Heavy metals impact at plants using photoacoustic spectroscopy technology with tunable CO2 laser in the quantification of gaseous molecules

Microchemical Journal 134 (2017) 390–399

Contents lists available at ScienceDirect

Microchemical Journal journal homepage: www.elsevier.com/locate/microc

Heavy metals impact at plants using photoacoustic spectroscopy technology with tunable CO2 laser in the quantification of gaseous molecules Cristina Popa ⁎, Mioara Petrus National Institute for Laser, Plasma and Radiation Physic, Laser Department, 409 Atomistilor St., PO Box MG-36, 077125 Magurele, Romania

a r t i c l e

i n f o

Article history: Received 26 June 2017 Received in revised form 6 July 2017 Accepted 6 July 2017 Available online 08 July 2017 Keywords: CO2 laser photoacoustic spectroscopy Heavy metals metabolism Plantlets Ammonia Ethylene Plant growth regulator

a b s t r a c t Heavy metals (HM) are common pollutants and received considerable attention since they may also have impacts on human via the food chain, drinking water, air inhalation or may be absorbed through the skin when they enter into contact with humans in agriculture and in manufacturing, pharmaceutical, industrial or residential settings. In this research, the effects of some HM: Hg, Pb, Cd, Cu and Zn on common wheat seeds (Triticum aestivum), Spelt wheat (Triticum spelta), common corn (Zea mays), alfalfa (Medicago sativa) and green peas (Pisum sativum) were investigated using photoacoustics as a non-invasive analysis method, in the detection of two gaseous molecules: ethene (ethylene) and ammonia. From the point of view of toxicology and oxidation states, the determinations demonstrates that seeds/grains germinated with HM, determine a greater increase of ammonia vapors in the respiration of plants and a greater decrease of ethylene vapors in the respiration of vegetation. In the same time, the investigations presented in this paper showed that the photoacoustic technology with tunable CO2 laser, in the quantification of gaseous molecules, was able also to distinguish between seeds/grains germinated with HM and seeds/grains germinated with distilled water and can play an important role in testing the contaminated biological samples. © 2017 Elsevier B.V. All rights reserved.

1. Introduction HM are common pollutants with a high atomic weight and a density at least 5 times greater than that of water. The primary source of HM in the environment is from naturally occurring geochemical materials [1]. This occurrence may be enhanced by a human activity. It is difficult to eliminate a HM pollution after being released in the environment. HM may transport from water, air and soil to plants, engender impacts on the growth and development of plants [1,2]. These elements may also have impacts on human via the food chain, drinking water and inhalation. In recent years, there has been an increasing ecological and global public health concern associated with environmental contamination by HM. Also, human exposure has risen dramatically as a result of an exponential increase of their use in several industrial, agricultural, domestic and technological applications [1–3]. Some HM are essential in various biochemical processes (Co, Cu, Fe, Mn, Mo, Ni, V and Zn — necessary for the body in small amounts, but toxic in large quantities). Other HM such as Pb, Cd, Hg, and As (this element is a metalloid but it is usually defined as a HM) have harmful ⁎ Corresponding author. E-mail address: cristina.achim@inflpr.ro (C. Popa).

http://dx.doi.org/10.1016/j.microc.2017.07.006 0026-265X/© 2017 Elsevier B.V. All rights reserved.

effects on human organism and thus are considered “main threats” because they are very detrimental to plants and animals [4–6]. HM are considered systemic toxicants that are known to induce multiple organ damage, even at lower levels of exposure. They are also classified as human carcinogens according to the U.S. Environmental Protection Agency, and the International Agency for Research on Cancer [1]. So HM have received considerable attention. Usually, the impacts of HM on plant growth were studied by adding them to soil but it took a long time and the results reflected on the indirect impacts because of the interaction of HM with soil. In biological systems, HM have been reported to affect cellular organelles and components such as cell membrane, mitochondrial, lysosome, endoplasmic reticulum, nuclei, and some enzymes involved in metabolism, detoxification, and damage repair [7]. Metal ions have been found to interact with cell components such as DNA and nuclear proteins, causing DNA damage and conformational changes [7–9] that may lead to cell cycle modulation, carcinogenesis or apoptosis. This paper provides an analysis of common wheat seeds, Spelt wheat, common corn, alfalfa and green peas germinated with some HM like: cadmium (Cd), lead (Pb), mercury (Hg), copper (Cu) and zinc (Zn), in order to compare the variety of effects on different plant tissues.

C. Popa, M. Petrus / Microchemical Journal 134 (2017) 390–399

We have chosen to investigate plant-tissue responses by using photoacoustic spectroscopy technology with CO2 laser in the detection of ethylene and ammonia gases. From multiple research techniques developed for trace gas assessment (including gas chromatography, mass spectrometry, Fourier transform infrared spectroscopy or chemiluminescence), the photoacoustic spectroscopy technology with CO2 laser in the detection of gaseous molecules is distinguished by valuable and proven advantages including: high sensitivity and selectivity, have allowed detection of parts-per-billion ppbv or even sub-ppbv concentrations, high accuracy and precision, large dynamic range, multicomponent capability, none or only minor sample preparation, good temporal resolution, versatility, reliability, ease of use, and robustness [10]. Photoacoustic spectroscopy with tunable CO2 laser has known a great increase over the years, in principal by enlarging the application fields gaining importance when it is applied to: biology, physics, chemistry, medicine, atmosphere, military or engineering [11,12]. The number of detectable molecules is related to the spectral overlapping of the CO2 laser emission with the absorption bands of the trace gas molecules. Based on the mentioned above our method became one of the most sensitive techniques in the world, being able to measure gas concentrations at sub-ppb levels with partial pressure of 10−10 atm and a minimum detectable concentration of 0.9 ppbV [10–13]. This experimental research is devoted to analyze in the presence of synthetic air, at atmospheric pressure and at room temperature, the effects of Cd, Pb, Hg, Cu and Zn at germinated seeds/grains using photoacoustic spectroscopy with respect to the CO2 laser frequencies (as radiation source), in the detection of ethylene and ammonia molecules. Because HM induced toxicity and carcinogenicity involves many mechanistic aspects (some of which are not clearly elucidated or understood), we compared the results with seeds/grains germinated with distilled water. However, each metal is known to have unique features and physic-chemical properties that confer to its specific toxicological mechanisms of action.

