Aquacult Int (2007) 15:287-298 DOI 10.1007/s 10499-007-9080-7

ORIGINAI. l'API-.Il

Analysis of the off-flavor risk in carp production in ponds in Dombes and Forez (France)

D. Vallod • J. P. Cravedi • A. Hillenweck • J. Robin

Received: 8 August 2005/Accepted: 21 November 2006/Published online: 3 April 2007 © Springer Science+Business Media B.V. 2007

Abstract Off-flavors represent one of the most economically significant problems encountered in continental aquaculture. The appearance of a repulsive odor, or taste, in fish may cause a majorreduction in the consumption of the products. Carp farmers and processors are aware of this problem and wish to keep it under control. A first cross-disciplinary study was carried out in France in 2002-2003, following three approaches: (1) estimation of water quality and composition of the phytoplankton community in carp ponds, (2) sensory analysis of carp, (3) quantification of odoriferous volatile compounds in carp. The results show that strongly off-flavor carp were found in ponds colonized by cyanobacteria, mainly Anabaena spp. in summer. The quantification of odorous compounds was not effective in the fish samples, but sensory evaluation confirmed the existence of off-flavors in the carp tested. The most commonly used descriptors associated with geosmin, referred to a waste water, earthy/musty odor and taste, were those identified by the panel of testers. Such results led us towards a new strategy of carp fishing, stocking and processing during the summer.

Keywords Common carp • Cyanobacteria • Geosmin • Off-flavor • Sensory evaluation • Water quality

Introduction

Off-flavors represent one of the prevailing problems encountered in continental aquacul­ture from an economic viewpoint. The appearance of a repulsive odor or taste in fish may cause a major reduction in consumption or may make the products unacceptable for fish

D. Vallod (E l ) • J. Robin Department of Agro Systems, Environment, Productions, ISARA-Lyon, A G R A P O L E , 23, rue Jean Baldassini, F-69364 L Y O N Cedex 07, France e-mail: vallod@isara.fr

J. P. Cravedi • A. Hillenweck INRA, U M R 1089Xenobiotiques, 180 Chemin de Tournefeuille, BP 3, 31931 Toulouse Cedex, France

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processors and consumers. As a result, high losses in producers' earnings are common. If no product quality management is developed in the near future, economic repercussions may become serious.

Off-flavors in fish were studied in the U S A catfish industry (Van der Ploeg et al. 1992; Zimba and Grimm 2003) but also in tilapia (Yamprayoon and Noomhorm 2000), salmon (Farmer et al. 1995) and trout (Robertson and Lawton 2003). In most cases, the term off-flavor is linked to an earthy/musty odor and taste, due to high concentrations of geosmin or 2-methyl iso-borneol (MIB) in fish meat (Tucker 2000; Schrader and Rimando 2003). The presence of these two compounds in water and fish is attributed to cyano-bacteria and actinomycetes, which develop during the summer and the beginning of autumn. Oscillatoria chalybea (Van der Ploeg et al. 1995; Schrader et al. 1998; Tucker 2000) and Anabaena spp. (Lovell et al. 1986; Matsumoto and Tsuchiya 1988; Rosen et al. 1992; Van der Ploeg et al. 1992) are frequently at the origin of off-flavors in catfish.

Common carp are one of the main aquatic species bred in the world. Their total annual production reached 3.2 million tonnes in 2002 (Food and Agriculture Organization, F A O , 2002). In Europe there is a traditional consumption in the winter, mainly at Christmas time. However, the market is not well developed, and the economic value is kept at a relatively low level, mainly because of the negative image of the carp, a big fish with a muddy odor/ taste, numerous intermuscular bones and a low filleting yield (Vallod 1995; Sehgal and Sehgal 2002). In order for this situation to be improved, processing methods were developed and French fishpond farmers decided to change consumers' habits by also selling carp during the summer. It required the development of new harvesting practices, and made it necessary to solve taste problems commonly encountered at this season. To do so, a cross-disciplinary study was carried out in 2002-2003 in Dombcs and Forcz ponds to obtain some first data on off-flavors in carp. The aims of the study were (1) to examine the sensory characteristics of carp produced in ponds where the risk of algae blooms is high in summer; (2) to link off-flavors with the presence of cyanobacteria known for their capacity to produce odorous compounds, and (3) to identify by chemical analysis an odorous compound in off-flavor carp meat and correlate its presence with the results of sensory evaluations.

