DocumentJa

Recent developments and improvements in

ornamental fish packaging systems for air transport

Lian Chuan Lim1, Philippe Dhert2 & Patrick Sorgeloos3

1Freshwater Fisheries Centre, Agri-food & VeterinaryAuthority of Singapore, Sembawang Research Station, Lorong

Chencharu, Singapore2INVE Technologies NV, Oeverstraat 7, Baasrode, Belgium3Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Gent, Belgium

Correspondence: Lian Chuan Lim, Freshwater Fisheries Centre, Agri-food & VeterinaryAuthority of Singapore, Sembawang Research

Station, Lorong Chencharu, Singapore 769194, Singapore. E-mail: [email protected]

Abstract

Current ornamental ¢sh packaging systems are char-acterized by veryhigh ¢sh loading densities and highmetabolic wastes in the transport water after ship-ment. They focus mainly on management of thequality of transport water. Recent studies usingthe guppyas a model ¢sh showed that post-shipmentmortality could be reduced through enhancementof the stress resistance of the ¢sh, and hence em-phases should also be placed on the preparationof the ¢sh for transport and recovery of the ¢sh aftershipment. Farmers can contribute signi¢cantly byapplying nutritional prophylaxis before harvesting.Exporters may use the salinity stress test to identify¢sh lots of good quality for transport, apply healthprophylaxis to eradicate parasites and optimize othertechniques such as starvation of the ¢sh or additionof salt to the transport water to enhance the stressresistance of the ¢sh. Importers may adopt properacclimation procedure and allow ¢sh to recover inlow salinity water to reduce post-shipment mortality.As themain bulkof post-shipmentmortality is stress-mediated and occurs during the 1-week recoveryperiod, the industry should consider revising thebasis of the current warranty system for theircustomers, from death on arrival to cumulativemortality at 7 days post shipment (or death after7 days, DA7), in order to cut down ¢sh losses aftershipment.

Keywords: air transport, ¢sh packaging, nutri-tional prophylaxis, ornamental ¢sh, post-shipmentmortality, stress resistance

Introduction

In the ornamental ¢sh business, the ability to meetthe customers’needs for high-quality ¢sh is always acritical factor. As most of the ornamental ¢sh pro-duced in Southeast Asia are destined for export, the¢sh must not only be attractive but also robust towithstand long air transportation. Since the 1950swhen polyethylene bags were ¢rst used for live ¢shtransport, the polyethylene-bag transport systemhas greatly reduced the shipping weight of ornamen-tal ¢sh consignments, and made them feasible for airtransport. Nevertheless, the freight cost of ¢sh con-signments is still a major cost of the ornamental ¢shbusiness. For consignments from Asia to the USA,shipping may cost more than the ¢sh in the consign-ment (Lim & Chua 1993). Hence, the use of modernpackaging technology for air transport to increase¢sh loading density and improve post-shipment sur-vival is critical to the industry.The key limiting factor to increase the ¢sh loading

density in a live-¢sh transport system is the dete-rioration of the quality of transport water due to ac-cumulation of metabolic wastes. A variety oftechniques have been developed to manage the qual-ity of transport water during transport. They includestarving ¢sh before packaging (Phillips & Brockway1954; Nemato 1957), lowering the temperature oftransport water (Phillips & Brockway 1954; Norris,Brocato, Calandrino & McFarland 1960; Lim & Chua1993;Teo & Chen1993), addition of anaesthetics (Ta-kashima,Wang, Kasai & Asakawa 1983; Teo, Chen &Lee1989; Guo,Teo & Chen1995a, b), ion exchange re-sin (Amend, Croy, Goven, Johnson & McCarthy1982;

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Bower & Turner 1982; Teo et al. 1989; Lim & Chua1993), bu¡ers (McFarland & Norris1958; Amend et al.1982; Teo et al.1989) or drugs in the transport water(Ling, Chew, Lim, Koh & Lim 2000).Singapore is the top ornamental ¢sh exporting

country in the world, with a 26% share in the globalexport market in 1998 (Anonymous 2000). The cur-rent ornamental ¢sh packaging practice focusesmainly on the control of the quality of transportwater. For ornamental ¢sh consignments, the indus-try standard for warranty is 5% death on arrival(DOA); that is, exporters are expected to compensatecustomers for losses exceeding this standard.This pa-per presents an overview of ornamental ¢sh packa-ging systems for air transport, with special referenceto that used by Singapore exporters. It also reportsrecent developments in research on ¢sh packagingtechnology, discusses possible improvements to thepackaging system and proposes a new warranty sys-tem for the industry.

Fish packaging system

The system used for packaging ornamental ¢sh forair transport is a closed one in which all factors tomeet the requirements of the ¢sh for survival areself-sustained. It involves packing the ¢sh in sealedpolyethylene bags ¢lled with water that is usuallypre-treated with chemicals or drugs, and over-satu-rated with pure oxygen. After packing, the bags with¢sh are placed in a styrofoam (polystyrene) box,usually four to eight bags to a box, to provide thermalinsulation to prevent sudden changes in temperatureof the transport water, especially when the consign-ments are in the cargohold of aircraft (21^22 1C) dur-ing air transport.The volume of transport water limits the number

