E ﬀ ect of Water pH on the Uptake of Acidic (Ibuprofen) and Basic (Propranolol) Drugs in a Fish Gill Cell Culture Model

: Water pH is predicted to a ﬀ ect the uptake of ionizable pharmaceuticals in ﬁ sh. The current study used an in vitro primary ﬁ sh gill cell culture system to assess the e ﬀ ect of pH values in the range of 4.5 − 8.75 on the uptake rates of the base propranolol (pKa 9.42) and the acid ibuprofen (pKa 4.59). The rate-limiting step in the uptake was the di ﬀ usive supply ﬂ ux of the unionized form from the water to the apical membrane, with subsequent rapid transfer across the epithelium. Computed uptake rate based on the unionized fraction best described the uptake of propranolol and ibuprofen over the range of pH values 5 − 8 and 6 − 8.75, respectively. For ibuprofen, the computed uptake rate overestimated the uptake below pH 6 where the unionized fraction increased from 4% at pH 6 to 55% at pH 4.5. As the unionized fraction increased, the uptake rate plateaued suggesting a saturation of the transport process. For both drugs, large variations in the uptake occur with only small ﬂ uctuations in pH values. This occurs between pH values 6 and 8, which is the pH range acceptable in regulatory test guidelines and seen in most of our freshwaters.

Effect of Water pH on the Uptake of Acidic (Ibuprofen) and Basic (Propranolol) Drugs in a Fish Gill Cell Culture Model 1. INTRODUCTION Human and veterinary pharmaceuticals are biologically active compounds with concentrations in global freshwater environments being measured at the pg−μg/L range 1 and accumulation in biota being observed. 2,3 This raises concerns about the effect these compounds may have on aquatic organisms because many human drug targets are evolutionarily conserved in fish and aquatic invertebrates. 4−7 Adverse effects have been observed for a number of drugs when internal concentrations in fish exceed the human therapeutic plasma concentrations, 8 for example, ethinyl oestradiol, 9 fluoxetine, 10,11 glucocorticoids, 12−15 ibuprofen, 16 propranolol, 17 and salbutamol. 18 Identification and characterization of environmental risks posed by chemicals currently involve lengthy and costly acute and chronic fish toxicity studies, 19 and there is a need to develop alternative methods, whether computational such as QSARs 20 or in vitro techniques. 21,22 This is extremely pertinent to pharmaceuticals because there are over 11,000 small molecule drugs and approximately 2500 approved (https://www.drugbank.ca/stats), most of which are legacy compounds with little environmental impact data. 6 As part of a risk assessment, information on a chemical's absorption, distribution, metabolism, and excretion (ADME) is needed to determine its bioaccumulative properties. 23 There is a paucity of data for drug ADME for fish, 24 with absorption and metabolism processes representing the largest factors of uncertainty in current accumulation models. 2,25,26 Many bioaccumulation assessments are based on computer modeling tools whose parameters are largely derived from data for lipophilic neutral compounds that passively diffuse across lipid membranes and undergo little to no metabolism. 25 About 78− 95% of existing pharmaceuticals are ionizable. 27−29 The degree of ionization of a given compound at a given pH is determined by its acid dissociation constant (pKa), and this impacts the biopharmaceutical properties of a drug influencing solubility, lipophilicity, permeability, and protein binding, which in turn underpins the drug ADME characteristics. 27 In combination, these processes result in a certain internal dose, the dynamic intracellular speciation of which is the driver for potential toxicity.
In the aquatic environment, water pH can vary from 3− 10, 30,31 with the majority of lotic waters being in the range of pH 6−8 (e.g., pH values of 90% of the European rivers are in the range of 6.88−8.35 32 ). Testing guidelines for OECD 203 Fish, 33 acute toxicity test, OECD 305 34 bioaccumulation in fish: aqueous and dietary exposure and OECD 236 35 Fish Embryo Acute Aquatic Toxicity (FET) Test protocols reflect these environmental values, and tests are accepted in the pH range of 6.0−8.5. Recent amendments to OECD protocols 36 acknowledge the difficulty in testing ionizable compounds and provide guidance. This means that the extent to which a given drug is ionized will vary depending on the surrounding pH, with the potential to greatly influence bioaccumulation and effects. Recent early fish larval embryo toxicity studies have shown that pH influences drug uptake and toxicity 36−38 and similarly, pH affects the uptake of weak bases more than salinity in estuarine fish. 39 These studies suggest that the unionized drug species is more readily bioavailable and has the greatest influence on predicting toxicity. Models predicting the uptake of other ionizable organic chemicals, such as chlorinated phenols, 40 indicate that the unionized fraction is also more readily bioavailable. 41−46 However, the decrease in the extent of ionization was not proportional to the uptake rate, suggesting that the ionic species may contribute to diffusion across the membrane 41,45,47 and indicating that the real situation is more nuanced.
