![Background
Australian farmers and their workers are exposed to a
wide variety of pesticides [1]. Pesticides are substances
that destroy, repel or attack pests that have a negative ef-
fect on productivity and profitability of a farming enter-
prise. Pesticide groups include herbicides, insecticides,
fungicides and rodenticides. Organophosphate (OP) insec-
ticides are widely used for animal husbandry practices
(Naphthalophos sheep drench), public health (head lice,
locust control), and in crop production (Dimethoate for
insect control). The OP class of insecticides effect the
nervous system with poisonings most common among
agricultural workers and children [2, 3]. OPs are regularly
used as a form of pest control on most farms and farmers
have enhanced their understanding of pesticide use and
handling. However, many are still not meeting the full
range of personal protection standards in their own
handling and application [4]. Routes of human exposure
include dermal absorption, inhalation and ingestion [5].
Symptoms of OP toxicity are caused by inhibition of the
enzyme acetylcholinesterase (AChE). Inhibition of AChE
results in the subsequent accumulation of acetylcholine at
the cholinergic synapses of nerves causing uncontrolled
firing of the synapse [1, 6]. Subclinical effects of chronic
OP exposure such as AChE inhibition may be detected
early by biological tests. Recovery from AChE depression
is prolonged, due to the irreversible binding of OPs to
the red blood cell and its subsequent 120 day life cycle
[7]. Monitoring exposure to organophosphates involves
the measurement of peripheral cholinesterase enzymes that
are inhibited by organophosphates. These include; erythro-
cyte cholinesterase (EAChE) and serum cholinesterase
(SAChE). Organophosphates have been associated with
chronic neurological disease such as impaired memory,
impaired fine motor skills control and Parkinson’s disease
[8]. The evidence suggests that whilst an acute episode
experience may not have occurred, exposure over a long
period of time (such as in workers dipping sheep) may re-
sult in neurological conditions such as Parkinson’s disease
and neuropsychiatric conditions [6, 8, 9]. The window for
exposure to toxicants may occur years before the onset of
neurological symptoms [8, 10, 11], this is particularly
important for sheep and crop producers exposed to long-
term, low levels of OPs. The Western region of the state of
Victoria in Australia has been a centre of sheep production,
since settlement, and a large number of farmers have been
exposed to OPs through routine sheep dipping (lice con-
trol), drenching (control internal parasites), jetting (blow fly
control) and spraying of pastures (insect control). The
National Centre for Farmer Health (NCFH) is in a unique
position to monitor these farmers and workers, and work
with farmers to incorporate cholinesterase testing as part of
their assessments during peak periods and provide much
needed data for them on exposures in this area. Additional
practices such as mixing contaminated laundry and storing
pesticides in the home are not only common causes of
exposure to the farmer and farm worker, but may also be
placing other household members at risk, particularly
children [12]. Work completed by the program Sustainable
Farm Families™ [13] has identified that there is strong
interest from farm men and women who wish to investigate
exposures and possible health impacts of agricultural
chemical use.
The acute effect of OP exposure is well documented
in Australia and overseas for humans, pests and animals
[3, 6, 9]. However, the extent to which asymptomatic
monitoring is taking place is not well known or docu-
mented. This research builds on work conducted by the
NCFH. It utilises the data collection methods of the
Sustainable Farm Families™ which includes health behav-
iours and conditions, with the expectation that this
monitoring EAChE testing will be integrated into this
procedure, providing the springboard to break through
barriers that are currently inhibiting best practice chem-
ical handling safety in Australia.
Understanding the mode of action of a chemical is a
critical component of the risk assessment process.
Cholinesterase activity is a direct link to exposure and
this pilot study will produce valuable data on possible
exposures for farmers routinely using OP’s for animal
husbandry or crop production.
