臨床環境医学（第32巻第2号）

59

Original article

Urinary neonicotinoids in rural populationof the Philippines and comparison with paired
hair samples
Kumiko Taira1), Alexandre Aebi2), Gaétan Glauser3), Edward A. D. Mitchell2),
Yoshinori Ikenaka4), 5), Francisco Sanchez-Bayo6), Andrea Tapparo7),
Elizabeth Lumawig-Heitzmann8), Jean-Marc Bonmatin9), Anton Safer10),
Maarten Bijleveld van Lexmond11), Florencia Claveria12), Yoshiko Aoyama13),
Yuichi Miyabara14) and Makiko Komori1)
1) Adachi Medical Center, Tokyo Women’s Medical University, Japan
2) Laboratory of Soil Biodiversity, University of Neuchâtel, Switzerland
3) Neuchâtel Platform of Analytical Chemistry, University of Neuchâtel, Switzerland
4) Hokkaido University, Japan
5) Northwest University, South Africa
6) The University of Sydney, Australia7) Department of Chemical Sciences, University of Padua, Italy
8) Marinduque Biological Field Station, Philippines
9) CNRS Center for Molecular Biophysics, France10) Heidelberg University, Germany11) Task Force on Systemic Pesticides, Switzerland
12) De La Salle University-Manila, Philippines
13) Aoyama Allergy Clinic, Japan14) Institute of Mountain Science, Shinshu University, Japan

Abstract
Neonicotinoid insecticides, the most widely used category of insecticides for 20 years, have been
detected in various human tissues including blood, cerebrospinal fluid, urine, and hair. We collected
spot urine from 99 volunteers in three islands of the Philippines, and compared their neonicotinoid concentrations with those obtained in paired hair samples from our previous study. Geometric means ofconcentration in urine (ng/L) were 0.048 for imidacloprid, 0.030 for clothianidin, 0.044 for thiamethoxam, 0.007 for acetamiprid, 0.005 for thiacloprid, 0.036 for dinotefuran, 0.026 for nitenpyram, and
0.006 for N-desmethyl acetamiprid. The total concentrations of the former five neonicotinoids in urine

受付：2023年９月25日 採用：2024年２月20日
責任著者：平 久美子
東京女子医科大学附属足立医療センター麻酔科〒123-8558 東京都足立区江北4-33-1
VFG03077@nifty.com

Jpn J Clin Ecol（Vol.32 No.2 2023）

60

samples were lower in children (6-16 years old, n=28) than in adults (18-39 yo, n=42) and seniors (40-83
yo, n=29) (p=0.030), but not in hair. Imidacloprid concentrations were lower in hair (p=0.014) in adultwomen with dyed hair (n=9) as compared to women with un-dyed hair (n=18), but not in urine (p=0.71).
We also analyzed strong ions in urine by ion chromatography and found an increase in urinary strong
ion difference (SIDu) in five urine samples, suggesting possible renal metabolic acidosis. Our results
suggest that urine is an appropriate matrix to investigate human exposure to neonicotinoids, while hair
may be better suited to identify chronic exposure especially in children.
(Jpn J Clin Ecol 32 : 59－76, 2023)《Keywords》neonicotinoids, hair, urine, Philippines, children

要旨

ネオニコチノイドは世界で最も多く使われている殺虫剤で、ヒトの尿および毛髪から検出される。2017
年にフィリピンの三つの島の99人のボランティア（成人男性44人、成人女性27人、 6 -16歳の子供28人の随時尿と毛髪を採取しネオニコチノイドを分析した。同時に尿中電解質の測定も行った。各ネオニコチノ
イドの尿中濃度（ng/L）と毛髪中濃度（ng/kg）の幾何平均は、イミダクロプリド0.048 vs 0.076、クロチ
アニジン0.030 vs 0.036、チアメトキサム0.044 vs 0.021、アセタミプリド0.007 vs 0.005、チアクロプリド
0.005 vs 0.005だった。成人女性の毛髪中イミダクロプリド濃度は、染髪（n＝ 9 ）で染髪なし（n＝18）と比べ有意に低かった（p＝0.014）。尿中 strong ion differences（SIDu＝Na＋K−CI）は５検体で異常高値を示した。
（臨床環境 32：59－76，2023）《キーワード》ネオニコチノイド、尿、毛髪、フィリピン、子供

