A q uacultural Engineering 3 ( t 984) 289-301

The Utilization of Clam Waste Meal as a Protein Source for Rainbow Trout Salmo gairdneri

*C.L. Goodrich, *S.M. Barnett, +G. Levine and *K.L. Simpson

* Department of Food Science and Technology', Nutrition and Dietetics, University of Rhode Island, Kingston, Rhode Island 02881. USA Blount Seafood Corp., Warren, Rhode Island 02885. USA

A BS TRA CT SolM waste recovered from clam processing wash water was evahtated lbr its utilizatkm as a replacement protebz for fish meal. Three diets, diets 1 and 2 fi)rmulated to simulate the Oregon Moist Pellet {OMP) and diet 3 {Rangen), were fed to rabzbow trout (Salmo gairdneri)for eight weeks. The effects o f the clam waste meal on fish growth, food conversion, carcass composition attd whole body fatO' acM content were determhwd. The clam waste diet supported mcLt'imttm growth attd food conversiott. Carcass attalysis o f moisture, proteht and lipid contents were similar among the moist feeds. The d#tary levels o f 20 : 5w3 + 22 : 6w3 and total poO'unsaturated fatty acids (PUFAs} were significantly higher bz the clam waste diet pellet.

INTRODUCTION Traditionally, fish meal has been the dominant protein source in fish leeds. Fish meal is becoming increasingly expensive and in short supply; therefore, a strong economic incentive exists to find alternative protein sources (Fowler and Banks, 1976; Foltz et al., 1982; Jackson er al., 1982). Plant proteins are commercially available and are less expensive as fish meal supplements. However, many factors affect the nutritional quality o f plant proteins, and problems such as inferior amino acid composition, antinutritional and toxic factors and processing conditions must be overcome when using these ingredients in diet formulations 289 Aquacultural Engineering 0144-8609/84/$03.00 Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain

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(Rumsey, 1973). Other protein feedstuffs of animal origin have successfully replaced fish meal. Disappointingly, most of these sources are scarce and/or as expensive as fish meal (Viola et al.. 1981, 1982). The dam processing industry generates an appreciable amount of waste. Wash water in clam processing plants contains small particles of clam tissue and soluble solids. Elimination of this organic waste not only magnifies disposal problems, but also results in the non-utilization of a valuable protein resource (Joh and Hood, 1979). There is a need to develop new treatment methods and productive means for the utilization of clam wastes. Clam waste has been shown to have a relatively high protein content necessary for its inclusion in fish feeds (67.9% on a dry weight basis; Hang e t a l . , 1980). The present study was conducted to evaluate clam waste meal as a fish meal replacement in salmonid diets. The effects of the clam waste on fish growth, food conversion, carcass composition and whole body fatty acid content were determined.

MATERIALS AND METHODS Three diets, two simulating the OMP, a commercial moist fish feed (Table 1), and Rangen, a commercial dry feed, were used in the present study. The moist diets differed only in the replacement of fish meal with clam waste meal.
Source of clam waste meal

Solid waste was obtained from a commercial clam processor located in Warren, Rhode Island. The material was collected by the sedimentation of clam processing wash water. Recovered waste was freeze-dried, ground and stored at --20C under nitrogen. Duplicate samples of the clam waste meal were subjected to proximate analysis, the results of which are shown in Table 2. Protein, crude fat, moisture, ash and crude fiber were determined by standard methods (AOAC, 1975). Proteins were acid hydrolyzed in 6-0 N HCI by the procedure of Spackman et al. ( 1958) as modified by Niederwieser and Pataki ( 1971). The amino acid composition (Table 2) was measured by ion-exchange chromatography using an Nc-2P Technicon auto-analyzer (cf. Seidel et al., 1980).

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TABLE 1 Composition of OMP Diets

hzgredien t Perce, tage o f total diet Diet 1 Diet 2

Fish meal (Menhaden) Clam waste meal Cottonseed meal Wheat germ meal Dried whey Corn distillers dried solubles Wet fish component a Fish oil (Menhaden) Soybean oil Choline chloride (70% liquid) OMP vitamin premix b OMP mineral premix c

29.0 15.0 8.9 5-0 4.0 30-0 3.0 3-0 0.5 1.5 O-I

29-0 15,0 8-9 5.0 4.0 30.0 3.0 3.0 0.5 1.5 O. I

a Ground, pasteurized flounder. b Contains in mg kg-~ premix: L-ascorbic, 59400; D-biotin, 40: viu, min BI 2.4.0: alpha-tocopherol acetate. 33 440 [U" folic acid, 847; myoinositol, 17 600; menadione sodium bisulfite. 1199; niacin. 12540: D-calcium pantothenate, 7652: pyridoxine HCI: 1430: riboflavin, 3520: thiamine mononitrate, 1712. c Contains in g kg-1 premix: ZnSO4, 185: MnSO,;, 207: FeSO,:.7H20, 50: CuSO4, 4.0: KIO3, 0 4 : CoSO4.7H~O, 25.

