Simultaneous removal of structurally different pesticides in a biomixture: Detoxification and effect of oxytetracycline

Chemosphere 169 (2017) 558e567

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Simultaneous removal of structurally different pesticides in a biomixture: Detoxification and effect of oxytetracycline
nica Lizano-Fallas, Alejandra Huete-Soto, Mario Masís-Mora, Vero Juan Salvador Chin-Pampillo, Elizabeth Carazo-Rojas, Carlos E. Rodríguez-Rodríguez*
n en Contaminacio
n Ambiental (CICA), Universidad de Costa Rica, 2060 San Jos
Centro de Investigacio e, Costa Rica

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Removal of structurally diverse pesticides was simultaneously assayed in biomixtures.
Herbicides and some fungicides were mostly removed after 70 d (optimum period).
Neonicotinoid insecticides and triazole fungicides were not significantly removed.
Co-application of oxytetracycline affected only removal of carbendazim and metalaxyl.
Ecotoxicity towards Daphnia magna was not reduced but phytotoxicity decreased.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 July 2016 Received in revised form 28 October 2016 Accepted 20 November 2016

The biopurification systems (BPS) used for the treatment of pesticide-containing wastewater must present a versatile degrading ability, in order to remove different active ingredients according to the crop protection programs. This work aimed to assay the simultaneous removal of several pesticides (combinations of herbicides/insecticides/fungicides, or insecticides/fungicides) in a biomixture used in a BPS over a period of 115 d, and in the presence of oxytetracycline (OTC), an antibiotic of agricultural use that could be present in wastewater from agricultural pesticide application practices. The biomixture was able to mostly remove the herbicides during the treatment (removal rates: atrazine z linuron > ametryn), and suffered no inhibition by OTC (only slightly for ametryn). Two fungicides (carbendazim and metalaxyl) were removed, nonetheless, in the systems containing only fungicides and insecticides, a clear increase in their half-lives was obtained in the treatments containing OTC. The neonicotinoid insecticides (imidacloprid and thiamethoxam) and the triazole fungicides (tebuconazole and triadimenol) were not significantly eliminated in the biomixture. Globally, the total removal of active ingredients ranged from 40.9% to 61.2% depending on the system, following the pattern: herbicides > fungicides > insecticides. The ecotoxicological analysis of the process revealed no detoxification towards the microcrustacean Daphnia magna, but a significant decay in the phytotoxicity towards Lactuca sativa in some cases, according to seed germination tests; in this case, OTC proved to be partially responsible for the phytotoxicity. The patterns of pesticide removal and detoxification provide inputs for the improvement of BPS

Handling Editor: A Adalberto Noyola Keywords: Antibiotics Biopurification system Degradation Pesticides Toxicity

* Corresponding author. E-mail address: [email protected] (C.E. Rodríguez-Rodríguez). http://dx.doi.org/10.1016/j.chemosphere.2016.11.106 0045-6535/© 2016 Elsevier Ltd. All rights reserved.

