Biodegradation of phytosanitary products in biological wastewater treatment

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Biodegradation of phytosanitary products in biological wastewater treatment A. Massot b, K. Este`ve a, P. Noilet c, C. Me´oule d, C. Poupot a, M. Mietton-Peuchot a,* a

Univ. de Bordeaux, ISVV, EA 4577, INRA, USC 1219 OENOLOGIE, 210 chemin de Leysotte, 33882 Villenave d’Ornon, France Amarante Process, 210 rue de Leysotte, 33882 Villenave d’Ornon, France c Bucher Vaslin, 49290 Chaloˆnnes-sur-Loire, France d Agroenvironnement, Pian sur Garonne, 33490 St Macaire, France b

article info

abstract

Article history:

Agricultural activity generates two types of waste: firstly, biodegradable organic effluents

Received 12 October 2011

generally treated by biological processes and, secondly, phytosanitary effluents which

Received in revised form

contain residues of plant protection products. The latter are collected and treated. Current

20 December 2011

technological solutions are essentially based on concentration or physicalechemical

Accepted 28 December 2011

processes. However, recent improvements in the biodegradability of pesticides open the

Available online 11 January 2012

way to the consideration of alternative, biological, treatment using mixed liquor from wastewater plant activated sludge. The feasibility of the biological treatment of viticultural

Keywords:

effluents has been evaluated by the application of pesticides to activated sludge. The

Phytosanitary

necessity for selection of a pesticide-resistant biomass has been highlighted. The elimi-

Effluent

nation of the phytosanitary products shows the potential of a resistant biomass in the

Pesticides

treatment of pesticides. The aerated biological storage ponds at three wineries, followed by

Biological treatment

a sand or reed-bed filter, were used for the treatment of the total annual volume of the viticulture effluents and validate the laboratory experiments. The results show that the biological purification of pesticides by activated sludge is possible by allowing approximately 8 days for biomass adaptation. Stability of purification occurs between 20 and 30 days. ª 2012 Elsevier Ltd. All rights reserved.

1.

Introduction

The nature of the phytosanitary effluents resulting from wineyard applications is very variable as over 350 active molecules may be applied. One of the main characteristics of these effluents is their toxicity in terms of the environment. Existing techniques for the remediation of such polluted water are based on physical and/or electrochemical treatments (Olette et al., 2008). The only feasible option for the treatment of such biologically persistent wastewater is the

use of advanced technologies based on chemical oxidation, such as the Advanced Oxidation Processes (AOPs), widely recognized as highly efficient (Lucas et al., 2009; Oller et al., 2011). In some cases it has been found that the toxicity of the original effluent actually increases and peaks during early pre-treatment due to the formation of toxic intermediates (Este`ve et al., 2009b). Winemaking generates significant volumes of wastewater. Depending on their origin, these effluents are described as winemaking effluents or viticultural effluents. The winemaking effluents emanate from the

* Corresponding author. Tel.: þ33 5 57 57 58 70. E-mail addresses: [email protected] (A. Massot), [email protected] (P. Noilet), [email protected] (C. Me´oule), [email protected] (M. Mietton-Peuchot). 0043-1354/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2011.12.055

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Nomenclature ML3, 5-day biochemical oxygen demand ML3, Chemical oxygen demand %, Inhibition concentration of the biotest ML3, Extracellular Polymeric Substances ML3, Half saturation constant for microbial growth KLapure water T1, Mass transfer coefficient in pure water KLasludge T1, Mass transfer coefficient of the sludge Kow e, Octanolewater partition coefficient MLVSS ML3, Mixed liquor volatile suspended solids MS ML3, Dry matters ML3, Oxygen concentration [O2]

