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2019
Dependence of welding fume particle toxicity on electrode type and current intensity assessed by microalgae
growth inhibition test
Kirichenko, K
Elsevier BV
Tieteelliset aikakauslehtiartikkelit
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CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/
http://dx.doi.org/10.1016/j.envres.2019.108818
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Dependence of welding fume particle toxicity on electrode type and current intensity assessed by microalgae growth inhibition test
Konstantin Yu Kirichenko, Alexander M. Zakharenko, Konstantin S. Pikula, Vladimir V. Chaika, Zhanna V. Markina, Tatiana Yu Orlova, Stanislav A. Medvedev, Greta Waissi, Aleksey S. Kholodov, Aristides M. Tsatsakis, Kirill S. Golokhvast
PII: S0013-9351(19)30615-2
DOI: https://doi.org/10.1016/j.envres.2019.108818 Reference: YENRS 108818
To appear in: Environmental Research Received Date: 21 August 2019
Revised Date: 28 September 2019 Accepted Date: 9 October 2019
Please cite this article as: Kirichenko, K.Y., Zakharenko, A.M., Pikula, K.S., Chaika, V.V., Markina, Z.V., Orlova, T.Y., Medvedev, S.A., Waissi, G., Kholodov, A.S., Tsatsakis, A.M., Golokhvast, K.S., Dependence of welding fume particle toxicity on electrode type and current intensity assessed by microalgae growth inhibition test, Environmental Research (2019), doi: https://doi.org/10.1016/
j.envres.2019.108818.
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© 2019 Published by Elsevier Inc.
Dependence of welding fume particle toxicity on electrode type and current intensity assessed by microalgae growth inhibition test
Konstantin Yu. Kirichenkoa, Alexander M. Zakharenkoa, Konstantin S. Pikulaa,*, Vladimir V. Chaikaa, Zhanna V. Markinaa,b, Tatiana Yu. Orlovab, Stanislav A. Medvedevc Greta Waissid, Aleksey S. Kholodova, Aristides M. Tsatsakis a,e,f, Kirill S. Golokhvast g
a Far Eastern Federal University, 690950, Vladivostok, Russian Federation
b National Scientific Center of Marine Biology FEB RAS, 690014, Vladivostok, Russian Federation
c Stock company Izumrud, 690105, Vladivostok, Russian Federation
d University of Eastern Finland, School of Pharmacy, POB 1627, 70211, Kuopio, Finland
e University of Crete, School of Medicine, Laboratory of Toxicology, 71003, Heraklion, Greece
f I.M. Sechenov First Moscow State Medical University, Moscow 119048, Russia
g Pacific Geographical Institute FEB RAS, 690014, Vladivostok, Russian Federation
* Corresponding author Konstantin S. Pikula
Researcher, Education and Scientific Center of Nanotechnology
Far Eastern Federal University, Sukhanova Street, 8, Vladivostok 690950, Russian Federation Tel +7(423)2652429; Fax +7(423)2432315; mob +79149632194
Email: k.pikula@mail.ru
ABSTRACT
Welding fumes are a major source of metal oxide particles, ozone, carbon monoxide, carbon dioxide, nitrogen oxides, and many other toxic substances. Hazardous properties and the level of toxicity of welding fumes depend mostly on the welding electrode type and the welding regime parameters. The specific objective of this study was to evaluate the aquatic toxicity of metal welding fume particles in vivo on microalga Heterosigma akashiwo. The quantity and size of particles were measured by flow cytometry using a scattering laser light with a wavelength of 405 nm. The number of microalgae cells after 72 hours and 7 days exposition with welding fume particle suspensions was evaluated by flow cytometry. Morphological changes of the microalga were observed by optical microscopy. The toxic effect was demonstrated as a significant reduction of cell density after exposure of microalgae to welding fume particles. The greatest impact on the growth of microalga was caused by particles with high rutile content. It was shown that the adverse effect of metal oxide particles depends more on the chemical composition of particles in welding fume while the number and dispersity of particles had no noticeable toxic influence on microalgae.
The findings of this research confirm the fact that the toxicity of welding fume particles can be significantly reduced by using rutile-cellulose coated electrodes.
Keywords: ecotoxicology; flow cytometry; metal oxides; microalgae; welding fumes.
1. Introduction
Anthropogenic impact on the environment due to technological progress is constantly increasing, which is caused, inter alia, by the synthesis of new substances and compounds. The hazardous substances such as nanoparticles can enter the environment either directly during their formation and use or as a result of fuel and waste combustion, during welding and related processes.
