Evaluation of Developmental Toxicity, Developmental Neurotoxicity, and Tissue Dose in Zebrafish Exposed to GenX and Other PFAS
2020; National Institute of Environmental Health Sciences; Volume: 128; Issue: 4 Linguagem: Inglês
10.1289/ehp5843
ISSN1552-9924
AutoresShaza Gaballah, Adam Swank, Jon R. Sobus, Xia Meng Howey, Judith E. Schmid, Tara Catron, James McCord, Erin P. Hines, Mark J. Strynar, Tamara Tal,
Tópico(s)Atmospheric chemistry and aerosols
ResumoVol. 128, No. 4 ResearchOpen AccessEvaluation of Developmental Toxicity, Developmental Neurotoxicity, and Tissue Dose in Zebrafish Exposed to GenX and Other PFAS Shaza Gaballah, Adam Swank, Jon R. Sobus, Xia Meng Howey, Judith Schmid, Tara Catron, James McCord, Erin Hines, Mark Strynar, and Tamara Tal Shaza Gaballah https://orcid.org/0000-0002-9452-6091 Oak Ridge Institute for Science and Education, Integrated Systems Toxicology Division (ISTD), National Health and Environmental Effects Research Laboratory (NHEERL), Office of Research and Development (ORD), U.S. Environmental Protection Agency (EPA), Research Triangle Park, North Carolina, USA Search for more papers by this author , Adam Swank Research Cores Unit, NHEERL, ORD, U.S. EPA, Research Triangle Park, North Carolina, USA Search for more papers by this author , Jon R. Sobus https://orcid.org/0000-0003-0740-6604 Exposure Methods and Measurement Division, National Exposure Research Laboratory, ORD, U.S. EPA, Research Triangle Park, North Carolina, USA Search for more papers by this author , Xia Meng Howey Oak Ridge Institute for Science and Education, Integrated Systems Toxicology Division (ISTD), National Health and Environmental Effects Research Laboratory (NHEERL), Office of Research and Development (ORD), U.S. Environmental Protection Agency (EPA), Research Triangle Park, North Carolina, USA Search for more papers by this author , Judith Schmid Toxicology Assessment Division, NHEERL, ORD, U.S. EPA, Research Triangle Park, North Carolina, USA Search for more papers by this author , Tara Catron https://orcid.org/0000-0003-1193-9366 Oak Ridge Institute for Science and Education, Integrated Systems Toxicology Division (ISTD), National Health and Environmental Effects Research Laboratory (NHEERL), Office of Research and Development (ORD), U.S. Environmental Protection Agency (EPA), Research Triangle Park, North Carolina, USA Search for more papers by this author , James McCord https://orcid.org/0000-0002-1780-4916 Exposure Methods and Measurement Division, National Exposure Research Laboratory, ORD, U.S. EPA, Research Triangle Park, North Carolina, USA Search for more papers by this author , Erin Hines https://orcid.org/0000-0002-2458-6267 National Center for Environmental Assessment, ORD, U.S. EPA, Research Triangle Park, North Carolina, USA Search for more papers by this author , Mark Strynar https://orcid.org/0000-0003-3472-7921 Exposure Methods and Measurement Division, National Exposure Research Laboratory, ORD, U.S. EPA, Research Triangle Park, North Carolina, USA Search for more papers by this author , and Tamara Tal Address correspondence to Tamara Tal, Department of Bioanalytical Ecotoxicology, Helmholtz Centre for Environmental Research–UFZ, Permoserstraße 15, 04318, Leipzig, Germany. Telephone: 49 341 235 1524. Email: E-mail Address: [email protected] https://orcid.org/0000-0001-8365-9385 ISTD, NHEERL, ORD, U.S. EPA, Research Triangle Park, North Carolina, USA Search for more papers by this author Published:9 April 2020CID: 047005https://doi.org/10.1289/EHP5843Cited by:2AboutSectionsPDF Supplemental Materials ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail AbstractBackground:Per- and polyfluoroalkyl substances (PFAS) are a diverse class of industrial chemicals with widespread environmental