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Critical Review
Nitrate: an Environmental Endocrine Disruptor? A Review of Evidence and Research Needs Rikke Poulsen, Nina Cedergreen, Tyrone B Hayes, and Martin Hansen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06419 • Publication Date (Web): 01 Mar 2018 Downloaded from http://pubs.acs.org on March 1, 2018
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Nitrate: an Environmental Endocrine Disruptor? A
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Review of Evidence and Research Needs
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Rikke Poulsen†,*, Nina Cedergreen†, Tyrone Hayes‡ and Martin Hansen†,‡,§,∇,*
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†
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Thorvaldsensvej 40, 1871 Frederiksberg, Denmark. ‡Laboratory for Integrative Studies in
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Amphibian Biology, Molecular Toxicology, Group in Endocrinology, Energy and Resources
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Group, Museum of Vertebrate Zoology, and Department of Integrative Biology, University
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of California, Berkeley, CA 94720, United States. §Department of Environmental and Civil
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Engineering, University of California, Berkeley, CA 94720, United States. ∇Department of
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Department of Plant and Environmental Sciences, University of Copenhagen,
Environmental Science, Aarhus University, 4000 Roskilde, Denmark.
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KEYWORDS. Nitrate; endocrine disruption; environmental toxicology; hormone homeostasis;
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physiological endpoints.
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ABSTRACT. Nitrate is heavily used as an agricultural fertilizer and is today a ubiquitous
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environmental pollutant. Environmental endocrine effects caused by nitrate have received increasing
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attention over the last 15 years. Nitrate is hypothesized to interfere with thyroid and steroid hormone
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homeostasis and developmental and reproductive endpoints. The current review focuses on aquatic
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ecotoxicology with emphasis on field and laboratory controlled in vitro and in vivo studies.
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Furthermore, nitrate is just one of several forms of nitrogen that is present in the environment and
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many of these are quickly interconvertable. Therefore the focus is additionally confined to the
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oxidized nitrogen species (nitrate, nitrite and nitric oxide). We reviewed 26 environmental toxicology
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studies and our main findings are: (1) Nitrate has endocrine disrupting properties and hypotheses for
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mechanisms exist, which warrants for further investigations; (2) there are issues determining actual
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nitrate-speciation and abundance is not quantified in a number of studies, making links to speciation-
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specific effects difficult; and (3) more advanced analytical chemistry methodologies are needed both
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for exposure assessment and in the determination of endocrine biomarkers.
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INTRODUCTION
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In recent decades the influence of humans has caused mass extinctions of plants and animal species,
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contaminated oceans and fresh waters and polluted the atmosphere 1–3. Tripling the human population
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in a single human-lifetime, food, water and energy demands are currently enormous 4. By-products of
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fertilizers, pesticides and pharmaceuticals that we rely on for health and to enhance food production,
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are dispersed into the environment. Many of these products are toxic or mimic natural biomolecules
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causing endocrine disruption in humans and wildlife 5–8.
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Nitrogen is a macronutrient essential for any type of life and one of the most plentiful
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elements on the planet. However, because the major form is non-reactive molecular nitrogen (N2),
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only a very small percentage is readily available for biological uptake 9,10. The available forms include
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inorganic species like nitrate (NO3-), nitrite (NO2-), ammonia (NH3) and ammonium (NH4+) as well as
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organic compounds (e.g. amino acids, urea and nucleic acids). Nitrogen is cycled between these
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different chemical forms in the biosphere and in the full assessment of environmental consequences of
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increased nitrogen emission, it is important to consider all reactive species and their mixtures. In the
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current review we focus on the oxidized species; nitrate and nitrite as well as their metabolite, nitric
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oxide.
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The double Nobel Prize awarded Haber-Bosch process, which converts atmospheric nitrogen
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to ammonia as a feedstock for agricultural nitrate-fertilizers, kick-started the Agricultural Revolution
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11
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of nitrate as an agricultural fertilizer and due to other atmospheric emissions of the nitrogen oxides
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and of ammonia 12. This has also lead to an increase in the nitrogen concentrations in surface waters 13.
