Urinary Concentrations of Neonicotinoid Insecticides and Tubular Biomarkers, and Clinical Symptoms in Chronic Kidney Disease Patients, Their Family Members and Others in Dry-zone of Sri Lanka: a Small Scale Field- based Case-Control Study

Kumiko Taira (  VFG03077@nifty.com ) Tokyo Women's Medical University Medical Center East: Tokyo Joshi Ika Daigaku Higashi Iryo Center https://orcid.org/0000-0001-6988-5060 Tomonori Kawakami Toyama Prefectural University Toyama Campus: Toyama Kenritsu Daigaku Toyama Campus Sujithra Kaushaliya Weragoda National Water Supply and Drainage Board H.M.Ayala S. Herath National Water Supply and Drainage Board Yoshinori Ikenaka Hokkaido University Faculty of Veterinary Medicine Kazutoshi Fujioka Albany College of Pharmacy and Health Sciences Madhubhani Hamachandra Arizona State University Nirmalie Pallewatta University of Colombo Faculty of Science Yoshiko Aoyama Aoyama Allergy Clinic Mayumi Ishizuka Hokkaido University Jean-Marc Bonmatin CNRS Campus Orleans Makiko Komori Tokyo Women's Medical University Medical Center East Department of Anesthesiology

those of organophosphate insecticides, and exert a serious impact on ecosystems in many countries [62,63].
Neonicotinoids exposure may cause renal dysfunction, which supposed to be caused by human nAChR modulation. The acute toxicity of neonicotinoids for humans had been said to be not as strong as organophosphates; therefore, the use of neonicotinoids is growing rapidly as alternatives to organophosphates. Neonicotinoids formulation containing imidacloprid, acetamiprid and thiacloprid caused acute intoxication by ingestion such as cardiovascular symptoms (tachycardia, bradycardia, arrhythmia, hypertension, hypotension), neurological symptoms (low Glasgow Coma Scale, unconsciousness, sleepiness, dizziness, convulsion, excitation), respiratory symptoms (dyspnea, tachypnea, respiratory arrest, cough, cyanosis), gastrointestinal symptom (nausea, vomiting, diarrhea), secretion symptoms (diaphoresis, anhydrosis, excessive discharge of saliva and bronchial secretion, mouth dryness), pupil symptoms (mydriasis, miosis, abnormal light re ex), abnormal body temperature (fever, low body temperature), skeletal muscle symptoms (muscle weakness, muscle spasm, high creatinine kinase in blood test), metabolic acidosis, and death [65,66]. Metabolic acidosis is commonly caused by renal tubular disorders. An in vivo study showed that nicotine caused CKD by direct effects on tubular protein reabsorption via α7-nAChR [67]. Another in vitro study showed nicotine induced podocyte apoptosis through reactive oxygen species generation and association [68]. In addition, the pesticide formula contains some additives as surfactants and solvents, which are more toxic than the active substances [69][70][71]. Common neonicotinoid formulations contain renal toxic additives, such as dimethyl sulfoxide, N-methylpyrrolidone, diethylene glycol, propylene carbonate and mineral oil. Multiple acute kidney injury (AKI) episodes may cause CKD as the nal stage of chronic renal pathological conditions [72].
Subacute and chronic neonicotinoids exposure may also cause tubular disorders. We previously reported in our experience that the consecutive intake of tea beverage and/or fruits contaminated with neonicotinoids, may cause similar symptoms as acute intoxication. Some typical symptoms were signi cantly found in patients with neonicotinoids detection in urine, i.e. general fatigue, headache, chest pains, palpitation, stomachache, muscle pain/ spasm/ weakness, shoulder stiffness, cough, fever (> 37°C), and nger tremor, as well as electrocardiographic abnormalities [73][74][75]. In those cases, oliguria and the increase of urine Cystatin-C were found [75]. It was assumed oliguria was caused by renal hypoperfusion by nAChR action and increase of urine Cystatin-C was caused by direct or indirect tubule action by neonicotinoids. These patients also complained of dizziness upon standing, skin eruptions, sleeplessness, edema, low urine volume, high urine volume, constipation, diarrhea, skin itching, appetite loss, reduced body weight, and increased body weight. In addition to those symptoms, altered consciousness/dreamy state, recent memory loss with compulsive behaviors, agitation/fear/anger, sudden change of senses of smell, auditory or visual hallucinations, and abnormal behavior [75], which had been reported in a case study of myasthenia gravis patients anti-nAChR antibody were positive [76] were also complained of occasionally. We hypothesized that neonicotinoids caused those symptoms through nAChR action, because all those symptoms were almost always reversible in neonicotinoid intoxication. Chronic occupational exposure of neonicotinoid formulations containing imidacloprid caused renal disorders, such as hematuria and interstitial nephritis, as well as liver dysfunction and leukoclastic vasculitis [77]. In animal study, e.g. 0.2 and 0.4 mg/kg/day thiamethoxam for 15 days to male mice caused renal pathological change in parenchyma [78].
Neonicotinoids absorbed via the intestines and lungs, pass through blood brain barrier and are mainly excreted in the urine [79,80]. They do not bio-accumulate since they are water soluble; however, their concentrations in tissues may remain at a steady state, or even increase, through continuous exposure [79,81]. An active neonicotinoid metabolite, N-desmethyl-acetamiprid (DMAP) and dinotefuran pass through placenta and were detected in the urine of newborns [82]. As the route of neonicotinoid exposure in agricultural areas, occupational use and food/beverage intake can be considered.
There is scienti c evidence that neonicotinoids and the metabolites are frequently detected in human urine samples from healthy volunteer as well as the patients with neonicotinoid intoxication [74,75,79,81,82]. DMAP is one of the most frequently detected metabolite of acetamiprid. Spot urine is one of the most popular matrices to use in screening human environmental chemical exposure, such as heavy metals [25], water-soluble pesticides [74,83] [90], caffeine [91], and personal care products [10,92]. Whether urine is an appropriate sample for evaluating neonicotinoid exposure in CKDu patients or not is unknown, because of their low urine concentration ability.
To elucidate the neonicotinoids exposure in people living in CKDu-epidemic area by urine analysis can be the rst step of an appropriate pesticide regulation to reduced the patients suffered by CKDu.

