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Frontiers of Environmental Science & Engineering

ISSN 2095-2201

ISSN 2095-221X(Online)

CN 10-1013/X

Postal Subscription Code 80-973

2018 Impact Factor: 3.883

Front. Environ. Sci. Eng.    2021, Vol. 15 Issue (1) : 6    https://doi.org/10.1007/s11783-020-1298-x
RESEARCH ARTICLE
Assessing combined toxic effects of tetracycline and P25 titanium dioxide nanoparticles using Allium cepa bioassay
Barsha Roy1, Khushboo Kadam2, Suresh Palamadai Krishnan1, Chandrasekaran Natarajan2, Amitava Mukherjee2()
1. School of Biosciences and Technology, VIT, Vellore 632014, India
2. Centre for Nanobiotechnology, VIT, Vellore 632014, India
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Abstract

• UVA pre-irradiation to TiO2 NPs enhanced its toxicity toward plant A. cepa.

• UVA TiO2 NPs increased intracellular ROS, resulting in more cell damage.

• Cell death enhanced cell permeability and increased uptake of NPs.

• Being highly toxic (EC50 = 0.097 µmol/L), TC did not increase ROS generation.

• Even at a low dose, TC enhanced the toxic potential of TiO2 NPs significantly.

Usage of titanium dioxide nanoparticles (TiO2 NPs) and tetracycline (TC) has increased significantly in the present era. This leads to their release and accumulation in the environment. Both the compounds, individually, can have adverse toxic effects on the plants. Their binary mixtures can increase this degree of damage. The present study aimed to evaluate the toxicity of both the contaminants in individual and binary mixtures in Allium cepa. Further, the toxicity of TiO2 NPs upon UVA pre-irradiation was also measured. Results showed that UVA pre-irradiated NPs (UVA-TiO2 NPs) had a significant decrease in cell viability than their non-irradiated counterparts (NI-TiO2), denoting an increase in photocatalytic activity upon UVA pre-irradiation. Very low concentrations of TC (EC10 = 0.016 µmol/L) mixed with TiO2 NPs significantly increased the toxicity for both UVA-TiO2 and NI-TiO2 NPs. Intracellular ROS generation was significantly high for UVA-TiO2 NPs. However, TC did not have any effects on ROS production. Both the compounds exhibited genotoxic potential in A. cepa. Different chromosomal abnormalities like anaphase bridges, telophase bridges, laggard chromosomes, binucleate cells, etc. were observed. The binary mixture of UVA-TiO2 NPs and TC showed the highest chromosomal aberrations (64.0%±1.26%) than the mixture with NI-TiO2 or the individual contaminants. This decreased significantly after recovery (46.8%±1.92%), denoting the self-repair processes. This study proved that UVA-TiO2 NPs were more toxic and could be enhanced further when mixed with a sub-lethal concentration of TC. This work will help to assess the risk of both compounds in the environment.

