Commercial application of polychaete sand filters for ...era.daf.qld.gov.au/id/.../2/Commercial_application... · Commercial application of polychaete sand filters for wastewater

Commercial application of polychaete sand filters for wastewater remediation and broodstock feeds. Palmer, P.J. (2011) Landcare Sustainable Practices Grant No. SEQC1418. Technical Report: p1 - 34 Department of Employment, Economic Development and Innovation (DEEDI), Bribie Island Research Centre (BIRC), PO Box 2066 Woorim, Queensland, 4507 Australia. Email: [email protected] Photographs taken by Paul Palmer Keywords: Mariculture; Wastewater; Bioremediation; Polychaetes; Sand filtration; Water qualities; Nutrients; Fatty acids.

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Landcare Sustainable Practices Grant No. SEQC1418 Technical Report

___________________________________________________________________________________ © The State of Queensland, Department of Employment, Economic Development and Innovation, 2011

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Commercial application of polychaete sand filters for wastewater remediation and broodstock feeds Paul J. Palmer

Department of Employment, Economic Development and Innovation (DEEDI), Bribie Island Research Centre (BIRC), PO Box 2066 Woorim, Queensland, 4507 Australia. Email: [email protected] ___________________________________________________________________________________ Table of Contents

Summary ....................................................................................................................3 Introduction................................................................................................................4 Materials and methods ...............................................................................................5 Results......................................................................................................................10

Sand beds and water volumes treated ..................................................................10 Water qualities and nutrients ...............................................................................12 Polychaete survival, biomass production and harvest .........................................23 Polychaete biomass analyses ...............................................................................24

Discussion................................................................................................................28 Conclusion ...............................................................................................................32 Acknowledgments....................................................................................................33 References................................................................................................................33

List of Tables Table 1. Particle size characteristics* of sand products used to construct beds at different farms.5 Table 2. Water quality parameters monitored, sampling periodicity and methods of analysis. ...10 Table 3. Water quality measurements for inflows during the tidal sampling program. ................21 Table 4. Survival estimates and experimental harvest (1 m2) results for polychaete sand filter

beds at Farms A, B and C...........................................................................................................23 Table 5. Proximal analyses for worm biomass from different beds at each farm...........................25 Table 6. Total lipid, cholesterol and phospholipids analyses expressed as a % of dry sample for

worm biomass from different beds at each farm......................................................................25 Table 7. Fatty acid analyses expressed as a % of dry sample for worm biomass from different fed

and unfed1 beds at each farm.....................................................................................................26 Table 8. Mean (± se) representations (% of dry matter) of the 12 most prevalent fatty acids

detected in worm biomass samples across all 6 beds in the study...........................................27 Table 9. Mean arachidonic acid (AA): eicosapentaenoic acid (EPA): docosahexaenoic acid (DHA)

ratios in worm biomass from different fed and unfed1 beds using AA as the equal comparator. .................................................................................................................................27

Table 10. Amino acid analyses expressed as a % of dry sample for worm biomass from different fed and unfed1 beds at each farm...............................................................................................27

Table 11. Mean (± se) representations (% of dry matter in descending order) of the amino acids detected in worm biomass samples across all 6 beds in the study...........................................28

List of Figures Figure 1. Construction of sand beds at Farm A (A: installing subsurface bed drainage; B: final

layout; C: filtered water channels; D: operational with bird netting in place). ......................6 Figure 2. Daily fish meal feeding rates applied to Beds 1 and 3 at Farm A.......................................7 Figure 3. Construction of sand bed at Farm B (A: excavating the pond; B: placing sand over

drainage pipes; C: shade-house rain cover installed; D: external view of completed facility).........................................................................................................................................................8



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Figure 4. Sand bed retrofitted into existing facilities at Farm C (A: sand bed being flooded with pond water; B: sampling inflow). ................................................................................................9

Figure 5. Pond-water volumes filtered at each farm on a square metre of sand basis ...................11 Figure 6. Sand beds at Farm A after normal operations (A: benthic algal growth; B: worms

feeding on the surface) and Farm B after a period of sub-optimal operation (C: undisturbed surface; D: reduced sediments at depth)...................................................................................12

Figure 7. Water temperatures of pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Means (± se) shown for beds (n=4) at Farm A...13

Figure 8. Salinities of pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Means (± se) shown for beds (n=4) at Farm A.....................13

Figure 9. Dissolved oxygen and pH levels in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se) levels for fed and unfed beds (n=2) shown for Farm A..........................................................................................15

Figure 10. Total nitrogen and total phosphorus levels in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se) levels for fed and unfed beds (n=2) shown for Farm A. .................................................................................16

Figure 11. Total suspended solids levels in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se, n=4) levels shown for Farm A...................................................................................................................................17

Figure 12. Chlorophyll a levels in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se, n=4) levels shown for Farm A.........17

Figure 13. Visual evidence of the difference between unfiltered (left) and filtered (right) pond water.............................................................................................................................................18

Figure 14. Total ammonia nitrogen and nitrite nitrogen in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se) levels for fed and unfed beds (n=2) shown for Farm A. (Note the smaller scale for Farm A nitrite nitrogen).......................................................................................................................................19

Figure 15. Nitrate nitrogen levels in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se, n=4) levels shown for Farm A.......................................................................................................................................................20

Figure 16. Phosphate phosphorus levels in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se, n=4) levels shown for Farm A...................................................................................................................................20

Figure 17. Sulphide levels in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se, n=4) levels shown for Farm A..................20

Figure 18. Flow rates of pond-sourced inflows and filtered outflows from polychaete sand filter beds operated at Farms B and C during a period of intensive water sampling and over an entire artificial tide. ....................................................................................................................21

Figure 19. Water quality parameters prevailing in filtered outflow from polychaete sand filter beds operated at Farms B and C during a period of intensive water sampling and over an entire artificial tide. ....................................................................................................................22

Figure 20. Harvesting Perinereis helleri from sand beds (A: dredge harvester; B: demonstration harvest; C: before separation from mucus-laden sand; D: worm biomass after separation).......................................................................................................................................................24

Figure 21. Bait worm fishery production in Queensland from 2000 to 2009 (modified from Queensland Government Fisheries data)..................................................................................32



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Summary Prototype sand-worm filtration beds were constructed at two prawn farms and one fish farm to assess and demonstrate their polychaete (marine worm) production and wastewater remediation capacities at semi-commercial scale. Wastewater treatment properties were monitored and worms produced were assessed and either sold for bait or used by the farms’ hatcheries as broodstock (prawn or fish breeder) feed. More than 34 megalitres of prawn- and fish-pond water was beneficially treated in the 116-319-d trial. The design of the polychaete-assisted sand filters (PASFs) constructed at each farm affected their water handling rates, which on average ranged from 315 to 1000 L m-2 d-1 at the three farms. A low profile design incorporating shallow bunded ponds made from polyethylene liner and timber stakes provided the easiest method of construction. This simple design applied at broad scale facilitated the highest quantities of treated water and the greatest worm production. Designs with higher sides increased the head pressure above the sand bed surface, thus increasing the amount of water that could be treated each day. Most water qualities were affected in a similar way to that demonstrated in the previous tank trials: dissolved oxygen, pH, total suspended solids and chlorophyll a levels were all consistently significantly lowered as pond water percolated through the sand bed, and dissolved forms of nitrogen and phosphorus were marginally increased on several occasions. However, unlike the previous smaller-scale tank trials, total nitrogen (TN) and total phosphorus (TP) levels were both significantly lowered by these larger-scale PASFs. The reasons for this are still unclear and require further research. Maximum TN and TP removals detected in the trial were 48.8% and 67.5%, respectively, and average removals (in unfed beds) at the three farms ranged from 20.0 to 27.7% for TN and from 22.8 to 40.8% for TP. Collectively, these results demonstrate the best suspended solids, chlorophyll and macronutrient removal capacities so far reported for any mariculture wastewater treatment methodology to date. Supplemental feeding of PASFs with fish meal was also investigated at one farm as a potential means of increasing their polychaete biomass production. Whilst fed beds produced higher biomass (152 ± 35 g m-2) compared with unfed beds (89 ± 17 g m-2) after 3.7 months of operation, the low number of replicates (2) prevented statistically significant differences from being demonstrated for either growth or survival. At harvest several months later, worm biomass production was estimated to be similar to, or in slight excess of, previously reported production levels (300-400 g m-2). Several qualities of filtered water appear to have been affected by supplemental feeding: it appeared to marginally lower dissolved oxygen and pH levels, and increased the TN and TP levels though not so much to eliminate significant beneficial water treatment effects. Periodic sampling during an artificial-tide demonstrated the tendency for treated-water quality changes during the first hour of filtration. Total nitrogen and ammonia peaked early in the tidal flow and then fell to more stable levels for the remainder of the filtration period. Other dissolved nutrients also showed signs of this sand-bed-flushing pattern, and dissolved oxygen tended to climb during the first hour and become more stable thereafter. These patterns suggest that the routine sampling of



