A FUR-MIDABLE PROBLEM: PATTERNS OF LEAD AND MERCURY EXPOSURE

IN NEW ORLEANIAN CATS AND DOGS

AN HONORS THESIS

SUBMITTED ON THE TWENTY-SEVENTH DAY OF APRIL, 2021

TO THE DEPARTMENT OF ECOLOGY AND EVOLUTIONARY BIOLOGY

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

OF THE HONORS PROGRAM

OF NEWCOMB-TULANE COLLEGE

TULANE UNIVERSITY

FOR THE DEGREE OF

BACHELOR OF SCIENCES

WITH HONORS IN ECOLOGY AND EVOLUTIONARY BIOLOGY

BY

_________________________________

Caroline Camus

APPROVED:

Jordan Karubian, Ph. D.

Director of Thesis

Alex Gunderson, Ph.D.

Second Reader

Laura McKinney, Ph.D.

Third Reader

ii

Caroline Camus. A Fur-midable Problem: Patterns of Lead and Mercury Exposure in

New Orleanian Cats and Dogs.

(Professor Jordan Karubian, Ecology and Evolutionary Biology)

Anthropogenic activity has greatly accelerated the bioavailability of toxic heavy

metals, resulting in increased exposure, especially in urban centers. Lead and mercury, in

particular, pose a significant health risk to both humans and animals, but whereas impacts

are relatively well-studied in humans, far less is known about contamination levels and

potential impacts on animals, particularly pets. To fill this knowledge gap, we compared

baseline blood lead and total mercury concentrations of 23 domestic cats, Felis catus, and

30 domestic dogs, Canis lupus, from the city of New Orleans, LA. Based on these

species’ documented differences in physiology, behavior, diet, and environment, we

predicted domestic cats would present with relatively lower lead and higher total mercury

concentrations, whereas domestic dogs would present with the inverse. Consistent with

this prediction, dogs had higher blood lead concentrations, and cats had higher blood total

mercury concentrations. Also, pets’ home address, categorized by zip code, was

correlated with pets’ blood lead, but not their blood total mercury concentrations, and

both toxins were present in every pet sampled, though often at low concentrations.

Obtaining additional data on baseline heavy metal concentrations in pets and exploring

impacts for health and well-being represents a clear priority for future work.

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ACKNOWLEDGEMENTS

This piece would not exist without the support of an extraordinary group of people.

I would first like to express my sincerest gratitude to Dr. Jordan Karubian, whose

support, expertise, and advice were invaluable throughout this process. His unwavering

faith in my work continually inspired me to think more deeply about the natural world.

I want to thank Annelise Blanchette, Dr. Myra Finkelstein, Dr. Carl Lamborg, Dr. Craig

LaMarsh, Dr. Melinda Camus, the Southern Animal Foundation staff, and the Karubian

lab partners for their diligent efforts in helping me collect, analyze, and interpret my data.

I also wish to acknowledge the many New Orleanian residents and their companion

animals who allowed me to learn from them. Their interest, and the financial support of

the Newcomb-Tulane College Dean’s Office, Newcomb College Institute, and the Morris

Animal Foundation helped make my goals a reality.

In addition, I would like to thank my parents and brother for their compassion and

motivation during this process and throughout my life. Finally, I would like to thank my

best friends, William Driscoll and Johnathan Michka, for entertaining my incessant

discussions and always providing comic relief, even on the toughest days. I dedicate this

project in loving memory of my cat, Loretta, who inspired this work and is missed daily.

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TABLE OF CONTENTS

Page No.

ABSTRACT ........................................................................................................................ ii

ACKNOWLEDGEMENTS ............................................................................................... iii

TABLE OF CONTENTS ................................................................................................... iv

LIST OF TABLES ............................................................................................................... v

LIST OF FIGURES ............................................................................................................ vi

1 INTRODUCTION .......................................................................................................... 1

2 METHODS ..................................................................................................................... 4

2.1 Study site ........................................................................................................... 4

2.2 Sample collection .............................................................................................. 4

2.3 Pet survey .......................................................................................................... 5

2.4 Blood lead evaluation ........................................................................................ 5

2.5 Blood total mercury evaluation ......................................................................... 5

2.6 Basophilic stippling analysis ............................................................................. 6

2.7 Statistical analysis .............................................................................................. 6

