Biofilms represent a nexus for the degradation of plastic materials in aquatic environments. The purpose of this study was to examine the influence of anthropogenic pollution on biofilm composition and function in a freshwater river (Saint-Lawrence River, Québec, Canada). Newly formed biofilms were collected on ceramic tiles at 2 combined sewers and street runoffs sites, one downstream of a large city (2 million inhabitants) and one site located 8 km downstream a municipal effluent dispersion plume. The data revealed that biofilms from the rainfall overflow sites were most contaminated with Nanoplastics while biofilms from the municipal effluent dispersion plume contained significantly more lipids. Biofilms from the overflow sites also exhibited increased esterase and biodegradation index compared to the other sites. However, no signs of oxidative stress were observed in biofilms suggesting that urban pollution was not deleterious to biofilm communities. In fact, 16S taxonomic analysis of biofilms revealed an increased presence of bacteria species usually associated with the plastisphere and known to degrade plastics in aquatic environments. In conclusion, biofilms could represent a sink to plastic pollution in urban environments.

Biofilms are critical mediators of contaminant fate in aquatic environments, acting as sites for the accumulation and potential degradation of plastic materials amongst other contaminants. However, the ecological impacts of urban pollution on freshwater biofilm composition and function remain poorly understood. In this study, we examined how anthropogenic contamination, including nanoplastics, alters biofilm communities in the Saint-Lawrence River (Québec, Canada). To test this, freshwater mussels and bare terracotta tiles for biofilm colonization were placed together in cages at three sites along the Saint-Lawrence River: a combined sewers and street runoffs site, a site downstream of a large city (2 million inhabitants), and a site 8 km downstream of a municipal effluent dispersion plume. The experiment involved two replicate cages per site and a 3-month exposure period. Biofilms were harvested at the end of the experiment to determine the levels of plastic-related contaminants (plastic nanoparticles), functional activity (esterase activity, lipids, oxidative stress, and plastic biodegradation capacity), and community composition by 16S rRNA sequencing. In parallel, the digestive gland of mussels was sampled to assess the microbiome’s capacity to degrade plastics, oxidative stress, and heterotrophic bacterial load from undsinfected wastewaters. The data revealed that biofilms from the rainfall overflow site were most contaminated with nanoplastics, while those from the municipal effluent dispersion plume contained significantly more lipids. Biofilms from the overflow site also exhibited increased esterase activity and biodegradation index compared to the other sites. However, no signs of oxidative stress were observed in biofilms from the overflow site compared to those of the municipal effluent plume site. Microbial community composition showed only a marginal shift among sites (PERMANOVA, F = 1.69, p = 0.066), while differences in community dispersion were highly significant (PERMDISP, p < 0.001), reflecting increased heterogeneity at urban-impacted locations. Thus, urban pollution did not uniformly impair biofilm communities; instead, it resulted in site-specific ecological responses, including elevated oxidative stress at the municipal effluent plume site and substantial community heterogeneity and instability at the overflow site. Additionally, microbial taxonomic analysis of biofilms revealed an increased presence of bacterial species typically associated with the plastisphere and known to degrade plastics in aquatic environments. In mussels, the biodegradation index and bacterial load were significantly increased at the overflow and downstream effluent sites, respectively. In conclusion, both mussels and biofilms may represent critical compartments in plastic pollution dynamics in urban environments as evidenced by their increased capacity to degrade plastics.

Small plastic polymers have the potential to bioaccumulate in tissues and initiate toxicity, raising concerns about the long-term impacts on filter feeders such as freshwater mussels. The purpose of this study was to examine the toxicity of two common plastic polymers (polyvinyl chloride—PVC; polyethylene terephthalate—PET) and the plasticizer dibutylphthalate (DBP) to quagga mussels, also called Dreissena bugensis. Mussels were exposed to 5, 50, and 100 µg/l of the above compounds for 96 h at 15 °C. They were then analyzed for total plastic accumulation, esterase, peroxidase, lipids, and protein aggregation in soft tissues. The data revealed that DBP reduced survival at concentrations > 5 µg/l and all died at 100 µg/l. Estimated bioavailability factors of 90, 40, and 1580 for PVC, PET, and DBP were determined. These differences could be explained by particle size and form for PVC (0.5 µm fiber) and PET (2 µm diameter). DBP tissue levels were also detected in mussels exposed to PVC and PET, suggesting its presence in plastics. Esterase activity was strongly increased in DBP (ester)-treated mussels and was slightly decreased in mussels exposed to ester-containing PET. The biomarker data also revealed an increase in lipids, peroxidase, and protein aggregation in a concentration-dependent manner. It is concluded that these compounds are bioavailable to mussels and the changes in esterase activity could be a factor leading to oxidative stress and protein aggregation in mussel tissues.

