Marine primary production is a globally important component of the Earth's carbon cycle. This article describes several approaches for measuring marine primary production, including description of whether the approaches estimate of net or gross rates of production. Both incubation-dependent and–independent methodologies are described. Among the methodologies presented are those that rely on isotopes as tracers of both carbon and oxygen, in addition to approaches that rely on assessment of time-varying changes in concentrations of bioelements or particles in the upper ocean. Moreover, we highlight approaches that leverage remote and autonomous sensing platforms for their ability to provide high temporal and spatial resolution estimates of primary production.

The most probable number (MPN) assay is used to enumerate viable phytoplankton in the 10–50 μm size range for Type Approval of ballast water management systems. However, its validation has been questioned by the United States Coast Guard, which does not accept it as an alternative to its required method. To address key elements of method validation, three research laboratories used the same experimental protocols to enumerate viable organisms from uniformly viable and heat-killed cultures of three species of phytoplankton. Statistical analysis of the ratio of MPN estimates of viable organisms to measurements from flow cytometry was consistent with predictions based on perfect adherence to the method’s assumptions. These include reliable detection of all viable organisms and no false-positive results. The validation confirmed established estimates of the MPN method’s precision and demonstrated reproducibility, having been transferred to and implemented by two of the laboratories for the first time.

Ballast water management systems (BWMS) must be tested to assess their compliance with standards for the discharge of organisms, for example in the ≥ 10- and < 50-μm size category, which is dominated by phytoplankton. Assessment of BWMS performance with the vital stains fluorescein diacetate + 5-chlorofluorescein diacetate, required by regulations in the USA, is problematic in the case of ultraviolet-C (UVC) radiation. This is because UVC targets nucleotides-and thus reproduction, hence viability-rather than membrane integrity, which is assayed by the stains. The Serial Dilution Culture-Most Probable Number (SDC-MPN) method, long used to enumerate fragile phytoplankton from natural communities, is appropriate for counting viable phytoplankton. We developed QA/QC "best practice" criteria for its application as a robust and repeatable assay of viable cells in cultures of phytoplankton before and after experimental treatment, then constructed dose-response curves for UVC-induced loss of viable cells in 12 species of phytoplankton from seven divisions. Sensitivity to UVC, expressed as the dose required to reduce viability by 99%-the criterion for type approval of treatment systems-varied more than 10-fold and was not correlated with cell size. The form of the dose-response curves varied between taxa, with most having a threshold dose below which there was no reduction in viability. Analysis of the patterns of growth indicates that if recovery from treatment occurred, it was complete in 1 or 2 days in > 80% of cases, long before the assays were terminated. We conclude that the SDC-MPN assay as described is robust and adaptable for use on natural phytoplankton.

The shipping industry is critical to international commerce; however, contemporary shipping practices involve uptake and discharge of ballast water, which introduces the potential for transfer of nonindigenous, invasive species among geographically distinct habitats. To counteract this hazard, regulations for ballast water management have been implemented by the International Maritime Organization (IMO) and by regulatory agencies such as the United States Coast Guard (USCG). IMO and USCG discharge standards are numerically identical, but involve different definitions of treatment end points, which are based on fundamentally different biological assays for quantification of ballast water treatment effectiveness. Available assays for quantification of the responses of organisms in the 10-50 μm size range include vital stains based on fluorescein diacetate (FDA), sometimes used in combination with 5-chloromethylfluorescein diacetate (CMFDA), observations of motility, and the most probable number dilution culture method (MPN). The mechanisms and implications of these assays are discussed relative to the Type Approval process, which quantitatively evaluates compliance with ballast water discharge standards (BWDSs) under controlled shipboard and land-based tests. For antimicrobial processes that accomplish treatment by preventing subsequent replication of the target species, the FDA/CMFDA and MPN methods can yield dramatically different results. An important example of a treatment process that is affected by the choice of assay is ultraviolet (UV) irradiation. Results of laboratory and field experiments have demonstrated UV-based technologies to be effective for accomplishing the objectives of ballast water treatment (inactivation of cellular reproduction), when the MPN assay is used as the basis for evaluation. The FDA, CMFDA, motility, and MPN methods are subject to well recognized sources of error; however, the MPN method is based on a response that is consistent with the objectives of ballast water management as well as the mechanism of action of UV-based inactivation. Complementary assays are available for use in compliance testing; however, the development of relevant indicative tests remains as a research priority. Historical lessons learned from applications of vital stains (and other indirect methods) for quantification of microbial responses to UV irradiation in other settings also support the use of assays that provide a direct measure of growth and reproduction, such as MPN. Collectively, these observations point to the use of MPN assays as the standard for type testing, especially when UV-based treatment is employed.

