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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.

AbstractNatural 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.

Topics: Biosolids (57%), Sludge (57%), Mineralization (soil science) (55%), Sewage (52%)

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
More filters

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.
Abstract: The fate and persistence of natural estrogens from livestock manure and the interactions of these compounds with veterinary antibiotics (VAs) have not been well studied. We therefore employed 14C-labeling to explore the mineralization, degradation, and residual distribution of 17β-estradiol (E2) in swine manure in the absence and presence of six categories of VAs at concentrations of 10 and 100 mg/kg. After 16 days of incubation, 94% of the E2 dissipated, of which 28% was mineralized to 14CO2, 18% was transformed into organic-extractable E1 (9%) and other unknown metabolites (9%), and 48% into non-extractable residues (NER). VAs inhibited, enhanced or had no effect on E2 mineralization or its degradation to E1 and other metabolites. Principal component analysis showed that the overall effect of VAs was not necessarily related to their physicochemical properties or concentrations. However, high doses of macrolides inhibited E2 mineralization in manure and increased the retention of E2 and its metabolites in both free and NER forms. Our study demonstrates that considerable amounts of E2 and NER are retained in manure, despite nearly complete mineralization. Thus, VAs administered to livestock may increase the persistence of natural estrogens in manure and, accordingly, the environmental risks posed by these compounds.

6 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
Abstract: To provide the first nationwide reconnaissance of the occurrence of pharmaceuticals, hormones, and other organic wastewater contaminants (OWCs) in water resources, the U.S. Geological Survey used five newly developed analytical methods to measure concentrations of 95 OWCs in water samples from a network of 139 streams across 30 states during 1999 and 2000. The selection of sampling sites was biased toward streams susceptible to contamination (i.e. downstream of intense urbanization and livestock production). OWCs were prevalent during this study, being found in 80% of the streams sampled. The compounds detected represent a wide range of residential, industrial, and agricultural origins and uses with 82 of the 95 OWCs being found during this study. The most frequently detected compounds were coprostanol (fecal steroid), cholesterol (plant and animal steroid), N,N-diethyltoluamide (insect repellant), caffeine (stimulant), triclosan (antimicrobial disinfectant), tri(2-chloroethyl)phosphate (fire retardant), and 4-nonylphenol (nonionic detergent metabolite). Measured concentrations for this study were generally low and rarely exceeded drinking-water guidelines, drinking-water health advisories, or aquatic-life criteria. Many compounds, however, do not have such guidelines established. The detection of multiple OWCs was common for this study, with a median of seven and as many as 38 OWCs being found in a given water sample. Little is known about the potential interactive effects (such as synergistic or antagonistic toxicity) that may occur from complex mixtures of OWCs in the environment. In addition, results of this study demonstrate the importance of obtaining data on metabolites to fully understand not only the fate and transport of OWCs in the hydrologic system but also their ultimate overall effect on human health and the environment.

6,628 citations



Journal ArticleDOI
TL;DR: The occurrence of certain natural and synthetic steroidal estrogens in the final effluent from STW has been demonstrated and the response of adult male and female roach following exposure to 17β-estradiol was compared to the response to the alkylphenolic xenoestrogen.
Abstract: The occurrence of certain natural and synthetic steroidal estrogens in the final effluent from STW has been demonstrated. 17β-Estradiol and estrone were present at concentrations in the tens of nanograms per liter range, and the synthetic estrogen 17α-ethynylestradiol was also identified, albeit in the low nanogram per liter range. The findings from subsequent in vivo tank trial experiments, in which adult male rainbow trout (Oncorhynchus mykiss) and adult roach (Rutilus rutilus) were exposed for 21 days via the water to environmentally relevant concentrations of 17β-estradiol and estrone are presented. In addition, the response of adult male and female roach following exposure to 17β-estradiol (1, 10, and 100 ng/L) was compared to the response to the alkylphenolic xenoestrogen, 4-tert-octylphenol (1, 10 and 100 μg/L). Plasma levels of vitellogenin were determined using previously validated radioimmunoassays in order to measure the estrogenic response of the fish to the varying concentrations of the compo...

1,321 citations


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

  • ...abnormal secondary sexual characteristics in male and juvenile female fish,([10,11]) for example, the production of an egg yolk protein...

    [...]


Journal ArticleDOI
TL;DR: This review tries to present part of the knowledge that is currently available with regard to the occurrence of pharmaceutical residues in aquatic matrices, the progress made during the last several years on identification of such compounds down to trace levels, and of new, previously unidentified, pharmaceuticals such as illicit drugs, metabolites, and photo-products.
Abstract: Pollution from pharmaceuticals in the aquatic environment is now recognized as an environmental concern in many countries. This has led to the creation of an extensive area of research, including among others: their chemical identification and quantification; elucidation of transformation pathways when present in wastewater-treatment plants or in environmental matrices; assessment of their potential biological effects; and development and application of advanced treatment processes for their removal and/or mineralization. Pharmaceuticals are a unique category of pollutants, because of their special characteristics, and their behavior and fate cannot be simulated with other chemical organic contaminants. Over the last decade the scientific community has embraced research in this specific field and the outcome has been immense. This was facilitated by advances in chromatographic techniques and relevant biological assays. Despite this, a number of unanswered questions exist and still there is much room for development and work towards a more solid understanding of the actual consequences of the release of pharmaceuticals in the environment. This review tries to present part of the knowledge that is currently available with regard to the occurrence of pharmaceutical residues in aquatic matrices, the progress made during the last several years on identification of such compounds down to trace levels, and of new, previously unidentified, pharmaceuticals such as illicit drugs, metabolites, and photo-products. It also tries to discuss the main recent findings in respect of the capacity of various treatment technologies to remove these contaminants and to highlight some of the adverse effects that may be related to their ubiquitous existence. Finally, socioeconomic measures that may be able to hinder the introduction of such compounds into the environment are briefly discussed.

664 citations


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

  • ...Both antimicrobials have been detected in surface water following wastewater effluent discharge and following surface runoff from soils amended with biosolid.([31,32,36,37])...

    [...]


Journal ArticleDOI
TL;DR: The main outcome of the study was that a common municipal STP with an activated sludge system for nitrification and denitrification including sludge recirculation can appreciably eliminate natural and synthetic estrogens.
Abstract: The fate of the highly potent endocrine disrupters estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2) was investigated in mechanical and biological sewage treatment as well as in sewage-sludge treatment at a municipal German sewage treatment plant (STP). The main outcome of the study was that a common municipal STP with an activated sludge system for nitrification and denitrification including sludge recirculation can appreciably eliminate natural and synthetic estrogens. As a consequence, the endocrine effects of biota in the receiving waters should be significantly reduced. All estrogen concentrations decreased gradually along the treatment train. In the STP effluent, the steroid estrogen concentrations were always below the quantification limit of 1 ng/L. The elimination efficiency of the natural estrogens (E1 and E2) exceeded 98%, and EE2 was reduced by more than 90%. The natural estrogens were largely degraded biologically in the denitrifying and aerated nitrifying tanks of the activate...

538 citations


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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.