Materials and methods

Ponds

Four ponds (Table 1) were investigated. Their selection was based on the trophic level, the quantity of organic matter and phosphorus in sediment and the presence of cyanobacteria blooms in summer. Three ponds (CI , C2 and C3) were potentially presenting a risk of off-flavor conditions, while pond C4 was apparently free of this problem.

Table 1 Main characteristics of the four ponds studied

Characteristics CI C2 C3 C4

Water surface (ha) 14 22 5 7

Stocking density (kg ha" 1) 52 61 69 43

Production (kg ha" 1) 360 540 550 230

Supplementary feeding No Yes Yes No

Aeration No Yes Yes No

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Sampling

Water quality was analyzed every second week from June 2002 to November 2002, between 10 a.m. and 12 p.m. The whole water layer was collected with a vertical plastic water bottle (1 m), in four stations, and then it was mixed in one composite sample for each date.

The sediment was sampled in June and October with an Ekman Bottom Grab Sampler. The final sample was composed of samples from three areas of each pond.

The phytoplankton was sampled by filtration of 2501 of water with a net of 10 mri mesh. The sample was concentrated in a 125 ml bottle and preserved with Lugol's solution.

The fish were sampled each month with specific fishing nets. Each time, eight carp were slaughtered and then filleted. The fillets of each fish were skinned and frozen separately at —20°C. One fillet was used for the chemical analysis of odorous volatile compounds, the other one for sensory evaluation.

Physicochemical and biological analysis

The following water parameters were measured:

- in situ: T°C and dissolved oxygen ( W T W multi 197i), - in the laboratory: ammonium ion (NF EN ISO 11732), total nitrogen (NF EN 25663),

total phosphorus (NF EN ISO 11885) and total suspended solids (NF EN 872).

Total nitrogen (NF X31-111), assimilable phosphorus (Joret-Hebert' method) and organic matter (NF X31-109) were measured after the sediment had been reduced on a 2 mm mesh and dried at 40°C.

The density and the composition of the algal community were estimated by cell count under an inverted microscope.

The phytoplankton biomass was estimated by the calculation of chlorophyll-a concentrations (Parsons and Strickland 1963).

Sensory evaluation

The sensory panel consisted of 12 trained persons (eight women, four men, all aged between 30 years and 65 years). This group attended six training sessions to define relevant terms for carp flavor characterization. The sensory evaluation was performed using a descriptive profile test (norm NF V 09-015). The samples were evaluated by odor and taste with the following descriptors: chlorophyll-grassy, chemical (phenols), sour, woody, waste water, earthy/musty, sweet and acid. The level of intensity of off-flavor odor and taste was also graded.

Sensory analyses were conducted on portions of minced fish (20 g). The samples were thawed at 4°C, aluminum wrapped and then cooked in a convection oven for 8 min. They were classified for each descriptor using a 7-point hedonic scale (0 = on-flavor; 3 = slight off-flavor; 7 = very strong off-flavor).

Twenty carp of 800 g were batched for 4 days in 500 1 water tanks with 35 u.g 1~' of geosmin at 20°C so that we could have a comparison with a reference sample, artificially tainted with geosmin. After this uptake phase, they were placed in clear running water to depurate. Two carp were sampled every other day, slaughtered, then filleted. The fillets were frozen until sensorial analyses were conducted.

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Chemical analysis of volatile compounds

The quantification of volatile compounds in fish meat concerned geosmin and M I B and was performed by gas chromatography coupled to mass spectrometry (GC/MS) , using iso-bornyl acetate as an internal standard.

Analyses were carried out on the dorsal anterior muscle (10 g). Each sample was thawed at room temperature, spiked with iso-bornyl acetate (1 u,g kg" 1 ) and homogenized in 30 ml pentane using a Polytron homogenizer. Two successive extractions were carried out, and the solvent was filtered into a microfiber glass filter. The filtrate was concentrated to 1 ml under a slight stream of nitrogen before G C / M S analysis.

The samples were analyzed using a Trace GC system coupled to a Polaris Q mass spectrometer. The system was operated in electron impact mode. The separation was achieved with a 25 m x 0.22 mm i.d. B P X 5 column coated with 5% phenyl-methyl-polysiloxane (film thickness 0.25 u,m). Oven temperature was programmed from 50°C (holding time 3 min) to 250°C (holding time 5 min) with an 18°C m i n " 1 gradient. Injector temperature was set at 250°C, and flow rate was maintained at 1 ml m i n " 1 using helium as a carrier gas. In these conditions, the retention time of M I B and geosmin was 8.45 min and 10.90 min, respectively.

