The integrated culture of seaweed, abalone, fish and clams in modular intensive land-based systems: II. Performance and nitrogen partitioning within an abalone (Haliotis tuberculata) and macroalgae culture system

Aquacultural Engineering 17 (1998) 215 – 239

The integrated culture of seaweed, abalone, fish and clams in modular intensive land-based systems: II. Performance and nitrogen partitioning within an abalone (Haliotis tuberculata) and macroalgae culture system Amir Neori a,*, Norman L.C. Ragg b, Muki Shpigel a a

Israel Oceanographic and Limnological Research, National Center for Mariculture, P.O. Box 1212, Eilat 88112, Israel b Department of Zoology, Uni6ersity of Canterbury, Pri6ate Bag 4800, Christchurch, New Zealand Received 12 December 1996; accepted 7 September 1997

Abstract A pilot-scale system for the intensive land-based culture of abalone was established using an integrated design aimed at eliminating the dependence on external food sources, whilst reducing water requirements and nutrient discharge levels. The system was the first and simplest trial in a series of progressive complexity of the concept of integrated culture of seaweed, abalone, fish and clams in modular and intensive land-based facilities. Relative sizes of the modules, their stocking densities and the rate of nutrient supply were determined based on earlier results to be optimal. Effluents from two abalone (Haliotis tuberculata) culture tanks drained into macroalgae (Ul6a lactuca or Gracilaria conferta) culture and biofilter tanks, where nitrogenous waste products contributed to the nutrition of the algae; net algal production from each algal tank was harvested and used to provide a mixed diet for the abalone. Excess algal yield was used elsewhere. The system was monitored to assess productivity and nitrogen partitioning over a year, while improvements were made based on the accumulating results. Total annual N-budgets were combined with mean production figures to determine a suitable ratio of abalone biomass to algal culture vessel productivity, towards commercial application of the concept. The abalone grew on average 0.26% and 0.25% body weight/d in the two culture tanks; reduced growth and increased food conversion ratios (food eaten/biomass gain; w/w) were associated with high summer water temperatures (max. 26.9°C). U. lactuca showed reliable growth and filtration performance (mean production of 230 g fresh weight/m2/d, removing on average 58% of nitrogen supplied). Conversely, G. conferta growth was highly erratic and was deemed unsuitable for the current application.

* Corresponding author. Tel: + 972 7 6361445/25; fax: +972 7 6375761; e-mail: [email protected] 0144-8609/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0144-8609(98)00017-X

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It is estimated that 1 kg of abalone biomass would require food supplied by 0.3 m2 of U. lactuca culture, reducing N inputs required by 20% and N in effluent by 34% when compared to the two organisms grown in monoculture. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Seaweed; Abalone; Nitrogen recycling; Modular intensive land-based system

1. Introduction Over exploitation by heavy artesanal fishing of the European abalone (or Ormer) Haliotis tuberculata in the northern limits of its range, the British Channel Islands and the French Brittany coast, and the subsequent depletion in natural stocks during the second half of the twentieth century have resulted in increasingly strict fishery legislation and reduced landings (Mgaya and Mercer, 1994). The continued demand for abalone can not be met by the fishery. Therefore the feasibility of culturing abalone has received some attention within the regions that previously supported its main fisheries (Hayashi, 1982; Hahn, 1989; Mgaya, 1995). The high value of H. tuberculata led to its introduction as a mariculture species into Ireland in 1976 (Mgaya and Mercer, 1994), and in 1993 at land-based facilities of the Israeli National Center for Mariculture (NCM), on the Gulf of Aqaba, Red Sea (Shpigel and Neori, 1996; Shpigel et al., 1996). The development of commercial abalone culture is frequently limited by the need to acquire sufficient quantities of suitable dietary seaweed. Natural populations of brown or red algae are usually required, which are often in short supply (Mercer et al., 1993). However, large quantities of the ubiquitous chlorophyte Ul6a lactuca L. can be produced in seaweed culture systems, which serve as biofilters and are associated with intensive seawater fishponds (Neori et al., 1996). Ul6a sp. biofilters have been successfully integrated into a number of other experimental and commercial mariculture systems, efficiently removing dissolved inorganic nitrogen from the effluent water (Ryther et al., 1975; Tenore, 1976; Vandermeulen and Gordin, 1990; Hirata and Kohirata, 1993). While reported as being effective in its application as a biofilter, particularly in land-based systems, the produced biomass of Ul6a sp. has been of limited commercial value (Kissil et al., 1992; Arieli et al., 1993). The valuable rhodophyte Gracilaria sp. has also been cultured in mariculture biofilters in Eilat and elsewhere (Ryther et al., 1975; Neori, 1991; Buschmann et al., 1994). One proposed way of increasing the economic viability of seaweed biofilters has been to feed the biomass produced to commercially valuable macroalgivores, particularly abalone (Tenore, 1976). H. tuberculata was introduced for this purpose in Eilat (Shpigel et al., 1996). Subsequent feeding trials revealed that H. tuberculata displayed improved growth performance when fed a diet of Ul6a sp. supplemented with Gracilaria conferta, compared to monospecific diets of either alga (Shpigel, 1995). A novel bioengineering concept of a self-sustaining, self-cleaning, modular, integrated, land-based abalone and seaweed culture unit was first outlined by

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Shpigel and Neori (1996), as the simplest of several combinations of progressive complexity. The proposed pilot-scale, two-organism, abalone-seaweed system was subsequently constructed at the NCM as a first step toward ultimately developing a polyculture system for four organisms (seaweed, abalone, fish and clams). The performance of the simple two-organism system, with regard to abalone growth parameters and nutrient regimes, is described in the present report. Inorganic nitrogen is the main nutrient that, when added to pristine coastal seas, causes marine eutrophication (McCarthy, 1980). Furthermore, the most costly component of diets used in aquaculture is protein, which also is a major determinant of the nutritional value in diets of the abalone H. tuberculata (Mai et al., 1994). Recycling of nitrogen and reduction of its release to the environment are major anticipated benefits of the proposed polyculture concept. N-budget was, therefore, selected as the optimal measurement criterion for this aspect of the experimental system.

