May | June 2013

Controlling mycotoxins with binders

The International magazine for the aquaculture feed industry

International Aquafeed is published six times a year by Perendale Publishers Ltd of the United Kingdom.All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies, the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis of information published. ©Copyright 2013 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any form or by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058

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26 | InternatIonal AquAFeed | May-June 2013

Feed ingredientsand feedpricesareincreasing; it is becoming harderto maintain the nutritional balanceof the feed without increasing too

much the feed price. Now, the use ofingredients from less stringent quality islikely to increase. Though plant materialsare usually more reasonable in price thananimalproducts,theycanpresentproblemsthroughthepresenceofnaturallyoccurringcontaminants.Indeed,contaminationoffeedcommoditiesbymicroorganismsandmyco-toxins is the first negative factor impactinganimal feed quality. Numerous researcheshave studied thedecreaseof performanceswithcontaminatedfeeds.

Lipopolysaccharides (LPS), also known asendotoxins, are present in the cell mem-brane of gram negative bacteria. They area structural component of the cell wall andarecontinuouslyreleasedintheenvironmentat cell death and during cell growth or divi-sion. Therefore, endotoxins are omnipres-ent in feed, water and fish gut which hasshowntobeanimportantbacterialreservoir.Endotoxinsactasneurotoxiccompoundsandhaveimmunosuppressiveeffectonfish.

Mycotoxinsareadiversegroupofpoten-tial toxic metabolites produced by a vari-ety of fungal species that often contami-nate feedstuffs and consequently fish diets.Mycotoxinscanvary inshapeandsize.Theyareheat stable and resist toextrusionproc-ess. For the ones that have been identified,it isknownthatafewpartsperbillion(ppb)already impact animal growth performances.Mycotoxins effects are specie dependent;cross contamination of different mycotoxinsincreases the damage caused (synergy) andresults in uncharacteristic symptoms, thus

making it difficult to diagnosemycotoxicosis.Evenifextensivestudiesaredoneinthisfield,manymycotoxinseffectsremainunknown.

PreventionBecause of their effect on the immune

system and fish performances, the presenceoftoxinsimpairsthefarmeconomicperform-

ances. Strategies of prevention and controlexist.

In order to avoid deleterious effects ofmycotoxinsonfish,thebest istoavoidcon-taminationoftheplantswithmouldsthroughadapted cultural practices. During harvestandstorage,mycotoxinsproductionmustbeprevented by reducing mould stress condi-

Controlling mycotoxins with bindersby Adrien Louyer, aquaculture supervisor, Olmix Asia Pacific, Marie Gallissot, technical supervisor, Olmix SA, Dr Nguyen Van Nguyen, director of The Research Center for Fish Nutrition and Fishery Postharvest Technology - RIA2.

Table 1: Formulated diets with different Mt.X+ doses (values are expressed as a % on an as fed basis)

Feed Ingredients D0 (0% Mt.X+) D0.05 (0.05% Mt.X+)

D0.15 (0.15% Mt.X+)

Fish meal 65% 17.00 17.00 17.00

Soybean meal 28.00 28.00 28.00

Cassava meal 18.75 18.75 18.75

rice bran 35.00 35.00 35.00

DCP 0.3 0.25 0.15

Premix- M-V 0.30 0.30 0.30

Fish oil 0.50 0.50 0.50

lysine (lys) 0.10 0.10 0.10

Methionine (Met) 0.05 0.05 0.05

Mt.X+ 0.00 0.050 0.15

total 100.00 100.00 100.00

Proximate composition (% as fed basis)

Dry matter 89.22 89.22 89.22

Moisture 10.78 10.78 10.78

Crude protein 28.55 28.55 28.55

Crude fat 5.48 5.48 5.48

Crude fibre 5.86 5.86 5.86

Crude ash 8.84 8.84 8.84

nitrogen free extract 40.34 40.34 40.34

Gross energy (kcal.g-1) 3.63 3.63 3.63

Fishmeal 65 percent (Vietnam, Kien Giang); Soybean meal 47 percent (India); Fish Oil (Chile fish oil), cassava meal (Vietnam, Tay Ninh), Rice bran (Vietnam, Tien Giang), Lysine and Methionine (Japan)

16 | InternatIonal AquAFeed | May-June 2013

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May-June 2013 | InternatIonal AquAFeed | 17

tions such as quick temperatureor humiditychange. Unfortunately, even with the bestmanagement procedure, it is extremely diffi-culttototallyavoidmycotoxincontamination.

Additionally, it is very hard to manageendotoxin ingestion by the fish. Endotoxinsfoundinthefishintestinearebroughtbycon-taminatedfoodandwaterorcanbeliberatedfromintestinalgram-negativebacteria.

Oneofthebestsolutionstocontrolthesetoxinsistouseawidespectrumbinderinthefeed.Olmix,aFrenchcompany,hasdevelopeda patented hybridmaterial calledAmadeite®(Figure 1): a clay which interlayer space hasbeen extended by the insertion of algaepolysaccharides (ulvans). The adsorption oftoxinsinthismaterialisacomplexmechanisminvolving the surface area of montmorillonite,the polyanionic structure of ulvans and thescaffold structure formed in the interlayerspace.Basedonthisuniqueingredient,awidespectrumtoxinbinder,MT.X+wascreated.

