Considering the concerns about the safety of the use of formaldehyde as antifungal agent, the effects of three alternative chemicals (potassium sorbate, copper hydroxide and magnesium chloride) were tested in the artificial incubation of signal crayfish (Pacifastacus leniusculus) eggs. Eight treatments were performed during 15 min every other day: formaldehyde at 3000 ppm (control), potassium sorbate at 5000 and 10000 ppm, copper hydroxide at 40, 60, 80 and 200 ppm, and magnesium chloride at 10000 ppm. Eggs were incubated in a flow through system at a density of 20 eggs·cm−2. After 61 days of incubation, the highest efficiencies were obtained with 200 ppm of copper hydroxide (77.6% of survivors to stage 2) with no significant differences from the control (74.3%). Lower concentrations of copper hydroxide resulted in high egg mortality whereas potassium sorbate and magnesium chloride were ineffective to avoid fungal growth and total egg mortality took place. From the obtained results, copper hydroxide baths at 200 ppm could be considered as a good alternative to formaldehyde

A. González

J.D. Celada

J.M. Carral

M. Sáez-Royuela

P.M. Melendre

R. González

V. García

Knowledge and Management of Aquatic Ecosystems

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Knowledge and Management of Aquatic Ecosystems (2009) 394-395, 16 http://www.kmae-journal.orgc© ONEMA, 2010DOI: 10.1051/kmae/2010010Antifungal treatments in artificial incubation of crayfisheggs (Pacifastacus leniusculus, Astacidae): Searchingfor alternatives to formaldehydeJ.M. Carral(1), A. González(1), J.D. Celada(1), M. Sáez-Royuela(1),P.M. Melendre(1), R. González(1), V. García(1)Received September 29, 2009 / Reçu le 29 septembre 2009Revised February 3rd, 2010 / Révisé le 3 février 2010Accepted February 6, 2010 / Accepté le 6 février 2010ABSTRACTKey-words:antifungaltreatment,artificialincubation,astacid crayfishConsidering the concerns about the safety of the use of formaldehydeas antifungal agent, the effects of three alternative chemicals (potassiumsorbate, copper hydroxide and magnesium chloride) were tested in the ar-tificial incubation of signal crayfish (Pacifastacus leniusculus) eggs. Eighttreatments were performed during 15 min every other day: formaldehydeat 3000 ppm (control), potassium sorbate at 5000 and 10 000 ppm, cop-per hydroxide at 40, 60, 80 and 200 ppm, and magnesium chloride at10 000 ppm. Eggs were incubated in a flow through system at a den-sity of 20 eggs·cm−2. After 61 days of incubation, the highest efficiencieswere obtained with 200 ppm of copper hydroxide (77.6% of survivors tostage 2) with no significant differences from the control (74.3%). Lowerconcentrations of copper hydroxide resulted in high egg mortality whereaspotassium sorbate and magnesium chloride were ineffective to avoid fun-gal growth and total egg mortality took place. From the obtained results,copper hydroxide baths at 200 ppm could be considered as a good alter-native to formaldehyde.RÉSUMÉTraitements antifongiques lors d’incubations artificielles d’œufs d’écrevisse (Pacifastacusleniusculus, Astacidae) : recherche d’alternatives au formaldéhydeMots-clés :traitementantifongique,incubationartificielle,écrevisseastacidéConsidérant les préoccupations sur l’emploi sécurisé du formaldéhyde commeantifongique, les effets de trois alternatives chimiques (sorbate de potassium,hydroxyde de cuivre et chlorure de magnésium) ont été testés en incubationartificielle d’œufs d’écrevisse signal (Pacifastacus leniusculus). Huit traitementsdifférents ont été effectués pendant 15 minutes chaque jour : formaldéhyde à3000 ppm (contrôle), sorbate de potassium à 5000 et 10 000 ppm, hydroxyde decuivre à 40, 60, 80 et 200 ppm, et chlorure de magnésium à 10 000 ppm. Lesœufs ont été incubés à une densité de 20 œufs·cm−2 dans un système d’eaucourante. Après 61 jours d’incubation, les plus fortes efficacités ont été obtenuesavec 200 ppm d’hydroxyde de cuivre (77.6 % de survivants au stade 2) sansdifférence avec le contrôle (74.3 %). Les plus faibles concentrations en hydroxydede cuivre ont entraîné une plus forte mortalité des œufs alors que le sorbatede potassium et l’hydroxyde de cuivre étaient inefficaces à éviter la croissance(1) Dpto. de Producción Animal, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain,jmcarl@unileon.esArticle published by EDP SciencesJ.M. Carral et al.: Knowl. Managt. Aquatic Ecosyst. (2009) 394-395, 16fongique et une mortalité totale des œufs intervenait. À partir des résultats obte-nus, des bains d’hydroxyde de cuivre à 200 ppm peuvent être considérés commeune bonne alternative au formaldéhyde.INTRODUCTIONIn astacid crayfish, advances in artificial incubation techniques (AI) provide reliable options forits application under culture conditions (see review by González et al., 2009). Depending onstripping time and temperature, artificial incubation covers long periods, over 100 days (Pérezet al., 1998), during which nonviable eggs can be invaded by fungi, usually