Flow-through imaging and automated analysis of oil-exposed early stage Atlantic cod (Gadus morhua).

Toxicology studies in early fish life stages serve an important function in measuring the impact of potentially harmful substances, such as crude oil, on marine life. Morphometric analysis of larvae can reveal the effects of such substances in retarding growth and development. These studies are labor intensive and time consuming, typically resulting in only a small number of samples being considered. An automated system for imaging and measurement of experimental animals, using flow-through imaging and an artificial neural network to allow faster sampling of more individuals, has been described previously and used in toxicity experiments. This study compares the performance of the automated imaging and analysis system with traditional microscopy techniques in measuring biologically relevant endpoints using two oil treatments as positive controls. We demonstrate that while the automated system typically underestimates morphometric measurements relative to analysis of manual microscopy images, it shows similar statistical results to the manual method when comparing treatments across most endpoints. It allows for many more individual specimens to be sampled in a shorter time period, reducing labor requirements and improving statistical power in such studies, and is noninvasive allowing for repeated sampling of the same population.

Catch my drift? Between-farm dispersal of biofouling waste from salmon pen net cleaning: Potential risks for fish health.

Biofouling is a serious challenge for global salmon aquaculture and farmers have to regularly clean pen nets to avoid impacts on stock health and farms' structural integrity. The removed material is released into the surrounding environment. This includes cnidarian species such as hydroids, whose nematocyst-bearing fragments can impact gill health and fish welfare. There is also increasing evidence of the association of parasites and pathogens with biofouling organisms and cleaning fragments. It is unknown whether and how far local current regimes disperse biofouling material and whether this material reaches and interacts with adjacent pens or even neighbouring farms downstream, or wild fish populations in surrounding environments. We focussed on the cnidarian hydroid Ectopleura larynx, one of the most abundant biofouling species on Norwegian aquaculture installations. Using a 3D hydrodynamic model parameterised with physical and biological properties of hydroid particles (derived via field and laboratory studies), we simulated the dispersal of net cleaning waste from two Norwegian salmon farms. Our results demonstrate that net cleaning waste is extensively dispersed throughout neighbouring pens, and even to adjacent aquaculture facilities. Salmon were exposed to concentrations of biofouling particles up to 41-fold elevated compared to background concentrations, and for up to 30.5 h. Maximum dispersal distance of hydroid particles was 5.5 km from the point of release, achieved largely within 48 h. Least-cost distance calculations show that this distance exceeds the nearest-neighbour distance of 70 % of Norway's salmon farms (654 farms). Our study provides some evidence that actions taken to manage biofouling at salmon farms may affect neighbouring farms and surrounding natural environments. The results highlight the potential risks associated with net cleaning: the dispersal of harmful cnidarian particles, associated pathogens, and non-indigenous species, thus underlining the need for novel farming or net cleaning technologies that prevent the release of potentially harmful cleaning waste.

Effects of mine tailing exposure on the development of early life stages of the marine copepod Calanus finmarchicus.

The demand for mineral resources is increasing mining activities worldwide. In Norway, marine tailing disposal (MTD) is practiced, introducing mineral particles into fjord ecosystems. We investigated the effects of two concentrations (high and low) of fine tailings from a CaCO3 processing plant on early life stages of the marine copepod Calanus finmarchicus. Results show that the exposure did not significantly impact hatching success or development in non- and early feeding life stages. However, feeding stage nauplii ingested tailings, which caused a significantly slower development in later nauplii stages in high exposure groups, with most individuals being two stages behind the control group. Further, high mortality occurred in late nauplii and early copepodite stages in low exposure groups, which could be caused by insufficient energy accumulation and depleted energy reserves during development. Individuals exposed to high exposure concentrations seemed to survive by arresting development and potentially by reduced activity, thereby conserving energy reserves. In nature, slower development could affect lipid storage buildup and reproduction.

Reducing oil droplet sizes from a subsea oil and gas release by water jetting a laboratory study performed at different scales.

