Xizhi Huang

Hemocyte responses of the thick shell mussel Mytilus coruscus exposed to nano-TiO2 and seawater acidification

With increasing production from nanotechnology industries, nanomaterials are inevitably released into the aquatic environment, thereby posing a potential risk to aquatic organisms. Thus, concerns have been raised on the potential ecotoxicological effect of nanoparticle. Furthermore, the ecotoxicological consequences caused by the interaction of nanoparticles with other environmental stresses, such as seawater acidification on marine animals, have not been evaluated. In particular, whether acidification enhances the susceptibility to nanoparticles in bivalves needs to be evaluated. In the present study, we investigated the combined effects of low pH and nanoscale titanium dioxide (nano-TiO2) on some immune parameters of hemocytes in the mussel Mytilus coruscus by flow cytometry under six combinations of two pH values (7.3 and 8.1) and three nano-TiO2 concentrations (0, 2.5, and 10mgL-1) for 14 d. Afterward, the mussels were shifted to normal conditions without nano-TiO2 at pH 8.1 for 7 d further to test their recovery from the multiple stresses. Total hemocyte count (THC), phagocytosis (Pha), esterase (Est), and lysosomal content (Lyso) decreased under low pH and high nano-TiO2 concentration conditions, whereas hemocyte mortality (HM) and reactive oxygen species (ROS) increased with nano-TiO2 concentrations under low pH conditions. The interactive effects between pH and nano-TiO2 were observed at the latter part of the exposure experiment (7 and 14 d) in most hemocyte parameters. Nano-TiO2 influenced the immune functions of mussel more severely than low pH. Slight recovery from the combined stresses was observed for HM, THC, Pha, and Lyso, but significant carry-over effects of nano-TiO2 and low pH were still observed. This study demonstrated that both low pH and high concentration of nano-TiO2 had negative effects on mussels, and these effects still acted for some time even though the mussels were already out of such stressors.

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.

CO2-induced pH reduction increases physiological toxicity of nano-TiO2 in the mussel Mytilus coruscus.

The increasing usage of nanoparticles has caused their considerable release into the aquatic environment. Meanwhile, anthropogenic CO2 emissions have caused a reduction of seawater pH. However, their combined effects on marine species have not been experimentally evaluated. This study estimated the physiological toxicity of nano-TiO2 in the mussel Mytilus coruscus under high pCO2 (2500–2600 μatm). We found that respiration rate (RR), food absorption efficiency (AE), clearance rate (CR), scope for growth (SFG) and O:N ratio were significantly reduced by nano-TiO2, whereas faecal organic weight rate and ammonia excretion rate (ER) were increased under nano-TiO2 conditions. High pCO2 exerted lower effects on CR, RR, ER and O:N ratio than nano-TiO2. Despite this, significant interactions of CO2-induced pH change and nano-TiO2 were found in RR, ER and O:N ratio. PCA showed close relationships among most test parameters, i.e., RR, CR, AE, SFG and O:N ratio. The normal physiological responses were strongly correlated to a positive SFG with normal pH and no/low nano-TiO2 conditions. Our results indicate that physiological functions of M. coruscus are more severely impaired by the combination of nano-TiO2 and high pCO2.

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.

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.

Combined effects of ZnO NPs and seawater acidification on the haemocyte parameters of thick shell mussel Mytilus coruscus

Flow cytometry was used to investigate the immune parameters of haemocytes in thick-shell mussel Mytilus coruscus exposed to different concentrations of ZnO nanoparticles (NPs) (0, 2.5, and 10mgl-1) at two pH levels (7.3 and 8.1) for 14days following a recovery period of 7days. ZnO NPs significantly affected all of the immune parameters throughout the experiment. At high ZnO NPs concentrations, total haemocyte counting, phagocytosis, esterase, and lysosomal content were significantly decreased whereas haemocyte mortality and reactive oxygen species (ROS) were increased. Although low pH also significantly influenced all of the immune parameters of the mussels, its effect was not as strong as that of ZnO NPs. Interactive effects were observed between pH and ZnO NPs in most haemocyte parameters during the exposure period. Although a slight recovery from the stress of ZnO NPs and pH was observed for all immune parameters, significant carry-over effects of low pH and ZnO NPs were still detected. This study revealed that high concentration of ZnO NPs and low pH exert negative and synergistic effects on mussels, and these effects remain even after the mussels are no longer exposed to such stressors.

