By Lynn Panganiban,1 Nelia
Cortes-Maramba,1 Carissa Dioquino,1 Maria Lurenda Suplido,1
Herbert Ho,2 Ana Francisco-Rivera,3 and Ailyn Manglicmot-Yabes1
1National Poison Control and Information
Service and 2Department of Pharmacology, University of
the Philippines-College of Medicine, Manila, Philippines;
3Environmental and Occupational Health Office, Department
of Health, Manila, Philippines
Ethylenebisdithiocarbamates (EBDCs) are metabolized into
ethylenethiourea (ETU), a possible human carcinogen and
an antithyroid compound. In this study our goal was to
correlate ETU levels with the incidence of thyroid land
disorders among banana plantation workers exposed to EBDC.
We randomly selected 57 directly exposed orkers and 31
indirectly exposed workers from four banana plantations
and 43 workers from an organic farm; all subjects underwent
complete medical examinations and laboratory tests. Results
showed a higher mean thyroid-timulating hormone measurement
among exposed workers compared with the control group,
although the levels were well within normal range. Nine
of the exposed farmers had abnormal thyroid ultrasound
findings, consisting mostly of solitary nodules, compared
with three among the control group. Analysis of variance
showed significantly different blood ETU levels among
the directly exposed, indirectly exposed, and control
groups (p < 0.001), but ETU levels in urine were not
significantly different (p = 0.10). Environmental ETU
levels were below the U.S. Environmental Protection Agency
remediation levels. Among farmers with solitary thyroid
nodules, we found a very good direct correlation between
the size of the nodule and blood ETU level. In this study
we showed that blood ETU is a more reliable biomarker
for EBDC exposure than urinary ETU; therefore, the determination
of blood ETU should be part of medical surveillance efforts
among workers exposed to EBDC to detect occurrences of
thyroid gland disorders. Key words: banana plantations,
biologic monitoring, blood ethylenethiourea, environmental
monitoring, ethylenebisdithiocarbamate exposure, thyroid
gland disorders. Environ Health Perspect 112:42-45 (2004).
[Online 22 October 2003]
Ethylenebisdithiocarbamates (EBDCs) such as maneb and
mancozeb have been extensively used in Philippine banana
plantations for the past 40 years. These fungicides are
applied through aerial or backpack spraying. EBDCs (zineb
and maneb) are broken down into ethylenethiourea (ETU),
which is a Type IIB carcinogen and an antithyroid compound
[International Agency for Research on Cancer (IARC) 1991].
Furthermore, some ethylenebisdithiocarbamates contain
ETU in their formulated products.
Animal studies using several mammalian
species showed that ETU is rapidly absorbed from the gastrointestinal
tract and cleared from the body. In one study (Kato et
al. 1976), after only 5 min, ETU appeared in the blood
of rats administered an oral dose of 100 mg 14C-ETU/kg
body weight. Within 48 hr, 82-99% of the oral dose was
eliminated via urine and about 3% was eliminated via feces.
Another study found that approximately 70% of an oral
dose of ETU was eliminated in urine and 1% in feces (Newsome
1974; Ruddick et al. 1976). Comparable results were found
for mice, whereas in monkeys 55% was eliminated via urine
within 48 hr and < 1.5% was eliminated via feces (Allen
et al. 1978). ETU and its metabolites have been found
to have a half-life of about 28 hr in monkeys, 9-10 hr
in rats, and 5 hr in mice [International Programme on
Chemical Safety (IPCS) 1988].
Regarding the effects of dithiocarbamates
on organ systems, studies done on exposed rats showed
thyroid hyperplasia, which is largely reversible on cessation
of exposure [Blackwell-Smith et al. 1953; Food and Agriculture
Organization/World Health Organization (FAO/WHO) 1965,
1971; Seifter and Ehrich 1948]. Male rats given ziram,
a metallobisdithiocarbamate, developed C-cell thyroid
carcinoma (IPCS 1988).
In humans, urinary excretion of ETU among pesticide formulation
workers varies according to the type of work. Aprea et
al. (1998) reported that employees engaged in formulating
80% mancozeb excreted 65.3 µg ETU/g creatinine.
