Fate of steroid hormones and endocrine activities in swine manure disposal and treatment facilities

w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 8 9 5 e9 0 6

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Fate of steroid hormones and endocrine activities in swine manure disposal and treatment facilities Sarah Combalbert a, Virginie Bellet b, Patrick Dabert c,d, Nicolas Bernet a, Patrick Balaguer b, Guillermina Hernandez-Raquet a,e,* a

INRA, UR050 Laboratoire de Biotechnologie de l’Environnement, Avenue des Etangs, F-11100 Narbonne, France IRCM, Institut de Recherche en Cance´rologie de Montpellier, INSERM U896, Universite´ Montpellier 1, CRLC Val d’Aurelle Paul Lamarque, Montpellier F-34298, France c CEMAGREF, UR GERE, 17, avenue de Cucille´, CS 64427, F-35044 Rennes cedex, France d Universite´ europe´enne de Bretagne, France e Laboratoire d’Inge´nierie des Syste`mes Biologiques et des Proce´de´s, UMR5504, UMR792, CNRS, INRA, INSA, 135 Avenue de Rangueil, 31077 Toulouse cedex 4, France b

article info

abstract

Article history:

Manure may contain high concern endocrine-disrupting compounds (EDCs) such as steroid

Received 25 July 2011

hormones, naturally produced by pigs, which are present at mg L
1 levels. Manure may also

Received in revised form

contain other EDCs such as nonylphenols (NP), polycyclic aromatic hydrocarbons (PAHs)

29 November 2011

and dioxins. Thus, once manure is applied to the land as soil fertilizer these compounds

Accepted 30 November 2011

may reach aquifers and consequently living organisms, inducing abnormal endocrine

Available online 8 December 2011

responses. In France, manure is generally stored in anaerobic tanks prior spreading on land; when nitrogen removal is requested, manure is treated by aerobic processes before

Keywords:

spreading. However, little is known about the fate of hormones and multiple endocrine-

Manure

disrupting activities in such manure disposal and treatment systems. Here, we determined

Endocrine disruptors

the fate of hormones and diverse endocrine activities during manure storage and treat-

Steroid hormones

ment by combining chemical analysis and in vitro quantification of estrogen (ER), aryl

Endocrine receptors

hydrocarbon (AhR), androgen (AR), pregnane-X (PXR) and peroxysome proliferator-acti-

Aerobic treatment

vated g (PPARg) receptor-mediated activities. Our results show that manure contains large

Anaerobic digestion

quantities of hormones and activates ER and AhR, two of the nuclear receptors studied. Most of these endocrine activities were found in the solid fraction of manure and appeared to be induced mainly by hormones and other unidentified pollutants. Hormones, ER and AhR activities found in manure were poorly removed during manure storage but were efficiently removed by aerobic treatment of manure. ª 2011 Elsevier Ltd. All rights reserved.

1.

Introduction

Currently, there is increasing concern about the presence of contaminants known as endocrine-disrupting compounds

(EDCs) in the environment (McLachlan, 2001). EDCs interfere with the normal functioning of the endocrine system affecting negatively the reproduction and development of wildlife and humans, even at very low concentrations (ng L
1 to mg L
1)

* Corresponding author. INRA, UR050 Laboratoire de Biotechnologie de l’Environnement, Avenue des Etangs, F-11100 Narbonne, France. Tel.: þ33 5 61 55 99 77; fax: þ33 5 61 55 97 60. E-mail addresses: [email protected], [email protected] (G. Hernandez-Raquet). 0043-1354/$ e see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2011.11.074

