Environmental DNA Metabarcoding: A Promising Tool for Ballast Water Monitoring.

Nonindigenous species are introduced worldwide with ballast water (BW). To prevent further introductions, oceanic BW exchange and BW treatment systems are utilized, but their performance needs to be evaluated. To that aim, characterizing BW communities is essential but usually relies on exhaustive sampling and morphological taxonomic identification, which does not always allow fine-scale taxonomic resolution. Through the analysis of BW samples from 11 vessels arriving to the Chesapeake Bay (USA), we evaluated the potential of environmental DNA (eDNA) metabarcoding for BW monitoring by assessing whether the impact of BW management type could be identified, analyzing the influence of BW sampling access locations on communities, and comparing the accuracy of eDNA for taxonomic assignment and identification of nonindigenous taxa. We found that (1) different sampling access locations of the same tank resulted in different communities, (2) communities from treated and exchanged BW differ, (3) signals of source port and of ocean exchange are observed, (4) eDNA metabarcoding results in more diversity than morphological taxonomy, and (5) the nonindigenous copepod Oithona davisae, not reported before in the Chesapeake Bay, is detected. Overall, this study highlights the potential of eDNA metabarcoding for BW monitoring, but more comprehensive sampling will be needed to optimize the approach.

Ballast Water Exchange and Invasion Risk Posed by Intracoastal Vessel Traffic: An Evaluation Using High Throughput Sequencing.

Ballast water remains a potent vector of non-native aquatic species introductions, despite increased global efforts to reduce risk of ballast water mediated invasions. This is particularly true of intracoastal vessel traffic, whose characteristics may limit the feasibility and efficacy of management through ballast water exchange (BWE). Here we utilize high throughput sequencing (HTS) to assess biological communities associated with ballast water being delivered to Valdez, Alaska from multiple source ports along the Pacific Coast of the United States. Our analyses indicate that BWE has a significant but modest effect on ballast water assemblages. Although overall richness was not reduced with exchange, we detected losses of some common benthic coastal taxa (e.g., decapods, mollusks, bryozoans, cnidaria) and gains of open ocean taxa (e.g., certain copepods, diatoms, and dinoflagellates), including some potentially toxic species. HTS-based metabarcoding identified significantly differentiated biodiversity signatures from individual source ports; this signal persisted, though weakened, in vessels undergoing BWE, indicating incomplete faunal turnover associated with management. Our analysis also enabled identification of taxa that may be of particular concern if established in Alaskan waters. While these results reveal a clear effect of BWE on diversity in intracoastal transit, they also indicate continued introduction risk of non-native and harmful taxa.

Design and installation of ballast water sample ports: Current status and implications for assessing compliance with discharge standards.

To verify ships' compliance with ballast water regulations, samples may be collected and tested for viable organisms. This task is completed using a sample probe, which is placed in the ballast discharge pipe through a sample port (a flanged opening). To collect representative samples, the placement of the sample port and the size of the sample probe must be appropriate for the shipboard piping arrangement and ballast water flows. The placement of sample ports was evaluated on 72 ships to assess the current condition of ballast water sampling installations against available guidance. Few ships (15%) had sample ports fully aligned with International Organization for Standardization (ISO) standard 11711-1. While current configurations may present challenges in collecting representative samples, these installations likely occurred before the ISO standard was available. Future installations should be in accordance with the standard to facilitate representative sampling.

Characteristics of global port phytoplankton and implications for current ballast water regulations.

The International Maritime Organization and U.S. Coast Guard have implemented regulations to reduce introductions of non-indigenous species via ballast water (BW). For phytoplankton, regulations limit discharges to <10 live/viable cells mL-1 (size: 10-50 µm), ignoring other size fractions. Additionally, challenge conditions of 100 (shipboard) and 1000 (land-based) cells mL-1 are required in BW management system certification testing. How these requirements correspond to natural phytoplankton populations is poorly resolved. We analyzed phytoplankton samples from 31 major ports to evaluate: a) how natural communities compare to challenge requirements and b) abundances of unregulated size fractions (i.e., <10 and ≥50 µm). None of the ports met land-based challenge conditions, and only 32% met requirements for shipboard testing. Approximately 71% of organisms ≥50 µm were centric diatoms, also unregulated by current protocols. This study demonstrates that current regulations do not consider natural phytoplankton populations, limiting control efforts for potentially harmful non-indigenous species.

Metabarcoding quantifies differences in accumulation of ballast water borne biodiversity among three port systems in the United States.

Characterizing biodiversity conveyed in ships' ballast water (BW), a global driver of biological invasions, is critically important for understanding risks posed by this key vector and establishing baselines to evaluate changes associated with BW management. Here we employ high throughput sequence (HTS) metabarcoding of the 18S small subunit rRNA to test for and quantify differences in the accumulation of BW-borne biodiversity among three distinct recipient port systems in the United States. These systems were located on three different coasts (Pacific, Gulf, and Atlantic) and chosen to reflect distinct trade patterns and source port biogeography. Extensive sampling of BW tanks (n = 116) allowed detailed exploration of molecular diversity accumulation. Our results indicate that saturation of introduced zooplankton diversity may be achieved quickly, with fewer than 25 tanks needed to achieve 95% of the total extrapolated diversity, if source biogeography is relatively limited. However, as predicted, port systems with much broader source geographies require more extensive sampling to estimate diversity, which continues to accumulate after sampling >100 discharges. The ability to identify BW sources using molecular indicators was also found to depend on the breadth of source biogeography and the extent to which sources had been sampled. These findings have implications both for the effort required to fully understand introduced diversity and for projecting risks associated with future changes to maritime traffic that may increase source biogeography for many recipient ports. Our data also suggest that molecular diversity may not decline significantly with BW age, indicating either that some organisms survive longer than recognized in previous studies or that nucleic acids from dead organisms persist in BW tanks. We present evidence for detection of potentially invasive species in arriving BW but discuss important caveats that preclude strong inferences regarding the presence of living representatives of these species in BW tanks.

