Gaultney Beverly Teal

UNIVERSITY OF CINCINNATI

Date:

I, ,

hereby submit this original work as part of the requirements for the degree of:

in

It is entitled:

Student Signature:

This work and its defense approved by:

Committee Chair:

2/19/2010 402

15-Jan-2010

Beverly Teal Gaultney

Master of Science

Industrial Hygiene (Environmental Health)

Determination of Urinary 2-naphthol Concentration in Rubber Manufacturing

Workers

Glenn Talaska, PhD

Paul Succop, PhD

Andrew Maier, PhD, MS

Glenn Talaska, PhD

Paul Succop, PhD

Andrew Maier, PhD, MS


1

Determination of Urinary 2-Napthol Concentration in Rubber Manufacturing Workers

A thesis submitted to the Graduate School of the University of Cincinnati

In partial fulfillment of the requirements for the degree of

Master of Science in Industrial Hygiene (Environmental Health)

in the Department of Environmental Health

of the College of Medicine

by


B.S. University of Georgia

December 2005

Committee Chair: Glenn Talaska, Ph.D.

2

ABSTRACT

Polycyclic aromatic hydrocarbons (PAHs), which consist of 2 or more fused benzene rings, are formed by

the incomplete combustion of fossil fuels. Although PAHs are an exposure concern in many industrial

sectors, one industry of particular interest is the rubber industry. PAHs make up around 20% of the total

weight of rubber products including tires. For tire manufacturing, PAH exposures arise when they are

added as extender oils and they may also be present when carbon black is used to produce the master

batch material (IARC, 1982). Such exposures have been of public health interest because epidemiology

studies of workers exposed to PAHs have identified an increase risk of developing cancers of the lungs,

urinary bladder, skin, and prostate (Bofetta et al 1997; Constantino et al 1995). Naphthalene is the simplest

and most volatile member of the PAHs. The major route of elimination of naphthalene is via the formation of

the hydroxylated metabolites 1- and 2- naphthol which are excreted through the urine (Onyemauwa et al

2009; Serdar et al 2004; Preuss et al 2003). The hypothesis of this thesis is that Post-shift urinary 2-

naphthol levels are significantly higher than pre-shift urinary 2-naphthol levels in rubber workers exposed to

naphthalene.

3

4

Acknowledgements

Thank you to Dr. Paul Succop, Dr. Andrew Maier, and Dr. Glenn Talaska. It was a privilege to have your

assistance and advice during the completion of this thesis. I am glad that such a skilled group offered its

time to helping me better this body of work. Each of you has helped me, and I appreciate all of your time

and effort.

5

1.0 INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs), which consist of 2 or more fused benzene rings, are formed by

the incomplete combustion of fossil fuels. PAHs are common environmental pollutants due to the wide

range of sources that generate them, including cigarette smoke, cooked foods, forest fires, motor

vehicle exhaust, air traffic, and nearby industrial plants. Workers are also exposed to PAHs present in

raw materials used in the production of many products such as distillation and crystallation of coal tar,

coking plants, intermediates of the pharmaceutical and chemical industries, graphite electrode plants,

production and installation of fireproofing materials, and numerous other industries which have lower

levels of PAH exposures (US EPA 1998 and Preuss et al 2003).

Although PAHs are an exposure concern in many industrial sectors, one industry of particular interest is

the rubber industry. PAHs make up around 20% of the total weight of rubber products including tires.

For tire manufacturing, PAH exposures arise when they are added as extender oils and they may also be

present when carbon black is used to produce the master batch material (IARC, 1982). PAHs are also

formed during the incomplete combustion of organic rubber products particularly during vulcanization

and curing (Talaska et al 2002, Jonsson et al 2008). Such exposures have been of public health interest

because epidemiology studies of workers exposed to PAHs have identified an increase risk of developing

cancers of the lungs, urinary bladder, skin, and prostate (Bofetta et al 1997; Constantino et al 1995). This

increased risk of cancer among other PAH-exposed workers is consistent with the increased incidence of

cancer among rubber workers, which has been well documented (Monson and Nakano 1976a, Monson

and Nakano 1976b, Delzell and Monson 1981, Fine and Peters 1976, and IARC 1982).

