Individual and binary toxicity of anatase and rutile nanoparticles towards Ceriodaphnia dubia

Accepted Manuscript Title: Individual and binary toxicity of anatase and rutile nanoparticles towards Ceriodaphnia dubia Author: V. Iswarya M. Bhuvaneshwari N. Chandrasekaran Amitava Mukherjee Dr. Senior Professor and Deputy Director PII: DOI: Reference:

S0166-445X(16)30225-9 http://dx.doi.org/doi:10.1016/j.aquatox.2016.08.007 AQTOX 4460

To appear in:

Aquatic Toxicology

Received date: Revised date: Accepted date:

9-6-2016 5-8-2016 8-8-2016

Please cite this article as: Iswarya, V., Bhuvaneshwari, M., Chandrasekaran, N., Mukherjee, Amitava, Individual and binary toxicity of anatase and rutile nanoparticles towards Ceriodaphnia dubia.Aquatic Toxicology http://dx.doi.org/10.1016/j.aquatox.2016.08.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Individual and binary toxicity of anatase and rutile nanoparticles towards Ceriodaphnia dubia

V. Iswarya, M. Bhuvaneshwari, N. Chandrasekaran, Amitava Mukherjee*

Centre for Nanobiotechnology, VIT University, Vellore, India

Corresponding Author: *Dr. Amitava Mukherjee Senior Professor & Deputy Director Centre for Nanobiotechnology VIT University, Vellore-632014 Email: [email protected];[email protected] Phone

No:

+91

416

2202620;

Fax

1   

No:

+91

416

2243092.

Highlights: 

Individual, binary toxicity of anatase and rutile NPs studied on Ceriodaphnia dubia



Anatase and rutile phases showed differential effect upon variation in irradiation



Mixture induced antagonistic at visible and additive effect at UV-A irradiation



Marking-Dawson model fitted more appropriately than Abbott model.



Agglomeration played a major role in the toxicity induced by the mixture.

 

Abstract: Increasing usage of engineered nanoparticles, especially Titanium dioxide (TiO2) in various commercial products has necessitated their toxicity evaluation and risk assessment, especially in the aquatic ecosystem. In the present study, a comprehensive toxicity assessment of anatase and rutile NPs (individual as well as a binary mixture) has been carried out in a freshwater matrix on Ceriodaphnia dubia under different irradiation conditions viz., visible and UV-A. Anatase and rutile NPs produced an LC50 of about 37.04 and 47.95 mg/L, respectively, under visible irradiation. However, lesser LC50 values of about 22.56 (anatase) and 23.76 (rutile) mg/L were noted under UV-A irradiation. A toxic unit (TU) approach was followed to determine the concentrations of binary mixtures of anatase and rutile. The binary mixture resulted in an antagonistic and additive effect under visible and UV-A irradiation, respectively. Among the two different modelling approaches used in the study, Marking-Dawson model was noted to be a more appropriate model than Abbott model for the toxicity evaluation of binary mixtures. The agglomeration of NPs played a significant role in the induction of antagonistic and additive effects by the mixture based on the irradiation applied. TEM and zeta potential analysis confirmed the surface interactions between anatase and rutile NPs in the mixture.  Maximum uptake was noticed at 0.25 total TU of the binary mixture under visible irradiation and 1 TU of anatase NPs for UV-A irradiation. Individual NPs showed highest uptake under UV-A than visible irradiation. In contrast, binary mixture showed a difference in the uptake pattern based on the type of irradiation exposed.

2   

Keywords: Agglomeration, Crystalline phases, Binary mixture, Mortality, Uptake.

3   

1. Introduction Engineered nanoparticles production and utilization are projected to reach about a trillion dollar (US) business in the upcoming future (Nel et al., 2006). In a survey conducted by Piccinno et al. (2012) on the industrial usage of NPs, titanium dioxide nanoparticles (TiO2 NPs) were noted to be the most produced NPs in Europe and worldwide with a production of about 550 and 3000 tons per year, respectively. Among the overall production, TiO2 NPs have been widely used for cosmetic purposes, including sunscreens (70–80%) than other applications such as coatings and plastics (20%), paints (10–30%), cement (1%) and other uses (10%). The total global production of TiO2 NPs was estimated to reach about 2.5 million metric tons per year around 2025, as predicted by Robichaud et al. (2009). The crystalline nature of TiO2 NPs can be used to differentiate them into three various forms namely, anatase, rutile, and brookite. Among these three forms, anatase and rutile (tetragonal in structure) were signified as the most active TiO2 forms than the brookite, an orthorhombic form. Thus, anatase and rutile forms have been widely used for both industrial and commercial purposes (Cho et al., 2013). Based on the type of application and function it serves, anatase or rutile NPs were selected for a particular purpose. Due to the higher occurrence in nature, rutile NPs have been utilized as a white pigment in paints, plastics, food products (Winkler, 2003; Weir et al., 2012), as UV absorbers in sunscreens and cosmetics (Mueller and Nowack, 2008). While, anatase NPs have been used in various environmental oriented applications such as semiconductor catalysts in water treatment (Velhal et al., 2012; Lazar et al., 2012), and reactors in the photodegradation of wastes and pollutants in industries (Montazer and Seifollahzadeh, 2011; Mahlambi et al., 2015). It was also frequently used in self-cleaning products, solar cells, light emitting diodes, sunscreens (EPA, 2009), disinfectants (Kuhn et al., 2003), and in cancer treatment (Fujishima et al., 2000; Fujiwara et al., 2015). Photocatalytic property (reactive oxygen species generation) of anatase and higher refractive index of rutile NPs make their usage inimitable. Massive handling of TiO2 NPs led to their inadvertent discharge into the environment, especially the aquatic bodies (Kaegi et al., 2008). Therefore, risk assessment of TiO2 NPs towards various aquatic organisms becomes indispensable. Freshwater crustaceans like daphnids, in particular, play a significant role in the maintenance of aquatic system since it serves as a vital food source 4   

in the aquatic food chain (Lampert and Sommer, 2007). Due to the ease of availability of daphnids, they have been widely used in ecotoxicological studies of nanoparticles. Literature available on the toxicity on crustaceans of TiO2 NPs concerning their crystalline form has not been well studied so far. Although an enormous toxicity experiments were carried out on daphnids for the anatase (Dabrunz et al., 2011; Hund–Rinke and Simon, 2006) and P25 (Jascobasch et al., 2014; Li et al., 2011) NPs, the data remains inconclusive. On the other hand, only a handful of information is present for rutile NPs (Bang et al., 2011; Marcone et al., 2012). In our previous toxicity study towards Chlorella sp. due to anatase and rutile NPs, algal cells followed a different cellular/mechanistic pathway (anatase-nucleus specific; rutile-organelle specific) due to the variance in crystallinity in response to NPs (Iswarya et al., 2015). Campos et al. (2013) analyzed the effect of various forms of TiO2 NPs on microalgae, Chlorella vulgaris, and their further consequences on Daphnia magna. They observed that the aggregation of TiO2 NPs with the microalgae led to the depletion of food available to the daphnia and in turn induced impairment in their growth and reproduction. In addition to the variation in crystalline structure, the type of irradiation i.e. whether UV (A, B, C) or visible light was used also influences the toxicity of TiO2 NPs. Amiano et al. (2012) and Clemente et al. (2014) revealed the importance of irradiation while testing the toxicity of TiO2 NPs due to variation in the photocatalytic activity upon irradiation. Photocatalytic differences, in turn, have an impact upon their toxicity under different irradiation condition. Hence, exploring the differences in toxicity of TiO2 NPs in relation with crystallinity and irradiation becomes pertinent. It is very natural for the aquatic system to get consistently contaminated with a mixture of nanoparticles from various anthropogenic sources rather than individual NP types. Most of the ecotoxicological studies conducted so far have addressed the impact of individual NPs alone upon various aquatic organisms, which contradicts the actual environmental scenario. Jak et al. (1998) emphasized the necessity to evaluate the difference in toxicity of individual NPs with that of a NP mixture. Toxicity of the mixture may/may not differ from that of the individuals. Recent studies on NP toxicity pointed out the emergence of works that assess the NP toxicity studies for a NP mixture (Tong et al., 2015; Li et al., 2015). Yu et al. (2016) examined the dual effect of CeO2/TiO2 NPs and CeO2/ZnO NPs on Nitrosomonas europaea bacteria. They had noticed 5   

antagonistic and synergistic cytotoxic effects on exposure to the binary combinations of TiO2 and ZnO NPs, respectively, when co-exposed with CeO2 NPs. In some cases, both additive and antagonistic effects were observed at certain combinations/concentrations of a binary mixture of anatase and rutile NPs towards the freshwater algae, Chlorella sp. (Iswarya et al., 2015). Hai– Zhou et al. (2012) evaluated the individual and binary toxic effect of CuO and ZnO NPs towards Daphnia magna. A binary mixture of NPs showed higher mortality than individual NPs, which indicated the higher toxicity of binary NPs. The foremost objective of the present study was to investigate the toxic response of a freshwater crustacean, Ceriodaphnia dubia, upon exposure to different crystalline phases of TiO2 NPs, such as anatase and rutile NPs, both individual as well as a mixture in a freshwater matrix. A toxic unit (TU) approach was employed to determine the concentrations of binary mixtures (detailed in section 2.6), which have been extensively used in most of the ecotoxicological studies of chemical mixtures (Cao et al., 2007; Naddy et al., 2015; Hu et al., 2014). Since TiO2 NPs are photocatalytic in nature, the impact of irradiation on their toxicity was evaluated by exposing the individual as well as binary NPs under two different, environmentally relevant irradiation conditions such as UV-A and visible light. Furthermore, two modeling approaches such as Marking–Dawson and Abbott’s modeling have been followed to differentiate the mode of the binary mixture into antagonistic, additive or synergistic (detailed in section 2.7). Stability analysis (Dynamic Light Scattering, DLS) evaluated the aggregation potential of NPs in sterile lake water. Besides, interactions between anatase and rutile NPs in the mixture have been assessed with various techniques (zeta potential and TEM). NP uptake by Daphnia has been evaluated to examine their potential risk in the aquatic system. 2. Materials and Methods: 2.1 TiO2 nanoparticles and preparation Two different crystalline phases of TiO2 nanoparticles viz., anatase with a reported size of <25 nm (CAS No: 1317-70-0, 99.7% trace metal basis) and rutile with a reported size of <100 nm (~10 nm Diam. × 40 nm L, CAS No: 1317-80-2, 99.5% trace metals basis) were procured from Sigma-Aldrich, Missouri, USA in powder form.

