Adult exposure to insecticides causes persistent behavioral and neurochemical alterations in zebrafish

Journal Pre-proof Adult exposure to insecticides causes persistent behavioral and neurochemical alterations in zebrafish

Andrew B. Hawkey, Lilah Glazer, Cassandra Dean, Corinne N. Wells, Kathryn-Ann Odamah, Theodore A. Slotkin, Frederic J. Seidler, Edward D. Levin PII:

S0892-0362(19)30153-9

DOI:

https://doi.org/10.1016/j.ntt.2019.106853

Reference:

NTT 106853

To appear in:

Neurotoxicology and Teratology

Received date:

12 November 2019

Revised date:

20 December 2019

Accepted date:

20 December 2019

Please cite this article as: A.B. Hawkey, L. Glazer, C. Dean, et al., Adult exposure to insecticides causes persistent behavioral and neurochemical alterations in zebrafish, Neurotoxicology and Teratology (2020), https://doi.org/10.1016/j.ntt.2019.106853

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© 2020 Published by Elsevier.

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Hawkey et al, 2019

Adult Exposure to Insecticides Causes Persistent Behavioral and Neurochemical Alterations in Zebrafish Andrew B. Hawkey,* Lilah Glazer,*,† Cassandra Dean,* Corinne N. Wells,* Kathryn-Ann Odamah,* Theodore A. Slotkin,# Frederic J. Seidler,# and Edward D. Levin*,#,1 *

Department of Psychiatry and Behavioral Sciences, Duke University School of Medicine,

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†

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Durham, NC, 27710, USA

School of Biological and Chemical Sciences, Queen Mary University of London, London,

Department of Pharmacology and Cancer Biology, Duke University School of Medicine,

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#

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E1 4NS, UK. Email: [email protected]

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Durham, NC, 27710, USA

To whom correspondence should be addressed at Department of Psychiatry and Behavioral

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Sciences, Box 104790, Duke University Medical Center, Durham, NC 27710, USA, Phone: 1-

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919-681-6273; Fax: 1-919-681-3416. Email: [email protected]

Running Head: Pesticide neurotoxicity in adult zebrafish Keywords: zebrafish; DDT; aging; neurobehavioral toxicology, anxiety-related behavior

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Abstract Farmers are often chronically exposed to insecticides, which may present health risks including increased risk of neurobehavioral impairment during adulthood and across aging. Experimental animal studies complement epidemiological studies to help determine the cause-and-effect relationship between chronic adult insecticide exposure and behavioral dysfunction. With the zebrafish model, we examined short and long-term

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neurobehavioral effects of exposure to either an organochlorine insecticide, dichlorodiphenyltrichloroethane (DDT) or an organophosphate insecticide chlorpyrifos (CPF). Adult fish were exposed continuously for either

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two or 5 weeks (10-30nM DDT, 0.3-3uM CPF), with short- and long-term effects assessed at 1-week post-

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exposure and at 14 months of age respectively. The behavioral test battery included tests of locomotor activity,

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tap startle, social behavior, anxiety, predator avoidance and learning. Long-term effects on neurochemical

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indices of cholinergic function were also assessed. Two weeks of DDT exposure had only slight effects on locomotor activity, while a longer five-week exposure led to hypoactivity and increased anxiety-like diving

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responses and predator avoidance at 1-week post-exposure. When tested at 14 months of age, these fish showed

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hypoactivity and increased startle responses. Cholinergic function was not found to be significantly altered by DDT. The two-week CPF exposure led to reductions in anxiety-like diving and increases in shoaling responses

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at the 1-week time point, but these effects did not persist through 14 months of age. Nevertheless, there were persistent decrements in cholinergic presynaptic activity. A five-week CPF exposure led to long-term effects including locomotor hyperactivity and impaired predator avoidance at 14 months of age, although no effects were apparent at the 1-week time point. These studies documented neurobehavioral effects of adult exposure to chronic doses of either organochlorine or organophosphate pesticides that can be characterized in zebrafish. Zebrafish provide a low-cost model that has a variety of advantages for mechanistic studies and may be used to expand our understanding of neurobehavioral toxicity in adulthood, including the potential for such toxicity to influence behavior and development during aging. 2

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1. Introduction Since the introduction of synthetic insecticides in the mid-20th century, their use has been ubiquitous in pest control. At present, there are an array of effective compounds available, utilizing multiple chemical classes and mechanisms of action. While these compounds have had remarkable benefits for improving food production and vector control of insect-borne diseases,

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the risks they pose as contaminants are increasingly recognized as major concerns (Oberemok,

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2015). Through the 20th century, some of the most prominent insecticides have been banned or restricted due to accumulated evidence for health or environmental effects and neurotoxicity,

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including organochlorines such as dichlorodiphenyltrichloroethane (DDT) and organophosphates

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such as chlorpyrifos (CPF) (Abreu-Villaca, 2017; Banks, 2005; Hellou, 2012). Although bans

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have suppressed organochlorine use, particularly DDT which was broadly banned in the US in 1972, it has remained a prominent pesticide for the control of mosquito-borne diseases through

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much of the world (Bouwman, 2011). Despite domestic use bans, organophosphates remain

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heavily used in agriculture (Trasande, 2017). Due to their pervasive use historically, now banned

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or restricted compounds may pose a persistent public health issue as long-term effects across the lifetime of those exposed.

While the bulk of neurotoxicity testing for pesticides has focused on exposures during early development (Abreu-Villaca, 2017), existing data suggests that adults, particularly those subjected to occupational exposures, are also at risk for a number of long-term health problems, including adverse neurobehavioral effects. For example, agricultural workers with a history of exposures to one or more pesticides show deficits on a range of neuropsychological assessments (Malekirad, 2013; Muñoz-Quezada, 2016) including impaired psychomotor speed and dexterity, mood, visuospatial memory, and working memory. Furthermore, emerging data suggests that 3

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long-term development into aging may also be impacted by adult pesticide exposures (Hernández, 2016), including enhancements in neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease (Hayden, 2010; Jokanović, 2018; Kamel, 2007; Mostafalou, 2018). For instance, Hayden et al (2010) surveyed residents of an agricultural community in Utah over the age of 65, and found elevated risk for Alzheimer’s and non-Alzheimer’s dementia among individuals with a history of pesticide exposures, particularly organophosphates. These

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latter findings are particularly concerning for organochlorine and organophosphate pesticides, as

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the primary exposed populations for DDT are now largely elderly, and the exposed populations

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for CPF contain a large segment entering the age of elevated risk for age-related declines and

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diseases. However, at present, the short and long-term effects of adult exposures to individual pesticides and their relevant mechanisms of action have yet to be fully evaluated.

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In order to evaluate the risks posed by adult pesticide exposures, animal models are

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critical for the demonstration of key features, outcomes and mechanisms of toxicity for further study. With respect to pesticide exposures early in development, zebrafish have been shown to

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be a strong complementary model of neurotoxicity, detecting effects which mirror those

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generated by rodent models (Aldridge, 2005; Eddins, 2010). Adult zebrafish may be similarly valuable for assessing pesticide effects on mature nervous systems. As vertebrates, zebrafish share many similarities with humans, including overlaps in genetics, physiology, general nervous system organization, and mechanisms relevant to pharmacology, toxicity and neurobehavioral function (Guo, 2004; Lieschke, 2007). Zebrafish also display an array of behaviors that can be used to probe neurological functions, including locomotion, reflexive startle responses, avoidance- and anxiety-like responses, prosocial behaviors and learning (Kalueff, 2013; E. D. Levin, & Cerutti, D. T. , 2009). Furthermore, their small size, low cost and dense housing allow

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zebrafish to be practically maintained for life-span length studies which are difficult to reasonably conduct with rodent models. Given these strengths, zebrafish may be ideal for assessing the long-term neurotoxic potential of insecticides in adult vertebrates. The current study utilized adult zebrafish to identify neurobehavioral effects which may be produced by adult exposure to DDT or CPF. Adult zebrafish were chronically exposed to DDT or CPF and then assessed with a battery of neurobehavioral tests for locomotor activity,

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reflexive behaviors, reward, anxiety, predator avoidance and recognition memory. In this study,

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multiple sub-experiments were performed to assess pesticide effects at a range of doses and

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durations of exposures. Adult fish (ages 6-8 months) were exposed to DDT (10-30nM) or CPF

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(0.1-3µM) for either two or 5 weeks. Behavioral testing was performed at 1-week post-exposure (short-term effects) and/or at 14 months of age (effects evident later in development). The latter

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testing window of 14 months of age was selected to detect long-term effects of adult DDT or

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CPF exposure shortly before the onset of age-related declines in zebrafish health and numbers, which we have observed to occur between 18 and 24 months (data not shown).