The Stain-Motility (S-M) and Most Probable Number (MPN) methodologies may be used for enumerating viable organisms >= 10 mu m to <50 mu m in discharge water for administrative type approval of a ballast water management system (BWMS). Only MPN is suitable for assessing the efficacy of disinfection using UV radiation - a chemical-free treatment technology - but the U.S. Coast Guard has not approved an MPN-based approach as an alternate to their required S-M method. Approval depends on a demonstration of equivalence, but a framework for comparative validation is not well established. The purpose of this study is to provide such a framework. It is shown that the requirement for 5 consecutive successful results in BWMS type approval testing fundamentally changes the relationship between a method's precision and its effectiveness in ensuring compliance with regulations. False approval due to random measurement error is effectively eliminated for both methods because it requires 5 consecutive underestimates, and false rejection due to a single erroneously high measurement is more likely for the method with wider confidence limits, imposing an extra margin of safety for MPN. These results reverse conventional interpretations of efficacy based on method precision alone. Sources of systematic error (bias) are reviewed and methods for estimating the errors are described. If combined bias is positive (overestimation), either method would yield type-approval results fully compliant with regulations. Subject to similar negative bias, the less precise method (generally, MPN) would be more protective of the environment. An illustrative analysis of 64 paired MPN and S-M counts from BWMS trials indicates that neither method was significantly biased relative to the other. Considered in the framework for method validation described here, available evidence strengthens arguments that in a BWMS type approval testing regime, the efficacy of the MPN method is equivalent to that of Stain-Motility. (C) 2018 The Author. Published by Elsevier B.V.

The fluorescent stain Nile Red has been used extensively for the quantification of lipids in phytoplankton, including microalgae, because it preferentially stains neutral lipids and it is economical and sensitive to use for screening purposes. Although its basic application has not changed for several decades, recent improvements have been made to improve its utility across applications. Here we describe additional refinements in its application and interpretation as a high-throughput method for the rapid quantification of neutral lipids in liquid cultures of marine phytoplankton. Specifically we address (1) interspecies comparisons, (2) fluorescence excitation and emission wavelengths, and (3) the time course of the Nile Red signal in the context of using bulk or cell-specific fluorescence to quantify neutral lipids of live or preserved cells. We show that with proper caution in its interpretation across species and physiological states the quantity of lipid in hundreds of small volume samples can be reliably assessed daily using a refined Nile Red protocol.

