Gene-TEQ--a standardized comparative assessment of effects in the comet assay using genotoxicity equivalents

Existing methods for the comparison of genotoxic effects in the comet assay bear considerable disadvantages such as the problem to link information about concentration dependence and severity of effects. Moreover, given the lack of standardized protocols and the use of various standards, it may be extremely difficult to compare different studies. In order to provide a method for standardized comparative assessment of genotoxic effects, the concept of genotoxicity equivalents (Gene-TEQ) was developed. As potential reference compounds for genotoxic effects, three directly acting (N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl-methanesulfonate, and N-methyl-N-nitrosourea) and three indirectly acting (cyclophosphamide, dimethylnitrosamine, and 4-nitroquinoline-oxide) genotoxic substances were compared with respect to their cytotoxic (neutral red) and genotoxic (comet assay) concentration-response profiles in the permanent fish cell line RTL-W1. For further comparison, two sediment extracts from the upper Danube River were investigated as environmental samples. Based on the results of cytotoxicity and genotoxicity testing, MNNG was selected as the reference compound. At several exposure levels and durations, genotoxic effects of both the other pure substances and the environmental samples were calculated as percentages of the maximum MNNG effect and related to the absolute MNNG effect (EC values). Thus, genotoxicity equivalent factors (Gene-TEQs) relative to MNNG could be calculated. Gene-TEQs can easily be applied to pure substances, mixtures and field samples to provide information about their toxicity relative to the reference compound. Furthermore, the Gene-TEQ concept allows a direct comparison of environmental samples from different laboratories.     

Preparation and measurement methods for studying nanoparticle aggregate surface chemistry

Despite best efforts at controlling nanoparticle (NP) surface chemistries, the environment surrounding nanomaterials is always changing and can impart a permanent chemical memory. We present a set of preparation and measurement methods to be used as the foundation for studying the surface chemical memory of engineered NP aggregates. We attempt to bridge the gap between controlled lab studies and real-world NP samples, specifically TiO(2), by using well-characterized and consistently synthesized NPs, controllably producing NP aggregates with precision drop-on-demand inkjet printing for subsequent chemical measurements, monitoring the physical morphology of the NP aggregate depositions with scanning electron microscopy (SEM), acquiring "surface-to-bulk" mass spectra of the NP aggregate surfaces with time-of-flight secondary ion mass spectrometry (ToF-SIMS), and developing a data analysis scheme to interpret chemical signatures more accurately from thousands of data files. We present differences in mass spectral peak ratios for bare TiO(2) NPs compared to NPs mixed separately with natural organic matter (NOM) or pond water. The results suggest that subtle changes in the local environment can alter the surface chemistry of TiO(2) NPs, as monitored by Ti(+)/TiO(+) and Ti(+)/C(3)H(5)(+) peak ratios. The subtle changes in the absolute surface chemistry of NP aggregates vs. that of the subsurface are explored. It is envisioned that the methods developed herein can be adapted for monitoring the surface chemistries of a variety of engineered NPs obtained from diverse natural environments.     

Toxicological assessment of TiO2 nanoparticles by recombinant Escherichia coli bacteria

Rapid and efficient methods to assess nanoparticle toxicity are desired in current research. Here we showed that Escherichia coli labeled by green fluorescent protein can be a good model bacterium for assessing acute toxicity of TiO(2) (about 50% inhibition ratios after 135 min exposure). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that TiO(2) nanoparticles (NPs) can influence certain protein expression in the recombinant bacterium, and the obvious effects in repressed expression and elevated expression were observed in 30/40, 10/20 μg mL(-1) treated cells, respectively. However, the GFP expression (27 kD) was not influenced by introduced TiO(2) NPs. The change of the fluorescence intensity may be caused by the damage in folding and chromophore formation of the GFP post-translational modification due to generated reactive oxygen species. Furthermore, TiO(2) NPs at higher concentrations decreased their toxicity because of aggregation. 20 μg mL(-1) humic acid (HA) introduced to the medium can decrease the fluorescent inhibition owing to the barrier of steric hindrance it provides between NPs and cells.     

