Plastic pollution presents a global environmental concern with potentially widespread ecological, socio-economic and health implications. Methodological advances in microplastic extraction, ...quantification and identification from sediments have been made. However, integrating these fragmentary advances into a holistic, cost-effective protocol and applying it to organic rich sediments with fine grain size remains a challenge. Nonetheless, many hot spots of microplastic contamination such as harbour and estuarine sediments are characterised by such sediments. We conducted a series of experiments to integrate methodological advances, and clarify their applicability to organic rich sediments with fine grain size. The resulting protocol consisted of three stages. First, pre-treatment with Fenton’s reagent was found to be efficient in reducing organic matter content, compatible with later Fourier Transform-Infrared Spectroscopy (FT-IR) for polymer identification, although it did affect the size of polyethylene (PE) and polyethylene terephthalate (PET). Secondly, a novel density separation column with a top overflow (the OC-T) obtained recovery rates above 90% for microplastics present in a ZnCL2 solution. Finally, automated epifluorescence microscopic image analysis of Nile Red stained filters with selected validation of polymer identities using FT-IR revealed 91.7% of stained particles to be plastics. A case study on estuarine sediments demonstrated a high extraction efficiency with quantification possible down to 125 μm and detection possible down to 62.5 μm. This makes this protocol suitable for large scale monitoring of microplastics in sediments of estuarine origin provided polymer specific recovery rates, background contamination and uncertainty in Nile Red identification is accounted for. Subject to further validation, the protocol could also offer a solution to similar organic rich sediments with fine grain size, such as some soils and sludge, to improve our ability to conduct cost-effective, large scale monitoring of microplastic contamination.
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•Methodological advances were integrated into a holistic microplastic lab protocol.•The protocol targets cost-effective, large scale monitoring of microplastic pollution.•A new density separation column (OC-T column) obtained high extraction efficiency.•Validation of Nile Red fluorescent stained particles revealed 91.7% to be plastics.•Microplastic quantification down to 125 μm was possible in an estuarine case study.
We developed a three-stage protocol, integrating current fragmentary methodological advances for microplastic extraction, identification and quantification. A combination of fluorescent quantification with selected polymer identification for particles down to 125 μm makes this protocol suitable for cost-effective, repeatable, large scale monitoring of microplastic contamination in organic rich sediments.
The effect of temperature on the fluorescence of carbon dot colloidal solution in glycerol has been studied. The dots were prepared by pyrolysis of a Nile Red laser dye using mesoporous silica as a ...template. It is established that the temperature dependence of the intensity of dominant emission peak is nontrivial: as the temperature of the solution rises, the initial quenching of the fluorescence is then replaced by its enhancement to the initial value. A three-level model of electronic states is considered, within which the observed temperature dependence of fluorescence is quantitatively described and the energies of nonradiative deactivation and activation of the emissive state are estimated. The nature of the electronic state responsible for the nonradiative activation of the emissive state is discussed. The possible influence of hydrogen bonds in the solute-solvent system on the temperature behavior of the CD fluorescence is also considered. A pronounced solvatochromic effect is noted in the fluorescence spectra of Nile Red based carbon dots in different solvents.
•Nile Red laser dye is used to synthesize fluorescent C-dots by template method.•Nontrivial PL intensity temperature dependence of C-dot glycerol solution is revealed.•The dependence can be described within a three-level model of electronic states.•H-bond formation in the solute-solvent system influences the PL temperature behavior.
•Toxicity of fluorescent-labelled PE-MPs and their leachate is evaluated in this study.•Nile red dye leachate induces toxicity in Physalaemus cuvieri tadpole.•Fluorescent-labelled PE-MPs and Nile red ...dye leachate reduces antioxidant activity of tadpoles.•Anticholinesterasic effect is observed in tadpoles exposed to fluorescent PE-MPs and their leachate.•We recommend the use of purification steps and leachate controls.
The toxicity of microplastics (MPs) has been demonstrated in several previous studies, exposing different model organisms to fluorescent-labeled particles. However, many of these studies did not report the use of control groups for unbound and leaching dyes, which limits the conclusions about the bioaccumulative potential of these micromaterials and their (eco)toxicological effects. Thus, considering the need to evaluate the possibility of the toxicity of MPs resulting from the leaching of their dyes, we used Physalaemus cuvieri tadpoles to investigate whether the leachate of fluorescently labeling polyethylene MPs (PE-MPs) with Nile red dye induces behavioral changes, redox imbalance, anticholinesterasic effect, as well as changes in the growth/development of animals. By observing that the response of P. cuvieri tadpoles exposed to fluorescent PE-MPs and their respective leachate were similar, we confirmed the hypothesis that, alone, the fluorescent PE-MPs leachate can induce changes in antioxidant activity (inferred by SOD and CAT activity), in the mechanisms that mediate/regulate the production of NO and MDA, as well as in the animals' AChE activity. These results suggest that the Nile red dye eluted from PE-MPs can induce toxicological effects. Furthermore, we observed that the Nile red dye, when binding to the lipid components of the feed (and later being ingested by the animals), can induce interpretive biases on the bioaccumulative potential of PE-MPs. Therefore, for the evaluation of the (eco)toxicity of PE-MPs, it is important that extensive post-labeling cleaning steps of the PE-MPs (e.g.: via dialysis) and monitoring of the elution of fluorescent probes not bound to these particles be carried out before being added to the exposure water and that control groups for unbound and leaching dyes be established in future experimental designs, accounting for fluorescence induced by any other confounding factors.