Experimental study in the Mekong Delta

TheobjectiveofthisstudywastoevaluatetheeffectsofMT.X+ongrowthperformanc-esandfeedutilizationfortracatfishjuveniles.

Material and MethodsLocation and set up

Theexperimentwasdone inacommercialfarm intheMekongDeltaduringtwomonths.1,080 healthy catfish fingerlings (initial weightaround 30 g) obtained from a local supplierwere used for the test. They were raised infloatingcages(hapas),of2x2x2metres,inwhichpangasiuswererandomlyallocated(120fishperhapas). The cages were in the same pond toavoid water difference. Daily water exchangewasdonewithatidalsystem.Fishwereacclimat-edforaweekbeforethebeginningofthetrial.

Experimental designThree iso-nitrogenous and iso-energetic

diets were formulated (Table 1). Controldiet, Experimental diet 1 and Experimentaldiet 2 respectively contained 0, 0.05 and0.15% of MT.X+. The diets were producedby RIA2 feedmill, using extrusion processwithpelletsizeof5±1mm.Thedietswererandomlyallocatedtocages.Threereplicates

perdietweredone.Fish were fed adlibitum twice a dayandexcessfeedwasremoved from thecages 20 minutesafterfeeding.

To check themycotoxin contami-nation of the feedused, mycotoxinanalysis has beendone byHPLCMS/MS method in anindependent labo-ratory, LDA 22, inFrance.

Water qual-ity was watched byrecording daily dis-solvedoxygen(DO),temperature (T°C),pH, nitrites (NO2)and ammonia(NH3). DO, NO2andNH3wereana-lysed by commercialaquariatestkit.

Proximate com-position of the dietswas analysed accord-ing to the AOACprocedures. Theparameters usedto evaluate growthperformance andfeed utilization wereexpressed as DailyWeightGain(DWG),FeedConversionRate(FCR) and SurvivalRate(SUR).

Data from eachtreatmentweresub-jected to one-wayANOVA (differ-ences were consid-ered significant atp < 0.05) and toa Duncan multiplerange of tests byusingRsoftware.

Table 2: Contamination level of the feed for the most common mycotoxins

MYCotoXIn leVel

t-2 toxin < 0.01ppm

Deoxynivalenol (Don) <0.01 ppm

Zearalenone <0.01 ppm

Fumonisins (B1+B2) 0.025 ppm (B1:0.015+ B2:0.010)

aflatoxins <0.004 ppm (aFB1:<0.001)

ochratoxin α <0.001 ppm

16 | InternatIonal AquAFeed | May-June 2013 May-June 2013 | InternatIonal AquAFeed | 17

FEATURE

46 | InternatIonal AquAFeed | May-June 2013

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ResultsMycotoxin analysis

Among the 44 different mycotoxins thatwere tested, levels of the most commonmycotoxinsaredisplayedinTable2.Thecon-taminationlevelwasverylowforthissample.

Water quality analysisWater quality parameters are displayed in

Table3.During all the timeof theexperimentammonia(NH3)washigherthantheVietnameseacceptedlimit(2mg/linsteadof<0.3mg/l).NO2washigherthantheVietnameselimitduringthelast month of the experiment (2 mg/l insteadof <1 mg/L). Moreover, it was observed thatthefishdensityoutsidethehapaswasveryhigh.The water was probably heavily loaded withpathogens.

Zootechnical performancesGrowthperformances, feedefficiencyandsur-

vival ratearepresented inTable4.After60daysof feeding period, no significant difference wasobservedonsurvivalrate(±88%)andfeedintake(± 104 g/fish). However, the final body weightwas significantly different between fish fed 0.15%MT.X+andfishfedcontroldiet(82.66and70.05g/fish, respectively). As a consequence, FCR wassignificantlylowerforfishfedD0.15comparedtofishfedD0.05orD0(2.01,2.62and2.57,respectively).

DiscussionFish fedeither controldiet,MT.X+0.05%

orMT.X+0.15%hadsimilarsurvivalrateandfeedintake.However,FeedConversionRatio,DailyWeightGainandFinalWeightweresig-nificantlydifferentamongdiets.FishfedMT.X+0.15% had significantly better performancesthan fish fed control diet (-0.5 point in FCR,

+18% in final weight). The supplementationwith 0.05% of MT.X+ did not impact theperformancesincomparisonofcontrolgroup.

Several factors can explain the obtained results:

Mycotoxin contamination was very lowin this experiment. We cannot exclude thepossibility that the real contamination washigher than measured, due to uncertaintylinked with the analysis (sampling method,unknown toxins). However, as we observe

a dose-dependent effect of MT.X+ in thisexperiment,thispossibilityislikelydismissed.

