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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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1 Effects of glyphosate and its formulation,
2 Roundup®, on reproduction in zebrafish (Danio
3 rerio).
4 Tamsyn. M. Uren Webster1*, Lauren V. Laing1, Hannah Florance1 and Eduarda M. Santos1*
5 1. Biosciences, College of Life & Environmental Sciences, Geoffrey Pope Building, University
6 of Exeter, Exeter, EX4 4QD
7 * Corresponding authors
8 9 10 11 12
16 ABSTRACT
17 Roundup®, and its active ingredient glyphosate, are among the most widely used herbicides
18 worldwide, and may contaminate surface waters. Research suggests both Roundup® and
19 glyphosate induce oxidative stress in fish, and may also cause reproductive toxicity in
20 mammalian systems. We aimed to investigate the reproductive effects of Roundup® and
21 glyphosate in fish, and the potential associated mechanisms of toxicity. To do this, we conducted
22 a 21-day exposure of breeding zebrafish (Danio rerio) to 0.01, 0.5 and 10 mg/L (glyphosate acid
23 equivalent) Roundup® and 10 mg/L glyphosate. 10 mg/L glyphosate reduced egg production, but
24 not fertilisation rate in breeding colonies. Both 10 mg/L Roundup® and glyphosate increased
25 early-stage embryo mortalities and premature hatching. However, exposure during
26 embryogenesis alone did not increase embryo mortality, suggesting that this effect was caused
27 primarily by exposure during gametogenesis. Transcript profiling of the gonads revealed 10
28 mg/L Roundup® and glyphosate induced changes in the expression of cyp19a1 and esrl in the
29 ovary, and hsd3b2, cat and sodl in the testis. Our results demonstrate that these chemicals cause
30 reproductive toxicity in zebrafish, although only at high concentrations unlikely to occur in the
31 environment, and likely mechanisms of toxicity include disruption of the steroidogenic
32 biosynthesis pathway and oxidative stress.
39 INTRODUCTION
41 Glyphosate is extensively used worldwide, topping lists of agricultural herbicide usage in
42 Europe [1] and the US [2]. It is a broad-spectrum, post emergence herbicide, which acts by
43 binding phosphoenolpyruvate, the substrate of EPSP synthase, and subsequently inhibiting
44 aromatic amino acid synthesis via the shikimate pathway in plants [3, 4]. Glyphosate is generally
45 applied as part of a formulated product, the most widely used of which are the Roundup®
46 herbicides. Roundup® formulations contain glyphosate in the form of an isoproylamine salt,
47 which aids solubility but does not affect its properties as the active ingredient, together with
48 various adjuvants which enhance its herbicidal properties. One of the most important and
49 commonly used adjuvants is polyethoxylated tallow amine (POEA), a surfactant that enhances
50 penetration of glyphosate through the plant cuticle [5, 6]. Glyphosate and Roundup® are also
51 extensively used as domestic and urban-area weed-killers [2]. Commercial glyphosate
52 formulations vary in composition with country and purpose and the properties of these
53 formulations, including their toxicity, can be compared using the concentration of glyphosate
54 present, expressed as glyphosate acid equivalent (a.e.).
55 Glyphosate is known to strongly adsorp to soil, where it is subject to microbial degradation.
56 This is one of glyphosate's advantageous herbicidal properties, limiting agricultural input to
57 surface waters in ideal conditions. However, pulses of contamination can be expected when
58 rainfall occurs directly after application and when flood events increase river sediment load [6].
59 Urban runoff and wastewater treatment effluent also account for considerable glyphosate input
60 into rivers [7]. Despite its widespread use, concentrations of glyphosate, or its associated
61 formulation components, are not routinely monitored in surface waters. However, glyphosate
concentrations worldwide have been regularly reported to occur up to ~10-15 |ig/L in rivers [e.g.8, 9]. Considerably higher peaks in concentration, in the high |ig/L range, have also been measured, but are mainly associated with direct aquatic application, and in isolated wetland environments [6, 10].
Although the target mechanism of action of glyphosate and glyphosate-based formulations is specific to plants, they have been shown to induce diverse biological effects in a range of nontarget organisms. In fish, much of previous research assessing effects of Roundup® and glyphosate has focused on their induction of oxidative stress through ROS generation and/or interference with cellular antioxidant production. Short-term exposures (up to 6 days) to 1-20 mg/L of several Roundup® formulations in a number of fish species altered levels of cellular antioxidants and induced oxidative damage of DNA, lipids and proteins [e.g. 11, 12-15]. Environmentally relevant concentrations of Roundup®, glyphosate and POEA have also induced DNA damage in blood and liver cells of eel and catfish after up to 9 days exposure [16-19]. Other studies have found that Roundup®, and in some cases glyphosate, induce other effects in fish including neurotoxicity and immunotoxicity [e.g. 20, 21, 22]. Similar evidence of Roundup® and glyphosate toxicity has been found for other vertebrate species, and demonstrated effects include occurrence of developmental abnormalities, especially in amphibians [23, 24]. Roundup® formulations have largely been found to be more toxic than pure glyphosate. The inherent toxicity of POEA [17, 23], and potentially other formulation components, is likely to contribute to this, although a modulating effect on glyphosate toxicity is also possible. Few studies, however, have directly compared equivalent concentrations of Roundup® and glyphosate.
