THE POTENTIAL ENVIRONMENTAL IMPACT OF DISCARDED CIGARETTE

WASTE IN MARINE SEDIMENTS

A Thesis

Presented

to the Faculty of

California State University Dominguez Hills

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

in

Biology

by

Ian C. King

Spring 2018

ACKNOWLEDGEMENTS

I want to thank Dr. Varenka Lorenzi and Mary Blasius for their help in performing this

thesis project. I would like to thank Dr. John Thomlinson and Dr. Patrick Still for being

on my thesis committee and for helping to edit this work. The IIRMES staff and

volunteers who helped me carry out this project are also thanked. This research was

funded by a grant from the University of California Tobacco-Related Disease Research

Program awarded to Rich Gossett at IIRMES (award #24XT-0015).

ii

TABLE OF CONTENTS

PAGE

ACKOWLEDGMENTS…………………………………………………………………..ii

LIST OF FIGURES………………………………………………………………………vi

CHAPTER

TABLE OF CONTENTS……………………………………………...............................iii

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

ABSTRACT……………………………………………………………………………..vii

1. INTRODUCTION……………………………………………………………………...1

Background……………………………………………………………………….1 Literature review………………………………………………………………….3 Research plan……………………………………………………………………..8

2. METHODLOGY……………………………………………………………….............9

Cigarette extractions………………………………………………………………9

Sediment GC/MS analysis……………………………………………………….15

Sediment exposures……………………………………………………………...11 Cigarette GC/MS analysis………………………………………………………..14

Beach Samples…………………………………………………………………...17

3.RESULTS……………………………………………………………………………...18

Cigarette samples………………………………………………………………...18 Sediment samples…………………………………..…………………………….19 Alkanes……………………………………………………………………..........20 Pyridine derivatives………………………………………………………...........23 Other Chemicals…………………………………………………………………28 Beach Samples…………………………………………………………………...30

iii

CHAPTER PAGE

4. DISCUSSION…………………………………………………………………………31

Cigarette samples………………………………………………………………...31 Pyridine Derivatives……………………………………………………………Alkanes...…………………………………………………………………………35

...32

Other Chemicals………………………………………………………………….37

5. CONCLUSIONS……………………………………………………………………...39

REFERENCES…………………………………………………………………………..49

iv

LIST OF TABLES

PAGE

1. Library ID and CAS codes for hydrophobic chemicals found in 11 or more cigarette samples……………………………...41

2. Hydrophobic Chemicals found in both cigarette samples and exposure tanks……………………………………………………45

v

LIST OF FIGURES

PAGE

1. Concentrations of alkanes in exposure tanks…………………………………….22

2. Chemical structure of four pyridine derivatives…………………………………24

3. Concentrations of pyridine derivatives in cigarette samples………….................24

4. Concentration of nicotine in exposure tanks……………………………………..25

5. Concentration of myosmine in exposure tanks………………………..................26

6. Concentration of β-nicotyrine in exposure tanks………………………………...27

7. Concentration of cotinine in exposure tanks…………………………..................28

8. Concentration of 2,4-xylenol in exposure tanks………………………………....29

9. Concentration of nicotine in beach sediment samples…………………………...30

vi

ABSTRACT

Information on the long-term impacts of discarded cigarettes within marine

sediment is lacking. This study analyzed chemicals that leached from cigarette butts into

marine sediment, and determined which ones could be detected over an extended period

of time. Solvent extraction and gas chromatography/mass spectrometry (GC/MS) analysis

of cigarette butts generated a list of non-polar chemicals that could possibly leach out.

Cigarette butts were mixed into marine sediments in 10 tanks filled with sea water, and

sediment samples were removed periodically over 60 days. Sediment was solvent

extracted and analyzed via GC/MS. Chemicals extracted from sediment were compared

to those extracted from cigarette butts, with 35 chemicals identified and present in both.

Some tobacco alkaloids (nicotine, cotinine, myosmine, and β-nicotyrine) were the most

prevalent cigarette constituents being detected every day of sampling. A large number of

linear alkanes were identified, but only a handful drove the concentrations in the

sediment. As alkanes were also detected in sediment before cigarette butt introduction,

tobacco alkaloids were considered the best markers for measuring impacts of cigarette

butts within aquatic sediments.

1

CHAPTER 1

INTRODUCTION

Cigarettes may provide enjoyment and stress relief to some people, but the

amount of harm cigarettes can cause to those who smoke and others around them is far

greater. Approximately 30% of deaths due to coronary heart disease can be directly

linked to smoking,21 while second-hand smoke increases the chance of developing lung

cancer in individuals who live in close proximity to smokers.5 Even once the cigarettes

have been discarded, the chemicals within them can cause detrimental effects. There is

evidence that ingestion of discarded cigarette butts can cause vomiting and convulsions in

children and pets.19 Studies such as this are important in understanding the detrimental

effects cigarettes can cause besides the well-documented and more predictable effects

like cancer and pulmonary disease. However, the environmental side effects of cigarette

butt toxicity are often overlooked.

Background

Cigarette butts are the most identifiable litter found in the oceanic costal

environment, with over two million found during a single international coastal cleanup in

2014.20 While these numbers suggest that there are plenty of discarded cigarette butts to

be ingested by both domestic animals and wildlife, in aquatic environments these

cigarettes can cause harm in a different way.19,24 Submerged cigarette butts can leach out

their chemical components into the water, thus allowing them to spread out further than if

2

they were stationary. These chemicals have the potential to negatively affect organisms

by inducing lethal and possibly sub-lethal effects on aquatic species such as the fathead

minnow (Pimephales promelas) and marine topsmelt (Atherinops affinis).28 These sub-

lethal effects can range in intensity, and they include behavioral changes, immobilization,

DNA damage, and reduced growth.1,17,28,36 These types of effects need to be studied

further in order to understand the impact that these chemicals can have on organisms in

the aquatic environment. This knowledge has the potential to influence the regulation of

discarded cigarette butts and even restrict the use of cigarettes in general.

Nicotine (originally used as an herbicide) is probably the most highly researched

chemical constituent of cigarettes, and it is the one that has raised the most public

awareness. A study by Konar (1969) demonstrated that nicotine could be lethal to

freshwater catfish (Heteropneustes fossilis). The catfish exposed to nicotine had their

opercular muscles paralyzed by the chemical and thus suffered eventual asphyxiation.

Further studies by Konar (1977) confirmed these results with other fish species including

the Gangetic leaffish (Nandus nandus) and rohu (Labeo rohita). Additionally, the study

also examined the effects of nicotine on aquatic insects such as diving beetles (Dytiscus

spp.) and water sticks (Ranatra filiformis). While these organisms exhibited more

tolerance to the nicotine than the fish species, they too eventually became lethargic and

died. Nicotine can cause many lethal and sub-lethal effects. However, it is not the only

dangerous chemical found in cigarette butts.

3

Literature Review

After studies performed by Konar in the 1970s, very little research has been

performed to further elucidate the potential harm that chemicals in cigarette butts can

have on aquatic species. It was not until the 2000s that additional studies to elucidate the

effects of cigarette butts and their chemicals on aquatic environments were performed.

One of the first studies to examine how cigarette butts can affect the aquatic environment

was performed by Kathleen Register who used Daphnia magna as the test species.

Groups of Daphnia were exposed in Petri dishes to three different types of leachates from

either smoked filters with no tobacco, smoked filters with remnant tobacco or unsmoked

cigarette filters.24 Daphnia populations exposed to the smoked filters without tobacco for

48 hours all died at concentrations of ≥ 2 cigarettes per liter, whereas 20% of them died at

concentrations of 1 cigarette per liter.24 Exposures to smoked filters with tobacco were

able to produce death in 100% of the Daphnia at 48 hours, even at a concentration of 0.5

cigarettes per liter equivalent.24 The Daphnia exposed to unused filters however, had

death rates less than 50% after the 48 hour period even when using solutions that were 16

cigarettes per liter. This information helps to show that not only is the tobacco in

cigarettes the most lethal component to aquatic organisms, but the change in chemical

composition in the filter via combustion can also lead to lethal results even when tobacco

is no longer present.