2. Ethylene and ammonia at plants Have you ever wondered what do humans and vegetation may have in common? We cannot see it or smell it, but plants, fruits, and humans generate some particular gases in specific circumstances. These gases are ethylene and ammonia. In humans, ethylene is a marker of oxidative stress and ammonia is a product of urea (marker of kidney insufficiency), these gases being detected in the exhaled air [10,13]. In plants, ethylene is a colorless gas that is naturally produced by all tissues and is transported by diffusion into the plant. Acts a growth regulator and not only plays an important role in various aspects of plant growth and development, including seed germination, organ senescence, and fruit development, but also regulates stress responses to environmental challenge [14,15]. Ethylene regulates many diverse metabolic and developmental processes in plants. It has been shown that ethylene is produced from essentially all parts of higher plants, including leaves, stems, roots, flowers, fruits, tubers, and seedlings [16]. Dimitry Neljubov [17] discovered for the first time the active role of ethylene (1901, 1911) showing that the etiolated epicotyl of the pea seedling was very sensitive to illuminating gas and demonstrated that ethylene was the effective constituent [17]. The numerous papers [18– 21] which have been published on this subject report a great variety of effects on different plant tissues. Ethylene has been found to break the rest period of dormant tissues with the result that growth commences earlier [22]. In 1924, Denny [23,24] showed that ethylene accelerated the yellowing of citrus fruits and increased their respiration. Since then ethylene has been found to accelerate ripening processes in many other fruits, including tomatoes, bananas, melons, persimmons, apples and pears [24–27].

391

Environmental clues can induce the biosynthesis of the plant hormone. Flooding, drought, chilling, wounding, and pathogen attack can induce ethylene formation in the plant. The biosynthesis of the ethylene [16,19,28] inside plant tissues depend on the activities of certain enzymes, the rate of outward diffusion and the rate of metabolization: it starts with the conversion of the amino acid methionine into Sadenosyl-L-methionine (SAM or AdoMet) by the enzyme Met Adenosyltransferase. SAM is then converted into 1-aminocyclopropane-1-carboxylic-acid (ACC) by the enzyme ACC synthase (ACS). The activity of ACS is the rate-limiting step in ethylene production; therefore regulation of this enzyme is the key for the ethylene biosynthesis. The final step requires oxygen and involves the action of the ACC-oxidase enzyme (ACO), formerly known as the Ethylene Forming Enzyme (EFE) [19, 28]. Ethylene biosynthesis can be induced by endogenous or exogenous ethylene. The action of ethylene is not only controlled by endogenous ethylene concentrations in tissues, but also by the tissue sensitivity. It is widely assumed that molecules involved in ethylene perception and in the transduction of the signal probably controls how much ethylene is required to evoke a physiological response [16–29]. Because of the unique effects of ethylene, more precise studies of its influence on various aspects of plant metabolism are needed and for that we have chosen to investigate in addition the ammonia responses of plant tissues. In plants, ammonia is a common byproduct of the metabolism of nitrogenous compound and is involved (with proteins) in plant urea metabolism. The urea pathway has been documented not only in mammals and amphibians but in many other organisms as well, including birds, invertebrates, insects, plants, yeast, fungi, and even microorganisms [30]. Ammonia is smaller, more volatile and more mobile than urea. If allowed to accumulate, ammonia would raise the pH in cells to toxic levels. Therefore, many organisms convert ammonia to urea (a plant metabolite), even though this synthesis has a net energy cost. In recent years various molecular players of plant urea metabolism have been investigated: active and passive urea transporters, the nickel metallo enzyme urease catalyzing the hydrolysis of urea, and three urease accessory proteins involved in the complex activation of urease [31, 32]. The degradation of plants derived from protein breakdown has long been discussed as a possible additional metabolic source for urea, but an enzymatic route for the complete hydrolysis of ureides without a urea intermediate has recently been described for Arabidopsis thaliana [30–32]. A common consequence of stress conditions (e.g. HM) is given by an increased production of reactive oxygen species (ROS) which can lead to non-specific oxidation of proteins and lipids and can cause damage to DNA in plant cells. As a result, the plant tissues affected by oxidative stress, generally contain high concentrations of protein and fat and the effects of the stress are shown in concentrations of ammonia and ethylene [33]. Several studies [34–43] have demonstrated that ROS production and oxidative stress play a key role in the toxicity and carcinogenicity of metals such as arsenic, Cd, chromium, Pb and Hg. Because of their high degree of toxicity, these five elements rank among the priority metals that are of great public health significance. They are all systemic toxicants that are known to induce multiple organ damage, even at lower levels of exposure. According to the United States Environmental Protection Agency (U.S. EPA), and the International Agency for Research on Cancer (IARC), these metals are also classified as either “known” or “probable” human carcinogens based on epidemiological and experimental studies showing an association between exposure and cancer incidence in humans and animals [1,34–43]. 3. Biological materials and experimental method 3.1. Biological sample collection In this research, for the photoacoustic analysis we used approximately 6 g of seeds/grains and the germination was carried out at

392

C. Popa, M. Petrus / Microchemical Journal 134 (2017) 390–399

room temperature. The concentration of ethylene and ammonia were evaluated for common wheat seeds, Spelt wheat (also known as hulled wheat), common corn, Alfalfa and green peas. The seeds and grains were grown in particular colorless polycarbonate containers used to analyze biological samples [44,45], with a specific volume of 0.83 cm3/g (see Fig. 1 a). For every polycarbonate containers was added 6 g of seeds or grains and a total of 10 mL of HM (see Table 1) or a total of 10 mL of distilled water (selected as a Control). After the germination of seed/grains, the containers with plants were introduced into small glass cuvette (see Fig. 1 b, c) with volume of 150 cm3, connected to the photoacoustic detector. HM have been obtained from Research and Development National Institute for Metals and Radioactive Resources, Bucharest, Romania and in Table 1 are presented the concentrations for each element. 3.2. Photoacoustic spectroscopy technology with tunable CO2 laser The photoacoustic signal at a particular operating frequency [12], including the contributions of the resonance, is given by: S ¼ αcPL R;

