Aquatic Toxicology 146 (2014) 259–260
Contents lists available at ScienceDirect
Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox
Commentary
Are some invertebrates exquisitely sensitive to the human pharmaceutical Fluoxetine? John P. Sumpter ∗ , Luigi Margiotta-Casaluci Institute for the Environment, Brunel University, Uxbridge, Middlesex UB8 3PH, UK
1. Background It is now obvious that a wide range of different human pharmaceuticals, and probably also their metabolites/degradation products, are present in the aquatic environment worldwide. Therefore just about all aquatic organisms will be exposed to these biologically active chemicals, most of which have very specific modes-of-action, at least in humans. Trying to determine whether or not these pharmaceuticals present a threat to aquatic organisms has become a major focus of ecotoxicological research in the last decade. With so many different pharmaceuticals in use, and so many aquatic species likely to receive exposure, this is a daunting task. Concentrations of human pharmaceuticals in the aquatic environment are becoming clearer. Of course these will vary depending on many factors, with the amount used by patients and the ease of metabolism and degradation in the environment being major ones. The highest concentrations routinely expected are probably a few gs/L in effluents of wastewater treatment works and tens to hundreds of ngs/L in rivers. Concentrations in the coastal marine environment are less well documented, but would be expected to be considerably less than those in rivers receiving effluents. Thus the key question is whether or not chronic exposure to such concentrations has adverse effects on aquatic organisms. 2. Some surprising results? Recently, a number of papers have been published that present results suggesting that some aquatic, especially invertebrate, species, are extremely sensitive to the human pharmaceutical fluoxetine. The following two papers stand out in this regard: Di Poi, C., Darmaillacq, A-S, Dickel, L., Boulouard, M. and Bellanger, C. 2013. Effects of perinatal exposure to waterborne fluoxetine on memory processing in the cuttlefish Sepia officinalis. Aquat. Toxicol. 132–133, 84–91. Franzellitti S., Buratti, S., Valbonesi, P. and Fabbri, E. 2013. The mode of action (MOA) approach reveals interactive effects of
∗ Corresponding author. E-mail address: [email protected] (J.P. Sumpter). 0166-445X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.aquatox.2013.11.021
environmental pharmaceuticals on Mytilis galloprovincialis. Aquat. Toxicol. 140–141, 249–256. In the first of these two papers (Di Poi et al., 2013), it is stated that “The lowest observed effect concentration of this anti-depressant on learning and retention was 1 ng/L, which is under the range of environmental concentration”. Perhaps even more startling results are presented in the other paper (Franzellitti et al., 2013). These authors present results suggesting that a concentration of fluoxetine of 0.3 ng/L had a number of biochemical and molecular effects on mussels, leading them to suggest that fluoxetine has “the potential to affect the ability of animals to elaborate strategies of defences or adaptation”. Thus, both papers provide results suggesting that fluoxetine affects aquatic invertebrates at concentrations well within the environmental range. If true, widespread effects of fluoxetine on aquatic organisms would presumably be anticipated. 3. The mode-of-action (MOA) of fluoxetine The antidepressant fluoxetine is a selective serotonin reuptake inhibitor (SSRI). It increases serotonin (5-HT) levels in nerve synapses by inhibiting the serotonin transporter (SERT), and hence serotonin reuptake. To date, all authors reporting effects of fluoxetine on non-target (aquatic) organisms appear to think that this MOA applies to the effects they report. In fact, Franzellitti et al. (2013) explicitly state that they believe this. Given this situation, it seems relevant and appropriate to utilise all the information on the pharmacodynamics of fluoxetine in patients, as discussed by Rand-Weaver et al. (2013). In patients taking the drug for its anti-depressant effects, blood or plasma fluoxetine concentrations are usually in a range of 50–500 g/L. Brain concentrations of fluoxetine and its metabolites steadily increase for several weeks after patients begin taking the drug, and the full beneficial effects are not realised for at least a month. If fluoxetine was as potent in aquatic organisms as it was in patients, then it could be argued that blood concentrations in the former need to be similar to human therapeutic concentrations before effects occur. Currently no data are available on blood or plasma concentrations of fluoxetine in any aquatic organisms, so validating (or not) the read-across hypothesis is not possible (Rand-Weaver et al., 2013). Doing so would be especially difficult for invertebrates, particularly those without a circulatory
260
J.P. Sumpter, L. Margiotta-Casaluci / Aquatic Toxicology 146 (2014) 259–260
