BY Mathew Ross

ASSESSMENT OF INTERNATIONAL PRACTICES ON THE USE OF FISH IN

TOXICITY TESTING AND ADAPTATIONS FOR SOUTH AFRICAN

CONDITIONS

Mathew Ross

A DISSERTATION SUBMITTED IN FULFILMENT

OF THE REQUIREMENTS FOR THE

DEGREE OF

MAGISTER SCIENTIAE

AQUATIC HEALTH

IN THE FACULTY OF SCIENCE

AT THE

RAND AFRIKAANS UNIVERSITY

NOVEMBER 2004

SUPERVISOR: PROF. V. WEPENER

CO SUPERVISOR: PROF. G.J. STEYN

CO SUPERVISOR: PROF. H.H. DU PREEZ

TABLE OF CONTENTS

List of Tables ix

List of Figures x

Acknowledgments xii

Summary xiii

Opsomming xv

Chapter 1

Introduction

1.1 Background 3 1.2 Objectives of the study 5 1.3. Choice of fish species 7 1.4. Literature overview 8 1.5. References 10

Chapter 2

Maintenance and breeding of B. trimaculatus and B. argenteus (Cyprinidae) to determine their suitability for use in routine laboratory toxicity tests

2.1. Background 15 2.2. Barbus trimaculatus 15 2.2.1. Introduction 15 2.2.1.1. Natural history 15 2.2.1.2. Background on captive breeding and use of B. trimaculatus 17 2.2.2. Environmental requirements and procedures for maintenance of B. trimaculatus 18 2.2.2.1. Water temperature 18 2.2.2.2. Water chemistry 18 2.2.2.2.1. pH 18 2.2.2.2.2. Total water hardness 19 2.2.2.3. Photoperiod 19 2.2.2.4. Aquaria design 19 2.2.2.4.1. Aquaria 19 2.2.2.4.2. Stocking densities of fish in aquaria 20 2.2.2.4.3. Substrate 22

iii

TABLE OF CONTENTS

2.2.2.4.4. Filtration 22 2.2.3. Barbus trimaculatus brood stock management 23 2.2.3.1. Age of brood stock 23 2.2.3.2. Conditioning of brood stock 24 2.2.3.2.1. Temperature 24 2.2.3.2.2. Food 24 2.2.3.2.3. Photoperiod 24 2.2.4. Breeding B. trimaculatus 25 2.2.4.1. Breeding system design 25 2.2.4.2. Gender determination of adult fish 26 2.2.4.3. Gender ratios 26 2.2.4.4. Procedure for breeding B. trimaculatus 26 2.2.5. Larvae and juvenile care 27 2.2.5.1. Care of free-swimming larvae 28 2.2.5.2. Care of juveniles 28 2.3. Barbus argenteus 29 2.3.1. Introduction 29 2.3.1.1 Natural history 30 2.3.1.2. Background on captive breeding and use of B. argenteus 31 2.3.2. Environmental requirements and procedures for maintenance of B. argenteus 31 2.3.2.1. Gender determination of adult fish 31 2.4. Results and discussion 32 2.4.1. Spawning tank design and breeding of fish 32 2.5. Conclusions and recommendation 34 2.7 References 36

Chapter 3

Maintenance and breeding of O. mossambicus, T. sparrmanii and P. p. philander (Cichlidae) to determine their suitability for use in routine laboratory toxicity tests

3.1. Background 39 3.2. Oreochromis mossambicus 39 3.2.1. Introduction 39 3.2.1.1. Natural history 39 3.2.1.2. Background on captive breeding and use of O. mossambicus 42 3.2.2. Environmental requirements and procedures for maintenance of O. mossambicus 43 3.2.2.1. Water temperature 43 3.2.2.2. Water chemistry 43 3.2.2.2.1. pH 43 3.2.2.2.2. Salinity 43 3.2.2.2.3. Total water hardness 44 3.2.2.3. Photoperiod 44 3.2.2.4. Aquaria design 44 3.2.2.4.1. Aquaria 44 3.2.2.4.2. Stocking densities of fish in aquaria 44

TABLE OF CONTENTS

3.2.2.4.3. Substrate 45 3.2.2.4.4. Filtration 45 3.2.2.4.5. Maintenance 46 3.2.3. Oreochromis mossambicus brood stock management 46 3.2.3.1. Age of brood stock 46 3.2.3.2. Conditioning of brood stock 46 3.2.3.2.1. Temperature 46 3.2.3.2.2. Food 47 3.2.4. Breeding O. mossambicus 47 3.2.4.1. Breeding system design 47 3.2.4.2. Gender ratios 47 3.2.4.3. Procedure for breeding O. mossambicus 47 3.2.5. Embryo and larvae care 48 3.2.5.1. Embryo care 48 3.2.5.2. Care of free-swimming larvae and juveniles 50 3.2.6. Conclusions and recommendations 50 3.3. Tilapia sparrmanii 52 3.3.1. Introduction 52 3.3.1.1. Natural history 52 3.3.1.2. Background on captive breeding and use of T. sparrmanii 54 3.3.2. Environmental requirements and procedures for maintenance of T. sparrmanii 55 3.3.2.1. Water temperature 55 3.3.2.2. Water chemistry 55 3.3.2.2.1. pH 55 3.3.2.2.2. Total water hardness 55 3.3.2.3. Photoperiod 56 3.3.2.4. Aquaria design 56 3.3.2.4.1. Aquaria 56 3.3.2.4.2. Stocking densities of fish in aquaria 56 3.3.2.4.3. Substrate 56 3.3.2.4.4. Filtration 57 3.3.2.4.5. Maintenance 57 3.3.3. Tilapia sparrmanii brood stock management 57 3.3.3.1. Age of brood stock 57 3.3.3.2. Conditioning of brood stock 58 3.3.3.2.1. Temperature 58 3.3.3.2.2. Food 58 3.3.3.2.3. Photoperiod 58 3.3.4. Breeding T. sparrmanii 58 3.3.4.1. Breeding system design 58 3.3.4.2. Gender ratios 60 3.3.4.3. Procedure for breeding T. sparrmanii 60 3.3.5. Embryo and larval care 61 3.3.5.1. Embryo care 61 3.3.5.2. Care of free-swimming larvae and juveniles 62 3.3.6. Conclusions and recommendations 62 3.4. Pseudocrenilabrus philander philander 64 3.4.1. Introduction 64 3.4.1.1. Natural history 64 3.4.1.2. Background on captive breeding and use of P. p. philander 66 3.4.2. Environmental requirements and procedures for maintenance of P. p. philander 67

TABLE OF CONTENTS

3.4.3. Procedure for breeding P. p. philander 67 3.4.4. Embryo and larval care 68 3.4.4.1. Embryo care 68 3.4.4.2. Care of free-swimming larvae and juveniles 69 3.4.5. Conclusions and recommendations 69 3.5. Conclusions and recommendations for the use of O. mossambicus, T. sparrmanii and P. p. philander as routine toxicity testing species 70 3.6. References 71

Chapter 4

Maintenance and breeding of Poecilia reticulata (Poeciliidae) to determine its suitability for use in routine laboratory bioassays

4.1. Background 75 4.2. Introduction 75 4.2.1. Natural history of P. reticulata 75 4.2.2. Background on captive breeding and use of P. reticulata 78 4.3. Environmental requirements and procedures for maintenance of P. reticulata 79 4.3.1. Water temperature 79 4.3.2. Water chemistry 80 4.3.2.1. pH 80 4.3.2.2. Salinity 80 4.3.2.3. Total water hardness 80 4.3.3. Aquaria design 81 4.3.3.1. Aquarium size and stocking densities of fish 81 4.3.3.2. Filtration 81 4.3.3.3. Maintenance 82 4.4. Poecilia reticulata brood stock management 83 4.4.1. Age of brood stock 83 4.4.2. Conditioning of brood stock 84 4.4.2.1. Temperature 84 4.4.2.2. Food 85 4.5. Breeding P. reticulata 86 4.5.1. Breeding system design 86 4.5.2. Gender ratios 89 4.5.3. Procedure for breeding P. reticulata 89 4.6. Larvae and juvenile care 90 4.7. Conclusions and recommendations 91 4.8. References 94

TABLE OF CONTENTS

Chapter 5

Maintenance and breeding of D. rerio (Cyprinidae) to determine its suitability for use in routine laboratory toxicity tests

5.1. Introduction 99 5.1.1. Aims and objectives 99 5.1.2. Natural history 99 5.1.3. Background on captive breeding and use of D. rerio 103 5.1.4. Important documentation 107 5.2. Environmental requirements and procedures for maintenance of D. rerio 109 5.2.1. Water temperature 109 5.2.2. Water chemistry 111 5.2.2.1. pH 111 5.2.2.2. Conductivity 114 5.2.2.3. Total water hardness 114 5.2.2.4. Reconstituted water 116 5.2.3. Photoperiod 116 5.2.4. Aquaria design 117 5.2.4.1. Aquaria 117 5.2.4.2. Stocking densities of fish in aquaria 118 5.2.4.3. Substrate 119 5.2.4.4. Filtration 120 5.2.4.5. Maintenance 128 5.2.4.5.1. General 128 5.2.4.5.2. Water changes 129 5.2.4.5.3. Maintenance of flow-through systems 129 5.2.4.5.4. Maintenance of filters 129 5.2.4.5.5. Periodic sterilisation 130 5.3. Danio rerio brood stock management 131 5.3.1. Age of brood stock 131 5.3.2. Conditioning of brood stock 132 5.3.2.1. Temperature 132 5.3.2.2. Food 132 5.3.2.3. Photoperiod 134 5.4. Breeding D. rerio 134 5.4.1. Breeding system design 135 5.4.2. Gender ratios 136 5.4.3. Procedure for breeding fish 137 5.5. Embryo, larvae and juvenile care 138 5.5.1. Embryo care 138 5.5.2. Care of free-swimming larvae and larvae 138 5.6. Troubleshooting unexpected spawning results 139 5.6.1. A general decrease in fecundity amongst all of the spawning groups 140 5.6.2. A general decrease in fecundity amongst individual spawning groups 142 5.7. Conclusions and recommendations 143 5.8. References 145

vii

TABLE OF CONTENTS

Chapter 6

Toxicity assessment of D. rerio, P. reticulata, B. trimaculatus, B. argenteus, O. mossambicus, T. sparrmanii and P. p. philander

6.1. Introduction 153 6.1.1. Background 153 6.1.2. Uptake and effects of chemicals on fish 156 6.1.3. The use of fish in standard toxicity tests 160 6.1.4. Objectives 167 6.2. Materials and methods 167 6.2.1. Apparatus and test conditions 167 6.2.2. Handling and placing test organisms within the testing beakers 169 6.2.2.1. Danio rerio 170 6.2.2.2. Barbus argenteus and B. trimaculatus 170 6.2.2.3. Poecilia reticulata 171 6.2.2.4. Oreochromis mossambicus, T. sparrmanii and P. p. philander 171 6.2.3. Statistical analysis of the data 171 6.3. Results and discussion 173 6.3.1. Test conditions 173 6.3.2. Handling and placing test organisms within the test beakers 173 6.3.2.1. Danio rerio 173 6.3.2.2. Barbus argenteus and B. trimaculatus 174 6.3.2.3. Oreochromis mossambicus, T. sparrmanii and P. p. philander 174

6.3.3. Relative sensitivities to K2Cr2O7 175 6.4. Conclusions and recommendations 182 6.5. References 183

Chapter 7

General conclusions, recommendations and scope for future research

7.1. General conclusions 189 7.2. References 199

Appendix A 203

Appendix B 206

viii

LIST OF TABLES

List of Tables

Table 2.1: Synonyms, their authors, status and current validity of B. trimaculatus (adapted from Fishbase, 2004). 16 Table 2.3: Synonyms, their authors, status and current validity of B. argenteus (adapted from Fishbase, 2004). 29 Table 2.2: Summary of recommended ranges of physical and chemical water quality parameters of the system water conducive to successful maintenance and breeding of B. trimaculatus and B. argenteus. 35 Table 3.1: Synonyms, their authors, status and current validity of O. mossambicus (adapted from Fishbase, 2004). 40 Table 3.2: Summary of recommended ranges of physical and chemical water quality parameters of the system water conducive to successful maintenance and breeding of O. mossambicus. 51 Table 3.3: Synonyms, their authors, status and current validity of T. sparrmanii (adapted from Fishbase, 2004). 53 Table 3.4: Summary of recommended ranges of physical and chemical water quality parameters of the system water conducive to successful maintenance and breeding of T. sparrmanii. 63 Table 3.5: Synonyms, their authors, status and current validity of P. philander philander (adapted from Fishbase, 2004). 64 Table 3.6: Summary of recommended ranges of physical and chemical water quality parameters of the system water conducive to successful maintenance and breeding of P. p. philander. 69 Table 4.1: Synonyms for P. reticulata, their status and current validity (adapted from Petrovický, 1998 and Fishbase, 2004). 76 Table 4.2: Summary of recommended ranges of physical and chemical water quality parameters of the system water conducive to successful maintenance and breeding of P. reticulata. 91 Table 5.1: Synonyms for D. rerio, their status and present validity (adapted from Fishbase, 2004). 100 Table 5.2: The correlation between an increase in pH of a system and the subsequent increase of toxic NH3 at 26 °C (adapted from Wilkerson, 2001). 112

Table 5.3: Typical water hardness ranges measured in both mg/l CaCO3 as well as °dH (adapted from Sandford, 2003). 115 Table 5.4: Summary of water chemistry parameters and physical requirements for the maintenance of D. rerio. 120 Table 5.5: Recommended routine maintenance schedule. 131 Table 5.6: Summary of the most important physical and water chemistry parameters required for successful culturing of D. rerio. 144 Table 6.1: Comparative matrix denoting which species of fish differ significantly (P<0.05) from one another in terms of sensitivity to K2Cr2O7. 180

LIST OF FIGURES

List of Figures

Figure 2.1: Barbus trimaculatus. 17 Figure 2.2: Breeding tank for B. trimaculatus showing the basic features within the tank. 20 Figure 2.3: Breeding tank for B. trimaculatus showing the basic features of the filtration system. 23 Figure 2.4: Artificial plants made to create shelter and security for fish. 25 Figure 2.5: Barbus argenteus (Photograph by R. Bills). 30 Figure 3.1: Oreochromis mossambicus. 41 Figure 3.2: The funnel system used for the artificial incubation of mouthbrooder-type embryos. 49 Figure 3.3: Tilapia sparrmanii (Photograph by R. Bills). 54 Figure 3.4: Breeding tank design for T. sparrmanii. 59 Figure 3.5: Incubation chamber for artificially incubating embryos of T. sparrmanii. 62 Figure 3.6: Pseudocrenilabrus philander philander. 66 Figure 4.1: Poecilia reticulata male (left) and female (right). 77 Figure 4.2: Corydoras aeneus – ideally suited to keeping the bottom of the breeding aquarium free from surplus food. 82 Figure 4.3: Adult female P. reticulata showing symptoms of abdominal dropsy, which include protruding scales. 84 Figure 4.4: Breeding cages used for P. reticulata breeding showing how the cages rest on the sides of the tank allowing the upper surface to be above the water line. 87 Figure 5.1: Danio rerio adult male. 101 Figure 5.2: A group of adult male D. rerio (left) showing the obvious golden sheen. The females (right) are more silver in colour, with distended abdomens – particularly prior to spawning. 101 Figure 5.3: Typical scenario of nitrogen cycling within a new aquarium system (adapted from Wilkerson, 2001). 122 Figure 5.4: A double sponge air-driven filter (‘Oxy Plus Bio Filter II’). It is useful for filtering aquarium water containing embryos and very young fish. 123 Figure 5.5: An air-driven box-type corner filter with filter media. 124 Figure 5.6: An external canister filter. This filter works by siphoning water from the aquarium, forcing it through a filter medium, before pumping it back into the aquarium. 125 Figure 5.7: A flow-through system with a series of tanks that all share a common filter. 126 Figure 5.8: The common filter system shared by a series of 10 tanks that form part of a flow-through system. 127 Figure 5.9: The outlets of all of the tanks in a flow-through system need to be fitted with a mesh to stop any fish from being sucked into the drain pipe and landing in the filter. 127 Figure 5.10A and 5.10B: Breeding cage design and placement. 136 Figure 6.1: Interrelationships between the major environmental factors that can affect a fish community (adapted from Lloyd, 1992). 155

LIST OF FIGURES

Figure 6.2: Uptake, accumulation and loss processes for a toxicant in the ambient water with fish (adapted from Connell et al, 1999). 159

Figure 6.3: Relative sensitivities of different fish species to K2Cr2O7. Bars represent mean LC50 values (± standard error). The numbers of tests on which the means are based are presented in parenthesis in the legend. 176

Figure 6.4: Comparison of the mean LC50 values (± standard error) when using early larval stages of D. rerio in USEPA dilution water and ISO dilution water. 177 Figure 6.5: Results of the tests done with D. rerio of six weeks old at 21 °C and 25 °C. The bars represent the mean LC50 values (± standard error) at the two different temperatures. 179

ACKNOWLEDGMENTS

Acknowledgments

This study was funded by Rand Water and could not have been possible without the willing support offered by the staff of Scientific Services, Analytical

Services, Hydrobiology Division of Rand Water, Vereeniging. For this I am very grateful. A further acknowledgement goes to the Rand Afrikaans

University (University of Johannesburg) for the use of their equipment as well as facilities that made this study possible. I am grateful to the staff and friends of the Zoology Department of RAU for their support throughout the duration of the project, especially the aquarium staff, Moses Mathonsi and

Solomon (Solly) Tsabalala for their willingness to help with the maintenance and upkeep of the equipment used for the study. A special thank you goes to

Prof. Hein du Preez (Rand Water), Prof. Gert Steyn (Ecodynamics) and Prof.

Victor Wepener (RAU) for their guidance and the role that they played in the formulation and structure of the study as well as for their support and willingness to help wherever possible. I am also very grateful to my family who have always supported me, both financially as well as emotionally, throughout my whole university career and encouraged me wherever possible

– this would not have been possible without them. A special thank you goes to my uncle ‘Chappy’. Without his initial financial support, I would not be where I find myself today. Lastly, the support and assistance of my life partner, Tahla Ansara, will never be forgotten. Your help, support, love and encouragement were an endless source that fuelled my enthusiasm for the project, both on the hobby as well as the professional level – I will always be indebted to you.

xii

SUMMARY

Summary

This study was initiated after a clear need to test and establish a more user- friendly fish species for use in routine laboratory bioassays was identified.

This led to a literature review of current toxicity testing species of fish being used, internationally as well as nationally, and identifying which species could possibly be the most suited for use in South African laboratories. From this literature review, it was evident that much emphasis is placed on the practicability of the chosen fish species, and the fact that it can easily be bred within the laboratory, as well as the particular fish specie’s general sensitivity to various toxicants over a wide range of concentrations. The objective of this study is therefore to profile various species of fish to determine which species would be most suited to routine toxicity testing under South African laboratory conditions. This will be done through assimilation of available literature as well as personal communications with people with various expertise and experience in working with the particular fish species. This choice will then be based on the ability of the particular species of fish to ‘balance’ amenability to laboratory conditions with general sensitivity to toxicants. Various indigenous as well as exotic species were therefore selected and tested for suitability for routine testing. Exotic species included Poecilia reticulata and Danio rerio, while the indigenous species tested were Barbus trimaculatus, Barbus argenteus, Tilapia sparrmanii, Oreochromis mossambicus and

Pseudocrenilabrus philander philander. Breeding experiments were conducted with all of the abovementioned species and, based on these results; recommendations are made as to which species of fish showed the highest degree of amenability to maintenance within the laboratory. These

xiii

SUMMARY recommendations are also based on cost and space economy, as well as ease and reliability of routine breeding and fecundity under laboratory conditions. The species ultimately recommended for fulfilling these criteria was D. rerio. All of the fish species concerned were then exposed to different concentrations of the same reference toxicant – potassium dichromate

(K2Cr2O7) to determine their acute 96 h LC50 values, and therefore relative sensitivities to this reference toxicant. Statistical analysis of the results of the sensitivity comparisons showed that the recommended species (D. rerio), after the breeding and maintenance suitability tests, was found not to differ significantly, in terms of sensitivity, from the indigenous fish tested under the same conditions. Therefore, based on these results, D. rerio was recommended as the most suitable testing fish species for use in routine laboratory toxicity tests within South African laboratories.

xiv

OPSOMMING

Opsomming

Die behoefte aan ‘n meer geskikte vis spesie vir roetine laboratorium toksisiteitstoetse het gelei tot hierdie projek. Daar bestaan ‘n defnitiewe behoefte vir ‘n spesie wat makliker aangehou en geteel kan word onder laboratorium toestande. ‘n Uitgebreide nasionale en internasionale literatuur soektog is onderneem en die visspesie wat potesieel gebruik kan word onder

Suid Afrikaanse laboratorium toestande is geïdentifiseer. Verskeie inheemse en uitheemse visse is sodoende gekies en vir hul toepasbaarheid in roetine toetsings ge-evalueer. Die uitheemse visspesies wat geselekteer is, is

Poecilia reticulata en Danio rerio, en die inheemse vis spesies het Barbus trimaculatus, Barbus argenteus, Tilapia sparrmanii, Oreochromis mossambicus en Pseudocrenilabrus philander philander ingesluit. Al hierdie spesies was gevolglik aangehou en geteel onder laboratorium toestande en die mees geskikte spesie was vir roetine laboratorium toksisiteitstoetse aanbeveel. Op grond van die volgende faktore is die mees geskikte toets spesie geselekteer: koste; benodigde spasie; gemak van roetine teel en vrugbaarheid. Daar is gevind dat die sebravis, D. rerio, die beste aan hierdie vereistes voldoen het.

Daarna was al die spesies aan verskillende konsentrasies van dieselfde verwysingstoksikant, nl. kalium dichromaat (K2Cr2O7) blootgestel ten einde die verskillende akute 96 uur LC50 waardes te bepaal. Statistiese analises het getoon dat die aanbevole spesie (D. rerio) vir aanhouding en teel, nie beduidend verskil (in terme van akute 96 uur LC50 waardes) het van die inheemse visse wat getoets is nie. Die enigste visspesie wat statisties verskil

OPSOMMING het in terme van sensitiwieteit was P. p. philander. Hierdie spesie was minder sensitief as al die ander spesies. Die bogenoemde resultate het dus getoon dat D. rerio die mees geskikste spesie is, en daarvoor aanbeveel word vir roetine laboratorium analise.

xvi

ASSESSMENT OF INTERNATIONAL PRACTICES ON THE USE OF FISH IN TOXICITY TESTING AND ADAPTATIONS FOR SOUTH AFRICAN CONDITIONS

CHAPTER 1

INTRODUCTION

CHAPTER 1

1.1 Background

Toxicity tests using a variety of fish species have long been the mainstay of aquatic toxicity evaluations, with the acute fish test being the initial step in testing environmental chemicals (Nagel, 2002). One of the problems, however, in conducting these toxicity tests is the reliable supply of healthy test organisms (Lloyd, 1992; Landis & Yu, 1999). Personal communications with various toxicity testing laboratories throughout South Africa that do require the routine use fish for acute toxicity tests, have indeed revealed a definite need for a fish species to be selected that is more easily maintained and cultured within the laboratory. Toxicity tests that require the use of fish as a test organism are routinely requested, but due to the unavailability of the required numbers of the correct age group of healthy fish, the tests are often postponed or simply not done (pers. com. du Preez1, 2002; Jooste2, 2002 and

Slabbert3, 2002). The indication that many problems are encountered with culturing of the fish makes the correct choice of fish species vital in enhancing the more widespread application of the fish acute toxicity test.

The idea of the use of indigenous fish species for research purposes is not new, with the favoured indigenous species used for physiological, behavioural, histopathological, bioaccumulation studies as well as other research disciplines being Oreochromis mossambicus (Mozambique tilapia)

(Skelton, 2001; van Dyk, 2001 and Kruger, 2002). Other species that have been successfully bred within the laboratory for research purposes include

1 Dr. H. du Preez, Hydrobiology, Rand Water, Vereeniging. 2 Dr. S. Jooste, Resource Quality Services, Pretoria. 3 L. Slabbert, Environmentek, CSIR, Pretoria.

CHAPTER 1

Tilapia sparrmanii (banded tilapia) (Wepener, 1990; Grobler-van Heerden,

1991; du Preez & van Vuren, 1992 and du Preez et al., 1993) and Clarias gariepinus (sharptooth catfish) (Viljoen, et al., 2003). A selection of these species will therefore be used as comparisons for other selected species as they have been successfully cultured before within the laboratory. Research on indigenous fish species has, however, in the majority of cases, been performed on wild-caught specimens such as Labeo capensis (Orange River mudfish) (Pieterse, 1986). The use of laboratory-cultured, indigenous specimens specifically for routine toxicity tests has been relatively rare in the past, mainly due to the problems experienced with breeding sufficient numbers of test organisms of specific species.

From the literature it is evident that some of the most critical (amongst others) criteria that test organisms are required to fulfil are (adapted from Connell, et al., 1993 and Landis & Yu, 1999):

· Small size; thereby being able to be maintained in relatively small

aquaria. This is important due to the space constraints typical of most

laboratories.

· Widely available test species are preferred.

· No aggressive tendencies brought on by high stocking densities,

meaning that more fish can be maintained in fewer aquaria.

· Be able to reproduce uniformly in relatively high numbers so that

routine toxicity tests can be done on fish of similar ages, reducing the

variability of the results of the tests.

CHAPTER 1

· Be able to reproduce on demand, to have the numbers of fish available

to fit into the proposed schedule of the toxicity tests.

· Be able to reproduce easily with relatively little induction of their

spawning cycles. There is therefore no need for a laboratory

technician to be a specialist in fish culturing in order to breed the fish.

· Ease of maintenance. This is important when considering the time

needed to be devoted to the culturing of the test organisms.

· No specialist water chemistry parameters or habitats are necessary for

successful culturing of the organisms. This reduces the time demands

for maintenance of the culture.

· Show a response to toxicants over a wide range of concentrations.

· The history of the culture of the test organisms should be known, which

can only be certain if the test organisms are cultured within the

laboratory.

The chosen fish species therefore is required to successfully fulfil these criteria. From this list of criteria, it is therefore clear that the choice of fish species is limited to relatively few species of fish, not only nationally, but internationally as well.

1.2 Objectives of the study

The objective of this study is to identify various species of fish commonly used internationally as toxicity test organisms, and to use the data available from the literature to compare these species to fish that are used locally. This will

CHAPTER 1 be done to assess the potential of fish species that are available locally to be used for routine toxicity testing in South African laboratories.

The specific objectives of this dissertation are to:

· Assimilate the available literature on the use of fish presently being

used for toxicity testing, including various national and international

guidelines on toxicity testing.

· Profile the maintenance and breeding of various fish species (both

presently being used in fish toxicity tests and proposed indigenous

species) to verify their individual suitability for toxicity testing under

South African laboratory conditions.

· Use all toxicity data collected, as well as data derived from testing and

comparing different species relative sensitivities to one another, to

recommend the most suitable fish species for toxicity testing.

· Compile a laboratory procedure detailing the design of a system,

husbandry and breeding of the proposed fish. This will potentially allow

laboratories to establish their own self-sustaining fish culturing facility

for routine toxicity testing.

This study follows the format of the maintenance and breeding of selected fish species under laboratory conditions (Chapters 2, 3, 4 and 5). Chapter 6 documents the relative sensitivities of the various fish species to one reference toxicant. This then follows with a general conclusion and recommendations for further studies in Chapter 7.

CHAPTER 1

1.3. Choice of fish species

The various fish species evaluated for suitability as toxicity-testing species included a fish species currently being used for routine testing internationally included the zebrafish (Danio rerio). This fish species was chosen for this study as it is one of the important fish used internationally for routine toxicity testing. The D. rerio acute toxicity test is also a test that is endorsed by the

International Organization for Standardization (ISO, 1996). As this test is also endorsed for use in South African laboratories, it would therefore be appropriate to use this fish for testing under South African laboratory conditions to conform to international standards. This is important when the quality of products exported to other countries (especially Europe) needs to be screened to conform to international standards. Fish species that are currently being used for routine testing nationally include the exotic Poecilia reticulata (guppy) and indigenous O. mossambicus (Mozambique tilapia)

(pers. com. du Preez4, 2002; Jooste 5, 2002 and Slabbert6, 2002). Poecilia reticulata was therefore included as a standard fish species to compare the data to that that were collected from the other fish species that are included in this study. This species was also selected as a comparative indigenous representative that data could be compared to. Another indigenous fish species chosen for this study was Barbus trimaculatus (threespot barb). This species was chosen due to its widespread distribution in both temperate as well as tropical waters. It is therefore a species that is exposed to a wide range of toxic pollutants from different industrial sectors of the country. Its

4 Dr. H. du Preez, Hydrobiology, Rand Water, Vereeniging. 5 Dr. S. Jooste, Resource Quality Services, Pretoria. 6 L. Slabbert, Environmentek, CSIR, Pretoria.

CHAPTER 1 widespread distribution also meant that it was easily obtainable. Barbus argenteus (rosefin barb) was also included for this study as this species is confined to a relatively local distribution whose waters receive pollutants from an important industry. Tilapia sparrmanii (banded tilapia) and

Pseudocrenilabrus philander philander (southern mouthbrooder) were also included in this study as these fish are found within waters that drain the greater Gauteng region, which is a major industrial area of the country. After being maintained and bred in the laboratory, those fish were screened for their relative sensitivities to a reference toxicant – potassium dichromate

(K2Cr2O7). After these aspects had been explored for all of the representative species, conclusions regarding which species would be best suited to routine toxicity bioassays within a laboratory could be reached, and suitable recommendations made.

1.4. Literature overview

It is evident from the literature that the majority of international toxicity testing laboratories using fish in routine freshwater toxicity tests, as well as the majority of studies undertaken by universities and other institutions using fish in toxicity studies, are using fish that are bred within the laboratory or institution. This method of acquiring testing organisms is favoured over wild- caught testing individuals from indigenous waters or receiving waters, or populations of testing organisms that are purchased from commercial hatcheries. This is because the laboratory is able to keep accurate records as to the origin of the testing organisms, age class and health status of the organisms. This ensures the quality control of the results reported from the

CHAPTER 1 tests done by the laboratories. The trend towards making use of fish species that show a high degree of amenability to laboratory conditions, with relatively high fecundities, is gaining popularity within the international toxicity testing fraternity over the fish that are representatives of the receiving waters of the toxicants and is emphasised throughout the literature. The preferred fish species that are used in toxicity testing because of the practicability of the particular species is inevitably favoured by the countries that fall under the natural distributions of the species. If, however, the particular country does not have such a species of fish found within its indigenous waters, then the use of exotic species of fish is advocated. The data collected by the use of exotic species has gained popularity as reliable data representative of the receiving waters of the country; therefore the use of exotic species – for practicable and economic reasons – is widely accepted throughout the international toxicity testing community. This is especially true for species such as Oryzias latipes (Japanese medaka), Pimephales promelas (fathead minnow), D. rerio (zebrafish) and P. reticulata (guppy) – all relatively small species of fish that are easily maintained and bred under laboratory conditions, yet show relatively good sensitivity to a wide range of toxicants.