Among those formulating 35% mancozeb, urinary excretion
was 36.6 µg/g creatinine. Those engaged in maintenance
and internal transport of materials excreted 10.3 µg/g
creatinine (Aprea et al. 1998). Kurttio et al. (1990a,
1990b) reported an ETU excretion rate of 6-10 ng/hr among
potato farmers during the first 60 hr after the cessation
of exposure; the excretion rate diminished thereafter
to 0.2 ng/hr over a 22-day observation period. The estimated
half-life for eliminating ETU through the kidneys ranged
from 32 to 100 hr (Kurttio et al. 1990a, 1990b). Studies
on workers exposed to bisdithiocarbamates (thiram) in
Russia revealed an increased incidence of thyroid gland
disorders (7.6%) compared with a nonexposed group (1.0%),
with one documented case of a malignant lesion of the
thyroid (IARC 1991).
There are limited data in literature that
indicate the sensitivity of ETU as a biomarker for EBDC
exposure, and there are no reported studies that used
blood ETU for monitoring. Furthermore, few epidemiologic
studies have investigated the effects of EBDC exposure
on the thyroid gland (Houeto et al. 1995; Smith 1984;
Steenland et al. 1997; Brucker-Davis 1998). In the present
cross-sectional study, we investigated the possible correlation
between the levels of blood ETU and urinary ETU and the
incidence of thyroid gland disorders among banana plantation
workers in the Philippines.
Materials and Methods
We randomly selected 88 workers, 21-53 years
of age, with a 3-year history of direct or indirect exposure
to EBDCs from four plantations using large amounts of
dithiocarbamates for the past 20 years. The 57 directly
exposed workers included mixers, assistant mixers, sprayers,
flagmen, and clean-up laborers. The 31 indirectly exposed
workers consisted of supervisors, maintenance crew, and
research aides. The last exposure of these workers occurred
between 1 and 9 days before the study was conducted. We
randomly selected 43 control workers from an organic farm.
These workers had no exposure to EBDCs and resided at
least 50 km away from the banana plantations.
EBDCs were applied through aerial and backpack
spraying on a weekly and daily basis, respectively. Other
fungicides such as chlorothalonil, propanil, and bitertanol
were used at the same time or interchangeably with EBDCs.
The primary routes of exposure to farmers were inhalation
and skin contact.
Assessment. This cross-sectional study was conducted on
11-13 October 1999 among the EBDC-exposed workers and
7-8 December 2000 for organic-farm workers. We collected
a demographic profile, nutritional history, work background,
and exposure data using face-to-face interviews.
Physical examination centered on the thyroid
gland. Thyroid-stimulating hormone (TSH) and free thyronine
(T4) determinations were performed using immunoradiometric
assay and radioimmunoassay methods, respectively, by the
Medical Research Laboratory of the Philippine General
Hospital. We used the SpectraScreen TSH and T4 assays
(both from Ortho-Clinical Diagnostics, Rochester, NY,
USA), which use an "antibody sandwich" method
with the TSH and T4 in the middle. The test kit can detect
at least 5 µIU/mL TSH and up to 200 µIU/mL
T4. Free T4 was determined only among subjects with elevated
TSH levels. The Bureau of Research and Laboratory of the
Department of Health analyzed iodine in urine using an
acid-digestion method and urinary creatinine using Jaffe's
reaction. The urine, blood, and environmental samples
were analyzed for the presence of ETU by the Toxicology
Laboratory of the Department of Pharmacology, University
of the Philippines College of Medicine, using HPLC with
an ultraviolet detector. A modified extraction procedure
yielded a recovery rate of 90-100% ETU, with a detection
limit of 0.2 ppb (Yabes A, Liwag E, Pinzon L. Unpublished
data). Thyroid gland ultrasound was performed by a trained
radiologist at the Tagum Regional Hospital. A fine needle
aspiration biopsy was performed on subjects who had palpable
Environmental samples were collected from
air through 4-8 hr continuous monitoring using 0.37-mm
polyvinyl chloride filters and an air sampling device.
Soil samples were collected from the top down to 0.5 m
from the surface. All biologic and environmental samples
were stored on dry ice and sent to the respective laboratories
24-48 hr after collection. Good laboratory practice and
quality control measures were observed during the conduct
of the tests.
