Validation of an adenosine triphosphate (ATP) model for 10-50 µm plankton.

A recent model demonstrated that the adenosine triphosphate (ATP) content of spherical aquatic organisms with a 10 to 50 µm diameter is between 0.16 and 19.9 pg cell-1. Here, the model is validated by comparing microscopy-based counts with ATP concentrations from a commercial ATP kit. The measured ATP content of both freshwater and marine organisms 10 to 50 µm size range falls in the 0.16 to 19.9 pg cell-1 model range. On average, freshwater organisms contain 0.33 pg ATP cell-1, have a spherical equivalent diameter (SED) of 13 µm, while marine organisms have 0.89 pg ATP cell-1 and a SED of 18 µm. In addition, their 13 to 18 µm size is within the 10 to 50 µm ballast water size range and in agreement with the 15 µm mean SED of a coastal plankton size-distribution model. This study concludes that the ATP-model is reliable, emphasizing the need for caution when converting three-dimensional biomass proxies into linear cell concentrations.

Human fatalities related to a Phaeocystis harmful algal bloom in the North Sea.

On May 11, 2020, five surfers drowned after asphyxiation in a massive foam bank near the Dutch coast. We present a detailed account of the event and an examination of factors that govern local foam events, that are traditionally caused by Phaeocystis globosa (Prymnesiophyceae). The data support the hypothesis that the foam originated from a Phaeocystis bloom which if correct would make this accident the first report of human fatalities due to a harmful algal bloom in The Netherlands, and the first globally due to a Phaeocystis bloom. The rarity of massive foam banks and the absence of local real-time plankton monitoring makes it difficult to accurately predict dangerous situations as in 2020. As a prevention, surfers and other water sportsmen need to apply good seamanship during stormy and foamy conditions.

Suspended matter filtration causes a counterintuitive increase in UV-absorption.

In water treatment, filtration is often a first step to avoid interference of chemical or UV-disinfection with suspended matter (SPM). Surprisingly, in testing a ballast water filter with 25 and 40 µm mesh screens, UV-absorption (A, 254 nm) of filtered water increased with the largest increase in the finest screen. The hypothesis that filtration partly removes large particles and partly replaces them with small unfiltered ones, leading to an overall increase in absorption, was tested by measuring particle counts, particle-size distributions (PSD) and by modeling the Mass Normalized Beam Attenuation Coefficient (A/SPM) before and after filtration. An independent model verification was made by measuring and modeling A/SPM of three differently sized Arizona test dust suspensions. It is concluded that filtration is a good pretreatment for chemical disinfection systems because it removes the suspended matter mass, but that the production of smaller particles increases UV-absorption and hence may reduce disinfection performance.

False-positive enterococci counts in seawater with the IDEXX Enterolert-E most probable number technique caused by Bacillus licheniformis.

Enterolert-E is an easy-to-use method for the enumeration of enterococci in water samples as an indicator of fecal pollution. This most probable number technique replaced the laborious and more time-consuming MEA-BEA plating method, and it is used extensively in ballast water testing and monitoring. In spring 2018, the Control Union Water ballast water test facility measured high enterococci concentrations in Wadden Sea water without any correlation with polluted freshwater input. By isolating bacteria from samples incubated in Enterolert-E culture medium, followed by analyses of colony morphology and DNA, it is shown that these erroneously high concentrations were caused by Bacillus licheniformis, a gram-positive rod-shaped chlorine-resistant bacterium. It is concluded that control analyses or the MEA-BEA method or dilution to reduce salinity must be performed when high enterococci concentrations are measured in water samples that are not suspected to be polluted.

Operational and environmental factors affecting disinfection byproducts formation in ballast water treatment systems.

