, A., 1974. The distribution of macrofauna on a Malayan Science and Agrochemestry Devision, Koninklijk Instituut Vor De mangrove shore. Journal of Animal Ecology 43:51-69. Tropen, Amsterdam, Mauritskade. pp. 110. Schumacher, B. A., 2002. Methods for determination of total organic carbon (TOC) in soils and sediments. In: Ecological Risk Assessment Support Center Office of Research and Development (ERASC), pp. Authors 23, US Environmental Protection Agency. •Dewiyanti Irma, Syiah Kuala University, Department of Marine Sudjadi, M., Widjik, I. M., Soleh, M., 1971. Soil analysis guidance: the Sciences, Indonesia, Banda Aceh, Darussalam, Putroe Phang soil fertility [Penuntun Analisa Tanah: Bagian Kesuburan Tanah]. Street, number 1, 23111, e-mail: [email protected] pp. 166, Bogor Soil Research Institute, Bogor, Indonesia. •Karina Sofyatuddin, Syiah Kuala University, Department of Sukardjo, S., 1994. Soils in the mangrove forests of the Apar Nature Marine Sciences, Indonesia, Banda Aceh, Darussalam, Putroe Reserve, Tanah Grogot, East Kalimantan, Indonesia. Southeast Asian Phang Street, number 1, 23111, e-mail: [email protected] Studies 32(3):385-398. Tee, G. A. C., 1982. Some aspect of the mangrove forest at Sungai Buloh, Selangor II. Distribution patternand population dynamic of tree dwelling fauna. Mal Nat J 35:267-277.

Dewiyanti, I., Karina, S., 2012. Diversity of Gastropods and Bivalves in mangrove ecosystem rehabilitation areas in Aceh Besar and Banda Aceh Citation districts, Indonesia. Aquaculture, Aquarium, Conservation & Legislation 5(2):55-59. Editor Ionel-Claudiu Gavriloaie, I. Valentin Petrescu-Mag Received 21 February 2012 Accepted 01 March 2012 Published Online 15 May 2012 Funding Directorate General of Higher Education through Syiah Kuala University Conflicts / Competing No disclosures Interests

AACL Bioflux Volume 5 | Issue 2 Page 59 http://www.bioflux.com.ro/aacl Aquaculture, Aquarium, Conservation & Legislation OPEN ACCESS International Journal of the Bioflux Society Research Article

Usage of biotic indices in evaluating the impact of the urban centres on the quality of the water in rivers

Adrian Sinitean, Milca Petrovici

Department of Biology and Chemistry, Faculty of Chemistry, Biology, Geography, West University of Timișoara, Timişoara, Romania

Abstract. The quality of the river waters of the Crişul Repede River and the influence of the powerful urban and industrial centre Oradea upon these have been studied by means of collecting quantitative samples of benthos, for three consecutive years, in a seasonal manner. The calculation of the biotic indices that consider the abundance of benthic macroinvertebrates indicating the presence of high qualitative waters (Ephemeroptera, Plecoptera and Trichoptera) and of those indicating strong loading of the waters and of the under layer of the water ecosystem with organic matter (Diptera – Chironomidae Family), highlighted an advanced degradation of the water quality, from the upstream towards the downstream. Equally, the intensity of the process of degradation of the water quality has been established, which, in the case of this river, is very strong and visible by the increasing prevalence of groups that do not require special ecological conditions, proving to be resistive and even proliferating in the presence of intense pollution of the water.

Key Words: biotic indices, EPT, Chironomidae, macroinvertebrates, water quality.

Corresponding Author: M. Petrovici, [email protected]

Introduction The present study approaches the matter of evaluating the impact of the urban and industrial centre Oradea on the water quality of The equilibrium of the lentic water ecosystems is very delicate, the Crişul Repede River, taking in consideration the modifica- because the surface area has a relative stability, resisting only to tion of the values of the most utilizated biotic indices (Wallace slight modifications of the condition parameters. The macrozoo- et al 1996). To this effect, the following biotic indices were benthic organisms are completely exposed to the modifications taken in account: EPT Count, EPT/Total Count, Chironomidae of the environment, not disposing of various possibilities of Count, EPT abundance/Chironomidae abundance, % abundance leaving the affected environment and of settling in a new habi- of Chironomidae larvae. tat (Petrovici 2009). Any modification of the physicochemical factors produces corresponding modifications in the structure and in the specific composition of the biocenosis (Filimonet al Material and Method 2010). The proportion of the different species is also affected, The Crişul Repede River (length = 107 km) flows in the north- as well as their distribution in space and their dynamics in time, western region of Romania, being the major tributary of the and their interspecific relations. Tisa river. The catchment area is of about 1,437 km2, and it One of the major causes of the modification of these physico- is irregular, because its tributaries streaming from the Meseş chemical factors is the location on the river streambed of urban – Plopiş Mountains are almost non-existent (Petrovici 2009). and / or industrial centres that are performing caption and dis- Two sites were selected on the reaches of the river, in order to charging of residual waters in the river, as well as the way of investigate the changes that are induced on the water quality usage of the site and the intensity of agricultural activities on by the city of Oradea, an important urban and industrial centre the site (Getwongsa et al 2010). (over 200,000 inhabitants). The fist site, Fughiu site, is situ- The evaluation of the impact that an urban and industrial cen- ated at about 10 km upstream of the city (140 m altitude), in tre has on the water quality, analysing both the modification of a highly agricultural region. The width of the streambed is of the physicochemical factors as well as the changes that appear 50-60 m, and its average depth is of 40 cm. The average water in the structure of the benthic macro invertebrate community, speed is of 0.8 m/second, and the sediment is equally made out has been the topic of many studies so far, carried out on vari- of rocks and grit, that have allowed accumulation of slopes of ous rivers (Badea et al 2010; Marin et al 2011; Rácz et al 2010; sandy sediments. Near the banks, the sediment is mostly made Răescu et al 2011; Stoian et al 2009, Vincze et al 2011). out of sand, with thick slopes of ooze. The second site, Cheresig

AACL Bioflux Volume 5 | Issue 2 Page 60 http://www.bioflux.com.ro/aacl Sinitean et al 2012 site (94 m altitude), is situated at 30 km downstream of the city, index were encountered during the entire period, in the spring at a distance of 2 km of the state border line. The width of the season, followed by a decrease of the number of individuals of streambed is of 60-70 m, and the depth and the average water the three groups. speed is of 50-60 cm and respectively 0.9 m/second. The un- At the Cheresig site, the EPT indices have low values (oscillat- der layer is made mostly of sand and the grit is present only in ing between 1 and 11), except for the summer of 1998, when the a small proportion, with many oozy deposits. value 33 was recorded, after the identification in the samplings The samplings were collected in a seasonal manner (spring, of a record number of Ephemeroptera (22). These were identi- summer, autumn), in the period 1996-1998, by using a Surber fied to be part of the Baëtis vernus species that did not present sampler (1060 cm2 surface, mesh size 250 µm). Three samplings strict ecological criteria, supporting a higher degree of degra- (replicates) were collected from each site, in order to be able to dation of the water quality (Găldean 1992; Hellawell 1986). calculate an average value. The samplings were entrenched, in The same pattern of variation presents the EPT/T Count index formaldehyde 8%. Afterwards, the samplings were sorted, and (Figure 1, up-right - Figure 1.b), but on another scale of values, the respective organisms were sorted and numbered, then sepa- because the sum of the individuals from the three groups indi- rated according to the reign, except the midges, that were sorted cating the presence of high qualitative waters is divided, in this only by family-level (Ord. Diptera – Chironomidae Family). case, at the number of individuals present in the whole macro In order to evaluate the water quality, the following biotic in- zoobenthic community. Therefore, this index becomes very low dices were taken in account (Hellawell 1986): and has a constant value (lower than 0.05) on the Cheresig site, Ephemeroptera Plecoptera Trichoptera Count (EPT Count): where the reduced number of Ephemeroptera and Trichoptera Count of the number of individuals in the three generally pol- individuals is divided to the great number of Chironomidae and lution-sensitive orders – Ephemeroptera (mayflies), Plecoptera Oligochaeta, which are numerically dominating the benthic com- (stoneflies), and Trichoptera (caddisflies). A high variety is good. munity. The great number of Chironomidae on the Cheresig site EPT/Total Count: (EPT/T Count) EPT count divided by the total (Figure 1, middle-left - Figure 1.c), indicated a heavy loading of number of individuals in the sample. A higher number is good. the underlayer of the Crişul Repede River with organic substanc- Chironomidae Count (Ch Count): Total of individual Chironomidae es, that was also certified by (Răescuet al 2011) for the Cerna (midge larvae) collected in sample. River, after its waters had travel through a large urban centre. EPT abundance/Chironomidae abundance (EPT/Ch Count): EPT The model of the variation of the number of Chironomidae count divided by Chironomidae count. A lower Chironomidae (Figure 1, middle-left - Figure 1.c) and of the total number of abundance is good. macro zoobenthic organisms (Figure 1, down-right - Figure Total Count (T Count): total of macroinvertebrates individuals. 1.f) are similar, only the scale of values being different. This Percent (%) of abundance of Chironomidae larvae (Ch abund): can be explained by adding it at the number of Chironomidae Compares the number of Chironomidae to the total number and Oligochaeta present on both sites. The increased affluence of organisms in the sample. (The number of organisms in the of the two groups underlines once more the strong influence Chironomidae family is divided by the total number of organ- on the water quality of the Crişul Repede River that the city of isms in the sample to calculate a percent composition). A low Oradea has, both groups being characteristic to heavy-loaded percentage is good. water ecosystems and to sedimentary organic matter. Figure 1, middle-right, indicates this, with very low values of the Results and Discussions EPT/Ch Count index on the Cheresig site, in comparison with the values of the same index, but on the site situated upstream of the The sorting of the biological material collected (54 benthos sam- city. This index is considered by Hershey and Lamberti (2001) plings) up to a reign or family-level, proved that in the benthos the most popular community level metric used in biomonitor- from the Fughiu site, all groups of macroinvertebrates specific to ing. In the case of the Crişul Repede River, after the river trav- this particular river sector are present. Thereby, we encountered els through the city of Oradea, the value of the index decreases alongside Plecoptera, Trichoptera, Ephemeroptera (groups that very much (even by a few hundred times, e.g. the spring and are taken in account when calculating the EPT Count, EPT/T the autumn of 1996 and, respectively, the autumn of 1997). A Count, ETP/Ch Count indices), the presence of Chironomidae, decrease of this index has been tracked down by (Răescu et al Oligochaeta, Acarina, Coleoptera, Nematoda, Collembola, Odonata, 2011; Dumbravă-Dodoacă & Petrovici 2010; Wang et al 2012) Isopoda, Hirudina, Turbellaria, Gastropoda, Lamellibranchiata too, after the river has travelled through an urban centre, but in and Amphipoda. All these groups were taken in account when this case the index decreased only by a few times, from values calculating the EPT/T Count and T Count indices. of 0.17 and 0.28 in the upstream sector, to values of 0.56 and In the benthos of the Crişul Repede River present on the 0.67 in the downstream sector. The differences presented can Cheresig site, we encountered only the following: Chironomidae, be related to the differences of the impact that the two cities Trichoptera, Ephemeroptera, Oligochaeta, Nematoda, Acaria, had on the water ecosystems. Thereby, the city of Oradea had a Coleoptera, Isopoda, Hirudina, Turbellaria, Gastropoda and major, overwhelming impact on the water quality of the Crişul Lamellibranchiata. On this site, the Chironomidae and the Repede River, as it is an important urban and industrial centre, Oligochaeta compose about 94% of the total benthic macroin- while the city of Băile Herculane has only a smaller impact on vertebrate community. the water quality of the Cerna river, as it has fewer inhabitants The calculation of the EPT indices for the two stations (Figure and its industrial sector is not as developed as the one of Oradea. 1, up-left), indicates higher values of this index at the site situated upstream of the city than at the site situated downstream, during the entire period of study. The highest values of the

AACL Bioflux Volume 5 | Issue 2 Page 61 http://www.bioflux.com.ro/aacl Sinitean et al 2012

a) b)

c) d)

e) f)

Figure 1. Season variation of the biotic indices at the Fughiu and Cheresig sites, Crişul Repede River, 1996-1998 (For abscissa: the seasons are Spring = March-May, Summer = June-August, Autumn = September-November. For ordinate: values of biotic indices: a) EPT Count indices; b) EPT/T indices; c) Ch Count Indices; d) EPT/Ch Count indices; e) Ch abund indices; f) T Count indices)

Conclusions The calculation of the biotic indices that consider the abundance industrial centre Oradea. Equally, the indices calculated have of benthic macroinvertebrates indicating the presence of high highlighted the intensity of the process of degradation of the qualitative waters and of those indicating strong loading of the water quality, which, in the case of this particular river, is very waters and of the underlayer of the water ecosystem with organ- strong and visible by the increasing prevalence of groups that ic matter is an appropriate method of studying the water qual- do not require special ecological conditions, proving to be re- ity level of a water ecosystem. In the case of the Crişul Repede sistive and even proliferating in the presence of intense pollu- River, this analysis highlighted an advanced degradation of the tion of the water. water quality, once the river has travelled through the urban and

AACL Bioflux Volume 5 | Issue 2 Page 62 http://www.bioflux.com.ro/aacl Sinitean et al 2012 References Rácz, B., Kotroczó, Z., Vincze, G., Dobi, L., Posta, J., 2010. Biotic index at secondary education level and its adaptation in the sustain- Badea, A. B., Gagyi-Palffy, A., Stoian, L. C., Stan, G., 2010. Preliminary able development (Hungary). Studia Universitatis “Vasile Goldiş” studies of quality assessment of aquatic environments from Cluj sub- Seria Ştiinţele Vieţii 20(4):67-74. urban areas, based on some invertebrates bioindicators and chemical indicators. Aquaculture, Aquarium, Conservation & Legislation Răescu, C. S., Dumbravă-Dodoacă, M., Petrovici, M., 2011. Macro- 3(1):35-41. zoobenthic community structure and dynamics in Cerna River (western Romania). Aquaculture, Aquarium, Conservation & Legislation Dumbravă-Dodoacă, M., Petrovici, M., 2010. The influence of the an- 4(1):79-87. thropic activities on the benthonic macroinvertebrates communities existing in the Jiu and Jiul de Vest rivers, south-west of Romania. Stoian, L. C., Gagyi-Palffy, A., Stan, G., 2009. Preliminary aspects re- Aquaculture, Aquarium, Conservation & Legislation 3(2):133-140. garding the use of some invertebrate bioindicator species in the ecological study of an aquatic lotic ecosystem. Aquaculture, Aquarium, Filimon, M. N., Borozan, A., Sinitean, A., Popescu, R., Torok-Oance, Conservation & Legislation 2(3):331-337. R., Verdes, D., 2010. Bacteriological studies on Timis and Bega rivers with a role in appreciating pollution. Annals of the Romanian Vincze, G., János I., Hörcsik Z.T., Kotroczó Z., Szabó S., 2011. Water Society for Cell Biology 15(2):217-220. quality assessments on a natural wetland (Igrice-Marsh) on the basis of chemical parameters and macroinvertebrate taxa. Studia Găldean, N., 1992. Contribution to the zoogeography of the mayflies Universitatis “Vasile Goldiş” Seria Ştiinţele Vieţii 21(4):901-905. (Insecta: Ephemeroptera) of Romania. Travaux du Museum National D’Histoire Naturelle Grigore Antipa 32:425-443. Wallace, J. B., Grubaugh, J. W., Whiles, M. R., 1996. Biotic indices and stream ecosystem processes: results from an experimental study. Getwongsa, P., Hanjavanit, C., Sangpradub, N., 2010. Impacts of agri- Ecological Applications 6(1):140-151. cultural land use on stream benthic macroinvertebrates in tributaries of the Mekong River, northeast Thailand. Advances in Environmental Wang, B. X., Liu, D. X., Liu, S. R. Zhang, Y., Lu, D. Q. Wang, L. Z., Sciences 2(2):97-112. 2012. Impacts of urbanization on stream habitats and macroinvertebrate communities in the tributaries of Qiangtang River, China. Hellawell, J. M., 1986. Biological indicators of freshwater pollution Hydrobiologia 680(1):39-51. and environmental Management. Elsevier Applied Science, London. Hershey, A. E., Lamberti, G. A., 2001 Aquatic insect ecology. In: Ecology and classification of North American freshwater invertebrates 2nd edn. Thorp, J. H., Covich, A. P. (eds), pp. 733-775, Authors Academic Press, San Diego. •Adrian Sinitean, West University of Timișoara, Faculty of Marin, A. A., Dumbravă-Dodoacă, M., Petrovici, M., Herlo, G., 2011. Chemistry, Biology, Geography, Dept. of Biology and Chemistry, The human impact on benthic community structure and dynamics 16A Pestalozzi St., 300115, Timişoara, Romania, European of different ecosystems from Lunca Mureşului Nature Park (West Union, e-mail: [email protected] of Romania). Aquaculture, Aquarium, Conservation & Legislation 4(1):72-78. •Milca Petrovici, West University of Timișoara, Faculty of Chemistry, Biology, Geography, Dept. of Biology and Chemistry, Petrovici, M., 2009. Evaluarea calităţii apei râului Crişul Repede uti- lizând larvele de efemeroptere (Insecta: Ephemeroptera) ca bioin- 16A Pestalozzi St., 300115, Timişoara, Romania, European dicatori. Editura Universitatii din Oradea, Oradea. Union, e-mail: [email protected]

Sinitean, A., Petrovici, M., 2012. Usage of biotic indices in evaluating the impact Citation of the urban centres on the quality of the water in rivers. Aquaculture, Aquarium, Conservation & Legislation 5(2):60-63. Editor Ştefan C. Vesa Received 28 March 2012 Accepted 1 May 2012 Published Online 18 May 2012 Funding None reported Conflicts/ Competing None reported Interests

AACL Bioflux Volume 5 | Issue 2 Page 63 http://www.bioflux.com.ro/aacl Aquaculture, Aquarium, Conservation & Legislation OPEN ACCESS International Journal of the Bioflux Society Research Article

Evidence of WSSV transmission from the rotifer (Brachionus plicatilis) to the black tiger shrimp (Penaeus monodon) postlarvae and means to control rotifer resting eggs using industrial disinfectants

1Valeriano Corre, Jr, 2Joseph Faisan, Jr, 1,3Rhea J. Carton-Kawagoshi, 1Bessie J. Elle, 1Rex F. Traifalgar and 4Christopher M. Caipang

1 College of Fisheries and Ocean Sciences-Institute of Aquaculture, University of the Philippines Visayas, Iloilo, Philippines; 2 Aquaculture Department, Southeast Asian Fisheries Development Center, Iloilo, Philippines; 3 Department of Aquatic Biosciences-Laboratory of Applied Phycology, Tokyo University of Marine Science and Technology, Tokyo, Japan; 4 BioVivo Technologies AS, Bodø, Norway.

Abstract. Rotifers are considered possible vectors of the white spot syndrome virus (WSSV) and have been implicated in its recurrence in pond-cultured shrimp. However, direct evidence of the transmission and the pathogenicity of this virus from rotifers to shrimp has been lacking. In the present study, the pathogenicity of WSSV transmitted from infected rotifers (Brachionus plicatilis) to post larval black tiger shrimp (Penaeus monodon) was investigated. Results show that WSSV transmitted from infected rotifers to post-larval P. monodon caused an 82% cumulative mortality as compared to a 9% mortality in the non-infected control group. We also investigated the possibility of industrial disinfectants (sodium hypochlorite, granulated calcium hypochlorite and formalin) as possible means to inhibit the viability of rotifer resting eggs, considered a biological reservoir of WSSV in earthen ponds. Among the disinfectants that were tested, granulated calcium hypochlorite at 5 mg/L was the most effective. The present study provides direct evidence of the high pathogenicity of WSSV transmitted from rotifers to post larval P. monodon and shows the potential use of granulated calcium hypochlorite in pond disinfection. This treatment could be a promising strategy to inhibit the spread and recurrence of WSSV outbreaks in P. monodon culture.

Key Words: rotifers, resting eggs, WSSV, disinfectants.

Corresponding Authors: R. J. Carton-Kawagoshi, [email protected]; C. M. Caipang, [email protected]

Introduction These resting eggs can stay viable for months and even years in sediments of shrimp culture ponds, serve as biological reservoir of the virus and could be a probable cause of WSSV The white spot syndrome virus (WSSV) of the genus Whispovirus, recurrence. family Nimaviridae is the major cause of mass mortalities and Since the first report of WSSV occurrence in the Philippines economic losses in shrimp aquaculture. This virus is highly in 2000 (Magbanua et al 2000), the virus has been implicated pathogenic that could kill all infected shrimp within 7 to 10 in yearly crop losses of pond cultured P. monodon. days post infection (Chou et al 1995; Lightner 1996). Other Rotifers could be a possible vector and reservoir of WSSV in than shrimp, WSSV is also known to infect other crustacean earthen ponds but information on the transmission and patho- species including crabs and crayfishes (Wanget al 1998; Lo genicity of WSSV from infected rotifers to P. monodon has et al 1996). been lacking. Also, there is a need to develop a disinfection Recently, Yan et al (2004) reported that rotifers, commonly method to kill and eliminate rotifers resting eggs to reduce found in the rearing water of cultured shrimp are also host of the risk and recurrence of this disease in P. monodon culture. WSSV and a possible vector in the transmission of this virus. The present study detected WSSV in water rotifer and exam- Further, the work of Zhang et al (2006) provided direct evi- ined the transmission and pathogenicity of WSSV from in- dence of the transmission of WSSV from infected Brachionus fected rotifers to post larval P. monodon. In addition the ef- urceus to the larvae of Penaeus chinensis. Rotifers also posses ficacies of 3 different commercially available disinfectants the unique ability to produce resting eggs enclosed in a hard (NaOCl, Ca(OCl) , H CO ), to destroy the resting eggs of ro- protective casing that serves as a protective barrier against 2 2 tifers were also tested. adverse environmental conditions and chemical treatments.

