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Analysis of the Seasonal Variation in Biochemical Composition Of Annls Limnol. 35 (4) 1999 : 223-231 Analysis of the seasonal variation in biochemical composition of Daphnia magna Straus (Crustacea : Branchiopoda : Anomopoda) from an aerated wastewater stabilisation pond H.-M. Cauchie1 M.-F. Jaspar-Versali2 L. Hoffmann3 J.-P. Thome1 Keywords : Daphnia magna, lipids, proteins, carotenoids, chitin, biochemical composition, waste stabilisation pond, aquacultu• re. The biochemical composition of Daphnia magna Straus, the dominant planktonic crustacean of the waste stabilisation pond of Differdange (Grand-Duchy of Luxembourg), was quantitatively determined from October 1993 to July 1994. Over the sampling period, the average composition (mean ± S.D.) was 271 ± 64 mg proteins.g-1 dry weight (DW), 100 ± 28 mg lipids.g-1 (DW), 96 ± 58 fig carotenoids.g-1 (DW), 49 ± 14 mg chitin.g"1 (DW) and 125 ± 78 mg ash.g"1 (DW). The seasonal variations of the bio• chemical composition were related to several ecological variables (water temperature, dissolved oxygen concentration, pH, wa• ter transparency, chlorophyll a concentration and D. magna biomass). The chitin content was positively correlated to the water temperature as a result of the strong influence of this later variable on the moulting rate of the daphnids and, subsequently, on the chitin synthesis by these organisms. The carotenoid content was positively correlated to the water transparency as a result of their photoprotective role in daphnids. The fluctuations of the lipid, protein and ash levels in D. magna depended to the food availa• bility. Despite a seasonal variation in the biochemical composition, D. magna appeared to have adequate lipid and protein levels to be used in aquaculture. Its carotenoid content is similar to fish meals used to color salmonid flesh and these organisms could be used for this purpose. The prospect of using D. magna for chitin extraction is worth considering with respect to its significant chitin content, especially if highly valuable applications are aimed. Analyse de la variation saisonnière de la composition biochimique de Daphnia magna Straus (Crustacea : Branchiopoda : Anomopoda) vivant dans un étang de lagunage aéré Mots-clés : Daphnia magna, lipides, protéines, caroténoïdes, chitine, composition biochimique, étang de lagunage, aquaculture. La composition biochimique de Daphnia magna Straus, le crustacé planctonique dominant dans l'étang de lagunage de Differ• dange (Grand-Duché de Luxembourg), a été déterminée quantitativement d'octobre 1993 à juillet 1994. Pendant cette période, la composition moyenne a été caractérisée par des teneurs s'élevant à (moyenne ± écart-type) 271 ± 64 mg protéines.g"1 masse sèche (MS), 100 ± 28 mg lipids.g"1 (MS), 96 ± 58 ug caroténoïdes.g"1 (MS), 49 ± 14 mg chitine.g"1 (MS) and 125 ± 78 mg cendres.g- 1 (MS). Les variations saisonnières de la composition chimique des daphnies ont été mises en relation avec plusieurs variables écologiques (température de l'eau, concentration en oxygène dissous, pH, transparence de l'eau, concentration en chlorophylle a et biomasse de D. magna). La teneur en chitine s'est avérée positivement corrélée à la température de l'eau en raison de l'influence marquée de celle-ci sur le rythme de mue des daphnies et, par conséquent, sur la synthèse de chitine chez ces organismes. Le contenu en caroténoïdes s'est révélé positivement corrélé à la transparence de l'eau en raison du rôle de photoprotection de ces pigments. Les fluctuations des teneurs en lipides, protéines et cendres chez D. magna dépendent de la disponibilité en nourritu• re dans le milieu. Malgré les variations saisonnières de sa composition biochimique, D. magna s'avère avoir une teneur en lipides et en protéines adéquate pour son utilisation en aquaculture. Sa teneur en caroténoïdes est similaire à celle des régimes utilisés pour colorer la chair des salmonidés. Les daphnies peuvent donc être utilisées à cet effet. La perspective d'utilisation de D. ma• gna comme source commerciale de chitine est particulièrement prometteuse compte tenu de sa teneur en chitine, en particulier si on envisage des applications à haute valeur ajoutée. 1. Université de Liège, Institut de Zoologie, Laboratoire d'Ecologie Animale et d'Ecotoxicologie, quai Van Beneden, 22, B-4020 Liège, Belgique. 2. Université de Liège, Institut de Chimie, Groupe de Recherche "Chitin-Chitosan", B6, Sart-Tilman, B-4000 Liège, Belgique. 3. Université de Liège, Institut de Botanique, Laboratoire d'Algologie, de Mycologie et de Systématique Expérimentale, B22, Sart Tilman, B- 4000 Liège, Belgique. Article available at http://www.limnology-journal.org or http://dx.doi.org/10.1051/limn/1999031 224 H.-M. CAUCHIE, M.-F. JASPAR-VERSALI, L. HOFFMANN, J.P. THOME (2) 1. Introduction 2.2. Biochemical analysis of Daphnia magna From July 1993 to June 1994, zooplankton samples Due to its ability to survive in highly eutrophic were collected on fourteen occasions. Crustaceans we• conditions, Daphnia magna Straus is commonly found re concentrated by pumping and filtering large vo• in waste stabilisation ponds (Dinges 1973, Dor et al. lumes of water over a Nylon net (mesh size =100 um). 