Vet-MARTI Veterinary Marine Animal Research, Teaching and Investigation. Port Curtis, Gladstone South Central Queensland FINAL REPORT.

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1 Vet-MARTI Veterinary Marine Animal Research, Teaching and Investigation GREEN TURTLE DISEASE INVESTIGATION at Port Curtis, Gladstone South Central Queensland FINAL REPORT November 2015 Dr Mark Flint 1,2 BVSC, BSC(HONS), MAPPLSC, MPHIL, PHD 1 Vet-MARTI unit School of Veterinary Science The University of Queensland Gatton, Queensland, School of Forest Resources and Conservation The University of Florida The Florida Aquarium s Center for Conservation 529 Estuary Shore Lane Apollo Beach, Florida, FLINTM@UFL.EDU 1

2 This report should be cited as: Flint, M. (2015). Green turtle disease investigation at Port Curtis, Gladstone South Central Queensland- Final Report. Report produced for the Ecosystem Research and Monitoring Program Advisory Panel as part of Gladstone Ports Corporation s Ecosystem Research and Monitoring Program. This report has been produced for the Ecosystem Research and Monitoring Program Advisory Panel as part of Gladstone Ports Corporation s Ecosystem Research and Monitoring Program. This publication has been compiled by Dr Mark Flint, Vet-MARTI, School of Veterinary Science, The University of Queensland. Gladstone Ports Corporation Disclaimer: Except as permitted by the Copyright Act 1968, no part of the work may in any form or by any electronic, mechanical, photocopying, recording, or any other means be reproduced, stored in a retrieval system or be broadcast or transmitted without the prior written permission of Gladstone Ports Corporation. This document has been prepared with all due diligence and care, based on the best available information at the time of publication, and the information contained herein is subject to change without notice. The copyright owner shall not be liable for technical or other errors or omissions contained within the document. The reader/user accepts all risks and responsibility for losses, damages, costs and other consequences resulting directly or indirectly from using this information. Any decisions made by other parties based on this document are solely the responsibility of those parties. Information contained in this document is from a number of sources and, as such, does not necessarily represent the policies of GPC. Enquiries about reproduction, including downloading or printing the web version, should be directed to ermp@gpcl.com.au 2

3 Executive summary Findings A previous study of green sea turtles in Queensland established baseline blood reference ranges (Flint et al., 2010a) and normal variance in pathology (Flint et al., 2010b) of functionally healthy coastal sea turtle populations during a period ( ) when no adverse natural or anthropogenic events occurred at the study sites to skew baseline values. In July 2011, a study was undertaken of green sea turtles in Port Curtis after considerable concern was expressed by the public, scientists, and commercial interests that health issues were arising from environmental catastrophes and anthropogenic activities. Several issues were reported and the report concluded that a combination of environmental and anthropogenic factors contributed to the elevated mortality rates (Flint et al., 2015). In May 2014, the population of Port Curtis green sea turtles was reassessed to determine the state of health of these animals as indicators of environmental recovery and/or stressor persistence. Complete ante-mortem examinations were conducted on a subset (n = 99) of Port Curtis green sea turtles. Of these 99 animals, 47% were juveniles, 27% were sub-adults and 26% were adults. Of the sampled animals, all except three were in good body condition. This is consistent with normal wild population figures. However, examination of bloods indicated several health anomalies are present among this population. Blood biochemistry performed on the 99 captured turtles indicated a high proportion of those sampled had chronic changes consistent with renal and hepatic insufficiencies. Haematology performed on the sampled turtles (n = 94) indicated 6% had minor white cell anomalies consistent with parasitism and infection. Of the 99 sampled turtles, three were recaptured animals from the 2011 investigation. These animals had a lower growth rate and subclinical blood biochemical anomalies when compared to average southern Great Barrier Reef green sea turtles. The findings from the current study suggested that: o The green turtle population of Port Curtis has made a significant, but not complete, recovery since the 2011 and subsequent environmental and anthropogenic stressors that resulted in elevated stranding and disease prevalence. o Subclinical pathology may be present. 3