AACL Bioflux Volume 5 | Issue 1 Page 64 http://www.bioflux.com.ro/aacl Corre et al 2012 Materials and Methods DNA samples were tested for the presence of WSSV using a PCR technique based on the protocol developed by Kimura et al (1996). Artificial infection of rotifer (Brachionus plicatilis) with WSSV The first primer pair was 5’-ATCATGGCTGCTTCACAGAC- WSSV was prepared from a stored, deep frozen (-80˚C freezer) and 5’-GGCTGGAGAGGACAAGACAT-3’ with a product tissue of a P. monodon infected with WSSV. One gram of in- fragment of 982 bp. Nested-step PCR was conducted when fected shrimp gill was macerated with phosphate buffered sa- negative result is found from the first-step PCR using the fol- line (PBS, pH 8.2) and passed through a cellulose filter (0.45 lowing primer pairs 5’-TCTTCATCAGATGCTACTGC- 3’ µm, Millipore, USA). The resulting filtrate was centrifuged at and 5’-TAACGCTATCCAGTATCACG- 3’ which amplified a 10,000 xg at 4˚C for 10 minutes (Eppendorf, Germany) before 570 bp fragment. The PCR products were detected by electro- being injected intramuscularly into the 2nd or 3rd pleopod of phoresis of 10 µL of the reaction solution in 1.5% agarose gel apparently healthy shrimp (1.02 g, WSSV- negative). Antibiotic for 30 minutes. This was stained in 1 µg/mL ethidium bromide or antimycotic treatment was omitted as the filter can effective- solution for 5 minutes and de-stained in DDW for 10 minutes. ly exclude bacteria into the filtrate. Dead shrimp with evident After destaining, the gel was viewed under UV trans-illuminator white spots in the carapace of the cephalothorax area were col- (Syngene) to check DNA amplification bands positive for WSSV. lected, confirmed by PCR for WSSV infection and stored at Rotifer resting egg collection -80˚C. Viral inoculum used in the rotifer infection experiment was prepared in a similar manner as described above in the Rotifer resting eggs were collected from sediment samples of preparation of WSSV inoculum for shrimp infection. a shrimp farm in Leganes, Iloilo, Philippines that had a recent Newly-hatched rotifers were collected in a 1-L beaker. The ro- incident of WSSV outbreak. Following the method of Aparici et tifer stock was then divided into two containers with a density al (2001), deep sediment cores each measuring 10 cm in length of 500 ind mL-1 each as the infected and non-infected stock. For were taken by a core sampler and placed in sterile plastic bot- the infected stock rotifers (positive control), 5 mL of the filtered tles for analysis. Collected sediments were dried at room tem- WSSV supernatant was added to the first container and aerated perature, sieved through a 25 μm mesh net and particles passing for 2 hours. For the non-infected rotifers (negative control), the the sieved were collected. After sieving, three composite sam- 3 other container was added with 5 mL PBS. The two containers ples of 10 cm each were taken to which 1.75M sucrose solu- were covered with foil to prevent contamination. After 2 hours, tion was added to make a 40-mL final volume. The soil-sugar rotifers were collected and washed three times with sterile sea- mixture was centrifuged for 5 minutes (600 rpm). The result- water (SSW). Sterile seawater was prepared by autoclaving for ing supernatant was decanted and placed in sterile Petri dish 15 minutes at 15 psi. Samples for both infected and non-infected where rotifer resting eggs were collected using a capillary tube. rotifers were collected for PCR detection. Collected eggs were also checked by one-step PCR to confirm the presence of WSSV in these diapause eggs. Pathogenicity and transmission of WSSV from infected rotifers to P. monodon larvae Destruction of rotifers resting eggs using industrial disinfectants Three commercially available industrial disinfectants or bioc- To assess the transmission and pathogenicity of WSSV from ides such as commercial bleach sodium hypochlorite (NaOCl), rotifers to shrimp, 200 P. monodon postlarvae (PL 21, 12.75 granulated calcium hypochlorite (Ca(OCl) ) and formalin (H CO) mm) were equally distributed in ten 5-L round glass aquarium 2 2 were tested on their efficacies to destroy rotifer resting eggs at at a density of 20 post larvae per aquarium. The experiment concentrations of 1, 5 and 10 mg L-1. was conducted in two experimental treatments consisting of These biocides were first diluted with sterile sea water (SSW) shrimp co-cultured with WSSV-negative rotifers and shrimp to obtain the desired concentration. This experiment was con- co-cultured with WSSV infected rotifers. Each treatment was ducted using a 12-well micro titer plate. Five resting eggs were replicated five times. Seawater used (28.8˚C, 32 ppt) was first inoculated per well and triplicate run was performed on each passed through a sand filter then to a UV lamp for disinfec- treatment. After 24 hours, viability of the resting eggs was mon- tion. Aeration was provided and each aquarium was covered itored and expressed as percent disruption. with foil to prevent cross-contamination. Monitoring was done twice daily with dead and moribund larvae collected and Statistical analyses stored in -80˚C. Mortalities were pooled and expressed as % Data from the non-viability experiment computed in percent- cumulative mortality. After 9 days, the remaining shrimp were ages were first transformed to arcsin prior to testing for one- collected and processed for PCR analysis. The experiment was way analysis of variance (ANOVA). The Duncan’s Multiple conducted at the Infection Building of SEAFDEC-Aquaculture Range Test was conducted to test differences between means. Department, Tigbauan, Iloilo. All statistical analyses were done using SPSS 10.0. WSSV detection by PCR Samples were collected and DNA was extracted following the Results and Discussion procedures of Caipang et al (2011). Tissue sample was homog- White spot syndrome virus (WSSV) is a devastating disease enized and centrifuged for 15 minutes at 12,000 rpm (4˚C). threatening the sustainability and economic viability of shrimp The supernatant was collected, added with 100% ethanol and culture. In the previous decades, collective knowledge in under- concentrated by centrifugation for 10 minutes at 12,000 rpm standing the biology and pathogenecity of this virus on shrimp (4˚C). The supernatant was decanted and the resulting DNA have significantly increased, but were not enough to develop a o pellet was solubilized in 1x TE buffer and stored at -20 C for technology for the full control of WSSV outbreaks in crustacean subsequent PCR analyses.

AACL Bioflux Volume 5 | Issue 2 Page 65 http://www.bioflux.com.ro/aacl Corre et al 2012 culture. Recently there is a growing interest in understanding the mechanism and ecological dynamics of viral survival in the aquatic ecosystem. Recurrence of viral outbreaks are common in shrimp ponds that have undergone thorough preparations that includes drying, liming and bottom soil desiccation, conditions considered unfavorable to virus survival. These observations led to a hypothesis of the existence of a non-crustacean host that may serve as a reservoir of WSSV in an aquatic environment. However, information on other organisms, serving as a host for WSSV had been few. Recent works indicate that rotifers, a common constituent of the shrimp pond zooplankton community could be a host and a reservoir of WSSV. The rotifer, Brachiounus urcerus and their resting eggs collected from sediments of a shrimp farm strick- en with WSSV in China were found to be positive with WSSV (Yan et al 2004). Results of the present study are in agreement Figure 1. Mean cumulative mortality per day of penaeid shrimp post with these findings. In the present study, WSSV syndrome virus larvae exposed to WSSV fed with WSSV negative or positive rotifers was detected in the rotifer, Brachiounus plicatilis after expo- Brachionus plicatillis sure to tissue homogenates of WSSV infected shrimp. Further, diapause eggs of this rotifer species were also found to harbor WSSV, confirming the previous evidences that rotifers is a host and a vector in the transmission of WSSV. Additional evidence was also shown that rotifer cellular membrane contains receptors for the binding of WSSV (Yan et al 2007b). Binding to cellular receptors is the initial step in the process of viral entry to the host cell. Transmission and infectivity of WSSV from infected rotifers to a crustacean hosts were documented by Yan et al (2007) and Zhang et al (2006). The work of Zhang et al (2006) demonstrated that WSSV from the rotifer, Brachionus urcerus is highly infective but were not pathogenic to the larvae of Penaeus chinensis. In the present study, WSSV transmitted from infected Figure 2. Detection of WSSV in hatched rotifer samples and shrimp postlarvae (PL) by two-step PCR result: M, Marker; Lane 1, positive rotifers, Brachionus plicatilis to post-larval P. monodon were control; Lane 2 rotifer (negative); Lane 3, shrimp (negative); Lane 4, found to be highly pathogenic. These contrasting findings are rotifer (positive); Lane 5, shrimp (positive); Lane 6, negative control not yet fully understood, but this could be due to the differences in the species of rotifer host, species of shrimp and the differ- Highest percentage (100%) of disruption was obtained by using ent shrimp larval stages used in these experiments. To the best granulated chlorine at higher concentrations (5 and 10 mg L-1). of our knowledge, the present study is the first to document However, this was not significantly different if bleach solutions the high pathogenicity of WSSV transmitted from rotifers to P. at 5 mg L-1 (96%) and 10 mg L-1 (86%) were used. On the oth- monodon post larvae. er hand, the percentage of resting eggs disruption by formalin Rotifers immersed in a WSSV inoculum prior to feeding to at 5 to 10 mg L-1 was comparable with that of bleach at all test shrimp (PL 21) showed higher cumulative mortalities (81% in concentrations. At 1 mg L-1 of formalin, lowest percentage of test as opposed to 45% in negative control) than shrimp fed with disruption was observed. uninfected rotifer (Figure 1). While this was not shown in the Chlorine, commonly applied as sodium hypochlorite and gran- first-step PCR, a further test using nested PCR analysis showed ulated chlorine, is one of the arthropod pesticides along with that rotifers exposed to the WSSV inoculum and shrimp larvae trichlorophon that is used extensively for the control of zoo- fed with the infected rotifers were WSSV-positive (Figure 2). planktons in shrimp farms (Funge-Smith & Briggs 1998; Schuur One-step PCR detected WSSV in pre-disinfected samples showed 2003). At dosages as low as 1 ppm, it can significantly increase that 4 out of 9 replicates were WSSV-positive with a band size the hatching rate of B. plicatilis resting eggs after 1 hour of ex- of 211 base pairs (Figure 3). Detection of WSSV using one-step posure due its action on the cyst covering and the elimination PCR of pooled samples indicates the high titer of the virus in of bacterial contaminants (Balompapueng et al 1997). However, the resting egg samples. As pooled samples were used, one or at higher concentrations, it renders the resting egg non-viable more of the 9 resting eggs used may be WSSV-positive. due to oxidation of live tissue (Gray et al 2006). For Artemia Surface disinfection was initially used to further verify whether sp., 90% of the isolated cysts became non-viable after expo- the virus is also present within the egg or just along the surface. sure to 53 ppm of hypochlorite solution for 24 hours (Sano et However, disinfectants used at high concentrations in this study al 2004) while 500 to 1000 ppm was required to prevent hatch- were found to make the eggs non-viable. All three disinfectants ing of the diapausing invertebrate eggs in a non-ballasted ship or biocides tested were effective in destroying the rotifer rest- (Gray et al 2006). ing eggs after 24 hours (Figure 4).

AACL Bioflux Volume 5 | Issue 2 Page 66 http://www.bioflux.com.ro/aacl Corre et al 2012

Figure 3. Detection of WSSV in pre-disinfected samples containing 9 resting eggs by one-step PCR result: Lane 1-9, rotifer samples, +, positive control; -, negative control; M, marker

industrial disinfectants already used to disinfect shrimp ponds can control the resting egg stage of the rotifers, thus reducing the risk of WSSV outbreak. However, proper disinfection strategies in shrimp farms should also include pond sediments and more studies are recommended in this area.

Acknowledgements

We would like to thank UP Visayas for funding this research project and to SEAFDEC-AQD for allowing us to use their research facilities. Special thanks also go to Ms. Mila Castanos for correcting this manuscript. Figure 4. Mean non-viable resting (Brachionus plicalitis) eggs after 24 h treatments with three disinfectants References

In this study, the range of effective concentration (5-10 mg/L) Aparici, E., Carmona, M. J., Serra, M., 2001. Variability for mixis obtained was lower but was within the range (1-30 mg/L) of initiation in Brachionus plicatilis. Hydrobiologia 446/447:45-50. dosages typically used in the pre-stocking treatment of shrimp Caipang, C.M.A., Aguana, M.P.N., 2011. Conventional PCR assays farms (Boyd 1995; Gräslund & Bengtsson 2001). The different for the detection of pathogenic Vibrio spp. in shrimp aquacul- effects of the high dosage concentrations on the resting eggs ture in the Philippines. Aquaculture, Aquarium, Conservation & can be attributed to the methods used in the isolation process. Legislation 4:339-350. Generally, the presence of sediments would require higher con- Chou, H.Y., Huang, C.Y., Wang, C.H., Chiang, H.C., Lo, C.F., 1995. centration of hypochlorite than just the isolated resting eggs as Pathogenicity of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan. Diseases of Aquatic the sediments present a physical barrier to the action of the dis- Organisms 23:165-173. infectant. It also generates a high chlorine demand (Sano et al Balompapueng, M.D., Munuswamy, N., Hagiwara A., Hirayama, K., 2004). Hence, results of this study may underestimate the ef- 1997. Effect of disinfectants on the hatching of marine rotifer rest- fective dose that is applicable in farm conditions. At the farm ing eggs Brachionus plicatilis Muller. Aquatic Research 28:559-565. scale, its efficacy may not be replicated as sediments present Boyd, C.E., 1995. Chemistry and efficacy of amendments used to treat numerous matrices where the resting eggs may reside; hence, water and soil quality imbalance in shrimp ponds. In: Bowdy, C.L., decreasing the contact between the disinfectant and the eggs. Hopkins, J.S. (eds.) Swimming through troubled water. Proceedings Further tests using this disinfectant along with other manage- of the special session on shrimp farming, Aquaculture ’95. World ment practices must be done in the field to be able to obtain Aquaculture Society, Baton Rouge, Louisiana. relative success in its application. Eckmann, R., 1985. Histopathological alterations in the intestine of whitefish Coregonus sp. larvae reared on zooplankton from Lake Conclusion Constance. Diseases of Aquatic Organisms 1:11–17. Gräslund, S., Bengtsson, T.S., 2001. Chemicals and biological products used in south-east Asian farming, and their potential impact on Results of this study suggest that the rotifer, Brachionus plica- the environment-a review. The Science of the Total Environment tilis is a vector for the transmission of WSSV in P. monodon 280:93-131. and the WSSV from infected rotifers is highly pathogenic to Gray, D., Duggan, I., MacIsaac, H.J., 2006. Can sodium hypochlorite post-larval stage of this shrimp. Also, commercially available reduce the risk of species introductions from diapausing invertebrate eggs in non-ballasted ships? Marine Pollution Bulletin 52:689-695.

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Lightner, D.V., 1996. A handbook of shrimp pathology and diagnostic Strinyalucksana, A.K., Srisala, J., McColl, K., Nielsen, L., Flegel, T.W., procedures for diseases of cultured penaeid shrimp. World Aquaculture 2006. Comparison of PCR testing methods for white spot syndrome Society, Baton Rouge, LA. p 305. virus (WSSV) infections in penaeid shrimp. Aquaculture 255:95-104. Lightner, D.V., Redman, R.M., Poulos, B.T., Nunan, L.M., Mari, J.L., Venegas, C., Nonaka, L., Nushiake, K., Shimizu, K., Nishizawa, T., Hasson, K.W., 1997. Risk of spread of penaeid shrimp viruses in Muroga, K., 1998. Pathogenicity of penaeid, rod-shaped DNA vi- the Americas by the international movement of live and frozen rus (PRDV) to Kuruma prawn in different developmental stages. shrimp. Revue Scientifique et Techique (International Office of Fish Pathology 34:19–23. Epizootics) 16:146-160. Tanasomwang, V., Muroga, K., 1988. Intestinal microflora of larval Lightner, D.V., Hason, K., White, B., Redman, R., 1998. Experimental and juvenile stages in Japanese flounder (Paralichthys olivaceus). infection of western hemisphere penaeid shrimps with Asian white Fish Pathology 23(2):77–83. spot syndrome virus and Asian yellow head virus. Journal of Aquatic Wang, Y.C., Lo, C.F., Chang, P.S., Kou, G.H., 1998. Experimental in- Animal Health 10:271–281. fection of white spot baculovirus in some cultured and wild deca- Lo, C.F., Ho, C.H., Peng, S.E., Chen, C.H., Hsu, C.H., Chiu, Y.L., pods in Taiwan. Aquaculture 164:221–231. Chang, C.F., Liu, K.F., Su, M.S., Wang, C.H., Kou, G.H., 1996. Yan, D.C., Dong, S.L., Hang, J., Yu, X.M., Feng, M.Y., Liu, X.Y., 2004. Infection of white spot syndrome baculovirus (WSBV) detected in White spot syndrome (WSSV) detected by PCR in rotifer and roti- cultured and captured shrimps, crabs and other arthropods. Diseases fer resting eggs from shrimp pond sediments. Diseases of Aquatic of Aquatic Organisms 27:215–225. Organisms 59:69–73. Magbanua, F.O., Natividad, K.T., Migo, V.P., Alfafara, C.G., de la Pena Zhang, J.S., Dong, S.L., Tian, X.L., Dong, Y.W., Liu, Y.W., Yan, D.C., F.O., Miranda, R.O., Albaladejo, J.D., 2000. White spot syndrome 2006. Studies on the rotifer (Brachionus urceus Linnaeus, 1758) virus (WSSV) in cultured Penaeus monodon in the Philippines. as a vector in white spot syndrome virus (WSSV) transmission. Diseases of Aquatic Organisms 42:77-82. Aquaculture 261:1181-1185. Marcus, N.H., 1989. Abundance in bottom sediments and hatching requirements of eggs of Centropages hamatus (Copepoda: Calanoida) from the Alligator Harbor Region, Florida. The Biological Bulletin Authors (Woods Hole)176:142-146. •Valeriano Corre, Jr, Institute of Aquaculture, College of Fisheries Natividad, K.D., Nomura, N., Matsumura, M., 2008. Detection of white and Ocean Sciences, University of the Philippines in the Visayas, spot syndrome virus DNA in pond soil using a 2-step nested PCR. Miag-ao, Iloilo 5023 Philippines, email: [email protected]. Journal of Virulogical Methods 149:28-34. •Joseph Faisan, Jr. Aquaculture Department, Southeast Asian OIE, 2006. Manual of Diagnostic Tests for Aquatic Animals (5th ed.). OIE, Paris. (http://www.oie.int/eng/normes/fmanual). Fisheries and Development Center, Tigbauan, Iloilo 5021 Philippines, email: [email protected]. Perez, F., Volckaert, F.A.M., Calderon, J., 2005. Pathogenicity of white spot syndrome virus on postlarvae and juveniles of Penaeus •Rhea Joy Carton-Kawagoshi. Laboratory of Applied Phycology, (Litopenaeus) vannamei. Aquaculture 205:586–591. Department of Aquatic Biosciences, Tokyo University of Marine Pramod Kiran, R.B., Rajendran, K.V., Jung, S.J., Oh, M.J., 2002. Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108- Experimental susceptibility of different life-stages of the giant 8477 Japan, email: [email protected]. freshwater prawn, Macrobrachium rosenbergii (de Man), to white •Bessie J. Elle. Brackishwater Aquaculture Center, Institute spot syndrome virus (WSSV). Journal of Fish Diseases 25:201–208. of Aquaculture, College of Fisheries and Ocean Sciences, Sano, L.L., Mapili, M.A., Krueger, A., Garcia, E., Gossiaux, D., Phillips, University of the Philippines in the Visayas, Leganes, Iloilo K., Landrum, P.F., 2004. Comparative efficacy of potential chemi- 5003 Philippines, email: [email protected]. cal disinfectants for treating unballasted vessels. Journal of Great Lakes Research 30:201–216. •Rex F. Traifalgar. Institute of Aquaculture, College of Fisheries Schuur, A.M., 2003. Evaluation of biosecurity applications for inten- and Ocean Sciences, University of the Philippines in the Visayas, sive shrimp farming. Aquacultural Engineering 28:3-20. Miag-ao, Iloilo 5023 Philippines, email: [email protected]. Skliris, G.P., Richards, Randolph, H., 1998. Assessment of the suscepti- •Christopher M. Caipang. BioVivo Technologies AS, Bodø, bility of the brine shrimp Artemia salina and rotifer Brachionus plica- Norway, email: [email protected]. tilis to experimental nodavirus infections. Aquaculture 169:133–141.

Corre, Jr, V., Faisan, Jr, J., Carton-Kawagoshi, R. J., Elle, B. J., Traifalgar R. F., Caipang, C. M., 2012. Evidence of WSSV transmission from the rotifer (Brachionus Citation plicatilis) to the black tiger shrimp (Penaeus monodon) postlarvae and means to control rotifer resting eggs using industrial disinfectants. Aquaculture, Aquarium, Conservation & Legislation 5(2):64-68. Editor Ştefan C. Vesa Received 26 April 2012 Accepted 21 May 2012 Published Online 08 June 2012 Funding In-house Research Fund of the UP Visayas Conflicts/ Competing None reported Interests

AACL Bioflux Volume 5 | Issue 2 Page 68 http://www.bioflux.com.ro/aacl Aquaculture, Aquarium, Conservation & Legislation OPEN ACCESS International Journal of the Bioflux Society Research Article

The influence of abiotic factors on suppliers of organic matter in the peloidogenesis process from Lake Techirghiol, Romania

1,2Liana M. Gheorghievici, 1Iulia Pompei, 1Gheorghe Gheorghievici, 1Iosif Tănase

1 National Institute of Rehabilitation, Physical Medicine and Balneoclimatology, Bucharest, Romania, EU; 2 Bucharest University, Faculty of Biology, Bucharest, Romania, EU.

Abstract. The purpose of the study was the determination of the current situation of the Techirghiol ecosystem which is unique in Romania because of its hypersaline character and because of the large productivity in the formation of the sapropelic mud with therapeutic properties. A number of 107 taxonomic units have been identified in the phytoplankton. Most of the algae species belong to the diatoms group, followed in descending order by representatives of Chlorophyceae, Dinophyceae, Euglenophyceae, Chrysophyceae, Cryptophyceae and Xantophyceae. The number of taxa identified in the phytoplankton varied inverse proportionally with the water mineralization degree, unlike the values of - 2- the phytoplanktonic biomass, that have risen along with the water salinity. The high concentrations of Cl and SO4 ions have been associated with a diverse phytoplankton and superior values of the microalgae biomass only in the year 2007. The report of the Na+> Mg2+> Ca2+ cations, generally valid for any sampling point, has created optimal conditions for the development of the phytoplankton, and especially the calciphi- + - lic alga Cladophora crystallina (Roth) Kütz. The concentrations of the nitrogen compounds (N-NH4 , N-NO3 ) have been sufficient for the insurance of the nutritional necessities of the phytoplankton, especially for the diatoms that have registered the highest diversity and the highest biomass along microalgae.

Key Words: salt, lake, algae, mud, biomass.

Rezumat. Scopul studiului a fost determinarea situaţiei actuale a ecosistemului Techirghiol, unic în România prin caracterul hipersalin şi prin marea productivitate în formarea nămolului sapropelic cu proprietăţi terapeutice. S-a identificat compoziţia fitoplanctonului din lacul Techirghiol în anii 2004, 2007, 2009 şi 2010 (prin evaluarea numărului de specii şi a biomasei lor) dar şi influenţa variaţiilor factorilor hidro- chimici asupra acesteia şi, implicit, asupra potenţialului peloidogenetic al acestui lac. În fitoplancton s-au identificat 107 unitaţi taxonomice. Majoritatea speciilor de microalge au făcut parte din grupul diatomeelor, urmate în ordine descrescătoare de Chlorophyceae, Dinophyceae, Euglenophyceae, Chrysophyceae, Cryptophyceae and Xantophyceae. Numărul taxonilor identificaţi în fitoplancton a variat invers proporţional cu gradul de mineralizare al apei, spre deosebire de valorile biomasei fitoplanctonice, care au crescut odată cu salinitatea apei. Concentraţiile - 2- mărite ale ionilor de Cl şi SO4 s-au asociat cu un fitoplancton diversificat şi cu valori superioare ale biomasei microalgelor doar în anul 2007. Raportul cationilor Na+> Mg2+> Ca2+, general valabil pentru orice punct de prelevare, a creat condiţii optime pentru dezvoltarea fitoplanctonu- + - lui şi, în special, a algei calcifile Cladophora crystallina (Roth) Kütz. Concentraţiile compuşilor cu azot (N-NH4 , N-NO3 ) au fost suficiente pentru asigurarea necesităţilor nutritionale ale fitoplanctonului, în special pentru diatomee care au înregistrat cea mai mare diversitate şi cea mai mare biomasă dintre microalge.

Cuvinte cheie: sărat, lac, alge, nămol, biomasă.

