Health of Domestic Mallards (Anas platyrhynchos domestica

Jul 8, 2014 - Department of Biological Sciences, University of Alberta, Z-708, 11455 Saskatchewan Drive, Edmonton, Alberta Canada T6G 2E9. ‡ Ecosyst...
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Health of Domestic Mallards (Anas platyrhynchos domestica) Following Exposure to Oil Sands Process-Affected Water Elizabeth M. Beck,† Judit E. G. Smits,‡ and Colleen Cassady St. Clair*,† †

Department of Biological Sciences, University of Alberta, Z-708, 11455 Saskatchewan Drive, Edmonton, Alberta Canada T6G 2E9 Ecosystem and Public Health, Faculty of Veterinary Medicine, University of Calgary, TRW 2D20, 3280 Hospital Drive NW, Calgary, Alberta Canada T2N 4Z6

‡

S Supporting Information *

ABSTRACT: Bitumen extraction from the oil sands of northern Alberta produces large volumes of process-affected water that contains substances toxic to wildlife. Recent monitoring has shown that tens of thousands of birds land on ponds containing this water annually, creating an urgent need to understand its effects on bird health. We emulated the repeated, short-term exposures that migrating water birds are thought to experience by exposing pekin ducks (Anas platyrhynchos domestica) to recycled oil sands process-affected water (OSPW). As indicators of health, we measured a series of physiological (electrolytes, metabolites, enzymes, hormones, and blood cells) and toxicological (metals and minerals) variables. Relative to controls, juvenile birds exposed to OSPW had higher potassium following the final exposure, and males had a higher thyroid hormone ratio (T3/T4). In adults, exposed birds had higher vanadium, and, following the final exposure, higher bicarbonate. Exposed females had higher bile acid, globulin, and molybdenum levels, and males, higher corticosterone. However, with the exception of the metals, none of these measures varied from available reference ranges for ducks, suggesting OSPW is not toxic to juvenile or adult birds after three and six weekly, 1 h exposures, but more studies are needed to know the generality of this result.

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INTRODUCTION The Alberta oil sands underlie an area of 140 000 km2 with bitumen deposits that comprise one of the largest crude oil reserves in the world.1 Bitumen is a mixture of organic compounds and trace metals that can be upgraded into more valuable forms of fuel such as crude oil.1 Approximately 20% of this resource can be extracted via surface mining; bitumen is recovered from oil-impregnated sand using Clark’s hot water separation process which requires large amounts of water (the typical ratio is 3:1).2 Some of this water is reused in the mining process, but the remainder has accumulated over the past four decades; 64 of these oil sands process-affected water (OSPW) ponds currently exist, ranging in size from less than 0.01 to over 10 km2 and with a total surface area of 182 km2.3,4 These ponds may contain residual bitumen, fine clay particulate, and several other mining byproducts including polycyclic aromatic hydrocarbons, naphthenic acids, salts, ammonia, and trace metals.2 Although the specific constituents of OSPW ponds vary with age and operator-specific mining procedures, many are toxic to wildlife, including invertebrates,5 amphibians,6 fish,7 mammals,8 and birds.9 The oil sands industry is therefore obliged by federal and provincial laws to mitigate the risks that process-affected water ponds pose to wildlife, particularly birds.10,11 In addition to resident birds, over one million migratory water birds pass through the oil sands region each spring and fall traveling to © 2014 American Chemical Society

and from the Peace-Athabasca Delta, a globally significant staging area that hosts waterfowl from across North America.9 OSPW ponds are attractive to waterfowl as they migrate because they afford short-term opportunities to rest and refuel, with a very small proportion using them as long-term opportunities to nest. This issue of bird landings and protection attracted minimal public attention until mass mortalities of migrating flocks occurred in 2008 and 2010,12 prompting provincial regulators to implement a standardized regional program to monitor bird contacts with OSPW ponds.3 In 2012, over 20 000 such contacts were reported, highlighting the incomplete solution afforded by the legally imposed bird deterrent systems put in place by industry.3 This situation creates an urgent need to understand the biological effects of OSPW pond contaminants on waterfowl. That petroleum products are toxic to birds is well documented, but research primarily addresses exposure to compounds associated with conventional oil.13 Mortality is likely for birds that come into contact with crude oil or residual bitumen (as occurs in the oil sands), because both adhere to Received: Revised: Accepted: Published: 8847