2. Materials and methods

2.1. Organisms Haliotis tuberculata were introduced into land-based facilities at the NCM from Guernsey, UK, in 1993 (Shpigel et al., 1996). Ul6a lactuca L. was produced from vegetative thalli isolated from the Red Sea and cultured in biofilters (Vandermeulen and Gordin, 1990). Gracilaria conferta cultured in the second biofilter had been collected on the Mediterranean coast of Israel (Levy and Friedlander, 1990).

2.2. System design A two-organism (seaweed and abalone) culture system was built as diagrammed in Shpigel and Neori (1996). The experimental system was designed to allow an evaluation of the biological-chemical practicality of the abalone-seaweed integrated culture concept, not specifically to maximize performance. Therefore, a large margin of error was allowed, by using low stocking densities of the animals and relatively large seaweed biofilters. The sizes of the abalone and the seaweed culture vessels were adjusted with the following considerations: 1. A low abalone density (far below the 35 kg/m3 found practical in our regular abalone culture systems); 2. Maximal consumption of seaweed expected, with a food conversion ratio (FCR) of 25 kg fresh seaweed per 1 kg of abalone growth (Shpigel et al., 1996); 3. Minimal seaweed productivity expected (only 0.5 kg fresh weight m − 2 week − 1; Friedlander et al., 1987, 1991; Neori, 1991; Neori et al., 1991, 1996). Abalone were cultured in two similar bottom-draining square 600-l PVC tanks (labelled A and B) of 1.0-m side length. The tanks were elevated, allowing their effluents to drain into the seaweed biofilters described below. A removable screen (1-cm mesh) covered the whole area 10 cm above the flat bottom, and retained the

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abalone while allowing faeces and detritus to drain from the tank; water was drained via a removable, 40-mm ID, 60-cm tall stand-pipe, fitted to a hole at the bottom and covered by a 5-mm mesh at the top. Two perforated horizontal aeration tubes on the bottom below the screen kept food algae in suspension. Eight 160-mm diameter PVC half-pipes were stacked on the screen and provided, combined with the tank walls, approximately 5.2 m2 of wet surface area available for abalone attachment. The abalone tanks were each initially stocked with 235 H. tuberculata of 30 – 60-mm shell length (4.1–32.6 g wet weight) with a total biomass of 2.2 kg (Table 1). Two seaweed biofilters, identical to those reported in Neori et al., (1996), were installed, one stocked with U. lactuca and the other with G. conferta. They were made of round-bottom elongated (3× 1.1 m) fiberglass tanks, bottom-aerated and with a useable water volume of approximately 1500 l. U. lactuca and G. conferta were stocked at 1.5 kg and 12.0 kg m − 2 (fresh weight) respectively, following the recommendations of Neori (1991) and Neori et al. (1991). The only nutrient source for the whole system was mineral fertilizer (solutions of ammonium sulfate and disodium phosphate), supplied directly to both biofilters by continuous dripping. The N supply rate was initially about 5.6 g N m − 2 day − 1. This ammonia-N flux was estimated to provide an optimal combination of good seaweed growth, about 50% removal of ammonia-N and a moderate N-content in the seaweed (Cohen and Neori, 1991; Neori et al., 1991). From July 1995 onward ammonia-N was supplied to the seaweed at only about 4.0 g m − 2 day − 1, in an effort to improve the fraction of N removed by the biofilters. The influx of orthophosphate was maintained throughout the year at 0.6 g P m − 2 day − 1. In addition, the U. lactuca biofilter received the entire effluents from abalone tank A and the G. conferta biofilter from abalone tank B.

2.3. System monitoring The integrated culture system was monitored for 1 year, beginning in March 1995.

2.3.1. Abalone At the time of initial stocking, seventy-five animals in each tank were randomly tagged. They were wet weighed (after inverting each animal on absorbent paper to remove excess water from the mantle cavity) and shell length measured at 2–3month intervals. Length is not a particularly useful growth parameter in abalone. It was measured and presented below only to allow the readers comparison with previous studies were weight was not used. The tagged abalone were assumed to constitute a sub-sample representative of the tank population. Wet weight figures were used to calculate for each time interval specific growth rates (SGR%; Eq. (1)), the percent body weight gain per day (Shpigel et al., 1996). SGR% = 100 ×([ ln {Wt} − ln {W0}]/t)

(1)

May 1995 100 0.47

286

392

April 1995 235 2.30

4.62

1067

2.67

787

Animals were added before sampling dates.

Abalone restocking Date Individuals Kilograms

Abalone Standing stock (kg) Growth (mg N/tank/d)

Water flow rate (l h−1)

January 1996

March 1996

June 1995 80 0.93

170

5.16

833

October 1995 591 4.52

517

8.34

510

332

8.96

525

April 1995 235 2.19

150

2.28

520

April 1995

August 1995

April 1995

June 1995

Tank B

Tank A

Sampling date

Table 1 Basic information on the abalone culture tanks and seaweed fresh-weight yield in two biofilter tanks

May 1995 100 0.48

203

4.06

1075

June 1995

June 1995 80 0.77

315

8.5

531

January 1996

October 1995 276 2.17

552

8.91

508

March 1996

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where W0 is the wet weight of an animal at the beginning of each monitoring interval and Wt is the weight after t days of growth, at the end of the interval. The mean daily net biomass gain of the abalone (DB) in each tank was estimated for each day of intensive sampling by Eq. (2). DB =(B0 ×SGR%/100) − Mw

(g/d, fw)

(2)

Where B0 is total biomass at the start of the day and Mw the weight of dead animals removed (dead animals were removed on a weekly basis and their shell lengths used to predict equivalent live weight using linear regression equations fitted to the loge length:loge weight relationship in the tagged animals). On three occasions during the observation period (in May, June and October 1995), based on the observation that U. lactuca production (Table 2) by far exceeded consumption by the existing stock, and to offset the mortalities, additional abalone were stocked (Table 1); B0 was corrected accordingly.