The negative effect of nanoparticles included in welding fumes (WF) can lead to cell membranes damage and formation of reactive oxygen species that cause oxidative stress into the cells of living organisms (Pikula et al. 2019a). Hazardous properties and the level of toxicity of this type of nanoparticles depend not only on their chemical composition but also on their size, as well as on the welding electrode type and the welding regime parameters (Kauppi et al., 2015; Mehrifar et. al., 2019). Due to a lack of comprehensive studies oriented to WF risk assessment, more works should be focused on obtaining new information related to the welding process impact to the environment.
Aquatic organisms are widely used in toxicology and ecotoxicology because of their sensitivity, ubiquity, and simplicity of cultivation. Microalgae Heterosigma akashiwo is a widespread and well-studied species (Kempton et al., 2008; Shikata et al., 2008). Our previous study has shown significant sensitivity of H. akashiwo to carbon and silicon nanoparticles exposure and emphasized the role of metal impurities included in the samples (Pikula et al., 2018). We have recently evaluated the suitability of the used culture of microalgae H. akashiwo as a test-organism by the assay with toxicant potassium dichromate (Pikula et al., 2019b).
The present study explores the effects of dispersity of nanoparticles under 1 µm, formed by the welding process, on microalga H. akashiwo. Welding fume particles obtained from four different types of electrodes with alterations in welding process voltage were investigated. Our results will greatly contribute to the field of WF environmental risk assessment.
2. Materials and methods 2.1 Materials
The welding aerosol sample characteristics were previously studied by Kirichenko et al.
(2017a, b). Eight samples of welding fume particles were obtained from four different electrodes in the two most common operating conditions with currents of 100 and 150 amperes (A).
Suspensions of WF samples obtained as a result of working with electrodes No. 3, 4, 5, and 9 at a current of 100 A and samples 3.1, 4.1, 5.1, and 9.1 obtained with the same electrodes at a current of 150 A were used in microalgae toxicity bioassay. Welding fume samples collected from 0.07 m3 of working zone air three meters apart from the center of welding. The three-meter distance was previously reported as a point of a maximum concentration of nanoparticles in the air of the working zone (Kirichenko et al., 2017b). The particles were filtered through 0.45 µm nylon filters.
The quantitative characteristics of particle suspensions were measured using CytoFLEX flow cytometer (Beckman Coulter, USA) with a violet laser at 405 nm wavelength. The cytometer was calibrated with a mixture of fluorescent particles (Megamix-Plus SSC and Megamix-Plus FSC (BioCytex, France).
2.2 Microalgae bioassay
The culture of microalga H. akashiwo (Y. Hada) Y. Hada ex Y. Hara & M. Chihara 1987 (Raphydophyceae) was provided by The Resource Collection “Marine biobank” of the National
Scientific Center of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences (NSCMB FEB RAS). Microalgae cells were cultivated with Guillard’s f/2 medium (Guillard and Ryther, 1962). Filtered seawater (pore diameter of the filter was 0.22 µm) with salinity 33 ± 1 ‰ and pH 8.0 ± 0.2 was used. The cultivation was carried out at a temperature of 20 ± 2 oC, with an illumination intensity of 70 PAR (µmol of photons m-2 s-1), with a light cycle of 12:12 hours. For tests, algal cultures were selected in the exponential growth phase with a density of 1.5 × 104 cellsml-1. The duration of the experiments was seven days. The algae grow-inhibition tests were conducted according to the OECD guidance test No.201 (OECD, 2011) with minor modifications.
For microalgae cultivation, we used 24-well plates with WF suspended in f/2 media at concentrations of 1, 10, 100 mg/l. For the control group microalgae cells cultivated in wells with only f/2 medium. All assays for each concentration of WFs were conducted in four replications. The volume of microalgae aliquots in each replication was 2 ml. Statistically significant reduction of algal cell number compared to control after 72 hours and 7 days of exposition with WF samples has been assumed as the main criterion of toxic effect.