The current understanding of the mechanisms of neutral/ unionizable and ionizable pharmaceutical uptake in fish and how water chemistry influences this process are still poorly understood. For example, the relative contribution from paracellular and transcellular transport and the role of passive diffusion versus carrier-mediated transport are the subject of debate. 48 Knowledge of the involved processes is fundamental to develop a mechanistic understanding of the risk of pharmaceuticals in the aquatic environment and identify which drugs have the highest priority for assessment. 6 We have helped to develop an in vitro model of a fish gill cell culture system (FIGCS) for toxicological and physiological studies. 22 This system uses a double seeding technique onto inserts to develop a three-compartment model, the apical and basolateral compartments and the double cell layer (Figure 1). The epithelium forms exceptionally tight junctions 22 and tolerates the application of water to the apical surface. The dynamic nature of the FIGCS setup provides important advantages over 2D scaffold systems, which reach the steady state even for charged chemicals. 50 The advantage of using an in vitro model for the fish gill is the reduction in the number of fish used, and FIGCS typically produces 24−48 inserts from two donor fishes. The FIGCS has been used to measure drug uptake from the water across the epithelium 51 and to develop a model based on chemical descriptors to describe drug uptake. 52 In the latter study, water chemistry was kept constant with a pH value of 7.6 and the uptake of 10 drugs, including acidic, basic, and neutral drugs monitored, and the model that best-described the uptake showed a positive correlation to drug solubility (Log S) and a negative correlation to pKa. 52 The current study uses the same in vitro model but focuses on the basic drug propranolol (pKa 9.42) and acid ibuprofen (pKa 4.59) uptake in the pH range of 4.5 to 8.75, with the aim to assess how drug ionization influences gill uptake and the hypothesis that the unionized fraction is more readily bioavailable. Temperature was maintained at 14°C with 14 h light/10 h dark cycle, and fish were fed a 1% (w/w) ration of trout pellets daily.
The double-seeded primary gill cell cultures are prepared and maintained according to protocols described in Schnell et al. 22 Briefly, on the first day, the cells collected from the gills of the first donor fish were seeded onto a permeable polyethylene terephthalate (PET) membrane inserts with 0.4 μm pores having an area of 0.9 cm 2 (Transwell System, Corning) and maintained at 18°C in media (L-15 (Invitrogen, cat. No. 21083−027) containing 5% FBS (Sigma-Aldrich, cat. No. F7524) and a pH of 7.7. On the second day, the cells from a second donor were seeded on top of the first to generate the double seeded gill cell culture. This system has an apical compartment where the cells are in direct contact with the media and a basalolateral compartment below ( Figure 1). The transepithelial resistance was monitored daily using an epithelial tissue voltohmmeter (EVOMX) with STX-2 chopsticks (World Precision Instruments).
2.2. pH Monitoring. Artificial freshwater (AFW) was prepared according to OECD 203 Test Guidelines 39 [2 mM CaCl 2 ; 0.5 mM MgSO 4 ; 0.8 mM NaHCO 3 , 77.1 μM KCl (total ionic strength ca. 10 mM)], and pH was adjusted to 5, 6, 7, 8, and 9 with NaOH or HCl. Buffered AFW water was  22 that forms two layers of cells 57 that are bathed in the L15 medium from the basolateral compartment and exposed to water on the apical surface. In the current study, the bulk water was buffered with 30 mM MES and 10 mM bis-tris propane to maintain the pH at 4.5−8.75 over 24 h ( Figure S2). This meant that the ibuprofen-ionized fraction varied between 45−100% and propranolol between 82.4−100%. The intracellular pH has been measured at pH 7.43, 60 and the L15 medium has a pH value of 7.7. At present, it is unknown how the cells influence the pH of the boundary layer in a buffered system. The red arrow represents the diffusion of the drug (image of propranolol is shown) through the bulk media, boundary layer, and interaction with the apical membrane. The blue arrow indicates a potential uptake route via a paracellular pathway and the green arrow via a transcellular route; because the cell preparation consists of two layers, it cannot be discounted that uptake across the first layer is transcellular and then paracellular across the second layer, or vice versa. Circles highlight the point at which uptake across the lipid bilayer occurs either through facilitated uptake or passive diffusion.
