Racial disparities affect the association between gestational urinary phthalate mixtures and infant genital measures.

Background: Phthalates are ubiquitous anti-androgenic endocrine disrupting chemicals found in personal care products, medications, and many plastics. Studies have shown a racial disparity in phthalates exposure among U.S. women, which may also impact fetal development. Methods: We conducted a prospective cohort study of gestational exposure to a phthalates mixture in a racially-diverse population to determine their association with genital development. Mid-gestation (18-22 weeks) urine was collected from 152 women who self-identified as non-Hispanic Black and 158 women who self-identified as non-Hispanic White in Charleston, South Carolina between 2011 and 2014. We measured eight phthalate monoester metabolites in urine using liquid chromatography tandem-mass spectrometry. Mid-gestational penile dimensions were measured using ultrasound and anogenital distances were measured postnatally. We used Bayesian kernel machine regression to estimate the associations among the mixture of phthalate metabolites and mid-gestation penile dimensions and postnatal anogenital distance measures among singleton male (n = 179) and female (n = 131) infants, adjusted for urinary specific gravity, maternal age, body mass index, education level, cigarette smoking, and gestational age at enrollment or birth weight z-score. Results: We found a stronger association between greater phthalates and decreased anopenile distance among infants born to women who self-identified as Black. Mono (2-ethylhexyl) phthalate (MEHP) was the driving mixture component among Black women, and monobutyl phthalate (MBP) and monoethyl phthalate (MEP) were drivers among White women. We also identified a non-linear association between phthalates and lesser ultrasound penile volume among women who self-identified as Black with monoisobutyl phthalate (MiBP) and MBP being most important. We also found an association between greater phthalates and shorter anoclitoral distance among infants born to women who self-identified as Black, with MEP and monobenzyl phthalate (MBzP) contributing most to this association. Conclusion: Our results suggest a disparity in the association between gestational exposure to a mixture of phthalates and fetal genital development among women who self-identified as Black compared to White.

Using Plasma Vitellogenin in Loggerhead Sea Turtles to Assess Reproductive Maturation and Estrogen-Like Contaminant Exposure.

Vitellogenin (VTG), an egg yolk precursor, is abnormally produced by male and juvenile oviparous species after exposure to estrogens. Plasma VTG in loggerhead sea turtles (Caretta caretta) helped us understand their reproductive maturation and investigate it as a biomarker of contaminant exposure. The presence of VTG was screened in plasma from 404 loggerheads from the northwestern Atlantic Ocean using a freshwater turtle antibody in western blots. The concentrations of VTG were semiquantified using band intensities calibrated to results from a loggerhead antibody enzyme-linked immunoassay. The detection and concentrations of VTG were in (from highest to lowest): nesting females, in-water adult females, subadult females, smaller females, unknown sex, and males. Loggerheads from this region begin vitellogenesis at ≅77 cm straight carapace length. We classified VTG expression as abnormal in nine male or juvenile turtles. Organochlorine contaminant (OC) concentrations were measured in blood and/or fat biopsies of some turtles. One abnormal VTG female had the second highest fat polychlorinated biphenyl (PCB) and 4,4'-dichlorodiphenyldichloroethylene concentrations compared among 43 VTG-negative juveniles. The nine VTG-abnormal turtles had average blood PCB concentrations 8.5% higher, but not significantly different, than 46 VTG-negative juveniles (p = 0.453). In turtles less than 77 cm, blood PCB concentrations were significantly, but weakly, correlated with semiquantified VTG concentrations (tau = 0.1, p = 0.004). Greater blood OC concentrations were found in adult females than in males, which motivated the creation of a conceptual model of OC, VTG, and hormone concentrations across a reproductive cycle. A decision tree is also provided incorporating VTG as a sexing tool. Abnormal VTG expression cannot conclusively be linked to endocrine disruption caused by these OC concentrations. Studies should further investigate causes of abnormal VTG expression in wild sea turtles. Environ Toxicol Chem 2023;42:1309-1325. © 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Assessing Reliability of Non-targeted High-Resolution Mass Spectrometry Fingerprints for Quantitative Source Apportionment in Complex Matrices.