To determine the number of cells in each measurement, a blue laser (488 nm) of CytoFLEX flow cytometer was used as a source of excitation light. A homogeneous population of registered events was selected on the FSC/SSC dot cytogram (forward scattering to side scattering ratio), then from that population there were selected only those events that have an intense fluorescence in the PC5.5 emission channel (690 nm), which tallies the emission of chlorophyll a. Propidium iodide (PI) was used for the determination of live and dead cells (Suman et al., 2015). The mechanism of PI action is incorporation between DNA or RNA base pairs, whereupon the dye increases its fluorescence intensity by 20–30 times (Suzuki et al., 1997). Since PI is not able to penetrate the intact membranes of living cells, therefore the cells, which fluorescent intensity in the ECD emission filter (610 nm) dramatically increased as compared to the control can be determined as dead ones. The algae cells were stained with PI for 10 minutes at a concentration of 20 µmol. The calculation of live microalgae cells was performed by identifying cells that have chlorophyll a autofluorescence and by the exclusion of dead cells from counting. The number of cells was calculated as a percentage from the control group.
Morphological changes of microalgae cells were observed by optical microscope Axio Observer A1 (Carl Zeiss, Germany).
2.3 Statistical analysis
Statistical analyses were performed in the software package STATISTICA 10 (StatSoft, Inc., USA). One-way ANOVA test was used for analysis. A value of р ≤ 0.05 was considered statistically significant.
3. Results and Discussion
The measurement showed that the highest content of nanoparticles was in samples 4, 4.1, 5, 5.1, and 9.1 (Table 1). In the samples 9 and 9.1, there were detected the largest number of particles the size ranges 300-500 nm (Fig. 1). According to the data of Table 1, welding with a higher strength of current produces more WF nanoparticles compared to welding with the same electrode, but with less current.
Table 1
The quantity and chemical composition of WF particles
Sample Electric current intensity, А
Number of particles in size
range 100–900 nm per 100 µl Chemical composition*
3 100 41023 Al – 0.5%; Cr – 15.0%; Ti – 18.2%; Co – 0.46%.
3.1 150 72019 Mn – 2.1%; Si – 0.5%; Ni – 24.5%; Cr – 15.0%; Ti –
20.5%; V – 1.3%.
4 100 89706 Ba – 47.29%; Zn – 2.88%; Mn – 5.79%; Ti – 9.13%;Si –
18.36 %; Al – 1.48%; Mg – 1.05%.
4.1 150 130340 Ba – 37.11%; Zn – 2.13%; Mn – 5.25%; Ti – 9.13%; Si
– 18.36%; Al – 1.48%; Mg – 1.05%.
5 100 227007 Mg – 1.21%; Al – 1.48%; Si – 3.71%; Ti – 3.45%; Cr –
0.07%; Mn – 0.74%; S- 0.42%; Co – 0.18%.
5.1 150 124541 Mg – 1.01%; Al – 1.68%; Si – 3.51%; Ti – 3.12%; Cr – 0.03%; Mn – 0.83%; S – 0.42%; Co – 0.21%.
9 100 47633 Al – 0.88%; Mn– 0.40%; Si – 0.30%; Ti – 0.39%.
9.1 150 598981 Mn – 0.40 %; Si – 0.95%; Ti – 0.51%; Al – 0.48%.
*Kirichenko et al. (2017a, b)
Fig. 1 Particle size distribution in the samples of WF suspensions.
Filtered sea water was used as control.
The results of bioassay showed that the toxic effect of WF particles on microalgae growth rate depends not only on the initial composition of used electrode but also on the electric current intensity at which the work was performed (Fig. 2a, b). Welding fume samples 3 and 3.1, obtained by working with a rutile-covered electrode, showed the highest effect on the growth of microalgae (Fig. 2c, d).
Fig.2 The number of H. akashiwo cells after 72-h (a, b) and 7-days (c, d) exposure of WF particles at concentrations 1, 10, and 100 µl·ml-1
According to these data, we can infer that the toxicity of WF was mostly related to the ionic and chemical composition of the emitted nanoparticles but not to their concentration. In support of this conjecture, the sample with the highest content of nanoparticles (samples 5, 4.1, 9.1) showed no pronounced impact on the growth of microalgae (Table 1).
The most significant morphological changes were captured for the cells exposed to the samples 3 and 3.1 obtained from welding with rutile-covered electrodes (Fig. 3a, Fig. 4a). Irregular shape, softness, multiple protrusions, and folds of algae cell membranes were detected (Fig. 3a, Fig.
4a). The depletion of plastids and an increase in the size of nuclei were shown for sample 4 (Fig.
3b) and for sample 3.1 (Fig. 4c).
In general, the cells were enlarged, and multiple vesicles were present under the membranes (Fig. 4a, 4b). At the same time, even deformed cells did not detach their flagella and remained motile. Agglomeration of WF particles with microalgae cells has not been observed.