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Table 1. Summary of Health Assessment Measurements and Statistical Analysis Used to Evaluate the Effects of Repeated Exposure to OSPW on Domestic Ducksa diagnostic body condition biochemistry electrolytes metabolites enzymes endocrinology hematology

metals and minerals

statistical analysis and factorsb

measurements weight sodium, potassium, chloride, bicarbonate, anion gap Calcium, phosphorus, glucose, cholesterol, bile acid, uric acid, total protein, albumin, globulin, albumin/globulin ratio (A/G) creatine kinase (CK), aspartate aminotransferase (AST), γ-glutamyl transferase (GGT), glutamate dehydrogenase (GLDH) thyroid (triiodothyronine (T3), thyroxine (T4)) and adrenal (corticosterone) hormones packed cell volume (PCV), total and differential (heterophils, lymphocytes, monocytes, eosinophils, and basophils) white blood cell count (WBC) antimony, arsenic, barium, beryllium, bismuth, cadmium, chromium, cobalt, copper, iron, lead, magnesium, manganese, molybdenum, nickel, selenium, strontium, thallium, tin, vanadium, zinc

LMM (A): bird ID, sex, exposure (B, 1−6), treatment LMM (J and A): bird ID, sex, exposure (J: B, 1−3; A: B, 1−3, 6), treatment, weight (A only)

LMM (A) and Logistic Regression (A - EOS): bird ID, sex, exposure (4−6), treatment, weight perMANOVA* (A - Exposure 6): sex, treatment

a

Samples were collected from juvenile (J; n = 36) and adult (A; n = 29) ducks prior to any exposure (B), and following each of six exposures (1−6). LMM - Linear Mixed Effect Model; perMANOVA - permutational multiple analysis of variance; EOS, Eosinophils; *To meet analytical volume requirements samples were pooled (n = 11) within sex and experimental group. b

between pekin ducks repeatedly exposed to OSPW and those similarly exposed to control water. Because of the short duration of contact and characteristics of the water used, we hypothesized that birds would not show adverse physiological effects from this contact.

feathers, destroying their waterproofing and insulating properties and hindering thermoregulation, buoyancy, and flight.13,14 Additional toxic effects stem from ingestion of oil, bitumen and other chemical compounds during preening or through contaminated food or water.13 Ingestion of small amounts of bitumen can induce a range of toxicity affecting the gastrointestinal, hematological, immunological, neurological, reproductive, and developmental systems.13 More subtle effects include hormonal disruptions and behavioral changes. The effects on birds of exposure to recycled OSPW, which does not contain fresh tailings or residual bitumen, are rarely addressed, although this type of water can also be found in reclaimed wetlands.15 A single experiment with mallard ducklings reared on wetlands containing oil sands effluent suggested that such water is not acutely toxic.16 This possibility is supported by the relative rarity of mortalitiestypically less than 1% of the number of contacts.3 The purpose of the present study was to assess the effects of repeated, transient exposure to recycled OSPW on the health of waterfowl. The pekin duck (Anas platyrhynchos domestica), a domestic subspecies of the mallard (Anas platyrhynchos) was used as an experimental model. As there is no single measurement that can be used as an indicator of “health,” we evaluated a range of physiological parameters. Plasma biochemical analyses (i.e., electrolytes, metabolites, and enzymes) are routinely used in the veterinary profession as indicators of “healthy” vs “diseased” states; changes reflect disturbances to critical nutritional or metabolic functions, and provide information on the functional capacity of organ systems (e.g., kidney, liver).14 The endocrine system was assessed using measurement of thyroid hormones, which regulate critical physiological processes including basal metabolic rate, thermoregulation, growth, and reproduction, and corticosterone, which is associated with the stress response.17 Hematological evaluations (i.e., total and differential leukocyte counts) are used to identify anemia, which has been identified as a toxic effect of oil exposure,13 as well as secondary effects such as stress, infection, and inflammation.18 Analysis of metals and minerals included a wide range of elements, many of which are listed by the USEPA as priority pollutants.19 In summary, in order to assess the adverse effects of OSPW on waterfowl, we compared a variety of health indicators

