A preliminary evaluation of physiological filtration variables for Crassostrea cortesiensis and Anadara tuberculosa farmed in shrimp aquaculture effluents.

Emilio Pea-Messina1, Luis Rafael Martnez-Crdova2; Luis Fernando BckleRamrez3, Manuel Alberto Segovia-Quintero3, Jos Antonio ZertucheGonzlez4. 1. Direccin de Fortalecimiento de la Investigacin Cientfica, Universidad Autnoma de Nayarit. pemess1@hotmail.com. 2. Departamento de Investigacines Cientficas y Tecnolgicas de la Universidad de Sonora. 3. Centro de Investigacin Cientfica y Educacin Superior de Ensenada, B.C. 4. Centro de Investigaciones Oceanogrficas. Universidad Autnoma de Baja California. Correspondence: Dr. Luis Rafael Martnez-Cordova. Departamento de Investigacines Cientficas y Tecnolgicas de la Universidad de Sonora. P.O. Box, 1819, Hermosillo, Sonora, 83000 Mexico. lmtz@guaymas.uson.mx

Abstract An experimental study was done in the facilities of the Universidad de Nayarit, Mexico to evaluate the main filtration variables (filtration rate, clearance rate and assimilation efficiency) of Crassostrea cortesiensis and Anadara tuberculosa farmed in shrimp aquaculture effluents at three different flux velocities (1.5, 3.0 and 4.5 L h-1). The filtration rates for both species (0.124, 0.328 and 0.402 L h-1 for C. cortesiensis; 0.093, 0.189 and 0.345 for A. tuberculosa), were relatively low as compared to the reported for similar or related species. The clearance rates were higher for C. cortesiensis ( 20.04, 52.92 and 64.70 L h-1) as compared to A. tuberculosa (10.96, 22.95, and 42.12 L h-1); in both cases values were in the range reported previously for the last species. The assimilation efficiency for both mollusks (over 92 % for C. cortesiensis and over 95% for A. tuberculosa), was very high and greater than the found by other authors for the same or related species. The three filtration variables (filtration rate, clearance rate and assimilation efficiency) were better at the highest effluent flux velocities. These preliminary results strongly suggest that both species are good candidates to be considered for use as biofilters in aquaculture bioremediation.

Introduction One of the major challenges of world aquaculture is to produce more in a lower space without an important and/or progressive impact to the environment, which may affect the quality and diversity of natural ecosystems in one or more of their biotic and/or abiotic components (Phillips, 1998). The main impacts in aquaculture are related to the uncontrolled discharge of organic and inorganic wastes coming from unconsumed supplemental feeds, fertilizers and additives used to increase the natural productivity of the system (Tacon, 2002). Bivalve mollusks are mostly filterfeeders; this means they have the capacity to remove suspended organic and inorganic solids from water column and reject them as pseudo feces after obtain the nutrients. This capacity makes the bivalve mollusk potentially useful as biological filters in the bioremediation of effluents, which represents one of the most important alternatives to minimize the aquaculture impacts (Miranda-Baeza & Ramos Brito, 2009). Mollusks are preferred for bioremediation over other organism because of their adaptability and resistance to management, inclusively in poor quality waters (MartinezCordova & Martinez-Porchas, 2006). There are many studies about the successfully grow out of mollusks in aquaculture effluents of fishes (Shpigel & Blaylock, 1991; Jones & Iwama, 1991; Stirling & Okimus, 1995; Shpigel, Gasith, & Kimmel, 1997; Lefebvre, Barrill & Clerck, 2000), and shrimp (Jakob, Pruder, & Jaw-Kai, 1993; Jones & Preston, 1999, Miranda, 2005; Martnez-Crdova & Martnez Porchas, 2006). A study conducted by Figueras (1989) estimated that a suspended culture of about 800,000 mussels can filtrate 70x 106 liters of water per day, producing up to 180 tons of biodeposits during the farming cycle.

