environmental management of aquaculture efluente. development of biological indicators and...

Environmental Management of

Aquaculture Effluent:

Development of

Biological Indicators and Biological

Filters

Adrian B. Jones

Environmental Management of Aquaculture Effluent:

Development of Biological Indicators and Biological Filters

A Thesis

submitted by

Adrian B. Jones B.Sc. (Hons)

The University of Queensland, Australia

to the

Department of Botany

The University of Queensland

AUSTRALIA

in fulfilment of the requirements for

the degree of Doctor of Philosophy within

The University of Queensland

July 1999

STATEMENT

The work presented in this thesis is, to the best of my knowledge and belief, original, except

as acknowledged in the text, and the material has not been submitted, either in whole or in

part, for a degree at this or any other University.

Signed................................................

ACKNOWLEDGMENTS

This thesis was initiated from a Fisheries Research Development Corporation (FRDC) grant

to Dr Nigel Preston (CSIRO) and Moreton Bay Prawn Farm. Additional funding was

provided through grants from the Australian Research Council (ARC) to Dr William C.

Dennison, the CRC for Aquaculture and a University of Queensland Postgraduate Research

Scholarship.

Its hard to believe its finally finished. From the early days at CSIRO and Moreton Bay

Prawn Farm laying concrete slabs and besser brick raceways to cruising Moreton Bay in

thunder storms, the all-nighters at Straddie and then the endless days and nights in front of

the computer. None of it would have been possible without the support and friendship of

many people, mostly from the Marine Botany Group at UQ.

Dr. Bill Dennison, who developed my interest in marine research through his amazing

enthusiasm in the field, for his advice and suggestions, and for his ability to make you realise

that its not all as bad as it seems when youre in the depths of confusion.

To everyone else in Marine Botany who provided help with field work, help with

interpretation and presentation of results, proof reading of manuscripts, and general

friendship and support. In particular, thanks to Cindy Heil, Michele Burford, Mark

ODonohue and Joelle Prange who reviewed various sections of the thesis. Also a special

mention to Ros Murrell who has tirelessly helped me to track down a certain person when

times were desperate. You were my link to the outside world during those last six months.

ACKNOWLEDGMENTS viii

Dr. Nigel Preston, CSIRO Cleveland Marine Laboratories, for his constant encouragement

regarding my writing abilities, his invaluable help regarding the initial planning of the thesis

topic, his timely pep talks and last minute editing.

Theresa Mitchell, for her help, efficiency, support and advice regarding anything and

everything to do with forms, policies, scholarships and finally the thesis submission

procedures. Life at Botany just isnt the same without you! Jan Stewart, for conducting the

isotopic analyses, and Gordon Moss for analysing the amino acid samples.

Sabine Roberts who read early drafts of all the chapters, picking my grammar to pieces and

helping get my thoughts on the right track. Thankyou so much for all your help,

encouragement and friendship throughout my PhD.

My parents who put up with me over the last 4 years constantly complaining about how this

didnt work and that didnt work, and no, I dont know when I will finish it all. Thankyou

for being there to listen to my complaining, and for your support and most importantly, for

never pressuring me.

Finally to Tracey, who despite my constant rebuttal continued to maintain that somehow I

would finish it before our flight left. Thankyou for your support during my constant state of

stress and panic, especially when I was having serious doubts as to how I would manage to

finish it on time. Thankyou for putting up with no end of complaints and irrational

ACKNOWLEDGMENTS ix

behaviour, and for always being there and for accepting my stream of unfulfilled promises.

Thankyou for your marathon proof reading / printing / collation efforts at the end, especially

amongst the continual printer jams and photocopying nightmares and my accompanying

frustration and rampage. Thankyou for somehow managing to keep me together! Most

importantly, thankyou for your love, companionship, support and understanding.

ABSTRACT

Rapid global expansion of the aquaculture industry has prompted the need for development of techniques for

effective environmental management. In intensively farmed regions, aquaculture effluent has resulted in

environmental degradation of receiving waters. The issues to be addressed include analysis of effluent water

quality, determination of the ecological impact of effluent on the ecosystem, and development of remediation

strategies to reduce these impacts. Physical and chemical water quality analyses can identify elevated

concentrations of suspended solids, chlorophyll a, water column nutrients and other components of aquaculture

effluent, however, additional biological sampling is required to provide meaningful information about the

ecological impacts of effluent discharge on receiving waters.

Analyses of the amino acid composition, tissue nitrogen content and stable isotope ratio of nitrogen (d15N) in

seagrasses, mangroves and macroalgae were developed as biological indicators to determine the influence of

shrimp farm effluent on a coastal ecosystem. Different responses in these biological parameters revealed that

the impacts of aquaculture effluent on receiving waters were qualitatively different to the impacts of sewage

effluent. The impacts were also spatially more extensive than identified by water quality analyses, which

revealed no elevation in the concentration of water column nutrients, chlorophyll a concentration or total

suspended solids further than 400 m from the mouths of the creeks receiving the sewage and aquaculture

effluent. The maximum d15N of the mangroves, seagrass and macroalgae associated with the treated sewage

discharge was 19.6, which was significantly higher than the influence of the shrimp effluent (7.6). A d15N

value of 4.5, which is elevated relative to unimpacted sites, indicated that the impacts extended up to 4 km

from the mouths of the creeks. Differences in the concentrations of the amino acids proline, serine, glutamine

and alanine in the seagrass and macroalgae were suggested to reflect the source (aquaculture or sewage) of the

nutrients taken up by the plants.

To reduce the environmental impacts, effluent treatment techniques using biological filters were investigated.

Filtration by oysters (Saccostrea commercialis) significantly reduced the concentrations of chlorophyll a

(phytoplankton), bacteria, total nitrogen, total phosphorus and total suspended solids to 5%, 32%, 67%, 63%

and 11% of the initial concentrations, respectively. However, oyster excretion increased the concentrations of

the dissolved nutrients, ammonium (from 18 to 51 M), nitrate / nitrite (from 1.0 to 13 M), and phosphate

(from 0.5 to 3.3 M), however macroalgal (Gracilaria edulis) absorption significantly reduced these

concentrations to 2.3%, 2.2% and 4.8%, respectively. The ratio of ammonium to nitrate / nitrite in the effluent

was also significantly reduced, which has positive implications for recycling of wastewater back into shrimp

production ponds, and reducing impacts on receiving waters.

The efficiency and condition of the oysters and macroalgae was reduced by fouling from the high concentration

of suspended particulates in the effluent. Several novel techniques such as dissolved free amino acid

composition, pigment concentrations, PAM fluorescence, tissue nitrogen and d15N were used to assess the

condition of the macroalgae. It was observed that an intermediate reduction in the concentration of suspended

ABSTRACT xii

particulates resulted in the best growth and condition of the biofilters. The concentration of particulates in this

treatment (11 nephelometric turbidity units) provided sufficient particulates for oysters to filter, and a source for

regeneration of nutrients for macroalgal uptake, as well as reducing the effects of photoinhibition which can

occur in Gracilaria spp. at relatively low light intensities.

The problems associated with fouling were successfully mitigated by incorporating natural sedimentation prior

to oyster filtration, and subsequent macroalgal absorption. This combined system of treatment proved effective

at optimising the performance of the biological filters to improve the water quality of the effluent. Using this

combination of polyculture, it was estimated that up to 18 kg N ha-1 d-1 and 15 kg P ha-1 d-1 could be removed

from commercial shrimp ponds.

The water quality of aquaculture effluent and its impact on the receiving waters will vary due with differing

environmental conditions, as well as the type of aquaculture being conducted. Regardless, this thesis has

demonstrated that filtration / absorption by various marine organisms can be effective tools for monitoring and

reducing the environmental impacts of aquaculture effluent.