In South Africa, the use of indigenous species for toxicity testing has shown promise, however, laboratory-reared cultures are still relatively rare and their use limited. Laboratories still favour the use of P. reticulata as the standard testing organism, but species such as O. mossambicus are used, with limited scope, however, due to problems with space economy and fecundities of this species.

CHAPTER 1

1.5. References

Connell, D., Lam, P., Richardson, B. and Wu, R. (1993). Introduction to

ecotoxicology. Blackwell Science Ltd., Oxford. 170 p.

Du Preez, H.H. and van Vuren, J.H.J. (1992). Bioconcentration of atrazine in

the banded tilapia, Tilapia sparrmanii. Comparative Biochemistry and

Physiology 101C (3): 651-655.

Du Preez, H.H., van Rensburg, E. and van Vuren, J.H.J. (1993). Preliminary

laboratory investigation of the bioconcentration of zinc and iron in

selected tissues of the banded tilapia, Tilapia sparrmanii (Cichlidae).

Bulletin of Environmental Contamination and Toxicology 50: 674-

681.

Grobler-van Heerden, E., van Vuren, J.H.J. and du Preez, H.H. (1991).

Bioconcentration of atrazine, zinc and iron in the blood of Tilapia

sparrmanii (Cichlidae). Comparative Biochemistry and Physiology

100C (3): 629-633.

Kruger, T. (2002). Effects of zinc, copper and cadmium on Oreochromis

mossambicus free-embryos and randomly selected mosquito

larvae as biological indicators during acute toxicity testing. M.Sc.

Thesis, Rand Afrikaans University.

CHAPTER 1

International Organization for Standardization. (1996). Water Quality –

Determination of the acute lethal toxicity of substances to a

freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei,

Cyprinidae)] – Part 1: Static method. ISO Report 7346-1 Second

edition. International Organization for Standardization, Switzerland.

Landis, W.G. and Yu, M. (1999). Introduction to environmental toxicology

– Impacts of chemicals upon ecological systems. CRC Press,

Florida, USA. 328 p.

Lloyd, R. (1992). Pollution and freshwater fish. Blackwell Scientific

Publications Ltd., Oxford. 176 p.

Nagel, R. (2002). DarT: The embryo test with the zebrafish Danio rerio – a

general model in ecotoxicology and toxicology. Altex 19, Supplement

1/02: 39-48.

Pieterse, G.M. (1986). Aspekte van die histomorfologie en histochemie

van die testis van Labeo capensis (Cyprinidae). M.Sc. Dissertation,

Rand Afrikaans University.

Skelton, P. (2001). A complete guide to the freshwater fishes of southern

Africa. Struik, Cape Town. 395 p.

Van Dyk, J.C. (2001). Histological changes in the liver of Oreochromis

mossambicus (Cichlidae) after exposure to cadmium and zinc.

M.Sc. Dissertation, Rand Afrikaans University.

CHAPTER 1

Viljoen, A., Steyn, G.J., Van Vuren, J.H.J., Wade, P. (2003). Zinc effects on

the embryos and larvae of the sharptooth catfish, Clarias gariepinus

(Burchell, 1822), Bulletin of Environmental Contamination and

Toxicology, 70, pp 1022–1027.

Wepener, V. (1990). Die effek van swaarmetale by variërende pH op die

bloedfisiologie en metaboliese ensieme van Tilapia sparrmanii

(Cichlidae). M.Sc. Dissertation, Rand Afrikaans University.

ASSESSMENT OF INTERNATIONAL PRACTICES ON THE USE OF FISH IN TOXICITY TESTING AND ADAPTATIONS FOR SOUTH AFRICAN CONDITIONS

CHAPTER 2

MAINTENANCE AND BREEDING OF B. TRIMACULATUS AND B. ARGENTEUS (CYPRINIDAE) TO DETERMINE THEIR SUITABILITY FOR USE IN ROUTINE LABORATORY TOXICITY TESTS

CHAPTER 2

2.1. Background

In this part of the dissertation, two Cyprinidae species (B. trimaculatus and B. argenteus) were selected for testing their suitability in terms of amenability to maintenance, as well as ease of breeding and fecundities under routine laboratory conditions. A laboratory procedure recommending guidelines to the individual maintenance and breeding requirements of this fish species was compiled. This was based on available literature and experimentally determining suitable maintenance and breeding systems, as well as investigating their particular spawning cues and environmental requirements.

Based on the breeding results and the degree to which they fulfil the criteria of a suitable toxicity testing species (see 1.1. - Background), conclusions are drawn and recommendations given regarding whether or not they will be suitable candidates for use in routine toxicity tests.

2.2. Barbus trimaculatus

2.2.1. Introduction

2.2.1.1. Natural history

The classification of B. trimaculatus is as follows (Axelrod & Schultz, 1990;

Fishbase, 2004):

Phylum: Chordata Subphylum: Craniata Superclass: Gnathostomata Class: Osteichthyes Subclass: Actinopterygii Superorder: Teleostei Order: Cypriniformes Suborder: Cyprinoidea

CHAPTER 2

Family: Cyprinidae Genus: Barbus (Linnaeus, 1758) Species: Barbus trimaculatus (Peters, 1852)

From the literature, it is, however, evident that there are several synonyms for

B. trimaculatus (Table 2.1).

Table 2.1: Synonyms, their authors, status and current validity of B. trimaculatus (adapted from Fishbase, 2004).

Synonym Author Status Valid

Barbus trimaculatus Peters, 1852 Original comb. Yes Barbus kurumani Castelnau, 1861 Jnr synonym No Barbus breijeri Weber, 1897 Jnr synonym No Barbus katangae Boulenger, 1900 Jnr synonym No Barbus decipiens Boulenger, 1907 Jnr synonym No

Barbus trimaculatus is a benthopelargic, tropical freshwater species found between 9 °S and 30 °S of the equator, typically in shallow water near river outlets or close to swampy areas. It is a hardy species, commonly occurring in a wide variety of habitats, especially where there is vegetation (pers. obs.), and feeds on insects and other small aquatic organisms (Skelton, 2001). It breeds in summer, with shoals of ripe adults moving upstream when rivers are in spate after rain. Large females are capable of producing up to 8 000 eggs in a single spawning (Skelton, 2001).

The dorsal fin is made up of three true spines with eight branched rays, whilst the anal fin consists of three unbranched segmented rays, with five branched rays. There are 31-33 scales in the lateral line series, with 14 scales around the caudal peduncle. It has a robust body with two long barbels extending from the mouth. It is silver in colour, tinged with gold when in breeding

CHAPTER 2 condition, usually with three clear black spots on the body and base of the caudal peduncle (Figure 2.1). It attains a maximum length of 150 mm SL

(Skelton, 2001).

Figure 2.1: Barbus trimaculatus.

Barbus trimaculatus is found in the associated rivers of the Komati and Vaal rivers. Elsewhere, it is found at the east coast from Ruvuma, Tanzania to

Umvoti (Kwazulu/Natal), as well as in the Orange, Cunene and Congo

(Zambian) river systems (Skelton, 2001).

2.2.1.2. Background on captive breeding and use of B. trimaculatus

Barbus trimaculatus has the potential to become a popular aquarium fish due to its small size, non-aggressive, social behaviour as well as its lively swimming pattern (pers. obs.; Skelton, 2001). It is commonly used as a baitfish (especially for tigerfish - Hydrocynus vittatus) as well as a fodder fish for bigger predatory angling species of fish, such as largemouth bass

(Micropterus salmoides) (Skelton, 2001). As far as could be ascertained, B. trimaculatus has been bred under laboratory conditions (pers. com. Vlok,

CHAPTER 2

20027). This, however, was done as a hobby, rather than for routine laboratory work.

2.2.2. Environmental requirements and procedures for maintenance

of B. trimaculatus

2.2.2.1. Water temperature

The mean water temperature of the dam where the original group of B. trimaculatus was collected was 23 ± 1 °C. The fish were collected at the onset of summer when they were thought to be actively breeding. The system water where these fish were housed in the laboratory was therefore set at this temperature. This temperature was, indeed, conducive to the temperature range that supported breeding activity of the fish, as they started to spawn.

2.2.2.2. Water chemistry

2.2.2.2.1. pH

The pH of the system water ranged between 7.37 and 8.34. The group of B. trimaculatus was maintained and bred within this pH range, therefore no further experimentation was undertaken to determine the limits of the pH range for this species of fish.

7 Dr. W. Vlok, University of the North, Polokwane.

CHAPTER 2

2.2.2.2.2. Total water hardness

The total water hardness for the system water that B. trimaculatus were housed and bred in ranged between 52 and 87 mg/l (CaCO3), which is within the range of ‘moderately soft’ water (USEPA, 1993). This total hardness range of the system water was conducive to suitable conditions for breeding activity, maintenance as well as successful rearing of the larval and juvenile fish, therefore no further experimentation was undertaken to determine the limits to the suitable water hardness range.

2.2.2.3. Photoperiod

This species of fish was maintained and bred using a 14/10 h light/dark cycle.

As these fish are known to spawn in the summer months (Skelton, 2001), this light cycle was chosen as a representation of the light cycle typical of the summer months. No other photoperiod was investigated as the fish bred whilst exposed to this light cycle.

2.2.2.4. Aquaria design

2.2.2.4.1. Aquaria

The collected group of B. trimaculatus were housed within a 1 000 l tanks for acclimation purposes to the laboratory conditions. The dimensions of these tanks were 1 250 mm (l) x 800 mm (b) x 1 000 mm (h). The tanks were blackened on three sides (fibreglass) with only the front being left clear

(glass). A relatively large group of fish was housed together to reduce the stress that the fish inevitably experienced from being wild-caught individuals

CHAPTER 2

(pers. obs.). This will reduce the stress experienced by the fish as they are naturally a shoaling species (Skelton, 2001). Maintaining this species in a relatively large group, as opposed to small groups or singly therefore is a closer simulation of their natural conditions, therefore giving the fish a sense of ‘security’ (pers. obs.). Having the three sides of the housing tank blackened also reduced stress to the fish, making them less likely to be disturbed by movements outside of the tank (pers. obs.). The tanks were also covered with nets to stop the fish from jumping out of them.

The breeding tank was a 250 l all-glass aquarium with dimensions: 1 000 mm

(l) x 500 mm (b) x 500 mm (h) (Figure 2.2). For a detailed description, refer to section 2.2.4. - Breeding system design.

Return pipe from the submersible pump, returning filtered water to the tank.

Artificial ‘plants’ within the system.

Position of air stone.

Relatively large-stoned gravel.

Figure 2.2: Breeding tank for B. trimaculatus showing the basic features within the tank.

2.2.2.4.2. Stocking densities of fish in aquaria

The 1 000 l tanks housed 50 adult B. trimaculatus. This translates to one fish in 20 l of water. Two large airstones were also placed at either end of the

CHAPTER 2 tank with the purpose to increase aeration/circulation of the system water.

This stocking density was worked out from the recommendation guideline given by Sandford (2003). The author recommends that each 2.5 cm standard length of fish housed in the system be allowed 75 cm2 of water surface area. The average length of the group of fish was 7.5 cm. This meant that each of the fish required at least 225 cm2 of surface area. The housing tank had a surface area of 10 000 cm2. This meant that the system could safely house 44 individuals, with a mean length of 75 cm each. The author also indicates that the amount of dissolved oxygen available for the fish to use within the water is a more critical factor than water volume when the stocking capacity of an aquarium is calculated, therefore it was deemed necessary to increase the aeration/circulation of the water (thereby increasing the contact time of the water with air) so that the tank could house up to 50 individuals.

The breeding tank, with the dimensions of 1 000 mm (l) x 500 (b) x 500 mm

(h), had a surface area of 5 000 cm2. By the same calculation as above, this system could safely house 22 fish. For breeding purposes, however, the tank was under-stocked to reduce the levels of ammonia and nitrates within the system water. Only eight males and four females were placed within this tank at any one time. Under-stocking the tank with fish did prove successful as a higher degree of fatalities was recorded within this tank when stocking density was increased.

CHAPTER 2

2.2.2.4.3. Substrate

No bottom gravel substrate was placed in the 1 000 l housing tank. This facilitated the sanitation of the system as solid wastes were easily siphoned from the bottom of the tank. This did not have any apparent observable detrimental effects on the fish. The breeding tank had a substrate of relatively large-stoned gravel (± 10 mm) which allowed the embryos to sink in between the gaps surrounding the stones. This allowed them to fall out of the reach of the adults that would otherwise have eaten them (also see section 2.2.4.1. -

Breeding system design).

2.2.2.4.4. Filtration

A flow-through filter was built within one side of the aquarium, which was designed to direct the water through different compartments containing different filter media, before a submersible pump returned it to the aquarium.

The filtration series consisted of a mechanical stage made up of densely packed shade cloth to remove solids from the water. The second stage of the filter was the biological stage that consisted of a commercially available biological media - Kaldness®. This stage of the filter was fitted with an air stone to maximise oxygenation throughout the media, as well as to constantly move the Kaldness® substrate to maintain the health of the bacterial colonies growing on and within it. The submersible pump, returning the water to the aquarium, was placed in a position that minimised the risk of larval and juvenile fish being sucked up by it. The aquarium was then also fitted with a hang-on external power filter (Aquaclear® 300) at the opposite end of the built

CHAPTER 2 in filter (Figure 2.3). For a comprehensive explanation of the processes and principles of filtration, refer to Chapter 5, section 5.2.4.4. - Filtration.

External hang-on power filter – Aquaclear® (model 300).

Mechanical stage of filter.

Biological stage of filter showing the aerated Kaldness® biological media.

Filter chamber housing submersible pump.

Figure 2.3: Breeding tank for B. trimaculatus showing the basic features of the filtration system.

2.2.3. Barbus trimaculatus brood stock management

2.2.3.1. Age of brood stock

Individual fish that displayed sexual dimorphism were used for breeding. The age that this differentiation occurred was not established as a group of individual fish with mixed ages was initially collected in the field. Only the fish that showed obvious sexual dimorphism were used for breeding purposes

(see section 2.2.4.2. - Gender determination of adult fish).

CHAPTER 2

2.2.3.2. Conditioning of brood stock

2.2.3.2.1. Temperature

The water temperature was kept relatively constant at 23 ± 1 °C. This temperature was representative of the waters where the fish were originally collected from during early summer (when they were thought to breed

(Skelton, 2001)). Keeping the brood stock at this temperature was conducive to inducing spawning activity amongst the fish, as well as the successful raising of larval and juvenile fish.

2.2.3.2.2. Food

Adult fish were conditioned for breeding purposes by adhering to a regular feeding routine of twice per day with a good quality commercial flake food

(TetraMin®) supplemented once daily with frozen bloodworms, Daphnia and brine shrimp (only enough that the fish will actively consume within five minutes).

2.2.3.2.3. Photoperiod

The brood stock fish were maintained at a 14/10 h light/dark cycle. This photoperiod was chosen as it represented a typical summer photoperiod.

This photoperiod did indeed induce spawning behaviour amongst the fish.

CHAPTER 2

2.2.4. Breeding B. trimaculatus

2.2.4.1. Breeding system design

Relatively large stoned gravel was used as a substrate within the aquarium and artificial plants were made by cutting 0.5 m lengths of 10 mm nylon rope.

The one end was melted and tied into a knot to prevent fraying. The rest of the rope was then unravelled and ‘teased’ to spread it out freely (Figure 2.4).

The entire length of the rope is frayed to resemble a plant.

Knotted ends of the rope are threaded through a ringed suction cup.

Knotted ends of the rope are melted and knotted to prevent further fraying.

Figure 2.4: Artificial plants made to create shelter and security for fish.

Several strands of wool were then also tied in with the knot on the one end.

The knotted end was then threaded through a ringed suction cup and stuck onto the bottom glass pane of the aquarium. Cutting different lengths of the rope, as well as tying a few pieces together varied this theme, and by placing them at different points within the aquarium also created different degrees of cover for the fish. Three sides as well as the top and half of the front of the

CHAPTER 2 aquarium were covered by black plastic to minimise the disturbance of the fish.

2.2.4.2. Gender determination of adult fish

Observing the fish from the side through a glass aquarium differentiated sexually mature male and female B. trimaculatus from one another. Male fish are more slender and are generally smaller than the more round-bodied and relatively larger female fish. The female fish are more fuller-bodied due to the presence of ripe ovaries. If the fish’s state of general health and their nutritional status were both conducive to the breeding condition of the fish, this method of gender differentiation was found to be reliable. If the general health and nutritional status of the fish is not optimal, then the females do not produce the high quantities of eggs that give it the fuller body proportion (pers. obs.). Only adult fish that were successfully differentiated from one another in terms of gender were used for breeding purposes.

2.2.4.3. Gender ratios

Eight males and four females, showing sexual dimorphism, were transferred from the larger housing tank into the breeding aquarium, until there was a final count of 24 ripe adult fish in this breeding aquarium.

2.2.4.4. Procedure for breeding B. trimaculatus

The spawning group was allowed to acclimate to the smaller size of the breeding tank for two to three days. After the acclimation period, half of the water was removed from the spawning tank, taking the opportunity to vacuum

CHAPTER 2 the gravel to remove debris and dirt from it. After three days of the reduced water volume, the water was replaced with distilled water by slowly siphoning through 5 mm air tubing. The distilled water was slightly colder (± 2 °C) than the tank water. The idea of this was to simulate rainfall, and a river system in spate, thus giving the fish the environmental cues to induce spawning activity.

During this period, the spawning group was fed copious amounts of live food

(frozen bloodworms, Daphnia and brine shrimp) and high quality flake food

(TetraMin®). The shade cloth in the first stage of the filter was greatly reduced to allow free passage of the larval fish through it. The airflow was also stopped within the biological stage of the filter for the same reason. Later on, however, the fish were cued to spawn by simply doing a water change with borehole water that was slightly colder than the aquarium water.

2.2.5. Larvae and juvenile care

From three to four days after the spawning induction procedure, larval fish were removed with a fine-meshed net from the filter as they collected there, with the majority being in the filter chamber that housed the pump. A proportion of the batch of larval fish was used for toxicity tests (see Chapter 6) with the remainder being housed in a rearing tank that formed a flow-through system with the breeding tank. The rearing tank (that had a volume of 10 l) was placed on top of the breeding tank. A water line (5 mm diameter) was diverted from the submersible pump within the breeding tank to supply the rearing tank with water circulation at a rate of 10 l per hour. A hole was cut into the side of the rearing tank which governed the water level of this rearing tank. A small-gauge (500 µm mesh size) mesh was placed over this hole to

CHAPTER 2 stop the larval fish from being flushed out of the rearing tank. The water was then allowed to overflow through this hole back into the breeding tank. This process ensured that the water within the rearing tank was of the same quality as the breeding tank that the larval fish originated from. This meant that the larval fish were never subject to water chemistry changes and the associated fatalities expected from changing the water chemistry of such young fish

(pers. obs.).

2.2.5.1. Care of free-swimming larvae

The larval fish were left in the rearing tank for at least two weeks, being fed microworms (see Appendix A – Procedure for culturing microworms

(Anguillula silusiae).). There were also up to five aquatic snails (Physidae) put into the container together with the larval fish to consume the leftover culture media from the microworms that would inevitably remain in the water.

After two weeks, the (now juvenile) fish were fed on finely crushed flake food

(TetraMin Baby®) and then moved to a 100 l growing tank.

2.2.5.2. Care of juveniles

The juvenile fish were moved to a larger (100 l) aquarium fitted with an air- driven sponge filter. They were fed on TetraMin Baby® until they had grown sufficiently to have their diet supplemented by Daphnia (approximately four weeks). As the fish continued to grow, putting a proportion of the juvenile fish into other tanks thinned their numbers out. The final stocking density of the fish was approximately 15 fish per 100 l of system water when they were approximately eight weeks old.

CHAPTER 2

2.3. Barbus argenteus

2.3.1. Introduction

The classification of B. argenteus is as follows (Axelrod & Schultz, 1990 and

Fishbase, 2004):

Phylum: Chordata Subphylum: Craniata Superclass: Gnathostomata Class: Osteichthyes Subclass: Actinopterygii Superorder: Teleostei Order: Cypriniformes Suborder: Cyprinoidea Family: Cyprinidae Genus: Barbus (Linnaeus, 1758) Species: Barbus argenteus (Günther, 1868).

From the literature, it is however evident that there are several known synonyms for B. argenteus (Table 2.3). The current name of this species of fish is set to change in the near future to B. crocodiliensis (pers. com. Bills 8,

2004).

Table 2.3: Synonyms, their authors, status and current validity of B. argenteus (adapted from Fishbase, 2004).

Synonym Author Status Valid

Barbus argenteus Günther, 1868 Original comb. Yes Puntius argenteus Günther, 1868 New comb. No Barbus crocodiliensis* Fowler, 1934 Jnr synonym No

(*) Barbus crocodiliensis is soon to become the official name that B. argenteus will be known by in the future.

8 R. Bills, SAIAB, Grahamstown.

CHAPTER 2

2.3.1.1 Natural history

The dorsal fin of B. argenteus consists of three spines, followed by seven to eight soft rays, while the anal fin has three soft spines followed by five soft rays. There are 27-32 scales in the lateral line series, with 14 around the caudal peduncle. The mouth has two pairs of well-developed barbels. The body is generally a silvery colour, with a light olive dorsal surface. A vague stripe along the caudal peduncle is often present. The fins turn an orange-red in mature specimens (Figure 2.5) (Skelton, 2001). The maximum size recorded for this species of fish is 197 mm total length (TL) (Fishbase, 2004).

Figure 2.5: Barbus argenteus (Photograph by R. Bills9).

Barbus argenteus is found in the tropical (12 °S to 27 °S) escarpment streams of the Incomati and Phongola systems, as well as in the Cunene and Cuanza

(where the type specimen is described from) rivers in Angola, where it inhabits pools and riffles in clear rocky streams where it feeds on aquatic and flying insects. It has also been known to take trout flies (Skelton, 2001).

9 R. Bills, SAIAB, Grahamstown.

CHAPTER 2

2.3.1.2. Background on captive breeding and use of B. argenteus

This species of fish has the potential to be used as an ornamental fish for the larger aquarium (Skelton, 2001). Subsistence anglers have also been seen to be catching B. argenteus (pers. obs.). As far as could be ascertained, this species of fish has not been successfully bred under laboratory conditions.

2.3.2. Environmental requirements and procedures for maintenance

of B. argenteus

From experimentally determining the environmental requirements and procedures for maintenance of this species of fish, it was found that the majority of the parameters and protocols used for B. trimaculatus were suitable for the successful maintenance and breeding of B. argenteus as well.

Therefore, for the procedures and protocols for maintenance and breeding of fish, refer to section 2.2.2. - Environmental requirements and procedures for maintenance and breeding of B. trimaculatus. Methodologies differing from that of B. trimaculatus to specifically accommodate B. argenteus gender determination of adult fish are given in section 2.3.2.1. (Gender determination of adult fish).

2.3.2.1. Gender determination of adult fish

Sexes were separated by careful observation of the fish as well as a ‘milking’ procedure of the individual fish, which proved to be very successful with this particular species of fish. Ripe males in breeding condition released milt with the gentle application of pressure in a milking fashion to the vent. Ripe females were separated by observation of body proportions relative to the

CHAPTER 2 males. Females were on average bigger than the males, as well as being more fuller-bodied due to the ripening of ova within the abdomen. Many ripe females released eggs when gentle pressure was applied to the vents in a milking fashion.

2.4. Results and discussion

2.4.1. Spawning tank design and breeding of fish

The spawning tank used for both of the Barbus species worked relatively well, with the fecundities of both of the species meeting the relatively high expectations of a single adult female capable of producing up to 8,000 eggs at one time (Skelton, 2001). The initial protocol, to adjust water chemistry, volume and temperature (to simulate a rainfall event), was feasible due to the fact that the literature referred these fish species to spawn after rains when rivers are typically in spate. This meant that the spawning tank’s volume was dropped to about two thirds of its total volume and filling it up with distilled water with ± 2 °C colder temperature difference. The idea was to give the fish the impression that it had rained – the water volume increased considerably, the conductivity of the water also dropped quite considerably, together with the temperature. Initially following this protocol worked relatively well, but was time consuming to necessitate the acclimation of the fish to the new conditions. Running the filter system with the decreased volume of water was also not possible, that is why an additional external hang-on power filter was used to run during these periods. The potential breeding group of fish were then placed into the spawning tank and allowed a few days to acclimate to

CHAPTER 2 their new surrounding. Only once the distilled water was added, and the internal filter initiated, did the fish spawn. Spawning was therefore either induced by the change in temperature, or the water volume, or the softening of the water by the addition of the distilled water, or the increase in the current within the spawning tank brought on by the pump that drove the internal filter.

It could also quite possibly have been due to the increase in water quality by

- the more efficient NH3 and NO3 removal from the water when the internal filter was initiated. It was only when, after a routine water change that decreased the water’s temperature slightly, and the fish spawned unexpectedly, that it was realised that it was unnecessary to alter the volume of the water, together with the conductivity and hardness, with the addition of the distilled water. Thereafter, the fish would spawn after every routine water change, but they did, however, require a substantial resting period (two to three weeks) in between consecutive spawning episodes. A typical spawning from both barb species delivered on average between 800 and 1 000 larvae.

This resting period, even though not actually tested for feasibility based on numbers of offspring produced, was typically at least two weeks. Trying to induce spawning before two weeks after the previous spawning episode did not produce the high number of offspring as when the resting period was at least two weeks.

The way that the spawning tank was designed allowed for the collection of the free-swimming larval fish as they collected in the filter system. It did not, however, allow for the collection of embryos. This meant that, even though

CHAPTER 2 the larval fish were counted, the fecundity of the spawning fish themselves could not be accurately determined.

2.5. Conclusions and recommendation

A relatively high degree of fish maintenance knowledge and experience is necessary for the successful maintenance of these species of fish as they are highly susceptible to stresses inevitably induced by the routines of a typical laboratory. These species are therefore not entirely suited to an average routine laboratory looking to use them only for routine toxicity tests. The breeding tank used for breeding both of the barb species did also take up a lot of space within the culturing room, making the breeding of the barb species relatively inefficient in terms of space efficiency. This, in turn, translated to the culturing operation being relatively expensive. Even though the breeding of these barb species is a relatively time consuming and costly undertaking, the practise of breeding these and other similar species should not be ruled out.

The breeding of these species, however, seems to be more important for conservation purposes rather than for routine, commercial toxicity testing.

This is especially true when the time and economics involved for breeding these fish species are taken under consideration.

The most important water quality parameters of the system water in which the fish were successfully maintained and bred are given in Table 2.2. Aged

(dechlorinated) tap water was used throughout the experimentation within the maintenance tanks as well as the breeding tanks. The fish were successfully maintained and bred within water of this quality, with the range of values

CHAPTER 2 being based on the results of chemical analysis of the water over a six-month period. The water quality parameters given in Table 2.2 are therefore proposed as guideline values, with the actual extremes of the water quality parameters suitable for these species of fish not having being tested.

Table 2.2: Summary of recommended ranges of physical and chemical water quality parameters of the system water conducive to successful maintenance and breeding of B. trimaculatus and B. argenteus.

Water quality parameter Recommended range

Water temperature 22-26 °C Total water hardness 3-30 °dH (± 50-500 mg/l CaCO3) pH 7.0-8.5 Photoperiod 14/10 h light/dark

CHAPTER 2

2.7 References

Axelrod, H.R and Schultz, L.P. (1990). Handbook of tropical aquarium

fishes. T.F.H. Publications, New Jersey. 728 p.

Fishbase. (2004). Froese, R. and Pauly, D. (Editors). World Wide Web

electronic publication. www.fishbase.org, version (06/2004).

Sandford, G. (2003). Aquarium owner’s manual. Dorling Kindersley

Limited, London. 256 p.

Skelton, P. (2001). A complete guide to the freshwater fishes of southern

Africa. Struik, Cape Town. 395 p.

USEPA. (1993). Methods for measuring the acute toxicity of effluents

and receiving waters to freshwater and marine organisms. Weber,

C.I. (Ed). Environmental Monitoring Systems Laboratory – Cincinnati.

Office of Research and Development, U.S. Environmental Protection

Agency, Cincinnati, Ohio 45268. EPA-600/4-90/027F.

ASSESSMENT OF INTERNATIONAL PRACTICES ON THE USE OF FISH IN TOXICITY TESTING AND ADAPTATIONS FOR SOUTH AFRICAN CONDITIONS

CHAPTER 3

MAINTENANCE AND BREEDING OF O. MOSSAMBICUS, T. SPARRMANII AND P. P. PHILANDER (CICHLIDAE) TO DETERMINE THEIR SUITABILITY FOR USE IN ROUTINE LABORATORY TOXICITY TESTS

CHAPTER 3

3.1. Background

This part of the dissertation deals with experimentally determining the most practical breeding methodologies for O. mossambicus, T. sparrmanii and P. p. philander with the purpose of determining their suitability for routine toxicity testing within the laboratory. This will be done by determining the amenability shown by each of the individual species of fish to laboratory conditions. Their ease and reliability of their breeding habits, together with their fecundities will be determined, and a proposed laboratory guideline document compiled detailing their environmental requirements and breeding procedures. The conclusion whether or not these species of fish are recommended for use in routine toxicity testing will be based on their amenability to laboratory conditions, ease of breeding and the fecundity of each species concerned.

3.2. Oreochromis mossambicus

3.2.1. Introduction

3.2.1.1. Natural history

The classification of O. mossambicus is as follows (Axelrod & Schultz, 1990 and Fishbase, 2004):

Phylum: Chordata Subphylum: Craniata Superclass: Gnathostomata Class: Osteichthyes Subclass: Actinopterygii Superorder: Teleostei Order: Perciformes Suborder: Percoidea

CHAPTER 3

Family: Cichlidae Genus: Oreochromis (Linnaeus, 1758) Species: Oreochromis mossambicus (Peters, 1852)

From the literature, it is however evident that there are several synonyms for

O. mossambicus (Table 3.1).

Table 3.1: Synonyms, their authors, status and current validity of O. mossambicus (adapted from Fishbase, 2004).