In Japan, it was estimated that in a period of 9 months 420,000 oysters produced 16 tons of biodeposits (Pillay, 1992). To assess the effectiveness of a particular mollusk as biofilter, it is necessary to investigate its performance under conditions simulating water effluents, evaluating those variables considered the most important as indicators of that effectiveness. Evaluation of filtration rate (FR) in bivalve mollusks is fundamental in studies about energy budget and growth estimations. This indicator has been motive of some controversy due to the great variety of methods used for its determination, which can be influenced by flux velocity through containers with one or more experimental individuals (Hildreth & Crisp, 1976) or by size and shape of mollusks (Riisgrd, 2001). Clearance rate (CR) (Winter, 1978) is an indicator derived from filtration rate, but considering the particles concentration at the efflux of control, excluding the sedimented matter by the effect of empty valves. The Assimilation efficiency (AE%) is a useful tool in understanding the nature and quality of suspended solids when used as food for filtering organisms. The method proposed by Conover (1966) assumes that the inorganic compounds ingested by filterfeeders are not assimilated in the digestive tract, which means it can be calculated as a reason of the proportion of both organic and inorganic compounds in the control flux and in fecal matter at the end of the experiment. The present study was focused on evaluating the above mentioned filtration variables for Anadara tuberculosa, and Crassostrea cortesiensis, farmed in laboratory, using effluents from a shrimp farm in Nayarit, Mexico.

Methods and materials

The investigation was conducted in the Aquaculture Laboratory of the National Fisheries Engineering School of the Universidad de Nayarit, Mexico. A system of 36 aquariums with respirometers adapted to each of them, with manual flux regulators, was designed for the experiment. The system was similar to other developed and successfully used in the laboratory of microalgae in the Universidad Autnoma de Sinaloa, Mexico.

Experimental organisms Some individuals of each one of the two species used for the study were collected from its natural habitat. A. tuberculosa was obtained directly from salt marshes in El Pozo and El Rey estuaries during a low tide. C. cortesiensis was obtained by scuba diving in the tidal inlet of estuary San Cristobal, both sites in the coast of Nayarit, Mexico. Organisms were washed and brushed to make them free of epicomensals. After that, they were acclimated to experimental conditions during 15 days (darkness, and temperature between 29 and 30C).

Experimental design A single factor experimental design with 10 replicates was performed to estimate: filtration rate (FR), clearance rate (CR), and assimilation efficiency (AE %) of each one of the mollusks evaluated. Treatments consisted of 3 effluent flux velocities (1.5, 3.0 and 4.5 L-h-1). For each determination 30 organisms of each species was selected, dried and weighed. Ten of them were assigned to each treatment putting one by respirometer. For the control

treatment, one pair of valves of the specific mollusk was put in each respirometer. Filtration rate (FR) defined as the volume of water (containing the suspended particles used by mollusks for feeding) filtered by organisms in a determined time, was estimated for each species and each water flux, from the following equations suggested by Hidreth & Crisp (1976)

(1)

FR1 = (C1 C2) F C1 FR2 = (C1 C2) F C2

(2)

Where F is the flux velocity through the respirometers measured as L h-1, C1 is the particles concentration at the exit of control respirometer (with only valves of the mollusks to evaluate the sedimentation of particles) and C2 is the particles concentration at the exit of the respirometers containing the experimental organisms. Both indicators, FR1 and FR2, are widely used to measure the filtration efficiency in bivalves, although the second appears to be more precise. In FR2, the difference between particles before and after filtration is calculated in relation of the filtered matter along the time and not as a function of the whole matter present in the control flux. In order to have an indicator of the filtration efficiency among species, the filtration rates were normalized with respect to the drained flesh weight of each organism. In this way, the normalized filtration rates NFR1 (from FR1) and NFR2 (from FR2), were obtained.