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use and their relevance as devices for wastewater treatment according to specific pesticide application programs. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction In general, pest management in agriculture mainly includes the use of pesticides. Given their associated toxicity to non-target organisms, the pesticides may pose a threat in different ecosystems. In particular, occurrence of pesticides in the environment may be related to diffuse or point source contamination (Karanasios et al., 2012). The latter includes the contamination derived from leakages or improper handling of pesticide application equipment and the incorrect disposal of pesticide residues or equipment washing waters (De Wilde et al., 2007). Biopurification systems (BPS) represent a biotechnological approach for the detoxification of pesticides present in wastewater produced in agricultural activities, and therefore their goal is the treatment of point sources of contamination (Castillo et al., 2008). Removal of pesticide residues is expected to occur faster in BPS than in soil, thanks to the presence of the biomixture, the biologically active component of BPS. The biomixture is composed of three materials: a lignocellulosic substrate, employed to enhance the colonization and activity of ligninolytic fungi, widely described as capable to transform organic pollutants (Yang et al., 2013), including pesticides (Mir-Tutusaus et al., 2014; RodríguezRodríguez et al., 2013); soil, commonly pre-exposed to the target pesticides, which provides an adapted microbial community (Sniegowski et al., 2012); and finally a humic-rich component to enhance the retention of the pesticides in the matrix (Karanasios et al., 2012). Agrochemicals employed for pest control also include antibiotics (Vidaver, 2002), often applied on crops in the same manner as pesticides. Therefore, antibiotic-containing wastewaters are also produced in agricultural activities and they could be potentially disposed in BPS. However, antibiotics could negatively affect the degrading capacity of the biomixture through the inhibition of some microbial populations, as these compounds may alter several microbial-mediated processes in the environment such as the degradation of organic matter and key reactions in the biogeochemical cycles of N and S (Kümmerer, 2009). Taking into account that pesticide application programs include the use of several pesticides through the crop production cycle (according to diverse approaches of pest removal), it is desirable for the biomixtures to express a versatile degrading capacity to remove different active ingredients. This work aimed to evaluate the removal capacity of a biomixture during the simultaneous application of different pesticides (herbicides, fungicides and insecticides). The effect of oxytetracycline, an antibiotic of agricultural use, was assayed in the biomixture performance at a relevant BPS concentration. Ecotoxicological assays (acute toxicity on Daphnia magna and seed germination tests) were also conducted in order to better estimate the potential detoxification that takes place during the treatment process. The work yields relevant information on the application scope and design of biomixtures. 2. Materials and methods 2.1. Chemicals and reagents Analytical

standards

atrazine

(1-chloro-3-ethylamino-5-

isopropylamino-2,4,6-triazine), ametryn (2-(ethylamino)-4-(isopropylamino-6-(methylthio)-1,3,5-triazine)), linuron (3-(3,4dichlorophenyl)-1-methoxy-1-methylurea), metalaxyl (methyl N(methoxyacetyl)-N-(2,6-xylyl)-DL-alaninate), carbendazim (methyl benzimidazol-2-ylcarbamate), tebuconazole ((RS)-1-p-chlorophenyl-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)pentan-3ol), triadimenol ((1RS,2RS;1RS,2SR)-1-(4-chlorophenoxy)-3,3dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol), imidacloprid ((E)-1(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine) and thiamethoxam ((EZ)-3-(2-chloro-1,3-thiazol-5-ylmethyl)-5methyl-1,3,5-oxadiazinan-4-ylidene(nitro)amine) were obtained from Chem Service Inc. (West Chester, Pennsylvania, USA). Commercial formulations of atrazine (Atranex®, 90% w/w), ametryn (Agromart®, 50% w/v), linuron (Afalon®, 45% w/v), carbendazim (Agromart®, 50% w/v), metalaxyl (Abak®, 24% w/v), tebuconazole/ triadimenol 3:1 (Silvacur® Combi 30 EC, 22.5% and 7.5% w/v, respectively), imidacloprid (Manager®, 35% w/v), thiamethoxam (Engeo®, 24.7% w/v) and OTC ((4S,4aR,5S,5aR,6S,12aS)-4-(dimethylamino)-3,5,6,10,11,12a-hexahydroxy-6-methyl-,12-dioxo-1,4, 4a,5,5a,6,12,12a-octahydrotetracene-2-carboxamide; Terramicina Agrícola®, 5% w/w) were acquired from a local store. Carbofuran-d3 (surrogate standard, 99.5%) and linuron-d6 (internal standard, 98.5%) were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Solvents and extraction chemicals are listed in Ruiz-Hidalgo et al. (2014). 2.2. Experimental set-up The removal of pesticides was assayed in a biomixture containing coconut fiber, compost and soil pre-exposed to carbofuran at a volumetric composition of 45: 13: 42, respectively (pH 6.4; C 4.83%; N 0.32%; C/N 15.2; P 0.22%; Ca 0.48%; Mg 0.71%; K 0.19%; S 0.07%; Fe 31 192 mg kg1; Cu 94 mg kg1; Zn 91 mg kg1; Mn 521 mg kg1; B 66 mg kg1; EC 0.6 mS cm1). The biomixture composition was previously optimized in order to maximize the removal of carbofuran and to reduce the residual toxicity of the matrix (Chin-Pampillo et al., 2015). The removal assays were performed in buckets (14 cm radius, 29 cm height) containing 10 L (~7.7 kg) of the biomixture. Four buckets were prepared; one (M) was spiked with a mixture of the commercial formulations of herbicides (atrazine, ametryn and linuron), fungicides (carbendazim, metalaxyl, tebuconazole and triadimenol) and insecticides (imidacloprid and thiamethoxam), to give a final nominal concentration of 25 mg kg1 each (except triadimenol, 8.3 mg kg1 which is contained in the same formulation of tebuconazole at a lower concentration). The second bucket was prepared using the same pesticide mixture and concentrations, plus Terramicina Agrícola 5WP® added at a final concentration of OTC of 17 mg kg1 (M þ O). A third bucket was spiked with the mixture of insecticides and fungicides (IF), while the forth contained the insecticides, fungicides and OTC (IF þ O). All of the buckets were incubated in static conditions at 25  C until the end of the assay; water was added when necessary in order to keep constant water content in the matrix. Duplicate biomixture samples were periodically withdrawn from every single system during a period of 115 d to determine the concentration of pesticides (5 g), and to perform ecotoxicological

