Balancing protein ingredients in carp feeds to limit discharge of phosphorus and nitrogen into water bodies

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1 Blackwell Science, LtdOxford, UK FISFisheries Science Blackwell Science Asia Pty Ltd Multi-ingredient less-polluting carp diets P Jahan et al /j x Original Article226233BEES SGML FISHERIES SCIENCE 2003; 69: Balancing protein ingredients in carp feeds to limit discharge of phosphorus and nitrogen into water bodies Parveen JAHAN, Takeshi WATANABE, Viswanath KIRON* AND Shuichi SATOH Department of Aquatic Biosciences, Tokyo University of Fisheries, Minato, Tokyo , Japan ABSTRACT: Through a series of experiments, it has been demonstrated successfully that an appropriate choice of ingredients is primary for achieving a reduction in phosphorus (P) and nitrogen (N) loading from carp culture. In this final study, four multi-ingredient diets were examined. In addition to the basal ingredients (fish meal 15%, corn gluten meal 8%, defatted soybean meal 10%, blood meal 3 5%), three of the test diets (PF, MM, and SP) contained 6% each of either poultry feather meal, meat meal or soy protein concentrate, respectively, and the fourth diet (PMS) had each of the three aforementioned ingredients included at 3%. These diets had crude protein < 35%, digestible energy > 3.5 kcal/g, available P between 0.65 and 0.66% and total P ranging from 1.26 to 1.36%. The diets were fed to juvenile carp during a 12-week feeding trial. The highest growth was obtained for fish fed the MM diet. The absorption and retention rates of P were higher for the PF diet, whereas these values for N did not show treatment-related differences. The total discharges per ton fish production were estimated to range between 7.8 and 9.2 kg for P and between 39.1 and 40.7 kg for N. These were lower than the figures for Lake Kasumigaura, a main carp culture site, indicating that proper balancing of protein ingredients aids in efficiently lowering emissions from aquaculture. KEY WORDS: alternative protein ingredients, aquatic environment, carp feed, nitrogen, phosphorus, waste discharge. INTRODUCTION World aquaculture production has increased the demand for fish meal, which, in turn, has been linked ironically to aquaculture effluents. 1 4 Many commercial fish feeds typically contain 30 70% fish meal by weight, 2 meaning that a high amount of phosphorus (P) in the form of tricalcium phosphate, which is scarcely absorbed by fish, 3 is discharged into the water. 4 Of the effluents from aquaculture systems, nitrogen (N) and P are two elements that, in excess, lead to eutrophication and a consequent change in the aquatic ecosystem. 5 Considering the rising cost and uncertain availability of fish meal, as well as the need to formulate diets that help to keep P excretion by fish at minimum levels, the replacement of fish meal with low P (mainly the unavailable portion)-containing alternative protein sources in aquatic feed seems to be one of the few remaining options. 1 3 Cho et al. 6 reported that using high protein ingredients that have a high percentage of digestible P may *Corresponding author: Tel: Fax: vizi@tokyo-u-fish.ac.jp Received 6 March Accepted 17 September help to reduce the unavailable P concentration of the feed. On another front, Rumsey noted that the complete replacement of fish meal with any of the alternative plant or animal protein ingredients negatively affected fish growth due to imbalances in the amino acid profile and the presence of some antinutritional factors derived from plant sources. 2 However, a proper combination of various alternative protein ingredients helps to replace fish meal, in addition to lowering waste loading. Watanabe et al. 7 introduced less N-polluting diets in Lake Kasumigaura, a main carp culture site in Japan, and the present series of studies aimed at the development of a low P-polluting feed for this species. Previous experiments 8 11 examined the efficacy of blood meal, defatted soybean meal, poultry feather (PF) meal, meat meal (MM) and soy protein (SP) concentrate as the main ingredients for the partial replacement of fish meal and, in turn, limiting the unavailable P portion in carp feed. The formulated diets were effective at reducing waste discharge; however, the high inclusion of single ingredient, such as blood meal and PF meal, decreased the dietary methionine content 8,9 and inclusion of SP concentrate increased the hardness of the pellets, thereby affecting the overall feed per-