The main objective of subsea mechanical dispersion (SSMD) is to reduce the oil droplet sizes from a subsea oil release, thereby influencing the fate and behaviour of the released oil in the marine environment. Subsea water jetting was identified as a promising method for SSMD and imply that a water jet is used to reduce the particle size of the oil droplets initially formed from the subsea release. This paper presents the main findings from a study including small-scale testing in a pressurised tank, via laboratory basin testing, to large-scale outdoor basin testing. The effectiveness of SSMD increases with the scale of the experiments. From a five-fold reduction in droplet sizes for small-scale experiments to more than ten-fold for large-scale experiments. The technology is ready for full-scale prototyping and field testing. Large-scale experiments performed at Ohmsett indicate that SSMD could be comparable to subsea dispersant injection (SSDI) in reducing oil droplet sizes.

Monitoring ocean water quality by deployment of lumpfish (Cyclopterus lumpus) eggs: In situ bioaccumulation and toxicity in embryos.

Fish embryos can bioaccumulate and are particularly sensitive to a wide range of contaminants, which makes them suitable sentinels for environmental biomonitoring. However, fish embryos are very rarely utilized in environmental monitoring surveys, possibly due to their fragility and seasonality. In the present work, we assessed the applicability of caged lumpfish (Cyclopterus lumpus) eggs for in situ biomonitoring of exposure and effects of organic contaminants focusing on polyaromatic hydrocarbons and phenolic compounds. Fertilized eggs (1 dpf) were transplanted for 17-19 days at different locations that differed in terms of contaminant load, depths and weather conditions, namely at three stations close to the city of Trondheim (two harbour areas and a one in the Fjord) and three stations at a coastal aquaculture facility. High survival upon retrieval after deployment showed that lumpfish eggs are relatively robust and survive encaging in different environments. Bioaccumulation of organic contaminants (PAHs and phenolic compounds) was measured and potential effects on hatching, development, survival and larvae morphometry were determined. Chemical analyses showed that especially PAHs were effectively accumulated in eggs in contaminated sites, with concentrations of Æ©PAHs being 15 - 25 times higher in harbour areas compared to those at the aquaculture facility. A higher incidence of embryonic deformations was observed in the most polluted deployment location, but larvae morphometry revealed no evidence of toxicity related to pollutant body burden. In conclusion, the in-situ exposure method was proven to work well, making it attractive for implementations in environmental monitoring programs.

Contrasting phytoplankton-zooplankton distributions observed through autonomous platforms, in-situ optical sensors and discrete sampling.

Plankton distributions are remarkably 'patchy' in the ocean. In this study, we investigated the contrasting phytoplankton-zooplankton distributions in relation to wind mixing events in waters around a biodiversity-rich island (Runde) located off the western coast of Norway. We used adaptive sampling from AUV and shipboard profiles of in-situ phytoplankton photo-physiology and particle identification (copepods, fecal pellets and the dinoflagellate Tripos spp.) and quantification using optical and imaging sensors. Additionally, traditional seawater and net sampling were collected for nutrient and in-vitro chlorophyll a concentrations and phytoplankton and meso-zooplankton abundances. Persistent strong wind conditions (~5 days) disrupted the stratification in offshore regions, while stratification and a subsurface chlorophyll maximum (SCM) were observed above the base of the mixed layer depth (MLD ~30 m) in inshore waters. Contrasting phytoplankton and zooplankton abundances were observed between inshore (with the presence of a SCM) and offshore waters (without the presence of a SCM). At the SCM, phytoplankton abundances (Tripos spp., the diatom Proboscia alata and other flagellates) were half (average of 200 cell L-1) of those observed offshore. On the contrary, meso-zooplankton counts were ~6× higher (732 ind m-3 for Calanus spp.) inshore (where a SCM was observed) compared to offshore areas. In parallel, fecal pellets and ammonium concentrations were high (>1000 ind m-3 for the upper 20 m) at the SCM, suggesting that the shallow mixed layer might have increased encounter rates and promoted strong grazing pressure. Low nutrient concentrations (< 1µM for nitrate) were found below the MLD (60 m) in offshore waters, suggesting that mixing and nutrient availability likely boosted phytoplankton abundances. The size of the absorption cross-section (σPII') and yield of photosystem II photochemistry under ambient light (φPII') changed according to depth, while the depth-related electron flow (JPII) was similar between regions, suggesting a high degree of community plasticity to changes in the light regime. Our results emphasize the importance of using multiple instrumentation, in addition to traditional seawater and net sampling for a holistic understanding of plankton distributions.

