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1.
The nematode Caenorhabditis elegans receives increasing attention in sediment ecotoxicology and new toxicity tests with sensitive test parameters are under development. In this study, the motility of C. elegans could be measured for the first time online in sediment, using the Multispecies Freshwater Biomonitor. Whereas single nematodes could not be recorded, groups of 10 nematodes gave typical locomotive signals in different media (water, agar, sediment) with comparable precision and accuracy. The results of this study encourage to develop a new rapid online whole-sediment toxicity test with behaviour as sensitive test parameter.  相似文献   

2.
This work assesses the ecotoxicological effects of polluted sediment after a decontamination treatment process using a new sediment washing technique. Sediment samples were collected from four sites in Marghera Port industrial channels (Venice, Italy). Ecotoxicological evaluations were performed with Vibrio fischeri and Crassostrea gigas bioassays. Whole sediment and elutriate were deemed as the most suitable environmental matrices for this study. Toxicity scores developed in the Lagoon of Venice for V. fischeri on whole sediment and for C. gigas on elutriate were considered for the final ranking of samples. Ecotoxicological results showed that the treated sediment samples presented both acute and sub-chronic toxicities, which were mainly attributed to the presence of some remaining chemicals such as metals and polyaromatic hydrocarbons. The acute toxicity ranged from low to medium, while the sub-chronic one from absent to very high, suggesting that treated sediments could not be reused in direct contact with seawater.  相似文献   

3.
Abstract

This study investigated the toxicity of various concentrations of technical resmethrin and Scourge® on adult and larval Palaemonetes pugio, a common grass shrimp species. Two types of tests were conducted for each of the resmethrin formulations using adult and larval grass shrimp life stages, a 96-h static renewal aqueous test without sediment, and a 24-h static nonrenewal aqueous test with sediment. For resmethrin, the 96-h aqueous LC50 value for adult shrimp was 0.53 μg/L (95% confidence interval (CI): 0.46–0.60 μg/L), and for larval shrimp was 0.35 μg/L (95% CI: 0.28–0.42 μg/L). In the presence of sediment, technical resmethrin produced a 24-h LC50 value for adult shrimp of 5.44 μg/L (95% CI: 4.52–6.55 μg/L), and for larval shrimp of 2.15 μg/L (95% CI: 1.35–3.43 μg/L). For Scourge®, the 96-h aqueous LC50 for adult shrimp was 2.08 μg/L (95% CI: 1.70–2.54 μg/L), and for larval shrimp was 0.36 μg/L (95% CI: 0.24–0.55 μg/L). The 24-h sediment test yielded an LC50 value of 16.12 μg/L (95% CI: 14.79–17.57 μg/L) for adult shrimp, and 14.16 μg/L (95% CI: 12.21–16.43 μg/L) for larvae. Adjusted LC50 values to reflect the 18% resmethrin concentration in Scourge® are 0.37 μg/L (adult), 0.07 μg/L (larvae) for the 96-h aqueous test, and 2.90 μg/L (adult), 2.6 μg/L (larvae) for the 24-h sediment test. Larval grass shrimp were more sensitive to technical resmethrin and Scourge® than the adult life stage. The results also demonstrate that synergized resmethrin is more toxic to P. pugio than the nonsynergized form, and that the presence of sediment decreases the toxicity of both resmethrin and Scourge®  相似文献   

4.
尹倩  张薛  陆韻  赵璇 《环境工程学报》2014,8(4):1692-1698
大型蚤是一种国际公认的标准实验生物,广泛应用于污水、地表水等水质毒性检测。毒性水平较低的城市二级出水,对大型蚤往往无急性毒性效应,而具有慢性毒性效应,但慢性毒性检测周期过长,因此探索一种更灵敏的指标,实现快速检测,对于控制二级出水的水质风险具有重要意义。本研究考察了大型蚤在短期暴露于城市二级出水条件下,其体内乙酰胆碱酯酶、超氧化物歧化酶、过氧化氢酶、ATP酶、羧酸酯酶和碱性磷酸酯酶的酶活变化特征,从中筛选出对二级出水毒性响应灵敏的标志酶指标。实验结果表明,碱性磷酸酯酶、过氧化氢酶对二级出水毒性响应相对较灵敏,具有成为标志酶的潜力,研究结果为城市二级出水生物毒性评价方法优化提供新的思路。  相似文献   

