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Ecotoxicol Environ Saf
2021 Dec 01;225:112775. doi: 10.1016/j.ecoenv.2021.112775.
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Microplastics from miscellaneous plastic wastes: Physico-chemical characterization and impact on fish and amphibian development.
Bonfanti P
,
Colombo A
,
Saibene M
,
Motta G
,
Saliu F
,
Catelani T
,
Mehn D
,
La Spina R
,
Ponti J
,
Cella C
,
Floris P
,
Mantecca P
.
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Microplastic pollution represents a global problem with negative impacts on aquatic environment and organisms' health. To date, most of the laboratory toxicological studies on microplastics (MPs) have made use of single commercial micro and nano-polymers, which do not reflect the heterogeneity of environmental MPs. To improve the relevance of the hazard assessment, micrometer-sized plastic particles of miscellaneous non-reusable waste plastics, with size <100 µm and <50 µm (waste microplastics, wMPs), were characterized by microscopic and spectroscopic techniques and tested on developing zebrafish and Xenopus laevis by FET and FETAX assays respectively. Moreover, the modalities of wMP interaction with the embryonic structures, as well as the histological lesions, were explored by light and electron microscopy. We have shown that wMPs had very heterogeneous shapes and sizes, were mainly composed of polyethylene and polypropylene and contained metal and organic impurities, as well as submicrometric particle fractions, features that resemble those of environmental occurring MPs. wMPs (0.1-100 mg/L) caused low rate of mortality and altered phenotypes in embryos, but established species-specific biointeractions. In zebrafish, wMPs by adhering to chorion were able to delay hatching in a size and concentration dependent manner. In Xenopus embryos, which open stomodeum earlier than zebrafish, wMPs were accumulated in intestinal tract, where produced mechanical stress and stimulated mucus overproduction, attesting an irritation response. Although wMP biointeractions did not interfere with morphogenesis processes, further studies are needed to understand the underlying mechanisms and long-term impact of these, or even smaller, wMPs.
Fig. 1. Polymer characterization of wMPs. Panel A: superimposed ATR-FTIR spectra of polyethylene (left) and polypropylene (right) granulates obtained from each of the six dimensional fractions of wMPs. Panel B: Raman spectroscopy analysis of wMPs. On the right: examples of raw Raman spectra of the analyzed particles before baseline subtraction, many of them showing wide fluorescence peaks in addition to the Raman lines. From top to bottom: a) polyethylene, b) polyethylene-octene copolymer, c) polyethylene with phthalocyanine blue additive, d) polypropylene in fraction F1, e) metal oxide (rutile) containing particle in fraction F2. On the left: summary of the chemical composition of the wMP particles in fractions F1 and F2.
Fig. 2. Morphlological characterization of wMPs. Representative light (A) and fluorescence microscope images (B) that highlight heterogeneity in terms of shapes, colors, sizes and optical properties of the particles contained in F1 and F2 fractions. LM=light microscope, BF=bright field. Filter setting for fluorescence microscope: UV= 358 nm, and GFP= 488 nm, DsRed= 563 nm. Scale bar = 50 µm.
Fig. 3. Electron Microscopy analysis of wMPs. Panel A: SEM representative images at low and high magnification of F1 and F2 particles. It is possible to observe heterogeneous shapes and dimensions and the irregular surface of wMPs with the presence of sub-micrometric particles deposited on them (arrows). The high magnification of F2 corresponds to the white dotted area in low magnification image. Panel B: TEM images of F1 and F2 fractions ultrafine slices and corresponding EDX analysis in STEM mode (square selection and elemental maps). F1 fraction shows the presence of nanoparticles inside material of min Feret diameter around 100–200 nm (arrows) and the F2 fraction shows particles of min Feret diameter of 1 µm or less. The elemental composition of both fractions confirm the presence of Ti, Pb, Fe and Si in nano-form (F1 fraction) or distributed in the material (Al, Pb, Fe and Si in F2 fraction).
Fig. 4. Embryotoxicity of wMPs evaluated by FET. Panel A: mortality and malformation rates in 96 hpf embryos after exposure to F1 and F2 fractions (0.1–100 mg/L). On the right, representative stereomicroscopy images of a control embryo and a F2 exposed embryo at the end of assay (96 hpf). Panel B: daily and cumulative percentage of hatched control and wMPs exposed zebrafish embryos during FET. On the right, fluorescence stereomicroscopy representative images of control and F2 exposed zebrafish embryos at 24 hpf obtained by merging the red and green channels with bright field photographs. In wMP treated embryos, chorion surface is less smooth and transparent than in control and fluorescent wMP spots are observable. All values in the graphs are given as mean ± SE of three independent assays. * p < 0.05, * * p < 0.01 indicate statistical difference from control (Chi-squared test). Scale bars = 500 µm.
Fig. 5. Embryotoxicity of wMPs evaluated by FETAX. Mortality and malformation rates (A) and head-tail length (B) in 96 hpf embryos after exposure to F1 and F2 fractions (0.1–100 mg/L). All values are given as mean ± SE of three independent assays. (*) statistically different from control (p < 0.05, ANOVA + Fisher LSD Method). Panel C shows representative stereomicroscopic images of control and 100 mg/L F1 exposed embryos at the end of FETAX (stage 46–96 hpf), showing debris in the intestinal loops (arrows) of the latter. In the magnification of intestine of the 100 mg/L F1 exposed embryo observed at fluorescence stereomicroscopy (merge of the green and red fluorescence channels with the bright field photographs), it was ascertained that the most of the debris is represented by fragments of wMPs that emit fluorescence when excited in the GFP (green) or DsRed (red) channels. Scale bars = 500 µm.
Fig. 7. SEM analysis of intestinal tube isolated from control and F1 and F2 wMP exposed Xenopus embryos. Representative images of intestinal epithelium from a control Xenopus embryointestine (A, B) where the columnar morphology of enterocytes and the characteristic brush border facing to the lumen can be appreciated. In the representative images of the intestine isolate from embryos exposed to F1 (C, E, G, I) and F2 (D, F, H, L), the wMP fragments are crammed into the intestinal lumen and contact the microvilli, causing mechanical damage and hypersecretion of mucus that envelops them (white and black asterisks). In G the detail of a fibrous wMPs is visible that deepens in the epithelium. The dotted lines in E and L mark the wMP particles.
Figure S1. Waste microplastic (wMP) granulates. Large plastic granulates obtained by mechanical fragmentation of collected plastic waste (A), fine plastic granules with size less than 3 mm (B) achieved by additional milling and residual wMP fractions recovered after sieving (C). It is appreciable the decreasing graininess in all fractions. The two finest wMP fractions named F1 and F2 were used for chemical-physical and toxicological analyses.
Figure S2. Head-tail length in 96 hpf zebrafish embryos after exposure to F1 and F2 fractions (0.1-100 mg/L). All values are given as mean ± SE of three independent assays. (*) statistically different from control (p < 0.05, ANOVA + Fisher LSD Method).