To prevent the worldwide spread of invasive aquatic species, the ballast water of ships may be disinfected with either physical or chemical treatment systems. Excess chemicals, such as chlorine, are neutralized before the ballast water can be discharged. Unfortunately, disinfection byproducts (DBPs) formed during treatment are not neutralized and remain potentially toxic. In this study, DBPs obtained from land-based tests of seven different ballast water treatment systems (BWTSs) have been statistically analyzed. Effect of operational factors (treatment type, holding time, source of carbon and active substance dosages) and environmental variables (salinity, pH, temperature, organic matter) were related to the formation of DBPs, such as trihalomethanes (THMs), haloacetic acids (HAAs), haloacetonitriles (HANs) and aldehydes. THMs and HAAs were the groups with major occurrences and concentrations detected in all BWTSs. Treatment type and source of carbon were the operational factors with major significance on DBP production, especially in chlorination systems. Salinity is the main variable determining DBP composition, as it differs between brominated-DBPs and chlorinated-DBPs. Concentration and type of organic matter (dissolved and particulate) have also a significant influence on the formation of total DBPs. According to the specific group of DBPs, some factors get significance. For instance, THMs are significantly affected by pH, and the production of aldehydes correlates positively with oxidant dose.

Abiotic drivers of interannual phytoplankton variability and a 1999-2000 regime shift in the North Sea examined by multivariate statistics.

The Dutch coastal zone is a region of the North Sea with a marked interannual and long-term abiotic and phytoplankton variability. To investigate the relationship between abiotic variability and phytoplankton composition, two routine water monitoring data sets (1991-2005) were examined. Multivariate statistics revealed two significant partitions in the data. The first consisted of interannual abiotic fluctuations that were correlated to Rhine discharge that affected the abundance of summer and autumn diatom species. The second partition was caused by a shift in the abiotic data from 1998 to 1999 that was followed by a shift in phytoplankton composition from 1999 to 2000. Important factors in the abiotic shift were decreases in suspended matter (SPM) and phosphate (DIP) concentrations, as well as in pH. The decrease in SPM was caused by a reduction in wind speed. The increase in water column daily irradiance from the decrease in SPM led to increases in the abundance of winter-spring species, notably the prymnesiophyte Phaeocystis globosa. Because wind speed is related to the North Atlantic Oscillation (NAO) index it was possible to correlate NAO index and P. globosa abundance. Only five abiotic variables representing interannual and long-term variability, including Rhine discharge and NAO index, were needed to model the observed partitions in phytoplankton composition. It was concluded that interannual variability in the coastal phytoplankton composition was related to year-to-year changes in river discharge while the long-term shift was caused by an alternating large-scale meteorological phenomenon.

Assessment of imaging-in-flow system (FlowCAM) for systematic ballast water management.

Assessing the disinfection of ballast water and its compliance with international standards requires determining the size, viability, and concentration of planktonic organisms. The FlowCAM (Flow Cytometer and Microscope) is an Imaging Flow Cytometry designed to obtain the particle concentration, images, and quantitative morphologic information. The objective in this paper is to establish the basis for transforming the FlowCAM from being a laboratory analyzer into a tool for systematic monitoring of ballast water. The capacity of the FlowCAM was evaluated by analyzing artificial microbeads, phytoplankton monocultures, and real seawater samples. Microbead analyses reported high accuracy and precision in size and concentration measurements. Monoculture analyses showed the effect of disinfection treatments in cell appearance and growth. Low concentration and heterogeneity of particles in real seawater analyses require the comprehensive observation of images by experts. Additionally, some physical characteristics of the device must be improved. The optimization of device configuration enables the quick transferring of files and information between parties involved in ballast water management. FlowCAM may become a feasible technology for this after the device and protocols are adapted.

Immediate ecotoxicological effects of short-lived oil spills on marine biota.

Marine environments are frequently exposed to oil spills as a result of transportation, oil drilling or fuel usage. Whereas large oil spills and their effects have been widely documented, more common and recurrent small spills typically escape attention. To fill this important gap in the assessment of oil-spill effects, we performed two independent supervised full sea releases of 5 m(3) of crude oil, complemented by on-board mesocosm studies and sampling of accidentally encountered slicks. Using rapid on-board biological assays, we detect high bioavailability and toxicity of dissolved and dispersed oil within 24 h after the spills, occurring fairly deep (8 m) below the slicks. Selective decline of marine plankton is observed, equally relevant for early stages of larger spills. Our results demonstrate that, contrary to common thinking, even small spills have immediate adverse biological effects and their recurrent nature is likely to affect marine ecosystem functioning.

A comparison of six different ballast water treatment systems based on UV radiation, electrochlorination and chlorine dioxide.