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assays (80 g). Each sample consisted of a composite sample obtained by collecting subsamples (at least six) at different depths in the bucket (superficial, 5 cm and 15 cm).

2.3.2. Ecotoxicity assays Toxicity tests were performed on sample elutriates, which were prepared according to the protocol EPA-823-B-01-002 (EPA, 2001). Briefly, a mixture of distilled water and sample at a ratio 4 mL/g was mechanically shaken for 1 h and centrifuged for 10 min at 3500 rpm; the resultant supernatant was used as elutriate.

2.3. Analytical procedures 2.3.1. Extraction and quantification of pesticides Extraction of pesticides was carried out following a method described by Ruiz-Hidalgo et al. (2014), which employs a mixture of water and acidified acetonitrile (formic acid 1% v/v) as extractant. Carbofuran-d3 and linuron-d6 were added as surrogate and internal standard, respectively. Analyses were performed by LC-MS/MS using ultra high performance liquid chromatography (UPLC-1290 Infinity LC, Agilent Technologies, CA) coupled to a triple quadrupole mass spectrometer (model 6460). Chromatographic separation was done at 40  C by injecting 6 mL samples in a Poroshell 120 EC-C18 column (100 mm  2.1 mm i.d., particle size 2.7 mm), and using acidified water (formic acid 0.1% v/v, A) and acidified methanol (formic acid 0.1% v/v, B) as mobile phases. The mobile phase flow was 0.3 mL min1 at the following conditions: 30% B for 3 min, followed by a 15 min linear gradient to 100% B, 4 min at 100% B and 0.1 min gradient back to 30% B, followed by 4 min at initial conditions. Selected transitions, limits of detection (LOD) and limits of quantification (LOQ) for the analytes are shown in Table 1. Conditions of the mass spectrometry detector are described in ChinPampillo et al. (2015). Removal values for each pesticide were determined as percentages with respect to the initial concentration quantified in the samples; total pesticide removal values were analogously determined considering the sum of selected pesticide concentrations. Removal data was modeled according to a first order model. Removal rate constants were analyzed by means of ANOVA tests to compare regression lines using the STATGRAPHICS Centurion software (version XVII, Statpoint Technologies, Inc.).

2.3.2.1. Immobilization of Daphnia magna. The acute toxicity tests on D. magna (EPA, 2002) were performed by triplicate, using glass vials (25 mL) and moderately hard reconstituted water without B12 vitamin complex for dilutions. Five daphnid neonates (less than 24 h) were placed in each vial and then exposed to 10 mL of proper dilutions of the elutriates, in darkness at (23 ± 1)  C. The bioassay endpoint was the immobility at 24 h and 48 h of exposure, which is assumed as equivalent to mortality. EC50, the concentration producing 50% of immobilization in the daphnids, was determined using the TOXCALC e Toxicity Data Analysis Software (Tidepool Scientific Software, CA, USA). Toxicity results were expressed as toxicity units (TU), calculated according to the expression: TU ¼ (EC50)1$100. 2.3.2.2. Seed germination tests. The phytotoxicity of the matrix during the treatment was monitored by seed germination tests with lettuce (Lactuca sativa var. Georgia; donation by School of Agronomy, University of Costa Rica); among several crops, lettuce was determined as the most sensitive to veterinary antibiotics (Pan and Chu, 2016). The relative seed germination (SG) and relative root elongation (RE) were determined using 10 seeds exposed to the elutriates, after 6 d of incubation at 22  C, and using equations (1) and (2) (US Department of Agriculture and US Composting Council, 2001). These parameters were determined by comparison to germination controls obtained by exposure to distilled water. All the tests were performed in triplicate. The germination index (GI) was calculated according to equation (3) (US Department of Agriculture and US Composting Council, 2001).