2.3.2. Algal production and abalone feeding Abalone in both tanks were fed a mixture of fresh seaweed from the biofilters of both systems. The seaweed was fed to the abalone in considerable excess (1–2 times the weight of corresponding abalone biomass, maintained by supplying additional seaweed every 2 – 3 d), to provide good shade for the animals as well as to ensure that food was continuously available. The experiment was interrupted for 2 months following the November 22nd 1995 earthquake in the Gulf of Aqaba; however, the animals were fed seaweed from other tanks and the monitoring of the animals continued. Table 2 Seaweed fresh-weight yield (g/m2/d) in two biofilter tanks in 1995 and 1996 Date

Ul6a lactuca

Gracilaria conferta

Late April 1995 Early May 1995 May-June 1995 Late June 1995 Early June –late July 1995 Early July 1995 Late July 1995 Late August 1995 September-October 1995 Late October 1995 October-November 1995 Mid-November 1995 Mid-January 1996 Late January 1996 Early February 1996 February-March 1996 Mid-March 1996 Late March 1996

210 403 286 412 — 194 295 303 284 196 155 168 81 52 104 85 95 402

−46 261 268 — 170 — — −57 −34 — 14 −330 −39 11 197 −136 — —

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Biofilters were drained every 2 weeks, the seaweed weighed and re-stocked at the original weight. The net yield of algae, including weight of algae harvested for feeding, was used to calculate the mean daily seaweed production for each 2-week period. The abalone tanks were drained weekly by removing the stand pipe; the debris (assessed visually to be of negligible quantity) that settled to the space between the bottom and the screen above was flushed and cleaned from the bottom; dead animals were removed and their shell length measured; uneaten algae were removed and weighed; the quantity of algae ingested was determined by difference from the total weight fed. Ingestion rate was estimated by dividing the seaweed weight, ingested during the time interval between two weighings, and the mean abalone biomass for that interval (estimated using SGR% and initial abalone biomass measurements for that interval). Food conversion ratio (FCR) was determined by the fresh weight of ingested seaweed divided by the abalone biomass gain over the same time interval. Algal growth inside the abalone tanks, and consequent possible nutrient recycling in situ, was not assessed, hence the estimates of ingestion rate and food conversion ratio are considered ‘apparent’. Annual values of SGR% and FCR were calculated by the sums of ingested seaweed and abalone growth, adjusted for mortalities and animal stocking.

2.3.3. Nutrient analyses Sixteen individuals of H. tuberculata grown under conditions similar to those described above were sampled at 3-month intervals during 1994; individual wet weight was measured, the animals were then rinsed in fresh water and freeze-dried. Each dry abalone was homogenized in a mill and its total nitrogen content was determined by the Kjeldahl method. Fifteen samples of both seaweed species were taken between April 1994 and April 1996 and treated in the same way as the abalone samples. The mean nitrogen content of the animals and DB were used to estimate the amount of nitrogen incorporated into abalone tissue during any specific 24-h period. Similarly, nitrogen incorporation into algal tissue was determined as the mean tissue nitrogen content× mean daily production. The form, quantity and diurnal fluctuation of nitrogen and phosphorous flowing into and out of each tank were determined at approximately 2-month intervals by intensive sampling days from one morning (08:30) to the next (P was analyzed in only about half of the days). Each such day was divided to four 6-h sampling periods. Each water sample from a tank was collected during the entire 6-h period in a separate covered 20-l PVC container via a drip siphon from the effluent stand-pipe. The collected water, typically 5 l, was sub-sampled for analysis. All sampling and storage vessels for the sampling were pre-soaked for 24 h in 1.0 N HCl and then rinsed well with de-ionized water. Total nitrogen (TN) and total phosphorous (TP) were analyzed by a Technicon Autoanalyzer II using standard methods and following the modified persulphate oxidation procedure as described by Neori et al. (1996). Total dissolved nitrogen

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and phosphorous (TDN and TDP) were analyzed similarly, only following filtration through acid-washed Whatman GF/C filters. Sub-samples of these filtrates were deep-frozen, for subsequent determination of the mineral forms of N (DIN) and P by standard Autoanalyzer methods (Krom et al., 1985). Suspended particulate nitrogen (PN) was determined by the difference between TN and TDN, and dissolved organic nitrogen (DON) was estimated by subtracting DIN from TDN. Flowmeter readings were used to determine mean flow rate of water through the tanks during each sample day (Table 1). Water flow and nutrient concentration were used to determine the overall rate of input and output of nutrients for each tank and biofilter. Net abalone production of N and P and their net uptake by the biofilters were calculated as the difference between absolute levels entering and leaving each tank. Complete system nitrogen budgets were constructed for each 24-h sampling period with the absolute nitrogen quantities, according to the scheme in Fig. 1. Nitrogen partitioning was also standardized by biofilter water surface area or abalone biomass to give comparable nitrogen budgets for the biofilters and abalone tanks separately.

3. Results

3.1. Temperature The greatest diurnal variations experienced by the abalone, up to 5.5°C, occurred during the spring (Fig. 2). Annual temperature extremes were 16.0°C and 26.9°C. The algal biofilters, being downstream and with a longer residence time, experienced larger diurnal temperature variations all year round.