Diet formulation
T h e OMP diets (I and 2) were m i x e d and pelleted in a H o b a r t c o m mercial m e a t g r i n d e r using a 3 / 3 2 in die. T h e pellets were blast frozen and stored at - - 2 0 C until fed. R a n g e n (diet 3) was p u r c h a s e d f r o m Zeigler B r o t h e r s Inc. ( G a r d n e r s , P e n n s y l v a n i a ) and used as supplied.

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TABLE 2(a) Proximate Composition of Clam Waste Meal

Component Crude protein (N x 6.__) Crude fat Ash Moisture Crude fiber NFE a

Percott 70.0 9.8 11 -1 3.0 0-3 5-8

a Nitrogen-free extractive determined by difference.

TABLE 2(b) Amino Acid Content of Clam Waste Meal

Amino acM Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Histidine lsoleucine Leucine

g 100 g-1 sample 3.6 6. I 5.9 0.4 7.8 1-4 1.5 3-0 4.5 6.1 1-6 2.5 3.2 3.7 3.6 2.6 3-4

Lysine
Methionine Phenylalanine Proline Serine Threonine Tryptophan a Tyrosine Valine a Not determined.

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Diet analysis
The proximate composition of the fish diets was determined by AOAC (11975) methods. The total caloric contents were calculated using energy values of 3.9, 8-0 and 1-6 kcal g-~ for protein, fat and carbohydrate, respectively (National Academy of Sciences, 1973). Dietary amino acids were analyzed as described above. Lipids were extracted by the method of Btigh and Dyer (1959) as modified by Kates (1972) for fatty acid analysis, blethyl esters were prepared by saponification with 0-5N potassium hydroxide and methylation with 14% boron trifluoride-methanol (Morrison and Smith, 1964). Fatty acid methyl esters (FAblE) were analyzed on a single column Varian Aerograph 1700 gas-liquid chromatography unit operated at 200C isothermally and equipped with a flame ionization detector. The FAMEs were separated on a 10% SP-2330 column supplied with 30 ml rain -~ nitrogen as the carrier gas. A 37- ethylene glycol succinate polyester-Z (EGSP-Z) column was used to confirm fatty acids of similar retention time on the SP-2330 column. FAMEs were identified with an electronic integrator (Hewlett Packard 3380 A) supplied with the relative retention times of methyl ester standards (cf. Schauer er al., 1980), Cod liver oil was used as a secondary reference standard (Ackman and Burgher, 1965).

Feeding study Rainbow trout S a l m o g a i r d n e r i were purchased from the Plymouth Rock Trout Co. (Plymouth, Massachusetts). Following an acclimation period of four weeks, fish averaging 70.9 g were counted into groups of 25 and randomly distributed into each of six tanks. Rearing units were 45-gallon circular fiberglass tanks supplied with an average water flow of 4 liters min-L Oxygen content, temperature and water pH were monitored biweekly. The mean dissolved oxygen level was 7.4 ppm, temperature ranged between 12 and 15C and the mean pH was 5.9. Each diet was fed to fish in duplicate tanks for the duration of eight weeks. Fish were fed twice daily, six days a week, with food offered as long as the fish continued to actively feed. The fish were weighed every two weeks and average weight data were subjected to analysis of variance to determine if differences in means were statistically significant

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at the 5% level. The tables used for statistical analysis were from Snedecor and Cochran (1967). F o o d consumption and mortality were recorded daily. Carcass analysis At the termination of the study, five fish were randomly selected from each group for proximate analysis and whole body lipid analysis. Fish samples from each tank were pooled and homogenized in a Waring blender. Moisture and ash contents were determined immediately: the remainder o f the sample was lyophilized and stored under nitrogen for crude lipid and protein analysis. Whole body lipids of the wet tissue homogenates were extracted and fatty acids analyzed by gas-liquid chromatography as described above.

RESULTS AND DISCUSSION The proximate composition of the fish diets is given in Table 3. The protein content was 38% with the exception of diet 1 which provided 33% protein. Crude fat contents ranged from 7.9% in diet 3 to 13.25'; in diet 1. The total caloric value was similar among the three feeds.

