Does ketoprofen or diclofenac pose the lowest risk to fish?
Highlights
► Ketoprofen bioconcentrates poorly (<0.05) from water to trout blood plasma. ► No pharmacological responses were identified by microarray or qPCR at 100 μg/L. ► Based on single drug exposure data, ketoprofen is less likely to affect wild fish. ► Bioconcentration factors for NSAIDs were much higher in effluent-exposed fish. ► Single drug exposures may underestimate field bioconcentration and thus risks.
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently found in surface waters. Several NSAIDs are well known to the general public as they are frequently used to treat common symptoms, such as fever, pain conditions and inflammation. The mechanism of action is not entirely known, though the primary targets are the prostaglandin G/H synthases 1 and 2 (ptgs1 and ptgs2), also known as cyclooxygenase 1 and 2. Both ptgs1 and ptgs2 are involved in the arachidonic acid pathway, and inhibited activities of the enzymes leads to reduced peripheral prostaglandin synthesis. Prostaglandins are pain-receptor sensitizers, hence the inhibition of their synthesis provides the analgesic effects of NSAIDs. In addition, prostaglandins suppress blood coagulation and regulate vasodilatation and vascular permeability. However, not all NSAIDs are used for the same indications; ibuprofen, for example, is most commonly used for light pain conditions and for its antipyretic effects, whereas diclofenac and ketoprofen are more commonly used to suppress inflammation, stronger pain as well as for rheumatic diseases. Although ketoprofen and diclofenac are used in comparable manners there are still differences in their pharmacokinetic and pharmacodynamic properties. Both are considered non-selective blockers of ptgs1 and ptgs2, though diclofenac is slightly more selective for ptgs2, whereas ketoprofen tends to be more selective for ptgs1. Diclofenac has a higher lipophilicity (log P of 4.26) compared to ketoprofen (3.61), which in the ecotoxicological context discussed below would suggest a higher potential for bioconcentration for diclofenac [1], [2], [3].
One of the most substantial environmental effects caused by any pharmaceutical is the dramatic decline in vulture populations in India and Pakistan. Diclofenac was given to cattle in order to relieve pain during the last time of their lives. Carcasses of treated livestock were subsequently eaten by Gyps vultures, which rapidly developed visceral gout and renal failure, a known side-effect of high doses of diclofenac in mammals [4], [5]. Subsequently, in 2006 the use of diclofenac for veterinary purposes was banned in India, Nepal and Pakistan [6]. However, the recommended alternative, meloxicam, is expensive and other alternatives, such as ketoprofen, is often used instead [7]. Unfortunately, the use of ketoprofen has recently been reported to cause mortalities in Gyps vultures. Captive and wild-caught vultures were fed ketoprofen-treated cattle-tissue containing concentrations similar to the levels found in deceased cattle available to wild vultures [7]. These findings stand in some contrast to previous indications that ketoprofen is rapidly eliminated from livestock tissues and does not cause mortality after exposure to Gyps vultures and other scavenging birds at therapeutic doses [8]. Also, ketoprofen-related mortality has been reported in male eider ducks given the drug [9]. The symptoms were identical to those found in the Gyps vultures exposed to diclofenac, i.e. renal failure and visceral gout.
The occurrences of both ketoprofen and diclofenac in effluents from sewage treatment plants (STPs) are typically at similar levels, up to approximately 1 μg/L [10], [11], [12], [13]. Several studies have reported effects on fish at these concentrations of diclofenac, ranging from cytological and histological effects [14], [15], [16], [17] to effects on gene expression [15], [18]. Reported bioconcentration factors (BCFs) for diclofenac in rainbow trout varies between studies [10], [11], [16], [18], [19]. However, the most recent studies are in relatively close agreement, showing a stable BCF from water to blood plasma of approximately 4 [18], [19]. We have recently shown effects on the global hepatic gene expression at exposure concentrations of diclofenac down to 1.6 μg/L, even though the corresponding plasma concentration of exposed fish was relatively low (approximately 6 ng/mL) in comparison with the human therapeutic plasma concentrations (HTPC) of >420 ng/mL [20], [21]. The number of affected genes as well as their fold change increased with an increasing exposure concentration up to a blood plasma concentration just below the HTPC [18]. Based on its frequent occurrence in the environment together with its risks to affect wildlife, diclofenac was very recently included in the substance priority list within the Water Framework Directive [22].
As ketoprofen and diclofenac often are used for similar indications, we wanted to investigate whether the use of ketoprofen could pose lower risks for fish. However, to the best of our knowledge, studies on the effects of ketoprofen exposure on fish are very scarce. Thibaut et al. [23] have studied the potential of ketoprofen impact on metabolism in carp liver in vitro and there are a few studies on the bioconcentration potential of ketoprofen, though the BCF to fish blood plasma is reported to vary quite much from 0.1 to 48 [10], [11]. Here we studied the water to plasma BCF of rainbow trout during controlled flow-through exposure experiments with multiple water concentrations of ketoprofen. Measurements of bioconcentration to blood plasma provide several advantages to whole body bioconcentration (traditional BCF). Importantly, HTPCs are most often available, therefore data on fish plasma levels provide an opportunity for read across between species and thus an estimate of the risk for a pharmacological response in the fish [2], [3], [24]. Another reason to focus on blood plasma levels is that they conceptually provide a better measure of exposure at the targets compared with whole body levels as some substances are mainly stored in fat and thus not readily available. We have also put our findings in perspective by performing a meta-analysis including previously reported data on BCFs between water and blood plasma for three different NSAIDs. Finally, similar to our previous study on diclofenac [18] we searched for effects on global hepatic gene expression in rainbow trout exposed to ketoprofen as an additional strategy to determine if water concentrations found in the environment are likely to evoke detectable pharmacological responses in exposed fish.
Section snippets
Fish exposure and sampling
Ketoprofen (purity ≥ 98%) (Sigma–Aldrich, Steinheim, Germany) was dissolved in water (500 mg/L concentrated solution), stirred vigorously and diluted stepwise to obtain stock concentrations (0.5 mg/L, 5 mg/L and 50 mg/L). Juvenile rainbow trout (Oncorhynchus mykiss) of both sexes (age: approximately six months, weight: 39.4 ± 6.6 g) were obtained from Vänneåns fiskodling AB, Sweden, and kept in 500 L holding tanks with sand-filtered, aerated fresh water in a flow-through system for one week prior to the
Bioconcentration
Ketoprofen concentration was successfully measured in water and blood plasma samples, with stable and reproducible results and R2 above 0.99 for all standard curves. No ketoprofen was detected in the instrumental or procedural blanks. Absolute recoveries of the solid phase extraction of the fish plasma and water samples were 127% (17% RSD, n = 6, spiking level 100 ng) and 103% (8% RSD, n = 6, spiking level 100 ng) respectively. The following results are all displayed in the order of low, intermediate
Discussion
In the present study we show that waterborne ketoprofen bioconcentrates considerably less in fish than diclofenac under controlled laboratory conditions. Measured plasma levels of ketoprofen in fish at an exposure concentration about 100 times higher than levels found in undiluted sewage effluents reached less than 1‰ of human therapeutic plasma levels. In accordance no effects on the global hepatic gene expression were found, in sharp contrast to previous reports for diclofenac [15], [18].
Acknowledgements
The authors thank Lina Gunnarsson, Bethanie Carney-Almroth and Hannah Svedlund for help with experiments and analyses. This research was supported by the Swedish Foundation for Strategic Environmental Research (MISTRA), the Swedish Research Council (VR) and the Adlerbertska Research Foundation.
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