Effective management of contaminated sites requires differentiating and deconvoluting contaminant source impacts in complex environmental systems. The existing source apportionment approaches that use targeted analyses of preselected indicator chemicals are limited whenever target analytes are below the detection limits or derived from multiple sources. However, non-targeted analyses that leverage high-resolution mass spectrometry (HRMS) yield rich datasets that deeply characterize sample-specific chemical compositions, providing additional potential end-members for source differentiation and apportionment. Previous work demonstrated that HRMS fingerprints can define sample uniqueness and support accurate, quantitative source concentration estimates. Here, using two aqueous film-forming foams as representative complex sources, we assessed the qualitative fidelity and quantitative accuracy of HRMS source fingerprints in increasingly complex background matrices. Across all matrices, HRMS-derived source concentration estimates were 0.81 ± 0.11-fold and 0.64 ± 0.24-fold of actual in samples impacted solely by analytical matrix effects (MEs) or by sample processing recovery and analytical MEs, respectively. Isotopic internal standards were not easily paired to individual unidentified non-target features, but bulk internal standard-based abundance corrections improved apportionment accuracy in higher matrix samples (to 0.90 ± 0.12-fold of actual) and/or informed concentration estimate relative errors. HRMS fingerprint mining could identify, based on the dilution behavior, effective individual chemical end-members across 16 homologous series. Although method development is needed, the results further demonstrate the potential applications of non-targeted HRMS data for source apportionment and other quantitative outcomes.

Levels and profiles of perfluorinated alkyl acids in liver tissues of birds with different habitat types and trophic levels from an urbanized coastal region of South Korea.

Contamination status and characteristics of perfluorinated alkyl acids (PFAAs) including perfluorinated carboxylic acids (PFCAs) and perfluorinated sulfonic acids (PFSAs) was examined using liver tissue of birds - black-tailed gulls (Larus crassirostris), domestic pigeons (Columba livia var. domestica), pacific loons (Gavia pacifica), herons (Ardea cinerea), and egrets (Egretta garzetta and Ardea alba) - with different trophic levels, habitat types and migratory behaviors from an industrialized coastal region of South Korea. A wide range of PFAAs (1.09 ng/g to 1060 ng/g; median = 52.6 ng/g) were detected in bird livers from the Korean coasts with high detection frequency. Accumulation features of PFAAs in birds indicated that primarily trophic position and secondly habitat type influence the levels and composition of PFAAs, e.g., relatively high PFAA levels and high composition of odd-numbered long carbon chain PFCAs (perfluoroundecanoic acid (PFUnDA) and perfluorotridecanoic acid (PFTriDA)) and PFOS in higher trophic and marine birds. The prevalence of long carbon chain (≥14) PFCAs likely implies a wide use of fluorotelomer-based substances in Korea. Interspecies comparison in the accumulation profile of persistent organic pollutants (including polychlorinated biphenyls (PCBs), organochlorine pesticides, polybrominated diphenylethers (PBDEs), and PFAAs) reveals relatively high load of PFAAs in inland (pigeons) and estuarine (egrets/herons) species compared to marine bird species, indicating wide use of PFAAs in the terrestrial environment.

Steroid hormone profiles and body conditions of migrating male humpback whales (Megaptera novaeangliae).

Simultaneous analysis of multiple steroid hormones from remotely obtained blubber biopsies has the potential to concurrently provide information regarding stress and reproductive status from free-swimming cetaceans, while also investigating correlations between hormone concentrations and other health biomarkers. In this study we measured blubber concentration profiles of eight reproductive and adrenal steroid hormones (17α-hydroxy-progesterone, testosterone, androstenedione, progesterone, cortisol, 11-deoxy-corticosterone, oestrone, and oestradiol) together with body condition, as determined by the inverse Adipocyte Index, of 101 male humpback whales. Whales were sampled randomly at two time points, while migrating to and from their northeast Australian breeding grounds, allowing for intra- and inter-seasonal profile analysis. Testosterone, progesterone and cortisol together with androstenedione 17α-hydroxyprogesterone, and oestrone concentrations (the latter quantified for the first time in live biopsied male humpback whales) decreased between the northward and southward migrations. Decreasing testosterone levels during the height of humpback whale conceptions suggests asynchronicity between blubber testosterone levels and the expected peak of male fertility. Statistically significant relationships between levels of certain steroid analytes were observed and appeared to change between the early and late breeding seasons. During the northward migration, testosterone, progesterone, androstenedione, oestrone and 17α-hydroxyprogesterone levels were positively correlated. Cortisol concentrations correlated positively with those of testosterone during the northward migration, but negatively during the southward migration. Androstenedione and testosterone were positively correlated with adiposity during the late breeding season. These hormone-hormone and hormone-adiposity correlations may be reflective of the activation of certain steroid hormone synthesis pathways, or alternatively, of concomitant physiological stimuli. As steroid hormones work in concert, information on multiple steroid hormones is needed to interpret endocrinological status and understand the relationships between these compounds and ancillary health markers. This study provides steroid hormone profiles of wild male humpback whales, as well as the first insight into seasonal male endocrinology as a function of adiposity.

