Urinary Phthalate Metabolite Concentrations in Common Bottlenose Dolphins (Tursiops truncatus) Under Professional Care

Ammonium urate nephrolithiasis frequently develops in common bottlenose dolphins (Tursiops truncatus) managed under human care but is rare in free-ranging common bottlenose dolphins. In other species, the dietary cation–anion difference (DCAD) can affect ammonium urate urolith formation by increasing proton excretion as ammonium ions. Therefore, differences in diet between the 2 dolphin populations could affect urolith formation, but the DCAD of most species consumed by free-ranging and managed dolphins is unknown. To compare the nutrient composition of diets consumed by free-ranging and managed bottlenose dolphins, samples (n = 5) of the 8 species of fish commonly consumed by free-ranging bottlenose dolphins in Sarasota Bay, FL, and the 7 species of fish and squid commonly fed to managed bottlenose dolphins were analyzed for nutrient content. Metabolizable energy was calculated using Atwater factors; the DCAD was calculated using 4 equations commonly used in people and animals that use different absorption coefficients. The nutrient composition of individual species was used to predict the DCAD of 2 model diets typically fed to managed common bottlenose dolphins and a model diet typically consumed by common bottlenose dolphins in Sarasota Bay. To mimic differences in postmortem handling of fish for the 2 populations of bottlenose dolphins, “free-ranging” samples were immediately frozen at −80°C and minimally thawed before analysis, whereas “managed” samples were frozen for 6 to 9 mo at −18°C and completely thawed. “Free-ranging” species contained more Ca and P and less Na and Cl than “managed” fish and squid species. As a consequence, the DCAD of both model managed dolphin diets obtained using 3 of the 4 equations was much more negative than the DCAD of the model free-ranging bottlenose dolphin diet (P < 0.05). The results imply that managed bottlenose dolphins must excrete more protons in urine than free-ranging bottlenose dolphins, which will promote nephrolith formation. The nutrient composition of the free-ranging bottlenose dolphin diet, determined for the first time here, can be used as a guide for feeding managed bottlenose dolphins, but research in vivo is warranted to determine whether adding more cations to the diet will prevent urolith formation in managed dolphins.

Signature whistle use by temporarily restrained and free-swimming bottlenose dolphins, Tursiops truncatus

TPS 632: Health effects of flame retardants and plasticizers, Johan Friso Foyer, Floor 1, August 27, 2019, 3:00 PM - 4:30 PM Background: Urinary phthalate metabolites and psychological stress in pregnancy have been individually associated with preterm birth (PTB), but no study has examined the joint effect of these two environmental exposures. We hypothesized that there would be stronger associations between phthalate exposure and PTB in mothers with higher stress in pregnancy compared to mothers with lower stress. Methods: We addressed this question using data from The Infant Development and the Environment Study (TIDES), a prospective birth cohort conducted at four US sites (N=785). Urinary phthalate metabolite concentrations were measured in samples collected in the first and third trimesters of pregnancy. Stressful life events (SLE) during gestation were assessed by a questionnaire administered in the third trimester. PTB was defined as delivery before 37 weeks completed gestation (n=71). We examined associations between urinary phthalate metabolite concentrations at each visit in association with PTB using logistic regression models adjusted for maternal race, age, pre-pregnancy body mass index, specific gravity, and gestational age at sample collection. Results: Summed di-2-ethylhexyl phthalate (ΣDEHP) metabolites measured in urine samples from the third trimester, but not the first trimester, were associated with an increased odds ratio (OR) of PTB (OR=1.38, 95% confidence interval [CI] =1.01, 1.89). In models stratified by SLE, associations between third trimester ΣDEHP concentrations and PTB were significant only for women experiencing 1 or more SLE during pregnancy (OR: 2.05, 95% CI: 1.28, 3.29) but not for women with no SLE during pregnancy (OR: 0.99, 95% CI: 0.63, 1.55); the interaction was marginally significant (p=0.05). Conclusions: We observed an association between urinary ΣDEHP levels and PTB that was modified by psychological stress in pregnancy. Additional research to understand the joint effects of chemical as well as non-chemical exposures is necessary in order to advance the state of the science on how the environment impacts pregnancy.