occurrence. Exposure to long-chain PFAS is associated with developmental toxicity, prompting their replacement with short-chain and fluoroether compounds. There is growing public concern over the safety of replacement PFAS.Objective:We aimed to group PFAS based on shared toxicity phenotypes.Methods:Zebrafish were developmentally exposed to 4,8-dioxa-3H-perfluorononanoate (ADONA), perfluoro-2-propoxypropanoic acid (GenX Free Acid), perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid (PFESA1), perfluorohexanesulfonic acid (PFHxS), perfluorohexanoic acid (PFHxA), perfluoro-n-octanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), or 0.4% dimethyl sulfoxide (DMSO) daily from 0–5 d post fertilization (dpf). At 6 dpf, developmental toxicity and developmental neurotoxicity assays were performed, and targeted analytical chemistry was used to measure media and tissue doses. To test whether aliphatic sulfonic acid PFAS cause the same toxicity phenotypes, perfluorobutanesulfonic acid (PFBS; 4-carbon), perfluoropentanesulfonic acid (PFPeS; 5-carbon), PFHxS (6-carbon), perfluoroheptanesulfonic acid (PFHpS; 7-carbon), and PFOS (8-carbon) were evaluated.Results:PFHxS or PFOS exposure caused failed swim bladder inflation, abnormal ventroflexion of the tail, and hyperactivity at nonteratogenic concentrations. Exposure to PFHxA resulted in a unique hyperactivity signature. ADONA, PFESA1, or PFOA exposure resulted in detectable levels of parent compound in larval tissue but yielded negative toxicity results. GenX was unstable in DMSO, but stable and negative for toxicity when diluted in deionized water. Exposure to PFPeS, PFHxS, PFHpS, or PFOS resulted in a shared toxicity phenotype characterized by body axis and swim bladder defects and hyperactivity.Conclusions:All emerging fluoroether PFAS tested were negative for evaluated outcomes. Two unique toxicity signatures were identified arising from structurally dissimilar PFAS. Among sulfonic acid aliphatic PFAS, chemical potencies were correlated with increasing carbon chain length for developmental neurotoxicity, but not developmental toxicity. This study identified relationships between chemical structures and in vivo phenotypes that may arise from shared mechanisms of PFAS toxicity. These data suggest that developmental neurotoxicity is an important end point to consider for this class of widely occurring environmental chemicals. https://doi.org/10.1289/EHP5843IntroductionPer- and polyfluoroalkyl substances (PFAS) are a structurally diverse class of industrial chemicals that contain aliphatic chains with all or some of the carbons bonded to fluorines (-CnF2n-) and carboxylic acid or sulfonic acid terminal moieties (OECD 2018). There are 4,370 unique PFAS structures (OECD 2018) with 602 compounds currently in commercial use in the United States (U.S. EPA 2019). PFAS have flame-retardant, water-resistant, and surfactant-like properties (Banks et al. 1994; Kissa 2001). This class of compounds is therefore widely used as protectants in paper and packaging products, water- and grease-repellent textiles, nonstick cookware coatings, and firefighting foams (Lindstrom et al. 2011). PFAS are extremely stable due to the carbon–fluorine bond strength (Banks et al. 1994; Kissa 2001). Based on their structurally inherent thermal and chemical stability, PFAS persist in the environment where they are generally resistant to biodegradation, photooxidation, direct photolysis, and hydrolysis (Schultz et al. 2003). As a result, they are widely detected in the environment (Dauchy et al. 2019; Pan et al. 2018), wildlife (Cui et al. 2018; Escoruela et al. 2018; Route et al. 2014), drinking water (Guelfo and Adamson 2018; Guelfo et al. 2018), and humans (Daly et al. 2018; Hurley et al. 2018; Jain 