Effect of antibiotic treatment on the growth and

survival of juvenile northern Chilean scallop,

Argopecten purpuratus Lamarck (1819), and

associated microflora in experimental cultures

Jorge Fierro1 & Doris Oliva2

1Pesquera San Jose¤ S.A., Coquimbo, Chile2Departamento de Biolog|¤ a & Ciencias Ambientales, Facultad de Ciencias, Universidad deValpara|¤ so,Valpara|¤ so, Chile

Correspondence: D Oliva, Departamento de Biolog|¤ a & Ciencias Ambientales, Facultad de Ciencias, Universidad deValpara|¤ so,Valpara|¤ so,

Chile. E-mail: doris.oliva@uv.cl

Abstract

Juvenile northern Chilean scallops of 937 � 55 mmshell height were exposed to ¢ve di¡erent concentra-tions of chloramphenicol (CHL) (5, 10, 25, 50 and100 mgmL�1), plus a control without antibiotics. Todetermine the e¡ect of CHL on the accompanyingmi-cro£ora, the number of colony-forming units (CFU)that grew on TGE culture medium was counted inthe seawater of containers with juveniles, and in con-tainers with microalgae used as food. Both were ex-posed to the same concentrations of CHL. Thegrowth rates of juveniles treated with CHL and thecontrol without antibiotic showed highly signi¢cantdi¡erences (P50.0001). The growth rate was inver-sely proportional to the CHL concentration. The con-trol sample presented the highest growth rate(84.4 � 14.3 mmday�1), followed by the sample trea-tedwith 5 mgmL�1 (64.2 � 14.3 mmday�1).The sur-vival in the control and in the treated samples with5^50 mgmL�1was rather high, with a mean value of95%. Only the sample treated with100 mgmL�1hada low survival (36.7%). The CFU count was larger inthe containers with juveniles, when compared withthe ones with food.The CFUcount tended to decreasewith increasing CHL concentration in the juveniles.

Keywords: chloramphenicol,Argopecten purpuratus,hatchery

Introduction

Since the1950, antibiotics have been used in shell¢shaquaculture to control bacterial growth (Loosano¡ &

Davis 1963; Walne 1974; Bourne, Hodgson & Whyte1989; Fitt, Heslinga & Watson 1992). However, thesecomponents may be toxic to the cultured organisms,due to their interference with protein synthesis(Alderman1988; Burka, Hammell, Horsberg, Johnson,Rainnie & Speare1997). Also, the antibiotics a¡ect theaccompanying micro£ora that usually grow with thecultured organisms and form part of their diet (Moal,Corre, Nicolas & Samain1995).At present, chloramphenicol (CHL) is one of the

most used antibiotics in bivalve shell¢sh aquacul-ture (Uriarte, Rupp & Abarca 2001). In pectinides,it has been used in the larval culturing of Pectenmaximus (Delaunay, Marty, Moal & Samain 1992;Robert, Nicolas & Miner 1995), Placopecten magel-lanicus (Naidu, Fournier, Marsot & Worms 1989),Patinopecten yessoensis (Cochard, Comps, Buestel &Paquotte 1991) and Argopecten purpuratus (Uriarte,Farias & Castilla 2001). This antibiotic has a wideantimicrobial spectrum; it attacks Gram-negativeand Gram-positive bacteria, inhibiting proteinsynthesis.In Chile, A. purpuratus is an important commercial

shell¢sh species. The national aquaculture produc-tion of this species in 2006 reached19.426 t, the thirdlargest after China and Japan. There are a number ofcommercial hatcheries of this species, principally inthe northern part of the country, and although theuse of antibiotics is decreasing, they are still beingused without knowing their real e¡ects.From1996, Chile prohibited the use of CHL in ani-

mals meant for human feeding. In fact, the certi¢ca-tion for ¢sh products export today, requires the

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absence of CHL in the samples. These regulations arein agreement with the international tendencies to-wards the increasing restriction in the use of wide-spectrum antibiotics (Dang, Ren, Song, Son & An2008; Grave, Hansenn, Kruse, Bangen & Kristo¡er-sen 2008; Richmond 2008).The present paper studies the e¡ect of CHL on the

growth and survival of the Chilean scallopA. purpur-atus juveniles and on their accompanyingmicro£ora.