Developmental evidence for foraging traditions in wild bottlenose dolphins

For the first time we report on an aggressive interaction between wild bottlenose dolphins (Tursiops truncatus) and estuarine dolphins (Sotalia guianensis) observed in Baia Norte, southern Brazil. Three bottlenose dolphins aggressively herded a Sotalia guianensis calf, which was the main target of the aggressive and threatening behaviours of the bottlenose dolphins. Another two to four adult S. guianensis were involved in the interaction and were constantly chased by the bottlenose dolphins. After approximately two hours, the bottlenose dolphins left the calf and the area, and no dead or wounded animals were seen afterwards. We provide a detailed spatial and temporal description of the interaction, and discuss the potential causes of this event.

We examined the seasonality of reproduction in captive and wild bottlenose dolphins, Tursiops truncatus . Stranding records of neonatal dolphins and observed births from a long-term field study were used to estimate peak periods of birth for wild populations, and information from captive-breeding colonies was used to assess the effects of latitude and origin of breeding females on timing of birth for dolphins in captivity. Bottlenose dolphins showed diffuse peaks and considerable flexibility in their seasonality of reproduction. There was no relationship between latitude and seasonality of reproduction or synchrony of births. However, there was a correlation between origin of population and seasonality of reproduction in both wild and captive dolphins. We suggest that adaptations to local environmental conditions have the strongest influence on seasonality of reproduction in these populations of bottlenose dolphins.

Occurrence of triclosan in plasma of wild Atlantic bottlenose dolphins ( Tursiops truncatus) and in their environment

Stroke is a significant global health issue, resulting in substantial mortality and morbidity. Environmental exposures are increasingly recognized as essential contributors to cerebrovascular risk profiles. Phthalates are ubiquitous synthetic plasticizers with well-established endocrine-disrupting properties and have been associated with various vascular and metabolic disturbances. However, their specific contribution to stroke pathogenesis, particularly concerning potential sex-specific vulnerability patterns, remains poorly understood. A cross-sectional analysis was conducted on 8184 participants (4140 women and 4044 men) from the US National Health and Nutrition Examination Survey to evaluate the associations between urinary phthalate metabolite concentrations and stroke prevalence. The weighted quantile sum (WQS) regression methodology assessed the effects of cumulative exposure to the phthalate mixture. An integrative approach was used, incorporating network toxicology, protein-protein interaction mapping, and the Friends algorithm, to identify molecular targets related to stroke that may be affected by phthalate exposure. Molecular docking analyses were performed to characterise binding affinities between key phthalate metabolites and identified protein targets. We analyzed single-cell RNA sequencing data to determine cell-type-specific expression patterns of implicated molecular targets. Sex-stratified analyses revealed that phthalate exposure was positively associated with stroke risk in women but not in men. Specifically, higher urinary concentrations of mono(carboxynonyl) phthalate (MCNP) were significantly associated with self-reported stroke in women. WQS regression confirmed cumulative effects, with MCNP identified as the primary contributor. Network and docking analyses revealed strong interactions between MCNP and vascular proteins KDR, AKT1, and MAPK8, which were predominantly expressed in endothelial cells. These findings suggest that phthalate exposure may increase stroke risk in a sex-specific manner through disruption of an endothelial-specific KDR-AKT1-MAPK8 signaling pathway. This work highlights a novel mechanism of environmentally mediated cerebrovascular risk and provides potential targets for therapeutic intervention, particularly in women.

