gaultney beverly teal
TRANSCRIPT
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
Beverly Teal Gaultney
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
Beverly Teal Gaultney
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.
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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
5 ND #VALUE! ND 179 PRE
+/-
7.5 ND
6 ND #VALUE! ND 179 POST 15 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
27 ND #VALUE! ND 215 PRE
+
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
35 ND #VALUE! ND 135 PRE
+/-
13.5 ND
36 ND #VALUE! ND 135 POST 14.5 ND
38 2966441 2.373907083 230 POST
+
15 0.012724142
39 ND #VALUE! ND 230 PRE 15 ND
42 4474218 3.825519523 202 PRE
-
12.5 0.024483325
43 ND #VALUE! ND 202 POST 15 ND
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
49 ND #VALUE! ND 143 PRE 15 ND
50 ND #VALUE! ND 126 PRE
+/-
15 ND
51 ND #VALUE! ND 126 POST 10 ND
19
52 5309173 4.629372523 2 PRE
-
15 0.024813437
54 ND #VALUE! ND 2 POST 15 ND
56 742203 0.232521752 <LOD 65 POST
+/-
15 0.001246317
58 ND #VALUE! ND 65 PRE 15 ND
59 ND #VALUE! ND 62 POST
+/-
15 ND
60 ND #VALUE! ND 62 PRE 11 ND
61 ND #VALUE! ND 36 POST
-
15 ND
62 4008394 3.377047426 36 PRE 15 0.018100974
63 2952728 2.360704891 23 PRE
-
15 0.012653378
64 ND #VALUE! ND 23 POST 15 ND
65 ND #VALUE! ND 30 PRE
+/-
15 ND
66 ND #VALUE! ND 30 POST 15 ND
69 ND #VALUE! ND 6 POST
+/-
15 ND
70 ND #VALUE! ND 6 PRE 4 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
83 ND #VALUE! ND 41 PRE
+/-
15 ND
86 ND #VALUE! ND 41 POST 15 ND
87 ND #VALUE! ND 4 POST
-
15 ND
88 8691446 7.885656153 4 PRE 15 0.042267117
90 ND #VALUE! ND 67 PRE
+/-
15 ND
91 ND #VALUE! ND 67 POST 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
98 ND #VALUE! ND 17 POST 15 ND
99 1264395 0.735262146 58 POST
+
15 0.003941005
102 ND #VALUE! ND 58 PRE 15 ND
107 ND #VALUE! ND 112 PRE
+/-
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
117 ND #VALUE! ND 110 POST
+/-
15 ND
119 7448 -0.474863689 <LOD 110 PRE 15 <LOD
120 ND #VALUE! ND 90 PRE
+
15 ND
122 1096201 0.573333358 <LOD 90 POST 12.5 0.003669333
20
123 161735 -0.326323866 <LOD 128 PRE
+/-
15 <LOD
125 ND #VALUE! ND 128 POST 15 ND
126 2741090 2.156950388 14 POST
+
15 0.011561254
128 ND #VALUE! ND 14 PRE 15 ND
129 ND #VALUE! ND 84 PRE
+
10.5 ND
132 4317974 3.675095598 84 POST 11 0.026754696
133 ND #VALUE! ND 103 PRE
+/-
15 ND
134 ND #VALUE! ND 103 POST 15 ND
139 1254370 0.725610577 144 POST
+
12.5 0.004643908
140 ND #VALUE! ND 144 PRE 5 ND
141 ND #VALUE! ND 154 POST
+/-
15 ND
143 436851 -0.061455913 <LOD 154 PRE 10 <LOD
145 ND #VALUE! ND 95 POST
+/-
15 ND
147 ND #VALUE! ND 95 PRE 15 ND
148 4043984 3.411311702 86 POST
+
12.5 0.021832395
152 ND #VALUE! ND 86 PRE 2.5 ND
153 ND #VALUE! ND 161 PRE
+/-
15 ND
156 ND #VALUE! ND 161 POST 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