The objective of this study was to compare the cytotoxicity of monomeric and aggregated cadmium telluride quantum dots (CdTe QD) in human hepatoma (HepG2) and rainbow trout hepatocytes (RTH). Hepatocytes were exposed to concentrations of monomeric CdTe QDs (4 nm diameter) and isolates of different size aggregates for 48 h. The results revealed that the added Cd concentration in the cell culture media increased with the additions of both the monomeric and aggregated QDs where most (72%) of the total Cd was between 100 and 450 nm diameter size range as determined by ultrafiltration. CdTe QDs were cytotoxic to both cell types with an estimated 48 h-EC50 of 3.6 and 7.3 mg/L Cd for monomeric CdTe QDs for the HEPG2 and trout hepatocytes respectively. For the aggregated QDs, analysis of the concentration-response slopes revealed that HepG2 cells were able to significantly discriminate between 2 size ranges: nanoparticles < 4.6 nm and aggregates between 4.6 and 450 nm with the < 4.6 nm group being more toxic than the latter. The RTH model discriminated between 3 distinct size ranges in decreasing order of toxicity: 6.8 nm and smaller > 6.9-50 nm > 50-450 nm. In all cases, the toxicity of QD aggregates decreased with increasing size of the aggregates.

Plastic nanoparticles (NPs) released from plastic breakdown pervade aquatic ecosystems, raising concerns about their long-term toxic effects in aquatic organisms. The purpose of this study was to examine the sublethal toxicity of polyethylene (PeNPs) and polypropylene (PpNPs) nanoparticles of the same size (50 nm diameter) in Hydra vulgaris. Hydras were exposed to increasing concentrations of PeNPs and PpNPs (0.3–10 mg/L) for 96 h at 20 °C. Toxicity was determined based on the characteristic morphological changes and gene expression analysis of genes involved in oxidative stress, DNA repair, protein salvaging and autophagy, neural activity and regeneration. The data revealed that PpNPs produced morphological changes (50% effects concentration EC50 = 7 mg/L), while PeNPs did not. Exposure to both nanoplastics produced changes in gene expression in all gene targets and at concentrations less than 0.3 mg/L in some cases. PpNPs generally produced stronger effects than PeNPs. The mode of action of these plastic polymers differed based on the intensity of responses in oxidative stress (superoxide dismutase, catalase), DNA repair of oxidized DNA, regeneration and circadian rhythms. In conclusion, both plastics’ nanoparticles produced effects at concentrations well below the appearance of morphological changes and at concentrations found in highly contaminated environments.

The cumulative effects of pollution of intertidal clam populations should be investigated to ensure the sustainable perennity of our resources. The purpose of this study was to examine the health status of intertidal clams and tissue levels of essential and non-essential elements at sites under anthropogenic stress. Clams were collected at two anthropized sites, a St. Lawrence Estuary (SLE) beluga high-residency area and reference site in the Saguenay–St. Lawrence Marine Park (Québec, Canada). Clam health status was determined by the condition factor (CF: wet weight/shell length ratio), growth index (GI: shell length/age), air survival time and weight loss index (WLI). Elemental analysis was also performed in soft tissues. The data revealed that clams from at least one of the harbor/marina sites had reduced CF, GI and WLI. Air survival time was not affected at the anthropized sites but was significantly higher at the St. Lawrence Estuary beluga high-residency area. The clams were contaminated by Ag, Al, Cd, Cu, Hg and V, with a decrease in essential cations (K, Ca, Mg) suggesting altered osmoregulation. Although the individual metals in tissues were not found at harmful concentrations based on reported data, the combined effects of non-essential elements could not be excluded. More research will be needed to better understand the cumulative effects of various stressors, such as low salinity, algal toxins and elemental composition, on clam health status.