Introduction

pollinators in general5,6), and is of toxicological

Neonicotinoids have been the most widelyconcern for humans7-11). The European commu-

used class of insecticides for 20 years1). Five neo-nity has restricted the use of three major com-

nicotinoids are registered as pesticides world-

pounds i.e., imidacloprid, clothianidin, and thia-

wide (imidacloprid, acetamiprid, thiacloprid, thi-methoxam12), the registration of thiacloprid has

amethoxam, clothianidin), and otherrecently expired13), and the revision of their reg-neonicotinoids and related insecticides (e.g. ni-

istration is underway in the USA and Austra-

tenpyram, dinotefuran, cycloxaprid, paichong-lia14,15).

ding, imidaclothiz, flupyradifurone, sulfoxaflor,

Documenting the levels of neonicotinoid con-triflumezopyrim, and flupyrimin) are also fre-tamination in the general population has be-

2,3)quently used in some countries . Being water

come an important issue of public health, espe-

soluble, systemic, and long-acting, neonicotinoids

cially given the suspected risks these pesticides

are widely used for agriculture, forestry, horti-

pose on neurodevelopment11), as well as the doc-

culture, turf, termite control for wooden building

umented subacute and chronic health effects,

materials, and for protection against parasites

which include neuronal, reproductive, musculo-in domestic animals4). Their strong and persis-

skeletal, cardiovascular, gastrointestinal, liver

tent nicotinic acetylcholine receptor (nAChR)

and renal toxicity in addition to acute health ef-

competitive modulator action is identified as afects7-10,16-20). As the human urinary excretion

causal factor for the decline of honeybees andhalf-lives of some neonicotinoids, i.e. imidaclo-

臨床環境医学（第32巻第2号）

prid and N-desmethyl-acetamiprid (DMAP), the61

In an earlier study, we detected five neonicoti-

phase 1 metabolite of acetamiprid, exceed 24

noids (thiamethoxam, clothianidin, imidacloprid,

hours21), continuous environmental exposure toacetamiprid, and thiacloprid) in the hair of theneonicotinoids through food22,23), beverages24,25),

Philippine general population30). At the same

and/or air26) may cause an increase of tissues

time, we collected paired spot urine samples

concentration in the human body.

with the goal to understand the relationship be-

Neonicotinoids and their metabolites havetween neonicotinoid concentrations in urine andbeen detected in urine (Supplemental Table 1),

hair for surveying human exposure. In urine,

hair, whole blood, serum, plasma, cerebrospinal

neonicotinoids and their metabolites have been

fluids, saliva, semen, breast milk, and teeth

detected in the general population including(Supplemental Table 2) from the general popula-pregnant women, in Japan, China, Sri Lanka,

tion. A recent study showed very high within-

US, Korea, Vietnam, India, Kuwait, Saudi Ara-

and between-individual variability in urinary

bia, Greece, Thailand, Ghana, Switzerland, andconcentrations of neonicotinoids and their me-

Germany (Supplemental Table 1), but not yet intabolites in consecutive collection for up to 44

the Philippines.

27)

days except for DMAP . Another study suggest-

The aim of the present study was to analyze

ed there exists a positive relationship between

urinary neonicotinoid concentrations by HPLC-

the neonicotinoid concentration in urine, blood

MS/MS in Philippine populations, and to com-28)

and cerebrospinal fluid . Urinary concentrationpare them with concentrations in paired haircan be an appropriate biomarker of short-term

samples30）. Additionally, we analyzed strong ionsbrain neonicotinoid exposure, but the negative(e.g., sodium, potassium, and chloride) in urine,

prediction value for chronic exposure is un-

and calculated SIDu31,32), in order to screen for

known. Bio-distribution of neonicotinoids andrenal tubular acidosis which may be caused bytheir metabolites into hair after oral intake has

renal tubular dysfunction.

29)

been reported , and residues in hair could thus
be better indicators of chronic exposure, when

Methods

occupational direct contamination or the effectStudy design, ethics approval and sample col-of hair dye are eliminated.

lection

Neonicotinoids exposure may cause metabolic
19,20)

This study was carried out as an academic. Basedproject of the Task Force on Systemic Pesticides

on urinary biomarkers such as L-FABP and

Public Health Working Group (Chair. K. Taira).

