Aquatic invasive species are among the worst threats to marine biodiversity. The main vector for the spread of these aquatic invasive species is ships’ ballast water. Because of this, the International Maritime Organization (IMO) adopted the Ballast Water Convention. Part of this convention is the D-2 ballast water performance standard, which sets limits to the amount of viable organisms allowed to be in ballast water upon discharge. The limits of the D-2 standard are: 1. less than 10 viable organisms/m3 in the size class =50 µm; 2. less than 10 viable organisms/mL in the size class =10 - <50 µm; 3. limits on the abundance of toxigenic Vibrio cholera, Escherichia coli and intestinal enterococci. In order to meet this standard, manufacturers developed different types of Ballast Water Treatment Systems (BWTSs). These BWTSs need to be approved according to IMO regulations by an independent party. Several approval tests were performed at the Royal Netherlands Institute of Sea Research (NIOZ). The focus of this thesis was to test the effects of various ballast water treatment methods on the survival of phytoplankton and bacteria. Different methods are used to reduce the numerical abundance of organisms, most notably Ultraviolet-radiation (UV) and ‘active substances’ (chemicals). Both treatment methods were considered in this thesis. To measure the efficacy of different BWTSs, methods had to be developed that are applicable to all types of treatments. The standard IMO regulations state that treated ballast water has to be stored in the dark in simulated ballast water tanks for five days before being tested against the D-2 standard. However, it was questionable if this time period would be sufficient to account for delayed effects of the disinfectant and possible recovery of organisms. In other words, it was not known if re-growth of micro-organisms could occur after this standardized five day period. Therefore, in the present thesis, the possibility of re-growth was examined by executing long term incubation experiments under light-dark conditions simulating the post-discharge situation in the open sea. Phytoplankton and bacterial abundance, composition and diversity were monitored by a range of analytical techniques, including classical microscopy, flow cytometry and molecular fingerprinting. In a first series of experiments, UV and chlorine dioxide (CD) treated water was incubated for 20 days under favorable conditions with respect to irradiance and nutrient availability to stimulate the growth of micro-organisms that had survived the treatment. After both treatments, re-growth of phytoplankton occurred (Chapter 2). This suggests that currently approved BWTSs meet the IMO D-2 standard, but do not completely eliminate the potential spread of aquatic organisms through ballast water. To identify the species that re-grew after ballast water treatment, UV treated samples were incubated and monitored for phytoplankton abundance and species composition. Microscopy showed that ballast water treatment changed the species composition and that certain species were more likely to re-grow after treatment (Chapter 3). However, microscopy was not always able to identify the exact species. Because of this the application of flow cytometry, microscopy and DNA-sequencing as methods of species identification were investigated. Flow cytometry provided fast quantification of phytoplankton, but could only provide a rough indication of phytoplankton diversity. Microscopy provided a more qualitative method of identification, but could not always identify the phytoplankton to the species level. DNA-sequencing provided accurate species identification but proved to be time-consuming and only identified one or two of the most dominant species in the sample. The most common re-growing species after UV treatment proved to be Thalassiosira weissflogii. This indicates that some species are more likely to survive ballast water93treatment than others and that ballast water treatment may apply selective force to create resistant species (Chapter 4). In the follow-up experiment, phytoplankton re-growth was monitored in six BWTSs; three systems were based on UV, two based on electrochlorination (EC) and one based on chlorine dioxide (CD). All BWTSs incubation experiments were performed for 20 days with treated ballast water, during which growth, photosynthetic efficiency and phytoplankton species composition were followed. The three UV systems all showed the same pattern after the initial UV exposure, notably a gradual decrease in phytoplankton abundances followed by re-growth. Treatments using 200 % or 400 % of the normal UV dose reduced phytoplankton numbers more strongly, but did not prevent their re-growth. Results of EC and CD BWTSs were comparable to each other. However, UV and active substance-based treatment systems showed significantly different responses. Both types of systems showed an immediate reduction in phytoplankton photosynthetic efficiency. However, for UV treatment systems phytoplankton abundances decreased over several days while for chlorine-based treatment systems the drop in phytoplankton abundance was immediate. The species composition of re-growing phytoplankton also differed between UV and EC treatment. Overall, all BWTSs reduced phytoplankton abundances to below the values of the D-2 standard, which represents a reduced risk of future aquatic invasions through ballast water. However, all (but one) re-growing species were smaller than 10 µm, which means they are not covered by the D-2 standard (Chapter 5). To assess possible environmental risks associated with BWTSs that use ‘active substances’, a BWTS that uses a chemical mixture known as Peraclean® Ocean (PO) was evaluated. The residual of PO is acetate that might be present in concentrations exceeding 100 mg/L in discharged ballast water. To study the potential environmental impact of PO, microbial dynamics and acetate degradation were measured during incubation of discharge water following PO treatment. In addition, microbial dynamics and acetate degradation were studied at different temperatures in dark microcosms that simulated enclosed ballast water tanks. After about nine days bacteria abundances greatly increase in PO treated waters to almost ten times of initial control abundances. Furthermore, bacterial diversity was also altered by the changes in water chemistry. Breakdown of acetate occurred faster at higher temperatures. At the lowest temperatures almost no acetate breakdown occurred, but even at the highest temperature the acetate pool was not depleted. This implies that not all acetate will be broken down in ballast water tanks, even during long voyages in warm waters. It was concluded from this study that regular discharge of acetate-containing ballast water in harbors and bays may stimulate growth of heterotrophic bacteria, causing oxygen depletion and changes in the microbial community, especially in colder regions (Chapter 6). The D-2 standard does not consider total heterotrophic bacterial abundances. Increases in bacterial abundance as shown for this BWTS are allowed under current IMO regulations. The potential harmful effects on the ecosystem presented by the discharge of bacteria-rich ballast water demonstrate the necessity to include total heterotrophic bacteria in the D-2 standard. In conclusion, the present thesis has revealed two major shortcomings in the ballast water regulations and particularly in the D-2 standard. It is recommended that the D-2 standard is amended to include limit values for viable phytoplankton and zooplankton organisms < 10 µm as well as total heterotrophic bacteria. |
