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Source: Archives of Environmental Health, May-June 1990 v45 n3 p175(5).

Title: Plasma chloroform concentrations in swimmers using indoor swimming
pools.

Author: Gabriella Aggazzotti, Guglielmina Fantuzzi, Pier Luigi Tartoni and
Guerrino Predieri

Abstract: Chloroform (CHCl3) is produced by chemical reactions when sodium
hypochlorite (NaOCl) interacts with organic molecules in chlorinated swimming
pool water. Chloroform levels build up both in the water and in the air of
indoor swimming pools where the vapors are contained. The level of
concentration in these situations is conditioned on may factors, including the
number of persons using the pool, the level of turbulence produced by the
swimmers and water circulators, and the ventilation of the building. Although
visitors and swimmers who do not do vigorous exercise inhale chloroform, the
maximum uptake is reserved for swimmers who breath under the stress of
exertion for extended periods of time. Although chloroform is classified as a
carcinogen on the basis of animal research, little is known about the
epidemiologic implications for humans. This study evaluates the level of
chloroform in the air and water of three different swimming pools, which were
measured at 18 different times. The blood of 127 volunteers (81 males and 46
females), who frequently attended the pools, was also collected and analyzed.
Similarly, the blood of 40 volunteers (controls), who had no know occupational
exposure to chloroform, was also collected. The local drinking water supply
was free of chloroform. None of the controls had chloroform levels greater
than the detection level of the equipment. The blood of all 127 exposed
volunteers had measurable levels of chloroform. Analysis showed that the blood
chloroform level in this group was strongly correlated with the level of
chloroform in the air and water, the number of swimmers in the pool, time
spent swimming, and intensity of their exercise. (Consumer Summary produced by
Reliance Medical Information, Inc.)

Subjects: Chloroform - Toxicology
Swimming pools - Health aspects
Pollutants - Measurement
Chloroform - Measurement
Pollutants - Toxicology
Chloroform - Physiological aspects

Electronic Collection: A9405815
RN: A9405815
 

Full Text COPYRIGHT 1990 Heldref Publications

Plasma Chloroform Concentrations in Swimmers Using Indoor Swimming Pools

CHLOROFORM ([CHCI.sub.3]) is present in indoor swimming pools as a consequence
of water chlorination with sodium hypochlorite (NaOCI). As a lipophilic
volatile substance, [CHCI.sub.3] can be released from water into environmental
air, where its concentration depends on several factors, among which the most
important are the number of swimmers in the water and the turbulence caused by
their movements. [1-4] Chloroform in air can be inhaled by swimming pool
visitors, but is mainly inhaled by agonistic swimmers who breathe under stress
for a long time directly at the surface of the water.

Chloroform is a class 2B carcinogen with sufficient evidence in experimental
animals but inadequate epidemiological evidence. [5] Thus, it is advisable to
look for biological markers of exposure in humans.

Very little information is available on [CHCI.sub.3] metabolism in humans, and
no byproducts have been found in human fluids. In addition, few studies have
reported on the level of [CHCI.sub.3] in human blood, and many difficulties
have been encountered when determining the actual level of human exposure via
inhaled air and drinking water in various circumstances. [6-10]

Because indoor swimming pool visitors are exposed to [CHCI.sub.3] during a
precise period of time at a known environmental concentration, they form an
available population to obtain more detailed information about nonoccupational
exposure to [CHCI.sub.3].

The aims of this study were to evaluate the level of [CHCI.sub.3] in blood of
exposed (indoor swimming pools visitors and swimmers) and nonexposed subjects,
and to verify if individual and/or environmental variables can influence blood
[CHCI.sub.3] concentration.

Materials and methods

Sampling. Three indoor swimming pools in Modena were visited on 18 occasions
from November 1987 to April 1988. During every visit, the [CHCI.sub.3]
concentration was determined in pool water and in environmental air within the
breathing zone of all subjects at the swimming pool. A total of 127 volunteer
subjects (81 males and 46 females) who regularly attended a swimming pool were
examined. Informed consent was obtained from each person. Blood samples were
collected from each subject at the time he or she was ready to leave the
swimming pool environment.

