Fig 1.
A) Scanning electron microscopy (SEM) images reveal the shape of the polystyrene nanoplastics used in this study (scale bar = 300 nm). B) Size (diameter) distribution of the nanoplastics from SEM image.
Table 1.
Study parameters.
Table 2.
Sample collection time points.
Fig 2.
Image processing and analysis.
(A) Sequential processing of larvae images- i) Acquisition of large images of larvae at various Z levels using different filters (BF: Bright-field image and TRITC: Fluorescence image), ii) Superimposition of BF and TRITC images of larvae at multiple Z levels, iii) Superimposition of images found from step A(ii). (B) Fluorescence intensity measurement- i) Splitting of the superimposed image from step A (iii) into red, green, and blue channels, ii) Isolation of the red channel image, identification of larvae as the region of interest (ROI), conversion into gray scale image, and application of a threshold and normalization via the control group to quantify fold change in fluorescence intensity.
Fig 3.
Bioaccumulation of NPs in C. hippurus through trophic transfer at different exposure periods.
Superimposed bright field and fluorescence images demonstrate the presence of red fluorescent compared to the control in (A) B. plicatilis following waterborne exposure and (B) C. hippurus via trophic transfer after 3 h and various exposure periods, respectively. (C) Fluorescence intensity (fold change as compared to the control) of the red fluorescent nanoplastics accumulated in C. hippurus via trophic transfer.
Fig 4.
Effect of depuration period on the retention of NPs in C. hippurus following trophic transfer exposure for various durations.
(A) Superimposed bright field and fluorescence images show the presence of red florescence nanoplastics in C. hippurus undergoing depuration. (B) Fluorescence intensity (FI) (fold change) of nanoplastics accumulated in C. hippurus after various depuration periods compared to the control, and % decrease in FI after depuration from corresponding post-exposure FI.
Fig 5.
Fluorescent and bright-field images showing the biodistribution of NPs in C. hippurus after trophic transfer and depuration.
A) Superimposed fluorescent and bright-field images of C. hippurus after (i) 48 hours of NP exposure via dietary intake, followed by (ii) a 72-hour depuration period. B) 3D z-stacked fluorescent images of the gut area indicated by the white dotted line in Fig 5A (i & ii).
Fig 6.
Effects of trophic transfer of NPs on the growth of C. hippurus larvae.
(A) Length and (B) eye diameter measurements after different exposure durations and subsequent depuration periods.
Fig 7.
Histopathological changes in the intestine of C. hippurus larvae following exposure to NPs via trophic transfer and subsequent depuration.
H&E-stained intestine sections of: (A) Control group, and larvae exposed to nanoplastics for (B) 24 h, (C) 48 h, (D) 72 h, (E) 96 h, and (F) 24 h of exposure followed by 72 h of depuration. Integration of villi (IV), shortening of villi (SV), and degradation of villi (DV).