Enrichment characteristics of microplastics in Antarctic benthic and pelagic fish and krill near the Antarctic Peninsula.

Global microplastic pollution has garnered widespread attention from researchers both domestically and internationally. However, compared to other regions worldwide, little is known about microplastic pollution in the marine ecosystems of the Antarctic region. This study investigated the abundance and characteristics of microplastics (MPs) in the gills and intestines of 15 species of Antarctic fish and Antarctic krill (Euphausia superba). The results indicate that the abundance of MPs in Antarctic fish and E. superba ranged from 0.625 to 2.0 items/individual and 0.17 to 0.27 items/individual, with mean abundances of 0.93 ± 0.96 items/individual and 0.23 ± 0.44 items/individual, respectively. Antarctic fish ingested significantly more MPs than E. superba. There was no significant difference in the abundance of MPs between the gills and intestines of Antarctic fish. However, the quantity of pellet-shaped MPs in the gills was significantly higher than in the intestines. The depth of fish habitat influenced the quantity and size of MPs in their bodies, with benthic fish ingesting significantly fewer MPs than pelagic fish. Pelagic fish ingested significantly more MPs sized 1-5 mm than benthic fish. Additionally, analysis of the characteristics of MPs revealed that fiber-shaped MPs were predominant in shape, with sizes generally smaller than 0.25 mm and 0.25-0.5 mm. The predominant colors of MPs were transparent, red, and black, while the main materials were polypropylene (PP), polystyrene (PS), polyamide (PA), and polyvinyl chloride (PVC). Compared to organisms from other regions, the levels of MPs in Antarctic fish and E. superba were relatively low. This study contributes to a better understanding of the extent of MP pollution in Antarctic fish and E. superba, aiding human efforts to mitigate its impact on the environment.

Ceratophyllum demersum alleviates microplastics uptake and physiological stress responses in aquatic organisms, an overlooked ability.

It has been demonstrated that microplastics (MPs) may be inadvertently ingested by aquatic animals, causing harm to their physiological functions and potentially entering the food chain, thereby posing risks to human food safety. To achieve an environmentally friendly and efficient reduction of MPs in freshwater environments, this experiment investigates the depuration effect of C. demersum on MPs using three common aquatic animals: Macrobrachium nipponense, Corbicula fluminea, and Bellamya aeruginosa as research subjects. The amounts of MPs, digestive enzyme activity, oxidative stress index, and energy metabolism enzyme activity in the digestive and non-digestive systems of three aquatic animals were measured on exposure days 1, 3, and 7 and on depuration days 1 and 3. The results indicated that the depuration effect of C. demersum and the species interaction were significant for the whole individual. Concerning digestive tissue, C. demersum was the most effective in purifying B. aeruginosa. When subjected to short-term exposure to MPs, C. demersum displayed a superior depuration effect. Among non-digestive tissues, C. demersum exhibited the earliest purifying effect on C. fluminea. Additionally, C. demersum alleviated physiological responses caused by MPs. In conclusion, this study underscores C. demersum as a promising new method for removing MPs from aquatic organisms.

Microplastics footprint in nature reserves-a case study on the microplastics in the guano from Yancheng Wetland Rare Birds National Nature Reserve, China.

Bio-ingestion of microplastics poses a global threat to ecosystems, yet studies within nature reserves, crucial habitats for birds, remain scarce despite the well-documented ingestion of microplastics by avian species. Located in Jiangsu Province, China, the Yancheng Wetland Rare Birds Nature Reserve is home to diverse bird species, including many rare ones. This study aimed to assess the abundance and characteristics of microplastics in common bird species within the reserve, investigate microplastic enrichment across different species, and establish links between birds' habitat types and microplastic ingestion. Microplastics were extracted from the feces of 110 birds, with 84 particles identified from 37.27% of samples. Among 8 species studied, the average microplastic abundance ranged from 0.97 ± 0.47 to 43.43 ± 61.98 items per gram of feces, or 1.5 ± 0.87 to 3.4 ± 1.50 items per individual. The Swan goose (Anser cygnoides) exhibited the highest microplastic abundance per gram of feces, while the black-billed gull (Larus saundersi) had the highest abundance per individual. The predominant form of ingested microplastics among birds in the reserve was fibers, with polyethylene being the most common polymer type. Significant variations in plastic exposure were observed among species and between aquatic and terrestrial birds. This study represents the first quantitative assessment of microplastic concentrations in birds within the reserve, filling a crucial gap in research and providing insights for assessing microplastic pollution and guiding bird conservation efforts in aquatic and terrestrial environments.