A flow-through imaging system for automated measurement of ichthyoplankton.

Microscopic imaging and morphometric measurement of fish embryos and larvae is essential in environmental monitoring of fish populations and to evaluate larvae development in aquaculture. Traditional microscopy methods require time-consuming, repetitive work by human experts. We present a method for fast imaging and analysis of millimetre-scale ichthyoplankton suspended in seawater. Our system can be easily built from common and off-the-shelf components and uses open-source software for image capture and analysis. Our system obtains images of similar quality to traditional microscopy, and biological measurements comparable to those by human experts, with minimal human interaction. This saves time and effort, while increasing the size of data sets obtained. We demonstrate our approach with cod eggs and larvae, and present results showing biologically relevant endpoints including egg diameter, larval standard length, yolk volume and eye diameter, with comparison to similar measurements reported in the literature. • High throughput, microscope-scale imaging of fish eggs and larvae • Automated measurement of biologically relevant endpoints • Easily built from off-the-shelf components and open-source software.

Automated morphometrics on microscopy images of Atlantic cod larvae using Mask R-CNN and classical machine vision techniques.

Measurements of morphometrical parameters on i.e., fish larvae are useful for assessing the quality and condition of the specimen in environmental research or optimal growth in the cultivation industry. Manually acquiring morphometrical parameters from microscopy images can be time consuming and tedious, this can be a limiting factor when acquiring samples for an experiment. Mask R-CNN, an instance segmentation neural network architecture, has been implemented for finding outlines on parts of interest on fish larvae (Atlantic cod, Gadus morhua). Using classical machine vision techniques on the outlines makes it is possible to acquire morphometrics such as area, diameter, length, and height. The combination of these techniques is providing accurate-, consistent-, and high-volume information on the morphometrics of small organisms, making it possible to sample more data for morphometric analysis.•Capabilities to automatically analyse a set of microscopy images in approximately 2-3 seconds per image, with results that have a high degree of accuracy when compared to morphometrics acquired manually by an expert.•Can be implemented on other species of ichthyoplankton or zooplankton and has successfully been tested on ballan wrasse, zebrafish, lumpsucker and calanoid copepods.

Atlantic cod (Gadus morhua) embryos are highly sensitive to short-term 3,4-dichloroaniline exposure.

3,4-dichloroaniline (3,4-DCA) is one of the most widely produced anilines world-wide, used in plastic packaging, fabrics, pharmaceuticals, pesticides, dyes and paints as well as being a degradation product of several pesticides. 3,4-DCA has been detected in freshwater, brackish and marine environments. Although freshwater toxicity thresholds exist, very little toxicological information is available on marine and cold-water species. In this study, we exposed Atlantic cod (Gadus morhua) embryos (3-7 days post fertilization) to 3,4-DCA concentrations ranging from 8-747 µg/L for 4 days followed by a recovery period in clean sea water until 14 days post fertilization (dpf). The cod embryos were significantly more sensitive to acute 3,4-DCA exposure compared to other species tested and reported in the literature. At the highest concentration (747 µg/L), no embryos survived until hatch, and even at the lowest concentration (8 µg/L), a small, but significant increase in mortality was observed at 14 dpf. Delayed and concentration-dependent effects on surviving yolk-sac larvae, manifested as cardiac, developmental and morphometric alterations, more than a week after exposure suggest potential long-term effects of transient embryonic exposure to low concentrations of 3,4-DCA.

