Environment and shipping drive environmental DNA beta-diversity among commercial ports.

The spread of nonindigenous species by shipping is a large and growing global problem that harms coastal ecosystems and economies and may blur coastal biogeographical patterns. This study coupled eukaryotic environmental DNA (eDNA) metabarcoding with dissimilarity regression to test the hypothesis that ship-borne species spread homogenizes port communities. We first collected and metabarcoded water samples from ports in Europe, Asia, Australia and the Americas. We then calculated community dissimilarities between port pairs and tested for effects of environmental dissimilarity, biogeographical region and four alternative measures of ship-borne species transport risk. We predicted that higher shipping between ports would decrease community dissimilarity, that the effect of shipping would be small compared to that of environment dissimilarity and shared biogeography, and that more complex shipping risk metrics (which account for ballast water and stepping-stone spread) would perform better. Consistent with our hypotheses, community dissimilarities increased significantly with environmental dissimilarity and, to a lesser extent, decreased with ship-borne species transport risks, particularly if the ports had similar environments and stepping-stone risks were considered. Unexpectedly, we found no clear effect of shared biogeography, and that risk metrics incorporating estimates of ballast discharge did not offer more explanatory power than simpler traffic-based risks. Overall, we found that shipping homogenizes eukaryotic communities between ports in predictable ways, which could inform improvements in invasive species policy and management. We demonstrated the usefulness of eDNA metabarcoding and dissimilarity regression for disentangling the drivers of large-scale biodiversity patterns. We conclude by outlining logistical considerations and recommendations for future studies using this approach.

Global marine biosecurity and ship lay-ups: intensifying effects of trade disruptions.

Recent global trade disruptions, due to blockage of the Suez Canal and cascading effects of COVID-19, have altered the movement patterns of commercial ships and may increase worldwide invasions of marine non-indigenous species. Organisms settle on the hulls and underwater surfaces of vessels and can accumulate rapidly, especially when vessels remain stationary during lay-ups and delays. Once present, organisms can persist on vessels for long-periods (months to years), with the potential to release propagules and seed invasions as ships visit ports across the global transportation network. Shipborne propagules also may be released in increasing numbers during extended vessel residence times at port or anchor. Thus, the large scale of shipping disruptions, impacting thousands of vessels and geographic locations and still on-going for over two years, may elevate invasion rates in coastal ecosystems in the absence of policy and management efforts to prevent this outcome. Concerted international and national biosecurity actions, mobilizing existing frameworks and tools with due diligence, are urgently needed to address a critical gap and abate the associated invasion risks.

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.

Formation of Brominated Organic Compounds and Molecular Transformations in Dissolved Organic Matter (DOM) after Ballast Water Treatment with Sodium Dichloroisocyanurate Dihydrate (DICD).

Estuarine water treated with a ballast water management system (BWMS) using a solution of dissolved dichloroisocyanurate dihydrate (DICD) resulted in the formation of newly described brominated disinfection byproducts (Br-DBPs). Analysis of dissolved organic matter (DOM) in untreated water with ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) identified 3897 m/z ions and their exact molecular formulas. After DICD treatment, a total of 213 halogenated molecular ions with relative abundance of at least 1% were assigned and confirmed using isotope simulation. Halogenated ions were assigned in four DBP elemental groups including CHOBr (180), CHONBr (13), CHOCl (16), and CHOBrCl (4). Forty-nine of the 197 brominated formulas have not been previously reported. We also were able to tentatively assign possible structures to the formula C3HBr3N2 due to very limited isomeric possibilities. The tentatively assigned compound found at 6.4% relative abundance was identified as either tribromoimidazole or tribromopyrazole. Our results show the formation of complex halogenated DBPs that are formed in the treatment of water with a novel BWMS that employs granular DICD as a biocide. The toxicological and mutagenic properties as well as the fate of these newly identified brominated DBPs are unknown.

Potential application of SMART II for Vibrio cholerae O1 and O139 detection in ship's ballast water.

Ballast water is used to safely stabilize and operate shipping vessels worldwide, in a multitude of aquatic settings, including inland, coastal and open oceans. However, ballast water may pose ecological, public health, and/or economic problems as it may serve as vehicles of transmission of microorganisms. Current ballast water regulations include limits of Escherichia coli, Enterococcus spp. and toxigenic Vibrio cholerae. Several United States Environmental Protection Agency approved standard operating protocols (SOPs) exist for detection of E. coli and Enterococci, yet none exists for V. cholerae. Current V. cholerae detection methods include colony blot hybridization, direct fluorescent antibody test (DFA), and/or polymerase chain reaction (PCR), which can be time consuming and difficult to perform. This study utilizes Cholera SMART II to determine its potential use in detection of V. cholerae. Validation of this method would help provide quick and accurate analysis for V. cholerae in ballast discharge waters in the field.

Long-term algal toxicity of oxidant treated ballast water.

National and international regulations require that ships' ballast water is treated to minimize the risk of introducing potentially invasive species. A common approach employed by commercial ballast water management systems is chlorination. This study presents the algal toxicity findings for three chlorination-based BWMS and their implications to environmental safety of port waters receiving treated ballast water from ships. Discharged treated ballast water from all three BWMS was toxic to algae with IC25s (25% growth inhibition) ranging from 9.9% to 17.9%, despite having total residual oxidant concentrations below 0.02 mg/l, based on Whole Effluent Toxicity assays. When held at 4 °C, some of the ballast water samples continued to exhibit toxic effects with no observed effect concentrations as low as 18% after a 134 day holding time. Thirteen individual disinfection by-products were measured above the detected limit at the time of discharge. No correlation between DBPs and algal toxicity was observed.