Tetrabromobisphenol a and its alternative tetrachlorobisphenol a induce oxidative stress, lipometabolism disturbance, and autophagy in the liver of male Pelophylax nigromaculatus.

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) have been widely used as flame retardants. However, their potential health risks to organisms have raised concerns, particularly for liver toxicity. Present study aimed to explore the toxic effects of TCBPA and TBBPA on black-spotted frogs (Pelophylax nigromaculatus) liver oxidative stress, autophagy, and lipid accumulation. After exposure to 0.001, 0.01, 0.1, and 1 mg/L TBBPA and TCBPA for 14 days, the content of cholesterol and triglyceride were significantly elevated. In addition, the malondialdehyde level rose greatly in dose dependent. However, the glutathione level declined in high TBBPA groups (0.01 and 0.1 mg/L). Furthermore, expressions of Beclin1, Atg5, and Atg7 were significantly increased, while p62 was markedly declined, respectively. Results obstained suggested that TBBPA and TCBPA exposure induced liver toxicity in black-spotted frog. This study provided insights into the toxicity mechanism of bisphenol flame retardants in amphibians and will aid in the ecological risk assessment of flame retardants.

Habitual feeding patterns impact polystyrene microplastic abundance and potential toxicity in edible benthic mollusks.

That increasing microplastics (MPs, <5 mm) eventually end up in the sediment which may become a growing menace to diverse benthic lives is worthy of attention. In this experiment, three edible mollusks including one deposit-feeding gastropod (Bullacta exarate) and two filter-feeding bivalves (Cyclina sinensis and Mactra veneriformis) were exposed to polystyrene microplastic (PS-MP) for 7 days and depurated for 3 days. PS-MP numbers in the digestive system and non-digestive system, digestive enzymes, oxidative stress indexes, and a neurotoxicity index of three mollusks were determined at day 0, 3, 7, 8 and 10. After seven-day exposure, the PS-MP were found in all three mollusks' digestive and non-digestive systems. And PS-MP in M. veneriformis (9.57 ± 2.19 items/individual) was significantly higher than those in C. sinensis (3.00 ± 2.16 items/individual) and B. exarate (0.83 ± 1.07 items/individual) at day 7. Three-day depuration could remove most of the PS-MP in the mollusks, and higher PS-MP clearance rates were found in filter-feeding C. sinensis (77.78 %) and M. veneriformis (82.59 %) compared to surface deposit-feeding B. exarate (50.00 %). The digestive enzymes of B. exarate significantly reacted to PS-MP exposure, while oxidative responses were found in C. sinensis. After three-day depuration, the changes of digestive enzymes and the oxidative states were fixed, but neurotoxicity induced by PS-MP was not recoverable. Besides, it is noteworthy that changes of digestive enzymes and acetylcholinesterase are related to feeding patterns.

Fate of Microplastic Fibers in the Coelomic Fluid of the Sea Cucumber Apostichopus japonicus.

Sea cucumbers are economical and ecologically important species, playing a crucial role in nutrient recycling in the ocean and providing valuable bioactive compounds for nutrition. Previous research has demonstrated that microplastic fibers, which are widely recognized as emerging contaminants, are transferred to the perivisceral coelomic fluid during respiration in sea cucumbers; however, their fate in sea cucumbers is still not well understood. We tracked the status of sea cucumbers (Apostichopus japonicus) with polyester microplastic fibers in their coelomic fluid in clean water. The results showed that after transferring sea cucumbers to clean water, the number of microplastic fibers transferred significantly decreased in the coelomic fluid, but at least one microplastic fiber was found up to 60 days. In addition, sea cucumbers recovered from the effect of microplastic fiber transfer, as indicated by enzyme levels and histological observations. Furthermore, single microplastic fiber transfer over a 60-day farmed period did not significantly affect the growth of sea cucumbers. However, repetitive microplastic fiber transfer (i.e., twice and thrice a week over 60 days) significantly decreased the growth rate (p < 0.05). Accordingly, increasing microplastic fibers in sea cucumber habitats pose a threat to sea cucumbers because they can disrupt development. Thus, farmers are advised to select locations for farming sea cucumbers where low microplastic fiber concentrations are expected. Environ Toxicol Chem 2023;42:205-212. © 2022 SETAC.

