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Effects of glyphosate and its formulation, Roundup , on reproduction in zebrafish (Danio rerio). Tamsyn M Uren Webster, Lauren V Laing, Hannah Florance, and Eduarda M. Santos Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 23 Dec 2013 Downloaded from http://pubs.acs.org on December 30, 2013
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Environmental Science & Technology
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Effects of glyphosate and its formulation,
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Roundup®, on reproduction in zebrafish (Danio
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rerio).
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Tamsyn. M. Uren Webster1*, Lauren V. Laing1, Hannah Florance1 and Eduarda M. Santos1*
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1. Biosciences, College of Life & Environmental Sciences, Geoffrey Pope Building, University
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of Exeter, Exeter, EX4 4QD
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* Corresponding authors
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ABSTRACT
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Roundup®, and its active ingredient glyphosate, are among the most widely used herbicides
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worldwide, and may contaminate surface waters. Research suggests both Roundup® and
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glyphosate induce oxidative stress in fish, and may also cause reproductive toxicity in
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mammalian systems. We aimed to investigate the reproductive effects of Roundup® and
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glyphosate in fish, and the potential associated mechanisms of toxicity. To do this, we conducted
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a 21-day exposure of breeding zebrafish (Danio rerio) to 0.01, 0.5 and 10 mg/L (glyphosate acid
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equivalent) Roundup® and 10 mg/L glyphosate. 10 mg/L glyphosate reduced egg production, but
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not fertilisation rate in breeding colonies. Both 10 mg/L Roundup® and glyphosate increased
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early-stage embryo mortalities and premature hatching. However, exposure during
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embryogenesis alone did not increase embryo mortality, suggesting that this effect was caused
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primarily by exposure during gametogenesis. Transcript profiling of the gonads revealed 10
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mg/L Roundup® and glyphosate induced changes in the expression of cyp19a1 and esr1 in the
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ovary, and hsd3b2, cat and sod1 in the testis. Our results demonstrate that these chemicals cause
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reproductive toxicity in zebrafish, although only at high concentrations unlikely to occur in the
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environment, and likely mechanisms of toxicity include disruption of the steroidogenic
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biosynthesis pathway and oxidative stress.
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Environmental Science & Technology
INTRODUCTION
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Glyphosate is extensively used worldwide, topping lists of agricultural herbicide usage in
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Europe [1] and the US [2]. It is a broad-spectrum, post emergence herbicide, which acts by
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binding phosphoenolpyruvate, the substrate of EPSP synthase, and subsequently inhibiting
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aromatic amino acid synthesis via the shikimate pathway in plants [3, 4]. Glyphosate is generally
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applied as part of a formulated product, the most widely used of which are the Roundup®
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herbicides. Roundup® formulations contain glyphosate in the form of an isoproylamine salt,
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which aids solubility but does not affect its properties as the active ingredient, together with
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various adjuvants which enhance its herbicidal properties. One of the most important and
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commonly used adjuvants is polyethoxylated tallow amine (POEA), a surfactant that enhances
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penetration of glyphosate through the plant cuticle [5, 6]. Glyphosate and Roundup® are also
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extensively used as domestic and urban-area weed-killers [2]. Commercial glyphosate
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formulations vary in composition with country and purpose and the properties of these
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formulations, including their toxicity, can be compared using the concentration of glyphosate
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present, expressed as glyphosate acid equivalent (a.e.).
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Glyphosate is known to strongly adsorp to soil, where it is subject to microbial degradation.
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This is one of glyphosate’s advantageous herbicidal properties, limiting agricultural input to
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surface waters in ideal conditions. However, pulses of contamination can be expected when
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rainfall occurs directly after application and when flood events increase river sediment load [6].
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Urban runoff and wastewater treatment effluent also account for considerable glyphosate input
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into rivers [7]. Despite its widespread use, concentrations of glyphosate, or its associated
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formulation components, are not routinely monitored in surface waters. However, glyphosate
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concentrations worldwide have been regularly reported to occur up to ~10-15 µg/L in rivers
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[e.g.8, 9]. Considerably higher peaks in concentration, in the high µg/L range, have also been
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measured, but are mainly associated with direct aquatic application, and in isolated wetland
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environments [6, 10].
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Although the target mechanism of action of glyphosate and glyphosate-based formulations is
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specific to plants, they have been shown to induce diverse biological effects in a range of non-
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target organisms. In fish, much of previous research assessing effects of Roundup® and
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glyphosate has focused on their induction of oxidative stress through ROS generation and/or
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interference with cellular antioxidant production. Short-term exposures (up to 6 days) to 1-20
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mg/L of several Roundup® formulations in a number of fish species altered levels of cellular
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antioxidants and induced oxidative damage of DNA, lipids and proteins [e.g. 11, 12-15].
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Environmentally relevant concentrations of Roundup®, glyphosate and POEA have also induced
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DNA damage in blood and liver cells of eel and catfish after up to 9 days exposure [16-19].
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Other studies have found that Roundup®, and in some cases glyphosate, induce other effects in
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fish including neurotoxicity and immunotoxicity [e.g. 20, 21, 22]. Similar evidence of Roundup®
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and glyphosate toxicity has been found for other vertebrate species, and demonstrated effects
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include occurrence of developmental abnormalities, especially in amphibians [23, 24]. Roundup®
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formulations have largely been found to be more toxic than pure glyphosate. The inherent
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toxicity of POEA [17, 23], and potentially other formulation components, is likely to contribute
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to this, although a modulating effect on glyphosate toxicity is also possible. Few studies,
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however, have directly compared equivalent concentrations of Roundup® and glyphosate.