4 Recommendations Recommendations suggested here are not presented as an obligation to any organisation or group. They merely report a collection of steps that summarise the current situation and present options for further monitoring. For continued monitoring and response in the Gladstone region: o No intervention is required at this stage. o Surveillance (diagnosis, documentation and quantification of existing disease syndromes and abnormal events should be identified as they arise) of this population should be continued. o Any signs of increased disease prevalence and/or stranding is treated as a disease outbreak and monitored accordingly. A rapid response to any abnormal events is essential if diagnoses are to be made. Specifically, comprehensive physical and necropsy examinations should be conducted within 24 hours of any elevated mortality event on any affected species. Sampling for toxins (inert storage) and other causative agents needs to be undertaken in both the animal and the environment during this response. Archiving samples may be required until funding is available or analyses. o Accordingly, trigger points need to be determined after discussions with the Department of Environment and Heritage Protection and any other interested appropriate parties as to what is an increased mortality. Inclusion of these agencies may contribute to a Queensland wide response strategy being implemented. o On-going population studies should be undertaken that include longitudinal measures (weighing individuals and assessing body condition over time). Several research facilities, such as EHP and CSIRO, have work in place of this nature that collects morphometrics, population structure, and breeding status data. Health surveillance data including serial assessment of population health, specific disease diagnoses and environmental monitoring should be included in this collected dataset. 4

5 Contents Executive summary... 3 Findings... 3 Recommendations... 4 Contents... 5 Terminology... 6 Acknowledgements... 7 Introduction... 8 Objectives... 8 Methods... 9 Study site and animal selection... 9 Recent site history... 9 Gender, maturity and breeding status Turtle health Clinical assessment Blood collection and preparation Blood biochemistry Haematology Recaptures Statistical analyses Results and Discussion External health assessments and morphology Population assessment Recaptures Clinical Pathology Population Assessment Biochemistry Haematology Recaptures Veterinary interpretation of findings Population Assessment Recaptures Morphology Clinical Pathology Recommendations Immediate Ongoing Outcome References

6 Terminology Term Definition (Blood and Studdert, 2000) Acute Having severe signs and a short course of hours Biochemistry (blood) Cachexic Catabolism Chronic Environmental stressor Haematology Phagocytic Sub-clinical Blood chemistry of living organisms and their vital organs In a marked state of health or nutritional disorder Destructive breakdown of complex compounds into simple compounds Persisting for a long time, 3 months or greater with little change in clinical signs. Factor within the local habitat acting on an organism Blood leukocyte types and frequencies (-study of) Any cell that ingests other cells and foreign particles Without clinical manifestation; either due to early disease stage or mild disease 6

7 Acknowledgements This disease investigation was conducted by The University of Queensland s Veterinary-Marine Animal Research, Teaching and Investigation (Vet-MARTI; unit for the Gladstone Port Corporation as part of a Queensland Department of Environment and Heritage Protection (EHP) studies in Port Curtis. This investigation was conducted under agreement and support by the Ecosystem Research and Monitoring Program Advisory Panel (ERMPAP) to meet conditional requirements of the ERMP. Sample collection was facilitated by EHP Chief Scientist Dr Col Limpus and Research Scientist Dr Ian Bell, and The University of Queensland EnTox staff Assoc Prof Caroline Gaus and Mr C. Alex Villa. James Cook University students assisted in the capture and measurements of the turtles. QPWS Gladstone generously allowed us access to their facility to assess the caught turtles. Blood biochemistry was performed by Mr Brian Bynon in the Clinical Pathology Laboratory in the School of Veterinary Science at The University of Queensland. Prof Paul Mills (Vet-MARTI, UQ) and Dr Col Limpus (EHP) offered constructive comments on this report. Laboratory sample processing was paid for by the Gladstone Ports Corporation. All contributions are gratefully acknowledged. 7