Naturally occurring organobromine compounds (OBCs) including polybrominated dibenzo-p-dioxins in the marine sponge Hyrtios proteus from The Bahamas.

Halogenated natural products (HNPs) were identified from organic extracts of the marine sponge Hyrtios proteus from The Bahamas using gas chromatography with electron capture negative ion mass spectrometry and non-targeted gas chromatography with electron ionization mass spectrometry. The HNPs found have similar properties to anthropogenic persistent organic pollutants (POPs). Two ortho-methoxy brominated diphenyl ethers (MeO-BDEs) 2'-MeO-BDE 68 and 6-MeO-BDE 47 were the most abundant compounds. Fourteen other MeO-BDEs were detected along with several polybrominated dibenzo-p-dioxins (PBDDs) (1,3,7-triBDD, 1,3,6,8-tetraBDD and 1,3,7,9-tetraBDD) and MeO-PBDDs. Further analysis of a higher trophic level octopus (Octopus maya) from the same FAO fishing area showed that the major HNPs detected in Hyrtios proteus were also predominant. Moreover, HNPs were more than 30-fold higher in abundance than the major POPs in the octopus, i.e., polychlorinated biphenyls. Hence, Caribbean marine organisms, including those potentially used for food, harbor relatively high concentrations of HNPs.

Maternal Food and Beverage Consumption Behaviors and Discrepant Phthalate Exposure by Race.

Background: Differential exposure to endocrine-disrupting chemicals, including phthalate diesters, may contribute to persistent racial/ethnic disparities in women's reproductive health outcomes. We sought to characterize sources of gestational exposure to these agents that may differ according to maternal race. Methods: We enrolled pregnant Black (n = 198), including African American, and White (n = 197) women during the second trimester, and measured eight phthalate monoester metabolites in urine. We assessed confounder-adjusted associations between multiple food and beverage consumption habits, summarized using a principal component analysis, as predictors of maternal urinary phthalate metabolite levels, stratified by race. Results: Whites reported significantly greater unprocessed food consumption (42.5% vs. 32.0%; p < 0.001) and storage of food in clear unbreakable plastic containers (66.5% vs. 49.3%; p < 0.001) than Blacks, while Blacks consumed more canned fruits and vegetables (23.5% vs. 12.2%; p < 0.001) than Whites. Using plastics for food storage, microwaving in plastic containers, and using hard plastic water bottles was associated with urinary phthalate concentrations, especially DEHP metabolites (e.g., mean difference = 5.13%; 95% CI: 3.05, 7.25). These associations were driven primarily by Black pregnant women. Conclusions: Targeted interventions to reduce maternal exposure to phthalates need to be designed with specific attention to differences in food and beverage consumption behaviors among Black and White women.

Association between gestational phthalate exposure and newborn head circumference; impacts by race and sex.

