Causing Feet Abnormalities in the Cape Wagtail (Motacilla Capensis)

THE SEARCH FOR THE REASON(S) CAUSING FEET ABNORMALITIES IN THE CAPE WAGTAIL (MOTACILLA CAPENSIS)

TRACY LINDA MUNDAY

DISSERTATION

Submitted in the Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE

ZOOLOGY

in the

FACULTY OF SCIENCE

at the

UNIVERSITY OF JOHANNESBURG

Supervisor: Professor Victor Wepener Co-supervisor: Professor Gerhard Verdoorn

May 2006

My thesis is dedicated to my late beloved friend and mentor, my Granny Thelma, for her love and her adoration of Cape Wagtails.

Table of contents

Acknowledgements ...... i Summary ...... ii Opsomming ...... iv List of tables...... vii List of figures ...... viii List of appendices...... xii List of abbreviations...... xiii

Chapter One...... 1 1.1. Motivation ...... 1 1.2. Birds as indicators ...... 1 1.3. The Cape Wagtail (Motacilla capensis) ...... 3 1.4. Biomarkers and their function...... 4 1.5. Aims ...... 4 1.6. Objectives...... 4 1.7. Hypotheses ...... 5 1.8. Brief dissertation outline ...... 5 1.9. References ...... 6

Chapter Two ...... 7 2.1. Review...... 7 2.1.1. Cape Wagtails (Motacilla capensis)...... 7 2.1.1.1. General background and classification ...... 7 2.1.1.2. Location and habitat...... 8 2.1.1.3. Nesting, roosting and breeding ...... 8 2.1.1.4. Diet...... 9 2.1.1.5. The importance of their feet...... 9 2.1.1.6. Cape Wagtail status at present ...... 10 2.1.2. Birds as biomonitors...... 11 2.1.3. Xenobiotics: Pesticides and chemicals – action and toxicity ...... 12 2.1.3.1. General overview...... 12 2.1.3.2. Uptake...... 13 2.1.3.3. The stages and effects of toxicity...... 13 2.1.3.4. Organochlorines (OCs) ...... 15

2.1.3.5. Organophosphates (OPs) ...... 16 2.1.3.6. Secondary poisoning...... 17 2.1.3.7. Neurotoxicity ...... 18 2.1.4. Biomarkers: testing and response ...... 19 2.1.4.1. Non-destructive biomarkers...... 19 2.1.4.2. Blood...... 20 2.1.4.3. Biomarker types...... 21 2.1.5. Heavy metal accumulation and feather analysis...... 28 2.1.5.1. Heavy metals and their effects...... 28 2.1.5.2. Feathers and feather analysis ...... 30 2.1.6. The aspect of viral infection ...... 33 2.1.7. Skeletal abnormalities...... 33 2.1.8. Mites and infestation ...... 34 2.1.8.1. General introduction ...... 34 2.1.8.2. Types of bird mites ...... 36 2.1.8.3. Mites previously associated with Cape Wagtails...... 36 2.1.8.4. The Scaley-leg Mite (Knemidocoptes sp.) ...... 36 2.1.8.5. Knemidocoptes mutans ...... 38 2.1.8.6. Knemidocoptes jamaicensis...... 40 2.2. Conclusion...... 43 2.3. References ...... 45

Chapter Three ...... 52 3.1. Introduction ...... 52 3.2. Materials and methods...... 57 3.2.1. Site selection...... 57 3.2.2. Site descriptions...... 57 3.2.2.1. Western Cape sites...... 57 3.2.2.2. Eastern Cape sites ...... 58 3.2.2.3. Mpumalanga sites ...... 58 3.2.2.4. Gauteng sites...... 58 3.2.3. Sampling technique ...... 58 3.2.4. Bird in hand ...... 59 3.2.5. Biomarker analysis ...... 60 3.2.5.1. δ-Amino–levulinic acid dehydratase (ALA-D) ...... 60 3.2.5.2. Acetylcholinesterase (AChE)...... 61 3.2.5.3. Catalase (CAT) ...... 61

3.2.5.4. DNA damage ...... 62 3.2.5.5. Protein concentration ...... 63 3.2.6. Metal analysis...... 63 3.2.7. Statistical analysis...... 64 3.3. Results ...... 65 3.3.1. Spatial variation (biomarker response and metal accumulation)...... 65 3.3.1.1. Evaluation of metal levels against ecological quality objectives (EcoQOs)...... 72 3.3.1.2. Toxic units (TUs)...... 73 3.3.1.3. Relationships between biomarker response and metal exposure ...... 78 3.3.2. Variation between healthy and affected birds (biomarker response and metal accumulation) ...... 80 3.3.2.1. Toxic units (TUs)...... 85 3.4. Discussion ...... 85 3.4.1. Spatial differences in biomarker responses and metals ...... 85 3.4.1.1. Biomarkers...... 85 3.4.1.2. Metals in feathers...... 89 3.4.1.3. Metals in plasma ...... 97 3.4.2. Summary of the differences between healthy and affected Cape Wagtails...... 102 3.4.2.1. Biomarkers...... 102 3.4.2.2. Metals in feathers...... 103 3.5. References ...... 105

Chapter Four ...... 111 4.1. Introduction ...... 111 4.2. Materials and methods...... 114 4.2.1. Site selection and descriptions...... 114 4.2.2. Field work...... 114 4.2.3. Mite collection...... 114 4.2.4. Laboratory work ...... 114 4.2.4.1. Histopathology...... 114 4.2.4.2. Mite identification...... 115 4.3. Results ...... 115 4.3.1. Sampling observations...... 115 4.3.2. Histopathological examination of the ankle (tibia – tarsal joint)...... 117 4.3.3. Mite identification ...... 120 4.4. Discussion ...... 123 4.5. References ...... 128

Chapter Five ...... 130 5.1. Conclusions and recommendations ...... 130 5.1.1. Conclusions ...... 130 5.1.2. Recommendations ...... 133

Appendices ...... 134

ACKNOWLEDGEMENTS

My deepest thanks and appreciation go to:

• South Africa – Netherlands Research Programme on Alternatives in Development (SANPAD) and the National Research Foundation (NRF) for their financial support throughout this research.

• The University of Johannesburg for the use of its facilities and equipment.

• Professor Victor Wepener, my supervisor, for his never-ending patience and expert advice.

• Professor Gerhard Verdoorn, my co-supervisor, for his expert guidance and the incredible value of his wide array of networks among the birding fraternity of South Africa.

• Dr Truuske Gerdes (Virology Department, Onderstepoort) for efficiently examining samples at the prospect of a viral infection.

• Dr Emily Lane (Specialist Wild and Domestic Animal Pathologist) for her much-appreciated histopathological examination and report.

• Professor Avenant-Oldewage for her professional assistance with the histopathological interpretation.

• Dr Eddie Ueckermann (ARC) for his expert identification of the mites.

• The Western Cape Nature Conservation Board (CapeNature), the Department of Economic Affairs, Environment and Tourism (Eastern Cape), Mpumalanga Parks Board and Gauteng Nature Conservation for their issuing of permits required for the research undertaken.

On a more personal note, warm thanks go to:

• All those bird-loving people around the country who reported sightings of Cape Wagtails to me, which were appreciated and very useful.

• My dad for his unconditional love, limitless encouragement and above all else – for giving me the confidence to believe in myself.

• My mom and my sister for understanding the importance of this project to me and for their support and love.

• To my friend and fellow researcher, Jono, for his endless advice and assistance throughout my research.

SUMMARY

Keywords: Motacilla capensis, Cape wagtails, feet abnormalities, biomarkers, metals, Knemidocoptes jamaicensis

During the last decade, Cape Wagtails (Motacilla capensis) have been observed with several forms of feet abnormalities, such as missing toes and clubbed feet. The aim of this research was to find the reason for these abnormalities. The initial hypothesis was that ‘Feet abnormalities in the Cape Wagtail (M. capensis) are caused by the internal action of contaminants (e.g. pesticides and metals) through direct contact and/or secondary poisoning’. Wagtails were caught with mist nets and/or ground traps depending on the area and accessibility of the sampling sites. Wagtails with feet abnormalities, as well as those without, were caught at six localities around South Africa.

Indicators of pollutant exposure and effect were studied. The bioaccumulation of certain metals (measures of exposure) in the feathers such as aluminium (Al), arsenic (As), cadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), Pb, nickel (Ni), manganese (Mn), silver (Ag), strontium (Sr) and zinc (Zn) and Al, As, calcium (Ca), Cd, Cr, Co, Cu, Fe, Pb, Ni, Mn, Ag and Zn in the blood plasma were analyzed on the Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Biomarker testing (measures of effect) was carried out in the blood such as acetylcholinesterase (AChE) which tested for nerve transmission inhibition, δ-aminolevulinic acid dehydratase (ALA-D) which tested for lead (Pb) bioaccumulation, catalase (CAT) which tested for the invasion of oxyradicles and oxidative stress and DNA damage which tested for DNA band length alteration caused by stress due to xenobiotics.

Biomarker outcomes and metal analysis results were carried out on a site comparison basis. Acetylcholinesterase, ALA-D and DNA damage were found not to be linked to the occurrence of feet deformities at these sites. However, CAT revealed stress at all the sites which may have indicated the presence of a stress (or stresses) which could have been caused by feet abnormalities or other factors. A non-metric multidimensional scaling graph (NMDS) ordination suggested that biomarkers were not grouped according to sites that had affected birds and those that did not, but were rather assorted. Consequently, few real differences in biomarker responses were noted between healthy and affected birds. Catalase enzyme activity and DNA damage were suggested to be indicative of feet abnormalities although results were not convincing.

Secunda displayed the highest concentrations of metals in the feathers when compared to the other sites. Most metals were higher than reference values for contaminated areas and recent South African- reported concentrations. Almost all of the metals tested at each site exceeded the Ecological Quality

Objectives (EcoQOs) for metals in bird feathers, based on Weavers (Ploceus sp.) in the Gauteng region. Zinc levels at all the sites posed some concern. Chromium and Ni were suspected to possibly trigger feet and toe abnormalities in Cape Wagtails.

Levels of various metals in the plasma were higher at sites that had no deformities. This suggested that these metal concentrations did not cause or influence feet or toe deformities. Calcium levels were found to be lower at sites with affected birds which indicated that the deformities may perhaps be caused by decreased Ca concentrations. Metal concentrations were higher than the uncontaminated reference levels, but were lower than contaminated site reference values.

The NMDS ordinations suggested that no real significant differences were evident between metal groupings in the feathers and plasma. Secunda revealed the highest toxic unit (TU) which supported the fact that Secunda had the highest concentrations in the feathers and plasma for most metals. However, individual metal levels in the feathers and plasma were found to be generally higher in healthy birds than in affected birds. This indicated that it was doubtful that metal exposure (as reflected in feathers and plasma) caused or contributed to any feet or toe deformities, such as in the case of Cr, Ni and Zn. The TUs at Paarl and Somerset West in healthy birds proved to be higher than in affected birds, although Port Elizabeth was the opposite.

No significant differences were found to indicate spatial variation analysis of the affected birds versus the healthy birds. This led to the investigation of external factors, for instance parasitism, being the cause of the deformities. Further research, sampling and identification subsequently led to the discovery of the Scaley-leg mite (Knemidocoptes jamaicensis) in the dermal and epidermal layers of affected legs. All life stages were found on the host (the Cape Wagtails) which suggested that the mites reproduced and metamorphosized successfully on the birds. Pathologically speaking, infection by this mite is called Scaley-leg disease or knemidokoptic mange. In addition to this, a few wagtails were caught with artificial materials such as threads and fishing line entwined around their toes and feet which is also believed to be a contributing factor.

In this research, mite infestation was shown to have a detrimental effect on the legs and feet of wagtails. This could potentially have further negative effects on the continued survival of the Cape Wagtail. It is essential that it is understood why Cape Wagtails, in particular, are highly vulnerable to infection by K. jamaicensis. Further research is required to establish the precise action of this particular species of mite and how it can cause such severe and unsightly feet and toe abnormalities, as well as the time frame associated with the effects of infestation. Specific suggestions have been made in this study but these questions require further investigation.

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OPSOMMING

Sleutelwoorde: Motacilla capensis, kwikstertjie, pootabnormaliteite, biomerkers, metale, Knemidocoptes jamaicensis

Gedurende die laaste dekade is kwikstertjies (Motacilla capensis) opgemerk met verskeie vorme van pootabnormaliteite soos horrelpote en die afwesigheid van tone. Hierdie navorsing was daarop gemik om die oorsaak van sodanige abnormaliteite op te spoor. Die aanvanklike hipotese was dat ‘Pootabnormaliteite by die kwikstertjie (M. capensis) veroorsaak word deur die interne werking van kontaminante (bv. insekdoders en metale) deur direkte kontak en/of sekondêre vergiftiging’. Kwikstertjies is gevang met newelnette (“mist nets”) en/of grondwippe na gelang van die area en toeganklikheid van die steekproeflokaliteite. Kwikstertjies met pootabnormaliteite, asook dié daarsonder, is gevang in ses lokaliteite regdeur Suid-Afrika.

Indikators van besoedelingsblootstelling en -uitwerking is bestudeer. Die bioakkumulasie van sekere metale (maatstawwe van blootstelling) in vere soos aliminium (Al), arseen (As), kadmium (Cd), chroom (Cr), kobalt (Co), koper (Cu), yster (Fe), lood (Pb), nikkel (Ni), mangaan (Mn), silwer (Ag), stronsium (Sr) en sink (Zn), sowel as Al, As, kalsium (Ca), Cd, Cr, Co, Cu, Fe, Pb, Ni, Mn, Ag en Zn in die bloedplasma, is ontleed op die induktief gekoppelde plasma-massaspektrometer (IKP-MS). Biomerkertoetsing (maatstawwe van uitwerking) is op die bloed uitgevoer soos byvoorbeeld asetielcholienesterase (AChE) wat getoets het vir die inhibering van senuweegeleiding, δ- aminolevuliensuurdehidratase (DALA-D) wat getoets het vir Pb-bioakkumulasie, katalase (KAT) wat getoets het vir die indringing van oksiradikale en oksidatiewe stres, en DNA-skade wat getoets het vir DNA-bandlengteverandering veroorsaak deur stres weens xenobiotika.

Biomerkeruitkomste en metaalontledingsresultate is uitgevoer op ’n lokaliteitsvergelykingsgrondslag. Daar is gevind dat asetielcholienesterase, DALA-D en DNA-skade nie gekoppel was aan die voorkoms van pootgebreke by hierdie lokaliteite nie. KAT het egter stres aangedui by al die lokaliteite, wat verder kon gedui het op die teenwoordigheid van ’n streskomponent of -komponente wat kon veroorsaak word deur pootabnormaliteite of ander faktore. ’n Niemetriese multidimensionele skalerings- (NMDS) grafiek-ordinate het daarop gedui dat biomerkers nie gegroepeer was volgens die lokaliteite met geaffekteerde voëls en gesonde voëls, maar eerder as ’n sortering. Gevolglik is min werklike verskille in biomerkerreaksies opgemerk tussen gesonde en geaffekteerde voëls. Katalase- ensiemaktiwiteit en DNA-skade was dalk aanduidend van pootabnormaliteite, hoewel die resultate nie oortuigend was nie.

Vergeleke met ander lokaliteite, het Secunda het die hoogste konsentrasies van metale in vere getoon. Die meeste metale was hoër as die verwysingswaardes vir gekontamineerde areas en onlangse Suid- Afrikaans gerapporteerde konsentrasies. Bykans al die metale waarvoor by elke lokaliteit getoets is, het die ekologiese kwaliteitsmikpunte (EcoQOs) vir metale in voëlvere oorskry, gegrond op wewervoëls in die Gauteng-streek. Sinkvlakke by al die lokaliteite was rede tot kommer. Daar was aanvanklik vermoed dat chroom en nikkel moontlik poot- en toonabnormaliteite by kwikstertjies veroorsaak.

Vlakke van verskeie metale in die plasma was hoër in lokaliteite waar voëls geen abnormaliteite getoon het nie. Dit het daarop gedui dat hierdie metaalkonsentrasies nie die oorsaak van poot- of toonabnormaliteite was of dit beïnvloed het nie. Daar is gevind dat kalsiumvlakke laer was by lokaliteite met geaffekteerde voëls, wat daarop gedui het die misvorming veroorsaak kon wees deur verminderde Ca-konsentrasies. Metaalkonsentrasies was hoër as die ongekontamineerde verwysingsvlakke, maar laer as die verwysingswaardes van gekontamineerde lokaliteite.

Die NMDS-ordinate het gesuggereer dat daar nie werklik betekenisvolle verskille geblyk het tussen metaalgroeperings in vere en plasma nie. Secunda het die hoogste toksiese eenheid (TE) getoon, wat die feit ondersteun dat Secunda die hoogste konsentrasies in vere en plasma vir die meeste metale gehad het. Daar is egter gevind dat die individuele metaalvlakke in vere en plasma oor die algemeen hoër was in gesonde as in geaffekteerde voëls. Dit het daarop gedui dat dit te betwyfel was dat metaalbioakkumulasie in vere en plasma poot- of toongebreke veroorsaak of daartoe bygedra het, soos in die geval van Cr, Ni en Zn. Die TE’s by Paarl en Somerset-Wes by gesonde voëls het geblyk hoër te wees as by geaffekteerde voëls, hoewel die teenoorgestelde die geval was by Port Elizabeth.

Geen betekenisvolle verskille is gevind wat ruimtelike variasieanalise van die geaffekteerde voëls teenoor die gesonde voëls aangedui het nie. Dit het gelei tot die ondersoek van eksterne faktore, byvoorbeeld parasitisme, as die oorsaak van misvorming. Verdere navorsing, steekproefneming en identifikasie het gevolglik gelei tot die ontdekking van die skubpoot-myt (Knemidocoptus jamaicensis) in die dermale en epidermale lae van misvormde pote. Alle lewenstadia is op die gasheer (kwikstertjie) aangetref, wat daarop gedui het dat die myte suksesvol op die voëls voortplant en metamorfose ondergaan. Patologies gesproke staan infeksie deur hierdie myt bekend as skubpootsiekte of knemidokoptiese skurfte (omloop of brandsiekte). Daarbenewens is ’n paar kwikstertjies gevang wat kunsmatige stowwe soos tou en vislyn om hul pote en tone gekoek gehad het, wat ook as ’n bydraende faktor beskou is.

In hierdie navorsing is aangetoon dat mytinfestering ’n nadelige uitwerking op die bene en pote van kwikstertjies het. Dit kan potensieel ’n verdere negatiewe uitwerking op die voortbestaan van

kwikstertjies hê. Dit is noodsaaklik om te verstaan waarom kwikstertjies in die besonder hoogs kwesbaar is vir infeksie deur K. jamaicensis. Verdere navorsing word vereis om die presiese werking van die hierdie besondere spesie myt te bepaal, hoe dit sulke ernstige en onooglike poot- en toonabnormaliteite veroorsaak, asook die tydsduur wat met die effek van die infestasie verband hou. Spesifieke voorstelle word in hierdie studie aan die hand gedoen, maar daar is vrae wat verdere ondersoek vereis.

LIST OF TABLES

Table 1. Average dissimilarity based on biomarker responses among groups ...... 67

Table 2. Average dissimilarity based on metal analysis results...... 71

Table 4. Toxic Units calculated for each site...... 73

Table 5. Toxic Units for healthy and affected Cape Wagtails...... 85

Table 8. Condition of Cape Wagtail (M. capensis) legs, feet and toes found at several sites (percentage (%) of occurrences)...... 117

Table 9. Seasonal variation and the occurrence of healthy and affected birds ...... 122

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LIST OF FIGURES

Figure 1. Map showing positions of sampling sites. Numbers on the provincial maps represent sites (1 – Paarl, 2 – Somerset West, 3 – Port Elizabeth, 4 – Secunda, 5 – Dullstroom and 6 – Alberton and Rietvlei (Gauteng))...... 59

Figure 2. Biomarker responses in A – AChE, B - ALA-D, C – CAT and D – DNA ABPL at various sites. Bars indicate mean and standard error. Sites with significant differences (p<0.05) are indicated by letters (site 1 – Paarl, site 2 – Somerset West, site 3 – Port Elizabeth, site 4 – Secunda, site 5 – Dullstroom and site 6 – Alberton and Rietvlei, collectively named Gauteng)...... 66

Figure 3. A –NMDS ordination and B – hierarchical cluster analysis plot depicting biomarkers at various sites (site 1 – Paarl, site 2 – Somerset West, site 3 – Port Elizabeth, site 4 – Secunda, site 5 – Dullstroom and site 6 – Alberton and Rietvlei, collectively named Gauteng). The 2-dimensional stress after 10 iterations was 0.00. Numbers of groupings in Figure A assist in representation of Simper analysis and ANOSIM...... 67

Figure 4. Metal levels in the feathers (A – Al, B – As, C – Cd, D – Cr, E – Co and F – Cu) from different sites around South Africa. Bars indicate mean and standard error. Sites with significant differences (p<0.05) are indicated by common letters above the graph (site 1 – Paarl, n = 18, site 2 – Somerset West, n = 14, site 3 – Port Elizabeth, n = 44, site 4 – Secunda, n = 9, site 5 – Dullstroom, n = 4 and site 6 – Alberton and Rietvlei, collectively named Gauteng, n = 2)...... 69

Figure 5. Metal levels in the feathers (A – Fe, B – Pb, C – Mn, D – Ni, E – Ag and F – Sr) from different sites around South Africa. Sites with significant differences (p<0.05) are indicated by common letters above the graph. Bars indicate mean and standard error. Sites with significant differences (p<0.05) are indicated by letters (site 1 – Paarl, n = 18, site 2 – Somerset West, n = 14, site 3 – Port Elizabeth, n = 44, site 4 – Secunda, n = 9, site 5 – Dullstroom, n = 4 and site 6 – Alberton and Rietvlei, collectively named Gauteng, n = 2)...... 70

Figure 6. Zinc levels in the feathers from different sites around South Africa. Bars indicate mean and standard error (site 1 – Paarl, n = 18, site 2 – Somerset West, n = 14, site 3 – Port Elizabeth, n = 44, site 4 – Secunda, n = 9, site 5 – Dullstroom, n = 4 and site 6 – Alberton and Rietvlei, collectively named Gauteng, n = 2)...... 71

viii

Figure 7. A -NMDS and B – hierarchical cluster analysis plot depicting the metals found in feathers at various sites (site 1 – Paarl, site 2 – Somerset West, site 3 – Port Elizabeth, site 4 – Secunda, site 5 – Dullstroom and site 6 – Alberton and Rietvlei, collectively named Gauteng). The 2-dimensional stress after 10 iterations was 0.01. Numbers of groupings in Figure A assist in representation of simper analysis and ANOSIM...... 72

Figure 8. Metal levels in the plasma from different sites around South Africa (A – Al, B – As, C – Cd, D – Ca, E – Cr and F – Co). Bars indicate mean and standard error (site 1 – Paarl, n = 2, site 3 – Port Elizabeth, n = 4, site 4 – Secunda, n = 2 and site 5 – Dullstroom, n = 2)...... 75

Figure 9. Metal levels in the plasma from different sites around South Africa (A – Cu, B – Fe, C – Pb, D – Mn, E – Ni and F – Ag). Bars indicate mean and standard error. Metals with significant differences (p<0.05) among sites are indicated by z next to the graph letter (site 1 – Paarl, n = 2, site 3 – Port Elizabeth, n = 4, site 4 – Secunda, n = 2 and site 5 – Dullstroom, n = 2)...... 76

Figure 10. Zinc levels in the plasma from different sites around South Africa. Bars indicate mean and standard error. Metals with significant differences (p<0.05) among sites are indicated by z next to the graph letter (site 1 – Paarl, n = 2, site 3 – Port Elizabeth, n = 4, site 4 – Secunda, n = 2 and site 5 – Dullstroom, n = 2)...... 77

Figure 11. A -NMDS and B – hierarchical cluster analysis plot depicting the metals found in the plasma at various sites (site 1 – Paarl, site 3 – Port Elizabeth, site 4 – Secunda and site 5 – Dullstroom). The 2-dimensional stress after 10 iterations was 0.00. Numbers of groupings in Figure A assist in representation of simper analysis and ANOSIM...... 78

Figure 12. Relationship between CAT enzyme activity (µmol H2O2/mg protein.min) and average base pair length of DNA...... 79

Figure 13. Relationship between ALA-D enzyme activity (units per milliliter of red blood cells per hour) in the blood and Pb concentrations (µg/g dw) in the feathers...... 79

Figure 14. Relationship between ALA-D enzyme activity (units per milliliter of red blood cells per hour) in the blood plasma and Pb concentrations (µg/l) in plasma...... 79

Figure 15. Biomarker responses in A – AChE, B - ALA-D, C – CAT and D – DNA ABPL in healthy (represented as H - no feet abnormalities) and affected Cape Wagtails (represented by A - feet deformities). Bars indicate mean and standard error. Sites and bird conditions with significant

differences (p<0.05) are indicated by (site 1 – Paarl, site 2 – Somerset West and site 3 – Port Elizabeth)...... 81

Figure 16. Metal levels in the feathers of healthy (no feet deformities – H) and affected (feet abnormalities – A) Cape Wagtails from different sites around South Africa (A – Al, B – As, C – Cd, D – Cr, E – Co and F – Cu). Bars indicate mean and standard error. Metals with significant differences (p<0.05) among sites and conditions are indicated by next to the graph letter (site 1 – Paarl, n = 18H and 3A, site 2 – Somerset West, n = 8H and 1A and site 3 – Port Elizabeth, n = 35H and 10A)...... 82

Figure 17. Metal levels in the feathers of healthy (no feet deformities – H) and affected (feet abnormalities – A) Cape Wagtails from different sites around South Africa (A – Fe, B – Pb, C – Mn, D – Ni, E – Ag and F – Sr). Bars indicate mean and standard error (site 1 – Paarl, n = 18H and 3A, site 2 – Somerset West, n = 8H and 1A and site 3 – Port Elizabeth, n = 35H and 10A)...... 83

Figure 18. Zinc levels in the feathers of healthy (no feet deformities – H) and affected (feet abnormalities – A) Cape Wagtails from different sites around South Africa. Bars indicate mean and standard error (site 1 – Paarl, n = 18H and 3A, site 2 – Somerset West, n = 8H and 1A and site 3 – Port Elizabeth, n = 35H and 10A)...... 84

Figure 19. A – healthy feet of the Cape Wagtail; B, C and D – ‘whitish’ encrustations along both legs with the proliferation of excess tissue around the ankle area in D, and; E – encrustations along the leg...... 115

Figure 20. A – encrustations along the right leg and a deformed left leg; B and C – affected foot. . 116

Figure 21. A, B and C – thread entwined around the feet; D – thread entwined around the foot and an affected foot with encrustations and raised epidermal scales, and; E – thread entwined around one foot and the other a deformed foot...... 116

Figure 22. Arthropods (Scaley-leg mite, K. jamaicensis) in a small section (10x magnification) of white encrustations along the legs. Blocks indicate the position of the burrowing mite...... 117

Figure 23. Cross section (4-6 µm) of the ankle (tibia - tarsal joint) showing the appearance of healthy tissue. Sections were processed routinely and stained with Haemotoxylin and Eosin (Bancroft and Cook, 1982)...... 118

Figure 24. Cross section (4-6 µm) of the ankle (tibia - tarsal joint) showed mite presence. Sections were processed routinely and stained with Haemotoxylin and Eosin (Bancroft and Cook, 1982). .... 119

Figure 25. Line diagrams of K. jamaicensis found on the leg region of the Cape Wagtail (M. capensis). A – adult mite with A1 – front leg and A2 – back leg and; B - larva...... 120

Figure 26. Life stages of K. jamaicensis found in the encrustations along the legs of the Cape Wagtail (M. capensis). Figures A - nymph, B - larva, C - adult male and D - adult female...... 121

LIST OF APPENDICES

Appendix 1. Mean and standard errors of biomarkers at the study sites...... 134

Appendix 2. Mean and Standard error of metal concentrations in the feathers at each study site .... 135

Appendix 3. Mean and standard error of metal concentrations in the plasma for all study sites ...... 136

Appendix 4. Mean and standard error for biomarkers at sites 1 (Paarl), 2 (Somerset West) and 3 (Port Elizabeth) for healthy (H) and affected (A) birds ...... 137

Appendix 5. Mean and standard errors for metal concentrations in the feathers at sites 1 (Paarl), 2 (Somerset West) and 3 (Port Elizabeth) for healthy (H) and affected (A) birds ...... 138

xii

LIST OF ABBREVIATIONS

AChE - acetylcholinesterase CAT - catalase DNA ABPL(s) - DNA average base pair length(s) ALA-D - δ-aminolevulinic acid dehydratase ALA - aminolevulinic acid RBC(s) - red blood cell(s) WBC(s) - white blood cell(s) M. capensis - Motacilla capensis K. jamaicensis - Knemidocoptes jamaicensis Al - aluminium As - arsenic Cd - cadmium Ca - calcium Cr - chromium Co - cobalt Cu - copper Fe - iron Pb - lead Ni - nickel Mn - manganese Ag - silver Sr - strontium Zn - zinc Hg - mercury Se - selenium USEPA - United States Environmental Protection Agency EcoQO(s) - Ecological Quality Objective(s) TU(s) - Toxic Unit(s) NMDS - non-metric multidimensional scaling graph ICP-MS - Inductively Coupled Plasma Mass Spectrometry OC(s) - organochlorine(s) OP - organophosphorus / OP(s) - organophosphate(s) Primer - Plymouth Routines in Marine Environmental Research B. hagedash - Bostrychia hagedash A. tristis - Acridotheres tristis P. major - Parus major P. caeruleus - Parus caeruleus P. domesticus - Passer domesticus dw - dry weight

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CHAPTER ONE

1.1. MOTIVATION

Birds are a highly specialized class of vertebrates with characteristics and unique features that make them vulnerable to the intake and severity of toxins (Walker, 1983). The class of birds ‘Aves’, is characterized by the presence of feathers, a feature which is shared by no other animal group (Burger, 1993).

According to BirdLife South Africa (2005), there are four good reasons for conserving birds:

Birds are of great value in South Africa’s rich biodiversity (Worldwide, the combined value of 17 various ecosystems, such as pollination and water catchment, is estimated to be worth R97 - R163 trillion rand per annum which is about twice the world’s gross national product). The bird watching field is on the increase. The expenditure of bird watchers in South Africa alone is about R73 – R163 million per year. Birds are of great economic, cultural, ethical and spiritual value. Birds are good indicators of the state of the environment and, in general, places that are rich in bird variety are also rich in other forms of biodiversity.

1.2. BIRDS AS INDICATORS

Over the last few years, birds have served as early warning indicators of the state of the environment in terms of xenobiotics, such as pesticides. Xenobiotics are defined as those chemicals that are not naturally present, or do not occur, in high concentrations in the natural environment and are normally introduced by man-controlled activities (Connell et al., 1999). Birds serve as useful indicators because they are visible, sensitive to toxins, occupy a high position in the food chain and are of sufficient public interest. Many toxic compounds may be stored in their feathers, body tissues and organs. Metals are mostly sequestered in the feathers while there is still active blood supply, thus making them good indicators of exposure and excellent bioindicators of the ecosystem in general (Burger, 1993).

Based on past research and present studies, pesticides appear to have a higher bioaccumulation rate and are more acutely toxic to birds than to mammals. Walker (1983)

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

suggested that biochemical and physiological differences between the two groups may be responsible for this. Evidence suggests that birds have less effective detoxification and defence mechanisms against foreign chemicals than mammals do, such as:

1. Birds have relatively high body temperatures (41-42 ºC), compared with a lower temperature of 37 ºC in mammals. The maintenance of high body temperatures requires the frequent intake of food and in general, therefore, more pesticides may be accumulated. 2. Birds have smaller livers than mammals of a similar body size and, consequently, their detoxification rates are lower. 3. Birds are egg-laying biota. Eggs provide an excretory route for liposoluble xenobiotics and some compounds may reach high concentrations in the egg yolk, which may yield toxic effects in future generations. 4. Urine moves into the cloaca which allows for the reabsorbtion of xenobiotics from the cloaca itself. In mammals, urine is independent of the faeces. 5. The coccygeomesenteric vein which links the hepatic portal vein to the renal portal vein is found only in birds. Blood from the gastrointestinal tract enters the vein from which it is directed towards the kidneys and liver. In mammals, however, blood travels directly to the liver and not to, or via, the kidneys. The fact that birds have the ability to direct blood from the gut to the kidneys via the renal portal system increases the possibility of high concentrations of orally induced pollutants reaching the kidneys (Walker, 1983).

The factors mentioned above make avian species more vulnerable to pollutant uptake and effects. There is also evidence suggesting that birds are deficient concerning specific enzymes that aid with the detoxification of accumulated toxins (Walker, 1983). Birds also have lower levels of ‘A’ esterase than mammals which, according to Walker (1983), might explain why birds are particularly vulnerable to organophosphorus (OP) pesticides such as diazon and pirimiphos-methyl, as well as various organochlorine (OC) compounds.

In discovering the impact a pollutant might have on the environment, the main goal of an ecotoxicologist according to Fossi and Leonzio (1994), is to determine the effect of a contaminant on natural communities. In this case, it is expected that a contaminant(s) is responsible for the impact on Cape Wagtails (discussed in section 1.3 below). The effects of insecticides on birds in forests and agricultural areas have been investigated extensively in

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

the past in laboratories, but there has been little studied or recorded about such effects in suburban areas (Decarie et al., 1993).

1.3. THE CAPE WAGTAIL (MOTACILLA CAPENSIS)

Cape Wagtails (M. capensis) are insectivorous, passerine birds first described by Linnaeus in 1766 (Hockey et al., 2005). They were once common birds in the gardens of South Africa and were often seen patrolling garden lawns, school fields and golf course greens in search of food. Recently, however, it is thought that they have been affected by the use of various garden chemicals such as pesticides. Population numbers and sighting frequencies have decreased and Skead (1954 in Hockey et al., 2005) suggested that the decline in Cape Wagtail numbers during the 1940s was due to tarsal joint disease which is similar to poultry Scaley-leg. Fluctuations in population numbers may, over the last few years, have been due to the competition presented by House Sparrows (Passer domesticus) (Winterbottom, 1968 in Hockey et al., 2005).

In the last decade, M.capensis have been observed with missing toes and clubbed feet. Various reasons have been suggested for the abnormalities. Firstly, it is thought that secondary poisoning by pesticides may be a cause (Masterson, 1976 and Vernon, 1972 in Hockey et al., 2005). Secondly, the deformities could be caused by strangulation and consequently necrosis due to nesting materials such as threads and hair. However, Steyn (1996 in Hockey et al., 2005) reported that many Cape Wagtails had been observed with encrustations along the legs and feet and had been seen to lose toes, feet and parts of the leg. The cause of this was suggested to be the result of the entanglement of threads (Niven, 1981 in Hockey et al., 2005). Thirdly, abnormalities could be caused by microbial infections from the surfaces and substrates they walk on, such as hot asphalt (tar). Lastly, the hint at the possibility of a parasitic infection has also been put forward (Skead, 1954 in Hockey et al., 2005).

So, the question remains as to exact reason why Cape Wagtails are vulnerable to these feet abnormalities?

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

1.4. BIOMARKERS AND THEIR FUNCTION

A biomarker is a xenobiotically induced variation in cellular or biochemical components or processes, structures or functions that is measurable in a biological system or sample. They serve as endpoints which measure the effects of toxic compounds in biota and are useful tools in research because they can measure responses and effects in living organisms. They can be used to assess the overall state of an organism, which reveals the present state of the environment it resides in (Connell et al., 1999).

The ideal biomarker is specific for a chemical, detectable in small quantities, measured by non-invasive techniques, inexpensive and associated with prior exposure and with a good positive predictive value to a specific observed state. Exposure biomarkers can give information regarding the contamination of tissues by foreign compounds (Travis, 1993).

1.5. AIMS

The ultimate aim of this research was to find the reason(s) for the feet abnormalities in the Cape Wagtail (M. capensis) and to establish the frequency of the deformity at various sites.

1.6. OBJECTIVES

1. To collect blood and feather samples from Cape Wagtails at various sites around the country.

2. To explore the spatial variation of biomarker responses (AChE, ALA-D, CAT in different blood components and DNA damage in erythrocytes).