Methods
We conducted a small-scale eld-based screening survey in Wilgamuwa Divisional Secretariat in the Matale District of Central Province and Anuradhapura city in North Central Province in Sri Lanka, both included CKDu-epidemic areas (prevalence of CKDu are more than 10%), to con rm the evidence of neonicotinoid exposure. Matale District has 484,531 population in 2012 and the percentage of households reporting at least one member diagnosed with CKD who reside in the household between 2009 and 2018 was 16.7% [93]. Anuradhapura has 854,602 population in 2013, the percentage of households reporting at least one member diagnosed with CKD who reside in the household between 2009 and 2018 was 18.9% [93]. Unpublished database by Water Supply Scheme in Sri Lanka indicated 10,288 CKDu patients (1.2%) was identi ed and the prevalence varied from 0 to 16.47 % in 692 areas in Anuradhapura in 2013. In the urban area with a clean water supply, the prevalence of CKDu is low and in the agricultural area with ground water use, it is high.
We collected spot urine samples and compiled questionnaires of health and symptoms from CKD patients, their family members, and neighbors in two different seasons. We investigated mainly the next ve points.
(1) Urinary metal/metalloids exposure analysis: We found that arsenic, cadmium, lead, and chromium concentrations in human urine samples from CKDu-epidemic areas were not signi cantly different from those from not-CKDu-epidemic areas (Already published by Herath et al. in 2018 [25]).
(2) Basic urine analysis and the assessment of renal tubular activity by new urine biomarkers: Human liver-type fatty acid-binding protein (L-FABP) is cytotoxic oxidation products secreted from proximal tubules under ischemia and oxidative stress, and can be a biomarker for the early detection of CKD and AKI in humans [94,95]. In the absence of renal diseases, L-FABP secreted from the liver into the blood crosses the glomerular barrier and then is reabsorbed by the proximal tubular cells. As a result, L-FABP hardly appears in urine. When proximal tubule damage is active, L-FABP appears in the urine; but when proximal tubule change is not active and in high urine volume, L-FABP would stay in the normal ranges. The reference value of L-FABP is no more than 8.4 µg/g Cre. The result of L-FABP analysis in urine was partially published with the metal/metalloids exposure analysis report [25]. No relationships between the L-FABP concentration and concentrations of arsenic, cadmium, lead, and chromium in urine was observed.
Cystatin-C is a low molecular weight protein (~ 13.3 kDa) originating from the cell, secreted in the urine from glomerulus constantly and reabsorbed from the proximal tubule [96]. Creatinine is another small molecule (Mw 113.1 g/mole) that originates from the skeletal muscle, and is secreted in the urine from the glomerulus but is not reabsorbed from the tubule. Urine creatinine adjusted Cystatin-C elevation suggest renal tubular disorders, and signi cant correlation was observed in CKDu patients [13]. The reference value of urine Cystatin-C is no more than 70 µg/g Cre. Whether L-FABP can be good biomarkers of CKD, as well as Cystatin-C and UACR (creatinine adjusted albumin concentration in urine) in these areas was investigated.
(3) Acute occupational neonicotinoid exposure: It can be identi ed by a spot urine neonicotinoid analysis, ideally just after the neonicotinoids were applied in the area [97]. The schedule of when farmers apply neonicotinoids for rice cropping are not strictly determined by the almanac. Traditionally, there are two seasons for rice cropping in Sri Lanka, Yala (from April till August) and Maha (from September to January). They determine the beginning of the seasons by the rain, and then start rice seed sowing and spraying pesticides. Originally, Maha season falls during the northeast monsoon, but recently frequent draught diminished the farmers time for rice cropping [98]. As a result, we went to urine sampling in late May and mid December in 2015. Additionally, a few o cial records of pesticide registration for rice cropping in Sri Lanka were tried to obtain; and we interviewed two clerks in the pesticide shops in CKDu-epidemic area to know what kinds of pesticides were sold in each season.
(4) Environmental neonicotinoid exposure: It also can be identi ed by spot urine neonicotinoid analysis. Concerning the environmental source of exposure, drinking water, tea, rice, vegetables and fruits could be considerable.
Inclusion of the families of CKD patients and neighbors in the same district can be helpful, because in general urine concentrating ability of CKDu patients and xenobiotics excreting ability of CKD patients are supposed to be compromised or lost. At the same time, tea leaves that the participants daily consumed, and water samples that they were drinking were collected. They traditionally drink milk tea with spice many times every day. Tea leaves and water samples were analyzed at Hokkaido University. Ten tea leaves samples that 10 CKD patients daily consumed had been analyzed and no neonicotinoid was detected from all 10 tea leaves samples. The detailed results have already been published [100]. Their daily drinking water was also analyzed in Toyama Prefectural University, but no neonicotinoid was detected (not published data).
(5) Effect of renal tubule activity on urinary neonicotinoid concentration: When urine concentrating ability is compromised or lost by impairment of renal tubule activity, urinary xenobiotic concentration would become lower.
Relationship between Cystatin-C and neonicotinoid concentration in urine was investigated.
(6) Symptoms related to neonicotinoids intoxication: Neonicotinoid exposure may cause many clinical symptoms. Systemic analysis of clinical symptoms related to neonicotinoid exposure can be helpful to assess the probability of neonicotinoid exposure.

