The Comet Assay, a Sensitive Biomarker of Water Quality Improvement Following Adoption of Beneficial Agricultural Practices?

Numerous actions have been undertaken by farmers to attenuate the impact of agricultural activities on aquatic ecosystems. The identification of biomarkers that respond quickly to water quality improvement could facilitate the assessment of adopted alternative practices and help maintain mobilization among stakeholders. We evaluated the potential of the comet assay, a biomarker of genotoxic effects, using a freshwater mussel, Elliptio complanata, as a model animal. The frequency of DNA damage was assessed in hemocytes of mussels collected from a pristine habitat and caged for 8 weeks in the Pot au Beurre River, a tributary of the fluvial Lake St.-Pierre (Quebec, Canada) impacted by agricultural activities. We found that the level of DNA damage naturally induced in mussel hemocytes was low and showed very limited variations over time. Compared with these baseline levels and to laboratory controls, we observed a doubling in DNA alterations in mussels exposed to agricultural runoff in the third branch of the Pot au Beurre River. The genotoxic response was significantly lower in mussels caged in the first branch of the Pot au Beurre River, where longer stretches of shoreline have been restored as buffer strips. Glyphosate, mesotrione, imazethapyr, and metolachlor were the main discriminant pesticides between these two branches. Metolachlor was found in sufficient concentrations to induce DNA damage, but it is more likely that the observed genotoxicity was the result of a "cocktail effect," that is, the cumulative contribution of coexisting genotoxicants including the above-mentioned herbicides and ingredients in their formulation. Our findings suggest that the comet assay is a sensitive tool for the early detection of changes in water toxicity following the adoption of agricultural beneficial practices. Environ Toxicol Chem 2023;42:2201-2214. © 2023 Crown copyright and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article is published with the permission of the Controller of HMSO and the King's Printer for Scotland.

Early developmental toxicity of Atlantic salmon exposed to conventional and unconventional oils.

Atlantic salmon is an important species for Canadian culture and economy and its importance extends beyond Canada to Scandinavia and Western Europe. However, it is a vulnerable species facing decline due to habitat contamination and destruction. Existing and new Canadian pipeline projects pose a threat to salmonid habitat. The effects of diluted bitumen (dilbit), the main oil circulating in pipelines, are less studied than those of conventional oils, especially during the critical early embryonic developmental stage occurring in freshwater ecosystems. Therefore, this study aimed to compare the effects of water-accommodated fractions (WAF) of the Clearwater McMurray dilbit and the Lloydminster Heavy conventional oil on Atlantic salmon embryos exposed either from fertilization or from eyed stage. The dilbit contained the highest concentrations of low molecular weight (LMW) compounds (including BTEX and C6-C10), while the conventional oil contained the highest concentrations of PAHs. The Clearwater dilbit caused a higher percentage of mortality and malformations than the conventional oil at similar WAF concentrations. In addition, the embryos exposed from fertilization suffered a higher mortality rate, more developmental delays, and malformations than embryos exposed from the eyed stage, suggesting that early development is the most sensitive developmental stage to oil exposure. Gene expression and enzymatic activity of the detoxification phase I and II enzymes (CYP1A and GST) were measured. Data showed increases in both cyp1a expression and GST activity with increasing WAF concentrations, while gst expression was not affected by the exposures. Also, gene expression of proteins involved in the biotransformation of vitamin A and DNA damage repair were modified by the oil exposures. Overall, this study indicates that Atlantic salmon is mostly affected by oil exposure at the beginning of its development, during which embryos accumulate deformities that may impact their survival at later life stages.

The effects of dissolved petroleum hydrocarbons on benthic organisms: Chironomids and amphipods.

