Use of waste materials to prevent tetracycline antibiotics toxicity on the growth of soil bacterial communities

Use of waste materials to prevent tetracycline antibiotics toxicity on the 1 growth of soil bacterial communities 2 3 Santás-Miguel, Vanesa1, 2,*, Fernández-Sanjurjo, Maria José3, Núñez-Delgado, Avelino3, 4 Álvarez-Rodríguez, Esperanza3, Díaz-Raviña, Montserrat4, Arias-Estevez, Manuel1, 2, 5 Fernandez-Calviño, David1, 2. 6 7 1Área de Edafoloxía e Química Agrícola. Facultade de Ciencias. Universidade de Vigo. As Lagoas 1, 8 32004 Ourense. Galiza * Corresponding author: [email protected] 9 2CITACA-Clúster de Investigación e Transferencia Agroalimentaria do Campus Auga, Universidad de 10 Vigo, 32004-Ourense, España. 11 3 Departamento de Edafoloxía e Química Agrícola, Escola Politécnica Superior de Enxeñaría, 12 Universidade de Santiago de Compostela. Lugo. Galicia 13 4 Departamento de Bioquímica del Suelo, Instituto de Investigaciones Agrobiológicas de Galicia 14 (IIAG/CSIC). Santiago de Compostela. Galicia 15 16 ABSTRACT 17 The increase of concentrations of tetracycline antibiotics in agricultural soils worldwide is of 18 special concern, due to its potential toxic effects on soil bacterial communities. In the present work, 19 the reuse of two waste/by-product materials as soil amendments was tested as a preventive practice 20 for reducing tetracycline antibiotics toxicity in soils. Pine bark (PB), with high percentage of 21 organic carbon, and crushed mussel shell (CMS), a frequent natural liming material, were added 22 to 4 soils in doses 0, 6, 12 and 48 g of by-product per kg-1 of soil (dry weight) of each one 23 (separately). The soils and soil-waste mixtures were then spiked with tetracycline (TC), 24 oxytetracycline (OTC) and chlortetracycline (CTC). After one day of incubation, the bacterial 25 growth was estimated in soils and soil-mixtures using the leucine incorporation technique. The 26 addition of PB to the soils showed two different behaviors, depending on the antibiotics. The 27 toxicity of TC and OTC decreased with the addition of PB (toxicities going from 6 to 25% and 28 from 5 to 36%, respectively). However, CTC toxicity did not change, or even increased in response 29 to the PB amendment. Regarding soil amendment with CMS, it was not effective to prevent the 30 toxicity of any of the three antibiotics studied. 31 32 Keywords: Crushed mussel shell; leucine incorporation; pine bark; oxytetracycline; 33 chlortetracycline 34 35 36 1. Introduction 37 In recent years, the spread of antibiotics in the environment has raised special concern. Antibiotics 38 are widely used for the medical treatment of microbial infectious diseases, and therefore tons of 39 antibiotics are administered annually to humans and animals (FEDESA, 2001; Boxal et al., 2003; 40 Kumar et al., 2005). Within the latter, which are called veterinary antibiotics (VA) (Cabello, 2006), 41 those used with livestock are of main relevance. In general, these antibiotics are rapidly excreted 42 from the treated body, since they are poorly absorbed by the animal intestine, and between 30-43 90% are excreted in the form of the original compounds, some of which are bioactive (Sarmah et 44 al., 2006). Thus, manure and slurry used as fertilizers on agricultural land are often contaminated 45 with veterinary antibiotics, with repeated applications of them being the main route of entry of 46 antibiotics into soil ecosystems (Hamscher et al., 2002; Thiele-Bruhn, 2003). Once in the soil, 47 antibiotics are potentially dangerous for soil microorganisms (Baguer et al., 2000) since they can 48 cause changes in microbial structure, affecting their biodiversity (Hammesfahr et al., 2008), and 49 these antibiotics can also cause changes in the various environmental functions of the affected 50 microorganisms (Zielezny et al., 2006; Aminov and Mackie, 2007; Liu et al., 2009; Toth et al., 51 2011; Ma et al., 2016; Santás-Miguel et al., 2020a; Santás-Miguel et al., 2020b). Tetracyclines are 52 the veterinary antibiotics most used in the European Union (33.4% of the total) (ESVAC, 2016), 53 with three of them (tetracycline, oxytetracycline and chlortetracycline) being the most consumed. 