1987, Hathaway & Stefan 1995). In such ponds, this At the laboratory, 2 mm long D. magna specimens we• planktonic species is thought to improve the biological re selected, killed with 4 % formaldehyde and imme• quality of the effluent by extensive grazing on suspen• diately rinsed during 10 minutes in distilled water. In ded bacteria, algae and detritus. The standing crops of order to prevent the alteration of the carotenoids, the Daphnia magna are often considerable, notably becau• daphnids were freeze-dried in the dark. The samples se of the absence of predators, and its annual produc• were kept at 4°C until biochemical analysis. tion reach from 100 to 500 g dry weight.nr3. year1 in For protein and chitin analyses, the freeze-dried ma• some facilities (Sevrin-Reyssac 1992, Cauchie et al. terial was first decalcifided by means of HC10.5 N du• 1995). At present, these crustaceans are mainly used, ring 4 hours. Proteins were then extracted in the resi• alive or preserved, as food for fish in aquaculture (Ed• due using a NaOH 0.5 N solution at 100°C for 6 hours wards & Pullin 1990, Sevrin-Reyssac 1992). and were assayed by the Lowry method (Lowry et al. In 1988, Daphnia magna was introduced in the was• 1951) as modified by Schacterle & Pollack (1973). te stabilisation pond of Differdange (Grand-Duchy of The amount of protein removed by the HC1 treatment Luxembourg) in order to enhance the wastewater treat• was estimated spectrophotometrically in HC1 extracts ment. Since 1992, large amounts of daphnids have by the method described by Scopes (1974). Total pro• been regularly harvested in spring and in summer to be tein content was estimated by adding HC1-soluble pro• sold as aquarium fish food. The aim of this study was tein estimates to NaOH-soluble protein concentra• tions. Chitin was enzymatically determined according to determine the seasonal variation of the daphnid bio• to Jeuniaux (1963, 1965) in the insoluble residue re• chemical composition in order to evaluate the pros• maining after the successive HC1 and NaOH treat• pect of using them for commercial applications such ments. The residue was incubated in a solution of puri• as fish meal preparation or chitin extraction. Chitin, fied chitinase (Sigma 6137; 1 mg.mr1 distilled water) poly-P-l,4-D-glucosamine, and its deacetylated deri• at 37°C during 8 hours. The supernatant was then in• vative, chitosan, have recently found numerous appli• cubated in a solution of N-acetylglucosaminidase cations in various fields with a great added value, in• (lobster serum diluted ten times) during 4 hours at cluding pharmaceutical and biomedical sectors, cos• 37°C. The N-acetylglucosamine monomers liberated metics and wastewater treatment (Muzzarelli 1996). by the enzymatic hydrolysis of chitin were measured The basic biochemical composition of Daphnia ma• by the colorimetric method of Reissig et al. (1955). gna (proteins, lipids, chitin, ash and carotenoids) was Total lipid extraction was performed on freeze-dried investigated from October 1993 to July 1994. The sea• daphnids using a chloroform : methanol (2 :1) mix sonal variation of the composition was analysed in re• (Bligh & Dyer 1959). The chloroform layer was sepa• lation to physico-chemical variables in a redundancy rated and dried at 50°C under dry nitrogen flow. Lipids analysis. were assayed colorimetrically in the residues using the sulfophospho-vanilline method (Barnes & Blackstock 2. Material and methods 1973). 2.1. Site description The total carotenoid content of the daphnids was de• termined following a method similar to that of Herring The studied waste stabilisation pond is located near (1968a). The pigments were extracted in 90 % ethanol the town of Differdange in the Grand-Duchy of during 24 hours at 20°C in the dark. Absorption scans Luxembourg (49°32'N - 5°55'E). It is a roughly rectan• of the supernatant were performed between 350 and 2 gular pond (59,000 m , 2.3 m mean depth) equipped 700 nm with a precision of 1 nm using a Beckman DU with two surface aerators (35 kW capacity each) col• 650 spectrophotometer. The total carotenoid content lecting the waste waters of the town (15,000 inhabi• was calculated using the average OD of the superna• tant-equivalents) after primary treatment. In terms of tant between 450 and 480 nm assuming an extinction biomass, the zooplankton community is dominated by coefficient of 2,500 as an approximate value for caro• crustaceans, namely the anomopod Daphnia magna tenoids (Zagalsky et al. 1967). The, wavelengths cor• (Cauchie et al. 1995). responding to maximal absorbance were identify in or- (3) BIOCHEMICAL COMPOSITION OF DAPHNIA MAGNA 225 der to determine, if possible, the nature of the carote- From October to March, the chitin level of noids on the basis of their specific absorbance profiles D.
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