of ¢sh that can be packed. The loading density of ¢shis expressed as biomass of ¢sh (g) per unit volume ofwater (L). The loading density used by Singapore ex-porters generally increases with increasing bodyweight of the ¢sh (Fig. 1), as large individuals con-sume less oxygen and produce less nitrogenouswastes per unit weight than small ones (Fry & Norris1962). For consignments from Singapore to Europewith a 30-h transit time, the average loading densityranges from 22 g L�1for neon tetra, Paracheirodon in-nesi (Myers) (average weight 0.22 g) to 272 g L�1 forgold¢sh, Carassius auratus (L.) (average body weight13.8 g). They are correspondingly higher than thosefor food¢sh juveniles, such as cyprinids and perch of

the same size (Fig.1). Higher loading densities may beused if the transit time is shorter. Consignments toAsia with a transit time of 12^14 h, for instance, al-low density increases of about 50%. Fish densitiesare decreased byabout 20% for shipments to theUni-ted States (transit time 36^40 h).The most important factor in live-¢sh transport is

anadequate supplyof dissolved oxygen. In Singapore,the volume of pure oxygen supplied to the transportbag used to be up to six times the volume of transportwater, and it has now been reduced to three to fourtimes the volume of water. Even at these reduced vo-lumes, dissolved oxygen content is never a limitingfactor. Several ¢sh packaging experiments have re-corded over-saturated oxygen content of above10mg L�1 (at 25 1C,100% saturation58.26mg L�1)after 24^48-h shipment, even when there was high¢sh mortality of up to 20% (Froese 1988; Teo et al.1989). Because of the high loading densities used,the volume of oxygenused for packaging ornamental¢sh is very high compared with the volume of waterused for food¢sh, such as cyprinid and perch juve-niles (Berka 1986). The oxygen-to-¢sh ratio, ex-pressed in volume of oxygen (mL) to the total weightof ¢sh (g), is a good measure of the actual amount ofoxygen used in relation to the ¢sh biomass. For fresh-water ornamental ¢sh shipments from Singapore toEurope, the ratios range from 14 in large-sized ¢shsuch as gold¢sh to 120 in small-sized ¢sh such asneon tetra (Fig. 2). All the ratios for ¢sh from Singa-pore are similar to those used for cyprinid and perchjuveniles, indicating that in terms of ¢sh biomass, theamounts of oxygen used by the two groups of ¢sh aresimilar.

Figure 1 The average loading densities of eight commonfreshwater ornamental ¢sh used by Singapore exporters.The standard densities used for food¢sh juveniles (cypri-nid and perch) in similar systems are shown for purposesof comparison (data derived from Berka1986).

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Overview of the current industrypractice

Conditioning of ¢sh for packaging

Ornamental ¢sh are conditioned before packaging inthree stages, namely, prophylactic treatment, starva-tion and pre-packaging. Prophylactic treatment isusually performed on ¢sh that are possibly infectedwith parasites or other pathogens, especially thosereared in earthen ponds enriched with organic man-ure. The period of treatment varies from 1 day to aweek or more, depending on the species and condi-tion of the ¢sh. Fish after prophylactic treatment arehealthier, and hence more likely to withstand trans-port stress during the journey to their destination.Before packaging, ¢sh are starved to prevent regurgi-tation of partially digested food material and to re-duce the amount of excreta during transport.Singapore exporters starve their ¢sh for 1 or 2 daysfor small ¢sh suchas guppyand neon tetra and 2daysor more for larger ¢sh such as gold¢sh and koi, Cypri-nus carpio L. Counting of ¢sh is usually performed be-fore packaging. After counting, the ¢sh are pre-packed in polyethylene bags that are then placed inan air-conditioned room at 22^23 1C to enable themto acclimate to the packaging conditions, such ascon¢nement, crowding, high pressure and lowwatertemperature.

Quality control

The packaging process initiates severe stress in ¢sh(Nikinmaa, Soivio, Nakari & Lindgren 1983; Maule,Schreck, Bradford & Barton 1988; Schreck, Solazzi,Johnson & Nickelson 1989; Barton & Iwama 1991),

andwhen ¢sh are unable to recover homeostasis dur-ing and after the process, mortality may ensue. It istherefore imperative for exporters to examine andevaluate the quality of their ¢sh before packaging,and ship only healthy and good-quality ¢sh withhigh stress resistance to increase survival aftertransport.The current industry practice is to screen ¢sh,

starting from the time of counting ¢sh to the actualpackaging, based on visual inspection for pathologi-cal signs, suchas dark bodycolour, closing of ¢nnage,cloudy eyes, lack of appetite and lethargy or even thepresence of external parasites. Pre-packaging is animportant means of quality control, and ¢sh that failto adapt to the packaging conditions and show signsof sickness are eliminated from the shipment toreduce the risk of mass mortality.

Control of temperature of transport water

The objective of live ¢sh transport is to maximizeloading density and concurrently to maintain the¢sh in good condition on arrival. As the metabolismduring transport is about three times higher than theroutine metabolism (Froese1988), the existing packa-ging system focuses on lowering themetabolic rate ofthe ¢sh, to reduce oxygen consumption and accumu-lation of acidity, carbon dioxide and ammonia in thetransport water. Highwater temperature reduces theloading density of ¢sh due to the increased metabolicrate of ¢sh. It results in faster bacteria growth andlower dissolved oxygen levels, leading to increasedwaste production and decreased availability of oxy-gen to the ¢sh. Hence, the temperature of transportwater is usually reduced to the level that the ¢sh cantolerate, that is, 22 1C for tropical ¢sh and 15^18 1Cfor temperate species. After pre-packaging, ¢sh areallowed to acclimate to the packaging temperaturein an air-conditioned room for 4^6 h. Thereafter,they are packed in water that has been cooled to thedesired temperature at packaging.During the winter months in importing countries,

exporters usually attach a heat pack to the under-side of the cover of the packaging box to preventcold-shock to the ¢sh. The heat generated from theheat pack induced by rocking movement will in-crease the temperature in the packaging system. Inthis case, the loading density of ¢sh is reduced ac-cordingly, due to the anticipated increase in tempera-ture.