system similar to that of higher vertebrates. Nevertheless, some comparisons can be made, as demonstrated below. Franzellitti et al. (2013) claim that a concentration of fluoxetine of 0.3 ng/L affected mussels. They used a semi-static set-up, with 10 mussels in each 10 L of water, and an exposure period of 7 days. Seawater was renewed each day, so 3 ng fluoxetine (0.3 ng/L) was added to each vessel containing mussels each day. This procedure was repeated 7 times, so a total of 21 ng fluoxetine was added to each vessel during the experimental period. If there was no loss of fluoxetine during the experiment, which is unlikely, all of the drug was taken up by the mussels, and it was evenly distributed between the mussels, then each mussel would have contained a maximum of 2.1 ng fluoxetine at the end of the experiment. Compare this value to the human therapeutic concentration of 50–500 ng/ml, which is required to produce any effect in patients. Although this comparison is very difficult, if not impossible, to make with any confidence, it nevertheless seems likely that fluoxetine would need to be much more potent in mussels than humans to account for the effects reported by Franzellitti et al. (2013). From the pharmacological point of view, investigating the following points would help to elucidate if fluoxetine is (or is not) more potent in molluscs than in humans: (a) the affinity of fluoxetine to the molluscan SERT; (b) the degree of binding of fluoxetine to molluscan blood (or hemolymph) proteins, and the consequent concentrations of free fluoxetine; (c) the binding of fluoxetine to potential off-targets in molluscs. Unfortunately, at the moment no experimental data are available for any of these points. 4. Other surprising results? Although the two papers discussed above have reported effects of fluoxetine at extremely low concentrations, there have been other reports of fluoxetine causing effects on invertebrates at quite low concentrations. For example, Lazzara et al. (2012) reported that both 20 and 200 ng fluoxetine/L caused gamete release from mussels, and Gular and Ford (2010) reported that swimming behaviour of an amphipod was affected by 100 ng/L, although in that study higher concentrations did not lead to significant effects. However, these reports are in contrast to others, which suggest that much higher concentrations of fluoxetine are required to produce effects
on invertebrates. For example, hundreds of gs/L were needed to cause foot detachment from the substrate in five species of marine mollusc (Fong and Molnar, 2013), and a similar concentration was required to affect reproduction of mussels (Bringolf et al., 2010). 5. Will the ‘low concentration’ results be substantiated? It is not possible to answer this question presently, although it needs to be answered. In both reports discussed in this ‘Commentary’ (Di Poi et al., 2013; Franzellitti et al., 2013), the results from only one experiment are provided, so it is currently unknown if the results will be repeatable in the same laboratory, let alone other, independent laboratories. In neither study were concentrations of fluoxetine measured either in the water or the test organisms. In one study only a single concentration of fluoxetine was utilised (Franzellitti et al., 2013), and in the other two (Di Poi et al., 2013); thus, it is unclear whether or not the effects reported would be concentration-related, as we would expect any effect of a drug to be (as they are in patients). Results such as those reported by Di Poi et al. (2013) and Franzellitti et al. (2013) have major regulatory implications, and hence it is incumbent on scientists to do their research to the highest standards possible, in order that the most appropriate decisions are made to protect aquatic wildlife. References Bringolf, R.B., Heltsey, R.M., Newton, T.J., Eads, C.B., Fraley, S.J., Shea, D., Cope, W.G., 2010. Environmental occurrence and reproductive effects of the pharmaceutical fluoxetine in native freshwater mussels. Environ. Toxicol. Chem. 29, 1311–1318. Di Poi, C., Darmaillacq, A.-S., Dickel, L., Boulouard, M., Bellanger, C., 2013. Effects of perinatal exposure to waterborne fluoxetine on memory processing in the cuttlefish Sepia officinalis. Aquat. Toxicol. 132–133, 84–91. Fong, P.P., Molnar, N., 2013. Antidepressants cause foot detachment from substrate in five species of marine snail. Mar. Environ. Res. 84, 24–30. Franzellitti, S., Buratti, S., Valbonesi, P., Fabbri, E., 2013. The mode of action (MOA) approach reveals interactive effects of environmental pharmaceuticals on Mytilus galloprovincialis. Aquat. Toxicol. 140–141, 249–256. Gular, Y., Ford, A.T., 2010. Anti-depressants make amphipods see the light. Aquat. Toxicol. 99, 397–404. Lazzara, R., Blazquez, M., Porte, C., Barata, C., 2012. Low environmental levels of fluoxetine induce spawning and changes in endogenous estradiol levels in the zebra mussel Dreissena polymorpha. Aquat. Toxicol. 106–107, 123–130. Rand-Weaver, M., Margiotta-Casaluci, L., Patel, A., Panter, G.H., Owen, S.F., Sumpter, J.P., 2013. The read-across hypothesis and environmental risk assessment of pharmaceuticals. Environ. Sci. Technol. 47, 11384–11395.