2018).Since the voluntary phaseout of perfluoro-n-octanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) in the early 2000s, time trends of National Health and Nutrition Examination Survey (NHANES) PFOS and PFOA serum levels are generally indicative of reduced human exposures (Jain 2018). Despite reductions, exposures are still widespread, with PFAS detectable in 95% of NHANES subjects (2013–2014) (CDC 2019) and in pregnant women, maternal serum levels for PFOS (35.3 ng/mL) and PFOA (5.6 ng/mL) have been reported (Fei et al. 2007). Of additional concern, an examination of these compounds in U.S. children 3–11 years of age, most of whom were born after PFOS and PFOA were phased out of use, revealed detectable levels of 14 PFAS, including PFOS and PFOA, in more than 60% of study subjects (Ye et al. 2018). A longitudinal study in Finnish children and adolescents showed that although serum levels of PFOS, PFOA, perfluorohexanesulfonic acid (PFHxS), and perfluorohexanoic acid (PFHxA) decreased over the study period, calculated body burdens generally remained constant and, in some cases, increased (Koponen et al. 2018). In humans, PFAS exposure has been associated with reduced birth weight (Apelberg et al. 2007; Fei et al. 2007), although weak associations with low birth weight or conflicting data have also been reported (Manzano-Salgado et al. 2017; Shoaff et al. 2018; Whitworth et al. 2012). In animal studies, early life stage exposure to PFOS or PFOA have been linked to developmental toxicity in chickens and mice (Jiang et al. 2012; Tucker et al. 2015), immunotoxicity in mice (reviewed by DeWitt et al. 2009), and developmental (Huang et al. 2010; Padilla et al. 2012; Truong et al. 2014) and reproductive toxicity in zebrafish (Jantzen et al. 2017).To address toxicity concerns, longer alkyl chain PFAS like PFOS and PFOA have been replaced with shorter alkyl chain compounds such as perfluorobutanesulfonic acid (PFBS) or large fluoroether PFAS such as perfluoro-2-propoxypropanoic acid (GenX) and 4,8-dioxa-3H-perfluorononanoate (ADONA). Alternative chemistries that retain the long-chain character, such as ADONA, were engineered with ether linkages and sites of hydrogenation in efforts to reduce biological half-lives (Fromme et al. 2017). Replacement PFAS are therefore increasingly detected in the environment, including in surface water (De Silva et al. 2011; McCord et al. 2018; Pan et al. 2018; Strynar et al. 2015; Wang et al. 2016) and drinking water (Kaboré et al. 2018; McCord et al. 2018). Environmental screening efforts have also identified relevant exposures to PFAS by-products, such as sulfonated fluorovinyl ethers (i.e., PFESA compounds), that are not strictly chemicals of commerce (McCord et al. 2018; Strynar et al. 2015). Growing concern over the safety of GenX and other replacement PFAS has unsurprisingly led to a greater demand for toxicity data (Blum et al. 2015; Borg et al. 2017; Scheringer et al. 2014). However, traditional mammalian toxicity assays can be costly and time consuming, and it is challenging to test multiple chemicals and concentrations of chemicals in parallel. Because PFAS exposures have been historically linked to complex toxicity outcomes involving whole organisms (e.g., developmental toxicity) or specific organ systems (e.g., immunotoxicity), the use of a rapid in vivo animal screening system is justified.The zebrafish is a widely used in vivo model for toxicity testing (Hamm et al. 2019; Padilla et al. 2012). Development is rapid, with organogenesis complete by 3 d post fertilization (3 dpf). The zebrafish genome contains orthologs for ∼70% of human genes (Howe et al. 2013) and ∼86% of the genes that are known human drug targets (Gunnarsson et al. 2008). Zebrafish developmental toxicity testing