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Journal ArticleDOI

17 β-estradiol mineralization in human waste products and soil in the presence and the absence of antimicrobials.

TL;DR: It is unlikely for doxycycline and norfloxacin, or their mixtures, to have a significant effect on E2 mineralization in human waste products and soil, however, the potential for E2 to be persistent in biosolids, with and without the presence of antimicrobials, is posing a challenge for biosolid disposal to agricultural lands.
Abstract: Natural steroidal estrogens, such as 17 β-estradiol (E2), as well as antimicrobials such as doxycycline and norfloxacin, are excreted by humans and hence detected in sewage sludge and biosolid. The disposal of human waste products on agricultural land results in estrogens and antibiotics being detected as mixtures in soils. The objective of this study was to examine microbial respiration and E2 mineralization in sewage sludge, biosolid, and soil in the presence and the absence of doxycycline and norfloxacin. The antimicrobials were applied to the media either alone or in combination at total rates of 4 and 40 mg kg-1, with the 4 mg kg-1 rate being an environmentally relevant concentration. The calculated time that half of the applied E2 was mineralized ranged from 294 to 418 days in sewage sludge, from 721 to 869 days in soil, and from 2,258 to 14,146 days in biosolid. E2 mineralization followed first-order and the presence of antimicrobials had no significant effect on mineralization half-lives, except for some antimicrobial applications to the human waste products. At 189 day, total E2 mineralization was significantly greater in sewage sludge (38 ±0.7%) > soil (23 ±0.7%) > biosolid (3 ±0.7%), while total respiration was significantly greater in biosolid (1,258 mg CO2) > sewage sludge (253 mg CO2) ≥ soil (131 mg CO2). Strong sorption of E2 to the organic fraction in biosolid may have resulted in reduced E2 mineralization despite the high microbial activity in this media. Total E2 mineralization at 189 day was not significantly influenced by the presence of doxycycline and/or norfloxacin in the media. Antimicrobial additions also did not significantly influence total respiration in media, except that total CO2 respiration at 189 day was significantly greater for biosolid with 40 mg kg-1 doxycycline added, relative to biosolid without antimicrobials. We conclude that it is unlikely for doxycycline and norfloxacin, or their mixtures, to have a significant effect on E2 mineralization in human waste products and soil. However, the potential for E2 to be persistent in biosolids, with and without the presence of antimicrobials, is posing a challenge for biosolid disposal to agricultural lands.

Summary (2 min read)

INTRODUCTION

  • Estrogens are excreted by vertebrates in both urine and feces with an estimated global rate of 29,500 kg of natural estrogens being excreted by humans each year. [1].
  • Drinking water contaminated with estrogens could pose health risk to humans. [7, 12].
  • Antimicrobial additions to soil and other media has shown to reduce reduced E2 mineralization in some cases. [20, 23, 26].
  • Doxycycline is commonly used to treat wide array of infections in humans (urinary tract, intestine and eye) and is also increasingly used as an anti-cancer therapeutic. [28].
  • Tetracycline has shown to decrease soil bacterial diversity following its application to a plantsoil system. [24].

Chemicals and Media

  • Analytical grade 17 β-estradiol (≥98% pure), doxycycline (≥98% pure) and norfloxacin (≥98% pure) were purchased from Sigma-Aldrich Chemical Company (Saint Louis, MO, USA).
  • Sewage sludge was obtained from facultative lagoon-based sewage treatment facility in southern Manitoba, Canada that services approximately 8000 people.
  • The soil was sampled (0-15 cm) from the Ap-horizon of a Newdale Clay Loam, which is recognized as the provincial soil of Manitoba and represents a large area of agricultural land in this province.
  • Hormones were below detection limits in both samples.

