Chronic exposure to styrene and a number of other volatile organic
compounds has been linked to the occurrence of neurologic and behavioral
deficits, including increased reaction time (Kishi et al. 2000), visual
system disturbances (Iregren et al. 2002), and neurophysiologic alterations
(e.g., Harkonen et al. 1978; Lilis et al. 1978; Seppalainen and Harkonen
1976; Stetkarova et al. 1993). In addition, a number of dosimetric
studies have quantified the relationship between inhaled styrene and
markers of exposure: urinary concentrations of styrene (Ghittori et
al. 1987; Gobba et al. 1993; Imbriani et al. 1986, 1990; Pezzagno et
al. 1985) or mandelic acid, its major excretory metabolite (Campagna
et al. 1995; De Rosa et al. 1993; Fields and Horstman 1979; Severi
et al. 1994). The findings in this literature suggest that styrene
may exert a variety of effects on the nervous system and that sufficient
dosimetric information exists to develop quantitative relationships
between these effects and conditions of exposure. The purpose of this
study was to perform a meta-analysis of these observations, to quantify
the relationship between exposure (estimated from the biomarkers) and
effects on two measures of central nervous system function: reaction
time and color vision. An effort was made to assess the importance
of styrene-related deficits to real-world task performance. An insufficient
number of reports were found for meta-analyses of other neurotoxic
effects.
Behavioral Outcomes
Reaction time has been a popular variable for assessment of impaired behavioral
task performance for several reasons, including ease of measurement, adversity
of effect, and sensitivity to drugs and toxic chemicals. Reaction time
tasks are usually divided into simple reaction time (SRT), in which the
subject must simply react to a predefined stimulus as quickly as possible,
and choice reaction time (CRT), in which the subject must first select
between options before deciding whether or in what way to respond.
Long-term exposure to a number of solvents has been reported to also
produce deficits in the performance of screening tests for perception
of color or visual contrast (Geller and Hudnell 1997; Iregren et al.
2002). Perhaps the most prevalent agent in this growing literature is
styrene, for which numerous published studies have reported that exposure
was associated with visual deficits, in particular, an acquired impairment
of color perception. Color vision was usually assessed using the Lanthony
desaturated D-15d color vision test (Lanthony 1978), a hue discrimination
test designed to grade the loss of color discrimination from mild to
moderate. Performance on the test was usually quantified using the color
confusion index (CCI) scale (Geller 2001). Details of testing procedure
and scoring are reviewed in Geller and Hudnell (1997).
The literature reporting the effects of long-term styrene exposure on behavioral
performance is diverse. Critical experimental factors such as group sizes,
analytical approaches, and methodologic details vary greatly across studies.
A meta-analysis of the literature involving a quantitative treatment of
the combined data from a number of individual reports can help unify the
literature by improving
a) accuracy, by minimizing the impact of
single, perhaps anomalous reports;
b) precision, by including a
large number of subjects; and
c) generality, by aggregating a variety
of studies with differing subject populations and exposure histories.
A meta-analysis of the effects of exposure to styrene and
other organic solvents on CCI was recently reported by Paramei
et al. (2004). This analysis featured means from various studies
that were converted to Z-scores to place them on a common
scale of measurement for comparison and to help assess the
possibility that an effect might have been statistically significant.
High variance was observed between the results of different
studies after transformation, and the authors argued that no
reliable conclusions could be drawn about the effects of styrene
on CCI. The Z-score transform, however, compares the
magnitude of each particular effect (the mean) with the variance
for that measure. This transform conflates the measure of magnitude
of an effect with its stability, so a transformed score may
be larger or smaller depending on the variance of the group.
In the present work, our intent was to evaluate the magnitude
of styrene effects on CCI and reaction times. Thus, values
were not converted to Z-scores but were expressed as
a percentage of baseline. The dose levels were expressed as
inhaled-air styrene concentration multiplied by the work-years
of exposure. These data were then fitted to a dose-effect regression
equation. In this analysis, magnitude estimates were not conflated
with variances, and the variation among the reported data was
used to compute confidence limits and test the fitted result
for statistical significance (Benignus et al. 1998).