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In addition to behavioral tests, we evaluated whole brains for two biomarkers of

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cholinergic presynaptic innervation: the activity of choline acetyltransferase (ChAT) and the concentration of presynaptic high-affinity choline transporters (hemicholinium-3 [HC3] binding). ChAT is the enzyme that synthesizes ACh, but is not regulated by nerve impulse activity, so that its presence provides an index of the number of acetylcholine synapses (Slotkin, 2008). In contrast, HC3 binding to the choline transporter is directly responsive to neuronal activity (Klemm, 1979), so that comparative effects on HC3 binding and ChAT enable the characterization of both the density of cholinergic innervation and presynaptic impulse activity. For the DDT studies, evaluations were made in the groups receiving 5 weeks of treatment,

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measured at the end of the behavioral studies. For CPF, neurochemistry was performed on the groups receiving 2 weeks of treatment, and again, was measured after the completion of the behavioral measures.

Prior studies in rodents have shown that organochlorine and

organophosphate pesticides target cholinergic function (Abreu-Villaca, 2017).

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2. Materials and Methods 2.1 Fish Housing and Husbandry

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All experiments were conducted using a local colony of AB* wild-type strain of

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zebrafish, maintained and bred in the Levin Laboratory at Duke University. The experimental

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procedures were approved by the Duke University Institutional Animal Care and Use Committee. Prior to exposure adult zebrafish were held in mixed (females and males) groups at a

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density of ≤5 fish/l in 3 or 10 l tanks kept on a recirculating flowing water system (Aquatic

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Habitats, Inc., Apopka, FL, USA; Aquatic Enterprises, Inc., Bridgewater, MA, USA). System water was a mixture of sea salt (Instant Ocean, 0.5 parts per thousand) and buffer (Seachem

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Neutral Regulator, 125 mg/l) in de-ionized water. Water chemistry, salinity and temperature were monitored weekly. Illumination was set to 14:10 h light:dark cycle and water temperature was kept at 28 ± 1C. The fish were fed three times daily; morning and afternoon with brine shrimp (Artemia salina) hatched in-house over 24 h (eggs from Brine Shrimp Direct, Ogden, UT, USA); and noon feeding with solid pellet food GEMMA Micro 300 micro-pellets (Skretting USA, Tooele, UT, USA).

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2.2 Chemicals Dimethyl Sulfoxide ReagentPlus®, ≥99.5% (DMSO; CAS# 67-68-5, Lot# SHBG9650V) was purchased from Sigma-Aldrich (St. Louis, MO, USA). 4,4′-Dichlorodiphenyltrichloroethane (DDT) and chlorpyrifos were purchased from Chem Service, Inc., (West Chester, PA, USA). These compounds dissolved in 100% DMSO stocks which were then delivered into exposure tanks at 10uL DMSO/L of system water (0.001%). Dose ranges were selected based on prior

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findings or initial screening during pilot testing. These doses were selected because they were

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below those that would increase lethality or impair the clinical health of the fish.

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2.3 Exposure of adult zebrafish

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Adult fish at 6-8 months of age were exposed to either 0.001% DMSO alone (vehicle control) or one of the below concentrations of DDT or CPF in 0.001% DMSO. Exposures were

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conducted in glass tanks (Deep Blue Professional, City of Industry, CA, USA) containing 3.5

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liters of system water. Each tank was equipped with its own aeration tube and mini-pump, heater and thermometer, and the water was maintained at 26 ± 1 C. Each tank contained 10 fish, with a

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target of 2 cohorts and 3-4 replicate tanks being generated for each experiment (except for set 1, which contained a single replicate tank of each). The fish were allowed to acclimate to the tanks for 1 week prior to exposure. Exposure water was a 1:1 mixture of fresh system water and water pulled from an established flow-through housing system. This was done to supplement the stilltanks with nitrifying microbes and prevent the buildup of ammonia released by uneaten food and other waste. Exposure water was replaced once per week and the dosing was renewed with each water change. At the end of the exposure period, the fish were transferred to clean glass tanks

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with non-dosed system water and remained in these tanks until the completion of the first behavioral testing battery.

2.4 Relevance of the model and doses Adult zebrafish were selected for use due to their neurophysiological and genetic

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similarity to humans as vertebrates, allowing testable predictions to be made as to the impacts of

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pesticide exposures in mammalian species. Additionally, zebrafish were selected for their practical advantages for long-term testing, including low cost and dense housing, which make

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these studies more feasible. Doses were selected according to prior data or tolerability testing (as

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needed), to identify does ranges below the threshold for over toxicity (either behavioral or lethal)

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in adults. These doses are not modelled directly off of human exposure levels, but are scaled to this toxicity threshold. As a result, they represent physiologically-relevant doses which are likely

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to go unnoticed or untreated, due to their subtle acute effects.

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2.5 Experimental Design

A series of cohorts and exposures were generated in order to identify appropriate dose ranges and durations of exposure for each compound (doses and sample sizes, see Table 1). All fish in a given testing cohort came from the same egg collection (ie. - were age matched), whereby egg traps were placed into tanks of mixed-sex fish (multiple tanks of 15-30 fish represented per egg lay) to allow heterogenous egg-laying and fertilization. At 6 months of age, the resulting fish were eligible to be randomly assigned to treatment conditions and begin an exposure series, with the exact timing of exposure subject to the availability of exposure 8

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facilities. To begin this series, a preliminary study evaluated the tolerability and behavioral effects of a range of DDT doses (10-100nM) using a two-week exposure period. This consisted of one tank (n=10) per condition at the doses 10, 30 and 100uM. The findings of this initial study indicated that survival was impaired at a DDT concentration of 100nM, but not for doses of 30nM or below. Two-week exposures at these non-lethal doses produced minimal behavioral effects at a 1-week follow-up. Therefore, set 2 was generated with an extended exposure period

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of 5 weeks, utilizing a range of doses 3-60nM (cohort 1: 3-30uM, cohort 2: 10-60). The range for

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analysis was determined to be 10-30nM, as only one replicate cohort of 3uM was produced and

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60nM led to reduced survival. Set 2 fish were tested at 1-week post-exposure and again at 14

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months of age, then subjected to brain tissue removal and neurochemical analysis. Following this general design, the CPF study proceeded with an assessment of adult fish exposed to CPF at 0.3-

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3uM for two weeks (set 3) and five weeks (set 4) with testing at 1-week post-exposure, and 14

behavioral testing.

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months of age. Brain tissue from set 3 was collected for neurochemical analysis following

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2.6 Behavioral Testing

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In each phase of testing, a series of behavioral assays was conducted to evaluate several emotional, social and cognitive functions. Each assay was conducted on a separate day between 10 AM and 5 PM, and testing times were counterbalanced across all experimental groups. Fish were allowed to acclimate to the testing room for 30-60 min prior to testing. An HD camcorder (VIXIA HFR700; Canon Inc., Tokyo, Japan) was used for video recording in all assays, and the videos were fed to the EthoVision XT® software (Noldus Information Technology, Wageningen, The Netherlands) for fish tracking position and activity analysis. An inclusion criterion was used for the live tracking data, whereby all tracked videos were required to contain a tracked object in

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96% or more of captured images for inclusion in the study, based on an autogenerated “subject not found” score in the Ethovision software. At the end of the first behavior test battery, the fish were transferred from still tanks to a recirculating flowing water system (Aquatic Habitats, Inc., Apopka, FL, USA) until the second round of testing. Adult behavioral assays were conducted as described in Glazer et al. (2018). 2.7 Novel Tank Dive Test

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Adult zebrafish were tested for novel environment response based on the method

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developed in our laboratory (Levin, 2007) with modifications (Glazer, 2018). Briefly, at the

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beginning of each trial fish were individually placed into a narrow trapezoidal 1.5-l tank

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(Aquatic Habitats) filled with 10 cm of system water. The tank was video recorded from the side

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for 5 min. Measurements extracted were total locomotor activity in cm for each min of testing

2.8 Startle Tap Test

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and the mean distance from the tank floor in cm per min.