Recently, the U.S. Coast Guard (USCG) rejected the Serial Dilution Culture-Most Probable Number (SDC-MPN) method for enumerating viable phytoplankton cells in ballast water discharge as an alternate to their prescribed method — the Environmental Technology Verification (ETV) Protocol. This method distinguishes living from dead organisms using vital stains and motility. Succinctly, the USCG position has been that the ETV Protocol is a reliable and repeatable efficacy test and the SDC-MPN method is not. New evidence and an expanded consideration of published research supports a fundamentally different assessment. A peer-reviewed quantitative evaluation of ETV vital stains for 24 species of phytoplankton has conclusively established that the ETV Protocol, even with observations of motility, is not reliable for all species. In contrast, published results suggest that errors in the method were small for the limited number of locations studied to date. It is possible that the communities tested in these were dominated by species that can be classified accurately using vital stains. Even so, it must be acknowledged that the reliability and accuracy of vital stains is untested for thousands of species of phytoplankton. Introduced in 1951, the SDC-MPN method for phytoplankton is an established approach for use with multi-species communities. As applied to ballast water testing, SDC-MPN is much less vulnerable to methodological uncertainties than has been assumed. Notably, all species of phytoplankton need not be cultured in the conventional sense. Rather, a single viable cell in a dilution tube need grow only enough to be detected — a requirement known to have been met by otherwise uncultured species. Further, delayed restoration of viability after treatment with ultraviolet radiation (UV) is not a problem: organisms repair UV damage quickly or not at all, consistent with the assumptions of the test. Two critical methodological failures could compromise protection of the environment in ballast water testing: living organisms that do not stain or move, and viable organisms that do not grow to detection in the MPN cultures. These can be assessed with complementary measurements, but importantly, the relative protection of each method can be evaluated by comparing counts of living cells from the ETV Protocol with counts of viable cell from SDC-MPN in untreated samples. Available evidence provides no basis for concluding that either method is consistently less protective. However, as applied in ballast water testing, the statistical estimate of MPN is less precise. On this basis, SDC-MPN is worse for a single test. But, counter-intuitively, it is more protective of the environment when five consecutive tests must be passed for type approval, because the likelihood of one false rejection out of five tests is higher and five false passes would be exceedingly rare. Addressing only the science, we conclude that both the ETV Protocol and the SDC-MPN method, though imperfect, are currently appropriate for assessing the efficacy of ballast water management systems in a type-approval testing regime. In closing, we show proof of concept for a rapid assay of viability, benchmarked against SDC-MPN, that could be well suited for routine assessment of treatment system performance.

Regulations for ballast water treatment specify limits on the concentrations of living cells in discharge water. The vital stains fluorescein diacetate (FDA) and 5‐chloromethylfluorescein diacetate (CMFDA) in combination have been recommended for use in verification of ballast water treatment technology. We tested the effectiveness of FDA and CMFDA, singly and in combination, in discriminating between living and heat‐killed populations of 24 species of phytoplankton from seven divisions, verifying with quantitative growth assays that uniformly live and dead populations were compared. The diagnostic signal, per‐cell fluorescence intensity, was measured by flow cytometry and alternate discriminatory thresholds were defined statistically from the frequency distributions of the dead or living cells. Species were clustered by staining patterns: for four species, the staining of live versus dead cells was distinct, and live‐dead classification was essentially error free. But overlap between the frequency distributions of living and heat‐killed cells in the other taxa led to unavoidable errors, well in excess of 20% in many. In 4 very weakly staining taxa, the mean fluorescence intensity in the heat‐killed cells was higher than that of the living cells, which is inconsistent with the assumptions of the method. Applying the criteria of ≤5% false negative plus ≤5% false positive errors, and no significant loss of cells due to staining, FDA and FDA+CMFDA gave acceptably accurate results for only 8–10 of 24 species (i.e., 33%–42%). CMFDA was the least effective stain and its addition to FDA did not improve the performance of FDA alone.

Discharge standards for ballast water treatment (BWT) systems are based on concentrations of living cells, for example, as determined with vital stains. Ultraviolet radiation (UV) stops the reproduction of microorganisms without killing them outright; they are living, but not viable, and ecologically as good as dead. Consequently, UV-treated discharge can be compliant with the intent of regulation while failing a live/dead test. An alternative evaluation of BWT can be proposed based on the assessment of viable, rather than living, cells in discharge water. In principle, the serial dilution culture-most probable number (SDC-MPN) method provides the appropriate measure for phytoplankton. But, the method has been criticized, particularly because it is thought that many phytoplankton species cannot be cultured. A review of the literature shows that although SDC-MPN has been used for more than 50 years-generally to identify and count phytoplankton species that cannot be preserved-its application to enumerate total viable phytoplankton seems to be new, putting past criticisms of the method in a different light. Importantly, viable cells need to grow only enough to be detected, not to be brought into sustained culture, and competition between species in a dilution tube is irrelevant as long as the winner is detectable. Thorough consideration of sources of error leads to recommendations for minimizing and quantifying uncertainties by optimizing growth conditions and conducting systematic comparisons. We conclude that with careful evaluation, SDC-MPN is potentially an effective method for assessing the viability of phytoplankton after BWT.