8 Introduction There are six species of sea turtles in Australia (Healey, 1997), all of which are reportedly found within the Port Curtis region, with the green sea turtle, Chelonia mydas, being the most abundant and resident within Port Curtis waterways (Limpus et al., 2013a, b, c, d, e, f). A previous study of green sea turtles in Queensland established baseline blood reference ranges (Flint et al., 2010a) and normal variance in pathology of a functionally healthy coastal sea turtle populations during a period ( ) when no adverse natural or anthropogenic events occurred at the study sites to skew baseline values (Flint et al., 2010b). In response to a dramatic increase in stranding numbers for the Rockhampton region (23 latitude block) in July 2011, an investigation into the health of green sea turtles was undertaken at the Boyne River estuary within Port Curtis. A subsample of the population was subject to clinical examination and blood sampling for routine blood profiles, and 12 deceased turtles underwent a thorough necropsy examination. This population of green turtles was found to be in a poor body condition and a range of infectious and non-infectious conditions were identified in the unhealthy turtles, including likely hepato-renal insufficiency (up to 81%, 27/33 based on clinical pathology), and cachexia (92%, 11/12), parasitism (75%, 9/12), cardiopulmonary anomalies (42%, 5/12), gastroenteritis (25%, 3/12), masses (25%, 3/12) and mechanical impediments (17%, 2/12 based on necropsy). Overall, there was no evidence to indicate a unifying disease as a primary cause of the mass mortality. Recent adverse weather events, historic regional contamination and nearby industrial activities were identified as potential causative factors (Flint et al., 2015; Gaus et al., 2012b). In May 2014, Port Curtis green sea turtles were again sampled, including turtles from the same area as those sampled in 2011, to determine the state of health of these animals as indicators of environmental recovery and/or stressor persistence. Interim reports were made available. Objectives The objectives of this study were to: 1. Determine the health status of Port Curtis green sea turtles for Compare findings with previous results for Port Curtis (2011) and Queensland ( ). 8

9 Methods Study site and animal selection Investigations to assess the clinical health of green turtles were undertaken over a 2 day period in early May 2014 in Port Curtis. This served as a reassessment of the health of turtles for comparison with the 2011 study undertaken at the Boyne River Estuary at Boyne Island, approximately 26 km south-east of Gladstone and at the Pelican Banks in north-eastern Port Curtis. The 2011 study was in response to an elevated concentration of green turtle strandings in the 23 latitude block that includes Port Curtis and Port Alma. Live green turtles (n = 99) were collected from the intertidal and sub-tidal waters and underwent morphometric assessment, health assessment and blood sampling. Turtles were collected in shallow inter-tidal and shallow sub-tidal waters via the turtle rodeo technique (Limpus, 1978). Recent site history The study area had recently undergone several regional and localised environmental events: Following a period of prolonged drought, a strong La Niña event on the southwestern Pacific Ocean with associated cyclone activity brought heavy, prolonged rainfall to most of coastal eastern Queensland during December 2010 and January 2011, producing the wettest summer on record to that date for Queensland (BOM, 2012). This resulted in extreme flooding of a number of rivers along the Queensland coast, including the Fitzroy, Calliope and Boyne Rivers, and had significant impacts on the local coastal environment, contributing to reductions in biomass of seagrass meadows in the Port Curtis region through increased turbidity and settling of silt on seagrass meadows (McCormack et al., 2013). The year 2011 saw a dramatic increase in the number of stranded marine turtles along the Queensland coast; peaking in April, with reported stranding numbers in the Rockhampton region (23 S latitude block which includes Port Curtis) almost six times higher than in previous years (Meager and Limpus, 2012). On May 21, 2011, dredging of the port for new shipping channels associated with port development on the northern aspect of the bay began (approximately six weeks prior to the initial sea turtle health investigation but after the initial escalation of stranding events). This included dredging and land reclamation activities being undertaken for the construction of multiple liquefied natural gas processing and export terminals in the Western Basin area of the port (Gladstone Ports Corporation, 2011). Stage 1A of the dredging activities ended in September The bund wall around a land reclamation site in the Western Basin at the Port of Gladstone was completed on 21 July 2011 and the pumping of dredge spoil into this reclamation area began on 6 September The bund wall was 9