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Sensorimotor startle response and habituation were tested using a custom-built apparatus and a protocol developed in our laboratory (Crosby, 2015; Eddins, 2010) with modifications

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(Glazer, 2018). Briefly, the apparatus consisted of eight clear cylindrical arenas arranged in a 2 × 4 setup and video recorded from above. The testing sequence consisted of a 30-s acclimation period followed by 10 consecutive taps at 1 min intervals. The taps were generated by 24-volt DC push solenoids located under each arena and activated by the EthoVision XT® software. Measurements extracted were total locomotor activity in cm during the 5-s period immediately before (pre) and after (post) each tap.

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2.9 Shoaling assay Individual social interaction was tested using the Multiple Use Partitioned Experimental Tank (MUPET), a custom-built adult behavior testing tank. The MUPET was situated on two metal bars and a light box (Huion Technology, Shenzhen, China) was placed underneath the tank bottom providing even light throughout the tank. Black acrylic partitions were used to create two adjacent lanes across the tank width. Two 19.5-inch LCD monitors flanked the narrow ends of

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the two lanes. A digital video camcorder was placed above the tank.

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The test procedure was based on a protocol developed in our laboratory (Oliveri, 2015),

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with modifications (Glazer, 2018). Briefly, adult fish were singly isolated for 30 min before

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being netted into the MUPET lanes described above. Behavior was recorded for a 7 min session

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consisting of a 2-min habituation period followed 5 min in which a video recording of a zebrafish shoal was played on one of the monitors. Measurements extracted were total locomotor

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activity in cm per min and the mean distance in cm per min to the side of the tank on which the video was displayed. A pre-post video difference value was calculated for each treatment group

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by subtracting the average distance from the tank side in the first four minutes after the video

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started playing from the average distance in the two minutes before the video began. 2.10 Predator avoidance assay Threat recognition and evasion behavior were tested using the same testing apparatus and set-up described in the previous section (Shoaling assay). The test procedure was based on a protocol developed in our laboratory (Oliveri, 2015), with modifications (Glazer, 2018). Briefly, individual fish were placed in the MUPET lanes and recorded for 9-min consisting of one min acclimation followed by 8-min of alternating minute-long stimulus/no stimulus (NS) events. The stimulus was a power point presentation showing either a blue slow-growing dot (4-s) or a red 11

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fast-growing dot (1-s) appearing repeatedly on one of the screens. These stimuli are 2dimentional representations of a large entity, such as predator, approaching the fish at either slow (blue) or fast (red) speeds. The blue dot appeared in the first two stimulus events and the red dot appeared in the last two stimulus events. Measurements extracted were total locomotor activity in cm per min, and the mean distance in cm per min to the side of the tank on which the stimulus was displayed. An avoidance response value was calculated for each stimulus by subtracting the

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average distance from the tank side during each NS period from the preceding period when the

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stimulus was present.

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2.11 Novel Place Recognition

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During the course of conducting this study, our lab piloted an additional behavioral test in order to assess learning and memory, and tested fish from Set 2 (5-week DDT exposure) and Set

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3 (2-week CPF exposure) prior to brain tissue collection (14-15 months old). Due to technical

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difficulties, this could not be assessed in Set 4. This test was a novel place recognition task, adapted from Cognato (2012) and based on the common novel object recognition task for rodents

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(Bevins, 2006). The apparatus for NPR was a Plexiglas plus maze consisting of a central hub (10cm x 10cm) with four arms (30cm x 10cm) radiating out from it. The walls of the arms had replaceable covers which could be used to show distinct visuospatial cues. For this version of the test, three of the four arms had matching covers with a black and white stripe pattern, with the fourth “novel” arm having a distinct blue background with green dots. Testing consisted of a 10 min familiarization session in the striped arms only (a removable opaque panel blocked off the “novel arm”), a two- hour retention period, and a 10 min recognition memory testing in the full

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maze accessible. Recognition memory was assessed as a preference between exploring the novel and familiar portions of the maze.

2.12 Neurochemical analysis Fish designated for brain tissue dissection were briefly anesthetized in ice water, weighed and culled by severing of the spinal cord using scissors. Whole brain tissue was then dissected,

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placed on a small piece of aluminum foil and weighed. The foil was then folded, flash frozen in

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liquid N2 and placed in -80°C until neurochemical analysis. For ChAT activity, assays were

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conducted on individual brains, so that determinations could be made of treatment effects for

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each sex (n=6-13 for each treatment group and sex). For HC3 binding, there was insufficient

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tissue to run assays on individual brains, so samples were prepared as pools of 4-6 brains for each sample, containing both males and females in each sample (n=4-6 for each treatment

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group). The assay techniques were identical to those used previously (Dam et al., 1999) and

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accordingly, will be described only in brief.

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Tissues were thawed and homogenized (Polytron) in 99 volumes of ice-cold 10 mM sodium-potassium phosphate buffer (pH 7.4). Aliquots of the homogenate were assayed for ChAT using 50 µM [14C]acetyl-coenzyme A[14C]acetyl-coenzyme A (PerkinElmer Life Sciences, Boston, MA, USA; specific activity 6.7 mCi/mmol) as a substrate and activity was determined as the amount of labeled ACh produced relative to tissue protein. For HC3 binding measurements, the cell membrane fraction was prepared from the same tissue homogenate and aliquots were assayed for using a ligand concentration of 2 nM [ 3H]HC3 (PerkinElmer Life Sciences; specific activity, 125 Ci/mmol) with or without 10 µM unlabeled

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HC3 (Sigma Aldrich), to displace specific binding. Ligand binding was calculated relative to the membrane protein concentration.

2.13 Statistical Analysis All statistical analyses were performed in SPSS v.21 (IBM Corp.). Significance was set at

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p < 0.05 for all analysis of variance (ANOVA) and post hoc comparisons. For each set of tests,

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the sequence of analysis proceeded as follows. A mixed factorial ANOVA was performed to detect main effects of time (e.g. 1 minute blocks across a testing session), treatment and, in the

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phase of testing (e.g. pre-stimulus vs during stimulus) or stimulus type (e.g. fast or slow cue), as

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required per test. Sex and housing tank identities were included as covariates. Main effects of a

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single factor (e.g. treatment) were investigated using post hoc T-tests with Tukey’s correction for multiple testing, collapsed across all other factors (time, phase of testing, etc). Follow-up tests

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were also performed on interactions at p < 0.10 (Snedecor & Cochran, 1967) using post hoc T-

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tests with Tukey’s correction for multiple testing at each of the relevant secondary units (e.g. Ttests of treatment in each time block in a session). A cut-off of p < 0.05 (two-tailed) was used as

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the threshold for significance of all main effect tests and post hoc comparisons. Violations of homogeneity of variance were corrected using the Greenhouse-Geisser correction (Greenhouse, 1959). This correction generally results in degrees of freedom that are not whole numbers. Summaries of treatment groups and sample sizes are shown in Table 1. A summary of statistical results is found in Table 2.