Complete mitochondrial genome of the top hat blenny, Omobranchus fasciolatoceps Richardson (Blenniiformes: Blenniinae) in Southern China Coast.

The complete mitochondrial genome sequence of Omobranchus fasciolatoceps was firstly described in this article. The total length of mitogenome was 16,569 bp. It contains 13 protein-coding genes, 22 tRNA genes, and two ribosomal RNA genes. The overall base composition of H-strand was 29.04% A, 27.14% C, 27.89% T, and 15.93% G, with an A＋T bias of 56.93%. The phylogenetic analysis results showed that the O. fasciolatoceps was most closely related to O. elegans.

Synthesized effects of medium-term exposure to seawater acidification and microplastics on the physiology and energy budget of the thick shell mussel Mytilus coruscus.

Ocean acidification (OA) and microplastics (MPs) contamination are two results of human excises. In regions like estuarine areas, OA and MPs exposure are happening at the same time. The current research investigated the synthesized effects of OA and MPs exposure for a medium-term duration on the physiology and energy budget of the thick shell mussel Mytilus coruscus. Mussels were treated by six combinations of three MPs levels (0, 10 and 1000 items L-1) × two pH levels (7.3, 8.1) for 21 d. As a result, under pH 7.3, clearance rate (CR), food absorption efficiency (AE), respiration rate (RR), and scope for growth (SFG) significantly decreased, while the fecal organic dry weight ratio (E) significantly increased. 1000 items L-1 MPs led to decrease of CR, E, SFG and increase of AE under pH 8.1. Interactive effects from combination of pH and MPs were found in terms of CR, AE, E and RR, but not for SFG of M. coruscus.

Effects of Ocean Acidification and Microplastics on Microflora Community Composition in the Digestive Tract of the Thick Shell Mussel Mytilus coruscus Through 16S RNA Gene Sequencing.

Ocean acidification and microplastic pollution is a global environmental threat, this research evaluated the effects of ocean acidification and microplastics on mussel digestive tract microbial community. The 16S rRNA gene was sequenced to characterize the flora. Species diversity in the samples was assessed by clustering valid tags on 97% similarity. Bacteroidetes, Firmicutes and Proteobacteria were the three most abundant genera in the four groups, with Bacteroidetes showing the highest diversity. However, no differences in flora structure were evident under various treatments. Phylogenetic relationship analysis revealed Bacteroidetes and Firmicutes had the highest OTU diversity. The weighted UniFrac distance, principal coordinate analysis (PCoA), unweighted pair group method with arithmetic mean (UPGMA) cluster tree and analysis of molecular variance (AMOVA) evaluation results for all samples also showed that changes in pH and microplastics concentration did not significantly affect the microbial community structure in the mussel digestive tract. The results presented the no significant effects of ocean acidification and microplastics intake on mussel intestinal diversity.

Antioxidant responses of triangle sail mussel Hyriopsis cumingii exposed to toxic Microcystis aeruginosa and thermal stress.

Harmful algal blooms (HABs) and thermal stress as climate changes become more common in global water ecosystem, especially under eutrophic habitats. Here our study examined the combined impacts of bloom forming cyanobacteria Microcystis aeruginosa and thermal stress on the antioxidant responses of the ecologically important species triangle sail mussel Hyriopsis cumingii. The differential responses of a series of enzymes, e.g. superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST), as well as signal metabolites including reactive oxygen species (ROS), malondialdehyde (MDA) and glutathione (GSH) involved in antioxidant defense mechanisms were analyzed during 14 d exposure to toxic cyanobacterium M. aeruginosa and 7 d depuration period. The activities of SOD and GPx as well as the content of ROS and MDA in H. cumingii increased, while CAT activity reduced due to M. aeruginosa exposure. Thermal stress resulted in decrease of CAT, the accumulation of GSH and the enhance of GST and SOD. Meanwhile, the interactive effects among M. aeruginosa, thermal stress and time were also observed on most parameters except for GST activity. The total amount of microcystins (MC) in sail mussels increased with concentrations of exposed M. aeruginosa, independently of the presence or absence of thermal stress. Although around 50% of MC in mussels dropped in the depuration period, most parameters showed alterations because of cyanobacteria exposure and thermal stress. Overall, these findings suggested that toxic cyanobacteria or thermal stress induces oxidative stress and severely affects the enzymes activities and intermediates level associated with antioxidant defense mechanisms in sail mussels respectively. More importantly, the toxic impacts on sail mussels could be intensified by their combination.

