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Neurobehavioral Effects of Pesticides:
State of the Art
C. Colosio
1,*
, M. Tiramani
1
, M. Maroni
1,2
1
International Centre for Pesticides and Health Risk Prevention, Via Magenta 25, 20020 Busto Garolfo, Milan, Italy
2
Department of Occupational Health, University of Milan, Milan, Italy
Received 20 August 2002; accepted 9 April 2003
Abstract
The authors have reviewed the literature on neurobehavioral toxicity of pesticides to assess the status of knowledge on
this matter. Some data suggest that exposure to DDT and fumigants may be associated with permanent decline in
neurobehavioral functioning and increase in psychiatric symptoms, but, due to the limited numberof studies available and
the scarce knowledge on exposure levels, no firm conclusion can be drawn. Data on subjects acutely poisoned with
organophosphorous compounds suggest that an impairment in neurobehavioral performance and, in some cases,
emotional status may be observed as a long-term sequela, but the possibility still remains that these effects were only an
aspecific expression of damage and not of direct neurotoxicity. Studies carried out on subjects chronically exposed to
organophosphates, but never acutely poisoned, do not provide univocal results but the slight changes consistently
observed in sheep dippers suggest the need of focusing on activities characterized by relatively higher exposure levels. In
general, the main limits of existing knowledge are the variability of the testing methods used, which makes it difficult to
compare the results of single studies, and the scarce knowledge on exposure levels. A promising approach may be the
conduction of prospective longitudinal or cohort studies, where exposure and dose assessment can be more easily
controlled, or the evaluation of cohorts of workers a priori selected for the availability of environmental and biological
monitoring data. The follow up of the populations under study may give an answer at the problem of the prognostic
significance of the observed changes. Also the protocols used to assess neurobehavioral functioning need to be
standardized.
#2003 Elsevier Science Inc. All rights reserved.
Keywords: Pesticides; Neurobehavioral effects; Neurobehavioral testing; Review
INTRODUCTION
Pesticides differ from other chemical substances
because they are toxic chemicals deliberately spread
into the environment with the aim of controlling unde-
sired living species. Since their toxicity may be not
completely specific for the target organisms, their use
may pose a risk to human health.
The mechanism of action of pesticides frequently
involves a neurotoxic effect: organophosphorous
compounds and carbamates act through the inhibition
of central nervous system cholinesterase (Jeyaratnam
and Maroni, 1994; Machemer and Pickel, 1994);
pyrethroids affect the sodium channels of the nerve
membrane, keeping them open for more than the few
milliseconds needed for the generation of the action
potential (He, 1994); organochlorinated compounds in
general act as central nervous system stimulants, but
the mechanism of action varies for the different active
ingredients (Tordoir and Van Sittert, 1994); morpho-
line derivatives alter the balance between excitatory
and inhibitory threshold in neurons, impairing the
function of the nervous system (Barbieri and Ferioli,
1994), while formamidines have an agonistic action on
the alpha-2 catecholamine receptor (Xue and Loosly,
1994).
The absorption of high doses of neurotoxic active
ingredients gives rise to well known pictures of acute
NeuroToxicology 24 (2003) 577–591
*
Corresponding author. Tel.: þ39-0331-406611/406615;
fax: þ39-0331-568023.
E-mail address: claudio.colosio@icps.it (C. Colosio).
0161-813X/$ – see front matter #2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S0161-813X(03)00055-X
poisoning, characterized by impairment of the nervous
system function. In some cases, acute poisoning may
be followed by delayed sequelae, the most studied
being the so-called ‘‘organophosphates induced
delayed neuropathy’’ (OPIDN), that can be demon-
strated in hens and may also occur in man from one to
three weeks after a severe acute exposure to certain
OPs. The disease is characterized by a flaccid weakness
of the distal limb muscle and is attributed to the
capacity of some OP compounds to link a specific
nervous system molecular target, the neurotoxic ester-
ase (NTE) (Lotti and Moretto, 1999).
The almost ubiquitous presence of pesticide residues
in the living and working environments gives rise to the
question of whether or not prolonged exposure to
neurotoxic compounds at concentrations lower than
those able to cause acute effects may have adverse
consequences on health. Since CNS and some specific
CNS functions including behavior may be a sensible
target for the effects of other neurotoxicants such as
solvents and metals (Mergler et al., 1999; Noraberg and
Arlien-Soborg, 2000; White and Proctor, 1997), study-
ing selected neurobehavioral effects in subjects
exposed to neurotoxic pesticides may contribute to
provide an answer to this question.
This paper aims at reviewing the studies published
in the literature on neurobehavioral effects of
pesticides and critically evaluating the information
available for some groups of active ingredients. Area
of uncertainty and specific research needs are also
underlined.
ORGANOCHLORINE COMPOUNDS
Available data regarding organochlorine compounds
are summarized in Table 1.
In a USA study, 859 children were tested at the ages
of 3, 4, or 5 years to evaluate the effects of DDT
absorbed either through transplacental route or during
breast-feeding (Gladen and Rogan, 1991). This study
indicated that DDT did not affect either psychomotor
and mental behavioral patterns, or school performance
in English and mathematics, even when the provisional
tolerated daily intake was exceeded. Six hundreds and
seventy children out of the 858 studied by Gladen were
tested at birth and at the ages of 6, 12, 18 and 24 months
(Rogan and Gladen, 1991). Exposures to polichlori-
nated biphenyls and DDE were taken into account. A
small delay in motor maturation was observed at 24
months in PCB exposed children, while DDE exposure
was not associated with any neurobehavioral effect. A
study carried out in Costa Rica on 27 retired men
engaged in malaria control activities exposed to DDT
for at least 2 years, suggested that prolonged occupa-
tional exposure to DDT was associated with permanent
decline in neurobehavioral functioning and increase of
neuropsychological and psychiatric symptoms (van
Wendel de Joode and Wesseling, 2001). This study
however did not provide information on the exposure
levels of the studied workers.