Impact of zinc oxide nanoparticles and ocean acidification on antioxidant responses of Mytilus coruscus

Increased production of engineered nanoparticles (NPs) has raised extensive concerns about the potential toxic effects on marine organisms. Extensive evidences documented the impact of ocean acidification (OA) on the physiology and fitness of bivalves. In the present study, we investigated the biochemical responses of the mussel Mytilus coruscus exposed to both nano-ZnO and low pH relevant for ocean acidification conditions for 14 d followed by a 7-d recovery period. Most biochemical indexes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), acid phosphatase (ACP) and alkaline phosphatase (ALP)) measured in gills and hemocytes were increased when the mussels were subject to low pH or high concentration of nano-ZnO, suggesting oxidative stress responses. No significant interactions between the two stressors were observed for most measured parameters. After a 1 week recovery period, low pH and nano-ZnO had less marked impact for SOD, GPx, ACP and ALP in hemocytes as compared to the end of the 14 d exposure. However, no recovery was observed in gills. Overall, our results suggest that both low pH and nano-ZnO induce an anti-oxidative response in Mytilus coruscus with gills being more sensitive than hemocytes.

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.

Short-Term Exposure of Mytilus coruscus to Decreased pH and Salinity Change Impacts Immune Parameters of Their Haemocytes

With the release of large amounts of CO2, ocean acidification is intensifying and affecting aquatic organisms. In addition, salinity also plays an important role for marine organisms and fluctuates greatly in estuarine and coastal ecosystem, where ocean acidification frequently occurs. In present study, flow cytometry was used to investigate immune parameters of haemocytes in the thick shell mussel Mytilus coruscus exposed to different salinities (15, 25, and 35‰) and two pH levels (7.3 and 8.1). A 7-day in vivo and a 5-h in vitro experiments were performed. In both experiments, low pH had significant effects on all tested immune parameters. When exposed to decreased pH, total haemocyte count (THC), phagocytosis (Pha), esterase (Est), and lysosomal content (Lyso) were significantly decreased, whereas haemocyte mortality (HM) and reactive oxygen species (ROS) were increased. High salinity had no significant effects on the immune parameters of haemocytes as compared with low salinity. However, an interaction between pH and salinity was observed in both experiments for most tested haemocyte parameters. This study showed that high salinity, low salinity and low pH have negative and interactive effects on haemocytes of mussels. As a consequence, it can be expected that the combined effect of low pH and changed salinity will have more severe effects on mussel health than predicted by single exposure.

Synergistic effects of nano-ZnO and low pH of sea water on the physiological energetics of the thick shell mussel Mytilus coruscus

In order to investigate the ecotoxicological effects of nano-ZnO particles and seawater acidification on marine bivalves, the thick shell mussels, Mytilus coruscus were subjected to joint treatments with different nano-ZnO concentrations (0 [control], 2.5 [medium] and 10 mg L-1 [high]) under two pH levels (7.7 [low]and 8.1 [control]) for 14 days. The results showed that respiration rate (RR), absorption efficiency (AE), clearance rate (CR), O:N ratio and scope for growth (SFG) were significantly reduced with nano-ZnO concentration increase, but ammonium excretion rate (ER) was increased. Low pH significantly reduced CR, RR, SFG, and O:N ratio of the mussels especially under high nano-ZnO conditions, and significantly increased ER. Principal component analysis (PCA) showed consistent relationships among most tested parameters, especially among SFG, RR, O:N ratio and CR under the normal pH and 0 nano-ZnO conditions. Therefore, seawater acidification and nano-ZnO interactively impact the ecophysiological responses of mussels and cause more severe effects when they appear concurrently.