The potential of Roundup® to disrupt the endocrine system in vitro has been demonstrated in mammalian cell lines. In mouse Leydig cells, sub-lethal concentrations of Roundup® (from 25
85 mg/L) altered the transcription and activity of steroidogenic acute regulatory protein (StAR),
86 resulting in disruption of progesterone production [25]. A number of studies demonstrated
87 consistent inhibition of aromatase activity by Roundup® in various human cell lines. The
88 concentrations required to cause these effects varied depending on the cell type and formulation,
89 but included from 4 mg/L in liver cells and 72 mg/L in placental and embryonic cells [26-28].
90 Less consistently, and to a smaller extent than Roundup®, aromatase inhibition by glyphosate
91 alone has also been reported (approximately 10 fold higher concentrations) [28]. Additionally,
92 both glyphosate and Roundup® were reported to reduce testosterone production in rat testicular
93 cells at concentrations from 0.36 mg/L [29], but it is difficult to relate these results to the
94 potential effects of these chemicals in vivo. Few studies have investigated the effects of
95 glyphosate and its commercial formulations on the endocrine system in vivo. Drakes treated with
96 5 and 100 mg Roundup®/kg (body weight) exhibited reduced levels of testosterone,
97 corresponding with alterations in testis structure. In rats, maternal and juvenile treatment from 5
98 and 50 mg/kg (body weight) of Roundup® impaired male reproductive development, with effects
99 including alteration in testis structure, sperm production and sex steroid production [30-32].
100 Reproductive effects of Roundup® and glyphosate in fish have seldom been investigated and
101 are far from clear. While no evidence of altered gonadal development was evident in juvenile
102 stickleback exposed to 0.1-100 |ig/L glyphosate [33], treatment with 3.6 mg/L Roundup® had
103 some negative impact on offspring production in Silver catfish [34]. The mechanisms
104 contributing to this reproductive effect have not been investigated.
105 Given the extensive usage of glyphosate based herbicides, there is a clear potential for the
106 environmental exposure of fish populations to glyphosate together with associated formulation
107 products, which may modify its toxicity. This study aimed to examine the effects of Roundup®
formulation on reproduction in fish and to determine to what extend these effects were associated with the toxicity of glyphosate alone. To do this, we conducted a 21 day reproductive test in breeding colonies of zebrafish, to determine if reproduction, embryo development and embryo survival, were affected by exposure to 0.01, 0.5 and 10 mg/L (glyphosate acid equivalent) Roundup® and 10 mg/L glyphosate. The two lower Roundup® concentrations included in this study were chosen to represent concentrations that can be expected to occur in the environment regularly (0.01 mg/L) and during occasional peak contamination events (0.5 mg/L). The highest concentration tested (10 mg/L) is unlikely to occur in surface waters, and was included to facilitate the analysis of the mechanisms of toxicity. We included a treatment group exposed to 10 mg/L glyphosate alone to allow for a direct comparison of its mechanisms of toxicity with the equivalent a.e. concentration of Roundup®. We hypothesised that the mechanisms of toxicity resulting in effects on reproduction might include oxidative stress and disruption of steroid biosynthesis, and to investigate this we conducted transcript profiling of a suite of genes involved in these processes in the gonads.
MATERIALS AND METHODS
Fish maintenance
Colonies of 4 male and 4 female adult (20 week old) WIK strain zebrafish were established in individual 15 L glass tanks and allowed to breed naturally during a 7 day acclimation period. Fish were maintained according to Paull et al. [35] and a full description of husbandry procedures is provided in the supporting information.
131 Chemical exposures
132 Chemical exposure was conducted via a flow through system for a period of 21 days in
133 accordance with OECD guidelines for fish reproductive tests, preceded by a 10 day pre-exposure
134 period [36]. The treatment groups consisted of three concentrations of Roundup®; 0.01, 0.5 and
135 10 mg/L glyphosate acid equivalent (using Roundup® GC liquid glyphosate concentrate
136 containing 120 g/L glyphosate acid; Monsanto, Cambridge, UK); 10 mg/L glyphosate (analytical
137 grade; Molekula, Wimborne, UK); and a control group. Each treatment group was comprised of
138 three replicate breeding colonies (4 males and 4 females) in 15L tanks. Water samples were
139 collected from each tank on days 7, 14 and 21 of the exposure period and stored at -20 °C prior
140 to chemical analysis. Details of the analytical chemistry procedures are provided in supplemental
141 material.
