Quantification of Ingestion of Microplastics by Marine Medaka (Oryzias melastigma) Using Fluorescence and C-14 Isotope Radiotracer
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摘要: 微塑料已在多种海洋生物体内检出，造成不同程度的毒性效应，但由于技术限制，关于海洋鱼类对小粒径微塑料摄入和排出过程的定量研究仍比较缺乏.该研究针对生物体内小粒径微塑料定量示踪的技术难题，提出荧光和放射性同位素示踪法，并对比了两种方法的检测限、灵敏度和定性定量的方便程度等；同时以PS（聚苯乙烯，polystyrene）为微塑料代表，采用荧光法和C-14同位素法定量研究了PS微塑料（< 1 μm）在海水青鳉（marine medaka，Oryzias melastigma）成鱼和仔鱼中的摄入和排出情况，以及摄食行为对微塑料赋存状态的影响.结果表明：①荧光法适用于直观观察微塑料在生物体内的分布及高浓度暴露时的荧光定量，而C-14同位素法因具有更低的检测限和高的灵敏度，在复杂介质中的定量检测更具优势.②海水青鳉成鱼和仔鱼摄入微塑料的量随着培养时间而变化，且均在24 h摄入较多微塑料，成鱼（以鱼湿质量计）摄入的微塑料含量[（246.8±38.1）mg/g]显著（P < 0.05）高于仔鱼[（4.32±0.77）mg/g].③微塑料在海水青鳉体内主要分布部位为肠道（99.9%），极少量在鱼鳃（0.07%）和体表（0.03%）中，表明摄食是微塑料进入鱼体的主要途径；在不喂食72 h后，微塑料在肠道内仍有一定量残留[（1.29±0.52）mg/g]，鱼鳃中微塑料则完全排出至检测限以下.研究显示，海水青鳉通过对水中悬浮状态微塑料的摄入，将海水中的微塑料由初始悬浮分散态变成粪便团聚体沉入水底，在很大程度上改变了微塑料在环境中的赋存形态，由此对微塑料环境过程和生态效应产生的未知影响值得进一步关注.
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关键词:
- 海水青鳉(Oryzias melastigma) /
- 微塑料 /
- 定量 /
- 摄食 /
- 排出
Abstract: Microplastics have been detected in various marine organisms and have caused significant toxic effects. Due to the limitations of detection and quantification methods, the dynamics of marine fish ingestion and egestion of microplastics are still not well understood. With the aim to overcome the difficulties of quantifying microplastics in organisms, especially for microplastics of small sizes and low concentrations, this study proposed two methods using fluorescence and C-14 isotope tracer. The advantages of these two methods in terms of detection limit, sensitivity, qualitative and quantitative aspects were compared. Moreover, these two methods were used to quantify PS microplastics (< 1 μm) ingested by marine medaka (Oryzias melastigma) and the influence of ingestion/egestion process on microplastics in seawater. The results showed that fluorescence labeling was suitable for mapping and quantification of high-concentration microplastics in organisms, while the C-14 isotope tracer was more sensitive to microplastics in low-concentrations or complex environmental matrices. Large ingestion was observed during the incubation experiments and reached a maximum amount at 24 h. The microplastics concentration in adult medaka ((246.8±38.1) mg/g in wet weight) was significantly higher than that of juvenile fish ((4.32±0.77) mg/g in wet weight). The distribution of microplastics in adult medaka were detected highest in the gut of marine medaka (99.9%), followed by 0.07% in the gill and 0.03% in the body, indicating food ingestion was the dominant way of microplastic accumulation. After 72 h of egestion, microplastics could still be detected in the gut of marine medaka ((1.29±0.52) mg/g), but no microplastics could be detected in the gill and body, indicating potential ecological risks for marine organisms. It is worth noting that the ingestion of microplastics by marine medaka could significantly change the exiting state of microplastics from dispersion into aggregation, which may change the environmental fate of microplastics in marine systems. The unknown outcomes caused by such alteration on behavior and ecological effects of microplastics deserve more attention.-
Key words:
- marine medaka (Oryzias melastigma) /
- microplastics /
- quantification /
- ingestion /
- egestion
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图 1 PS微塑料浓度与荧光强度的标准曲线
Figure 1. Standard curve of PS microplastics concentrations with their fluorescence intensity
图 2 海水青鳉成鱼和仔鱼摄食24 h后体内PS微塑料含量
注：不同字母表示数据之间具有显著性差异(P < 0.05).
Figure 2. The amount of PS microplastics in adult and juvenile marine medaka after the 24 h incubation
图 3 海水青鳉仔鱼在不同微塑料浓度下培养72 h摄入的PS微塑料含量随培养时间的变化情况
注：不同小写字母或大写字母表示数据之间具有显著性差异(P < 0.05).
Figure 3. The amount of PS microplastics in juvenile marine medaka under different PS concentration during the 72 h incubation
图 4 荧光显微镜下观察海水青鳉摄入和排出后PS微塑料在鱼鳃、肠道和鱼体中的分布情况
Figure 4. Distribution of microplastics in the gill, gut and body of marine medaka after ingestion and egestion periods under the fluorescence microscope
图 5 海水青鳉摄入和排出后鱼鳃、肠道和鱼体的PS微塑料含量
注：不同小写字母或大写字母表示数据之间具有显著性差异(P < 0.05).
Figure 5. The amount of PS microplastics in the gill, gut and body of marine medaka after ingestion and egestion
图 6 微塑料在海水青鳉成鱼摄食前后的状态以及粪便的荧光信号表征和扫描电镜形貌表征
Figure 6. Status of microplastics in seawater before and after ingestion by marine medaka and characterization of feces with fluorescence microscope and scanning electron microscope
表 1 混合消解液成分表
Table 1. Ingredients of the liquid digestion mixture
成分 含量 Tris-HCl 400 mmol/L EDTA-2Na 60 mmol/L NaCl 105 mmol/L SDS 10 g H2O 1 L 
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