3 RESULTS ....................................................................................................................... 8

3.1 Between species ................................................................................................. 8

3.2 Environmental correlates ................................................................................... 8

3.3 Basophilic stippling ........................................................................................... 8

4 DISCUSSION ............................................................................................................... 10

REFERENCES .................................................................................................................. 15

TABLES AND FIGURES ................................................................................................. 18

APPENDICES ................................................................................................................... 24

v

LIST OF TABLES

TABLE 1. New Orleanian pets’ blood lead and total mercury concentrations

TABLE 2. AIC model selection results

TABLE 3. Significance values of pets’ blood lead and total mercury concentrations by

zip code and sex

TABLE 4. Contingency analysis summary

vi

LIST OF FIGURES

FIGURE 1. Map of home addresses for animals included in this study superimposed on a

map of soil lead concentrations of New Orleans post-Katrina, with permission from

Mielke et al. 2017. The pets’ home addresses are represented with circles (closed = cats,

open = dogs). All blood samples were collected at SAF, represented with a star.

FIGURE 2. Log-transformed blood lead (in ng/ml) and blood total mercury

concentrations (in ng/g) of New Orleanian domestic cats and dogs. Dogs have higher

blood lead concentrations relative to the cats, but cats have higher blood total mercury

concentrations relative to the dogs.

1

1 INTRODUCTION

Although heavy metals are naturally occurring elements originating in the Earth’s crust,

anthropogenic activity has accelerated their dispersal, resulting in environmental

pollution and associated health concerns (Tchounwou et al. 2012, Jaishankar 2014). Lead

(Pb) and mercury (Hg), in particular, pose a significant worldwide health risk despite

recent mitigation and rehabilitation initiatives (Järup 2003). For example, extensive

research on human subjects and lab animals such as Rhesus monkeys (Macaca mulatta;

Lasky et al. 2005), Wistar rats (Rattus norvegicus; Fioresi et al. 2014), and mallard ducks

(Anas platyrhynchos; Żarski et al. 2017) illustrate the metals’ detrimental effects on

development, cognition, behavior, reproduction, and physiological function (Park &

Zheng 2012). On account of lead and mercury’s historical use in many manufacturing,

farming, and household products, coupled with their spread via fossil fuel emissions,

these metals are pervasive in many urban areas (Tchounwou et al. 2012, Driscoll et al.

2013). These toxins present shared concerns for city-dwelling humans and animals, but

far less is known about their impact on urban animals, particularly pets.

The existing literature focused on pets primarily addresses the most common

sources of lead and mercury and how to detect toxicosis, likely because it was intended

for diagnostic purposes. In contrast, exposure levels among wildlife and pets are far less

documented than in humans, despite these animal populations’ high susceptibility.

Relatively more is known about exposure and impacts on wildlife. Acute exposure to lead

via leaded fishing gear or ammunition is relatively common in Common Loons (Gavia

immer; Grade et al. 2017) and the California condor (Gymnogyps californianus;

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Finkelstein et al. 2012). Chronic lead exposure has been recorded in New York City’s

rock pigeons (Columba livia; Fayme & Calisi 2016), New Orleans’s northern

mockingbirds (Mimus polyglottus; McClelland et al. 2019), and Washington, D.C.’s

passerine birds (Roux & Marra 2007). Mercury affects a wide variety of wild organisms

even in remote locations, such as sharp-skinned hawks (Accipiter striatus; Rimmer et al.

2009), wild mink (Mustela vison; Klenavic et al. 2008), and polar bears (Ursus

maritimus; Dietz et al. 2013), targeting organisms at the top of the food chain most

(Kessler 2013, Faganeli et al. 2013). These toxins are also likely to affect domestic

animals such as dogs and cats, but much less is known about exposure in these groups

(below). This knowledge gap limits the ability of veterinarians, pet owners, ecologists,

and government officials to evaluate and address potential health risks.

