Use of TLM derived models to estimate toxicity of weathered MC252 oil based on conventional chemical data and the potential impact of unresolved polar components.

Target lipid model (TLM) and toxic unit (TU) approaches were applied to ecotoxicity and chemistry data from low-energy WAFs (LE-WAFs) of source and weathered crude oils originating from the Deepwater Horizon oil spill. The weathered oils included artificially weathered oils and naturally weathered samples collected in the Gulf of Mexico after the spill. Oil weathering greatly reduced the concentrations of identified LE-WAF components, however, the mass of uncharacterized polar material (UPC) in the LE-WAFs remained largely unchanged during the weathering process. While the TLM-derived calculations displayed a significant decrease in toxicity (TUs) for the heavily weathered oils, copepod toxicity, expressed as LC10-based TUs, were comparable between LE-WAFs of fresh and weathered oils. The discrepancy between observed and predicted toxicity for the LE-WAFs of artificially weathered oils may be related to limitations by the chemical analyses or increased toxicity due to generation of new unknown compounds during the weathering process.

A simple protocol for estimating the acute toxicity of unresolved polar compounds from field-weathered oils.

Crude oil spilled at sea is chemically altered through environmental processes such as dissolution, biodegradation, and photodegradation. Transformation of hydrocarbons to oxygenated species increases water-solubility. Metabolites and oxidation products largely remain uncharacterized by common analytical methods but may be more bioavailable to aquatic organisms. Studies have shown that unresolved (i.e. unidentified) polar compounds ('UPCs') may constitute > 90% of the water-accommodated fraction (WAF) of heavily weathered crude oils, but still there is a paucity of information characterizing their toxicological significance in relation to other oil-derived toxicants. In this study, low-energy WAFs (no droplets) were generated from two field-weathered oils (collected during the 2010 Deepwater Horizon incident) and their polar fractions were isolated through fractionation. To allow establishment of thresholds for acute toxicity (LC50) of the dissolved and polar fraction of field collected oils, we concentrated both WAFs and polar fractions to beyond field-documented concentrations, and the acute toxicity of both to the marine copepod Acartia tonsa was measured and compared to the toxicity of the native WAF (non-concentrated). The difference in toxic units (TUs) between the total of the mixture and of identified compounds of known toxicity (polycyclic aromatic hydrocarbons [PAHs] and alkyl phenols) in both WAF and polar fractions was used to estimate the contribution of the UPC to overall toxicity. This approach identified that UPC had a similar contribution to toxicity as identified compounds within the WAFs of the field-weathered oils. This signifies the relative importance of polar compounds when assessing environmental impacts of spilled and weathered oil.

Effects of petrogenic pollutants on North Atlantic and Arctic Calanus copepods: From molecular mechanisms to population impacts.

Oil and gas industries in the Northern Atlantic Ocean have gradually moved closer to the Arctic areas, a process expected to be further facilitated by sea ice withdrawal caused by global warming. Copepods of the genus Calanus hold a key position in these cold-water food webs, providing an important energetic link between primary production and higher trophic levels. Due to their ecological importance, there is a concern about how accidental oil spills and produced water discharges may impact cold-water copepods. In this review, we summarize the current knowledge of the toxicity of petroleum on North Atlantic and Arctic Calanus copepods. We also review how recent development of high-quality transcriptomes from RNA-sequencing of copepods have identified genes regulating key biological processes, like molting, diapause and reproduction in Calanus copepods, to suggest linkages between exposure, molecular mechanisms and effects on higher levels of biological organization. We found that the available ecotoxicity threshold data for these copepods provide valuable information about their sensitivity to acute petrogenic exposures; however, there is still insufficient knowledge regarding underlying mechanisms of toxicity and the potential for long-term implications of relevance for copepod ecology and phenology. Copepod transcriptomics has expanded our understanding of how key biological processes are regulated in cold-water copepods. These advances can improve our understanding of how pollutants affect biological processes, and thus provide the basis for new knowledge frameworks spanning the effect continuum from molecular initiating events to adverse effects of regulatory relevance. Such efforts, guided by concepts such as adverse outcome pathways (AOPs), enable standardized and transparent characterization and evaluation of knowledge and identifies research gaps and priorities. This review suggests enhancing mechanistic understanding of exposure-effect relationships to better understand and link biomarker responses to adverse effects to improve risk assessments assessing ecological effects of pollutant mixtures, like crude oil, in Arctic areas.