In summary, no firm conclusions can be drawn on
neurobehavioral toxicity of DDT and other organo-
chlorine compounds, and further research is needed.
Table 1
Summary of studies on organochlorinated compounds
Number of subjects
(job for workers)
Functions tested Observed effect(s) Author (year) Comments
27 male (public health
DDT workers)
Cognitive, motor and
sensory functions
Overall poor
performance
van Wendel
de Joode and
Wesseling (2001)
Data on exposure
levels lack
27 male (controls) Verbal attention Other active
ingredients involved
Visuo-motor speed
Neuropsychological and
psychiatric symptoms
1 female (occasional
exposure to lindane)
Hospitalization Central nervous system
toxicity; symptoms
lasted 20 months
Hall and
Hall (1999)
Acute poisoning case
859 DDT exposed children
tested at the age
of 3–5 years
Mental behavior patterns
(McCarthy and Bayley
scores; school performances)
No observed effects Gladen and
Rogan (1991)
Transplacental and
cumulative exposure
through breast milk tested
670 PCBs and DDE exposed
children (follow up at
birth and at 6, 12, 18
and 24 months)
Bayley scales of infant
development (MDI and PDI)
Small delay in motor
maturation (PCBs)
(24 months PDI);
no effect of DDE
Rogan and
Gladen (1991)
Transplacental and
cumulative exposure
through breast milk tested
578 C. Colosio et al. / NeuroToxicology 24 (2003) 577–591
ORGANOPHOSPHOROUS COMPOUNDS
Organophosphorous compounds represent the che-
mical group of pesticides most extensively investigated
for their neurobehavioral toxicity. The first reports of
neurobehavioral changes, such as memory impairment,
confusion, anxiety, drowsiness, labile emotion, fatigue,
depression, irritability in subjects heavily exposed to
OP compounds date back to the 1950s and 1960s (Dille
and Smith, 1964; Gerson and Shaw, 1961; Grob et al.,
1950; Metcalf and Homes, 1969; Durham et al., 1965).
Even if these studies were not adequate to reach
conclusions, they have for the first time underlined
the possibility of development of ‘‘chronic OP-induced
neuropsychiatric disorders’’ (COPIND). After these
early studies, two main lines of study have been
pursued: the onset of the neurobehavioral impairment
as a consequence of an acute OP poisoning and the
occurrence of neurobehavioral changes as a conse-
quence of prolonged exposure without preceding epi-
sodes of acute poisoning.
Neurobehavioral Effects After Acute OP Poisoning
The studies published on neurobehavioral effects in
subjects with a previous episode of acute OP poisoning
are summarized in Table 2.
A reduction of verbal attention, visual memory,
motricity and affectivity was demonstrated through
neurobehavioral testing with the WHO protocol core
test battery (Maroni et al., 1986; WHO, 1986) and
supplementary tests in a group of 38 subjects with a
previous unintentional episode of acute occupational
OP poisoning with hospitalization. Neurobehavioral
outcomes were compared with a control group com-
posed by 38 never poisoned subjects, living in the same
community (Rosenstock et al., 1991). The study sug-
gested that even a single episode of OP intoxication, for
which medical treatment was needed, was associated
with a persistent decline in neuropsychological
functions. The authors underlined that one of the most
important drawbacks in these studies is the inability to
identify the contribution of pesticide exposures other
than clinically significant acute poisoning. These
findings are similar to those obtained by Savage
et al. (1988) on a group of 100 previously poisoned
subjects, who showed no clinical neurological effects
but differences in several neuropsychological per-
formance abilities, namely intellectual functioning,
abstraction, flexibility of thinking and simple motor
skills. The authors of the study underlined the need
of carrying out an evaluation of both neurological and
neuropsychological parameters when examining sub-
jects with previous acute poisoning.
Also a study carried out on a group of 128 subjects
included in the register of poisonings of the California
State for an acute OP episode showed a reduction
in sustained visual attention, vibrotactile sensitivity,
symbol digit tests. Interestingly, the most heavily
poisoned subjects (hospitalized cases) showed the
highest level of neurobehavioral impairment (Steenland
et al., 1994).
Thirty-six male workers were evaluated between
10 and 34 months after hospitalization for acute orga-
nophosphate poisoning and compared to an age and
sex-matched community reference group (McConnell
et al., 1994). The main finding of this study consisted
of an abnormal vibrotactile threshold in one fourth of
patients previously poisoned with methamidophos.
It should be noted that some of the above mentioned
studies have been criticized and their conclusions
questioned on the basis of unclear selection of subjects,
inappropriate control group, and unknown significance
of some of the neurobehavioral tests used (Lotti, 1992).
The possibility of a neurobehavioral impairment
consequent to a previous episode of acute poisoning
has also been confirmed by a follow-up study carried
out on the subjects involved in the Tokio subway Sarin
poisoning episode (Yokoyama et al., 1998), and by a
significant number of published case reports.
In summary, the evidence of a neurobehavioral
impairment in previously poisoned subjects is rather
consistent. The highest levels of neurobehavioral
impairment are observed in the most heavily poisoned
subjects (those hospitalized and treated with atropine
and antidotes). On the contrary, a clear reduction of
AChE activity without overt signs of poisoning has
shown to be inadequate to cause neurobehavioral effect
(Ames et al., 1995). As for single OP compounds, a
group of toxicologists and medical experts, after
reviewing the scientific literature on chlorpyrifos, con-
cluded that there was no clear evidence of long-term
effects other than cases of OPIDN from suicidal inges-
tion. As for long-term exposure, the authors concluded
that the peripheral and central nervous system was
not affected, unless an acute and severe poisoning
occurred. With respect to neurobehavioral effects, they
were defined as unlikely to occur (Clegg and Van
Gemert, 1999).
