Aquatic Ecotoxicities Of Several Manufactured Nanomaterials

Nanotechnology has been expected to be the next technical revolution which will bring us large advantages in our everyday life in the coming years. Manufactured nanomaterials (MNMs, e.g. nanoparticles, nanotubes, nanowires, fullerenes and its derivatives, and other nanoscale materials) with at least one critical dimension below 100 nm, specially engineered for applications, are the building blocks of this new industry. MNMs display novel physicochemical characteristics, such as nanoscale size effects, quantum effects, increased surface area and higher surface curvature as well as unique electric, thermal, mechanical, and imaging properties, which is highly desirable for applications within the medical, industrial, commercial, and environmental sectors. The commercial manufacture of MNMs is already under way, for example the nanoscale zinc oxide, titanium dioxide, carbon nanotubes and fullerenes are being produced by tons each year. It has been reported that the market for nanotechnologies is estimated at US$700 billion by 2008 and more than US$1 trillion by 2015. However, with the rapid development of nanotechnology, there is a higher risk for people and environment exposed to more and more nanotechnology-based products. People began to worry that the risks of MNMs in terms of their environmental impact may outweigh their benefits. Thus, interests in the broad implication of MNMs have grown.It is well known that aquatic environment is the foundation to sustain living organisms. However, aquatic environments may suffer MNMs pollution coming from consumer products (e.g., sunscreens and cosmetics) as well as the accidental releases during the processes of production, transportation and disposal operations, MNMs is potentially dangerous in terms of causing damage to aquatic organisms (e.g., phytoplankton, zooplankton, and fishes etc.), and eventually becoming a hazard to human through food webs. Unfortunately, up to now, very few studies on the MNMs toxicity have been conducted especially with the testing organism of aquatic species, and no chronic, full, or multigenerational life cycle studies appear in the literature.In this study, a series of ecotoxicological tests on MNMs were designed and conducted as an initial step to address the potential environmental impacts of manufactured nanomaterials. The main contents and results of the experiments were:⑴The growth inhibition test (OECD 201 guidelines with slight changes) using the green alga Scenedesmus obliquus and the immobilization test (OECD 202 guidelines with slight changes) using the daphnia Daphnia magna as the testing organism were performed to compare the different ecological effects of several nanoparticles (i.e., metal oxide nanoparticles: nTiO2, nZnO and nAl2O3; carbon nanoparticles: fullerene(C60), single wall carbon nanotube(SWCNT) and multiply wall carbon nanotube (MWCNT) ); the dose—effect relationship was determined and their LC50 or EC50 were calculated respectively. The results showed that:①These six nanomaterials with different chemical compositions and/or nanostructure were all able to inhibit the growth of Scenedesmus obliquus and the behavior of Daphnia magna to varying degrees, displaying obvious ecotoxicities with a dose-depending property. The 96 h EC50 of nZnO, nTiO2, nAl2O3, C60, SWCNT and MWCNT on the growth of Scenedesmus obliquus were 1.049, 15.262, >1000, 13.122, 22.633, 15.488 mg/L, respectively. Based on the 96 h EC50, the toxicity of these six MNMs was in the order of nZnO > C60, nTiO2, MWCNTs, SWCNTs > nAl2O3. The 48 h immobilization EC50 of nZnO, nTiO2, nAl2O3, C60, SWCNT and MWCNT on Daphnia magna were 0.622, 35.306, 114.357, 9.344, 1.306 and 8.273 mg/L, respectively. Based on this result, the toxicity of such MNMs was in the order of nZnO > SWCNTs >C60, MWCNTs >nTiO2 > nAl2O3. Apparently, nZnO suspension was the most toxic to both of the two testing organisms, and Daphnia magna was more susceptible to their effects. For the three carbon nanomaterials (C60, SWCNT and MWCNT), their toxic effects to both algae and daphnia were generally similar, their EC50 values varing only within one order of magnitude. However, when the fact was taken into consideration that C60, when coming into contact with water, can form stable nanoscale suspended aggregates (nC60) whose concentration can reach up to a high level (>20 mg L-1) and whose properties differ from those of the pristine C60, C60 should be regarded as one of the most environmentally relevant form of carbon nanomaterials.