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MATERIALS AND METHODS Bird Acquisition and Housing. Thirty-six pekin ducklings were obtained from a commercial hatchery (Golden Feather Hatchery, Chilliwack, BC, Canada) in July 2011. The ducklings were held in a field camp (Lewyk Campground, Fort McMurray, AB, Canada) in a shed with heat lamps overnight until they were 35 days old, when they were moved to wire dog kennels (each approximately 3 × 3 × 2 m). Ducks were fed approximately 250 g each day of 21% protein unmedicated Grower Crumbles ration (Hi-Pro Feeds Inc., Sherwood Park, AB, Canada). In late October 2011, the birds were moved to a private farm east of Edmonton for overwinter maintenance. Adult ducks were fed a 17% protein grower/finisher nonmedicated ration (Hi-Pro Feeds Inc.); food and well water were provided ad libitum. Experimental Protocol. To test whether effects of exposure to OSPW differed depending on life stage, birds were exposed both as juveniles and as adults. In the first trial (September 2011), we randomly assigned juvenile birds into a control (n = 11) or treatment group (n = 25). Birds remained in their experimental groups for the duration of the two-year experiment, with the exception of eight birds that shed their identification bands between trials. For the second trial (July 2012), these birds were randomly reassigned in a manner that equalized sexes between controls (n = 14) and treatments (n = 15). Control groups were exposed to local well water obtained from each of the study areas, and treatment groups to OSPW obtained from the recycled water pond at Shell Canada’s Muskeg River Mine (57°15′18.87″N, 111°29′59.69″W). For each exposure we placed birds individually in 60 L plastic tubs containing approximately 15 L of either OSPW or control water. All exposures were 6 h, except for the final exposure in trial one, which was eight. To ensure ducks were ingesting the water, they were offered approximately 100 mL of chopped greens (lettuce, spinach, chard, etc.) floated on the surface of 8848

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Table 2. Mean (± SE) Blood Biochemistry and Endocrinology Concentrations from the Final Exposure Periods in Juvenile and Adult Ducks Exposed to OSPW and Control Water juvenile analyte Na (mmol/L) K (mmol/L) Cl (mmol/L) HCO3 (mmol/L) anion gap (mmol/L) Ca (mmol/L) P (mmol/L) glucose (mmol/L) cholesterol (mmol/L) GGT (U/L) GLDH (U/L) CK (U/L) AST (U/L) total protein (g/L) albumin (g/L) globulin (g/L) A/G ratio uric acid (μmol/L) bile acid (μmol/L) corticosterone (nmol/L) T3 (nmol/L) T4 (nmol/L) thyroid (T3/T4)

control (n = 11) 147.2 2.85 103.5 20.2 26.6 2.923 2.676 9.84 5.295 2.5 1.8 1268.6 7.7 36 17.4 18.6 0.9 347.6 5.9 65.221 1.86 14.738 0.135

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.3 0.1 0.3 0.4 0.6 0.04 0.04 0.3 0.15 0.3 0.3 183.7 1.2 0.6 0.3 0.3 0.01 44.7 0.7 5.3 0.1 1.4 0.01

adult

OSPW (n = 25)

effect size (%)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 20 0.2 −6 6 0.3 −3 −2 −5 10 25 −7 19 2 −1 6 −6 −14 9 −19 10 −2 16

147.3 3.43 103.7 19 28.2 2.933 2.585 9.61 5.03 2.8 2.3 1179.5 9.2 36.8 17.2 19.6 0.9 299.3 6.4 52.703 2.047 14.498 0.158

0.4 0.1 0.4 0.4 0.4 0.02 0.04 0.15 0.11 0.2 0.2 133.7 0.9 0.5 0.3 0.4 0.02 14.8 0.6 5.3 0.1 1.12 0.013

control (n = 14) 145.1 2.55 103.7 20.1 24 3.81 1.631 9.27 4.714 29.5 1 405.4 8.1 45.1 17.6 27.4 0.7 239.4 6.5 74.903 2.007 7.869 0.397