应用小鼠淋巴细胞彗星试验检测地下水遗传毒性

应用小鼠淋巴细胞彗星试验对长治市5个点位地下水水质作遗传毒性研究。结果表明,各水样有机浓集物均可对小鼠淋巴细胞DNA产生不同程度的遗传损伤,随着剂量的增加,Olive尾矩与阴性对照组(0.72±0.09)相比均有显著性差异(P0.05,P0.01)。在试验浓度范围内,以彗星尾部DNA含量、彗星尾长、尾矩和Olive尾矩所指示的DNA损伤程度随剂量增大而逐渐增加,存在显著的剂量-效应关系,其相关系数0.815。     

Genotoxicity evaluation of water soil leachates by Ames test, comet assay, and preliminary Tradescantia micronucleus assay

Combining genotoxicity/mutagenicity tests and physico-chemical methodologies can be useful for determining the potential genotoxic contaminants in soil samples. The aim of our study was to evaluate the genotoxicity of soil by applying an integrated physico-chemical-biological approach. Soil samples were collected at six sampling points in a Slovenian industrial and agricultural region where contamination by heavy metals and sulphur dioxide (SO₂) are primarily caused by a nearby power plant. The in vitro alkaline version of the comet assay on water soil leachates was performed with Caco-2 and HepG2 cells. A parallel genotoxicity evaluation of the samples was performed by Ames test using Salmonella typhimurium and the Tradescantia micronucleus test. Pedological analyses, heavy metal content determination, and different physico-chemical analyses, were also performed utilizing standard methodology. Water leachates of soil samples were prepared according to standard methods. Since only a battery of biotests with prokaryotic and eukaryotic organisms or cells can accurately estimate the effects of (geno)toxicants in soil samples and water soil leachates, a combination of three bioassays, with cells or organisms belonging to different trophic levels, was used. Genotoxicity of all six water soil leachates was proven by the comet assay on both human cell lines, however no positive results were detected by bacterial assay, Ames test. The Tradescantia micronucleus assay showed increase in micronuclei formation for three samples. According to these results we can assume that the comet assay was the most sensitive assay, followed by the micronucleus test. The Ames test does not appear to be sensitive enough for water soil leachates genotoxicity evaluations where heavy metal contamination is anticipated.     

Evaluation of genotoxicity of coal fly ash in Allium cepa root cells by combining comet assay with the Allium test

Fly ash is a by-product of coal-fired electricity generation plants. Its utilization and disposal is of utmost importance. Using onion (Allium cepa) root tip system, the present study was carried out to evaluate the potential toxic and genotoxic effects of fly ash, collected from a thermal power plant in West Bengal, India. Prior to testing, the collected fly ash sample was mixed with sand in different proportions. Allium bulbs were allowed to germinate directly in fly ash and after five days the germinating roots were processed for the Allium test. Additionally, the Allium test was adapted for detecting DNA damage through comet assay. The results from the Allium test indicate that fly ash at 100% concentration inhibits root growth and mitotic indices; induces binucleated cells as a function of the proportion, but is not toxic at very low concentration. In the comet assay, a statistical increase for DNA strand breaks was found only at higher concentrations. The sample was analyzed by flame atomic absorption spectrometer for Zn, Pb, Cu, Ni, Cd and As, whose presence could partly be responsible for the toxicity of fly ash. The study concludes that the classical Allium test can give a more comprehensive data when done in combination with the comet assay, which is faster, simpler and independent of mitosis. Also when fly ash is used for other purposes in combination with soils, it should be judiciously used at very low concentrations in order to protect the ecosystem health from any potential adverse effects.     

Effects of selected metal oxide nanoparticles on Artemia salina larvae: evaluation of mortality and behavioural and biochemical responses

The aim was to investigate the toxicity of selected metal oxide nanoparticles (MO-NPs) on the brine shrimp Artemia salina, by evaluating mortality and behavioural and biochemical responses. Larvae were exposed to tin(IV) oxide (stannic oxide (SnO₂)), cerium(IV) oxide (CeO₂) and iron(II, III) oxide (Fe₃O₄) NPs for 48 h in seawater, with MO-NP suspensions from 0.01 to 1.0 mg/mL. Mortality and behavioural responses (swimming speed alteration) and enzymatic activities of cholinesterase, glutathione-S-transferase and catalase were evaluated. Although the MO-NPs did not induce any mortality of the larvae, they caused changes in behavioural and biochemical responses. Swimming speed significantly decreased in larvae exposed to CeO₂ NPs. Cholinesterase and glutathione-S-transferase activities were significantly inhibited in larvae exposed to SnO₂ NPs, whereas cholinesterase activity significantly increased after CeO₂ NP and Fe₃O₄ NP exposure. Catalase activity significantly increased in larvae exposed to Fe₃O₄ NPs. In conclusion, swimming alteration and cholinesterase activity represent valid endpoints for MO-NP exposure, while glutathione-S-transferase and catalase activities appear to be NP-specific.     