Antioxidant responses of triangle sail mussel Hyriopsis cumingii exposed to harmful algae Microcystis aeruginosa and high pH.

In lakes and reservoirs, harmful algal blooms and high pH have been deemed to be two important stressors related to eutrophication, especially in the case of CO2 depletion caused by dense blooms. However, the effects of these stressors on the economically important shellfish that inhabit these waters are still not well-understood. This study evaluated the combined effects of the harmful algae Microcystis aeruginosa (0%, 50%, and 100% of total dietary dry weight) and high pH (8.0, 8.5 and 9.0) on the antioxidant responses of the triangle sail mussel H. cumingii. The mussels were exposed to algae and high pH for 14 d, followed by a 7-day depuration period. Reactive oxygen species (ROS) in the mussel hemolymph, antioxidant and detoxifying enzymes, such as glutathione-S-transferase (GST), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) in the digestive glands were analyzed during the experimental period. GST, SOD and GPx activity levels and the content of GSH increased following exposure to toxic M. aeruginosa, whereas CAT activity was inhibited. pH showed no significant effects on the immune defense mechanisms and detoxification processes. However, a high pH could cause increased ROS and MDA levels, resulting in oxidative injury. After a 7-day depuration period, exposure to toxic M. aeruginosa or high pH resulted in latent effects for most of the examined parameters. The treatment group exposed to the highest pH (9.0) displayed an increased oxidation state compared with the other pH treatments (8.0 and 8.5) for the same concentrations of toxic M. aeruginosa. The trends observed for ROS, MDA, GPx, GST, SOD and GSH levels indicated that a high density of toxic algae could result in severe and continuous effects on mussel health.

Effects of antibiotics on nitrogen uptake of four wetland plant species grown under hydroponic culture.

To investigate the effects of antibiotics on nitrogen removal and uptake by wetland plants, four typical macrophyte species, Cyperus alternifolius L., Typha angustifolia L., Lythrum salicaria L., and Acorus calamus L., were grown in hydroponic cultivation systems and fed wastewater polluted with 10 µg L-1 Ofloxacin (OFL) and Tetracycline (TET). Biomass production, nitrogen mass concentration, chlorophyll content, root exudates, and nitrogen removal efficiency of hydroponic cultivation were investigated. The results indicated that in all hydroponic systems, NH4+-N was entirely removed from the hydroponic substrate within 1 day and plant nitrogen accumulation was the main role of the removed NO3-. OFL and TET stimulated the accumulation of biomass and nitrogen of A. calamus but significantly inhibited the NO3--N removal ability of L. salicaria (98.6 to 76.2%) and T. augustifolia (84.3 to 40.2%). This indicates that A. calamus may be a good choice for nitrogen uptake in wetlands contaminated with antibiotics. OFL and TET improved the concentrations of total organic carbon (TOC), total nitrogen (TN), organic acid, and soluble sugars in root exudates, especially for oxalic acid. Considering the significant correlation between TOC of root exudates and nitrogen removal efficiency, the TOC of root exudates may be an important index for choosing macrophytes to maintain nitrogen removal ability in wetlands contaminated with antibiotics.

Complete mitochondrial genome of the deep pugnose ponyfish Secutor ruconius (Perciformes: Leiognathidae) in the East China Sea.

The complete mitochondrial genome sequence of Secutor ruconius is firstly described in this article. The total length of mitogenome is16,465 bp. It contains 13 protein-coding genes, 22 tRNA genes, and 2 ribosomal RNA genes. The overall base composition of H-strand is 31.58% A, 28.65% C, 25.23% T, and 14.53% G, with an A + T bias of 56.81%. The phylogenetic analysis result showed that the S. ruconius, Zebrasoma flavescens, and Pristipomoides multidens were closely related.

Complete mitochondrial genome of the glowbelly Acropoma japonicum (Perciformes: Acropomatidae) in the East China Sea.