Contents lists available at ScienceDirect
Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox
Commentary
Are some invertebrates exquisitely sensitive to the human pharmaceutical Fluoxetine? John P. Sumpter ∗ , Luigi Margiotta-Casaluci Institute for the Environment, Brunel University, Uxbridge, Middlesex UB8 3PH, UK
1. Background It is now obvious that a wide range of different human pharmaceuticals, and probably also their metabolites/degradation products, are present in the aquatic environment worldwide. Therefore just about all aquatic organisms will be exposed to these biologically active chemicals, most of which have very specific modes-of-action, at least in humans. Trying to determine whether or not these pharmaceuticals present a threat to aquatic organisms has become a major focus of ecotoxicological research in the last decade. With so many different pharmaceuticals in use, and so many aquatic species likely to receive exposure, this is a daunting task. Concentrations of human pharmaceuticals in the aquatic environment are becoming clearer. Of course these will vary depending on many factors, with the amount used by patients and the ease of metabolism and degradation in the environment being major ones. The highest concentrations routinely expected are probably a few gs/L in effluents of wastewater treatment works and tens to hundreds of ngs/L in rivers. Concentrations in the coastal marine environment are less well documented, but would be expected to be considerably less than those in rivers receiving effluents. Thus the key question is whether or not chronic exposure to such concentrations has adverse effects on aquatic organisms. 2. Some surprising results? Recently, a number of papers have been published that present results suggesting that some aquatic, especially invertebrate, species, are extremely sensitive to the human pharmaceutical fluoxetine. The following two papers stand out in this regard: Di Poi, C., Darmaillacq, A-S, Dickel, L., Boulouard, M. and Bellanger, C. 2013. Effects of perinatal exposure to waterborne fluoxetine on memory processing in the cuttlefish Sepia officinalis. Aquat. Toxicol. 132–133, 84–91. Franzellitti S., Buratti, S., Valbonesi, P. and Fabbri, E. 2013. The mode of action (MOA) approach reveals interactive effects of
∗ Corresponding author. E-mail address: [email protected] (J.P. Sumpter). 0166-445X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.aquatox.2013.11.021
environmental pharmaceuticals on Mytilis galloprovincialis. Aquat. Toxicol. 140–141, 249–256. In the first of these two papers (Di Poi et al., 2013), it is stated that “The lowest observed effect concentration of this anti-depressant on learning and retention was 1 ng/L, which is under the range of environmental concentration”. Perhaps even more startling results are presented in the other paper (Franzellitti et al., 2013). These authors present results suggesting that a concentration of fluoxetine of 0.3 ng/L had a number of biochemical and molecular effects on mussels, leading them to suggest that fluoxetine has “the potential to affect the ability of animals to elaborate strategies of defences or adaptation”. Thus, both papers provide results suggesting that fluoxetine affects aquatic invertebrates at concentrations well within the environmental range. If true, widespread effects of fluoxetine on aquatic organisms would presumably be anticipated. 3. The mode-of-action (MOA) of fluoxetine The antidepressant fluoxetine is a selective serotonin reuptake inhibitor (SSRI). It increases serotonin (5-HT) levels in nerve synapses by inhibiting the serotonin transporter (SERT), and hence serotonin reuptake. To date, all authors reporting effects of fluoxetine on non-target (aquatic) organisms appear to think that this MOA applies to the effects they report. In fact, Franzellitti et al. (2013) explicitly state that they believe this. Given this situation, it seems relevant and appropriate to utilise all the information on the pharmacodynamics of fluoxetine in patients, as discussed by Rand-Weaver et al. (2013). In patients taking the drug for its anti-depressant effects, blood or plasma fluoxetine concentrations are usually in a range of 50–500 g/L. Brain concentrations of fluoxetine and its metabolites steadily increase for several weeks after patients begin taking the drug, and the full beneficial effects are not realised for at least a month. If fluoxetine was as potent in aquatic organisms as it was in patients, then it could be argued that blood concentrations in the former need to be similar to human therapeutic concentrations before effects occur. Currently no data are available on blood or plasma concentrations of fluoxetine in any aquatic organisms, so validating (or not) the read-across hypothesis is not possible (Rand-Weaver et al., 2013). Doing so would be especially difficult for invertebrates, particularly those without a circulatory
260
J.P. Sumpter, L. Margiotta-Casaluci / Aquatic Toxicology 146 (2014) 259–260
