Toxicity Of Water Dispersible Graphene Oxide (GO) To Four Species Of Algae

Graphene oxide (GO), as one of the significant graphene derivatives, has been applied in extensive fields. On account of its unique 2D shape, together with special physicochemical characteristics, some graphene and derivatives have become potential candidates in biomedicine. As the rapid development of nanotechnology and increasing GO application, we are worrying about their negative effects on the survival of aquatic organisms when they are released into water environment, and then influence the balance of ecosystem. Algae act as primary producer in aquatic ecosystem, whose diversity and production can directly impact on the functions and structures of system. The growth, antioxidant enzymes and organelles damages of algae have been widely used in the toxicity research of nanomaterial, such as Ag-NP, PbS-NP and carbon nanotubes (CNTs). However, the present studies on the toxicity of GO mostly focus on bacteria and mammalian cell. Very limited information is available on the algal toxicity of GO.This study used two species of marine microalgae-- Dunaliella salina, Nannochloropsis oceanic and two fresh water microalgae-- Scenedesmus obliquus, N. limnetica as tested plants. The toxicity of GO in four species of algae was investigated. Firstly, we got the growth kinetics of microalgae exposed to GO and calculate the values of 72h EC50 for toxicity grading of GO. Secondly, the physiological and biochemical responses (GSH and other related enzymes, MDA, photosynthetic pigments) and bioactive compounds (total protein, carbohydrate and total lipids) were also measured. Thirdly, we try to explore the effects of three kinds of environmental factors (temperature, salinity and pH) on GO’s toxicity. At last, through a couple of verification tests, the underlying toxicity mechanisms were discussed. The main conclusions are as follows:(1)GO can cause obvious toxic effects towards microalgae. The main experiment phenomena and analysis results are as follows:①It is obvious that GO has significant inhibition effect on the growth of all microalgae studied. D. salina seems to be most sensitive to GO which exhibited the lowest EC50 value of 13.04 mg/L among four algal species. The 72h EC50 values for GO to N. oceanic, S. obliquus, N. limnetica were 79.10,25.63 and 48.44 mg/L respectively. According to grading standard for acute aquatic toxicity, it is possible to classify GO as slightly toxic.②A 72-h exposure experiment revealed a substantially higher oxidative stress in GO treated algal cells by the changing of cellular reduced glutathione (GSH) contents and related enzymes (GPx, GR, GST) activities. As the concentration of GO increasing, the activity/contents of GPx and GSH had a significant decline. In contrast, there was an obvious growth in the activity of GR and GST.③The induction of MDA (18.03,10.88,4.48,15.06 times respectively than control) further indicated severe membrane damage and oxidative stress in 100mg/L GO treated algal cells④Synthesis of photosynthetic pigments(Chla, Chlb, Car) in lOmg/L GO treated microalgae was promoted, which suggested the enhancement of photosynthesis. While, microalgae exposed to 100mg/L GO might lose their self-regulation, and photosynthetic pigments were damaged or their synthesis was inhibited, which lead to the decrease of contents.⑤The contents of total protein and total lipids in all GO treated four microalgae strains increased apparently. Also 10mg/L can promote the synthesis of carbohydrates(2)Three kinds of environmental factors (temperature, salinity and pH) caused obvious effects on GO’s toxicity:①High temperature can decrease GO’s toxicity on D. salina and S. obliquus through changing the stability of GO and promoting GO’s aggregation. The density of 100mg/L GO treated algal cells under 32℃ reached to 1.59 and 2.69 times of those in 11℃.②GO aggregated seriously under high salinity, then weakened the toxicity of GO. The maximum values of D. salina cell density and biomass (when the salinity is 50) were 2.64 and 1.97 times than those with the salinity of 10.③Lower pH (close to pHpzc) can promote both the aggregation of GO and the generation of polymers in larger fractal dimension which were easy to settle. Therefore, the toxicity of GO on S. obliquus declined as pH (6.5-8.5) decreasing. On account of complicated compounds in seawater and the effects of pH on D. salina, GO had the weakest toxicity when pH=7.5.(3)The toxicity of 10mg/L and 100mg/L GO on D. salina was related to its shading effects which enhanced under higher concentration of GO. Growth inhibition ratios of 100mg/L GO treated D. salina was 84% and 23% respectively. Mn and Zn in the leachates of 150mg/L GO did not inhibit the growth of D. salina. GO aggregation and GO coatings on cell surface suggest that GO have physically blocked lights to algae. Most of the algal cells incubated on the GO aggregates lost their cellular integrity through some close interaction between cell membrane and GO. GO can enter into the gap between cell walls and plasma membrane. And some serious damage and changes were induced, including the broken of cell walls or cell membrane, the injury of chloroplast, the agglutination of nuclear chromation and the inhibition of cell division and so on. In conclusion, the toxicity mechanisms of GO on microalgae mainly contain oxidative stress, shading effects, aggregation and sorption, and the entrance into algal cells.