TABLE OF CONTENTS

Statement v

Acknowledgments vii

Abstract xi

Table of Contents xiii

List of Tables xviii

List of Figures xx

List of Plates xxiii

CHAPTER 1. INTRODUCTION 1

1.1 Aquaculture 1

1.2 Environmental Impacts 2

1.3 Biological Indicators 4

1.4 Biological Treatment Options 5

1.4.1 Oysters 6

1.4.2 Macroalgae 9

1.5 Polyculture / Integrated Aquaculture 12

1.6 Thesis Aims 13

1.7 Thesis Overview 14

1.7.1 Chapter Outline 16

1.9 Publication Status of Thesis Chapters 18

CHAPTER 2 ASSESSING ECOLOGICAL IMPACTS OF SHRIMP

AND SEWAGE EFFLUENT: BIOLOGICAL INDICATORS WITH

STANDARD WATER QUALITY ANALYSES 19

Abstract 19

2.1 Introduction 20

2.2 Materials and Methods 25

TABLE OF CONTENTS xiv

2.2.1 Study Region 25

2.2.2 Experimental Design 26

2.2.3 Collection 27

2.2.4 Analytical Procedures 27

2.2.5 Statistical Analysis 30

2.3 Results 31

2.3.1 Physical and Chemical Water Quality Analyses 31

2.3.1.1 Salinity 31

2.3.1.2 Nutrients 31

2.3.1.3 Phytoplankton 32

2.3.1.4 Suspended Solids and Secchi Depth 34

2.3.1.5 Sediment Organic Content 34

2.3.2 Bioindicators 35

2.3.2.1 Tissue Nitrogen Content 35

2.3.2.2 d15N Stable Isotope Ratio of Nitrogen 36

2.3.2.3 Free Amino Acid Composition 40

2.4 Discussion 45

2.4.1Water Quality Parameters 45

2.4.1.1 Effluent Composition 45

2.4.1.2 Phytoplankton Biomass and Productivity 46

2.4.2 Biological Indicators 47

2.4.2.1 Tissue N Content 47

2.4.2.2 d 15N Isotopic Signature 48

2.4.2.3 Amino Acid Composition 51

2.4.3 Comparison of Impacts 55

2.4.4 Conclusion 57

2.4.5 Application for other types of Aquaculture 58

2.4.6 Remediation Options 59

TABLE OF CONTENTS xv

CHAPTER 3 OYSTER FILTRATION OF SHRIMP FARM EFFLUENT,

THE EFFECTS ON WATER QUALITY 63

Abstract 63

3.1 Introduction 64




3.3 Results 70

3.3.1 Suspended Solids 70

3.3.2 Organic content 70

3.3.3 Chlorophyll a 72

3.3.4 Bacteria 72

3.3.5 Total Nutrients 72

3.4 Discussion 73

3.4.1 Scaling Up Calculations 75

3.4.2 Summary 75

CHAPTER 4 THE EFFICIENCY AND CONDITION OF OYSTERS AND

MACROALGAE USED AS BIOLOGICAL FILTERS OF SHRIMP POND

EFFLUENT 77

Abstract 77

4.1 Introduction 78



4.2.1.1 Filtration Efficiency Experiments 81

4.2.1.2 Biofilter Condition Experiments 83


4.3 Results 90

4.3.1 Filtration Efficiency Experiments 90

4.3.1.1 Continual Flow 90

4.3.1.2 Recirculating Experiments 92

TABLE OF CONTENTS xvi

4.3.2 Biofilter Condition Experiments 97

4.4 Discussion 103

4.4.1 Efficiency of Biofilters 103

4.4.2 Condition of Biofilter Organisms 110

4.4.3 Conclusion 115

CHAPTER 5 IMPROVEMENTS IN WATER QUALITY OF AQUACULTURE

EFFLUENT AFTER TREATMENT BY SEDIMENTATION, OYSTER

FILTRATION AND MACROALGAL ABSORPTION 117

Abstract 117

5.1 Introduction 118




5.3 Results 126

5.3.1 Suspended Solids 126

5.3.2 Organic content 126

5.3.3 Chlorophyll a 128

5.3.4 Bacteria 129

5.3.5 Dissolved Oxygen 131

5.3.6 Total Nitrogen 132

5.3.7 Total Phosphorus 133

5.3.8 Ammonium 134

5.3.9 Nitrate / Nitrite 136

5.3.10 Phosphate 137

5.3.11 Nutrient Uptake Rates and Ratios 137

5.4 Discussion 140

5.4.1 Sedimentation 140

5.4.2 Oyster Filtration 141

5.4.3 Macroalgal Absorption 144

5.4.4 Nutrient Regeneration 147

5.4.5 Conclusions 149

TABLE OF CONTENTS xvii

CHAPTER 6 CONCLUSION 151

6. 1 Downstream Impacts 151

6.2 Efficiency of Biological Filters 152

6.3 Condition of Biofilters 153

6.4 Scaling up for Commercial Treatment 155

6.5 Other Potential Biofilters 156

6.6 Management Implications and Potential Problems with

Biofiltration / Polyculture 157

6.7 Benefits of Polyculture or Integrated Aquaculture 158

6.8 Future Research 159

6.9 Summary 160

BIBLIOGRAPHY 163

APPENDIX 1 FACTORS LIMITING PHYTOPLANKTON BIOMASS IN THE

BRISBANE RIVER AND MORETON BAY 192

APPENDIX 2 PHOTOSYNTHETIC CAPACITY IN CORAL REEF SYSTEMS:

INVESTIGATIONS INTO ECOLOGICAL APPLICATIONS FOR THE

UNDERWATER PAM FLUOROMETER 203

LIST OF TABLES

Table 2.1. Results of traditional water quality monitoring for the creek with shrimp farm

effluent and sewage treatment effluent. DIN = Dissolved Inorganic Nitrogen; DIP =

Dissolved Inorganic Phosphorus; Chl a = Chlorophyll a; Phyto Prod = phytoplankton

productivity; TSS = total suspended solids; VSS = volatile suspended solids; Secchi =

secchi disc depth. Only one replicate measurement was recorded for Secchi disk depth

and salinity (Practical Salinity Scale). 33

Table 2.2. Correlations (r2) between the concentration of phytoplankton (chlorophyll a) and

phytoplankton productivity (14C uptake) and various water quality parameters. DIN =

Dissolved Inorganic Nitrogen; DIP = Dissolved Inorganic Phosphorus; Phyto Prod =

phytoplankton productivity (mg C m-3 h-1); Chl a = Chlorophyll a (g L-1); TSS = total

suspended solids (mg L-1); VSS = volatile suspended solids (mg L-1); ISS = inorganic

suspended solids (mg L-1); Secchi = secchi disc depth (m). Numbers in bold type

indicate significant correlations (r2 0.6). 35

Table 2.3. Results of bioindicator monitoring for the creek with shrimp farm effluent and

sewage treatment effluent. d15N = Nitrogen stable isotope ratio; %N = Tissue N content;

nd = no data (no plants were present). 38

Table 2.4. Results of bioindicator monitoring for the shrimp and sewage creeks. % refers to

percentage of total free amino acid pool. SER = serine; ALA = alanine; GLN =

glutamine; PRO = proline; Total aa = total concentration of free amino acids

(mol g wet-1); nd = no data (no plants were present). 42

Table 3.1 Combinations of live and dead oysters (Saccostrea commercialis) used in

experiments to determine the effects of oyster density on the water quality of shrimp

pond effluent. 68

Table 3.2 Concentration of various water quality parameters before and after filtration by

oysters at 3 different densities (see Table 3.1). Values for control (no oysters) and shells

(dead shells only) are also given. Values in brackets are concentrations expressed as a

percentage of the inflow value. Values in italics are standard errors. 71

Table 4.1 Water quality parameters after filtration by oysters under flow through conditions

in raceways. Total N = total Kjeldahl nitrogen; Total P = total phosphorus. 92

LIST OF TABLES xix

Table 4.2 Water quality parameters after filtration by oysters after the first circuit during

recirculating flow in raceways. TSS = total suspended solids; Organic = organic

component of TSS (loss on ignition); Inorganic = inorganic component of TSS. 94

Table 4.3 Changes in the free amino concentration and composition of macroalgae for

various treatments in laboratory settling experiments. % refers to percentage of total

free amino acid pool. CIT = citrulline; GLU = glutamate; ALA = alanine; GLN =

glutamine; PHE = phenylalanine; SER = serine; Total aa = total concentration of free

amino acids (mol g wet-1). 101

Table 5.1 Percentage of original concentrations of various water quality parameters after

settling, filtration by oysters and filtration by macroalgae. * p 0.05; ** p 0.01; *** p 0.001. Percentage of highest concentration represents the final concentration as a

percentage of the highest recorded concentration after sedimentation and oyster

filtration. The percent of initial concentration represents the final concentration as a

percentage of the initial concentration in the untreated effluent. The only differences

between the two values are for the dissolved inorganic nutrients (NH4+, NO3

-, & PO43-).

128

Table 5.2 Nutrient uptake and release rates for sedimentation, oyster filtration and

macroalgal absorption. Negative symbols represent nutrient uptake, and positive

represent nutrient release. The top value for each treatment is the gross value, the

middle value is the control and the bottom value (in bold type) is the net value after

correction for nutrient changes in the control tanks. The last row of results represent the

rates of macroalgal nutrient uptake over the first hour, when nutrient concentrations

were still saturating uptake kinetics. 136

LIST OF FIGURES

Figure 2.1 Map of study sites in Moreton Bay, including the location of shrimp and sewage

effluent discharges. 27

Figure 2.2. Map showing the values of %N in seagrass (Zostera capricorni), macroalgae

(Catenella nipae), and mangroves (Avicennia marina) at the study sites (see Fig. 2.1 for

site references). 39

Figure 2.3. Map showing the values of d15N in seagrass (Zostera capricorni), macroalgae

(Catenella nipae), and mangroves (Avicennia marina) at the study sites (see Fig. 2.1 for

site references). 40

Figure 2.4. Map showing the amino acid composition of seagrass (Zostera capricorni) at the

study sites (see Fig. 2.1 for site references). 43

Figure 2.5. Map showing the amino acid composition of macroalgae (Catenella nipae) at the

study sites. Pie graphs have been reduced to quarters for layout purposes. The

remaining three quarters of the pie graphs not represented are a continuation of the

other amino acid category (not serine or alanine) (see Fig. 2.1 for site references). 44

Figure 2.6. Conceptual model of the two creeks and the range and type of impacts from the

different effluent sources. 61

Figure 3.1. Location map of Moreton Bay Prawn Farm near Brisbane, Australia. 66

Figure 3.2. Schematic representation of tank and waterflow layout. 67

Figure 4.1 Diagrammatic representation of experimental setup, a) single raceway with

baffles and oyster trays, and b) laboratory settling experiment. NTU = nephelometric

turbidity units. The oysters used in the experiments were Sydney Rock oysters,

Saccostrea commercialis and the macroalgae was Gracilaria edulis. Effluent was from

an intensive Penaeus japonicus shrimp farm. 84

Figure 4.2 Impacts of effluent on biofilters: a) Oyster mortality (%) from upper, middle and

lower trays after 2 weeks at low, medium and high oyster stocking densities in raceways

supplied with unsettled shrimp effluent, and b) change in dissolved nutrient

concentrations after passing effluent through low, medium and high macroalgal stocking

densities in raceways supplied with unsettled shrimp effluent. Positive change

represents an increase, negative change represents a decrease. 91

LIST OF FIGURES xxi

Figure 4.3 Particle size distribution, a) before and after control and oyster treatment

raceways during single continuous flow, b) before and after consecutive circuits through

oyster treatment raceways (linear scale), and c) before and after consecutive circuits

through oyster treatment raceways (log scale). 93

Figure 4.4 Concentrations of water quality components before and after consecutive circuits

through oyster treatment raceways, a) bacterial numbers, b) chlorophyll a concentration,

and c) total suspended solids (TSS). 96

Figure 4.5 Growth of oysters and macroalgae after 8 weeks in tanks supplied with shrimp

effluent pre-settled for 0, 1, 6 & 24 h. a) change in oyster growth rate expressed as

changes in oyster volume (cm3 oyster -1), and b) macroalgal biomass. n.d. = no data. 98