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treated water undertaken at mid-inflow during the majority of the wider study would likely have overestimated the levels of TN and dissolved nutrients discharged from the beds, and hence underestimated the PASFs treatment efficacies in this regard. Analyses of polychaete biomass collected from each bed in the study revealed that the worms were free from contamination with the main prawn viruses that would create concerns for their feeding to commercial prawn broodstock in Australia. Their documented proximal and nutritional contents also provide a guide for hatchery operators when using live or frozen stock. Their dry matter content ranged from 18.3 to 22.3%, ash ranged from 10.2 to 14.0%, gross energy from 20.2 to 21.5 MJ kg-1, and fat from 5.0 to 9.2%. Their cholesterol levels ranged from 0.86 to 1.03% of dry matter, whilst total phospholipids range from 0.41 to 0.72%. Thirty-one different fatty acids were present at detectable (≥0.005% of dry matter) levels in the sampled worm biomass. Palmitic acid was by far the most prevalent fatty acid detected (1.21 ± 0.18%), followed by eicosapentaenoic (EPA) (0.48 ± 0.03%), stearic (0.46 ± 0.04%), vaccenic (0.38 ± 0.05%), adrenic (0.35 ± 0.02%), docosadienoic (0.28 ± 0.02%), arachidonic (AA) (0.22 ± 0.01%), palmitoleic (0.20 ± 0.04%) and 23 other fatty acids with average contents of less than 0.2% of dry matter. Supplemental feeding with fish meal at one farm appeared to increase the docosahexaenoic acid (DHA) content of the worms considerably, and modify the average AA : EPA : DHA from 1.0 : 2.7 : 0.3 to 1.0 : 2.0 : 1.1. Consistent with previous results, the three most heavily represented amino acids in the dry matter of sampled worms were glutamic acid (8.5 ± 0.2%), aspartic acid (5.5 ± 0.1%) and glycine (4.9 ± 0.5%). These biomass content results suggest that worms produced in PASF systems are well suited to feeding to prawn and fish broodstock, and provide further strong evidence of the potential to modify their contents for specific nutritional uses. The falling wild-fishery production of marine bloodworms in Queensland is typical of diminishing polychaete resources world-wide and demonstrates the need to develop sustainable production methods here and overseas. PASF systems offer the dual benefits of wastewater treatment for environmental management and increased productivity through a valuable secondary crop grown exclusively on waste nutrients.

Introduction There is presently strong international demand for industrial-scale methods that can beneficially treat eutrophic brackish water. Such methods are of particular interest to mariculture operations like prawn and fish farms. They routinely use natural coastal waters in their broad-scale production ponds, and release nutrient-enhanced waters after various passive treatment processes such as settlement. To improve production efficiencies and to adhere to regulatory environmental frameworks, mariculture farms have an ongoing need to remove and potentially utilise nutrients and suspended particles from large volumes of supply- and waste-waters. Many operators are also seeking methods that can treat pond discharge to a suitable level for on-farm recirculation, so that they can potentially operate independent of adjacent waterways. To address this need for large-scale brackish-water treatment systems, pilot investigations into polychaete-assisted sand filters (PASFs) began at the Bribie Island Research Centre in 2005. The research was expanded in 2007 through funding by the



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National Landcare Programme (Project No. 60945) where the concept was tested at small scale at a commercial prawn farm. Those investigations demonstrated that passive down-flow PASFs could beneficially treat large volumes of prawn- and fish-pond water, and could simultaneously produce polychaete biomass as a useful and valuable by-product (see Palmer 2008, 2010). The next step in these developments has been to test the concept at larger scale, and in different localities and commercial environments. Three marine prawn or fish farms in the Pumicestone and Burnett Regions were selected for the construction and testing of up-scaled PASF prototypes. During 2010 these prototypes were jointly operated by farmers and DEEDI officers to collaboratively build a knowledge base that could later be extended to industry. The objectives of this work were to investigate, monitor and compare operational practicalities, water treatment efficacies and worm biomass productivities at each farm.

Materials and methods In consultation with each of the farm managers, sand bed construction designs were developed to suit the specific site characteristics and future envisaged needs of the different facilities. All farms opted for the use of water-proof polyethylene plastic pond liners. Localised commercially available bedding sand products were used at each site (Table 1). Specifications of the sand bed and drainage characteristic were as previously described by Palmer (2008, 2010). In brief, this entailed 200 mm deep beds drained at 900 mm intervals with a network of 60 mm corrugated slotted pipe covered with gauze sock. Two or three tidal simulations were applied to each bed daily. Pond water inflow rates and pumping periods were adjusted to maximise the volumes filtered, but also so that the bed had time to drain completely between tides. Since freshwater can adversely affect marine worms, all bed designs and management procedures had to account for operations during excessive rainfall events. Table 1. Particle size characteristics* of sand products used to construct beds at different farms.

Sieve size (mm)

Farms A and C (% passing)

Farm B (% passing)

4.750 100 - 2.360 99.9 - 1.180 95.4 - 1.0 - 92

0.85 - 86 0.600 76.6 73 0.425 - 58 0.300 18.8 42 0.212 - 21 0.150 0.5 3 0.106 - 0 0.075 0.1 0

* Modified from commercial product data sheets. One-month old cultured Perinereis helleri juveniles were stocked into the sand beds at Farms A and B in December 2009, and at Farm C in February 2010. A stocking rate of approx 2000 m-2 was used for all beds in the study. Stocking was timed to occur three days after water filtration activities began, and it involved transferring nursery sand (containing juveniles) into recently drained beds according to a stratified



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plan for even distribution. From that point on the filtration beds were operated according to the preset artificial-tidal regime. Farm A – Bullock Creek Prawn Farm at Donnybrook Farm A had been operated for many years as a relatively small semi-intensive prawn farm selling its product directly to the public. At the time of the project’s inception, the owners, Mr Peter Spindler and Mrs Judy Butler, were reducing their commitment to prawn farming and seeking a less intensive business option. Their interest in producing live worms for the bait industry in an extensive outdoor setting drove the design characteristics of the facility developed at that site. Project funds partially contributed to the construction of ten 60 m2 (6 x 10 m) sand beds of which four were studied in detail as part of the project. The beds were constructed on the bottom of an unused prawn pond using methods that involved low profile free-standing sides supported by regular timber posts (see Figure 1). It was envisaged that this method could easily be implemented at large scale on relatively flat ground and without significant earthworks. Having been used to produce a crop of worms the previous year, the sand used at Farm A had been harvested, washed and fully dried prior to repeating the process for the study.

Pond 3 Pond 4

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Figure 1. Construction of sand beds at Farm A (A: installing subsurface bed drainage; B: final layout; C: filtered water channels; D: operational with bird netting in place). Project activities at Farm A included monitoring water volumes and qualities over a four-month period from the start of operations on 6 Dec 2009. Water was pumped onto each bed from the monk drain of a medium-density Penaeus monodon growout pond according to three tidal simulations per day (pumping from 1-4 am, 8-11 am and



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6-9 pm). During excessive rainfall the beds were kept covered with brackish pond water using external standpipes on the drainage outlet. Two of the four experimental sand beds at Farm A (Ponds 1 and 3; see Figure 1) were also provided with supplemental feed based on the recommendations for marine worm culture by Poltana (2007). This was undertaken to assess the effects of this approach on water qualities and worm survival and production. Accordingly, fish meal was spread evenly over beds 1 and 3 on a daily basis using the rates described in Figure 2.

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Figure 2. Daily fish meal feeding rates applied to Beds 1 and 3 at Farm A. Farm B – Sunrise Seafoods at Baffle Creek Farm B is a large semi-intensive prawn farm and hatchery operated by Mr and Mrs Ting and Alice Ko, Mr Eric Ko and their family. The farm has been successfully producing P. monodon for many years and markets much of its product through large supermarket chains. The company’s interest in producing marine worms for use in their hatchery as broodstock feed was central to their involvement in the project, and their desire to work towards improved conditioning systems for prawn broodstock drove the design characteristics of the facility developed at that site. Project funds contributed to the construction of a fully covered 57.6 m2 sand bed built into the bottom of a shallow lined pond (see Figure 3). It was envisaged that this design could operate under a consistent management regime, without the need for modifications due to inclement weather conditions. It was also considered to be a replicable and practical design should the company decide to investigate the longer-term holding of prawn broodstock in the future. Project activities at Farm B included the routine monitoring of water volumes and qualities over a four-month period from the start of operations on 4 Dec 2009. Water was pumped onto the bed from the corner of the primary discharge settlement pond according to two tidal simulations per day. Pump-period durations of each tide varied from 1 to 4 h during the trial, due to various management decisions based on the general premise of maximum water handling with intermittent dry times.