The single-ion monitoring technique (based on ions mlz = 95 and mlz = 112 for M I B and geosmin, respectively) was used for quantification.

Calibration curves were determined for geosmin and M I B at concentrations ranging from 3 ng m l " ' to 100 ng ml"" 1 . A linear correlation was observed between the area on the profile and the concentration of geosmin ( j = 208.67A- - 3485.5, R2 = 0.9978) and M I B (y = 288.28A- - 263.62, R2 = 0.9997). In these analytical conditions, the limit of quantification was found to be 1 ug kg""1 for each compound.

Results

Sediment and water quality

The sediment of ponds C I , C2, C3 contained larger quantities of phosphorus and organic matter (Fig. 1) than C4 (Kruskal-Wallis and Tukey test, P < 0.05).

There were very few differences in water temperature in each pond over the time period (Fig. 2). However, C I had lower water temperature in August, probably due to the shade from the surrounding trees. Total nitrogen values in the water (Fig. 3) were acceptable (N < 2 mg l " 1 ) during all the survey, even if pond C3 was found to have the lowest value of total N. Total phosphorus values were regularly lower than 0.2 mg l " " 1 in P, but CI and C2 had the highest values in July (Fig. 4).

Biological analyses

From July 2002 to the end of October 2002, algal biomass and the composition of the algal community evolved differently in the four ponds (Fig. 5). Pond C I was found to have an abundant algal biomass (> 100 j.ig l - 1 ) from mid-July to mid-October, with a maximum concentration in the first days of August (253 ug l " 1 ) . At the same time, cyanobacteria were dominant in the algal community, represented mainly by two species: Anabaena spiroides until the end of August, then Microcystis aeruginosa from the end of August to

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Nitrogen (mg/g) Phosphorus (mg/g) Organic Matter (%}

Fig. 1 Results of sediment analysis (mean value of the spring and autumn analyses)

Fig . 2 Seasonal evolution of Water water temperature in the ponds Temperature (°C)

the end of October. In pond C2, cyanobacteria were also the dominant algal group, with two species: Anabaena spiroides and Anabaena flos-aquae, during July (maximum 398 ug 1 '). At the end of August, the algal community was more diversified, as cyano­bacteria were disappearing progressively. Three cyanobacteria (Microcystis aeruginosa,

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Anabaena spiroides and A. flos-aquae) were die main species observed in the algal community in pond C3, with a predominance of Microcystis. The algal biomass fluctuated between 100 ug 1 _ 1 and 250 ug In pond C4, Chlorophyta and Diatoma were the main algae present in the algal community all summer long. The algal biomass was lower than in the three other ponds (25-110 ug

Sensory analysis

Six samples were undoubtedly characterized as off-flavor by the panel of testers: three samples from pond C I (7/18, 8/8 and 11/11), two from pond C2 (7/18 and 11/11) and one from pond C3 (10/30). Off-flavor carp were never found in pond C4. Significant differ­ences (Kruskal-Wallis, P < 0.05) were found for odor and taste between the different samples of ponds C I , C2 and C3 (Table 2). The following descriptors contributed to the differences in the off-flavor odor and taste: waste water, earthy/musty and overall intensity of off-flavors. Other descriptors were marginal. A statistical analysis with Fizz software, using a hierarchical ascending classification and a principal components analysis (Fig. 6), showed that the descriptors "earthy/musty" and "waste water" were correlated with the overall intensity of off-flavor odor and taste. During all the summer, carp from pond C I were off-flavor, especially in July and August. The intensity of the off-flavor was lower at the end of the season. Ponds C2 and C3 seemed to be characterized by occasional off-flavor problems. The sensorial evaluation of artificial geosmin-batched carp confirmed that "waste water" and "earthy/musty" were the significant descriptors for odor and taste. It also showed (Fig. 7) that a 10-day depuration phase is required in the experimental conditions to decrease the sensory evaluation below the sensory threshold (value 3).

Volatile compounds

Neither geosmin nor 2-methyl iso-borneol was measurable in the carp muscles. However, unquantifiable traces of geosmin were detected in some samples (Fig. 8). In addition, a contaminant having a retention time close to that of geosmin (11.04 min) and giving a signal at the same characteristic ions as for geosmin (m/z = 97, 112, 126 and 182) was observed on the chromatogram. These results suggest that the detected contaminant could be related to geosmin, but further analyses are needed to identify this unknown compound.