After this study, it was not until 2011 that another key study was published that

could show the effects of cigarette butts on aquatic organisms. Slaughter et al. (2011)

performed one of the first studies to look at the potential effects cigarette butts could have

4

on aquatic vertebrate species. Their experiment consisted of placing marine topsmelt

(Atherinops affinis) and freshwater fathead minnow (Pimephales promelas) into tanks

that had been treated with cigarette butt leachates. These leachates were made of either

unsmoked cigarette filters that had no tobacco, smoked cigarette filters with no tobacco,

or smoked cigarette filters with remnant tobacco.28 The parameter used to measure

toxicity in these experiment was LC50, defined as the concentration of cigarette leachate

required to cause 50% mortality in the population exposed. The leachate made with

cigarettes containing remnant tobacco had the highest toxicity with an LC50 of 1

cigarette butt per liter.28 Smoked filters and unsmoked filters showed higher LC50 values

of 1.8 and 5.1 cigarette butts per liter respectively for marine topsmelt, while the

freshwater fathead minnow had LC50 values of 4.3 and 13.5 cigarette butts per liter

respectively.28 These findings illustrate that smoked cigarette filters with remnant tobacco

are the most toxic to marine and freshwater vertebrate species followed by smoked filters

with no tobacco.28 However, although the LC50 values of unsmoked filters with no

tobacco were larger than the smoked samples, the fact remains that these samples still

caused lethal effects in the fish.28 This study shows that regardless of the condition

discarded cigarettes are in, they can cause lethal effects, as the chemicals within them are

still toxic when they enter the water column.

While Slaughter et al. (2011) produced strong evidence for the lethal impact of

cigarette butts on fish species, it neglected to look at possible effects at lower

concentrations of exposure. In 2015, a study was performed to help elucidate the

potential sub-lethal effects, such as behavioral modifications, that could occur at lower

5

concentrations of cigarette butts.1 They exposed three different Australian snail species to

varying levels of a cigarette butt solution that included 5%, 10%, 25%, and 100%

concentrations of the leachate. This study showed some interesting species-specific

patterns in snail survivorship. While complete mortality was seen across all three snail

species in the 100% leachate solution after 8 days, lower concentrations of the leachate

showed markedly different results.1 All of the species were able to survive for at least 24

hours at concentrations of 25% leachate or lower.1 After 150 hours, all species showed a

drop in survival in 25% leachate, however, Austrocochlea porcata also incurred a drop in

survival at 10% leachate.1 When looking at the sub-lethal response of the snails at lower

concentrations, the results varied among species. The species that took the longest to

respond by moving out of the tank was A. porcata, which was also the species that

exhibited the lowest survival.1 Nerita atramentosa escaped the tank the soonest, and it

had the highest survival.1 These results show that while at high concentrations cigarette

butts are almost undoubtedly lethal to these marine species, the way that different species

react to cigarette butt chemicals at lower concentrations can be extremely variable, with

consequences to survivorship.1

In order to broaden the scope of the study, more scientists began to look past

mortality as a consequence of the chemicals in discarded cigarette butts, and to focus

instead on other aspects, such as the negative effects cigarettes can have on embryonic

development. A study by Lee and Lee (2015) helped to address this issue by examining

the effects of cigarette butts on the development of medaka (Oryzias latipes) embryos.

The embryos were treated with four different types of leachate made from smoked

6

tobacco cigarettes, cigarettes with unsmoked tobacco, smoked filters with no tobacco,

and unsmoked filters with no tobacco. These different treatments were then made into

multiple leachates at varying concentrations ranging from 0.2 cigarette pieces per liter to

20 cigarette pieces per liter. Samples with smoked tobacco exhibited the greatest effects

on development, followed by unsmoked tobacco, smoked filters, and then unsmoked

filters having the least toxic effects.13 Compared to the control, developmental effects

could be seen at low concentrations of solution such as an increase in heart rate and

accelerated development at only 0.2 pieces of smoked tobacco cigarettes per liter.13

Unsmoked cigarette filters did not seem to differ from the control.13 From this work, it

can be seen that smoked cigarette butts can cause problems not only to adult individuals,

but also at earlier developmental stages.

All these studies looked at cigarette butts as a whole in terms of the effects they

cause to aquatic organisms, yet the studies did not identify the specific chemicals in

cigarette butts that cause these effects. A more recent study was designed to identify the

specific chemicals found in cigarette butt leachate, and it found many toxic chemicals

including benzyl alcohol, hexanoic acid, and nicotine.27 An interesting finding within this

study was the occurrence of chemical compounds with structures similar to nicotine such

as 2,3ʹ-dipyridyl and quinolone.27 These chemicals are suspected to be derivatives of

nicotine caused by smoking the tobacco. The similarity these chemicals share with

nicotine has led researchers to hypothesize that they possess similar eco-toxic properties

to nicotine.27 While over 4000 identified chemical components of cigarette butts and

7

cigarette smoke have been identified in the past,22 this study narrows down the list to

chemicals that leach out of cigarette butts into aquatic environments.27

While the breakthroughs in these studies have led to a much greater understanding

of how cigarettes affect the environment, they have only scratched the surface. One area

that needs more study is the ability of chemical constituents of cigarettes to bind to

sediments. Studies on this topic so far have shown that chemicals, nicotine in particular,

affect organisms primarily in the water column.36 These studies, however, have been

primarily focused on polar, hydrophilic chemicals and not on non-polar, hydrophobic

ones. Non-polar chemicals manufactured in the past such as

dichlorodiphenyltrichloroethane (DDT) and polychlorinated biphenyls (PCBs), were able

to accumulate in aquatic sediments and thus affect the environment for years after

introductions ceased.15,31 Non-polar chemicals such as DDT derivatives have the ability

not only to last a long time in the environment, but also to bioaccumulate in animal

tissues as they are taken up by organisms further up the food chain.18 An additional study

by Grung et al. (2016) showed that road runoff could introduce non-polar polycyclic

aromatic hydrocarbons (PAHs) into sediment in freshwater ponds. These PAHs caused

DNA damage to Phoxinus phoxinus (common minnow) living in sedimentation ponds

along roadsides.4 These studies illustrate that long-term bioaccumulation of non-polar

chemicals from discarded cigarette butts could also be possible.

Wright et al. (2015) investigated whether cigarette butts were toxic to marine

ragworms (Hediste diversicolor) by grinding up smoked cigarette filters and mixing them

with sediment in concentrations of 0.5 to 8 cigarette filters per liter. Worms were exposed

8

to this sediment for periods of 96 hours and 28 days to determine the short-term and long-

term effects, respectively, caused by the chemicals within the sediment.36 They found no

significant effects on relative growth rate or DNA damage in the worms, although there

was an inhibition of burrowing activity to worms exposed to 8 cigarette filters per liter in

the sediment after 96 hours of exposure. While the results from this study suggest that

chemicals from cigarettes do not have strong lethal effects on the organisms in the

sediment, it should be noted that the maximum concentration used in this study was 8

cigarette filters per liter without any excess tobacco. Ragworms in the environment could

be exposed to higher concentrations for longer periods of time.

Research Plan

These studies have all attempted to elucidate the harmful effects of discarded

cigarette butts on the aquatic environment. However, very little work has been done on

the long-term consequences of the chemicals present within cigarette butts, especially in

sediments versus the water column. This study hypothesizes that chemicals within

cigarette butts can bind to sediments within the ocean, in a manner similar to PCB and

DDT for an extended period of time. In order to test this, samples of marine sediment

were exposed to discarded cigarette butts and then solvent extracted. Two primary

questions frame this project:

1) Can chemicals that leach out of cigarettes bind to marine sediment?

2) Can cigarette chemicals bind to sediment for an extended period of time?