Figure 2 The average oxygen to ¢sh ratios of eight com-mon freshwater ornamental ¢sh used in the Singaporepackaging system. The ratios used for cyprinid and perchjuveniles in similar systems are shown for purposes ofcomparison (data derived from Berka1986).

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Control of metabolic wastes

As ornamental ¢sh are packed in a small volume ofwater at a high loading density, the metabolic wasteaccumulates rapidly in the transport water. Ammo-nia and carbon dioxide are the two major metabolicwastes produced in transport water. Ammonia accu-mulates in transport water due to the excretion from¢sh and the bacterial action on ¢sh excreta and dead¢sh. There are two forms of ammonia: the ionized(NH4

1) ammonia and the unionized or free ammonia(NH3). Only the unionized ammonia is able to passtissue barriers and is toxic to ¢sh. Fish may succumbif the toxic unionized ammonia in the tissue is abovetolerable levels. Cation exchange resins such as clin-optilolite, a natural zeolite, is commonly used to re-move ammonia from the transport water. The resinshave the ability to absorb ammonia by selective ionexchange. They are either wrapped in a net bagor added directly into the transport water at 15^20 g L�1of water. They do not control the accumula-tion of carbon dioxide in the transport water.Bacterial growth is another major source of meta-

bolicwastes. Bacteria not only increase the ammoniaload and compete with ¢sh for oxygen in the trans-port water, but also weaken or cause diseases. Drugssuch as neomycin sulphate, methylene blue and acri-£avine are added to the transport water to controlbacterial growth.

Addition of salts to transport water

Osmoregulatory dysfunction is common in ¢sh thatare exposed to transport stress, and the addition ofsalt to transport water is e¡ective in reducing the os-moregulatory dysfunction and other physiologicalresponses to stress (Johnson & Metcalf 1982; McDo-nald & Milligan1997), resulting in reduced ¢sh mor-tality (Tomasso, Davis & Parker 1980; McDonald &Milligan 1997). In Singapore, coarse salt containing95^98% sodium chloride is commonly used to

reduce e¡ects of stress of the ¢sh. It is added directlyto transport water at 0.5^3.0%, and the concentra-tion used varies with species and exporters.

Recent developments and possibleimprovements

Current ornamental ¢sh packaging practices focuson minimizing stress imposed on the ¢sh throughthe control of metabolic rate and the removal of me-tabolicwaste from the transport water.There is insuf-¢cient attention to enhancement of the stressresistance of the ¢sh, which would enable the ¢sh toovercome the stressful conditions during transporta-tion better. To improve the post-shipment perfor-mance of the ¢sh, e¡orts from di¡erent sectors of theornamental ¢sh industry should be made to reducethe stress imposed on the ¢sh and enhance theirstress resistance.

Characteristics of transport water and ¢shmortality pattern after shipment

A survey on the packaging of guppy was conductedin Singapore to determine the quality of transportwater and the ¢sh mortality pattern after transport.A total of 104 bags were checked for the quality oftransport water after a 40-h simulated shipment.Fish from 51bags were stocked in aquaria to monitortheir post-shipment mortality for 1 week. The waterquality of the transport water after the 40-h ship-ment was characterized by relatively high tempera-ture, super-saturated dissolved oxygen (at 27.5 1C,100% saturation57.90mg L�1), low pH and hightotal ammonia and unionized ammonia (Table1).After 40-h simulated shipments, the mean ¢sh

mortality at unpacking was only 2.6% (Table 2). Themedian mortality indicated that in half of the bags,the mortality was 0.5% or less. The mean cumulativemortality at 7 days post shipment varied from 0% to

Table 1 Quality parameters of the transport water of guppy after 40-h simulated shipments

Parameter N Mean7SD Median Minimum Maximum

Temperature (1C) 104 27.3470.22 27.40 26.90 27.70

Dissolved oxygen (mgL�1) 104 18.5071.80 19.27 12.61 20.00

pH 104 6.1770.16 6.20 5.90 6.40

Total NH3 (mgL�1) 88 28.2678.56 25.68 13.60 53.05

Unionized NH3 (mgL�1) 88 0.03170.006 0.031 0.018 0.047



26%, with a mean mortality of 10.8% and a medianmortality of 9.5%. The high variation is attributableto the varying quality of ¢sh acquired from di¡erentfarms (Table 2). Transportation of freshwater drum,Aplodinotus grunniens Ra¢nesque or largemouthbass, Micropterus salmoides Lacepde resulted in ¢shmortality that occurred either immediately or secon-darily due to osmoregulatory dysfunction or infec-tious diseases (Johnson & Metcalf 1982; Carmichael,Tomasso, Simco & Davis 1984). At present, industrypractice is for exporters to provide a warranty of 5%DOA to their customers. Exporters are expected tocompensate customers for losses exceeding the stan-dard. The mortality pattern of ornamental ¢sh re-£ects the fact that only a small portion of the ¢sh iskilled directly by the reductions in the quality oftransport water. Most post-shipment mortality wasdue to osmoregulatory dysfunction or stress-mediated diseases and occurred during the ¢rst weekafter transport. It is therefore suggested that the in-dustry revises the basis of the warranty system totheir customers from DOA to cumulative mortalityat 7 days post shipment (or death after 7 days, DA7)to cut down mortality after arrival to customers.

