Optimised effluent treatment by stabilised trout faeces

Aquaculture 249 (2005) 125 – 144 www.elsevier.com/locate/aqua-online

Optimised effluent treatment by stabilised trout faeces Alexander Brinker a,b,c,T, Wolfgang Kopped, Roland Ro¨scha a

Fisheries Research Station of Baden-Wu¨rttemberg, Untere Seestrasse 81, 88085 Langenargen, Germany b Limnological Institute, University of Constance, 78457 Konstanz, Germany c Institute for Lake Research of the Environmental Protection Agency Baden-Wu¨rttemberg, Argenweg 50/1, 88085 Langenargen, Germany d Nutreco Aquaculture Research Centre, Sjb hagen 3, 4001 Stavanger, Norway Received 20 July 2004; received in revised form 27 October 2004; accepted 17 December 2004

Abstract The efficiency of mechanical effluent treatment depends on the ratio of particle bound waste load to total waste load and on the size distribution of the suspended solids. We tested an innovative approach to increase mechanical treatment efficiency by adding small amounts of indigestible binders to trout feed. The hypothesis was that the addition of binders would enhance faecal stability and improve shear resistance. This should slow the breakdown of faeces by water turbulences resulting in larger particles. These are easier to remove by filtration and should be more resistant to leaching than small particles, so more soluble waste remains particle-bound and removable. In two separate trials, portion size trout were fed diets containing indigestible binders, while control groups received the same basal diet without binder. The fish were reared in 500 L circular tanks. The effect of each binder on the stability (viscosity and elastic resistance) of dissected faeces was measured by a rheometer. Both stability parameters increased significantly by the addition of the binders. Addition of Guar gum increased the viscosity of the faeces best by 183% and 140% in Trial 1 and Trial 2 respectively, while Alginate increased elastic resistance best by 173% and 125%. The digestibility of the diets was not affected. The dissected faeces were exposed to controlled turbulence in the laboratory. The particle size distributions of the suspensions were measured using a laser-based technique applying the dtime-of-transitionT theory. The Guar gum and Alginate treatments led to significantly larger particles compared to the control. Based on these data the beneficial effect of the Guar gum binder on post-filtration suspended solid load (100 Am gauze) was calculated to be 40.2% and 18.2% for Trials 1 and 2 respectively. In a leaching experiment, faecal particles were suspended in distilled water and allowed to leach for 1 h. The solids were fractionated by sieving and their relative phosphorus and nitrogen content was determined. The relative phosphorus and nitrogen content of the particles increased significantly with increasing size class. In a second experiment, larger particles of the Guar gum treatment yielded significantly higher total amounts of phosphorus and more dry matter remained solid-bound compared to the control.

T Corresponding author. Fisheries Research Station of Baden-Wu¨rttemberg, Untere Seestrasse 81, 88085 Langenargen, Germany. Tel.: +49 7543 930824; fax: +49 7543 930820. E-mail address: [email protected] (A. Brinker). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.12.029

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The present study demonstrates promising potential for binders as tools in significantly reducing the effluent load of fish farms without affecting feed efficiency. D 2005 Elsevier B.V. All rights reserved. Keywords: Effluent treatment; Binder; Guar gum; Rheology; Faeces; Leaching; Shear resistance

1. Introduction The production of fish in flow-through fish farms causes environmental impact with feed-derived wastes. Essentially, these are the end products of fish metabolism in solid (faeces) and dissolved form (Summerfelt, 1999; Davenport et al., 2003). Effluent load can be reduced by supplying highly digestible feeds, by avoiding feeding losses, by optimising rearing conditions, and by treatment of effluent (Talbot and Hole, 1994; Bergheim and Asgard, 1996; Cho and Bureau, 1997; Cripps and Bergheim, 2000; Kaushik, 2000; Dumas and Bergheim, 2001; Kaushik, 2000; Boyd, 2003). Although such methods have proven very effective in terms of waste reduction (Bergheim and Brinker, 2003; Davenport et al., 2003), their potential for further improvement is now rather low, due to physical, biological and economic constraints. Hence substantial progress requires new approaches, a view that has motivated the present work. A survey of drum filtration efficiency in flowthrough fish farms showed remarkable variation (Cripps, 1994). The variability of waste removal efficiency indicates how much removal potential is often lost (Table 1). This can be attributed to differences in the size distribution of the suspended particles and in the ratio of particle-bound to dissolved loads. Table 1 Percentages of particle-bound wastes in total load and a review of removal efficiency of drum filtration for flow-through trout farms (cf. Cripps, 1994; Cripps and Bergheim, 2000)

Total phosphorus Total nitrogen BOD5 Solids

Particle-bound fraction (%)

Minimum efficiency removal values (%)

Maximum efficiency removal values (%)

Up to 90 Up to 32 More than 80 –

47 7 21 19

84 32 80 91

In typical flow-through trout farm effluent, most of the total phosphorus load, most of the biodegradable matter, and much of the total nitrogen load is carried in suspended particles (Cripps and Bergheim, 2000; Dumas and Bergheim, 2001, Table 1). Properties such as particle size and stability, which are crucial in determining mechanical treatment efficiency, depend on complex parameters including the origin of the organic wastes and various physical properties of the water (Brinker and Ro¨sch, in press). A large proportion of unfilterable small particles and dissolved waste derives from the degradation of suspended faecal particles into finer and more soluble ones (Summerfelt, 1999). Such disintegration is promoted by shear forces in turbulent water zones, for example, those generated by fish, pumps, and falls (McMillan et al., 2003; Brinker and Ro¨sch, in press). Therefore, enhancing the shear resistance of the faeces may significantly inhibit particle breakdown. Binders that enhance the viscosity, elastic resistance, and structure stability of digesta and faeces may be used as food ingredients in order to control the stability of the faecal particles (Onsoyen et al., 1992; USDA/CSREES, 1997). Only very small doses of indigestive binder should be necessary, because the additive will be concentrated along the intestine. This is beneficial on both economic and biological grounds because both costs and potential negative side effects on digestibility are minimized. More stable faeces are thought to possess improved resistance to shear forces and should therefore be more effectively removed by mechanical treatment (Dumas and Bergheim, 2001). Larger particles also have an improved resistance to leaching due to reduced water contact area. Thus a higher percentage of soluble wastes should remain particle bound. The present study investigates the effect of binders on the mechanical stability of faecal particles, specifically their resistance to shear forces.