Cystatin C, we have indeed found a correlation

After obtaining the ethic committee approval of

between neonicotinoids exposure and renal tu-

Tokyo Women’s Medical University (No.4521)

bular dysfunction in Sri Lanka where chronic

with that of Marinduque Biological Field Station

kidney disease with unknown etiology was prev-

in Philippines (No.1), the written informed con-acidosis by renal tubular dysfunction

10)

alent . Previous reports suggested an adequate

sent for sampling from each participant, spot

response to non-renal metabolic acidosis should

urine samples (10 mL) and hair samples (ca. 100

be a negative urinary strong ion differencemg) were collected at three locations on three(SIDu = NaU + KU - CIU)31,32), while renal meta-different Philippine islands from randomly cho-bolic acidosis by acute kidney injury caused highsen local volunteers: children (more than 5 years

31,32)

SIDu value above 40 mEq/L.

old), adult men and women (no less than 18

62

Jpn J Clin Ecol（Vol.32 No.2 2023）

were recorded at the same time. No participant
had a history of chronic renal failure. All methods were performed in accordance with the relevant guidelines and regulations.
Urine samples were frozen at -20 ˚C immediately after collection, transferred to Hokkaido
University, and stored in a freezer at -20 ˚C until they were analyzed. Hair samples were kept
in zip locked bags, transported in a cooler to the
University of Neuchâtel and stored in a freezer
at -20 ˚C until analysis. The datasets generated
and/or analyzed during the current study areavailable from the corresponding author upon
reasonable request.
Chemicals and reagents
For the urine samples analyzed at Hokkaido
University, acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram thiacloprid, thiamethoxam, N - desmethyl-acetamiprid, and N Figure 1. Geographic data of the three sampling places. Modified from Fig. 1 ino u r p r e v i o u s p a p e r 30) b a s e d o n
Google map（https://www.google.
co.jp/maps）on 23 December 2023.

desmethyl-acetamiprid-d3 were purchased from
Hayashi Junyaku (Osaka, Japan). PL Pesticides
surrogate mix VII (Neonicotinoid-II 7mix), 10
ng/ml in acetonitrile, mixture of acetamiprid d6, nitenpyram - d3, clothianidin - d3, dinotefu-

years old). Geographic data of the three sam-ran - d3, imidacloprid - d4, thiacloprid - d4, thia-

pling place are shown in Figure 1. Samples were

methoxam - d4, were also purchased fromcollected between September and November

Hayashi Junyaku (Osaka, Japan). Acetonitrile,

2017 before the application of insecticides to thedichloromethane formic acid, ammonium acetate

crops in the respective areas. The sample-set

and distilled water were of HPLC grade andsize was determined by the carrying capacity ofpurchased from Kanto Chemical Co. Inc. (Tokyo,

urine samples from Philippines to Japan. All

Japan).

participants who consented to participate in thisFor the hair samples analyzed at the Univer-research, were informed about the purpose, ben-sity of Neuchâtel, analytical standards (99.6 toefits, risks, and other options in a written infor-

99.9% purity) of the five neonicotinoids (acet-mation sheet, which they signed if they agreed

amiprid, clothianidin, imidacloprid, thiacloprid

with the rules. When volunteers were minors or

and thiamethoxam) were purchased from Sig-children, written informed consent was obtained

ma-Aldrich (Germany). Isotopically labelled neo-from the next of kin, caretakers, or guardians on

nicotinoids were purchased from CDN Isotopes

their behalf. The participants’ age, sex, occupa-(Canada) and used as internal standards. Chro-tion, and whether their hair was dyed or notmatography grade solvents (acetonitrile, ethyl

臨床環境医学（第32巻第2号）

63

Table 1. Compound-specific technical data of urine and hair analyses by HPLC-MS/MS.

a

LOQ, limit of quantification; b RSD, relative standard deviation; c MRM, multiple reaction monitoring; d ESI, electrospray ionization; e DMAP, N-desmethyl-acetamiprid; f N.A., not available.

acetate, methanol) were purchased from Fisher

quantitation (LOQs) of each analyte were calcu-

Scientific (UK). Magnesium sulphate, sodium

lated as the lowest points on standard curves

chloride, trisodium citrate and sodium hydrogen(Table 1) with relative standard deviations lesscitrate sesquihydrate were purchased from Sig-

than 15% (n=5) and signal-to-noise ratios of 10 :ma-Aldrich. Isolute PSA and ZeoPrep 90 C18

1.

bulk phases were purchased at Biotage (Sweden)

Major ions, i.e. Na+, K+, Ca2+, Mg2+, NH4+, Cl-,and ZeoChem (Switzerland), respectively. Ultra-

NO3-, SO42-, and PO43- were analyzed with an Ion

pure water was obtained from a Milli-Q system

Chromatograph (Dionex® ICS-1500). Details are(Millipore, France) and solvents (acetonitrile)shown in Supplemental Text 2 in additional fileand additives (formic acid, ammonium formate)

1. SIDu and Na/K concentration ratio were cal-for LC-MS/MS were obtained from VWR (Aus-culated by urinary Na+ (unit: mEq/L, NaU), K+tria).