5.
Freshwater sediments with low levels of anthropogenic contamination and a broad range of geochemical properties were investigated using various sediment-contact tests in order to study the natural variability and to define toxicity thresholds for the various toxicity endpoints. Tests were performed with bacteria (Arthrobacter globiformis), yeast (Saccharomyces cerevisiae), nematodes (Caenorhabditis elegans), oligochaetes (Lumbriculus variegatus), higher plants (Myriophyllum aquaticum), and the eggs of zebrafish (Danio rerio). The variability in the response of some of the contact tests could be explained by particle size distribution and organic content. Only for two native sediments could a pollution effect not be excluded. Based on the minimal detectable difference (MDD) and the maximal tolerable inhibition (MTI), toxicity thresholds (% inhibition compared to the control) were derived for each toxicity parameter: >20% for plant growth and fish-egg survival, >25% for nematode growth and oligochaete reproduction, >50% for nematode reproduction and >60% for bacterial enzyme activity.  相似文献   

6.
Microcosm experiments have been carried out with whole natural meiobenthic communities to look at the effects of TBT sediment contamination on the community structure of the dominant nematode component of the meiobenthos. TBT has a high affinity for aquatic sediments, yet this is the first study of the effects of this contaminant in sediment on natural benthic communities. Three communities were studied from contrasting locations in south-west England: the intertidal of the Lynher estuary (muddy sediment) and the Exe estuary (sandy sediment) and the subtidal (50m depth) at Rame Head off Plymouth (muddy sand). Fresh sediment with natural meiobenthic communities was incubated for 2 months with TBT-contaminated sediment (three dose levels) in bottles. Nematodes were identified and enumerated and subjected to multivariate data analysis. The sandy Exe estuary fauna was significantly affected by TBT-contaminated sediment at all three doses (0.3, 0.6 and 0.9 microg g(-1) dry wt (as Sn) sediment), whereas the offshore fauna from Rame Head was significantly affected only at the highest dose. The muddy Lynher estuary meiofauna was affected (somewhat peculiarly) at the medium dose level only. Meiobenthic nematodes may not be as sensitive to TBT-contaminated sediment as other infaunal benthos but exhibited responses to levels of contamination still persisting in some UK estuaries and harbours. Comparing the effects of TBT with those of copper and zinc in the same laboratory experiments, our observations suggest that the relative impact of TBT on meiobenthic community structure is not as great as these contaminants in marine sediments. Although there are very few observations of TBT toxicity in sediment, it appears that TBT is toxic at much lower concentrations in seawater (ppb) than it is in sediment (ppm).  相似文献   

7.

Goal, Scope and Background

Lumbriculus variegatus Müller (Oligochaeta), a common freshwater sediment-dweller, has frequently been used in toxicokinetic studies, although has been less used in ecotoxicity tests.

Methods

For the first time the Multispecies Freshwater Biomonitor® (MFB) was applied in a short-term whole-sediment toxicity test. The MFB automatically and quantitatively recorded the spontaneous locomotory behaviour of Lumbriculus variegatus in exposures with two compartments, water and sediment. The study questioned, whether the animals altered their locomotion depending on the compartment which was spiked with lead (Pb).

Results and Discussion

As in the exposures to Pb-contaminated water/clean sediment, the animals exposed to Pb-contaminated sediment/clean water showed higher activities in intermediate Pb-concentrations. This indicates, that spontaneous locomotory activity is affected by Pb-concentrations at sublethal levels regardless of whether the Pb-concentration is found in the water or in the sediment, because these animals use both environmental compartments simultaneously. However, within the same Pb-levels, the animals showed higher locomotory activity in contaminated water compared with contaminated sediment. This indicates a possible tendency to withdraw from (‘avoidance’) contaminated water into the clean sediment compartment, whereas there was no withdrawal from contaminated sediment into clean water. The latter might be explained by the fact that withdrawal from sediment to water might increase the risk of predation and drift in nature, whereas retracting to sediment might provide shelter.

Conclusions

The study showed that spontaneous locomotory responses of L. variegatus to Pb depend on whether the water or sediment is contaminated. The study also concluded that the Multispecies Freshwater Biomonitor® can be applied effectively in sediment toxicity testing.