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1.1. Background: faecal stability and inclusion of binder The idea of using binders to increase faecal stability and thereby facilitate mechanical effluent treatment was discussed by Spyridakis et al. (1989), Dias and Huelvan (1998), and McMillan et al. (2003). Although the potential of this approach was recognised, not much experimental work has been done so far. In a research project certain binders were added to basal diets at concentrations ranging from 3% to 19% (USDA/CSREES, 1997). The main conclusion of this study was that adding binder to the feed was not practical as a means of improving waste removal. However, the authors also pointed out that the large variations in their data may have hampered their efforts to isolate the effects of dietary binder. In general, chyme or faeces are viscoelastic bodies rather than simple viscous liquids. Such materials have properties of both liquids (viscosity) and solids (elasticity). The viscoelastic properties can be determined by dynamic measurements (Boltzmann, 1876) whereby a sinusoidal strain (r) is applied to the material, and the sinusoidal response (stress, c) is measured. Both strain and response share the same frequency but have a phase difference (d). The inphase modulus GV (elastic modulus) characterizes the elasticity of the substance, whereas the out-of-phase modulus GW (loss modulus) characterizes its viscosity. Most technical knowledge of binder effects on viscoelastic properties is derived from simple solvent–binder mixtures. Chyme however, is a complex matter made up of diverse constituents—for example innate hydrocolloids and solutes. These compounds may interact with the binder and progressively change the freedom of movement of the binder molecules or prevent their full hydration. Both effects modify viscosity or gelation properties of polysaccharide binders (Morris et al., 1981; Onsoyen et al., 1992). Additionally, it is nearly impossible to model the interaction of the binder with the chyme in the course of the alimentary tract due to the dynamic processes of digestion, absorption, and pH-change. Clearly, an approach combining fundamental research as well as empirical observations from the food sector is required. The viscous behaviour of the binders used in this study is strongly concentration-dependent, with two markedly different phases separated by a distinct

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threshold concentration (Morris et al., 1981; Phillips, 1986). At low (dilute) concentrations the polymer chains are separated by solvent, and viscosity increases linearly with concentration according to Einstein’s theory (Einstein, 1906). As the binder passes the threshold to high concentrations the polysaccharide chains begin to entangle, and the viscosity increases exponentially (Morris et al., 1981). Experiments on foodstuffs, which are comparable to chyme and faeces in many ways, found that the most effective concentration of polysaccharide binders lies within the entanglement domain where the dimensionless overlap factor is slightly above unity (Fox, 1992). The threshold value determined for a high viscosity Guar gum was c0.2% and for a comparable Alginate it was c0.6% (Morris et al., 1981; Phillips, 1986). In the alimentary tract of trout, food is diluted several times by water intake (Tytler et al., 1990; Loretz, 2001). Hence Guar gum and Alginate have to be dosed in levels that account for this dilution as well as for the concentration effects of digestion and absorption. What matters is the final concentration after water absorption and compaction of faeces in the very distal intestine have been finished and the pellet is ready to be expelled (Windell and Norris, 1969). As a positive consequence, the viscosity that binders impart to chyme during the main digestion processes is minimal. In contrast, Alginbind (threshold level N1.0%, Morris et al., 1981) was used at much lower concentrations as the main focus concerning this binder was gelation. Beside the binding polymers, Alginbind contains a considerable quantity of divalent cations, whose action enhances gelation and final gel strength (Onsoyen et al., 1992; Sobeck and Higgins, 2002).

2. Materials and methods 2.1. Diets and binder—preliminary trials In three preliminary trials (data not shown) different dietary binders were tested for their efficiency at different concentrations (Table 2). The diets, extruded 3.0 or 4.5 mm pellets, were fed to groups of portionsized trout for at least 5 weeks. The basal diet compositions were comparable to commercial diets. To examine the binder effects on faecal stability, faeces from three different sources were isolated and

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Table 2 Basic data of the tested binders Dietary binder

Product specification

Characteristics (solubility, viscosity, gel strength, digestibility)

Costs

Inclusion level [range]

Lignin sulphonate

Not known

Cold water-soluble, medium viscosity, no gel strength, indigestible

Very low

Not known

Algae meal

ALGINBIND (p.c. 5221025), Algae a.s.

Cold water-soluble, low viscosity, medium gel strength, partly digestible

Low

0.1–2%

Modified (non-gelatinised) starch

Not known

Cold water-soluble, very low viscosity, very low gel strength, low digestibility

Very low

Not known

Sodium-Alginate

ALGINATE (Scogin HV Alginate-2205000), FMC BioPolymer

Cold water-soluble, medium viscosity, medium to high gel strength, indigestible

High

1%

Fish gelatine

´ gerWarfe´lag Akureyringa hf., U Akureyri Fishing and Processing plc.

Cold water-soluble, very low viscosity, high gel strength, digestible

Medium

1%

Guar gum

Equivalent to GOMME DE GUAR HV 105 (Code: 3303), SEAH International

Cold water-soluble, very high viscosity, no gel strength, indigestible

Medium

0.1–1.0%

Resistant starch

POVEX, Lyckeby Sta¨rkelsen

Cold water-soluble, low viscosity, low gel strength, indigestible

Low

2%

Powdered cellulose

ARBOCEL B 600, J. Rettenmaier and So¨hne GmbH

Water-insoluble, no viscosity, no gel strength, synergistic effects with hydrocolloids, indigestible

Low

2%

Values arranged according to the supplied product specifications and data from Onsoyen et al. (1992), USDA/CSREES (1997) and Semke (2001).

inspected macroscopically. Intestinal faeces were obtained by collecting the lowermost faecal pellet after intestinal dissection, expelled faeces were collected from the tank bottom, and expelled faeces were gathered from tank effluent using a special faeces collector (Fassbender, 1990). In the latter case no contamination of faeces with uneaten feed was possible as during feeding the faeces collector was closed and afterwards all uneaten pellets were removed from the tank. The effects of several macroscopically promising binders on faecal stability were assessed qualitatively. For each treatment, duplicate samples (2.5 g) were suspended in water (200 ml in 500 ml PE flasks) by controlled mechanical agitation (shaker SM25: about 90 r.p.m. for 1200 s); then the sedimentation performance of the suspension was tested in Imhoff cones. The clearance of particles from the water column was scored subjectively.

Special attention was paid to possible side effects of the binder, specifically to those affecting feed performance. Measurements were made of digestibility (crude protein, crude lipid), specific growth rate, and feed conversion. Faeces obtained during these trials were used to assess the laser particle-sizing method described below. These laser data were subject to a power analysis to determine the sample sizes required for subsequent trials. Only those binders that yielded the most stable faeces (Fig. 1) without influencing nutrient digestibility, growth and feed conversion were employed in the subsequent feeding experiments. These binders were: i) Guar gum: A linear polysaccharide (galactomannan) derived from the endosperm of the Indian cluster bean (Cyamoposis tetragonolobus). It is based on a back-

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A) Basal diet

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B) plus Guar gum

Fig. 1. Macroscopic differences in faeces from trout fed a basal diet (A) and from trout fed the same diet but with inclusion of Guar gum as binder (B). Faeces were obtained by intestinal dissection. Note that the binder-containing faeces (B) are distinctly more structured than the more pulpy faeces of the basal diet (A).

bone of h(1Y4)-linked d-mannose residues with single linked a(1Y6)-d-galactose. The polymer shows differing degrees of polymerisation and viscosity is a property of the hydroxyl groups (Fox, 1992). ii and iii) Alginate and Alginbind: These two binders share the same binding component, the alginic acid (h(1Y4)-d-mannuronic acid (M-block) and its C-5 epimer a(1Y4)-l-guluronic acid (G-block)). Both are extracted from seaweed. Alginate is the purified salt (sodium alginate) of alginic acid whereas Alginbind is dried algae (Ascophyllum nodosum) containing 18–23% alginic acid. Alginate has a medium to high gel strength (35–45 according to Fira method). The alginic acid of Alginbind has a M/G ratio of 1.85 which indicates a medium gel strength but performance is enhanced by the presence of calcium ions—a natural constituent of the algae—which support gelation (Onsoyen et al., 1992). In aqueous solution the viscoelastic properties of Guar gum differ from those of Alginate or Alginbind in that Guar gum shows no true gelation (Fox, 1992). At the same concentration, the viscosities of Alginate and Alginbind are much lower than that of Guar gum, but these binders have the