Samplings to evaluate particle concentration were done 30 minutes after put the mollusks in respirometers when valves opening were confirmed. Samples were taken at 0, 4, 8 and 12 hours. Concentration was measured according to procedure 8006 of Standard Methods (Clesceri; Greenberg; & Eaton, 1999). This spectrophotometric method is sensible to detect concentration of suspended solids between 5 to 750 mg L-1. A calibration curve was made from a saturated solution, doing dilutions up to approximately 1%. Clearance rate (CR) was estimated according to the procedure proposed by Winter (1878), which consist of a multiplication of the filtration rate (FR) by the concentration of particles in the effluent of control. Since CR must be expressed in terms of mgh-1, it was necessary the measurement of organic fraction of suspended solids in the entering of respirometers. Assimilation efficiency (AE%) was evaluated by the method suggested by Conover (1966), by the equation:

(3)

AE % = (F-E) x 100 (1-E) F

Where F is the rate between organisms weight and feed supplied, and E is the rate among organism weight and feces dry weight. Feces were collected by a siphon tube, concentrated in a glass, filtered through GFC WHATMAN filters 47 mm and dried in oven at 60 C during 24 h to know dry weight. After that filters were incinerated in a muffle at 450 C during 12 h to know the organic fraction. The same procedure was used to determinate dry weight and organic fraction of feed supplied the mollusk.

In order to correlate the results of filtration variables to physiological stage of the organisms, condition index (CI) of mollusks was evaluated by the equation of Crosby & Gale (1990) as followed:

(4)

CI = (DW * 1,000)/(TMW - VDW)

Where DW is the dry weight of the organism, TMW is the total moist weight (g) and VDW is the dry weight of valves.

Results Flesh dry weight of experimental organisms in each treatment is shown in Table 1. The wide differences among treatments in the case of C. cortesiensis was mostly due to the diversity of forms of the shells, which make difficult to select organisms of the same weight. Particle concentration of shrimp aquaculture effluents used in the experiments maintained similar levels for the two species and the two fluxes proven as shown in table 2.

Filtration Rates. For C. cortesiensis, no significant differences in the filtration rate FR1 were observed among flux velocities, although a slightly higher value was found at the lowest velocity (1.5 Lh -1). With respect to FR2, an important increment was observed at the two highest flux velocities (3 and 4.5 L h-1) (Table 3 and Figure 1). A similar tendency was found for the normalized filtration rates,

higher values of both NFR1 and NFR2, were found at the higher flux velocities (Table 4 and Figure 2), For A. tubeculosa, significant differences were found on the filtration rate (both FR1 and FR2) among the flux velocities. For both parameters, the filtration rate was increasing as the flux velocity does it (Table 5 and Figure 3). The same tendency was found when data was normalized (Table 6 and Figure 4).

Clearance Rates. For C. cortesiensis clearance rates (CR1 and CR2) were significantly higher at the flux velocities of 3 and 4.5 L h-1 as compared to that of 1.5 L h-1. (Table 7 and Figure 5). The clearance rates for A. tuberculosa (both CR1 and CR2) increased significantly as flux velocity increased. In this case each velocity was significantly different from the two other (Table 8 and Figure 6).

Assimilation Efficiency The assimilation efficiency for C. cortesiensis as well as for A. tuberculosa, recorded values extremely high and no significant differences were found between species neither among effluent flux velocities (Table 9).

Condition Index

The condition index of C. cortesiensis was significantly higher as compared to A. tuberculosa, as shown in Table 10.

