Exposure of sunflower plants to 150 mg/kg AgNPs or Ag+ in soil resulted ina significant increase in
vitamin A and E concentrations (Fig. 6.14). The greatest increase was with AgNPs > Ag+ > Control. The
exposure of sunflower plants to AgNPs and Ag+ stimulated the production of ascorbate oxidase
resulting in ROS production, which can lead to a production of L-ascorbic acid. , Vitamins A and E (alpha-tocopherol) act as a defence in addition to ascorbate oxidase, which oxidises L-ascorbic acid to dehydroascorbic acid, and therefore the amount of dehydroascorbic acid increased and total ascorbic acid decreased (Shimada and Ko, 2008). Zengin and Munzuroglu (2005) reported a significant increase in the antioxidant vitamins A and E in the bean plant (Phaseolus vulgaris)
following exposure to heavy metals Pb (1.5, 2, 2.5 mM), Cu (0.1, 0.2, 0.3 mM), Cd (0.05, 0.06 and 0.08 mM) and Hg (0.02, 0.04 and 0.06), for 10 days, which is in agreement of my findings.
Vitamin C (Fig. 6.15) is a free radical scavenger against O3 (Gupta et al., 1991). It could also act as an
antioxidant in photosynthetic organisms and reduce H2O2 (Pehlivan, 2017). Exposure of Bacopa
monnieri to different Hg concentrations for 14 days resulted in a significant increase in vitamin C
(Sinha et al., 1996). This finding with vitamin C is in agreement with my studies on exposure of
sunflower to AgNPs/Ag+. Zengin (2013) measured the stress effect from exposure of the bean plant
(Phaseolus vulgaris L.) to several heavy metals, namely, Ni, Co, Cr, Zn, on the concentration of
vitamins A, E, and C in plant leaves. He found that the amounts of these vitamins were significantly increased in combination with carotenoids, while chlorophyll content decreased, which is consistent with our findings. Collin et al. (2007) reported that vitamins C and E can act as chain-breaking
scavengers for proxy radicals and as a synergist with vitamin E because vitamin C can donate an H atom to vitamin E-derived phenolate radicals, thus regenerating its activity. Similar findings were observed in my studies also.
Vitamin E (alpha-tocopherol) can quench singlet oxygen (1O2) and acts as a chain-breaking
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components by physically quenching and reacting chemically with 1O2. Scavenging of 1O2 oxygen by
vitamin E in chloroplasts results in formation of α-tocopherol quinone, which is involved in cyclic electron transport in thylakoid membranes (Collin et al., 2007).
6.7
Conclusions
Exposure of sunflower plant to AgNPs and Ag+ (from AgNO
3) causes oxidative stress similar to that
caused by other heavy metals such as Cd, Pb, Hg, and Ni. The stress of exposure to such heavy metals can lead to ROS and elevated activity in defence enzymes (Cat, SOD, GST, and GPx) in addition to increased activity of peroxidases (LPO, pyrogallol peroxidase and guaiacol peroxidase). The major impact of the two Ag forms, AgNPs and Ag+, on the sunflower plant is to reduce total
protein and inhibit chlorophyll synthesis in the leaf, which can result in reduced growth and yield of the crops. Overall, the responses of the parameters measured were most marked in those plants exposed to Ag+ > AgNPs.
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Chapter 7
Biochemical Toxicity of Silver Nanoparticles and Ag
+(AgNO
3) to
Aporrectodea caliginosa Earthworm, Using Filter Paper as a Matrix
7.1
Abstract
Earthworms A. caliginosa were exposed to varying concentrations of AgNPs (0, 0.3, 3, 30, 300 mg/l)
or Ag+ (from AgNO3) (0, 0.03, 0.3, 3, 10 mg/l) in moistened filter paper in Petri dishes for 24 and 48 h
(n = 4). The filter paper contact test was used as a rapid screening of sub-clinical toxicity of AgNPs and Ag+ toxicity to A. caliginosa and also as a practice run to get familiar with earthworm
homogenisation and enzyme analyses for the main earthworm soil study described in Chapter 8. The sub-clinical toxicity caused by these chemicals through dermal uptake was assessed by measuring the effect on selected antioxidant enzymes, namely, superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase, in addition to lipid peroxidation (LPO). Based on the results, the enzyme activities and LPO were slightly higher in earthworms exposed to Ag+than in earthworms exposed to AgNPs, and greater at 48 h than 24 h.
7.2
Introduction
Many have shown that the release of AgNPs used in different industries can have an adverse impact on the environment (McGillcuddy et al. 2017; Wilson 2018). Among such industries, two major ones
used the most AgNPs in their products, namely, the clothing industry and the medical field. Some of these AgNPs will likely end up in sewage treatment plants and the sludge could be released into the environment as fertiliser in some plantations. The AgNPs release Ag ions that are toxic to soil microorganisms and other organisms that come in contact with soil (McShan et al., 2014). Another
impact of AgNPs is that they are phytotoxic to some plants, such as Phaseolus radiatus, where
seedling growth is adversely affected on exposure to AgNPs in soil (Lee et al., 2012).
This study was conducted to quickly screen the potential effects of AgNPs on A. caliginosa
earthworms and to compare these with exposure to Ag+ (AgNO
3) using filter paper as a matrix and
more importantly as a trial to get familiarised with biochemical methodologies to be used in the main earthworm study described in Chapter 8. The toxicities of AgNPs and Ag+ were evaluated by
75
chemicals, using filter paper as a matrix. I used filter paper as a matrix in this short-term study to quickly scan the potential range of AgNPs/Ag+ concentrations that could be used in the A. caliginosa
soil study in Chapter 8. The antioxidant enzymes that were monitored (CAT, GPx, SOD, GST) are those involved in detoxification of xenobiotics to minimise the oxidative stress caused by ROS (Lionetto et al., 2012). Lipid peroxidation (LPO) was also measured. Malondialdehyde (MDA), 4-
hydroxy-nonenal (HNE) and the F2-isoprostane 15(S)-8-iso-prostaglandin F2α (15(S)-8-iso-PGF2α) are the most frequently measured biomarkers of lipid peroxidation (Tsikas, 2017). MDA is the prototype of the so-called thiobarbituric acid reactive substances (TBARS).
The use of biochemical markers is preferred to characterise the potential hazard of AgNPs and these could be assayed by different techniques (Ray et al., 2009). The antioxidant enzymes in A. caliginosa
homogenate were measured following exposure to 300, 30, 3, 0.3 μg/ml AgNPs and 10, 3, 0.3, 0.03 μg/ml Ag+ respectively for 24 and 48 h. The selection of these concentrations was based on the Petri dish LD50 studies of AgNPs and Ag+ to A. caliginosa carried out in our laboratory a few years ago
(Zhan, 2012). As mentioned above, the objective of this study was to test the potential doses of AgNPs and Ag+ to be used and to become familiar with the earthworm homogenisation technique and also the antioxidant enzymes and LPO analyses in preparation for the main earthworm experiment in soil.
7.3
Materials and methods
7.3.1
Chemicals and reagents
Trisodium citrate and sucrose di-potassium hydrogen phosphate were purchased from BDH (UK). All the other chemicals, including AgNO3, were supplied from Aldrich-Sigma (St. Louis, MO, USA).