Descriptive analyses of bacterial communities in marine sediment microcosms spiked with fish wastes, emamectin benzoate, and oxytetracycline.

In marine sediments surrounding salmon aquaculture sites, organic matter (OM) enrichment has been shown to influence resident bacterial community composition; however, additional effects on these communities due to combined use of the sea-lice therapeutant emamectin benzoate (EMB) and the widely used antibiotic oxytetracycline (OTC) are unknown. Here, we use sediment microcosms to assess the influence of OM, EMB, and OTC on benthic bacterial communities. Microcosms consisted of mud or sand sediments enriched with OM (fish and feed wastes) and spiked with EMB and OTC at environmentally-relevant concentrations. Samples were collected from initial matrices at the initiation of the trial and after 110 days for 16 S rRNA gene sequencing of the V3-V4 region and microbiome profiling. The addition of OM in both mud and sand sediments reduced alpha diversities; for example, an average of 1106 amplicon sequence variants (ASVs) were detected in mud with no OM addition, while only 729 and 596 ASVs were detected in mud with low OM and high OM, respectively. Sediments enriched with OM had higher relative abundances of Spirochaetota, Firmicutes, and Bacteroidota. For instance, Spirochaetota were detected in sediments with no OM with a relative abundance range of 0.01-1.2%, while in sediments enriched with OM relative abundance varied from 0.16% to 26.1%. In contrast, the addition of EMB (60 ng/g) or OTC (150 ng/g) did not result in distinct taxonomic shifts in the bacterial communities compared to un-spiked sediments during the timeline of this experiment. EMB and OTC concentrations may have been below effective inhibitor concentrations for taxa in these communities; further work should explore gene content and the presence of antibiotic resistance genes (ARGs) in sediment-dwelling bacteria.

Drug and pesticide usage for sea lice treatment in salmon aquaculture sites in a Canadian province from 2016 to 2019.

We used 4 years of publicly available data (2016-2019) on chemical usage at salmon sites with information on production, stocking, locations and environmental conditions to explore patterns of anti-sea lice treatments in a Canadian province. Results show that sequential chemical treatments are prevalent, emamectin benzoate (EMB) with azamethiphos being the most used combination with a decrease in ivermectin usage. Relatively high rates of usage of EMB per fish biomass may point to potential lice resistance patterns with information needed on mechanisms and local populations. Lower or no chemical usage at some sites indicate less sea lice infestations likely influenced by localized site conditions (coves), and a lessened need for medication due to the usage of cleaner fish and possibly other non-chemical methods (not documented in this report). The year/climate influenced chemical input only in sites with higher treatment levels likely due to effects on sea lice growth and reproduction. Observed differences between years are warmer surface temperature in the fall, a higher freshwater input in spring, and stronger wind conditions for 2017 and 2018 with more medication usage for these two years. The lack of significant effect of site distances calculated in zones of influence based on 24 h potential connectivity patterns highlight the need to refine the resolution of hydrodynamic processes.

Linking the presence of visual indicators of aquaculture deposition to changes in epibenthic richness at finfish sites installed over hard bottom substrates.

Effects of finfish aquaculture on benthic communities at hard bottom sites can be assessed using visual indicators of organic enrichment, namely bacterial mats, opportunistic polychaetes, and/or barren substrates (i.e., with no visible epifauna). Under some regulatory frameworks, the presence of visual indicators beyond a certain threshold proportion (e.g., > 70%) of mandatory sampling stations at a site signals an unacceptable degree of benthic organic loading. However, relationships between visual indicator presence and other biological characters such as epibenthic taxon richness are unclear, and should be examined to advise on the validity of existing legislative frameworks. Here, we used video data collected for regulatory purposes before and after aquaculture production to document (1) change in epibenthic taxon richness (TR) and its discriminatory power in determining aquaculture impact and (2) the association between TR change and the presence of visual indicators. Despite low richness values overall, our results show a significant post-production decrease in TR in the near-cage area, which was predicted to be affected by aquaculture. Decreases in TR were associated with visual indicator presence, validating the use of a suite of visual indicators to detect organic deposition. Importantly, visual indicators should be considered together in the context of regulation, given that relationships between TR and indicators were not linear when the latter are considered individually.

