Effects of two Bisphenol A replacement compounds, 1,7-bis (4-hydroxyphenylthio)-3,5-dioxaheptane and Bisphenol AF, on development and mRNA expression in chicken embryos.

Concerns about the estrogenic properties of Bisphenol A (BPA) have led to increased efforts to find BPA replacements. 1,7-bis(4-Hydroxyphenylthio)-3,5-dioxaheptane (DD-70) and 4,4'-(hexafluoroisopropylidene) diphenol (bisphenol AF, BPAF) are two potential chemical substitutes for BPA; however, toxicity data for these chemicals in avian species are limited. To determine effects on avian embryonic viability, development, and hepatic mRNA expression at two distinct developmental periods (mid-incubation [day 11] and term [day 20]), two egg injection studies were performed. Test chemicals were injected into the air cell of unincubated, fertilized chicken eggs at concentrations ranging from 0-88.2 µg/g for DD-70 and 0-114 µg/g egg for BPAF. Embryonic concentrations of DD-70 and BPAF decreased at mid-incubation and term compared to injected concentrations suggesting embryonic metabolism. Exposure to DD-70 (40.9 and 88.2 µg/g) and BPAF (114 µg/g) significantly decreased embryonic viability at mid-incubation. Exposure to DD-70 (88.2 µg/g) decreased embryo mass and increased gallbladder mass, while 114 µg/g BPAF resulted in increased gallbladder mass in term embryos. Expression of hepatic genes related to xenobiotic metabolism, lipid homeostasis, and response to estrogen were altered at both developmental stages. Given the importance of identifying suitable BPA replacements, the present study provides novel, whole animal avian toxicological data for two replacement compounds, DD-70 and BPAF. DATA AVAILABILITY: Data, associated metadata, and calculation tools are available from the corresponding author (doug.crump@canada.ca).

Extracts of Passive Samplers Deployed in Variably Contaminated Wetlands in the Athabasca Oil Sands Region Elicit Biochemical and Transcriptomic Effects in Avian Hepatocytes.

Recent contaminant monitoring in boreal wetlands situated in Alberta's Athabasca oil sands region revealed increased concentrations of polycyclic aromatic compounds (PACs) in passive sampling devices deployed in wetlands close to bitumen surface mining operations. In this study, graded concentrations of semipermeable membrane device (SPMD) extracts, collected from 4 wetlands with variable burdens of PACs, were administered to chicken and double-crested cormorant (DCCO) embryonic hepatocytes to determine effects on 7-ethoxyresorufin-O-deethylase (EROD) activity and mRNA expression. Concentrations and composition of PACs detected in SPMDs varied among sites, and the proportion of alkyl PACs was greater than parent compounds at all sites. ΣPACs was the highest in SPMDs deployed within 10 km of mining activity (near-site wetland [5930 ng SPMD-1]) compared to those ∼50 km south (far-site wetland [689 ng SPMD-1]). Measures of EROD activity and Cyp1a4 mRNA expression allowed the ranking of wetland sites based on aryl hydrocarbon receptor-mediated end points; EROD activity and Cyp1a4 mRNA induction were the highest at the near-site wetland. ToxChip PCR arrays (one chicken and one DCCO) provided a more exhaustive transcriptomic evaluation across multiple toxicological pathways following exposure to the SPMD extracts. Study sites with the greatest PAC concentrations had the most genes altered on the chicken ToxChip (12-15/43 genes). Exposure of avian hepatocytes to SPMD extracts from variably contaminated wetlands highlighted traditional PAC-related toxicity pathways as well as other novel mechanisms of action. A novel combination of passive sampling techniques and high-throughput toxicity evaluation techniques shows promise in terms of identifying hotspots of chemical concern in the natural environment.

Athabasca Oil Sands Petcoke Extract Elicits Biochemical and Transcriptomic Effects in Avian Hepatocytes.