Data analysis. Descriptive statistics, Student t-test,
analysis of variance, Fisher's exact test, Pearson's correlation
analysis, and regression analysis were performed using
the Statistical Package for Social Sciences (SPSS) for
Windows, version 10 (SPSS, Chicago, IL, USA).
The number, age, and sex of the persons
examined in directly exposed, indirectly exposed, and
control groups are summarized in Table 1. Despite attempts
to select participants within the same age range, the
exposed male workers were significantly older than their
control counterparts. In general, there appeared to be
a higher intake of seaweed, other seafood, and cassava
among the exposed group, while the diet of the control
group consisted mainly of vegetables. There was no significant
difference between the two groups in the intake of fish.
Comparison of the exposed and control groups using Fisher's
exact test showed no significant differences regarding
history of known thyroid gland disorders and diabetes
mellitus in first degree relatives (p = 0.227 and p =
Work history and exposure.
More than 83% of workers in both exposed
and control groups were engaged in their particular type
of work for more than 5 years and had been living in the
same area during their period of employment.
Exposed workers reported handling different types of EBDCs,
mostly maneb and mancozeb. It is noteworthy that 27.2%
of the exposed group did not know the types of pesticides
to which they were exposed. Half of those who were able
to name the pesticides had been exposed to multiple chemicals.
Daily utilization of personal protective equipment by
workers ranged from 13.6 to 73.9%, depending on the type
used. Patient records showed that almost 56% of the workers
handled EBDCs on a daily to weekly basis. Many workers
(43.2%) were not able to quantify the duration of their
Physical examination findings.
We found enlarged thyroid glands in 17
workers from the EBDC-using plantations and only 2 among
the organic-farm workers. There was no significant difference
in the neck diameter of the two groups (p = 0.07) after
controlling for variables such as age, sex, height, and
weight. We found no significant difference in the physical
diagnosis of goiter in the two groups for both female
(p = 0.16) and male (p = 0.79) workers.
Thyroid function tests and ultrasound
TSH levels were higher among both types
of exposed workers compared with those of organic-farm
workers (p = 0.34) (Table 1). Compared with control values,
TSH values were elevated in three exposed workers (5.1,
5.8, and 6.2 mIU/L), although their free T4 levels were
within normal limits (12.9-15.5 pMol/L). After controlling
for age, we found no statistically significant difference
in the TSH levels between the exposed and control groups
(p = 0.24).
The mean level of urinary iodine among the exposed group
was not significantly different from that of the control
group after correcting for dietary differences (p = 0.45).
Nine of the exposed workers and three organic-farm
workers had abnormal thyroid ultrasound results. Of the
exposed workers, five showed normal-sized thyroid glands
with solitary nodules. Among the control group, two had
diffuse thyroid parenchymal disease and one with a small
solitary nodule (Table 2). Results of the Fisher's exact
test on the proportion of exposed and control farmers
with abnormal thyroid ultrasound findings showed no significant
difference (p = 1.000). Age and dietary preferences had
insignificant influence on the development of goiter among
the study groups at p = 0.421 and p = 0.559, respectively.
Two women with palpable nodules in the exposed
group underwent fine needle aspiration biopsy, and cytology
results showed colloid goiter.
Biologic and environmental monitoring.
Significant differences were found in both
arithmetic and geometric mean blood ETU levels of directly
exposed, indirectly exposed, and control groups (p 0.001).
In contrast, both the arithmetic and geometric means of
urinary ETU levels among the three groups were not statistically
different (p = 0.10). When corrected for age and diet,
blood ETU levels remained significantly different among
the three groups (p 0.001). Urinary ETU levels also remained
insignificant after correction for age and diet (p = 0.67).
Blood ETU correlated poorly with urinary ETU levels (p
= 0.156, r2 = 0.024).
ETU was detected in both soil and air samples
from all plantations (Table 1). The Student t-test showed
significant differences in the ETU levels of air and soil
from the two areas (p 0.001 and p = 0.003, respectively).
Temperature and humidity levels in both study and control
areas were not statistically different (p = 0.09 and p
= 0.13, respectively).