Materials and methods

The experiment was performed in March1993, in theEstacio¤ n Costera de Investigaciones Marinas (ECIM),of the Ponti¢ciaUniversidad Cato¤ lica de Chile, locatedin Las Cruces, central Chile (33130 0S,71138 0W). Juve-niles of A. purpuratus with a mean shell height of937 � 55 mm were used. These juveniles were ob-tained from a mass culture reared for optimizationof the culture technology.The juveniles were exposed to ¢ve di¡erent con-

centrations of CHL (5, 10, 25, 50 and 100 mgmL�1),plus a control group without an antibiotic. Each con-centration was administered daily to three plasticcontainers with 30 individuals each. The seawaterused was ¢ltered at 0.5 mm, irradiated with UV lightand replaced every 2 days. The containers were aera-ted and the temperature was maintained at22 � 2 1C. The photoperiod was 12 h of light and12 h of darkness. Food was provided daily and con-sisted of a diet of150000 cells mL�1day�1of micro-algae of the species Isochrysis galfana (clone T-iso),Pavlova lutherii, Chaetoceros calcitrans and Chaeto-ceros mullerii in equal proportions.The experiment was continued for 2 weeks. The

survival of the juveniles was veri¢ed every 2 days,and their growth was measured on day 7 and at theend of the experiment.To evaluate the e¡ect of CHL on the accompanying

micro£ora, the same concentrations of CHL added tothe juveniles were tested in triplicate in containerswith only microalgae in the same concentration andcomposition as the juvenile scallop diet. Additionally,a sample of microalgae without an antibiotic and asample with only the ¢ltered seawater were used ascontrols. These containers were placed together withthe containers with juveniles under the same condi-tions. After 24 h, samples of 2mLwere taken fromallthe receptacles (scallop juveniles, microalgae, micro-algae control and ¢ltered seawater control) and werediluted in150mL of autoclaved ¢ltered seawater. Thediluted samples were passed through a polyamide

membrane ¢lter with 0.2 mm pores using a vacuumpump.The ¢lters were deposited in petridishes on cel-lulose disks with Bacto m-TGE (Difco, Detroit, MI,USA), which is a non-selective liquid medium inwhich most bacteria, yeasts and other fungi cangrow. The plates were incubated at 26 � 2 1C for24 h and the colony-forming units (CFU) that grewon the plates were counted.In order to determine signi¢cant di¡erences be-

tween the treatments, an analysis of variance andan a posteriori Tukey’s test were carried out. The sur-vival data were transformed using the arc sine func-tion of the square root of the probability.

Results

The statistical test used showed highly signi¢cantdi¡erences in the growth rates of the juveniles keptin di¡erent concentrations of CHL and in the controlgroup without an antibiotic (P50.0001). Only be-tween the treatments of 50 and100 mgmL�1was nosigni¢cant di¡erence detected (Tables 1 and 2). The

Table 1 Growth rate (mmday�1) and survival of juveniles(mean value � SD of three replicates) of Argopecten purpur-atus exposed to di¡erent chloramphenicol (CHL) doses(mgm�1)

CHL dose (lgmL� 1) Growth rate (lmday� 1) Final survival (%)

0 84.36 � 6.23 97.8 � 1.9

5 64.19 � 14.33 98.9 � 1.9

10 47.9 � 13.34 96.7 � 3.3

25 14.87 � 10.72 90.0 � 5.8

50 1.36 � 6.2 93.3 � 0.0

100 3.05 � 4.72 36.7 � 33.8

Table 2 Analysis of variance and Tukey test for thegrowth rate and survival (%) of juveniles of the Chileannorthern scallop Argopecten purpuratus exposed to di¡erentconcentrations (0, 5,10, 25, 50,100 mgmL�1) of cloramphe-nicol (CHL)

Source d.f. F P4F r2

Growth rate (mm day�1) 5 594 0.0001 0.87

Tukey test 456 tc 5 4.05 a5 0.05

Survival (%) 5 9.54 0.0007 0.80

Tukey test 12 tc 5 4.75 a5 0.05

CHL concentration lgmL� 1 0 5 10 25 50 100

Growth rate (mm day�1) A B C D E E

Survival (%) A A A A A B

Concentrations with the same letter are not signi¢cantly di¡erent.tc, critical value.