ability to form strong gels (Onsoyen et al., 1992). The gel forming capacity is positively correlated with the relative abundance of G-blocks as well as with G-block length. The mechanism of gelation involves the formation of stable intermolecular djunction zonesT between structurally and conformationally regular chain sequences (Morris et al., 1981). Nevertheless, gelation is strongly supported by the availability of calcium ions as a sequestrant (Onsoyen, 1992). Alginbind, which contains up to 2.6% calcium ions, was applied in low doses to test whether this ingredient (Onsoyen, 1992; Sobeck and Higgins, 2002) really can compensate for the reduced concentration of alginic acid compared to Alginate. 2.2. Rearing of fish Rainbow trout (Oncorhynchus mykiss, all female, Hofer strain) were reared in six circular green fibreglass tanks (diameter: 1 m, height: 0.7 m, in approximately 0.5 m3 water). Average rearing density was about 30 kg m
3 and 38 kg m
3 in Trial 1 and Trial 2, respectively. The fish were of conventional, unspecified microbiological status. Water free of fish pathogens was supplied from a groundwater well. The inlet water was pre-treated using fine bubble aeration to strip elementary nitrogen and carbon dioxide and enrich it with oxygen to near saturation. Water was delivered to each tank through a vertical PVC inlet pipe with a

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458 angle. The inlet was tangentially orientated near the tank wall to generate a gentle circular flow in which the fish could orientate. The swirling dtea cupT effect generated by this set-up (Goldsmith and Wang, 1993) moved all faeces to the tank’s central drain, which was shielded by a perforated plate (F 0.2 m, borings F 0.01 m). Therefore, almost no tank cleaning was necessary. For each tank the water flow was adjusted to 7–9 L min
1. The photoperiod was fixed at 12L/12D (lights on between 07:00 h and 19:00 h without twilight). Oxygen concentration (F 0.1 mg O2 L
1), pH (F0.1) and temperature (F0.1 8C) of the water were measured in the outlet of each tank daily about half an hour after the end of hand feeding. Further water parameters (cf. Table 3) were determined according to German standard methods for the analysis of water, waste water and sludge, modified by Gewa¨sserschutzkommission (2000). Samples were tested as described above at the beginning and the end of each trial and water parameters (Table 3) were all within the recommended range for rainbow trout (Boyd, 1982; Alana¨ra¨ and Bra¨nna¨s, 1996; Wedemeyer, 1996; Summerfelt, 1999). Seventy-five fish per tank in Trial 1 (31.03.2002 to 26.04.2002) and 99 trout per tank in Trial 2 (20.10.2002 to 28.01.2003) were fed the experimental diets. The initial average weight was 184 g in Trial 1 and 191 g in Trial 2. 2.3. Diet composition and feeding of fish Six isoproteic and isoenergetic diets were formulated, one without binder and the others with binder

(Table 4). They contained balanced levels of essential amino acids, fatty acids, vitamins and minerals exceeding the levels recommended by National Research Council (1993). The diets were extruded (maximum extrusion values at feed matrix: 120 8C, 22 bar) and sized to F 4.5 mm. The binders were added on top of the basal mix for maintaining maximum homogeneity between treatments. Dilution effects were minimal as seen from chemical analyses of the finished feeds (Table 4). The fish were fed 6 days a week (Monday through Saturday) with a daily amount of 1.2% body weight. Approximately 40% of the daily ration was dispensed manually between 07:30 h to 09:00 h under continuous observation of the animals’ intake behaviour. The remaining feed was supplied by an automatic feeder, which fed continuously until 18:00 h. This feeding protocol produces a faecal pellet on the verge of excretion at around 10:00 h. 2.4. Digestibility, specific growth rate, feed conversion For digestibility measurements, after 10 days of feeding in the time from 09:30 to 12:00, 54 trout per treatment in Trial 1 and 75 in Trial 2 were anaesthetized with clove oil (concentration: 0.1 mL L
1, exposure time: ca. 60 s); their faeces were stripped from the rectum, immediately frozen in liquid nitrogen, lyophilised, and homogenised. Dry matter content, protein, fat, phosphorus and yttrium oxide content were determined. The apparent digestibility of protein, fat, and phosphorus was

Table 3 Basic water parameters during both trials Oxygen [mg L
1]

pH

Temperature [8C]

NH4–N [Ag L
1]

NH3–Na [Ag L
1]

NO2–N [Ag L
1]

NO3–N [Ag L
1]

8.0 6.2–11.5

8.1 7.8–8.4

12.5 11.3–14.1

164.8 39–233

4.6 1.1–6.5

2.6 0.8–6.6

1889 1841–1929

Chloride [mg L
1]

Sulphate [mg L
1]

PO4–P [Ag L
1]

Conductivity [AS cm
1]

Acid binding capacity [mmol L
1]

Earthy base ions [mmol L
1]

7.4 7.0–7.7

18.8 17.5–20.2

72 54–81

580 569–587

6.5 6.3–6.7

19.7 19.4–19.9

Bold numbers: average values. Plain numbers: range of values. a Calculated according to Trussel (1972).

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Table 4 Composition of the experimental diets Unit

Diet 1

Diet 2

Diet 3

Diet 4

Diet 5

Diet 6

BD

GG 0.1

GG 0.3

AB 0.3

AB 0.6

AT 1.0

kg kg
1 kg
1 kg
1 kg
1 kg
1 kg
1 kg
1 kg
1 kg
1 kg
1 kg
1 kg
1 kg
1 kg
1

305.1 200.0 192.8 119.0 6.6 168.0 2.4 2.4 3.0 0.6 0.1 – – – 1000

305.1 200.0 192.8 119.0 6.6 168.0 2.4 2.4 3.0 0.6 0.1 1.0 – – 1001

305.1 200.0 192.8 119.0 6.6 168.0 2.4 2.4 3.0 0.6 0.1 3.0 – – 1003

305.1 200.0 192.8 119.0 6.6 168.0 2.4 2.4 3.0 0.6 0.1 – 3.0 – 1003

305.1 200.0 192.8 119.0 6.6 168.0 2.4 2.4 3.0 0.6 0.1 – 6.0 – 1006

305.1 200.0 192.8 119.0 6.6 168.0 2.4 2.4 3.0 0.6 0.1 – – 10.0 1010

Chemical composition (dry matter) Crude protein g kg
1 Crude lipid g kg
1 Total phosphorus g kg
1

478.1 263.1 13.1

481.9 256.7 13.7

477.7 265.6 13.6

482.8 277.2 14.1

479.2 267.5 13.9

477.1 270.4 13.7

Fish meal Soybean meal Corn gluten meal Wheat Mono-Ca-Phosphate Fish Oil Mineral premixa Vitamin premixa Lysine*HCl Carophyll pink Yttrium oxideb Guar gum (GG) Alginbind (AB) Alginate (AT) Sum

a b

g g g g g g g g g g g g g g g


1

According to National Research Council (1993). Yttrium oxide was added as marker for digestibility measurements.

calculated using the following equation (Guillaume et al., 2001): Apparent digestibility coefficient ðADC½%Þ    Y2 O2ðdietÞ % nutrient ðfaecesÞ ¼100
100   : ð1Þ Y2 O2ðfaecesÞ % nutrient ðdietÞ Values for each parameter were determined in duplicate. Dry matter content was determined as the ratio of wet to dry weight after lyophilisation (F0.1 mg). Crude protein was analysed according to Commission Directive 93/28/EEC, Kjehldahl method, but with selenium as a catalyst (Anonymous, 1993). Crude lipid was analysed according to Commission Directive 84/4/EEC, method B, but with diethyl ether as a solvent (Anonymous, 1986). Phosphorus and yttrium were determined by Jordforsk, 2s, Norway. The samples were first digested in 10 mL 6 M nitric acid (p.a.) and 0.5 mL hydrogen peroxide (p.a.) in a microwave oven (140–160 8C, duration: 20 min), then diluted with distilled water, and finally analysed on an ICP-AES (Inductively Coupled Plasma-Atomic Emission Spectroscopy).