Fig. 3 H. akashiwo cells after 72 hours of exposure with samples 3 (a), 4 (b), 5 (c), and 9 (d).
n – nucleus.
Mixotrophic microalgae H. akashiwo has an ability to consume specific species of bacteria, and even with a lack of lighting and nutrients, algal cells did not absorb non-specific bacterial species and microorganisms (Seong et al., 2006). Therefore, it can be assumed that the algae would not absorb WF particles and all the observed effects produced by physical interaction of cells with the particles.
The presence and number of thecal vesicles or alveoli is a specific characteristic of H. akashiwo. The proliferation of vesicles (Fig. 4a, d) and the formation of noticeable protrusions on the cell surface (Fig. 4b, c) also should be considered as a reaction to WF particle influence. Part of the cells after exposure of sample 5.1 (Fig. 5) showed the deformation of cell walls, but flow cytometry did not reveal any significant changes in the algae growth rate.
Fig.4 H. akashiwo cells after 72 hours of exposure with samples 3.1 (a-c), and 4.1 (d).
a – alveoli, p – protrusions of the cell wall, n – nuclei.
Fig.5 H. akashiwo cells after 72 hours of exposure with samples 5.1 (a) and 9.1 (b)
It is important to emphasize that microalgae cells initially have a significant advantage over animal cells due to the protection of cell wall, while animal cells are surrounded only by a plasma membrane (Hoec et al., 1995). Thus, it can be expected that the effect of WF on animal cells would be even more significant and pronounced.
Dysfunction, deformation, and violation of the cell wall integrity are very dangerous for cell life or the health of organisms. In animal cells, the intact cell membrane is a vital factor because the membranes accomplish the barrier, mechanical, and matrix properties of organisms. Even the smallest defects in cell membrane structure can cause severe diseases in humans, such as Alzheimer's disease, and adrenoleukodystrophy (Wells et al., 1995; Ginsberg et al., 1998). Such defects lead to changes in the elasticity of membrane, disappearances of lipid microdomains, changes in permeability for cations, and variations in other characteristics of cell membranes (Buchet et al., 2000). Such changes lead to various diseases or make the cell easily susceptible to the attack of pathogens. There is evidence that defective regeneration of a partially damaged cell membrane in animals leads to the development of autoimmune disease (Chakrabarti et al. 2003).
In the current study, the highest toxicity was found in WF with a high content of Ti and Cr, which agrees to earlier data of TiO2 nanoparticles toxicity (Aruoja et al., 2015; Ma et al., 2019). In addition, it has been previously shown that Cr, V, Mo, and Ti are present in WF in the ionic form that is more accessible to living organisms as compared to oxides or pure metals (Wang et al., 2017;
Zeidler-Erdely et al., 2019). Thus, the development of recommendations intended to reduce the adverse environmental and health impact of welding is an important issue for future research.
4. Conclusion
The influence of welding fume particles on microalga H. akashiwo was detected as growth rate inhibition, death, and deformation of cell membranes. Particles formed by welding with rutile- coated electrodes were the most toxic due to their high chromium (15%) and titanium (18.2–20.5%) content. Moreover, these particles had 24.5% of nickel, 1.3% vanadium, 2.1% manganese, and 0.5% silicon. Particles formed by welding with electrodes having rutile-cellulose coating among all studied samples were the least toxic since they did not contain chromium and the content of titanium in them was 40.1-46.6 times less than in particles with rutile coating.
Our findings confirm the fact that environmental and human hazards from welding processes and welding fume toxicity can be significantly reduced by using rutile-cellulose coated electrodes.
Acknowledgements
This work was supported by the Russian Science Foundation (project No. 15-14-20032-P).
This work was supported by the grant from the President of the Russian Federation for young candidates of sciences (PhD) MK-2461.2019.5.
The authors are grateful to the FEFU Collective Use Center for providing scientific equipment. Authors are also thankful to Dr. Muhammad Amjad Nawaz (Education and Scientific Center of Nanotechnology, Far Eastern Federal University, Vladivostok, Russian Federation) for his help in copyediting and English language editing of this manuscript.
Conflict of interest
The authors did not report any conflict of interest.
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Highlights
• Chemical content of welding fume particles effects the toxicity more than their size
• Toxic effect of welding fumes mostly depends on Ti and Cr content in emissions
• Usage of rutile-cellulose coated electrodes can reduce welding fume toxicity
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
OThe authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