system similar to that of higher vertebrates. Nevertheless, some comparisons can be made, as demonstrated below. Franzellitti et al. (2013) claim that a concentration of fluoxetine of 0.3 ng/L affected mussels. They used a semi-static set-up, with 10 mussels in each 10 L of water, and an exposure period of 7 days. Seawater was renewed each day, so 3 ng fluoxetine (0.3 ng/L) was added to each vessel containing mussels each day. This procedure was repeated 7 times, so a total of 21 ng fluoxetine was added to each vessel during the experimental period. If there was no loss of fluoxetine during the experiment, which is unlikely, all of the drug was taken up by the mussels, and it was evenly distributed between the mussels, then each mussel would have contained a maximum of 2.1 ng fluoxetine at the end of the experiment. Compare this value to the human therapeutic concentration of 50–500 ng/ml, which is required to produce any effect in patients. Although this comparison is very difficult, if not impossible, to make with any confidence, it nevertheless seems likely that fluoxetine would need to be much more potent in mussels than humans to account for the effects reported by Franzellitti et al. (2013). From the pharmacological point of view, investigating the following points would help to elucidate if fluoxetine is (or is not) more potent in molluscs than in humans: (a) the affinity of fluoxetine to the molluscan SERT; (b) the degree of binding of fluoxetine to molluscan blood (or hemolymph) proteins, and the consequent concentrations of free fluoxetine; (c) the binding of fluoxetine to potential off-targets in molluscs. Unfortunately, at the moment no experimental data are available for any of these points. 4. Other surprising results? Although the two papers discussed above have reported effects of fluoxetine at extremely low concentrations, there have been other reports of fluoxetine causing effects on invertebrates at quite low concentrations. For example, Lazzara et al. (2012) reported that both 20 and 200 ng fluoxetine/L caused gamete release from mussels, and Gular and Ford (2010) reported that swimming behaviour of an amphipod was affected by 100 ng/L, although in that study higher concentrations did not lead to significant effects. However, these reports are in contrast to others, which suggest that much higher concentrations of fluoxetine are required to produce effects
on invertebrates. For example, hundreds of gs/L were needed to cause foot detachment from the substrate in five species of marine mollusc (Fong and Molnar, 2013), and a similar concentration was required to affect reproduction of mussels (Bringolf et al., 2010). 5. Will the ‘low concentration’ results be substantiated? It is not possible to answer this question presently, although it needs to be answered. In both reports discussed in this ‘Commentary’ (Di Poi et al., 2013; Franzellitti et al., 2013), the results from only one experiment are provided, so it is currently unknown if the results will be repeatable in the same laboratory, let alone other, independent laboratories. In neither study were concentrations of fluoxetine measured either in the water or the test organisms. In one study only a single concentration of fluoxetine was utilised (Franzellitti et al., 2013), and in the other two (Di Poi et al., 2013); thus, it is unclear whether or not the effects reported would be concentration-related, as we would expect any effect of a drug to be (as they are in patients). Results such as those reported by Di Poi et al. (2013) and Franzellitti et al. (2013) have major regulatory implications, and hence it is incumbent on scientists to do their research to the highest standards possible, in order that the most appropriate decisions are made to protect aquatic wildlife. References Bringolf, R.B., Heltsey, R.M., Newton, T.J., Eads, C.B., Fraley, S.J., Shea, D., Cope, W.G., 2010. Environmental occurrence and reproductive effects of the pharmaceutical fluoxetine in native freshwater mussels. Environ. Toxicol. Chem. 29, 1311–1318. Di Poi, C., Darmaillacq, A.-S., Dickel, L., Boulouard, M., Bellanger, C., 2013. Effects of perinatal exposure to waterborne fluoxetine on memory processing in the cuttlefish Sepia officinalis. Aquat. Toxicol. 132–133, 84–91. Fong, P.P., Molnar, N., 2013. Antidepressants cause foot detachment from substrate in five species of marine snail. Mar. Environ. Res. 84, 24–30. Franzellitti, S., Buratti, S., Valbonesi, P., Fabbri, E., 2013. The mode of action (MOA) approach reveals interactive effects of environmental pharmaceuticals on Mytilus galloprovincialis. Aquat. Toxicol. 140–141, 249–256. Gular, Y., Ford, A.T., 2010. Anti-depressants make amphipods see the light. Aquat. Toxicol. 99, 397–404. Lazzara, R., Blazquez, M., Porte, C., Barata, C., 2012. Low environmental levels of fluoxetine induce spawning and changes in endogenous estradiol levels in the zebra mussel Dreissena polymorpha. Aquat. Toxicol. 106–107, 123–130. Rand-Weaver, M., Margiotta-Casaluci, L., Patel, A., Panter, G.H., Owen, S.F., Sumpter, J.P., 2013. The read-across hypothesis and environmental risk assessment of pharmaceuticals. Environ. Sci. Technol. 47, 11384–11395.