2 Multi-ingredient less-polluting carp diets FISHERIES SCIENCE 227 formance. 11 The use of various ingredients in limited amounts could be more appropriate than using large proportions of a single component. We have been able to keep dietary fish meal levels even at 15% by using one of the above protein ingredients without hampering fish growth. 9,10 This prompted us to formulate diets containing 15% fish meal and a combination of all or some of the above mentioned protein ingredients in order to restrict P and N discharge from carp culture. MATERIALS AND METHODS Ingredients and composition of the experimental diets Four experimental diets (PF, MM, SP and PMS) were formulated as shown in Table 1 for the present feeding trial. The main protein source was 15% fish meal and the alternative protein sources in common were corn gluten meal (8%), defatted Table 1 Ingredients and nutrient contents of the experimental diets Diets PF MM SP PMS Ingredients (%) Fish meal Corn gluten meal Defatted soybean meal Blood meal (spray dried powder) Polutry feather meal (heat dried powder) Meat meal Soy protein concentrate (aquafeed grade) Wheat flour (low grade) Defatted rice bran Rye Available phosphorus from ingredients (%) NaH 2 PO 4 2H 2 O Oil mix Chromium oxide mix Others Nutrients Moisture (%) Crude protein (%) Methionine (% protein) Cystine (% protein) Crude lipid (%) Crude ash (%) Gross energy (kcal/g) Digestible energy (kcal/g) Total phosphorus (%) Available phosphorus 13 In diet (%) % Total phosphorus Chilean jack mackerel meal. 2 Product of China, sourced from Sakamoto Feed (Chiba, Japan). 3 Kanematsu (Chiba, Japan). 4 AP301; American Protein Corporation (Ames, IA, USA). 5 Japan Farm (Chiba, Japan). 6 Product of Japan, sourced from Nippon Formula Feed (Yokohama, Japan). 7 DANPRO TM ; Central Soya European Proteins (Aarhus Olie, Aarhus, Denmark). 8 Calculated based on the water-extractable phosphorus (P) content of each ingredient. 9 Beef tallow : soybean oil = 3 : 2 v/v. 10 Chromium oxide : dextrin = 1 : 1 w/w. 11 Phosphorus-free mineral mix 1.0%; vitamin mix 1.5%; vitamin E (50%) 0.1%; choline chloride 0.5%. 12 Calculated value. 13 Water-extractable P content that is available to carp. PF, MM and SP, diets that contained 6% each of either poultry feather meal, meat meal or soy protein concentrate, respectively; PMS, a diet that had each of the three aforementioned ingredients included at 3%.

3 228 FISHERIES SCIENCE P Jahan et al. soybean meal (10%) and blood meal (3 5%). The PF, MM and SP diets contained 6% of either PF meal, MM or SP concentrate, respectively, whereas the PMS diet contained 3% each of all three ingredients. Wheat flour, defatted rice bran and rye were used as the carbohydrate sources and binders and the lipid source was a mix of soybean oil and beef tallow (3 : 2 v/v). The available P content originating from the ingredients was less than the requirement levels ( %); hence, monosodium phosphate was supplemented to make up the deficit. 12 A chromium oxide mix was the digestibility indicator and a P-free mineral mix was the source of other minerals. The dietary nutrient contents are also presented in Table 1. It should be noted that a Feed Standard (crude protein < 35% and digestible energy > 3.5kcal/g), stipulated by the Prefectural Government, is followed for carp culture in Lake Kasumigaura. 7 Because the standard for comparison is data from this lake, all diets in the present study were prepared to comply with that standard. Methionine and crude lipid contents were similar and met the requirements of carp. 7,13 The total P contents varied slightly ( %), whereas available P (water-extractable P) contents were adequate in all diets. 12 five fish from each tank were sampled and stored at -20 C for fish whole-body analysis. Analytical methods and calculations Water-extractable P contents of diets, the proximate composition and P contents of diets and fish whole body, and the chromium oxide, P and N contents of the diets and feces were all measured using methods described previously. 10 Dietary and fecal gross energy contents were determined by an autocalculating bomb calorimeter (CA-4P; Shimadzu, Kyoto, Japan). The protein efficiency ratio, net protein utilization rate, retention of P and N and total P and N loading (T-P and T-N, respectively) were calculated according to the methods used previously. 10 Statistical analysis The data were subjected to one-way analysis of variance (ANOVA) and Scheffés test was used to evaluate differences between means of duplicate groups. Differences were considered significant at P < Fish and feeding Juvenile common carp (Cyprinus carpio) were purchased from a local seed supplier and kept on a commercial diet (Nippon Formula Feed, Yokohama, Japan) in 100 L indoor polycarbonate tanks prior to the trial. At the start of the feeding trial, 25 fish, weighing 5.1 ± 0.6 g on average, were stocked into eight 60 L glass tanks. Duplicate groups of fish were fed each of the test diets three times a day, 6 days a week, to near satiation for 12 weeks. The average water temperature during the trial was 22.0 ± 2.1 C. The fish were individually weighed at 4-weekly intervals for feed performance calculations. Fish were starved for 24 h and anesthetized with ethylene glycol monophenyl ether (300 p.p.m.) before being weighed. Fish rearing facilities and fish husbandry techniques were the same as those used in our previous experiment. 