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relative abundance C1 biomass (ug L"1) relative abundance C2 biomass (ug L"1)

Table 2 Average notes (on the 7-point scale) on intensity for the carp samples tested. Samples with a value >3.5 are considered as off-flavor (underlined)

Descriptors CI C2 C3 C4

7/18 8/8 9/23 11/11 7/18 8/8 9/23 11/11 7/15 8/9 9/24 10/30 7/15 8/9 9/24 10/30

Significant descriptors

Significance level a < 0.01%

Waste water odor 3.75 3.50 2.25 2.92 3.83 1.83 2.42 3.08 2.00 2.00 1.83 3.67 1.75 2.50 2.17 1.42 Earthy/musty odor 3.42 4.00 2.08 2.17 2.83 2.50 2.25 3.42 2.33 2.25 1.92 3.50 2.00 2.00 2.08 1.75 Overall intensity

of bad odor 4.17 4.75 2.58 2.67 3.83 2.25 2.33 3.83 2.83 2.42 2.33 3.92 1.92 2.67 2.17 1.75

Waste water taste 4.25 4.25 2.83 3.67 3.42 2.17 2.42 3.33 2.42 2.75 1.92 4.58 1.58 2.75 2.00 1.00 Chemical taste 2.33 3.25 1.92 1.92 2.08 1.50 1.25 2.08 2.25 1.75 1.92 2.50 1.33 1.75 1.75 1.25 Earthy/musty taste 3.58 4.42 2.75 3.42 3.75 2.33 2.92 3.58 2.75 2.83 2.33 3.25 1.92 2.00 1.58 1.50 Sour taste 2.92 3.58 2.17 3.00 2.42 2.33 2.17 2.67 2.42 1.83 2.25 2.67 2.17 2.58 1.42 1.25 Overall intensity

of bad taste 4.25 5.25 3.42 3.58 4.42 2.42 3.00 4.58 2.25 2.75 2.75 5.33 3.25 2.75 2.17 1.67

Non-significant descriptors

Significance level a > 0.05%

Chemical odor (a = 0.60%) 2.17 2.42 1.17 1.33 2.17 1.50 1.25 1.67 2.08 1.50 1.75 1.83 1.42 1.75 1.83 1.92 Acid taste (a = 2.10%) 2.17 2.67 2.00 1.92 2.92 1.83 2.33 2.33 1.75 1.83 2.50 3.00 2.17 2.33 1.67 1.17 Sweety taste (a = 6.10%) 1.58 1.67 2.08 2.42 1.83 1.75 2.50 1.83 1.50 1.92 1.83 2.25 1.58 1.50 2.50 1.67 Sour odor (a = 7.70%) 2.33 2.33 1.58 1.83 2.42 1.25 1.75 2.25 1.75 2.00 1.33 2.17 1.58 1.92 1.67 1.50 Woody taste (a = 14.10%) 2.83 2.58 2.08 2.7 2.17 1.92 2.33 2.50 1.92 1.83 2.25 2.58 2.33 2.08 1.67 1.92 Woody odor (a = 35.50%) 2.50 2.67 2.00 1.92 1.67 1.83 1.92 2.58 2.17 1.83 1.92 2.25 2.08 2.17 2.17 1.92 Grassy odor (a = 52.10%) 2.08 1.42 1.92 1.58 1.92 1.92 2.17 1.58 1.42 2.17 2.08 1.58 1.75 2.08 1.92 1.83 Grassy taste (a = 56.20%) 2.08 1.83 2.08 1.83 2.33 2.00 2.17 1.67 1.58 2.00 2.25 2.08 2.00 2.42 2.42 2.50

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Axis 2

Discussion

The present study provides the first multi-criteria comparative evaluation of off-flavor in carp from French ponds. Physicochemical and biological surveys confirm that ponds C I , C2 and C3 are potential sites for off-flavor conditions and confirm that pond C4 is a relevant control pond. These results are validated by the sensory analysis. Off-flavor carp are found in ponds when cyanobacteria are in abundance. This is confirmed by the com­parison between the three ponds C I , C2 and C3 with control pond (C4). In this pond, cyanobacteria were never observed, and no off-flavor fish was detected.