9

CHAPTER 2

METHODOLOGY

Experimental procedures presented in this thesis consisted of several steps, which

included analysis of the hydrophobic chemicals found in cigarette butts, sediment

exposures to discarded cigarette butts, and analysis of the chemicals found in that

sediment.

Cigarette Extractions

Cigarettes were collected from the campus of California State University Long

Beach (CSULB). When possible, cigarette butts were described based on brand name and

type (for example menthol, light). Previous studies have shown that chemical

compositions, such as those of pyridines, vary among different types of cigarettes,12 and

it is expected that a variety of cigarettes would be discarded in nature. A requirement of

cigarettes chosen was that they needed to have been smoked, as previous studies have

shown that the most potent chemicals from cigarettes come after combustion has

occurred.24 The cigarettes were collected in this way to ensure they had been smoked. If

the cigarettes had been only burned, this would have caused chemicals changes, but the

chemicals would have been lost in the cigarette smoke to the atmosphere. Smoking the

cigarettes pulls the chemicals into the filter and allows them to be stored there, so that

even if the remnant tobacco is all gone, the cigarette will still have the chemicals created

from the combustion of the tobacco. For the purposes of this experiment, one discarded

10

cigarette butt was placed into each cellulose thimble for the extraction of the chemicals

within the cigarettes. Sodium sulfate was mixed with the cigarettes in order to absorb any

traces of water.

Dichloromethane (DCM) was tested as a solvent for extracting chemicals, but it

caused the extracts to solidify into a rubbery material when concentrated down, thus

making it impossible to perform gas chromatography/mass spectrometry (GC/MS)

analysis on them. A possible interpretation is that the strong solvent altered the

plasticizers or other chemicals present in the cigarette filters causing them to precipitate.

Hexane was therefore chosen to be the extracting solvent since it did not present the same

problem. Cellulose thimbles containing the cigarettes and sodium sulfate were spiked

with PAHs recovery surrogates (AccuStandard) and then placed into soxhlet extractors

that were connected to flasks containing 200 mL of hexane. The recovery surrogates

consisted of known chemicals, including d10-Naphthalene and d10-Phenanthrene that

could be detected with the GC/MS. Adding these recovery surrogates acted as an

additional control to ensure that the samples of interest were being extracted accurately.

The combined soxhlets and flasks were placed onto a heating rack with condensers on top

where the extraction was allowed to run overnight. As the heating rack evaporated the

hexane, it would then condense down into the chamber containing the cigarette sample.

Once enough hexane had filled the chamber, it would flow back down into the flask and

the process would repeat. In the morning, the thimbles were discarded and all of the

solvent was collected into the flasks.

11

The flasks were then placed into a rotary evaporator (Rotovap) with a water bath

set at 37 in order to further concentrate the samples. They were then transferred with

disposable glass pipettes into smaller pear flasks. The original flasks were rinsed with

hexane 3 times in order to ensure complete transfer of the chemicals.

The pear flasks were placed back onto the Rotovap to concentrate the chemicals

to approximately 1 mL in volume. Once this volume was obtained, they were removed

and transferred into 2 mL glass vials. The pear flasks were rinsed with hexane 3 times

and the rinses transferred into the vials. The samples were then evaporated to 1 mL in

volume under a gentle flow of nitrogen gas. After this was completed, the vials were

ready for GC/MS analysis. Before running the samples on the instrument, they were

spiked with an internal standard solution containing deuterated PAH’s (AccuStandard) to

use for the quantification of the chemicals.

Sediment Exposures

Sediment to be used in the exposure experiments were collected from the western

side of Catalina Island and inside of Catalina Harbor because it was more sheltered from

the surge. The samples were taken from Catalina because the contamination would likely

be small due to a lack of major rivers, urban runoff, storm drains, or industrial outputs

commonly found along the coast of Los Angeles County. In addition, there are fewer

people on the west side of the island than on the more touristic east side of Catalina, so

the introduction of cigarettes to this environment would be minimized.

12

Sediment was collected with a bucket from the shallow sandy bottom, stored in

ice chests, and transported to a cold room located inside of the marine lab at CSULB.

Sediment samples were sieved with a 1 mm sieve to remove the large shells and pebbles

from the sediment to make samples more homogeneous across treatments.

Eleven tanks (approximately 4 L in volume) were set up for the purpose of this

experiment, 10 for cigarette exposure and one as a negative control tank. Tanks were

cleaned with dish soap, then rinsed with methanol followed by a rinse with deionized

water to remove any contaminants already present.

Cigarette butts for the experiment were collected from the CSULB upper campus

area both off the ground and from cigarette waste containers. A total of 500 cigarette

butts of various brands was collected; they were stored in two closed jars.

Each tank in the experiment contained 2040 g of sediment and 2 L of clean sea

water which was obtained from the marine lab at CSULB. Each tank was equipped with

an air stone for constant oxygenation. These tanks were then allowed to settle in the cold

room (16 °C) for approximately 2 hours before removing the first 40 g of sediment for

Day 0 analysis. This sample was obtained in order to differentiate between contaminants

introduced by the cigarette butts and contaminants that were already present in the

sediment prior to exposure. The ratio of grams of sediment to cigarettes was set at 40:1

with a total of 50 cigarettes for each tank (2000 g of sediment.) Cigarettes were pushed

into the sediment to ensure introduction of their chemical components into the sediment.

Tanks were placed back into the cold room and a plastic lid and aluminum foil were

placed over each tank to reduce water evaporation and reduce the dispersion of the most

13

volatile cigarette constituents. A wire mesh covering was placed on top of the sediment

on Day 5 to ensure that the cigarettes could not float up after it was found that many of

the cigarettes did not stay buried due to the air flow from the stone.

Following the introduction of cigarette butts, approximately 40 grams of

sediment, without cigarette butts, was removed on Day 5, 10, 20, 30, 45, and 60.

Removed sediment samples were stored at -20 °C in small glass jars until it was time to

extract them. One cigarette was removed and discarded from each tank every time the

sediment was removed in order to keep the ratio of sediment to cigarettes consistent at

40:1.

Extraction of contaminants from within the sediment samples was done with 200

mL of hexane per sample via Soxhlet extraction. Approximately 15 g of sediment was

removed from each glass jar and placed into a cellulose thimble along with 10 g of

sodium sulfate. The dry/wet weight ratio of the sediment was calculated by drying

approximately 5 g of sediment for each sample.

Cellulose thimbles containing the sediment and sodium sulfate were placed into

Soxhlets inserted on top of the flasks containing hexane, and then spiked with PAH

recovery surrogates. The Soxhlet extraction and concentration of the extracts by Rotovap

were performed as described in section 2.1. The samples were evaporated under a gentle

flow of nitrogen gas to bring them all to the same final volume of 1 mL. These vials were

then stored in the refrigerator until they were run through the GC/MS.

14

Cigarette GC/MS Analysis

GC/MS method offers a precise ion analysis in detecting small molecular weight

and highly volatile chemicals based on their characteristic ions.7 Concentrated samples

were injected using an autosampler (7683B series, Agilent Technologies, Santa Clara,

California, USA) onto an Agilent gas chromatograph (GC; 6890N series) equipped with a

mass selective detector (MSD; Agilent 5973 inert series). The GC column employed was

an Agilent DB-5 fused silica capillary (0.25 mm ID x 60m) with 0.25 µm film thickness.

The temperature profile of the GC oven was programmed to ramp up from 45 °C to 125

°C at 20 °C/min, then to 295 °C at 2.5 °C/min and held for 10 min. Injector and transfer

line temperatures were set at 285 °C and 300 °C, respectively. The source and quadrupole

temperatures were set at 230 °C and 150 °C, respectively. Helium was used as the carrier

gas at a flow velocity of 40 cm/sec. The MSD was used in the Electron Ionization (EI)

mode and scanned from 45-500 amu at a rate of 1.66 scans/sec. Data were acquired and

analyzed with the software associated with the GC/MS system (Environmental

ChemStation, Agilent Technologies).