3.2. Abalone performance Initial abalone growth rates differed between the two tanks, but converged during April – May 1995 to subsequently follow similar patterns. The best growth (both SGR% and shell length) occurred in the spring, and it progressively declined towards an autumn minimum (Fig. 3). By February 1996 growth rate had begun to increase. Over 1 year, the tagged animals grew with an average SGR% of 0.262 9 0.033%/d (n =28) in tank A and 0.2519 0.037%/d (n= 31) in tank B. FCR values (Fig. 4) were low (i.e. efficient) and relatively stable during the spring, but increased (i.e. became less efficient) in summer to maxima in October of 4093.3 in tank A and 29 92.5 in tank B. Annual FCRs of 20 and 17 were calculated for the two abalone tanks, using the overall growth (corrected for mortalities and animal restocking) and seaweed ingestion values. Abalone mortality (Fig. 5) was high following initial stocking of the systems, with up to 13.6% of the animals dying within 1 month. Subsequent mortality decreased during the summer and rose in autumn 1995 and again in spring 1996. Cumulative annual mortality was 32.8% and 39.6% in tanks A and B, respectively.

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223

Fig. 1. Schematic partitioning of nitrogen within an integrated abalone/algal biofilter culture system. Dotted arrows represent recycling of nitrogen within the system.

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A. Neori et al. / Aquacultural Engineering 17 (1998) 215–239

Fig. 2. Daily variation between minimum and maximum water temperatures measured over a year in abalone culture vessels and seaweed biofilters.

However, the interpretability of these annual figures in relation to Fig. 5 is compromised by the periodic restocking of the tanks, as reported in Table 1. The high mortality rates in the early months are offset in the overall annual rate by the larger animal numbers later. The dead animals were generally found in areas where the abalone tended to crowd, notably in the darkest corners of the tank; aggressive behaviour, the use of the radula to inflict foot lesions on conspecifics, was observed within these stacks.

3.3. Algal production Production of Ul6a lactuca was seasonally-dependent (Table 2). Production was lower in winter than in the rest of the year, averaging 2929 5 g fresh weight m − 2 d − 1 (529 1 g dry weight m − 2 d − 1) in the summer, and 839 9 g fresh weight m − 2 d − 1 (159 1 g dry weight m − 2 d − 1) in winter. Production rates in the spring and autumn showed greater variability; annual maximum and minimum values of 412 g fresh weight m − 2 d − 1 and 52 g fresh weight m − 2 d − 1 were recorded. Gracilaria conferta yield was erratic and consistently lower than U. lactuca (Table 2). Algal stocks within the G. conferta biofilter repeatedly crashed during the monitoring period. A cessation in net growth was followed by frond fragmentation and washout, and then a take over by opportunistic chlorophytes, predominantly Ul6a spp. and Enteromorpha spp (see also in Friedlander et al., 1987, 1991; Ugarte and Santelices, 1992; Buschmann et al., 1994). When production could be sustained, in April–June 1995, it averaged 231931 g fresh weight m − 2 d − 1.

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225

Neither seaweed tank functioned following the strong Gulf of Aqaba earthquake of 22 November 1995, until January 1996; subsequent G. conferta production was too low and erratic to permit harvesting and feeding to the abalone, and therefore the abalone did not receive this algae in 1996 (see Table 3).

3.4. Nitrogen partitioning 3.4.1. Abalone tanks Mean N content of abalone was 1.64 9 0.07% of wet weight (12.029 0.09% of soft tissue dry weight), in U. lactuca 0.8190.05% of wet tissue (4.69 0.1% of dry weight) and in G. conferta 0.81 9 0.06% in wet tissue (5.89 0.26% of dry weight). These values were used to calculate budgets in N units (Table 3). The significant inputs of N to the abalone tanks were abalone protein (taken into account in the abalone growth figures) and seaweed protein. Each abalone tank received seaweed biomass from the biofilters downstream. Overall, the A abalone tank received from 58% to 100% of its seaweed N as U. lactuca and the rest as G. conferta, and the B tank received from 64% to 100% of its N as U. lactuca and the rest as G. conferta (Table 3).

Fig. 3. (a) Mean specific growth rates (SGR) and (b) mean daily shell length growth increments (9S.E.) of samples of 75 tagged H. tuberculata grown in vessels A and B.

0 356 70 150 30 506 100 147 29 249 49 396 78 −110 −22 392 1351

N Outputs Abalone growth % of input total Effluents % of input total N output total % of input total Deficit (out-in) % of input total Abalone growth Total, mg N/tank Seaweed input Total, mg N/tank

April 1995

N Inputs Inflowing water Ul6a lactuca % of input total Gracilaria conferta % of input total N input total % of input total

Date:

Tank A

3119

286

−59 −9

62 9 554 82 616 91

0 394 58 281 42 675 100

June 1995

2508

170

−251 −52

33 7 202 42 235 48

0 310 64 176 36 486 100

August 1995

2043

517

4 2

62 25 187 76 249 102

0 245 100 0 0 245 100

January 1996

4803

332

−76 −14

37 7 423 79 460 86

0 536 100 0 0 536 100

March 1996

1379

150

−392 −65

66 11 147 24 213 35

0 430 71 175 29 605 100

April 1995

Tank B

2509

203

−427 −69

50 8 141 23 191 31

0 394 64 224 36 618 100

June 1995

2010

315

−74 −31

37 16 125 53 162 69

0 237 100 0 0 237 100

January 1996

4776

avg. 552

54 10

62 12 528 99 590 110

0 536 100 0 0 536 100

March 1996

24 498

2918

62 14 284 59 346 72

0 382 81 112 19 494 100

2722

sum 324

−148 −28

Average SD

1222

135

161 27

32 8 161 25 165 27

103 18 106 18 146 0

Table 3 Daily N budgets for abalone tanks A and B on dates of intensive sampling during 1995–1996. Figures represent biomass-specific rates (microgram N/g abalone /d), and the corresponding percentages relative to the total N input to the tank

226 A. Neori et al. / Aquacultural Engineering 17 (1998) 215–239

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227

Fig. 4. (a) Mean ingestion rates and (b) calculated food conversion ratios (FCR, fresh weight of algae ingested/abalone weight increase) of H. tuberculata grown in vessels A and B. Symbols as in Fig. 3.