Ontheotherhand,waterqualityandpondman-agementshowedtobepoor.Inacontextofexcessammonia and nitrite concentrations, frequent andimportantwaterexchangeandprobablehighpatho-genload,fishundergostressandimmunosuppression.MT.X+,bybindingendotoxins and supporting theimmunesystemhelpsfishtocopewiththesestres-sors.Betterprotected,fishfedMT.X+bettervalorizethefeedandshowimprovedgrowthperformances.

MT.X+:improveprotection,improveper-formances.

ReferencesHalver,J.E.andHardy,R.W.(2002),FishNutrition,ElsevierScience,pp.601-618.

RoederD.J.,1989:“Endotoxic-lipopolysaccharide-specificbindingproteinsonlymphoidcellsofvariousanimalspecies:associationwithendotoxin

susceptibility”,InfectionandImmunityjournal,57(4):1054-1058.

NayakS.K.,etal2008;“EffectofendotoxinontheimmunityofIndianmajorcarp,Labeorohita’,FishandShellfishImmunology,24(4):394-399.

NRC(NationalResearchCouncil)(2011),Nutrientrequirementsoffishandshrimp,NationalAcademyPress,Washington,D.C.,pp.233-247.

RodriguezM.A.,etal2012:“MycotoxinDetoxification:SciencevsMarketing,AllAboutFeed,Mycotoxinspecialp24-26.

Tapia-Salazar,M.etal.2010.Mycotoxinsinaquaculture:Occurrenceinfeedscomponentsandimpactonanimalperformance.En:Cruz-Suarez,L.E.,Ricque-Marie,D.,Tapia-Salazar,M.,Nieto-López,M.G.,Villarreal-Cavazos,D.A.,Gamboa-Delgado,J.(Eds),AvancesenNutriciónAcuícolaX-MemoriasdelDécimoSimposioInternacionaldeNutriciónAcuícola,8-10deNoviembre,SanNicolásdelosGarza,N.L.,México.ISBN978-607-433-546-0.UniversidadAutónomadeNuevoLeón,Monterrey,México,pp.514-546.

Springetal2005.Mycotoxinarisingthreattoaquaculture,NutritionalBiotechnologyintheFeedandFoodIndustriesp323-331

HavenaarRetal,2006:”Efficacyofsequestrant/chelatorAmadeite,inthebindingofmycotoxinsduringtransitthroughadynamicgastrointestinalmodel(TIM)simulatingtheGIconditionsofpigs;Theworldmycotoxinforum-Thefourthconference,November6-82006,Cincinnati,Ohio,USA.

AOAC(1992):AOACOfficialMethod992.15CrudeProteininMeatandMeatProductsIncludingPetFoodsCombustionMethodFirstAction1992http://www.aoac.org/omarev1/992_15.pdf

http://thuvienphapluat.vn/archive/Thong-tu/Thong-tu-45-2010-TT-BNNPTNT-dieu-kien-co-so-vung-nuoi-ca-tra-tham-canh-vb109053t23.aspx

Table 3: average water quality parameters during the time of the experiment

Parameters level Maximum limit [10]

pH 6± 1 7-9

toC 28 ± 1 28-300 C

nH3 (mg/l) 2 ±0 ≤ 0,3

no2 (mg/l) 0-2 ±1 0.01 -1

Do (mg/l) 4-6 ±1 ≥ 2,0

Table 4: Zootechnical performances of pangasius (Pangasius hypophthalmus) fed with diets containing different levels of Mt.X+

D0 D0.05 D0.15

Survival rate (%) 88.7 a ± 7.6 88.8a ± 4.5 88.3a ± 5.4Final body weight (g/fish) 70.05a ± 7.6 70.86ab ± 4.5 82.66b ± 5.4

Daily weight gain (g/day) 0.64b ± 0.15 0.66ab ± 0.05 0.85a ± 0.08

Feed intake (g/fish) 101a ± 9.8 106a ± 3 106a ± 8.1

FCr 2.57 ab ± 0.38 2.62a ± 0.27 2.01b ± 0.04

Figures are presented as mean ± SD, values in the same row with different superscript letters are significantly different (p< 0.05)

Figure 1: The interlayer space of Montmorillonite is multiplied by 10 thanks to the intercalation of green algae polysaccharides, the ulvans. The interlayer space

is enlarged from 0,3-0,4 nm to 3-4 nm allowing to capture 2 nm molecules such as Trichothecenes or fumonisins

More InforMatIon:Adrien Louyer, alouyer@olmix.comMarie Gallissot, mgallissot@olmix.comDr Nguyen Van Nguyen, nguyenria2@gmail.com

This article was first published on www.aquafeed.com

18 | InternatIonal AquAFeed | May-June 2013

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May-June 2013 | InternatIonal AquAFeed | 19

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They are what they eat Enhancing the nutritional value of live feeds

with microalgae

Controlling mycotoxins with binders

Ultraviolet water disinfection for fish

farms and hatcheries

Niacin – one of the key B vitamins for sustaining

healthy fish growth and production

Volume 16 I s sue 3 2 013 - mAY | J uNe

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