Environmental Science & Technology pubs.acs.org/est Article prepared with the addition of 30 mM 2-(N-morpholino ethanesulfonic acid) (MES) (MES hydrate BioUltra, Sigma-Aldrich, cat. # 69890-10 g) and 10 mM 1,3-bis[tris-(hydroxymethyl) methylamino] propane (bis-tris propane) (Sigma-Aldrich, cat. # B6755-25G) and also adjusted to pH 4.5, 5, 5.5, 6, 7, 8, or 9 with NaOH or HCl. Following the formation of a tight epithelium, a TEER value >5000 Ω cm −2 , inserts were prepared for exposure by washing with phosphatebuffered saline. Buffered AFW suitability was assessed by monitoring the stability of transepithelial resistance and MTT 3-((4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assay, and over the 24 h, pH did not affect the cell viability ( Figure S1). The pH was monitored with a PerpHecT ROSS Micro Combination pH Electrode (Thermo Scientific, cat. # 8220-BNWP) and a Corning pH meter 140. This was also used to monitor the pH of various solutions in the absence of the FIGCS. Propranolol and ibuprofen uptake (appearance of radiolabel in the basolateral compartment following an application to the apical compartment) and intracellular accumulation (accumulation of radiolabel in the cells following an application to the apical compartment) were measured over a 24 h period. The exposure water or media used in experiments with radiolabelled propranolol or ibuprofen were prepared the day before and stored at 18°C prior to use.
The FIGCS uptake and intracellular accumulation of 3 Hpropranolol were assessed at 12.5 nM 3 H-propranolol in AFW and Buffered AFW at pH 5, 6, 7, 8, and 9. The uptake of 14 Cibuprofen was assessed at 1.5 μM 14 C-ibuprofen in Buffered AFW at pH 5, 6, 7, 8, and 9 as well as at 3 μM 14 C-ibuprofen in buffered AFW at pH 4.5, 5, 5.5, and 6.0. Upon the formation of a tight epithelium, media were aspirated and cells were washed with PBS prior to the addition of 800 μL of pH-adjusted AFW or buffered AFW containing radiolabel to the apical compartment and addition of 1 ml of media to the basolateral compartment, and cells were incubated for 24 h at 18°C.
Water or the medium (10 μL) was taken from the apical and basolateral compartments, respectively, for the assessment of uptake for propranolol exposure or 20 μL for ibuprofen experiments at 0, 1, 2, 3, 4, 6, and 24 h. In addition, cell-free propranolol or ibuprofen exposure was conducted under the same conditions to assess potential adhesion of the radiolabel to the plastic and insert and this accounted for <5%, similar to that previously measured. 38 Following incubation, the apical and basolateral compartments were aspirated, and the apical compartment was washed 3 times with 500 μL of PBS. To sample intracellular accumulation, the inserts were dried for 15−20 min and then incubated with 500 μL of DMSO for 1 h. The DMSO was then agitated by pipetting up and down 25 times, and the samples were taken and used to calculate the internal concentration of the pharmaceutical in the cells.
All experiments consisted of three biological replicates (inserts prepared from separate fish), and each biological replicate consisted of two to three separate inserts.
2.4. Radiolabeled Counting. Ecoscint A scintillation fluid (2 mL) (National Diagnostics, cat. # LS-273) was added to each sample and shaken before radioactivity, which was measured using a Beckmann Coulter LS6500 Multipurpose Scintillation Counter machine. Background radioactivity, measured in 2 mL of scintillation fluid containing 20 μL of radiolabel-free pH-adjusted AFW, buffered AFW, media, or L15-ex media (approximately 10 dpm), was deducted from all following calculations.