Figure 4.6 Response of macroalgae to 8 weeks in tanks supplied with shrimp effluent pre-

settled for 0, 1, 6 & 24 h. a) macroalgal growth expressed as number of news shoots per

tank, and b) concentration of the photosynthetic pigments, chlorophyll a (CHL) and

phycoerythrin (PE). 99

Figure 4.7 Macroalgal nitrogen content (a) and d15N (b) after 8 weeks in tanks supplied with

shrimp effluent of different settlement times, a) %N, and b) d15N. 100

Figure 4.8 The response of electron transport rate (ETR) versus photosynthetically active

radiation (PAR) in macroalgae incubated in seawater (control) or shrimp effluent

(settled 24 h plus oyster filtered for 12 h). 102

Figure 5.1 Design of integrated treatment system stocked with oysters (40 g Saccostrea

commercialis), and macroalgae (Gracilaria edulis). 124

Figure 5.2 Changes in total suspended solids (A) and phytoplankton biomass (chlorophyll a)

(B) from sedimentation, oyster filtration and macroalgal absorption. Standard error bars

have been plotted, but are too small to be visible. 127

Figure 5.3 Concentration of particles settled per litre from sedimentation and oyster

filtration. 129

Figure 5.4 Changes in the organic content of the a) total suspended solids (TSS) and b)

settled particles in the effluent water from sedimentation, oyster filtration and

macroalgal absorption. Standard error bars have been plotted, but are too small to be

visible. 130

Figure 5.5 Changes in bacterial numbers from sedimentation, oyster filtration and macroalgal

absorption. 131

LIST OF FIGURES xxii

Figure 5.6 Changes in water column dissolved oxygen concentrations from sedimentation,

oyster filtration and macroalgal absorption. Standard error bars have been plotted, but

are too small to be visible. 132

Figure 5.7 Changes in water column total N (A) and P (B) concentrations from

sedimentation, oyster filtration and macroalgal absorption. Standard error bars have

been plotted, but are too small to be visible. 133

Figure 5.8 Changes in water column NH4+, NO3

-, PO43- concentrations from sedimentation,

oyster filtration and macroalgal absorption. Standard error bars have been plotted, but

are too small to be visible. 135

Figure 5.9 Changes in water column total N: P ratio (A) and DIN: DIP ratio (B) from

sedimentation, oyster filtration and macroalgal absorption. Standard error bars have

been plotted, but are too small to be visible. 139

Figure 6.1 Diagrammatic design of water flow for typical untreated shrimp farms (left) and a

design to incorporate physical (sedimentation) and biological (oyster filtration and

macroalgal absorption) treatment (right). 162

LIST OF PLATES

Plate 1.1 Ponds at Moreton Bay Prawn Farm, an intensive shrimp farm (Penaeus japonicus)

near Moreton Bay, Queensland, Australia. 2

Plate 1.2 Shrimp Farm plume discharging into Moreton Bay, Queensland, Australia. 3

Plate 1.3 High phytoplankton concentration in plume from shrimp farm discharging into

Moreton Bay, Queensland, Australia. 3

Plate 1.4 Penaeus japonicus from ponds at Moreton Bay Prawn Farm, Queensland,

Australia. 5

Plate 1.5 Sydney Rock Oysters (Saccostrea commercialis) cultured in Moreton Bay,

Queensland, Australia. 7

Plate 1.6 Gracilaria edulis collected from Moreton Bay, Queensland, Australia. 10

Plate 4.1 Raceways constructed at Moreton Bay Prawn Farm, Queensland, Australia. 82

Plate 4.2 Control raceway on the left with no oysters and treatment stocked at low density

55 g oysters. Demonstrates changes in water clarity (reduction in suspended solids) with

the oyster tray clearly visible in the raceway stocked with oysters, but not in the control

raceway. 104

Plate 4.3 First chamber (foreground) and second chamber (background) of an oyster

treatment raceway showing the improvement in water clarity (reduction in suspended

solids) within the raceway. 105

Plate 4.4 Fouling of oysters by settling particulates in raceways. 108

Plate 5.1 Experimental setup with sedimentation drum (background) and control, oyster,

macroalgal filtration tanks (foreground). 124

Plate 5.2 Water samples collected: a) before sedimentation; b) after sedimentation;

and c) after biofiltration. 150

CHAPTER 1

INTRODUCTION

1.1 Aquaculture

The UN Food and Agricultural Organisation has estimated that by 2020 more than 50% of

fisheries production will need to come from aquaculture due to human population growth,

continuing demand for seafood, and static or declining natural fish harvests. However, in

many countries aquaculture practices have already resulted in the destruction of coastal

vegetation, salinisation of land, pollution of waterways and massive crop losses (Phillips et

al., 1993). Further expansion using current technologies is simply not justifiable or

sustainable. If the level of demand for seafood is to be met the only alternative is to develop

new technologies that require less space and have minimal adverse environmental impacts.

Penaeid prawn (shrimp) farming has been one of the most economically successful of all

intensive aquaculture industries. In the early days of shrimp farming and other forms of

aquaculture, the perception was that they were completely clean industries (Weston, 1991).

Recent reviews of intensive shrimp aquaculture have emphasised the need for more effective

controls on the quality of effluent water discharged into the environment (Phillips et al.,

1993; Primavera, 1994).

Shrimp farming can be separated into extensive, semi intensive and intensive culture systems

(Macintosh & Phillips, 1992). Extensive culture systems have large pond sizes (>5 ha),

relatively low stocking densities (20 per m2), aeration, and formulated high protein feed pellets (Plate 1.1). Intensive farming

CHAPTER 1 2

is becoming more prominent, increasing the potential for environment impact from shrimp

farming (Phillips et al., 1993).

Plate 1.1 Ponds at Moreton Bay Prawn Farm, an intensive shrimp farm (Penaeus japonicus) near Moreton Bay,

Queensland, Australia.

1.2 Environmental Impacts

Intensive shrimp aquaculture systems rely on high protein feed pellets to produce high rates

of growth, but a large proportion of the pellets are not assimilated by the shrimps (Primavera,

1994). Approximately 10% of the feed is dissolved and 15% remains uneaten. The

remaining 75% is ingested, but 50% is excreted as metabolic waste, producing large amounts

of gaseous, dissolved and particulate waste (Lin et al., 1993). Subsequently, the effluent

contains elevated concentrations of dissolved nutrients (primarily ammonia), plankton and

other suspended solids (Ziemann et al., 1992). The dissolved nutrients and organic material

in shrimp ponds stimulate rapid growth of bacteria, phytoplankton, and zooplankton (Lin et

al., 1993). These untreated wastes are usually discharged directly into the environment,

where they may enhance eutrophication, organic enrichment and turbidity of the local

waterways (Plates 1.2 & 1.3) (Eng et al., 1989; O' Connor et al., 1989; Prakash, 1989).

INTRODUCTION 3

Plate 1.2 Shrimp Farm plume discharging into Moreton Bay, Queensland, Australia.

Plate 1.3 High phytoplankton concentration in plume from shrimp farm discharging into Moreton Bay,

Queensland, Australia.

CHAPTER 1 4

Australia has a small but expanding coastal aquaculture industry. From 1984 to 1998, the

shrimp farming sector rose from 15 to 2 000 t. The industry is well placed to take advantage

of developments in integrated aquaculture systems, such as the use of natural biofilters and

recirculating systems. In southeast Queensland, there are two shrimp species farmed,

Penaeus monodon Fabricius and P. japonicus Bate (Plate 1.4), with stocking of post larvae in

October and harvest the following April to June. With increasing development of the

industry, concerns have risen about the impact of effluent from the farms. The effluent from

shrimp farms discharging into Moreton Bay, Queensland often has concentrations of

dissolved nitrogen and phosphorus which are 60 fold higher than receiving waters,

chlorophyll a concentrations 200 fold higher, and total suspended solids (TSS) 20 fold higher

(Jones et al., in prep a; Chapter 2). Australian waters are relatively low in nutrients

compared with other coastal waters (State of the Environment Council, 1996), and therefore

impacts may be potentially more acute. In Moreton Bay, background concentrations of water

quality parameters are: NH4+ < 2 M; NO3

- / NO2- ~ 0.1 M; PO4

3- ~ 0.2 M;

chlorophyll a < 1 g L-1; TSS < 20 mg L-1.

1.3 Biological Indicators

Due to the close proximity of shrimp farm discharges to several other point and non-point

nutrient sources (ie. sewage effluent, agricultural runoff), it can be difficult to determine the

impacts of aquaculture on the environment (Grant et al., 1995). Techniques are needed to

distinguish the effect of each source and its range of impact so that appropriate discharge

limits can be applied.

INTRODUCTION 5

Plate 1.4 Penaeus japonicus from ponds at Moreton Bay Prawn Farm, Queensland, Australia.