Observational and experimental studies report associations between gestational phthalate exposure and fetal development, yet few data exist to characterize phthalate effects on head circumference (HC) or to estimate the impact of race or sex. To address this data gap, we enrolled 152 African American and 158 white mothers with uncomplicated singleton pregnancies from the Charleston, South Carolina (USA) metropolitan area in a prospective birth cohort. Study participants provided up to two urine specimens during mid and late gestation, completed a study questionnaire, and allowed access to hospital birth records. We measured eight phthalate monoester metabolites using liquid chromatography with tandem mass spectrometry, and calculated molar sums of phthalate parent diesters. After specific gravity correction, we tested for associations between phthalates and neonatal HC (cm) and cephalization index (cm/g) using multiple informant linear regression with inverse probability weighting to account for selection bias between repeated urine sampling, adjusted for maternal race, age, body mass index, education, and smoking. We explored interactions by maternal race and infant sex. A doubling of urinary monoethyl phthalate (MEP) concentration was associated with a -0.49% (95%CI: -0.95%, -0.02%) smaller head circumference, although seven other phthalate metabolites were null. There were no statistically significant associations with cephalization index. HC was larger for whites than African American newborns (p < 0.0001) but similar for males and females (p = 0.16). We detected interactions for maternal race with urinary monobutyl phthalate (MBP; p = 0.03), monobenzyl phthalate (MBzP; p = 0.01), monoethylhexyl phthalate (MEHP; p = 0.05), monomethyl phthalate (MMP; p = 0.02), and the sum of dibutyl phthalate metabolites (∑DBP; p = 0.05), in which reduced HC circumference associations were stronger among whites than African Americans, and interactions for sex with MBP (p = 0.08) and MiBP (p = 0.03), in which associations were stronger for females than males. Our results suggest that gestational phthalate exposure is associated with smaller neonatal HC and that white mothers and female newborns have greater susceptibility.

Biomonitoring of emerging DINCH metabolites in pregnant women in charleston, SC: 2011-2014.

Due to the mounting evidence that phthalates, specifically di-2-ethylhexyl phthalate and dibutyl phthalate, produce adverse endocrine effects in humans and wildlife, the use of other chemicals as replacements has increased. One of the most commonly encountered phthalate replacements is di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH). Currently, little is known about the prevalence of human exposure, bioactivity, and endocrine disrupting potential of DINCH. We sampled urine from 100 pregnant women during the second trimester of pregnancy living in Charleston, SC between 2011 and 2014 and measured the following DINCH metabolites by LC-MS/MS: cyclohexane-1,2-dicarboxylic acid-mono(hydroxy-isononyl) ester (OH-MINCH), cyclohexane-1,2-dicarboxylic acid-mono(oxo-isononyl) ester (oxo-MINCH), and cyclohexane-1,2-dicarboxylic acid-monocarboxy isooctyl ester (cx-MINCH). These metabolites were also tested on human estrogen receptor alpha and progesterone receptor beta transactivation assays in vitro. OH-MINCH was detected in 98% of urine samples. The specific gravity-adjusted median (interquartile range) OH-MINCH concentration was 0.20 (0.25) ng/mL, and concentrations were significantly higher in African American women compared to Caucasian women (p = 0.01). DINCH metabolite concentrations were consistent between years, and they did not exhibit estrogenic or progestogenic activity in vitro. Human exposure to these emerging compounds should continue to be monitored, especially in vulnerable populations, to ensure the replacement of phthalates by DINCH is not a case of regrettable substitution.

Androstenedione and testosterone but not progesterone are potential biomarkers of pregnancy in Humpback Whales (Megaptera novaeangliae) approaching parturition.

The blubber steroid hormone profiles of 52 female humpback whales migrating along the east coast of Australia were investigated for seasonal endocrine changes associated with reproduction. Individuals were randomly sampled during two stages of the annual migration: before reaching the breeding grounds (northward migration; June/July), and after departing from the breeding grounds (southward migration; September/October). Assignment of reproductive status of the sampled individuals was based on season, single-hormone ranks and multi-variate analysis of the hormonal profiles. High concentrations of progesterone (>19 ng/g, wet weight), recognised as an indicator of pregnancy in this species, were only detected in one sample. However, the androgens, testosterone and androstenedione were measured in unusually high concentrations (1.6-12 and 7.8-40 ng/g wet weight, respectively) in 36% of the females approaching the breeding grounds. The absence of a strong accompanying progesterone signal in these animals raises the possibility of progesterone withdrawal prior to parturition. As seen with other cetacean species, testosterone and androstenedione could be markers of near-term pregnancy in humpback whales. Confirmation of these androgens as alternate biomarkers of near-term pregnancy would carry implications for improved monitoring of the annual fecundity of humpback whales via non-lethal and minimally invasive methods.

Remote blubber sampling paired with liquid chromatography tandem mass spectrometry for steroidal endocrinology in free-ranging bottlenose dolphins (Tursiops truncatus).