Monitoring cholinesterase levels during agricultural
health screening and assessment is a key objective of
Agrisafe™ clinics in Australia. However, its incorporation
into such clinics will depend on health professional’s skills
and knowledge. This study will further prepare health care
providers for integration of correct cholinesterase testing
procedures into point of care (POC) health checks. This
work will further highlight pesticide exposure as a risk for
farmers, workers and their families, leading to an easily
adopted method that will quantify the environmental
exposures providing early detection. Research has also
shown links between cholinesterase activity and vascular
complications in diabetic patients [14]. Diabetes is a
disease also of increasing concern to the farming and rural
population in Australia [15].
Aims
The Cholinesterase Research Outreach Project (CROP)
will use clinical sampling to determine the level of expos-
ure associated with the routine use of OPs by Victorian
farmers and/or their workers. This preliminary study will
examine the methodology of testing for EAChE activity at
POC and provide evidence for future work by identifying
the exposure patterns and pathways of individuals most at
risk in farming communities. It is anticipated that CROP
will inform future research studying pesticide exposure in
farming communities.
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![Methods/design
Evaluation design
To evaluate if a difference in cholinesterase activity was
present in farmers before and after exposure, sample size
is determined at the power of 80 % (β = 0.2) with α set
at 0.05 for an effect size of one standard deviation. This
requires the sample size to be a minimum of 30 farming
individuals exposed to pesticides. By considering the
probable retention rate (≥85 %) for a 7-month study,
based on previous research in similar populations, [16]
the target recruitment number is fixed at a target of 50
farmers.
Sample size and inclusions
Consent will be obtained from 80 participants recruited
via existing contacts and industry groups, letterbox
drops and newspaper articles from a number of farming
and community groups, specifically;
Mixed farming enterprises and sheep dipping/spraying
contractors (n = 50) who are using OPs and a conveni-
ence group of non-farming individuals (n = 30) from the
rural community of Hamilton, Australia.
Participants need to have been farming for more than
5 years, be aged between 18 and 75 years, speak English,
and not had a previous known chemical incident. Ques-
tionnaires will be used to determine whether or not they
are the primary pesticide user. Any non-farming individ-
uals tested must also be aged between 18 and 75 and
have not had a previous chemical incident. Participants
from both groups will be self-reported not pregnant, not
suffering from a known chronic disorder, not taking
anti-inflammatory drugs or supplements and not exer-
cising excessively throughout the study.
Anthropometry
The simplicity of anthropometry allows it to be used in
population-based studies to assess body-composition
changes over time, as well as in clinical and field situa-
tions where access to technology is limited such as in
this study. Height, weight, waist and hip circumference
has previously been included as part of an initial POC
assessment for farm men and women and agricultural
workers. Participants have found these measurements
helpful and easy to understand and also cite having a
free health assessment as a reason to attend [13]. Add-
itionally, (whilst not the focus of this study) if used in
combination with blood biochemistry and body fat per-
centage, anthropometry can identify distinct fat distribu-
tion changes that occur in the elderly [17]. Weight is
measured to the nearest 0.05 kg using electronic scales
taken in light clothing with shoes removed and pockets
emptied and prior to breakfast. Height is measured using
a portable stadiometer to the nearest 0.1 cm with shoes
removed and weight distribute evenly on both feet.
Physical assessments and health condition data
A short health history and health behaviours (smoking
and alcohol consumption habits) will be obtained from
participants with baseline physical assessments of blood
pressure measurement (repeated twice), pulse rate, body
fat percentage (bioelectrical impedance), respiratory
function test (PiKo meter) also measured (see Table 1).