Traditional water quality analyses provide little information as to the impact of nutrients on

the biota in the ecosystem (Lyngby, 1990). As a result there is a lack of data on the

ecological impact of aquaculture effluent (Gowen et al., 1990). The use of biological

indicators can provide information as to the nutrient source, the bioavailability of the

nutrients, and the integration of short lived nutrient pulses (Lyngby, 1990; Horrocks et al.,

1995; Jones et al., 1996; Udy & Dennison, 1997b; Jones et al., 1998; Appendix 1).

1.4 Biological Treatment Options

Concerns about the possible adverse impacts of aquaculture discharge have become a risk

factor for the industry (Braaten, 1991). This has prompted efforts to develop cost-effective

methods of effluent treatment. In addition to prohibitive costs, because of the large volume

of effluent, sewage treatment practices have proved inefficient due to the high suspended

solid load (Tetzlaff & Heidinger, 1990) and the high salinity of aquaculture effluent. There

are a number of commercially available bacterial systems to promote nitrification and

subsequent denitrification to remove nitrogen from the effluent. However, the effectiveness

of such systems for treating shrimp pond effluent have yet to be examined rigorously.

CHAPTER 1 6

The use of filter feeding bivalves such as oysters to consume phytoplankton, zooplankton,

and bacteria (Lin et al., 1993), and macroalgae to assimilate the remaining dissolved nutrients

(Haines, 1975) may prove to be an efficient and economically viable alternative for

improving the water quality of shrimp farm discharges (Hopkins et al., 1993a; Lin et al.,

1993). In addition to filtering organic food particles, oysters can also improve the quality of

pond effluent by reducing the concentration of inorganic suspended solids.

1.4.1 Oysters

The oyster industry in Moreton Bay, Queensland is based on the Sydney Rock Oyster

Saccostrea commercialis (Iredale & Roughley) (Plate 1.5), an estuarine species native to Port

Stevens, N.S.W., Australia and found from Victoria, Australia to Thailand (Angell, 1986).

This species is considered marketable between 29 and 40 g (bottle oysters) and 40 to 67 g

(plate oysters) whole weight (Witney et al., 1988).

Culture of oysters on traditional leases from spat to marketable size takes two to three years

(Witney et al., 1988). Local availability of oysters is seasonal, with oysters being fat and

ready for sale in summer, while in winter when phytoplankton concentrations are lower

(Dennison et al., 1993), they are lean and growth is substantially slower. The use of land

based aquaculture systems has been trialed to improve productivity and year-round

marketability, but most attempts have been relatively unsuccessful, primarily due to the high

cost and unreliability of mass algal culture. In an attempt to find alternative sources of

microalgae as food for enhanced oyster production, shrimp farm effluent has been trialed

(Wang & Jakob, 1991; Hopkins et al., 1993a; Jakob et al., 1993; Lin et al., 1993). Very fast

INTRODUCTION 7

rates of growth has been observed for oysters grown under controlled conditions with shrimp

pond effluent (Jakob et al., 1993).

Plate 1.5 Sydney Rock Oysters (Saccostrea commercialis) cultured in Moreton Bay, Queensland, Australia.

Oysters are suspension feeders and use their gills to filter phytoplankton, zooplankton,

bacteria and other microscopic particles. Bivalves can remove phytoplankton from the water

with high efficiency (Jrgensen, 1966), but their filtering ability is affected by a number of

factors including the water flow rate (Walne, 1972), temperature (Loosanoff & Tommers,

1948), salinity (Djangmah, 1979), reproductive effort, and silt concentration (Loosanoff &

Tommers, 1948; Angell, 1986). A temperature of approximately 30C (Angell, 1986) and

salinity of 35 (Nell & Gibbs, 1986) is optimal, although the survival range for

S. commercialis is 15 50.

Shrimp pond effluent water typically has elevated concentrations of total suspended solids, a

large fraction being small inorganic clay minerals (Smith, 1996). In waters with high

concentrations of silt, oyster pumping and therefore feeding can be greatly inhibited

(Loosanoff & Tommers, 1948), or they may even cease pumping entirely.

CHAPTER 1 8

To overcome the problems of oyster fouling, sedimentation ponds could be used to remove

the larger settleable particles prior to oyster filtration (Wang, 1990). The remaining particles

are either motile, or are small particles (

INTRODUCTION 9

0.5 g d-1, from seed size (0.04 g) to market size (55.0 g), in just 4 months (Jakob et al., 1993).

The authors state that it was clearly shown that undiluted, semi-intensive, marine shrimp

pond water provides all the requirements for the very rapid growth of the American oyster

C. virginica from 0.05 g spat through 78 g adults (Jakob et al., 1993). Evidence that the

quality of oysters remains high in aquaculture was observed by Lam & Wang (1989) who

used shrimp pond water to produce excellent quality half-shell oysters, grown from 0.1g to

54.2 g in 198 days with 96% survival.

The use of oysters as biofilters can improve the quality of water leaving aquaculture ponds,

and potentially provide a secondary cash crop. After filtration by oysters most of the

nutrients (those bound up in phytoplankton and other suspended solids), are deposited as

faeces and pseudofaeces, while the rest are incorporated into oyster tissue. However, oysters

can also contribute significant amounts of ammonia to the effluent through excretion (Srna &

Baggaley, 1976). Ammonia toxicity to shrimp is one of the primary reasons farmers

undertake water exchange (Kou & Chen, 1991), and therefore it must be removed before

effluent water can be recycled back into production ponds. Consequently, removal of these

deposited nutrients from the system entirely will require either physical removal of the settled

sediment, denitrification, or assimilation of the dissolved nutrients (from the remineralisation

of faeces and pseudofaeces) by macroalgae such as Gracilaria spp. (Funge-Smith & Briggs,

1998).

1.4.2 Macroalgae

Macroalgae can absorb significant quantities of dissolved inorganic and organic nutrients,

usually with a preference for NH4+ (D'Elia & DeBoer, 1978; Haines & Wheeler, 1978;

Hanisak & Harlin, 1978; Harlin, 1978). The ability of macroalgae to rapidly take up nutrients

CHAPTER 1 10

for growth, and store luxury reserves in the form of amino acids and pigments makes them

ideal for stripping nutrients from aquaculture effluent (Haines, 1975). Additionally,

macroalgae are known to absorb and store heavy metals (Burdin & Bird, 1994), which may

be a potential pollutant in shrimp pond effluent.

Removal of nutrients by macroalgae is also efficient as harvesting is relatively simple, and

provides an additional cash crop (Hopkins et al., 1995b). Macroalgae can also assimilate

metabolic wastes from mariculture animals, which is beneficial to shrimp production ponds if

the wastewater is to be recycled (Qian et al., 1996). Macroalgae can be used to ensure

complete removal of inorganic nitrogenous excreta from the bivalves (Mann & Ryther,

1977). In particular, commercial red seaweeds such as species from the genera Chondrus,

Gracilaria, Agardhiella and Hypnea are candidates as a final polishing step to leave the

effluent virtually free of inorganic nitrogen (Ryther et al., 1975) (Plate 1.6).

Plate 1.6 Gracilaria edulis collected from Moreton Bay, Queensland, Australia.

Certain species of red macroalgae (Rhodophyta), in particular those from the genera

Gracilaria, Gelidium, and Hypnea are harvested commercially. These species contain

INTRODUCTION 11

sulfated galactan agar and carrageenin which are widely used in the pharmaceutical, cosmetic

and food industries (Raven et al., 1987). Nutrients are generally the limiting factor to

macroalgal growth in natural systems, and attempts have been made to culture them in land

based aquaculture systems. The wastewater from aquaculture effluent contains sufficient

nutrients to sustain the high growth rates required without fertilisation, but the high

concentrations of suspended solids can foul the macroalgae and reduce light availability

(Briggs & Funge-Smith, 1993).

There are potentially considerable economic benefits to be gained from growing macroalgae

in shrimp pond effluent. The growth of Hypnea musciformis in the effluent from a tropical

mariculture system has been estimated as producing a gross harvest value of $107 250 ha-1

annually (Roels et al., 1976). H. musciformis cultured in aquaculture effluent grew at 64.5 g

wet wt d-1, compared to deep water growth of 12.1 g wet wt d-1 (Haines, 1975). Percent

carrageenin yields however were lower, ie., 16% dry wt versus 29% for the deep water,

however the total production of carrageenin is approximately 3 times greater from the

aquaculture effluent (Haines, 1975).

The use of bivalves and / or macroalgae to treat the effluent from shrimp farms has been

investigated in a number of studies (Wang & Jakob, 1991; Hopkins et al., 1993a; Jakob et al.,

1993; Shpigel et al., 1993b; Jones & Preston, 1999; Chapter 3). Using oysters (to filter

phytoplankton, bacteria and other suspended solids), and macroalgae (to take up dissolved

nutrients) can potentially improve the quality of shrimp pond effluent. In addition to the

environmental benefits for receiving waters, there are also economic gains resulting from the

conversion of high cost uneaten and dissolved feed pellets into two additional marketable

crops (Wang, 1990).

CHAPTER 1 12

1.5 Polyculture / Integrated Aquaculture

Polyculture is defined as the culture of several different organisms in the one culture unit. In

contrast, integrated aquaculture is the co-culture of different organisms, but in discrete culture

units (Chien & Tsai, 1985). These techniques are regarded as being more ecologically sound

methods of aquaculture (Mackay & Lodge, 1983), with a more efficient use of resources, and

a higher resilience against environmental fluctuation (Chien & Liao, 1995).

Despite the advantages of these types of combined aquaculture, there may be some problems

associated with management of several organisms all with differing culture requirements.