Clear and present danger? The use of a yeast biosensor to monitor changes in the toxicity of industrial effluents subjected to oxidative colour removal treatments

Discharges of coloured effluents into surface waters provide conspicuous evidence of the impact of industry on the environment. The textile industry is an obvious candidate for sources of such discharges. Conventional treatment methods appear to alleviate this situation by removing colour, however the affect on toxicity is less obvious. The objective of this study was to examine the changes in effluent toxicity during the course of two alternative wastewater treatment methods, ozonation and electrochemical oxidation, using a novel toxicity biosensor, GreenScreen EM. The biosensor is capable of measuring both general acute toxicity (cytotoxicity), and more specifically genotoxicity, that is damage to a cell's DNA structure, replication or distribution, caused by substances that may be mutagenic and/or carcinogenic. The biosensor utilises a modified strain of the brewers yeast Saccharomyces cerevisiae, incorporating a gene encoding green fluorescent protein (GFP) linked to the inducible promoter of the DNA damage responsive RAD54 gene. Upon exposure to a genotoxin, the production of GFP is up-regulated in parallel with RAD54, and the resulting cellular fluorescence provides a measure of genotoxicity. Acute toxicity is simultaneously determined by monitoring relative total growth of the cell culture during incubation. The results presented in this paper show that a reduction in colouration does not necessarily correspond to a reduction in effluent toxicity.     

Genetic damage induced by lead chloride in different tissues of fresh water climbing perch Anabas testudineus (Bloch)

The present investigation was undertaken to study the induction of DNA damage by lead chloride (PbCl(2)) in freshwater climbing perch Anabas testudineus using alkaline single cell gel electrophoresis (comet assay). Based on the LC(50) values of lead chloride of A. testudineus three different concentrations viz., 0.1, 1.0 and 2.0 mg/L were selected to expose fish. The DNA damage was observed in the gill, kidney and liver tissue as the percentage of DNA in comet tails and comet heads in the tissue of the exposed fish. DNA damage at different concentrations showed sensitivity to particular tissue. The liver tissue exhibited significantly (p < 0.01) higher DNA damage, followed by kidney and gill. However, the DNA damage was found to be dose dependent; at 2 mg/L of PbCl(2) the tail and head DNA of liver tissue were 57.84% and 39.49%, in kidney tissue the values were 52.36% and 44.97% whereas in gill tissue the values were 48.86% and 48.96% respectively. The current study explored the utility of the comet assay for in vivo laboratory studies using A. testudineus species for screening the genotoxic potential of lead chloride.     

A yeast-based cytotoxicity and genotoxicity assay for environmental monitoring using novel portable instrumentation

An assay capable of simultaneously measuring both general toxicity and more subtle genotoxicity, in aqueous environmental samples, is described. The assay uses eukaryotic (yeast) cells, genetically modified to express a green fluorescent protein (GFP) whenever DNA damage, as a result of exposure to genotoxic agents, is repaired. A measure of the reduction in cell proliferation is used to characterise general toxicity producing familiar EC(50) and LOEC data. The assay protocol has been developed for proposed use in the field and hence employs dedicated, portable instrumentation, the development of which is described. A range of environmentally relevant substances has been evaluated using the assay, including solutions of metal ions, solvents and pesticides. Preliminary data comparing the yeast assay's response to that of a standard Daphnia test in the analysis of the toxicity of 34 varied industrial waste effluents are also presented. The sensitivity to a wide range of substances and effluents suggests the assay should be useful for environmental toxicity monitoring.     