ð1Þ

where S[V] is the voltage measured as peak-to-peak value, α[cm−1 atm−1] is the absorption coefficient at the laser wavelength, c [atm] represents the trace gas concentration, PL[W] is the unchopped cw laser beam power and R [V cm/W] is the responsivity of the photoacoustic cell (or the cell calibration constant). The photoacoustic signal is usually linearly proportional with the incident laser power and the absorption coefficient over several orders of magnitude. It becomes nonlinearly for optical densities at which the deviation from the linear behavior is higher than 3% [12], when the saturation effect is arising either due to a large concentration of the measured analyte, or a high level of the laser power. Though, according with the performance of the detection system and with the restrictions imposed by the signal linearity, the photoacoustic method proves a large dynamical range such as 7 orders of magnitude [11,12]. Moreover, Eq. (1) reveals an essential feature of the photoacoustic detection, the fact that photoacoustic spectroscopy technique is a zero-baseline technique, meaning that no signal is generated in the absence of the absorbing molecules along the optical path [12]. The photoacoustic spectroscopy of CO2 laser set-up used for the determination of the ethylene and ammonia molecules is schematically presented in Fig. 2 and extensively described in four previous papers [10–13].

Table 1 Heavy metal concentrations. No.

Metal

Metal concentration

UM

Initial standard Merck METAL 1 g/L

1. 2. 3. 4. 5.

Zn 2 μM Cu 2 μM Cd 2 μM Pb 2 μM Hg 2 μM

0.1315 0.1220 0.2260 0.4171 0.4000

mg/L mg/L mg/L mg/L mg/L

ZnCl2 CuCl2 CdCl2 Pb(NO3)2 Hg(NO3)2

In order, to summarize, the photoacoustic detection scheme consists of a tunable continuously wave (cw) frequency stabilized CO2 laser (emitting radiation in the 9.2–10.8 μm region on 73 different vibrational-rotational lines, with a maximum power of 6.5 W), a lens, a chopper, a photoacoustic cell (the heart of the photoacoustic spectroscopy setup), a powermeter, a lock-in amplifier, an acquisition board and a computer for data acquisition and processing. The detection chain is accompanied by a complex gas handling system built for a proper manipulation of the gases molecules under study, from the gas bottle to the photoacoustic cell. The gas handling system ensuring gas purity in the cell and it can be used to pump out the cell, to introduce the biological sample in the cell, monitor the total and partial pressures of gas mixtures and also, can perform several functions without necessitating any disconnections [46]. Practically, the cw, tunable CO2-laser beam is chopped, focused by a ZnSe lens, and introduced in the cell. The light beam was modulated by a high quality, low vibration noise and variable speed (4–4000 Hz) mechanical chopper model DigiRad (30 aperture blade), operated at the appropriate resonant frequency of the cell (564 Hz). The laser beam diameter is typically 6.2 mm at the point of insertion of the chopper blade and is nearly equal to the width of the chopper aperture. An approximately square waveform was produced with a modulation depth of 100% and a duty cycle of 50% so that the average power measured by the powermeter at the exit of the cell is half the cw value. By enclosing the chopper wheel in housing with a small hole (10 mm) for the laser beam to enter and exit, reduces chopper-induced sound vibrations in air that can be transmitted to the microphone (sensitivity 20 mV/Pa) detector as noise interference. A compatible phase reference signal is provided for use with a lock-in amplifier (time constant 1 s sensitivity 1–100 mV). The resonance frequency corresponds to the resonant excitation of the first longitudinal mode of the cell (depends on its length). The acoustic resonator is characterized by the quality factor Q, which is defined as the ratio of the resonance frequency to the frequency bandwidth between half power points. The amplitude of the microphone signal is 1/View the MathML source of the maximum amplitude at

Fig. 1. (a) Polycarbonate containers used to germinate seeds; (b) The glass cuvette with germinated common wheat seeds; (c) The sample is placed in a cuvette which is connected to the cell.

C. Popa, M. Petrus / Microchemical Journal 134 (2017) 390–399

393

Fig. 2. Photoacoustic spectroscopy with tunable laser.

these points, because the energy of the standing wave is proportional to the square of the induced pressure. For our photoacoustic cell, the profile width at half intensity was 35 Hz, yielding a quality factor Q = 16.1 at a resonance frequency f0 = 564 Hz. Photoacoustic spectroscopy of CO2 laser set-up presents a very elegant way to determine trace gas emissions. It is fast (seconds) and sensitive (sub ppb level) and it allows near on-line measurements and multi-component analysis [47]. The calibration measurements are extensively presented in [10] for both ammonia and ethylene and were experimentally determined using commercially prepared, certified gas mixtures containing 0.96 ppmV ethylene diluted in pure nitrogen and 10 ppmV ammonia diluted in pure nitrogen. The absorption coefficients of ethylene and ammonia at different CO2 laser wavelengths were precisely measured previously [12,48] and the CO2 laser was kept tuned at the 10P (14) line (10.53 μm) where ethylene exhibit a strong peak, corresponding to an absorption coefficient of 30.4 cm − 1 atm − 1 and at 9R(30) CO2 laser line (9.22 μm), where the ammonia absorption coefficient has the maximum value of 57 cm− 1 atm− 1. We examined this reference mixture at a total pressure of approximately 1013 mbar and a temperature of 23 °C, using the commonly accepted values: 30.4 cm−1 atm−1 (for ethylene) and 57 cm−1 atm−1 (for ammonia). To analyze the plant tissue response from the small glass cuvette, we evacuate the extra gas and then we flushed the system with pure nitrogen at atmospheric pressure for few minutes. After the system was cleaned, we transferred the gas from the sample by using a synthetic air flow near atmospheric pressure (around 1014 mb with a responsitivity of 375 cmV/W). Because ammonia is a highly adsorbing compound and the results of successive measurements are often altered by the molecules previously adsorbed on the pathway and cell wall, an intensive cycle of N2 washing was performed between biological samples, in order to have a maximum increase of 10% for the background photoacoustic signal (to ensure the quality of each determination). Ammonia molecule exhibits a very dense spectrum of rotational-vibrational absorption lines no attempt has been undertaken to make a detailed assignment of the structures in the photoacoustic spectrum.

