and Kevin Rummel provided assistance in many of the analytical techniques used in our .... Bird, 1990), or by catching prey on the wing (Rudolph, 1983).
METAL EXPOSURE AND EFFECTS IN AMERICAN KESTREL (Faico sparverius) NESTLINGS RAISED ON A SMELTER-IMPACTED SUPERFUND SITE by TOBIAS JOHN McBRIDE, B.S. A THESIS IN ENVIRONMENTAL TOXICOLOGY Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Approved
May, 2002
ACKNOWLEDGMENTS
I want to express my gratitude to the numerous individuals who were instmmental in assisting me in the completion of this thesis. The members of my committee, Mike Hooper, George Cobb and Scott McMurry, provided outstanding support and assistance throughout the process. All of you were very approachable for every question, idea or concem I had over this period. I want to especially acknowledge Blakely Adair, for her inmiense assistance in Montana, as well as at home. Your help in the field, in the lab, and in the writing of this thesis has been greatly appreciated, and your friendship has made this process easier. Thanks to Dr. Dale Hoff, and to Dr. Bill Olsen for providing funding, instruments, ideas, and comments during our investigations. Catherine Polydore, Craig McFarland, and Kevin Rummel provided assistance in many of the analytical techniques used in our analysis. In addition, Gabi Rutz and Lisa Podhaisky helped in compiling and checking the data, while Debi Agenbroad and Misti Porter provided much needed assistance in the field collections. Thanks especially to the U.S. EPA - Region 8, U.S. Fish and Wildlife Service, and to NIEHS - ES 04696 for providing financial support for this investigation. Finally, I want to acknowledge my family. Ellen, I would have never gotten this far without your support and understanding during these difficult years. Charles, you were instmmental in developing my curiosity of science and my love for nature. Mom, you have always pushed me to do my best. Thanks to all of you for a lifetime of support.
II
TABLE OF CONTENTS
ACKNOWLEDGMENTS
ii
ABSTRACT
vi
USTOFTABLJES
viii
UST OF FIGURES
x
L INTRODUCTION
1
American Kestrels
1
Avian Toxicology
3
Lead and Lead-Shot Exposure
5
Mining and Smelting Wastes
9
Reclamation and Remedial Action
12
Anaconda Superfund Site
13
Wildlife Concerns in Remediation
14
Primary Research Objectives
15
n. METHODOLOGY
16
Daily Nest Box Monitoring
16
Addled Egg Collection
22
Nestling Monitoring And Measurement
22
Esophageal Constriction
23
Nestling Sampling
23
Blood Sample Collection
25
Sample Digestion and Metal Analysis
26
Sample Digestion
26
Inductively Coupled Plasma-Atomic Emission (ICP-AE) Analysis
27
Graphite Fumace-Atomic Absorption (GF-AA) Analysis
28
Instmment Detection Limits (IDE)
28
Effects Assessment Methodology
29
Porphyrin Extraction
29
111
HPLC and Equipment
31
Porphyrin Recovery/Detection Limits
31
5-Aminolevulinic Acid Dehydratase (ALAD) Extraction
32
6-Aminolevulinic Acid Dehydratase (ALAD) Activity Determination....33 Data Analysis
34
Summary Statistics
34
Exposure Estimation
35
m. RESULTS
37
Nest Box Placement And Use
37
1999 Field Season
37
2000 Field Season
39
Exposure Assessment
39
Food Item Metal Analysis
39
Pellet Metal Analysis
45
Egg Metal Analysis
47
Blood Metal Analysis
47
Liver and Kidney Metal Analysis
58
Fecal/Urate Metal Analysis
61
Effects Assessment - Biochemical And Cellular
61
Porphyrin Analysis
61
ALAD Analysis
64
Hematology
68
Tissue Weights
68
Effects Assessment - Reproductive Demographics
71
Nesting and Hatching Success
71
Hatchling and Fledgling Success
74
Egg Measurement
75
Morphological Measurement
75
IV DISCUSSION
92
IV
Exposure Assessment
93
Effects Assessment
107
V. CONCLUDING STATEMENT
116
BIBUOGRAPHY
118
ABSTRACT
Nestling American kestrels (Falco sparverius) inhabiting nest boxes placed throughout the Anaconda Smelter Superfund Site, Montana, were monitored for reproductive success and growth over two successive breeding seasons. A gradient of decreasing soil contaminant concentrations occurred with increasing distance away from the smelter stack. The contaminants of concem (COCs; arsenic, cadmium, lead, copper, and zinc) were determined in food items, pellets, fecal/urate, blood, kidney and liver tissues. Hepatic and renal porphyrin profiles were characterized, and erythrocyte ALAD inhibition assessed, for use as indicators of health effects. Site wide in 1999,43 chicks hatched (81%) with 41 surviving to fledging age (95.3% fledging efficiency). In 2000, 34 chicks hatched (59%) with 32 surviving to fledging age (94% fledging efficiency). Nestling growth and demographics did not appear related to tissue contaminant levels. Active boxes were grouped into two site types (Smelter HiU and Opportunity Ponds) based on their proximity to the smelter site. In 1999, Smelter HiU birds had higher blood lead, liver cadmium, lead, and copper, and kidney cadmium. In 2000, only blood samples were collected, with notably increased lead and cadmium from Smelter Hill birds. Inhibition of erythrocyte ALAD activity at the highest blood lead concentrations indicated lead concentrations reached a threshold level sufficient to initiate biochemical
VI
effects. An increase in liver 4-carboxyl porphyrin at the highest liver lead levels likewise suggested sufficient exposure to initiate dismption of heme synthesis. Food items were collected from each active nest. COC concentrations were higher in samples from Smelter Hill boxes, with notable increases seen in arsenic and lead. Contaminant levels in food items were sufficiently high for accumulation in nestling tissues, with elevated levels occurring in nestlings closer in proximity to the smelter.
VII
LIST OF TABLES
1. American Kestrel Nest Boxes Deployed on The Anaconda Smelter Site
17
2. Detection and Reporting Limits for Metals Data in Collected Samples
30
3. Egg and Nestling Data for Occupied Nest Boxes, 1999
38
4. Egg and Nestling Data for Occupied Nest Boxes, 2000
40
5. Food Item Metal Concentrations, 1999
41
6. Food Item Metal Concentrations, 2000
43
7. Pellet Metal Concentrations Collected from Nest Boxes, 1999
46
8. Intra-Clutch Variability for Sample Metals and Effects Endpoints
48
9. Unincubated Egg Metal Concentrations, 1999
49
10. Blood Metal Concentrations from Day 25 Post-Hatch Nestlings, 1999
50
11. Blood Metal Concentt-ations by Box By Age, 2000
52
12. Blood Metal Concentrations from Day 25 Post-Hatch NestUngs, 2000
56
13. Liver Metal Concentrations, 1999
59
14. Kidney Metal Concentrations, 1999
60
15. Fecal/Urate Metal Concentrations, 1999
62
16. Hepatic Porphyrin Profiles, 1999
63
17. Renal Porphyrin Profiles, 1999
66
18. ALAD Activities and Packed CeU Volumes, 2000
67
19. Nestling Hematology, 1999
69
20. Tissue Weights, 1999
70
21. Nesting and Demographic Parameters, 1999 and 2000
72
viii
22. Unincubated and Addled Egg Weights And Measurements, 1999 and 2000
76
23. Body Weight and Morphological Measurements Recorded on Days 5, 10, 20, and 25 Post-Hatch, 1999
77
24. Body Weight and Morphological Measurements of Day 25 NestUngs, 1999
84
25. Body Weight and Morphological Measurements Recorded on Days 5, 10, 20, and 25 Post-Hatch, 2000
86
26. Body Weight and Morphological Measurements of Day 25 Nestiings, 2000
90
27. Estimated Total Metal Amount in Food Items by Box
98
28. Estimated Total Metal Amount in Pellet and Fecal/Urate by Box
99
29. Mean Prey Metal Concentration, and Estimated Total Daily Intake and Dose
IX
101
LIST OF HGURES
1. Map of Anaconda Superfund Site
19
2. Map Indicating Site Delineation Used for Comparative Analysis
21
3. Food Item Metal Concentration (Mean ± SD) by Prey Type
44
4. Mean (+ SD) Blood Metal Concentrations by Box By Age
54
5. Hepatic Porphyrin (4-CP) as a Function of Log Lead Concentration
65
6. Nestling Growth Curve Estimates Using Nonlinear Logistic Regression
91
7. Blood Lead Concentrations by Box Mean (Both Years)
104
8. Hepatic Porphyrin (4-CP) Versus Hepatic Lead Concentration
108
9. Nest Box Mean (± SE) Blood ALAD Activity as a Function of Blood Lead
110
10. Nestling Blood ALAD Activity Versus Lead Concentration
Ill
CHAPTER I INTRODUCTION
Though human health concems are historically the principal motivation in the remediation processes for hazardous waste sites, ecological concems have risen in importance, with wildlife evaluations being performed on increasing numbers of contaminated sites for the specific purpose of incorporating the ecosystem into risk assessments. Exposure routes, biomarkers of exposure and effects, population demographics, and ecosystem response are assessment methods commonly used in the evaluations. Though the scope of such research may be burdensome, well-directed studies on selected species, using appropriate endpoints, produce valuable data for site management decision-making (Hooper and LaPoint, 1994). This thesis examined the utility of a top-level avian predator, the American kestrel {Falco sparverius), in assessing accumulation and effects of exposure to environmental contaminants resulting from historic copper smelting practices.
American Kestrels American kestrels are the smallest falcon species found in North America (Brown and Amadon, 1968). They are ubiquitous throughout the United States, and maintain the highest populations of all falcons within their range (Balgooyen, 1976). Kestrels are generally found in open habitats, where they hunt by perching in exposed areas (Bird, 1988), or hovering above open grasslands, prior to direct dives upon their prey (Gard and Bird, 1990), or by catching prey on the wing (Rudolph, 1983). Being opportunistic
feeders, kestrels regularly select large invertebrates (grasshoppers, beetles, dragonflies), rodents, passerines, reptiles or amphibians, depending on availability (MeuUer, 1987, Bohall-Wood and CoUopy, 1987). They are sexually dimorphic, both in size and coloration (Bird, 1988). Overall, adult kestrels are solitary except during breeding, when male kestrels will defend home ranges and nesting sites against competing males (Balgooyen, 1976). Old woodpecker holes and natural tree cavities are regularly used as nest sites, though kestrels are also known to utilize bams and eaves of urban buildings (Hamerstrom et al., 1973). A general scarcity of suitable nesting sites seems to be a principle-limiting factor to kestrel populations (Cade, 1982). In as much, kestrels will readily use nest boxes (Hamerstrom et al., 1973), with competition with other cavity nesters like starlings (Stumus vulgaris), flickers (Colaptes auratus), mountain bluebirds (Sialia currucoides), and screech owls {Otus asio) noted as being major factors in reduced box utilization (Balgooyen, 1976; Curley et al., 1987; Craft and Craft, 1996). Seasonally monogamous, males establish potential nesting sites early, with the female's choice of mate based on nest site characteristics as well as the fitness of the male (Brown and Amadon, 1968). Females lay an average of 4 to 5 eggs per clutch (Anderson et al., 1997), delaying incubation so all eggs hatch synchronously (Balgooyen, 1976). Incubation is 28 to 30 days, with the females remaining on the nest except for quick feeding forays. Though males may assist in incubation at times, much of their effort is in providing food for the female, as well as for the subsequent clutch (Brown and Amadon, 1968). NestUngs are semi-altricial at birth, achieving full body mass by approximately 17-20 days, and will remain in the nest for 30-35 days (Bird, 1988).
Fledglings continue to be dependent on their parents for food, and will often remain in the nest vicinity for several weeks (Lett and Bird, 1987; Balgooyen, 1976).
Avian Toxicology The study of environmental contaminant impacts on wildlife is a relatively new science. For many, the first infroduction to environmental toxicology was through the writings of Rachel Carson's 'Silent Spring' in 1962, detaihng her observations of pesticide effects (specifically, dichlorodiphenyltrichloroethane or DDT) on the native birds in her region. Subsequent investigations, brought to light the potential of DDT to bioaccumulate through food sources, causing significant detrimental effects in North American predatory bird populations, most notably bald eagles (Haliaeetus leucocephalus; Bowerman et al., 1998), ospreys, and other birds of prey. Likewise, metallic contaminants have caused serious environmental concems, though the ensuing effects are varied and investigations as to the toxicity of metals in birds are extremely wide-ranging. Generally, field-based avian research of metal contamination has been directed on specific issues often involving unusual natural concentrations (Elliott and Scheuhammer, 1997), metaUic spills or releases (Pain et al., 1998), lead-shot exposure (Pain, 1996; Kendall et al., 1996), or mining and smelter wastes (Henny et al., 1991; Blus et al., 1999). High northern latitude sea birds have shown increased tissue concentrations due to exposure to high concentrations of cadmium, copper, arsenic and selenium from ocean water and food sources (Tmst, 2000; Elliott and Scheuhammer, 1997; Elliott et al., 1992).
Spectacled eiders {Somateria fischeri) were found with hepatic copper concentrations of 559 jUg/g and cadmium concentrations of 33.8 jUg/g (Tmst, 2000), though with no identifiable ill health effects. Metallothionein, an inducible protein utilized for natural cellular metal homeostasis and sequestration, acts as a cellular protective mechanism from some non-essential metals. Metallothionein induction has been seen in a number of avian species after cadmium exposure (Fumess, 1996; Scheuhammer, 1988; Osbom, 1978). Exposure pathway for high natural cadmium concentrations in upland game birds in Norway (Myklebust and Pedersen, 1999) and Colorado (Larison et al., 2000) has been identified as the result of accumulation of cadmium in willows. Willow leaves and buds {Salix spp.) biomagnify cadmium up to two orders of magnitude above background concentrations, exposing species that utilize the plants as a primary food source (notably the ptarmigan) to dangerous levels of cadmium. Cadmium accumulation in Norwegian willow ptarmigan {Lagopus lagopus) was identified in kidney and liver tissues, though kidney concentrations were 7 to 10 times higher (Myklebust and Pedersen, 1999). Histopathological examination revealed that 57% of white-tailed ptarmigans {Lagopus leucurus) living in Colorado exhibited renal damage (Larison et al., 2000). Cadmiuminduced dismption of calcium re-uptake in the kidneys has caused osteoporosis, thereby increasing occurrence of bone breaks. An industrial mercury release into Minamata Bay, Japan, in the late 1950s resulted in significant human health effects (Hosokawa, 1993), as a result of contaminated food sources. Increased mortality had been noted in many of the native piscivorous bird species, resulting from severe neurological impairment indicative of
mercury poisoning (Boening, 2000). Mercury accumulation in aquatic environments generally results in biomagnifications in native fish populations. Subsequentiy, piscivorous birds are at great risk for accumulation of toxic levels (Sepulveda et al., 1999; Frederick et al., 1999; Bouton et al., 1999). One of the most important bird breeding sites in western Europe, the Doiiana National Park, was severely impacted by the accidental release of millions of cubic feet of acidic mine waste in the spring of 1998 (Pain et al., 1998). A byproduct of the AznalcoUar pyrite mine near Seville, Spain, metal contaminated wastes broke into the estuarine marshes in Dohana resulting in large-scale kills of nesting waterfowl and shorebirds. Another unintended pollutant, selenium was found in high concentrations in Califomia's Kesterson reservoir in the 1980s, from agricultural drain waters, where it increased exposure to wildlife (Paveglio et al., 1997). The propensity of selenium to accumulate in tissues (Yamamoto et al., 1998), and cause detrimental health and population level effects has been documented in the surrounding avian wildlife (Hoffman etal., 1988).