can be completed in a matter of days by directly exposing the developing organism to xenobiotics. Post-hatch, automated locomotor behavior tests can be used to assess swimming behavior in response to a variety of stimuli as a functional neurodevelopmental outcome. One major limitation of the zebrafish model for toxicity testing relates to chemical dosimetry. Zebrafish embryos are exposed to xenobiotics via immersion. In most studies, nominal waterborne concentrations are generally reported when making determinations on compound toxicity (i.e., positive or negative for toxicity). However, based on physicochemical properties like LogP and differences in exposure parameters (e.g., static vs. semi-static exposures), both of which can affect the uptake, distribution, metabolism, and elimination of test chemicals, the internal tissue dose does not generally reflect nominal exposure media concentrations (Brox et al. 2014, 2016; Kirla et al. 2016; Souder and Gorelick 2017).The developmental toxicity and developmental neurotoxicity of a subset of PFAS, such as PFOS and PFOA, have been previously evaluated in zebrafish (Hagenaars et al. 2011; Huang et al. 2010; Jantzen et al. 2016; Khezri et al. 2017; Spulber et al. 2014; Ulhaq et al. 2013a, 2013b). PFOS exposure results in failed swim bladder inflation, abnormal ventroflexion of the tail (Hagenaars et al. 2011; Huang et al. 2010; Jantzen et al. 2016; Ulhaq et al. 2013a), and hyperactivity (Hurley et al. 2018; Khezri et al. 2017; Spulber et al. 2014), whereas results for PFOA exposures are quite mixed for both developmental toxicity and behavior (Hagenaars et al. 2011; Huang et al. 2010; Jantzen et al. 2016; Khezri et al. 2017; Padilla et al. 2012; Truong et al. 2014; Ulhaq et al. 2013a, 2013b). However, because replacement PFAS such as GenX and ADONA are detected in the environment yet lack adequate data on their potential toxicity, the goal of this study was to assess the developmental toxicity, developmental neurotoxicity, and tissue doses of multiple aliphatic PFAS (e.g., PFOS, PFOA, PFHxS, and PFHxA), several emerging replacement PFAS (e.g., GenX and ADONA), and a polymer production by-product [e.g., perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid (PFESA1)] in parallel, using zebrafish as a test organism. In addition, the potential of sulfonic acid PFAS with varying alkyl chain lengths to elicit similar toxicity phenotypes was assessed.MethodsZebrafish HusbandryAll procedures involving zebrafish were approved by the U.S. Environmental Protection Agency (EPA) National Health and Environmental Effects Research Laboratory Institutional Animal Care and Use Committee and carried out in accordance with the relevant guidelines and regulations. Embryos were obtained from a mixed wild-type (WT) adult zebrafish line (Danio rerio) that was generated and maintained as previously described (Phelps et al. 2017). Briefly, to maintain genetic diversity, a minimum of one WT line (AB and/or Tupfel long fin WT strains) was added one time per year. Zebrafish adults were housed in 6-L tanks at an approximate density of 8 fish/L. Adults were fed Gemma Micro 300 (Skretting) once daily and shell free E-Z Egg (Brine Shrimp Direct) twice daily Mondays through Fridays. Both food sources were fed once daily on weekends. U.S. EPA WT zebrafish were maintained on a 14 h:10 h light cycle at 28.5°C and bred every 2–3 weeks. For embryo collection, 60–100 adults were placed in 10- or 20-L angled static breeding tanks overnight. The following morning, adults were transferred to new angled bottom tanks containing fish facility water, and embryos were collected 30–40 min later.Chemical PreparationADONA [Chemical Abstracts Service Registry No. (CASRN): 958445-44-8; Catalog No. NaDONA] was purchased