Has a pyramidal structure with the N atom sitting at the top and the three hydrogen atoms situated equidistant from the nitrogen. The vibrational band active in the considered spectral region is the ν2 mode where the N atom moves down along the symmetry axis while the hydrogens move in phase upward and slightly away from the axis. Ethylene or ethene, is an unsaturated hydrocarbons with a planar structure. Having a C_C double bond, it is the lightest member of the alkenes group. The absorption of H2C_CH2 in the spectral region considered in this study is produced mainly by the excitation of the ν7 vibrational mode in which the four hydrogen atoms move in phase perpendicular to the molecular plane (“butterfly”-vibration). The well-defined structures are produced by the excitation of rotational-vibrational transitions in the ν7 vibration band. The close coincidence between the large absorption peak and the 10P (14) laser transition is often used for calibration

Table 2 Important parameters of ethylene and ammonia detection. Work parameters

Specifications

Plant sample pressure Total amount of HM used for germination Total amount of distilled water used for germination CO2 laser line for ethylene detection CO2 laser line for ammonia detection Synthetic air flow

≈1014 mb 10 mL

Nitrogen flow Working temperature Polycarbonate containers total volume Glass cuvette total volume Photoacoustic total volume Responsivity of the photoacoustic cell Plant sample time analysis

10 mL 10P(14); λ = 10.53 μm; α = 30.4 cm−1 atm−1 9R(30) λ =9.22 μm, α = 57 cm−1 atm−1 Linde gas: 20% oxygen, 80% nitrogen (impurities: hydrocarbons max. 0.1 ppmV, nitrogen oxides max. 0.1 ppmV) Linde gas 6.0, purity 99.9999% ≈23–25 °C 0.83 cm3/g 150 cm3 1000 cm3 375 cmV/W) ≈3 min

394

C. Popa, M. Petrus / Microchemical Journal 134 (2017) 390–399

Fig. 3. Ethylene production for germinated green peas (Pisum sativum) in HM compared with germinated green peas in distilled water (Control).

purposes in the case of systems employing line-tunable CO2 lasers although care has to be taken with saturation effect. 4. Results and discussion 4.1. Results for plant tissue respiration Regarding the results for plant tissue respiration, HM in different doses may cause metabolic disorders and growth inhibition for most of the plant species. This experimental research is devoted to analyze in the presence of synthetic air, at atmospheric pressure and at room temperature, the effects of Cd, Pb, Hg, Cu and Zn at germinated seeds/ grains using photoacoustic spectroscopy with respect to the CO2 laser frequencies. Ethylene and ammonia molecules from biological samples were measured after the germination of the seeds/grains with toxic elements and we compare the results with seeds/grains germinated with distilled water in order to analyze the plant growth regulator and degradation plant tissues. Toxicity was established by adding 10 mL of the respective HM compound solution (Cd, Hg, Pb, Cu and Zn) at the various concentrations (see Table 1) into the polycarbonate containers used to germinate 6 g of seeds (common wheat seeds, Spelt wheat, common corn, alfalfa and green peas). Distilled water was used as the control treatment and the solutions were replenished every day. The photoacoustic response of ethylene and ammonia molecules was made for peas and corn at: 92 h, 116 h and 260 h while for alfalfa and common wheat seeds were made at: 96 h, 104 h and 125 h. For Spelt wheat the research were conducted at 72 h 80 h and 96 h. The following important parameters (see Table 2) were used throughout the experiments for the detection of the gases molecules.

Fig. 3 shows the ethylene level of green peas after 92, 116 and 260 h of germination with HM compared to the ethylene concentrations of green peas germinated with distilled water. For green peas (germinated with distilled water) the ethylene molecules starts from about 85 pppb at 92 h and is reached at a concentration of about 45 ppb (at 260 h) while for the case of grains germinated with HM, the concentrations of ethylene is inhibited more for: Pb, Cu, Cd, Hg, Zn. Fig. 4 shows the ammonia concentration of green peas after 92, 116 and 260 h of germination with HM compared to the ammonia concentrations of green peas germinated with distilled water. For ammonia vapors in the respiration of grains with distilled water, it starts at a concentration of about 115 ppb at 92 h and reach a concentration of about 80 ppb at 260 h. The concentration of ammonia at green peas germinated with different HM (unlike ethylene concentration) increases more for: Pb, Cd, Hg, Cu, Zn. Fig. 5 shows the emission of ethylene (92 h, 116 and 260 h) in common corn germinated with HM correlated with emissions of ethylene for grains germinated only with distilled water. In the case of common corn, the ethylene starts at a concentration of about 14 ppb at 92 h after germination with distilled water and reached a concentrations of about 11 ppb at 260 h while for grains germinated with HM the concentration of ethylene is inhibited more for: Pb, Hg, Cd, Zn, Cu. Fig. 6 shows the emission of ammonia (92 h, 116 and 260 h) in common corn germinated with HM correlated with emissions of ammonia for grains germinated only with distilled water. Ammonia vapors in the respiration of common corn plants germinated with distilled water, starts of about 220 ppb at 92 h after germination and reach an ammonia concentration of about 260 ppb at 260 h.

Fig. 4. Ammonia production for germinated green peas (Pisum sativum) in HM compared with germinated green peas in distilled water (Control).

C. Popa, M. Petrus / Microchemical Journal 134 (2017) 390–399

395

Fig. 5. Ethylene production for germinated common corn (Zea mays) in HM compared with germinated common corn in distilled water (Control).

Fig. 6. Ammonia production for germinated common corn (Zea mays) in HM compared with germinated common corn in distilled water.