3. To explore the spatial variation of metal exposure by making use of feathers and plasma of Cape Wagtails.

4. To investigate and compare biomarker responses and metal levels in healthy and affected birds.

5. To determine whether there is a causal link between the severe feet abnormalities and biomarker responses and metal exposure.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

6. To gain insight into the biology and effect of the Scaley-leg mite (K. jamaicensis) in Cape Wagtails.

1.7. HYPOTHESES

Hypothesis One

‘Feet abnormalities in the Cape Wagtail (M. capensis) are caused by the internal action of contaminants (e.g. pesticides and metals) through direct contact and/or secondary poisoning’

The initial hypothesis (one) was rejected due to the results revealed by the biomarker responses and metal analysis in Chapter three. No significant inhibition of the enzymes tested revealed differences between healthy and affected birds. It was concluded, therefore, that it was highly unlikely that pesticides caused the deformities. As far as metals were concerned, levels in healthy and affected birds were compared. No real significant trends or differences could be established and it was concluded that none of the common heavy metals tested had a noteworthy influence on the feet abnormalities. The basic contaminant - induced outcomes had been tested for and the hypothesis was thus rejected. This led to the formulation of the next hypothesis, that an external factor such as a parasite infestation could be responsible for the deformities.

Hypotheses Two

‘An external factor, such as parasitism, is responsible for the feet deformities observed in the Cape Wagtail (M. capensis)’

1.8. BRIEF DISSERTATION OUTLINE

Chapter two serves as an introduction to all aspects of the subject matter related to this dissertation. Chapter three presents the results obtained with regards to the biomarker responses and metal exposure as reflected in metal bioaccumulation in feathers and plasma. Results were obtained for various sampling sites and a comparison between healthy and affected birds was carried out based on the same results. Chapter four considers feet abnormalities in the Cape Wagtail (M. capensis) and the Scaley-leg mite (K. jamaicensis), in

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

general, and explains how they are inter-linked. Lastly, chapter five serves as a summary for all the results and offers several recommendations obtained from this dissertation. A list of appendices contains the descriptive statistics for all analysis.

1.9. REFERENCES

Bird Life South Africa. 2005. Why Join Bird Life South Africa? Pp 3. Burger, J. 1993. Metals in Avian Feathers: Bioindicators of Environmental Pollution. Review Environmental Toxicology 5: 203-311. Connell, D., Lam, P., Richardson, B. and Wu, R. 1999. Introduction to Ecotoxicology. Blackwell Science Pty Ltd. United Kingdom. Pp 170. Decarie, R., DesGranges, J.L., Lepine, C. and Morneau, F. 1993. Impact of Insecticides on the American Robin (Turdus migratorius) in a Suburban Environment. Environmental Pollution 80: 231-238. Fossi, M.C. and Leonzio, C. 1994. Non-destructive Biomarkers in Vertebrates. Lewis Publishers. United States of America. Pp 345. Hockey, P.A.R., Dean, W.R.J. and Ryan, P.G. 2005. Roberts Birds of Southern Africa. VIIth edition. Tien Wah Press. Singapore. Pp 1296. Travis, C.C. 1993. Use of Biomarkers in Assessing Health and Environmental Impacts of Chemical Pollutants. Plenum Press Publishers. New York. Pp 284. Walker, C.H. 1983. Pesticides and Birds – Mechanisms of Selective Toxicity. Agriculture, Ecosytems and Environment 9: 211-226.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

CHAPTER TWO

2.1. REVIEW

2.1.1. CAPE WAGTAILS (MOTACILLA CAPENSIS)

2.1.1.1. GENERAL BACKGROUND AND CLASSIFICATION

Class: Aves Subclass: Neornithes Superorder: Neognathae Order: Passeriformes Suborder: Passeri Family: Motacillidae Subfamily: Motacillinae Genus: Motacilla Species: Motacilla capensis

Wagtails are a group of long-tailed birds whose Latin name ‘Motacilla’ is derived from their characteristic ‘wagging and moving of their tails’ up and down as they feed (Hockey et al., 2005). Their tails are known to aid in direction change (Engelbrecht et al., 2005). The wagtail family Motacillidae, at present, is subdivided into one genus with six species.

In South Africa there are three resident wagtail species and a further two species from the north that visit South Africa as non-breeding visitors in the summer. The most common wagtail species is the Cape Wagtail, known as the ‘Kwikstertjie’ in Afrikaans, ‘Um-Vemve’ in Zulu and ‘Mo-Tjoli’ in Sotho (Prozesky, 1983). It is about 175 mm `in length, grayish- brown above and white below with a black(ish) band or ‘bib’ between the chest and throat area, and white outer tail feathers (Winterbottom, 1967).

The Cape Wagtail is mentioned numerous times in Khoi, San and Zulu Folklore (Dunning, 1947 in Hockey et al., 2005) and the Cape Wagtail is viewed by the Xhosa people as the ‘bird of cattle’ and the ‘bird of good fortune’ and are therefore treasured by these people (Godfrey, 1941 in Hockey et al., 2005).

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

2.1.1.2. LOCATION AND HABITAT

Wagtails are most common south of 23 ºS and are found in a range of habitats from natural rural habitats to highly crowded urban locations. They naturally occur along watercourses such as rivers, lakes and dams as well as along the coast and are always associated with a water source and fairly open habitats. Wagtails are especially common around sewerage works (Engelbrecht et al., 2005). They are resident in most areas but some individuals may disappear from the highveld in winter and visit areas in KwaZulu-Natal and Zululand (Winterbottom, 1967). These birds show seasonal altitudinal migration tendencies, according to Engelbrecht et al. (2005).

Wagtails are fairly sedentary and remain in the same area for most of their lives, but it was recorded that an individual was once found 450 kilometers from where it was ringed (Engelbrecht et al., 2005).

2.1.1.3. NESTING, ROOSTING AND BREEDING

Nests are built in a clump of vegetation relatively close to the ground and often overhanging water. They normally consist of grass, pine needles, cotton, roots, feathers, hair and other artificial materials such as string and sundry fibres. The same nest can be used as many as six times by the same breeding pair which are known to mate for life, although some pairs build a new nest for every clutch of eggs. The clutch size may be two to four depending on the conditions at the time and the eggs take approximately two weeks to hatch. The young are cared for by both parents. Youngsters normally fledge about 15-16 days after hatching but if they are disturbed they may leave the nest earlier. Mortality amongst youngsters is high suggesting that normally only one out of every three eggs layed will survive to become an adult wagtail. Even then, the fledging faces challenges such as food, water and habitat shortages. To compensate for any loss, a pair of wagtails can breed up to eight times a year (Winterbottom, 1967).

Along the west coast of the Southern Cape most active nests are found around August, and in Cape Town around September. In the Eastern Cape, September is the peak time for breeding (Winterbottom, 1967). The Cape Wagtail is a monogamous breeder and will defend its territory, which may range between 15-40 hectares, although smaller territories have been recorded in urban areas (Engelbrecht et al., 2005).

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Cape Wagtails may congregate in very large groups known as roosts and up to 2 000 birds have been recorded at one roost (Winterbottom, 1967). They roost in reed beds and trees such as palms (Winterbottom, 1967), but they often roost in and around city centres (Skead, 1995, Winterbottom, 1964 and Keith et al., 1992 in Hockey et al., 2005). They may also roost singly, but communal roosting is popular during the winter months in numbers generally between 150 and 200. Before roosting, which occurs just before sunset, wagtails engage in a behaviour known as pre-roost gathering when they are getting ready to settle down for the evening. Roosts are a form of feeding congregation as reported by Taylor (2003). In recent years, such as in Tableview in the Western Cape, birds have been seen to gather in car parks and roost in conifer and palm trees. It has also been reported that Cape Wagtails are commonly found in and around shopping malls (Williams, 2004).

Christensen and Mortimer (2004) reported that a roost of Cape Wagtails occurs at Somerset West Mall. They noted that at about 19:20 (about 10 minutes after sunset) Cape Wagtails made a quick dash for the trees. By 19:28 there were hardly any wagtails left to be seen and by 19:33 all was quiet. As stated by Christensen and Mortimer (2004), ‘some years ago the confiding little Cape Wagtails were disappearing from our gardens and parks – how strange that they should make a return to a mall car park!’.

2.1.1.4. DIET

Wagtails (Motacilla) are insectivores, feeding mainly on insects such as flies, mosquitoes, termites and ants (Winterbottom, 1967), lawn crickets and worms (Engelbrecht et al., 2002). When they occupy areas of human inhabitance, they will eat mielie meal, porridge, bread crumbs, cake crumbs, raw meat, fat, suet and even grated cheese (Winterbottom, 1967). The diet of the Cape Wagtail as stated by Winterbottom (1967), is beneficial and not, by any means a concern or threat to humans.

2.1.1.5. THE IMPORTANCE OF THEIR FEET

Passerine species possess feet and toes with pads and folds separated by furrows. Covering the pads are numerous papillae considered to be important in heat regulation. If there are few papillae, contact with the substrate is reduced which lowers the rate of heat flow from the bird to the external environment and visa versa (Lennerstedt, 1975b in Lennerstedt (1), 1975). The top surface of the papillae makes contact with the bark when the bird perches on

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

a branch or twig. It is evident that the form and function performed by the papillae differs from species to species depending on the habitat they occupy. Passerine birds use trees for protection and surveying their habitat for food and water and, through this, their feet are often used which makes the feet vulnerable to various pressures and forces (Lennerstedt (2), 1975).

Steen and Steen (1965 in Frost and Siegfried, 1975) discovered the role of the unfeathered region of passerine bird legs. Their primary function is to assist in heat regulation. As air flows over the unfeathered region of the leg, heat is lost. In areas of warm temperatures, birds become heat stressed and sometimes heat loss mechanisms can become over-worked. However, the bare leg as in the Cape Wagtail has been shown to aid greatly in the loss of heat from the body to the external environment (Frost and Siegfried, 1975).

2.1.1.6. CAPE WAGTAIL STATUS AT PRESENT

In the past, the migration of Cape Wagtails into urban areas was influenced by the feeding opportunities presented by open watered lawns rich in food (Hayman and Arlott, 1994). In the quest to have lush and healthy lawns, garden owners, golf course managers and school field maintenance staff use pesticides and insecticides to get rid of insects and other pests. In 1966, Mr Marius Verster reported that in only a few months Cape Wagtail numbers in Beaufort West had decreased to the point where they had almost disappeared. He suggested that the reason for this may have been due to poisoning through lawn insecticides. He stated that it is ‘an alarming thought that the endearing little wagtail may be threatened with extinction over perhaps a wider area than may be suspected’ (Verster, 1966).

A speculation for the reason behind the diminishing numbers may be due to secondary poisoning (Masterson, 1976 and Vernon, 1972 in Hockey et al., 2005) which is caused by the extensive intake of harmful pesticides which causes internal damage to the bird. This possibly affects nerve transmission and thereby results in feet and toe deformities. Birds lose a lot of heat through their legs which is very important for birds that swim or stand in water such as the wagtail. If feet are deformed this may interrupt in the heat balance of the bird (Schmidt-Nielsen, 1997).

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Population numbers and areas of Cape Wagtails have changed considerably over the past few years due to human interference such as habitat destruction and infrastructure construction (Engelbrecht et al., 2005).

According to a report by Engelbrecht et al. (2005), the Cape Wagtail is a flag-ship species for many conservation efforts especially concerning the careful application and use of pesticides. There was a sharp decline in wagtail numbers during the 1950s and 1960s which was believed to be a result of the vast use of pesticides and insecticides, which was never proven to be true (Steyn, 1995 in Hockey et al., 2005). Rather, it was suggested that the diminishing numbers may be linked with urbanization during that time. Along with this came the change in biology of the bird due to the need for it to adapt to the change in the environment, which included a change in feeding type from an insectivore to an omnivore. It was also suggested that this caused a bottleneck in the population but gradually, as time progressed, their numbers increased. So, it is a question whether the drop in numbers was due to urbanization or the use of pesticides (Engelbrecht et al., 2005).

2.1.2. BIRDS AS BIOMONITORS

The use of birds as biomonitors has been prevalent since the early sixties when it became obvious that bird populations declined with human influence and associated pollutants (Denneman and Douben, 1992). Birds have been widely used to monitor the various levels of pollutants in the environment (Lock et al., 1991), especially heavy metals, due to their relatively high niche status in the food chain and wide range of distribution (Kim et al., 1995).

Birds are useful bioindicators as they are common, widespread, diurnal and easy to observe and monitor over several years (Burger, 1993), and they are representative of their area or territory (Battaglia et al., 2005). They consume a wide range of food types; insectivorous, omnivorous and piscivorous. They are sensitive; show early warning signs at very low levels of toxicants; show an effect of an accumulation of toxicants; and, the public shows a keen interest in them. The disadvantage of using birds, however, is that they are mobile and may be endangered which makes collection of samples difficult or even impossible. They usually feed over a very large area making the identification of point-source pollution difficult. Birds, however, are effective as they integrate the time versus space over which they feed. Birds are thus not advisable for the location of point source pollution but rather an area of

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

pollution (Burger, 1993). But, contradicting this, Dauwe et al. (2002) said that some birds are viewed as excellent biomonitors of point-source contamination.

Birds of prey have been used extensively in the past as monitors of environmental pollution because they are highly sensitive to chemical changes (Dauwe et al., 2003). Today in many areas, organisms are frequently exposed to varying mixtures of pesticides and mobile species, especially birds, are exposed to these conditions as they fly from area to area in search of food and water (Walker, 1998). In the past, passerines such as the Great Tit (Parus major) and Blue Tit (Parus caeruleus) have proved to be good biomonitors (Dauwe et al., 2002).

During the seasonal cycle, according to an article by van den Brink et al. (1997), birds undergo several changes in physiological condition. During times when food is scarce, the birds’ nutritional reserves are exhausted as any fat stores present are activated. Organochlorine pesticides are lipid soluble and, therefore, concentrations will increase as fat reserves decrease. So, as recommended by van den Brink et al. (1997), when working out pollution levels in avian species, it is necessary to take into account various ecological and physiological factors which could influence the bird such as feeding and breeding. Therefore, energy consumption and different activities carried out by the bird during the breeding season could result in fluctuating concentrations of OC pollutants within its body (Van den Brink et al., 1997).

2.1.3. XENOBIOTICS: PESTICIDES AND CHEMICALS – ACTION AND TOXICITY

2.1.3.1. GENERAL OVERVIEW

Pesticide use is common and occurs throughout the world. The by-products of OC pesticides negatively affect non-target species which may be in the vicinity of the action of the pesticides (Ownby et al., 2004). These compounds are highly lipophilic (i.e. they may sequester in inert tissues such as fat) (Connell et al., 1999) and resist metabolic breakdown and are, therefore, strongly biomagnified through trophic levels reaching higher levels at top predator position (Van den Berg et al., 1994 in Van Wyk, 2001).

Apart from urban area activities, agricultural practices are a major source of foreign compounds. In an effort to grow as many crops in as little time as possible and to make sure

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

a crop is of good quality, size and appearance, most farmers make use of pesticides and often do not realize the potentially detrimental effect they may have on the ecosystem (Connell et al., 1999). Some OC pesticides are non - biodegradable, remaining unchanged in their form for long periods of time. Due to this, natural ecosystems experience interference especially those biota high up in the food chain as they ingest a cumulative amount of pesticide residues in the food they eat (Jenkins, 2005).

2.1.3.2. UPTAKE

Birds with contaminated feathers might also ingest chemicals via preening of their feathers. Dermal exposure can occur by direct contact with the chemical or by water uptake resulting in absorption through the eyes or skin surface. Inhalation of the pesticide itself can be a significant route of uptake by biota (Rainwater et al., 1995).

Species such as thrushes (Turdus sp.) that search the top layer of mulch for seeds and food are also vulnerable to pollutant uptake. Chemical consumption may occur via four pathways:

• orally, whereby the bird ingests the contaminant directly; • through dead or infected insects; • through pesticide containing particles ; and • through contaminated water sources from irrigation water or run-off water which the bird might drink (Rainwater et al., 1995).

2.1.3.3. THE STAGES AND EFFECTS OF TOXICITY

The toxicity of a chemical is dependent on several factors and interconnected stages:

firstly, various factors may influence the delivery of the toxicant to its site of action or target site such as absorption, storage, activation, distribution or detoxification; secondly, the reaction with the primary target site or organ; thirdly, the biochemical and physiological responses elicited by the compound; and lastly, the expression of the toxicity in the organism (Connell et al., 1999).

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

The acute effects of certain pesticides towards non-target species comprise of two parts; firstly, the toxicity of the pesticide concerned and, secondly, the exposure by the organism to the contaminant (Kjaer and Jepson, 1994).

Certain xenobiotics are potent teratogens especially in avian species which cause malformation of embryos, resulting in congenital defects and diseases. Deformities have been reported in species such as cormorants and terns from the Laurentian Great Lake in Canada which were exposed to halogenated compounds such as dioxins (incinerated form of polychlorinated biphenyls (PCB) and OC pesticides which produce a toxic compound) (Connell et al., 1999).

There is continuing concern about the potential indirect effects of pesticides in the food chain. The impact of pesticides on the environment may reduce the numbers of weeds and insects available for food consumption by larger vertebrates but affects the productivity and breeding success of those biota that feed on these affected food resources. Indirect affects may occur due to pesticides (Boatman et al., 2004).

During the past 20 to 30 years, the increase in agricultural practices has accelerated due to the high demand for food to feed growing human populations. As a result, many avian species such as the Lapwing (Vanellus vanellus), Skylark (Alauda arvensis), Whitethroat (Sylvia communis), Swallow (Hirundo rustica), Linnet (Acanthis cannabina) and Partridge (Perdix perdix) have decreased in numbers (Cordi et al., 1996). It was shown that passerine species which forage or nest close to agricultural areas are at a greater risk of being affected by OP pesticides than those that do not. This statement was substantiated by the pattern of plasma cholinesterase (ChE) reactivations among various avian species that were tested (Maul and Farris, 2004).

Many effects of xenobiotic compounds have been reported in the past but none have been well researched and sufficient. These propositions included behavioural and chronic effects on the central nervous system; mutagenic, carcinogenic and teratogenic effects on the immune system; disruption to hormones; damage to the retina and reproductive systems and on lipid metabolism, to name a few (Johnson, 1993 in Travis, 1993).

The effect of a pesticide on organisms depends on the composition and potency of each pesticide. Some are highly toxic, causing nerve transmission interference and even

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

unexpected behaviour while others cause endocrine disruption that affects the hormone levels in the body. A bird that is particularly vulnerable to the effects of pesticides is the Peregrine Falcon (Falco peregrinus). Literature explains that in the 1960s and 1970s, the Peregrine Falcon approached extinction because of the severe effects of OC pesticides (Jenkins, 2005).

The effects of pesticides have found to be variable amongst biota of the same species. Each species responds differently and, therefore, a large number of different responses can be observed (Moore, 1965 in Steyn et al., 1985). Biota may be exposed to a variety of chemical compounds at one time which raises the question whether the toxic effects of pollutants are additive or do they just enhance the toxicity effect of the pollutants synergism(Johnston et al., 1993)?

In the late 1940s and early 1950s, it was noticed that pesticides had a profound effect on the breeding rate and population numbers of various falcon, hawk and eagle species (Jenkins, 2005), and it has been found that there is a correlation between declining Helmeted Guineafowl (Numida meleagris) populations and the use of pesticides (Crowe and Ratcliffe, 2001).

Some poisons are less detrimental but have long term residual effects. Biota can experience acute toxicity through direct contact with the contaminant (Henriques et al., 1997 in Van Wyk, 2001).

2.1.3.4. ORGANOCHLORINES (OCS)

On of the most famous and well known OCs is DDT as it was the most useful insecticide known in the history of man. DDT is a type of OC compound used long ago for the control of insects (Brown, 1978). It is estimated that around four billion pounds of DDT have been used since 1940. DDT affects the nervous system of biota where it interferes with nerve transmission between neurons causing muscles to twitch which often leads to convulsions and even death (Ware, 1991). High levels of DDT residue in birds are mobilized if the bird is starved or forced to be unnaturally active. This transforms the stored adipose or fat tissue and releases the OCs which had been stored into the blood circulatory system where the toxic compound settles in the phospholipids of the nervous system thus affecting nerve

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

transmission (Brown, 1978). The uptake of OC pesticides by organisms causes various behavioural and toxicological alterations (Cobb et al., 1992).

2.1.3.5. ORGANOPHOSPHATES (OPS)

Organophosphates were used instead of OCs for pest control because they are known to have a shorter life span in the environment and a lower potential to accumulate in a food chain (Rainwater et al., 1995). They have been used extensively in the past in agriculture, industry, government and military applications and are still widely used today. Organophosphates are a massive and complex group of compounds with many uses, including the control of pests. Organophosphates are linked to their ability to inhibit AChE which is a vital enzyme required for nerve functioning in the insect kingdom and various classes of higher order biota (Ozmen et al., 1999).

The central nervous system is a target organ for OPs where it affects neurotransmission by inhibiting AChE activity. Normal signaling between neurons and muscles is disturbed either by binding directly to acetylcholine (ACh) receptors and muscle cells or by actually blocking the action of AChE. The result is disruption of the nervous system and uncontrolled muscular contraction causing muscle fatigue, paralysis and, in extreme cases, death (Connell et al., 1999).

Some of the most widely used OPs are azinphos-methyl, chlorfenvinphos, chlorpyrifos, demeton, diazinon, dichlorvos, fenitrothion, malathion, parathion, phosalone, temephos and terbufos. It should be noted that not all OPs are used as pesticides. Some are used as defoliants, for example tributyl phosphorotrithioate (DEF), which is a strong inhibitor of cholinesterases (Lagadic et al., 2000).

Some OPs such as diazinon, monocrotophos and cyanophos are strongly selective towards avian species (Walker, 1983). They consist mainly of phosphate and may also be called as organic phosphates, phosphorus insecticides or phosphate insecticides. They are all derived from phosphoric acid which boosts their hazardous effects on biota. They are generally the most toxic of all the pesticides.

Compared to other animal groups, birds are very sensitive to OP pesticides because they have a very low level activity of ‘A esterase’s’ which function to hydrolyze any OPs that the

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

bird may accumulate. Acute and subacute toxicities of several OP pesticides have been determined in adult passerine birds but, according to a report by Cordi et al. (1996), few studies have been done on the responses elicited by OP compounds on nestling, passerine birds.

Birds are vulnerable to the long term effects of various OPs such as the anticholinesterase compounds. Compounds such as TOCP, haloxon, carbophenothion, EPN and mipafox have delayed neurotoxic effects that can be observed as a quick developing paralysis which may first appear in a few days or even a few weeks after exposure to these OPs (Johnson, 1975 in Walker, 1983). It is associated with demyelination of nerves and the phosphorylation of a protein termed ‘neurotoxic esterase’. Species that have been known to be vulnerable to this are the chicken (Gallus domesticus), the pheasant (Phasianus colchicus) and the Mallard Duck (Anas platyrhynchos) (Walker, 1983).

Birds associated with golf courses are often exposed to OPs where such pesticides are used for pest control. They thus may be directly exposed to the chemical or may accumulate it via secondary poisoning. From 1970 to 1985 a study was completed on 16 golf courses where OPs were used. A total of 900 avian deaths were reported on these golf courses during these 15 years (Rainwater et al., 1995).

Acute poisoning with OPs compounds has been observed in ring-necked pheasants. Symptoms included regurgitation, goose-stepping, ataxia, wing drooping, wing spasms, anal sphincter tenesmus, diarrhoea, myasthenia, dyspnea, prostration and death with wing-beat convulsions. With chronic toxicity a reduction in activity, fluffing of the feathers and lethargy were observed (Brown, 1978).

2.1.3.6. SECONDARY POISONING

Birds can act as very efficient vectors for pollutants. Species that prey on other biota are vulnerable to secondary poisoning (Walker, 1983). The aspect of secondary poisoning comes in when studying the bioaccumulation of pesticides in birds. According to a study done on the effect of OPs and polychlorinated biphenyls in household composts and earthworms (Wagman et al., 1998), it was mentioned that worms are known to accumulate lipophilic substances through their epidermis and into their intestine.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

A study was done on the exposure of OP pesticides to birds on a coastal South Carolina golf course (Rainwater et al., 1995) and it was discovered that the birds that foraged on the ground took up OPs both orally and dermally. It suggested that the primary route of exposure to anti-ChE pesticides was through the ingestion of dead or struggling insects that had been affected or had taken up OPs as well as OP containing particles or granules. It was reported that an area that had been sprayed with OPs still had mole crickets (Scapteriscus borellia) emerging from the ground three days after exposure to the chemical. On the other hand, a study was carried out by Kendall et al. (1993 in Rainwater et al., 1995), where avian species were observed feeding on a grass area on invertebrates that had been sprayed with diazinon. None of the birds appeared to be affected after ingestion of the chemical and no change in behaviour was observed (Rainwater et al., 1995).

2.1.3.7. NEUROTOXICITY

Presently, there is great interest in the neurotoxic effects caused by environmental contaminants. Firstly, various epidemiological, clinical and laboratory studies show that the nervous system is a popular target site for many xenobiotics and, secondly, there is little information available concerning the neurotoxicity caused by environmental contaminants (MacPhail, 1992).

Neurotoxic chemicals produce a wide range of responses by biota due to the extreme complexity of the functioning and structures associated with the nervous system. Research on various neurotoxic compounds concerning the various mechanisms and effects caused by these agents to the nervous system has been limited. Due to the vast range of effects and lack of research understanding, the development of biomarkers for the study of neurotoxic effects is also limited (Anger, 1993 in Travis, 1993).

Due to the diversity of the nervous system, in Travis (1993) it is said that the most widely used biomarker for neurotoxicity is behavioural tests. A few nervous system functions assessed included cognitive, motor (coordination, speed, response speed, steadiness and grip strength), sensory and affective deficits which are known to be associated with exposure to contaminants.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

2.1.4. BIOMARKERS: TESTING AND RESPONSE

Biota reveal specific responses and act as indicators or markers of changes to the environment when they are exposed to various xenobiotics. The responses observed from them are known as biomarkers, which help reflect chronic exposure to contaminants and have been proved to be important tools in ecotoxicological studies (Connell et al., 1999). Biomarkers must be sensitive, specific and suitable for the purpose of the study as well as harmless to obtain so as not to disrupt the species concerned (Walker, 1995). It is important that more than one biomarker is tested to monitor the situation as accurately as possible (Connell et al., 1999).

Toxicants can inhibit enzymes at very specific sites, such as the esteratic site of AChE. Some effects are toxicant specific as in OPs where they cause the inhibition of AChE while others are non-specific in their approach (Huggett et al., 1992). Two biomarkers used in this study are a direct enzyme inhibition (Huggett et al., 1992), namely ALA-D and AChE.

Blood holds a variety of biomarkers that may yield responses in biota especially avian species. For this reason, blood is generally used as the site of biomarker testing and also because the organism does not need to be sacrificed for the extraction of it. The level of response by the tested biota may be molecular, biochemical, cellular, histological, physiological or behavioural (Fossi and Leonzio, 1994).

2.1.4.1. NON-DESTRUCTIVE BIOMARKERS

Non-destructive biomarkers have been extensively used in the past. Their techniques include blood esterases, hormones, vitamin A in plasma and DNA alteration in blood cells. They are carried out using non-invasive techniques and when working with rare and endangered biota this is the preferred technique. Non-destructive biomarkers can be used in many assessments, one of them being the identification of ‘species at risk’. This assessment is based on the assumption that different species react differently to some contaminants and thus elicit various responses (Fossi and Leonzio, 1994).

A study done by Cordi et al. (1996) showed the importance of non-destructive biomarkers because they allow repeated protocols to be performed on the same individual after a few weeks. This allows populations of birds during the research period, some of which are

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

endangered, not to be sacrificed although data can still be obtained from live specimens (Cordi et al., 1996).

The advantages of non-destructive biomarkers according to Fossi and Leonzio (1994) are:

1. Non-destructive sampling allows for repeated sampling and measurements to be done on the same individual, provided that enough time is given for the individual to recover before the next sampling. 2. Biomarker responses can be compared more accurately to the exposure and concentration of a toxicant. 3. This type of sampling does not involve the killing of the specific biota and so animals are not lost from the population. This is a very important factor if one is working with rare or endangered organisms. 4. Long term studies on the development stages and changes observed, measured and recorded can be carried out. 5. This technique provides detection of changes at an individual level.

2.1.4.2. BLOOD

Blood is the best biological material for non-destructive biomarker analysis (Fossi and Leonzio, 1994). Blood is easily obtained with minimal work and inexpensive equipment and the animal can be released relatively unharmed and unstressed. However, careful attempts have to be made to preserve the blood samples and prevent clotting. Knowledge of and experience in blood extraction is important to prevent harm to, or even killing specimens (Fossi and Leonzio, 1994).

In birds, blood can be obtained via non-destructive techniques using capillary tubes or a hypodermic needle. Blood is normally extracted from the brachial vein on the wing, the jugular vein in the neck or the femoral artery in the leg. Sampling from the neck must be done with incredible sensitivity and is generally not advised as one careless move can be fatal (Fossi and Leonzio, 1994). It is interesting to note that birds have been known to tolerate a greater blood loss than mammals (Evans, 1987).

Blood is a mass distribution system around the body performing many functions such as delivering heat, force and various solutes to areas where they are required. Blood transports

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

nutrients which are absorbed from the digestive tract and, in the same manner, pollutants are also transported in this way (Schmidt-Nielsen, 1997). Blood consists of a fluid part known as the plasma, corpuscles which are comprised of erythrocytes (or red blood cells - RBCs) and leucocytes (white blood cells or WBCs) (Sturkie, 1953). Erythrocytes in birds are oval shaped and different to those of mammalian blood which are nucleated and larger.

According to Fossi and Leonzio (1994), blood as a non-destructive biomarker is good for the assessment of heavy metals as well as OCs in birds whereas feathers are appropriate for only heavy metals. Both blood and feathers have been successfully used in the past as biomarkers and are viewed as fairly reliable. It should be mentioned that blood has been used in the past to evaluate levels of OCs in human blood (Fossi and Leonzio, 1994).

2.1.4.3. BIOMARKER TYPES

Catalase (CAT)

Erythrocytes serve as an important source of antioxidants in the blood system. Catalase is one of the components of this antioxidant system (Rauchova et al., 2005) and is an enzyme which consists of hematin-containing particles (Huggett et al., 1992) It is a biological catalyst which lowers the activation energy required for a chemical reaction to proceed and, therefore, increases the reaction rate without itself being used up in the process. Hydrogen peroxide, which acts as the substrate for the reaction, is a poisonous by-product of metabolic processes that may damage cells if it is not removed. In the reaction, CAT speeds up the breakdown reaction of hydrogen peroxide into water and oxygen gas (Anon(a), 2003). Catalase removes hydrogen peroxide from cells during basal aerobic metabolism or after a pollutant induced oxyradical reaction (Khessiba et al., 2005).

Catalase activity may serve as a measure of oxidative stress as well as an antioxidant defence system for biota, as mentioned above. When the oxidative stress of an organism increases, the activity of CAT increases (Kale et al., 1999). Red blood cells often undergo varying levels of oxygen but the cell is capable of reducing any damage caused (Rauchova et al., 2005). Catalase protects the cell membrane from degradation and damage. At the same time, if catalyse activity increases, it indicates that the cell is protecting itself against the effect of oxyradicles in its surrounding environment (Khessiba et al., 2005). Popular sites of CAT presence are the liver and kidney and in subcellular organelles such as peroxisomes.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Hydrogen peroxide that cells generate cannot be broken down by CAT unless the hydrogen peroxide diffuses into the peroxisomes (Khessiba et al., 2005).

In a study performed by Khessiba et al. (2005), when mussels were exposed to lindane - a well-known environmental contaminant - CAT activity was higher in the sample mussels than in the control mussels. It was reported that sex, age, condition and season all influenced CAT activity in mussels and may, therefore, interfere in biomarker responses.

Protein concentration

Protein concentration is a non-specific biomarker as it will not isolate the type of toxicant causing the decrease in synthesis rate (growth) (Fossi and Leonzio, 1994), but it will certainly indicate the presence of a foreign compound, most likely a xenobiotic substance or combination of substances. Cell growth is made possible due to protein synthesis because without protein growth is inhibited (Fossi and Leonzio, 1994).

Protein concentration in blood plasma can be affected by infections, inflammation, as well as the general well-being of the organism. As stated by Grasman et al. (2000), protein concentration is an important indicator of health status in free-living biota. It provides information regarding the health and physiological status of living organisms (Grasman et al., 2000). Oxidative stress may be responsible for protein inactivation (Guecheva et al., 2003).

Protein concentration values are used to express CAT (Cohen et al., 1969) (mentioned above), AChE enzyme (Ellman et al., 1961 and Venter, 1990 in Venter et al., 2003) and ALA-D (Hodson, 1976) (both mentioned below) enzyme activities on a comparable scale. Therefore, this biomarker will serve to make enzyme biomarker values understandable and comparable.

δ-Aminolevulinic acid dehydratase (ALA-D) and lead (Pb)

δ-Aminolevulinic acid dehydratase is a relatively new biomarker in the scientific and ecotoxicological field (Gonzalez and Tejedor, 1992 in Lagadic et al., 2000) and is a cytosolic enzyme in many tissues. It is active in the manufacturing of haemoglobin by catalyzing the formation of porphobilinogen, which is a precursor of haeme. δ-Aminolevulinic acid

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

dehydratase is the second enzyme in the haeme biosynthesis pathway and is one of the most sensitive indicators of Pb accumulation in the body. According to a study done by Mehdi et al. (2000), Pb concentration in blood is regarded as the most reliable indicator of Pb exposure. Proteins in the ALA-D fraction of the erythrocyte supernatant have the highest affinity for Pb among all of the erythrocyte compounds (Mehdi et al., 2000).

δ-aminolevulinic acid dehydratase protocols are relatively easy to carry out, inexpensive and accurate and represent a biomarker that is strongly recommended for Pb exposure. According to Scheuhammer (1989 in Lagadic et al., 2000), a great advantage of the ALA-D protocol is that the animal concerned need not be sacrificed (Lagadic et al., 2000).

Lead enters the food chain through anthropogenic sources such as agricultural runoff, industrial effluent, storm water runoff and natural processes such as erosion and volcanism. Lead has many industrial uses such as in paint and gasoline. Birds can be exposed to Pb due to exhaust fumes or through the inhalation of contaminated food particles (Grue et al., 1984). It should also be noted out that Pb arsenate is still widely used as a pesticide (Burger, 1993). In vertebrates, Pb causes neurobehavioural, hematologic, nephrotoxic and reproductive effects. Lead has been known to generally inhibit action of the central nervous system as well as the immune system (Truscott, 1970 in Grue et al., 1984). It is, therefore, a neurotoxin and in nature has been known to cause lowered egg production and thus reduced hatching rates as well as lowered growth rates which all lead to a decrease in survival rate (Burger, 1993).

Biota which are exposed to Pb experience a decrease in erythrocyte ALA-D depending on the concentration of the metal. The enzyme becomes inhibited under certain concentrations of Pb. Blood lead concentrations have been found to be directly proportional or related to Pb concentrations. In the past, ALA-D has been used on passerine birds nesting along highways. δ-Aminolevulinic acid dehydratase is often inhibited at maximum levels before other signs of toxicity become obvious, showing that ALA-D is a good biomarker of Pb exposure but not necessarily for other chemicals (Huggett et al., 1992).

Lead accumulated from the environment will inhibit the activity of ALA-D in the erythrocytes (Mehdi et al., 2000). A study completed by Mehdi et al. (2000) showed that the activity of ALA-D decreased as the Pb concentration in the blood increased. Red blood cell

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

ALA-D levels can be inhibited by Pb at levels as low as 10-15 µg/dL (Fossi and Leonzio, 1994).

Lead has been found to be a 10-100 fold more potent inhibitor of ALA-D activity than Cu, Cd and mercury (Hg) and inhibition has been frequently used as a detector of environmental contamination in avian species. δ-Aminolevulinic acid dehydratase is rarely inhibited by other metals and is, therefore, a relatively specific biomarker for Pb exposure (Eeva et al., 2000). It must be mentioned that blood is a popular site for ALA-D testing (Tanhuanpaa et al., 1999). Measuring ALA-D activity in blood is a more sensitive indicator of Pb contamination than in liver.

δ-Aminolevulinic acid dehydratase inhibition is a sensitive protocol which is dose dependent and specific for Pb. It is clear that ALA-D has been widely used in past research as an avian biomarker in ecotoxicological investigations (Tanhuanpaa et al., 1999) and has proved to be relatively successful. Part of the ALA-D protocol described by Hodson (1976) includes the collection and analysis of a hematocrit. Hematocrit measures the amount of erythrocytes found in the volume of total blood (Nando, 2005). This measure shows the efficiency and success of oxygen uptake and transfer to the surrounding tissues. A low hematocrit value in humans may be an indication of anaemia, blood loss and destruction of RBCs, leukaemia, malnutrition or arthritis. A high hematocrit value, however, may be indicative of dehydration, erythrocytosis (excessive RBC production) or polycythemia vera. Nutrition, seasonal cycles, xenobiotics and stress can all alter the hematocrit of a species (Nando, 2005).