(unit: mEq/L, KU), and Cl- (unit: mEq/L, ClU) as
follows:
SIDu = NaU + KU − ClU

Sample analysis
Urine analysis was performed according to a
33)

Na/K concentration ratio = NaU / KU

previously established method . Details are

Hair samples were analysed according to theshown in Supplemental Text 1 in additional filemethod of Bonmatin et al. (2021)30). Details are

1. Seven neonicotinoids (thiamethoxam, thiaclo-shown in Supplemental Text 3. Five neonicoti-prid, acetamiprid, imidacloprid, clothianidin, ni-noids (thiamethoxam, thiacloprid, acetamiprid,

tenpyram, and dinotefuran) and a metabolite,

imidacloprid, clothianidin) were targeted. Limits

DMAP, were analyzed by LC-MS/MS. For massof quantitation (LOQs) were defined as the con-

spectrometry, multiple reaction monitoring

centrations which gave S/N ratios (signal to(MRM) was programmed. The precursor andnoise ratio) of 10 in hair samples (Table 1). Pre-

product ions are shown in Table 1. Limits ofcision and accuracy were calculated from aJpn J Clin Ecol（Vol.32 No.2 2023）

64

blank hair sample (n=5) spiked at 1 ng/kg. As

Mindanao, between August 23th and November

the blank hair sample, we used a hair sample

10th 2017 (Table 2). All participants resided infrom the Philippine study which contained norural agricultural areas. While all adult men

30)

detectable trace of neonicotinoids . The hair

had an occupation related to potential pesticide

data were published alongside with results of

exposure (hereafter “pesticide-exposed”), such as

soil and water neonicotinoid analyses of the cor-farmers, gardeners, and farmworkers in fruit

30)

responding area .

plantation and citrus orchards, this proportion
was much lower in adult women (44/44 = 100%
vs. 8/27 = 29.6%; Chi-square test p < 0.0001; Ta-

Statistical Analyses
All sample analytical results were sent to De-ble2).

partment of Anesthesiology, Adachi Medical
Center, Tokyo Women’s Medical University fordata analysis. Relevant information about age,

Analyses of urine and hair – general results
In urine, at least one of the seven neonicoti-sex, occupation, pesticides use, and hair dye

noids or DMAP could be quantified in 78.8%

were combined with the sample analytical re-(78/99) of samples. Of these positive samples,

sults for statistical analysis. As previous reports

imidacloprid could be quantified in 60.2% of thesuggested residue data for urine and hair fol-samples (47/78, geometric mean = 0.048 ng/L),

30,34)

lowed a gamma distribution, they were log-

followed by thiamethoxam (39.7%), dinotefurantransformed for statistical analysis. The average(20.5%), clothianidin (17.9%), acetamiprid

concentrations are, therefore, represented by the(12.8%), DMAP (7.7%), nitenpyram (2.6%), andgeometric mean. In order to enable calculation

thiacloprid (1.3%) (Table 3). In 77 of 99 hairof geometric means of the neonicotinoid concen-

samples (77.8%), at least one of five neonicoti-

trations, <LOQ measurements was substituted

noids was detected above quantification limits.

by half of LOQ for consistency with our previous

Of these positive samples, imidacloprid was30)report . The detection threshold for the analy-

present in 76.6% of the samples (59/77, geomet-

sis was set at 50%. For comparison of geometricric mean = 0.076 ng/kg), followed by clothianidin

mean of two groups, a t-test was used. One-way(58.7%), thiamethoxam (39.0%), acetamiprid

ANOVA was used to compare hair and urine(16.8%), and thiacloprid (11.7%) (Table 3).

residue levels among people of the three age

Overall, the total concentrations of five neo-groups. The correlation between individual hairnicotinoids in urine and in hair were not corre-

and urine concentration was tested by a Spear-

lated (r=-0.008, p=0.94). Of the 99 participants,

man’s rank correlation test when an analyte wascases with both urinary and hair detections

detected no less than 50% both in urine and inabove the LOQ in the same person were foundhair. All statistical analyses were performed inin 25.3% of people for imidacloprid, 11.1% for

StatPlus version 7.3.32 (AnalystSoft Inc.2020).

thiamethoxam, 9.1% for clothianidin, and 53.3%
for the total concentration of five neonicotinoids.