2.5. Calculations and Statistical Analysis. The diffusive flux, J dif , from the bulk solution to the apical membrane was calculated (eq 1) based on Fick's first law: where D is the aqueous diffusion coefficient for these compounds, ca. 4 × 10 −10 m 2 s −1 , 53 c is the concentration in mol m −3 of the unionized fraction assuming that it is this form that is more readily available to cross the membrane 44 calculated from the pKa for ibuprofen, which is 4. The primary gill cell culture pharmaceutical uptake rate was calculated based on the disappearance of the radiolabel from the apical compartment or appearance in the basolateral compartment (eq 2).
where Δ dpm is the difference between the disintegrations per minute at t = 0 and t = n in the apical or basolateral compartment, S.A is the specific activity of the radiolabel (dpm pmol −1 ), t is the time of the flux measurement calculated over the linear component of the temporal disappearance from the apical compartment or appearance in the basolateral compartment, and cm 2 represents the surface area of the epithelium (0.9 cm 2 ). Intracellular radiolabel concentrations are expressed as a % of the total radiolabel added to the apical compartment at t = 0. Significant differences between the uptake rates and the logtransformed values for the fraction of internalized radiolabeled drug at different pH values were compared using a one-way ANOVA followed by a Tukey's multiple comparison, p < 0.05 (GraphPad Prism v 8.2).

RESULTS AND DISCUSSION
Disturbances in the pH of the ambient water have a marked effect on acid−base balance in fish; for example, the expired water from across the gills is acidified when the fish are exposed to high external pH (9.91) and conversely alkalinized during low external pH (3.88) exposure. 61 The gills also play a major role in internal pH regulation via acid−base equivalent exchange 62 that may affect external water pH and the species of ionizable compounds. The pH monitoring of the apical compartment following exposure to weakly buffered AFW adjusted to pH 5 to 9 showed the ability of the FIGCS to modify water pH where an adjustment to pH 5 resulted in an apical pH of 6.91 after 24 h; pH 6 to pH 7.3; pH 7 to 7.64; pH Environmental Science & Technology pubs.acs.org/est Article 8 to 7.72 and pH 9 to 7.83 ( Figure S2). However, ion movement to counterbalance the abrupt pH change, that is, to maintain homeostasis, occurred over the first few hours ( Figure S2A). This equates to the excretion of ∼5 × 10 15 molecules of base and acid equivalents respectively, over the first 4 h ( Figure S3). The net effect is that cytosolic pH moves toward the attainment of equilibrium with the bulk water, where the external pH is balanced with the internal pH, which was calculated to be approximately pH 7.5 ( Figure S3). This estimate of intracellular pH is slightly higher than the intracellular pH value of 7.43 measured by Part and Wood 63 using a fluorescent intracellular pH probe. However, that measurement may not be representative of the cells used in the present studies, as the data by Part and Wood 63 were recorded in single-seeded cells plated on a glass coverslip in media and hence not in the FIGCS epithelium. Buffering of the AFW with 30 mM MES and 10 mM bis-tris stabilized the pH ( Figure  S2B), [original pH: final pH; 5: 5.14; 6: 5.99; 7: 6.96; 8: 7.95 except for pH 9 where there was a linear drop to pH 8.49, and the average pH 8.75 value was used in subsequent % ionization calculations] allowing for the assessment of the influence of pH on drug uptake. The primary gill cell epithelium is grown on a PET insert in a static system. 22 The apical compartment contains the exposure media (0.8 mL), and there is evidence of a "glycocalyx-like" covering on the cell membrane. 60 The primary cell culture method utilizes a double seeding epithelium, and the epithelium consists of two cellular layers approximately 10 −5 m in thickness. 60 Paracellular transport is unlikely to significantly contribute to uptake because the epithelium forms exceptionally tight junctions, which is relatively impermeable to mannitol (permeability 0.1 ± 0.01 × 10 −6 cm s −1 ), 51 a compound with a molecular mass of 182.17 g mol −1 , smaller than both ibuprofen (206.29 g mol −1 ) and propranolol (259.34 g mol −1 ), 51 and previously, propranolol permeability was 27× greater than that of mannitol (2.7 ± 0.2 × 10 −6 cm s −1 ) in the FIGCS. 51 In addition, in the human gut cell line, Caco-2, where the TEER ranges from 150 to 400 Ω.m 264 (all FIGCS inserts in this study have TEER values >5000 Ω.m 2 ), the paracellular permeability of propranolol has been measured at 0.22 × 10 −6 cm s −1 and the transcellular pathway is proposed as the favored uptake route. 65 Thus, in our system, the compounds have to come in contact with the apical membrane, enter the cell, and pass across the cytosol and the basolateral membrane of the first cell layer before experiencing the same resistance across the second cell layer before finally being deposited into the basolateral compartment across the PET insert, which possesses pores of 0.4 μm in diameter ( Figure 1).