Table 1 Selected transitions and other parameters in the detection of herbicides, insecticides and fungicides in the biomixtures, using the dynamic multiple reaction monitoring (dMRM) method. Compound

Transition Precursor ion

Product ion

Ametryn

228

Atrazine

216

Carbendazin

192

Imidacloprid

256

Linuron

249

Metalaxyl

280

Tebuconazole

308

Thiamethoxam

292

Triadimenol

296

Linuron-d6 (i.s.)

255

Carbofuran-d3 (s.s.)

225

186 96 174 96 160 132 209 175 182 160 220 192 70 125 211 181 70 99 160 185 165 123

Q: quantification transition, q: qualifier transition. i.s.: internal standard; s.s.: surrogate standard. LOD: limit of detection; LOQ: limit of quantification.

Fragmentor (V)

Collision energy (V)

Retention time (min)

Type of transition

LOD (mg kg1)

LOQ (mg kg1)

106

17 25 17 25 17 33 13 17 13 17 9 17 21 40 9 21 9 13 17 13 9 21

7.96

Q q Q q Q q Q q Q q Q q Q q Q q Q q Q q Q q

7.9

15.7

10.5

20.7

2.1

4.2

11.2

22.2

21.0

41.2

16.0

31.4

16.7

32.8

24.7

48.2

9.0

17.8

e

e

e

e

106 94 72 94 94 106 82 72 92 86

9.45 1.45 2.56 11.14 9.99 13.55 1.76 12.33 11.14 7.67

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SG ¼

seeds germinated  100 seeds germinated in control

(1)

RE ¼

mean root length  100 mean root lenght in control

(2)

GI ¼

ðSGÞ  ðREÞ 100

(3)

561

3. Results and discussion 3.1. Removal of herbicides The elimination of herbicides was evaluated in the presence of fungicides and insecticides (M and M þ O systems). The biomixture was able to remove the triazine atrazine with a half-life of 21.7 d (M); this behavior was not significantly affected (p > 0.05) by the co-application of OTC at a biomixture relevant concentration (M þ O), that resulted in a half-life of 23.4 d (Fig. 1A). The removal achieved was faster than reported in soil, where half-life values range from 29 d to 75 d (Lewis et al., 2016); nonetheless, it was slower than elimination reported in other biomixtures (half-lives from 10 d to 16 d) (Chu and Eivazi, 2015; Tortella et al., 2013a). Ametryn elimination was achieved with a half-life of 40.3 d (Fig. 1B), slower than atrazine. This herbicide also belongs to the group of triazines; the structural difference between both compounds is the substitution of a eCl present in atrazine, for a eSCH3 group in ametryn, which may be responsible for the variation in their transformation rates. In the presence of OTC, the elimination of ametryn was slightly but not significantly delayed (p > 0.05) to a half-life of 46.2 d, which suggests some degree of inhibition in the microbial populations in charge of ametryn transformation. Previous reports of ametryn removal in biomixtures could not be found in specialized literature, nonetheless the elimination in soil takes place with half-lives from 37 d to 120 d (Lewis et al., 2016). Considering that accelerated triazine degradation and mineralization have been described (including the case of atrazine) (Mor
an et al., 2006; Zablotowicz et al., 2006), it is expected that the use of triazine primed soil or successive triazine applications on the biomixture could enhance the removal of these herbicides, resulting in shorter half-lives in the matrix. The removal of linuron followed the same pattern of atrazine (though this compound is structurally different to triazines), in terms of half-life values and the neglected effect exerted by OTC (Fig. 1C). No significant difference was observed among the halflives of 21.9 d and 23.2 d (p > 0.05) in the absence and presence of OTC, respectively. The biomixture improved linuron removal, as the typical half-life values reported in soil are over 48 d (Lewis et al., 2016). Similarly, the performance was better compared to another biomixture used for the treatment of several pesticides, in which linuron half-life was longer than three months (Spliid et al., 2006). Shorter mineralization half-lives (6 de25 d) have been reported in biomixtures prepared with linuron primed-soil or after successive linuron applications (Sniegowski et al., 2011). Overall, the herbicides were successfully eliminated in the biomixture, and OTC did not elicit a significant adverse effect on herbicide removal in the presence of insecticides and fungicides. 3.2. Removal of insecticides The insecticides were not significantly removed in the biomixture, either in the M or IF systems and regardless of the