54 The maintenance of the correct growth of the bacterial communities in the soil is of vital 55 importance, since it contributes to maintaining the recycling of nutrients and the renewal of organic 56 matter (Thiele-Bruhn et al., 2012), which are crucial aspects for the maintenance and sustainability 57 of an optimal soil fertilization. However, there are few studies evaluating the effect of tetracycline 58 antibiotics on the growth of soil bacterial communities (Rousk et al., 2008; Rousk et al., 2009; 59 Santás-Miguel et al., 2020a; Santás-Miguel et al., 2020b), even if is clearly relevant, as these 60 studies showed negative effects of TC, OTC and CTC on the growth of these communities of 61 microorganisms. Therefore, finding appropriate means to prevent the toxicity of tetracycline 62 antibiotics on soil bacterial communities can be considered an important objective for soils 63 polluted with these substances. 64 A good alternative to minimize the negative effect of contaminants on soil is the use of bio-65 adsorbents materials, due to their low cost and adsorption capacity for toxic substances 66 (Fernández-Calviño et al., 2016; Fernández-Pazos et al., 2013; Romar-Gasalla et al., 2018). Pine 67 bark (PB) and crushed mussel shell (CMS) are abundant waste/by-products with potential for 68 retaining pollutants (Nehrenheim and Gustafsson, 2008; Gundogdu et al., 2009; Cutillas-Barreiro 69 et al., 2014; Farouq and Yousef, 2015). In addition, PB is mainly characterized by having high 70 organic carbon content (Cutillas-Barreiro et al., 2014), while CMS is characterized by their 71 potential for increasing soil pH (Garrido-Rodríguez et al., 2013). For organic pollutants such as 72 antibiotics, and specifically for tetracyclines, PB was much more effective than mussel shell as 73 regard its adsorption potential (despite similar surface areas), which was attributed to the organic 74 matter content of the pine bark (Conde-Cid et al., 2020a), whose interactions occur mainly through 75 cation exchange, through the formation of ternary complexes and through hydrogen bonds. 76 However, the effect of the addition of PB and CMS on soil bacterial communities has been poorly 77 studied (Fernández-Calviño et al., 2015; Fernández-Calviño et al., 2018; Santás-Miguel et al., 78 2018), but with promising results. Specifically, Fernández-Calviño et al. (2018) found that the 79 amendment of mining soils with PB, CMS and a mixture of both had positive effects on microbial 80 growth due to increases in organic matter and pH, respectively. However, the effect on soil 81 bacterial communities due to the amendment with PB and CMS in agricultural soils as a preventive 82 tool against antibiotics pollution was not studied yet. 83 In view of this background, the main hypothesis of the current work is that adding 84 appropriate doses of PB and CMS to soils polluted with tetracycline antibiotics, the effects due to 85 the toxicity of these compounds could be decreased. Therefore, the main objective of this work is 86 to evaluate the effect of PB and CMS amendments on the toxicity exerted by tetracycline 87 antibiotics (tetracycline, oxytetracycline and chlortetracycline) on the growth of bacterial 88 communities in soils. 89 90 2. Material and methods. 91 2.1. Chemicals 92 Tetracycline hydrochloride (TC, CAS. 64-75-5; ≥95% in purity), Oxytetracycline hydrochloride 93 (OTC, CAS 2058-46-0; ≥95% in purity) and Chlortetracycline hydrochloride (CTC, CAS 64-72-94 2; ≥97% in purity) were supplied by Sigma–Aldrich (Steinheim, Germany). Talc (CAS 14807-96-95 6) was supplied by Sigma–Aldrich (Steinheim, Germany). 96 97 2.2. Soil, pine bark and crushed mussel shell 98 Four agricultural soils were selected for this study, using a pool of previously studied soils 99 characterized by Conde-Cid et al. (2018). The main characteristics of the selected soils are shown 100 in Table S1 (Supplementary Material). Briefly, the pH measured in water (pHW) varied between 101 4.9 and 5.7, while the pH measured in KCl (pHKCl) ranged from 4.1 up to 4.5. The total organic 102 carbon values varied between 1.1 and 3.8% and the effective cation exchange capacity (eCEC) 103 between 4.1 and 6.8 cmolc kg-1. 104 The pine bark (PB) used was supplied by Geolia (Madrid, Spain) and the crushed mussel shell 105 (CMS) was supplied by Abonomar S.L. (Illa de Arousa, Galicia, Spain). Both materials were 106 ground and grinded by a mesh with 2 mm light. The PB and the CMS samples were previously 107 analyzed by Romar-Gasalla et al. (2018), and their main characteristics and analytical 108 methodologies used are shown in Table S2 (Supplementary Material). Briefly, PB presented an 109 acid pH (4.0), organic carbon content of 486 g kg-1 and nitrogen content of 0.8 g kg-1. The CMS 110 batch used presented a strongly alkaline pH of 9.4 and a total carbon concentration of 124 g kg-1 111 (mainly as inorganic carbon ≥ 99%), both due to the high presence of carbonates. 