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(Brody and Rudel, 2003; Sumpter, 1998). EDCs include naturally produced substances such as steroid hormones and phyto-estrogens as well as wide range of chemicals including synthetic hormones, polycyclic aromatic hydrocarbons (PAHs), polychloro-biphenyls (PCBs), dioxins, furans, nonylphenol (NP), pharmaceuticals and pesticides (Andersen et al., 1999). Most of the known EDCs alter the endocrine system by direct interaction with the estrogens receptors (ER) (Kuiper et al., 1998). However, other hormones (e.g androgens and progestagens) and EDCs may also interact with other nuclear receptors involved in the control of the endocrine system (Le Maire et al., 2010; Tabb and Blumberg, 2006). For instance, some compounds interact with the androgen receptor (AR) inducing androgenic or antiandrogenic responses (Blankvoort et al., 2005; Fang et al., 2003). They may also activate the pregnane-X receptor (PXR, also known as Steroid Xenobiotic Receptor, SXR) implicated in xenobiotics detoxification (Di Masi et al., 2009). Some EDCs bind the aryl hydrocarbon receptor (AhR), also called dioxin-like receptor, implicated on xenobiotic and hormone metabolism (Denison and Nagy, 2003; Tian et al., 2002). Others induce the proliferation of peroxysome via the peroxysome proliferatoractivated receptor gamma (PPARg) involved in lipid metabolism (Casals-Casas and Desvergne, 2011; Le Maire et al., 2009). Therefore, in assessing the potential endocrine disruption exerted by EDCs present in the environment, the activation of different nuclear receptors must be considered. Among the EDCs, steroid hormones are of particular concern because of their capacity to induce strong endocrine responses (Andersen et al., 2003; Desbrow et al., 1998). Natural hormones belong to three different families: estrogens (estrone, E1, alpha (aE2) and beta (bE2) estradiol, estriol, E3), androgens (testosterone, T) and progestagens (progesterone, Pg). These compounds are naturally produced by humans and animals where they are implicated in inter-cell communication; thereafter, they are excreted in urine and faeces either as biologically active free forms or as inactive forms conjugated with glucuronide and/or sulfate groups (Sandor and Mehdi, 1979). However, after their excretion, conjugated forms are easily transformed into biologically active compounds by microorganisms (Dray et al., 1972). Hence, hormones, along with their associated endocrine-disrupting activity, enter the environment mainly through effluents from wastewater treatment plants (WWTP) as well as through runoff from soils receiving sewage sludge, animal excreta or manure. Different studies have identified animal breeding activities as a major source of hormones to the environment. Indeed, hormones have been detected at environmentally significant levels in farm effluents (Bushee´ et al., 1998; Fine et al., 2003; Hanselman et al., 2006; Raman et al., 2004), surface water and groundwater impacted by livestock effluents (Chen et al., 2010; Matthiessen et al., 2006). Animals may also be in contact with other EDCs such as PAHs and dioxins that may be present in animal feed and indoor farm dust (Ciganek and Neca, 2008; Ciganek et al., 2002); EDCs such as phthalates and bisphenol A have also been detected in manure samples, probably issued from materials coating the inner surface of food containers or manure-storage tanks (Fromme et al., 2002). Rearing livestock involves the use of detergents for cleaning purposes; some of them may release EDCs such as NP (Tolls et al., 1994). Several

studies have highlighted the estrogenic effects on wildlife of manure, farm effluents or runoff from manure spread soils (Irwin et al., 2001; Kjaer et al., 2007). However, so far, studies related to the presence of endocrine-disrupting activity in manure have focused almost exclusively on ER activation. Only two studies assessed AR and progesterone-receptor activation by manure extracts (Burnison et al., 2003; Lorenzen et al., 2004) demonstrating the presence in manure of a compound (equol) able to weakly bind AR receptor while no progesterone-receptor activation was detected. Animal manure is managed in a wide range of systems depending on the species bred, animal density and country’s regulations. In the more basic systems, manure is stored under the animal units; it can also be stored in heaps (solid waste) or disposed in lagoons (Burton and Turner, 2003). The information concerning the presence of hormones in such systems shows great variability in hormone levels. In swine and cattle waste, between 46 to 34,326 ng L
1 and from 130 to 6800 mg kg
1 dry weight (mg kg
1 dw) were reported in, respectively, the liquid and solid fractions. Lagoons receiving poultry litter displayed hormone levels varying from 41 to 7817 ng L
1 (Combalbert and Hernandez-Raquet, 2010). The efficiency of these various manure-storage systems in reducing hormone levels has been assessed by chemical analysis or by measuring estrogenic activity using different in vitro tests. Hormone concentrations seem to be reduced in successive lagoons receiving animal waste (Fine et al., 2003; Hutchins et al., 2007; Zheng et al., 2008). However, in such systems it is difficult to distinguish hormone degradation from dilution. More complex manure-treatment facilities have been installed in large-scale farms or in regions with high livestock production. In such manure-treatment processes, little is known about the fate of hormones and endocrine-disrupting activities. Clearly, there is a lack of accurate information about the fate of hormones and endocrine-disrupting activity in manure management systems. This article provides integrated screening of hormones and endocrine-disrupting activity in pig manure storage and biological treatment facilities used in France. France is Europe’s third biggest producer of pork (Burton and Turner, 2003); this activity is associated with an annual production of 28 Mtons of manure containing hormones and other EDCs. On 85% of French pig farms, manure is stored in an anaerobic disposal system prior to spreading. On the remaining farms, manure is treated by aerobic processes sometimes with combined composting of solid manure. For this study, hormone concentrations were determined by chemical analysis in both the liquid and solid fractions of swine manure throughout the whole manure storage and treatment processes. Simultaneously, the fate of multiple endocrine activities was assessed by in vitro bioluminescent assays for ER, AR, AhR, PPARg and PXR receptors.

2.

Material and methods

2.1.

Reagents

The steroids estrone (E1), a-estradiol (aE2), b-estradiol (bE2), estriol (E3), progesterone (Pg) and 17b-testosterone (T) were purchased from SigmaeAldrich (purity > 98%; St. Quentin

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Fallavier, France). The deuterated estrogens bE2-d4, T-d3 and ethinylestradiol (EE2-d4) (isotopic purity > 99%), used as internal standards (IS), were supplied by Cluzeau Info Labo (Ste. FoyeLa-Grande, France). All organic solvents were HPLC grade and were obtained from Atlantic Labo (Floirac, France).