from Wellington Laboratories (Table 1). GenX Free Acid (CASRN: 13252-13-6; Catalog No. 2121-3-13), PFHxA (CASRN: 307-24-4; Catalog No. 2121-3-39), PFHxS (CASRN: 3871-99-6; Catalog No. 6164-3-X4), PFOA (CASRN: 335-67-1; Catalog No. 2121-3-18), PFOS (CASRN: 1763-23-1; Catalog No. 6164-3-08), perfluorobutanesulfonic acid (PFBS; CASRN: 375-73-5; Catalog No. 6164-3-09), and perfluoroheptanesulfonic acid (PFHpS; CASRN: 375-92-8; Catalog No. 6164-3-2S) were purchased from Synquest. Perfluoropentanesulfonic acid (PFPeS; CASRN 2706-91-4; Catalog No. 6164-3-2U) was synthesized for the study by Synquest Laboratories and chlorpyrifos (CASRN: 2921-88-2; Catalog No. 45395) was purchased from Sigma-Aldrich. PFESA1 (CASRN: 29311-67-9) was obtained from Chemours (Table 1). Stock solutions (20 mM or 25 mM) were prepared either by mixing liquid chemical or dissolving neat chemical into molecular-grade dimethyl sulfoxide (DMSO) (>99.9%) or deionized (DI) water, and aliquots were stored at −80°C. For each experiment, 250× working solutions were prepared by thawing single-use stock solution aliquots and performing semi- or quarter-log serial dilutions in DMSO or DI water in a 96-well polycarbonate microtiter plate. Stock plates containing 250× working solutions were sealed (Biorad; Catalog No. MSB1001) and stored at room temperature in the dark and used for the duration of each study (maximum storage time of 5 weeks).Table 1 Test chemicals.Table 1 has six columns, namely, chemical, name, CASRN, MW (gram per mole), LogP (OPERA), and company, catalog number.ChemicalNameCASRNMW (g/mol)LogPa (OPERAb)Company, catalog no.4,8-Dioxa-3H-perfluorononanoateADONA958445-44-8400.053.96Wellington Laboratories, NaNODAPerfluoro-2-propoxypropanoic acidGenX Free Acid13252-13-6330.053.21Synquest, 2121-3-13Perfluorobutanesulfonic acidPFBS375-73-5300.13.10Synquest, 6164-3-09Perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acidPFESA129311-67-9444.126.02Obtained from ChemoursPerfluoroheptanesulfonic acidPFHpS375-92-8450.122.83Synquest, 6164-3-2SPerfluorohexanoic acidPFHxA307-24-4314.052.78Synquest, 2121-3-39Perfluorohexanesulfonic acidPFHxS3871-99-6438.213.87Synquest, 6164-3-X4Perfluoro-n-octanoic acidPFOA335-67-1414.073.79Synquest, 2121-3-18Perfluorooctanesulfonic acidPFOS1763-23-1500.132.77Synquest, 6164-3-08Perfluoropentanesulfonic acidPFPeS2706-91-4350.113.18Synquest, 6164-3-2UNote: CASRN, Chemical Abstracts Service Registration Number; MW, molecular weight.aPartition coefficient.bOPEn structure-activity/property Relationship App (OPERA) ( https://comptox.epa.gov/dashboard).Study DesignIn Study 1 (Figure 1), the developmental toxicity and developmental neurotoxicity and the media and internal tissue doses of ADONA, GenX Free Acid, PFESA1, PFHxA, PFHxS, PFOA, and PFOS were determined, using DMSO as a vehicle. All chemicals except PFESA1 were tested in parallel and shared the same DMSO control samples for all three assays. PFESA1 was obtained subsequently from Chemours and therefore had unique, experiment-specific control data. In Study 1, GenX Free Acid diluted in DMSO was determined to be unstable, resulting in a null data set that was therefore excluded. In Study 2 (Figure 1), zebrafish were exposed to GenX Free Acid diluted in DI water and evaluated in the developmental toxicity (DevTox) and developmental neurotoxicity (DNT) assays. Measured media and tissue doses were also obtained. Last, in a sulfonic acid PFAS follow-up study (Study 3) (Figure 1), the ability of PFBS (4-carbon), PFPeS (5-carbon), PFHxS (6-carbon), PFHpS (7-carbon), or PFOS (8-carbon) exposure to cause developmental toxicity or developmental neurotoxicity was assessed. All chemicals tested in Study 3, except PFPeS, were exposed in parallel and have shared DMSO control data. PFPeS