Liquid chromatography tandem mass spectrometry allows for the measurement of steroid hormone suites in the blubber of marine mammals. By combining this technology with minimally invasive techniques such as remote biopsy, endocrine profiles can be assessed, allowing for studies of hormonal profile variation over time. In this study, we explored associations among different steroidogenic pathways and seasonal differences in blubber hormone profiles of free-ranging common bottlenose dolphins along the coast of South Carolina, USA. Male dolphins experience a peak in testosterone, androstenedione, progesterone, and 17-hydroxyprogesterone in the spring, likely related to an upregulation of the androgen steroidogenic pathway during mating season. We also observed increased cortisol concentrations during summer compared to winter. Among females, there was an increase in androstenedione with elevated progesterone concentrations indicative of pregnancy, highlighting another potential endocrine marker for pregnancy in free-ranging dolphins. This work emphasizes the importance of selecting the appropriate season for studies on endocrine status to effectively uncover physiological variation or disruption in free-ranging cetaceans.

Racial disparity in maternal phthalates exposure; Association with racial disparity in fetal growth and birth outcomes.

Experimental and observational data implicate phthalates as developmental toxicants. However, few data are available to assess the maternal risks of gestational exposure by race and infant sex. To begin to address this data gap, we characterized associations between maternal urinary phthalate metabolites and birth outcomes among African American and white mothers from a southeastern U.S. population. We enrolled pregnant African American (n = 152) and white (n = 158) women with singleton live births between 18 and 22 weeks gestation. We measured phthalate metabolites (mono-n-butyl phthalate (MBP), monoisobutyl phthalate (MiBP), monobenzyl phthalate (MBzP), mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP), monoethyl phthalate (MEP), monomethyl phthalate (MMP), and the sums of DEHP (ΣDEHP) and DBP (ΣDBP) metabolites) in up to two gestational urine specimens from mothers, and evaluated confounder-adjusted associations per natural log unit greater concentration with birth weight for gestational age z-score, small for gestational age (SGA; <10th %tile), preterm birth (PTB; <37 weeks gestation), and low birth weight (LBW; <2500 g). We also tested for interactions by maternal race and infant sex. We found that lower z-scores were associated with greater MiBP (ß = -0.28; 95% CI: -0.54, -0.02) and MMP (ß = -0.30; 95% CI: -0.52, -0.09) concentrations, while MEP interacted with race (p = 0.04), indicating an association among whites (ß = -0.14; 95% CI: -0.28, 0.001) but not among African Americans (ß = 0.05; 95% CI = -0.09, 0.19). Greater MiBP (OR = 2.82; 95% CI: 1.21, 6.56) and MEOHP (OR = 2.80; 95% CI: 1.05, 7.42) were associated with an overall higher SGA risk, greater MEHP was associated with higher SGA risk (p = 0.10) in whites (OR = 3.26 95% CI: 0.64, 16.56) but not in African Americans (OR = 0.71 95% CI: 0.07, 7.17), and the associations for MiBP (p = 0.02) and ΣDBP (p = 0.02) varied by infant sex. We detected interactions for PTB in which African Americans were at higher risk than whites for greater MiBP (p = 0.08) and MEP (p = 0.02) although lower risk for greater MEHP (p = 0.09). Greater MEP was associated with an overall higher LBW risk (OR = 1.33; 95% CI: 0.95, 1.86), and males were at higher risk than females with greater MBP (p = 0.002), MiBP (p = 0.02), MBzP (p = 0.01), MEP (p = 0.002), MMP (p = 0.09), and ΣDBP (p = 0.01) concentrations. Overall, our results suggest that gestational phthalate exposure is associated with adverse maternal birth outcomes, and that the effects vary by maternal race and infant sex.

Environmental contaminants in coastal populations: Comparisons with the National Health and Nutrition Examination Survey (NHANES) and resident dolphins.