6   

Stock suspensions (200 mg/L, i.e. 2.504 mM) of both anatase and rutile NPs were prepared in deionized water. Then, they were sonicated for about 15 min with the help of an ultrasonicator (130 W, 20 kHz, Sonics, USA) to ensure uniform dispersions of nanoparticles, which were utilized for further experiments. 2.2 Preliminary characterization of pristine nanoparticles Electron microscopic analysis was performed to determine the primary size and shape of anatase and rutile nanoparticles using high-resolution transmission electron microscope (HRTEM, FEI TecnaiG2 T20 S-Twin). The average size of individual nanoparticles was calculated by measuring about 100 nanoparticles randomly from the TEM images, which was analyzed with the help of an imaging software, ImageJ. The initial hydrodynamic size of TiO2 nanoparticles (anatase and rutile) was analyzed in deionized water by dynamic light scattering (DLS) analysis method with the help of 90 Plus Particle Size Analyzer (Brookhaven Instruments Corp., USA). 2.3 Isolation and identification of Ceriodaphnia dubia Freshwater crustaceans were isolated from the VIT Lake (situated on the VIT University premises), Vellore, Tamil Nadu, India, by following the protocol described by Pakrashi et al. (2013). Freshwater collected from the VIT Lake was initially checked for the presence of daphnids. Daphnids, which were predominant in the lake water were separated using a Pasteur pipette (5 mL) and added to the beakers containing sterile lake water (prepared as described in section 2.4). Then, they were regularly subcultured under specific conditions to confirm their species uniformity. Isolated daphnids were further evaluated under a phase contrast microscope (100X, Zeiss Axiostar, USA) and identified as Ceriodaphnia dubia by morphological identification. Daphnia cultures were well maintained in the sterile lake water at 26±2C under specific photoperiod conditions (16h light:8h dark) using an ambient fluorescence light (TL-D Super 80 linear fluorescent tubes, Philips LifeMax, 18W, intensity of 1250 lux) as a light source. Freshwater algae, Chlorella sp., were given as a feed to the daphnids twice a week.

7   

2.4 Experimental setup Freshwater was collected from the VIT Lake situated in VIT University, Vellore, India and filtered with Whatman No. 1 filter paper to remove the debris and suspended particles. Then, the filtered lake water was sterilized and used for the toxicity experiments (Pakrashi et al., 2011). In this toxicity study, freshwater was chosen as a test matrix in order to stimulate the lake water environment in the toxicity assessments. The physicochemical parameters of the sterile lake water were examined and found to be: temperature: 24±1.4C, pH: 7.2±0.4, conductance: 2.45±0.23 mS/cm, total dissolved solids: 1730±100 mg/L, and total organic carbon (TOC):14.6±0.5 mg/L. Metal ions in the lake water were quantified by ICP-OES (Inductively coupled plasma-optical emission spectrometry, PerkinElmerOptima 5300 DV, USA). The water was noted to contain trace amounts of few metal ions such as, Al3+ : 0.247 mg/L, Cr6+ : 0.002 mg/L, Fe : 0.048 mg/L, Mn2+ : 0.011 mg/L, Ni : 0.004 mg/L, and Ti : 0.006 mg/L along with some inorganic ions. Acute toxicity tests of nanoparticles were carried out on Ceriodaphnia dubia by following the standard protocol of OECD 202 (OECD, 2004). Toxicity of anatase and rutile NPs were tested under different irradiation conditions such as visible and UV-A light using a fluorescence lamp (TL-D Super 80 linear fluorescent tubes, Philips, 18W, intensity of 1250 lux, 0.18mW/cm2) and UV-A lamp (Wavelength of 365 nm, Philips, 18W, intensity of 0.23 mW/cm2) as the light sources, respectively. The volume of the experimental matrix used for the toxicity study was 20 mL, such that the height of the water column was about 1.5 cm in a 100-mL beaker. The distance of the lamp was about 36 cm from the sample to be irradiated. Visible and UV-A lights were used to determine the influence of irradiation type on the toxicity of TiO2 NPs, as TiO2 NPs were photocatalytic in nature. Photoperiodic conditions (16:8 h, light: dark) were maintained for all the toxicity experiments. Neonates were not fed during the test period of about 48 h. 2.5 Lethal concentration evaluation Prior to the toxicity study of binary mixture of NPs, lethal concentration of individual NPs was analyzed in order to follow the TU method. Lethal concentration (LC) of anatase and rutile NPs were determined by exposing the less than 24-h old neonates to various concentrations of NPs (0, 2, 4, 8, 16, 32, 64 and 128 mg/L; i.e. 0, 25.04, 50.08, 100.16, 200.32, 400.64, 801.28 μM) for 8   

about 48 h under visible and UV-A irradiations. Ten healthy neonates were added to 20mL of the test solution, i.e., sterile lake water containing TiO2 NPs. After the exposure period of 48 h, daphnids were checked for their mobility after a gentle agitation for about 5 s. The immobile daphnids were considered as dead. Lethal concentrations (LC10, LC50, and LC90) of anatase and rutile NPs were calculated using a software, EPA probit analysis, version 1.5 (USEPA, Cincinnati, OH, USA). 2.6 Toxicity of anatase and rutile phases as individual NPs and binary mixture Based on the LC50 values observed, the toxicity of individual (anatase and rutile) NPs, as well as their binary mixture, were assessed at various toxic units (TU) like 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, and 1.75 TUs. The toxic unit approach proposed by Sprague and Ramsay (1965) has been widely used in ecotoxicological studies to determine the effect of different toxicants in a mixture (Ferreira et al., 2008; Naddy et al., 2015; Hu et al., 2014). Toxic units for individual NPs and mixture were calculated using the Eq. (1) and (2), respectively. TU = Concentration of NPs/LC50 of NPs TU = TU = TUA+ TUR

(For single NPs)

(1)

(For mixture of NPs) (2)

Where, TU is the total toxic unit, TUA and TUR were the toxic units of individual anatase and rutile NPs, respectively. For the binary mixture, toxic units were calculated at their equitoxic proportions (Cao et al., 2007). For example, one total TU contains 0.5 TU of anatase NPs and 0.5 TU of rutile NPs in the mixture. The concentrations of anatase and rutile NPs used in the toxicity assessment of both individual NPs and binary mixture were calculated for their respective toxic units. As the LC50 values varied under different irradiation conditions, the concentration of anatase and rutile NPs for toxic units was found to be varied. Thus, the concentration of anatase and rutile NPs (both individual and mixture) corresponding to their respective TUs were calculated for both visible and UV-A irradiation and represented in Table 1 and 2, respectively. According to the concentration of each NPs measured in the mixture, their actual TUs were recalculated and added together to obtain the total mixture TUs, which was to be considered.

9   

Ten healthy neonates of less than 24-h old were exposed to the test solution, i.e., sterile lake water, containing TiO2 NPs (anatase, rutile, and binary mixture) at various TUs under different irradiation conditions, such as visible and UV-A irradiation. Then, samples were maintained under photoperiodic conditions for about 48 h at 26±2C. After the exposure period of 48 h, mortality of daphnids was determined according to the acute immobilization test (OECD 202, 2004). All the toxicity experiments were repeated at least five times (n=5) to ensure the reproducibility of the results. 2.7 Antagonistic–synergistic modeling for binary mixture Statistical modellings, such as Marking–Dawson (1975) and Abbott model (1925), have been employed to characterize the toxic effects of anatase and rutile NPs in a mixture as antagonistic, synergistic, and/or additive. Two-way ANOVA was followed for both models to calculate the statistical differences at p<0.01 with the help of graph pad prism, v 5.0 (GraphPad Software, Inc., San Diego, California, USA). 2.7.1 Marking–Dawson model The effect of concentration on a mixture was predicted by the concentration addition model as proposed by Marking and Dawson (1975). In Marking–Dawson model, the sum of the biological effects (S, LC50 values) produced by the individual NPs in a mixture were calculated using the formula (3), S = Am/Ai + Rm/Ri

(3)

Where, Am and Rm represent the LC50 values of anatase and rutile NPs in the binary mixture, respectively. These LC50 values were the concentration of anatase and rutile NPs present in a particular toxic unit of the mixture which produced 50% mortality. Using the toxic unit formula (Eq. 1 and 2), the concentrations of anatase and rutile NPs were calculated from the specified toxic unit of the mixture. Ai indicates the LC50 of individual anatase and Ri for individual rutile NPs. From the calculated S value, additivity Index (AI) has been derived using the Eq. (4) and (5), AI = 1/S -1 (S≤1)

(4)

10   

AI = S(-1) + 1 (S≥1)

(5)

If the AI value is negative, then the toxic action is said to be antagonistic. However, the action is observed to be synergistic, when the AI value is positive, and it can be said to be simply additive, when AI is equal to zero. In case, AI is not statistically significant from zero; it is also said to be additive. 2.7.2 Abbott’s modeling Independent action of anatase and rutile NPs in a binary mixture were predicted using a model proposed by Abbott (Chesworth et al. 2004; Teisseire et al. 1999). Using Abbott’s model, the expected toxicity (Cexp) of the binary mixture was computed from the Eq. (6) using the mortality (%) induced by individual nanoparticles (anatase and rutile NPs). Expected toxicity is the percentage mortality predicted from the mortality (%) observed for the individual NPs (anatase and rutile NPs). Cexp = A+B(AB/100)

(6)

Where, A and B indicate the toxicity, i.e., mortality (%) caused by individual anatase and rutile NPs, respectively. Then, the ratio of inhibition (RI) was computed using the Eq. (7) to compare the expected toxicity with the observed toxicity for a binary mixture. RI = observed toxicity/expected toxicity (Cexp)

(7)

Here, the observed toxicity represents the percentage mortality observed for binary mixtures. Comparing RI value with 1, the interactive effect between anatase and rutile NPs in a mixture can be categorized into antagonistic, synergistic, and/or additive. The interactive effect of the binary mixture was found to be antagonistic, when the RI value is less than 1(<1); whereas, it is said to be synergistic when RI>1, and additive, when RI= 1. The interactive effect from additivity computed from the RI was considered to be statistically significant only when the mean RI value was significant from 1 ± SD.