was synthesized for this study and tested separately, with an experiment-specific DMSO control.Figure 1. Study design. Zebrafish were semi-statically exposed to test PFAS daily, from 0–5 dpf. At 6 dpf. developmental toxicity, developmental neurotoxicity, and PFAS tissue concentrations were assessed. Test PFAS included in Study 1, solubilized in DMSO (final concentration 0.4% DMSO), are highlighted in light blue. Because GenX Free Acid was not stable in DMSO, the compound was retested in all three assays using DI water as a diluent in Study 2 (highlighted in blue). In Study 3, a set of sulfonic acid aliphatic PFAS solubilized in DMSO were tested in the DevTox and DNT assays (shown in green). Note: ADONA, 4,8-dioxa-3H-perfluorononanoate; DevTox, developmental toxicity; DI, deionized; DMSO, dimethyl sulfoxide; DNT, developmental neurotoxicity; dpf, days post fertilization; GenX Free Acid, perfluoro-2-propoxypropanoic acid; PFAS, per- and polyfluoroalkyl substances; PFBS, perfluorobutanesulfonic acid; PFESA1, perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid; PFHpS, perfluoroheptanesulfonic acid; PFHxA, perfluorohexanoic acid; PFHxS, perfluorohexanesulfonic acid; PFOA, perfluoro-n-octanoic acid; PFOS, perfluorooctanesulfonic acid; PFPeS, perfluoropentanesulfonic acid.Chemical ExposuresAt 0 dpf, zebrafish embryos were bleached as previously described (Tal et al. 2017). A single embryo at the dome-to-epiboly stages (Kimmel et al. 1995) was placed into each individual well of a 96-well plate containing a 40-μm nylon mesh filter (Millipore, Catalog No. MANMN4010) with 400μL of 10% Hanks’ balanced salt solution (HBSS) per well. Filter inserts containing zebrafish embryos were transferred to 96-well culture trays (Millipore, Catalog No. MAMCS9610) containing 250μL of 10% HBSS (Westerfield 2007) and 1μL of 250× working solutions per well. A final concentration of 0.4% DMSO was used for all exposure groups and as a vehicle control. In the case of GenX Free Acid in Study 2 (Figure 1), DI water was used as a vehicle control. Daily, from 1–5 dpf, plates underwent 100% media changes to refresh chemical dosing solutions by blotting (Brandel; Catalog No. FPXLR-196) and transferring mesh inserts containing zebrafish to new bottom plates (Millipore; Catalog No. MAMCS9610). To minimize evaporation, plates were sealed (Biorad; Catalog No. MSA5001) and wrapped with parafilm. Plates were maintained on a 14 h:10 h light cycle at 26.0°C and scored daily for death, malformations, hatching, and swim bladder inflation. At 6 dpf, plates were evaluated by two independent observers and DevTox or DNT assays were performed or media and tissue were collected for analytical chemistry analyses as described below.Developmental Toxicity AssayIn Study 1 (Figure 1), zebrafish were exposed, as described in the “Chemical Exposures” section, to 0.04, 0.1, 0.4, 1.1, 3.1, 9.3, 27.2, or 80.0μM PFOS, PFOA, PFHxS, PFHxA, or ADONA, or 0.4% DMSO. Six 96-well plates were tested with a single chemical concentration included on each microtiter plate. Subsequently, as part of Study 1, zebrafish were exposed to 0.04, 0.1, 0.4, 1.1, 3.1, 9.3, 27.2, or 80.0μM PFESA1, or 0.4% DMSO. The number of biological replicates per study and additional experimental details are shown in Table 2. In Study 2, GenX Free Acid diluted in DI water was tested by exposing zebrafish to 0.04, 0.1, 0.4, 1.1, 3.1, 9.3, 27.2, or 80.0μM of the compound or DI water. In a follow-up study to assess the toxicity of aliphatic sulfonic acid PFAS (Study 3), a higher starting concentration was used to increase the likelihood of observing both malformations with shorter-chain compounds and malformations at multiple test concentrations. Zebrafish were exposed to 1.7, 3.1, 5.5, 9.8, 17.6, 31.4, 56.0, or 