BACKGROUND: People living in coastal communities are at risk for exposure to environmental hazards, including legacy chemicals. We can use databases such as NHANES to assess whether contaminants in coastal communities are present in higher levels than in the United States overall. We can use information from studies of local animal populations to assess which of these contaminants could have been transferred to people from their shared environment. OBJECTIVE: Our objectives were to examine the POP profiles in human populations in areas where there are published POP profiles in resident dolphins and to compare our results with data from NHANES and the dolphin studies. METHODS: We identified three areas where POPs have been analyzed in local resident dolphin populations (total N =73). We identified human communities in the same areas, and asked 27 eligible adults to read and sign a consent form, complete a questionnaire about demographics and seafood consumption, provide nine 10-mL blood samples, and provide one sample of seafood (N = 33). Blood and seafood were analyzed for a suite of POPs similar to those analyzed in published dolphin population studies. We compared the results from human blood analyses with NHANES and with data from the published reports of dolphin studies. RESULTS: Levels and proportions of specific POPs found in people and animals reflect POPs found in the local environment. Compared with the nationally representative data reported in NHANES, the levels of many POPs found in high levels in dolphins were also higher in the corresponding human communities. CONCLUSIONS: Contaminants measured in marine animals, such as dolphins, can be used to identify the types and relative levels of environmental contaminants expected to occur in people sharing the same environment. Likewise, contaminants measured in coastal human populations can provide insight into which contaminants may be found in nearby animal populations.

Liquid chromatography tandem mass spectrometry for the quantification of steroid hormone profiles in blubber from stranded humpback whales (Megaptera novaeangliae).

Marine mammal blubber is known to have quantifiable concentrations of steroid hormones and is increasingly chosen as a matrix for the detection of these reproductive and stress biomarkers. Steroid hormones act through complex cascades, often in concert, yet studies conducted on cetaceans have rarely measured more than two steroids simultaneously. Due to the role of steroid hormones in multiple physiological processes, and variability in concentration among individuals, data on single compounds are often difficult to interpret. Here a liquid chromatography tandem mass spectrometry method for the simultaneous analyses of multiple steroid hormones in cetacean blubber was validated and applied to samples from 10 stranded humpback whales (Megaptera novaeangliae). Progesterone, 17α-hydroxyprogesterone, testosterone, androstenedione, oestrone, oestradiol, cortisone, cortisol, corticosterone and 11-deoxycorticosterone were reliably (relative standard deviation on six replicates <15%) and accurately (recovery of an amended sample between 70% and 120%) quantified, but not 11-deoxycortisol. With the exception of progesterone, testosterone, oestradiol and cortisol, these compounds were quantified for the first time in humpback whales. Given that blubber is frequently collected from free-swimming cetaceans in ongoing research programs, the technique developed here could substantially strengthen understanding and monitoring of the physiological condition of these species.

Temporal trends of persistent organic pollutants in Arctic marine and freshwater biota.

More than 1000 time-series of persistent organic pollutants (POPs) in Arctic biota from marine and freshwater ecosystems some extending back to the beginning of 1980s were analyzed using a robust statistical method. The Arctic area encompassed extended from Alaska, USA in the west to northern Scandinavian in the east, with data gaps for Arctic Russia and Arctic Finland. The aim was to investigate whether temporal trends for different animal groups and matrices were consistent across a larger geographical area. In general, legacy POPs showed decreasing concentrations over the last two to three decades, which were most pronounced for α-HCH and least pronounced for HCB and ß-HCH. Few time-series of legacy POPs showed increasing trends and only at sites suspected to be influenced by local source. The brominated flame retardant congener BDE-47 showed a typical trend of increasing concentration up to approximately the mid-2000s followed by a decreasing concentration. A similar trend was found for perfluorooctane sulfonic acid (PFOS). These trends are likely related to the relatively recent introduction of national and international controls of hexa- and hepta-BDE congeners and the voluntary phase-out of PFOS production in the USA in 2000. Hexabromocyclododecane (HBCDD) was the only compound in this study showing a consistent increasing trend. Only 12% of the long-term time-series were able to detect a 5% annual change with a statistical power of 80% at α < 0.05. The remaining 88% of time-series need additional years of data collection before fulfilling these statistical requirements. In the case of the organochlorine long-term time-series, 45% of these would require >20 years monitoring before this requirement would be fulfilled.

Skin Transcriptomes of common bottlenose dolphins (Tursiops truncatus) from the northern Gulf of Mexico and southeastern U.S. Atlantic coasts.