BOD COD EC50 EPS Ks

washing down of cellars and cellar equipment (harvesters, wine press, tanks, filters.). Winemaking wastewater is characterized by high concentrations of biodegradable organic matter. The Chemical Oxygen Demand (COD) is high (ranging from 3 to 30 g L1), the pH is in the region of 4e6 (except during descaling), and the measured Suspended Solids (SS) are generally between 1 and 2 mg L1 (Racault et al., 1995). The bulk of wastewater (typically over 80%) is generated during the 3-month production period (Oller et al., 2011). Effluent production is characterized by significant seasonal fluctuation with a prominent peak during the grape harvest with very little waste being generated from May to August (in the northern hemisphere) (Racault, 2003). Two types of treatment are privileged: aerobic biological treatment by aerated storage or activated sludge, and anaerobic digestion (Massot, 2007; Agustina et al., 2008). In the case of viticultural or other agricultural effluents, the techniques involve concentration processes (flocculation, reverse osmosis) prior to either elimination as final waste or chemical, photo-chemical or electrochemical degradation followed by complementary treatment (activated carbon) or a biological process with a specific biomass. Nowadays, the majority of organic phytosanitary products used in agriculture are largely biodegradable. Biodegradability of a target substance is determined in the light of three main ecological considerations (Pitter and Chudoba, 1990): Is bacterial adaptation necessary? What are the rate and the degree of biodegradation under given conditions? Is it possible to achieve final mineralization or are toxic compounds produced? To answer these questions, many testing strategies have been evolved (Lapertot and Pulgarin, 2006; Nsabimana et al., 1996). Chemicals entering the secondary treatment stage may be removed through sorption into the activated sludge, air stripping by the forced injection of air, and biotransformation/biodegradation. Zipper et al. (1999) show that activated sludge degrades certain pesticides (86e98%) within 7 days. Similar results are obtained for other pesticides (Sahinkaya et Dilek, 2005). Katsoyiannis and Samara (2005) investigated the distribution of nineteen organochlorine pesticides between the dissolved and the sorbed phases of wastewater and sludge. Biotransformation is considerable for compounds with a moderate octanolewater partition coefficient log Kow (3e3.5), while it is dramatically reduced for compounds with log Kow (4e6) (Byrns, 2001; Chen

OUR OURex OURend SS S/X S TOC X Yx/S a m mmax qc

ML3T1, Oxygen uptake rate ML3T1, Exogenous respiration rate ML3T1, Endogenous respiration rate ML3, Suspended Solids T1, Initial substrate to micro-organisms ratio ML3, Substrate concentration ML3, Total organic carbon ML3, Biomass concentration e, Conversion yield e, Corrective factor T1, Specific growth rate T1, Maximum specific growth rate T, Solid retention time

et al., 2007). The relative distribution of POPs between the sludge and the effluent treatment streams shows different behaviors in different compounds, with accumulation in sludge varying between 39% and 98%. The first part of this study relates to the determination of the biological and kinetic constants of the activated sludge for viticultural effluents containing three plant protection products commonly used in viticulture. The degradation of these molecules is monitored over a period of time (200 h) and the resulting data are used to estimate the hydraulic retention time of the biological treatment. In the second part of the study real pesticide effluents are treated in three winery wastewater treatment plants. The biological treatments are successive, bearing in mind that the production of winemaking effluents is very low during the summer months. The relatively low volume of agricultural effluents (0.2e0.3 m3 ha1 year1) was stored in a special tank and could thus be set aside for treatment during this period. The work provided answers to the question of whether phytosanitary effluents may be treated in a winery wastewater plant. For the total annual volume of the viticultural effluents treated, the aerated biological storage ponds of the wineries were used, followed by filtration in a sand or reed-bed filter. Effluents from wineries are essentially produced during the harvest period. Pesticide-contaminated effluents are treated at other periods using the same process.

2.

Material and methods

2.1.