Microcosm Experiments

  • The impact of antibiotics on E2 mineralization in these media was quantified using a factorial design of antimicrobial additions plus controls without antimicrobials.
  • Both radiolabeled and analytical grade E2 was applied and the E2 experiment was terminated at 189 days at which time E2 mineralization had slowed to <0.1% per day for all treatments.
  • In a parallel experiment, only analytical E2 was applied, but the same factorial design plus control was utilized to quantify the impact of antibiotics on microbial respiration (CO 2 production) over 189 days.
  • Microcosms were pre-incubated at 20˚C for seven days prior to E2 applications.
  • Antibiotic stock solutions were prepared in milli-Q water that was acidified with 0.5 M HCl (pH 4) to help dissolve doxycycline and/or norfloxacin.

Statistical Analyses

  • The effect of media and antibacterial treatment on total E2 mineralization or total respiration was analyzed by ANOVA using PROC MIXED in SAS ver. 9.4 (SAS Institute Inc., 2013).
  • All data respected normality based on Shapiro-Wilks statistic ≥ 0.9.
  • Dissipation kinetics of E2 for each media was generated and compared using PROC NLIN in SAS.
  • The calculated half-life is equivalent to the time that 50% of the radioactivity would have been mineralized in a media.
  • The Tukey multiple comparison procedure was used for pairwise comparisons of treatment means.

RESULTS AND DISCUSSION

  • Specifically, when norfloxacin or doxycycline were applied together, or when no antimicrobials were added, cumulative respiration was significantly greater in the sewage sludge than soil; however, when either norfloxacin or doxycycline were applied alone, sewage sludge and soil had a similar cumulative respiration (Fig. 3 ).
  • Regardless of whether norfloxacin or doxycycline was applied alone or in combination, the antimicrobials did not supress microbial respiration and, consistently, antimicrobial applications had no pronounced effect on E2 mineralization.
  • Biosolid showed a very small total E2 mineralization relative to sewage sludge and soil, yet this medium had a much greater microbial respiration.
  • 3 LECO model CHN 600 carbon-hydrogen-nitrogen determinator.
  • 8 Aqua regia digestion followed by Inductively Coupled Plasma Optical Emission Spectroscopy.

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1"
2"
17 Β-Estradiol mineralization in human waste products and soil in the presence and 3"
absence of antimicrobials 4"
5"
6"
INOKA AMARAKOON
1
, ANNEMIEKE FARENHORST
1,
*, KARIN ROSE
1
, ANNE 7"
CLAEYS
2
and BRUNA ASCEF
3
8"
9"
10"
1
Department of Soil Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada 11"
2
Department of Biologie, Instit
1
t Universitaire de Technologie de Perpignan, Perpignan, 12"
Pyrénées Orientales, France 13"
3
Department of Pharmacy, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 14"
Diamantina, Minas Gerais, Brazil 15"
"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""
*Address correspondence to, Dr. Annemieke Farenhorst, Department of Soil Science, Faculty of
Agricultural and Food Sciences,University of Manitoba, 380 Ellis Building, Winnipeg,
Manitoba, R3T 2N2, Canda; Tel: (204) 474 6858; FAX: (204) 474 7642
E-mail: annemieke.farenhorst@umanitoba.ca
"

2"
"
16"
17"
18"
19"
ABSTRACT 20"
21"
Natural steroidal estrogens such as 17 β-estradiol (E2), as well as antimicrobials such as 22"
doxycycline and norfloxacin, are excreted by humans and hence detected in sewage sludge and 23"
biosolid. The disposal of human waste products on agricultural land results in estrogens and 24"
antibiotics being detected as mixtures in soils. The objective of this study was to examine 25"
microbial respiration and E2 mineralization in sewage sludge, biosolid and soil in the presence 26"
and absence of doxycycline and norfloxacin. The antimicrobials were applied to the media either 27"
alone or in combination at total rates of 4 and 40 mg kg
-1
, with the 4 mg kg
-1
rate being an 28"
environmentally relevant concentration. The calculated time that half of the applied E2 was 29"
mineralized ranged from 294 to 418 d in sewage sludge, from 721 to 869 d in soil and from 30"
2,258 to 14,146 d in biosolid. E2 mineralization followed first-order and the presence of 31"
antimicrobials had no significant effect on mineralization half-lives, except for some 32"
antimicrobial applications to the human waste products. At 189 d, total E2 mineralization was 33"
significantly greater in sewage sludge (38 ±0.7%) > soil (23 ±0.7%) > biosolid (3 ±0.7%), while 34"
total respiration was significantly greater in biosolid (1,258 mg CO
2
) > sewage sludge (253 mg 35"
CO
2
) soil (131 mg CO
2
). Strong sorption of E2 to the organic fraction in biosolid may have 36"
resulted in reduced E2 mineralization despite the high microbial activity in this media. Total E2 37"
mineralization at 189 d was not significantly influenced by the presence of doxycycline and/or 38"