Subjects and sample collection
This study was conducted as a part of the Sri Lanka CKDu survey by Professor Kawakami Tomonori, Toyama Prefectural University. After ethic committee's approval of Tokyo Women's Medical University (No. 2810R2) and obtaining written informed consent from participants, in May 2015, approx. 50ml of spot urine samples were collected from 33 residents in Wilgamuwa and Anuradhapura, and in December 2015, 59 residents in Anuradhapura, who had ever been diagnosed as CKD, the family, or healthy individuals. Systemic questions to each participant about physical and psychological conditions were also performed by a trained staff, and recorded in the documents. That include typical symptoms observed in subacute neonicotinoid intoxication (general fatigue, headache, chest pains, palpitation, stomachache, muscle symptoms (muscle pain/ spasm/ weakness, shoulder stiffness), cough, fever (> 37°C), nger tremor, and recent memory loss with food diary), and other symptoms previously observed in subacute neonicotinoid intoxication patients (altered consciousness/dreamy state, recent memory loss with compulsive behaviors, agitation/fear/anger, sudden change of senses of smell, auditory or visual hallucinations, abnormal behavior, dizziness upon standing, skin eruptions, sleeplessness, edema, low urine volume, high urine volume, constipation, diarrhea, skin itching, appetite loss, reduced body weight, and increased body weight). Recent memory loss with food diary was diagnosed when the patient could not recall or ll out a questionnaire of recent meals asking what (s)he ate in the previous three days.
Each urine sample was divided into four plastic tubes, one is analyzed on the day of sampling by staff, and the rest of three samples were kept in a refrigerator.
Then one out of three samples was send to Hokkaido University, (Sapporo, Hokkaido, Japan) and kept in a freezer at -20°C until LC-MS/MS analysis. Another one out of three samples were sent to a commercial laboratory IKAGAKU (Kyoto, Japan) to quantify urinary Cystatin-C and creatinine. The last one was used to analyze L-FABP and trace minerals, and the method and the result was reported in the previous literature [25].
The use of pesticides in CKDu affected area was surveyed by the interview of the clerks in the pesticide shops. Our research group members interviewed clerks from two shops (A and B), who sold pesticides to farmers in Anuradapura. At the same time, a few o cial records of pesticide registration for rice cropping in Sri Lanka were tried to obtain. Urine Analysis 1. Simple urine chemistry analysis on the day of sampling.