Estimates of Exposure
In the neurobehavioral reports, four different measures were
used to quantify the level of exposure to styrene:
a)
concentration of styrene in inhaled air (personal monitors) given
as a time-weighted average (TWA),
b) concentration of
styrene in urine,
c) concentration of mandelic acid in
urine as a fraction of creatinine in urine, and
d) concentration
of mandelic acid in urine. Individual-subject data were given
via scatter plots in some studies, whereas only group means were
provided in others. In the interest of homogeneity, we computed
group means from individual-subject data and used them to express
inhaled-air styrene concentration. We also analyzed individual-subject
exposure concentrations as a check on the results obtained from
means.
Table
1
|
To pool the data on effects of exposure across studies, it
was necessary to convert data from the reported measurement
of exposure to one common scale (inhaled-air styrene concentration).
To estimate inhaled-air styrene concentration from the measures
supplied by the original authors, we found representative publications
in which the various methods of reporting urinalyses were standardized
and tested (Table 1). In these publications, styrene measurements
from personal monitors were recorded for a work shift, and
then urine was sampled after exposure. We digitized plotted
data for individual subjects and pooled them into common databases,
one for each of the three methods of urine analysis. We then
fitted regression equations to the pooled data to predict inhaled-air
styrene concentration from the various observed urine measures.
In neurobehavioral studies, exposure to styrene was usually
estimated at the end of a work shift. It was implicitly assumed
by the authors of the reviewed articles that this single measure
was a representative measure of the workers’ exposure
history. This measure of exposure would underestimate historical
exposure in cases where there have been improvements in environmental
controls in the workplace (Gong et al. 2002).
Duration of exposure in work-years was given in all of the
neurobehavioral reports, but always as group means, even when
individual-subject data were given for styrene concentration.
The mean duration of exposure and mean concentration, although
fixed for a particular report, varied across reports. Thus,
when pooling data from several reports, we were able to analyze
for the effects of a combination of concentration and duration
of exposure, expressed as the product of concentration and
time (ppm work-years) (Cavalleri et al. 2000; Haber 1924; Miller
et al. 2000).
Measures of Adverse Outcomes
In each neurobehavioral study, tests were given at a fixed time
after the last exposure, usually the morning after the work shift
of the previous day; occasionally a weekend elapsed between the
last exposure and behavioral testing. This delay was intended
to avoid possible acute effects of the parent compound or its
metabolites.
Most of the reports did not specify whether the personnel
administering the tests were aware of the group to which each
subject belonged (Tables 2, 3). Such “nonblind” procedures
have the potential to overestimate the effects of a toxicant
in laboratory experiments (Benignus 1993).
Four publications produced a total of seven data points for
CRT (Table 2). For SRT, three articles yielded six data points
(Table 3). Some studies provided data on both SRT and CRT.
Only those studies of color vision that employed the Lanthony
desaturated D-15d test were used (Table 4). The data were individual
CCI and styrene exposure estimates from five studies. The illuminants
used in testing were of intensity 1,000-1,200 lux with spectral
distribution specified as “daylight illumination.” Color
vision testing was done in the morning, before the work shift
in four of five of the references used. CCI was reported as
a raw score in three of the five studies; in the other two,
CCI was adjusted for individual age and alcohol consumption.
In the three studies reporting raw CCI scores, subjects were
eliminated from analyses if criteria were exceeded for age,
disease, alcohol consumption, or drug use.
When data were graphically presented in publications, graphs
were digitized as previously described (Benignus et al. 1998).
Graphs were scanned and imported into digitization software (UN-SCAN-IT;
Silk Scientific, Orem, UT), which produced a table of
x,y-coordinates
for each data point. Because some of the points on a plot were
hidden behind others, the number of digitized data points was
always slightly less than the number of points that the original
authors reported. The number of digitized points was used and
reported in the present work. When data were given numerically
by the authors, these were used directly.