Recommendations and Perspectives

More emphasis should be given to the interactions of water/sediment in sediment ecotoxicity tests to better simulate field conditions and increase ecological realism in risk assessment, especially as quantitative recording methods exisit.
  相似文献   

8.
Wu Q  Qu Y  Li X  Wang D 《Chemosphere》2012,87(11):1281-1287
Here we investigated whether the assay system (10-d) in Caenorhabditis elegans can be used to evaluate chronic toxicity of chromium (Cr(VI)) at environmental relevant concentrations ranging from 5.2 μg L−1 to 260 μg L−1. The results indicated that lethality, locomotion behavior as revealed by head thrash, body bend, and forward turn, metabolism as revealed by pumping rate and mean defecation cycle length, intestinal autofluorescence, and ROS production were severely altered in Cr chronically exposed nematodes at environmental relevant concentrations. The most surprising observations were that head thrash, body bend, intestinal autofluorescence, and ROS production in 13 μg L−1 Cr exposed nematodes were significantly influenced. The observed adverse effects of Cr on survival, locomotion behavior, and metabolism were largely due to forming severe intestinal autofluorescence and ROS production. Therefore, our findings demonstrate the usefulness of chronic toxicity assay system in C. elegans in evaluating the chronic toxicity of toxicants at environmental relevant concentrations.  相似文献   

9.
The study of the effect of the sorption of linear alkylbenzene sulfonates (LAS) on the bioavailability to marine benthic organisms is essential to refine the environmental risk assessment of these compounds. According to the equilibrium partitioning theory (EqP), the effect concentration in water-only exposure will be similar to the effect concentration in the sediment pore water. In this work, sorption and desorption experiments with two marine sediments were carried out using the compound C12-2-LAS. The effect of the sediment sorption on the toxicity of benthic organisms was studied in water-only and in sediment bioassays with the marine mud shrimp Corophium volutator. In addition, three common spiking methods were tested for its application in the toxicity tests, as well as the stability of the surfactant during the water-only and sediment-water test duration. LC50 values obtained from water-only exposure showed a good correspondence with the pore water concentrations calculated from the sorption and desorption isotherms in the spiked sediments.  相似文献   

10.
Toxicity evaluation is an important segment in sediment quality monitoring in order to protect aquatic organisms and human health. The purpose of this study is to assess the toxicity of sediments from three sediment cores in Yangtze River Estuary, China, using the zebrafish (Danio rerio) embryo tests. Fertilized zebrafish eggs were exposed to both whole sediments and sediment organic extracts prepared from collected sediments, in order to provide a comprehensive and realistic insight into the bioavailable toxicity potential of the sediments. As end points, development parameters (mortality, hatching rate, and abnormality) in the developing embryos were recorded during the 96-h exposure. The results showed that some samples increased mortality, inhibited the hatching of embryos, and induced morphological abnormalities. The embryonic toxicities presented serrated changes and irregular distribution with depth, which may be related to hydrodynamic effect and unstable environmental input. However, lethal and sub-lethal effects were more significant at the sub-surface sediments (10~40 cm), which indicated that the pollution is more serious in recent decades.  相似文献   

11.
Due to hydrophobic and persistent properties, polycyclic aromatic hydrocarbons (PAHs) have a high capacity to accumulate in sediment. Sediment quality criteria, for the assessment of habitat quality and risk for aquatic life, include understanding the fate and effects of PAHs. In the context of European regulation (REACH and Water Framework Directive), the first objective was to assess the influence of sediment composition on the toxicity of two model PAHs, benzo[a]pyrene and fluoranthene using 10-day zebrafish embryo-larval assay. This procedure was undertaken with an artificial sediment in order to limit natural sediment variability. A suitable sediment composition might be then validated for zebrafish and proposed in a new OECD guideline for chemicals testing. Second, a comparative study of toxicity responses from this exposure protocol was then performed using another OECD species, the Japanese medaka. The potential toxicity of both PAHs was assessed through lethal (e.g., survival, hatching success) and sublethal endpoints (e.g., abnormalities, PMR, and EROD) measured at different developmental stages, adapted to the embryonic development time of both species. Regarding effects observed for both species, a suitable artificial sediment composition for PAH toxicity testing was set at 92.5 % dry weight (dw) silica of 0.2–0.5-mm grain size, 5 % dw kaolin clay without organic matter for zebrafish, and 2.5 % dw blond peat in more only for Japanese medaka. PAH bioavailability and toxicity were highly dependent on the fraction of organic matter in sediment and of the K ow coefficients of the tested compounds. The biological responses observed were also dependent of the species under consideration. Japanese medaka embryos appeared more robust than zebrafish embryos for understanding the toxicity of PAHs following a sediment contact test, due to the longer exposure duration and lower sensitivity of sediment physical properties.  相似文献   