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Even though nitrogen levels in surface waters during pre-industrial times cannot be precisely
. Subsequently, environmental nitrogen emissions have increased tremendously both due to the use
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estimated 14 it is unarguable that present day nitrate-levels (typically ranging 1-30 mg/L NO3-N
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[nitrate-as-nitrogen] in surface waters 15–21) are likely above the concentrations where most aquatic
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life evolved 22. The habitat of much aquatic wildlife in minor water bodies such as ponds and creeks,
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where nitrate concentrations typically are higher, may reach several hundred mg/L NO3-N in extreme
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cases 21.
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During the past decades, deviations in reproductive hormone levels and sex ratios in wildlife
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have been reported in organisms ranging from alligators, newts, fish and frogs to small crustaceans
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8,21,23–28
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discharged from urban waste water treatment plants 30. In addition, more apparent suspects have been
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identified such as estrogens from human contraceptives 31, hormone residues in livestock waste 32,33 or
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even phytoestrogens released into agricultural drainage systems during soy and red-clover
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productions 34. Currently, nitrate is recognized as a pollutant partly due to a combined act with
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phosphorus for eutrophication of aquatic environments 35,36, however the long-term effects on wildlife
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have received limited attention. Relationships between vertebrate hormonal changes and water nitrate
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concentrations have, however, been observed and have last been reviewed by Guillette and Edwards
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(2005)24.
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. Many suspects have been implicated, such as pesticides 7, biocides 29 and chemicals
An endocrine disruptor is defined by the U.S. E.P.A. as “an exogenous agent that interferes
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with the production, release, transport, metabolism, binding, action, or elimination of natural
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hormones in the body responsible for the maintenance of homeostasis and the regulation of
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developmental processes”37. Since the review by Guillette and Edwards and raised hypotheses24,
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experimental evidence for the endocrine disruptive potential of nitrate in both vertebrates and
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invertebrates has increased. The present review compiles the existing scientific data and examines
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how nitrate can act as an environmentally relevant endocrine disruptor with multiple ways of affecting
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the endocrine system, leading to new hypotheses (as we previously have reported38). Our aim is two-
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fold: 1) to report and assess studies, where endocrine effects of nitrate and/or its metabolites have
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been investigated on aquatic organisms and ecosystems, and 2) to give an overview of the possible
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molecular initiating events and key cellular events which may link nitrate and/or its metabolites to
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endocrine disrupting outcomes. Both field and laboratory-controlled in vitro and in vivo studies have
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been conducted and several different animal species have been investigated. The current review
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focuses on aquatic ecotoxicology and therefore emphasizes aquatic organisms.
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EFFECTS ON STEROIDOGENESIS AND FECUNDITY
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Steroid hormones are essential for reproduction, behavior, and metabolism via genomic processes, e.g.
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regulation of gene transcription39,40. In addition, steroid hormones also act via rapid non-genomic
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processes, such as the acrosome reaction41. Hence, quantification of this hormone class is a much-
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used biomarker of endocrine effects in ecotoxicology. The hypothalamus-pituitary-adrenal and
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hypothalamus-pituitary-gonadal axes tightly regulate steroidogenesis, the biosynthesis of steroid
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hormones. An overview of the in-vivo investigations targeting nitrate and nitrite affecting the
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steroidogenesis in aquatic living organisms is displayed in Table 1.
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Circulating steroid hormone levels. Numerous studies have explored how nitrate may affect
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steroidogenesis (Table 1) by using the circulating levels of steroid hormones as toxicological endpoint.
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Hamlin et al. (2008)42 investigated effects of 30-day chronic nitrate exposure (1.5, 11.5 and 57 mg/L
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NO3-N) on levels of cortico- and sex-steroids in female Siberian sturgeons (Acipenser paeri).