Bioreactor and activated sludge

The mixed liquor was collected from the aeration tank of a winemaking biological treatment plant (Bordeaux, France). The mixed liquor was retained in a 30L bioreactor (BRAUN BIOSTAT). Mixing inside the bioreactor was performed by a turbine with six Rushton blades and was aerated by means of a perforated crown. Several probes were associated in order to monitor various parameters: pH, oxygen, and temperature level. These sensors controlled the pumps, making it possible to feed the medium in substrate, to inject a base and an acid to control the pH, or to recycle the sludge from the settling tank. All the parameters such as air flow rate, dissolved oxygen concentration (4 mg L1), stirring velocity (50 rpm), pH (7),

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temperature (20  C) and the flow rate of the pumps, were monitored. The bioreactor was supplied with diluted wine as the composition of this substrate was similar with the one of a traditional winemaking effluent. The hydraulic residence time was 10 days. In the experiments, samples taken from the reactor at various time intervals throughout the degradation process were analyzed for biomass by means of measurement of suspended solids (SS)-EN 872, mixed liquor volatile suspended solids (MLVSS), dry solids content (NF T 90-029), and turbidity. In parallel with the bioreactor, a jar-test provided with aeration systems allowed work to be undertaken simultaneously on six 1-L samples with different kinetic parameters.

2.2.

Reagents and chemicals

The tested pesticides were selected from chemical substances commonly used in vineyards. Good agricultural practices (GAP) recommend that, in order to promote best practices, two rinses of the pulverization tank must be carried out in the vineyard prior to washing down the application equipment. Thus, in order to simulate a real agricultural effluent, the pesticide concentrations were defined as follows: 0.43 g L1 pyrimethanil (C12H13N3), 0.017 g L1 flufenoxuron (C21H11ClF6NO4) and 0.13 g L1 dimetomorph (C21H22ClNO4). The pesticide samples were provided by BASF France. Each pesticide was investigated separately. The proteins and humic substances used for calibration ranges were bovine serum albumin (SIGMA) and humic acids (FLUKA).

2.3.

Analysis

The biomass was observed using a Hund H 500 microscope, coupled with a COHU Monochrome camera. Archimed (Microvision Instruments) image analysis software permitted the identification of the main micro-organisms. To estimate the efficiency of the pilot unit, physical-chemical parameters were evaluated: chemical oxygen demand (COD) (NF T 90-101), total organic carbon (TOC), and active compound concentrations. Total organic carbon (TOC) was measured by a direct method developed by HACH Lange. The protocol is described in method 10173 provided by HACH LANGE. The method established by Dubois et al. (1956) was used to analyze the polysaccharides content. The proteins and humic substances were measured by the Lowri method modified by Frolung and Keiding (1994). The methods for the determination of the EPS have been detailed in a previous paper (Este`ve et al., 2009b). The samples of mixed liquor were filtered with a glass filter (GFC Watman) prior the extraction of the active molecules. Ethyl-acetate extraction is used for pyrimethanil and dimetomorph. The molecules were checked by High Performance Liquid Chromatography (HPLC) with a diode array UVeVisible detector (280 nm). The separation of the products was carried out on a TSK gel ODS-80TS non-polar column (T ¼ 35  C; acetonitrile/water-80/20; 0,2 mL/min). For the industrial tests, mean samples (1 L during 24 h) were collected after the filter in 1 L glass vials and stored at 4  C until the analysis of the pesticides concentrations by the laboratory LDA Drome (26), France. The ecotoxicity tests were carried out according to standard EN 11348-3 for the experiments at the laboratory

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scale (determination of the inhibitor effect of wastewater samples on the Vibrio fischeri luminescence e Part 2: Method using dehydrated bacteria) and for industrial experiments with daphnia magna test (NF EN ISO 6341 e test of inhibition of the mobility of daphnia magna e test of acute toxicity) and algae test (NF ISO 8692 e test of inhibition of the growth of the fresh water algae with unicellular green algae). The toxicity is evaluated with different dilutions of the toxic effluent. When the initial concentration is known, the EC can be calculated as the concentration of the toxic molecule to obtain a given percentage of the inhibition (50 or 70). For industrial effluents where the initial concentrations are unknown, the EC50 is the value of the ratio: volume of the effluent*100/total volume (%) to obtain 50% of the inhibition of the micro-organisms involved in the test. Respirometric essays are used to calculate different parameters: the mass transfer coefficient (kLa), the oxygen uptake rate (OUR), the maximum growth rate (mmax), the half saturation constant for microbial growth (Ks) and the conversion yield (Ys/x). The methods are described in supplementary data.