Management can be more complex with respect to stocking densities, culture techniques and

associated infrastructure, harvesting procedures, and effluent flow management (Chien &

Liao, 1995). Specific problems for intensive shrimp farming relate to fouling effects from the

high concentrations of suspended solids on secondary crop species (and potential biofilters)

such as oysters and macroalgae (Ziemann et al., 1992; Funge-Smith & Briggs, 1998).

Although the use of these and other biological treatment techniques for facilitating water

recycling are ecologically sound, much research is needed to improve the efficiency of these

systems (Lin, 1995).

Effective management of aquaculture effluent can be separated into identification of

downstream impacts, and effective farm management to reduce these impacts. Identification

of impacts to receiving waters may be accomplished with a combination of water and

sediment water analyses with biological indicators to elucidate ecological impacts. Effective

on-farm management of effluent can probably be accomplished by a combination of physical

and biological treatment techniques.

INTRODUCTION 13

1.6 Thesis Aims

Characterise the components of shrimp pond effluent, and their concentrations relative to

the receiving waters,

Develop the use of various marine plants as bioindicators to determine the effects of

prawn farm effluent on receiving waters,

Determine the viability of oysters and macroalgae as biological treatment organisms for

shrimp pond effluent,

Determine the differences in biological filter performance with changes in density, size,

and water flow regimes,

Identify problems associated with maintaining oysters and macroalgae in the high

suspended solids environment and optimise techniques to minimise the impact on their

growth, condition, and effectiveness as biofilters,

Design an integrated system to produce the greatest improvements in water quality, while

maintaining the condition of the biological filter organisms.

CHAPTER 1 14

1.7 Thesis Overview

Despite several reported cases of large scale environmental degradation linked to aquaculture

effluent, there has been no successful determination of the ecological impacts, and certainly

no techniques to distinguish these downstream impacts in relation to other nutrient inputs.

Techniques to improve effluent discharge water quality, including the use of bivalves to filter

aquaculture effluent have been undertaken on a small scale by the industry in other regions of

the world. However, there has been a distinct lack of quantitative data to determine the most

efficient use of these techniques and the ecophysiological responses of the biofilter

organisms. This thesis has addressed these shortcomings.

Bioindicator techniques were developed to investigate the ecological impacts of aquaculture

effluent and biofilter organisms were employed, not only to mitigate these impacts, but also

to provide an efficient use of resources by producing secondary crops from aquaculture farm

effluent. The research is this thesis has been conducted using techniques to look at

ecophysiological responses of organisms and ecological changes in the system. This

contrasts much of the published material in this area, which has been conducted purely at an

applied level.

Evaluation of ecological impacts from shrimp farming was conducted using biological

indicator techniques (tissue nitrogen content, d15N isotopic signatures, amino acid

composition, phytoplankton productivity) in conjunction with more traditional water quality

parameters (nutrient concentrations, suspended solids, chlorophyll a) to determine

ecophysiological changes in the biota in the receiving waters. Rates of isotopic fractionation

of nitrogen in the effluent and the changes in the dissolved free amino acid composition of

the macroalgae incubated in shrimp effluent under controlled laboratory conditions provided

INTRODUCTION 15

some of the background responses used for determining the spatial range of impacts of

shrimp effluent in receiving waters. These biological or ecological health indicators provided

direct measures of the influence of aquaculture discharge.

The effects of different sizes and stocking densities of oysters and different densities of

macroalgae on the water quality (total and dissolved nutrients, chlorophyll a, bacteria, total

suspended solids, organic versus inorganic particulates, and particle size distribution) of

shrimp effluent was determined for a variety of effluent flow regimes and during different

stages of the shrimp growout season. In particular, analysis of the particle size distribution of

the effluent provided information into the mechanisms by which oysters remove particulates

from the water column, especially the small inorganic clay particles that are difficult to

remove by sedimentation or mechanical filtration.

The effects of different concentrations of suspended solids from shrimp effluent on oyster

and macroalgal condition was determined by physiological responses in the organisms. This

information facilitated estimates of the optimum concentration of suspended particulates for

efficient filtration performance by oysters and macroalgae, while minimising sedimentation

time and / or mechanical filtration costs. A variety of novel techniques such as dissolved free

amino acid composition, pigment concentrations, PAM fluorescence, tissue nitrogen and

d15N were used to assess the condition of the macroalgae. These techniques also provide

information about the bioavailability of the nutrient profile from shrimp effluent, i.e. whether

it is suited for uptake by biofilter organisms (e.g. oysters and macroalgae).

Higher effluent flow rates are likely to improve biofilter condition, but may reduce the

filtration performance. In an attempt to improve the condition and performance of the

CHAPTER 1 16

biofilters, experiments were conducted to recirculate the effluent though the biofilter

organisms several times to test the possibility of increasing the effluent flow rate, without

sacrificing improvements in water quality.

The combined efficiency of sedimentation, followed by oyster filtration of particulates and

macroalgal absorption of dissolved nutrients proved to be an effective technique for

improving shrimp pond effluent water quality. After treatment in this polyculture system, the

effluent proved suitable for reuse in shrimp production ponds. The rates of nutrient

regeneration from settled particulates, oyster excretion rates, nutrient uptake rates (bacteria,

phytoplankton and macroalgae) and loss of N to the atmosphere via volatilisation and

denitrification were determined directly, or inferred by difference.

1.7.1 Chapter Outline

Chapter 2

Investigations were conducted to determine the impact of effluent from a local shrimp farm

on the biota and integrity of the receiving waters in Moreton Bay. Results were compared

with data from other unimpacted regions in Moreton Bay and with a nearby sewage treatment

plant. Several bioindicator techniques were utilised to characterise the impacts of the

effluent.

Chapter 3

Experiments were conducted to determine if oysters would be successful at improving the

water quality of shrimp pond effluent, and to assess the optimal stocking density of the

oysters to produce the greatest improvements in water quality.

INTRODUCTION 17

Chapter 4

The filtering efficiency of macroalgae and different sized oysters in raceways with flow

through effluent supply, and recirculating supply were conducted. Issues regarding fouling of

oysters and macroalgae were investigated to determine the maximum concentration of

suspended solids that the oysters and macroalgae could tolerate without adversely impacting

their health, survival and filtration efficiency.

Chapter 5

The overall efficiency of a polyculture treatment system was tested using sedimentation

followed by oyster filtration and macroalgal absorption.

Chapter 6

The conclusions of the study and areas of potential future research and comparisons with the

results of other studies are discussed.

CHAPTER 1 18

1.9 Publication Status of Thesis Chapters

Chapter 2

Jones, A.B., O'Donohue, M.J., Udy, J. & Dennison, W.C. (2001) Assessing ecological impacts of

shrimp and sewage effluent: Biological indicators with standard water quality

analyses. Estuarine, Coastal and Shelf Science 52, 91109.

Presented at the Australian Marine Science Association annual conference,

Adelaide, Australia, July 1998.

Chapter 3

Jones, A.B. & N.P. Preston (1999) Oyster filtration of shrimp farm effluent, the effects on

water quality. Aquaculture Research 30, 51-57.

Chapter 4

Jones, A.B., N.P. Preston & W.C. Dennison (in review) The efficiency and condition of oysters

and macroalgae used as biological filters of shrimp pond effluent. Aquaculture

Research.

Chapter 5

Jones, A.B., Dennison, W.C. & Preston, N.P. (2001) Integrated treatment of shrimp effluent

by sedimentation, oyster filtration and macroalgal absorption: a laboratory scale study.

Aquaculture 193 (1-2), 155-178.

Presented at the World Aquaculture Society Meeting, Sydney, Australia, May 1999.

CHAPTER 2

ASSESSING ECOLOGICAL IMPACTS OF SHRIMP AND SEWAGE EFFLUENT:

BIOLOGICAL INDICATORS WITH STANDARD WATER QUALITY ANALYSES

Abstract

Despite evidence linking shrimp farming to several cases of environmental degradation, there remains a lack of

ecologically meaningful information about the impacts of effluent on receiving waters. The aim of this study

was to determine the biological impact of shrimp farm effluent, and to compare and distinguish its impacts from

a nearby treated sewage discharge. Assessment of impacts was conducted using both water quality / sediment

analyses and biological indicators. Water quality and sediment parameters measured included chlorophyll a,

total suspended solids, volatile suspended solids, dissolved nutrients, salinity, and sediment organic content.

Biological indicator monitoring consisted of analysis of amino acid composition, tissue nitrogen (N) content and

stable isotope ratio of nitrogen (d15N) in seagrasses, mangroves and macroalgae. The study area consisted of

two tidal creeks, one receiving effluent from a sewage treatment plant (sewage creek) and the other receiving

effluent from an intensive shrimp farm (shrimp creek). The creeks discharged into Moreton Bay, a sub tropical

coastal embayment on the east coast of Australia. Water quality in both creeks was significantly modified, but

changes were indistinguishable from unimpacted eastern Moreton Bay levels further than 750 m from the c reek

mouths. Biological indicators, however, detected significant impacts up to 4 km beyond the creek mouths. The

shrimp creek was more turbid due to clay minerals with a relatively high dissolved NH4+ (3.8 M)

concentration, whereas the sewage creek had a higher percentage of organic material (35%) and dissolved

nutrient concentrations were higher, particularly NO3- / NO2

- (65 M) and PO43- (31 M). The sewage creek did

not support high phytoplankton productivity (18-20 mg C m-3 h-1), in spite of high nutrient concentrations.