Table 1 Data collection description and timeline for farmer and non-farmer groups
Variable Baseline (T0) Visit 1 (T1, 1 mth) Visit 2 (T2, 2 mth) Visit 3 (T3, 3 mth)
Body weight ✓ X X X
Height ✓ X X X
Waist circumference ✓ X X X
Hip circumference ✓ X X X
Body fat % ✓ X X X
Blood pressure ✓ X X X
Pulse rate ✓ X X X
Fasting blood glucose ✓ X X X
Fasting blood cholesterol ✓ X X X
Fasting triglycerides ✓ X X X
HDL cholesterol ✓ X X X
LDL cholesterol ✓ X X X
Erythrocyte Cholinesterase (EAChE) ✓ ✓ ✓ ✓
Description of monthly chemical use ✓ ✓ ✓ ✓
Kessler (K10) ✓ X X X
Chemical Usage Survey ✓ X X X
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![Biochemical analysis of EAChE activity using a 10 μl
capillary blood sample (measured by Test-mate ChE
Cholinesterase tester system Model 400), and EAChE
field Assay kit - to establish a baseline reading, will be
used (Fig. 1). The Test-mate field assay testing system
is based on the Ellman method [18]. Blood glucose and
lipids (LDL, HDL) and total cholesterol is also taken
after 10-hour fast using the same blood sample.
Waist circumference is measured to the nearest
0.1 cm at the end of a normal expiration using a con-
stant tension “Figure Finder Tape Measure”™ [19]. Body
Mass Index will be calculated to classify obesity and
overweight cut off points for future reference using the
WHO parameters [20].
Survey methodology
Trained research staff will collect all anthropometric and
behavioural data. Sociodemographic data are collected
directly from the participant including age, gender,
country of origin, using the Victorian Department of
Health service coordination tools (SCOT) [21]. The type
of farming undertaken and residential postcode will also
be collected from the participants.
A validated questionnaire, the Kessler 10 (K10) will
be used to measure psychological distress. The K10 is a
10-item questionnaire intended to yield a global meas-
ure of distress based on questions about anxiety and
depressive symptoms that a person has experienced in
the most recent 4 week period [22]. This survey has been
used extensively in Australia and provides a comparison
to the general population. Given the relationship between
organophosphates, neuropsychological and psychiatric
functioning a baseline was deemed important [23, 24]. It
also forms part of the usual farmer health assessment.
Occurrence of illness & injury
Occurrence of illness or injury experienced within the
last 3 months will be measured using a questionnaire de-
scribing 33 symptoms of chemical exposure, in random
order of severity. The illness and injury data will be
standardised and analysed accordingly.
Monitoring agrichemical use
In addition to completion of the agrichemical use survey
of 13 questions, self-reported chemical use is recorded
during each visit. Participants are provided with counsel-
ling regarding their EAChE levels in accordance with
AgriSafe guidelines (Fig. 2). The USA’s Department of
Pesticide Regulation specifies that a drop to 70 % or
lower in red blood cell AChE or to 60 % or lower in
plasma cholinesterase relative to the individual’s baseline
is an indication for immediate removal of the individual
from all exposure to organophosphate and carbamate
pesticides [25]. Participants will be provided with a sum-
mary of their EAChE baseline and subsequent results at
the conclusion of the study. This methodology reflects
recent exposures and is used to provide feedback at the
time of testing. However, it is noted that the level of
current exposure is not a measure of long-term exposure
Fig. 1 Point of care schedule for cholinesterase assessment
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![and that the clinical parameters around long-term
exposure are not well defined.
Data entry, handling and statistical analysis
All data will be managed and analysed within the statis-
tical program SPSS (IBM Corp. Released 2012 IBM
SPSS Statistics for Windows, Version 21.0. Armonk,
NY). SPSS is a statistical package widely used in quanti-
tative social science research and is suitable for multi-
variate analysis. Data will be analysed using repeated
measures (subjects as own controls) within the General
Linear Model. Correlation coefficients between pesti-
cide exposure and metabolic variables will be calculated
using Pearson’s and Spearman’s correlation as appropri-
ate. P values will be considered statistically significant
at p < 0.05.
Consent and ethics
All adults participating in the study are to be provided
with a plain language statement and will provide in-
formed written consent. Ethics approval has been
granted to the project by Deakin University Human
Research Ethics Committee (HREC 2013-100 dated
18/06/2013). All researchers involved in data collection
have a Victorian Police check undertaken.