10 alleged to be leaking sometime between July and September (Diggles and Diggles, 2014; IRP, 2014; Pattison-Sowden, 2014) with increased harbour water turbidity noted in early September 2011 (IRP, 2014). Actions commenced to reduce sediment leakage from the area. In 2012, seagrass meadows were noted to be recovering; and continue to recover as per State Government and GPC reports. In January 2013, a flood event caused by Cyclone Oswald impacted the area with effects anecdotally reported to be greater than the 2011 event. In May 2014, sea turtles were assessed for recovery in the period following these events. Gender, maturity and breeding status Gender, maturity and breeding status were determined using a combination of morphological measurements and gonad examination via ultrasound (Limpus et al., 2005). Turtle health Each selected turtle was clinically assessed and a blood sample collected from the cervical sinus fossa (external jugular fossa) (Flint et al., 2010a; Owens and Ruiz, 1980). Clinical assessment Turtles in good body condition, displaying no clinically apparent abnormalities (Herbst and Jacobson, 2003), including neurological deficits (Chrisman et al., 1997) or lesions of the carapace or plastron (Flint et al., 2010a), were classified as clinically healthy. Animals being of average body condition were classed as healthy or poor depending on lesions and other recorded abnormalities. Turtles in poor body condition and/ or exhibiting clinical abnormalities were classified as clinically unhealthy and examined in detail to determine cause of disease. Blood collection and preparation Blood samples were collected via the external jugular, prepared and examined using previously described techniques (Flint et al., 2010a; Owens and Ruiz, 1980), with minor modifications. In summary, blood samples (>20 ml) were collected from turtles using a 18 G 38 mm needle attached to a 50 ml syringe. For the blood biochemistry and haematology, voided samples were transferred to a heparinised evacuated tube, and then placed in coolers (4ºC), separated and serum frozen at -80ºC until assessment of blood biochemistry and haematology, which occurred within 6 months of collection. 10

11 Blood biochemistry Whole blood samples warmed to room temperature were run on a calibrated (using coefficients of variance) autoanalyser (VetScan VSZ) in accordance with test methodology outlined by the manufacturer. The following blood variables were measured: albumin, amylase, aspartate transaminase (AST), creatine kinase, cholesterol, creatinine, protein, gamma-glutamyl transpeptidase, globulin, alkaline phosphatase (ALP), alanine aminotransferase (ALT), urea, glucose, total bilirubin, sodium, calcium, magnesium potassium, triglyceride, chloride, bicarbonate, and phosphorous. Haematology Whole blood samples warmed to room temperature were run on a calibrated (using coefficients of variance) autoanalyser (VetScan HM II) in accordance with test methodology outlined by the manufacturer. Haematological measures (of leukocytes) were taken for thrombocytes, lymphocytes, heterophils, eosinophils, basophils and monocytes and used to estimate total white cell counts (TWCC) (Flint et al., 2010a; Work et al., 1998). Recaptures Of the 55 turtles captured in 2011 three of these animals were recaptured among the 99 turtles sampled in Statistical analyses Blood biochemistry and haematology results were compared with established reference intervals derived from healthy green sea turtles sourced from Queensland coastal waters using previously described calculators and methodologies (Flint et al., 2010a; Pesce et al., 2005; Solberg, 2006). Proportions of turtles with values outside the presented reference intervals were counted and presented using descriptive statistics for interpretation. 11