Since available data, including sarin poisoning cases,
indicate that a severe clinical poisoning is necessary
for the occurrence of neurobehavioral impairment,
a further question concerns the specificity of the
observed effects. Interestingly, such effects are very
C. Colosio et al. / NeuroToxicology 24 (2003) 577–591 579
similar to those observed in subjects with a previous
episode of CO poisoning with CNS anoxia (Raub et al.,
2000), or even in subjects with a previous CNS trau-
matic injury (Deb et al., 1999). Also in the experi-
mental model, long-term sequelae seem to be
secondary to convulsions or anoxia associated with
the severity of cholinergic syndrome and not directly
related to AChE inhibition (Lotti, 1992). Although
recent studies on chlorpyrifos have pointed out a
neurobehavioral effect in rats repeatedly exposed to
low doses (Socko et al., 1999) and an in vitro inhibitory
effect on brain cells associated with oxidative stress
Table 2
Studies on subjects with a previous episode of acute OP poisoning
Number of workers (job) Functions tested Observed effect(s) Author (year) Comments
18 (9 male, 9 female)
(Tokyo subway
sarin poisoning)
Medical follow up
of acute poisoning
Reduction in psychomotor
performance (digit symbol)
Yokoyama
et al. (1998)
Long-term sequelae of
Tokio subway sarin
acute poisoning casesGeneral health, chronic
fatigue, posttraumatic stress
disorder checklist: impaired
36 male workers
(evaluated 10–34
months after
hospitalization
for OP poisoning)
Questionnaire Abnormal vibrotactile
threshold in one forth of the
previously poisoned cohort
McConnell
et al. (1994)
Higher prevalence of
reported history of
exposure to solvents
among the previously
poisoned group
Vibrotactile threshold
34 male (community-
based reference
group)
128 male (OP poisoned
(1982–1990) in
California)
Definite cases (83) Reduction in sustained
visual attention, vibrotactile
sensitivity, symbol digit tests
Steenland
et al. (1994)
Particularly affected
heavily poisoned
(hospitalized) subjects
Probably cases (45)
90 male
(non-poisoned)
Neurologic function and
eight computerized
neurobehavioral tests
36 male
(Nicaragua workers)
Three groups: never
poisoned (I); poisoned
with other than
methamidophos (II);
poisoned with
methamidophos (III)
Abnormal vibrotactile
threshold in group III
McConnell
et al. (1994)
–
36 male (controls) Vibrotactile threshold
8 poisoned cases
(chlorpyrifos)
Medical follow up of
acute poisoning
Deficit of concentration,
word-finding and memory
in five out of eight
patients, reversible in four
Kaplan
et al. (1993)
Case reports
38 male (previous
acute poisoning,
hospitalization)
Neurobehavioral tests
(WHO protocol)
Reduction of verbal
attention; visual memory,
motricity; affectivity
Rosenstock
et al. (1991)
Unclear selection of
subjects and control
groups
38 male (controls) Unclear interpretation
of some neurobehavioral
changes
19 farm workers
acutely poisoned
No formal neurological
examination or testing
Blurred vision, night
sweats, headaches,
weakness, nausea
Whorton and
Obrinsky (1983)
Raw observational study
100 male þfemale
(previous OP
poisoning)
Neurologic functions No neurological effects Savage et al.
(1988)
Unclear selection of
subjects and control
groups
100 male þfemale
(controls)
Neurophisiological testing
(Wechsler adult intelligence
scale, Helsted-Reiten
battery and Minnesota
multiphasic personality
inventory)
Differences in the
neurophysiological tests:
intellectual functioning,
abstraction, flexibility of
thinking and simple motor
skills (distress)
Unclear interpretation
of some neurobehavioral
changes
580 C. Colosio et al. / NeuroToxicology 24 (2003) 577–591
(Garcia et al., 2000), the possibility still remains that
the long-term sequelae after acute OP poisoning are
only an unspecific expression of the CNS anoxic
damage.
Neurobehavioral Effects After Prolonged
Low-Level Exposure
The main studies on workers with prolonged expo-
sure to OPs in the absence of a documented episode of
acute poisoning are collected in Table 3. They will be
analyzed according to the exposure context.
Exposure in Agriculture
Durham et al. (1965) and co-workers did not find any
change in reaction time in workers chronically exposed
to OP compounds if compared to unexposed control
subjects. Analogous results were obtained by Rod-
nitzky et al. (1975) in a group of 12 farmers and 11
professional sprayers. Levin et al. (1976) studying a
group of 13 professional applicators and 11 farmers
found increased levels of anxiety, impaired vigilance
and reduced concentration in professional applicators
but not in agricultural workers. The authors could not
rule out the possibility that the findings may have been
produced by factors other than exposure. Maizlish et al.
(1987) in a group of 99 pest control workers, 56 of
which exposed to diazinon, failed to show any sig-
nificant behavioral change between pre- and post-shift
examination, while a significantly lower performance
was observed in the applicators when compared to
controls. The findings did not correlate with duration of
exposure. Since estimated cumulative exposure may
not be a valid risk indicator, particularly on intermit-
tent-exposure conditions, the study could not definitely
rule out the presence of long-term neurobehavioral
effects.
Daniell et al. (1992) studied a group of 49 agricul-
tural applicators and 40 unexposed control subjects. A
questionnaire was administered prior and after a 6-
month application season; in both exposed and controls
neurobehavioral functions were tested through a com-
puterized battery of tests. The study did not show any
clear evidence of clinically significant decrement in
neuropsychological performance following a 6-month
period of pesticide exposure, and only one subtest of
the applied battery (symbol digit substitution) showed
a statistically significant across-season change. Since
one control out of four reported a previous pesticide
exposure, the criteria for selection of control subjects
may be questioned.
Stokes et al. (1995) showed a significant increase in
hand vibration threshold in a group of 90 male pesticide
applicators compared to 68 unexposed control subjects.