Correlation studies. ETU in blood and urine
was poorly correlated with environmental ETU levels. The
same is true with urinary iodine and TSH levels. Furthermore,
urinary and blood ETU levels were not correlated with
TSH and time of spraying. Neither was there a relationship
between TSH and spraying time. However, there was a strong
correlation between the size of solitary nodules and blood
ETU levels (p = 0.001) (Table 3).
Investigation of TSH levels of EBDC-exposed
workers has been advocated by previous studies because
ETU is a known inhibitor of thyroid peroxidase activity
(Hurley et al. 1998; IARC 1991; Marinovich et al. 1997;
Steenland et al. 1997). The present study among banana
plantation workers showed differences in TSH levels among
the three study groups (directly exposed, indirectly exposed,
and control). This finding is similar to those of Steenland
et al. (1997), whose study involved 49 heavily exposed
workers who sprayed EBDCs on tomatoes; TSH was significantly
higher in the study group (2.13 ± 0.15 mIU/L )
compared with the control group (1.6 ± 0.19 mIU/L),
although both were within normal range (Steenland et al.
1997). In contrast to the study of Steenland et al. (1997),
in the present study we did not find any correlation between
age and TSH levels. Furthermore, we found no correlation
between the time of exposure and TSH levels. This can
be explained by the fact that workers from a number of
plantations sprayed EBDCs on a daily basis, indicating
The significant differences in the urinary iodine excretion
levels observed among the three groups in the study can
be explained primarily by the dietary preferences of these
groups; these differences were no longer found after correcting
for dietary preferences (p = 0.45). However, the increased
urinary iodine excretion can be an effect of ETU, as seen
in animal studies. Using rodent studies, Hurley et al.
(1998) demonstrated that ETU can have the effect of inhibiting
the iodide pump. The decreased iodide uptake leads to
an increase in urinary iodine excretion. This may also
be the explanation for the direct correlation between
TSH levels and urinary iodine excretion among the workers
in the study.
Because this is a cross-sectional study,
it is difficult to ascertain the real prevalence of thyroid
gland disorders and actual finding of clinical hypothyroidism
among the workers. Because of the strenuous nature of
the farmer's work and the course of the illness itself,
it is possible that affected workers no longer perform
these jobs. However, even if the presence of thyroid gland
disorders in both exposed and control groups is not statistically
different, the prevalence rates of thyroid gland disorders
in these groups are higher than the national prevalence
rates. The 1993 national nutrition survey of the Philippine
Food and Nutrition Research Institute reported the prevalence
rates of 0.2 for nodular goiters and 3.2 for diffuse goiters
per 100 people in the study area (Velandria et al. 1997).
This study showed a prevalence rate of 6.8/100 people
for nodular goiters among exposed workers, which was three
times higher than the observed prevalence rate in control
workers. In contrast, the prevalence of diffuse goiters
among the exposed group was lower (1.1/100 people) compared
with the control group (4.5/100 people) and the national
average (6.0/100 people) (Velandria et al. 1997).
Our finding of solitary nodules among the exposed group
is significant because, in 95% of cases, thyroid cancer
presents as a nodule or lump in the thyroid gland, usually
solitary (Greenspan 1994; Studer and Gerber 1991). In
a review of literature, Houeto et al. (1995) reported
that a number of studies showed ETU to cause thyroid neoplasm
in animals. Only one study in humans showed an increasing
incidence of thyroid cancer in several geographic areas
of the United States in relation to the degree of dithiocarbamate
exposure, determined by sales and crop production statistics
for dithiocarbamate-containing pesticides; this is unlike
the present study in which we used biomarkers of exposure.
Thus, it is important that workers with thyroid gland
disorders are monitored regularly for the development
of cancer in later years.
All results of environmental monitoring
in the present study were below the U.S. Environmental
Protection Agency (EPA) guidelines for remediation for
industries (U.S. EPA 2000). The air ETU values were much
lower than findings in previously published studies in
which ambient air monitoring of ETU during spraying of
EBDCs revealed levels ranging from 400 to 5,200 ng/m3
(Kurttio et al. 1990a, 1990b). Likewise, air monitoring
done in an industrial formulating plant in Italy showed
ETU levels ranging from 200 to 1,300 ng/m3 (Aprea et al.
