To cite this paper: Margas, M., A.I. Piotrowicz-Cieślak, A. Ziółkowska and B. Adomas, 2016. Tetracycline accumulation in pea seedlings and its effects on proteome and enzyme activities. AbstractAmong antibiotics, tetracyclines are the most commonly used and detected in the environment. In this study, the amount of tetracycline taken up from soil by pea seedlings was analyzed, identified its main site of accumulation in plants and determined also changes in the protein profile of pea. The study demonstrates that pea seedlings take up tetracycline from soil and transport the drug via roots to over-ground parts and then accumulate it in the youngest parts, such as upper stem and leaves. After the taken up of drug, the activity of guaiacol peroxidase is modified and changes in the profile of proteins, as determined by two-dimensional gel electrophoresis occur. The majority of proteins (∼40%) visualized possessed molecular weight between 25 and 37 kDa. Only 8% of the proteins had molecular weight lower than 20 kDa, and 2% greater than 75 kDa. The number of spots in the control samples was 194, which is less by 49 than at the concentration of 150 mg kg -1 of soil. Isoflavone reductase was present only in seedlings growing with tetracycline. Tetracycline uptake from soil modify mainly the changes in biochemical processes connected with protein.

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Antibiotics are a new type of contaminants found in the environment. They are increasingly used in farm animal production systems and may accumulate in crops, limiting the plant growth rate and nutritive value. The aim of this study was to determine the effects of tetracycline (TC) on physiological and biochemical properties of pea seedlings. The presence of TC in the soil during 24 hours did not result in any distinct changes of the seedlings. However, after five days (120 h) of soil TC action, the seedling appearance and metabolic activities were significantly affected. Leaves lost their green coloration as a result of a 38% degradation of their chlorophyll. Total protein was isolated from shoots of pea grown for 120 h in TC-supplemented perlite (250 mg × L−1) or perlite with no TC (control plants). The 2D electrophoretic maps of proteins from non-TC shoots contained 326 spots, whereas maps of shoot proteins from TC-treated seedlings contained only 316 spots. The identity of 26 proteins was determined. The intensity of most proteins (62%) increased. This was particularly visible with diphosphate kinase, superoxide dismutase [Cu-Zn], peroxiredoxin, and glutathione S-transferase. A distinctly increased quantity of a protein involved in photosynthesis (photosystem II stability/assembly factor HCF136) was also noted. One protein was detected only in shoots of TC-treated plants (as opposed to controls); however, it could not be identified. Moreover, at the highest concentration of TC (250 mg × L−1 of perlite), a sharp increase in free-radical content was observed along with the amount of callose deposited in vascular bundles of leaves and roots and the occurrence of masses of dead cells in roots. It was found, therefore, that tetracycline which has been known for inhibiting predominantly the attachment of aminoacyl-tRNA to the ribosomal acceptor in bacteria can disturb diverse metabolic pathways in plants.

The objective of this study was to propose a method for efficient degradation of tetracycline as a water contaminant. UV-C rays, ozonation, and iron chelates were used for removal of tetracycline from water. Aqueous solution of tetracycline (5 9 10 -5 M) was exposed to UV-C rays (in two doses-6 and 12 W), ozonation (at 6-12 mg ozone), or iron chelates: iron(III) sodium ethylenediaminetetraacetate, iron(III) trisglycinate, and iron(III) citrate. For each of iron compounds, three doses were studied: 2.5 9 10 -5 M, 5 9 10 -5 M, 10 9 10 -5 M. The experiments have shown that aqueous solution of tetracycline (5 9 10 -5 M) is immediately degraded as a result of ozonation with 12 mg ozone. Absorbance of tetracycline decreased from A = 0.78 to A = 0.35 after 20-min ozone treatment of sample. The fluorescence spectra revealed the presence of two ozone-induced TC degradation products with fluorescence maxima at 523 and 531 nm appearing immediately after the ozonation treatment. On the other hand, iron(III) sodium ethylenediaminetetraacetate and iron(III) trisglycinate gave rise to a single TC degradation product with a fluorescence maximum at 531 nm, observed after 10 days of the experiment. On application of iron(III) trisglycinate, at any studied concentration, tetracycline becomes degraded faster-in 4 days. Iron(III) citrate degraded 90 % of tetracycline, when used at the level 10 9 10 -5 M. The biggest changes in tetracycline concentration were obtained as a result of ozonation and iron(III) citrate treatments.

Iron ions can be used to degrade tetracycline dispersed in nature. Studies of absorption and fluorescence spectra and quantum chemistry calculations showed that iron is more readily released from Fe(III)-citrate than from Fe(III)-EDTA, so Fe(III)-citrate (Fe(III)-Cit) is more suitable for tetracycline (TC) degradation. At 30 °C, a severe degradation of TC by Fe(III)-Cit occurred as early as after 3 days of incubation in the light, and after 5 days in the dark. In contrast, the degradation of TC by Fe(III)-EDTA proceeded very slowly in the dark. By the fifth day of incubation of TC with Fe(III)-Cit in darkness, the concentrations of the former compound dropped by 55% and 75%, at 20 °C and 30 °C, respectively. The decrease in tetracycline concentrations caused by Fe(III)-EDTA in darkness at the same temperatures was only 2% and 6%, respectively. Light increased the degradation rates of TC by Fe(III)-EDTA to 20% and 56% at 20 °C and 30 °C, respectively. The key role of the light in the degradation of tetracycline by Fe(III)-EDTA was thus demonstrated. The TC degradation reaction showed a second-order kinetics. The rate constants of Fe(III)-Cit-induced TC degradation at 20 °C and 30 °C in darkness were k = 4238M−1day−1 and k = 11,330M−1day−1, respectively, while for Fe(III)-EDTA were 55 M−1day−1 and 226 M−1day−1. In light, these constants were k = 15,440M−1day−1 and k = 40,270 M−1day−1 for Fe(III)-Cit and k = 1012 M−1day−1 and 2050 M−1day−1 at 20 °C and 30 °C; respectively. A possible reason for the higher TC degradation rate caused by Fe(III)-Cit can be the result of its lower thermodynamical stability compared with Fe(III)-EDTA, which we confirmed with our quantum chemistry calculations. Two quantum chemistry calculations showed that the iron complex with EDTA is more stable (the free energy of the ensemble is 15.8 kcal/mol lower) than the iron complex with Cit; hence, Fe release from Fe(III)-EDTA is less effective.