In a study done on Barn Swallows (Hirundo rustica), Pb concentrations in the feathers of adults nesting along a highway were higher than those of adults living in rural areas. But, it was noted that concentrations in the feathers of nestling Barn Swallows were similar between the two locations. The activity of ALA-D in RBCs were found to be lower in the highway colony when compared to the rural living colony (Grue et al, 1984)

DNA damage

Damage to DNA is a useful parameter for measuring the genotoxic properties of various pollutants and is a good indicator of gene induction by genotoxic agents (Feige et al., 1996). DNA is critical in any species of biota as inherited mutations can be indicative of debilitating

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

illnesses (Wallace, 1999 in Karanjawala and Lieber, 2004). The manufacturing mutant DNA proteins in subsequent generations can have serious consequences on the organism itself. Karanjawala and Lieber (2004) said that when DNA damage is high, a general decline in the population numbers of the species concerned can be observed.

When an organism is exposed to pollutants, a cascade of events may occur resulting in the mutation of DNA and diseases as a result of gene damage (Fossi and Leonzio, 1994). Types of structural alterations that may occur on the DNA due to the presence of a toxicant may be strand breakage, changes of bases, sister chromatid exchange as well as adducts. Most of these, as stated in Fossi and Leonzio (1994), can be detected and observed in blood or skin samples. According to Feige et al. (1996), there are many ways that a cell may respond to damage. If serious damage is experienced, the cell may die. On the other hand, the cell may develop survival strategies to combat the damage. This may lead to mutations in the genome or the cell may repair the DNA itself. If the DNA damage is replicated in future cell generations the result may be instability of the genome. This can occur via simple mutations at a point along the DNA strand, insertions, deletions, duplications or inversions. Serious DNA damage can result in chromosomal rearrangements or even loss of one or more chromosomes (Feige et al., 1996). DNA damage that is not repaired often results in fixed genetic information that is passed onto daughter cells (Musquiz, 2003). Inadequate oxygen levels or oxidative stress can also cause DNA damage (Guecheva et al., 2003).

DNA strand breakage can be assessed in a wide range of tissues but nucleated avian RBCs are preferred (Fossi and Leonzio, 1994). If an organism is stressed and the DNA bands on a DNA gel are studied, shorter average base pair lengths (ABPLs) will be evident. Base lengths will be long in an organism that is not stressed. This is due to pollutants or a combination of pollutants breaking the phosphorus bonds in the DNA helix and causing shorter bands to appear. This is an excellent tool for comparing one site to another and concluding which sites’ biota are under more stress.

Methods used to measure the effect of genotoxic chemicals are based upon three assumptions:

direct measurement of DNA structural damage; observation of their effects by measuring the rate of DNA repair directly or indirectly; and

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

the production of mutations on the genome of the exposed biota.

DNA damage can occur due to three factors:

1. wear and tear; 2. interaction with physical agents such as Ultra violet (UV) light and ionizing radiation; and 3. interaction with chemical agents (Huggett et al., 1992).

In addition, it was suggested in Musquiz (2003) that various OCs may cause DNA damage in individuals that are already stressed. It was reported that this field requires further investigation.

Acetylcholinesterase (AChE)

Cholinesterase is a class of ‘B’ esterases which are characterized by the presence of a binding site for the choline head and esteratic site. Two types of ChE exist, namely AChE and butyrylcholinesterase (BChE). Acetylcholinesterase which has a high affinity for ACh will be the main focus in this review (Fossi and Leonzio, 1994).

Acetylcholinesterase is found mainly in plasma and RBCs as well as nervous tissue. According to Fossi and Leonzio (1994), AChE is not found at a significant level in bird erythrocytes. In nervous tissue, AChE is vital for neurotransmission by the hydrolysis of ACh. Organophosphorus pesticides and carbamates are known to inhibit the activity of AChE. Calcium and Mg ions have been known to encourage activity. The inhibition of AChE may occur at high substrate concentrations due to the binding of cationic head of one (ACh) to its binding site and the ester head of another molecule to the esteratic site. Acetylcholinesterase can exist in various forms such as globular or asymmetric, each of which occurs as a monomer, dimmer and tetramer forms (Fossie and Leonzio, 1994).

Plasma ChE enzyme activity is a widely used method for measuring the exposure of OP pesticides in birds (McInnes et al., 1996 in Maul and Farris, 2004). Knowledge of AChE activity accumulated with the introduction of OPs and carbamates (Huggett et al., 1992) and a test to assess the exposure of birds to these chemicals involves AChE, which is inhibited by these pesticides. The substrate, ACh, cannot be hydrolyzed causing disturbance to nerve

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

functioning. Acetylcholinesterase is regarded as a very important biomarker of OP pesticides and carbamate pesticides which are difficult and expensive to analyze chemically (Connell et al., 1999).

Inhibition of AChE is linked directly with the effects of a toxicant which cause reversible and irreversible binding to the esteratic site and the potential of causing cholinergic effects. Acetylcholinesterase is responsible for hydrolyzing ACh into choline and acetic acid. In an article by Najimi et al. (1997), it was said that AChE enzyme is inhibited by some pesticides (as mentioned above) and metals. There has been past research on the lethal and sub-lethal effects of ChE inhibiting compounds on non-target species, in particular birds. In a report on the monitoring of nestling songbirds to OP pesticides it was said that a large number of OP pesticides inhibit ChE enzymes and, thus, measurement of ChE activity or ChE inhibition in blood plasma or brain tissue in avian species can be considered to be a good bioindicator of exposure to OPs compounds (McInnes et al., 1995).

The tissue of choice for the measurement of AChE activity is the brain but blood can also be utilized and has yielded positive results in past avian studies. It is said that the effects on blood are more transient (Fossi and Leonzio, 1994). Various studies have been done on a wide range of bird species for the detection of OP insecticides and carbamates using blood plasma. Temperature is critical when measuring ChE activity (Lagadic et al., 2000). When working with AChE, it must be remembered that ChE is an enzyme and it may be inhibited by temperature fluctuations. Therefore, it is essential that it is kept cold so that the enzyme is preserved and activity is inhibited. In this way, the compound will remain stable for several months (Lagadic et al., 2000).

Martin et al. (1995) completed a study on grasshoppers exposed to pesticides and the effect they have on AChE activity in the brain. It was discovered that the mean AChE activity of birds exposed to areas with extensive pesticide application had lower AChE activities than birds exposed to areas of little pesticide application. At the same time, birds which ingested food that had been in high application rate areas had a lower AChE activity than those that fed in low application rate areas (Martin et al., 1995). Decreased AChE activity in the chicks exposed to contaminated food showed that the birds received the primary intake of pesticides from their food rather than from ambient atmosphere. It was stated in the report that the dermal uptake of pollutants occurred through the feet which could have been responsible for a decrease in the AChE activity of the chicks in the study (Martin et al., 1995).

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Decarie et al. (1993) completed a study on the effects of insecticide application on the American Robin in a suburban environment. All the birds tested showed a decrease in AChE activity in the brain as well as in the blood plasma.

It was stated by Mineau (2003), that for carbamate pesticides and OPs pesticides the main field strategy for detection was the measure of ChE in either the blood or brain of the test specie. He said that ChE inhibition was the ‘golden standard’ of biomarker testing as it was easy to test but that a new cause for concern was whether these tests could detect any new types of pesticides on the market (Mineau, 2003).

2.1.5. HEAVY METAL ACCUMULATION AND FEATHER ANALYSIS

2.1.5.1. HEAVY METALS AND THEIR EFFECTS

According to Burger and Gochfeld (1995), heavy metals enter the ecosystem through air, water, soil and biota and are derived from agricultural runoff, industrial effluent, mining and mineral processing, storm water runoff, natural erosion of bedrock, transport in the atmosphere and various biogeochemical cycles. Concentrations of various metals depend on how often they are used, how they are used and how much of them are used. Heavy metals are associated with areas and activities of hazardous waste and, therefore, birds are vulnerable to exposure because of their mobility. Heavy metal consumption in birds is known to cause impaired growth and development as well as reproductive problems (Burger and Gocheld, 1995). Because of this, metals have been suggested to be a major contributing effect to the survival and reproduction of avian species (Swiergosz et al., 1997).

Heavy metals have been studied extensively in the past (Burger and Gochfeld, 1995) and measuring heavy metal levels in feathers is a useful tool but in many cases no concrete evidence is provided from such data as the stress caused by metals can be due to direct or indirect causes such as a decreased amount of food (Eeva et al., 2000).

Most heavy metals are toxic. Lead is one of the most widespread and dangerous and poisoning is common (Lagadic et al., 2000). Heavy metals are associated with industrial effluent which often results in contamination of the surrounding environment. Indicators of heavy metal exposure are vital as they serve as an early warning system for ecosystem interference (Dauwe et al., 2002). Heavy metals, whether they are essential or not, have the

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

ability to be toxic above a certain threshold concentration. The threshold value depends on the bird species concerned, metal type, bioavailability rate, type of exposure of the metal as well as characters of the birds concerned such as their age, sex, genetic make up and overall general health. Some birds have the ability to better regulate metals in the body compared to others (Burger, 1993).

Heavy metals can pose a serious threat to the natural ecosystem. Heavy metals accumulated by various bird species depend on the food they eat, the intensity, timing and frequency of exposure to contaminants as well as various physiological features (Kim et al., 1995). Secondary toxicosis is common in birds, especially birds of prey, because they feed on biota which have died or are weak due to primary toxicosis (Battaglia et al., 2005).

Metal levels in biota can also reflect trophic levels. The higher up in the food chain and the more the organism eats, the higher the metal levels will be. Birds are often high up in the food chain and if heavy metals are present they will be accumulated. Heavy metal levels can often reflect the human activities which surround the habitat of the organism (Burger and Gochfeld, 1995).

Different metals have greater affinities for certain areas in the body than others (Burger, 1993). Movement of Hg around the body has been studied in detail but little is known about most other metals (Burger, 1993). Up until 2002, only Hg accumulation in avian species had been studied (Goede and de Bruin, 1984; Furness et al., 1986; Hahn et al., 1993; Monteiro and Furness, 2001 in Dauwe et al., 2003), but for most other metals little data exists on the effect of external contamination of feathers (Dauwe et al., 2003). Burger (1993) completed a study where Hg levels in healthy terns were compared to terns with defects of the eyes, beak and skeleton. It was concluded that chlorinated hydrocarbons were the cause of the reproductive abnormalities (Burger, 1993).

Ohlendorf et al. (1986) carried out a study on the embryonic mortality and abnormalities of nesting aquatic birds and the apparent impacts of selenium (Se) from irrigation water in the San Joaquin Valley, California. In the study, 347 nest sites were monitored. Of the nests, 40.6% had at least one dead embryo and 19.6% had at least one embryo or chick which had a clearly observable abnormality. Deformities sometimes numbered one or more including missing or malformed eyes, beaks, wings, legs and feet. Brain, heart, liver and skeletal abnormalities were also noted. The study concluded that these deformities were due to the Se

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

concentrations present in the drain water where they were nesting (Ohlendorf et al., 1986). Denneman and Douben (1992) completed a study on Barn Owls which revealed that the main threat of heavy metals to the owls resulted from their diet.

2.1.5.2. FEATHERS AND FEATHER ANALYSIS

Feathers are keratinaceous structures unique to birds which provide protection, thermoregulation and flight as well as camouflage and the defence of territories. Keratin is an inert, insoluble, amorphous, proteinaceous matrix rich in disulfide bonds that are quickly reduced to sulfhydryl groups which become available to bind to metals. The affinity of air borne contaminants and metals depends on the affinity they have for the protein (keratin) in the feathers. Interestingly, feathers account between 5% and 12% of a birds body mass (Burger, 1993).

Feathers have been used as biomonitoring tools for the last twenty years but few studies are reported to have investigated the heavy metal levels in feathers of passerines (Dauwe et al., 2002). Feathers play a role in both the storing and eliminating of metals and do not only measure internal contamination but also external contamination which may result due to airborne contaminants which adhere to the external surfaces of the feathers. This may lead to the incorrect readings of metal concentrations (Battaglia et al., 2005). When monitoring heavy metals in bird feathers, it is important that a distinction is made between exogenous and endogenous metal levels (Denneman and Douben, 1992).

Feathers or parts of it are regarded as ideal for determining heavy metals levels, especially in research cases where no birds or organs from dead birds are available, as they accumulate toxic compounds in proportion to the blood levels at the time of feather formation (Burger and Gochfeld, 1995). Burger and Gochfeld (1995) ‘encourage further work in examining levels of heavy metals in avian feathers. Only with an accumulation of data from various bird species can we begin to understand metal levels in birds and their use as indicators of environmental exposure’.

Feathers show the immediate chemical presence and levels in birds and are viewed to be an excellent tool for integrating the exposure of chemicals versus time. More importantly, obtaining feathers is a non-destructive and non-invasive technique of sampling as a small sample of feathers can be plucked from a bird with little harm done. Feather regrowth takes

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

about two to four weeks and the bird can be released again with as little stress caused as possible (Burger and Gochfeld, 1995). Birds shed their feathers on a regular basis and in dealing with rare or endangered birds this is an ideal non-invasive monitoring technique (Denneman and Douben, 1992). Feather analysis has proved to be a good sampling technique (Hahn et al., 1993; Denneman and Douben, 1993 in Dauwe et al., 2003) as metals bind to the protein molecules during growth periods when the feather is connected to the blood stream of the bird via blood vessels (Burger, 1993).

Birds may sequester heavy metals in their feathers as a form of elimination from the body. It is reported that metal concentrations are generally represented at a higher concentration in feathers than in the blood or other tissues (Dauwe et al., 2002). However, blood levels only reflect the heavy metals at the time of feather formation. When the feather is fully formed the blood supply to the area seizes and the feather remains for 6–12 months before the bird sheds it during moulting (Burger and Gochfeld, 1995).

Dauwe et al. (2002) carried out a study on Great Tit (P. major) and Blue Tit (P. caeruleus) feathers where they were shown to be suitable indicators of heavy metal pollution. Feathers amongst the test species depicted differences in contamination levels amongst different sites. All metal levels were higher at the polluted sites (Dauwe et al., 2002). Although there are differences between Great Tits (P. major) and Blue Tits (P. caeruleus) in terms of biology, ecology, behaviour and metabolic rate, no significant differences among metal levels were reported for birds living in the same area. In addition, no gender or age-related differences were noted. It was concluded that feathers are an important target site for heavy metals (Dauwe et al., 2002).

Birds are exposed mainly to pollutants via the food they eat, respiratory exposure to various airborne contaminants and the preening of feathers when an oily secretion is applied which forms part of airborne deposition. Insects provide food for many bird species and in many food webs insects are important links in the transport of heavy metals (Dauwe et al., 2002). Air-borne contamination occurs at a high rate in urban areas where highways and air-borne particles exist such as fumes from cars. Newly formed feathers are expected to have the least amount of external contamination as they have not been exposed for a long time. Thus, it is expected that feathers that have been on the bird for the longest period of time have the greatest levels of external contamination and the greatest bioaccumulation rates (Burger, 1993).

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

A great advantage is that feathers can easily be stored over long periods of time at room temperature and this will not alter heavy metal presence or levels at the time of collection (Burger, 1993). There are several disadvantages to using feathers. Metals might not be deposited or deposited equally amongst feathers. Metals may be primarily from the feathers or may move from a different tissue. Metal levels can also vary among different feathers (Burger, 1993). Variation with and among feathers may occur but there is substantial evidence of the levels of Hg in different feather types. Variation of Hg levels among one sample species are much less on body or contour feathers and should rather be used for analysis rather than tail or wing feathers (Burger, 1993). Optimally, feathers should be collected when they are fully formed and the blood supply of the body is no longer connected. The stage of feather development can affect the amount or levels of metals sequestered (Burger, 1993) and it was, therefore, suggested that feathers should be collected from various regions of the body as this would be the best representative of the levels of metals and contaminants accumulated by the bird (Burger, 1993).

A variety of factors could be responsible for the differences in metals in bird feathers such as the time of feather collection (which is very important), sample size, the sex and age of the bird feathers collected as well as whether a live or dead specimen was used. Seasons can affect the stage of moult of the bird as well as the length of time the feathers have taken to form, as longer exposure time results in more bioaccumulation (Burger, 1993).

During moulting, birds sequester metals in their feathers while the blood supply is still connected. The first feathers to moult generally have the highest concentration of metals. It has been suggested that the levels of metals in the feathers do not only reflect the metals accumulated through food but also reflect the accumulation of metals since the first moult (Burger, 1993). Differences in the accumulation of metals by different feathers may also be due to the pigments and colours in each feather. Metals are accumulated differently in white or lightly-coloured feathers such as breast feathers, as opposed to darkly pigmented ones (Burger, 1993).

If feathers are used to assess metal levels in feathers, significant correlations should be found in organs such as the kidney, brain, blood, liver, muscle and bone (Burger, 1993). For many metals, levels are higher in the feathers than in other areas but for some metals such as Cd, Cu, Fe and Mn, levels appear higher in other tissues than in the feathers. For most metals,

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

however, there is no significant difference between levels in the feathers and levels in the tissues (Burger, 1993).

Feathers are useful indicators of historical pollution level trends and for comparing the levels of metals in various bird species from different regions. This can help with establishing the levels of metals in individual bird species and their potential impact on the bird, as well as the reaction of the metals present in the environment (Burger, 1993).

In conclusion, feather analysis is an important and very useful biomonitoring technique which helps to establish toxic effects. One can draw conclusions and or develop hypotheses about the cause of defects or abnormalities by comparing levels in healthy birds with that of affected birds (Burger, 1993).

2.1.6. THE ASPECT OF VIRAL INFECTION

Viruses are not cells but particles of nucleic acid, either DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) containing genetic information, surrounded by a protein coat and sometimes by a membrane. Viruses are smaller than bacteria and are essentially parasites which rely on the host for survival (Ingraham and Ingraham, 2000). Viruses cannot reproduce themselves and so as they enter their host they instruct it to make more virus using its metabolic machinery. When this occurs, only a small part of the host’s energy is used and the host will not be severely affected. In some cases, the virus may affect a host cell so badly that it may die. This implies that viruses are obligate intracellular parasites and are capable of infecting birds, animals, plants and various micro organisms. Many diseases are caused by viruses such as smallpox, polio, AIDS and yellow fever (Ingraham and Ingraham, 2000).

There is a proposed idea that a viral infection may be responsible for the feet deformities in Cape Wagtails.

2.1.7. SKELETAL ABNORMALITIES

Vitamin C is a critical factor in bone development as it is critical in the production of hydroxyproline which is a vital component of the collagenous matrix in bones. Vitamin deficiency may be due to diet, oxygen decrease, increased or decreased absorption. Cadmium has been known to cause a decrease in vitamin C in the mullet (Mugil cephalus)

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

because it increases or decreases absorption. Several OCs have been shown to decrease bone structure by interfering with vitamin C metabolism. It is interesting to note that some fish species could not synthesize vitamin C and are, therefore, very sensitive to a deficiency. Cadmium, Zn and Pb are known to displace Ca from its binding site resulting in a weakening of the bone structure (Huggett et al., 1992).

Muscular tetany may also yield vertebral deformities. This can be caused by OP and OC pesticides as well as parasitic infection and acute temperature changes. In certain fish species, OPs cause spinal fractures by inhibiting AChE, which leads to the build up of ACh at the nerve endings followed by muscular tetany. Organophosphates have been known to cause both muscular tetany and vitamin C deficiency (Huggett et al., 1992). Could the aspect of skeletal deformities in fish assist with the understanding of it, in Cape Wagtails?

Vertebral abnormalities caused by contaminants can be caused in two ways. Firstly, in an acute method which causes neurotoxic titanic contractions of skeletal muscle and, secondly, chronic exposures which may alter bone composition and make the bones more fragile. Other biological aspects that were found to be influenced in fish were swimming performance which was interrupted, a decreased ability to escape predators and altered feeding behaviour. Other effects found included a decreased ability to fight for a mate, decreased territorial defence and general physiological weakness (Huggett et al., 1992).

A Hadeda ibis was reported to have only one toe on each foot. The bird failed to walk properly but otherwise the bird appeared healthy. It was suggested that the missing toes were caused by ‘entangled nest material as a youngster or from wire or possibly a gin trap’ (Visagie and Visagie, 2005).

2.1.8. MITES AND INFESTATION

2.1.8.1. GENERAL INTRODUCTION

Mites are viewed as the most diverse and abundant of all the arachnids but because they are so small (often less than one millimeter in length), they are seldom seen. Mites are some of the oldest terrestrial organisms with fossils found from the early Devonian period, about 400 million years ago. About 45 000 mites have been described up to now which is only 5% of the total amount of mites that are thought to exist today. Mites have been successful as

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

biota as they have managed to invade a wide range of terrestrial habitats as well as aquatic ecosystems which fluctuate in extreme temperatures (Walter et al., 1996).

Mites are the most abundant ectoparasites found on birds (Boyd, 1951). Mites belong to the arthropod group which are morphologically very similar but vary greatly in the habitats they occupy. There are many types of bird mites that belong to a group known as the mesostigmatid mites. Bird mites are known to be host specific and are closely associated with dwellings of house sparrows (P. domesticus), pigeons (Columba sp) and starlings (Lamprotornis sp) (Gojmerec and Pellitteri, 2005). They are often colourless in appearance and are difficult to identify with the naked eye. They are, however, more visible if they have ingested a blood meal which makes them appear red to black (Anon, 2003).

Mites are tiny chelicerate arthropods with hexapod larvae, a discrete gnathosoma, and a loss of primary segmentation (Walter et al., 1996). Mites have three pairs of legs when they are in the larva stage and four pairs in the nymph and adult stages (Anon, 2003). The legs of these mites are equal in length and are divided into two pairs of legs towards the front of the body and two pairs which extend towards the posterior area of the body. They have well developed plates on the ventral and dorsal surfaces. Coxae are close together, lateral spiracles are present and the body is covered with many short hairs (Gojmerec and Pellitteri, 2005).

Bird mites generally have a life cycle of about seven days if conditions are favourable. The life cycle consists of four basic stages, these are, the egg, larva, nymph and adult which, completes their metamorphosis (Anon, 2003).

Infestations are more likely associated with birds that roost communally. Mites are known to thrive in the nesting materials of birds where they feed on unfeathered nestlings as well as adult birds. Contact between hosts is of utmost importance when parasites are transferred (Boyd, 1951). The mites inject saliva into their host which causes severe rashes and irritations to the host with intense itching. Mites infecting birds are known to be most active during late spring and early summer (Anon, 2003) and temperature is said to be a major contributing factor to the distribution of mites on a host (Boyd, 1951).

There are many types of mites which affect the feathered and unfeathered regions of birds. Most mites occupy the surface layer of the skin which leads to thickening of the skin and

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

‘flaking’ (Pesek, 2000). Scratching of the bites sometimes results in secondary infections (Anon, 2003). Mites that feed on host tissues can affect the health of the host species (Proctor and Owens, 2000) and some mites can transmit disease and thus serve as vectors of disease (Apperson and Waldvogel, 2002). Anaemia can result and even death from asphyxiation. Some mites are also known to excrete noxious substances which can disable their hosts (Proctor and Owens, 2000).

2.1.8.2. TYPES OF BIRD MITES

Bird mites, or otherwise known as ‘tropical fowl mites’ or starling mites’, describe the mite known as Ornithonyssus bursa from the mite family, Macronyssidae. This mite is widely distributed throughout the warmer regions of the world and it is a parasite which feeds on the blood of common garden birds such as sparrows, starlings, pigeons, Indian Mynahs (Acridotheres tristis), domestic birds as well as a range of other wild birds (Anon, 2003). Ornithonyssus sylviarum or the ‘northern fowl mite’ (NFM) is a mite common around domestic fowl, pigeons, starlings and house sparrows (Apperson and Waldvogel, 2002).

2.1.8.3. MITES PREVIOUSLY ASSOCIATED WITH CAPE WAGTAILS

Two mites have been found on Cape Wagtails. According to Zumpt (1961), the mites known as Ptilonyssus motacillae Fain and Boydaia nigra Fain have been recorded. Boydaia nigra Fain, belongs to the Ereynetidae family and are free-living mites. In South Africa this mite was found in the nasal cavities of Cape Wagtails in and around Gauteng and particularly Johannesburg.

2.1.8.4. THE SCALEY-LEG MITE (KNEMIDOCOPTES SP.)

Classification

Class: Arachnida Subclass: Acari Superorder: Parasitiformes Order: Astigmata Family: Knemidocoptidae Genus: Knemidocoptes

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Scaley-leg in birds is caused by microscopic burrowing mites, which belong to the genus Knemidocoptes - first described by Furstenberg in 1870. Several species of this genus have been found on passerine, psittacine and gallinaceous birds which include domestic birds (SCWDS Briefs, 1996), where they cause a condition known as Scaley-leg disease. The infection has become a serious problem in the industry (Carothers et al., 1974). The cause of this disease was thought for many years to be a type of fungus because of the appearance of the lesions that result (Butcher and Beck, 1996). This ectoparasitic mite feeds on the host’s tissues and is able to live and reproduce by burrowing into the cornified epithelium of areas such as the legs or feet or any unfeathered region of the bird (Kirmse, 1996 in Latta and O’Connor, 2001) and by feeding on keratin in these areas (Butcher and Beck, 1996). Scaley- leg is known to be highly contagious and mites can easily be transmitted between birds (SCWDS Briefs, 1996). Butcher and Beck (1996), suggested that the susceptibility of biota to this mite may be genetically linked. In a study done in Arizona, the disease was identified in the House Sparrow (Passer domesticus), Cassin’s Finch (Carpodacus cassinii) and the evening Grosbeak (Hesperiphona vespertina) (Carothers et al., 1974).

Mites of the order Astigmata lack tracheal systems and respiration occurs through their soft and thin tegument. They lack claws but have sucker-like structures on their pretarsi. Some of the mites that belong to this order are both medically and economically significant (Schmidt and Roberts, 2000).

Symptoms include ‘powdery’ type lesions on the feet and legs and sometimes the beak, white scabs and roughened skin, crusty-like growths on the legs with raised scales. If the infestation advances, severely disfigured legs covered with scabs is observed (SCWDS Briefs, 1996). The scabs develop due to the mites burrowing into the epithelium of the skin tissue which causes the proliferation of excess tissue (Herman et al, 1962 in Carothers et al., 1974).

Knemidocoptes sp. form tunnels which end in so-called ‘pouches’ where both the adults and larval forms can be found (Yunker and Ishak, 1957 in Carothers et al., 1974). Knemidocoptes sp. can cause severe abnormalities and serious disorders to their hosts (Krantz, 1978) and in extreme cases the legs can be deformed or lost (SCWDS Briefs, 1996). Most of the cases observed show that the mite only remains just under the surface of the skin and does not penetrate any deeper. The host’s skin tissue responds to the action of the mite by growing lesions and proliferations of the infected skin area. The mite also causes the overgrowth of

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

the toe nails which results in an inability to perch properly. The pathological condition referred to by this mite is known as Scaley–leg disease (Latta and O’Connor, 2001), or otherwise known as knemidokoptic mange, as is reported by Carothers et al. (1974). Gangrene can also develop around the leg area due to the infection and pressure exerted on the leg by the mites (Butcher and Beck, 1996).

It has been suggested by Yunker and Ishak (1957 in Carothers et al., 1974), that the mites complete their life cycle on one host and populations will feed and reproduce on one host as long as it lives.

It was shown by Latta and O’Connor (2001) that direct physical contact between host species may not be necessary for the transmission of the mite (Pulson, 1964 and Kirmse, 1966 in Latta and O’Connor, 2001), but any close contact between birds will result in mite transmission (Wichmann and Vincent, 1958 in Latta and O’Connor, 2001). This is ultimately the primary route of transmission between hosts. Mites also walk from host to host (Proctor and Owens, 2000).

2.1.8.5. KNEMIDOCOPTES MUTANS

Scaley-leg is a disease of chickens and various wild birds such as Guineafowl that occur world wide where ever host birds are present (Baker, 1999). These mites burrow themselves in the non-feathered regions of the bird namely; the cere, beak, eyes, vent and legs, causing lesions which develop slowly over time and may appear healthy for a long period until symptoms start appearing. It has been suggested that these mites are predominantly found in the nests of birds and, thus, this is predominantly the route of transmission and infection. Mite infestation is common in canaries (Serinus canaria) and finches and is often called ‘Tasselfoot’ or ‘hyperkeratosis’ (Irons, 1997).

Symptoms are known to develop very slowly and include irritation, inflammation, epidermal vesicle formation, serous exudation and spongy encrustation (Baker, 1999). Birds which have just been infected with Scaley-leg usually show raised or protruding scales out of the legs, often with a white crusty appearance on the legs and feet (Pesek, 2000). Scaley-leg is extremely irritating to the infected bird and in extreme cases lameness may occur and birds may appear to be crippled (Howell et al., 1983). Claws often become overgrown and cracked and affected birds are unable to perch properly. Secondary infection and arthritis is known to

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

follow infection (Pesek, 2000). According to Davies (2001), if the infection is left untreated, the legs and feet become ‘quite grotesque in appearance’. If the host is left untreated, crusts cover and distort the leg area and lameness as well as loss of digits will follow (Baker, 1999).

Knemidocoptes mutans spends its life cycle on the bird, metamorphosizing into its different life stages (Pesek, 2000). With reference to Dubinin (1953 in Zumpt, 1961), the life cycle of K. mutans includes a larval stage followed by two nymphal stages and an adult male or female stage. It is mentioned that sexual dimorphism can even be observed as early as the larval stage. This species of mite is spread by direct contact between birds (Pesek, 2000).

Carothers et al. (1974) reported an incidence rate of 25% of foot abnormalities among a population of grosbeaks in the spring of 1964. In 1967, 14% of the birds at the same locality had foot abnormalities. In 1973, another 7% of Evening Grosbeaks were said to have “varying stages of diseased feet’. The declining evidence of foot abnormalities was suggested to be because heavily infected birds were removed from the population during survey periods (Carothers et al., 1974).

Macro and micro examinations were carried out on the birds with diseased feet to confirm that the cause was Knemidokoptes sp. of mite and not a fungus as was previously thought and reported (Carothers et al., 1973 in Carothers et al., 1974). In all the cases of mite infestation, only one Evening Grosbeak was found to be infected along the beak region. In all the other infected individuals, symptoms appeared along the feet and unfeathered region of the tarsometatarsus. It was said that the lesions began forming at the proximal end of the tarsometatarsus and extended distally until the foot and exposed leg were surrounded by thick exudates. In many of the cases, the disease was so far advanced in its infection, that parts of the foot were breaking off and the birds had problems in walking and perching (Carothers et al., 1974). Birds that were heavily infected were forced to use the tarsometatarsus to support their body weight while they curled their badly deformed foot up towards their body. This caused some claws to become extremely long as they were not being used. It is important to note that none of the birds in the study by Carothers et al. (1974), showed any serious physiological consequences of being infected with Scaley-leg. For example, there were no significant differences in body weight between infected birds and healthy birds.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

A study by Mainka et al. (1994) was undertaken at the Mai Po Nature Reserve in Hong Kong. Wild passerine birds were observed to have a skin disease characterized by crusty, proliferative lesions on the beak, legs and feet. It was reported that Eurasian Tree Sparrows (Passer montanus) were the most heavily infected, with 58 of the 411 sparrows caught being infected with the skin disease. It was reported that adult birds were more often infected than youngsters and males were more affected than females. The juveniles showed lesions on the feet while adults showed lesions on the beak and legs. Just as it was stated by Carothers et al. (1974), it was also shown in Mainka et al. (1994) that the aspect of the mite infection did not cause a significant alteration of body weight.

Pence et al. (1999) stated that migratory American Robins (Turdus migratorius) had lesions that ranged from Scaley hyperkeratosis of the feet and legs to severe lesions with the loss of toes and even the foot. It was suggested that knemidokoptic mange can cause host morbidity and even mortality but the exact impact of a species such as the American Robins remains unknown (Pence et al., 1999)

2.1.8.6. KNEMIDOCOPTES JAMAICENSIS

Knemidocoptes jamaicensis has been recorded thus far in Denmark, Canada, Austria, Mexico, South Africa, Sri Lanka, the West Indies, the USA and England (Baker, 1999). This is a burrowing mite belonging to the family Knemidocoptidae which causes wart-like skin proliferations and Scaley-leg disease. Knemidocoptes jamaicensis (Turk, 1950 in Literak et al., 2005) has been found in chaffinches (MacDonald, 1962 in Literak et al., 2005). K.jamaicensis was first described when found on a Golden Thrush (Tardus aurantiacus) in Jamaica (Literak et al., 2005). Since then, this mite has been collected from over 30 species of passerine birds mainly in North America, but also in South Africa, Sri Lanka and Europe (Fain and Elsen, 1967 in Literak et al., 2005). The discovery of K. jamaicensis was first described by Fain and Elsen (1967, in Literak et al., 2005) when they completed the revision of the mites of the genus Knemidocoptes.

The areas that are infected eventually become distorted and covered with thick, nodular, spongy crusts (Schmidt and Roberts, 2000). Schmidt and Roberts (2000) reported that mites burrow under the scales of the lower legs and feet. Both adults and nymphs burrow under the skin of the bird and cause skin nodules on the surface of the dermis. The leg swells as a consequence and the leg appears encrusted (Proctor and Owens, 2000). In a study done by

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Benkman et al. (2005), it was said that K jamaicensis can cause the loss of digits and feet (Pence et al., 1999) and some infections may be lethal to the host (Latta and Faaborg, 2001 in Latta and O’Connor, 2001), as the bird is often unable to remove the mites. In their study, male crossbill birds were more vulnerable to infections than females in terms of the occurrence and severity of the infection (Benkman et al., 2005). Literak et al. (2005) completed a parasitological examination of the encrustations on the legs which revealed the presence of the mite K. jamaicensis. The infection may be fatal either directly or by secondary infections that often involve internal organs. The mite infection is highly contagious.

In a study done by Latta and O’Connor (2001), mites were common on birds that roosted communally but were rarely found on birds that roosted alone. In a report by Fain and Elsen (1967 in Latta and O’Connor, 2001), ten species of birds from the western hemisphere were found to have this mite. They were the Great-crested Flycatcher (Myiarchis crinitus L.), Black-capped Chickadee (Poecile atricaapillus L.), White-breasted Nuthatch (Sitta carolinensis Latham), Bare-eyed Robin (Tardus nudigenis Lafresnaye), White-chinned Thrush (Tardus aurantius Gmelin), Gray Catbird (Dumetella carolinensis L.), Red-winged Blackbird (Agelaius phoeniceus L.), Brewer’s Blackbird (Euphagus cyanocephalus wagler), Common Gackle (Quiscalus quiscula L.) and the Brown-headed Cowbird (Molothrus ater Boddaert). Since then, some additional species have been added to the list such as the Eastern Towhee (Pipilo erythrophthalamus L.) by Pence (1970) and the American Crow (Corvus brachyrhynchos) by Brehm and Pence in (1972) in North America (Latta and O’Connor, 2001).

In a further study Latta and O’Connor (2001), mites were found in leg lesions of eight species of birds inhabiting the Dominican Republic. The birds were the Hispaniolan Pewee (Contopus hispaniolensis), Northern Mockingbird (Mimus polyglottos), Cape May Warbler (Dendioca tigrina), Prairie Warbler (Dendroica discolour), Palm Warbler (Dendroica palmarum), Green-tailed Warbler (Microligea palustris), Black-crowned Palm Tanager (Phanicophilus palmarum) and the Greater Antillean Bullfinch (Loxigilla iolacea). This study investigated the habit preferences of the mites and it was found that a high infestation occurred in the desert thorn scrub. Few mites were associated with a forest habitat at a higher altitude although most of the mite-infested bird species were found across a few of the habitat types observed. Mites have been found in birds associated with North America and the Caribbean Islands of Hisponiola as well as migratory birds from the Neartic and

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Neotropic regions. Two additional reports have been made from the Caribbean, Jamaica and Trinidad both of which have resident thrush infestations (Fain and Elsen, 1967 in Latta and O’Connor, 2001).

Latta and O’Connor (2001) discovered that K. jamaicensis has the potential to affect any passerine bird species. Their results show that infections can affect large bird populations. The infestation rates at different localities and habitat types may be due to environmental factors as well as host factors.

In a publication by Latta and O’Connor (2001), the infestation by the mites on the Palm Warbler was found to be associated with rainfall. Higher rates of infestation were associated with regions of lower rainfall. It is thus assumed that a habitat with low rainfall and a dry climate is optimal for mite survival, as it was suggested by Van Riper (1991 in Latta and O’Connor, 2001) that K. jamaicensis requires a specific microclimate in which to survive. It has also been suggested that rainfall might be important in terms of infestation as a dryer climate may exert more physiological stress (Esch et al., 1975 in Latta and O’Connor, 2001) on the host, making it weaker and more vulnerable to infection (Deerenberg et al., 1997 and Saino et al., 1997 in Latta and O’Connor, 2001). Higher infestations may have occurred in the desert thorn scrub because birds that roost communally in open habitats but not in dense forests (Latta, 2000 in Latta and O’Connor, 2001). This could be a huge aid in the transmission of mites between host individuals.