Data were gathered from each subject about the possibility of exposure outside
the swimming pool (e.g., occupational expoure or handling of solvents at home
while engaged in hobby activities). The type of activity practiced in the
swimming pool was recorded; therefore, all subjects were classified into three
main groups: (1) agonistic swimmers (n = 102) who trained for competitions
every day, (2) nonagonistic swimmers (n = 16) who attended swimming courses
twice a week, and (3) visitors (n = 9) who were present but who did not swim.

Every subject was asked about the frequency of attendance and length of time
spent at the swimming pool during a week. The length of the swimming session
was determined, and the lapse of time between the end of the sport activity
and the blood sampling was registered for swimmers only.

A control group of 40 volunteers who had no known occupational and
environmental exposure and who never attended an indoor swimming pool were
examined to evaluate [CHCI.sub.3] in blood. Modena is supplied with drinking
water treated with chlorine dioxide ([CIO.sub.2]) and is free from
[CHCI.sub.3] and other halogenated organic compounds. However, we analyzed
drinking water samples before, during, and after this study to confirm the
absence of [CHCI.sub.3].

Blood was drawn by a physician at the swimming pool (from 1 to 40 min) after
exposure ceased (i.e., 1-40 min after exposure). A [5-cm.sup.3] volume of
blood was drawn from a cubital vein with a [10-cm.sup.3] syringe and
immediately placed in a capped disposal tube that contained the anticoagulant
[K.sub.3] EDTA. The tubes were shaken gently, chilled on ice, and kept cold
until analyzed. The samples were centrifuged for 10 min at 1000 g) as soon as
possible; plasma was then drawn using an Eppendorf micropipette and
transferred into [3.7-cm.sup.3] borosilicate glass vials with hole caps sealed
with Teflon-faced silicon-rubber septa (Supelco).

During each session, three air samples were collected at the same level above
the water surface, i.e., 150 cm in height at the edge of the pool, and three
samples of water were obtained at three different points in the pool at a
depth of 20 cm near the edge of the pool. Environmental air samples were
collected within the breathing zone. Environmental air and water samples were
collected in screw-capped [40-cm.sup.3] glass vials with silicone-faced septa.
Immediately prior to sampling, vials that were to be used for water collection
received 5 mg of sodium thiosulfate to quench residual chlorine reactions.
Septa and screw caps were washed in water, acetone, n-pentane, and then dried
at 105 [+ or -] 1 [degrees] C for 4 h. Vials and all pieces of glassware were
washed in water, acetone, n-pentane, and were then placed in a muffle furnace
at 400 [+ or -] 5 [degrees] C for 2 h to purge any volatile contaminants.

Determination of [CHCI.sub.3] in plasma. Analyses were performed on the plasma
aliquots. Samples were analyzed by a head-space gas chromatographic technique,
in accord with a previous report, using a glass steel column packed with 10%
OV-1 on Chromosorb WAW. [11]

Determination of [CHCI.sub.3] in wateer. Samples were analyzed by a head-space
gas chromatographic technique. An autosample DAN-NI HSS 3950 and a VAR-IAN
3400 gas chromatograph equipped with a [sup.63.Ni] electron-capture detector
and a VOCOL capillary column (length = 30 m, ID = 0.53 mm, film thickness = 30
[micrometer] [SUPELCO]) were used. The carrier gas (He) flow rate was 8
ml/min; make-up gas, 20 ml/min; initial column temperature 50 [degrees] C (1
min); final temp, to 100 [degrees] C (7 min at 6 [degrees] C/min); injector
temperature, 150 [degrees] C, detection temperature, 260 [degrees] C; detector
ECD, range 10, attenuation 32. Calibration was performed by the external
standard method. This method is preferred by most workers when studying the
presence of [CHCI.sub.3] in waters by head-space gas chromatography [12-13].
Precision, calculated from 5 duplicate determinations on 5 different days, was
2.8% (coefficient of variation [CV]). The detection limit was calculated
during 30 different series of [CHCI.sub.3] determinations of blank value
(vials with [CHCI.sub.3]-free water). The mean blank concentration was 2.9
nmol/l and had a standard deviation of 0.3 nmol/l. Based on 2.5 times the
standard deviation, the detection limit was 0.8 nmol/l.

Determination of [CHCI.sub.3] in environmental air. Samples were injected
directly into the gas chromatograph using a gas-tight syringe (Hamilton).
Calibration was performed by the external standard method. Precision,
calculated as before, was 3.5% as C.V.

