Ministry of Agriculture & Rural Development
PROGRESS REPORT
Intensive in-pond floating raceway production of marine finfish (CARD VIE 062/04)
MILESTONE REPORT NO.5
Development of a zero-discharged system
Report Author: Michael Burke, Tung Hoang & Daniel Willet

1
, and T. Hoang
2
1
Department of Primary Industries and Fisheries, Bribie Island Aquaculture Research Centre, Bribie Island,
Queensland, Australia.
2
Nha Trang University, International Centre for Research and Training, NHATRANG City, Vietnam

Correspondence: Daniel Willett, Bribie Island Aquaculture Research Centre, PO Box 2066 Bribie Island,
Queensland, 4507 Australia.
EXECUTIVE SUMMARY
A major problem with intensified pond-based aquaculture production systems has been
managing water quality and discharge quotas due to the accumulation of waste nutrients.
This is exacerbated in the current CARD project which demonstrated the very high
production capability of in-pond raceways in excess of 35 ton/ha of combined mulloway
and whiting. While the current operation managed water quality through exchanging water
(approximately 10% per day on average – see MS No.4), it is recognised that with water
conservation issues and environmental nutrient discharge impacts, flushing pond water to
waste is a less desirable solution. One of the original goals of this project was to
investigate strategies that limited water discharge to show that raceway production of fish
could be sustainable. This report summarises details of water remediation strategies
investigated to progress towards zero water discharge.
Waste sumps were installed into the raceways as a proposed means for collecting and
concentrating uneaten feed and faeces, thereby reducing nutrients entering the ponds. A
trial tested the effectiveness of these solids traps by comparing Total Solids, TN and TP
collected in the sump with those flowing out of the raceway through the end screens.

to a bio-floc community was also achieved by applying this carbon dose in a replicated
continuous-flow treatment system. The bio-floc community was characterised by lower,
stable pH (8.0-8.2) and DO (6.9-8.8) levels, increased biomass and a decreased proportion
of phytoplankton present. This demonstrated that effluent treated in an external bio-floc
pond would be suitable for recirculation, and a schematic of a proposed integrated
production system is presented.
Of the wastewater remediation strategies investigated in this project, it is evident that bio-
floc treatment was the most promising technology to progress towards zero water
discharge.

INTRODUCTION
A major goal of this CARD project was to develop a pond-based fish production system
that is both sustainable and profitable, designed to increase production and improve stock
4

management efficiencies and ultimately make better use of existing unprofitable
aquaculture pond infrastructure in Australia and Vietnam. The development of low-cost in-
pond Floating Raceways (FRs) in this project has demonstrated an innovative approach to
larval rearing, juvenile nursery and fish growout. As reported in Milestone No.4, the FR
system within a pond a Bribie Island Aquaculture Research Centre demonstrated
production capability in excess of 35 ton/ha of combined mulloway and whiting.

An inherent problem of any pond-based production system is the accumulation of residual
organic matter (uneaten feed, faeces) and toxic inorganic nitrogen (specifically ammonia).
Even the best practices cannot avoid this since it has been shown that fish and shrimp only
assimilate on average about 25% of ingested food – the rest being excreted into the water
column predominately as ammonia (Boyd & Tucker 1998; (Funge-Smith and Briggs 1998;
Hargreaves 1998). This feeds phytoplankton blooms which are at best only a partial
nutrient sink in ponds stocked at densities above 5 ton/ha (Avnimelech 2003; Brune et al.
2003). Dense phytoplankton blooms can cause lethal DO and pH fluctuations and their

nutrients entering the ponds.

Methods: Plastic stormwater drain sumps were inserted into the tail end floor of each
raceway as a solids trap (Fig 1). These sumps were connected via a flexible hose to a pump
on a timer which periodically (twice daily) pumped collected waste to a holding tank for
evaluation of nutrient content. On monthly occasions between February and October 2006,
water leaving the raceways through the end screen was also sampled and nutrient data was
compared with that from the sump waste to determine differences. Water quality analyses
evaluated Total Solids (TS), Total Nitrogen (TN) and Total Phosphorous (TP), and were
determined using validated laboratory protocols based on standard methods (American
Public Health Association 1989) and nutrient analysis equipment at BIARC. 6Figure 1. Design and configuration of the solids trap inserted within the nursery raceways.
A plastic grate cover (not shown) prevented fish from entering the sump.