Ames et al. (1995) among 135 agricultural applica-
tors, selected a group of 45 subjects that showed
reduction of acetylcholinesterase or butiryl cholines-
terase activity without any overt symptom of poison-
ing. The whole group underwent a battery of 27
neurological tests. When the findings obtained in the
group with a previous episode of cholinesterase inhibi-
tion were compared with those obtained in the group
that never suffered any decrease in cholinesterase
activity, the authors did not find any significant differ-
ence for all but one (serial digit performance) of the
investigated parameters. However, the difference
observed in serial digit performance showed a better
performance in the cholinesterase inhibited group. The
study would suggest that in presence of AChE inhibi-
tion, but without symptomatic poisoning, no neurobe-
havioral sequelae are expected.
A study carried out by London et al. (1997) on 231
sprayers and 115 internal controls, who underwent a
complete clinical and neurological examination and
neurobehavioral testing, did not show any appreciable
difference either in neurological or neurobehavioral
parameters. However, the authors reported the pre-
sence of confounding factors and the estimation of
the exposure levels, done through a job-exposure
matrix, was not reliable.
Analogous results were obtained by Bazylewicz-
Walckzak et al. (1999), who evaluated 25 females
engaged in pesticide application in greenhouses and
25 paired controls through the application of the
neurobehavioral core test battery, without showing
any difference between pre- and post-exposure mea-
surements. However, the authors showed longer reac-
tion times, reduced motor steadiness, tension,
depression and fatigue in the exposed group when
compared to controls.
Fiedler et al. (1997) evaluated neuropsychological
and psychiatric effects due to chronic use of OPs in 57
male tree fruit farmers, compared with 42 age-matched
male cranberry/blueberry growers and hardware store
owners. The study revealed statistically significant
slowing of reaction time in the exposed subjects, while
all other measures of cognitive function (concentration,
visuomotor skills, memory, expressive language,
mood) and emotional distress did not differ between
exposure groups.
In conclusion, there is not a firm and consistent
evidence that OP compounds are able to cause neuro-
behavioral effects after long-term low-dose exposure,
C. Colosio et al. / NeuroToxicology 24 (2003) 577–591 581
Table 3
Studies on OP exposed subjects without a previous episode of acute poisoning
Number of workers (job) Functions tested Observed effect(s) Author (year) Comments
Exposure in agriculture
26 female (Greenhouse
workers)
Neurobehavioral core
test battery
Longer reaction times Bazylewicz-
Walckzak
et al. (1999)
No differences
pre- and post-exposure
25 female (controls)
Reduced motor steadiness
Tension, depression and
fatigue in the exposed
57 male (tree
fruit farmers)
Cognitive functions
(concentration, visuomotor
skills, memory, expressive
language, mood)
Statistically significant
slowing in reaction time
Fiedler
et al. (1997)
Cognitive functions and
emotional status not
differing between
the groups
42 male (cranberry/
blueberry
growers) Emotional status
231 male (sprayers) Clinical neurologic
examination
No significant neurologic
and neurobehavioral effects
London
et al. (1997)
Long-term exposure
determined using a
job-exposure matrix 22
cases of previous acute
poisoning reported
115 male (internal
controls) Vibration sense
Presence of confounders
Neurobehavioral tests
The same group of
subjects selected
by Stephens
Selected 30 subjects
after dipping (10 most
symptomatic, 10 least
symptomatic and
10 unexposed)
Significant differences in
two-point discrimination
(dorsum hands and foots)
Beach
et al. (1996)
Subtle differences, clinical
significance unclear, levels
of exposure unknown
Mean calf circumferences No observed effects on
short-term memory and
learning
45 male (previous
moderate
cholinesterase
inhibition)
Previous RBC
cholinesterase at 70% or
less than baseline or
PChe at 60% or less
than baseline
One out of 27 neurological
test (serial digit performance)
significantly different, but in
direction opposite than
hypothesized
Ames
et al. (1995)
The study suggest that even
with ACHE inhibition,
but without poisoning,
no neurobehavioral
sequelae are expected90 male (no previous
cholinesterase inhibition
(agricultural
applicators))
90 male (pesticide
applicators)
Symptom questionnaire
measurement
Significant increase in
hand vibration threshold
in applicators
Stokes
et al. (1995)
Vibration threshold
sensitivity
68 male (unexposed
control subjects)
Dimetildithiophosphate
49 male (agricultural
applicators)
Questionnaire prior and
before application
season (6 months)
Only one subtest showed
statistically significant
exposure related across-season
change (symbol digit
substitution)
Daniell
et al. (1992)
No clear evidence of
clinically decrement in
neuropsychological
performance
One control out of four
reported a previous
pesticide exposure
40 male (controls)
99 pest control
workers
Neurological screening
examination
No adverse DETP related
changes in pre- and post-
shift neurobehavioral function
Maizlish
et al. (1987)
No difference pre- and post-
exposure, slight differences
exposed vs. controls56 of which exposed
to diazinon
Symptom questionnaire
Poorer performances in the
exposed group, not supported
by regression analysis with
duration of exposure
Sine estimated cumulative
exposure may be not a good
indicator in intermittent-
exposure conditions, the
study suggest that is not
an effect, if any
Tests of concentration,
eye-hand coordination,
patter recognition, visual
memory, finger tapping
Diethylthiophosphate
(DETP) determination
582 C. Colosio et al. / NeuroToxicology 24 (2003) 577–591
even if these effects are neither been consistently nor
extensively investigated (Goran and Jamal, 1997;
Jamal, 1995). The main limits of the available studies
are uncertainty on quantitative exposure definition,
inappropriate selection of control subjects, lack of
consistency among the results of different studies,
and difficulties in the interpretation of some results
in terms of health outcomes.