12.
The sole routine testing of the standard earthworm Eisenia fetida for the terrestrial risk assessment of pesticides has been under much debate since other soil invertebrates may be more sensitive than this standard test species. However, the very low availability of laboratory toxicity data for taxa other than E. fetida has greatly hampered sensitivity comparisons. In the present study, the relative tolerance (Trel) approach was used to enable comparing toxicity thresholds obtained from the US-EPA ECOTOX database, for main terrestrial taxonomic groups and pesticidal types of action (insecticides, fungicides, herbicides, and other) separately. Analyses confirmed previously reported lower and higher sensitivity of collembolans to fungicides and insecticides, respectively. However, various other discrepancies in susceptibility relative to E. fetida were encountered as indicated by species sensitivity distributions and/or calculated 95% confidence intervals of Trel values. Arachnids and isopods were found to be more sensitive to insecticides, and nematodes to fungicides, as compared to E. fetida. Implications of study findings for the terrestrial risk assessment of pesticides are discussed.  相似文献   

13.
The objective of this study was to test the hypotheses that (1) free-living marine nematodes respond in a differential way to diesel fuel if it is combined with three trace metals (chromium, copper, and nickel) used as smoke suppressants and that (2) the magnitude of toxicity of diesel fuel differs according to the level of trace metal mixture added. Nematodes from Sidi Salem beach (Tunisia) were subjected separately for 30 days to three doses of diesel fuel and three others of a trace metals mixture. Simultaneously, low-dose diesel was combined with three amounts of a trace metal mixture. Results from univariate and multivariate methods of data evaluation generally support our initial hypothesis that nematode assemblages exhibit various characteristic changes when exposed to different types of disturbances; the low dose of diesel fuel, discernibly non-toxic alone, became toxic when trace metals were added. For all types of treatments, biological disturbance caused severe specific changes in assemblage structure. For diesel fuel-treated microcosms, Marylynnia bellula and Chromaspirinia pontica were the best positive indicative species; their remarkable presence in given ecosystem may predict unsafe seafood. The powerful toxicity of the combination between diesel fuel and trace metals was expressed with only negative bioindicators, namely Trichotheristus mirabilis, Pomponema multipapillatum, Ditlevsenella murmanica, Desmodora longiseta, and Bathylaimus capacosus. Assemblages with high abundances of these species should be an index of healthy seafood. When nematodes were exposed to only trace metals, their response looks special with a distinction of a different list of indicative species; the high presence of seven species (T. mirabilis, P. multipapillatum, Leptonemella aphanothecae, D. murmanica, Viscosia cobbi, Gammanema conicauda, and Viscosia glabra) could indicate a good quality of seafood and that of another species (Oncholaimellus mediterraneus) appeared an index of the opposite situation.  相似文献   

14.
Mutatox test: a new test for monitoring environmental genotoxic agents   总被引:3,自引:0,他引:3  
In this study, Yamaska River water and Milli-Q water and organically extracted sediment extracts were used to evaluate the sensitivity of a new genotoxicity screening test, the Mutatox test. Also in this study, the samples were tested for acute and chronic toxicity using the following screening test procedures: Microtox, Daphnia magna, Ceriodaphnia reticulata and ATP-TOX Systems. The Mutatox test is based on the use of a dark mutant strain of Photobacterium phosphoreum and is sensitive to chemicals which are (1) DNA damaging agents (2) DNA intercalating agents, (3) DNA synthesis inhibitors and (4) direct mutagens. In this study, the Mutatox test was found to be a simple-to-perform sensitive procedure which added greater scope to the battery of tests approach. Preliminary indications are that this procedure may prove to be one of the more responsive and valuable tests in the 'battery of tests' approach to environmental screening.  相似文献   