Results & Discussion: Nutrient analyses showed some small differences in concentration
between water pumped from the sump and water leaving the raceways through the end
screen (Table 1.) The greatest difference was with TS, where the sump captured on average
16% more solids than water discharged from the pond. Differences in TN and TP between
sump and raceway screen were smaller but still showed a marginally greater average
nutrient removal via the sump. This data cannot be statistically validated however because
monthly data from the raceway was from a single water sample (due to budgetary
constraints) whereby no measure of error rate can be determined. Regardless, the sump
was designed to trap and concentrate solids into a thick sludge that could be periodically
removed from the pond. It was clear that only a slightly more concentrated effluent was
captured by the sumps and their role in preventing nutrients entering the pond from the

Total Phosphorous 0.78 0.83 Strategy 2: Evaluation of Harpoon Weed
Summary: The concept of using seaweeds as biofilters for removing waste nutrients from
fish and shrimp aquaculture operation is well known, with a seminal review by Neori et al
(2004) describing the state of the art of this technology. Presently, the most commonly
proposed and researched biofilters are green seaweeds from the genus Ulva and the red
seaweed Gracilaria. Yet, in practice most seaweed-based remediation systems have proven
not to be economically viable, mainly due to the low value of the produced seaweed and
the high labour and area requirements for its cultivation. Other physical impediments to the
culture of seaweeds in effluent from aquaculture ponds include their susceptibility to
epiphytism (Friedlander et al., 1987), infestation by grazers such as amphipods, and
8

competition for available nutrients with phytoplankton (Palmer 2005). These difficulties
are compounded by the accumulation of effluent particulate matter on the seaweed’s
surfaces. The result therefore in practice, is that growth rate of the seaweeds (and their
corresponding value as a nutrient sink) is very often limited and nutrient removal
efficiencies are below optimum rates achieved in scaled trials under more favourable
conditions (Palmer 2005; previous BIARC research).

The present CARD project proposed to investigate the performance of the red seaweed
Asparagopsis armata (also known as Harpoon Weed) as a sink for waste nutrients
generated in raceway production system. This species was selected on the basis of new
work by Schuenhoff & Mata (2004) which suggested that it had considerably greater
market value than other seaweeds due to its high concentration of halogenated organic
metabolites. Once extracted, these halogenated compounds are used for antifouling and in
the cosmetic industry as fungicides. Schuenhoff & Mata (2004) suggest that these
compounds are also responsible for limiting epibiota and epiphytes in culture – an

forms available for direct plant uptake. Current work at BIARC, outside of the CARD
project, is assessing the role of polychaete-aided sand filtration as one such pre-treatment
option (Palmer 2007).

Strategy 3: Bacterial nutrient processing
Background: There is now recognition that promoting a swing from autotrophic
(phytoplankton-based) to heterotrophic (bacterial-based) processing of residual pond
nutrients has many advantages for water remediation. Sewage effluent treatment has long
employed bacterial digestion of organic matter in activated sludge systems (Arundel 1995)
and more recent studies have shown that suspended growth systems, where heterotrophic-
dominated processes regulate water quality, have great application for limited-water-
exchange shrimp and tilapia production (Avnimelech 1999; Burford, et al. 2003; Erler et
al. 2005). In aquaculture, these heterotrophic-dominated growth systems are generally
termed Bio-floc systems.

10

The challenge is to determine the best configuration for incorporating biofloc treatment as
part of the raceway production system. Two approaches are possible: in-pond biofloc
treatment or external biofloc treatment as part of a recirculating system.

Most studies on using bio-floc water remediation for aquaculture have advocated floc
formation within the culture pond as a supplementary source of dietary protein
(Avnimelech 1999; McIntosh et al. 2001; Erler et al. 2005) in addition to controlling water
quality. While increased feed utilisation is ideal, the excessive turbidity and high oxygen
demand created by bio-flocs may have a negative effect on fish cultured within floating
raceways. The high DO demands of the floc colony in addition to those of the cultured
species means that cultured stock are even more vulnerable in the event of any aeration
failure, especially in intensive production systems such as floating raceways. High
suspended solids levels can foul the gills of cultured animals and lead to bacterial,


While a quantitative rationale for estimating C additions was described by (Avnimelech
1999), his equation was based on total ammonia nitrogen (TAN) residue. A complication is
that TAN is not the only form of nitrogen available to heterotrophic bacteria. Dissolved
organic nitrogen (DON) in particular, but also nitrite and nitrate can constitute a varying
but substantial portion of bio-available N in aquaculture wastewater (Preston et al. 2000)
and bacteria may scavenge these in addition or in preference to ammonia (Jorgensen et al.
1994). Therefore, C additions based solely on TAN level may be under-dosing.