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Elevated plasma concentrations of sex steroids (17β-estradiol, testosterone and 11-ketotestosterone)
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were detected, while cortisol and glucose levels were unaffected. Unfortunately, the effect on 11-
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ketotestosterone could not be replicated in a follow-up experiment. Increased 11-ketotestosterone was,
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however, also observed in male fathead minnows (Pimephales promelas) exposed to nitrate (0, 11.3
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and 56.5 mg/L NO3-N) from 61 mg/L NO2-N). 24 hr exposure to 457 mg/L NO2-N significantly affected swim bladder non-inflation and number of axial
Concentrations are high compared to observed environmental levels.
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malformations Investigate the effects of 45-days nitrite (2.0 mg/L NO2-N) exposure on endocrine functions of juvenile Labeo rohita and elucidate the possible counteracting role of dietary scavengers of superoxides and NO-radicals. *
Weight gain, plasma concentration of triiodothyronine (T3) and thyroxine (T4), but also cortisol, testosterone and estradiol (Table 1).
Nitrate exposure significantly decreased serum concentration of T3 and T4, but also weight gain. This could be reversed by Vitamin E and to some extent tryptophan.
Notice that this is a study of nitrite, which is more potent than nitrate. pH variation 7.4–8.6. Immunoassay used for steroid and thyroid hormone measurements.
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Investigate hormonal responses to 27days elevated nitrate (5, 10 and 102 mg/L NO3-N) by juvenile Atlantic salmon (Salmo salar) *
Plasma concentration of nitrate and of thyroid hormones (T4 and T3). Testosterone and 11-ketotestonerone also measured (Table 1).
No significant differences were observed.
Hormone concentrations as sole endpoint. Immunoassay used for thyroid hormone measurements.
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Investigate effect of nitrate on zebrafish (Danio rerio) based on acute (96 hr, 1076-2209 mg/L NO3-N) and chronic (23 days, 50, 100, 200, 400 mg/L NO3-N) bioassays in early life stages.
Mortality, growth and body condition factor.
Chronic assay: survival and all growth parameters were significantly decreased at 400 mg/L. Also morphological abnormalities were observed at this concentration.
Inclusion of sodium control treatment (n=1) of similar ionic strength to the 400 mg/L treatment also showed large decrease in survival. Ionic strength alone may therefore have influenced the survival parameter. This control treatment was however not replicated. Control treatment unfortunately also contained 7 mg/L NO3-N.
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Investigate actute and subchronic toxicity of ammonia (2.5 to 30 mg/L NH3-N), nitrite (13 to 152 mg/L NO2-N), and nitrate (20 to 176 mg/L NO3-N) exposure in the rare minnow (Gobiocypris rarus).
Body length, hatchability, malformation rate, swim bladder size as a measurement of development and mortality
All compounds affected growth, development, and survival dosedependently with potency following the order ammonia > nitrite > nitrate. Maximum allowable toxicant concentration is 3.05 mg/L NH3-N, 16.3 mg/L NO2-N and 34.5 mg/L NO3-N.
Water quality monitored but not reported in paper. Tap water used for control with unknown background level.
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It is important to notice potency differences between nitrite and nitrate. Unlike nitrite, nitrate failed to increase fluorescence associated with NOgeneration in ex vivo exposure of perinatal daphnids. Sodium nitroprusside was used as NO-donor. Ecdysteroid measurements were performed by radioimmunoassay, however information on origin, specificity and cross-reactivity
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Invertebrates Investigate if nitrite and nitrate undergo intracellular conversion to nitric oxide and if the three compounds interfere with embryo development and fecundity in Daphnia magna exposed through four brood cycles to the NO-donor sodium nitroprusside (0.05-0.75 mg /L N), nitrite (0.04-1.1 mg/L NO2-N) and nitrate (15250 mg/L NO3-N). *
Fluorescence detection of NO, number of offspring, developmental abnormalities, ecdysteroid measurements.
Developmental toxicity was observed and the response to nitrite and nitrate qualitatively mimicked the response to NO, with nitrite being more potent than nitrate (factor of ~200).
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of antibody is lacking.
1215 1216
LOEC, lowest observed effect concentration. NOEC, no observed effect concentration.
1217 1218
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