3"
"
norfloxacin in the media. Antimicrobial additions also did not significantly influence total 39"
respiration in media, except that total CO
2
respiration at 189 d was significantly greater for 40"
biosolid with 40 mg kg
-1
doxycycline added, relative to biosolid without antimicrobials. We 41"
conclude that it is unlikely for doxycycline and norfloxacin, or their mixtures, to have a 42"
significant effect on E2 mineralization in human waste products and soil. However, the potential 43"
for E2 to be persistent in biosolids, with and without the presence of antimicrobials, is posing a 44"
challenge for biosolid disposal to agricultural lands. 45"
46"
Keywords: 17 β-estradiol, antimicrobial, doxycycline, norfloxacin, sewage sludge, biosolid, 47"
soil, mineralization, respiration. 48"
49"
50"
INTRODUCTION 51"
52"
17 β-estradiol (E2) is a natural steroidal estrogen produced by vertebrates. Estrogens are excreted 53"
by vertebrates in both urine and feces with an estimated global rate of 29,500 kg of natural 54"
estrogens being excreted by humans each year.
[1]
17 β-estradiol has been frequently detected in 55"
human waste products such as in sewage sludge and biosolid at rates ranging up to 230 µg kg
-1
.
56"
[2-5]
The disposal of such human waste products to agricultural land enhances soil nutrients and 57"
carbon content, but also introduces estrogens that can contaminate surface waters via runoff
[6,7]
58"
and groundwater via leaching.
[8,9]
Contamination of surface waters with estrogens at low 59"
concentrations has been shown to induce vitellogenesis and other abnormal secondary sexual 60"
characteristics in male and juvenile female fish
[10,11]
, for example the production of an egg yolk 61"

4"
"
protein precursor has been observed in male fish exposed to E2 concentrations as low as 25 ng L
-
62"
1
.
[12]
Drinking water contaminated with estrogens could pose health risk to humans.
[7,12]
63"
64"
Bacteria that can degrade E2 in soil and human waste products include the genera Aminobacter, 65"
Escherichia, Pseudomonas, Sphingomonas (Proteobacteria), Corynebacterium, Microbacterium, 66"
Nocardioides, Rhodococcus (Actinobacteria), and Flavobacterium (Bacteroidetes).
[13-19]
67"
Microbial degradation is a main path of estrogen degradation
[20-22]
, hence antimicrobial toxicity 68"
towards these bacteria may result in increased E2 persistence in soil and human waste products. 69"
For example, chlortetracycline, sulfamethazine and tylosin applied to soil at concentrations as 70"
low as 2 mg kg
-1
significantly reduced the transformation of E2 to estrone (E1).
[23]
71"
Antimicrobials affect bacterial compositions with the impact on specific species being depended 72"
on the types and concentrations of antimicrobial present, as well as the duration of exposure and 73"
the level of bacterial resistance to the antibiotics.
[24]
74"
75"
E2 mineralization can be quantified by using radiolabeled [4-
14
C] E2 in microcosm experiments 76"
[21,25,26]
, whereby the recovery of
14
CO
2
indicates that the steroid molecule has been inactivated 77"
because of ring cleavage.
[27]
Antimicrobial additions to soil and other media has shown to 78"
reduce reduced E2 mineralization in some cases.
[20,23,26]
For example, the addition of 40 mg kg
-1
79"
tetracycline to manure decreased total E2 mineralization by 14% relatively to manure without 80"
antibiotics added, but E2 mineralization in soils was not impacted.
[26]
81"
82"
"In this study, we examine the impact of the antimicrobials doxycycline and norfloxacin on E2 83"
mineralization in media. Doxycycline, which selectively inhibits the 30S ribosomal subunit in 84"