The oil sands industry in Canada, produces heavy unconventional oils, diluted for transport and called diluted bitumen. However, despite advances in our knowledge of the ecotoxicological risk that these products represent, their effects on benthic organisms following a spill are still largely unknown. In order to fill these gaps, this study aims to determine the lethal and sublethal effects of two diluted bitumens (Bluesky and Cold Lake) and one conventional oil (Lloydminster) for two freshwater benthic invertebrates: Chironomus riparius and Hyalella azteca. The objective of this study is to assess the toxicity of dissolved hydrocarbons, resulting from the physical dispersion of oil, immediately after a spill on the benthic invertebrates. To this end, organisms were exposed for 7 days for chironomids and 14 days for amphipods to a fraction containing soluble hydrocarbons (WAF: water accommodated fraction; 10 g/L, 18 h of agitation, followed by 6 h of sedimentation) with natural or artificial sediment. After exposure, the effects of hydrocarbons were determined using size, mortality, and antioxidant capacities. Dissolved hydrocarbons induced mortality for both species, but these hydrocarbons disappeared very quickly from the water column, regardless of the oil type. The amphipods were sensitive to both types of oil while the chironomids were only sensitive to diluted bitumens. The presence of a natural sediment seems to provide a protective role against dissolved hydrocarbons. The antioxidant enzymes measured (CAT, SOD and GPx) do not appear to be relevant biomarkers for the exposure of these organisms to diluted bitumen.

Comparative toxicity of conventional and unconventional oils during rainbow trout (Oncorhynchus mykiss) embryonic development: From molecular to health consequences.

Canadian freshwater ecosystems are vulnerable to oil spills from pipelines, which contain mostly diluted bitumen. This study aimed to compare the toxicity of a dilbit and a conventional oil on developing rainbow trout. A total of five exposure scenarios were performed, from 10 to 43 days, using water-accommodated fraction (WAF) with an initial loading of 1:9 oil to water ratio (w/v) in a range of dilutions from 0.32 to 32% WAF, respectively, with TPAH and VOC concentrations from 2.41 to 17.5 µg/L and 7.94-660.99 µg/L, and with or without a recovery period. Following the five exposures, several endpoints were examined, including survivorship, morphometrics, gene expression, and enzymatic activity. Significant mortality rates were measured for the highest WAF concentration of the dilbit in all five exposures (60-100% mortality at 32% WAF). In comparison, the highest WAF concentration of the conventional oil induced significant mortality in three out of the five exposure (from 35 to 100% mortality at 32% WAF). Hatching delays were noted in embryos exposed to both oils. Developmental delays were observed in dilbit-exposed embryos and are suspected to be an indicator of reduced survivorship after hatching. The induced expression of cyp1a remained a reliable biomarker of exposure and of fish malformations, though it did not always predict mortality. Using CYP1A activity in combination with cyp1a may bring more insights in studies of oil risk assessment. This study demonstrates that dilbits are more toxic to early life stages compared to conventional oils and highlights the need to consider the most sensitive stage of development when performing risk assessment studies on oils.

Comparative developmental toxicity of conventional oils and diluted bitumen on early life stages of the rainbow trout (Oncorhynchus mykiss).

Petroleum hydrocarbons are widely used and transported, increasing the risks of spills to the environment. Although conventional oils are the most commonly produced, the production of unconventional oils (i.e. diluted bitumen or dilbit) is increasing. In this study, we compared the effects of conventional oils (Arabian Light and Lloydminster) and dilbits (Bluesky and Clearwater) on early life stages of a salmonid. To this end, aqueous fractions (WAF: water accommodated fraction) of these oils were extracted using mountain spring water. Rainbow trout (Oncorhynchus mykiss) larvae were exposed to 10 and 50% dilutions of these WAFs from hatching (340 DD; degree days) until yolk sac resorption (541 DD). Exposure to WAFs increased skeletal malformations (both dilbits) and hemorrhage (both conventional oils and Bluesky) and decreased head growth (Arabian Light). In addition, increases in EROD activity and DNA damage were measured for all oils and an increase in cyp1a gene expression was measured for Arabian Light, Bluesky and Clearwater. The PAH and C10C50 concentrations were positively correlated to total larval EROD activity, whereas concentrations of total hydrocarbons, VOCs, PAHs, and C10C50 were positively correlated to cyp1a expression. Total hydrocarbon, VOC, and C10C50 concentrations were also negatively correlated to larval growth. This study supports that petroleum hydrocarbons are toxic to early developmental stages of rainbow trout and show that their degree and spectrum of toxicity depends on their chemical composition.