12 Results and Discussion External health assessments and morphology 2014 Population assessment Of the 99 sampled animals, 47% of those with records were juveniles, 27% were subadults and 26% were adults. These figures demonstrate an expected age class distribution for a coastal foraging area. Of these animals, 3% (3/99; one juvenile and two sub-adults) were in apparent poor condition. This is consistent with functional wild population morbidity figures and is not indicative of any acute or chronic negative environmental events. The poor conditioned animals had corresponding high proportions of blood anomalies in biochemistry in 67% (2/3, 95% CI ) of the cases. Outwardly, the green sea turtle population of Port Curtis appeared normal. Recaptures Table 1. Clinical health and morphometric data of the three juvenile green sea turtles caught in Port Curtis in 2011 and Body Condition CCL (cm) Weight (kg) Turtle K98432 Good Good QA23287 Good Good QA23288 Good Good The three turtles caught in 2011 and recaptured in 2014 were classed as being in good condition each time and all increased in curved carapace length (between 0.13 cm and 1.0 cm per year) and weight (between 0.23 kg and 0.63 kg per year) over the 3 year period (Table 1). Clinical Pathology 2014 Population Assessment The serum biochemical and haematological pathologies found in this investigation indicate potentially chronic disease processes of sub-clinical significance were active among the examined animals. These anomalies were not evident on external examination. Biochemistry In contrast to the low number of green sea turtles with outward indicators of poor health, several prevalent biochemical anomalies were detected in the blood analysis of the 99 examined animals. Low creatinine was detected in 60% (60/99; 95% CI ) of the examined turtles; as was phosphorous in 42% (42/99; 95% CI ); AST in 19% (19/99; 95% CI ); total bilirubin in 16% (16/99; 95% CI ); and creatinine kinase in 15% (15/99; 95% CI ). 12

13 Possible causes for these recorded blood biochemical anomalies include renal compromise (low creatinine and AST) and hepatic compromise (low bilirubin and creatinine kinase). Recovering from starvation/ cachexia (low phosphorous) is likely associated with these findings. All results are suggestive of pathology of the excretory system. However, it should also be noted that renal and hepatic diseases result from a very wide range of causes and there may not necessarily be a single or common cause of disease in populations of wild animals. The three identified poor condition animals had at least four parameters outside of the normal range. These parameters included creatinine kinase, total protein, albumin, globulin, and estimated lymphocytes and monocytes, indicating illhealth. However, for the majority of animals with abnormal biochemistry parameters, no overt clinical manifestation was noted on physical examination. Haematology Ninety nine blood smears were examined. Of the 99 examined bloods, 6 (6/99; 95% CI ) animals had total white cell counts that were marginally elevated. None of these were considered to be clinically significant. Of the examined elevated bloods, heterophilia and eosinophilia were the common presentations which are indicative of an inflammatory response and parasitic infections, respectively. Recaptures Lymphocytes, heterophils, eosinophils, creatinine kinase and urea were clinically lower in 2014 compared with Monocytes, albumin, total protein and globulins were clinically lower in 2011 compared with 2014 (Table 2). All are indicative of a return to values similar to those expected of a healthy population. 13

14 Table 2. Haematology and plasma biochemistry for three juvenile green sea turtles caught in Port Curtis in 2011 and Grouped QA23287 QA23288 K Haematology Estimated Thrombocytes Estimated Lymphocytes Estimated Heterophils Estimated Eosinophils Estimated Monocytes Estimated Basophils Estimated total WCC Biochemistry Albumin ALP ALT AST Total Bilirubin Calcium Cholesterol Creatinine Kinase Creatinine GGT Glucose Magnesium Phosphorous Total Protein Tri-glycerine Urea Sodium Potassium Chloride Bicarbonate Globulins