Lead and Lead-Shot Exposure Released by power plants, industrial and gasoline emissions, lead has become the most ubiquitous toxic metal in the environment, able to be detected in fauna in virtually every area of the worid (Pain, 1996). Concems over lead in birds were not expressed until acute poisoning was recognized to result from lead use in firearm munitions and
fishing sinkers (Bellrose, 1959); consequently, a majority of lead research has emphasized artifactual lead intake and exposure. Filter feeders, such as swans, geese, cranes and some species of duck utilize aquatic plants as a considerable food source, eating large amounts of associated sediment. Waterfowl and shorebirds can be exposed to lead shot remaining in lake and marshland sediment (Grand et al., 1998), as a result of accidental ingestion (Pain, 1996). Eighteen percent of 39 salvaged mallards {Anas platyorhynchus) analyzed from Canada contained elevated (>10 ug/g) bone lead concentrations (Tsuji and Karagatzides, 2001). A risk assessment of lead effects in upland bird species concluded that lead may accumulate in tissues following exposure to lead shot (Kendall et al., 1996). The variability of uptake between species has been explained by different exposure and depuration mechanisms (Burger, 1995). Japanese quail dosed with lead showed significantly decreased rates of weight gain and initiation of anemia at the level of 500 ppm in the diet (Morgan et al., 1975), though bobwhite quail {Colinus virginianus) dosed at levels of 2000 ppm did not show any significant growth effects (Damron and Wilson, 1975). Game birds may ingest lead shot in heavily hunted areas (Burger et al., 1997), for use as grit, resulting in prolonged erosion of the pellet within the gizzard. Mourning doves {Zenaida macroura) dosed with a single #8 lead shot showed a ten-fold increase of renal lead and a two-fold increase of hepatic lead within three weeks, with significant erosion of the pellet prior to excretion (Mam et al., 1988). Human health concems have become recognized, since game birds are harvested and subsequently consumed by hunters (Burger et al., 1997), intensifying the potential for increased lead ingestion. In
addition, passerines and even mammals are at risk for accidental ingestion of spent shot in heavily hunted areas. Lewis et al. (2001), investigating a firearm training facility, documented 11 of 22 local species (birds and mammals) as having liver or kidney tissue lead concentrations above 1 ppm, with 6 of those species above 2 ppm lead. Scavengers and raptors are exposed to lead contamination through ingestion of incapacitated waterfowl suffering lead toxicosis, as well as ingestion of lead shot from upland game birds and mammals that have been injured or killed during hunting (Kendall et al., 1996). American kesfrels were dosed for two weeks with either a #9 lead shot placed inside a feeder mouse, or a meal of lead-spiked mallard homogenate (Stendell, 1980). Both routes of exposure resulted in significantly increased lead in kestrel liver, while ingestion of the lead shot significantiy increased bone lead concenfration. Lead toxicosis and exposure to lead shot have been documented in many wild raptors, including eagles, goshawks, vultures and kites. (Wayland et al., 1999; Miller et al., 1998; Gamer, 1991; Piatt et al., 1999; Mateo et al., 2001). Studies of expelled pellets collected from two wild raptor populations in Spain, found lead shot artifacts in 6% and 11% of those collected (Mateo et al., 2001). Critical body threshold ranges in Falconiformes have been suggested for lead, with subclinical effects levels (generally indicated by a biochemical perturbation) expressed as 0.2 to 1.5ppm in blood, 2 to 4ppm in liver and 2 to 5ppm in kidney (Franson, 1996). Bald eagles administered with 10 lead shot per day in food items, had increased blood lead (0.8ppm) within 24 hours, and exhibited 80% inhibition of 6-aminolevulinic acid dehydratase (ALAD) activity. Within one week, blood lead levels had increased to over 3ppm, with a significant decrease in percent
hematocrit and hemoglobin concentration (Pattee et al., 1981). Individual responses to lead may be quite variable, as three eagles dosed with the 10 lead shot per meal died within 20 days, while two survived at least 125 days (Pattee et al., 1981). Lead is well known for its effects on the vascular, nervous, renal, reproductive and hematopoietic systems, as well as its promotion of behavioral abnormalities (Eisler, 1988; Burger, 1995). Severe toxicosis may produce peripheral neuropathies (Piatt et al., 1999), regenerative anemia (Gamer, 1991), renal lesions (Pattee et al., 1981), decreased liver and kidney weights, and alterations in growth rate (Hoffman et al., 1985). 5-Aminolevulinic-acid dehydratase (ALAD) is a widely studied heme-related enzyme that is altered by metal contamination. ALAD condenses two aminolevulinic acid molecules to porphobilinogen, a carboxylated pyrrole intermediate in heme biosynthesis. The zinc-dependant enzyme is easily inhibited by lead substitution and has been extensively characterized as a sensitive indicator of lead exposure (Hoffman et al., 1981, Scheuhammer, 1987; Goering et al., 1986; Pain et al., 1996). Decreased red blood cell ALAD activity has been correlated to lead accumulation in a wide variety of birds such as raptors (Franson et al., 1983; Hoffman et al., 1985), waterfowl (Dieter and Finley, 1979; Pain et al., 1996), game birds (Scheuhammer and Wilson, 1990) and passerines (Beyer et al., 2000), with inhibition occurring at blood lead ranges of 0.2 to 1.5 ppm. Effects of lead on the heme biosynthesis pathway have been examined using porphyrin profile changes and ALAD enzyme inhibition as endpoints. Porphyrins are the molecular precursors of heme, the prosthetic group in a variety of proteins found in all eukaryotic ceMs, such as hemoglobin, cytochrome P450 and cytochrome C. Metals react with
specific heme-synthesis enzymes producing characteristic alterations of the porphyrin profile (Woods, 1996; Fowler and Mahaffey, 1978). Porphyrin concentrations have historically been analyzed in Hver and kidney tissues; however, urine and fecal matter may offer a reliable non-lethal substrate (Martinez et al., 1983; Akins et al., 1993) that can be easily collected. Porphyrin increases were significantly correlated with hepatic arsenic concentrations of spectacled eiders (Tmst, 2000) in western Alaska. In leadexposed American kestrel nestlings raised near the Kellogg-Smelterville Superfund site in Coeur d'Alene, Idaho, a 55% inhibition of erythrocyte ALAD in conjunction with significant decreases (when compared to reference area nestlings) in hemoglobin concentration and hematocrit levels were documented. Starlings inhabiting the Anaconda Smelter site in Montana showed porphyrin increases in kidneys of nestlings that accompanied elevated lead levels in that tissue. High concentrations of zinc in kidneys, however, appear to modulate the porphyrin increases (Hooper et al., 2001).
Mining and Smelting Wastes Mining activities generate enormous amounts of waste products in a variety forms, such as waste rock, processed tailings, slag, flue dust and liquid leachates. Generally, much of the resulting contamination is due to increased accessibility of metal in the rock, made available through the mining and milling processes. Furthermore, chemical additives used in the purification process may contribute additional contaminants, or may increase availability of the mineral contaminants by alteration of their chemical nature. Investigators on mine waste contaminated sites are typically
confronted with polluted pond or stream systems from leachate, highly concentrated waste rock, contaminated tailings, and aerially deposited pollutants. Mining and smelting activities have occurred in Coeur d'Alene River Basin in northem Idaho since 1880's, depositing 72 million tons of tailings washed into the river and lake system (URS, 2001). Lead exposure has been examined in waterfowl, passerines (Blus et al., 1995; Blus et al., 1999; Johnson et al., 1999), and raptors (Henny et al., 1991; Henny et al., 1994) inhabiting the site. Lead concentrations, in sediments consumed secondarily by waterfowl feeding in the river basin, are sufficiently increased to be implicated in the deaths of 219 of 285 scavenged waterfowl carcasses from the area. Dosing studies using collected sediment significantly depressed ALAD activity and elevated free erythrocyte protoporphyrin (protoporphyrin) concentrations in nestling mallard ducks (Hoffman et al., 2000). Nestling American kestrels, raised downstream from the mining site, had significantiy elevated blood lead levels and decreased ALAD activities compared to reference nestlings (Henny et al., 1994). Heavy metal exposure and associated adverse health effects in pied flycatchers {Ficedula hypoleuca) were documented from mining and smelting facilities in Finland (Eeva and Lehikoinen, 1996) as well as in northem Sweden (Nyholm, 1998). Breeding performance in Tengmalm's owls {Aegolius funereus) was similarly reduced with decreasing distance from the smelting facility (Homfeldt and Nyholm, 1996). Great and blue tits (Parus major and Parus caeruleus) had increased cadmium and copper accumulation, with blue tits also showing increased lead accumulation in feathers (Eens
10
et al., 1999), resulting from aerially deposited pollutants from a waste incinerator in northem Belgium. Wintering golden {Aquila chrysaetos) and bald eagles trapped from throughout central Idaho were examined for lead and mercury contamination (Craig and Craig, 1998). Essentially all (100% and 99.6% respectively) adult blood tested had detectable lead, with 46% (golden) and 61% (bald) showing elevated levels (> 0.2 ppm). Although there was no source specific contamination, the area is a historical mining region and has heavy use by hunters. Historic gold and silver mining near Leadville, Colorado, has contaminated the Arkansas River (Custer and Custer, 2000). Nest box studies of small passerines in the region demonsfrated elevated lead concentrations in nestiing tree swallow {Tachycineta bicolor) Uvers that corresponded to levels found in collected food items. A 50% inhibition of ALAD activity was coixelated with the highest lead exposures. The leadmining district in southeastem Missouri (Niethammer et al., 1985) and the zinc-mining district at Palmerton, Pennsylvania (Beyer et al., 1985), have also been assessed for lead impacts on birds. In Missouri, the mine drainage flows into the Big River system where green-backed herons {Butorides striatus) showed increased lead and cadmium levels compared to non-contaminated sites (Niethammer et al., 1985). Songbirds collected 2 km from the Palmerton smelter had carcass concentrations of 56 ppm lead, as well as exhibited a 50% reduction in erythrocyte ALAD activity when compared to songbirds collected 12 km away.
11
Reclamation and Remedial Action The methods used in mining and refinement of metalliferous products have historically caused severe pollution, often requiring remedial action for both health risks and aesthetic interests. Smelting facilities, in particular, are confronted with the difficulties of handling enormous amounts of slag, leachate, and tailings wastes. Additionally, the smelting process generates substantial amounts of aerial pollutants, creating an additional wide-ranging contamination concem. Many of these sites encompass enormous amounts of land, precluding them from the realistic hope of simply removing the contaminated matrix for storage elsewhere. Unlike many organic or industrial contaminants, metals have an extremely slow degradation half-life and few metabolic processes to assist in decreasing their terrestrial concentration over time, although chemical form (i.e., ionic state, organic configuration) may influence accessibility. In general, the two primary methods of treatment involve dilution and stabilization. Mixing of the topsoil through tillage can decrease high metal concentrations accessible to flora and fauna at the soil's surface. Addition of lime, organic matter and chemical fertilizers is often necessary in highly acidic soils with low organic content, while addition of non-contaminated cover soils acts as a barrier against wind and water erosion in the least hospitable and most contaminated areas. Vegetative stabilization of tilled or cover soils, however, is generally seen as the only mechanism for sustainable, long-term rehabilitation (Tordoff et al., 2000). Site investigations are often performed before full-scale remedial action, as differing soil properties, land use, and aesthetic requirements will determine the most
12
responsible corrective measures for separate portions of a site (EPA, 1997). Specific data requirements are mandated when sites are placed under the auspices of the Environmental Protection Agency (EPA) Comprehensive Environmental Response, Compensation and Liability Act (CERCLA). Ecological Risk Assessments, specific for superfund sites, are an integral part of the CERCLA process (EPA, 1997).
Anaconda Superfund Site The Anaconda Smelter Superfund Site (NPL# MTD093291656), in and around the city of Anaconda, Deer Lodge County, Montana, encompasses approximately 100 square miles of the Clark Fork River valley that have been affected by over 100 years of copper milling and smelting operations. The area consists of agricultural, pasture, range, forest, riparian and wetland areas that contain large volumes of wastes, slag, and tailings, as well as soil, ground water, and surface water contaminated from copper and other metal ore milling, smelting, and refining operations conducted on site by the Anaconda Mining Company (purchased in 1977 by the Atlantic Richfield Company or ARCO). Aerial stack emissions and stream discharges have transported metal-contaminated wastes over an expansive area. These wastes include approximately 230 million cubic yards of concentrated mine taiUngs, 30 million cubic yards of furnace slags, 500,000 cubic yards of flue dust, and many square miles of contaminated soils (CDM 1997). Extensive phytotoxicity effects at the Anaconda site have left vast areas either denuded or severely stressed in their vegetative components. The site was placed on the EPA's National Priorities List (NPL) and under the authority of CERCLA in 1983. The
13
contaminants of concem (COC) at the site are arsenic (As), cadmium (Cd), lead (Pb), copper (Cu) and zinc (Zn) in a variety of forms and concentrations.
Wildlife Concems in Remediation The EPA's Baseline Environmental Risk Assessment (BERA) for the Anaconda site identified wildlife receptors having the potential for deleterious exposures to the contaminants (CDM, 1997). Risks to wildlife were identified based on models evaluating contaminant uptake from foraging, food chain transfer, drinking water and direct ingestion of contaminated soils (Sheppard et al., 1995; Traas et al., 1996). Modeled species for the site, selected as representatives of feeding and habitat guilds (CDM, 1997), were white-tailed deer {Odocoileus virginianus), deer mouse {Peromyscus maniculatus), American robin {Turdus migratorius), red fox {Vulpes vulpes) and American kestrel. The risk assessment investigations conducted on the Anaconda Smelter Site initiated additional studies of actual health effects endpoints in wildlife inhabiting the impacted areas, including the present study of American kestrels. American kestrels have been used in environmental contaminants studies due to their position at the top of trophic food webs, their ubiquitous occurrence in North America, their willingness to occupy nest boxes, and their tolerance of monitoring and handling procedures (Craft and Craft, 1996). The placement of artificial nest boxes for enhancing natural populations within a study area has been found to be advantageous when investigating localized contaminant zones (Hooper and LaPoint, 1994). Because American kestrels were employed as a sentinel species used in the bioaccumulation
14
modeling in the US EPA's Baseline Ecological Risk Assessment (BERA), and because the BERA predicted notable accumulation of all five COCs, field data on the species were obtained to allow regulators to scmtinize the accuracy of this model.
