Miljø- og Fødevareudvalget 2015-16
MOF Alm.del Bilag 509
Offentligt
1645441_0001.png
Organophosphate
metabolites in urine
samples from
Danish children
and women
Measured in the Danish DEMOCOPHES
population
[Serietitel og årstal]
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0002.png
Title:
Organophosphate metabolites in urine samples
from Danish children and women
-
Measured in the Danish
DEMOCOPHES population
Edited by:
Thit Aarøe Mørck
1
Helle Raun Andersen
2
Lisbeth E. Knudsen
1
1
University
2
University
of Copenhagen
of Southern Denmark
Published by:
The Danish Environmental Agency
Strandgade 29
1401 København K
www.mst.dk
Year:
2015
ISBN nr.
[xxxxxx]
Disclaimer:
When the occasion arises, the Danish Environmental Protection Agency will publish reports and papers concerning
research and
development projects within the environmental sector, financed by study grants provided by the Danish
Environmental Protection Agency. It should be noted that such publications do not necessarily reflect the position or
opinion of the Danish Environmental
Protection Agency.
However, publication does indicate that, in the opinion of the Danish Environmental Protection Agency, the content
represents an important contribution to the debate surrounding Danish environmental policy.
Sources must be acknowlegded.
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
Contents
Foreword .................................................................................................................. 4
Abbreviations ............................................................................................................ 5
Summary and conclusion .......................................................................................... 6
Resume og konklusion ............................................................................................... 8
1.
2.
Introduction ..................................................................................................... 10
Methods ............................................................................................................ 11
2.1 Recruitment of study participants ....................................................................................... 11
2.2 Urine samples – dialkylphosphate (DAP) analysis ............................................................. 11
2.3 Hair and blood samples ........................................................................................................ 13
2.4 Questionnaires ...................................................................................................................... 13
2.5 Statistical analysis ................................................................................................................. 13
Results ............................................................................................................. 14
3.1 Dialkylphosphate concentrations ......................................................................................... 14
3.2 DAP concentration in relation to age ................................................................................... 16
3.3 Impact of Socioeconomic status ...........................................................................................18
3.4 Food frequency questionnaire ..............................................................................................18
3.5 Urban and rural differences ................................................................................................. 21
Discussion ........................................................................................................ 23
Conclusion ....................................................................................................... 28
3.
4.
5.
Referencer .............................................................................................................. 28
6.
References ....................................................................................................... 30
Organophosphate metabolites in urine samples from Danish children and women
3
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
Foreword
The project ”DAP metabolites in Danish Children and Women” was conducted in 2014-2015 as part
of the project “Pesticide Measurements in urine - establishment of analysis method and analysis of
urine samples from Danish mothers and children collected as part of DEMOCHOPHES ” financed
by the Danish Environmental Agency.
This report describes the concentrations of six dialkylphosphate metabolites (DAPs) used as
biomarkers for organophosphate exposure. The DAPs have been measured in Danish school
children and mothers participating in the European project DEMOCOPHES co-funded by the
LIFE+ programme of EU (GD environment – LIFE09 ENV/BE/000410) and the Danish
Environmental Agency, the Danish Veterinary and Food Administration and the Danish Health and
Medicines Authority.
The project is a collaboration between Environmental Medicine, Institute of Public Health at
University of Southern Denmark and section of Environmental Health at the Institute of Public
Health, University of Copenhagen. The samples were analyses by the Flemish Institute for
Technological Research NV (VITO) in Belgium in collaboration with professor Greet Schoeters.
We would like to thank the families participating in DEMOCOPHES and the collaborating schools
taking part in the recruiting process.
4
Organophosphate metabolites in urine samples from Danish children and women
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
Abbreviations
C, creatinine
COPHES, Consortium to perform human biomonitoring on a European scale
DAP, dialkyl phosphate
DEAP, diethyl alkylphosphates
DEDTP, Diethyl dithiophosphate
DEMOCOPHES, Demonstration of a study to coordinate and perform human biomonitoring on a
European scale
DEP, Diethyl phosphate
DETP, Diethyl thiophosphate
DMAP, dimethyl alkylphosphates
DMDTP, Dimethyl dithiophosphate
DMP, Dimethyl phosphate
DMTP, Dimethyl thiophosphate
GM, geometric mean
LOD, limit of detection
NAAP, paracetamol
NHANES, National Health and Nutrition Examination Survey (US)
OP, organophosphate
PFBBr, pentafluorobenzylchloride
POPs, Persistent organic pollutants
SES, socioeconomic status
2.4-DCP, 2,4-dichlorophenol
2.5-DCP, 2,5-dichlorophenol
2-PP, 2-phenylphenol.
Organophosphate metabolites in urine samples from Danish children and women
5
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
Summary and conclusion
Background
The use of organophosphate insecticides (OPs) in agriculture and as biocides in Denmark is very
restricted due to their relatively high acute toxicity and neurotoxic effects. However, the population
might still be exposed by ingestion of imported food items. Exposure to organophosphates can be
estimated by measurements of dialkylphosphate (DAP) metabolites and in studies from the US
urinary concentrations of DAPs have been associated with adverse neurobehavioural outcomes.
Children with increased OP exposure may have higher risk of ADHD, and prenatal OP exposure was
reported to be associated with structural brain anomalies and neurobehavioral deficits at school
age. Urinary concentrations of DAP metabolites have only been measured in the Danish population
in one study previously.
Objectives
The objectives of the present study was to investigate the urinary concentration of six unspecific
DAP metabolites, which are commonly used as biomarkers for organophosphate exposure to
pesticides such as dichlorvos, fenthion, dimethoat, malathion and chlorpyrifos and to compare the
found concentrations with one previous Danish study and concentrations measured in other
countries.
Methods
The urinary concentrations of Dimethyl phosphate (DMP), Dimethyl thiophosphate (DMTP),
Dimethyl dithiophosphate (DMDTP), Diethyl phosphate (DEP), Diethyl thiophosphate (DETP),
Diethyl dithiophosphate (DEDTP) were analyzed in 144 Danish school children and 145 mothers.
The study persons were part of a large EU pilot project called DEMOCOPHES (Demonstration of a
study to coordinate and perform human biomonitoring on a European scale), with focus on
harmonization of human biomonitoring in Europe and which was ongoing in Europe from 2010 to
2012. In Denmark mother child pairs were recruited from an urban and a rural area and urine, hair
and blood samples were collected from September to December 2011 The urine samples from the
biobank of DEMOCOPHES in Denmark were used for the DAP analyses in the present study.
Results
At least one of the six DAP metabolites was detected in more than 90%, and four metabolites was
detected in more than 30%, of both children and mothers. There was a tendency of higher DAP
concentrations in children compared to their mothers. Furthermore, there was a tendency of higher
concentrations in younger mothers and in children from families with higher socioeconomic status.
The exposure source of the organophosphates in this study was difficult to determine as the study
was not initially designed for this purpose. DAP concentrations were generally lower in participants
from the rural compared to the urban area and the concentrations of the methylated DAPs were
lower in children who often were eating homegrown fruit and vegetables, though only statistically
significant for DMP. The levels of total DAP and DMAPs were lower in the investigated population
than in Danish children investigated in 2007-08 indicating a decline in the total OP exposure level.
However, the concentration of DEAPs was not similarly reduced. In general, the DAP levels were
comparable with concentrations measured in other European countries in recent years, but the
levels of total DAPs and DEAPs were higher than levels found in the US.
The concentration of the individual DAP metabolites were significantly correlated with each other
in both mothers and children and in the mothers they were also significantly correlated with other
6
Organophosphate metabolites in urine samples from Danish children and women
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
chemicals associated with pesticide exposure (2.4-DCP, 2.5-DCP and 2-PP) measured in the same
urine sample residues.
Conclusion
The findings of relatively high detection frequency of the DAP metabolites DEP, DETP, DMP and
DMTP in a Danish group of children and their mothers, clearly indicate that there is still a
widespread exposure to organophosphate pesticides in the Danish population, even though there
have been major restrictions on their use in agriculture. The concentrations of the metabolites DEP
and DETP found in the children and women of the present study are in line with what was found in
Europe and previous measurements in Denmark. The methylated metabolites seem to have
decreased in Denmark compared to previous measurements in 2007-08 and levels found in other
European countries before 2007. However, the levels of total DAPs and DEAPs in the present study,
as well as the levels found in other European countries, are higher than concentrations found in the
biomonitoring program NHANES in the US, indicating higher exposure to some organophosphates
in Europe. As the exposure to OPs has been associated with adverse health effects in some studies
from the US population, there may also be a risk of adverse effects in Europe and Denmark, as we
have found even higher levels. Although we do not know the specific organophosphates responsible
for the relatively high concentrations of DEAPs in Denmark, chlorpyrifos is likely to be an
important contributor since it is often found in samples of imported fruit and vegetables, which is
considered the main exposure source for OPs in Denmark. Thus, further studies of potential adverse
health effects related to organophosphate exposure in European populations are needed. Also
identification of the main dietary exposure sources and related OPs is warranted in order to
introduce adequate measures to reduce the exposure - especially for vulnerable population groups
as children and pregnant women.
Organophosphate metabolites in urine samples from Danish children and women
7
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
Resume og konklusion
Baggrund
Brugen af organofosfat-insekticider i landbruget og som biocider i Danmark er meget restriktiv på
grund af deres relativt høje akutte toksicitet og neurotoksiske effekter. Den danske befolkning kan
dog stadig være eksponeret for organofosfater via indtagelse af importerede fødevarer. Man kan
estimere befolkningens udsættelse for organofosfater ved at måle dialkylfosfat (DAP) metabolitter i
urinen. I undersøgelser fra USA er der fundet sammenhæng mellem urinkoncentrationen af DAP og
negativ påvirkning af nervesystemets udvikling. Børn med højere organofosfateksponering synes at
have øget risiko for at udvikle ADHD, og prænatal organofosfateksponering er fundet associeret
med strukturelle ændringer i hjernen og adfærdsmæssige vanskeligheder i skolealderen.
Koncentrationen af DAP-metabolitter er kun blevet målt i ét studie tidligere i Danmark.