Corresponding Author: L. M. Gheorghievici, [email protected]

Introduction the benefit of mankind (Charlier & Chaineux 2009; Sava et al 2009; Silaghi et al 2011). The important role of algae in the salt water ecosystems is well Lake Techirghiol, a fluvial marine bank type of lake, situated known, they contributing to the biological balance and repre- in the south-east region of Romania, on the Black Sea littoral, senting the basis of the primary productivity in aquatic basins. enters the category of pelogenous lakes, in which mud with They are involved in water purification, mud formation and of- therapeutic properties is formed, used empirically at the begin- fer the oxygen needed in the process of respiration (Doroftei ning, in an unorganized mode, though gradually being proven et al 2010). Nowadays, many scientists from around the world the effect it has in the maintaining and improvement of health try to find solutions not only for the exploration, but also for (Gheorghievici 2010; Kerkmann 2010). the exploitation of this valuable marine flora and other ma- The catchment area of Lake Techirghiol is of 150 km2, the rine related natural resources in various activity domains, for water volume is 4.18 mil.m3 and the maximum depth 9.75 m. AACL Bioflux Volume 5 | Issue 1 Page 69 http://www.bioflux.com.ro/aacl Gheorghievici et al 2012 The water supply resources of the lake are ensured by both Techirghiol has been designated protected natural area of na- the superficial discharge and the underground water contribu- tional interest (Law 462/2001), Ramsar site (2006) and Water tion. It is also supplied by the shore springs (sources), which Direction Dobrogea has elaborated a management plan of the bring and amount of fresh water, active mostly at the back of Protected Area Lake Techirghiol 2008-2013 (www.scribd. the lake (Environment Report, Techirghiol City Hall, 2009). com⁄doc.⁄37636154⁄Draft-Management Plan of the Protected The lake water from Techirghiol is highly concentrated, bro- Area Lake Techirghiol 2008-2013). minated, chorinated, sodic, hypertonic (Testing Report no. As a consequence, such lakes as lake Techirghiol, require the 350/03.08.2010, I.N.R.M.F.B.). maintenance of some biological, chemical, limnogenetical, lim- The cenosis of this lake are poor as a number of species, but nogeological, morphometrical standards, which may be differ- rich as a number of individuals. Through the decomposition ent from the general recommedations for freshwater and sa- (mainly anaerobic) of the rich vegetal and animal biomass, the line lakes, but non-therapeutical (Bulgăreanu 1996; Poplăcean formation of salted peloid deposits is reached, which is used 2010), because it has been observed a differentiation between with success in balneotherapy (Godeanu 2002). the plankton, microbenthos, macrophytes and cormophytes spe- The purple water, described by Olga Bonciu in 1955 and 1957 cies present in the pelogenous lacustrine ecosystems (usually on the shore of Lake Techirghiol (Bonciu et al 1963) and I. therapeutic) and the non-pelogenous ones (Ionescu et al 1998; Ţuculescu in 1965, biotope that contained numerous sulfur Vincze et al 2011). autotrophic photosynthetic bacteria (species of Cromatium, Rhabdocromatium and Thiopolycoccus) and had represented Material and Methods one of the most productive factors in the genesis of the mud (Samson et al 1984), has disappeared. It was not present on the The study was conducted in the years 2004-2010 in the north entire surface of the lake, during the development period of the east side of lake Techirghiol with salt water, where peloido- study (2004, 2007, 2009, 2010). genesis process is intense, from three stations. A number of 45 The important phenomenon that interfered in the life of Lake samples of phytoplankton (5 parallel samples/ sampling point/ Techirghiol was either a concentration of the water due to the year) from surface layer (1m depth) were filtered with plank- physical and geographic particular conditions (being the only tonic net, Wisconsin type, n020. The concentrated samples littoral lake that does not communicate at the surface with the were introduced in plastic containers, preserved immediately Black Sea), or a decrease of the mineralization of the water with Lugol solution (a proportion of 3 mL of fixing solution and even the mud, due to the inflow of fresh water in the lake. for 1000 mL of sample), and then labeled. The algological ma- The variations of the mineralization level of the lake water, the terial was analyzed on a Fuchs-Rosenthal chamber; for accu- imbalances from the ecosystem induced by the anthropic fac- racy, three duplicate counting were made in chamber (both the tor through the uncontrolled utilization of the north part of the upper and the lower chamber) for each sample of phytoplank- lake with salted water, for bathing and those provoked by the ton. Algal species were determined in accordance with Van abiotic factors (precipitation, wind, discharge of fresh water), Landingham (1982), Anagonstidis & Komárek (1988), Kramer have diminished the pelogenous potential of the lake through & Lang-Bertalot (1988), Sládeček (1989), Prescott et al (1996), the decrease of the biomass contribution of the main providers Godeanu (2002), Pârvu (2003), Sigee (2005), Anastasiu (2008), of organic materia for the formation of mud, Cladophora crys- Hamed (2008), Zgrundo et al (2009). tallina (Roth) Kütz and Artemia salina L. For the estimation of the microalgae biomass, the individual 3 -3 In 1931, when macrophytic alga Cladophora has disappeared, volumes of cell (µm ) and biomass as wet weight (mg•dm ) due to the water salinity of 110 g•dm-3 and the water densi- for each species were calculated according to approximate geo- ty that has decreased from 1.075-1.076 g•cm-3 to 1.060-1.065 metrical figures (Hillebrandtet al 1999). g•cm-3, the pelogenous capacity of lake Techirghiol has been Macrophytes were sampled after transects method (I.E.P, Wiser 2 questioned, although Paul Bujor sustained that, in lack of spe- D3.2-1, 2009) using 0.25 m enclosure system. Biomass dry cies of the Cladophora type, and therefore of the organic sub- weight of 120 samples (20 sample/ transects; 3 transects/ sam- stance that can produce hydrogen sulfide through its putre- pling point) was determined after filamentous thalli were dried faction, mud can still be formed through the reduction of the at 75°C for 48 hours (Sfriso et al 1991) and weighed. The abun- sulfates (Bujor 1928). dance for each taxon was calculated as the number of cell per On the other side, the utilization of the sulfur sapropelic mud, cubic decimeter. highly hydrated, with aqueous, chlorinated, sodic magnesiac so- Samples for the hydrochemical analysis have been taken in the lution (Analysis Report I.N.R.M.F.B./05.10.2010), in the treat- same time and from the same locations as the phytoplankton ment of different diseases (rheumatism diseases, gynecological samples; the pH has been determined electrometrically, with diseases, diseases of the central and peripheral nervous system, pH primary standard solutions, the dissolved oxygen has been determined through the Winkler method, and the SO 2- ion by respiratory diseases etc), which is used in Techirghiol Balneary 4 Sanatorium and also in other balneary stations from Romania gravimetric method with barium chloride solution in acid me- (Bavaru et al 2011), has lead to the graduate depletion of the dium (HCl). The chlorine concentration has been determined through the volumetric method with AgNO , in the presence mud reserves and also to the modification of the characteristics 3 of the ferric alum indicator, in acid medium (HNO ), the Ca2+ of the ecosystem that generates it. 3 2+ + For the conservation of the aquatic ecosystem Techirghiol and and Mg ions through complexometric method, and Na flame- photometric (JENCONS spectral device). HCO has been de- the assurance of the continuity of the necessary conditions for 3 the achievement of the peloidogenesis process, and also for termined volumetric, in the presence of metilorange indicator. the regeneration of the used mud returned in the lake, actions For the determination of the pollution microbiological indica- have been initiated for the protection and improvement of the tors water samples have been taken in sterile recipients, ac- ecosystem through legislative and institutional measures; lake cording to SR EN ISO 19458:2007; the membrane filtration AACL Bioflux Volume 5 | Issue 2 Page 70 http://www.bioflux.com.ro/aacl Gheorghievici et al 2012 method has been utilised according to SR EN ISO 6222:2004 1976-1982 (Trică 1988), when there have been determined val- for the identification of the total number of bacteria that devel- ues of 109-1010 order. There is a similarity between the present op at 37°C, SR EN ISO 9308-1:2004 for the coliform bacteria and anterior data (Ţuculescu 1965) only for the biotop-water and Escherichia coli, SR EN ISO 7899-2:2002 for the intesti- shore with green algae. nal enterococci. Results were expressed as colony forming units The Cladophora population from lake Techirghiol has its ori- (CFU) per 1 cm3 or 100 cm3 of water. gins in the marine flora of the golfs that gave birth to the littoral lake with a salinity of de 14-16 g•dm-3, flora which was Results similar to the one existing in the Black Sea. As a consequence of the intense evaporation and the semi-arid climate, the salin- The structure of the phytoplankton in lake Techirghiol in the ity of lake Techirghiol grew a lot because of the accumulation years 2004, 2007, 2009 and 2010, has been diverse, a number of mineral salts, macrophytic green alga Cladophora passing of 69 taxa being identified and organised in 8 classes (Table through an adaptation process. 2). Diatoms (Bacillariophyceae) present the highest diver- At first, theCladophora population has diminished its biomass, sity (63 taxa), being followed by Chlorophyceae (23 taxa), until the year 1931 when it totally dissapeared, in the same year, Cyanobacteria (21 taxa), Dinophyceae (9 taxa), Euglenophyceae the lake water salinity reaching record values of 110 g•dm-3. (6 taxa), Chrysophyceae (3 taxa), Cryptophyceae (2 taxa), and In the year 2007, we have calculated a medium dry biomass of Xantophyceace being represented by only one taxon. the Cladophora species in the Sanatorium point of the littoral Diatoms and Cyanobacteria were the groups with the highest zone of 536.2 g•m-2 , and in the year 2009, also in the littoral diversity from the phytoplankton. From the Bacillariophyceae, zone but in the Boathouse point, the medium dry biomass of the Pennatae subclass was represented in lake Techirghiol by 13 the Cladophora was of 328.6 g•m-2 (see Table 4). species of the Nitzschia genus, and 10 species of the Navicula The most representative anions for lake Techirghiol are Cl- and genus. 2- SO4 , the concentration of the chlorine ion has varied between The Pinnularia, Surirella, Amphora, Achnanthes, Cocconeis the interval 30.77 g•dm-3- 34.38 g•dm-3, from one sampling point genera have been identified with three species each, Rhopalodia, to another, demonstrating a good homogenity on an horizontal Stauroneis, Gyrosigma, Fragilaria, Cymbella with two species gradient (see Table 5). each, and Rhoicospaenia, Synedra, Amphiprora, Gomphonema, 2- The SO4 has registered a maximum concentration in the Tower Diploneis, Caloneis have included only one species. From TV sampling point from the central part of the lake, its concen- the subclass Cetricae, only three types have been identified: tration variations being a result of the distribution mode of the Thalassiosira, Melosira with two species and Cyclotella with organic matter and the densities of the bacterial populations in one species. the pelogenous zone of the lake. From the Chlorophyceae class, 27 genera have been identified in The HCO3 ions proves the afflux of fresh water in lake Techirghiol; the plankton, most of them being represented by only one species it has presented an unspectacular variability on all three sam- (Asterococcus, Carteria, Chlamydomonas, Dictyosphaerium, pling locations, determined by the biological processes. Dunaliella, Gonium, Keratococcus, Oocystis, Pediastrum, The main cations (Na+, Ca2+, Mg2+) have manifested a limited Scenedesmus, Schroederia, Selenochloris, Ulotrix, Urospora), dynamics of the concentrations in the analyzed sampling points, an exception being made by Chlorella and Cladophora, which following the one of the anions to which it relates. have included two species. The Enteromorpha genus has proven + The distribution of the ammonium nitrogen (N-NH4 ) shows a maximum diversity in the green algae groups, and was present in reduced spatial or temporal variability; in Tower TV sampling the plankton of lake Techirghiol by three species (see Table 1). point, in the year 2010, even becoming analytically undetect- For the determination of time and space variability of the struc- able. In reverse, the concentration of the nitrate nitrogen (N- ture of the phytoplankton, the Shannon index and the Margalef - NO3 ) varied more in time (with maximum values in 2010), and index have been calculated; the evaluation of the variability less in space (except for the year 2007). of the phytoplankton’s structure from Lake Techirghiol is pre- The dissolved oxygen presented a concentration over 5.00 sented in Table 2. mg•dm-3 in the sampling point from the central part of the lake, The numeric abundance of the microalgae in the years 2004, in inferior values in the littoral sampling points Sanatorium 3 6 2007, 2009, and 2010 has varied between 1.1•10 and 1.9•10 and Boathouse, where there are bathing areas and numerous -3 4 -3 cells•dm (with a medium value of 8.3•10 cells•dm (see Table clumps of Cladophora. The oxygen saturation of the lake wa- -3 3), and the wet biomass from 0.002 to 10.4 mg•dm (medium ter has registered values under 50% in the years 2004 (37.55%) -3 value being 1.23 mg•dm ) (see Table 4). and 2007 (26.32%) and above 50% in the years 2009 (51.52%) The most frequently identified species in the phytoplankton and 2010 (62.84%). from lake Techirghiol between the years 2004-2010 have been Fragilaria ulna (Nitzsch) Lange-Berthalot (52.48% in the Sanatorium prelevation point, in 2010), Amphora coffeaformis Discussion var. acutiuscula (Kütz) Hustedt (51.67% in the Boathouse prel- It can be concluded that the pronounced prevalence of the di- evation point, in 2009) and Navicula salinarum Grunow (40.57% atoms was typical for the phytoplankton of lake Techirghiol in in the Sanatorium prelevation point, in 2004). all of the studied years. In medium, the Bacillariophyceae have The microalgae density has registered values in the interval contributed with 54.08% to the total specific diversity, their prev- 1.8•104 (2010) – 1.9•106 (2007) cel•dm-3; these values are close alence being maximum (70%), in June 2007, and a bit reduced to the ones recorded in 1977 – 7.1•105 cel•dm-3 (Trică 1977), in the years 2010 (52.63%), 2009 (50.94%), and 2004 (45.76%). but inferior to the existing data in bibliography from the period

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Table 1a. The qualitative composition of the phytoplankton in Lake Techirghiol 2004, 2007, 2009, 2010.

May 2004 June 2007 June 2009 June 2010 Taxa S B S B S B S B T Euglenophyceae Euglena pisciformis Klebs + - + + - - - - + Eutreptia viridis Perty - - + + + - - - - Trachelomonas euristoma Stein - - + + - - - - - T. hispida (Perty) Stein em. Deflandre - + + + - - - - - T. incerta Lemmermann - - + + - - - - + T. regularis Skvortzov - - + + - - - - - Bacillariophyceae Achnanthes brevipes (Agardh) - - + + + - + - + A. lanceolata var. elliptica Cleve - - + + - - + - - A. longipes (Agardh) - - + + - + - - - Amphiprora paludosa W.Sm. - - + + - - - - - Amphora coffeaformis (Agardh) + - + + - - - + - A. coffeaformis var. acutiuscula (Kütz) Hustedt - ++ + + - +++ + + + A. commutata Grűnow - - + + + - + - - Caloneis amphisbaena (Bory) Cleve + ------+ - Cocconeis pediculus (Ehr.) - - + + + - + - + C. placentula (Ehrenberg) + - + + - - - - - C. scutellum (Ehrenberg) - - + + - - - - + Cyclotella meneghiniana Kützing - - + + + + - - - Cymbella gracilis (Rabenh) Cleve - - + + - - - + + C. ventricosa (Kützing) - - + + - - - - - Diploneis interrupta (Kütz.) Cleve - - + + - - + - + Fragilaria - - + + + - - + + fasciculata (Agardh) Lange-Berthalot F. ulna (Nitzsch.) Lange-Berthalot + - + + - ++ +++ ++ ++ Gomphonema angustatum (Kütz.) Rabenhorst - - + + - + - + - Gyrosigma spenceri W. Smith (Griffith & Henfrey) + - + + - + - + + G. strigile (W. Smith) Cleve - - + - - - - + - Melosira moniliformis (O. Müller) Agardh + - + + - - + + - M. moniliformis var. subglosa Grunow - - + + - - - - - Navicula cincta (Her.) Kütz + + + + - - + + + N. cryptocephala Kützing - - - - + + - + + N. exigua (Greg.) O.Müller - - + + - - - + - N. hennedyi W. Smith + + - + - - - - - N. menisculus Schumann - - + + - - + - - N. placentula (Ehr.) Grunow - - + + - - - + - N. placentula (Ehr.) Grunow var. rostrata A. Mayer - - + + ++ - - - + N. pupula Kützing - - + + + + - + - N. rhyncocephala Kütz. - - + + - - + - + N. salinarum Grunow ++ - + + - - - - + Nitzschia apiculata (Greg.) Grunow - - + + - - - - + N. acicularis Kützing Smith - - + + - + + + - N. closterium (Ehr.) W. Smith + - - + - - + - + N. distans Gregory - - + + - - - - +

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Table 1b. The qualitative composition of the phytoplankton in Lake Techirghiol 2004, 2007, 2009, 2010.

May 2004 June 2007 June 2009 June 2010 Taxa S B S B S B S B T N. lanceolata W. Smith - - + + - - - - - N. longissima f. parva Grunow - - + - + - + - - N. longissima var. reversa Grunow + + + + + + + + - N. lorenziana Grunow - + + - + - - + - N. recta Hantzsch - - + + - - - - + N. subtilis (Kütz.) Grunow + + ------N. tryblionella Hantzsch - - - - + + + + - N. tryblionella var. victoriae Grunow + + + ------Pinnularia microstauron (Ehr.) Cleve - - - + + + + + + P. microstauron var. ambigua Meister + + + + + + + - + P. viridis (Nitzch) Ehr. - - + - - - - - + Pleurosigma angulatum (Queck) W.Smith + + - - + - - - + P. angulatum var. strigosum (W.Sm.) Cleve - - - - + + - - - P. delicatulum W. Smith - - - + - - - - - P. elongatum W. Smith + + - - - - - + - Rhoicosphaenia curvata (Kütz.) Grunow + + - - - - - + - Rhopalodia gibba (Ehr.) O. Muller - + ------R. gibba var. ventricosa (Ehr.) Grunow + - - - - + - - - Stauroneis anceps (Ehr.) - - - + - - + - - S. salina W. Smith - + - + - + - - + Surirella ovata Kützing + + + + + - - - - S. spiralis Kützing + ------S. striatula Turp. - + - - - + - - - Synedra tabulata (C. Agardh) Kütz. + + + ++ + + + + + Thalassiosira excentrica (Grun.) Jorg - - + + - - - - - T. parva Pr.-Lavrenko - + - - + - - - - Chlorophyceae Asterococcus superbus (Cienkowski) Sherfell - - + + - - - + - Carteria multifilis (Fres.) Dill. - - - - + - - - - Chlamydomonas incerta Pascher - - + ------Chlorella ellipsoidea Gerneck - + + + - + - - - C. vulgaris Beyerinck - - + + + - - - - Cladophora crystallina (Roth) Kütz. + + - - - + + + - C. vagabunda (L.) Hoek + + + + + + + + - Dictyosphaerium ehrenbergianum Nagela - - - - + - - - - Dunaliella salina Teodoresco + + - - + + - - - Enteromorpha intestinalis (L.) Link - - - + - - + - - E. linza (L)J. Agardh - - + - + - - - - E. maeotica Pr.-Lavrenko + + + ------Gonium pectorale O. F. Műller - - + + - - + - + Keratococcus suecicus Hindak + - - - - - + - - Oocystis elliptica W. West + + - - + - - - - Pediastrum tetras (Ehrenberg) Ralfs - - + - - + - + - Rhizoclonium hieroglyphicum (Ag.) Kűtzing - + ------R. riparium (Roth) Kűtz. ex.Harv. + + + + - - - + - Scenedesmus quadricauda (Turp.) Brébisson + + - - - - + + + Schroederia setigera (Schröd.) Lemmermann + + - - - - + + -

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Table 1c. The qualitative composition of the phytoplankton in Lake Techirghiol 2004, 2007, 2009, 2010.

May 2004 June 2007 June 2009 June 2010 Taxa S B S B S B S B T Selenochloris bicaudata Pascher - - - - + + - - - Ulotrix zonata (Weber & Möhr) Kűtz. + + - - - - + - - Urospora penicilliformis (Roth) Areschoug + - - + - - + - - Chrysophyceae Chrysamoeba scherffelli (Pascher) Matvienko - - + + - - - - - Dendromonas cryptostylis Skuja + - + - - + - - - Desmarella moniliformis Kent + - - - + - - - - Cryptophyceae Chroomonas caudata L. Geitler - - - + - - - - - Chryptomonas ovata Ehrenberg - - - - - + + - - Xantophyceae Gloeobotrys chlorinus A. Pascher - - + - - + - - - Dinophyceae Amphidinium klebsii Kofoid et Swezy - - + + - - - - - Glenodiniopsis steinii Woloszynska - + + + - - + - - G. uliginosa (Schilling) Woloszynska - - - - + - - - - Gymnodinium eurytopum Skuja - + - + - - + - + G. fuscum (Ehr.) Stein + + ------Oxyrrhis marina Dujardin - - + - + + - - - Peridinium cinctum (O.F. Mull.) Ehr. + + - + + - + - + Woloszynskia leopoliensis Thompson - + + ------W. negleta (Schilling) Thompson + - - - - + - - - (-) = absent taxon; (+) = present taxon; (++) = abundant taxon (26-50%) ; (+++)= common taxon (51-75%)

S – Sanatorium sampling point ; B – Boathouse sampling point; T- Television tower sampling point

Table 2. Time (2004, 2007, 2009, 2010) and space (three sampling points) variability of phytoplankton structure in Lake Techirghiol Spatial Temporal Structure of phytoplankton variability variability Average number of species per sample 36 58 Number of species present in all 75 2 sampling points Number of samples containing all species 0 0 Figure 2. Variation of phytoplankton structure in lake Techirghiol Margalef Index 7.23-7.79 4.54-11.03 depending on Cl- and SO42- concentrations (number of taxa, biomass) Shannon Index 2.40-2.64 1.65-3.39

Figure 3. Variation of phytoplankton structure in lake Techirghiol Figure 1. Relationship between structure of phytoplankton and depending on the dissolved oxygen (number of taxa, biomass) mineralization of the lake Techirghiol

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Table 3. Numerical abundance percentage of phytoplankton taxa in Lake Techirghiol

Taxa May 2004 June 2007 June 2009 June 2010 S B S B S B S B T Euglenophyceae 2.29% 0.88% 2.09% 1.77% 0.28% 0.00% 0.00% 0.00% 1.04% Bacillariophyceae 90.88% 48.28% 93.43% 95.13% 95.80% 98.12% 97.37% 98.35% 97.12% Chlorophyceae 3.98% 8.74% 2.10% 1.58% 2.52% 0.93% 1.30% 1.65% 0.52% Chrysophyceae 1.14% 0.00% 0.52% 0.23% 0.28% 0.19% 0.00% 0.00% 0.00% Cryptophyceae 0.00% 0.00% 0.00% 0.23% 0.00% 0.19% 0.26% 0.00% 0.00% Xantophyceae 0.00% 0.00% 0.27% 0.00% 0.00% 0.19% 0.00% 0.00% 0.00% Dinophyceae 1.71% 5.23% 1.6% 1.12% 1.12% 0.38% 1.04% 0.00% 1.32% Total 100.00% 100.00% 100.00% 100.00% 100.00% 100.00 % 100.00% 100.00% 100.00% S - Sanatorium sampling point ; B - Boathouse sampling point; T - Television tower sampling point Table 4. Dry biomass(g•m-2) of the species Cladophora crystallina (Roth) Kütz

Sanatorium June 2007 Distance Transects 1/1 Transects 1/2 Transects 1/3 from shore 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 7.5 m 540 823.1 820.2 669.2 478.2 613.4 802.1 456.4 702.4 334.2 678.4 565.3 645.8 334.2 778.4 15 m 742.7 567.2 965.2 778.2 468.6 312.1 545.2 789.3 376.9 763.9 873.2 878.3 489.2 678.9 367.5 22.5 m 426.9 647.2 552.1 832.9 582.1 577.5 387.9 396.2 531.8 584.3 484.1 484.1 614.7 476.4 594.7 30 m 317.4 330 432.1 328.1 301.6 341.2 367.3 449.6 256.4 252.7 524.6 224.6 164.9 587.4 284.5 Average 506.7 591.9 692.4 652.1 457.6 461.1 525.6 522.9 466.9 483.8 640.1 538.1 478.6 519.2 506.3 Boathouse June 2009 Distance Transects 1/1 Transects 1/2 Transects 1/3 from shore 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 7.5 m 367.4 389.4 424.5 334.7 556.2 467.9 642.8 285.2 445.7 461.8 556.9 641.2 423.1 478.9 167.3 15 m 456.3 256.3 412.6 389.3 184.9 456.9 293.5 676.4 579.3 223.1 117.4 289.3 165.2 445.6 189.7 22.5 m 189.3 229.4 256.2 474.2 371.1 443.8 467.2 332.1 233.6 541.4 367.9 373.2 273.2 124.2 245.7 30 m 167.3 187.4 131.9 94.6 45.8 188.3 161.1 412.9 352.7 88.1 152.7 465.6 343.7 112.5 117.8 Average 295.1 265.6 306.3 323.2 289.5 389.2 391.2 426.6 402.8 328.6 298.7 440.1 301.3 290.3 180.1