In conclusion, the abundance of mites and the infection rate depends on the host species being available and abundant as well as various ecological conditions that need to be suitable to the mite itself and to promote its transmission. It was mentioned, however, that more work needs to be done with respect to these mites to determine their habitat preferences as well as their biology (Latta and O’Connor, 2001).

The aspect of mites and birds is a growing field of interest but the results obtained thus far are conflicting. Providing the evidence that mites cause negative effects to the health and reproductive success of the host is often unclear (Proctor and Owens, 2000).

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

2.2. CONCLUSION

This research project involves many topics which need to be integrated and investigated. Walker (1983) reported that there is a shortage of information regarding the chronic toxicity of xenobiotics in avian species and further research is needed to understand their effects on biota and their immediate environment. Physiological and biochemical aspects concerning the toxicity of xenobiotics are a ‘field ripe for exploitation’ (Walker, 1983).

Birds are highly specialized vertebrates with unique features and characteristics making them particularly vulnerable to the intake and severity of toxins (Walker, 1983), which may be stored in their feathers, body tissues and organs (Burger, 1993). To support this, evidence suggests that birds have less effective detoxification and defence mechanisms than mammals (Walker, 1983).

For the last few years, birds have assisted in indicating the state of the environment in terms of pollutants such as pesticides and metals (Burger, 1993), which are commonly used worldwide. However, many pesticides negatively affect non-target species which may be in the vicinity of the action when the pesticides are applied (Ownby et al., 2004). According to an article by MacPhail (1992), there is advancing interest in the aspect of neurotoxic effects on birds caused by environmental pollutants which is the target area for most toxicants. This is a growing field as little is known about the neurotoxicity implications of pesticides. Reports and articles indicate that further work is needed to establish the trends and details of pesticide action in the environment (MacPhail, 1992).

Birds show specific responses, known as biomarkers, which serve as indicators of changes in the environment when exposed to various xenobiotics. Biomarkers have proved to be important tools in ecotoxicological studies (Connell et al., 1999), and their use has increased in the last few decades and is now considered to be powerful and useful tools for detecting the impact of foreign compounds on ecosystems (Fossi and Leonzio, 1994). It is stressed in many written works that more than one biomarker should be tested so that the situation can be monitored accurately (Connell et al., 1999). Non-destructive biomarkers, which have been extensively used in the past, are favoured (Fossi and Leonzio, 1994) as protection of the test species is vital and ensured in this way.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

The Cape Wagtail has been described as ‘one of our best known birds and a general favourite that is protected everywhere by common consent’. It is a bird species of which there is a lot still to be learnt and discovered (Engelbrecht, 2005). Cape Wagtails are a good species for biomonitoring purposes as they catch a wide range of prey (worms, insects, crustaceans and tadpoles) and are sedentary i.e. they don’t migrate and are normally limited to a specific area. This implies that they are representative of their area or territory (Battaglia et al., 2005) and may prove to be excellent biomonitors of a point source contamination (Dauwe et al., 2002).

Steen and Steen (1965, in Frost and Siegfried, 1975) discovered that the role of the unfeathered region of passerine bird legs is to assist in heat regulation. This is why the aspect of feet deformities in the Cape Wagtail is of utmost concern and an explanation needs to be discovered.

The main aim of an ecotoxicologist according to Fossi and Leonzio (1994) is to find out the effect of a contaminant on natural communities. In the case of this research, it is expected that a contaminant(s) is responsible for the impact on the Cape Wagtails, M.capensis. If conclusive, this research may serve as a precursor for similar abnormalities that may occur in other bird species. This is expected if the abnormality is caused by something which is not specific to the Cape Wagtail. It must be noted that this might not be the case because the deformity may be limited to only this species. It would be of interest to establish whether birds such as the Cape Wagtail, which is of a lower trophic level, can be used as a monitor of environmental chemical change (Walker, 1998).

At this stage, few suggestions have been made for the reason behind the clubbed and missing toes of the Cape Wagtail. Could this be due to secondary poisoning through the use of garden pesticides, or is it due to the artificial nesting material used by wagtails, such as threads, which may get caught up around their toes and feet, or is it more likely to be a microbial infection or even a parasitic infestation – who knows?

The Scaley-leg mite Knemidocoptes sp. has symptoms and responses which are closely elicited by the Cape Wagtails. They indeed show lost toes and feet (SCWDS Briefs, 1996) and some appear to be lame and crippled (Howell et al., 1983).

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

It must be noted that little data is available for Cape Wagtails specifically, especially concerning the aspects discussed in this review. This study will be used as a baseline study for future investigations into the Cape Wagtail.

This research topic integrates a wide range of fields, scientific literature and discoveries. By incorporating many of them, and investigating all the observations and findings by past and present scientists, it may, hopefully, be possible to find the reason for the missing toes and clubbed feet of the Cape Wagtail. It would be of great interest to the scientific world and general public, especially bird lovers across the country.

2.3. REFERENCES

Anonymous. 2003. Bird Mites. Available from: http://www.medent.usyd.edu.au/fact/birdmite.html. (Accessed: November 2005). Anonymous (a). 2003. Catalase Lab. Available from: http:www.galaxynet.com. (Accessed: November 2005). Apperson, C.S. and Waldvogel, M. 2002. Residential, Structural and Community Pests. Available from: http://www.ces.ncsu.edu/depts/ent/notes/Urban/mites.htm (Accessed: November 2005). Baker, A.S. 1999. Mites and Ticks of Domestic Animals – An Identification Guide and Information Source. The Natural History Museum. London. Pp 240. Battaglia, A., Ghidini, S., Campanini, G. and Spaggiari, R. 2005. Heavy Metal Contamination in Little Owl (Athene noctua) and Common Buzzard (Buteo buteo) from Northern Italy. Ecotoxicology and Environmental Safety 60: 61-66. Benkman, C.W., Colquitt, J.S., Gould, W.R., Fetz, T., Keenan, P.C. and Santisteban, L. 2005. Can Selection by an Ectoparasite Drive a Population of Red Crossbills from its Adaptive Peak? Evolution 59(9): 2025-2032. Boatman, N.D., Brickle, N.W., Hart, J.D., Milsom, P., Morris, A.J., Murray, A.W.A., Murray, K.A. and Robertson, P.A. 2004. Evidence for the Direct Effects of Pesticides on Farmland Birds. Ibis 146: 131-143. Boyd, E.M. 1951. The External Parasites of Birds: A Review. The Wilsok Bulletin 63(4): 363-369. Brown, A.W.A. 1978. Ecology of Pesticides. John Wiley and Sons. Canada. Pp 525. Burger, J. 1993. Metals in Avian Feathers: Bioindicators of Environmental Pollution. Review Environmental Toxicology 5: 203-311. Burger, J. and Gochfeld, M. 1995. Biomonitoring of Heavy Metals in the Pacific Basin Using Avian Feathers. Environmental Toxicology and Chemistry 14(7): 1233-1239.

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Butcher, G.D. and Beck, C. 1996. Knemidokoptic Mange in Pet Birds: Scaly Face and Scaley-Leg Disease. University of Florida. Gainesville. Pp 2. Carothers, S.W., Sharber, N.J. and Foster, G.F. 1974. Scaley-Leg (Knemidokoptiasis) in a Population of Evening Grosbeaks. The Wilson Bulletin 86 (2): 121-124. Christensen, F. and Mortimer, J. 2004. Wagtail Wonderland in the Mall. Promerops 258. Pp 1. Cobb, G.P., Norman, D.M. and Kendall, R.J. 1992. Organochlorine Contaminant Assessment in Great Blue Herons Using Traditional and Nonlethal Monitoring Techniques. Environmental Pollution 83: 299-309. Cohen, G., Dembiec, D. and Marcus, J. 1969. Measurement of Catalase Activity in Tissue Extracts. Analytical Biochemistry 34: 30-38. Connell, D., Lam, P., Richardson, B. and Wu, R. 1999. Introduction to Ecotoxicology. Blackwell Science Pty Ltd. United Kingdom. Pp 170. Cordi, B., Fossi, C. and Depledge, M. 1996. Temporal Biomarker Responses in Wild Passerine Birds Exposed to Pesticide Spray Drift. Environmental Toxicology and Chemistry 16(10): 2118- 2124. Crowe, T. and Ratcliffe, C. 2001. Farming it Out: The Decline of Helmeted Guineafowl in KwaZulu- Natal. Africa-Birds and Birding 6(2): 4. Dauwe, T., Bervoets, L., Ellen, J., Rianne, P., Ronny, B. and Marcel, E. 2002. Great and Blue Tit Feathers as Biomonitors for Heavy Metal Pollution. Ecological Indicators 1: 227-234. Dauwe, T., Bervoets, L., Pinxten, R., Blust, R. and Eens, M. 2003. Variation of Heavy Metals within and Among Feathers of Birds of Prey: Effects of Molt and External Contamination. Environmental Pollution 124: 429-436. Davies, G.S. 2001. The Chook Shed – External Parasites. Available from: http://www.members.iinet.net.au/-greggles1/pest.html. (Accessed November 2005). Decarie, R., DesGranges, J.L., Lepine, C. and Morneau, F. 1993. Impact of Insecticides on the American Robin (Turdus migratorius) in a Suburban Environment. Environmental Pollution 80: 231-238. Denneman, W.D. and Douben, P.E.T. 1992. Trace Metals in Primary Feathers of the Barn Owl (Tyto alba guttatus) in the Netherlands. Environmental Pollution 82: 301-310. Eeva, T., Tanhuanpaa, S., Lehikoinen, E., Nikinmaa, M., Rabergh, C. and Airaksinen, S. 2000. Biomarkers and Fluctuating Asymmetry as Indicators of Pollution-Induced Stress in Two Hole- Nesting Passerines. Functional Ecology 14 (2): 235-243. Engelbrecht, D., Matlamela, P. and Watkins, J. 2005. Little Man on Campus. Birds and Birding 10(2): 6. Evans, P.G.H. 1987. Electrophoretic Variability of Gene Products. Academic Press Publishers. London. Pp 105-162. Feige, U., Morimoto, R.I., Yahara, I. and Polla, B.S. 1996. Stress-Inducible Cellular Responses. Birkhauser Publishers. Berlin. Pp 492.

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Fossi, M.C. and Leonzio, C. 1994. Non-destructive Biomarkers in Vertebrates. Lewis Publishers. United States of America. Pp 345. Frost, P.G.H. and Siegfried, W.R. 1975. Use of Legs as Dissipaters of Heat in Flying Passerines. Zoologica Africana 10(1): 101-102. Gojmerec, W.L. and Pellitteri, P.J. 2005. Controlling Nuisance Mites. Entomolgy: University of Wisconsin-Madison. A2206. RP-12-91-.5M-15-H. Pp 2. Grasman, K.A., Armstrong, M., Hammersley, D.L., Scanlon, P.F. and Fox, G.A. 2000. Geographic Variation in Blood Plasma Protein Concentrations of Young Herring Gulls (Larus argentatus) and Caspian Terns (Sterna caspia) from the Great Lakes and Lake Winnipeg. Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology 125(3): 365-375. Grue, C.E., O’Shea, T.J. and Hoffman, D.J. 1984. Lead Concentrations and Reproduction in Highway-Nesting Barn Swallows. The Condor 86: 383-389. Guecheva, T.N., Erdtmann, B., Benfato, M.S. and Henriques, J.A.P. 2003. Stress Protein Response and Catalase Activity in Freshwater Planarian Dugesia (Girardia) schubarti Exposed to Copper. Ecotoxicology and Environmental Safety 56(3): 351-357. Hayman, P. and Arlott, N. 1994. Birds of Southern Africa. Struik Publishers. Cape Town. Pp 432. Hockey, P.A.R., Dean, W.R.J. and Ryan, P.G. 2005. Roberts Birds of Southern Africa. VIIth edition. Tien Wah Press. Singapore. Pp 1296. Hodson, P.V. 1976. &-Amino Levulinic Acid Dehydratase Activity of Fish Blood as an Indicator of a Harmful Exposure to Lead. J. Fish. Res. Board Can. 33: 268-271. Howell, C.J., Walker, J.B. and Nevill, E.M. 1983. Bosluise, Myte en Insekte van Huisdiere in Suid- Afrika. Deel 1. Beskrywing en Biologie. Wetenskaplike Pamplet: No 393. Entomologie Departement. Onderstepoort. Pp 71. Huggett, R.J., Kimerle, R.A., Mehrle, P.M. and Bergman, H.L. 1992. Biomarkers: Biochemical, Physiological, and Histological Markers of Anthropogenic Stress. Lewis Publishers. United States of America. Pp 347. Ingraham, J.L. and Ingraham, C.A. 2000. Introduction to Microbiology. Second Edition. Brooks/Cole Publishers. United States of America. Pp 804. Irons, P. 1997. South African Birdnet Digest. Pp 1. Jenkins, A. 2005. The Price of Perfect Produce. Africa Birds and Birding 10 (1): 1. Johnston, G., Walker, C.H. and Dawson, A. 1993. Interactive Affects between EBI Fungicides (Prochloraz, Propiconazole, Chlorpyrifos, Diazinon and Malathion) in the Hybrid Red-Legged Partridge. Environmental Toxicology and Chemistry 13(4): 615-620. Kale, M., Rathore, N., John, S. and Bhatnagar, D. 1999. Lipid Peroxidative Damage on Pyrethroid Exposure and Alterations in Antioxidant Status in Rat Erythrocytes: A Possible Involvement of Reactive Oxygen Species. Toxicology Letters 105: 197-205. Karanjawala, Z.E. and Lieber, M.R. 2004. DNA Damage and Aging. Mechanisms of Aging and Development 125: 405-416.

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Khessiba, A., Romeo, M. and Aissa, P. 2005. Effects of Some Environmental Parameters on Catalase Activity Measured in the Mussel (Mytilus galloprovincialis) Exposed to Lindane. Environmental Pollution 133 (2): 275-281. Kim, E.Y., Ichihashi, H., Saeki, K., Atrashkevich, G., Tanabe, S. and Tatsukawa, R. 1995. Metal Accumulation in Tissues of Seabirds from Chaun, Northeast Siberia, Russia. Environmental Pollution 92(3): 247-252. Kjaer, C. and Jepson, P.C. 1994. The Toxic Effects of Direct Pesticide Exposure for a Nontarget Weed-Dwelling Chrysomelid Beetle (Gastrophysa polygoni) in Cereals. Environmental Toxicology and Chemistry 14(6): 993-999. Krantz, G.W. 1978. A Manual of Acarology. Second Edition. Oregon State University Book Stores. Corvallis. Pp 395 – 396. Lagadic, L., Caquet, T., Amiard, J.C. and Ramade, F. 2000. Use of Biomarkers for Environmental Quality Assessment. Technique et Documentation Publishers. Paris. Pp 324. Latta, S.C. and O’Connor, B.M. 2001. Patterns of Knemidokoptes jamaicensis (Acari: Knemidokoptidae) Infestations among Eight New Avian Hosts in the Dominican Republic. Journal of Medical Entomology 38(3): 437-440. Lennerstedt, I (1). 1975. Pattern of Pads and Folds in the Foot of European Oscines (Aves, Passeriformes). Zoologica Scripta 4: 101-109. Lennerstedt, I (2). 1975. A Functional Study of Papillae and Pads in the Foot of Passerines, Parrots, and Owls. Zoologica Scripta 4:111-123. Literak, I., Smid, B., Dusbabek, F., Halouzka, R. and Novotny, L. 2005. Co-Infection with Papillomavirus and Knemidocoptes jamaicensis (Acari: Knemidocoptidae) in a Chaffinch (Fringilla coelebs) and a Case of Beak Papillomatosis in Another Chaffinch. Vet. Med – Czech 50 (6): 276-280. Lock, J.W., Thompson, D.R., Furness, R.W. and Bartle, J.A. 1991. Metal Concentrations in Seabirds of the New Zealand Region. Environmental Pollution 75: 289-300. MacPhail R.C. 1992. Principles of Identifying and Characterizing Neurotoxicity. Toxicology Letters 64-65: 209-215. Mainka, S.A., Melville, D.S., Galsworthy, A. and Black, S.R. 1994. Knemidocoptes Sp on Wild Passerines at the Mai Po Nature Reserve, Hong Kong. Journal of Wildlife Diseases 30(2): 254-256. Martin, P.A., Johnson, D.L. and Forsyth, D.J. 1995. Effects of Grasshopper Control Insecticides on Survival and Brain Acetylcholinesterase of Pheasant (Phasianus colchicus) Chicks. Environmental Toxicology and Chemistry 15(4): 518-524. Maul, J.D. and Farris, J.L. 2004. Monitoring Exposure of Passerines to Acephate, Dicrotophos, and Malathion Using Cholinesterase Reactivation. Environmental Contamination and Toxicology 73: 682-689.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

McInnes, P.F., Andersen, D.E., Hoff, D.J., Hopper, M.J. and Kinkel, L.L. 1995. Monitoring Exposure of Nestling Songbirds to Agricultural Application of an Organophsophorus Insecticide Using Cholinesetase Activity. Environmental Toxicology and Chemistry 15(4): 544-552. Mehdi, J.K., Al-Imarah, F.J.M. and Al-Suhail, A.A. 2000. Levels of Some Trace Metals and Related Enzymes in Workers at Storage-Battery factories in Iraq. Eastern Mediterranean Health Journal 6(1): 66-82. Mineau, P. 2003. Avian Species. Encyclopedia of Agrochemicals. John Wiley and Sons. Canada. Pp 1-27. Musquiz, D. 2003. Cave and Cliff Swallows as Indicators of Exposure and Effects of Environmental Contaminants on Birds from the Rio Grande, Texas. Master of Science. Wildlife and Fisheries Sciences. Pp 71. Najimi, S., Bouhaimi, A., Daubeze, M., Zekhnini, A., Pellerin, J., Narbonne, J.F. and Moukrim, A. 1997. Use of Acetylcholinesterase in Perna perna and Mytilus galloprovincialis as a Biomarker of Pollution in Agadir Marine Bay (South of Morocco). Environmental Contamination and Toxicology 58: 901-908. Nando, R. 2005. Medical Encyclopedia (Medline Plus) - University of Chicago Medical Centre. Available from: Akademie-de. www.free-definition.com/Hematocrit.html (Accessed: June 2005). Ohlendorf, H.M., Hoffman, D.J., Saikithomas, M.K. and Aldrich, W. 1986. Embryonic Mortality and Abnormalities of Aquatic Birds: Apparent Impacts of Selenium from Irrigation Drain Water. Science of the Total Environment 52(1-2): 1-155. Ownby, D.R., Trimble, T.A., Cole, K.A. and Lydy, M.J. 2004. Pesticide Residues in Water, Sediment, and Fish at the Sparta, IL, USA, National Guard Armory. Environmental Contamination and Toxicology 73: 802-809. Ozmen, M., Sener, S., Mete, A. and Kucukbay, H. 1999. In Vitro and in Vitro Acetylcholinesterase - Inhibiting Effect of New Classes of Organophosphorus Compounds. Environmental Toxicology and Chemistry 18(2): 241-246. Pence, D.B., Cole, R.A., Brugger, K.E. and Fischer, J.R. 1999. Epizootic Podoknemidokoptiasis in American Robins. Journal of Wildlife Diseases 35 (1): 1-7. Pesek, L. 2000. Mites. Available from: http://www.Birdsnways.com/wisdom/ww48eiv.htm. (Accessed: December 2005). Proctor, H.C. and Owens, I. 2000. Mites and Birds: Diversity, Parasitism and Coevolution. Tree 15(9): 358-364. Prozesky, O.P.M. 1983. A Field Guide to the Birds of Southern Africa. Collins Publishers. London and Johannesburg. Pp 350. Rainwater, T.R., Leopold, V.A., Hooper, M.J. and Kendall, R.J. 1995. Avian Exposure to Organophosphorus and Carbamate Pesticides on a Coastal South Carolina Golf Course. Environmental Toxicology and Chemistry 14(12): 2155-2161.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Rauchova, H., Vokurkova, J. and Koudelova, J. 2005. Developmental changes of Erythrocyte Catalase Activity in Rats Exposed to Acute Hypoxia. Institute of Physiology. First Faculty of Medicine. Charles University. Albertov 5, 12800 Prague 2, Czech Republic. Pp 16. Schmidt, G.D. and Roberts, L.S. 2000. Foundations of Parasitology. Sixth Edition. McGraw Hill Publishers. United States of America. Pp 670. Schmidt-Nielsen, K. 1997. Animal Physiology Adaptation and Environment. Fifth Edition. Cambridge University Press. United Kingdom. Pp 607. SCWDS Briefs. 1996. Scaley-Leg Mites in Wild Birds 11, 4. Steyn, P.J., Reinecke, A.J. and Venter, J.M. 1985. The Weakening of Eggshells of the Laughing Dove, Streptopelia senegalensis. South African Journal of Zoology 21(3): 233-236. Sturkie, P.D. 1953. Avian Physiology. Comstock Publishing Associates. New York. Pp 423. Swiergosz, R., Sawicka-Kapusta, K., Nyholm, N.E.I., Zwolinska, A. and Orkisz, A. 1997. Effects of Environmental Metal Pollution on Breeding Populations of Pied and Collared Flycatchers in Niepolomice Forest, Southern Poland. Environmental Pollution 102: 213-220. Tanhuanpaa, S., Eeva, T., Lehikoinen, E. and Nikinmaa, M. 1999. Developmental Changes in 7- Ethoxyresorufin-O-Deethylase (EROD) and δ-Aminolevulinic Acid Dehydratase (ALA-D) Activities in Three Passerines. Comparative Biochemistry and Physiology Part C 124: 197- 202. Taylor, J. 2003. Cape Wagtail Group Behaviour. Promerops 253: 1. Travis, C.C. 1993. Use of Biomarkers in Assessing Health and Environmental Impacts of Chemical Pollutants. Plenum Press Publishers. New York. Pp 284. Van den Brink, N.W., van Franeker, J.A. and de Ruiter-Dukman, E.M. 1997. Fluctuating Concentrations of Organochlorine Pollutants during a Breeding Season in Two Antarctic Seabirds: Adelie Penguin and Southern Fulmar. Environmental Toxicology and Chemistry 17(4): 702-709. Van Wyk, E., Bouwman, H., van der Bank, H., Verdoorn, G.H. and Hofmann, D. 2001. Persistent Organochlorine Pesticides Detected in Blood and Tissue Samples of Vultures from Different Localities in South Africa. Comparative Biochemistry and Physiology Part C 129: 243-264. Venter, E.A., Slabbert, J.L., Joubert, A., Vorster, A. and Barnhoorn, I. 2003. Biomarker Assays for the Detection of Sub-Lethal Toxicity in Fish. Operational Manual. Environmentek, CSIR. WRC report No. 952/2/03. Report to the water research commission on the project ‘Biomarker Assays for the Detection of Chronic Toxicity in the Aquatic Environment’. Pp 49. Verster, M. 1966. Disappearance of the Cape Wagtail. Ostrich. Pp 3. Visagie, R. and Visagie, S. 2005. Digitally Disadvantaged Hadeda. Africa Birds and Birding 10(2):1. Wagman, N., Strandberg, B., van Bavel, B., Bergqvist, PA., Oberg, L. and Rappe, C. 1998. Organochlorine Pesticides and Polychlorinated Biphenyls in Household Composts and Earthworms (Eisenia foetida). Environmental Toxicology and Chemistry 18(6): 1157-1163.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Walker, C.H. 1983. Pesticides and Birds – Mechanisms of Selective Toxicity. Agriculture, Ecosytems and Environment 9: 211-226. Walker, C.H. 1995. Biochemical Biomarkers in Ecotoxicology – Some Recent Developments. The Science of the Total Environment 171: 189-195. Walker, C.H. 1998. The Use of Biomarkers to Measure the Interactive Effects of Chemicals. Ecotoxicology and Environmental Safety 40: 65-70. Walter, D.E., Krantz, J. and Lindquist E. 1996. Acari: The Mites. Available from: http://tolweb.org/tree?group=Acari&contgroup=Arachnida. (Accessed: October 2005). Ware, G.W. 1991. Fundamentals of Pesticides: A Self-Instruction Guide. Thomson Publications. United States of America. Pp 307. Williams, T. 2004. Birds of Shopping Malls. Promerops 257. 1. Winterbottom, J.M. 1967. The Farmer’s Birds. Maskew Miller Publishers. Cape Town. Pp 186. Zumpt, F. 1961. The Arthropod Parasites of Vertebrates in South Africa, South of the Sahara (Ethiopian Region). Volume 1: Chelicerata. Number 1. Volume IX. Publications of the South African Institute for Medical Research. Johannesburg. Pp 457.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

CHAPTER THREE

Non - destructive Biomarker Responses and Metal Analyses in the Blood and Feathers of the Cape Wagtail (Motacilla capensis).

3.1. INTRODUCTION

The use of birds as biomonitors has been prevalent since the early sixties when it became obvious that bird populations declined with human influence and associated pollutants (Denneman and Douben, 1992). Over the last few years, birds have served as early warning signs of the state of the environment in terms heavy metals (Lock et al., 1991) and xenobiotics. Birds serve as useful indicators because they are common, widespread (Burger, 1993), visible and easy to observe and monitor over several years and sensitive to toxins (Burger, 1993 and Kim et al., 1995). They occupy a high position in the food chain, have a wide range of distribution (Kim et al., 1995), are representative of their area or territory (Battaglia et al., 2005) and are of sufficient public interest. Toxic compounds may be stored in their feathers, body tissues and organs. Metals are mostly sequestered in the feathers while there is still active blood supply, thus making them good indicators of exposure and excellent bioindicators of the ecosystem in general (Burger, 1993). Today, organisms are frequently exposed to varying mixtures of pesticides and mobile species and especially birds which are exposed to these conditions as they fly from area to area in search of food and water (Walker, 1998).

Cape Wagtails (M. capensis), belonging to the family ‘Motacillidae’, are insectivorous, passerine birds first described by Linnaeus in 1766 (Hockey et al., 2005). They were once common birds in the gardens of South Africa and were often seen patrolling garden lawns, school fields and golf course greens in search of food. Recently, however, it is thought that they have been affected by the use of various garden chemicals such as pesticides. The Cape Wagtail is a flag-ship species for many conservation efforts concerning the application and use of pesticides (Engelbrecht et al., 2005), A sharp decline in wagtail numbers during the 1950s and 1960s was believed to be a result of the vast use of pesticides and insecticides, but in Steyn (1995 in Hockey et al., 2005) it was said that this was never proven to be entirely true. However, in the last decade, M. capensis have been observed with abnormalities around the feet region such as missing toes and clubbed feet. Various reasons have been suggested for this such as the possibility of secondary poisoning caused by the extensive intake of

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

harmful pesticides affecting nerve transmission and internal damage. Secondary poisoning is common where chemicals may target biota such as birds (e.g. wagtails), that feed on dead or dying food species and are thereby also poisoned, often causing fatalities (Masterson, 1976 & Vernon, 1972 in Hockey et al., 2005)

Pesticide use is common and occurs throughout the world. However, the by-products of pesticides negatively affect non-target species which may be in the vicinity of the action of the pesticides (Ownby et al., 2004).

Most compounds are highly lipophilic (Connell et al., 1999) and resist metabolic breakdown and, therefore, are strongly biomagnified through trophic levels (Van den Berg et al., 1994 in van Wyk, 2001). The effects of pesticides have been found to be variable amongst biota of the same species and for different species (Moore, 1965 in Steyn et al., 1985) which depends on the composition and potency of the pesticide. Some are highly toxic causing nerve transmission interference and even unexpected behaviour, while others cause endocrine disruption that affects the hormone levels in the body (Jenkins, 2005). Some poisons are less detrimental but have long term residual effects (Henriques et al., 1997 in van Wyk, 2001). Avian species are vulnerable to pollutant uptake and consumption may occur via four pathways. Firstly, orally, whereby the bird ingests the contaminant directly or secondly, through inhalation or through dermal exposure and absorption through the eyes and skin (Rainwater et al., 1995); thirdly, through eating dead or infected insects (secondary poisoning) and; fourthly, through contaminated water sources from irrigation water or runoff water which the bird may drink (Connell et al., 1999). Birds with contaminated feathers might also ingest chemicals through preening (Rainwater et al., 1995).

Biota reveal specific responses and act as indicators or markers of changes to the environment when they are exposed to various xenobiotics. The responses observed from them are known as biomarkers. A biomarker is a xenobiotically induced variation in cellular or biochemical components or processes, structures or functions that is measurable in a biological system or sample (Connell et al., 1999). Biomarkers serve as endpoints which measure the effects of toxic compounds on biota and help reflect chronic exposure to them. They are useful tools in ecotoxicological studies because they can be used to assess the overall state of an organism, which reveals the present state of the environment it resides in (Connell et al., 1999). Biomarkers must be sensitive and detectable in small quantities, specific for chemicals, suitable for the purpose of the study, measured by non-invasive

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

techniques, harmless to obtain so as not to disrupt the species concerned and inexpensive (Travis, 1993 and Walker, 1995). It is important that more than one biomarker is tested to monitor the situation as accurately as possible (Connell et al., 1999). Non-destructive biomarkers which make use of non-invasive techniques have been extensively used in the past due to the many advantages presented by them. When working with rare and endangered biota this is the preferred technique (Fossi and Leonzio, 1994). Blood is ultimately the best biological material (Schmidt-Nielsen, 1997) for non-destructive testing (Fossi and Leonzio, 1994) as it holds a variety of biomarkers. Blood is a mass distribution system around the body which delivers various solutes to areas where they are required but, in the same manner, pollutants are also transported in this way (Schmidt-Nielsen, 1997). Blood is easily obtained with minimal work and inexpensive equipment and the animal can be released relatively unharmed and unstressed provided the person undertaking the work is experienced in the field (Fossi and Leonzio, 1994). In this study four biomarkers were assessed in blood of M. capensis namely, AChE, ALA-D, CAT and DNA damage.

Acetylcholinesterase has a high affinity for ACh which is found mainly in plasma and RBCs as well as in nervous tissue where AChE is vital for neurotransmission (Fossi and Leonzio, 1994). It is responsible for hydrolyzing the substrate ACh into choline and acetic acid and is inhibited by various pesticides and metals (Najimi et al., 1997). It is, therefore, an important biomarker of pesticides and metals. When ACH is not hydrolyzed, nerve functioning is disturbed (Connell et al., 1999). At the same time, plasma ChE enzyme activity is widely used for measuring the exposure of OP pesticides in birds (McInnes et al., 1996 in Maul and Farris, 2004). Cholinesterase (ChE) activity or ChE inhibition in avian blood plasma can be considered to be a good bioindicator of exposure to OPs compounds (McInnes et al., 1995). The tissue of choice for the measurement of AChE activity is the brain but blood can also be utilized and has yielded positive results in past avian studies (Fossi and Leonzio, 1994).

δ-Amino–levulinic acid dehydratase has been widely used in past research as an avian biomarker in ecotoxicological investigations (Tanhuanpaa et al., 1999) and has proved to be relatively successful. δ-Amino–levulinic acid dehydratase is a cytosolic enzyme in many tissues and is active in the manufacturing of haemoglobin by catalyzing the formation of porphobilinogen, which is a precursor of heme. Birds can be exposed to Pb through the food chain in a variety of routes in their natural environment (Grue et al., 1984). In vertebrates, Pb acts as a neurotoxin (Burger, 1993) and has been known to generally inhibit action of the central nervous system as well as the immune system (Truscott, 1970 in Grue et al., 1984).

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Biota exposed to Pb experience a decrease in erythrocyte ALA-D depending on the concentration of Pb (Huggett et al., 1992) or an inhibition of it (Mehdi et al., 2000) at maximum levels before other signs of toxicity become obvious (Huggett et al., 1992). δ- Amino–levulinic acid dehydratase is rarely inhibited by other metals and is, therefore, in blood a relatively specific and sensitive biomarker for Pb exposure (Tanhuanpaa et al., 1999 and Eeva et al., 2000). It is viewed as a more reliable indicator in blood (Mehdi et al., 2000) than in the liver.

Catalase is a component of the antioxidant system in erythrocytes (Rauchova et al., 2005) and it protects the cell membrane from degradation, damage (Khessiba et al., 2005) and oxidative stress (Kale et al., 1999). It is a biological catalyst of a reaction where the poisonous by-product, hydrogen peroxide (the substrate), is removed from cells during basal aerobic metabolism or after a pollutant induced oxyradical reaction (Khessiba et al., 2005) into water and oxygen gas (Anon (a), 2003). When oxidative stress in an organism increases the activity of CAT increases indicating the cells are protecting themselves against the effect of oxyradicles in their surrounding environment (Khessiba et al., 2005).

When an organism is exposed to pollutants, a cascade of events may occur resulting in the mutation of DNA and diseases as a result of gene damage (Fossi and Leonzio, 1994). Damage to DNA is a useful parameter for measuring the genotoxic properties of various pollutants and is a good indicator of gene induction by genotoxic agents (Feige et al., 1996). Karanjawala and Lieber (2004) found that when DNA damage is high, a general decline in the population numbers of the species concerned can be observed. According to Feige et al. (1996) if cells are seriously damaged the cells may die. However, cells may develop survival strategies to combat the damage which may lead to mutations in the genome, or the cell may repair the DNA itself. If the DNA damage is replicated in future cell generations the result may be instability of the genome. DNA damage that is not repaired often results in fixed genetic information that is passed on to daughter cells (Musquiz, 2003). Inadequate oxygen levels or oxidative stress can also cause DNA damage (Guecheva et al., 2003). If an organism is stressed and the DNA bands are studied, shorter ABPLs will be evident. Base lengths will be long in an organism that is not stressed. This is due to pollutants or a combination of pollutants breaking the phosphorus bonds in the DNA helix and causing shorter bands to appear.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Heavy metals can enter the ecosystem in a variety of ways and birds are vulnerable to their intake because of their mobility and relatively high position in the food chain (Burger and Gochfeld, 1995). The accumulation of heavy metals by various bird species depends on the food they eat, the intensity, timing and frequency of exposure to contaminants as well as various physiological features (Kim et al., 1995). Secondary toxicosis is common in birds that feed on primary toxicosis victims (Battaglia et al., 2005). Some birds have the ability to better regulate metals in the body compared to others (Burger, 1993). Metal intake has been suggested to be a major contributing effect to the survival and reproduction of avian species (Swiergosz et al., 1997). Indicators of heavy metal exposure are vital as they serve as an early warning system for ecosystem interference (Dauwe et al., 2002). Heavy metals, whether they are essential or not, have the ability to be toxic above a certain threshold concentration which depends on the bird species concerned, metal type, bioavailability rate, type of exposure as well as characters of the bird concerned such as their age, sex, genetic make up and overall general health (Burger, 1993).

Feathers are made of keratin protein which readily binds to metals. Metals bind to keratin during growth periods when the feather is connected to the blood stream of the bird via blood vessels (Burger, 1993). Feathers have been used as biomonitoring tools for the last twenty years but few studies are reported to have investigated the heavy metal levels in feathers of passerines (Dauwe et al., 2002). Feathers play a role in both the storing and eliminating of metals (Battaglia et al., 2005) and are regarded as ideal for determining immediate heavy metal level and presence (Burger and Gochfeld, 1995), as well as integrating the exposure of metals over time. More importantly, obtaining feathers is a non- destructive and non-invasive technique. Feather regrowth takes about two to four weeks and the bird can be released again with as little stress caused as possible (Burger and Gochfeld, 1995). Feathers should be collected from various regions of the body to ensure the best representation of the levels of metals accumulated by the bird (Burger, 1993). Feathers that have been on the bird for the longest period of time have the greatest levels of external contamination and bioaccumulation rates (Burger, 1993). Birds can be exposed to heavy metals via the food they eat, such as insects which are important links in the transport of heavy metals through the food web (Dauwe et al., 2002), through contaminated air they breathe and through preening. A variety of factors could be responsible for the differences in metal levels in bird feathers such as the time of feather collection, sample size and the sex and age of the feathers collected (Burger, 1993). It is interesting to note that for most metals there is no significant difference between levels in the feathers versus those in the tissues

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

(Burger, 1993). One can draw conclusions and or develop hypotheses about the cause of defects or abnormalities by comparing levels in healthy birds with that of affected birds (Burger, 1993).

The objectives of this chapter are twofold. The first objective is to explore the spatial variation of biomarker responses (AChE, ALA-D, CAT and DNA damage in different blood components) and metal exposure as reflected in metal bioaccumulation in the feathers and plasma of Cape Wagtails. The second objective is to determine whether or not a relationship exists between affected feet and both biomarker responses and metal concentrations.