The spread of aquatic invasive species through ballast water is a major ecological and economical threat. Because of this, the International Maritime Organization (IMO) set limits to the concentrations of organisms allowed in ballast water. To meet these limits, ballast water treatment systems (BWTSs) were developed. The main techniques used for ballast water treatment are ultraviolet (UV) radiation and electrochlorination (EC). In this study, phytoplankton regrowth after treatment was followed for six BWTSs. Natural plankton communities were treated and incubated for 20 days. Growth, photosystem II efficiency and species composition were followed. The three UV systems all showed similar patterns of decrease in phytoplankton concentrations followed by regrowth. The two EC and the chlorine dioxide systems showed comparable results. However, UV- and chlorine-based treatment systems showed significantly different responses. Overall, all BWTSs reduced phytoplankton concentrations to below the IMO limits, which represents a reduced risk of aquatic invasions through ballast water.

Assessment of didecyldimethylammonium chloride as a ballast water treatment method.

Ballast water-mediated transfer of aquatic invasive species is considered a major threat to marine biodiversity, marine industry and human health. A ballast water treatment is needed to comply with International Maritime Organization (IMO) ballast water discharge regulations. Didecyldimethylammonium chloride (DDAC) was tested for its applicability as a ballast water treatment method. The treatment of the marine phytoplankton species Tetraselmis suecica, Isochrysis galbana and Chaetoceros calcitrans showed that at 2.5 µL L(-1) DDAC was able to inactivate photosystem II (PSII) efficiency and disintegrate the cells after 5 days of dark incubation. The treatment of natural marine plankton communities with 2.5 µL L(-1) DDAC did not sufficiently decrease zooplankton abundance to comply with the IMO D-2 standard. Bivalve larvae showed the highest resistance to DDAC. PSII efficiency was inactivated within 5 days but phytoplankton cells remained intact. Regrowth occurred within 2 days of incubation in the light. However, untreated phytoplankton exposed to residual DDAC showed delayed cell growth and reduced PSII efficiency, indicating residual DDAC toxicity. Natural marine plankton communities treated with 5 µL L(-1) DDAC showed sufficient disinfection of zooplankton and inactivation of PSII efficiency. Phytoplankton regrowth was not detected after 9 days of light incubation. Bacteria were initially reduced due to the DDAC treatment but regrowth was observed within 5 days of dark incubation. Residual DDAC remained too high after 5 days to be safely discharged. Two neutralization cycles of 50 mg L(-1) bentonite were needed to inactivate residual DDAC upon discharge. The inactivation of residual DDAC may seriously hamper the practical use of DDAC as a ballast water disinfectant.

Microbial dynamics in acetate-enriched ballast water at different temperatures.

The spread of invasive species through ships' ballast water is considered as a major ecological threat to the world's oceans. For that reason, the International Maritime Organization (IMO) has set performance standards for ballast water discharge. Ballast water treatment systems have been developed that employ either UV-radiation or 'active substances' to reduce the concentration of living cells to below the IMOs standards. One such active substance is a chemical mixture known as Peraclean(®) Ocean. The residual of Peraclean(®) Ocean is acetate that might be present at high concentrations in discharged ballast water. In cold coastal waters the breakdown of acetate might be slow, causing a buildup of acetate concentrations in the water if regularly discharged by ships. To study the potential environmental impact, microbial dynamics and acetate degradation were measured in discharge water from a Peraclean(®) Ocean treatment system in illuminated microcosms. In addition, microbial dynamics and acetate degradation were studied at -1, 4, 10, 15 and 25°C in dark microcosms that simulated enclosed ballast water tanks. Acetate breakdown indeed occurred faster at higher temperatures. At 25°C the highest bacteria growth, fastest nutrient and oxygen consumption and highest DOC reduction occurred. On the other hand, at -1°C bacterial growth was strongly delayed, only starting to increase after 12 days. Furthermore, at 25°C the acetate pool was not depleted, probably due to nutrient and oxygen limitation. This means that not all acetate will be broken down in ballast water tanks, even during long voyages in warm waters. In addition, at low temperatures acetate breakdown in ballast water tanks and in discharged water will be extremely slow. Therefore, regular discharge of acetate enriched ballast water in harbors and bays may cause eutrophication and changes in the microbial community, especially in colder regions.

Impact of a simulated oil spill on benthic phototrophs and nitrogen-fixing bacteria in mudflat mesocosms.