Table 5. Physico-chemical parameters of water from lake Techirghiol ( mean ±S.D) (30-50 cm depth) ( INRMFB analysis bulletins)

Sampling station Parameter S B T pH 8.38(±0.16) 8.35 (±0.18) 8.14 (±0.06) Cl-1 (mg·dm-3) 31172.52 (±3178.19) 33588.43 (±6148.84) 30768.77 (±3724.94) - -3 N-NO3 (mg·dm ) 6.7 (±2.01) 7.6 (±2.20) 8.76 (±1.12) - -3 HCO3 (mg·dm ) 668.03 (±15.72) 694.23 (±7.26) 658.32(±19.95) 2- -3 SO4 (mg·dm ) 6548.45 (±963.85) 6723.52 (±947.20) 7867.8 (±243.45) Na1+ (mg·dm-3) 20107.68 (±2097.11) 21598.48 (±2704.37) 20802.83 (±1042.13) Ca2+ (mg·dm-3) 330.8 (±92.35) 356.05 (±99.71) 235.63 (±17.08) Mg2+ (mg·dm-3) 2472.12 (±84.92) 2679.35 (±19.07) 2498.87 (±)

+ -3 N-NH4 (mg·dm ) 1.8 (±0.35) 2.1 (±0.2) B.L.D. -3 Dissolved O2 (mg·dm ) 3.65 (±1.11) 3.9 (±0.56) 5.2 (±0.56) Mineralization (mg·dm-3) 62.66 (±5.32) 67 (±8.14) 70.95 (±7.55) S - Sanatorium sampling point ; B - Boathouse sampling point; T - Television tower sampling point

B.L.D. - below the limit of analitycal detection

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Table 6. Density and biomass variability of phytoplakton groups in lake Techirghiol in 2004, 2007, 2009, 2010 (means)

2004 2007 2009 2010 Density Biomass Density Biomass Density Biomass Density Biomass (cell·dm-3) w.s. (cell·dm-3) w.s. (cell·dm-3) w.s. (cell·dm-3) w.s. (mg·dm-3) (mg·dm-3) (mg·dm-3) (mg·dm-3) Euglenophyceae 2·103 0.02 6.5·103 0.8 2·103 0.08 absent absent Bacillariophyceae 2.45·105 6.8 1.89·106 10.4 7.88·104 4.7 9.14·103 5.7 Chlorophyceae 1.7·104 1.3 1.1·104 1.8 3.7·103 0.88 6.3·103 1.2 Chrysophyceae 2·103 0.17 3·103 0.34 2·103 0.09 absent absent Cryptophyceae absent absent 3·103 0.02 3·103 0.01 2·103 0.008 Xantophyceae absent absent 2·103 0.006 2·103 0.002 absent absent Dinophyceae 1.3·103 0.03 5.7·103 0.08 3.7·103 0.02 1.1·103 0.01 Total 2.79·105 8.32 1.9212·106 13.446 9.52·104 5.782 1.854·104 6.918 w.s.= wet substance

Table 7. Microbiological indicators of pollution levels in Techirghiol lake ( 30-50 cm depth)

Pollution indicator May 2004 June 2007 June 2009 June 2010 bacteria S B S B S B S B T Mesophilic aerobic >100 CFU / >100 CFU / >100 CFU/ >100 CFU/ >100 CFU >100 CFU 65 CFU / 23 CFU / 15 CFU/ bacteria to 37°C cm3 cm3 cm3 cm3 / cm3 / cm3 cm3 cm3 cm3 >100 CFU 47 CFU >100 CFU >100 CFU 109 CFU >100 CFU 121 CFU 69 CFU >100 CFU Coliform bacteria /100 cm3 /100 cm3 /100 cm3 /100 cm3 /100 cm3 /100 cm3 /100 cm3 /100 cm3 /100 cm3 172 CFU 47 CFU 0 CFU 0 CFU 0 CFU 0 CFU 0 CFU 0 CFU 0 CFU Escherichia coli /100 cm3 /100 cm3 /100 cm3 /100 cm3 /100 cm3 /100 cm3 /100 cm3 /100 cm3 /100 cm3 20 CFU 12 CFU 2 CFU 2 CFU 0 CFU 2 CFU 0 CFU 0 CFU 0 CFU Intestinal enterococci /100 cm3 /100 cm3 /100 cm3 /100 cm3 /100 cm3 /100 cm3 /100 cm3 /100 cm3 /100 cm3

CFU - colony forming units; S - Sanatorium sampling point ; B - Boathouse sampling point; T - Television tower sampling point

In descending order, come the green algae (Chlorophyceae), 2004, 2007), Dinophyceae (2009), and Cryptophyceae (2010) which have contributed with only 18.28% to the total spe- (see Table 6). cific diversity (recording a maximum of 22.03% in the year For the phytoplanktonic biomass from lake Techirghiol, bibli- 2004, intermediate values in the years 2009 (20.75%), 2010 ography data show increased values in the period 1976-1978; (22.00%), and a minimum of 12.63% in June 2007), and classes from the year 1978, microalgae had a reduced biomass con- Dinophyceae (7.39%), Euglenophyceae (3.50%), Chrysohyceae tribution to the sapropelic mud formation, until the year 1981. (2.33%), Cryptophyceae (1.16%) and Xantophyceae (0.78%). Starting with the year 1987, the phytoplanktonic biomass has The values of the Margalef index indicate a variability of the registered a new rising tendency, but never have they reached phytoplanktonic density, more pronounced in time then in space, the phytoplanktonic biomass values calculated in 1953 by I. and the diversity of the species from the phytoplankton identified Ţuculescu (see in 1965). through the Shannon index, varied also less from one sampling Not even the phytoplanktonic biomass values determinated in point to another and more from a year to another. the years 2004, 2007, 2009 and 2010 haven’t reached the ones The composition of the phytoplankton from lake Techirghiol has from 1953 (Ţuculescu 1965); the most important biomass con- modified in time. If in the year 1954, I. Ţuculescu appreciated tribution the phytoplankton has registered in the year 2007 (13.4 that in the plankton of lake Techirghiol, from the microphytes, w.s.(mg•dm-3)) when even its numeric abundance was maximum, the most numerous are the Bacillariophyceae, followed by the and the lowest was in 2009 ( 5.8 w.s.(mg•dm-3)). Cyanobacteria group and other unicelllular algae, and in the year 1977 the highest density was manifested by Bacillariophyceae The effect of the water quality of the structure of the and Euglenophyceae classes followed in descending order by phytoplankton Cyanobacteria and algae from the Chlorophyceae class (Trică The results registered during the study period reveal the ten- 1977), the study which we realised between the years 2004- dency to associate the decrease of the mineralization values 2010 reflects another structure of the phytoplankton from lake of the lake water (medium values for three sampling points in Techirghiol; numeric dominants in the microalgae group have the years 2004, 2007, 2009, and 2010), with an increase of the been the diatoms, but those that follow in numeric abundance number of taxa identified in the phytoplankton. have been the Clorophyceae, Euglenophyceae (in the years

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The biomass values of these microalgae have a proportional the Clorophyceae, Euglenophyceae (in the years 2004, 2007), variation with the ones of the water mineralization; the highest Dinophyceae (2009) and Cryptophyceae (2010). quantity of organic substance was present in the phytoplankton The phytoplanktonic biomass was maximum in the year 2007; in the year 2007, when even the mineral content of the lake wa- this maximum value is close to the one obtained in the years ter was maximum for the entire study interval (see Figure 1). 1976-1978 (Trică 1977), but never has there been reached the - 2- The high concentrations of the Cl and SO4 ions have been as- phytoplanktonic biomass values calculated in the year 1953 sociated with a large number of species in the phytoplankton by I. Ţuculescu. and superior values of the microalgae biomass only in the year 2007; in 2004 the minimum concentration of these ions corre- References spond with intermediate values of the number of taxa and phy- Anagonstidis, K., Komárek, J., 1988. Modern approach to classification toplankton biomass, and in 2009 and 2010 the concentrations system of cyanophytes. Oscillatoriales. Archiv für Hydrobiologie, of the main anions and the structure of the phytoplankton var- Suppl., 80 Algological Studies, 50/53:327-372. ied chaotic (see Figure 2). + 2+ 2+ Anastasiu, P., 2008. Taxonomie vegetală, Editura Universităţii din The report of the cations Na > Mg > Ca , was generally for Bucureşti, pp.58-129. any sampling point, during the realizing of the study; it creat- Bavaru, A., Bercu, R., Bavaru, E., Barbeş, L., 2011. Present aspects on ed optimum conditions for the development of the phytoplank- environment protection and sustainable development on Dobrudja ton, and especially, the calciphilic alga Cladophora crystallina Littoral zone. Studia Universitatis “Vasile Goldiş” Seria Ştiinţele (Roth) Kütz, that supplied an important quantity of biomass to Vieţii 21(3):665-673. the peloidogenesis process in 2007. For this species, the ni- Bonciu, O., Bonciu, C., Petrovici, M. M., 1963. Experimental research trogen compounds are often quoted as being limited nutrients on the pathogenicity of certain ferrouginous bacteria. Archives (Dodds & Gudder 1992). Roumaines de Pathologie Expérimentales et de Microbiologie + - The concentration of the nitrogen compounds (N-NH4 ,N-NO3 ), 22:1039-1044. have been sufficient for the insurance of the nutritional necessi- Bujor, P., 1928. Nouvelle contribution á l’étude de la biologie du lac ties of the phytoplankton, especially for the diatoms that have salé du Tékirghiol. 5 Congr. Intern. Thalassiothérapie, Iassy. registered the highest diversity and biomass from the microal- Bulgăreanu, V. A. C., 1996. Protection and management of anthro- gae (see Table 6). posaline lakes in Romania. Lakes and Reservoires: Research and The oxygen concentration from the epilimnion varied indepen- Management 2:211-229. dently from the number of taxonomic unities and the biomass Charlier, R. H., Chaineux, M.-C. P., 2009. The healing sea: A sustain- value from the phytoplankton in the years 2004, 2007, 2009 able coastal ocean resource: Thalassotherapy. Journal of Coastal and 2010 (see Figure 3). Research 25(4):838-856. The values of the oxygen saturation from the lake water (medium Dodds, W. K., Gudder, D. A., 1992. The ecology of Cladophora. Journal values) under 50% from 2004 and 2007 indicate a certain level of Phycology 28:415-427. of pollution, the results of the microbiological analysis reflecting Doroftei, E., Antofie, M. M., Sava, D., Arcuş, M., 2010. Cytological this fact (see Table 7). In 2009 and 2010, Techirghiol ecosystem features of some macrophytic algae from Mediterranean Sea. Annals has been characterized by the superior self-purification capac- of the Romanian Society for Cell Biology 15(1):316-323. ity, according to the oxygen saturation of the water above 50%. Gheorghievici, L. M., 2010. The contribution of the micro- and macrophytes to the genesis of the therapeutic mud from Lake Techirghiol, Romania. BALWOIS, 25-29 May 2010, Ohrid, Republic of Macedonia. Conclusions Godeanu, S. P., 2002. Determinatorul ilustrat al florei şi faunei României, The number of taxa identified in the phytoplankton varied in- Vol.II, Editura Bucura Mondi, Bucharest, pp. 116-151. verse proportionally with the mineralization level of the water, Hamed, A. F., 2008. Biodiversity and distribution of blue-green algae/ unlike the values of the phytoplanktonic biomass, that increased Cyanobacteria and Diatoms in some of the Egyptian water habitats along with the water salinity. in relation to conductivity. Australian Journal of Basic and Applied - 2- Sciences 2(1):1-21. The increased concentrations of the Cl and SO4 ions have been associated with a diverse phytoplankton and with superior val- Hillebrandt, H., Dűrselen, C.-D., Kirschtel, D., Pollingher, U., Zohany, ues of the microalgae biomass only for the year 2007. T., 1999. Biovolume calculation for pelagic and benthic microalgae. The report of the Na+> Mg2+> Ca2+ cations, generally valid for Journal of Phycology 35(2):403-424. any sampling point, has created optimal conditions for the de- Ionescu, V., Năstăsescu, M., Spiridon, L., Bulgăreanu, V. A. C., 1998. velopment of the phytoplankton, and especially the calciphilic The biota of Romanian saline lakes on rock salt bodies: A review. alga Cladophora crystallina (Roth) Kütz. International Journal of Salt Lake Research 7:45-80. + Kerkmann, G. R., 2010. Techirghiol Lake – Present, Past and Future. The concentrations of the nitrogen compounds (N-NH4 ,N- - BALWOIS, 25-29 May 2010, Ohrid, Republic of Macedonia. NO3 ) have been sufficient for the insurance of the nutritional necessities of the phytoplankton, especially for the diatoms that Krammer, K., Lang-Bertalot, H., 1988. Bacillariophyceae. Nitzschiaceae. have registered the highest diversity and the highest biomass Gustav Fisher Verlag, Stuttgart, New York, pp. 821. from the microalgae. Pârvu, M., 2003. Botanică sistematică, vol. I, Editura Gloria, Cluj The 2004-2010 interval is characterised by a modification of the Napoca, pp.122-152. taxonomic structure of the phytoplankton of lake Techirghiol. Poplăcean, M., 2010. Enzymological characterization of the sapropel- Numeric dominant in the plankton was still the diatoms group, ic muds in the lakes Ocna Sibiului. Extreme Life, Biospeology & as in the precedent studies, but those that follow this time are Astrobiology 2(1):1-6.

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Prescott, L. M., Harley, J. P., Klein, D. A., 1996. Microbiology. Third Zgrundo, A., Dziengo-Czaja, M., Bubak, I., Bogoczewicz-Adamczak, Edition. Wm. C. Brown Pub. Dubuque, Iowa, pp. 390-414. B., 2009. Studies on the biodiversity of contemporany diatom assem- Samson S., et al., 1984. Studies regarding the physical and chemical blages inthe Gulf of Gdansk. International Journal of Oceanography aspects of the peloidogenesis process of the therapeutic lake mud, and Hydrobiology 37(Suppl.2):139-153. in exploration. Archives of INRMFB. ***Institute of Environmental Protection, 2009. WISER (Water bod- Sava, D., Rotaru-Stăncic, M., Doroftei, E., Arcuş, M., 2009. Pharmaceutical ies in Europa: Integrative System to assess Ecological status and importance of some multicellular red algae species from the Romanian Recovery), D3.2-1:2009. Black Sea shore. Annals of the Romanian Society for Cell Biology ***SR EN ISO 19458: 2007 Water quality – Sampling for microbio- 14(2):297-300. logical analysis. Sfriso, A., Raccanelli, S., Pavoni, B., Marcomini, A., 1991. Sampling ***SR EN ISO 6222: 2004 Water quality – Enumeration of cultur- strategies for measuring macroalgal biomass in the shallow wa- able microorganisms. Colony count by inoculation in a nutrient ter of the Venice lagoon. Environmental Technology 12:263-269. agar culture medium. Sigee, D. C., 2005. Freshwater microbiology: Biodiversity and dynamic ***SR EN ISO 9308-1: 2004/ AC: 2009 Water quality. Detection and interactions of microorganisms in the aquatic environments. John enumeration of Escherichia coli and coliform bacteria – Part 1: Wiley and Sons, pp 544. Membrane filtration method. Sládeček, V., 1989. Atlas of Frehwater Saprobic Organism, Hokurykan ***SR EN ISO 7899-2: 2002 Water quality. Detection and enumera- Co., Ltd. tion of intestinal enterococci – Part 2: Membrane filtration method. Silaghi, C. N., Crăciun, A. M., Cristea, V., 2011. Matrix Gla protein: the inhibitor of vascular and osteoarticular calcifications. Human & Veterinary Medicine 3(3):178-190. Authors Trică, V., 1977. Les corrélation entre les conditions de milieu et les or- •Liana Gheorghievici, National Institute of Rehabilitation, Physical ganismes qui prennent part á la formation des boues des lacs salés Medicine and Balneoclimatology, Ion Mihalache Blvd, 11A, de Techirghiol et d`Amara – Roumanie. Hydrobiology, Tom 15, 011171, Bucharest, Romania, [email protected] Editura Academiei RSR, Bucharest. •Iulia Pompei, National Institute of Rehabilitation, Physical Trică, V., 1988. Influenţa unor agenţi de dezinfecţie folosiţi în stabili- Medicine and Balneoclimatology, Ion Mihalache Blvd, 11A, mente balneare asupra caracteristicilor biologice ale ecosistemului 011171, Bucharest, Romania Techirghiol. Studii şi Cercetări de Biologie 40(2):139-145. Ţuculescu, I., 1965. Biodinamica Lacului Techirghiol. Biocenozele şi •Gheorghe Gheorghievici, National Institute of Rehabilitation, Geneza Nămolului. Editura Academiei RSR, Bucharest. Physical Medicine and Balneoclimatology, Ion Mihalache Blvd, Van Landingham, S. L., 1982. Guide to the identification, environ- 11A, 011171, Bucharest, Romania mental requirements and pollution tolerance of blue green algae. •Iosif Tănase, National Institute of Rehabilitation, Physical EPA 600/ 3-82-073. Medicine and Balneoclimatology, Ion Mihalache Blvd, 11A, Vincze, G., János, I., Hörcsik, Z. T., Kotroczó, Z., Szabó, S., 2011. 011171, Bucharest, Romania. Water quality assessments on a natural wetland (Igrice-marsh) on the basis of chemical parameters and macroinvertebrate taxa. Studia Universitatis “Vasile Goldiş” Seria Ştiinţele Vieţii 21(4):901-905.

Gheorghievici, L. M., Pompei, I., Gheorghievici, G., Tănase, I., 2012. The influence of abiotic factors on suppliers of organic matter in the peloidogenesis process from Citation Lake Techirghiol, Romania. Aquaculture, Aquarium, Conservation & Legislation 5(2):69-78. Editor Ştefan C. Vesa Received 23 February 2012 Accepted 24 March 2012 Published Online 12 June 2012 Funding None reported Conflicts/ Competing None reported Interests

AACL Bioflux Volume 5 | Issue 2 Page 78 http://www.bioflux.com.ro/aacl Aquaculture, Aquarium, Conservation & Legislation OPEN ACCESS International Journal of the Bioflux Society Research Article

Current and future prospects of commercial fish farming in Zambia

Confred G. Musuka, Felix F. Musonda

The Copperbelt University, School of Natural Resources, Kitwe, Zambia.

Abstract. According to the survey conducted, there were 15 land based commercial fish farms in Lusaka, Copperbelt and Northern provinces and 10 cage culture operators, mainly based on Lake Kariba. Fish production per hectare ranged between 13 and 18 tonnes while total production was estimated to be approximately 4971.37 metric tons. The weight of fish at harvest from ponds and tanks ranged from 250g to 350g per piece while under cage culture, where fish were regularly fed with floating pellets, ranged from 400 to 450g. Fish stocking density by most farmers was 30,000 fingerlings per hectare and between 12,000 and 15,000 for cage culture. Those who operated fish hatcheries employed a technology that manipulated genetic materials of Oreochromis andersonii and O. niloticus, resulting in production of all-male tilapia, simply referred to as “super males”. Environmental considerations remained a great challenge to the development of both land based commercial fish farms and cage culture on Lake Kariba. Higher fees were charged to do environmental impact assessment (EIA) studies before commencement of operation.

Key Words: current, future, prospects, commercial, fish farming, Zambia.

Corresponding Authors: C. G. Musuka, [email protected]

Introduction farming in Zambia using semi-structured questionnaires (see the Annex 1) that were administered personally to respondents Zambia’s rich endowment of water in form of rivers, lakes and (i.e. commercial fish farmers, fish seed producers and fisher- swamps provides the foundations for supporting significant eco- ies officers). Data collection included the following: aquacul- nomic growth and development (Mudenda 2006; Musumali et ture production statistics; fish feed/seed availability as well al 2009). These natural resources are ideally suited to aquacul- as institutional and regulatory policy frame work supporting ture production (ACF/FSRP 2009). Aquaculture development aquaculture development. Other relevant information was programs have the potential to create new jobs, improve food obtained from the Department of Fisheries (DoF) headquar- security among poor households, remove variability in terms ters and provincial fisheries offices. Field data analyses were of household income flow, and increase farm level efficiency done using Statistical Package for Social Sciences (SPSS 12.0) and sustainability (Kaliba et al 2007). while Microsoft Excel was used to prepare graphs and tables. The potential for aquaculture that has been ascertained for commercial pond fish farming development exceeds 38,000 hectares in all provinces of Zambia (NADP 2010). Commercial Results fish farming is a principal economic activity that is focussed on It was established that fish production per hectare ranged be- making money (profit) by actively participating in the market tween 13 and 18 tonnes while total production from land based through the sale of all kinds of fish products and other related commercial fish farming was estimated to be approximately farm produce. Commercial fish farming is usually very large, 4,453.21 metric tons in 2010 (Figure 1), with 51% production intensive and involves large investment. It is market oriented being recorded in the Copperbelt Province (Figure 2) while and may include processing for export (ACF/FSRP 2009). In Lusaka and Southern recorded 34% and 11%, respectively. Zambia, it is operated at two levels: land based (i.e. use of ponds On the other hand, fish production from cage culture was es- and tanks with and without recirculatory system) and cage aq- timated to be approximately 518.16 metric tons in 2010 com- uaculture. These practices involve higher stocking densities of pared to 387 metric tons recorded in 2009 (Figure 3). mono-sex species, reliance on feed and oxygen supplementa- Fish cultivation was centered on Nile Tilapia (Oreochromis tion in some cases. niloticus (Linnaeus, 1758)), O. andersonii (Castelnau, 1861), O. macrochir (Boulenger, 1912) and Tilapia rendalli (Boulenger, Materials and methods 1897) (Figure 4). The government was promoting aquaculture as a business in A survey was conducted between December 2010 and April higher potential zones where specific aquaculture production 2011 to assess current and future prospects of commercial fish systems were to be marched with available resources (Figure 5).

