DOI: 10.5061/dryad.2jm63xt0c
title: Developmental exposure to the Fox River PCB mixture modulates behavior in juvenile mice

description: Developmental exposures to PCBs are implicated in the etiology of
 neurodevelopmental disorders (NDDs). This observation is concerning given
 the continued presence of PCBs in the human environment and the increasing
 incidence of NDDs. Previous studies reported that developmental exposure
 to legacy commercial PCB mixtures (Aroclors) or single PCB congeners found
 in Aroclors caused NDD-relevant behavioral phenotypes in animal models.
 However, the PCB congener profile in contemporary human samples is
 dissimilar to that of the legacy Aroclors, raising the question of whether
 human-relevant PCB mixtures similarly interfere with normal brain
 development. To address this question, we assessed the developmental
 neurotoxicity of the Fox River Mixture (FRM), which was designed to mimic
 the congener profile identified in fish from the PCB-contaminated Fox
 River that constitute a primary protein source in the diet of surrounding
 communities. Adult female C57BL/6 J mouse dams (8-10 weeks old) were
 exposed to vehicle (peanut oil) or FRM at 0.1, 1.0, or 6.0 mg/kg/d in
 their diet throughout gestation and lactation, and neurodevelopmental
 outcomes were assessed in their pups. Ultrasonic vocalizations (USVs) and
 measures of general development were quantified at postnatal day (P) 7,
 while performance in the spontaneous alternation task and the 3-chambered
 social approach/social novelty task was assessed on P35. Triiodothyronine
 (T3) and thyroxine (T4) were quantified in serum collected from the dams
 when pups were weaned and from pups on P28 and P35. Developmental exposure
 to FRM did not alter pup weight or body temperature on P7, but USVs were
 significantly decreased in litters exposed to FRM at 0.1 or 6.0 mg/kg/d in
 the maternal diet. FRM also impaired male and female pups'
 performance in the social novelty task. Compared to sex-matched vehicles,
 significantly decreased social novelty was observed in male and female
 pups in the 0.1 and 6.0 mg/kg/d dose groups. FRM did not alter performance
 in the spontaneous alternation or social approach tasks. FRM increased
 serum T3 levels but decreased serum T4 levels in P28 male pups in the 1.0
 and 6.0 mg/kg/d dose groups. In P35 female pups and dams, serum T3 levels
 decreased in the 6.0 mg/kg/d dose group while T4 levels were not altered.
 Collectively, these findings suggest that FRM interferes with the
 development of social communication and social novelty, but not memory,
 supporting the hypothesis that contemporary PCB exposures pose a risk to
 the developing brain. FRM had sex, age, and dose-dependent effects on
 serum thyroid hormone levels that overlapped but did not perfectly align
 with the FRM effects on behavioral outcomes. These observations suggest
 that changes in thyroid hormone levels are not likely the major factor
 underlying the behavioral deficits observed in FRM-exposed animals.
description: <strong>Fox River PCB Mixture</strong>
 The Fox River PCB Mixture (FRM) is a synthetic PCB mixture
 containing 35% Aroclor 1242, 35% Aroclor 1248, 15% Aroclor 1254, and 15%
 Aroclor 1260 by weight (22). All Aroclors for this study, including
 Aroclor 1242 (catalog #: C-242N, lot #: 0114-A), Aroclor 1248 (catalog #:
 C-248N, lot #: 1063-24B), Aroclor 1254 (catalog #: C-254N, lot #:
 124-191-B) and Aroclor 1260 (catalog #: C-260N, lot #: 021-020-1A) were
 purchased from AccuStandard, Inc. (New Haven, CT, USA). The FRM was
 prepared by adding 525 mg of Arclor1242, 525 mg of Aroclor 1248, and 225
 mg of Aroclor 1254 to a vial containing 225 mg of Aroclor 1260. The
 mixture was kept at 30 °C for 5 min and homogenized by shaking. 
