Lincoln University Digital Thesis Copyright Statement The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). This thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: you will use the copy only for the purposes of research or private study you will recognise the author's right to be identified as the author of the thesis and due acknowledgement will be made to the author where appropriate you will obtain the author's permission before publishing any material from the thesis. THE FATE OF VETRAZIN@ (CYROMAZINE) DURING WOOLSCOURING AND ITS EFFECTS ON THE AQUATIC ENVIRONMENT THE FATE OF VETRAZIN® (CYROMAZINE) DURING WOOLSCOURING AND ITS EFFECTS ON THE AQUATIC ENVIRONMENT A thesis submitted in fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY in AQUATIC TOXICOLOGY at LINCOLN UNIVERSITY P.W. Robinson 1995 " ; " i Abstract of a thesis submitted in partial fulfllment of the requirement for the Degree of Doctor of Philosophy THE FATE OF VETRAZIN® (CYROMAZINE) DURING WOOLSCOURING AND ITS EFFECTS ON THE AQUATIC ENVIRONMENT by P.W. Robinson A number of ectoparasiticides are used on sheep to protect the animals from ill health associated with infestations of lice and the effects of fly-strike. Most of the compounds currently in use are organophosphate- or pyrethroid-based and have been used for 15-20 years, or more. In more recent times, as with other pest control strategies, there has been a tendency to introduce 'newer' pesticides, principally in the form of insect growth regulators (IGRs). Vetrazin® was the first IGR­ based compound to be registered for use as an ectoparasiticide in Australia and New Zealand. The active ingredient of Vetrazin® is cyromazine, a triazine-based compound. Prior to the introduction of Vetrazin®, and indeed more recently with other 'new' pesticides, little was, or is known regarding the fate of these compounds during woolscouring. Additionally, given the present practice for woolscours to discharge their aqueous effluents to the aquatic environment, little was, or is known regarding their effects on these receiving waters. In addition, cyromazine is a chemically 'unique' compound, apparently possessing chemical and environmental stability, as well as being highly polar. This uniqueness required the development of specialised techniques for its analysis. Its environmental stability and polarity also suggested that it could become a significant aquatic contaminant, certainly in localised situations. This thesis outlines the development of a clean-up method for the analysis of cyromazine residues in woolscouring waste waters and on greasy (shorn) and scoured wool. A clean-up method using Extract-Clean lld ion-exchange cartridges and analysis by both gas chromatography with nitorgen­ phosphorus detector (GCINPD) and gas chromatography/mass spectrometry (GC/MS) is described. The fate of cyromazine residues during the scouring of greasy wool was investigated by both laboratory and industrial pilot plant trials. Cyromazine was shown to be readily removed in the first three scouring bowls of a woolscour. A simple model is described that can be used to predict cyromazine effluent concentrations based on a known content for greasy wool. These effluent 11 concentrations can then be used to assess compliance with a proposed environmental quality standard (EQS) established for cyromazine, based on aquatic bioassays. Aquatic bioassays were conducted with both Chironomus zealandicus (Diptera: Chironomidae) larvae and eggs and Deleatidium sp. (Ephemeroptera: Leptophlebiidae) nymphs. Cyromazine possessed negligible acute toxicity to both organisms, with the concentration required for 50% mortality (LCso) being greater than 100 mg/I. However, significant chronic toxicity to C. zealandicus was observed, with a' lowest observable effect concentration (LOEC) of 25 ~gI1 being recorded. A proposed environmental quality standard (EQS) value of 1.0 ~g/l has been suggested, a figure supported by limited aquatic toxicity studies performed elsewhere. Cyromazine was shown to dominate approximately 30% of the fly-strike control market in New Zealand. This high usage, plus a moderate EQS value, suggests that a minimum seven week withholding period be instigated between· dipping for fly-strike control and shearing of treated sheep. Limited toxicity testing was carried out on another IGR-based ectoparasiticide, Zenith®, containing diflubenzuron. This testing, combined with published aquatic toxicity data, suggests than an EQS value for this compound be set at 0.01 ~g/l and a withholding period for dipped sheep be set at 18 weeks. Such a long withholding period is unlikely to be attainable in practice such that, if highly aquatically toxic compounds