Table 3 (Continued )
Number of workers (job) Functions tested Observed effect(s) Author (year) Comments
13 professional
applicators
Beck depression
Inventory (22 multiple
choice items)
Elevated levels of anxiety,
impaired vigilance and
reduced concentration in
commercial sprayers but
not in farmers
Levin
et al. (1976)
The authors underline that
anxiety may have been
produced by other factors
than exposure
11 farmers
Structured interview
PChE but not AChE
depressed in the
exposed groups
24 unexposed control
subjects PChE and AChE
determination
12 farmers Memory No difference exposed
vs. controls in all the
parameters taken
into account
Rodnitzky
et al. (1975)
Many of the workers taken
into account had used many
different active ingredients
11 professional
applicators
Vigilance
Signal processing time
Language
Proprioception
AChE
(A) Sheep dipping
612 male (sheep dippers) Administered a
standard neuropathy
symptom questionnaire
Neurologic symptoms
(primarily sensory type) in
the highly exposed subjects
Pilkington
et al. (2001)
Data on the levels of
exposure retrospectively
collected through
questionnaire
53 male (non-dipper-
farmers)
No clear effects on thermal
or vibration sensory thresholds
107 male (ceramic
workers)
146 male
(sheep farmers)
Subjects selected based
on presence of symptoms
after dipping
Significant cognitive and
neuropsychological effects
Stephens
et al. (1995)
No association presence of
symptoms and chronic
outcomes143 male (controls)
Measured
neurophisiological testing
Several since several of the
quarry workers selected as
controls reported to have
helped on sheep farms
during sparing time
Urine alkylphosphates
38 male (sheep dippers
exposed to diazinon)
Measured AChE activity
and dialkylphosphate
excretion
Significant AChE inhibition
(not at levels associated
with toxicity), increase in
alkylphosphate excretion
Rees (1996) Dippers are significantly
exposed
24 completed the study Handling of concentrate
dip is the main source of
exposure
(B) Other exposures
191 male (current and
former termiticide
chlorpyrifos
applicators)
189 controls
Neurologic function No major differences
exposed vs. controls
Steenland
et al. (2000)
In recently exposed
subjects, biological
monitoring data show
significant occupational
exposure
Biological monitoring
(urine 3,5,6-TCP) Reduction of nerve conduction
velocity, tremor, visual motor
skills, memory impairment,
emotional status, fatigue
reported in the exposed group
a
Other active ingredients
involved
59 male (quinalphos
and other pesticide
manufacture)
17 controls
Physical examination,
testing battery, digit span,
digit symbol test, Bourdon
Weirsma vigilance test
Altered plantar and
ankle reflexes
Srivastava
et al. (2000)
AChE activity not different
in exposed and controls
AChE activity
Memory, learning and
vigilance affected
Higher level of education
in controls
a
Eight subjects with a previous episode of acute poisoning had a pattern of low performance on a number of tests.
C. Colosio et al. / NeuroToxicology 24 (2003) 577–591 583
Since some studies (Bazylewicz-Walckzak et al.,
1999; Beach et al., 1996; Maizlish et al., 1987) did
not show neurobehavioral changes between pre- and
post-exposure determinations nor association between
changes and biological monitoring indicators, but sug-
gested slight differences when comparing exposed and
controls, it might be hypothesized that such effects
build up over time and a one-season exposure only is
not sufficient to cause an appreciable deterioration. In
future, research should be more focused on prolonged
exposures, with particular attention for highly exposed
subjects and on the definition of reliable indicators of
prolonged exposure.
Exposure in ‘‘Sheep Dipping’’
Apart from agriculture, a significant exposure to
pesticides may be present in other rural activities.
One of these activities is the so-called ‘‘sheep dipping’’
(Table 3(A)). It consists of dipping sheep in a pesticide
aqueous solution to prevent sheep scab, the allergic
disease caused by the mite Psoroptes Ovis, and other
external sheep parasitosis. The treatment is usually
made with organophosphorous compounds, but in
some cases also pyrethroids can be used.
A study carried out on a group of 146 sheep farmers
and 143 unexposed control subjects showed a signi-
ficant cognitive and neuropsychological deficit in
farmers. Recent exposure was measured through the
determination of alkylphosphate concentration in
urine. No association was observed between the pre-
sence of OP-related symptoms and long-term out-
comes (Stephens et al., 1995). This suggests that
neurobehavioral effects, if present, may occur inde-
pendently of symptoms that might immediately follow
an OP overexposure. In this study, the selection of
control subjects may be questioned, since several of the
quarry workers selected as controls reported to have
helped on sheep farms during sparing time. At the
comparison of the ten most symptomatic and the ten
less symptomatic subjects selected by Stephens et al.
with 10 unexposed healthy control subjects, Beach
showed a reduction in two-points discrimination (dor-
sum, hands and foots) and mean calf circumferences.
The study did not show any effect on short-term
memory and learning (Beach et al., 1996).
Pilkington et al. (2001) evaluated a group of 612 OP
exposed dippers, and compared the results of a stan-
dard neuropathy symptom questionnaire with those
obtained from a control group of 53 non-dipper-farm-
ers and 107 ceramic workers. The study showed an
increase in neurological symptoms in the dippers,
primarily of sensor type, the significance of which
was dependent on few individual workers with extre-
mely high exposures. The study did not show any
significant association between exposure and sensory
thresholds. Data on exposure levels were collected
through an exposure history questionnaire.
The possibility of a neurological impairment of
dippers related to pesticide exposure has generated a
serious concern over the last years. Certainly, dippers
are to be considered as a high exposure group (Rees,
1996), and dip concentration is the most important
determining factor of OP exposure, since the excretion
of OP metabolites in the exposed dippers increases
with the increase of the concentration of the solution
used (Buchanan et al., 2001). In some cases dippers
develop flue like symptoms at the end of the work-shift.
This has been matter of further concern, because mild
acute OP poisoning may be difficult to diagnose
(Goran and Jamal, 1997) and such ‘‘dipper flu’’ cases
might, in fact, be unrecognized mild OP poisonings
that are erroneously interpreted as flue (Murray et al.,
1992).