 The FRM was dissolved in peanut oil at a stock concentration of
 20 mg/ml and stored in amber glass vials at -20 °C. A 12.5 µL or 125 µL
 aliquot of 20 mg/ml peanut oil/FRM stock was mixed into 10 grams of peanut
 butter with a metal spatula for 5 min daily before use.  Organic peanut
 butter (Trader Joe’s, Monrovia, CA) and organic peanut oil (Spectrum
 Organic Products, LLC, Melville, NY) were purchased from Trader Joe’s
 (Seattle, WA).
 <strong>Animals</strong> All
 procedures involving animals were conducted in accordance with the NIH
 Guide for the Care and Use of Laboratory Animals and were approved by the
 University of Washington, Seattle Institutional Animal Care and Use
 Committee. C57Bl/6J WT mice were purchased from Jackson
 Labs (Sacramento, CA, United States). All animals were housed in clear
 plastic shoebox cages containing corn cob bedding and maintained on a 12 h
 light and dark cycle at 22 ± 2°C with 40–50% humidity. Feed (Diet 5010
 before mating, Diet 5021 during and following mating, LabDiet, Saint
 Louis, MO, United States) and water were available <em>ad
 libitum</em>. <strong>PCB Exposure
 Paradigm</strong> A schematic of the experimental
 paradigm is shown in Figure 2 of the publication. Adult female nulliparous
 C57BL/6 mice (8-10 weeks old) were randomly divided into one of four
 experimental groups using a random number generator (GraphPad Prism 8
 software- San Diego, CA, RRID:SCR_002798): (1) vehicle (peanut oil in
 peanut butter); (2) FRM at 0.1 mg/kg/d; (3) FRM at 1 mg/kg/d; or (4) FRM
 at 6.0 mg/kg/d. All animals were weighed daily before feeding and the
 amount of peanut butter was adjusted accordingly to account for weight
 changes throughout pregnancy and lactation. Two weeks prior to mating,
 dams were housed two per cage, and PCB dosing was initiated. Individual
 animals were placed in a clean cage containing a sterile weigh boat
 holding the FRM-peanut butter mixture. Animals were monitored to ensure
 the dose was fully consumed before they were placed back in the home cage.
 An age-matched male was placed in the cage for 7 d during mating. During
 this period, females were checked for the presence of a copulatory plug,
 which was considered gestational day 0. After mating, dams were housed
 singly and with their pups after parturition. At postnatal day 2 (P2),
 pups were culled or cross-fostered within dose groups to ensure all
 litters consisted of 6–8 pups. One male and one female pup from each
 litter were euthanized on P4 to collect brains for caspase-3 analyses
 (these data will be reported in a companion paper). After weaning at P28,
 pups were group-housed with same-sex littermates. One male and one female
 pup from each litter and their respective dams were euthanized on P28; on
 P35, one male and one female pup from each litter were euthanized to
 collect brain tissues for immunohistological analyses of neuroinflammation
 and neurogenesis (these data will be reported in a companion paper) and
 serum for thyroid hormone measurement. The pups euthanized on P35 were
 tested in several behavioral paradigms before euthanasia (Fig 2B of
 publication). <em>Metadata for the dose groups
 for each litter can be found in the file</em>:
 PCB-MB_Metadata <strong>Behavioral
 Tests</strong> Ultrasonic vocalizations were
 recorded using an Avisoft UltraSoundGate microphone and Avisoft Recorder
 USGH software (version 4.2, Avisoft Bioacoustics, Glienicke, Germany).
 Spectrograms were analyzed using Avisoft SASLab Pro software (version 5.2,
 Avisoft Bioacoustics) as previously described  (37, 38). Since ultrasonic
 vocalizations can vary within a litter (39), all pups in the litter (n=6-8
 pups per litter) were analyzed. Briefly, pups at P7 were removed from
 their home cage, and placed singly in a separate container with corn cob
 bedding within a sound-attenuating chamber equipped with an Avisoft
 UltraSoundGate microphone. Ultrasonic vocalizations were recorded for a
 total of 3 min. After each recording period, body mass and temperature
 were recorded, after which pups were returned to their home cage. The
 temperature of the room was maintained at 22 ± 2°C. An experienced
 individual blinded to exposure group manually quantified ultrasonic calls.