3.

Results and discussion

3.1. Characterization of the initial mixed liquor from the winemaking activated sludge plant The treatment of wastewater by activated sludge is based on the development of appropriate bacterial aggregates and other associated organisms (biological flocs). Ciliated protozoa play an essential role in the whole process by removing dispersed bacteria through grazing, which may otherwise result in high turbidity in the effluents. The presence of toxic substances in the influent may induce changes in the activated sludge ecosystems, thus affecting its activity and the biological performance of the wastewater treatment plant (Papadimitriou et al., 2007). Whereas the nature of the biomass is relatively well known in a context of industrial or urban waste treatment (Watanabe et Hino, 1996; Snaidr et al., 1997; Eschenhagen et al., 2003; Lin et al., 2003; Brose´us et al., 2009), the available literature offers relatively few references on the treatment of winemaking effluents. According to Petruccioli et al. (2002), the effluent may be degraded by populations of the bacteria Pseudomonas and Agrobacterium as well as by the yeasts Saccharomyces and Candida. A first observation is that during the treatment of winemaking effluents, the biomass is essentially composed of Protozoa: Flagelates, Sarcodines and Ciliates. Some Metazoa such as Rotifers or Nematodes are also present. In addition, Ciliates constitute the most varied microfauna. Due to the continuous supply of winemaking effluents, the significant presence of oenological micro-organisms was observed, in accordance with the observations of Petruccioli et al. (2002). Moreover, these observations are reminiscent those made by Drakides (1978) in the activated sludge of traditional wastewater units, or by Canler et al. (1999). The presence of Monas and Actinopodes is an assurance of the specific nature of winemaking effluents. Respiration and kinetic parameters are defined using successive additions of 1 mL of winery effluent at a concentration of 230 g COD L1 to the bioreactor containing 15 L of

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activated sludge at 0.6 g SS L1. The respiration rate corresponding to the consumption of 1 mL of wine remained constant at 0.22 mg L1 min1. When the aeration restarted, the calculated KLa remained constant at 0.23 min1. To date, there is no data available in the literature on the values of the maximum specific growth of micro-organisms (mmax) and the half saturation coefficient (Ks) for activated sludge supplied with a winemaking effluent. The tests set up allowed a first estimation of these parameters by following the evolution of the concentration of the suspended solids. Two types of sludge were produced in the bioreactor fed with two different loads (Si): Si ¼ 0.6 g COD L1 d1 and Si ¼ 1.2 gCOD L1 d1. These mixed liquors were distributed in the aerated jar-test and fed with different initial concentrations in substrate (S0). Fig. 1 illustrates the evolution of suspended solids over time. The increase in biomass concentration in each jar-test was quasi linear. This was even more significant when the concentration S0 was high: increase of 15% for S0 ¼ 92 mg L1 and of 31% for S0 ¼ 920 mg L1, with Si ¼ 0.6 kg COD m3 d1. At S0 ¼ 920 mg L1, suspended solids increased by 31% and 42% for Si ¼ 0.6 kg COD m3 d1 and Si ¼ 1.2 kg COD m3 d1 respectively. The graphs ln(Xt/X0) ¼ F (t) (X corresponds to the concentration in suspended solids) lead to the value of the growth rate (m) (Fig. 2). The layout of 1/m versus 1/S0 allows calculation of mmax and Ks. The values obtained for mmax, Ks and Yx/S at 0.6 kg COD m3 d1 were respectively 0.033 h1, 79 mgL1, and 0.4. With 1.2 kg COD m3 d1, the biological constants were 0.047 h1, 130 mg L1, and 0.56. The values of the biological constants Ks and Yx/S determined for a winemaking effluent were close to those found for a synthetic effluent with glucose (Gaudy and Srinivasaraghaven, 1974). However, mmax was 10 times lower when the substrate feeding the bioreactor was wine. This could be due to the biodegradability of the wine compounds being modified in the presence of polyphenols.