Effects of mine tailing exposure on early life stages of cod (Gadus morhua) and haddock (Melanogrammus aeglefinus).

Mining and processing of minerals produce large quantities of tailings as waste. Some countries, including Norway, allow disposal of mine tailings in the sea. In this study we investigated the impacts of tailings from a calcium carbonate (CaCO3) processing plant on early live stages of haddock (Melanogrammus aeglefinus) and Atlantic cod (Gadus morhua). Fish eggs (3 days post fertilisation; dpf) were exposed for 48 h to three concentrations of tailings, nominally 1 mg L-1 (low, L); 10 mg L-1 (medium, M) and 100 mg L-1 (high, H); with L and M representing concentrations occurring at tailing release points. Results show that tailings rapidly adhered to eggs of both species, causing negative buoyancy (sinking of eggs) in M and H exposures. While tailings remained on egg surfaces in both species also after exposure termination, adhesion seemed more pronounced in cod, leading to larger impacts on buoyancy even after exposure. Tailing exposure further induced early hatching and significantly reduced survival in M and H exposed embryos in both fish species, and in cod from the L exposure group. Moreover, tailing exposure caused reduced survival and malformations in larvae, potentially related to premature hatching. This study shows that mineral particles adhere to haddock and cod eggs, affecting egg buoyancy, survival and development.

The hidden influence of large particles on ocean colour.

Optical constituents in the ocean are often categorized as water, phytoplankton, sediments and dissolved matter. However, the optical properties of seawater are influenced, to some degree, by scattering and absorption by all particles in the water column. Here we assess the relevant size ranges for determining the optical properties of the ocean. We present a theoretical basis supporting the hypothesis that millimetre-size particles, including zooplankton and fish eggs, can provide a significant contribution to bulk absorption and scattering of seawater and therefore ocean color. Further, we demonstrate that existing in situ instruments are not capable of correctly resolving the impact of such large particles, possibly leading to their optical significance being overlooked. These findings refresh our perspective on the potential of ocean color and invite new applications of remote sensing for monitoring life close to the ocean surface.

Large-scale basin testing to simulate realistic oil droplet distributions from subsea release of oil and the effect of subsea dispersant injection.

Small-scale experiments performed at SINTEF, Norway in 2011-12 led to the development of a modified Weber scaling algorithm. The algorithm predicts initial oil droplet sizes (d50) from a subsea oil and gas blowout. It was quickly implemented in a high number of operational oil spill models used to predict fate and effect of subsea oil releases both in academia and in the oil industry. This paper presents experimental data from large-scale experiments generating oil droplet data in a more realistic multi-millimeter size range for a subsea blow-out. This new data shows a very high correlation with predictions from the modified Weber scaling algorithm both for untreated oil and oil treated by dispersant injection. This finding is opposed to earlier studies predicting significantly smaller droplets, using a similar approach for estimating droplet sizes, but with calibration coefficients that we mean are not representative of the turbulence present in such releases.

The influences of phytoplankton species, mineral particles and concentrations of dispersed oil on the formation and fate of marine oil-related aggregates.

The formation and fallout of oil-related marine snow have been associated with interactions between dispersed oil and small marine particles, like phytoplankton and mineral particles. In these studies, the influences of phytoplankton species, mineral particle concentration, and oil concentration on the aggregation of oil in seawater (SW) were investigated. The experiments were performed in a low-turbidity carousel incubation system, using natural SW at 13 °C. Aggregation was measured by silhouette camera analyses, and oil compound group distribution and depletion by gas chromatography (GC-FID or GC-MS). Aggregates with median sizes larger than 500 µm in diameter were measured in the presence of dispersed oil and the phytoplankton species Thalassiosira rotula, Phaeocystis globosa, Skeletonema pseudocostatum, but not with the microalgae Micromonas pusilla. When mineral particles (diatomaceous earth) were incubated at different concentrations (5-30 mg/L) with dispersed oil and S. pseudocostatum, the largest aggregates were measured at the lower mineral particle concentration (5 mg/L). Since dispersed oil rapidly dilutes in the marine water column, experiments were performed with oil concentrations of from 10 mg/L to 0.01 mg/L in the presence of S. pseudocostatum and diatomaceous earth. Aggregates larger than 500 µm was measured only at the highest oil concentrations (10 mg/L). However, oil attachment to the marine particles were also measured at low oil concentrations (≤1 mg/L). Depletion of oil compound groups (n-alkanes, naphthalenes, PAHs, decalins) were measured at all oil concentrations, both in aggregate and water phases, with biodegradation as the expected main depletion process. These results showed that oil concentration may be important for oil-related marine snow formation, but that even oil droplets at low concentrations may attach to the particles and be transported by prevailing currents.