Fig. 1. Removal of herbicides in a biomixture during simultaneous application of insecticides and fungicides. Systems lacking OTC (;, solid line); systems containing OTC (D, dotted line). Herbicides: atrazine (A), ametryn (B) and linuron (C). Solid and dotted lines represent the fitting of the curve to a first order model. Values plotted are the means ± SD for duplicate biomixture samples withdrawn from each treatment.

presence of OTC (Fig. 2). Both neonicotinoid compounds have been regarded as highly recalcitrant pesticides, with reports of half-lives in soil of 174 d for imidacloprid and 39 de121 d for thiamethoxam (Lewis et al., 2016). The failure of the proposed biomixture to eliminate these compounds remarks the importance of developing biotechnological strategies for the removal of neonicotinoid pesticides; in particular, the use of imidacloprid or thiamethoxam primed-soil or the use of microbial degrading consortia or highly efficient degrading strains (Pandey et al., 2009; Phugare et al.,

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Fig. 2. Concentration profile of the insecticides imidacloprid (A) and thiamethoxam (B) in a biomixture during simultaneous pesticide application. Systems containing herbicides þ fungicides þ insecticides in the absence of OTC (;, M) and during OTC co-application (D, M þ O); systems containing insecticides þ fungicides in the absence of OTC (C, IF) and during OTC co-application (B, IF þ O). Values plotted are the means ± SD for duplicate biomixture samples withdrawn from each treatment.

2013), are suggested approaches in the design of new biomixtures. 3.3. Removal of fungicides The benzimidazole carbendazim was removed by the biomixture at different extents depending on the presence of OTC and other pesticides. In the IF system, its half-life was quite low, 8.9 d (Fig. 3A), showing a faster removal than usually reported in soils (half-lives: 22 de40 d, Lewis et al., 2016), and similar to the behavior observed in other biomixtures in which the half-life was reduced from 10.0 d to 6.2 d after successive applications of the pesticide (Tortella et al., 2013b). Nonetheless, in IF þ O the half-life significantly increased (p ¼ 0.0002) by three-fold to 26.8 d, thus indicating an inhibition effect mediated by the antibiotic. In the system containing also the herbicides but not OTC (M), the half-life of carbendazim was 22.1 d (Fig. 3B); in this case, the removal delay (compared to IF) was apparently caused by the presence of the herbicides. The ability to transform carbendazim has been described in some phototrophic microorganisms (Rajasekhar et al., 2000); therefore, the presence of herbicides, typically toxic to phototrophic metabolism (De Lorenzo et al., 2001) could partially explain the inhibition in carbendazim removal observed in this work. The co-application of OTC in the M þ O system did not result in further removal inhibition (half-life 23.6 d), contrary to the findings in the systems lacking herbicides. The phenylamide fungicide metalaxyl exhibited a behavior similar to that of carbendazim. In the IF system its half-life was of 6.5 d, which considerably increased (p ¼ 0.0002) during the coapplication of OTC to 17.5 d (Fig. 3C). When herbicides were also included (M), the half-life slightly increased from 6.5 d to 8.8 d; nonetheless, in M þ O, this value curiously decreased with respect to the IF þ O system from 17.5 d to 12.9 d (Fig. 3D); despite these results, metalaxyl removal in M and M þ O was not statistically different (p > 0.05). In every case, the biomixture performance was highly efficient with respect to metalaxyl removal in soils, where half-lives of 36 de39 d have been reported (Lewis et al., 2016). Faster removal was achieved in another biomixture made of straw and compost, with a half-life of 3 de4 d after a second application (Coppola et al., 2011) or using a biomixture prepared with metalaxyl primed materials (De Wilde et al., 2010); nonetheless, when using non-primed materials, as in the case of the present work, its half-lives increased up to 26 d.