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.

Do oil droplets and chemical dispersants contribute to uptake of oil compounds and toxicity of crude oil dispersions in cold-water copepods?

Accidental crude oil spills to the marine environment cause dispersion of oil into the water column through the actions of breaking waves, a process that can be facilitated using chemical dispersants. Oil dispersions contain dispersed micron-sized oil droplets and dissolved oil components, and the toxicity of oil dispersions has been assumed to be associated primarily with the latter. However, most hydrophobic, bioaccumulative and toxic crude oil components are retained within the droplets which may interact with marine filter-feeders. We here summarize the findings of 15 years of research using a unique methodology to generate controlled concentrations and droplet size distributions of dispersed crude oil to study effects on the filter-feeding cold-water copepod Calanus finmarchicus. We focus primarily on the contribution of chemical dispersants and micron-sized oil droplets to uptake and toxicity of oil compounds. Oil dispersion exposures cause PAH uptake and oil droplet accumulation on copepod body surfaces and inside their gastrointestinal tract, and exposures to high exposure (mg/L range) reduce feeding activity, causes reproductive impairments and mortality. These effects were slightly higher in the presence of chemical dispersants, possibly due to higher filtration of chemically dispersed droplets. For C. finmarchicus, dispersions containing oil droplets caused more severe toxic effects than filtered dispersions, thus, oil droplets contribute to the observed toxicity. The methodology for generating crude oil dispersion is a valuable tool to isolate impacts of crude oil microdroplets and can facilitate future research on oil dispersion toxicity and produce data to improve oil spill models.

Chemical and toxicological characterisation of residues from offshore in-situ burning of spilled fuel oils.

Burn residues collected after large scale experimental in situ burns performed in the North Sea were characterised with emphasis on chemistry and acute toxicity. Low-energy water accommodated fractions (WAFs) of three marine fuels (Ultra Low Sulphur Fuel Oil (ULSFO), bunker oil (IFO180), and marine gas oil (MGO) and their field-generated residues from in situ burning (ISB). were prepared to evaluate the potential impact of ISB residue to the environment. The toxicity effects on primary consumers were assessed by testing on early life stage (nauplii) of the marine copepod Calanus finmarchicus. Toxicity studies showed that ISB decreased the acute toxicity of the WAFs compared to the initial oils. WAF of MGO had highest toxicity, and ISB residue of MGO seems to be more toxic than WAFs of fresh ULSFO and IFO180. Additive toxicity expressed as toxic unit (TU) based on the chemical composition also indicated that the toxicity of WAFs from ISB residues were lower than for the initial oils. The 2-3 ring PAHs seem to contribute most to the TU. Overall, the three offshore burns reduced the total mass of PAHs in the water accommodated fractions by >90 % compared to the released unburned oils and caused a reduction of the acute toxicity to copepod nauplii.

Application of chemical herders do not increase acute crude oil toxicity to cold-water marine species.