6

PAH exposures are typically in the form of complex mixtures, but in some industries individual chemicals

may predominate or may be able to be used as a marker for a broader PAH exposure. Naphthalene is

the simplest and most volatile member of the PAHs. It consists of 2 fused benzene rings, and in most

indoor and outdoor environments it is most frequently encountered as a vapor, although it may also be

bound to particulate to some degree (Price et al 2008). Cigarette smoke has been found to be one of the

most significant sources of naphthalene exposure in the general population (Hoffman et al 2001), and

the EPA confirms that the environmental exposure to naphthalene is high compared to that of the other

PAHs, with the main route of exposure being inhalation (Price et al 2008; Wilhelm et al 2008). In

workplaces where PAH exposure is present, naphthalene is generally the most abundant compound

present in the PAH emissions (Rappaport et al 2004; Preuss et al 2003). The amount of naphthalene

exposure increases when PAH containing materials are exposed to heat, and this is of particular interest

in the rubber manufacturing industry in the curing department where the rubber is heated to around

130°C until the curing is complete (Fine and Peters, 1976). Rubber may continue to off gas naphthalene

during the inspection process as it continues to cool. The major route of elimination of naphthalene is

via the formation of the hydroxylated metabolites 1- and 2- naphthol which are excreted through the

urine (Onyemauwa et al 2009; Serdar et al 2004; Preuss et al 2003).

In 2000, the National Toxicology Program published the results of two-year inhalation cancer bioassay in

F344/N rats. They concluded that “under the conditions of this 2-year inhalation study, there was clear

evidence of carcinogenic activity* of naphthalene in male and female F344/N rats based on increased

incidences of respiratory epithelial adenoma and olfactory epithelial neuroblastoma of the nose” (NTP,

2000). In addition a 1992 2-year inhalation study of male and female B6C3F1 mice found that there was

“some evidence of carcinogenic activity” in the female was “ based on increased incidences pulmonary

7

alveolar/bronchiolar adenomas” (NTP, 1992). The results of these rodent studies increased concern

regarding the carcinogenic potential of naphthalene in the scientific community and led to a

reclassification of naphthalene as a possible human carcinogen by the EPA and IARC (Preuss et al 2004;

Preuss et al 2003; Rappaport et al 2004; EPA 2003; IARC 2002). Prior to the publication of these studies,

naphthalene was not considered a significant concern for cancer in the occupational environment

because it was thought to be non-carcinogenic, tolerable levels above set exposure limits were too high

to be exceeded in the workplace, and sampling at workplaces revealed levels below current

occupational exposure limits, which were based on non-cancer effects (Preuss et al 2003). As the

potential carcinogenicity of naphthalene and its underlying mode of action have been actively debated,

more emphasis has been given to sampling the compound in the workplace where it was found to be

one of, if not the most, abundant chemical associated with PAH exposure. There is an increasing need

to develop robust monitoring approaches for exposures to naphthalene based on the increased

regulatory and public emphasis on naphthalene and its abundance in processes that involve PAHs.

Urinary Naphthols have been suggested as a biomarker of general PAH inhalation exposure due to their

specificity of reflecting inhalation based exposures. Urinary naphthols were not found to correlate with

dietary habits, as 1HP is, but were correlated with smoking indicating an inhalation route specific marker

(Yang et al 1999; Kang et al 2002; Kim et al 2001; Onyemauwa et al 2009). With bladder cancer being a

prevalent illness in the rubber industry the fact that inhaled naphthalene is metabolism in the urinary

bladder makes biomonitoring of interest for researchers.

In 2008 a study entitled “Polycyclic Aromatic Hydrocarbon Exposure, Urinary Mutagenicity, and DNA

Adducts in Rubber Manufacturing Workers” DNA Adducts were seen in the urothelial cells and in the

8

peripheral blood mononuclear cells of PAH-exposed rubber workers. These adducts did not correlate to

levels of 1-hydroxypyrene, a common biomarker for PAH exposure, or with measures of urinary

mutagenicity. The results from this study suggest that some other DNA-reactive agent may better

correlate to the observed adduct formation in these workers (Peters et al 2008). As an alternative to

PAHs as reflected by 1-hydroxypyrene, naphthalene may be an inhalation exposure in these workers

that would correlate better with the urinary mutagenicity and the presence of urothelial cell adducts.

Since the peripheral blood mononuclear cells and urothelial cell adducts were not correlated to each

other, it may suggest that there is a specific bladder carcinogen in the rubber industry which remains

unidentified (Peters et al 2008). Since naphthalene’s major route of elimination is through oxidized

metabolites, 1- and 2-naphthol, excreted through the urine one or both of these metabolites may be

measured to determine if there is a possible correlation between their concentration in urine and levels

of urothelial cell DNA adducts. As a first step in investigating this possibility we evaluated whether there

is an increase in mean levels of 2-naphthol from pre-shift to post-shift samples to confirm this

metabolite as a valid biomarker for naphthalene exposure in rubber workers. This information may be

used in future studies to examine correlations between exposure, adducts and urinary mutageniticty as

described in the report by Peters et al (2008) and to compare differences in biomarker levels between

differing departments within the rubber working process as a means to identify areas with greatest

potential exposures.