Quantitative analysis of neonicotinoids and a metabolite by LC-ESI/MS/MS
Materials Acetamiprid, dinotefuran, imidacloprid, nitenpyram and thiacloprid were purchased from Kanto Chemical Corp.

Urine sample preparation
Urine was thawed, stirred, and allowed to stand for some time and the supernatant was used. Puri cation of the urine was performed by solid phase extraction (SPE). One hundred µL of internal standard mixture (each 10 ppb) was added to 100 µL of each urine sample, and then 2800 µL of distilled water was added to the sample. Two types of SPE cartridges were used for puri cation: an InertSep Pharma SPE column (60 mg/3 ml) (GL Science, Tokyo, Japan) pre-conditioned with 3 mL of an acetonitrile/dichloromethane (1/1) mixture followed by 3 ml of distilled water; and an InertSep PSA SPE column (100 mg/1ml) (GL Science) pre-conditioned with 1 mL of the acetonitrile/dichloromethane (1/1) mixture. Prepared samples were loaded on the pre-conditioned InertSep Pharma and washed with 0.5 mL of distilled water. The InertSep Pharma (top) was combined with the InertSep PSA (bottom) and 3 ml of the acetonitrile/dichloromethane (1/ 1) mixture were used to elute the target chemicals. After concentrating and dry-solidifying with a centrifugal concentrator (CVE-200D with UT-2000, Eyela, Tokyo, Japan), the samples were reconstituted with 100 µL of 3% methanol in distilled water and transferred to vials for analysis.
Seven neonicotinoids and DMAP were analyzed in each sample. A LC-ESI/MS/MS system (Agilent 6495B, Agilent Technologies, Santa Clara, CA, USA) equipped with a Kinetex Biphenyl column (2.1 mm ID × 100 mm, ϕ2.6 µm, Phenomenex, Torrance, CA, USA) was used for quantitative analysis. Mobile phases used were 0.1% formic acid + 10 mM ammonium acetate in aqueous solution (A) and 0.1% formic acid + 10 mM ammonium acetate in methanol (B). The gradient was linearly programmed as: t = 0 to 1 min: 5% B, t = 6 min: 95% B, t = 6 to 8 min: 95% B at a ow rate of 0.5 ml/min. The column oven temperature was 60°C. For mass spectrometry, multiple reaction monitoring (MRM) was programmed. The MRM transition of precursor and product ions are shown in Table 1. The recovery e cient of each neonicotinoid and its metabolites ranged from 80 to 120 %. The reproducibility of the analysis system was con rmed in the duplicate analyses of each sample, with a relative standard deviation (RSD) of 10% for all the compounds.

Quanti cation of neonicotinoids and a metabolite
Seven neonicotinoids and DMAP were analyzed in each sample. Six deuterium-labeled neonicotinoids were used as internal standards. Quanti cation of the neonicotinoids and a metabolite was carried out by the internal standard method. Five calibration points were set at 0.5, 1.25, 2.5, 3.75 and 5 ppb, whereas the internal standard was used to 5 ppb at all calibration points.
Quality control and quality assurance A mixture of six deuterium-labeled neonicotinoids was spiked into samples as an internal standard prior to sample preparation and extraction. Quantitation was performed using ve calibration points and the average coe cients of determination (r 2 ) for the calibration curves were ≥ 0.995. The analytical method was checked for precision and accuracy. Limits of quanti cation (LOQs) were calculated based on 3SD/S (SD is the standard deviation of the response of seven replicate standard solution measurements and S is the slope of the calibration curve). Recovery % and LOQs (µg/L) of the analytes are given in Table 1.

Results
The demographic data of the volunteers of urine samples (Table 2) Overall, 15 spot urine samples were collected from 15 CKD patients previously diagnosed by doctors, and resided in CKDu-epidemic areas (local prevalence more than 10%) in Wilugamuwa and Anuradhapura. We could not con rm through a medical exam, whether the CKD patients were CKDu or not. Seventy-seven urine samples were collected from non-CKD participants including 15 CKD patients' family members (CKD family members) and 62 healthy individuals (neighbors). Most of the CKD patients were male, 75 %, (in non-CKD participants 35.1%, p < 0.001, Chisquare test) and the age was older, 54.9 ± 13.1 years old (non-CKD participants 40.5 ± 17.7 years old (mean ± SE), p = 0.009, t-test). 62 neighbors resided in Wilugamuwa or Anuradhapura, but whether their working place was CKDuepidemic areas or not was not con rmed. The status of pesticides applied onto the rice paddies In summary, we could not nd any concrete evidence of a large scale application of neonicotinoid insecticides on rice paddies in the studied area in May 2015 (Yala season) or in December 2015 (Maha season). In both season, rice seed sowing seems to be performed and pesticides applied to rice paddies, because signi cant crops were reported in Anuradhapura and Matale district by the national record [98,99]. Whether any pesticide was applied to rice paddies or not was not con rmed. Unfortunately, we could not obtain a list of the registered pesticides in Wilgamuwa or in Anuradhapura. We could only obtain a list of the registered pesticides in other CKDu-epidemic districts, Kandy and in Negombo (Supplemental Table 1). Among the category of neonicotinoids, imidacloprid was registered in Kandy, while imidacloprid and thiamethoxam in Negombo.
An interview with a clerk in shop A in Mihintale, Anuradapura revealed that glyphosate was commonly used from April to May, MCPA, 3-4 DPA (propanil) and Gulliver (azimslfuron) from June to July, and Avimavar (imidacloprid), Mospilan (acetamiprid) and Marshal (carbobulfan) in August. However, from September to next March no speci c pesticide was sold (Supplemental Table 2 Basic urinary ndings The result of the urine analysis is shown in  Table 3).

Discussion
The urine neonicotinoids and a metabolite analysis revealed the environmental neonicotinoids exposure, such as by food intake or by pesticide drift, seems to be common in Wilgamuwa and in Anuradhapura. Among them, the Phase-I metabolite of acetamiprid, DMAP, was detected from almost all participants. The source of exposure does not seem to be tea leaves [100] nor drinking water (not published data). Presumably, rice, vegetables, fruits, or milk are suspected to be potential dietary sources of acetamiprid exposure. Dinotefuran and thiacloprid which was not registered in Sri Lanka in 2015 were also detected. It suggests dinotefuran and thiacloprid were contaminated in the imported food or domestic food by use of illegally imported pesticides.
Symptoms CKD patients frequently complained of might suggest the pathology of CKDu. 1. Fever: 46.7% of them complained of fever. It can be one of the nicotinic symptoms but another possibility is that it might be a symptom of infection or immunological disturbance. Farther investigation is needed. 2. Neurological symptoms: CKD patients might be exposed to neurotoxic substances chronically or routinely. They complained of symptoms not only that was common in CKD, such as high volume urine, appetite loss, reduced body weight, and constipation but also fever and symptoms suggesting neurological and neurobehavioral disorder such as nger tremor, and abnormal behavior. It is well known that a variety of xenobiotics including organophosphate insecticides, neonicotinoid insecticides and herbicides have neurotoxicity. The off-target toxicity is common in xenobiotics, such as pharmaceuticals and pesticides, because they are low molecules with high a nity to strands of amino acids and nucleotides, e.g. enzymes, ion channels, receptors, and genomes. Reportedly, organophosphate acetylcholine esterase inhibitors, have secondary targets, e.g. neuropathy target esterases (NTE), fatty acid amide hydrolase (FAAH), KIAA1363, and monoacylglycerol lipase (MAGL), and cause off-target toxicity, e.g. organophosphateinduced delayed neuropathy [101]. Organophosphate insecticides, profenofos and diazinon seem to be the rst line insecticides in Sri Lanka [57]. The herbicide glyphosate has also secondary off-target toxicity in the mammalian brain [102]. It may cause limbic encephalopathy after occupational exposure [103]. Our results indicate that CKD patients appear to have acute and chronic intoxication of xenobiotic neurotoxicants.
The renal tubular biomarkers L-FABP as well as urine Cystatin-C in addition to other biomarkers previously investigated in the area [13,14] would be useful for early diagnosis of CKD in CKDu-epidemic area and searching the therapeutic approach with the strategy for prevention. Higher L-FABP and urine Cystatin-C values are frequently observed in the CKD patients in CKDu-epidemic areas, as well as UACR. Additionally, we found that several participants in CKD family members and neighbors with high L-FABP and urine Cystatin-C; suggesting that they were under diagnosed. At the same time, we observed very low level of Cystatin-C with low creatinine and L-FABP levels in several neighbors. This phenomenon is very rare in Japan [75], which suggests an extremely high intake of water that, in turn, causes a low concentration of these biomarkers, urine was sampled during diuretic period of acute kidney injury, or possibly there were problems in the sample preservation.
Relationship between clinical category (CKD or not), urine Cystatin-C level, urine neonicotinoids and DMAP level and clinical symptoms in this study were summarized in Table 9. CKD patients in CKDu-endemic area were characterized by high urine Cystatin-C and seven symptoms (high volume urine, appetite loss, reduced body weight, constipation, fever, nger tremor, and abnormal behavior) but not by urinary neonicotinoids and DMAP concentration.
However, we must keep vigilant concerning the occupational exposure of neonicotinoids and pesticide drift exposure as one of the risk factor of CKDu, because neonicotinoids themselves and their formula have renal toxicity and not so low concentration was observed in some CKD patients for DMAP and imidacloprid. It is known that urinary excretion of DMAP and imidacloprid is slower and more persistent than other neonicotinoids [79]. Lia et al. suggested that the high variance of detected creatinine corrected level of urinary neonicotinoids [104], but the level of DMAP was rather consistent. CKD patients frequently complained of typical symptoms previously observed in subacute neonicotinoid intoxication in Japan [75]. The interview of the clerks of pesticide shops, and Sri Lanka government data [98,99] suggested that neonicotinoids might be applied in the CKDu-epidemic area preceded by our urine sampling. In a previous study, lower urinary neonicotinoid concentration in CKDu patients than in healthy volunteers living in non-CKDu epidemic area was reported [39]. More comprehensive biomonitoring of pesticides in farmers is needed. As shown in Fig. 2, low neonicotinoids concentration in urine might not always mean low level exposure of neonicotinoids in the patients with high urine Cystatin-C. To assess the xenobiotic pathogenicity to the kidneys, quanti cation of a peak dose of xenobiotic exposure is essential. Alternatively, hair and blood analysis to evaluate xenobiotic exposure in epidemiological setting could be considerable.
We also found that acidic urine was prevalent in this area. It might be caused by high consumption of tea drinks.
Black tea leaves contain many organic acidic compounds, such as gallic acid, epigallocatechin gallate and other catechins [105]. Recent literature suggests that CKDu may have the background of gene polymorphism [50]. It is reasonable that a group in population is more sensitive to xenobiotic nephrotoxins, especially when other risks of acute kidney injury coexisting, e.g. dehydration in hot temperature, physical labor, low urine pH, hypertension and old age. Although we found that the participants from whose urine clothianidin and imidacloprid detected, aluminum and manganese was more quanti ed in this area, the clinical signi cance was unknown because the detection rate of the two neonicotinoids were rather low. Farther investigation is needed.
The limitation of this study is, the sample size was small, CKD diagnosis was not certi ed by physician directly, the history of pesticides exposure in participants could not investigated thoroughly, and no repetition of sampling was performed. Whether seasonal change of neonicotinoids detection in urine was caused by the method of farming or food intake was unknown. Anyways, urine detection of nephrotoxic pesticides from the people in CKDu-epidemic area were not ignorable towards sustainable agriculture is desired. We recommend that occupational and environmental exposure to neurotoxic pesticides through diet and application of pesticide formulations should be kept as low as possible in CKDu-epidemic area.

Conclusion
We conducted a small-scale eld-based case-control study of urinary neonicotinoids/a metabolite and symptoms in 15 CKD patients, 15 CKD patient's family and 62 neighbors, in the Dry-zone of Sri Lanka in 2015. In the urine, Ndesmethyl-acetamiprid (DMAP, the rst metabolite of acetamiprid) was detected at the highest rate, followed by dinotefuran and thiamethoxam; and the detection levels in the CKD patients were lower than non-CKD participants.
CKD patients exhibited more symptoms and their complaints were more signi cant than non-CKD participants.
Those include neurological/psychological symptoms, e.g. nger tremor and abnormal behavior, and common symptoms of CKDu, e.g. general fatigue, high volume urine, appetite loss, and reduced body weight. CKD patients in this area appeared to have intoxication of neurotoxic xenobiotics including pesticides. Urine detection of nephrotoxic pesticides from the people in CKDu-epidemic area were not ignorable toward sustainable agriculture is desired.

Declarations Competing interests
The authors declare that they have no competing interests.

Funding
This study is partially supported by Japan Endocrine-disruptor Preventive Action,