Aquaculture Research, 2009, 40, 1358^1362 Antibiotic treatment on juvenile northern Chilean scallop J Fierro & D Oliva

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control juveniles without antibiotic showed thegreatest growth rate,84.4 � 16.2 mmday�1, followedby the juveniles of the 5 mmmL�1 treatment, with64.2 � 14.3 mmday�1. The rest of the treatmentsshowed lower growth rates, especially the 50 and100 mmday�1 treatments, with growth rates of1.4 � 6.2 and 3.1 � 4.7 mmday�1respectively.Figure 1 shows the shell height and survival of

scallop juveniles in the di¡erent treatments after 6and14 days.There is an inverse relationship betweenshell height and antibiotic concentration.The controlwithout antibiotics presented the largest shell height.By day14, the di¡erences between the treatments in-creased. The survival rate was similar between treat-ments on days 6 and 14. Nevertheless, in the100 mgmL�1 treatment the survival was only36.7 � 33.8% at the end of the experiment. The restof the treatments and the control without antibioticsended with survival rates that were around 95%.There were signi¢cant di¡erences (P 50.0007) onlybetween the 100 mgmL�1 treatment and the rest ofthe treatments.On day 6 of the experiment a discoloration in the

digestive gland of the juveniles that received CHLwas noted. It was more evident in the treatmentswith greater concentrations. On day14, at the end ofthe experiment, those juveniles of the 100 mgmL�1

treatment had a translucent digestive gland.The plates with TGE medium seeded with the

water of the containers showed large deviations in

the CFU counts, which impeded the discovery of sig-ni¢cant di¡erences between the treatments. How-ever, after 24 h, a signi¢cant increase in the CFUcount was observed in the juvenile containers, show-ing a decreasing tendency in the CFU count with anincrease in the CHL concentration (Fig. 2).The containers with microalgae and the control

samples with ¢ltered seawater did not show signi¢-cant di¡erences in the CFU counts. In these contain-ers, the micro£ora growth rate was much lower thanthe rates observed in the containers with juveniles(Fig. 2). Finally, there was no colony growth on theplates seeded with the autoclaved water used in sam-ple dilutions, con¢rming its sterility at the time ofseeding.

Discussion

The results showed that after14 days of experimenta-tion, CHL signi¢cantlya¡ected the growth rate of theA. purpuratus juveniles. This was evident even at thelowest CHL concentration (5 mgmL�1). This con-£icts with the results of Montes and Lara (1988) onA. purpuratus juveniles culturing, where growthrates of 56 mmday�1 were obtained using up to100 mgmL�1of CHL.The same concentration drasti-cally decreased the juvenile growth and producedcomplete discoloration of the digestive gland, whichcould be related to the decrease in the growth rate.

Figure 1 Shell heightand survival of juvenilesof Argopecten purpuratusat di¡erent chloramphe-nicol concentrations.

Antibiotic treatment on juvenile northern Chilean scallop J Fierro & D Oliva Aquaculture Research, 2009, 40, 1358^1362

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The similar survival percentages observed in thecontrol sample and in the samples treated with 5^50 mgmL�1 of CHL indicate that this antibiotic didnot contribute to an increase in juvenile survival;on the contrary, it seems to be toxic in concentrationsof 100 mgmL�1, where the survival was drasticallyreduced.The accompanying micro£ora increased consider-

ably only in the containers with juveniles, comparedwith the containers with microalgae and the controlsamples of ¢ltered seawater. This suggests that prob-ably the juveniles provide metabolites that enrich themedium, promoting the growth of the microorgan-isms. On the other hand, the microalgae did not con-tribute to the growth of the micro£ora, because theCFUcount for those containers did not present signif-icant di¡erences with the ¢ltered seawater.The number of CFUmL�1 that grew on the