Nutritional prophylaxis and harvestingat farm

Ornamental ¢sh are often reared under intensiveconditions and are subjected to physiological chal-lenges from changes in water chemistry and cultureprocedures to behavioural interactions among ¢sh(Wedemeyer1997). Such chronic stress may manifestas health problems and physiological disturbance.Control of the stress response of ornamental ¢sh toprepare them for transport should therefore start atthe farming stage. Assuming that the primary causeof post-shipment mortality is partly associated withthe low stress resistance of ¢sh, use of stress resis-

tance enhancement techniques before the transpor-tationprocess may reduce post-shipment mortalityofthe ¢sh.Vitamin C, ascorbic acid, is an essential dietary nu-

trient for aquatic organisms. It assists in maintainingnormal growth and collagen formation, improvingdisease resistance and reducing stress (Menasveta1994). Nutritional prophylaxis using vitamin C sup-plementationworked well in enhancing the stress re-sistance of the guppy (Lim, Dhert, Chew, Dermaux,Nelis & Sorgeloos 2002b; Lim, Wong, Koh, Dhert &Sorgeloos 2000). Supplementing the diet of guppieswith vitamin C at 2000mg kg�1 for 10 days signi¢-cantly improved the cumulative mortality of the ¢shat 7 days post shipment, from 23% in the control ¢shto 8% in the vitamin C-supplemented ¢sh (Fig. 3). As¢sh were stressed during transport, the lower cumu-lative mortality in the vitamin C-supplemented ¢shcould be attributed to the enhanced stress resistanceof the ¢sh. Guppies fed the vitamin C supplementwere also more resistant to disease. When the ¢shwere exposed to Tetrahymena, vitamin C supplemen-tation reduced cumulative mortality at 7 days postshipment from 90% in the control ¢sh to 14% in thevitamin C-supplemented ¢sh (Fig. 3). Similarly, Li &Lovell (1985) reported that feeding mega-doses of vi-taminC at 3000mg kg�1completely protected chan-nel cat¢sh, Ictalurus punctatus (Ra¢nesque) againstexperimental Edwardsiella ictaluri Hawke (entericsepticaemia) infections. The bene¢cial e¡ects of vita-min C in enhancing the disease resistance were alsorecorded in other aquaculture species such as turbotlarvae, Scophtalmus maximus (L.) (Merchie, Lavens,Dhert, Gomez, Nelis, De Leenheer & Sorgeloos 1996),postlarvae of Penaeus monodon (Fabricius) (Kontara,Merchie, Lavens, Nelis, De Leenheer & Sorgeloos1997) and Atlantic salmon, Salmo salar L. (Waagb�,Glette, Raa-Nilsen & Sandnes1993). Hence, ¢sh farm-ers can contribute to the success of ornamental ¢shtransport by use of nutritional prophylaxis using

Table 2 Cumulative post-shipment mortality (%) of guppyduring the1-week recovery period after 40-h simulated shipment(¢sh density100 ¢sh L�1or 35 g L�1; number of bags551)

Dayafter unpacking

Parameters 0 1 2 3 4 5 6 7

Mean7SD 2.6373.52 4.0275.96 4.8776.36 6.3976.83 7.7476.95 8.7677.14 10.1476.85 10.8477.19

Median 0.5 1.25 2.5 4.5 6.25 7.00 9.00 9.50

Minimum 0 0 0 0 0 0 0 0

Maximum 9.90 22.77 24.75 24.75 24.75 24.75 24.75 26.25



dietary vitamin C supplementation to enhance stressresistance of their ¢sh before capture for shipment.During ¢sh collection for shipment, farmers

should take precautions to avoid injury to the ¢sh, ex-posure of the ¢sh to air for a prolonged period or over-crowding. These stress factors may be super-imposedby capture and, if not controlled, could be more se-vere than the e¡ects of transport (Iversen, Finstad &Nilssen1998).

Health prophylaxis

Parasitic infection is common in ornamental ¢sh. Inmany cases, the infection is mild and asymptomatic,and the infection alone may not kill the ¢sh. How-ever, elevation of corticosteroid (mainly cortisol) le-vels in plasma as a primary response to stress can beimmuno-suppressive, resulting in increased vulner-ability of the ¢sh to pathogens (Barton & Iwama1991). Hence, an apparently harmless infection mayturn lethal when ornamental ¢sh are stressed andwhen immune function is suppressed during trans-port. Parasitic infection may also pre-dispose ¢sh tosecondary bacterial infections that further compro-mise the immune system and aggravate the problemof post-shipment mortality. A recent challenge testusing guppies that were conditioned in the hatcheryfor 1 month demonstrated that the ¢sh, when chal-lenged with Tetrahymena, exhibited a signi¢cant de-crease in its stress resistance without displayingvisible signs of infection (Lim et al. 2000). However,