Discussion. The values of filtration rate found in the present study for the two species, were relatively low as compared to the reported previously for the same or other bivalve mollusks used as biofilter. Fonseca-Garca, Nieves-Soto, & GonzlezVega (2006), in an experimental study with A. tuberculosa, found filtration rates as high as 0.903 Lh-1 working at effluent flux velocities between 1.8 to 6 Lh-1. This value is twice bigger than the highest found in our study. It is necessary however to point out that the total suspended solids in the effluent used in the present study (estimated in around 300,000 particles/ml), was more than three times the used in the other (79,000 to 82,000 particles/ml). It is also relevant to mention that a great percentage of the TSS in our effluent was organic, which probably influenced the filtration rate because an enough amount of nutrients could be obtained by the mollusks with a low volume of water filtered. This finding is confirmed by clearance rates we found for A. tuberculosa, which were in the range reported by Fonseca et al (op. cit.) for the same species. In both studies the flux velocities were similar. C. cortesiensis had a clarification rate higher than A. tuberculosa, which reveal a greater capacity to remove material from effluents rich in organic matter such as aquaculture effluents. The assimilation efficiency recorded for both species was greater than the reported for other mollusks. Wong et al found an assimilation efficiency from 60 to 75 % for Mitilus edulis and from 40 to 75% for Perna viridis, depending on the density of food. The efficiency at which filterfeeders retain particles from water column, may vary with many factors such as environmental conditions, concentration

and composition of suspended materials and other. The variations are frequently influenced by the size of the particles (Barker-Jrgensen, 1990). As conclusions of the present study, it can be established that in spite of the relatively low filtration rate found for both species, they are capable to remove a great amount of suspended solids from effluents, and assimilate high percentages of the organic fraction of these solids. Both, the clearance rate and the assimilation efficiency were better at the highest flux velocities. These preliminary results clearly suggest that C. cortesiensis and A. tuberculosa are good candidates to be used as biofilter for the bioremediation of aquaculture effluents. More studies including evaluations in shrimp farms are now in progress to complement the information and confirm without doubts this fact.

Acknowledgements Special thanks to the National Fisheries Engineering School of the Universidad Autonoma de Nayarit, Mexico, with a special mention to the Director: M Sci. Geronimo Rodriguez Chavez for the facilities and cooperation to support the experimental assays. Our sincerely recognition to M. Sci. Delia Domnguez Ojeda, Ing. Ral Tapia Varela, M. Sci. Humberto Gonzalez Vega, the students Jess Prez Jimenez and Rosa Elena Silva for their invaluable help to this work.

References

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Table 1. Flesh dry weight (means SD) of mollusks used for the three treatments Flesh dry weight (g) Flux velocity Crassostrea cortesiensis Anadara tuberculosa 1.5 Lh -1 3.404 0.111 1.978 0.046 3 Lh -1 3.612 0.075 1.960 0.098 4.5 Lh -1 4.103 0.094 2.091 0.098

Table 2. Particle concentration (means SD) .in shrimp aquaculture effluents used in experiment for the two species of mollusks. Concentration ( mg L-1) Flux velocity Crassostrea cortesiensis Anadara tuberculosa 1.5 Lh -1 167.99 1.197 176.93 0.774 3 Lh -1 167.11 1.224 176.50 0.720 4.5 Lh -1 167.79 1.495 174.59 0.792

Table 3. Means D.E. of filtration rates (FR1 and FR2). for Crassostrea cortesiensis at three effluent flux velocities. Flux velocity FR1 FR2 1.5 Lh -1 0.1196 0.063 a 0.1243 0.076 b 3 Lh -1 0.08243 0.043 a 0.32832 0.167 a 4.5 Lh -1 0.09107 0.059 a 0.40192 0.250 a

Table 4. Means Standard deviation of normalized filtration rates (TFN1 and (TFN2). for Crassostrea cortesiensis at three effluent flux velocities.

Flux velocity NFR1 NFR2

1.5 Lh -1 0.03444 0.021 b 0.03822 0.024 b

3 Lh -1 0.08243 0.043 a 0.0937 0.052 a

4.5 Lh -1 0.09107 0.059 a 0.1022 0.071 a

Table 5. Means standard deviation of filtration rate (FR1 and FR2). for Anadara tuberculosa at three effluent flux velocities. Flux velocity 1.5 Lh -1 3 Lh -1 4.5 Lh -1