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Figure 1. Fish production from land-based commercial farmers

Figure 5. Map showing higher potential zones for aquaculture in Zambia (Source: NADP 2010)

Discussion Findings of this study agree with a report by FAO (2004b) and ACF/FSRP (2009) that observed the concentration of land based commercial fish farmers in areas around Lusaka, Copperbelt and Northern provinces where ideal conditions for such business existed. However, during the survey, only 15 such farmers were recorded, compared to earlier reports by FAO (2004) and Mudenda (2006), which indicated that there were at least Figure 2. Fish production from commercial fish culture between 20 and 24 commercial fish farmers in Zambia. It was observed that the substantial decline in number of commercial operators over the years may have been attributed to: - poor performance of the country’s economy as well as lack of incentives from government to promote investment and growth of this sector; - fragmented support to aquaculture by both the government and private sector to ensure growth, which has contributed to its poor development; - higher operation costs; inability of the individual fish farmers to access investment loans from financial institutions due to lack of collateral; lending institutions normally prefer to lend to the agriculture sectors, which they are more knowledgeable of; - the sector previously operating without an enabling policy Figure 3. Fish production from cage culture and legal framework needed to promote its sustainable growth; - lack of quality affordable feed/inadequate hatcheries that produce quality fish seed leading to high fish yields; fish farmers also found it difficult to meet costs of feed, fingerlings and tools as a result of indirect expenses arising from transport and communication costs; - so much red tape in registering a fish farming company; - inability of potential farmers to raise funds to hire consultants to conduct environmental impact assessment (EIA). The entry into the sector of five new investors (Savanna and Eureka farms in Lusaka, Revendel Tilapia Enterprises in Luanshya, Aqua and St. Patrick fish farm in Kafue) showed an increase in investment, implying that fish production from aquaculture could be increased 10-fold, contributing significantly Figure 4. Percentage distribution of cultured fish species to national food security, important domestic provider of much needed high-quality animal protein and other essential nutrients (generally at affordable prices to the poorer segments of

AACL Bioflux Volume 5 | Issue 2 Page 80 http://www.bioflux.com.ro/aacl Musuka et al 2012 the community) and/or a provider of employment opportunities genetic materials of O. andersonii and O. niloticus, resulting and cash income (Tacon 2001; DoF 2010). in production of all-male tilapia, simply referred to as “super In Zambia, cage culture, a specialized type of fish farming was males”, which avoids the common inconvenience resulting practiced at commercial level by hospitality business owners, from premature pond fish reproduction, a problem that most the majority of whom were also Kapenta fishers on Lake Kariba. farmers were grappling with. The introduction of cage culture on Kariba holds a great poten- Most land-based fish farmers used both on-farm feed and com- tial for growth of aquaculture in Zambia. The practice has the plete diets (either extruded or floating pellets) to feed their fish potential of producing large quantities of fish for export and either by broadcasting or through the use of feed rings twice or domestic markets (FAO 2004a). Cage fish farmers have contin- more per day. Zambia has at least 2 to 3 commercial fish feed ued expanding with new cages introduced in Mkushi (Central producers. The price of aqua feed supplied by Tiger Animal Province) and expansion in both cage numbers and fish pro- Feed and National Milling Corporation (NMC) ranged between duction on Lake Kariba. Observations by FAO (2004) revealed $15.63 and $20.83 per fifty (50) kg bag. However, according to that for cage culture in Africa to succeed, five key constraints Machena & Moehl (2001) feed remains one of the most promi- of seeds, feed, finance, skills/information and marketing have nent barriers to expanded aquaculture production, especially to be addressed comprehensively. medium- and large-scale production. Unless affordable feed During the study, 10 cage culture operators (with 42 active cag- costs can be maintained, farm-raised products cannot compete es out of a total number of 51) were recorded, most of which with those coming from capture fisheries, unless there is sig- were concentrated in Siavonga and Sinazongwe districts on nificant value added through the production of luxury items. Lake Kariba with few found elsewhere. However, an earlier re- The weight of fish at harvest for ponds and tanks was from port by Maguswi (2003) observed that there were 4 commercial 250g to 350g per piece while under cage culture where fish enterprises practicing cage aquaculture on Lake Kariba. They were regularly fed with floating pellets, the weight at harvest each used 44 cages 6 m x 6 m x 6 m (216 m3) and 10 pens to ranged from 400g to 450g. The fish after harvest was transport- grow Nile tilapia bream (O. niloticus) and used commercial ed to the market (wholesalers and retailers) using refrigerated feeds such as pellets. Stocking density was around 20 kg/m3, trucks, where they were sold whole/round and in some cases while average production was 3.5 tonnes per cage measuring processed as fillets between $2.30 and $3.13 per kg. 216 m3. But Blow & Leonard (2007) indicated the existence The study also revealed an increase in number of fish farm em- of only three small cage farms in Zambia operating on Lake ployees by 10%. Mudenda (2006) reported that commercial Kariba in the Siavonga area that were established in the 1990s fish farmers employed over 310 workers of which 69 were full and whose production was not more than 10 tonnes per an- time and the rest were indirect workers. To date only 15% of num of whole fish. The authors reported that all three farms employees at commercial fish farms were on permanent basis had 30 square cages of around 40 m3, with wooden walkways. while 85% were contractual workers. It was also observed that Production nets were nylon and made in Zimbabwe or import- the majority of the workforce employed on permanent basis, ed from abroad. No predator nets were used. The authors fur- showed commitment and motivation in discharging their duties ther reported that the three cage sites were located in shallow compared to those who were indirectly employed. The study (<5 m deep) inshore areas and were close enough to land to further agrees with Mudenda (2006) on the aspect of most, if not have walkways out to the sites. Juveniles were transferred to all the workers in commercial aquacultures being functionally the cages from pond sites, where they were on-grown to mar- literate, although less than 1% of commercial fish farm work- ket size of around 350 g. ers had received specialist training in aquaculture at any level. Fish stocking density by most commercial farmers was 30,000 Environmental considerations remained a great challenge to de- fingerlings per hectare, which was equivalent to 3 fish per2 m . velopment of both land based commercial fish farms and cage Mudenda (2006) reported that the recommended stocking den- culture on Lake Kariba. The Zambia Environmental Management sity for breams was 2.5 fingerlings per square metre of pond. Agency (ZEMA) (formally Environmental Council of Zambia) Cage farmers stocked between 12,000 and 15,000 mono sex or was cited as the major hindrance to establishment of new fish sex reversed Nile Tilapia fingerlings meant to improve yield. farms. Higher fees were charged by ZEMA for carrying out en- The average pond size was about half a hectare (5000 m2), al- vironmental briefs or environmental impact assessment (EIA) though some farms had ponds below that size. studies before commencement of operation. The concerned The study concurred also with FAO (2005) who reported that farmer was required by law to hire consultants (whose costs the concentration of fish cultivation was centered on O. niloti- were not less than $5,000) to conduct an EIA, an exercise per- cus, O. andersonii, O. macrochir and T. rendalli to some ex- ceived to be expensive. If ZEMA determined that a project was tent. These contributed about 99% of production while other likely to have a significant impact on the environment, it may fish species such as carp (Cyprinus carpio Linnaeus, 1758) and require the preparation of an environmental impact assessment the red swamp crayfish (Procambarus clarkii (Girard, 1852)) (EIA). In addition, EIAs are obligatory for all projects speci- contributed only 1%. Nile Tilapia was the most favoured spe- fied in the Second Schedule, including all projects located in cies because of its relatively faster growth, and flexible feeding or near environmental sensitive areas such as areas support- habits in crowded conditions, particularly in cages. Not until ing populations of rare and endangered species or major water such a time when the conclusion of evaluation of substituting it catchment areas. with a suitable native species O. niloticus will remain the main From this study the authors further learnt that conducting an species for cage aquaculture. Some farms operated fish hatch- environmental project brief was far much cheaper than EIA. eries in which they employed a technology that manipulated For example, Zambia National Service (ZNS) owned seventeen

AACL Bioflux Volume 5 | Issue 2 Page 81 http://www.bioflux.com.ro/aacl Musuka et al 2012 hectares Chanyanya farm, only prepared an environmental brief to aquaculture development. NADS was based on four funda- as opposed to conducting an EIA before commencing operations. mental principles: A project brief is a report on preliminary conditions of possible - to promote aquaculture development as a business (profit) in impacts of a project on the environment and constitutes the first higher potential zones (HPZs) where specific aquaculture pro- phase of the EIA process. A project brief is required for all production systems were to be marched with available resources jects listed in the First Schedule to the Regulations, including (Figure 5); fish farms with a production of 100 tonnes or more per year. A - stakeholders were expected to contribute to development where project brief was also required for the introduction of alien spe- they had a comparative advantage; cies of flora and fauna into the local ecosystem. - aquaculture was considered a profitable venture; Other concerns raised by stakeholders included issues in which - producers required having a say in the management of the sec- they urged government to provide an enabling environment in tor, with clearly outlined roles and responsibilities. terms of regulations and legal support that promoted the com- The strategy’s final goal was to have a healthy and dynamic aq- mercialization of aquaculture to ensure availability of credit fa- uaculture sector in Zambia. It acknowledged 9 key priority areas cilities, good quality fish seed and affordable feed. According for growth of aquaculture: (1) suitable production systems, (2) to FAO (2004) the challenge to government and regional or- availability and cost of inputs (feed, seed capital), (3) exten- ganisations was to identify bottlenecks to development and sion or outreach (4) research and technological innovation, (5) make short and effective interventions where necessary. As for education and training, (6) marketing, (7) producer organisa- Zambia, a good national policy was lacking to promote sustain- tion, (8) regulations and control, and (9) monitoring and evalu- able aquaculture development from the time of its inception in ation (NADP 2010). the early fifties except for one that supported only the fisheries from natural waters, lakes, rivers and swamps. The aquaculture Conclusion sector operated without an enabling policy and legal framework that caused it to lag behind in terms of development compared The conclusion that can be drawn from this study is that envi- with other agriculture activities such as crops and livestock pro- ronmental considerations have remained a great challenge to the duction that received yearly incentives (farmer input support development of commercial fish farming in Zambia. However, programme and free animal vaccines) from the central govern- the current trends indicate that investment in the sector was be- ment. FAO (2005) further reported that a policy and regula- ing recorded in the country although the growth of aquaculture tory framework which addressed issues of resource allocation, was perceived to be relatively slow. Fish farming had great po- cumulative environment impact, and input as well as product tential for reducing poverty in the country by increasing fish quality was also needed for this sector to grow. production for food security and income generation amongst A need therefore, arose for the amendment of the Fisheries Act, households, thereby contributing directly to the achievement Cap 200 of 1974, the country’s major piece of legislation gov- of the Millennium Development Goals (MDGs). Through co- erning the fisheries sector by parliament that did not contain any ordinated support to the sector it was possible for fish farming substantive provisions relating to aquaculture (Mutuna 2009). to grow to unprecedented levels. The Fisheries Act was amended in 2007 to help strengthen provisions for the development of the sector. Some of the provisions Acknowledgement in the amended Act describe the license procedure to engage in The authors wish to acknowledge the Copperbelt University and set up an aquaculture facility and address issues such as the for financial and material support rendered towards conducting protection of the aquatic environment, fish movement and fish of this study. Special thanks to go to various members of staff disease (i.e. Zambia had a liberal policy with respect to translo- in the Department of Fisheries and all the cooperating partners cation and introductions, provided they were closely monitored too numerous to mention by names. Without their support we with strict measures to prevent escapes into the natural waters), would have faced a lot of challenges to accomplish the task. environmental impact assessments and genetically modified organisms. The legal framework as contained in the Fisheries Act concerning regulation and control fish escapees from fish References farms, cages, fish pens or any other aquaculture facility into the Agriculture Consultative Forum/Food Security Research Project (ACF/ natural fisheries will be followed. The same legal frameworks FSRP), 2009. The Status of fish population in Zambia’s water bodies will apply for regulating the introduction and use of exotic fish Blow, P., Leonard, S., 2007. A review of cage aquaculture: sub-Saharan species and use of genetically improved fish species in aquacul- Africa. In: Cage aquaculture – Regional reviews and global over- ture. The amended legislation also provides for a definition of view. Halwart M., Soto D., Arthur J. R. (eds), pp. 188–207. FAO Fisheries Technical Paper. No. 498. Rome, Italy. aquaculture, the preparation of aquaculture development plan, the declaration of aquaculture development areas and estab- Do, F., 2010. National Aquaculture Development Plan. Overcoming the slow growth of Aquaculture in Zambia 2010-2015. lishment of a Technical Aquaculture Committee (FAO 2005) In order to address the ambiguity of aquaculture objectives, FAO, 2004a. Regional Technical Expert Workshop on Cage Culture the Department of Fisheries embarked on the preparation of a in Africa (20-23 October, 2004), Entebbe, Uganda. FAO Fisheries Proceeding 6. ISSN 1813-3940, 113 pp. National Aquaculture Development Strategy (NADS) in 2004 with the support of Food and Agriculture Organization (FAO) FAO, 2004b. Report of the Workshop on the Promotion of Sustainable Commercial Aquaculture in Zambia and Malawi. Lusaka, Zambia, of the United Nations to refocus development and management 2-4 October 2002. of aquaculture so as to commit government to remove obstacles FAO Fisheries Report. No. 733. Rome, Italy, 46 pp.

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FAO, 2005. Aquaculture production, 2003. Year book of Fishery Statistics Mutuna, C. L., 2009. Fisheries development facilitation manual for field - Vol.96/2. Food and Agriculture Organization of the United Nations, workers and community leaders. DoF/PLARD. pp 157. Rome, Italy. 195 pp. National Aquaculture Development Plan (NADP), 2010. Overcoming Kaliba, A. R., Ngugi, C. C., Mackambo, J. M., Osewa, K. O., Senkondo the slow growth of aquaculture in Zambia 2010-2015. Department E., Mnembuka B. V.,Amisah S., 2007. Potential effect of aquaculture of Fisheries, Chilanga, pp 153. promotion on poverty reduction in Sub-Saharan Africa. Aquacult Tacon, A. G. J., 2001. Increasing the contribution of aquaculture for Int 15:445–459. food security and poverty alleviation. In: Aquaculture in the Third Machena, C., Moehl, J., 2001. Sub-Saharan African aquaculture: regional Millennium. Subasinghe, R. P., Bueno P., Phillips, M. J., Hough, summary. In: Aquaculture in the Third Millennium. Subasinghe, R. C., McGladdery, S. E., Arthur, J. R. (eds). Technical Proceedings P., Bueno, P., Phillips, M. J., Hough, C., McGladdery, S. E., Arthur, of the Conference on Aquaculture in the Third Millennium, Bangkok, J. R. (eds). Technical Proceedings of the Conference on Aquaculture Thailand, 20-25 February 2000. pp.63-72. NACA, Bangkok and in the Third Millennium, Bangkok, Thailand, 20-25 February 2000. FAO, Rome, Italy. pp. 341-355. NACA, Bangkok and FAO, Rome, Italy. Maguswi, C. T., 2003. National Aquaculture Sector Overview - Zambia. National Aquaculture Sector Overview Fact Sheets. FAO Inland Authors Water Resources and Aquaculture Service (FIRI). Rome, Italy. •Confred G. Musuka, The Copperbelt University, School of Mudenda, C. G., 2006. Economic perspective of aquaculture develop- Natural Resources, Department of Zoology and Aquatic Sciences, ment strategy of Zambia. Consulant Report, Development Consultant, Jambo Drive, Riverside, Kitwe, Zambia, e-mail: Confred.mu- Lusaka, Zambia. Development Consultant. TCP/ZAM/3006. pp 99. [email protected], [email protected] Musumali, M. M., Heck, S., Husken, S. M. C., Wishart, M., 2009. •Felix F. Musonda, The Copperbelt University, School of Natural Fisheries in Zambia: an undervalued contributor to poverty reduction. Resources, Department of Zoology and Aquatic Sciences, The WorldFish Center/The world Bank. Policy Brief 1913, pp 16. Jambo Drive, Riverside, Kitwe, Zambia, e-mail: felix.muson- [email protected]

Annex 1

Current and future prospects of commercial fish farming in Zambia

(1) DATE: ______/______/ ______

(2) NAME OF OPERATOR: ______

(3) DESIGNATION (OPTION): ______

(4) TYPE OF AQUACULTURE:______(A) LAND BASED (B) CAGE CULTURE

(5) LOCATION OF FARM/CAGES:______

(6) HOW LONG HAVE YOU BEEN IN OPERATION?:______(A) BELOW 6 MONTHS (B) 6 MONTHS TO 1 YEAR (C) 2-5 (D) 6- 10 YEARS (E) >10 YEARS

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(7) HOW DID YOU FINANCE THE BUSINESS? (A) USED OWN RESOURCES (B) BANK LOAN (C) GRANT AID (D) OTHER(S)/SPECIFY: ______

(8) DID YOU DO AN ENVIRONMENTAL IMPACT ASSESSMENT (EIA) BEFORE COMMENCEMENT OF OPERATION? ______(A) YES (B) NO

(9) IF YES, WHO DID IT? ______(A) SELF (B) CONSULTANT (C) OTHERS

(10) WHAT WAS THE COST OF EIA? ______(A) BELOW $1000 (B) $1200-3000 (C) $3100-5900 (D) ABOVE $6000

(11) IF ANSWER IS NO TO QUESTION 6, WHAT DID YOU DO? ______(A) NOTHING (B) ENVIRONMENTAL BRIEF (C) OTHERS/SPECIFY: ______

(12) WHAT FISH SPECIES DO YOU CULTURE? ______(A) RED BREASTED BREAM (B) THREE SPOTTED BREAM (C) NILE TILAPIA (D) AFRICAN CATFISH (E) OTHERS ______

(13) WHAT IS THE SOURCE OF YOUR FINGERLINGS? ______(A) BREED OWN JUVENILES (B) BUY FROM LOCAL SEED PRODUCERS (C) IMPORT FROM OTHER COUNTRIES (D) OTHER(S)/SPECIFY______

(14) WHY IS THIS SPECIES MOST PREFERRED ______(A) FAST GROWTH (B) IMPROVED GENETIC MATERIAL (C) MARKET DEMAND (D) OTHER(S) SPECIFY: ______

(15) DO YOU EXPERIENCE ANY FISH DISEASES? ______(1) YES (2) NO

(16) IF ANSWER IS YES TO QUESTION ABOVE, WHAT TYPE OF INFECTIONS HAVE YOU EXPERIENCED? ______(A) FUNGAL (B) BACTERIAL (C) ECTOPARASITES (D) ENDOPARASITES (E) OTHER(S)/SPECIFY ______

(17) WHAT DO YOU DO WITH THE INFECTED FISH? ______(A) NOTHING (B) REMOVE AND THROWN AWAY (C) QUARINTINE THEM (D) APPLY TREATMENT TO SICK FISH (E) OTHER(S)/SPECIFY: ______

(18) WHO SUPPLIES FEED FOR YOUR FISH? ______(A) OWN FEED PRODUCTION (B) TIGER ANIMAL FEED (C) NATIONAL MILLING CORPORATION (D) OTHER(S)/ SPECIFY______

(19) WHAT IS THE PRICE OF FEED/50KG? ______(A) BELOW $10 (B) $10-14 (C) $15-19 (D) ABOVE $20

(20) HOW DO YOU FEED THE FISH ______(A) BROADCASTING (B) USE FISH FEEDERS (C) OTHERS SPECIFY:______

(21) HOW OFTEN DO YOU FEED YOUR FISH PER DAY? ______(A) ONCE (B) TWICE (C) MORE THAN TWICE

(22) WHAT QUALITY STANDARDS HAVE YOU PUT IN PLACE TO ENSURE FEED WASTAGE IS KEPT TO MINIMUM? ______(A) NOTHING (B) USE OF FISH EXTRUDED DIETS (C) USE OF FLOATING FEED (D) OTHER(S)/ SPECIFY______

STATUS OF AQUACULTURE OPERATION:

(A) LAND BASED

(1) WHAT IS YOUR FARM SIZE? ______(A) BELOW A HA (B)1-5HA (C) 6-10HA (D) 11-19HA (E) >20HA

(2) WHAT IS THE TOTAL VALUE OF YOUR FARM? ______(A) BELOW $10000 (B) $15000-25000 (C) $26000-50000 (D) >$50000

(3) NUMBER AND SIZES OF AQUACULTURE FACILITIES ______

(4)WHO CONSTRUCTED THE FACILITIES AT FARM?______(A) SELF (B) HIRED CONSTUCTORS (C) OTHER(S) SPECIFY______

(5) WHAT WAS THE CONSTRUCTION COST/FACILITY? ______

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(6) FISH STOCKING DENSITY/M2 ______

(7) FISH FARM PRODUCTION CYCLE:______(A) 4-6 MONTHS (B) 7-9 MONTHS (C) 12 MONTHS (D) MORE THAN 12 MONTHS

(8) PRODUCTION/FACILITY/UNIT TIME: ______

(9)TOTAL FARM OUTPUT: ______TONS/YEAR

(B) CAGE PRACTICE

CAGE STOCKING DENSITY NUMBER NAME(S) OF SPECIES PRODUCTION/CAGE/ No. (KG/M3) STOCKED STOCKED UNIT TIME Type Size Number 1 2 3 4 5

CAGE TYPE: (1) PLASTIC CIRCLE CAGES (2) SQUARE CAGES (3) WOODEN CAGES

(1) WHERE ARE THE CAGES LOCATED? (A) INSHORE (B) OFFSHORE (C) OTHERS (SPECIFY):______

(2) HAVE YOU EXPERIENCED FISH ESCAPE? ______(A) YES (B) NO

(3) IF YES EXPLAIN WHAT CAUSED THE ESCAPE ______

(4) DO YOU USE PREDATOR NETS AROUND YOUR CAGES? ______(A) YES (B) NO

(5) IF NO WHAT MEASURES HAVE YOU PUT IN PLACE TO PREVENT FUTURE ESCAPE OF FISH? ______

(6) WHAT IS YOUR FISH MARKET SIZE? ______(A) 200-249G (B) 250-299G (C) 300-349G (D) ABOVE 350G

(7) WHERE DO YOU SALE YOUR FISH AFTER HARVEST? ______(A) LOCALLY (B) SURROUNDING TOWNS WITHIN THE PROVINCE (C) LUSAKA (D) OTHER(S)/SPECIFY: ______

(8) HOW DO YOU SALE YOUR FISH? _____ (A) WHOLE/ROUND/GUTTED FISH (B) FRESH FILLETS (C) SUPER- CHILLED FILLETS (D) FROZEN FILLETS (6) SMOKE DRIED

(9) TO WHO DO YOU SALE YOUR FISH (A) WHOLESALE RS (B) RETAILERS (C) BOTH (D) OTHER(S)/SPECIFY______

(10) AT WHAT PRICE DO YOU SALE YOUR FISH/KG ______(A) $1- 2 (B) $2.5- 3 (C) $3.5-4 (D) ABOVE $4.5

(11) HOW DO YOU TRANSPORT YOUR FISH TO THE MARKET? ______(A) ICED SACKS/SEALED BOXES IN OPEN VANS (B) REFRIGERATED TRUCKS (C) OTHER(S)/SPECIFY: ______

(12) DO YOU HAVE FISH PROCESSING FACILITIES? ______(A) YES (B) NO

(13) IF ANSWER IS YES WHAT TYPE IF ANY? ______(A) COLD ROOMS (B) DRY SMOKING KILNS (C) OTHER(S)/ SPECIFY: ______(14) HOW DO YOU RATE YOUR PRODUCTION COST? ______(A) BELOW $1/KG (B) $1-$3/KG (C) ABOVE $4/KG

(15) NO. OF EMPLOYEES: ______(A) 1-5 (B) 6-14 (C) 15-29 (D) ABOVE 30

(16) WHAT IS THEIR CONDITION OF SERVICE? ______(A) PERMANENT (B) CONTACT (C) CASUAL (D) OTHER(S)/SPECIFY ______

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(17) WHAT PERCENTAGE OF EMPLOYEES ARE WOMEN? ______(A) BELOW 15% (B) 16-29% (C) 30-49% (D) ABOVE 50%

(18) WHAT JOBS DO THEY DO? ______(A) NET MENDING (B) FISH PROCESSING (C) HATCHERY OPERATIONS (D) OTHER(S)/SPECIFY: ______

OTHER REGULATORY FRAMEWORKS AND LEGAL ISSUES

(1) HOW DO YOU RATE POLICIES AND LEGAL FRAME WORKS ______(A) DOES NOT EXIST (B) VERY POOR (C) POOR (D) GOOD) (E) VERY GOOD (F) EXCELLENT

(2) DO YOU HAVE ANY DESIGNATED ZONES FOR AQUACULTURE OPERATIONS? ______(A) YES AND (B) NO (C) IF NO, STATE HOW THIS COULD BE DONE: ______

(3) HAVE YOU ATTENDED ANY FISH FARMING TRAINING? ______(A) YES (B) NO

(4) IF ANSWER TO QUESTION ABOVE IS YES, STATE TYPE OF COURSE AND ITS DURATION: ______

(5) WHERE DID YOU TRAIN: ______

(6) WHAT DO YOU THINK ARE THE MAIN REASONS FOR THE GROWTH OF THE INDUSTRY? YES/NO RANK FAVOURABLE FISH PRICE POSSIBILITY OF MAKING MONEY GOVERNMENT PROMOTION AVAILABILITY OF CREDIT AVAILABILITY OF FISH FEED AVAILABILITY OF FISH SEED OTHERS SPECIFY RANK = (1) VERY POOR (2) POOR (3) FAIR (4) GOOD (5) VERY GOOD

(7) WHAT DO YOU THINK ARE THE MAIN CONSTRAINTS FOR THE GROWTH OF THE INDUSTRY?