Chemical herders may be used to sequester and thicken surface oil slicks to increase the time window for performing in situ burning of spilled oil on the sea surface. For herder use to be an environmentally safe oil spill response option, information regarding their potential ecotoxicity both alone and in combination with oil is needed. This study aimed at assessing if using herders can cause toxicity to cold-water marine organisms. Our objective was to test the two chemical herders Siltech OP-40 (OP-40) and ThickSlick-6535 (TS-6535) with and without oil for toxicity using sensitive life stages of cold-water marine copepod (Calanus finmarchicus) and fish (Gadus morhua). For herders alone, OP-40 was consistently more toxic than TS-6535. To test herders in combination with oil, low-energy water accommodated fractions (LE-WAFs, without vortex) with Alaskan North Slope crude oils were prepared with and without herders. Dissolution of oil components from surface oil was somewhat delayed following herder application, due to herder-induced reduction in contact area between water and oil. The LE-WAFs were also used for toxicity testing, and we observed no significant differences in toxicity thresholds between treatments to LE-WAFs generated with oil alone and oil treated with herders. The operational herder-to-oil ratio is very low (1:500), and the herders tested in the present work displayed acute toxicity at concentrations well above what would be expected following in situ application. Application of chemical herders to oil slicks is not expected to add significant effects to that of the oil for cold-water marine species exposed to herder-treated oil slicks.

The impact of exposure timing on embryo mortality and the partitioning of PAHs when cod eggs are exposed to dispersed and dissolved crude oil.

During sub-sea oil spills to the marine environment, oil droplets will rise towards the sea surface at a rate determined by their density and diameter as well as the vertical turbulence in the water. Micro-droplets (< 50 µm) are expected to have prolonged residence times in the water column. If present, pelagic fish eggs may thus be exposed to dispersed oil from subsurface oil spills for days, and the contribution of these micro-droplets to toxicity is not well known. The purpose of this work was to investigate to what extent timing of exposure and the presence of oil micro droplets affects PAH uptake and survival of pelagic Atlantic cod eggs. A single batch of eggs was separated in two groups and exposed to dispersions and corresponding water-soluble fraction at 3-7 days (Early exposure) and 9-13 days (Late exposure) post fertilization. Partitioning of PAHs between crude oil microdroplets, water and eggs was estimated as well as the contribution of oil droplets to PAH body residue and acute and delayed mortality. Timing of oil exposure clearly affects both the mortality rate and the timing of mortality. Even though the body residue of PAHs were lower when embryos were exposed in the later embryonic stage, mortality rate increased relative to the early exposure indicating that critical body residue threshold is stage specific. Although our results suggest that the dissolved fraction is the dominating driver for toxicity in cod embryos exposed to oil dispersions, crude oil micro droplets contribute to increased mortality as well.

Oil-mediated oxidative-stress responses in a keystone zooplanktonic species, Calanus finmarchicus.

The copepod Calanus finmarchicus is an ecologically important species in the North Atlantic, Norwegian and Barents seas. Accidental or continuous petroleum pollution from oil and gas production in these seas may pose a significant threat to this low trophic level keystone species. Responses related to oxidative stress, protein damage and lipid peroxidation were investigated in C. finmarchicus exposed to a water-accommodated fraction (WAF) of a naphthenic North Atlantic crude oil. The exposure concentration corresponded to 50% of the 96 h LC50, and samples were obtained at 0, 24, 48, 72 and 96 h after exposure initiation. Gene expressions (superoxide dismutase, catalase, glutathione S-transferase, glutathione synthetase, heat shock protein 70 and 90, ubiquitin and cytochrome P-450 330A1), enzyme activities (superoxide dismutase, catalase, glutathione S-transferase) and concentrations of total glutathione and malondialdehyde were analyzed. Gene expression analyses showed no differences between controls and the exposed animals, however significantly higher glutathione S-transferase activity and malondialdehyde concentrations were found in the exposed group, suggests lipid peroxidation as main toxic effect.

Exposure to low environmental copper concentrations does not affect survival and development in Atlantic cod (Gadus morhua) early life stages.

In this study we investigated potential impacts of Cu exposure at low, environmentally relevant, concentrations on early live stages of Atlantic cod (Gadus morhua). Cod embryos and larvae were exposed to 0.5 µg/L (low), 2 µg/L (medium), and 6 µg/L (high) Cu from 4 to 17 days post fertilisation (dpf). Hatching success, mortality, oxygen consumption, biometric traits, and malformations were determined. A dynamic energy budget (DEB) model was applied to identify potential impacts on bioenergetics. A positive correlation was found between Cu exposure concentrations and Cu body burden in eggs, but not in larvae. The tested concentrations did not increase mortality in neither embryos nor larvae, or larvae deformations. Further, the DEB model did not indicate effects of the tested Cu concentrations.