Synonym Author Status Valid

Chromis niloticus (non Linnaeus, 1758) Miss identification No Oreochromis mossambica Peters, 1852 Misspelling No Chromis mossambicus Peters, 1852 Original comb. No Tilapia mossambica Peters, 1852 New comb. No Tilapia mossambica mossambica Peters, 1852 New comb. No Sarotherodon mossambicus Peters, 1852 New comb. No Tilapia mossambicus Peters, 1852 New comb. No Oreochromis mossambicus Peters, 1852 New comb. Yes Chromis niloticus mossambicus Peters, 1852 New comb. No Oreochromis mossambica Peters, 1852 Misspelling No Chromis mossambicus Peters, 1852 Misspelling No Tilapia dumerilii Steyndachner, 1864 Jnr synonym No Chromis dumerilii Steyndachner, 1864 Jnr synonym No Tilapia vorax Pfeffer, 1893 Jnr synonym No Chromis vorax Pfeffer, 1893 Jnr synonym No Chromis natalensis Weber, 1897 Jnr synonym No Tilapia natalensis Weber, 1897 Jnr synonym No Tilapia kafuensis (non Boulenger, 1912) Miss identification No Tilapia arnoldi Gilchrist & Thomson, 1917 Jnr synonym No

The dorsal fin consists of 15 to 17 spines, with a further 10 to 13 branched rays. The anal fin has three spines, together with between nine and twelve branched rays. There are between 30 and 32 scales in the lateral line series.

This species of fish has a moderately deep body and caudal truncate. Its head profile is straight in juveniles as well as in the females. It is, however, concave in mature males. The jaws have three to five rows of slender teeth, being bicuspid in the outer row. Jaws of older males become enlarged and teeth project forward. There are between 16 and 20 gill rakers on lower limb

CHAPTER 3 of the first arch. Juveniles are silvery, with six to seven vertical bars, with three spots along their flanks. Adults are silvery olive to deep blue-grey, and the dorsal and caudal fins have red margins. Breeding males develop a deep greyish black colour, with the lower head and throat being white. It attains about 400 mm SL (Skelton, 2001) (Figure 3.1).

Figure 3.1: Oreochromis mossambicus.

It is found in the east coastal rivers from the lower Zambezi system south to the Bushman’s system of the Eastern Cape. South of the Phongola system, it is naturally confined to closed estuaries and coastal reaches of rivers. It is widely dispersed beyond this range to inland regions and to the southwest and west coastal rivers including the lower Orange River, and rivers of

Namibia. It has, however, been introduced to tropical and warm temperate localities throughout the world. Oreochromis mossambicus occurs in all but fast-flowing rivers; thriving in standing waters. This species of fish is tolerant of fresh, brackish or marine waters and even higher salinity concentrations

CHAPTER 3 and it is able to survive lower temperatures (below about 15 °C) in brackish or marine waters. It, however, does prefer warm water temperatures (above 22

°C) and is tolerant of temperatures of up to about 42 °C. It feeds on algae, especially diatoms, and detritus, but large individuals may take insects and other invertebrates. Oreochromis mossambicus breeds in summer, with the females raising multiple broods every three to four weeks during a season.

The males construct a saucer-shaped nest on sandy bottoms and female mouth-broods the eggs, larvae and small juveniles. Juveniles shoal in shallow water and grows rapidly and may mature and breed within a year.

Growth of young fish may be prone to stunting under adverse or crowded conditions (Skelton 2001).

3.2.1.2. Background on captive breeding and use of O. mossambicus

Oreochromis mossambicus is widely used in aquaculture and commercial subsistence fisheries and is a valued angling species, with the South African angling record being 3.265 kg, the Zimbabwean record is 2.181 kg, and the

Malawian record being 0.64 kg. It is a popular species of fish used by the scientific fraternity, used extensively in biological, physiological toxicological and behavioural research (Skelton, 2001; Kruger, 2002) and it has been extensively studied in terms of histological damage from toxicants (van Dyk,

2002). Oreochromis mossambicus has been shown to breed successfully under laboratory conditions; however it is a species of fish that is not entirely suited to maintenance under the typical problems of space constraints of the average South African laboratory (pers. obs.).

CHAPTER 3

3.2.2. Environmental requirements and procedures for maintenance

of O. mossambicus

3.2.2.1. Water temperature

Oreochromis mossambicus is tolerant of temperatures between 15 °C and 42

°C (Skelton, 2001). Breeding as well as successful rearing of larval and juvenile fish has been undertaken at temperatures between 24 °C and 27 °C

(pers. obs.). This is therefore the temperature that this species of fish was maintained and bred in with successful results.

3.2.2.2. Water chemistry

3.2.2.2.1. pH

The pH range of the system water was between 7.0 and 8.5. This pH range was conducive to successful maintenance, breeding of the fish as well as raising of larval and juvenile fish.

3.2.2.2.2. Salinity

The addition of 0.5 g/l NaCl to the system water was found to increase the fish’s vitality and vigour. The fish were also found to breed more readily if the

NaCl was added to the water as opposed to when it was not added. The

NaCl added was in the form of non-iodated coarse rock salt.

CHAPTER 3

3.2.2.2.3. Total water hardness

The system water had a total hardness range of between 3 °dH and 30 °dH.

The fish were maintained and bred successfully, and the larval and juvenile fish were raised successfully within this total water hardness range.

3.2.2.3. Photoperiod

The photoperiod was maintained at a constant 14/10 h light/dark cycle. This particular light cycle was found to be conducive to successful breeding of the fish.

3.2.2.4. Aquaria design

3.2.2.4.1. Aquaria

A group of sexually mature adult fish each measuring between 100 mm and

150 mm were maintained together in a 1,000 l glass-fronted aquarium that allowed the viewing of the fish from the front. The remaining three sides of the aquarium were made from fibreglass that was pigmented brown. This tank was used to minimise the disturbance to the fish from passing aquarium personnel (pers. obs.). A few pieces (±200 mm long) of 50 mm PVC piping were placed inside the tank that were utilised as hiding places for the fish

(pers. obs.).

3.2.2.4.2. Stocking densities of fish in aquaria

A group of 20 sexually mature fish each measuring between 100 mm and 150 mm were maintained together within the 1,000 l tank. This relatively low

CHAPTER 3 stocking density of the fish (see Chapter 2, section 2.2.2.4.2. – Stocking densities of fish in aquaria) was observed to minimise aggression that the fish showed towards one another. This aggression that was induced by a higher stocking density of the fish resulted in inevitable fatalities of subordinate fish

(pers. obs.).

3.2.2.4.3. Substrate

No substrate in terms of gravel was added to the tank. The only addition to the bottom of the tank were a few pieces of 50 mm PVC piping which allowed fish to seek shelter and to act as spawning sites for the breeding fish (pers. obs.).

3.2.2.4.4. Filtration

The tank formed part of a flow-through filtering system, which was filtered by an initial settling tank to allow the settling of solid wastes and then by a gravel bed, which was the biological component of the filter. Water was then returned to the tank by a submersible pump within the gravel bed at a flow rate of approximately 600 l/h. Due to the relatively low stocking density of the fish, and the relatively hardy nature of the fish (pers. obs), this filtration method was conducive to maintaining the water quality required by the fish to maintain good health and allow for successful breeding. For a comprehensive explanation of filtration processes, refer also to Chapter 5, section 5.2.4.4. -

Filtration.

CHAPTER 3

3.2.2.4.5. Maintenance

Water changes should be done at least every second week with water of similar salinity and temperature to the system water. This water should be free of chlorine and chlorine compounds (typical of municipal water). The filter also needs to be periodically cleaned out (especially the mechanical component). For a maintenance schedule applicable to the maintenance of all fish, refer to Chapter 5, section 5.2.4.5. - Maintenance.

3.2.3. Oreochromis mossambicus brood stock management

3.2.3.1. Age of brood stock

Sexually mature adults should be used for breeding purposes. These are typically from approximately one year old, where laboratory-reared fish will be between 100 mm and 150 mm in size). Older, bigger fish will however have higher fecundities than the smaller fish. The brood stock should, however, be of uniform size to minimise aggression between individuals. Bigger fish are more dominant (especially the males) and exert their dominance onto the smaller males in the form of aggression. This often leads to the death of the smaller fish (pers. obs.).

3.2.3.2. Conditioning of brood stock

3.2.3.2.1. Temperature

The temperature was kept at a constant 27 ± 2 °C. This temperature was a criterion to stimulate prolonged breeding activity of the fish.

CHAPTER 3

3.2.3.2.2. Food

The fish were fed a diet consisting of frozen Daphnia, bloodworms and ox heart. They were then also fed several times daily on a pellet food (Tetra

DoroMin®) with just enough that the fish could consume it all within five minutes. This diet was sufficient to maintain the fish in good health as well as successful breeding of the fish.

3.2.4. Breeding O. mossambicus

3.2.4.1. Breeding system design

There was no specific breeding system design that differed from the tank that the adult fish were maintained in. The fish were merely left within this tank and allowed to breed naturally (see section 3.2.2.4. - Aquaria setup).

3.2.4.2. Gender ratios

Four male fish were put together with 16 females. This relatively high female to male gender ratio was maintained to reduce aggression in between the male fish. There were, however, enough males to breed with all of the females, therefore this gender ratio is recommended for breeding this species of fish.

3.2.4.3. Procedure for breeding O. mossambicus

The group of breeding fish were left undisturbed with only the female fish being monitored for extended buccal cavities. This indicated that they were brooding a batch of embryos within their mouths. The observation of the

CHAPTER 3 female fish was possible by merely viewing them through the front glass pane of the tank. There was no actual procedure for breeding the fish – a group of adult fish were merely maintained in a tank together in a tank that contained a number of pieces of 50 mm PVC piping of approximately 20 mm in length.

This method was successful, as at any one time, there were up to four females brooding embryos. Females of approximately 120 mm – 150 mm typically yielded up to 400 embryos at a time. This meant that up to 1 200 embryos could be collected every few days. Removing the embryos from the mouths of the females was done so that a record could be kept of the numbers of embryos being produced. Removal of the embryos also allowed the female fish to breed again without the delay of having to incubate and rear the embryonic and larval fish. There was therefore a quicker turn around time per female if the embryos were removed and artificially incubated (pers. obs.).

3.2.5. Embryo and larvae care

3.2.5.1. Embryo care

Embryos were removed from the mouths of the female fish by holding open their mouths and rinsing the embryos into a net suspended in the water. This was facilitated by the relatively large size of the female fish. The embryos were then removed from the system and incubated in a funnel-type incubation system designed to allow flow of water over the embryos at all times. This maximised oxygenation as well as the flushing of waste products from the embryos (Figure 3.2).

CHAPTER 3

System water leaves the incubation funnel via the top.

System water enters the incubation funnel through a flow regulator at the bottom.

Figure 3.2: The funnel system used for the artificial incubation of mouthbrooder-type embryos.

This was a successful way of incubating the embryos as it is a simulation of the water flow over the embryos induced by the brooding female fish as it pumps water into and out of the buccal cavity containing the embryos. A typical success rate of 95 % hatching rate was recorded when using this incubation method. As the embryos are negatively buoyant, they do not spill out with the water as it exits the tube through an outlet pipe at the top. The up flow of water through the funnel keeps the embryos circulating within the bottom portion of the funnel. Many hundreds of embryos could successfully be incubated at one time within these funnels.

After the incubating embryos had hatched (approximately six days after removal from the female fish), they remained suspended within the water column, with their movements governed by the current of the incoming water.

As the larval fish began to develop swim bladders (within approximately five

CHAPTER 3 days after being put into the incubation funnels) with the consequence that they were able to start gaining control over their buoyancy, they began to start utilising the upper part of the funnel. Therefore, the more the larval fish developed the further up within the funnel they were able to inhabit. The consequence of this was that these larval fish spilled out with the out flowing water through the outflow situated at the top of the funnel. This out flow pipe initially drained the water from the funnels into a larger container. This container had an outlet that was covered by a screen to stop the larval fish from escaping.

3.2.5.2. Care of free-swimming larvae and juveniles

Once all of the larval fish had spilled out of the funnels and into the larger container (approximately 12 days after the embryos were removed from the female), they were removed and reared in aquaria that were fitted with air- driven sponge filters. A proportion of these now-juvenile fish were used for toxicity testing and the remainder of them reared to adulthood. These juveniles were fed small amounts of TetraMin® Baby and Daphnia, until they were big enough to feed on crushed flake food (TetraMin®).

3.2.6. Conclusions and recommendations

A summary of the most important physical environmental features as well as the chemical water parameter ranges is given in Table 3.2.

CHAPTER 3

Table 3.2: Summary of recommended ranges of physical and chemical water quality parameters of the system water conducive to successful maintenance and breeding of O. mossambicus.

Water quality parameter Recommended range

Water temperature 24-27 °C Total water hardness 3-30 °dH (± 50-500 mg/l CaCO3) pH 7.0-8.5 Salinity 0.5 g/l NaCl Photoperiod 14/10 h light/dark Stocking density 20 fish [100-150 mm (SL)]/ 1 000 l

This species of fish was successfully maintained, bred and the embryos, larval and juvenile fish successfully reared within the water quality and environmental ranges given in Table 3.2. These values must, however, only be viewed as guideline values as they are by no means the extremes of the values that the fish are able to withstand. Further experimentation is necessary to establish the environmental parameter extremes that these fish are successfully maintained.

Oreochromis mossambicus was found to breed reliably under the conditions provided (see Table 3.2). The frequencies of the spawning of each female may, however, have been improved by placing one male with two females within their own breeding tank that had a gravel substrate and refuge for the females to escape the possible aggression shown by the male. The capacity of such a tank should be at least 250 l to reduce the aggression of the species by providing enough space for them to interact naturally, as placing a breeding trio into a tank with a lesser capacity was shown to induce aggression from the male, which often caused the death of females. The incubation method used for the incubation of the embryos of O. mossambicus was found to be very successful. This method does, however, require

CHAPTER 3 specialist apparatus, which does take up a lot of space within the laboratory and for these reasons, this species is not recommended for routine toxicity testing.

3.3. Tilapia sparrmanii

3.3.1. Introduction

3.3.1.1. Natural history

The classification of T. sparrmanii is as follows (Axelrod & Schultz, 1990 and

Fishbase, 2004):

Phylum: Chordata Subphylum: Craniata Superclass: Gnathostomata Class: Osteichthyes Subclass: Actinopterygii Superorder: Teleostei Order: Perciformes Suborder: Percoidea Family: Cichlidae Genus: Tilapia (Linnaeus, 1758) Species: Tilapia sparrmanii (Smith, 1840)

From the literature, it is however evident that there are several known synonyms for T. sparrmanii (Table 3.3). The dorsal fin of T. sparrmanii has 13 to 15 spines followed by nine to eleven soft rays, whilst the anal fin consists of three spines, followed by nine to ten soft rays. There are between 27 and 29 scales in the lateral line series. The body shape is variable, usually being moderately deep, with an ovoid, straight or concave predorsal profile. The caudal fin is truncate. The mouth of T. sparrmanii is small, with fine bicuspid teeth in three rows. There are nine to twelve short, well-spaced gill rakers on

CHAPTER 3 the first gill arch. The colour of T. sparrmanii is variable, being predominantly deep olive green with eight to nine dark vertical bars on the body. There are two bars between the eyes, with a well developed “tilapia spot” - a dark spot on gill cover surrounded by iridescent green or blue scales. There is also iridescent blue along the lower jaw. Breeding males have a bright red margin to the dorsal and caudal fins with a grey-black throat and chest (Figure 3.3).

Juveniles have characteristic light “bubbles” behind the tilapia mark on the soft dorsal fin. It attains about 230 mm SL, with the South African and

Zimbabwean angling records being 0.445 kg and 0.54 kg, respectively

(Skelton, 2001).

Table 3.3: Synonyms, their authors, status and current validity of T. sparrmanii (adapted from Fishbase, 2004).

Synonym Author Status Valid

Chromis niloticus (non Linnaeus, 1758) Miss identification No Tilapia sparrmanni Smith, 1840 Misspelling No Tilapia sparrmanii Smith, 1840 Original comb. Yes Chromis sparrmanii Smith, 1840 New comb. No Tilapia sparmanii Smith, 1840 Misspelling No Tilapia sparmanni Smith, 1840 Misspelling No Chromys sparmanni Smith, 1840 New comb. No Tilapia sparrmani Smith, 1840 Misspelling No Chromis sparrmanii Smith, 1840 New comb. No Chromis moffatii Castelnau, 1861 Jnr synonym No Tilapia melanopleura (non Deméril, 1861) Miss identification No Chromis ovalis Steyndachner, 1866 Questionable No Tilapia ovalis Steyndachner, 1866 Questionable No Tilapia fouloni Boulenger, 1905 Jnr synonym No Tilapia deschauenseei Fowler, 1931 Jnr synonym No Tilapia sparmani David, 1935 Misspelling No

Tilapia sparrmanii occurs from the Orange River and Kwazulu-Natal south coast northwards up to the upper reaches of the southern Congo tributaries,

Lake Malawi and the Zambezi system. It is extensively translocated south of the Orange system in the Cape (Skelton, 2001), where it is tolerant of a wide

CHAPTER 3 range of habitats but prefers quiet, or standing waters, with submerged or emergent vegetation. It is an omnivore, feeding on any available foods such as algae, soft plants, small invertebrates and even small fish.

Figure 3.3: Tilapia sparrmanii (Photograph by R. Bills10).

The males construct a simple saucer-shaped nest in which the females lay the eggs. The nest is then guarded and tended by both parents. The parents may move the eggs or larvae to alternative nests, probably for safety and sanitation reasons. Newly hatched larvae attach to a substrate by head glands and wriggle constantly for aeration, then, after seven to eight days, the larvae are free-swimming but remain in a shoal guarded by the parents for several weeks (Skelton, 2001).

3.3.1.2. Background on captive breeding and use of T. sparrmanii

Tilapia sparrmanii is distributed as forage fish for bass and forms a common component of subsistence fisheries. It is an occasional angling target

10 Roger Bills, SAIAB, Grahamstown.

CHAPTER 3

(Skelton, 2001). They also have the potential as an ornamental aquarium species (pers. obs). This species has been used for bioaccumulation studies within the laboratory (Grobler-van Heerden et al., 1991; du Preez & van

Vuren, 1992; du Preez et al., 1993) where it was not bred within the laboratory itself. The individual fish were collected as adults from a provincial hatchery.

Tilapia sparrmanii has, however, been bred successfully within the laboratory for toxicological studies (Wepener, 1990).

3.3.2. Environmental requirements and procedures for maintenance

of T. sparrmanii

3.3.2.1. Water temperature

Tilapia sparrmanii were bred and the larval and juvenile fish successfully reared at temperatures between 24 °C and 27 °C.

3.3.2.2. Water chemistry

3.3.2.2.1. pH

The pH range of the system water was between 7.0 and 8.5, with this pH range being conducive to successful maintenance, breeding of the fish as well as raising of larval and juvenile fish.

3.3.2.2.2. Total water hardness

The system water had a total hardness range of between 3 °dH and 30 °dH.

The fish were maintained and bred successfully, and the larval and juvenile fish were raised successfully within this total water hardness range.

CHAPTER 3

3.3.2.3. Photoperiod

The photoperiod was maintained at a constant 14/10 h light/dark cycle. This particular light cycle was found to be conducive to successful breeding of the fish.

3.3.2.4. Aquaria design

3.3.2.4.1. Aquaria

A group of fish were maintained in a 1,000 l all-glass aquarium. This tank was part of a flow-through system that was filtered by a common filter. The filter was packed relatively densely with shade cloth, which acted as both a mechanical as well as a biological filter. The filtered water was then returned to the holding tank by a submersible pump situated within the filter at a flow rate of 1,000 l/hr.

3.3.2.4.2. Stocking densities of fish in aquaria

Approximately 50 adult fish were maintained within the 1,000 l tank. That translated to one fish per 20 l of water.

3.3.2.4.3. Substrate

No substrate was put into the maintenance stock tank. This was done to facilitate cleaning of the system as well as to inhibit spawning behaviour of the fish.

CHAPTER 3

3.3.2.4.4. Filtration

The maintenance system was part of a flow-through system that was filtered by a common filter that was packed densely with shade cloth. This acted as both a mechanical and a biological filter. For a comprehensive explanation of the filtration process applicable to all fish, refer to Chapter 5, section 5.2.4.4. -

Filtration.

3.3.2.4.5. Maintenance

A 20 % water change was done of the entire system every two weeks to facilitate the removal of the nitrates that would have built up within the system.

It is important to ensure that the water that replaces the removed water is of the same temperature and is free of chlorine and chlorine compounds.

3.3.3. Tilapia sparrmanii brood stock management

3.3.3.1. Age of brood stock

Only adults showing the signs of sexual maturity in terms of colour intensification should be used for breeding purposes. If the fish are kept under conditions conducive to breeding, then colour intensification brought on by reproductive maturity can be seen within one to one and a half years (pers. obs.).

CHAPTER 3

3.3.3.2. Conditioning of brood stock

3.3.3.2.1. Temperature

Fish that were required to breed were kept at a constant temperature of 25

°C. This temperature was conducive to stimulating spawning behaviour, and is therefore recommended as the temperature that is suitable for breeding as well as raising larvae and juvenile fish.

3.3.3.2.2. Food

The fish were fed a diet consisting of frozen Daphnia, bloodworms and ox heart. They were then also fed several times daily on a pellet food (Tetra

DoroMin®). This diet was sufficient to maintain the fish in good health as well as successful breeding of the fish. This diet can therefore be recommended for maintenance and breeding of this species of fish.

3.3.3.2.3. Photoperiod

The breeding fish were kept at a constant photoperiod of a 14/10 h light/dark cycle. This was kept constant throughout the study. It was not found that this species of fish needed manipulation of the photoperiod to induce spawning.

3.3.4. Breeding T. sparrmanii

3.3.4.1. Breeding system design

Breeding tanks were all-glass aquaria with a 100 l capacity (500 mm (L) X

500 mm (B) X 400 mm (H)) with three sides of the tank painted black. These

CHAPTER 3 aquaria had relatively fine gravel as a substrate, fitted with under gravel filters.

A corner box-type filter containing filter wool and activated carbon was also placed within each tank. Two fist-sized roundish rocks were placed next to one another with a piece of slate resting on the top of them to create a type of cave effect in each tank (Figure 3.4).

Artificial plants acting as refuge for the fish.

Two fist-sized rocks covered by a piece of flat slate creating further ref uge for the fish.

Figure 3.4: Breeding tank design for T. sparrmanii.

Further refuge was created by cutting pieces of 10 mm nylon rope, tying the one end in a knot and burning the end to stop it unravelling. The opposite end of the rope was unravelled, and “teased” to make it spread out evenly. The knotted end was then threaded through a ringed suction cup and secured onto the glass of the tank at various locations (Chapter 2, Figure 2.4).

CHAPTER 3

3.3.4.2. Gender ratios

One male fish was put into the breeding tank and allowed two weeks to acclimate to the new tank environment. This time was also for the male fish to establish a potential breeding site and begin nest construction. After these two weeks, three female fish were put into the tank with the male. The male fish was then allowed to single out a suitable mate, usually acting overly aggressively towards the other females. These subordinate females were removed from the tank and placed into a separate tank where they were allowed to recuperate from the injuries inflicted by the aggression of the male.

The final gender ratio was therefore one male to one female.

3.3.4.3. Procedure for breeding T. sparrmanii

Male fish were selected from the maintenance group by colour distinction and placed singly within the spawning tanks. They were given at least two weeks to acclimate to the spawning tank environment before three females were placed into each tank with them. The behaviour of the males was carefully monitored for aggression shown towards any of the females. Subordinate females were removed from the spawning tanks and returned to the stock group of fish. If a particular male was overly aggressive towards all of the females, and that particular male did not select a suitable mate, it was removed, and another male was selected from the stock group of fish. If pairing did occur between the brood fish, they were closely monitored, but disturbed as little as possible. When a male had selected a suitable mate, and breeding did commence, there were on average 400-500 eggs laid with

50-60 % of these being successfully raised to the juvenile stage when left with

CHAPTER 3 the breeding pair. This was initially done, but it was found that only one out of every six broods was successfully raised in this manner. Therefore, as soon as eggs were laid, they were removed and artificially incubated. The parent fish were subsequently returned to the stock group of fish. The spawning tanks were then cleaned and the procedure repeated.

The removal of the breeding pair of fish and returning them to the maintenance group of fish was done to neutralise any territory that the male had established. After the removal of the eggs, the male fish pursued and aggressively attacked the female fish, often killing it. Indeed, any sort of disturbance of the breeding pair led to the male fish acting aggressively towards the female, often killing it. Removal of the breeding pair and re- establishing another breeding pair was time consuming, but necessary to ensure the survival of the fish. This method was successful in ensuring the survival of the female fish, therefore it is recommended when this species of fish is to be bred under laboratory conditions.

3.3.5. Embryo and larval care

3.3.5.1. Embryo care

The eggs were usually laid on the pieces of slate, or on the rocks. These were then removed from the tanks with the eggs still attached to them and placed into a container that allowed water to pass over the embryos as part of a flow-through system (Figure 3.5). Two to three days later, after the embryos hatched, the rock or piece of slate was removed, washed, and returned to the

CHAPTER 3 spawning tanks. The water containing the larvae was periodically cleaned of debris, but otherwise left alone until they were free-swimming (approximately

10 days after hatching). As much as 90 % of the viable embryos were raised to juvenile stages when this method of artificial incubation was used.

Out flow covered with a mesh to stop free-swimming larvae from escaping.

Tilapia sparrmanii embryos, approximately six days old.

The in flow pipe situated to create a down flow of the water. This facilitated circulation within the container.

Figure 3.5: Incubation chamber for artificially incubating embryos of T. sparrmanii.

3.3.5.2. Care of free-swimming larvae and juveniles

The free-swimming larvae were then fed small amounts of TetraMin® Baby.

A proportion of the juveniles were used for toxicity tests (see chapter 6), with the remainder of the juveniles being reared on TetraMin® flake food, frozen bloodworms, Daphnia and brine shrimp through to adulthood.

3.3.6. Conclusions and recommendations

A summary of the most important physical environmental features as well as the chemical water parameter ranges is given in Table 3.4. This species of fish was successfully maintained, bred and the embryos, larval and juvenile fish successfully reared within these water quality and environmental ranges.

CHAPTER 3

Table 3.4: Summary of recommended ranges of physical and chemical water quality parameters of the system water conducive to successful maintenance and breeding of T. sparrmanii.

Water quality parameter Recommended range

Water temperature 24-27 °C Total water hardness 3-30 °dH (± 50-500 mg/l CaCO3) pH 7.0-8.5 Photoperiod 14/10 h light/dark Stocking density of breeding fish one male to one female in 100 l

These values must, however, only be viewed as guideline values as they are by no means the extremes of the values that the fish are able to withstand.

Further experimentation is necessary to establish the environmental parameter extremes that these fish are successfully maintained.

The aggression displayed by the males of this species necessitated the isolation of breeding pairs. This aggression also meant that the maintenance and routine catching and separating of the male and female fish was a very time-consuming procedure. This aspect of this species character also meant that the males killed many females before suitable pairs were established – even with the least disturbance. The space needed to maintain separate breeding pairs, as well as the time and effort required to maintain healthy and productive pairs, means that this species of fish is not recommended for use in routine toxicity tests where large numbers of embryos are required on a regular basis. To increase the frequency of breeding as well as to possibly reduce the aggression shown by the male fish, it is recommended that the fish be housed in bigger aquaria with more refuge provided for the subordinate females. This will, however, further compound the problem of space economy for the laboratory.

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3.4. Pseudocrenilabrus philander philander

3.4.1. Introduction

3.4.1.1. Natural history

The classification of P. p. philander is as follows (Axelrod & Schultz, 1990 and

Fishbase, 2004):

Phylum: Chordata Subphylum: Craniata Superclass: Gnathostomata Class: Osteichthyes Subclass: Actinopterygii Superorder: Teleostei Order: Perciformes Suborder: Percoidea Family: Cichlidae Genus: Pseudocrenilabrus (Weber, 1897) Species: Pseudocrenilabrus philander philander (Weber, 1897)

From the literature, it is however evident that there are several known synonyms for P. p. philander (Table 3.5).

Table 3.5: Synonyms, their authors, status and current validity of P. philander philander (adapted from Fishbase, 2004).

Synonym Author Status Valid

Haplochromis moffati (non Castelnau, 1861) Miss identification No Astatotilapia moffati (non Castelnau, 1861) Misspelling No Haplochromis moffattii (non Castelnau, 1861) Original comb. No Chromis philander Weber, 1897 New comb. No Pseudocrenilabrus philander Weber, 1897 Misspelling No Hemihaplochromis philander Weber, 1897 Misspelling No Haplochromis philander Weber, 1897 New comb. No Pseudocrenilabrus philander philander Weber, 1897 New comb. Yes Haplochromis philander philander Weber, 1897 New comb. No Tilapia cabrae (non Boulenger, 1899) Jnr synonym No Haplochromis desfontainesii (non Boulenger, 1899) Miss identification No Tilapia lucullae (non Boulenger, 1913) Questionable No Pseudocrenilabrus natalensis Fowler, 1934 Questionable No

CHAPTER 3

The dorsal fin of P. p. philander consists of 13 to 16 spines, followed by nine to eleven soft rays, whilst the anal fin has three spines, followed by seven to nine soft rays. There are between 27 and 30 scales in the lateral line series.

The chest scales are not markedly differentiated from the body scales. The body of P. p. philander is stout and the caudal fin is rounded. The mouth is small and horizontal. Females are light brown with dark vertical bars and light yellowish fins, whilst the males’ colours differ with locality. The body is usually a mesh of iridescent light blue and yellow, with an oblique bar through the eye and an iridescent blue lower jaw. The dorsal fin has a red tip, black submarginal band and iridescent blue blocks. The pelvic fins are black, whilst the caudal and anal fins have iridescent blue and red blocks. The anal fin has an orange tip. All the colours are accentuated during breeding (Figure 3.6).

This species attain 130 mm TL (Skelton, 2001). It is found from the Orange

River and southern Kwazulu-Natal northwards throughout the region, extending to southern Congo tributaries and Lake Malawi, where it occurs in a wide variety of habitats from flowing waters to lakes and isolated sinkholes.