FR1 FR2

0.098 0.053 b 0.093 0.050 c

0.097 0.050 b 0.189 0.089 b

0.316 0.146 a 0.345 0.172 a

Table 6. Means Standard deviation of normalizad filtration rate (TFN1 and TFN2). for Anadara tuberculosa at three effluent flux velocities. Flux velocity TFN1 TFN2 1.5 Lh -1 0.052 0.032 c 0.049 0.031 c 3 Lh -1 0.097 0.050 b 0.105 0.056 b 4.5 Lh -1 0.168 0.096 a 0.184 0.110 a

Table 7. Means standard deviation of the clearance rates (CR1 and CR2) for Crassostrea cortesiensis at three effluent flux velocity. Flux velocity CR1 CR2 1.5 Lh -1 18.00 10.62 b 20.04 12.74 b 3 Lh -1 46.57 23.08 a 52.92 28.21 a 4.5 Lh -1 57.68 34.66 a 64.70 41.73 a

Table 8. Means Standard deviation of clearance rates (CR1 and CR2) for Anadara tuberculosa at three effluent flux velocities. Flux velocity CR1 CR2 1.5 Lh -1 11.62 5.631 c 10.96 5.252 c 3 Lh -1 21.39 9.373 b 22.95 10.674 b 4.5 Lh -1 38.54 17.987 a 42.12 20.895 a

Table 9. Means standard deviations of assimilation efficiency of Crassotrea cortesiensis and Anadara tuberculosa at three effluent flux velocities. Assimilation efficiency % Species Crassostrea cortesiensis Anadara tuberculosa 1.5 Lh -1 92.65 1.67 95.25 0.69 3 Lh-1 94.20 2.03 96.71 0.27 4.5 Lh-1 92.04 3.63 96.80 0.54

Table 10. Means standard deviation of the condition index of C. cortesiensis and A. tuberculosa.

Crassostrea cortesiensis Anadara tuberculosa

289.21 22.34 106.79 28.22

Crassostrea cortesiensis

0.5

0.4

-1 -1 FR )) TF(L ( Lh h

0.3

0.2

0.1

0.0
1.5 3
-1 -1 Flujo L h Flux (L( h ) )

4.5

TF1 FR1 TF2 FR2

Figure 1. Tendency of filtration rate (FR1 and FR2) of Crassostrea cortesiensis at different effluent velocities.

Crassostrea cortesiensis
0.5

0.4

-1 -1 NF R( (L T F N Lh h ))

0.3

0.2

0.1

0.0

1.5

3
-1 -1) ) Flujo(L (L Flux hh

4.5

NFR TFN1 1 NFR TFN2 2

Figure 2. Tendency of normalized filtration rates (NFR1 and NFR2) for Crassostrea cortesiensis at 3 effluent flux velocities.

Anadara tuberculosa

0.45

0.40

0.35

0.30

R.(L h .F (L h ))

-1 -1

0.25

Figure 3. Tendency of filtration rate (FR1 and FR2) for Anadara tuberculosa according to the effluent flux velocities.

Anadara tuberculosa
0.45 0.40 0.35 0.30
-1 -1 NFR (L T F N( Lh h ) )

0.25 0.20 0.15 0.10 0.05 0.00

1.5

3 Flujo (L -1 ) Flux (L hh )
-1

4.5

NFR1 TFN1 NFR2 TFN2

Figure 4. Tendency of the normalized filtration rates (NFR1 and NFR2) of Anadara tuberculosa according to the effluent flux velocities.

Crassostrea cortesiensis

90

80

70

CR (mg T C (m g h h) )

-1 -1

60

50

40

Figure 5. Tendency of the clearance rates (CR1 and CR2) for Crassostrea cortesiensis at three different flux velocities.
Anadara tuberculosa

55 50 45 40
-1 -1 CR T C(mg (m gh h))

35 30 25 20 15 10 5
1.5 3
-1 Fujo Lh h-1 Flux ( (L ))

4.5

CR1 TC1 CR2 TC2

Figure 6. Tendency of the clearance rate of Anadara tuberculosa according to the effluent flux velocity.