Formål
Formålet med dette studie var at måle urinkoncentrationen af seks uspecifikke DAP-metabolitter,
som bliver brugt som biomarkører for eksponering for organofosfat-insekticider som dichlorvos,
fenthion, dimethoat, malathion and chlorpyrifos. Formålet var desuden at sammenligne de målte
koncentrationer med koncentrationer fra et tidligere studie lavet i Danmark, samt koncentrationer
målt i andre lande.
Metoder
Urin koncentrationen af Dimethyl phosphate (DMP), Dimethyl thiophosphate (DMTP), Dimethyl
dithiophosphate (DMDTP), Diethyl phosphate (DEP), Diethyl thiophosphate (DETP og Diethyl
dithiophosphate (DEDTP) blev analyseret i 144 danske skolebørn og 145 mødre. Forsøgspersonerne
var en del af et større EU pilotprojekt omhandlende harmonisering af human biomonitering i
Europa kaldet DEMOCOPHES (Demonstration of a study to coordinate and perform human
biomonitoring on a European scale). Projektet kørte i Europa fra 2010 til 2012. Mor-barn par blev
rekrutteret i Danmark i to områder, som henholdsvis repræsenterede et urbant og et ruralt område.
Urin, hår og blodprøver blev indsamlet mellem september og december 2011. Urinprøver fra den
indsamlede biobank fra den danske del af DEMOCOPHES blev brugt til analyse af DAP
metabolitterne i dette studie.
Resultater
Mindst en af de seks DAP metabolitter kunne måles i mere end 90% af forsøgspersonerne og fire af
metabolitterne blev detekteret i mere end 30 % af både børn og mødre. Der var en tendens til højere
DAP-koncentrationer i børnene sammenlignet med deres mødre. Desuden var der en tendens til
højere koncentrationer i de yngste grupper af børn og mødre, samt i børn fra familier med højere
socioøkonomisk status. Kilder til eksponeringen for organofosfater var svær at identificere ud fra
dette studie, eftersom DEMOCOPHES projektet ikke initialt var designet til at undersøge
udsættelsen for disse stoffer. DAP-koncentrationerne var generelt højere i deltagere fra byområdet
sammenlignet med deltagere fra det mere landlige område og koncentrationen af de methylerede
DAP’er (DMAPs) var højere i børn, der ikke spiste hjemmedyrkede fødevarer særlig ofte, dette var
dog kun signifikant for DMP. Det samlede niveau af DAP-metabolitter og DMAP-metabolitter var
lavere i dette studie sammenlignet med danske børn undersøgt i 2007-08, hvilket indikerer at der
er sket et samlet fald i organofosfat-eksponeringen i Danmark. Koncentrationen af de ethylerede
DAP’er (DEAPs) ser dog ikke ud til at være reduceret. Generelt var DAP koncentrationerne
sammenlignelige med målinger fra andre Europæiske lande, men det samlede niveau af alle DAP-
8
Organophosphate metabolites in urine samples from Danish children and women
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
metabolitter og DEAP-metabolitter var højere end koncentrationer fundet i USA i
biomonitoreringsprogrammet NHANES. Koncentrationerne af de individuelle DAP metabolitter var
signifikant korreleret med hinanden i både børn og mødre, og de var desuden korreleret med
koncentrationerne af andre kemikalier, som også er associeret med pesticid eksponering (2,4-DCP,
2,5-DCP and 2-PP), som er målt tidligere i samme urinprøver.
Konklusion
Den relative høje detektionsfrekvens af DAP metabolitterne DEP, DETP, DMP and DMTP i danske
børn og deres mødre, er en tydelig indikation på at der stadig i Danmark er en væsentlig udsættelse
for organofosfat-pesticider på trods af nationale restriktioner på området.
De koncentrationer vi har målt i børn og mødre er generelt sammenlignelige med niveauer målt
tidligere i Danmark samt målinger i andre Europæiske lande. Koncentrationen af de methylerede
metabolitter ser dog ud til at være faldet siden målinger foretaget i Danmark i 2007-08 samt
Europæiske målinger foretaget før 2007. Det totale DAP niveau samt niveauet af DEAP i dette
studie, såvel som andre Europæiske studier, er højere end koncentrationer målt i det amerikanske
biomoniteringsprogram NHANES, hvilket indikerer at eksponeringen for nogle organofosfater er
højere i Europa end i USA. Eftersom organofosfat-eksponering i USA er fundet associeret med
negative helbredseffekter, er der en risiko for, at vi i Europa og Danmark også kan have en forhøjet
risiko for negative effekter ved det eksisterende eksponeringsniveau. Selvom vi ikke ved hvilke
specifikke organofosfater, der er ansvarlige for den relativt høje koncentration af ethylerede
metabolitter i Danmark, er det sandsynligt at chlorpyrifos bidrager væsentligt, da det er det
organofosfat, som oftest måles i prøver af importeret frugt og grønt, som antages at være den
vigtigste eksponeringskilde for organofosfater i Danmark. Der er derfor behov for flere studier af
potentielle negative helbredseffekter forbundet med det nuværende eksponeringsniveau for
organofosfater i Europa. Desuden er de vigtigste eksponeringskilder i kosten og hvilke
organofosfater de bidrager med vigtigt at få undersøgt således at nødvendige foranstaltninger kan
foretages for at reducere eksponeringen, - især i særligt sårbare grupper som børn og gravide.
Organophosphate metabolites in urine samples from Danish children and women
9
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1. Introduction
Organophosphates (OPs) are a group of insecticides widely used in agriculture and as biocides
throughout the world. Because of their relatively high acute toxicity and neurotoxic properties,
more bans or restrictions of use have been implemented in Europe and the US in the recent years
accompanied of declining use. In Denmark it has only been legal to use few OPs after 2010.
Dimethoate was banned in August 2013 and azamethiphos is still allowed in certain products
(plates and cans) against ants and flies and is, to our knowledge, the only OP still in use in DK.
Despite these reductions, residues of organophosphates are among the most frequently detected
pesticides in imported food items on the Danish marked (Fødevareinstituttet 2014) and this is likely
an important source of exposure for the Danish population. An important tool in the investigation
of exposure to chemical substances is human biomonitoring, where the actual concentrations of the
chemical of interest, or its metabolites, can be measured in suitable matrices. For OPs, the urinary
concentration of specific or unspecific common metabolites, have often been used. In previous
studies form different countries, metabolites of organophosphates have been detectable in urine
samples from 70 - 90 % of the populations (Barr et al. 2004; Heudorf et al. 2004) with a tendency
to decreasing levels in the most recent studies (Clune et al. 2012). The exposure level in the Danish
population has only been measured in one previous study (Andersen et al. 2012) which
demonstrated widespread exposure among school children in samples collected between 2007 and
2008. The urinary concentrations seemed to be higher than in samples collected in the same period
among children of similar age in the US (Centers for Disease Control and Prevention 2009).
In 2011 the European pilot project DEMOCOPHES was conducted in 17 European project countries,
with the purpose to exploit the possibility of a harmonized human biomonitoring setup across
Europe. In all countries morning urine and hair samples were collected from school children and
their mothers in two areas, representing an urban and a rural area, respectively. In Denmark, a
blood sample was also collected from the majority of the participants. The urine samples were
analyzed for phthalates, cadmium and cotinine, and the hair samples was analyzed for mercury. In
Denmark a biobank was set up and additional biomarkers were analyzed. The blood was analyzed
for persistent organic pollutants (POPs) and the urine was further analyzed for parabens, phenols
and paracetamol. In the present study, the urine samples were analyzed for six unspecific DAP
metabolites that are common metabolites for multiple OPs. Since OPs, like other pesticides used
nowadays, are metabolized and excreted within few days, measurement of these unspecific common
organophosphate metabolites are more useful to estimate the total OP exposure level in a
population than measurements of specific metabolites of individual organophosphates. This
approach has been used in several other studies which allow comparison with levels found in other
countries.
Thus, the aim of this project was to investigate the urinary concentration of six unspecific DAP
metabolites, which are commonly used as biomarkers for OPs such as dichlorvos, fenthion,
dimethoat, malathion and chlorpyrifos and to compare the levels with one previous Danish study
and concentrations measured in other countries.
10
Organophosphate metabolites in urine samples from Danish children and women
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
2. Methods
2.1
Recruitment of study participants
The study persons of the present study was the Danish participation in the EU pilot project
DEMOCOPHES/COPHES (Consortium to perform human biomonitoring on a European scale) and
the recruitment was therefore performed in compliance with the COPHES protocol (Becker et al.
2014; Joas et al. 2012; Mørck et al. 2015a). In Denmark, the recruitment was done via local schools
in two selected areas, representing urban and rural communities, respectively. An invitation to
participate in the project was sent out to all parents of children in the age-group of 6 to 11 years of
age and the project was also presented at parent meetings. Mothers could sign up for participation
and book an appointment for sampling on our project webpage, where they also could find
additional information about DEMOCOPHES. To reach the required number of participants the
sampling was expanded to include additional schools in each area and articles in local newspapers
were used to increase local attention to the project. The two areas of recruitment in Denmark were
Gentofte (urban area), and Viby Sjælland (rural area). The areas were selected according to
population density, where < 150 inhabitants/km2 was defined as rural. The following inclusion
criteria were used: the child should be living with the mother a minimum of 16 days a month, the
child and mother should have lived in the area for a minimum of 5 years, have sufficient Danish
language knowledge and have normal kidney function and no metabolic disturbances. The goal of
DEMOCOPHES was to reach 120 mother-child couples. In Denmark, 75 mother-child couples from
the urban area and 70 couples from the rural area were recruited, resulting in 145 mother-child
pairs in total. The children were equally distributed in age, gender and urban/rural location. All
participants received written information about the study and gave informed consent before
participating (for the child, all holders of custody should sign). Each mother-child pair received a
voucher for two cinema tickets as a reward for their participation. The study was approved by the
local regional ethics committee (H-3-2011-075 and H-1-2014-004) and the Danish data protection
agency (2011-41-6607 and 2011-41-6766).
On the day of sampling, the completed questionnaire was handed in along with first morning urine
samples. At the appointment hair samples were taken, and blood was drawn from the cubital vein.
