University of Plymouth PEARL https://pearl.plymouth.ac.uk 04 University of Plymouth Research Theses 01 Research Theses Main Collection 2001 DEVELOPMENT OF CULTURE AND TOXICITY TESTING METHODS FOR THE FRESHWATER COPEPOD BRYOCAMPTUS ZSCHOKKEI BROWN, REBECCA JAYNE http://hdl.handle.net/10026.1/2085 University of Plymouth All content in PEARL is protected by copyright law. Author manuscripts are made available in accordance with publisher policies. Please cite only the published version using the details provided on the item record or document. In the absence of an open licence (e.g. Creative Commons), permissions for further reuse of content should be sought from the publisher or author. DEVELOPMENT OF CULTURE AND TOXICITY TESTING METHODS FOR THE FRESHWATER COPEPOD BRYOCAMPTUS ZSCHOKKEI by REBECCA JAYNE BROWN A thesis submitted to the University of Plymouth in partial fulfilment for the degree of DOCTOR OF PfflLOSOPHY Department of Biological Sciences Faculty of Science In collaboration with Brixham Environmental Laboratory, AstraZeneca UK Limited August 2001 90 0498290 X UNIVERSITV or PLTMOUTH Hem No, ' Date 7 N'GV 2001 3 Class No. Com. No. PLYMU •iRY DEVELOPMENT OF CULTURE AND TOXICITY TESTING METHODS FOR THE FRESHWATER COPEPOD BRYOCAMPTUS ZSCHOKKEI by Rebecca Jayne Brown ABSTRACT The aim of this thesis was to evaluate the use of a meiofaunal copepod as a test species for assessing the developmental and reproductive effects of toxicants relevant to freshwater ecosystems. The harpacticoid copepod Bryocamptus zschokkei was chosen as a candidate test species as it possesses several attributes (widespread distribution, small size and fast development times) that are considered pre-requisites for toxicity test organisms, and has previously been shown to be a sensitive component of the stream community to contaminant exposure. Prior to conducting toxicity tests with B. zschokkei, studies were performed to evaluate the efifects of water hardness and food quality on the development and reproduction of this copepod. These data were then used to define optimal culture conditions. Bryocamptus zschokkei was insensitive to water hardness at <150 mg 1"' (as CaCOa), reflecting its range of tolerance in the field and suggesting the potential for toxicity testing across a range of hardness levels. Food quality afifected development and reproduction: beech leaves {Fagus sylvatica L.), conditioned for 2 weeks supported optimal overall development to adult and reproduction of B. zschokkei and were chosen for use in subsequent culturing and testing regimes. Development and reproduction assays for assessing the effects . of environmental parameters on B. zschokkei were combined to produce the first full life-cycle toxicity test for a freshwater copepod. This life-cycle test was relatively quick (6 weeks at 20°C) and is highly reproducible. The effects of three reference chemicals, the trace metal zinc, the pesticide lindane, and the moulting hormone 20-hydroxyecdysone (20-HE), were measured using this toxicity test. There was no effect of 20-HE (0-269 ng I"') on the life cycle of B, zschokkei. Bryocamptus zschokkei was, however, relatively sensitive to zinc and