The complete mitochondrial genome sequence of Acropoma japonicum is first described in this article. The total length of mitogenome is16,973 bp. It contains 13 protein-coding genes, 23 tRNA genes, and 2 ribosomal RNA genes. The overall base composition of H-strand is 27.64% A, 29.49% C, 26.84% T, and 16.03% G, with an A＋T bias of 54.48%. The phylogenetic analysis result showed that the A. japonicum and Lutjanus peru had a close relationship.

Complete mitochondrial genome of the skinnycheek lantern fish Benthosema pterotum (Perciformes: Myctophidae) in the East China Sea.

The complete mitochondrial genome sequence of Benthosema pterotum is first described in this article. The total length of mitogenome is 18,052 bp. It contains 13 protein-coding genes, 22 tRNA genes, and two ribosomal RNA genes. The overall base composition of H-strand is 27.83% A, 30.88% C, 25.61% T, and 15.69% G, with an A＋T bias of 53.43%. The phylogenetic analysis result showed that the B. pterotum and Electrona carlsbergi were close relationship.

Salinity mediates the toxic effect of nano-TiO2 on the juvenile olive flounder Paralichthys olivaceus.

Increased production of engineered nanoparticles has raised extensive concern about the potential toxic effects on marine organisms living in estuarine and coastal environments. Meanwhile, salinity is one of the key environmental factors that may influence the physiological activities in flatfish species inhabiting in those waters due to fluctuations caused by freshwater input or rainfall. In this study, we investigated the oxidative stress and histopathological alteration of the juvenile Paralichthys olivaceus exposed to nano-TiO2 (1 and 10 mg L-1) under salinities of 10 and 30 psu for 4 days. In the gills, Na+-K+-ATPase activity significantly deceased after 4 days 10 psu exposure without nano-TiO2 compared with 1 day of acclimating the salinity from the normal salinity (30 psu) to 10 psu. Under this coastal salinity, low concentration (1 mg L-1) of nano-TiO2 exerted significant impacts. In the liver, the activities of superoxide dismutase, catalase, the levels of lipid peroxide and malondialdehyde increased with nano-TiO2 exposed under 30 psu. Such increase indicated an oxidative stress response. The result of the integrated biomarker responses showed that P. olivaceus can be adversely affected by high salinity and high concentration of nano-TiO2 for a short-term (4 days) exposure. The histological analysis revealed the accompanying severe damages for the gill filaments. Principal component analysis further showed that the oxidative stress was associated with the nano-TiO2 effect at normal salinity. These findings indicated that nano-TiO2 and normal salinity exert synergistic effects on juvenile P. olivaceus, and low salinity plays a protective role in its physiological state upon short-term exposure to nano-TiO2. The mechanism of salinity mediating the toxic effects of NPs on estuarine fish should be further considered.

Oxidative stress induced by titanium dioxide nanoparticles increases under seawater acidification in the thick shell mussel Mytilus coruscus.

Biochemical responses of the mussel Mytilus coruscus exposed to different concentrations of titanium dioxide nanoparticles (nano-TiO2) (0, 2.5, 10 mg L-1) and two pH levels (pH 8.1 and pH 7.3) for 14 days. Mussel responses were also investigated after a 7 days recovery period (pH 8.1 and no nanoparticle). Exposure to nano-TiO2 led changes in antioxidant indexes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione (GSH)), biotransformation enzyme activity (GST) and malondialdehyde level (MDA) in gills and digestive glands. An increase in MDA level and a decrease in SOD and GSH activities were observed in gill of mussels exposed to 10 mg L-1 nano-TiO2. This effect was more severe in mussels kept at pH 7.3 as compared to pH 8.1. A different response was observed in the digestive gland as SOD, CAT and GSH levels increased in mussels exposed to nano-TiO2. These contrasting results in digestive glands and gills were only evident at high concentration of nano-TiO2 and low pH. A 7 days recovery period was not sufficient to fully restore SOD, GPx, GST, GSH and MDA levels to levels before exposure to nano-TiO2 and low pH. Overall, our results confirmed that seawater acidification modulates effects of nanoparticles in mussels, and that gills are more sensitive to these stressors as compared with digestive glands.

Adsorption and degradation of 14C-bisphenol A in a soil trench.