On the other hand, the triazoles tebuconazole and triadimenol were not significantly eliminated in the biomixture (Fig. 3E and F), even in the absence of OTC. The half-lives reported for both compounds are high, from 65 d to 250 d and from 47 d to 365 d for tebuconazole and triadimenol, respectively (Lewis et al., 2016), which makes them highly persistent in soils. Moreover, tebuconazole has been regarded as responsible to decrease biomass and ~ oz-Leoz et al., 2011). Further microbial activity in soils (Mun research should be conducted to find biological approaches to degrade triazoles. 3.4. Global performance of the biomixture and detoxification analysis Overall, the biomixture was capable to remove up to 61% of the total pesticides, depending on the mixture of agrochemicals and the co-application of OTC (Fig. 4), as follows: M: 61.2%, M þ O: 56.0%, IF: 54.4%, IF þ O: 40.9%. In the case of the M system, the final removal of herbicides and fungicides reached 98% and 60%, respectively. The additional removal achieved from 70 d to 115 d of treatment was quite low, and basically corresponded to the elimination of herbicides, given that the amount of fungicides and insecticides remained unchanged after 70 d. Global removal was slightly lower in the M þ O system, nonetheless the main effect of OTC co-application was displayed as a delay in the onset of removal during the first 14 d of treatment. The net final removal of herbicides was the same with respect to M, therefore the difference in total elimination was due to a decrease in fungicides removal (54% in M þ O versus 60% in M). Considering that the biomixture failed to remove the neonicotinoid insecticides, the systems lacking herbicides yielded lower relative elimination after 115 d; nevertheless, the highest removal of fungicides (69%) was exhibited in the IF system. As described above for the M systems, the OTC also delayed the removal (of fungicides) during the first 14 d of treatment. The inhibitory effect of the antibiotic appeared at short-term, however, during longterm treatment processes such as those expected in BPS, this effect seemed to be minimized. This could be partially explained by the high sorption of OTC to soils (Rabølle and Spliid, 2006) that might reduce its bioavailability during the aging of the biomixture; moreover, sorption could be increased in this matrix due to its high concentration of Fe, as OTC sorption by ternary complex formations

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Fig. 3. Removal of carbendazim (A, B) and metalaxyl (C, D) in biomixtures containing insecticides þ fungicides (A or C) or herbicides þ fungicides þ insecticides (B or D). Systems lacking OTC (C, solid line) and containing OTC (B, dotted line). Solid and dotted lines represent the fitting of the curve to a first order model. Concentration profile of tebuconazole (E) and triadimenol (F) in systems containing herbicides þ fungicides þ insecticides in the absence of OTC (;, M) and during OTC co-application (D, M þ O) or systems containing insecticides þ fungicides in the absence of OTC (C, IF) and during OTC co-application (B, IF þ O). Values plotted are the means ± SD for duplicate biomixture samples withdrawn from each treatment.

with humic acids has been demonstrated in the presence of polyvalent metal cations (MacKay and Canterbury, 2005). Similarly, transformation of the antibiotic in the matrix cannot be discarded. Long-term assays usually report the recovery of microbial communities that were initially inhibited by the antibiotics (Ding

and He, 2010), including pesticide-degrading communities that may resume their removal activity after a period of adaptation; this phenomenon is coincident with the findings of this study in terms of the degrading ability of the biomixture. In a previous report, Huete-Soto et al. (2017), demonstrated that concentrations of OTC

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Fig. 4. Total removal of pesticides during simultaneous application in a biomixture. Systems containing herbicides þ fungicides þ insecticides in the absence of OTC (M) and during OTC co-application (M þ O); systems containing insecticides þ fungicides in the absence of OTC (IF) and during OTC co-application (IF þ O). Stacked bars: Fungicides (black); insecticides (light grey); herbicides (dark grey).