10 Collection of feces and fish samples During the 8th week, feces were collected every day using the TUF column and pooled for each tank. 14 Samples were vacuum freeze-dried and stored for further analyses. At the start of the trial, 25 fish from stock were collected and at the end of the trial RESULTS Fish growth and feed performance Figure 1 shows the average body weights recorded during the 12-week feeding period. Fish fed the SP diet had the lowest growth, even during the first 4 weeks, but there were no marked differences among the rest. From the 8th week, fish fed the MM diet started to show better growth, followed by the PF and PMS groups. This trend continued until the end of the trial and, finally, the MM diet performed the best. Feed performances determined after the 12- week feeding period are summarized in Table 2. Differences in the final average body weights of fish were not significant (P > 0.05), except for the SP group, the values for which were significantly lower compared with the MM diet. Moreover, a similar trend was observed for percentage body weight gain, whereas no significant differences were recorded for daily feed consumption rates. The feed gain ratio obtained for the SP diet group was significantly inferior to the ratios obtained for the PF and MM groups, but matched the PMS diet group. However, the protein efficiency ratio and net protein utilization rates were not significantly different among the treatment groups.

4 Multi-ingredient less-polluting carp diets FISHERIES SCIENCE 229 Fish whole-body composition The level or type of alternative protein ingredients did not influence the proximate composition and P contents of fish whole body at the end of the experiment (Table 3). Fish from all experimental groups showed lower moisture and higher crude protein and crude lipid contents when compared with the initial fish. Crude ash and P contents were similar among all groups. Fish fed the SP and PMS diets showed slightly higher crude protein values that were not significantly different (P > 0.05) from the rest. Absorption and retention rates of dietary phosphorus and nitrogen Table 4 shows the P and N absorption rates determined for all diet groups. Fish fed the PF diet showed the highest P absorption rate followed by the SP diet group. The value obtained for the MM group was significantly lower than values obtained for the PF and SP groups. Moreover, available P, as based on P absorption values (data not shown), was identical with values estimated by measuring the dietary water-extractable P content (Table 1). For N absorption, fish fed the PF diet showed the lowest rate and this was significantly different (P < 0.05) from that of SP group, which gave the highest value. The retention rates of P and N are also shown in Table 4. The P retention of fish fed the PF diet was significantly higher than for the other diets. The lowest value was for the PMS diet. The retention rates, calculated as a percentage of absorbed P, were not significantly different among the treatments and the highest value was that for the MM diet. However, no significant differences were observed in N retention values among the dietary treatments when calculated either way. Estimation of total phosphorus and nitrogen loading Fig. 1 Growth of carp and changes in water temperature during the feeding trial. The T-P values (kg/ton fish production), as based on P absorption and retention, produced after Table 2 Growth of carp and feed performance at the end of the 12-week feeding trial Diet group PF MM SP PMS Initial body weight (g) 5.1 ± ± ± ± 0.6 Final body weight (g) 81.4 ± 18.4 ab 94.1 ± 15.6 b 71.7 ± 18.0 a 84.2 ± 17.7 ab % Body weight gain 1486 ab 1729 b 1297 a 1539 ab Daily feed consumption rate 2.58 a 2.60 a 2.65 a 2.63 a Feed/gain ratio 1.05 a 1.04 a 1.10 b 1.07 ab Protein efficiency ratio 2.73 a 2.75 a 2.62 a 2.68 a Net protein utilization rate 46.6 a 47.0 a 47.0 a 46.5 a Where appropriate; data are the mean ± SD (n = 25). Values within the same row not sharing common superscript letters are significantly different at P < PF, MM and SP, diets that contained 6% each of either poultry feather meal, meat meal or soy protein concentrate, respectively; PMS, a diet that had each of the three aforementioned ingredients included at 3%.