143 Reproductive test and embryo exposures
144 Group spawning occurred daily at dawn and eggs were collected 1 hour post fertilisation (hpf),
145 rinsed thoroughly to remove detritus and incubated in water containing the same chemical
146 exposure concentrations as their tank of origin, at 28 °C. Exposure water for the embryo
147 experiments was made according to the ISO 7346-3:1996 guidelines [37], fully oxygenated and
148 supplemented with 2.5 |il/L of the antifungal agent Methylene Blue (Interpet; Dorking, UK) to
149 avoid mortalities caused by fungal infections. The eggs from each colony were examined using
150 light microscopy between 2 ^ and 3 ^ hours after dawn, when all fertilised eggs had reached at
151 least the 16-cell stage during early cleavage [38], and the total number of fertilised and
152 unfertilised eggs were quantified on each day throughout the pre-exposure and exposure periods.
153 During the 21-day chemical exposure, fertilised eggs displaying cellular necrosis were counted
and recorded as early-stage mortalities (<3.5 hpf). Fifty fertilised eggs from each tank were selected randomly and incubated in 50 ml exposure water until 72 hpf. During this period, embryo mortality was recorded at 24, 54 and 72hpf and embryo hatching was recorded at 54 and 72 hpf.
In order to determine if the observed effects of Roundup® and glyphosate on embryos were due to the effects of exposure during gametogenesis or during embryogenesis, embryos collected from a control population were exposed to a range of concentrations of glyphosate and Roundup® as above. Chemical treatment was initiated between 10-20 minutes post fertilisation. In addition to the exposure concentrations used for the adult exposures, embryos were also treated with higher concentrations (50, 100, 250, 500 and 1000 mg/L a.e. Roundup® and glyphosate) to determine the concentration thresholds for embryo mortalities and developmental toxicity. Experiments were conducted in triplicate; each replicate contained 50 embryos and observations of mortalities and hatching were performed as described above.
Sampling
All fish were humanely sacrificed on day 21 of the exposure period by a lethal dose of benzocaine (0.5 g L-1; Sigma-Aldrich) followed by destruction of the brain, in accordance with UK Home Office regulations. Wet weight and fork length were recorded and the condition factor (k= (weight (g) x 100)/ (fork length (cm))) was calculated for individual fish. Livers were dissected and weighed, and the hepatosomatic index (HSI) (liver weight (mg)/ total weight (mg)) x 100)) was determined for individual fish. Gonads were dissected, weighed and one gonad from each fish was snap frozen in liquid nitrogen and stored at -80°C prior to transcript profiling. The remaining gonad was fixed in Bouin's solution (Sigma-Aldrich) for histological analysis. The
177 gonadosomatic index (GSI; gonad weight (mg)/ total weight (mg)) x 100)) was determined for
178 both males and females.
180 Transcript profiling and histological analysis
181 Transcript profiling of genes encoding steroidogenic enzymes, sex steroid receptors and
182 antioxidant enzymes, was conducted using RT-QPCR in the gonads of exposed fish according to
183 [39]. Histological analysis of the gonads was conducted according to [40]. A full description of
184 these methodologies is presented in the supporting information.
186 Statistical analysis
187 Statistical analyses were conducted with SigmaStat (version 12.0). Before analysis,
188 proportional data (embryo survival and hatching) were subjected to variance-stabilising square-
189 root or arcsine transformations as appropriate. All reproductive output and sampling data met
190 assumptions of normality and equal variance. Outliers in transcript expression data were
191 identified and removed according to Chauvenet's criterion [41] prior to statistical analysis.
192 Transcript expression data that did not meet normally-distributed criteria was log transformed
193 before statistical analysis. All data was analysed using single factor one way analysis of variance
194 (ANOVA), followed by the Holm-Sidak post hoc test using a pairwise comparison method. Data
195 were considered to be significant when P < 0.05.
197 RESULTS
199 Water chemistry
The mean measured concentrations of glyphosate in the tank water were between 88-140 % of the nominal values for all treatments (quantification of glyphosate in tanks receiving 0.01 mg/L Roundup® was below the detection limit of our method), and are presented in Table S2.
Morphometric parameters
The mean mass and length of male and female fish were 375.0 ± 6.3 mg/ 32.6 ± 0.2 mm and 402.6 ± 9.3 mg/ 31.7 ± 0.2 mm respectively. There were no significant differences in size or condition factor (mean 1.08 and 1.25 for males and females, respectively) between treatment groups. Additionally, we observed no alteration of general health or behaviour in any colony. The GSI of females was significantly lower in the fish treated with 10 mg/L glyphosate compared to the control group (Figure 1c). There was no significant difference in the GSI of males between treatment groups, or in the HSI of males or females.