Discussion
Organophosphates are still used widely with the actions
and attitudes of farmers and workers using these pesti-
cides is vital to their wellbeing and that of their families.
It is common for mild to chronic depression of EAChE
activity to be reported as ‘normal’ due to the wide refer-
ence range for EAChE activity [26]. It is important that
farmers and agricultural workers who are routinely using
OPs establish their baseline EAChE activity and have
access to regular EAChE activity checks for comparison
with baseline. Routine monitoring of EAChE may allow
for early recognition of frequent and continuous
low-level exposure to OPs. This work will provide an
evidence base, leading to research to further quantify
pesticide exposure both on the farm and in the home of
farming families and highlight the importance of sustain-
ing and providing a safe work environment for farming
communities.
Abbreviations
AChE: Acetylcholinesterase; EAChE: Erythrocyte Acetylcholinesterase;
CROP: Cholinesterase Research Outreach Project; OP: Organophosphate;
K10: Kessler 10; LDL: Low Density Lipoproteins; HDL: High Density
Lipoproteins; NCFH: National Centre for Farmer Health; POC: Point of Care;
SCOT: Service Coordination Tools; BMI: Body Mass Index; mth: Month.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SB was an initiator of the original Sustainable Farm Families program,
contributed to the design of the study, and specific health assessment
protocols. JC, SB and PAL participated in the design of the study and
drafting of the manuscript. All authors read and approved the final
manuscript
Acknowledgements
The work is supported by a Faculty Research Grant provided by Deakin
University Faculty of Health. The authors would like to also acknowledge the
support of Western District Health Service.
Received: 11 February 2014 Accepted: 21 July 2015
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- 1. See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/280872529 Cholinesterase Research Outreach Project (CROP): measuring cholinesterase activity and pesticide use in an agricultural community Article in BMC Public Health · August 2015 DOI: 10.1186/s12889-015-2076-8 · Source: PubMed CITATIONS 14 READS 188 3 authors: Some of the authors of this publication are also working on these related projects: Shhh hearing in a farming environment View project Farming Fit View project Jacquie Cotton Deakin University 11 PUBLICATIONS 56 CITATIONS SEE PROFILE Paul Lewandowski Deakin University 56 PUBLICATIONS 1,223 CITATIONS SEE PROFILE Susan Brumby Deakin University 97 PUBLICATIONS 708 CITATIONS SEE PROFILE All content following this page was uploaded by Susan Brumby on 19 August 2015. The user has requested enhancement of the downloaded file.
- 2. STUDY PROTOCOL Open Access Cholinesterase Research Outreach Project (CROP): measuring cholinesterase activity and pesticide use in an agricultural community Jacqueline Cotton1,2* , Paul Lewandowski2 and Susan Brumby1,2 Abstract Background: Australian farmers and their workers are exposed to a wide variety of pesticides. Organophosphate (OP) insecticides are a widely used class of pesticide used for animal husbandry practices (Naphthalophos for sheep dipping, jetting and drench), crop production for pest control (Dimethoate) and in public health (Maldison for head lice). Acute poisonings with this class of insecticide are reported among agricultural workers and children around the globe, due to the inhibition of acetylcholinesterase (AChE). Less is known about chronic exposures. Regular monitoring of erythrocyte AChE will enable farmers to identify potential exposure to organophosphate insecticides and take action to reduce exposures and improve their health and safety practices. This study aims to assess and improve the integration of AChE monitoring into routine point of care health clinics, and provide farming and non-farming people with a link between their AChE activity and their household chemical and agrichemical use. Methods/Design: The research will target individuals who work on mixed farming enterprises and routinely using OPs (n = 50) and non-farmers (n = 30). Baseline data are collected regarding demographic, health conditions and behaviours, Kessler 10 (K10) scores, chemical use and personal protection. Baseline anthropometric measures include height, weight, hip and waist circumference, body fat analysis and, biochemical analysis of fasted total serum cholesterol, triglycerides, low-density cholesterol (LDL), high-density