Chromatograms and mass spectra were obtained for each cigarette sample. As

each main peak in the chromatogram was analyzed, the library database included with the

software was used to produce a list of possible chemical identities based on the

percentage match with the mass spectrum of a known compound in the library. The

closest chemical match was recorded for each peak along with the main fractionation ions

and the retention time. The original threshold for accepting chemicals using this library

database was set at a match of 50% or higher. However, this parameter was determined to

15

be too inaccurate to determine the precise identity of the chemicals. The parameter was

changed to a 70% library match for the final list of cigarette chemicals.

Sediment GC/MS Analysis

Sediment chromatograms were also obtained after the samples were run on the

GC/MS with the same method described above. These chromatograms were organized

first by number of days after cigarette introduction, and then by tank number. Each peak

in the chromatogram was matched to its most likely chemical identity based on the

library search.

Many of these chemicals were low in concentration and hard to distinguish within

the background noise in the chromatograms, making it difficult to identify the chemicals

by library search alone. In order to narrow down the number of chemicals, an extracted

ion chromatogram of the total chromatogram was performed for each main peak

identified. This was accomplished by first using the GC/MS data to obtain the main

fractionation ions for each peak in the sediment. These ions were compared to those

found on the cigarette chromatograms to establish if the correct chemical had been

identified with that peak. If the chemicals were properly identified, and found to meet the

threshold of presence in 5 or more cigarette samples, then the chemical was retained on

this list, otherwise it was discarded. Following this confirmation, each chemical had its

ions extracted from the chromatogram across all of the Day 0 tanks to find out if these

chemicals were already present in the sediment before the start of the experiment. The

chemicals there were not present in Day 0 tanks had their ions compared to peaks across

16

the chromatograms of the exposed sediment samples that supposedly contained that

specific chemical based on the library search. This step allowed for the chemicals found

within the cigarette butts to be compared across the different sediment samples to see if

the sediment was able to bind these specific cigarette chemicals based on their ions.

Using the ions also offered a more accurate result than simply relying on the library

database matches especially for compounds present at lower concentrations. However,

many of these chemicals contained very similar ions. Additionally, using the library

search and the ions to identify the chemicals only showed what chemicals they might be

without offering certainty on the identity nor information about their concentration.

Therefore, chemical standards were purchased to help verify many of the chemicals

found during the ion analysis stage.

The standard for alkanes (Sigma) contained all linear alkanes from C9 to C34 and

was run on the GC/MS together with some of the extract to identify the exact retention

time of the various alkanes present in the standard mix. A quantification method based on

main fragmentation ions, alkane retention times, and the Sigma standard was used in

order to positively identify the alkanes across the rest of the tanks and days. Using this

quantification method, the alkane concentrations in the sample could be quantified by

measuring the area under the peak of the specific compound and of the internal standard

and comparing it with the calibration curve.

A second standard solution was made of a combination of chemicals that were

tentatively identified with the library search and included: nicotine (AccuStandard), beta-

nicotyrine (Cayman Chemicals), myosmine (Cayman Chemicals), 2,4-xylenol (Sigma),

17

cotinine (Cayman Chemicals), p-xylene (Sigma), and 4-phenylmorpholine (Sigma).

These standards were run on the GC/MS to identify retention times. A quantification

method based on the main fragmentation ions, retention times, and an internal standard

allowed for positive identification of these chemicals in both cigarettes and sediment

samples. The samples were quantified by measuring the area under the chromatogram

peaks. These were then compared to the calibration curve, in a manner similar to the

alkane analysis.

Beach Samples

In order to compare the concentrations of chemicals found in the sediment

exposed to cigarette butts to the types of concentration found in nature, samples of

sediment were collected from sandy beaches in Long Beach. These locations were as

follows: Alamitos Beach at the very end of the beach towards downtown, Alamitos

Beach at Molino Avenue near a storm drain outflow, Belmont Pier near a dry storm drain

on the beach, Alamitos Bay near a buried storm drain, and Mothers Beach on the beach.

These samples were put through the same extraction process as the samples exposed to

cigarettes and then analyzed using the alkane and the cigarette chemical cocktail

quantification methods developed earlier.

18

CHAPTER 3

RESULTS

Cigarette Samples

A total of 23 cigarette samples were extracted for this project to develop a list of

hydrophobic chemicals that might be found within cigarettes (Table 1). The chemicals

were identified using the library database search of the Environmental ChemStation

software. Identities with lower than a 70% match with a chemical in the library database

were excluded. Duplicate chemicals were deleted, and individual lists of chemicals for

each cigarette were compiled to ascertain how many cigarettes held each chemical. A

total of 695 individual chemicals were found within the 23 cigarette butts after the 70%

cut off. Out of the 695 chemicals, six were found in all 23 samples post-quantification

methods (Table 2). A total of 77 chemicals were present in 11 or more samples, and 156

chemicals were found in five or more samples. The majority of the chemicals found

across the cigarettes (n=617) were present in fewer than five samples. Chemicals found in

only ten or less samples were discarded from the list of possible chemicals found in

cigarette butts. Of the 78 chemicals found in 11 or more samples, only the identity of the

alkanes and the chemicals present in the cigarette cocktail were verified and quantified

using the calibration solutions. The rest were only identified via ion comparisons after the

initial library search.

19

Sediment Samples

The sediment obtained for this experiment was composed of sand (66.65%), silt

(29.95%), and clay (6%). The grain size composition was obtained using a Saturn

Digisizer Particle Size Analyzer (Micromeritics) at IIRMES. Two duplicate samples of

sediment were processed to obtain the concentration of total organic carbon (TOC). The

average TOC was 0.24% as measured with a Costech Elemental Combustion System at

IIRMES.

A library database search was used to identify the chemicals found in the

chromatograms of the sediment tanks during the first round of analysis. There were 261

individual chemicals in sediment exposed to cigarette butts that were not found in the

control tanks or the Day 0 tanks. Chemicals were identified based on whether or not they

had a 50% or higher probability of their mass spectrum matching one of the chemicals in

the software chemical library. Chemicals were organized first by day of exposure and

then by the tank number they were sampled from. However, eight samples which

included tanks 1, 3, and 5 of Day 20 as well as tanks 1, 4, 5, 8, and 9 of Day 30 were

excluded from this comparison. These samples had cigarette butts left inside the sediment

after being taken from the tanks, and therefore would bias the results of the experiment

because they were exposed to the cigarette for more than 20 or 30 days, respectively. The

rest of the sediment sample extracts were compared to the ones from the cigarette

samples and with the negative controls to see which chemicals came from the cigarettes

specifically as opposed to chemicals present in the sediment before cigarette introduction.

The total number of identified chemicals, based on the library search, was reduced from

20

261 to 58. At this point, it was noted that several of the chemicals appeared multiple

times on a chromatogram for the same tank. Part of this occurred due to the fact that the

library search would often assign the same name for similar chemicals, or that the same

peak would appear at different points in the chromatogram in different GC runs because

the retention time shifted. An example of this was one peak that had the name tocopherol,

and a peak preceding it that had the name vitamin E, which is the common name for

tocopherol. Following further analysis via comparison to fragmentation ions and

reference chemical standards, the correct chemicals were identified and the number of

chemicals within sediment was narrowed down to 35 (Table 2). These identified

chemicals were divided into three groups: alkanes, pyridine derivatives, and “other

chemicals.”

Alkanes

The largest group of similar chemicals found during the analysis of the peaks

were the alkanes (n=15). Alkanes were found in the chromatograms for each tank on all

sampling days and in the cigarette samples as well. These alkane peaks were also found

in the Day 0 samples and the control tank for each day. However, while they were found

in the negative controls, the alkanes were present in higher concentrations after the

introduction of cigarettes in the tanks, thus leading to a need to properly quantify them in

the sediment samples. The majority of alkanes had the same main fragmentation ions (57,

71, 85) so it was impossible to use the library search to identify them unequivocally. We

used a standard solution of linear alkanes from C7 to C34 (Sigma) to produce a

21

calibration curve and a quantification method that provided the retention time for each of

these alkanes. The alkane quantification methods offered much clearer results because

they could help to distinguish between the different alkanes in the sediment samples.