Fig. 1 e Suspended solids versus time for activated sludge produced with two different loads Si (0.6 g COD LL1 dL1 and Si [ 1.2 g COD LL1 dL1) and with increasing concentrations (S0) of wine (A: 92 mg LL1, -: 230 mg LL1, :: 345 mg LL1, x: 460 mg LL1, -: 690 mg LL1, C: 920 mg LL1).

Fig. 2 e Evolution of log (Xt/X0) versus time used to calculate the respirometric parameters for the biological treatment of wine effluent with two different loads (Si [ 0.6 g COD LL1 e lines in the graph e and S0 [ 1.2 g COD LL1 e dashes -). and with increasing concentrations (S0) of wine (A: 92 mg LL1, -: 230 mg LL1, :: 345 mg LL1, x: 460 mg LL1, +: 690 mg LL1, C: 920 mg LL1).

3.2. Characterization of the mixed winemaking and pesticide effluent following biological treatment in the pilot unit Loading the bioreactor with a toxic effluent requires the study of biomass adaptability to phytosanitary products. 250 mL of a solution of phytosanitary product (0.43 g L1 pyrimethanil (C12H13N3), 0.017 g L1 flufenoxuron (C21H11ClF6NO4), and 0.13 g L1 dimetomorph (C21H22ClNO4)) were introduced, together with 750 mL of mixed liquor, into the jars of the aerated and mixed jar-test. For a daily addition of winemaking effluent into the reactors, the microscopic observation of sludge did not show qualitative modifications of the biomass. The microfauna (Protozoa: Flagelates, Sarcodines, Ciliates.) does not change with the addition of the molecules even if the bacteria seem to be deflocculated which suggests its possible adaptation to the phytosanitary products. Three reactors were supplied at determined times with different substrates. Results are shown (in Figs. 3 to 6) for the three reactors: the pilot with wine only, for wine with dimethomorph added (Ci ¼ 32 mg L1), and for wine with pyrimethanil added (Ci ¼ 107 mg L1). The initial COD for the three samples was fixed at 10 g L1 and the initial concentration in biomass at 0.5 g L1 in order to obtain a ratio of S0/X0 ¼ 20. In the presence of pesticide, the determination of mmax and Y brought to light significant decreases in their values due to the inhibitory effect of the pesticide: mmax was 0.016 h1 for dimethomorph and 0.012 h1 for pyrimethanil (Si ¼ 0.6gCOD L1 d1) (Fig. 3). The yield coefficient (Y ) values exhibited a similar trend to that of the specific maximum growth rate. The calculated conversion yields were 0.3 and 0.2 respectively. Evolutions in

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Fig. 3 e Evolution of log (Xt/X0) versus time used to calculate the respirometric parameters of the biological treatment of pyrimethanil (A) (Si [ 0.43 g LL1) and dimethomorp (-) (Si [ 0.13 g LL1). Comparison with the treatment of wine effluent (:).

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Fig. 5 e Evolution of mixed liquor volatile suspended solids (MLVSS) versus time during the biological treatment of three effluents: wine effluent (A), pyrimethanil effluent : (Si [ 0.43 g LL1) and dimethomorph - (Si [ 0.13 g LL1).

COD concentration, in volatile suspended solids, and in respiration rate are represented in Figs. 4e6. After a short adaptation stage, a decrease was observed in COD coupled with an increase in volatile suspended solids and the oxygen uptake rate. The evolution of the COD curves was similar for the different pesticides: the decrease phase was much more pronounced when wine was the only substrate. After nine days, the pilot showed that the wine was completely consumed by the biomass whereas a residual COD was quantified in the reactors containing the mixtures of wine and pesticides. The increase in MLVSS confirms the selection of an active biomass capable of treating the effluents. This is confirmed by the reduction of the COD and of the oxygen uptake rate. The monitoring of the evolution of effluent toxicity permitted monitoring of the elimination of the phytosanitary products. Table 1 presents the results of the toxicity of the synthetic effluents for three molecules after nine days. The results are

