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

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.

Figures

Table 1. Selected physical and chemical properties of sewage sludge, biosolid and soil ___________________________________________________________________________ Parameter Sewage sludge Biosolid Soil

Table 3. First-order dissipation models of 17 β-estradiol radioactivity in human waste products and soil, and the calculated days after application that half of initial radioactivity is mineralized in these media in the presence and absence of antimicrobials. Different letters indicate significantly different mineralization half-lives (HL).

Table 2. Treatment effects of media and antimicrobials on total 17 βestradiol mineralization at 189 d.

Full text

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"

Frequently asked questions

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.