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The potential of Roundup® to disrupt the endocrine system in vitro has been demonstrated in
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mammalian cell lines. In mouse Leydig cells, sub-lethal concentrations of Roundup® (from 25
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mg/L) altered the transcription and activity of steroidogenic acute regulatory protein (StAR),
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resulting in disruption of progesterone production [25]. A number of studies demonstrated
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consistent inhibition of aromatase activity by Roundup® in various human cell lines. The
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concentrations required to cause these effects varied depending on the cell type and formulation,
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but included from 4 mg/L in liver cells and 72 mg/L in placental and embryonic cells [26-28].
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Less consistently, and to a smaller extent than Roundup®, aromatase inhibition by glyphosate
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alone has also been reported (approximately 10 fold higher concentrations) [28]. Additionally,
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both glyphosate and Roundup® were reported to reduce testosterone production in rat testicular
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cells at concentrations from 0.36 mg/L [29], but it is difficult to relate these results to the
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potential effects of these chemicals in vivo. Few studies have investigated the effects of
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glyphosate and its commercial formulations on the endocrine system in vivo. Drakes treated with
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5 and 100 mg Roundup®/kg (body weight) exhibited reduced levels of testosterone,
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corresponding with alterations in testis structure. In rats, maternal and juvenile treatment from 5
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and 50 mg/kg (body weight) of Roundup® impaired male reproductive development, with effects
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including alteration in testis structure, sperm production and sex steroid production [30-32].
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Reproductive effects of Roundup® and glyphosate in fish have seldom been investigated and
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are far from clear. While no evidence of altered gonadal development was evident in juvenile
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stickleback exposed to 0.1-100 µg/L glyphosate [33], treatment with 3.6 mg/L Roundup® had
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some negative impact on offspring production in Silver catfish [34]. The mechanisms
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contributing to this reproductive effect have not been investigated.
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Given the extensive usage of glyphosate based herbicides, there is a clear potential for the
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environmental exposure of fish populations to glyphosate together with associated formulation
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products, which may modify its toxicity. This study aimed to examine the effects of Roundup®
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formulation on reproduction in fish and to determine to what extend these effects were associated
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with the toxicity of glyphosate alone. To do this, we conducted a 21 day reproductive test in
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breeding colonies of zebrafish, to determine if reproduction, embryo development and embryo
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survival, were affected by exposure to 0.01, 0.5 and 10 mg/L (glyphosate acid equivalent)
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Roundup® and 10 mg/L glyphosate. The two lower Roundup® concentrations included in this
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study were chosen to represent concentrations that can be expected to occur in the environment
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regularly (0.01 mg/L) and during occasional peak contamination events (0.5 mg/L). The highest
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concentration tested (10 mg/L) is unlikely to occur in surface waters, and was included to
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facilitate the analysis of the mechanisms of toxicity. We included a treatment group exposed to
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10 mg/L glyphosate alone to allow for a direct comparison of its mechanisms of toxicity with the
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equivalent a.e. concentration of Roundup®. We hypothesised that the mechanisms of toxicity
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resulting in effects on reproduction might include oxidative stress and disruption of steroid
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biosynthesis, and to investigate this we conducted transcript profiling of a suite of genes
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involved in these processes in the gonads.
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MATERIALS AND METHODS
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Fish maintenance
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Colonies of 4 male and 4 female adult (20 week old) WIK strain zebrafish were established in
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individual 15 L glass tanks and allowed to breed naturally during a 7 day acclimation period.
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Fish were maintained according to Paull et al. [35] and a full description of husbandry
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procedures is provided in the supporting information.
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Chemical exposures
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Chemical exposure was conducted via a flow through system for a period of 21 days in
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accordance with OECD guidelines for fish reproductive tests, preceded by a 10 day pre-exposure
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period [36]. The treatment groups consisted of three concentrations of Roundup®; 0.01, 0.5 and
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10 mg/L glyphosate acid equivalent (using Roundup® GC liquid glyphosate concentrate
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containing 120 g/L glyphosate acid; Monsanto, Cambridge, UK); 10 mg/L glyphosate (analytical
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grade; Molekula, Wimborne, UK); and a control group. Each treatment group was comprised of
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three replicate breeding colonies (4 males and 4 females) in 15L tanks. Water samples were
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collected from each tank on days 7, 14 and 21 of the exposure period and stored at -20 °C prior
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to chemical analysis. Details of the analytical chemistry procedures are provided in supplemental
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material.
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Reproductive test and embryo exposures
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Group spawning occurred daily at dawn and eggs were collected 1 hour post fertilisation (hpf),
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rinsed thoroughly to remove detritus and incubated in water containing the same chemical
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exposure concentrations as their tank of origin, at 28 °C. Exposure water for the embryo
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experiments was made according to the ISO 7346−3:1996 guidelines [37], fully oxygenated and
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supplemented with 2.5 µl/L of the antifungal agent Methylene Blue (Interpet; Dorking, UK) to
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avoid mortalities caused by fungal infections. The eggs from each colony were examined using
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light microscopy between 2 ½ and 3 ½ hours after dawn, when all fertilised eggs had reached at
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least the 16-cell stage during early cleavage [38], and the total number of fertilised and
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unfertilised eggs were quantified on each day throughout the pre-exposure and exposure periods.
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During the 21-day chemical exposure, fertilised eggs displaying cellular necrosis were counted
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and recorded as early-stage mortalities (