9

2.0 HYPOTHESIS

Post-shift urinary 2-naphthol levels are significantly higher than pre-shift urinary 2-naphthol levels in rubber

workers exposed to naphthalene.

10

3.0 MATERIALS AND METHODS Materials used to perform analysis of urine samples for 2-naphthol included:

• 0.1M Sodium Acetate (Pure Sodium Acetate (1M solution =82.03g/l) was diluted 1:10 by adding

8.203 g and adding to 100ml water to the graduated cylinder yielding a 0.1M

solution=8.203g/100ml) (Fisher Scientific, Pittsburgh, Pennsylvania)

• β-glucuronidase/arylsulfatase (G-0876 Type h-2 from Helix pomatia, 105,000 β-glucuronidase

units/ml and 4,300 arlysulfatase units/ml , Sigma)

• 2-naphthol (CAS#135-19-3, Alfa Aesar, 98+% 2-Naphthol, stock #: A14564, lot#H4478A, Fisher

Scientific, Pittsburgh, Pennsylvania )

• HPLC Grade Methanol (CAS#67-56-1, Fisher Scientific, Pittsburgh, Pennsylvania)

• Milli-Q (ultra pure) water (double deionized water)

• Analytical nitrogen evaporator with nitrogen tank with nitrogen regulator turned to pressure of

approximately 50psi

• Water bath (contained at bottom of analytical nitrogen evaporator)

• Refrigerator

• 37°C Incubator (Fischer Scientific Model 630D, Fisher Scientific, Pittsburgh, Pennsylvania)

• Shaking platform

• High Performance Liquid Chromatograph (HPLC) (2695 separation module, Waters) with

Fluorescence detector (730 Water Data Module fluorescence detector) connected to a PC with

Empower 2 HPLC software

11

• Jack Berberich’s Milk crate

• 50ml Screw cap tubes (samples were received in these)

• 25ml glass Scintillation vials (Fisher Scientific, Pittsburgh, Pennsylvania)

• Gilson Micropipettes (20ul, 5ml) (Fisher Scientific, Pittsburgh, Pennsylvania)

• Pipette tips (Fisher Scientific, Pittsburgh, Pennsylvania)

• Test tube racks (Fisher Scientific, Pittsburgh, Pennsylvania)

• Styrofoam holder for 25ml vials

• 20ml syringes (Fisher Scientific, Pittsburgh, Pennsylvania)

• 0.45 um filters (Fisher Scientific, Pittsburgh, Pennsylvania)

• 3ml syringes (Fisher Scientific, Pittsburgh, Pennsylvania)

• C18 Sep Pak Cartridges (Part No. WAT020515 Waters Corporation, Milford, Massachusetts)

• Tape (Fisher Scientific, Pittsburgh, Pennsylvania)

• Sharpie Markers (Fisher Scientific, Pittsburgh, Pennsylvania)

• Plastic tray (Fisher Scientific, Pittsburgh, Pennsylvania)

• Weigh boat (Fisher Scientific, Pittsburgh, Pennsylvania)

• Balance ( Denver Instruments model TR403, Fisher Scientific, Pittsburgh, Pennsylvania)

Sample Acquisition

A total of 159 samples were received from Drs. Bo A.G. Jonsson and Roel Vermeulen. The samples were

collected in 1997 from a group of rubber manufacturing workers from several Dutch facilities which

manufactured rubber tires and belts, general rubber goods, and a retreading company. The urine samples

collected were from exposed males with a mean age of 38.5 years. Urine samples were collected pre-shift

on the Sunday prior to other sampling, and post-shift samples were collected either on Wednesday or

Thursday of the same week at 4pm which was at the end of the workday (Peters et al, 2008). All samples

12

were received via a FedX shipment from Dr. Bo A.G. Jonsson in good condition, still frozen, caps secured,

labels adhered and legible, with a large amount of dry-ice left in each of the two containers used to ship the

samples. Dr. Glenn Talaska contacted Dr. Vermeulen to verify which samples were those of non-smokers,

and these were selected as the samples used in the current study. A total of 86 individual, 43 pairs of pre

and post, samples were analyzed in the study. To avoid knowledge of which samples were pre- and post-

shift, another student, Mr. John Jaskolka, covered the post and pre shift label indications and identification

number of each sample with tape. The samples were then relabeled with a new number corresponding to

the order in which they were unpacked by the student. The original numbering and pre/post shift

information was recorded by the student, and the new numbering system was used throughout the

experiment to ensure that the investigator, Ms. Beverly Gaultney, and advisor, Dr. Glenn Talaska, were

blinded in regards to the sample status (pre or post shift as well as department).