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A B

C D

Figure 3. Construction of sand bed at Farm B (A: excavating the pond; B: placing sand over drainage pipes; C: shade-house rain cover installed; D: external view of completed facility). In addition to the routine fortnightly/monthly monitoring regime, one entire tidal period was intensively studied to assess the sequential changes in water flows and qualities as the sand filter began to operate, as it reached its full flow capacity, and as it ran down to cessation at low tide. This was undertaken on the 16 March 2010 with a 2-h pump cycle and after an 8-h dry period. Farm C – Creel Seafoods at Ningi Farm C is an ex-prawn farm that is slowly building its capacity for marine fish production. The operator, Mr Andrew Lancaster, is interested in producing estuarine finfish that can supplement the wild catch that is presently being processed at the site. The company’s desire to beneficially treat its aquaculture pond discharge and also produce marine worms for bait and broodstock feeds drove their interest in the project. Late in the project’s planning phase, one of the original participants unfortunately became unable to take part, and at that stage Creel Seafoods agreed to fill this position. The availability and suitability of unused facilities at the site drove the design characteristics of worm beds that were developed. Project funds contributed to the construction of two 59.3 m2 sand beds built into the bottom of pre-existing concrete tanks (see Figure 4). Only one bed could be stocked with juveniles worms at that late stage so only one was studied in the project. However, unlike the other prawn farms in the study which harvest their stock before winter, this fish farm operates its ponds over a more extended time frame, which presented an opportunity to monitor that PASF over a much longer operational period.



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A B

Figure 4. Sand bed retrofitted into existing facilities at Farm C (A: sand bed being flooded with pond water; B: sampling inflow). Project activities at Farm C included the routine monitoring of water qualities over a six-month period and water volumes over a ten-month period from the start of operations on 31 Jan 2010. Water was pumped onto the bed from the corners of two alternative fish ponds: initially from a pond containing several hundred cobia (Rachycentron canadum) up to 3 Mar 2010, and thereafter from a pond containing several hundred thousand sand whiting (Sillago ciliata). At the start of the trial, two 1-2 h pump periods (commencing at 6 am and 6 pm) provided two evenly spaced simulated tides per day, but to increase water handling from the 1 April 2010 this was changed to three 2-h durations (commencing at 6 pm, 12 midnight and 6 am). This regime provided three simulated tides and an extended dry-bed period each afternoon. Since this bed was not protected from rainfall, an external standpipe was raised to maintain brackish-water cover during inclement weather. In addition to the routine fortnightly/monthly monitoring regime, one entire tidal period was also intensively studied at Farm C. This was undertaken on the 14 July 2010 and, as with Farm B, a 2-h pump cycle was used after an 8-h dry period. Water quality monitoring Water volumes handled by each sand filter were measured regularly with permanently-fixed inline flow meters (BIL 25 or 40 mm, accurate to 0.0001 m3) on inflow and (where possible) discharge pipes. Routine water samples taken from the inflow and discharge pipes of each sand filter were assessed according to the methods described in Table 2. The timing of this fortnightly or monthly sampling was standardised at midway through daytime inflow periods. Parameters assessed during the intensive tidal monitoring undertaken at Farms B and C included water flow rates (L min-1) and all water qualities measured in the routine monthly sampling program except TSS and Chla1. Sampling of the filtered outflow was undertaken 5, 30, 60, 90 and 120 min after starting the inflow pump, and then at hourly intervals thereafter until the outflow stopped. The inflow for this intensive tidal study was sampled during the first inflow hour.

1 These parameters can only be reliably tested in unfrozen samples stored on ice for less than 24 h; the remote nature of Farm B prevented sample delivery to the laboratory within this time frame.



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Table 2. Water quality parameters monitored, sampling periodicity and methods of analysis. Parameter (unit of measurement) Periodicity Method of analysis Temperature (ºC) Monthly In situ measurement - YSI 556 Multi-probe system Salinity (g L-1) Monthly In situ measurement - YSI 556 Multi-probe system Dissolved oxygen (DO) (mg L-1) Monthly In situ measurement - YSI 556 Multi-probe system pH Monthly In situ measurement - YSI 556 Multi-probe system Total nitrogen (TN) (mg L-1) Fortnightly Frozen sample - NATA approved laboratory Total ammonia nitrogen (TAN) (mg L-1) Monthly Fresh sample - Palintest Photometer 5000 Nitrite nitrogen (NO2N) (mg L-1) Monthly Fresh sample - Palintest Photometer 5000 Nitrate nitrogen (NO3N) (mg L-1) Monthly Fresh sample - Palintest Photometer 5000 Total phosphorus (TP) (mg L-1) Fortnightly Frozen sample - NATA approved laboratory Phosphate phosphorus (PO4P) (mg L-1) Monthly Fresh sample - Palintest Photometer 5000 Total suspended solids (TSS) (mg L-1) Monthly Fresh (<24 h on ice) sample - NATA approved lab. Chlorophyll a (Chla) (µg L-1) Monthly Fresh (<24 h on ice) sample - NATA approved lab. Sulphide (S) (mg L-1) Monthly Fresh sample - Palintest Photometer 5000 Worm biomass studies Polychaete survival was estimated at each farm by sieving all worms from a randomly selected full-depth 1 m2 transect (200 mm x 5,000 mm) of sand from each experimental bed. This was undertaken on 23 March 2010, 29 March 2010 and 5 July 2010 at Farms A, B and C, respectively. In each case the worms sampled were purged for 24 hr in clean seawater at ambient temperature, counted and weighed en masse. A live sample of purged worms from each bed was sent to the Tropical and Aquatic Animal Health Laboratories at Oonoonba for testing for endemic marine prawn viruses. Nested polymerase chain reaction (PCR) was used to test for Gill-associated virus (GAV), Infectious hypodermal and haematopoietic necrosis virus (IHHNV) and Mourilyan virus (MoV). The remainder of each worm sample was frozen (-80°C) at BIRC for later biomass analyses at the Health and Nutritional Biochemistry Laboratory at Yeerongpilly, Brisbane. Proximal analyses and nutritional components tested included profiles for amino acids, fatty acids and phospholipids, as well as dry matter, ash, nitrogen, phosphorus, gross energy, fat, total lipid and cholesterol. Statistical analyses were undertaken using GenStat (2011). Comparisons of water quality parameters over time used repeated measures analysis of variance (ANOVA) with protected least significance difference testing between means. One-way ANOVA was applied to data involving worm biomass weights, and percentage survival data were analysed using a generalised linear model with the binomial distribution and logit link (McCullagh and Nelder, 1989).

Results

Sand beds and water volumes treated In total, over 34 megalitres of pond water was treated by sand beds during the trial. During the first four months of operations at each farm, more pond water was filtered through experimental beds at Farm A (9.6 megalitres) than at the other two farms (Figure 5). However this was due to that farm having a much greater sand bed area (4 x 60 m2 beds studied). Although the very practical design at Farm A facilitated the construction of a much greater area of sand bed (600 m2 in total at the farm), the lower profile sides reduced the head pressure that could be generated to push water through the bed. As a result, a lower rate of treatment per square metre of sand bed



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could be achieved. During the 127-d monitoring period at Farm A, the average amount of pond water filtered by each square metre of sand each day was 315 L.

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‐2) Farm A Bed 1

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Farm A Bed 4Farm B

Farm C

Figure 5. Pond-water volumes filtered at each farm on a square metre of sand basis Unfortunately, the sand bed at Farm B was compromised some time in early April when the pump intake became lodged in anaerobic silt. No water volumes or samples taken thereafter were included in the study, but up until the 30/3/10 that PASF had performed well and had filtered over 5.7 megalitres. During the 116-d monitoring period at Farm B the average pondwater filtration rate was 857 L m-2 d-1. The sand bed at Farm C treated the most pond water during the trial, both in terms of total volume filtered (18.8 megalitres) and total volume per square metre of sand (0.317 megalitres). Its much longer term of operation (319 d) contributed to this. On average that bed treated about 59,000 L per day which equated with about 1000 L for each square metre of sand each day. Unlike the sand bed management applied in the previous trials, where routine raking of bed surfaces was undertaken during the first month to break surface crusts and maintain water flows, no disturbances were generally applied to the beds in this trial. Raking was undertaken only once after 40 days at Farm B, but in this case it was later found that the outlet flow meter was the point of flow restriction causing slower than usual filtration rates. After the blocked flow meter was removed flow dynamics appeared to return to normal, and since the pond was not regularly overflowing, the inflow meter readings were used as a proxy for outflow measurements. Outflow meter blockages also occurred at Farm C, necessitating a similar approach of inflow readings as a proxy for that of the outflow. The sand beds at all farms in the trial developed a surface layer of filtrate and benthic algae on which the worms appeared to graze. At depth the beds developed the typical mottled pattern of aerobic and anaerobic sediments which has been demonstrated in previous work under normal operating conditions. The worm’s burrowing activities aerated portions of the sediment by allowing water to flow more freely into pockets under the surface. Sprinkling fish meal on the flooded bed encouraged the worms to feed on the surface (Figure 6). This was in contrast to the bed at Farm B after it was discovered to have handled a significant amount of anaerobic sludge. In this case all of the worms had moved to the very surface of the bed and were obviously showing



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signs of stress, and the sediment was more heavily reduced at depth and produced a mild sulphur dioxide odour when disturbed (Figure 6).