Mother-child pairs participating in the supplementary study on pain and self-medication were
interviewed at the end of the visit. Sampling was conducted in parallel in the urban and the rural
area from September to December 2011 to minimize seasonal variation.
2.2
Urine samples – dialkylphosphate (DAP) analysis
Urine was collected in 750 mL polyethylene containers, which were delivered to the participant’s
home the day prior to their appointment. The containers were prewashed in 10% nitric acid (>3
hours) and rinsed twice in purified water. The participants collected the total volume of the first
morning urine void on the day of their appointment. To avoid decomposition of samples these were
kept as cold as possible in the refrigerator or outdoors at the participants’ home until the
appointment. After collection of the samples, the urine was stored in a cooling box (4
o
C). At the
laboratory the filled containers were weighed and 2 mL urine were transferred to 4 mL glass tubes
with screw caps packed with aluminum foil and stored at -20
o
C until further analyses.
At the Flemish Institute for Technological Research NV (“VITO”) in Belgium, the urine samples
were analyzed for six unspecific OP metabolites. The analyzed metabolites were:
Dimethyl phosphate (DMP), Dimethyl thiophosphate (DMTP), Dimethyl dithiophosphate
(DMDTP), Diethyl phosphate (DEP), Diethyl thiophosphate (DETP), Diethyl dithiophosphate
Organophosphate metabolites in urine samples from Danish children and women
11
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0012.png
(DEDTP). The urine concentration of these metabolites was determined with a GC/MS method that
was developed and validated in-house at VITO. For a brief description of the method, urine samples
were thawed and isotope-labeled internal standards were added to the samples. The samples were
acidified with HCl and liquid/liquid extracted twice with a mixture of diethylether and acetonitrile
(1:1). The organic layer was evaporated till dryness and the residue was dissolved in dehydrated
acetonitrile. The OP metabolites were derivatized with pentafluorobenzylchloride (PFBBr). Hexane
and water were added and the metabolites were transferred to the hexane fraction by shaking the
tubes. The hexane fraction was concentrated and analyzed with GC/MS in SIM mode as follows:
The alkyl phosphates are quantified with the internal standard method using the characteristic ions
of the native and isotope labeled internal standard.
Samples were analyzed in sequences of 20 samples. Every sequence contains the necessary quality
control samples: a procedural blank, a matrix spike (addition to one urine sample in the sequence),
analysis in duplo (repetition of the analysis of one of the samples in the sequence) and calculation of
the recovery of the isotopically labeled internal standards in each sample. The measured
metabolites are listed in table 1 below, where examples of mother compounds and limit of detection
(LOD) are also shown.
DAPs are stable in urine under storage at -20
C
(Tarbah et al. 2004). Six urine samples collected
in 2007-08 and analyzed at Center of Disease Control in the US were re-analyzed at the VITO-
laboratory in Belgium immediately before the analysis of the samples in this study and the results
were very similar. This indicates that storage for up to six years at -20
C
does not affect the
concentration. It also shows that results obtained in different laboratories are comparable.
Dialkyl phosphate
metabolites (DAPs)
Dimethyl alkylphosphates
(DMAPs)
DMP
Azinphos-methyl, chlorpyrifos-methyl,
dichlorvos, dimethoate, fenthion, malathion,
phosmet, pirimiphos-methyl
1.49
Organophosphate mother
compounds
Limit of detection
(g/L)
DMTP
Azinphos-methyl, chlorpyrifos-methyl,
dimethoate, fenthion, malathion, phosmet,
pirimiphos-methyl
0.30
DMDTP
Diethyl alkylphosphates
(DEAPs)
DEP
DETP
DEDTP
Azinphos-methyl, dimethoat, malathion,
phosmet
0.30
Chlorpyrifos, coumaphos, diazinon, disulfoton,
ethion, parathion, phorate, terbufos
Chlorpyrifos, coumaphos, diazinon, disulfoton,
ethion, parathion, phorate, terbufos
Disulfoton, ethion, phorate, terbufos
0.30
0.30
0.30
TABEL 1
EXAMPLES OF ORGANOPHOSPHATE MOTHER COMPOUNDS OF THE MEASURED DAP METABOLITES AND LOD OF
THE METABOLITES.
The results were expressed in μg/l for each of the six metabolites if the values were above LOD. For
samples below LOD, a value of LOD/√2 was assigned. To account for urinary dilution, the DAP
12
Organophosphate metabolites in urine samples from Danish children and women
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0013.png
concentrations were adjusted for urinary creatinine content in the samples by the following
formula:
µ����
��������
�������� ( ) × 1000 ( )
µ����
����
����
��������
����������������
(
) =
��������
����������������
����
����������������
��������
����������������
(
) × ��������
����������������
(���������������� )
����
where UE
crea
is the urinary concentration adjusted for creatinine, UC is the measured urinary
concentration of each compound, UC
crea
is the urinary concentration of creatinine, and MW
crea
is the
molecular mass (113 mg/mmol) for creatinine. Urinary concentrations of creatinine were
determined previously by enzymatic detection on an Abbott Architect C8000 system.
Concentrations of the metabolites were converted to nmol/L (or nmol/g creatinin) by using their
molecular weights (MW) as indicated below. Then DEAP (sum of diethyl alkylphosphates:
diethylphosphate (DEP, MW: 154.1 ) anddiethylthiophosphate (DETP, MW: 170.2and DMAP (sum
of dimethyl alkylphosphates: dimethylphosphate (DMP, MW:126.0) anddimethylthiophosphate
(DMTP, MW: 142.1) were calculated. Dimethyldithiophosphate (DMDTP) and
diethyldithiophosphate were not included in the summed variables due to none or very low
detection frequency. Finally, the total DAP (dialkylphosphate) concentration was calculated as
DEAP plus DMAP.
2.3
Hair and blood samples
In the Danish part of DEMOCOPHES, hair and blood samples were also collected. Hair samples
were analyzed in a laboratory that participated actively in DEMOCOPHES and showed successful
results in the hair analysis external quality assessment exercises (Esteban et al. 2015). The
measurement of mercury in hair was determined on a Flow Injection Mercury System 400 (Perkin
Elmer, Waltham, MA) in the 3 cm of the hair closest to the scalp according to the method previously
described (Grandjean et al. 1992). The blood samples were analyzed for the persistent organic
pollutants: polychlorinated biphenyls, perfluorinated alkyl substances and polybrominated
diphenyl ethers (Mørck et al. 2014; Mørck et al. 2015a; Mørck et al. 2015b).
2.4
Questionnaires
The basic questionnaire and the urine-sampling related questionnaire developed within the
COPHES framework were translated into Danish to ensure that there was no language barrier.
Information on living conditions of the participants, dietary exposure and use of specific personal
products and socio demographics were included in the questionnaire. The questionnaires were
filled in at home and handed in at the day of the sampling with an on-site dialogue on potential
non-response questions which were then resolved. Since measurement of organophosphates was
not included in the original DEMOCHOPHES project, the questionnaire was not developed to
investigate exposure sources for this group of chemicals. However, details on the consumption of
rice, cereal and homegrown food as well as education, household income and living area were
included in the present study.
2.5
Statistical analysis
Correlations between mothers and children and between the different chemicals were analyzed by
Spearman’s rho. The concentration variables were log-transformed for further statistical analyses to
reach normal distributions of the values. Associations between DAP concentrations and different
variables were analyzed by linear regression models using both unadjusted models (crude) and
models adjusting for different covariates (e.g., household income and educational level) as
indicated in the Tables. A p-value of < 0.05 was used to determine statistical significance.
Organophosphate metabolites in urine samples from Danish children and women
13
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0014.png
3. Results
3.1
Dialkylphosphate concentrations
DAP metabolites was measured in 145 mothers and 144 children. The children were aged 6-11 years
old (8.5 ±1.7) and the mothers were between 31 and 52 years old (40.8 ± 4.3). The majority of the
mothers have taken a higher education (127 out of 145) and 81% of the participants have a
household incomes above the median household income in Denmark in 2011.
The geometric mean (GM), median and 95th percentile of the summed DAP metabolites (DMAP,
DEAP and DAP) can be seen in table 2 below. Statistical significant differences and correlations
between mothers and children are indicated with bold numbers. The concentrations and detection
frequencies of the individual metabolites can be seen in tables 3 and 4, for children and mothers,
respectively.
Concentration
(nmol/L)
DMAP
Children
Mothers
DEAP
Children
Mothers
DAP
Children
Mothers
Creatinine corrected
(nmol/g creatinine)
DMAP
Children
Mothers
DEAP
Children
Mothers
DAP
Children
Mothers
GM [95% CI]
57.7 [48.2-68.4]
45.5 [37.9-54.3]
35.9 [30.8-41.7]
29.6 [25.1-34.5]
111 [96.7-126]*
84.8 [72.7-98.2]
Median (p95)
59.5 (318)
50.7 (245)
37.8 (150
29.8 (135)
106 (387)
92.3 (386)
Spearman's ρ
0.121
0.107
0.086
60.4 [49.5-72.5]*
47.3 [39.8-55.5]
37.5 [32.4-43.6]
30.8 [26.9-35.2
116 [99.8-133]**
88.1 [77.6-100]
63.5 (378)
48.2 (251)
39.3 (151)
31.6 (122)
106 (515)
81.9 (286)
0.228**
0.091
0.203*
TABLE 2
GEOMETRIC MEANS (GM) WITH 95% CONFIDENCE INTERVALS (CI), MEDIANS AND 95 PERCENTILES OF THE
SUMMED METABOLITES DMAP, DEAP AND DAP IN CHILDREN (N=144) AND MOTHERS (N=145). THE CORRELATION
COEFFICIENT SPEAMAN'S RHO IS SHOWN FOR CORRELATIONS BETWEEN MOTHERS AND CHILDREN.
VALUES BELOW LOD WERE SET TO LOD/√2, DAP WAS CALCULATED AS THE SUM OF DEAP (SUM OF DIETHYL
ALKYLPHOSPHATES): DEP+ DETP AND DMAP (SUM OF DIMETHYL ALKYLPHOSPHATES): DMP+DMTP. SIGNIFICANT
DIFFERENCES BETWEEN MOTHERS AND CHILDREN BY PAIRED T-TEST AND SIGNIFICANT CORRELATIONS
MEASURED BY SPEARMAN'S RHO ARE MARKED IN BOLD. * SIGNIFICANCE LEVEL P<0.05, ** SIGNIFICANCE LEVEL
P<0.01.