Common bottlenose dolphins serve as sentinels for the health of their coastal environments as they are susceptible to health impacts from anthropogenic inputs through both direct exposure and food web magnification. Remote biopsy samples have been widely used to reveal contaminant burdens in free-ranging bottlenose dolphins, but do not address the health consequences of this exposure. To gain insight into whether remote biopsies can also identify health impacts associated with contaminant burdens, we employed RNA sequencing (RNA-seq) to interrogate the transcriptomes of remote skin biopsies from 116 bottlenose dolphins from the northern Gulf of Mexico and southeastern U.S. Atlantic coasts. Gene expression was analyzed using principal component analysis, differential expression testing, and gene co-expression networks, and the results correlated to season, location, and contaminant burden. Season had a significant impact, with over 60% of genes differentially expressed between spring/summer and winter months. Geographic location exhibited lesser effects on the transcriptome, with 23.5% of genes differentially expressed between the northern Gulf of Mexico and the southeastern U.S. Atlantic locations. Despite a large overlap between the seasonal and geographical gene sets, the pathways altered in the observed gene expression profiles were somewhat distinct. Co-regulated gene modules and differential expression analysis both identified epidermal development and cellular architecture pathways to be expressed at lower levels in animals from the northern Gulf of Mexico. Although contaminant burdens measured were not significantly different between regions, some correlation with contaminant loads in individuals was observed among co-expressed gene modules, but these did not include classical detoxification pathways. Instead, this study identified other, possibly downstream pathways, including those involved in cellular architecture, immune response, and oxidative stress, that may prove to be contaminant responsive markers in bottlenose dolphin skin.

Urinary Phthalate Metabolites in Common Bottlenose Dolphins (Tursiops truncatus) From Sarasota Bay, FL, USA.

Phthalates are chemical additives to common consumer goods including cleaning products, cosmetics, personal care products, and plastic. Because they are not chemically bound to these products and are widely used, the potential for environmental contamination is significant. Phthalates and their metabolites have been associated with endocrine disruption and reproductive impairment, among other adverse health effects, in laboratory animals and human epidemiologic studies. Common bottlenose dolphins (Tursiops truncatus) are vulnerable to environmental pollutants due to their apex position in the food chain, long life spans, and habitat overlap with developed coastal areas. The objective of this study was to quantify phthalate metabolite concentrations in urine collected from bottlenose dolphins in Sarasota Bay, Florida, during May 2016 (n = 7) and May 2017 (n = 10). Screening of nine phthalate monoester metabolites in bottlenose dolphin urine was performed by liquid chromatography tandem mass spectrometry using methods adapted from those used for analyzing human samples. At least one phthalate metabolite was detected in 71% of the dolphins sampled across both years, with the highest concentrations detected for monoethyl phthalate (MEP; GM = 5.4 ng/ml; 95%CI: 1.3-22.0 ng/ml) and mono-(2-ethylhexyl) phthalate (MEHP; GM = 1.9 ng/ml; 95%CI: 1.1-3.2 ng/ml). These data demonstrate exposure to two of the most commonly used phthalates in commercial manufacturing, diethyl phthalate (DEP) and di-2-ethylhexyl phthalate (DEHP). This study establishes methods for urinary detection of phthalate metabolites in marine mammals and provides baseline data to address a significant and growing, yet poorly understood, health threat to marine wildlife.

Influence of race on prenatal phthalate exposure and anogenital measurements among boys and girls.