Calculating real-time (i.e. on-the-day) bio-available N levels is difficult (particularly for
DON which requires laboratory digestion and analysis) whereas daily in-the-field testing
of TAN is standard practice, so we acknowledge the validity of Avnimelech’s (1999)
suggestion to use TAN as a convenient reference to gauge C requirements. The objective
of this study was to refine C dosing requirements based on real-time TAN readings for
more complete nutrient assimilation in discharged wastewater. A further objective was to
assess the ability to convert plankton-dominated wastewater into a bio-floc community
using these established C dose rates, within pilot-scale external treatment ponds.

Methods: A series of experiments were carried out at BIARC during 2006. The wastewater
source was the discharge from the sumps of the FRs containing the mulloway and whiting.
Molasses (37.5% C) was the carbohydrate source used to adjust substrate C:N ratios in
both experiments because it contains simple sugars, negligible nitrogen, is readily available
and relatively inexpensive.

Experiment 1
This trial investigated the effect of molasses addition at two application rates on
wastewater nutrient levels over a 48 hour period. Nine 3L tanks were filled with common
12

wastewater and supplied with continuous aeration to ensure thorough mixing. The

x 12.5

According to this equation, 12.5 g C is needed to convert 1 g bio-available N into bacterial
biomass. Given that molasses is 37.5% C, 33.3 g of molasses is needed to convert 1 g bio-
available N.

A stock solution of molasses was prepared (100 g molasses L
-1
= 37.5 g C L
-1
) to aid
addition to the experimental tanks. Molasses 1 treatment was a single molasses dose based
on N
ww
= the real-time TAN level measured in the wastewater immediately prior to filling
experimental tanks. 'Molasses 2' treatment was based on double the amount of Molasses 1
to account for the extra ‘unmeasured’ bio-available N present. No molasses was added to
the Control treatment.

After molasses addition, two 50mL water samples (one filtered [0.45um] & one unfiltered)
were taken from each tank at regular intervals (0, 3, 6, 12, 24, 48 hrs). Nutrient
concentrations in the water samples were measured including Total Nitrogen [TN], Total
Phosphorus [TP], Total Ammonium Nitrogen [TAN], Nitrate/Nitrite [NOx], and Dissolved
Inorganic ortho-Phosphate [DIP]), Dissolved Organic Nitrogen [DON] and Dissolved
Organic Phosphorus [DOP]. Measurements were conducted using validated laboratory
13

protocols based on standard methods (American Public Health Association 1989) on a
Flow Injection analyser at BIARC. Data was statistically analysed using Arepmeasures
with treatment and time as parameters on Genstat 8

prescribed C:N ratios (as determined in Experiment 1), and averaged 200 ml of Molasses
every 2 days.

Weekly monitoring involved assessing untreated (influent) and treated discharged water
quality. A YSI multiprobe meter measured the Standard parameters (pH, temperature,
salinity, dissolved oxygen [DO]) during the experiment. Methods for determining nutrient
concentrations, total suspended solids [TSS], and Chlorophyll A [Chl-a] were as
described for Experiment 1.

Measurements assessed differences between bio-floc treatment and standard
phytoplankton-dominated PSP treatment. In addition, differences between the (untreated)
influent and post-treatment water were measured to assess the efficiency within each
treatment system. Changes in water quality parameters were statistically analysed using
Arepmeasures with treatment type and time as parameters on Genstat 8
th
Ed Software.

Results:

Experiment 1
Results for each constituent tested are described in detail in the paragraphs below and
displayed graphically in Figure 4.

Nitrogen
TAN levels in the un-dosed Control treatment increased significantly (p>0.01) during the
trial period. In contrast, at just three hours after a single addition of C, TAN levels in the
two molasses treatments had fallen by over 35% and were significantly (P>0.01) lower
than the control. By six hours TAN removal remained consistent between the two molasses
treatments with over 65% of TAN removed from the water. However, beyond six hours
TAN in the lower dose (Molasses 1) treatment began to rise again, suggesting the

completely eliminating DIP (93% removal). DIP levels also began to rise significantly
after 24 hours in Molasses 1 and 48 hours in Molasses 2 as seen in the TAN levels.

DOP levels were significantly (p>0.05) lower in the Control samples but the level of C
dose did not significantly (p<0.05) effect the response.

16

Similarly to TN levels, C addition did not significantly effect (p<0.05) TP levels during the
experimental period. Again suggesting the C addition can significantly influence the
nutrient processes without affecting the nutrient budget.