Primary Research Objectives The objective of this study was to assess COC exposure and effects in nestling American kestrels inhabiting the Anaconda Smelter Superfund Site. Exposure was assessed via maternal input of contaminants into eggs, food item metals in samples collected using esophageal constriction techniques, and metals accumulation in the nestling. Effects assessments focused on biochemical, physiological and morphological effects in the nestling, and reproductive demographics at individual nest box and local population levels. To attain our objective, three main assessments were regarded: 1. Do metal concentrations in prey items or tissues correspond to soil concentrations on the exposure area? 2. Do individual health effects correspond to metal concentrations in tissues? 3. Do reproduction and population demographics correspond to metal concentrations in tissues?
15
CHAPTER n METHODOLOGY
Nest box-based studies of American kestrels were performed during the spring and summer breeding seasons of 1999 and 2000. Exposure and effect assessments were made of nestiings, and reproductive success quantified for each nest. In 1999, a representative nestling was collected from each nest to investigate metal and arsenic (further included in all referrals to 'metals') exposure and effects at the tissue level. Based on the findings of this first year study, the focus of the year 2000 investigation shifted away from nestling collections and focused on blood sample-based exposure and effect assessments. Reproductive demographic information was collected for both study years.
Daily Nest Box Monitoring In the spring of 1999, 50 kestrel nest boxes, and in the spring of 2000, 49 kestrel nest boxes were monitored throughout the Anaconda Smelter site study area. Latitudinal and longitudinal readings were recorded for all boxes (Table 1), and a brief description of all locations was recorded. Lat/long coordinates were converted to the universal transverse mercator (UTM) grid system, and metric distance and direction of all boxes from the smelter smoke stack were calculated. The nest boxes were placed from 2.5km southwest of the stack, to 16km northeast of the stack, throughout areas that represented a gradient of potential contaminant exposure and habitat types (Figure 1). Of the fifty
16
Table 1. American Kestrel Nest Boxes Deployed on the Anaconda Smelter Site.Coordinates, direction and distance from the Smelter smoke stack, and brief descriptions of location. (* - Lat. and Long, coordinates map estimated).
Box
Lat.
Long.
Dist. from Stack (m)
1 2 *3 4 5 *6 *7
46-06.00N 46-05.93N 46-06.87N 46 06.23N 46-06.33N 46-06.33N 46-07.00N 46-06.86N 46-06.99N 46-07.15N 46-07.20N 46-06.84N 46-06.65N 46-06.59N 46.06.17N 46-06.15N 46-05.60N 46-08.08N 46-07.47N 46-08.16N 46-07.84N 46-07.22N 46-08.64N 46.08.64N 46-07.88N 46-07.52N 46-07.01N 46-08.86N 46-09.02N 46-08.ION 46-07.53N
112-56.41W 112-55.13W 112-54.84W 112-54.51W 112-54.5 IW 112-54.12W 112-54.76W 112-55. IIW 112-55.50W 112-55.22W 112-55.4IW 112-53.91W 112-53.30W 112-52.92W 112-53. lOW 112-52.42W 112-52.09W 112-54.94W 112.53.01W 112-53.43W 112-52.87W 112-51.99W 112-53.49W 112-51.87W 112-52.09W 112-51.22W 112-51.19W 112-51.12W 112-50.39W 112-49.74W 112-49.76W
2558 SW 1737 SW 44 NE 1218 SE 1046 SE 994 SE 295 NE 348 NW 890 NW 745 NW 1378 NW 1198 SE 2017 SE 2519 SE 2568 SE 3374 SE 4229 SE 2289 NW 2625 NE 3036 NE 3141 NE 3736 NE 3750 NE 5067 NE 4035 NE 4827 NE 4712 NE 6067 NE 7004 NE 6967 NE 6665 NE
*8 *9 10 *11 12 13 14 15 16 17 18 *19 20 *21 22 23 24 *25 26 27 *28 *29 30 31
Description Telephone pole. SW of Smelter hill. Telephone pole. S of Smelter hiU. High voltage tower. Topof smelter hill. Telephone pole. Geyser gulch. Cottonwood tree. Geyser gulch. Wooden post. Smelter hill aqueduct. Telephone pole. Smelter hill water tower. Cottonwood tree. Walker gulch, W of stack Small pine tree. Slag Gulch. Telephone pole. W of lab facility. Power line pole. W of main slagheap. Metal trellis. SW of Anaconda pond. Power line pole. S of Anaconda pond. Power line pole. W of Mill Creek Road. Telephone pole. Mill Creek town site. Telephone pole. E of Mill Creek town site. Cottonwood tree. SEof Mill Creek town site. Cottonwood tree. SEof Anaconda landfill. Power line pole. HWY 1,N of Anaconda Pond. Telephone pole. E of Galen road. Power line pole, E of HWY 48. Cottonwood tree. SE comer of triangle wastes. Telephone pole. Mile marker 1 of Galen Road. Cottonwood tree. E of airport entrance, Hwy 48. Telephone pole. WofA-cell. Wooden post. SW comer of A-ceU. Power line pole. NW of Country Club road. Telephone pole. HWY 48, NW of Opp. Ponds Cottonwood tree. Nbermof C-ceU. Dead snag. Middle of C Cell. A-frame stmcture. S berm between B & C-cells.
17
Table 1. (Continued) Box
Lat.
Long.
32 33 *34 35 36 37 *38 39 40 *41 *42 43 44 45 46 47 48 49 50
46-07.48N 46-07.92N 46-08.73N 46.09.39N 46-09.5 IN 46-09.24N 46-09.56N 46-08.83N 46-08.53N 46-08.33N 46-07.84N 46-07.06N 46-06.46N 46-05.25N 46-lO.OlN 46-10.31N 46-10.71N 46-12.41N 46-12.69N
112-48.66W 112-48.66W 112-48.84W 112-49.65W 112-48.99W 112-48.38W 112-47.87W 112-48.19W 112-48.08W 112-47.95W 112-48.06W 112-47.94W 112-47.99W 112^7.93W 112-48.08W 112-48.3 IW 112^8.15W 112-45.53W 112-45.63W
Dist. from Stack (m) 8047 NE 8205 NE 8478 NE 8177 NE 9005 NE 9427 NE 10286 NE 9319 NE 9248 NE 9286 NE 8925 NE 8897 NE 8854 NE 9382 NE 10493 NE 10576 NE 11198 NE 15805 NE 16052 NE
Description 1Cottonwood tree. SE comer of C-cell. 1Cottonwood tree. E slope of C-cell. :Decant tower. D-cell. 1Cottonwood tree. NW of D-cell. (Cottonwood tree. N of D-cell. 'Telephone pole. E of D-cell. 'Telephone pole. E of Box 37. 'Telephone pole. E of D-cell. 'Telephone pole. E of D-cell. 'Telephone pole. W of Pond entrance from 1-90. 'Telephone pole. E of D-cell. ]Power line pole. S entrance to Warm Springs WMA. ]Power line pole. Road from Opportunity, W of 1-90. ]Power line pole. W of 1-90, S of HWY 1. (Cottonwood tree. SW of HWY 48, W of 1-90. (Cottonwood tree. SW comer behind State Hospital. 'Telephone pole. W of State Hospital. ' Telephone pole. S of Lampert Ranch. ^Wooden post. Lampert Ranch.
18
19
boxes, 70% (35/50) were placed on man-made structures (telephone poles, power poles, metal stmctures, etc.), with the remainder affixed to selected trees. Beginning in May, boxes were checked every two to six days for nesting activity by kesfrels or other species. Activity was measured on a four-point scale indicative of no avian nesting activity (0) to a full nesting attempt (3). For comparative analysis purposes, all nest boxes were combined into two groupings based on their proximity to Smelter Hill: Boxes 01 through 27 were grouped as 'Smelter Hill' sites, while boxes 28 through 50 were grouped as 'Opportunity Ponds' sites (Figure 2). The Smelter Hill site was delineated based upon areas of greatest soil COC concentrations according to EPA soil data (CDM, 1997), and by a visual determination, from aerial maps, of the area of greatest plant sfress surrounding the main smelter facility. In the 1999 field season, the first egg of each active nest was marked with a pencil for identification, and the nest was allowed to remain undisturbed for six days. Upon reinspection, the marked egg was removed (in 1999, one entire clutch was removed from one box to examine intra-clutch variability), wrapped in clean Kimwipes®, and placed in a clean vial. Eggs were then weighed, and length and width measured in triplicate. An incision along the egg's air cell allowed for removal of contents. Egg contents were placed into a new I-Chem® metals-certified-clean vial and immediately frozen at -20°C for metal analysis. In the 2000 field season, nest boxes that showed activity were monitored every three days to check for initiation of clutch. Upon the first egg being laid, the box was left unchecked for four to six days, to allow for undisturbed completion of the clutch.
20
21
Addled Egg Collection The lack of either parent in the vicinity and/or the clutch cold to touch, for two consecutive monitoring sessions identified nests as "abandoned." The clutch was collected, and placed in certified clean vials. Any eggs remaining in the nest five days post-hatching were deemed "unhatched," and collected in the same manner. Eggs were then weighed, and length and width measured in triplicate. An incision along the egg's air cell allowed for removal of contents for determination of viability, or aging of embryo at death. Contents were placed into a new certified vial and immediately frozen at -20°C for metal analysis. Egg shells were retained for future assessments as needed.
Nestiing Monitoring and Measurement Clutches were monitored approximately every three days until hatch (roughly 30 days). Kestrels delay incubation until the clutch is near completion; therefore, all eggs typically hatch within one day of each other (Bird, 1988). Clutch age (post-hatch) was recorded using the hatch date of the last chick. Nestlings were monitored every five days through day 25 post-hatch. Body weight was taken with a Pesola® scale and recorded to the nearest gram. Measurements were taken of bill depth and width, as well as tarsus and third toe length. Nestlings were visually inspected for any obvious signs of ectoparasites, general health status, or other notable observations. Nestlings found dead in the nest were retrieved and, based on tissues available, analyzed for metals.
22
Esophageal Constriction Contaminant exposure and food item preferences were determined by collection of prey items. Nestlings were fitted temporarily with esophageal constriction devices (Hoff, 1992; Mellot and Woods, 1993), preventing food items from entering the crop and allowing for easy removal. The devices were left in place for up to two hours depending on the time required for the adults to make a feeding foray back to the nest. Adult activity at the nest boxes was monitored from at least 50m away, to prevent interference of normal adult behavior. All food items obtained from the collecting session, including items in the nest prior to constrictor fitting, nestling crop contents and those found in the nest when the session ended, were stored frozen in certified clean vials at -20°C. All samples from nest mates collected on a single date were pooled in the same vial, to ensure adequate mass for analysis.
Nestling Sampling In the 1999 field season, a representative nestling from each nest was retrieved at day 25 post-hatch, euthanized, dissected, and tissues collected. In the 2000 field season, only non-lethal blood samples were collected using venipuncture techniques on all chicks in each box. At day 25 post-hatch, a randomly selected nestling was removed for tissue collection (an entire clutch was removed from one nest box (Box 19) to examine intraclutch variation in study endpoints). Each nestling was euthanized by CO2 asphyxiation, and a blood sample was collected via cardiac puncture. An aliquot of the sample was
23
stored in a certified clean vial for metal analysis, with the remainder stored appropriately for hematological and biochemical analyses. Red blood cell (RBC) and white blood cell (WBC) counts of each individual were determined with the use of a Hausser Scientific® hemacytometer. White blood cells were quantified by counting the number of cells in the four outer squares of the hemacytometer grid. The cell concentration was calculated as foUows: Cell count/ml = Total cell count * 2500 * dilution factor Red blood cells were quantified by counting the number of cells in five squares in the large middle square of the grid. Cell concentration was calculated as follows: Cell count/ml = Total cell count * 50,000 * dilution factor A minimum of 27 white cells and 146 red cells, per count, were needed for reliability in cell counts. The lower half of the large, primary lobe of the liver was removed and placed into a certified vial, weighed, and frozen for metal analysis. The remaining upper section, and the entire secondary lobe of the liver was removed, weighed and separated into two aliquots. These remaining tissues were wrapped in hexane-rinsed aluminum foil and frozen at -80°C for biochemical analysis. The right portion of the kidney was removed for metals analysis. The left portion was divided into upper and lower portions and maintained separately at -80°C for biochemical analysis. Small representative portions of the following tissues were fixed in phosphate buffered formalin for histopathology analysis (not reported in this thesis): liver, kidney.
24
brain, eye, heart, brachial plexus, cecae, stomach, intestine, femoral muscle, femoral bone, spleen, bursa, thyroid, thymus, lung, intestine, pancreas, adrenal and gonad. Fecal/urate samples were collected from each individual by pinching directly behind the cloacal opening during dissection. All samples were collected and maintained in acid-cleaned glass vials. Several adult kestrel pellets were collected intermittentiy from within nest boxes prior to and during nest initiation. Many pellets were collected from nest boxes that subsequentiy resulted in no nest attempt. All samples were collected and maintained at -20°C in sealed plastic bags.
Blood Sample Collection Blood sample collections in 2000 were attempted with every nestling at day 10 and day 25 (post-hatch). Unsuccessful collections from day 10 were re-attempted at day 17. Blood samples were collected non-lethally via jugular veni-puncture. The nestlings were weighed and measured just prior to sample collection. Individuals were then held ventrally, allowing the thumb and forefinger to expose the jugular between the feather tracts of the neck. The skin was cleaned with alcohol swabs, and a heparinized Ice syringe with a 27-gauge needle was used to withdraw the blood sample. Samples collected never exceeded 1% of body weight, so as to reduce any detrimental health effects (McGuill and Rowan, 1989). The samples were separated into aliquots of approximately 150-200 ju.1 for use in ALAD activity determinations, with the remaining sample collected in certified clean vials for metals analysis. Samples were packed in ice.
25
and immediately retumed to lab facilities where they were stored appropriately. Packed cell volumes (PCVs) of the samples were determined using 100 jLil hematocrit capillary tubes and a hematocrit centiifuge. Remaining blood for ALAD analysis was frozen at -80°C.
Sample Digestion and Metal Analysis Sample Digestion Sample preparation procedures were slight modifications of published methods (Adair et al., 1999; Cobb et al., 2000a). All digestion procedures were validated with standard reference materials (SRMs) before sample preparation began (not reported in this thesis). Samples were prepared in batches of no more than 40 analytical samples, along with Quality Assurance (QA) samples including one blank, two standard reference materials, and two spiked blanks. All QA materials were analyzed in the same manner as field collected samples. Under fume hood, 5ml Optima® grade nitric acid was added to each sample, and allowed to pre-digest for at least an hour (50ml acid was used for food item carcass digestion to ensure complete digestion, and allowed to pre-digest over night). Each digest was covered with a watch glass, and placed on hot plates adjusted to approximately 120°C. The contents were swirled regularly in order to allow acid to react completely with the sample. Upon complete digestion, watch glasses were removed and the nitric acid was evaporated to approximately 1-2 ml for organ tissues and 10-25 ml for carcasses. Samples were removed from heat and allowed to cool to room temperature at
26
which time 1.5 ml of 30% hydrogen peroxide was added. After initial reaction, the samples were covered and replaced on the hot plates until fully reacted with the peroxide. The digests were removed from the hot plate and allowed to cool again to room temperature. Samples were transferred to appropriately sized volumetric flasks (10, 25, 50 ml) depending on final volume of sample remaining. Those samples with excessive sediment, lipid, or other indigestible matter were filtered using glass fiber filters. Volumetric flasks were diluted to final volume using MilliQ quality water. Diluted digests were transferred to 15 ml or 50 ml Falcon® centrifuge tubes and kept undisturbed until instmmental analysis.