Each fish was weighed (F1 g) immediately after killing. From the mean weight at the beginning and end of the trial, the specific growth rate of the fish, SGR, was calculated as   SGR % day
1 lnðmean final weightÞ
lnðmean initial weightÞ ¼  100: ð2Þ tðfinal dateÞ
tðinitial dateÞ Feed conversion ratio, FCR, was calculated as FCR ¼

Feed ½kg : Weight gain ½kg

ð3Þ

2.5. Sampling of faeces Due to necessary investments in time for particle size measurements and rheological measurements trout were sampled in weekly groups at the end of the experiment. The fish were sampled daily from two randomised tanks from 09:30 to 11:30. They were randomly selected, anaesthetized with clove oil (dose: 0.1 mL L
1; exposure time: 60 s), and killed by a sharp blow on the head. The lowermost faecal pellet

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was removed by intestinal dissection. Only mucus sheathed faecal pellets were used. The faeces were kept in aluminium dishes, hermetically sealed with a plastic film to prevent dehydration, and cooled to 4 8C to slow down microbial degradation. All measurements were performed within 8 h of dissection. The faeces and intestine were inspected for irritation of intestinal mucosa, exudative enteritis, and haemorrhagic enteritis. 2.6. Rheological measurements Rheological measurements were carried out on the faeces of 15 trout per treatment in Trial 1 and 25 trout per treatment in Trial 2. Three to five pellets, constituting a minimum volume of about 3 cm3 per measurement, were merged and transferred to the rheometer (Paar Physica-Physica UDS 200). The measuring system applied was a MP 313 (plate: F 50 mm, 08) with a gap width of 1 mm. The shear stress factor was 2.037 and shear rate factor was 2.617. For the time sweep a deformation with an amplitude of c=60% at a frequency of 1 Hz was used. The duration of measurement was 12 s. For the studies in the frequency domain the samples were probed by sinusoidal excitation at frequencies of 50, 32.1, 20.6, 13.2, 8.47, 5.43, 3.49, 2.24, 1.43, 0.92, 0.59, 0.38, 0.24, 0.16 and 0.10 Hz. In each run the deformation amplitude was 40% and the duration of measurement was 30 s. In the sample compartment the temperature was set to 4 8C and the air humidity was adjusted to 100% saturation. All measurements were deformation-controlled. Each measurement started with a time sweep of 50 single deformations, followed by a frequency sweep after a 60 s delay. 2.7. Particle size distribution (PSD) For particle size measurements, 15 trout from Trial 1 and 30 from Trial 2 were sampled as described above. At first, 2 g of faecal pellets from the control group, i.e. fish fed the diet without binder, were broken under defined conditions until the PSDs achieved resembled those found in effluents of flow-through trout farms. This was done according to Brinker et al. (2005) using water turbulence caused by a constant air stream from below through a 2 L volume of distilled water. Thus air

pressure and agitation duration settings of 0.05 MPa and 480 s respectively were determined and used for all further experiments. 2 g (F0.01 g) of wet faeces were used in Trial 1 and 3 g (F0.01 g) in Trial 2. Particle sizes were determined using a non-invasive laser particle sizer (GALAI: CIS-1) equipped with a flow controller (GALAI: LFC-100) and a flow-through cell (GALAI: GM-7) according to Brinker et al. (2005). As the upper size range of the laser particle sizer is 600 Am, all values were corrected by the percentage of particles greater 600 Am. This fraction of oversized particles was determined by sieving (Brinker et al., 2005). 2.8. Effect of particle size on leaching The general effects of size-dependent nutrient leaching were tested during a previous trial in 2001 in which nitrogen and phosphorus content of particles of certain size classes was determined following controlled leaching in distilled water for 1 h. Feeding and rearing conditions were as described in Sections 2.2 and 2.3. Diet composition was soybean meal: 291 (g kg
1), corn gluten 400 (g kg
1), wheat 69 (g kg
1), monoCa-phosphate 6 (g kg
1), fish oil 224 (g kg
1), premix 9 (g kg
1), and yttrium oxide 0.1 (g kg
1). The applied binders had no effect on the faeces stability (details not shown). The faeces were collected and broken as described in Section 2.5. The suspension was allowed to leach for 60 minF5 min. Then the particles were separated by sieving into size classes (N0.45 Am, F 50 mm, Sartorius 11106-50-N; N32, N64, N100, N200, N400, and N600 Am, Polyester-gauze (PES), F 68 mm; Franz Eckert GmbH/Germany) and immediately frozen with liquid nitrogen. Dry matter for each size class was determined according to Brinker et al. (2005). The filter and the particle-filtrate were transferred into a closed microwave-pressure-digestion system (MARS 5, CEM, Kamp Lintfort) using XP1500 vials with 100 mL volume. A solution of 10 mL distilled water, 10 mL hydrogen peroxide (30%, p.a.) and 2 mL sulphuric acid (95–97%; p.a.) served as digestion reagent. Within 4 min the reagent was heated to 107 8C (boiling point of H2O2); this temperature was held for 5 min. Then the mixture was further heated to 200 8C within 14 min and held at this temperature for 9 min. Afterwards the phosphorus and organic nitrogen content of the solution were determined according to dGerman standard methods for the analysis of water, waste water and

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sludge’ using the modifications of the Gewa¨sserschutzkommission (2000). Trial 2 examined whether the addition of a binder significantly influences the retention potential of suspended faecal particles. Leaching conditions were as above, but the digestion process was modified because in rare cases the PES-gauze reacted pyrophoricly with the acid, thereby destroying the XP1500 vial in a vigorous explosion! To avoid this, the digestion was carried out in two steps. In step one, all faeces were removed from the PES-gauze (reagent: 10 mL distilled water, 10 mL H2O2; First stage: heating to 107 8C within 4 min, temperature held for 5 min. Second stage: heating to 180 8C within 12 min, temperature held for 12 min). Then the PES-gauze was removed, and any adhering reagent was washed into the vial using 10 mL distilled water. In step 2, the components were digested (added reagent: 3 mL H2SO4; protocol: heating to 200 8C within 14 min, temperature held for 15 min). 2.9. Data analysis Differences in the digestibility of nutrients and dry matter content were tested by analysis of variance (ANOVA). Due to the hierarchical design of Trial 2, a nested ANOVA was applied with the variable Tank as a random factor. The rheological data of the time sweep measurements were analysed using repeated measurement ANOVA with the variable Measuring point as a random block factor. Post-hoc comparisons were made by Tukey’s HSD test (Hayter, 1984). Measuring points one to five were removed from the analysis because of possible undetected faecal air inclusions which were eliminated by the first five agitations. The rheological data for frequency sweep measurements were analysed using nested analysis of covariance (ANCOVA) with the variable Tank as a random factor (Sokal and Rohlf, 2003). The dependent variable Viscosity and the independent variable Frequency were log10 transformed to meet the assumptions of the model. Diet was included as main factor, and the interaction of the variables Diet log 10 Frequency was used to check for treatment-dependent differences in slopes. The PSD data were arranged into size classes (d i+1=1.26d i, d=upper diameter of class) according to Patterson et al. (1999). All data were converted into cumulative volume data assuming a sphere as basic shape. Inference statistics were done for cumulative