14
Organophosphate metabolites in urine samples from Danish children and women
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0015.png
%>LOD
Concentrations (µg/L)
DMP
DMTP
DMDTP
DEP
DETP
DEDTP
69
76
1.4
96
40
0
GM [95% CI]
Min
P5
P50
P95
Max
3.97 [3.27-4.74]
2.42 [1.89-3.09]
-
4.54 [3.89-5.27]
0.59 [0.47-0.73]
-
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
1.1
<LOD
<LOD
4.0
3.2
<LOD
4.8
<LOD
<LOD
25.2
23.2
<LOD
19.7
5.2
<LOD
53.5
57.5
6.2
33.6
57.9
<LOD
Creatinine corrected concentrations (µg/g creatinine)
DMP
DMTP
DMDTP
DEP
DETP
DEDTP
4.15 [3.41-5.04]
2.52 [1.91-3.31]
-
4.75 [4.11-3.31]
0.61 [0.50-0.77]
-
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
1.22
<LOD
<LOD
4.53
3.42
<LOD
5.00
<LOD
<LOD
26.5
29.29
<LOD
18.67
5.66
<LOD
67.92
88.96
9.59
32.03
57.12
<LOD
TABLE 3
URINARY CONCENTRATIONS OF DIALKYL PHOSPHATE METABOLITES CHILDREN (N=144). DETECTION FREQUENCY
(%), GEOMETRIC MEAN WITH 95% CONFIDENCE INTERVALS (CI), MIN, 5, 50 AND 95 PERCENTILES ARE SHOWN
Organophosphate metabolites in urine samples from Danish children and women
15
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0016.png
%>LOD
Concentrations (µg/L)
DMP
DMTP
DMDTP
DEP
DETP
DEDTP
54
73
2.1
97
34
0
GM [95% CI]
Min
P5
P50
P95
Max
2.69 [2.30-3.16]
2.17 [1.61-2.86]
-
3.78 [3.21-4.40]
0.52 [0.41-0.65]
-
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
1.1
<LOD
<LOD
2.3
3.1
<LOD
4.0
<LOD
<LOD
15.7
24.3
<LOD
16.0
6,2
<LOD
78.7
58.8
1.7
36.5
56.1
<LOD
Creatinine corrected concentrations (µg/g creatinine)
DMP
DMTP
DMDTP
DEP
DETP
DEDTP
2.80 [2.37-3.31]
2.26 [1.72-2.87]
-
3.93 [3.40-4.52]
0.54 [0.43-0.66]
-
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
1.14
<LOD
<LOD
2.88
3.22
<LOD
4.12
<LOD
<LOD
16.22
22.6
<LOD
12.57
5.3
<LOD
61.87
61.87
0.96
29.6
29.46
<LOD
TABLE 4
URINARY CONCENTRATIONS OF DIALKYL PHOSPHATE METABOLITES IN MOTHERS (N=145). DETECTION
FREQUENCY (%), GEOMETRIC MEAN WITH 95% CONFIDENCE INTERVALS (CI), MIN, 5, 50 AND 95 PERCENTILES ARE
SHOWN
The mean concentration of total DAPs and DMAP was significantly higher in the children compared
to their mothers for creatinine (C) corrected concentrations (DAP:
p=0.003,
DMAP:
p=0.038)
and
for DAP also for the non-corrected values (p=0.010). The same tendency was seen for all
metabolites (see tables 3 and 4), although only significant at p < 0.05 level for DMP (C corrected
and non-corrected). The urinary concentrations of total DAP were significantly correlated between
mothers and children in C corrected values (see table 2). DMAP alone (see table 2) and the
metabolite DMTP (Spearman’s ρ: 0.218, p=0.009) were also significantly correlated between
mothers and children.
3.2
DAP concentration in relation to age
The urinary concentrations of DAPs showed a trend of higher concentrations in younger age groups
compared to the older age-groups for both the children (figure 1) and the mothers (Figure 2). For
the children, the association was significant for the summed DAP (p=0.002) and DMAP (p=0.009)
and for the metabolites DMP (p=0.003) and DMTP (p=0.037) for C corrected values. The same
trend was also seen for the mothers, where the concentrations of DMAP (uncorrected) were
negatively associated with age (p=0.011). However, this association disappeared after correction for
creatinine. For the individual metabolites, the concentrations of DMP, DMTP and DEP seemed
higher in the younger age group of the mothers compared to the older age groups, but the
differences was only significant for DMP (p=0.036) before C-correction. No differences in urine
DAP concentrations were seen between boys and girls in this study.
16
Organophosphate metabolites in urine samples from Danish children and women
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0017.png
FIGURE 1
MEAN CONCENTRATIONS WITH 95%CONFIDENCE INTERVALS OF DAP, DMAP AND DEAP IN THE DIFFERENT AGEGROUPS
OF THE CHILDREN (N=144)
Organophosphate metabolites in urine samples from Danish children and women
17
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0018.png
FIGUR 2
MEAN CONCENTRATIONS WITH 95%CONFIDENCE INTERVALS OF DAP, DMAP AND DEAP IN THE DIFFERENT AGEGROUPS
OF THE MOTHERS (N=144)
3.3
Impact of Socioeconomic status
For the children, a significant positive association was found between the uncorrected total
concentrations of DAP (p=0.040) and DMAP (p=0.024) and educational level of the mother. This
was also significant for the DM metabolites alone (DMP:
p=0.019
and DMTP:
p=0.048,
DMAP:
p=o.024)
and for the DETP metabolite in mothers (p=0.029). The significance disappeared when
the concentrations were corrected for creatinine, except for DETP in mothers (p=0.038). For
children the concentration of DETP (creatinine corrected) also increased with increasing monthly
household income (p=0.017). The concentrations of DAP, DMAP and DEAP (nmol/ g creatinine) I
mothers and children in relation to educational level of the mother and household income can be
seen in table 5 below.
3.4
Food frequency questionnaire
Among potential relevant questions in relation to OP exposure, the questionnaire contained
information on the frequency of eating rice, cereals and homegrown food. Homegrown food was
explained in the questionnaire as: fruit and vegetables, inclusive potatoes, grown in your own,
friends’, or family’s garden or from a local producer nearby. For the children 37% ate rice several
times a week or more, 64% ate cereal daily and 33% reported to eat homegrown food 2-3 times a
month or more. In mothers these numbers were 34%, 52% and 35%, respectively. For the intake of
rice and cereal no significant association with DAP metabolites – neither for individual metabolites
18
Organophosphate metabolites in urine samples from Danish children and women
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
nor for total-DAPs, DMAP, or DEAPs was found. For the intake of homegrown food, the
concentration of DMP in children eating homegrown food 2-3 times a month or more was
significantly lower than children eating homegrown food once a month or less. The difference was
found in both creatinine corrected (p=0.027) and non-corrected (p=0.020) values and persisted
after adjusting for age, gender, household income and education of the mother. No difference was
seen for other metabolites or total-DAPs, DMAP, or DEAPs. For the mothers, intake of homegrown
food was not significantly associated with DAP concentrations. The concentrations of the summed
variables DMAP, DEAP and DAP in relation to intake of rice, cereal and homegrown food can be
seen in table 5 below.
Organophosphate metabolites in urine samples from Danish children and women
19
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0020.png
Children (GM [95%CI in nmol/g creatinine])
Variables
Household income
Categories
< 450.000 a year
N
49
DMAP
67.5
[46.9-93.7]
> 450.000 a year
91
58.3
[45.1-73.2]
Educational level*
ISCED 2-4
17
53.7
[29.05-99.5]
ISCED 5 or 6
126
61.8
[50.3-75.5]
Rice
Several times a week or
more
Once a week or less
53
71.0
[52.3-96.5]
91
55.0
[43.1-70.6]
Cereal
Daily
93
62.0
[48.3-79.2]
Several times a week or
more
Homegrown food
2-3 times a month or
more
Once a month or less
50
55.6
[40.5-77.4]
47
51.6
[36.0-74.8]
97
65.2
[52.0-81.5]
DEAP
34.1
[26.4-43.3]
40.4
[33.9-48.2]
34.6
[24.6-49.0]
38.3
[32.6-44.7]
39.7
[32.5-49.2]
36.3
[29.4-44.1]
38.0
[31.3-45.7]
35.6
[28.0-45.1]
41.4
[31.3-56.4]
35.8
[30.2-42.2]
DAP
118
[89.0-152]
118
[99.7-139]
105
[68.8-160]
118
[102-137]
128
[104-158]
109
[91.1-160]
120
[101-145]
105
[83.0-134]
109.2
[83.8-145]
119
[102-140]
Mothers (GM [95%CI in nmol/g creatinine])
N
50
DMAP
48.2
[35.9-63.0]
91
47.0
[38.4-57.9]
17
45.0
[31.6-79.5]
127
46.7
[39.6-55.9]
49
42.6
[32.3-56.3]
95
49.2
[40.3-58.9]
75
44.2
[35.5-54.8]
69
49.9
[40.0-62.9]
50
46.6
[35.2-61.8]
95
47.7
[39.1-57.9]
DEAP
30.4
[23.7-38.5]
31.2
[26.0-37.3]
30.1
[19.8-43.9]
30.9
[27.1-35.8]
32.2
[26.6-38.9]
30.3
[25.3-36.4]
30.9
[25.4-38.1]
31.0
[25.9-37.0]
33.1
[26.9-40.7]
29.6
[24.8-35.2]
DAP
88.8
[70.5-110.4]
88.1
[75.4-103]
91.6
[66.4-128]
87.4
[76.8-101]
83.3
[68.2-101]
89.8
[76.5-104]
84.2
[70.0-100]
91.3
[76.9-109]
89.4
[73.0-108.9]
87.4
[74.2-101]
TABEL 5
THE CONCENTRATIONS OF THE SUMMED VARIABLES DMAP, DEAP AND DAP IN RELATION TO HOUSEHOLD INCOME, EDUCATIONAL LEVEL OF THE MOTHER AND INTAKE OF RICE, CEREAL AND
HOMEGROWN FOOD, RESPECTIVELY. * EDUCATIONAL LEVEL ISCED 2-4: FINISHED 10TH GRADE, HIGH SCHOOL LEVEL, OR SUPPLEMENTS TO HIGH SCHOOL. ISCED 5 AND 6: SHORT, MIDDLE OR LONG
UNIVERSITY DEGREE AND PH.D. DEGREE
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0021.png
3.5
Urban and rural differences
When comparing the two areas, we generally found highest concentrations of DAPs in participants
from the urban area. The concentration of total DAPs before C correction (p=0.049), the summed
DMAP (p=0.029), and the metabolite DMTP alone (p=0.019) were significantly higher in the
children from the urban area compared to children from the rural area. It should be noted that the
rural area in this study (Viby-Sjælland) is not an agricultural region but rather a village area. The
same tendency was found for DMP, however this was only borderline significant (p<
0.10)
(see
figure 1). In mothers, the differences in total DAP were not significantly different between the areas,
but significantly higher levels of DEAP (p=0.017) and the individual metabolite DEP (p=0.012)
were found in urban living mothers compared to rural mothers in creatinine corrected values. The
differences in DEP (before C correction) and DETP were also borderline significant. The differences
between areas remained after adjusting for age, educational level of the mother, and household
income. The differences in DAP metabolites between the two areas may be partly explained by the
frequency of intake of homegrown food as 40% of children and mother in the rural area reported to
eat homegrown food 2-3 times a month or more compared to 27% and 29% of children and mothers
in the urban area.