TABLE 3

Proximate Composition of Diets (All Values on As-fed Basis)

Diet Moisture Chide number (%) prote#z a (%) Crude fat (%) Ash (%) Crude fiber (%) NFE b Total cah~ric vahte ( kcal 1 O0 g-l diet)

l 2 3

28.8 24.8 10.2

32-6 37-5 37.7

13.2 11.9 7-9

8.1 6-1 8-8

1-7 1.7 2-2

15.6 18-0 33.2

257,7 270.3 263.3

a N x 6-25. b Nitrogen free extractives determined by difference.

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TABLE 4

Amino Acid Composition ot" Diets [g 100 g-~ Diet, As-fed Basis)
Diet I Diet 2 Diet 3 Requirement a

Amino acid Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Histidine Isole ucine Let, cine Lysine Methionine ~ Phenylalanine c Proline Serine Threouine Tryptophan a Tyrosine Valine

2.0 2.9 2.5 0-I 4.4 2.1 0.9 1.3 2.1 2-0 0-7 1-4 2.0 1.3 1.3 0.9 1-6

1.6 2.4 2.3 0.1 4.2 1.6 0.7 1-0 2-3 2-1 0.7 1.2 1.9 1.6 1.4 1.0 1.2

2.3 2.1 3.0 4-6 2.2 1-0 0.8 2.7 2.0 0.6 1.6 1.8 2.1 1.2 0.9 1.4

2.4

0.7 0.9 1.6 2.0 0.5 2.1

0.9 0.2 1.3

a Values for Chinook salmon fingerlings receiving 40% protein (Nat. Acad. Sci., 1973). t, Ii1 the presence of 1% cystine. c In the absence of tyrosine. a Not determined.

No a p p a r e n t d i f f e r e n c e s were s h o w n in the essential a m i n o acid c o m p o s i t i o n o f the diets (Table 4). T h e essential amino acid requirem e n t s for C h i n o o k salmon were m e t (Nat. Acad. Sci., 1973) w i t h the e x c e p t i o n o f arginine and isoleucine in diet 3 and valine in diet 2. Precautions were n o t t a k e n t o p r e v e n t cystine f r o m o x i d a t i o n in acid h y d r o l y s a t e s ; t h e r e f o r e , with partial d e c o m p o s i t i o n it can be supposed that the c o n t e n t o f this a m i n o acid was originally higher (Moore, 1963). All o f the diets were d e f i c i e n t in p h e n y l a l a n i n e ; h o w e v e r , t y r o s i n e has a sparing a c t i o n o n p h e n y l a l a n i n e (Satia and B r a n n o n , 1975).

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X

12C

BC

4 Time (weeks)

Fig. 1.

Growth curves of rainbow trout fed diets for eight weeks. Curves (from top to bottom) represent fish fed diets 2, 1. and 3 respectively.

No mortalities were observed throughout the eight week feeding study. A growth-response curve for each diet (Fig. 1) was obtained by plotting the mean weight of replicate fish treatments measured initially and biweekly thereafter. Differences in growth rates among the diets became apparent after the fourth week. The best overall growth performance was observed in fish fed the clam waste meal diet. Analysis of fish weights showed statistical differences between diet treatments at the 5% level. Fish fed diets 1 and 2 had significantly higher weight gains than fish fed diet 3. Weight gain data between the two OMP fommlations did not show statistical differences. Food conversions, on a dry weight basis, of fish f e d diets I and 2, were significantly better than those fed diet 3 (Table 5). Good food conversion values and tow incidence of several diseases have been observed in fish culture on the OMP (Ghittino, 1972).

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TABLE 5 Average Body Weights, Percent Gain, Food Conversion. and Feeding Rates for Rainbow Trout Fed Diets For Eight Weeks
Diet mlmber Average body weight (g) Inisial Final Percent gain Food Feediw rate b conversion a {% body weight)

1 "~ 3

70.8 71.5 70.4

140.1
151.5

126.0

97.9 111.9 79.0

1-18 1.01 1.38

1.9 1-9 i.9

a Food conversion = food, dry weight (g)/weight gain (g). b Calculated on a dry weight basis. Weight and food conversion are the mean values for replicate tanks.