Inductively Coupled Plasma-Atomic Emission (ICP-AE) Analysis In 1999, all samples were analyzed to determine the concentration of five COCs: arsenic (As), cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn). Copper and Zn were analyzed using the ICP in 2000. Instmmental analysis was conducted with a Leeman® DRE Inductively Coupled Plasma Spectrophotometer with auto-sampler. All data were captured by Leeman® instmment control software. Spectral emissions for As (197.198 nm), Cd (214.438 nm), Cu (324.754 nm), Pb (220.353 nm), and Zn (213.856 nm) were detected and corrected for background interference. Five point calibration curves were developed for each analyte to provide quantitative instmmental analyses using traceable standards for all standard preparations. Complete calibrations were performed daily, and calibration checks were performed after analysis of every 10 digested samples. Intermittent checks were made to ensure that deviations from the full calibration curve
27
were less than fifteen percent. Two standard reference materials, two spiked tissue samples, and a method blank were analyzed with every batch of 40 samples or less.
Graphite Fumace-Atomic Absorption (GF-AA) Analysis Graphite Fumace Atomic Absorption (GEAA) was used to obtain more sensitive results for As, Cd, and Pb in samples collected during the 2000 field season. GFAA analyses were performed with a Perkin Elmer® AAnalyst 600 spectrophotometer with auto sampler. All data were captured by Perkin Elmer® AAWinLab (version 3.71) instmment control software. Each specfral absorbance for As, Cd and Pb was corrected for background interference. Five-point calibration curves were developed for each analyte to provide quantitative instmmental analyses using traceable standards for all standard preparations. Complete calibrations were performed daily, and calibration checks were performed after analysis of every 10 digested samples. Intermittent checks were made to ensure that deviations from the full calibration curve were less than fifteen percent. Two standard reference materials, two spiked tissue samples, and a method blank were analyzed along with every batch of 40 samples or less.
Instmment Detection Limits (IDL) The IDL values on the ICP were as follows: As (0.0679 ^g/ml), Cd (0.00502 /zg/ml), Cu (0.0103 Mg/ml), Pb (0.0308 fig/ml), and Zn (0.0156 iig/ml). The IDL values on the GFAA were the lowest standard concentrations, which were 0.005 M.g/ml for As and Pb and 0.0000lp.g/ml for Cd. The DDL for each element was defined as the mean
28
blank response -i- 8 times the standard deviation of the blank signal. The standard deviation was added to represent potential instmment noise. Analysis performed in 1999 using inductively coupled plasma (ICP) analysis resulted in "below detection level" findings in some tissues, particularly with the As, Cd and Pb determinations. To improve resolution, year 2000 analyses employed graphite fumace atomic absorption, dropping detection levels substantially and placing greater confidence in a finding of "below detection level." For statistical analysis, "Reporting Limits" of one-half the detection limits were included (Table 2) for all samples found below the detection limit for that analyte. All analytical data were expressed as jxg analyte/gram tissue, wet weight.
Effects Assessment Methodology Porphyrin Extraction All tissue samples were wrapped in aluminum foil, and maintained at -80°C until immediately prior to analysis. Tissue porphyrins were extracted using a modification of the extraction procedure of Kennedy and James (1993). Briefly, measured tissue was combined with a 1:1 mixture (6 ml) of IN HCl: acetonitrile. Samples were homogenized, then centrifuged at 3000-x g (15 minutes on a Beckman® AUegra 6R centrifuge, FuUerton, CA), and the resultant supernatant saved. The pellet was re-homogenized with 6 ml of fresh 1:1 HCl: acetonitrile and centrifuged as before. The supematants were combined and diluted with approximately 70 mL HPLC grade water. The sample was concentrated using a Waters® C-18 Sep-Pak solid phase extraction cartridge, eluted with acetonitrile (10 mL), placed in a 35°C water bath, and evaporated to complete dryness
29
Table 2. Detection and Reporting Limits for Metals Data in Collected Samples. Detection limits were based on analytical capabilities and mean sample weight for each sample type. Reporting limits were established as half the detection limits. Year /
Average Sample Mass
As
5.666 1.306 12.938 1.586 0.881 0.684 0.415
0.120 0.038 0.052 0.428 0.771 0.993 0120
0.009 0002 0.004 0.032 0.057 0.073 0.006
0.054 0.038 0.024 0.194 0.350 0.451 0.120
0.018 0.097 0.008 0.065 0116 0150 0.241
0.028 0.119 0.012 O098 0177 0.228 0.376
Egg Blood Liver Kidney Fecal/Urate
5.666 1.306 12.938 1.586 0.881 0.684 0.415
0.060 0019 0.026 0.214 0.385 0496 O060
0.004 0.001 O002 0016 0.028 0.037 0.003
0.027 0.019 0.012 0.097 0.175 0.225 0.060
0.009 0.038 0.004 0.032 O058 0.075 O120
O014 0.060 0.006 0.049 O089 0114 0188
Detection Limits 2000 Blood Food
0.479 5.552
0104 O023
0.005 OOOl
0.104 0.023
0.214 0.046
0326 0.070
Reporting Limits 2000 Blood Food
0.479 5.552
0.052 0.011
0.003 OOOl
0.052 OOll
0.107 0.023
0163 0.035
Sample Type Detection Limits 1999 Food Pellet Egg Blood Liver Kidney Fecal/Urate Reporting Limits 1999 Food Pellet
30
Detection Limits (Mg/g) Cd Pb Cu
Zn
with nitrogen gas. The dried porphyrin samples were reconstituted with IN HCl (500 txL), filtered (0.45 |.im), and stored at approximately 4°C until and during analysis.
HPLC and Equipment Porphyrin analysis was performed using reverse-phase HPLC (Woods et al., 1993). The 8-, 7-, 6-, 5-, 4-, and 2- carboxylporphyrins were separated using a Waters® 6980 separations unit with an Alltech® Econosphere-C18 5iu,m HPLC column, and equipped with a fluorescence detector (Waters model 474) at excitation/emission wavelengths of 390/614nm. HPLC grade methanol and a sodium phosphate buffer (0.5 M, pH 3.5) were used as mobile phases. A 30-minute mobile phase gradient established by Rummel (Rummel, 2000), characterized for separation of porphyrin peaks, was used during each injection (100 |J,L) of sample. Peaks were quantified using a Waters® Millennium32 chromatograph control software package retuming a calculated amount of each porphyrin in pmol/100 [iL of sample. Dilutions of porphyrin standards (Porphyrin Products®) were used to establish a standard curve prior to each sample batch.
Porphyrin Recovery/Detection Limits Critical values were established for HPLC analysis of porphyrin profiles. Recovery was established utiUzing homogenized chicken liver apportioned into 0.3 g aliquots and frozen at -20°C. One reference aliquot and one reference aliquot spiked with 100 \xl of porphyrin standard were analyzed with each sample run. Eleven aliquots were spiked with the 2-pmol/lOO \i\ standard (resulting in 0.4 pmol/injection), which
31
resulted in a 47.03% recovery rate. However, seven samples were spiked with the 5 pmol/lOOp-l standard (1.0 pmol/injection), resulting in a mean 97.73% recovery rate, illustrating an adequate recovery at pertinent sample concentrations. Instmment baseUne was established using blank injections (IN HCl) from representative sample mns. Each blank injection was quantified using the calibration curve established for each specific mn. Areas of baseline response were assessed at the same retention times of all six porphyrin peaks, and the six responses were averaged to establish a mean 'Baseline' area for the mn. A value four times the area of 'Baseline' was chosen as the Instmment Detection Limit (IDL). IDL values were compared with areas quantified for each peak established for the lowest, 0.4 pmol standard. Mean area values for these standards were generally twice the DDL value, ensuring detection of peaks quantified at 0.4 pmol. A Practical Detection Limit (PDL) was established at 0.4 pmol / injection, as this concentration was the lowest standard used in each calibration curve. Any values quantified below this limit were designated Below Detection Limits (BDL), and assigned a value of 0.2 pmol/injection (half the detection limit) for quantification purposes.
5-Aminolevulinic Acid Dehydratase (ALAD) Extraction Blood samples collected for ALAD determination were maintained at -80°C until just before extraction. An ahquot of 175 p.] of blood was added to 5035-pl ultra-pure (MilliQ) water and placed in a covered ice bath for 10 minutes, allowing the red blood
32
cells (RBC) to lyse. The sample was separated into three separate aliquots of 740 fi\ of lysate. Mixtures were allowed to equilibrate for 5 minutes in a 38°C water bath.
5-Aminolevulinic Acid Dehydratase (ALAD) Activity Determination The assay was started by the addition of 500-pl of 6-aminolevulinic acid (ALA; 100 mM) in sodium phosphate buffer, pH 6.4, and incubated in the 38°C water bath for one hour, at the end of which, enzyme activity was halted by the addition of 500 [il of 612mM trichloroacetic acid (TCA) solution. Precipitated proteins were separated by centrifugation (2000 rpm for 10 minutes), and 100 jU.1 aliquots of the supernatant solution were pipetted into the appropriate weUs of a 96-well microtiter plate. One hundred ptl of Ehrlich's indicator reagent was then added to each well, covered and immediately analyzed. Color production was assessed using a Molecular Devices® SPECTROMax Plus 96-well spectrophotometric plate reader controlled by Molecular Devices® SOFTmax Pro. Absorbance was read every minute for 10 minutes, at 555 nm (kinetic mode). Enzyme activity was calculated using the following equation: Activity = (11,580 * Max. Absorbancesss) / Hematocrit Where 11,580 is a conversion factor based on the molar extinction coefficient specific to the porphobilinogen / p-dimethylaminobenzaldehyde complex (62,000) and stoichiometric calculations and dilution factors. Max. Absorbance, measured in absorbance units, is the output of the spectrophotometer and Hematocrit is expressed as a percent. ALAD activity units were expressed as: nmoles ALA * min" * ml RBC" .
33
Data Analysis Data generated from the field and laboratory portions of the study were recorded on activity-specific data collection forms, subjected to daily intemal QA verification and later transferred to spreadsheets or directly to a Microsoft® Access database, where they were verified by a formal QA audit.
Summary Statistics Demographic and growth data were evaluated to literature-based benchmark data for uncontaminated, confrol or reference animals in the same or similar species to determine how the nesting statistics discovered compare to what might be considered normal. Exposure and effects data were compared within the study based on their location in relation to the Smelter Hill area and the more distant Opportunity Ponds area. Metal concentrations, biochemical endpoints, and morphological measurements were expressed on a per clutch or per site basis as means ± standard deviation. All significance tests were performed using Minitab® (version 13.31) statistical package or SigmaStat® (version 2.0) statistical package, and an alpha value (a) of 0.05 was established as our critical probability value for significance. Associations between metal concentrations in food items and tissues and between metal levels and effects endpoints were examined using a Pearson product moment correlation analysis. Further comparisons of tissue metal concentrations, biochemical endpoints, and morphological measurements between sites were examined using a Student's t-test. If data normality testinc' failed, non-parametric data sets were examined using a Mann-Whitney rank-sum
34
test for comparison between sites. A two-way ANOVA was mn to evaluate differences in age-wise blood metal accumulation between sites (2000 only), and a Tukey HSD posthoc test used to examine all pairwise comparisons among means. Linear regression equations were calculated to further examine relationships between multiple tissue metals concentrations, as well as between tissue metals and effects endpoints. Finally, nestiing growth curves were estimated in SigmaPlot® 2000, using a nonlinear 4-parameter logistic curve estimation: y = yo + a/l+(x/xo)'' Difference between sites was calculated by adding the sums of squares and degrees of freedom for both curves (Separate), and comparing those to the equation sums of squares and degrees of freedom of all nestlings grouped together (Combined). An F value was calculated using the equation:
F=
\^^c0KUTied
^'^iepo'alej'y^^coKUKed "'^caxUxed '
^^iepis'ii^]
^^lepwnH
Exposure Estimation In assessing risks to wildlife, exposure estimates are calculated based on a potential dose of contaminant to the individual. The U.S. Environmental Protection Agency (EPA) uses specific guidelines to quantify risk, utilizing several equations to calculate dose exposure estimations. A preliminary assessment of daily dietary exposure of the five COCs to 25 day-old kestrels (mean body weight - 120g) inhabiting the Superfund site was calculated, based on assumptions and calculations for adult American
35
kestrels from the EPA's Wildlife Exposure Factors handbook. Contaminant concentrations of collected food items were back calculated against their mass, to determine total COC /xg amounts. Food items from both years were separated by site and by food type (rodent, invertebrate, avian), and a mean sample weight and mean total jxg value was determined for each food type. A combined mean COC /i.g/g concentration, as well as individual food type )Ltg/g means was established. Taking the percent occurrence of each type in all collected samples produced an estimated proportion of food items ingested. Over both years, rodent items were collected in 66% of all samples, whereas invertebrates were found in 23% and avian tissue in 12%. The average concentrations for each prey type, multiplied by its percent occurrence, were added together for a proportioned mean prey concentration. Using a food ingestion rate calculation of 0.30 g/g body weight- a day (EPA, 1993), 25 day-old kestrels have an estimated daily intake of 36 g of food (proportioned to an average of 24 g rodent, 8 g invertebrate and 4 g avian tissue a day). Using this food intake estimate, a daily contaminant intake may be calculated to express a total /xg COC intake through food items per day. To compare the intake levels to reference toxicity values associated with adverse effects, an estimated average daily dose {fig contaminant/g nestiing body weight - day) is established based on the mean mass of the birds. Subsequently, we then are able to compare our estimated dose to detected tissue concentrations, as well as to associated health effect endpoints.
36
CHAPTER m RESULTS
Nest Box Placement and Use 1999 Field Season Between May 12 and June 21, kestrels initiated nesting attempts in 19 boxes, with 16 (84%) of these located in boxes found on man-made stmctures. Kestrels occupied 38% (19/50) of available boxes, as indicated by the presence of at least one egg. Preincubation removal of an entire clutch of eggs left 18 nests with success potential. Successful nests, with at least one live fledging age chick, occurred in 14 of the 18 initiated nests (78%; Table 3). Kestrel activity was well dispersed, resulting in a good distribution of nesting throughout the entire study area. Of the active nests, 47% (boxes 1, 2, 7, 12, 13, 14, 19, 20, and 27) were classified as Smelter HiU associated boxes while 53% (boxes 28, 30, 32, 35, 39,40,43, 44,45,49) were considered Opportunity Ponds associated boxes. Collections in 1999 included 26 food items, 27 egg, 17 blood and 17 nestling samples. One egg and 1 nestling were collected from each nest box. One entire clutch of eggs (box 40) and an entire nest of 25-day nestiings (box 19) were collected to evaluate intra-clutch variability of measured endpoints. Two nest mortalities were also collected.