0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 6 12 18 24 30 36 42 48
HOURS
TAN mg/L
Control
Molasses 1
Molasses 2

0.0
0.1
0.2
0.3

Molasses 2
Molasses 1
Control0
4
8
12
16
20
0 6 12 18 24
Hours
TN mg/L
Control
Molasses 1
Molasses 2
0.0
1.0
2.0
3.0
4.0
5.0
0 6 12 18 24
Hours
TP mg/L
Control
Molasses 1
Molasses 2


pH
PSP
BFP
6
8
10
12
14
16
18
20
22
123456789101112
Week
DO mg/L
PSP
BFP
Figure 5: Water Quality measurements for pH and Dissolved Oxygen (DO)

Temperature and Salinity remained within biological limits for both systems. As expected,
the temperature was similar in both systems (15.3 – 21.0
O
C) on most occasions. Salinity
showed significant (p>0.01) fluctuations over time for both treatments due to rain events.
The salinity of the BFP was significantly(p<0.01) lower than PSP on a number of
occasions probably due to the more effective mixing of rain water which can float on top
of still seawater in the PSP.

Nutrient Analyses
In general, both treatments significantly (p<0.05) lowered the dissolved nutrients levels

UNTREATED
PSP
BFP
0.0
0.2
0.4
0.6
0.8
123456789101112
WEEK
TAN mg/L
UNTREATED

123456789101112
WEEK
TP mg/L
BFP
UNTREATED
PSP
0
20
40
60
80
123456789101112
WEEK
TSS mg/L
BFP
UNTREATED
PSP
0.0
0.5
1.0
1.5
2.0
123456789101112
WEEK
DON mg/L
BFP
PSP
UNTREATED
0
20

TSS, another indicator of water column biomass, confirmed the trend that the BFP
treatment significantly (p<0.01) increased biomass (TSS levels) present compared to both
Untreated and PSP samples. Figure 6 displays results for all nutrients.

Interestingly, Chlorophyll A (ChlA) levels in the BFP treatment were significantly
(p<0.05) higher than ChlA levels present in Untreated samples on most occasions and was
significantly higher than the PSP treatment during the final three weeks (See Figure 6). A
heterotrophic community in a BFP treatment might be expected to have less photosynthetic
material (ChlA) than the phytoplankton dominated communities present in the untreated
water or PSP system. However, others have observed that C addition did not affect ChlA
levels in production system (Avnimelech 2001; Erler et al. 2005; Hari et al. 2006). The
higher ChlA levels in the BFP treatment can be explained by the retention of
phytoplankton within the floc material and thus within the system (i.e. concentrating the
phytoplankton). Hargreaves (2006) described suspended organic material in BFPs as
primarily made up of senescing algal cells colonised by bacteria. It is therefore, more
appropriate to look at the proportion of phytoplankton within the whole community
structure. Although the ChlA levels are higher in the BFP system, the community structure
has a lower proportion of phytoplankton than the PSP (See Figure 7).

Phytoplankton biomass can be estimated from the ChlA levels using the relationship: 1 mg
ChlA = 200mg dry weight (Pagand et al. 2000). Estimates of the contribution by
phytoplankton to the TSS levels recorded for each system were calculated. The graphs
below demonstrate the difference in community structure achieved by the applied
treatment. The PSP community was dominated by phytoplankton (57%) with a low
percentage (43%) of other particulates (including bacteria, and zooplankton etc.). In
20

contrast, the BFP community had a relatively low percentage of phytoplankton (41%) and
was dominated by other particulates (59%) presumably bacterial biomass.


Other
Phytplankton
Figure 7: Proportion of phytoplankton present during the experimental period

Discussion: Increasing the C dose in BFPs to 30g C L
-1
achieves almost complete
elimination of dissolved nutrients within 12 hours and extends the period before a
significant remineralisation or release of these dissolved nutrients occurs. This suggests
that with higher C dosing, treatment systems require only 12 hours retention time to
process available dissolved nutrients and exceeding 24 hours will complicate the system
with remineralisation and reduce efficiency. The data also suggests that carbon plays a part
in the processing of DON, however the data was inconclusive and further work in this area
is required.