Petroleum coke or "petcoke" is a granular carbonaceous material produced during the upgrading of heavy crude oils, including bitumen. Petcoke dust was recently reported as an environmental contaminant in the Athabasca oil sands region, but the ecotoxicological hazards posed by this complex bitumen-derived material-including those to avian species-have not been characterized. In this study, solvent extracts (x) of delayed and fluid petcoke (xDP and xFP) were prepared and dissolved in dimethyl sulfoxide. A water-accommodated fraction of delayed petcoke (waDP) was also prepared. Graded concentrations of xDP, xFP, and waDP were administered to chicken and double-crested cormorant hepatocytes to determine effects on 7-ethoxyresorufin-O-deethylase (EROD) activity, porphyrin accumulation, and mRNA expression. Polycyclic aromatic compounds (PACs) were characterized, and xDP, xFP, and waDP had total PAC concentrations of 93 000, 270, and 5.3 ng/mL. The rank order of biochemical and transcriptomic responses was xDP > xFP > waDP (e.g., EROD EC50s were lower for xDP compared to xFP and waDP). A total of 22, 18, and 4 genes were altered following exposure to the highest concentrations of xDP, xFP, and waDP, respectively, using a chicken PCR array comprising 27 AhR-related genes. To provide more exhaustive coverage of potential toxicity pathways being impacted, two avian ToxChip PCR arrays-chicken and double-crested cormorant-were utilized, and xDP altered the expression of more genes than xFP. Traditional PAC-related toxicity pathways and novel mechanisms of action were identified in two avian species following petcoke extract exposure. Extrapolation to real-world exposure scenarios must consider the bioavailability of the extracted PACs compared to those in exposed organisms.

Use of a Novel Double-Crested Cormorant ToxChip PCR Array and the EROD Assay to Determine Effects of Environmental Contaminants in Primary Hepatocytes.

In vitro screening tools and 'omics methods are increasingly being incorporated into toxicity studies to determine mechanistic effects of chemicals and mixtures. To date, the majority of these studies have been conducted with well-characterized laboratory animal models. In the present study, well-established methods developed for chicken embryonic hepatocyte (CEH) studies were extended to a wild avian species, the double-crested cormorant (DCCO; Phalacrocorax auritus), in order to compare the effects of several environmental contaminants on cytotoxicity, ethoxyresorufin O-deethylase (EROD) activity, and mRNA expression. Five organic flame retardants and one plasticizer decreased cormorant hepatocyte viability in a similar manner to that observed in previous studies with CEH. EROD activity was induced in a concentration-dependent manner following exposure to two dioxin-like chemicals and the calculated EC50 values were concordant with domestic avian species from similar species sensitivity categories. Transcriptomic effects were determined using a novel DCCO PCR array, which was designed, constructed and validated in our laboratory based on a commercially available chicken PCR array. The DCCO array has 27 target genes covering a wide range of toxicity pathways. Gene profiles were variable among the 10 chemicals screened; however, good directional concordance was observed with regard to results previously obtained in CEH. Overall, the application of well-established methods (i.e., CEH and chicken PCR array) to the double-crested cormorant demonstrated the portability of the techniques to an indicator species of ecological relevance.

Biochemical and Transcriptomic Effects of Herring Gull Egg Extracts from Variably Contaminated Colonies of the Laurentian Great Lakes in Chicken Hepatocytes.

Determining the effects of complex mixtures of environmental contaminants poses many challenges within the field of ecotoxicology. In this study, graded concentrations of herring gull egg extracts, collected from five Great Lakes breeding colonies with variable burdens of organohalogen contaminants (OHCs), were administered to chicken embryonic hepatocytes to determine effects on 7-ethoxyresorufin-O-deethylase (EROD) activity, porphyrin accumulation, and mRNA expression. EROD activity and porphyrin accumulation permitted the ranking of colonies based on the efficacy of eliciting an aryl hydrocarbon receptor-mediated response. An avian ToxChip polymerase chain reaction (PCR) array provided more exhaustive coverage in terms of potential toxicity pathways being affected, including xenobiotic and lipid metabolism and the thyroid hormone pathway. Herring gull eggs from Channel Shelter Island (CHSH, Lake Huron) and Gull Island (GULL, Lake Michigan) had among the highest OHC burdens, and extracts elicited a biochemical and transcriptomic response greater than that of extracts from the other three, less polluted colonies. For example, EROD EC50 values and porphyrin ECthreshold values were lower for CHSH and GULL extracts than for the other colonies. Extracts from CHSH and GULL altered 15 and 13 of 27 genes on the PCR array compared to no more than eight genes for the less contaminated sites. The combination of a well-established avian in vitro assay, two well-characterized biochemical assays, and the avian ToxChip PCR array permitted the geographical discrimination of variably contaminated herring gull eggs from the Great Lakes. Such high-throughput assays show potential promise as cost-effective tools for determining toxic potencies of complex mixtures in the environment.