BACKGROUND: Select phthalates have antiandrogenic activity, which raises concern for adverse developmental outcomes given widespread exposure of pregnant women. Investigators have reported associations between maternal urinary phthalates and altered anogenital distance (AGD), a marker of in utero androgen activity, among offspring. However, data assessing the impact of race on these associations is sparse. OBJECTIVES: To evaluate associations between prenatal phthalate exposure and AGD in a racially diverse newborn population. METHODS: We prospectively collected second trimester urine from 187 African American and 193 white mothers, and used liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to measure eight phthalate metabolites and calculate molar sums. We measured anopenile (APD) and anoscrotal (ASD) distances of 171 boys and anoclitoral (ACD) and anofourchette (AFD) distances of 128 girls at delivery. We collected sociodemographic and clinical data from questionnaires and delivery records. RESULTS: We identified a statistically significant inverse association for mono-2-ethylhexyl phthalate (MEHP) and APD in boys (B=-1.57mm, p=0.02), which was stronger for African Americans (B=-2.07mm, p=0.04) than for whites (B=-1.23mm, p=0.22), although the racial interaction was not statistically significant (p=0.56). We found a longer ASD for higher molar sums of dibutyl phthalate (∑DBP; B=0.99mm, p=0.04), with stronger associations for whites (B=1.30mm, p=0.04) than for African Americans (B=0.39mm, p=0.59), again without a statistically significant racial interaction (p=0.34). Among girls, we found inverse associations for tertiles of MEHP with AFD and ACD, and statistically significant race-based interactions, in which ACD was longer for whites and shorter for African Americans, following exposure to monoethyl phthalate (MEP; p=0.01) and ∑DBP (p=0.08). CONCLUSIONS: Our findings suggest race and sex play important roles in phthalate-associated reproductive developmental toxicity, with important implications for designing future investigations and health interventions.

Prevalence and predictors of phthalate exposure in pregnant women in Charleston, SC.

Phthalates are plasticizers commonly detected in human urine due to widespread exposure from PVC plastics, food packaging, and personal care products. Several phthalates are known antiandrogenic endocrine disruptors, which raises concern for prenatal exposure during critical windows of fetal development. While phthalate exposure is ubiquitous, certain demographics are subject to greater or lesser exposure. We sampled urine from 378 pregnant women during the second trimester of gestation living in Charleston, SC, and measured eight urinary phthalate metabolites as biomarkers of phthalate exposure: monobutyl phthalate (MBP), monobenzyl phthalate (MBzP), mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), monoethyl phthalate (MEP), monoisobutyl phthalate (MiBP), and monomethyl phthalate (MMP). Demographic data was collected from questionnaires administered at the time of specimen collection. All phthalate metabolites were detected in over 93% of urine samples. On average, concentrations were highest for MEP (median = 47.0 ng/mL) and lowest for MMP (median = 1.92 ng/mL). Sociodemographic characteristics associated with elevated phthalate concentrations included being unmarried, less educated, having a low income, high body mass index (BMI), and/or being African American. After racial stratification, age, BMI, education, and income were significantly associated with phthalate concentrations in African American women. Marital status was associated with phthalate concentrations in Caucasian women only, with greater concentrations of MBP, MEHHP, MiBP, and MMP in unmarried versus married women. Results of this cross-sectional study provide evidence for significant racial and demographic variations in phthalate exposure.

Rapid and reliable steroid hormone profiling in Tursiops truncatus blubber using liquid chromatography tandem mass spectrometry (LC-MS/MS).

Monitoring of marine mammal steroid hormone status using matrices alternative to blood is desirable due to the ability to remotely collect samples, which minimizes stress to the animal. However, measurement techniques in alternative matrices such as blubber described to date are limited in the number and types of hormones measured. Therefore, a new method using bead homogenization to QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction, C18 post extraction cleanup and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and applied to the measurement of hormone suites in bottlenose dolphin blubber. Validations were conducted in blubber from fresh dead stranded bottlenose dolphin. The final method consisting of two LC separations and garnet bead homogenization was tested for extraction efficiencies. Steroids were separated using a biphenyl column for reproductive hormones and C18 column for corticosteroids. Three hormones previously noted in blubber, testosterone, progesterone, and cortisol, were quantified in addition to previously unmeasured androstenedione, 17-hydroxyprogesterone, 11-deoxycortisol, 11-deoxycorticosterone, and cortisone in a single sample (0.4 g blubber). Extraction efficiencies of all hormones from blubber ranged from 84% to 112% and all RSDs were comparable to those reported using immunoassay methods (< 15%). The method was successfully applied to remote biopsied blubber samples to measure baseline hormone concentrations. Through this method, increased coverage of steroid hormone pathways from a single remotely collected sample potentially enhances the ability to interpret biological phenomena such as reproduction and stress in wild dolphin populations. Graphical abstract The steroid hormone profile is quantifiable from a single sample of bottlenose dolphin blubber using liquid chromatography tandem mass spectrometry. This profile can be applied to remotely collected dart biopsies and be used to determine reproductive or stress status of a wild-living dolphin.