Mangroves and macroalgae in the sewage creek were highly enriched with sewage nitrogen (indicated by high

d15N), as was seagrass at the creek mouth. The d15N of seagrasses, mangroves and macroalgae ranged from

10.4-19.6 at the site of sewage discharge to 2.9-4.5 at the reference site, 4 km from the creek mouths. The

d15N values of seagrass (4.5) and mangroves (3.4) at the reference site were higher than values reported for

oligotrophic areas of Moreton Bay, but the d15N of macroalgae (2.9) was close to unimpacted eastern

Moreton Bay values. Macroalgae derive nutrients from the water column, whereas seagrass and mangroves take

up nutrients from the sediment. Therefore, deposition of effluent derived N into the sediments is implicated in

the elevated d15N values of the seagrass and mangroves at the reference site. The free amino acid concentration

and composition of seagrass and macroalgae was used to distinguish uptake of sewage and shrimp derived N.

Proline (seagrass) and serine (macroalgae) were high in sewage impacted plants and glutamine (seagrass) and

alanine (macroalgae) were high in plants impacted by shrimp effluent. The d15N and amino acid composition

indicated sewage N extended further from the creek mouths than shrimp N. This analysis of physical / chemical

and biological indicators was able to distinguish the composition and subsequent impacts of aquaculture on the

receiving waters.

CHAPTER 2 20

2.1 Introduction

Aquaculture is a rapidly expanding industry, and its effluent can be a major source of

pollution in marine ecosystems (Chua et al., 1989; Twilley, 1989; Gowen et al., 1990;

Braaten, 1991; Holmer, 1991; Phillips et al., 1991; Macintosh & Phillips, 1992; Pruder, 1992;

Raa & Liltved, 1992; Wu et al., 1994; Samocha & Lawrence, 1997; Hargreaves, 1998).

Environmental studies into the effects of shrimp aquaculture are limited and have mostly

focussed on in-pond water quality, with little research conducted into the ecological impacts

of wastewater on receiving waters (Pillay, 1992).

The monitoring of traditional water quality parameters has identified that downstream

impacts of shrimp effluent, and other forms of aquaculture, are only measurable in close

proximity to the discharge point. Hensey (1991) observed that environmental monitoring of

aquaculture effluent using water quality sampling techniques showed no impacts. Samocha

& Lawrence (1997) observed large diurnal fluctuations in water quality parameters measured

downstream of shrimp farm discharge points, and that no increase in total suspended solids

(TSS) or nutrient concentrations could be measured further than 400 m from the farms

discharge. It is possible, however, that sediment impacts such as increased organic matter

and anoxia, may extend further (up to 1 km) than water column impacts (Wu et al., 1994).

With the current projected expansion of shrimp farming in most coastal areas of the world,

large scale increases in nutrients and suspended solids in the receiving waters are likely.

Elevated loadings of particulate material to receiving waters have immediate effects on the

receiving environment such as reduced light penetration and smothering of benthic fauna and

flora (Abal et al., 1994). In addition, particle loading may also contribute to longer term

ASSESSING ECOLOGICAL IMPACTS 21

changes through initial downstream settling, with resuspension into the water column at a

later time.

Increases in the concentration of NH4+ in receiving waters from shrimp farms have been

observed by many researchers, and as a result nutrient enrichment of poorly flushed

embayments may occur (Gowen & Rosenthal, 1993). In some instances the level of impact

has been sufficient to result in feedback which affects the aquaculture operation itself

(Gowen et al., 1990). Evidence suggests that serious shrimp farm production losses resulting

from the outbreak of disease in Asia and Latin America, are due to the environmental impacts

of shrimp culture (Phillips et al., 1993). In addition to impacts on the aquaculture operation

itself, shrimp farming has been linked to several cases of environmental degradation,

however, despite this type of evidence, there is still a lack of quantitative data on the

ecological impacts to receiving waters (Phillips et al., 1993).

The need for data on the ecological impact of aquaculture effluent has been identified

(Gowen et al., 1990), and it has been shown that physical and chemical water quality

monitoring techniques cannot provide this information. Bioindicators have long been used to

determine ecological impacts of point source discharges (Worf, 1980; Kramer, 1994). For

example, marine macrophytes can be used to provide insights into the ecological impacts of

nutrients and suspended particulates by measuring changes in plant distributions,

morphology, pigment concentrations and total tissue N (Lyngby, 1990; Alamoudi, 1994;

Horrocks et al., 1995; Abal & Dennison, 1996; Udy & Dennison, 1997b). The morphology

of seagrasses can change with reduced light availability, as a consequence of elevated

concentrations of suspended solids in the water column (Abal et al., 1994). Seagrass

CHAPTER 2 22

distribution and depth penetration are also reduced as a consequence of reduced light

availability in shallow estuarine systems (Dennison et al., 1993; Abal & Dennison, 1996).

Recently, marine plants have been used to detect and integrate the long term effects of small

and /or pulsed nutrient inputs in well flushed oceanic systems (Costanzo, 1996), and elucidate

the possible sources of the nutrient inputs (Jones et al., 1996). Macrophyte amino acid

concentrations and composition have been shown to change with various N sources in both

controlled laboratory experiments (Nasr et al., 1968; Di Martino Rigano et al., 1992; Jones et

al., 1996) and field surveys (Udy & Dennison, 1997b). In particular, accumulation of the

amino acids alanine, glutamine, proline and serine in plants (both terrestrial and marine) has

been associated with N uptake, with different amino acids responding to different N sources

(Steward and Pollard, 1962; Silveira et al., 1985; Lawlor et al., 1987; Kiladze et al., 1989; Di

Martino Rigano et al., 1992; Vona et al., 1992; Heuer & Feigin, 1993). Stable isotope ratios

of nitrogen (d15N) have been used widely in marine systems as tracers of discharged nitrogen

from point and diffuse sources, including sewage effluent (Rau et al., 1981; Wada et al.,

1987; Van Dover et al., 1992; Macko & Ostrom, 1994; Cifuentes et al., 1996; McClelland &

Valiela, 1998). Elevated d15N signatures in seagrass, mangroves and macroalgae have been

attributed to plant assimilation of N from treated sewage effluent (Wada et al., 1987; Grice et

al., 1996; Udy & Dennison, 1997b; Abal et al., 1998). This study, however, appears to be the

first to use all these techniques to study the extent of impacts from aquaculture effluent.

In coastal marine systems a variety of point source inputs from aquaculture ponds, sewage

treatment plants, fertiliser plants, agriculture and urban runoff can make it difficult to

determine responsibility for ecological impacts (Grant et al., 1995). With increasing conflict


between users of coastal resources, it has become essential to determine the specific influence

of each source (Teichert-Coddington, 1995).

To assess the potential impact of aquaculture effluent, comparisons between the water

volumes and water quality parameters of aquaculture effluent and treated sewage effluent

have been conducted (Bergheim & Selmer-Olsen, 1982; Muir, 1982; Solbe, 1982; Macintosh

& Phillips, 1992; Paez Osuna et al., 1997). However, there are very few studies comparing

the impacts on the receiving waters (Pearson & Rosenberg, 1978; Cifuentes et al., 1996).

Despite the differences in the two forms of waste, Pearson & Rosenberg (1978) hypothesised

that the impacts of these two sources on receiving sediments would be similar.

Shrimp pond effluent has higher concentrations of suspended solids and phytoplankton

(Ziemann et al., 1992), but lower concentrations of nutrients than sewage effluent (Muir,

1982). Dissolved nutrients in shrimp effluent are predominantly NH4+, whereas sewage

effluent is proportionally higher in NO3-, and PO4

3- (Macintosh & Phillips, 1992). Shrimp

effluent is typically produced in large volumes (Macintosh & Phillips, 1992), which can

equate to up to 40% of the total inputs of N and P in some localised areas (Bergheim &

Selmer-Olsen, 1982). Sewage is freshwater, whereas the salinity of shrimp effluent is

typically 35-36 on the practical salinity scale. These differences may have a considerable

impact on the fate of organisms in the receiving waters when effluent is released into shallow

tidal estuaries. Both sewage and aquaculture effluent can be discharged intermittently,

resulting in large diel fluctuations in water quality. Difficulties in monitoring these variable

discharges can be overcome by the use of biological indicators, which integrate the impacts

of these effluents over time (Costanzo, 1996). Unlike traditional chemical analyses of water

CHAPTER 2 24

column nutrients, these biological indicators reflect the availability of biologically available

nutrients (Lyngby, 1990) which provides more ecologically meaningful information.

The aims of this study were to assess the influences on the receiving environment of

wastewater discharges to a shallow estuarine system. Changes in receiving water and

sediment quality analyses were compared with biological impacts measured as a consequence

of shrimp farm and sewage effluent discharges. The region of influence of these two

pollutant sources is defined, and mechanisms are suggested which may aid in discerning the

relative impacts of these two discharges on a common receiving environment.


2.2 Materials and Methods

2.2.1 Study Region

Moreton Bay is a shallow coastal embayment on the east coast of Australia. The western side

of the bay receives a variety point and non point source inputs including agricultural runoff,

sewage and aquaculture effluent. The eastern bay is well flushed and influenced by oceanic

waters. In eastern Moreton Bay, background concentrations of water quality parameters are:

NH4+ < 2 M; NO3

- ~ 0.1 M; PO43- ~ 0.2 M; chlorophyll a < 1 g L-1; TSS < 20 mg L-1,

and typical d15N values for mangroves, seagrass and macroalgae in the eastern bay are 2-3

(Abal et al., 1998).