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volume percentages at 100 Am and 600 Am, respectively. Data were checked for homoscedasticity using Bartlett’s test (Sachs, 1997). In cases involving heteroscedasticity, Welch’s ANOVA was used, otherwise standard ANOVA was applied (Sachs, 1997). In the first case, post-hoc comparisons of treatment to control were done with sequentially Bonferroni-corrected Wilcoxon’s signed-rank tests (Rice, 1989), in the second case Dunnett’s control test was applied (Dunnett, 1955). The retention potential depending on binder treatment for dry matter, phosphorus and nitrogen was tested the same way. The correlations between stability and particle size were checked using reduced major axis regressions (Sokal and Rohlf, 2003). The restricted residual maximum likelihood method was used to fit the present models for random effects (Smyth and Verbyla, 1996). All descriptive statistics and linear regression analyses were calculated according to Sachs (1997). All data analyses were done with JMP (SAS Institute) Version 5.0.1.2 with the exception of the reduced major axis regressions (RMA), which were done with RMA Software version 1.14b.

3. Results 3.1. Digestibility, specific growth rate, feed conversion The fish grew at similar rates in both trials. In Trial 1 specific growth rate (SGR) was 1.13% day
1F 0.069% day
1 (average tank meanFS.D.), in Trial 2 it

Table 5 Effect of binder treatment on apparent digestibility coefficients (ADC) for protein, lipid, and phosphorus (meanFS.E.) Diet

Basal diet + Guar gum (0.3%) + Alginate (1.0%)

Trial 2 Protein ADC (n=12)

Lipid ADC (n=12)

Phosphorus ADC (n=12)

89.7%aF0.17% 89.1%aF0.23%

95.7%aF0.25% 94.6%aF0.45%

46.7%aF0.23% 51.5%aF0.90%

89.4%aF0.09%

95.9%aF0.13%

50.4%aF0.36%

Means within a column that do not share a common superscript letter are different ( pb0.05).

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was 1.11% day
1F0.082% day
1. Average feed conversion ratio in Trial 1 was 0.90F0.046 and in Trial 2 it was 0.73F0.026. The final weights of the last group of fish to be killed ranged from 257 to 292 g in Trial 1 and from 417 to 490 g in Trial 2. The fish developed normally and there were no visible signs of intestinal pathology that could have been attributed to the inclusion of dietary binder. At most two fish per treatment including the control showed slight intestinal irritations (rubor). During both trials three individual fish, all fed on different diets, showed signs of haemorrhagic enteritis. In Trial 1 digestibility of protein, lipid, and phosphorus was not affected by the binder addition with mean values of 87.0%F0.9% (FS.D.), 90.8%F

1.2%, and 50.7% F3.7%, respectively. This result was confirmed in Trial 2 (Table 5). The increase of yttrium oxide in faeces was 4.6fold F0.06. 3.2. Rheological measurements At least three replicates per treatment in Trial 1 and nine replicates in Trial 2 were measured. The data for the Guar gum (0.1%) treatment were discarded because of a control problem in the rheometer. The addition of binders improved the viscosity and the elastic modulus of fish faeces significantly (Fig. 2, Table 6). The absolute effect was similar in both trials but the relative effect was most pronounced in Trial 1,

Trial 1 Basal diet;

160

Guar gum (0.3%);

Alginbind (0.3%);

120

100

100

80

80

60

60

40

40

Viscosity

20

20 100

200

300

400

500

100

600 500

N=16

450

200

350

300

300

250

250

200

200

150

150

100

100

400

500

600

500

600

Elastic modulus

400

350

300

N=29

450

Elastic modulus

400 Elastic modulus [Pa]

Alginate (1.0%)

N=29

140

Viscosity

120

500

Alginbind (0.6%);

160

N=16

140 Viscosity [Pa*s]

Trial 2

50

50 100

200

300 Time [s]

400

500

600

100

200

300

400

Time [s]

Fig. 2. Viscosity and elastic modulus of trout faeces depending on binder inclusion (meanFaverage standard error of the mean). Note the remarkably fast decay of the elastic modulus of the faeces containing Alginate.

A. Brinker et al. / Aquaculture 249 (2005) 125–144

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Table 6 Adjusted mean values of viscosity and elastic modulus of fish faeces from trout fed the basal diet or the basal diet with binder treatment Diet

Trial 1

Trial 2

Viscosity Basal diet +Guar gum (0.3%) +Alginbind (0.3%) +Alginbind (0.6%) +Alginate (1.0%)

Elastic modulus

Mean

Impr. %

Mean

38.6a [Pa s] 109.3b [Pa s] 59.6c [Pa s] 72.4d [Pa s] 77.4d [Pa s]

– +183 +54 +88 +100

110.7a 252.5b 197.2c 235.2b 302.5d

[Pa] [Pa] [Pa] [Pa] [Pa]

Viscosity

Elastic modulus

Impr. %

Mean

Impr. %

Mean

Impr. %

– +128 +78 +112 +173

49.4a [Pa s] 118.3b [Pa s] – – 72.5c [Pa s]

– +140 – – +47

161.2a [Pa] 284.6b [Pa] – – 362.6c [Pa]

– +76 – – +125

Impr. (improvement): percentage improvement compared to basal diet. Means within a column that do not share a common superscript letter are different ( pb0.05).

where the basal diet led to less stable faeces than in Trial 2 (Fig. 2). Compared to the basal diet, the addition of Guar gum led to the highest increase of viscosity (Trial 1: 183%; Trial 2: 140%) and Alginate led to the highest increase of elastic modulus (Trial 1: 173%; Trial 2: 125%). If average improvements of both viscoelastic parameters are considered, Guar gum performed best (Trial 1: 155.5% Trial 2: 108.5%), followed by Alginate (Trial 1: 136% Trial 2: 86.5%). As expected, all viscoelastic functions decayed over time due to viscoelastic relaxation. This was most pronounced for the elastic modulus data of the Alginate treatment.

In Trial 1 the Alginbind treatment had two inclusion levels. Viscosity as well as elastic modulus increased significantly with increasing inclusion level (Table 6). The frequency sweep measurements show a clear shear thinning of all treatments (Fig. 3). These measurements were used to detect differences in the negative slopes of the linear fits because a lower negative slope indicates a higher structural resistance to mechanical stress. According to the statistical analysis (ANCOVA-model: pb0.00001, r 2adjusted= 0.982) no significant difference in the slopes ( pN0.25) was detected.

3.5 Basal diet;

Log10 viscosity [Pas]

3.0

n=10

Guar gum (0.3%); n=9 Alginate

2.5

(1.0%); n=10

2.0 1.5 1.0 0.5 0.0 -1.0

-0.5

0.0

0.5

1.0

1.5

2.0

Log10 frequency [Hz] Fig. 3. Frequency sweep data of the rheological measurements. Data points of Guar gum are displaced horizontally by 3% and of Alginate by 6%.

