Study on Multi-Dimensional Optimization of Detailed Laboratory Analysis Method for Ship Ballast Water

Ballast water can bring aquatic organisms into foreign ecosystems and cause the risk of biological invasion. Therefore, it needs to be treated before discharge, and the treated water needs to be analyzed to determine whether it meets the specified standard. At present, there is no unified method for the detection of plankton worldwide, and there are some problems in the existing methods, such as unreasonable and inaccurate. In this study, neutral red (NR), 5-chloromethyfluorescein diacetate (CMFDA) and fluorescein diacetate (FDA) were used to dye the 10-50 µm plankton of three common phyla in ballast water, and were determined by fluorescence microscope. The results showed that for 10-50 µm plankton, the staining effect of double fluorescence staining was obvious, while the fluorescent dyes could only stain part of marine diatoms, and the sample could not be fixed. The optimal dyeing concentration of neutral red was 1/5000, the dyeing time was 20 min, and the overall dyeing efficiency was above 99%. Fixing agent was used after neutral red staining, when fixing with Lugol’s solution, the lower the temperature, the better the preservation effect. At -4 °C, the storage time of the sample could be increased to 12 h, and the proportion of plankton was more than 96%. In conclusion, neutral red staining can be used as a supplement to fluorescence staining, and the the storage time of samples can be prolong by pretreatment and fixation the samples.

Simultaneous Detection of Viability and Concentration of Microalgae Cells Based on Chlorophyll Fluorescence and Bright Field Dual Imaging

Ship ballast water contains high concentration of plankton, bacteria, and other microorganisms. If the huge amount of ballast water is discharged without being inactivated, it will definitely spell disaster to the marine environment. Microalgae is the most common species exiting in ballast water, so the detection of the concentration and viability of microalgae is a very important issue. The traditional methods of detecting microalgae in ballast water were costly and need the help of bulky equipment. Herein, a novel method based on microalgae cell intracellular chlorophyll fluorescence (CF) imaging combines with cell bright field (BF) microscopy was proposed. The geometric features of microalgae cells were obtained by BF image, and the cell viability was obtained by CF image. The two images were fused through the classic image registration algorithm to achieve simultaneous detection of the viability and concentration of microalgae cells. Furthermore, a low-cost, miniaturized CF/BF microscopy imaging prototype system based on the above principles was designed. In order to verify the effectiveness of the proposed method, four typical microalgae in ballast water (Platymonas, Pyramimonas sp., Chrysophyta, and Prorocentrum lima) were selected as the samples. The experimental results show that the self-developed prototype can quickly and accurately determine the concentration and the viability of microalgae cells in ship ballast water based on the dual images of BF and CF, and the detection accuracy is equivalent to that of commercial microscope. It was the first time to simultaneously detect the viability and concentration of microalgae cells in ship ballast water using the method that combining the fluorescence and bright field images; moreover, a miniaturized microscopic imaging prototype was developed. Those findings expected to contribute to the microalgae detection and ship ballast water management.

An Assessment of Progress in the Implementation of the BWM Convention on Ships as an Important Element in Protecting Aquatic Ecosystems

The purpose of this study was to assess progress in the implementation of rules for the control and handling of ballast water and sediments, regulated by the International Convention on the Control and Handling of Ship Ballast Water and Sediments (BWM Convention). The survey was conducted in 2018-2019 among seafarers responsible for handling ballast tanks. Analysis of the survey data revealed that despite the introduction of water treatments, ballast water continues to be a vector for the transfer of non-native organisms. This is due to the low effectiveness of the methods used, and the fact that 8% of the respondents answered that no BWT systems were used on the ships they manage. Despite this, some seafarers (4.7%) indicated that increasing environmental awareness and adherence to BWM principles should result in improved protection of aquatic ecosystems. Therefore, the global problem concerning the transport of non-native organisms via ballast water has still not been solved effectively. In the near future, improved BWT systems should be introduced, which after proper training of the crew will act as a barrier to the transfer of non-native organisms in ballast water.