A B

C D

Figure 6. Sand beds at Farm A after normal operations (A: benthic algal growth; B: worms feeding on the surface) and Farm B after a period of sub-optimal operation (C: undisturbed surface; D: reduced sediments at depth). Despite remedial actions on the compromised bed at Farm B, involving the installation of air lifts on each drainage pipe designed to increase water flow through the sand to oxidise the sulphides, the bed did not recover after a further 10 days of recirculated operation (no further organic inputs). At that time it was drained, limed, and dried, and no worms were harvested.

Water qualities and nutrients Operations at Farms A and B were restricted to summer conditions so remained in the 24-29 °C range of water temperatures. The PASF at Farm C was operated through summer and winter and so had a broader range of water temperatures (16-29 °C). Although the filtered outflow was significantly (P<0.001) cooler than the pond water inflow at Farm A, this difference did not occur at Farms B or C (Figure 7).



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Water temperature (°C)

Farm A In Farm A Out

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Figure 7. Water temperatures of pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Means (± se) shown for beds (n=4) at Farm A. A similar range of salinities prevailed at the same times of year at each of the farms (Figure 8). They were particularly high at the beginning of 2010 but by March had declined to more moderate levels (approx 25 ppt) where they persisted for the remainder of the trial. Salinities were not affected by supplemental feeding at Farm A, or by the sand filtration process at any of the farms.

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Salinity (ppt)




Figure 8. Salinities of pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Means (± se) shown for beds (n=4) at Farm A. Dissolved oxygen levels at Farm A were much lower (P<0.001) in filtered outflow than in pond inflow. This significant DO reduction (3-4 mg L-1) was also demonstrated in most samples taken at Farms B and C (Figure 9). Feeding the beds with fish meal appears to have lowered (P<0.05) the DO of outflow water at one time point (7 Jan 2010 at Farm A). The data presented for Farms B and C may also have slightly underestimated the DO drop through the beds, since the readings were not taken directly adjacent to the outflow stream outlet.



14

The pH levels of filtered outflow were also significantly lower (P<0.001) than the pond-sourced inflow at Farm A, and again, this pattern was reflected in the majority of results at Farms B and C (Figure 9). Although the fed beds consistently gave the lowest pH values at each time point in the study at Farm A this supplemental feeding did not significantly (P=0.08) affect the results. The sand bed filters at Farm A had a pronounced effect on TN over time (P<0.001) and there was a significant interaction with supplemental feeding (P<0.05). Total nitrogen levels in filtered outflows were significantly reduced (P<0.05) for both fed and unfed beds, and outflow TN from beds receiving supplemental feed was higher (P<0.05) than that from unfed beds (Figure 10). The average TN removals at Farm A were 14.6 % and 27.7 % by fed and unfed beds, respectively, whilst the maximum TN removal recorded at that farm was 48.8 % (by unfed Bed 2 on 24 Dec 2009). Similarly, TP levels in the filtered outflow from Farm A were lower (P<0.05) than the pond-sourced inflow for all samples taken after mid-January 2010 (Figure 10). Samples taken before this showed no TP differences (P>0.05) between inflows and outflows, except the first sample taken on 10 December 2009 which showed an anomaly of much higher (P<0.05) outflow than inflow. Averaged data from Farm A showed that the mean TP level in the outflow from unfed beds (0.082 mg L-1) was lower (P<0.05) than all mean inflow levels (0.123-0.124 mg L-1), and was also lower (P<0.05) than the mean outflow level from fed beds (0.115 mg L-1). The average TP removals at Farm A (excluding the first sampling point2) were 14.1 % and 37.2 % by fed and unfed beds, respectively, whilst the maximum TP removal recorded at that farm was 52.7 % (by unfed Bed 2 on 21 Jan 2010). These general patterns of nitrogen and phosphorus removal were also evident in the results from Farms B and C (Figure 10). Average TN removals were 20.0 % and 22.5 % at Farms B and C, respectively, whilst the maximum TN removals at these two farms were 46.9 % (on 19 Jan 2010) and 40.8 % (on 12 May 2010). Average TP removals were 22.8 % and 40.8 % at Farms B and C, respectively, whilst maximum TP removals at these respective farms were 55.7 % (on 16 Feb 2010) and 67.5 % (on 9 June 2010). Levels of TSS were not affected (P>0.05) by supplemental feeding, but were significantly (P<0.05) reduced by sand bed filtration at Farm A. This reduction was also very evident in all of the results from Farms B and C (Figure 11). On most occasions the TSS levels in the filtered outflow were lower than the detection levels of the testing laboratory (<5 mg L-1) and thus were assigned a zero in the analyses. Chlorophyll a levels were not significantly affected by the supplemental feeding at Farm A, but were shown to be significantly reduced (P<0.001) by the sand filtration process on all but the first sample date. Again, the results at Farm B and C reflected this pattern of pronounced Chla removal (Figure 12). Average removal rates were 66.6 %, 57.3 % and 81.7 % at Farms A, B and C, respectively, whilst maximum removal rates were 80.0 % (on 4 Feb 2010), 84.4 % (on 2 Feb 2010) and 93.3 % (on 21 July 2010) at these respective farms.

2 TP data collected for the first sample day is discounted as an anomaly in percentage removal summaries, since outflow levels were uncharacteristically much higher than the inflow.



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Figure 9. Dissolved oxygen and pH levels in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se) levels for fed and unfed beds (n=2) shown for Farm A.


_______________________________ © The State of Queensland, Department of Employment, Economic Development and Innovation, 2011

16

____________________________________________________

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Figure 10. Total nitrogen and total phosphorus levels in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se) levels for fed and unfed beds (n=2) shown for Farm A.



17

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Total suspended solids (m

g L‐1) Farm A In Farm A Out



Figure 11. Total suspended solids levels in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se, n=4) levels shown for Farm A.

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Chlorophyll a

(µg L‐1)




Figure 12. Chlorophyll a levels in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se, n=4) levels shown for Farm A. The ability of the polychaete sand filter system to remove particulate matter including silt and plankton from pond water was also easily demonstrated at all farms through simple visual comparison (Figure 13). These visual differences were apparent on a continuous basis at all farms and across a range of different pond bloom conditions. Total ammonia nitrogen (TAN) levels were not significantly affected (P=0.07) by supplemental feeding at Farm A, but were affected (P<0.05) by the sand filtration process to varying degrees over time during the study. Whilst the outflow at Farm A was more consistently higher than the inflow, this pattern was not seen in the results from the other farms (Figure 14).



18

Figure 13. Visual evidence of the difference between unfiltered (left) and filtered (right) pond water. Time had a less pronounced effect on nitrite nitrogen levels at Farm A and it did not significantly interact with supplemental feeding or sand filtration. Although the mean level of nitrite nitrogen in outflows from fed beds (0.0165 mg L-1) was higher than the levels of inflows and the outflow from unfed beds (0.0035-0.0067 mg L-1) no consistent patterns were apparent in nitrite nitrogen results at all three farms (Figure 14). Nitrate nitrogen levels ranged from 0 to 0.2 mg L-1 across all the farms in the study, and there were no consistent patterns produced (Figure 15). Although time had the most pronounced effect on nitrate nitrogen levels at Farm A (P=0.21) this and the effects of supplemental feeding and sand filtration were not significant (P>0.05). Phosphate phosphorus was not significantly affected by supplemental feeding at Farm A but there was a significant interaction (P=0.01) of time and sand filtration. Although outflow levels were always above inflow levels at that farm, this was only significant (P<0.05) on the first sample day (10 Dec 2009). Phosphate levels in the outflow were also consistently higher than the inflow at Farm B, but this pattern did not hold at Farm C (see Figure 16). Supplemental feeding at Farm A did not significantly influence the sulphide levels detected in the trial. Time was the only factor that produced a significant effect (P=0.03) with a somewhat inconsistent increasing trend at Farm A over time. Sulphide levels ranged from 0 to 0.13 mg L-1 in the pond water inflows and from 0.06 to 0.14 mg L-1 in the sand bed outflows across all the farms. There were no clear patterns of sulphide occurrence or production in any of the farms’ results (Figure 17).


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Figure 14. Total ammonia nitrogen and nitrite nitrogen in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se) levels for fed and unfed beds (n=2) shown for Farm A. (Note the smaller scale for Farm A nitrite nitrogen).


19



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Nitrate nitrogen (mg L‐1)

Farm A In Farm A OutFarm B In Farm B OutFarm C In Farm C Out

Figure 15. Nitrate nitrogen levels in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se, n=4) levels shown for Farm A.

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Phosphate phosphorus (m

g L‐1) Farm A In Farm A Out

Farm B In In Farm B Out


Figure 16. Phosphate phosphorus levels in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se, n=4) levels shown for Farm A.

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Sulphide (m

g L

‐1)

Farm A In Farm A Out Farm B In

Farm B Out Farm C In Farm C Out

Figure 17. Sulphide levels in pond-sourced inflow and filtered outflow from polychaete sand filter beds operated at Farms A, B and C. Mean (± se, n=4) levels shown for Farm A.