compared with the initial toxicity. The EC50 of the initial effluents are similar (25e35%). After nine days, the increase of EC50 for the effluents with flufenoxuron and dimethomorph (58 and 53% respectively) is linked to the degradation of the molecules by the biomass. The EC50 of the effluent with pyrimethanil is not modified after 9 days. Two possibilities could be given: the molecule is not degraded by the biomass or the molecule still very toxic after nine days even if this concentration is reduced. The initial concentrations of pyrimethanil and dimetomorph were respectively 107 mg L1 and 32 mg L1. From the first day, the concentrations decrease and remain stable during two days, equal to 25 and 35 mg L1. This initial reduction probably corresponds to the adsorption of a part of the pesticide by the biomass. The micro-organisms of the biomass then degrade this adsorbed fraction. After 9 days of treatment the efficiency was 60% and 74% for dimethomorph and pyrimethanil respectively. After thirty days of treatment, pesticide concentrations were measured in the effluent and in

Fig. 4 e Soluble COD versus time during the biological treatment for 2 effluents with active molecules (pyrimethanil : (Si [ 0.43 g LL1) and dimetomorph (Si [ 0.13 g LL1)) and for the standard effluent (A wine).

Fig. 6 e Evolution of oxygen uptake rate (rO2) versus time during the biological treatment of three effluents: wine effluent (A), pyrimethanil effluent (:) and dimethomorph (-) effluent.

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Table 1 e Effluent toxicity (EC50) measured with the determination of the inhibitor effect of wastewater samples on the Vibrio fischeri luminescence. (EN 11348-3). Initial toxicity (EC50i) and residual toxicity after 9 days (EC50f) of biological treatment for three active molecules mixed with winemaking effluents. Phytosanitary products Initial EC50 i (%)a EC50 f after 9 days (%) EC50 f/EC50 i

Dimethomorph

Pyrimethanil

Flufenoxuron

29.40 52.20 1.78

36.40 39.90 1.10

26.80 57.70 2.15

a EC50 ¼ volume of the effluent*100/(volume of water þ volume of effluent).

the sludge. Residual concentrations in the sludge showed that the active substances were more or less concentrated. Over 99.94% of the dimethomorph was removed from the effluent according to the measurements of residual concentrations in the sludge (<0.25 mg L1). The concentration of pyrimethanil in the effluent was reduced by 98.2%, the substance being almost entirely concentrated in the sludge. This result explains the observed stability of the toxicity during nine days for pyrimethanil. Treatment experiments (data not shown) have been carried out with a typical annual effluent from vineyard pulverizations (Este`ve et al., 2009b). Fourteen molecules were mixed at the mean concentration corresponding to a double rinse of the pulverization material and then injected into the activated sludge plant together with the winemaking effluent. The biomass was observed and treatment efficiency was monitored over 29 days. In the case of the mixed active molecules, the toxic effect on a population of sensitive microorganisms resulted in a significant increase in Chemical Oxygen Demand (COD) and Total Organic Carbon (TOC). The resistant biomass then adapted, which in turn led to a reduction in COD and TOC. Adaptation time was observed to be in the region of 7e8 days. The subsequent reduced COD conformed with the European regulations, which made it possible to determine the limiting values for rejection and also to monitor agricultural effluents. The toxic effect on microorganisms is produced by lysis of certain micro-organisms, which results in the release of soluble microbial products. Polysaccharides, proteins and humic substances are polymers produced by bacteria. Measurements showed a release of EPS at 7 days of treatment, which could be correlated with cell lysis. The EPS concentration increases from 25 mg/L until 260 mg/L after 7 days. Data are detailed in the paper of Esteve et al. (2009b). The presence of toxic molecules causes a loss of micro-organisms viability and provokes the cell lysis thus inducing the release of EPS. Microscopic observations revealed the presence of numerous free bacteria in the supernatant. The phenomenon of deflocculation is to be taken into account when considering the purification of toxic molecules by the activated sludge plant: it will be necessary either to carry out flocculation prior to rejection into the environment or to have additional and complementary treatment such as sand filtration or use of a reed-bed filter. Analysis of the sludge was performed with a synthetic effluent (data not shown) (Este`ve et al, 2009b). The active compounds were found to be more or less concentrated in the activated sludge. Cyprodinil and Pyrimethanil were entirely concentrated in the sludge. These two products were the only ones under test that are part of the anilinopyrimidine family. They apparently act by inhibiting methionine biosynthesis. The accumulation of pesticide