Smaller alkanes (tridecane and below) were excluded from the analysis, as these alkanes

could not be clearly detected in the chromatograms. The main alkanes identified typically

ranged from tetradecane on the lighter side to dotriacontane on the heavier side with

approximately 20 alkanes being quantified in this manner, but only 15 being found in

sediment samples and cigarette samples. Besides the 15 linear alkanes, many complex

alkanes were found based on the library search, such as cyclo-alkanes and 1-iodo-

hexadecane, for example. These alkanes were not present in the reference standard

solution and could not be quantified with the same method. Comparison to their extracted

main ion was therefore used to help organize them. The main peaks of these more

complex alkanes were examined across the chromatogram at the points where they had

initially been identified using ion integration. Some of the cyclo-alkanes had 55 instead

of 57 as their main ion, thus making it possible to differentiate them from possible linear

alkanes that appeared close together on the chromatogram. The ion integration

identification of the complex alkanes made it clear that these chemicals were not present

in the cigarette samples, and were therefore excluded from the lists of chemicals found in

the exposed sediment. Concentrations of alkanes in tanks with cigarettes increased during

the 60 day period (Fig.1), indicating that alkanes were leaching from the cigarette butts

into the tanks. In order to confirm this, Day 0 samples had their concentrations compared

to the concentrations of alkanes from all other days of the study.

22

Fig. 1. Average total concentration of linear alkanes in the sediment exposed to cigarette butts for each day of the sampling period. The points represent average concentrations across 10 tanks in ng/g of dry sediment and the error bars represent standard error of the mean.

After adjusting for each sediment sample dry:wet weight ratio, the results

provided in ng of alkane/gram of dry sediment could then be used to compare the

concentrations across samples. The alkane values were averaged across tanks for each

sampling day to see if there was an overall increase or decrease in their concentrations.

An increase was denoted beginning from Day 0 (19.373 ng/g) to Day 5 (55.244 ng/g),

and continuing up to Day 20 (108.554 ng/g). Between Day 20 and Day 30 (52.682 ng/g)

the alkanes showed a decrease with the average concentration reaching values similar to

Day 5. A slight increase was then observed between Day 30 and Day 45 (60.72 ng/g),

followed by the largest increase occurring between Day 45 and Day 60 (258.834 ng/g).

The mean concentrations of the alkanes on different days were significantly different

23

from one another (ANOVA, p<0.05). Individually, the alkanes showed a similar trend,

albeit only in the larger alkanes (pentacosane to tetratriacontane) suggesting that the

larger alkanes were the ones most likely leaching from the cigarettes and contributing to

the increased values in the sediment after Day 0. Alkanes such as nonadecane showed a

slight increase from Day 0 (4.565 ng/g) to Day 20 (17.56 ng/g). However, once the

experiment reached Day 60 (4.845 ng/g), the concentration had decreased almost all the

way back to the Day 0 concentration. For alkanes such as pentacosane however, the

concentration increased overall from Day 0 (20.81 ng/g) to Day 60 (85.94 ng/g). This

trend of increasing concentration over time was seen in all the alkanes larger than

pentacosane.

Pyridine Derivatives

Another set of conspicuous peaks were 5 chemicals that were grouped together as

derivatives of pyridines (Fig. 2). These chemicals were not only in every cigarette sample

(Fig. 3), but also in nearly every tank exposed to cigarette butts. These chemicals all had

peaks on the chromatogram that occurred during the first 30 minutes of the GC/MS run,

and one of them (3-(1-methyl-2-pyrrolidinyl)- (s) Pyridine) (common name nicotine),

was found to be present in every tank across every sampling day. Additionally, nicotine

appeared to decrease rather than increase across the 60 days in terms of concentration

(Fig. 4).

24

Fig. 2. The chemical structures of the four quantified pyridine derivatives found in the cigarette and tank samples. A. Nicotine B. Myosmine C. β-nicotyrine D. Cotinine a

While nicotine leaching out of the cigarette butts had already increased to an

average concentration of 19034 ng/g on day 5, by Day 60 the concentration was an

average of 4457 ng/g, a statistically significant decrease (ANOVA, p<0.05).

Fig. 3. Average concentrations (in ng/g of cigarette) of the pyridine derivatives extracted from smoked cigarette butts (N=23). Nicotine was present in much higher concentrations (A) than the other 3 alkaloids (B). Error bars represent standard error of the mean. Nicotine was also the only pyridine derivative to be found in Day 0 samples (9 out of 10

tanks), however, this could be attributed to nicotine being in such high concentrations

that some carry over from one sample to the next could have taken place while running

24000

21000

18000

15000

12000

9000

Con

cent

ratio

n (n

g/g)

6000

3000

0 0 10 20 30 40 50 60 70 80

Days

Fig. 4. Average total concentration of nicotine in the sediment exposed to cigarette butts for each day of the sampling period. The points represent average concentrations across 10 tanks in ng/g of dry sediment and the error bars represent standard error of the mean.

25

on the GC/MS. The trace amounts detected at Day 0 were minimal compared to the

elevated concentrations in exposed tanks, and if subtracted from the total would not

significantly affect the overall concentrations.

The other pyridine derivatives showed similar results to those of nicotine.

Pyridine,3-(1-methyl-1H pyrrol-2-yl) (common name nicotyrine), pyridine,3-(3,4 -

dihydro-2H-pyrrol-5-yl) (common name myosmine), and 2-pyrrolidinone, 1-methyl-5-(3 -

pyridinyl)-, (S)- (common name cotinine) all appeared in the same number of tanks.

These three chemicals were not found in tanks 1 and 10 at day 45, while day 60 lacked

these chemicals in tanks 1,5 and 10. These three chemicals also exhibited overall

50

300

250

Con

cent

ratio

n (n

g/g)

200

150

100

0 0 10 20 30 40 50 60 70 80

Days

Fig. 5. Average total concentration of myosmine in the sediment exposed to cigarette butts for each day of the sampling period. The points represent average concentrations across 10 tanks in ng/g of dry sediment and the error bars represent standard error of the mean.

26

decreases after day 5, although in a different pattern than nicotine. Myosmine first

increased from day 5 (137 ng/g) to day 30 (217 ng/g), then decreased to day 60

(112 ng/g)(Fig. 5).

Beta-nicotyrine on the other hand, had a steady decrease from day 5 (145 ng/g) to

day 60 (34.2 ng.g) (Fig. 6).

300 C

once

ntra

tion

(ng/

g) 250

200

150

100

50

0 0 10 20 30 40 50 60 70 80

Days

Fig. 6. Average total concentration of β-nicotyrine in the sediment exposed to cigarette butts for each day of the sampling period. The points represent average concentrations across 10 tanks in ng/g of dry sediment and the error bars represent standard error of the mean.

27

Cotinine showed a very different pattern from the first two by initially decreasing from

Day 5 (139 ng/g) to Day 10 (62.0 ng/g), and then increasing to day 20 (94.8 ng/g)(Fig. 7).

After Day 20, cotinine decreased in concentration to Day 60 (16.7 ng/g). All three of

these pyridine derivatives decreased significantly over time (ANOVA, p <0.05). One last

pyridine derivative that could not be quantified was 2,3ʹ-dipyridyl (common name

bipyridine), because a chemical standard was not available. This chemical appeared in all

tanks across all days except for tank 10 on Day 45, and it appeared in all cigarette

samples.

300

250

Con

cent

ratio

n (n

g/g)

200

150

100

50

0 0 10 20 30 40 50 60 70

28

80

Days

Fig. 7. Average total concentration of cotinine in the sediment exposed to cigarette butts for each day of the sampling period. The points represent average concentrations across 10 tanks in ng/g of dry sediment and the error bars represent standard error of the mean.