100.0μM of PFBS, PFHxS, PFHpS, or PFOS or 0.4% DMSO. Subsequently, zebrafish were exposed to 1.7, 3.1, 5.5, 9.8, 17.6, 31.4, 56.0, or 100.0μM PFPeS, or 0.4% DMSO. Chlorpyrifos was used as positive control for malformations (8.0μM) and lethality (80.0μM) (Padilla et al. 2012; Tal et al. 2017). To conduct DevTox assay assessments, at 6 dpf, two independent observers evaluated zebrafish larvae for survival, hatching, swim bladder inflation, and malformations, including curved body axis, shortened trunk, pericardial edema, yolk sac edema, necrotic yolk sac, pectoral fin abnormalities and head/jaw abnormalities. Directly after assessments, data were reviewed and, in the case of discrepancies, consensus calls were reached. Toxicity values were assigned to descriptive data (i.e., normal=0, abnormal=20, severely abnormal=50, and dead=100), modified from a previously described approach (Padilla et al. 2012). Briefly, animals with a single malformation were scored as abnormal, whereas animals with ≥2 malformations were scored as severely abnormal. A study inclusion criterion based on a previously published study (Padilla et al. 2012) was applied where microtiter plates with >15% abnormal or dead DMSO or DI water control larvae were excluded (one plate from Study 3 was excluded).Table 2 Study-specific metrics.Table 2 has eight columns, namely, study, name, diluent and virgule or vehicle, assay, concentrations tested (micromolar), exposure replicate number, control replicate number, and number of 96 well plates chemicals tested across.StudyNameDiluent and/or vehicleAssayConcentrations tested (μM)Exposure replicatea (n)Control replicatea (n)96-well plates chemicals tested acrossb (n)1ADONADMSODevTox0.04, 0.1, 0.4, 1.1, 3.1, 9.3, 27.2, 80.062166DNT4.4, 7.9, 14.0, 25.1, 44.8, 80.02439417Chemistry25.1, 44.8, 80.05531GenX Free AcidDMSODevTox, DNT, ChemistryNot stable in DMSO; results not reported; see Study 21PFESA1DMSODevTox0.04, 0.1, 0.4, 1.1, 3.1, 9.3, 27.2, 80.06441DNT4.4, 7.9, 14.0, 25.1, 44.8, 80.0243397Chemistry25.1, 44.8, 80.04531PFHxADMSODevTox0.04, 0.1, 0.4, 1.1, 3.1, 9.3, 27.2, 80.062166DNT4.4, 7.9, 14.0, 25.1, 44.8, 80.02439417Chemistry25.1, 44.8, 80.04421PFHxSDMSODevTox0.04, 0.1, 0.4, 1.1, 3.1, 9.3, 27.2, 80.062166DNT4.4, 7.9, 14.0, 25.1, 44.8, 80.02439417Chemistry14.0, 25.1, 44.84421PFOADMSODevTox0.04, 0.1, 0.4, 1.1, 3.1, 9.3, 27.2, 80.062166DNT4.4, 7.9, 14.0, 25.1, 44.8, 80.02439417Chemistry25.1, 44.8, 80.04421PFOSDMSODevTox0.04, 0.1, 0.4, 1.1, 3.1, 9.3, 27.2, 80.062166DNT0.2, 0.3, 0.6, 1.0, 1.8, 3.12439417Chemistry1.0, 1.8, 3.14422GenX Free AcidDI waterDevTox0.04, 0.1, 0.4, 1.1, 3.1, 9.3, 27.2, 80.06441DNT4.4, 7.9, 14.0, 25.1, 44.8, 80.0241614Chemistry25.1, 44.8, 80.04423PFBSDMSODevTox1.7, 3.1, 5.5, 9.8, 17.6, 31.4, 56.0, 100.0101406DNT5.5, 9.8, 17.6, 31.4, 56.0, 100.025327103PFPeSDMSODevTox1.7, 3.1, 5.5, 9.8, 17.6, 31.4, 56.0, 100.06441DNT3.1, 5.5, 9.8, 17.6, 31.4, 56.02418643PFHxSDMSODevTox1.7, 3.1, 5.5, 9.8, 17.6, 31.4, 56.0, 100.0101406DNT3.1, 5.5, 9.8, 17.6, 31.4, 56.025327103PFHpSDMSODevTox1.7, 3.1, 5.5, 9.8, 17.6, 31.4, 56.0, 100.0101406DNT1.7, 3.1, 5.5, 9.8, 17.6, 31.425327103PFOSDMSODevTox1.7, 3.1, 5.5, 9.8, 17.6, 31.4, 56.0, 100.0101406DNT0.5, 1.0, 1.7, 3.1, 5.5, 9.82532710Note: ADONA, 4,8-dioxa-3H-perfluorononanoate; DevTox, developmental toxicity; DI, deionized; DMSO, dimethyl sulfoxide; DNT, developmental neurotoxicity; GenX Free Acid, perfluoro-2-propoxypropanoic acid; PFBS, perfluorobutanesulfonic acid; PFESA1, perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid; PFHpS, perfluoroheptanesulfonic acid; PFHxA, perfluorohexanoic acid; PFHxS, perfluorohexanesulfonic acid; PFOA, perfluoro-n-octanoic acid; PFOS, perfluorooctanesulfonic acid; PFPeS, perfluoropentanesulfonic acid.aReplicate numbers indicate