11   

2.8 Physiochemical characterization of NPs in the experimental matrix 2.8.1 Stability of NPs in the test matrix Colloidal stability of anatase and rutile NPs both individually and in the mixture was determined by evaluating their hydrodynamic size in the sterile lake water at various time intervals. The hydrodynamic size of TiO2 nanoparticles (individually as well as a mixture) in the sterile lake water was analyzed under both visible and UV-A irradiation after 0 and 4 h using particle size analyzer (90 Plus, Brookhaven Instruments, USA). The TUs employed in the hydrodynamic size analysis were 0.25, 1, and 1.75 TU. As the size of nanoparticles reached micron size after 4 h, the experiment was discontinued due to the limitations of the particle size analyzer, which is inappropriate to measure beyond the micron size range of 6µm. 2.8.2 Sedimentation Assay The sedimentation rates of TiO2 NPs (individual and mixture) at different TUs such as 0.25, 1, and 1.75 TU were evaluated at various time intervals using a UV–vis spectrophotometer (Allouni et al., 2009). To 30 mL of the sterile lake water, TiO2 NPs were added and kept under visible and UV-A irradiation similar to the other experimental conditions. The concentration of TiO2 NPs in the undisturbed upper layer was analyzed at various time intervals such as 0, 4, 24, and 48 h. Anatase and rutile NPs showed a characteristic UV peak at 314 and 294 nm, respectively. The concentration of TiO2 NPs was analyzed at specific wavelengths using a UV–vis spectrophotometer (Model U2910, HITACHI, Japan). 2.8.3 Surface interaction studies between anatase and rutile NPs in a mixture Any changes in morphology of NPs due to the interaction between anatase and rutile NPs in a mixture were analyzed using electron microscopy. A binary mixture of 1.75 total TU (both visible and UV-A) containing anatase and rutile NPs was added to the sterile lake water. Then a drop of the binary mixture was coated on to a copper grid and analyzed by HRTEM (FEI Technai G2 T20 S-Twin TEM).

12   

The surface interaction between the TiO2 NPs (anatase, rutile, and binary mixture) and experimental matrix was evaluated by measuring the change in their zeta potential during the exposure period of 48 h. Anatase, rutile, and the binary mixture of NPs were interacted with the sterile lake water at a concentration of 1.75 total TU for about 48 h under both visible and UV-A irradiation. During the interaction, their zeta potential was measured at different time intervals such as 0 and 48 h with the help of a zeta analyzer (NanoBrook 90Plus PALS Particle Size Analyzer, Brookhaven Instruments, USA). 2.9 Biouptake studies NP uptake by daphnia was initially verified with the help of phase contrast microscopy. After the 48-h exposure to different forms of TiO2 NPs viz., anatase, rutile, and binary mixture (1.75 TU), neonates were removed from the solution and gently placed on the microscopic slide. Then, they were observed under a phase contrast microscope (DM2500, Leica, Germany) at 100X magnification. Surplus care has been taken such that the neonates did not get dried during the time of microscopic observation. NP biouptake was further confirmed by analyzing Ti quantitatively using an atomic absorption spectrometer (AAS). Uptake of TiO2NPs (anatase, rutile, and binary mixture) by the daphnids under different exposure conditions was checked at various toxic units (TU) such as 0.25, 1, and 1.75 TU. After the exposure period of 48 h under visible and UV-A irradiation, organisms were collected and washed with de-ionized water thrice to remove the loosely bound nanoparticles. After washing, the daphnids were dried at 60C in a hot-air oven. Their dry weight was measured and noted. Then, they were acid digested with 2 mL of concentrated nitric acid and diluted with de-ionized water. Acid-digested samples were analyzed for Ti content using a graphite-furnace AAS (AAanalyst400, PerkinElmer) at a specific lamp wavelength of 364.27 nm. The limit of detection for Ti is 45 pg/L. The Ti taken up by the organisms were calculated based on their dry weight and represented in mg/Kg. Then, the relative uptake of Ti was calculated and expressed in L/Kg. The relative uptake was determined from the ratio of the concentration of Ti in test organism (mg/Kg) to the concentration of Ti in TiO2 NPs added to the experimental matrix (mg/L). 2.10 Statistical analysis 13   

Two-way ANOVA (p<0.01) was employed to calculate the statistical differences among the various TiO2 NPs (anatase, rutile, and binary mixture) and different irradiation (visible and UVA) conditions. In addition, one-way ANOVA was used to find out the difference between the control and NP-treated daphnids at the statistical level of significance, p<0.05. The statistical software, graph pad prism, v 5.0 was used to calculate ANOVA. 3. Results 3.1 Preliminary characterization of pristine nanoparticles Transmission electron micrographs (Fig. 1) revealed that anatase and rutile NPs were spherical and rod-like in shape, respectively. Anatase NPs (Fig. 1A) appeared to contain smaller agglomerates, in which, individual NPs were present in the size range of 5 to 12 nm with an average size of 9.5±1 nm. The average size of rod-shaped rutile nanoparticles (Fig. 1B) was around 26±3 nm in length and 4±0.5 nm in breadth. The initial hydrodynamic sizes of anatase and rutile NPs in de-ionized water were observed to be 307.07±37.9 and 218.92±1.92 nm, respectively. A polydispersity index of about 0.14 and 0.25 was noted for anatase and rutile NPs, respectively, indicating that the NPs were monodispersed and highly stable in de-ionized water. 3.2 Lethal concentration evaluation The lethal concentrations (LC10, LC50, and LC90) of anatase and rutile NPs towards C. dubia under visible and UV-A irradiation were calculated and represented in Fig 2. Anatase NPs showed LC50 values of about 37.041.93 and 22.560.54 mg/L under visible and UV-A irradiation, respectively. However, rutile NPs produced an LC50 effect at the concentrations of 483.64 mg/L under visible light and 23.760.57 mg/L under UV-A irradiation. Significant differences were observed (p<0.01) between the lethal concentrations of anatase and rutile NPs only under visible irradiation, but not with UV-A irradiation. Both anatase and rutile NPs showed a significantly lesser LC50 value (p<0.01) under UV-A irradiation than for visible conditions. 3.3 Toxicity of anatase and rutile phases as individual NPs and binary mixture.

14   

Toxicity of anatase and rutile NPs as individual NPs and binary mixture towards C. dubia were tested at various toxic units such as 025, 0.5, 0.75, 1, 1.25, 1.5, and 1.75 TU. The concentrations of NPs corresponding to the total TUs have been portrayed in Table 1 and Table 2. Mortality induced by anatase and rutile NPs (both individual and binary mixture) under visible and UV-A irradiation are represented in Fig 3A and 3B, respectively. Upon different irradiation (visible and UV-A) conditions, a toxic unit-dependent increase in the mortality was observed for both individual (anatase and rutile) and binary mixture-treated organisms. The toxicity was statistically significant (p<0.05) as compared to the control under both irradiation conditions at all the TUs tested, except at 0.25 TU only under visible irradiation conditions. Anatase NPs exhibited mortality of about 72±3.74% and 80±4.47% under visible and UV-A irradiation, respectively at 1.75 TU. While rutile NPs induced a mortality of 86±2.45% under visible and 80±4.47% upon UV-A irradiation at 1.75 TU. At all the toxic units, mortality induced by anatase NPs didnot show any significant differences (p>0.01) from the rutile NP-induced mortality under both visible and UV-A conditions, excluding 1.25 and 1.75 TU at visible irradiation. At 1.75 total TU, binary mixture showed 64±2.45% and 66±2.45% mortality under visible and UV-A irradiation, respectively. A remarkable reduction in the toxicity was noted for the binary mixture rather than individual NPs under visible irradiation. However, significant differences (p<0.05) were noted only at 1.5 total TU while comparing with anatase NPs and at 1.25 to 1.75 total TU in comparison with rutile NPs. Upon UV-A irradiation, the binary mixture did not induce any significant (p>0.05) change in the mortality as compared to individual NPs at all the toxic units tested, except 1.75 total TU. As there is no significant variation in the LC50 of individual NPs upon UV-A irradiation, the binary mixture didnot induce an antagonistic effect as observed in the visible irradiation. Comparing the toxic effects of TiO2 NPs under visible and UV-A irradiation, individual NPs (both anatase and rutile NPs) did not induce any significant variation in their mortality (p>0.01), except with 0.5 TU of rutile NPs. However, significant differences (p<0.01) were observed among the binary mixtures at all the TUs used, except 1 TU. 3.4 Antagonistic–synergistic modeling for binary mixture 15   