residues observed after biological treatment in the concentrated sludge from the settling tanks has been observed in scientific researches and described by Harrison et al. (2006). Measurements of the residual toxicity make it possible to visualize the adaptation phase of the biomass, when toxicity increases in the bioreactor (Este`ve et al., 2009a and 2009b). This phase is followed by a continuous decrease in toxicity; which confirms that pesticide degradation has taken place. The results make it possible to guarantee that any resulting metabolites are no more toxic than the initial active substances.

3.3. Successive stages of biological treatment for viticultural and winemaking effluent in three winery wastewater treatment plants Three winery wastewater treatment plants were chosen for the experiments in real conditions. Thirteen pesticides were used to treat the vines throughout the year. A detailed list is given in Table 2. Viticultural effluent was stored in a special tank in which it was pre-treated (coagulation-flocculation) in order to reduce mineral elements such as copper, sulfur and aluminum. When the volume and the charge of the effluent from the winery are low, the annual volume of pesticide effluent is injected into the aerated biological treatment. The toxic effect of the molecules is reduced by the dilution in the mixed liquor. For the three wastewater treatment plants, the volume of the viticulture effluent is less than 1/30 of the volume of the biological treatment. The outflow of the unit was closed for the degradation of the organic pesticides. The mixed liquor was aerated for 30 days and then allowed to settle. The treated effluent was gradually discharged to the sand filter. The effluent percolated through the sand in aerobic conditions. During the biological treatment of the pesticides, the relatively small volume of the winery effluents could be added, thus providing nutriments for the biomass. The COD of the mixed liquor supernatants during treatment remained below 250 mg L1 for 30 days. It seems that the biomass did not produce excessive soluble exopolymers (SEP) during the treatment of the pesticides and was active in the biodegradation of toxic molecules. The concentration of the total suspended solids was relatively high in the mixed liquor supernatant (50 mg L1). The presence of the pesticides provoked a deflocculated growth of the biomass. These suspended solids will be eliminated by the tertiary treatment (sand or reed-bed filter). The efficiency of the wastewater treatment plants was measured by monitoring residual concentrations of the pesticides and effluent toxicity. Good results were obtained in terms of yield and the removal of

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Table 2 e Concentrations of active molecules in the effluents before the biological treatment and at the outlet of three wastewater treatment plants (biological treatment D sand filter). Plant 1

Plant 2

Plant 3

After treatment (mg/L)

Efficiency (%)

After treatment (mg/L)

Efficiency (%)

After treatment (mg/L)

Efficiency (%)

<0.26 <0.027 <0.009 <0.005

>99.6% NS >92.7% >99.8% / >91.6% / / >99.9% NS 46% 99% / /

<1 <0.1 / <0.02 <0.02 / / <0.02 <4 <0.1 / / 0.28 <0.02

>99.9% >87% / >99.3% >97.1% / / >93.8% NS 97.3% / / 80.6% NS

5.42 / / / / / <0.004 <0.004 55.6 / <0.006 <0.004 / <0.004

96.5% / / / / / >72% >56% 62.7% / >94% >62% / >78%

Phosphoric acid AMPA (metabolite of Glyphosate) Carbendazim Cymoxanil Cyprodinil Diethofencarb Fenbuconazol Fenhexamid Fosetyl-Al Glyphosate Iprovalicarb Pyraclostrobin Spiroxamine Trifloxystrobin

<0.009 / / <1.06 <0.027 0.19 0.018 / /

NS: not significant - ND: not detected.