cholesterol (HDL) and blood glucose. Analysis of erythrocyte cholinesterase (EAChE) activity is also conducted using a finger prick test. Testing of EAChE is then repeated in all participants every 3 weeks for a maximum of three times over a period 10 weeks. Participants are provided with full feedback and counselling about their EAChE activity after each reading and a detailed summary provided to all participants at the completion of the study. Data will be analysed using repeated measures within a general linear model. Discussion: This work will provide an evidence base and recommendations for the integration of EAChE monitoring into Australian rural health clinics, leading to research which will further quantify pesticide exposure both on the farm and in the home, highlighting the importance of sustaining and providing a safe work and home environment for farming communities. Trial registration: ACTRN12613001256763 Keywords: Farmer, Organophosphate, Pesticides, Safety, Chemical, Acetylcholinesterase * Correspondence: [email protected] 1 National Centre for Farmer Health, Western District Health Service, PO Box 823, Hamilton, Vic 3300, Australia 2 School of Medicine, Deakin University, 75 Pigdons Road, Waurn Ponds, Vic 3216, Australia © 2015 Cotton et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cotton et al. BMC Public Health (2015) 15:748 DOI 10.1186/s12889-015-2076-8
- 3. Background Australian farmers and their workers are exposed to a wide variety of pesticides [1]. Pesticides are substances that destroy, repel or attack pests that have a negative ef- fect on productivity and profitability of a farming enter- prise. Pesticide groups include herbicides, insecticides, fungicides and rodenticides. Organophosphate (OP) insec- ticides are widely used for animal husbandry practices (Naphthalophos sheep drench), public health (head lice, locust control), and in crop production (Dimethoate for insect control). The OP class of insecticides effect the nervous system with poisonings most common among agricultural workers and children [2, 3]. OPs are regularly used as a form of pest control on most farms and farmers have enhanced their understanding of pesticide use and handling. However, many are still not meeting the full range of personal protection standards in their own handling and application [4]. Routes of human exposure include dermal absorption, inhalation and ingestion [5]. Symptoms of OP toxicity are caused by inhibition of the enzyme acetylcholinesterase (AChE). Inhibition of AChE results in the subsequent accumulation of acetylcholine at the cholinergic synapses of nerves causing uncontrolled firing of the synapse [1, 6]. Subclinical effects of chronic OP exposure such as AChE inhibition may be detected early by biological tests. Recovery from AChE depression is prolonged, due to the irreversible binding of OPs to the red blood cell and its subsequent 120 day life cycle [7]. Monitoring exposure to organophosphates involves the measurement of peripheral cholinesterase enzymes that are inhibited by organophosphates. These include; erythro- cyte cholinesterase (EAChE) and serum cholinesterase (SAChE). Organophosphates have been associated with chronic neurological disease such as impaired memory, impaired fine motor skills control and Parkinson’s disease [8]. The evidence suggests that whilst an acute episode experience may not have occurred, exposure over a long period of time (such as in workers dipping sheep) may re- sult in neurological conditions such as Parkinson’s disease and neuropsychiatric conditions [6, 8, 9]. The window for exposure to toxicants may occur years before the onset of neurological symptoms [8, 10, 11], this is particularly important for sheep and crop producers exposed to long- term, low levels of OPs. The Western region of the state of Victoria in Australia has been a centre of sheep production, since settlement, and a large number of farmers have been exposed to OPs through routine sheep dipping (lice con- trol), drenching (control internal parasites), jetting (blow fly control) and spraying of pastures (insect control). The National Centre for Farmer Health (NCFH) is in a unique position to monitor these farmers and workers, and work with farmers to incorporate cholinesterase testing as part of their assessments during peak periods and provide much needed data for them on exposures in this area. Additional practices such as mixing contaminated laundry and storing pesticides in the home are not only common causes