The phenomenon of subsurface chlorophyll maximum layers (SCMLs) is not a unique ecological response to environmental conditions; rather, a broad range of interacting processes can contribute to the formation of persistent layers of elevated chlorophyll a concentration (Chl) that are nearly ubiquitous in stratified surface waters. Mechanisms that contribute to the formation and maintenance of the SCMLs include a local maximum in phytoplankton growth rate near the nutricline, photoacclimation of pigment content that leads to elevated Chl relative to phytoplankton biomass at depth, and a range of physiologically influenced swimming behaviors in motile phytoplankton and buoyancy control in diatoms and cyanobacteria that can lead to aggregations of phytoplankton in layers, subject to grazing and physical control. A postulated typical stable water structure characterizes consistent patterns in vertical profiles of Chl, phytoplankton biomass, nutrients, and light across a trophic gradient structured by the vertical flux of nutrients and characterized by the average daily irradiance at the nutricline. Hypothetical predictions can be tested using a nascent biogeochemical global ocean observing system. Partial results to date are generally consistent with predictions based on current knowledge, which has strong roots in research from the twentieth century.

An equation for the rate of photosynthesis as a function of irradiance introduced by T. T. Bannister included an empirical parameter b to account for observed variations in curvature between the initial slope and the maximum rate of photosynthesis. Yet researchers have generally favored equations with fixed curvature, possibly because b was viewed as having no physiological meaning. We developed an analytic photosynthesis‐irradiance equation relating variations in curvature to changes in the degree of connectivity between photosystems, and also considered a recently published alternative, based on changes in the size of the plastoquinone pool. When fitted to a set of 185 observed photosynthesis‐irradiance curves, it was found that the Bannister equation provided the best fit more frequently compared to either of the analytic equations. While Bannister's curvature parameter engendered negligible improvement in the statistical fit to the study data, we argued that the parameter is nevertheless quite useful because it allows for consistent estimates of initial slope and saturation irradiance for observations exhibiting a range of curvatures, which would otherwise have to be fitted to different fixed‐curvature equations. Using theoretical models, we also found that intra‐ and intercellular self‐shading can result in biased estimates of both curvature and the saturation irradiance parameter. We concluded that Bannister's is the best currently available equation accounting for variations in curvature precisely because it does not assign inappropriate physiological meaning to its curvature parameter, and we proposed that b should be thought of as the expression of the integration of all factors impacting curvature.

As bivalve aquaculture continues to grow, it is imperative to understand the spatially-explicit interactions between farmed bivalves and the environment. However, the ability of models to represent a large number of bivalve ecophysiology and environmental variables-in 3-D spatially-explicit domains-has been limited by computational constraints. To overcome some of these computational limitations, we developed an optimized hybrid model by two-way coupling a state-of-the-art Eulerian model (Regional Ocean Modeling System; ROMS) that simulates physical, planktonic and sediment dynamics, with an individual-based model (IBM) that simulates shellfish ecophysiology (SHELL-E). The IBM model efficiently represents sparsely-distributed variables that do not occur in every grid cell of the domain, and simplifies the representation of complex life-history and physiological processes, like spawning events. We applied the hybrid model to a mussel farm in Ship Harbour (Eastern Canada) and compared model results against measurements of physical variables, water samples (chlorophyll, nutrients, oxygen and suspended sediments) and mussel size distributions. The hybrid model reproduced the main dynamics of the physical, planktonic and sediment Eulerian variables, as well as the bivalve ecophysiology IBM variables. Prognostic limitations estimated by the model suggested that mussels were temperature-stressed during parts of the summer, and food-limited during parts of the winter. We also used the hybrid model to estimate the production carrying capacity of the farm and we found that the farm is not overstocked. However, we also found that the estimation of carrying capacity strongly depends on the inferred natural mortality, which is difficult to estimate accurately. This work aims to transfer sound and open-source oceanographic tools (i.e. ROMS) into the applied fields of aquaculture research and management. (C) 2013 Authors. Published by Elsevier B.V. All rights reserved.