Other Chemicals

In addition to the alkanes and pyridines, there were also several other chemicals

(n=17) that we identified and did not fit into those two categories. One such chemical was

2,4-xylenol, a derivative of a phenol. This chemical was present in all the cigarette

samples and all the tank samples. However the concentrations of 2,4-xylenol were lower

than both the alkanes and pyridines (Fig.8). The pattern for 2,4-xylenol over the period of

the experiment was a decrease similar to the pyridine derivatives. The concentration of

2,4-xylenol started at 16.5 (ng/g) on day 5 and then deceased to 3.99 (ng/g) by day 60

(ANOVA, p <0.05).

22

20

18

C

once

ntra

tion

(ng/

g)

16

14

12

10

86

4 2 0

Days

Fig. 8. Average total concentration of 2,4-xylenol in the sediment exposed to cigarette butts for each day of the sampling period. The points represent average concentrations across 10 tanks in ng/g of dry sediment and the error bars represent standard error of the mean.

0 10 20 30 40 50 60 70

29

Other chemicals in this category include menthol, bicyclo(3.1.1) heptane,2,6,6 -

trimethyl)-,(1.alpha.,2.beta.,5.alpha.), and Vitamin E (Table II). Menthol appeared in 44

of 52 samples, Vitamin E appeared in 36 of 52 samples, and bicyclo(3.1.1) heptane,2,6,6 -

trimethyl)-,(1.alpha.,2.beta.,5.alpha.) appeared in 17 of 52 samples. Also of interest is the

chemical 4-phenylmorpholine, one of the chemicals included in the mixed standard

solution for the quantification method used to identify nicotine and other pyridine

derivatives. This chemical was identified in the cigarettes and sediment based on the

library search but with different names assigned to the same peak across different

samples. A standard of this chemical was purchased to determine whether this was the

chemical being represented by those specific peaks. The use of the chemical standard

confirmed that the peak seen in the sediment exposure tanks was not 4 -

phenylmorpholine.

Conc

entra

tion

(ng/

g) 400

300

200

100

0

Mothers Beach Alamitos Bay Alamitos Beach Belmont Pier Beach Molino

Fig. 9. Average total concentration of nicotine in the sediment samples from public beaches around Long Beach, California. The bars represent average concentrations in duplicate samples in ng/g of dry sediment and the error bars represent standard error of the mean.

The nicotine at this site was detected at a concentration of 150 ng/g. Alkanes were

found at all the beach locations with the largest concentration found at the beach at

Molino Ave. (269 ng/g). The alkane concentrations at all the other beach sites were found

to be less than 80 ng/g, with the smallest site having a concentration of 38.8 ng/g

(Mothers Beach).

30

Beach Samples

Of beach sites sampled, some including Alamitos beach at Molino Ave. had

cigarette butts visible on the beach near the sampling area. However Alamitos beach at

Molino Ave. was the only location that had nicotine present (Fig.9).

31

CHAPTER 4

DISCUSSION

Cigarette Samples

The chemicals found in this experiment consisted of those found via a solvent

extraction, and therefore excluded all the hydrophilic chemicals that may have been

present within the cigarettes. Many of the chemicals only appeared in one cigarette out of

the 23 analyzed. The majority of chemicals that were found in four or fewer of the

cigarette samples were not found in the sediment tanks. Out of the 35 chemicals

positively identified as being in the sediment samples, 34 of them were found in 5 or

more cigarette samples. This helps to illustrate the fact that while many of the cigarette

brands may be different in their composition, the majority of them will release a small

number of similar chemicals into the sediment. This was clear from the fact that nicotine

and its metabolites appeared in every cigarette sample.

One interesting result of this experiment was that, while many chemicals were

extracted from the cigarettes, many of them were not found in the sediment. This could

mean that they did not successfully bind to the sediment or that they could not be

identified due to them being at such low concentrations. When using the library search

function, it is difficult to identify chemicals that are low in concentration as the

background noise will cover up these chemicals quite easily. The sediment extracts could

not be cleaned before running them on the instrument because the chemicals of interest

could have been lost in the process. Another possible explanation is that some of the

32

chemicals leaching out of the cigarette butts are more hydrophilic in nature.27 Some of

the chemicals could also be so hydrophobic that they do not leach out of the cigarette

butts at all.23 If this is correct, only 35 of the 695 chemicals were of the appropriate

hydrophobic nature to leach out of the cigarette butts and bind to the sediment, though

there could be several more that were lost in the background noise of the sediment

chromatograms.

Pyridine Derivatives

The first chemical to examine is cotinine, which had the lowest concentration of

all the pyridine derivatives. Nicotine is first metabolized to cotinine by the enzyme

cytochrome P450 2A6 (CYP2A6.) This enzyme will convert nicotine into nicotine-

Δ1′(5′)-iminium, followed by a conversion into cotinine by a cytoplasmic aldehyde

oxidase.6 In humans, this all takes place typically in the liver, which would explain why

the concentrations of cotinine were so low compared to nicotine in this study. The

cotinine detected in this study would have had to come from either the individual who

smoked the cigarette, or from microbes present in the sediment that were metabolizing

the nicotine into cotinine.6

Despite its similarity to nicotine as its primary metabolite, cotinine is also not

nearly as potent as nicotine in terms of toxicity. A study by Vlasceanu et al. (2015)

helped to elucidate that nicotine was potentially more toxic and faster acting on Daphnia

magna than cotinine. Cotinine in this study required 48 hours to reach 50% lethality in

Daphnia magna while nicotine achieved this after only 24 hours.34 Additionally, in

33

humans cotinine has been found to lower sperm motility and fertilizing capacity.29 As all

sexually reproducing male animals produce sperm in order to reproduce, it is possible

that these effects on sperm caused by cotinine could occur in marine species as well.

While cotinine had the lowest concentration in this category of chemicals, the

highest concentration belonged to nicotine itself. Nicotine has been shown to be highly

toxic to marine species,9 as well as being a major contributor towards increased blood

pressure, DNA damage, and diabetes.30 As nicotine and other tobacco alkaloids affect

organs that are similar in humans and aquatic organisms (e.g. nervous and circulatory

systems), there is a strong chance that these chemicals could have a similar impact on

organisms living in the sediment as well. Nicotine has long been the primary culprit in

most studies focusing on the addiction to cigarettes, so it comes as no surprise that it had

such a large peak in the chromatograms in this experiment as well. What is a surprise is

that previous studies such as Konar (1977) showed the potential for lethality of nicotine

in the water column without testing nicotine’s presence in sediment. Yet nicotine was

able to bind to the sediment in all the tanks across all the days of sampling. This may be

due in part to nicotine’s ability to be released from cigarettes quickly when exposed to

water. A study by Green et al. (2014), showed that after approximately 1440 minutes, a

smoked cigarette butt would release all of its remaining nicotine into water, with 50%

released after a mere 27 minutes. This suggests that all the nicotine from the cigarette

samples in this current study would have been released into the sediment after just one

day. It is probable that binding to the sediment occurred due to the sheer volume of

nicotine being released as the average amount of nicotine in 50 cigarette butts was

34

approximately 7,108 µg/g (average concentration calculated from the 23 cigarette

samples). This is even more probable as the water octanol partition coefficient (Kow) of

nicotine is only 1.17. Generally speaking, when a compound has a Kow of this level, it is

considered to be mostly hydrophilic. In fact, nicotine has a water solubility of 106 mg/L.a

Nicotine therefore may not have been binding to the sediment at all, but instead to water

trapped within the sediment. As this water started to flow out of the sediment into the

water of the tank, this could have caused the decrease in nicotine that was seen.

When looking at pyridine derivatives in the beach samples, only one collected on

Alamitos Beach near Molino Avenue had nicotine present (Fig. 9). The samples of beach

sediment were collected during a dry season when stormwater runoff, the primary

delivery system of cigarette butts to the ocean, was low. If sampling had been performed

after a rainy day, it is likely that the number of cigarette butts would have been much

greater and more data could have been obtained. The one beach sample that displayed

nicotine in the sediment was collected next to a large storm drain, and a few cigarette

butts were seen around it. The cigarettes that were observed were not in wet sand and

were on the surface, which would indicate that nicotine can still seep into and bind to

sediment. However, it cannot be excluded that more cigarette butts could have been

buried in the sediment. This can only be inferred, and it will require further testing in the

future to confirm.