Temporal Bacterial Surveillance of Salmon Aquaculture Sites Indicates a Long Lasting Benthic Impact With Minimal Recovery.

Coastal aquaculture has experienced substantial growth in the last few decades and associated impacts on natural environments are of increasing importance. Understanding both the effects of aquaculture on marine ecosystems and the processes of recovery during fallowing periods is crucial for the development of a more environmentally sustainable industry. Because bacteria are sensitive to environmental change, surveying fluctuations in bacterial communities is a promising tool for monitoring the status of benthic environments. Here, we used 16S rRNA gene high-throughput sequencing to characterize bacterial communities in flocculent matter samples collected over a period of 3 years and at various distances from cages (0-200 meters) at production and fallow (3-35 months) salmon aquaculture sites in southern Newfoundland to evaluate the environmental impact of aquaculture on predominantly hard-bottom substrates. Bacterial composition analysis revealed four clusters, three of which (defined as "recently disturbed," "intermediate impact," and "high impact") differed markedly from a fourth "low impact" cluster that contained far-field samples collected >500 m from cages. Samples within the high impact group were most often collected directly under cages, whereas those in the intermediate impact group were mainly sampled from 20 to 40 m from cages. Large scale phylum shifts (increases of Bacteroidetes, Firmicutes, Spirochaetes, and decreases in Proteobacteria and Epsilonbacteraeota) and a decline in bacterial diversity were observed in the high impact cluster, indicating significant ecological change. Samples from sites of different fallow duration were found in the high impact cluster, indicating a lack of recovery, even after 35 months of fallowing. Finally, we identified 28 genera as bacterial biomarkers, specific to one or more clusters, including genera associated with organically enriched environments and previously reported in the context of aquaculture impacts. Tracking the relative abundance of biomarkers in relation to different lengths of fallowing in the three more impacted clusters showed that these markers remained significantly above low impact cluster levels at all times, further pointing toward incomplete recovery. Our results suggest that coastal aquaculture on hard-bottom substrates is prone to long lasting impacts on bacterial communities, especially below cages, and that effects can be accurately tracked using bacterial community profiles or specific biomarkers.

The usage of visual indicators in regulatory monitoring at hard-bottom finfish aquaculture sites in Newfoundland (Canada).

Finfish aquaculture can be installed over hard and patchy substrates where grab sampling is challenging and use of video can be an appropriate tool to document benthic changes. Video monitoring can show visual indicators of enrichment, namely flocculent matter, Beggiatoa-like mats, and opportunistic polychaete complexes (OPC). We examined factors influencing presence of indicators using 52 video monitoring reports collected in Newfoundland, Canada. The main driving factor was distance to cage, with indicators showing a higher probability of occurrence within 10m from cages due to low current velocities. Indicators were less prevalent on sites dominated by hard substrates while OPC in particular were restricted to depths >35m. Beggiatoa-like bacteria covered a larger surface than the two other indicators; however, our results suggest the necessity of amalgamating information related to all the indicators (including bare stations that could indicate anoxia) to establish a more accurate evaluation of aquaculture impact.

The Impact of Egg Ozonation on Hatching Success, Larval Growth, and Survival of Atlantic Cod, Atlantic Salmon, and Rainbow Trout.

The direct exposure of fish eggs to ozonated water has generated interest as a means of ensuring pathogen-free eggs without the use of harsh chemicals. However, there are numerous knowledge gaps, including safe contact times, exposure levels, and potential long-term effects on aquaculture species in both freshwater and seawater. The effect of different ozone (O3) doses (0.5-1.0, 1.5-2.0, and 2.5-3.0 mg of O3/L for 90 s) on recently fertilized eggs of Atlantic Cod Gadus morhua and eyed eggs of Atlantic Salmon Salmo salar and Rainbow Trout Oncorhynchus mykiss was evaluated in comparison with the effects of two commercial disinfectants: Perosan (0.004 mg/L) and Ovadine (100 mg/L). The impact of ozone application was evaluated based on hatching success, larval nucleic acid concentration, larval growth, and survival. Overall, results indicated that ozonation of Atlantic Cod eggs at a dose less than 3.0 mg/L for 90 s produced no negative effect on the larvae up to 30 d posthatch. Furthermore, ozonation of Atlantic Salmon and Rainbow Trout eggs generated no negative effect on the larvae, based on monitoring until 85% yolk sac re-absorption (16 d posthatch).