14
*
Urban
Rural
12
Mean concentration with 95 %CI
10
8
6
4
2
0
DMP DMTP
Children
DEP
DETP
DMP DMTP
Mothers
DEP
DETP
*
FIGUR 3
MEAN (95% CI) CONCENTRATIONS OF CREATININE CORRECTED VALUES IN MOTHERS AND CHILDREN FROM URBAN
AND RURAL AREAS.
The correlation between the individual DAP metabolites was significant for DEP, DETP, DMP,
DMTP in mothers. In children significant correlations were only found between the related
metabolites – DEP/DETP and DMP/DMTP, respectively. Furthermore, the concentrations of the
individual DAPs in the mothers significantly correlated with the levels of some other industrial
chemicals 2,4-dichlorophenol (2,4-DCP), 2,5- dichlorophenol (2,5-DCP) and 2-phenylphenol (2-PP)
measured in the same participants. No significant correlations were found between DAP
concentrations and paracetamol (NAAP) or mercury in hair for neither children nor mothers.
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0022.png
DAPs
DMP
Children
DMP
DMTP
1.000
0.567
**
1.000
0.192
*
0.114
0.042
0.131
DMTP
DEP
DETP
Other chemicals measured in DEMOCOPHES
2,4-DCP
2,5-DCP
2-PP
NAAP
Mercury
0.032
0.113
-0.018
0.022
-0.065
-0.089
0.066
-0.008
0.060
0.015
DEP
DETP
Mothers
DMP
DMTP
1.000
0.450
**
1.000
1.000
0.477
**
1.000
0.109
0.056
0.032
-0.006
0.078
-0.006
0.144
0.063
-0.055
-0.067
0.290
**
0.409
**
0.226
**
0.453
**
0.076
0.305**
0.179*
0.294**
0.178*
0.313**
0.002
0.015
-0.02
0.015
DEP
DETP
1.000
0.624
**
1.000
0.320**
0.246**
0.265**
0.254**
0.223**
0.213*
0.018
0.041
-0.031
-0.016
TABEL 6
CORRELATIONS BETWEEN THE MEASURED DAPS, 2,4-DCP, 2,5-DCP AND 2-PP IN CHILDREN (N=144) AND MOTHERS
(N=145). * P<0.05, **P<0.01
22
Organophosphate metabolites in urine samples from Danish children and women
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
4. Discussion
Although the urine samples analyzed in this study were collected after organophosphates were
almost phased out for agricultural and biocidal use in Denmark, most urine samples had detectable
concentrations of at least one DAP metabolite. The metabolite DEP was detected in 96% of the
samples from the children and 97% of the samples from the mothers and the concentration of DEP
and the total concentration of DEAPs was at the same level as in urine samples collected among
Danish children of similar age in 2007-08 (Table 7). The levels were similar to DEP and DEAP
concentrations reported in other European studies performed in recent years but higher than levels
reported in studies from the US (Table 7 and 8). In contrast, the concentration of methylated
metabolites, DMAPs, and the total DAP concentration was markedly lower than in the Danish
samples from 2007-08 and lower than levels measured in Germany, France, and the Netherlands
between 2002 and 2007 but higher than among Spanish children from the Valencia region
measured in 2010 (Table 7 and 8).
Overall, the DAP levels found in this study and in other European studies seem to be higher than
levels measured in the US National Health and Nutrition Examination Survey (NHANES) and for
DEP and the total DEAPs, the concentration seem to be higher in Denmark than in most other
countries with available data (Table 7 and 8).
To enable comparison with other studies, the results are presented both as
g
or nmol per L of urine
and as
g
or nmol per gram of creatinine (to account for inter-individual differences in urine
dilution). In adults, the daily mass of excreted creatinine is considered to be fairly constant (Mage et
al. 2008). The creatinine excretion in children is more variable because of their growth but despite
that, presentation of creatinine corrected concentrations has been s recommended for both adults
and children. However, for children creatinin variability has to be taken into consideration if the
urine concentrations are used for calculation of the daily dose of contaminants (Mage et al. 2008).
In studies from both the US, Australia, and Canada, the main source of organophosphate exposure
in the general population has been demonstrated to be the diet (Curl et al. 2015; Oates et al. 2014;
Ye et al. 2015). This is also likely the case in Europe inclusive Denmark, although no specific studies
on this issue have been published. In our study, DAP levels were in general lower among those
living in the more rural area and among those reporting to eat homegrown fruit or vegetables 2-3
times a month or more although the later association was only statistically significant for DMP. In a
study from Australia, higher DAPs were found in children from rural areas compared to urban areas
(Babina et al. 2012) probably due to agricultural use of OPs in the rural area. The associations
between the levels in mothers and children, which were found statistically significant for DAP and
DMAP, also indicates that the exposure is related to a common source within the family, very likely
from the consumption of the same diet containing more or less organophosphate contaminated
food.
According to results from the Danish monitoring program of pesticide residues in food samples on
the Danish marked for the 2004-2011 period, foreign produced fruit and vegetables most often
contained detectable pesticide residues and most often contained concentrations above the
Maximum Residue Limits (MRLs) (Petersen et al. 2013) especially those produced outside EU.
Chlorpyrifos was the second most frequently detected pesticide and was found in approximately
10% of 12.500 samples of fruit and vegetables analyzed. For some commodities the detection
Organophosphate metabolites in urine samples from Danish children and women
23
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
frequency of chlorpyrifos was especially high e.g., 63% for mandarins/clementines, 43% for
oranges, 37% for grapefruit, 36% for lemons, 25% for apples, 19% for peaches and 12% for table
grapes. Compared to the period 1998-2003, the detection frequency of several OPs, inclusive
chlorpyrifos-methyl and chlorpyrifos, was reported to have increased. Malathion was also often
detected during the 2004-2011 period but in less than 5% of the samples and the detection
frequency was unchanged compared to 1998-2003. For cereals, pesticide residues were found in
39% of the samples. Primiphos-methyl was detected in 5.2% of the samples and was the second
most detected pesticide in cereals (Petersen et al. 2013). Thus, this data support that intake of
imported fruit and vegetables is likely to be the main source for OP exposure in this cohort. The
data also indicate that chlorpyrifos may contribute considerably to the relatively high level of DEP
and DEAPs seen in this study.
The higher concentrations of DAPs in the children than their mothers are in accordance with other
studies as for example NHANES (Barr et al. 2004)(see also Table 6) and related to a relatively
higher food intake (food per kilogram of bodyweight) in children than adults which is more
pronounced for the youngest children. The positive association between socioeconomic status (SES;
maternal education level and household income) and urinary DAP concentrations among the
children might be explained by dietary habits related to SES as both education level and income are
determinants of fruit and vegetable intake (Groth et al. 2001).
Health benefits of eating fruits and vegetables are well-documented but whether pesticide residues
in non-organic produce can compromise the health beneficial effects are discussed (Baranski et al.
2014; Forman and Silverstein 2012). However, it is noteworthy that the levels in the Danish and
European studies in general are higher than in the US studies in which neurobehavioural outcomes
have been associated with DAP concentrations in urine samples. A cross-sectional study based on
data from the NHANES has demonstrated that, within the range of exposure in the general US
population in 2002-04, the odds of ADHD for 8- to 15-year old children increased 55% with a 10-
fold increase in urinary concentrations of DMAPs (Bouchard et al. 2010). Among 5 years old
children of farm workers in Salinas Valley in California (the CHAMACOS cohort) a 10-fold increase
in child urinary DEAP concentration doubled the odds in an ADHD indicator variable although this
association was not seen for the total DAP concentration (Marks et al. 2010). The cross-sectional
study design of these studies does not allow conclusion about causality but effects on attention and
other neurobehavioural outcomes have also been reported from longitudinal studies, especially
related to prenatal exposures. In longitudinal birth cohort studies based on the CHAMACOS-cohort
and residents of New York City, maternal exposure to chlorpyrifos and other organophosphate
insecticides in pregnancy was associated with neurobehavioural deficits in the children at least
through 7 years of age (Bouchard et al. 2011; Eskenazi et al. 2007; Marks et al. 2010; Rauh et al.
2011; Rauh et al. 2006). Additionally, prenatal exposure to chlorpyrifos was associated with
structural brain anomalies at school-age including a thinner cortex and disruption of normal sexual
dimorphisms in brain structure demonstrated by magnetic resonance imaging (Rauh et al. 2012).