Unlike ethylene concentration, the concentration of ammonia increases for grains germinated with HM more for: Pb, Cd, Hg, Cu, Zn. Fig. 7 shows the emission of ethylene (96 h, 104 and 125 h) in alfalfa seeds germinated with HM and correlated with emissions of ethylene for seeds germinated only with distilled water. For alfalfa seeds it starts at a concentration of about 130 ppb at 96 h to reach a concentration of about 50 ppb (at 125 h) in the case of ethylene emission from germinated alfalfa with distilled water. The

concentration of ethylene is inhibited for seeds germinated with HM, more for: Zn, Cd, Hg, Cu, Pb. Fig. 8 shows the emission of ammonia (96 h, 104 and 125 h) in alfalfa seeds germinated with HM and correlated with emissions of ammonia for seeds germinated only with distilled water. Ammonia vapors in the respiration of alfalfa seeds with distilled water, it starts at a concentration of about 60 ppb at 96 h and reach a concentration of ammonia of about 127 ppb at 125 h. The concentration

Fig. 7. Ethylene production for germinated Alfalfa (Medicago sativa L.) in HM compared with germinated Alfalfa in distilled water (Control).

396

C. Popa, M. Petrus / Microchemical Journal 134 (2017) 390–399

Fig. 8. Ammonia production for germinated Alfalfa (Medicago sativa L.) in HM compared with germinated Alfalfa in distilled water (Control).

Fig. 9. Ethylene production for germinated common wheat seeds (Triticum aestivum) in HM compared with germinated common wheat seed in distilled water(Control).

of ammonia for seeds germinated with HM increases more for: Pb, Cu, Hg, Cd, Zn. Fig. 9 shows the emission of ethylene (96 h, 104 and 125 h) in common wheat seed germinated with HM and correlated with emissions of ethylene for seeds germinated only with distilled water. As can be seen, in the case of common wheat seeds germinated with HM, the concentration of ethylene is inhibited when we added 10 mL of HM. It starts with a concentration of about 40 ppb (at 96 h) and reach a concentration of 30 ppb at 125 h for germination with distilled water

case while, for HM the ethylene concentration is inhibited more for: Cu, Pb, Hg, Cd, Zn. Fig. 10 shows the emission of ammonia (96 h, 104 and 125 h) in common wheat seed germinated with HM and correlated with emissions of ammonia for seeds germinated only with distilled water. Ammonia levels from the respiration of common wheat seeds germinated in distilled water is started from a concentration of about 50 ppb at 96 h and reaches a concentration of about 175 ppb at 125 h of germination. Unlike ethylene concentration, the ammonia concentration increases more for: Cu, Cd, Pb, Hg, Zn.

Fig. 10. Ammonia production for germinated common wheat seeds (Triticum aestivum) in HM compared with germinated common wheat seed in distilled water(Control).

C. Popa, M. Petrus / Microchemical Journal 134 (2017) 390–399

397

Fig. 11. Ethylene production for germinated Spelt wheat (Triticum spelta) seeds in HM compared with germinated Spelt wheat seeds in distilled water (Control).

Fig. 11 shows the emission of ethylene (72 h, 80 and 96 h) in Spelt wheat seeds germinated with HM and correlated with emissions of ethylene for seeds germinated only with distilled water. For germinated Spelt wheat seeds with HM, the concentration of ethylene is inhibited in time for 10 mL of HM. It starts with a concentration of about 30 ppb at 72 h and reaches a concentration of about 43 ppb at 96 h after germination with distilled water. The concentration of ethylene is inhibited more for: Cu, Hg, Zn, Pb, Cd. Fig. 12 shows the emission of ammonia (72 h, 80 and 96 h) in Spelt wheat seed germinated with HM and correlated with emissions of ammonia for seeds germinated only with distilled water. Ammonia vapors it starts from a concentration of about 58 ppb at 72 h and reaches about 25 ppb at 96 h for the respiration of wheat plants germinated only with distilled water. For ammonia rate at seeds germinated with HM, increases more for: Hg, Cd, Cu, Pb, Zn. The mechanisms of HM toxicity are poorly understood and it has been speculated that some HM (including: Cd, Hg, Pb) causes damage to cells primarily through the generation of ROS [1,49], which causes single-strand DNA damage and disrupts the synthesis of nucleic acids and proteins. As an observation of our results of interest about the effect and the metabolism degree of HM on seeds or grains at different plantlets, we see that HM affecting the plant tissue and the ethylene plant growth regulator is inhibited when we compared to the ammonia level from the respiration of plantlets. Seems that HM acts as stress to plantlets and affect the plant physiology. Other previous studies [49–56] reported that HM (in biological systems) affect cellular organelles and components such as cell membrane, mitochondrial, lysosome, endoplasmic reticulum, nuclei, and some enzymes involved in metabolism, detoxification, and damage repair.

Metal ions have been found to interact with cell components such as DNA and nuclear proteins, causing DNA damage and conformational changes that may lead to cell cycle modulation, carcinogenesis or apoptosis. Several other studies [50–63] have demonstrated that ROS production and oxidative stress play a key role in the toxicity and carcinogenicity of metals. Because of their high degree of toxicity, these elements rank among the priority metals that are of great public health significance. They are all systemic toxicants that are known to induce multiple organ damage, even at lower levels of exposure. So, our findings confirm previous determinations that the synthesis of nucleic acids and proteins at plants is disrupted and that this is unlikely to be a consequence of germination with HM, affected the germination and growth. Although report exist over effect of the metal toxicity on plants, very few report exist on how HM affect seed physiology. In this research our attention was focused to the effect and to the metabolism degree of different HM on seeds for different plantlets that can affect the germination and growth level. Practically, the aim of our experiments was to monitor the ethylene and ammonia emissions in plantlets growth with distilled water at room temperature together with the effect of HM at different germinated seeds/grains. 4.2. Discussion Abiotic stress factors including salinity, drought, extreme temperatures, chemical toxicity and oxidative stress from the environment are the major causes of worldwide crop loss that pose serious threats to agricultural produce. With the ongoing technological advancements in

Fig. 12. Ammonia production for germinated Spelt wheat seeds (Triticum spelta) in HM compared with germinated Spelt wheat seeds in distilled water.