Despite β-nicotyrine having lower concentration in sediment compared to

nicotine, it does play an interesting role in regards to nicotine metabolism. The enzyme

CYP2A6 that helps metabolize nicotine into cotinine can be inhibited and inactivated by

35

β-nicotyrine.11 Hypothetically, if a cigarette that contributed nicotine into the sediment

contributed β-nicotyrine as well, enzyme deactivation in the microbes could maintain the

concentration of nicotine, which would otherwise metabolize into cotinine, in the

environment for a longer period of time. As β-nicotyrine is a common metabolite of

nicotine which is found in all cigarettes, then β-nicotyrine should be considered a good

maker for cigarette butt pollution within sediments.35

Myosmine on the other hand, does exhibit lethal effects similar to nicotine, albeit

much less powerful. Myosmine was found to both decrease the viability of cells and

increase lactate dehydrogenase in a study performed by Kondeva-Burdina et al. (2010).

Since lactate dehydrogenase acts as an inhibitor on the conversion of pyruvate to lactate

at high concentrations, myosmine essentially makes it difficult to continue any form of

exercise. Myosmine is not as toxic as nicotine: 2mM myosmine decreased cell viability

by 38%, while much smaller doses of nicotine (250 µM) decreased cell viability by

74%.10 It should also be noted that myosmine is not solely coming from tobacco, but can

also come from other sources such as apples, cocoa, and even milk.33 While myosmine

can come from other sources, the traces of it in sediment will be highly connected to

cigarette butts, especially if nicotine and other common cigarette chemicals are present as

well. Therefore myosmine would constitute a good indicator for cigarette butt pollution.

Alkanes

The simple linear-alkanes made up the largest single group of chemicals in the

tanks, yet their role is anything but simple. While alkanes were found across the majority

36

of tanks and days, they were also found across most of the Day 0 tanks and the tank 11

negative control. This indicates that at least a portion of the alkanes were not introduced

into the tanks by the cigarettes, but instead they were already present. Alkanes are

characteristic constituents of crude oil, diesel, and fuel oil, and therefore since the

sediment samples came from a harbor, it is likely that either the boats or the oil fields

present in the area contributed to the alkane contamination in the sediment. However, as

can be seen in figure 1, the concentrations of alkanes in the tanks increased during the 60

day exposure. While the alkanes may already have been present in the sediment, the

cigarette butts did contribute to their presence as well. This is further supported by the

fact that alkanes were present in the extracted cigarette butts.

It should be noted that the main increase in total alkane concentration came from

alkanes with chains longer than pentacosane. When observing the alkanes individually,

most of them exhibited decreases instead of increases over time. This could be attributed

to the fact that alkanes are more insoluble in water the heavier they are.25 While smaller

alkanes such as tetradecane may be slightly soluble in water, thus freeing

themselves from the sediment, the heavier ones will tend to stay bound to the sediment. A

second point to consider is that the alkanes can be metabolized by microorganisms living

within the sediment. Smaller alkanes are more readily metabolized, but larger alkanes

such as pentacosane and triacontane will not be metabolized as quickly due to their large

molecular weight.25

Cigarettes could act as vectors for how some alkanes may be introduced into the

environment. This amount of contamination may be quite small compared to oil or other

37

industrial outputs, but alkanes being contributed by cigarette butts to sediment are still

toxic to marine life. A study by Fisk et al. (1999) found that Japanese medaka (Oryzias

latipes) eggs suffered mortality when exposed to concentrations of 9600 ng/mL of the

polychlorinated n-alkane C10H15.5Cl6.5. When this same concentration of alkane was

administered to medaka larvae instead of eggs, sublethal effects such as lethargy were

experienced.2 While the alkanes identified in this study were not exactly the same as the

ones from Fisk et al.’s 1999 study, the potential for harm from alkanes is still present. It

should also be noted that these chemicals can stay in the environment for an extended

period of time. Turner et al. (2014) found that while the alkane concentrations

contributed to wetlands in Louisiana by the Deepwater Horizon oil spill in

2010 decreased to less than 10% of their peak values, the concentrations of these alkanes

in 2012 were still 15x greater than the original concentrations before the oil spill. Added

to the result of this study that alkanes were found to persist for the whole 60 days, this

illustrates the potential harm cigarettes can cause to the marine environment by acting as

a source for alkane pollution.

Other Chemicals

In the category of “other chemicals,” the most well known is probably menthol.

Menthol is added to cigarettes to change the flavor and make them easier to smoke.14

Besides its ability to make cigarettes more appealing, menthol can also act as an inhibitor

of the CYP2A6 enzyme.11 However, menthol is approximately 1000 times less powerful

as an inhibitor than β-nicotyrine.11 However, menthol can also be lethal to fish by causing

38

hypertrophy and aneurisms at dosages as low as 80 mgL-1 .16 While menthol appeared

regularly in the sediment samples, cigarettes are not the only source of menthols.

Toothpaste, lip balm, aftershave, and peppermint are all products that have menthol in

them, and these can also be easily transported to the ocean. While menthol can be toxic at

times, the presence of multiple other sources indicate that it would not be the best

indicator of cigarette butt pollution.

The other chemical in this category frequently detected in the exposed sediment

was 2,4-xylenol, a phenol derivative. This chemical was found in every cigarette sample

and tank sample, although its concentration was significantly lower than any of the

pyridine derivatives and large alkanes. While 2,4-xylenol is capable of exhibiting acutely

toxic effects such as impaired motility at concentrations as low as of 2.1 µM,26 the fact

that xylenols can come from many different sources such as paint and cleaning solvents

indicates that 2,4-xylenol would not be a good indicator of cigarette butt pollution.

39

CHAPTER 5

CONCLUSIONS

The less-polar organic chemicals within cigarette butts extracted and identified in

this study were not only able to bind to the sediment within the exposure tanks, but many

of them were able to do so for the entire 60 day period. The work here has shown

specifically which chemicals have the potential to exhibit long-term toxic effects to the

aquatic environment. Even more importantly it has helped to identify a set of chemicals

consisting of nicotine, cotinine, myosmine and β-nicotyrine, which can be used as

markers to help detect cigarette butt pollution within sediment in the aquatic

environment. It can be seen from this work that cigarette butts should be better regulated

in terms of their disposal, as the chemicals they leach out can have lasting effects on the

organisms that they come in contact with. In the future, different types of sediment

should be put through the same tests to see what kinds of effect this can have on the

ability of the cigarette chemicals to bind, especially if the sediment is one that can house

more biological diversity. Future work can also be done to understand how long these

chemicals can bind to sediment in order to realize how many generations of organisms

living in the sediment they can impact. It is noted however, that since nicotine and the

other pyridine derivatives can be metabolized within organisms, these effects are likely to

be more acutely toxic rather than chronic. Additionally, the bioaccumulation of these

40

specific chemicals, especially nicotine and its metabolites, up the food chain needs to be

further studied so as to understand the impact these chemicals can have long after

organisms from within the sediment have absorbed them.