 The total number of calls per pup over the 3 min recording period was
 averaged across all pups within the litter. Each litter was assigned a
 single total number of ultrasonic vocalizations (e.g., the average USV
 value of each litter was the statistical unit of measure). One-way ANOVA
 was used to detect differences in calls with FRM dose as the variable (p
  0.05). Multiple comparisons were assessed using the Tukey
 <em>post-hoc</em> test (p  0.05). <em>Filename:</em>
 PCB-MB_USVS_Data_Masterfile <em>USV Test
 outputs: </em><em> </em>Each row is reflective of the
 behavioral output of one pup from a litter. The cohort, litter, dose
 group, pup number, and sex is given in the first 4 rows. For dose group,
 the vehicle group is represented by 0. Each pup was seperated and placed
 in the soundproof chamber individually. The number of ultrasonic
 vocalizations were measured for each file and are presented as minute 1,
 minute 2, and minute 3. The sum of these calls is then reported to get a
 representation of the total calls over the entire 3 minute
 trial.  Spatial memory was assessed at P35 using the
 spontaneous alternation task (y-maze) performed as previously described in
 the manuscript. The Y-shaped apparatus was made of non-reflective matte
 white finished acrylic ¼” thick (P95 White, Tap Plastics, Sacramento, CA,
 USA). The 3 arms (33.5 cm length x 6 cm width x 23 cm height) were
 separated by 120° between each other. Subjects were placed at the end of
 the designated start arm facing the center of the Y, and the sequence of
 entries into each arm during the 8 min test period was recorded by a
 camera mounted above the apparatus. An investigator blinded to exposure
 group scored transitions between the three arms. The percentage of
 spontaneous alternations was calculated as the number of triads (entries
 into each of the three different arms of the maze in a sequence of three
 without returning to a previously visited arm) relative to the number of
 entries. Two-way ANOVA was used to detect differences in alternation with
 dose and sex as the variables (p  0.05). Multiple comparisons were
 assessed using the Tukey <em>post-hoc</em> test (p
  0.05). <em>Filename</em>:
 PCB-MB_YMaze_Data_Masterfile <em>Y-Maze Test
 Outputs</em>: Each row is reflective of one pup from a litter. One
 male pup and one female pup per litter underwent this behavioral test. The
 respective cohort, litter, dose group, and sex is given in the first 4
 rows. For dose group, the vehicle group is represented by 0. Columns
 describe the behavioral outcomes including: the number of triads (see
 description above), the number of entries into each individual arm (start,
 left, and right), the sum of total entries into all arms, and the
 percentage of spontaneous alternation (%SAT)(also described
 above).  Sociability and social novelty were measured
 using the social approach and social novelty test at P35. Mice were given
 3 h rest between the spontaneous alternation task and the social
 approach/social novelty task to reduce the potential effects of stress on
 the latter task.  For this test, we used a three-chambered social box
 constructed from white plastic with removable doors as described
 previously (42, 43). Mice were moved into the behavioral testing room (lux
 level 110) in their home cage and allowed at least 30 min to acclimate
 prior to testing. The sociability assay consisted of one 5 min period and
 three 10-min periods for a total of 35 min. First, animals were placed in
 the center of the three-chambered arena for a 5 min acclimation period.
 Second, in the habituation phase, the doors were removed, and mice were
 allowed free exploration of the entire arena for 10 min. If an animal
 failed to explore during this time, they were removed from the study.
 Third, animals were allowed to interact for 10 min with either an empty,
 upside-down wire pencil cup (object) or an identical cup containing a
 novel age- and sex-matched “stranger mouse”. The two cups were placed on
 opposite sides of the arena and the middle chamber was left empty.
 Placement of the empty cup and the cup with the mouse was counterbalanced
 between the left and right sides of the apparatus to eliminate side bias.