Saprolegnia spp.and other Oomycetes (Edgerton et al., 2002). Their hyphae can spread to the surroundinghealthy ones (Celada et al., 2004; Melendre et al., 2006; Sáez-Royuela et al., 2009) causing animportant decrease of efficiency rates. Periodical removal of dead eggs has shown positiveeffect on the hatching rate (Carral et al., 2004; Policar et al., 2006) but it is laborious andmanipulation can cause damages in healthy ones, giving rise to new losses (Sáez-Royuelaet al., 2009). Thus, the use of antifungal treatments would be advisable, and several studieshave been addressed to find effective chemicals.For many years malachite green has been the most effective fungicide used in aquaculturebut since 1991 and 1997 is not permitted for aquaculture in USA (Schereier et al., 1996)and EU (Celada et al., 2004) respectively, because of its teratogenic and carcinogenic effects(Andersen et al., 2006; Sapkota et al., 2008). After a series of trials testing chemicals used inother aquatic species, Celada et al. (2004) and Melendre et al. (2006) reported formaldehydeas the most effective antifungal. Although formaldehyde is approved for use in fish culture inUSA and EU, there is concern about user safety because of its suspected carcinogenicity andits potential adverse effects on the aquatic environment (Arndt et al., 2001; Gieseker et al.,2006).Considering the convenience of antifungal agents alternative to formaldehyde, the aim of thisstudy was to obtain data on the possible effectiveness of other chemicals. Three were se-lected: potassium sorbate (E202) is used to inhibit molds and yeasts in many foods for foodpreservation and extension of shelf-life (Dorko et al., 1997), copper hydroxide (Cu(OH)2) iscommonly used to prevent some fungal diseases of fruit and vegetable crops, and magne-sium chloride (MgCl2) was proposed by Rantamäki et al. (1992) to prevent transmission of thecrayfish plague fungus (Aphanomyces astaci).MATERIALS AND METHODSEggs from 70 berried Pacifastacus leniusculus females from a crayfish farm were detachedin the laboratory by sliding blunt forceps smoothly from the base to the tip of the pleopods.Prior to artificial incubation, embryonic phases were identified following Celada et al. (1985,1987) being at phase IX (embryo with naupliar appendages).A total of 6400 eggs were pooled and artificially incubated in the experimental devices de-scribed by Carral et al. (1992) and Sáez-Royuela et al. (2009) up to stage 2 juvenile pro-duction. Artesian well water was supplied in open system at a flow rate of 0.5 L·min−1(12.5 mL·cm−2·min−1). The parameters of incoming water quality were: pH 7.9, hardness5.2 odH (calcium: 32.3 mg·L−1), dissolved oxygen about 8 mg·L−1, nitrite < 0.015 mg·L−1,ammonium < 0.02 mg·L−1, total dissolved solids 110.5 mg·L−1, and total suspended solids< 0.5 mg·L−1. Temperature was 10±1 ◦C until eggs reached the eye stage (phase XIII). There-after, it was raised to 15.5 ± 1 ◦C up to final stage 2 juvenile production.Chemicals used were formaldehyde (Proquiman S.L.) at 3000 ppm, magnesium chloride(MgCl2, Sigma-Aldrich) at 10 000 ppm, potasium sorbate (E202, Coralim Aditivos) at 5000and 10 000 ppm, and copper hydroxide (Cu(OH)2, Funguran-OH 50 WP, Nufarm España S.A.)16p2J.M. Carral et al.: Knowl. Managt. Aquatic Ecosyst. (2009) 394-395, 16Table ISurvival rates in artificial incubation receiving eight antifungal treatments. Values followed by differingletters were significantly different (P < 0.05) from the others in the same column. SEM = standard errorof mean.Tableau ITaux de survie en incubation artificielle selon huit traitements antifongiques. Les valeurs suivies de diffé-rentes lettres sont significativement différentes (P < 0,05) des autres de la même colonne. SEM = erreur-standard de la moyenne.Chemical agent Concentration Application form Stage 1 Stage 2(ppm) (% ± SEM) (% ± SEM)Formaldehyde (control) 3000 Peristaltic pumps 83.7 ± 0.4a 74.3 ± 2.8a5000 0 0Potassium sorbate 10 000 Peristaltic pumps 0 040 0 0Copper hydroxide 60 Static baths 0 080 10.3 ± 3.1b 2.5 ± 1.5b200 86.2 ± 1.5a 77.6 ± 5.6aMagnesium chloride 10 000 Peristaltic pumps 0 0at 40, 60, 80 and 200 ppm. Treatments were administered during 15 min every other day upto the beginning of hatchings. Solutions of formaldehyde, potasium sorbate and magnesiumchloride were discharged to the incoming water flow using peristaltic pumps. Because its lowsolubility in water, copper hydroxide treatments were performed in static baths.Each treatment was performed by quadruplicate at an egg density of 20 cm−2 (one com-plete layer, eggs contacting to others). Throughout incubation eggs were daily checked bycareful ocular inspection and any presence of fungal growth was noted. Dead eggs were notremoved. The number of stage 1 (after hatching) and stage 2 (after first moult) juveniles wasquantified. Collection of stage 2 juveniles was carried out daily following the recommendationof Melendre et al. (2007), because final survival is reduced the longer they remain in the