Reproductive test and embryo exposures
During the 10 day pre-exposure period, there was no difference in cumulative egg production between the treatment groups (P=0.468). During the exposure period, colonies in the control group consistently spawned the greatest number of eggs per female, while those treated with 10 mg/L glyphosate spawned the least. From day 10 of the exposure period, cumulative egg production was significantly reduced in colonies exposed to 10 mg/L glyphosate compared to the controls, and this difference intensified throughout the remainder of the exposure period. At the end of the 21 day exposure, cumulative egg production was significantly lower in colonies exposed to 10 mg/L glyphosate compared to the control, and also compared to the 10 and 0.01 mg/L Roundup® groups (Figure 1a,b). Additionally, egg output significantly correlated (R2=
223 0.79; P= 0.043) with female GSI across all treatment groups. Fertilisation rate remained
224 consistently high throughout the exposure period with no significant differences between
225 treatment groups and an overall mean value of 83.4%.
226 There was a significant increase in embryo mortalities occurring before 3.5 hpf in embryos
227 from both the 10 mg/L Roundup® and glyphosate treatment groups (Figure 2a). Additionally,
228 there was a significant correlation between early embryo mortality and the concentration of
229 Roundup® (R2= 0.52; P=0.008). There were no significant differences between treatments in
230 embryo mortality between the start of epiboly (3.5 hpf) and the end of somitogenesis at 24 hpf
231 (Figure 2b). However, there was a significant increase in the percentage of embryos that had
232 hatched at 54 hpf in groups treated with 10 mg/L Roundup® and 10 mg/L glyphosate compared
233 to the control group (Figure 2b).
234 For embryos originating from a control population, exposure to glyphosate and Roundup® at
235 the concentrations used in the adult reproductive test (0, 0.01, 0.5 and 10 mg/L Roundup® and 10
236 mg/L glyphosate) did not result in increased mortality rate at either 3.5 hpf or 24 hpf (Figure
237 2a,b), but there was a significant increase in 3.5-24 hpf mortality in embryos exposed to
238 concentrations >100 mg/L glyphosate and > 500 mg/L Roundup® (Figure S4a). We also
239 observed evidence of developmental delay and abnormalities from concentrations > 50 mg/L
240 glyphosate and >250 mg/L Roundup® at 24 hpf. There was a trend towards increased hatching at
241 54 hpf in groups exposed to 10 and 50 mg/L Roundup® and glyphosate, and there was a
242 significant correlation between hatching rate at 54hpf and exposure concentration of Roundup®
243 up to 50 mg/L (R2= 0.27; P=0.04) (Figure S4b). For embryos exposed to > 100 mg/L Roundup®
244 and glyphosate, we found evidence of progressive delay in development and hatching with
245 increasing concentration.
Gonad transcript profiling
In the ovary, the transcript encoding aromatase (cyp19a1) was significantly up-regulated in the 10 mg/L Roundup® treatment group compared to the controls. Estrogen receptor 1 (esr1) in the 10 mg/L Roundup® group was significantly up-regulated compared to the 10 mg/L glyphosate group. There were similar, but not statistically significant, decreasing trends in expression of other steroidogenic enzymes including cytochrome P450, subfamilies 17 and 11 (cyp17a1, cyp11a1) and 3P-hydroxysteroid dehydrogenase (hsd3b2) in groups exposed to both Roundup® and glyphosate. In contrast, for the antioxidants glutathione peroxidase (gpx1a), catalase (cat) and glutathione-S-transferase pi (gstp1) non-significant, increasing trends in transcript expression were observed (Figure 3 a, Figure S1a).
In the testis, hsd3b2 was significantly up-regulated following exposure to 10 mg Roundup®/L compared to all other treatment groups. The expression pattern of steroidogenic acute regulatory protein (star), cyp17a1, cyp11a1 and the androgen receptor (ar) additionally appeared to follow an expression pattern similar to hsd3b2 across treatment groups. cat was significantly up-regulated in groups exposed to both 10 mg/L Roundup® and 10 mg/L glyphosate compared to those treated with 0.5 mg/L Roundup®. In addition, sod1 was significantly up-regulated in the 10 mg/L compared to 0.5 mg/L Roundup® groups (Figure 3b, Figure S1b).
Gonad Histology
Histological examination of females from all treatment groups showed that the ovaries of all individuals contained oocytes at all stages of development (oogonia, primary oocytes, cortical alveoli stage oocytes, secondary oocytes and mature vitellogenic oocytes) and the majority
269 contained recent post-ovulatory follicles. We found evidence of ovarian abnormalities in 9.1,
270 18.2, 9.1, 50.0 and 63.6 % of females in the control, 0.01 mg/L Roundup®, 0.5 mg/L Roundup®,
271 10 mg/L Roundup® and 10 mg/L glyphosate treatment groups, respectively (Figure S3). The
272 majority of abnormalities were relatively mild and included accumulation of eosinophilic fluid
273 and presence of abnormal tissue. In addition, the proportion of fish containing atretic oocytes in
274 their ovaries also appeared to be increased (Figure S2).