Lead and mercury each have multiple chemical forms. Of these forms, pets most

often interact with inorganic lead and methylmercury. Inorganic lead particles are inhaled

or ingested via the air, paint, soil, consumer products, or wild prey with elevated lead

concentrations (Tarragó & Brown 2017, Buenz 2016). Clinicians diagnose lead poisoning

in dogs more often than in cats, suggesting dogs are exposed to this toxin at greater

frequencies (Blakley 2013a). Dog’s propensity to dig in the dirt, chew baseboards, and

ingest leaded consumer products likely contribute to their elevated exposure (Lundgren

2004, Newman 2007). In contrast, methylmercury is most commonly ingested from

marine fish and is found in some pets’ commercial diets (Driscoll et al. 2013, Luippold &

Gustin 2016). While dog foods are of negligible concern, Luippold and Gustin (2016)

advised that certain wet-based cat foods contain exceedingly high concentrations of total

mercury. However, evaluating a pets’ exposure to both of these metals is challenging

3

because the source is often unknown, clinical signs vary, and the available diagnostics

generally lack sensitivity and specificity. For example, the presence of hematologic

abnormalities, such as basophilic stipples in pets’ erythrocytes, represent a highly

contested method of diagnosing lead poisoning since its often suggestive but not

confirmatory of the condition (Blakley 2013a, Weiss & Tvetden 2004, Valenciano et al.

2014).

Indeed, to our knowledge, there are no baseline surveys available comparing lead

contamination in dogs and cats, and work comparing these species’ mercury exposure is

limited to a single study (Sekai et al. 1995; below). To fill this knowledge gap, we

compared blood lead and total mercury concentrations in domestic cats, Felis catus, and

dogs, Canis lupus from the city of New Orleans, LA (USA). Our overarching hypothesis

was that lead and mercury exposure would be widespread among domestic cats and dogs

but that patterns of exposure would differ between the two species. Based on their

divergent physiologies, behaviors, diets, and environments, we predicted that domestic

cats would present with relatively low lead and higher total mercury concentrations,

whereas domestic dogs would present with higher lead and lower total mercury

concentrations. In addition, we used our data to gain preliminary insights into the

behavioral, dietary, and environmental correlates of exposure as well as evaluated the

effectiveness of a disputed lead toxicity diagnostic: basophilic stippling analysis. Our

findings are consistent with chronic, low-level exposure, highlight some potential sources

of exposure, and suggest that additional testing and studies on diagnostic techniques and

health impacts are warranted.

4

2 METHODS

2.1 Study site:

The domestic cats and dogs sampled for this study resided in the Greater New Orleans

Area (in Orleans Parish, Jefferson Parish, & St. Bernard Parish, specifically) and were

patients of the Southern Animal Foundation (SAF), a veterinary clinic located in the

Lower Garden District. The majority of pets lived in the Uptown neighborhood, but

animals living in the French Quarter, Marigny, the Lower 9th Ward, Lakeshore, and

Gentilly neighborhoods were also represented. A published census-tract-based survey of

New Orleans demonstrates that these neighborhoods differ in soil lead concentration

(Mielke et al. 2017) (Fig. 1).

2.2 Sample collection:

Between August and November 2019, veterinarians from SAF collected approximately

2.0 ml of whole blood in a tan (K2EDTA) low lead Vacutainer® tube from 23 cats (12

females, 11 males) and 30 dogs (14 females, 15 males, 1 unknown). The pets enrolled in

the study belonged to New Orleans residents, who offered their verbal consent during

their pet’s annual wellness examination. Upon informed consent, veterinarians sampled

the cat’s blood from an accessible peripheral vein using proper manual restraint, as

specified by Tulane University’s IACUC and SAF’s SOP. The blood tubes were stored at

-20°C pending analysis. Prior to freezing, two blood smears per pet were made of

peripheral blood and were stained with modified Wright’s stain (Wescor, Inc. Logan, UT,

USA).

5

2.3 Pet Survey:

At SAF, pet owners completed a survey detailing their animal’s signalment, living

arrangement, and behavior. Participants were briefed on the study’s objectives, via a

standardized letter (Appendix A), prior to completing the questionnaire (Appendix B).

2.4 Blood Lead Evaluation:

Whole blood samples were analyzed following published methods (Finkelstein et al.

2003) under established trace metal clean techniques (Flegal & Smith 1992).

