Evaluation of extraction and analysis protocols for polystyrene microplastics ingested by copepods using Pseudodiaptomus annandalei

• Main Authors: Ariana Chih-Hsien Liu, 劉芝仙
• Other Authors: Chih-hao Hsieh
• Format: Others
• Language: en_US
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/44ehv4

碩士 === 國立臺灣大學 === 海洋研究所 === 106 === 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.