Bisphenol A (BPA) has caused widespread concern among scholars as a result of its estrogenic toxicity. It exists mainly in natural waters, sediments, and soil, as well as sewage and wastewater sludge. Considering that BPA is a common environmental pollutant that is removed along with chemical oxygen demand (COD), nitrogen, and phosphorus in drainage treatment systems, it is important to research the fate of BPA in sewage treatment systems. In this research, laboratory batch experiments on soil degradation and adsorption were conducted with 14C-BPA, aiming to discuss the transport and degradation characteristics of BPA in both simulated facilities and a soil trench. Based on the experimental results, the Freundlich model could be applied to fit the isothermal adsorption curve of the BPA in soil. A low mobility characteristic of BPA was discovered. The mineralization rate of BPA was fast and that of the reaction showed small fluctuations. After degradation, 21.3 and 17.7% of the BPA groups (the experimental group treated with ammonia oxidase (AMO) inhibitor and the control group) were converted into 14CO2, respectively. This indicates that the nitrification and degradation of BPA had a certain competitive relationship. Besides, nitrification did not significantly affect the soil residue of BPA. Through the soil trench test, the average removal rate of BPA in the soil trench was 85.5%. 14CO2 was discharged via the mineralization of BPA, accounting for 2.5% of the initial input. BPA easily accumulated in the bottom soil of the soil trench. BPA and its metabolites in the effluent accounted for 14.5% of the initial dosage. The residual extractable BPA and its metabolites in the soil accounted for 51.3%, and the remaining part of the unextractable residue represented 19.8% of the initial radioactive dosage.

Defense Responses to Short-term Hypoxia and Seawater Acidification in the Thick Shell Mussel Mytilus coruscus.

The rising anthropogenic atmospheric CO2 results in the reduction of seawater pH, namely ocean acidification (OA). In East China Sea, the largest coastal hypoxic zone was observed in the world. This region is also strongly impacted by ocean acidification as receiving much nutrient from Changjiang and Qiantangjiang, and organisms can experience great short-term natural variability of DO and pH in this area. In order to evaluate the defense responses of marine mussels under this scenario, the thick shell mussel Mytilus coruscus were exposed to three pH/pCO2 levels (7.3/2800 µatm, 7.7/1020 µatm, 8.1/376 µatm) at two dissolved oxygen concentrations (DO, 2.0, 6.0 mg L-1) for 72 h. Results showed that byssus thread parameters, such as the number, diameter, attachment strength and plaque area were reduced by low DO, and shell-closing strength was significantly weaker under both hypoxia and low pH conditions. Expression patterns of genes related to mussel byssus protein (MBP) were affected by hypoxia. Generally, hypoxia reduced MBP1 and MBP7 expressions, but increased MBP13 expression. In conclusion, both hypoxia and low pH induced negative effects on mussel defense responses, with hypoxia being the main driver of change. In addition, significant interactive effects between pH and DO were observed on shell-closing strength. Therefore, the adverse effects induced by hypoxia on the defense of mussels may be aggravated by low pH in the natural environments.

Antioxidant response of the hard shelled mussel Mytilus coruscus exposed to reduced pH and oxygen concentration.

Ocean acidification (OA) and hypoxic events are increasing worldwide problems, their interactive effects have not been well clarified, although their co-occurrence is prevalent. The East China Sea (the Yangtze River estuary area) suffers from not only coastal hypoxia but also pH fluctuation, representing an ideal study site to explore the combined effect of OA and hypoxia on marine bivalves. We experimentally evaluated the antioxidant response of the mussel Mytilus coruscus exposed to three pH levels (8.1, 7.7 and 7.3) at two dissolved oxygen (DO) levels (2.0mgL-1 and 6.0mgL-1) for 72h. Activities of superoxide dismutase, catalase, glutathione peroxidase, acid phosphatase, and alkaline phosphatase and levels of malondialdehyde were measured in gills and hemolymph. All enzymatic activities in hemolymph and gills followed a similar pattern throughout the experiment duration. Generally, low DO showed greater effects on enzyme activities than elevated CO2. Significant interactions between DO, pH and time were only observed at superoxide dismutase and catalase in both tissues. PCA revealed positive relationships between most enzyme activities in both gills and hemolymph with the exception of alkaline phosphatase activity and the level of malondialdehyde in the hemolymph. Overall, our results suggested that decreased pH and low DO induced similar antioxidant responses in the hard shelled mussel, and showed an additive effect on most enzyme activities. The evaluation of multiple environmental stressors, a more realistic scenario than single ones, is crucial to predict the effect of future global changes on coastal species and our results supply some insights on the potential combined effects of reduced pH and DO on marine bivalves.