Results

The relationships between the geometric mean

Demographic dataof each neonicotinoid concentration in urine andA total of 99 sets of urine and hair samples

hair are shown in Figure 2.

were collected from 99 volunteers in the three

Urinary ion analysis was performed in 93

Philippine islands i.e., Luzon, Marinduque andsamples (27 of 28 samples in children, 43 of 44

臨床環境医学（第32巻第2号）

Table 2. Demographic data of the participants.

ahttps://www.facebook.com/dacentralphilippines/posts/planting-calendar-heres-your-easy-guide-onwhen-is-the-best-time-of-the-year-to-/3133675680047184/ b M.A. Gutierrez et al. The Rice Planting Window
In The Philippines: An Analysis Using Multi-Temporal Sar Imagery. The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-4/W19, 2019. c Department ofAgriculture, Bureau of Plant Industry, Davao National Crop Research, Development and Production Support
Center, Bago Oshiro, Davao City. ‘Lakatan’ production. https://library.buplant.da.gov.ph/images/1640927353Lakatan%20Production%20Guide.pdf d Agricultural Training Institute, Production and Processing Calamansi. https://ati2.da.gov.ph/ati-4b/content/sites/default/files/2022-12/calamansi_final.pdf
eFarmers, gardeners, farmworkers and drivers in fruit plantation and citrus orchards. f Students, housewife,and house keepers.

65Jpn J Clin Ecol（Vol.32 No.2 2023）

66

Table 3. Results of neonicotinoid detection in urine (μg/L) and hair (μg/kg).

ageometric mean.

in adult men, and 22 of 27 in adult women) due

Difference among islands and age groups

to the too low volume of six samples. All tested

In Luzon and Mindanao Islands, imidacloprid

urinary ions were positive (Table 4). Urine Na

was the most frequently detected neonicotinoid

concentrations were lower than 15mEq/L in two

in both urine and hair samples, while in Marin-

participants from Mindanao. SIDu concentra-

duque Island the most frequently detected com-

tions were higher than 40 mEq/L in five partici-

pounds were thiamethoxam in urine and clo-

pants, two of which were adult men from Luzon

thianidin in hair (Figure 3, Supplemental Table

and Mindanao, two were adult women from Lu-

4). The total concentration of five neonicotinoids

zon and one was a child from Marinduque (fur-in hair was significantly higher in Mindanao Is-

ther details are shown in Supplemental Table 3).

land than in the other two islands (p<0.001, one

No significant correlation was observed between

way ANOVA). However, no such difference was

SIDu and the concentration of neonicotinoids in

observed for urine (Supplemental Table 4).

urine or hair. The Na/K concentration ratio was

The total concentrations of neonicotinoids in

5.19 ± 3.31 in adult men, 5.04 ± 2.06 in adulturine samples were lower in children (6 to 16

women, and 7.06 ± 4.35 in children (mean ± SD).

years old, n = 28) than in adults (17 to 39 years

臨床環境医学（第32巻第2号）

67

Figure 2. Comparison of neonicotinoid concentrations in urine and hair. The diameter of bubble represents the geometric mean of each neonicotinoid concentration. The center of bubble isplaced on the value of > LOQ detection of each neonicotinoid (%).
Table 4. Results of urinary ion level in participants (n=93)

Jpn J Clin Ecol（Vol.32 No.2 2023）

68

Figure 3. Comparison of neonicotinoid detection in soil, hair, and urine in the three islands. The
data of soil and hair were reposted from a previous study [Ref.30]. The number of soil
samples and participants were 20 and 31 in Luzon, 21 and 31 in Marinduque, and 17 and 37
in Mindanao, respectively.
old, n = 42) and seniors (40 to 83 year old, n =cide-exposed workers (n=42) and others (n=42)29) (p=0.030, p=0.044, respectively, one way(t-test, p=0.88, p=0.76, respectively).

ANOVA). By contrast, no significant difference

Imidacloprid concentrations were lower in

was found for imidacloprid and for the total con-hair (p=0.014) in adult women with dyed haircentrations of five neonicotinoids in hair (Table(n=9) as compared to women with un-dyed hair

5). The SIDu and Na/K concentration ratio did(n=18). The total concentration of five neonicoti-

not differ between children and either adults ornoids in hair was also lower in women with dyedseniors (p=0.92, p=0.20, respectively, one way

hair than with un-dyed hair (p=0.017) (Supple-

ANOVA).