Pseudocrenilabrus philander philander, however, usually favours vegetated zones. This species preys on insects, shrimps and even small fish. It breeds from early spring to late summer.

CHAPTER 3

Figure 3.6: Pseudocrenilabrus philander philander.

The males establish and defend a territory, construct a simple cleared nest and then attract ripe females. The females then lay eggs in the nest, which are fertilised by the male and then collected by the female. She then withdraws to a quiet nursery area and broods the embryos, larvae and juveniles within her mouth until they are able to fend for themselves. Several broods may be raised in a season. Pseudocrenilabrus philander philander have been known to live for four to five years (Skelton, 2001). Threats to isolate populations of this species of fish include habitat changing and insecticide poisoning as well as the introduction of fishes into springs and sinkholes (Skelton, 2001).

3.4.1.2. Background on captive breeding and use of P. p. philander

This species of fish has the potential to be used as an aquarium species due to its relatively small size. It is also used for behavioural and evolutionary research (Skelton, 2001). It has also become popular as a mosquito control

CHAPTER 3 fish in temperate waters (pers. com. Steyn11, 2004). Pseudocrenilabrus philander philander has been bred under laboratory conditions for the purpose of hobby interest (pers. obs.).

3.4.2. Environmental requirements and procedures for maintenance

of P. p. philander

From experimentally determining the environmental requirements and procedures for maintenance and breeding of this species of fish, it was found that the majority for the parameters and protocols used for T. sparrmanii were suitable for successful maintenance and breeding of P. p. philander.

Therefore, for the procedures and protocols for maintenance and breeding of this fish species, refer to section 3.3.2. - Environmental requirements and procedures for maintenance of T. sparrmanii as well as sections 3.3.3. -

Tilapia sparrmanii brood stock management and 3.3.4. - Breeding T. sparrmanii. Deviations from this protocol will be dealt with under the relevant headings.

3.4.3. Procedure for breeding P. p. philander

Individual males (measuring approximately 60 mm) were selected from the stock tank and placed singly into the spawning tanks (see section 3.3.4.1. -

Breeding system design). After at least two weeks of acclimation to these conditions, three females (measuring approximately 50-60 mm) were placed with each male. Subordinate and harassed females were removed and replaced from the stock tank, until each male had three compatible females.

11 Prof. G.J. Steyn – Rand Afrikaans University.

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Once the male had chosen suitable mates, and they were observed to settle without any further aggression, the females were monitored closely for not feeding, as this was an indication that they were brooding embryos. If this was the case, the embryos were removed by opening up the females’ mouths and rinsing the embryos into a net submersed in the water. By keeping two to three females with each male, a batch of embryos was collected about once a week. The fecundity of this species was relatively low though. A female measuring 55 mm only produced 100 embryos at a time. These fish were found to periodically spawn in a tank with no substrate, but the frequencies of breeding as well as the fecundity were both relatively poor to make this method a viable one.

3.4.4. Embryo and larval care

3.4.4.1. Embryo care

The embryos were left with the female for approximately six days until the throat of the female darkened in colour. This indicated that the larvae had hatched. Only then were the larvae removed from the females’ mouths. This was relatively difficult die to the small size of the female fish. The larvae were then artificially incubated within the same funnel-type incubators used for the

O. mossambicus until they were free-swimming (Figure 3.2). This took approximately 10 days.

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3.4.4.2. Care of free-swimming larvae and juveniles

A proportion of the juveniles were used for toxicity tests, whilst the rest of them were reared to adulthood. These juvenile fish were fed on TetraMin

Baby® flake food as well as Daphnia, bloodworms and brine shrimps as they got big enough to accept it (approximately three weeks old).

3.4.5. Conclusions and recommendations

A summary of the most important physical environmental features as well as the chemical water parameter ranges is given in Table 3.6.

Table 3.6: Summary of recommended ranges of physical and chemical water quality parameters of the system water conducive to successful maintenance and breeding of P. p. philander.

Water quality parameter Recommended range

Water temperature 24-27 °C Total water hardness 3-30 °dH (± 50-500 mg/l CaCO3) pH 7.0-8.5 Photoperiod 14/10 h light/dark Stocking density for breeding fish 2-3 females to one male in 100 l

This species of fish was successfully maintained, bred and the embryos, larval and juvenile fish successfully reared within these water quality and environmental ranges. These values must, however, only be viewed as guideline values as they are by no means the extremes of the values that the fish are able to withstand. Further experimentation is necessary to establish the environmental parameter extremes that these fish are successfully maintained.

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This species of fish showed a relatively low fecundity, which would necessitate the maintenance of a large quantity of brood stock to deliver the large amount of embryos on a regular basis, which is required by a laboratory doing routine toxicity tests. The fact that this is also an aggressive species of fish means that small breeding groups have to be housed in separate aquaria.

This makes this species of fish unsuitable for routine use in toxicity tests due to the factor of space economy. The artificial incubation of this species of fish also relies on specialist apparatus, which also takes up a lot of room within the laboratory. This species of fish is therefore not recommended as a suitable candidate to be used by the laboratory doing routine toxicity tests.

3.5. Conclusions and recommendations for the use of O.

mossambicus, T. sparrmanii and P. p. philander as routine

toxicity testing species

The overall aggressive behaviour shown by these three species of fish necessitates the separation of breeding pairs. This means that, in order to produce the relatively high number of larval and juvenile fish of uniform age and health, the laboratory needs to maintain relatively high numbers of breeding pairs. This, coupled to the relatively low fecundities shown by all of the fish tested, means that a relatively high number of individual aquaria that will take up a lot of space within the laboratory – a luxury that a laboratory does not necessarily have. Therefore, these species of fish are not recommended for use in routine toxicity tests unless the laboratory has the space required to culture these fish, as well as the laboratory staff skilled enough to deal with the aggressive behaviour of the fish.

CHAPTER 3

3.6. References

Axelrod, H.R and Schultz, L.P. (1990). Handbook of tropical aquarium

fishes. T.F.H. Publications, New Jersey. 728 p.

Du Preez, H.H. and van Vuren, J.H.J. (1992). Bioconcentration of atrazine in

the banded tilapia, Tilapia sparrmanii. Comparative Biochemistry and

Physiology Vol. 101C (3): 651-655.

Du Preez, H.H., van Rensburg, E. and van Vuren, J.H.J. (1993). Preliminary

laboratory investigation of the bioconcentration of zinc and iron in

selected tissues of the banded tilapia, Tilapia sparrmanii (Cichlidae).

Bulletin of Environmental Contamination and Toxicology 50: 674-

681.

Fishbase. (2004). Froese, R. and Pauly, D. (Editors). World Wide Web

electronic publication. www.fishbase.org, version (06/2004).

Kruger, T. (2002). Effects of zinc, copper and cadmium on Oreochromis

mossambicus free-embryos and randomly selected mosquito

larvae as biological indicators during acute toxicity testing. M.Sc.

Dissertation, Rand Afrikaans University.

Grobler-van Heerden, E., van Vuren, J.H.J. and du Preez, H.H. (1991).

Bioconcentration of atrazine, zinc and iron in the blood of Tilapia

sparrmanii (Cichlidae). Comparative Biochemistry and Physiology

Vol. 100C (3): 629-633.

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Van Dyk, J.C. (2001). Histological changes in the liver of Oreochromis

mossambicus (Cichlidae) after exposure to cadmium and zinc.

M.Sc. Dissertation, Rand Afrikaans University.

Wepener, V. (1990). Die effek van swaarmetale by variërende pH op die

bloedfisiologie en metaboliese ensieme van Tilapia sparrmanii

(Cichlidae). M.Sc. Dissertation, Rand Afrikaans University.

ASSESSMENT OF INTERNATIONAL PRACTICES ON THE USE OF FISH IN TOXICITY TESTING AND ADAPTATIONS FOR SOUTH AFRICAN CONDITIONS

CHAPTER 4

MAINTENANCE AND BREEDING OF POECILIA RETICULATA (POECILIIDAE) TO DETERMINE ITS SUITABILITY FOR USE IN ROUTINE LABORATORY BIOASSAYS

CHAPTER 4

4.1. Background

This part of the dissertation describes the maintenance and breeding procedures for P. reticulata (guppy) under laboratory conditions with the specific aim of experimentally determining if this species of fish is suited for being used in routine toxicity bioassays. This is done by comparing that which is cited in the literature to what is practical to the laboratory situation.

The amenability to maintenance, the ease at which this species breeds as well as its fecundity under laboratory conditions in relation to the space and cost required to culture it will determine the degree to which this species of fish is suited to being used as a routine toxicity testing species.

Recommendations are then made for the culturing of this species, based on the results obtained from the actual culturing of this species under laboratory conditions.

4.2. Introduction

4.2.1. Natural history of P. reticulata

The classification of P. reticulata is as follows (Axelrod & Schultz, 1990 and

Fishbase, 2004):

Phylum: Chordata Subphylum: Craniata Superclass: Gnathostomata Class: Osteichthyes Subclass: Actinopterygii Superorder: Teleostei Order: Cyprinodontiformes Suborder: Poeciliodea Family: Poeciliidae Genus: Poecilia (Bloch & Schneider) Species: Poecilia reticulata (Peters, 1859)

CHAPTER 4

From the literature, it is however evident that there are several synonyms for

P. reticulata from its time of discovery by Peters in 1859 (Table 4.1).

Table 4.1: Synonyms for P. reticulata, their status and current validity (adapted from Petrovický, 1998 and Fishbase, 2004).

Synonym Author Status Valid

Poecilia reticulata Peters, 1859 Original combo Yes Poecilioides reticulatus Peters, 1859 New combo No Lebistes reticulatus Peters, 1859 New combo No Haridichthys reticulatus Peters, 1859 New combo No Girardinus reticulatus Peters, 1859 New combo No Acanthophacelus reticulatus Peters, 1859 New combo No Poecilia reticulatus Peters, 1859 Misspelling No Lebistes poeciloides De Filippi, 1861 Junior synonym No Lebistes poecilioides De Filippi, 1861 Junior synonym No Girardinus guppyi Günther, 1866 Junior synonym No Acanthophacelus guppii Günther, 1866 Junior synonym No Heterandria guppyi Günther, 1866 Junior synonym No Girardinus petersi Junior synonym No Girardinus poecilioides Junior synonym No Poecilia poecilioides Junior synonym No

There are between seven and nine branched dorsal fin rays, and eight to nine branched anal fin rays, with 26-28 scales in the lateral line series. Females have a typical rounded abdomen and caudal fin and the more slender male has a well-developed gonopodium, long caudal peduncle and variable

(usually delta-shaped) caudal fin. The males’ colours are extremely variable, often spectacular, in combinations of iridescent red, blue, turquoise and yellow. Black spots and stripes are often present in certain strains of the species. Colours of wild fish are less extravagant. Gravid females have a dark spot above the vent. Males attain 30 mm total length (TL) with the females attaining 60 mm TL (Skelton, 2001) (Figure 4.1).

CHAPTER 4

Figure 4.1: Poecilia reticulata male (left) and female (right).

The type locality of P. reticulata is located in Caracas in Rio Guaire. Its natural range is in Venezuela, Trinidad, and Barbados, as well as the

Caribbean Islands, but has been introduced into many localities of the world for mosquito control (Axelrod & Schultz, 1990). Feral populations are reported from coastal reaches of Kwazulu-Natal rivers from Durban southwards, as well as from the Kuruman Eye and Lake Otjikoto in Namibia

(Skelton, 2001). It was first introduced and released in 1912 in Gauteng and

Kwazulu-Natal rivers for mosquito control. Most feral populations, however, are from private aquarium releases (Skelton, 2001). It is naturally found in brackish and seawater around the islands of Martinique and St. Thomas

(Petrovický, 1998).

Poecilia reticulata is capable of having broods approximately every four weeks, with the brood size averaging 45, although there have been records of females having up to 187 young (Axelrod & Schultz, 1990), other accounts claim larger females having as much as 250 young at a time (Petrovický,

1998). These reports, however, come from fish populations that are kept outdoors in earth dams. It is a euryhyaline species of fish (Chiyokubo, et al.,

CHAPTER 4

1998) that requires relatively warm temperatures (optimally 23-24 °C) and quiet vegetated water for survival in natural systems. Temperatures of between 10 °C and 32 °C, however, are tolerated. It feeds naturally on

Daphnia, mosquito larvae and small worms (Coffey, 1986; Axelrod & Schultz,

1990 and Skelton, 2001).

4.2.2. Background on captive breeding and use of P. reticulata

Poecilia reticulata is an extremely popular aquarium fish species worldwide amongst professional and novice aquarists alike, due to its widespread cultivation, availability and relatively small cost. It is also a desirable species for the community tank due to its peaceful nature, small size and the ease at which it is bred in the home aquarium. The males are very attractively coloured, with almost every conceivable colour variation being produced by commercial breeders (Coffey, 1986; Axelrod & Schultz, 1990; Sandford,

2003).

Poecilia reticulata is used for toxicity tests in Brazil as well as some European countries (OECD, 1992; ISO, 1996; Slabbert et al., 1998), as well as being regarded as the ‘standard’ fish for use in tests locally in commercial testing laboratories performing routine toxicity tests due to its amenability to culturing under laboratory conditions (DWAF, 1992). It is cited in the literature as a

‘beginner’ fish, often being the first species of fish to reproduce within the home aquarium (Axelrod & Schultz, 1990; Axelrod, 1995; Hemdal, 2003;

Sandford, 2003) as well as being one of the most common fish used for laboratory toxicity studies (Khangarot & Ray, 1990). This, however, has

CHAPTER 4 proven to be more of a challenge locally than what the literature implies, when the demand for young fish is required in relatively large numbers by commercial testing laboratories. After consultation with various people currently within the field of commercial toxicity testing, the consensus indicated the shortcomings of P. reticulata as a testing species used for routine testing. These factors included their lack of reproduction, as well as the relatively low fecundity shown by this species under laboratory conditions.

This species of fish seemed to also be relatively difficult to maintain under laboratory conditions (pers. comm. Jooste 12 (2002); du Preez13 (2003);

Slabbert14 (2003).

4.3. Environmental requirements and procedures for maintenance

of P. reticulata

4.3.1. Water temperature

Poecilia reticulata can tolerate temperatures ranging from 14 °C to 32 °C, but are best maintained in optimum temperatures of between 22 °C and 26 °C

(Axelrod & Schultz, 1990). Keeping the temperature at 25 ± 1 °C yielded relatively good results in terms of maintenance and breeding, as well as growth of the larval and juvenile fish. This temperature is therefore recommended for successful maintenance and breeding of this species of fish.

12 Dr S. Jooste, Resource Quality Services, Pretoria. 13 Dr H. du Preez, Hydrobiology, Rand Water, Vereeniging. 14 L. Slabbert, Environmentek, CSIR, Pretoria.

CHAPTER 4

4.3.2. Water chemistry

4.3.2.1. pH

The pH value of the system water is recommended to be between 7.0 and 8.5

(Sandford, 2003). The pH of the system water ranged between 7.37 and

8.34. These pH values were found to be conducive to successful maintenance and breeding of P. reticulata (pers. obs.).

4.3.2.2. Salinity

It is recommended that one heaped teaspoon of non-iodised coarse rock salt

(NaCl) is added for every 10 l of system water (Petrovický, 1998). Fifty grams of non-iodised coarse rock salt (NaCl) was indeed added to the system water

(100 l) throughout the duration of this study (0.5 g/l). If the NaCl was not added to the system water, it was found that the vitality of the fish was not as good as when the NaCl was added (pers. obs.).

4.3.2.3. Total water hardness

The recommended total water hardness (°dH) for P. reticulata (as for all

Poeciliidae) is as high as 30 °dH (Sandford, 2003). This is categorised on the

°dH scale as ‘very hard’ water. Successful maintenance and breeding, however, of this species was achieved at water hardness values of between

52 and 87 mg/l CaCO3 (3-6 °dH), which in turn translates to ‘moderately soft’ water. These were the total water hardness values of the standard municipal water used throughout the duration of this project.

CHAPTER 4

4.3.3. Aquaria design

4.3.3.1. Aquarium size and stocking densities of fish

The choice of aquarium size is of relatively minor importance to the successful maintenance and breeding of P. reticulata as they can be successfully bred in as little as 20 l of water (Hemdal, 2003). The surface area of the aquarium, however, will govern the number of adult fish suitable for that particular aquarium (Sandford, 2003). An adult fish (measuring approximately 25 mm) will require a surface area of at least 75 cm2. This translates then to 33 fish within an aquarium with a surface area of 2 500 cm2 (50 cm x 50 cm). This value will also, however, be strongly governed by the filtration capacity of the aquarium. This was the size of the tanks that the fish were maintained and bred in with relatively successful results (for these results, see section 4.5. -

Breeding P. reticulata).

4.3.3.2. Filtration

Good filtration is of utmost importance to the successful maintenance of P. reticulata as they are very prone to bacterial infections that are relatively difficult to treat successfully (pers. obs.). The filtering capacity of the particular filter system used within the tank must be established - usually stated on the packaging by the manufacturer. This value, however, should be regarded as an overestimated guideline value. Removal of uneaten food is of utmost importance as rotting organic matter inevitably increases the overall heterotrophic bacterial content of the water, leading to bacterial infections within the fish. For this reason, the use of a bottom-feeding fish such as

CHAPTER 4

Corydoras sp. (Callichthyidae) (Figure 4.2) is advocated to feed on the food that the other fish do not eat. Avoiding over feeding the fish is also very important in maintaining good water quality as uneaten food quickly rots within the tanks, producing toxic ammonia.

Figure 4.2: Corydoras aeneus – ideally suited to keeping the bottom of the breeding aquarium free from surplus food.

4.3.3.3. Maintenance

Organic matter should be removed by siphoning from the bottom of the tank on a routine basis at least twice per week. The water removed in this way should be replaced with water of similar temperature and salinity and should be free of chlorine and chlorine compounds. Filter media also needs to be routinely cleaned of the build up of organic matter. This media should be rinsed in clean system water of similar temperature and chemistry; otherwise, the nitrifying bacteria within the media will be destroyed with the consequence of decreased biological filtration ability. This would be detrimental to the fish within the system. For further schedules of routine maintenance and procedures, refer to the maintenance schedules described in Chapter 5.

CHAPTER 4

4.4. Poecilia reticulata brood stock management

4.4.1. Age of brood stock

Bigger females of between one and two years old typically produced more offspring than the younger females of under a year old. The older females were, however, more susceptible to bacterial infections than the younger fish and therefore did not produce larval fish as consistently as the younger females. Therefore, the use of adult females between the ages of one year and 18 months is recommended as the most reliable breeders. As soon as the larger females begin to show signs of bacterial infections of the abdomen

(abdominal dropsy), they should be discarded and replaced by fresh brood stock (Figure 4.3).

As soon as male fish have well-developed tails (approximately three to five months old), they can be used for breeding (pers. obs.). Only actively swimming males should be used for breeding, as these individuals will produce the best results (pers. com. Kirsten, 200215). Males that develop too large a tail that begins to hinder their swimming potential should be discarded and replaced with younger males.

15 A. Kirsten, commercial ornamental fish farmer, Modimolle (Nylstroom).

CHAPTER 4

Figure 4.3: Adult female P. reticulata showing symptoms of abdominal dropsy, which include protruding scales.

4.4.2. Conditioning of brood stock

The condition of the fish refers to their general health and nutritional status.

Only fish that are well conditioned in terms of these two aspects can be expected to reproduce successfully (Hemdal, 2003). Throughout this study, these aspects were found to be important. If the brood stock were not adequately conditioned, they were reluctant to breed, and if they did breed, they had relatively low fecundities.

4.4.2.1. Temperature

Brood stock should be kept consistently in temperatures of between 24 °C and 26 °C for optimum conditioning. Breeding activity was induced and juveniles were successfully raised within this temperature range.

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4.4.2.2. Food

Brood stock fish should be fed several times daily on a good quality commercial dry flake food such as TetraMin®, as well as live food such as bloodworm, Daphnia and brine shrimp, either fresh or in the frozen form

(which is more convenient for storage). Only feed fish enough that they can consume within five minutes. Leftover food should be routinely removed from the bottom of the tanks or bottom-feeding fish such as Corydoras sp.

(Callichthyidae) (Figure 4.2) are placed into the tanks to consume the leftover food, thereby facilitating the sanitation of the tanks. It is necessary to remove the leftover food, as rotting food within the tank is a major source of ammonia, which is poisonous to the fish.

The conditioning of P. reticulata is therefore a relatively simple matter as it is not necessary to manipulate any environmental parameters, such as photoperiods and water chemistries. To ensure optimal fecundities from the fish, close attention must be focussed on nutrition of the fish as well as maintenance of good water quality. Poor water quality and poor nutritional status will inevitably stress the fish, making them susceptible to diseases.

This will lead to overall decrease in fecundities of the fish. Choosing of suitable individual fish to be used as brood stock is also an important factor in ensuring optimal fecundities of the fish. Male fish with larger caudal fins that hinder their swimming ability will inevitably decrease the incidences of successful matings between male and female fish. This will decrease the fecundities of the fish as well.

CHAPTER 4

4.5. Breeding P. reticulata

4.5.1. Breeding system design

Five breeding tanks of 100 l capacity with dimensions 500 mm (L) x 500 mm

(B) x 400 mm (H) were used for breeding this species of fish. Temperature was kept between 24 °C and 26 °C. There was no gravel substrate added to the bottom of the tanks for one group of fish, but gravel with an under gravel filter was placed in with another group of fish to determine which system setup gave the best results. All tanks were, however, fitted with a double sponge air-driven filter (Oxy Plus Bio Filter II – Unipet), as well as a large corner box- type filter (Marltons) to ensure the maintenance of good water quality.

Breeding cages were constructed from 50 % shade cloth stretched and sewn around a frame made from 5 mm grade 316 stainless steel round bar (joined by welding the pieces together using grade 316 stainless steel welding rods) of dimensions 450 mm (L) x 250 mm (W) x 300 mm (H). The breeding cages were designed to rest on the sides of the tanks so that the tops of the cages were out of the water (Figure 4.4). Five such breeding systems were set up for the purpose of determining their suitability. Three to five males and 10 females were placed in each with the result that there were approximately 1

250 larval fish produced per month.

CHAPTER 4

Breeding cage placement in the tank.

Rods resting on the sides of the tank to keep it in place.

Water level kept below the top edge of the breeding cage to prevent jumping fish from escaping.

Figure 4.4: Breeding cages used for P. reticulata breeding showing how the cages rest on the sides of the tank allowing the upper surface to be above the water line.

The breeding system designed for P. reticulata yielded better results than the commercially available breeding traps (pers. obs.). This can be attributed to the larger size of the breeding cage in comparison to the commercial traps.

This larger size allowed for more fish to be placed together in a single breeding cage, thereby reducing the stress placed on the fish brought on by the isolation of single individuals. The reduction of stress placed on the fish is important to the successful maintenance and breeding of all fish (pers. obs.).

CHAPTER 4

Reducing the stress that a fish is subjected to also reduces their susceptibility to diseases brought on by parasitic and bacterial infections. The breeding system used for this study also allowed more water circulation throughout the breeding cage than what the commercially available breeding traps did. This, in turn, allowed for increased water quality within the breeding cage, thereby further reducing the chance of a further stress factor placed on the fish.

Left over food and fish wastes are siphoned from the tanks three times per week with the water removed being replaced with aged tap water of similar salinity (0.5 g/l). Two Corydoras sp. (Callichthyidae) - a bottom-feeding fish species - were put into each breeding tank to consume any left over food that fell through the nets of the breeding cages. The incorporation of this species of fish to aid in sanitation of the breeding tanks greatly reduced the time and effort required to maintain the sanitation of the individual tanks. They also were never observed to consume any of the larval and juvenile fish. This was proved by comparing the numbers of larval fish collected from tanks that had the Corydoras sp. in them, to the numbers of larval fish collected from tanks where the Corydoras sp. were absent. There was consistently found to be no difference in the numbers of larval fish collected, therefore the use of this particular species of fish can be advocated to aid in the sanitation of the breeding tanks without the risk of losing larval fish through them being eaten by the Corydoras sp.

CHAPTER 4

4.5.2. Gender ratios

Five groups of up to 30 mixed-sex adults were maintained for breeding purposes. A gender ratio of four males to 10 females was found to work relatively well. Males would constantly harass the females, so by having a higher male to female ratio just places unnecessary stress on the females, with the consequence that they will not breed to their full potential. This gender ratio is therefore recommended.

4.5.3. Procedure for breeding P. reticulata

A breeding tank, complete with a breeding cage, is prepared and allowed sufficient time to circulate and complete the nitrogen cycle (see Chapter 5 for a comprehensive description of the nitrogen cycle) before broodstock adults are placed into it. The adult fish are placed into the breeding cage in the correct gender ratios. After one or two days, the males should be starting to pursue the females. If this behaviour is observed, larval fish can be expected approximately two weeks later. The entire breeding group can be kept together with the larval fish being routinely removed, or, alternatively, the obviously pregnant females can be removed and place into their own tank fitted with a breeding cage. This method is more desirable as the males will then not harass the pregnant females. Results that are more consistent, in terms of successful batches of larval fish, are obtained in this manner.

Commercial breeding cages are available that are useful for placing only one or two pregnant females into it. These traps were assessed for suitability, but were found to be unsuitable for relatively large-scale production of fish

CHAPTER 4 typically required by a routine toxicity-testing laboratory. On an individual basis, they were also not as successful as the larger breeding cages as they seemed to sometimes place undue stress on the female fish due to the small size of the trap. These traps also do not allow adequate water circulation, thereby greatly reducing the water quality within them. This, in turn, makes the female relatively susceptible to bacterial infections. The use of these commercial breeding traps is therefore not recommended for use in the laboratory where large numbers of larval fish are required.

Even though this species of fish did breed readily under the laboratory conditions, their relatively low fecundities make it necessary to maintain a relatively large number of tanks to successfully breed this species in adequate numbers necessary to do routine fish bioassays.

4.6. Larvae and juvenile care

The breeding tanks were inspected every day for larval fish, with the numbers being recorded. The larval fish were caught from the tanks in the morning and transferred to rearing tanks that had a gravel substrate fitted with under gravel filters. Larval and juvenile fish were initially fed on a finely crushed flake food (TetraMin® Baby) until they were big enough to feed on Daphnia and later on, bloodworms. Larval fish were reared in tanks with a gravel substrate fitted with under gravel filters to maintain a more stable environment with superior water quality in a relatively small tank, as this type of set up does not require the high degree of water changes than the breeding tanks needed. This meant fewer disturbances to the larval fish. Larval and juvenile

CHAPTER 4 fish of this species are very susceptible to ailments brought on by unfavourable tank conditions – fin rot in particular – the caudal fin of the juvenile fish become ‘pointed’ in the middle, with the fishes condition deteriorating rapidly from then onwards. The incidence of bacterial infections was greatly reduced by placing greater emphasis on sanitation and filtration of the water. The juvenile fish showed better growth rates and earlier sexual maturity if the males and females were separated from one another as early as possible. Early sexing of the fish is possible if viewed from the side in a glass aquarium. The females have the rounded anal fin, whereas the males have an elongated, straight anal fin. A proportion of the larval were used for toxicity tests (see Chapter 6) with the balance being reared to adulthood in a 2

000 l tank, to be used as breeding stock later on.

4.7. Conclusions and recommendations

The water quality and physical environmental parameter ranges given in

Table 4.2 are to be seen as guideline values.

Table 4.2: Summary of recommended ranges of physi cal and chemical water quality parameters of the system water conducive to successful maintenance and breeding of P. reticulata.

Water quality parameter Recommended range

Water temperature 22-26 °C Total water hardness 3-30 °dH (± 50-500 mg/l CaCO3) pH 7.0-8.5 Salinity 0.5 g/l NaCl

Even these values are based on available literature as well as personal observations throughout the study, they are by no means representative of

CHAPTER 4 the parameter extremes that the fish can be maintained in. They were, however, the parameters that the fish seemed to be most productive in.

Further experimentation regarding this is necessary to conclude more accurate parameter extremes.

Poecilia reticulata has a biology that is amenable to laboratory conditions, however, their maintenance and breeding is not as simple as the readily offered anecdote of ‘just add water’ (Editor - Tropical Fish Hobbyist Magazine,

200416). This species of fish proved to be a reliable breeder, but were very susceptible to diseases created by ‘less than perfect’ water conditions, making the maintenance very laborious and time consuming. These are two aspects, which a laboratory doing routine toxicity tests commercially tries to minimise due to economic constraints. The literature cites larger females of this species of fish as being capable of producing up to 250 young at one time. This, however, refers to females that are raised in ponds outdoors, not laboratory stock raised in aquariums. The females raised within the laboratory gave birth to a maximum of 100 young at any one time – making the fecundity of laboratory-raised guppies relatively low. This aspect makes it necessary to maintain a relatively large stock of breeding fish to produce the large amounts of juveniles typically required by a toxicity-testing laboratory.

This species of fish is therefore only recommended as a suitable toxicity testing species if the laboratory has ample space for the culturing of the fish, as well as staff with the expertise of dealing with a fish species that is

16 ‘Letters to the editor’ - Tropical Fish Hobbyist Magazine, June, 2004.

CHAPTER 4 relatively susceptible to diseases as well as the high degree of maintenance required by this species.

CHAPTER 4

4.8. References

Axelrod, H.R. (1995). Breeding aquarium fishes – a complete

introduction. T.F.H. Publications, New Jersey. 128 p.

Axelrod, H.R. and Schultz, L.P. (1990). Handbook of tropical aquarium

fishes. T.F.H. Publications, New Jersey. 718 p.

Chiyokubo, T., Shikano, T., Nakajima, M. and Fujio, Y. (1998). Genetic

features of salinity tolerance in wild and domestic guppies (Poecilia

reticulata). Aquaculture 167: 339-348.

Coffey, D. (1986). The encyclopaedia of aquarium fish. The Rainbird

Publishing Group. 224 p.

DWAF. (1992). Toxicity assessment using Poecilia reticulata (Guppy). In

Department of Water Affairs and Forestry - Analytical methods

manual, TR151. Department of Water Affairs and Forestry, Pretoria,

South Africa.

Fishbase. (2004). Froese, R. and Pauly, D. (Editors). World Wide Web

electronic publication. www.fishbase.org, version (06/2004).

Hemdal, J.F. (2003). Aquarium fish breeding. Barron’s Educational Series,

New York. 169 p.

CHAPTER 4

International Organization for Standardization (ISO). (1996). Water Quality –

Determination of the acute lethal toxicity of substances to a

freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei,

Cyprinidae)] – Part 1: Static method. ISO Report 7346-1 Second

edition, International Organization for Standardization, Switzerland.