Formation and fate of oil-related aggregates (ORAs) in seawater at different temperatures.

In this study, the formation and fate of oil-related aggregates (ORAs) from chemically dispersed oil in seawater (SW) were investigated at different temperatures (5 °C, 13 °C, 20 °C). Experiments in natural SW alone, and in SW amended with typical marine snow constituents (phytoplankton and mineral particles), showed that the presence of algae stimulated the formation of large ORAs, while high SW temperature resulted in faster aggregate formation. The ORAs formed at 5 °C and 13 °C required mineral particles for sinking, while the aggregates also sank in the absence of mineral particles at 20°. Early in the experimental periods, oil compound accumulation in ORAs was faster than biodegradation, particularly in aggregates with algae, followed by rapid biodegradation. High abundances of bacteria associated with hydrocarbon biodegradation were determined in the ORAs, together with algae-associated bacteria, while clustering analyses showed separation between bacterial communities in experiments with oil alone and oil with algae/mineral particles.

Interaction between microalgae, marine snow and anionic polyacrylamide APAM at marine conditions.

When an oil field ages and the pressure in the reservoir decreases, or for oil fields with heavy oil, there may be a need for enhanced oil recovery (EOR) technologies. Polymer injection is a water-based EOR method where the viscosity of the water injected for pressure support is increased by mixing with a high concentration polymer solution. In this project, the potential fate of a synthetic anionic polyacrylamide (APAM) in seawater was investigated, since these EOR polymers may enter the marine environment with the produced water (PW). The main objective of the study was to determine if the APAM will interact with cells or aggregates (marine snow) of microalgae, resulting in potential polymer transport from the euphotic zone to the seabed. Three different species of microalgae with different degree of autotrophy (autotroph, mixotroph and heterotroph) were exposed to fluorescence-tagged APAM. Attachment to algal cells or aggregates formed by active or heat-inactivated algae were analysed by fluorescence microscopy and fluorometry. Our results suggested that attachment of APAM to cells of the algal species included in his study was negligible. A carousel system with natural seawater (SW) was used for formation of algal aggregates, one of the key components of marine snow. When aggregates of the diatom Thalassiosira rotula were formed in the presence of the fluorescence-tagged APAM, and at SW temperatures relevant for the Norwegian Continental Shelf, the polymer was nearly exclusively measured in the water phase after separation from the aggregates. The aggregate measurements therefore confirmed the results from the attachment studies, and we found no evidence of accumulation of APAM in aggregates formed from algae. Marine snow from algae is therefore not expected to significantly contribute to sedimentation of APAM dissolved in the water column.

Shedding from chemically-treated oil droplets rising in seawater.

The degree to which droplet shedding (tip-streaming) can modify the size of rising oil droplets has been a topic of growing interest in relation to subsea dispersant injection. We present an experimental and numerical approach predicting oil droplet shedding, covering a wide range of viscosities and interfacial tensions. Shedding was observed within a specific range of droplet sizes when the oil viscosity is sufficiently high and the IFT is sufficiently low. The affected droplets are observed to reduce in size, as smaller satellite droplets are shed, until the parent droplet reaches a stable size. Shedding of smaller droplets is related to the viscosity-dominated modified capillary number (Ca'), especially for low dispersant dosages recommended for subsea dispersant injection. This, in combination with the IFT-dominated Weber number (We), characterise droplets into three possible states: 1) stable (Ca' < 0.21 &We<12); 2) tip-streaming (Ca' > 0.21 &We<12); 3) unstable and subject to total breakup (We>12).