5"
"
bacteria, belongs to the tetracycline group of antimicrobials. Doxycycline is commonly used to 85"
treat wide array of infections in humans (urinary tract, intestine and eye) and is also increasingly 86"
used as an anti-cancer therapeutic.
[28]
Norfloxacin belongs to the fluoroquinolone group of 87"
antimicrobials and is primarily used to treat urinary tract infections and respiratory diseases in 88"
humans by selectively inhibiting DNA replication in target bacteria.
[29]
Both doxycycline and 89"
norfloxacin are readily excreted by humans and have been detected in sewage sludge and 90"
biosolid at concentrations of up to 1.5 mg kg
-1
for doxycycline and 11 mg kg
-1
for norfloxacin
[30-
91"
34]
, and in agricultural soils following biosolid application at concentrations of up to 15 µg kg
-1
92"
for doxycycline and 45 µg kg
-1
for norfloxacin.
[32,35]
Both antimicrobials have been detected in 93"
surface water following wastewater effluent discharge and following surface runoff from soils 94"
amended with biosolid.
[31,32,36,37]
Tetracycline and fluoroquinolone compounds, either alone or 95"
in combination, have reported toxicity to photosynthetic aquatic organisms such cyanobacteria. 96"
[38]
Tetracycline has shown to decrease soil bacterial diversity following its application to a plant-97"
soil system.
[24]
Fluoroquinolones have shown to inhibit the growth of Azopirillum brasilense, a 98"
nitrogen-fixing bacteria in soil.
[39]
99"
100"
The objective of this study was to assess the individual and combined effects of doxycycline and 101"
norfloxacin on microbial respiration and E2 mineralization in sewage sludge, biosolid and soil. 102"
We tested the hypothesis that when present at an environmentally relevant concentration of 4 mg 103"
kg
-1
doxycycline and norfloxacin individually or combined will not inhibit microbial activity to 104"
the extent of significantly reducing E2 mineralization in media, but that at a ten times greater 105"
concentration rate, E2 mineralization will be reduced in one or more of the media tested. 106"
107"

Citations
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Journal ArticleDOI
Yujie He1, Ting Wang, Feifei Sun1, Lianhong Wang1, Rong Ji1 
TL;DR: It is demonstrated that considerable amounts of E2 and NER are retained in manure, despite nearly complete mineralization, and VAs administered to livestock may increase the persistence of natural estrogens in manure and, accordingly, the environmental risks posed by these compounds.

15 citations

Journal ArticleDOI
TL;DR: A microcosm protocol that quantified the mineralization of E2 in soils under field temperatures concluded that incubations at 20°C give a reasonable representation of E 2 mineralization occurring under field conditions to be expected in a typical Prairie summer season.
Abstract: Mineralization studies of natural steroid hormones (e.g., 17β-estradiol, E2) are performed in environmental incubators, usually under a constant temperature such as 20°C. In this paper, we present a microcosm protocol that quantified the mineralization of E2 in soils under field temperatures. The nine agricultural soils tested had a wide range of soil organic carbon (1.1 to 5.2%) and clay (9 to 57%) contents. The calculated time over which half of the applied E2 was mineralized (E2-½) ranged from 299 to 910 d, and total E2 mineralization at 48 d (E2-TOT48) ranged from 4 to 13%. In subsequent laboratory incubations, the same soils were incubated under a constant temperature of 20°C, as well as under cyclic temperatures of 14.5°C (14 h) and 11.5°C (10h), which was within the temperature extremes observed in the field microcosms. E2-½ ranged from 157 to 686 d at 20°C and from 103 to 608 d at the cyclic temperatures, with the E2-TOT48 ranging from 6 to 21% at 20°C and from 7 to 30% under cyclic temper...