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3.3. Particle size distribution

show that in Trial 2 larger particles were more abundant than in Trial 1. This agrees with the smaller relative improvement in viscoelastic properties observed in Trial 2 (cf. Section 3.2). In both trials, the addition of Guar gum yielded the best particle size improvements. However, the results in Trial 2 were not entirely clear-cut because in the

All particle size distributions (PSD) from faeces of fish fed diets with binders showed a shift to larger particles (Fig. 4). The effect increased with the concentration of the respective binder (Fig. 4, Table 7). The PSD data for the control groups

BD AB1 AB2

Cumulative volume percentage

1.0 Basal diet (BD); n=8 Guar gum (GG1)(0.1%); n=7 Guar gum (GG2)(0.3%); n=7 Alginbind (AB1)(0.3%); n=7 Alginbind (AB2)(0.6%); n=9 Alginate (AT)(1.0%); n=6

0.8

0.6

AT GG1 GG2

0.4

0.2

Trial 1 0.0 8

16

32

64

128

256

512

Cumulatice volume percentage

1.0 Basal diet; n=16 Guar gum (0.3%); n=15 Alginate (1.0%); n=14

0.8

0.6

0.4

0.2

Trial 2 0.0 8

16

32

64

128

256

512

Particle size [µm] Fig. 4. Volume-dependent cumulative size distributions of suspended particles after breakdown by defined hydro-mechanical stress depending on binder inclusion (meanFS.E.).

A. Brinker et al. / Aquaculture 249 (2005) 125–144

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Table 7 Percentages of total particle volume below 100 Am and below 600 Am of suspended fish faeces particles originating from fish fed the same basal diet but with different binders Diet

Trial 1

Trial 2

Below 100 Am Basal diet + Guar gum (0.1%) + Guar gum (0.3%) + Alginbind (0.3%) + Alginbind (0.6%) + Alginate (1.0%)

Below 600 Am

Below 100 Am

Below 600 Am

Cum. %

Impr. %

Cum. %

Impr. %

Cum. %

Impr. %

Cum. %

Impr. %

38.8 27.8s 23.2s 36.3ns 35.1ns 26.9s

–
28.3
40.2
6.4
9.5
30.6

92.0 75.2s 69.4s 90.4ns 89.1ns 79.8s

–
18.3
24.6
1.7
3.2
13.3

35.1 – 28.7s – – 26.9s

– –
18.2 – –
23.3

93.4 – 79.7s – – 90.7s

– –
14.7 – –
2.9

Impr. (improvement): percentage improvement compared to the control (basal diet without binder) calculated with respect to remaining waste load after filtration at 100 Am and 600 Am. Cumulative means within a column marked by the superscript s show significant lower values ( pb0.05) compared to basal diet whereas superscript ns does not.

range below 128 Am Alginate performed better. Generally, the effect of the binder was more pronounced with increasing particle size (Fig. 4, Table 7). The difference in cumulative particle sizes between the basal diet and the Guar gum (0.3%) treatment was 15.6% (Trial 2: 6.4%) at 100 Am and 22.6% (Trial 2: 13.7%) at 600 Am. In Trial 2 the functions of Alginate- and Guar gum-derived PSDs crossed. In Trial 1, by contrast, the respective functions developed in parallel. Table 7 shows the percentage of total particle volume below 100 Am and 600 Am for each treatment. Guar gum and Alginate led to a much greater shift towards large particles than Alginbind. From the filtration potential associated with a suspension of these size characteristics, the effect of particle enlargement on the remaining effluent load can be calculated by the ratio of binder treatment to control: the addition of Guar gum (0.3%) decreases the remaining effluent load after removal at 100 Am by 40.2% (Trial 2: 18.2%) and at 600 Am by 24.6% (Trial 2: 14.7%), whereas in case of Alginate (1.0%) the remaining load decreases for 100 Am by 30.6% (Trial 2: 23.3%) and for 600 Am by 13.3% (Trial 2: 2.9%) (Table 7). The rheological data correlate significantly with the observed PSDs. Plots of the adjusted means (population marginal means) of viscosity, elastic modulus, and their merged values against the cumulative percentage at 100 Am revealed a significant negative linear correlation in all three cases. The

average of both improvement data shows the best correlation (r 2=0.734; pb0.01) compared to viscosity (r 2=0.613; pb0.05) or elastic modulus (r 2=0.632; pb0.05) alone. 3.4. Effect of particle size on leaching process Leaching experiments with faecal suspensions from an earlier trial showed a significant increase of nitrogen and phosphorus content with increasing particle size (Fig. 5), thereby indicating a higher retention potential associated with larger particles. Investigation of binder-induced leaching effects requires faeces with comparable properties (apart from random variation in dry weight and nutrient content). For each binder treatment, the dry matter content of faeces from 75 trout per tank was determined in duplicate. Dry matter content for the control was 11.4%F0.2% (meanFS.E.), for the Guar gum (0.3%) treatment 11.6%F0.3%, and for the Alginate (1.0%) treatment 10.9%F0.1% with no statistically significant difference (ANOVA, pN0.1). As previously shown, there were no apparent differences in nutrient digestibility (cf. Section 3.1). Samples of 3 g faeces—15 for control and Guar gum (0.3%), 16 for Alginate (1.0%)—were allowed to leach for 1 h. The recovered solids showed no significant difference in nitrogen or phosphorus content (Table 8). A significantly higher dry matter (+5%) and particulate phosphorus (+15%) content compared to the basal diet was observed for faeces

18 N=125

17

Nitrogen

16 15 14 13 12 11

Y = 10.8 + 0.0087 X

10

2

R = 0.946; p<0.0001

9 0

100

200

300

400

500

600

Lower particle size class [µm]

Particulate phosphorus [µg PO4-P/mg SS]

A. Brinker et al. / Aquaculture 249 (2005) 125–144

Particulate organic nitrogen [µg NH4-N/mg SS]

138

23 22 21 20 19 18 17 16 15 14 13 12

N=125

Phosphorus

Y = 14.3 + 0.013 X 2

R = 0.625; p<0.05 0

100

200

300

400

500

600

Lower particle size class [µm]

Fig. 5. Particulate organic nitrogen and phosphorus content in suspended solids (SS) with increasing particle size after leaching of 1 h (meanFS.E.).

from the Guar gum treatment. The Alginate treatment did not differ from the control.

4. Discussion Most of the current knowledge of dietary binder effects in fish originates from investigations on wet feed where binders were used in high concentrations to stabilise the pellets (Storebakken, 1985; Spyridakis et al., 1989; Morales et al., 1991; Robinson, 1998). Meanwhile, due to an increased preference for extruded feeds binders have widely lost their former importance for salmonid diets. 4.1. Digestibility, specific growth rate, feed conversion The composition of the basal experimental diet was comparable to that of modern commercial trout feeds. It led to fast growth and a good feed conversion. In

both trials SGR was nearly the same, whereas feed conversion ratio was somewhat lower in Trial 2. This difference may be due to some feed losses, which became obvious during hand feeding in Trial 1. These feed losses were probably compensated for SGR in Trial 1 by the generally faster growth rate of smaller trout (Guillaume et al., 2001). Neither of the experimental trials were designed to test for SGR or FCR depending on diet and thus they did not meet the essential requirements for this purpose (Ruohonen, 1998). However, Storebakken (1985) found no negative effects on SGR or FCR using distinctly higher binder concentrations (2.5% of Guar gum or Alginate per dry weight of feed). In this study no intestinal disorders or malnutrition occurred. This was as expected because of the very low binder concentrations. Binders are considered potentially harmful to fish, but tangible evidence is scarce (Morales et al., 1991; Tacon, 1992). Alginate administered in large quantities (up to 10%) caused stomach wall changes in rainbow trout (Storebakken,