mental Table 5).
According to the result above, we classified all

Differences between exposure categories

participants into four categories, i.e., Category A

The total concentration of the eight analyzed(children, n=28), Category B (adults and seniors

neonicotinoids in urine was significantly higher

with neither hair dye nor pesticide exposure,in pesticide-exposed workers (n=52) than in oth-n=13, M/F=1/12), Category C (adults and seniors

ers (n=47, not pesticide-exposed workers andwith no hair dye but pesticide-exposed workers,

children) (t-tests, p=0.030) (Table 6). The concen-

n=44, M/F=39/5), and Category D (adults andtration of imidacloprid and of the total concen-

seniors with dyed hair, n=14, M/F=5/9). Assum-

tration of five neonicotinoids in hair did not dif-

ing that hair neonicotinoid detection provides

fer between pesticide-exposed workers andevidence for chronic exposure, sensitivity andothers despite the fact that the maximum con-specificity of urinary neonicotinoid detection for

centrations were far higher in pesticide-exposed

hair neonicotinoid detection was examined (Sup-workers than in others. The SIDu and Na/K

plemental Table 6). Only in Category A, positive

concentration ratio did not differ between pesti-

likelihood ratio of urinary DMAP detection for臨床環境医学（第32巻第2号）

69

Table 5. Comparison of urine and hair analysis of each age groupageometric mean; b one way ANOVA test.

hair acetamiprid detection was more than 10

centrations in urine, and that an effect of hair

(13.0). In all categories, negative likelihood ra-

dye on the neonicotinoid concentration in hairtios were no less than 0.1. Likelihood ratios

can not be ruled out. Additional strong ion anal-

above 10 and below 0.1 are considered to provide

ysis in urine raises the hypothesis that substan-strong evidence to rule in or rule out diagnoses

tial renal tubular dysfunction might be preva-

35)

respectively in most circumstances .

lent in Philippines.
In Philippine rural area, most men work in

Discussion

agricultural settings, e.g. rice, banana, and hor-

This is the first analysis of human urinary ne-

ticultural fruits and vegetables. Neonicotinoids

onicotinoid contamination in the Philippines,

are routinely used as spray applications, coatedwhich reveals that it is ubiquitous in the Philip-

seeds, granular and drench formulations 36).

pines rural area. Our results suggest an effect ofClean drinking water supply is insufficient, es-

age and occupational pesticide use on the con-pecially in low-income class37). Even though theJpn J Clin Ecol（Vol.32 No.2 2023）

70

Table 6. Comparison of neonicotinoids and DMAP concentration between pesticide related
workers and others.

ageometric mean; b comparison of neonicotinoid or metabolite concentration between pesticide related
workers and others (t-test, two-tailed); c N.C., not calculable due to a low sample number.

information about the type of products andam, clothianidin, imidacloprid, acetamiprid andquantities used in particular crops was not

dinotefuran are registered as insecticides in theavailable, the lower frequency of detection andPhilippines40), but not thiacloprid, and shipment

median value of urinary DMAP in this study

volume data for this substance were not avail-(6.1%, <0.01 ng/L) as compared to previous stud-able. Nevertheless, thiacloprid was also detected38)

ies conducted in Japan (100%, 0.46 ng/L) , Gha39)na (94.7%, 0.41 ng/L) , and Sri Lanka (92.4%,
10)

in human samples in this study.
Being less intrusive and easy to collect, urine

by a laboratory with the same

may be the preferable matrix for screening pop-

method may suggest that the use of acetamiprid

ulations highly or chronically exposed to neonic-

in the three studied islands in the Philippines

otinoids and other pollutants, and urinary con-

was uncommon in the season when samples

centrations can be good biomarkers of brain

were collected. Products containing thiamethox-

neonicotinoid exposure for a short time. The de-

0.52 ng/L)

臨床環境医学（第32巻第2号）

71

tection rate and median concentrations or geo-easier to preserve and transport than urine

metric mean may be useful to compare the de-samples. However, the preparation for analysis

gree of neonicotinoid exposure between groups.

of hair samples is more laborious than for urine.

Lower urinary neonicotinoid concentrations in

Although the stability of pesticide residues inchildren than in adults and seniors suggest thathair is not known, it is presumed to be stable forthe age of participants seems to be an importantmany days if not months, based on the physico-factor when evaluating urinary neonicotinoid

chemical characteristics of neonicotinoids andconcentration.

their documented long-term persistence in the

Equal or higher levels of neonicotinoid concen-

environment2).