Tris(2-butoxyethyl)phosphate and triethyl phosphate alter embryonic development, hepatic mRNA expression, thyroid hormone levels, and circulating bile acid concentrations in chicken embryos.

The organophosphate flame retardants tris(2-butoxyethyl) phosphate (TBOEP) and triethyl phosphate (TEP) are used in a wide range of applications to suppress or delay the ignition and spread of fire. Both compounds have been detected in the environment and TBOEP was recently measured in free-living avian species. In this study, TBOEP and TEP were injected into the air cell of chicken embryos at concentrations ranging from 0 to 45,400 ng/g and 0 to 241,500 ng/g egg, respectively. Pipping success, development, hepatic mRNA expression of 9 target genes, thyroid hormone levels, and circulating bile acid concentrations were determined. Exposure to the highest doses of TBOEP and TEP resulted in negligible detection of the parent compounds in embryonic contents at pipping indicating their complete metabolic degradation. TBOEP exposure had limited effects on chicken embryos, with the exception of hepatic CYP3A37 mRNA induction. TEP exposure decreased pipping success to 68%, altered growth, increased liver somatic index (LSI) and plasma bile acids, and modulated genes associated with xenobiotic and lipid metabolism and the thyroid hormone pathway. Plasma thyroxine levels were decreased at all TEP doses, including an environmentally-relevant concentration (8 ng/g), and gallbladder hypotrophy was evident at ≥ 43,200 ng/g. Tarsus length and circulating thyroxine concentration emerged as potential phenotypic anchors for the modulation of transthyretin mRNA. The increase in plasma bile acids and LSI, gallbladder hypotrophy, and discoloration of liver tissue represented potential phenotypic outcomes associated with modulation of hepatic genes involved with xenobiotic and lipid metabolism.

Tris(1,3-dichloro-2-propyl) phosphate perturbs the expression of genes involved in immune response and lipid and steroid metabolism in chicken embryos.

We previously demonstrated that in ovo exposure to the flame retardant tris(1,3-dichloro-2-propyl) phosphate (TDCPP) decreased plasma thyroxine levels, reduced growth parameters, and decreased gallbladder size in chicken embryos. In the current study DNA microarrays were used to evaluate global mRNA expression in liver tissue of male chicken embryos that exhibited the above mentioned effects. Injected doses were dimethyl sulfoxide vehicle control, 7.6 or 45 µg TDCPP/g egg. TDCPP caused significant changes in the expression of five genes at the low dose and 47 genes at the high dose (False Discovery Rate p ≤ 0.1, fold change ≥ 1.5). The gene expression analysis suggested a compromised immune function, a state of cholestatic liver/biliary fibrosis, and disrupted lipid and steroid metabolism. Circulating bile acid levels were elevated, which is an indication of liver dysfunction, and plasma cholesterol levels were reduced; however, hepatic bile acid and cholesterol levels were unaltered. Interactome analyses identified apolipoprotein E, hepatocyte nuclear factor 4 alpha, and peroxisome proliferator-activated receptor alpha as key regulatory molecules involved in the effects of TDCPP. Our results demonstrate a targeted effect of TDCPP toxicity on lipid metabolism, including cholesterol, that helps explain the aforementioned phenotypic effects, as chicken embryos are highly dependent on yolk lipids for growth and maintenance throughout development. Finally, our results are in concordance with the literature that describes TDCPP as a cancer-causing agent, since the majority of dysregulated genes were involved in cancer pathways.

1,2-Dibromo-4-(1,2-dibromoethyl)-cyclohexane and tris(methylphenyl) phosphate cause significant effects on development, mRNA expression, and circulating bile acid concentrations in chicken embryos.