Coastal and estuarine ecosystems are highly susceptible to crude oil pollution. Therefore, in order to examine the resilience of benthic phototrophs that are pivotal to coastal ecosystem functioning, we simulated an oil spill in tidal mesocosms consisting of intact sediment cores from a mudflat at the mouth of the Colne Estuary, UK. At day 21, fluorescence imaging revealed a bloom of cyanobacteria on the surface of oiled sediment cores, and the upper 1.5 cm thick sediment had 7.2 times more cyanobacterial and 1.7 times more diatom rRNA sequences when treated with oil. Photosystem II operating efficiency (Fq'/Fm') was significantly reduced in oiled sediments at day 7, implying that the initial diatom-dominated community was negatively affected by oil, but this was no longer apparent by day 21. Oil addition significantly reduced numbers of the key deposit feeders, and the decreased grazing pressure is likely to be a major factor in the increased abundance of both diatoms and cyanobacteria. By day 5 concentrations of dissolved inorganic nitrogen were significantly lower in oiled mesocosms, likely resulting in the observed increase in nifH-containing, and therefore potentially dinitrogen-fixing, cyanobacteria. Thus, indirect effects of oil, rather than direct inhibition, are primarily responsible for altering the microphytobenthos.

Central role of dynamic tidal biofilms dominated by aerobic hydrocarbonoclastic bacteria and diatoms in the biodegradation of hydrocarbons in coastal mudflats.

Mudflats and salt marshes are habitats at the interface of aquatic and terrestrial systems that provide valuable services to ecosystems. Therefore, it is important to determine how catastrophic incidents, such as oil spills, influence the microbial communities in sediment that are pivotal to the function of the ecosystem and to identify the oil-degrading microbes that mitigate damage to the ecosystem. In this study, an oil spill was simulated by use of a tidal chamber containing intact diatom-dominated sediment cores from a temperate mudflat. Changes in the composition of bacteria and diatoms from both the sediment and tidal biofilms that had detached from the sediment surface were monitored as a function of hydrocarbon removal. The hydrocarbon concentration in the upper 1.5 cm of sediments decreased by 78% over 21 days, with at least 60% being attributed to biodegradation. Most phylotypes were minimally perturbed by the addition of oil, but at day 21, there was a 10-fold increase in the amount of cyanobacteria in the oiled sediment. Throughout the experiment, phylotypes associated with the aerobic degradation of hydrocarbons, including polycyclic aromatic hydrocarbons (PAHs) (Cycloclasticus) and alkanes (Alcanivorax, Oleibacter, and Oceanospirillales strain ME113), substantively increased in oiled mesocosms, collectively representing 2% of the pyrosequences in the oiled sediments at day 21. Tidal biofilms from oiled cores at day 22, however, consisted mostly of phylotypes related to Alcanivorax borkumensis (49% of clones), Oceanospirillales strain ME113 (11% of clones), and diatoms (14% of clones). Thus, aerobic hydrocarbon biodegradation is most likely to be the main mechanism of attenuation of crude oil in the early weeks of an oil spill, with tidal biofilms representing zones of high hydrocarbon-degrading activity.

CURRENT KNOWLEDGE OF THE LIFE CYCLES OF PHAEOCYSTIS GLOBOSA AND PHAEOCYSTIS ANTARCTICA (PRYMNESIOPHYCEAE)(1).

Despite continuous efforts since the 1950s and more recent advances in culturing flagellates and nonflagellate cells of the prymnesiophyte Phaeocystis, a number of different life-cycle models exist today that appear to apply for P. globosa Scherff. and P. antarctica G. Karst., both spherical colony formers. In one such model, this life cycle consists of three different flagellates and one nonmotile cell stage that is embedded in carbohydrate matrix-forming colonies of different sizes and forms. Recently, noncolonial aggregates of diploid nonmotile cells attached to surfaces of diatoms were put forward as a new stage in the sexual life cycle of P. antarctica. However, it can be discussed that these "attached aggregates" (AAs) are an intermediate between motile diploid flagellates, with their well-known tendency to adhere to surfaces, and the young spherical colony with its diploid nonmotile cells, which in nature is commonly found attached to diatoms. A life-cycle model pertaining to both P. globosa and P. antarctica is presented.