The subsequent experiment included the application of C at this higher dose rate to
demonstrate the effect on a phytoplankton-dominated waste-stream in a continuous flow
pilot-scale treatment system. By applying the higher C dose and BFP principles to
phytoplankton-dominated influent we demonstrated a clear shift to a bio-floc community.
A Bio-floc community can be characterised by the following criteria:
o Low levels of photosynthesis occurring indicated by lower and more stable pH
levels due to the release of carbon dioxide into the water column and lower DO
levels due to uptake of available oxygen (Hargreaves 2006).
o High nutrient levels (Burford, Thompson et al. 2003)
o High levels of organic matter (which can be measured by TN & TP) and low levels
of dissolved nutrients due to assimilation (Avnimelech 2003; Ebeling, et al. 2006).
21

o A high level of water column suspended material and a low proportion of
phytoplankton present in the community biomass (Burford et al. 2003).

limited TAN and NO
2
, and while DO levels can be maintained. This trial demonstrates that
those conditions can be achieved with bio-floc treatment. High TSS can be detrimental to
22

fish health as discussed earlier so the preferred production model would be an external
biofloc treatment as part of a recirculating system. For most effective performance, a
means to separate or exclude bio-flocs from the supernatant would permit the return of
treated water back to the production pond without a high BOD or TSS load. Schneider et
al. (2007) also reported a similar conclusion when trying to apply a bacteria reactor to clear
Recirculating Aquaculture System wastewater. Such a bio-floc exclusion device needs
further research but may be in the form of a mechanical particle filter such as a screen or
drum filter. Figure 8 shows a schematic representation of the proposed recirculating
system, which offers scope to grow and additional crop of prawns (or similar detritivore)
within the bio-floc pond, which graze on the nutrient-rich bio-flocs and have the added
benefit of helping to keep flocs in suspension.

Supernatant
returned to
production pond
Floc
excluder

Figure 8. Schematic representation of the proposed recirculating system, with external bio-
floc pond for water treatment.

Conclusion
Of the wastewater remediation strategies investigated in this project, it is evident that bio-
floc treatment, particularly as a component of an integrated recirculating production

Organic-rich wastewater
removed from raceways
to Bio-floc Pond
23

Development of Vietnam. The research team would like to thank the Queensland
Department of Primary Industries and Fisheries, in particular Adrian Collins, Ben Russell
and Blair Chilton for their efforts in establishing the project. We also thank our
Vietnamese research colleagues ably led by Dr Tung Hoang (Director, International Centre
for Research & Training, Nha Trang University) for their valuable help and support
throughout this project.

References

American Public Health Association (1989). Standard Methods for the examination of
water and wastewater. L. S. Clesceri, A. E. Greenberg and R. R. Trussell.
Washington, Port City Press: 10-31 - 10-35.
Avnimelech, Y. (1999). Carbon/nitrogen ratio as a control element in aquaculture systems.
Aquaculture 176(3-4): 227-235.
Avnimelech, Y. (2003). "Control of microbial activity in aquaculture systems: active
suspension ponds." World Aquaculture Dec: 19-21.
Boyd, C. E. (1995). Chemistry and efficacy of amendments used to treat water and soil
quality imbalances in shrimp ponds. In: Swimming through troubled water -
Proceedings of the special session on shrimp farming, San Diego, The World
Aquaculture Society.
Boyd, C. E. (2002). Understanding pond pH. Global Aquaculture Advocate June: 74-75.
Brune, D. E., G. Schwartz, et al. (2003). Intensification of pond aquaculture and high rate
photosynthetic systems. Aquacultural Engineering 28(1-2): 65-86.
Burford, M. A., P. J. Thompson, et al. (2003). Nutrient and microbial dynamics in high-
intensity, zero-exchange shrimp ponds in Belize. Aquaculture 219(1-4): 393-411.

253.
McIntosh, D., T. M. Samocha, et al. (2001). Effects of two commercially available low-
protein diets (21% and 31%) on water and sediment quality, and on the production of
Litopenaeus vannamei in an outdoor tank system with limited water discharge.
Aquacultural Engineering 25(2): 69-82.
Neori, A., T. Chopin, et al. (2004). Integrated aquaculture: rationale, evolution and state of
the art emphasizing seaweed biofiltration in modern mariculture. Aquaculture 231(1-
4): 361-391.
Obaldo, L. G. and D. H. Ernst (2002). Zero-exchange shrimp production. Global
Aquaculture Advocate June: 56-57.
Pagand, P., J P. Blansheton, et al. (2000). The use of high rate algal ponds for the
treatment of marine effluent from a recirculating fish rearing system. Aquaculture
Research 31: 729-736.
Palmer, P. J., Ed. (2005). Wastewater remediation options for prawn farms. Brisbane,
DPI&F Publications. 93pp.
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