1,2-Dibromo-4-(1,2-dibromoethyl)-cyclohexane (DBE-DBCH; formerly abbreviated as TBECH) and tris(methylphenyl) phosphate (TMPP; formerly abbreviated as TCP) are additive flame retardants that are detected in the environment and biota. A recent avian in vitro screening study of 16 flame retardants identified DBE-DBCH and TMPP as important chemicals for follow-up in ovo evaluation based on their effects on cytotoxicity and mRNA expression in avian hepatocytes. In this study, technical mixtures of DBE-DBCH and TMPP were injected into the air cell of chicken embryos at concentrations ranging from 0 to 54,900ng/g and from 0 to 261,400ng/g, respectively, to determine effects on pipping success, development, hepatic mRNA expression, thyroid hormone levels, and circulating bile acid concentrations. Both compounds were detectable in embryos at pipping and the ß-DBE-DBCH isomer was depleted more rapidly than the α-isomer in tissue samples. DBE-DBCH had limited effects on the endpoints measured, with the exception of the up-regulation of two phase I metabolizing enzymes, CYP3A37 and CYP2H1. TMPP exposure caused embryonic deformities, altered growth, increased liver somatic index (LSI) and plasma bile acid concentrations, and altered mRNA expression levels of genes associated with xenobiotic and lipid metabolism and the thyroid hormone pathway. Overall, TMPP elicited more adverse molecular and phenotypic effects than DBE-DBCH albeit at concentrations several orders of magnitude greater than those detected in the environment. The increase in plasma bile acid concentrations was a useful phenotypic anchor as it was associated with a concomitant increase in LSI, discoloration of the liver tissue, and modulation of hepatic genes involved with xenobiotic and lipid metabolism.

Toxicity Screening of Bisphenol A Replacement Compounds: Cytotoxicity and mRNA Expression in Primary Hepatocytes of Chicken and Double-Crested Cormorant.

A market for bisphenol A (BPA) replacement compounds has emerged as a result of restrictions on the use of BPA. Some of these compounds have been detected in the environment; however, little is known about their toxicological properties. In the present study, an avian in vitro toxicogenomic approach was used to compare the effects of 5 BPA alternatives. Cell viability and mRNA expression were compared in primary embryonic hepatocytes of chicken (CEH) and double-crested cormorant (DCEH) exposed to 4,4'-(propane-2,2-diyl) diphenol (BPA), bis (4-hydroxyphenyl) methane (BPF), bis (3-allyl-4-hydroxyphenyl) sulfone (TGSH), 7-bis (4-hydroxyphenylthio)-3,5-dioxaheptane (DD-70), 2,2-bis (4-hydroxyphenyl) hexafluoropropane (BPAF), and 4-hydroxyphenyl 4-isoprooxyphenylsulfone (BPSIP). Changes in gene expression were determined using 2 polymerase chain reaction (PCR) arrays: 1) species-specific ToxChips that contain genes representing toxicologically relevant pathways, and 2) chicken-specific AestroChip that measures estrogen responsive genes. In CEH and DCEH, BPA alternatives TGSH, DD-70, and BPAF were most cytotoxic. Some of the replacement compounds changed the expression of genes related to xenobiotic metabolism, bile acid, and cholesterol regulation. The rank order based on the number of genes altered on the chicken ToxChip array was TGSH > DD-70 > BPAF = BPF > 17ß estradiol (E2) > BPSIP > BPA. On the cormorant ToxChip array, BPSIP altered the greatest number of genes. Based on the chicken AestroChip data, BPSIP and BPF were slightly estrogenic. These results suggest that the replacement compounds have comparable or even greater toxicity than BPA and act via different mechanisms. Environ Toxicol Chem 2021;40:1368-1378. © 2021 Her Majesty the Queen in Right of Canada. Reproduced with the permission of the Minister of Environment and Climate Change Canada.

Extracts from Dated Lake Sediment Cores in the Athabasca Oil Sands Region Alter Ethoxyresorufin-O-deethylase Activity and Gene Expression in Avian Hepatocytes.

Increases in oil sands mining operations in the Athabasca oil sands region have resulted in increased concentrations of polycyclic aromatic compounds (PACs) and heavy metals in aquatic systems located near surface mining operations. In the present study, sediment cores were collected from 3 lakes with varying proximity to surface mining operations to determine the differences in PAC concentrations. Sediment cores were separated into 2 sections-current mining (top; 2000-2017) and premining (bottom; pre-1945)-and extracts were prepared for in vitro screening using a well-established chicken embryonic hepatocyte (CEH) assay. Concentrations and composition of PACs varied between sites, with the highest ∑PACs in Saline Lake, 5 km from an active oil sands mine site. The proportion of alkylated PACs was greater than that of parent PACs in the top sediment sections compared with the bottom. Ethoxyresorufin-O-deethylase activity in CEH permitted the ranking of lake sites/core sections based on an aryl hydrocarbon receptor-mediated end point; mean median effect concentration values were lowest for the top cores from Saline Lake and another near-mining operations lake, referred to as WF1. A ToxChip polymerase chain reaction (PCR) array was used to evaluate gene expression changes across 43 target genes associated with numerous toxicological pathways following exposure to top and bottom sediment core extracts. The 2 study sites with the greatest ∑PAC concentrations (Saline Lake and WF1) had the highest gene expression alterations on the ToxChip PCR array (19 [top] and 17 [bottom]/43), compared with a reference site (13 [top] and 7 [bottom]/43). The avian in vitro bioassay was useful for identifying the toxicity of complex PAC extracts associated with variably contaminated sediment cores, supporting its potential use for hotspot identification and complex mixture screening. EnvironToxicol Chem 2021;40:1883-1893. © 2021 SETAC.