21

The flow rates that were measured during intensive sampling at Farms B and C during one entire artificial tide are presented in Figure 18. Despite using similar sized pumps, the shorter distance between the pump and the sand bed at Farm B created lower resistance in the pipe and produced a faster pumping rate than at Farm C (222 L min-1 at Farm B compared with 182 L min-1 at Farm C). Farm C also demonstrated a slower filtration rate through the sand bed (maximum of 70.6 L min-1 versus 108 L min-1) which produced a longer period of water discharge from the bed (370 min verses 70 min at Farm B).

0

50

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250

0 31 60 91 121 152 182 213 244Time after inflow started (min)

Flow rate (L min

‐1) Inflow

OutflowFarm B

0

50

100

150

200

250

0 60 121 182 244 305 366Time after inflow started (min)

Flow rate (L min

‐1)

InflowOutflow

Farm C

Figure 18. Flow rates of pond-sourced inflows and filtered outflows from polychaete sand filter beds operated at Farms B and C during a period of intensive water sampling and over an entire artificial tide. Inflow water quality parameters measured at the start of each tidal simulation are presented in Table 3, and were assumed to remain relatively constant during the entire inflow period at each Farm. Outflow measurements are provided in Figure 19. Levels of both TN and TAN in the filtered outflow showed distinct peaks during the

arm C for the first hour or so. After these peaks TN and TAN levels declined ).

es om starting levels, but this was again less pronounced at Farm C. At both farms DO

first hour after the filter began to operate (Figure 19). At both farms TN peaked for a short period at a higher level than the inflow levels (see also Table 3). TAN remained above the inflow level for the whole tidal flow at Farm B, but this only occurred at Fsignificantly and remained more constant for the rest of the tidal period (Figure 19Nitrate nitrogen also appeared to peak during the first hour at Farm B, but this was not detected at Farm C. Other dissolved nutrients (PO4P and NO2N) showed declinfrlevels in the outflow rose sharply during the first hour after the start of the filtration period, whilst salinity was unaffected, and pH and temperature generally showed veryslow inclines. Table 3. Water quality measurements for inflows during the tidal sampling program.

Parameter Farm B Farm C Total nitrogen (mg L-1) 1.8 1.3 Total phosphorus (mg L-1) 0.13 0.12 Phosphate phosphorus (mg L-1) 0 0.066 Total ammonia nitrogen (mg L-1) 0.03 0.05 Nitrite nitrogen (mg L-1) 0.014 0.128 Nitrate nitrogen (mg L-1) 0.028 0.180 pH 7.68 7.88 Dissolved oxygen (mg L-1) 6.5 7.0 Temperature (°C) 24.7 18.6 Salinity (g L-1) 21.9 34.0



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O2N

and NO3N (mg L‐1)

TN TP PO4P

TAN NO2N NO3N

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40

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ity (ppt)

alin

nd SapH DO

Temp Sal

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Figure 19. Water quality parameters prevailing in filtered outflow from polychaete sand filter beds operated at Farms B and C during a period of intensive water sampling and over an entire artificial tide.


__________________________________________________________________________________© The State of Queensland, Department of Employment, Economic Development and Innovation, 2011

23

Polychaete survival, biomass production and harvest Despite the acknowledged imprecision associated with enumerating juveniles involved with stocking procedures (Palmer 2010), survival estimates for polychaetes stocked into each of the sand filtration beds in the trial were undertaken under the assumption that 2000 juveniles m-2 were in fact stocked. Quantities of worms recovered from the 1 m2 transects at Farm A and B in March 2010, and at Farm C in July 2010 are presented in Table 4. After 3.7 months in the filtration beds, fish-meal-supplemented worms had a mean (±se) density of 152 ± 35 g m-2, compared with 89 ± 17 g m-2 for un-supplemented worms. Analysis of these data suggests that the supplemental feeding at Farm A did not significantly (P=0.25) affect the wet weights of worms harvested from the experimental transects. However, with only two replicates of each treatment the statistical power of this analysis was low. The overall average weight experimentally harvested at Farm A (120 ± 34 g m-2) was similar to the production measured over a similar time frame at Farm B (Table 4), where no supplemental feeding had occurredFarm C also appears to have been tracking along a similar production pathway with afurther (approx) 50 g m-2 produced over an additional 1.5-month period. Survival estimates at Farm A were also not significantly affected by supplemental feeding (27.8 ± 5.7 % verses 34.2 ± 4.0 % for fed and unfed beds, respectively). The low number of replicates again likely reduced the potential to demonstrate the statistical significance these results. The overall mean survival at Farm A (31.0 ± 4.8 %) was however substantially lower than the survival estimates for Farms B and C (see Table 4). Table 4. Survival estimates and experimental harvest (1 m2) results for polychaete sand filter beds at Farms A, B and C. Farm Bed Polychaete age

(months) Harvested

quantity (g) Harvested number

Survival (%)

_

.

1 4.7 116.7 442 22.1 2 4.7 71.9 605 30.3 3 4.7 187.0 670 33.5

A

4 4.7 105.3 763 38.2 B 1 5.0 124.0 1510 75.5 C 1 6.4 175.5 1009 50.5

Although no worms were harvested from Farm B at the end of the trial, due to operational failures (see Materials and Methods section), final harvest estimates at Farms A and C3 were similar to or in slight excess of those production levels reportein previous work (300-400 g m-2: Palmer 2010). Commercial harvests of worms for both bait and broodstock feeds proceeded at both of these farms and were complete bJanuary 2011. Harvest equipment developed and demonstrated in the work (Figure 20) produced high-quality products that were well accepted in several local and interstate markets. Worms harvested from the trial were fed to broodstock black tigerprawns (Penaeus monodon) and sand whiting (Sillago ciliata) at two different commercial hatcheries with excellent overall spawning results in both cases.

d

y

3 Final harvest figures from each farm were treated as commercial in confidence.



24

A B

C D

Figure 20. Harvesting Perinereis helleri from sand beds (A: dredge harvester; B: demonstration harvest; C: before separation from mucus-laden sand; D: worm biomass after separation).

olychaete biomass analyP ses Endemic marine prawn viruses The live worm samples provided from each farm for analysis tested negative for

f f f arm A, and one sample from e bed and ther sam ken at the sa time as the Farm A ples, from at BIRC wand raw-sea ater source as also test ve. In all c the PCR positive controls were positive and the negative controls were negative. Proximal and nutritional components

GAV, IHHNV and MoV. This included one sample rom each oC. ur

our beds at Feac f thh o s at s B Farm A f ple ta

me sam a bed hich treated a range of pond- w s, w negati ases

from

ls than otal

The moisture content of frozen/thawed worm samples submitted for analysis ranged from 77.7 to 81.7%. The percentage of ash in the dry matter of samples ranged 10.2 to 14%, and for gross energy from 20.23 to 21.45 MJ kg-1. Variable levels of crude fat (hexane extract) were present in the dry matter of samples from different farms and beds, whereas the levels of nitrogen and phosphorus were more consistent(see Table 5). Total lipid results (Table 6) generally reflected higher percentages of dry matter compared with the levels of crude fat determined in the proximal analyses4, but a similar pattern across farms where worms from beds at Farm A had higher leveat the other farms, and where Farm C was the lowest. Neither cholesterol nor t

4 Crude fat analyses involved hexane soluble material extracted under reflux, whereas total lipid analyses are more rigorous and include free fatty acids, triacyglycerols and membrane bound phospholipids.



25

phospholipids levels reflected this pattern. Ch terol levels ranged from 0.86% to 1.03% of dry matter, whilst total phospholipids range from 0.41% to 0.72%. Of the phospholipids present phosphatidylcholine was most heavily represented, followed by lyso-phosphatidylcholine and then phosphatidyl-ethanolamine (Table 6). Sphingomyelin was only detected at low levels in one sample from Farm A, and levels of phosphatidylinositol and phosphatidylserine were consistently below the detection level of the analysis (<0.005%). Table 5. Proximal analyses for worm biomass from different beds at each farm. Farm sample

Bed treatment1

Dry matter (%)

Ash (%)

Gross energy (MJ kg-1)

Fat (%)

Nitrogen (%)