2.2.

Sites studied

The fate of steroids as well as endocrine-disrupting activities was studied in two different types of pig manure management systems: manure-storage facilities (here after called ‘storage systems’) and aerobic manure-treatment facilities (here after called ‘treatment systems’). In the storage systems, manure is kept in outside tanks for 4e6 months without any further treatment before the manure is spread on farmland. In these tanks, anaerobic conditions prevailed. Three different swine farms using such a system were studied (Sites S1eS3). On all of them, pigs were raised and fattened in three separate breeding units according to their stage of development: a swine nursery (SN) with post-weaned piglets, growingfattening (GFS) and gestating sow (GS) buildings. Urine, faeces, bedding material, residues of animal food and cleaning wastewater from each breeding unit composed the raw manure (RM). RM fell through the pigboards and was kept below the animal houses in a pre-storage chamber for about 3e4 weeks. Manure from the different buildings arrived successively by a series of connecting-channels to the anaerobic tank where it was stored for up to six months. In the treatment systems, the manure is treated aerobically with the main objective to remove nitrogen by nitrificationedenitrification processes. Two farms using such a treatment were considered (T1 and T2, Supporting Information 1): On farm 1 (T1), manure was stored for a short time (less than 15 days, SM) and treated straightaway in an aerobic basin (AB). The solids content in AB was of 47  2 g L
1 and the hydraulic and solid retention times were both about 40 days. After treatment, the solid and liquid fractions of manure are separated by settling. There is no sludge recirculation to the AB. The solid fraction constituting the sludge for spreading (SS) is stored in an anaerobic tank (four to six months) until spread on land while the liquid fraction was stored (six to twelve months) in a large non-aerated lagoon (LAG) for irrigation. On farm 2 (T2), the solid and liquid fractions contained in the stored manure are first separated by centrifugation before going to the aerobic basin, the residual solid fraction (RSS) thus obtained, containing a high amount of phosphorus, is finally exported to be used as fertilizer. The manure liquid fraction (solid content of 16  0.3 g L
1) is treated as on farm 1 (T1). In all farms studied, pigs are fed with commercial or farm made cereal formula consisting of wheat and corn (about 55%), whole wheat (20%), bean forage (10%), soybeans and colza (10%), plus minerals (5%). All the farms are situated in the Brittany region of France; in this region 60% of the French swine manure is produced (around 13,106 m3 per year).

2.3.

Sample collection and conditioning

2.3.1.

Storage systems

The three farms using a manure-storage system (S1eS3) were monitored via 4 sampling periods carried out a little less than

897

once a month from July to December 2008. One composite raw manure (RM) sample (about 30 L) was taken from the prestorage chamber (or in the respective connecting channel) that was emptied on the sampling date. One composite sample of stored manure (SM, about 30 L) was also taken on each occasion from the anaerobic storage tank. In order to obtain the most representative samples possible, great effort was made to homogenize the manure. All samples were taken after 1 h homogenization of manure by resident mechanical agitation. Both RM and SM were transported at 4  C to the CEMAGREF laboratory (Rennes, France; 3 h transport).

2.3.2.

Treatment systems

For the two farms studied (T1 and T2), samples were taken in spring and autumn 2009 in the different compartments of the system (Supporting Information 1): stored manure (SM), aerobic basin (AB), sludge for spreading (SS), lagoon (LAG) and, for site T2 only, the residual solid fraction (RSS). Every precaution was taken to get the most representative sample (about 30 L) from each compartment. SM and SS were sampled as described above for SM from the anaerobic systems; AB samples were naturally mixed by the aeration system. LAG samples were taken from 4 different areas of each compartment. Composite samples of RSS (10 kg) were obtained by carefully mixing 15 elementary 1 kg samples taken by digging about 0.5 m3 at three different depths and along the length of a manure heap of about 30 m3. All samples were transported at 4  C to the laboratory. Once in the laboratory, all liquid manure samples were separated by a double centrifugation (4  C, 11,100  g, 20 min). Pellets were frozen at
20  C and freeze-dried (Thermo Electro-Corporation). Supernatants were filtrated on Whatman GF-A and GF-F glassefiber filters before freezing (
20  C). Solid samples were directly frozen at
20  C.

2.4.

Manure characterization

2.4.1.

Physico-chemical analysis

The main characteristics of the manure samples, including pH, dry matter, organic matter and total Kjeldahl nitrogen (TKN), were measured by standard methods for wastewater analysis (APHA, 1992).

2.4.2.