FLOW CYTOMETRIC APPLICABILITY OF FLUORESCENT VITALITY PROBES ON PHYTOPLANKTON1.

The applicability of six fluorescent probes (four esterase probes: acetoxymethyl ester of Calcein [Calcein-AM], 5-chloromethylfluorescein diacetate [CMFDA], fluorescein diacetate [FDA], and 2',7'-dichlorofluorescein diacetate [H2 DCFDA]; and two membrane probes: bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4 (3)] and SYTOX-Green) as vitality stains was tested on live and killed cells of 40 phytoplankton strains in exponential and stationary growth phases, belonging to 12 classes and consisting of four cold-water, 26 temperate, and four warm-water species. The combined live/dead ratios of all six probes indicated significant differences between the 12 plankton classes (P < 0.01) and between individual species (P < 0.05). No specific differences were observed among strains of one species, among species or strains from different origin, nor between cells in exponential and stationary growth phase except for FDA. FDA showed a significant (P < 0.05) drop of <20% in fluorescence intensity in stationary cells. Of the four esterase probes, the live/dead ratios of FDA and CMFDA were not significantly different from each other, and both performed better than Calcein-AM and H2 DCFDA (P < 0.001). Of the two membrane probes, DIBAC4 (3) stained rhodophytes and euglenophytes much better than SYTOX-Green. The 13 algal strains best stainable (high live/dead ratios) among all six probes belonged to nine genera from six classes of phytoplankton. In conclusion, FDA, CMFDA, DIBAC4 (3), and SYTOX-Green represent a wide choice of vitality probes in the study of phytoplankton ecology, applicable in many species from different algal classes, originating from different regions and at different stages of growth.

Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment.

The bacterial diversity of a chronically oil-polluted retention basin sediment located in the Berre lagoon (Etang-de-Berre, France) was investigated. This study combines chemical and molecular approaches in order to define how the in situ petroleum hydrocarbon contamination level affects the bacterial community structure of a subsurface sediment. Hydrocarbon content analysis clearly revealed a gradient of hydrocarbon contamination in both the water and the sediment following the basin periphery from the pollution input to the lagoon water. The nC17 and pristane concentrations suggested alkane biodegradation in the sediments. These results, combined with those of terminal-restriction fragment length polymorphism analysis of the 16S rRNA genes, indicated that bacterial community structure was obviously associated with the gradient of oil contamination. The analysis of bacterial community composition revealed dominance of bacteria related to the Proteobacteria phylum (Gamma-, Delta-, Alpha-, Epsilon- and Betaproteobacteria), Bacteroidetes and Verrucomicrobium groups and Spirochaetes, Actinobacteria and Cyanobacteria phyla. The adaptation of the bacterial community to oil contamination was not characterized by dominance of known oil-degrading bacteria, because a predominance of populations associated to the sulphur cycle was observed. The input station presented particular bacterial community composition associated with a low oil concentration in the sediment, indicating the adaptation of this community to the oil contamination.

Water management strategies against toxic Microcystis blooms in the Dutch delta.

To prevent flooding of the Dutch delta, former estuaries have been impounded by the building of dams and sluices. Some of these water bodies, however, experience major ecological problems. One of the problem areas is the former Volkerak estuary that was turned into a freshwater lake in 1987. From the early 1990s onward, toxic Microcystis blooms dominate the phytoplankton of the lake every summer. Two management strategies have been suggested to suppress these harmful algal blooms: flushing the lake with fresh water or reintroducing saline water into the lake. This study aims at an advance assessment of these strategies through the development of a mechanistic model of the population dynamics of Microcystis. To calibrate the model, we monitored the benthic and pelagic Microcystis populations in the lake during two years. Field samples of Microcystis were incubated in the laboratory to estimate growth and mortality rates as functions of light, temperature, and salinity. Recruitment and sedimentation rates were measured in the lake, using traps, to quantify benthic-pelagic coupling of the Microcystis populations. The model predicts that flushing with fresh water will suppress Microcystis blooms when the current flushing rate is sufficiently increased. Furthermore, the inlet of saline water will suppress Microcystis blooms for salinities exceeding 14 g/L. Both management options are technically feasible. Our study illustrates that quantitative ecological knowledge can be a helpful tool guiding large-scale water management.