lindane compared with other freshwater crustaceans although sensitivity depended on the chemical and the duration of exposure. A model of'equiproportional development' was used to aid interpretation of the mechanism of toxicity of lindane, which was found to act by significantly prolonging the development time to adult. Reproductive endpoints (numbers of eggs and nauplii per female) were the most sensitive measure of zinc and lindane exposure, with lowest observed effect concentrations (LOEC) of 0.48 mg Zn 1"* and 32 fig lindane 1"*, respectively. An increase in abortion frequency, observed for these contaminants, may have potential as a biomarker of stress for this copepod. In conclusion, it is proposed that toxicity tests with B. zschokkei should be included in contaminant assessment procedures for freshwater systems as they would increase the choice and ecological relevance of current testing regimes. in LIST OF CONTENTS COPYRIGHT STATEMENT i TITLE PAGE ii ABSTRACT iii LIST OF CONTENTS iv LIST OF HGURES viu LIST OF TABLES xi ACKNOWLEDGEMENTS xiv AUTHOR'S DECLARATION xv CHAPTER 1: GENERAL INTRODUCTION 1 7.7 The Role of Meiofauna in Stream Ecosystems 2 7.2 Pollution and Meiofauna J 7.5 Single-Species Tests 5 1.4 Marine Harpacticoid Copepods as Model Test Organisms 8 7.5 Freshwater Copepods as Test Organisms 10 1.6 Bryocamptus zschokkei (Schmeil, 1893) as a Representative Meiofaunal Test Species 13 7.7 Outline of Thesis 16 CHAPTER 2: INFLUENCE OF WATER HARDNESS ON THE POST- EMBRYONIC DEVELOPMENT OF BRYOCAMPTUS ZSCHOKKEI, 21 2 ABSTRACT 22 2.7 Introduction 22 2.2 Materials and Methods 25 2.2.1 Copepod Cultures 25 2.2.2 Conditioning Leaves 26 2.2.3 Test Medium 27 2.2.4 Post-Embryonic Development 28 2.2.5 Statistical Analyses 29 2J Results 30 2.3.1 Handling Frequency 30 2.3.2 Water Hardness 31 2.3.3 Sex Ratio 39 2.4 Discussion 40 2.4.1 Handling Frequency 40 2.4.2 Water Hardness 40 2.4.3 Copepod Development Times 42 2.4.4 Sex Ratio 45 2.5 Summary 47 IV CHAPTER 3: INFLUENCE OF WATER HARDNESS ON REPRODUCTIVE PARAMETERS OF BRYOCAMPTUS ZSCHOKKEL 48 3 ABSTRACT 49 3.1 Introduction 49 3.2 Materials and Methods 57 3.2.1 Test Medium 51 3.2.2 Experimental Animals 51 3.2.3 Experimental Design 51 3.2.4 Abbreviated Life Table 52 3.2.5 Statistical Analyses 55 3.3 Results. 55 3.3.1 Survival 55 3.3.2 Fecundity 57 3.3.3 Brood Interval and Embryonic Development 57 3.3.4 Abbreviated Life-Table 59 3.4 Discussion 61 3.4.1 Water Hardness 61 3.4.2 Fecundity 62 3.4.3 Embryonic Development 64 3.4.4 Individual variability 65 3.4.5 Life-Tables 66 3.4.6 Hatching Success 68 3.5 Summary 70 CHAPTER 4: EFFECT OF DETRITUS QUALITY ON THE DEVELOPMENT AND REPRODUCTION OF BRYOCAMPTUS ZSCHOKKEL 71 4 ABSTRACT 72 4.1 Introduction 73 4.1 Materials & Methods 76 4.1.1 Grazing Experiment 76 4.1.2 Scanning Electron Microscopy 77 4.1.3 The Effects of Detritus Type on Demographic Parameters 78 4.1.4 The Effects of Detritus Quality on Demographic Parameters 79 4.1.5 Demographic Parameters 80 4.1.6 Statistical Analysis 82 4.2 Results 83 4.2.1 Grazing Experiment 83 4.2.2 The Influence of Different Detritus Types 83 4.2.2.1 Microbial Community 83 4.2.2.2 Development 84 4.2.2.3 Reproduction 89 4.2.3 The