Summary of Methods

Urinary levels of 2-naphthol were determined using the solid phase extraction method of Jongeneelen et al

(1987), and the HPLC analysis was carried out as in the methods used by Kim et. al, 2001. In summary,

the urine was adjusted to a pH of 5.0 (+/- 0.05) by adding aliquots of 1N HCl. Next, sodium acetate and β-

glucuronidase/arylsulfatase were added and incubated on a shaking grid for 4 hours at 37°C. Following

incubation SepPak cartridges were primed to remove contaminates using methanol and water; the

prepared samples were then passed through the primed cartridges. Milli-Q water and methanol were once

more passed through the cartridges. The sample was then eluted with methanol, and dried using a nitrogen

evaporator and water bath set at 60°C. Samples were resuspended with methanol and filtered into brown

glass HPLC vials. The HPLC analysis was performed to detect the excitation/emission wavelengths for 2-

napthol at 227/355 nm (Kim et.al, 2001). The amount of 2-naphthol was estimated using a calibration curve

13

developed with standard 2-napthol solutions of varying concentrations and the determination of the peak

levels seen in the HPLC analysis was assessed using the excitation/emission wavelengths of 227/355 nm.

Sample Preparation

As noted above, Ms. Beverly Gaultney, and advisor, Dr. Glen Talaska, were blinded in regards to the

sample status (pre or post shift as well as the department where each subject worked). Urine samples were

removed from the freezer and placed in the refrigerator for 4 hours to thaw. The volume of each urine

sample was recorded. The pH of each urine sample was then determined using a calibrated pH meter.

Each urine sample was then adjusted to a pH of 5.0 (+/- 0.05) by adding 1N HCl one drop at a time while

agitating the pH probe. The resultant pH of each sample was recorded.

Five ml (per 15ml of urine) of 0.1M Sodium acetate and 8.75ul (per 15ml of urine) of β-

glucuronidase/arylsulfatase were added to each sample. Samples with less than 15ml of urine contained

proportionally adjusted amounts of Sodium acetate and β-glucuronidase/arylsulfatase. The samples were

then incubated with agitation at 37°C for 4 hours. Once incubation was complete, the samples were

refrigerated at 41°F overnight.

Hydrolysis

The C18 Sep Pak Cartridges were primed to remove contaminants. Each Sep Pak Cartridge was attached

to a 20mL syringe, with the plunger of each syringe removed. The syringes plus Sep Pak Cartridges were

Cartridge Priming

14

held upright and 5 ml of HPLC grade methanol was added to each syringe with a pipette. The plungers

were then added to the syringes and the methanol was pushed through the Sep Pak Cartridges at a rate of

2.5ml/minute. The syringe plungers were then removed and 10ml of Milli-Q water was added to each

syringe using a pipette. All plungers were then replaced, and the Mill-Q water was pushed through the Sep

Pak Cartridges at a rate of 2.5ml/min. This step of washing with 10ml of Milli-Q water was then repeated for

a second time. Each syringe was labeled with a unique sample number.

After samples were incubated and cartridges were primed, the samples were then loaded onto the Sep Pak

Cartridges. Syringes labeled with the corresponding number to each sample were used. The plungers were

removed from each syringe and the entire volume of each sample was emptied into the syringe with the

corresponding sample number. The syringe plungers were then added to the syringes and the urine sample

was pushed through the Sep Pak Cartridges at the rate of 2.5ml/min. This waste was not collected and was

allowed to flow down the drain the laboratory’s sink. Some cartridges became clogged due to sediments in

the urine and would not allow the entire sample volume to be pushed through a single Sep Pak Cartridge.

These samples were then added to a second labeled syringe and Sep Pak cartridge with the corresponding

sample number and the letter B. When required a 3rd, 4th, and 5th syringe and Sep Pak Cartridge were used

and labeled with the sample number plus the letter C, D, or E respectively. After the entire sample was

loaded onto the cartridge, the cartridge was washed to remove polar contaminants. The syringe plungers

were removed with care taken to remember which plunger corresponded to each sample/syringe. This was

accomplished by removing the plungers and placing them upside down (plunger facing upward, and the

bottom of the handle placed on the counter top) on the counter in the same pattern that the samples were

arrayed. A pipette was used to add 8ml of Milli-Q Water to each syringe and the plungers were then placed

Sample Loading

15

in each syringe. The water was pushed through at a rate of 2.5ml/min and the waste was collected in a tray

and transferred to a waste beaker.