TABLE 6 Moisture, Protein, Lipid, and Ash Contents of the Carcasses of Rainbow Trout Fed for Eight Weeks
Diet Jmmber Moisture % in whole bodies offish Protein Lipid .4 sh

I 2 3 Initial

685 68-5 69.6 71.2

16-8 16.6 17.5 17-0

11.2 11-3 9-9 7.9

2.3 2-5 2-5 2-4

Carcass analysis o f m o i s t u r e , p r o t e i n and lipid c o n t e n t s at the termination o f the feeding s t u d y were the same for diets 1 and 2 as s h o w n in Table 6. Fish fed diet 3 had a slightly higher carcass p r o t e i n c o n t e n t . T h e m o i s t u r e and c r u d e lipid levels were inversely related, with moisture c o n t e n t increasing and lipid c o n t e n t decreasing, as previously d o c u m e n t e d ( J a u n c e y , 1982). Ash c o n t e n t in the fish carcasses did not vary b e t w e e n the diet t r e a t m e n t s . T h e fatty acid c o m p o s i t i o n o f fisil b o d y lipids reflected the f a t t y acid c o m p o s i t i o n o f the diets as s h o w n in Table 7. T h e r e was a positive

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TABLE 7

Fatty Acid Composition of Dietary l_ipids and Total Body Lipidsa ~Percent)

Fatta, acid Diec

12 0 14 0 15 "0 16 0 16" lw7 16 " 2w4 18 0 18' lw9 18 " 2w6 18 awa " " 18 4w3 20 I w9 20 2w6 20 3w6 20 4w6 20 3-W . ~" 22 1 22 5w3 22 6w3 24"1 Total saturated fatty acids 0.06 5-85 0.42 19.53 7.51 0-60 3.10 19.49 ._.78 5.10 ,.15
_

Diet 1 Fish
0-19 3-51 0.30 16-86 7.28 4.99 26.12 19.o9 1.18 4.59 9 a "~ 0-. 0.29 0.67 3.02 I .79 0-75 7.86 0.38 25.85

Diet 2 Diet
3.8l 0.28 18.66 7.28 0.31 3.61 16-53 25.57 4.64 2.52 0-43 0.29 9.90 0-83 0.60 4.50 0.43 ..6-40 "~ "

Diet 3 Fish Diet

2.04 4.40 0.44 22-23 5.19 0.33 5-59 20-08 _ _ "~6"-~8 3.71 0.82 0.08 0.12 3.55 1-24 0.15 3.76 0-07 34.70

Fish
0.90 3.35 0.34 18.23 8.54 5-40 33.09 13-43 0.80 3.42 0.88 0.43 0.27 1.52 1.74 0.32 7.13 0-40 28.22

0.54 6-71 1.63 0.38 3.87 0.34 28.96

0.20 2.75 0.27 18.15 7-13 5-78 25-18 18.,.~ "~" 1.10 4.49 1.05 0.33 0-54 3.29 1.45 1.04 8-65 0-48 27.15

a Average analysis of duplicate tanks.

c o r r e l a t i o n b e t w e e n d i e t a r y levels o f 14 0, 15 O, 16 0, 18 " I, 18 ~ 18" 4, 2 0 4, 20 5, 22 1, 22 5, 22 " 6 a n d 2 4 1 a n d fish lipid levels f o r all e x p e r i m e n t a l g r o u p s . The total saturated fatty acid ( S F A ) content in fish b o d y tipids

r e m a i n e d fairly c o n s t a n t at 27% r e g a r d l e s s o f t h e high S F A level in d i e t 3 ( 3 4 - 7 % ) . T h i s f i n d i n g is c o n s i s t e n t w i t h t h a t o f Y u e t al. ( 1 9 7 7 ) . T h e y r e p o r t t h a t p a r t i a l r e p l a c e m e n t o f h e r r i n g oil b y a n i m a l l a r d , w h i c h c o n t a i n s a high p e r c e n t a g e of SFAs, does not increase the s a t u r a t i o n of

Utilization of" elalq'z waste meal as protein source/br rainbow trout

TABLE 8 Essential Fatty Acid Contents (%1 of Diets
Diet number Total w3 PUFA
(~)

299

20 : 5 w 3 + 22 . 6w3
,-"