Effects on Apical Outcomes of Regulatory Relevance of Early-Life Stage Exposure of Double-Crested Cormorant Embryos to 4 Environmental Chemicals.

Environmental risk assessment is often challenged by a lack of toxicity data for ecological species. The overall goal of the present study was to employ an avian early-life stage toxicity test to determine the effects of 4 chemicals (benzo[a]pyrene [BaP], chlorpyrifos, fluoxetine hydrochloride [FLX], and ethinyl estradiol [EE2]) on an ecologically relevant avian species, the double-crested cormorant (Phalacrocorax auritus), and to compare our results with those we previously reported for a laboratory model species, Japanese quail. Chemicals were dissolved in dimethyl sulfoxide and administered via air cell injection to fertilized, unincubated double-crested cormorant eggs at 3 nominal concentrations, the highest selected to approximate the 20% lethal dose. Of the 4 chemicals, only chlorpyrifos and FLX were detected in liver tissue of embryos at midincubation (day 14) and termination (day 26; 1-2 d prior to hatch); EE2 and BaP were not detectable, suggesting embryonic clearance/metabolism. No apical effects were observed in double-crested cormorant embryos up to the highest concentrations of chlorpyrifos (no-observed-effect level [NOEL] = 25 µg/g) or FLX (NOEL = 18 µg/g). Exposure to EE2 reduced embryonic viability and increased deformities at a concentration of 2.3 µg/g (NOEL = 0.18 µg/g), and BaP decreased embryonic viability (median lethal dose = 0.015 µg/g; NOEL = 0.0027 µg/g). Compared with Japanese quail, double-crested cormorant were more sensitive with regard to embryolethality and deformities for EE2 and embryolethality for BaP, whereas they were less sensitive to embryonic deformities associated with chlorpyrifos exposure. These data reinforce the idea that standardized toxicity tests using a laboratory model species may not always be protective of wild birds, and thus they stress the importance of developing such alternative testing strategies (e.g., the EcoToxChip Project) for ecologically relevant species to augment risk assessment efforts. Environ Toxicol Chem 2021;40:390-401. © 2020 SETAC.

A rapid method of preparing complex organohalogen extracts from avian eggs: Applications to in vitro toxicogenomics screening.

Double-crested cormorants are piscivorous birds that breed in variably contaminated colonies across the Laurentian Great Lakes of North America. Collection and preparation of environmentally relevant extracts from eggs that contain variable concentrations of organohalogen contaminants represents a minimally invasive approach to characterize potential effects of exposure using in vitro bioassays. In the present study, a rapid, efficient lipid freeze-filtration extraction method was used to prepare extracts from double-crested cormorant eggs collected from 5 breeding colonies that had variable organohalogen contaminant burdens. Extracts, solubilized in dimethyl sulfoxide, were administered to chicken embryonic hepatocytes (CEHs) to determine effects on cell viability, 7-ethoxyresorufin-O-deethylase (EROD) activity, and messenger RNA expression using a chicken ToxChip polymerase chain reaction (PCR) array. The EROD median effect concentration (EC50) values were lower for extracts with greater organohalogen contaminant burdens and thus permitted an initial ranking of colonies based on the efficacy of eliciting an aryl hydrocarbon receptor-mediated response. The ToxChip PCR array data provided a more exhaustive, pathway-based evaluation of extract effects; variability in the transcriptomic profiles was associated with organohalogen contaminant burdens. For example, extracts from Mud Island (Detroit River, MI, USA) had among the highest organohalogen contaminant burdens and elicited a greater biochemical (EROD EC50 = 0.005) and transcriptomic response (22/43 genes altered on the array) in CEHs compared with the least contaminated site, which was Mandarte Island (BC, Canada; EROD EC50 = 0.172; 8/43 genes altered). Avian eggs represent a useful biomonitoring tool for determining complex mixture effects, and the combination of a rapid extraction method, an in vitro bioassay, and targeted endpoint evaluation (biochemical and transcriptomic) shows great promise as an environmental effects monitoring approach. Environ Toxicol Chem 2019;38:811-819. © 2019 Crown in the right of Canada. Published by Wiley Periodicals Inc. on behalf of SETAC.