3.2. MATERIALS AND METHODS

3.2.1. SITE SELECTION

Sites (see Figure 1) were chosen based on regular sightings, abundance and roost reports. Cape Wagtails (M. capensis) were caught at seven different sites; two sites in the Western Cape, one site in the Eastern Cape, two sites in Mpumalanga and two sites in Gauteng. No uncontaminated reference site was selected in this study as the primary aim was to compare healthy Cape Wagtails to affected ones, irrespective of the state of each site.

3.2.2. SITE DESCRIPTIONS

3.2.2.1. WESTERN CAPE SITES

Paarl sewerage works (site 1) This site is situated in Paarl. Wagtails feed on the vast lawns in and around the sewerage works and they also collect insects off the surface of the turning tanks and the run-off thereof. A total of 19 individuals were sampled at this site.

Somerset West Mall (site 2) Wagtails roost in two tall trees alongside the shopping mall. The birds spend most of the day in and around the mall picking up scraps off the floors of the restaurants, while others feed on neighbouring garden lawns. A total of 9 individuals were sampled at this site.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

3.2.2.2. EASTERN CAPE SITES

Port Elizabeth (site 3) Wagtails roost communally in numbers of between 250-350 in Brazillian Pepper trees which line the shopping centre car park. Wagtails fly to surrounding areas and gardens during the day and return each evening before sunset to roost. A total of 47 individuals were sampled at this site.

3.2.2.3. MPUMALANGA SITES

Secunda (site 4) Birds roost communally in two trees on the peripheral of a petrol station. About 100 birds are present at this site. They fly off at sunrise to feed and return just before sunset. A total of 9 individuals were sampled at this site.

Dullstroom (site 5) Wagtails exist naturally in a farm surrounding. They spend the day picking insects off large grassy areas and alongside edges of watercourses and dams, and then roost together in and amongst the reeds at the dam edge. A total of 4 individuals were sampled at this site.

3.2.2.4. GAUTENG SITES

Rietvlei (site 6) Birds exist close to Rietvlei dam in an area dominated by reeds and watercourses surrounded by grassveld. A total of 1 individual was sampled at this site.

Alberton (site 6) Wagtails feed on garden lawns in residential areas and roost in a small shopping centre car park in the evenings. A total of 1 individual was sampled at this site.

3.2.3. SAMPLING TECHNIQUE

Wagtails (M. capensis) were caught with mist nets as well as ground flap traps where cultivated meal worms served as bait. Methods used depended on the lay out and

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

accessibility of the sampling area. For the purpose of this study, it was not necessary to determine the age or sex of the Cape Wagtails sampled.

N Rietvlei 64

1 2 3 Figure 1. Map showing positions of sampling sites. Numbers on the provincial maps represent sites (1 – Paarl, 2 – Somerset West, 3 – Port Elizabeth, 4 – Secunda, 5 – Dullstroom and 6 – Alberton and Rietvlei (Gauteng)).

3.2.4. BIRD IN HAND

The general health condition and external appearance of the bird was carefully observed and noted. Abnormalities on the legs, feet or toes were noted and photographed. Three cover feathers were consistently collected from various areas on the breast and placed in labeled brown paper envelopes. A blood volume of 200-250 µl was extracted from each bird via the brachial vein using capillary tubes (van Wyk, 1992 in van Wyk et al., 2001). Cape Wagtails were then immediately released with as little stress as possible caused to the individuals. Blood was immediately transferred to clean labeled eppendorph tubes and kept at 4 ºC until analysis.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

3.2.5. BIOMARKER ANALYSIS

3.2.5.1. δ-AMINO–LEVULINIC ACID DEHYDRATASE (ALA-D)

δ-Amino–levulinic acid dehydratase protocol was taken from Hodson (1976). It is a colorimetric detection method that measures the activity of the enzyme ALA-D (Schaller and Berlin, 1984 in Venter et al., 2003) which catalyses the manufacturing of porphobilinogen (PBG) - which is a precursor of haemoglobin (Hb) - from aminolevulinic acid (ALA) (Schmitt et al., 1984 in Venter et al., 2003).

When the bird was in hand, a capillary tube was used to take a small blood sample for hematocrit readings that were required for the final calculation of this biomarker. The protocol was carried out on whole blood within three days after the extraction to preserve the activity of the ALA-D enzyme.

A 0.1 M phosphate buffer was made up. From this buffer, two different solutions were established. The first solution had triton X-100 solution added to it to make a 0.2% Triton X- 100 solution. The second solution was made by taking 100 ml of the phosphate Triton X-100 solution and adding 6.7 mg ALA. Samples were performed in duplicates and three clean labeled eppendorphs were used for each sample. One tube had 100 µl of the phosphate Triton X-100 solution which acted as the control. The other two tubes had 20 µl of sample and 100 µl of the phosphate Triton X-100 ALA solution added into each. Eppendorphs were mixed on a mixer and incubated for two hours at 11 ºC. The reaction was stopped with 300 µl of a 4 g trichloroacetic acid and 2.7 g mercuric chloride mixture made up to 100 ml with distilled water. Tubes were centrifuged for five minutes at 5 000 rpm and 70 µl of supernatant was extracted and transferred to a 96 well Microplate. A volume of 60 µl of Ehrlich’s Reagent was added to each well and wells were left to react for 15 minutes.

Thereafter, colour development absorbency was read at 490 nm on an Elx 800 (Bio-tek) Universal Microplate Reader against the blank and the ALA-D activity (units per milliliter of red blood cells per hour) was calculated as follows:

ALA D Activity (units per milliliter of red blood cells per hour) = D x A T x H

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

D = dilution coefficient = 50 A = absorbance after 2hrs (average of 2 determinations) T = incubation time = 2hrs H = hematocrit ÷ 100

After the ALA-D protocol was carried out, blood samples were separated using a centrifuge at 6 500 rpm. Red blood cells were preserved in Henrikson buffer or stabilizing medium (HB). Separated components were frozen in clean labeled eppendorphs until further biomarker testing.

3.2.5.2. ACETYLCHOLINESTERASE (ACHE)

Blood plasma was used for this biomarker taken from the protocol by Ellman et al. (1961) and Venter (1990, in Venter et al., 2003). It is a colorimetric detection method which depicts the enzyme action of AChE when ACh is broken down into thiocholine and acetic acid.

A volume of 210 µl of a 0.09 M potassium phosphate buffer, 10 µl 30 mM s-acetylcholine

iodide (C7H16INOS), which acts as the substrate for the reaction, and 10 µl of a 10 mM

Ellman’s Reagent (2,2’-Diinitro-5-5’-dithio-dibenzoic acid – C14H8N2O8S2) were added to each well of a 96 well microplate, depending on how many samples were being tested. Samples were done in triplicates. Wells were mixed properly and incubated for five minutes at 37 ºC. Between 2.5 µl – 5 µl of sample was added to each well depending on how much sample was available for testing. Wells were mixed again and the simultaneous absorbencies

of all wells were read on an Elx 800 (Bio-tek) Universal Microplate Reader at 405 nm every two minutes for 12 minutes. Acetylcholinesterase activity (Absorbency/min/mg protein) was calculated as follows:

AChE (Absorbency/min/mg protein) = [gradient (absorbency)/5 µl (amount of sample used)] / protein conc.

Unit - Nmol/min/protein (mg) - measured per minute

3.2.5.3. CATALASE (CAT)

Catalase activity was measured using blood plasma according to the protocol taken from Cohen et al. (1969). A 0.01 M (pH = 7) phosphate buffer was made up and diluted to a

1:100 solution. A volume of 60 µl of a 30% Hydrogen peroxide (H2O2) was added to 100 ml

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

of phosphate buffer. A volume of 3,0024ml of sulphuric acid (H2SO4) was added to 7 ml of

distilled water and 0,0316 g potassium permanganate (KMnO4) was added to 100 ml of distilled water. In a 96 well Microplate, the first three wells served as blanks with 250 µl of distilled water in each. A standard was set up in the next three wells with 102 µl phosphate

buffer, 19 µl sulphuric acid (H2SO4) and 130 µl potassium permanganate (KMnO4). In the next few wells, 10 µl of sample was added to each followed by 93 µl of a 30% hydrogen

peroxide (H2O2) solution. Wells were mixed and, after three minutes, 19 µl of sulphuric acid

(H2SO4) was added to discontinue the reaction followed by 130 µl of potassium

permanganate (KMnO4). Absorbency was read at 490 nm on an Elx 800 (Bio-tek) Universal Microplate Reader within 30-60 seconds after adding the last chemical. Finally, CAT

activity (µmol H2O2/mg protein.min) was calculated as follows:

K = log (So/S3) x 2.3/t

K = first order reaction rate constant

So = substrate concentration at zero time

S3 = substrate concentration at 3 minutes T = 3 mins (time interval over which the reaction occurs and is measured)

3.2.5.4. DNA DAMAGE

DNA damage was carried out according to the protocol by De Coen (1999), with a few changes. Red blood cells were used for DNA analysis which had been stored in eppendorph tubes. The protocol recommends that one transfers a measured amount of RBCs cells to a clean eppendorph. However, more DNA was obtained when the RBCs remained in the original collecting eppendorph for the duration of DNA extraction.

After thawing, extraction buffer (7.3 g NaCl, 18.5 g EDTA, 6.05 g Tris base, 5 g sarcosyl solution (dodecyl sulphate or N-lauroysarcosinate) was added to each sample and mixed gently. RNAse A (5 µl) was added to each sample and placed on ice. RNase A was used instead of RNase as it has less DNA contaminants. Following this, proteinase K (10 µl) was added to separate the proteins from the DNA helix and incubated for 30 minutes at 55ºC. One volume (De Coen, 1999) PCI was added to each sample, mixed lightly and centrifuged for one minute at 12 000 rpm. The DNA containing top layer was pipetted out into clean eppendorphs. A further one volume of PCI was added to the original tubes, centrifuged and

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

the top layer was decanted once again into the new tubes. One volume of chloroform was added to the original containing tubes and the DNA layer decanted off once again.

In the 0.5% Agarose gels, 15 µl – 20 µl bromophenol blue was pipetted carefully into the first well. A volume of between 15 µl and 20 µl of DNA molecular weight marker II was pipetted into the second well followed by 15 µl – 20 µl of the sample DNA into the remaining wells. Lastly, 5 µl of a 40% sucrose solution was pipetted into each well before the gels were inserted into the electrophoresis container.

After 3-4 hours of running at 70 V, 200 ml of TBE buffer (40.87 g Tris base, 6.955 g Boric acid, 2.302 g EDTA dissolved in 200 ml and made up to 2.25 L with distilled water) was added to 20 µl of syber gold and poured over the gels under dark conditions. The stain recommended was syber green, although syber gold nucleic acid gel stain (Roche) was used as it is a more sensitive stain which, in practice, it proved to be. After 30 minutes, the gel was examined under Ultraviolet transilluminator capture system (UVP). DNA bands were studied and photos were taken with an image capture system (Grab-IT version 2.5) and SigmaScan.

Analysis was done using OptiQuant Acquisition and analysis program and DNA band ABPLs were calculated for each sample.

3.2.5.5. PROTEIN CONCENTRATION

Protein concentration (total protein) was determined using a colourmetric method (test kit) by Roche and calculated as follows:

Total Protein = Standard concentration (51.7 g/l) x (absorbency test/absorbency standard)

3.2.6. METAL ANALYSIS

The protocol for digestion was followed according to Dauwe et al. (2002 and 2004). Feathers were washed alternately three times with double distilled water (milliQ water) and 1 mol-1 acetone and placed in clean labeled falcon tubes. Feathers underwent microwave digestion; three minutes at 10% power followed by two minutes at 20% power and lastly two minutes

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

at 30% power. Feather digestions were left overnight. Sample blanks without feathers were also prepared.

A volume of 0.5 ml of 30% hydrogen peroxide (H2O2) and 1.5 ml of 65% nitric acid (HNO3) were added to each falcon tube and left overnight to digest.

Samples were prepared for the ICP-MS. A 250 times’ dilution was found to be optimum for metal readings. The measurements for each feather were as follows; 50 µl indium which served as an internal standard in each feather sample, 200 µl digestion and 1% nitric acid

(HNO3) to make up 50 ml total solution, which acted as the dilutant. The solution was mixed well and 10 ml of the feather digestion solution was decanted into clean, labeled falcon tubes to be used at the ICP-MS. Each sample was analyzed by ICP-MS within a week after dilution.

3.2.7. STATISTICAL ANALYSIS

Primer (Plymouth Routines in Marine Environmental Research) program v5.0 (Plymouth Marine Laboratory) was used to perform multivariate analysis. Bray-Curtis similarity measures with square root transformation were used to draw up similarity matrixes for biomarker and metal (feather and plasma) data. Hierarchical cluster analysis, NMDS (non- metric multi-dimensional scaling) and ANOSIM (analysis of similarities) revealed any significant differences between sites (p<0.05). Stress values were indicated on the Hierarchical Cluster Analysis Graphs and the interpretation of them was as follows: less than 0.05 indicated an excellent representation, less than 0.01 indicated a good ordination, less than 0.02 revealed a useful ordination and less than 0.3 indicated a poor representation (Clarke and Warwick, 2001). Simper analysis with a 90% cut off with no transformation was used to describe similarity percentages and species contributing to these similarities.

SPSS 12.0.1. for windows compared means using one way ANOVA (test of homogeneity of variances). Post hoc tests (Scheffe - parametric or Dunnette – T3 - non parametric) were used to revealed significant (p<0.05) differences among sites as well as within sites for all biomarkers and metals tested. When the means were compared using the independent samples T Test, any significant (p<0.05) differences between affected birds and healthy birds with regards to all biomarkers and metals became evident. Correlations (regression analysis)

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

with linear regression were performed between various metals and biomarkers known to have a corresponding relationship.

Graph Pad Prism 4 represented the data in a graphical format with means (x) and standard errors (standard error of the mean value, SE) calculated for each set of data displayed in bars on the graphs.

3.3. RESULTS

3.3.1. SPATIAL VARIATION (BIOMARKER RESPONSE AND METAL ACCUMULATION)

Sample sizes varied at each site and the blood volume extracted from each individual was inconsistent. The sequence of biomarker testing was influenced, firstly, by the component of blood required by each assay and, secondly, by the volume of blood obtained from each bird. δ-Amino–levulinic acid dehydratase was carried out first on each sample as whole blood was required. Following this, blood samples were separated and AChE and CAT were performed on the plasma while DNA damage was performed using the RBC component. Hence, the number of biomarker results was always higher for ALA-D and DNA damage.

Biomarker responses are presented graphically in Figure 2. Acetylcholinesterase revealed no significant differences (p<0.05) among sites, although activity was higher at sites 1 (Paarl), 3 (Port Elizabeth) and 4 (Secunda) when compared to site 5 (Dullstroom), thus showing inhibition of this enzyme at those sites. Sites 1, 3 and 4 were evidently similar in mean activity which ranged from 6.44 (Absorbency/min/mg protein) (site 1) to 8.85 (Absorbency/min/mg protein) (site 3). In graph B, ALA-D displayed significant differences (p<0.05). Site 3 was significantly different from sites 4 and 5. Both sites 4 and 5 (both Mpumalanga) were notably much lower in ALA-D activity than sites 1, 2 (Somerset West) and 3, thus showing possible inhibition of that enzyme at those sites. In addition to this, mean enzyme activity at sites 1, 2 and 3 were very similar, as they were at sites 4 and 5. Catalase indicated no significant differences (p<0.05) among sites. Mean activity was within

similar ranges between 2.61 (µmol H2O2 /mg protein.min) at site 2 and 3.53 (µmol H2O2 /mg protein.min) at site 1. In graph D, DNA ABPL revealed significant differences (p<0.05) between sites 1 and 2, as well as among sites 3, 4 and 5. Mean ABPLs were within similar

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

ranges with site 1 being the lowest (6647.59) and site 6 being the highest (9667.98). Small sample size should be taken into account.

A B

12 1.1 11 1.0 10 0.9 9 n= 16 0.8 A,B 8 0.7 n= 7 7 n= 8 0.6 n= 37 n= 2 6 n= 4 0.5 5 0.4 4 0.3 3 A 0.2 B 2 n= 4 0.1 n= 7 1 n= 4 0 0.0 Site 1 Site 3 Site 4 Site 5 AChE (absorbency/min/mg protein) Site 1 Site 2 Site 3 Site 4 Site 5 ALA-D milliliter per (units RBC/hr) of Sampling sites Sampling sites

D C A,B, 5 10000 C,D n= 3 n= 1 B C D 4 A,B, 7500 n= 4 C,D n= 40 n= 9 n= 4 3 n= 8

/mg protein.min) 5000 2 n= 2 n= 21

2 2 DNA ABPL 2500

mol H mol 1 γ

0 0 CAT ( CAT Site 1 Site 2 Site 3 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Sampling sites Sampling sites

The relationship of biomarker responses among sites are presented in Figure 3. The NMDS ordination (Figure 3A) and hierarchical cluster analysis plot (Figure 3B) yielded Paarl (group 1) as more similar to Dullstroom, Port Elizabeth and Secunda (group 2) than it is to Gauteng and Somerset West (group 3). The stress value of 0.00 is indicative of the ordinations being an excellent representation of the data. An analysis of similarities (ANOSIM) among groups showed that the groupings were significant (p< 0.017) based on biomarkers compared at all sites. Simper analysis revealed that group 3 (Gauteng and Somerset West) had a similarity percentage of 99.23% while group 2 (Dullstroom, Port Elizabeth and Secunda) had a

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

similarity of 98.95%, due to the DNA ABPLs. The average dissimilarities and the biomarkers contributing to the dissimilarities among groupings are presented in Table 1.

Table 1. Average dissimilarity based on biomarker responses among groups

Group 1 Group 2 (Dullstroom, Port Group 3 (Gauteng (Paarl) Elizabeth and Secunda) and Somerset West) Group 1 (Paarl) - Group 2 (Dullstroom, 7.77% (*DNA ABPLs Port Elizabeth and responsible for the _ Secunda) dissimilarity) Group 3 (Gauteng 18.19% (*DNA ABPLs 10.59% (*DNA ABPLs and Somerset West) responsible for the responsible for the _ dissimilarity) dissimilarity)

Stress: 0 1 Paarl

2 Port Elizabeth Secunda Dullstroom

3 Alberton & Rietvlei Somerset West A 92

Similarity

100 Paarl

Secunda Dullstroom Port Elizabeth Port Somerset West Somerset

&Alberton Rietvlei B Figure 3. A –NMDS ordination and B – hierarchical cluster analysis plot depicting biomarkers at various sites (site 1 – Paarl, site 2 – Somerset West, site 3 – Port Elizabeth, site 4 – Secunda, site 5 – Dullstroom and site 6 – Alberton and Rietvlei, collectively named Gauteng). The 2- dimensional stress after 10 iterations was 0.00. Numbers of groupings in Figure A assist in representation of Simper analysis and ANOSIM.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Figures 4, 5 and 6 represent metal levels in the feathers. Significant differences (p<0.05) were observed for Cr (Figure 4D) and Ni (Figure 5D). As far as Cr was concerned, site 1 (Paarl) was significantly different from sites 3 (Port Elizabeth), 4 (Secunda) and 5 (Dullstroom). Site 3 was materially different from sites 4 and 5. In terms of Ni, site 1 was substantially different from sites 3, 4 and 5 and site 3 was significantly different from site 4. Some metals followed a particular trend with sites 1, 2 (Somerset West) and 3 being lower in metal levels such as in Cu (Figure 4F), Mn (Figure 5C) and Ag (Figure 5E), than at sites 4, 5 and 6. In conjunction with this, some metals showed lower levels at sites 1, 2 and 3 and higher levels at sites 4 and 5, but lower levels at site 6 in the case of Al (Figure 4A), As (Figure 4B) and Pb (Figure 5B). Some metals showed the opposite effect with sites 1, 2 and 3 having higher Cr (Figure 4D) and Ni (Figure 5D) levels than sites 4, 5 and 6. Cadmium (Figure 4C), Co (Figure 4E), Fe (Figure 5A), Sr (Figure 5F) and Zn (Figure 6) levels varied among sites and no pattern could be distinguished. In many cases (eight metals), Secunda (site 4) had the highest levels amongst the sites. In eight of the metals, sites 5 or 6 had the lowest metal levels but for the remaining five metals, sites 2 and 3 shared the lowest levels.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

A B 1200 30 1100 1000 900 800 20 700 600 Al 500 As 400 10 300 200 100 0 0 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Sampling sites Sampling sites

C D A,B,C 12 550 11 500 10 450 9 8 400 7 350 A,D,E 6 300 Cd 5 Cr 250 4 200 3 150 B,D 2 100 C,E 1 50 0 0 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Sampling sites Sampling sites

E F

1.5 140 130 120 110 100 1.0 90 80 70 Co Cu 60 0.5 50 40 30 20 10 0.0 0 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Sampling sites Sampling sites

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

A B

3500 100 3000 75 2500 2000 50 1500 g/g dry weight) g/g dry weight)

µ µ 1000 25 500 Pb ( ( Fe 0 0 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Sampling sites Sampling sites

C D 225 130 A,B,C 120 200 110 175 100 90 150 A,D 80 125 70 60 100 50 g/g dry weight) g/g dry weight) 75 40 B,D C µ 30 µ 50 20 Ni ( Mn ( Mn 25 10 0 0 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Sampling sites Sampling sites

F E 5.5 40 5.0 4.5 4.0 30 3.5 3.0 20 2.5 2.0 g/g dry weight) g/g dry weight) µ 1.5 µ 10 1.0 Sr (

Ag ( 0.5 0.0 0 Site 1 Site 3 Site 4 Site 5 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Sampling site Sampling sites

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

4000

3000

2000

g/g dryweight) µ 1000

Zn ( 0 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Sampling sites

The relationship of metal exposure (as reflected in metal bioaccumulation in the feathers) among sites is presented in Figure 7. Analysis of similarities (ANOSIM) among groups showed a significance level of p=0.067 which indicated no significant differences among site groupings for metal levels found in the feathers. The NMDS ordination (Figure 7A) and hierarchical cluster analysis plot (Figure 7B) illustrated that Paarl (site 1), Somerset West (site 2), Port Elizabeth (site 3) and Gauteng (site 6) (all group 1) had an average similarity percentage of 84.59% with Fe, Zn and Al responsible for the similarity grouping. Secunda (site 4) and Dullstroom (site 5) (both group 2) had an average similarity of 73.96% where Fe, Zn and Al were also responsible for the grouping. Average dissimilarity between groups one and two appear in Table 2.

Table 2. Average dissimilarity based on metal analysis results Group 1 Group 2 (Paarl, Somerset West, Port (Secunda and Dullstroom) Elizabeth and Gauteng) Group 1 (Paarl, Somerset West, Port _ Elizabeth and Gauteng)

Group 2 22.91% (*Zn, Al, Fe and Cr were (Secunda and Dullstroom) responsible for the dissimilarity) _

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Stress: 0 Paarl Somerset West Port Elizabeth 1

Alberton & Rietvlei

Secunda

2 Dullstroom A

Similarity 95

100

Paarl Secunda Dullstroom Port Elizabeth Somerset West Somerset Rietvlei & Alberton B

3.3.1.1. EVALUATION OF METAL LEVELS AGAINST ECOLOGICAL QUALITY OBJECTIVES

(ECOQOS)

Table 3 presents the EcoQOs for three Weaver (Ploceus sp.) species in Gauteng (Meyer, 2005). The EcoQOs were used as they were the closest source of comparison data to the

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Cape Wagtail. Metal concentrations (µg/g dry weight) at each site during the sampling periods are presented. Generally most metals in the feathers of Cape Wagtails at all sites exceeded the EQOs (red highlighted values indicate present metal concentrations in the feathers at the specific sites which exceed the EcoQOs). Very few metal concentrations at some sites (Cu at site 2, Pb at sites 2, 3 and 6, and Sr at site 5) were below the EQOs (yellow highlighted values indicate current metal concentrations which are below the EcoQO target level).

Table 3. Ecological Quality Objectives of several heavy metals in the feathers of three Weaver species (Ploceus sp.) in Gauteng (Red or yellow highlighted values indicate metal concentrations which are above or below the target levels (EcoQOs) respectively) Metal concentration (µg/g dw) EQO 1 2 3 4 5 6 Target (Paarl) (Somerset (Port (Secunda) (Dullstroom) (Gauteng) Level West) Elizabeth) Metal Al 199.744 496.96 430.94 450.49 915.76 809.91 375.91 Cd 1.613 8.26 3.44 7.67 9.51 5.72 2.11 Co 0.158 1.23 0.59 0.75 0.74 0.75 0.37 Cr 7.190 497.49 312.69 274.37 58.29 40.85 104.35 Cu 33.560 43.29 30.53 38.78 105.91 81.04 80.04 Fe 374.960 2893.57 1730.26 1870.11 1990.78 1513.51 1490.3 Mn 21.883 63.72 38.14 51.61 96.67 66.46 65.15 Ni 9.299 188.18 117.67 110.91 44.07 36.24 41.52 Pb 44.831 47.48 20.49 30.2 79.21 59.86 31.1 Sr 18.440 23.54 20.62 34.15 24.63 12.37 21.08 Zn 708.220 2634.14 2128.32 1740.4 2820.33 971.51 1797.59

3.3.1.2. TOXIC UNITS (TUS)

The combined toxicity of metal exposure was expressed as a standardized toxicity unit (TU), which represents the toxicity of several metals as a portion of the bird’s threshold concentration (Sprague, 1970 in Yoo et al., 2003). The TUs were calculated as follows; TU

= concentration of metal tested per site/LD50 (Parametrix, 2002) for birds for that specific metal. All individual metal TU values are added and a TU is obtained for each site.

Table 4. Toxic Units calculated for each site Site TU TU ranking of sites (order increases further down the table) Paarl (site 1) 30.721 Site 4 (Secunda) Somerset West (site 2) 27.741 Site 1 (Paarl) Port Elizabeth (site 3) 21.475 Site 6 (Gauteng) Secunda (site 4) 39.636 Site 2 (Somerset West) Dullstroom (site 5) 18.853 Site 3 (Port Elizabeth) Gauteng (site 6) 30.459 Site 5 (Dullstroom)

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Site 4 (Secunda) revealed the highest TU of all the sites. The lowest TU was observed at site 5 (Dullstroom). The TUs in this study did not correspond with the metal groupings presented in the NMDS ordination (see Figure 7A) or in groupings in the biomarker NMDS ordination (see Figure 3A). Neither did the TUs of affected and healthy birds (Table 5) relate to the site groupings in Figures 3A and 7A

Only four sites (Paarl, Port Elizabeth, Secunda and Dullstroom) could be considered for the analysis in Figure 8, 9 and 10 due to a shortage of plasma samples. Toxic units were not calculated for plasma samples as the sampling size was too small. Figures 8, 9 and 10 represent metal levels in plasma samples. Figure 9E (Ni) and Figure 10 (Zn) revealed significant differences (p<0.05) among sites. In the case of sites 4 (Secunda) and 5 (Dullstroom), Al (Figure 8A), Ca (Figure 8D), Co (Figure 8F), Pb (Figure 9C), Mn (Figure 9D), Ni (Figure 9E) and Zn (Figure 10), were generally much higher than at sites 1 (Paarl) and 3 (Port Elizabeth). However, Figure 9B (Fe) appeared to be the opposite, where sites 1 and 3 were greater in Fe levels than sites 4 and 5. In Figure 8C (Cd), Figure 9A (Cu) and Figure 9F (Ag), there was a noticeable difference with regards to site 1 as levels at this site were much greater than sites 3, 4 and 5. In terms of As (Figure 8B) and Cr (Figure 8E), levels varied from site to site and no trend could be established. Sites 1 (Paarl) and 4 (Secunda) normally depicted the highest metal levels. For As (Figure 8B), Cd (Figure 8C), Cu (Figure 9A), Fe (Figure 9B) and Ag (Figure 9F), site 1 displayed the highest levels of the respective metal. For Al (Figure 8A), Ca (Figure 8D), Co (Figure 8F), Fe (Figure 9B) and Zn (Figure 10) site 4 portrayed the highest levels in each case. For the remaining metals, site 5 showed the highest levels in Cr (Figure 8E), Mn (Figure 9D) and Ni (Figure 9E). Site 3 generally had the lowest levels as was illustrated by As (Figure 8B), Cd (Figure 8C), Cr (Figure 8E), Co (Figure 8F), Cu (Figure 9A), Ag (Figure 9F) and Zn (Figure 10). Site 1 also represented the lowest levels in many of the metals as in Al (Figure 8A), Ca (Figure 8D), Pb (Figure 9C), Mn (Figure 9D) and Ni (Figure 9E). At site 4, however, Fe (Figure 9B) was the lowest.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

A B

7.5 0.11 0.10 0.09 0.08 5.0 n= 2 0.07 n= 2 n= 2 n= 2 g/l) g/l) 0.06 µ µ 0.05 n= 2 Al ( 2.5 ( As 0.04 n= 4 0.03 0.02 n= 4 n= 2 0.01 0.0 0.00 Site 1 Site 3 Site 4 Site 5 Site 1 Site 3 Site 4 Site 5 Sampling sites Sampling sites

C D

0.25 1000

0.20 750 0.15 g/l) g/l) n= 2 n= 2 µ n= 2 µ 500 0.10 Cd ( Cd Ca ( Ca 250 0.05 n= 2

n= 2 n= 4 n= 2 0.00 n= 4 0 Site 1 Site 3 Site 4 Site 5 Site 1 Site 3 Site 4 Site 5 Sampling sites Sampling sites

E F

0.3 0.020 n= 2

n= 2 0.015 0.2 n= 2 g/l) g/l) µ µ 0.010 n= 2 n= 4 n= 2 n= 2 Cr ( 0.1 n= 4 ( Co 0.005

0.0 0.000 Site 1 Site 3 Site 4 Site 5 Site 1 Site 3 Site 4 Site 5 Sampling sites Sampling sites

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

A B 8 45 7 40 6 35 30 5 n= 2

g/l) g/l) 25 µ 4 n= 2 µ 20 n= 4 3 Fe ( Cu ( Cu 15 2 10 1 n= 4 5 n= 2 n= 2 n= 2 0 n= 2 0 Site 1 Site 3 Site 4 Site 5 Site 1 Site 3 Site 4 Site 5 Sampling sites Sampling sites

C D

0.6 0.75

0.5

n= 2 0.4 0.50 n= 2 g/l) g/l) n= 2 n= 2 µ µ 0.3

Pb ( 0.2 ( Mn 0.25

n= 4 0.1 n= 4 n= 2 n= 2 0.0 0.00 Site 1 Site 3 Site 4 Site 5 Site 1 Site 3 Site 4 Site 5 Sampling sites Sampling sites

E z F 0.5 0.14 0.13 n= 2 0.12 0.4 0.11 n= 2 0.10 n= 2 0.09 0.3 g/l) 0.08 g/l) µ

µ 0.07 0.06 0.2 Ni ( Ag ( Ag 0.05 0.04 0.1 n= 4 0.03 n= 2 0.02 0.01 n= 2 n= 2 n= 4 0.0 0.00 Site 1 Site 3 Site 4 Site 5 Site 1 Site 3 Site 4 Site 5 Sampling sites Sampling sites

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

n= 2 50 n= 2

g/l) µ

Zn ( Zn 25

n= 2 n= 4 0 Site 1 Site 3 Site 4 Site 5

Sampling sites

The NMDS ordination (Figure 11A) and hierarchical cluster analysis plot (Figure 11B) portrayed a significance level of p>0.05 and, therefore, no significant differences occurred among sites. Secunda (site 4) and Dullstroom (site 5) (group 2), both in Mpumalanga, were fairly similar with a similarity percentage of 97.57%. In terms of metals in the plasma, Paarl (site 1) and Port Elizabeth (site 3) (group 1) had a similarity percentage of 77.79%. Calcium and Zn, in both cases, were responsible for the similarity groupings. To add to this, the two groupings showed a dissimilarity of 67.73%, again because of Ca and Zn.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Stress: 0 Paarl 1

Port Elizabeth

Dullstroom 2

Secunda A

Similarity 90

100 Paarl Secunda

Dullstroom Port Elizabeth Port

3.3.1.3. RELATIONSHIPS BETWEEN BIOMARKER RESPONSE AND METAL EXPOSURE

Pearson’s correlation analyses was conducted to determine if there were any relationships between the biomarkers. No significant correlations were found. However, the known relationships that may exist among biomarkers are presented in Figures 12, 13 and 14 to illustrate the trend but non-significant correlations.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

7.5

5.0 r2= -0.057 /mg protein.min) 2 O 2 2.5

mol H mol γ

0.0 ( CAT 5000 6000 7000 8000 9000 10000 11000 DNA ABPL

Figure 12. Relationship between CAT enzyme activity (µmol H2O2/mg protein.min) and average base pair length of DNA.

Referring to Figure 12 regression analysis was performed on an expected relationship between CAT enzyme activity and DNA ABPL. Although the expected negative correlation was present, it was not significant.

2.25 2.00 1.75 1.50 1.25

1.00 r2= -0.047 0.75 0.50 0.25 0.00 0 100 200 300 400 500 600 ALA-Dmilliliter per (units of RBC/hr) Pb (µg/g dw) Figure 13. Relationship between ALA-D enzyme activity (units per milliliter of red blood cells per hour) in the blood and Pb concentrations (µg/g dw) in the feathers.

2.0

1.5

1.0 r2 = -0.252

0.5

0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 ALA-D (units per milliliter of RBC/hr) Pb (µg/l)

Figure 14. Relationship between ALA-D enzyme activity (units per milliliter of red blood cells per hour) in the blood plasma and Pb concentrations (µg/l) in plasma.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Referring to Figures 13 and 14, regression analysis was performed on an expected relationship between ALA-D enzyme activity in the blood plasma and Pb concentrations in the feathers and blood plasma. Although the expected negative correlation was present, it was also not significant. In terms of an approximate threshold value, Figure 13 revealed that at Pb concentrations above 100 µg/g dw in the feathers, ALA-D activity was generally below 0.5 units per milliliter of red blood cells per hour while Figure 14 showed that at Pb concentrations above 0.2 µg/l in the plasma, ALA-D activity was generally below 0.5 units per milliliter of red blood cells per hour.

3.3.2. VARIATION BETWEEN HEALTHY AND AFFECTED BIRDS (BIOMARKER RESPONSE AND

METAL ACCUMULATION)

In the previous few paragraphs, spatial differences were investigated and used to compare birds sampled at different sites in terms of their biomarker responses and metal exposure. In the following section, biomarker responses as well as metal levels in the feathers of healthy and affected birds are assessed within sites and among sites. Only those sites that had wagtails with healthy feet and affected feet (site 1 – Paarl, site 2 – Somerset West and site 3 – Port Elizabeth) were included in the following analysis (Figures 15, 16, 17 and 18).

Figure 15 represents the biomarker responses in healthy and affected wagtails. Graph A showed that affected birds at sites 1 (Paarl) and 3 (Port Elizabeth) had higher AChE activities than healthy birds but small sample size should be considered. No significant (p<0.05) differences were revealed. In graph B, healthy birds at site 1 had a higher ALA-D activity than affected birds. At site 3, healthy bird ALA-D enzyme activity was relatively similar to the activity elicited by affected birds. Mean ALA-D activity for healthy birds at site 2 (Somerset West - 0.61 units per milliliter of red blood cells per hour) was similar to that at sites 1 (0.75 units per milliliter of red blood cells per hour) and 3 (0.65 units per milliliter of red blood cells per hour). No significant (p<0.05) differences were observed. Catalase activity revealed no significant differences (p<0.05) between healthy and affected birds. At sites 1 and 3, healthy birds had a considerably lower activity than affected birds.