Effects of marine mine tailing exposure on the development, growth, and lipid accumulation in Calanus finmarchicus.

Marine tailing disposal (MTD) is sometimes practiced as an alternative to traditional mine tailing deposition on land. Environmental challenges connected to MTD include spreading of fine particulate matter in the water column and the potential release of metals and processing chemicals. This study investigated if tailing exposure affects the marine copepod Calanus finmarchicus, and whether effects are related to exposure to mineral particles or the presence of metals and/or processing chemicals in the tailings. We investigated the impacts of three different tailing compositions: calcium carbonate particles with and without processing chemicals and fine-grained tailings from a copper ore. Early life stages of C. finmarchicus were exposed over several developmental stages to low and high suspension concentrations for 15 days, and their development, oxygen consumption and biometry determined. The data was fitted in a dynamic energy budget (DEB) model to determine mechanisms underlying responses and to understand the primary modes of action related to mine tailing exposure. Results show that copepods exposed to tailings generally exhibited slower growth and accumulated less lipids. The presence of metals and processing chemicals did not influence these responses, suggesting that uptake of mineral particles was responsible for the observed effects. This was further supported by the applied DEB model, confirming that ingestion of tailing particles while feeding can result in less energy being available for growth and development.

Cellular level response of the bivalve Limecola balthica to seawater acidification due to potential CO2 leakage from a sub-seabed storage site in the southern Baltic Sea: TiTank experiment at representative hydrostatic pressure.

Understanding of biological responses of marine fauna to seawater acidification due to potential CO2 leakage from sub-seabed storage sites has improved recently, providing support to CCS environmental risk assessment. Physiological responses of benthic organisms to ambient hypercapnia have been previously investigated but rarely at the cellular level, particularly in areas of less common geochemical and ecological conditions such as brackish water and/or reduced oxygen levels. In this study, CO2-related responses of oxygen-dependent, antioxidant and detoxification systems as well as markers of neurotoxicity and acid-base balance in the Baltic clam Limecola balthica from the Baltic Sea were quantified in 50-day experiments. Experimental conditions included CO2 addition producing pH levels of 7.7, 7.0 and 6.3, respectively and hydrostatic pressure 900 kPa, simulating realistic seawater acidities following a CO2 seepage accident at the potential CO2-storage site in the Baltic. Reduced pH interfered with most biomarkers studied, and modifications to lactate dehydrogenase and malate dehydrogenase indicate that aerobiosis was a dominant energy production pathway. Hypercapnic stress was most evident in bivalves exposed to moderately acidic seawater environment (pH 7.0), showing a decrease of glutathione peroxidase activity, activation of catalase and suppression of glutathione S-transferase activity likely in response to enhanced free radical production. The clams subjected to pH 7.0 also demonstrated acetylcholinesterase activation that might be linked to prolonged impact of contaminants released from sediment. The most acidified conditions (pH 6.3) stimulated glutathione and malondialdehyde concentration in the bivalve tissue suggesting potential cell damage. Temporal variations of most biomarkers imply that after a 10-to-15-day initial phase of an acute disturbance, the metabolic and antioxidant defence systems recovered their capacities.

Predation risk alters life history strategies in an oceanic copepod.