Khangarot, B.S. and Ray, P.K. (1990). Acute toxicity and toxic interaction of

chromium and nickel to common guppy Poecilia reticulata (Peters).

Bulletin of Environmental Contamination and Toxicology 44:832-

839.

Organization for Economic Cooperation and Development (OECD). (1992).

OECD guidelines for testing of chemicals. Fish, acute toxicity test.

Guideline 203. OECD

Petrovický, I. (1998). Aquarium fish of the world. Caxton Editions, London.

303 p.

Sandford, G. (2003). Aquarium owner’s manual. Dorling Kindersley

Limited, London. 266 p.

Skelton, P. (2001). A complete guide to the freshwater fishes of southern

Africa. Struik Publishers (Pty) Ltd. 395 p.

Slabbert, J.L., Oosthuizen, J., Venter, E.A., Hill, E., du Preez, M. and

Pretorius, P.J. (1998). Development of guidelines for toxicity

bioassaying of drinking and environmental waters in South Africa.

Report to the Water Research Commission, Project No. 358/1/98.

Division of Water Environment and Forestry Technology, CSIR.

CHAPTER 4

ASSESSMENT OF INTERNATIONAL PRACTICES ON THE USE OF FISH IN TOXICITY TESTING AND ADAPTATIONS FOR SOUTH AFRICAN CONDITIONS

CHAPTER 5

MAINTENANCE AND BREEDING OF D. RERIO (CYPRINIDAE) TO DETERMINE ITS SUITABILITY FOR USE IN ROUTINE LABORATORY TOXICITY TESTS

CHAPTER 5

5.1. Introduction

5.1.1. Aims and objectives

Danio rerio is the species of fish favoured overall by the international toxicity testing fraternity due to its amenability to laboratory conditions (Westerfield,

2002; ISO, 1996). This chapter therefore aims to test this amenability to conditions found within a ‘typical’ South African laboratory and to provide a comprehensive laboratory manual to the maintenance and breeding of this fish species. This laboratory manual was compiled after a literature review was conducted on D. rerio, with the concepts and expectations introduced in the literature being tested for applicability to conditions commonly encountered in South African laboratories. Results from these concept and expectation reviews that were tested within the laboratory are given, and recommendations are made as to which methods are most applicable to

South African laboratories. Final recommendations are then also made ascertaining the suitability of D. rerio as a routine toxicity testing species of fish.

5.1.2. Natural history

The classification of D. rerio is as follows (Axelrod & Schultz, 1990 and

Fishbase, 2004):

Phylum: Chordata Subphylum: Craniata Superclass: Gnathostomata Class: Osteichthyes Subclass: Actinopterygii Superorder: Teleostei Order: Cypriniformes Suborder: Cyprinoidea

CHAPTER 5

Family: Cyprinidae Genus: Danio (Hamilton-Buchanan) Species: rerio (Hamilton-Buchanan, 1822)

From the literature, it is however evident that there are several known synonyms for D. rerio (Table 5.1).

Table 5.1: Synonyms for D. rerio, their status and present validity (adapted from Fishbase, 2004).

Name Author Status Valid

Perilampus striatus McClelland, 1839 Junior synonym No Cyprinus chapalio Hamilton-Buchanan, 1822 Junior synonym No Cyprinus rerio Hamilton-Buchanan, 1822 New combination No Brachydanio rerio Hamilton-Buchanan, 1822 New combination Yes Danio lineatus Day, 1868 Junior synonym No Danio rerio Hamilton-Buchanan, 1822 New combination Yes

Danio rerio is a tropical, vigorous swimming cypriniform representative of the family Cyprinidae (Laale, 1977). Its maximum size is 60 mm (total length), but rarely exceeds 45 mm in length (Laale, 1977; ISO, 1996). It has a slim, compressed cylindrical body with seven to nine dark blue horizontal stripes on a silver body. These stripes run from the operculum to into the caudal and anal fins (Figure 5.1). The colour can, however, vary with the aquarium background and location (Laale, 1977) with more pronounced patterning becoming evident in a darker environment. Two pairs of small barbules are present on the lower jaw (Coffey, 1986; ISO, 1996). There is no lateral line present (Laale, 1977). Males are slimmer than females and possess a golden sheen. Females are more silvery and the abdomen is distended, particularly prior to spawning (ISO, 1996) (Figure 5.2).

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Figure 5.1: Danio rerio adult male.

Besides differences in body form, female D. rerio have blue stripes on the anal fin with silver stripes interspersed. In the male, the lighter stripes are gold (Axelrod & Vorderwinkler, 1978).

Figure 5.2: A group of adult male D. rerio (left) showing the obvious golden sheen. The females (right) are more silver in colour, with distended abdomens – particularly prior to spawning.

Danio rerio naturally inhabits fast flowing streams from Bengal to the

Coromandel Coast of India (Axelrod & Schultz, 1990; Axelrod, 1995), but also occurs in slower streams, canals, ditches and ponds (Rahman, 1989). They also inhabit slow moving to stagnant standing water bodies, particularly rice-

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CHAPTER 5 fields (Talwar & Jhingran, 1991), where they naturally feed on worms, small crustaceans (Mills & Vevers, 1989) as well as insect larvae (Shrestha, 1990).

Danio rerio are typical egg-laying r-strategists (Schäfers et al., 1993). This means that they are egg scatterers (Axelrod & Vorderwinkler, 1978) and have a relatively high rate of reproduction with low investment of energy in the individual offspring. Under natural conditions, spawning activity is cued by environmental factors such as increasing day lengths, water temperature, and food sources typical of the onset of spring. Due to this, female D. rerio begin to develop distended abdomens caused by the development of eggs. After a period of total darkness (night), the initial appearance of light, and the persistent rubbing of the female by the male, induces the female to spawn.

After inducing the female to spawn through tactile behaviour, the males promptly fertilise the eggs. It is important to note that adults will readily consume their own fertilised eggs before they fall out of reach (Axelrod, 1995;

Astrofsk? et al., 2002). The eggs then fall freely through the water column, where they undergo developmental stages. Larvae then begin to hatch, under optimal conditions, on day three or four post-fertilisation. This development period may be longer, depending on the temperature of the ambient water. After hatching, the larvae adhere motionless to a substrate, where they undergo further development. By day four or five post-fertilisation, the larvae are free-swimming, and now actively search for food, which consists of plankton and other microscopic organisms. Growth is then rapid, with juvenile fish becoming sexually mature within approximately two to four months, depending on density of the fish and the rate of feeding (Astrofsk? et al., 2002). The eggs are laid in large numbers within a period of 5 to 45 days

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CHAPTER 5 following the previous oviposition, with the optimum being from 5 to 10 days

(Laale, 1977). The maximum number of eggs obtained from a single gravid female may be as high as 1,500 to 1,800. The number laid generally by one female varies between 150 and 400 eggs per spawning (Laale, 1977).

5.1.3. Background on captive breeding and use of D. rerio

Danio rerio are very popular as an aquarium fish (Arunachalam et al., 2000), with selective breeding producing veil-tail as well as long-fin varieties

(Astrofsk? et al., 2002). Danio rerio are, based on worldwide sales, amongst the top ten most popular and available freshwater fish species, as they are amongst the easiest fish to keep in the aquarium (Ford, 1981; Axelrod, 1995) and must be one of the most well loved of tropical fish (Coffey, 1986) due to their active swimming behaviour and vibrant nature. Next to the guppy, this species is the best seller in fish of this class amongst aquarium enthusiasts, due to it being inexpensive, hardy, and easy to breed (Axelrod & Schultz,

1990). Danio rerio are also often the first egg laying fish species to be bred by the budding aquarist (Axelrod & Vorderwinkler, 1978). They are a sociable and peaceful fish by nature (Axelrod & Schultz, 1990). Danio rerio are also not susceptible to stress induced by a high stocking density. Low stocking densities, in fact, tend to induce aggressive tendencies within a population of

D. rerio (Brand et al., 1995).

This species of fish have been extensively studied since the 1930’s (Laale,

1977) and are becoming increasingly popular as a model system (=organism) for developmental biology (Valdesalice & Cellerino, 2003) with extensive

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CHAPTER 5 studies already done on various aspects of its biology. It was introduced as early as 1965 as a model for carcinogen studies at the National Cancer

Institute (Stanton, 1965). In the early 1970’s, D. rerio was identified as a vertebrate model to isolate mutations in genetic screens using systematic mutagenesis protocols (Astrofsk? et al., 2002; Hemdal, 2003). More recently, it has also become an important model system for the study of developmental biology (Meyer et al., 1993) and biological problems relating to human disease, including development and organogenesis (Zon, 1999; Barbazuk et al., 2000; Barut et al., 2000; Beckwith et al., 2000; Dodd et al., 2000; Neely et al., 2002). Furthermore, fish eggs and larvae have been employed extensively in toxicity tests as indicators of pollution, and the effects of environmental agents on specific developmental events having been studied

(Laale, 1977). The vast scope of studies encompassing so many aspects of the D. rerio’s life history has seen it become the ‘white rat’ of fish species.

The International Organisation for Standardisation (ISO) issued a document as early as 1976, detailing proposals for screening chemicals and commercial products for their acute toxicity to freshwater fish. The test species recommended was Brachydanio (=Danio) rerio (Ford, 1981). A literature review on the use of D. rerio in fisheries research by Laale (1977) collated the findings of 450 publications, showing the immense scope of literature available on D. rerio. This fish species is currently being used both as a sentinel species to screen for compounds with toxic effects and as a model organism for the in-depth analysis of the effects of particular compounds, such as dioxin. The eggs and early embryos of D. rerio can be used to

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CHAPTER 5 determine both mortality rates and frequencies of abnormalities in developing embryos. The exposure of adult D. rerio to toxicants is also used as a model for detection of tumorigenic compounds. Cell lines have been established from various D. rerio tissues and used in analysis of cellular responses to toxins (Astrofsk? et al., 2002). Other investigations have dealt with growth rates and mortality, feeding and distribution, as well as morphological adaptations in polluted test systems.

Danio rerio is suitable for such studies as it is easily obtainable, inexpensive, readily maintained and cared for, and under appropriate conditions, will provide large numbers of emersible, non-adherent and transparent eggs

(Laale, 1977) and for this reason, it is one of the most common test fish in

Europe today (Schäfers et al., 1993). Furthermore, a fish suitable for the test should be easy to keep and to handle in the laboratory, should grow rapidly, show early sexual maturity, deliver gametes throughout the year, and respond to a wide range of toxicant concentrations. Danio rerio has been found to meet these requirements (Bresch, 1993). This species has been shown to produce a large number of embryos on demand, and this is an aspect that makes them favourable for use for laboratory research, as minimal space is required for breeding and maintenance (Goolish et al., 1998; Zon, 1999).

Twelve-month old D. rerio females spawned on average every 1.9 days

(Laale, 1977), with sexual maturity potentially being reached as early as 74-75 days old (Eaton & Farley, 1974a; Nagel, 1993). They are also a hardy species of fish, not generally succumbing to diseases due to them generally not being very susceptible to handling stresses, as well as the stresses

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CHAPTER 5 induced by the inevitable movement of laboratory staff performing routine maintenance throughout the fish culturing facility. This aspect makes them relatively easy to maintain and to work with.

Danio rerio are comparable to other species of fish internationally accepted for toxicity testing in terms of sensitivity to various toxins and environmental pollutants (Nagel, 1993). For example, the acute toxicity of 3,4-dichloroaniline in adults and juveniles is similar for both P. reticulata and D. rerio. The 96 h

LC50 values are 8.5 mg/l in D. rerio and 8.7 – 9.0 mg/l in P. reticulata. The

LC50-values for feeding larval fish were shown to be at 8.4 mg/l for 17-day-old

D. rerio and 9.1 mg/l for newborn guppies (Schäfers et al., 1993). The acute toxicity (96 h) of potassium dichromate in synthetic soft water was in the range of 84.4-117.3 mg/l for D. rerio (Sissino et al., 2000). This fish species has also proven to be a suitable test species for aspects of chronic toxicity testing on fish since complete life-cycle tests can only be carried out within a reasonable period of time with small, rapidly growing warm water fish. Danio rerio is such a fish (Nagel, 1993).

Future research involving toxicity testing and toxicant effects on D. rerio involve the combination of toxicology and genetics, which should permit the isolation of mutations that compensate for defects induced by toxins. This will allow for a better understanding of the toxic mechanisms of action (Astrofsk? et al., 2002).

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CHAPTER 5

5.1.4. Important documentation

Due to the popularity of the use of D. rerio in laboratory studies, there is a vast amount of literature available in the form of various scientific publications, extensive web sites, as well as ISO and OECD guidelines (ISO, 1996; OECD,

1992). The following literature is a guide to some of the more important publications to date, covering different aspects of the D. rerio’s biology.

1. Westerfield, M. (2002). The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), 4th edition. University of Oregon Press, Eugene, OR, USA.

This document is a publication by the University of Oregon describing the husbandry of D. rerio within the laboratory. It is a web-based document with various hyperlinks to other documents that is also available in a printed version It can be downloaded from the following web address: http://zfin.org/zf_info/zfbook/zfbk.html. This is a fully comprehensive guide for laboratories doing routine work on the D. rerio from a developmental studies perspective, covering a very broad spectrum of topics relating to the use of D. rerio in various laboratory procedures. Various links to laboratories and scientists doing routine work on D. rerio are also available here. It contains much useful information, but it is technically presented, aiming at laboratory technicians already familiar with fish husbandry. It therefore has limited application to smaller laboratories requiring only a regular supply of embryos for routine work.

2. Astrofsk?, K.M., Bullis, R.A. and Sagerström, C.G. (2002). Biology and management of the zebrafish. In Laboratory animal medicine, 2nd edition. Fox, J., Anderson, L., Loew, F. and Quimby, F. (Eds). Elsevier Science, USA. pp 862-883.

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This chapter of the book Laboratory animal medicine describes the background, husbandry and diseases of D. rerio. It is a comprehensive overview on the husbandry of fish in general with specific emphasis on D. rerio. It is a useful document to a smaller laboratory with laboratory technicians that have limited knowledge in the field of aquarium maintenance and fish husbandry. Some of the commonly encountered diseases affecting

D. rerio are also described. A very useful document that is easy to follow and understand.

3. Eaton, R.C. and Farley, R.D. (1974a). Spawning cycle and egg production of zebrafish, Brachydanio rerio, in the laboratory. Copeia 1:195-204.

This document describes fecundity and spawning procedures of D. rerio. All technical aspects as well as statistical analyses of fecundities are described, making the usefulness of this document to smaller laboratories only interested in producing a relatively small number of embryos, very limited.

4. Laale, H.W. (1977). The biology and use of zebrafish, Brachydanio rerio in fisheries research. A literature review. Journal of Fisheries Biology 10: 121-173.

This is a comprehensive literature review of the biology of D. rerio under laboratory conditions. A vast amount of background information on the use of

D. rerio within laboratories is given, making this document important to gain a useful perspective to the scope of the use of D. rerio in research.

5. International Organization for Standardization. (1996). Water Quality – Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] – Part 1: Static method. ISO Report 7346-1 Second edition, International Organization for Standardization, Switzerland.

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This is set of guidelines set by the ISO for toxicity testing aimed mainly at the

European toxicity testing community. The husbandry section to this document is an overview of the procedures to follow; however, the finer points of the husbandry of D. rerio are not elaborated on. This document is therefore aimed at laboratories that are already efficient is fish maintenance, and its application to smaller laboratories with inexperienced staff is therefore limited.

5.2. Environmental requirements and procedures for maintenance

of D. rerio

5.2.1. Water temperature

Temperature is critically important in the survival, development, growth, and successful reproduction of fish and other organisms in biological systems

(Hawkins & Anthony, 1981; Astrofsk? et al., 2002). When the water temperature is abruptly raised or lowered, fish show an internal shock reaction

(Astrofsk? et al., 2002), with temperature being perhaps the most potent of all environmental factors controlling and governing the metabolism of aquatic animals (Hawkins & Anthony, 1981). The magnitude of this effect depends on the strain, its recent thermal history, and the magnitude of the thermal change. As a rule, a change in temperature should be limited to ±1.5 °C /day

(Astrofsk? et al., 2002).

Danio rerio are capable of withstanding temperature ranges of 15.5 °C to 43.3

°C (Axelrod, 1967). The optimal temperature for D. rerio is 25 °C to 29 °C, with the higher temperatures (28.5 ± 1 °C) recommended when stimulating

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CHAPTER 5 egg laying/reproductive behaviour, and to facilitate development of fertilised embryos (Astrofsk? et al., 2002; Westerfield, 2002). Keeping fish outside of this temperature range is not recommended as growth and development is impaired (Westerfield, 2002). Reducing the temperature of the incubation water of embryos will lengthen their developmental period, thereby increasing time taken for hatching.

A change in temperature affects the degree of tolerance of the fish to other factors, such as fluctuating pH, conductivity, dissolved oxygen levels, and other water chemistry parameters. Increases in temperature (within the tolerance range) have the most distressing effect on fish. It speeds up metabolism of the fish (therefore excitability and movement of the fish) and increases oxygen demand, at the same time decreasing the oxygen carrying capacity of the water (Astrofsk? et al., 2002 and Westerfield, 2002). An increase in temperature also increases the amount of un-ionised ammonia

(NH3) within a system (Lloyd, 1981). This can become detrimental to the fish if the water is inadequately filtered. Larvae are usually less tolerant to temperature changes than their respective adult forms. The limits of temperature tolerance are highly variable among populations and between seasons (Astrofsk? et al., 2002).

Temperature change (usually temperature increases) is often a factor in the initiation of reproductive activity (Astrofsk? et al., 2002). If necessary, D. rerio can be conditioned at 26 ± 1 °C with the temperature then being increased to

28 ± 1 °C to induce spawning. This, however, is very seldom necessary due

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CHAPTER 5 to the large number of embryos normally produced by brood stock in good condition.

Temperature is maintained by heating of the environment such as an insulated, heated room (environmentally controlled room) or the use of submersible aquarium heaters. The volume of water to be heated and the difference between both ambient and water temperatures needs to be taken into consideration when choosing the wattage of a submersible heater (refer to manufacture’s specifications for this information). It is advisable to make use of heaters with thermostatic controls that can be easily set, without the removal of the heating unit from the glass tube being necessary.

5.2.2. Water chemistry

5.2.2.1. pH

Danio rerio can tolerate a pH range of 6.6–8.2 (ISO, 1996) but the preferred pH range being 6.8–7.2 with pH 7.0 being optimal (Astrofsk? et al., 2002;

Sandford, 2003). Acidity or alkalinity extremes can cause direct physical damage to skin, gills and eyes of fish, whereas prolonged exposure to sub- lethal pH levels can cause stress, increase mucous production and encourage epithelial hyperplasia with sometimes-fatal consequences to the fish, as this hinders gaseous exchange between the gill lamellae and surrounding water.

Fish also have to maintain their own internal pH levels, with even minor shifts in blood pH levels within the body of the fish being potentially fatal. Extreme external or water pH can influence and affect blood pH of fish, resulting in

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CHAPTER 5 either acidosis of alkalosis of the blood (Fishdoc, 2004). The pH of an aquarium system will also influence the proportion of toxic non-ionised ammonia in an ammonia solution. As the pH of the water becomes more alkaline (>8.0), the non-ionised ammonia (NH3) increases (Lloyd, 1981) which is toxic to the fish. A low pH (<5.0) inhibits the activity of nitrifying bacteria, which tends to increase total ammonia levels due to accumulation, and a pH value this low will probably kill the fish (Table 5.2). In closed, recirculating systems (typical of an aquarium), the pH will gradually decrease due to the production of acids during the nitrification process as the bacteria within the bio filter convert ammonia to nitrate.

Table 5.2: The correlation between an increase in pH of a system and the subsequent increase of toxic NH3 at 26 °C (adapted from Wilkerson, 2001). pH % ionised (non-toxic) % free (toxic) ammonia – + ammonia NH3 (ammonium – NH4 )

7.5 98.7 1.3 7.8 96.2 3.8 7.9 95.3 4.7 8.0 94.1 5.9 8.1 92.7 7.3 8.2 91.1 8.9 8.3 89.0 11.0 8.4 86.5 13.5

Another process affecting pH of a system is the denitrifying process itself.

This is due to the fact that for every milligram of NH3 that is consumed by the denitrifying bacteria within a system, 8.64 mg of alkalinity, in the form of

? hydrogen carbonate (HCO3 ), is consumed, affecting the pH stability of a

? closed system over time due to the pH buffering capacity of the HCO3 being slowly lost over time (Fishdoc, 2004). This, together with the pH decreasing

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CHAPTER 5 in poorly aerated systems due to the production and accumulation of carbon dioxide (CO2) created by respiring fish and aerobic bacteria, ultimately leading to increasingly acidic conditions. The presence of photosynthesising plants and algae within the system also causes the pH of a system to fluctuate. The photosynthetic process absorbs CO2 from the system in the presence of light, thereby increasing the pH of the system during the day (or light cycle), and these same plants and algae release CO2 in the absence of light when normal respiration continues during the night (or dark cycle). This pH fluctuation is

? relatively minimal in properly buffered water, but as the HCO3 is continually being removed from the water during the denitrifying process with the system losing its buffering potential, the influence of photosynthesis on pH fluctuations within the system become increasingly more severe, which in turn, becomes increasingly more detrimental to the fish within the system.

Fish can acclimate to a differing pH values over an extended period of time

(as long as the pH falls within the limits of the pH tolerances of that particular fish species) but continual fluctuations of pH, however, are likely to be stressful and harmful to the fish (Fishdoc, 2004). The pH of the water has a direct influence on various toxicants in a system, which in turn, affects the fish. As the pH of a system becomes more acidic, common metallic contaminants such as zinc, copper, iron, and aluminium become more soluble in water under acidic conditions, where they can be taken up and metabolised by the fish. Therefore, toxicity associated with exposure to these elements is more common in water systems maintained at a lower pH value (Astrofsk? et al., 2002). Testing the pH value of the system water should be undertaken on

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CHAPTER 5 a weakly basis, more frequently if the system has been disturbed or medicated (Fishdoc, 2004).

5.2.2.2. Conductivity

Conductivity is an indicator of the total amount of dissolved ions in a solution that includes sodium and other ionised minerals. It is a direct measure of the amount of electric current that a particular aqueous solution can conduct.

Since direct measurement of salinity is not easily measured, conductivity is a convenient method to imprecisely measure the salinity of the water system and allows monitoring of changes in salinity due to water changes or evaporative loss. Danio rerio generally have a tolerance to a wide range of conductivity values (3–500 µs/cm) wherein they will grow and breed optimally

(Astrofsk? et al., 2002).

5.2.2.3. Total water hardness

The amount of calcium and magnesium salts in the water is referred to as the water hardness. Other cations also contribute to the total hardness of the water, but these are usually present only in very small quantities within normal fresh water. Commercially available test kits tend to measure hardness in terms of how much calcium carbonate (CaCO3) is present in the water. Water quality reports usually express hardness levels in terms of parts per million

(ppm) or milligrams per litre (mg/l) of calcium carbonate (CaCO3) (Astrofsk? et al., 2002). Another common measurement of total water hardness is German hardness measured as °dH. Table 5.3 shows different degrees of water

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CHAPTER 5 hardness, and how the different measurement values compare to one another.

Table 5.3: Typical water hardness ranges measured in both mg/l CaCO3 as well as °dH (adapted from Sandford, 2003).

Water type CaCO3 (mg/l)* °dH*

Soft 0-50 3° Moderately soft 50-100 3°-6° Slightly hard 100-200 6°-12° Moderately hard 200-300 12°-18° Hard 300-45 18°-25° Very hard >450 >25°

(* To convert °dH to mg/l CaCO3, multiply by 17.9 (Fishdoc, 2004).

Danio rerio are generally considered to be a “hard” water species with optimum calcium and magnesium levels between 80 and 200 mg/l (4–12 °dH)

(Astrofsk? et al., 2002) but will tolerate, with no detrimental effects, water hardness as much as 300 mg/l CaCO3 (18 °dH) (ISO, 1996) that is the tolerance range of most fish. Very soft water (0–10 mg/l) can be detrimental to young developing larvae since they rely on the water for essential mineral uptake during the early, growing phases of life. Low water hardness or calcium levels have also been found to be associated with low embryo survival rates and increased susceptibility to other environmentally induced disease because of poor water quality. The water hardness affects fish health because it influences osmoregulation. As hard water is more concentrated in salts than soft water, there is more of an osmotic balance between the body fluids of the fish and its surrounding water. This means that the fish will not have to work as hard at osmoregulation. This is particularly important when fish are stressed by bacterial infections and other conditions that disrupt

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CHAPTER 5 cellular integrity. Conditions such as these often lead to cells and tissues of the fish not being able to maintain osmoregulation, and the cells flood with water causing abnormalities and, often irreversible, damage to the tissues

(Fishdoc, 2004).

? As the nitrification process continually removes HCO3 ions from the system, thereby reducing the pH and the buffering capacity of the water, total water hardness needs to be monitored at least on a monthly basis. If the general hardness is found to fall below the optimum values, a carbonate buffer can be added to the water.

5.2.2.4. Reconstituted water

If there is any doubt regarding the quality of tap water, then reconstituted water should be used. Reconstituted water is made up with distilled or deionised water with a pH of 7.8 ± 0.2 and a calcium hardness of approximately 250 mg/l (expressed as calcium carbonate) in distilled or deionised water (ISO, 1996):

294.0 mg/l CaCl2.2H2O

123.3 mg/l MgSO4.7H2O

63.0 mg/l NaHCO3 5.5 mg/l KCl

5.2.3. Photoperiod

Zebra fish spawning activity is controlled by photoperiodism. The optimal light/dark cycle being 14:10 h, respectively. Spawning activity is induced by

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CHAPTER 5 the onset of the light cycle, with spawning typically being complete within two hours of the lights having come on.

5.2.4. Aquaria design

5.2.4.1. Aquaria

Fish can be maintained in glass aquaria with dimensions suited to the number of fish required. Aquarium sizes of 20 l or more are suitable when breeding the fish as well as for housing the early developmental stages of the young fish. Aquaria of 50 l however, are recommended with dimensions (L x B x H) of 500 mm x 500 mm x 200 mm. The large number of embryos resulting from the spawned fish is normally high enough to warrant the use of the larger volume of water. If smaller aquarium sizes are to be used, care must be taken to thin out the embryos, before they hatch, to avoid overcrowding when the larvae hatch. When the fish are approximately 10 mm long (± four weeks), they should be thinned out enough to a stocking density not exceeding two or three fish per litre of water to grow to adulthood. This will ensure no stunting of growth due to overcrowded conditions within the tank

(see also section 5.5 Embryo and juvenile care). As fish grow, it is recommended that they be housed in larger aquaria of 100 l or more to ensure optimum growth. Refer also to Table 5.4 for a summary of water chemistry parameters and physical requirements for the maintenance of D. rerio.

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5.2.4.2. Stocking densities of fish in aquaria

When stocking a tank, the most critical factor is surface area, rather than the total volume of water. The aquarium depth is irrelevant; it is the water/air interface at the surface that determines the amount of dissolved oxygen needed to support life. For example: a 160 l tank, if 100 cm x 40 cm x 40 cm

(L x B x H) will have a surface area of 4000 cm2; alternatively, it may be 75 cm x 40 cm x 52 cm (L x B x H) with a surface area of 3000 cm2. Although both tanks hold the same amount of water, the one with the larger surface area will support more fish. To find the correct stocking level, first calculate the surface area by multiplying the tank width by its length. Then establish the adult length (excluding the caudal fin) of the fish that are to be maintained in the tank, then for each 2.5 cm of fish, 75 cm2 of tank space is needed (Sandford,

2003). An aquarium with dimensions of 50 cm (length) x 50 cm (width) x 40 cm (height) has a water volume of 100 l, and a surface area of 2500 cm2. As adult D. rerio attain an average length of 2.5 cm, it means that each adult fish should have at least 75 cm2 of surface area to accommodate it. Therefore, dividing 2 500 by 75, gives the number of fish that can safely be accommodated in an aquarium with these dimensions as approximately 33.

This is not a hard, fast rule, and densities of fish can be slightly increased with increased aeration and filtration to remove the added ammonia and other waste product build ups, as well as providing the water with increased oxygen

(see also section 5.2.4.4. - Filtration).

When stocking an aquarium with fish, the stocking density also largely depends on the nature of the fish. High stocking densities, for the only reason

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CHAPTER 5 that they are not shoaling fish by nature, stress many fish. This is true for many popular aquarium species (Sandford, 2003). Other species may be excessively stressed by not being kept in a school of the same species of fish due to their natural schooling behaviour. Therefore, the stocking density of a particular fish species within a system should not only be determined by the filtration, oxygen-carrying capacity of the water and size of the aquarium.

Relatively small, shallow aquaria may be used for D. rerio, as high stocking densities do not excessively affect them in terms of inducing stress or aggressive behaviour (Brand et al., 1995).

It is important to note that these calculations are for aerated, filtered systems.

They are not intended for non-aerated systems. Non-aerated systems with no filtration are not recommended to house fish.

5.2.4.3. Substrate

No substrate on the bottom of the tanks is required, or recommended. This aids in the routine cleaning and maintenance of the tanks. Danio rerio also do not require a substrate to induce or aid in spawning activity. Refer also to

Table 5.4 for a summary of water chemistry parameters and physical requirements for the maintenance of D. rerio.

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Table 5.4: Summary of water chemistry parameters and physical requirements for the maintenance of D. rerio.

Parameter Measurement

Temperature. 24 – 28.5°C. pH. 6.8 – 7.2. Conductivity. 3 – 500 µs/cm. Total water hardness. 80 – 200 mg/l CaCO3 (4 – 12°dH). Photoperiod. 14:10 (light: dark cycle). Stocking density of fish. Adult: 75 cm2 (surface area) per fish. Young: 2 – 3 fish /l. Substrate. None.