15 Veterinary interpretation of findings 2014 Population Assessment Considering the vital data gained from the three recaptured turtles, the outward apparent good health of the population examined, the depressed renal and hepatic blood chemistry markers in a considerable proportion of the population, and the subset of the blood smears that have been examined to date that indicated an infectious agent was present, the health of green sea turtles sampled in Port Curtis in May 2014 can be summarised as follows. Based on the body condition of the 99 animals examined in 2014, the green turtle population of Port Curtis has made a significant recovery from the 2011 and 2013 population health assessments where environmental and anthropogenic (commercial) stressors were speculated to result in elevated stranding and disease prevalence throughout Queensland. However, blood biochemical and haematological analyses indicated potentially chronic changes occurring within the population at the time of the 2014 sampling. Although reduced prevalence of animals with clinical pathologies were evident when comparing the examined periods of 2014 to 2011, these changes may be due to a population under persistent duress or still recovering from the 2011 and 2013 flooding events. Given the known history of this resident population based on elevated blood levels in 2011 that suggested environmental stressors, renal and hepatic issues are likely caused by toxic insults; either of a natural or anthropogenic origin. The extent to which these conditions are affecting the population (> 60% of examined animals) gives confidence this is operating at a regional level with common causative factors acting across all age classes. For green sea turtles, this is likely in the water or the food source (sea grass meadows). What cannot be concluded is whether the blood levels detected in 2011 were the result of an acute event (that caused the spike in mortalities) or preexisted as a localised chronic condition. Extrapolating from other hotspot populations at the mouths of Queensland s major water catchments, this was likely an acute event. For green turtles sampled in the Boyne Estuary in 2011, Gaus et al. (2012a) found a range of metals (arsenic, cadmium, cobalt, mercury, nickel, selenium, and vanadium) were present at levels clearly above those reported for green turtles and other marine megafauna species from other locations, and were near or above acute tissue based effect concentrations reported across various vertebrate taxa. It is unknown at this time whether these pollutants have persisted and, if so, at what concentration. Knowing current toxic loads, currently being examined by EnTox at The University of Queensland, will inform whether the green sea turtles examined in May 2014 were still under a regime of increased environmental stressors or whether recovery from a removed stressor is continuing at least 1 year after the most recent insult. Recaptures Examining three turtles from the pool of 155 turtles sampled in 2011 and 2014 creates limitations. It is difficult to make inferences on the clinical health of less than 2% of a subpopulation which was randomly sampled from within a large resident population. 15

16 The interpretations presented here should be acknowledged as limited to these three animals and inferences to the entire population should only be made using a larger dataset. Morphology These three turtles maintained an outward appearance of being healthy with a consistently good body condition score. However, their growth rates were notably lower than that of green sea turtles of the same size class for each reported southern Great Barrier Reef population (Limpus, 2008); and their blood chemistry and haematology suggest clinical anomalies when compared. Clinical Pathology In contrast to each turtle demonstrating outward indicators of good health and body condition, several biochemical anomalies were detected in the blood analysis of these animals in 2011 with evidence that these anomalies were greatly reduced but persisting in a subclinical form in The anomalies detected in 2011 were consistent with environmental stressors causing subclinical pathologies, assigned to natural and anthropogenic events (Flint et al., 2015). Elevated protein fractions (total protein, albumin and globulins) in 2011 were indicative of stressors on the hepatic system commonly caused by a toxic or contaminant insult. In 2014, these had resolved to within normal limits. Similar patterns were seen for creatinine kinase which is indicative of several physiological processes, including renal function but most commonly used to indicate catabolism (burning muscle and fat) caused by limitations in nutrition; and urea which is indicated of a renal-function impediment/ challenge caused by toxic or contaminant insults. Given the known history of this resident population based on elevated blood values in the 2011 sampling, renal and hepatic issues are likely caused by toxic insults; either of a natural or anthropogenic origin. For these three turtles, total white cell counts did not vary and were not significantly elevated at either sampling period. Although within normal limits, there was a lymphocytosis in 2011 indicative of an immune challenge that had resolved by 2014; however a low-grade monocytosis was detected in the recaptured animals in The overall clinical picture of these three turtles in 2014 is one of recovering animals that may still be under the influence of chronic insidious pathologies. Examination of a larger number of samples at the population is required before conclusions are drawn. 16