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3. Results 3.1 DDT Effects 3.1.1 Set 1: 2-week DDT exposure, 1-week post-test Behavioral endpoints 1-week after DDT exposure were generally unaffected by twoweeks of DDT treatment, with novel tank dive, shoaling and predator avoidance showing no

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effects of treatment, while the tap startle test did show minor treatment effects (Fig. 1). In the tap

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test, pre-tap activity showed significant effects of tap, F(5.89, 153.25) =2.86, p < 0.05, and

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treatment, F(2, 26) =3.86, p < 0.05, whereby fish with prior exposure to 30uM DDT had reduced

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pre-tap activity (Fig. 1). No other treatment effects or interactions were observed. The three remaining tests were not significantly affected by DDT treatment. In the novel

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tank dive test, main effects of time were observed for locomotor activity, F(2.47, 61.69) = 21.21,

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p < 0.05, and distance from the bottom, F(2.33, 58.21) = 10.71, p < 0.05, whereby each increased across the session. No other effects or interactions were observed. On the shoaling assay, a main

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effect of time was noted for locomotor activity, F(2.63, 63.10) = 10.19, p < 0.05, and for distance

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from the video screen, F(1, 24) = 4.32, p < 0.05, which was significantly reduced after the onset of the shoaling video (p< 0.05). No other main effects or interactions were observed. On the predator avoidance assay, main effects of time were detected for locomotor activity, F(4.79, 119.82) = 2.56, p < 0.05, and distance from the video screen, F(3.91, 97.98) = 4.67, p < 0.05. A main effect of predator stimulus (present/absent) was also observed, F(1, 25) = 22.39, p < 0.05, as well as an interaction of stimulus (present/absent) and predator speed (slow/fast), F(1, 25) = 5.62, p < 0.05. Distance from the screen was enhanced by the presence of the predator stimulus

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(p < 0.05) and by increased cue speed (p< 0.05). No other main effects or interactions were observed.

3.1.2 Set 2: 5-week DDT exposure, 1-Week post-test The longer 5-week DDT exposure led to multiple distinct behavioral effects at 1-week post-

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exposure (Fig. 2), including reduced activity in min 3-5 in the novel tank session (Fig. 2a),

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reduced distance from the bottom in the novel tank (Fig. 2b), and enhanced sensitivity to the

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faster predator stimulus (Fig. 2c and d). Shoaling and tap startle responses were not found to be

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affected at this time point.

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In the novel tank dive test, main effects were observed for locomotor activity, including time, F(2.86, 320.29) = 22.02, p < 0.05, and treatment, F(2.86, 320.29) = 22.02, p < 0.05, as was a

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trending time by treatment interaction, F(5.72, 320.29) = 1.87, p = 0.08. Post hoc comparisons showed that fish treated with 30nM DDT treatment were hypoactive relative to controls in the

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latter portions of the session (p = 0.09, 0.008 and 0.02 in minutes 3, 4 and 5 respectively) (Fig.

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2a). Additionally, fish from the 30nM group were significantly less active than the 10nM group in minute 4 (p < 0.05). No other effects or interactions were observed. For distance from the bottom, main effects were also observed for time, F(2.66, 297.54) = 15.38, p < 0.05, and treatment, F(2, 112) = 4.82, p < 0.05. Post hoc comparisons showed that fish exposed to 30nM DDT swam significantly closer to the bottom than controls (p < 0.05) (Fig. 2b). A similar trend between 30nM and 10nM approached significance (p = 0.057). On the tap test, significant main effects of tap were noted on pre-tap activity for tap, F(6.50, 697.25) =8.09, p < 0.05, and for the magnitude of the startle response, F(6.50, 702.37) =6.08, p < 16

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0.05. No significant treatment effects or interactions were detected on these outcomes. For posttap activity, a main effect of treatment was detected, F(2, 109) = 3.93, p < 0.05, whereby fish exposed to 30nM DDT had lower average post-tap activity than those exposed to 10nM DDT (p < 0.05). Neither DDT group was significantly different from controls in post hoc testing. On the shoaling assay, a main effect of time was noted for locomotor activity, F(2.70, 288.62) = 8.69, p <0.05. No treatment effects or interactions were observed.

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On the predator avoidance assay, a main effect on locomotor activity was observed for time,

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F(5.42, 537.08) = 11.97, p < 0.05, as was a time by treatment interaction, F(10.85, 537.08) =

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1.89, p < 0.05. Post hoc comparisons showed that fish exposed to 30nM DDT were less active

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during the first presentation of the fast predator stimulus (p< 0.05) (Fig. 2c). A similar trend

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between 30nM and 10nM approached significance (p = 0.08). For distance from the video screen, main effects were observed for the predator stimulus (present/absent), F(1, 25) = 22.39, p

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< 0.05, and cue speed (slow/fast), F2, 98) = 26.32, p < 0.05, as well as trending interactions of stimulus by treatment, F(1, 98) = 2.60, p = 0.08, and cue-speed by treatment, F(1, 98) = 2.84, p=

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0.06. Post hoc comparisons (Fig. 2d) showed that fish treated with 30nM DDT stayed further

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from the screen than controls when the fast-cue was present (p < 0.05). No other treatment effects or interactions were observed.

3.1.3 Set 2: 5-week DDT exposure, 14 months of age When tested again at 14 months of age, fish with prior 5-week DDT exposure no longer showed behavioral effects relative to controls on the novel tank diving test or the predator avoidance assay. However, two novel treatment effects were apparent at this time point (Fig. 3), in the tap test (Fig. 3a) and shoaling assays respectively (Fig. 3b). 17

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In the novel tank dive test, main effects on locomotor activity were observed for time, F(2.68, 262.24) = 6.50, and treatment, F(2, 98) = 3.11, p < 0.05. The 30nM DDT group was significantly less active than the 10nM group (p < 0.05), although neither group significantly differed from controls (p > 0.05). There was also a main effect of time on distance from the bottom, F(2.56, 169.09) = 7.58, p < 0.05. On the tap test, a significant main effect of tap was observed on pre-tap activity, F(7.13,

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699.29) = 2.12, p < 0.05, post-tap activity, F(7.00, 686.44) = 3.34, p < 0.05, and the magnitude

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of startle, F(7.18, 703.12) = 4.89, p < 0.05. Additionally, there were tap by treatment interactions

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for post-tap activity, F(14.01, 686.44) = 2.69, p < 0.05, and magnitude of startle, F(14.35,

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703.12) = 1.84, p< 0.05. Post hoc comparisons showed a significantly higher post-tap activity for fish in to 30nM DDT group relative to controls and 10nM DDT following tap 1 (p < 0.05), as

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well as elevated magnitudes of startle relative to controls and 10nM DDT on tap 1 (p < 0.05)

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(Fig. 3a). Additionally, a trend for increased startle response on tap 2 relative to controls approached significance (p = 0.056).

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In the shoaling assay, main effects on locomotor activity were observed for time, F(3.06,

Jo

315.17) = 7.68, p < 0.05, and treatment, F(2, 104) = 6.06, p < 0.05. Post hoc comparisons showed that fish in the 10nM DDT group were hypoactive relative to controls (p < 0.05) (Fig. 3b). A similar trend in the 30nM DDT group failed to reach significance (p = 0.14). With respect to distance from the video screen, significant main effects were observed for the shoaling video (present vs absent), F(1, 103) = 27.41, p < 0.05, and treatment, F(2, 103) = 3.67, p < 0.05), whereby fish in the 10nM DDT group generally remained closer to the screen than controls regardless of the presence of the video. A similar trend between the 30nM and 10nM groups

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approached significance (p = 0.06). No effect was seen on the magnitude of the shoaling response. On the predator avoidance assay, there were main effects of time, F(3.94, 397.89) = 4.99, p < 0.05, on locomotor activity, as well as distance from the screen, (3.71, 374.27) = 3.35, p < 0.05. There was also a significant main effect of the predator stimulus (present/absent), F(1, 101) =

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11.00, p < 0.05, and a significant stimulus x cue speed interaction, F(1, 101) = 7.63, p < 0.05. In the novel place recognition task, no significant effects of treatment or interactions were

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detected. Exploration of the novel arm, measured as cumulative time spent in that arm, showed a

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main effect of time, F(1, 83) = 8.35, p < 0.05, whereby exploration increased from the first five-

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minutes to the second five-minutes. Among controls, this led to a shift from a general avoidance

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the novel arm (15.6% +/- 2.9, chance at 25%) to chance levels of exploration for the novel arm (27.0% +/- 3.1, chance at 25%). Prior DDT treatment did not significantly alter this pattern.

