以安氏偽鏢水蚤評估橈足類體內聚苯乙烯微粒之萃取與分析設計

Abstract

浮游動物體內的塑膠微粒之量化是重要的環境問題但是在技術上有困難。本研究探討浮游動物的塑膠微粒攝食狀況及量化技術，透過浮游動物攝食塑膠微粒實驗，以有效率的方法萃取生物體內的塑膠微粒。問題的癥結是塑膠微粒在浮游動物體內之實際數量與萃取後偵測到的數量不同，此因依據塑膠微粒濃度與顆粒大小不同，在萃取過程與計數過程中都有不同的損失和誤差值。因此，為了解浮游動物體內塑膠微粒的萃取率、萃取檢量線與偵測範圍，以安氏偽鏢水蚤 (Pseudodiaptomus annandalei ) 進行塑膠微粒餵食實驗。此研究採用直徑9 μm、20 μm與 25 μm的聚苯乙烯塑膠微粒顆粒，在九種不同濃度：介於1-10000 顆/ mL下餵食。 此研究測試了兩種對於萃取塑膠微粒較溫和且有效率的方式：蛋白酶K (proteinase-K) 和次氯酸鈉 (sodium hypochlorite, NaOCl)，其中蛋白酶K的萃取率最高。蛋白酶K混合液 (500 μg / mL) 和次氯酸鈉在50±2℃ 下反應三個小時。浸泡萃取液的塑膠微粒標準品過濾於玻璃纖維濾紙 (GF/F) 上，觀察後發現這個萃取方式對塑膠微粒並沒有任何損害。 透過餵食實驗估算浮游動物攝食量，而浮游動物樣本按上述方法萃取，其萃取後的顆粒過濾在玻璃纖維濾紙上，在螢光顯微鏡下計數。攝食實驗及塑膠微粒萃取結果顯示：25-μm顆粒的攝食量低，推測已超出攝食大小範圍，故後續分析終止。在優化萃取法下，20-μm顆粒的萃取率平均值約90.6%。20-μm顆粒的攝食量與萃取量皆可直接在螢光顯微鏡下計算總數量。攝食量與實驗之塑膠微粒濃度呈正相關，推估出的檢量線可以回推攝食量 (linear regression, r2 = 0.9997, slope= 0.9454)。 9-μm塑膠微粒因計數困難，因此以消化道內的螢光量來代表，萃取量用測線採樣計算法來估算。在固定的水蚤姿勢及拍攝角度下，樣本在螢光顯微鏡下記錄與計數。優化萃取法下，攝取量與實驗之塑膠微粒濃度呈正相關，並估算出與20-μm顆粒相似趨勢的檢量線 (linear regression, r2=0.772)。 為分析自然樣本，我們測試臺灣海峽及東海南部豐度穩定的隨機濾食型橈足類。以擬哲水蚤科(Paracalanidae copepodids) 和哲水蚤科(Calanidae copepodids)為研究物種(體長大於300 μm)。優化萃取法下，我將玻璃纖維上的殘留物以常見分析微小顆粒之方法進一步定性與定量。我嘗試視覺特徵法、拉曼光譜分析法、流式顯微攝像、尼羅紅螢光染色和螢光顯微鏡。其中效果最好的是螢光顯微鏡與拉曼光譜分析法的組合，可以分析到小於50-μm的塑膠微粒。然而，儘管已用優化萃取法與量化法，我們並沒有在野外樣本中找到塑膠微粒。此可能是分析流程效率不夠高或野外浮游動物攝食濃度低於可偵測範圍。

Being able to quantify microplastics in the gut of zooplankton is a critical environmental concern. The amount of extracted microplastics may be less than the real amount in the gut of zooplankton, due to loss during the extraction and quantification procedures. These losses could vary depending on the size and concentration of microplastics. To tackle this difficulty, I tried various protocols to extract and quantify microplastics in copepods through feeding experiments. In this study, adult Pseudodiaptomus annandale were fed with polystyrene beads of 9-25 μm, using a series of concentration from 1- 10000 beads/ mL. To optimize extraction protocol, I tried proteinase-K and sodium hypochlorite to digest biological materials. I found that proteinase-K enzyme extraction method is the most efficient, with no noticeable impact on microplastics. Extracts from digestion were then filtered on a glass filter. The results of feeding experiments indicate that ingestion of 25-μm beads was low, suggesting that this size was out of feeding range and thus no further analysis was carried out. Using a series of beads concentration for the 20-μm beads, I found that the ingestion amount and extraction amount are correlated. My optimized enzyme extracting protocol had an extraction rate at an average of 90.6% for 20-μm beads. I established the extraction calibration curve (linear regression, r2=0.9997, slope= 0.9454), which allows to back-calculate the ingestion amount from the bead counts in the extract. Due to difficulty in counting 9-μm beads, ingestion amount was estimated by the intensity of luminescent light within the gut area while extraction amount was estimated through a transect subsampling procedure. Ingestion in the gut and transect sampling on the filter were analyzed to establish the extraction calibration curve. I found that the ingestion amount and extraction amount are correlated for 9-μm beads, which allows establishing the extraction calibration curve (linear regression, r2=0.772). To investigate the samples in natural systems, I analyzed the filter-feeding copepods that are abundant in the southern East China Sea. Paracalanidae and Calanidae copepodids (body length > 300 μm) were chosen for examination. The optimized enzyme extracting protocol was applied before testing identification and quantification methods on the extracts of copepods. I tried visual characterization, spectrometry instruments, flow cytometer and microscope (FlowCAM®), fluorescent Nile red dye, and fluorescent microscope; these methods are commonly used in larger organisms. Methods capable of analyzing particles below 50 μm include fluorescent microscope and micro-RAMAN. Despite using the optimal enzyme extracting protocol and quantification methods, I did not find any microplastic from the extract. This result may be because my protocol has low extraction efficiency for microplastics or the concentration of microplastics inside the gut of those copepod specimens are too low to be detectable.