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.5 0.1 0.7 0.8 1 0.5 0.2 0.7 0.4 19.3 0.2 45.9 0.9 2.2 0.5 1.8 0.03 27.8 1.4 8.5 0.16 1.6 0.1

OSPW (n = 15) 146.9 2.67 105 21.1 23.6 4.634 2.068 8.71 4.189 20 1.4 476.5 10.1 47.5 18.3 29.1 0.6 259.1 9.1 100.671 1.597 10.941 0.264

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.6 0.1 0.7 0.5 0.7 0.8 0.3 0.9 0.3 8.2 0.3 81.3 0.9 2.7 0.9 1.8 0.02 25.7 0.9 20.2 0.14 1.7 0.1

effect size (%)

reference range

1 5 1 5 −2 22 27 −6 −1 −32 40 18 25 5 4 6 −4 8 41 34 −20 39 −34

130−16024 2−424 100−12024 20−3024 1529 2−328 1−328 7−1828 3−628 0−1027 0.05), nor did survival. Seven birds died over the course of the two trials, but their deaths were attributed to intraspecific aggression in five cases, predation in one case, and in the last case, of an unknown cause. Clinical Biochemistry. Significant differences between experimental groups were identified for only potassium, bicarbonate, cholesterol, uric acid, bile acids, globulins, and GGT (Table 2 and SI Table S3). As an interaction (exposure × experimental group), potassium levels in juveniles were 20% greater in treatment birds than controls, but only following the final exposure (SI Figure S2; β = 0.19 ± 0.09, DF = 104, p = 0.026). In contrast, in adults, baseline potassium levels were 12% higher than controls, but this difference decreased to 5% by the final exposure (SI Figure S2; β = 0.053 ± 0.03, DF = 113, p = 0.037). Bicarbonate levels in treatment juveniles were similar at baseline but were 6% lower than controls following the final exposure (SI Figure S2; β = −0.58 ± 0.3, DF = 104, p = 0.04). In adults they were 7% less than controls at baseline, and 5% greater by the end of the trial (SI Figure S2; β = 0.36 ± 0.2, DF = 113, p = 0.03). Significant interactions existed between experimental group and exposure for cholesterol in juveniles (β = −0.12 ± 0.05, DF = 104, p = 0.03), and uric acid in adults (β = 6.8 ± 2.2, DF = 112, p = 0.0024). Concentrations in both were higher in treatment than in control birds at baseline (6%, 47% respectively), but by the final exposure this ratio had reversed or diminished (SI Figures S2, S3; −5%, 8% respectively). In adults, we also found differential effects of experimental group by sex for bile acids (β = −5.8 ± 1.9, DF = 25, p = 0.0061) and globulins (β = −4.4 ± 1.8, DF = 25, p = 0.02). Specifically, OSPW treated females had higher levels of bile acids (37%) and

Table 3. Mean (± SE) Hematology of Adult Ducks Exposed to OSPW and Control Water from the Final Exposure Perioda analyte PCV (%) WBC (×109/L) basophils (×109/L) eosinophils (×109/L) heterophils (×109/L) lymphocytes (×109/L) monocytes (×109/L)

control (n = 14)

OSPW (n = 15)

effect size (%)

reference means26

43.6 ± 1 19.4 ± 2.3

43.5 ± 1.1 15.7 ± 1.4

−0.4 −19

36.15 19.5

0.342 ± 0.1

0.307 ± 0.1

−10

1.13

0.365 ± 0.1

0.078 ± 0.03

−79

0.12 12.11

13.87 ± 1.9

11.637 ± 1.3

−16

4.251 ± 0.4

3.176 ± 0.4

−25

0.476 ± 0.1

0.522 ± 0.1

10

6.035 0.12

a

Effect size is described as the percentage difference relative to control ([(OSPW - Control)/Control]*100), and is based off raw data. 8850