Mean activity at site 2 (2.61 µmol H2O2/mg protein.min) and 3 (2.36 µmol H2O2/mg protein.min) was similar for healthy birds. In Graph D, DNA ABPL displayed significant differences (p<0.05) at site 3 between healthy birds and affected birds. At sites 1 and 2, healthy birds and affected birds had very similar – almost equal - DNA ABPL, although small sample size should be taken into account.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

A B 20 1.1 1.0 0.9 15 0.8 0.7 n= 6 n= 7 0.6 n= 30 n= 7 10 n= 2 n= 1 0.5 n= 17 0.4 5 0.3 n= 3 0.2 n= 1 0.1 0 0.0 1H 1A 3H 3A 1H 1A 2H 3H 3A AChE (absorbency/min/mgprotein) RBC/hr) of milliliter per (units ALA-D Sampling sites and condition Sampling sites and condition

C D 7.5 10000 n= 2 n= 1 7500 n= 33 5.0 n= 1 n= 7 n= 7 n= 1

n= 3 5000 /mg protein.min) 2 O 2 2.5 n= 3 DNA ABPL n= 2 n= 18 2500 mol H mol γ 0.0 0 ( CAT 1H 1A 2H 3H 3A 1H 1A 2H 2A 3H 3A Sampling sites and condition Sampling sites and condition

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

A B

650 17.5 600 550 15.0 500 450 12.5 400 350 10.0 300 7.5 250 g/g dry weight) g/g dry weight) 200 µ

µ 5.0 150 100 Al ( 2.5 50 ( As 0 0.0 1H 1A 2H 2A 3H 3A 1H 1A 2H 3H 3A Sampling sites and condition Sampling sites and condition

C D 11 650 600 10 550 9 500 8 450 7 400 6 350 5 300 250

4 g/g dry weight) g/g dry weight) 200 µ µ 3 150 2 100 Cr ( ( Cd 1 50 0 0 1H 1A 2H 2A 3H 3A 1H 1A 2H 2A 3H 3A Sampling sites and condition Sampling sites and condition

E F

1.75 110 100 1.50 90 1.25 80 70 1.00 60 0.75 50 40 g/g dry weight) g/g dry weight)

µ 0.50 µ 30 0.25 20 Co ( Co Cu ( Cu 10 0.00 0 1H 1A 2H 2A 3H 3A 1H 1A 2H 2A 3H 3A Sampling sites and condition Sampling sites and condition

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

B A 3500 150 3000 2500 100 2000 1500 g/g dry wieght)

g/g dry wieght) 50 µ µ 1000 500 Fe ( ( Pb 0 0 1H 1A 2H 2A 3H 3A 1H 1A 2H 2A 3H 3A Sampling sites and condition Sampling sites and condition

C D 80 350 70 300 60 250 50 200 40 150 30 g/g dry wieght) g/g dry wieght) µ 100 20 µ

Ni ( 50 Mn ( 10 0 0 1H 1A 2H 2A 3H 3A 1H 1A 2H 2A 3H 3A Sampling sites and condition Sampling sites and condition

E F 5 50 4 40 3 30 2 20 g/g dry wieght) g/g drywieght) µ µ 1 10 Sr ( Ag ( Ag 0 0 1H 3H 3A 1H 1A 2H 2A 3H 3A Sampling sites and condition Sampling sites and condition

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

4500

4000 3500 3000 2500 2000

g/g dry weight) 1500 µ 1000

Zn ( Zn 500 0 1H 1A 2H 2A 3H 3A

Sampling sites and condition

Metal levels in the feathers of healthy and affected birds are depicted in Figures 16, 17 and 18. At site 1 (Paarl), most metals such as Al (Figure 16A), As (Figure 16B), Cd (Figure 16C), Co (Figure 16E), Cu (Figure 16F), Fe (Figure 17A), Pb (Figure 17B), Mn (Figure 17C), Ni (Figure 17D) and Sr (Figure 17F) had higher levels for healthy birds than affected birds. In only two cases, as in Figure 16D (Cr) and Figure 18 (Zn), the opposite was observed where levels were higher in affected birds. At site 2 (Somerset West), it was common for the level of the respective metal to be higher in affected birds, such as in Al (Figure 16A), Cd (Figure 16C), Cr (Figure 16D), Co (Figure 16E), Fe (Figure 17A), Pb (Figure 17B), Mn (Figure 17C), Ni (Figure 17D), Sr (Figure 17F) and Zn (Figure 18). Copper (Figure 16F) levels were no different in healthy and affected birds. A consistent pattern was observed at site 3 (Port Elizabeth) where healthy birds were normally higher in the respective metal levels such as As (Figure 16B), Cd (Figure 16C), Cr (Figure 16D), Co (Figure 16E), Cu (Figure 16F), Fe (Figure 17A), Pb (Figure 17B), Mn (Figure 17C), Ni (Figure 17D) and Sr (Figure 17F), than affected birds. The reverse occurred in Al (Figure 16A) and Ag (Figure 17E) where affected birds had higher levels of these metals than healthy birds did. In terms of Zn (Figure 18), levels were almost equal for affected and healthy birds. In most cases, healthy birds at site 1 had the highest respective metal level and healthy birds residing at site 2 had the lowest levels.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

3.3.2.1. TOXIC UNITS (TUS)

Looking at the TUs (Table 5) between healthy and affected birds at each site, affected birds at sites 1 (Paarl) and 2 (Somerset West) were higher than affected birds. However, at site 3 (Port Elizabeth) the opposite was observed. Healthy birds had a higher TU than affected birds. In terms of viewing all the sites together, site 1 affected birds had the highest TU, while healthy birds at site 2 had the lowest TU.

Table 5. Toxic Units for healthy and affected Cape Wagtails Site Condition TU TU ranking of sites (order increases further down the table) Paarl (site 1) Healthy (1N) 32.341 Site 1 (Affected-1A) Affected (1A) 50.726 Site 1 (Healthy-1N) Somerset West (site 2) Healthy (2N) 18.471 Site 3 (Healthy - 3N) Affected (2A) 27.372 Site 2 (Affected - 2A) Port Elizabeth (site 3) Healthy (3N) 28.724 Site 3 (Affected - 3A) Affected (3A) 23.039 Site 2 (Healthy - 2N)

3.4. DISCUSSION

3.4.1. SPATIAL DIFFERENCES IN BIOMARKER RESPONSES AND METALS

3.4.1.1. BIOMARKERS

Biomarkers carried out in this study gave an indication of the responses elicited by Cape Wagtails in their respective sampling areas. However, when interpreting biomarker responses it is important to realize that the exact cause and effect of a potential contaminant cannot always be established or linked (Musquiz, 2003). Acetylcholine esterase, ALA-D and CAT are enzymes and are, for example, strongly influenced by temperature (Ingraham and Ingraham, 2000). It should not be ignored that, during sampling, temperatures may have affected the preservation of the enzymes activity and possibly influenced the results presented in section 3.3. In conjunction with this, small sample size at some sites may also have affected the results. Most importantly, little data is available for small passerines on the aspects covered in this dissertation, especially for Cape Wagtails. This makes it difficult to compare the results obtained with other research studies and to come to sensible conclusions.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Acetylcholinesterase (AChE)

Pollutants exert their neurotoxic actions by inhibiting AChE (Fisher et al., 1999). In other research, AChE tests were carried out on various pesticides in birds and most results revealed an inhibition of the enzyme or variable effects of AChE activity (Lagadic et al., 2000). Consequently, AChE served as a baseline screening for the presence of pesticides in this study. Wagtails characteristically feed on open lawns of school fields and golf course greens (Hayman and Arlott, 1994). Generally, these vast expanses of lawns are treated with various pesticides and it was, therefore, prudent to carry out the protocol of AChE. It was suggested in a study by Martin et al. (1995) that AChE inhibition in pheasant (Phasianus colchicus) chicks occurred due to dermal uptake through the feet. It is not unlikely that this occurred with the wagtails.

At site 5 (Dullstroom), lower enzyme activity prevailed than at sites 1 (Paarl), 3 (Port Elizabeth) and 4 (Secunda) (see Figure 2A, section 3.3). Although site 5 (Dullstroom) is in a rural area where Cape Wagtails occur naturally, the surrounding area is dominated by farming activities (Anon (a), 2006). Pesticide use is expected to be common in the area. Wagtails may have been vulnerable to pesticide intake via air transport or water (Gil and Sinfort, 2005). To support this, Decarie et al. (1993) discovered that insecticide spraying caused a decreased AChE activity in the plasma of the American Robin. At sites 1 (Paarl), 3 (Port Elizabeth) and 4 (Secunda) wagtails roost in and around a sewerage works, a shopping centre car park and a fuel station respectively, and are expected to be less exposed to pesticides than the birds at site 5 (Dullstroom). It is interesting to note that higher enzyme activities occurred at some of the sites which had affected birds (such as at sites 1 (Paarl) and 3 (Port Elizabeth)) as well as at site 4 (Secunda), which had healthy birds. Correlation analyses revealed that there is no evident relationship between AChE activity, altered nerve stimulation and the occurrence of deformities.

δ-Aminolevulinic acid dehydratase (ALA-D)

Most heavy metals are toxic but Pb is one of the most widespread and dangerous (Lagadic et al., 2000). In vertebrates, Pb acts as a neurotoxin and it has been known to inhibit the action of the central nervous system (Burger, 1993 and Truscott, 1970 in Grue et al., 1984). It was, therefore, important to include this biomarker in this research. Biota exposed to Pb experience a decrease or inhibition in erythrocyte ALA-D, depending on the Pb

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

concentration (Lagadic et al., 2000). However, in this study, no significant correlation was found between the activity of ALA-D and various Pb concentrations (see Figures 13 and 14, section 3.3). Although correlation was not significant, it is evident that there was a clear threshold concentration in the feathers and plasma where ALA-D levels decreased. One should keep in mind that Pb sensitivity varies from species to species (Lagadic et al., 2000). Increased Pb levels in erythrocytes cause a decrease in ALA-D activity (Mehdi et al., 2000).

Lower ALA-D activity was observed at the Mpumalanga sites (sites 4 - Secunda and 5 - Dullstroom), relative to the Cape sites (sites 1 - Paarl, 2 – Somerset West and 3 – Port Elizabeth) (see Figure 2B, section 3.3). At the same time, site 3 (Port Elizabeth) was significantly different from sites 4 (Secunda) and 5 (Dullstroom). These results may be due to the heavy industrial practices occurring at site 4 (Secunda) (Anon (b), 2006) and the surrounding agricultural practices (Anon (a), 2006) at site 5 (Dullstroom). This is a conflicting result as one would expect lower activities at sites 1 (Paarl), 2 (Somerset West) and 3 (Port Elizabeth) due to the much greater levels of exhaust fumes which are rich in Pb (Grue et al., 1984). To coincide with this, Grue et al. (1984) reported that ALA-D activity in RBC was lower in highway nesting birds (urban) than in birds in rural habitats. These results show that sites with affected birds (sites 1 - Paarl, 2 – Somerset West and 3 – Port Elizabeth) have higher ALA-D activities than those sites with healthy birds. This, therefore, supports the suggestion that Pb bioaccumulation does not cause or influence feet deformities.

Catalase (CAT)

Catalase activity is expected to be higher in stressful environments (Sanchez et al., 2005) but, under very stressful conditions, a threshold value may be reached where enzyme activity decreases (Moolman, 2004). Sites 2 (Somerset West) and 3 (Port Elizabeth) revealed slightly lower CAT activity than site 1 (Paarl) (see Figure 2C, section 3.3). It is unfortunate that results were not obtained for sites 4 (Secunda) or 5 (Dullstroom) as a means of an enzyme activity comparison, because the sites represented had affected birds while those sites that are not shown did not have affected birds. The wagtails at these three sites may be experiencing oxidative stress. Kale et al. (1999) reported that, when the oxidative stress of an organism increases, it indicates that the cell is protecting itself against the effect of oxyradicals in its surrounding environment (Khessiba et al., 2005), and the activity of CAT increases (Kale et al., 1999). It is difficult to draw conclusions from these results as not all sites are represented. Catalase activity was inhibited in gastropods (Moolman, 2004) and fish

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

(Pandey et al., 2003) in areas of high metal levels. This was true if one considers the levels of Al, As, Cd, Co, Cu, Fe, Pb and Mn (section 3.3.1. Figure 4 and 5) where these metals correlated with the activity of the enzyme at the three sites represented (section 3.3.1. Figure 2C). With reference to Meyer (2005), CAT activity in Weavers (Ploceus sp.) from Barberspan, Rietvlei and Roodekrans in 2005 was lower than the activity recorded in this study.

DNA damage

Gene damage may result when an organism is exposed to pollutants (Fossi and Leonzio, 1994) and DNA damage increased after exposure to heavy metal pollution (Pastor et al., 2001a and 2001b). Therefore, this biomarker was considered. DNA damage was reflected by short fragmented DNA strands and shorter ABPLs (Fossi and Leonzio, 1994). However, DNA damage, expressed as shorter ABPLs, did not correlate with increased metal levels in this study. Site 1 (Paarl) displayed the shortest DNA ABPL which may indicate that the wagtails at this site were more stressed when compared to the wagtails at the other sites (see Figure 2D, section 3.3). Site 6 (Gauteng) had the longest DNA ABPL which indicated that the birds were least stressed at this site, although small sample size should be considered. In Meyer (2005), DNA ABPL in Weavers (Ploceus sp.) from Barberspan, Rietvlei and Roodekrans in 2005 was above 10 000 ABPL, but in this study all sites revealed ABPL that were shorter thus showing that the organisms from the sampling sites in this study were more stressed than the sites studied in Meyer (2005). In a study by Wepener et al. (2005), it was reported that fresh water mussels in a polluted site had longer DNA lengths than an unpolluted site, which shows that the DNA repair system was induced. This normally occurs when the self repairing potential of DNA might affect the understanding of the stress caused by genotoxic agents (Connell et al., 1999). A study done on Cave Swallows (Petrochelidon fulva) and Cliff Swallows (Petrochelidon pyrrhonota) revealed that at a site with agricultural and industrial contamination, and possibly genotoxic chemicals, DNA damage occurred (Musquiz, 2003). This could support the fact that site 1 (Paarl) has shorter DNA ABPLs compared to other sites.

In terms of significant differences among sites with affected birds and those without, sites 1 (Paarl) and 2 (Somerset West), which both had affected birds, were significantly different to site 3 (Port Elizabeth), which had affected birds, and sites 4 (Secunda) and 5 (Dullstroom) both of which did not have affected birds. Therefore, one can interpret these results and

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

conclude that, although there are significant differences among sites, there is no clear difference between DNA damage in affected birds and healthy birds.

The NMDS ordination and cluster analysis revealed specific groupings according to the biomarkers tested at each site. It is interesting to note that in groupings 2 and 3, sites where no deformities were recorded were similar in biomarkers to sites that had deformities, such as in group 2; Port Elizabeth (site had affected birds), Dullstroom (healthy birds) and Secunda (healthy birds).

Because both DNA damage and CAT are both influenced by oxidative stress (Guecheva et al., 2003 and Kale et al., 1999), regression analysis (Figure 12, section 3.3) was carried out based on the data obtained. However, no significant relationship was revealed. This may have been because of small sample sizes or alteration of the enzymes and proteins during sampling due to temperature fluctuations (Ingraham and Ingraham, 2000).

In summary, decreased AChE activity (as indicator of exposure to organic pesticides) was independent of the occurrence of feet abnormalities and the two were not found to be related. Lead accumulation in feathers and plasma and ALA-D enzyme activity was not found to be linked to the feet and toe deformities. Catalase activity at sites 1 (Paarl), 2 (Somerset West) and 3 (Port Elizabeth) revealed a possible stress amongst the birds at those sites, although no comparison could be made to sites with healthy birds. In terms of DNA damage, site 1 (Paarl) appeared to be the most stressed site while site 6 (Gauteng) appeared to be the least stressed. There seemed to be no difference between DNA damage in sites that had affected birds and those that did not, therefore indicating that the feet abnormalities and DNA damage are not linked. When considering the integrated biomarker responses the resultant NMDS ordination did not reveal different patterns among sites that had birds with deformities and those sites without affected birds. The biomarkers used in this study did not distinguish between birds with and without feet deformities.

3.4.1.2. METALS IN FEATHERS

Metal levels in feathers can be used as indicators of local exposure (Dauwe et al., 2002). Wagtails have small territories in urban areas (Engelbrecht et al., 2005) which make them good bioindicators of point-source contamination (Eens et al., 1999). It is difficult to know what the threshold value of the Cape Wagtail is for a specific metal. One can only rely on the

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

data that is available on studies performed on birds of similar character, size and feeding type.

It is very difficult to explain the results obtained as little information has been published on the metal levels in bird feathers. As a consequence, various authors’ works have been integrated in the following few paragraphs to seek explanations for the results obtained (see Table 6). A study completed on Weaver (Ploceus sp.) feathers from various sites in Gauteng was used as the closest comparison for the data recorded in this research (Meyer, 2005). Therefore, Weaver (Ploceus sp.) EcoQO concentrations were regarded as reference values for passerine birds in this study.

For most metals, site 4 (Secunda) presented the highest metal levels. These results were anticipated, possibly because to the fact that Secunda is a highly industrialized area (Anon (b), 2006), when compared to the other sites. For most metals, sites 5 (Dullstroom) or 6 (Gauteng) displayed the lowest metal levels. The results obtained are regarded with caution as sample size was relatively small (n = 6 for both sites).

Aluminium (Al)

Current Al levels at all sites exceed the EcoQO target level. Levels of Al in this study were higher (other than site 6 – Gauteng) than those recorded (430 µg/g dry weight (dw)) in Weaver (Ploceus sp.) feathers (Meyer, 2005). Eens et al. (1999) reported values at a polluted site (81.2 µg/g dw for Blue Tits (P. caeruleus) and 88.3 µg/g dw for Great Tits (P. major)) which were much lower than the results presented (see Figure 4A, section 3.3). This suggests that the sampling sites in this study were either more contaminated if one refers to past studies or that prevailing natural background levels of Al were higher. Sampling size was small and this should be taken into account.

Arsenic (As)

If one considers As concentrations at a polluted site (16.3 µg/g dw - Dauwe et al., 2004), then concentrations at sites 1 (Paarl), 2 (Somerset West), 3 (Port Elizabeth) and 6 (Gauteng) were below these values but sites 4 (Secunda) and 5 (Dullstroom) were above these values (see Figure 4B, section 3.3). These values were expected for Secunda due to the high industrial activities (Anon (b), 2006) which occur at that site. At site 5 (Dullstroom), high As

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

levels may be due to the pesticides used in the adjacent farming areas (Gregory et al., 1996 in Sarkar et al., 2005). High As levels can cause growth and reproductive problems as well as muscle inco-ordination amongst other symptoms (Stanley et al., 1994; Whitworth et al., 1991 and Camardese et al., 1990 in USEPA, 2005). Arsenic levels were higher than the results obtained in Weavers (Ploceus sp.) (Meyer, 2005).

Cadmium (Cd)

Cadmium is said to be highly toxic at unnatural levels. It is potentially mutation causing with various lethal and sub-lethal affects at very low doses (Eisler 1985a in USEPA, 2005). It can affect enzyme activities (USEPA, 2005) but it was not apparent in this study. Current Cd levels at all sites exceeded the EcoQOs target levels. Cadmium values (2.6 µg/g dw for Great tits (P. major) and 3 µg/g dw in Blue tits (P. caeruleus)), at a waste incinerator in Burger (1993), are amongst the highest levels reported for bird species. Burger and Gochfield (1995) presented a value of 0.1 µg/g dw at two industrial and agricultural sites. With referral to works by these two authors, the results presented (see Figure 4C, section 3.3) were much higher. In addition to this, results (2.2 µg/g dw) by Meyer (2005) for Weavers (Ploceus sp.) were lower than the results presented in this dissertation (except at site 6 – Gauteng). However, if one considers the study by Dauwe et al. (2004) at a polluted site, the values obtained in this study were lower. This suggests that the sites sampled in this study were not as polluted as the sites studied by Dauwe et al. (2004), but were more polluted than the sites sampled in Burger (1993) and Burger and Gochfield (1995).

Chromium (Cr)

Chromium concentrations at all six sites were above the EcoQOs target concentrations. Burger and Gochfield (1995) reported values of between 9.6 µg/g dw and 13.7 µg/g dw at two polluted (industrial and agricultural) areas. The results in this study were much higher (see Figure 4D, section 3.3). Chromium results of between 4 µg/g and 18 µg/g were obtained by Meyer (2005) in Weaver (Ploceus sp.) birds which were much lower than the levels found in this research. To support this, Cr levels ranged from 0.5 µg/g dw to 2.4 µg/g dw for birds in an area dominated by agricultural and industrial contaminants (Musquiz, 2003). Again, the results in this study were much higher which suggests that the sites sampled were either fairly contaminated or natural background levels of Cr were higher.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Cobalt (Co)

Current Co concentrations at all sites exceeded the EcoQO target level. Movalli (2000) measured a mean Co value of 0.9 µg/g dw in Laggar Falcons (Falco biarmicus jugger) for the studied sites. Site 1 (Paarl) was above this value but sites 2 (Somerset West), 3 (Port Elizabeth), 4 (Secunda), 5 (Dullstroom) and 6 (Gauteng) were all below this value (see Figure 4E, section 3.3). However, levels in this study were higher than Co levels reported in Weavers (Ploceus sp.) (Meyer, 2005) which nearly reached 0.125 µg/g dw.

Copper (Cu)

At all sites except at site 2 (Somerset West), current Cu levels were above the EcoQO target value. Eens et al. (1999) obtained values of 13.8 µg/g dw for Great tits (P. major) and 24 µg/g dw for Blue tits (P. caeruleus) at a contaminated site (a waste incinerator). Copper results obtained were higher than those by Eens et al. (1999) as well as Meyer (2005) (see Figure 4F, section 3.3). In Dauwe et al. (2004), Cu values were 67.7 µg/g dw in the immediate vicinity of a pollution emission. Sites 1 (Paarl), 2 (Somerset West) and 3 (Port Elizabeth) were below these values of Dauwe et al. (2004), while sites 4 (Secunda), 5 (Dullstroom) and 6 (Gauteng) were above.

Iron (Fe)

At present, Fe levels exceed the EcoQO target concentration at all sites. Site 1 (Paarl) presented much higher levels of Fe when compared to the other sites (see Figure 5A, section 3.3). In addition, levels reported (480 µg/g dw) for Weavers (Ploceus sp.) by Meyer (2005) were lower than the Fe levels in this study.

Lead (Pb)

Current Pb concentrations at sites 2 (Somerset West), 3 (Port Elizabeth) and 6 (Gauteng) did not exceed the target level of the EcoQO, however, sites 1 (Paarl), 4 (Secunda) and 5 (Dullstroom) were above the EcoQO target level. Lead levels and ALA-D activities can be compared. Enzyme activity at sites 1 (Paarl), 2 (Somerset West) and 3 (Port Elizabeth) were higher with respect to the other sites but depicted lower Pb levels (see Figure 2B). However, if one looks at the lower ALA-D activity at sites 4 (Secunda) and 5 (Dullstroom), higher

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

levels of Pb were presented for those sites. Therefore, by looking at these results one can conclude that Pb levels influence ALA-D activity which is supported by the works of Lagadic et al. (2000). In a study by Eens et al. (1999) at a polluted site, Pb values between 16.28 µg/g dw in Great tits (P. major) and 64 µg/g dw in Blue tits (P. caeruleus) were obtained. These results were reported to be amongst the highest levels reported for bird species (Burger, 1993). In addition, Dauwe et al. (2004) reported a level of 60.7 µg/g dw in the immediate vicinity of a pollution emission. Results were below 64 µg/g dw (Eens et al., 1999) but higher than 16.28 µg/g dw (Eens et al., 1999) except at site 4 (Secunda) where levels were greater (see Figure 5B, section 3.3). This can only suggest that site 4 (Secunda) is fairly polluted when compared to the other sites of this study. This was inevitable as Secunda (site 4) is an industrial hub with daily doses of potentially toxic emissions (Anon (b), 2006). To support this, the results obtained were lower than those presented by Meyer (2005) for Weavers (Ploceus sp.) (about 70 µg/g dw) except at site 4 (Secunda).

Manganese (Mn)

Manganese levels at all sites exceeded the EcoQO target concentration. Results were much higher than the results (22.901 µg/g dw – 35.99 µg/g dw) reported by Burger and Gochfield (1995) for two polluted (industrial and agricultural) sites as well as a value of 15 µg/g dw (Meyer, 2005) (see Figure 5C, section 3.3). This indicates that the sites sampled were either fairly contaminated or natural background levels of Mn were higher.

Nickel (Ni)

Little information has been reported on Ni concentrations in bird feathers. Current Ni concentrations at all sites exceeded the EcoQO target level. All sites presented, revealed a higher Ni level than those results (9.1 µg/g dw) reported by Dauwe et al. (2004) for a polluted site and in Weavers (Ploceus sp.) (2.5 µg/g dw) by Meyer (2005) (see Figure 5D, section 3.3). This indicates that the sites sampled were either fairly contaminated or natural background levels of Ni were higher.

Silver (Ag)

Silver concentrations of 1.84 µg/g dw were reported by Dauwe et al. (2004) for a site in the immediate vicinity of a pollution emission were lower than the results presented in Figure 5E

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

(section 3.3). This suggests that the sites may be fairly contaminated when compared to other studied sites.

Strontium (Sr)

There is little data available on Sr levels in bird feathers. At present, Sr levels at all the sites, except at site 5 (Dullstroom), were above the EcoQO target concentration. Levels in this study were higher than in Weaver (Ploceus sp.) birds (7.5 µg/g dw) reported by Meyer (2005) (see Figure 5F, section 3.3). It is interesting to note that Sr levels appeared to influence the AChE activity (see Figure 2A, section 3.3) at each site.

Zinc (Zn)

Currently, the Zn levels at all the sites exceed the EcoQO target level. Eens et al. (1999) and Dauwe et al. (2004) reported values of between 172.7 µg/g dw and 443.9 µg/g dw in contaminated areas. The results presented in Figure 6 are much higher than these values. Zinc levels in this study were also higher when compared to the levels in Weavers (Ploceus sp.) as reported by Meyer (2005) which were about 250 µg/g dw. High levels of Zn can cause mortality, decreased growth, damage to the pancreas and weight loss in birds (Eisler, 1993 and NAS 1980 in USEPA, 2005). Decreased growth and weight loss was evident at sites 5 (Dullstroom) and 6 (Gauteng) when compared to the health condition of the Wagtails caught at the remaining sites. The levels of Zn at all sites was of great concern, unless natural background levels of Zn were high at the sites sampled.

Table 6 represents the reference values used (in the above paragraphs) to compare the values obtained in this study to the values reported by other researchers for polluted and unpolluted sites. Rankings (indicated by symbols) show the comparison results among sites.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Table 6. Grading the metal results (µg/g dw) in the feathers (S1 = site 1 (Paarl), S2 = site 2 (Somerset West), S3 = site 3 (Port Elizabeth), S4 = site 4 (Secunda), S5 = site 5 (Dullstroom) and S6 = site 6 (Gauteng)) against the results reported by other researcher (symbols (-, + and ++) indicate that the results in this study were lower, higher and much higher respectively than those obtained by other researchers (dw represents dry weight) Al As Cd Cr Co Cu Fe S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 Weaver + + + + + - ++ ++ ++ ++ ++ ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + (Ploceus sp.) birds (closest South African comparison) Meyer (2005) (µg/g dw) Eens et al. (1999) Dauwe et al. (2004) Burger (1993), Burger and Burger and Gochfield Dauwe et al. (2004) / Eens Gochfield (1995) / Dauwe et al. (1995) / Musquiz (2003) et al. (1999) (2004) Contaminated + + + + + + - - - + + - ++ ++ ++ ++ ++ ++ + + + + + + ------+ + + ------areas /- /- /- /- /- /- / / / / / / / / / / / / (µg/g dw) + + + + + + + + + + + + Movalli (2000) Unknown ------+ ------sites (µg/g dw)

Table 6. Continued

Pb Mn Ni Ag Sr Zn S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 Weaver (Ploceus sp.) birds - - - + - - + + + + + + + + + + + + ------+ + + + + + + + + + + + (closest South African + + + + + + comparison) Meyer (2005) + + + + + + (µg/g dw) Eens et al. (1999) – below Burger and Gochfield Dauwe et al. (2004) Dauwe et al. (2004) Eens et al. (1999) and 64 µg/g dw but above (1995) Dauwe et al. (2004) 16.28 µg/g dw - 9 / Dauwe et al. (2004) Contaminated areas 9 9 9 9 9 9 + + + + + + + + + + + + + + + + + + ------+ + + + + + (µg/g dw) / - / / / / / + + + + + + - - - - - + + + + + +

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Chromium and Ni revealed significant differences (p<0.05) among sites. For both metals, site 1 (Paarl) was significantly different from sites 3 (Port Elizabeth), 4 (Secunda), and 5 (Dullstroom). The reason for the high metal levels, as far as Cr and Ni are concerned, and the significant differences are unclear. Site 3 (Port Elizabeth) was significantly different to site 4 (Secunda) for Ni (see Figure 5D, section 3.3). This was not expected as site 4 (Secunda) is a heavy industrial area (Anon (b), 2006). However, this may indicate that Ni has an influence on the deformities as site 3 (Port Elizabeth) had affected birds but site 4 (Secunda) did not. As far is Cr (see Figure 4D, section 3.3) was concerned, site 3 (Port Elizabeth) was significantly different from sites 4 (Secunda) and 5 (Dullstroom). This too was not expected but Cr may be contributing to the deformities, as both sites 4 (Secunda) and 5 (Dullstroom) did not have affected birds, but site 3 (Port Elizabeth) did. In conclusion, the role that Cr and Ni may have on the feet abnormalities requires further investigation.

Referring to the EcoQOs (Table 3, section 3.3), all the selected metals (Al, Cd, Co, Cr, Fe, Mn, Ni and Zn) exceed the target level at all sites. The remaining metals (Cu, Pb and Sr) exceed the levels at some sites and not at others (Cu and Pb levels do not exceed the target level at site 2 (Somerset West), Pb levels do not exceed the target level at sites 3 (Port Elizabeth) and 6 (Gauteng) and Sr levels at site 5 (Dullstroom) are not above the EcoQOs).

Referring to Figure 7 (section 3.3), at Secunda and Dullstroom (group 2 in Figure 7A) no affected birds were sampled. In group 1, all sites except site 6 (Gauteng) had affected birds. One can only deduce from these results that the groupings were a result of similar metal levels and not because of the presence of affected or healthy birds. This can be seen in Figures 4, 5 and 6. where most metals (such as Al, As, Cu, Pb, Mn and Ag) were higher at both sites 4 (Secunda) and 5 (Dullstroom) where healthy birds were sampled. This indicates that for those metals it is highly unlikely that they contribute to the occurrence of feet abnormalities. Chromium and Ni are the only metals with higher levels at sites 1 (Paarl), 2 (Somerset West) and 3 (Port Elizabeth) where affected birds were sampled compared to the lower levels at sites 4 (Secunda) and 5 (Dullstroom).

In conclusion, Al (except at site 6 – Gauteng), As, Cd, Cr, Co, Cu, Fe, Pb (at site 4 – Secunda), Mn, Ni, Sr and Zn levels were above the values reported by Meyer (2005). No values were available for Ag. In terms of the contaminated reference values, Al, As (at sites 4 – Secunda and 5 – Dullstroom), Cu (at sites 4 – Secunda, 5 – Dullstroom and 6 – Gauteng),

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Mn, Ni, Ag and especially Zn were higher then the reference values. Cadmium, Co and Cu revealed mixed results for some sites. Chromium and Ni displayed significant differences among sites and could have contributed to the deformities. Similarity groupings were suggested because of similar metal levels rather than because of metal level differences between affected and healthy birds.

3.4.1.3. METALS IN PLASMA

Very little data exists concerning the levels of various metals in the blood plasma of birds especially Al, Cr, Fe, Mn, Ni and Ag. It should be kept in mind throughout the discussion that in terms of the sites being represented in the figures, sites 1(Paarl) and 3 (Port Elizabeth) had affected birds while sites 4 (Secunda) and 5 (Dullstroom) did not. Aluminium, Ca, Cu, Pb, Mn, Ni and Zn were all higher at sites 4 (Secunda) and 5 (Dullstroom) than at sites 1 (Paarl) and 3 (Port Elizabeth) which shows that these high metal levels did not influence feet abnormality occurrences. Site 4 (Secunda) displayed the highest Al, Ca, Co, Pb and Zn levels. High levels at sites 4 (Secunda) and 5 (Dullstroom) were possibly due to heavy industrial emissions at Secunda and farming activities in the Dullstroom area (Anon (b), 2006 and Anon (a), 2006). Site 1 is situated at Paarl Bird Sanctuary close to Paarl Sewage Works. A wide variety of waterfowl are attracted to this area (Hester, 2001). The reason for site 1 (Paarl) showing the highest As, Cd, Cu, Fe and Ag levels is not clear. It is interesting to note that site 3 (Port Elizabeth), which had the highest rate of deformities, did not have the highest plasma metal level compared to other sites indicating that it is highly unlikely that metals accumulated in the plasma have any influence on feet deformities. In terms of a comparison between metal levels in the feathers and plasma, only Al, Pb and Mn were higher at sites 4 (Secunda) and 5 (Dullstroom) in both the feathers and plasma when compared to the other sites.

Arsenic (As)

Arsenic levels in an uncontaminated area were recorded to be 0.02 µg/l (Burger and Gochfield, 1997 in Benito et al., 1999). All sites were above this metal concentration (see Figure 8B, section 3.3) Contradicting this, Benito et al. (1999) recorded levels of up to 0.181 µg/l at a toxic spill. It is noticeable that the As levels at each site in this study were below those concentrations.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Cadmium (Cd)

In an uncontaminated site, levels of Cd were around 0.001 µg/l (Garcia-Fernandez et al., 1995 in Benito et al., 1999). All study sites revealed higher Cd levels (see Figure 8C, section 3.3). In a contaminated site, Benito et al. (1999) documented concentrations of between 0.0001 µg/l and 0.029 µg/l. Concentrations at sites 1 (Paarl), 4 (Secunda) and 5 (Dullstroom) were greater than the values in Benito et al. (1999), although site 3 (Port Elizabeth) was lower. Site 1 (Paarl) was quite a lot higher than other sites which may reveal an abnormally high level of Cd, however, if one refers to the levels of this metal in the feathers, site 1 (Paarl) does not display the highest levels when compared to the other sites. Cadmium is highly toxic with potential mutation causing severe affects (at relatively low concentrations) such as mortality, increased muscle contractions, enzyme activity interference, disruption to the respiratory system and decrease in growth rate (USEPA, 2005), although none of these symptoms could be positively linked to the observations during sampling.

Calcium (Ca)

Leg disorders can be caused by nutrient imbalances. Calcium is one of the most abundant minerals in bone (Waldenstedt, 2006) and Ca is essential for bone formation. Deficiencies often result in skeletal disorders (Williams et al., 2000). This may apply to the results revealed at sites 1 (Paarl) and 3 (Port Elizabeth) where less Ca concentrations existed in an area where affected birds were prevalent rather than at sites 4 (Secunda) and 5 (Dullstroom), where no deformities were found but with higher Ca concentrations (see Figure 8D, section 3.3). This leads to the suggestion that Ca may be a contributing factor to the deformities in this case. In ring doves (Columba palumbus), Ca levels were reported to be in the range of 0.085 µg/l and 126 µg/l (Scheuhammer, 1996). All sites studied, except site 1 (Paarl), had much greater Ca levels than in the ring doves, especially sites 4 (Secunda) and 5 (Dullstroom). Lead and Zn are known to displace Ca from it’s binding site which weakens the bone structure (Huggett et al., 1992). This may be the case as high Pb (Figure 9C, section 3.3) and Zn (Figure 10, section 3.3) levels are associated with lower Ca (Figure 8D, section 3.3) levels.

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Cobalt (Co)

At a toxic spill site, Co levels of up to 0.11 µg/l were found (Benito et al., 1999). All sites studied were below these recorded Co concentrations (see Figure 8F, section 3.3).

Copper (Cu)

Levels of 1.530 µg/l were documented in a study by Benito et al. (1999) in an area affected by a toxic spill. In addition to these values, van Eeden and Schoonbee (1996) revealed a Cu concentration of 1.15 µg/l at a contaminated site. All the sites studied were below this value (see Figure 9A, section 3.3) except site 1 (Paarl) which indicated an abnormally high level of Cu suggesting toxic concentrations if one refers to the values obtained for contaminated sites by other authors. A possible explanation is that the lawns on which the wagtails feed is watered with sewage recycled water which may have an unusually high Cu concentration. This essential metal is needed by birds but can be extremely toxic above a threshold value and various sub-lethal affects have been observed (Ramey and Sterner, 1995; Martinez and Diaz, 1996 and Ewing et al., 1998 in Benito et al., 1999). High levels of Cu can cause lowered egg production, decreased growth as well as development abnormalities (USEPA, 2005).

Lead (Pb)

In an uncontaminated area, Pb levels were about 0.062 µg/l in ducks (Dieter et al., 1976 in Benito et al., 1999). The results obtained for each site were all above this Pb concentration (see Figure 9C, section 3.3). In a study by Benito et al. (1999), Pb levels were between 0.002 µg/l and 0.454 µg/l in the vicinity of a toxic spill. Site 4 (Secunda) was above this value but other sites were below. In a sample of Mallards, Pb levels were at a concentration of 0.5 µg/l. This level is regarded as causing Pb poisoning in swans (Blust et al., 1991 in Benito et al., 1999). Sites were just below the Pb level but at site 4 (Secunda) the concentration was very close to a poisoning level (0.47 µg/l). It is possible for a small bird such as the wagtail that Pb poisoning might occur at a lower concentration. If this is so, birds at site 4 (Secunda) might be heavily intoxicated with Pb. High levels of Pb are known to interrupt the functioning of the nervous system, decreased growth, sterility and developmental retardation (USEPA, 2005).

