Transmission of stony coral tissue loss disease (SCTLD) in simulated ballast water confirms the potential for ship-born spread.

Stony coral tissue loss disease (SCTLD) remains an unprecedented epizootic disease, representing a substantial threat to the persistence and health of coral reef ecosystems in the Tropical Western Atlantic since its first observation near Miami, Florida in 2014. In addition to transport between adjacent reefs indicative of waterborne pathogen(s) dispersing on ocean currents, it has spread throughout the Caribbean to geographically- and oceanographically-isolated reefs, in a manner suggestive of ship and ballast water transmission. Here we evaluate the potential for waterborne transmission of SCTLD including via simulated ballast water, and test the efficacy of commonly-used UV radiation treatment of ballast water. Two species of reef-building corals (Orbicella faveolata and Pseudodiploria strigosa) were subjected to (1) disease-exposed or UV-treated disease-exposed water, and (2) a ballast hold time series of disease-exposed water in two carefully-controlled experiments to evaluate transmission. Our experiments demonstrated transmission of SCTLD through water, rather than direct contact between diseased and healthy corals. While UV treatment of disease-exposed water led to a 50% reduction in the number of corals exhibiting disease signs in both species, the statistical risk of transmission and volume of water needed to elicit SCTLD lesions remained similar to untreated disease-exposed water. The ballast hold time (24 h vs. 120 h) did not have a significant effect on the onset of visible disease signs for either species, though there appeared to be some evidence of a concentration effect for P. strigosa as lesions were only observed after the 120 h ballast hold time. Results from both experiments suggest that the SCTLD pathogens can persist in both untreated and UV-treated ballast water and remain pathogenic. Ballast water may indeed pose a threat to the continued spread and persistence of SCTLD, warranting further investigation of additional ballast water treatments and pathogen detection methods.

Sample port design for ballast water sampling: Refinement of guidance regarding the isokinetic diameter.

By using an appropriate in-line sampling system, it is possible to obtain representative samples of ballast water from the main ballast line. An important parameter of the sampling port is its "isokinetic diameter" (DISO), which is the diameter calculated to determine the velocity of water in the sample port relative to the velocity of the water in the main ballast line. The guidance in the U.S. Environmental Technology Verification (ETV) program protocol suggests increasing the diameter from 1.0× DISO (in which velocity in the sample port is equivalent to velocity in the main line) to 1.5-2.0× DISO. In this manner, flow velocity is slowed-and mortality of organisms is theoretically minimized-as water enters the sample port. This report describes field and laboratory trials, as well as computational fluid dynamics modeling, to refine this guidance. From this work, a DISO of 1.0-2.0× (smaller diameter sample ports) is recommended.

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.

3D imaging provides a high-resolution, volumetric approach for analyzing biofouling.

A volumetric approach for determining the fouling burden on surfaces is presented, consisting of a 3D camera imaging system with fine (5 µm) resolution. Panels immersed in an estuary on the southwest coast of Florida, USA were imaged and the data were used to quantify seasonal changes in the biofouling community. Test panels, which were submerged in seawater for up to one year, were analyzed before and after gentle scrubbing to quantify the biovolume of the total fouling community (ie soft and hard organisms) and the hard fouling community. Total biofouling ranged from 0.01 to 1.16 cm(3) cm(-2) throughout the immersion period; soft fouling constituted 22-87% of the total biovolume. In the future, this approach may be used to inform numerical models of fluid-surface interfaces and to evaluate, with high resolution, the morphology of fouling organisms in response to antifouling technologies.

Validation trials of a shipboard filter skid (p3SFS) demonstrate its utility for collecting living zooplankton.

Relatively large volumes of water-on the order of cubic meters-must be sampled and analyzed to generate statistically valid estimates of sparsely concentrated organisms, such as in treated ballast water. To this end, a third prototype of a shipboard filter skid (p3SFS) was designed and constructed. It consisted of two housings (each containing a 35 µm mesh filter bag) and its own pump and computer controller. Additionally, the skid had a drip sampler, which collected a small volume (∼ 10 L) of whole (unfiltered) water immediately upstream of the housings. Validation of the p3SFS occurred in two segments: (1) land-based trials, in which the collection of organisms ⩾ 50 µm (nominally zooplankton) by the p3SFS was compared to a plankton net, and (2) shipboard trials, in which ballast water was sampled aboard a ship. In both types of trials, the data collected showed the filter skid to be an appropriate flow-through sampling device.

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.

Ciliate ingestion and digestion: flow cytometric measurements and regrowth of a digestion-resistant Campylobacter jejuni.

We measured ingestion and digestion rates of the pathogenic bacterium Campylobacter jejuni by a freshwater ciliate Colpoda sp. to determine whether Campylobacter is able to resist protist digestion. Campylobacter and the nonpathogenic bacterium Pseudomonas putida LH1 were labeled with a 5-chloromethylfluorescein diacetate, which fluoresces in intact and active cells but fades when exposed to low pH environments, such as protistan food vacuoles. Ingestion and digestion rates were measured via flow cytometry as the change in ciliate fluorescence over time, which corresponded to the quantity of intracellular bacteria. The rate of Campylobacter ingestion exceeded the digestion rate. Ciliates retained labeled Campylobacter 5 h after ingestion was stopped. In contrast, ciliates grazing upon P. putida returned to baseline fluorescence within 5 h, indicating that P. putida were completely digested. The ability of intracellular Campylobacter to remain viable after ingestion was tested by sorting individual ciliates and bacterial cells into Campylobacter-selective media. Campylobacter growth occurred in 15% (± 5 SE) of wells seeded with highly fluorescent ciliates, whereas only 4% (± 1) of wells seeded with free-living Campylobacter exhibited growth. A key advantage of this approach is that it is rapid and should be applicable to other phagocytotis studies.