The nitrogen leaving in the effluents of the abalone tanks originated from feeding activity of the animals (metabolic by-products, undigested material and algal cell contents not ingested), as negligible quantities of N came in with the fresh seawater (Table 3). The nitrogen supplied as seaweed protein was either incorporated into abalone biomass, washed out of the abalone tank into the biofilter or was unaccounted for (deficit). During most of the days monitored, much of the N entering the abalone tanks (up to 69%) remained unaccounted for, whereas on two occasions N surpluses of 2% and 10% were measured (Table 3). On average, of the N that entered the abalone tanks only 149 8% was incorporated into abalone biomass over the entire monitoring period. Of the rest of the N that entered the abalone tanks, 59 9 25% flowed out into the seaweed biofilters with the effluent and 28927% was not found (Table 3). Total N in the abalone effluents averaged for the entire nine intensive sampling data sets (five for tank A and four for tank B) was 3019 73 mg N/g abalone/d (Tables 3 and 4). This TN value was comprised of ammonia-N (629 12 mg N/g abalone/d), DON (145 9 63 mg N/g abalone/d), and PN (101 9 55 mg N/g abalone/ d). The production rate of TN and its components did not show discernible

228

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Fig. 5. Monthly abalone mortality, expressed as a percentage of the standing stock present at the beginning of each month, in abalone culture vessels A and B.

significant diurnal patterns (Table 4). Oxidized nitrogen was not detected in any of the samples from tanks A and B, suggesting that either nitrification did not occur there or that the nitrification was tightly coupled with denitrification, which consumed all the oxidized N.

3.4.2. Seaweed biofilters Smaller fractions of the N supplied to the seaweed exited the biofilters in the effluents in summer than in winter (Table 5). U. lactuca, on 3 out of 4 days monitored, incorporated larger fractions of the N entering the biofilter into seaweed Table 4 Quantity and forms of nitrogen production measured in abalone tank effluent water during 6 h sections of 24 h monitoring periods; figures represent mean microgram N/g abalone biomass, produced during 6 h (9 S.E.; 5 dates in tank A and 4 dates in tank B) Form of N in abalone effluent:

Ammonia-N Dissolved organic N Particulate N Total N

Sample period: 08:30–14:30

14:30 – 20:30

20:30 – 02:30

02:30 – 08:30

18 ( 911) 38 (946) 9.3 (915) 64 (9 38)

11 52 31 91

19 26 23 65

14 26 37 78

(911) (9101) (9 51) (9 97)

(914) ( 944) (9 26) (949)

( 911) ( 927) ( 986) ( 983)

Total/d 62 (912) 145 (9 63) 101 (9 55) 301 (9 73)

−1059 −18

Deficit (out-in) % of input total 47

1687 29 3107 53 4794 82

Outputs Algal harvest % of input total Effluents % of input total N-output Total % of input total

Filtration efficiency in biofilters (% of input total)

221 4 5632 96 5853 100

Inputs From abalone tank % of input total Added nutrients % of input total N-input-total % of input total

59

−496 −8

3316 52 2619 41 5935 92

853 13 5578 87 6431 100

66

−444 −10

2437 56 1505 34 3942 90

348 8 4038 92 4386 100

42

−1075 −24

834 18 2649 58 3483 76

520 11 4038 89 4558 100

Date: April 1995 June 1995 August 1995 January 1996

Ul6a

42

979 18

3231 61 3049 58 6280 118

1263 24 4038 76 5301 100

76

−4243 −74

130 2 1370 24 1500 26

111 2 5632 98 5743 100

88

−3718 −64

1365 24 686 12 2051 36

191 3 5578 97 5769 100

29

316 7

1587 36 3122 71 4709 107

355 8 4038 92 4393 100

3

−187 −3

0 0 5419 97 5419 97

1568 28 4038 72 5606 100

March 1996 April 1995 June 1995 January 1996 March 1996

Gracilaria

Table 5 Daily N budgets for biofilter tanks stocked with Ul6a lactuca and Gracilaria conferta on dates of intensive sampling during 1995–1996. Figures represent for each tank area-specific rates (mg N/m2 of biofilter tank/d), and the corresponding percentages relative to the total N input to the tank A. Neori et al. / Aquacultural Engineering 17 (1998) 215–239 229

230

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Table 6 Overall N budget for the integrated culture system (two abalone vessels and two seaweed biofilters). Annual averages, calculated from Tables 2 and 4 mgN/m2/d Input

SE

% of N input

4734

779

105

44

Seaweed Total harvest Fed to animals within the system Unused (export)

1621 878 743

1139 394

34 19 16

Effluents

2614

1293

55

Outputs Abalone growth

Deficit (out-in)

−1273

100 2.2

−27

production than did G. conferta (Table 5). Overall, nitrogen filtration efficiency of the biofilters was highest in summer (Table 5). Although the G. conferta biofilter occasionally removed N more efficiently than the U. lactuca biofilter, it incorporated a smaller fraction of N into a harvestable biomass and created larger N-deficits. Generally, the U. lactuca tank removed 58% of the N input to the system, while its total harvest contained about half the average inorganic N input. The algal harvest from the G. conferta tank contained only about a quarter of inorganic N that supplied large fractions of unaccounted-for N were associated with visual observations of frond fragmentation in this seaweed. Most of the N budget was comprised of ammonia, the inorganic form supplied to the biofilters, and the other N forms were inconsequential. DON and PN (data not shown) were sometimes removed and sometimes produced in the biofilters, but in small quantities (B 10% of the overall N budget). Oxidized N (data not shown) was sporadically produced in both biofilters in small quantities (up to 5% of the overall N budget).