5.2.4.4. Filtration

The most pronounced and damaging changes to water quality originate within the aquarium inhabitants themselves. In particular, water quality is impaired by the end products of nitrogen metabolism. These include ammonia (either

+ as the gas NH3 or as ammonium ions NH4 ) urea, uric acid and other nitrogenous substances including proteins and amino acids. Ammonia, especially, is one of the most harmful substances to aquatic life, having a variety of detrimental effects (Hawkins & Anthony, 1981). These toxic nitrogenous compounds are metabolised by bacteria such as Nitrosomonas sp., Nitrobacter sp. and Nitrocystis sp., which normally reside in filter mediums within the working filter systems of an aquarium (Wickins & Helm, 1981). The process of bacteria converting toxic NH3 stepwise to less harmful nitrate is known as nitrification (Fishdoc, 2004). Nitrosomonas sp. converts the toxic ammonia into less dangerous nitrites, and then Nitrobacter sp. and Nitrocystis sp convert the nitrites into less dangerous nitrates. The nitrates are then taken up and metabolised by aquarium plants. If insufficient or no plants are in the aquarium, these nitrates need to be removed by doing routine partial

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CHAPTER 5 water changes of the aquarium water with fresh water (see also section

5.2.4.5.4. - Maintenance of filters).

A further explanation of this process is given in Figure 5.3, where the diagram represents the natural cycles of the bacteria colonising a typical new biological filter, and how the presence or absence of certain bacteria affect

NH3 and NO2 levels within the system. As a new system is set up, denitrifying bacteria are absent from the biological filter, but as the system ages, toxic

NH3 produced by tank inhabitants triggers the colonisation of Nitrosomonas sp. which breaks down the NH3 and converts it to NO2. This NO2, however, is also toxic to fish, but another bacteria (Nitrobacter sp.) then colonises the

- system to break the NO2 down, converting it to NO3 , which is less harmful to fish. As can be seen from the diagram, there is a latent period within a new system for the colonisation of the specific bacterial colonies, so there are periods where both the NH3 and the NO2 concentrations rise to dangerous

- levels. The NO3 is then finally removed from the system by live plants or by periodic water changes. This scenario is not only true for a new system, but also for a system that has its biological filter cleaned by a way that is detrimental to the bacterial colonies within the filter media. Only until the bacteria are able recolonise the filter again, will NH3 and NO2 removal be effective once more (Wilkerson, 2001).

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12 NO2 concentration,

removed by 10 - NO3 concentration,

NH3 6 concentration,

Level (mg/L) removed by 4

0 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 Time (days)

Figure 5.3: Typical scenario of nitrogen cycling within a new aquarium system (adapted from Wilkerson, 2001).

The smaller tanks that are used for spawning and for housing the very young fish should be fitted with air-driven sponge filters (Figure 5.4). These filters only release the bubbles into the water column on the surface of the water thereby being a relatively passive, yet effective form of filtration, suitable for very young fish that would otherwise be possibly harmed by large rising bubbles. The double sponge filter, as opposed to the single sponge version, is recommended to facilitate maximum filtration. The sponges do, however, need to be rinsed out periodically as needed and replaced when necessary.

As the sponges act as biological filters, it is recommended that they be rinsed in clean tank water of the same temperature. Do not use fresh tap water to rinse the sponges, as the chlorine within it and the possible temperature difference between the tank water and the tap water will adversely affect the denitrifying bacteria colonised within the filter sponge. This will (temporarily)

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CHAPTER 5 reduce the capability of the filter to remove ammonia and other wastes from the tank water. It must be remembered that sponge filters serve as biological filters only, so fish faeces and leftover food will need to be siphoned from the bottom of the tank at least every second day.

Air riser tube (outlet).

Air inlet.

Open-celled filter sponges.

Figure 5.4: A double sponge air-driven filter (‘Oxy Plus Bio Filter II’). It is useful for filtering aquarium water containing embryos and very young fish.

Making use of air-driven, box-type, corner-filters can adequately filter the smaller tanks housing fish, as they grow bigger (Figure 5.5). These filters should contain filter floss and activated carbon as mechanical/biological and chemical filter mediums, respectively. A few stones placed at the bottom within the box filter will keep the filter from floating to the surface of the water, and the stones will act as a “reservoir” for the denitrifying bacteria. This is

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CHAPTER 5 essential when the filter floss is replaced, as bacteria that had colonised the stones before will quickly spread throughout the new filter media. Fish faeces and leftover food will also need to be siphoned from the bottom of the tank at least every second day. The disadvantage to using this type of filter is that smaller fish can become trapped within the filter, or damaged by the course rising bubbles.

Air inlet.

Filter floss.

Stone medium acting as a reservoir for denitrifying bacteria when the filter floss gets changed.

Figure 5.5: An air-driven box-type corner filter with filter media.

Larger tanks with a volume of in excess of 100 l will require more efficient filtration devices. This is achieved by making use of external hang on filters, such as ‘Aqua Clear’ hang-on filters or equivalent, or external canister filters, such as ‘Hydro Prime External Canister Filter’ or equivalent (Figure 5.6).

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These types of filters both actively pump water from the aquarium, force it through a filter medium (typically course sponge, zeolite or activated carbon) and then return the filtered water back to the tank. Manufacture’s directions must be followed when choosing a filter to suite the volume of an aquarium as well as the stocking density that will be maintained within that aquarium.

Water inlet.

Water outlet.

Sealed lid.

Filter medium.

Water pump.

Figure 5.6: An external canister filter. This filter works by siphoning water from the aquarium, forcing it through a filter medium, before pumping it back into the aquarium.

Alternatively, fish can be maintained in an adequately filtered flow-through system with the advantage that good water quality can be maintained without the time needed to give each individual tank the attention with regards to water changes, siphoning off debris and cleaning individual filters (Figure 5.7).

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A flow-through system has a water pump that supplies a series of individual tanks with water that is filtered through a common filter. This water moves through the outlets of the individual tanks into a drainpipe, which empties into the filter (Figure 5.8). This method, however, has the disadvantage of being unable to curb the spread of diseases from one tank to the next. For this reason, it is advisable not to house all of D. rerio stock as part of the same system thereby limiting the losses of stock due to the outbreak of a disease.

The drains of the individual tanks need to be fitted with a mesh that will stop smaller fish being sucked through the drain and landing up inside the filters

(Figure 5.9). These drains need to be fitted to the outlets of the tanks with an open-ended fitting to stop any siphoning action of the water out of the tanks if the pumps stop. The open-ended pipefitting is also necessary to avoid air locks within the system that would otherwise restrict water movement through the pipe.

Water inlets.

Water outlets.

Individual tanks.

Figure 5.7: A flow-through system with a series of tanks that all share a common filter.

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Location of water pump.

Second filter with shade cloth filter medium.

Return pipe from water pump back to the system.

First filter with shade cloth filter medium.

Inlet pipes collecting water from all the tanks to be filtered.

Figure 5.8: The common filter system shared by a series of 10 tanks that form part of a flow-through system.

Open-ended pipefitting connected to outlet.

Water level.

Mesh covering the holes of the outlet.

Figure 5.9: The outlets of all of the tanks in a flow-through system need to be fitted with a mesh to stop any fish from being sucked into the drain pipe and landing in the filter.

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5.2.4.5. Maintenance

Daily maintenance is important in any fish culturing facility to ensure proper working order of all equipment to minimise the risk of unnecessary fish deaths due to equipment failure. Observing the behaviour of fish on a daily basis is also useful for the early detection of diseases.

5.2.4.5.1. General

Fish need to be fed daily (see also section 5.3.2.2. - Food) and checked for signs of disease, such as unusual swimming behaviour and refusal of food, so that appropriate steps can be taken in the case of morbidity or mortality of fish. Any dead fish are to be removed and disposed of. Any nets or other equipment used should be sterilised on an ongoing basis. It is recommended that nets be kept in a 10 % formalin solution to ensure that diseases are not transferred between tanks. It is essential to rinse the nets thoroughly in clean water to remove all the formalin before using the net in an aquarium; otherwise, the formalin will have detrimental effects to the fish. This formalin solution should be changed regularly. Filters should also be checked on a daily basis to ensure that they are working correctly. Air blowers also need to be checked and kept in good working order. Electrical timer units controlling the light cycles within the facility need to be checked at least on a weekly basis. This is imperative due to the fact that spawning and reproductive activity is controlled primarily by the lighting regime in D. rerio, and an alteration in the photoperiod will decrease fecundity. Fish that have been exposed to a mistimed photoperiod can take some time to acclimate to ideal photoperiod conditions once more.

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5.2.4.5.2. Water changes

At least a 10% water change is required in all tanks weekly to ensure that nitrates and other undesired wastes are removed (see also section 5.2.4.5.4. -

Maintenance of filters). Water that is removed from the aquaria should be replaced with water of the same chemical properties and temperature as the tank water. If tap water is to be used, it needs to be dechlorinated by adding chlorine neutralisers (sodium thiosulphate) such as Tetra Aquasafe® or equivalent commercially available chlorine neutralising product. It is essential to follow manufacturer’s directions for use. Chlorine neutralisation can also be achieved by heavily aerating the fresh tap water in an open container for at least 48 hours prior to being used.

5.2.4.5.3. Maintenance of flow-through systems

Flow-through systems also require at least a 10 % water change at least once every two weeks to remove nitrates and other undesired wastes from the water. If need be, solid wastes are siphoned from the bottom of all the individual tanks. Inlets, as well as outlets, of all individual tanks need to be checked daily for blockages to ensure adequate water flow and waste removal of the system.

5.2.4.5.4. Maintenance of filters

Filters also need to be checked for adequate flow rates of water or air, and cleaned on a regular basis. Filter media needs to be replaced when necessary (see also section 5.2.4.4. - Filtration). Do not allow filter medium to accumulate too much decomposing organic matter. This is because

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CHAPTER 5 heterotrophic bacteria attracted to the filter by the accumulation of decomposing organic material, under conditions of low oxygen saturation of the water, will switch to nitrate reduction, with the consequence that nitrites are released back into the water. This, in turn, is detrimental to the well being of the fish (Fishdoc, 2004). It is important to realise that in order for the biological filtration to remain optimum, it requires a sustainable amount of nitrates. This means that too frequent water changes, thereby removing too much of the nitrates from the water, can be just as deleterious as not removing enough. If too much of the nitrate content is removed, the denitrifying bacteria will not be able to sustain the optimal level of ammonia removal from the system. This situation must be avoided in the aquarium, as it is potentially harmful to the fish (see also section 5.2.4.5.2. - Water changes). Temperature shocks brought on by water changes with water of a different temperature than the tank water is also harmful to the denitrifying bacteria within the filters and care should be taken to avoid this.

5.2.4.5.5. Periodic sterilisation

It is recommended that tanks are emptied, scrubbed using a nylon pot scourer

(e.g. Scotchbrite®) and sterilised regularly to further reduce the risk of the spread of disease. Mixing a 10 % bleach (sodium hypochlorite) solution and leaving it in the tank for 24 hours is a suitable method of sterilisation.

Alternatively, tanks can be sterilised with a solution of 10 % formalin (40 % formaldehyde solution) and left overnight. After sterilisation, tanks need to be thoroughly rinsed out with clean water to minimize the danger of toxic residues remaining behind that would otherwise be detrimental to the fish.

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Table 5.5 is a guideline time schedule for routine maintenance of aquaria and aquarium systems.

Table 5.5: Recommended routine maintenance schedule.

Activity Application Time period

Feed live food* as well as flake Spawning groups. 3-4 times daily. food. Check outlets of tanks in flow- through systems. All flow-through systems.

Feed all fish. All fish. Twice daily.

Observe fish for any signs All fish/systems. Daily. of disease.

Siphon solid wastes from bottom All systems. Every second day. of tanks. Check heaters, water pumps All systems. and blowers for working order.

Do partial water changes. All systems. Weekly. Check pH and NH3 content of water. All systems.

Clean filter mediums. All systems. Every two weeks.

Store nets and siphon pipe All nets and siphon pipe- Routinely. ends in a sterilising medium. ends.

Sterilise aquaria. All aquaria, routinely. Periodically.

(*Live foods (bloodworms, Daphnia or brine shrimp) is best fed to the fish whilst still alive (fresh), but for the sake of convenience, they can be frozen and fed to the fish on demand).

5.3. Danio rerio brood stock management

5.3.1. Age of brood stock

Danio rerio are relatively short-lived, and after longer than two years, show a steady decrease in fecundity and show an increase to susceptible of diseases. Even though they become sexually mature after 74 to 75 days

(Eaton and Farley, 1974b), a spawning group of fish should be at least one

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5.3.2. Conditioning of brood stock

Food, temperature and light cycles are factors of importance in preparing the fish for spawning (Laale, 1977). Male and female fish can be separated for the conditioning period to ensure maximum fecundity, although not always necessary due to the high number of embryos normally produced. Towards the end of the two-week conditioning period, males develop a golden yellow colour on their ventral surfaces, and females become distended with ova (see

Figure 5.2). Females without distended abdomens after the conditioning period should not be used, as they are not carrying ova in sufficient numbers.

5.3.2.1. Temperature

Temperature is to be kept between 26 °C and 28.5 °C to ensure optimal conditioning and fecundity. Keeping fish outside of the range of between 25

°C and 29 °C is not healthy for the fish (Westerfield, 2002).

5.3.2.2. Food

Fish are conditioned for at least two weeks prior to spawning induction by feeding them twice daily with live food, such as bloodworms, water fleas, brine shrimp or mosquito larvae. This is fed in conjunction with a high quality dry flake food, such as TetraMin® also fed twice daily. Storage instructions on the packaging of dry flake food must be adhered to, to retain the vitamin and other nutritional component integrity. For both live food as well as dry flake

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CHAPTER 5 food, feed as much as the fish will consume within three to five minutes.

Supplementing feed with live foods is important for breeding fish to build up protein reserves needed when spawning, as well as getting nutrients that are lacking in commercially prepared dry foods. Live foods also have the advantage of remaining alive in the aquarium if not eaten straight away by the fish, thereby not fouling the water. Live foods also are available in a frozen form as well as freeze-dried form. Freeze-dried foods undergo this procedure to safeguard against possible transmission of parasites and diseases.

Freezing live foods does make it lose some of its original nutritional value

(Hemdal, 2003), however, freezing the live foods does have the advantage of being able to be stored and used when needed. Do not overfeed with frozen live foods, as well as dry flake foods, as uneaten food will settle on the bottom of the aquarium where it quickly begins to decay and foul the water, producing toxic ammonia and other by-products, as well as attracting bacteria that may potentially pose a threat to the fish. This is not only a waste of food, but the uneaten food will also need to be removed by siphoning it out of the aquarium. Adding a few aquatic snails to the aquarium will help in cleaning the bottom of the tank as they scavenge the left over food. Certain bottom- feeding species of fish (e.g. Corydoras sp. or Ancistrus sp. (pers. obs.) will also help in keeping the bottom of the aquarium free from decaying food therefore helping to maintain good water quality, their application, however, may be limited in larger commercial laboratories.

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5.3.2.3. Photoperiod

Fish being conditioned should be kept under a strict light /dark cycle of 14/10 h, respectively.

5.4. Breeding D. rerio

Fish may be bred when embryos are needed, allowing the group of breeding fish to recuperate their protein reserves for further conditioning before being bred again. This period is typically one to two weeks to ensure optimal fecundity. Alternatively, a spawning group of fish can be kept in the same breeding cage and spawned every day if they are fed very well. The maximum egg production is obtained when male and female fish are left together continuously (Eaton & Farley, 1974a). The choice of which method to use is therefore up to the laboratory to decide according to the number and frequency of embryos needed. Leaving a spawning group together continuously, however, presents a problem in feeding the fish. Any food that isn’t eaten straight away by the fish inevitably falls through the spawning cage out of the reach of the fish and, due to the fact that the fish are being fed two to three times daily, presents the problem of fouling the water very quickly with toxicants given off by the decomposing food. This also leads to an increase in time required for maintenance of water quality within the aquaria.

Scavenger organisms, such as snails or bottom-dwelling catfishes, should not be used in the spawning tanks as these organisms consume many of the embryos that lie on the bottom of the aquarium.

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5.4.1. Breeding system design

Since adult D. rerio eat their eggs (Laale, 1977; Axelrod & Schultz, 1990), a method of separating the spawning fish from the eggs needs to be devised.

This is done with the use of plastic mesh breeding cages, with a mesh size big enough to allow the eggs to fall freely through it, but not too big so as to allow the adult fish to escape through it. A mesh size of 2 mm is adequate.

The breeding cage is designed and constructed so that it can easily be suspended from the top of the tank with the lower surface immersed for at least 3 cm within the water. A breeding cage design that has proven to be adequate is one made from a plastic fruit basket, measuring 350 mm x 250 mm x 120 mm, with a 50 % shade cloth sewn around the entire outer surface

(Figure 5.10A and 5.10B). The fish avidly eat the eggs that have just been spawned and are moving down through the water column, so the depth of the water that the cages are in has to be limited. The upper edges of the cage must be above the water level so as not to allow the adult fish to escape into the tank. It is recommended that a mesh top be put onto the breeding cage, as some of the fish may escape from jumping out. Alternatively, a deeper breeding cage can be used (e.g. 350 mm x 250 mm x 220 mm). Breeding cages may alternatively be constructed out of high-grade stainless steel with stainless steel mesh size of 2 mm. The eggs that are spawned and fall through the mesh of the breeding cage, settle on the bottom of the aquarium where they undergo further development until hatching approximately three days afterwards.

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Shallow spawning tank.

Breeding cage. Note depth of water (±3 cm).

Double sponge filter.

Rods inserted through the netting to hold breeding cage at the desired depth within the spawning tank.

Figure 5.10A and 5.10B: Breeding cage design and placement.

5.4.2. Gender ratios

The breeding cage is suspended in the spawning tank, and the brood fish are placed in it in a ratio of 1.5 males to 1 female. This is to ensure maximum fertilization of the eggs. If fertilization is low, the ratio of males to females can be increased. Thirty fish can comfortably fit into a breeding cage of

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CHAPTER 5 dimensions 350 mm x 250 mm x 120 mm (L x B x H). Do not stock a breeding cage with these dimensions with a higher density of breeding fish than this for the sake of overcrowding the fish. Too many fish will restrict the movement of breeding pairs that is needed during courtship behaviour.

5.4.3. Procedure for breeding fish

The proposed spawning group of fish are placed into the spawning tank at least two days prior to spawning induction. This is done to reduce the stress placed on the spawning group that would, in turn, reduce the potential reproductive output of the group as a whole. The fish are fed on the afternoon prior to spawning (see also section 5.3.2.2. Food) and given enough time to digest the food so that fish wastes resulting from this feeding can be siphoned out of the tank. At least 10 % of the water from the tank should be removed from the spawning tank during this siphoning process, and should be replaced by fresh water. The bottom of the spawning tank is kept as clean as possible to minimise the risk of fungi from growing on the embryos. The spawning group of fish are then placed within a breeding cage in the same spawning tank (see also section 3.4.2. - Gender ratios) one to two hours before the onset of the dark cycle. Spawning activity is typically over by one to two hours after the lights have turned on. The breeding cage containing the adult fish is then removed to another tank to spawn again the next morning, or the fish are released from the spawning cage into another tank where they undergo further conditioning to be ready to spawn at a later stage. If fish are expected to spawn again the next day, it is essential that they be fed well (see also section 5.3.2.2. - Food). When transferring fish from one tank to another,

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CHAPTER 5 temperature changes as well as drastic changes in pH and water hardness and conductivity need to be avoided. This will place stress on the group of breeding fish, and they will not reproduce optimally.

5.5. Embryo, larvae and juvenile care

5.5.1. Embryo care

The embryos are then visible on the bottom of the tank where they can be left in gently circulating water until they hatch within 72-96 hours. Fungus amongst the embryos can be treated with a solution of methylene blue by adding 5-10 drops of methylene blue solution (Rid-All® Methylene Blue 1%

B.V.F.) to 100 litres of tank water. The cleaner the bottom of the tank is, the smaller the threat of fungal infections will be. Generally, only unfertilised eggs will be susceptible to fungal infections and should be removed as soon as possible. The number of unfertilised eggs can be reduced by ensuring the correct gender ratios in the breeding cages, and that the breeding fish are in good overall condition.

5.5.2. Care of free-swimming larvae and larvae

Soon after hatching, the larvae will adhere motionless to the sides of the tank for a further 24-48 hours after which they will become free-swimming. Free- swimming larvae can be fed (twice daily) on microworms (refer to Appendix A

- Procedure for culturing microworms (Anguillula silusiae). Alternatively, hard- boiled egg yolk (Appendix B - Preparation of egg yolk as a starting food for feeding larval fish.) as a starting food until the larvae are big enough to eat

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CHAPTER 5 finely ground flake food, or a suitable commercially prepared baby food, such as TetraMin Baby®. It is best at this stage in their development to keep the larvae at high stocking densities in smaller tanks, as it will be easier for them to find food. Their food can be supplemented by feeding newly hatched brine shrimp nauplii, or day-old water fleas.

As the fish grow bigger, they are separated into smaller batches, with each batch being put into its own tank to reach a final density of about two fish per litre of water. This, however, can be exceeded with increased aeration and filtration of the water, as well as by the system being part of a flow-through system.

5.6. Troubleshooting unexpected spawning results

The reproductive output of D. rerio, as with all fish, is greatly influenced by condition of the breeding fish themselves. Any one, or in combination with one another factors of water quality variables, physical condition, feeding regime, quality and type of food, age of the fish, and many more variables play a substantial role in influencing the condition of the fish, and therefore the success of a particular spawn. No two consecutive spawns, even if the procedures followed are the same, and the same spawning group is used, can yield exactly the same results. It is for this reason that spawns can be widely classified into ‘successful spawns’ and ‘unsuccessful spawns’, with the latter being of concern in the following chapter. A well-conditioned spawning group of approximately 30 breeding fish can be expected to yield once-off spawns in excess of 2,000 embryos. A similar sized spawning group

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5.6.1. A general decrease in fecundity amongst all of the spawning

groups

If the fish are still actively swimming and feeding normally, yet a general decrease of fecundity is evident amongst all of the spawning groups within the same room, the light cycle regime may be uncoordinated. Often a power failure will result in a mistimed photoperiod leading to a shift in the light vs. dark cycle in relation to the actual time of day. By doing so, the fish often receive a longer than normal dark cycle. This will reduce their spawning activity over the next few days as they acclimate to the regular lighting regime once again (also see section 5.3.2.3. - Photoperiod).

If an environmental room is used to maintain water temperature, thermostatic controls need to be checked and replaced if it is found that the temperature has increased or decreased. Immersible aquarium heaters need to be checked for correct working condition on a daily basis. Reducing the temperature by just a few degrees Celsius will influence the fecundity of the fish. Dramatic increases in temperature will induce a stress response from the fish, and spawning activity will cease. This type of stress response is often coupled to a dramatic increase in ammonia released from the fish, therefore if this is found to be the case, a partial water change is necessary to reduce the possibility of ongl term effects from the increase in ammonia.

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Stress responses due to a dramatic increase in temperature are often coupled to an increase of mucous shed by the fish that is visible in and on the surface of the water. Dramatic increases in temperature should be reversed immediately by reducing the water to optimal temperatures. Filters also need to be cleaned out due to the elevated ammonia levels, as well as the mucous typically shed by the fish following such an incident. Acclimation of the fish to spawning condition once again may take some time following a dramatic temperature shock, specifically after a sharp increase in temperature (see also section 5.2.1. - Water temperature as well as section 5.3.2.1. -

Temperature). Air blowers used for aeration of aquaria within the laboratory also need to be checked for contaminants such as fumes or oils.

New batches of fish food should also be checked for expiry date. Many commercial flake foods or dry fish foods contain vitamins that have a limited shelf life before they begin to deteriorate. Many of the vitamins are also light sensitive, being broken down in the presence of light. It is therefore essential to follow manufacturer’s instructions regarding storage instructions and expiry dates.

The water quality of the water that is used in the routine water changes in all the aquaria needs to be tested to see if it has changed at all from the previous water quality data (see also section 5.2.2. - Water chemistry).

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5.6.2. A general decrease in fecundity amongst individual spawning

groups

If spawning groups in individual tanks show a lowered fecundity, the age of the breeding fish needs to be taken into account. After about two years of age, the spawning group will show a general decrease in fecundity. Fish getting this old need to be discarded and replaced with a younger group of fish (see also section 5.3.1. - Age of brood stock).

If individual aquarium heaters are used for each of the aquaria, they need to be checked for correct working order (see also section 5.2.1. - Water temperature as well as section 5.3.2.1. - Temperature).

Feeding the fish correctly is an important factor to consider as well, and careful attention needs to be given to this aspect (see section 5.3.2.2. - Food).

The outbreak of a disease is always a possibility and careful observation of feeding habits and general appearance and change in habits of the fish is a clear indication of this. Diseased fish should be quarantined and treated accordingly.

Fish that have been bred continuously showing a decreased fecundity should be given a break from spawning. The sexes should be separated; water temperatures lowered by one or two degrees Celsius, and attention given to their feeding regimes (see also section 5.3.2.2. - Food). If, after a two-week period of this “conditioning”, the fish are still not producing optimally, they

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CHAPTER 5 should be replaced with new brood stock (see also section 5.3.2. -

Conditioning of brood stock).

Poor water quality can also play an important role in the general overall health of the fish. Fish kept in poor quality water will not channel energy into reproduction, but rather into survival (see also section 5.2.4.4. - Filtration and section 5.2.2. - Water chemistry).

5.7. Conclusions and recommendations

Danio rerio can therefore be seen as a very useful tool (=organism) for experimental work within the laboratory, as they are relatively easy to breed, producing a relatively high number of embryos on a reliable, regular basis.

Their international popularity as a test organism is evident by the vast amount of important literature available describing the biology and other aspects of the life history. Danio rerio is also very adaptable to various basic aquarium systems, so a laboratory does not need to have specialised culturing and rearing facilities to successfully culture this species of fish. They also, due to their small size, require relatively little space within the laboratory for successful culturing. Danio rerio is tolerant of a wide range of water chemistry and physical parameters, as well as being a relatively hardy species of fish where common diseases are concerned.

Table 5.6 is a summary of the most important physical and water chemistry parameters that are required for successful culturing of D. rerio.

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Table 5.6: Summary of the most important physical and water chemistry parameters required for successful culturing of D. rerio.

Aspect Parameters

Light cycle. 14:10 (Light: Dark cycles). Tank size. At least 100l for adult fish (500mm x 500mm x 400mm – LxBxH). Stocking density. At least 75cm2 surface area for each adult fish (±30 fish / 100l tank). Temperature. 26°C - 29°C. pH. 6.5 - 7.5. Conductivity. 3 – 500 µs/cm. Water hardness. 80 – 200mg/l CaCO3 (4 – 12°dH). Food. Frozen bloodworms, Daphnia and brine shrimp, and flake food. Feeding routine. At least 2 to 3 times per day. Maintenance. Filter maintenance and maintenance of sanitary conditions are very important. Periodic sterilisation of nets and tanks also very important for disease prevention. Age of brood stock. Between one and two years.

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5.8. References

Arunachalam, M., Johnson, J.A., Sathyanarayanappa, S.N.,

Sankaranarayanan A. and Soranam, R. (2000). Cultivable and

ornamental fishes from Hemavathi and Ekachi rivers, South Karnataka.

In Endemic fish diversity of western Ghats. Ponniah, A.G. and

Gopalakrishnan, A. (Editors.). NBFGR-NATP Publication. National

Bureau of Fish Genetic Resources, Lucknow, U.P., India. 1,347 p.

Astrofsk?, K.M., Bullis, R.A. and Sagerström, C.G. (2002). Biology and

management of D. rerio. In Laboratory animal medicine, 2nd edition.

Fox, J., Anderson, L., Loew, F. and Quimby, F. (Eds.). Elsevier Science,

USA. pp 862-883.

Axelrod, H.R. (1995). Breeding aquarium fishes. A complete

introduction. T.F.H Publications, New Jersey. 125 p.

Axelrod, P.H. (1967). Breeding aquarium fishes. Book 1. T.F.H

Publications, New Jersey. 149 p.

Axelrod, H.R. and Schultz, L.P. (1990). Handbook of tropical aquarium

fishes. T.F.H. Publications, New Jersey. 718 p.

Axelrod, H.R. and Vorderwinkler, W. (1978). Encyclopaedia of tropical

aquarium fish, with special emphasis on techniques of breeding,

24th edition. T.F.H. Publications, New Jersey. 631 p.

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Barbazuk, W.B., Korf, I., Kadavi, C., Heyen, J., Tate, S., Wun, E., Bedell, J.

A., McPherson, J.D. and Johnson, S.L. (2000). The syntenic

relationship of D. rerio and human genomes. Genome research, 10(9):

1351-1358.

Barut, B.A., Korf, I., Zon, L.I. (2000). Realizing the potential of D. rerio as a

model for human disease. Physiological genomics, 2: 49-51.

Beckwith, L.G., Moore, J.L., Tsao-Wu, G.S., Harshbarger, J.C. and Cheng,

K.C. (2000). Ethylnitrosourea induces neoplasia in zebrafish (Danio

rerio). Laboratory investigation, 80: 379-385.

Brand, M., Beuchle, D., Endres, F., Haffter, P., Hammerschmidt, M., Mullins,

M., Schulte-Merker, S., Nüsslein-Volhard, C., Lück, R., Jürgen, K. and

Schwartz, S. (1995). Keeping and raising zebrafish (Danio rerio) in

Tübingen. The zebrafish science monitor, 3(5): 2-7. Institute of

Neuroscience, University of Oregon Press, Eugene, OR.

Bresch, H. (1993). Some remarks to a long-term toxicity test in fish for

ecological purposes. In Fish: ecotoxicology and ecophysiology.

Braunbeck, T., Hanke, W. and Segner, H. (Eds.). V.C.H. Publishers,

Cambridge, England. 418 p.

Coffey, D.J. (1986). The encyclopaedia of aquarium fish. Treasure Press,

London. 224 p.

Connell, D., Lam, P., Richardson, B. and Wu, R. (1999). Introduction to

ecotoxicology. Blackwell Science, UK. 362 p.

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Dodd, A., Curtis, P.M., Williams, C and Love, D.R. (2000). Zebra fish:

bridging the gap between development and disease. Human molecular

genetics, 9(16): 2443-2449.

Eaton, R.C. and Farley, R.D. (1974a). Spawning cycle and egg production of

zebrafish, Brachydanio rerio, in the laboratory. Copeia, 1: 195-204.

Eaton, R.C. and Farley, R.D. (1974b). Growth and the reduction of

depensation of zebrafish, Brachydanio rerio, reared in the laboratory.

Copeia, 1: 204-209.

Fishdoc. (2004). Fish diseases diagnosis and fish disease treatments.

World Wide Web publication. www.fishdoc.co.uk/water.

Fishbase. (2004). Froese, R. and Pauly, D. (Editors). World Wide Web

electronic publication. www.fishbase.org, version (06/2004).

Ford, D. (1981). Small Aquaria. Aquarium Design. In Aquarium systems.