when the ¢sh were stressed during a 40-h simulatedair transport, the challenged groups su¡ered heavymortality of 24% at the time of unpacking, and 75%at 7 days post shipment. This is in contrast to control¢sh that had almost 100% survival at 7 days postshipment.Wise, Schwedler & Otis (1993) showed thathandling, transport and con¢nement stress in-creased the mortality rate of channel cat¢sh thatwere challenged with Edwardsiella ictaluri. A similarexperiment on guppies demonstrated that treatmentwith chlorine dioxide at 20mg L�1 in the transportwater reduced post-shipment mortality signi¢cantly,both at the time of unpacking (4%) and at 7 days postshipment (31%). It is therefore critical for exporters totreat their ¢sh for pathogenic infections beforepackaging.Several protocols have been developed in Singa-

pore to treat common bacterial pathogens such asAeromonas and parasites such asTetrahymena, Gyro-dactylus spp. and Cestodes in guppy and Hexamita,Costia and Trichodina in angel¢sh. Most treatmentsinvolve bathing or feeding the ¢sh with appropriatechemicals or drugs before packaging and treating¢sh with chemicals or drugs in the transport water(Table 3). In all cases, post-shipment survival rates ofthe treated ¢sh are improved. In the latest develop-ment, the treatment protocols forTetrahymena infec-tion in guppy and Hexamita infection in angel¢shhave been simpli¢ed (Ling et al. 2000). Fish are trea-ted directly in the pre-packaging and transport waterwith appropriate drugs (Table 3), and hence the needfor tank-space and manpower to maintain the ¢sh isreduced.

Starvation

Starving ¢sh before live-¢sh transport has been usedto improve the survival of ¢sh during transport sincethe beginning of the 19th century (McCraren 1978).Its primary objectives are to reduce metabolic rateand improve the quality of transport water. The me-tabolism of blue gourami was found to decrease withincreasing duration of starvation (Chow, Chen & Teo1994a), but in guppy, starvation for 2^5 days beforepackaging did not a¡ect the oxygen consumption ofthe ¢sh (Teo & Chen, 1993). A recent study showedthat starvation enhanced the stress resistance of theguppy. The stress resistance of guppies starved for 1day was signi¢cantly higher than that of ¢sh starvedfor 2 days, which in turnwas higher than that of non-starved ¢sh or ¢sh starved for half a day or 3 days

Figure 3 Cumulative mortality of vitamin C-supplemen-ted guppies and the control ¢sh during the1-week recov-ery period after 40-h simulated shipment. Two batches of¢sh were tested, with one batch infected with Tetrahyme-na. Values represent the mean of three replicates (Inf-contr: infected ¢sh fed control diet; Inf-vit C: infected ¢shfed vitamin C supplement; Hlt-contr: healthy ¢sh fed con-trol diet; Hlt-vit C: healthy ¢sh fed vitamin C supplement).



(Lim et al. 2000). Subsequent ¢sh packaging experi-ments revealed that while starvation had no e¡ecton mortality at unpacking, guppies starved for a daybefore shipment had signi¢cantly higher survivalthan control ¢shat 7 days post shipment (Fig.4). Star-vation for half a dayor for 2 or 3 days did not improvepost-shipment survival. The same study found thatthere were no signi¢cant di¡erences in all para-meters in the transport water between the controlgroup and the group starved for 1 day. These resultsindicated that reduced ¢shmortality was attributableto the increased stress resistance of the ¢sh. Hencefor ornamental ¢sh transport, the optimal durationfor starving small ¢sh such as guppy should be lim-ited to 1 day only, as prolonged starvation may haveadverse e¡ects on the stress resistance of the ¢sh,and hence increase post-shipment mortality.

Quality control: evaluation of stressresistance

The existing industry practice for quality controlbased on visual inspection is unreliable, as ¢sh with

low stress resistance are detectable only when theybegin to exhibit disease symptoms after shipment.To overcome this problem, a salinity stress test wasdeveloped to evaluate the stress resistance of the gup-py (Lim et al. 2000). It entails exposure of 10 guppiesto osmotic shock in 500mL saline water made up ofpre-aerated water and salt (sodium chloride). The

Table 3 Improvements of post-shipment survival of guppy and angel¢sh through health prophylaxis

Fish species Targeted pathogens Treatment protocols Survival rate at 7 days post shipment Sources

Guppy Gyrodactylus and

Cestode

Bathe fish in 20mgL�1 formalin,

2mgL� 1 acriflavine and 2% salt

solution for 1 h before packaging and

add 2mgL� 1 acriflavine and 2% salt

solution in transport water during

transportation (36h)

Increases from 78% in control fish

to 99% in treated fish

Ling, Lim & Cheong

(1996)

Tetrahymena and

Aeromonas sp.

One-day bath in 10mgL� 1

chloramphenicol and 2% salt

solution, followed by 1-h bath in

0.1mgL� 1 malachite green,

50mgL� 1 formalin and 2% salt

solution, and then add 0.1mgL�1

malachite green and 2% salt to

transport water during transportation

(36h)

Increases from 67% in the control

group to 91% in the treated group

Loo, Ling & Lim

(1998)

Tetrahymena Add 20mgL�1 chlorine dioxide

to pre-packaging water (6 h) and

transport water (40h)

Improves from 61% in control fish

to 89% in treated fish.