5 230 FISHERIES SCIENCE P Jahan et al. feeding the experimental diets are presented in Table 5. The apparent values, derived from P absorption, do not consider non-fecal excretions and ranged between 6.7 and 8.2 kg/ton fish production. The lowest value was that of the PF diet. The more accurate estimation, taking into consideration both fecal and non-fecal excretion and based on P retention, showed higher T-P rates ranging from 7.8 to 9.2 kg/ton fish production. Table 5 also shows the T-N values (kg/ton fish production) based on N retention rates. Despite the fact that fish were fed different combinations of alternative protein ingredients, the T-N values were similar for all groups and ranged from 39.1 to 40.7 kg/ton fish production. DISCUSSION The main principles behind the development of low-polluting aquatic feeds are the selection of Table 3 Proximate composition and phosphorus content in the whole body of fish Initial Diet group* PF MM SP PMS Proximate composition (%) Moisture Crude protein Crude lipid Crude ash Phosphorus (%) * Values at the end of the 12-week feeding period. Values within the same row are not significantly different at P > PF, MM and SP, diets that contained 6% each of either poultry feather meal, meat meal or soy protein concentrate, respectively; PMS, a diet that had each of the three aforementioned ingredients included at 3%. highly digestible and easily available ingredients and their scientific formulation through proper combination after considering the physiological requirements of the culture species All these relate to the reduction of fish meal in aquatic feeds; however, previous studies have shown that the main disadvantage of fish meal replacers is an inadequate methionine content that decreases fish growth. 2,7 10 In the present experiment, the use of multiple ingredients provided a sufficient amount of the amino acid, despite the lower fish meal level. In order to achieve better fish growth and limit pollution due to feeds, dietary nutrients should be supplied in adequate amounts in a highly available (or digestible) form. In the present case, it should be mentioned that stomachless carp should be provided with dietary P that is mostly in the waterextractable form 16 or else it may hamper growth. 17,18 In the present study, available P and digestible energy contents in the diets were identical and adequate for carp. All diet groups showed better absorption rates of P and N, indicating that the combinations of the alternative protein ingredients were effective for increasing the availability of P, N and energy in the diets. These helped to achieve efficient feed per gain ratios in the present experiment. This result agrees with results of previous studies on rainbow trout 19 and carp 20 and shows that a better feed gain ratio could be expected in fish fed diets containing the required levels of digestible energy, digestible protein and available P. Moreover, protein efficiency ratios and net protein utilization rates did not vary much among the diets. Despite this, fish fed the high plant protein-based diet (SP) showed a slightly inferior growth performance when compared with the rest. Although the reason for this is not clear, these results were in accordance with our earlier observation. 11 The overall growth performance in the present study was better than in fish grown on popular commercial carp diets described in previous studies 17,18 and matched our earlier records 8 11 on alternative protein ingredients. Table 4 Diet group Absorption and retention rates of dietary phosphorus and nitrogen in carp P absorption rate (% dietary P) P retention rate N retention rate N absorption rate % Dietary P % Absorbed P (% dietary N) % Dietary N % Absorbed N PF 52.0 b 44.7 b 85.9 a 89.7 a 37.5 a 41.8 a MM 46.3 a 40.2 a 86.8 a 91.1 ab 38.1 a 41.8 a SP 50.9 b 41.0 a 80.6 b 92.3 b 38.4 a 41.6 a PMS 48.4 ab 39.9 a 82.4 a 90.3 a 37.7 a 41.7 a Values within the same column not sharing common superscript letters are significantly different at P < P, phosphorus; N, nitrogen; PF, MM and SP, diets that contained 6% each of either poultry feather meal, meat meal or soy protein concentrate, respectively; PMS, a diet that had each of the three aforementioned ingredients included at 3%.