Table 2 Toxicity of elutriates from biomixtures employed in the simultaneous removal of pesticides, as determined with an acute test on D. magna. Combination of pesticides applied in the biomixture

Time of treatment in the biomixture (d)

Toxicity of the elutriate after 48 h of exposure (TU)

Insecticides/Fungicides/Herbicides (M)

0 14 28 70 115 0 14 28 70 115 0 14 28 70 115 0 14 28 70 115

90 (66e127) 135 (100e182) 361 (261e521) 88a 51 (36e71) 44 (33e57) 122 (87e192) 53a 55 (39e77) 82 (61e111) 108 (84e139) 226 (182e281) 226 (182e281) 118 (64e207) 230 (168e344) 42 (32e54) 198 (106e326) 95a 89 (66e147) 190 (119e365)

Insecticides/Fungicides/Herbicides þ OTC (M þ O)

Insecticides/Fungicides (IF)

Insecticides/Fungicides þ OTC (IF þ O)

TU ¼ (EC50)1$100. a Confidence intervals (95%) could not be determined.

ranging from 0.1 mg kg1 to 1000 mg kg1 increased microbial respiration rates in the same biomixture used in this work (with respect to a biomixture without OTC); in that study, lower OTC

concentrations (0.1, 1 and 10 mg kg1) produced slightly higher respiration rates than the control (<10%), while higher doses (100, 500 and 1000 mg kg1) resulted in more accelerated respiration

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Fig. 5. Seed germination test with lettuce (Lactuca sativa) as an indicator of phytotoxicity of elutriates from biomixtures employed in the simultaneous removal of pesticides. (A), (C), (E), (G) Root elongation: the black line within the box denotes the median, the red line marks the mean, inferior and superior boundaries of the box indicate the 25th and 75th percentiles respectively, C denote 5th and 95th percentiles; mean values significantly different from the treatment “t0” are marked with an asterisk (*); mean values significantly different from the control are marked with an “a” (p < 0.05); t0-t115 denotes the time of treatment in the biomixture. (B), (D), (F), (H) Relative seed germination (SG, C), relative root elongation (RE, B) and germination index (GI, bars). Mean values significantly different from t0 are marked with an asterisk (*) (p < 0.05). None of the SG values was significantly different from the value at time zero. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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rates (up to 36% higher than the control). In this respect, the antibiotic does not produce an inhibition in the microbial activity (determined as respiration); moreover, some stress-linked stimulation is achieved in the presence of OTC. Culture-independent analysis of the bacterial and fungal communities in this biomixture by PCR-DGGE suggest that no significant alterations in the DGGE profiles are produced by the co-application of OTC; changes in microbial communities (DGGE profiles) along the treatment time are mostly ascribed to the aging of the matrix, irrespective of the
nez-Gamboa presence of the antibiotic or other pesticides (Jime et al., unpublished results). Ecotoxicological assays were performed in order to estimate the detoxification potential of the treatments, which is in the end the goal of BPS. Acute toxicity tests on D. magna revealed a similar pattern in all of the systems (Table 2), in which no clear evidence of detoxification could be determined. Moreover, final toxicity values were higher than the initial ones in most cases. The apparent lack of detoxification towards D. magna may be partially due to the persistence of tebuconazole (EC50 2.79 mg L1; Lewis et al., 2016), which is, among the studied pesticides, one of the most toxic to this microcrustacean. The other most toxic pesticides for D. magna were linuron and carbendazim (EC50 0.31 mg L1 and 0.15 mg L1, respectively, Lewis et al., 2016), nonetheless, they were mostly removed in the treatment process. Metabolites such as 2aminobenzimidazole and 3,4-dichloroaniline have been reported for carbendazim and linuron in soil, respectively (Lewis et al., 2016), both of higher water solubility than the parental compounds; therefore, if produced in the biomixtures, these metabolites could be more easily extracted in the elutriates, and in the case of 3,4dichloroaniline, this transformation product exhibits higher toxicity to D. magna (EC50 0.12 mg L1) than linuron itself. In this respect, the persistence in the toxicity and time-variations could be ascribed to the formation of temporary transformation products of high toxicity and interactions among these intermediate compounds with the non-removed pesticides, which may result in different cumulative and synergistic effects, as it has been described for several pesticide mixtures (Cedergreen, 2014; Deneer, 2000). Interestingly, final toxicity values in the M systems (with or without OTC) were lower than those obtained in the respective IF systems; this finding could be ascribed to toxicity antagonist effects caused by the presence of the herbicides contained in the M systems, as previously demonstrated in the interactions between different pesticides including triazine herbicides (Gomez-Eyles et al., 2009; Hern
andez et al., 2013). All and all, data suggest that pesticide removal behavior and toxicity are dependent on complex interactions among the matrix, the degrading microbiota and the pollutants, driven by the chemical environment modeled by the mixture of xenobiotics applied in the biomixture. Contrasting results were achieved when the phytotoxicity was analyzed by means of seed germination tests using lettuce (Fig. 5), through the parameters SG, RE and GI. Although the SG was not affected in any of the systems, significant differences were observed in M and M þ O in the other parameters with respect to the time of pesticide application (t0), which revealed that detoxification took place along the treatment. Increased RE and GI values were subsequently obtained, up to peaks at times 70 d (M) and 28 d (M þ O). In the M þ O system, lower SG produced lower RE and GI values compared to the M system. In general, the detoxification proved to be more extensive in the M systems, thus suggesting some inhibition effect due to the presence of OTC. This finding is supported by the reports of adverse effects of OTC over the growth of terrestrial and aquatic plants such as alfalfa (Medicago sativa L.) (Kong et al., 2007) and duckweed (Lemna minor) (Pro et al., 2003). The decrease in the phytotoxicity of the matrix correlates with the high removal efficiency observed for herbicides, which are likely to