 Finally, the empty cup was replaced with an identical cup containing a
 novel age- and sex- matched WT “stranger mouse” and the test subject was
 given 10 min to interact with the novel and familiar stranger mice. The
 placement of the novel and familiar mouse was counterbalanced between the
 left and right sides. Both the area and the cups were cleaned with 70%
 ethanol and allowed to dry completely between each subject to eliminate
 scent cues. Animals used as stranger mice were habituated to sitting under
 the wire cup for 30 min every day for 3 days prior to the testing phase to
 avoid erratic behavior during the testing phase of the assay. Behavior was
 recorded using Noldus EthoVision XT (version 11.0), an automated tracking
 and analysis software (Noldus Information Technology Inc., Leesburg, VA)
 and analyzed by an investigator blind to exposure group. Paired t-tests
 were used to detect within-group differences in sniff time, number of
 interactions, and time in chamber (p  0.05). One-way ANOVA was used to
 detect between-group differences in the time spent in the middle chamber
 (p  0.05). Two-way ANOVA was used to detect between-group differences in
 the number of entries with dose and sex as the variables (p  0.05).
 Multiple comparisons were assessed using the Tukey
 <em>post-hoc</em> test (p  0.05). <em>Filename</em>:
 PCB-MB_SApproach_Data_Masterfile <em>Social Approach Test
 Outputs</em>: Each row is reflective of one pup from a litter. One
 male pup and one female pup per litter underwent this behavioral test. The
 respective cohort, litter, dose group, and sex is given in the first 4
 rows. For dose group, the vehicle group is represented by 0. The following
 7 columns describe behavioral outcomes related to the chambers the mouse
 explores including the total entries into each individual chamber (left,
 middle, and right), the sum of entries into all three chambers, and the
 time spent in each individual chamber. The final 6 column refer to
 behavioral outcomes related to the direct interactions the experimental
 mouse had with the cup and the stranger mouse including, the number of
 interactions with each item, the sum of the interactions for both items,
 the time spent interacting with each item.  <em>Filename:</em>
 PCB-MB_SNovelty_Data_Masterfile <em>Social Novelty Test
 Outputs</em>: Each row is reflective of one pup from a litter. One
 male pup and one female pup per litter underwent this behavioral test. The
 respective cohort, litter, dose group, and sex is given in the first 4
 rows. For dose group, the vehicle group is represented by 0. The following
 7 columns describe behavioral outcomes related to the chambers the mouse
 explores including the total entries into each individual chamber (left,
 middle, and right), the sum of entries into all three chambers, and the
 time spent in each individual chamber. The final 6 column refer to
 behavioral outcomes related to the direct interactions the experimental
 mouse had with the "familiar" stranger mouse (same mouse from
 previous trial - "Fam. Mus") and the "novel" stranger
 mouse ("Novel Mus") including, the number of interactions with
 each mouse, the sum of the interactions for both mice, the time spent
 interacting with each mouse. <strong>Thyroid
 Hormone Assay</strong> Levels of triiodothyronine
 (T3) and thyroxine (T4) were measured in maternal serum collected from
 dams (which were 4 months old at the time of collection) at the conclusion
 of the FRM dosing period, and in serum from P28 and P35 pups. Blood was
 collected during the second half of the animals’ 12-hour light cycle.
 Animals were anesthetized with 4% isoflurane in medical grade oxygen and
 once determined to be properly anesthetized as determined by lack of
 response to toe pinch and tail pinch, blood was collected via cardiac
 stick using a 5 ml luer lock syringe (Becton Dickinson, San Jose, CA)
 fitted with a 30 gauge BD PrecisionGlide Needle (Becton Dickinson, San
 Jose, CA). Blood was transferred to a BD vacutainer blood collection tube
 (4 mL, Becton Dickinson, San Jose, CA) and 30 min after collection,
 centrifuged at 2000 rcf for 10 min at 4°C. The supernatant (serum), was
 collected and placed into 2 ml cryogenic vials (ThermoFisher) and frozen
 at -80°C. T3 (Invitrogen, Carlsbad, CA, USA; Cat# EIAT3C) and T4 levels
 (Invitrogen; Cat# EIAT4C) were determined using enzyme‐linked
 immunosorbent assays (ELISA) per the manufacturer’s instructions.