15.
The goal of this study was to contribute to understanding of the mechanisms behind sensitivity differences between early and late instar larvae of Chironomus riparius and to address the influence of the differences in standard testing approaches on the toxicity evaluation. A 10-day contact sediment toxicity test was carried out to assess sensitivity to cadmium exposure in relation to different age and laboratory culture line origin of test organisms. Chironomid larvae of early (OECD 218 method) and late instar (US-EPA600/R-99/064 method) differed substantially in sensitivity of traditional endpoints (OECD: LOEC 50 and 10 μg Cd/g dry weight (dw); US-EPA: LOEC?>?1000 and 100 μg Cd/g dw for survival and growth, respectively). Bioaccumulated cadmium and metallothioneins (MTs) concentrations were analyzed to investigate the role of MTs in reduced sensitivity to cadmium in late instar larvae. Metallothioneins were induced after treatment to greater Cd concentrations, but their levels in relation to cadmium body burdens did not fully explain low sensitivity of late instars to cadmium, which indicates some other effective way of detoxification in late instars. This study brings new information related to the role of MTs in age-dependent toxicant sensitivity and discusses the implications of divergence in data generated by chironomid sediment toxicity tests by standardized methods using different instars.  相似文献   

16.

To examine pesticide mixture toxicity to aqueous organisms, we assessed the single and combined toxicities of thiamethoxam and other four pesticides (chlorpyrifos, beta-cypermethrin, tetraconazole, and azoxystrobin) to the rare minnow (Gobiocypris rarus). Data from 96-h semi-static toxicity assays of various developmental phases (embryonic, larval, juvenile, and adult phases) showed that beta-cypermethrin, chlorpyrifos, and azoxystrobin had the highest toxicities to G. rarus, and their LC50 values ranged from 0.0031 to 0.86 mg a.i. L?1, from 0.016 to 6.38 mg a.i. L?1, and from 0.39 to 1.08 mg a.i. L?1, respectively. Tetraconazole displayed a comparatively high toxicity, and its LC50 values ranged from 3.48 to 16.73 mg a.i. L?1. By contrast, thiamethoxam exhibited the lowest toxic effect with LC50 values ranging from 37.85 to 351.9 mg a.i. L?1. Rare minnow larvae were more sensitive than embryos to all the pesticides tested. Our data showed that a pesticide mixture of thiamethoxam–tetraconazole elicited synergetic toxicity to G. rarus. Moreover, pesticide mixtures containing beta-cypermethrin in combination with chlorpyrifos or tetraconazole also had synergetic toxicities to fish. The majority of pesticides are presumed to have additive toxicity, while our data emphasized that the concurrent existence of some chemicals in the aqueous circumstance could cause synergetic toxic effect, leading to severe loss to the aqueous environments in comparison with their single toxicities. Thence, the synergetic impacts of chemical mixtures should be considered when assessing the ecological risk of chemicals.

  相似文献   

17.
《Chemosphere》2013,93(5):805-812
Chronic toxicity and bioaccumulation of decamethylcyclopentasiloxane (D5) to Hyalella azteca was examined in a series of spiked sediment exposures. Juvenile H. azteca were exposed for 28 d (chronic) to a concentration series of D5 in two natural sediments of differing organic carbon content (O.C.) and particle size composition. The chronic, LC50s were 191 and 857 μg D5 g−1 dry weight for Lakes Erie (0.5% O.C.) and Restoule (11% O.C.) respectively. Inhibition of growth only occurred with the L. Restoule spiked sediment with a resultant EC25 of 821 μg g−1 dw. Lethality was a more sensitive endpoint than growth inhibition. Biota sediment accumulation factors (BSAFs, 28 d) were <1 indicating that D5 did not bioconcentrate based on lipid normalized tissue concentrations and organic carbon normalized sediment concentrations. Organic carbon (OC) in the sediment appeared to be protective, however normalization to OC did not normalize the toxicity. Normalization of D5 concentrations in the sediments to sand content did normalize the toxicity and LC50 values of 3180 and 3570 μg D5 g−1 sand dw were determined to be statistically the same.  相似文献   