Two tidal creeks in close proximity (1.5 km apart) were studied in Moreton Bay, Australia

(Fig. 2.1). One creek received discharge (18000 m3 d-1 containing 2.0 mg N L-1 and

0.2 mg P L-1, which equates to 36 kg N d-1 and 3.6 kg P d-1) from a shrimp farm (Jones,

unpub. data). The creek was 2-3 m deep, approximately 1 km in length, and the shrimp farm

discharge was 500 m from the mouth of the creek. The intensive shrimp farm (6 ha of ponds)

was stocked with Penaeus japonicus (35 animals m-2). Ponds were routinely flushed (~20%

per day) and water discharged into the creek on low tide. Except during the effluent

discharge, the creek runs dry at low tide. The other creek (Eprapah Creek) received

discharge (2400 m3 d-1 containing 4.5 mg N L-1 and 8.0 mg P L-1, which equates to

10.8 kg N d-1 and 19.2 kg P d-1) from a sewage treatment plant (Redland Shire Council, pers.

comm.). The creek was approximately 2-5 m deep, 15 km in length, has a standing body of

water at low tide, and the sewage discharge point was 2 km from the mouth. The sewage

treatment plant serviced approximately 14 000 people and utilised secondary (activated

sludge) treatment techniques. Both creeks were tidally flushed, and had virtually no

freshwater flow during the study period (Autumn, 1997).

CHAPTER 2 26

2.2.2 Experimental Design

Three sites were chosen in each creek, the first at the nutrient source (discharge site), the

second approximately mid way between the nutrient source and the mouth (middle site), and

the final at the mouth of the creek (mouth site) (Fig. 2.1). A site was positioned midway

between both creeks and in close proximity to the shore (midway site). Several more sites

were selected in Moreton Bay in a radiating pattern out from the creek mouths (Oyster Point,

Sewage Plume and Cox Bank), including a reference site located approximately 4 km from

the creek mouths. The creek banks at low tide extended the mouth of the sewage creek as far

as the sewage plume site. At eight of the sites, traditional water quality parameters (dissolved

N & P, total suspended solids, volatile suspended solids, sediment organic content, secchi

depth, chlorophyll a, and physico-chemical parameters) were determined.

Brisbane

MoretonBay

0 0.5 1.0

kilometres

N

Eprapah Ck

ReferenceSite

SewageTreatment

Plant

ShrimpFarm

OysterPoint

DischargeSite

DischargeSite

MiddleSite

MiddleSite

Mouth Site

Mouth Site

MidwaySite

Cox BankSite

SewagePlumeSite

Oyster PointSite

VictoriaPoint

PointHalloran

Coochie-mudloIsland

Nutrient SourceSampling Site


Figure 2.1 Map of study sites in Moreton Bay, including the location of shrimp and sewage effluent discharges.

Bioindicators were utilised at eleven sites with macroalgae and phytoplankton at all sites,

mangroves at the creek sites, and seagrass at the bay sites. Amino acid composition was

determined for macroalgae and seagrass, and the d15N signature and total tissue N was

determined for all bioindicator species.

2.2.3 Collection

Samples of seagrass (Zostera capricorni), mangrove (Avicennia marina), and macroalgae

(Catenella nipae) were collected, placed on ice and returned to the laboratory and prepared

for analysis of %N, d15N and amino acids. In the case of the seagrass and mangroves, the

second youngest leaves were chosen, and for the macroalgae a single mangrove

pneumatophore covered in macroalgae was collected for each replicate. Three replicates for

each plant type were collected at each site.

2.2.4 Analytical Procedures

Salinity was measured with a Horiba U-10 water quality meter (California, U.S.A.) and

expressed on the Practical Salinity Scale.

Chlorophyll a concentration was determined by filtering a known volume of water sample

through Whatman GF/F filters, which were immediately frozen. Acetone extraction and

calculation of chlorophyll a concentration was performed using the methods of Clesceri et al.

(1989), and Parsons et al. (1984).

Light-saturated phytoplankton productivity (potential productivity in mg C m-3 h-1) was

determined in the laboratory using the 14C-bicarbonate incorporation method

CHAPTER 2 28

(Parsons et al., 1984). One hundred millilitres of water from each site was dispensed to three

120 ml polycarbonate bottles. A common dark control was established for each site by

combining 33 ml of sample from each replicate into a fourth bottle wrapped in foil. Aqueous

14C sodium bicarbonate (4 Ci) was added and bottles were incubated at a light intensity of 1100

to 1200 E m-2 s-1. A recirculating water bath and perspex heat shields maintained temperatures

at ambient levels. After approximately two hours, water samples were filtered through 0.4 m

polycarbonate filters (Poretics). The filters were placed into 5 ml scintillation vials and two

drops of 5N HCl were added to each vial to drive off any remaining 14CO2. Four millilitres of

scintillation fluid was added to each vial, and radioactivity as disintegrations per minute (DPM)

determined using a scintillation counter (Packard Tricarb 1600TR, Meriden, Connecticut,

U.S.A.). Total CO2 concentration in samples was determined from carbonate alkalinity using

the method of Parsons et al. (1984).

Total suspended solids concentrations were determined using the methods of

Clesceri et al. (1989). A known volume of water was filtered onto a pre-weighed and pre-

dried (110 C; 24 h) Whatman GF/C glass fibre filter. The filter was then oven dried at 60 C

for 24 h and total suspended solids calculated by comparing the initial and final weights.

Volatile suspended solids were determined as loss on ignition by combusting samples in a

muffle furnace for 12 h at 525 C (Clesceri et al., 1989). The organic content of the sediment

was determined from 10 cm deep core samples collected using 50 mL cut-off syringes. The

sediment sample was combusted in a muffle furnace at 525 C for 12 h and the proportion of

organic material determined by loss on ignition (Clesceri et al., 1989).

Dissolved inorganic nutrients (NH4+, NO3

-/NO2-, and PO4

3-) were determined by filtering

water samples through Whatman GF/F glass fibre filters and freezing them immediately on


dry ice. Samples were analysed within two weeks by the NATA accredited Queensland

Health Analytical Services Laboratory in accordance with the methods of Clesceri et al.

(1989) using a Skalar autoanalyser (Norcross, Georgia, U.S.A.).

For analysis of plant total tissue N, d15N and amino acids, tissue was rinsed in distilled water

to remove nutrients and sediment from the thallus surface, and then prepared for analysis.

For calculation of total tissue N content and the d15N isotopic signature, samples were oven

dried to constant weight (24 h at 60 C), ground and three sub-samples were oxidised in a

Roboprep CN Biological Sample Converter (Europa Tracermass, Crewe, U.K.). The

resultant N2 was analysed by a continuous flow isotope ratio mass spectrometer (Europa

Tracermass, Crewe, U.K.). Total %N of the sample was determined, and the ratio of 15N to

14N was expressed as the relative difference between the sample and a standard (N2 in air)

using the following equation (Peterson & Fry, 1987):

d15N = (15N/14N (sample) / 15N/14N (standard) 1) x 1000 ()

For amino acid analysis, approximately 1.0 g wet weight of plant tissue was weighed and

placed in 5 mL of 100% methanol (analytical reagent grade) for 24 h to extract amino acids.

The methanol extract was filtered through Millipore Millex - HV13 (0.45 mm) filters and

injected into a post column derivatisation HPLC amino acid analyser (Beckman System

6300, Fullerton, California, U.S.A.), for detection of ninhydrin positive free amino acid

groups at 570 nm. Results were calculated and expressed as mol g-1 wet weight. As well as

detecting free amino acids, this technique also measures the concentration of free NH4+ in

plant tissue. Changes in amino acid composition were used to infer nutrient source (either

shrimp or sewage effluent). This technique was based on responses observed under ambient

field, as well as controlled laboratory conditions using artificial nutrient additions (Jones et

al., 1996; Udy & Dennison, 1997a).

CHAPTER 2 30

2.2.5 Statistical Analysis

For all sampling techniques, three replicates were analysed and means and standard errors

were calculated. Differences between treatments were tested for significance using one way

analysis of variance (ANOVA) and Tukey's Test for multiple comparison of means at a

significance level of 0.05 using Minitab 12.1 software (State College, Pennsylvania, U.S.A.).


2.3 Results

2.3.1 Physical and Chemical Water Quality Analyses

The concentrations of dissolved nutrients, chlorophyll a, phytoplankton productivity, total

suspended solids, volatile suspended solids, and sediment organic content were different

between the sewage and shrimp creek discharge sites. However, these parameters failed to

detect an impact at the midway site (750 m beyond the mouths of the creeks), with values not

significantly higher than at the reference site (4 km from the creek mouths) (Table 2.1).

2.3.1.1 Salinity

All sites in the shrimp creek and in the bay were close to full salinity seawater (35-36). At

the sewage creek discharge, the salinity was 29 as a consequence of freshwater inputs from

the sewage effluent. Salinity increased downstream to 35 at the mouth site.

2.3.1.2 Nutrients

The concentrations of dissolved nutrients (NH4+, NO3

- / NO2- and PO4

3-) for each creek were

highest at the discharge sites, and declined rapidly towards the mouth site. In particular,

NH4+ concentration decreased to near eastern Moreton Bay concentrations at both creek

mouth sites. The concentration of dissolved nutrients at the midway site (750 m from the

creek mouths) was not significantly different (p > 0.05) from the reference site (~4 km from

the creek mouths) (Table 2.1). The dissolved nutrient ratios were significantly different

(p < 0.05) between the two discharge sites. At the sewage creek discharge site, NH4+ : NO3

- /

NO2- was 0.45, compared to 3.8 for the shrimp creek discharge site. The ratio of DIN (NH4

+

+ NO3- / NO2

-) to DIP (PO43-) at the sewage creek discharge site was 3.0, compared to 24 at

the shrimp creek discharge site. The concentrations of dissolved nutrients at the midway and

CHAPTER 2 32

reference sites were at eastern Moreton Bay levels, and the relative ratios of the dissolved

nutrients were similar to the shrimp creek.