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differences were most pronounced in baseline samples but declined thereafter. Decreases in potassium were found in adult pigeon guillemots sampled in sites affected by the Exxon-Valdez oil spill,33 while other studies found no effect in mallard ducklings31 or adults34 fed crude oil. Elevations in potassium can be associated with severe renal disease,24 which was not present in our birds based on normal uric acid levels. Further, we did not expect differences to be present in baseline samples if they occurred because of experimental manipulation. Similarly, bicarbonate levels following final exposures were higher in controls for juvenile birds and higher in OSPW treated birds for adults; however, none of the bicarbonate values were sufficiently decreased or increased to indicate metabolic acidosis or alkalosis, respectively. Biochemical evaluation of the kidney found little evidence change (phosphorus, potassiumas above). Uric acid levels in adults were higher in OSPW treated birds at baseline, but this difference decreased with repeated exposure, and both experimental groups had mean uric acid levels within normal reference intervals.14 Oil-induced changes to uric acid concentrations are not well described, although one study found elevated concentrations in oiled female mallards postoiling while no effect was seen in males.35 Other studies have found no such differences in pigeon guillemots,36 or mallards exposed to crude oil.37 There was no increase in AST or GLDH, enzymes that are released as a result of hepatocyte injury. Adults exposed to OSPW had lower GGT levels following the final exposure. Decreased GGT activity has been reported following fuel oil exposure in female yellow-legged gulls (Larus michahellis), which, researchers hypothesized, may be attributable to birds’ reduced ability to increase GGT in response to a physiological condition, such as egg production.38 However, we saw no such sex difference and GGT is primarily a nonparametric analyte, for which increases are more significant than decreases.17 Bile acids were also higher in adult females exposed to OSPW; increases are both sensitive and specific to hepatic disease but in concentrations much higher than seen in this study.17 One study found no effect on bile acids in pigeon guillemot (Cepphus columba) sampled following the Exxon-Valdez oil spill,33 while another found statistical differences which were again not supported by falling outside biological reference intervals.36 Cholesterol levels were lower in OSPW treated juveniles following the final exposure. Previous work has shown decreased,39 increased,40 and unchanged30,31 cholesterol in response to oil exposure suggesting other factors are involved in the regulation of cholesterol levels in these birds. Evaluation of plasma proteins was not supportive of toxic effects on the production of albumin or immune globulins. In adults, female treatment birds had higher globulins compared to controls. Elevated globulins fractions are seen with inflammation, increased antibody production, and preceding egg production in females.24 Both of these factors may have contributed to our results as female adults laid eggs intermittently and many birds had inflammatory foot lesions. Laboratory evaluation of hormone levels revealed no differences between experimental groups for T3 or T4. We did find a higher thyroid hormone ratio in juvenile males, and higher corticosterone in adult males exposed to OSPW. Previous studies in birds exposed to crude oil have described increases in corticosterone, or T4, while others described no endocrine related changes.13 A series of field experiments on the effects of OSPW in tree swallows did find elevated plasma

exposure four, and 10% higher in treatment birds following the last exposure (SI Figure S3; exposure × experimental group: β = 0.24 ± 0.09, DF = 54, p = 0.0016). Metals and Minerals. The multivariate analysis of metals and minerals revealed no differences between experimental groups (SI Table S4; F1 = 0.96, p = 0.47). However, univariate models showed that treatment birds had 244% higher levels of vanadium than controls (Figure 1; F1 = 8.33, p = 0.032), and, as an interaction, molybdenum levels in females were 28% higher in treatment than in control birds, as compared to males, where levels in treatment birds were 20% less than controls (Figure 1; F1 = 7.057, p = 0.048).

