Article pubs.acs.org/est
Several Synthetic Progestins with Different Potencies Adversely Affect Reproduction of Fish Tamsin J. Runnalls,*,† Nicola Beresford,† Erin Losty,† Alexander P. Scott,‡ and John P Sumpter† †
Institute for the Environment, Brunel University, Kingston Lane, Uxbridge, Middlesex, UB8 3PH, U.K. CEFAS Weymouth Laboratory, Barrack Road, Weymouth, Dorset, DT4 8UB, U.K.
‡
S Supporting Information *
ABSTRACT: Synthetic progestins are widely used as a component in both contraceptives and in hormone replacement therapy (HRT), both on their own and in combination with EE2. Their presence in the environment is now established in wastewater effluent and river water and this has led to concerns regarding their potential effects on aquatic organisms living in these waters. We carried out in vivo experiments to determine the potencies of four different synthetic progestins on the reproductive capabilities of the fathead minnow (Pimephales promelas). We then performed a series of in vitro assays to try and determine the reason for the effects seen in the in vivo experiments. In the first experiment, fathead minnow exposed to a single concentration of 100 ng/L of either Levonorgestrel or Gestodene stopped spawning almost completely. The same nominal concentration of Desogestrel and Drospirenone did not affect reproduction (21 d NOECs of 100 ng/L). The second experiment investigated two progestins of different potency: Gestodene at 1, 10, and 100 ng/L and Desogestrel at 100 ng/L, 1 μg/L, and 10 μg/L. Gestodene concentrations as low as 1 ng/L had significant effects on reproduction over 21 d, whereas concentrations of Desogestrel at or above 1 μg/L were required to significantly reduce egg production. The synthetic progestins also masculinized the female fish in a concentration-dependent manner. Results from yeast-based in vitro assays demonstrated that the progestins are all strongly androgenic, thereby explaining the masculinization effects. The results strongly suggest that synthetic progestins merit serious consideration as environmental pollutants.
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INTRODUCTION It is now well established that there are many different pharmaceuticals entering the aquatic environment via wastewater effluents, with some possibly causing significant effects on aquatic organisms. Steroid hormones are examples of some of these pharmaceuticals. A great deal of research has been focused on the synthetic estrogen ethinylestradiol (EE2) over the last 15 years, whereas in contrast, there are only limited data available on the effects of synthetic progestins, which are very widely used in combination with EE2 (in contraceptives), and on their own (in hormone replacement therapy). These medicines usually contain the synthetic progestin component at doses from 3 to 100 times that of EE2, depending on the formulation and therapeutic application.1 Their prescribed usage in the UK (in 2006) was almost 2 orders of magnitude higher than the amount of EE2,2 which is well-known to cause significant effects to aquatic organisms at low concentrations.3 Synthetic progestins (also known as progestogens or gestagens) mimic the effects of the natural hormone progesterone, which is a C-21 steroid hormone involved in regulating the menstrual cycle, pregnancy, and embryogenesis in humans and other species. They work as contraceptives by suppressing ovulation (by their antigonadotropic properties). In humans, progestins are widely known to have interactions not only with the progesterone receptor (PR), but also with © 2013 American Chemical Society
other steroid hormone receptors, for example the androgen, estrogen, glucocorticoid, and mineralocorticoid receptors. All progestins bind to the PR and affect the human uterine endometrium, but each progestin has a distinct profile of activity in other target tissues. Some of the first progestins were introduced for clinical use over 40 years ago and had undesirable side effects in humans (e.g., androgenic activity). The newer progestins have far fewer side effects, as they are more progesterone receptor specific.4,5 The first measurements of a synthetic progestin in river water samples were made over two decades ago.6 Since then other synthetic progestins, including Norethindrone and Levonorgestrel, have been reported to be present in STW effluents at concentrations up to 188 ng/L,7−11 while river concentrations are significantly lower, due to the dilution of effluent in receiving waters.12 Nevertheless, a concentration of 38 ng/L has recently been reported in the Langat River, Malaysia.11 It is very likely that a number of synthetic progestins will be present simultaneously in the aquatic environment, and hence the total concentration of synthetic progestins to which aquatic Received: Revised: Accepted: Published: 2077
November 29, 2012 January 25, 2013 January 28, 2013 January 29, 2013 dx.doi.org/10.1021/es3048834 | Environ. Sci. Technol. 2013, 47, 2077−2084
Environmental Science & Technology
Article
dissolved oxygen (8 ± 1 mg/L), nitrite, nitrate, ammonia, pH, KH (carbonate hardness), and GH (general hardness). Tank concentrations of progestin were also measured. The photoperiod was maintained at 16 h light:8 h dark throughout, incorporating 20 min dawn/dusk transition periods. All tubing was either glass or medical grade silicone. Experiment 1 (the potency study) involved four different progestins (Levonorgestrel, Gestodene, Desogestrel, and Drospirenone), each at a nominal concentration of 100 ng/L, and Experiment 2 (a concentration−response experiment) involved using Gestodene at nominal concentrations of 1, 10, 100 ng/L and Desogestrel at 0.1, 1, and 10 μg/L. All Levonorgestrel (Sigma-Aldrich, UK, CAS 797-63-7), Drospirenone (USPharmacopeia, USA, CAS 67392-87-4), Gestodene (Steraloids Inc., USA, CAS 60282-87-3), and Desogestrel (Sigma-Aldrich, UK, CAS 54024-22-5) (Supporting Information Figure S1) stock solutions were prepared weekly, in 2.5-L amber bottles, using double distilled water. Concentrated stock solutions (Masters) were made up in ethanol and stored at 4 °C, and these were used each time the dosing stock solutions were made, to ensure reproducibility among dosing stocks. These dosing