of exposure to the farmer and farm worker, but may also be placing other household members at risk, particularly children [12]. Work completed by the program Sustainable Farm Families™ [13] has identified that there is strong interest from farm men and women who wish to investigate exposures and possible health impacts of agricultural chemical use. The acute effect of OP exposure is well documented in Australia and overseas for humans, pests and animals [3, 6, 9]. However, the extent to which asymptomatic monitoring is taking place is not well known or docu- mented. This research builds on work conducted by the NCFH. It utilises the data collection methods of the Sustainable Farm Families™ which includes health behav- iours and conditions, with the expectation that this monitoring EAChE testing will be integrated into this procedure, providing the springboard to break through barriers that are currently inhibiting best practice chem- ical handling safety in Australia. Understanding the mode of action of a chemical is a critical component of the risk assessment process. Cholinesterase activity is a direct link to exposure and this pilot study will produce valuable data on possible exposures for farmers routinely using OP’s for animal husbandry or crop production. Monitoring cholinesterase levels during agricultural health screening and assessment is a key objective of Agrisafe™ clinics in Australia. However, its incorporation into such clinics will depend on health professional’s skills and knowledge. This study will further prepare health care providers for integration of correct cholinesterase testing procedures into point of care (POC) health checks. This work will further highlight pesticide exposure as a risk for farmers, workers and their families, leading to an easily adopted method that will quantify the environmental exposures providing early detection. Research has also shown links between cholinesterase activity and vascular complications in diabetic patients [14]. Diabetes is a disease also of increasing concern to the farming and rural population in Australia [15]. Aims The Cholinesterase Research Outreach Project (CROP) will use clinical sampling to determine the level of expos- ure associated with the routine use of OPs by Victorian farmers and/or their workers. This preliminary study will examine the methodology of testing for EAChE activity at POC and provide evidence for future work by identifying the exposure patterns and pathways of individuals most at risk in farming communities. It is anticipated that CROP will inform future research studying pesticide exposure in farming communities. Cotton et al. BMC Public Health (2015) 15:748 Page 2 of 6
- 4. Methods/design Evaluation design To evaluate if a difference in cholinesterase activity was present in farmers before and after exposure, sample size is determined at the power of 80 % (β = 0.2) with α set at 0.05 for an effect size of one standard deviation. This requires the sample size to be a minimum of 30 farming individuals exposed to pesticides. By considering the probable retention rate (≥85 %) for a 7-month study, based on previous research in similar populations, [16] the target recruitment number is fixed at a target of 50 farmers. Sample size and inclusions Consent will be obtained from 80 participants recruited via existing contacts and industry groups, letterbox drops and newspaper articles from a number of farming and community groups, specifically; Mixed farming enterprises and sheep dipping/spraying contractors (n = 50) who are using OPs and a conveni- ence group of non-farming individuals (n = 30) from the rural community of Hamilton, Australia. Participants need to have been farming for more than 5 years, be aged between 18 and 75 years, speak English, and not had a previous known chemical incident. Ques- tionnaires will be used to determine whether or not they are the primary pesticide user. Any non-farming individ- uals tested must also be aged between 18 and 75 and have not had a previous chemical incident. Participants from both groups will be self-reported not pregnant, not suffering from a known chronic disorder, not taking anti-inflammatory drugs or supplements and not exer- cising excessively throughout the study. Anthropometry The simplicity of anthropometry allows it to be used in population-based studies to assess body-composition changes over time, as well as in clinical and field situa- tions where access to technology is limited such as in this study. Height, weight, waist