17 Recommendations Recommendations suggested here are not presented as an obligation to any organisation or group. Based on these findings, the following recommendations are proposed for continued monitoring and response in the Gladstone region: Immediate Assessment of the May 2014 bloods for heavy metal levels (currently being conducted by EnTox at UQ as a doctoral study). Surveys of forage and habitat resources should be performed to investigate any nutritional deficits noted in cohorts of these animals. Ongoing Serial surveillance of the resident population of green sea turtles should be undertaken annually for the next 5-10 years to assess recovery of the population from a medical standpoint. Although the examined animals appear externally normal, this investigation is indicating a subclinical stressor is still in effect. The impact of this subclinical stressor should be determined through assessing the fecundity of this local foraging population. Serial water and soil testing in the ilk to that being conducted by Port Curtis Integrated Monitoring Program, should continue to be undertaken to develop and use baseline data under a range of seasonal environmental conditions (precipitation, season and anthropogenic effects). Testing should include but not be limited to, detection of potential contaminants (pesticides, heavy metals, biological anomalies, fungi, viruses and bacteria), as this is a likely origin or at least an important aetiological factor. Suspected disease syndromes as they arise, or are identified, should be rigorously diagnosed and described. Outcome If the outlined recommendations are required, conducted and met, management and any operational plans undertaken by any organisation or group may require further revision based on findings. 17

18 References Blood, D.C., Studdert, V.P., Saunders Comprehensive Veterinary Dictionary 2nd Edition. W.B. Saunders, Bath. BOM, Record-breaking La Nina events. An analysis of the La Nina life cycle and the impacts and significance of the and La Nina events in Australia. Bureau of Meteorology, Melbourne. Chrisman, C.L., Walsh, M., Meeks, J.C., Zurawka, H., LaRock, R., Herbst, L.H., Neurological examination of sea turtles. Journal of American Veterinary Medical Association 211, Diggles, B., Diggles, G., Submission to the independent review into the leaking bund wall at the Port of Gladstone, Banskia Beach. Flint, M., Eden, P.A., Limpus, C.J., Owen, H., Gaus, C., Mills, P.C., Clinical and pathological findings in green turtles (Chelonia mydas) from Gladstone, Queensland: investigations of a stranding epidemic. EcoHealth 12, Flint, M., Morton, J.M., Limpus, C.J., Patterson-Kane, J.C., Murray, P.J., Mills, P.C., 2010a. Development and application of biochemical and haematological reference intervals to identify unhealthy green sea turtles (Chelonia mydas). The Veterinary Journal 185, Flint, M., Patterson-Kane, J.C., Limpus, C.J., Mills, P.C., 2010b. Health surveillance of stranded green turtles in southern Queensland, Australia ( ): an epidemiological analysis of causes of disease and mortality. EcoHealth 7, Gaus, C., Grant, S., Jin, N.L., Goot, K., Chen, L., Villa, A.C., Neugebauer, F., Qi, L., Limpus, C.J., 2012a. Investigation of contaminant levels in green turtles from Gladstone, In: Report to Gladstone Ports Corporation Limited and Department of Environment and Resource Management. National Research Centre for Environmental Toxicology (Entox), p Gaus, C., Grant, S., Jin, N.L., Goot, K., Chen, L., Villa, C.A., 2012b. Investigation of contaminant levels in green turtles from Gladstone. National Research Centre for Environmental Toxicology (Entox), Brisbane. Gladstone Ports Corporation, Briefing - Western basin dredging and disposal project. Environmental impacts., Gladstone, Queensland. Healey, J., Encyclopaedia of Australian Wildlife, 1st ed. Reader's Digest Pty Ltd, Sydney. Herbst, L.H., Jacobson, E.R., Practical approaches for studying sea turtle health and disease, In: The Biology of Sea Turtles Volume II. CRC Press, New York, pp IRP, Independent review of the bund wall at the Port of Gladstone, April 2014, Gladstone. Limpus, C., The Reef: uncertain land of plenty., In: Exploration North - a natural history of Queensland. Lloyd O'Neill Pty Ltd, Sydney, p Limpus, C.J., A biological review of Australian marine turtles. 2. Green turtle, Chelonia mydas (Linnaeus). Queensland Environmental Protection Agency, Brisbane. Limpus, C.J., Limpus, D.J., Arthur, K.E., Parmenter, C.J., Monitoring green turtle population dynamics in Shoalwater Bay: Great Barrier Reef Marine Park Authority & Environmental Protection Agency Queensland Parks and Wildlife Service, p