trations in hair than in urine, and the unsatis-

To explain the high neonicotinoid detection infactory positive and negative likelihood ratio of

hair, two factors can be considered, i.e., lipophi-

urinary neonicotinoid detection for hair detec-

licity and melanin binding. This is consistenttion in this study suggests that neonicotinoids

with the binding properties of 75 chemicals toresidue analysis in hair should be preferred

keratin45). Lipophilic substances are more likely

when aiming to assess chronic human exposure

to bind to keratin and other proteins. Althoughto pesticides, especially in children, in accor-

the lipophilicity of neonicotinoids is not very

dance with previous studies

41-44). Residues inhigh (Table 1), neonicotinoid binding to high af-

hair seems to be better indicators of chronic en-

finity proteins such as melanin may explainvironmental exposure, when occupational direct

their accumulation in hair. Melanin is a naturalcontamination or the effect of hair dye were ex-pigment found in the eyes, skin, nails and hair,cluded (i.e., excluding people with dyed hairand reportedly binds to a variety of drugs andfrom the analysis). Contamination during hairchemicals46-52). The structural characteristics ofsampling is deemed unlikely in this study be-

drugs and chemicals for melanin binding arecause we observed no significant difference inpresumably related to basicity, hydrophobicity,

imidacloprid concentration between the hair ofand charge-transfer49-59). Reilly et al.49) studied

pesticide-exposed workers and others, althoughchemical structures contributing to melanin

neonicotinoid concentrations in urine were muchbinding in silico and found that the most con-higher in pesticide-exposed workers. In real life

tributing structural characteristics were themonitoring surveys using hair, non-dietary con-number of rigid bonds and rings, which is relat-

tamination of hair (e.g., adsorption from parti-

ed to pi-pi interactions between the aromaticcles suspended in the air or in the rain) shouldmelanin structure and drugs. Neonicotinoids

nevertheless be considered. To clarify the signifi-

have rigid structures such as the chloro-pyridyl

cance of such differences and the possible bias

ring, the thiazole ring and the tetrahydrofuran

caused by non-dietary contamination, controlled

ring, suggesting they can likely form melanin-

toxicological studies in experimental animals

drug complexes.

with biomonitoring of hair, plasma and tissue

Taken together, these results suggest that ne-

samples including brain could be useful to deter-

onicotinoid detections in spot urine is preferable

mine the benefits of hair neonicotinoid analysis

to detect acute exposure in the general popula-to assess brain exposure to neonicotinoids.

tion, while the analysis of hair is more appropri-

An additional advantage of hair sampling is it

ate for studies of chronic exposure. However, in

is fast and easy, and samples are smaller andboth cases the results will not yet allow re-Jpn J Clin Ecol（Vol.32 No.2 2023）

72

searchers to assess personal exposure levels pre60)

cisely. Recently, Loser et al.

result of the 24h urine in the INTERMAP study

showed that imi-

from Japan, China, UK, and US (3.89 ± 2.11;

dacloprid, acetamiprid, clothianidin andmean ± SD; N = 4,680, age 49.17 ± 5.47 yrs)65).

thiacloprid have agonistic action on a model ofThis result might be caused by the comparative-

human developing neurons, i.e., dopaminergic

ly higher amount of water and salt intake toneurons generated from LUHMES neuronal pre-adapt to the hot climate in the Philippine. Fur-cursor cells and that SH-SY5Y neuroblastoma

ther investigation is needed to better under-

cells can be used as an established model ofstand and interpret such patterns.

nAChR signaling. They also showed that one of

A limitation of this study is that two recent

the imidacloprid metabolites, desnitro-imidaclo-

neonicotinoids, dinotefuran and nitenpyram,prid has strong binding affinity and efficacy on

and DMAP, one of major neonicotinoid metabo-

the human developing neurons, which was com-lites detected in urine were not analyzed in hair.

61)

62)

parable to nicotine . Nimako et al.

showed in

Our results may therefore have under-estimatedmice, that continuous low dose imidacloprid ex-

the total neonicotinoid exposure. Furthermore,posure caused higher desnitro-imidacloprid con-blood was not analyzed. Besides, the relatively

centration in liver, brain, testis, lung and kidneysmall number of participants in this study limits

than in blood, but no such pattern was visible

the power of statistical tests.