The samples were then eluted into labeled 25 ml glass scintillation vials, by removing the syringe plunger

and adding 10 ml of methanol to each syringe with Sep Pak cartridge still attached. The methanol was

pushed through the Sep Pak cartridges at a rate of 2.5 ml/min. For sample numbers with multiple Sep Pak

cartridges, 10 ml of methanol was pushed through the Sep Pak cartridge labeled with the letter A as

described previously. The methanol and sample in the scintillation vial were then drawn up using a pipette

and then place into the syringe with the Sep Pak cartridge labeled with the letter B, the methanol was then

pushed through the Sep Pak cartridge at a rate of 2.5ml/min and collected in the scintillation vial. This

process was repeated until all Sep Pak cartridges corresponding to the sample number were eluted into the

vial. The solvent was then evaporated by placing the glass scintillation vials in a water bath at 60°C and

under the gentle flow of nitrogen. This process took on average 30 minutes. While the solvent was

evaporating in the nitrogen flow and water bath, 0.45 um syringe filters were washed (1 for each sample) by

drawing 5ml of methanol into a 3ml syringe and attaching it to each filter. The methanol was pushed

through and then air was pushed through twice to dry the filters. Once the solvent was evaporated, the

samples were removed from the water bath and resuspended by adding 2ml of HPLC grade methanol to

each scintillation vial. The 3ml syringes were then used to draw up the resuspended sample and methanol

in the vials. A 0.45 um filter was then added and the sample was pushed through into a 2ml brown glass

HPLC vial labeled with the sample number. A new filter and syringe were used for each sample.

High Pressure Liquid Chromatography (HPLC) Waters 2695 separation module with 730 Water Data

Module fluorescence detector)

16

After all other preparation steps were complete the samples were run through the High Performance Liquid

Chromatograph using the excitation/emission wavelengths of 227/355 nm (Kim et.al, 2001). The flow rate

was set to 0.8ml/min and the solvent flow was set to 38% Water and 62% Methanol with the column

temperature set to 35°C with a range of 5°C. To determine a standard curve and the peak time for 2-

naphthol elution a standard diluted to a concentration of 18.8 ng 2-naphthol/ul methanol was run at (5ul,

10ul, 20ul, 30ul, 40ul, 50ul,1.88ul twice, 3.76 ul twice, 5.64 ul twice, 7.52 ul twice, 9.4ul twice, and 18.8 ul

twice). The 2-naphthol standard was diluted by weighing out 18.8mg of dry 2-naphthol standard on a scale,

then placing the dry 2-naphthol into a 25ml glass scintillation vial and adding 10ml of methanol to yield

1.88ug/ul, another 10 ml of methanol was added to yield 18.8 ng/ul. After running the standards through the

HPLC machine with the above settings, 2-naphthol was found to peak at 4.1 minutes. The standard curve

was created in Microsoft Excel by taking the results from the lower end of the curve (1.88ul twice, 3.76 ul

twice, 5.64 ul twice, 7.52 ul twice, 9.4ul twice, and 18.8 ul twice) and entering the amount of standard

injection as the y value and the area produced at each of these levels as the x values. The function linest of

Excel was used with a constant equal to the area of that sample and the stats =1. To determine the

correlation coefficient between the areas and levels the stat/correlation function was used with array 1

being set as the levels and array 2 set as the areas. The results yielded a correlation coefficient of 0.9997.

The slope and y intercept were determined by the Excel program with the y=to the areas and the x=to the

levels to yield an equation for the standard curve line. The x value, the area produced by each sample run,

was then entered manually after the sample run for each sample and the y-value was calculated by excel

using this equation to yield a result of the amount of 2-naphthol in each sample. The equation produced by

this process in the format y=mx+b was y=1038691*(area produced from HPLC curve)+(500684.7131).

For the sample run the following settings were used: the integration was set to inhibit integration from a

start time of 0.017min to 2.3 minutes and again from 6.0 minutes to 25 minutes. The component settings

17

were set to a migration time for 2-naphthol set to 4.0 minutes with a MT Window of 1.0 minutes, and the

Peak Match was set to Closet. The Y Value was set as the area, the X Value as the amount, the fit set to

linear, and the weighting set to none.

4.0 RESULTS

The data revealed a possible work-related exposure to naphthalene in the rubber workers as post shift

levels in 17 sample pairs were elevated when compared to the pre shift samples, while only 7 of the data

sets revealed a decrease in 2- naphthol levels in the post- versus pre shift samples. Although 17 of the

pairs showed an increase in the 2-napthol recovery levels in the post shift compared to the pre shift

sample, this was not an adequate number of pairs to statistically determine a difference between pre and

post shift samples; therefore, contrary to the hypothesis, the differences between the pre shift and post

shift samples was not statistically significant.