②The experimental results confirmed that, the current standard ecological effects testing schemes and test protocols with some revision could meet the particular requirements of examing the ecological effects of nanoparticles, and could be adapted to compare their ecotoxicities. According to a new OECD guideline (i.e., OECD No.23), in the algal test, poorly water-soluble MNMs and/or its aqueous dispersions may absorb photosynthetically active light and hence limit the growth of the algal cultures, lead to an overestimation of toxicity. In practice, it is better to change the incubation system, such as shortening the light path, increasing the light intensity, and maintaining a good turbulence, to compensate for the internal light absorption, in turn to minimise the dispersion effect. Observing Daphnia magna is also difficult in the test media with high concentration of MNMs particles. Placing test vessels on a light box or transferring the contents of the test vessels to shallow containers for scoring may help.⑵nZnO and C60 were found to be more poisonous or environmentally relevant in previous tests. Thus, nZnO and C60 were selected for the toxicity mechanic exploration. One of the most widely used tools, the early life stage (ELS) test using zebrafish (Danio rerio) embryo was chosen in this part of study. Early life stage parameters such as zebrafish embryo survival, hatching rate, heartbeat and pericardial edema were measured within 96 h of exposure. And activity of Superoxide Dismutase (SOD) and Lipid Peroxidation of zebrafish embryos exposed to nanoparticles were determined to investigate the oxidative damage induced by nanoparticles. The results showed that:①nZnO was very toxic to zebrafish embryos and larvae with dose-depending and time-depending property. nZnO delayed zebrafish embryo and larva development, decreased their survival and hatching rate, and caused teratogenesis. The 96 h LC50 of nZnO was >100 mg/L for the zebrafish survival, and the 84 h EC50 was 1.174 mg/L for the zebrafish embryo hatching rate.②The release of zinc ions in the nZnO particle suspension was detected, and it was confirmed that the released Zn2+ might play a role in the acute toxicity of nZnO suspension on zebrafish embryos and larvae, which suggested that the stability of chemical compositions of nanomaterials itself should be considered as an important factor which affects their potential environmental impacts and biological effects. However, Zn2+ positive control displayed a less developmental toxicity than that of nZnO suspensions under the same experimental conditions, which indicated that there might be some other factors also affecting the developmental toxicity of nZnO particles. Experiments using filtered solution from nZnO suspensions as incubation solution were conducted, and the toxicity of filtrates was found to be significantly less than that of nZnO particles suspension. So it was supposed that ZnO nanoparticles accumulated/adhered on the surface of zebrafish embryos should be responsible for the more toxicity of nZnO particles suspension. ZnO have been now well recognized as photocatalyst, which can generate reactive oxygen species (ROS) when they are suspended in water and irradiated with ultraviolet (UV) light. However, the ROS was not found in nZnO particle suspensions in this experiment. Therefore, the determined developmental toxicity of nZnO particles suspension could not be directly linked to the ROS.③The zebrafish 96-h embryo-larval bioassay was also conducted to better understand the potential ecotoxicological impacts of C60 nanoparticles released to aquatic environments. In this investigation, I firstly compared the developmental toxicity of water-soluble C60 aggregates (nC60) produced through three methods and a hydroxylated C60 derivative (fullerol, C60(OH)16-18). The three methods are solvent exchange using toluene, acetone, and THF (nC60/TTA), mixing C60 in a toluene solution with water through sonication (nC60/toluene) and extended mixing of C60 in water without the use of organic solvents (nC60/aq). Within 96 h of exposure, nC60/toluene at 38 mg L-1, fullerol at 50 mg L-1 and nC60/aq at 5 mg L-1 did not show any toxicity to zebrafish embryos. In contrast, nC60/TTA delayed zebrafish embryo and larva development, decreased its livability and hatching rate, and caused pericardial edema. The 96 h LC50 of nC60/TTA on the survival of zebrafish embryos was 2.561 mg/L, and the 84 h EC50 of nC60/TTA on the hatching of zebrafish embryos was 2.572 mg/L.