The Search for the Reason(s) Causing Feet Abnormalities in the Cape Wagtail (Motacilla capensis)

Zinc (Zn)

In Benito et al. (1999), Zn levels in birds were between 0.3 µg/l and 8.6 µg/l in an area affected by a toxic spill. All sites displayed higher Zn levels than those researched by Benito et al. (1999) (see Figure 10, section 3.3). Zinc is an essential element required by birds. If the threshold concentration is exceeded, sub-lethal effects have and can be observed (Ramey and Sterner, 1995; Martinez and Diaz, 1996 and Ewing et al., 1998 in Benito et al., 1999). Increased Zn levels can cause decreased growth rates, decreased ability to gain weight and mortality (Eisler 1993 and NAS 1980 in USEPA, 2005).

Table 7 represents the reference values used (in the above paragraphs) to compare the values obtained in this study to the values reported by other researchers for polluted and unpolluted sites. Rankings (indicated by symbols) show the comparison results among sites.

Referring to the NMDS ordination (Figure 11, section 3.3), Secunda (site 1) and Dullstroom (site 5) were grouped together while Paarl (site 1) and Port Elizabeth (site 3) were grouped. The groupings were due to Ca and Zn. Calcium may be lower at sites 1 (Paarl) and site 3 (Port Elizabeth) because of the presence of feet deformities, compared to sites 4 (Secunda) and 5 (Dullstroom) where no deformities occur. Most metals (Al, Co, Co, Pb, Mn, Ni and Zn) were higher at sites 4 (Secunda) and 5 (Dullstroom) where no deformities occurred. Iron is the only metal where both sites 1 (Paarl) and site 3 (Port Elizabeth) together were higher in levels than at sites 4 (Secunda) and 5 (Dullstroom) together. One can, therefore, conclude that it is unlikely that feet abnormalities are related to metal levels in the plasma.

Site 4 (Secunda) had the highest TUs (Table 4). This was expected as this site is an industrial area with potentially more toxic emissions than any other site studied. In addition to this, birds at site 4 were higher in metal levels than was the case at other sites, in the feathers as well as the plasma. Site 1 (Paarl) revealed the second highest toxic unit. This was not expected but if one considers the results obtained for the metals, particularly in the feathers, this site was generally higher than all the sites, or at least most of them.

In summary, As, Cd and Pb were higher than the reference values for uncontaminated sites. Arsenic, Cd (at sites 1 – Paarl, 4 – Secunda and 5 – Dullstroom), Cu (at site 1 – Paarl), Pb (at site 4 – Secunda) and Zn were higher than the reference values used for various contaminated sites. Calcium (at site 3 – Port Elizabeth) was higher than the reference value

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Table 7. Grading the metal results (µg/l) in the plasma (site 1 = Paarl, site 2 = Somerset West, site 3 = Port Elizabeth, site 4 = Secunda, site 5 = Dullstroom and site 6 = Gauteng) against the results reported by other researchers (symbols (-, + and ++) indicate that the results in this study were lower, higher and much higher respectively than those obtained by other researchers Al Cd Ca Co Cu Pb Zn Site Site Site Site Site Site Site Site Site Site Site Site Site Site Site Site Site Site Site Site Site Site Site Site Site Site Site Site 1 3 4 5 1 3 4 5 1 3 4 5 1 3 4 5 1 3 4 5 1 3 4 5 1 3 4 5 Uncontaminated + + + + + + + + ------+ + + + - - - - (Benito et al., 1999) Contaminated - - - - + - + + ------+ - - - - - ++ - + + + + (Benito et al., 1999) Unknown ------+ ++ ++ ------(Scheuhammer, 1996)

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so it was unlikely that a shortage of Ca in the birds contributed to feet abnormalities. Sites 4 (Secunda) and 5 (Dullstroom) were much higher than the reference value of Ca. Most metals (Al, Cu, Co, Pb, Mn, Ni and Zn) were higher at sites 4 (Secunda) and 5 (Dullstroom) where no deformities occurred, demonstrating that it is unlikely that metals in the plasma trigger feet deformities. Iron, however, was the only metal where both sites 1 (Paarl) and site 3 (Port Elizabeth) together were higher than at sites 4 (Secunda) and 5 (Dullstroom) together.

3.4.2. SUMMARY OF THE DIFFERENCES BETWEEN HEALTHY AND AFFECTED CAPE

WAGTAILS

There is little, if any, data provided by other researchers that can be used to compare the results obtained in this study. No past literature has been found that covers the aspects and tests that were carried out in this study on birds with deformities of the same nature. This makes it difficult to evaluate and comment on, or take a stance on, the results presented.

3.4.2.1. BIOMARKERS

Acetylcholinesterase (AChE)

Affected birds at both sites 1 (Paarl) and 3 (Port Elizabeth) had higher enzyme activities than healthy birds at those sites. There was noticeably more inhibition of the enzyme in healthy birds compared to affected ones (see Figure 15A, section 3.3). From these results, it could be deduced that AChE enzyme activity and nerve interruption was independent of the occurrence of feet abnormalities.

δ-Aminolevulinic acid dehydratase (ALA-D)

δ-Aminolevulinic acid dehydratase enzyme activities were lower in affected birds compared to healthy birds at site 1 (Paarl). However, at site 3 (Port Elizabeth), enzyme activities were almost similar for affected and healthy wagtails. Site 2 (Somerset West) could not be commented on as no affected samples were represented (see Figure 15B, section 3.3). It can be suggested that the reason for the decreased enzyme activity in healthy birds was due to higher Pb levels. But, if this was so, enzyme activity for all the birds (both healthy and affected) at site 1 (Paarl) would be the same. However, this is not the case. At site 3 (Port

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Elizabeth) enzyme activities were very similar and thus it can be suggested that ALA-D activity and Pb accumulation have little to do with the incidence of feet deformities.

Catalase (CAT)

Catalase activity was expected to be higher in more stressful environments (Sanchez et al., 2005). This may be applicable for affected birds at sites 1 (Paarl) and 3 (Port Elizabeth) which had higher enzyme activities than healthy birds (see Figure 15C, section 3.3). The activity of CAT is known to increase when an organism is experiencing oxidative stress (Kale et al., 1999). When this happens, it indicates that cells are protecting themselves against the effect of oxyradicles in their surrounding environment (Khessiba et al., 2005). From these results, one can suggest that affected birds were more stressed than healthy birds. In this case, CAT activity is related to the presence of feet abnormalities. Site 2 (Somerset West) could not be commented on due to a lack of data.

DNA damage

DNA damage at sites 1 (Paarl) and site 2 (Somerset West) was very similar between healthy and affected birds. However, affected birds had slightly shorter DNA ABPLs than healthy birds (see Figure 15D, section 3.3). This was expected as affected birds would be more stressed and thus show shorter DNA ABPLs, which they did. DNA damage between healthy and affected birds at site 3 (Port Elizabeth) was significantly different and, again, affected birds had shorter DNA ABPLs. It could be concluded that DNA damage and ABPL between affected and healthy birds could be linked to the occurrence of birds with affected legs and those without.

In conclusion, the biomarker responses investigated did not seem to alter between affected and healthy birds, however, there appeared to be a distinction between ABPLs.

3.4.2.2. METALS IN FEATHERS

Aluminium and Zn (at sites 1 – Paarl, 2 – Somerset West and 3 – Port Elizabeth), Cr (at sites 1 – Paarl and 2 – Somerset West) and Ag (site 3 – Port Elizabeth only) revealed higher levels in affected birds compared to healthy birds. These metals could be linked to the feet and toe deformities in those areas. However, if one looks at the levels of those metals in the feathers,

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it is unlikely that they contributed to the deformities. This is because site 4 (Secunda) had no feet deformities but it presented higher Al, Ag and Zn levels than at the sites mentioned. Chromium may contribute to the abnormalities because levels in the feathers at sites 1 (Paarl) and 3 (Port Elizabeth) were significantly higher than sites 4 (Secunda) and 5 (Dullstroom).

Arsenic, Cd, Co, Fe, Pb, Mn, Ni and Sr at sites 1 (Paarl) and 3 (Port Elizabeth) and Cu at sites 1 (Paarl), 2 (Somerset West) and 3 (Port Elizabeth) presented results that showed that healthy birds had higher levels of these metals than affected birds. This implies that it is very unlikely that these metals triggered or contributed towards the feet deformities found at these sites. At site 2 (Somerset West), As, Cd, Co, Fe, Pb, Mn, Ni and Sr were higher in affected birds than in healthy birds. This suggested that these metals may be contributing to the deformities at this site, but this is questionable when one remembers that sites 1 (Paarl) and 3 (Port Elizabeth) were the opposite.

Affected birds at site 1 (Paarl) revealed the greatest TUs. This was unexpected considering the metal results between affected and healthy birds discussed in the paragraphs above. Healthy birds at site 1 (Paarl) were second in order in terms of its TU. It is not easy to draw conclusions from this. It must be remembered that TUs look at the combined metal exposure (Sprague, 1970 in Yoo et al., 2003) and not at individual metal levels (discussed above). Considering each metal individually appeared to be clearer and more easily explained. In Table 4 (section 3.3), site 1 (Paarl) had the highest TU (ignoring Secunda as no affected birds were found there). In Table 5 (section 3.3), site 1 (Paarl), both healthy and affected birds were the highest TUs amongst the sites and conditions. Site 2 (Somerset West), in Table 4 (section 3.3) was the second highest in terms of TUs (ignoring Gauteng as no affected birds were found at that site). In Table 5 (section 3.3) site 3’s (Port Elizabeth) healthy birds were higher than site 2 (Somerset West) affected birds which indicates that there is a difference between affected and healthy birds in terms of their TUs at site 3 (Port Elizabeth). This suggested that metal levels as a whole were not contributing or causing feet abnormalities in the wagtails.

In conclusion, the combined toxicity of metal exposure (the TU values) were generally higher in affected birds than in healthy birds, however, studying metals separately revealed that higher concentrations were found predominantly at sites with healthy birds but it is

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doubtful that metal exposure reflected in the feathers had any influence on the feet and toe deformities found in the Cape Wagtails at the three sites studied.

To conclude this chapter, if one considers at the discussed results on a site versus site basis, AChE, ALA-D and DNA damage were not linked to the occurrence of feet deformities. However, CAT revealed stress at the sites represented which may have been caused by feet abnormalities or other factors. In terms of metals in the feathers, Zn levels at all sites was a cause for concern. Most metals were higher than reference values for contaminated sites. Various metals in the plasma were higher than the reference values used, which requires further study. Iron was discovered to potentially cause or influence deformities. If one refers to the discussion between affected birds and healthy birds, CAT enzyme activity and DNA damage was suggested to be indicative of feet abnormalities. However, spatial differences did not reveal evidence that DNA damage was indicative of feet abnormalities. Metal levels were generally higher in healthy birds than in affected birds which indicated, that it is doubtful that metal exposure is responsible for deformities, or even contributes to them.

Based on the results discussed in this chapter, there is not enough substantial evidence to suggest that pesticides, metals and other contaminants in both the feathers and plasma contribute, trigger or cause feet and toe deformities in Cape Wagtails.

3.5. REFERENCES

Anonymous. 2003. Catalase Lab. Available from: http:www.galaxynet.com. (Accessed: November 2005). Anonymous (a). 2006. The Complete Africa Travel Guide and Reservation Service for your African Safari. Available from: www.go2africa.comsouth-africa/panorama-west (Accessed: March 2006). Anonymous (b). 2006. Sasol Watch. Available from: www.sasolwatch.com (Accessed: March 2006). Battaglia, A., Ghidini, S., Campanini, G. and Spaggiari, R. 2005. Heavy Metal Contamination in Little Owl (Athene noctua) and Common Buzzard (Buteo buteo) from Northern Italy. Ecotoxicology and Environmental Safety 60: 61-66. Benito, V., Devesa, V., Munoz, O., Suner, M.A., Montoro, R., Baos, R., Hiraldo, F., Ferrer, M., Fernandez, M. and Gonzalez, M.J. 1999. Trace Elements in Blood Collected from Birds Feeding in the Area Around Donana National Park Affected by the Toxic Spill from the Aznalcollar Mine. The Science of the Total Environment 242: 309 – 323.

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Burger, J. 1993. Metals in Avian Feathers: Bioindicators of Environmental Pollution. Review of Environmental Toxicology 5: 203-311. Burger, J. and Gochfeld, M. 1995. Biomonitoring of Heavy Metals in the Pacific Basin Using Avian Feathers. Environmental Toxicology and Chemistry 14(7): 1233-1239. Clarke, K.R. and Warwick, R.M. 2001. Change in Marine Communities: An approach to Statistical analysis and Interpretation. Second Edition. PRIMER-E: Plymouth. Cohen, G., Dembiec, D. and Marcus, J. 1969. Measurement of Catalase Activity in Tissue Extracts. Analytical Biochemistry 34: 30-38. Connell, D., Lam, P., Richardson, B. and Wu, R. 1999. Introduction to Ecotoxicology. Blackwell Science Pty Ltd. United Kingdom. Pp 170. Dauwe, T., Bervoets, L., Ellen, J., Rianne, P., Ronny, B. and Marcel, E. 2002. Great and Blue Tit Feathers as Biomonitors for Heavy Metal Pollution. Ecological Indicators 1: 227-234. Dauwe, T., Janssens, E., Bervoets, L., Blust, R. and Eens, M. 2004. Relationships between Metal Concentrations in Great Tit Nestlings and their Environment and Food. Environmental pollution 131: 373-380. Decarie, R., DesGranges, J.L., Lepine, C. and Morneau, F. 1993. Impact of Insecticides on the American Robin (Turdus migratorius) in a Suburban Environment. Environmental Pollution 80: 231-238. De Coen, W.M.I. 1999. Energy Metabolism, DNA Damage and Population Dynamics of the Water Flea Daphnia magna (straus) Under Toxic Stress. Unpublished thesis. University of Ghent. Belgium. Denneman, W.D. and Douben, P.E.T. 1992. Trace Metals in Primary Feathers of the Barn Owl (Tyto alba guttatus) in the Netherlands. Environmental Pollution 82: 301-310. Eens, M., Pinxten, R., Verheyen, R.F., Blust, R. and Bervoets, L. 1999. Great and Blue Tits as Indicators of Heavy Metal Contamination in Terrestrial Ecosystems. Ecotoxicology and Environmental Safety 44: 81-85. Eeva, T., Tanhuanpaa, S., Lehikoinen, E., Nikinmaa, M., Rabergh, C. and Airaksinen, S. 2000. Biomarkers and Fluctuating Asymmetry as Indicators of Pollution-Induced Stress in Two Hole-Nesting Passerines. Functional Ecology 14 (2): 235-243. Engelbrecht, D., Matlamela, P. and Watkins, J. 2005. Little Man on Campus. Birds and Birding 10(2): p 6. Feige, U., Morimoto, R.I., Yahara, I. and Polla, B.S. 1996. Stress-Inducible Cellular Responses. Birkhauser Publishers. Berlin. Pp 492. Fisher, T.C., Crane, M. and Callaghan, A. 1999. An Optimized Microtiterplate Assay to Detect Acetylcholinesterase Activity in Individual Chironomus riparius Meigen. Environmental Toxicology and Chemistry 19(7): 1749-1752. Fossi, M.C. and Leonzio, C. 1994. Non-destructive Biomarkers in Vertebrates. Lewis Publishers. United States of America. Pp 345. Gil, Y. and Sinfort, C. 2005. Emission of Pesticides to the Air During Sprayer Application: A

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Bibliographic Review. Atmospheric Environment 39: 5183-5193. Grue, C.E., O’Shea, T.J. and Hoffman, D.J. 1984. Lead Concentrations and Reproduction in Highway-Nesting Barn Swallows. The Condor 86: 383-389. Guecheva, T.N., Erdtmann, B., Benfato, M.S. and Henriques, J.A.P. 2003. Stress Protein Response and Catalase Activity in Freshwater Planarian Dugesia (Girardia) schubarti Exposed to Copper. Ecotoxicology and Environmental Safety 56(3): 351-357. Hayman, P. and Arlott, N. 1994. Birds of Southern Africa. Struik Publishers. Cape Town. Pp 432. Hester, A. 2001. Southern African Birding. Paarl Bird Sanctuary. Available at: www.sabirding.co.za/birdspot/10335.asp (Accessed: March 2006). Hockey, P.A.R., Dean, W.R.J. and Ryan, P.G. 2005. Roberts Birds of Southern Africa. VIIth edition. Tien Wah Press. Singapore. Pp 1296. Hodson, P.V. 1976. &-Amino Levulinic Acid Dehydratase Activity of Fish Blood as an Indicator of a Harmful Exposure to Lead. J. Fish. Res. Board Can. 33: 268-271. Huggett, R.J., Kimerle, R.A., Mehrle, P.M. and Bergman, H.L. 1992. Biomarkers: Biochemical, Physiological, and Histological Markers of Anthropogenic Stress. Lewis Publishers. United States of America. Pp 347. Ingraham, J.L. and Ingraham, C.A. 2000. Introduction to Microbiology. Second Edition. Brooks/Cole Publishers. United States of America. Pp 804. Jenkins, A. 2005. The Price of Perfect Produce. Africa Birds and Birding 10 (1): 1. Kale, M., Rathore, N., John, S. and Bhatnagar, D. 1999. Lipid Peroxidative Damage on Pyrethroid Exposure and Alterations in Antioxidant Status in Rat Erythrocytes: A Possible Involvement of Reactive Oxygen Species. Toxicology Letters 105: 197-205. Karanjawala, Z.E. and Lieber, M.R. 2004. DNA Damage and Aging. Mechanisms of Aging and Development 125: 405-416. Khessiba, A., Romeo, M. and Aissa, P. 2005. Effects of Some Environmental Parameters on Catalase Activity Measured in the Mussel (Mytilus galloprovincialis) Exposed to Lindane. Environmental Pollution 133 (2): 275-281. Kim, E.Y., Ichihashi, H., Saeki, K., Atrashkevich, G., Tanabe, S. and Tatsukawa, R. 1995. Metal Accumulation in Tissues of Seabirds from Chaun, Northeast Siberia, Russia. Environmental Pollution 92(3): 247-252. Lagadic, L., Caquet, T., Amiard, J.C. and Ramade, F. 2000. Use of Biomarkers for Environmental Quality Assessment. Technique et Documentation Publishers. Paris. Pp 324. Lock, J.W., Thompson, D.R., Furness, R.W. and Bartle, J.A. 1991. Metal Concentrations in Seabirds of the New Zealand Region. Environmental Pollution 75: 289-300. Martin, P.A., Johnson, D.L. and Forsyth, D.J. 1995. Effects of Grasshopper Control Insecticides on Survival and Brain Acetylcholinesterase of Pheasant (Phasianus colchicus) Chicks. Environmental Toxicology and Chemistry 15(4): 518-524. Maul, J.D. and Farris, J.L. 2004. Monitoring Exposure of Passerines to Acephate, Dicrotophos, and

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Malathion Using Cholinesterase Reactivation. Environmental Contamination and Toxicology 73: 682-689. McInnes, P.F., Andersen, D.E., Hoff, D.J., Hopper, M.J. and Kinkel, L.L. 1995. Monitoring Exposure of Nestling Songbirds to Agricultural Application of an Organophsophorus Insecticide Using Cholinesetase Activity. Environmental Toxicology and Chemistry 15(4): 544-552. Mehdi, J.K., Al-Imarah, F.J.M. and Al-Suhail, A.A. 2000. Levels of Some Trace Metals and Related Enzymes in Workers at Storage-Battery factories in Iraq. Eastern Mediterranean Health Journal 6(1): 66-82. Meyer, I..J. 2005. The Use of Bioaccumulation in Weaver Feathers and Biomarkers as Bioindicators of Metal Contamination. Unpublished Thesis. University of Johannesburg. Johannesburg, South Africa. Moolman, L. 2004. The Use of Selected Freshwater Gastropods as Biomonitors to Assess Water Quality. Unpublished Thesis. University of Johannesburg. Johannesburg, South Africa. Movalli, P.A. 2000. Heavy Metal and Other Residues in Feathers of Lagger Falcon Falco biarmicus jugger from Six Districts of Pakistan. Environmental Pollution 109: 267-275. Musquiz, D. 2003. Cave and Cliff Swallows as Indicators of Exposure and Effects of Environmental Contaminants on Birds from the Rio Grande, Texas. Master of Science. Wildlife and Fisheries Sciences. Pp 71. Najimi, S., Bouhaimi, A., Daubeze, M., Zekhnini, A., Pellerin, J., Narbonne, J.F. and Moukrim, A. 1997. Use of Acetylcholinesterase in Perna perna and Mytilus galloprovincialis as a Biomarker of Pollution in Agadir Marine Bay (South of Morocco). Environmental Contamination and Toxicology 58: 901-908. Ownby, D.R., Trimble, T.A., Cole, K.A. and Lydy, M.J. 2004. Pesticide Residues in Water, Sediment, and Fish at the Sparta, IL, USA, National Guard Armory. Environmental Contamination and Toxicology 73: 802-809. Pandey, S., Parvez, S., Sayeed, I., Haque, R., Bin-Hafeez, B. and Raisudden, S. 2003. Biomarkers of Oxidative Stress: A Comparative Study of River Yamuna Fish Wallago attu. The Science of the Total Environment 309: 105 – 115. Parametrix. 2002. Ecological Screening-Level Risk Assessment of the Lower Ottawa River. Available at: www.epa.gov/glnpo/sediment/ottawa River/ra2001/%23Download (Accessed: February 2006). Pastor N., Lopez – Lazaro, M., Tella, J.L., Baos, R., Forrero, M.G., Hiraldo, F. and Cortes, F. 2001a. DNA Damage in Birds after the Mining Waste Spill in Southwestern Spain: A Comet Assay Evaluation. Journal of Environmental Pathology, Toxicology and Oncology 20 (4): 317 – 324. Pastor N.M., Tella, J.L., Baos, R., Hiraldo, F. and Cortes, F. 2001b. Assessment of Genotoxic Damage by the Comet Assay in White Storks (Ciconia ciconia) after the Donana Ecological Disaster. Mutagenesis 16 (3): 219 – 223. Rainwater, T.R., Leopold, V.A., Hooper, M.J. and Kendall, R.J. 1995. Avian Exposure to

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Organophosphorus and Carbamate Pesticides on a Coastal South Carolina Golf Course. Environmental Toxicology and Chemistry 14(12): 2155-2161. Rauchova, H., Vokurkova, J. and Koudelova, J. 2005. Developmental Changes of Erythrocyte Catalase Activity in Rats Exposed to Acute Hypoxia. Institute of Physiology. First Faculty of Medicine. Charles University. Albertov 5, 12800 Prague 2, Czech Republic. Pp 16. Sanchez, W., Palluel, O., Meunier, L., Coquery, M., Porcher, J. and Ait-Aissa, S. 2005. Copper- induced Oxidative Stress in Three-spined Stickleback: Relationship with Hepatic Metal Levels. Environmental Toxicology and Pharmacology 19: 177 – 183. Sarkar, D., Datta, R. and Sharma, S. 2005. Fate and Bioavailability of Arsenic in Organo-arsenical Pesticide Applied Soils. I: Incubation Study. Chemosphere 60: 188-195. Scheuhammer, A.M. 1996. Influence of Reduced Dietary Calcium on the Accumulation and Effects of Lead, Cadmium, and Aluminium in Birds. Environmental Pollution 94 (3): 337-343. Schmidt-Nielsen, K. 1997. Animal Physiology Adaptation and Environment. Fifth Edition. Cambridge University Press. United Kingdom. Pp 607. Steyn, P.J., Reinecke, A.J. and Venter, J.M. 1985. The Weakening of Eggshells of the Laughing Dove, Streptopelia senegalensis. South African Journal of Zoology 21(3): 233-236. Swiergosz, R., Sawicka-Kapusta, K., Nyholm, N.E.I., Zwolinska, A. and Orkisz, A. 1997. Effects of Environmental Metal Pollution on Breeding Populations of Pied and Collared Flycatchers in Niepolomice Forest, Southern Poland. Environmental Pollution 102: 213-220. Tanhuanpaa, S., Eeva, T., Lehikoinen, E. and Nikinmaa, M. 1999. Developmental Changes in 7- Ethoxyresorufin-O-Deethylase (EROD) and δ-Aminolevulinic Acid Dehydratase (ALA-D) Activities in Three Passerines. Comparative Biochemistry and Physiology Part C 124: 197-202. Travis, C.C. 1993. Use of Biomarkers in Assessing Health and Environmental Impacts of Chemical Pollutants. Plenum Press Publishers. New York. Pp 284. USEPA (United States Environmental Protection Agency). 2005. Ecological Risk Assessment. Available from: www.epa.gov/region5/superfund/ecology/html/toxprofiles.htm (Accessed: February 2006). Van Wyk, E., Bouwman, H., van der Bank, H., Verdoorn, G.H. and Hofmann, D. 2001. Persistent Organochlorine Pesticides Detected in Blood and Tissue Samples of Vultures from Different Localities in South Africa. Comparative Biochemistry and Physiology Part C 129: 243-264. Venter, E.A., Slabbert, J.L., Joubert, A., Vorster, A. and Barnhoorn, I. 2003. Biomarker Assays for the Detection of Sub-Lethal Toxicity in Fish. Operational Manual. Environmentek, CSIR. WRC report No. 952/2/03. Report to the Water Research Commission on the Project ‘Biomarker Assays for the Detection of Chronic Toxicity in the Aquatic Environment’. Pp 49. Waldenstedt, L. 2006. Nutritional Factors of Importance for Optimal Leg Health in Broilers: A

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Review. Animal Feed Science and Technology 126: 291-307. Walker, C.H. 1995. Biochemical Biomarkers in Ecotoxicology – Some Recent Developments. The Science of the Total Environment 171: 189-195. Walker, C.H. 1998. The Use of Biomarkers to Measure the Interactive Effects of Chemicals. Ecotoxicology and Environmental Safety 40: 65-70. Wepener, V., van Vuren, J.H.J., Chatiza, F.P., Mbizi, Z., Slabbert, L. and Masola, B. 2005. Active Biomonitoring in Freshwater Environments: Early Warning Signals from Biomarkers in Assessing Biological Effects of Diffuse Sources of Pollutants. Physics and Chemistry of the Earth 30: 751 – 761. Williams, B., Waddington, D., Solomon, S. and Farquharson, C. 2000. Dietary Effects on Bone Quality and Turnover, and Ca and P Metabolism in Chickens. Research in Veterinary Science 69: 81-87. Yoo, L.J., Stevens, J.A. and Landrum, P.F. 2003. Development of a New Bioaccumulation Testing Approach: The Use of DDE as a Challenge Chemical to Predict Contaminant Bioaccumulation. ERDC/TN EEDP-01-05.

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CHAPTER FOUR

The Link between Feet Abnormalities in the Cape Wagtail (M. capensis) and the Scaley-leg Mite (K. jamaicensis) Infestation.

4.1. INTRODUCTION

Cape Wagtails (M. capensis) are passerine birds first described by Linnaeus in 1766 (Hockey et al., 2005). Wagtails are most common south of 23 ºS and are found in a range of habitats from natural rural areas to highly crowded urban locations. They occur naturally along watercourses such as rivers, lakes and dams as well as along the coast and are always associated with a water source and fairly open habitats. They are especially common around sewerage works, are fairly sedentary and remain in the same area for most of their lives (Engelbrecht et al., 2005). It has also been reported that Cape Wagtails are commonly found in and around shopping malls (Williams, 2004). Cape wagtails may roost singly or communally (Winterbottom, 1967). Single roosting involves a nest built in a clump of vegetation relatively close to the ground and often overhanging water. It normally consists of grass, pine needles, cotton, roots, feathers and other artificial materials such as string, hair and sundry fibres. Communal nesting, otherwise known as roosting, involves the congregation of very large groups of up to 2 000 birds (Winterbottom, 1967). Wagtails roost in reed beds and trees (Winterbottom, 1967) and often in and around city centres (Skead, 1995; Winterbottom, 1964 and Keith et al., 1992 in Hockey et al., 2005). Communal roosting is popular during the winter months in numbers generally between 150 and 200 birds. Roosts are a form of feeding congregation as reported by Taylor (2003). In recent years, such as in Tableview in the Western Cape, birds have been seen to gather in car parks and roost in conifer and palm trees.

Wagtails are insectivores, feeding mainly on insects such as flies, mosquitoes, termites and ants (Winterbottom, 1967), lawn crickets and worms (Engelbrecht et al., 2005). When they occupy areas of human inhabitance they also eat mielie meal, porridge, bread and cake crumbs, raw meat, fat, suet and even grated cheese (Winterbottom, 1967).

In the last decade, M. capensis have been observed with abnormalities such as missing toes and clubbed feet. Steen and Steen (1965 in Frost and Siegfried, 1975) discovered the role of the unfeathered region of passerine bird legs, which is primarily to assist in heat regulation.

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Undoubtably, this is a cause for concern. Various reasons have been suggested for the abnormalities. Firstly, it is thought that secondary poisoning by insecticides may be a cause (Masterson, 1976 and Vernon, 1972 in Hockey et al., 2005). This causes internal damage to the bird and may possibly affect nerve transmission (Schmidt-Nielsen, 1997) thereby, resulting in feet and toe deformities. Secondly, the deformities could be caused by strangulation and consequently necrosis due to nesting materials such as threads and hair. Interestingly, Steyn (1996 in Hockey et al., 2005) reported that many Cape Wagtails had been observed with encrustations along the legs and feet and had been seen to lose toes, feet and parts of the leg. The cause of this was suggested to be caused by the entanglement of threads (Niven, 1981 in Hockey et al., 2005). Thirdly, abnormalities could be caused by microbial infections from the surfaces and substrates they walk on, such as hot asphalt (tar). Lastly, the hint of the possibility of a parasitic infection has also been put forward (Skead, 1954 in Hockey et al., 2005). So, the question remains as to exact reasons why Cape Wagtails are vulnerable to these feet abnormalities?

The discovery of K. jamaicensis was first described by Fain and Elsen (1967) as reported in Literak et al. (2005) from the Golden Thrush (Tardus aurantiacus) in Jamaica. Since then, this mite has been collected from over 30 species of passerine birds (Fain and Elsen, 1967 in Literak et al., 2005). Latta and O’Connor (2001) discovered that K. jamaicensis has the potential to affect any passerine bird species. Thus far K. jamaicensis has been recorded in Denmark, Canada, Austria, Mexico, South Africa, Sri Lanka, the West Indies, the USA, England (Baker, 1999), the Caribbean Islands as well as in migratory birds from the Neartic and Neotropic regions (Fain and Elsen, 1967 in Latta and O’Connor, 2001).

Knemidocoptes jamaicensis belongs to the mite family Knemidocoptidae. It is a burrowing mite which causes wart-like skin proliferations and lesions which are pathologically called Scaley-leg disease (Literak et al., 2005 and Latta and O’Connor, 2001), or knemidokoptic mange (Carothers et al., 1974). For many years, a type of fungus was thought to be the cause of the lesions (Butcher and Beck, 1996). This ectoparasitic mite feeds on the host’s tissues and is able to live and reproduce by burrowing into the cornified epithelium of the skin tissue and feeding on keratin (Butcher and Beck, 1996) which causes proliferation of excess tissue (Herman et al., 1962 in Carothers et al., 1974) around areas such as the legs, feet or any unfeathered region of the bird (Kirmse, 1996 in Latta and O’Connor, 2001). Most of the cases observed show that the mite remains just under the surface of the skin and does not penetrate any deeper (Latta and O’Connor, 2001). Over time, the host’s infected areas

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become distorted and covered with thick, nodular, spongy crusts caused by both adults and nymphs burrowing under the dermis of the scales of the lower legs and feet (Schmidt and Roberts, 2000). The leg swells as a consequence and the leg appears encrusted (Proctor and Owens, 2000). Benkman et al. (2005) reported that K. jamaicensis can cause the loss of digits and feet (Pence et al., 1999) and that in the advanced stages of infection severely disfigured legs covered with scabs is observed. In extreme cases the legs can be deformed or lost (SCWDS Briefs, 1996). Gangrene can also develop around the infected area due to the pressure exerted on the leg by the mites (Butcher and Beck, 1996). Some infections may be lethal to the host (Latta and Faaborg, 2001 in Latta and O’Connor, 2001). Butcher and Beck (1996) suggested that the susceptibility of biota to this mite may be genetically linked.

Direct physical contact between host species is the primary route of transmission (Proctor and Owens, 2000). Mites have also been known to walk from host to host. Infection is highly contagious and mites are common on birds which roost communally rather than on those birds that roost solitary. Infections can affect large populations of birds and infestation rates may be due to environmental factors such as habitat type and rainfall as well as host factors (Latta and O’Connor, 2001). In a study regarding Palm Warblers it was concluded that a habitat with low rainfall and a dry climate was optimal for mite survival (Van Riper, 1991 in Latta and O’Connor, 2001). A dryer climate may exert more physiological stress (Esch et al., 1975 in Latta and O’Connor, 2001) on the host, making it weaker and more vulnerable to infection (Deerenberg et al., 1997 and Saino et al., 1997 in Latta and O’Connor, 2001).

So, in conclusion, the abundance of mites and the infection rate depends on the host species being available and abundant as well as various ecological conditions which need to be suitable for the mite itself and to promote its transmission. More work needs to be done with respect to these mites to determine their habitat preferences as well as their biology (Latta and O’Connor, 2001). The symbiosis between mites and birds is a growing field of interest but results obtained thus far are conflicting. Providing the evidence that mites cause negative effects to the health and reproductive success of the host is often unclear (Proctor and Owens, 2000).

The objective of this chapter is to gain insight into Scaley-leg mite (K. jamaicensis) infestation and histopathology in Cape Wagtails (M. capensis).

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4.2. MATERIALS AND METHODS

4.2.1. SITE SELECTION AND DESCRIPTIONS

Refer to Chapter three, section 3.2.1 and 3.2.2 and Figure one.

4.2.2. FIELD WORK

Sampling techniques and observations were carried out as mentioned in Chapter three, section 3.2.3. and 3.2.4.

4.2.3. MITE COLLECTION

Affected birds at all sites displayed white encrustations along the legs. Port Elizabeth (site 1) was chosen as the site for mite collection because of the high incidence (17%) of white encrustations along the legs and feet abnormalities (one, more or all digits had been lost) of the wagtails in that area.

For mite collection, encrustations on the leg were scraped carefully off with a blade and observed under a field dissection microscope. Mites were collected and placed in a 70% ethanol solution for preservation (Baker, 1999 and Boyd, 1951).

4.2.4. LABORATORY WORK

4.2.4.1. HISTOPATHOLOGY

No birds were sacrificed during this research. Only those individuals that were mortally injured during the sampling process were used for histological examination.

One affected specimen was used for the purpose of an histological study. A swollen and affected ankle was dehydrated and embedded in paraffin wax according to standard followed procedures and cross sections (4-6 µm) were made using a microtome. Sections were processed routinely and stained with Haemotoxylin and Eosin (Bancroft and Cook, 1982) and observed under a compound light microscope.

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4.2.4.2. MITE IDENTIFICATION

Mites were permanently mounted onto slides according to standard followed procedures (Baker, 1999) and identified.

4.3. RESULTS

4.3.1. SAMPLING OBSERVATIONS

Figure 19A shows the appearance of healthy Cape Wagtail (M. capensis) legs. Figures 19B, C, D and E reveal areas of the legs that display off-white ‘powdery’ type encrustations and scabs. Leg scales are raised and the contour of the leg appears irregular (see Figure 19D) and enlarged (swollen). In Figure 19D the proliferative excess tissue (spongy white accumulation) around the ankle area should be noted.

A B C

D E

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Figure 20A shows a bird with encrustations along the right leg and a deformed left leg with all digits lost. Figures 20B and C reveal variations of affected feet. Figure 20B shows a foot with only one digit (back toe) while Figure 20C shows a foot with two digits missing.

A B C

Figure 20. A – encrustations along the right leg and a deformed left leg; B and C – affected foot.

Figure 21 demonstrates the result of threads entwined around the feet. Figures 21A, B and C show artificial threads entwined around the feet and toes. Figure 21C reveals a badly affected foot. Figure 21D shows an encrusted leg with raised epidermal scales and entwined thread around the foot with only one digit present. Figure 21E presents one foot with entwined thread and the other that was affected with only a stump observed.

A B C

D E

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Table 8 displays the sampling results at each site. The condition of the leg and feet areas are presented as a percentage of occurrences at that specific site.

Table 8. Condition of Cape Wagtail (M. capensis) legs, feet and toes found at several sites (percentage (%) of occurrences) Site Healthy (legs Encrustations along Affected (one or Threads entwined appeared the legs (indicating both feet were round feet (one, normally as in mite presence as in affected as in two or both feet as Figure 19A) Figures 19B, C, D Figures 20A, B, in Figures 21A, B, and E) C, 21D and E) C, D and E) Paarl (site 1) 84.2 0 10.5 5.3 Somerset West (site 2) 88.9 11.1 0 0 Port Elizabeth (site 3) 78.7 10.6 8.5 2.2 Secunda (site 4) 100 0 0 0 Dullstroom (site 5) 100 0 0 0 Gauteng (site 6) 100 0 0 0

Sighting reports from people around the country were recorded over a period of one year. Out of the total of 169 reports, 38.4% reported healthy wagtails, 46.8% reported affected birds and 14.8% of the reports were ‘unknown’ reports (observers were unable to see the state of the feet).