incu-bators. Survival rates were calculated as the percentage of juveniles from the initial numberof eggs.Prior statistical analysis arc-sine transformation of percentages was made. Results were ex-amined by analysis of variance (one-way ANOVA) using the SPSS 16.0 computer program(SPSS Inc., Chicago, USA). Mean comparison was tested using the Duncan’s test. The sig-nificance level was P < 0.05.RESULTSArtificial incubation lasted for 61 days. Hatchings began on day 43, stage 2 juveniles wereobtained from day 51 and the first moult period lasted for 10 days.Survival rates in each treatment are presented in Table I. The highest survival rate to stage 2(77.6%) was obtained with copper hydroxide at 200 ppm for 15 minutes every other day,with no significant difference from the formaldehyde control (74.3%). In both cases no fungalgrowth was observed. However, on eggs treated with copper hydroxide at 40 and 60 ppmfungi were observed from day 14 onwards, whereas at 80 ppm from day 19. In the later case,a low production of stage 2 juveniles was obtained (2.5%).Earlier fungal growth was observed in eggs treated with potassium sorbate and magnesiumchloride, from day 5 onwards, affecting all the eggs and causing total mortality.DISCUSSIONCopper hydroxide is a widely used fungicide-bactericide approved for both organic and con-ventional agricultural production of vegetable crops for control of diseases (Rice et al., 2007).16p3J.M. Carral et al.: Knowl. Managt. Aquatic Ecosyst. (2009) 394-395, 16Although there are no references of its use in aquatic species it was the only one, among thechemicals tested as alternative to formaldehyde, effective to prevent fungal growth on cray-fish eggs. As it has been proved with other antifungal tested (Celada et al., 2004; Melendreet al., 2006; Policar et al., 2006; Sáez-Royuela et al., 2009), artificial incubation of astacidcrayfish eggs calls for higher concentrations than those recommended for other species. Inthe case of copper hydroxide, the lower concentrations tested did not avoid the presenceof fungus which were observed after 14–19 days of incubation, resulting in poor survival tostage 2 juveniles (2.5% at 80 ppm) or total mortality (at 40 and 60 ppm). However at 200 ppm,juvenile production was high (77.6%) and similar to the obtained with formaldehyde.Because their low water solubility, copper complexes, including Cu(OH)2, tend to precipitateand thus administration by means of peristaltic pumps does not guarantee the concentrationreceived on eggs, as Melendre et al. (2006) pointed out for the copper sulphate. Thus, theapplication of these chemicals should be restricted to static baths leading to additional manwork throughout the AI period.Considering that Saprolegnia spp. and the genus Aphanomyces belong to the same family(Diéguez-Uribeondo et al., 2006), magnesium chloride was tested. The concentration usedwas the proposed by Rantamäki et al. (1992) as effective to reduce the sporulation process ofA. astaci, but it did not prevent the fungal growth on eggs and total mortality was registered.The same occurs with potassium sorbate (E202) widely used to inhibit molds and yeasts inmany foods, even when it was applied at higher concentrations. Considering this, both agentsshould be rejected as antifungal in astacid artificial incubation, at least at the concentrationstested.Other antifungal agents effective on artificial incubation of crayfish eggs, such as formalde-hyde, have also disinfection effects allowing producing specific virus-free juveniles (Edgertonand Owens, 1997) or faster growth juveniles (González et al., 2009). Considering the po-tent biocidal properties of cooper, alone or in complexes, which determine their wide currentusages as antibacterial, antifungal and antiviral (Borkow and Gabbay, 2009), it would be ex-pected a similar performance.To sum up, copper hydroxide at 200 ppm administered in 15 min static baths every otherday can be considered as effective alternative to formaldehyde. Further research on accurateconcentration of this copper compound and others commonly used in aquatic species, suchas copper sulphate, should be developed.ACKNOWLEDGEMENTSFunding of this study was the Plan Nacional de I+D+i, Ministerio de Eduación y Ciencia, Spain,Research Project AGL2005-01127. We should also like to thank the Quiñón S.A. crayfish farmfor their collaboration.REFERENCESAndersen W.C., Turnipseed S.B. and Roybal J.E., 2006. Quantitative and confirmatory analyses of mala-chite green and leucomalachite green residues in fish and shrimp. J. Agric. 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Antifungal treatments in artificial incubation of crayfish eggs (Pacifastacus leniusculus, Astacidae): Searching for alternatives to formaldehyde

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Antifungal treatments in artificial incubation of crayfish eggs (Pacifastacus leniusculus, Astacidae): Searching for alternatives to formaldehyde

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