To implement the Marking–Dawson model, LC50 of the binary mixture was calculated for both visible and UV-A irradiation. Under visible irradiation, binary mixture exerted an LC50 (S) value of about 1.4±0.05 total TU, i.e., 25.93±1.01 mg/L of anatase NPs (0.7±0.03 TU) and 33.6±1.31 mg/L of rutile NPs (0.7±0.03 TU). Further, the additive index (AI) value was calculated from the obtained S value using Eq. 5. AI value was noted to be – 0.4±0.05, i.e., less than additivity indicating the antagonistic effect between anatase and rutile NPs when they are present together as a mixture under visible irradiation. On the other hand, an LC50 (S) value of about 0.99±0.12 total TU, i.e., 11.21±1.36 mg/L of anatase NPs (0.5±0.06 TU) and 11.92±1.43 mg/L of rutile NPs (0.5±0.06 TU) were noted for the binary mixture upon treatment with UV-A irradiation. An AI value of about 0.01±0.12 was obtained upon treatment with UV-A irradiation indicating the additive effect of anatase and rutile NPs in a binary mixture. Abbott’s modeling was also performed to determine the type of interaction between anatase and rutile NPs in a mixture and details are provided in Table 3. The ratio of inhibition (RI) was calculated for the binary mixture and observed to be in the range of 0.9 – 1.3 (visible) and 0.8 – 1.2 (UV-A). These RI values were found to be statistically insignificant from 1, and thus, was said to demonstrate an additive effect. Both Marking–Dawson and Abbott’s models revealed that anatase and rutile NPs exert an additive effect as a binary mixture under UV-A irradiation. At the same time, the results from the two models did not corroborate with each other in the case of visible irradiation and provided a different result. 3.5 Physiochemical characterization of NPs in the experimental matrix 3.5.1

Stability of the NPs in the test matrix

Colloidal stability of anatase, rutile, and binary mixture in the sterile lake water was analyzed by measuring their hydrodynamic size at different time intervals (0 and 4 h). The hydrodynamic size of TiO2 NPs (anatase, rutile, and mixture) in the sterile lake water was analyzed at 0.25, 1, and 1.75 TUs and expressed as an effective diameter (Fig. 4). The effective diameter of TiO2 NPs (both individual as well as a binary mixture) has significantly (p<0.01) increased from 0 h to 4 h at all the concentrations (0.25, 1, and 1.75 TU) tested in the study, under both visible and UV-A irradiation. 16   

Under visible irradiation, the size of anatase NPs at 0 h in the sterile lake water was found to be 550.78±44.3, 723.83±32.62, and 839.06±49.77 nm for 0.25, 1, and 1.75 TU, respectively. After 4 h of interaction under visible irradiation, the size of anatase NPs was further increased to 939.33±67.22 (0.25 TU), 2004.98±203.06 (1 TU) and 2357.29±98.24 (1.75 TU). Similarly, the effective diameters of anatase NPs were found to be increased under UV-A irradiation from 544.93±4.82 (0 h) to 1069.84±92.14 (4 h) for 0.25 TU, 666.16±5.72 (0 h) to 2133.55±78.02 (4 h) for 1 TU, and 783.77±37.98 (0 h) to 2179.33±230.91 (4 h) nm for 1.75 TU. Significant differences in the size of anatase NPs (0 and 4 h) were not observed (p>0.01) while comparing visible and UV-A irradiation. Rutile NPs attained a size of about 405.922±1.06, 741.88±36.99, and 1197.17±114.84 in the sterile lake water at 0 h under visible irradiation, for 0.25, 1, and 1.75 TU, respectively. It was later increased to 1417.62±124.6 (0.25 TU), 4640.10±346.63 (1 TU), and 5714.70±415.36 nm (1.75 TU) after 4-h irradiation with visible light. Likewise, rutile NPs showed an increase in the effective diameter of 369.54±120.69, 535.28±11.76, and 982.47±79.98 nm (0 h) to 1015.08±45.36, 3975.04±219.31, and 6111.59±283.21 nm (4 h) for 0.25, 1, and 1.75 TU, respectively, under UV-A irradiation. Comparing visible and UV-A irradiation, only 0.25 and 1 TU showed a significant difference (p<0.01) between their sizes in the sterile lake water. Binary mixture showed an effective diameter of about 495.09±2.32 for 0.25 TU, 788.465±103.92 for 1 TU, 1014.31±74.03 nm for 1.75 TU at 0 h under visible irradiation. Then, the size of the binary mixture was rapidly increased to 933.95±30.8, 3568.31±104.66, and 4430.25±81.63 nm for 0.25, 1, and 1.75 TU, respectively, after 4-h exposure to visible irradiation. Similarly, under UV-A irradiation, the effective diameter of the binary mixture significantly increased from 499.24±32.53, 720.19±33.56, and 842.58±75.33 nm to 1280.45±88.74, 3628.80±12.12 and 4078.24±265.08 nm (0 – 4 h) for 0.25, 1, and 1.75 TU, respectively. While comparing the size of mixture noted at 0 and 4 h, significant differences were not observed (p>0.01) under visible and UV-A irradiation at all the TUs studied (excluding 0.25 TU). Among the individual NPs, i.e., different phases of TiO2 NPs (anatase and rutile), the effective diameter of the rutile NPs was rapidly increased than for anatase NPs at 4 h. These size differences were found to be statistically significant (p<0.01) at all the concentrations under both visible and UV-A (except 0.25 TU) irradiation. Comparing the binary mixture (anatase+rutile) 17   

with anatase NPs, the size of NPs in the mixture was quite high at 4 h, in comparison with anatase NPs and was statistically significant (p<0.01) at 0.25, 1, and 1.75 TUs under all the irradiation conditions except at 0.25 TU (UV-A). In contrast, the size of rutile NPs at 4 h was rapidly increased in the sterile lake water when compared to the size increment for mixture under both visible (0.25, 1, and 1.75 TU) and UV-A (only at 1.75 TU) systems. Therefore, the rutile NPs were unstable in the sterile lake water at highest TUs rather than anatase NPs and binary mixture. The polydispersity index (PDI) of TiO2 NPs (individual NPs and binary mixture) is represented in Table S1. PDI values were noted to be within the range of 0.2 to 0.5 for all the forms of TiO2 NPs, excluding 1.75 TU of rutile NPs (PDI: 0.71) after 4 h of exposure period under both irradiation conditions. The high PDI indicated the unstable, polydispersed nature of NPs due to the agglomeration of NPs in the test matrix used. 3.5.2

Sedimentation assay

Sedimentation profiles of anatase and rutile NPs in the sterile lake water are represented in Fig 5. As time progressed, the absorbance ratios (A/A0) of both anatase and rutile NPs were found to decrease rapidly under the tested conditions (visible and UV-A irradiation). A toxic unit (TU)dependent increase in the sedimentation of TiO2 NPs (anatase and rutile NPs) was observed under both visible and UV-A irradiation. Under visible irradiation, the absorbance ratios of 0.25 TU of anatase and rutile NPs were found in the range of 0.8 to 0.7 till 24 h. After 24 h, a significant sharp decrease in the absorbance ratio was noted due to the lesser availability of NPs in the experimental matrix used. Thus, TiO2 NPs at 0.25 TU were relatively stable till 24 h under visible irradiation. These results were well corroborated with the DLS findings. In contrast, the absorbance ratios of anatase and rutile NPs (0.25 TU) in the lake water were decreased from 1 to 0.57 and 0.56 at 4 h, which further declined to 0.21 and 0.16 at 24 h, respectively, under UV-A irradiation. Rutile NPs further showed a continuous decrease in the A/A0 contradicting with the results for anatase NPs, which remained same after 24 h. At higher toxic units i.e. 1 and 1.75 TUs, A/A0 of anatase and rutile NPs in the sterile lake water was reduced significantly till 24 h, irrespective of the irradiation conditions tested. Even though a 18   

decrease in the absorbance was noted after 24 h, the differences were not significant at p<0.05. These results indicated that the sedimentation of TiO2 NPs was faster at higher toxic units, irrespective of their crystalline form. Lower toxic units (0.25 TU) impact the sedimentation rate of NPs based on their crystalline nature and irradiation condition. Sedimentation rate of TiO2 NPs (both anatase and rutile NPs) were significantly higher under UV-A irradiation than visible at all the toxic units (0.25, 1, and 1.75 TU) of anatase NPs and only at 0.25 TU of rutile NPs. It clearly discloses that UV-A irradiation significantly influenced the sedimentation rate of NPs. 3.5.3. Anatase – rutile interactions The surface interaction between anatase and rutile NPs in the sterile lake water was determined by analyzing the changes in the morphology (TEM) and surface charge of NPs with the help of transmission electron microscopy and zeta potential analysis, respectively. 3.5.3.1 Transmission electron microscopy TEM images of the binary mixture under visible (Fig 6A, B, and C) and UV-A (Fig 6D, E, and F) irradiation revealed that rutile NPs were capped over the anatase NPs due to the interparticle interaction between them. Natural colloids present in the sterile lake water amplified the interaction among them. As noted in Figures 6C and F, spherical-shaped anatase NPs was enclosed by the rutile NPs. Thus, the surface interaction between anatase and rutile NPs in a mixture was confirmed, revealing the electron transfer among the phases. 3.5.3.2 Zeta potential analysis Zeta potential of anatase, rutile, and binary mixture (1.75 TU) in the sterile lake water was analyzed and represented in Fig 7. All the forms of TiO2 NPs imparted a positive charge in the lake water at 0 h. Irrespective of irradiation, a significant decrease in the zeta potential was observed at 48 h for both anatase and rutile NPs, without any change in the charge of NPs. In contrast, the zeta potential of the binary mixture decreased rapidly within 0 to 48 h from 28.89±0.41 to -24.09±2 mV under visible irradiation and 10.47±0.16 to -27.98±1.21mV under UV-A irradiation. The decline in the zeta potential was statistically significant at p<0.01. A transition in the zeta potential from positive to negative charge for the binary mixture confirmed 19   