Microelectrophoretic study of environmentally induced DNA damage in fish and its use for early toxicity screening of freshwater bodies

This study investigates the potential of the comet and micronucleus assays of fish DNA as a means of screening the toxicity of aquatic environments. Catla catla and Cirrhinus mrigala collected from the River Chenab in Pakistan were used as a case study for the application of comet and micronucleus techniques. Comet and micronucleus assays were used to compare DNA damage in C. catla and C. mrigala collected from polluted areas of the River Chenab and farmed fish. Atomic absorption spectrophotometry showed an acute level of toxicity from Cd, Cu, Mn, Zn, Pb, Cr, Sn, and Hg in river water. Comet assay showed significant (p < 0.05) DNA damage in C. catla representing 17.33 ± 2.42, 11.53 ± 2.14, and 14.17% DNA in the comet tail, averaged from three sites of the polluted area of the river. Tail moment was observed as 10.06 ± 2.71, 3.11 ± 0.74, and 14.70 ± 1.89, while olive moment was 8.85 ± 1.84, 3.83 ± 0.76, and 7.11 ± 0.73, respectively. Highly significant (p < 0.01) damage was reported in C. mrigala as 37.29 ± 2.51, 34.96 ± 2.53, and 38.80 ± 2.42% DNA in comet tail, tail moment was 23.48 ± 3.90, 19.78 ± 4.26, and 14.30 ± 1.82, and olive moment was 16.22 ± 2.04, 13.83 ± 1.96, and10.99 ± 0.90. Significant (p < 0.05) differences were observed in genotoxicity between farmed and polluted area fish. Micronucleus assay showed a similar picture of significant difference in respect to single and double micronucleus induction: i.e., 23.20 ± 4.19 and 2.80 ± 1.07‰ in C. catla and 44.80 ± 3.73 and 06.20 ± 0.97‰, respectively, in C. mrigala. Nuclear abnormalities were found as 6.00 ± 0.84 and 09.60 ± 1.72/thousand cells, respectively, in both species. The results of this study suggest that these novel fish DNA damage assays can be used as an expedient toxicity screening for aquatic environments.     

A flounder (Paralichthys olivaceus) gill cell line as in vitro acute assay system of nonylphenol cytotoxicity

A cell line (FG cells) derived from a gill of the flounder, Paralichthys olivaceus were used to determine the cytotoxic effects of nonylphenol (NP). Cytotoxicity was measured by three endpoint systems: neutral red (NR) uptake assay, methyl thiazolyl tetrazolium (MTT) assay and cell protein assay. The result showed that NP was cytotoxic to FG cells at all tested concentrations, and toxicity increased as the concentration of NP was progressively increased. The 24 h-IC₅₀ values of NP were 39.81, 37.76 and 38.22 ??mol/L for NR uptake, MTT assay and cell protein assay, respectively. Moreover, the morphological changes of FG cells were also studied at the concentration of 30 ??mol/L for 24 h. Cells morphology were markedly altered by NP observed under a scanning electron microscopy, as evidenced by swelling cells, two and more nucleolus and an increased number of lipid particles. This would suggest that the FG cell line is a suitable bioindicator for the screening of the acute toxicity of NP.     

Use of two population metrics clarifies biodiversity dynamics in large-scale monitoring: the case of trees in Japanese old-growth forests

An alkaline comet assay and a micronucleus test were carried out on erythrocytes of the European chub, Squalius cephalus L., collected in spring and autumn in 2005 and 2006 at three sampling sites in River Sava, near Zagreb, Croatia. The results of comet assay showed the lowest genotoxic influence at the least polluted site, while higher DNA damage was observed at the polluted sites. Although the basal levels of DNA damage were elevated, a clear gradation of DNA damage was found due to pollution intensity in all sampling periods. The lowest cytogenetic damage as revealed by the micronucleus test (MNT) was observed as well at the least polluted site. High variations in MN frequency were observed between sampling periods, although the number of micronucleated erythrocytes was consistently the highest one at the polluted site. The comet assay as a biomarker of genotoxic effect exhibited higher sensitivity in discriminating the genotoxic capacity of studied polluted sites while the MNT was less sensitive. However, both tests should be used together in biomonitoring studies because they can reveal different aspects of DNA damage; comet assay, the early event of genotoxic exposure, and MNT, its final result as a mutagenic potential.     