The detection limit was calculated during 30 different series of [CHCI.sub.3]
determinations of blank value (vials with environmental air). The mean blank
concentration was 41.9 [nmol/m.sup.3], with a standard deviation of 3.4
[nmol/m.sup.3]. Based on 2.5 times the standard deviation, the limit of
detection was 8.4 [nmol/m.sup.3]. Quantitative analysis was performed by
Chromato-Integrator Merck Hitachi D2000.

The identity of [CHCI.sub.3] was confirmed by gas chromatography-mass
spectrometry (GC-MS). We examined both standard samples of [CHCI.sub.3] in
n-pentane and standard examples of [CHCI.sub.3]-fortified human plasma at
increasing concentrations (i.e., 0.8-8 376 [micrometer]ol/l) after extraction
with n-pentane. The identification of [CHCI.sub.3] is based on retention times
measured on a total ion-current chromatogram and on mass chromatograms of the
molecular ion and the most significant fragments of [CHCI.sub.3] (m/z 47-50,
82-87, and 117-124). [11]

Statistical analyses. The correlations between parameters were evaluated by
Spearman's rank correlation coefficient. As the distribution of parameters
after log-transformation was not closer to Gaussian, no-log transformation was
applied before using the parametric tests. Linear and multiple regression
analyses were performed, and the linearity of the regression model was
controlled. Moreover, to evaluate the ifluence of some parameters on
[CHCI.sub.3] content in plasma, the analysis of covariance was performed.
Differences among mean values were evaluated by the Student-Newman-Keuls test.

These tests were always checked with regard to the homogeneity of variance
according to the Cochran and Bartlett Tests.

Results

Concentrations of [CHCI.sub.3] in water samples and environmental air samples
(mean values of three samples) in indoor swimming pools during each sampling
session are given in Table 1. As the three air samples collected during every
session at different points at the same level above the water surface were
very similar, the mean value was considered as representative of the
environmental concentration. Mean values of [CHCI.sub.3] in plasm samples
collected during the same session are also reported.

Although no plasma sample from the 40 nonexposed control subjects showed
[CHCI.sub.3] levels [is greater than] 0.8 nmol/l (detection limit), all
samples taken from the 127 exposed subjects showed [CHCI.sub.3] values ranging
from 0.8 nmol/l to 25.1 nmol/l.

Levels of [CHCI.sub.3] in pool waters ranged from 142 to 394 nmol/l. Mean
levels of [CHCI.sub.3] in environmental air ranged from 553 to [ 5 445
[nmol/m.sup.3], depending on the level of [CHCI.sub.3] and the number of
swimmers in the pool.

Figure 1 shows the distribution of [CHCI.sub.3] in 127 plasma samples. The
geometric mean was 6.9 nmol/l, and the median was 7.5 nmol/l. Of interest is
that 53.3% of the samples showed levels from 0.8 to 8.0 nmol/l, whereas 13% of
samples showed a [CHCI.sub.3] concentration [is greater than] 16.0 nmol/l.

Table 2 shows the correlation coefficients between some variables. The
[CHCI.sub.3] level in plasma was significantly correlated to four variables:
(1) concentration in water, (2) time spent swimming, (3) number of swimmers,
and (4) most significant of all, [CHCI.sub.3] concentration in environmental
air. No significant correlation was found between [CHCI.sub.3h in plasma and
the time since exposure ceases. As expected, mean [CHCI.sub.3] values in
environmental air were correlated to mean levels in water and the number of
swimmers. Unexpectedly, the mean level of [CHCI.sub.3] in water correlated to
the number of swimmers. Perhaps this finding is due to the fact that swimmers
contribute to the total amount of organic substance that is responsible for
[CHCI.sub.3] and another trihalomethanes in pool water when continuous
chlorination with NaOCI is performed.

Chloroform levels in plasma are also influenced by the variable "intensity of
the sport activity." When covariance analysis was applied, the plasma
concentration was considered as a dependent variable, whereas [CHCI.sub.3]
values in environmental air and intensity of the sport were considered
independent parameters. Chloroform in air was accounted for as a covariate and
intensity of the sport as a factor. The total explained variance by this
statistical model was 67.43%: 61.69% accounted for [CHCI.sub.3] in
environmental air (F = 232.97; p [is less than] .001), whereas 5.74% accounted
for intensity of the sport (F = 10.83, p [is less than] .001).

