Asellus Aquaticus and Pacifastacus Leniusculus
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University of Plymouth PEARL https://pearl.plymouth.ac.uk 04 University of Plymouth Research Theses 01 Research Theses Main Collection 2004 Biomarkers of chemical impacts in the freshwater invertebrates Asellusaquaticus and Pacifastacus leniusculus O'NEILL, ANNE JENNIE http://hdl.handle.net/10026.1/1687 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. Biomarkers of chemical impacts in the freshwater invertebrates Asellus aquaticus and Pacifastacus leniusculus by ANNE JENNIE O'NEILL A thesis submitted to the Uiii^ersit'y of Plymouth in partial fulfilm_ent-for the-clegre^^:of- DOCTOR OF PHILOSOPHY School of Biological Sciences Faculty of Science In collaboration with the Environment Agency December 2004 University of Pljmiouth Library Item No. Shelfmark . ft,.-, ^ ^ Biomarkers of chemical impacts in the freshwater invertebrates ^5^//H5 aquaticus and Pacifastacus leuiusciilus Anne Jennie 0*Neill Abstract The international scientific community has grown increasingly concerned that exposure to low levels of anthropogenic chemicals may disturb hormone function and/or sub lethal toxicity to other cells or organ systems in vertebrate and invertebrate species. This research programme was designed to develop, evaluate, and apply a range of biological and chemical indicators in the laboratory and/or field to assess the impact of sublethal toxicants including potential endocrine disaipting chemicals in the aquatic invertebrates, Asellus aquaticus (isopod) and Pacifasiacus leniiisciiius (decapod). Laboratory exposures of A. aquaticiis to environmentally realistic concentrations of known contaminants present within the river systems have demonstrated a link between health status and chemical exposure. Exposure to the organoiins, tributyltin and triphenyltin, and the pesticides, dichlorvos and endosulphan, demonstrated that adults were adversely affected at the biochemical (esterase activities) and physiological level (ventilation rates), as well as disrupting embryo development during in vitro investigations. Field studies demonstrated that cholinesierase and carboxyleslerase activities, ventilatory rates, growth, sex ratios, number of copulatory pairs and ovigerous females, fecundity, and embryonic growth, development and survival were all adversely affected to some extent in the isopod at sites downstream of industrial and domestic effluent discharges in comparison to control populations. Some links were established between the biological parameters measured and sediment faecal sterol content and/or water physico-chemical parameters and comparisons of multiple biomarkers did demonstrate differences between organisms collected at reference and contaminated sites which may be an indicator of environmental stress. A preliminary field investigation of P. lenittscultis collected from sites of mild sewage pollution indicated statistically significant changes in some responses at the cellular (cell viability), molecular (micronucleus formation), and physiological (heart rate) level. The significance of these changes at tb^:- population level was not determined, however, there is evidently a need to conduct laboratory exposures of P. leniusculits to sewage effiuent and its individual components to determine whether .he responses observed in the field are indicative of sewage contamination. In the present study, the sublethal responses of A. aquaticits and P. len 'utscidits to range of endocrine disrupting chemicals has been inve.stigated in laboratory and field settings and although cause and effect could not be demonstrated conclusively, this study has gont some way in identifying possible causative agents of these adverse effects in invertebrate populations. CONTENTS Abstract //V Chapter I: Introduction 1 1.1. Crustacean endocrinology- I 1.1.1. Moulting and limb rcgcneraiion 4 1.1.2. Reproduciion 7 1.1.3. Pigmeni hormones 13 1.1.4. Meiabolic hormones 14 1.2. Assessment of aquatic pollution 15 1.3. Environmental monitoring and the use of bioniarkcrs 16 1.4. Choice of test organisms 1}< 1.4.1. Aselhts aqiiaticus 20 1.4.2. Pacifastacus leniuscultis 20 1.5. Choice of sample sites 21 1.6. Molecular and cellular biomarkers 22 1.6.1. Cholincsicrasc and carboxylesierase aciiviiy 22 1.6.2. Lysosomal neutral red retention 26 1.6.3. Micronucleus assay 27 1.7. Physiological bioniarkers 28 \ J.