Synthetic lipids as a biocide candidate for disinfection of ballast water.

The objective of this study is to propose the use of specific synthetic lipid as an active substance (biocide) in the control of harmful aquatic microorganisms, such as pathogens and non-indigenous species, transported in ships' ballast water. The biocide candidate, without metal or halogen components, was produced from a sub-product of the edible oil industry, the lecithin. Laboratory assays were conducted with phytoplankton, zooplankton, and marine bacteria to evaluate the efficiency of the biocide. The study also considers specific biocide's characteristics related to environmental risks, such as chemical composition, persistence, bioaccumulation, and toxicity. Results showed that, in the first 24 h of treatment, the biocide effectively reduced the concentration of the planktonic micro-organisms to very low levels. Additionally, a preliminary risk evaluation pointed that biocide candidate has a low residual toxicity, also a low potential for persistence and bioaccumulation in the environment.

Potential effects of LNG trade shift on transfer of ballast water and biota by ships.

As the US natural gas surplus grows, so does the prospect of establishing new trade partnerships with buyers abroad, a process that has major consequences for global ship movement and ballast water delivery. Since US annual imports of liquefied natural gas (LNG) peaked in 2004-2007, the country is rapidly transitioning from net importer to net exporter of LNG. Combining multiple datasets, we estimated changes in the associated flux of ships' ballast water to the US during 2015-2040, using existing scenarios for projected exports of domestic LNG by ships. Our analysis of the current market (2015) scenario predicts an approximate 90-fold annual increase in LNG-related ballast water discharge to the US by 2040 (42millionm3), with the potential to be even greater under high oil prices. We also described changes in geographic connectivity related to trade direction. These findings highlight how 21st century global energy markets could dramatically alter opportunities for seaborne introductions and invasions by nonnative species.

Evaluating the combined effects of ballast water management and trade dynamics on transfers of marine organisms by ships.

Global trade by merchant ships is a leading mechanism for the unintentional transfer of marine organisms, including non-indigenous species, to bays and estuaries worldwide. To reduce the likelihood of new invasions, ships are increasingly being required to manage their ballast water (BW) prior to discharge in coastal waters. In the United States, most overseas arrivals have been required to manage BW discharge since 2004, primarily through ballast water exchange (BWE), which flushes out ballast tanks in the open ocean (>200 miles from shore). Studies have found BWE to generally reduce the abundance of organisms, and the amount of water exchanged has been estimated at 96-100%. Despite its widespread use, the overall effect of this management strategy on net propagule supply through time has not been explored. Here, temporal changes in zooplankton concentrations and the volume of BW discharged in Chesapeake Bay, U.S. were evaluated, comparing pre-management era and post-management era time periods. Chesapeake Bay is a large port system that receives extensive BW discharge, especially from bulk cargo vessels (bulkers) that export coal overseas. For bulkers arriving from overseas, mean zooplankton concentrations of total and coastal indicator taxa in BW did not decline between pre- (1993-2000) and post management (2012-2013) eras, when controlling for season and sampling method. Moreover, bulkers discharged 21 million tonnes (82% of total for Chesapeake Bay) of overseas BW in 2013, representing a 374% increase in volume when compared to 2005. The combination of BW discharge volume and zooplankton concentration data indicates that (a) net propagule supply by bulkers has increased since BWE began in Chesapeake Bay; and (b) changes in vessel behaviour and trade have contributed strongly to this outcome. Specifically, the coal-driven increase in BW discharge volume from 2005-2013, concurrent with the onset of BWE regulations, worked to counteract intended results from BW management. A long-term analysis of bulker arrivals (1994-2013) reveals a 20-year minimum in arrival numbers in 2000, just when the implementation of BWE began. This study underscores the need to consider shifts in trade patterns, in order to advance and evaluate effective management strategies for biological invasions.

Difficulties in obtaining representative samples for compliance with the Ballast Water Management Convention.

As implementation of the Ballast Water Convention draws nearer a major challenge is the development of protocols which accurately assess compliance with the D-2 Standard. Many factors affect the accuracy of assessment: e.g. large volume of ballast water, the shape, size and number of ballast tanks and the heterogeneous distribution of organisms within tanks. These factors hinder efforts to obtain samples that truly represent the total ballast water onboard a vessel. A known cell density of Tetraselmis suecica was added to a storage tank and sampled at discharge. The factors holding period, initial cell density and sampling interval affected representativeness. Most samples underestimated cell density, and some tanks with an initial cell density of 100 cells ml(-1) showed <10 cells ml(-1) at discharge, i.e. met the D-2 standard. This highlights difficulties in achieving sample representativeness and when applied to a real ballast tank this will be much harder to achieve.