Hormone analysis

For extracting free hormones contained in the solid fraction of manure, one gram of dried samples was added with IS and extracted by accelerated solvent extraction (ASE) using a mixture of methanol/acetone (MeOH/Ace; 50:50 v:v). For liquid samples, after IS addition, free and conjugated hormones were extracted by solid phase extraction (SPE). Sample volumes were 20 mL for raw and stored manure, 30 mL for aerobic sludge (AS) or sludge for spreading (SS) and, 70 mL for lagoon (LAG), as described by Combalbert et al. (2010). Conjugated estrogens were subjected to solvolysis and enzymatic hydrolysis, as previously described (Labadie and Budzinski, 2005). Extracts were further purified using 500 mg LC-NH2 columns (Waters). Hormones were derivatized and quantified by gas chromatography/mass spectrometry (GC/MS; Labadie and Budzinski, 2005) using a GC autosytem XL (Perkin Elmer, Waltham, MA, USA) coupled to a Turbo Mass

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Gold mass spectrometer (Perkin Elmer). Identification of compounds and quantification were performed as described by Combalbert et al. (2010). Both liquid and solid manure samples were extracted in triplicate.

(RLU). Sample activities were expressed as the percentage of the maximal activity obtained with reference ligand at saturating concentration: 10 nM E2 (MELN) and dioxin (HAhLP), 1 mM SR12813 (HG5LN GAL4-PXR) and rosiglitazone (HG5LN GAL4-PPARg), respectively.

2.4.3. Endocrine activity measurements 2.4.3.1. Manure samples extraction. For assessing endocrine activities, solid and liquid manure samples were extracted as above for hormone analysis, but without adding IS. For solid samples, in order to maximize the recovery of the overall endocrine potency, after the MeOH/Ace extraction, a further extraction was performed once with a mixture of hexane/ acetone (Hex/Ace) 50:50 at 120  C and 100 bars. Each extraction cycle was made up of one warming step of 6 min, followed by two static extraction steps of 5 min each and a final nitrogen purge. This methodology makes it possible to extract the less polar EDCs such as PAHs, NP or PCBs (HernandezRaquet et al., 2007; Trably et al., 2004). The extracts (20 mL each) were gathered together and evaporated to dryness. The final extract was dissolved in 500 mL of MeOH and used directly for the measurement of endocrine activity. For liquid samples, in order to recover all the potentially EDCs contained in such samples, one further elution with diethylether was carried out; the purification step using LC-NH2 columns was not performed. Each fraction was recovered separately, evaporated under a gentle nitrogen stream at 35  C and suspended in 500 mL of MeOH (four fractions for each sample: Triethylamine-MeOH/H2O, MeOH, diethylether and hexane). Both liquid and solid manure samples were extracted in duplicate for the measurement of the estrogenic activity.

2.4.3.2. Generation of stably transfected reporter cell lines. Reporter cell lines were used to evaluate the presence in manure of chemicals able to bind ER, AhR, PXR and PPARg receptors and, by this way, to determine the contamination of manure with EDCs. The stably transfected luciferase reporter cell lines were obtained as already described (Le Maire et al., 2006; Pillon et al., 2005; Seimandi et al., 2005). Briefly, MELN cell line was obtained by transfecting ERa positive breast cancer MCF-7 cell line with an estrogen responsive element cloned upstream of the luciferase reporter gene construct. HAhLP cell line, used to detect dioxin receptor-mediated activity, was obtained by transfecting HeLa cells with the CYP1A1-Luc plasmid (Pillon et al., 2005). HG5LN GAL4-PXR and -PPARg cells, used to detect PXR- and PPARg-like activities, were HeLa cells stably transfected by a Gal4-responsive reporter gene and Gal4 DNA-Binding-Domain (DBD)-hPXR Ligand Binding Domain (LBD) or -hPPARg (LBD) expressing plasmids, respectively.

2.4.3.3. Luciferase assay. Cells were seeded in 150 ml test culture medium at a density of 5  104 cells per well in 96-well white opaque tissue culture plates (Greiner Bio-One, Courtaboeuf, France). Eight hours after cell plating, 50 mL of samples diluted in culture medium were added in each well. Cells were incubated with samples for 16 h. Experiments were performed in quadruplicate. At the end of incubation, effector-containing medium was removed and replaced by 0.3 mM luciferin containing test culture medium. Then, luminescence was measured for 2 s and expressed as relative luminescence units

3.

Results and discussion

3.1.

Storage systems

3.1.1.

Manure physico-chemical parameters

Manure is a slightly alkaline matrix with pH fluctuating from 7 to 8. Dry matter, organic matter and nitrogen contents were extremely variable depending on the origin of the manure and on the particular sampling campaign (Supporting Information 2). Overall, the organic matter represented about 60% of the dry matter while nitrogen concentration varied between 1.5 and 7 g L
1 (mean for raw and stored manures of 3.5 g L
1). These are typical values for swine manure (Martinez-Suller et al., 2008).

3.1.2.