Based on these studies, chlorpyrifos was recently categorized as a human developmental
neurotoxicant (Grandjean and Landrigan 2014). These findings are also supported by results from
experimental studies of low dose gestational exposure to chlorpyrifos (De Felice et al. 2014; Mullins
et al. 2015), parathion (Levin et al. 2010) or diazinon (Slotkin et al. 2008) showing long-lasting
cognitive effects in the offspring. Most reviews of neurodevelopmental effects of OPs in humans
suggest that exposure during pregnancy, at levels found among groups of the general population,
may have negative effects on children’s neurodevelopment (Gonzalez-Alzaga et al. 2014; Munoz-
Quezada et al. 2013; Ross et al. 2013) while others find the evidence less convincing (Burns et al.
2013; Reiss et al. 2015). The discrepancy is likely related to the large variability in study designs and
in methodologies used for assessing exposure and neurodevelopmental outcomes across studies as
well as differences in the procedure used for including studies in the reviews. However, the OP
exposure has been declining in the US, and a recent study found no negative impact of gestational
OP-exposure on neurobehavioral outcomes in early infancy (four weeks of age) (Yolton et al. 2013).
24
Organophosphate metabolites in urine samples from Danish children and women
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
This might indicate that the exposure level in the general US population has reached a no-adverse-
effect-level. Hopefully this cohort will be followed further during childhood where more sensitive
neurobehavioural test can be performed. To our knowledge no European studies have investigated
associations between urinary DAP concentrations and health outcomes, however, as the European
levels exceeds the US levels where adverse health outcomes have been found in some studies, such
studies are recommended.
We have previously measured the urinary concentrations of the 2,4-DCP and 2,5-DCP and 2-PP in
the same batch of urine samples (Frederiksen et al. 2013). 2,4-DCP, 2,5-DCP and 2-PP are
intermediates in the production of dichlorophenols pesticides (e.g., phenoxy acid herbicides such as
2.4-D and 2,4,5-T, some OPs such as dichlofention and prothiofos, and p-dichlorobenzene used as
insecticide) but also degradation products of these pesticides as well as of chlorinated phenols used
as biocides and for disinfection (e.g., triclosan) (Bomhard et al. 2002; Casas et al. 2011; Wei et al.
2014). The concentrations found in the mothers in this study significantly correlate with the DAP
metabolites measured in the present study. This indicates that some exposure sources of 2,4-DCP,
2,5-DCP and 2-PP are similar to OP exposure and might be related to intake of fruits and vegetables
imported from countries with less restrictions on pesticide use in agriculture.
The present study has some limitations. First, the study was not planned and designed to
investigate determinants of OP exposure and therefore the questionnaire did not include detailed
questions on relevant food items or other potential exposure sources such as use of pesticides in the
homes or gardens. However, since OPs were almost phased out in Denmark in 2011, information
about residential pesticide use seem less important. The finding, that intake of home-grown food
was associated with reduced urinary DAP concentrations may be an indication of lower intake of
imported fruit and vegetables although we cannot make final conclusion on this.
The use of unspecific DAP metabolites as exposure biomarkers does not allow us to identify which
OPs that constitute the majority of the exposure and although the total level seem higher than in
studies from the US, the composition of the exposure may differ. Besides, part of the DAPs
measured may be from intake of OP metabolites preformed in food items and in the environment.
This will cause an overestimation of the exposure to the parent toxic compound and hamper the
conclusion on the health risk associated with the exposure level. However, the same approach has
been used in studies reporting associations between urinary DAP concentrations and adverse health
outcomes (Eskenazi et al. 2014). Although some OPs are more potent than others, they all target the
same organ (nervous system) and share common mechanisms (e.g., cholinesterase inhibition)
indicating additive effects and therefor the total exposure level seem to be of most relevance for
public health. From a precautionary point of view, a risk assessment assuming that the exposure
consists of the most potent OPs identified from the food monitoring program would be obvious.
Formation of DAPs from other chemicals or endogenous compounds would also affect the urinary
concentrations and cause an overestimation of the OP exposure. However, in intervention studies
where groups of individuals have been offered organic food, a marked reduction in urinary DAP
concentrations were reported (Bradman et al. 2015; Oates et al. 2014) indicating that OPs are the
main source for these metabolites. The herbicide glyphosate, which is also an organophosphate,
does not seem to contribute to the DAP concentrations, as this compound is rapidly converted to
aminomethylphosphonic acid (AMPA) and glufosinate to 3-(methylphosphinyl) propionic acid (3-
MPPA). These very polar metabolites are excreted in the urine (Motojyuku et al. 2008; Raina-
Fulton 2014).
An additional limitation is the use of spot urine samples. Since OP and their metabolites have short
half-lives of few days, DAP concentrations in spot urine samples will only reflect exposures
occurring few days prior to sampling. Accordingly, the within-individual variation is high, and
sometimes higher, than between individuals (Attfield et al. 2014; Bradman et al. 2013; Spaan et al.
2015). Therefore, these measurements should only be used to compare OP exposure at population
level whereas for individual exposure information repeated sampling or analysis of pooled urine
samples collected during a period would be more valid.
Organophosphate metabolites in urine samples from Danish children and women
25
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0026.png
Years of
sampling
Denmark, DGCC (6-11 years), n=174
Spain (6-11 years), n=125
France, ENNS (18-74 years), n=392
Australia (2.5-6 years) n urban area = 115
n periurban area= 111
n rural area = 114
Israel (20-74 years), n=247
USA NHANES ( > 6 years), n=3016
(6-11 years), n=576
USA NHANES ( > 6 years), n=2491
(6-11 years), n=310
USA NHANES ( > 6 years), n=2634
(6-11 years), n=350
USA NHANES ( > 6 years), n=2591
(6-11 years), n=385
USA, NYC HANES (aged > 20 years), n= 876
Puerto Rica (pregnant women), n=54 (154
samples)
Ecuador (6-9 years old), n=92
2007-08
2010
2006-07
2003-06
Value
DMP
DMTP
DMDTP
DEP
DETP
DEDTP
Reference
Studies presenting creatinin corrected concentrations (
g/g creatinine)
M
M
M
M
6.37
<LOD
8.04
-
-
-
10.0
< LOD
1.93
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
1.65
4.14
<LOD
5.95
6.8
24.8
37.3
5.2
0.93
2.16
1.98
3.41
1.75
2.78
2.00
4.28
< LOD
1.0
0.70
0.36
<LOD
0.54
7.2
6.9
7.2
0.2
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
3.83
2.28
3.66
4.1
15.4
24.7
1.5
< LOD
0.89
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
0.52
< LOD
0.53
0.84
<LOD
1.12
2.7
7.9
13.8
0.5
0.57
0.64
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
0.24
0.31
<LOD
0.02
-
-
-
0.02
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
(Andersen et al. 2012)
(Roca et al. 2014)
(Fréry et al. 2011)
(Babina et al. 2012)
2011
2001-02
2003-04
2005-06
2007-08
2004
2010-12
2008
M
M
M
M
M
M
M
M
(Berman et al. 2013)
(CDC 2013 )
(CDC 2013 )
(CDC 2013 )
(CDC 2013 )
(McKelvey et al. 2013)
(Lewis et al. 2014)
(Harari et al. 2010)
Studies presenting volume based concentrations (
g/L urine)
Denmark, DGCC (6-11 years), n=174
Germany, GerES IV (3-14 years), n=599
Canada, CHMS (6-79 years), Males (n=2653)
Females (n=2814)
Canada, MIREC, (pregnant women), n=1850
Caribbean Islands (pregnant women), n=150
Ecuador (pregnant women), n=26 (incl. 16 rose
workers)
2007-08
2003-06
2007-09
2008-11
2011-12
2011
M
M
GM
M
M
M
8.40
15.2
3.02
2.91
3.25
1.40
3.41
5.60
15.9
2.07
1.99
3.43
0.80
40.6
<LOD
0.5
<LOD
<LOD
0.39
-
0.45
5.16
6.0
2.38
2.22
2.38
1.50
0.92
1.02
1.0
<LOD
<LOD
0.65
< LOD
0.41
<LOD
0.02
<LOD
<LOD
<LOD
-
0.38
(Andersen et al. 2012)
(Schulz et al. 2012)
(Haines and Murray
2012)
(Colapinto et al. 2015)
(Forde et al. 2015)
(Handal et al. 2015)
TABEL 7 URINARY CONCENTRATIONS OF DIALKYL PHOSPHATE METABOLITES (DAPS) IN DIFFERENT STUDIES CONDUCTED AFTER 2000. VALUES ARE PRESENTED AS MEDIAN (M)
OR GEOMETRIC MEAN (GM). LOD BETWEEN 0.01 AND 0.5 DEPENDENT ON CHEMICAL, LABORATORY, AND YEAR OF ANALYSIS.
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0027.png
Years of
sampling
Danish children (6-11 years old),
n=174
The Netherlands, Generation R
Study (pregnant women), n=106
Canada, CHMS (6-79 years)
USA, CHAMACOS, Salinas valley,
(5-years old), n=274
USA, NYC HANES (aged > 20
years), n= 876
USA, HOME-study (pregnant
women), n=350
Ecuador (6-9 years old), n=92
2007-08
2002-06
2007-09
2004-05
2004
2003 - 06
2008
Values
DEAP
DMAP
DAP
Reference
Studies presenting creatinine corrected concentrations (nmol/g creatinine)
M (5-95 pcts)
M (5-95 pcts)
GM (95% CI)
GM (95% CI)
M (95 pcts)
GM (95% CI)
M (5-95 pcts)
36.1 (5.0-255.9)
33.4 (8.9-158.4)
25.0 (23.0-27.2)
11.1 (9.1-13.5)
6.4 (90.0)
9.4 (8.0-11.1)
6.6 (1.8-58.5)
96.7 (10.0-891.6)
204.6 (37.1-611.5)
57.2 (50.1-65.3)
114 (95.8-136.0)
57.8 (992.6)
46.4 (40.2-53.7)
23.7 (5.2-267.1)
141.4 (22.4-1070.9)
261.7 (50.3-698.2)
93.2 (84.3-103.1)
147 (124.2-173.9)
83.1 (1003.9)
73.5 (64.8-83.4)
32.2 (7.2-343.0)
(Andersen et al.
2012)
(Spaan et al. 2015)
(Ye et al. 2015)
(Quiros-Alcala et al.
2011)
(McKelvey et al.