1998). Two factors that may account for this difference
are the time of monitoring in relation to spraying and
the prevailing meteorologic conditions. In these previous
studies (Aprea et al. 1998; Kurttio et al. 1990a, 1990b),
ambient air monitoring was conducted while the workers
were spraying. In the present study, environmental monitoring
was done 1-9 days after spraying.
In the present study, soil ETU levels were
higher on the banana plantations than on the organic farm,
which we attribute to EBDC use. The elevated air ETU and
the presence of ETU in the soil on the organic farm are
possibly due to spray drift coming from an adjacent plantation
as enhanced by prevailing metereologic conditions.
The present study revealed generally higher levels of
urinary ETU (mean, 244.01 ppb) among backpack sprayers
with an acute exposure to EBDCs as well as chronic exposure
for at least 3 years. Previous studies showed varying
levels of urinary ETU among farmers using EBDCs (Aprea
et al. 1996, 1998; Kurttio et al. 1990a, 1990b; Steenland
et al. 1997). In their study among potato field applicators,
Kurttio et al. (1990b) showed levels ranging between <
0.20 and 23.00 ppb after a 4-hr single exposure to EBDCs
using a tractor-pulled, pump-operated spray. In contrast,
Steenland et al. (1997) showed mean urinary ETU levels
of 58 ppb among 49 applicators. The difference in the
recovery rate of the HPLC procedure [50% for Kurttio et
al. (1990b) and 92% for this study] and the difference
in the limit of detection [10 ppb for Steenland et al.
(1997) and 0.2 ppb for our study) could account for the
higher urinary ETU levels observed among Filipino workers.
To provide more evidence for this assumption, it is necessary
to quantify the EBDC exposure of farmers into milligrams
per kilogram per day. The presence of urinary ETU among
the organic-farm workers can be explained by exposure
from nearby plantations that use aerial spraying and also
by possible use of pesticides by neighbors.
Kurttio et al. (1990b) stated that urinary
ETU is not the ideal choice for biologic monitoring of
EBDC or ETU exposure because of ETU's biological properties
and because of the complexity of the biotransformation
of EBDC to ETU. In this study we showed blood ETU levels
to be significantly elevated among the directly exposed
farmers compared with the indirectly exposed and control
groups. This relationship was not demonstrated by urinary
ETU levels among the three groups. Furthermore, urinary
ETU levels poorly correlated with blood ETU levels. Therefore,
blood ETU levels may be a better biomarker than urinary
ETU for monitoring exposure to EBDCs.
The significant elevation of ETU in urine and blood and
low environmental ETU levels indicate that the exposure
of the workers to ETU was mainly from the direct handling
of EBDCs rather than from ambient air. This is supported
by the finding of no correlation between blood and urinary
ETU levels and environmental ETU levels.
Although blood ETU levels decrease several days after
cessation of exposure to EBDCs and the half-life of ETU
is approximately 28 days, the daily exposure of farmers
in this study through backpack spraying and weekly exposure
through aerial spraying creates a steady-state concentration
of ETU in the blood. The persistently high levels of ETU
in the blood can increase the likelihood for the rapid
growth of the thyroid nodule. This can explain the strong
direct correlation between the size of thyroid nodules
and blood ETU levels. With increasing size of the nodule
(> 4 cm), there is a greater chance for the nodule
developing into cancer. The assumption of persistently
high blood ETU levels in the workers should be confirmed
by collecting repeated samples over several months.
In this study we did not find significant differences
in thyroid function tests of exposed and control groups
after controlling for age and dietary preferences. However,
we found a higher prevalence of solitary nodules in exposed
workers as detected by ultrasound. Furthermore, findings
showed that soil and ambient air ETU levels were below
the U.S. EPA remediation levels (U.S. EPA 2000). Blood
ETU levels were significantly higher among the exposed
group compared with the control group; this test is a
more reliable biomarker for EBDC exposure than urinary
ETU. Also, the size of the thyroid nodule is strongly
correlated with blood ETU level.
Health monitoring of individuals exposed to EBDCs should
include checking for thyroid gland disorders, particularly
nodular goiters. We recommend that blood ETU levels be
determined on workers who are directly exposed to EBDCs.
Workers with solitary nodules should be followed closely
to rule out the possibility of thyroid cancer.