The appearance of the drug in the basolateral compartment and disappearance from the apical compartment are influenced by the pH of the apical water, with greater uptake into the basolateral compartment of propranolol in alkaline conditions and ibuprofen in acidic conditions over 24 h (Figure 2). The influence of pH on propranolol uptake was evident when compared to the uptake in AFW water where the pH fluctuated ( Figure S2A) and was stable ( Figure S2B) and the rate in the weakly buffered AFW was greater at an initial pH 5, 6, and 7 and was less at pH 8 and 9 ( Figure S4). The greatest uptake of the drug is thus observed in apical water conditions where the fraction of unionized drug is the greatest. These observations corroborate previous studies that have shown that ionic drug species are considered poorly permeable in fish 38,51 including propranolol in a previous study with the FIGCS 51 and other ionizable organic compounds such as chlorinated phenols in vivo. 42 The predicted uptake based on the unionized fraction at each pH is in good agreement with the experimental propranolol uptake data and the ibuprofen data between pH values 6−8.75 and 5−8, respectively (Figures 3  and 4). Thus, up to a certain concentration, the overall disappearance from the apical compartment and appearance in the basolateral compartment are reasonably well approximated by the diffusive flux of the unionized fraction from the water phase to the apical surface; the overall driving force is the Environmental Science & Technology pubs.acs.org/est Article concentration gradient of the unionized form between the apical and basolateral compartments. This implies that the various processes involved in getting the compound into/ through the cell layer are faster than the initial diffusion step. There is a caveat because the gills are known to alter the pH of the aqueous boundary layer, which may differ from that of the bulk water 66 (Figure 1). However, the extent to which the buffering capacity of the bulk water influences this process is uncertain and the pH of the FIGCS boundary layer is currently unknown. The adjustment of the pH of the bulk water in response to the pH-adjusted AFW ( Figure S2A) indicates that it is plausible that at high pH, the excretion of the acid equivalent will reduce the boundary layer pH, and at low pH, the excretion of the base will increase the boundary layer pH. This would in effect increase the fraction of ionized acidic and basic drug at the apical membrane interface at a bulk water pH where we see the greatest influx. If this process remains significant in our pH-buffered setup, then it could be a contributing factor to the observed plateau in the uptake rate of ibuprofen at low pH (Figure 4), and a similar tendency was observed for propranolol at high pH ( Figure 3). Evidently, the knowledge of the extent and timescale over which the cells alter the boundary layer pH is of fundamental importance for predicting the branchial drug bioavailability.
The computed uptake rate overestimates the observed uptake rate at pH 4.5−6 for ibuprofen where the fraction of unionized drug ranged between 45−96% ( Figure 4A), and instead of increasing the uptake as the computation predicts, the uptake rate plateaus; there is also a hint of a plateau in the uptake of propranolol (Figure 3), but more data points are required for verification. Apart from the possible influence of the local pH in the boundary layer (see above), a plateau of this nature is associated with a substrate saturation of the transport site and suggests that drug movement across the cell membranes is in part via transport proteins. There is a disparity between the calculated uptake rate of propranolol based on the disappearance from the apical compartment and that based on appearance in the basolateral compartment at higher pH (Figure 3), and a potential explanation may be the higher accumulation of propranolol in the cell ( Figure 5) as pH rises  Environmental Science & Technology pubs.acs.org/est Article with the rate-limiting step being the extrusion across the basolateral membrane. A similar observation is made with cells exposed to 1.5 μM Ibuprofen ( Figure 4B); however, the internal ibuprofen concentration was not measured at the end of the measured uptake rate, but at 24 h, the internal concentrations between the pH treatments did not differ ( Figure 5). There is much debate as to the role of passive and facilitated transport-mediated drug uptake processes across biological membranes, 49 with advocates that suggest that facilitated transport processes can account for the observed accumulation 67 and others that favor a passive uptake pathway. 68 For example, Zheng et al 69 identified that passive diffusion can account for the transport of propranolol across the mammalian cell lines MDCK, MDCK-MDR1, and Caco-2; in contrast, other mammalian in vitro studies indicated that both propranolol and ibuprofen are substrates for putative drug transporters, which are notable members of the solute carrier 22 family. 49,70 In fish, Mihaljevićet al 71 and Dragojevićet al 72 have shown that a number of drugs and hormones are substrates for orthologues to members of the SLC22 family of proteins, thus providing evidence of a potential transport route. A number of rainbow trout cell lines, derived from different tissues including the gill, 73 and intact gills express transcripts for ABC transporters. 