Bis-(3-allyl-4-hydroxyphenyl) sulfone decreases embryonic viability and alters hepatic mRNA expression at two distinct developmental stages in chicken embryos exposed via egg injection.

Concerns surrounding the toxicological effects and environmental prevalence of bisphenol A (BPA) have increased efforts to identify suitable safer replacement alternatives. Bis-(3-allyl-4-hydroxyphenyl) sulfone (TGSH) represents a potential BPA alternative; however, exposure and ecotoxicological data are scarce. To determine effects on embryonic viability, development, and hepatic mRNA expression at 2 distinct developmental periods (midincubation [day 11] and pipping [days 20-21]), TGSH was injected into the air cell of unincubated, fertilized chicken embryos at 4 concentrations ranging from 0 to 180 µg/g egg. Concentrations of TGSH increased in a dose-dependent manner in whole-embryo homogenates, and the estimated median lethal dose (LD50) based on embryonic viability at midincubation was 66 µg/g (95% confidence interval = 31-142 µg/g), which is similar to the BPA LD50 (∼ 67 µg/g) reported in a previous study. Modulation of hepatic gene targets from a chicken ToxChip polymerase chain reaction (PCR) array was observed at both developmental stages. At midincubation, 21/43 genes on the PCR array were significantly altered (by >1.5-fold) in the 180 µg/g dose group, whereas 9 and 6/43 were altered at pipping in the 9.2 and 48 µg/g groups, respectively. Predominant toxicity pathways included xenobiotic metabolism, lipid homeostasis, bile acid synthesis, and cell cycle regulation. The estrogen-responsive gene apolipoprotein II was significantly up-regulated in liver tissue of midincubation embryos at 180 µg/g; however, neither apolipoprotein II nor vitellogenin II were altered at the other concentrations or developmental time points. Given the importance of identifying suitable BPA replacement alternatives, the present study provides novel, whole-animal toxicological data for a BPA replacement alternative that has an effect on embryonic viability similar to that of the compound it could replace. Environ Toxicol Chem 2018;37:530-537. © 2017 SETAC.

Design and Operation of the National Survey of Children's Health, 2011-2012.

Objectives This report presents the development, plan, and operation of the 2011-2012 National Survey of Children's Health, a module of the State and Local Area Integrated Telephone Survey, conducted by the National Center for Health Statistics. Funding was provided by the Maternal and Child Health Bureau, Health Resources and Services Administration. The survey was designed to produce national and state prevalence estimates of the physical and emotional health of children aged 0-17 years, as well as factors that may relate to child well-being including medical homes, family interactions, parental health, school and after-school experiences, and neighborhood characteristics.

Bisphenol S alters embryonic viability, development, gallbladder size, and messenger RNA expression in chicken embryos exposed via egg injection.

Amid concerns about the toxicological effects and environmental prevalence of bisphenol A (BPA), efforts to find suitable, safer replacement alternatives are essential. Bisphenol S (BPS) is a potential chemical substitute for BPA; however, few studies are available confirming that it has a more desirable ecotoxicological profile. In the present study, BPS was injected into the air cell of unincubated, fertilized chicken embryos at 6 concentrations ranging from 0 µg/g to 207 µg/g egg to determine effects on pipping success, development, hepatic messenger ribonucleic acid (mRNA) expression, thyroid hormone levels, and circulating bile acid concentrations. Concentrations of BPS increased in a dose-dependent manner in whole-embryo homogenates, and exposure to the highest dose, 207 µg/g, resulted in decreased pipping success (estimated median lethal dose = 279 µg/g; 95% confidence interval = 161-486 µg/g). Exposure to BPS also reduced growth metrics including embryo mass and tarsus length, whereas the most pronounced phenotypic effect was the concentration-dependent, significant increase in gallbladder size at concentrations ≥52.8 µg/g. These adverse phenotypic outcomes were associated with the modulation of gene targets from a chicken ToxChip polymerase chain reaction array, which are involved with xenobiotic metabolism, lipid homeostasis, bile acid synthesis, and the thyroid hormone pathway. Expression levels of 2 estrogen-responsive genes, apolipoprotein II and vitellogenin, were too low at the sampling time point assessed (i.e., pipping embryos) to quantify changes, and no effects were observed on circulating free thyroxine or bile acid concentrations. The present study provides novel, whole-animal toxicological data for a BPA replacement alternative that is not well characterized. Environ Toxicol Chem 2016;35:1541-1549. © 2015 SETAC.