Lethal and sublethal effects of diluted bitumen and conventional oil on fathead minnow (Pimephales promelas) larvae exposed during their early development.

The increasing extraction of bitumen from the oil sands region in Canada is creating a need for transport. Spills from current and projected pipelines represent a significant environmental risk, especially for freshwater ecosystems. The toxicity of diluted bitumen (dilbit) on freshwater fish is largely unknown. This study assessed the toxicity of two dilbits (Clearwater McMurray and Bluesky) and compared their toxicity to a conventional oil (Lloydminster Heavy) on fathead minnow (Pimephales promelas) larvae. Larvae were exposed to various concentrations of the water-accommodated fraction (WAF) of the oils during 7 days from hatching. In the WAF treatments, the concentrations of volatile organic compounds (VOCs), including benzene, toluene, ethylbenzene, xylene (BTEX), hydrocarbons containing 6 to 10 carbon atoms (C6-10), and polycyclic aromatic hydrocarbons (PAHs) and their alkylated forms were measured. Both dilbits contained higher concentrations of light components, while the conventional oil contained the highest concentrations of PAHs and alkylated PAHs. The Clearwater McMurray dilbit induced a higher mortality, with a maximum of 65.3%, while the other oils induced a similar mortality up to 16.5% and 18.6% for Lloydminster and for Bluesky, respectively. All three oils induced an increase in gene expression of the phase I detoxification enzyme (cyp1a) with increasing total hydrocarbon concentrations. All three exposures induced a similar increase in glutathione S-transferase (GST) activity, but no change in gst gene expression. For the Bluesky and Lloydminster exposures, an increase in malondialdehyde concentration was also observed, suggesting a rate limiting capacity of GST and phase II enzymes to perform the biotransformation of the PAH metabolites. Overall, this study brings new insights on the toxicity of dilbits in comparison to conventional oils on early life stages of North American freshwater fish and demonstrated that dilbits can be more toxic than conventional oils, depending on their composition and diluent proportions.

Fate and Fathead Minnow Embryotoxicity of Weathering Crude Oil in a Pilot-Scale Spill Tank.

For several years now, the Natural Resources Canada research facility at CanmetENERGY Devon (AB, Canada) has been performing experiments in a pilot-scale spill tank using 1200 L of river water to examine the physical and chemical behaviors of various crude oil/water mixtures under varying water temperature regimes. Because oil toxicity can be modulated by weathering of the petroleum products, the present study aimed to assess changes in fish embryotoxicity to mixed sweet blend crude oil as it weathered at air and water temperatures of 14 °C and 15 °C, respectively, for 28 d. The physicochemical behavior of the oil was also monitored. Water samples were taken from the spill tank 5 times during the 28-d experiment on days 1, 6, 14, 21, and 28 and were used to perform toxicity exposures using fathead minnow embryos (Pimephales promelas). For each water sampling day, newly fertilized embryos were exposed to a serial dilution of the spill tank water, noncontaminated river water (used in the spill tank), and a reconstituted water laboratory control. Embryos were raised until hatching. Although mortality was not significantly altered by the oil contamination over the time period, malformation occurrence and severity showed concentration-dependent responses to all contaminated water collected. The results suggest that days 14, 21, and 28 were the most toxic time periods for the fish embryos, which corresponded to increasing concentrations of unidentified oxidized organic compounds detected by a quadropole-time-of-flight system. The present study highlights a novel area for oil research, which could help us to better understand the toxicity associated with oil weathering for aquatic species. Environ Toxicol Chem 2021;40:127-138. © 2020 Her Majesty the Queen in Right of Canada. Reproduced with the permission of the Minister of Natural Resources Canada.