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DISCUSSION The objective of this study was to determine whether repeated, short-term exposure to OSPW would adversely affect the health of ducks, as determined by measurements of physiological indicators. Using captive pekin ducks as a model, we did not find consistent or strong evidence of such effects based on body mass; measures of biochemical, endocrinological, or hematologic analytes; or metal or mineral levels. As our intention was to evaluate for effects on health, rather than simply numeric differences between control and treatment groups, this conclusion is based not only on statistical analyses, but also on comparison of our results to published reference intervals.24−29 Reference intervals, or reference ranges, are critical in interpreting the biological relevance of differences in health variables, as many biological measures will vary substantially depending on the age, sex, and reproductive status of the source population.24 When specific intervals for pekin ducks were not available, comparisons were made using those for other avian species, preferably those with a close taxonomic relationship, as is practiced in avian medical and health assessments. Where individual and mean values for both control and experimental groups were within the most appropriate reference intervals that could be compiled, any statistical differences between groups were not considered indicative of an adverse effect on the health of the birds. For similar reasons, the possibility of variability within the data masking differences among treatments was not considered to be of concern. We found no evidence that exposure to OSPW influenced the body mass of adult birds. Reduction of growth and weight in birds as a result of oil toxicity are well-established in the literature,16,30,31 although mechanisms are not described. The birds in this study were fed ad libitum; results might have been different under conditions of restricted food intake. Further, direct comparisons between the literature and the present study are not appropriate due to differences in the type of oil used, the exposure conditions, and biological differences between species, and even subspecies.13 For example, A. platyrhynchos weigh 1.2 kg on average,14 while A. platyrhynchos domesticas have been domesticated and bred for meat production, and weigh 3.2 kg on average.32 Clinical biochemistry analysis included evaluation of plasma electrolytes, metabolites, and enzymes. We found no effect on glucose, calcium/phosphorus regulation, and no evidence of muscle injury (creatine kinase). Electrolyte and acid−base balance analytes were generally unaffected (sodium, chloride, and anion gap) with the exception of potassium and bicarbonate. In juvenile OSPW treated birds, we found that potassium levels following the final exposure were higher than controls, whereas in adults 8851

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T3 and T4,41 as well as elevated corticosterone in males nestlings on one of two experimental wetlands.42 Methodological differences between that work and our study, namely study species, age of birds, and exposure type and duration, limit the comparisons that can be made. Increased corticosterone in the OSPW treated males suggests a higher stress level, perhaps in response to the difference in water quality. A study of longer-term exposure and chronic stress, such as could be determined from feather corticosterone analysis would be required to provide more compelling evidence of increased stress than is possible through the measurement of plasma corticosterone, as a short-term indicator. No indication of toxicity was apparent in blood analysis (PCV, total or differential WBC counts). Monocyte numbers were lower in treatment birds following the fourth exposure, but comparable by the final exposure. The mean value of OSPW treated birds was not; however, low enough to be of clinical concern, and no evidence of oil-induced changes specific to monocyte numbers has been reported.43 Examination of metals and minerals found higher blood vanadium levels in all treatment birds, and higher molybdenum in female treatment birds. Reference intervals are not available to help assess the biological significance of these elevations; however, dietary studies on vanadium have shown that lowlevel exposure is not acutely toxic to mallards.44,45 At higher levels, this metal tends to accumulate primarily in the liver and kidney,45,46 or in some cases, the bone and liver.44 A single study examined concentrations of vanadium in the liver and kidney of oiled seabirds as a biomarker for exposure but found no differences from control birds,47 although, another study found that vanadium accumulation in wild birds in Japan was reflective of other types of environmental contamination.46 Vanadium is a metal found in high concentrations in petroleum,44 therefore, in the current context at the levels observed, we suggest it could be a biomarker of exposure, but likely not toxicity. Molybdenum is an essential micronutrient that has been shown to have adverse effects on growth, reproduction, and survival, but only with high dietary concentrations ranging from 200 to 6000 mg/kg.48 While few toxic effects from repeated short-term exposure to OSPW were identified in this study, this information can be broadly generalized. There is huge variation in the exposure risk of different OSPW ponds; they vary in their size, chemical constituents, toxicity, accessibility, and attractiveness to birds. Following bitumen extraction, remaining liquid waste is pumped to the tailing’s disposal site.1 As fresh tailings are delivered into ponds, they spontaneously divide into three layers: solids that settle to become mature fine tailings (MFT), suspended fine particles such as silt and clay, and a water layer available for recycling.1 As well, residual bitumen mats can accumulate at the edges of pond surfaces.2 The water layer is extracted and stored in recycled water ponds until its reuse in the bitumen extraction process. This recycled water generally does not contain visible bitumen, but each time the water goes through the extraction process, its chemistry changes: for example, dissolved salts accumulate.2 High concentrations of trace metals, ammonia, and total dissolved solids are also possible causes of health concerns across OSPW ponds,2 as well as the potential for chronic effects on aquatic and terrestrial organisms inhabiting reclaimed wetlands.2 Our research investigated the effects of short-term exposure to recycled OSPW on birds, while early toxicological research focused mainly on the acute toxicity of polycyclic aromatic