Translocation of Sb and Ti in an undisturbed floodplain soil after application of Sb2O3 and TiO2 nanoparticles to the surface

The pore water transport of antimony and titanium, applied as nanoparticles (NPs), was studied by spiking stable suspensions of two different nanomaterials on the surface of an undisturbed floodplain soil. For preparation of stable dispersions, two different strategies were followed. (i) Comparable to those used in industrial applications: titanium dioxide nanoparticles, with an average diameter of 99 nm, were prepared by high-energy ball milling in water, whereas for (ii) antimony trioxide (Sb(2)O(3); average diameter 121 nm) a dispersing agent (sodium salt of poly[(naphthaleneformaldehyde)sulfonate] (pNFS) in water) was used. The upper 17 cm of a floodplain soil (river Rhine, Germany) was sampled using the minimally invasive sediment or fauna incubation experiment (SOFIE? two compartment cell; 3 l volume each), which preserved the pore system of the soil. The cells were equipped with 450 and 100 nm filter probes at different depths providing a non-invasive sampling of the pore water. The pore water was sampled at different times (T = 0, 24, 48, 96 and 196 h) and analysed by inductively coupled plasma quadrupole mass spectrometry (ICP-QMS). Sb and Ti were transported via the pore water of the floodplain soil to a depth of 14 cm, corresponding to the maximum cell depth. The highest Sb concentration in the pore water was detected after 24 h at a depth of 5.5-8 cm. Although the spiked concentration was higher for Ti than for Sb, the total Ti concentration in the pore water of the spiked cell was lower. This indicates a stronger agglomeration of TiO(2) NPs or a more intensive interaction of Ti with the solid matrix and a faster transport of Sb towards deeper soil layers. The results show that metal(loid)s from metal oxide NPs are transported in the soil pore water and, hence, have the potential to act as the source of contamination of deeper soil layers after soil surface contamination.     

饮用水消毒副产物基因毒性与致癌性研究进展

简述了饮用水消毒副产物（DBPs）的基因毒性与致癌性的研究进展。从Ames试验、SOS/umu试验、彗星试验、微核试验及一些新颖的致突变试验结果对DBPs基因的毒性,以及从毒理学实验、流行病学研究和致癌风险评估3个方面对DBPs的致癌性进行了分析和总结,以期为今后饮用水DBPs毒性效应及其致毒机理研究提供参考,进而促进饮用水质量管理与立法的发展。     

Acute and reproductive toxicity of nano-sized metal oxides (ZnO and TiO₂) to earthworms (Eisenia fetida)

An increase in nanomaterial applications will likely lead to an increased probability of environmental exposures, raising concerns regarding the safety of these materials. Recent studies have indicated that manufactured nanomaterials, such as metal oxides, have the potential to be harmful to aquatic and terrestrial organisms. The majority of nano-metal oxide research addressing potential toxicological issues has been focused in aquatic environments with very little terrestrial data. This study characterized the acute and reproductive toxicity of zinc oxide (ZnO) and titanium dioxide (TiO(2)) to earthworms (Eisenia fetida) in a terrestrial system. Following a 14 d exposure, nano-sized ZnO on filter paper was acutely toxic to E. fetida, while nano-sized TiO(2) did not exhibit acute toxicity. In contrast, neither nano-sized ZnO nor TiO(2) exhibited acute toxicity to earthworms in sand. Both nano-sized ZnO and TiO(2), following a 4 week exposure, caused reproductive effects in earthworms in artificial soil. Overall, nano-sized ZnO exhibited greater toxicity than nano-sized TiO(2) in Eisenia fetida.     

Combining passive sampling and toxicity testing for evaluation of mixtures of polar organic chemicals in sewage treatment plant effluent

Effluent from sewage treatment plants has been associated with a range of pollutant effects. Depending on the influent composition and treatment processes the effluent may contain a myriad of different chemicals which makes monitoring very complex. In this study we aimed to monitor relatively polar organic pollutant mixtures using a combination of passive sampling techniques and a set of biochemistry based assays covering acute bacterial toxicity (Microtox), phytotoxicity (Max-I-PAM assay) and genotoxicity (umuC assay). The study showed that all of the assays were able to detect effects in the samples and allowed a comparison of the two plants as well as a comparison between the two sampling periods. Distinct improvements in water quality were observed in one of the plants as result of an upgrade to a UV disinfection system, which improved from 24x sample enrichment required to induce a 50% response in the Microtox assay to 84x, from 30x sample enrichment to induce a 50% reduction in photosynthetic yield to 125x, and the genotoxicity observed in the first sampling period was eliminated. Thus we propose that biochemical assay techniques in combination with time integrated passive sampling can substantially contribute to the monitoring of polar organic toxicants in STP effluents.     

