螢光染色法定量土壤塑膠微粒之研究:消化試劑與分離萃取程序之優化

Abstract

塑膠微粒污染是廣受關注的環境議題，其中土壤被視為其主要的匯集處之一。然而，土壤基質的複雜性，特別是有機質的干擾，使塑膠微粒的定量分析極具挑戰。本研究旨在建立並優化一套使用尼羅紅染色法結合螢光顯微鏡來定量土壤中小型塑膠微粒的分析方法。為此，本研究優化前處理流程，首先比較三種消化試劑，包括過氧化氫、硝酸與芬頓試劑去除土壤背景螢光之效率，並評估其對聚丙烯（PP；<100 µm）和聚氯乙烯（PVC；<150 µm）等塑膠微粒完整性的影響，接著探討氯化鈣與氯化鋅等高密度溶液對塑膠微粒的萃取回收率。實驗結果顯示，硝酸是去除土壤背景螢光最有效的試劑，在壤土和黏土中的去除率分別高達 89%與 82%，且對 PP 與 PVC 的表面形態和化學結構僅造成輕微影響。使用氯化鈣（1.4 g/cm³）溶液進行兩次萃取，在 PP 與 PVC 的回收率分別達到約 78-80%及 61-70%，考量其效率與較低的環境毒性，氯化鈣被選定為較佳萃取液。本研究應用此優化後的方法分析三處環境樣品，結果顯示農田土壤的塑膠微粒濃度最高（1.04×10⁵ 顆粒/公斤），其次為掩埋場（7.10×10⁴ 顆粒/公斤），公園則最低（4.00×10⁴ 顆粒/公斤）。在所有樣品中以粒徑小於 100 µm 的微粒占多數。綜合上述，本研究提供了一套經過系統性驗證的分析方法，可作為快速評估土壤環境中小型塑膠微粒污染的實用工具。

Microplastic pollution is a widespread environmental issue, with soil emerging as a major sink for these contaminants. The quantification of microplastics in soil, however, is challenging due to complex matrix interference, particularly from soil organic matter. This study aimed to develop and optimize a protocol for quantifying small-sized microplastics in soil using fluorescence staining with Nile red followed by fluorescence microscopy. To achieve this, the method was systematically optimized by comparing three digestion reagents, including H₂O₂, HNO₃, and Fenton's reagent, for their effectiveness in reducing background fluorescence and their impact on the integrity of polypropylene (PP；< 100 µm) and polyvinyl chloride (PVC；< 150 µm) microplastics. Additionally, the extraction efficiency using different high-density salt solutions including CaCl2 and ZnCl2 was evaluated. Results demonstrated that HNO₃ was the most effective and consistent reagent, achieving up to 89% removal of background fluorescence in loam and 82% in clay, with only minor effects on the morphology and chemical property of PP and PVC. A double extraction with CaCl2 (1.4 g/cm³) solution achieved recoveries of approximately 78-80% for PP and 61-70% for PVC, and was selected over the more toxic ZnCl2. The optimized method, involving HNO₃ digestion and CaCl2 extraction, was then applied to environmental samples. The highest microplastics concentration was found in farmland soil (1.04×10⁵ particles/kg), followed by a landfill site (7.10×10⁴ particles/kg) and a park (4.00×10⁴ particles/kg), with particles smaller than 100 µm being dominant in all samples. In conclusion, this study provides a systematically validated protocol that serves as a practical and rapid tool for assessing small microplastic contamination in complex soil environments.