Ling et al. (2000)

Angelfish Hexamita, Costia

and Trichodina

Bathe fish in 2mgL�1 acriflavine

and 2% salt solution for 3 days, and

during the first 2 days, feed them with

dried pellet incorporated with 2%

metronidazole, twice a day (36h)

Increases from 3% in control group,

to 42% in group with bath treatment,

and to 96% in group given full

treatment

Ling & Khoo (1997)

Hexamita Add 3.5mgL� 1 metronidazole

(soluble form) in pre-packaging

water (6 h) and transport water

(40h)

Improves from 58% in the control

to 95% in treated group

Ling et al. (2000)

Figure 4 E¡ects of the duration of starvation on stressresistance of guppy and cumulative mortality of the ¢shat 7 days post shipment. Values represent the mean ofthree replicates. For ¢sh mortality, the vertical bar repre-sents standard deviation. Di¡erent alphabet letters indi-cate signi¢cant di¡erences between means (Po0.05).



mortality of the ¢sh is monitored and cumulativemortality is recorded at 3-min intervals over a 2-hperiod. Stress resistance is expressed as the stress in-dex, which is obtained as the sum of 40 cumulativemortality readings recorded over the test period. Ahigher stress index is a result of either earlier or high-er mortality or both, and indicates a lower stress re-sistance of the ¢sh. The optimal salinities for use inthe stress test are 35% for market-sized guppy and30% for guppy fry (Lim et al.2000). Optimal salinitylevels for testing juveniles of other species are 35%for molly, 25% for platy and swordtail and 15% forblack neon tetra, Hyphessobrycon herbertaxelrodiGe¤ ry (Lim, Cho, Dhert, Wong, Nelis & Sorgeloos2002a). Salinity was chosen for the stress test be-cause it is an important factor determining the os-moregulatory pattern of aquatic organisms (Limet al. 2000). The use of saline water as a test mediumhas the advantages of being easy and convenient toprepare, and it is more stable than other parameterssuch as temperature, pH and ammonia. Comparedwithvisual inspection, the stress test provides amoreobjective, reliable and quanti¢able measure of thephysiological conditions of the ¢sh. A similar testusing sodium nitrite as the test mediumwas also de-veloped for comparing the stress resistance between¢sh cultured in freshwater and those in brackishwater (Lim et al. 2000).

Temperature insulation using styrofoam box

Maintaining transport water temperature through-out transit is a major problem of the ¢sh packagingsystem. Recent water quality data showed that thetemperature of transport water increased from22^23 1C at packaging to 26.9^27.7 1C after 40-h si-mulated shipments (Table1), indicating poor thermalinsulation of the styrofoam boxes used by the indus-try. The temperature may drop below 20 1C or 15 1Cwhen the consignments are placed in unheated car-go holds (Chen & Teo 1990; Froese 1998). As the ma-jority of the ornamental ¢sh in the export trade are oftropical origin, and are relatively stenothermic, theyare readily stressed by any drastic change in the en-vironmental temperature and mortality will occur atsub-optimal temperature (Chow, Chen & Teo 1994b;Froese 1998). Hence, it is imperative that styrofoamboxes used for ornamental ¢sh packaging shouldprovide good insulation during the transit time. InSingapore, two sizes of styrofoam boxes, which mea-sure 48.5 cm�36.5 cm�36.5 cm (thickness: 2.0 cm)

and 60.5 cm�45.5 cm�30.5 cm (thickness: 2.0 or1.30 cm), respectively, are most commonly used. Fro-ese (1998) recommended that cube-shaped styro-foam boxes with at least 2.5-cm wall thicknessshould be used for ornamental ¢sh packaging to re-duce heat loss during transport. Further researchwork on improving the insulating property of styro-foam boxes to cut down ¢sh loss due to thermal stressshould be conducted.

Management of quality of transport water

Table 1 shows that the pH of transport water after40-h simulated shipment is below the acceptable levelof 7.0^8.5 for guppyandmanyother tropical ornamen-tal ¢sh (Sandford1995). The low pH readings re£ect ahigh level of carbon dioxide in the transport waterdue to the low carbon dioxide stripping e⁄ciency inthe closed packaging system (Colt & Orwicz1991). Athigh carbon dioxide and low pH levels, the oxygen-carrying capacity of blood is markedly reducedthrough the Bohr e¡ect, and ¢sh mortality may oc-cur even if oxygen levels are within acceptableranges (Hoar 1984). A sudden drop in pH may alsocause gill damage that may lead to osmoregulatorydysfunction (Tomasso et al. 1980). In addition, highconcentration of carbon dioxide may anaesthetize¢sh in the transport water (Bell 1964; Gebhards1965). Althoughbu¡ers suchas tris-bu¡erwere e¡ec-tive in stabilizing the pH of transport water (Teo et al.1989), the resulting higher pH may increase theamount of free ammonia. The application of bu¡ersin ¢sh transport is therefore limited.Ammonia toxicity to ¢sh depends upon the con-

centration of unionized ammonia, and its relativeproportion increases with increasing pH and tem-perature and decreasing salinity (Emerson, Russo,Lund & Thurston 1975). Despite the low pH of thetransport water, the unionized ammonia (mean0.029mg L�1; median 0.030mg L�1) exceeded theacceptable level of 0.02mg L�1 for intensive ¢sh cul-ture (Wedemeyer 1997). The high ammonia level intransport water may cause reduced excretion of am-monia by the ¢sh and consequently a build-up of am-monia in the ¢sh tissues (Chen & Teo 1990).Brockway (1950) suggested that ammonia may inhi-bit the ability of haemoglobin to combine with oxy-gen, altering the oxygen-carrying capacity of theblood.The current practices aimed at reducing the