Cites background from "17 β-estradiol mineralization in hu..."

  • ...recovery of (14)CO2 indicates that the steroid molecule has been inactivated because of ring cleavage.([17]) The time that 50% of the applied E2 is mineralized (E2-(1)/2) can be calculated from these laboratory data....

    [...]

References
More filters
Journal ArticleDOI
TL;DR: The draft genome sequence of strain SJTE-1 is reported and major findings from its annotation, which could provide insights into its biodegradation mechanisms are reported, are reported.
Abstract: Pseudomonas putida strain SJTE-1 can utilize 17β-estradiol and other environmental estrogens/toxicants, such as estrone, and naphthalene as sole carbon sources. We report the draft genome sequence of strain SJTE-1 (5,551,505 bp, with a GC content of 62.25%) and major findings from its annotation, which could provide insights into its biodegradation mechanisms.

27 citations

Journal ArticleDOI
Soul Chun1, Jaehoon Lee1, Roland Geyer1, David C. White1, D. Raj Raman1 
TL;DR: It is recommended that any study evaluating the fate and transport of estrogenic hormones in soil should include the effect of agricultural antibiotics because antibiotics and estrogenic hormone are commonly excreted together in environmental samples.
Abstract: A laboratory incubation study was conducted to investigate the effect of agricultural antibiotics (sulfamethazine, tylosin, and chlortetracycline) on the persistence and transformation of 17β-estradiol in Sequatchie loam. We measured concentrations of 17β-estradiol and its primary metabolite (estrone) in soils spiked with antibiotics and 17β-estradiol. Dehydrogenase activity (DHA) was also measured as an indicator of the total microbial activity of the soils. The presence of antibiotics significantly decreased transformation of 17β-estradiol to estrone. There was a positive correlation between the DHA and the concentrations of estrone in soil spiked with 17β-estradiol only, implying that the reaction is mainly catalyzed by dehydrogenases. However, the positive correlation was weakened in soil spiked with 17β-estradiol and antibiotics together. We recommend that any study evaluating the fate and transport of estrogenic hormones in soil should include the effect of agricultural antibiotics because antibioti...

24 citations


"17 β-estradiol mineralization in hu..." refers background in this paper

  • ...to soil and other media has shown to reduce E2 mineralization in some cases.([20,23,26]) For example, the addition of 40 mg kg¡1 tetracycline to manure decreased total E2 mineralization by...

    [...]

  • ...In another laboratory experiment of shorter duration (7 days), [23] the presence of a single antimicrobial (chlortetracycline, sulfamethazine, or tylosin) in a loam soil reduced the transformation of E2 to estrone (E1) when the antimicrobial was applied at 2 or 200 mg kg¡1....

    [...]

  • ...zine, and tylosin applied to soil at concentrations as low as 2 mg kg¡1 significantly reduced the transformation of E2 to estrone (E1).([23]) Antimicrobials affect bacterial compositions with the...

    [...]

Journal ArticleDOI
TL;DR: These studies build upon previously theorized interactions, yielding a more comprehensive model of binding between estrogens and HA, as well as demonstrate the value of the confluence of biochemical assay methods with analytical NMR techniques.

23 citations


"17 β-estradiol mineralization in hu..." refers background in this paper

  • ...and hence has a strong affinity for organic matter substances in biosolid.([44]) Desorption of E2 from biosolid has been reported to be low (e....

    [...]