Municipal wastewaters contain a peculiar mix of rare earth elements (REEs) dominated by gadolinium (Gd). The sublethal and lethal toxicity was examined in rainbow trout exposed to a representative REE mixture (municipal waswaters) composed of Gd (105 ng/L), Ce (9 ng/L), Nd (8 ng/L), Yb (6.2 ng/L) and Dy (4 ng/L) corresponding to total REEs loading of 137 ng/L. Juveniles were exposed to a mixture of REEs (Gd representing 80 % of the loading) at concentrations between 0.5 and 100X for 96 h at 15 °C. The data revealed that the 100X mixture was not acutely lethal but produced a range of sublethal effects at concentrations in the 1-10X range, which could be found in some municipal wastewaters. The sublethal effects involved changes in liver weight, heat stable metal binding proteins, protein aggregation, lipid peroxidation and DNA damage. Hence, these sublethal effects were observed in juvenile fish at environmentally relevant concentrations. This suggests that the REEs found in wastewaters could contribute to long-term negative impacts on fish populations.

The purpose of this study was to compare the toxicity of two major municipal effluents subjected to different treatment processes: a physico-chemically treated effluent (high-risk effluent) and a biofiltered/ UV-disinfected effluent (low-risk effluent). Juvenile fathead minnows were exposed to increasing concentrations of the high-risk and low-risk effluents in semi-static conditions for 16 weeks at 25 °C. At the end of the exposure period, juveniles were collected for immunocompetence (leukocyte density and phagocytosis), oxidative stress (catalase and superoxide dismutase activity) and DNA damage (COMET assay) assessments. The data revealed that DNA damage was the most sensitive biomarker, with increases at concentrations between 3.5% and 5%, and that the treatment processes had no influence on the intensity of genotoxic effects. A greater increase in oxidative stress enzymes was observed with the high-risk effluent as compared to the low-risk effluent, suggesting that oxidative stress was dependent on the treatment process applied. At the immunocompetence level, a biphasic pattern of response was found in both the high-risk and low-risk effluents. Indeed, in fish exposed to the low-risk effluent, an initial increase in leukocyte density and phagocytosis activity was followed by a subsequent decrease in these effects with increasing concentrations of the effluent. In the high-risk effluent, the initial increase and subsequent drop in leukocyte density occurred at lower concentrations while phagocytosis activity increased only at the highest concentration. In conclusion, the occurrence of DNA damage was not affected by the 2 types of effluents; however, oxidative stress and immunocompetence were more strongly influenced by the high-risk effluent than the low-risk one.

The cumulative effects of urban pollution and the presence of toxic phytoplankton in bivalves are not well understood. The purpose of this study was to determine the impacts of urban and algal pollution on wild Mya arenaria clams in the St. Lawrence Estuary. Clams were collected during low tide at sites differing in urban population (S1: 100; S2: 675; S3: 2,500; and S4: 50,000 inhabitants) in areas susceptible to toxic algal blooms and analyzed for immunocompetence (hemocyte density, viability, phagocytosis and oxidative burst/oxygen reactive species), energy reserves (sugars and proteins) and neural activity (acetylcholinesterase). Total phytoplankton counts in surface waters at the four sites, in decreasing order, were S3>>S1>S4 and S2. However, toxic phytoplankton species counts at the four sites were different: S4>>S1>S2>S3. The most abundant toxic species was Pseudonitzschia delicatissima, which is responsible for amnesic shellfish poisoning. Multiple regression analysis showed that toxic phytoplankton counts were significantly correlated with urban population size (β = 0.89) and with total phytoplankton (β =-0.25), suggesting that large populations favored algal bloom proliferation. Factorial analysis showed that toxic phytoplankton counts and population size were closely associated with reactive oxygen species production in hemocytes and increased AChE activity in clams, which is consistent with the cholinergic properties of some toxic algae. Total phytoplankton loadings were associated with increased energy reserves and hemocyte density. The data suggest that urban pollution contributes to algal production and that the effects of both could combine in clams, especially in stimulating AChE activity and the production of ROS in hemocytes.