Phosphorus(%)

oles

FarmB

A ed 1

Fed 22.3 10.2 21.45 7.6 10.59 0.78

FaBe

Unfed 20.1 12.2 7 8.1 9.98 0.76 rm A d 2

20.8

Farm A Bed 3

Fed 21.9 10.7 21.33 7.0 10.13 0.75

Farm A Bed 4

Unfed 21.6 11.4 21.04 9.2 10.74 0.79

Farm B

Unfed 18.3 14.0 20.23 6.1 11.17 0.82

Farm C

Unfed 20.5 12.3 20.3 5.0 11.55 0.78

1In relation to supplemental feeding

1 phosphor-lipids

A B C hosL D

Table 6. Total lipid, cholesterol and phospholip s analyses expressed as a % of dry sample for worm biomass from different beds at each farm. Farm Bed Total Cholest- Total PhosL PhosL PhosL P

id

sample treatment lipid erol

Farm A Fed 13.4 Bed 1

0.88 0.484 0.122 0.233 <0.005 0.128

Farm A Bed 2

Unfed 12.9

0.963

0.568

0.058

0.313

<0.005

0.198

Farm A Bed 3

Fed 12.6

0.858

0.621

0.125

0.334

<0.005

0.161

Farm A Bed 4

Unfed 12.5

0.973

0.627

0.038

0.356

0.036

0.196

Farm B Unfed 10.2 1.032 0.718 0.104 0.3

65

<0.005

0.249

Farm C Unfed 9.1 0.858

0.406

<0.005

0.156

<0.005

0.249

1In relation to supplemental feeding; PhosL A = Phosphatidylethanolamine; PhosL B = phosphatidylcholine; PhosL C = Sphingomyelin; PhosL D = Lyso-Phosphatidylcholine. Thirty-one different fatty acids were present at detectable levels (≥0.005%) in the dry matter of worm biomass sampled in the study (Table 7). The mean level of total fatty cids across alla beds in the study was 4.98 ± 0.47% of dry matter. In general the

ere

e later three.

pattern displayed by fats and total lipid analyses held true for most fatty acids, whFarm A generally had the highest levels followed by Farm B and then Farm C. Notable exceptions to this pattern were arachidic acid (20:0), gadoleic acid (20:1n-11) and behenic acid (22:0), but these all occurred at relatively low levels. Palmitic acid (16:0), stearidonic acid (18:4n-3) and docosahexaenoic acid (DHA) (22:6n-3) were noticeably higher in fed beds, as were oleic acid (18:1n-9), cis-5,11-eicosadienoic acid (listed as 20:1n-6) and docosapentaenoic acid (22:5n-6), though to a lesser degree in thes



26

d Table 7. Fatty acid analyses expressed as a % of dry sample for worm biomass from different feand unfed1 beds at each farm. Fatty acid

Farm A Fed bed 1

Farm A Unfed bed 2

Farm A Fed bed 3

Farm A Unfed bed 4

Farm B Unfed bed

Farm C Unfed bed

14:0 0.076 0.079 0.059 0.082 0.021 0.013 14:1n-5 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 15:0 0.058 0.059 0.053 0.063 0.028 0.019 16:0 1.736 1.291 1.651 1.178 0.802 0.598 16:1n-7 0.303 0.241 0.249 0.252 0.100 0.068 17:0 0.184 0.201 0.164 0.192 0.117 0.081 17:1n-8 5 .0 05<0.00 <0.005 <0 05 <0.0 <0.005 <0.005 18:0 0.570 0.468 0.52 7 6 0.45 0.382 0.327 18:1n-13? 5 0.202 0.188 00.194 0.20 .168 0.138 18:1n-9 0.339 0. 5 0.299 0.135 0. 0.12 085 064 18:1n-7 0.432 0.495 0.389 0.513 0.242 0.224 18:2n-6 0.199 0.096 0.169 0.089 0.100 0.078 19:0 0.021 0 0.018 0.016 < < 0.02 0.005 0.00518:3n-3 0.153 0.168 0.121 0.110 0.044 0.078 18:4n-3 0.052 0.018 0.046 0.017 < < 0.005 0.00520:0 0.042 0.046 0.038 0.036 0.043 0.090 20:1n-11 5 0.083 0.108 00.080 0.11 .094 0.050 20:1n-9 0.183 0.121 0.169 0.111 0.066 0.063 20:1n-7 0.016 0.029 0.015 0.039 <0.005 0.010 20:1n-6 0.153 0.093 0.153 0.083 0.041 0.032 2?

20:2n-6 0.157 0.123 0.140 0.105 0.077 0.070 20:3n-6 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 20:4n-6 0.246 0.201 0.224 0.209 0.231 0.197 20:3n-3 0.015 0.032 0.013 0.018 <0.005 0.010 20:4n-3 5 0 <0.0 05 <0<0.00 <0.0 5 05 <0.005 <0.0 .005 20:5n-3 0.473 0.538 0 0. 431 0.38.462 583 0. 1 22:0 0.018 <0.005 0.01 <0.005 <0.005 <0.005 5 22:1n-11 5 <0.005 <0.0 < 0 <0<0.00 05 0.005 <0.0 5 .005 22:1n-9 0.015 0.010 0.015 0.0 005 <0.0011 <0. 5 22:1n-63? 4 0.2 1 0.0.285 0.29 98 0.288 0.30 193 22:4n-6 0.307 0.376 0.291 0.4 0.411 0.296 03 22:3n-3 <0.005 .005 <0.0 < 0 <0<0 05 0.005 <0.0 5 .005 22:5n-6 0.043 0.014 0.042 0.0 005 0.011 14 <0.24:0 <0.005 .005 <0.0 < 005 <0<0 05 0.005 <0. .005 22:5n-3 0.118 0.142 0.115 0.1 0.100 0.089 50 22:6n-3 0.266 0.053 0.2 3 0.61 0.070 0.04 052 24:1n-9 0.032 <0.005 0.029 <0. 5 005 <0.0000 <0. 5 TOTAL 6.768 5.650 6.3 5 3.08 5.523 3.92 232 1In relation to supplemental feeding; 2cis-5,11-eicosadienoic acid; s-7,13-d osadieno acid;

tra of W.W.

ted

ortion

h

3ci oc ic?Likely identification from gas chromatography mass spectrometry and based on the specChristie, Scottish Crop Research Institute, Dundee, Scotland. The 12 most heavily represented fatty acids are listed in Table 8 in order of their meanoccurrence. Palmitic acid was by far the most prevalent fatty acid in the study. Its mean representation was more than double that of the next most heavily represenfatty acid, EPA. Considering the AA:EPA:DHA complex that is often considered in marine fish nutrition, arachidonic acid (AA) had the most stable dry matter propin worm samples taken across all six beds in the study (see standard errors in Table 8), so it was used for conversion to compare these ratios between different farms and treatments (Table 9). These results suggest that the supplemental feeding of beds witfish meal increased the DHA (docosahexaenoic acid) content of the worms considerably, but that this may have been at the expense of their EPA content.



27

les across all 6 beds in the study. atte

Table 8. Mean (± se) representations (% of dry matter) of the 12 most prevalent fatty acids detected in worm biomass samp

Dry m r % Lipid numbe mon r Com name Mean ± se

16 Palmitic ac 1id .209 0.184 20:5n-3 Eicosapen acid (Etaenoic PA) 0.478 0.030 18 Stearic aci 0 d .455 0.03718:1n-7 cenic a 0.383 Vac cid 0.05122:4n-6 Adrenic ac 0.347 id 0.02322:1n-61 sadie id 0 Doco noic ac .276 0.01720:4n-6 Arachidon A) ic acid (A 0.218 0.008 16:1n-7 mitoleic 0.202 Pal acid 0.03918:1n-131 0 .182 0.01018:1n-9 Oleic acid 0.174 0.04717 Margaric a 0 cid .157 0.01922:6n-3 Docosahex acid (D 0 aenoic HA) .124 0.0441See Table 7 fo cation

Mean donic ac ): eicosa noic aci ): doco oic aci ) in worm ss from different fed an d1 beds A as th compar

d trea A E D

r clarifi Table 9. arachi id (AA pentae d (EPA sahexaen d (DHAratios bioma d unfe using A e equal ator. Worm be tment A PA HA Across all beds ms (n = 2 0. at 3 far 6) 1 .19 57 Fed beds at Far 2) 1 1m A (n = 1 .99 .12 Unfed beds at F (n = 2) 2 0.arm A 1 .73 30 Unfed beds at F and C ( 1 0.arms B n = 2) 1 .90 22 1Fed and unfed with a ut supp feedin sh mea ively.

ino ac tents o s from bed in are presented in Table tamic acid, glycine and serine appeared to be y high fed b

fed be lutam ad the st repr ion of no acer w ycine was the most e between samples 11). Fcids, s from and/o C had r leve orm

. Ami nalyse sed as ry sam m b om different fed1 t each fa

o acid A ed 1

A bed 2

A ed 3

A bed 4

B d

C bed

refers to nd witho lemental g with fi l, respect The am id con f worm each the study10. Glu

in tlsligh e nr in u eds

than ds. G ic acid h highe esentat all ami ids in dry matt hilst gl variabl (Table or most amino a worm Farm B r Farm highe ls than w s from Farm A. Table 10 no acid a s expres a % of d ple for wor iomass frfed and un beds a rm. Amin Farm