plates was very low.The treatments with scallop juve-niles did not surpass 500 CFUmL�1 and the treat-ments with microalgae and controls did not reach100 CFUmL�1. These values are very similar to theones found by Jeanthon, Prieur and Cochard (1988)analysing the number of existing Vibrionaceans onthe seawater used in the Argenton Hatchery, France,for the culture of P. maximus. Subsequent analyses ofthe colonies obtained in our experiments, performedin the Laboratorio de Microbiolog|¤ a of the Ponti¢ciaUniversidad Cato¤ lica de Chile, indicated that these

colonies were con¢rmed to be of Vibrionacean andPseudomonacean bacteria. Apparently, these bacter-ia are not pathogenic for the juveniles. They couldeven be part of their usual micro£ora or contributeto their diet. Langdon and Newell (1990) found thatthe bacteria that grow accompanying two ¢lter fee-der bivalves (Crassostrea virginicay Geukensia demissa)in the estuary of Canary creek, USA, were an impor-tant food source, contributing up to 31% of theircarbon requirements and 71% of their nitrogenrequirements.Additionally, this work suggests that CHL may be

used on theA. purpuratus scallop as a way to preventthe proliferation of pathogenic bacteria; however, itsuse a¡ects the individuals in culture as the accompa-nying micro£ora. Because of this, it is advisableto use it in concentrations that do not exceed5 mgmL�1. This was the concentration used routi-nely at the ECIM between 1991 and 1994 for thenorthern Chilean scallop juvenile cultures. It isworth mentioning that with this concentration ofCHL, survival rates of 30% in mass larvae culturesand of 25% in juvenile cultures were obtained. Addi-tionally, even though this experiment showed thatCHL may have a certain degree of toxicity to the cul-tivated individuals, its use might prevent diseasesthat usually occur under the high-density culturingconditions in which shell¢sh are maintained in thehatcheries.

Figure 2 Colony-form-ing units (CFU) after 24 hin containers with juve-niles ofArgopecten purpur-atus and with microalgae,treatedwith di¡erent con-centrations of chloram-phenicol. FSW, Filteredsea water; AW, Auto-claved water used in sam-ple dilutions.

Aquaculture Research, 2009, 40, 1358^1362 Antibiotic treatment on juvenile northern Chilean scallop J Fierro & D Oliva

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Finally, we suggest that rotating the use of variousantibiotics, administered to the cultures alone ormixed, could increase the antimicrobial specter andavoid the developing of resistant bacterial strains.Nevertheless, the e¡ects of antibiotics on humanhealth and the enviroment are an increasing con-cern (HernaŁ ndez 2005; Lai, Hou, Su & Chen 2008).Therefore, the use of probiotics is a new approach inaquaculture to increase growth rates and improvethe nutritional status of the cultured animals byincreasing the amount of algynate lyase and otherextracelular enzymes (Doeschate & Coyne 2008).

Acknowledgments

We would like to thank our friends in the Estacio¤ nCostera de Investigaciones Marinas (ECIM), espe-cially Juan Carlos Castilla for the discussion of theexperiments, and L. R. DuraŁ n for improving the text.

References

Alderman D. (1988) Fisheries chemotherapy: a review. In:Recent Advances in Aquaculture,Vol.3 (ed. by J.F. Muir & R.Roberts), pp.1^61. Croom Helm, London.

Bourne N., HodgsonC. &Whyte N. (1989) Amanual for scal-lop culture in British Columbia. CanadianTechnical Reportof Fisheries and Aquatic Sciences1694,1^215.

Burka J.F., Hammell K.L., Horsberg T.E., Johnson G.R., Rain-nie D.J. & Speare D.J. (1997) Drugs in salmonid aquacul-ture ^ a review. Journal of Veterinary Pharmacology andTherapeutics 20,333^349.

Cochard J.C., CompsM., Buestel D. & Paquotte P. (1991) Intro-duction of the Japanese scallop Patinopecten yessoensis(Jay) into France and the hatchery production of the F1generation. In: World Aquaculture Workshops (ed. by S.Shumway & P. Sandifer), pp. 281^286.World AquacultureSociety, Louisiana State University, Baton rouge.