Whole blood. Blood (~0.1mL) was transferred from low lead Vacutainers into trace metal

clean micro-centrifuge tubes, weighed, and digested as follows: 0.250 mL of

concentrated HNO3 (optima, Fisher Scientific) was added to each sample, and samples

were digested cold for 10 hours. 0.125 mL 30% H2O2 was added and diluted with Milli-Q

water for an approximate final concentration of HNO3 of ~6%. Samples were vortexed

and left to sit overnight before analysis.

Blood lead analysis. Lead concentrations were determined by inductively coupled plasma

mass spectrometry (ICP-MS, Finnigan MAT Element magnetic sector), measuring

masses of 208Pb and 205Tl (used as an internal standard). Standard reference material

NIST SRM 955c was digested using the methods described above with an average

recovery (n = 3) of 87% ± 2.1% RSD.

6

2.5 Blood Total Mercury Evaluation:

Whole blood samples (~0.4 mL) were analyzed for total mercury in Dr. Tubbs’s

laboratory in the Ocean Sciences Department at the University of Santa Cruz, using a

Direct Mercury Analyzer-80 (Murata et al. 2019). Total mercury concentrations were

calibrated with aqueous standards (SPEX CertiPrep, Metuchen, NJ), and concentrations

were verified by standard reference material NIST SRM 955c with an average recovery

(n = 3) of 100% ± 2.7% RSD.

2.6 Basophilic stippling analysis:

All blood smears were evaluated using a 100X oil-immersion objective to search for the

presence of basophilic inclusions within erythrocytes. Each pet was given a score on a 0-

4 scale, based on the frequency of basophilic inclusions sited (0 = no stipples, 4 =

widespread stippling). In the absence of reticulocytosis, inclusions are suggestive of lead

poisoning in small animals (Blakley 2013a, Osweiler & Imerman 2012, Valenciano et al.

2014).

2.7 Statistical Analysis:

We operationalized our variables as follows: significant lead exposure = >50ng/ml, lead

toxicity = >350ng/ml, significant mercury exposure = >100ng/g, mercury toxicity =

>6000ng/g, as specified in Langlois et al. (2017) and Blakley (2013b), respectively. Prior

to analyses, the blood lead and blood total mercury data were checked for normality

(Shapiro-Wilk’s test) and homogeneity of variance (Levene’s test). The lead and total

mercury data were log-transformed to meet the assumptions of normality. To test for

7

differences in blood lead and blood total mercury concentrations between species,

separate unpaired two-sample t-tests were used for each contaminant type. We chose to

run separate tests instead of combining both in a single model including interaction terms

because there was no reason to believe that lead and mercury uptake mechanisms would

be related. One dog lacking these data was removed from the two-way ANOVA analyses

of differences between sex, species, and blood lead and blood total mercury

concentrations. AIC model selection was used to distinguish among a set of possible

models describing the relationship between blood lead or total mercury concentrations

and zip code, sex, and the environmental variables (house age, pet age, time spent

inside/outside, and pet weight) for each species. An ANOVA was run on the best fit

model for each species. Linear regression analyses were performed on untransformed

data to test for correlations between (1) dogs’ basophilic stippling scores and their blood

lead concentrations and (2) pets’ lifestyle characteristics and their blood lead and total

mercury concentrations. All analyses were performed in R (R Core Team, 2018).

8

3 RESULTS

3.1 Between species:

Species designation (cat vs. dog) was a significant predictor of blood lead (t = -3.50, p <

0.001) and blood total mercury concentrations (t = 5.18, p < 0.0001): dogs had higher

blood lead values and cats had higher blood total mercury concentrations (Table 1, Fig.

2). In both species, sex was not a significant predictor of their blood lead (F = 0.117, p =

0.735) or blood total mercury (F = 0.795, p = 0.377) concentrations.

One cat and five dogs had blood lead concentrations consistent with significant

lead exposure (>50 ng/ml), but none met the criteria for lead toxicosis (>350 ng/ml). One

cat, but no dogs, had blood total mercury concentrations consistent with significant

mercury exposure (>100 ng/g). Neither species displayed concentrations consistent with

mercury toxicosis (>6000 ng/g).