Combined releases of oil and gas under pressure; the influence of live oil and natural gas on initial oil droplet formation.

Both oil droplets and gas bubbles have simultaneously been quantified in laboratory experiments that simulate deep-water subsea releases of both live oil (saturated with gas) and additional natural gas under high pressure. These data have been used to calculate particle size distributions (50-5000 µm) for both oil and gas. The experiments showed no significant difference in oil droplet sizes versus pressure (from 5 m to 1750 m) for experiments with live oil. For combined releases of live oil and natural gas, oil droplet sizes showed a clear reduction as a function of increased gas void fraction (increased release velocity) and a weak reduction with increased depth (increased gas density/momentum). Oil droplets were reduced by a factor of 3 to 4 during simulated subsea dispersant injection (SSDI) and no significant effect of pressure was observed. This indicates that SSDI effectiveness is not dependent on water depth or pressure.

Quantification of oil droplets under high pressure laboratory experiments simulating deep water oil releases and subsea dispersants injection (SSDI).

Limited experimental and field data are available describing oil droplet formation from subsea releases at high pressure. There are also analytical challenges quantifying oil droplets over a wide size and concentrations range at high pressure. This study quantified oil droplets released from an orifice in seawater at low and high pressure (5 m and 1750 m depth). Oil droplet sizes were quantified using a newly developed sensor (Silhouette camera or SilCam). The droplet sizes measured during experiments at low and high pressure, using the same release conditions, showed no significant difference as a function of pressure. This lack of a pressure effect on oil droplet sizes was observed for both untreated oil and for droplet formation during subsea dispersant injection or SSDI. This strongly indicates that the effectiveness of SSDI is not influenced by water depth or pressure, at least for simulated subsea releases of oil alone (no gas).

Toward adaptive robotic sampling of phytoplankton in the coastal ocean.

Currents, wind, bathymetry, and freshwater runoff are some of the factors that make coastal waters heterogeneous, patchy, and scientifically interesting-where it is challenging to resolve the spatiotemporal variation within the water column. We present methods and results from field experiments using an autonomous underwater vehicle (AUV) with embedded algorithms that focus sampling on features in three dimensions. This was achieved by combining Gaussian process (GP) modeling with onboard robotic autonomy, allowing volumetric measurements to be made at fine scales. Special focus was given to the patchiness of phytoplankton biomass, measured as chlorophyll a (Chla), an important factor for understanding biogeochemical processes, such as primary productivity, in the coastal ocean. During multiple field tests in Runde, Norway, the method was successfully used to identify, map, and track the subsurface chlorophyll a maxima (SCM). Results show that the algorithm was able to estimate the SCM volumetrically, enabling the AUV to track the maximum concentration depth within the volume. These data were subsequently verified and supplemented with remote sensing, time series from a buoy and ship-based measurements from a fast repetition rate fluorometer (FRRf), particle imaging systems, as well as discrete water samples, covering both the large and small scales of the microbial community shaped by coastal dynamics. By bringing together diverse methods from statistics, autonomous control, imaging, and oceanography, the work offers an interdisciplinary perspective in robotic observation of our changing oceans.

Automatic determination of heart rates from microscopy videos of early life stages of fish.

Toxic effects of organic hydrophobic contaminants include impacts on fish heart rate (HR) and cardiac functioning. Thus, in ecotoxicology as well as aquaculture and even medicine, fish heart functioning plays an important role in application areas. The aim of this study was to assemble a pipeline of image processing and statistical techniques to extract HR information from microscopy videos of the embryo and larval stages of three species of fish (Atlantic cod, haddock, and Atlantic bluefin tuna). The method enables automatic processing for a large number of individuals, saving a significant amount of time compared with manual processing, while simultaneously eliminating the type of errors such a manual process might incur.