Normalization of data before pooling. Data
from different studies can be pooled only if the measurement
scales can be made comparable. Each datum was adjusted as follows:
in which
E is a normalized (effect) value,
D is
the value of the unadjusted dependent variable, and
B is
the value of a baseline condition. In studies where only means
were reported, the performance of the control group provided
baseline values. In some other studies reporting individual-subject
data, independent control groups were not studied, and the investigators
relied on a dose-effect analysis in which subjects with very
low exposures served as implicit controls. In the present work,
when individual-subject data were given, the mean of all data
from exposed to < 10 ppm styrene was used as a baseline. This
procedure was followed even if specific control groups were measured
to assure consistency and also to include the maximum number
of studies in the meta-analysis.
Fitting dose-effect curves. The data were
pooled after all useable data had been transformed by Equation
1 and all exposure data had been converted to inhaled-air styrene.
A linear regression equation of the form
was then fitted to the data. Here
E˜ is the estimated
value of the effect,
C is the concentration of styrene
in inhaled air (parts per million),
t is the duration
of exposure (work-years), and the βs
are empirical parameters fitted with a least-squares procedure
(Proc REG; SAS Institute Inc., Cary, NC). Equation 2 was fitted
first to assure that the intercept term, β
1,
was near zero and not statistically significant (which should
be the case for data adjusted by Equation 1). If the intercept
term was not statistically significant, Equation 2 was refitted
with only a slope (β
2)
term.
In cases where the regression lines were fitted to means
from various studies or groups, each mean was weighted by the
number of subjects for that mean. This was done by the “weight” statement
of Proc REG. This had the effect of giving the larger studies
(with smaller SEs) greater weight in the fitting procedure.
If a regression equation was found to be statistically significant,
the data were plotted with the effect on the y-axis and with
the product of styrene concentration (in parts per million)
and work-years on the x-axis. In general, when regression lines
are fitted to means instead of individual-subject data, estimated
lines are very nearly the same, but confidence limits will
be somewhat wider than if individual-subject data had been
available. This may be intuitively explained as due to the
loss of information when means are used. The effects were also
plotted separately as functions of styrene parts per million
alone with four lines for 2, 4, 6, and 8 work-years of exposure.
These lines were calculated by solving the regression equation
with styrene concentration as the independent variable for
each of the work-years of exposure (either 2, 4, 6, or 8).
Because some of the published reports gave SRT and CRT data
only as means ± SDs, all regression lines were fitted
to means, even for CCI, where individual-subject data were
available. For the CCI data, means were computed from the baseline-adjusted
individual-subject data by dividing the exposure range of each
report into two or three subranges and computing the means
of exposure and effect magnitude within the subranges. To assess
the effect of converting individual-subject data to means,
a regression line was also fitted to the individual-subject
data.
Estimating Exposure
We evaluated the relationship between styrene concentration
in inhaled air and styrene concentration in urine from the
pooled individual-subject data from five reports (Table 1).
These data are presented in Figure 1 along with a linear regression
line and 95% confidence limits (CLs). We evaluated the relationship
between inhaled-air styrene concentration and mandelic acid
in urine (expressed as milligrams per gram creatinine) from
the pooled data of three studies (Table 1) and the result is
given in Figure 2. Results from the only study (Fields and
Horstman 1979) that estimated inhaled-air styrene concentration
from mandelic acid (expressed in grams per liter) are shown
in Figure 3. Parameters and statistical tests for the three
regression lines are given in Table 5. All of the relationships
were statistically significant.
Effects of Styrene on Neurobehavioral Measures
Reaction time. In one case, Mutti et al. (1984)
found that urine samples were collected in the morning, just
before behavioral testing; in all other cases, urine samples
were taken at the end of a work shift. The data for exposures
for Mutti et al. (1984) were back adjusted to an end-of-shift
value, using a published elimination curve (Engstrom et al. 1976,
their Figure 1, group I).