the interparticle interactions between the anatase and rutile NPs in the sterile lake water when they were present together as a mixture. Within the individual NPs, the zeta potential of anatase NPs was decreased rapidly (p<0.01) than that of rutile NPs under both visible and UV-A irradiation. 3.6 Biouptake studies Microscopic analysis was performed to analyze any noticeable changes in the C. dubia after exposure to TiO2 NPs (individually and mixture) at different irradiations. A proper intestinal gut was noticed for the control (NP untreated) organisms as in Fig S1Aand S2A. Under visible irradiation, TiO2 NP uptake and their bioaccumulation were clearly observed as a black shade in the intestinal tract of the daphnids (Fig S1B–D), regardless of their form. Significant visible changes were not observed among all the types of NPs tested i.e. anatase, rutile, and binary mixture other than accumulation under visible irradiation. Accumulation resulted only when the nanoparticles were ingested by the neonates exposed to it. These microscopic images confirmed that NP ingestion by neonates was the primary cause for their corresponding toxicity. In contrast, differential action of individual NPs and mixture were noticed under UV-A irradiation. Accumulation of NPs in the intestinal tract, pigmentation at the carapace, and attachment of NP aggregates at the appendages were seen in anatase-treated daphnids (Fig S2B). While for rutile-treated daphnids, NP aggregates have adhered to the antennae of daphnids apart from the NP accumulation in the tract (Fig S2C). All the observations such as accumulation, NP adherence, and pigmentation were observed in the binary mixture-treated neonates (Fig S2 D and E). These findings supported the additive effect of the binary mixture as discerned from the mortality data. A slight crystallinity based difference was noted as a visible change on the microscope for UV-A irradiated samples. Further, uptake of TiO2 NPs (individual as well as a binary mixture) by C. dubia sp. was quantitatively measured at different TUs (025, 1, and 1.75 TU) and represented in Fig 10. At 1.75 TU, the relative Ti uptake by C. dubia was found to be 8563.74±313.93, 28237.52±655.38, and 4955.90±551.75 L/Kg for anatase, rutile, and mixture NPs, respectively, under visible irradiation. While for UV-A irradiation, the relative Ti uptake was noted to be 48905.31±788.69 L/Kg (anatase NPs), 77567.36±3147.10 L/Kg (rutile NPs), and 24026.54±369.21 L/Kg (binary 20   

mixture) at 1.75 TU. Maximum uptake was observed at 0.25 total TU of the binary mixture under visible irradiation and 1 TU of anatase NPs under UV-A irradiation. A toxic unit-dependent increase in the uptake was observed for the rutile NPs under both visible and UV-A irradiation. The differences were ascertained to be statistically significant except 1.75 TU at visible irradiation. Under visible irradiation, the relative uptake was found to decrease for binary mixtures as the toxic units were increased. They were statistically significant (p<0.01). In the case of anatase NPs (UV-A) and binary mixture (UV-A), the uptake was found to significantly (p<0.01) increase till 1TU, after which a rapid decrease was observed at 1.75 TU. Significant differences in the uptake were not noticed for the anatase NPs under visible irradiation (p>0.01). Comparing the differences in the uptake of individual NPs under visible and UV-A irradiation, the highest uptake of TiO2 NPs was observed under UV-A rather than visible irradiation. They were statistically different (p<0.01) at all the concentrations (0.25, 1, and 1.75 TU) except 0.25 TU of anatase NPs. In contrast, uptake was found to be dependent on the toxic unit of the binary mixture under visible irradiation condition. This monotonic response was not observed for the binary mixtures exposed to UV-A irradiation. The differences were statistically significant (p<0.01) for 0.25 and 1.75 TU. Among the individual forms of TiO2NPs, rutile NPs showed significantly (p<0.01) higher uptake than anatase NPs at all the concentrations tested under both visible and UV-A (except 1 TU) irradiation. Under visible irradiation, binary mixture showed significantly (p<0.01) higher uptake than individual forms of TiO2 NPs at all the TUs excluding 1.75 TU of anatase NPs. However, under UV-A irradiation, both anatase and rutile NPs showed higher uptake than their binary mixture at selected total TUs, 1 and 1.75 TU for anatase NPs and 0.25 and 1.75 TU for rutile NPs. 4. Discussion The impact of irradiation on the toxicity of two different crystalline phases (anatase and rutile NPs) was evaluated in the present study. Anatase NPs showed lesser LC50 values, which signify their higher toxicity towards C. dubia than rutile NPs under visible irradiation (Fig 2). Anatase NPs have higher band gap energy of about 3.18 eV than rutile NPs with 3.05 eV (evaluated in 21   

our previous study, Iswarya et al., 2015). Thus, anatase NPs possess higher photocatalytic activity than rutile NPs due to their higher band gap energy (Scanlon et al., 2013; Luttrell et al., 2014), which in turn reflects in the toxicity of NPs. A similar low EC50 pattern was observed by Bang et al. (2011) for 21-nm sized anatase NPs (0.42 mM) in comparison with 500-nm sized rutile NPs (5.94 mM) towards Daphnia magna under photoperiodic conditions tested in a reconstituted hard water matrix. In contrast, the LC50 of anatase and rutile NPs do not significantly vary under UV-A irradiation (Fig 2). In a study by Numano et al. (2014) on primary alveolar macrophage cell cultures, toxicity differences observed among the anatase and rutile NPs have been found to be insignificant under UV irradiation. Moreover, TiO2 (both anatase and rutile) NPs were noticed to be highly reactive under UV-A than visible irradiation. It was mainly due to the differences in their catalytic properties in the presence of light. The importance of illumination on the toxicity evaluation of TiO2 NPs has been addressed by Marconi et al. (2013) by testing on Daphnia similis in an OECD media. They acquired an EC50 of about 56.9 and >100 mg/L for the synthesized anatase and rutile NPs, respectively, under UV-A irradiation; but was undetermined under visible and dark conditions. Most of the toxicity reports on NPs towards crustaceans have been evaluated in artificial media, which differs a lot from the real freshwater environment. Only in few toxicity investigations, a real environmental matrix was used as a test media in which NP toxicity was noticed to be quite different from the literature available. For example, EC50 was noted to be 3.4 mg/L for Degussa P25 under UV-A on D. magna in a real river water sample (Amiano et al., 2012) and 8.26 mg/L for anatase NPs (25 nm) under photoperiodic light conditions, and the toxicity for the latter was tested on C. dubia in a lake water matrix (Dalai et al., 2013). A similar less LC50 value was obtained in the present study for both anatase and rutile NPs in comparison with the toxicity of NPs observed in an artificial test matrix. Gao et al. (2009) stated that interaction of freshwater constituents such as natural organic matter and ions with the NPs might influence their behavior and in turn their toxicity. These results implied the necessity of in-depth toxicity studies in real environmental matrix for the thorough understanding of NP toxicity. Mixture toxicity of anatase and rutile NPs was also studied on C. dubia, using the toxic unit (TU) approach. Based on the irradiation condition (visible or UV-A), binary mixture induced a variation in the toxicity. Under visible irradiation, a significant decrease in the mortality was 22   

noted at certain total TUs of the binary mixture when compared to individual NPs (Fig 3). This reduction could be due to the interaction between anatase and rutile NPs at the particular TUs tested. The antagonistic effect produced by the mixture at visible irradiation was further confirmed with the Marking–Dawson model rather than the Abbott model, which represented an additive effect. Cedergreen et al. (2008) analyzed the concentration addition (CA) and the independent action (IA) model for predicting the toxicity of various binary mixtures in several model organisms. These models exerted a variance in their assessment over the organisms tested. For single species systems such as algae and daphnia, CA model was predicted to be the best model rather than IA model. In contrast, an opposite effect was observed for multispecies system such as mixed bacterial cultures, where IA serves as a superior model in predicting the effects of mixtures. In some cases, both the models didnot predict the effects accurately. From these results, they concluded that the selection of model was not solely based on their accuracy. Among the two models applied in the present study, Marking–Dawson model was found to be more fitting for the data as it correlated with the antagonistic effect noticed in the mortality data. In contrast to the visible irradiation data, the binary mixture didnot exert any significant changes in the toxicity under UV-A (Fig 3B), as compared with the individual NPs. These results indicated the additive action of anatase and rutile NPs when they were co-exposed together. Simple additivity obtained under UV-A was further confirmed with both Marking–Dawson and Abbott models. Hence, the type of irradiation and crystalline pattern plays a major role in the toxicity of NPs, both individually as well as a mixture. With increase in time, the aggregation of NPs was observed in the sterile lake water for all forms (anatase, rutile, and mixture) of TiO2 NPs exposed (Fig 4). Under both visible and UV-A irradiation, higher aggregation of NPs was noted in the order of sequence, rutile > mixture > anatase NPs. DLS results also indicated that the size of the NPs didnot differ much at higher toxic units, even though there was a difference in the irradiation/light exposure. However, at a lower toxic unit, i.e., 0.25 TU of rutile and binary mixture, the aggregation was influenced by irradiation. These data was further validated with the sedimentation results (Fig 5). Aggregation of NPs occurred mainly due to the interaction between the NPs and colloids (natural organic matter (NOM) and other colloids such as ions etc.) available in the sterile lake water, as mentioned in the earlier reports (Labille et al., 2010; Sillanpää et al., 2011). It was also noted that the availability of individual NPs in the sterile lake water was higher under visible irradiation 23   