3.4.3. O6erall N budgets In the overall N budget of the abalone tanks and their seaweed biofilter tanks (Table 6), the seaweed harvest contained about one third of the total N input. However, only about half of this harvest was fed to the animals. These assimilated about 12% of the seaweed given to them, and therefore only 2.2% of the total N supplied was harvested as abalone biomass. There was, however, a large net seaweed surplus, about half of the overall production of algae in both biofilters. Nearly twice as much surplus was produced by U. lactuca than by G. conferta. About half of the N input of the entire four-tank system was released in the effluents, and about a quarter of the N was unaccounted for (deficit). The deficit N (which possibly was also released) within G. conferta biofilter was greater than in the U. lactuca biofilter.

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3.4.4. Phosphorous No phosphorous was detected in the influent or effluent water of either abalone tank. The biofilters (data not shown) both consistently removed less than 25% of the phosphorous added (with the exception of the G. conferta in January 1996, that removed 84.8% of phosphorous encountered over 24 h).

4. Discussion The concept of ecological sustainability in aquaculture refers to the maximization of internal feedback (e.g., recycling) within a culture system. This minimizes the inputs and the wasted outputs of resources (Dalsgaard et al., 1995), such as nutrients, water and energy, in effluent water. The results presented here show the potential of the integrated abalone-seaweed culture to be practical. A quantitative evaluation of the performance of each component of the system studied here will aid in the development and design of more practical facilities and techniques for integrated mariculture, based on internal nutrient recycling and leading to better effluent quality. The system incorporates a number of features that can increase the ecological sustainability of the proposed integrated culture system, as follows: (a) the use of the same water for both abalone and seaweed cultures reduces seawater requirements by half in this first trial; (b) biofiltration and recycling of the abalone nutrient excretions by the seaweed reduces both the nutrient input requirements and the overall environmental impact of the culture operation; (c) the use of biofiltergrown seaweed eliminates the need for a destructive harvest of natural seaweed beds; and (d) the chemical composition of the cultured seaweed, and hence their nutritional value to the algivores, is controllable. Following refinements to the integrated culture system that can arise from the present results, the incorporation of fish and bivalves will follow, according to the principles of the more complex designs proposed and outlined by Shpigel and Neori (1996).

4.1. Abalone performance Best H. tuberculata performance was seen in spring (March–May), when water temperatures corresponded closely to the summer temperatures considered optimal for growth in the Ormer’s natural range (Hayashi, 1982; Clavier and Richard, 1986). The initial differences observed in SGR% between the two abalone populations may be explained by differences in the timing of spawning, usually synchronous within a confined abalone population (A. Marshall, NCM, Israel, personal communication). From May to October, when daytime water temperatures in Eilat remained above 23.5°C, growth rates of the Ormer were low, reflected by increased FCRs. It has been suggested (Shpigel et al., 1996) that the elevated temperatures increase basal metabolic rate, thus reducing the energy available for somatic growth.

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Ingestion rates remained fairly constant throughout the year, in close agreement with rates recorded by Mercer et al. (1993). They reported 5–9% daily body weight food ingestion by H. tuberculata fed U. lactuca, suggesting that the animals in the present study were feeding to satiation. Overall growth performance, expressed as annual shell length growth increment, was inferior in the present study when compared to values previously reported in Eilat (Shpigel et al., 1996). Elsewhere, for H. tuberculata of similar size ranges grown in warm water culture (18 – 20.5°C), growth rates of 15–20.3 mm year − 1 were recorded (Forster, 1967; Hayashi, 1980, 1982; Mgaya and Mercer, 1995). In a parallel experiment H. tuberculata from the same stock as used in the present study showed growth rates corresponding to 21.8 9 2.7 mm year − 1, in a controlled aquarium environment (Ragg et al., unpublished data). In the present study abalone were stocked at densities (maximum 166 individuals m − 2) well below the levels that were suggested by Koike et al. (1979), and by Mgaya and Mercer (1995) as causing significant crowding pressure. However, the tank design in the present study allowed animals to move freely, hence the abalone, responding to the same stimuli, such as negative phototaxis (Mgaya and Mercer, 1994), and their gregarious nature (Douros, 1987) tended to crowd. This apparently induced local effects of severe crowding pressure, smothering and cannibalism (unpublished observation) and resource competition that is likely to interfere with growth (Koike et al., 1979; Mgaya and Mercer, 1995). Despite an apparently considerable scope for improving growth performance, mean annual SGR%s and FCRs were better here than the conservative estimates proposed by Shpigel and Neori (1996) (Table 7) and concurred with those found by Mercer et al. (1993). Smothering and aggressive behaviour between conspecifics are held partly responsible for observed abalone mortality, and initial high mortality is likely to be associated with handling stress incurred during stocking (Mgaya and Mercer, 1995), and high autumn mortality is attributed to the rapid decline in water temperature (Aviles and Shepherd, 1996).