275 Histological examination of males showed that testes of all individuals from all treatment
276 groups contained germ cells at all stages of spermatogenesis (including spermatogonia,
277 spermatocytes, spermatids and mature spermatozoa) (Figure S2). There were no abnormalities
278 and no differences between stages of development between treatment groups.
280 DISCUSSION
282 Reproductive effects on adult zebrafish
283 This study provides evidence that glyphosate caused a reduction in the number of eggs
284 spawned by female zebrafish exposed to high concentrations (10 mg/L) of glyphosate. However,
285 this concentration is well above concentrations measured to date in the environment and unlikely
286 to occur in aquatic systems, except when glyphosate is directly applied to control algal
287 populations. In addition, our study also showed an apparent reduction, albeit not significant, in
288 egg production in all three Roundup® treated groups. Therefore, the potential for adverse effects
289 of Roundup® on reproductive output and impact on wild populations cannot be ruled out. A
290 number of potential mechanisms may contribute to the observed effect of glyphosate on egg
291 production, including disruption of normal progression through oogenesis, inhibition of
ovulation and increased rate of oocyte atresia. In order to explore this, we conducted histological analysis of the gonads of exposed females and observed a trend towards an increase in the incidence of ovarian abnormalities as a result of exposure to both Roundup® and glyphosate. Ovarian follicle atresia is an apoptotic process leading to re-absorption of maturing oocytes rather than ovulation. It is a highly-regulated, natural process thought to have a role in maintaining ovarian homeostasis; however various environmental stressors, as well as disruption of the hormonal control of oogenesis and ovulation, have been shown to increase atresia [42]. We found atretic vitellogenic oocytes in all treatment groups, but this incidence tended to increase in both the 10 mg/L Roundup® and 10 mg/L glyphosate treatment groups. Similarly, in these groups we also found an increased trend in the incidence of abnormal ovarian tissue, including excess connective tissue and putative haemopoietic tissue. In some females treated with 0.01 and 10 mg/L Roundup® and 10 mg/L glyphosate we observed the presence of areas containing eosinophilic fluid. Previously, accumulated proteinaceous fluid in the ovary has been found to contain vitellogenin, and this has been associated with a disruption in the endocrine control of oogenesis in zebrafish through exposure to elevated levels of 17P-oestradiol [43].
It is important to note that despite the trends towards increased incidence of atretic follicles and ovarian abnormalities following exposure to glyphosate and Roundup®, the majority of fish were only moderately affected and their ovaries contained oocytes at all stages of maturation, including mature vitellogenic oocytes and post-ovulatory follicles. Moreover, we found no differences in the ovarian expression of bcl2-associated X protein (baxa) and tumour protein 53 (tp53), which are typical marker genes of apoptosis. This indicates that oocyte atresia was unlikely to be the major mechanism responsible for the decline in egg production rate induced by glyphosate treatment. Corresponding with this, a similar degree of atresia in fish exposed to
315 Roundup® was not accompanied by a significant decline in egg-production in this treatment
316 group. Therefore, we hypothesise that the observed decrease in egg production following
317 exposure to glyphosate was more likely to be due to a reduction in the number of follicles
318 undergoing oogenesis. The strong correlation between egg production and female GSI, including
319 a significant reduction in GSI in females exposed to 10 mg/L glyphosate, which indicates
320 reduced gonadal volume, provides support for this hypothesis.
321 Sex steroids are essential for the regulation of oogenesis, and alterations in sex steroid
322 biosynthesis may have contributed to the reduction of egg production in colonies exposed to
323 glyphosate. To test this hypothesis, we investigated the effects of glyphosate and Roundup® on
324 the expression of a number of transcripts encoding enzymes involved in steroid biosynthesis,
325 several of which have previously been shown to be targets of their toxicity [25-28]. We found a
326 significant increase in the expression of ovarian aromatase, an enzyme which catalyses the
327 conversion of testosterone to oestradiol in granulosa cells, in the gonads of females exposed to
328 10 mg/L Roundup®, and also an increasing trend in those exposed to 10 mg/L glyphosate.
329 Several previous studies have demonstrated that Roundup® disrupts both aromatase activity and
330 cyp19a1 expression levels in a number of human cell lines, and there is some evidence that
331 glyphosate can also inhibit aromatase activity, especially with the addition of small percentages
332 of Roundup®, which may facilitate its cellular entry [26-28]. Romano et al. [32] proposed
333 inhibition of aromatase as a causative mechanism for disruption of steroidogenesis and adverse
334 reproductive impacts in the male offspring of rats exposed to Roundup® during pregnancy. The
335 stimulatory effect of Roundup® on cyp19a1 expression observed in the present study contrasts
336 with the predominantly inhibitory effects found in the in vitro studies. This may reflect the
337 complex nature of feedback mechanisms governing steroid biosynthesis pathways in vivo or,
possibly, a compensatory transcriptional response to a potential inhibition of aromatase enzyme. Additionally, it is difficult to equate the concentrations used in the present study with those used in the in vitro studies. It is possible that differential stimulatory and inhibitory responses occur with concentration, and also with time. Although not significant, there were also similar decreasing trends in expression of steroidogenic enzymes, hsd3b2, cyp17a1 and cypllal, in females treated with both 10 mg/L of Roundup® and 10 mg/L glyphosate, indicating a possible wider effect on steroidogenic pathways.