Fed bFarm

UnfedFarm

Fed bFarm

UnfedFarm

Unfed beFarm

UnfedAlanine 4.259 5 9 5 89 8 4.37 4.14 4.27 4.5 3.86Arginine 3 3 3 8 7 6 4.47 4.43 4.22 4.33 4.99 4.53Aspartic acid 9 7 2 8 99 3 5.38 5.35 5.16 5.54 5.7 5.92Cystine 0.765 0.780 0.785 0.773 0.785 0.872 Glutamic acid 8.093 8.517 7.849 8.624 9.432 8.789 Glycine 3.795 4.732 3.724 4.711 5.586 6.914 Histidine 1.387 1.310 1.361 1.350 1.480 1.436 Isoleucine 2.198 2.160 2.200 2.201 2.500 2.422 Leucine 3.842 3.790 3.891 3.877 4.435 4.357 Lysine 3.706 3.513 3.698 3.719 4.335 4.020 Methionine 1.194 1.098 1.253 1.169 1.180 1.379 Phenylalanine 2.116 2.040 2.105 2.099 2.376 2.297 Proline 2.554 2.595 2.522 2.652 2.792 2.813 Serine 2.279 2.459 2.255 2.491 2.826 2.581 Threonine 2.609 2.585 2.555 2.606 2.804 2.795 Tryptophan 0.685 0.614 0.705 0.629 0.679 0.697 Tyrosine 1.952 1.805 1.889 1.903 2.153 2.037 Valine 2.496 2.412 2.410 2.480 2.759 2.581 1In relation to supplemental feeding



28

ids

se

Table 11. Mean (± se) representations (% of dry matter in descending order) of the amino acdetected in worm biomass samples across all 6 beds in the study. Amino acid mean Glutamic acid 8.551 0.227 Aspartic acid 0.117 5.530 Glycine 4.910 0.490 Arginine 0.109 4.500 Alanine 4.252 0.098 Leucine 4.032 0.116 Lysine 3.832 0.121 Threonine 2.659 0.045 Proline 2.655 0.050 Valine 2.523 0.054 Serine 2.482 0.086 Isoleucine 0.058 2.280 Phenylalanine 2.172 0.054 Tyrosine 1.957 0.050 Histidine 1.387 0.025 Methionine 1.212 0.039 Cystine 0.793 0.016 Tryptophan 0.668 0.015

Discussion The water treatment efficacies demonstrated by PASFs in the present study were etter than expected from previous work at smaller scales (Palmer, 2010). TSS and

eds that did not receive supplemental feeding) were 20-28% and 23-41%, spectively. Additionally, these removal efficiencies were likely underestimated in

iscussed below).

sults c vo tr s si s mercially proven me tilised tralia. ample, n et a

ted al effic in set t pond to 60% SS, ur TN, and 30% fo By com on, the e remo ficaci

n in esent s see par above better e bes efficacies of settleme ds. Th achiev g shor atmen

aller po of ove rm area with s ant val by-promass) ction. T PASF r also co favou ith o

porary water treatmen iques s fluidis i hich , TAN biologi ygen d but ha wn little TN or TP

ids l. 2008

p ylene ( ) liners e outeof po his is n ry to co the san and as d wataterials, and to separate the cultured worms a ds from rlying

nts. The H liners’ erm du y and v ity to b ked inpletely water-proof seal makes it an ideal choice for

bChla were typically removed with a very high level of efficiency (up to 100% for TSS; and up to 93% for Chla) as was previously demonstrated. However, nitrogen and phosphorus removal appears to have been substantially improved in these larger-scale PASFs. Up to 49% TN, and up to 68% TP were removed from treated wastewaters during the trial, where the average N and P removals across the entire trial (for brethe trial due to the tidal patterns of dissolved nutrient flushing demonstrated in the intensive sampling programs (d These re om fapare ura ithbly w ea intment ettl t baemen ns h i whic the only com thod u in Aus For ex Presto l. (2000) repor remov iencies tlemen s of up for T p to 20% fo up to r TP. paris averag val ef es of PASFs show the pr tudy ( agraph ) were than th t removal nt pon is was ed usin ter tre t times, sm rtions rall fa s, and ignific uable duct (worm bio produ hese esults mpare rably w ther contem t techn uch as ed sand biof lters wreduce TSS and cal ox emand ve shoremoval (Dav on et a ). All farmers preferred to use high density olyeth HDPE for th r membrane

mnds. T ecessa ntain d bed sociate er

handling nd be unde sedime DPE long-t rabilit ersatil e wor to any shape and welded for a com



29

re must be taken when

s and harp-ed ols so that the liner is not punctured.

howed e desig racteristics of the ponds affect their water abilities. allowe plemented at Farm A could only filter 315 , compared with up to 1000 L m-2 d-1 using the deeper design. None of the

uld handle es dem ted in the previous smaller scale experiments -2 d-1) (Pal 10), al if this was necessary longer pumping times sumably h een use r handling and nutrient results show that

should be e ed whe apolating results from small scale trials to large ystems, and suggests that further optimisation work should be undertaken at

e.

tly, the da ented he intensive sampling of outflows during entire s sugge t the levels of TN removed from pond waters during the underes d by th ightly monitoring program. Routinely, samples taken m y through the inflow period, which is when the

N levels in the outflow were shown to peak. This is likely due to the flushing of dissolved nutrients that have been remineralised during the low tide period when no

ng through the sand bed. Bacterial breakdown of organic deposits

s

ords, greater efficiencies

t eventually floated and accumulated in the corners of e

this use. The main drawbacks of this method of construction are that heavy machinery(bobcats) invariably cannot be operated over it, and that causing shovel other s ged to

y sThe stud how th n chahandling The sh r design imL m-2 d-1

beds co the rat onstra(1500 m

remer 20 though

could p ave b d. The watecaution xercis n extrscale slarge scal Importan ta pres from ttidal period

erests tha

study w timate e fortnfortnightly were idwaT

water is percolatiapparently continues during these low flow conditions, and its by-products of dissolved nutrients are concentrated in the sediments whilst little flushing is taking place. As suggested in the earlier work (Palmer 2010), this predictable nutrient-concentration change presents the opportunity to optimise nutrient recovery and recirculated culture systems. For example, by selectively directing PASF-filtered waters towards macrophyte (eg. seaweed) culture beds when dissolved nutrients are high, maximum growth of plant biomass and nutrient recovery could be achieved. Then, by alternatively directing the PASF-filtered waters back to source culture pondwhen more desirable (lower nutrient) water qualities prevail, the most effective

ilution of eutrophic conditions could be achieved. In other wdwould be possible by selectively redirecting different stages of the tidal outflow. Since automatic timers control the pumps that drive the PASF tidal periods, linkages to water controlling devices (eg. solenoid valves) that could provide this functional partitioning of filtered waters could be simply achieved. One feature of the beds that was noticeable in the present study was the periodic growth of benthic algae on the surface of the beds, particularly after the beds had been submerged without drying for several days. At times this was necessary to avoid the lethal effect on the worms of freshwater from rainfall. Vulnerable (uncovered) beds (at Farm A and C) were kept submerged using external standpipes during wet weather, and although this permitted continued water filtration, it often caused a

roliferation of benthic algae thapthe beds. By comparison, dry-weather operations incorporated the complete drainagof beds two or three times each day. In this case pumping rates and timers were adjusted so that an extended low tide period (beds completely drained) occurred on fine-weather afternoons. Two or three hours of direct sunlight tended to bake the surface sand of the bed dry, which helped to reduce the growth of algae and prevent



30

ong-term operational plans for the PASF system involve the reuse of the same sand d in

a

ies experienced at Farm B towards the later part of the prawn production ason demonstrated the need for well-engineered mechanisms in this relatively harsh

levels

space and the creation of low oxygen conditions. More direct management is

estowed upon growout ponds in terms of water qualities, and they are also less polychaetes

study n

s

e

sms of meters and lead to lse readings. To address this, meters on the bed inflows provided the volume

cept

cess

or

the proliferation of a range of other sub-tidal organisms that would have otherwise colonised the bed. Leach year. After each crop of worms has grown to full capacity, beds are harvestea process that washes and cleans the sand and piles it on one side of the lined pond for complete drainage. It is then dried, respread evenly across the ponded area, plumbedwith drainage pipes and restocked with juvenile worms. Since Farm A had producedpilot crop of worms the season before the present study, the results from this farm demonstrates the water treatment efficacies that can be expected when reusing sand inthis way. The difficultseand dynamic environment. Fouling organisms like tube worms and barnacles slowly build on most solid surfaces that are in regular contact with pond waters, which creates shifting weight balances that can unsettle structures with unsteady foundations. In this case the pump intake sank into anaerobic mud causing contamination of the sand bed. Depending on design characteristics it may also be advisable to draw water for PASFsystems directly from growout ponds rather than from settlement ponds. High of inorganic silt are not tolerated for long periods by the worm beds due to cloggingof pore bimpacted by heavy rainfall events which can adversely affect the culturedthrough lowered salinities. Furthermore, the occasional pump failures during the highlighted the pitfalls of completely automatic systems. Whilst the worm beds catolerate prolonged periods of exposure, regular observation and maintenance is an absolute requirement of all aquaculture systems. Another difficulty experienced during the study was the reliability of flow meterused to measure PASF discharge. The gelatinous material (biofilm) that normally builds on the insides of PASF drainage pipes and other surfaces in contact with thfiltered discharge from the sand beds caused clogging and meter failure. Strainers in the meters became blocked with this material and it was considered that removal of the strainers could further compromise the inside mechanifameasurements for the majority of the study. Since the beds seldom overflowed, exwhen excessive rainfall was being channelled to waste, inflow meters were considered to be an acceptable measure of water filtered in the trial. The supplemental feeding of beds at Farm A was studied to identify any water-quality-related issues that may be caused by trying to maximise polychaete productivity whilst also treating pond water. The results suggest that supplemental feeding with fish meal can increase the levels of nitrogen and phosphorus in the filtered water, but that even with this level of supplemental feeding the PASF procan still remove significant amounts of N and P from culture pond discharge. Whilstthere may be several other more sustainable organic materials that could be used fthis purpose with less impact on discharge qualities, the selection of fish meal as a