Influence of Detritus Quality 92 4.2.3.1 Microbial Community 92 4.2.3.2 Development 95 4.2.3.3 Reproduction 97 4.3 Discussion 100 4.3.1 Grazing Experiment 100 4.3.2 The Influence of Different Types of Detritus 101 4.3.3 The Influence of Detritus Quality 104 4.4 Summary 106 V CHAPTER 5: THE EFFECTS OF THE CRUSTACEAN MOULTING HORMONE 20-HYDROXYECDYSONE ON THE DEVELOPMENT AND REPRODUCTION OF BRYOCAMPTUS ZSCHOKKEL 108 5 ABSTRACT 109 5.1 Introduction 109 5.2 Materials and Methods 112 5.2.1 Test Substance 112 5.2.2 Chronic Toxicity 113 5.2.3 Statistical Analysis 114 5.3 Results. 115 5.3.1 Long-Term Survival 115 5.3.2 Development 115 5.3.3 Reproduction 117 5.4 Discussion 121 5.4.1 Effects of20-Hydroxyecdysone 121 5.4.2 Solvent EfiFects 122 5.4.3 Full Life-Cycle Test 123 5.5 Summary 124 CHAPTER 6: THE EFFECTS OF ZINC ON THE DEVELOPMENT AND REPRODUCTION OF BRYOCAMPTUS ZSCHOKKEI 125 6 ABSTRACT 126 6.1 Introduction 126 6.2 Materials and Methods 128 6.2.1 Test Chemical 128 6.2.2 Acute Toxicity 129 6.2.3 Chronic Toxicity 130 6.2.4 Statistical Analysis 132 6.3 Results 132 6.3.1 Analytical Chemistry 132 6.3.2 Acute Toxicity 133 6.3.3 Long-term Survival 133 6.3.4 Development 134 6.3.5 Reproduction 135 6.4 Discussion 140 6.4.1 Acute Toxicity 140 6.4.2 Long-term Survival 143 6.4.3 Development 144 6.4.4 Reproduction 146 6.5 Summary 148 VI CHAPTER 7: THE EFFECTS OF LINDANE ON THE DEVELOPMENT AND REPRODUCTION OF BRYOCAMPTUS ZSCHOKKEI 149 7 ABSTRACT 150 7.7 Introduction J 50 7.2 Materials and Methods 755 7.2.1 Test Substance 153 7.2.2 Acute Toxicity 153 7.2.3 Chronic Toxicity 155 7.2.4 Chemical Analysis 155 7.2.5 Statistical Analysis 156 7.3 Results 757 7.3.1 Analytical Chemistry 157 7.3.2 Acute Toxicity 157 7.3.3 Long-term Survival 159 7.3.4 Development 162 7.3.5 Reproduction 163 7.4 Discussion 165 7.4.1 Acute Toxicity 165 7.4.2 Long-Term Survival 168 7.4.3 Development Times 168 7.4.4 Reproduction 171 7.5 Summary 7 75 CHAPTER 8: GENERAL DISCUSSION 176 8 DISCUSSION 177 8.1 Laboratory Culture 777 8.2 Small Size and Short Generation Time 7 78 8.3 Multiple Life-Stages 181 8.4 Ecological Relevance 182 8.5 Relative Sensitivity 183 8.6 Standardisation and Reproducibility 186 8.7 Conclusions and Recommendations 189 88 Summary 797 REFERENCES: 193 VII LIST OF FIGURES Figiu-e 1.1: The morphologically distinct A) naupliar and B) copepodid stages of Bryocamptus zschokkei 17 Figure 1.2: Male Bryocamptus zschokkei (left) clasping the female (right) by her caudal rami 18 Figure 1.3: Female Bryocamptus zschokkei carrying her egg sac externally 19 Figure 2.1: Effect of water hardness on A) naupliar development [Dn] and B) copepodid development [Dc] in Bryocamptus zschokkei at 'low-handling frequency' 32 Figure 2.2: Time taken (mean ± ISE) for male and female Bryocamptus zschokkei to moult to adult 35 Figure 2.3: The mean (± ISE) duration of individual copepodid stages for Bryocamptus zschokkei .36 Figure 2.4: An interaction plot showing the proportion of total development spent in each copepodid stage at different hardness in the Mow handling frequency' experiment 37 Figure 2.5: An interaction plot showing the proportion of total development spent in each copepodid stage high vs.