3.2 Environmental correlates:

The best-fit models did not include the environmental variables (Table 2). Zip code and

sex were not significant predictors of blood lead or blood total mercury concentrations in

cats. Zip code, but not sex, was a significant predictor of blood lead and blood total

mercury concentrations in dogs (Table 3). None of the environmental variables were

correlated with blood lead or total mercury concentrations (Table 4).

3.3 Basophilic stippling:

9

Basophilic inclusions were present in the erythrocytes of 0 cats and 4 dogs, who scored a

1, 1, 2, and 3 on the 0-4 stippling scale, respectively (Appendix C). Basophilic stippling

score did not predict the dogs’ blood lead concentrations (R2 = 0.02, p = 0.20).

10

4 DISCUSSION Domestic animals can serve as powerful sentinels of human health, but baseline

measurements illustrating the frequency and chronicity of their exposure to toxic heavy

metals, are largely non-existent (Bischoff et al. 2010, Beck et al. 2020, Esposito et al.

2018). This represents an important blind spot because dogs and cats, like humans, are

likely to experience widespread heavy metal pollution, particularly in metropolitan areas

(Esposito et al. 2018, Tomza-Marciniak et al. 2012). To address this knowledge gap, we

collected and analyzed the blood lead and total mercury concentrations of New Orleanian

pets. By comparing these data between species, and analyzing our findings alongside

various correlates of exposure, our goal was to shed light on the pervasiveness and

potential dispersal mechanisms of these toxins on urban companion animals and

contribute to a growing body of knowledge on the effects of anthropogenic activity on the

health of urban animals, which in turn can benefit pets and people.

Our results support our main hypothesis that species designation (dog vs. cat)

significantly affect pets’ baseline heavy metal concentrations. Due to the feasibility and

relative expense of these analyses, pets are typically tested for lead or mercury only after

displaying significant clinical signs or having a known source of exposure (Blakley

2020a, Gribbles Vet. Path. 2021). Indeed, to our knowledge, no studies comparing dogs

and cat’s baseline blood lead concentrations exist. A slightly larger number of studies

exist comparing dogs and cats’ total mercury concentrations. For example, Sekai et al.

(1995) found that cats living in Japan had significantly higher hair total mercury

concentrations than dogs, particularly when cat owners prepared their pets’ meal.

11

In general, the pets’ blood lead concentrations were consistent with chronic but

low-level exposure to lead and mercury. For example, the mean lead concentrations of

8.76 ng/ml for cats and 25.82 ng/ml for dogs are 85% and 55% lower, respectively than

those recorded for free-living northern mockingbirds (Mimus polyglottos; McClelland et

al. 2019) and 99% lower than those recorded for Cuban anoles (Anolis segrei;

unpublished data) sampled from the same areas where these pets live. This may reflect

the fact that pets sampled in the current study spend relatively little time outdoors and

that they are eating foods unlikely to have appreciable concentrations of lead. Also, only

pets in seemingly good health with invested owners that used veterinary services, were

included in this study. However, even low concentrations of lead may be hazardous to

animals and blood lead and total mercury concentrations are best interpreted with the

patient’s clinical signs and history of exposure in mind, making it difficult to evaluate a

population’s baseline values without extensive life history information (Plumlee 2004).

Given the scope of this study and because normal values for domestic animals are not

widely available, we did not measure the pets’ erythrocyte ALAD activity, urinary ALA

levels, or zinc protoporphyrin levels (Plumlee 2004). In addition, we avoided hair and

fecal analysis, since these diagnostics are considered minimally useful in animals

(Plumlee 2004).

Even with extensive analyses of each pets’ lifestyle, health history, clinical signs,

and diet, clinicians struggle to discern and eliminate the exact sources of pets’ heavy

metal exposures. To shed light on this question, we conducted contingency analyses

between various environmental variables and pets’ heavy metal concentrations. The

significant association between home address, categorized by zip code, and blood lead

12

concentrations in dogs, but not cats, aligns with the idea that dust and soil contamination

represent a significant source of inorganic lead exposure among dogs. Dogs generally

spend more time outside than cats, and previous work has shown that these New Orleans

neighborhoods vary strongly in soil lead concentrations (Mielke et al. 2017), also

consistent with this idea. In contrast, the lack of relationship between location and

exposure to either heavy metal is consistent with the idea that food contamination, likely

unaffected by home address, is organisms’ main source of methylmercury exposure

(Mielke et al. 2017, Tchnounwou et al. 2012, Blakley 2020b).