The nitrogen cycle is fundamental to Earth's biogeochemistry. Yet major uncertainties of quantification remain, particularly regarding the global oceanic nitrogen fixation rate. Hydrogen is produced during nitrogen fixation and will become supersaturated in surface waters if there is net release from diazotrophs. Ocean surveys of hydrogen supersaturation thus have the potential to illustrate the spatial and temporal distribution of nitrogen fixation and to guide the far more onerous but quantitative methods for measuring it. Here we present the first transect of high resolution measurements of hydrogen supersaturations in surface waters along a meridional 10,000 km cruise track through the Atlantic. We compare measured saturations with published measurements of nitrogen fixation rates and also with model-derived values. If the primary source of excess hydrogen is nitrogen fixation and has a hydrogen release ratio similar to Trichodesmium, our hydrogen measurements would point to similar rates of fixation in the North and South Atlantic, roughly consistent with modeled fixation rates but not with measured rates, which are lower in the south. Possible explanations would include any substantial nitrogen fixation by newly discovered diazotrophs, particularly any having a hydrogen release ratio similar to or exceeding that of Trichodesmium; undersampling of nitrogen fixation south of the equator related to excessive focus on Trichodesmium; and methodological shortcomings of nitrogen fixation techniques that cause a bias toward colonial diazotrophs relative to unicellular forms. Alternatively, our data are affected by an unknown hydrogen source that is greater in the southern half of the cruise track than the northern.

This paper describes an investigation of the carbon footprint associated with plastic trays, used as packaging for foodstuffs (e.g., mushrooms). In recent years there has been an increase in both consumer and legislative pressure on the packaging sector to reduce the environmental impact of its products, which are often only single use items. Using data from a plastics manufacturer, a cradle-to-grave study was conducted for trays produced from recycled polyethylene terephthalate, calculating their product carbon footprint and analysing how various parameters affect the carbon footprint. A model based on a spreadsheet analysis was developed, which allows the product carbon footprint to be determined using production batch data. It was found that the cradle-to-grave carbon footprint of 1 kg of recycled polyethylene terephthalate trays containing 85% recycled content was 1.538 kg CO(2)e. The raw material, manufacturing, secondary packaging, transport and end-of-life stages each contributed 45%, 38%, 5%, 3% and 9% of the total life cycle greenhouse gases respectively. The recycled content of raw material was found to have a significant effect on product carbon footprint: a 24% decrease in tray carbon footprint could be obtained by manufacturing trays from 100% recycled content, compared to the current recycled content level of 85%. A reduction in tray weight was found to give almost an equivalent proportionate reduction in carbon footprint, with 20% and 30% tray weight reductions resulting in product carbon footprint reductions of 18.7% and 28% respectively. Transport was found to only contribute a minor amount of the greenhouse gases (3%) and hence improving transport efficiency had very little effect on the carbon footprint. The effect of end-of-life treatment was also found to be relatively small. The worst case scenario of no recycling taking place in the end-of-life stage results in the carbon footprint of the trays increasing by 2.7%, while increasing the recycling rate from 23.7% to 32% and 50%, results in the carbon footprint decreasing by 1% and 3% respectively. In both the extrusion and thermoforming processes, the specific manufacturing carbon footprints arising from consumption of electricity, chilled water energy and compressed air were found to decrease logarithmically with production speed. The greatest reductions in the carbon footprint of recycled polyethylene terephthalate trays can be achieved in the raw material and manufacturing life cycle stages. The proportion of recycled raw material should be maximised while extrusion and thermoforming process speeds should be optimised as significant manufacturing energy reductions can be attained when the speeds of both processes are increased. Tray light-weighting should be implemented to as great an extent as possible without compromising tray structural integrity while high recycling rates in the end-of-life stage should continue to be targeted. (C) 2013 Elsevier Ltd. All rights reserved.