63)

for imidacloprid itself. Wang et al.

quantified

desnitro-imidacloprid in human urine, while64)

Conclusion

did not identify the urinary ex-

We reported urinary neonicotinoids concentra-

cretion of desnitro-imidacloprid after micro dosetion in the Philippine general population for theof imidacloprid intake. Continuous exposure to

first time. The frequency of detection of neonic-

neonicotinoids and their metabolites mightotinoid of hair and urine were comparable, buttherefore cause accumulation in tissue as well

the positive and negative likelihood ratio was

as in hair, even in the case of relatively low uri-unsatisfactory. Hair can be an essential matrix

Wrobel et al.

nary concentration, and therefore might induce

to screen chronic neonicotinoid exposure, espe-

negative effects for neurodevelopment.

cially for children. Future analytical and epide-

Considerable increases of SIDu were observed

miological studies are required to clarify the po-

in approximately 5% of Philippine participants.

tential links between chronic exposure to

If environmental exposure of neonicotinoids due

neonicotinoids and potential human healthto occupational use, drift inhalation, drinking

problems.

water and food contamination, represents risk ofchronic kidney disease, a global investigation isurgently needed. Abnormal decreases of urine+

Acknowledgements
We would like to thank Dr. Kazutoshi Fujioka

Na concentration were observed in two partici-

at Albany College of Pharmacy and Health Sci-

pants, but the clinical significance of this obser-

ences, USA, who gave us many insightful sug-vation is unknown. Interestingly, the urine ion

gestions to finalize this study.

analysis of spot urine revealed that the Na/K ratio among Philippine people (5.19 ± 3.31 in adultFundingmen, 5.05 ± 2.06 in adult women, and 7.06 ± 4.35

This study was supported by the Triodos

in children; mean ± SD), were higher than theFoundation’s Support Fund for Independent Re-

臨床環境医学（第32巻第2号）

quantification of neonicotinoid metabolites in humansearch on neonicotinoids and humans, a scientif-

urine by liquid chromatography coupled with mass

ic investigation into the neurotoxicological effects of neonicotinoid insecticides, awarded to
the Public Health Working Group of the Task

73

spectrometry. PLoS One. 8: e80332, 20139)

Marfo JT, Fujioka K, et al. Relationship between
Urinary N-Desmethyl-Acetamiprid and Typical

Force on Systemic Pesticide (Chair Dr. K. Taira),

Symptoms including Neurological Findings: A

and Grants-in-Aid for Scientific Research from

Prevalence Case-Control Study. PLoS One. 10:

the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) awarded
to Dr. K. Taira (No.15K00559). The funders had
no role in the study design, data collection or
analysis.

e0142172, 201510) Taira K, Kawakami T, et al. Urinary concentrationsof neonicotinoid insecticides were related to renal
tubular dysfunction and neuropsychological complaints in Dry-zone of Sri Lanka. Sci Rep. 11: 22484,
2021
11) Cimino AM, Boyles AL, et al. Effects of Neonicotinoid

Competing interests
Authors have no competing interest to disclose.
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Additional file（http://jsce-ac.umin.jp/200725/
files_jjce/32-2/index.html）

Supplemental Text 1. Details of urine analysis.
Supplemental Text 2. Details of ion analysis
in urine.
Supplemental Text 3. Details of hair analysis.
Supplemental Table 1. Neonicotinoids andmetabolites quantification in urine from thegeneral population.

57) Kronstrand R, Anderson MC, et al. Incorporation of

Supplemental Table 2. Neonicotinoids andselegiline metabolites into hair after oral selegiline

metabolites quantification in body fluid except

intake. J Anal Toxicol. 25: 594-601, 2001
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61) Loser D, Grillberger K, et al. Acute effects of theurine and in hair from the general population.
Supplemental Table 3. Profiles of two participants urinary Na were less than 15 mEq/L, andFive Participants Urinary Strong Ion Difference
were more than 40 mEq/L.
Supplemental Table 4. Comparison of urine
and hair analysis of the three islands.
Supplemental Table 5. Comparison of urine
and hair analysis of each age group.
Supplemental Table 6. Comparison of hair neonicotinoid concentration in adult women be-Jpn J Clin Ecol（Vol.32 No.2 2023）

76

tween dyed and un-dyed hair groups.
Supplemental Table 7. Relationships between
neonicotinoids detection in urine and in hair.

Declarations
Ethics approval and consent to participate
This study was reviewed and approved by the
ethic committee of Tokyo Women’s Medical University (Approval number 4521). Written informed consent to participate in this study was
obtained from each participant.
Consent for publication
Not applicable.
Data availability statement
The data that support the findings of thisstudy are available from the corresponding author at VFG 03077@nifty.com, upon reasonable
request.
Authors’ contribution
KT, EADM, AT, FSB, AS, MBL, JMB, FC, YA
and MK contributed to the substantial conception and designed the work.
EL and FC contributed to the sample acquisition.
GG, YM and YI contributed to the sample
analysis.
KT, AA, GG, EADM, FSB, AT, JMB, AS contributed to the interpretation of data.
KT, AA, GG, EADM, YI, FSB, JMB, AS, andYM drafted the manuscript and revised it.