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Neurochemical assays of tissue from fish with 5-week DDT exposure yielded no significant

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effects of treatment on either ChAT activity or HC3 binding (Fig. 4).

3.2 CPF Effects

3.2.1 Set 3: 2-Week Exposure to CPF, 1-week post-exposure Two-week CPF exposure in adulthood led to multiple behavioral effects at 1-week postexposure (Fig. 5). This included reduced diving in the novel tank dive test (Fig. 5a), and enhanced shoaling responses (Fig. 5b). Tap startle and predator avoidance appeared to be unaffected by this exposure.

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In the novel tank diving test, there was a main effect of time on locomotor activity, F(2.53, 346.11) = 17.33, p < 0.05, and main effects of time, F(2.62, 359.15) = 8.52, p < 0.05, and treatment, F(3, 137) = 3.10, p < 0.05, on distance from the bottom. Post hoc tests revealed that fish exposed to 3.0uM CPF were a greater distance from the floor than controls (p< 0.05), while a similar trend for the 1.0uM group approached significance (p= 0.08). No other treatment effects or interactions were observed.

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In the tap test, main effects of tap were observed for pre-tap activity, F(7.31, 1037.65) =

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2.99, p < 0.05, post-tap activity, F(7.56, 1073.56) = 2.00, p < 0.05, and the magnitude of the

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startle response, F(7.43, 1055.78) = 2.06, p < 0.05. A main effect of treatment was also observed

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significant differences between groups.

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for post-tap activity F(3, 142) = 4.35, p < 0.05, however, post hoc comparisons revealed no

In the shoaling assay, a main effect on locomotor activity was observed for time, F(2.61,

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359.59) = 9.89, p < 0.05, as was a main effect on distance from the video screen for the shoaling

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video (present/absent), F(1, 137) = 7.00, p < 0.05, and a significant video (present/absent) by treatment interaction, F(3, 137) = 4.83, p < 0.05. Post hoc comparisons showed a trend towards

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increased distance from the screen prior to the video presentation for fish exposed to 3.0uM CPF (p < 0.06) relative to controls. A main effect of treatment was also observed on the magnitude of the shoaling response, F(1, 137) = 4.83, p < 0.05. Post hoc comparisons showed that the 3.0uM CPF group had a significantly higher approach score than controls (p < 0.05) and a trend compared to the low dose group which approached significance (p = 0.074) (Fig. 5b). The moderate dose group of 1.0uM CPF showed a similar trend relative to controls (p = 0.055) without a confounding pre-video position effect.

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On the predator avoidance assay, a main effect on distance from the screen was observed for the predator stimulus (present/absent), F(1, 133) = 29.20, p< 0.05, as well as a stimulus by speed by treatment interaction, F(1, 133) = 3.11, p < 0.05. Post hoc comparisons showed that fish exposed to the higher dose of 3uM CPF remained farther from the video screen than those exposed to the moderate dose of 1.0uM CPF (p < 0.05) after the removal of the fast

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stimulus. No other treatment effects or interactions were observed.

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3.2.2 Set 3: 2-Week Exposure to CPF, 14 months of age

Although the 2-week CPF exposure led to multiple distinct behavioral effects at 1-week

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post-exposure, similar effects were not observed at 14 months of age. Analyses of brain

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was impacted at this time point.

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tissue (Fig. 7) showed that although no behavioral effects were evident, cholinergic function

In the novel tank dive test, a main effect of time was observed for locomotor activity,

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F(3.01, 358.57) = 8.15, p < 0.05, and distance from the bottom, F(2.56, 304.96) =3.34, p <

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0.05. No treatment effects or interactions were observed. In the tap test, no main effects or interactions were detected. In the shoaling assay, there was a main effect of time for activity (3.06, 371.84) = 3.92, and treatment, F(3, 122) = 4.83, p < 0.05, and a trending time x treatment interaction, F(9.14, 371.84) = 1.87, p = 0.053. However, no post hoc pairwise comparisons reached significance. For distance from the video screen, there was a significant main effect of the shoaling video (present/absent), F(1, 122) = 16.98, p < 0.05, and a trending video x treatment interaction, F(3, 122) = 2.17, p = 0.096, although no pairwise comparisons reached significance in post hoc testing (p > 0.05). On the predator avoidance assay, there were significant main effects of time, F(4.78, 573.54) = 3.90, p < 0.05, on locomotor activity, 21

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as well as a main effect of time on distance from the video screen, F(4.59, 550.94) = 4.63, p< 0.05, but no treatment effects or interactions. There were also main effects of predator stimulus (present/absent), F(1, 120) = 23.98, p < 0.05, and an interaction of predator stimulus (present/absent) and cue speed (slow/fast), F(1, 120) = 5.90, p < 0.05. No treatment effects or interactions were observed. For the magnitude of the avoidance response, a significant effect of cue speed was detected, F(1, 120) = 5.90, p< 0.05, but no treatment effects or interactions

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were observed.

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In the novel place recognition task, no significant effects of treatment or interactions were

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detected. Analyses of locomotor activity also showed a main effect of time, F(1, 63) = 6.47, p

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< 0.05, whereby exploration increased from the first five-minutes to the second five-minutes.

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Exploration of the novel arm, measured as cumulative time spent in that arm, also showed a main effect of time, F(1, 63) = 7.40, p < 0.05, whereby exploration increased from the first

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five-minutes to the second five-minutes. Among controls, this led to a shift from approximately chance level preference for the novel arm (27.0% +/- 7.02, chance at 25%) to

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a distinct preference for the novel arm (41.7% +/- 8.0, chance at 25%).

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CPF had no discernible effect on ChAT activity but there was a collective, significant (p < 0.05) decline in HC3 binding, comparing all three CPF dosing groups to the control (Fig. 7).

3.2.3 Set 4: 5-Week CPF Exposure, 1-week post-exposure Five weeks of CPF exposure in adulthood did not produce significant behavioral effects relative to controls at 1-week post-exposure, although a dose-interaction was apparent on

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predator avoidance, whereby fish with higher dose exposures showed reduced avoidance of the screen during red trials relative to fish exposed to lower doses (Fig. 6a). In the novel tank dive test, a main effect of time was observed for locomotor activity, F(2.45, 220.83) = 6.37, p < 0.05, and distance from the bottom, F(2.62, 236.49) =3.26, p < 0.05. No treatment effects or interactions were observed. In the tap test, a main effect of time, F(6.95, 645.95) = 2.21, p < 0.05, was detected for pre-tap activity. No other effects or

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interactions were detected. In the shoaling assay, there was a main effect of time for activity

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(3.25, 286.24) = 8.12. For distance from the video screen, there was a significant main effect

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of the shoaling video (present/absent), F(1, 88) = 5.51, p < 0.05). On the predator avoidance

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assay, no significant treatment effects were detected for locomotor activity. For distance from

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the screen (Fig. 6a), there were main effects of stimulus (present/absent), F(1, 88) = 32.67, p< 0.05, cue-speed (slow/fast), F(1, 88) = 8.02, p< 0.05, and treatment, F(3, 88) = 3.91, p<

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0.05, as well as interactions of cue-speed by stimulus, F(1, 88) = 5.04, p< 0.05, and cuespeed (slow/fast) by treatment, F(1, 88) = 3.33, p< 0.05. Fish treated with the higher dose of

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3.0uM CPF remained closer to the screen during the “fast” stimulus phase of testing (whether

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the stimulus was present or absent) (p < 0.05) than fish exposed to either 0.3uM or 1.0uM CPF. These fish did not significantly differ from controls. No treatment effects or interactions were observed on the magnitude of predator avoidance.