YES/NO RANK SHORTAGE OF FISH SEED LACK OF CREDIT FACILITIES COST OF CREDIT LACK OF MARKETS GOVERNMENT REGULATIONS GOVERNMENT TAXATION LACK OF TECHNOLOGICAL KNOWLEDGE LACK OF SKILLED LABOUR OTHERS SPECIFY

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Musuka, C. G., Musonda, F. F., 2012. Current and future prospects of commercial fish Citation farming in Zambia. Aquaculture, Aquarium, Conservation & Legislation 5(2):79-87. Editor Ştefan C. Vesa and Ionel-Claudiu Gavriloaie Received 05 March 2012 Accepted 30 May 2012 Published Online 22 June 2012 Funding Copperbelt University. No grant number reported by authors Conflicts/ Competing None reported Interests

AACL Bioflux Volume 5 | Issue 2 Page 87 http://www.bioflux.com.ro/aacl Aquaculture, Aquarium, Conservation & Legislation OPEN ACCESS International Journal of the Bioflux Society Research Article

Histopathological study of parasitic infestation of skin and gill on Oscar (Astronotus ocellatus) and discus (Symphysodon discus)

1Forough Mohammadi, 1Seyed M. Mousavi, 2Annahita Rezaie

1 Department of Fisheries, Faculty of Marine Natural Resources, Khorramshahr University of Marine Science and Technology, Khorramshahr, Iran; 2 Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.

Abstract. Histopathology provides a rapid method to detect effects of irritants and pathogens in different organs and it can be considered as the indicator for abnormal condition for fish environment. The present study was initiated to record the histopathological lesions of gill and skin associated with external parasites in two common aquarium fish, Oscar (Astronotus ocellatus) and Discus (Symphysodon discus) fish. Twenty Oscar and twenty Discus were obtained from aquarium shops and wet mount was prepared from skin and gill mucosa and histopathological study was performed on tissue samples of gills and skin on tissue sections which were stained with haematoxilin-eosin. Based on the results, Dactylogyrus sp. was the most prevalent parasites in Oscar and Discus fish. Ichthyophthirius multifliis, Trichodina sp. and Gyrodactylus sp., Epistylis sp. and Vorticella sp. were seen in the skin and gill of fish. In histopathological examination, fusion of secondary lamella associated with hyperplasia, aneurysm, edema, purulent bronchitis were seen. Sections of Ichthyobodo sp. and purulent bronchitis are rare and in skin. Dermatitis was observed. Histopathological lesions in Oscar were in high rate in comparison with lesions which were seen in Discus and they are in relation to parasitic infestation.

Key Words: histopathology, Oscar fish, Discus fish, gill, skin.

Corresponding Authors: S. M. Mousavi, [email protected] Introduction 1831)) and Discus (Symphysodon discus Heckel, 1840) belong to Cichlidae family and they have been kept in aquariums for Ornamental fish culture has rapidly developed in different many years and they are considered the “Kings and Queens” countries. Parasitic infestation is the most important disease of the tank. The present study was initiated to record the his- affecting ornamental fish and it causes economical losses for topathological lesions of gill and skin associated with external this growing industry in intensive culture systems. Fish may parasites in these two common aquarium fish, Discus and Oscar. be infected by the parasites as final or intermediate hosts in a parasitic life cycle (Hoffman 1999; Smith & Roberts 2010). Parasites of fish can either be external or internal. Parasitic Material and Methods infections often give an indication of the quality of water, This research was conducted as from april 2010 to may 2011. since parasites generally increase in abundance and diversity Twenty Oscar and Twenty Discus were obtained from aquari- in more polluted waters (Poulin 1992; Noga 2010). Parasites um shops and transferred to the Fisheries laboratory. Biometry are capable to cause damage to the fish through injury to the was performed and wet mount was prepared from skin and gill tissues or organs. Fish parasites resulted in economic losses mucosa. For histopathological study, the fish were euthanized or mortality, treatment expenses, growth reduction during and and tissue specimens of gills and skin were excised, rinsed in after outbreak of disease and these interact with expanding of normal saline and fixed in formalin buffer 10% for 24 h. After ornamental fish culture. There are no specific pathogonomic fixation, the tissues were dehydrated in an alcohol series of clinical signs for parasitic diseases in fish, although a group ascending concentration (70%, 80%, 90% and 100%, respec- of clinical signs may be specific for some parasitic infesta- tively), embedded in paraffin and sectioned at 5 µm. The tissue tion (Reavill & Roberts 2007). Most external parasites can be sections were stained with haematoxilin-eosin (H&E) and were readily identified on direct observation and wet mount prepa- examined by light microscope. rations. But some parasitic infections need another paraclini- cal examination to be confirmed. Histopathology provides a rapid method to detect effects of irritants and pathogens in Results different organs (Johnson et al 1993) and it can be consid- Table 1 shows the frequency of parasites which were observed ered as the indicator for abnormal condition for fish environ- on wet mount in Oscar fish and Discus fish. Based on the results, ment (Roberts 2001). Oscar (Astronotus ocellatus (Agassiz, Dactylogyrus sp. was the most prevalent in Oscar fish (Figure

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1A). Infection with Ichthyophthirius multifiliis is approximate- also penetrated to secondary lamella (Figure 7). These sections ly prevalent and it was seen in skin and gill of four Oscar fish were detected in 25% and 10% of Oscar and Discus, respective- (Figure 1B). Trichodina sp. and Gyrodactylus sp. were detected ly. Purulent bronchitis with accumulation of inflammatory cells in gill and skin of two fish (Figure 1C). Epistylis sp. was ob- was distinguished in 10% of both species. served in the skin and gill of one case (Figure 1D) and Vorticella Histopathological study of fish skins revealed dermatitis in 15% sp. was seen in the skin and gill of one case. of Discus fish and 35% of Oscar fish (Figure 8).

Figure 2. Hyperplasia (H) and fusion of secondary lamella (F) Figure 1: Wet Mount preparation: A) Dactylugyrus sp. on of Oscar fish is obvious (H&E, Bar 50µm). the gill, eye spots is clearly visible (Magnification × 40); B) Ichthyophthirius multifiliis on skin (Magnification × 40); C) Trichodina sp. on skin (Magnification× 40); D)Epistylis sp. on skin (Magnification ×40)

Based on results which were shown in table 1, in the wet mount of skin and gills of Discus fish different parasites were found, such as Dactylogyrus sp., Trichodina sp. and Vorticella sp.; based on the results, Dactylogyrus sp. was the most prevalent parasite in Discus fish (frequency of 35%).Trichodina sp. was reported in gill and skin of 25% of Discus fish and Vorticella sp. which is a sessile ciliata was found in 10% of Discus fish. Also, there is not any observation for I. multifiliis, Gyrodactylus sp. and Epistylis sp. in Discus fish. In comparison, parasitic infestation in Oscar fish was more prevalent than in Discus fish. Histopathological examination of Oscar and Discus gills and skin revealed different lesions which are summarized in Table Figure 3. Aneurism (A) in secondary lamella of Oscar fish (H&E, 2. Hyperplasia was observed in 95% and 35% of Oscar and Bar 20µm). Discus gill, respectively, and it was characterized by prolif- eration of epithelial, connective and inflammatory cells which were accumulated dorsal part of lamella (Figure 2). Aneurism in secondary lamella was detected in 90% and 30% of Oscar and Discus, respectively (Figure 3). Fusion of secondary lamella due to hyperplasia was seen in 55% and 25 % of Oscar and Discus, respectively (Figure 2). Edema was seen in 50% of Oscar and it was characterized by more free spaces between gill structures. Epitheliocystis was basophilic ball shape structures which was attached to endothelial of secondary lamella capillaries (Figure 4). These structures were seen in 35% of Oscar gill. Ichthyobodo sp. was observed in two shapes, free with flagella and attached to epithelial cells and without flagella and pear shape (Figure 5). This infestation was seen in 25% and 5% of Oscar and Discus, respectively. Sections of Dactylogyrus sp. were seen in 25% of Oscar gills. The attachment place of para- Figure 4. Epitheliocystis in secondary lamella. Note the baso- site showed necrosis of lamella (Figure 6). Sections of different philic ball shape attached to capillaries wall (E) and hyperplasia stages of I. multifiliis have been seen between the lamella and (arrow) (H&E, Bar 10µm).

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Table 1. Frequency of parasitic infestation in gill and skin in Oscar and Discus (%)

Parasite infestation Oscar gill Oscar skin Discus gill Discus skin Dactylogyrus sp. 50 20 35 35 Ichthyophthirius multifiliis 20 20 0 0 Gyrodactylus sp. 5 10 0 0 Trichodina sp. 5 10 25 25 Epistylis sp. 5 5 0 0 Vorticella sp. 5 5 10 10 Table 2. Frequency of histopathological effects on gill and skin in Oscar and Discus (%)

Gill and skin histopathological lesion Oscar frequency Discus frequency Hyperplasia 95 35 Aneurism 90 30 Fusion of secondary lamella 55 25 Edema 50 0 Epitheliocystis 35 0 Ichthyobodo sp. 25 5 Dactylogyrus sp. section 25 0 Ichthyophthirius multifiliis 25 10 Purulent bronchitis 10 10 Dermatitis 35 15

Figure 5. Ichtiobodo sp. attached to epithelial cells of second- Figure 7. Trophont stage of I. multifiliis (arrows) in Oscar fish ary lamella (arrows) and they are pear shape. Also hyperplasia (H&E, Bar 20µm) (H) is obvious (H&E, Bar 10µm)

Figure 8. Dermatitis in Oscar. Note to dermal edema and vacu- Figure 6. Section of Dactylogyrus sp. in gill of Oscar. Note oles in epidermal cells (blue arrows) and penetration of inflam- the necrosis of attachment place of parasite (H&E, Bar 50µm) matory cells between epithelial and dermal cells (black arrows) (H&E, Bar 50µm) AACL Bioflux Volume 5 | Issue 2 Page 90 http://www.bioflux.com.ro/aacl Mousavi et al 2012 Discussions fresh water leech. Epistylis sp. and the related species Vorticella sp., are sessile and stalked ciliated protozoans generally found Fish gills participate in many important functions such as respi- attached to vegetation or crustaceans (Saglam & Sarieyyupoglu ration, osmoregulation and excretion and remain in close con- 2002). In high organic water, they proliferate and attach to fish tact with external environment and it is particularly sensitive to and eggs. They frequently affect goldfish and many species of changes in the quality of the water (Mazon et al 2003; Camargo bottom-dwelling freshwater fish.Epistylis sp. first appeared on & Martinez 2007). Gills are generally considered as a good in- the tips of both dorsal and pectoral fin spines. Then, Epistylis dicator for water quality and it will be a model for studies of colonies spread down the spines and eventually covered much environmental impacts. of the anterior region of the body. Epithelial tissue and under- In the current study, Dactylogyrus sp. was found as the most lying bone are eroded away at the site of attachment. Both have common parasite in Oscar and Discus fish. Jalali & Barzegar been isolated from ulcerated areas in association with Aeromonas (2005) reported that this parasite is commonly seen in fresh wa- bacteria (Noga 2010). The infected fish showed lethargy and ter fish in intensive culture.Dactylogyrus sp. was found in two erosion on gill filament. The infected fish were seen with other Cyprinidae fish (Alburnus alburnus and Carassius carassius) in parasites in this study, so it is suggested that the symptoms ap- freshwaters from Turkey (Koyun 2011). The oviparous dacty- peared in this fish, are mainly caused by other parasites. However logyrids are primarily gill parasites of freshwater fish but they this ciliophora is potentially harmful if it is in large number es- may be seen in skin of freshwater fish (Noga 2010). The fish pecially to gill tissue where gas exchange may be impeded by infected with dactylogyrus showed clinical symptoms including the large numbers of parasites physically covering the gills. It the lethargy, unilateral swimming and erosion on gill filament is not known to kill or damage fishes, but may slightly reduce and scale loss (Longshaw & Feist 2001; Baker et al 2007). Gill their productivity (Bunkley Williams & Williams 1994). filament fusion, secondary filament hyperplasia and aneurism The histopathological lesions which were observed in this were reported in fishes which were infected by Dactylogyrus study include lamellar fusion, hyperplasia, purulent bronchi- sp. after Jalali & Barzegar (2005), which is in agreement with tis and aneurysm. Some alternations in blood vessel may oc- results obtained from this study. Infection with Gyrodactylus cur when fishes suffer from severe types of stress. In this case, sp. was only seen in Oscar fish. The viviparous gyrodactyl- damaged pillar cells can result in an increased blood flow inside ids are skin and gill parasites of both freshwater and marine the lamellae and cause blood congestion or even an aneurysm fish. Various species of Gyrodactylus are pathogenic to Eels, (Rosety-Rodriguez et al 2002). Hyperplasia of epithelial cells Salmonids, Cyprinids, Ictalurids, Cichlids and Pleuronectids and fusion of some secondary lamella are examples of defense (Noga 2010). Histopathological lesions of Gyrodactylus sp. on mechanisms of gills. gill and skin are the same as Dactylogyrus sp. (Jalali & Barzegar Purulent bronchitis was found in 10% of Oscar and Discus fish. 2005; Noga 2010). This is a chronic alteration and in this case, a lot of inflamma- I. multifiliis was observed in 20% of Oscar fish but was not tory cells (lymphocytes and macrophages) can found among la- found in Discus fish. Ich is one of the most common diseases of mella (Ferguson et al 2006). Especially, eosinophilic granular freshwater fish and virtually all freshwater fish are susceptible cells were seen. These granular cells were similar to submucosal to infection and up to 100% mortality may occur (Noga 2010). layer cells of intestine. These types of cells increase in chronic Stress plays a major role in Ich epidemia. Outbreaks are also diseases and maybe are inflammatory cells. Secretion of these more severe at high fish density. In current study, all infected cells in the gills justified that these are destruction and corrup- fish showed white nodules on body and some of them showed tion response to different chemicals and pathogens (Kantham lethargy, increased mucus production and gill filaments hypere- & Richards 1995). mia which is in agreement to results of Smith & Roberts (2010). Edema was found in 50% of Oscar fish. This alteration usually The epithelial cell erosion and ulceration that has been resulted occurs when the environment is not normal (Roberts 2001). By from the entrance of parasites into skin and exit from the host removing of fish pathogens from gill and supplying better en- are probably at least as damaging as its feeding activity while vironmental condition, severe lamellar edema may be treated it is on the host. Lesions produced by the parasites may also (Nero et al 2006; Schlacher et al 2007). lead to secondary microbial infections (Noga 2010). Dermatitis In this study, epitheliocystis was found in 35% of Oscar fish. and purulent bronchitis which were seen in this study may be Hyperplasia and aneurism were observed in the gill tissue of affected by Ich infection in Oscar and Discus. all of these infected cases. Epitheliocystis disease is a common Trichodina sp. was also found in Oscar and Discus fish. Clinical condition that has been observed in various teleosts as an in- signs due to infestation by Trichodina sp. were scale loss and cidental finding (Bradley et al 1988; Turnbull 1993; Groff et lethargy. Noga (2010) and Baker et al (2007) found lethargy al 1996; Polkinghorne et al 2010). This incidental form of the in fishes that were infected withTrichodina sp.. Many trichod- disease has been characterized as a nonlethal or chronic con- inid species infest marine and freshwater fish (Noga 2010, dition because of the apparent mild infection and associated Thilakarante 2003). They have a similar morphology which host response that is typically absent or limited to a mild epi- includes cilia, a circular shape and a ring of hook-like denti- thelial hyperplasia (Roberts 2001; Noga 2010; Polkinghorne cules. The parasites are commonly found on skin and gills of et al 2010). A severe form of the disease has also been docu- fish from waters with high organic loads. In a heavily infected mented and characterized as a hyperinfection because of se- fish, their adherence and suction on the epithelium may cause vere infection and the associated host response, which has been enough damage to produce the clinical signs of anorexia and described as a diffuse, severe, proliferative epithelial hyperpla- weight loss (Noga 2010). sia often with branchial lamellar fusion and excessive mucus Epistylis sp. and Vorticella sp. were found in both Oscar and production (Bradley et al 1988; Groff et al 1996). Progression Discus fish. Saglam & Sarieyyupoglu (2002) reportedEpistylis on from the chronic to the proliferative form of the disease may AACL Bioflux Volume 5 | Issue 2 Page 91 http://www.bioflux.com.ro/aacl Mousavi et al 2012 be secondary to stressful conditions, such as low temperature Jalali, B., Barzegar, M., 2005. Dactylogyrids (Dactylogyridae: Monogenea) (Crespo et al 1990). on common carp (Cyprinus carpio L.) in freshwaters of Iran and de- Epitheliocystis histologically affected chloride and epithelial scription of the pathogenicity of D. sahuensis. Journal of Agricultural cells and caused the single cell swelling (Bradley et al 1988). Science and Technology 7:9-16. In the most samples that infected with Epitheliocystis, eosino- Johnson, L. L., Stehr, C. M., Olson, O. P., Myers, M. S., Mccain, B. philic granular cells were observed, that is compatible with re- B., Chan, S. L., Varanasi, U., 1993. Chemical contaminants and hepatic lesions in winter flounder (Pleuronectes americanus) from sults reported by Groff et al (1996) on Acipenser transmonta- the northeast cost of the United States. Environmental Science and nus Richardson, 1836. Technology 27:2759-2771. Infestation with Ichthyobodo sp. was 25% in Oscar fish and 5% Kantham, K. P. L., Richards, R. H., 1995. Effect of buffers on the gill in Discus fish. Infestation withIchthyobodo sp. caused hyperpla- structure of common carp, Cyprinus carpio L., and rainbow trout, sia of epithelial cells and fusion of secondary lamella. In almost Oncorhynchus mykiss. Journal of Fish Disease 18:411-423. all of infected fish withIchthyobodo sp., hyperplasia, fusion and Koyun, M., 2011. Seasonal distribution and ecology of some Dactylogyrus aneurism were observed (Molnar & Baska 1993; Noga 2010). species infecting Alburnus alburnus and Carassius carassius Dermatitis was the histopathological alterations found in skin (Osteichthyes: Cyprinidae) from Porsuk River, Turkey. African of fish. The skin lesions are common in fish and the fish skin is Journal of Biotechnology 10(7):1154-1159. constantly exposed to the water environment and external en- Longshaw, M., Feist, S., 2001. Parasitic diseases. In: BSAVA man- vironment, is susceptible to the toxins, environmental pollution ual of ornamental fish health. Wildgoose W. H. (ed). 2nd edition, and pathogens (Abalaka et al 2010). Gloucester: BSAVA, pp. 167–83. Mazon, A. F., Pinheiro, G. H. D., Fernandes, M. N., 2002. Hematological Conclusions and physiological changes induced by short-term exposure to cop- per in the freshwater fish Prochilodus scrofa. Brazilian Journal of Histopathological lesions in Oscar were in high rate in com- Biology 62(4):621-631. parison with lesions which were seen in Discus and they are in Molnar, K., Baska, F., 1993. Scientific report on intensive training relation to parasitic infestation. course on parasites and parasitic disease of freshwater fishes of Iran. November 15-25, Fisheries Comany of Iran. Acknowledgements Nero, V., Farwell, A., Lister, G., VanDer Kraak, L. E. J., Lee, T., Van Meer, M., Dixon, D. G., 2006. Gill and liver histopathological chang- This work was financially supported by the Khorramshahr es in yellow perch (Perca flavescens) and goldfish (Carassius au- University of Marine Science and Technology under research ratus) exposed to oil sands process-affected water. Ecotoxicology project no.36. and Environmental Safety 63:365–377. Noga, E. J., 2010. Fish disease: diagnosis and treatment. 2nd edition, References Blackwell Publishing, 519 pp. Abalaka, S., Yakasai, F. M., Doguwar, G. B. N., Makonjuola, K. H., Polkinghorne, A., Schmidt-Posthaus, H., Meijer, A., Lehner, A., Vaughan, 2010. Histopathological changes in the gills and skin of adult Clarias L., 2010. Novel chlamydiales associated with epitheliocystis in a gariepinus exposed to Ethanolic extract of Parkia biglobosa pods. leopard shark Triakis semifasciata. Diseases of Aquatic Organisms Basic and Applied Pathology 3:109–114. 91:75-81. Baker, D. G., Kent, M. L., Fournie, J. L., 2007. Parasites of fishes. In: Poulin, R., 1992. Toxic pollution and parasitism in freshwater fish. Flynn’s parasites of laboratory animals. Baker D. G. (ed). 2nd edi- Parasitology Today 8(2):58–61. tion. Hoboken, Blackwell, 840 pp. Reavill, D. R., Roberts, H. E., 2007. Diagnostic cytology of fish. Veterinary Bradley, T. M., Newcomer, C. E., Maxwell, K. O., 1988. Epitheliocystis Clinics of North America Exotic Animal Practice10:207–234. associated with massive mortalities of cultured lake trout, Salvelinus Roberts, R. J., 2001. Fish Pathology. 3rd edition, Roberts R. J., Saunders namaycush. Diseases of Aquatic Organisms 4:9-18. W. B. (eds), 472 pp. Bunkley Williams, L., Williams, E. H., 1994. Diseases caused by Rosety-Rodríguez, O. M., Rosety, F. J., Rosety, M. J. A., Carrasco, Trichodina spheroidesi and Cryptocaryon irritans (Ciliophora) in C., 2002. Morpho-histochemical changes in the gills of turbot, wild and coral reef fish. Journal of Aquatic Animal Health 6:360-361. Scophthalmus maximus L., induced by sodium dodecyl sulfate. Camargo, M. M. P., Martinez, C. B. R., 2007. Histopathology of gills, Ecotoxicology and Environmental Safety 51:223-228. kidney and liver of a Neotropical fish caged in an urban stream. Saglam, N., Sarieyyupoglu, M., 2002. A study on Tetrahymena pyri- Neotropical Ichthyology 5(3):327-336. formis (Holotrichous) and Epistylis sp. (Peritrichous) found on fresh- Crespo, S., Grau, A., Padrós, F., 1990. Epitheliocystis disease in the cul- water leech, Nephelopsis obscura. Pakistan Journal of Biological tured amberjack, Seriola dumerili Risso (Carangidae). Aquaculture Sciences 5(4):497-498. 90:197–207. Schlacher, T. A., Julie, A., Mondon, R., Connolly, M., 2007. Estuarine Ferguson, H., Bjerkas, E., Evensen, O., 2006. Systemic Pathology of fish health assessment. Evidence of wastewater impacts based on Fish. 2nd Edition. Scotian Press, pp. 120-125. nitrogen isotopes and histopathology. Marine Pollution Bulletin 54:1762–1776. Groff, J. M., LaPatra, S. E., Munn, R. J., Anderson, M. L., Osburn, B. I., 1996. Epitheliocystis infection in cultured white sturgeon Smith, S. A., Roberts, H. E., 2010. Parasites of fish. In: Fundamentals of (Acipenser transmontanus): antigenic and ultrastructural similari- Ornamental Fish Health. Roberts H. E. (ed). Hoboken (NJ): Wiley- ties of the causative agent to the chlamydiae. Journal of Veterinary Blackwell, pp. 102-112. Diagnostic Investigation 8:172-180. Thilakarante, I. D. S. I. P., Rajapaksha, G., Hewakopara, A., Rajapakse, Hoffman, G. L., 1999. Parasites of North American freshwater fishes. R. P. V. J., Faizal, A. C. M., 2003. Parasitic infections in freshwa- 2nd edition, Ithaca (NY), Cornell University Press, 539 pp. ter ornamental fish in Sri Lanka. Diseases of Aquatic Organisms 54:157-162.