Values of total N in water of the four ponds were low compared to those observed in catfish ponds, from 4.3 mg l - 1 to 10.6 mg 1 _ 1 (Zimba et al, 2003). Large amounts of phosphorus were observed to be trapped in the sediment of C I , C2 and C3 and frequently

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Fig . 7 Kinetics of depuration of 35 ug l " 1 geosniin-batched carp. Values of the 7-points hedonic scale are on the K-axis. The value 3 represents a subtle perception of off-flavor

Fig . 6 Plan 1-2 from the principal components analysis (BiPlot constant: 9.58811) for the odor and taste descriptors for all the samples of carp tested. Axis 1 = 60.5% — Axis 2 = 11.3%. Dotted line groups on-flavor carp, black line groups off-flavor carp

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Fig. 8 Chromatogram showing the presence of a contaminant in the analyses of geosmin in carp

released into the water during the summer, when temperature exceeded 20°C and O2 presented stratification in the water column. This release of phosphorus from sediment induced important an phosphorus content in the water (0.4 mg l - 1 ) , similar to those measured by Zimba et al. (2003) in catfish ponds with off-flavor problems (0.33-0.71 mg l™1). In pond C3, aeration had an influence on the release of phosphorus by modifying the stratification conditions of 0 2 . In those cases, the quantities of phosphorus were lower than in C I and C2 and steadily present during all the season. Associated with the ability of some cyanobacteria to fix atmospheric nitrogen, the high concentrations of phosphorus allowed the development of an important biomass of cyanobacteria (>100 pig l - 1 ) . Anabaena spiroides and A. flos-aquae became rapidly dominant in the algal community. Pond C4 presented a more regular evolution of chemical and biological characteristics, with low concentrations of phosphorus in sediment and water on the one hand and dominance of Chlorophyta on the other. In ponds C2 and C3, Anabaena sp., and particularly Anabaena spiroides, could be responsible for the appearance of off-flavors in fish by the secretion of geosmin in water (Matsumoto and Tsuchiya 1988; Wu et al. 1991; Van der Ploeg et al. 1992; Peterson et al. 1995). In pond C3, the cyanobacteria community was dominated by Microcystis aeruginosa. This species is not known as a geosmin producer, and pond C3, except at the end of October, did not present off-flavor carp.

The results of sensorial analysis showed that strongly off-flavor carp were found in ponds colonized by cyanobacteria during the summer. The most significant descriptors were always correlated (except in one case) with the observation of off-flavor in carp and were the same for odor and taste—waste water and earthy/musty. This relationship could be explained by the sensory panel's similarity in their appreciation by nose and mouth and by the subtlety of their differentiation. It confirms the advantage of working with a trained

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panel, even if, in this case, the threshold for odorous compounds can be lower than the quantification threshold by G C / M S . Human geosmin threshold is 1.5 p.p.b. in trout (Robertson and Lawton 2003) and catfish (Grimm and Zimba 2003) and 1.5-2.6 p.p.b. in tilapia (Yamprayoon and Noomhorm 2000), but no indication exists for carp. The trained testers may have a higher sensitivity, as, for M I B in catfish, 0.1 p.p.b. and 0.7 p.p.b., respectively,were soon noticed (Zimba and Grimm 2003). The artificial geosmin-batched carp and off-flavor carp from studied ponds had a very similar evaluation of off-flavor. This suggested that geosmin could be responsible for the off-flavor situations. Moreover, an analysis of geosmin in water of pond C2 in November showed a value of 2.5 ng

The negative quantification of geosmin in carp meat could be linked to the presence of a contaminant. This compound may be a biodégradation or a biotransformation product from geosmin, but neither its chemical structure nor its capacity to be odorous or not is known. Degradation of geosmin and M I B in water is a slow process, and bacteria are the main agents responsible (Lawton et al. 2003). Some authors assert that geosmin is a more stable compound than M I B (Saito et al. 1999), while others (Lawton et al. 2003) claim geosmin is biodegraded within 3 days and M I B in 5-14 days. Are trained testers less sensitive to these biological degradation processes than chemical analyses are?

The results of this study suggest a relationship between the presence of Anabaena spp. and the presence of geosmin in the off-flavor carp that were tested. They confirm that off-flavor conditions may last over long periods (ex Pond CI ) , so that pond fish farmers must develop new strategies for fishing and stocking carp during the summer before processing and selling the fish.

Acknowledgement This study was carried out with funding from the French Ministry of Agriculture, OFIMER and Rhône-Alpes Region. The authors would like to thank the fish farmers for their collaboration, Laurent Debrauwer (INRA, Toulouse) for his help in mass spectrometry analyses and Jean-Christophe Perrin (Les Maisons du Goût, Bourg en Bresse) for his help in sensorial analyses.

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