Journal ArticleDOI
TL;DR: Surprisingly, the increase in estrogen sorption to the soil solid phase either through short-term or long-term organic waste application does not control estrogen mineralization.
Abstract: Natural and synthetic estrogens can reach agricultural soils with manures, biosolids, or wastewater. This study evaluates (i) the effects of long-term field application of such organic soil amendments and (ii) the short-term effects of 14 different organic amendments in one agricultural soil on mineralization and sorption of (14)C-labeled 17beta-estradiol, estrone, and 17alpha-ethinylestradiol. Long-term organic waste applications resulted in increasing soil organic carbon (SOC) contents, causing increased estrogen sorption. The mineralization of the estrogens was enhanced by up to 147% or depressed by up to 50%, depending on site and organic waste, but not related to changes in sorption parameters. Short-term organic waste amendments directly increased estradiol mineralization to up to 70% in the treated soil compared with 5% in the untreated control. Estradiol sorption increased with the amount of incorporated organic waste, but the log K(oc) values of 3.1-3.2 L kg(-1) for organic wastes showed a lower sorption potential for estrogens compared with 3.5 L kg(-1) in the untreated soil. The effects of organic waste amendments on estrogen behavior depend on amendment type and aging. Short-term organic waste applications to soil resulted in enhanced microbial estrogen mineralization likely due to cometabolic processes. In soils with a long history of organic waste amendments, the controlling factor for estrogen sorption is an increased SOC content. The observed positive or negative effects on estrogen mineralization in these soils are not well understood. Surprisingly, the increase in estrogen sorption to the soil solid phase either through short-term or long-term organic waste application does not control estrogen mineralization.

17 citations

Journal ArticleDOI
TL;DR: The effects of perhexiline on the sensitivity of platelets to the antiaggregatory effects of nitric oxide are reported, prompted by the group’s previous observation that in a multivariate analysis, per hexiline treatment predicted greater platelet nitricoxide sensitivity in patients with angina.
Abstract: In this issue, Willoughby and colleagues report the effects of perhexiline on the sensitivity of platelets to the antiaggregatory effects of nitric oxide. Nitric oxide potently inhibits the aggregation of platelets via a cyclic GMP dependent mechanism. This group of workers has previously shown that platelets from patients with both stable angina and unstable coronary syndromes exhibit impaired ex vivo sensitivity to the antiaggregatory effects of the nitric oxide donor sodium nitroprusside, this abnormality being particularly marked in patients with unstable coronary syndromes. Platelet nitric oxide resistance may play an important role in platelet thrombus formation in unstable coronary syndromes. Previous work by the group has suggested that this resistance to the antiaggregatory effects of nitric oxide on platelets might occur from one or both of two mechanisms. First, by increased nitric oxide clearance as a result of increased superoxide production. Second, via a reversible inhibition of platelet guanylate cyclase. Platelet NO resistance has also been documented in association with insulin resistance in obesity. In certain situations, including heart failure and coronary artery disease there may also be ‘resistance’ to the vascular smooth muscle effects of nitric oxide. Because this resistance results in a parallel shift of the dose-response relation it is only identified by examining the vascular response to submaximal doses of NO donors. Nitric oxide resistance may contribute to the endothelial dysfunction seen in these conditions. It is not known whether the mechanisms responsible for vascular smooth muscle and platelet nitric oxide resistance are the same, and whether the two phenomena parallel each other. The present study was prompted by the group’s previous observation that in a multivariate analysis, perhexiline treatment predicted greater platelet nitric oxide sensitivity in patients with angina. Perhexiline is an antianginal now little used in Europe because of reports of hepatotoxicity and neurotoxicity in the 1970s and 1980s. Nevertheless, several reports have shown it to be a potent anti-anginal, both as monotherapy and as an adjunct to conventional antianginal therapy. It is metabolized by a P450 enzyme system for which there is polymorphic variation, and toxicity can be avoided by measuring plasma levels and maintaining them within the range

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Q1. What are the contributions in this paper?

In the presence and absence of antimicrobials, Amaro et al. this paper showed that Estradiol mineralization occurs in human waste products and soil in the presence of antimicrobial agents.