19 Limpus, C.J., Parmenter, C.J., Chaloupka, M., 2013a. Monitoring of Coastal Sea Turtles: Gap Analysis 1. Loggerhead turtles, Caretta caretta, in the Port Curtis and Port Alma Region. Report produced for the Ecosystem Research and Monitoring Program Advisory Panel as part of Gladstone Ports Corporation s Ecosystem Research and Monitoring Program. Limpus, C.J., Parmenter, C.J., Chaloupka, M., 2013b. Monitoring of Coastal Sea Turtles: Gap Analysis 2. Green turtles, Chelonia mydas, in the Port Curtis and Port Alma Region. Report produced for the Ecosystem Research and Monitoring Program Advisory Panel as part of Gladstone Ports Corporation s Ecosystem Research and Monitoring Program. Limpus, C.J., Parmenter, C.J., Chaloupka, M., 2013c. Monitoring of Coastal Sea Turtles: Gap Analysis 3. Hawksbill turtle, Eretmochelys imbricata, in the Port Curtis and Port Alma Region. Report produced for the Ecosystem Research and Monitoring Program Advisory Panel as part of Gladstone Ports Corporation s Ecosystem Research and Monitoring Program. Limpus, C.J., Parmenter, C.J., Chaloupka, M., 2013d. Monitoring of Coastal Sea Turtles: Gap Analysis 4. Olive ridley turtle, Lepidochelys olivacea, in the Port Curtis and Port Alma Region. Report produced for the Ecosystem Research and Monitoring Program Advisory Panel as part of Gladstone Ports Corporation s Ecosystem Research and Monitoring Program. Limpus, C.J., Parmenter, C.J., Chaloupka, M., 2013e. Monitoring of Coastal Sea Turtles: Gap Analysis 5. Flatback turtles, Natator depressus, in the Port Curtis and Port Alma Region. Report produced for the Ecosystem Research and Monitoring Program Advisory Panel as part of Gladstone Ports Corporation s Ecosystem Research and Monitoring Program. Limpus, C.J., Parmenter, C.J., Chaloupka, M., 2013f. Monitoring of Coastal Sea Turtles: Gap Analysis 6. Leatherback turtles, Dermochelys coreacea, in the Port Curtis and Port Alma Region. Report produced for the Ecosystem Research and Monitoring Program Advisory Panel as part of Gladstone Ports Corporation s Ecosystem Research and Monitoring Program. McCormack, C.V., Rasheed, M.A., Davies, J., Carter, A., Sankey, T.L., Tol, S., Long term seagrass monitoring in the Port Curtis Western Basin: quarterly seagrass assessments and permanent transect monitoring progress report November 2009 to November James Cook University, Cairns, p. 88. Meager, J.J., Limpus, C.J., Marine wildlife stranding and mortality database annual report III. Marine Turtle. Conservation Technical and Data Report 2012 (3):1-46. Owens, D.W., Ruiz, G.J., New methods of obtaining blood and cerebrospinal fluid from marine turtles. Herpetologica 36, Pattison-Sowden, M., Dredging facts from the fiction: GPC responds to claims, In: The Gladstone Observer. APN Group, Gladstone, pp. Pesce, A.J., Horn, P.S., Lewis, D., Reference Interval, Draft ed. Solberg, H.E., RefVal 4.11, Rykkinn. Work, T.M., Raskin, R.E., Balazs, G.H., Whittaker, S.D., Morphologic and cytochemical characteristics of blood cells from Hawaiian green turtles. American Journal of Veterinary Research 59,