Environmental genotoxicity and cytotoxicity studies in mussels before and after an oil spill at the marine oil terminal in the Baltic Sea

Environmental genotoxicity and cytotoxicity effects in the gills of mussels Mytilus edulis, from the Baltic Sea areas close to the Būtingė oil terminal (Lithuania) before and after accidental oil spill in 31 January 2008 were studied. Mussels from the oil spillage zones were collected in 12 days, in 3 and 6 months after the spill to determine the effects of the spill. Mussels sampled in 2006–2007 were used for the assessment of the background levels of genotoxicity and cytotoxicity in the Būtingė oil terminal area. Comparison of the responses in M. edulis before and after the oil spill revealed significant elevation of frequencies of micronuclei (MN), nuclear buds (NB) and fragmented-apoptotic (FA) cells. Environmental genotoxicity and cytotoxicity levels in mussels from the Palanga site before the accident (in June 2007) served as a reference. Six months after the accident, in July 2008, 5.6-fold increase of MN, 2.9-fold elevation of NB, and 8.8-fold elevation of FA cells were observed in mussels from the same site.     

Association of textile industry effluent with mutagenicity and its toxic health implications upon acute and sub-chronic exposure

Complex industrial discharges pose certain risks to the ecosystem. This study was aimed at identifying acute and sub-chronic toxicological effects of the textile industry wastewater. The textile wastewater was evaluated for the metals and organic pollutants by atomic absorption spectrophotometer and GC-MS respectively. In vitro genotoxicity and mutagenicity were assessed by Comet assay in peripheral lymphocytes isolated from Ovis aries and Ames test in Salmonella typhimurium strains TA-100 and 102 respectively. Physiological and behavioral changes along with systemic toxicity were determined in Rattus norvegicus albinus following acute and sub-chronic exposure. High amount of heavy metals such as Cr, Pb, Hg, As, and Cd were detected in textile wastewater. Organic pollutants such as 25-deacetoxy cucurbitacin-b, E-14-Hexadecenal, 11-Tricosene, and phthalates were also found. In vitro genotoxicity assessment in lymphocytes showed statistically significant DNA damaging potential of textile wastewater. Textile wastewater also showed significantly higher (p? 0.05) mutagenic potential in Salmonella TA-100 and TA-102 strains than sodium azide and 2-amino anthracycline. Acute exposure of textile wastewater to Rattus norvegicus was associated with several physiological changes and behavioral symptoms. Sub-chronic exposure of textile wastewater in Rattus norvegicus instigated the degeneration and necrosis of epithelial cells in renal tubules, hydropic degeneration and necrosis of hepatocytes, peri-bronchiolar infiltration and emphysema of the alveoli, and the degradation of myocardial cells. This study concludes that the textile wastewater may cause genotoxicity and mutagenicity, result in physiological and behavioral changes upon acute exposure, and inflict various pathological lesions upon sub-chronic exposure.     

Effects of aqueous suspensions of titanium dioxide nanoparticles on Artemia salina: assessment of nanoparticle aggregation, accumulation, and toxicity

Aquatic stability and impact of titanium dioxide nanoparticles (TiO₂ NPs, 10–30 nm) were investigated using Artemia salina. Acute exposure was conducted on nauplii (larvae) and adults in seawater in a concentration range from 10 to 100 mg/L TiO₂ NPs for 24 and 96 h. Rapid aggregation occurred in all suspensions of TiO₂ NPs to form micrometer size particles. Yet, both nauplii and adults accumulated the aggregates significantly. Average TiO₂ content in nauplii ranged from 0.47 to 3.19 and from 1.29 to 4.43 mg/g in 24 and 96 h, respectively. Accumulation in adults was higher ranging from 2.30 to 4.19 and from 4.38 to 6.20 mg/g in 24 and 96 h, respectively. Phase contrast microscopy images revealed that Artemia were unable to excrete the particles. Thus, the TiO₂ aggregates filled inside the guts. No significant mortality or toxicity occurred within 24 h at any dose. Lipid peroxidation levels characterized with malondialdehyde concentrations were not statistically different from those of the controls (p?>?0.05). These results suggested that suspensions of the TiO₂ NPs were nontoxic to Artemia, most likely due to the formation of benign TiO₂ aggregates in water. In contrast, both mortality and lipid peroxidation increased in extended exposure to 96 h. Highest mortality occurred in 100 mg/L TiO₂ NP suspensions; 18 % for nauplii and 14 % for adults (LC₅₀?>?100 mg/L). These effects were attributed to the particle loading inside the guts leading to oxidative stress as a result of impaired food uptake for a long period of time.