4.3.2. HISTOPATHOLOGICAL EXAMINATION OF THE ANKLE (TIBIA – TARSAL JOINT)

Figure 22. Arthropods (Scaley-leg mite, K. jamaicensis) in a small section (10x magnification) of white encrustations along the legs. Blocks indicate the position of the burrowing mite.

Mites were found burrowing in the white lesions along the legs (Figure 22). Mites were abundant in such tissue, especially around the ankle joint area as could be seen in Figure 19D. The left leg of the bird displayed the accumulation of proliferative tissue around the ankle joint which was abundant in mites (K. jamaicensis) and characteristic of its presence.

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Figure 23 shows the healthy leg tissue of the Cape Wagtail with no mite infestation present.

Outer keratin layer

Dermis

Epidermis

Blood vessel

containing

erythrocytes

Pathological examination (E. Lane, pers. comm.1) of the cross section (4-6 µm) of a swollen and affected ankle revealed severe, chronic and prominent inflammation with lymphocyte (WBC) infiltration, as well as moderate oedema, hemorrhage and hyperplastic dermatitis with intracorneal mites (see Figure 24.). These findings were consistent with mite infestation as cross sections of arthropods could be observed. In the whitish material found along the legs, sections of arthropods (suspected to be mites) were also observed. Arthropods had citinized mouthparts and muscle attachments found predominantly in areas of serocellular crusting (or white encrustations as seen in Figure 22).

Knemidocoptes jamaicensis existed in proliferative lesions along the legs (keratin layer of connective tissue) of each section. Mites appeared to be encapsulated in a granuloma of connective tissue. Cell positions had been disturbed and some appeared ‘squashed’ and out of place. Active inflammation was observed with infiltration of lymphocytes (WBCs) and erythrocytes escaping blood vessels (hemorrhage). Moderate oedema was also observed in some of the cross sections, as in Figure 24B.

1 Dr. Emily Lane : Specialist Wild and Domestic Animal Pathologist

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Female K. jamaicensis with egg containing yolk droplets

Infiltration of erythrocytes and lymphocytes

K. jamaicensis encapsulated in granulomas Outer keratin layer Epidermis

Dermis showing hyperplastic dermatitis

Dermal and epidermal layer Granuloma of connective tissue surrounding the mite

K. jamaicensis (mite) Outer keratin layer Moderate oedema

Infiltration of eosinophils

(lymphocytes) and erythrocytes B (haemorrhage)

Figure 24. Cross section (4-6 µm) of the ankle (tibia - tarsal joint) showed mite presence. Sections were processed routinely and stained with Haemotoxylin and Eosin (Bancroft and Cook, 1982).

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4.3.3. MITE IDENTIFICATION

The mites were identified (E. Ueckermann, pers. comm.2) as Knemidocoptes jamaicensis. Larvae, nymphs and adult male and females were found. Cape Wagtails were, therefore, diagnosed pathologically to have Scaley-leg disease (Literak et al, 2005 and Latta and O’Connor, 2001), or otherwise known as knemidokoptic mange (Carothers et al., 1974).

A 500µm

B 500µm

Figure 25. Line diagrams of K. jamaicensis found on the leg region of the Cape Wagtail (M. capensis). A – adult mite with A1 – front leg and A2 – back leg and; B - larva.

2 Dr. Eddie Ueckermann : Plant Protection Research Institute

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In Figure 25, Figure A displays the appearance of an adult mite. Four pairs of legs show claws at the end of each digit. Figure B shows the larval form with three pairs of legs with sucker–like organs at each end. Both adult and larval forms have a pair of setae at the posterior region of the body. Mites measure about 500 µm in length from the anterior head region to the posterior region.

D C

Figure 26. Life stages of K. jamaicensis found in the encrustations along the legs of the Cape Wagtail (M. capensis). Figures A - nymph, B - larva, C - adult male and D - adult female

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All life stages (see Figure 26) of K. jamaicensis were found in the encrustations along the legs and amongst the proliferative tissue, especially around the ankle area. This yielded the idea that the complete life cycle of K. jamaicensis was successful on one host.

In past studies, mite infestation rates have been associated with weather patterns. Temperature and rainfall patterns for each sampling area were reported in Table 9 along with the percentage of healthy and affected birds sampled as well as those birds with encrustations along the legs and thread entwined around their feet.

Table 9. Seasonal variation and the occurrence of healthy and affected birds Sites Month a Average a Min and Sampling sampled (mm) annual max annual Percent Percent Percent Percent (winter / rainfall temperatures (%) (%) (%) (%) summer) (minimum (ºC) healthy encrusted thread affected and birds legs entwined maximum) around feet Paarl (site 1) April (late 15 (Feb & Jan) 9 -26 84.2 0 0 15.79 summer) – 105 (June) Winters are cold & wet (Sept – April) Somerset April (late 15(Feb & Jan) 9 -26 87.5 12.5 0 0 West (site 2) summer) – 105 (June) Winters are cold & wet (Sept – April) Port April (late 33 (February)- 16 -26 87.2 (late 10.3 0 2.6 Elizabeth summer) 58 (Sep) (summer) summer) (late (late (late (site 3) and June 7 ºC -20 ºC 37.5 summer) summer) summer) (winter) (winter) (winter) 12.5 12.5 37.5 (winter) (winter) (winter) Secunda September About 620 Average of 19 100 0 0 0 (site 4) (summer) (Sept – March) (8 -26) Dullstroom August About 620 Average of 19 100 0 0 0 (site 5) (winter) (Sept – March) (8 -26) Gauteng March 6 (July & Aug) 4-26 100 0 0 0 (site 6) (summer) – 123 (Jan)

a World Travels (2006).

At Paarl (site 1), sampling occurred during summer when rainfall was low with relatively high maximum temperatures. A high percentage of healthy birds and a relatively high percentage of affected birds were recorded at this site. No birds with encrusted legs or thread entwined around the digits were found. At site 2 (Somerset West), the situation was similar. Sampling was carried out in the summer months when rainfall was low and temperatures were relatively high. A high percentage of healthy birds were observed while a relatively high rate of encrusted legs and no deformities or thread entwined feet were found. At site 3 (Port Elizabeth), sampling was carried out in both summer and winter seasons. Higher

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rainfall with low temperatures occurs in winter months. A considerably higher portion of healthy birds was found in summer compared to winter. The percentage of encrusted birds was relatively similar, although in winter it was slightly higher. A much higher rate of deformities was found during winter when compared to summer. During winter, more birds were found with thread entwined around the feet. At the Mpumalanga sites (sites 4 – Secunda and 5 – Dullstroom), where high rainfall and high temperatures are characteristic of the summer months, no deformities could be reported and all birds were healthy. Sampling was completed during summer at Secunda and during winter at Dullstroom. In Gauteng, sampling was carried out in summer when very high rainfall and high temperatures were experienced. All the birds sampled in this area were healthy.

4.4. DISCUSSION

Mites are reported to be the most abundant ectoparasites found on birds (Boyd, 1951). Consequently, it was not surprising to discover K. jamaicensis was responsible for causing the feet and toe deformities observed in the Cape Wagtail. Knemidocoptes jamaicensis has the potential to affect any passerine bird (Latta and O’Connor, 2001).

One questions how mite infestation along the legs and feet can result in such serious deformities. When the mites attach to their host they inject saliva into their target organ (in this case the legs and feet) and cause severe rashes and intense itching (Anon, 2003). When observing infected wagtails, no birds appeared to persist in ‘attending to’ the infected area. However, this is not to say they do not do so. Anonymous (2003) reported sightings of affected individuals and suggested that secondary infection often resulted when infected areas were scratched. Gangrene has also been reported in some affected individuals (Butcher and Beck, 1996). The infection may be potentially fatal either directly or indirectly through secondary infections that often involve internal organs (Literak et al., 2005). At site 3 (Port Elizabeth) a few individuals were seen with severely swollen (Proctor and Owens, 2000) and infected feet. Tibia-tarsal joints were also often observed to be infected. However, no fatalities were observed at any of the sampling sites.

Legs of the Cape Wagtails (M. capensis) sampled revealed ‘dirty’ white ‘powdery’ type encrustations and lesions (see Figures 19B, C, D and E, section 4.3) with crusty (SCWDS Briefs, 1996) distorted thick, nodular and spongy-like growths (Schmidt and Roberts, 2000).

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Growths, or what appears to be more like ‘scabs’ as described in SCWDS Briefs (1996), were ‘roughened’ in appearance. Leg scales were observed to be raised as was seen by researchers in SCWDS Briefs (1996) and the contour of the leg appeared irregular and enlarged (swollen). Proliferative excess tissue around the ankle area (see Figure 19D, section 4.3) was also evident.

This species of mite is known to feed-off and burrow into the cornified epithelium of the hosts unfeathered leg and feet regions (Kirmse, 1996 in Latta and O’Connor, 2001), which in this case is the Cape Wagtail. They feed on keratin in these areas (Butcher and Beck, 1996) and the scabs (proliferative tissue) that develop along the legs are reported to be caused by the burrowing adults and nymphs (Proctor and Owens, 2000). In this study, adults were observed to have claws on each leg extension which may assist in movement and attachment to the host. Larvae were seen to have sucker-like organs (Albert et al., 2001) which may help in attachment or for feeding. Both adult and larval forms posses a pair of setae at the posterior end of the body which may assist in sensory functions (see Figure 25). The scabs are thought to be a form of protection for the mites (Albert et al., 2001). The host responds by growing lesions around the infected area (Latta and O’Connor, 2001) and producing proliferative excess tissue (Herman et al., 1962 in Carothers et al., 1974). Similar observations were noted in the wagtails that were sampled (see Figures 19B-E and 21D, section 4.3) particularly around the ankle area of the wagtail in Figure 19D (section 4.3).

Somerset West (site 2) and Port Elizabeth (site 3) revealed an occurrence of birds with encrusted legs (11.1% and 10.6% respectively). This suggests that these birds were showing early signs of infection.

The mites appeared to remain only in the shallow areas of the epidermal layer towards the skin surface. To support this, Latta and O’Connor (2001) suggested that the mite rarely penetrates very deeply into the skin along the leg and feet areas.

Benkman et al. (2005) proposed that birds are unable to remove the mites from their bodies. This appeared to be the case with the Cape Wagtails. As the infestation advanced, the legs and feet became unsightly with severe abnormalities (Krantz, 1978). In some of the far- advanced infected individuals, lameness of the legs (Baker, 1999) and arthritis (Pesek, 2000) can set in and subsequently severely disfigured and deformed legs and loss of digits or even feet (SCWDS Briefs, 1996 and Pence et al., 1999) were observed (see Figures 20A to C and

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21C, D and E, section 4.3). In a cross-section of a mite infested ankle area, granulomas observed along the dermal layers were indicative of mite presence. A thin layer of connective tissue enveloped each mite. The granuloma caused the movement and dispersion of healthy cells to smaller areas where they appeared ‘squashed’ and compacted. Infiltration of eosinophils and erythrocytes (haemorrhage), which is typical of active inflammation in an area, was observed. This is characteristic of a host’s immune response (the Cape Wagtails) to a foreign compound (the mites) (Mader, 2001).

No references were found that explain how the mite infestation can cause such severe abnormalities or toes and feet to become dismembered. It is postulated that as the mites burrow in the keratin and dermal layers from where they feed and over time, the host’s protective response is to form a granuloma around each mite. However, as time passes the mite uses its claws to erode the granuloma away. By that stage the host’s immune system has begun to repair the damaged cells and more dermal cells are produced. The mite once again burrows into the repaired cells where it feeds. Each time this happens, the leg or foot is increasingly eroded until eventually sections become so thin that they have no support and are severed. Of the birds sampled, 10.5% at site 1 (Paarl) and 8.5% at site 3 (Port Elizabeth) were affected. Paralyzed birds were observed at site 2 (Somerset West) where wagtails appeared to be fine externally but could not fly or walk naturally. Knemidocoptes jamaicensis is also known to cause the claws to become cracked (Pesek, 2000) and overgrown which makes it difficult for the birds to perch (Latta and O’Connor, 2001), but this was not observed.

In a recent study, it was found that K. jamaicensis was predominantly found on birds which roost communally rather than individually (Latta and O’Connor, 2001). This may be one of the reasons why Cape Wagtails are particularly vulnerable to infection by this mite. Scaley- leg disease caused by K. jamaicensis is highly contagious (SCWDS Briefs, 1996) and, as the wagtails often roost communally, transmission from bird to bird would be fairly rapid. Wagtails roosted in numbers of between 60 and (about) 400 at some of the sites sampled (Somerset West - site 2 and Port Elizabeth - site 3). It was clear that these birds relied on the close contact by other individuals for warmth as they huddled close together in cold and windy weather (especially at site 3 – Port Elizabeth), thus making it an ideal opportunity for K. jamaicensis to be transmitted from one wagtail to another in a very short space of time. Another possibility as to why Cape Wagtails are severely vulnerable to infection has been suggested by Butcher and Beck (1996) to be genetically linked. This remains unknown and

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requires further investigation. It would be beneficial to understand why Cape Wagtails are seriously prone to infection by this mite compared to other birds of a similar biology, character and feeding habit.

Mites have been reported to be most active during late spring and early summer (Anon, 2003). Temperature is a very important factor that determines the prevalence and rate of infection (Boyd, 1951) as well as other environmental factors and host factors (Latta and O’Connor, 2001). In a study of Palm Warblers, infestation rates were associated with rainfall patterns. Lower rainfall and a drier climate were associated with a higher rate of mite infection (Latta and O’Connor, 2001), which was suggested to be due to a drier climate which could possibly exert more physiological stress (Esch et al., 1975 in Latta and O’Connor, 2001) on the host, making the bird weaker and more prone to infection (Deerenberg et al., 1997 and Saino et al., 1997 in Latta and O’Connor, 2001). However, looking at the results (see Figure 14, section 4.3), the opposite was found when compared to the Latta and O’Connor (2001) study. Results obtained at site 3 (Port Elizabeth’s) provides convincing evidence in this respect because sampling occurred during both winter and summer months. During winter (high rainfall and low temperatures) less healthy birds and more affected birds (with encrusted legs and deformities) were found. However, during summer (low rainfall and high temperatures) more healthy birds were found with a lower rate of individuals displaying encrusted legs or deformities. From this, it is postulated that infections may be higher and more prone to occur during times of high rainfall and lower temperatures. Following this, it was suggested that the reason for the higher rate of infection was because the wagtails huddle closer when it is cold in an effort to keep each other warm. This would promote mite transmission and thus infection.

The abundance and success of the mite is dependent on the availability of the host species in sufficient numbers as well as various ecological conditions which are optimal for the mite and its transmission (Latta and O’Connor, 2001). In terms of the Cape Wagtails, they are available in large numbers where they roost and are readily exposed to mite infection making them ideal candidates for infection by this mite.

Latta and O’Connor (2001) reported that more work is needed to determine the habitat preferences as well as the detail of the mite’s biology. Little evidence has been provided for the negative health or reproductive effects of such mites on birds. However, in this study, the serious chronic effects of this mite on the legs and toes of the host were quite obvious.

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Some of the wagtails sampled had artificial threads (predominantly cotton) tightly entwined around their feet and toes (see Figure 21). It was suggested that Cape Wagtails are vulnerable to picking these up as they walk along substrates in search of food. It is easier for a bird to pick up such material if they walk rather than hop as they frequently do. The threads appeared to be so tightly wound around the toes that the bird was unable to free itself from them. Eventually after some time, the threads ceased blood supply to the suffocating, open, cut and ripped areas and secondary, possibly bacterial infections began to set in. At site 1 (Paarl) 5.3% of the birds and 2.2% of the birds at site 3 (Port Elizabeth) were found with thread entwined around their feet. At site 3 (Port Elizabeth), the occurrence of threads might be explained by the association with a busy shopping centre complex where threads and cotton could occur more often than on garden lawns.

One could question why a high percentage (11.1%) of birds at site 2 (Somerset West) showed encrusted legs and none with deformities. In addition, no wagtails were sampled at site 1 (Paarl) with encrustations along the legs while 10.5% had deformities. One should bear in mind that sampling size was small and not representative of the population at those sites. In addition to this, climatic conditions may not have been optimal for the mites’ optimum reproduction or survival and its effect on the host may have ceased during these unfavourable conditions.

The life cycle of the mite consists of four stages; an egg, larva, nymph and adult which completes their metamorphosis (Anon, 2003). All four stages were located and identified at site 3 (Port Elizabeth) on the sampled Cape Wagtails. Some mites in the studied cross sections of an infected ankle revealed an egg with yolk droplets. All this suggested that the mites have an optimal microclimate on the Cape Wagtails where they reproduce successfully thereby ensuring the survival of their species.

The mites appear to occur naturally on the wagtails. It is postulated that these parasites and the effect they have on their hosts (the Cape Wagtails), is merely a form of natural selection and survival of the strongest genes. The Cape Wagtail may be particularly genetically weak and vulnerable to the infection and effects of this mite. It would be interfering with natural processes if one had to find a way of eradicating the mites from these birds. It is simply a parasitic symbiotic relationship taking place and unfortunately the Cape Wagtail is severely and distressingly affected by it.

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4.5. REFERENCES

Albert, O.B., Fernandez, J.C., Esch, G.W. and Seed, J.R. 2001. Parasitism: The Diversity and Ecology of Animal Parasites. Cambridge University Press Publishers. United Kingdom. Pp 566. Anonymous. 2003. Bird Mites. Available from: http://medent.usyd.edu.au/fact/birdmite.html. (Accessed: November 2005). Baker, A.S. 1999. Mites and Ticks of Domestic Animals – An Identification Guide and Information Source. The Natural History Museum. London. Pp 240. Bancroft, J.D. and Cook, H.C. 1982. Theory and Practice of Histological Techniques and their Diagnostic Application. Churchill Livingston Publishers. Edinburgh. Pp 25-254. Benkman, C.W., Colquitt, J.S., Gould, W.R., Fetz, T., Keenan, P.C. and Santisteban, L. 2005. Can Selection by an Ectoparasite Drive a Population of Red Crossbills from its Adaptive Peak? Evolution 59(9): 2025-2032. Boyd, E.M. 1951. The External Parasites of Birds: A Review. The Wilsok Bulletin 63(4): 363-369. Butcher, G.D. and Beck, C. 1996. Knemidokiptic Mange in Pet Birds: Scaly Face and Scaley-Leg Disease. University of Florida. Gainesville. Pp 2. Carothers, S.W., Sharber, N.J. and Foster, G.F. 1974. Scaley-Leg (Knemidokoptiasis) in a Population of Evening Grosbeaks. The Wilson Bulletin 86 (2): 121-124. Engelbrecht, D., Matlamela, P. and Watkins, J. 2005. Little Man on Campus. Birds and Birding 10(2): 6. Frost, P.G.H. and Siegfried, W.R. 1975. Use of Legs as Dissipaters of Heat in Flying Passerines. Zoologica Africana 10(1): 101-102. Hockey, P.A.R., Dean, W.R.J. and Ryan, P.G. 2005. Roberts Birds of Southern Africa. VIIth edition. Tien Wah Press. Singapore. Pp 1296. Krantz, G.W. 1978. A Manual of Acarology. Second Edition. Oregon State University Book Stores. Corvallis. Pp 395 – 396. Latta, S.C. and O’Connor, B.M. 2001. Patterns of Knemidokoptes jamaicensis (Acari: Knemidokoptidae) Infestations Among Eight New Avian Hosts in the Dominican republic. Journal of Medical Entomology 38(3): 437-440. Literak, I., Smid, B., Dusbabek, F., Halouzka, R. and Novotny, L. 2005. Co-Infection with Papillomavirus and Knemidocoptes jamaicensis (Acari: Knemidocoptidae) in a Chaffinch (Fringilla coelebs) and a Case of Beak Papillomatosis in Another Chaffinch. Vet. Med – Czech 50 (6): 276-280. Mader, S.S. 2001. Biology. Seventh Edition. McGraw-Hill Companies Publishers. New York. Pp 939. Pence, D.B., Cole, R.A., Brugger, K.E. and Fischer, J.R. 1999. Epizootic Podoknemidokoptiasis in American Robins. Journal of Wildlife Diseases 35 (1): 1-7. Pesek, L. 2000. Mites. Available from: http://www.Birdsnways.com/wisdom/ww48eiv.htm.

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(Accessed: December 2005). Proctor, H.C. and Owens, I. 2000. Mites and Birds: Diversity, Parasitism and Coevolution. Tree 15(9): 358-364. Schmidt, G.D. and Roberts, L.S. 2000. Foundations of Parasitology. Sixth Edition. McGraw Hill Publishers. United States of America. Pp 670. Schmidt-Nielsen, K. 1997. Animal Physiology Adaptation and Environment. Fifth Edition. Cambridge University Press. United Kingdom. Pp 607. SCWDS Briefs. 1996. Scaley-Leg Mites in Wild Birds 11, 4. Taylor, J. 2003. Cape Wagtail Group Behaviour. Promerops 253 1. Williams, T. 2004. Birds of Shopping Malls. Promerops 257: 1. Winterbottom, J.M. 1967. The Farmer’s Birds. Maskew Miller Publishers. Cape Town. Pp 186. World Travels. 2006. South Africa Climate and Weather. Available at: www.worldtravels.com/Travelguide/Countries/South+Africa/Climate (Accessed: March 2006).

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CHAPTER FIVE

5.1. CONCLUSIONS AND RECOMMENDATIONS

5.1.1. CONCLUSIONS

Initially it was assumed that, because of the feeding habitats of the Cape wagtails, that ‘feet abnormalities in the Cape Wagtail (M. capensis) were caused by the internal action of contaminants (e.g. pesticides and metals) through direct contact and/or secondary poisoning’. However, following appropriate biomarker response testing and metal analysis, it was apparent that these factors had little influence on the occurrence or frequency of the deformities. The initial hypothesis was therefore rejected.

The fundamental aim of this research was to find the reason(s) for the feet abnormalities in the Cape Wagtail (M. capensis) and to establish the frequency of the deformities at various sites. Macro pathological observations led to the formulation of an alternative hypothesis, which stated that ‘an external factor, such as parasitism, is responsible for the feet deformities observed in the Cape Wagtail (M. capensis)’.

This hypothesis was accepted following the discovery of K. jamaicensis in the white, powdery encrustations along the leg regions of several subjects. The manner in which the mite causes such severe deformities as presented in this dissertation is not completely understood, although various suggestions have been put forward following this research as well as by various other authors. In terms of the frequency of deformities, Paarl (site 1) and Port Elizabeth (site 3) presented the highest rate of deformities while Somerset West (site 2), Secunda (site 4), Dullstroom (site 5) and Gauteng (site 6) recorded no deformities. Thread entwined around the feet and toes was also found to cause deformities, albeit in lesser numbers.

In terms of the supplementary objectives, the first objective was to collect blood and feather samples from Cape Wagtails at various sites around the country. This objective was completed successfully. Blood and feather samples were collected from three coastal sites (Paarl, Somerset West and Port Elizabeth) and three inland sites (Secunda, Dullstroom and Gauteng). Blood samples were very small which limited assays and responses that could have been tested on each bird.

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The second objective was to explore the spatial variation of biomarker responses (AChE, ALA-D, CAT in different blood components and DNA damage in erythrocytes). Little substantial data from fellow researchers was attained to explain the biomarker results in this study. Acetylcholinesterase, ALA-D and DNA damage were not found to be linked to the occurrence of feet deformities. However, CAT revealed stress at all the sites sampled which may have been caused by the feet abnormalities or other unknown factors. The NMDS ordination showed significant differences among biomarkers at all sites. Sites with affected birds and those without were grouped together suggesting that biomarker responses did not vary between healthy and affected birds.

The third objective was to explore the spatial variation of metal exposure as reflected in feathers and plasma of Cape Wagtails. In terms of metals in the feathers, most were higher than the reference values used for contaminated sites and almost all the current concentrations at each site exceeded the target levels set by EcoQOs for Weavers (Ploceus sp.) in Gauteng. Zinc levels at all sites were high when compared to the concentrations recorded by fellow researchers. Secunda (site 4) displayed the highest levels in most of the metals. At the same time, Secunda (site 4) revealed the highest TU which was expected due to the surrounding industrial activities. This result supported the fact that Secunda (site 4) displayed the highest metal concentrations in most of the metals in the feathers. Chromium and Ni were suspected to possibly trigger feet and toe abnormalities in Cape Wagtails. But, if one considers the levels of these metals in the blood plasma it could be seen that this was not the case because both of these metals at sites 4 (Secunda) and 5 (Dullstroom) were higher than at sites 1 (Paarl) and 3 (Port Elizabeth). The NMDS ordination revealed no significant differences among all the metals analyzed at each site. Sites with affected birds and those without were grouped together indicating that metal concentrations were not different in affected versus healthy birds. Various metals were notably higher at the Cape Sites (sites 1 – Paarl, 2 – Somerset West and 3 – Port Elizabeth) compared to the Highveld or Mpumalanga sites (sites 4 - Secunda and 5 -Dullstroom) such as in the case of Cr and Ni. However, the reverse was true for Al, As, Cu, Mn and Ag.

Limited data was found in the literature to explain the levels of various metals in the plasma. Most metals were higher than the reference values used. The NMDS ordination revealed groupings of sites with affected birds and those with healthy birds. Because no significant differences were reported, it was doubtful that metals in the plasma were indicative of exposure to pollutants that may have caused feet abnormalities. In general, most metals in

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the plasma were higher at Secunda (site 4) and Dullstroom (site 5) which had healthy birds sampled, compared to Paarl (site 1) and Port Elizabeth (site 3) which had affected birds. This suggested that it was doubtful that metals in the plasma could not be responsible for the deformities.

The fourth objective was to investigate and compare biomarker responses and metal levels in healthy birds and affected birds. This objective linked in with the first proposed hypothesis which stated that ‘Feet abnormalities in the Cape Wagtail (M. capensis) are caused by the internal action of contaminants (e.g. pesticides and metals) through direct contact and/or secondary poisoning’. If one refers to the discussion between affected birds and healthy birds, increased CAT enzyme activity and DNA damage was associated with birds that displayed feet abnormalities although results were conflicting. Metal levels were generally higher in healthy birds than in affected birds which indicated that it was doubtful that metal exposure was responsible for the deformities, or even contributed to them. Biomarker responses were not convincingly different between affected and healthy birds. This statement satisfied objective five, which was to determine whether there is a causal link between the severe feet abnormalities and biomarker responses and metal exposure. If one considers the TU results (combined toxicity of metals) at Paarl (site 1), affected birds displayed a higher TU value than affected birds at that site. Somerset West (site 2) was similar, while Port Elizabeth (site 3) was the reverse. However, if one considers each metal individually, healthy birds generally had higher concentrations in the feathers than affected birds. This can only suggest that the birds (both affected and healthy) at Paarl (site 1) were exposed to higher metal concentrations than birds at other sites and that metals in the feathers cannot be linked to the presence of feet or toe deformities in these birds.

The last objective was to gain insight into the biology and effect of the Scaley-mite (K. jamaicensis) in Cape Wagtails. It has seldom been observed that a parasite can cause such obvious and severe effects as observed on Cape Wagtails. It was evident in this research that the mites caused serious, unsightly deformities which interrupted the bird’s normal daily activities such as feeding which may lead to impaired breeding success. Weather conditions at the time were suggested to influence the rate of mite transmission as well as the prevalence of deformities. Mites and deformities were more prevalent in areas of high rainfall and low temperatures, although this contradicts theories of past researchers. The behavioral characteristics of the wagtails in these areas huddling together to seek warmth can suggest a rapid rate of transmission. Much research is needed to focus on the action of this

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mite and to establish the time frames of infection to visual encrustations along the legs, followed by the unsightly appearance of missing toes and, at times, feet. One needs to understand why the Cape wagtail is so vulnerable to infection by this mite. Subsequently, this objective was met with the observations made in this study as well as the evidence provided by previous researchers.

5.1.2. RECOMMENDATIONS

• Sample sizes in most cases were relatively limited. It is recommended that an increased number of samples be obtained to yield more accurate data.

• The blood sample volume extracted from each bird was minimal to ensure that the least possible stress was caused to the individual. But, at the same time, this limited the number of biomarkers that could be measured from each bird. In general, further non- destructive scientific analysis is required to build up data on this bird, such as additional biomarker response testing.

• Additional sites should be sampled to achieve a comprehensive understanding of the status of Cape Wagtails (M. capensis).

• In this study, AChE activity was used as a first screening test for the exposure to pesticides. However, it is advised that more samples should be considered for this testing in conjunction with pesticide level analysis to verify the accuracy of the data obtained.

• Fundamental ecological research needs to be carried out to establish why Cape Wagtails are particularly and severely vulnerable to the Scaley-leg mite when compared to other bird species with similar characteristics.

• Additional research needs to be undertaken on K. jamaicensis to determine the time period for the mite to attach to its host (Cape Wagtail), and then to produce such severe abnormalities and to cause feet and toes to fall off.

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APPENDICES

Appendix 1. Mean and standard errors of biomarkers at the study sites Graph Biomarker Site Mean (x) Standard error (SE) A AChE 1 6.44 2.08 3 8.85 2 4 7.49 1.48 5 2.44 0.32 B ALAD 1 0.68 0.15 2 0.62 0.44 3 0.65 0.08 4 0.14 0.03 5 0.1 0.02 C CAT 1 3.53 0.85 2 2.61 1.98 3 2.64 0.5 D DNA ABPL 1 6647.59 110.52 2 9524.01 125.35 3 7752.56 194.14 4 7652.4 160.9 5 7887.03 105.28 6 9667.98 *

* represents no standard error available as the number of samples was one (applies to all the following appendices)

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Appendix 2. Mean and Standard error of metal concentrations in the feathers at each study site

Graph Metal Site Mean (x) Standard error (SE) Graph Metal Site Mean (x) Standard error (SE)

A Al 1 496.96 81.21 H Pb 1 47.48 9.25

2 430.94 127.55 2 20.49 5.3

3 450.49 48.29 3 30.2 4.16

4 915.76 259.1 4 79.21 19.12

5 809.91 211.24 5 59.86 29.81

6 375.91 188.7 6 31.1 14.43

B As 1 10 2.27 I Mn 1 63.72 9.92

2 9.96 5.27 2 38.14 8.64

3 9.9 1.48 3 51.61 6.18

4 18.01 6.04 4 96.67 26.36

5 17.44 8.92 5 66.46 31.04

6 11.06 * 6 65.15 31.16

C Cd 1 8.26 1.67 J Ni 1 188.18 13.88

2 3.44 0.87 2 117.67 21.1

3 7.67 1.42 3 110.91 15.3

4 9.51 2.49 4 44.07 12.15

5 5.72 2.63 5 36.24 20.51

6 2.11 0.74 6 41.52 30.51

D Cr 1 497.47 49.05 K Ag 1 2.39 *

2 312.69 75.17 3 2.06 0.64

3 274.37 36.19 4 3.58 0.85

4 58.29 18.79 5 3.5 1.84

5 40.85 24.49 L Sr 1 23.54 4.49

6 104.35 76.3 2 20.62 4.54

E Co 1 1.23 0.19 3 34.15 3.32

2 0.59 0.14 4 24.63 7.48

3 0.75 0.12 5 12.37 1.75

4 0.74 0.16 6 21.08 7.22

5 0.75 0.52 M Zn 1 2634.14 744.44

6 0.37 0.06 2 2128.32 601.54

F Cu 1 43.29 5.53 3 1740.4 173.86

2 30.53 10.14 4 2820.33 727.92

3 38.78 5.71 5 971.51 314.65

4 105.91 25.51 6 1797.59 834.82

5 81.04 35.57

6 80.04 57.05

G Fe 1 2893.57 368.53

2 1730.26 357.75

3 1870.11 250.25

4 1990.78 488.41

5 1513.51 729.76

6 1490.3 284.61

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Appendix 3. Mean and standard error of metal concentrations in the plasma for all study sites Standard Standard Graph Metal Site Mean (x) error (SE) Graph Metal Site Mean (x) error (SE) A Al 1 1.18 0.11 H Fe 1 29.48 16.96 3 1.63 1.22 3 21.67 20.41 4 5.04 0.38 4 1.53 0.22 5 4.86 3.72 5 5.41 5.06 B As 1 0.08 0.04 I Pb 1 0.11 0.06 3 0.03 0.03 3 0.16 0.15 4 0.07 0.01 4 0.47 0.04 5 0.05 0.04 5 0.37 0.3 C Cd 1 0.14 0.16 J Mn 1 0.18 0.09 3 0.01 0.01 3 0.21 0.08 4 0.03 0.01 4 0.47 0.99 5 0.05 0.05 5 0.5 0.33 D Ca 1 90.64 17.06 K Ni 1 0.11 0.01 3 140.78 59 3 0.15 0.03 4 616.4 221.61 4 0.37 0.09 5 601.25 533.94 5 0.38 0.15 E Cr 1 0.15 0.13 L Ag 1 0.14 0.16 3 0.11 0.02 3 0.01 0 4 0.14 0 4 0.01 * 5 0.23 0.05 5 0.02 0.01 F Co 1 0.01 * M Zn 1 14.83 4.75 3 0 * 3 11.82 9.95 4 0.02 0 4 59.36 16.42 5 0.01 0.01 5 51.49 27.84 G Cu 1 4.16 5.1 3 0.24 0.05 4 0.62 0.06 5 0.89 0.54

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Appendix 4. Mean and standard error for biomarkers at sites 1 (Paarl), 2 (Somerset West) and 3 (Port Elizabeth) for healthy (H) and affected (A) birds Graph Biomarker Status Mean (x) Standard error (SE) A AChE 1H 5.18 2.34 1A 10.23 * 3H 8.97 1.82 3A 11.99 6.16 B ALAD 1H 0.75 0.16 1A 0.26 * 2H 0.61 0.44 3H 0.65 0.1 3A 0.65 0.18 C CAT 1H 2.81 0.62 1A 5.7 * 2H 2.61 1.98 3H 2.36 0.37 3A 4.38 2.93 D DNA ABPL 1H 6658.74 126.95 1A 6569.59 * 2H 9557.64 209.14 2A 9456.75 * 3H 7879.19 225.98 3A 7155.59 210.251

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Appendix 5. Mean and standard errors for metal concentrations in the feathers at sites 1 (Paarl), 2 (Somerset West) and 3 (Port Elizabeth) for healthy (H) and affected (A) birds

Standard error Standard error Graph Metal Status Mean (x) (SE) Graph Metal Status Mean (x) (SE) A Al 1H 521.51 97.04 H Pb 1H 112.32 36.76

1A 382.38 73.27 1A 50.48 19.87 2H 319.6 80.75 2H 19.43 5.89 2A 401.49 * 2A 28.99 * 3H 401.12 41.12 3H 29.17 4.3 3A 432.14 88.8 3A 23.16 4.38 B As 1H 10.07 2.74 I Mn 1H 65.81 12.02 1A 9.71 4.01 1A 53.97 5.08 2H 9.96 5.27 2H 36.83 9.69 3H 10.61 1.8 2A 48.66 * 3A 7.95 2.33 3H 50.28 5.8 C Cd 1H 8.76 1.96 3A 38.24 6.02 1A 5.77 0.93 J Ni 1H 272.96 52.25 2H 3.39 0.98 1A 195.54 29.94 2A 3.88 * 2H 124.41 146.91 3H 7.77 1.57 2A 146.91 *

3A 7.31 3.43 3H 126.14 15.78 D Cr 1H 493.19 58.06 3A 55.07 22.81 1A 516.01 91.51 K Ag 1H 2.39 * 2H 306.84 84.97 3H 1.38 0.79 2A 359.48 * 3A 2.89 1.63

3H 317.57 41.63 L Sr 1H 25.26 5.35 3A 131.81 54.78 1A 15.41 2.74 E Co 1H 1.3 0.23 2H 19.8 5.06 1A 0.88 0.16 2A 27.25 * 2H 0.55 0.15 3H 39.81 6.17 2A 0.86 * 3A 32.2 5.74 3H 0.95 0.18 M Zn 1H 2685.88 702.62 3A 0.47 0.11 1A 4114.89 2054.65 F Cu 1H 52.6 10.08 2H 1291.29 387.97 1A 37.52 7.57 2A 1738.14 * 2H 30.51 11.71 3H 1889.04 285.35 2A 30.68 * 3A 1927.74 335.13 3H 90.24 18.66 3A 37.14 13.85 G Fe 1H 2933.21 440.68 1A 2706.58 495.22

2H 166.68 398.56 2A 2262.89 * 3H 1925.24 252.96 3A 1070.21 256.71

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