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Turnbull, J. F., 1993. Epitheliocystis and salmonid rickettsial septice- •Seyed Mohammad Mousavi, Department of Fisheries, Faculty mia. In: Bacterial diseases of fish. Inglis V., Roberts R. J., Bromage of Marine Natural Resources, Khorramshahr University of N. R. (eds.), Halsted Press, New York, pp. 237-254. Marine Science and Technology, Khorramshahr, Iran, P.O. Box: 669, Post Code No.: 43175-64199, Tel. No.: 06324236409, Fax Authors No.: 06324230551, Mobile Tel. No.: 09166112522, e-mail: sm- [email protected] •Forough Mohammadi, Department of Fisheries, Faculty of Marine Natural Resources, Khorramshahr University of Marine •Annahita Rezaie, Department of Pathobiology, Faculty of Science and Technology, Khorramshahr, Iran, P.O. Box: 669, Veterinary Medicine, Shahid Chamran University of Ahvaz, Post Code No.: 43175-64199, Tel. No.: 06324236409, Fax Ahvaz, Iran., Mobile Tel. No.: 09166112522, e-mail: a.rezaie@ No.: 06324230551, Mobile Tel. No.: 09163319073, e-mail: scu.ac.ir [email protected]

Mohammadi, F., Mousavi, S. M., Rezaie, A., 2012. Histopathological study of parasitic Citation infestation of skin and gill on Oscar (Astronotus ocellatus) and discus (Symphysodon discus). Aquaculture, Aquarium, Conservation & Legislation 5(2):88-93. Editors Ionel-Claudiu Gavriloaie and Ştefan C. Vesa Received 02 February 2012 Accepted 23 April 2012 Published Online 24 June 2012 Funding Khorramshahr University of Marine Science and Technology, Research Project No.36 Conflicts/ Competing None reported Interests

AACL Bioflux Volume 5 | Issue 2 Page 93 http://www.bioflux.com.ro/aacl Aquaculture, Aquarium, Conservation & Legislation OPEN ACCESS International Journal of the Bioflux Society Research Article

Production of Aphanius mento (Heckel, 1843) under controlled conditions

1Soner Sezen, 2Murtaza Olmez

1 Ministry of Food, Agriculture and Livestock Mediterranean Fisheries Research Production and Training, Antalya, Turkey. 2 Suleyman Demirel University Egirdir Fisheries Faculty, Isparta, Turkey.

Abstract. In this study, it was aimed to determine the adaptation capability to controlled conditions, reproductive behaviors, and production of Aphanius mento caught from Kirkgoz Springs, Antalya. Fish material for broodstock were caught using fine mesh tulle net and bag net with 2.5 mm mesh size. Twenty five female and 10 male breeding fish acclimated to experimental conditions in Aquarium Department Unit of Ministry of Food, Agriculture and Livestock Mediterranean Fisheries Research Production and Training Institute were spawned by providing egg collectors to the tank. Aphanius mento eggs were spherical, transparent and sticky. Examination with the microscope revealed that the eggs were full of pale yellow egg yolk with 7-8 lipid droplets and had fibrous sticking filaments on the surface. The average egg diameter was 1.59±0.50 mm (n=8). Hatching were observed at 11th day at 22.5±1.0ºC water temperature whereas at 8th day at 27.0±0.45ºC. Total length of newly hatched larvae was 5.1±0.07 mm, and swimming were competed at 4th day. Average size of one-week larvae was 5.6±0.2 mm. At this stage fins were fully developed, the larvae were able to swim freely and take exogenous food. Mean length of the fish was 2.7±0.2 cm on 7th month and a juvenile fish had an appearance of morphologically mature fish. Key Words: Cyprinodontidae, Aphanius mento, reproductive characteristics, larval development. Ozet. Bu calismada, Antalya Ili Kirkgoz Kaynagi’ndan yakalanan Aphanius mento turunun kontrollu ortam kosullarına uyumu, uretimi ve ureme davranislarinin belirlenmesi amaclanmistir. Damizlik olarak kullanilan erkek ve disi baliklar Kirkgoz Kaynagi’ndan ince gozlu tul ag ve 2,5 mm goz acikliginda torbali aglarla yakalanmistir. Gida Tarim ve Hayvancilik Bakanligi Akdeniz Su Urunleri Arastirma Uretim ve Egitim Enstitusu Akvaryum Birimi’nde hazirlanan ortam kosullarina uyumu gerceklestirilen 25 adet disi - 10 adet erkek damizlik balik, yumurta kolektorleri bulunan ureme tanklarina konularak, yumurtlamalari saglanmistir. Aphanius mento turunun yumurtalari kuresel sekilli, seffaf ve yapiskan ozel- likte olup, mikroskop altinda incelemede 7-8 adet yag damlacigi icerdigi, solgun sari renkli yumurta sarisi ile dolu oldugu ve disinda ipliksi yapisma flamentleri icerdigi gorulmustur. 8 adet disi baliktan alinan yumurtalarda ortalama yumurta capi 1,589±0,50 mm olarak olculmustur. Aphanius mento yumurtalarinda, 22,5±1,0ºC su sicakliginda dollenmeden sonra 11. gunde, 27,0±0,45ºC’ de ise 8. gunde larva cikisi gozlenmi- stir. Yumurtadan yeni cikmis larvanin total boyu 5,1±0,07 mm olarak olculmus, butun larvalar 4. gunde serbest yuzmeye gecmistir. 1 haftalik larvalarin boy ortalamasi 5,6±0,2 mm olup, yuzgecler tam olarak gelismistir, larvalar serbest olarak yuzmekte ve disaridan yem alabilmektedir. 7. ayda baliklar ortalama 2,7±0,2 cm’e ulasmis, yavru balik morfolojik olarak ergin balik gorunumu kazanmistir. Anahtar Kelimeler: Cyprinodontidae, Aphanius mento, ureme ozellikleri, larval gelisim. Copyright: 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. Corresponding Authors: M. Olmez, [email protected] Introduction al 2011) and limited competition ability with other introduced species such as Gambusia (Oltra & Todoli 2000). For instance, Fish play a significant role in examination of biological devel- Aphanius almiriensis, A. baeticus, A. burduricus, A. iberus, A. opment from experimental and conceptual perspectives due richardsoni, A. sirhani, A. splendens, A. transgrediens have to their enormous number of species among the animal king- been reported as Aphanius species which are under the threat dom and various living styles. Beside this, there is an increas- (Anonymous 2010). Investigation of reproductive characteris- ing importance of knowing egg number of fish prior to spawn- tics of fish defines what should be done for the species future. ing and survival rate after hatching because the early phase of Therefore, investigations of biological aspects of fish in arti- development has a great impact on annual production volume ficial and natural ecosystems are of significance for more effi- (Sahandi 2011). For this reason, egg hatching and first feeding cient protection of the environment (Blaustein & Byard 1993). times, larval metabolism and relationship between chemical During the acclimation, adaptation and domestication following and physical factors that affect fish larvae populations can be transfer from the natural environment, certain sets of criteria regarded as approaches to better understand the fish population should be considered. In this context, selection of local species (Bagenal & Braum 1978). has advantages such as becoming familiar with the artificial As other Eurasian fish populations (Györeet al 2011; Lenhardt conditions faster resulting in less specific problems and more et al 2011), some Aphanius species populations belonging to economic production. Moreover, fish caught from the wild for Cyprinodontidae have greatly decreased due to anthropogenic formation of broodstock can have difficulties with adaptation drying processes of their habitats, pollutants, new parasites from to the artificial environment and even die due to starvation and South East Asia (Paladini et al 2011; Ebrahimzadeh Mousavi et

AACL Bioflux Volume 5 | Issue 1 Page 94 http://www.bioflux.com.ro/aacl Sezen et al 2012 stress resulting from limited feeding activity. Thus, some species may be started to get domesticated at early stages. Monitoring feeding activities of fish, determination of most appropriate culture form, good facility design, and controlling the reproduction are major strategies in aquaculture (Liao & Huang 2000). In 1955 the number of cultured exotic and aquarium species were about 410 and 40 respectively whereas today these figures are expressed in thousands (Fossa 2003). Almost every day a new species is discovered and transferred to culture environments to introduce to aquarium world. People are more interested in these new species rather than those which are easily accessible. Hence, potential species for aquarium sector should be investi- Figure 2. Male and female members of Aphanius mento used gated without damaging to the nature and introduced to sector by in the experiment rearing in captivity. In this way, it would be possible to bringing value-added to an economically unexploited living organism. In adaptation of species to the artificial condition and determi- Scientific studies inAphanius genus have concentrated mainly on nation of reproductive behaviors, 10 male with 4-6 cm length determination faunistic and biological aspects, and the degree of and 1.1-3.7g body weight, and 25 female with 4.5-7.6 cm length kinship among the species members (Wildekamp 1993; Frenkel and 1.3-6.5 g body weight were used. The broodstock used were & Goren 1997; Wildekamp et al 1999; Oltra & Todoli 2000; caught from Kirkgoz Springs, Antalya, using fine mesh tulle net Hrbek & Meyer 2003; Hrbek & Wildekamp 2003; Bardakci et and bag nets with 2.5 mm mesh size. The seized fish were then al 2004; Kucuk & Ikiz 2004; Sari et al 2007; Guclu & Kucuk transferred using 10L plastic bags including 1/3 water and 2/3 2008, 2011; Karsli & Aral 2010). Nevertheless, their produc- pure oxygen (Berka 1986) to tank system in the Aquarium Unit. tion in the controlled conditions is extremely important in terms The system consisted of two fiber tanks one (1.20x1.20x0.50 of protection of ecological balance as well as ensuring the spe- m) for keeping the fish and the other (240 L) for filtration. cies continuity. Therefore the present study was planned to ac- Within the filtration tank, there were three floors shelves. Bio- climate Aphanius mento in view to investigate its potential to balls and coral parts and volcanic stones were placed into the adapt to artificial production conditions, and to determine their first floor whereas zeolite and staple fiber into second and thirds behavior and production potential. floors respectively. A bath treatment using 10 g/L stock solution of methylene blue + formaldehyde at 12 mL/ton dose was Materials and Methods applied against possible invasion of parasite and fungi (Noga 2000; Sahandi & Hajimoradloo 2011). To ease the acclimation The study was carried out between March 2010 and March of fish, pebbles and aquatic plants growing in the natural envi- 2011 at the Aquarium Unit of Mediterranean Fisheries Research ronment of fish were placed into the culture tank. Production and Training Institute under the auspices of Ministry Tank water temperature was kept at the same level as the natu- of Food, Agriculture and Livestock. The study material was the ral water at the beginning, and then gradually increased to 25°C matured male and female individuals of Aphanius mento caught using an electrical heater. PVC knee pipes were also provided from Kirkgoz springs, Antalya (Figs 1 and 2). to the tank to ensure shelter and reduce aggressive behavior of the males. Once the fish were adapted, all the materials were taken out from the tank and spawning time was tried to get controlled. Feeding was done thrice a day using live and dry feeds. Tank water was renewed weekly at a rate of 30%. Water temperature, dissolved oxygen and pH were monitored whereas nitrite and nitrate weekly. A stable artificial photoperiod was applied over the study (14L: 10D) (Frenkel & Goren 1997). First feeding at the artificial conditions was done with Daphnia sp. and Palaemon sp.. During the acclimation period, commercial diets (Table 1) were also used together with the live food organisms. After acclimation, a combination of live and artificial dry feed was given thrice a day. Larvae after hatching were fed with rotifer for one week, and then artemia and rainbow trout starter feed. Raffia and stable fiber as egg collector were placed on the tank bottom for the ac- Figure 1. Kirkgoz springs, Antalya - Turkey (Guclu 2003). climated broodstock to release their eggs easily (Ozen & Timur 1999). The existence of released eggs on the collectors were For acclimation and adaptation of the species, firstly information periodically checked. The collected eggs were treated with 2 regarding to natural living systems, water quality and environ- mg L-1 methylene blue against fungi (Noga 2000). For obser- mental factors was gathered (Liao & Huang 2000). Accordingly, vation of the development of fertilized eggs and measurements data of previous studies on characterization of population fea- a binocular microscope was used while for measuring egg di- tures (Guclu & Kucuk 2008, 2011) were accepted as a basis to ameters and larval lengths on the pictures taken a software, the present investigation. Image J, was used.

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Table 1. The nutrient content of commercial feeds used infeed- of the staple-raffia with eggs from the tank and replaced with ing broodstock (%)* new ones, the female were seen to lay the eggs again. By this Nutrients content Feeds way, it was determined that the female does not lay the eggs at Tetra Cichlid Sera Discus Trout Pellet once but several times. Feed Feed Diet (2 mm) Based on spawning period in the nature, the microscopic examination of the eggs collected was done in May 2010. The ex- Crude protein 31.6 46.2 50 amination revealed that the eggs are spherical, transparent and Crude fat 2.75 6.2 15 sticky, and full of egg yolks with 7-8 oil droplets, and have ad- Crude cellulose - 3.7 3 hesion filaments on the surface. Average diameter of the eggs Crude ash 5.04 11.2 13 from eight females was 1.589±0.50 mm. The raffias with eggs that were not used for measurement were (*) Declared values by the producers kept in 10L incubation aquarium. The eggs that were examined under the microscope were hatched out in 11 days at 22.5±1.0ºC Results and Discussion while those kept in 10L aquarium in 8 days at 27.0±0.45ºC (216.9 degree days). Newly hatched larvae tended to stay at the Some physical and chemical characters of water samples taken aquarium bottom; at three days after hatch (DAH) they started from the spring where A. mento occur and experimental unit to swim freely and at 4 DAH all larvae completed to pass free are given in Table 2. swimming stage. Average body length at 7 DAH was 5.6±0.2 mm and the fins had not been completely formed at that time. Table 2. Some chemical parameters of water used in the experiment During this period, the larvae could freely swim; take the rotifer and artemia easily. From this stage on, feeding was contin- Parameters Kirkgoz springs Experiment unit ued with live organisms together with the commercial dry foods and on seventh month average length reached 2.7±0.2 cm and Temperature (ºC) 14-17 18.5-28.5 morphologically resemble to adult individuals. pH 7.6 7.8 In the present study, centered on secondary freshwater fishes, Dissolved oxygen (mg L-1) 5 8.4 reproductive features and larval development of A. mento under Oxygen saturation (%) 50 89 the artificial conditions were tried to be determined by observa- Electrical conductivity (25 tions and measurements. We think the reasons of color chang- 809 744 ºC μS cm-1) es and tendency of hiding observed at the beginning of the acclimation period are stress factors. However, we also consider Alkalinity (mg L-1 CaCO ) 252 229 3 that a short-term-adaptation period observed in this study was probably due to very close water quality of the experiment unit to the spring water. Quality parameters of the water used in the The minimum and maximum water temperatures in the ex- experiment unit were always kept below the critical thresholds periment unit were recorded as 18.5ºC in winter and 28.5ºC in for fish health by setting up a filter system and regular renewal -1 summer, pH 7.5 and 8.7, dissolved oxygen 3.8 and 6.8 mg L , with fresh water. while nitrite and nitrate were never reached the threshold levels. In our study, spawning behaviors displayed by A. mento are Matured female and male individuals were collected from the similar to those observed in Fundulus luciae by Byrne (1978). places which are shallow, plentiful with aquatic plants and slow In both species, the male adopts and protects his territory and in water flow, using tulle nets. During the collection, body color then first approaches the female, displays assault-like behav- particularly in males, got darkened but once arrival at the ex- ior, tries to attract her to the collector to let it lay and the inter- periment unit, the darkening disappeared. During the acclima- course finally results in releasing the eggs. On the other hand, tion period, fish appeared to hide between the plants and peb- Fundulus waccamensis males approaches to incoming female bles in the tank. to the nest by swimming up and down, show their body color After the acclimation, feeding with daphnia and bait shrimp help and fin glows, lead the female to the nest and then spawning the fish getting used to commercial dry food. Two weeks after takes place (Shute et al 1983). acclimation matured male and female started to show reproduc- Guclu (2003) reported that natural spawning time of A. mento tive behaviors and some eggs were seen on the plant leaves that occurs between February and July but we observed it can takes had been previously placed into the tank. To take the spawning place even in November under controlled conditions. A. mento time under control, the materials onto which fish can lay the resembles to Cyprinodontiformes species, which are annual fish eggs were taken out. Only knee pipes were left as shelter for species, in terms of spawning conditions and place. For exam- timid females especially in the case of fight between the males. ple, A. mento experienced 25ºC water temperature in the ex- But it was seen that the males tended to adopt these shelters. periment tank during spawning, while the spawning tempera- Staple fiber and raffia were put into the tank for the fish to lay ture for Fundulus luciae was reported as 20-25ºC (Byrne 1978). their eggs on after color differentiation and chasing behaviors. In Likewise, staple - raffia egg collectors used in the present ex- this way the male were forced to select these materials as nest. periment resemble to the mop that was used by Byrne (1978). Dominant males became apparent with their darkening color The eggs can be found in clusters including 2-4 eggs on the mop and fight off the other males, and they started to attract the fe- and raffias (Byrne 1978). Guclu (2003) determined the egg di- males with assault-like movements. From this moment on, reg- ameter of A. mento as 1034.783±50.262 µm. The minimum di- ular egg control on the stable-raffia was started. Once removal ameter was reported to be 36 µm whereas the maximum 1756

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µm. Considering the spawning time, the egg diameters between of endemism, but not yet in danger of extinction, even so under this and our study are quite similar. The examination revealed the threat of environmental pollutions. The data obtained are of that the eggs are spherical, transparent and sticky, and full of importance since they are the first in the field of the production egg yolks with 7-8 oil droplets, and have adhesion filaments in controlled conditions. Moreover, a production model and a on the outer. Average diameter of the eggs from eight females database about the reproductive biology of this species, which was 1.589±0.50 mm. Where as in Fundulus luciae, average di- would be used in aquarium sector because of their colorful and ameter of mature eggs are 1.96 mm (Byrne 1978). The eggs of attractive appearance have been tried to be formed. Furthermore, F. luciae are slightly elliptic, sticky and have 21.8 oil droplets with the production in captivity the chances of extinction of the which are concentrated around the bottom (Byrne 1978). The species shall be reduced. egg diameter of Cynolebias viarius are 1.7 mm and they are transparent, covered with a spherical unicellular membrane and References has a homogenous egg yolk except in the region of oil droplets Abraham, B. J., 1985. Species Profiles: Life Histories and Environmental (Arezo et al 2005). On the other hand, Adinica xenica eggs are Requirements of Coastal Fishes and Invertebrates (Mid-Atlantic) yellow in color, their cell membranes have sticky filaments and Mummichog and Striped Killifish. Biological Report 82(11.40). the diameters can change between 1.70 and 1.90 depending on Anonymous, 2010 http://www.earthsendangered.com/list_html.asp. the water salinity. Size of oil droplets may change according as species genetic features as well as the age of the embryo Arezo, M. J., Pereioro, L., Berois, N., 2005. Early development in the annual fishCynolebias viarius. Journal of Fish Biology 66:1357–1370. (Cunningham & Balon 1985). Newly fertilized eggs of F. waccamensis are colorless, spherical and 2-3 mm in diameter and Armstrong, P. B., Child, J. S., 1965. Stages in the normal development of Fundulus heteroclitus. The Biological Bulletin 128(2):143-168. have countless oil droplets (Shute et al 1983). F. heteroclitus eggs are spherical in shape, 2.0 mm in diameter and clear yel- Bagenal, T. B., Braum, E., 1978. Eggs and Early Life History. In: Methods for Assessment of Fish Production in Freshwaters, Blackwell low-amber in color. F. majalis eggs are spherical, 2.0-3.0 mm, Science Publication, pp.165-201, Oxford, UK. translucent, yellow-amber and slightly sticky (Abraham 1985; Bardakci, F., Tatar, N., Hrbek, T., 2004. Genetic relationships between Armstrong & Child 1965). Anatolian species and subspecies of Aphanius Nardo, 1827 (Pisces, In the present investigation, hatching occurred in 11 days at Cyprinodontiformes) based on RAPD markers. Biologia, Bratislava, 22.5±1.0ºC. This is on par with about 216.9ºC days. The differ- 59(5):559-566. ences between the results resulted from the difficulties experi- Berka, R., 1986. The transport of live fish. A review. EIFAC Tech. enced during the stabilization of water temperature. Iwamatsu Pap., 48, 52 p. (2004) reported that hatching occurs in 9 days at 26±0.1ºC in Blaustein, L., Byard, R., 1993. Predation by a cyprinodontid fish, Japanese killifish (Oryzias latipes). Of Cyprinodontiformes Aphanius mento, on Culex pipiens: effects of alternative prey and species, in A. xenica hatching last for 9-10 days at 26-28ºC vegetation. Journal of the American Mosquito Control Association (Cunningham & Balon 1985). Whereas in F. luciae, eggs hatch 9(3):356-358. 10-14 days at 20-25ºC after fertilization (Byrne 1978). The eggs Byrne, D. M., 1978. Life History of the Spotfin Killifish, Fundulusluciae of F. heteroclitus hatch out within 16 days at 20ºC (Armstrong (Pisces: Cyprinodontidae), in Fox Creek Marsh, Virginia. Estuaries & Child 1965). We can conclude from these reports that em- 1(4):211-227. bryologic development and hatching duration can display varia- Cunningham, J. E. R., Balon, E. K., 1985. Early ontogeny of Adinia tion depending on fish species and water temperature. Similarly, xenica (Pisces, Cyprinodontiformes): 1. The development of em- Markofsky & Matias (1977), who discuss the effects of water bryos in hiding. Environmental Biology of Fishes 14(213):115-166. temperature on embryological development stated that a 5ºC Ebrahimzadeh Mousavi, H. A., Behtash, F., Rostami-Bashman, M., increase in temperature could reduce the hatching down to 10 Mirzargar, S. S., Shayan, P., Rahmati-holasoo, H., 2011. Study of days (Arezo et al 2005). Argulus spp. infestation rate in Goldfish,Carassius auratus (Linnaeus, In the current study, total length of newly hatching larvae of 1758) in Iran. Human & Veterinary Medicine 3(3):198-204. A. mento was measured as 5.1±0.07 mm. At this stage, it was Fossa, A. S., 2003. Man-Made Fish: Domesticated Fishes And Their seen that posterior, dorsal and anterior ventral sides of the head Place in The Aquatic Trade and Hobby. OFI Journal, Issue 44, pp. are colored and tail fin is not developed yet. In F. heteroclitus, 1, 3-4, 6-10, 12-16. a member of Cyprinidontiformes order, newly hatched larval Frenkel, V., Goren, M., 1997. Some environmental factors affecting length is 4.0-7.7 mm (mean 5.0 mm), whereas in F. luciae 7.0- the production of Aphanius dispar (Rüppell, 1828). Hydrobiologia 11.0 mm (Abraham 1985). In F. waccamensis, coloration has 347:197-207. been reported on the head, dorsal and abdomen regions. In ad- Guclu, S. S., 2003. An investigation of features feeding, growth and dition, larval body length at hatching in this species is 8.1 mm reproduction of Aphanius mento (Heckel in: Russegger, 1843) in Spring of Kirkgoz (Antalya). Suleyman Demirel University while in Fundulus diaphanus 5.3-6.4 mm (Shute et al 1983). Graduate School of Applied and Natural Sciences, Master thesis, When A. mento is compared with other species, larval length 43 pp, Isparta [in Turkish]. at hatching in zebra fish (Danio rerio) is 3.1 mm, in medaka Guclu, S. S., Kucuk, F., 2008. Population Age, Sex Structure, (Oryzias latipes) 3.8-4.2 mm, in red head goldfish (Carassius Growth and Diet of Aphanius mento Heckelin: Russegger, 1843 auratus) 2.54±0.01 mm, and inkalikoryukin goldfish 4.3-4.4 (Cyprinodontidae:Teleostei), at Kirkgoz Spring, Antalya-Turkiye. mm (Kimmel et al 1995; Iwamatsu 2004; Karsli et al 2007; Turkish Journal of Fisheries and Aquatic Sciences 8:269-274. Savas et al 2006). Guclu, S. S., Kucuk, F., 2011. Reproductive biology of Aphanius men- In the present study, some information has been gathered based to (Heckel in: Russegger, 1843) (Osteichthyes:Cyprinodontidae) in on obsevations and examinations of reproductive traits and larval Kirkgoz spring (Antalya-Turkey). Turkish Journal of Fisheries and developments A. mento, which has a significant place in terms Aquatic Sciences 11:323-327.