are going to be used as ectoparasiticides on sheep, then woolscours are going to have to invest in technology to remove pesticides from their effluents. Keywords: Vetrazin®, cyromazine, Zenith®, diflubenzuron, insect growth regulators (IGR), sheep ectoparasiticides, fly-strike, sheep dipping, withholding periods, Chironomus zealandicus, Deleatidium sp., environmental quality standard (EQS), aquatic bioassays, toxicity, wool, woolscouring. iii ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES ix LIST OF FIGURES xii LIST OF ABBREVIATIONS xiv 1. INTRODUCTION 1 2. REVIEW OF THE LITERATURE 4 2.1 Fly-strike control 4 2.2 Scouring of wool and environmental contamination 6 2.3 Cyromazine 8 2.3.1 Fly-strike control 9 2.3.2 Mode of action 10 2.3.3 Cyromazine - degradation/detoxification 15 2.3.4 Cyromazine - environmental toxicity 20 2.3.5 Cyromazine resistance 23 2.4 Toxicology 25 2.4.1 Effects of chemicals on organisms 32 2.5 Bioassay procedures 32 2.5.1 Bioassay test procedures 33 2.5.2 Test organisms 35 2.5.3 Water quality 36 2.5.4 Test protocol 37 2.6 New Zealand freshwater chironomids 39 2.6.1 Phylogeny and taxonomy 39 2.6.2 Chironomid life cycle 40 2.6.3 Toxicity testing and rearing of chironomids 43 IV ·2.7 Analysis of pesticides in woolscouring emuents and on wool 46 2.7.1 Extraction of solid samples 49 2.7.2 Extraction of liquid samples 50 2.7.3 Clean-up of extracts 51 2.7.4 Analysis of samples 54 3. CLEAN-UP OF WOOLSCOURING WASTE WATERS 56 3.1 Introduction 56 3.2 Materials and equipment 57 3.2.2 Chemicals 57 3.2.3 Equipment 57 3.3 Chemical analyses 58 3.3.1 GC method 58 3.3.2 Sensitivity and linearity 60 3.3.3 Internal and external calibration 63 3.4 Clean-up techniques 66 3.5 Summary and conclusions 78 4. THE BEHAVIOUR OF CYROMAZINE IN AQUEOUS SOLUTIONS 80 4.1 Materials and methods 80 4.1.1 Adsorption/desorption behaviour 80 4.1.2 Simulated laboratory scouring trials 81 4.1.3 Fate of cyromazine during woolscouring 82 4.2 Results 84 4.2.1 Adsorption/desorption behaviour 84 4.2.2 Simulated laboratory scouring trials 86 4.2.3 Fate of cyromazine during woolscouring 91 4.3 Discussion 99 4.3.1 Adsorption/desorption behaviour 99 v 4.3.2 Simulated laboratory scouring trials 99 4.3.3 Fate of cyromazine during woolscouring 100 5. THE TOXICITY OF CYROMAZINE AND DIFLUBENZURON TO CHIRONOMUS ZEALANDICUS (CHIRONOMIDAE) AND DELEATIDIUM SP. (LEPTOPHLEBIIDAE) 102 5.1 Introduction 102 5.2 Materials 103 5.2.1 Chemicals 103 5.2.2 Tanks 103 5.2.3 Test insects 103 5.3 Methods 105 5.3.1 Acute toxicity tests 106 5.3.2 Chronic bioassays 107 5.3.3 Vulnerable life stage 107 5.3.4 Microscopic examination 108 5.4 Results 108 5.4.1 Acute toxicity tests 108 5.4.2 Chronic tests 114 5.4.3 Vulnerable life stage 120 5.5 Discussion 121 5.6 Conclusions 126 5.7 Observations on the biology of Chironomus zealandicus in laboratory cultures 127 6. GENERAL DISCUSSION AND RECOMMENDATIONS 132 6.1 Analytical method 132 6.2 Determination of an EQS for cyromazine 132 6.3 Limits for cyromazine in wool scouring effiuents 133 VI 6.4 Withholding periods for cyromazine dipped sheep prior to shearing 135 6.5 Determination of an EQS for diflubenzuron 137 6.6 Withholding periods for diflubenzuron dipped sheep prior to shearing 137 6.7 Recommendations 138 7. SUMMARY AND FUTURE WORK 139 7.1 Summary 139 7.1.1 Analysis of cyromazine 139 7.1.2 Fate of cyromazine in a woolscour 139 7.1,.3 Rearing and bioassay procedures for Chironomus zealandicus 139 7.1.4 Toxicity testing of diflubenzuron 140 7.1.5 On-farm use and recommendations 140 7.2 Future work 140 8. REFERENCES 142 9. ACKNOWLEDGEMENTS 160 10. APPENDIX 1 161 10.1 The use of ectoparasiticides on sheep inNew Zealand: results from an on-farm survey. 161 10.2 Introduction 161 10.3 Methods 161 10.4 Results and discussion 162 10.4.1 General farm information 162 10.4.2 Shearing practices 164 10.4.3 Dipping practices 166 10.4.4 Health and safety 171 vii 10.4.5 Disposal of dip solutions and containers 173 10.5 Conclusions 173 11. APPENDIX 2 175 11.1 Survey form 175 viii List of tables Table 2.1 Solubility of pesticides in water. 9 Table 2.2 Comparative mammalian toxicity of cyromazine and other chemicals used as sheep-dips. 10 Table 2.3 Species which have been shown to be susceptible to the toxicity of cyromazine. 11 Table 2.4 Physical effects of cyromazine on larvae of Lucilia cuprina. 12 Table 2.5 Calculated half-lives (at 20 °C) for the hydrolysis of s-triazines (Burkhard, 1979a; Burkhard and Guth, 1981). 18 Table 2.6 Cyromazine toxicity (LDso) to wildlife as determined by Ciba (Anon, 1979). 20 Table 2.7 Concentration (ppm) of cyromazine, CGA 19255 or diflubenzuron in the diet of Aedes aegypti larvae responsible for 100% mortality (Miller et ai., 1981).