stock solutions were dosed at 12 mL/h (0.2 mL/min), using a Watson Marlow (Cornwall, UK) multichannel peristaltic pump, into glass mixing vessels (each supplying 8 individual tanks). In these, dosing stocks mixed with dilution water before delivery (at 125 mL/min) to each tank to produce the desired concentrations. Ethanol concentrations within each tank were no greater than 0.00003%. Flow rates and dosing efficiency were monitored daily to ensure that the chemicals entered the fish tanks at the expected rates to produce the desired tank concentrations. Each day the spawning tiles, grids, and dishes were removed from each tank and the number of eggs laid (including those stuck to the tile, attached to the grid, and those fallen through into the glass collection tray) were counted. At termination of an experiment, fish were terminally anaesthetized using buffered MS-222 (100 mg/L; Sigma, Poole, UK). Blood samples were collected via the caudal peduncle using 75-μL heparinized capillary tubes and decanted into eppendorf tubes containing aprotinin (Sigma), and stored on ice until centrifugation at 7000g for 5 min. The resulting plasma was withdrawn, snap frozen, and stored at −80 °C until analysis for steroid levels (11KT and T for males and E2 for females) using radioimmunoassays (RIA).18 Briefly, prior to RIA, plasma samples (10 μL) were placed in 1.5-mL eppendorf tubes to which were added 100 μL of distilled water and 1 mL of ethyl acetate. The tubes were sealed, mixed thoroughly, and then centrifuged to separate the two phases. The water phase was frozen by placing each tube for a brief interval on a block of dry ice, and the ethyl acetate was then poured into a borosilicate glass tube. The ethyl acetate was blown down under a stream of nitrogen in a heating block at 45 °C. The residue (containing the steroids) was redissolved in 1 mL of buffer and-100 μL aliquots were used for the RIAs. Plasma vitellogenin (VTG) concentrations were also measured using a kit designed specifically for the fathead minnow (Biosense Laboratories AS, Bergen, Norway). The detection limit was 2.5−5 ng/mL. Plasma samples were diluted 1:50, 1:5000, and 1:500 000 and assayed in duplicate according to the manufacturer’s protocol. Inter assay variation was 6.91%. Secondary sexual characteristics present on any of the female fish (normally a specifically male characteristic) were visually
organisms are exposed could be higher than the concentration of any individual progestin. A few reports have recently been published regarding the effects of progestins on aquatic wildlife.1,14,15 It is very important, however, to determine if all progestins will cause problems in the environment, or if some will be of more concern than others. There are only a handful of publications currently available on the effects of synthetic progestins in fish, the most relevant to our work being the data from Zeilinger et al.,1 who reported reduced fertility of fathead minnow at concentrations of Levonorgestrel as low as 0.8 ng/L and Drospirenone at a concentration of 6.5 μg/L. Along with that paper, previous work that we have published reported significant effects of Norethindrone on reproduction in the fathead minnow and medaka in the low ng/L range.14 Fick et al.13 also found that Levonorgestrel bioaccumulated in the blood of rainbow trout exposed to sewage effluent to concentrations four times that of the human therapeutic level. Kvarnryd et al. 15 recently looked at the effects of Levonorgestrel on sexual differentiation, reproductive organ development, and fertility in the frog, Xenopus. They reported effects on oviduct and ovarian development and fertility, resulting in sterile females when the water concentration was in the hundreds of ng/L range. Another recent study by Lorenz et al.16 also looked at the effects of Levonorgestrel on sexual development in Xenopus, and found effects were mediated by negative feedback on pituitary gonadotrophins and by modulating gonadal steroidogenesis at concentrations approximately 100-fold higher than those detected in the environment.
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MATERIALS AND METHODS The in vivo studies investigated the effects of four different synthetic progestins on reproduction of fathead minnow (Pimephales promelas), using the well established pair-breeding test,17 to determine any differences in potency between the different chemicals. Next, two concentration−response experiments were conducted, again using the pair-breeding assay, to determine effect concentrations of two of the progestins with very different potencies. In vitro experiments were also conducted to help explain the reasons for the differences in potency, and consisted of screening a selection of widely used synthetic progestins through a bank of recombinant yeast assays (estrogen, androgen, and progesterone). Research Organisms. Adult fathead minnow (Pimephales promelas) (6−12 months old) were obtained from a breeding stock maintained at Brunel University. Fish were fed three times per day, once with adult brine shrimp (Tropical Marine Centre, Gamma irradiated) and twice with flake food (Tetramin, Tetra, Southampton, UK). Fish were not fed on sampling days. Experimental Design. Each in vivo experiment employed a continuous flow-through system,17 incorporating 8-L glass tanks, which ensured a complete change of dechlorinated tap water (5- and 10-μm carbon-filtered) at least every 2 h. Every tank contained a pair of fish (1 male and 1 female) and there were 8 pairs of fish for each concentration of the test chemical (and the controls), along with a glass dish, grid, and tile to spawn on. The protocol consisted of a 21-day pre-exposure period, a 3-d transition (when dosing of the synthetic progestins started), and a further 21 days of exposure to the different chemicals. Parameters monitored within the tanks throughout the studies were temperature (25 ± 1 °C) and 2078
dx.doi.org/10.1021/es3048834 | Environ. Sci. Technol. 2013, 47, 2077−2084
Environmental Science & Technology
Article
Table 1. Measured Concentrations of Progestins in the Water at Termination of Experiment 1a Tank 1 2 3 4 5 6 7 8 average standard deviation a
Control
Gestodene
Desogestrel
Drospirenone
Levonorgestrel