398

C. Popa, M. Petrus / Microchemical Journal 134 (2017) 390–399

industrialization and urbanization process, release of toxic contaminants like HM in the natural resources has become a serious problem worldwide. Metal toxicity affects crop yields, soil biomass and fertility. HM can obstruct the plant germination, growth and production mainly associated with the physiological, biochemical and genetic elements of the plant system. The major effects of HM on seeds are manifested by overall abnormalities and decrease in germination, reduced root and shoot elongation, dry weight, total soluble protein level, oxidative damage, membrane alteration, altered sugar and protein metabolisms, nutrient loss all contributing to seed toxicity and productivity loss [1,50,57]. The HM toxicity on green peas manifested by decreased ethylene rate was reported in the order of Pb b Cu b Cd b Zn b Hg, while for ammonia was manifested by increased rate in the order of: Pb N Cd N Hg N Cu N Zn. The HM toxicity on common corn manifested by decreased ethylene rate was reported in the order of: Pb b Hg b Cd b Zn b Cu, whereas for ammonia was manifested by increased rate in the order of: Pb N Cd N Hg N Cu N Zn. For alfalfa plantlets, the metabolism of HM was manifested also by decreased ethylene rate in the order of: Zn b Cd b Hg b Cu b Pb while for ammonia the effect of HM was manifested by increased rate in the order of: Cu N Cd N Pb N Hg N Zn. For common wheat plantlets, it was reported that toxic effect of HM is as follows: ethylene molecules were inhibited in the order of: Cu b Pb b Hg b Cd b Zn while ammonia molecules were in higher concentrations in the order of: Cu N Cd N Pb N Hg N Zn. For Spelt wheat plantlets, ethylene molecules were inhibited as follow: Cu b Hg b Zn b Pb b Cd and ammonia molecules were in higher concentrations as follow: Hg N Cd N Cu N Pb N Zn. Oxidative stress and protein damage seems to be a key piece in the toxicity and carcinogenicity of HM. When oxidants exceed the antioxidant defense, biological systems suffer oxidative stress, with damage to biomolecules and functional impairment. Our measurements are based on the photoacoustic detection of two gases molecules from the respiration of seeds/grains germinated with HM and are in good agreement with those reported in the literature [1,49–63]. Our data supports also a deregulation in the synthesis of proteins by monitoring the ammonia molecules from plantlets tissue respiration and suggests new markers that may contribute to a better understanding of HM effect.

5. Conclusions The use of related gases molecules in the respiration of plant tissue for seeds or grains germinated with HM is theoretically reasonable; metabolic changes occur in plantlets with HM that inevitably lead to the production of abnormal metabolites. These molecules are transported through diffusion and the metabolites will then be discharged into the respiration of plantlets as components of each biological sample. In the current study we analyzed the ethylene and ammonia gases molecules at seeds/grains germinated with HM and we compared the results with the signal respiration of seeds/grains germinated only with distilled water. From the results of this research, the ethylene molecules at germinated seeds/grains with HM were identified in smaller concentrations when we compared to the ammonia molecules at germinated seeds/ grains with HM. The results also reveals that the ethylene levels can be considered as the measure of plant growth regulator and the ammonia levels can be considered as a by-product of protein damage. In conclusion, the data from this research support the hypothesis that oxidative damage and tissue damage seems to be a key piece in the toxicity and carcinogenicity of HM.

Based on a non-invasive analytical method, stable in biological materials and easy to measure; we conclude that photoacoustic spectroscopy technology with tunable CO2 laser appeared to distinguish biological samples germinated with HM from biological samples germinated with distilled water. Although plant defense strategies exist to cope with HM toxicity via reduced uptake into the cell, sequestration into vacuoles by the formation of complexes, binding by phytochelatins, synthesis of osmolytes, activation of various antioxidants to combat ROS, altered expression of enzymes, over expression of genes exist, mechanisms by which germinating seeds combat HM stress remains largely unknown [49–63]. The future scope of this research remains in understanding the biochemistry of HM toxicity in germinating seeds. Understanding such strategies in seeds to overcome such stress and manipulation of pathways and biomolecules involved will lead to better agricultural produce despite HM toxicity from contaminated soil. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments We gratefully acknowledge the assistance provided by the Dr. Marian Mogildea from the Institute for Space Science (ISS), BucharestMagurele, Romania. In addition, we acknowledge the financial support of the National Authority for Research, and Innovation, in the form of a research grant conducted through the Partnerships in priority areas – project no. PNII-PT-PCCA-2013-4-0608, Integrated system for monitoring and bioremediation of metal/radionuclides contaminated area (Acronym: IMONBIO) contract funded by C72/2014, to Nucleus programme-contract no. 4N/2016 and to Space Technology and Advanced ResearchESA, project (C3 2016) no. 603, Development of a New Instrument for Monitoring of the Astronauts Health (Acronym: IMAH). References [1] P.B. Tchounwou, C.G. Yedjou, A.K. Patlolla, D.J. Sutton, Heavy metals toxicity and the environment. Molecular, clinical and environmental toxicology, Experientia Suppl. 101 (2012) 133–164. [2] J.H. Duffus, Heavy metals-a meaningless term? Pure Appl. Chem. 74 (5) (2002) 793–807. [3] H. Bradl (Ed.), Heavy Metals in the Environment: Origin, Interaction and Remediation Volume London, Academic Press, 2002. [4] B.T. Tangahu, S.R.S. Abdullah, H. Basri, M. Idris, N. Anuar, M. Mukhlisin, A review on heavy metals (As, Pb, and Hg) uptake by plants through phytoremediation, Int. J. Chem. Eng. 2011 (2011), 939161. http://dx.doi.org/10.1155/2011/939161 (31 pp). [5] G.U. Chibuike, S.C. Obiora, Heavy metal polluted soils: effect on plants and bioremediation methods, Appl. Environ. Soil Sci. 2014 (2014), 752708. http://dx.doi.org/10. 1155/2014/752708 (12 pages). [6] D. Beyersmann, A. Hartwig, Carcinogenic metal compounds: recent insight into molecular and cellular mechanisms, Arch. Toxicol. 82 (8) (2008) 493–512, http://dx. doi.org/10.1007/s00204-008-0313-y. [7] L.W. Chang, L. Magos, T. Suzuki, Toxicology of Metals, CRC Press, Boca Raton. FL, USA, 1996. [8] S. Wang, X. Shi, Molecular mechanisms of metal toxicity and carcinogenesis, Mol. Cell. Biochem. 222 (2001) 3–9. [9] D. Beyersmann, A. Hartwig, Carcinogenic metal compounds: recent insight into molecular and cellular mechanisms, Arch. Toxicol. 82 (8) (2008) 493–512. [10] C. Popa, M. Petrus, A.M. Bratu, Ammonia and ethylene biomarkers in the respiration of the people with schizophrenia using photoacoustic spectroscopy, J. Biomed. Opt. 20 (5) (2015), 057006. [11] D.C. Dumitras, S. Banita, A.M. Bratu, R. Cernat, D.C.A. Dutu, C. Matei, M. Patachia, M. Petrus, C. Popa, Ultrasensitive CO2 laser photoacoustic system, Infrared Phys. Technol. J. 53 (5) (2010) 308–314. [12] D.C. Dumitras, D.C. Dutu, C. Matei, A.M. Magureanu, M. Petrus, C. Popa, Laser photoacoustic spectroscopy: principles, instrumentation, and characterization, J. Optoelectron. Adv. Mater. 9 (12) (2007) 3655–3701. [13] C. Popa, IR spectroscopy study of the influence of inhaled vapors/smoke produced by cigarettes at active smokers, J. Biomed. Opt. 20 (5) (2015), 051003. [14] C. Popa, D.C. Dumitras, M. Paţachia, S. Banita, Testing fruits quality by photoacoustic spectroscopy assay, Laser Phys. 24 (10) (2014)http://dx.doi.org/10.1088/1054660X/24/10/105702.