Lipid Classes and Fatty Acids in Ophryotrocha cyclops, a Dorvilleid from Newfoundland Aquaculture Sites.

A new opportunistic annelid (Ophryotrocha cyclops) discovered on benthic substrates underneath finfish aquaculture sites in Newfoundland (NL) may be involved in the remediation of organic wastes. At those aquaculture sites, bacterial mats and O. cyclops often coexist and are used as indicators of organic enrichment. Little is known on the trophic strategies used by these annelids, including whether they might consume bacteria or other aquaculture-derived wastes. We studied the lipid and fatty acid composition of the annelids and their potential food sources (degraded flocculent organic matter, fresh fish pellets and bacterial mats) to investigate feeding relationships in these habitats and compared the lipid and fatty acid composition of annelids before and after starvation. Fish pellets were rich in lipids, mainly terrestrially derived C18 fatty acids (18:1ω9, 18:2ω6, 18:3ω3), while bacterial samples were mainly composed of ω7 fatty acids, and flocculent matter appeared to be a mixture of fresh and degrading fish pellets, feces and bacteria. Ophryotrocha cyclops did not appear to store excessive amounts of lipids (13%) but showed a high concentration of ω3 and ω6 fatty acids, as well as a high proportion of the main fatty acids contained in fresh fish pellets and bacterial mats. The dorvilleids and all potential food sources differed significantly in their lipid and fatty acid composition. Interestingly, while all food sources contained low proportions of 20:5ω3 and 20:2ω6, the annelids showed high concentrations of these two fatty acids, along with 20:4ω6. A starvation period of 13 days did not result in a major decrease in total lipid content; however, microscopic observations revealed that very few visible lipid droplets remained in the gut epithelium after three months of starvation. Ophryotrocha cyclops appears well adapted to extreme environments and may rely on lipid-rich organic matter for survival and dispersal in cold environments.

A new annelid species from whalebones in Greenland and aquaculture sites in Newfoundland: Ophryotrocha cyclops, sp. nov. (Eunicida: Dorvilleidae).

A new species of Ophryotrocha was discovered on whalebones in Greenland (120 m depth) and at finfish aquaculture sites in Newfoundland (30-70 m depth), where it is considered to be a bioindicator of aquaculture-related organic enrichment. Phylogenetic analyses based on three genes (COI, 16S mitochondrial and H3 nuclear genes) show close affinities with O. lobifera and O. craigsmithi, two species also found on both whalebones and at aquaculture sites (North Sea), and with O. shieldsi from aquaculture sites in Tasmania. The new species is named Ophryotrocha cyclops sp. nov. due to the juxtaposed, quasi-fused central eyes that give the impression of a single eye in live specimens.

Effects of chronic exposure to the aqueous fraction of produced water on growth, detoxification and immune factors of Atlantic cod.

The biggest discharge from the offshore oil industry is produced water (PW). As new technologies emerge to remove oil from such discharges, the question remains as to the effect that the water soluble fraction of contaminants present in PW may have on the biota surrounding the areas of discharge. We investigated the effects of 8 weeks of intermittent exposure to environmentally relevant concentrations (100 or 1000mg/L) of the aqueous fraction of PW (AFPW) on growth parameters, food consumption, respiratory burst activity of head kidney leukocytes (RB), activity of antioxidant enzymes and mRNA expression of immune- and detoxification-related genes of Atlantic cod. No significant effects of AFPW were seen on growth parameters, food consumption and/or RB. Furthermore, the activity of antioxidant enzymes and the expression of CYP1A, GST and UGT were not impacted by AFPW treatment. The mRNA expression of some immune related genes was affected in a similar manner as what has been described in Atlantic cod exposed to full PW suggesting that short chain soluble compounds present in PW might be responsible for its immunomodulatory effect. Traditionally used biomarkers of toxicant exposure such as phase I (CYP1A) and phase II (GST, UGT) genes do not seem to be reliable indicators of exposure to AFPW. This study confirms the fact that some immune related genes are affected by soluble components of PW and that further investigation on potential increased disease susceptibility is warranted.