Premature feather loss among common tern chicks in Ontario: the return of an enigmatic developmental anomaly.

In July 2014, we observed premature feather loss (PFL) among non-sibling, common tern Sterna hirundo chicks between two and four weeks of age at Gull Island in northern Lake Ontario, Canada. Rarely observed in wild birds, to our knowledge PFL has not been recorded in terns since 1974, despite the subsequent banding of hundreds of thousands of tern chicks across North America alone. The prevalence, 5% of chicks (9/167), and extent of feather loss we report is more extreme than in previous reports for common terns but was not accompanied by other aberrant developmental or physical deformities. Complete feather loss from all body areas (wing, tail, head and body) occurred over a period of a few days but all affected chicks appeared vigorous and quickly began to grow replacement feathers. All but one chick (recovered dead and submitted for post-mortem) most likely fledged 10-20 days after normal fledging age. We found no evidence of feather dystrophy or concurrent developmental abnormalities unusual among affected chicks. Thus, the PFL we observed among common terns in 2014 was largely of unknown origin. There was striking temporal association between the onset of PFL and persistent strong southwesterly winds that caused extensive mixing of near-shore surface water with cool, deep lake waters. One hypothesis is that PFL may have been caused by unidentified pathogens or toxins welling up from these deep waters along the shoreline but current data are insufficient to test this. PFL was not observed among common terns at Gull Island in 2015, although we did observe similar feather loss in a herring gull Larus argentatus chick in that year. Comparison with sporadic records of PFL in other seabirds suggests that PFL may be a rare, but non-specific, response to a range of potential stressors. PFL is now known for gulls, penguins and terns.

Determination of the estrogenic and antiestrogenic effects of environmental contaminants in chicken embryo hepatocyte cultures by quantitative-polymerase chain reaction.

A method was developed to measure the estrogenic and antiestrogenic effects of various chemicals and organochlorine extracts in chicken embryo primary hepatocyte cultures. Messenger RNAs (mRNAs) for the estrogen-inducible egg yolk proteins, vitellogenin II (VTGII), and very low-density lipoprotein apoprotein II (apoII), were measured by multiplex quantitative reverse transcription-polymerase chain reaction (Q-PCR). After 48 h of exposure, both VTGII and apoII mRNA levels were induced by moxestrol (1-1,000 nM), 17 beta-estradiol (10-1,000 nM), o,p'-DDT (apoII: 1,000 and 10,000 nM, VTGII: 10,000 nM), 4-tertoctylphenol ([OP]; apoII: 20 and 50 microM, VTGII: 10-50 microM), and methoxychlor ([MXCL]; apoII: 5-50 microM, VTGII: 20 and 50 microM). Tamoxifen (100 and 1,000 nM) induced apoII mRNA only, and bisphenol A (BPA) was not estrogenic. Inhibition of moxestrol-mediated VTGII or apoII mRNA induction by MXCL, o,p'-DDT and tamoxifen indicated that these chemicals were also antiestrogenic at concentrations similar to those which caused estrogenic responses. Organochlorine extracts prepared from herring gull embryo yolk sacs obtained from three Great Lakes sites and one Atlantic coast site (reference site) did not show any estrogenic activities. However, the same extracts from all three Great Lakes sites had antiestrogenic activities. These results indicate that wild birds may be susceptible to the estrogenic or antiestrogenic effects of environmental contaminants.

Design and operation of the National Survey of Children with Special Health Care Needs, 2009-2010.