C. Popa, M. Petrus / Microchemical Journal 134 (2017) 390–399 [15] M.E. Saltveit, Effect of ethylene on quality of fresh fruits and vegetables, Postharvest Biol. Technol. 15 (1999) 279–292. [16] S.F. Yang, N.E. Hoffman, Ethylene biosynthesis and its regulation in higher plants, Annu. Rev. Plant Physiol. 35 (1984) 155–189, http://dx.doi.org/10.1146/annurev. pp.35.060184.001103. [17] F.E. Huelin, J. Barker, The Effect of Ethylene on the Respiration and Carbohydrate Metabolism of Potatoes New Phytologist, 38, 2006 2. [18] D.L. Ketring, P.W. Morgan, Ethylene as a component of the emanations from germinating peanut seeds and its effect on dormant Virginia-type seeds, Laiit Pliysiol. 44 (1969) 326–330. [19] S. Banita, C. Popa, M. Paţachia, D.C. Dumitraş, Ethylene concentration at fruits under aerobic vs. anaerobic conditions, U.P.B Sci. Bull. A 75 (3) (2013) 217–226. [20] C. Popa, A.M. Bratu, D.C. Dumitraş, M. Paţachia, S. Băniţă, Photoacoustic detection of ethylene concentration in cherry tomatoes, J. Optoelectron. Adv. Mater. 16 (2014) 82–86. [21] C. Popa, D.C. Dumitraş, M. Paţachia, S. Băniţă, Improvement of a photoacoustic technique for the analysis of non-organic bananas during ripening process, Rom. J. Physiol. 60 (7–8) (2015) 1132–1138. [22] G.A. Vacha, R.B. Harvey, The use of ethylene, propylene and similar compounds in breaking the rest period of tubers, bulbs, cuttings and seeds, Plant Physiol. 2 (1927) 187–193. [23] S.P. Burg, The physiology of ethylene formation, Annu. Rev. Plant Physiol. 13 (1962) 265–302, http://dx.doi.org/10.1146/annurev.pp.13.060162.001405. [24] F.E. Denny, Effect of ethylene upon respiration of lemons, Bot. Gaz. 77 (1924) 322–329. [25] H.S. Wolfe, Effect of ethylene on ripening bananas, Bot. Gaz. 92 (1931) 336–337. [26] E.L. Overholser, A study of the harvesting and storage of gravenstein apples, Amer. Soe. Hort. Sci. Proc. 24 (1927) 252–255. [27] F.W. Allen, The influence of ethylene gas treatment upon the coloring and ripening of apples and pears, Amer. Soc. Hort. Sci. Proc. 27 (1930) 43–50. [28] K.L.C. Wang, H. Li, J.R. Ecker, Ethylene biosynthesis and signaling networks, Plant Cell 14 (Suppl) (2002) s131–s151, http://dx.doi.org/10.1105/tpc.001768. [29] C.S. Barry, M.I. Llop-Tous, D. Grierson, The regulation of 1-aminocyclopropane-1carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato, Plant Physiol. 123 (2000) 979–986. [30] W. Sakami, H. Harrington, Amino acid metabolism, Annu. Rev. Biochem. 32 (1) (1963) 355–398, http://dx.doi.org/10.1146/annurev.bi.32.070163.002035. [31] C.P. Witte, Urea metabolism in plants, Plant Sci. 180 (3) (2011) 431–438. [32] http://nzic.org.nz/ChemProcesses/production/1A.pdf. [33] P. Sharma, A.B. Jha, R.S. Dubey, M. Pessarakli, Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions, J. Bot. 217037 (2012)http://dx.doi.org/10.1155/2012/217037 (26 pp). [34] C.G. Yedjou, P.B. Tchounwou, Oxidative stress in human leukemia cells (HL-60), human liver carcinoma cells (HepG2) and human Jerkat-T cells exposed to arsenic trioxide, Met. Ions Biol. Med. 9 (2006) 298–303. [35] G.C. Yedjou, P.B. Tchounwou, In vitro cytotoxic and genotoxic effects of arsenic trioxide on human leukemia cells using the MTT and alkaline single cell gel electrophoresis (comet) assays, Mol. Cell. Biochem. 301 (2007) 123–130. [36] P.B. Tchounwou, J.A. Centeno, A.K. Patlolla, Arsenic toxicity, mutagenesis and carcinogenesis — a health risk assessment and management approach, Mol. Cell. Biochem. 255 (2004) 47–55. [37] P.B. Tchounwou, A. Ishaque, J. Schneider, Cytotoxicity and transcriptional activation of stress genes in human liver carcinoma cells (HepG2) exposed to cadmium chloride, Mol. Cell. Biochem. 222 (2001) 21–28. [38] A. Patlolla, C. Barnes, J. Field, D. Hackett, P.B. Tchounwou, Potassium dichromate-induced cytotoxicity, genotoxicity and oxidative stress in human liver carcinoma (HepG2) cells, Int. J. Environ. Res. Public Health 6 (2009) 643–653. [39] A. Patlolla, C. Barnes, C. Yedjou, V. Velma, P.B. Tchounwou, Oxidative stress, DNA damage and antioxidant enzyme activity induced by hexavalent chromium in Sprague Dawley rats, Environ. Toxicol. 24 (2009) 66–73.