4.2. Algal production Algal biomass production in the U. lactuca biofilter was highly seasonal, the growth rate of U. lactuca appearing to be predominantly dependent upon water temperature and light, in agreement with the findings of Vandermeulen and Gordin (1990), Israel et al. (1995) and Neori et al. (1996). Constant daily yields accompanied the more stable water temperatures during winter and mid-summer, with production levels comparable to those of U. lactuca grown in other biofilters (Neori et al., 1991, 1996) and using artificial inorganic nitrogen and phosphorous sources (DeBusk et al., 1986; Israel et al., 1995). The annual mean seaweed production of 230 g fresh weight (42 g dw) m − 2 d − 1 obtained here exceeds that of almost all known intensive terrestrial and marine plant cultures (Lapointe et al., 1976). Gracilaria spp. is highly sensitive to temperature (Edding et al., 1987; Friedlander et al., 1987, 1991; Levy and Friedlander, 1990; Ugarte and Santelices, 1992); optimum growth of G. conferta occurs in cultures of 20–26°C (Levy and Friedlan-

Yield of Ul6a lactuca (kg fresh weight d−1)/kg N added to system Biofilter surface area required to support this production Yield of abalone (kg fresh weight d−1)/kg N added to system Volume of water required to support this production of abalone 25 m3

2.6

250 m2

65

Predicted by Shpigel and Neori (1996)

SGR = 0.3% d−1; stocked at 35 kg m−3

25.7 m3

SGR= 0.3% d−1; stocked at 35 kg m−3

Receives a net N-flux of 4 g m−2 d−1; yield 0.23 kg m−2 d−1 U. lactuca yield of 0.23 kg m− 2 d−1 FCR= 20

Calculated from mean values Assumptions found in the present study

Removes 55% of ammonia-N 54.9 at flux of 4 g m−2 d−1; yield 0.25 kg m−2 d−1 U. lactuca yield of 0.25 kg 239 m2 m−2 d−1 FCR=25 2.7

Assumptions

Table 7 Comparison between the yields, and corresponding system dimensions, predicted for an integrated H. tuberculata/U. lactuca system per kg of nitrogen input, using the figures proposed by Shpigel and Neori (1996) and the mean annual yields found possible in the present study

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der, 1990) supplemented with nutrients in a single weekly pulse (Friedlander et al., 1991; Levy and Friedlander, 1994). It is therefore suggested that stress imposed by summer and winter temperature extremes, as well as large diurnal ranges, combined with the continuous presence of nutrients, favouring the development of fouling chlorophytes, were responsible for the poor performance of G. conferta in the present study.

4.3. Nutrient partitioning 4.3.1. Abalone tanks On an annual average, dissolved N formed about two-thirds of the total N excretions in the abalone tanks (disregarding the N deficit). This value is exactly as we had reported for marine fish in intensive fishponds (Krom and Neori, 1989) and integrated fish-seaweed ponds (Neori et al., 1996), and with about similar deficits. In most of the nitrogen budgets of the abalone vessels presented here a large proportion of the nitrogen remained unaccounted for. These deficits are attributed to several possible sources of inaccuracy: 1. Analytical errors in determining the volumes of water (estimated at  10%); 2. Budgets were constructed for specific 24-h periods, while considerable day-today variability may exist; 3. Macroalgae show variable levels of tissue nitrogen, depending on ambient conditions, particularly the level of dissolved inorganic nitrogen in the water, and also light and temperature (Friedlander et al., 1987; Vandermeulen and Gordin, 1990; Cohen and Neori, 1991; Pedersen, 1994). In the present study, average values were used to represent the nitrogen content of either algal species throughout the monitoring period, hence no accommodation was made for possible variations in the amount of nitrogen within food algae or the subsequent uptake or loss of nitrogen by the algae within the abalone vessels. 4. Tightly-coupled nitrification-denitrification in animal digestive tract, faeces or in corners of the rectangular vessels. Such efficient coupling that leads to the complete removal of the oxidized N as soon as it is produced has been known for highly organic flooded soils and sediments (Reddy and Patrick, 1984). It could explain both the absolute lack of oxidized N in the abalone tanks (as opposed to its sporadic detection in the waters of the seaweed tanks) and the large N-deficit there. 5. Fouling organisms growth on the solid surfaces and on the shells. 6. Solid waste drained only during routine maintenance and evaluated visually to be negligible. Microorganisms probably could not substantially affect the nitrogen budget of the abalone tanks. Vigorous aeration and bottom-draining prevented the formation of dead spaces, where organic matter and bacteria could accumulate (Dvir, 1995), and abalone grazing kept all hard surfaces visibly free of fouling. It is possible that the amount of nutrients lost during the weekly cleaning of the tanks was much more than we assessed.

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Diurnal variation in composition of nitrogenous abalone effluent was on average limited to ammonia, most probably a net result of periods of greatest abalone activity (Barkai and Griffiths, 1987; Peck et al., 1987; Fleming et al., 1996) on the one hand, and daylight uptake of ammonia by the uneaten seaweed (Cohen and Neori, 1991) on the other hand. Un-ingested algal cell contents liberated by abalone radula scraping action may account for the persistently high presence of DON and PN in tank effluents. Average ammonia production rate of 62 mg N/live g/d in the present study appear higher than the 36 mg N/live g/d, which can be calculated for a 9.3-g (4 g dw) animal by the equation given in Peck et al. (1987): ln U =0.656 ln W − 0.914

(3)

U = ammonia excretion in mmol N/h and W= dw of whole animal. This is not surprising, considering the markedly higher temperatures in our study.