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The temperature dependence of the fluorescence of a colloidal solution of carbon dots in glycerol is studied. The dots are obtained by the pyrolysis of Nile Red laser dye using mesoporous silica as a ...matrix. To obtain a colloidal solution of individual dots, the matrix material is dissolved in hydrofluoric acid, followed by repeated cleaning of the dots with deionized water. The optical absorption spectrum of the dots demonstrates the presence in their composition of aromatic
sp
2
-hybridized carbon atoms, as well as CO and CN molecular groups. The fluorescence spectrum of the colloidal solution when excited by light with λ
exc
= 405 nm consists of two emission bands. It is established that the intensity
I
of the main (long-wavelength) fluorescence band of the synthesized dots increases with increasing solution temperature
T
in a wide temperature range from 270 to 415 K. The nature of the dependence
I
(
T
) indicates the occurrence of two competing processes: thermally induced fluorescence quenching and its enhancement. A three-level model of electronic states is considered, within which the observed temperature dependence of fluorescence is quantitatively described and the energies of nonradiative deactivation and activation of the emissive state are estimated. The nature of the electronic state responsible for the nonradiative activation of the emissive state is discussed.
The principal objective of this study was to develop a novel method based on confocal microscopy and a solvatochromic fluorescent dye, Nile red (NR) to quantify the main types of lipids in a single ...mammalian oocyte and embryo. We hypothesize that NR staining followed by the decomposition of NR-spectra identifies and quantifies the triglycerides, phospholipids, and cholesterol in a single oocyte and embryo. We analyzed the lipid droplets in porcine oocytes and pre-implantation embryos up to the hatched blastocyst stage developed
in vivo and in cultured blastocysts. The emission spectrum of NR-stained mixture of different lipid types is a convolution of several component spectra. The principal component analysis (PCA) and a multivariate curve resolution-alternating least squares method (MCR-ALS) allowed to decompose the emission spectrum and quantify the relative amount of each lipid type present in mixture. We reported here that the level of the triglycerides, phospholipids and cholesterol in lipid droplets significantly decreases by 17.7%, 26.4% and 23.9%, respectively, from immature to mature porcine oocytes. The content of triglycerides and phospholipids remains unchanged in droplets of embryos from the zygote up to the morula stage. Then the triglyceride level decreases in the blastocyst by 15.1% and in the hatched blastocyst by 37.3%, whereas the amount of phospholipids decreases by 10.5% and 12.5% at the blastocyst and hatched blastocyst stages, respectively. In contrast, the content of cholesterol in droplets does not change during embryo cleavage. The lipid droplets in the blastocyst produced
in vivo contain lower amounts of triglycerides (by 26.1%), phospholipids (by 14.2%) and cholesterol (by 34.8%) than those in the blastocyst cultured in NCSU-23 medium. In conclusion, our new technique is suitable to quantify the content of triglycerides, phospholipids and cholesterol in individual mammalian oocytes and embryos. Our findings indicate an important role for lipids during porcine oocyte maturation and early embryonic development, and suggest an altered lipid metabolism in cultured embryos.
The expansion of polyhydroxyalkanoates (PHAs) into the biodegradable polymers market is mainly prevented by their production process which is still complicated with a low efficiency, resulting in ...relatively expensive products. In this study, we developed a method that used the lipophilic fluorescent probe Nile Red (1 mg l⁻¹ solution in DMSO) directly into the culture broth to stain the PHA inclusions inside bacterial cells followed by detection of the emitted fluorescence by both microscopic and spectrometric techniques. Epifluorescence microscopy provides a rapid tool to distinguish producing from non-producing bacterial species and the relative fluorescence intensity (FI) determined at the maximum of emission spectra in the wavelength region of 560–710 nm (λₑₓ: 543 nm), allows a fast assessment of the cultural conditions that may enhance PHA production yield. During two-step cultivation in 500-ml flasks with glucose as the sole carbon source, the method aimed to select bacterial strains efficient for PHA synthesis among a marine collection. Subsequently, the NR assay was used to determine the C₀/N₀ ratio of the producing media that may improve the polymer yield as well as to follow the time course of fermentation. Characterization by GC–MS and DSC confirmed the production of the P(3-HB) homopolymer.
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