In spite of the great attention raised in the commu-
nity, the investigations carried out on dippers suffer the
same limitations of other similar studies, mainly in the
definition of the levels of exposure and in the selection
of the control groups. Thus no firm conclusions can be
drawn on the real occurrence of such effects and on the
causative factors. The slight changes consistently
shown by different studies indicate the need of better
studies on this topic, addressing the open questions
concerning the dose–effect relationship, the type of
involved compounds, and their mechanism of action.
Other Exposures
Steenland et al. (2000) studied 191 current and
former termiticide applicators and 189 unexposed
control subjects (Table 3(B)). In 65 recently exposed
subjects an average urinary 3,5,6-trichloropyridinol
(3,5,6-TCP) excretion of 629.5 mg/l was measured,
versus 4.5 mg/l measured in the US general population:
these data suggest a significant occupational exposure.
Clinical examination did not show any difference
between exposed and control subjects. On the other
hand, significant differences were reported in the
exposed group in nerve conduction velocity, arm/hand
tremor, vibrotactile sensitivity, vision, smell, visual/
motor skills, and neurobehavioral skills. Also symp-
toms such as memory problems, emotional status, and
fatigue were significantly more frequent in the exposed
subjects.
584 C. Colosio et al. / NeuroToxicology 24 (2003) 577–591
A study carried out on 59 subjects engaged in the
industrial production of quinalphos and 17 controls
showed altered plantar and ankle reflexes and reduction
in memory, learning and vigilance in the exposed
subjects, whose levels of acetylcholinesterase activity
did not differ from those measured in control subjects
(Srivastava et al., 2000). However, it must be noted that
the level of education was higher in the control group
(47.1% of graduated and above versus 25.5%), and this
difference may have affected the observed difference
between exposed and controls.
These papers are in line with some of the agricultural
studies above discussed and do not seem to add sub-
stantially new information.
SYNTHETIC PYRETHROIDS
Synthetic pyrethroids are insecticides becoming
more and more important in agriculture, public health
and for the struggle against vector born disease in the
developing world because of their low acute toxicity
and very short environmental half-lives. They also find
a significant use indoors (home, restaurants, offices and
also airplanes), therefore the definition of the health
risk associated with the use of these compounds is of
great public health interest. No extended studies are
available suggesting the capacity of pyrethroids to
impair neurobehavioral function in low-dose exposure
conditions. Two papers based on single case reports
(Altenkirch et al., 1996; Muller-Mohhnssen, 1999)
(Table 4) suggested the possibility of some neurobe-
havioral effects after acute poisoning episodes. How-
ever, the observed effects were various, the number of
cases very small, and the information on health status
were collected only through telephone interviews or
questionnaire. Finally, acute pyrethroid poisoning is a
very unlike event. In conclusion, this class of chemicals
does not seem to be of particular relevance for a
possible neurobehavioral toxicity.
FUMIGANTS
One of the most toxic fumigants is methyl bromide, a
compound widely used for soil fumigation and seed
treatment.
Two studies suggested the capacity of methyl bro-
mide and another fumigant, sulfuryl fluoride, to cause
neurobehavioral effects (Table 5). Calvert et al. (1998)
studied a group of 123 fumigation workers through the
measurement of nerve conduction, vibration, neurobe-
havioral, visual, and olfactory functions. The study
showed a reduction of the performance at the pattern
memory tests only in sulfuryl fluoride workers, but
exposure levels of these subjects had not been mea-
sured. A second study carried out on 15 fumigation
workers, only five of whom were considered as
‘‘chronically exposed’’, measured neurological func-
tions through the WHO neurobehavioral test battery
and the determination of other parameters (dynamome-
try, vibration, peripheral nerve conduction and psycho-
logical functions). Insomnia, headache, paresthesiae,
mood changes, loss of memory, increased threshold
for vibration, reduction of dynamometric test, and
increased score for negative auto-perception were more
frequent in the exposed group, who also showed a
significant increase in blood methyl bromide in the
post-exposure samples (Acuna et al., 1997).
Due to the limited number of studied subjects, it is
almost impossible to draw conclusions on the neuro-
behavioral toxicity of these compounds; however in
view of the relative frequency of acute poisoning with
fumigants, this issue would deserve further attention.
Table 4
Studies on neurobehavioral effects of pyrethroids
Number of subjects Functions tested Observed effect(s) Author (year) Comments
4 previous acute and
subacute pyrethroids
poisoning (case reports)
Follow up of acute
poisoning cases
Reduced intellectual performance,
visual disturbances, dysacousia,
tinnitus
Muller-Mohhnssen
(1999)
Cause–effect
relationship not
demonstrated
Sensomotor polyneuropathy
Vegetative nervous disorders
Opportunistic infections
23 (male þfemale) acute
pyrethroids in-home
poisoning case
(case reports)
Follow up of acute
poisoning cases
Nine out of 23 presented
severe somatic or
psychiatric disorders
Altenkirch
et al. (1996)
Cause–effect
relationship not
demonstrated
Study aimed at multiple
chemical sensitivity
C. Colosio et al. / NeuroToxicology 24 (2003) 577–591 585
MIXTURES
In several retrospective studies, the active ingredi-
ents used by the studied subjects could not be defined,
and, given that the use of many different active ingre-
dients is very common in agriculture, data were
referred to ‘‘use of mixtures’’. Also the so-called ‘‘Gulf
War syndrome’’ has been sometimes attributed to
exposure to mixtures of pesticides.
A reduction in cognitive tests was observed when
comparing 96 Hispanic adolescents working in agri-
culture with 51 contemporaries engaged in non-agri-
cultural activities (Rohlman et al., 2001). Agricultural
workers were supposed to be exposed to pesticides, but
qualitative and quantitative data on exposure were
lacking, and, at the same time, a comparability of
educational and cultural status of exposed and controls
was not demonstrated (Table 6).