112 113 2.3. Experimental design 114 Each soil was divided in 7 portions (72 g dry weight each) using 500 mL polypropylene bottles 115 with lids. Three portions were amended with PB (6, 24 and 48 g kg-1), other three portions with 116 CMS separately (6, 24 and 48 g kg-1) and the last one was not amended (acting as control, without 117 any waste/by-product). The doses of the different by-products added to soils were calculated in 118 dry weight. The doses of 6, 24 and 48 g kg-1 corresponded to 12, 48, and 96 Mg ha-1 (considering 119 an effective soil depth of 20 cm and a bulk density of 1 kg dm-3). These concentrations were 120 satisfactorily used in previous works testing the effects of different by-products on soils (Ramírez-121 Pérez et al., 2013; Fernández-Calviño et al., 2015; Conde-Cid et al., 2020b, 2020c; Santás-Miguel 122 et al., 2020c). Soils and waste/by-products were mixed using a spatula and shaking the bottles by 123 hand, resulting in a total of 28 soil/amendment mixtures (4 soils x 7 portions). The procedure has 124 been carried out in the same way as in the previous studies, due to comparative purposes 125 (Fernández-Calviño et al., 2015; 2018). Once these soil mixtures were homogenized, the samples 126 were moistened at 60-80% of water holding capacity, and incubated for 30 days at 22 ºC in the 127 dark. After this time, soil pH was measured in water (1:10 soil:water ratio), and the 128 soil/amendment mixtures were spiked with antibiotics. Briefly: different amounts of tetracycline, 129 oxytetracycline and chlortetracycline were added separately to the 28 soil mixtures using talc as a 130 carrier. For this, each soil mixture was divided into 24 polypropylene tubes (15 mL), and then 12 131 mg of the talc and antibiotic mixtures were added, reaching a final concentration of 0, 0.5, 2, 7.8, 132 31.3, 125, 500 and 2000 mg antibiotic per kg-1 of soil in the mixtures. These concentrations of the 133 antibiotics tested have been successfully used in previously studies (Santás-Miguel et al., 2020a, 134 2020b, 2020c). The use of high concentrations of antibiotics to cause almost complete inhibition 135 of bacterial communities is necessary for the correct estimation of toxicity indices in a more 136 reliable way (Fox and Landis, 2006). This process has been carried out in triplicate, resulting in a 137 total of 672 microcosms (3 grams each). 138 The mixtures spiked with the different concentrations of each antibiotic were incubated for 1 day 139 at 22 ºC in the dark, and subsequently bacterial community growth was estimated using the 3H 140 Leucine incorporation method (Bååth, 1994; Bååth et al., 2001). Briefly, 1 g of soil (fresh weight) 141 was mixed with 10 mL of distilled water using a multivortex shaker for 3 min at maximum 142 intensity, followed by low-speed centrifugation at 1000 x g for 10 min, thus creating a bacterial 143 suspension in the supernatant. An aliquot (1 mL) of this suspension was transferred to 2 mL micro-144 centrifugation tubes. Then, 2 μL of [3H]Leu (3.7 MBq mL−1 and 0.574 TBq mmol−1; Perkin Elmer, 145 USA) were added with non-labeled Leu to each tube, resulting in 410 nM Leu in the bacterial 146 suspensions. After incubation for 2 h at 22 °C, bacterial growth was stopped using 75 μL of 100% 147 trichloroacetic acid. The amount of Leu incorporated into bacteria cells was determined using 148 scintillation liquid counting (Tri-Carb 2810 TR). 149 150 2.4. Data analysis 151 The bacterial growth data estimated as a function of antibiotics concentration were 152 normalized dividing all values by the control (sample without antibiotic) for each soil, waste dose 153 and antibiotic, in order to allow comparison among different dose-response curves. 154 Dose-response curves were plotted (relative bacterial growth vs log antibiotic 155 concentration) using data obtained for each soil mixture and antibiotic. Using these curves, a 156 toxicity index (log IC50) was calculated. Log IC50 (logarithm of concentration that inhibits 50% of 157 bacterial growth) was calculated using the following logistic model (1): 158 Y= c/[1+eb(a-x)] (1) 159 where Y is Leu incorporation for each antibiotic concentration, x is the logarithm of the 160 concentration of antibiotic added, a is the value of log IC50, b is a parameter related with the slope 161 of inhibition curves, and c is the bacterial growth without antibiotic added (control sample). High 162 log IC50 values indicate that the toxicity of the antibiotics added is low as regards the growth of 163 the bacterial communities, while low log IC50 values implicate high toxicity of the antibiotics 164 added. The differences between the pH values obtained after adding the different doses of each 165 by-products were estimated by analysis of variance (ANOVA) followed by Dunnett's posthoc test. 166 This test allowed the comparison of the statistical differences between the control soils (without 167 by-product) and the soils amended with different doses of by-products. 