37
Table 3. Egg and Nestiing Data for Occupied Nest Boxes, 1999. Egg and nestiing success or subsequent fate is tabulated, by nest box. Eggs
Survived to Fledging Age
Removed For Analysis
Actually Fledged
1 0 1 0 1 1 1 0 1
0 0 0 1 0 0 0 0 0
0 2 0 1 0 0 0 1 0
0 0 0 0 0 1 0 0 0
3 0 3 0 4 2 4 0 4
3 0 3 0 4 2 4 0 4
1 0 1 0 1 1 4 0 1
2 0 2 0 3 1 0 0 3
K28 K30 K 32*** K35 K39 K40** K43 K44 K45 K49
529 529 603 529 529 529 610 617 512 526
4 5 1 4 5 4 4 4 5 4
1 1 0 1 1 4 0 1 1 1
0 0 0 0 0 0 0 0 1 0
0 0 1 0 0 0 0 0 0 0
0 0 0 0 2 0 0 0 1 1
3 4 0 3 2 0 4 3 2 2
2 4 0 3 2 0 4 3 1 2
1 1 0 1 1 0 1 1 1 1
1 3 0 2 1 0 3 2 0 1
Totals
72
17
2
5
5
43
41
17
24
38
Unhatched
Missing
'" All nestUngs collected for analysis ^* Clutch of eggs collected for analysis ^** Unsuccessful nesting attempt
Number Hatched
4 2 4 2 5 4 5 1 5
Abandoned
Removed For Analysis
602 621 527 531 518 602 523 601 526
Nest Initiation
KOI K 02*** K07 K12*** K13 K14 K19* K 20*** K27
Box No.
No. Laid
Nestlings
2000 Field Season Between May 18 and June 12, fourteen clutches (29% occupancy) were initiated in 49 (one box was removed due to constmction work) potential nest boxes. Of the 14 clutches, 9 (64.3%) were initiated in boxes located on man-made stmctures. Nine clutches successfully hatched at least one nestiing (9/14 or 64%; Table 4). Of active nests, 43% (boxes 1, 4, 17, 23, 24, and 26) were classified as Smelter Hill associated boxes while 57% (boxes 32, 34, 35, 37, 41, 43,46 and 50) were considered Opportunity Ponds associated boxes.
Samples collected in 2000 included 18 food items and 56
blood samples. Unhatched and abandoned eggs (6 and 13, respectively) were collected as in 1999.
Exposure Assessment Food Item Metal Analysis In 1999, 26 samples of nestling food items were collected through the use of esophageal constriction devices, or collected from within the nest box where deposited by an adult (Table 5). Seven samples (27%) contained invertebrate items (grasshopper and dragonfly), while two samples (8%) contained avian tissue (unidentified passerine). Nineteen samples (73%) included mammaUan tissue (vole, deer mouse, etc.). Detectable arsenic concentrations were found in all but two of the samples, with values ranging from below the level of detection ( .
X
o JQ
10-
to O
0 0.3
0.4
0.5
0.6
0.7
0.8
0.9
Lead Cpncentratipn (ug/g)
Figure 3. Hepatic Porphyrin (4-CP) as a Function of Log Lead Concentration. Thirty three percent of the variability of the hepatic 4-CP could be explained by the concentration of total lead in the liver (non-detectable lead and 4-CP outlier removed). Porphyrin responses show a distinct increase in response to increased hepatic lead concentration.
65
Table 17. Renal Porphyrin Profiles, 1999. N is total sample number; n is number with detectable levels. ND is not detected Carboxyl Porphyrins (pmol/g) 2 Total
Box No.
Chick
4
K19 K19 K19 K19
A B C D
20384 23.894 23.452 25.064
ND 10.377 ND 14.328
30.375 43.514 32.652 39.392
K19 Intra-Clutch
N=4
23.199 1.996 4
12.353 2.793 2
36.483 6.052 4
KOI K07 K13 K14 K19 K27
A C C B Mean B
31.434 26.464 28.353 20.560 23.199 21.153
8.895 7.907 7.870 8.768 12.353 8.930
67.727 43.663 44.761 38.277 36.483 30.082
K28 K30 K35 K39 K43 K44 K45 K49
C D C A A C B B
22.600 29.630 21.317 22.091 64.916 26.962 25.316 21.885
ND 10.594 9.786 13.702 18.078 7.738 12.278 6.606
28.757 40.224 61.256 63.166 92.847 42.689 60.040 37.453
All Sites
N=14
Mean SD n
27.563 11.289 14
10.269 3.143 13
49.102 17.720 14
Smelter Hill Sites
N=6
Mean SD n
25.194 4.297 6
9.120 1.655 6
43.499 13.001 6
Opportunity Pond Sites
N=8
Mean SD n
29.340 14.665 8
11.254 3.877 7
53.304 20.379 8
Mean SD n
66
Table 18. ALAD Activities and Packed Cell Volumes, 2000. Assessed on a box mean basis by age. Values were associated by site. ALAD activity was generally higher in younger nestiings, when compared to Day 25 activity. Age (Post-hatch) Day 10 Smelter Hill Associated Sites
Kestrel Box No.
PCV (%)
ALAD (nmol/min* RBC)
201 217 Mean SD n
36.5 34.7 35.6 1.3 2
135.25 114.40 124.83 14.74 2
Opp. PondsAssociated Sites
234 235 246 250 Mean SD n
36.0 40.0 35.8 32.0 35.9 3.3 4
119.19 133.36 149.33 129.51 132.85 12.51 4
201 217 224 Mean SD n
35.0 37.0 34.5 35.5 1.3 3
119.63 123.30 102.09 115.01 11.34 3
243 Mean n
36.2 36.2 1
119.17 119.17 1
201 217 224 Mean SD n
39.5 34.0 40.5 38.0 3.5 3
96.84 89.60 85.23 90.55 5.86 3
234 235 243 246 250 Mean SD n
34.5 40.5 34.8 36.0 36.0 36.4 2.4 5
134.30 113.04 120.66 131.12 109.33 121.69 10.91 5
Day 17 Smelter Hill Associated Sites
Opp. PondsAssociated Sites Day 25 Smelter Hill Associated Sites
Opp. PondsAssociated Sites
67
between sites, as activity on Smelter Hill sites averaged 90.6 ± 5.9, while the Opportunity Ponds box mean was 121.7 ± 10.9 (p = 0.014). The mean ALAD activity from Smelter Hill nestiings was 74.5% of the mean activity level found in Opportunity Ponds nestiings.
Hematology Counts of both white and red cells were conducted from blood samples collected from all dissected individuals. Two separate cell counts were conducted for leucocytes, and a mean cell count was used to calculate white cells per ml of whole blood. Intraclutch variability (Box 19; Table 8) was remarkably small, with mean WBC count of 3115 ± 331.2 and RBC count of 5.421 x 10^ ± 0.400 x 10^ cells/ml (CV of 11 and 7%, respectively; Table 19). Wide variation between boxes was noted however, as the WBC count for all sites averaged 6989 ± 5479 cells/ml. The elevated variability is predominantiy due to a single individual (Chick A of Box 43), whose WBC count of 23,594 cells/ml was twice the next highest value. This same individual, conversely, was found to have the lowest RBC count (2.04 x 10^ cells/ml), strongly depressed as compared to the site mean of 6.19 x 10^ ± 1.51 x 10^ cells/ml. Nevertheless, no significant hematological differences were noted between Smelter Hill and Opportunity Ponds sites.
Tissue Weights Tissue weights of five organ types were analyzed from nestiings collected in 1999 (Table 20). There were no significant tissue weight differences between Smelter Hill
68
Table 19. Nestiing Hematology, 1999. Red and white blood cell counts were performed on samples collected from each retrieved nestiing using a hemacytometer. Box No. K K K K
19 19 19 19
Chick
Hemacytometer count (cells/ ul) WBC RBC (10")
A B C D
BoxK19 Intra-clutch
Mean SD n
2679 3445 3278 3056
5.09 5.33 6.00 5.27
3115 331.2 4
5.42 0.40 4
KOI K07 K 13 K 14 K 19 K27
A C C B Mean B
8750 3657 4625 6313 3115 3722
7.91 4.51 7.31 7.78 5.42 6.21
K28 K30 K35 K39 K43 K44 K45 K49
C D C A A C B B
8500 5473 4938 5445 23594 12688 2889 4139
6.89 5.57 6.38 5.77 2.04 6.58 5.71 7.18
All Sites
Mean SD n
6989 5479 14
6.19 1.51 14
Smelter Hill Sites
Mean SD n
5030 2041 6
6.52 1.38 6
Opportunity Pond Sites
M can SD n
8458 6835 8
5.77 1.61 8
69
Table 20. Tissue Weights, 1999. Tissues were weighed at time of collection. N is the total number of chicks measured while n is the number positive for the endpoint. NC is not collected. Tissue weight (g) Liver Brain Spleen
Box No.
Chick
Kidney
K19 K19 K19 K19
A B C D
1.37 NC 1.52 1.25
3.80 3.93 3.68 3.92
2.11 2.12 2.23 2.39
0.087 0.138 0.151 0.128
0.226 0.402 0.387 0.291
Box K 19 Litra-Clutch
N=4
1.38 0.138 3
3.83 0.119 4
2.21 0.133 4
0.126 0.028 4
0.327 0.083 4
KOI K07 K13 K14 K19 K27
A C C B Mean B
1.25 NC 1.80 1.42 1.38 1.14
4.42 5.07 4.47 3.50 3.83 3.22
2.36 2.33 2.52 2.20 2.21 2.26
0.303 0.362 0.192 0196 0126 0133
0.453 0.459 0.387 0.346 0.327 0.422
K28 K30 K35 K39 K43 K44 K45 K49
C D C A A C B B
1.69 1.38 1.38 1.20 1.67 1.79 1.55 1.54
5.09 4.40 3.82 4.33 4.76 4.99 5.32 3.78
2.29 2.31 2.28 2.46 2.26 2.32 1.92 2.43
0.272 0.183 O150 O160 0.724 0461 0.265 0.164
0.228 0.349 0.393 0.401 0.286 0.573 NC 0.401
All Sites
N=14
Mean SD n
1.48 0.217 13
4.36 0.650 14
2.30 0.141 14
0.264 0.164 14
0.386 0.086 13
Smelter Hill Sites
N=6
Mean SD n
1.40 0.248 5
4.08 0.691 6
2.31 0.118 6
0.219 0.095 6
0.399 0.055 6
Opportunity Pond Sites
N=8
Mean SD n
1.53 0.195 8
4.56 0.577 8
2.29 0.164 8
0.297 0.201 8
0.376 0109 7
Mean SD n
70
Bursa
and Opportunity Ponds-associated nestlings. Individual kidney, liver, brain, spleen and bursa weights were assessed, yielding only one significant outiier in the spleen weight category (chick A of box 43) whose 0.724 g spleen weight was approximately three times greater than the population mean. Notably, this was the same individual identified previously (Chick A of Box 43) with the highest WBC counts and low RBC count. All other tissue weights for this individual were well within the normal range.
Effects Assessment - Reproductive Demographics Nesting and Hatching Success In 1999, 72 eggs were laid among the 19 initiated nests (Table 21). Removal of 17 unincubated eggs for analysis (4 from box 40 and one each from 13 additional boxes) left 55 eggs available for hatching. Two eggs were identified as missing during incubation monitoring, although no indication of nest parasitism or predation was found at any time at any box. Accounting for sampling and unknown causes of egg removal left 53 eggs by which hatching and nesting statistics were based. Four nests, with a total of 5 eggs, were identified as abandoned, three of these occurring soon after a late-season cold weather event in early June (boxes 12, 20 and 32), the other being the last clutch initiated in the summer (box 2). Another five eggs from four individual successful boxes remained unhatched and were collected five days after the remainder of their associated clutches hatched. When processed, three of the eggs appeared to be cracked slightly, allowing the contents to dry completely. No evidence of development was found in these eggs, while one egg (box 45) contained a fetus estimated
71
Table 2 1 . Nesting and Demographic Parameters, 1999 and 2000. Focus of Statistic
Nest Box Statistics
Statistic
Nest boxes available Initiated kestrel clutches Percent occupancy
1999
2000
50
49
19 38.0% (19/50)
14 28.6% (14/49)
Egg Statistics Total eggs laid Number of eggs/initiated clutch (mean ± s.d.) Eggs removed for chemical analysis Eggs missing from nest during incubation Eggs reaching hatch age Eggs abandoned Eggs un-hatched Eggs hatched Hatching success Successful nests (nests with live nestiing) Initiated clutches (corrected for removed clutch) Nesting success (successful nests/clutches initiated)
72 58 3.79 ± 1.32 4.14 ±0.66 N=19 N=14 17 0 2 5 53 53 5 13 5 6 43 34 81.1% 64.2% (43/53) (34/53) 14 9 18 14 77.8% 64.3% (14/18) (9/14)
Nestling Statistics Nestlings Missing/dead nestiings Hedglings (survival to 25 days post hatch) Fledging efficiency (fledglings/nestlings) Nestlings collected for analysis Actual number of fledged nestiings
43 2 41 95.3% (41/43) 17 24
34 2 32 94.1% (32/34) 0 32
General Statistics Number of eggs/successful nest (mean ± s.d.) Number of chicks/successful nest (mean ± s.d.) Nesting efficiency (fledglings/eggs at hatch)
72
4.43 + 051 3.78 + 0.83 N=14 N=9 3.07 ± 0 8 3 3.78 ±0.83 N=14 N=9 77.4% 60.4% (41/53) (32/53)
to have been approximately 20-23 days into the incubation process (Pesenti et al., 2001). The remaining egg (box 49) was in normal condition, but appeared to be unfertilized, due to the absence of a blastocyst. An overall hatching success of 81.1 % of available eggs (43/53) was achieved. There were 14 successful nests with live nestiings, giving a nesting success (successful nests/occupied boxes corrected for removal of the box 40 clutch) of 77.8% (14/18; Table 21). An average of 4.43 eggs were laid per successful nest, while 3.79 eggs were laid per occupied nest, the reduction due in equal measure to abandoned clutches of single eggs during inclement weather and unhatched eggs that were either infertile or cracked. In 2000, a total of 58 eggs were laid in 14 nest boxes (Table 21). No eggs were collected based on the previous year's findings. Five eggs from four nests were missing at different times during incubation leaving 53 eggs upon which nesting and hatching statistics are based. Three clutches with a combined total of thirteen eggs were abandoned (4,4 and 5 eggs in boxes 26, 37 and 41, respectively). A nest predator was indicated in box 41, as small holes were found in all five eggs in the box, suggesting talon or beak punctures. Six eggs from two boxes (a full clutch of two from box 4, and a full clutch of four eggs from box 23) were collected as unhatched while no unhatched eggs from successful nests were discovered. All six eggs were discovered to have slight cracks in the shells, which allowed the contents to dehydrate early in the incubation process. Overall, nine boxes were successful (nesting success of 64.3%), with a total of 34 of 53 eggs hatching (64.2%). An average of 4.14 eggs per clutch were laid in occupied boxes, with an average of 3.78 eggs laid per clutch in successful boxes.