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Györe, K., Józsa, V., Wilhelm, S., 2011. Monitoring of the fish com- Paladini, G., Huyse, T., Shinn, A. P., 2011. Gyrodactylus salinae n. sp. munity in the Hungarian reach of River Tisza in 2009. Studia (Platyhelminthes: Monogenea) infecting the south European tooth- Universitatis “Vasile Goldiş” Seria Ştiinţele Vieţii 21(4):793-801. carp Aphanius fasciatus (Valenciennes) (Teleostei, Cyprinodontidae) Hrbek, T., Meyer, A., 2003. Closing of the Tethys Sea and the Phylogeny from a hypersaline environment in Italy. Parasites & Vectors 4:100. of Eurasian Killifishes (Cyprinodontiformes: Cyprinodontidae). Sahandi, J., 2011. Reproduction of Persian Gulf anemone fish (Amphiprion Journal of Evolutionary Biology 16:17-36. clarkii) in captive system. Aquaculture, Aquarium, Conservation & Hrbek, T., Wildekamp, R. H., 2003. Aphanius villwocki, a new spe- Legislation 4(5):704-708. cies from the Sakarya Basin of central Anatolian plain, Turkey Sahandi, J., Hajimoradloo, A., 2011. Hole-in-head disease: New method (Teleostei:Cyprinodontiformes). Ichthyological Exploration of of treatment in flower horn ornamental fish. Human & Veterinary Freshwaters 14 (2):137-144. Medicine 3(2):165-169. Iwamatsu, T., 2004. Stages of normal development in the medaka Oryzias Sari, M, H., Ilhan, A., Yesilova, B., 2007. Some biological features of latipes. Mechanisms of Development 121:605–618. mediterranean toothcarp Aphanius fasciatus (Valenciennes, 1821). Karsli, Z., Aral, O., Sahin, D., Dogan, G., 2007. Reproduction, embryo Ege University Faculty of Fisheries Journal of Fisheries and Aquatic and larvae development of red carp Oranda (Carassius auratus L., Sciences 24(3-4):311-314 [in Turkish]. 1758). Turkish Journal of Aquatic Life 3-5 (5-8):643-650 [in Turkish]. Savas, E., Sener, E., Yildiz, M., 2006. Investigation of embryologic Karslı, Z., Aral, O., 2010. Population Age, Sex Structure and Growth and larval development of the gold fish (Carassius sp.). Journal of of Aphanius danfordii (Boulenger, 1890) to Sirakaraağaçlar Stream, the Faculty of Veterinary Medicine, Istanbul University 32(3):7- Turkey. Journal of Animal and Veterinary Advances 9(10):1427-1431. 19 [in Turkish]. Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, Shute, P. W., Lindquist, D. G., Shute, J. R., 1983. Breeding behavior T. F., 1995. Stages of embryonic development of the zebrafish. and early life history of the waccamaw killifish, Fundulus wacca- Developmental Dynamics 203:255-310. mensis. Environmental Biology of Fishes 8(3-4): 293-300. Kucuk, F., Ikiz, R., 2004. Fish fauna of streams discharging to Antalya Wildekamp, R. H., 1993. A World of Killies, Atlas of the Oviparous Bay. Ege University Faculty of Fisheries Journal of Fisheries and Cyprinodontiform Fishes of the World, Vol.I, “The Genera Adamas, Aquatic Sciences 21(3-4):287-294 [in Turkish]. Adinia, Aphanius, Aphyoplatys and Aphyosemion”, Published by the American Killifish Association, Inc., 311p, U.S.A. Lenhardt, M., Gacic, Z., Vukovic-Gacic, B., Poleksic, V., Visnjic-Jeftic, Z., Kolarevic, S., Jaric, I., 2011. Ecological status of serbian rivers Wildekamp, R. H., Kucuk, F., Unlusayin, M., Neer, W. V., 1999. based on an ichthyological assessment. Studia Universitatis “Vasile Species and Subspecies of the Genus Aphanius Nardo 1897 Goldiş” Seria Ştiinţele Vieţii 21(4):855-860. (Pisces:Cyprinodontidae) in Turkey. Turkish Journal of Zoology Liao, I. C., Huang, Y. S., 2000. Methodological approach used for the 23:23-44. domestication of potential candidates for aquaculture. In: Recent advances in Mediterranean aquaculture finfish species diversifica- tion, CIHEAM-IAMZ, 1999/05/24-28, 97-107. Authors Markofsky, J., Matias, J. R., 1977. The effects of temperature and sea- •Soner Sezen, Ministry of Food, Agriculture and Livestock son collection on the onset and duration of diapause in embryos of Mediterranean Fisheries Research Production and Training, the annual fishNothobranchius guentheri. Journal of Experimental Antalya Turkey. Tel: (+90)242 2510585/145, Fax: (+90)242 Zoology 202:49-56. 2510584; GSM: 05056539617, e-mail: [email protected] Noga, E. J., 2000. Fish Disease: Diagnosis and Treatment. Iowa State •Murtaza Olmez, Suleyman Demirel University Egirdir Fisheries Universty Press. ISBN 0-8138-2558-x, pp.536, USA. Faculty, 32500 Egirdir, Isparta, Turkey. Tel: (+90)246313447/1308, Oltra, R., Todoli, R., 2000. Reproduction of the endangered killifish Fax: (+90)246313452; GSM: 05334224835, e-mail: murtazaol- Aphanius iberus at different salinities. Environmental Biology of [email protected] Fishes 57:113–115. Ozen, M. R., Timur, G., 1999. A study on the embryonic and larval development of the artificially propagated eggs ofAlburnus orontis and Phoxinellus handlirschi. Turkish Journal of Biology 23:339- 356 [in Turkish].

Sezen, S., Olmez, M., 2012. Production of Aphanius mento (Heckel, 1843) under Citation controlled conditions. Aquaculture, Aquarium, Conservation & Legislation 5(2):94- 98. Editors Ştefan C. Vesa and I. Valentin Petrescu-Mag Received 13 April 2012 Accepted 09 May 2012 Published Online 24 June 2012 Funding None reported Conflicts/ Competing None reported Interests

AACL Bioflux Volume 5 | Issue 2 Page 98 http://www.bioflux.com.ro/aacl Aquaculture, Aquarium, Conservation & Legislation OPEN ACCESS International Journal of the Bioflux Society Communication

Trends in the marine aquarium trade: the influence of global economics and technology

1,2Andrew L. Rhyne, 1Michael F. Tlusty

1New England Aquarium, Prescott Marine Laboratory, Boston, Massachusetts, United States of America; 2Roger Williams University, Department of Biology and Marine Biology, Bristol, Rhode Island, United States of America.

Abstract. The marine aquarium hobby is a global industry with trade chains on six continents. This industry is undergoing rapid changes in hobbyist preferences that are in part driven by global economies and technology. In an effort to better assess the past trends and help provide insight into future growth and demand, we assessed three different trade data sources, the United States-State of Florida Marine Life Landings, CITES stony coral imports, and United States Fish and Wildlife Service’s Law Enforcement Information Systems import data for ‘live rock’. Data for the past 10 years were analyzed and a clear trend of growth followed by decline was observed with the peak and degree of decline varying for the different trade categories. While the global economic downturn decreased trade volumes across the board other forces have dramatically affected the importation of coral reef products. ‘Live Rock’ and Florida produced ‘live sand’ dramatically decreased two years prior to the global economic recession. The decline of these trade categories coincides with rapid changes in technology used to maintain captive reef aquariums which allowed for the advent of the smaller “nano” reef tanks. Importing and exporting countries, CITES and other international government bodies, concerned Non-Governmental Organizations and scientist would greatly benefit from a deeper understanding of this industry from a producer and consumer standpoint in order to better predict how the marine aquarium hobby will be shaped by external forces in the future.

Key Words: reef aquarium, coral trade, policy, import regulations, LED lighting, nano-reef.

Corresponding Author: A. L. Rhyne, [email protected]

Introduction The marine aquarium hobby has seen rapid changes over the Wood 2001; Wabnitz et al 2003). Recently the trade in coral past 50 years (see Bruckner 2000; Wood 2001; Wabnitz et al reef species has come under the scrutiny from scientists (Smith 2003; Carpenter et al 2008; Rhyne et al 2009; Murray et al et al 2008; Smith et al 2009; Tissot et al 2010) and in the United 2012). Changes in technology and economic livelihoods have States, there has already been one legislative call for a ban on affected the ability of consumers to procure and maintain ma- the importation of non-native species (H.R. 669) (Smith et al rine aquarium species in captivity. For example, the routes of 2009). This action was proposed based on the assumed rapid major international airlines control the ability to move live fish increase in trade volumes that have occurred over the past few shipments from remote island atolls to the U.S. markets (Kinch decades, lack of regulations in exporting countries and import- & Teitelbaum 2010; Teitelbaum et al 2010). It can be said that ing counties alike, and an overall lack of understanding of the the advent of modern aviation transport methods made the trade trade data. Because the understanding of this trade data is poor, in coral reef species a global industry. Furthermore, rapid chang- anticipated future demands based on how new technology or es in husbandry technologies have allowed for marine aquarist the global economic situations cannot be accurately forecasted. to move from fish only tanks to aquariums that replicate living ecosystems (reef aquariums). Recently, these tanks were min- The aquarium hobby has been termed a luxury hobby, and there- iaturized to mini-reefs. These advances have been observed in fore it can be inferred that the economic health of the consumer the hobbyist literature, trade shows and online discussion boards will directly affect the size of the hobby. The U.S. is the major (Riddle 2008; Adams 2010; Joshi 2010). destination port for coral reef wildlife, and economic health was greatly affected by the global recession of 2007-2009 (Bricker et The rapid increase in the volume of coral reef species entering al 2011). Thus it would be expected that a corresponding down markets into the US, and EU has been the cause of great concern turn in reported landings and imports of marine aquarium spe- for governments, scientists and conservation community. Early cies would be observed at the point of the global crisis. If so, in 2000’s the United States Coral Reef Task Force and UNEP- these declines should be observed across the board, from fish, WCMC took up the international trade of corals as a key issue invertebrates, corals and live rock and should match the down- of concern (www.coralreef.gov/international). The result was a turn in the global economy. several landmark publications about the trade (Bruckner 2000;

AACL Bioflux Volume 5 | Issue 2 Page 99 http://www.bioflux.com.ro/aacl Rhyne & Tlusty 2012 Table 1. Statistical values for the commodity groups presented in Figure 2. The coefficient of variation (c.v.) = s/m*100, and larger values indicated greater variability. Positive kurtosis values indicate a peaked distribution, where positive skewness indicates a larger positive (right) tail of the distribution. The percent ratio of the minimum to the maximum value indicates how small the lowest point of the trade was compared to the peak

Fish Inverts - SD Plants Live Rock Sand Sand Dollars Corals

c.v. 23.54 28.24 37.06 53.45 54.3 26.22 28.39 Kurtosis 1.6 -1.16 -1.14 0.19 -0.9 -0.64 -0.66 Skew 0.78 -0.64 0.31 1.08 -0.12 0.04 0.4 Min/Max 41.19 41.49 31.5 23.73 9.59 39.03 40.52

Materials and Methods The second major factor affecting the aquarium industry is technology. Technological advances are well documented in In order to elucidate both the effects of the global economic the hobby magazines as reviewed by leading hobby experts in recession and advances in husbandry technology have had on the topic of lighting (Riddle 2008; Joshi 2010) and filtration the aquarium trade we compared the landings and import data (Adams 2010) as well as the public aquarium community or from four major sources. 1) stony corals imports into the United professional hobbyists (Leewis & Janse 2008). Perhaps the States from 2000 until 2009 from the CITES database, 2) land- most significant change in husbandry technology that affects the ings reports by fisherman from the Florida marine aquarium size and accessibility of coral reef tanks is lighting. Changes in fishery from 2000 until 2011 (fish, plants, invertebrates (minus aquarium lighting have allowed for an increase in the number sand dollars), sand dollars (representing the curio trade), and of manufactures offering packaged mini or nano-reef aquari- sand), 3) import data for “live rock” reported by importers to um system (Figure 1) that are directly marketed to consumers USFWS on wildlife declaration forms mandated by law and held for reef aquariums. Advances in technology will affect various in Law Enforcement Information Management Service database species differentially and therefore, if technology drives trade (LEMIS) from 2000-2011, 4) import data of all live “corals” re- trends, then the different categories of traded animals should ported by importers on wildlife declaration forms mandated by not be synchronous as expected if trade was dominated by a sin- law and held in USFWS LEMIS from 2000-2011. This limited gle causative agent (such as the global economy). Here, these our data sources to that of import data reported by the United two hypotheses were examined using data for imports of stony States Government and those reported to the State of Florida. and live corals, as well as domestic fishery production from the These are most likely to reflect trends in the actual number of state of Florida (invertebrates, live corals, marine fish, plants, individual animals imported or landed in the fishery. These sand dollars, and sand). data are utilized with the caveat that they each have inherent strengths and weaknesses. We acknowledge known issues with data reporting in the CITES database (Phelps et al 2010; Rhyne et al In Press), and likewise with the LEMIS database (Smith et al 2008; Rhyne et al 2012). While alternate databases are being developed (Rhyne et al 2012) for now data analyses will focus on the extant LEMIS and CITES databases.

To determine trends in these datasets we plotted the total number of landings or imports over time from 2000 until the most recent completed year. Landing trends were compared to each other from 2000 until 2007 (the beginning of the global recession), and from 2007 until the most recent full year of data. The slopes from these two periods were compared to each other and general trends were observed. Distributional statistics (coefficient of variation, skewness, kurtosis, and the ratio of the minimum to the maximum) on yearly import volumes were also calculated for each trade category to assess trends in trade volume.

Results and Discussion Trade data Assessing the yearly trends in the volume of animals entering the marine ornamental aquarium trade, each of the commodity groups increases in popularity, reaches a peak, and then declines. The peak year varied from 2002 in Florida marine fish to 2008 Figure 1. A nano-reef aquarium. Used with permission. Photographer, in the live invertebrates from Florida. Live rock and sand had Jake Adams, retains copyright. the highest coefficient of variation indicating the highest variability off the mean (Table 1). Live rock and fish had the most

AACL Bioflux Volume 5 | Issue 2 Page 100 http://www.bioflux.com.ro/aacl Rhyne & Tlusty 2012 peaked distributions (Table 1), and the positive skew indicated reviewed in the hobby literature and forums (Adams 2010). a larger tail above the mean (greater positive variation above While each of these lamps has been demonstrated to grow and the mean than below it). sustain corals, they have the disadvantage of requiring large amounts of energy, along with producing heat and inferred light As a whole, these variable trends across commodity groups in- creating a significant heat load. This is particularly of concern dicate that multiple factors affect the trade in coral reef organ- for smaller aquariums, as smaller volumes of water have less isms. The global economic crisis did greatly affect this trade thermal elasticity. Hobbyists can counter this by equipping as a number of local peaks occurred in 2008-2009 (Figure 2). their aquariums with heat exchangers to draw excess heat from However, the 2003 peak in the live rock trade, and then its pre- aquarium water. Additionally, the size of the HID and VHO cipitous decline to 23% of its peak value, along with a 91% re- lights limits the minimal size of the aquarium. The advent of the duction in the sand trade, indicate a non-economic driver that CPL lighting allowed for spectral specialization and thus hobby- significantly affected these items (Figure 2). As indicated in ists could create reef environments in small aquariums, termed the introduction, technological advances, primarily in lighting mini or nano reefs. Nano and mini reef aquariums are under will result in the declines for live rock and sand. 100L in size and have been featured in the hobbyist literature as small as 25L (Adams 2010). While CPL lighting allowed for Advances in lighting the advance the husbandry of corals a smaller more compact aquarium without the heat concerns of Reef aquarium lighting was primary based on two main tech- HID lighting, this now pales in terms of advances occurring in nologies, the Metal Halide or High Intensity Discharge (HID) technologies surrounding Light Emitting Diodes (LED) (Joshi and Very High Output Fluorescents (VHO) until the late 1990s 2010). Aquariums could now be lit with energy efficient LED when the Compact Fluorescents Lamps (CPL) gained market lights that produce very little direct thermal heat and indirect share. These three types of lighting have dominated the trade IR heat into the aquariums allowing for sustained coral growth from the advent of coral husbandry until recent. The general at rates similar to those of HID lighting. This has effectively trend in increasing specialization of lamp spectral output is well removed the limitation of heat dissipation on aquarium size and at the same time reduced the operating costs for aquarist.

Conclusions Moving trade monitoring forward The trade in coral reef organisms is complex and is affected by technology and global economic health. Here, we report on a correlation between the advent of small home reef aquaria (‘nano’ tanks), and a decline in the harvest of biological active coral rock and sand. While suggestive, in a pure sense, correlation does not imply causation. There are other factors that may influence the trade in corals. As an example, the quality of coral products may affect import levels. Unfortunately these data are not collected, but the rapid appearance in the trade and proportional increase of corals originating from Australia (Rhyne et al In Press), and the general impression these are of “high quality” may indicate market sensitivity to animal vigor. In addition, the global coral market is also affected by the increase in coral aquaculture. As discussed in Rhyne et al (In Press), aquaculture production of corals (Parks et al 2008) will result in a decrease in the trade statistics of wild coral imports. More importantly, when the aquaculture production of corals is domestic, it will affect trade statistics by decreasing import volumes. However, without a suitable means of tracking domestic aquaculture coral production, the trade statistics will merely show a decline in volume, and not a shift in source country production.

The future of the coral (Rhyne et al In Press) and marine tropical fish trade (Rhyne et al 2012) are rapidly evolving. Difficulties in monotypic data recording, type instead of species declara- Figure 2. Trends in the marine aquarium hobby as depicted by 10 tions and “hidden” domestic production (Rhyne et al 2012) will years of import data. Top, is 10 years of import data for corals and complicate a full understanding of this trade. Future manage- live rock imported into the United States and marine invertebrate ment adjustments need to fully address species and type vari- landings from the State of Florida, United States. Bottom, is 10 years ation and the vagaries in listing to this level of data, as well as of landing data from Florida for marine fish, ‘plants’, live sand, and prompt recording of trade emanating from all source countries. sand dollars (curio). The legend is below the y-axis on which the data These improvements are necessary to allow for a full valuation are graphed. of this biodiverse global trade.

AACL Bioflux Volume 5 | Issue 2 Page 101 http://www.bioflux.com.ro/aacl Rhyne & Tlusty 2012 Acknowledgements Phelps, J., Webb, E. L., Bickford, D., Nijman, V., Sodhi, N. S., 2010. Boosting cites. Science 330:1752-1753. Florida landing data was provided by Florida Fish and Wildlife Rhyne, A. L., Rotjan, R., Bruckner, A., & Tlusty, M. F., 2009. Crawling Commission as part of the Federal Trip Ticket Program. United to collapse: Ecologically unsound ornamental invertebrate fisher- States Fish and Wildlife Services kindly provided the import data ies. PloS One 4(12):e8413. for ‘Live Rock’. Rhyne is supported by A National Oceanic and Rhyne, A. L., Tlusty, M. F., Kaufman, L., In Press. Long-term trends Atmospheric Administration (NOAA) Coral Reef Conservation of coral imports into the united states indicate future opportunities Program Grant NA09NMF4000385 and is thankful to the Ocean for ecosystem and societal benefits. Conservation Letters. Foundation and the United States Department of Commerce Rhyne, A. L., Tlusty, M. F., Schofield, P. J., Kaufman, L. E. S., Morris, National Oceanic and Atmosphere Administration, Office of J. S., Bruckner, A., 2012. Revealing the appetite of the marine aquar- Habitat Conservation for the invitation to present at the 2011 ium fish trade: The volume and biodiveristy of fish imported into stony coral mariculture workshop, Bali Indonesia, the origin of the united states. PloS One DOI:10.1371/journal.pone.0035808. this research. Jake Adams kindly provided the use of the nano Riddle, D., 2008. Product review: Lighting for reef aquaria: Tips on -reef aquarium in Figure 1. taking light measurements. Advanced Aquarist Online Magazine VII:www.advancedaquarist.com/2008/2002/review. References Smith, K. F., Behrens, M., Schloegel, L. M., Marano, N., Burgiel, S., Daszak, P., 2009. Reducing the risks of the wildlife trade. Science Adams, J., 2010. Reefing like it’s 1999: How reef aquarium flow and 324:594-595. lighting has changed over the past decade. Advanced Aquarist Online Magazine IX:www.advancedaquarist.com/2010/2011/review2012. Smith, K. F., Behrens, M. D., Max, L. M., Daszak, P., 2008. U.S. Drowning in unidentified fishes: Scope, implications, and regula- Bricker, J., Bucks, B., Kennickell, A., Mach, T., & Moore, K., 2011. tion of live fish import. Conservation Letters 1:103-109. Surveying the aftermath of the storm: Changes in family finances from 2007 to 2009 Federal Reserve Board, Finance and Economics Teitelbaum, A., Yeeting, B., Kinch, J., Ponia, B., 2010. Aquarium trade Discussion Series: Working Paper 2011-17, March 2011. in the pacific. SPC Live Reef Fish Information Bulletin 19:3-6. Bruckner, A., 2000. New threat to coral reefs: Trade in coral organisms. Tissot, B. N., Best, B. A., Borneman, E. H., Bruckner, A. W., Cooper, Issues in Science and Technology Fall 2000:1–6. C. H., D’Agnes, H., et al., 2010. How U.S. Ocean policy and market power can reform the coral reef wildlife trade. Marine Policy Carpenter, K. E., Abrar, M., Aeby, G., Aronson, R. B., Banks, S., 34:1385-1388. Bruckner, A., et al., 2008. One-third of reef-building corals face elevated extinction risk from climate change and local impacts. Wabnitz, C., Taylor, M., Green, E., Razak, T., 2003. From ocean to Science 321(5888):560-563. aquarium. Cambridge, UK: UNEP-WCMC. 64 pp. Joshi, S., 2010. Quantitative comparison of lighting technologies: Metal Wood, E. M., 2001. Collection of coral reef fish for aquaria: Global halide, t5 fluorescent and led. Advanced Aquarist Online Magazine trade, conservation issues and management strategies. UK: Marine IX:www.advancedaquarist.com/2010/2012/aafeature2012. Conservation Society. Kinch, J., Teitelbaum, A., 2010. Proceedings of the sub-regional workshop on the marine ornamental trade in the pacific. Noumea, New Caledonia: SPC Aquaculture Technical Papers / Secretariat of the Authors Pacific Community. 69 pp. •Andrew L. Rhyne, Roger Williams University, Department Leewis, R. J., Janse, M. (Eds), 2008. Advances in coral husbandry in of Biology and Marine Biology, One Old Ferry Road, Bristol, public aquariums (Vol. 2). 438 pp. RI 02809 USA. (p) 401-254-5750; (f) 401-254-3310; e-mail: Murray, J. M., Watson, G. J., Giangrande, A., Licciano, M., Bentley, [email protected] M. G., 2012. Managing the marine aquarium trade: Revealing the •Andrew L. Rhyne, John H. Prescott Marine Laboratory, New data gaps using ornamental polychaetes. PloS One 7(1):e29543. England Aquarium, Central Wharf, Boston MA 02110; e-mail: Parks, J. E., Pomeroy, R. S., Balboa, C. M., 2008. The economics of [email protected] live rock and live coral aquaculture Marine ornamental species. •Michael F. Tlusty, John H. Prescott Marine Laboratory, New Blackwell Publishing Company, pp.183-206. England Aquarium, Central Wharf, Boston MA 02110. +1-617- 973-6715; e-mail: [email protected]

Rhyne, A. L., Tlusty, M. F., 2012. Trends in the marine aquarium trade: the influence Citation of global economics and technology. Aquaculture, Aquarium, Conservation & Legislation 5(2):99-102. Editor I. Valentin Petrescu-Mag Received 19 March 2012 Accepted 31 May 2012 Published Online 30 June 2012 Rhyne is supported by A National Oceanic and Atmospheric Administration (NOAA) Funding Coral Reef Conservation Program Grant NA09NMF4000385 Conflicts/ Competing None reported Interests

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