Table 8 Average values of recovered total dry matter (DM), particulate organic nitrogen (N), and particulate phosphorus (P) after suspending 3 g trout faeces samples for 1 h in distilled water depending on treatment (meanFS.E.) Diet

DM [mg]

Impr. %

NH4–N [mg]

Impr. %

PO4–P [mg]

Impr. %

Basal diet; n=15 + Guar gum (0.3%); n=15 + Alginate (1.0%); n=16

236.9F4.4 249.5sF3.3 238.0nsF3.0

– +5.1 +0.8

6.452F0.205 6.324nsF0.328 5.524nsF0.184

–
1.9
14.4

7.717F0.265 8.863sF0.353 7.872nsF0.219

– +14.9 +2.0

%P

%N

3.3%F0.89 3.6%nsF0.76 3.3%nsF0.12

2.7%F0.94 2.5%nsF0.76 2.3%nsF0.12

Values within a column marked by the superscript s show significant higher values ( pb0.05) compared to basal diet whereas superscript ns did not.

A. Brinker et al. / Aquaculture 249 (2005) 125–144

1985) and Guar gum induced slight pathological effects including intestinal damage in poultry (Anderson and Warnick, 1964; Maisonnier et al., 2002). Otherwise, non-starch polysaccharides are known to have health benefits in man and animals (Johnson, 1990). In fish, Alginate stimulates the immune system of salmon Salmo salar (Nordmo et al., 1995; Gabrielsen and Austreng, 1998). This study revealed no negative effect of binder inclusion on nutrient digestibilit. However, inappropriate concentrations could negatively affect digestion. Negative effects on macronutrient digestibility have been observed in fish, including rainbow trout, when binders comprising Guar gum and Alginate were added to the diet in high concentrations (Storebakken, 1985; Storebakken and Austreng, 1987; USDA/CSREES, 1997; Carre´, 2003). The effect was most pronounced for lipids, less pronounced for proteins, and nearly undetectable for carbohydrates. This is in agreement with investigations using broilers (Maisonnier et al., 2001) and dogs (Meyer and Doty, 1988). According to Storebakken (1985), Meyer and Doty (1988), Iji et al. (2001), and Maisonnier et al. (2001, 2002, 2003), anti-nutritional consequences in fish, broilers, and dogs are mainly due to physical effects, such as changes in viscosity and agglomeration of chyme components. Inhibition of enzymes and selective binding cannot be precluded, but were regarded as negligible by previous authors (Storebakken, 1985; Meyer and Doty, 1988; Iji et al., 2001; Maisonnier et al., 2001). Increases in viscosity and agglomeration lead to entrapment or enlargement of chyme particles including micelles, thereby inhibiting hydrolysis (Meyer and Doty, 1988). The same changes also increase the thickness of the progressively diminished stirring water layers surrounding the intestinal villi, thereby hampering absorption processes by the enterocytes (Johnson and Gee, 1981). Furthermore, they slow the transit of fluid through the alimentary canal and speed up the relative transit of particles, thereby reducing the time chyme particles are exposed to digestive enzymes (Amidon, 1985). The consequences for size and diffusion coefficients are mutually enhancing. The diffusion of small nutritive components like glucose, dipeptides and proteins is hampered, and that of the large micelles to the mucosa is seriously impaired, which means nutrient absorption is reduced (Phillips, 1986;

139

Meyer and Doty, 1988; Lund et al., 1989). In rats, reduced intestinal absorption decreases the pool of available bile salts because bile salt recovery is lowered and those remaining in the gut are further diluted by the increased water content of the chyme (Lund et al., 1989). This mechanism explains the strong effect of binders on the digestibility of lipids by fish as bile salts in fish not only act as emulsifiers but also activate the lipase–colipase complex (Guillaume et al., 2001). These considerations were a major factor in our strict control of binder concentrations to levels that impart high viscosity to the chyme only in the very distal intestine after digestion is finished (cf. 1.1). 4.2. Rheological measurements In the preliminary trials, all binders selected for the present investigation stabilised the faeces of rainbow trout at the macroscopic level. These findings were confirmed by the present study. The addition of all binders significantly increased the viscosity and the elastic modulus of the faeces. Guar gum in particular enhanced the viscosity by 183% (Trial 2: 140%) compared to the control. The observed improvement in elastic modulus was much lower, but this is not surprising as Guar gum forms only synergistic interactions with other hydrocolloids and possesses no true gelation properties (Fox, 1992). In contrast to Guar gum, the alginic acid based binders enhanced the elastic modulus more than viscosity. Alginate, however, had a much greater effect on the elasticity than Alginbind. Obviously, the increased availability of divalent cations—especially Ca2+—in Alginbind was not sufficient to compensate for the missing alginic acid. Hence, Alginbind was omitted from the second trial. In relative but not in absolute terms in Trial 2 the stability improvements due to the binders were slightly lower than those in Trial 1. Similarly, the faeces of the control group in Trial 2 were more stable than in the first trial. This is traced back to the fact that the lower the stability of the basic matter, the more pronounced the same binder improvements are. The reason for the higher faeces stability of the control in Trial 2 is unclear; it is conceivable that fish size significantly influences the consistency of the faeces. The trout in Trial 2 were distinctly larger than those in Trial 1 and larger fish may produce more stable faeces.

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With time and repetitive deformations, the viscosity and elastic modulus values of each binder treatment decay. The reduction in viscosity was relatively minor and more or less parallel for all treatments, but the decay in the elastic modulus values of the Alginate treatment was much more pronounced. In an aquaculture facility, faeces are continuously exposed to mechanical stress due to water turbulences, for example, those caused by moving fish or technical installations (Brinker and Ro¨sch, in press). Therefore, the slight decrease in stability observed with repeated deformations of the Guar gum treated faeces has important implications. According to Morris et al. (1981) the rheological properties of Guar gum solutions are very stable as the binder leads to exceptional structural integrity and shows continuous re-entanglement. The picture is different for the Alginate-bound faeces. Here the polysaccharide network appears to disintegrate more substantially with each deformation step. The initially promising high values of the elastic modulus decrease rapidly, indicating that recurrent hydrodynamic stress will break these structures. Thus, improvements due to the inclusion of Alginate may be lost relatively fast in a real-life situation. The negative slopes of the frequency sweeps of Trial 2 indicate shear thinning, i.e. a breakdown of the polysaccharide network due to increasing shear stress. The polysaccharides are disentangled and aligned parallel to the direction of the applied shear stress (Onsoyen et al., 1992). Because the slope obtained for the control group was similar, there is no evidence that the addition of binder had any effect on structural resistance. A comprehensive comparison indicated that concentrated polysaccharide solutions all show essentially the same shear thinning profile irrespective of chemical type, molecular weight, solvent environment, or concentration (Morris et al., 1981). Nevertheless it is somewhat surprising that compared to the control, the binder treatment did not increase the structural resistance. It is likely that water-soluble non-starch polysaccharides already present in the basal diet act as a kind of polymeric binder themselves and dominate the shear thinning profile of faeces, irrespective of the inclusion of special polysaccharide binders (Carre´, 2003). As the binders are indigestible, the accumulation of indigestible yttrium oxide (factor of 4.6) and the