Effect of the electrocoagulation process on the toxicity of gold mine effluents: A comparative assessment of Daphnia magna and Daphnia pulex.

Mine effluents must meet discharge criteria for both physicochemical parameters and toxicity. While chemical precipitation is efficient for the treatment of metallic elements in mine effluents, the removal of sulfates, as a source of salinity and potential toxicity, is limited by gypsum solubility. This study evaluated the efficiency of electrocoagulation (EC), an emerging process to treat mine water, in removing sulfates and acute toxicity in two gold mine effluents (E1 and E2), before and after treatment (Fe-electrodes, 30 min at 20 mA/cm2, and pH near neutrality). Standard toxicity tests were conducted on two daphnia species, Daphnia magna (standard test species) and Daphnia pulex (more common in cold climate). Four uncontaminated surface waters (S#1 to S#4), which originated from different watershed lithologies, were also used as dilution media with E1 to assess water quality effect on toxicity response. Statistical analyses using the Student's t-test showed no significant difference in immobility or mortality caused by surface waters on either D. magna or D. pulex species (p > 0.05). However, higher toxicity was observed with both daphnia when reconstituted hard water was used for testing of the treated effluent E2. The present study highlights the toxicity effect added by EC despite a sulfates-related salinity decrease of >7.5%. Further research should identify and confirm the potential sources of observed toxicity.

Toxicity and Subcellular Fractionation of Yttrium in Three Freshwater Organisms: Daphnia magna, Chironomus riparius, and Oncorhynchus mykiss.

The demand for rare earth elements (REEs) has increased since the 1990s leading to the development of many mining projects worldwide. However, less is known about how organisms can handle these metals in natural aquatic systems. Through laboratory experiments, we assessed the chronic toxicity and subcellular fractionation of yttrium (Y), one of the four most abundant REEs, in three freshwater organisms commonly used in aquatic toxicology: Daphnia magna, Chironomus riparius, and Oncorhynchus mykiss. In bioassays using growth as an end point, C. riparius was the only organism showing toxicity at Y exposure concentrations close to environmental ones. The lowest observable effect concentrations (LOECs) of Y assessed for D. magna and O. mykiss were at least 100 times higher than the Y concentration in natural freshwater. A negative correlation between Y toxicity and water hardness was observed for D. magna. When exposed to their respective estimated LOECs, D. magna bioaccumulated 15-45 times more Y than the other two organisms exposed to their own LOECs. This former species sequestered up to 75% of Y in the NaOH-resistant fraction, a putative metal-detoxified subcellular fraction. To a lesser extent, C. riparius bioaccumulated 20-30% of Y in this detoxified fraction. In contrast, the Y subcellular distribution in O. mykiss liver did not highlight any notable detoxification strategy; Y was accumulated primarily in mitochondria (ca. 32%), a putative metal-sensitive fraction. This fraction was also the main sensitive fraction where Y accumulated in C. riparius and D. magna. Hence, the interaction of Y with mitochondria could explain its toxicity. In conclusion, there is a wide range of subcellular handling strategies for Y, with D. magna accumulating high quantities but sequestering most of it in detoxified fractions, whereas O. mykiss tending to accumulate less Y but in highly sensitive fractions.

Toxicity of tailing leachates from a niobium mine toward three aquatic organisms.