The Student-Newman-Keuls test at p [is less than] .050 showed significant
differences in [CHCI.sub.3] mean plasma levels of agonistic swimmers (10.3 [+
or -] 5.7 nmol/l) v. nonagonistic swimmers (3.4 [+ or -] 1.3 nmol/l) and v.
visitors (2.5 [+ or -] 1.7 nmol/l); however, absolute differences are small.

Sessions where [CHCI.sub.3] mean values in environmental air exceeded 1 700
[nmol/m.sup.3] 7mean plasma [CHCI.sub.3] value = 13.7 [+ or -] 5.5 nmol/l)
were considered, and multiple regression analysis was performed accounting for
temporal variables (i.e., time spent at the swimming pool, time spent
swimming, time since cessation of exposure). A positice association was found
between [CHCI.sub.3] in plasma and the time (min) spent swimming ([R.sup.2]
adjusted = 0.229; F = 12.45; p = .001), whereas no correlation was found
between the time spent at the swimming pool and time since exposure ceased.

Discussion

Very few studies report [CHCI.sub.3] levels in blood of nonoccupationally
exposed individuals. Recently, Hajimiragha et al. [9] reported values that
ranged from [is less than] 0.1 to 1.7 [mu]g/l ([is less than] 0.8 to 14.2
nmol/l) in samples from 39 subjects who had no occupational exposure to
[CHCI.sub.3], whereas increased levels were found in 6 occupationally exposed
subjects, e.g., drycleaners and pressers.

In our study, plasma [CHCI.sub.3] in nonexposed subjects was never measured at
a level that exceeded 0.8 nmol/l; however, plasma [CHCI.sub.3] levels were
evident in all exposed subjects. Exposure via drinking water could be excluded
because water in Modena does not contain [CHCI.sub.3]; similarly, other
recognized possibilities of exposure could be excluded. Therfore, [CHCI.sub.3]
in blood is most probably related to exposure in an indoor swimming pool
environment.

[CHCI.sub.3] in water results from chlorination with NaOCI when organic
substance is present in pool water. According to the haloform reaction,
[CHCI.sub.3] is released in environmental air, where its concentration is
dependent on the level of organic substance in water and the number of
swimmers who produce turbulence. [1-4] Whether swimmers or visitors,
individuals at the indoor swimming pool inhale [CHCI.sub.3] during the time
spent in this environment. However, the greatest amount of [CHCI.sub.3] is
inhaled by those who swim for a long time under physical stress.

The inhaled [CHCI.sub.3] is rapidly absorbed in the lung, and the total amount
is directly proportional to the concentration of the inspired air, the
duration of exposure, the blood/air Ostwald solubility coefficient, the
solubility in the various body tissues (very high for fat tissue), and the
intensity of physical activity (increased pulmonary ventilation rate and
cardiac output). [14-15] Therefore, all these factors can influence the level
of [CHCI.sub.3] in blood.

Our study showed that the [CHCI.sub.3] concentration in blood varies with
water and environmental air values, number of swimmers, length of the time
spent swimming, and the intensity of the sport activity. No significant
correlation with time since cessaton of exposure was found, perhaps because
many individual variables can influence the kinetics of [CHCI.sub.3]
absorption, distribution, and clearance. Some of these variables are difficult
to control in this type of study, e.g., total fat tissue content of the body
and the pulmonary ventilation in exposed subjects.

Chloroform exposure in indoor swimming pools represents an additional source
that must be accounted for when exposure levels for the general population are
considered. As reported previously, [CHCI.sub.3] is classified as a
carcinogenic substance as a result of tests with animals. For this reason,
more information about human exposure to [CHCI.sub.3] is required. Swimming
pools visitors represent a special population to be studied to acquire more
information about [CHCI.sub.3] intake, uptake, and elimination because
environmental conditions are well known and defined. Blood sampling represents
a first step to evaluate [CHCI.sub.3] exposure. However, satisfactory
biological monitoring will have to include other biological measurements,
e.g., breath (alveolar air) sampling. Studies are in progress on the kinetics
of [CHCI.sub.3] after exposure at low levels by taking samples of blood and
alveolar air from exposed subjects at different moments after the end of
exposure.

References

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[3] Chambon P, Taveau M, Morin M, Chambon R, Vial J. Survey of trihalomethane
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