moisture content of the faecal pellets (about 89%) can be used to calculate the final concentrations of binders. For Guar gum this was 0.2% and for Alginate it was 0.5%. As intended, these concentrations were within the range of the threshold values for useful binding effects whereas the corresponding values for the alginic acid of Alginbind (0.3% or 0.6%, respectively) were distinctly below its threshold binding level (cf. 1.1). 4.3. Particle size distribution The presence of dietary binder should result in faeces more resistant to turbulence and thus in larger particles. This hypothesis was supported by the size distributions observed in the current study. When unstabilised (control) and stabilised faeces (binder treatment) were exposed to hydro-mechanical stress, the latter retained significantly larger particles. With respect to shear forces, adhesive, flexible, and cohesive or non-brittle materials are most resistant. In rheological terms viscosity covers for adhesiveness, elastic modulus for flexibility and both properties influence cohesion. Viscosity, the elastic modulus, and the merged values of both parameters were all found to correlate significantly with enlargement of particle size. Following the break-up of faeces with a defined shear stress, binder treated faeces retained higher percentages of suspended solids larger than 100 Am. Nevertheless, the merged values showed the highest correlation and significance. This shows that considerable collinearity between both rheological parameters exists and that each parameter contributes to the overall shear resistance. All binders effected some improvement in faecal stability and shifted the particle size distribution (PSD) to larger particles, but the shift was only statistically significant for Alginate and Guar gum treatments. As with the stability measurements, the effects of binders on particle size increased with concentration. With respect to both rheological parameters the Guar gum treatment performed best and inclusion of Guar gum led to the greatest improvement in particle sizes. In accordance with the stability data, in Trial 2 the particles of the control remained larger than those of the control in Trial 1. This outcome reflects the dependence of the shear resistance and thereby of the particle size on faecal stability. In consequence,

A. Brinker et al. / Aquaculture 249 (2005) 125–144

despite the same absolute stability improvements in both trials the binder-induced particle size improvements were somewhat smaller in Trial 2. With respect to particle size distribution relative improvements are more important than absolute ones. From the smallest to the largest size class the binder-induced consequences on cumulative particle volume increased continuously. This suggests that along the size spectrum the binder-induced enhanced resistance to shear stress is more or less the same. The only exception to this general rule was the crossing of the cumulative PSD of the Alginate and Guar gum treatments in Trial 2. This may be related to flocculation of organic particles, which is commonly induced by Alginate (Sobeck and Higgins, 2002). Flocculation is thought to be positively correlated with solid concentration due to the increased frequency of particle collision (Droppo et al., 1997). In Trial 2 the solid load of faecal suspension was 50% greater than in Trial 1. The increase was in response to the fact that the larger trout of this trial produced more faeces, and the precision of PSD measurement improves with the number of sized particles (Brinker et al., 2005). The increased encounter probability, especially that of the small most abundant particles, and the concurrent agglomeration of these particles can explain the superior performance of Alginate over Guar gum in Trial 2 for sizes below 128 Am. Prima facie the effect of the binders is most pronounced for large particles. In Trial 1 for example, at 600 Am the cumulative volume difference between control and Guar gum treatment is 22.6 basal points, whereas at 100 Am this difference is reduced to 15.6 basal points. The removal of large particles not only reduces overall solids considerably, it also eliminates an important source of potentially leachable material. Thanks to the binders, it remains bound up inside large particles, and its exposure to water in which it might be dissolved and becomes unrecoverable is minimized, thus significantly improving the water quality and optimising mechanical waste removal. Using the Guar gum data, the improvement for suspended solids was calculated as 40.2% in Trial 1 and 18.2% in Trial 2 following treatment by a 100 Am drum filter. Such a calculation is justified as in a previous investigation the laser-based PSDs had been very precise in forecasting the real removal efficiency of drum filtration (Brinker and Ro¨sch, in press).

141

Reduced filter gauze openings theoretically allow the removal of smaller solids and could therefore lead to similar improvements of removal efficiency, but unfortunately this would also increase investment and operational costs exponentially because of increased hydraulic loading and backwash requirements (Cripps, 1995; Summerfelt, 1999). The same applies to improvements in sedimentation and hydraulic residence times (Henderson and Bromage, 1988; Bergheim et al., 1998; Engle and Valderrama, 2003). In the field, other factors such as biological degradation, flocculation, or differences in discharge properties of the suspended particles may complicate the picture. A verification and quantification of the present results under commercial conditions will be presented in a future publication. With respect to current market prices and effectiveness Guar gum is by far the most economic binder option. 4.4. Effect of particle size on leaching Solids in fish farm effluents readily release nutrients and organic compounds into the water (Windell et al., 1978; Alsted, 1989; Chen et al., 2003). Leaching is mainly caused by physicochemical processes, which are controlled by water temperature, osmotic conditions, and the liquid–solid contact area (Phillips et al., 1993; van Rijn and Nussinovitch, 1997; Chen et al., 2003). For technical reasons water temperature and osmotic conditions can hardly be modified in flow-through aquaculture, but the liquid–solid contact area can be altered by simply changing the size of the suspended solids. The liquid–solid contact area (and thus the potential for leaching) decreases exponentially with increasing particle size. Hence, enlarging the suspended solids seems to be a promising way to slow leaching effectively. The results of a preliminary experiment supported this hypothesis. There was a significant positive correlation between the particle size classes and their respective nutrient concentrations after a defined exposure to leaching. Assuming the nutrients were randomly distributed within the faeces, the outcome can only be explained by an improved leaching resistance with increasing particle size. Almost no information on nutrient distribution within size

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classes of faecal particles is available. It has been suggested that nutrient levels in certain particle sizes could be elevated as a consequence of their dietary origin. However, Cripps (1995) found no evidence of this in a study of size-fractionated particles from the effluent of a trout farm. In this work the effect of particle enlargement on nutrient retention was analysed quantitatively. The leaching time of 1 h corresponds to a typical residence time of water in German flow-through fish farms. The faeces used for controls and treatments were comparable, with the same dry weights and according to digestibility studies, with nutrient contents that varied only by chance. Compared to the control, only the Guar gum treatment significantly enhanced phosphorus retention by about 15% and dry matter recovery by about 5%. No significant improvement in nitrogen recovery occurred. This is surprising as in the preliminary trial, nitrogen levels appeared to be more closely correlated with particle size than phosphorus. It might be possible that prior to leaching the nitrogen content in faeces from the control was elevated. This is supported by the distinctly lower nitrogen recovery from the Alginate treatment compared to the control. There is no obvious reason why a binder should increase leaching. The nutrient concentration independent of treatment did not differ significantly in the recovered faecal material. This is not surprising as for Guar gum the improved phosphorus retention was combined with higher overall recovery of organic matter, which masked the positive effect on phosphorus retention.

5. Summary The results obtained in this study provide strong evidence that the addition of dietary binders to fish feed significantly enhances the stability of fish faeces thus favouring the formation of large waste particles with high mechanical removal potential and a considerably improved leaching resistance. Both Alginate and Guar gum were capable of exerting this effect in a dose-dependent manner. The positive effects occurred without negative side effects on the health of the fish and without affecting the digestibility of macronutrients.

Acknowledgements The authors thank A. Roem for providing technical experience. The help of M. Rimbach, Z. Gregus, and G. Ritchie is greatly appreciated. We thank Ch. Friedrich and U. Jacobs for their support in the field of rheology. The paper benefited greatly from the comments of R. Eckmann and K. Brinker.

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