Baldi et al. (2001) published the results of a study
carried out on 528 agricultural workers directly
exposed to pesticides during mixing, preparation or
spraying of pesticides, 173 indirectly exposed (re-entry
workers), and 216 never exposed subjects. The study
showed cognitive effects in the workers, the most
impaired functions being information selection and
processing, but a dose–effect relationship could not
be shown since exposure was only assessed with a
questionnaire and the compounds to which the workers
would have been exposed could not be identified. Apart
from the limitations on the exposure assessment, a
further weakness of this study was that the psycholo-
gists in charge of the examination knew the exposure
status of the workers.
A very complex study was carried out by Cole et al.
(1997) through the application of the WHO neurobe-
havioral core test battery (Cassitto et al., 1990)ona
group of 142 Ecuadorian agricultural workers exposed
to 43 different active ingredients, mainly represented
by Mancozeb, carbamates and OPs. This study showed
that visual–spatial tasks were the most sensitive targets
of farm membership, however, it has to be noted that
the levels of exposure were assessed only through a
questionnaire, the presence of numerous possible con-
founders was acknowledged in the paper, and the
authors themselves underlined that cross-sectional stu-
dies cannot provide conclusive evidence of effects.
Gulf War Syndrome (GWS)
Many of the veterans who served in the 1991 Gulf
War reported a number of unexplained symptoms
during or after their service. Difficulty in memory
and attention were among the symptoms most com-
monly reported (Fiedler et al., 2000; Storzbach et al.,
2000, 2001), but in some cases these symptoms could
be part of the so-called ‘‘chronic fatigue syndrome’’
and, in other cases, of the ‘‘multiple chemical sensi-
tivity’’ syndrome (Kipen et al., 1999; Pollet et al.,
1998). Among the possible etiological factors, con-
sideration was given to low-level exposure to nerve
gases, but also to exposure to pesticides used for the
treatment of dresses, tents and sleeping bags and to
the antidotes provided to the troops during their stay
in the war area. In some studies, severe and mild–
moderate illness was positively associated with self-
reports of pyridostigmine bromide use, insect repellent
use, and psychological distress caused by threat of
biological or chemical weapons (Nisenbaum et al.,
2000). Table 6 summarizes the most recent literature
on this topic.
The body of available data confirms that GWS is a
poorly known entity, characterized by signs and symp-
toms which fluctuate depending on the studies, with
many organs apparently involved but without any
Table 5
Studies on neurobehavioral effects of fumigants
Number of subjects Functions tested Observed effect(s) Author (year) Comments
123 male Nerve conduction, vibration,
neurobehavioral, visual,
olfactory, and renal
function testing
Exposure to sulfuryl fluoride
associated with reduced performance
at the pattern memory test
Calvert
et al. (1998)
Levels of exposure
unknownFumigation workers
(methyl bromide,
sulfuryl fluoride) Impairment of olfactory function
120 male (controls)
15 male WHO neuro behavior Insomnia, headache, paresthesiae,
mood changes, loss of memory,
increase of threshold for vibration,
reduction of dynamometric test,
score for negative auto-perception
Acuna
et al. (1997)
Blood methyl
bromide increased
from 13.3 to
30 mg/dl after
exposure
Fumigation workers
(methyl bromide)
(5 chronically exposed)
Core test battery
10 controls
Dynamometric
Vibration assessment
Peripheral nerve conduction
Psychological functions
586 C. Colosio et al. / NeuroToxicology 24 (2003) 577–591
clinical evidence of organic injury. Moreover, signs and
symptoms overlap with many other chronic diseases
and there is a serious possibility that this syndrome may
not represent a single nosological entity (Knoke et al.,
2000). Numerous etiological hypotheses have been
formulated, namely stress, vaccinations, exposure to
neurotoxins, and exposure to weak uranium (Tourni e r
et al., 2002), however none of them has a solid scientific
support and a credible mechanistic explanation. Factor
analysis of self-reported symptoms did not identify a
unique Gulf War syndrome (Knoke et al., 2000), the
most commonly reported finding being psychological
distress. At the state of current knowledge, a bio-
psycho-social explanatory model may probably provide
the most likely interpretation of the nature of these
disturbances (Ferrari and Russel, 2001).
Table 6
Studies on neurobehavioral effects of mixtures
Number of subjects Functions tested Observed effect(s) Author (year) Comments
528 directly exposed A selected battery of nine
neuropsychological tests
(not computerized)
Cognitive effects Baldi
et al. (2001)
Qualitative and
quantitative data on
exposure lacks
Pesticide applicators
in vineyards
No dose–effect
relationship
173 indirectly exposed
(re-entry workers)
Study not carried
out in blind
216 never exposed
3531 male (Gulf
War veterans)
Questionnaire Multiple chemical sensitivity Reid
et al. (2001)
Quantitative and
qualitative exposure
data lack
Chronic fatigue Syndrome
Psychological morbidity
96 hispanic adolescents
working in agriculture
Computerized behavioral
assessment and research
system (BARS)
Reduction in the cognitive
tests in agriculture workers
Rohlman
et al. (2001)
Qualitative and
quantitative data on
exposure lack51 hispanic adolescents
not working in
agriculture
Educational and cultural
differences may explain
the difference
239 male (Gulf
War veterans)
Medical examination
Battery of 12 psychological
and 6 neurobehavioral tests
The majority of cases with
symptoms had no objective
evidence of neurobehavioral
deficit
Storzbach
et al. (2001)
Effects attributed to
low-level nerve gas
exposure
Evidence of psychological
distress
Data on the levels of
exposure lack
45 þ35 þ23 male
(Gulf War veterans)
Complete physical,
psychiatric and
neuropsychological
evaluation
Personality, negative coping
strategies, life stress after the
war are significant predictors
of current physical function
Fiedler
et al. (2000)
Risk not related to
chemical exposures
241 male (Gulf War
veterans, with
symptoms)
Questionnaire Increased distress and more
poorly neurobehavioral
performances in subjects
with symptoms
Storzbach
et al. (2000)
Psychological
differences have a
prominent role
113 (Gulf War veterans,
without symptoms)
12 physiological and
6 neurobehavioral tests
1935 male (Gulf
War veterans)
Questionnaire 15.7: chronic fatigue
syndrome
Kipen
et al. (1999)
–
1161 accepted to
enter the study
13.1: multiple chemical
sensitivity
3.3: both conditions
142 farm workers
72 controls
Neurobehavioral
core test battery
Visual–spatial tasks Cole
et al. (1997)
Exposure to 43 different
active ingredients, but
mainly to Mancozeb,
carbamates and OP,
estimated through
questionnaire
C. Colosio et al. / NeuroToxicology 24 (2003) 577–591 587
CONCLUSIONS AND PERSPECTIVES
Although most pesticides have a known neurotoxic
potential, the evidence of their capacity to cause neu-
robehavioral impairment in man in the absence of a
previous acute poisoning episode is very limited. Even
in the past-poisoned subjects, the often observed reduc-
tion of verbal attention, visual memory and attention,
motricity, affectivity, and flexibility of thinking, may
be the result of a generic brain injury, for example,
anoxia consequent to the cholinergic crisis, and not
necessarily to be related to a specific neurotoxicity of
these compounds. Solving this dilemma would signif-
icantly improve the knowledge on the neurobehavioral
effects of OPs, and the answer would have relevant
implications on risk assessment and risk management
activities.