than UV-A. As a high energizing radiation, UV-A light induces more reactive oxygen species (ROS) generation by TiO2 NPs than visible light, due to their higher photocatalytic activity upon irradiation (Chen and Mao., 2009; Guo et al., 2011). NOM and other humic substances present in the lake water undergo photolysis by the ROS and produces smaller particles and ions (Strome and Miller, 1978). He et al. (2015) stated that TiO2 NPs absorb these charged particles on their surface and result in the aggregation of NPs by electrostatic attraction, and in turn, cause higher sedimentation. They also observed that variation in the irradiation significantly influenced the sedimentation of TiO2 NPs in the presence of sulfosalicylic acid (SSA). Thus, the sedimentation of TiO2 NPs was high (especially at 0.25 TU) under UV-A than visible irradiation. Interaction of NOM with NPs was further evident from the decrease in zeta potential of TiO2 NPs over a period, irrespective of irradiation and crystalline form (Fig 7). Agglomeration of NPs in a binary mixture could be a major factor that helps in their interaction, as reported by Sun and Smirniotis (2003), and thus, displays toxicity differences in contrast to individual anatase or rutile NPs. TEM images further confirmed the interactions occurring between anatase and rutile NPs when co-exposed together. Anatase NPs were noticed to be encircled by the rutile NPs (Fig 6). Natural organic matter adsorbed on the NPs helped in the interaction over the other phase. Anatase–rutile interactions in the sterile lake water were evident by the change in the surface charge of NPs, i.e., positive to negative charge (Fig 7). However, they didnot show any change in the surface charge of individual NPs, except a decrease in the zeta potential. Though anatase and rutile NPs were of similar (+ve) charges in the lake water, natural colloids and ions in the lake water might have acted as intermediates and created an interfacial interaction over the phases. Such interactions stabilized the NPs by forming agglomerates and thereby obtained a negative charge. Li et al. (2009) stated that the higher amount of rutile nanorods in a mixture saturated the anatase NPs and produced a negative effect in the solar cell application. Under visible irradiation, due to the higher concentration of rutile NPs present in a mixture, rutile NPs interacts more with anatase NPs and masks the activity/toxicity of anatase NPs by inducing the agglomeration of NPs. This could be the probable reason that resulted in a reduction in the toxicity under visible irradiation. However, the agglomeration provoked an additive effect upon UV-A irradiation. In several studies reported on the mixed phase of TiO2 NPs (Connley et al., 2012; Sun et al., 2003; Zhang et al., 2008), it has been stated that the interfacial interaction results at the junction of the surface phases of NPs. 24   

Upon photoexcitation, the electrons were transferred among the phases at these junctions and promote charge separation, thereby, increasing the photocatalytic activity of TiO2 nanoparticles. Thus, the binary mixture showed an additive effect under UV-A irradiation. Bioaccumulation of NPs was evidenced as dark shades in the intestinal tract of daphnids with the help of microscopic studies (Fig S1 and S2). NPs in the intestine confirmed the ingestion of NPs by the daphnids. Along with accumulation, NP aggregation over the appendages and attachment of NPs to the antennae were also observed under UV-A irradiation (Fig S2). These differences were not observed under visible irradiation indicating the influence of irradiation over the crystallinity of TiO2 NPs. Li et al. (2011) observed similar NP attachment in different parts of C. dubia upon exposure to TiO2 NPs. Few researchers also reported that NP adhesion induces some physical changes such as molting inhibition (Dabrunz et al., 2011), swimming behavior (Noss et al., 2013), etc. Hence, NP adhesion to the surface and other physical changes also provoke their toxicity apart from the sorption and ingestion of NPs. Further, the amount of Ti taken up by the test organisms has been evaluated quantitatively to confirm the difference in the uptake of TiO2 NPs due to the variation in the irradiation (Fig 8). Under visible irradiation, uptake of TiO2 NPs was observed in the following sequence, mixture > rutile NPs > anatase NPs. This trend was followed only at the lower toxic units, 0.25 and 1 TU. For the binary mixture, Ti uptake by C. dubia was substantially decreased as the TUs increased, which further validated their antagonistic action under visible irradiation. An interfacial interaction between anatase and rutile NPs in the mixture (as stated earlier in surface interaction data) and subsequent agglomeration was the probable reason behind their decreased uptake. Similar observations (i.e., highest agglomeration, decreased uptake, and reduced mortality) were noted by Dalai et al. (2013) on C. dubia at higher concentrations (32 and 64 mg/L) of anatase NPs under photoperiodic conditions. Sharma (2009) stated that the aggregation of NPs influences their bioavailability to the aquatic organisms and in turn, affects their uptake and toxicity. Under UV-A irradiation, Ti uptake by C. dubia showed a non-monotonic response for all the forms of TiO2 NPs tested, excluding rutile NPs. Comparing the uptake data of visible irradiation with UV-A, individual NPs showed maximum uptake under UV-A than visible irradiation, which was mainly due to the photoactivation of NPs under UV light. In contrast, the binary mixture uptake was high under visible irradiation (only at 0.25 TU) and found to be 25   

irrelevant with the toxic units exposed. Other than NP agglomeration, several other physical changes such as molting, excretion of NPs (retention period of NPs in the gut), and ROS generation may influence the NP uptake (Kim et al., 2010; Dabrunz et al., 2011). 5. Conclusion The present study was focused on the toxicity evaluation of anatase and rutile NPs (individual as well as a mixture) on Ceriodaphnia dubia in a freshwater matrix under different irradiation conditions. Individually, anatase NPs were found to be highly toxic than rutile NPs under visible irradiation. However, under UV-A irradiation, they showed equal toxicity due to the photoactivation of NPs. The mixture toxicity of anatase and rutile NPs revealed an antagonistic effect for visible irradiation and additive effect for UV-A irradiation. Thus, the type of irradiation and crystalline form of the TiO2 NPs significantly influenced their toxicity both individually as well as in a mixture. Elevated aggregation of NPs was noted under both visible and UV-A irradiation in the sequential order: rutile > mixture > anatase NPs. Aggregation of NPs played a vital role in the antagonistic and additive effects produced by the mixture based on the irradiation applied. Individually, the highest uptake was noted at UV-A irradiation rather than visible irradiation, whereas binary mixture showed maximum uptake at 0.25 TU under visible irradiation. Since daphnia serves as a food for other organisms such as fish in the aquatic system, it may pose a severe threat to freshwater organisms. Acknowledgement We acknowledge LSRB–DRDO, Government of India, for providing the financial support throughout the present work. We also thank School of Advanced Sciences (SAS), VIT University, Tamil Nadu, Vellore, for the TEM facility used in the primary size characterization of NPs.

26   

References Abbott, W.S., 1925. A Method of computing the effectiveness of an insecticide. J Econ Entomol. 18, 265-267. Allouni, Z.E., Cimpan, M.R., Høl, P.J., Skodvin, T., Gjerdet, N.R., 2009. Agglomeration and sedimentation of TiO2 nanoparticles in cell culture medium. Colloids Surf B Biointerfaces. 68(1), 83-87. Amiano, I., Olabarrieta, J., Vitorica, J., Zorita, S., 2012. Acute toxicity of nanosized TiO2 to Daphnia magna under UVA irradiation. Environ Toxicol Chem. 31(11), 2564-2566. Bang, S.H., Le, T.H., Lee, S.K., Kim, P., Kim, J.S., Min, J., 2011. Toxicity Assessment of Titanium (IV) Oxide Nanoparticles Using Daphnia magna (Water Flea). Environ Health Toxicol. 26, 14-19. Campos, B., Rivetti, C., Rosenkranz, P., Navas, J.M., Barata, C., 2013. Effects of nanoparticles of TiO2 on food depletion and life-history responses of Daphnia magna. Aquat Toxicol, 130, 174-183. Cao, Q., Hu, Q.H., Khan, S., Wang, Z.J., Lin, A.J., Du, X., Zhu, Y.G., 2007. Wheat phytotoxicity from arsenic and cadmium separately and together in solution culture and in a calcareous soil. J Hazard Mater. 148(1), 377-382. Cedergreen, N., Christensen, A.M., Kamper, A., Kudsk, P., Mathiassen, S.K., Streibig, J.C., Sørensen, H., 2008. A review of independent action compared to concentration addition as reference models for mixtures of compounds with different molecular target sites. Environmental Environ Toxicol Chem. 27(7), 1621-1632. Chen, X., Mao, S., 2009. Titanium dioxide nanomaterials: synthesis, properties, modifications, and applications. Chem Rev 107(7), 2891–2959 Chesworth, J.C., Donkin, M.E., Brown, M.T., 2004., The interactive effects of the antifouling herbicides Irgarol 1051 and Diuron on the seagrass Zosteramarina(L.). Aquat.Toxicol. 66, 293– 305.