amount of metabolic wastes in transport water, such



as fasting, lowering the temperature of transportwater and use of clinoptilolite, are insu⁄cient to low-er the ammonia levels and stabilize the pH in trans-port water to acceptable levels. Anaesthetics havebeen shown to be e¡ective in lowering metabolicrates by reducing the motor activity of ¢sh (McFar-land 1960), and may be used to reduce metabolicwaste production further. Teo et al. (1989) reportedthat 2-phenoxyethanol was e¡ective in reducing theammonia excretion of the guppy. Guo et al. (1995a)found that 2-phenoxyethanol and quinaldine sul-phate signi¢cantly reduced ammonia and carbon di-oxide excretion by platy. Anaesthetics may be used tolimit the stress responses of ¢sh, but con£icting re-ports exist on this subject. Some researchers reportedthat rapid anaesthesia before handling was e¡ectivein reducing or even eliminating the stress responsesin many ¢sh species (Strange & Schreck1978;Tomas-so et al.1980, Davis, Parker & Suttle 1982, Limsuwan,Limsuwan, Grizzle & Plumb1983; Robertson,Thomas& Arnold1988). Guest & Prentice (1982) found that inblueback herring, Alosa aestivalis, the anaesthetized¢sh had higher survival than non-dosed ¢sh aftertransportation. Tomasso et al. (1980) and Carmichaelet al. (1984) reported decreases in stress response inhybrid striped bass and largemouth bass when the¢sh were anaesthetized before capture and kept se-dated during transport. Robertson et al. (1988), onthe contrary, found that this method was ine¡ectivein red drum, Sciaenops ocellatus (Linnaeus). Other re-searchers have also reported that ¢sh treatedwith se-dating doses did not exhibit lower stress responses(Strange & Schreck1978; Davis et al.1982).The use of anaesthetics in the transport of orna-

mental ¢sh has not been fully explored. Teo et al.(1989) reported zero mortality in a guppy packagingexperiment using 2-phenoxyethanol at 0.11 and0.22 g L�1 after a 20-h simulated shipment. Al-though the ¢sh loading density used in the experi-ment (50 L�1) was only half the standard density(100 L�1) used by the industry for a 40-h shipment,the results indicated that the potential application ofanaesthetics in ornamental ¢sh packaging is promis-ing, and that it warrants further research.

Enhancement of stress resistance of ¢shusing salts

During the air transportation process, ¢sh are con-stantly subjected to a series of stressors starting fromcapture at farms to packaging for air transport, the

adverse water quality and crowded and over-pressur-ized conditions during transport. It is well knownthat stress leads to osmoregulatory dysfunction (Har-rell & Moline 1992; Weirich, Tomasso & Smith 1992)and causes ¢sh mortality (Tomasso et al. 1980). Theaddition of salt to transport water has been found tobe e¡ective in reducing osmoregulatory disturbancesand ¢sh mortality in several food¢sh species (Car-neiro & Urbinati 2001). Similarly, when guppies wereheld in saline water for 40 h, their stress resistanceincreased with increasing salinity to 9%, which isclose to isotonic conditions (Lim et al. 2000, Fig. 5).Subsequent experiments showed that when guppieswere packed in saline water of 1%, 3% or 9% andstocked in their respective salinities for recovery(with 30% daily dilution with freshwater) after ship-ment, all three treatments displayed a signi¢cantlylower cumulative mortality than the control groupat 7 days post shipment (Fig.6).While there is clear evidence on the bene¢cial ef-

fects of salts in live-¢sh transport, there were consid-erable variations in the recommended amounts ofsalt used for di¡erent species. Johnson (1979) recom-mended a lowconcentrationof 2% salt as a guidelinefor live-¢sh transport, while Carneiro & Urbinati(2001) found that 6% salt wasmost e¡ective formini-mizing the physiological response of matrinxa� , Bry-con cephalus (Gˇnther), to transport stress.Weirich &Tomasso (1991) recommended that red drum juvenileshould be transported inwater that is nearly isosmo-tic to plasma (11%). Similarly, a concentration of10%salt was found to be e¡ective for hauling and trans-port of striped bass, Morone saxatilis (Walbaum) (Po-well1970; Mazik, Simco & Parker1991; Harrell 1992).

Figure 5 E¡ects of salt content on the stress resistance ofguppy and the total ammonia content in transport water.The value represents mean of four replicates. For total am-monia content, the vertical bar represents standard devia-tion. Di¡erent alphabet letters indicate signi¢cantdi¡erences between means (Po0.05).



In Singapore, the salt concentration used for packa-ging ornamental ¢sh is 0.5^3.0%, which is muchlower than 9% recommended by Cole,Tamaru, Bai-ley, Brown & Ako (1999). The experiment with guppyshowed a drastic increase in the total ammonia con-tent when the salinity of transport water was in-creased to 3% or 9%, due to a decrease in thee⁄ciency of clinoptilolite (Fig. 6). These results sug-gest that among the three e¡ective salinities rangingfrom 1% to 9%, guppies should be packed at 1%.Higher salinity should be avoided as it may result inundesirable quality of the transport water.