399

[40] G.C. Yedjou, P.B. Tchounwou, N-acetyl-cysteine affords protection against lead-induced cytotoxicity and oxidative stress in human liver carcinoma (HepG2) cells, Int. J. Environ. Res. Public Health 4 (2008) 132–137. [41] P.B. Tchounwou, C.G. Yedjou, D. Foxx, A. Ishaque, E. Shen, Lead-induced cytotoxicity and transcriptional activation of stress genes in human liver carcinoma cells (HepG2), Mol. Cell. Biochem. 255 (2004) 161–170. [42] D.J. Sutton, P.B. Tchounwou, Mercury induces the externalization of phosphatidylserine in human proximal tubule (HK-2) cells, Int. J. Environ. Res. Public Health 4 (2007) 138–144. [43] D. Sutton, P.B. Tchounwou, N. Ninashvili, E. Shen, Mercury induces cytotoxicity, and transcriptionally activates stress genes in human liver carcinoma cells, Int. J. Mol. Sci. 3 (2002) 965–984. [44] G. Mogildea, M. Mogildea, Experimental investigation of the microwave electrothermal thruster using metals as propellant, Optoelectron. Adv. Mater-RC 4 (2010) 1826–1829. [45] M. Mogildea, G. Mogildea, Experimental research for the mass flow control of the metal vaporized and ionized with microwave used in electric propulsion, Optoelectron. Adv. Mater-RC 12 (2010) 1157–1160. [46] D.C. Dumitras, A.M. Bratu, C. Popa, CO2 laser photoacoustic spectroscopy: principles. Intech, Croatia, Chapter I in “CO2 Laser-optimisation and Application”, 2012 (ISBN 979-953-307-712-2). [47] D.C. Dumitras, A.M. Bratu, C. Popa, CO2 laser photoacoustic spectroscopy: instrumentation and applications, Intech, Croatia, Chapter II in “CO2 Laser-optimisation and Application”, 2012 (ISBN 979-953-307-712-2). [48] D.C. Dumitras, D.C. Dutu, C. Matei, R. Cernat, S. Banita, M. Patachia, A.M. Bratu, M. Petrus, C. Popa, Evaluation of ammonia absorption coefficients by photoacoustic spectroscopy for detection of ammonia levels in human breath, Laser Phys. 21 (2011) 796–800. [49] Stohs Bagchi, Oxidative mechanisms in the toxicity of metal ions, Free Radic. Biol. Med. 18 (1995) 321–336. [50] S.K. Sethy, S. Ghosh, Effect of heavy metals on germination of seeds, J. Nat. Sci. Biol. Med. 4 (2) (2013) 272–275. [51] M.S. Ahmad, M. Ashraf, Essential roles and hazardous effects of nickel in plants, Rev. Environ. Contam. Toxicol. 214 (2011) 125–167. [52] H.I. Mohamed, Molecular and biochemical studies on the effect of gamma rays on lead toxicity in cowpea (Vigna sinensis) plants, Biol. Trace Elem. Res. 144 (2011) 1205–1218. [53] C. Wang, Y. Tian, X. Wang, J. Geng, J. Jiang, H. Yu, Lead-contaminated soil induced oxidative stress, defense response and its indicative biomarkers in roots of Vicia faba seedlings, Ecotoxicology 19 (2010) 1130–1139. [54] D. Singh, K. Nath, Y.K. Sharma, Response of wheat seed germination and seedling growth under copper stress, J. Environ. Biol. 28 (2007) 409–414. [55] S. Rahoui, A. Chaoui, E.J. El Ferjani, Membrane damage and solute leakage from germinating pea seed under cadmium stress, Hazard. Mater. 178 (2010) 1128–1131. [56] J. Zhang, W.S. Shu, Mechanisms of heavy metal cadmium tolerance in plants, Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 32 (2006) 1–8. [57] Y. Mami, G. Ahmadi, M. Shahmoradi, H.R. Ghorbani, Influence of different concentration of heavy metals on the seed germination and growth of tomato, Afr. J. Environ. Sci. Technol. 5 (6) (2011) 420–426. [58] R. Athar, A. Masood, Heavy metal toxicity; effect on plant growth and metal uptake by wheat and on free living Azotobacter, Water Air Soil Pollut. 138 (2002) 165–180. [59] R.B. Meagher, Phytoremediation of toxic elemen tal and organic pollutants, Curr. Opin. Plant Biol. 3 (2000) 153–162. [60] K.P. Singh, K. Singh, Stress physiological studies on seed germination and seedling growth of source wheat hybrids, Indian J. Physiol. 24 (1981) 180–186. [61] Ö. Munzuroglu, H. Geckil, Effects of metals on seed germination, root elongation and coleoptile and hypocotyl growth in Triticumaestivumand Cucumissativus, Ach. Environ. Contam. Toxicol. 43 (2000) 203–213. [62] N. Pandey, C.P. Sharma, Effects of heavy metals Cu, Ni and Cd on growth and metabolism of cabbage, Plant Sci. 163 (2000) 753–758. [63] P. Sharma, R.S. Dubey, Lead toxicity in plant, Braz. J. Plant Physiol. 17 (2005) 35–52.