Rapid and cost-effective tests for the evaluation of industrial and municipal effluents are urgently needed for environmental monitoring. In this context, peroxidase (PER) activity has been proposed as an early-warning biosensor for assessing the water quality of various wastewater discharges and leachates. The peroxidase-toxicity (Perotox) assay includes 0.1 µg/mL PER, albumin, DNA (for the DNA protection index), 0.001% monounsaturated Tween-80, and the substrates luminol and H2O2. The results revealed that an initial burst of luminescence was followed by a steady decrease in luminescence within the first minute, accompanied by periodic (cyclic) changes in the intermediate compound III (CIII) of PER. When urban effluents were added, PER activity was inhibited, with a concomitant increase in lipid peroxidation, indicating oxidative damage. The reduction in PER activity was also associated with the collapse in the periodic formation of CIII, alongside a steady increase in CIII over time. The addition of DNA to the reaction mixture helped mitigate the inhibition of PER by certain effluents, enabling the calculation of a DNA protection index. The levels of polystyrene (PS) in the organic fraction of the effluents were higher in the primary aeration lagoon (36 µg/L) compared to secondary lagoons and membrane filtration (< 16 µg/L). Data analysis revealed that PER activity was negatively correlated with population size (r = -0.34) and the levels of PS materials (r = -0.56). In conclusion, the Perotox assay is proposed as a rapid screening tool for identifying potentially toxic environmental complex mixtures, such as municipal effluents.

Municipal effluents are recognized as major sources of pollutants that could compromise fish health and reproduction. The purpose of this study was to examine and compare the reproductive toxicity of low-and high-risk municipal effluents to fathead minnows (Pimephales promelas) exposed for 12 weeks in the laboratory. After the exposure period, reproductive success was determined by following changes in the total number of eggs, egg hatchability/survival and time to hatch. In parallel, the expression of the following was also assessed in adults to gain insights into the pathways involved in toxicity: estrogen receptor alpha (ERα), androgen receptor (Ar1), pregnane X receptor (PXR1), vitellogenin (VTG), CYP3A4 and 17β-hydroxysteroid dehydrogenase (HSD). The results revealed that in the high-and low-risk effluents, egg laying followed a biphasic response, with an initial increase in egg laying followed by a decrease at higher concentrations and stronger amplitudes with the high-risk effluent. Hatching success (i.e., release of viable fish fry) was directly proportional to the decrease in egg production with no hatching of viable fish at 10% and 20% for both effluents. VTG gene expression was significantly increased in females, reaching levels 4 and 3 orders of magnitude greater than in the controls for the high-and low-risk effluent, respectively. VTG gene expression was also found in males but at lower expression levels than for the females. The expression of ERα was significantly correlated with VTG levels, which suggests the presence of estrogenic compounds in municipal effluents. This was further supported by the increased expression of CYP3A4, which is involved in the biotransformation of steroid-like and pharmaceutical compounds. In conclusion, municipal effluents have the capacity to reduce reproduction in fathead minnows and involve estrogenic effects. The high-risk effluent generally displayed stronger effects than the low-risk effluent.

Pharmaceutical, personal care and veterinary products, which have been found in wastewater and surface water, are likely to contaminate the aquatic environment, groundwater included. The purpose of this review is to examine current and new strategies for assessing the toxicological effects of this special class of xenobiotics on aquatic species. Aquatic sentinel species that bioaccumulate some of these drugs remain to be identified, but studies with mussels and plants have shown that some antibiotics significantly accumulate in tissues. Laboratory tests have been conducted with somesuccess on several aquatic species, including bacteria, plants, invertebrates (molluscs and arthropods) and fish, with commonly found drugs both individually and in mixtures. These toxicity tests generally indicate that acute lethal effects are not likely to occur in the environment but that chronic or long-term effects are possible. In an attempt to measure the effects of pharmaceuticals and personal care products, two types of biomarkers are proposed. The first class, known as integrative biomarkers, consists of measuring ecologically relevant biomarkers that encompass the effects of drugs, such as oxidative stress or DNA damage. Biomarkers that have been shown to predict changes at both the individual and population levels, and that respond to these products, are particularly useful for taking into account the final effects of pollution on feral aquatic organisms. The second class of biomarkers, known as drug target-specific biomarkers, measures the state/integrity of drug targets likely to impede the organism’s health and reproduction. For example, prostaglandin synthase produces prostaglandins necessary to assist spawning in bivalves, and its activity could be blocked by non-steroidal antiinflammatory drugs such as acetylsalicylate and ibuprofen. Finally, two case studies arepresented to exemplify the use of biomarkers to assess drug target specific interactions and tissue damage in aquatic species. In the first case study, primary cultures of rainbow trout hepatocytes were used to assess the cytotoxicity of carbamazepine (CBZ), a drug commonly found in municipal wastewater, at µg/L range, after exposure for 48 h at 18oC. Results showed that CBZ induced the activity of cytochromes P4503A4 and 2B6 (benzyloxyresorufin as the substrate), known biotransformation enzymes for this drug class (iminostillbene), and was highly correlated with lipid peroxidation and cell viabilityat environmentally relevant concentrations. Lipid peroxidation and cell viability are considered integrative biomarkers, while cytochrome P4503A4/2B6 activity is a targetspecific biomarker. The second case study concerns feral carp that had survived for four years in an aerated lagoon that treats domestic municipal effluent. Results showed that cytochrome P3A4 activity, as determined by dibenzyloxyfluorescein (another substrate specific for cytochromes P450 3A4, 3A5 and 2C9), was readily induced in the postmitochondrial supernatant of liver homogenates. ATP-dependent dopamine transport activity in synaptosome preparations of brain tissues was shown to be significantly reduced. Increased cytochrome P450-related activities and reduced dopamine uptake suggest the pharmacological effects of opiate-like substances. Preliminary findings thus indicate that some aquatic species are likely to accumulate some drugs and that they are likely to produce harmful effects on fish. Further research is needed to validate such biomarkers and to relate changes in drug targets with their residual levels in tissues.