3.2.4 Set 4: 5-Week CPF Exposure, 14 months of age Five weeks of CPF exposure led to low-dose CPF treatment effects at fourteen months of age, specifically on locomotor activity (Fig. 6c) and avoidance behaviors (Fig. 6b/d) in the predator avoidance assay. 23

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In the novel tank dive test (not shown), a significant time by treatment interaction was detected for locomotor activity, F(8.43, 174.32) = 1.97, p < 0.05). Post hoc comparisons during each minute of the session showed that fish exposed to 3.0uM CPF were hyperactive relative to those exposed to 0.3uM CPF during minute 3, and relative to fish exposed to either 0.3 or 1.0uM CPF in minute 5. No differences were detected relative to controls. In the tap test, main effects of tap were observed for pre-tap, F(7.40, 554.75) = 2.55, p <

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0.05, and startle magnitude , F(7.31, 548.27) = 2.81. No other effects or interactions were

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observed. On the shoaling assay, a main effect of time was noted for locomotor activity,

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F(3.66, 304.14) = 2.89, p < 0.05. No other main effects or interactions were observed.

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On the predator avoidance assay, a main effect on locomotor activity was observed for

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time, F(5.37, 365.23) = 4.89, p < 0.05, and treatment, F(1, 68) = 4.90, p < 0.05, as was a time by treatment interaction F(16.11, 365.23) = 2.07, p < 0.05. Post hoc comparisons showed that

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the low dose of 0.3uM CPF led to significant hyperactivity relative to controls (p < 0.05) in

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minutes 1 (baseline), 4 (slow 2) and 6 (fast 1). In minutes 1 and 6, these fish were hyperactive relative to all groups (p < 0.05). Similarly, a univariate analysis of total

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locomotor activity (Fig. 6c) showed a main effect of treatment, F(3, 74) = 4.90, p < 0.05, whereby fish exposed to 0.3uM CPF were hyperactive relative to controls and those exposed to the higher dose (3.0uM CPF) (p < 0.05), with a similar trend approaching significance relative to the moderate (1.0uM CPF) dose group (p = 0.06). For distance from the screen (Fig. 6b), a main effect of stimulus (present/absent) was observed, F(1, 68) = 32.39, p < 0.05, as well as an interaction of stimulus (present/absent) and predator speed (slow/fast), F(1, 68) = 18.40, p < 0.05, an interaction of predator speed (slow/fast) and treatment, F(3, 68) = 3.44, p < 0.05, and an interaction of treatment, stimulus and predator speed, F(1, 68) = 3.86, p < 24

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0.05. Post hoc comparisons showed that the low dose of 0.3uM CPF led to reduced distance from the screen in the blank period following the fast stimulus relative to all other groups (p < 0.05). With respect to the magnitude of predator avoidance (Fig. 6d), there was a significant main effect of stimulus speed, F(1, 68) = 18.40, p < 0.05, and a significant stimulus speed by treatment interaction, F(1, 68) = 3.86, p < 0.05, whereby fish exposed to

stimulus than all other treatment groups (p < 0.05).

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3.3 Age Effects on Behavior

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the low dose of 0.3uM CPF showed a greater recovery of exploration following the slow

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To aid in the interpretation of long-term effects on behavior, a secondary analysis was

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performed to identify age-related effects on behavior in among controls. For this, data from

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control fish tested at both time points (sets 2, 3 and 4) were pooled into a single group and behavior was assessed as a function of age (1-week post-test vs 14 months). As the identity

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of the fish could not be maintained between the two datasets, age was tested as a between-

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groups variable, rather than a repeated-measures variable. Covariates included sex and set.

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Findings are graphically displayed in Figure 8. In the novel tank diving test, main effects on locomotor activity were observed for time, F(3.00, 528.57) = 28.81, p < 0.05, and age, F(1, 176) = 14.41, p < 0.05, but no age effects were observed on distance from the bottom of the tank. Post hoc comparisons showed that older fish were generally more active than younger adult fish in the novel tank (Fig 8a). For the tap test (Fig 8b), main effects of tap were observed for pre-tap activity, F(7.59, 1427.38) = 4.60, p < 0.05, and startle magnitude, F(7.50, 1157.54) = 2.01, p < 0.05. No significant treatment effects or interactions were observed. In the shoaling assay (Fig 8c), a main effect of time was detected for locomotor activity, F(2.80, 461.88) = 2.78, as was a 25

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main effect of the video (present/absent) on distance from the screen, F(1, 165) = 10.48, p < 0.05. No effects of treatment of interactions were observed. On the predator avoidance assay, a significant main effect on locomotor activity was observed for time, F(5.12, 926.21) = 5.78, p < 0.05, as was a significant age by time interaction F(5.12, 926.21) = 3.78, p < 0.05. Post hoc comparisons showed that older fish were significantly hyperactive relative to younger fish during minutes 7 and 9, corresponding

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to the minutes after the removal of a fast-cue, while no other time points showed significant

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age differences. With respect to distance from the screen, there were main effects of cue-

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speed, F(1, 181) = 4.61, p < 0.05, stimulus (present/absent), F(1, 181) = 20.74, p < 0.05, and

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age F(1, 181) = 29.51, p < 0.05, as well as significant interactions of stimulus by age, F(1,

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181) = 12.82, p < 0.05 and cue-speed by age, F(1, 181) = 5.18, p < 0.05. Post hoc comparisons showed that older fish remained closer to the predator-paired screen in all

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phases of testing (slow-cue, post-slow-cue, fast-cue, post-fast-cue) (Fig. 8d). Additionally, the avoidance magnitude score (or change in position after removal of the predator stimulus)

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was analyzed across the two cue-speeds. A main effect of age was observed F(1, 181) =

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12.82, p < 0.05, whereby older fish showed a smaller change in position than younger adults, regardless of cue-speed (Fig 8d). 4. Discussion Adult exposures to the insecticides DDT and CPF led to behavioral effects in the zebrafish beyond the duration of exposure, even several months later in some cases. These compounds represent two historically significant classes, organochlorine and organophosphate insecticides, which exert their insecticidal properties by differing mechanisms of action (Fukuto, 1990; Kleanthi, 2008). Among adults, CPF was tolerated at a 100x higher concentration than 26

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DDT without increased incidence of lethality. Within the selected dose ranges, differences in the duration of exposure proved to be important factors in the persistence of behavioral toxicity. A brief exposure to DDT (two weeks) produced minor behavioral effects, while an extended exposure (5 weeks) led to multiple dose-dependent effects at a 1-week follow up and persisted even to later in adulthood. In a somewhat different case, a brief, two-week exposure to CPF led to multiple behavioral effects which appeared to be attenuated in a longer exposure. This was not

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to say that the shorter exposure was more impactful, however, as the short exposure effects did

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not persist into later adulthood, while the longer exposure led to novel effects on predator

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avoidance at each time point. At the neurochemical level, these compounds were also somewhat

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distinct. CPF appeared to produce long term deficits in presynaptic cholinergic function, while DDT did not. In the case of CPF, the fact that reductions were restricted to HC3 binding but not

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ChAT activity, indicates that CPF does not simply ablate cholinergic innervation (which would

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have reduced both indices), but rather that it reduces presynaptic impulse activity. Taken together, the present data suggest that organochlorine and organophosphate compounds may

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each exert effects on behavior and/or brain neurochemistry in adulthood, but that those effects

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are quite distinct, as would be expected from their different cellular targets and mechanisms of action (Abreu-Villaca, 2017).

Across the current study, multiple pesticide exposure effects were detected in baseline levels of locomotor activity. After a brief (2 week) exposure to DDT, fish showed a reduction in pre-tap activity in the tap startle test, which suggests that baseline activity, rather than stimulusinduced activity, was altered by DDT exposure. The longer exposure of DDT produced a degree of locomotor hypoactivity at both 1-week and 14 months of age follow-ups, although the task and dosimetry of the effect was somewhat different at each time point. In general, these data

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suggest that DDT exposures may contribute to lasting reductions in locomotor activity in the zebrafish. Adult exposures to CPF, by contrast, were not found to produce hypoactivity in either short or longer exposure models. CPF failed to significantly impact activity levels in the short term, although the longer exposure model did show some potential for low doses of CPF to produce hyperactivity at a later time point. In addition to locomotor activity, DDT exposures were shown to selectively impact

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reflexive startle responses in zebrafish. Tap startle responses were not significantly altered by

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DDT exposures at the 1-week follow-up, nor by any CPF exposures, although the 5-week

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exposure led to a dose-dependent increase in initial sensitivity to the tap later in development.