168 169 3. Results and Discussion 170 3.1. Changes in soils characteristics after the addition of pine bark and crushed mussel 171 shell 172 The amendments with PB and CMS caused changes in soil properties. The pHw values 173 measured in non-amended soils (control soils) varied between 4.9 and 5.7. After the addition of 174 PB to the soils, the pHw values decreased in 0.1 units for all PB doses, except in soil 1 (the one 175 with lower organic carbon content), for which pH values decreased in a range going from 0.2 units 176 (for PB dose of 6 g kg-1) up to 0.5 units (for PB dose of 48 g kg-1) (Table 1). In addition, the CMS 177 amendment caused marked and significant (P<0.05) changes in soil pHw (Table 2). Specifically, 178 the amendment with 6 g kg-1 of CMS caused that soil pHw increased between 0.1 and 0.5 units. 179 The amendments with doses of 24 g kg-1 and 48 g kg-1 of CMS caused increases in pH values 180 ranging between 0.3 and 1.5 units, and between 0.7 and 1.8 units, respectively. As in the case of 181 PB, the highest variations in pH values were found in the soil which shows the lowest carbon 182 content (soil 1). 183 Carrying out a theoretical calculation, the addition of PB to the soils contributes to increase 184 total carbon and nitrogen contents in 2.9 g kg-1 and 0.02 g kg-1 respectively (for PB dose of 6 g kg- 185 1), 11.7 g kg-1 and 0.08 g kg-1 respectively (for PB dose of 24 g kg-1), and 23.5 g kg-1 and 0.15 g 186 kg-1 respectively (for PB dose of 48 g kg-1), being mainly organic carbon (Cutillas-Barreiro et al., 187 2014). Due to the high C/N value of PB, its addition to the soil contributed to increase the overall 188 C/N ratio. The addition of CMS contributes to increase total soil carbon and nitrogen in 0.7 g kg-1 189 and 0.01 g kg-1 respectively (for CMS dose of 6 g kg-1), 3.0 g kg-1 and 0.05 g kg-1 respectively (for 190 CMS dose of 24 g kg-1), and 6.0 g kg-1 and 0.1 g kg-1 respectively (for CMS dose of 48 g kg-1), in 191 this case mainly as inorganic carbon (Ramírez-Pérez et al., 2013). 192 The changes in the chemical properties of the soils obtained in this study after the 193 amendment with PB and CMS are similar to those observed by other authors (Munksgaard and 194 Lottermoser, 2010; Álvarez et al., 2012; Paz-Ferreiro et al., 2012; Abd El-Azeem et al., 2013; 195 Garrido-Rodríguez et al., 2013; Pérez-Esteban et al., 2012). In addition to the modification of 196 chemical properties, the amendment with these materials may cause changes in the biological 197 properties of the soils. In this regard, increases in medium- and long-term microbial activity were 198 found in previous studies (Kokalisburelle et al., 1994; Paz-Ferreiro et al., 2012; Fernández-Calviño 199 et al., 2015; Fernández-Calviño et al., 2018), together with increases in soil fertility (Schulte and 200 Whitcomb, 1975; Álvarez et al., 2012; Paz-Ferreiro et al., 2012). 201 202 3.2. Effect of pine bark and crushed mussel shell amendments on the toxicity exerted by 203 tetracycline antibiotics on bacterial community growth 204 3.2.1. Pine bark effects 205 Fig. 1 show the inhibition curves corresponding to the different antibiotics (tetracycline, 206 oxytetracycline and chlortetracycline) obtained for 4 soils and 4 PB doses (0, 6, 24, and 48 g kg- 207 1). In general, two different behaviors were observed. 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Average values (n=3), with 562 coefficients of variation always <5% 563 0 g kg -1 PB 6 g kg -1 PB 24 g kg -1 PB 48 g kg -1 PB Soil 1 5.7 5.5 5.4 5.2 Soil 2 5.2 5.1 5.1 5.1 Soil 3 5.1 5.1 5.1 5.1 Soil 4 4.9 4.9 4.8 4.8 564 Table 2. 565 Soil pH values after amendment with different doses of CMS. Average values (n=3), with 566 coefficients of variation always <5% 567 0 g kg -1 CMS 6 g kg -1 CMS 24 g kg -1 CMS 48 g kg -1 CMS Soil 1 5.7 6.2 7.1 7.5 Soil 2 5.2 5.5 5.7 6.1 Soil 3 5.1 5.2 5.4 5.8 Soil 4 4.9 5.3 5.7 6.5 568 569