6 Multi-ingredient less-polluting carp diets FISHERIES SCIENCE 231 Table 5 Total phosphorus and nitrogen loading from carp estimated at the end of the trial Diet group T-P (kg/ton production) based on T-N (kg/ton production) based on P absorption P retention N retention PF 6.7 a 7.8 a 39.6 a MM 8.2 b 9.2 b 39.1 a SP 7.3 ab 8.7 ab 40.7 a PMS 7.9 b 9.2 b 40.2 a Values within the same column not sharing common superscript letters are significantly different at P < T-P, T-N, total phosphorus and nitrogen loadings, respectively; P, phosphorus; N, nitrogen; PF, MM and SP, diets that contained 6% each of either poultry feather meal, meat meal or soy protein concentrate, respectively; PMS, a diet that had each of the three aforementioned ingredients included at 3%. Fish body composition is greatly influenced by the variety of ingredients, as well as the amount of nutrients in the diets. Similar fish whole-body protein and P contents indicate that the various ingredients adequately supplied the nutrients and fish efficiently utilized them. In contrast, one of the ingredients in the present combination, namely corn gluten meal, may have given rise to an entirely different issue. Work on gilthead seabream reported that a 20% corn gluten diet produced yellow orange coloration in the opercula and at the base of the fins. 21 However, in our previous study, no pigmentation was observed in carp fed a 5% corn gluten diet. 11 In the present study, corn gluten was increased to 8% and no visible malcoloration was observed, suggesting that corn gluten meal at this level could still be used in carp feed without any negative effect on pigmentation. One of the major parameters used to evaluate the efficiency of aquatic feeds is the retention of dietary nutrients: better retention relates to less waste loading. In animals, the retention of any nutrient takes place after its absorption and its complete retention may never be possible. Dietary nutrients that are not digested or absorbed can never be retained in the fish body and are therefore discharged through the feces into the water. In fact, both the available and unavailable dietary nutrient amounts and fish growth have a direct effect on retention efficiency. 22,23 In the present study, because available P content was similar in all diets, the differences in P retention rates (as a percentage of total P) were probably due to variations in dietary unavailable P contents and feed gain ratios. Previous studies have noted that an excess level of dietary available P decreases the P retention rate (as a percentage of absorbed P) from the amount absorbed, increasing non-fecal P excretion. 10,18 All the diets in the present study contained an adequate level of available P that resulted in higher and insignificant differences in these P retention values, indicating low and similar non-fecal discharge. This is in good agreement with results from previous studies 9,23 and also suggests that, for a better P retention efficiency, feeds should only provide the level required by the species in question. In addition, the N retention rates were identical between the groups, indicating that the nutrients were in adequate amounts in the different diets. Therefore, the results indicate that a balanced combination of protein sources should be used in aquatic feed formulation for a better nutrient efficiency. In the present study, the T-P values, as based on the P absorption rate, were apparent and only took into account fecal excretion, whereas those based on P retention were the more accurate values because they considered both fecal and non-fecal excretions. Research on rainbow trout, 22 Atlantic salmon 24 and silver perch 25 has demonstrated that the major loss of P was as the particulate form via feces. Although non-fecal P was not determined in the present study, from the difference between apparent and accurate T-P values, it could be assumed that carp excrete P mostly through the feces, as already reported With reference to non-fecal P excretion, Lall 3 stated that fish excrete P through their gills and kidney and Sugiura et al. 15 showed that the main pathway of non-fecal P excretion is in the urine. Considering the amount excreted, Rodehutscord et al. 23 reported that nonfecal P excretion in fish takes place even at zero P intake and the rate remains unchanged until the P intake exceeds the required level. We also found that while carp were fed diets with available P that was above their requirement, the P loading was high. 10,11 However, the diets in the present study contained only adequate amounts of available P at uniform levels and, hence, non-fecal P was similar and minimal. It should be noted that the standard of our research is Lake Kasumigaura, where the T- P estimation is based on apparent values. In order to compare our data directly with those of the lake, we performed the apparent calculation prior to accurate determination. These values were useful in assuming the non-fecal loss as mentioned above. The T-P values (kg/ton production) obtained in the present study were much lower than the reported average values of Lake Kasumigaura, 26 which were 13 kg/ton production (fecal form only) and kg/ton production (both fecal and non-fecal forms). Furthermore, these values were also lower than the values produced from carp culture with commercial diets ( and

