The increasing presence of micro-and nano-sized plastics in the environment and food chain is of growing concern. Although mindful consumers are promoting the reduction of single-use plastics, some manufacturers are creating new plastic packaging to replace traditional paper uses, such as plastic teabags. The objective of this study was to determine whether plastic teabags could release microplastics and/or nanoplastics during a typical steeping process. We show that steeping a single plastic teabag at brewing temperature (95 °C) releases approximately 11.6 billion microplastics and 3.1 billion nanoplastics into a single cup of the beverage. The composition of the released particles is matched to the original teabags (nylon and polyethylene terephthalate) using Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The levels of nylon and polyethylene terephthalate particles released from the teabag packaging are several orders of magnitude higher than plastic loads previously reported in other foods. An initial acute invertebrate toxicity assessment shows that exposure to only the particles released from the teabags caused dose-dependent behavioral and developmental effects.

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Rapid three-dimensional imaging of embryos to better understand the complex process of morphogenesis has been challenging. Recently introduced iodine staining protocols (I 2 KI and alcoholic iodine stains) combined with microscopic X-ray computed tomography allows visualization of soft tissues in diverse small organisms and tissue specimens. I 2 KI protocols have been developed specifically for small animals, with a limited number of quantitative studies of soft tissue contrasts. To take full advantage of the low X-ray attenuation of ethanol and retain bound iodine while dehydrating the specimen in ethanol, we developed an ethanol I 2 KI protocol. We present comparative microscopic X-ray computed tomography analyses of ethanol I 2 KI and I 2 KI staining protocols to assess the performance of this new protocol to visualize soft tissue anatomy in late stage Japanese quail embryos using quantitative measurements of soft tissue contrasts and sample shrinkage. Both protocols had only 5% shrinkage compared with the original harvested specimen, supporting the use of whole mounts to minimize tissue shrinkage effects. Discrimination within and among the selected organs with each staining protocol and microscopic X-ray computed tomography imaging were comparable to those of a gray scale histological section. Tissue discrimination was assessed using calibrated computed tomography values and a new discrimination index to quantify the degree of computed tomography value overlaps between selected soft tissue regions. Tissue contrasts were dependent on the depth of the tissue within the embryos before the embryos were saturated with each stain solution, and optimal stain saturations for the entire embryo were achieved at 14 and 28 days staining for I 2 KI and ethanol I 2 KI, respectively. Ethanol I 2 KI provided superior soft tissue contrasts by reducing overstaining of fluid-filled spaces and differentially modulating staining of some tissues, such as bronchial and esophageal walls and spinal cord. Delineating the selected soft tissues using optimal threshold ranges derived from the quantitative analyses of the contrast enhancement in optimally stained embryos is possible. The protocols presented here are expected to be applicable to other organisms with modifications to staining time and contribute toward rapid and more efficient segmentation of soft tissues for three-dimensional visualization.

Avian heads are characterized as having two extensive air‐filled systems lined with epithelia; the paranasal and paratympanic sinuses. Many diverticula derived from the paratympanic sinus system are known to reticulate with each other to form a single merged pneumatic space within the adult braincase. However, the development of these complex branching and reticulating epithelia has not been examined in detail. In this study, we describe the comprehensive developmental pattern of the paratympanic sinus and its associated soft tissues in a model bird, Japanese quail (Coturnix japonica). The data are derived from three‐dimensional reconstructions based on histological sections and soft tissue enhanced micro‐CT data. Those data provide the foundation of the complex hierarchical developmental pattern of the paratympanic sinus system. Moreover, associations with other tissues help establish key morphologies that identify each pneumatic entity. This study clarifies the developmental relationships of the ventral portions of the paratympanic sinus system, the siphoneal diverticulum and marginal sinus, based on the ligaments associated with the Eustachian tube. In addition, detailed histological pneumatic morphologies reveal hitherto unknown epithelial diversity, which may be indicative of equally complex developmental processes. We use the pneumatization of the quadrate as an example to support a close relationship with vascular growth and pneumatic epithelia invasion into ossified bone. We confirm pneumatic diverticula never enter into cartilages, possibly due to the absence of vasculature in these tissues. Lastly, we use the concept of a morphogenetic tree as a tool to help present the complex developmental pattern of the paratympanic sinus system and apply it toward inferring pneumatic morphologies in a nonavian theropod braincase.

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