4.3.2. Seaweed biofilters The U. lactuca biofilter showed consistent performance throughout the year and most of the nitrogen removed in this biofilter was accounted for by subsequent gains in algal biomass. Biofiltration efficiency was highest in summer, corresponding to fastest U. lactuca growth. The filter removed approximately half of the nitrogen, encountered predominantly as ammonia-N, at an even rate over 24 h, as noted by Vandermeulen and Gordin (1990) and by Cohen and Neori (1991). The consistent biofiltration performance of U. lactuca is highlighted when comparing the present nitrogen removal efficiencies to those recorded by Cohen and Neori (1991) who, working at the same site, found mean removal rates of 49–56% of ammonia-N supplied at fluxes of 4.8 – 5.2 g m − 2 d − 1. Cohen and Neori (1991) also demonstrated that nitrogen filtration efficiency was enhanced as influent nitrogen flux decreased; however, there was a corresponding reduction in U. lactuca tissue nitrogen, which has been shown to reduce the dietary value for H. tuberculata (Ragg et al., unpublished data). The low removal efficiency of phosphorous in the biofilters measured here has also been noted in other seaweed biofilters (DeBoer et al., 1978; Neori et al., 1996). Macroalgae grown in artificially enriched media are typically supplied with inorganic nitrogen and phosphorous at molar ratios of 10–13:1, N:P (Vandermeulen and Gordin, 1990; Friedlander et al., 1991; Ugarte and Santelices, 1992; Israel et al., 1995). In the present system, inorganic nutrients were added in accordance with the higher Redfield ratio for phytoplankton cells, N:P= 16:1. Despite this elevated ratio, phosphorous removal efficiency was low. In the G. conferta biofilter, high nitrogen filtration efficiencies in spring 1995 could not be accounted for by a correspondingly high production of Gracilaria tissue. It is considered likely that nitrogen was removed from the water by autotrophic fouling organisms; apart from the visibly obvious presence of macroscopic chlorophytes, small quantities of oxidized nitrogen were frequently detected in the effluents of this tank. It is therefore possible that there, the same nitrificationdenitrification coupled processes competed with the G. conferta for ammonia, as found by Dvir (1995), and created large N-deficits. Gracilaria conferta has always

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been an inferior grower in Eilat, and therefore it is not dependable as a biofilter. However, with special care it can be cultured, as a supplement to the U. lactuca culture. In both biofilters a large proportion of the nitrogen supplied was removed as surplus seaweed production and only a very small fraction as abalone biomass (2.2%), implying that the ratio of abalone biomass to algal production unit size, and hence the abalone production, was far too low. The results of the present study make it possible to determine a more appropriate ratio and estimate the subsequent system productivity. Owing to the unreliable performance of the G. conferta biofilter, the following calculations are based on integrated abalone/Ul6a tanks, stocked with animals of the size range used here, typical of second-year growout H. tuberculata (Mgaya and Mercer, 1994). Assuming a steady annual ingestion rate of 5.9% body weight d − 1, and mean U. lactuca production of 230 g m − 2 d − 1 (49% N-filtration efficiency, 58% if N-recycling is excluded), and using the more conservative parameters of the abalone population from tank A (mean FCR=20; 45% of N entering the abalone tank is released in tank effluents), an appropriately proportioned system can be proposed. The productivity of such a system, standardized to 1 kg N input, is compared to the original model of Shpigel and Neori (1996) in Table 7. The performance of the experimental system studied here shows close agreement with the predicted models. This comparison provides also an indication of the benefits of the integration of seaweed and abalone culture units into a single system. If the U. lactuca and abalone were grown in separate systems, seawater supply would be doubled, the nitrogen leaving the abalone vessel would be dumped into the sea and a corresponding amount of nitrogen (up to 24% in the 27 March 1996 experiment) would have to be added to the U. lactuca culture. Hence, using this example, separating the culture units would result in the need to supply an additional 24% nitrogen to the algal unit and a similar (all the abalone effluents N) increase in nitrogen release to the environment. Although the production estimated possible by the data from the present study (Table 7) compares closely with the projected yields suggested by Shpigel and Neori (1996), it is unrealistic in the use of mean annual U. lactuca production to calculate the corresponding biofilter size needed to provide food for the abalone. In reality, if a single U. lactuca culture was used, the filter would have to provide sufficient production during minimum winter growth (mean 82 g Ul6a m − 2 d − 1), this would require a filter 2.8 times larger than proposed by the model. A more practical solution would be to introduce a second U. lactuca biofilter in series, to serve as a polishing filter, further reducing nutrient loading in system effluents, as successfully applied by Lapointe et al. (1976), Krom et al. (1995) and Neori et al. (1996); the second filter would also provide an additional source of U. lactuca biomass if the production from the first filter fails to meet demands. The level of inorganic nitrogen and N:P necessary to produce U. lactuca of optimum nutritional value, while minimizing the level of nutrients in the effluent, still needs to be determined and corrected for nitrogen supplied by abalone

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effluents. The commercial application of such a system would also benefit from the use of an improved abalone vessel design that does not permit excessive free movement of animals and subsequent crowding problems, as recommended by Fleming and Hone (1996), e.g. by use of suspended shelters or cages. An additional recent finding can allow a significant reduction in the ratio of algae biofilter area to kilogram of cultured abalone. Supplying ammonia-N influx at double the rates used here has increased significantly the seaweed protein content (see also in Cohen and Neori, 1991), a feature which has been shown by us to reduce the FCR for the abalone by about half (Ragg et al., in preparation). As G. conferta has been a useful dietary supplement it is suggested that the rhodophyte be grown in a separate temperature regulated culture, receiving weekly nutrient pulses. This can further reduce the area of seaweed biofilter per kilogram of reared abalone.

Acknowledgements The authors would like to offer special thanks to A. Marshall for valuable observations as system manager; our gratitude also to D. Ben-Ezra, R. Fridman and B. Simpson for their expert technical advice and assistance, to O. Dvir and I. Lupatsch who performed the chemical analyses, and to A. Colorni and R. Goldberg for critical reviews of the manuscript. The project was supported by the Israeli Ministry for Energy and Infrastructure and by a joint program of the EC and the Israeli Ministry for Science (Grant No. 4564192 to M.S. and A.N.).

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.