The slight changes sometimes observed in subjects
exposed to OP without any previous acute poisoning,
such as longer reaction time, emotional and intellectual
changes and fatigue, and the evidence of effects in
heavily exposed subjects, such as sheep dippers, are a
matter of concern and further research is recommend-
able to confirm or rule out the presence of a risk
of neurobehavioral toxicity consequent to repeated
low-dose OP exposure. The same applies to fumigant
exposure.
In spite of their great social and legal impact, the
effects attributed to mixed exposures, including the
Gulf War syndrome, are not sufficiently substantiated
in scientific terms to call for specific investigations: it
is, in fact, very difficult to search the cause of a
unspecified disease that may actually not even exist.
A disappointing feature common to the majority of
the studies on neurobehavioral effects is represented
by their inconclusiveness: in spite of the number of
available studies, very few conclusions can be made.
To improve our knowledge on this area and remove
the existing uncertainties, future studies should be
designed and conducted in a different way from the
past, taking in due account the need of combining
clinical or neurobehavioral testing with robust toxico-
logical assessments.
In most of the past studies, no dose measurement has
been performed and in some of them even the nature of
exposure has not been documented. When a dose was
measured, it was impossible to correlate the current
level of exposure with the observed effects, which were
very likely attributable to previous exposures.
Since the current dose does not necessarily correlate
with the historical exposure and past levels of exposure
are usually undeterminable, cross-sectional studies
may be inconclusive, if a preliminary check of avail-
able data is not carried out. A sound solution may be
the selection of cohorts of workers employed in plant
protection products’industries, for which historical
environmental and biological monitoring data are
available. In this case, the possibility of creating
job-exposure matrices would allow the researcher to
better categorize exposure classes and to identify a
dose–response relationship.
Promising results may be provided also by pros-
pective longitudinal or cohort studies where exposure
and dose assessment can be more easily controlled
(Rohlman et al., 2001). Such studies are obviously
more expensive and difficult to carry out and provide
results only after many years, however this is the toll to
pay if clear scientific results are sought.
The follow up of the cohort under study might allow
to answer the question of the prognostic significance of
the observed changes.
A further critical area is represented by testing
protocols: although many different procedures are
available for the assessment of human behavioral
functions, performance tests are displacing traditional
diagnostic tests. Performance tests include variables
such as attention, concentration, sensory function,
motor control, spatial relations, visuomotor coordina-
tion, memory, and affect (Fiedler et al., 1996). Each of
these functions may be assessed through different tests,
which may be organized in different batteries; even the
parameters of administration of the same test may vary
among different studies. The lack of standardization
makes impossible to repeat single studies and also to
compare similar studies made by different researchers:
since reproduction of results by independent groups is
the only scientific criterion to confirm the validity of
observations, this field of research condemns itself to
perennial uncertainty unless a clear definition of stan-
dardized testing methods and procedures is achieved.
Recently, performance tests have been computerized,
and these computerized test batteries seem adequate
for standardized and efficiently conducted field studies
(Fiedler et al., 1996). However, it must be taken into
account that, due to the need of alphabetic knowledge,
computerized tests may be not appropriate for studies
on low educated populations (Baldi et al., 2001).
The selection of the study population is another very
critical issue, because it poses relevant methodological
problems, especially in agricultural settings where a
low cultural profile of the subjects may influence
the outcomes of the study (Rohlman et al., 2001).
Given the difficulty of matching exposed farmers with
unexposed controls, cross-sectional studies ending with
588 C. Colosio et al. / NeuroToxicology 24 (2003) 577–591
exposed control comparisons ought to be carefully
designed and the comparability of the population
groups fully documented. The main variables to be
taken into account while planning a study on neuro-
behavioral toxicity are depicted in Table 7.
Since they are in the early phase of development,
children should be considered as a highly vulnerable
group and should deserve special attention. In a work-
shop held in Atlanta by the US Agency for Toxic
Substances and Disease Registry, approaches and
methods targeted to children and adults exposed to
toxic substances were evaluated, a list of basic func-
tions to be assessed during eight developmental periods
from birth to high school was discussed, and 10 specific
recommendations to establish a neurobehavioral
screening for pediatric populations were made (Kras-
negor et al., 1995). A test battery specifically targeted
to children has also been suggested (Davidson et al.,
2000). The poverty of studies on children in the
literature ought to be corrected taking also advantage
of the easier opportunity they may provide for long-
itudinal perspective studies.
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