We fitted equation 2 to the pooled mean data for CRT (seven
observations) from the studies in Table 2. We observed a statistically
significant linear relationship between the mean proportional
increase in CRT and cumulative styrene exposure. The intercept
term was not statistically significant, and the no-intercept
fitted equation was statistically significant, accounting for
91% of the variance (Table 6). The mean data along with the
fitted equation and 95% CLs are plotted in Figure 4. The size
of the plotted points reflects the relative number of subjects
used in computing each mean. Because one of the means in the
CRT data was collected at considerably higher exposure (1,336
ppm work-years) and therefore had a much greater effect magnitude
(Figure 4), concern arose that the fitted line may have been
heavily influenced by this point. We did an exploratory analysis
without the extreme point, and the results are given in Table
6 (labeled “CRT, exploratory”). Figure 5 gives
the effect magnitude as a function of styrene parts per million
for 2, 4, 6, and 8 work-years of exposure (calculated by setting
the work-years of exposure to either 2, 4, 6 or 8, and solving
for the effect of parts per million with the fitted regression
equation).
The relationship between SRT and styrene exposure was not
statistically significant in the pooled mean data from three
studies (Table 6).
Color confusion index. We fitted a regression equation
to mean data (as computed from the individual-subject data) to
keep the CCI results comparable with those of reaction time.
The intercept term was not statistically significant, and the
no-intercept form of the equation was statistically significant
and accounted for 35% of the variance (Table 6). Figure 6 is
a plot of the mean data along with the fitted line and its 95%
CLs. The size of the plotted points reflects the relative number
of subjects used in computing each mean. Equation 2 was also
fitted to the pooled individual-subject data (329 observations)
from the studies in Table 3. The intercept term was not statistically
significant and the no-intercept form was statistically significant
with the β
2 term
similar to that for the equation fitted to the means (Table 6).
Figure 7 is a plot of the individual-subject data and the fitted
line with 95% CLs. The confidence limit for individual-subject
data is somewhat narrower than for the means (Figure 6). The
scale of Figure 7 was kept the same as Figure 6 to facilitate
comparisons, even though some of the points were off scale.
Figure 8 gives the magnitude of effect plotted as a function
of styrene ppm for 2, 4, 6, and 8 work-years of exposure (calculated
by setting the work-years of exposure to either 2, 4, 6, or
8 and solving for the effect of parts per million with the
fitted regression equation). The scales were kept the same
as for Figure 5 to facilitate comparison with CRT results.
Estimates of Exposure
Inhaled-air styrene concentration was linearly related to
styrene concentration or its metabolites in urine (Figures
1-3). Inspection of these figures reveals that, although
the equations fit well, a number of individual-subject data
lay outside the confidence limits. One potential source of
such errors is the measurement of inhaled-air styrene, which
was usually made with dosimeters placed “near” the
subject’s personal exposure space and might not have
measured actual exposure. Another potential source of variance
involves differences in physical activity across subjects,
which would have affected the amount of styrene inhaled. Despite
the observed variability, the overall trend and the statistical
significance of the fitted lines in Figures 1-3 reveal that
all three biomarkers of styrene exposure provide reasonable
indicators of recent exposure.
Behavioral Effects
Choice reaction time. Cumulative styrene was associated
with increased CRT in a dose-related manner. Inspection of Figure
4 reveals that one point lies at a higher exposure value with
respect to the others. That point is the mean for one of four
groups from the same study, each exposed to a different amount
of styrene. These four means are represented as the smallest
four points in Figure 4. They form a series that is consistent
with the fitted dose effect function. Each of the points is the
mean of 18-28 subjects, for a total of 100 subjects. Despite
the fact that one point is outstanding in Figure 4, the fact
that it came from the only study with multiple exposure levels
makes the resulting fitted equation plausible. Furthermore, an
exploratory analysis, with the extreme point removed from the
data set, yielded only a slightly lower slope and a poorer fit.
The fitted line from the exploratory analysis was well within
the confidence limits of the line fitted to the whole data set.
More data at the upper end of exposure would improve the confidence
limits.
No significant effects were observed on SRT, perhaps because
the largest exposure for the SRT data was only about 250 ppm
work-years. Thus, there may not have been sufficient exposure
to produce a reliably detectable effect.