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Specifically, the highest dose (30nM DDT) group showed elevated post-tap activity and startle

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magnitude scores which habituated to control levels across the first few repetitions of the tap stimulus. This suggests that certain organochlorine effects may not be evident immediately after

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exposure, but may emerge later as adult maturation continues.

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DDT and CPF also caused different effects on emotional responses, which were measured in this study as social reward (approach of the shoaling stimulus), anxiety (diving in

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the novel tank), and fear (fleeing from predator stimulus). A 5-week DDT exposure at 30nM appeared to sensitize fish to the predator stimulus, leading to both a stronger spatial avoidance of the fast cue in general and a brief interruption in locomotor activity at its first presentation. This latter effect may reflect a form of freezing behavior, again indicating a stronger fear response. By contrast, a 5-week CPF exposure tended to lead to less avoidance of the screen. This was somewhat weak at a 1-week time point, only reaching significance for the high dose (3.0uM CPF) relative to lower doses of CPF, but not controls. By the 14 month age, the pattern was altered to favor the lowest dose of CPF (0.3uM CPF), and manifest as a greater return to the 28

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screen after the removal of the slow cue, and a greater tendency to approach the screen after the removal of the fast cue. The poor persistence of avoidance behaviors in this test may be related to the hyperactivity these fish also displayed, as increased movement could lead to greater exploration and inconsistent preference for the far side of the tank. Beyond fear-like responses, it was noted that a 2 week CPF exposure dose-dependently enhanced social approach in the shoaling assay and reduced anxiety-like diving responses in the

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novel tank diving test at 1-week follow-up, further suggesting that CPF exposures could disrupt

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inhibitory functions. For the highest dose group, the shoaling effect may be partly confounded

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with a trend in baseline exploration of the tank, although this appears to be unrelated to

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locomotor activity. Additionally, it should be noted that the magnitude of shoaling for controls in

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set 2 is less than half of the pooled average for this measure (see fig 8c and 5b), so it is not clear whether the size of the shoaling effect observed for set 2 is representative or may appear inflated

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due to the low control levels. It should also be noted that while DDT altered diving in the novel tank, this was only observed at doses that also reduced locomotor activity. Taken together, these

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data suggest that adult exposures to differing classes of insecticides may have very different

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consequences on affective behaviors and further that those effects may be heavily governed by the dose and duration of exposure. The present findings complement a range of similar studies investigating the neurotoxicity of pesticides. To date, animal models have shown that a diverse array of pesticides can pose a threat to the vertebrate nervous system (Abreu-Villaca, 2017), principally using models of vertebrate development like vertebrate cell cultures (Jameson, 2005), rodents (Aldridge, 2005; Lee, 2015), and fish (Hagstrom, 2018; Sledge, 2011). This includes a number of zebrafish studies documenting neurotoxicity for organochlorines, including DDT (Tiedeken, 29

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2008) and endosulfan (Silva, 2015; Stanley, 2009), and multiple organophosphates, including chlorpyrifos, diazinon and parathion (Eddins, 2010; Yen, 2011). Work in our lab and others have extended this to show that early life exposures to contaminants like organophosphates can produce long-term behavioral effects, some of which can be expressed much later in life and in diverse elements of behavior (Eddins, 2010; Glazer, 2018; Oliveri, 2015). The present study complements this prior work by showing that although

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the brain may be particularly sensitive to neurotoxic compounds during early development, a

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degree of neurotoxic risk may exist at any stage of life. This work also expands upon previous

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work by emphasizing the adaptability of the brain and behavior and the sometimes unexpected

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effects that adaptability may have on neurotoxicity in adulthood. Plasticity was observed in both

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a protective way, as with effects that appeared to fade with time, and in an adverse way, as noted by behaviors which emerged later as time and development carried forward. This study frames

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the prior developmental work on a longer timeline of development and emphasizes not only the hazards that insecticides may pose to a fetus or young child, but also adults who may come into

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contact with pesticides through occupational or environmental means.

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The present study suggests the potential for heterogeneity among adults with pesticide exposure and the need for comprehensive risk assessments in adulthood. Population and clinical health studies have previously noted neuropsychological and neurological risks associated with exposures to broad classes of chemicals, such as organochlorines or organophosphates (Alavanja, 2004; Hayden, 2010; Kamel, 2007; Muñoz-Quezada, 2016; Rohlman, 2011). Some of these studies have been able to obtain a positive identification of the pesticides in question, either by specific chemical or general chemical class. Unfortunately, the specific metrics of an exposure are often uncertain or difficult to gather from these epidemiological samples, such as 30

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specific compounds, potential for co-exposures, the doses of the exposure, and various temporal features. This is particularly true for studies with late career or retired farm workers (Hayden, 2010; Jokanović, 2018; Mostafalou, 2018), who may have decades of prior exposures with one or more insecticides that cannot be currently sampled beyond self-report. Given that the pesticide class, dose, and duration of exposure may meaningfully modulate the consequences of an exposure, the characterization of exposures may represent an important area for methods

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development in this field, and one that may allow clinical staff to better acknowledge and

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integrate exposure data into risk management and treatment for adults at risk for exposure.

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Additionally, the potential for interactions between pesticides of similar or differing mechanisms

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of action requires additional study and assessment. Future work with adult animal models, such as zebrafish, may allow some of the fundamental features of adult exposures to be explored more

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thoroughly, such as dose, timing, frequency of single pesticide exposures, as well as overlapping

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or sequential exposures to differing pesticides.

An additional concern may be the non-monotonicity of organophosphate effects, patterns

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which are often observed in environmental contaminants (Lagarde, 2015), and have been

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previously reported for zebrafish with developmental organophosphate exposures (Glazer, 2018; Oliveri, 2015). Although chronic CPF exposure led to diverse short-term behavioral effects and a persistent change in cholinergic function, multiple aspects of this study failed to show a consistent increase in effect with an increase in dose or length of exposure. Furthermore, it was observed that extending the exposure from two weeks to five weeks attenuated the initial behavioral effects of CPF, rather than replicating or making them more severe. This likely suggests another compensatory mechanism, like tolerance, which allows an organism to adapt to a chronic pharmacological stimulus and counteract its effects (Bushnell, 1994). Tolerance

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requires time to develop, and therefore may not reliably occur in short duration exposures. Future studies should more systematically explore the exposure parameters of adult organophosphate exposures using similar models and explore how compensatory processes may shape their shortand long-term consequences. Therefore, the present data suggest that organophosphates may have a complex neurobehavioral risk profile in adulthood, and that efforts to reduce the dose or length of exposure may have complex implications for outcomes like behavioral health.

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Another element of this project was to investigate the effects of adult pesticide exposure

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on aging in adulthood. To this end, the present study included a general assessment of age related

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changes in behavioral function. In general, these did not appear to reflect a decline in behavioral

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health, per se, but rather a shift in certain locomotor and exploratory behaviors. Older fish had a

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tendency to be more active in the novel tank diving test and less averse to the screen in the predator, including a general greater approach to the screen and a less pronounced avoidance

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response. In the present study, testing at older ages also coincided with a second exposure to each test, so there was some potential for carry-over effects, although it is not clear how

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substantial these effects may be after several months. The increase in activity due to age is

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consistent with the findings of pilot work done using single testing of individual groups at different ages (data not shown), and so seems to be due to age rather than repeated testing. Further analyses will be needed to more thoroughly show how repeated testing and/or age affect behavior in the MUPET testing apparatus. In any case, adult pesticide exposures did not exacerbate these age-related effects, suggesting that long-term effects on behavior may be interpreted as persistent or novel symptoms, rather than alterations of behavioral development. Further analyses will also be needed to determine the kinetic and temporal characteristics of the present exposure model. Exposure solutions were changed once per week, and the fish 32

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remained in that water until the next water change. It is not known how the concentrations of pesticide in the water and fish may differ across this period. So, it is not clear whether the exposures are continuous across multiple weeks, as intended, or whether pesticide availability in the water may be depleted over time through absorption and/or accumulation in the fish, or by any external process. Kinetic and temporal evaluations may be important for clarifying the relevance of this model to continuous, seasonal, or pulsatile forms of pesticide exposure.