Keywords Binary toxicity      Cell viability      Mitotic index      Chromosomal aberration      Reactive oxygen species      Nanoparticles internalization     
Corresponding Author(s): Amitava Mukherjee   
Issue Date: 30 July 2020
 Cite this article:   
Barsha Roy,Khushboo Kadam,Suresh Palamadai Krishnan, et al. Assessing combined toxic effects of tetracycline and P25 titanium dioxide nanoparticles using Allium cepa bioassay[J]. Front. Environ. Sci. Eng., 2021, 15(1): 6.
 URL:  
https://academic.hep.com.cn/fese/EN/10.1007/s11783-020-1298-x
https://academic.hep.com.cn/fese/EN/Y2021/V15/I1/6
Concentration
(mmol/L)
MHD of NPs at 0 h (nm) MHD of NPs+ TC at 0 h (nm) MHD of NPs at 4 h (nm) MHD of NPs+ TC at 4 h (nm)
0.782 (NI) 219±6.38 320±1.93 a 224±5.97 325±1.87 a
1.56 (NI) 220±2.86 322±0.71 a 224±1.21 327±0.59 a
3.13 (NI) 222±2.27 325±1.55 a 227±2.49 327±0.70 a
6.26 (NI) 224±1.70 325±0.85 a 228±1.60 326±1.10 a
0.782 (UVA) 178±1.93 276±3.53 a 182±1.22 282±2.68 a
1.56 (UVA) 180±2.00 285±2.56 a 185±1.01 286±1.02 a
3.13 (UVA) 182±2.22 287±2.10 a 186±2.24 288±2.35 a
6.26 (UVA) 180±2.06 288±4.19 a 185±1.76 287±3.21 a
Tab.1  MHDs of non-irradiated NPs (NI) and UVA pre-irradiated NPs (UVA) in the individual and binary mixture at 0 h and 4 h interval
Fig.1  Percentage decrease in cell viability for NPs individually and in binary mixtures (a) Non-irradiated NPs and (b) UVA pre-irradiated NPs. *** represents significance with respect to the control and a denotes significance between individual and binary mixtures. (***, a,p<0.001).
Treatment Concentration (mmol/L) %PP %MP %AP %TP %MI %CA
Control 89.4±1.84 6.65±0.85 1.93±1.16 1.99±1.36 14.3±1.38 0±0
TC 69.3±1.10 1.04±0.35 2.87±0.70 1.44±0.54 10.7±0.62# 25.3±1.46#
NPs (0.7825) 70.1±1.92 0±0 3.78±1.56 2.89±1.02 9.22±0.18# 23.2±1.98#
NPs (0.7825) + TC 57.8±4.24 0±0 0.58±0.53 0.95±0.94 5.65±0.52#, a 40.7±3.81#, a
NPs (1.565) 68.1±3.65 0±0 3.63±1.53 1.72±0.96 8.95±0.20# 26.5±2.37#
NPs (1.565) + TC 51.8±1.92 0.41±0.56 1.87±1.68 0.42±0.58 4.50±0.40#, a 45.5±2.79#, a
NPs (3.13) 67.3±1.72 0±0 3.59±0.80 1.48±1.62 7.86±1.42# 29.2±1.45#
NPs (3.13) + TC 49.3±3.56 0±0 0.90±0.83 1.49±0.97 3.32±0.21#, a 48.3±2.55#, a
NPs (6.26) 65.9±1.52 0±0 1.44±2.18 0.60±0.89 6.42±0.80# 32.1±2.08#
NPs (6.26) + TC 47.5±3.57 0.29±0.65 0.27±0.61 0.55±1.22 3.12±0.40#, a 51.4±2.40#, a
Tab.2  Percentage phase index (PP, MP, AP, and TP), mitotic index (%MI) and percentage chromosomal aberrations (%CA) of non-irradiated TiO2 NPs (individual and binary mixtures of NPs and TC)
Treatment Concentration (mmol/L) %PP %MP %AP %TP %MI %CA
Control 89.4±1.84 6.65±0.85 1.93±1.16 1.99±1.36 14.3±1.38 0±0
TC 69.3±1.10 1.04±0.35 2.87±0.70 1.44±0.54 10.7±0.62# 25.3±1.46#
NPs (0.7825) 64.4±2.07 0.33±0.66 0.90±0.61 0.56±0.64 5.38±0.45# 34.7±2.62#
NPs (0.7825) + TC 48.7±4.71 0.68±0.95 1.02±0.94 1.28±1.27 3.10±0.18#, a 48.3±4.24#, a
NPs (1.565) 57.1±3.14 0.23±0.51 1.36±1.24 1.12±1.11 4.91±0.17# 40.2±2.50#
NPs (1.565) + TC 43.9±3.19 0±0 0.92±0.85 1.15±1.15 2.88±0.17#, a 54.0±2.72#, a
NPs (3.13) 56.7±3.22 0±0 0.80±0.75 0.65±0.90 4.40±0.71# 41.8±3.61#
NPs (3.13) + TC 40.7±1.00 0±0 0.51±1.13 0±0 2.87±0.06#, g 58.8±0.62#, a
NPs (6.26) 52.2±1.80 0±0 1.55±0.49 1.33±0.85 3.87±0.57# 44.9±2.63#
NPs (6.26) + TC 30.6±2.15 0.66±0.60 2.41±1.67 2.36±1.55 2.84±0.08#, d 63.9±1.26#, a
Tab.3  Percentage phase index (PP, MP, AP, and TP), mitotic index (%MI) and percentage chromosomal aberrations (%CA) of UVA-pre-irradiated TiO2 NPs in an individual and binary mixture with the antibiotic TC
Fig.2  Normal cell division phases observed under the light microscope at 400× magnification (a) prophase, (b) metaphase, (c) anaphase, and (d) telophase.
Fig.3  Chromosomal aberrations observed under 400× magnification for non-irradiated and UVA pre-irradiated TiO2 NPs suspension-treated A. cepa roots: (a) anaphase bridge, (b) binucleate cell, (c) laggard chromosome, (d) telophase bridge; for the binary mixture of TC and non-irradiated and/ or UVA pre-irradiated TiO2 NPs suspension-treated A. cepa roots observed aberrations were: (e) chromosome break, (f) C- mitosis, (g) disturbed metaphase and (h) sticky anaphase.
Fig.4  Internalization of Ti inside root cells after treatment with individual TiO2 NPs suspension (NI and UVA) as well as with the binary mixture of TiO2 NPs and TC. *** (p<0.001) represents significant differences of Ti uptake in mg/g (mean value) between individual and binary mixture treatments, whereas a (p<0.001) denoted significance between the non-irradiated and UVA-pre-irradiated NPs.
Fig.5  Percentage cell viability for A. cepa roots treated with the binary mixture of TiO2 NPs (non-irradiated and UVA pre-irradiated) and tetracycline for 4 h (before the recovery phase) and followed by a 24 h post-treatment in Millipore water (after the recovery phase). The control sample was kept in Millipore water for 4 h followed by a 24 h post-treatment again in Millipore water. *** denoted significant differences between the mean values before and after the recovery phases (p<0.001).
Condition %MI (BR) %CA (BR) %MI (AR) %CA (AR)
Non-irradiated+ TC 3.12±0.40 51.4±2.40 4.45±0.73 26.7±1.87***
UVA pre-irradiated+ TC 2.84±0.08 64.0±1.26 3.61±1.92 46.8±1.92***
Tab.4  Percentage mitotic index (%MI) and percentage chromosomal aberrations (%CA) in the A. cepa roots treated with a binary mixture of TiO2 NPs (non-irradiated and UVA pre-irradiated) and tetracycline (pre- and post-recovery phases)
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