Hawkins, A.D. (Ed). Academic Press, London. 452 p.

Goolish, E.M., Evans, R., Okutake, K. and Max, R. (1998). Chamber Volume

Requirements for Reproduction of the Zebrafish, Danio rerio.

Progressive fish culture, 60: 127-132.

Hawkins, A.D. and Anthony, P.D. (1981). Aquarium Design. In Aquarium

systems. Hawkins, A.D. (Ed). Academic Press, London. 452 p.

Hemdal, J. F. (2003). Aquarium fish breeding. Barron’s Educational

Series, Inc. 169 p.

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International Organization for Standardization. (1996). Water Quality –

Determination of the acute lethal toxicity of substances to a

freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei,

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edition, International Organization for Standardization, Switzerland.

Laale, H.W. (1977). The biology and use of zebrafish, Brachydanio rerio in

fisheries research. A literature review. Journal of fisheries biology,

10: 121-173.

Lloyd, R. (1981). Freshwater Quality. In Aquarium systems. Hawkins,

A.D. (Ed). Academic Press, London. 452 p.

Meyer, A., Biermann, C.H. and Orti, G. (1993). The phylogenetic position of

the zebrafish (Danio rerio), a model system in developmental biology:

an invitation to the comparative method. Proceedings of the Royal

Society of London, 252: 231-236.

Mills, D. and Vevers, G. (1989). The Tetra encyclopaedia of freshwater

tropical aquarium fishes. Tetra Press, New Jersey. 208 p.

Nagel, R. (1993). Fish and environmental chemicals – a critical evaluation of

tests. In Fish. ecotoxicology and ecophysiology. Braunbeck, T.,

Hanke, W. and Segner, H. (Eds.). V.C.H. Publishers, Cambridge,

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Organization for Economic Cooperation and Development. (1992). OECD

guidelines for testing of chemicals. Fish, acute toxicity test.

Guideline 203. OECD.

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Society of Bangladesh. Department of Zoology, University of Dhaka.

364 p.

Sandford, G. (2003). Aquarium owner’s manual. Dorling Kindersley

Limited, London. 266 p.

Schäfers, C., Oertel, D. and Nagel, R. (1993). Effects of 3,4-dichloroaniline

on fish populations with differing strategies of reproduction. In Fish.

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Segner, H. (Eds.). V.C.H. Publishers, Cambridge, England. 418 p.

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Curriculum Development Centre, Tribhuvan University, Kathmandu,

Nepal. 645 p.

Sissino, C.L.S., Oliveira-Filho, E. C., Dufrayer, M. C., Moreira, J. C. and

Paumgartten, F. J. R. (2000). Toxicity evaluation of a municipal dump

leachate using zebrafish acute tests. Bulletin of environmental

contamination and toxicology, 64: 107-113.

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neoplasia in the aquarium fish, Brachydanio rerio. Journal of the

national cancer institute, 34: 117-130.

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Talwar, P.K. and Jhingran, A.G. (1991). Inland fishes of India and

adjacent countries. Volume 1. A.A. Balkema, Rotterdam. 541 p.

Valdesalici, S. and Cellerino, A. (2003). Extremely short lifespan in the

annual fish Nothobranchius furzeri. Proceedings of the Royal Society

of London, series B. Biological Sciences.

Westerfield, M. (2002). The zebrafish book. A guide for the laboratory

use of zebrafish (Danio rerio). 4th edition. University of Oregon Press,

Eugene, OR, USA.

Wickins, J.F. and Helm, M.M. (1981). Sea water treatment. In Aquarium

systems. Hawkins, A.D. (Ed.). Academic Press, London. 452 p.

Wilkerson, J.D. (2001). Clownfishes – a guide to their captive care,

breeding and natural history. T.F.H. Publications, Inc., Neptune City,

New Jersey. 240 p.

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research 9(2): 99-100.

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ASSESSMENT OF INTERNATIONAL PRACTICES ON THE USE OF FISH IN TOXICITY TESTING AND ADAPTATIONS FOR SOUTH AFRICAN CONDITIONS

CHAPTER 6

TOXICITY ASSESSMENT OF D. RERIO, P. RETICULATA, B. TRIMACULATUS, B. ARGENTEUS, O. MOSSAMBICUS, T. SPARRMANII AND P. P. PHILANDER

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6.1. Introduction

6.1.1. Background

Technological advancement up until the twenty-first century has, and continues to have, important positive consequences. This advancement, however, comes at the price of increasing pollution, especially to water systems throughout the world, in one form or another (Rand et al, 1995). The development of new sources of energy, that greatly enhanced (as it was intended) human welfare, is one important source of this pollution. Other sources of chemical pollution come from domestic and industrial waste discharges (petroleum, hydrocarbons, heavy metals, acids, alkalis, solvents, etc.) that may reach the groundwater through seepage of the pollutants or by rain containing soluble pollutants that enter the ground (Slabbert et al, 1998a).

Agricultural activities are also a contributing factor to water contamination in terms of pesticides, as well as nitrogenous and phosphorous compounds contained within fertilizers that find their way into water systems. These pollutants adversely affect aquatic ecosystems and may end up in the drinking water of terrestrial organisms, eventually becoming a direct threat to human lives. Therefore, in order to adequately protect aquatic ecosystems and human health from exposure to these pollutants, effective procedures are required to detect the presence of these chemicals so that corrective measures can be sought. Indeed, the state of the water quality of water sources throughout South Africa is deteriorating rapidly (Wepener, 1990).

Even though physico-chemical analytical procedures are accurate in determining the presence of certain chemicals within an aquatic ecosystem,

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CHAPTER 6 they cannot determine what the short, and long-term, effects of the chemicals may be on the aquatic organisms. They also cannot determine what the effects of a combination of a number of different chemicals on the aquatic ecosystem may be as well as not being able to possibly determine what the effects of possible unknown pollutants within effluents can be. Living organisms respond to the total effect of actual and potential disruptions in water, therefore the adoption of biological toxicity tests has become an approach to compliment chemical analysis in monitoring and controlling harmful chemicals in water (Lloyd, 1992; Slabbert et al, 1998a; Connell et al,

1999; Liu & Dutka, 1999).

In 1942, man knew only 600 000 chemicals, with that number having increased to almost 11 million by 1993 (Liu & Dutka, 1999). Up until 1999, there were vast numbers of chemicals in routine use by human society, with an estimation of around 70 000 chemicals commonly being utilised internationally for a wide variety of purposes and that the rate of introduction of new substances was between 200-1 000 compounds annually (Connell et al, 1999). Therefore, potentially there are currently up to 75 000 compounds in routine use worldwide. Indeed, in the period since World War II, acute toxicity tests have been used extensively to determine the effects of potentially toxic substances on aquatic organisms (Parrish, 1985). The manufacture of xenobiotic substances has increased markedly during the past

50 years (Connell et al, 1999; Liu & Dutka, 1999). Biological toxicity testing has therefore become a valuable component of effluent monitoring and control in many countries (Slabbert, et al, 1998b; Liu & Dutka, 1999).

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The interrelations between man’s various activities and the effect that these have on different components of the aquatic ecosystem on aquatic life are given in Figure 6.1.

LAND USE

Agriculture, forestry, land PLANTS drainage, urban development Chemical Water quality

inputs INVERTEBRATES WATER USE Water quantity Change in flow rate (canalisation, dams, water abstraction), effluent

disposal FISH COMMUNITY

POPULATION GROWTH

Change in physical Food demand, leisure, energy demand character (atmospheric inputs) FISHERY

SOURCES OF LOADS EFFECT ON WATER BODIES EFFECT ON LIVING ORGANISMS

Figure 6.1: Interrelationships between the major environmental factors that can affect a fish community (adapted from Lloyd, 1992).

This shows what effect the pressure of human population growth has on the aquatic environment. The increase in the population, with the associated increase in demand for food, leads to the development of more efficient agricultural practises. These developments include, amongst others, the increased land use, increased water abstraction from natural water sources, as well as the increased usage of agricultural chemicals such as fertilisers and pesticides. These factors have a detrimental effect on the water sources

– either physically or chemically. These effects negatively influence the water quality, thereby having the inevitable detrimental effect on the communities of aquatic organisms, including plants, invertebrates and fish.

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6.1.2. Uptake and effects of chemicals on fish

For a particular chemical to cause a toxic effect, or to trigger an adverse response on aquatic organisms, the chemical must make contact with its particular target site at a high enough concentration and for a sufficient amount of time. The concentration and time required to produce an adverse effect vary with the chemical, species of organism, and severity of the effect.

This contact-reaction between the organism and the chemical is called the exposure. As organisms transform chemicals to various metabolites after their uptake and absorption, the effects of the exposure may be reversible by normal repair mechanisms of the organism, such as by regeneration of damaged tissue and recovery from narcosis depending, of course, on the severity of the exposure (Rand et al, 1995). Sometimes, however, the effects of the toxicants on the fine, delicate organs of fish – such as the gills – are irreversible; with the effect that the fish cannot continue to effectively extract oxygen from the water any more. The damage caused by these toxicants may also lead to secondary infections by opportunistic bacteria that, together with the decreased oxygen-extraction efficiency, will inevitably lead to the fish’s death.

In order for fish to obtain oxygen from the small amounts that are dissolved as gas in the water, their gill structure consists of a very fine sieve through which water is pumped by muscular action of the mouth and pharynx. The primary lamellae or filaments are attached to the gill arches like teeth on a comb.

These lamellae are slightly covered so that their tips meet those of the adjoining gill arch, the whole system forming a folded filter. To increase the

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CHAPTER 6 efficiency of the filter, there are plate-like secondary lamellae on the upper and lower surface of the branchial lamellae, and the respiratory water has to pass between these plates. The structure of these plates can be compared to a sandwich consisting of a thin layer of epithelial cells on the outside and spaces through which the blood flows on the inside. Therefore, the dissolved oxygen in the water has to cross only a very short distance to get into the bloodstream. Added to this, the blood flows in the opposite direction to that of the water, which further increases the efficiency of oxygen extraction still further. Because of this arrangement, fish can extract up to 80 % of the oxygen dissolved in the respiratory water, but it is this very same efficiency that makes fish vulnerable to toxic substances in the water (Lloyd, 1992).

As the gills of fish are in direct contact with the surrounding water, chemicals in the water are in direct contact with the gills of fish; therefore, the gills can also absorb water-soluble chemicals, pass them into the bloodstream and circulate them through the body. Furthermore, if the concentrations of the toxic substances are high enough, the delicate cells of the gill secondary lamellae can be damaged with consequential disruption of the vital functions of respiration and salt regulation. The gills are of primary importance as a route whereby toxic chemicals in the water can be taken up by the fish.

Water-soluble chemicals may also enter an organism through the general body surface (dermal exposure) and through food that is ingested and therefore absorbed through the gastrointestinal tract (Lloyd, 1992; Rand et al,

1995).

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An important function of gills is to control the salt content of the body fluids.

Special cells regulate the active uptake of sodium and chloride and the excretion of hydrogen and bicarbonate ions. These activities can be disrupted by those chemicals such as zinc and copper, which have a direct affect on proteins in the cells. Indeed, morphological changes in the gills, as reflected by the decreases in the plasma sodium, potassium, calcium and chloride concentrations, although not lethal, have a significant effect on the respiration and osmoregulatory function of the gills (Wepener, 1990), possibly leading to secondary infections of the affected organs (normally the gills, liver and kidneys). This continued exposure does eventually lead to the decline in the affected fish’s overall health, possibly leading to death (Van Vuren et al,

1994). Also, some chemicals such as ammonia can increase the rate at which water enters the fish, leading to an increase in urine flow as the extra water is pumped out through the kidneys. It is possible that these effects on osmoregulation may be caused by a disruption of the joints between the cells of the gill epithelium, thus making this sheet of tissue less waterproof.

However, the concentrations of chemicals that have such a disruptive effect are probably close to, if not exceeding, those that are above the threshold

LC50 (Lloyd, 1992; Connell et al, 1999). These osmoregulatory factors are shown in Figure 6.2.

Oral exposure provides direct venous blood input of the chemical, which is subject to first-pass elimination in liver and gills, whereas chemicals absorbed dermally are subject to first-pass elimination in the kidneys and gills. Each

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CHAPTER 6 elimination pathway may produce a reduction in the amount of parent chemical and/or metabolite that reaches the systemic arterial circulation.

Equilibrium exchange with water

Water loss

Water uptake Storage in lipids

Gills

Circulatory fluid Metabolism & excretion

Gastrointestinal Food tract consumption

Excretion

Figure 6.2: Uptake, accumulation and loss processes for a toxicant in the ambient water with fish (adapted from Connell et al, 1999).

In contrast, chemicals absorbed across the gills enter the systemic circulation directly with no possibility of first-pass elimination and associated mitigation.

Thus, the route of exposure may affect kinetic factors such as absorption, distribution, biotransformation, and excretion, and may ultimately determine the toxicity of a chemical (Rand et al, 1995).

Rates and patterns of metabolism and excretion can substantially influence susceptibility with hydrophobic chemicals can be accumulated by organisms in various tissues, bio-transformed (metabolised), and excreted back into the water, which can be seen as a measure of an organism’s general extraction efficiency, through respiration, ingestion and surface absorption processes,

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CHAPTER 6 from the environmentally available portion. Differences in susceptibility to chemical agents among fish of different strains also result from genetic factors. Other factors, such as diet, also influence toxicity by producing changes in body composition, physiology and biochemical functions, as well as nutritional status of the organism (Rand et al, 1995).

6.1.3. The use of fish in standard toxicity tests

At low concentrations, chemicals can have a variety of different toxic actions that produce different toxic effects. Higher concentrations, however, have one common effect, that is, they cause fish to die. Standard tests have therefore been developed and are carried out on each chemical to obtain a concentration-response relationship so that the limiting amount of the substance that causes death can be calculated (Lloyd, 1992).

Standard acute toxicity tests have been developed to obtain the concentration-response relationship for fish exposed to chemicals. This is done to determine the immediate effects on test organisms of a short-term exposure to an effluent under specific experimental conditions to evaluate the potential for aquatic life present in the receiving water. Another reason is to compare the acute sensitivities of different species and the acute toxicities of different effluents, as well as to study the effects of various environmental factors on results of such tests (Rand & Petrocelli, 1985; APHA, 1992; OECD,

1992; EPA, 1993; ISO, 1996; ASTM, 1997). Acute tests are usually the first step in evaluating the effects of an effluent on aquatic organisms. Results of acute effluent tests might be used to predict acute effects likely to occur on

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CHAPTER 6 aquatic organisms in field situations as a results of exposure under comparable conditions, except that (1) motile organisms might avoid exposure when possible, (2) toxicity to benthic species might be dependent on settling of components of the effluent onto the substrate, and (3) the effluents might physically or chemically interact with the receiving water. An acute toxicity test does not provide information about whether delayed effects will occur, although a post-exposure observation period, with appropriate feeding if necessary, might provide such information.

Tests are carried out on each chemical so that the limiting amount of a substance that causes death can be calculated. A series of concentrations of a chemical is prepared in standard dilution water and these solutions are then transferred to testing vessels made from appropriate inert material such as laboratory glass. The concentrations are chosen from the result of a preliminary test (range finding test) with a few fish, which establishes the approximate range of concentrations of the chemical that begins to adversely affect the fish. A number of fish (usually between five and ten) are then placed into each testing vessel and their survival times are recorded. One testing vessel contains clean water with no added chemical in which all the fish are expected to survive. If more than ten percent of the fish die in this control vessel, the test is not valid because factors other than the chemical may be affecting the survival of the fish. The times at which individual fish die in each concentration of chemical are recorded. At fixed times during the test such as 6, 24, 48, 72 and 96 h after the start, the concentration causing 50 % mortality can be calculated; these are the LC50’s (the concentration lethal to

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50 % of the fish at each time interval). These values can then be plotted against the corresponding exposure periods and concentrations on a graph or inserted into a computer programme (e.g. Probit analysis) and the 96 h LC50 value is calculated (Lloyd, 1992). In doing so, other information regarding the tolerance of the test organism to the toxicant being used can also be extrapolated. Threshold concentrations (the concentration of a toxicant that an organism is able to metabolise and excrete it without apparent detriment to itself) can also be determined. This concentration is termed the ‘No observed effect concentration’ (NOEC) for that particular chemical. The concentration that affects 50 % of the test organisms (in a way other than death) can also be extrapolated from a dose-response curve, and is termed the EC50 (effect concentration) value (APHA, 1992; OECD, 1992; EPA, 1993; ISO, 1996;

ASTM, 1997).

According to Rand et al (1995) and Connell et al (1999), criteria to consider when selecting suitable test organisms are:

· A group of species that represents a broad range of sensitivities should be used whenever possible as sensitivities vary among species. · Widely available and abundant species should be considered. · Whenever possible, species should e studied that are indigenous to, or representative of the ecosystem that may receive the impact. · Species that are recreationally, commercially, or ecologically important should be included. · Species should be amenable to routine maintenance in the laboratory and techniques should be available for culturing and rearing them in the laboratory to facilitate both acute and chronic toxicity tests. · Species that are easily identified are preferred.

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· If there is adequate background information on a species (i.e., its physiology, genetics, and behaviour), the data from a test may be more easily interpreted.

To make all of the above criteria applicable to any species of fish would be greatly desirable, but on the other hand, it would also be very unrealistic to think that it is at all feasible. Indeed, according to Connell et al (1999) it is impossible to find a test species that is capable of fulfilling all of these criteria.

In the past, the most important criterion for choosing a particular species of fish for toxicity testing was its high degree of sensitivity to various chemical compounds. Besides sensitivity to test chemicals, other aspects have become important in selecting organisms to be used in toxicity testing due to practical reasons (Connell et al, 1999). One of the major hindrances facing the routine toxicity testing laboratory being the reliable source of readily available, healthy test fish, which are of proven similar ages and size (Lloyd,

1992; Landis & Yu, 1995). The most commonly used fish for toxicity tests are indeed those that can be readily obtained from a commercial supplier or which can be easily bred in the laboratory, and is a reliable indicator of the response of other organisms in the community (Lloyd, 1992; Connell et al, 1999).

Common species of freshwater fish that toxicity tests are routinely performed on are:

· Pimephales promelas (fathead minnow) (Parrish, 1985; EPA, 1993; Landis & Yu, 1995; ISO, 1996) · Oncorhynchus mykiss (rainbow trout) (Parrish, 1985; Lloyd, 1992; EPA, 1993; Landis & Yu, 1995; Rand et al, 1995)

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· Salvelinus fontinalis (brook trout) (Parrish, 1985; EPA, 1993; Landis & Yu, 1995) · Oncorhynchus kisutch (coho salmon) (Landis & Yu, 1995) · Carassius auratus (goldfish) (Landis & Yu, 1995) · Ictalarus punctatus (channel catfish) (Parrish, 1985; Landis & Yu, 1995) · Lepomis macrochirus (bluegill) (Parrish, 1985; Landis & Yu, 1995; Rand et al, 1995; ISO, 1996) · Lepomis cyanellus (green sunfish) (Landis & Yu, 1995) · Brachydanio (=Danio) rerio (zebrafish) (Lloyd, 1992; OECD, 1992; ISO, 1996) · Cyprinus carpio (common carp) (OECD, 1992) · Oryzias latipes (ricefish) (OECD, 1992; ISO, 1996) · Poecilia reticulata (guppy) (OECD, 1992; ISO, 1996; Slabbert, 1998a) · Cyprinodon variegates (sheepshead minnow) (Rand et al, 1995)

The rationale of the best test species to use has often been debated. Even though many test species are currently being used for screening tests, no single species can adequately cover a wide scope of biological categories, with some species being much more sensitive to certain classes of toxicants than others (Landis & Yu, 1995). On the other hand, according to Mount

(1980) living organisms will respond to every possible substance or mixture of substances at some level regardless of their chemical or physical characteristics. Therefore, the use of any species of fish as a test species will be relevant when determining what the effects of xenobiotics will be as any test species will show a response one way or another. Indeed, if acute toxicity tests yielded similar results for toxicants with the same modes of faction for different species in different continents, there would be no need for

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CHAPTER 6 developing countries to generate data for their native species (Sunderam et al, 1992).

In order for acute toxicity test to be comparable to one another, variations within each treatment group when doing screening tests need to be kept to a minimum, as many factors can cause variation within the same group of fish being tested. As species differ in susceptibility to various chemicals, the variation needs to be tested routinely to verify the accuracy and reliability of certain test methodologies, as well as the testing organisms. Factors affecting the variations in reliability of test results from tests carried out on a single species of fish may be size, sex, age or developmental stage, diet or nutritional status, state of health, reproductive state, etc. Larval or juvenile fish often appear more sensitive to toxicants than their respective adult stages. This has been attributed to the possible under development of certain detoxifying mechanisms within juvenile stages, as well as the relatively higher surface to volume ratio typical of the smaller larval or juvenile stages. There may also be differences in rates of excretion of toxicants between adult fish and juveniles. Interestingly, embryonic stages have proven to be more tolerant to toxicants due to the added protection of the selectively permeable membrane offered by the chorion (Rand et al, 1995; Connell et al, 1999).

Standards dictating the validity of routine toxicity bioassays are important to the quality control of laboratory performing those tests. There is in fact a national proficiency testing scheme run by the Aquatox Forum (Centre for

Scientific and Industrial Research - CSIR) that various toxicity testing

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CHAPTER 6 laboratories throughout the country partake. The various laboratories participate in this scheme to assess their own quality control in accordance to the rest of the participating toxicity testing laboratories (pers. com. Slabbert,

200217). There are indeed certain criteria that any toxicity test needs to adhere to in order to deem it valid, which include (according to ISO, 1996):

· The dissolved oxygen concentration in the test solutions remained at least above 60 %; · The concentrations of the test substance was not known (or suspected) to have declined significantly throughout the duration of the test; · The proportion of the control fish suffering mortality should not exceed 10 %;

· The LC50 of a reference test performed with a reference chemical

should be in reasonable agreement with results previously obtained by

the same laboratory under similar conditions.

It can therefore be seen that different species of fish react to similar xenobiotics in a similar way. The differing abilities of the different species of fish to metabolise similar xenobiotics more effectively than others however, determine whether or not they will be more resilient or more sensitive to that particular xenobiotic. It is the degree to which different species of fish react to the same xenobiotic, making them more resilient or sensitive, that is important to this study.

17 L. Slabbert – Environmentek, CSIR, Pretoria.

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6.1.4. Objectives

The objective of this chapter was to compare a variety of different species that will be subjected to screening tests with the same reference toxicant under similar conditions and using the same testing protocols, to determine relative interspecific sensitivities to the reference toxicant. This will be done to determine which of the species concerned are the most sensitive, by determining whether the sensitivities of the different fish species differ significantly from one another. Therefore, the objective is the exploration of one aspect (namely to show a response to a wide range of concentrations of toxicants) for the criteria of what makes a certain toxicity-testing organism suitable to be used in routine testing.

6.2. Materials and methods

6.2.1. Apparatus and test conditions

Young fish were obtained from in-house breeding (see Chapters 2, 3, 4 & 5).

After each species was successfully bred, a portion of the offspring was used in exposure tests to possibly distinguish differences in the degree of sensitivities between the different fish species. All tests were done as static acute toxicity tests, primarily based on the guidelines for fish acute toxicity tests stipulated by International Organization for Standardization (ISO, 1996) using an analytical grade potassium dichromate (K2Cr2O7) (Merck, Product code: AC004864.500, Batch no. 1013156) stock solution of 1,000 mg/l as the reference toxicant. The different testing concentrations were made up as a dilution of this stock solution, using the appropriate amount of standard

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CHAPTER 6 dilution water. All tests were done in standard low form, 600 ml borosilicate laboratory glass beakers, with the total test volume being made up to 400 ml in each beaker. Prior to use, as well as in between uses, all glassware was cleaned according to the method described by van Vuren et al (1994).

Testing was done in a climate-controlled environmental room at a temperature of 25 ± 1 °C with a 14/10 h light/dark cycle.

Different standard guidelines recommended standard dilution waters with different compositions. Therefore, a comparison of two different standard dilution waters (namely the dilution water components recommended in the

ISO (1996) guidelines as well as the USEPA (1993) guidelines for standard dilution water components) to determine if the different components within the two different dilution waters influenced the sensitivity of the fish to the reference toxicant.

All tests were done in duplicate with seven (excluding the control) concentrations of the reference toxicant. A range finding test was done initially to determine the range of concentrations that the definitive tests should include. Based on the results from the range finding test, the definitive test concentrations were typically 150, 175, 200, 225, 250, 275 and 300 mg/l of the K2Cr2O7. The beakers were inspected (and recorded) for mortalities of the test organisms every 24 h for the duration of the test (96 h), with the conductivity, total dissolved solids, pH, oxygen content and percentage oxygen saturation of the test solution monitored every 24 h for the duration of

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CHAPTER 6 the test. If the acceptable limits determining the validity of the test were not met, the test was discarded and repeated (ISO, 1996). These criteria include:

· The dissolved oxygen concentration in the test solutions during the test

was at least 60 %.

· The concentrations of the test substance were not known (or

suspected) to have declined significantly throughout the test.

· The mortality of the control fish did not exceed 10 % or one per testing

vessel.

· The 24 h – LC50 of the reference chemical (K2Cr2O7) previously

obtained by the same testing methodology within the same laboratory

is in reasonable agreement with the tests being performed.

6.2.2. Handling and placing test organisms within the testing

beakers

The most appropriate methods of handling and placing testing organisms within the testing beakers were found to be different for the different species of fish. This meant that, even though the testing procedures and protocols were the same for all species of fish, getting the individual fish within the different beakers differed. This was deemed necessary for the sole purpose of practicability. The way that the protocol was different for each of the testing species will therefore be discussed separately.

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6.2.2.1. Danio rerio

The tests for D. rerio were planned to commence on the morning of the third day of development of the embryos – meaning that they were due to hatch on that particular day, typically by mid-morning (see Chapter 5). The embryos were siphoned out from the spawning tank, with a 5 mm plastic tubing (airline tubing) fitted with a plastic pipette tip, into a holding beaker. They were then pipetted out of the holding beaker into the testing vessels using a 5 ml micropipette. These testing beakers had only the appropriate amount of dilution water at this stage. Once all of the beakers had 10 test organisms in each, the appropriate amount of K2Cr2O7 stock solution was added to make up the total 400 ml testing volume. Further tests with D. rerio were conducted under similar conditions except that the test organisms were six weeks old. A comparative test was then also done with six-week-old organisms at a temperature of 21 ± 1 °C.

6.2.2.2. Barbus argenteus and B. trimaculatus

Tests using B. argenteus and B. trimaculatus were done when the organisms were in the late larval stage. The free-swimming larvae were collected using a fine-meshed net and 10 individuals were placed into each of the testing beakers (see Chapter 2). The testing beakers only had the appropriate amounts of dilution water in them at this stage. When all the beakers were loaded with testing organisms, they were allowed at least an hour before they were inspected for swimming abnormalities and morbidity. Individuals exhibiting these symptoms were removed and replaced with new test

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organisms. Only then was the appropriate amount of K2Cr2O7 added to each beaker to make up the final testing volume of 400 ml.

6.2.2.3. Poecilia reticulata

Tests involving P. reticulata made use of five individuals per testing beaker.

The test organisms were typically between seven and fourteen days old (see

Chapter 4). Testing was also conducted at 25 ± 1 °C with the test organisms being placed into the testing beakers that were filled with dilution water only.

Only once all of the beakers were loaded with testing organisms, was the appropriate amount of K2Cr2O7 stock solution added to each to make up the final volume of 400 ml.

6.2.2.4. Oreochromis mossambicus, T. sparrmanii and P. p. philander

Five individuals of the free-swimming stage were used in each test beaker

(between seven and fourteen days old) where O. mossambicus, T. sparrmanii and P. p. philander were used as the testing organisms. Once again, testing was conducted at a temperature of 25 ± 1 °C, with the testing individuals being loaded into the testing beakers that only contained dilution water at that stage. Only once all of the test organisms were loaded into the beakers was the appropriate amount of K2Cr2O7 added to make up the final volume of 400 ml.

6.2.3. Statistical analysis of the data

The data that were collected from the exposure tests were analysed using the most appropriate statistical method for the specific toxicity data. The EPA

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Probit Analysis Programme used for calculating LC/EC values (version 1.5) was initially used to determine the LC50 values. If the Probit analysis was not suitable to calculate the LC50 value, then the data were analysed using the

Trimmed Spearman-Karber method (version November 1990) to determine the LC50 values. The requirements for the data obtained to be appropriately analysed using the Probit Method are (according to USEPA, 1993):

· The observed proportion mortalities must bracket 0.5.

· The log10 of the tolerance is assumed to be normally distributed.

· Two or more of the observed proportion mortalities must be between

zero and one.

The distribution of ensuing LC50 values were then analysed with SPSS

Version 11 using the Levene’s Test for homogeneity of variances, as well as the Kolmogorov-Smirnov Test for normal distribution of the test values. The data were found to be both normally distributed as well as homogenous. The standard error of the mean test recommended by Sprague & Fogels (1977) was used to determine whether LC50 values for the different species of fish and the same species of fish under different test conditions were significantly

2 different from one another according to the formula: f1.2= antilog v((log f1) +

2 (log f2) ), where f is the ratio of the upper or lower 95 % confidence limit of the

LC50 to the LC50. The two LC50’s were significantly different if the ratio of the higher LC50 to the lower LC50 was greater than f1.2 calculated for both the upper and lower 95 % confidence limits.

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6.3. Results and discussion

6.3.1. Test conditions

The rationale behind following the dilution water composition given by ISO

(1996) as apposed to the composition given by USEPA (1993) is that the ISO composition was developed specifically for D. rerio and more uniform results were obtained throughout the experiments for all the different species of fish.

It was therefore fair to assume that the more uniform results obtained from using this particular dilution water is more relevant for a comparative study.

6.3.2. Handling and placing test organisms within the test beakers

6.3.2.1. Danio rerio

Late embryo/early larval stages of D. rerio were used due to the fact that this stage was found to be the most practical stage to handle and load into the test vessels. By this late stage of development, it was relatively easy to determine the health and state of the embryos, with accurate predictions to their viability and hatching success. There was therefore no need for a net to be used.

Avoiding the use of a net, as well as handling free-swimming individuals, also reduced the stress experienced by the organisms. Not having to catch free- swimming individuals also greatly reduced the time needed to load all of the testing vessels, since all the embryos were easily sucked up together by the micropipette. As the embryos are negatively buoyant, they were allowed to settle to the tip of the micropipette and expelled within a single drop of water.