The pre and post shift differences, between the pairs were analyzed as a group, and the p-value of the

differences using a 2-sided student’s t-test was found to be p=0.4. A sign test revealed a p value of 0.053,

which was nearly statistically significant. The post-pre recovery differences were again analyzed after

taking the logarithm of both measurements. The p-value for the paired t-test is slightly smaller p= 0.3, but

still not significant. The post-pre differences were somewhat more normally distributed after the log

transformation. The results of the statistical analysis performed by Dr. Paul Succop are found as

Appendix A: Statistics Charts for Pre and Post Differences.

Table 1: contains the sample number, the pre or post shift identification, the area under the curve of the

HPLC peak identified as 2-naphthol, and the amount of 2-naphthol recovered in picograms or the notation

of <LOD (Limit of Detection, meaning a peak was observed but was under the limits set by the calibration

curve) or ND (Non Detect, meaning no peak was seen), and a calculated amount of 2-naphthol in urine

(ug/l).

18

Table 1: Results of Analysis

Sample ID

Area from HPLC Peak

Amount Recovered per HPLC injection volume 25ul (pg)

LOD/ND Value =0.28

Decoded Sample ID (Pre or Post) Change

Volume of Urine Analyzed (ml)

Calculated Amount (ug /l of urine)

2 ND #VALUE! ND 180 PRE

+/-

15 ND

4 ND #VALUE! ND 180 POST 15 ND


+/-

7.5 ND


9 2309273 1.741218533 195 PRE

+

15 0.009332931

10 16793551 15.68595846 195 POST 15 0.084076737

15 735564 0.226130054 <LOD 192 PRE

+

15 0.001212057

17 3374081 2.766362529 192 POST 15 0.014827703

19 4247202 3.606959849 146 PRE

-

15 0.019333305

20 576666 0.073150991 <LOD 146 POST 15 0.000392089

24 3800623 3.177015877 232 POST

+

15 0.017028805

26 ND #VALUE! ND 232 PRE 15 ND


+

15 ND

29 21128911 19.85982668 215 POST 15 0.106448671

30 10391432 9.522317825 113 POST

+

10 0.076178543

32 783238 0.272028202 <LOD 113 PRE 15 0.001458071


+/-

13.5 ND

36 ND #VALUE! ND 135 POST 14.5 ND

38 2966441 2.373907083 230 POST

+

15 0.012724142


42 4474218 3.825519523 202 PRE

-

12.5 0.024483325


44 398299 -0.098571854 <LOD 216 PRE

+

11 <LOD

47 1295146 0.764867674 216 POST 15 0.004099691

48 ND #VALUE! ND 143 POST

+/-

15 ND



+/-

15 ND


19

52 5309173 4.629372523 2 PRE

-

15 0.024813437


56 742203 0.232521752 <LOD 65 POST

+/-

15 0.001246317



+/-

15 ND



-

15 ND

62 4008394 3.377047426 36 PRE 15 0.018100974

63 2952728 2.360704891 23 PRE

-

15 0.012653378



+/-

15 ND



+/-

15 ND


74 731255 0.221981564 <LOD 15 POST

-

15 0.001189821

75 26679099 25.20327067 15 PRE 15 0.135089531

79 20020825 18.79301679 1 POST

+

15 0.10073057

80 ND #VALUE! ND 1 PRE 0.85 ND


+/-

15 ND



-

15 ND

88 8691446 7.885656153 4 PRE 15 0.042267117


+/-

15 ND


93 1006543 0.48701511 <LOD 28 POST

+

15 0.002610401

94 748752 0.238826802 <LOD 28 PRE 15 0.001280112

97 1994284 1.437962845 17 PRE

-

15 0.007707481


99 1264395 0.735262146 58 POST

+

15 0.003941005



+/-

15 ND

108 339197 -0.15547231 <LOD 112 POST 15 <LOD

111 10334224 9.467240818 182 POST

+

15 0.050744411

112 2757882 2.173116888 182 PRE 5 0.03476987


+/-

15 ND

119 7448 -0.474863689 <LOD 110 PRE 15 <LOD


+

15 ND

122 1096201 0.573333358 <LOD 90 POST 12.5 0.003669333

20

123 161735 -0.326323866 <LOD 128 PRE

+/-

15 <LOD


126 2741090 2.156950388 14 POST

+

15 0.011561254



+

10.5 ND

132 4317974 3.675095598 84 POST 11 0.026754696


+/-

15 ND


139 1254370 0.725610577 144 POST

+

12.5 0.004643908



+/-

15 ND

143 436851 -0.061455913 <LOD 154 PRE 10 <LOD


+/-

15 ND


148 4043984 3.411311702 86 POST

+

12.5 0.021832395

152 ND #VALUE! ND 86 PRE 2.5 ND


+/-

15 ND


158 6252048 5.537125513 92 POST

+

7.5 0.058915015

159 2090257 1.53036086 92 PRE 15 0.008202734

21

The change in individual urinary 2-naphthol levels is shown in Figure 1: Urinary 2-napthol levels (ug/l)

paired by pre and post shift samples, which plots each of the paired data sets that had either a change in

pre and post shift recoveries of urinary 2-naphthol. This presentation shows the trend (as reflected in the

near –significant sign test discussed above) of increased 2-naphthol levels in the post-shift samples.