affect plants at a higher extent. In the IF system, all the parameters were similar to the control (indexes around 100%) and their values were not significantly different with respect to the initial time of pesticide application. This finding indicates that the elutriates were not toxic to L. sativa, a trend that was maintained along the treatment. Clearly, the difference in IF compared to the M system, is the lack of herbicides which affect the normal germination in the latter. On the contrary, important inhibition was observed in the IF þ O system, showing RE and GI values of 38e57% and 32e54%, respectively. This observation confirms the deleterious effect of OTC on plant development; in this respect, among several veterinary antibiotics, tetracycline was the most phytotoxic towards several crops, including lettuce (Pan and Chu, 2016). As in other toxic samples, the stress response was also expressed through the formation of secondary roots and open seed leaves in some individuals. The toxicity observed in IF þ O slightly decreased during the treatment, though the point at 70 d was the only one significantly different to the initial elutriate. Given the difference in the findings from both ecotoxicological assays, the use of additional bioindicators from different trophic levels is highly suggested to obtain a more complete panorama of the residual toxicity in the matrix. 4. Conclusions The proposed biomixture was highly efficient in the elimination of the herbicides (>97% total removal) and some fungicides (metalaxyl and carbendazim), but it was not capable to remove triazole fungicides or neonicotinoid insecticides. OTC only inhibited the removal of metalaxyl and carbendazim (in IF systems), resulting in longer half-lives; however, by the end of the treatment the same net removal effect was achieved regardless of the co-application of the antibiotic. Similarly, the elimination of these two fungicides was influenced by the combination of pesticides also present in the biomixture. The removal process in the biomixture was partially successful to detoxify the matrix, as the acute toxicity towards D. magna was not removed in most cases, and even increased at some time points during the treatment; nonetheless, the phytotoxicity of the elutriates significantly dropped with respect to initial values in the M and M þ O systems; the presence of OTC resulted in important phytotoxicity towards the germination of lettuce seeds. Overall, the findings suggest that optimization of biomixtures should be performed in order to minimize the residual toxicity of the process, and to guarantee the simultaneous elimination of specific pesticide combinations according to pesticide application programs. Acknowledgements
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