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In summary, the present study examined the short- and long-term effects of

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organochlorine and organophosphate exposures on the adult zebrafish. This study showed that

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these compounds can exert monotonic or non-monotonic dose effects on a variety of behavioral

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endpoints, including locomotor activity, reflexive startle responses, prosocial responses, and

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affective responses. These effects are notably modulated by the duration of exposure and by the length of time since the end of exposure. Certain behavioral effects may persist in one form or

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another, be attenuated over time, or emerge over the course of continued development. Additionally, it was found that DDT and CPF produced quite contrasting profiles of behavioral

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change, with DDT being more associated with aversive symptoms, such as hypoactivity or

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enhanced stress responses, and CPF more associated with disinhibitory symptoms, such as boldness, enhanced social approach, hyperactivity, and reductions in the persistence of avoidance. This emphasizes the potential for heterogenicity of symptoms among those with occupational or environmental exposures to varying insecticides and the need for strong documentation of exposures in risk assessment and epidemiology. In general, these data support the growing concern for the consequences of chronic exposure to insecticides in adulthood and the potential for long term neurobehavioral effects. Future studies will be needed to elaborate on

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the present findings and to investigate the mechanisms that underlie them, as well as develop

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effective preventative and therapeutic tools that may offset these risks.

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Acknowledgments This work was supported by NIEHS ViCTER project grant 3R01ES024288-03S1. Lilah Glazer was supported by the Leon Golberg Post-doctoral Fellowship. The authors would also like to acknowledge the much needed technical support provided by Bani Bajaj, Jacky Zhang, Aryan Rai, Talia Shoval, Michael Armstrong, Ashley Ko and Samantha Skavicus. TAS and FJS were

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responsible for the neurochemical studies.

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Conflict of Interest Statement

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TAS has received consultant income in the past three years from Pardieck Law (Seymour, IN), Gjording Fouser (Boise, ID), Thorsnes Bartolotta McGuire (San Diego, CA), Walgreen Co.

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(Deerfield, IL) and Cracken Law (Dallas, TX).

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neuropsychological functioning in farm workers: a review. International journal of occupational and environmental health, 22(1), 68-79. Oberemok, V. V., Laikova, K. V., Gninenko, Y. I., Zaitsev, A. S., Nyadar, P. M., & Adeyemi, T. A. (2015). A short history of insecticides. Journal of Plant Protection Research, 55(3), 221-226.

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Snedecor, G. W., & Cochran, W. G. (1967). Statistical Methods. Ames, Iowa: Iowa State University Press. Stanley, K. A., Curtis, L. R., Simonich, S. L. M., & Tanguay, R. L. (2009). Endosulfan I and endosulfan sulfate disrupts zebrafish embryonic development. Aquatic toxicology, 95(4), 355-361.

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Figure Legends Fig. 1. Set 1 Results. Two weeks of DDT exposure at 30nM suppressed the average locomotor activity in the 5 sec prior to the tap stimulus (mean ± sem) at 1-week post-exposure. Asterisk (*) indicates significant difference from controls at the p < 0.05 level. Fig. 2. Set 2 Results: 1-week post-exposure. Five weeks of DDT exposure led to multiple effects

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at 1-week post-exposure (mean +/- SEM). Treatment with 30nM DDT led to reduced locomotion

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in the latter half of the novel tank diving test at multiple individual time points (A), a reduced average distance from the bottom in the novel tank, collapsed across time points (B), as well as

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reduced activity during the first presentation of the fast stimulus (C) and increased distance from

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the fast predator stimulus (D). Asterisk (*) indicates significant difference from controls at the p

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< 0.05 level. Pound sign (#) indicates marginal significance at p < 0.10. Fig. 3. Set 2 Results: 14 months of age. Five weeks of DDT exposure led to multiple effects at

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14 months of age (mean +/- SEM). Prior treatment with 30nM DDT significantly increased

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startle magnitudes to the initial taps in the tap test (A). Prior exposure to 10nM DDT led to a

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reduced total locomotor activity in the shoaling assay (B). Asterisk (*) indicates significant difference from controls at the p < 0.05 level. Pound sign (#) indicates marginal significance at p < 0.10.

Fig. 4. Neurochemistry results: DDT. Neurochemical effects of DDT exposure (mean +/- SEM): (A) ChAT activity, (B) HC3 binding. There were no significant treatment effects on either parameter. Fig. 5. Set 3 Results: 1 week post-exposure. Two weeks of CPF exposure led to multiple effects at 1-week post-exposure (mean +/- SEM). Treatment with 3.0uM CPF led to increased average

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distance from the bottom in the novel tank, collapsed across time points (A) and the magnitude of the approach response in the shoaling assay (B). Asterisk (*) indicates a significant difference from controls at the p < 0.05 level. Pound sign (#) indicates marginal significance at p < 0.10. Fig. 6. Set 4 Results. Five weeks of moderate CPF exposure led to multiple effects at 14 months of age (mean +/- SEM). Treatment with 3.0uM CPF altered distance from the screen at 1-week post-exposure (A). By contrast at 14 months, 0.3uM CPF led to reduced distance to the screen

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following removal of the fast predator stimulus (B), increased locomotor activity overall in the

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predator avoidance assay (C) and increased approach of the screen after the slow stimulus was

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removed (D). Asterisk (*) indicates significant difference from controls at the p < 0.05 level.

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Fig. 7. Neurochemistry results: CPF. Neurochemical effects of CPF exposure (mean +/- SEM):

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(A) ChAT activity, (B) HC3 binding. ChAT was unaffected by CPF, but HC3 binding was significantly reduced.

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Fig 8. Age-related effects on behavior. Pooled analysis of controls indicated that older fish

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showed higher average levels of locomotion relative to younger fish in the novel tank dive test,

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collapsed across time points (A). No age effects were evident on tap test performance (B), or during the shoaling assay (C). In the predator avoidance assay, older fish remained closer to the video screen (left axis), both when the predator cues were present and absent, as well as showed a reduced avoidance score, measured as a change in position after the predator cue was removed (right axis) (D). Asterisk (*) indicates significant difference from controls at the p < 0.05 level.

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Table Legends Table 1. Summary of treatments and sample sizes. Dose ranges and the number of fish (N) included in behavioral testing is presented for each set, and at each relevant time point. A degree of attrition was noted between the adult (1-week post-exposure) and older (14 months of age) time points, although the degree of this attrition did not appear to be related to the dose of drug.

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Table 2. Summary of treatment effects. Treatment effects are shown on the primary behavioral

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and neurochemical outcomes across the four sets of fish tested. Hyphen (-) indicates no significant effect. Asterisk (*) and carat (^) indicate significant (p < 0.05) or marginally

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significant (p < 0.10) differences relative to controls, respectively. Tilde (~) indicates a

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indicate that the test was not performed.

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significant effect relative to a treatment condition other than control (p< 0.05). Grayed out areas

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Fig. 1. Set 1 Results.

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Fig. 2. Set 2 Results: 1-week post-exposure

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Fig. 3. Set 2 Results: 14 months of age

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Fig. 4. Neurochemistry results: DDT.

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Fig. 5. Set 3 Results: 1-week post-exposure

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Fig. 6. Set 4 Results.

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Fig 7. Neurochemistry results: CPF

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Fig 8. Age-related effects on behavior.

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Table 1: Summary of Treatments and Sample Sizes

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Table 2: Summary of Findings

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Highlights

Chronic adult DDT exposure in zebrafish caused hypoactivity and increased anxiety-like behavior. Chronic adult DDT exposure in zebrafish was not found to significantly altered cholinergic function. Chronic adult CPF exposure in zebrafish caused hyperactivity and reduced anxiety-like behavior.

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Chronic adult CPF exposure in zebrafish caused persistent decrements in cholinergic presynaptic activity.

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