73
Hatchling and Fledgling Success In 1999,43 chicks hatched from the 14 successful nests, with an average of 3.07 chicks occurring per successful box (Table 21). Two nesthngs were later found dead (in boxes 28 and 45), one having been partially eaten by its nest-mates. Both individuals appeared to be mnts of the nest, as previous morphological measurements and body weights of both were consistentiy far below average until death. Thus, 41 chicks reached fledgling age (25 days post hatch), producing a fledging efficiency (fledglings/ hatchlings) of 95.3% (41/43), and a nesting efficiency (fledglings/eggs available) of 77.4% (41/53). Seventeen individuals were removed for tissue analysis, including the entire clutch from nest box 19, to investigate intra-clutch variability for all analytical endpoints. The 24 remaining fledglings were banded with USFWS numbered leg bands before fledge and allowed to fledge naturally. Nest checks between 30 and 35 days posthatch confirmed that all nestling birds greater than 25-days-old left the nest. In 2000, 34 nestlings hatched from 9 nests for a mean of 3.78 chicks per successful nest (Table 21). Nest box 32, which had two of four eggs missing during incubation, was found to have the remaining two nestlings missing between 5 and 10 days post hatch. It is likely that both events were due to avian or mammalian predation. All told, 32 of 34 nestiings survived to fledging age for a fledging efficiency of 94.1% and a nesting efficiency of 60.4% (32/53). No nestiings were collected for tissue analysis, thus the remaining 32 fledglings were banded with USFWS permanent identification leg bands and allowed to fledge undisturbed. As in 1999, nest checks
74
between 30 and 35 days post-hatch confirmed that all nestiing birds greater than 25-daysold left the nest.
Egg Measurement All unincubated and addled eggs collected were weighed and measured after coUection. Unincubated eggs collected in 1999 averaged 15.0±1.8 g, and a mean width and length of 23.3±1.1 and 35.0±1.6 mm respectively (Table 22). Addled eggs from 1999 and 2000 weighed considerably less (11.6±3.8 g and 10.4±4.4 g, respectively), though this was due to extensive dehydration of several of the eggs upon cracking of the shell during incubation. Nevertheless, width and length was similar to the unincubated lot, with 1999 means of 27.9±0.5 and 34.9±0.9 nun, and 2000 means of 28.2±1.0 and 34.5±2.0 mm.
Morphological Measurement Every five days during the 1999 season, beginning at day 5 post-hatch, all chicks in a clutch were monitored for body weight and morphological measurements (Table 23; Figure 3). In 1999, only chicks collected for analysis were measured at day 25, while all 2000 birds were monitored at that and younger ages. Mean (± S.D.) body weight at day 25 post-hatch in 1999 for aU boxes was 117± 7.86 g (Table 24). Intra-clutch variability, with a mean of 110 ± 9.12g for the box 19 nestiings, was similar to that between boxes. The four morphological measurements were similarly combined and compared. All
75
Table 22. Unincubated and Addled Egg Weights and Measurements, 1999 and 2000. Box No. 1 7 13 14 19 27 28 30 35 39 40 40 40 40 44 45 49 Mean SD n
1999 Unincubated (mm) (mm) (g) Weight Width Length 13.6044 28.37 33.44 13.2281 27.44 33.74 17.4497 29.81 36.53 14.9702 28.06 35.49 11.8913 26.66 31.16 17.4762 30.12 35.92 11.9454 26.85 31.77 16.4276 29.1 35.98 17.1653 29.7 35.9 13.97 27.39 34.72 16.2653 28.71 36.68 15.8217 28.87 36.25 15.9999 28.84 35.7 15.1179 28.47 34.41 13.6134 26.73 35.52 13.8758 27.72 34.54 15.8662 28.26 36.48 14.98 1.79 17
28.30 1.07 17
2 2 12 14 20 32 39 39 45
1999 Addled (mm) (g) Weight Width 13.7956 27.94 13.3386 28.07 10.8106 26.92 5.5978 28.47 15.347 28.21 14.9361 28.14 12.765 27.96 12.3077 28.17 5.0254 27.35
4 4 23 23 23 23 26 26 26 26 37 37 37 37 41 41 41 41 41
2000 Addled 6.7876 3.389 9.113 3.375 5.9975 3.3758 14.2217 14.0782 13.7854 11.9426 11.6488 9.9607 13.1447 4.6649 14.9197 14.6589 14.1827 13.7705 13.7367
26.85 27.39 27.51 28.27 26.97 28.01 27.76 27.79 28.00 26.90 27.98 27.36 28.30 27.40 29.52 29.90 29.36 29.79 29.93
35.02 33.76 31.73 30.71 31.96 31.62 34.80 34.22 33.40 32.43 35.00 37.01 34.69 35.86 35.96 36.01 37.35 36.43 36.54
Box No.
34.95 1.63 17
(mm) Length 34.52 33.3 35.4 36.21 35.92 34.7 35.61 34.38 34.21
1999
Mean SD n
11.55 3.79 9
27.91 0.48 9
34.92 0.94 9
2000
Mean SD n
1036 4.36 19
28.16 1.04 19
34.45 2.00 19
76
Table 23. Body Weight and Morphological Measurements Recorded on Days 5, 10, 20, and 25 Post-hatch, 1999. DayBox No. Post-Hatch KOI
K07
(g) Weight
N
1
Measurement (mm) Tarsus Third Toe Culmen Bill Width
5
3
Mean 49.00 SD 4.36 n 3
22.73 1.34 3
12.28 061 3
8.41 0.33 3
5.43 0.15 3
10
3
Mean SD n
31.27 1.15 3
17.49 1.16 3
9.47 0.40 3
5.90 0.14 3
15
3
Mean 119.00 SD 3.61 3 n
39.85 1.04 3
18.16 0.10 3
9.81 0.25 3
5.93 0.16 3
20
3
Mean 134.00 SD 7.21 3 n
39.73 0.51 3
17.49 0.24 3
1036 0.21 3
5.60 0.24 3
25
1
Mean 116.67 SD 1 n
42.04
19.86
10.91
5.66
1
1
1
1
84.00 4.00 3
5
3
Mean 35.67 SD 3.21 3 n
22.88 2.09 3
11.31 1.27 3
7.74 0.40 3
5.05 0.25 3
10
3
Mean 74.00 SD 5.29 3 n
33.14 1.72 3
16.57 1.15 3
9.46 0.37 3
5.37 0.16 3
15
3
Mean 99.33 SD 7.02 3 n
37.95 0.76 3
17.12 0.83 3
9.82 0.07 3
5.40 0.28 3
20
3
Mean 128.67 SD 5.03 3 n
39.01 1.65 3
17.33 013 3
1023 0.31 3
5.44 031 3
25
1
Mean 114.20 SD 1 n 77
39.92
18.63
1021
5.56
1
1
1
1
Table 23. (Continued) DayBox No. Post-Hatch K13
K14
N
(g) Weight
Tarsus
Measurement (mm) Third Toe Culmen Bin Width
5
4
Mean SD n
78.00 8.49 4
31.00 2.11 4
14.69 1.00 4
11.09 2.01 4
6.18 0.22 4
10
4
Mean 110.75 SD 6.60 n 4
36.11 3.77 4
19.12 0.91 4
10.06 0.11 4
6.21 0.41 4
15
4
Mean 129.00 SD 5.23 n 4
38.05 1.02 4
15.63 2.23 4
10.66 055 4
5.71 0.45 4
20
4
Mean 153.75 SD 4.79 n 4
37.56 0.47 4
16.42 0.16 4
11.02 0.29 4
5.45 0.34 4
25
1
Mean 132.51 SD n 1
40.67
17.57
11.36
5.72
1
1
1
1
5
2
Mean SD n
42.00 0.00 2
22.41 0.94 2
10.91 0.18 2
7.21 0.55 2
4.81 0.44 2
10
2
Mean SD n
91.00 1.41 2
31.58 021 2
16.94 0.18 2
8.74 0.11 2
5.47 0.18 2
15
2
Mean 119.50 SD 4.95 n 2
38.74 017 2
17.14 0.15 2
9.30 0.11 2
5.32 0.37 2
20
2
Mean 125.00 SD 8.49 n 2
40.42 0.45 2
19.21 021 2
9.78 0.28 2
5.36 0.42 2
25
1
Mean 109.26 SD
38.58
18.46
10.25
5.85
1
1
1
1
r.
1
78
Table 23. (Continued) DayBox No. Post-Hatch K19
K27
(g) Weight
N
Measurement (mm) Tarsus Third Toe Culmen Bill Width
5
4
Mean 52.50 SD 13.48 n 4
26.76 4.80 4
12.95 1.83 4
8.22 073 4
5.80 0.25 4
10
4
Mean 103.75 SD 16.38 4 n
34.07 2.97 4
16.50 2.33 4
9.13 0.25 4
6.03 0.12 4
15
4
Mean 118.25 SD 11.62 4 n
37.51 1.67 4
17.02 0.96 4
9.85 0.48 4
5.76 0.10 4
20
4
Mean 131.75 SD 8.26 4 n
37.01 1.55 4
15.51 054 4
9.92 0.32 4
5.51 0.10 4
25
4
Mean 110.44 SD 9.12 4 n
38.14 0.55 4
17.17 0.21 4
10.05 O30 4
5.41 0.13 4
5
4
Mean 44.00 SD 4.32 4 n
23.43 1.83 4
10.20 0.46 4
8.35 0.27 4
5.62 0.25 4
10
4
Mean 102.00 SD 3.65 4 n
36.57 1.01 4
17.27 0.72 4
9.28 0.29 4
5.73 0.07 4
15
4
Mean 124.75 SD 2.22 4 n
42.07 0.34 4
17.86 0.69 4
10.18 0.19 4
5.72 0.32 4
20
4
Mean 126.25 SD 6.13 4 n
41.91 1.49 4
17.62 0.45 4
10.56 0.24 4
5.60 0.20 4
25
1
Mean 107.56 SD 1 n
42.58
18.29
10.90
5.55
1
1
1
1
79
Table 23. (Continued) DayBox No. Post-Hatch K28
K30
(g) Weight
N
Measurement (mm) Tarsus Third Toe Culmen BiU Width
5
3
Mean SD n
31.67 18.01 3
19.80 6.58 3
9.95 1.06 2
7.31 1.33 3
4.81 1.56 3
10
2
Mean 97.00 SD 4.24 2 n
33.33 0.72 2
14.43 058 2
9.20 0.08 2
5.50 0.28 2
15
2
Mean 120.50 SD 0.71 2 n
36.93 2.84 2
16.01 2.05 2
9.76 0.31 2
6.31 0.41 2
20
2
Mean 135.50 SD 3.54 2 n
39.84 1.02 2
16.82 0.34 2
10.13 0.02 2
5.57 0.55 2
25
1
Mean 128.76 41.56 SD n i l
18.22
10.21
5.55
1
1
1
5
4
Mean 41.25 SD 5.56 n 4
23.37 1.85 4
11.03 1.19 4
7.79 0.74 4
5.73 0.20 4
10
4
Mean SD n
32.13 4.21 4
17.19 0.78 4
9.14 0.89 4
6.08 0.42 4
15
4
Mean 121.25 SD 6.40 n 4
40.12 1.34 4
19.72 2.03 4
9.13 0.71 4
6.54 0.36 4
20
4
Mean 135.75 SD 7.04 n 4
40.20 1.48 4
19.09 0.75 4
10.02 0.40 4
5.70 0.39 4
Mean 125.40 41.27 SD n i l 80
17.71
9.84
5.50
25
82.75 8.38 4
1
1
1
Table 23. (Continued) DayBox No. Post-Hatch K35
K39
(g) Weight
N
Measurement (mm) Tarsus Third Toe Culmen Bill Width
5
3
Mean SD n
42.33 3.51 3
24.22 0.48 3
11.49 0.50 3
8.22 0.02 3
5.55 0.18 3
10
3
Mean SD n
83.00 4.36 3
35.81 0.06 3
15.83 0.25 3
9.32 0.07 3
5.91 0.27 3
15
3
Mean 136.00 SD 6.00 n 3
41.16 1.01 3
17.81 0.20 3
9.93 0.06 3
5.79 0.14 3
20
3
Mean 125.00 SD 7.00 n 3
40.72 1.27 3
17.67 038 3
10.73 0.50 3
6.09 0.08 3
25
1
Mean 119.11 SD n 1
43.76
18.87
10.70
5.60
1
1
1
1
5
2
Mean 48.00 SD 0.00 n 2
25.16 1.10 2
12.88 014 2
8.13 0.33 2
5.51 0.33 2
10
2
Mean 101.00 SD 1.41 n 2
36.07 0.13 2
17.26 2
9.01 0.35 2
5.95 0.49 2
on
15
2
Mean 122.50 SD 0.71 n 2
40.97 0.89 2
18.37 2.42 2
8.86 0.66 2
6.57 0.09 2
20
2
Mean 131.00 SD 1.41 n 2
41.45 1.17 2
17.84 0.23 2
9.92 017 2
5.50 0.57 2
25
1
Mean 119.30 SD n 1
43.27
18.35
10.11
5.36
1
1
1
1
81
Table 23. (Continued) DayBox No. Post-Hatch K43
K44
(g) Weight
N
Measurement (mm) Tarsus Third Toe Culmen Bill Width
5
4
Mean SD n
35.00 4.83 4
18.11 1.58 4
9.07 0.89 4
7.44 035 4
4.97 0.37 4
10
4
Mean SD n
73.50 12.50 4
29.93 1.24 4
15.26 1.04 4
8.95 0.40 4
5.66 0.25 4
15
4
Mean 110.75 SD 13.30 n 4
37.73 0.57 4
17.83 0.80 4
9.67 0.34 4
5.63 0.29 4
20
4
Mean 139.50 SD 13.23 n 4
39.36 1.45 4
18.79 0.94 4
1010 0.14 4
5.69 0.29 4
25
1
Mean 114.99 SD n 1
41.96
17.65
9.79
5.39
1
1
1
1
5
3
Mean SD n
38.33 10.02 3
19.79 2.32 3
9.60 1.39 3
7.39 0.31 3
5.05 0.41 3
10
3
Mean SD n
89.33 10.26 3
33.68 2.63 3
15.35 1.25 3
8.66 0.52 3
5.57 0.17 3
15
3
Mean 112.00 SD 9.54 n 3
37.98 0.93 3
18.23 0.67 3
9.37 0.40 3
6.00 0.06 3
20
3
Mean 131.67 SD 8.14 n 3
39.19 061 3
17.17 0.47 3
9.79 0.48 3
5.90 012 3
25
1
Mean 126.69 SD n 1 82
38.48
18.01
10.21
6.12
1
1
1
1
Table 23. (Continued) DayBox No. Post-Hatch K45
(g)
N
Weight
Measurement (mm) Tarsus Third Toe Culmen Bill Width
5
2
Mean 22.00 SD 5.66 n 2
16.71 1.57 2
10
2
Mean SD n
55.50 0.71 2
24.44 2.55 2
15
2
Mean 71.00 SD 26.87 n 2
20
1
6.74 059 2
4.85 0.41 2
12.05 0.21 2
8.38 Oil 2
5.37 016 2
29.88 2.23 2
13.75 2.11 2
8.43 0.21 2
4.99 0.41 2
Mean 102.00 SD n 1
33.93
14.80
8.47
5.94
1
1
1
1
Mean 114.85 SD n 1
39.69
17.00
10.10
5.29
1
1
1
1
Mean 52.00 SD 0.00 n 2
27.67 1.99 2
15.36 0.68 2
8.07 0.52 2
5.77 OlO 2
10
Mean 103.00 SD 1.41 n 2
33.92 1.45 2
18.79 0.69 2
9.78 088 2
5.94 0.11 2
15
Mean 118.00 SD 2.83 n 2
42.27 0.76 2
16.56 0.03 2
9.80 0.28 2
5.69 0.16 2
20
Mean 138.50 SD 4.95 2 n
42.04 0.06 2
17.53 038 2
9.65 0.44 2
5.64 0.06 2
25
Mean 109.90 SD n 1 83
44.76
18.94
1014
5.91
1
1
1
1
25
K49
1
Table 24. Body Weight and Morphological Measurements of Day 25 Nestiings, 1999. Measurement (mm) Third Toe Culmen
Box No.