There is an urgent need to evaluate the toxicity of xenobiotics and environmental mixtures for preventing loss in water quality for the sustainability of aquatic ecosystems. A simple prebiotic chemical pathway based on malate formation from pyruvate (pyr) and glyoxalate (glyox) is proposed as a quick and cheap screening tool for toxicity assessment. The assay is based on the pyr and glyox (aldol) condensation reactions, leading to biologically relevant precursors such as oxaloacetate and malate. Incubation of pyr and glyox at 40–70 °C in the presence of reduced iron Fe(II) led to malate formation following the first 3 h of incubation. The addition of various xenobiotics/contaminants (silver, copper, zinc, cerium IV, samarium III, dibutylphthalate, 1,3-diphenylguanidine, carbon-walled nanotube, nanoFe2O3 and polystyrene nanoparticles) led to inhibitions in malate synthesis at various degrees. Based on the concentration inhibiting malate concentrations by 20% (IC20), the following potencies were observed: silver < copper ~ 1.3-diphenylguanidine ~ carbon-walled nanotube < zinc ~ samarium < dibutylphthalate ~ samarium < Ce(IV) < nFeO3 < polystyrene nanoplastics. The IC20 values were also significantly correlated with the reported trout acute lethality data, suggesting its potential as an alternative toxicity test. The pyr-glyox pathway was also tested on surface water extracts (C18), identifying the most contaminated sites from large cities and municipal wastewater effluents dispersion plume. The inhibition potencies of the selected test compounds revealed that not only pro-oxidants but also chemicals hindering enolate formation, nucleophilic attack of carbonyls and dehydration involved in aldol-condensation reactions were associated with toxicity. The pyr-glyox pathway is based on prebiotic chemical reactions during the emergence of life and represents a unique tool for identifying toxic compounds individually and in complex mixtures.

Quick and cheap biomarkers to evaluate water quality are urgently needed to accommodate remote and low-budget laboratories. Recently, three simple and cheap biomarkers were proposed for water quality assessments using two morphological biomarkers (condition factor-CF and growth index-GI) and a tolerance test to air emersion. A multiple regression model was used to find predictors of air survival in wild bivalve populations. The data revealed that the air lethal time (LT) was negatively related with weight loss (WL) at the 7 th day of exposure to air. This suggests that WL >25% at the 7 th day predicts LT below the natural variation range. The relationship was significant with the CF, GI and air temperature indicating that larger animals with sustained growth and in warmer air temperature resist less time to air exposure. Moreover, the relationship was also significantly related to oxidative stress (lipid peroxidation) and decreased temperature dependence of mitochondria activity. For intertidal Mya arenaria clams, a temperature of 28.4 ºC could harm clams between tides (LT=0.5 day) which is in agreement to the reported maximal temperature (28.7 ºC) of the southern limit of clam distribution in North America coast. This relationship was statistically stronger at polluted sites than in pristine sites. In conclusion, this study permits to determine the critical range of air exposure time and temperature range during drought and strong temperature changes caused by global warming at contaminated sites.