Figure 1: Urinary 2-napthol levels (ug/l) paired for each individual by pre and post shift samples

22

Figure 2 displays the mean values of the recovered amounts of pre and post shift samples above the limit

of detection. The values under the limit of detection and non detects were not included in this figure. The

25th quartile was found to be 0.008 ug/l for pre shift samples and 0.004 ug/l for post shift samples. The

75th quartile value for pre-shift samples was calculated as 0.02 ug/l and for post-shift samples the number

increased to a value of 0.05 ug/l.

Figure 2: Mean values of Recovered Pre and Post Shift Naphthol (ug/l)

23

Figure 3 displays the mean values of the urinary 2-naphthol levels in ug/l in pre and post shift samples

with the values under the Limit of Detection Value included in the calculated values as equal to one half

the limit of detection (LOD=0.28pg).

Figure 3: Calculated Mean values of Recovered Pre and Post Shift Naphthol (ug/l) including LOD values in calculations.

24

5.0 DISCUSSION AND CONCLUSIONS

The data revealed a possible work-related exposure to naphthalene in the rubber workers as post shift

levels in 17 sample pairs were elevated when compared to the pre shift samples, while only 7 of the data

sets revealed a decrease in 2- naphthol levels in the post- versus pre shift samples. Using a one-sided

significance sign test of post results greater than pre results p=0.053. This would indicate that the results

are extremely close to a statistically significant level of p=0.05. The difference in the two values is

approximately the difference between the odds of the data occurring to chance being 18.9 to 1 versus 20

to 1 odds at the p=0.05 level. These results support the workday exposure to naphthalene hypothesized

to be expected among the rubber workers.

The post shift samples ranged from non-detected and <limit of detection (=0.28pg recovered) to a

maximum recovered value of 0.11 ug/l urine and for pre shift samples the samples ranged from ND/LOD

to a maximum value of 0.14 ug/l urine with the next highest value equal to 0.03 ug/l for the pre shift

samples. It is possible that the outlier in the pre-shift samples which is higher than the post shift samples

may actually be a mislabeled post-shift sample as the post shift sample for the same individual decreased

to an amount of 0.0012 ug/l which would be more in line with the values collected as pre-shift samples.

Another possibility is that this individual may have smoked or been exposed to second hand smoke prior

to the pre- shift sample, but the individual was reported as a nonsmoker. With this data pair removed

from the data set, the post-shift average of the paired samples from the remaining rubber workers is

roughly twice that of the pre-shift averages. This adjusted data may be viewed below in Figure 4: Mean

Urinary Naphthol Levels with Outlying Pair Excluded. This figure does not include values at, or under, the

limit of detection.

25

Figure 4: Mean Urinary Naphthol Levels with Outlying Pair Excluded.

26

Figure 5 also excluded the outlying sample pair, and included the limit of detection values and those

values under the limit of detection were set equal to the LOD to calculate the mean urinary naphthol

levels. The figure displays an increase in the post shift recovery amounts of 2-naphthol when compared

to the pre shift recovery amounts.

Figure 5: Mean Urinary Naphthol Levels with Outlying Pair Excluded and Limit of Detection Values

Included in the Calculations.

27

The levels of urinary 2-naphthol recovered in the post shift samples were below those seen in non-

smokers in other industries with naphthalene exposure. The highest value recovered in this analysis for

the post-shift samples of 0.11ug/l was lower than the mean value obtained in studies from the following

industries: converter infeed, coal-tar distillation, coking plant, production of fire-proof materials, production

of graphite electrodes, shipyard workers, and steel workers. The results of these studies may be seen

below in Table 2: Comparison of urinary 2-naphthol levels recovered in non-smoking populations in

various exposed industries. In the production of fire-proof materials study, the production of graphite

electrodes, and the steel workers the values did include a range that encompasses lower values such as

the ones produced in this analysis (Preuss et al,2005; Onyemauwa et al, 2009; Kim et al 2001). This may

indicate that the department of an industry, processes involved in, and type of exposure could greatly

affect naphthalene exposure and thus the urinary 2-naphthol levels.