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This greatly reduced the potential dilution factor of conventional transferral of test organisms to the testing vessels using a net.

This particular design of test procedure was then compared to tests utilising older (six-week-old) fish to compare practicability as well as sensitivity of the older fish. A choice of which method was more suitable for routine testing was then based on this comparison. The time and expenses incurred by maintaining fish for the six weeks (or until adulthood), as well as the time needed to catch individual fish to load them into the individual testing vessels did not justify the relatively small difference in sensitivity shown by the older fish.

6.3.2.2. Barbus argenteus and B. trimaculatus

Tests involving B. argenteus and B. trimaculatus were done using the free- swimming stage due to the fact that the spawning tank was designed in such a way that it was more practical to catch the larval fish as apposed to collecting the embryos. After the organisms were loaded into the testing vessels, they were inspected for general vigour of the individuals to ensure that the transferral process by a net did not physically damage them.

Physically damaged individuals were removed and replaced by others. This ensured more uniform results of the tests.

6.3.2.3. Oreochromis mossambicus, T. sparrmanii and P. p. philander

Free-swimming individuals of the Cichlidae family (O. mossambicus, T. sparrmanii and P. p. philander) were used for the tests. When newly hatched

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CHAPTER 6 larvae were used, mortality was due more to the lack of circulation within the testing vessel than to the testing chemical itself. The embryos and sac-fry larvae of these species need constant water circulation and aeration, otherwise they tend to clump together and smother one another (Macintosh &

Little, 1995). This is shown by the way that the parents constantly fan the embryos and larval fish with their fins to induce water circulation (pers. obs.).

This became evident by the 100 % mortality of all of the larval fish within the test beakers, even in the control vessels, where there was no water circulation. When the larvae are allowed to mature to free-swimming stages, the stringent requirements in terms of oxygen saturation and water circulation are not as critical as for when they were early larval stages. Survival rates are therefore much better, with the mortalities of the individuals being a more accurate indication of the potency of the K2Cr2O7 than what previously would have been environmentally induced. The number of individuals in each test vessel was also kept to a minimum to also ensure adequate oxygenation within the test vessel throughout the duration of the tests.

6.3.3. Relative sensitivities to K2Cr2O7

The most sensitive species of fish used for the exposure tests is (by species comparison) D. rerio, followed then (in order of decreasing sensitivity to

K2Cr2O7) by P. reticulata, B. argenteus, B. trimaculatus, T sparrmanii, O. mossambicus and P. p. philander, respectively (Figure 6.3). These results show the cichlid species being generally more resilient to the toxicant. This is finding is in agreement with that of Dyer (1997) who also found cichlid species to be the most resilient family of fish that was tested during that study.

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300

275 ) 7 O

2 250 Cr 2 225

200

175

150 LC50 values (mg/L K

125

100

Fish species

D. rerio (n=10) T. sparrmanii (n=5) P. reticulata (n=10) O. mossambicus (n=6) B. argenteus (n=12) P. p. philander (n=5) B. trimaculatus (n=10)

Variations within intraspecific test results can be attributed to variations in handling methods of different ages of the test organisms, as well as natural variation occurring between different groups of the same species of fish (Rand et al, 1995). Handling of test organisms contributes to the stress endured by that particular individual, possibly adding to its sensitivity (pers. obs.). This possibility is reinforced by toxicity data in the form of proficiency testing scheme data routinely done by a commercial toxicity-testing laboratory on P.

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reticulata, which showed a variation of between 1.10 and 64.61 mg/l K2Cr2O3

(Rand Water, 2002). The large degree of variation shown by this particular species is a possible indication of the large intraspecific variability. The contributing factor of handling stress cannot be ruled out as well. However, considering the degree of experience within the field of laboratory-based toxicity tests that this particular laboratory has, this factor can be perceived as being less of a contributing factor than that of natural intraspecific variability shown by this species of fish.

250 ) 3

O 225 2 Cr 2 200

175

150

LC50 values (mg/L K 125

100 USEPA ISO Test condition (dilution water)

Figure 6.4: Comparison of the mean LC50 values (± standard error) when using early larval stages of D. rerio in USEPA dilution water and ISO dilution water.

The possibility that different dilution water constituents recommended by both

USEPA (1993) and ISO (1996) has an influence on the sensitivity of the same fish species of similar ages was investigated. It was found that the use of

USEPA ‘moderately hard’ standard dilution water made the fish more

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sensitive to K2Cr2O7. This can possibly be explained by the fact that USEPA dilution water was the first dilution water to be used at the onset of the study.

Therefore, human error can possibly be a contributing factor to sensitivity displayed by the fish. The lack of experience in handling the fish may have contributed to them being subjected to unnecessary stress, thereby increasing their sensitivity to the toxicant. This possibility is also supported by the high degree of variation shown within this specific test relative to the variation shown within the tests when ISO dilution water was used (Figure 6.4). The

ISO dilution water was also specifically recommended for D. rerio, so the osmotic balance of the water could possibly be a greater contributing factor to that particular species of fish. The possibility of this factor would then play a role in reducing the stress experienced by the test fish, thereby making it relatively more resilient to toxicants.

The influence of the temperature, at which a toxicity test is conducted, has on the sensitivity displayed by the fish during a toxicity test, was also investigated. Danio rerio individuals of similar ages (six weeks) and of similar genetic strain were simultaneously tested at different temperatures (Figure

6.5). The increase in sensitivity shown by the fish in the test conducted at 21

°C relative to the sensitivity shown by the fish in the test conducted at 25 ± 1

°C is in disagreement to the accepted fact that there is a positive correlation of toxicity with increased temperature (Dyer et al, 1997). These unexpected results can possibly be due to the fact that the fish used for the test at 21 °C were not allowed sufficient time to acclimate to the temperature decrease

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CHAPTER 6 from 25 °C (at which they were maintained) to 21 °C (at which the test was conducted).

300 ) 7

O 250 2 Cr

2 200

150

(mg/L K 100 50

LC 50

0 21 oC 25 oC Test condition (temperature)

Figure 6.5: Results of the tests done with D. rerio of six weeks old at 21 °C and 25 °C. The bars represent the mean LC50 values (± standard error) at the two different temperatures.

The fish experienced this decrease in temperature over a period of two hours.

This relatively drastic decrease in temperature would have placed stress upon the fish (Sandford, 2003), thereby possibly temporarily increasing their sensitivity to toxicants.

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Table 6.1: Comparative matrix denoting which species of fish differ significantly (P<0.05) from one another in terms of sensitivity to K2Cr2O7.

D. rerio A D. rerio B D. rerio C D. rerio D P. reticulata B. argenteus B. trimaculatus T. sparrmanii O. moss P. p. phil * ** D. rerio A D. rerio B 0 D. rerio C 0 0 D. rerio D 0 1 0 P. reticulata 0 0 0 0 B. argenteus 0 0 0 0 0 B. trimaculatus 0 1 0 0 0 0 T. sparrmanii 0 1 0 0 0 0 0 O. moss* 1 1 0 0 0 0 0 0 P. p. philander 1 1 0 1 1 1 0 0 0

(A) Exposure tests done on six -week old D. rerio at a temperature of 21 °C in ISO dilution water. 0 Not significantly different (B) Exposure tests done on early larval stage D. rerio at a temperature of 25 °C in USEPA dilution water. 1 Significantly different (C) Exposure tests done on six-week old D. rerio at a temperature of 25 °C in ISO dilution water. (D) Exposure tests done on early larval D. rerio at a temperature of 25 °C in ISO dilution water. *Oreochromis mossambicus **Pseudocrenilabrus philander philander

CHAPTER 6

Significant differences occur between D. rerio (early larval stage using USEPA dilution water) and the majority of the rest of the fish species and test conditions, as well as between P. p. philander and the majority of the rest of the fish species and test conditions (Table 6.1). Pseudocrenilabrus philander philander showed a markedly higher resilience to the toxic effects of K2Cr2O7.

This is probably attributed to this species of fish being able to metabolise and therefore excrete the K2Cr2O7 more efficiently than the rest of the fish species.

Danio rerio (early larval stage using USEPA dilution water) were shown to be statistically more sensitive than the majority of the rest of the species. This increased sensitivity may be due to this particular species of fish not being completely compatible with the chemical constituents and concentrations found within the USEPA dilution water, or the constituents of that particular dilution water differing too greatly from the water that the fish were maintained in (aged municipal water). This may have placed the test organisms under increased stress throughout the duration of the test thereby increasing their sensitivity to the chemical. Fish species of the Cichlidae family seemed to show greater resilience to the K2Cr2O7 when compared to the other fish species. This can probably be attributed to the fact that cichlids are classed as secondary freshwater fish (Skelton, 2001) with many members of the family Cichlidae being capable of withstanding salinity higher than seawater

(pers. com. Tetra®18). This possibly means that they are capable of metabolising and excreting inorganic salts more efficiently than other species of fish.

18 Tetra fish question forum, Germany (www.tetrafish.de/forum).

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6.4. Conclusions and recommendations

This chapter referred to different methods of placing the test organisms in the test vessels as well as different ways and life stages at which the different species of fish tested could most practically be utilised for routine toxicity tests. When looking at the requirements of a commercial toxicity testing laboratory, different factors invariably carry more weight than others. These factors include time and expenses. The testing method that is therefore the most user-friendly as well as the least time consuming, with the least amount of expenses to run them, would therefore be the most obvious choice. That is why the test utilising late embryo stages of D. rerio at 25 °C would be the test of choice due to sheer workability, being relatively cheaper than the other methods, and the fact that it is possible to do within one week’s notice. Even though the organisms used for this test were not shown to be the most sensitive to the K2Cr2O7, this seems a relatively small price to pay considering the many advantages to this test method as well as choice of test species.

The exotic species of fish used for this study showed comparable sensitivities to the reference toxicant as what the indigenous species did. They were more sensitive to that toxicant in many cases. It is therefore clear from the results obtained that it is unnecessary to focus on an indigenous representative of a fish species when the particular species is being considered for routine toxicity testing as it was shown that fish largely respond in a similar way to the same toxicant.

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6.5. References

APHA. (1992). Standard methods for the examination of water and

wastewater (18th edition). Greenberg, A.E., Clesceri, L.S., Eaton, A.D.

and Franson, M.H. (Editors). American Public Health Association,

American Water Works Association and Water Environment Federation.

1220 p.

ASTM. (1997). Standard guide for conducting toxicity tests on aqueous

ambient samples and effluents with fishes, macroinvertebrates, and

amphibians. E 1192-97. American Society for Testing and Materials.

Connell, D., Lam, P., Richardson, B. and Wu, R. (1999). Introduction to

ecotoxicology. Blackwell Science Ltd. Oxford. 170 p.

Dyer, S.D., Belanger, S.E. and Carr, G.J. (1997). An initial evaluation of the

use of Euro/North American fish species for tropical effects

assessments. Chemosphere 35 (11): 2767-2781.

International Organization for Standardization. (1996). Water Quality –

Determination of the acute lethal toxicity of substances to a

freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei,

Cyprinidae)] – Part 1: Static method. ISO Report 7346-1 Second

edition, International Organization for Standardization, Switzerland.

Landis, W.G. and Yu, M. (1995). Introduction to environmental toxicology

– Impacts of chemicals upon ecological systems. CRC Press,

Florida, USA. 328 p.

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Liu, D.L. and Dutka, B.J. (1999). An evaluation of the state of toxicity

assessment research and application in South Africa. Consultants

report for the 9th International Symposium on Toxicity Assessment,

Pretoria, South Africa, 26 September – 1 October 1999.

Lloyd, R. (1992). Pollution and freshwater fish. Blackwell Scientific

Publications Ltd., Oxford. 176 p.

Macintosh, D.J. and Little, D.C. (1995). Nile tilapia (Oreochromis niloticus) In:

Broodstock management and egg and larval quality. Bromage, N.R.

and Roberts, R.J. (Editors). Blackwell Scientific Publications Ltd.,

Oxford. pp 277-320.

Mount, D.I. (1980). Needs of toxicity tests to meet specific regulations. In

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Lexington Books, D.C. Heath and Company, Massachusetts. 416 p.

Organization for Economic Cooperation and Development. (1992). OECD

guidelines for testing of chemicals. Fish, acute toxicity test.

Guideline 203. OECD.

Parrish, P.R. (1985). Acute toxicity tests. In: Fundamentals of aquatic

toxicology – methods and applications. Rand, G.M. and Petrocelli,

S.R. (Editors). Hemisphere Publishing Corporation. 666 p.

Rand, G.M. and Petrocelli, S.R. (1985). Fundamentals of aquatic

toxicology – methods and applications. Hemisphere Publishing

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Rand, G.M., Wells, P.G. and McCarty, L.S. (1995). Introduction to aquatic

toxicology. In Fundamentals of aquatic toxicology: effects,

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Rand Water. (2002). Yearly data supplied from Rand Water proficiency

testing scheme (January 2000 to July 2002). Scientific Services,

Analytical Services, Hydrobiology, Rand Water Board, Vereeniging.

Sandford, G. (2003). Aquarium owner’s manual. Dorling Kindersley

Limited, London. 256 p.

Slabbert, J.L., Oosthuizen, J., Venter, E.A., Hill, E., du Preez, M. and

Pretorius, P.J. (1998a). Development of guidelines for toxicity

bioassaying of drinking and environmental waters in South Africa.

Report to the Water Research Commission, Project No. 358/1/98.

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Slabbert, J.L., Oosthuizen, J., Venter, E.A., Hill, E., du Preez, M. and

Pretorius, P.J. (1998b). Development of procedures to assess whole

effluent toxicity. Report to the Water Research Commission, Project

No. 453/1/98. Division of Water Environment and Forestry Technology,

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Skelton, P. (2001). A complete guide to the freshwater fishes of southern

Africa. Struik, Cape Town. 395 p.

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Sprague J.B. and Fogels, A. (1977). Watch the Y in Bioassay. Proceedings

from the third aquatic toxicology workshop. Halifax, N.S., Nov. 2-3,

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AR-77-1, Halifax. pp. 107-118.

Sunderam, R.I.M., Cheng, D.M.H. and Thompson, G.B. (1992). Toxicity of

endosulfan to native and introduced fish in Australia. Environmental

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USEPA. (1993). Methods for measuring the acute toxicity of effluents

and receiving waters to freshwater and marine organisms. Weber,

C.I. (Ed). Environmental Monitoring Systems Laboratory – Cincinnati.

Office of Research and Development, U.S. Environmental Protection

Agency, Cincinnati, Ohio 45268. EPA-600/4-90/027F.

Van Vuren, J.H.J, du Preez, H.H and Deacon, A.R. (1994). Effects of

pollutants on the physiology of fish in the Olifants River (eastern

Transvaal). Project K5/350. Report to the Water Research

Commission by the Department of Zoology, Rand Afrikaans University,

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Wepener, V. (1990). Die effek van swaarmetale by variërende pH op die

bloedfisiologie en metaboliese ensieme van Tilapia sparrmanii

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ASSESSMENT OF INTERNATIONAL PRACTICES ON THE USE OF FISH IN TOXICITY TESTING AND ADAPTATIONS FOR SOUTH AFRICAN CONDITIONS

CHAPTER 7

GENERAL CONCLUSIONS, RECOMMENDATIONS AND SCOPE FOR FUTURE RESEARCH

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7.1. General conclusions

It is clear from the literature that there is no complete consensus within the international toxicity-testing fraternity regarding what the qualifying criteria concerning the most suitable toxicity-testing fish species to be used. On one hand, the United States (through the USEPA (1993) Guidelines) advocates the use of fish species that have natural populations within their indigenous waters. This notion is completely feasible, as they do have indigenous fish that are capable of successfully fulfilling the criteria of a suitable toxicity testing species. Indeed, the fathead minnow (P. promelas) is regarded as the standard toxicity testing fish species used throughout the USA. This species of fish is deemed suitable due to its relatively large reproductive capacity and ease of maintenance and breeding to even the novelist aquarist. It is also a species of fish that is tolerant of a wide range of water chemistries and water temperatures to such an extent that personal communication with nature conservation authorities revealed that this particular species of fish has been blacklisted for importation to South Africa because of its reproductive potential within our natural waters. Therefore, if an indigenous fish can be found that is capable of matching the adaptability of the fathead minnow, then it would also be deemed a suitable testing species that can be used as a standard testing species. The Japanese medaka (O. latipes) is, however gaining popularity as a routine testing species amongst environmental researchers, which has been used as an attractive model test organism (Arcand-Hoy & Benson, 1998;

Lipscomb et al, 1998; Fournie et al, 1999; Scholz & Gutzeit, 2000). This species is, however, indigenous to Asian waters, with a distribution range incorporating Japan, Korea and adjacent coasts of China (Axelrod & Schultz,

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1990). The reasons for this is that O. latipes is a species that it is easily bred under laboratory conditions (Lipscomb et al, 1998; Sakamoto et al, 2001), is hardy, inexpensive and takes a wide variety of foodstuffs. This is emphasised by the fact that it is a species that is under consideration by the U.S. Army

Centre for Environmental Health Research, Fort Detrick, Maryland, USA, for use in the ‘Deintegrated Environmental Assessment Research Complex’

(Lipscomb et al, 1998). It is also claimed as being one of the easiest of the egg-laying species of fish to spawn within the aquarium (Coffey, 1986;

Axelrod & Schultz, 1990). Therefore, the trend to rather choose a fish species that is practicable in terms of ease of breeding and amenability to laboratory conditions is clear.

The European toxicity-testing fraternity, on the other hand, have not found a suitable indigenous species of fish that could successfully fulfil the criteria to be classed as a standard toxicity testing species. They therefore based their selection criteria on the practicability of working with the species within the laboratory rather than stipulating that the particular fish species had to be indigenous to their native waters. That is why the zebrafish (D. rerio) is used extensively throughout Europe as the standard toxicity testing species (from the ISO (1996) and OECD (1993) Guidelines). This species is, however, not the only species recommended as a suitable toxicity testing species. Other species recommended for testing do include native species as well as other various exotic species native to other continents, but D. rerio is the preferred species to work with within the laboratory. The Europeans have found the results of the toxicity tests using these exotic species quite capable of deriving

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The species of fish used (almost exclusively) throughout Japan and other

Asian countries, is O. latipes (Axelrod & Schultz, 1990), although D. rerio is also gaining popularity. This is largely due to O. latipes being indigenous to

Japanese, Korean and Chinese waters, as well as the high amenability to laboratory conditions and ease of breeding shown by this species (Sakamoto et al, 2001).

Locally, the guppy (P. reticulata) was also initially chosen on the basis of availability and ease of culture within the laboratory. There is also a vast amount of literature describing its maintenance and breeding as this species of fish is a particular favourite with the aquarist hobby fraternity, as well as this species being used internationally for laboratory tests, particularly in Europe and Brazil (Slabbert et al, 1998a). The choice of P. reticulata was therefore also one of practicability as it is easy to breed and maintain as well as being relatively sensitive (Slabbert et al, 1998a). The way in which the literature describes the ease of maintenance and breeding as well as the reproductive potential is, however, misleading and was found throughout the country to be an exaggeration of the potential of the species. This led to the search for an easier fish to culture and maintain within the laboratory that is suitable as a routine toxicity testing species.

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It is therefore clear that what is needed from a toxicity testing species deviates from what would traditionally and theoretically be seen as the most suitable toxicity testing species. This is due to the fact that toxicity testing laboratories do routine tests on a commercial basis and therefore the practicability of the proposed species of fish begins to outweigh the theory that the species needs to be an indigenous as well as a ‘super-sensitive’ species of fish to successfully derive water quality data of our local waters. The sensitivity and representivity of a species should not, however, be outweighed by the cost of culturing the organism, as this is the actual purpose of fish toxicity testing.

Taken at face value, the most suitable testing species would, indeed, be the most sensitive indigenous species of fish, but realistically, this is not always a feasible concept. The term ‘feasible’ here is used in the context of ‘what is practical and cost effective’, as this is probably one of the most important aspects to a routine laboratory. Realistically, a routine testing laboratory does not have the luxury of ample space, time and expertise required to culture the most sensitive (suitable) testing species. Indeed, the least amount of time and effort should be required to culture the organisms, and, as long as suitable or representative results are obtained, the most suitable testing species chosen to be cultured should be on the basis of these requirements.

It must be realised that the successful culturing of fish requires a certain degree of expertise that often takes a lifetime to accumulate. This ‘skill’ for the fish is not something that can be learnt overnight by reading up on the subject in a textbook, it is something that develops over time within a person with a passion for culturing the organisms and is, ultimately, a relatively time consuming past time. Therefore, if the choice of a testing species is because

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CHAPTER 7 it must be the most sensitive species, it must be realised that the most sensitive species will ultimately be the one that shows the least amenability to laboratory conditions. The most sensitive species would also be the most difficult as well as often the most expensive one to culture, and the culturing of such species would require the services of an expert in the field – only adding to the expense of the operation. These are all aspects that need to be critically considered as they are facets of routine toxicity tests that are, in reality, the ones where the most economical choice is the best. The next best choice to therefore possibly consider is a fish species that shows (and continues to show) a very high amenity to be successfully cultured on demand under laboratory conditions, with relatively little effort, expense, as well as space and time. This species is D. rerio. As long as this choice of species is capable of showing consistent, reliable as well as repeatable responses to a wide range of toxicants over a wide range of toxicant concentrations, and is relatively easy and cost effective to culture and maintain within a typical laboratory, then this choice of fish species warrants closer inspection. This should include making recommendations in terms of testing protocols and possibly testing by a wide range of laboratories throughout the country to determine if the proposed protocols produce acceptably similar and consistent results when carried out by different technicians. The large amount of literature and documentation available regarding this species, with reliable data already extensively evaluated by the international toxicity testing community (aspects recommended by Slabbert et al, 1998b), makes this an attractive testing species of fish. This study showed that the concept of developing a protocol for the use of indigenous fish in routine bioassays is

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CHAPTER 7 quite possibly not a priority at this stage as there are exotic species of fish that are realistically easier to culture and, at the same time show similar responses to toxicants. Therefore, if the particular species of fish successfully shows uniform results in terms of sensitivity and fecundities, then it should be considered. Many laboratories purchase their testing organisms on a routine basis from fish culturing farms that have the space as well as the expertise to do so, but this is not always the most desirable option. The source and therefore the health of the fish are often questionable as well – both important aspects to consider when using fish for toxicity testing (Slabbert et al, 1998b).

A laboratory that relies on the use of living organisms cannot afford to jeopardise the results of their tests due to an outbreak of a disease that could have been avoided. Routine toxicity test require that the age of the testing organism be known, and this is not always possible to be determined when the fish are brought in from an external source. The toxicity testing protocols also prefer that tests should be performed on organisms that are of uniform genetic stock. This again, is not always possible when purchasing fish from an external source. It is therefore desirable for the laboratory to be able to maintain and culture their own stocks of fish so that these variables can be narrowed to a certain degree so that more uniform results can be obtained from the tests performed.

As the use of fish taken directly from natural waters (especially from the receiving waters) is not advocated (Slabbert et al, 1998b) due to the lack of history, genetic uniformity and the irregular results obtained from the fish due to the stress factors placed on wild-caught specimens, the need to culture fish

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CHAPTER 7 within the laboratory is clear. If the collection of natural population (or a fish population is purchased from a commercial hatchery) is deemed necessary, then a thorough acclimation period (of up to one month) is recommended

(Harris et al, 1994; Nirmala et al, 1999; Djomo et al, 1995; van den Belt et al,

2002). This acclimation period places a time delay on the results obtained from the test – further escalating the cost of the test. The reality that all the fish culture stock within the same laboratory is kept under the specific conditions of that particular laboratory also warrants further explanation. An indigenous population of fish from one certain area will have, over time, adapted to the water chemistry and physical conditions found within the particular system that they are naturally found in. This is true for all fish.

There is therefore the possibility (over time) of that same population of fish being able to adapt to the conditions found within a typical laboratory.

Therefore, over time, the variations in sensitivity shown by fish populations within the same laboratory that share a similar genetic strain will eventually begin to narrow and show responses to similar toxicants that are similar to other closely-related species also maintained within that same laboratory.

Therefore, unless a fish population is consistently maintained and cultured under the conditions of where it was originally found, there will eventually be a shift in the sensitivity of the fish as it adapts to the conditions found within the laboratory in terms of water chemistry, environments, commercially prepared foods, etc. Having said this, however, different species of fish will always show variations in terms of their sensitivities to various toxicants, and this must not be overlooked. It does, however, imply that the use, and continued use, of an exotic fish species should not be ruled out as this species of fish

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For the reasons mentioned above, the use of the zebrafish (D. rerio) is advocated in this study. There is already a large database available in terms of biology and toxicity evaluations for this species of fish, and is seen by the international toxicity testing fraternity as a ‘white rat’ of laboratory studies.

This particular species of fish showed uniform results in spawning, with the added advantage that they were very reliable in the timing of the spawnings.

They therefore did not require to be spawned on an ongoing basis as their reliability meant they were only spawned a few days before they were required for tests (this time period is of course variable, and would be determined by the age that the testing organisms would be required to be).

Relatively minimal space is required for their successful maintenance and cultivation, with no special equipment being required to do so. This means that the cultivation is cost effective as well as a relatively simple practise, not requiring specialist training – but, having said this, it must be emphasised that the successful culturing of any species of fish requires a certain degree of knowledge and experience. The zebrafish showed sensitivities to the reference toxicant that were not significantly different to the other test species

– exotic as well as indigenous. The significantly different species of fish, in terms of sensitivity, was in fact an indigenous species that showed that it was significantly more resilient than the rest of species in question.

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This all shows that the fish species that is most practical to work with in terms of time and cost effectiveness, and still shows good results in terms of sensitivity to toxicants should be deemed suitable to be used for routine toxicity tests. The fact of it being indigenous or exotic should be of minor consequence, when selecting a test species for routine toxicity assessment.

Future scope for research includes:

· Reducing the space required to do the actual testing with the possibility

of performing the tests within micro plates. Preliminary tests were

performed during the course of this study using 24-well micro plates.

Three early-larval zebrafish individuals were placed within each well as

a trial acute toxicity test. The results obtained (even though not verified

due to the inability to determine the oxygen concentration within the

individual wells) were comparable to the tests undertaken in the 600 ml

beakers. The possible reduction in effort and cost when performing a

test at this reduced scale is clear when one considers the volume of

chemicals required for the preparation of dilution water and toxicants,

as well as the added glassware and the reduction of time that

performing the test on this scale. This is therefore definitely a

possibility to be explored in the future.

· Possibilities for doing chronic tests involving the use of embryos such

as the work done by Lange et al (1995) and Oberemm (2000) where an

early embryo test looks at deformations within the embryo as an

indication of toxicity should also be developed further.

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· The screening of more toxicants should also be done to test the

response of the zebrafish to a wide spectrum of toxicants with different

modes of action (i.e. organic as well as inorganic toxicants).

· Different laboratories should also perform these tests to determine if

the results are indeed repeatable when using fish from different

sources and kept under different conditions.

· The effort to find a suitable indigenous fish should be ongoing, with all

the different variables in terms of ease of culturing and amenability to

laboratory conditions scrutinised closely.

This type of research would be invaluable to the conservation of our indigenous fish as breeding protocols for one particular species of fish can possibly cover a broad spectrum of other indigenous fish. Endangered fish in particular would benefit greatly from this.

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7.2. References

Arcand-Hoy, L.D. and Benson, W.H. (1998). Fish reproduction: an

ecologically relevant indicator of endocrine disruption. Environmental

toxicology and chemistry, 17(1): 49-57.

Axelrod, H.R. and Schultz, L.P. (1990). Handbook of tropical aquarium

fishes. T.F.H. Publications, New Jersey. 718 p.

Coffey, D.J. (1986). The encyclopaedia of aquarium fish. Treasure Press,

London. 224 p.

Djomo, J.E., Garrigues, P. and Narbonnes, J.F. (1996). Uptake and

depuration of polycyclic aromatic hydrocarbons from sediment by the

zebrafish (Brachydanio rerio). Environmental toxicology and

chemistry, 15(7): 1177-1181.

Fournie, J.W., Hawkins, W.E. and Walker, W.W. (1999). Proliferative lesions

in swimbladder of Japanese medaka Oryzias latipes and guppy Poecilia

reticulata. Diseases of aquatic organisms, 38: 135-142.

Harris, G.E., Kiparissis, Y. and Metcalfe, C.D. (1994). Assessment of the

toxic potential of PCB congener 81 (3,4,4’,5-tetrachlorobiphenyl) to fish

in relation to other non-ortho-substituted PCB congeners.

Environmental toxicology and chemistry, 13(9): 1405-1413.

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International Organization for Standardization. (1996). Water Quality –

Determination of the acute lethal toxicity of substances to a

freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei,

Cyprinidae)] – Part 1: Static method. ISO Report 7346-1 Second

edition, International Organization for Standardization, Switzerland.

Lange, M., Gebauer, W., Markl, J. and Nagel, R. (1995). Comparison of

testing acute toxicity on embryo of zebrafish Brachydanio rerio and RTG-

2 cytotoxicity as possible alternatives to the acute fish test.

Chemosphere, 30 (11): 2087-2102.

Lipscomb, J.C., Confer, P.D., Miller, M.R., Stamm, S.C., Snawder, J.E. and

Bandiera, S.M. (1998). Metabolism of trichloroethylene and chloral

hydrate by the Japanese medaka (Oryzias latipes) in vitro.

Environmental toxicology and chemistry, 17(2): 325-332.

Nirmala, K., Oshima, Y., Lee, R., Imada, N., Honjo, T. and Kobayashi, K.

(1999). Transgenerational toxicity of tributyltin and its combined effects

with polychlorinated biphenyls on reproductive processes in Japanese

medaka (Oryzias latipes). Environmental toxicology and chemistry,

18(4): 717-721.

Oberemm, A. (2000). The use of a refined zebrafish embryo bioassay for the

assessment of aquatic toxicity. Laboratory animal 29 (7): 32-40.

Organization for Economic Cooperation and Development (OECD). (1992).

OECD guidelines for testing of chemicals. Fish, acute toxicity test.

Guideline 203. OECD.

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Sakamoto, T., Kozaka, T., Takahashi, A., Kawauchi, H. and Ando, M. (2001).

Medaka (Oryzias latipes) as a model for hypoosmoregulation of

euryhyaline fishes. Aquaculture 193: 347-354.

Scholz, S. and Gutzeit, H.O. (2000). 17-a-ethinylestradiol affects

reproduction, sexual differentiation and aromatase gene expression of

the medaka (Oryzias latipes). Aquatic toxicology 50: 363-373.