2013)
(Yolton et al. 2013)
(Harari et al. 2010)
Studies presenting volume based concentrations (nmol/L urine)
USA, NHANES (6-11 years old),
n=471
USA, NHANES (8-15 years old),
n=1139
USA, CHAMACOS, Salinas valley,
(pregnant women), n=348
USA, CHAMACOS, Salinas valley,
(5-years old), n=320
Canada, CHMS (6-11 years),
n=1035
Ecuador (pregnant women),
n=26 (incl. 16 rose workers)
1999-2000
2000-04
1999-2000
2004-05
2007-09
2011
Least-squares
GM
A
(95% CI)
GM (IQR
GM (95% CI)
GM(95% CI)
M (IQR)
GM
17.4 (11.1-27.3)
11.0 (2.1-35.0)
17.7 (16.1-19.4)
7.2 (6.0-8.7)
25.0 (10.5-51.3)
8.26
72.8 (54.3-97.5)
41.3 (10.1-130.7)
76.8 (69.3-85.0)
72.4 (61.0-86.0)
62.0 (18.7-192.8)
51.6
109.6 (83.3-144.3)
68.3 (24.4-186.0)
109.0 (99.4-119.6)
92.6 (78.6-109.0)
99.2 (34.3-273.3)
83.6
(Barr et al. 2004)
(Bouchard et al.
2010)
(Marks et al. 2010)
(Marks et al. 2010)
(Oulhote and
Bouchard 2013)
(Handal et al. 2015)
TABEL 8
URINARY CONCENTRATIONS OF DIALKYL PHOSPHATE METABOLITES (DAPS) IN DIFFERENT STUDIES. RESULTS ARE PRESENTED AS MEDIANS (M) WITH PERCENTILES (PCTS) OR GEOMETRIC MEANS (GM)
WITH CONFIDENCE INTERVALS (CI) OR INTERQUARTILE RANGE (IQR). DEAP (SUM OF DIETHYL ALKYLPHOSPHATES): DEP+ DEP+DETP; DMAP (SUM OF DIMETHYL ALKYLPHOSPHATES):
DMP+DMTP+DMDTP; DAP: DEAP + DMAP.
A
ADJUSTED FOR AGE, SEX, RACE/ETHNICITY, AND CONCENTRATIONS OF SERUM COTININE AND URINARY CREATININE.
Organophosphate metabolites in urine samples from Danish children and women
27
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0028.png
5. Conclusion
The findings of relatively high detection frequency of the DAP metabolites DEP, DETP, DMP and
DMTP in a Danish group of children and their mothers, clearly indicate that there is still a
widespread exposure to organophosphate pesticides in the Danish population, even though there
have been major restrictions on their use in agriculture and as biocides. The concentrations of the
metabolites DEP and DETP found in the children and women of the present study are similar to
levels found among Danish children in 2007-08 and as levels found in other recent European
studies. The methylated metabolites seem to have decreased in Denmark compared to previous
measurements in 2007-08 and levels found in other European countries before 2007. However, the
levels of total DAP and DEAP in the present study, as well as the levels found in other European
countries, are higher than concentrations found in the US in a national survey (NHANES),
indicating higher exposure to organophosphates in Europe compared to the US. As the exposure to
organophosphates has been associated with adverse effects in some studies from the US, there may
also be a risk of adverse effects in Europe and Denmark, as we have found even higher exposure
levels. Although we do not know the specific organophosphates responsible for the relatively high
concentrations of DEAPs in Denmark, chlorpyrifos is likely to be one of the most important since it
is often found in samples of imported fruit and vegetables – also in the latest published survey of
pesticide residues in food samples on the Danish marked (Fødevareinstituttet 2014). Thus, studies
of potential adverse health effects related to organophosphate exposure in European populations
are needed. Also identification of the main dietary exposure sources and the related OPs is
warranted in order to introduce adequate measures to reduce the exposure - especially for
vulnerable population groups.
Referencer
Strandgade 29
1401 København K
Tlf.: (+45) 72 54 40 00
www. mst.dk
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0029.png
Strandgade 29
DK - 1401 København K
Tlf.: (+45) 72 54 40 00
www. mst.dk
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0030.png
6. References
Andersen HR, Wohlfahrt-Veje C, Debes F, Nielsen F, Jensen TK, Grandjean P, et al. 2012.
Langtidseffekter af prænatal pesticideksponering. (Bekæmpelsesmiddelforskning fra Miljøstyrelsen
).
Attfield KR, Hughes MD, Spengler JD, Lu C. 2014. Within- and between-child variation in repeated
urinary pesticide metabolite measurements over a 1-year period. Environ Health Perspect 122:201-
206.
Babina K, Dollard M, Pilotto L, Edwards JW. 2012. Environmental exposure to organophosphorus
and pyrethroid pesticides in south australian preschool children: A cross sectional study. Environ
Int 48:109-120.
Baranski M, Srednicka-Tober D, Volakakis N, Seal C, Sanderson R, Stewart GB, et al. 2014. Higher
antioxidant and lower cadmium concentrations and lower incidence of pesticide residues in
organically grown crops: A systematic literature review and meta-analyses. The British journal of
nutrition:1-18.
Barr DB, Bravo R, Weerasekera G, Caltabiano LM, Whitehead RD, Jr., Olsson AO, et al. 2004.
Concentrations of dialkyl phosphate metabolites of organophosphorus pesticides in the u.S.
Population. Environ Health Perspect 112:186-200.
Becker K, Seiwert M, Casteleyn L, Joas R, Joas A, Biot P, et al. 2014. A systematic approach for
designing a hbm pilot study for europe. International Journal of Hygiene and Environmental
Health 217:312-322.
Berman T, Goldsmith R, Goen T, Spungen J, Novack L, Levine H, et al. 2013. Urinary
concentrations of organophosphate pesticide metabolites in adults in israel: Demographic and
dietary predictors. Environ Int 60:183-189.
Bomhard EM, Brendler-Schwaab SY, Freyberger A, Herbold BA, Leser KH, Richter M. 2002. O-
phenylphenol and its sodium and potassium salts: A toxicological assessment. Critical reviews in
toxicology 32:551-625.
Bouchard MF, Bellinger DC, Wright RO, Weisskopf MG. 2010. Attention-deficit/hyperactivity
disorder and urinary metabolites of organophosphate pesticides. Pediatrics 125:e1270-1277.
Bouchard MF, Chevrier J, Harley KG, Kogut K, Vedar M, Calderon N, et al. 2011. Prenatal exposure
to organophosphate pesticides and iq in 7-year-old children. Environ Health Perspect 119:1189-
1195.
Bradman A, Kogut K, Eisen EA, Jewell NP, Quiros-Alcala L, Castorina R, et al. 2013. Variability of
organophosphorous pesticide metabolite levels in spot and 24-hr urine samples collected from
young children during 1 week. Environ Health Perspect 121:118-124.
Bradman A, Quiros-Alcala L, Castorina R, Aguilar Schall R, Camacho J, Holland NT, et al. 2015.
Effect of organic diet intervention on pesticide exposures in young children living in low-income
urban and agricultural communities. Environ Health Perspect.
Burns CJ, McIntosh LJ, Mink PJ, Jurek AM, Li AA. 2013. Pesticide exposure and
neurodevelopmental outcomes: Review of the epidemiologic and animal studies. Journal of
toxicology and environmental health Part B, Critical reviews 16:127-283.
Casas L, Fernandez MF, Llop S, Guxens M, Ballester F, Olea N, et al. 2011. Urinary concentrations
of phthalates and phenols in a population of spanish pregnant women and children. Environ Int
37:858-866.
Strandgade 29
1401 København K
Tlf.: (+45) 72 54 40 00
www. mst.dk
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0031.png
CDC. 2013 Fourth national report on human exposure to environmental chemicals, opdated tables
september 2013. Washington, DC: Centers for Disease Control and Prevention.
Centers for Disease Control and Prevention C. 2009. Fourth national report on human exposure to
environmental chemicals. Atlanta, US.
Clune AL, Ryan PB, Barr DB. 2012. Have regulatory efforts to reduce organophosphorus insecticide
exposures been effective? Environ Health Perspect 120:521-525.
Colapinto CK, Arbuckle TE, Dubois L, Fraser W. 2015. Tea consumption in pregnancy as a predictor
of pesticide exposure and adverse birth outcomes: The mirec study. Environ Res 142:77-83.
Curl CL, Beresford SA, Fenske RA, Fitzpatrick AL, Lu C, Nettleton JA, et al. 2015. Estimating
pesticide exposure from dietary intake and organic food choices: The multi-ethnic study of
atherosclerosis (mesa). Environ Health Perspect 123:475-483.
De Felice A, Venerosi A, Ricceri L, Sabbioni M, Scattoni ML, Chiarotti F, et al. 2014. Sex-dimorphic
effects of gestational exposure to the organophosphate insecticide chlorpyrifos on social
investigation in mice. Neurotoxicology and teratology 46:32-39.
Eskenazi B, Marks AR, Bradman A, Harley K, Barr DB, Johnson C, et al. 2007. Organophosphate
pesticide exposure and neurodevelopment in young mexican-american children. Environ Health
Perspect 115:792-798.
Eskenazi B, Kogut K, Huen K, Harley KG, Bouchard M, Bradman A, et al. 2014. Organophosphate
pesticide exposure, pon1, and neurodevelopment in school-age children from the chamacos study.
Environ Res 134C:149-157.
Esteban M, Schindler BK, Jimenez-Guerrero JA, Koch HM, Angerer J, Rivas TC, et al. 2015.
Mercury analysis in hair: Comparability and quality assessment within the transnational
cophes/democophes project. Environmental research 141:24-30.
Forde MS, Robertson L, Laouan Sidi EA, Cote S, Gaudreau E, Drescher O, et al. 2015. Evaluation of
exposure to organophosphate, carbamate, phenoxy acid, and chlorophenol pesticides in pregnant
women from 10 caribbean countries. Environmental science Processes & impacts 17:1661-1671.
Forman J, Silverstein J. 2012. Organic foods: Health and environmental advantages and
disadvantages. Pediatrics 130:e1406-1415.
Frederiksen H, Nielsen JK, Mørck TA, Hansen PW, Jensen JF, Nielsen O, et al. 2013. Urinary
excretion of phthalate metabolites, phenols and parabens in rural and urban danish mother-child
pairs. Int J Hyg Environ Health 216:772-783.