and hip circumference has previously been included as part of an initial POC assessment for farm men and women and agricultural workers. Participants have found these measurements helpful and easy to understand and also cite having a free health assessment as a reason to attend [13]. Add- itionally, (whilst not the focus of this study) if used in combination with blood biochemistry and body fat per- centage, anthropometry can identify distinct fat distribu- tion changes that occur in the elderly [17]. Weight is measured to the nearest 0.05 kg using electronic scales taken in light clothing with shoes removed and pockets emptied and prior to breakfast. Height is measured using a portable stadiometer to the nearest 0.1 cm with shoes removed and weight distribute evenly on both feet. Physical assessments and health condition data A short health history and health behaviours (smoking and alcohol consumption habits) will be obtained from participants with baseline physical assessments of blood pressure measurement (repeated twice), pulse rate, body fat percentage (bioelectrical impedance), respiratory function test (PiKo meter) also measured (see Table 1). Table 1 Data collection description and timeline for farmer and non-farmer groups Variable Baseline (T0) Visit 1 (T1, 1 mth) Visit 2 (T2, 2 mth) Visit 3 (T3, 3 mth) Body weight ✓ X X X Height ✓ X X X Waist circumference ✓ X X X Hip circumference ✓ X X X Body fat % ✓ X X X Blood pressure ✓ X X X Pulse rate ✓ X X X Fasting blood glucose ✓ X X X Fasting blood cholesterol ✓ X X X Fasting triglycerides ✓ X X X HDL cholesterol ✓ X X X LDL cholesterol ✓ X X X Erythrocyte Cholinesterase (EAChE) ✓ ✓ ✓ ✓ Description of monthly chemical use ✓ ✓ ✓ ✓ Kessler (K10) ✓ X X X Chemical Usage Survey ✓ X X X Cotton et al. BMC Public Health (2015) 15:748 Page 3 of 6
- 5. Biochemical analysis of EAChE activity using a 10 μl capillary blood sample (measured by Test-mate ChE Cholinesterase tester system Model 400), and EAChE field Assay kit - to establish a baseline reading, will be used (Fig. 1). The Test-mate field assay testing system is based on the Ellman method [18]. Blood glucose and lipids (LDL, HDL) and total cholesterol is also taken after 10-hour fast using the same blood sample. Waist circumference is measured to the nearest 0.1 cm at the end of a normal expiration using a con- stant tension “Figure Finder Tape Measure”™ [19]. Body Mass Index will be calculated to classify obesity and overweight cut off points for future reference using the WHO parameters [20]. Survey methodology Trained research staff will collect all anthropometric and behavioural data. Sociodemographic data are collected directly from the participant including age, gender, country of origin, using the Victorian Department of Health service coordination tools (SCOT) [21]. The type of farming undertaken and residential postcode will also be collected from the participants. A validated questionnaire, the Kessler 10 (K10) will be used to measure psychological distress. The K10 is a 10-item questionnaire intended to yield a global meas- ure of distress based on questions about anxiety and depressive symptoms that a person has experienced in the most recent 4 week period [22]. This survey has been used extensively in Australia and provides a comparison to the general population. Given the relationship between organophosphates, neuropsychological and psychiatric functioning a baseline was deemed important [23, 24]. It also forms part of the usual farmer health assessment. Occurrence of illness & injury Occurrence of illness or injury experienced within the last 3 months will be measured using a questionnaire de- scribing 33 symptoms of chemical exposure, in random order of severity. The illness and injury data will be standardised and analysed accordingly. Monitoring agrichemical use In addition to completion of the agrichemical use survey of 13 questions, self-reported chemical use is recorded during each visit. Participants are provided with counsel- ling regarding their EAChE levels in accordance with AgriSafe guidelines (Fig. 2). The USA’s Department of Pesticide Regulation specifies that a drop to 70 % or lower in red blood cell AChE or to 60 % or lower in plasma cholinesterase relative to the individual’s baseline is an indication for immediate removal of the individual from all exposure to organophosphate and carbamate pesticides [25]. Participants will be provided with a sum- mary of their EAChE baseline and subsequent results at the conclusion of the study. This methodology reflects recent exposures and is used to provide feedback at the time of testing. However, it is noted that the level of current exposure is not a measure of long-term exposure Fig. 1 Point of care schedule for cholinesterase assessment Cotton et al. BMC Public Health (2015) 15:748 Page 4 of 6