7 232 FISHERIES SCIENCE P Jahan et al kg/ton production, respectively), as reported previously. 18 Watanabe et al. 7 have already stated that the quality of the protein source and the quantity of dietary protein directly influence T-N values in carp. We also found that a reduction of the best quality protein source (fish meal) in carp diet increased the T-N rate, despite a sufficient dietary crude protein content. 8 Another study 18 on commercial carp diets showed that the inferior feed gain ratio greatly affected the N retention rate and, thereby, T-N in a diet, despite the fact that it contained higher fish meal inclusion and was adequate in protein. Similarly, Ballestrazzi et al. 27 reported that post-prandial N (ammonia) discharge in sea bass fed plant proteins or high protein levels was higher than that in sea bass fed animal proteins or low protein levels. The protein levels of the experimental diets in the present study were approximately 35%, the optimum protein level reported for carp, 7 and the feed gain ratios were superior in all treatments. All these produced similar T-N rates, confirming the efficiency of the alternative protein sources. Moreover, the T-N values (kg/ton production) were lower than those of the current average value of 45 kg/ton production reported for Lake Kasumigaura 26 or than the values produced by feeding carp with commercial diets ( kg/ton production) in our earlier laboratory scale experiment. 18 When considering fish growth, body composition, nutrient retention efficiency and waste discharge loading, all experimental diets produced better results than those of the commercial carp diets. 17,18 Although previous studies 9 11 showed that PF meal, MM and SP concentrate were effective for the part replacement of fish meal, the present study demonstrated that the inclusion of these ingredients, in combination with other alternative protein sources, was more beneficial. Thus, the present study indicates that both nutritional and environmental benefits can be achieved by balancing the protein ingredients in aquatic feeds. The combined use of several protein sources helps to reduce the dependence on any single ingredient. The search for several more alternative proteins should continue and optimum inclusion levels for different fish species need to be determined so that dietary fish meal levels are lowered and complete ecofriendly aquafeeds are developed. REFERENCES 1. Hardy RW. Current issues in salmonid nutrition. In: Lim CE, Sessa DJ (eds). Nutrition and Utilization Technology in Aquaculture. AOCS Press, Champaign. 1995; Rumsey GL. Fish meal and alternate sources of protein in fish feeds. Fisheries 1993; 18: Lall SP. Digestibility, metabolism, and excretion of dietary phosphorus in fish. In: Cowey CB, Cho CY (eds). Nutritional Strategies and Aquaculture Waste. University of Guelph, Ontario. 1991; Ketola HG, Richmond ME. Requirement of rainbow trout for dietary phosphorus and its relationship to the amount discharged in hatchery effluents. Trans. Am. Fish. Soc. 1994; 123: Bergheim A, Åsgård T. 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8 Multi-ingredient less-polluting carp diets FISHERIES SCIENCE Takeuchi T, Hoshi M, Satoh S, Watanabe T, Takashima Y, Kawamata T. Effects of dietary digestible energy and available phosphorus contents on total amount of nitrogen excretion from carp. Suisanzoshoku 1993; 41: Rabaina L, Moyano FJ, Izquierdo MS, Socorro J, Vergara JM, Montero D. Corn gluten and meat and bone meals as protein sources in diets for gilthead seabream (Sparus aurata): Nutritional and histological implications. Aquaculture 1997; 157: Ketola HG, Westers H, Houghton W, Pecor C. Effect of diet on growth and survival of coho salmon and on phosphorus discharges from a fish hatchery. Am. Fish. Soc. Symp. 1991; 10: Rodehutscord M, Gregus Z, Pfeffer E. Effect of phosphorus intake on faecal and non-faecal phosphorus excretion in rainbow trout (Oncorhynchus mykiss) and the consequences for comparative phosphorus availability studies. Aquaculture 2000; 188: Johnsen F, Hillstad M, Austreng E. High energy diets for Atlantic salmon. Effect on pollution. In: Kaushik SJ, Luquet P (eds). Fish Nutrition in Practice. Institut National de la Recerche Agronomique, Paris. 1993; Kibria G, Nugegoda D, Fairclough R, Lam P. Effect of temperature on phosphorus losses and phosphorus retention in silver perch, Bidyanus bidyanu (Mitchell 1838), (Teraponidae) fed on artificial diets. Aquacult. Res. 1998; 29: Fisheries Science of Kasumigaura. An Official Publication of the Ibaraki Prefectural Institute of Inland Fisheries. Ibaraki, Japan Ballestrazzi R, Lanari D, D Agaro E, Mion A. The effect of dietary protein level and source on growth, body composition, total ammonia and reactive phosphate excretion of growing sea bass (Dicentrachus labrax). Aquaculture 1994; 127: