Chlordane induced neurotoxicosis in urban and suburban Detroit, Michigan striped skunks (Mephitis mephitis)

Chlordane induced neurotoxicosis in urban and suburban Detroit, Michigan striped skunks (Mephitis mephitis) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Chlordane induced neurotoxicosis in urban and suburban Detroit, Michigan striped skunks (Mephitis mephitis) John Buchweitz, Rachel Sheffler, Birgit Puschner, Scott Fitzgerald, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4546233/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Despite the ban of technical chlordane, contamination from this persistent organic pollutant threatens wildlife and human health nearly forty years since its last application. In this study, eight of seventeen skunks displaying illness and neurologic signs had brain tissue concentrations of combined oxychlordane, heptachlor epoxide, and trans-nonachlor exceeding a 1,000 ng/g wet weight diagnostic threshold for toxicosis. Liver tissue concentrations were ten-fold greater than brain when measured on a lipid weight basis, which can help predict lethal brain residues in skunks. The ongoing presence of chlordane in the environment is expected to cause further unintended consequences for wildlife across the Detroit Metropolitan area for decades to come. As with other pollutants in the River Rouge and River Raisin Areas of Concern, the presence of chlordane in the urban environment presents a significant risk for animal, human and ecological health. Health sciences/Diseases/Neurological disorders/Neurotoxicity syndromes Earth and environmental sciences/Environmental sciences/Environmental chemistry/Pollution remediation Wildlife Chlordane Persistent Organic Pollutant Organochlorine Great Lakes Neurotoxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Across the Great Lakes basin, decades of industrial discharges and agricultural runoff have contaminated soil sediments with persistent organic pollutants including organochlorine pesticides, polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), heavy metals, oil, and grease. 1 , 2 The vast freshwater ecosystem surrounds the state of Michigan and interconnects with the state's most urbanized and densely populated watersheds; these watersheds include the River Rouge and River Raisin which span the greater Detroit metropolitan area across Wayne, Oakland, and Washtenaw counties. 1 – 4 The River Rouge was designated an Environmental Protection Agency (EPA) Area of Concern (AOC) under the Great Lakes Water Quality Agreement of 1987. Multiple compounds were banned throughout the 1970s whose manufacture and use contributed to watershed contamination including PCBs, once used in coolants and lubricants for transformers and electrical equipment, and organochlorine pesticides including chlordane, dieldrin, and DDT previously used in insect control. Technical chlordane is a synthetic mixture of more than 140 components that was extensively used in the United States until it was banned in 1988. Due to its chemical stability, chlordane continues to persist in the environment today despite bans on its production and use in the United States and many countries globally. The composition of technical chlordane is 60% cis- and trans-chlordane and 40% related compounds including heptachlor, trans-nonachlor, cis-nonachlor, chlordene, and other minor constituents. 5 These compounds have environmental half-lives of 10–20 years, are resistant to environmental breakdown, and continue to pose a risk to wildlife and public health. 6 Mammalian, avian, and microbial metabolism favors the trans isomer of chlordane and results in the formation of the epoxide metabolites, oxychlordane and heptachlor epoxide, which accumulate in fat. 7 – 11 Before its ban in the United States in 1988, chlordane was widely used for termite control around the foundation of homes, for the control of scarab beetle populations on golf courses, and for agricultural pest management. 5 , 6 , 12 Termiticides applied around foundation walls remain within the upper 6–8 inches of soil without vertical migration to deeper layers. 6 Soil testing has shown low concentrations of oxychlordane and heptachlor epoxide with a greater presence of cis- and trans-chlordane. 12 , 13 In contrast, significant residues of oxychlordane and heptachlor epoxide have been documented in oriental scarab beetles ( Anomala orientalis ), other species of beetles, and earthworms. 12 In suburban and urban areas where chlordane was liberally used, it continues to contaminate habitat and food sources for wildlife with pesticide accumulation documented in the tissues of bats 12 , 14 , 15 , racoons 16 , 17 , cats 18 , dogs 18 , songbirds 7 , 12 , 19 , 20 , raptors 21 , and now skunks. Skunks that burrow under porches and areas near foundations while consuming insectivorous diets that include beetles, earthworms, bees, and caterpillars, can be predicted to have significant chlordane metabolite residues. 22 Chlordane and its metabolites are reported to cause neurotoxic effects such as tremors, salivation, ataxia, depression, vomiting, and seizures in man 23 , 24 , mammals 5 , 25 , and birds 7 , 19 . Specifically, oxychlordane, a metabolite of chlordane, is six times more toxic to birds, and heptachlor epoxide, the metabolite of heptachlor, is ten times more toxic to rats than their respective parent compound. 7 , 12 Herein, we employed the use of a highly sensitive and selective gas chromatography tandem mass spectrometry method to detect high concentrations of chlordane metabolites in skunk tissues collected from urban and suburban neighborhoods in Michigan. Our findings provide the first evidence of skunks succumbing to neurotoxicosis by chlordane exposure. Cases of neurotoxicosis in wildlife are likely under reported due to insufficient surveillance, an initial presumption of rabies or infectious disease, or lack of funding and access to comprehensive toxicology testing. But such information is necessary for assessing the potential threat consequent to the past use of chlordane in the urban environment. Materials and Methods 2.1 Clinical Sample Collection and Initial Diagnostic Workup Five female and twelve male skunks found dead or moribund were submitted to the Michigan Department of Natural Resources Wildlife Disease Laboratory (MDNR WDL) for necropsy (Lansing, MI). The skunks were assessed for age and reported as five adults and twelve juveniles. Brain stem and cerebellum were submitted to the United States Department of Agriculture, Wildlife Services or the Michigan Department of Health and Human Services (MDHHS) for routine rabies screening. Animals were screened for canine distemper virus and three animals were screened for highly pathogenic avian influenza virus through the diagnostic service at the Michigan State University Veterinary Diagnostic Laboratory (MSU VDL). Liver samples were analyzed for drugs, pesticides, and environmental pollutants using gas chromatography mass spectrometry (GC/MS), based on methods previously described by this laboratory. 15 Brain samples were also tested for desmethylbromethalin, a neurotoxic metabolite of the rodenticide bromethalin, by liquid chromatography tandem mass spectrometry (LC-MS/MS). Both brain and liver tissues were quantitatively assessed for organochlorine and PCB concentrations, with confirmed positive findings for chlordane and heptachlor metabolites from mass spectral screening. Control tissues from bovine brain and canine liver, obtained from other necropsy submissions to the veterinary diagnostic laboratory, tested negative for these compounds by GC/MS. 2.2 Pesticide Standards Organochlorine pesticide mix containing alpha-benzene hexachloride or hexachlorocyclohexane (BHC), beta-BHC, gamma-BHC (Lindane), delta-BHC, alpha (cis)-chlordane, gamma (trans)-chlordane, 4,4’ dichlorodiphenyltrichloroethane (DDT), 4,4’ dichlorodiphenyldichloroethylene (DDE), 4,4’ dichlorodiphenyldichloroethane (DDD), aldrin, dieldrin, endrin, endrin aldehyde, endrin ketone, endosulfan I (alpha), endosulfan II (beta), endosulfan sulfate, heptachlor, and heptachlor exo-epoxide was acquired from MilleporeSigma (Sigma-Aldrich Corporation-St. Louis, MO, USA). Oxychlordane, heptachlor exo-epoxide, and heptachlor endo-epoxide standards with chromatographic purity were purchased from Agilent Technologies (Santa Clara, CA, USA). PCB standards for congeners #52, 101, 118, 153, 138, 187, 183, 126, 180, 170, and 209 were obtained from ChemService (West Chester, PA, USA). 2.3 Solvents and Reagents Acetonitrile UV (chromatography grade) and isooctane (chromatography grade) were purchased from Burdick and Jackson (Muskegon, MI, USA). Hexane, isopropanol, and anhydrous sodium sulfate were purchased from VWR (Radnor, PA, USA). Glacial acetic acid was purchased from Fisher Scientific (Pittsburgh, PA, USA). Pure 100% proof ethyl alcohol was purchased from Koptec (King of Prussia, PA, USA). Solutions of 3:2 (v:v) hexane: isopropanol, 7.5% sodium sulfate in 18 ohm ultrapure, deionized water, 1% (v/v) acetic acid in acetonitrile UV and 80:20 isooctane: ethanol were prepared prior to extraction procedures. Ultrapure water was acquired from a Milli-Q system (MilliporeSigma, Burlington, MA, USA). 2.4 Equipment A Precellys® Evolution bead mill homogenizer (Bertin Corp, Rockville, MD, USA) was utilized to homogenize all tissue samples. An Agilent Technologies 7890A GC system coupled with an Agilent 7000 GC/MS Triple Quadrupole detector (Agilent, Santa Clara, CA, USA) was used to perform the GC-MS/MS analysis. The GC separation was conducted on a Phenomenex Zebron ZB-Multiresidue column (45m x 250 µm x 0.25 µm) using a 4 µL injection into an inlet operated in splitless mode (Phenomenex, Torrence, CA, USA). An Agilent 5062 − 3587 split/splitless glass cylinder with single taper liner was used with the glass wool removed. The inlet pressure was 13.4 psi with an initial temperature of 40°C held for 1 minute, then increased by 8°C/minute to 310°C and held for 14 minutes for a total analysis time of 49 minutes. The helium carrier gas flow rate was 1.2 mL/min. The triple quadrupole mass analyzer was operated in negative electron ionization mode at 70 eV maintaining a source temperature of 230°C. Nitrogen served as the collision gas with a flow rate of 1.5 mL/minute and helium was utilized as a quench gas at 2.25 mL/minute. Multiple reaction monitoring was initiated after a 15-minute solvent delay. Data acquisition and processing were performed using MassHunter software (Agilent, Santa Clara, CA, USA). 2.5 Lipid and QuEChERS Extraction Procedure Liver and brain tissue were preserved at -20°C with liver tissues thawed prior to use. Brain tissues were trimmed while frozen to preserve their structural integrity and ensure a representative sample. The following extraction procedure was applied to all samples and also used in the preparation of matrix-matched calibration standards. For lipid fraction assessment, each tissue underwent liquid-liquid extraction. One gram of tissue was combined with 8 mL of 3:2 hexane: isopropanol in a homogenizer tube with 100 µL of a 10 µg/mL PCB 209 internal standard. Matrix-matched standards were prepared with one gram of bovine brain or canine liver previously verified to be free of the analytes of interest. Matrix-matched standards were spiked with 100 µL of a 10 µg/mL PCB 209 internal standard and an appropriate amount of stock or working solution of organochlorine and polychlorinated biphenyls (see below). The tissue was homogenized, and the homogenate was transferred to a 50 mL round bottom glass tube. Two 8 mL 3:2 hexane:isopropanol washes of the homogenizer tube were added to the round bottom glass tube before adding 12 mL of 6.7% sodium sulfate solution in deionized, ultrapure water. Samples were vortexed for one minute and centrifuged at 1000 x g for five minutes. The hexane layer was transferred into pre-weighed 50 mL glass flat-bottom tubes and the solvent was evaporated under a stream of nitrogen with gentle heat in a 40°C water bath. After cooling to room temperature, the percent fat was calculated. A bovine adipose sample was used for quality control throughout the fat extraction. Samples and matrix-matched calibration standards were reconstituted in hexane and transferred to 10 mL volumetric flasks to a final volume of 10 mL. For pesticide analysis, a QuEChERS extraction was performed using 5 mL of extract and commercially available extraction and clean-up tubes. 26 A final volume of 5 mL of supernatant from the QuEChERS clean-up tube was transferred to a 16x125 glass culture tube with 50 µL of dimethylformamide. Samples and standards were dried to completeness under a stream of nitrogen and reconstituted in 500 µL 80:20 isooctane: ethanol. 2.6 Calibration Curve Construction and Analysis Neat standards were prepared by dilution of a standard stock solution. Working standard concentrations were 1, 10, 100, 200, 500, and 1000 ng/mL in 80:20 isooctane ethanol. PCB 209 was utilized as an internal standard for PCB quantitation. Samples were diluted in 80:20 isooctane ethanol in 1:10, 1:100, and 1:1,000 preparations as necessary. Matrix matched standards were similarly prepared in the presence of bovine brain or canine liver that underwent fat and QuEChERS extraction with the addition of pesticide concentrations consistent with the neat standard curve. 2.7 Statistical Analysis Statistical testing was performed in Graphpad Prism (Version 10.2.2; 2024). Numeric concentrations of summed cyclodienes were converted to categorical data for exposure based on the scheme, Low = concentrations 10,000 ng/g. Data were grouped by geolocation as either Wayne County or Other (Clinton, Ingham, Kent, Macomb). 2.8 Reporting Summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Results From July through November 2022, seventeen skunks were assessed by the MDNR WDL. Thirteen of the skunks (76%) presented for evaluation following observed neurologic signs including an inability to balance, altered mentation, tremors, convulsions, or were found dead. One animal (D) had signs consistent with an upper respiratory infection. Three additional animals (O, P, and Q) were submitted with nonspecific illness. Complete physical examinations by a licensed veterinary professional are not available and clinical signs were reported by either the Michigan Humane Society intake staff, MDNR Wildlife Division staff, or property owners where the animal was found. There were no significant findings on gross postmortem evaluation. Brains were submitted for histologic examination for nine skunks; three (F, J, and N) were reported to have mild to moderate lymphoplasmacytic encephalitis or meningoencephalitis and the remaining six brains had no significant findings. Initial diagnostic workup ruled out rabies virus, canine distemper virus, highly pathogenic avian influence virus, and bromethalin exposure. Additionally, drugs of abuse, strychnine, carbamate and organophosphorous pesticides, and other toxicants detected by GCMS were not observed in the samples. Of skunk liver samples, twelve (71%) were positive for at least one chlorinated pesticide by GCMS on initial screening. Fat content ranged 4.47-10.48% in brain and 2.97-22.83% in liver (Table 1). Concentrations of the three most abundant analytes and summed chlordane related compounds are presented in Table 2. All analyte concentrations are reported on a wet- (w.w.) and lipid weight (l.w.) basis. Four skunk brains did not contain individual analyte concentrations at or above the 10 ng/g reporting limit (J, O, P, and Q). Of all compounds analyzed, concentrations of oxychlordane, heptachlor epoxide, and trans-nonachlor were present in the highest concentrations. Oxychlordane was present in greatest abundance in the skunks with the highest total chlordane burden as noted in Figure 1. Skunks D, H, and M were found to have brain oxychlordane residues that exceeded the 10 µg/g (10,000 ng/g) w.w. lethal residue reported by Stickel et al. for birds. 7 The brain tissue of skunk C contained oxychlordane residues of 9,185 ng/g w.w. which was considerably increased with respect to previously reported lethal thresholds for birds; however, the additional brain burden of heptachlor epoxide with a concentration of 2,515 ng/g w.w. suggests a potentially lethal burden of chlordane metabolite in this animal’s brain. 7 The remaining eight skunks had total chlordane, nonachlor, and metabolite concentrations ranging from 62-3,782 ng/g w.w. in brain. Numeric concentrations of summed cyclodienes in brain tissue (w.w.) were converted to categorical data for exposure based on the scheme: low (10,000 ng/g) exposure groups and by county of origin (Wayne versus other counties). Chi-square for trends analysis revealed a statistically significant trend (p=0.018); however, a Fisher’s exact test (p=0.073) did not find an association between concentrations and represented counties. This is likely due to the small sample size of fewer than twenty animals. Concentrations of chlordane metabolites and trans-nonachlor were significantly greater in liver tissue when compared to brain tissue from the same individual. Summed chlordane, nonachlor, and metabolite concentrations in liver tissues ranged from 34-815,826 ng/g w.w. and at least one analyte was detected in all but one individual. Seven animals were reported to have total liver residue concentrations less than 1,000 (range 34-986), two less than 10,000 (3,477 and 9,232), five less than 100,000 (range 17,917-78,258) and three greater than 100,000 ng/g w.w. (range 185,648-815,826). When analytes were reported on a lipid weight basis for brain and liver, liver total chlordane and metabolite concentrations were approximately ten-fold greater than brain concentrations. A simple linear regression comparing brain and liver concentrations on a lipid basis yielded an r 2 =0.9581 compared to wet weight concentrations with r 2 =0.8080 as noted in Figure 2. Low concentrations (<500 ng/g w.w.) of dieldrin were detected in 24% of brains and 59% of livers. Two liver samples, H and M, had dieldrin concentrations of 887 and 2,417 ng/g w.w. respectively. Additionally, low concentrations (<500 ng/g w.w.) of DDE were detected in 47% of brains and 41% of livers. Skunks H and M also contained DDE concentrations of 619 and 1453 ng/g w.w. in brain tissue. Five other skunks had hepatic DDE concentrations ranging from 706-6806 ng/g w.w. while skunk H contained 11,975 ng/g w.w. DDE. DDD was detected in trace amounts in the liver of skunks C and I and at concentrations of 1,019 and 717 ng/g w.w. in skunks H and M respectively. DDD was not detected in brain tissue of any skunk at concentrations exceeding the 10 ng/g reporting limit.PCBs were detected in 47% of brain samples and 94% of liver samples. In brain, total PCB concentrations ranged from 14-135 ng/g w.w. or 206-3021 ng/g l.w. in the eight positive samples. Liver total PCB concentrations ranged from 19-640 ng/g w.w. or 510-11,138 ng/g l.w. in the sixteen positive samples. Negative samples did not have individual PCB congener concentrations exceeding the 10 ng/g reporting limit. PCB congeners #52, 101, and 126 were not detected in any of the seventeen brain or liver samples. Congeners #153, 138, and 180 were found in the highest concentrations in order of decreasing abundance. Accumulation of chlordane and its metabolites was not correlated with accumulation of PCB congeners in either liver or brain tissue with r 2 values of 0.0161 and 0.1754 respectively as described in Figure 3. Discussion Ten out of seventeen skunks (59%) were submitted from Wayne County, Michigan which encompasses the majority of the River Rouge Watershed. This region has been known for its historical contamination with PCBs and other pollutants (Figure 4). 3 The River Raisin and River Rouge Watersheds span the greater Detroit metropolitan area across Wayne, Oakland, and Washtenaw counties and are the most urbanized and densely populated watersheds in the state of Michigan. 1–4 Designated an Environmental Protection Agency (EPA) Area of Concern (AOC) under the Great Lakes Water Quality Agreement of 1987, the River Rouge prompted the MDNR and Michigan Water Resources Commission to adopt a 20-year remedial action plan beginning in 1985. 1,3 The river’s sediment contamination with polycyclic aromatic hydrocarbon (PAH), PCB, oil, grease, and heavy metals has led to ongoing monitoring studies, beneficial use impairments, and concerted clean-up efforts. 3 PCBs, dieldrin, and DDE are often analyzed together and are expected to co-accumulate due to common source of exposure in contaminated watersheds or through bioaccumulation within aquatic food webs. 20,27–30 Of interest to this study, the accumulation of PCBs, dieldrin, and DDE were not well correlated within brain nor liver tissue. Within brain, the correlation coefficients of simple linear regression analysis were: dieldrin vs DDE R 2 <0.01, PCBs vs DDE R 2 =0.5669, and PCBs vs dieldrin R 2 <0.01. Similarly, the correlation coefficients of simple linear regression in liver were: dieldrin vs DDE R 2 =0.0128, PCBs vs DDE R 2 =0.2280, and PCBs vs dieldrin R 2 =0.0379. While dieldrin, DDE, and PCBs are unlikely to have significantly contributed to the moribund status or death of these skunks, the occurrence of these analytes as persistent organic pollutants in the Detroit Metropolitan Area and their long-term implications for human and ecological health at low concentrations remains a concern. Lethal thresholds for PCB brain residues are reported as 310,000 ng/g w.w. for birds fed Aroclor 1254 at a rate of 1,500 μg/g feed dry weight. None of the skunks reported herein exceeded the lethal threshold for PCB residues in brain tissue with the greatest concentration reported at 135 ng/g w.w.. 30 For comparison, PCB concentrations in Japanese raccoon dogs ranged from 24-1,200 ng/g l.w. in liver while PCBs in skunks from this study ranged from 510-11,138 ng/g l.w.. The PCB congeners 138, 153, and 180 were found in greatest abundance and are suggestive of environmental contamination from Aroclors 1260 and/or 1254. 31 With concentrations 2,000 times lower than those reported by Stickel, et al. it is unlikely that the PCB residues contributed to the neurologic presentation of these skunks. 30 The finding of PCB residues in skunks residing in areas supplied by the River Rouge Watershed further reinforces that PCBs contaminate and persist in this region nearly 35 years after the remedial action plan was implemented. Previous reports indicate lethal brain dieldrin concentrations in birds of 10,000 ng/g w.w. and DDE of 500,000 ng/g w.w. for birds 7,32,33 , and acutely lethal brain dieldrin concentrations for dogs averaged 5,500 ng/g w.w. in dogs. 34 Additionally, concentrations of DDE ranged from 5.3-150 ng/g l.w. in liver of Japanese raccoon dogs. 16 Dieldrin and DDE did not exceed previously reported lethal brain concentrations in any of the submitted skunks. The skunks presented herein had hepatic DDE concentrations ranging from 188-175,364 ng/g l.w., and suggest either increased dietary exposure, or decreased clearance of DDE by skunks when compared to raccoon dogs. These findings of sublethal PCB, dieldrin, and DDE concentrations further support that the neurologic state of these animals were more likely attributed to the combined burdens of oxychlordane, heptachlor epoxide, and trans-nonachlor. Tissue chlordane and metabolite residue concentrations in skunks found in urban and suburban areas of Michigan far exceed previous reports in other small wildlife species. A 2007 analysis of ten raccoon dogs ( Nyctereutes procyonoides) collected outside of Tokyo found hepatic concentrations of oxychlordane ranging from 160-20,000 ng/g l.w., concentrations significantly lower than those reported here. 16 The racoon dogs in this study succumbed to traffic accidents; therefore, antemortem clinical signs were not reported. 16 Racoon dogs are opportunistic carnivores consuming small animal prey, fruits, vegetation, and insects similar to North American striped skunks ( Mephitis mephitis) with each species filling comparable ecologic niches. 16,22 Striped skunks are known to inhabit residential areas by building dens around porches, homes, and garages or sheds. 22 Due to the close proximity of skunk dens to the foundation of homes and garages, skunks and other small residential wildlife species have an increased risk of environmental exposure to technical chlordane residues that were once applied in these locations for termiticidal properties. 5 Skunks positive for hepatic oxychlordane residues were found to have concentrations 155-times greater (range 11,318-3,096,515 ng/g l.w.) than those reported in raccoon dogs. While impressive, and suggestive of oxychlordane toxicosis, diagnostic criteria for neurotoxicity and lethality are not available for liver tissue. There are few studies available specifically investigating the neurotoxic effects of chlordane and its metabolites in mammals. Case studies in humans, for which convulsions and tremors were observed, involved high-dose acute exposures as opposed to chronic environmental or dietary exposure. 23,24 Tremors were reported in rats and mice receiving high-dose chronic oral exposures of chlordane throughout a carcinogenicity bioassay. 25 Previous studies in laboratory animals may not observe neurotoxic effects due to insufficient dose, short study duration, or utilization of alternative end points such as feed refusal or severe weight loss. 5,35,36 This may be due in part to the steep dose-response curve reported by one study in rats which reported an oral toxic dose of 10 mg/kg body weight (BW) and NOEL dose of 1 mg/kg BW with toxicity defined as feed refusal and weight loss. 36 For this reason, postmortem tissue chlordane concentrations are difficult to interpret and diagnostic decision thresholds for neurotoxicity and lethality are not readily available for most species. Female rats receiving 10 mg/kg BW per day oxychlordane by oral gavage for 28 days were reported to contain adipose tissue oxychlordane concentrations of approximately 600,000 ng/g l.w. and liver concentrations of approximately 18,000 ng/g w.w. 36 Rats in the 10 mg/kg BW per day dosing group were lethargic and unkempt, but tremors and convulsions were not reported. We report skunk hepatic oxychlordane concentrations up to 707,000 ng/g w.w. (range 517-706,981 ng/g w.w.), nearly 40x greater than those experimentally produced in rats suggesting a greater dose, repeated exposure, increased storage efficiency or capacity of oxychlordane, or decreased metabolism and elimination or chlordane and related compounds in these skunks. Three skunks contained brain oxychlordane residues exceeding the 10,000 ng/g w.w. lethal threshold reported for birds. 7 The brain tissue of a fourth skunk contained oxychlordane residues of approximately 9,185 ng/g w.w. and is considerably increased in accordance with previous lethal thresholds reported for birds; the additional brain burden of heptachlor epoxide at 2,515 ng/g w.w. suggests a lethal brain chlordane metabolite burden for this animal. 7 Therefore, the death or moribund status of these four animals may be attributed to the neurotoxic effects of these analytes. Moreover, if an uncertainty factor of 10 is utilized to account for species differences, we may consider brain residues of 1,000 ng/g w.w. as potentially lethal. Four additional skunks had brain oxychlordane residues exceeding 1000 ng/g w.w. with a fifth considered marginal (753 ng/g). Although the lethal residues described are for oxychlordane individually, skunks were found to have significant burdens of heptachlor epoxide and trans-nonachlor as well. It is important to reiterate that oxychlordane is six times more toxic when compared to technical chlordane in birds and hepatachlor epoxide is 10 times more toxic than either isomer of chlordane in rats. 7,12 Therefore, the use of comprehensive organochlorine panels and summed chlordane and metabolite burdens provides a more accurate diagnostic approach than analysis of any one single analyte. Data have been presented and discussed throughout on a wet weight basis for ease of interpretation when comparing to previous studies and diagnostic criteria. It is of note that correction for lipid content of tissues and reporting on a lipid weight basis allows for more accurate comparison across tissues within individual animals and within and across species. Liver was shown to be predictive of brain chlordane concentrations on a lipid weight basis and can be utilized to predict neurotoxic chlordane concentrations in brain when diagnostic samples are limited, or brain tissue cannot otherwise be collected during postmortem examination. While there is strong evidence that toxicologically significant concentrations of chlordane and its metabolites accumulated in the brain tissue of multiple animals, statistically significant differences between groups in this population could not be elucidated. Nevertheless, there is evidence that animals which originated from the Detroit Metropolitan Area have higher brain chlordane concentrations than those from other counties in the state of Michigan (Figure 4). Animals in the highest exposure group originated from Redford and Dearborn Heights within Wayne County, Michigan. Additionally, one animal submitted from Grand Rapids in Kent County, Michigan fell within the intermediate exposure group. A statistically significant difference between population groups when comparing Wayne County to other Michigan counties could not be elucidated. The lack of statistically significant findings in this population are largely due to the small sample size of fewer than twenty animals. Inclusion of additional animals from the western and northern counties as well as the Upper Peninsula of Michigan will provide further insight to the correlation between chlordane metabolite accumulation in densely populated metropolitan areas when compared to rural communities of Michigan. Additionally, due to the circumstances by which animals are submitted for post-mortem examination, samples from this population are unlikely to be randomly selected and the population is not normally distributed violating the assumptions of multiple statistical models. For these reasons, it is not unexpected that a statistically significant difference could not be elucidated despite having strong evidence for toxicologically significant exposures. This study provides the first report of neurotoxicosis associated with chlordane exposure in skunks across the greater Detroit Metropolitan area. Herein, we have reported alarmingly high concentrations of oxychlordane and heptachlor epoxide, two known chlordane metabolites with neurotoxic potential Contamination of the Great Lakes and watersheds across the state of Michigan remains a focus for improved water quality. This study emphasizes a further concern for soil and insect contamination associated with these watersheds that provide habitat and food sources for Michigan’s wildlife, and an urban environment for human activities. A better understanding of the environmental persistence of technical chlordane components and metabolites in soil, water, and insect populations from areas with widespread historical use is critical to the management of this widely found pollutant. We have highlighted the need for more systemic, broad-scale research efforts to monitor technical chlordane in wildlife, domestic animals, and humans in urban settings to understand the nature and extent of pesticide pollution and mitigate risk associated with exposure to these compounds. Declarations Funding This study was supported by the Michigan State University College of Veterinary Medicine’s Edward K Sales Endowed Research Fund. Author Contributions RMS conducted the analytical analysis, data interpretation, and primary writing for the manuscript. BP provided data interpretation, writing, and editing support for the manuscript. SDF served as the pathologist for the study and provided written narrative for pathologic findings. JM provided support in coordinating animal collection, initial necropsy, and submission of samples for analysis. JM also provided support in editing. JPB coordinated direction of the manuscript, data interpretation and provided editing support. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Data Availability All associated data presented herein will be made available on reasonable request. Correspondence and request for materials should be addressed to JPB. References Ridgway, J., Cave, K., DeMaria, A., O’Meara, J. & Hartig, J. H. The Rouge River Area of Concern - A multi-year, multi-level successful approach to restoration of Impaired Beneficial Uses. Aquat. Ecosyst. Heal. Manag. 21, (2018). Bosko, T., Crumpacker, A., Kozel, L., Wojdakowski, N. & Lantinga, C. Data Evaluation Report for Lower Rouge River Sediment Investigation Detroit, Wayne County, Michigan . https://www.epa.gov/great-lakes-aocs/data-evaluation-report-lower-rouge-river-sediment-investigation (2010). Southeast Michigan Council of Governments & Michigan Department of Natural Resources. Remedial Action Plan For the River Rouge Basin . https://www.epa.gov/sites/default/files/2015-04/documents/1990_rouge-river-rap-update.pdf (1990). Bean, C., Schrameck, R. & Davidson, C. 1994 Rouge River Remedial Action Plan Update . https://www.epa.gov/sites/default/files/2015-04/documents/1994_rouge-river-rap-update.pdf . McGaughy, R. E., Foureman, G. L., & McClure, P.. Toxicological review of chlordane (technical). USEPA (1997). Bennett, G. W. et al. Persistence and distribution of chlordane and dieldrin applied as termiticides. Bull. Environ. Contam. Toxicol. 11, (1974). Stickel, L. F., Stickel, W. H., Dyrland, R. A. & Hughes, D. L. Oxychlordane, hcs-3260, and nonachlor in birds: Lethal residues and loss rates. J. Toxicol. Environ. Health 12, (1983). Beeman, R. W. & Matsumura, F. Metabolism of cis- and trans-Chlordane by a Soil Microorganism. J. Agric. Food Chem. 29, (1981). Polen, P. B., Hester, M. & Benziger, J. Characterization of oxychlordane, animal metabolite of chlordane. Bull. Environ. Contam. Toxicol. 5, (1970). Liu, X. et al. Enantioselective characteristics, bioaccumulation and toxicological effects of chlordane-related compounds in laying hens. Chemosphere 300, (2022). Street, J. C. & Blau, S. E. Oxychlordane: Accumulation in Rat Adipose Tissue on Feeding Chlordane Isomers or Technical Chlordane. J. Agric. Food Chem. 20, (1972). Stansley, W., Roscoe, D. E., Hawthorne, E. & Meyer, R. Food chain aspects of chlordane poisoning in birds and bats. Arch. Environ. Contam. Toxicol. 40, (2001). Suprock, J. F., Vinopal, J. H. & Inatome, M. Oxychlordane residues in soil. Bull. Environ. Contam. Toxicol. 25, (1980). Clark, D. R., Kunz, T. H. & Kaiser, T. E. Insecticides applied to a nursery colony of little brown bats (Myotis lucifugus): lethal concentrations in brain tissues. J. Mammal. 59, (1978). Buchweitz, J. P., Carson, K., Rebolloso, S. & Lehner, A. DDT poisoning of big brown bats, Eptesicus fuscus, in Hamilton, Montana. Chemosphere 201, (2018). Kunisue, T., Takayanagi, N., Tsubota, T. & Tanabe, S. Persistent organochlorines in raccoon dogs (Nyctereutes procyonoides) from Japan: Hepatic sequestration of oxychlordane. Chemosphere 66, (2007). Ford, W. M. & Hill, E. P. Organochlorine Residues in Mississippi Raccoons. J. Wildl. Manage. 54, (1990). Yamagishi, T., Kaneko, S., Miyazaki, T., Akiyama, K. & Horii, S. Oxychlordane, trans- nonachlor and cis-nonachlor residues in adipose tissues of dogs and cats collected in the Tokyo area. Bull. Environ. Contam. Toxicol. 26, (1981). Stansley, W. & Roscoe, D. E. Chlordane poisoning of birds in New Jersey, USA. Environ. Toxicol. Chem. 18, (1999). Wattigney, W. A., Irvin-Barnwell, E., Li, Z. & Ragin-Wilson, A. Biomonitoring of toxic metals, organochlorine pesticides, and polybrominated biphenyl 153 in Michigan urban anglers. Environ. Res. 203, (2022). Barbehenn, K. R. & Reichel, W. L. Organochlorine concentrations in bald eagles: Brainjbody lipid relations and hazard evaluation. J. Toxicol. Environ. Health 8, (1981). Dragoo, J. W. Nutrition and Behavior of Striped Skunks. Veterinary Clinics of North America - Exotic Animal Practice vol. 12 (2009). Olanoff, L. S., Bristow, W. J., Colcolough, J. & Reigart, J. R. Acute chlordane intoxication. Clin. Toxicol. 20, (1983). Aldrich, F. D. & Holmes, J. H. Acute Chlordane Intoxication in a Child. Arch. Environ. Health 19, (1969). National Toxicology Program. Bioassay of chlordane for possible carcinogenicity. Natl. Cancer Inst. Carcinog. Tech. Rep. Ser. 8, 1–123 (1977). Andreas Lehner, M. B. & John Buchweitz, M. J. Determination of Organochlorine Pesticides in Wildlife Liver and Serum Using Gas Chromatography Tandem Quadrupole Mass Spectrometry. J. Chromatogr. Sep. Tech. 06, (2015). Carpenter, S. K. et al. River otters as biomonitors for organochlorine pesticides, PCBs, and PBDEs in Illinois. Ecotoxicol. Environ. Saf. 100, (2014). Batt, A. L., Wathen, J. B., Lazorchak, J. M., Olsen, A. R. & Kincaid, T. M. Statistical Survey of Persistent Organic Pollutants: Risk Estimations to Humans and Wildlife through Consumption of Fish from U.S. Rivers. Environ. Sci. Technol. 51, (2017). Hargrave, B. T. et al. Organochlorine pesticides and polychlorinated biphenyls in the Arctic Ocean food web. Arch. Environ. Contam. Toxicol. 22, (1992). Stickel, W. H., Stickel, L. F., Dyrland, R. A. & Hughes, D. L. Aroclor 1254® residues in birds: Lethal levels and loss rates. Arch. Environ. Contam. Toxicol. 13, (1984). Risso, F. et al. A comprehensive approach to actual polychlorinated biphenyls environmental contamination. Environ. Sci. Pollut. Res. 23, (2016). Stickel, W. H., Stickel, L. F. & Spann, J. W. Tissue residues of dieldrin in relation to mortality in birds and mammals. in Chemical Fallout: Current Research on Persistent Pesticides. Proceedings of the First Rochester Conference on Toxicity 174–204 (1969). Stickel, W. H., Stickel, L. F., Dyrland, R. A. & Hughes, D. L. DDE in birds: Lethal residues and loss rates. Arch. Environ. Contam. Toxicol. 13, (1984). Harrison, D. L. & Manktelow, B. W. Dieldrin poisoning of dogs. N. Z. Vet. J. 8, (1960). Bondy, G. S. et al. Trans-nonachlor and cis-nonachlor toxicity in sprague-dawley rats: Comparison with technical chlordane. Toxicol. Sci. 58, (2000). Bondy, G. et al. Toxicity of the chlordane metabolite oxychlordane in female rats: Clinical and histopathological changes. Food Chem. Toxicol. 41, (2003). Tables Table 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files table1and2.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4546233","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":318522466,"identity":"ebd5cd99-0c53-4217-a1cf-e06f13066381","order_by":0,"name":"John Buchweitz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYBACAwYGNiAlIccP4TMTr8VYsoFELQyJGw4Qq8VcIvnZY54KC8bN144/k2CosE5sIKTFckaauTHPGQlms9s5ZhIMZ9IJazG4kcMmndsmwQbUwibB2HaYWC3/JHiMZ6c/k2D8R7SWBgkJA+kEMwnGBiK0WPY8M5P+c0zCQOJ2jrFFwrF0Y4JazNmTn0nOqKmr75+d/vDGhxprWYJaUEECacpHwSgYBaNgFOACAN26OBi9tWnCAAAAAElFTkSuQmCC","orcid":"","institution":"Michigan State University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Buchweitz","suffix":""},{"id":318522468,"identity":"a2a0dfcd-3ab7-49b7-80ed-d62083e227d3","order_by":1,"name":"Rachel Sheffler","email":"","orcid":"","institution":"Michigan State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rachel","middleName":"","lastName":"Sheffler","suffix":""},{"id":318522471,"identity":"85daed5e-1da4-4003-8f08-ca9ad3b0a021","order_by":2,"name":"Birgit Puschner","email":"","orcid":"","institution":"Michigan State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Birgit","middleName":"","lastName":"Puschner","suffix":""},{"id":318522472,"identity":"9326b3b0-91f6-4491-b200-58d3e49db411","order_by":3,"name":"Scott Fitzgerald","email":"","orcid":"","institution":"Michigan State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Scott","middleName":"","lastName":"Fitzgerald","suffix":""},{"id":318522473,"identity":"771056fe-eddc-4118-a881-c98eaa92563e","order_by":4,"name":"Julie Melotti","email":"","orcid":"","institution":"Michigan Department of Natural Resources","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Julie","middleName":"","lastName":"Melotti","suffix":""}],"badges":[],"createdAt":"2024-06-07 12:59:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4546233/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4546233/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59163821,"identity":"9f4b1a98-dc9d-4602-9379-64b5b71877b8","added_by":"auto","created_at":"2024-06-27 06:14:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":166615,"visible":true,"origin":"","legend":"\u003cp\u003eChlordane related compounds in brain and liver on a wet weight and lipid weight basis.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4546233/v1/3ebf0f90f38e4a66b63a8d63.png"},{"id":59162889,"identity":"b069a420-a1cd-4842-8a5b-69fe9924d28c","added_by":"auto","created_at":"2024-06-27 05:58:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":112955,"visible":true,"origin":"","legend":"\u003cp\u003eLiver versus brain total chlordane plus metabolite concentrations (ng/g) on a wet weight (w.w) (left) and lipid weight (l.w.) (right) basis.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4546233/v1/77299cbe7ebf60e07b722bf7.png"},{"id":59163242,"identity":"bee53c0b-348c-45f2-bd14-cf3d2335edf6","added_by":"auto","created_at":"2024-06-27 06:06:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":105324,"visible":true,"origin":"","legend":"\u003cp\u003eTotal PCB versus chlordane and chlordane metabolite concentrations in liver (left) and brain (right) l.w.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4546233/v1/dd5fbb75d5715c317d4ce187.png"},{"id":59162893,"identity":"3e92a527-ea33-445c-ba2f-3c774380bc6a","added_by":"auto","created_at":"2024-06-27 05:58:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":287948,"visible":true,"origin":"","legend":"\u003cp\u003eSkunk geographic distributionby Summed Brain Chlordane Compound Exposure Level (wet weight).\u003c/p\u003e\n\u003cp\u003eSummed oxychlordane, heptachlor epoxide, and trans-nonachlor concentrations were divided into three levels of exposure: low (\u0026lt;1,000 ng/g), intermediate (1,000-9,999 ng/g) and high (\u0026gt;10,000 ng/g). Individual animals are represented by pins on the map of Michigan based on their exposure level and county of origin. The River Raisin and River Rouge Areas of Concern are highlighted with shaded ovals overlapping the counties that contain these watersheds.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4546233/v1/e9932c76b2250870a59d0570.png"},{"id":74187923,"identity":"206ab9db-d621-4b91-bd88-ddde01d9a4ff","added_by":"auto","created_at":"2025-01-19 17:31:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1225482,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4546233/v1/ae201c25-bcdd-411a-bd0f-2266226a2233.pdf"},{"id":59162891,"identity":"f397b726-025b-4433-9f73-3769628bdd23","added_by":"auto","created_at":"2024-06-27 05:58:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":62286,"visible":true,"origin":"","legend":"","description":"","filename":"table1and2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4546233/v1/e8c6d02681accb111e6f2be4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chlordane induced neurotoxicosis in urban and suburban Detroit, Michigan striped skunks (Mephitis mephitis)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcross the Great Lakes basin, decades of industrial discharges and agricultural runoff have contaminated soil sediments with persistent organic pollutants including organochlorine pesticides, polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), heavy metals, oil, and grease.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e The vast freshwater ecosystem surrounds the state of Michigan and interconnects with the state's most urbanized and densely populated watersheds; these watersheds include the River Rouge and River Raisin which span the greater Detroit metropolitan area across Wayne, Oakland, and Washtenaw counties.\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e The River Rouge was designated an Environmental Protection Agency (EPA) Area of Concern (AOC) under the Great Lakes Water Quality Agreement of 1987. Multiple compounds were banned throughout the 1970s whose manufacture and use contributed to watershed contamination including PCBs, once used in coolants and lubricants for transformers and electrical equipment, and organochlorine pesticides including chlordane, dieldrin, and DDT previously used in insect control.\u003c/p\u003e \u003cp\u003eTechnical chlordane is a synthetic mixture of more than 140 components that was extensively used in the United States until it was banned in 1988. Due to its chemical stability, chlordane continues to persist in the environment today despite bans on its production and use in the United States and many countries globally. The composition of technical chlordane is 60% cis- and trans-chlordane and 40% related compounds including heptachlor, trans-nonachlor, cis-nonachlor, chlordene, and other minor constituents.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e These compounds have environmental half-lives of 10\u0026ndash;20 years, are resistant to environmental breakdown, and continue to pose a risk to wildlife and public health.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Mammalian, avian, and microbial metabolism favors the trans isomer of chlordane and results in the formation of the epoxide metabolites, oxychlordane and heptachlor epoxide, which accumulate in fat.\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBefore its ban in the United States in 1988, chlordane was widely used for termite control around the foundation of homes, for the control of scarab beetle populations on golf courses, and for agricultural pest management.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Termiticides applied around foundation walls remain within the upper 6\u0026ndash;8 inches of soil without vertical migration to deeper layers.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Soil testing has shown low concentrations of oxychlordane and heptachlor epoxide with a greater presence of cis- and trans-chlordane.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e In contrast, significant residues of oxychlordane and heptachlor epoxide have been documented in oriental scarab beetles (\u003cem\u003eAnomala orientalis\u003c/em\u003e), other species of beetles, and earthworms.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e In suburban and urban areas where chlordane was liberally used, it continues to contaminate habitat and food sources for wildlife with pesticide accumulation documented in the tissues of bats\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, racoons\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, cats\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, dogs\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, songbirds\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, raptors\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and now skunks. Skunks that burrow under porches and areas near foundations while consuming insectivorous diets that include beetles, earthworms, bees, and caterpillars, can be predicted to have significant chlordane metabolite residues.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eChlordane and its metabolites are reported to cause neurotoxic effects such as tremors, salivation, ataxia, depression, vomiting, and seizures in man\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, mammals\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, and birds\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Specifically, oxychlordane, a metabolite of chlordane, is six times more toxic to birds, and heptachlor epoxide, the metabolite of heptachlor, is ten times more toxic to rats than their respective parent compound.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Herein, we employed the use of a highly sensitive and selective gas chromatography tandem mass spectrometry method to detect high concentrations of chlordane metabolites in skunk tissues collected from urban and suburban neighborhoods in Michigan. Our findings provide the first evidence of skunks succumbing to neurotoxicosis by chlordane exposure. Cases of neurotoxicosis in wildlife are likely under reported due to insufficient surveillance, an initial presumption of rabies or infectious disease, or lack of funding and access to comprehensive toxicology testing. But such information is necessary for assessing the potential threat consequent to the past use of chlordane in the urban environment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Clinical Sample Collection and Initial Diagnostic Workup\u003c/h2\u003e \u003cp\u003eFive female and twelve male skunks found dead or moribund were submitted to the Michigan Department of Natural Resources Wildlife Disease Laboratory (MDNR WDL) for necropsy (Lansing, MI). The skunks were assessed for age and reported as five adults and twelve juveniles. Brain stem and cerebellum were submitted to the United States Department of Agriculture, Wildlife Services or the Michigan Department of Health and Human Services (MDHHS) for routine rabies screening. Animals were screened for canine distemper virus and three animals were screened for highly pathogenic avian influenza virus through the diagnostic service at the Michigan State University Veterinary Diagnostic Laboratory (MSU VDL). Liver samples were analyzed for drugs, pesticides, and environmental pollutants using gas chromatography mass spectrometry (GC/MS), based on methods previously described by this laboratory.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Brain samples were also tested for desmethylbromethalin, a neurotoxic metabolite of the rodenticide bromethalin, by liquid chromatography tandem mass spectrometry (LC-MS/MS). Both brain and liver tissues were quantitatively assessed for organochlorine and PCB concentrations, with confirmed positive findings for chlordane and heptachlor metabolites from mass spectral screening. Control tissues from bovine brain and canine liver, obtained from other necropsy submissions to the veterinary diagnostic laboratory, tested negative for these compounds by GC/MS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Pesticide Standards\u003c/h2\u003e \u003cp\u003eOrganochlorine pesticide mix containing alpha-benzene hexachloride or hexachlorocyclohexane (BHC), beta-BHC, gamma-BHC (Lindane), delta-BHC, alpha (cis)-chlordane, gamma (trans)-chlordane, 4,4’ dichlorodiphenyltrichloroethane (DDT), 4,4’ dichlorodiphenyldichloroethylene (DDE), 4,4’ dichlorodiphenyldichloroethane (DDD), aldrin, dieldrin, endrin, endrin aldehyde, endrin ketone, endosulfan I (alpha), endosulfan II (beta), endosulfan sulfate, heptachlor, and heptachlor exo-epoxide was acquired from MilleporeSigma (Sigma-Aldrich Corporation-St. Louis, MO, USA). Oxychlordane, heptachlor exo-epoxide, and heptachlor endo-epoxide standards with chromatographic purity were purchased from Agilent Technologies (Santa Clara, CA, USA). PCB standards for congeners #52, 101, 118, 153, 138, 187, 183, 126, 180, 170, and 209 were obtained from ChemService (West Chester, PA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Solvents and Reagents\u003c/h2\u003e \u003cp\u003eAcetonitrile UV (chromatography grade) and isooctane (chromatography grade) were purchased from Burdick and Jackson (Muskegon, MI, USA). Hexane, isopropanol, and anhydrous sodium sulfate were purchased from VWR (Radnor, PA, USA). Glacial acetic acid was purchased from Fisher Scientific (Pittsburgh, PA, USA). Pure 100% proof ethyl alcohol was purchased from Koptec (King of Prussia, PA, USA). Solutions of 3:2 (v:v) hexane: isopropanol, 7.5% sodium sulfate in 18 ohm ultrapure, deionized water, 1% (v/v) acetic acid in acetonitrile UV and 80:20 isooctane: ethanol were prepared prior to extraction procedures. Ultrapure water was acquired from a Milli-Q system (MilliporeSigma, Burlington, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Equipment\u003c/h2\u003e \u003cp\u003eA Precellys® Evolution bead mill homogenizer (Bertin Corp, Rockville, MD, USA) was utilized to homogenize all tissue samples. An Agilent Technologies 7890A GC system coupled with an Agilent 7000 GC/MS Triple Quadrupole detector (Agilent, Santa Clara, CA, USA) was used to perform the GC-MS/MS analysis. The GC separation was conducted on a Phenomenex Zebron ZB-Multiresidue column (45m x 250 µm x 0.25 µm) using a 4 µL injection into an inlet operated in splitless mode (Phenomenex, Torrence, CA, USA). An Agilent 5062 − 3587 split/splitless glass cylinder with single taper liner was used with the glass wool removed. The inlet pressure was 13.4 psi with an initial temperature of 40°C held for 1 minute, then increased by 8°C/minute to 310°C and held for 14 minutes for a total analysis time of 49 minutes. The helium carrier gas flow rate was 1.2 mL/min.\u003c/p\u003e \u003cp\u003eThe triple quadrupole mass analyzer was operated in negative electron ionization mode at 70 eV maintaining a source temperature of 230°C. Nitrogen served as the collision gas with a flow rate of 1.5 mL/minute and helium was utilized as a quench gas at 2.25 mL/minute. Multiple reaction monitoring was initiated after a 15-minute solvent delay. Data acquisition and processing were performed using MassHunter software (Agilent, Santa Clara, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Lipid and QuEChERS Extraction Procedure\u003c/h2\u003e \u003cp\u003eLiver and brain tissue were preserved at -20°C with liver tissues thawed prior to use. Brain tissues were trimmed while frozen to preserve their structural integrity and ensure a representative sample. The following extraction procedure was applied to all samples and also used in the preparation of matrix-matched calibration standards. For lipid fraction assessment, each tissue underwent liquid-liquid extraction. One gram of tissue was combined with 8 mL of 3:2 hexane: isopropanol in a homogenizer tube with 100 µL of a 10 µg/mL PCB 209 internal standard. Matrix-matched standards were prepared with one gram of bovine brain or canine liver previously verified to be free of the analytes of interest. Matrix-matched standards were spiked with 100 µL of a 10 µg/mL PCB 209 internal standard and an appropriate amount of stock or working solution of organochlorine and polychlorinated biphenyls (see below). The tissue was homogenized, and the homogenate was transferred to a 50 mL round bottom glass tube. Two 8 mL 3:2 hexane:isopropanol washes of the homogenizer tube were added to the round bottom glass tube before adding 12 mL of 6.7% sodium sulfate solution in deionized, ultrapure water. Samples were vortexed for one minute and centrifuged at 1000 x g for five minutes. The hexane layer was transferred into pre-weighed 50 mL glass flat-bottom tubes and the solvent was evaporated under a stream of nitrogen with gentle heat in a 40°C water bath. After cooling to room temperature, the percent fat was calculated. A bovine adipose sample was used for quality control throughout the fat extraction. Samples and matrix-matched calibration standards were reconstituted in hexane and transferred to 10 mL volumetric flasks to a final volume of 10 mL. For pesticide analysis, a QuEChERS extraction was performed using 5 mL of extract and commercially available extraction and clean-up tubes.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e A final volume of 5 mL of supernatant from the QuEChERS clean-up tube was transferred to a 16x125 glass culture tube with 50 µL of dimethylformamide. Samples and standards were dried to completeness under a stream of nitrogen and reconstituted in 500 µL 80:20 isooctane: ethanol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Calibration Curve Construction and Analysis\u003c/h2\u003e \u003cp\u003eNeat standards were prepared by dilution of a standard stock solution. Working standard concentrations were 1, 10, 100, 200, 500, and 1000 ng/mL in 80:20 isooctane ethanol. PCB 209 was utilized as an internal standard for PCB quantitation. Samples were diluted in 80:20 isooctane ethanol in 1:10, 1:100, and 1:1,000 preparations as necessary. Matrix matched standards were similarly prepared in the presence of bovine brain or canine liver that underwent fat and QuEChERS extraction with the addition of pesticide concentrations consistent with the neat standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical testing was performed in Graphpad Prism (Version 10.2.2; 2024). Numeric concentrations of summed cyclodienes were converted to categorical data for exposure based on the scheme, Low = concentrations \u0026lt; 1,000 ng/g, Intermediate = concentrations between 1,000–9,999 ng/g, High = concentrations \u0026gt; 10,000 ng/g. Data were grouped by geolocation as either Wayne County or Other (Clinton, Ingham, Kent, Macomb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Reporting Summary\u003c/h2\u003e \u003cp\u003eFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eFrom July through November 2022, seventeen skunks were assessed by the MDNR WDL. Thirteen of the skunks (76%) presented for evaluation following observed neurologic signs including an inability to balance, altered mentation, tremors, convulsions, or were found dead. One animal (D) had signs consistent with an upper respiratory infection. Three additional animals (O, P, and Q) were submitted with nonspecific illness. Complete physical examinations by a licensed veterinary professional are not available and clinical signs were reported by either the Michigan Humane Society intake staff, MDNR Wildlife Division staff, or property owners where the animal was found. There were no\u0026nbsp;\u003c/p\u003e\n\u003cp\u003esignificant findings on gross postmortem evaluation. Brains were submitted for histologic examination for nine skunks; three (F, J, and N) were reported to have mild to moderate lymphoplasmacytic encephalitis or meningoencephalitis and the remaining six brains had no significant findings. Initial diagnostic workup\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eruled out rabies virus, canine distemper virus, highly pathogenic avian influence virus, and bromethalin exposure. Additionally, drugs of abuse, strychnine, carbamate and organophosphorous pesticides, and other toxicants detected by GCMS were not observed in the samples. Of skunk liver samples, twelve (71%) were positive for at least one chlorinated pesticide by GCMS on initial screening. Fat content ranged 4.47-10.48% in brain and 2.97-22.83% in liver (Table 1). Concentrations of the three most abundant analytes and summed chlordane related compounds are presented in Table 2. All analyte concentrations are reported on a wet- (w.w.) and lipid weight (l.w.) basis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFour skunk brains did not contain individual analyte concentrations at or above the 10 ng/g reporting limit (J, O, P, and Q). Of all compounds analyzed, concentrations of oxychlordane, heptachlor epoxide, and trans-nonachlor were present in the highest concentrations. Oxychlordane was present in greatest abundance in the skunks with the highest total chlordane burden as noted in Figure 1. Skunks D, H, and M were found to have brain oxychlordane residues that exceeded the 10 \u0026micro;g/g (10,000 ng/g) w.w. lethal residue reported by Stickel et al. for birds.\u003csup\u003e7\u003c/sup\u003e The brain tissue of skunk C contained oxychlordane residues of 9,185 ng/g w.w. which was considerably increased with respect to previously reported lethal thresholds for birds; however, the additional brain burden of heptachlor epoxide with a concentration of 2,515 ng/g w.w. suggests a potentially lethal burden of chlordane metabolite in this animal\u0026rsquo;s brain.\u003csup\u003e7\u003c/sup\u003e The remaining eight skunks had total chlordane, nonachlor, and metabolite concentrations ranging from 62-3,782 ng/g w.w. in brain. Numeric concentrations of summed cyclodienes in brain tissue (w.w.) were converted to categorical data for exposure based on the scheme: low (\u0026lt;1,000 ng/g), intermediate (1,000 \u0026ndash; 9,999 ng/g), and high (\u0026gt;10,000 ng/g) exposure groups and by county of origin (Wayne versus other counties). Chi-square for trends analysis revealed a statistically significant trend (p=0.018); however, a Fisher\u0026rsquo;s exact test (p=0.073) did not find an association between concentrations and represented counties. This is likely due to the small sample size of fewer than twenty animals.\u003c/p\u003e\n\u003cp\u003eConcentrations of chlordane metabolites and trans-nonachlor were significantly greater in liver tissue when compared to brain tissue from the same individual. Summed chlordane, nonachlor, and metabolite concentrations in liver tissues ranged from 34-815,826 ng/g w.w. and at least one analyte was detected in all but one individual. Seven animals were reported to have total liver residue concentrations less than 1,000 (range 34-986), two less than 10,000 (3,477 and 9,232), five less than 100,000 (range 17,917-78,258) and three greater than 100,000 ng/g w.w. (range 185,648-815,826). When analytes were reported on a lipid weight basis for brain and liver, liver total chlordane and metabolite concentrations were approximately ten-fold greater than brain concentrations. A simple linear regression comparing brain and liver concentrations on a lipid basis yielded an r\u003csup\u003e2\u003c/sup\u003e=0.9581 compared to wet weight concentrations with r\u003csup\u003e2\u003c/sup\u003e=0.8080 as noted in Figure 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLow concentrations (\u0026lt;500 ng/g w.w.) of dieldrin were detected in 24% of brains and 59% of livers. Two liver samples, H and M, had dieldrin concentrations of 887 and 2,417 ng/g w.w. respectively. Additionally, low concentrations (\u0026lt;500 ng/g w.w.) of DDE were detected in 47% of brains and 41% of livers. Skunks H and M also contained DDE concentrations of 619 and 1453 ng/g w.w. in brain tissue. Five other skunks had hepatic DDE concentrations ranging from 706-6806 ng/g w.w. while skunk H contained 11,975 ng/g w.w. DDE. DDD was detected in trace amounts in the liver of skunks C and I and at concentrations of 1,019 and 717 ng/g w.w. in skunks H and M respectively. DDD was not detected in \u0026nbsp;brain tissue of any skunk at concentrations exceeding the 10 ng/g reporting limit.PCBs were detected in 47% of brain samples and 94% of liver samples. In brain, total PCB concentrations ranged from 14-135 ng/g w.w. or 206-3021 ng/g l.w. in the eight positive samples. Liver total PCB concentrations ranged from 19-640 ng/g w.w. or 510-11,138 ng/g l.w. in the sixteen positive samples. Negative samples did not have individual PCB congener concentrations exceeding the 10 ng/g reporting limit. PCB congeners #52, 101, and 126 were not detected in any of the seventeen brain or liver samples. Congeners #153, 138, and 180 were found in the highest concentrations in order of decreasing abundance. Accumulation of chlordane\u0026nbsp;and its metabolites was not correlated with accumulation of PCB congeners in either liver or brain tissue with r\u003csup\u003e2\u003c/sup\u003e values of 0.0161 and 0.1754 respectively as described in Figure 3.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTen out of seventeen skunks (59%) were submitted from Wayne County, Michigan which encompasses the majority of the River Rouge Watershed. This region has been known for its historical contamination with PCBs and other pollutants (Figure 4).\u003csup\u003e3\u003c/sup\u003e The River Raisin and River Rouge Watersheds span the greater Detroit metropolitan area across Wayne, Oakland, and Washtenaw counties and are the most urbanized and densely populated watersheds in the state of Michigan.\u003csup\u003e1\u0026ndash;4\u003c/sup\u003e Designated an Environmental Protection Agency (EPA) Area of Concern (AOC) under the Great Lakes Water Quality Agreement of 1987, the River Rouge prompted the MDNR and Michigan Water Resources Commission to adopt a 20-year remedial action plan beginning in 1985.\u003csup\u003e1,3\u003c/sup\u003e The river\u0026rsquo;s sediment contamination with polycyclic aromatic hydrocarbon (PAH), PCB, oil, grease, and heavy metals has led to ongoing monitoring studies, beneficial use impairments, and concerted clean-up efforts.\u003csup\u003e3\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePCBs, dieldrin, and DDE are often analyzed together and are expected to co-accumulate due to common source of exposure in contaminated watersheds or through bioaccumulation within aquatic food webs.\u003csup\u003e20,27\u0026ndash;30\u003c/sup\u003e Of interest to this study, the accumulation of PCBs, dieldrin, and DDE were not well correlated within brain nor liver tissue. Within brain, the correlation coefficients of simple linear regression analysis were: dieldrin vs DDE R\u003csup\u003e2\u003c/sup\u003e\u0026lt;0.01, PCBs vs DDE R\u003csup\u003e2\u003c/sup\u003e=0.5669, and PCBs vs dieldrin R\u003csup\u003e2\u003c/sup\u003e\u0026lt;0.01. Similarly, the correlation coefficients of simple linear regression in liver were: dieldrin vs DDE R\u003csup\u003e2\u003c/sup\u003e=0.0128, PCBs vs DDE R\u003csup\u003e2\u003c/sup\u003e=0.2280, and PCBs vs dieldrin R\u003csup\u003e2\u003c/sup\u003e=0.0379. While dieldrin, DDE, and PCBs are unlikely to have significantly contributed to the moribund status or death of these skunks, the occurrence of these analytes as persistent organic pollutants in the Detroit Metropolitan Area and their long-term implications for human and ecological health at low concentrations remains a concern.\u003c/p\u003e\n\u003cp\u003eLethal thresholds for PCB brain residues are reported as 310,000 ng/g w.w. for birds fed Aroclor 1254 at a rate of 1,500 \u0026mu;g/g feed dry weight. None of the skunks reported herein exceeded the lethal threshold for PCB residues in brain tissue with the greatest concentration reported at 135 ng/g w.w..\u003csup\u003e30\u003c/sup\u003e For comparison, PCB concentrations in Japanese raccoon dogs ranged from 24-1,200 ng/g l.w. in liver while PCBs in skunks from this study ranged from 510-11,138 ng/g l.w.. The PCB congeners 138, 153, and 180 were found in greatest abundance and are suggestive of environmental contamination from Aroclors 1260 and/or 1254.\u003csup\u003e31\u003c/sup\u003e With concentrations 2,000 times lower than those reported by Stickel, et al. it is unlikely that the PCB residues contributed to the neurologic presentation of these skunks.\u003csup\u003e30\u003c/sup\u003e The finding of PCB residues in skunks residing in areas supplied by the River Rouge Watershed further reinforces that PCBs contaminate and persist in this region nearly 35 years after the remedial action plan was implemented.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrevious reports indicate lethal brain dieldrin concentrations in birds of 10,000 ng/g w.w. and DDE of 500,000 ng/g w.w. for birds\u003csup\u003e7,32,33\u003c/sup\u003e, and acutely lethal brain dieldrin concentrations for dogs averaged 5,500 ng/g w.w. in dogs.\u003csup\u003e34\u003c/sup\u003e Additionally, concentrations of DDE ranged from 5.3-150 ng/g l.w. in liver of Japanese raccoon dogs.\u003csup\u003e16\u003c/sup\u003e Dieldrin and DDE did not exceed previously reported lethal brain concentrations in any of the submitted skunks. The skunks presented herein had hepatic DDE concentrations ranging from 188-175,364 ng/g l.w., and suggest either increased dietary exposure, or decreased clearance of DDE by skunks when compared to raccoon dogs. These findings of sublethal PCB, dieldrin, and DDE concentrations further support that the neurologic state of these animals were more likely attributed to the combined burdens of oxychlordane, heptachlor epoxide, and trans-nonachlor.\u003c/p\u003e\n\u003cp\u003eTissue chlordane and metabolite residue concentrations in skunks found in urban and suburban areas of Michigan far exceed previous reports in other small wildlife species. A 2007 analysis of ten raccoon dogs (\u003cem\u003eNyctereutes procyonoides)\u003c/em\u003e collected outside of Tokyo found hepatic concentrations of oxychlordane ranging from 160-20,000 ng/g l.w., concentrations significantly lower than those reported here.\u003csup\u003e16\u003c/sup\u003e The racoon dogs in this study succumbed to traffic accidents; therefore, antemortem clinical signs were not reported.\u003csup\u003e16\u003c/sup\u003e Racoon dogs are opportunistic carnivores consuming small animal prey, fruits, vegetation, and insects similar to North American striped skunks (\u003cem\u003eMephitis mephitis)\u003c/em\u003e with each species filling comparable ecologic niches.\u003csup\u003e16,22\u003c/sup\u003e Striped skunks are known to inhabit residential areas by building dens around porches, homes, and garages or sheds.\u003csup\u003e22\u003c/sup\u003e Due to the close proximity of skunk dens to the foundation of homes and garages, skunks and other small residential wildlife species have an increased risk of environmental exposure to technical chlordane residues that were once applied in these locations for termiticidal properties.\u003csup\u003e5\u003c/sup\u003e Skunks positive for hepatic oxychlordane residues were found to have concentrations 155-times greater (range 11,318-3,096,515 ng/g l.w.) than those reported in raccoon dogs. While impressive, and suggestive of oxychlordane toxicosis, diagnostic criteria for neurotoxicity and lethality are not available for liver tissue.\u003c/p\u003e\n\u003cp\u003eThere are few studies available specifically investigating the neurotoxic effects of chlordane and its metabolites in mammals. Case studies in humans, for which convulsions and tremors were observed, involved high-dose acute exposures as opposed to chronic environmental or dietary exposure.\u003csup\u003e23,24\u003c/sup\u003e Tremors were reported in rats and mice receiving high-dose chronic oral exposures of chlordane throughout a carcinogenicity bioassay.\u003csup\u003e25\u003c/sup\u003e Previous studies in laboratory animals may not observe neurotoxic effects due to insufficient dose, short study duration, or utilization of alternative end points such as feed refusal or severe weight loss.\u003csup\u003e5,35,36\u003c/sup\u003e This may be due in part to the steep dose-response curve reported by one study in rats which reported an oral toxic dose of 10 mg/kg body weight (BW) and NOEL dose of 1 mg/kg BW with toxicity defined as feed refusal and weight loss.\u003csup\u003e36\u003c/sup\u003e For this reason, postmortem tissue chlordane concentrations are difficult to interpret and diagnostic decision thresholds for neurotoxicity and lethality are not readily available for most species. Female rats receiving 10 mg/kg BW per day oxychlordane by oral gavage for 28 days were reported to contain adipose tissue oxychlordane concentrations of approximately 600,000 ng/g l.w. and liver concentrations of approximately 18,000 ng/g w.w.\u003csup\u003e36\u003c/sup\u003e Rats in the 10 mg/kg BW per day dosing group were lethargic and unkempt, but tremors and convulsions were not reported. We report skunk hepatic oxychlordane concentrations up to 707,000 ng/g w.w. (range 517-706,981 ng/g w.w.), nearly 40x greater than those experimentally produced in rats suggesting a greater dose, repeated exposure, increased storage efficiency or capacity of oxychlordane, or decreased metabolism and elimination or chlordane and related compounds in these skunks.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThree skunks contained brain oxychlordane residues exceeding the 10,000 ng/g w.w. lethal threshold reported for birds.\u003csup\u003e7\u003c/sup\u003e The brain tissue of a fourth skunk contained oxychlordane residues of approximately 9,185 ng/g w.w. and is considerably increased in accordance with previous lethal thresholds reported for birds; the additional brain burden of heptachlor epoxide at 2,515 ng/g w.w. suggests a lethal brain chlordane metabolite burden for this animal.\u003csup\u003e7\u003c/sup\u003e Therefore, the death or moribund status of these four animals may be attributed to the neurotoxic effects of these analytes. Moreover, if an uncertainty factor of 10 is utilized to account for species differences, we may consider brain residues of 1,000 ng/g w.w. as potentially lethal. Four additional skunks had brain oxychlordane residues exceeding 1000 ng/g w.w. with a fifth considered marginal (753 ng/g). Although the lethal residues described are for oxychlordane individually, skunks were found to have significant burdens of heptachlor epoxide and trans-nonachlor as well. It is important to reiterate that oxychlordane is six times more toxic when compared to technical chlordane in birds and hepatachlor epoxide is 10 times more toxic than either isomer of chlordane in rats.\u003csup\u003e7,12\u003c/sup\u003e Therefore, the use of comprehensive organochlorine panels and summed chlordane and metabolite burdens provides a more accurate diagnostic approach than analysis of any one single analyte. Data have been presented and discussed throughout on a wet weight basis for ease of interpretation when comparing to previous studies and diagnostic criteria. It is of note that correction for lipid content of tissues and reporting on a lipid weight basis allows for more accurate comparison across tissues within individual animals and within and across species. Liver was shown to be predictive of brain chlordane concentrations on a lipid weight basis and can be utilized to predict neurotoxic chlordane concentrations in brain when diagnostic samples are limited, or brain tissue cannot otherwise be collected during postmortem examination.\u003c/p\u003e\n\u003cp\u003eWhile there is strong evidence that toxicologically significant concentrations of chlordane and its metabolites accumulated in the brain tissue of multiple animals, statistically significant differences between groups in this population could not be elucidated. Nevertheless, there is evidence that animals which originated from the Detroit Metropolitan Area have higher brain chlordane concentrations than those from other counties in the state of Michigan (Figure 4). Animals in the highest exposure group originated from Redford and Dearborn Heights within Wayne County, Michigan. Additionally, one animal submitted from Grand Rapids in Kent County, Michigan fell within the intermediate exposure group. A statistically significant difference between population groups when comparing Wayne County to other Michigan counties could not be elucidated. The lack of statistically significant findings in this population are largely due to the small sample size of fewer than twenty animals. Inclusion of additional animals from the western and northern counties as well as the Upper Peninsula of Michigan will provide further insight to the correlation between chlordane metabolite accumulation in densely populated metropolitan areas when compared to rural communities of Michigan. \u0026nbsp;Additionally, due to the circumstances by which animals are submitted for post-mortem examination, samples from this population are unlikely to be randomly selected and the population is not normally distributed violating the assumptions of multiple statistical models. For these reasons, it is not unexpected that a statistically significant difference could not be elucidated despite having strong evidence for toxicologically significant exposures.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study provides the first report of neurotoxicosis associated with chlordane exposure in skunks across the greater Detroit Metropolitan area. Herein, we have reported alarmingly high concentrations of oxychlordane and heptachlor epoxide, two known chlordane metabolites with neurotoxic potential Contamination of the Great Lakes and watersheds across the state of Michigan remains a focus for improved water quality. This study emphasizes a further concern for soil and insect contamination associated with these watersheds that provide habitat and food sources for Michigan\u0026rsquo;s wildlife, and an urban environment for human activities. A better understanding of the environmental persistence of technical chlordane components and metabolites in soil, water, and insect populations from areas with widespread historical use is critical to the management of this widely found pollutant. We have highlighted the need for more systemic, broad-scale research efforts to monitor technical chlordane in wildlife, domestic animals, and humans in urban settings to understand the nature and extent of pesticide pollution and mitigate risk associated with exposure to these compounds.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Michigan State University College of Veterinary Medicine\u0026rsquo;s Edward K Sales Endowed Research Fund.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRMS conducted the analytical analysis, data interpretation, and primary writing for the manuscript. BP provided data interpretation, writing, and editing support for the manuscript. SDF served as the pathologist for the study and provided written narrative for pathologic findings. JM provided support in coordinating animal collection, initial necropsy, and submission of samples for analysis. JM also provided support in editing. JPB coordinated direction of the manuscript, data interpretation and provided editing support. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Conflicting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll associated data presented herein will be made available on reasonable request. Correspondence and request for materials should be addressed to JPB.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRidgway, J., Cave, K., DeMaria, A., O\u0026rsquo;Meara, J. \u0026amp; Hartig, J. H. The Rouge River Area of Concern - A multi-year, multi-level successful approach to restoration of Impaired Beneficial Uses. Aquat. Ecosyst. Heal. 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Manage. 54, (1990).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamagishi, T., Kaneko, S., Miyazaki, T., Akiyama, K. \u0026amp; Horii, S. Oxychlordane, trans- nonachlor and cis-nonachlor residues in adipose tissues of dogs and cats collected in the Tokyo area. Bull. Environ. Contam. Toxicol. 26, (1981).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStansley, W. \u0026amp; Roscoe, D. E. Chlordane poisoning of birds in New Jersey, USA. Environ. Toxicol. Chem. 18, (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWattigney, W. A., Irvin-Barnwell, E., Li, Z. \u0026amp; Ragin-Wilson, A. Biomonitoring of toxic metals, organochlorine pesticides, and polybrominated biphenyl 153 in Michigan urban anglers. Environ. Res. 203, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarbehenn, K. R. \u0026amp; Reichel, W. L. Organochlorine concentrations in bald eagles: Brainjbody lipid relations and hazard evaluation. J. Toxicol. 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Determination of Organochlorine Pesticides in Wildlife Liver and Serum Using Gas Chromatography Tandem Quadrupole Mass Spectrometry. J. Chromatogr. Sep. Tech. 06, (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarpenter, S. K. \u003cem\u003eet al.\u003c/em\u003e River otters as biomonitors for organochlorine pesticides, PCBs, and PBDEs in Illinois. Ecotoxicol. Environ. Saf. 100, (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBatt, A. L., Wathen, J. B., Lazorchak, J. M., Olsen, A. R. \u0026amp; Kincaid, T. M. Statistical Survey of Persistent Organic Pollutants: Risk Estimations to Humans and Wildlife through Consumption of Fish from U.S. Rivers. Environ. Sci. Technol. 51, (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHargrave, B. T. \u003cem\u003eet al.\u003c/em\u003e Organochlorine pesticides and polychlorinated biphenyls in the Arctic Ocean food web. Arch. Environ. Contam. Toxicol. 22, (1992).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStickel, W. H., Stickel, L. F., Dyrland, R. A. \u0026amp; Hughes, D. L. Aroclor 1254\u0026reg; residues in birds: Lethal levels and loss rates. Arch. Environ. Contam. Toxicol. 13, (1984).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRisso, F. \u003cem\u003eet al.\u003c/em\u003e A comprehensive approach to actual polychlorinated biphenyls environmental contamination. Environ. Sci. Pollut. Res. 23, (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStickel, W. H., Stickel, L. F. \u0026amp; Spann, J. W. Tissue residues of dieldrin in relation to mortality in birds and mammals. in \u003cem\u003eChemical Fallout: Current Research on Persistent Pesticides. Proceedings of the First Rochester Conference on Toxicity\u003c/em\u003e 174\u0026ndash;204 (1969).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStickel, W. H., Stickel, L. F., Dyrland, R. A. \u0026amp; Hughes, D. L. DDE in birds: Lethal residues and loss rates. Arch. Environ. Contam. Toxicol. 13, (1984).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarrison, D. L. \u0026amp; Manktelow, B. W. Dieldrin poisoning of dogs. N. Z. Vet. J. 8, (1960).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBondy, G. S. \u003cem\u003eet al.\u003c/em\u003e Trans-nonachlor and cis-nonachlor toxicity in sprague-dawley rats: Comparison with technical chlordane. Toxicol. Sci. 58, (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBondy, G. \u003cem\u003eet al.\u003c/em\u003e Toxicity of the chlordane metabolite oxychlordane in female rats: Clinical and histopathological changes. Food Chem. Toxicol. 41, (2003).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Wildlife, Chlordane, Persistent Organic Pollutant, Organochlorine, Great Lakes, Neurotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-4546233/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4546233/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDespite the ban of technical chlordane, contamination from this persistent organic pollutant threatens wildlife and human health nearly forty years since its last application. In this study, eight of seventeen skunks displaying illness and neurologic signs had brain tissue concentrations of combined oxychlordane, heptachlor epoxide, and trans-nonachlor exceeding a 1,000 ng/g wet weight diagnostic threshold for toxicosis. Liver tissue concentrations were ten-fold greater than brain when measured on a lipid weight basis, which can help predict lethal brain residues in skunks. The ongoing presence of chlordane in the environment is expected to cause further unintended consequences for wildlife across the Detroit Metropolitan area for decades to come. As with other pollutants in the River Rouge and River Raisin Areas of Concern, the presence of chlordane in the urban environment presents a significant risk for animal, human and ecological health.\u003c/p\u003e","manuscriptTitle":"Chlordane induced neurotoxicosis in urban and suburban Detroit, Michigan striped skunks (Mephitis mephitis)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-27 05:58:30","doi":"10.21203/rs.3.rs-4546233/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2ef90097-fe5c-4622-9b52-9c39a8abf223","owner":[],"postedDate":"June 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33680348,"name":"Health sciences/Diseases/Neurological disorders/Neurotoxicity syndromes"},{"id":33680349,"name":"Earth and environmental sciences/Environmental sciences/Environmental chemistry/Pollution remediation"}],"tags":[],"updatedAt":"2025-01-19T17:23:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-27 05:58:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4546233","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4546233","identity":"rs-4546233","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}