The differential regulation of ovarian esrl by Roundup® and glyphosate is interesting and may reflect the effect of other chemicals present in Roundup® formulation on this receptor. Increased esrl expression following Roundup® exposure may have resulted from compensatory mechanisms in the ovary to maintain or restore oestrogen signalling pathways. This may explain, at least in part, the differences in the effects of these chemicals on egg production, with glyphosate having a more pronounced effect than Roundup®. Using human liver HepG2 cells, Gasnier [26] showed that Roundup® and glyphosate antagonistically bind oestrogen receptors (ERa and ERfi), although Kojima [44] found no evidence of agonistic or antagonistic interaction with oestrogen receptors in Chinese hamster ovary cells. A recent study showed glyphosate actively bound oestrogen receptors and induced proliferative growth of oestrogen-dependent breast cancer cells, and also increased protein levels of ERa and ERp [45]. Taken together, our ovarian transcript profiling data suggests that Roundup® and glyphosate may have disrupted steroid hormone biosynthesis and also potentially modulated the biological effects of oestrogens via alterations in the expression of esrl, the predominant oestrogen receptor in the ovary.
Despite having no significant effect on egg production, it is interesting to note that exposure to 10 mg/L Roundup® also elicited alterations in gene expression often in the opposite direction of
361 those induced by exposure to the equivalent concentration of glyphosate alone. This might
362 suggest the presence of compensatory mechanisms ameliorating the adverse effects of
363 glyphosate when in the presence of the other constituents of Roundup®. A possible mechanism
364 could be increased synthesis of aromatase to maintain sex steroid ratios and oestrogen signalling
365 in the ovary in order to promote oogenesis, and maintain egg production.
366 There was no effect of exposure to Roundup® or glyphosate on fertilisation rate.
367 Corresponding with this, histological examination revealed no evidence of any disruption of
368 spermatogenesis, or abnormalities in the testis following exposure to glyphosate or Roundup®.
369 Therefore, we found no indication that these chemicals affect the ability of the sperm produced
370 to fertilise eggs. This contrasts with several previous in vivo studies that have found some
371 evidence that Roundup® disrupts spermatogenesis in rats, resulting in testis pathology, sperm
372 abnormalities and altered sperm production [30-32]. It is important to note, however, that our
373 experimental conditions are optimised to maximise reproduction and may not detect subtle
374 changes in sperm quality that may be sufficient to cause effects under the conditions found in the
375 natural environment.
376 Previous Roundup®-induced testicular toxicity has been associated with alterations in
377 steroidogenesis and sex steroid levels in rats and drakes [30-32, 46]. In the current study,
378 analysis of transcripts encoding steroidogenic enzymes in the testes showed that hsd3b2 was
379 significantly up-regulated in males exposed to 10 mg/L Roundup® compared to those exposed to
380 0.01 and 0.5 mg/L Roundup®, and 10 mg/L glyphosate (Figure 3b). Moreover, although not
381 statistically significant, the expression patterns of the other steroidogenic enzymes profiled (star,
382 cyp17a1 and cypllal), as well as ar, followed a similar expression pattern to hsd3b2 across
383 treatment groups. This pattern, of apparent down-regulation in the 0.5 mg/L Roundup® treatment
and up-regulation in the 10 mg/L Roundup® group was robust across tank replicates. Walsh et al. [25] found evidence that Roundup®, but not glyphosate, disrupted StAR and P450scc (Cyp11a1) in mouse testis cells, primarily through alteration of protein expression and activity, suggesting that such post-transcriptional regulatory changes should also not be ruled out. Additionally, we found 10 mg/L Roundup® significantly increased expression cat and sodl compared to the lower Roundup® treatments, and 10 mg/L glyphosate also significantly increased cat expression in the testis. Together, these changes in the transcription of antioxidant enzymes provide evidence that both Roundup® and glyphosate induce oxidative stress in the testis. Therefore, despite no apparent impacts on fertilisation success, we have found some evidence that high concentrations of Roundup® and glyphosate cause disruption of steroidogenesis and oxidative stress in the testis, suggesting that their potential to cause adverse impacts on male reproductive health should not be ruled out. It is interesting to note that exposure to 10 mg/L Roundup® elicited differential responses, in terms of the magnitude and direction of transcript expression changes, compared to 10 mg/L glyphosate, possibly suggesting greater compensatory mechanisms of response following exposure to Roundup®, similarly to that observed in females.