41

Table 1 Library ID and CAS codes for hydrophobic chemicals found in 11 or more cigarette samples

Library ID CAS

1-Bromo-11-iodoundecane 139123-69-6

2-Dodecen-1-yl(-)succinic anhydride 019780-11-1

2,3'-Dipyridyl 000581-50-0

Cotinine 000486-56-6

Heptacosane 000593-49-7

Stigmasterol 000083-48-7

Cyclohexanol, 5-methyl-2-(1-methylethyl)-, [1R-(1.alpha.,2.beta.,5.alpha.)]-

002216-51-5

Megastigmatrienone 038818-55-2

n-Hexadecanoic acid 000057-10-3

Octacosane 000630-02-4

Pentadecanoic acid 001002-84-2

Pyridine, 3-(3,4-dihydro-2H-pyrrol-5-yl)- 000532-12-7

9,12-Octadecadienoic acid (Z,Z)- 000060-33-3

Cyclotetradecane, 1,7,11-trimethyl-4-(1-methylethyl)-

001786-12-5

Eicosane 000112-95-8

Geranylgeraniol 024034-73-9

Octadecane 000593-45-3

Pentadec-7-ene, 7-bromomethyl- 1000259-58-5

Phenol 000108-95-2

1-Hexacosene 018835-33-1

11,13-Dimethyl-12-tetradecen-1-ol acetate 1000130-81-0

42

Table Continued

Library ID CAS

Indole 000120-72-9

Rishitin 018178-54-6

1-Docosene 001599-67-3

1H-Indene, 2,3-dihydro-1,1,3-trimethyl-3-phenyl- 003910-35-8

Triacontane 000638-68-6

17-Pentatriacontene 006971-40-0

2-Methyl-3-(3-methyl-but-2-enyl)-2-(4-methyl-pent-3-enyl)-oxetane

1000144-10-2

2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-, (all-E)-

000111-02-4

9,12,15-Octadecatrienoic acid, (Z,Z,Z)- 000463-40-1

Phenol, 2-methyl- 000095-48-7

Phenol, 4-methyl- 000106-44-5

2-Pentadecanone, 6,10,14-trimethyl- 000502-69-2

3-Hydroxy-.beta.-damascone 102488-09-5

4,8,13-Cyclotetradecatriene-1,3-diol, 1,5,9-trimethyl-12-(1-methylethyl)-

007220-78-2

Cyclohexene, 4-(4-ethylcyclohexyl)-1-pentyl- 301643-32-3

dl-.alpha.-Tocopherol 010191-41-0

Carotol 000465-28-1

Cholesterol 000057-88-5

Docosane 000629-97-0

Octatriacontyl pentafluoropropionate 1000351-89-1

43

Table Continued

Library ID CAS

Tricosane 000638-67-5

1-Nonadecene 018435-45-5

1-Nonadecene 018435-45-5

1,2-Benzenediol 000120-80-9

1,2-Benzisothiazole, 3-(hexahydro-1H-azepin-1-yl)-, 1,1-dioxide

309735-29-3

2,6,10-Dodecatrien-1-ol, 3,7,11-trimethyl- 004602-84-0

2,6,10,14,18-Pentamethyl-2,6,10,14,18-eicosapentaene

075581-03-2

2(1H)-Naphthalenone, octahydro-4a,7,7-trimethyl-, trans-

054699-31-9

7-Hydroxy-6-methoxy-2H-1-benzopyran-2-one 000092-61-5

Nonacosane 000630-03-5

Octadecane, 1-chloro- 003386-33-2

Tetradecanoic acid 000544-63-8

1H-Indole, 3-methyl- 000083-34-1

Dodecanoic acid 000143-07-7

Heneicosane 000629-94-7

Hexadeca-2,6,10,14-tetraen-1-ol,3,7,11,16-tetramethyl,, (E,E,E)

007614-21-3

Hexadecane, 2,6,10,14-tetramethyl- 000638-36-8

Hexanedioic acid, dimethyl ester 000627-93-0

Tetrapentacontane, 1,54-dibromo- 1000156-09-4

Vanillin 000121-33-5

44

Table Continued

1-Propanol, 2-[2-(benzoyloxy)propoxy]-, benzoate 020109-39-1

1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester

004376

2-Cyclohexen-1-one, 4-(3-hydroxy-1-butenyl)-3,5,5-trimethyl-

034318-21-3

2-Methoxy-4-vinylphenol 007786-61-0

2-Pentenoic acid, 5-(decahydro-5,5,8a-trimethyl-2-methylene-1-naphthalenyl)-3-methyl-, [1S-[1.alpha.(E),4a.beta.,8a.alpha.]]-

024470-48-2

Benzamide, N-propyl- 010546-70-0

Heptadecane 000629-78-7

Hexacosane 000630-01-3

Longifolenaldehyde 019890-84-7

Pentanedioic acid, dimethyl ester 001119-40-0

Pyridine, 3-(1-methyl-2-pyrrolidinyl)-, (S)- 000054-11-5

Triacontyl pentafluoropropionate 1000351-80-0

1-Phenanthrenecarboxylic acid, 1,2,3,4,4a,9,10,10a-octahydro-1,4a-dimethyl-7-(1-methylethyl)-, methyl ester, [1R-(1.alpha.,4a.beta.,10a.alpha.)]-

001235-74-1

2H-1,2-Oxazine, tetrahydro-2-methyl-6-(3-pyridinyl)-, (-)-

015769-88-7

4-n-Hexylthiane, S,S-dioxide 070928-52-8

Dotriacontyl pentafluoropropionate 1000351-81-4

Octacosyl heptafluorobutyrate 1000351-83-6

45

Table 2 Hydrophobic chemicals found in both cigarette samples and exposure tanks

Library/ID Tank 5

Tank 10

Tank 20

Tank 30

Tank 45

Tank 60

# Cigs containing chemical

Pyridine, 3-(1-methyl-2-pyrrolidinyl)-,(s)

10 10 7 5 10 10 23

2,3ʹ-Dipyridyl 10 10 7 5 9 10 23

2-Pyrrolidinone, 1-methyl-5-(3-pyridinyl)-, (S)-

10 10 7 5 8 7 23

Pyridine, 3-(3,4-dihydro-2H-pyrrol-5-yl)-

10 10 7 5 8 7 23

Pyridine, 3-(1-methyl-1H pyrrol-2-yl)

10 10 7 5 8 7 23

2,4-Xylenol 10 10 7 5 10 10 23

Heptacosane 10 10 7 5 10 10 23

Octacosane 1 2 4 5 10 10 23

Nonacosane 9 10 7 5 10 10 23

Triacontane 9 9 6 5 10 10 23

Dotraicontane 9 10 7 5 10 10 23

Tetratriacontane 8 9 3 3 9 9 22

Cyclohexanol, 5-methyl-2-(1-methylethyl)-, [1R-(1.alpha.,2.beta.,5.alpha.)] -

7 9 7 5 8 8 21

Phenol 5 3 0 0 0 0 20

Hexacosane 9 6 3 5 10 10 20

46

Table Continued

Library/ID Tank 5

Tank 10

Tank 20

Tank 30 Tank 45 Tank 60

# Cigs containing

chemical

Pentacosane 6 6 5 5 10 10 20

2-Cyclopenten-1-one,2,3-dimethyl

8 8 0 0 0 0 19

Phenol, 2-methyl 10 8 1 0 0 0 17

Bicyclo(3.1.1) heptane,2,6,6-trimethyl)-,(1.alpha.,2.beta., 5.alpha.)

3 4 5 2 2 1 17

Tocopherol derivative

3 6 2 5 10 10 16

2-Pentadecanone,6, 10,14-trimethyl

2 0 0 0 0 0 16

Tributyl acetylcitrate

2 2 3 2 4 2 14

Tetradecanoic acid

0 3 4 2 0 0 14

Tetracosane 0 0 0 3 10 10 13

Tricosane 5 3 5 5 10 10 12

Docosane 2 4 6 3 9 10 10

Heneicosane 2 6 5 3 8 9 10

Octanoic acid 2 0 0 0 1 2 10

Pentadecane 9 9 1 3 10 8 9

47

Table Continued

Library/ID Tank 5

Tank 10 Tank 20 Tank 30 Tank 45 Tank 60

# Cigs containing

chemical

Nonadecane 6 8 2 2 5 4 9

Eicosane 5 8 3 2 6 4 8

Octadecanoic acid

0 0 4 2 0 1 8

Glycerol tricaprylate

0 0 2 0 2 2 7

p-Xylene 0 0 0 4 4 3 7

Propanoic acid, 2-methyl-,1-(1,1-dimethylethyl)-2-methyl-1,3-propanediyl ester

5 2 8 0 0 0 4

.

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