Under the assumption that the metric of exposure can be separated
into discrete concentration (parts per million) and duration
(work-years) components, the lines in Figure 5 may be used
to estimate the magnitude of various exposure histories on
CRT. For example, 8 work-years at 150 ppm is estimated to produce
an increase in CRT of almost 50%. For 20 ppm, a contemporary
limit for occupational styrene exposure [American Conference
of Governmental Industrial Hygienists (ACGIH) 2000] for 8 work-years
is estimated to produce a 6.5% increase in CRT. By use of the
fitted dose-effect equation the increase in CRT can be estimated
for any combination of concentration and duration of exposure.
Importance of rapid reaction times. The importance
of reaction times has been discussed in a number of ergonomic
settings, among which perhaps the most quantitative is automobile
driving. For drivers in the United States, it has been estimated
that reducing the reaction time by 100 msec would reduce accident-related
property damage costs alone by $655,000,000 annually (1994
US$) and prevent 58,000-70,000 injuries per year (Blincoe 1994;
Kahane and Hertz 1998). For unexpected events, a decrease in
reaction time of 100 msec is about 7% of the normal reaction
time, and for expected events 100 msec is about 14% (Green
2000). Thus, changing reaction time by 7-14% has important
economic and personal implications. Styrene exposure to permissible
levels produced the magnitude of change upon which the above
economic estimates were based. In addition, consideration should
be given to the possibility that people sometimes have additionally
increased reaction times due to work-related fatigue and consumption
of ethanol and drugs.
Given the above information it would be possible to calculate
the benefit of any proposed changes in styrene exposure limits
if data were available on a) the number of workers exposed
to styrene, b) the distribution of exposure concentrations
and durations, and c) the duration of the effect after
cessation of workplace exposures. It would then be possible
to compare the cost of regulation to the benefit of such regulation
on a continuous dose-related scale. Presumably, the above data
could be found, except for estimates of the permanence of the
increased CRT after exposure cessation.
Color vision. Long-term exposure to styrene
was associated with increased errors in performing a color
discrimination/arrangement task in the pooled data from six
studies of occupationally exposed workers. The mean effect
size for CCI was estimated with closer confidence limits for
Figure 7 than for Figure 6 because of the use of individual-subject
data, but the regression lines for the two procedures were
similar. The individual-subject observations scattered widely
about the estimated line, lowering the variance accounted for.
For an exposure of 8 work-years to 150 ppm, the estimated increase
in CCI score was approximately 17% (Figure 8). This is lower
than for CRT, which was estimated at nearly 50%. For 20 ppm,
a typical limit (ACGIH 2000) for 8 work-years, there was an
estimated 2.23% increase in CCI.
Color vision deficiencies associated with exposure to styrene
and other solvents have been associated primarily with difficulty
in discriminating among colors at the “blue” end
of the spectrum. This is commonly referred to as a blue/yellow
deficit. This type of color vision deficit could be associated
with reduced function in the short-wavelength-sensitive (blue)
cones or their associated ganglion cells (Greenstein et al.
1990; Hood et al. 1984; Pachec-Cutillas et al. 1999). Why,
or if, these cones are actually more susceptible is not well
understood.
The measures of CCI show a relatively large variance among
individual subjects, as was observed in group mean data by
Paramei et al. (2004). Factors that could contribute to this
variance include use of incorrect chromaticity values derived
from the saturated Farnsworth-Munsell D-15 test, rather than
unsaturated D-15d test (Geller 2001; Geller and Hudnell 1997)
and differences between spectrum color profiles of light sources
labeled as “daylight” (Wyszecki and Stiles 1982).
Also important are the effects of luminance and practice that
were not necessarily constant across studies.
Importance of color-vision deficits. The importance
of impaired color perception is difficult to specify quantitatively.
The broad scope of tasks deleteriously affected by color confusion
should, by itself, give weight to the importance of deficits.