Fréry N, Saoudi A, Garnier R, Zeghnoun A, Falq G. 2011. Exposition de la population française aux
substances chimiques de l'environnement. Saint-Maurice: Institut de veille sanitaire 201158.
Fødevareinstituttet DoF. 2014. Pesticidrester i fødevarer 2013.
Gonzalez-Alzaga B, Lacasana M, Aguilar-Garduno C, Rodriguez-Barranco M, Ballester F, Rebagliato
M, et al. 2014. A systematic review of neurodevelopmental effects of prenatal and postnatal
organophosphate pesticide exposure. Toxicology letters 230:104-121.
Grandjean P, Nielsen GD, Jorgensen PJ, Horder M. 1992. Reference intervals for trace elements in
blood: Significance of risk factors. Scandinavian journal of clinical and laboratory investigation
52:321-337.
Grandjean P, Landrigan PJ. 2014. Neurobehavioural effects of developmental toxicity. Lancet
neurology 13:330-338.
Groth MV, Fagt S, Brondsted L. 2001. Social determinants of dietary habits in denmark. European
journal of clinical nutrition 55:959-966.
Haines DA, Murray J. 2012. Human biomonitoring of environmental chemicals--early results of the
2007-2009 canadian health measures survey for males and females. Int J Hyg Environ Health
215:133-137.
Handal AJ, Hund L, Paez M, Bear S, Greenberg C, Fenske RA, et al. 2015. Characterization of
pesticide exposure in a sample of pregnant women in ecuador. Archives of environmental
contamination and toxicology.
Harari R, Julvez J, Murata K, Barr D, Bellinger DC, Debes F, et al. 2010. Neurobehavioral deficits
and increased blood pressure in school-age children prenatally exposed to pesticides. Environ
Health Perspect 118:890-896.
Strandgade 29
DK - 1401 København K
Tlf.: (+45) 72 54 40 00
www. mst.dk
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0032.png
Heudorf U, Angerer J, Drexler H. 2004. Current internal exposure to pesticides in children and
adolescents in germany: Urinary levels of metabolites of pyrethroid and organophosphorus
insecticides. IntArchOccupEnviron Health 77:67-72.
Joas R, Casteleyn L, Biot P, Kolossa-Gehring M, Castano A, Angerer J, et al. 2012. Harmonised
human biomonitoring in europe: Activities towards an eu hbm framework. International Journal of
Hygiene and Environmental Health 215:172-175.
Levin ED, Timofeeva OA, Yang L, Petro A, Ryde IT, Wrench N, et al. 2010. Early postnatal
parathion exposure in rats causes sex-selective cognitive impairment and neurotransmitter defects
which emerge in aging. BehavBrain Res 208:319-327.
Lewis RC, Cantonwine DE, Anzalota Del Toro LV, Calafat AM, Valentin-Blasini L, Davis MD, et al.
2014. Urinary biomarkers of exposure to insecticides, herbicides, and one insect repellent among
pregnant women in puerto rico. Environ Health 13:97.
Mage DT, Allen RH, Kodali A. 2008. Creatinine corrections for estimating children's and adult's
pesticide intake doses in equilibrium with urinary pesticide and creatinine concentrations. Journal
of exposure science & environmental epidemiology 18:360-368.
Marks AR, Harley K, Bradman A, Kogut K, Barr DB, Johnson C, et al. 2010. Organophosphate
pesticide exposure and attention in young mexican-american children: The chamacos study.
Environ Health Perspect 118:1768-1774.
McKelvey W, Jacobson JB, Kass D, Barr DB, Davis M, Calafat AM, et al. 2013. Population-based
biomonitoring of exposure to organophosphate and pyrethroid pesticides in new york city. Environ
Health Perspect.
Motojyuku M, Saito T, Akieda K, Otsuka H, Yamamoto I, Inokuchi S. 2008. Determination of
glyphosate, glyphosate metabolites, and glufosinate in human serum by gas chromatography-mass
spectrometry. JChromatogrB AnalytTechnolBiomedLife Sci 875:509-514.
Mullins RJ, Xu S, Pereira EF, Pescrille JD, Todd SW, Mamczarz J, et al. 2015. Prenatal exposure of
guinea pigs to the organophosphorus pesticide chlorpyrifos disrupts the structural and functional
integrity of the brain. Neurotoxicology 48:9-20.
Munoz-Quezada MT, Lucero BA, Barr DB, Steenland K, Levy K, Ryan PB, et al. 2013.
Neurodevelopmental effects in children associated with exposure to organophosphate pesticides: A
systematic review. Neurotoxicology 39C:158-168.
Mørck TA, Erdmann SE, Long M, Mathiesen L, Nielsen F, Siersma VD, et al. 2014. Pcb
concentrations and dioxin-like activity in blood samples from danish school children and their
mothers living in urban and rural areas. Basic & clinical pharmacology & toxicology 115:134-144.
Mørck TA, Nielsen F, Nielsen JK, Jensen JF, Hansen PW, Hansen AK, et al. 2015a. The danish
contribution to the european democophes project: A description of cadmium, cotinine and mercury
levels in danish mother-child pairs and the perspectives of supplementary sampling and
measurements. Environmental research:96-105.
Mørck TA, Nielsen F, Nielsen JK, Siersma VD, Grandjean P, Knudsen LE. 2015b. Pfas
concentrations in plasma samples from danish school children and their mothers. Chemosphere
129:203-209.
Oates L, Cohen M, Braun L, Schembri A, Taskova R. 2014. Reduction in urinary organophosphate
pesticide metabolites in adults after a week-long organic diet. Environ Res 132:105-111.
Oulhote Y, Bouchard MF. 2013. Urinary metabolites of organophosphate and pyrethroid pesticides
and behavioral problems in canadian children. Environ Health Perspect 121:1378-1384.
Petersen A, Jensen BH, Andersen JH, Poulsen ME, Christensen T, Nielsen E. 2013. Pesticide
residues, results from the period 2004-2011.Danmarks Tekniske Universitet, Fødevareinstituttet.
Quiros-Alcala L, Alkon AD, Boyce WT, Lippert S, Davis NV, Bradman A, et al. 2011. Maternal
prenatal and child organophosphate pesticide exposures and children's autonomic function.
Neurotoxicology 32:646-655.
Raina-Fulton R. 2014. A review of methods for the analysis of orphan and difficult pesticides:
Glyphosate, glufosinate, quaternary ammonium and phenoxy acid herbicides, and dithiocarbamate
and phthalimide fungicides. J AOAC Int 97:965-977.
Strandgade 29
1401 København K
Tlf.: (+45) 72 54 40 00
www. mst.dk
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0033.png
Rauh V, Arunajadai S, Horton M, Perera F, Hoepner L, Barr DB, et al. 2011. 7-year
neurodevelopmental scores and prenatal exposure to chlorpyrifos, a common agricultural pesticide.
EnvironHealth Perspect.
Rauh VA, Garfinkel R, Perera FP, Andrews HF, Hoepner L, Barr DB, et al. 2006. Impact of prenatal
chlorpyrifos exposure on neurodevelopment in the first 3 years of life among inner-city children.
Pediatrics 118:e1845-e1859.
Rauh VA, Perera FP, Horton MK, Whyatt RM, Bansal R, Hao X, et al. 2012. Brain anomalies in
children exposed prenatally to a common organophosphate pesticide. Proceedings of the National
Academy of Sciences of the United States of America 109:7871-7876.
Reiss R, Chang ET, Richardson RJ, Goodman M. 2015. A review of epidemiologic studies of low-
level exposures to organophosphorus insecticides in non-occupational populations. Crit Rev Toxicol
45:531-641.
Roca M, Miralles-Marco A, Ferre J, Perez R, Yusa V. 2014. Biomonitoring exposure assessment to
contemporary pesticides in a school children population of spain. Environ Res 131C:77-85.
Ross SM, McManus IC, Harrison V, Mason O. 2013. Neurobehavioral problems following low-level
exposure to organophosphate pesticides: A systematic and meta-analytic review. Crit Rev Toxicol
43:21-44.
Schulz C, Wilhelm M, Heudorf U, Kolossa-Gehring M. 2012. Reprint of "update of the reference and
hbm values derived by the german human biomonitoring commission". Int J Hyg Environ Health
215:150-158.
Slotkin TA, Bodwell BE, Levin ED, Seidler FJ. 2008. Neonatal exposure to low doses of diazinon:
Long-term effects on neural cell development and acetylcholine systems. Environ Health Perspect
116:340-348.
Spaan S, Pronk A, Koch HM, Jusko TA, Jaddoe VW, Shaw PA, et al. 2015. Reliability of
concentrations of organophosphate pesticide metabolites in serial urine specimens from pregnancy
in the generation r study. Journal of Exposure Science and Environmental Epidemiology 25:286-
294.
Tarbah FA, Kardel B, Pier S, Temme O, Daldrup T. 2004. Acute poisoning with phosphamidon:
Determination of dimethyl phosphate (dmp) as a stable metabolite in a case of organophosphate
insecticide intoxication. J Anal Toxicol 28:198-203.
Wei Y, Zhu J, Nguyen A. 2014. Urinary concentrations of dichlorophenol pesticides and obesity
among adult participants in the u.S. National health and nutrition examination survey (nhanes)
2005-2008. Int J Hyg Environ Health 217:294-299.
Ye M, Beach J, Martin JW, Senthilselvan A. 2015. Associations between dietary factors and urinary
concentrations of organophosphate and pyrethroid metabolites in a canadian general population.
Int J Hyg Environ Health 218:616-626.
Yolton K, Xu Y, Sucharew H, Succop P, Altaye M, Popelar A, et al. 2013. Impact of low-level
gestational exposure to organophosphate pesticides on neurobehavior in early infancy: A
prospective study. Environ Health 12:79.
Strandgade 29
DK - 1401 København K
Tlf.: (+45) 72 54 40 00
www. mst.dk
 MOF, Alm.del - 2015-16 - Bilag 509: Rapport om undersøgelse der viser, at rester fra organofosfater findes i menneskers urin
1645441_0034.png
Strandgade 29
1401 København K
Tlf.: (+45) 72 54 40 00
www. mst.dk