Individual
(g) Weight
Tarsus
K19 K19 K19 K19
A B C D
103.24 114.13 121.62 102.76
37.62 37.89 38.89 38.16
17.11 17.15 16.96 17.45
9.87 9.92 1O50 9.89
5.53 5.27 5.32 5.50
Mean SD n
110.44 9.12 4
38.14 0.546 4
17.17 0.205 4
10.05 0.304 4
5.41 0.129 4
KOI K07 K13 K14 K19 K27
A C C B Mean B
116.67 114.20 132.51 109.26 110.44 107.56
42.04 39.92 40.67 38.58 38.14 42.58
19.86 18.63 17.57 18.46 17.17 18.29
10.91 10.21 11.36 1025 10.05 10.90
5.66 5.56 5.72 5.85 5.41 5.55
K28 K30 K35 K39 K43 K44 K45 K49
C D C A A C B B
128.76 125.40 119.11 119.30 114.99 126.69 114.85 109.90
41.56 41.27 43.76 43.27 41.96 38.48 39.69 44.76
18.22 17.71 18.87 18.35 17.65 18.01 17.00 18.94
1021 9.84 10.70 10.11 9.79 10.21 1010 10.14
5.55 5.50 5.60 5.36 5.39 6.12 5.29 5.91
Box K 19 Litra-Clutch
N=4
Bill Width
All Sites
N=14
Mean SD n
117.83 7.86 14
41.19 2.05 14
18.20 0.760 14
10.34 0.452 14
5.60 0.232 14
Smelter Hill Sites
N=6
Mean SD n
115.11 9.16 6
40.32 1.80 6
18.33 0.936 6
10.61 0.519 6
5.62 0154 6
Opportunity Pond Sites
N=8
Mean SD n
119.88 6.61 8
41.84 2.09 8
18.09 0.649 8
10.14 0.277 8
5.59 0.287 8
84
results appeared consistent, with any unusual outiiers identified as being the runted individuals acknowledged previously. Nestling measurements in 2000 were similarly performed, with weights and the four morphological measurements collected every five days through day 25 (Table 25; Figure 6). Mean body weight at day 25 post-hatch for Smelter Hill nestUngs (127 ± 1.71 g; Table 26) was notably greater (p=0.051) than Opportunity Ponds nesthngs (120 ± 10.2 g). Nestiing tarsus length was also significantiy higher in day 25 Smelter Hill nestlings (42.3 ± 0.91 mm; p=0.009) than in Opportunity Ponds nesthngs (41.0 ± 1.26 nrni). AU remaining measurements were consistent, with no notable differences between sites.
85
Table 25. Body Weight and Morphological Measurements Recorded on Days 5, 10, 20, and 25 Post-hatch, 2000. DayBox No. Post-Hatch
N
KOI
5
4
10
K17
Measurement (mm) Third Toe Culmen Bill Width
(g) Weight
Tarsus
Mean SD n
26.38 7.56 4
20.01 2.45 4
7.99 1.14 4
7.51 0.54 4
4.89 017 4
4
Mean SD n
73.50 16.03 4
33.28 2.88 4
14.99 1.20 4
8.89 0.41 4
5.87 019 4
15
4
Mean SD n
116.00 9.31 4
41.81 1.79 4
17.73 0.66 4
9.97 0.33 4
6.49 0.25 4
20
4
Mean SD n
127.50 7.55 4
43.37 0.90 4
18.08 0.44 4
1049 0.36 4
5.82 015 4
25
4
Mean SD n
126.25 8.54 4
43.33 0.74 4
18.61 0.69 4
10.48 0.25 4
5.90 0.21 4
5
4
Mean SD n
50.50 1.73 4
27.59 0.62 4
12.80 0.09 4
8.20 0.15 4
5.47 012 4
10
4
Mean SD n
99.50 1.91 4
38.62 0.54 4
16.59 033 4
9.22 0.29 4
6.02 0.06 4
15
4
Mean SD n
120.75 5.56 4
42.34 0.69 4
19.11 0.32 4
9.98 0.24 4
6.04 OlO 4
20
4
Mean SD n
132.75 5.50 4
40.24 1.61 4
19.07 0.89 4
1O60 0.21 4
5.88 0.09 4
25
4
Mean SD n
126.50 9.15 4
41.92 0.93 4
19.10 0.33 4
1087 0.54 4
5.97 0.07 4
86
Table 25. (Continued) DayBox No. Post-Hatch
N
(g) Weight
Tarsus
Measurement (mm) Third Toe Culmen Bill Width
5
3
Mean SD n
42.00 5.57 3
23.55 1.79 3
11.74 1.43 3
7.58 0.48 3
5.32 0.25 3
10
3
Mean SD n
86.67 3.06 3
35.42 1.93 3
15.18 1.31 3
8.92 0.42 3
5.96 0.24 3
15
3
Mean SD n
122.00 3.46 3
39.70 0.11 3
19.95 0.98 3
9.84 0.23 3
6.08 0.38 3
20
3
Mean SD n
125.00 4.00 3
40.81 1.29 3
18.22 0.17 3
10.10 0.23 3
5.94 0.34 3
25
3
Mean SD n
129.33 6.35 3
41.63 082 3
18.33 0.43 3
10.27 015 3
6.02 0.30 3
K32
5
2
Mean SD n
44.50 7.78 2
24.59 4.14 2
11.51 2.79 2
7.81 013 2
4.90 0.21 2
K34
5
4
Mean SD n
41.25 6.70 4
25.52 1.11 4
12.00 0.82 4
8.45 0.20 4
5.57 0.50 4
10
4
Mean SD n
91.50 8.39 4
37.41 1.62 4
17.13 0.38 4
9.37 0.16 4
5.73 0.42 4
15
4
Mean SD n
100.00 7.48 4
40.14 1.52 4
18.67 1.03 4
9.72 025 4
5.65 0.30 4
20
4
Mean SD n
105.00 6.73 4
41.30 1.87 4
18.25 0.56 4
10.12 0.13 4
5.76 0.30 4
K24
87
Table 25. (Continued)
Box No. K34 (continued)
K35
K43
DayPost-Hatch
N
25
4
5
Measurement (mm) Third Toe Culmen Bill Width
(g) Weight
Tarsus
Mean SD n
105.75 5.68 4
41.60 1.48 4
18.23 0.57 4
10.46 0.13 4
5.72 0.29 4
4
Mean SD n
43.50 4.12 4
25.19 0.72 4
11.83 0.35 4
8.34 055 4
5.52 0.38 4
10
4
Mean SD n
89.00 5.66 4
37.18 085 4
17.62 0.40 4
9.05 0.26 4
5.70 0.36 4
15
4
Mean SD n
105.25 2.06 4
41.04 0.79 4
18.76 0.44 4
9.69 021 4
5.70 0.33 4
20
4
Mean SD n
126.50 4.43 4
41.40 1.31 4
18.79 0.20 4
10.02 0.19 4
5.51 0.43 4
25
4
Mean SD n
115.50 3.42 4
42.54 0.20 4
18.19 0.47 4
10.21 017 4
5.51 0.26 4
5
5
Mean SD n
32.40 6.11 5
22.06 2.31 5
10.92 1.07 5
7.63 O20 5
5.07 0.35 5
10
5
Mean SD n
75.80 7.16 5
34.26 2.35 5
15.60 1.17 5
8.97 0.43 5
5.77 0.09 5
15
5
Mean SD n
103.40 8.88 5
39.53 1.30 5
17.84 0.29 5
9.97 0.58 5
5.76 0.25 5
20
5
Mean SD n
115.60 6.54 5
40.05 1.39 5
18.59 0.29 5
10.19 0.34 5
5.70 0.16 5
88
Box No. K43 (continued)
DayPost-Hatch 25
K46
N 5
Table 25. (Continued) (g) Weight Tarsus Mean 126.60 40.84 SD 9.53 1.14 n 5 5
Measurement (mm) Third Toe Culmen Bill Width 18.53 10.43 5.99 0.56 0.44 0.22 5 5 5
Mean SD n
44.75 .5.12 4
26.21 1.33 4
12.76 0.88 4
8.26 0.46 4
5.37 0.40 4
10
Mean SD n
84.25 7.27 4
37.59 1.77 4
17.78 0.78 4
9.36 0.18 4
5.89 031 4
15
Mean SD n
105.50 5.51 4
41.05 1.19 4
19.02 0.94 4
9.95 0.24 4
5.74 0.35 4
20
Mean SD n
107.50 8.70 4
41.85 1.28 4
18.35 0.57 4
10.25 0.33 4
5.71 0.32 4
25
Mean SD n
120.00 9.59 4
40.93 1.97 4
18.19 1.02 4
10.74 025 4
5.81 0.34 4
Mean SD n
36.50 2.52 4
23.30 1.11 4
11.80 073 4
7.55 0.14 4
5.19 0.20 4
10
Mean SD n
85.00 4.76 4
35.70 0.41 4
15.40 0.97 4
8.83 0.33 4
6.12 021 4
15
Mean SD n
98.00 4.32 4
37.32 1.36 4
17.33 2.11 4
9.78 034 4
6.23 0.54 4
20
Mean SD n
112.50 10.25 4
39.40 1.31 4
18.64 0.26 4
9.97 0.44 4
5.67 0.22 4
25
Mean SD n
132.25 5.68 4
39.11 091 4
19.64 0.27 4
10.41 039 4
5.83 0.17 4
K50
89
Table 26. Body Weight and Morphological Measurements of Day 25 Nestlings, 2000.
Box No.
(g) Weight
Tarsus
Measurement (mm) Third Toe Culmen
Bill Width
KOI K17 K24
Mean Mean Mean
126.25 126.50 129.33
43.33 41.92 41.63
18.61 19.10 18.33
10.48 1087 1027
5.90 5.97 6.02
K34 K35 K43 K46 K50
Mean Mean Mean Mean Mean
105.75 115.50 126.60 120.00 132.25
41.60 42.54 40.84 4093 39.11
18.23 18.19 18.53 18.19 19.64
10.46 10.21 10.43 10.74 10.41
5.72 5.51 5.99 5.81 5.83
All Sites
Smelter Hill Sites
Opportunity Pond Sites
N=8
Mean SD n
122.77 8.65 8
41.49 1.26 8
18.60 052 8
10.48 022 8
5.84 0.17 8
N=3
Mean SD n
127.36 1.71 3
42.29 0.91 3
18.68 039 3
1054 O30 3
5.96 0.06 3
N=5
Mean SD n
120.02 1021 5
41.00 1.26 5
18.55 0.62 5
10.45 019 5
5.77 0.18 5
90
180
15 O
Nonlinear logistical regression Day V Weight (Smelter Hill sites)
20
Age (days post-hatch)
25
30
•— Nonlinear logistical regression » Day V Weight (Opp. Ponds sites)
Figure 4. Nestling Growth Curve Estimates Using Nonlinear Logistic Regression. Growth curves compare box means of nestlings raised in Smelter Hill associated boxes to nestlings raised in Opportunity Ponds associated boxes. Nestlings from both years were grouped according to site. No significant difference was detected between curves.
91
CHAPTER IV DISCUSSION
American kestrels were found to readily nest throughout the site. Nesting pairs were documented across the area, inhabiting boxes in all habitats. The proportion of boxes used over both years (38% -1999; 29% - 2000) was slightiy low compared to similar experiments (Hamerstrom et al., 1973; Hoff, 1992; Rohrbaugh and Yahner, 1997; Craft and Craft, 1996), though few adult kestrels were seen throughout the study area that were not affiliated with a nest box. Commonly, nest habitation is low in the first year of box placement (Wheeler, 1992; Craft and Craft, 1996), while weather-related factors may have contributed to the decreased box utilization seen in 2000. Parental contribution of COCs in-ovo, was low, not approaching known toxic levels (Table 9). Clutch numbers appeared normal (Craft and Craft, 1996; Hamerstrom et al., 1973; Toland, 1985), and niunbers of unsuccessful eggs were not unusual in regard to surrounding soil COC concentrations (Table 21). An assessment of collected food items, however, demonstrated the potential for exposure to all five COCs in nestlings. Investigations of blood, liver and kidney COC concentrations indicated systemic accumulations in all tissues, the most notable being increased blood lead levels occurring during the early nesting period, a time of critical neurological development. Additionally, metal concentrations were found in higher concentrations for all matrices collected from sites closest to the smelter smoke stack, when compared to samples from more distant nest boxes. Biological markers indicated initiation of detrimental health effects in individuals with the highest blood lead levels. These health effects were not
92
correlated to other metal concentrations in the blood, demonstrating that lead appears to be the most critical element for exposure in nestiings raised on the site. Nevertheless, no effects were documented in nestiing growth, overt health effects, or fledging success in response to the exposure risks. Blood lead levels suggest the potential for developmental neurological repercussions from lead exposure.