Steroid hormones in raw manure

The hormone concentrations measured in the different samples of raw manure (RM) from different units displayed great variability inter-dates and inter-sites (Fig. 1). The total hormone concentrations found in raw manure from SN and GFS samples were, on average, around 5000 ng L
1. In contrast, much higher hormone concentrations were found in manure from the gestating sow units (>20,000 ng L
1). These observations corroborate previous studies reporting high hormone levels in lagoons receiving manure from gestating sows compared to that from finisher or nursery buildings (Fine et al., 2003; Hutchins et al., 2007). As previously observed (Combalbert et al., 2010), steroid hormones were detected mainly as free forms contained in the solid fraction of manure (Fig. 1) even though hormones are mainly excreted as conjugated hormones via urine (90%; Lange et al., 2002; Terqui, 1971). This certainly resulted from deconjugation of conjugated forms by bacteria present in manure (Dray et al., 1972). Free hormones are lipophilic compounds with an octanol/ water partition coefficient (log Kow) from 2.8 to 3.9, leading them to be adsorbed onto the solid organic matter (Lai et al., 2000). Concerning the hormone composition, whatever the type of manure, E1 was the main compound found, followed by aE2, bE2 and E3 (Supporting Information 3), as observed in previous studies (Furuichi et al., 2006; Hutchins et al., 2007; Combalbert et al., 2010). Besides free hormones, a very small fraction of conjugated forms of E1, aE2, bE2 and E3 were also detected in manure (data not shown). These conjugated forms reached levels similar to those of free hormones detected in the liquid phase of manure (Fig. 1). In stored manure (SM), the concentrations of steroid hormones displayed wide variation for the different sampling dates and sites; varying from 5494 to 31,681 ng L
1. Nevertheless, on average, levels of 13,202 ng L
1, 15,699 ng L
1 and 20,156 ng L
1 were measured in sites 1e3, respectively (Fig. 1); these are intermediate values in comparison to those observed in raw manure from SN, GFS and GS buildings. Conjugated hormones were detected in stored manure but

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899

Fig. 1 e Total concentrations of hormones in the studied farms using manure-storage systems. Hormones were measured in the solid fraction of manure as free forms (solid free forms) and in the liquid as free (liquid free forms) and conjugated forms (liquid conjugated forms) in manure from the three breeding units (swine nursery, growing-fattening pigs, gestating sows) and in the storage tank (stored manure). Error bars represent the standard deviation observed in triplicate measurements for overall samples of the respective breeding unit and site (n [ 3e15. For details see Supporting Information 2).

they represent a minor fraction of the total hormone load (<10%). Nevertheless, this persistence of conjugates in stored manure suggests that they were not completely hydrolyzed during manure storage under anaerobic conditions. Our results are in accordance with previous data (De Mes et al., 2008) that showed the persistence of conjugated hormones in up-flow anaerobic reactors treating concentrated black water. It probably resulted from the presence in manure of sulfate-conjugated forms that are more recalcitrant to biodegradation (D’Ascenzo et al., 2003; Hutchins et al., 2007).

3.1.3.

Fate of steroid hormones during manure storage

On the three farms studied, hormone concentration in manure tended to decrease over the storage period, except on the last sample from site 1 (Fig. 2). However, the high variations observed in dry matter concentrations in raw manure made it difficult to distinguish between hormone degradation and hormone dilution. Indeed, hormone concentrations globally followed the total solid content (Fig. 2). Since the manure-storage tanks were fed with variable volumes of raw manure from different buildings and containing variable hormone levels, it is not possible to conclude about hormone elimination during manure storage. Obviously, hormone levels calculated per gram of dry matter (Table 1) show less variation. Similarly, the increase observed on hormone levels from the last sampling campaign on farm 1 (S1) could be the result of feeding additional raw manure containing high levels of hormones (e.g from GS building) into the storage tank. During manure storage, E1, aE2, bE2 and E3 were the main compounds found in SM (Supporting Information 4). However no particular hormone makeup was observed that might indicate a potential biodegradation: e.g. predominance of E1 (the main metabolite of steroid hormones) at the end of the storage period (Li et al., 2008). So our results argue for the nondegradation of hormones during manure storage under anaerobic conditions. Such observations are in accordance with previous studies concerning hormone degradation under

anaerobic or anoxic conditions carried out at lab scale (Czajka and Londry, 2006; De Mes et al., 2008). Those studies showed that, despite hormone transformation occurred (transformation of bE2 into E1, E1 to a or bE2 and bE2 into aE2), a very low hormone removal (17%) was observed after one year incubation. Similarly, other studies carried out at full-scale anaerobic plants treating sewage sludge concluded that hormones were not removed (Andersen et al., 2003; Ternes et al., 2002). In contrast, one study reported 85% hormone degradation in sewage sludge treated under anaerobic conditions (Carballa et al., 2006). However, this last study focused only on E1 and bE2 analysis: so if transformation occurred via aE2, it was not measured. Moreover, the studied system was a controlled laboratory scale reactor optimized for biogas production. Consequently, it is difficult to compare the fate of hormones in such conditions with their fate observed in real manure-storage systems.