Acclimation and recovery of ¢sh aftershipment

On arrival, ornamental ¢sh should be allowed to re-cover from transport stress in tanks and acclimate tolocal water conditions before being distributed toretailers. The acclimation operation includes £oatingsealed bags in recovery tanks until the temperaturedi¡erence between the transport water and therecovery water is less than 2 1C. This may take about30min for a temperature di¡erence of 4^5 1C.The pHand hardness of the recovery water should also beclose to the optimal values for the respective species.The addition of salt to the recovery water to en-

hance the stress resistance of the ¢sh is e¡ective inreducing post-shipment mortality. Guppies stockedin freshwater for recovery after transport exhibited

no signi¢cant di¡erences in cumulative mortality at7 days post shipment among the three groups of ¢shpacked in saline water (1%, 3% and 9%) and thecontrol group (Fig. 6). However, when the ¢sh werestocked in their respective salinity of transport waterfor recovery (with 30% daily dilution with fresh-water), all three salinity treatments displayed a sig-ni¢cantly lower cumulative mortality than thecontrol group. Fish packed in 1% also had signi¢-cantly higher survival in water of the same salinitythan when placed in freshwater during recovery.These results suggest that the addition of salt, evenonly1%, is critical to the recovery of the guppy aftertransport. Similar bene¢cial e¡ects of salt for recover-ing andmaintaining osmotic homeostasis during therecovery stage from transport stress were reportedfor other ¢sh species. They include 5% for hybridstriped bass ¢ngerlings (female white bass, Moronechrysops (Ra¢nesque) � male striped bass,M. saxati-lis) raised in freshwater (Reubush & Heath1997) and10% for striped bass (Mazik et al.1991).

Conclusion

The current state of the art in the packaging of orna-mental ¢sh focuses mainly on the control of meta-bolic waste products to reduce the stress imposed onthe ¢sh during transport.The existing packaging sys-tem is characterized by a very high ¢sh loading den-sity, which helps to reduce the freight cost of the ¢shconsignment, but at the same time, leads to veryhighammonia and carbon dioxide, and low pH in thetransport water at unpacking. Deterioration of waterquality may cause severe stress to the ¢sh and wouldresult in about 10% cumulative mortality at 7 dayspost shipment.Stress resistance is an important factor determin-

ing the post-shipment performance of the ¢sh.However, insu⁄cient attention has been given toenhancing the stress resistance of the ¢sh to increasetheir chances of survival after transport. To increasethe loading density and reduce post-shipmentmortality of ornamental ¢sh, e¡orts should be madeto lower the stress responses of the ¢sh and enhancetheir stress resistance. As the e¡ects of transport ofornamental ¢sh extend beyond the actual transpor-tation period, emphasis should also be placed on thepreparation of the ¢sh for the transport and recoveryof the ¢sh after shipment. Control of the stress re-sponse of ornamental ¢sh should start on the farm.Farmers can contribute by applying nutritional

Figure 6 E¡ects of salt content in the transport water oncumulative mortality of guppy at 7 days post shipment.Two batches of ¢sh were tested and they were stocked infreshwater or inwater with initial salinity similar to theirtransport water (with daily 30% dilutionwith freshwater)during the1-week recovery period after shipment.The va-lue represents the mean of four replicates and its standarddeviation. Di¡erent alphabet letters within the samegroup of recovery water indicate signi¢cant di¡erencesbetween means (Po0.05).



prophylaxis before capture and minimize stress tothe ¢sh during the capture operation. Feeding theguppy with diets supplemented with vitamin C at2000mg kg�1 for 10 days enhances both disease re-sistance to Tetrahymena and the stress resistance ofthe ¢sh, leading to reduced post-shipment mortality.For exporters, the visual inspection for pathologi-

cal problems is not satisfactory, as parasitic infectioncould be mild and asymptomatic, and the ¢sh mayshow symptoms only after shipment. Exporters mayuse the salinity stress test to identify ¢sh lots of goodquality for transport to reduce ¢sh loss after ship-ment. Quality enhancement through health prophy-laxis to eradicate parasites would signi¢cantlyimprove the cumulative mortality of ornamental ¢shat 7 days post shipment. A new treatment protocolinvolving treatment in pre-packaging and actualtransport water has been developed to eliminate theneed of tankmaintenance, thus leading to a saving inmanpower and space. Recent experiments usingguppy suggested that starvation for 1 day is e¡ectivein enhancing their stress resistance. The ¢sh shouldbe starved for an optimal duration only, as prolongedstarvation leads to a decrease in the stress resistanceof the ¢sh. A low concentration of salt should be usedto enhance the stress resistance of guppy, both in thetransport water and in the recovery water after ship-ment. High salinity should be avoided as the salt con-tent reduces the e⁄ciencyof clinoptilolite and lowersthe quality of transport water.Data on the water quality of transport water sug-

gest that the current practice in reducing the meta-bolic wastes in transport water is insu⁄cient tolower ammonia and stabilize pH to acceptable levels.Use of anaesthetics to lower the metabolic rate andenhance the stress resistance of the ¢sh could be apromising solution, but it warrants further research.To lower ¢sh loss due to thermal shock or heat loss,further research on the design of styrofoam boxeswith better insulation properties is needed.Finally, as the mortality is stress-mediated and oc-

curs during the1-week recovery period, the industryshould consider revising the basis of the warrantysystem to their customers, from DOA to cumulativemortality at 7 days post shipment (or death after 7days, DA7) to reduce ¢sh losses after shipment.

Acknowledgments

This project was funded by theAgri-food &VeterinaryAuthority of Singapore (AVA) (Project FFC/E5/00).

The ¢rst author would like to thank his colleaguesin the AVA MrWong Chee Chye and Ms LimYu Qianfor their technical assistance during the course ofthis study, and Mr Ho Kian Heng of Sun Aquarium,Singapore for his support and co-operation duringthe survey on the packaging of guppy.

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