 Concentrations of T3 and T4 hormones in serum were measured at 450 nm
 absorbance on a spectrophotometer microplate reader (BioTek Instruments,
 Winooski, VT). Samples were run in duplicates, and values were normalized
 to blank controls within each plate. Two-way ANOVA was used to detect
 differences in T3 (pg/mL) or T4 levels (ng/mL) with dose and sex as the
 variables. Multiple comparisons were assessed using the
 Tukey <em>post-hoc test</em> (p 0.05). <em>Filename:</em>
 PCB-MB_Thyroid_Data_Masterfile <em>Thyroid Hormone
 Outputs</em>: Each row is reflective of one animal. One male pup and
 one female pup per litter were analyzed as well as the Dam. The respective
 timepoint (P28, P35, or 4mo for dams), cohort, litter, dose group, and sex
 is given in the first 4 rows. For dose group, the vehicle group is
 represented by 0. The mean concentration of T3 levels and T4 levels in the
 serum are presented for each animal. In some cases, one thyroid hormone or
 the other was not able to be measured due to lack of serum for analysis,
 and in these cases there is no value reported. 
description: # Developmental exposure to the Fox River PCB mixture modulates behavior
 in juvenile mice
 [https://doi.org/10.5061/dryad.2jm63xt0c](https://doi.org/10.5061/dryad.2jm63xt0c) ## Description of the data and file structure ``` Developmental exposures to PCBs are implicated in the etiology of neurodevelopmental disorders (NDDs). This observation is concerning given the continued presence of PCBs in the human environment and the increasing incidence of NDDs. Previous studies reported that developmental exposure to legacy commercial PCB mixtures (Aroclors) or single PCB congeners found in Aroclors caused NDD-relevant behavioral phenotypes in animal models. However, the PCB congener profile in contemporary human samples is dissimilar to that of the legacy Aroclors, raising the question of whether human-relevant PCB mixtures similarly interfere with normal brain development. To address this question, we assessed the developmental neurotoxicity of the Fox River Mixture (FRM), which was designed to mimic the congener profile identified in fish from the PCB-contaminated Fox River that constitute a primary protein source in the diet of surrounding communities. Adult female C57BL/6 J mouse dams (8-10 weeks old) were exposed to vehicle (peanut oil) or FRM at 0.1, 1.0, or 6.0 mg/kg/d in their diet throughout gestation and lactation, and neurodevelopmental outcomes were assessed in their pups. Ultrasonic vocalizations (USVs) and measures of general development were quantified at postnatal day (P) 7, while performance in the spontaneous alternation task and the 3-chambered social approach/social novelty task was assessed on P35. Triiodothyronine (T3) and thyroxine (T4) were quantified in serum collected from the dams when pups were weaned and from pups on P28 and P35. Developmental exposure to FRM did not alter pup weight or body temperature on P7, but USVs were significantly decreased in litters exposed to FRM at 0.1 or 6.0 mg/kg/d in the maternal diet. FRM also impaired male and female pups' performance in the social novelty task. Compared to sex-matched vehicles, significantly decreased social novelty was observed in male and female pups in the 0.1 and 6.0 mg/kg/d dose groups. FRM did not alter performance in the spontaneous alternation or social approach tasks. FRM increased serum T3 levels but decreased serum T4 levels in P28 male pups in the 1.0 and 6.0 mg/kg/d dose groups. In P35 female pups and dams, serum T3 levels decreased in the 6.0 mg/kg/d dose group while T4 levels were not altered. Collectively, these findings suggest that FRM interferes with the development of social communication and social novelty, but not memory, supporting the hypothesis that contemporary PCB exposures pose a risk to the developing brain. FRM had sex, age, and dose-dependent effects on serum thyroid hormone levels that overlapped but did not perfectly align with the FRM effects on behavioral outcomes. These observations suggest that changes in thyroid hormone levels are not likely the major factor underlying the behavioral deficits observed in FRM-exposed animals. ``` ### Files and variables #### File: PCB-MB\_Metadata.csv **Description:**  ##### Variables * Cohort: The group of dams/litters that were exposed at the same time.  * Litter: One dam and her pups are considered a litter and this acts as the statistical unit for behavioral tests.  * Dose: The concentration of Fox River PCB Mixture that dams were exposed to (mg/kg/d) via a peanut butter vehicle. #### File: PCB-MB\_Thyroid\_Data\_Masterfile.csv **Description:** Circulating thyroid hormones were measured in the serum collected from experimental animals at each timepoint. Blood was transferred to a BD vacutainer blood collection tube (4 mL, Becton Dickinson, San Jose, CA) and 30 min after collection, centrifuged at 2000 rcf for 10 min at 4°C. The supernatant (serum), was collected and placed into 2 ml cryogenic vials (ThermoFisher) and frozen at -80°C. T3 (Invitrogen, Carlsbad, CA, USA; Cat# EIAT3C) and T4 levels (Invitrogen; Cat# EIAT4C) were determined using enzyme‐linked immunosorbent assays (ELISA) per the manufacturer’s instructions. Concentrations of T3 and T4 hormones in serum were measured at 450 nm absorbance on a spectrophotometer microplate reader (BioTek Instruments, Winooski, VT). Samples were run in duplicates, and values were normalized to blank controls within each plate. ##### Variables * Timepoint: For dams, tissue was collected at 4 months of age and was reflective of postnatal day (P) 28 for pups, which was also the day of weaning. For pups, tissues were collected either at P28 or P35.  * Cohort: The group of dams/litters that were exposed at the same time * Litter:  One dam and her pups are considered a litter. These were all exposed to the same amount concentration of FRM.  * Animal: Describes whether the sample was collected from the dam, or a male or female pup from that litter.  * Dose: The concentration of Fox River PCB Mixture that dams were exposed to (mg/kg/d) via a peanut butter vehicle.  * T3 Conc Mean: The mean concentration of T3 levels in the serum are presented for each animal (pg/mL) . In some cases, T3 was not able to be measured due to lack of serum for analysis and in these cases there is no value reported and cells are left blank.  * T4 Conc Mean: The mean concentration of T4 levels in the serum are presented for each animal (ng/mL). In some cases, T4 was not able to be measured due to lack of serum for analysis and in these cases there is no value reported and the cells were left blank.  #### File: PCB-MB\_USVS\_Data\_Masterfile.csv **Description:** To assess early social communication, pups at P7 were removed from their home cage, and placed singly in a separate container with corn cob bedding within a sound-attenuating chamber equipped with an Avisoft UltraSoundGate microphone. Ultrasonic vocalizations were recorded for a total of 3 min. An experienced individual blinded to exposure group manually quantified ultrasonic calls. The total number of calls per pup over the 3 min recording period was averaged across all pups within the litter. Each litter was assigned a single total number of ultrasonic vocalizations (e.g., the average USV value of each litter was the statistical unit of measure). ##### Variables * Cohort: The group of dams/litters that were exposed at the same time * Litter: One dam and her pups are considered a litter and this acts as the statistical unit for behavioral tests.  * Dose (mg/kg/d): The concentration of Fox River PCB Mixture that dams were exposed to (mg/kg/d) via a peanut butter vehicle. * Pup: The number of the pup isolated from the dam and placed into the ultrasonic vocalization chamber.  * Sex: Male or female * Minute 1: Number of calls recorded during first minute of trial * Minute 2: Number of calls recorded during second minute of trial * Minute 3: Number of calls recorded during third minute of trial.  * Sum: Total number of calls recorded for the entire 3 minute trial.  #### File: PCB-MB\_SApproach\_Data\_Masterfile.csv **Description:** For this test, we used a three-chambered social box constructed from white plastic with removable doors as described previously (42, 43). Mice were moved into the behavioral testing room (lux level 110) in their home cage and allowed at least 30 min to acclimate prior to testing. The sociability assay consisted of one 5 min period and three 10-min periods for a total of 35 min. First, animals were placed in the center of the three-chambered arena for a 5 min acclimation period. Second, in the habituation phase, the doors were removed, and mice were allowed free exploration of the entire arena for 10 min. If an animal failed to explore during this time, they were removed from the study. Third, during the social approach trial, animals were allowed to interact for 10 min with either an empty, upside-down wire pencil cup (object) or an identical cup containing a novel age- and sex-matched “stranger mouse”. The two cups were placed on opposite sides of the arena and the middle chamber was left empty. Placement of the empty cup and the cup with the mouse was counterbalanced between the left and right sides of the apparatus to eliminate side bias. ##### Variables * Cohort: The group of dams/litters that were exposed at the same time * Litter: One dam and her pups are considered a litter and this acts as the statistical unit for behavioral tests.  * Dose: The concentration of Fox River PCB Mixture that dams were exposed to (mg/kg/d) via a peanut butter vehicle. * Sex: Male or female * Entries Left: Total entries made into the left chamber of the apparatus.  * Entries Middle: Total entries made into the middle chamber of the apparatus.  * Entries Right: Total entries made into the right chamber of the apparatus.  * Total Entries: Total entries made into all chambers throughout the 10 minute trial.  * Time Left: Total time spent in the left chamber of the apparatus.  * Time Middle: Total time spent in the middle chamber of the apparatus.  * Time Right: Total time spent in the right chamber of the apparatus.  * Interactions Cup: Total number of interactions with the empty cup.  * Interactions Str. Mus.: Total number of interactions made with the cup with the stranger mouse.  * Total Interaction: Total number of interactions with both the empty cup and the stranger mouse.  * Time Cup: Total time in seconds, spent interacting with the empty cup.  * Time Str. Mus: Total time in seconds, spent interacting with the stranger mouse.  #### File: PCB-MB\_SNovelty\_Data\_Masterfile.csv **Description:** Following the social approach task (see description above), the empty cup was replaced with an identical cup containing a novel age- and sex- matched WT “stranger mouse” and the test subject was given 10 min to interact with the novel and familiar stranger mice. The placement of the novel and familiar mouse was counterbalanced between the left and right sides to eliminate side bias. ##### Variables * Cohort: The group of dams/litters that were exposed at the same time * Litter: One dam and her pups are considered a litter and this acts as the statistical unit for behavioral tests.  * Dose: The concentration of Fox River PCB Mixture that dams were exposed to (mg/kg/d) via a peanut butter vehicle. * Sex: Male or female * Entries Left: Total entries made into the left chamber of the apparatus.  * Entries Middle: Total entries made into the middle chamber of the apparatus.  * Entries Right: Total time spent in the right chamber of the apparatus.  * Total Entries: Total entries made into all chambers throughout the 10 minute trial.  * Time Left: Total time spent in the left chamber of the apparatus.  * Time Middle: Total time spent in the middle chamber of the apparatus. * Time Right: Total time spent in the right chamber of the apparatus.  * Interations Fam. Mus: Total number of interactions with the familiar mouse.  * Interactions Novel Mus.: Total number of interactions with the novel mouse.  * Total Interactions: Total number of interactions with the familiar mouse and novel mouse.  * Time Fam. Mus.: Total time spent interacting with the familiar mouse.  * Time Novel Mus: Total time spent interacting with the novel mouse.  #### File: PCB-MB\_YMaze\_Data\_Masterfile.csv **Description:** Working spatial memory was assessed at P35 using the spontaneous alternation task (y-maze) performed as previously described in the manuscript. The Y-shaped apparatus has three arms that were separated by 120° between each other. Subjects were placed at the end of the designated start arm facing the center of the Y, and the sequence of entries into each arm during the 8 min test period was recorded by a camera mounted above the apparatus. An investigator blinded to exposure group scored transitions between the three arms. ##### Variables * Cohort: The group of dams/litters that were exposed at the same time * Litter: One dam and her pups are considered a litter and this acts as the statistical unit for behavioral tests.  * Dose (mg/kg/d): The concentration of Fox River PCB Mixture that dams were exposed to (mg/kg/d) via a peanut butter vehicle. * Sex: Male or female * Triads: Entries into each of the three different arms of the maze in a sequence of three without returning to a previously visited arm * Entries Start: Total entries made into the start arm making up the base of the Y.  * Entries Left: Total entries made into the left arm of the Y.  * Entries Right: Total entries made into the right arm of the Y.  * Total Entries: Total entries made into all arms during the task.  * % SAT: Number of triads relative to the total number of entries.  ## Code/software No code or software is necessary to view my data. All data is put into .csv files.  ## Access information Data was derived from the following sources: * [https://pmc.ncbi.nlm.nih.gov/articles/PMC11489981/](https://pmc.ncbi.nlm.nih.gov/articles/PMC11489981/)