Effects on embryo survival and development
We found evidence that treatment with both 10 mg/L Roundup® and glyphosate induce an increased rate of embryo mortality during very early development. We observed necrosis of the fertilised embryos during cleavage and early blastula stages, prior to progression to epibioly at ~3.5 hpf (as described by Kimmel et al. [38]). In order to assess if the early stage mortality was caused as a direct result of the chemical exposure on embryos or by the parental exposure, we exposed embryos originating from a control population of untreated adults and found that
407 concentrations of up to 10 mg/L of Roundup® and 10 mg/L glyphosate had no effect on embryo
408 survival at <3.5 or 3.5-24 hpf. This corresponds with previous work showing exposure of
409 zebrafish embryos to up to 10 mg/L glyphosate for 5 days had no effect on survival or
410 development [47]. We only found a significant increase in embryo mortality at concentrations of
411 100 mg/L glyphosate and 1000 mg/L Roundup®, which are 10 and 1000 times higher than the
412 concentrations used in the reproductive study. Moreover, this mortality predominantly occurred
413 between 3.5-24 hpf, rather than in the earlier stages of development. These high concentrations
414 of glyphosate, and to a lesser extent Roundup® formulation, result in a pronounced decrease in
415 pH in the exposure water (to 3.8 (100 mg/L glyphosate) and 4.9 (1000 mg/L Roundup®)), which
416 may be responsible for the embryo toxicity seen. Overall, these results suggest that the increase
417 in early stage mortalities observed in embryos originating from fish exposed to 10mg/L
418 Roundup® and glyphosate is attributable to potential damage of the gametes occurring during
419 gametogenesis and/or fertilisation, rather than as a result of direct embryo exposure.
420 Alternatively, it is possible that maternal transfer of glyphosate, Roundup® or formulation
421 products, via the yolk, might contribute to embryo exposure to these toxicants and the increased
422 mortality observed.
423 As discussed above, gonadal transcript profiling revealed significant up-regulation of
424 transcripts encoding antioxidant enzymes in response to exposure to 10 mg/L Roundup® and 10
425 mg/L glyphosate in the testes and increasing trends in transcripts encoding antioxidant enzymes
426 in the ovary. Oxidative stress induced in the testis by chemical exposure has been shown to cause
427 DNA damage in developing sperm [48]. Pérez-Cerezales et al. [49] showed that DNA damage in
428 rainbow trout sperm did not impair fertilisation success, but resulted in a high rate of embryo
429 mortality in early stages of embryogenesis, particularly during gastrulation. This is consistent
with our findings that fertilisation success was unaffected, but that an increased rate of embryo mortalities occurred during early stages of development and before transition to epiboly. Therefore, we hypothesise that oxidative stress generation in the testis during spermatogenesis is likely to be an important causative mechanism responsible for the increase in early-stage embryo mortality. Additionally, the increase in ovarian histological abnormalities and the increased trends in ovarian antioxidant transcript expression suggest similar damage during oogenesis is also possible, although oocytes are thought to have greater response and repair mechanisms to counter-act oxidative stress than sperm [49, 50]. DNA damage after spermiation cannot be ruled out, but probably has a minimal effect compared to damage during spermatogenesis, given the brief period of less than 65 seconds that sperm remains motile before fertilisation (Van Look et al., personal communication).
We found an increased percentage of hatching at 54 hpf in groups exposed to 10 mg/L Roundup® and 10 mg/L glyphosate. Additionally, embryos originating from the unexposed control population showed a significant increasing trend in hatching rate at 54 hpf with concentrations up to 50 mg/L Roundup®, as well as an apparent increase in hatching in those treated with 10 mg/L glyphosate. This suggests an independent impact of Roundup® and glyphosate on embryos, not entirely attributable to toxicity during gametogenesis. Hatching is variable, and dependent on a number of environmental factors. Various chemical and other environmental stressors, such as temperature, are known to affect developmental rate and, subsequently, time to hatch. However, in this study, observations at 24h, 48h, 54 and 72 hpf showed no obvious change in development rate between treatment groups, indicating that exposure to 10 mg/L Roundup® and 10 mg/L glyphosate induces premature hatching in zebrafish. At 72 hpf, more than 90 % of embryos from all treatment groups had hatched (both
453 those originating from exposed and non-exposed adults), and there were no obvious behavioural
454 or morphological differences between treatments. In natural populations, premature hatching
455 could potentially result in detrimental impacts for population sustainability, for example by
456 increasing the susceptibility to predation.
457 We found no obvious signs of developmental toxicity at exposure concentrations up to 10
458 mg/L Roundup® or glyphosate, which corresponds with the findings of Stehr [47]. We did find
459 evidence of developmental delay in embryos exposed to concentrations > 50 mg/L glyphosate
460 and >250 mg/L Roundup® and hypothesise that the increased toxicity of glyphosate may be
461 attributed to its greater acidity than the buffered Roundup® formulation. With the exception of
462 amphibians, which appear particularly sensitive [e.g. 23, 24], these results show that only
463 extremely high concentrations of Roundup® and glyphosate induce developmental toxicity in
464 zebrafish and are generally in accordance with evidence from other species, including rats [51]
465 and sea urchins [52].