27   

Cho, W.S., Kang, B.C., Lee, J.K., Jeong, J., Che, J.H., Seok, S.H., 2013. Comparative absorption, distribution, and excretion of titanium dioxide and zinc oxide nanoparticles after repeated oral administration. Part Fibre Toxicol. 10 (9). Clemente, Z., Castro, V.L., Jonsson, C.M., Fraceto, L.F., 2014. Minimal levels of ultraviolet light enhance the toxicity of TiO2 nanoparticles to two representative organisms of aquatic systems. J Nanopart Res. 16(8), 1-16. Connelly, K., Wahab, A.K., Idriss, H. (2012). Photoreaction of Au/TiO2 for hydrogen production from renewables: a review on the synergistic effect between anatase and rutile phases of TiO2. Materials for renewable and sustainable energy, 1(1), 1-12. Dabrunz, A., Duester, L., Prasse, C., Seitz, F., Rosenfeldt, R., Schilde, C., Schaumann, G.E., Schulz, R., 2011. Biological surface coating and molting inhibition as mechanisms of TiO2 nanoparticle toxicity in Daphnia magna. PLoS One. 6(5), e20112. Dalai, S., Pakrashi, S., Chandrasekaran, N., Mukherjee, A., 2013. Acute toxicity of TiO2 nanoparticles to Ceriodaphnia dubia under visible light and dark conditions in a freshwater system.PloS one, 8(4), e62970. Ferreira, A.L., Loureiro, S., Soares, A.M., 2008. Toxicity prediction of binary combinations of cadmium, carbendazim and low dissolved oxygen on Daphnia magna. Aquat.Toxicol. 89(1), 2839. Fujishima, A., Rao, T.N., Tryk, D.A., 2000. Titanium dioxide photocatalysis. Journal of Photochemistry and Photobiology C: Photochemistry Reviews. 1(1), 1-21. Fujiwara, R., Luo, Y., Sasaki, T., Fujii, K., Ohmori, H., Kuniyasu, H., 2015. Cancer Therapeutic Effects of Titanium Dioxide Nanoparticles Are Associated with Oxidative Stress and Cytokine Induction. Pathobiology. 82(6), 243-51. Gao, J.I.E., Youn, S., Hovsepyan, A., Llaneza, V.L., Wang, Y., Bitton, G., Bonzongo, J.C.J., 2009. Dispersion and toxicity of selected manufactured nanomaterials in natural river water samples: effects of water chemical composition. Environ Sci Technol. 43(9), 3322-3328.

28   

Guo, Y., Cheng, C., Wang, J., Wang, Z., Jin, X., Li, K., Kang, P., Gao, J., 2011. Detection of reactive oxygen species (ROS) generated by TiO2(R), TiO2(R/A) and TiO2(A) under ultrasonic and solar light irradiation and application in degradation of organic dyes. J Hazard Mater 192, 786–793. Hai-zhou, Z., Guang-hua, L., Jun, X., Shao-ge, J., 2012. Toxicity of nanoscale CuO and ZnO to Daphnia magna. Chemical Research in Chinese Universites. 28(2), 209-213. He, G., Chen, R., Lu, S., Jiang, C., Liu, H., Wang, C., 2015. Alternative assessment of nanoTiO2 sedimentation under different conditions based on sedimentation efficiency at quasi-stable state. J Nanopart Res, 17(11), 1-11. Hu, C.W., Zhang, L.J., Wang, W.L., Cui, Y.B., Li, M., 2014. Evaluation of the combined toxicity of multi-walled carbon nanotubes and sodium pentachlorophenate on the earthworm Eiseniafetida using avoidance bioassay and comet assay. Soil Biology and Biochemistry, 70, 123-130. Hund-Rinke, K., Simon, M., 2006. Ecotoxic effect of photocatalytic active nanoparticles (TiO2) on algae and daphnids. Environ Sci Pollut Res Int, 13(4), 225-232. Iswarya, V., Bhuvaneshwari, M., Alex, S.A., Iyer, S., Chaudhuri, G., Chandrasekaran, P.T., Bhalerao, G.M., Chakravarty, S., Raichur, A.M., Chandrasekaran, N., Mukherjee, A., 2015. Combined toxicity of two crystalline phases (anatase and rutile) of Titania nanoparticles towards freshwater microalgae: Chlorella sp. Aquat. Toxicol. 161, 154–169. Jacobasch, C., Völker, C., Giebner, S., Völker, J., Alsenz, H., Potouridis, T., Heidenreich, H., Kayser, G., Oehlmann, J., Oetken, M., 2014. Long-term effects of nanoscaled titanium dioxide on the cladoceran Daphnia magna over six generations. Environ Pollut. 186, 180-186. Jak, R.G., Maas, J.L., ScholtenTh, M.C., 1996. Evaluation of laboratory derived toxic effect concentrations of a mixture of metals by testing fresh water plankton communities in exposures. Water Res. 30, 1215–1227.

29   

Kaegi, R., Ulrich, A., Sinnet, B., Vonbank, R., Wichser, A., Zuleeg, S., Simmler, H., Brunner, S., Vonmont, H., Burkhardt, M., Boller, M., 2008. Synthetic TiO2 nanoparticle emission from exterior facades into the aquatic environment. Environ Pollut. 156(2), 233-239. Kim, K.T., Klaine, S.J., Cho, J., Kim, S.H., Kim, S.D., 2010. Oxidative stress responses of Daphnia magna exposed to TiO2 nanoparticles according to size fraction. Sci Total Environ, 408(10), 2268-2272. Kühn, K.P., Chaberny, I.F., Massholder, K., Stickler, M., Benz, V.W., Sonntag, H.G., Erdinger, L., 2003. Disinfection of surfaces by photocatalytic oxidation with titanium dioxide and UVA light. Chemosphere, 53(1), 71-77. Labille, J., Feng, J., Botta, C., Borschneck, D., Sammut, M., Cabie, M., Auffan, M., Rose, J., Bottero, J.Y., 2010. Aging of TiO2 nanocomposites used in sunscreen. Dispersion and fate of the degradation products in aqueous environment. Environ Pollut. 158(12), 3482-3489. Lampert, W., Sommer, U., 2007 Limnoecology. Oxford: Oxford University Press. 324. Lazar, M.A., Varghese, S., Nair, S.S., 2012. Photocatalytic water treatment by titanium dioxide: recent updates. Catalysts. 2(4), 572-601. Li, G., Richter, C.P., Milot, R.L., Cai, L., Schmuttenmaer, C.A., Crabtree, R.H., Brudvig, G.W., Batista, V.S., 2009. Synergistic effect between anatase and rutile TiO2 nanoparticles in dyesensitized solar cells. Dalton Trans.(45), 10078-10085. Li, L., Fernández-Cruz, M.L., Connolly, M., Conde, E., Fernández, M., Schuster, M., Navas, J.M., 2015. The potentiation effect makes the difference: non-toxic concentrations of ZnO nanoparticles enhance Cu nanoparticle toxicity in vitro. Sci Total Environ. 505, 253-260. Li, M., Czymmek, K.J., Huang, C.P., 2011. Responses of Ceriodaphnia dubia to TiO2 and Al2O3 nanoparticles: a dynamic nano-toxicity assessment of energy budget distribution. J Hazard Mater. 187(1), 502-508. Luttrell, T., Halpegamage, S., Tao, J., Kramer, A., Sutter, E., Batzill, M., 2014. Why is anatase a better photocatalyst than rutile?-Model studies on epitaxial TiO2 films. Sci Rep. 4, 4043.

30   

Mahlambi, M.M., Ngila, C.J., Mamba, B.B., 2015. Recent Developments in Environmental Photocatalytic Degradation of Organic Pollutants: The Case of Titanium Dioxide Nanoparticles—A Review. J Nanomater. 2015. Article ID 790173. Marcone, G.P., Oliveira, Á.C., Almeida, G., Umbuzeiro, G.A., Jardim, W. F., 2012. Ecotoxicity of TiO2 to Daphnia similis under irradiation. J Hazard Mater. 211, 436-442. Marking, L.L., Dawson, V.K., 1975.Method for assessment of toxicity or efficacy of mixtures of chemicals (No. 67).US Fish and Wildlife Service. Montazer, M., Seifollahzadeh, S., 2011. Enhanced Self‐cleaning, Antibacterial and UV Protection Properties of Nano TiO2 Treated Textile through Enzymatic Pretreatment. PhotochemPhotobiol. 87(4), 877-883. Mueller, N.C., Nowack, B., 2008. Exposure modeling of engineered nanoparticles inthe environment. Environ. Sci. Technol. 42, 4447–4453. Naddy, R.B., Cohen, A.S., Stubblefield, W.A., 2015. The interactive toxicity of cadmium, copper, and zinc to Ceriodaphnia dubia and rainbow trout (Oncorhynchusmykiss). Environ Toxicol Chem. 34(4), 809-815. Nel, A., Xia, T., Madler, L., Li, N., 2006. Toxic potential of materials at the nanolevel. Science 311, 622–627. Noss, C., Dabrunz, A., Rosenfeldt, R.R., Lorke, A., Schulz, R., 2013. Three-dimensional analysis of the swimming behavior of Daphnia magna exposed to nanosized titanium dioxide. PloS one, 8(11), e80960. Numano, T., Xu, J., Futakuchi, M., Fukamachi, K., Alexander, D.B., Furukawa, F., Kanno, J., Hirose, A., Tsuda, H., Suzui, M., 2014. Comparative study of toxic effects of anatase and rutile type nanosized titanium dioxide particles in vivo and in vitro, Asian Pac J Cancer Prev. 15(2), 929-35. OECD. (2004), Test No. 202: Daphnia sp. Acute Immobilisation Test, OECD Guidelines for the Testing

of

Chemicals,

Section

2,

http://dx.doi.org/10.1787/9789264069947-en 31   

OECD

Publishing,

Paris.