The ubiquitous oceanic copepod Calanus finmarchicus is the major link between primary producers and important fish stocks in the North Atlantic Ocean and adjacent seas. Despite over a century of research on growth and development of this key species, the effect of predation risk on these processes remains elusive. We tested how food level and chemical cues from a fish predator influence growth and development of C. finmarchicus, using a predator naïve laboratory population. Copepods reached adult stage earlier both in response to high food and to predator cues in our experiment. High food also increased growth and lipid accumulation. In contrast, perceived predation risk triggered reduced size and lipid fullness, indicating a decoupling of growth and development rates. Our results demonstrate that chemical predator cues can influence life history strategies in C. finmarchicus, and suggest that present and future patterns in oceanic zooplankton size and population dynamics may also reflect differences in predation risk.

Attachment of APAM to mineral particles in seawater.

Polymer injection is used in enhanced oil recovery (EOR) when an oil field ages and the pressure in the reservoir decreases, or for oil fields with heavy oil. By polymer injection, the viscosity of the water injected for pressure support is increased by mixing with a high concentration of a polymer solution. Polymers used in EOR operations are often high molecular weight polyacrylamides, including anionic polyacrylamide (APAM), which may subsequently enter the marine environment with produced water releases. Since seawater (SW) contains mineral particles (MPs) in low concentrations, and polymers like APAM are known to flocculate MPs, we investigated if APAM at different concentrations (0.5-10 mg/L) would attach and flocculate MPs, when these occurred in concentrations relevant for oceanic SW (1 mg/L). Two types of MPs, diatomaceous earth and kaolin, were exposed to fluorescence-tagged APAM (APAM-TAG). A low-energy carousel system with natural seawater (SW) was used for incubation of MPs and APAM-TAG at a temperature relevant for the Norwegian Continental Shelf (13 °C). Attachment to MPs and aggregates of these were analysed by fluorometry and fluorescence microscopy. Particle analyses showed that only minor fractions of the MPs aggregated. When samples were separated in steel filter with a mesh size of 20 µm, APAM-TAG was mainly measured in the flow-through fraction (<20 µm), and the results therefore showed that the polymer mainly remained in the water-phase, or was attached to small particles (<20 µm). For the small fraction of APAM attaching to aggregated MPs, attraction to kaolin was higher than to diatomaceous earth, and fluorescence microscopy analyses confirmed the presence of fluorescent particles at the higher APAM concentrations. MPs at concentrations relevant for oceanic SW are therefore not expected to significantly contribute to sedimentation of APAM dissolved in the water column.

Lipid metabolism in Calanus finmarchicus is sensitive to variations in predation risk and food availability.

Late developmental stages of the marine copepods in the genus Calanus can spend extended periods in a dormant stage (diapause) that is preceded by the accumulation of large lipid stores. We assessed how lipid metabolism during development from the C4 stage to adult is altered in response to predation risk and varying food availability, to ultimately understand more of the metabolic processes during development in Calanus copepods. We used RNA sequencing to assess if perceived predation risk in combination with varied food availability affects expression of genes associated with lipid metabolism and diapause preparation in C. finmarchicus. The lipid metabolism response to predation risk differed depending on food availability, time and life stage. Predation risk caused upregulation of lipid catabolism with high food, and downregulation with low food. Under low food conditions, predation risk disrupted lipid accumulation. The copepods showed no clear signs of diapause preparation, supporting earlier observations of the importance of multiple environmental cues in inducing diapause in C. finmarchicus. This study demonstrates that lipid metabolism is a sensitive endpoint for the interacting environmental effects of predation pressure and food availability. As diapause may be controlled by lipid accumulation, our findings may contribute towards understanding processes that can ultimately influence diapause timing.

Microplastic dispersal behavior in a novel overhead stirring aqueous exposure system.

Using nominal dose metrics to describe exposure conditions in laboratory-based microplastic uptake and effects studies may not adequately represent the true exposure to the organisms in the test system, making data interpretation challenging. In the current study, a novel overhead stirring method using flocculators was assessed for maintaining polystyrene (PS) microbeads (Ø10.4 µm; 1.05 g cm-3) in suspension in seawater during 24 h and then compared with static and rotational exposure setups. Under optimized conditions, the system was able to maintain 59% of the initial PS microbeads in suspension after 24 h, compared to 6% using a static system and 100% using a rotating plankton wheel. Our findings document for the first time that overhead stirring as well as other, commonly used exposure systems (static) are unable to maintain constant microplastic exposure conditions in laboratory setups whereas rotation is very effective. This suggests toxicological studies employing either static or overhead stirring systems may be greatly overestimating the true microplastic exposure conditions.