\. Vemilaiion rales 29 1.7.2. Hear! rate 30 1.8. Population measures 31 1.8.1. Pre-copulaiory pair formation and reproductive output 31 1.8.2. Viability of offspring 33 1.8.3. Sex determination and sex ratios (distribution) 34 1.8.4. Size spectra analysis 36 1.9. Assemblage measures 37 1.9.1. Ganwiams. AseUus ratios 37 1.10. Chemical markers 39 1.11. Aims and objectives 44 1.12. Outline of thesis 45 Chapter 2: Optuuisation of biomarkers techniques, materials and methods 48 2.1. Determination of sewage contamination in river sediment using markers of faecal sterols 48 2.1.1. Chemicals 48 2.1.2. Sample collection 49 2.1.3. Sediment particle size analysis 49 2.1.4. Elemental composition 51 2.1.5. Sediment extraction 51 2.1.6. Deierminalion of faecal sterol content 52 2.2. Collection and maintenance of organisms 53 2.2.1. Aselhts aqmticus 53 2.2.2. Pacifasiaciis leniusculus 54 2.3. Optimisation of biomarker techniques in Aselius aquatiats 54 2.3.1. Collection of haemolymph from A. aqtiaticit.s 54 2.3.2. Cholinestcrase and carboxylesterase assay 55 2.3.2.1, A. aqiiaiicus cholinesterase and carhnxyle. "rase assay 64 2.3.3. Ventilation rales 66 2.3.3.1. A. aquaiictis veniilaiion rate a.ssay 66 2.3.4. Population measures 67 IV 2.4. Optimisation of techniques in P. leniiiscultis 72 2.4.1. Choice of crayOsh amicoagulanis 72 2.4.2. Cholinesierase and carboxylesierase assay 73 2.4.2. L P. leniusculus cholinesterase and carboxylesterase assay 81 2.4.3. Lysosomal neutral red retention 82 2.4.3.1. P. lenittscitlus neutral red retention assay 83 2.4.4. Micronucleus assay 85 2.4.4.1. P. lenittsculits microniicleus assay 89 2.4.5. Heart rate 89 2.4.5.1. P. leniiisctilus heart rate assay 90 2.4.6. Population measures 90 2.5. Statistical analysis 91 Chapter 3: The biochemical, physiological anil developmeiUal effects of tributyltin oxide and tripheiiyltin oxide on the isopod Asellus aquaticus 94 3.L Introduction 94 3.1.1. Tribuiyllin 95 3.1.2. Triphenyltin 98 3.2. Methods 100 3.2.1. Chemicals 100 3.2.2. Collcclion of organisms 101 3.2.3. Dose seleciion 101 3.2.4. Experimental scenario 1: 24 hr exposure to I ugl 'TBTO and TPTO 102 3.2.5. Experimental scenario 2: 7 day exposure lo sublethal conceniralions of TBTO and TPTO ... 103 3.2.6. Experimental scenario 3: 7 day exposure to lethal conceniraiions of TBTO and TPTO 103 3.2.7. Ventilation rales 104 3.2.8. Esterase enzyme activity 104 3.2.9. Experimental scenario 4: In vitro embryo exposure to sublethal concentraiions of TBTO and TPTO 104 3.3. Results 105 3.3.1. TBTO exposures 105 3.3.1. J. Experimental scenario I 105 3.3.1.2. Experimental scenario 2 105 3.3.1.3. Experimental scenario 3 109 3.3.1.4. Experimental .scenario 4 112 3.3.2. TPTO exposures 114 3.3.2.1. E.rperimental scenario / 1 14 3.3.2.2. Experimental scenario 2 I 15 3.3.2.3. E.xperimental scenario 3 119 3.3.2.4. Experimental scenario 4 122 3.4. Discussion 124 3.5. Conclusion 130 Chapter 4: The biochemical, physiological and developmental effects of a and p endosulphan and dichlorvos on the isopod Asellns aqnaticns 131 4.1. Introduction 131 4.1.1. Endosulphan 132 4.1.2. Dichlorvos 134 4.2. Methods 136 4.2.1. Chemicals 136 4.2.2. Collection of organisms j37 4.2.3. Dose selection I A7 4.2.4. Experimental .scenario I: I hr in\itro exposure to x and [5 endosulphan and dichlorvos .... 139 4.2.5. Experimental scenario 2: 4 clay exposure lo sublethal concentrations of cc and p endosulphan and dichlorvos 139 4.2.6. Ventilation rales 140 4.2.7. Esterase enzyme activity 140 4.2.8. Experimental scenario 3: In vitro embryo exposure lo sublethal concentrations of x and p endosulphan and dichlorvos 140 4.3. Results 141 4.3.1. oc and p endosulphan exposures 141 4.3.1.1. Experimeniai scenario I 141 4.3.1.2. E-xpehmental scenario 2 143 4.3.1.3. Experimental scenario 3 146 4.3.2. Dichlorvos exposures 148 4.3.2.1. Experimenuil scenario I 148 4.3.2.2. Experimental scenario 2 150 4.3.2.3. Experimental scenario 3 153 4.4. Discussion 155 4.5. Conclusion 162 Chapter 5: Evalnalion of the toxic impact of known sewage discharges in the Folly/Worsley Brook using the isopod Asellus aquaticus 164 5.1. Introduction 164 5.2. Study-site 166 5.3. Field sampling and methods 167 5.3.1. Water quality and sediment analysis 167 5.3.2. .4. fi7//a//c«j sampling and methods 168 5.4. Results 169 5.4.1. Analysis of Environment Agency water quality databases to determine key contaminants ... 169 5.4.2. Sediment size analysis and faecal sterol determination 171 5.4.3. Ventilation rales 175 5.4.4. Cholinesterase and carboxylesierase activity 175 5.4.5. Gammarus : Aselliis vi\\:\os 176 5.4.6. Population measures 178 5.4.7. Correlations between biomarkers and chemical parameters 190 5.5. Discussion 193 5.5.1. Waterqualily 193 5.5.2. Sediment faecal sterol content 195 5.5.3. Ventilation rate 202 5.5.4. Cholincsterase and carboxylesterase activity 203 5.5.5.