2.3.1.3 Phytoplankton

Chlorophyll a concentration was not significantly different (p > 0.05) between the discharge

site in the shrimp creek (10.8 g L-1) and the discharge site in the sewage creek (11.1 g L-1).

However, at the creek mouth sites the concentration in the sewage creek (5.2 g L-1) was

significantly lower (p < 0.001) than in the shrimp creek (17.9 g L-1). The concentration of

chlorophyll a at the midway site (2.5 g L-1) was not significantly higher (p > 0.05) than the

reference site (1.8 g L-1) (Table 2.1).

Despite the relatively low NH4+ concentration at the shrimp creek discharge site, the

phytoplankton productivity (212 mg C m-3 h-1) was significantly higher (p < 0.001) than at

the sewage creek discharge site (20 mg C m-3 h-1). However, the high productivity at the

shrimp creek discharge site did not result in a significantly higher (p > 0.05) chlorophyll a

concentration. The concentration of chlorophyll a in the shrimp creek increased from

10.8 g L-1 at the discharge site to 17.9 g L-1 at the mouth site, probably due to increased

light availability resulting from the reduction in the concentration of inorganic and other

suspended solids. Phytoplankton productivity at the midway site (9 mg C m-3 h-1) was not

significantly different (p > 0.05) to the reference site (11 mg C m-3 h-1) (Table 2.1).

Table 2.1. Results of traditional water quality monitoring for the creek with shrimp farm effluent and sewage treatment effluent. DIN = Dissolved Inorganic Nitrogen;

DIP = Dissolved Inorganic Phosphorus; Chl a = Chlorophyll a; Phyto Prod = phytoplankton productivity; TSS = total suspended solids; VSS = volatile suspended solids;

Secchi = secchi disc depth. Only one replicate measurement was recorded for Secchi disk depth and salinity (Practical Salinity Scale).

Sampling

Site

Salinity

NH4+

(M)

NO3-/NO2

--

(M)

PO43-

(M)

DIN: DIP

Ratio

Chl a

(g L-1)

Phyto Prod

(mg C m-3 h-1)

TSS

(mg L-1)

VSS

(% of TSS)

Secchi

(m)

Sediment

%Organic

Shrimp Discharge 35 3.8a 1a 0.2a 24c 10.8a 212d 63.5a 19a 0.5 6.0a

Shrimp Middle 35.5 1.6a 0.4a 0.3a 7ab 11.9ab 87c 51.5ab 21a 0.7 6.1a

Shrimp Mouth 36 2.3a 0.3a 0.2a 13bc 17.9b 157b 40.2b 26a 0.5 8.3ab

Midway 36 0.8a 0.2a 0.4a 3a 2.5c 9a 18.2d 25a 1.0+ 7.1a

Sewage Discharge 29 29b 65b 31b 3a 11.1ab 20a 44.3b 35b 1.1 13.5c

Sewage Middle 33 5.4a 8a 5.5a 2a 9.2ac 20a 32.9bc 33b 1.0 7.5ab

Sewage Mouth 35 2.4a 2.9a 2.1a 3a 5.2ac 18a 32.5bc 28ab 1.1 5.6a

Reference 36 1.2a 0.5a 0.3a 6ab 1.8c 11a 20.2d 28ab 1.9 11bc

F Value 40*** 15*** 34*** 13*** 15*** 88*** 35*** 14*** 15***

* p < 0.05; ** p < 0.01; *** p < 0.001. abc Means with different letters are significantly different at p < 0.05.

CHAPTER 2 34

2.3.1.4 Suspended Solids and Secchi Depth

The concentration of total suspended solids (TSS) at the shrimp creek discharge site

(63 mg L-1) was significantly higher than the sewage creek discharge site (44 mg L-1). The

concentrations at the midway (18 mg L-1) and reference sites (22 mg L-1) were not

significantly different (p > 0.05) from each other, but were significantly (p < 0.05) lower than

both creek mouth sites indicating significant sedimentation or dilution (Table 2.1).

The organic fraction of the suspended solids (volatile suspended solids) was significantly

higher (p < 0.001) at the sewage discharge site (35%) compared to the shrimp discharge site

(19%). In the shrimp creek the concentration of organic particles increased towards the

mouth in proportion with the increasing chlorophyll a concentration (r2 = 0.60). In

comparison, the concentration of organic particles in the sewage creek decreased in

proportion with the concentration of chlorophyll a (r2 = 0.8) (Table 2.2).

Secchi disk depths did not vary along the length of either creek, from discharge site to mouth

site. The mean secchi depth in the sewage creek (~1.0 m) was approximately double the

depth in the shrimp creek (~0.6 m), but only half that of the reference site (1.9 m). The

secchi depth at the midway site was greater than 1 m, but water depth was too shallow to

obtain a measurement (Table 2.1).

2.3.1.5 Sediment Organic Content

The organic content of the sediment (loss on ignition) in the shrimp creek increased from

6.0% at the discharge site to 8.3% at the mouth site, probably due to sedimentation. In the

sewage creek, the organic content declined significantly (p < 0.001) from the discharge site


(13.5%) to the mouth site (5.6%), probably due to senescence and subsequent sedimentation

of phytoplankton and other organic particulates near the discharge site (Table 2.1).

Table 2.2. Correlations (r2) between the concentration of phytoplankton (chlorophyll a) and phytoplankton

productivity (14C uptake) and various water quality parameters. DIN = Dissolved Inorganic Nitrogen; DIP =

Dissolved Inorganic Phosphorus; Phyto Prod = phytoplankton productivity (mg C m-3 h-1); Chl a =

Chlorophyll a (g L-1); TSS = total suspended solids (mg L-1); VSS = volatile suspended solids (mg L-1); ISS =

inorganic suspended solids (mg L-1); Secchi = secchi disc depth (m). Numbers in bold type indicate significant

correlations (r2 0.6).

TSS

(mg L-1)

VSS

(mg L-1)

ISS

(mg L-1)

NH4+

(M)

NO3-/NO2

-

(M)

PO43-

(M)

DIN:

DIP

Salinity

Shrimp Creek

Chl a - 0.85 - 0.60 - 0.87 0.12 0.51 0.14 0.09 0.86

Phyto Prod 0.21 0.48 0.19 0.93 0.56 - 0.81 0.94 0.19

Sewage Creek

Chl a 0.60 0.80 0.37 0.66 0.63 0.66 0.04 - 0.85

Phyto Prod 0.27 0.51 0.11 0.34 0.32 0.35 0.25 - 0.60

2.3.2 Bioindicators

In contrast to the water quality parameters, the responses of the bioindicator parameters at

sites beyond the creek mouths were elevated compared to the reference site. For some of the

parameters, the reference site appeared to be influenced by nutrients from the discharges.

2.3.2.1 Tissue Nitrogen Content

The %N of the macroalgae was responsive to the nutrient sources, with the highest value at

the sewage creek discharge site (3.1%), which was significantly higher (p < 0.05) than the

shrimp discharge site (1.9%) (Table 2.3; Fig. 2.2). There was no decrease in the %N of the

macroalgae with distance from the discharge site in the shrimp creek. However, in the

sewage creek the macroalgae at the mouth site had a %N of 1.6%, which was not

CHAPTER 2 36

significantly elevated (p > 0.05) above the reference site (1.5%). The %N of the macroalgae

at the midway site (2.3%) was significantly (p < 0.05) elevated above values in the

macroalgae at both of the creek mouth sites (Table 2.3; Fig. 2.2).

The %N of seagrass leaves was significantly higher (p < 0.05) at the sewage creek mouth site

(2.7%) compared with the shrimp creek mouth site (2.3%). The next three sites distant from

the creek mouths (midway, Oyster Point, and Sewage Plume) were not significantly lower

than the shrimp creek mouth site (2.3%). The seagrass %N at the next most distant site (Cox

Bank) was 2.0%, which was not significantly higher (p < 0.05) than at the reference site

(1.7%) (Table 2.3; Fig. 2.2).

The %N of the mangrove leaves appears less sensitive to nutrient inputs, with none of the

mangroves at the other sites being significantly higher (p < 0.05) than the reference site

(1.7%) (Table 2.3; Fig. 2.2).

2.3.2.2 d15N Stable Isotope Ratio of Nitrogen

The d15N isotopic signatures of the seagrass, macroalgae and mangroves were significantly

different (p < 0.001) between sites (Table 2.3; Fig. 2.3). The highest d15N was in the

macroalgae at the sewage creek discharge site (19.6), and the lowest in the macroalgae at

the reference site (2.9). d15N in the macroalgae in the sewage creek decreased with

distance away from the source. The value at the discharge site in the shrimp creek was 7.1,

with no significant difference (p > 0.05) along the length of the shrimp creek to the mouth

(7.9). The d15N at the midway site (6.4) was not significantly lower (p > 0.05) than the


creek mouth sites, indicating influence of nutrients from the discharges. The d15N at the

reference site (2.9) was significantly lower (p < 0.001) than all other sites.

The d15N of seagrass leaves at the sewage plume site (8.0) was significantly higher

(p < 0.05) than all other sites. The d15N was not significantly different (p > 0.05) between the

two creek mouths (7.1 and 6.8), but both were significantly higher (p < 0.05) than the

midway (5.8), Oyster Point (4.7) and Cox Bank (5.5) sites, and the reference site

(4.5) (Table 2.3; Fig. 2.3).

The highest d15N of mangrove leaves was 10.4 at the sewage discharge site, compared with

7.7 at the shrimp discharge site. Despite the significant differences (p < 0.05) at the

source, the d15N values at the creek mouths were not significantly different (p > 0.05) from

each other (4.9 and 4.6). The mid

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