In vitro effect of produced water on cod, Gadus morhua, sperm cells and fertilization.

The in vitro effect of produced water released by oil and gas platforms was assessed by exposing cod sperm cells to realistic concentrations of this mixture (100, 200, 500 ppm). We investigated produced water impact on enzymes of the aerobic (citrate synthase) and glycolytic metabolism (lactate dehydrogenase), lipid catabolism (lipase), as well as an anti-oxidant enzyme (catalase). Fertilization rates, viability, respiration, ATP, and total motility duration were also evaluated. To explore correlations between these parameters, we have also tested the effect of conserving sperm for 24 h at 4 degrees C. After conservation, fertilization success was decreased but other parameters were not affected. Produced water did not result in a significant change in fertilization; a significant increase in sperm protein amounts and citrate synthase activity can be observed. No correlations are found between parameters showing that sperm viability and unchanged energy levels do not translate into equivalent fertilization capacity. To conclude, exposure of sperm to produced water resulted only in subtle effects on cells. These findings bring information on the effect of produced water on sperm itself rather than on spermatogenesis or testis development of an exposed fish.

The immune response of juvenile Atlantic cod (Gadus morhua L.) to chronic exposure to produced water.

Produced water (PW) is the main discharge from the offshore oil industry and contains oil-derived compounds such as poly-aromatic hydrocarbons, phenols, alkylphenols, and heavy metals. Studies suggest that PW discharges may affect the biota over larger areas from the oil drilling sites at sea than originally predicted. We investigated the effects of chronic exposure to PW on some aspects of juvenile Atlantic cod immunity, stress response and growth by intermittently exposing fish to 0, 100 or 200 ppm of PW for 22 weeks. No significant effects of PW were observed on growth, hepatosomatic index, condition factor or plasma cortisol. The respiratory burst (RB) of circulating leukocytes was significantly elevated in the 100 ppm group only, while the RB of head-kidney leukocytes was significantly decreased in both the 100 and 200 ppm groups. Significant up-regulation of the mRNA expression of beta-2-microglobulin, immunoglobulin-M light chain and interleukins-1beta and -8 was observed in the 200 ppm group, while the down-regulation of interferon stimulated gene 15 was obvious for both the 100 and 200 ppm groups. The results suggest that chronic exposure to environmentally relevant concentrations of PW causes modulations of the immune system of juvenile Atlantic cod with most immune parameters being stimulated, potentially resulting in an energetic cost that may be detrimental to the fish.

State of the marine environment at Little Bay Arm, Newfoundland and Labrador, Canada, 10 years after a "do nothing" response to a mine tailings spill.

In 1989, the tailings pond dam at the site of a former copper mine near Little Bay, Newfoundland and Labrador, Canada, ruptured and tailings spilled into Little Bay Arm. At the time, no action was taken to arrest the flow of tailings or to mitigate the effects of the spill. To date, no action has been taken to repair the dam and tailings continue to flow into Little Bay Arm. As a result, the marine environment around Little Bay Arm has become contaminated with heavy metals from the tailings. However, the tailings are not the only source of heavy metals to the ecosystem. An old slag heap and what is presumably concentrated copper ore spilled during the loading of ore freighters, are also contributing to the ecosystem's metal load. Marine sediment throughout the Arm contained elevated concentrations of Cu, Ni, Zn, As, V, Co, and Mn. Beach material also contained elevated concentrations of metals with material near the slag heap being the most contaminated. At this site, Cu concentrations were in excess of 5000 mg kg(-1) dry weight, Zn greater than 3000 mg kg(-1) and Co concentrations exceeded 700 mg kg(-1). The highest concentrations of metals in biota were found near the slag heap, near the tailings dam breach, and at the site of the former concentrate loading dock. Despite elevated metal concentrations, the tailings and nearby sediment were not devoid of life. Bivalves and seaweed were abundant in the area and there were no obvious signs of tissue damage or disease in soft shell clams (Mya arenaria) living in the tailings. These clams may be suffering from chronic exposure to the tailings, however, evidence of lipid peroxidation in the clams was inconclusive.