466 Overall, we have found evidence that both 10 mg/L Roundup® and 10 mg/L glyphosate have
467 similar adverse impacts on embryo survival and hatching, while 10 mg/L glyphosate reduces egg
468 production. We have found some evidence that these reproductive effects occur via multiple
469 mechanisms of toxicity which appear to differ, to some extent, between Roundup® and its active
470 ingredient glyphosate. These mechanisms may include disruption of the steroidogenic pathway
471 and sex steroid signalling, and generation of oxidative stress. This work demonstrates that both
472 glyphosate and Roundup® have a detrimental impact on a number of measures of reproductive
473 health in zebrafish, although only at very high concentrations that are unlikely to occur in the
474 environment, based on the currently available measurements. Given the growing concern over
475 potential reproductive effects of these compounds, and their extremely widespread usage, this
provides valuable mechanistic information for their environmental risk assessment, particularly when considering the potential effects of complex mixtures of environmental contaminants.
495 FIGURES
496 Figure 1. (A) Cumulative egg production during the 10 day pre-exposure and 21 day chemical
497 exposure periods (n=3 replicate colonies per treatment); (B) Mean number of eggs laid per
498 female per day throughout the 21 day exposure period (n=3 replicate colonies per treatment); and
499 (C) Mean gonad-somatic index of females in each treatment group (n=12 individual females per
500 treatment). Data plotted are mean values ± SEM. Asterisks indicate significant differences
501 between treatment groups (*P<0.05 **P<0.01 ***P<0.001).
Figure 2. Effects of Roundup® and glyphosate on embryo survival and development. Black bars represent embryos originating from exposed parental populations (n= 3 replicate colonies, for each colony data was collected every day for 21 days of exposure and averaged) and grey bars represent embryos originating from a control parental population (n=3 replicate exposures, each replicate containing 50 embryos). (A) Percentage of embryo mortalities that occurred before 3.5 hpf; (B) percentage of embryo mortalities that occurred between 3.5-24 hpf; and (C) percentage of embryos that had hatched at 54 hpf in each treatment group. Data plotted are mean values ± SEM. Asterisks represent significant differences from the control treatment (***P<0.001).
516 Figure 3. Transcript profiling of target genes in the ovary (A) and testis (B) following
517 exposure to Roundup® (R) and glyphosate (G). Data are presented as fold change relative to
518 expression in the control group, whereby red shading indicates up-regulation and green shading
519 represents down-regulation. Relative expression was calculated as ratio of target gene /rpl8
520 mRNA concentration. For each treatment, n= 6-8 fish. Individual data points classified as
521 outliers, and for which the expression was below the detection limit of the assay were excluded
522 from the analysis. Lettering indicates significant differences between treatment group, with
523 groups identified with different letters being significantly different from each other (P< 0.05).
ASSOCIATED CONTENT Supporting Information contains: Page S2: Supplemental Experimental Section
Page S4: Target genes, primer sequences and assay details for RT-QPCR analysis, Table S1.
Page S5: Measured concentrations of glyphosate in tank water, Table S2.
Page S6: Transcript profiling of target genes in the gonads, Figure S1.
Page S7: Gonad histology of control and exposed fish, Figure S2.
Page S8: Occurrence of ovarian histological abnormalities, Figure S3.
Page S9: Effects of glyphosate and Roundup® on embryos originating from a control population, Figure S4.
This material is available free of charge via the Internet at http://pubs.acs.org.
552 AUTHOR INFORMATION
553 Corresponding Authors
554 * Tamsyn M. Uren Webster
555 Biosciences, College of Life & Environmental Sciences, Geoffrey Pope Building, University of
556 Exeter, Exeter, EX4 4QD
557 tu202@exeter.ac.uk
558 Phone: +44 (0)1392 724677
559 Fax: +44 (0)1392 263434
561 * Eduarda M. Santos
562 Biosciences, College of Life & Environmental Sciences, Geoffrey Pope Building, University of
563 Exeter, Exeter, EX4 4QD
564 E.Santos@exeter.ac.uk
565 Phone: +44 (0)1392 264607
566 Fax: +44 (0)1392 263434
568 ACKNOWLEDGMENTS
569 We wish to thank Dr James Cresswell for advice on the statistical analysis and Dr Gregory
570 Paull for help with the fish husbandry and experimental design. This work was funded by a
571 Natural Environment Research Council CASE PhD studentship (Grant number NE/I528326/1)
572 and the Salmon & Trout Association.
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733 TOC/ABSTRACT ART
Roundup &glyphosate Reproductive output
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4 Gonad transcript profiling & histology « Embryo survival & hatching