Color information is important to persons who are driving;
making distance judgments; reading colored text on video monitors,
medicine bottles, food cans, and the like; scanning for objects
in a complex visual scene; or working with color-coded electrical
circuitry (Klein et al. 1999; McClure et al. 2000; Owsley et
al. 2001; Rubin et al. 1994, 1997, 2001). Congenital color
deficiencies are related to poorer school performance, slowed
CRT at traffic lights, difficulty with information processing
from color video monitors, and increased difficulty with color-coded
tasks (Cole and MacDonald 1988; Margrain et al. 1996; O’Brian
et al. 2002; Steward and Cole 1989). Although all of these
tasks are important, there is no obvious way of relating these
reported deficits to the magnitude of effects reported for
styrene. For such a relationship to be established, experimental
evidence is needed to relate CCI values to task performance.
It is possible to compare the effect of styrene exposure
with the effect of aging on CCI. The CCI of men increases with
age at the rate of about 10% of baseline every 13 years of
age (Iregren et al. 2002, their Figure 1 and Table 6). Thus,
the deficit in color perception caused by exposures to styrene
of 115 ppm for 8 work-years or 156 ppm for 6 work-years is
roughly equivalent to 13 years of additional age in visual
dysfunction. Eight work-years at 20 ppm (the ACGIH limit) would
produce a 2.23% deficit, which is roughly equivalent to 1.7
additional years of age.
Reversibility of Effects
It is not clear whether the effects of long-term styrene exposure
are reversible. All of the data analyzed in the present work
were collected at least 15 hr after the last exposure and therefore
are probably not due to the concurrent presence of styrene or
its metabolites in the blood. Some experimenters gave behavioral
tests to subjects both before and after a day’s exposure
and found no statistically significant differences for CRT (Jegaden
et al. 1993; Triebig et al. 1989) or for CCI (Triebig et al.
2001). This implies that the acute body burden of styrene and
its metabolites was not responsible for the effects and that
longer-term processes were acting.
A few of the experimenters tested subjects both before and
long times after exposure, to characterize the reversibility
of effects. For CCI, Triebig et al. (2001) reported that effects
were reversible after 4 weeks of vacation. This study had relatively
low exposures (even though the effect was large). Mergler et
al. (1996) reported that reduction of exposure due to workplace
improvements also reduced the CCI effect (and other behavioral
effects) after a period of 2 years. These data are scant and
spotty but suggest the possibility that at least some recovery
may occur from the effects of long-term exposure to styrene.
Possibility of Estimation Errors
It is possible that the concentration of styrene in the past
was greater, by an unknown amount, than indicated by contemporary
measures because workplace improvements may have been made (Gong
et al. 2002). If this were the case, then the noted effects would
have been due, in part, to higher styrene exposures in the past
and not to the possibly lower concurrent exposures. Although
this would have the effect of overestimating the magnitude of
effect for any indicated concentration of exposure, it would
not have affected the statistical significance of the finding
that styrene produces the indicated effect.
On the other hand, all of the available data in this analysis
were from studies of occupationally exposed workers. The risks
of chronic styrene exposure to the general population may have
been underestimated to the extent that healthy workers are
not representative of the general population. It is possible
that persons who are more susceptible to effects of styrene
exposure do not remain in positions where such exposure occurs--the
so-called “healthy-worker effect.” It is also possible
that nonworking persons such as the young or elderly might
be more susceptible to effects of styrene exposure than are
healthy workers.
Workplace styrene exposure can increase CRT and CCI. The magnitudes
of the effects are statistically significant linear functions
of parts per million work-years. The magnitude of each effect
is a continuous function and reaches socially important values
at realistic exposure concentrations.
Increased CRTs are associated with impairment of tasks performance,
such as driving, that can be monetized for benefit-cost analysis.
Increased color vision deficiencies are associated with difficulty
in the performance of many everyday tasks. The cost of these
increases is difficult to estimate. It appears that the effects
on CRT and CCI persist for some time after exposure ends, but
this conclusion is based on limited data.
The effects of styrene on CRT and CCI may have been overestimated
by an unknown amount in this meta-analysis because of a)
underestimates of past exposure and b) bias from experimenter
knowledge of subject exposure status while testing. On the
other hand, the potential for effects of styrene exposure in
the general population may have been underestimated because
of the healthy-worker phenomenon or because of the lack of
susceptible persons in the workplace, such as the young or
elderly.