31

n recently published feeding rates for polychaete culture systems lsewhere in the world (in Thailand: Poltana et al. 2007). However, that particular

nature and contents of this polychaete to particular purposes. In previous ork with P. helleri in PASF systems (Palmer 2008) it was shown that stocking

t of es in their

rce for it provided a suitable supplemental food which,

sed in minimal amounts, boosted the unit area production of polychaete biomass

ustralia. In Queensland the marine orm market for fishing bait and aquaculture feeds is supplied by both beachworm

in er

ulation structures and cosystems (Scaps 2004). To better meet the increasing demand for this valuable

present

er

supplemental feed, and the amounts of fish meal used in the present study were necessarily based oemethod uses clear water supplies which do not contain the natural organic inputs provided by eutrophic pond waters. As a consequence it likely that the levels of supplemental feeding applied in the present study could potentially be lowered to levels that do not affect nutrient outfall but which still enhance polychaete productivity. The nutritional results of this study provide further evidence of the ability to modify or tailor thewdensity affected its harvest size, and that whilst small worms have higher nitrogen, phosphorus and protein levels, larger worms have higher total lipid and fatty acid contents. In the present study it is further shown that feeding fish meal substantially increases their DHA content whilst marginally lowering their proportional contenEPA, and that production in PASFs at different farms can lead to differencnutritional content. Whilst fish meal is not seen as a sustainable nutrient soularge scale polychaete productionufrom PASFs. Dietary effects on the contents of closely related polychaetes have previously been documented (Nereis diversicolor: Costa et al. 2000), and de novo biosynthesis of essential fatty acids for mariculture broodstock from terrestrial plant products have been shown for others (eg. Nereis virens and Arenicola marina: Olive et al. 2002). This suggests that organic materials other than fish meal will also besuitable to boost the worm biomass production levels of PASF systems whilst still making the product suitable for marine fish and crustacean broodstock. The supply of bait worms into local and interstate markets is a relatively dynamic activity that supports several hundred people in Aw(Onuphidae) and bloodworm (Eunicidae) fisheries. Whilst the beachworm fishery Queensland has officially produced between 0.7 and 1.0 million worms per year ovthe last 10 years and has demonstrated a relatively steady output, the bloodworm fishery over the same period shows a steady decline from 1.3 million in 2000 to less than 600,000 in 2008 and 2009 (see Figure 21). Since the main users of this fishery-limited resource (recreational fishers and aquaculture seed producers) are on the increase, pressures on wild stocks are likely to rise in the future. The intensive wild harvest of marine worms in other regions of the world is known to be causing environmental damage in terms of habitats, popeproduct, culture enterprises have been developed in several countries including Australia. Species belonging to the same genus as the species studied in the work (Perinereis) are commercially farmed in Taiwan and Japan (Scaps 2003) and research into their various culture methods has also been conducted in several othcountries including China (Zheng 2000), Thailand (Poltana et al. 2007) and Italy (Prevedelli and Vandini 1997).



32

900,0

1,000,000

1,100,000

1,200,000

1,300,000harvested

500,000

600,000

700,000

800,000

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

Year

Numbe

00r

Beachworms

Bloodworms

Figure 21. Bait worm fishery production in Queensland from 2000 to 2009 (modified from Queensland Government Fisheries data). Other related species from the Family Nereididae have also been investigated andcommercialised including Nereis virens in Europe (Olive 1999) where the bait-wormarket was estimated to be worth 200 million euros in 2004 (Scaps 2004). In Australia, one commercial polychaete farm located in New South Wales has recently begun producing a species from the genus Diopatra (Safarik et al. 2006; O’Sulliva2007), and research is also underway in South Australia where surveys assessing thpotential to profitably cultivate marine worms for bait have returned enc

m

n e

ouraging sults (Davies et al. 2008). The present study has stimulated further investments in

s

d with new

of able

r

d

rethis industry in Queensland, and provides an alternative approach which broadly hasustainable mariculture development at its core.

Conclusion There is clearly an increasing need for the development of sustainable polychaete production systems to provide bait resources for recreational fishing and high quality feed resources for mariculture seed stock production. In Queensland environmental concerns for the expansion of the mariculture industry would be alleviated by effective effluent treatment mechanisms that could be implementedevelopments. Polychaete-assisted sand filters appear to offer both of these key attributes and can practicably be applied at large scales that suitably amplify both of these beneficial effects. This study has successfully demonstrated the application PASFs at large scale using materials that are readily available, affordable, and durunder relatively harsh environmental conditions. Future studies could look for furthewastewater treatment and polychaete production optimisations and seek to better understand the benthic ecology and nutrient cycling that occur in these enhanced sanbed systems.



33

Acknowledgments This research was jointly supported by the Queensland Government’s Department of Employment, Economic Development and Innovation (DEEDI) through their State Aquaculture research programme, and the Australian Government’s Department of Agriculture, Fisheries Forestry through their Caring For Our Country: Landcare Sustainable Practices Grants - Project No. SEQC1418. The author wishes to thank Richard Thaggard, Sizhong Wang and Trevor Borchert for technical support at BIRC and in the field. Also thanks to the Bullock Creek Prawn Farm, Sunrise Seafoods, and Creel Seafoods for fostering this work at their farms, and to David Mayer for biometry assistance and Ian Brock, Michael Gravel and Ian Anderson for worm biomass nutritional and disease analyses.

References Costa, P.F., Narciso, L., da Fonseca, L.C. 2000. Growth, survival and fatty acid profile of Nereis diversicolor (O.F. Muller, 1776) fed on six different diets. Bulletin of

9. Polychaete aquaculture and polychaete science: a mutual biologia 402: 175-183.

pecial

almer, P.J. 2010. Polychaete-assisted sand filters. Aquaculture 306: 369-377. Poltana, P., Lerkitkul, T., Pongtippatee-Taweepreda, P., Asuvapongpattana, S., Wongprasert, K., Sriurairatana, S., Chavadej, J., Sobhon, P., Olive, P.J.W.,

Marine Science 67 (1): 337-343. Davidson, J., Helwig, N., Summerfelt, S.T. 2008. Fluidized sand biofilters used to remove ammonia, biological oxygen demand, total coliform bacteria, and suspended solids from an intensive aquaculture effluent. Aquacultural Engineering 39: 6-15. Davies, S., Mair, G., Harris, J. 2008. Baitshop survey report 2008. Flinders University, Adelaide, South Australia. GenStat. 2011. GenStat for Windows, Release 11.1. VSN International Ltd., Oxford. McCullagh, P., Nelder, J.A. 1989. Generalised Linear Models 2nd edition. Chapman nd Hall, London. a

Olive, P.J.W. 199synergism. Hydro Olive, P.J.W., Craig, S., Cowin, P.B.D., Islam, M.D., Rutherford, G. 2002. The culture of Polychaeta as a contribution to sustainable production of aquafeeds. Aquaculture Europe 2001. Book of Abstracts European Aquaculture Society SPublication No. 32: 392-393. O’Sullivan, D. 2007. Farmed tube worms by the millions! Austasia Aquaculture, December 2007. Palmer, P.J. 2008. Polychaete-assisted sand filters – prawn farm wastewater remediation trial National Landcare Programme Innovation Grant No. 60945 Technical Report. 61 p. P



34

Withyachumnarnkul, B. 2007. Culture and development of the polychaete Perinereis roduction and Development 50 (1): 13-20.

ces in Polychaete Research. ber 2006: 337-341.

de la

in Annelids. Bulletin de la Societe Zoologique de

cf. nuntia. Invertebrate Rep Preston, N., Jackson, C., Thompson, P., Austin, M., Burford, M., Rothlisberg, P. 2000. Prawn farm effluent: composition, origin and treatment. Fisheries Research &Development Corporation: Technical Report No. 95/162. p. 1-71. Prevedelli, D., Vandini, R.Z. 1997. Survival and growth rate of Perinereis rullieri (Polychaeta, Nereididae) under different salinities and diets. Italian Journal of Zoology 64 (2): 135-139. Safarik, M., Redden, A.M., Schreider, M.J. 2006. Density-dependent growth of the

olychaete Diopatra aciculata. Scientific AdvanpDecem Scaps, P. 2003. The exploitation and aquaculture of marine polychaetes. BulletinSociete Zoologique de France 128 (1-2): 21-33.

caps, P. 2004. Commercial tradeSFrance 129 (1-2): 37-48. Zheng, J.B. 2000. A preliminary study on the rearing and propagation of Perinereis

untia. Journal of Jimei University Natural Science 02. n

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