DOI:

Pakrashi, S., Dalai, S., Humayun, A., Chakravarty, S., Chandrasekaran, N., Mukherjee, A., 2013. Ceriodaphnia dubia as a Potential Bio-Indicator for Assessing Acute Aluminum Oxide Nanoparticle Toxicity in Fresh Water Environment. PLoS ONE 8(9): e74003. Pakrashi, S., Dalai, S., Sabat, D., Singh, S., Chandrasekaran, N., Mukherjee, A., 2011. Cytotoxicity of Al2O3 nanoparticles at low exposure levels to a freshwater bacterial isolate. Chem Res Toxicol. 24(11), 1899-1904. Piccinno, F., Gottschalk, F., Seeger, S., Nowack, B., 2012. Industrial production quantities and uses of ten engineered nanomaterials in Europe and the world. J Nanopart Res. 14(9), 1-11. Robichaud, C.O., Uyar, A.E., Darby, M.R., Zucker, L.G., Wiesner, M.R., 2009. Estimates of upper bounds and trends in nano-TiO2 production as a basis for exposure assessment. Environ Sci Technol. 43(12), 4227-4233. Scanlon, D.O., Dunnill, C.W., Buckeridge, J., Shevlin, S.A., Logsdail, A.J., Woodley, S.M., Catlow, C.R.A., Powell, M.J., Palgrave, R.G., Parkin, I.P., Watson, G.W., Keal, T.W., Sherwood, P., Walsh, A., Sokol, A.A., 2013. Band alignment of rutile and anatase TiO2. Nat. Mater. 12, 798–801. Sharma, V.K., 2009. Aggregation and toxicity of titanium dioxide nanoparticles in aquatic environment—a review. J Environ Sci Health A Tox Hazard Subst Environ Eng. 44(14), 14851495. Sillanpää, M., Paunu, T. M., Sainio, P., 2011. Aggregation and deposition of engineered TiO2 nanoparticles in natural fresh and brackish waters. Journal of Physics: Conference Series. 304(1), 012018. Sprague, J.B., Ramsay, B.A., 1965. Lethal levels of mixed copper-zinc solutions for juvenile salmon. Journal of the Fisheries Board of Canada, 22(2), 425-432. Strome, D.J., Miller, M.C., 1979. Photolytic changes in dissolved humic substances. Proceedings-International Association of Theoretical and Applied Limnology. 20, 1248- 125; Sun, B., Smirniotis, P.G., 2003. Interaction of anatase and rutile TiO2 particles in aqueous photooxidation. Catal Today 88, 49–59. 32   

Sun, B., Vorontsov, V., Smirniotis, P.G., 2003. Role of platinum deposited on TiO2 in phenol photocatalytic oxidation. Langmuir 19, 3151–3156 Teisseire, H., Couderchet, M., Vernet, G., 1999. Phytotoxicity of diuron alone and in combination with copper or folpet on duckweed (Lemna minor). Environ.Pollut. 106, 39–45. Tong, T., Wilke, C.M., Wu, J., Binh, C.T.T., Kelly, J.J., Gaillard, J.-F., Gray, K.A., 2015. Combined toxicity of nano-ZnO and nano-TiO2: from single- to multinanomaterial systems. Environ. Sci. Technol. 49, 8113-8123. U.S. EPA. Nanomaterial Case Studies: Nanoscale Titanium Dioxide in Water Treatment and in Topical Sunscreen (Final). U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-09/057F, 2010 Velhal, S.G., Kulkarni, S.D., Jaybhaye, R.G., 2012. Novel nanoparticles for water and waste water treatment. Research Journal of Chemistry and Environment, 16(2), 95-103. Weir, A., Westerhoff, P., Fabricius, L., Hristovski, K., Von Goetz, N., 2012. Titanium dioxide nanoparticles in food and personal care products. Environ Sci Technol. 46(4), 2242-2250. Winkler, J., 2003. Production of Titanium Dioxide Pigments, European Coatings Literature. Vincentz, 37–40. Yu, R., Wu, J., Liu, M., Zhu, G., Chen, L., Chang, Y., Lu, H., 2016.Toxicity of binary mixtures of metal oxide nanoparticles to Nitrosomonas europaea. Chemosphere, 153, 187-197. Zhang, J., Xu, Q., Feng, Z., Li, M., Li, C., 2008. Importance of the relationship between surface phases and photocatalytic activity of TiO2. Angew Chem Int Ed Engl. 120(9), 1790-1793.

33   

Figure Legends: Fig 1: Transmission electron micrographs of different phases of TiO2 NPs, A) Anatase NPs, and B) Rutile NPs. Fig 2: Lethal concentration values (LC10, LC50, and LC90) for anatase and rutile NPs towards Ceriodaphnia dubia under visible and UV-A irradiation. Both anatase and rutile NPs showed lower LC values under UV-A in comparison to visible irradiation. Fig 3: Mortality induced by TiO2 NPs (anatase, rutile, and mixture) on Ceriodaphnia dubia under visible and UV-A irradiation. Fig 4: Aggregation profile of anatase and rutile NPs (both individually as well as binary mixture in the sterile lake water. The symbol * indicates the size of NPs observed at 4 h were statistically significant (p<0.05) to the initial size of NPs in the lake water noted at 0 h. Fig 5: Sedimentation assay of TiO2 NPs such as anatase and rutile NPs individually in the sterile lake water at different time interval (0, 4, 24, and 48 h). Fig 6: Transmission electron microscopic images illustrating the anatase–rutile interactions in a mixture under visible (A, B, C) and UV-A (D, E, F) in the sterile lake water. (A and D) Aggregation of NPs occurred owing to the interaction of anatase and rutile phases in the mixture. (B and E) Rod shaped rutile NPs covered the spherical shaped anatase NPs. (C and F) High resolution images of the mixture revealing the surface interactions between the phases. Note: yellow arrows – anatase NPs; Red arrows - rutile NPs. Fig 7: Zeta potential of TiO2 NPs (anatase, rutile, and mixture) in the sterile lake water over a period of 48 h. Asterisks (*) indicates the zeta potential of NPs observed at 48 h were statistically significant (p<0.05) to the zeta potential of NPs observed at 0 h. Fig 8: Uptake of TiO2 NPs (anatase, rutile, and mixture) by Ceriodaphnia dubia under different irradiation conditions were analyzed by AAS. Asterisk symbol (*) indicates that the uptake observed at 0.25 TU was statistically different (p<0.05) with the respect to the uptake noted at 1 TU. The symbol # indicates the uptake observed at1.75 TU was statistically different (p<0.05) from the uptake noted at 1 TU.

34   

Fig 1

35   

Fig 2

Fig 3

36   

Fig 4

37   

Fig 5

38   

Fig 6

39   

Fig 7

40   

Fig 8

41   

Table 1: Concentration and toxic unit (TU) of anatase and rutile NPs used in the toxic assessment of individual NPs as well as a binary mixture under visible light irradiation.

Individual NPs

Binary mixture

Total

Concentration of

Concentration

Concentration

Concentration

TU

anatase NPs, mg/L

of Rutile NPs,

of anatase NPs,

of rutile NPs,

(TU)

mg/L (TU)

mg/L (TU)

mg/L (TU)

LC50

37.04

48

0

0 (0)

0 (0)

0 (0)

0 (0)

0.25

9.26 (0.25)

12 (0.25)

4.63 (0.125)

6 (0.125)

0.5

18.52 (0.5)

24 (0.5)

9.26 (0.25)

12 (0.25)

0.75

27.78 (0.75)

36 (0.75)

13.89 (0.375)

18 (0.375)

1

37.04 (1)

48 (1)

18.52 (0.5)

24 (0.5)

1.25

46.30 (1.25)

60 (1.25)

23.15 (0.625)

30 (0.625)

1.5

55.56 (1.5)

72 (1.5)

27.78 (0.75)

36 (0.75)

1.75

64.82 (1.75)

84 (1.75)

32.41(0.875)

42 (0.875)

--

42   

Table 2: Concentration and toxic unit (TU) of anatase and rutile NPs used in the toxic assessment of individual NPs as well as a binary mixture under UV-A irradiation were represented in the table.

Individual NPs

Binary mixture

Total

Concentration of

Concentration

Concentration

Concentration

TU

anatase NPs, mg/L

of Rutile NPs,

of anatase NPs,

of rutile NPs,

(TU)

mg/L (TU)

mg/L (TU)

mg/L (TU)

LC50

22.56

23.76

0

0 (0)

0 (0)

0 (0)

0 (0)

0.25

5.64 (0.25)

5.94 (0.25)

2.82 (0.125)

2.97 (0.125)

0.5

11.28 (0.5)

11.88 (0.5)

5.64 (0.25)

5.94 (0.25)

0.75

16.92 (0.75)

17.82 (0.75)

8.46 (0.375)

8.91 (0.375)

1

22.56 (1)

23.76 (1)

11.28 (0.5)

11.88 (0.5)

1.25

28.20 (1.25)

29.70 (1.25)

14.10 (0.625)

14.85 (0.625)

1.5

33.84 (1.5)

35.64 (1.5)

16.92 (0.75)

17.82 (0.75)

1.75

39.48 (1.75)

41.68 (1.75)

19.74 (0.875)

20.79 (0.875)

43   

--

Table 3: Abbott Modelling representing the type of interaction between the anatase and rutile NPs in a binary mixture under different irradiation conditions (Visible and UV-A) in the sterile lake water.

Total TU of

Observed

Expected

Ratio of

Statistically

Mode of

mixture (TU)

toxicity (%)

toxicity (%)

Inhibition

Significant

interaction

(p<0.05) A) Visible Irradiation 0.25

8±3.74

7.6±4.65

1.05±0.49

NS

Additive

0.5

14±2.45

11.4±4.65

1.23±0.22

NS

Additive

0.75

26±2.45

22.4±5.27

1.16±0.11

NS

Additive

1

42±3.74

34±5.28

1.24±0.11

NS

Additive

1.25

46±2.45

43.6±5.38

1.06±0.06

NS

Additive

1.5

48±2

51.8±5.83

0.93±0.04

NS

Additive

1.75

64±2.45

60.8±6.4

1.05±0.04

NS

Additive

B) UV-A Irradiation 0.25

22±3.74

24±5.17

0.92±0.16

NS

Additive

0.5

28±3.74

32.6±4.24

0.86±0.12

NS

Additive

0.75

38±3.74

34.4±2.99

1.11±0.11

NS

Additive

1

46±2.45

48±3.73

0.96±0.05

NS

Additive

1.25

62±4.90

52±4.68

1.19±0.09

NS

Additive

44   

1.5

64±2.45

60±3.83

1.07±0.04

NS

Additive

1.75

66±2.45

66.4±3.06

0.99±0.04

NS

Additive

NS – Not significant at p<0.05

45