Microplastics in Small Waterbodies and Tadpoles from Yangtze River Delta, China

Although microplastic (MP) pollution in freshwater systems is gaining attention, our knowledge of its distribution in small waterbodies is scarce. Small waterbodies are freshwater habitats to many species, including amphibians, that are vulnerable to MP pollution. This study analyzed the distribution and characteristics of MPs in 25 small waterbodies from the Yangtze River Delta, China. MPs were detected in surface water, sediment, and tadpoles with abundances ranging from 0.48 to 21.52 items L^–1, 35.76 to 3185.33 items kg^–1, and 0 to 2.73 items individual^–1 (0 to 168.48 items g^–1), respectively. The dominant shape and polymer of MPs in water and tadpole samples were polyester (PES) fibers, and polypropylene (PP) fibers and fragments were dominant in sediment samples. In addition, MPs were primarily <0.5 mm in length in all samples. Tadpole length was positively correlated to the number of MPs detected. The abundance, shape, and polymer distribution of MPs in tadpoles resembled that of water rather than sediment, suggesting that tadpoles likely take up MPs from the surrounding water. This study demonstrated that MPs are abundant in these small waterbodies and are ingested by resident tadpoles. This may suggest a pathway of MP entry into aquatic and terrestrial food webs

Histology of retina.

<p>Each panel is a transverse section of a similar region of the retina. Paraffin embedded sections were stained with hematoxylin and eosin as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013287#s4" target="_blank">Materials and Methods</a>. Cell layers are indicated at right: GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS/OS– inner segment/outer segment of the photoreceptor cells; RPE/C, retinal pigment epithelium/cells. Arrowheads in center and right panel indicated regions where pigmented cells have been lost.</p

Quantifiable biomarkers in aging liver.

<p>(<b>A</b>) Detection of lipofuscin by autofluorescence (red channel). Nuclei were counterstained with DAPI (blue channel). (<b>B</b>) Quantification of lipofuscin deposits per field. Number of deposits per field was scored manually and areas were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013287#s4" target="_blank">Materials and Methods</a>. Error bars denote standard deviation. Significance was determined based on Student's <i>t</i>-test (**, <i>P</i><0.01). Each field is 2.13×10³ µm². (<b>C</b>) Area of lipofuscin deposits. (<b>D</b>) Detection of apoptotic cells based on TUNEL staining (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013287#s4" target="_blank">Materials and Methods</a>) and condensed nuclear morphology. Nuclei were counterstained with methyl green. Positive cells are denoted by arrowheads. (<b>E</b>) Quantification of apoptotic cells per field. Each field is 3.29×10⁴ µm². Error bars and significance as in panel B (**, <i>P</i><0.01).</p

Age-associated morphological changes in liver.

<p>(<b>A-B</b>). Paraffin-embedded sections were prepared and stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013287#s4" target="_blank">Materials and Methods</a>. (<b>A</b>) Hematoxylin and eosin staining. (a-h) Individual sections representing pathology observed in different age groups. Lettering and symbols denote spongiosis hepatis (SH), ballooning degeneration (BD), nuclear pyknosis (NP), perivascular inflammation (PVI), inflammatory infiltrate (II), macrophage aggregate (MA), and steatosis (St). (<b>B</b>) Gomori's Trichrome stain. Panels (a) through (d) correspond to serial sections of corresponding specimens in (<b>A</b>). (<b>C</b>) Toluidine blue-stained semithin sections from 24-month old individuals. Lettering denotes macrophages (M), necrotic hepatocyte (H), space once occupied by hepatocyte (Sp), and congested sinusoid (S). (<b>D</b>) Transmission electron micrographs of liver sections from same fish. Lettering and symbols denote vacuoles communicating with space of Disse (*), bile cholangiole (Ch), macrophage (M), necrotic or shrunken hepatocytes (H), and congested sinusoid (S).</p

BrdU incorporation and senescence-associated β-galactosidase activity in skin and skeletal muscle.

<p>Each panel is a transverse section showing skeletal muscle (at top) and skin (at bottom). Cryosections were prepared and stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013287#s4" target="_blank">Materials and Methods</a>. (<b>A</b>) Senescence-associated β-galactosidase detected by histochemical analysis using X-gal substrate (blue staining). Positive areas are denoted by white arrows. Tissue was counterstained with eosin. Insets show higher magnification views of boxed regions. (<b>B</b>) BrdU incorporation detected by immunofluorescence (green channel). Positive cells are denoted by white arrows. Nuclei were counterstained with 7-amino actinomycin D (7-AAD) (red channel). (<b>C</b>) BrdU-positive cells per field were quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013287#s4" target="_blank">Materials and Methods</a>. Error bars denote standard deviation. Significance was determined based on Student's <i>t</i>-test (**, <i>P</i><0.01). Each field is 2.26×10⁵ µm².</p

Histology and lipofuscin accumulation in heart.

<p>Paraffin-embedded sections were prepared and stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013287#s4" target="_blank">Materials and Methods</a>. (<b>A</b>) Gomori's Trichrome staining was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013287#s4" target="_blank">Materials and Methods</a>. Arrows in 40X images indicate lipofuscin granules. Open arrowhead (40X image of 24 month-old fish) indicates blue-stained connective tissue (CT). Insets show enlargements of boxed regions (<b>B</b>) Detection of lipofuscin by autofluorescence (red channel, deposits indicated by white arrows). Nuclei were counterstained with DAPI (blue channel).</p

Mean and Median Survival Time^a.

a<p>Abbreviations are: Std. error, Standard error; C.I., Confidence interval.</p

Testis of medaka following a 14 day EE2 exposure followed by active breeding with 3 females for 20 days.

<p>A: DMSO control male. B: Male exposed to 1.0 µg/L EE2. There is thickening of the interstitium, an increase in proportion of spermatocytes, minimal spermatozoa. C: Male exposed to 1.0 µg/L EE2. This organ has severe thickening of the interstitium with a focal area of basophilic cells center of field and a severe decrease in germinal epithelium. D: Male exposed to 10.0 µg/L EE2. Severe thickening of interstitium and severe loss of germinal epithelium are. There are, however, spermatocytes and spermatids present suggesting active spermatogenesis. E: Male exposed to 10.0 µg/L EE2. There is severe thickening of the interstitium with loss of germinal epithelium.</p

Top ranked IPA Molecular Network generated from differentially expressed genes unique to day 14 in the 10.0 µg/L EE2 treatment.

<p>Myc family of genes is central to this Molecular Network suggesting an important role in the observed changes in gene expression and histology on day 14 in the 10.0 µg/L treatment group.</p

IPA top associated molecular networks function generated from gene lists comprised of significant genes common to all sampling days in each treatment.

<p>Significantly different genes listed in bold.</p