3.1.4.

Presence and fate of endocrine activities in manure

Throughout manure storage, the liquid and solid fractions of manure samples were tested for their ability to induce ER, AhR, PXR, AR and PPARg receptors. Our results show that very weak PXR and PPARg activities were induced by manure but, due to their very low levels, it was not possible to estimate an EC50 value for such activities. No androgen receptor (AR) activity was detected, which is in accordance with the low testosterone levels found in manure. By contrast, ER and AhR activities were present in all SM samples, mainly in the solid fraction (Fig. 3). In order to assess the contribution of hormones to the measured estrogenic activity, the ER activity measured in manure by the MELN in vitro test (expressed as ng of bE2 equivalents per liter; ng eqE2 L
1) was compared to the theoretical activity estimated from the abundance of steroid hormones, as determined by chemical analysis and considering their relative estrogenic potencies (Pillon et al., 2005). For liquid samples, theoretical and measured ER activities are very similar (Fig. 3), which means that steroid hormones

900

40 000

35

S1

S2

S3 30

30 000

25 20

20 000

15 10

10 000

5

-1) -1 ) Total solid content Total solids (g.L(g.L

Hormone concentration (ng.L-1 )

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0

0

solid free forms

liquid free forms

liquid conjugated forms

Fig. 2 e Total hormone concentration found in stored manure (bars, left axis) and total solid content (bold line, right axis) measured in the different sampling campaigns (date 1e4) and sites studied (S1eS3). Error bars represent the standard deviation observed in triplicate (hormone concentrations) or duplicate (total solids) measurements.

explain most of the detected estrogenicity. However, in the solid fraction of manure, where most ER activity was found, a very different behavior was observed. Indeed, at sites 1 and 2, only about 15e51% of the measured ER activity could be explained by the hormone concentrations determined by chemical analysis. By contrast, at site 3, most of this activity (35e97%) was explained by the hormone levels measured chemically. Thus, our results suggest that others estrogenic compounds able to bind to ER and inducing an estrogenic response were present in the solid fraction of manure, particularly at sites 1 and 2. The high levels of AhR activity (up to 60 mg equivalent TCDD L
1,eqTCDD L
1; Fig. 3B); measured in the solid fraction of manure revealed also the presence of compounds binding to this receptor. During manure storage, the in vitro ER activity fluctuated greatly on the three farms. Moreover, on all sites, a large part of the estrogenicity failed to be removed during manure storage (Fig. 3). This recalcitrance was more noticeable when data were normalized by the dry matter content (eqE2 g
1 dw; Fig. 4), showing that ER activity was stable throughout the storage period. This is in accordance with the no significant removal of hormone measured by chemical analysis. AhR activity decreased during manure storage (Fig. 3). This trend was confirmed after normalization by the solid content (eqTCDD g
1 dw, Fig. 4), with lower AhR activity at the end of the storage period. Furuichi et al. (2006) showed that the estrogenic activity in pig wastewater was due to E1 (17e30%), bE2

Table 1 e Steroid hormone concentrations in stored manure samples throughout the storage period (date 1edate 4). Average ± standard deviation (n [ 3). Hormone concentrations (ng g
1 dw) Date Date Date Date

1 2 3 4

Site 1

Site 2

Site 3

652  28 535  78 429  25 1049  56

972  223 941  128 996  17 491  150

856  20 1126  3 504  23 430  8

(23e30%), E3 and aE2 (1e3%) and the phyto-estrogen equol (2e3%) (Furuichi et al., 2006). But a wide part of the estrogenic activity measured by these authors (35e57%) resulted from the presence of unidentified compounds. In that study, the elimination of estrogenic activity was between 19% and 50%, indicating that anaerobic conditions were not favorable to elimination of estrogenicity from pig wastewaters, which is consistent with our observations. To our knowledge, no study has dealt with AhR activity in manure so far. In order to identify the EDCs inducing the high levels of dioxin-like activity found in manure, dioxins and PAHs were measured in two manure samples displaying high AhR levels (data not shown). No dioxin were found and only very low levels of PAHs, accounting for less than 0.1% of the measured AhR activity. These data suggest that AhR activity was induced by other compounds that remain to be identified.

3.2.

Treatment systems

3.2.1.

Physico-chemical parameters

On the two farms studied, the physico-chemical parameters measured for the two sampling campaigns (spring and autumn) displayed a low standard deviation (Supporting Information 5). This reflects that this type of facility, functioning in a continuous mode, is more homogeneous than simple manure-storage systems. A comparison of the two aerobic treatment sites, T1 and T2, shows that physicochemical parameters were highly influenced by the separation of solid and liquid fractions of manure upstream of the aerobic basin. Indeed, the separation of these fractions removed a third of the dry and organic matter; the nitrogen content was less affected due to its high solubility.