18 . 3w3

I 2 3

2-40 2.64 0-95

1.40 1.71 0.58

0-67
0.55

0.29

fish lipids. Apparently, a mechanism may exist in fish to regulate and maintain a proper level of body lipid saturation. The total w3 PUFAs, 2 0 5 w 3 + 2 2 " 6 w 3 , and 1 8 3 w 3 contents were calculated for the diets, tile results of which are given in Table 8. Diet _ ~ is higher in total w3 PUFAs and 2 0 ,gw3 + .;_~" 6w3 contents than the other diet groups. Linolenic acid ( 1 8 : 3 w 3 ) levels are somewhat low in all three of the diets, However, Takeuchi and Watanabe (1977) showed that the replacement of 0 . 5 ' 5 linolenate with either 0.55~ eicosapentaenoate ( 2 0 : 5 w 3 ) o r 0.57, docosahexaenoate (22: 6w3) markedly increases the growth rate of rainbow trout. They demonstrated that 2 0 5 w 3 in addition to 2 " 6 w 3 has an EFA efficiency higher than 1 8 " 3 w 3 . The best weight gain was observed in fish fed diets containing 0.25% each o f 20 : 5w3 + 22 : 6w3, respectively, indicating an additive effect between these fatty acids on tile growth response of rainbow trout. It is postulated that the superior growth response shown in diet 2 was partially due to the higher 2 0 : 5 w 3 + 2 2 : 6 w 3 composition.

CONCLUSIONS This study shows that clam waste meal can be utilized by rainbow trout as a protein source. Tile production of clam waste meal to be used as a feed ingredient illustrates a potential solution to tile problem of waste management for the clam industry. Waste material which is unfit for human consumption can be recycled through fish, to be sold as an acceptable human food.

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ACKNOWLEDGEMENT

Part of this work was presented at the 43rd Annual Meeting o f the Institute o f F o o d Technologists, New Orleans, Louisiana, 19-22 June 1983. This study was s u p p o r t e d by Sea G r a n t contract no. N A 8 1 A A D-00073.

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Moore, S. (1963). The determination of cystine and cysteic acid. J. Biol. Chem., 238. 235-7. Morrison. W. R. & Smith, L. M. (1964). Preparation of fatty acid methyl esters and demethyl acetals from lipids with boron trit'luoride-methanol. J. Lipid Res.. 5, 600-8. National Academy of Sciences (1973). Nutrient requirements of trout, salmon, and catfish. Nutrient Requirements o]'Domestic Animals No. 11, Washington DC. Niederwieser, A. & Pataki, G. (1971). In: New Tech#tiques bt ,4taboo AcidPeptide and Protein Analysis, eds. A. Niederwieser and G. Pataki. Ann Arbor, Michigan. Rumsey. G. L. (1973). The protein situation in fish feeds and feeding. Americatt Fishes and US Trout News, 18 (7). 6-11. Satia. B. P. & Brannon, E. L. (1975). The value of certain fish-processing wastes and dogfish (Squahts suckle.vi) as food for coho salmon (O#tcorh.vtzchtts kistttch) fry. Prog. Fish-Cult. 37 (2). 76-80. Schauer, P. S.. John, D. M.. Olney, C. E. & Simpson. K, L. 11980). International study on Artentia. IX. Lipid level, energy content, and fatty acid composition of the cysts and newly hatched nauplii from five geographical strains of Artemia. In: The Brine Shrimp Artemia, Vol. 3, eds G. Persoone, P. Sorgeloos, O. Roels and E. Jaspers, Universa Press, Wetteren, pp. 365-73. Seidel, C. R. Kryznowek, J. & Simpson. K. g. (1980). International study on Artemia. XI. Amino acid composition and electrophoretic protein patterns of Artemia from five geographical locations. In : The Brine Shrimp Artcmia, Vol. 3, eds G. Persoone, R. Sorgeloos, O. Roels and E. Jaspers. Universa Press, Wetteren, pp. 375-82. Snedecor. G. W. & Cochran, W. G. (1967). Statistical Methods. Iowa State University Press, Ames. Spackman, O. H.. Stein, W. W. & Moore, S. 11958). Automatic recording apparatus for use in the chrotuatography of amino acids. Anal. Chem., 30, 1190-206. Takeuchi, T. & Watanabe, T. (1977). Effect of eicosapentaenoic acid and docosahexaenoi acid in pollock liver oil on growth and fatty acid composition of rainbow trout. Bull. Jp. Soc. Sci. Fish., 44 (6), 677-81. Viola, S., Rappaport, U., Arieli, Y., Amidan, G. & Mohady, S. (1981). Effects ofoil coated pellets on carp (Cyprinus carpio) in intensive culture. Aquaculture, 26, 49-65. Viola, S., Mokady. S., Rappaport, U. & Arieli, Y. (1982). Partial and complete replacement of fishmeal by soybean meal in feeds for intensive culture of carp. Aquaculture, 26. 223-6. Yu, T. C.. Sinnhuber, R. O. & Putnam, G. B. (1977). Effect of dietary lipids on the fatty acid composition of body lipids in rainbow trout (Sabno gairdneri). Lipids, 12 (6) 495-9.