Acute and long-term effects of anionic polyacrylamide (APAM) on different developmental stages of two marine copepod species.

The application of synthetic polymers such as anionic polyacrylamides (APAM) in enhanced oil recovery (EOR) may increase in the future. This can lead to environmental release through offshore produced water discharges with so far limited knowledge on impacts in marine ecosystems. We investigated impacts of APAM polymers on two marine copepod species. Acute effects of APAM were studied on different life stages of C. finmarchicus (three molecular sizes: 200 kDa, 2800 kDa and 8000 kDa) and Acartia tonsa (one molecular size: 2800 kDa). Further, effects on development and survival following long-term exposure (spanning over several life stages) to 200 kDa APAM were studied in C. finmarchicus. Results show that none of the APAM molecules caused mortality in acute exposure experiments in adult C. finmarchicus even at high exposure concentrations (≥1000 mg/L). Comparing toxicity of the 2800 kDa APAM between C. finmarchicus and the standard marine toxicity test copepod Acartia tonsa showed that the latter was slightly more sensitive. Early life stages of both copepods were more sensitive compared to later ones, and APAM exposure induced increased mortality and developmental delays. Effects were generally more pronounced for the larger polymers, most likely due to increased viscosity of the test dispersions leading to increased energy expenditures of the animals. However, significant effects were only observed at very high exposure concentrations that are probably higher than concentrations found in the environment.

Comparison of artificially weathered Macondo oil with field samples and evidence that weathering does not increase environmental acute toxicity.

Macondo source oils and artificially weathered oil residues from 150 °C+ to 300 °C+, including artificially photo oxidized oils, were prepared and used for generating low energy water accommodated fractions (LE-WAFs) in order to assess the impact of oil weathering on WAF chemistry composition and toxicity to marine organisms. Two pelagic species representing primary producers (the marine algae Skeletonema pseudocostatum) and invertebrates (the marine copepod Acartia tonsa) were tested. Obtained acute toxicity levels, expressed as EC/LC50 values, were in the same range or above the obtained maximum WAF concentrations for WAFs from most weathering degrees. Based on % WAF dilutions, reduced toxicity was determined as a function of weathering. The chemical compositions of all WAFs were compared to compositions obtained from water samples reported in the GRIIDC database using multivariate analysis, indicating that WAFs of photo oxidized and two field weathered oils resembled the field data the most.

The use of PAH, metabolite and lipid profiling to assess exposure and effects of produced water discharges on pelagic copepods.

Several laboratory studies have demonstrated that exposure to oil components cause toxicity to copepods, however, this has never been shown in natural populations of copepods. In the present study, we sampled copepods in an area of the North Sea with high density of oil production platforms discharging produced water. Environmental modelling was used to predict produced water and copepod trajectories prior to copepod sampling in situ. To maximise output from a minimal number of field samples, a novel and combined methodology was developed to allow exploitation of the same extract for several purposes; contaminant body burden, lipidomics, and metabolomics analysis. PAH body burdens were low compared to laboratory experiments where correlations between PAH body burden and acute toxicity, reproduction and molecular endpoints had been established. Still, station-specific PAH profiles strongly indicated copepod exposure to PW. NMR metabolomics, focusing on water-soluble metabolites, suggested no correlation between metabolites and stations. Interestingly, lipidomics analyses suggested site-specific fingerprints and profiles displayed for acyl-glycerols and wax esters. Potential effects of produced water exposure on lipid metabolism in copepods cannot be ruled out and deserves more attention. Our study exemplifies the importance of incorporating novel and improved analytical methodologies in environmental monitoring.

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.