Are early-pregnancy urinary bisphenol and phthalate metabolite concentrations associated with placental function markers, blood pressure (BP) trajectories during pregnancy and risk of gestational hypertensive disorders? Early-pregnancy bisphenols and phthalate metabolites were not consistently associated with maternal BP changes or gestational hypertensive disorders, but subclinical, statistically significant associations with placental angiogenic markers and placental hemodynamics were identified. In vitro studies suggest that bisphenols and phthalate metabolites may disrupt early placental development and affect the risk of gestational hypertensive disorders. Previous studies investigating effects of bisphenols and phthalate metabolites on gestational hypertensive disorders reported inconsistent results and did not examine placental function or BP throughout pregnancy. In a population-based prospective cohort study, bisphenol and phthalate metabolite concentrations were measured in a spot urine sample in early pregnancy among 1396 women whose children participated in postnatal follow-up measurements. After exclusion of women without any BP measurement or with pre-existing hypertension, 1233 women were included in the analysis. Urinary bisphenol and phthalate metabolite concentrations were measured in early-pregnancy [median gestational age 13.1 weeks, inter-quartile range 12.1-14.5]. Molar sums of total bisphenols and of low molecular weight phthalate, high molecular weight (HMW) phthalate, di-2-ethylhexylphthalate, and di-n-octylphthalate metabolites were calculated. Placental angiogenic markers (placental growth factor (PlGF), soluble fms-like tyrosine kinase (sFlt)-1), placental hemodynamic function measures (umbilical artery pulsatility index (PI), uterine artery resistance index (RI), notching and placental weight), and maternal BP were measured in different trimesters. Information on gestational hypertensive disorders was obtained from medical records. Each log unit increase in HMW phthalate metabolites was associated with a 141.72 (95% CI: 29.13, 373.21) higher early pregnancy sFlt-1/PlGF ratio (range in total sample 9-900). This association was driven by mono-[(2-carboxymethyl)hexyl]phthalate. In the repeated measurements regression models, each log unit increase in bisphenol A was associated with a 0.15 SD (95% CI: 0.03, 0.26) higher intercept and -0.01 SD (95% CI: -0.01, -0.00) decreasing slope of the umbilical artery PI Z-score and a -1.28 SD (95% CI: -2.24, -0.33) lower intercept and 0.06 SD (95% CI: 0.02, 0.11) increasing slope of the uterine artery RI Z-score. These associations remained significant after Bonferroni correction. Early-pregnancy bisphenols or phthalate metabolites showed no consistent associations with any other outcome. Information on a large number of potential confounders was available but was partly self-reported. Bisphenols and phthalate metabolites, which typically have a half-life of 24-48 h, were measured via single spot urine samples in early-pregnancy. In addition, at the current sample size, the study was powered to detect an odds ratio of 1.57 for gestational hypertension and 1.78 for pre-eclampsia, but was underpowered to perform multivariable analyses for these outcomes. Further studies combining data from different cohorts may be necessary to increase power. These limitations are possible sources of non-differential misclassification leading to bias toward the null. Bisphenols and phthalate metabolites were not associated with longitudinal changes in BP in pregnancy in our low-risk population. The observed subclinical associations of phthalates with the sFlt-1/PlGF ratio and of bisphenol A with placental hemodynamics may contribute to adverse pregnancy outcomes. Our results are therefore more supportive of an association of early pregnancy bisphenols and phthalate metabolites with risk for pre-eclampsia than with gestational hypertension. This analysis was supported by Grant (ES022972) from the National Institutes of Health, USA. The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health. The authors report no conflicts of interest.

Are preconception phthalate and phthalate replacements associated with sperm differentially methylated regions (DMRs) among men undergoing IVF? Ten phthalate metabolites were associated with 131 sperm DMRs that were enriched in genes related to growth and development, cell movement and cytoskeleton structure. Several phthalate compounds and their metabolites are known endocrine disrupting compounds and are pervasive environmental contaminants. Rodent studies report that prenatal phthalate exposures induce sperm DMRs, but the influence of preconception phthalate exposure on sperm DNA methylation in humans is unknown. An exploratory cross-sectional study with 48 male participants from the Sperm Environmental Epigenetics and Development Study (SEEDS). The first 48 couples provided a spot urine sample on the same day as semen sample procurement. Sperm DNA methylation was assessed with the HumanMethylation 450 K array. Seventeen urinary phthalate and 1,2-Cyclohexane dicarboxylic acid diisononyl ester (DINCH) metabolite concentrations were measured from spot urine samples. The A-clust algorithm was employed to identify co-regulated regions. DMRs associated with urinary metabolite concentrations were identified via linear models, corrected for false discovery rate (FDR). Adjusting for age, BMI, and current smoking, 131 DMRs were associated with at least one urinary metabolite. Most sperm DMRs were associated with anti-androgenic metabolites, including mono(2-ethylhexyl) phthalate (MEHP, n = 83), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP, n = 16), mono-n-butyl phthalate (MBP, n = 22) and cyclohexane-1,2-dicarboxylic acid-monocarboxy isooctyl (MCOCH, n = 7). The DMRs were enriched in lincRNAs as well as in regions near coding regions. Functional analyses of DMRs revealed enrichment of genes related to growth and development as well as cellular function and maintenance. Finally, 13% of sperm DMRs were inversely associated with high quality blastocyst-stage embryos after IVF. Our modest sample size only included 48 males and additional larger studies are necessary to confirm our observed results. Non-differential misclassification of exposure is also a concern given the single spot urine collection. To our knowledge, this is the first study to report that preconception urinary phthalate metabolite concentrations are associated with sperm DNA methylation in humans. These results suggest that paternal adult environmental conditions may influence epigenetic reprogramming during spermatogenesis, and in turn, influence early-life development. This work was supported by grant K22-ES023085 from the National Institute of Environmental Health Sciences. The authors declare no competing interests.