The aim of this research was to assess the ecotoxicity of leachates originating from a niobium mine located in Canada. These tailings contain considerable amounts of carbonates and phosphates and could potentially be used as fertilizer for agriculture. However, the presence of different contaminants linked with the ores mined, including rare earth elements and daughter elements of the uranium disintegration chain is of concern. Bioassays have been used to determine if the tailings leachates could be harmful. The assessment of the toxicity of progressive dilutions of five tailing leachates (808, 809, 810, 811 and 897) was performed on different organisms: phytoplankton Raphidocelis subcapitata and duckweed Lemna minor, based on their growth and chlorophyll a content, and water flea Daphnia magna based on their mobility, mortality and reproduction. Overall, the leachates showed higher toxicity to Raphidocelis subcapitata and Lemna minor, than toward Daphnia magna. Leachate 808 showed no toxicity to all organisms while leachate 810 showed significant effects to all species. The results can be explained by the leachate dissolved metal or nutrient concentrations, but also by the metal bioavailability which depends on pH and hardness. Generally, toxicity was observed in undiluted samples tested, which is not representative of the conditions that could occur in the environment. This supports the idea that these tailings could be used as fertilizer albeit more studies may be required, particularly to assess the toxicity of the tailings leachate for benthic organisms, the toxicity of the tailings for terrestrial organisms and the variations of soil and sediment physicochemical properties after tailing treatments.

Comparison of laboratory batch and flow-through microcosm bioassays.

Since 1997, we have been developing a protocol for ecotoxicological bioassays in 2-L laboratory microcosms and have applied it to the study of various pollutants and ecotoxicological risk assessment scenarios in the area of urban facilities and transport infrastructures. The effects on five different organisms (micro-algae, duckweeds, daphnids, amphipods, chironomids) are assessed using biological responses such as growth, emergence (chironomids), reproduction (daphnids) and survival, with a duration of exposure of 3 weeks. This bioassay has mainly been used as a batch bioassay, i.e., the water was not renewed during the test. A flow-through microcosm bioassay has been developed recently, with the assumption that conditions for the biota should be improved, variability reduced, and the range of exposure patterns enlarged (e.g., the possibility of maintaining constant exposure in the water column). This paper compares the results obtained in batch and flow-through microcosm bioassays, using cadmium as a model toxicant. As expected, the stabilization of physico-chemical parameters, increased organism fitness and reduced variability were observed in the flow-through microcosm bioassay.

Comparison of bioassays with different exposure time patterns: the added value of dynamic modelling in predictive ecotoxicology.

The purpose of this study was to compare Daphnia magna responses to cadmium between two toxicity experiments performed in static and flow-through conditions. As a consequence of how water was renewed, the two experiments were characterised by two different exposure time patterns for daphnids, time-varying and constant, respectively. Basing on survival, growth and reproduction, we addressed the questions of organism development and sensitivity to cadmium. Classical analysis methods are not designed to deal with the time dimension and therefore not suitable to compare effects of different exposure time patterns. We used instead a dynamic modelling framework taking all timepoints and the time course of exposure into account, making comparable the results obtained from our two experiments. This modelling framework enabled us to detect an improvement of organism development in flow-through conditions compared to static ones and infer similar sensitivity to cadmium for both exposure time patterns.

Assessment of contaminated sediments with an indoor freshwater/sediment microcosm assay.

This study was conducted to assess the feasibility of using a 2-L, indoor microcosm assay to evaluate five contaminated sediments (A, B, C, D, and E). Toxic potential was deduced in the light of general contamination of sediments, pollutant partitioning in microcosms, and biological responses of species (Pseudokirchneriella subcapitata, Lemna minor, Daphnia magna, Hyalella azteca, Chironomus riparius): E > A > B > C > D. Sediments mainly were contaminated by metals (lead and zinc). Organic pollutant contents varied among the sediments. The major polycyclic aromatic hydrocarbon (PAH) compounds were pyrene, fluoranthene, and phenanthrene. Sediments A, B, and C highly stimulated duckweed growth (> 700%) and impaired daphnid (< 20%) and amphipod survival (< 30%). Sediment D had no significant effect on pelagic and benthic organisms. Finally, sediment E, the most toxic, limited duckweed growth (inhibition of 82%) and impaired daphnid survival (0% of survival). Amphipods were impaired dramatically by this sediment (0% of survival), in contrast with chironomids, for which no toxic effect was measured. The 2-L, indoor microcosm assay successfully was applied to the assessment of those five contaminated sediments. Sediments A, B, C, and E should not be deposited in gravel quarries, and new, more sensitive endpoint measurements should be developed.