Many of the results were expected and served as controls (e.g., a lack of

association between the amount of time pets spend inside vs. outside and their blood total

mercury concentrations). However, some of the results were not anticipated and

disagreed with the literature such as the independence between pets’ blood lead

concentrations and the age of their home. Older homes contain significantly higher

amounts of lead paint and 90% of residential homes in New Orleans were constructed

before the EPA banned lead paint in 1978 (NOLA_Unleaded 2019, U.S. Environmental

Protection Agency n.d.). Children and domestic animals living in low-income, older

homes are at the highest risk for lead toxicity (Kim et al. 2002, Barlow et al. 2015,

Bischoff 2010). Without testing each of the pets’ homes for lead paint or possessing

information detailing the homes’ renovation history, it is challenging to offer an

explanation for the lack of this association.

We also evaluated the efficacy of the contested basophilic stippling analysis and

found no correlation between a dog’s blood lead concentrations and their stippling score.

Basophilic stippling generally suggests lead intoxication in small animals but can be

13

attributed to other causes, such as regenerative anemia (Valenciano et al. 2014). In

addition, the absence of stipples does not necessarily mean that lead intoxication is not

occurring (Wismer 2013). These limitations make it challenging to determine whether

only 7.5% of the pets had inclusions because the general population is not experiencing

lead exposure or because we lack a more specific and sensitive diagnostic (Osweiler and

Imerman 2012). This contributes to the body of literature hazarding clinicians against

administering potentially dangerous and expensive chelation therapy following basophilic

inclusion sightings alone (Osweiler and Imerman 2012, Valenciano et al. 2014).

In summary, our findings highlight the importance of using species-specific and

heavy metal-specific strategies to understand the scope of lead and mercury

contamination in pets, and urban wildlife more generally. Even at extremely low

concentrations, these heavy metals pose a significant health risk to companion animals,

urban wildlife, and people (U.S. Food and Drug Administration 2019, Tchounwou et al.

2012, Godwell et al. 2018, Ali et al. 2019). Because people’s symptoms and animals’

clinical signs in response of these toxins vary in strength, scope, and frequency, often not

appearing before the toxins have already caused damage, the importance of testing

organisms’ baseline concentrations frequently cannot be overstated (U.S. Center for

Disease Control and Prevention 2021, Agency for Toxic Substances and Disease Registry

2015). These data would likely encourage greater exploration of the mechanisms,

pathology, and consequences of sub-lethal heavy metal exposure, subsequently

improving organisms’ health outcomes, as well as allow concerned parties to alter their

behavior, diagnostics, and even laws, accordingly. An interdisciplinary OneHealth

approach, promoting discussions between urban ecologists, sociologists, veterinarians,

14

healthcare providers, and lawmakers is likely to prove useful in tackling these issues

moving forward.

15

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Valenciano, A., Cowell, R. Rizzi, T., & Tyler, R. (2014). Atlas of Canine and Feline Peripheral Blood Smears. (D. B. DeNicola, Ed.). Elsevier, Inc.

Żarski, J. F., Skibniewski, M., Skibniewska, E., Żarski, T. P., & Majdecka, T. (2017). The Presence of Mercury in the Tissues of Mallards (Anas platyrhynchos L.) from Włockławek Reservoir in Poland. Biological trace element research, 176(2), 384-390.

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TABLE 1

Table 1. New Orleanian domestic cats’ and dogs’ a) blood lead and b) total mercury concentrations

a) b)

Blood Pb in ng/ml Cats Dogs Blood Hg in ng/g Cats Dogs

n 23 30 n 23 30

min 0.34 0.90 min 0.21 0.06

25% 1.81 6.41 25% 1.42 0.37

50% 3.57 10.92 50% 4.28 0.67

75% 10.61 34.87 75% 6.39 1.04

max 54.67 158.31 max 188.14 2.64

mean 8.76 25.82 mean 14.24 0.81

SD 14.29 35.12 SD 38.58 0.66

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TABLE 2

Table 2. Results of AIC model selection for competing models. Models 1 and 3 include only fixed effects. Models 2 and 4 include fixed and random effects. AICs for the best-fit models are in bold.