OBJECTIVES: This report presents the development, plan, and operation of the 2009-2010 National Survey of Children with Special Health Care Needs, a module of the State and Local Area Integrated Telephone Survey. The survey is conducted by the Centers for Disease Control and Prevention's National Center for Health Statistics. This survey was designed to produce national and state-specific prevalence estimates of children with special health care needs (CSHCN), to describe the types of services that they need and use, and to assess aspects of the system of care for CSHCN. METHODS: A random-digit-dial sample of households with children under age 18 years was constructed for each of the 50 states and the District of Columbia. The sampling frame consisted of landline phone numbers and cellular(cell) phone numbers of households that reported a cell-phone-only or cell-phone-mainly status. Children in identified households were screened for special health care needs. If CSHCN were identified in the household, a detailed interview was conducted for one randomly selected child with special health care needs. Respondents were parents or guardians who knew about the children's health and health care. RESULTS: A total of 196,159 household screening interviews were completed from July 2009 through March 2011, resulting in 40,242 completed special-needs interviews, including 2,991 from cell-phone interviews. The weighted overall response rate was 43.7% for the landline sample, 15.2% for the cell-phone sample, and 25.5% overall.

In Ovo effects of two organophosphate flame retardants--TCPP and TDCPP--on pipping success, development, mRNA expression, and thyroid hormone levels in chicken embryos.

Tris(1-chloro-2-propyl) phosphate (TCPP) and tris(1,3-dichloro-2-propyl) phosphate (TDCPP) are organic flame retardants detected in the environment and biota for which avian toxicological data are limited. In this study, domestic chicken eggs were injected with TCPP or TDCPP (maximum dose = 51,600 and 45,000ng/g egg, respectively) to determine dose-dependent effects on pipping success, development, hepatic messenger RNA (mRNA) expression levels of genes associated with xenobiotic metabolism and the thyroid hormone (TH) pathway, and TH levels following 20-22 days of incubation. Neither compound reduced pipping success; however, TCPP significantly delayed pipping at 9240 and 51,600ng/g and reduced tarsus length at 51,600ng/g. TDCPP exposure resulted in significant decreases in head plus bill length, embryo mass, and gallbladder size at 45,000ng/g and reduced plasma free T4 levels at 7640ng/g. Type I deiodinase, liver fatty acid-binding protein, and cytochrome P450 (CYP) 3A37 mRNA levels were significantly induced by TCPP, whereas TDCPP induced CYP3A37 and CYP2H1. Chemical analysis of egg contents at incubation days 0, 5, 11, 18, and 19 revealed that > 92% of the injected TCPP or TDCPP concentration was detectable up to day 5; however, < 1% was detected by day 19. The observed phenotypic responses to TCPP and TDCPP exposure may be associated with disruption of the TH axis, which is critical for normal growth and development in birds. The effects of TDCPP on the gallbladder indicate that the disturbance of lipid metabolism is a likely mechanism of toxicity.

In ovo effects of perfluorohexane sulfonate and perfluorohexanoate on pipping success, development, mRNA expression, and thyroid hormone levels in chicken embryos.

Perfluoroalkyl acids (PFAAs), specifically perfluorinated sulfonates and carboxylates, are synthetic substances known for their chemical stability, resistance to degradation, and potential to biomagnify in food chains. The toxicological and biological effects of PFAAs in avian species are not well characterized, although there is some evidence to suggest that they can impact neurodevelopment and hatching success. Our laboratory recently reported significant effects of perfluorohexane sulfonate (PFHxS) and perfluorohexanoate (PFHxA) on messenger RNA (mRNA) levels of thyroid hormone (TH)-responsive genes in chicken embryonic neuronal cells. In this study, we determined in ovo effects of PFHxS and PFHxA exposure (maximum dose = 38,000 and 9700 ng/g egg, respectively) on embryonic death, developmental endpoints, tissue accumulation, mRNA expression in liver and cerebral cortex, and plasma TH levels. Pipping success was reduced to 63% at the highest dose of PFHxS; no effects were observed for PFHxA. PFHxS exposure (38,000 ng/g) decreased tarsus length and embryo mass. PFHxS and PFHxA accumulated in the three tissue compartments analyzed as follows: yolk sac > liver > cerebral cortex. Type II and type III 5'-deiodinases (D2 and D3) and cytochrome P450 3A37 mRNA levels were induced in liver tissue of chicken embryos exposed to PFHxS, whereas D2, neurogranin (RC3), and octamer motif binding factor 1 mRNA levels were upregulated in cerebral cortex. Plasma TH levels were reduced in a concentration-dependent manner following PFHxS exposure; PFHxA had no effect. This in ovo study successfully validated previous in vitro results concerning the modulation of TH-responsive genes and identified adverse effects associated with TH homeostasis in response to PFHxS treatment.