hydrocarbons (PAHs) and naphthenic acids (NAs) to aquatic organisms. Naphthenic acids in particular have been the focus of a large body of research and have caused adverse effects in fish,49−51 amphibians,52 and mammals.8 Only one study to date has examined the effects of NAs in birds. Gentes et al.53 found that a range of health indicator variables were unchanged in wild tree swallow nestlings experimentally exposed throughout most of their nestling period, although an increase in hepatic extramedullary erythropoiesis was noted. The toxicity of OSPW varies considerably and is a complex interaction between the compounds present in the water, the type, and age of the pond, as well as any remediation strategies that are occurring.2 Potential cumulative effects of other stressors, such as food shortages, or adverse weather are also likely to influence effects of OSPW.54 Given the variability in the character of ponds currently on the landscape, our work cannot rule out the possibility of adverse health effects on birds receiving short-term exposure to OSPW obtained from other sources. Further toxicity testing on a wider range of OSPW ponds, perhaps in a gradient of those considered least to most toxic would be a logical next step. Future studies could also replicate the different ways in which wild birds interact with their environment. Birds often feed on materials present in process-affected water ponds, creating additional pathways for contaminant exposure by ingestion of bio accumulated toxins in sediment,55 plants,56 and invertebrates.57 Ingestion of grit has also shown to be a significant route of contaminant exposure for NAs, and oil and grease in mallards.58 There are also species-specific differences in the ways birds interact with their environment. For example, birds that feed on the water’s surface interact differently with the pond than those that dive to feed. Since we did not replicate all possible exposure routes, the risk of adverse health effects associated with spending time on OSPW may be greater, and may vary by species. Additionally, not all health effects are detectable by blood analyses. For example, multiyear examination of tree swallows nesting on reclaimed wetlands has shown potential for negative reproductive effects through reduced brood sizes, increased nestling mortality, and reduced fledgling success.54 Waterfowl may also be sensitive to such effects, especially those that nest on or in proximity to OSPW ponds. It is, in fact, likely that the small percentage that become seasonal residents are at greater risk to toxicity associated with oil sands contaminants than those with transient contact, such as was modeled in these trials. For example, mercury measured in 2012 in the eggs of California and ring-billed gulls (Larus californicus and L. delawarensis) nesting downstream of the oil sands have increased significantly since the first years of sampling (1977 and 2009 respectively).59,60 The negligible adverse physiologic effects documented in this study should not be taken as an overall indicator of a general lack of risk from the oil sands; there is in fact a large body of literature detailing the toxic effects of bitumen and conventional oils on birds. However, the results of our study may help to evaluate the relative risk posed by different facets of the oils sands mining processes, with important implications for a holistic approach to bird protection. In particular, segregation of pond constituents (e.g., with technologies such as booming and skimming) and encouraging smaller-scale deterrent strategies targeting sites containing the more toxic derivatives of the oil sands extraction processes may be warranted. This approach could increase deterrent efficacy where it is most 8852

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needed, reduce the tendency for birds to habituate to deterrent activity, and reduce the exposure of humans and wildlife to high levels of noise pollution.3

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ASSOCIATED CONTENT

S Supporting Information *

Additional details for methods and results, supplemental figures and tables. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (780) 492-9685; fax: (780) 492-9234; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding for this research was provided by the Research on Avian Protection Project with funding from Alberta Justice and the Natural Sciences and Engineering Research Council. We thank all project staff and summer assistants, the Fedun Family, especially Owen, for housing the ducks and Chelsea Hoff at Shell Canada Albian Sands for assistance with water collection. We thank Christine Godwin Shepard for collection of 2011 field data. This project was approved by the Animal Care and Use Committee for Biosciences through the Universities of Calgary and Alberta Animal Care Committees.

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REFERENCES

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