Table 2: Comparison of post-shift urinary 2-naphthol levels recovered in non-smoking populations in various exposed industries Industry Source

Number of Participants

Mean Value of 2-naphthol (ug/l)

Range (ug/l)

Converter Infeed (Preuss et al 2005)

6 70.2 1.0-190.4

Coal-tar Distillation (Preuss et al 2005)

7 74.1 5.6-334.2

Coking Plant (Preuss et al 2005)

18 13.4 3.0-36.2

Production of Fireproof Materials (Preuss et al 2005)

28 14.0 <LOD-127.0

Production of Graphite Electrodes (Preuss et al 2005)

34 13.9 <LOD-212.8

Shipyard Workers (Kim et al 2001)

65 2.46 0.20-20.37

Steel Workers (Onyemauwa et al 2009)

57 7.12 NA

Current Analysis 43 0.03 <LOD-0.11

28

In summary, urinary naphthols are a promising biomarker for exposure to PAHs; however, many

questions remain regarding their use and further research is needed to determine if they are an

adequate marker of PAH exposure especially in an occupational setting.

29

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Appendix A: Statistics Charts for Pre and Post Differences.

t test and sign test for post - pre differences 311 13:51 Tuesday, July 14, 2009 The UNIVARIATE Procedure Variable: diff_recovered Moments N 43 Sum Weights 43 Mean 0.93630298 Sum Observations 40.261028 Std Deviation 6.58091931 Variance 43.308499 Skewness -0.2925947 Kurtosis 7.11031036 Uncorrected SS 1856.65348 Corrected SS 1818.95696 Coeff Variation 702.862159 Std Error Mean 1.00358079 Basic Statistical Measures Location Variability Mean 0.936303 Std Deviation 6.58092 Median 0.000000 Variance 43.30850 Mode 0.000000 Range 44.50310 Interquartile Range 2.09391 Tests for Location: Mu0=0 Test -Statistic- -----p Value------ Student's t t 0.932962 Pr > |t| 0.3562 Sign M 4.5 Pr >= |M| 0.1078 Signed Rank S 43.5 Pr >= |S| 0.2497 Quantiles (Definition 5) Quantile Estimate 100% Max 19.57983 99% 19.57983 95% 13.94474 90% 7.29412 75% Q3 2.09391 50% Median 0.00000 25% Q1 0.00000 10% -3.32696 5% -4.34937 1% -24.92327 0% Min -24.92327

33

t test and sign test for post - pre differences 312 13:51 Tuesday, July 14, 2009 The UNIVARIATE Procedure Variable: diff_recovered Extreme Observations ------Lowest------ ------Highest----- Value Obs Value Obs -24.92327 6 7.29412 36 -7.60566 3 9.24232 25 -4.34937 2 13.94474 38 -3.54552 39 18.51302 1 -3.32696 31 19.57983 40

34

t test and sign test for ln(post) - ln(pre) differences 441 13:51 Tuesday, July 14, 2009 The UNIVARIATE Procedure Variable: diff_ln_recovered Moments N 43 Sum Weights 43 Mean 0.30336206 Sum Observations 13.0445685 Std Deviation 1.92397563 Variance 3.70168222 Skewness -0.2275838 Kurtosis 0.30006635 Uncorrected SS 159.42788 Corrected SS 155.470653 Coeff Variation 634.217622 Std Error Mean 0.29340353 Basic Statistical Measures Location Variability Mean 0.303362 Std Deviation 1.92398 Median 0.000000 Variance 3.70168 Mode 0.000000 Range 8.76160 Interquartile Range 1.47168 Tests for Location: Mu0=0 Test -Statistic- -----p Value------ Student's t t 1.033941 Pr > |t| 0.3071 Sign M 4.5 Pr >= |M| 0.1078 Signed Rank S 27.5 Pr >= |S| 0.4706 Quantiles (Definition 5) Quantile Estimate 100% Max 4.26166 99% 4.26166 95% 3.52660 90% 2.50006 75% Q3 1.47168 50% Median 0.00000 25% Q1 0.00000 10% -2.55583 5% -2.80539 1% -4.49994 0% Min -4.49994

35

t test and sign test for ln(post) - ln(pre) differences 442 13:51 Tuesday, July 14, 2009 The UNIVARIATE Procedure Variable: diff_ln_recovered Extreme Observations ------Lowest----- -----Highest----- Value Obs Value Obs -4.49994 6 2.50006 18 -3.33801 3 2.57454 17 -2.80539 2 3.52660 25 -2.61466 39 4.20645 1 -2.55583 31 4.26166 40