18.
An invertebrate embryo toxicity test with the ampullariid snail, Marisa cornuarietis, to assess the toxicity of pesticides and heavy metals recently was established. Snail embryos were treated with atrazine (100, 1000, 10 000, and 30 000 μg/L), imidacloprid (10 000, 25 000, and 50 000 μg/L), Ni2+ (0.1, 1, 10, and 100 μg/L) or Zn2+ (100, 200, 500, 1000, 2000, and 5000 μg/L). The effect of these substances was examined by monitoring the following endpoints: mortality, formation of tentacles and eyes, heart rate, hatching, and weight after hatching. Effects in term of a significant delay on the formation of both tentacles and eyes were found after treatment with 100 μg/L Ni2+ or 200 μg/L Zn2+. The heart rate was shown to significantly decrease at 25 000 μg/L imidacloprid or 1000 μg/L Zn2+. At 100 μg/L atrazine, 10 μg/L Ni2+, or 1000 μg/L Zn2+ a significant delay in hatching became visible. No significant mortality was observed for the tested concentrations of atrazine, imidacloprid, or Ni2+, while 5000 μg/L Zn2+ resulted in 100% mortality after 10 d. The weight of freshly hatched individuals remained unaffected in all treatments. On the basis of the lowest observed effect concentrations (LOECs) recorded, we could show the M. cornuarietis embryo toxicity test (MariETT) to react up to three orders of magnitude more sensitive (for metals) and at least one order of magnitude more sensitive (for the tested organics) than the established Danio rerio embryo test.  相似文献   

19.
In total, 24 polycyclic aromatic hydrocarbons (PAHs) in both gas and particle phases and 35 nitro-PAHs in particle phase were analyzed in the exhaust from heavy-duty diesel vehicles equipped with after-treatment for particulate matter (PM) and NOX control. The test vehicles were carried out using a chassis dynamometer under highway cruise, transient Urban Dynamometer Driving Schedule (UDDS), and idle operation. The after-treatment efficiently abated more than 90% of the total PAHs. Indeed, the particle-bound PAHs were reduced by >99%, and the gaseous PAHs were removed at various extents depending on the type of after-treatment and the test cycles. The PAHs in gas phase dominated the total PAH (gas + particle phases) emissions for all the test vehicles and for all cycles; that is, 99% of the two-ring and 98% of the three-ring and 97% of the four-ring and 95% of the carcinogenic PAHs were in the gas-phase after a diesel particle filter (DPF) and not bound to the very small amount of particulate matter left after a DPF. Consequently, an evaluation of the toxicity of DPF exhaust must include this volatile fraction and cannot be based on the particle fraction only. The selective catalytic reduction (SCR) did not appear to promote nitration of the PAHs in general, although there might be some selective nitration of phenanthrene. Importantly the after-treatmtent reduced the equivalent B[a]P (B[a]Peq) emissions by >95%, suggesting a substantial health benefit.

Implications: This study demonstrated that after-treatments, including diesel particulate filters (DPF), diesel oxidation catalysts (DOC), and selective catalytic reduction (SCR), significantly reduce the emissions of PAHs from heavy-duty diesel engines. The gas-phase PAHs dominate the total PAH (gas + particle phases) emissions from heavy-duty diesel vehicles retrofitted with various DPFs and not bound to the very small amount of particulate matter left after a DPF. Consequently, an evaluation of the toxicity of DPF exhaust must also include this volatile fraction and cannot be based on the particle fraction only.

Supplemental Materials: Supplemental materials are available for this paper. Go to the publisher's online edition of the Journal of the Air & Waste Management Association.  相似文献   

20.
The present study evaluated phototoxicity of nanoparticulate ZnO and bulk-ZnO under natural sunlight (NSL) versus ambient artificial laboratory light (AALL) illumination to a free-living nematode Caenorhabditis elegans. Phototoxicity of nano-ZnO and bulk-ZnO was largely dependent on illumination method as 2-h exposure under NSL caused significantly greater mortality in C. elegans than under AALL. This phototoxicity was closely related to photocatalytic reactive oxygen species (ROS) generation by the ZnO particles as indicated by concomitant methylene blue photodegradation. Both materials caused mortality in C. elegans under AALL during 24-h exposure although neither degraded methylene blue, suggesting mechanisms of toxicity other than photocatalytic ROS generation were involved. Particle dissolution of ZnO did not appear to play an important role in the toxicity observed in this study. Nano-ZnO showed greater phototoxicity than bulk-ZnO despite their similar size of aggregates, suggesting primary particle size is more important than aggregate size in determining phototoxicity.  相似文献   

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