single animals except for chemistry samples comprising pools of 10 larvae.bIndicates total number of 96-well microtiter plates assessed for each study and/or assay. For Study 1 DevTox and DNT assays, ADONA, GenX Free Acid, PFESA1, PFHxA, PFHxS, PFOA, and PFOS were tested in parallel and shared the same 0.4% DMSO control samples. PFESA1 was obtained subsequently and had unique, experiment-specific control data. In Study 1, GenX Free Acid diluted in 0.4% DMSO was determined to be unstable, resulting in a null data set. In Study 2 (Figure 1), zebrafish were exposed to GenX Free Acid diluted in DI water and evaluated in the DevTox and DNT assays. Measured media and tissue doses were also obtained. Study 3 (Figure 1) examined the ability of PFBS (4-carbon), PFPeS (5-carbon), PFHxS (6-carbon), PFHpS (7-carbon), or PFOS (8-carbon) exposure to cause developmental toxicity or developmental neurotoxicity. All chemicals tested in Study 3, except PFPeS, were exposed in parallel and have shared DMSO control data. PFPeS was synthesized for this study and tested separately, with an experiment-specific DMSO control.Developmental Neurotoxicity AssayTo increase the likelihood of observing behavioral effects in morphologically normal larvae, the highest concentration evaluated in the DNT assay was the lowest observed effect concentration (LOEC) determined in DevTox assay. Zebrafish were exposed in parallel to 4.4, 7.9, 14.0, 25.1, 44.8, or 80.0μM PFOA, PFHxS, PFHxA, or ADONA or 0.2, 0.3, 0.6, 1.0, 1.8, or 3.1μM PFOS or 0.4% DMSO. Subsequently, as part of Study 1, exposure to 4.4, 7.9, 14.0, 25.1, 44.8, or 80.0μM PFESA1, or 0.4% DMSO was evaluated. In Study 2, zebrafish were exposed to 4.4, 7.9, 14.0, 25.1, 44.8, or 80.0μM GenX Free Acid, or DI water. In Study 3, to increase the likelihood of observing malformations with shorter-chain compounds at multiple test concentrations, the highest concentration evaluated was 100.0μM. Zebrafish were exposed to 5.5, 9.8, 17.6, 31.4, 56.0, or 100.0μM PFBS; 3.1, 5.5, 9.8, 17.6, 31.4, or 56.0μM PFHxS; 1.7, 3.1, 5.5, 9.8, 17.6, or 31.4μM PFHpS; or 0.5, 1.0, 1.7, 3.1, 5.5, or 9.8μM PFOS, or 0.4% DMSO. Subsequently, as part of Study 3, zebrafish were exposed to 3.1, 5.5, 9.8, 17.6, 31.4, or 56.0μM PFPeS, or 0.4% DMSO. Chemical exposures and assessments were performed daily, as described above. To evaluate swimming behavior in a light/dark behavior test, microtiter plates were placed in a dark, temperature-controlled behavior testing room set to 26.0°C for at least 2 h prior to testing. At the time of testing, microtiter plates were placed on a Noldus tracking apparatus. Locomotor activity was recorded (30 frames/s) for a total of 60 min consisting of a 20-min dark acclimation period (0 lux) that was not analyzed followed by a 40-min testing period consisting of a 20-min light period (5.0 lux) and 20-min dark period (0 lux). Videos were analyzed using Ethovision software (version 3.1; Noldus Information Technology) as previously described (Jarema et al. 2015). Locomotor activity was collected for each individual fish for each 2-min period (minimum distance moved, set to 0.135cm). Thus, for a 40-min test, 20 data points were collected per larvae. Based on microtiter plate inclusion criterion (i.e., <15% abnormal or dead control larvae), two plates from Study 1 were excluded. Four additional criteria for inclusion of individual larvae were applied. One, all larvae that were identified as abnormal, severely abnormal, or dead were excluded. Two, larvae with uninflated swim bladders were removed from analyses. Three, individual larvae that moved 50% death or malformations within the test group) were excluded from behavior analyses. Also as part of the DN
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