- 6. and that the clinical parameters around long-term exposure are not well defined. Data entry, handling and statistical analysis All data will be managed and analysed within the statis- tical program SPSS (IBM Corp. Released 2012 IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY). SPSS is a statistical package widely used in quanti- tative social science research and is suitable for multi- variate analysis. Data will be analysed using repeated measures (subjects as own controls) within the General Linear Model. Correlation coefficients between pesti- cide exposure and metabolic variables will be calculated using Pearson’s and Spearman’s correlation as appropri- ate. P values will be considered statistically significant at p < 0.05. Consent and ethics All adults participating in the study are to be provided with a plain language statement and will provide in- formed written consent. Ethics approval has been granted to the project by Deakin University Human Research Ethics Committee (HREC 2013-100 dated 18/06/2013). All researchers involved in data collection have a Victorian Police check undertaken. Discussion Organophosphates are still used widely with the actions and attitudes of farmers and workers using these pesti- cides is vital to their wellbeing and that of their families. It is common for mild to chronic depression of EAChE activity to be reported as ‘normal’ due to the wide refer- ence range for EAChE activity [26]. It is important that farmers and agricultural workers who are routinely using OPs establish their baseline EAChE activity and have access to regular EAChE activity checks for comparison with baseline. Routine monitoring of EAChE may allow for early recognition of frequent and continuous low-level exposure to OPs. This work will provide an evidence base, leading to research to further quantify pesticide exposure both on the farm and in the home of farming families and highlight the importance of sustain- ing and providing a safe work environment for farming communities. Abbreviations AChE: Acetylcholinesterase; EAChE: Erythrocyte Acetylcholinesterase; CROP: Cholinesterase Research Outreach Project; OP: Organophosphate; K10: Kessler 10; LDL: Low Density Lipoproteins; HDL: High Density Lipoproteins; NCFH: National Centre for Farmer Health; POC: Point of Care; SCOT: Service Coordination Tools; BMI: Body Mass Index; mth: Month. Competing interests The authors declare that they have no competing interests. Authors’ contributions SB was an initiator of the original Sustainable Farm Families program, contributed to the design of the study, and specific health assessment protocols. JC, SB and PAL participated in the design of the study and drafting of the manuscript. All authors read and approved the final manuscript Acknowledgements The work is supported by a Faculty Research Grant provided by Deakin University Faculty of Health. The authors would like to also acknowledge the support of Western District Health Service. Received: 11 February 2014 Accepted: 21 July 2015 References 1. Fragar L, Franklin R. The health and safety of Australia’s farming community. Moree: ACAHS & RIRDC; 2000. 2. Kwong TC. Organophoshate Pesticides: Biochemistry and clinical toxicology. Ther Drug Monit. 2002;21(1):144–9. 3. O’Malley M. Clinical evaluation of pesticide exposure and poisonings. Lancet. 1997;349(9059):1161. 4. Radcliffe J. Pesticide use in Australia. Parkville: Australian Acadamy of Technological Sciences and Engineering; 2002. 5. Jones A, Gray B, Schaefer M. Chemcert AgVet Chemical Users Course Resource Manual. Warragul: ChemCert Australia (Vic) Incorporated; 2011. 6. Banks CN, Lein PJ. A review of experimental evidence linking neurotoxic organophosphous compounds and inflammation. Neurotoxicology. 2012;33(3):575–84. 7. Quandt SA, Chen H, Grzywarcz JG, Vallejos QM, Galvan L, Arcury TA. Cholinesterase depression and its association with pesticide exposure across Fig. 2 Guide to severity of acute poisoning with anti-cholinesterase agents (Source: Modified from California Environmental Protection Agency 2002) Cotton et al. BMC Public Health (2015) 15:748 Page 5 of 6
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