Feasibility of potable water generators to meet vessel numeric ballast water discharge limits.

Ballast water is taken on-board vessels into ballast water tanks to maintain vessel draft, buoyancy, and stability. Unmanaged ballast water contains aquatic organisms that, when transported and discharged to non-native waters, may establish as invasive species. Technologies capable of achieving regulatory limits designed to decrease the likelihood of invasion include onboard ballast water management systems. However, to date, the treatment development and manufacturing marketplace is limited to large vessels with substantial ballast requirements. For smaller vessels or vessels with reduced ballast requirements, we evaluated the feasibility of meeting the discharge limits by generating ballast water using onboard potable water generators. Case studies and parametric analyses demonstrated the architectural feasibility of installing potable water generators onboard actual vessels with minimal impacts for most vessel types evaluated. Furthermore, land-based testing of a potable water generator demonstrated capability to meet current numeric discharge limits for living organisms in all size classes.

Evaluation of approaches to quantify total residual oxidants in ballast water management systems employing chlorine for disinfection.

With the maturation and certification of several ballast water management systems that employ chlorine as biocide to prevent the spread of invasive species, there is a clear need for accurate and reliable total residual oxidants (TRO) technology to monitor treatment dose and assure the environmental safety of treated water discharged from ships. In this study, instruments used to measure TRO in wastewater and drinking water applications were evaluated for their performance in scenarios mimicking a ballast water treatment application (e.g., diverse hold times, temperatures, and salinities). Parameters chosen for testing these technologies in the past do not reflect conditions expected during ballast water treatment. Salinity, temperature, and oxidant concentration all influenced the response of amperometric sensors. Oxidation reduction potential (ORP) sensors performed more consistently than amperometric sensors under different conditions but it may be difficult to correlate ORP and TRO measurements for the multitude of biogeochemical conditions found naturally in ballast water. N,N-diethyl-p-phenylenediamine (DPD) analyzers and amperometric sensors were also tested under intermittent sampling conditions mimicking a ballasting scenario, with cyclical dosage and discharge operations. When sampling was intermittent, amperometric sensors required excessive response and conditioning times, whereas DPD analyzers provided reasonable estimates of TRO under the ballasting scenario.

How many organisms are in ballast water discharge? A framework for validating and selecting compliance monitoring tools.

As regulations governing the discharge of living organisms in ships' ballast water enter into force, tools to rapidly and easily measure compliance with the discharge standards will be essential. To assess, validate, and select compliance tools, a framework-consisting of three parts-is presented: proof-of-concept, validation and verification, and final selection stages. Next, a case study describing the proof-of-concept stage is discussed. Specifically, variable fluorescence was evaluated as an approach for determining compliance with the discharge standard for living organisms ⩾10 µm and <50 µm (typically protists). Preliminary laboratory experiments were conducted, which were followed by an expert workshop to gauge the feasibility of this approach and propose hypothetical thresholds indicating when the discharge standard is undoubtedly exceeded. Subsequently, field trials were conducted to assess this approach and recommended thresholds. All results were favorable, indicating the validation and verification stages are merited to further evaluate fluorometers as compliance monitoring tools.

Stratification of living organisms in ballast tanks: how do organism concentrations vary as ballast water is discharged?

Vertical migrations of living organisms and settling of particle-attached organisms lead to uneven distributions of biota at different depths in the water column. In ballast tanks, heterogeneity could lead to different population estimates depending on the portion of the discharge sampled. For example, concentrations of organisms exceeding a discharge standard may not be detected if sampling occurs during periods of the discharge when concentrations are low. To determine the degree of stratification, water from ballast tanks was sampled at two experimental facilities as the tanks were drained after water was held for 1 or 5 days. Living organisms ≥50 µm were counted in discrete segments of the drain (e.g., the first 20 min of the drain operation, the second 20 min interval, etc.), thus representing different strata in the tank. In 1 and 5 day trials at both facilities, concentrations of organisms varied among drain segments, and the patterns of stratification varied among replicate trials. From numerical simulations, the optimal sampling strategy for stratified tanks is to collect multiple time-integrated samples spaced relatively evenly throughout the discharge event.

Enumerating sparse organisms in ships' ballast water: why counting to 10 is not so easy.

To reduce ballast water-borne aquatic invasions worldwide, the International Maritime Organization and United States Coast Guard have each proposed discharge standards specifying maximum concentrations of living biota that may be released in ships' ballast water (BW), but these regulations still lack guidance for standardized type approval and compliance testing of treatment systems. Verifying whether BW meets a discharge standard poses significant challenges. Properly treated BW will contain extremely sparse numbers of live organisms, and robust estimates of rare events require extensive sampling efforts. A balance of analytical rigor and practicality is essential to determine the volume of BW that can be reasonably sampled and processed, yet yield accurate live counts. We applied statistical modeling to a range of sample volumes, plankton concentrations, and regulatory scenarios (i.e., levels of type I and type II errors), and calculated the statistical power of each combination to detect noncompliant discharge concentrations. The model expressly addresses the roles of sampling error, BW volume, and burden of proof on the detection of noncompliant discharges in order to establish a rigorous lower limit of sampling volume. The potential effects of recovery errors (i.e., incomplete recovery and detection of live biota) in relation to sample volume are also discussed.