3.2.2. Fate of steroid hormones and endocrine activities in the aerobic treatment facilities For both sites studied, it was not possible to know the real manure fluxes and therefore to calculate mass balances. Nevertheless, the aerobic basin was continuously fed with

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901

Fig. 3 e A: Estrogenic activities (Theoretical, T and measured, M) and, B: dioxin-like activities measured in stored manure in the different sampling campaigns and sites studied. For the measured estrogenic activity (M in A) and dioxin-like (B) activity, error bars represent the standard deviation observed in duplicate in vitro measurements from duplicate samples (n [ 4). For the theoretical ER activity (T in A), errors bars represent the standard deviation of triplicate samples (n [ 3).

stored manure while, simultaneously, treated manure flowed out of the system. Consequently, we assumed that the changes in hormone concentration between SM and AB indicate the hormone’s actual fate. At both sites T1 and T2, more than 90% of the hormones present in the inlet were removed in the aerobic basin (AB) (Fig. 5). Whatever the process used or the stage in the process, most of the hormones was contained in the solid fraction of manure. Similarly, the hormones present in the aerobic basin were associated with the solid fraction which, after settling, constituted the sludge for spreading. Indeed, SS displayed hormone levels similar to those found in the solid fraction of manure from the aerobic basin (7552  264 ng L
1 and 1975  186 ng L
1 for sites 1 and 2, respectively) suggesting that no hormone removal occurred during anaerobic storage of SS. This is consistent with our previous observations in the storage systems. A very minor amount of hormones was measured in the lagoons, corresponding to about 5% of the inlet levels. However, in system T2, where a solideliquid separation occurred before aerobic treatment, a very high concentration of hormones was detected in RSS. Indeed, hormone levels measured in RSS (687  443 ng g
1 dw) were of the same order of magnitude as those measured in SM (944  279 ng g
1 dw). Consequently, in T1 where manure is

treated without any solideliquid separation step, the hormone removal observed corresponded to the net hormone elimination. In contrast, in T2, it corresponded mainly to the elimination of solids through the liquidesolid separation step. So, for better hormone degradation, manure should be treated in bulk, without solideliquid separation. Concerning the estrogenic and dioxin-like activities measured, respective concentrations between 44.9 and 47.7 mg eqE2 L
1 and from 40 to 62.4 mg eqTCDD L
1 were found in SM from both sites (Fig. 5B). As in the storage facilities, both activities were mainly found in the solid fraction of manure (data not shown). The observed estrogenic and dioxin-like activities were both higher than those measured in the storage facility. This is probably due to the higher solid concentration measured in the SM from the treatment systems (average of 45 g L
1 compared to 19 g L
1 in the storage facilities). The estrogenicity measured in SM from the treatment facilities was higher than the theoretical one. Indeed, we estimated that hormones contribute to only 20e39% of the total estrogenic activity measured, suggesting that other unidentified compounds were present in manure. During manure treatment, both the theoretical and measured estrogenic activities decreased by up to 90% (Fig. 5). This suggested that the unidentified estrogenic compounds were eliminated

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w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 8 9 5 e9 0 6

Fig. 4 e A: Measured estrogenic activities and, B: dioxin-like activities measured in stored manure in the different sampling campaigns and sites studied normalized by the dry weight content (ng gL1 dw). Error bars represent the standard deviation observed in duplicate in vitro measurements from duplicate samples (n [ 4).

as well as the hormones. In contrast to our study, Furuichi et al. (2006) reported that hormones contributed from 41% to 62% of overall estrogenic activity and that the estrogenic activity due to hormone concentrations decreased more rapidly than the estrogenic activity due to unknown compounds. However, in that study, manure had less total solids (4.6e10.4 g L
1) and only the liquid fraction of wastewater was analyzed, which may explain the different behavior observed (Furuichi et al., 2006). In the other hand, our data suggests that the aerobic basin was more efficient in eliminating estrogenic activity in comparison to Furuichi’s trickling filter, probably due to the short hydraulic retention time (3 days) applied by the authors. The elimination of hormones and estrogenic activity observed in the aerobic basin is consistent with other studies dealing with manure treatment (Ermawati et al., 2007; Shappell et al., 2007). Compared to the estrogenic activity, dioxin-like activity appeared to be more recalcitrant in both T1 and T2 facilities which displayed an average removal rate after the aerobic basin of, respectively, 44% and 83%. High AhR activity was measured on sludge for spreading from T2, which displayed dioxin-like activity similar to SM. As mentioned, this AhR activity was not induced by dioxins or PAHs but by unidentified compounds. It appeared that the AhR activity had also high affinity with the solid fraction. Indeed, in the treatment facility with solid phase separation (T2), most of the AhR and estrogenic activities were concentrated in RSS