④Further experiments strongly suggested that the mechanism contributed to the developmental toxicity of nC60/TTA was oxidative damage, which was induced by ROS(e.g., Superoxide anion free radical (O2?-) and hydroxyl radicals (·OH), et.al) generated in nC60/TTA suspension, on the zebrafish embryos or larvae. O2?- was detected in free embryos nC60/TTA suspension. SOD activity of zebrafish embryos was stimulated when exposed to low level of nC60/TTA while inhibited at higher concentration of nC60/TTA (≥0.670mg/L). Moreover, it was found there was a good dose—effects relationship between nC60/TTA and Lipid Peroxidation of zebrafish embryos. With nC60/TTA concentration increasing, Lipid Peroxidation of zebrafish embryos was more serious. However, these nC60/TTA toxicities were mitigated when an antioxidant (Vitamin C) was added. Thus, ROS generation could be an important toxic pathway of nC60/TTA.⑶Finally, the potentially toxicological effects of long term exposure at low concentration (0.04～1.0 mg/L) of nC60/aq on the antioxidant defense system in juvenile crucian (Carassius auratus) were studied with the consideration that the exposure level of MNMs in natural environment is usually low. The physiochemical indexes such as SOD, catalase (CAT) and Na+-K+-ATPase activity as well as reduced glutathione (GSH) contents in the tissue of brain, liver and gill of juvenile crucian were measured after 32 days exposure. This work seeks to contribute to a better understanding of the MNMs health effects that may be produced on exposed species in natural ecosystem. nC60/aq is a particularly important consideration because it can be produced through the extended mixing of C60 in water without the use of organic solvents, and nC60 has been proposed to be the most environmentally relevant form of carbon nanomaterials. The results indicated that:①Compared to the control, the body weight and the total length of juvenile crucian exposed to 1 mg/L nC60/aq for 32 days were decreased significantly (p<0.05).②Different tissue of juvenile crucian exposed to nC60/aq displayed different antioxidation mechanisms. For the brain tissue of juvenile crucian exposed to nC60/aq, SOD, CAT and Na+-K+-ATPase activity were not affected, whereas GSH contents decreased significantly with the increase of nC60/aq concentration. Therefore, GSH might be the main substance contributed to the detoxification of nC60/aq in the brain tissue of juvenile crucian. Different from the response of brain tissue, SOD, CAT and Na+-K+-ATPase activity as well as GSH contents in the liver of juvenile crucian were all changed significantly (p<0.05) in response to the stress of nC60/aq with varying concentrations from 0.04 mg/L to 1.0 mg/L, this result suggested that the liver might be one of important target organs of nC60/aq to juvenile crucian. In the gill tissue of juvenile crucian exposed to nC60/aq, Na+-K+-ATPase was stimulated significantly although it did not exert any influence in brain and liver. Because Na+-K+-ATPase is a very important protein to sustain the inner equilibrium of cell, the observed results suggested that the gill tissue injury might have occurred. Furthermore, the GSH contents in all different tissues decreased significantly (p<0.05) when juvenile crucian was exposed to nC60/aq with different concentrations (0.04 to 1.0 mg/L), and the SOD and CAT activities were stimulated significantly in liver tissue, which indicated that oxidative stress induced by long term exposure could be the main toxic mechanism of the nC60/aq to juvenile crucian.The above studies showed that MNMs released into the aquatic environment could do harm to all levels of the aquatic organisms including phytoplankton, zooplankton and nekton, lead to significantly acute and chronic damage, and bring short term and long term ecotoxicological effects to the aquatic ecosystem. Its aquatic ecotoxicity mechanisms were found to be related to the inherent properties of themselves (e.g., size effects, photosensitivity, high surface area and reactivity), and also depend to the testing organisms. Different organisms with variance physiology condition (e.g., physiological construction, function and sensibility, etc.) can develop different response to the MNMs exposure. The toxicity results shown in the above experiments highlight the need for further ecotoxicological research and safe disposal protocols improvement for such MNMs, so as to ensure that the development of nanotechnology will not result in adverse ecological effects and harm human health.