Model AIC logLik p-value

Lead Cats 1 57.63 -23.82

2 65.45 -22.72 0.82

Dogs 1 64.44 -18.71

2 66.77 -23.38 0.17

Mercury Cats 3 60.63 -25.31

4 70.11 -25.05 0.99

Dogs 3 47.42 -18.72

4 51.02 -15.51 0.27

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TABLE 3

Table 3. Significance values of blood lead and total mercury concentrations in pets by zip code and sex

F p

Lead Cats Zip Code 2.73 0.12

Sex 0.05 0.82

Dogs Zip Code 5.32 0.03

Sex 0.11 0.73

Mercury Cats Zip Code 0.61 0.45

Sex 0.04 0.85

Dogs Zip Code 5.92 0.02

Sex 2.13 0.16

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TABLE 4

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FIGURE 1

Fig. 1. Map of home addresses for animals included in this study superimposed on a map of soil lead concentrations of New Orleans post-Katrina, with permission from Mielke et al. 2017. The pets’ home addresses are represented with circles (closed = cats, open = dogs). All blood samples were collected at SAF, represented with a star.

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FIGURE 2

Fig 2. Log-transformed blood lead (in ng/ml) and blood total mercury concentrations (in ng/g) of New Orleanian domestic cats and dogs. Dogs have higher blood lead concentrations relative to the cats, but cats have higher blood total mercury concentrations relative to the dogs.

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APPENDIX A

Letter shared with New Orleans residents explaining the study design

Department of Ecology & Evolutionary Biology, Tulane University, New Orleans, LA 70118

Pilot Study: Investigation of Lead Levels in New Orleans’ Pets To Whom It May Concern:

Research demonstrates that New Orleans, along with other major cities, has measurably high levels of lead present throughout the environment, as a result of the widespread use of lead in products such as paint and gasoline. Although lead is no longer used in the manufacturing of these products lead persists in the environment and can potentially be inhaled or ingested by pets, but little is known about whether this may be occurring. Throughout this pilot study, we hope to better understand the degree to which dogs and cats living in New Orleans may be coming into contact with lead.

With your permission, if your pet’s blood is drawn today, a small portion of the blood sample will be reserved for analysis of lead concentration. This will require no additional sample from your pet and no additional cost to you. Additionally, we request permission to collect a small hair sample (one or two pieces of hair) for analysis of lead concentration. The results of these analyses will be evaluated in the context of the responses you provide in a short Patient Questionnaire. If you wish to receive the results, we are more than happy to send them to you via email. Please make sure to include your email information on the bottom of the survey if you wish to be contacted.

Please feel free to ask Dr. LaMarsh any additional questions, or contact Caroline Camus (ccamus@tulane.edu) or Dr. Jordan Karubian (jk@tulane.edu) at Tulane University. Thank you in advance for your participation in this important study and for helping to make New Orleans safer for all pets! Thank you, _ Caroline Camus Tulane University | Class of 2021 Department of Ecology & Evolutionary Biology ccamus@tulane.edu

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APPENDIX B

Patient Questionnaire completed by pet owners at SAF

Background Information:

Your Name:

Address:

City: State: Zip Code:

Approximate age of your home (years): How long has your pet resided at this home?

Patient Information:

Species: Cat Dog Please include approximate weight: __________ lbs.

Sex:

Male Male/Neutered Female Female/Spayed

Breed: Age/DOB:

Approximately how much of your pet’s life is spent… Indoors __________% Outdoors__________%

What is the primary make-up of your yard?

Grass Rock Dirt Other _______________________________

Please feel free to include any additional information below: Do we have permission to contact you with follow-up questions? If yes, what is the best method of contact?

Thank you for your participation! Please feel free to contact us with any questions: Caroline Camus: ccamus@tulane.edu Dr. Jordan Karubian: jk@tulane.edu

LAB USE ONLY: Sample Number: ________________ Date Received: ________________

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APPENDIX C

Blood smears exhibiting small (TOP: score = 1) and large amounts (BOTTOM: score = 3) of basophilic inclusions present in the erythrocytes of New Orleanian dogs