containing high solid levels (360 mg dry matter g
1 dw), with dioxin-like and estrogenicity levels up to, respectively,
1 555  280 ng eqTCDD g
1 dw and 200  215 ng eqE2 gdw. So, as for hormone removal, a higher elimination of estrogenic and AhR activities was obtained when manure was treated without separation. To our knowledge, the fate of AhR activity in waste management systems has been only studied in sewage treatment plants (Dagnino et al., 2010; Mnif et al., 2010) and sludge composting (Patureau et al., 2007) but not in livestock systems. The Dagnino’s and Mnif’s work showed that AhR activity in WWTP, as in manure, was mainly associated with the solid fraction and accumulated in sewage sludge. Nevertheless, high elimination rates of dioxin-like activity were measured in WWTP using enhanced aerobic treatment processes (Dagnino et al., 2010); in contrast, no elimination of AhR activity was reported during sludge composting (Patureau et al., 2007). It seems thus that the removal of dioxin-like activity is strongly affected in matrices containing high solid levels such as compost or manure.

3.3.

Ecotoxicological considerations

To estimate the potential impact of manure-spreading practices on the transfer of hormones and endocrine activity, we considered the EU policy related to spreading manure in nitrogen sensitive area: manure can be spread

w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 8 9 5 e9 0 6

903

Fig. 5 e A: Hormone concentration and, B: Estrogenic (ng eqE2 LL1) and dioxin-like (ng eqTCDD LL1) activities along the swine manure-treatment facilities (T1 and T2). Abbreviations are as described in Fig. 1. For RSS only, the estrogenic or dioxin-like L1 activities are expressed, respectively, in ng eqE2 gL1 dw and ng eqTCDD gdw (right axis).

at a maximum annual rate of 170 kg of organic-nitrogen per hectare (Nitrates Directive, 1991). Thus, as the average N concentration of stored manure was 2.4 g L
1, about 71 m3 of stored manure per hectare can be spread on land, corresponding to a release of about 900 mg eqE2 ha
1 and a dioxin-like activity of 1800 mg eqTCDD ha
1. In the case of aerobically treated residues, the average N concentration of sludge for spreading was 2.1 g L
1. This corresponds to the application of about 85 m3 of such sludge, releasing 400 mg eqE2 ha
1 and 3440 mg eqTCDD ha
1 for T1 and 60 mg eqE2 ha
1 and 1140 mg eqTCDD ha
1 for T2. Lagoon water must also conform to N regulation thresholds to be used for irrigation. Thus, as the lagoon water contains about 0.2 g L
1 of N, the use of this water for irrigation (850 m3 ha
1) is likely to release 1441 mg ha
1 of hormones, 598 mg ha
1 eqE2 and 2738 mg ha
1 of eqTCDD. The high levels of dioxin-like activity detected in manure are surprising. Nevertheless, our data showed that this activity was not induced by the presence of dioxins or PAHs in manure, but it resulted from unidentified compounds,

which can be eliminated, particularly, by aerobic biological treatment. It is difficult to assess the impact of such endocrine activities on the environment. Indeed, the activity measured by in vitro tests may be very different from in vivo responses, depending on the organism (Folmar et al., 2002; Routledge et al., 1998), and compound or mixture of pollutants considered (Hasselberg et al., 2008). In some cases, the estrogenic response is comparable in in vitro and in vivo measurements but in other cases, the in vivo response will be different than the in vitro response (Dobbins et al., 2008; Van den Belt et al., 2004). Moreover, the interactions between the different signaling pathways are very complex and remain partially unknown. For example, AhR ligands can modulate estrogen signaling pathway through direct association of AhR with ER (Ohtake et al., 2003, 2007). Such interactions can greatly modify the final response to EDCs of the endocrine system of living organisms. So, further research is needed to identify the potential impact on living organisms of endocrine activities resulting from animal waste.

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4.

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Conclusions

We have shown that the traditional spreading of stored pig manure could lead to the release of significant concentrations of hormones and estrogenic and dioxin-like activities in the environment. E1, aE2, bE2 and E3 were the main steroid hormones present in manure. These compounds, particularly active on the estrogen receptor ERa, are mainly present in the solid fraction of manure. This fraction also contains ERa- and AhR-activating compounds which remain to be identified. Aerobic treatments, implemented on-site to remove nitrogen from manure, displayed the ability to eliminate hormones efficiently while endocrine activities were only partially removed. But when centrifugation is performed upstream of the aerobic basin, the majority of hormones and endocrine activities were concentrated in the solid fraction. Nowadays, there is a lack of information about the mobility of these compounds and the fate of endocrine activities from this solid fraction after spreading on land. The present study provides complete data about the fate of steroid hormones during manure management. It also highlights the presence of unidentified compounds in manure with high estrogenic and dioxin-like potencies.

Acknowledgments The authors would like to thank the French National Agency for Research (ANR) and the French Agency for the Environment and Energy Management (ADEME) which funded the DIPERPHA project (ANR-07-SEST-006-01). We gratefully acknowledge the farm owners for their cooperation and assistance.

Appendix. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.watres.2011.11.074.

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