Dang H., Ren J., Song L., Son S. & An L. (2008) Dominanchloramphenicol-resistant bacteria and resistance genesin coastal marine waters of Jiazhou Bay, China.WorldJournal of Microbiology and Biotechnology 24, 209^217.

Delaunay F., Marty Y., Moal J. & Samain J.F. (1992) Growthand lipid class composition of Pecten maximus larvaegrown under hatchery conditions. Journal of Experimen-tal Marine Biology and Ecology163, 209^219.

Doeschate K.I. & CoyneV.E. (2008) Improved growth rate infarmed Haliotis midae through probiotic treatment. Aqua-culture 284,174^179.

FittW., Heslinga G. &WatsonT.C. (1992) Use of antibiotics inthe mariculture of giant clams (F. Tridacnidae). Aquacul-ture104,1^10.

Grave K., Hansenn M.K., Kruse H., Bangen M. & Kristo¡er-sen A.B. (2008) Prescription of antimicrobial drugs inNorwegian aquaculture with emphasis on‘‘new’’¢sh spe-cies. PreventiveVeterinaryMedicine 83,156^169.

HernaŁ ndez P. (2005) Responsible use of antibiotics in aquacul-ture. FAO Fisheries Technical Paper 469,96pp.

Jeanthon C., Prieur D. & Cochard J.C. (1988) Bacteriologicalsurvey of antibiotic-treated sea waters in a Pecten maxi-mus hatchery. Aquaculture 71,1^8.

Lai H.T., Hou J.H., Su C.I. & Chen C.L. (2008) E¡ects of chlor-amphenicol, £orfenicol and thiamphenicol on growth ofalgae Chlorella pyrenoidosa, Isochrysis galbanaandTetrasel-mis chui. Ecotoxicology and Environmental Safety 72, 329^334.

Langdon C.J. & Newell R.I. (1990) Utilization of detritus andbacteria as food sources by two bivalve suspension-feeders,the oyster Crassostrea virginica and the mussel Geukensiademissa.Marine Ecology Progress Series 58, 299^310.

Loosano¡ V. & Davis H. (1963) Rearing of bivalve mollusks.Advances in Marine Biology1,1^136.

Moal J., Corre S., Nicolas J.L. & Samain J.F. (1995) Bacteria asfood for bivalve larvae. 10th International Pectinid Work-shop, Cork, Ireland.

Montes E. & Lara E.E. (1988) Contribucio¤ n a la optimizacio¤ n decultivo masivo de post-larvas de ostio¤ n. Memorias del Sim-posio Internacional de los RecursosVivos y las Pesquer|¤ asen el Pac|¤ ¢co Sudeste, Chile.Vin� a del Mar 9^13 mayo de1988.

Naidu K.S., Fournier R., Marsot P. & Worms J. (1989) Cultureof the sea scallop, Placopecten magellanicus: opportunitiesand constraints. In: Cold-WaterAquaculture in Atlantic Ca-nada (ed. byA. Boghen), pp. 211^239, CIID, Honcton, NB,Canada.

Richmond R. (2008) Environmental protection: applyingthe precautionary principle and proactive regulation tobiotechnology.Trends in Biotechnology 26, 460^467.

Robert R., Nicolas J.L. & Miner P. (1995) Mortality controlof Pecten maximus larvae in hatchery. 10th InternationalPectinidWorkshop, Cork, Ireland, pp.51^52.

Uriarte I., Farias A. & Castilla J.C. (2001) E¡ect of antibiotictreatment during larval development of the Chilean scal-lop Argopecten purpuratus. Aquacultural Engineering 25,139^147.

Uriarte I., Rupp G. & Abarca A. (2001) Produccio¤ n de juve-niles de pect|¤ nidos iberoamericanos bajo condicionescontroladas. In: Los moluscos pect|¤ nidos de Iberoame¤ rica:Ciencia y Acuicultura (ed. by A.N. Maeda-Mart|¤ nez), pp.147^171. Editorial Limusa, Me¤ xico.

Walne P.R. (1974) Culture of Bivalve Molluscs. 50 Years’sExperience at Conwy. Fishing News (Books), UK,173pp.

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