Exposure Assessment Soil appraisals conducted by the Environmental Protection Agency (CDM 1997) showed a gradient of all five COC concentrations generally moving away from the Smelter Hill area. Nevertheless, extensive variation in vegetative cover, historic landuse, and soil contamination may be found within each foraging area, illustrating the difficulty in quantifying actual exposure of the five COCs to nestling kestrels, based exclusively on modeled exposures from soil contamination data. Further, due to the wide variations of COC concentrations throughout the site and surrounding area, other methods were essential for assessing actual frequency and magnitude of COC exposure in kestrels. Food items were therefore collected from nestiings at each nest box, providing a means to integrate the broad range of potential exposure levels by evaluating the prey items through which the COCs move from the soil. As all five COCs were detected at elevated levels in representative food items and kestrel tissues, it appears that risk from exposure is a reasonable concem for American kestrels at the Anaconda Smelter site. Analysis of prey item types indicates that kestrels appeared to rely most heavily upon rodents for nestUng food (66% for both years), with
93
invertebrate (23%; predominantly grasshopper) and passerine (12%) items comprising the remainder. While rodents may be the preferred prey for clutch feeding (Anderson et al., 1993), other studies have shown a higher reliance on invertebrate items (Balgooyen, 1976; Meyer and Balgooyen, 1987; Bohall-Wood and CoUopy, 1987). The possibihty exists that more invertebrate items were able to pass unobstructed by the esophageal constrictor, and thus not recovered. Based on nest box observations, many of the grasshoppers and passerine items collected appeared to be opportunistically captured by the female, as she stayed in close proximity of the nest box. The majority of nestiing food items are collected by the male, however, who regularly transferred items to the female for presentation to the clutch. Separation of prey item COC data into prey class demonstrated significant differences in contaminant levels. Arsenic and lead were detected in significantly higher concentrations in rodent tissue than grasshoppers (Figure 3). This finding is consistent with those of Erry et al. (1999) who found significant increases in liver and kidney arsenic in European kestrels {Falco tinnunculus), a predominantly rodent predator, when compared to the same tissues of a sparrowhawk (Accipiter nisus), a similarly sized avian predator, collected from the same contaminated sites. Subsequently, prey items favored by kestrels, specifically rodents, may result in increased exposure to specific contaminants. The comparative metal bioavailability in the two prey types has not been well assessed, and differential sequestration of metals in non-digested bone, hair and carapace deserves consideration. Homfeldt and Nyholm (1996) found pied flycatchers {Ficedula hypoleuca) accumulated higher levels of metal contaminants (As, Cd, Cu, Pb) than Tengmalm's owls {Aegolius funereus) along the
94
same heavy metal pollution gradient, suggesting greater metal loads and/or bioavailablity from the flycatcher's invertebrate prey items. As kestrels are known to be opportunistic hunters, selecting prey based on relative abundance (Balgooyen, 1976), it is likely that as prey species vary in abundance, nestling exposure levels will also vary. Further, Anderson et al. (1993) determined that the larger female nestiings have a significant competitive advantage in seizing smaller monopolizeable items (such as grasshoppers) when presented by adults, as compared to male nest mates. Potential exposure differences between nestling sexes were not notable in examination of blood metal (day 25 post-hatch) concentrations. As anticipated, average COC concentrations were elevated (arsenic and lead significantiy) in food samples collected from nestiings in closer proximity to Smelter EQU when compared to those from the Opportunity Ponds sites. In as much, the general lack of detectable levels of arsenic and cadmium in kestrel liver and kidney, and arsenic in blood on day 25 is notable. Similarly, in owl prey items (Homfeldt, 1996), increased contaminant levels were noted with decreasing distance to a smelter site, though the nestling owls did not have similar increases in tissue levels. Based on the poor movement of arsenic and cadmium from food items to blood to tissues, it is apparent that relatively efficient removal of arsenic and cadmium from the system occurs prior to absorption by the gut or movement into the blood. Metallothioneins in the epithelial lining of the intestine play an important role in regulating systemic trace mineral absorption. High concentrations of zinc and cadmium will cause metallothionein induction (Klasing, 1998), dramatically reducing systemic absorption. Scheuhammer
95
found cadmium absorption of Japanese quail {Cotumix cotumix) to be approximately 0.6% of available, at intake levels similar to those seen in our collected prey items (Scheuhammer, 1987). However, a dose-dependent increase in percent absorption was noted with increased exposure concentration (up to 2% at 50 ppm; Scheuhammer, 1987). Mean rodent carcass lead levels of 1.639 /xg/g in kestrel food items were comparable to carcass data from small mammal investigations performed concurrently with this study. In those examinations, meadow voles {Microtus pennsylvanicus) and deer mouse {Peromyscus maniculatus) carcasses, collected from several locations throughout the Superfund site, were assessed for metal burdens. Site averages for carcass lead levels varied from a low of 0.6 to a high of 2.5 \igjg lead in voles, while deer mouse carcasses were more variable with average lead concentrations ranging from 0.6 to a high of 17.5 /ig/g (Hooper et al., 2001). Kestrels feeding along the northem faces of Smelter Hill, where the ARCO (Atiantic Richfield Company) and ARTS (Anaconda Revegetation Treatability Studies) remediation sites were performed, will capture deer mice with lead levels averaging 4 to 17 ppm in the carcass. Prey items with lead levels in this range were found in box 13, at the base of the northeast face of Smelter Hill, in 1999 (Table 5). With much of the body-burden of lead known to be sequestered in low digestibiUty depots like hair and bone (Scheuhammer, 1987; Eisler, 1988), actual bioavailable lead from a prey item is decreased due to the expulsion of undigested material in regurgitated pellets. Carcasses of voles captured from lead acetatecontaminated orchards contained 38-ppm lead concentrations (Stendell, 1989). Despite exposure to higher carcass lead concentrations than those of our study, captive kestrels
96
maintained on orchard vole carcasses for 60 days accumulated only sub-lethal amounts of lead in their livers (I ppm wet wt.) by the end of treatment. Pellets collected from Stendell's treatment birds contained 130-ppm lead, further illustrating this conclusion. Estimated total ^g amounts of each COC for all food items in this study (based on metal concentration and tissue weight; Table 27), and in adult deposited pellets and nestiing fecayurate samples (Table 28), showed that regurgitated pellets collected from within nest boxes throughout the Superfund site contained approximately equivalent total fig amounts of Pb, As and Cu as detected in collected food items, suggesting that large proportions of these contaminants are unavailable for absorption. Similarly, fecal/urate samples contained approximately 25% to 35% of Cd, Pb, Cu and Zn levels of collected food items, although less than 1% of As. For raptors, a single pellet is generally produced in a 1:1 ratio per meal (Klasing, 1998). This suggests that kestrels regurgitate a large proportion of copper, lead and arsenic ingested in their prey, and thus, are never systemically exposed to total prey carcass concentrations. It should be noted that gastric acidity of falconiformes is approximately 1.7 pH (and 2.4 for owls), resulting in up to 93% digestibility of prey bone (Klasing, 1998). As noted in examination of the collected pellets in this study, the majority was comprised of well-homogenized portions of insect carapace and rodent hair, with very little discernible bone fragments found. Undoubtedly, pellet formation of indigestible material affords some protection from absorption of contaminants, though bone-sequestered contaminants are likely more available than those found in chitin-containing portions. This topic deserves further assessment under control conditions to allow better interpretation of field collected data.
97
Table 27. Estimated Total Metal Amount in Food Items by Box. Total metal amounts were calculated using measured sample weight against measured concentration. Mean concentration and weight were calculated for boxes with more than one food item. 1999 FOOD ITEM
2000 FOOD ITEM
Box No.
As
KOI
0.87
Amount (Mg) Cd Pb Cu 1.35 0.66 16.21
K07
3.72
OOl
3.22
31.88
107.0
K13
44.58
1.43
28.49 143.05
276.8
K14
10.94
064
5.59
301.9
A.mount (Mg) Cd Pb Cu
Zn
As
82.01
6.87
5.24
4.94
94.11
9.26
2.85
6.03
175.60 389.3
59.67
065
29.67
240.60 451.7
0.42
0.40
0.98
65.49
124.5
2.12
1.69
1.05
41.33
281.2
1.56
0.16
1.29
36.51
143.4
K46
1.52
0.15
0.81
48.25
279.1
K50
0.06
0.69
0.84
37.98
201.6 271.6
300.27
K17 K19
13.79
021
11.60
68.55
Zn 302.1
335.1
K24 K27
9.54
0.09
4.64
28.59
158.1
K28
0.84
014
0.97
28.18
45.60
K30
6.24
0.58
9.68
32.62
355.4
K34 K35
8.18
0.47
3.18
25.18
165.8
K39
2.08
0.08
5.18
27.38
144.1
K43
6.67
2.54
15.29
69.37
521.2
K44
3.79
0.56
6.99
41.15
159.2
K45
6.37
025
11.88
29.20
129.9
ALL
9.05
0.64
8.26
64.74
214.0
1018
1.48
5.70
92.48
SMELTER
13.91
0.62
9.03
98.09
2102
25.26
2.91
13.55
170.10 381.0
OPP PONDS 4.88
066
7.60
36.15
217.3
1.14
0.62
1.00
45.91
98
206.0
Table 28. Estimated Total Metal Amount in Pellet and Fecal/Urate by Box. Total metal amounts were calculated using measured sample weight against measured concentration. Mean concentration and weight were calculated for boxes with more than one collected sample. PELLET
FECAL / URATE Amount (/xg) Cd Pb Cu
As
Amount [/^g) Cu Pb Cd
135.4
055
OlO
3.75
23.69 105.1
275.5
0.69
013
3.06
56.34 83.72
K14
044
0.00
0.00
2.71
1.01
K19
0.01
015
0.40
4.03
9.59
033
018
1.01
13.62 50.38
0.42
053
2.17
28.49 83.70
O30
015
0.77
16.51 51.76
3.71
022
5.89
53.47 118.2
0.44
O09
4.01
19.82 72.94
036
O05
1.71
6.66
017
O08
0.24
18.75 4061
Box No.
As
KOI
2.26
0.37
11.46
70.45
87.84
K02
5.68
0.75
5.77
73.20
63.27
K07
27.71 0.08
21.33
90.16
K13
12.48 0.28
6.39
133.88
Zn
K20
25.73 0.27
13.29 209.29
107.8
K26
7.04
0.05
1.48
30.29
55.38
K27
3.54
0.22
3.68
29.88
76.53
K28 K30
30.02 0.02
7.90
179.12
25.99
K31
0.07
6.73
41.47
1077
K37
2.97
0.32
0.38
57.02
46.40
K39
0.41
0.04
0.59
49.54
26.70
K41
1.25
0.08
3.36
122.41
97.24
K43 K44
0.38
0.04
0.72
40.06
75.72
K45
1.05
0.12
15.54 130.52
126.0
K49 ALL SMELTER
Zn
19.24
0.19
7.05
89.80
86.46
0.67
015
2.09
22.19 57.84
12.06 0.29
9.06
91.02
114.5
0.40
on
1.64
20.08 49.96
OlO
5.03
88.59
58.40
O90
019
2.46
23.95 64.41
9.27
OPP PONDS 6.01
99
A dose estimation of daily COC exposure was calculated for full grown nestiings (avg. 125g body wt.) using a daily food ingestion rate of 0.3 g/g body wt. - day (McVey et al., 1993; Table 29). Appropriately proportioned contaminant levels were calculated based on percent food item selection, and a daily COC intake was calculated. Based on these results, full grown kestrel nestiings (identified as post day 17 nestlings) are exposed to a calculated daily dose of 0.316 fig lead per gram body weight per day, with a difference in dose of 0.468 and 0.187 /ig/g-day between Smelter Hill and Opportunity Ponds associated nestiings. Interestingly, we noted a 57% increase in blood lead concentration, and concurrently saw a 25% decrease in er3l;hrocyte ALAD enzyme activity in Smelter Hill associated nestlings (day 25 post-hatch), when compared to Opportunity Ponds birds. These results suggest that 0.5 ug/g is a lowest observable adverse effect level (LOAEL) for kestrels exposed to this lead dose in a biologically incorporated food item. Further considerations, not part of this report, are required to fully investigate these results, as well as estimate total COC ingestion amount over the entire nesting period based on a food intake rate for younger nestlings. Sequential blood sampling from nestiings in 2000 allowed the investigation of age-specific changes in kestrel contaminant levels (Figure 4). Blood cadmium and lead levels demonstrated significant increases in concentration with age in several nest boxes, particularly from the Smelter Hill area. Dietary metal concentrations from food items did not reflect a temporal increase in exposure level that might explain the increase with age. Further, food consumption rates, on a kg food / kg body wt basis, decrease with nestling age as nestiings approach fledging (Balgooyen, 1976), which would decrease exposure
100
Table 29. Mean Prey Metal Concentration, and Estimated Total Daily Intake and Dose. Prey - Avg. Concentration (/xg/g) As
Cd
Pb
Cu
Zn
Combined All
1.566
0.189
1.208
13.62
42.62
Smelter
3.194
0.266
1.857
22.27
46.97
Opp pond
0.467
0.126
0.743
8.149
39.64
Food Ingestion Rate 0.3
g/g-day
Estimated Daily Proportioned AU
Food Item Selection 0.232
1.054
15.82
Smelter
1.488 2.804
0.327
1.559
Opp pond
0.434
0.117
21.70
45.16 48.76
66% 23%
Rodent Li vertebrate
0.624
10.97
37.93
12%
Avian
Pb
Cu
Zn
Estimated Daily Intake
Estimated Daily Intake (/xg / -day) As
Cd
Combined
36g
All
56.38
6.79
43.50
490.2
1534
Smelter
114.98
9.58
66.85
1691
Opp pond
16.81
4.53
26.74
801.7 293.4
1427 Proportioned Estimated Daily Intake
Proportioned All
53.58
8.35
37.93
569.6
1626
23.8g
Rodent
Smelter
100.95
11.78
56.13
781.1
1755
8.3g
Invertebrate
Opp pond
15.62
4.22
22.45
395.0
1366
4.3g
Avian
Cu
Zn
Estimated Daily Dose (/xg /g b.w. -day) Pb Cd As Combined All
0.470
0.057
0.363
4.085
12.79
Smelter
0.958
0.080
0557
6.681
14.09
Opp pond Proportioned
0.140
0.038
0.223
2.445
11.89
All
0.446
0.070
0316
4.747
Smelter
0.841
0.098
0.468
6.509
13.55 14.63
Opp pond
0.130
O035
0.187
3.292
11.38
101
levels in the face of constant food item COC concentrations. As neither metal forms substantial blood depots, the levels likely reflected recent dietary exposure. Increases in blood levels of these metals with age might reflect increased efficiency in absorption from the GI ti-act. Altematively, it is known that lead accumulates in bones, hair and feathers (Scheuhammer, 1987; Eisler, 1988), particularly in developing animals (Burger and Gochfeld, 2000). Nestling kestrels reach approximate adult size by day 17-20 (Figure 6), with major bone and feather development occurring up until this same period. A likely explanation for lower Cd and Pb levels in younger birds was their sequestration into bone and feather depots during the period of greatest growth. Cessation of rapid bone growth and feather development in the older nestlings likely results in an increase in free circulating blood concentrations of the two contaminants. Spalding et al. (2000) noted similar increases in nestiing great egret {Ardea albus) blood mercury levels with incurring age, and similarly theorized that feather development acted to decrease mercury concentrations in blood, by providing a ready depot during growth. Kestrel nestlings in the vicinity of Smelter Hill were exposed to elevated cadmium levels via food items, which led to elevations in blood, liver and kidney tissues in the Smelter HiU associated nestlings. Maximum liver levels reached 0.378 /xg/g, or approximately 3% of the toxic threshold (13 /xg/g) suggested by Eisler (Eisler, 1985). Cadmium, therefore, does not appear to be an acute threat to nestling health. Further consideration of cadmium as a cumulative toxicant is not, however, unreasonable. Liver and kidney concentration account for -90% of the total body burden, bound to metallothionein (Scheuhammer, 1987). With a long biological half-life, cadmium tends
102
to accumulate with age, even in animals exposed to low background levels (Scheuhammer, 1987). Thus, nestiings and aduh birds remaining in the vicinity of the smelter for long periods could accumulate levels approaching a toxic threshold. Assessment of adult cadmium accumulation in the vicinity of the Smelter might be prudent in further assessments, as kestrels have been noted to be philopatric, retuming to the same area (even the same nest site) in consecutive years (Hamerstrom, 1973). Dietary lead exposure appears to be a more significant concem for nestiing kestrel health. While liver and kidney concentrations are not at or above published levels of concem, blood lead levels indicate the potential for deleterious health effects, made more significant due to the age of the nestiings. Blood lead levels in falconiformes are considered "sub-clinical" at levels between 0.2 and 1.5 /xg/g (Franson, 1996). These levels are elevated above background concentrations (defined as