Phthalates are endocrine-disrupting chemicals that are commonly used in plastic consumer products and food packaging, with growing evidence suggesting that they have a potential role in obesity. This study aimed to investigate the association between urinary concentrations of phthalate metabolites and both general and abdominal obesity among adult males in Spain. We analysed data from 1124 male participants of the Aragon Workers' Health Study (AWHS) collected between 2011 and 2014 in Zaragoza, Spain. Eleven urinary phthalate metabolites were measured and adjusted for creatinine levels. Multivariate logistic regression models were used to evaluate associations between phthalate exposure and general and abdominal obesity, controlling for dietary and lifestyle factors. Dose-response relationships were explored using restricted cubic spline models. Higher urinary concentrations of di(2-ethylhexyl) phthalate (∑DEHP) and two of its metabolites-mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP)-were significantly associated with general obesity. The adjusted odds ratios were: ∑DEHP [OR = 1.26; 95% CI: 1.01, 1.58], MEOHP [OR = 1.24; 95% CI: 1.00, 1.53], and MEHHP [OR = 1.26; 95% CI: 1.03, 1.55]. In contrast, mono-isobutyl phthalate (MiBP) was inversely associated with abdominal obesity [OR = 0.73; 95% CI: 0.57, 0.93]. These findings suggest a positive association between exposure to DEHP and its metabolites and general obesity. This highlights the potential importance of environmental exposures as modifiable factors in obesity prevention and supports the need for further investigation in nutritional and public health contexts.

Conveying identity is important for social animals to maintain individually based relationships. Communication of identity information relies on both signal encoding and perception. Several delphinid species use individually distinctive signature whistles to transmit identity information, best described for the common bottlenose dolphin (Tursiops truncatus). In this study, we investigate signature whistle use in wild common dolphins (Delphinus delphis). Acoustic recordings were analysed from 11 encounters from three locations in South Africa (Hout Bay, False Bay, and Plettenberg Bay) during 2009, 2016 and 2017. The frequency contours of whistles were visually categorised, with 29 signature whistle types (SWTs) identified through contour categorisation and a bout analysis approach developed specifically to identify signature whistles in bottlenose dolphins (SIGID). Categorisation verification was conducted using an unsupervised neural network (ARTwarp) at both a 91% and 96% vigilance parameter. For this, individual SWTs were analysed type by type and then in a 'global' analysis whereby all 497 whistle contours were categorised simultaneously. Overall the analysis demonstrated high stereotypy in the structure and temporal production of whistles, consistent with signature whistle use. We suggest that individual identity information may be encoded in these whistle contours. However, the large group sizes and high degree of vocal activity characteristic of this dolphin species generate a cluttered acoustic environment with high potential for masking from conspecific vocalisations. Therefore, further investigation into the mechanisms of identity perception in such acoustically cluttered environments is required to demonstrate the function of these stereotyped whistle types in common dolphins.

Increased levels of phthalates in very low birth weight infants with septicemia and bronchopulmonary dysplasia

The placental transcriptome serves as a mechanistic link between prenatal phthalate exposure and placental efficiency

Environment, endocrinology, and biochemistry influence expression of stress proteins in bottlenose dolphins