pesticides whatever the original composition of the effluent (three different geographical areas), whatever the effluent volume/basin volume ratio (1/30 to 1/300), and whatever the solid retention time (1e5 years). These industrial-scale results highlight the flexibility of the treatment system and the interest of the sand filter in providing an effluent free of toxicity for the environment. The retention rate for the majority of the molecules was higher than 85% (Table 2). A lower efficiency was observed for fenbuconazol, pyraclostrobin and trifloxystrobin. These results may be explained by the limits of analytical detection in laboratory conditions. As for fosetyl-Al and iprovalicarb, supplementary investigations are in progress in order to explain these low efficiency results. The toxicity of the raw effluents was high. The ecotoxicological tests of the treated effluent after 30 days showed a large reduction in toxicity (Table 3). The treated effluent was considered to be non-toxic to daphnia and algae. The sand filter was not efficient in terms of toxicity reduction at sites 1 and 2. However, the EC50 (algae test) of the supernatant for plants 2 and 3 was measured at 5.6. Downstream of the sand

Table 3 e Ecotoxicological tests of the effluents from the three experimental sites.

Site 1

Site 2

Site 3

Daphnia Magna e EC (I)50 24 h (%) Algaes ECr10 72 h (%) ECr50 72 h (%) Daphnia Magna e EC (I)50 24 h (%) Algaes ECr10 72 h (%) ECr50 72 h (%) Daphnia Magna e EC (I)50 24 h (%) Algaes ECr10 72 h (%) ECr50 72 h (%)

Raw effluent (%)

Treated effluent (%)

0.031 0.10 0.12 0.26 0.24 0.29 0.51 0.41 0.50

>90 >98 >98 >90 >98 >98 >90 >98 >98

filter, the EC50 was over 98%. The lower efficiency of the biological reactor for the two sites may be explained by the input of a larger volume of winery effluent toward the end of the treatment (20 days). It can thus be deduced that a continuous, low, flow rate of winery effluent is suitable for the provision of nutriments to the biomass for the degradation of pesticides. However, if the input exceeds a certain value, the efficiency of toxicity removal seems to be diminished.

4.

Conclusions

The feasibility of the biological treatment of viticultural effluents has been evaluated by the application of pesticides to activated sludge. The values of the biological constants Ks and Yx/S determined for a winemaking effluent were close to those found for a synthetic effluent with glucose. However, mmax was 10 times lower (0.033 h1) when the substrate feeding the bioreactor was wine. This could be due to the biodegradability of the wine compounds being modified in the presence of polyphenols. In the presence of pesticide, the determination of mmax and Y brought to light significant decreases in their values: mmax was 0.016 h1 for dimethomorph and 0.012 h1 for pyrimethanil (Si ¼ 0.6 g COD L1 d1). Over 99.94% of the dimethomorph was removed from the effluent according to the measurements of residual concentrations in the sludge (<0.25 mg L1). The concentration of pyrimethanil in the effluent was reduced by 98.2%, the substance being almost entirely concentrated in the sludge. The COD of the effluent decreases during the time and is less than 300 mg L1 after thirty days. The efficiency of the treatment is validated by the reduction in effluent toxicity and the elimination of the active molecules that compose the effluent. The treatment of real effluents by aerated biological storage ponds at three wineries, followed by a sand or reedbed filter shows the degradation of the different molecules exceeds 85% and the filtered effluent greatly reduced toxicity (>98%). Thus, it can be confirmed that biological treatment

1792

w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 1 7 8 5 e1 7 9 2

offers interesting prospects for an application to agricultural effluents. The process has been validated by the French regulations covering the treatment of viticultural effluents and is now marketed by the company Agro-Environnement (France).

Acknowledgments The authors wish to express their appreciation to the three wineries. The research work was supported financially by ADEME (environmental Agency), OSEO (Innovation Agency) and the CIVB (Bordeaux wine council).

Appendix. Supplementary data Supplementary data related to this article can be found online at doi:10.1016/j.watres.2011.12.055.

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