74 Further support for the involvement of carrier-mediated transport of ionizable compounds across the fish gill comes from a recent study accessing the pH-dependent uptake of chemicals from oil sands process-affected water using the rainbow trout gill cell line RT-gill W1 grown on inserts. 75 Cyclosporine A, an inhibitor of the solute carrier and ATPbinding cassette transport family of proteins, reduced the appearance of chemical species in the basolateral compartment. 75 This is of interest because the TEER of RT-gill W1 grown on the inserts is ∼25 Ω cm 2 , indicating a relatively leaky epithelium that would presumably allow paracellular movement of chemicals down a concentration gradient. 75 In a previous study, numerous SLC and ABC transporter blockers including cyclosporine A, cimetidine, MK571, and quinidine reduced the propranolol transport across the FIGCS epithelium into the basolateral compartment. 51 The breadth of proteins inhibited by these compounds suggests that different carrier-mediated transport processes may be involved in the uptake of a single compound. However, it is not possible to definitively identify which transport processes are involved, and thus, Stott et al. 51 proposed that propranolol is likely to be taken up by the FIGCS via both passive and facilitated routes. A combination of uptake processes is not uncommon, and in the case of salicylic acid, a mixture of both passive and facilitated uptake accounts for the transport across Caco-2 cells. 76 It is clear that the processes by which these drugs traverse the gill epithelium require further investigation. Protonation and deprotonation reactions are fast, and once inside the cytosol, an intracellular pH of 7.43 63 would indicate that both drugs are practically 100% ionized. The concept of ion trapping of ionizable organic compounds has recently been recognized as an important consideration for an aquatic hazard assessment of these compounds 47 and would maintain an external concentration gradient for the unionized fraction (until eventual equilibrium is attained). For propranolol, at the end of the 24 h exposure, the internal concentration reached a maximum of 4% of that added to the apical compartment ( Figure 5A). However, this fraction was dependent on the external pH and thus may represent the larger mass of unionized compound moving across the epithelium at the higher pH values. For ibuprofen, the intracellular fraction was constant over the pH range but was relatively low, ∼0.15% of total mass ( Figure 5B,C). The intracellular levels of drugs were measured at 24 h, and further time course experiments assessing intracellular concentrations are required to ascertain the extent to which ionizable drugs are trapped within the gills in the short term. The radiolabeled drugs do accumulate in the basolateral compartment (Figure 2), which suggests that the drug readily moves across this epithelium. Ionizable drugs are not predicted to cross phospholipid bilayers, and thus, the transfer from the cytosol to the basolateral compartment is again likely via a transport-mediated process. This process is faster than diffusion and thus only plays a role in limiting the overall rate once the transporters become saturated. This implies that such transporters are less active on the apical side. This line of reasoning has been proposed as an explanation for the pH-dependent transport of salicylic acid across Caco-2 cells, where the drug is actively transported out of the cell across the basolateral membrane. 76

ENVIRONMENTAL IMPLICATIONS
There is a precipitous drop in the drug uptake rate by the FIGCS between ∼82.4−100% ionization for propranolol and 92−100% ionization for ibuprofen ( Figure 3B and Figure 4C). This occurs over the pH range of 6−8, which is the pH range of the majority of global freshwaters. 33 The pH in freshwater will vary either temporally or spatially in certain circumstances, such as during algal blooms with diurnal variations in water pH. 77 Water pH will also alter with anthropogenic input of acid mine drainage and treated wastewater. Fishes are also known to alter the pH of the surrounding water 62 as well as the microclimate at the water/gill epithelium interface. Thus, our observation that there is a large variation in uptake at different pH values and that a rapid drop-off in uptake occurs between pH 6 and 8 may have significant consequences for predicting the uptake of acidic and basic drugs and for future models aimed at predicting drug ADME dynamics in fish and other organisms. While the FIGCS results cannot be translated quantitatively to in vivo processes in fishes, our findings provide important insights into literature data. For example, the observed large effect of pH on drug uptake may explain variation between studies assessing the effects of drugs on aquatic wildlife and between individual fish within studies. Indeed, in vivo studies of ibuprofen exposure record a very wide range of internal concentrations in the blood plasma, 16 and similarly for propranolol. 17 Historic regulatory testing guidelines for acute toxicity (OECD203), 33 bioaccumulation (OECD 305), 34 and the fish embryo test (OECD 236) 35 also state that the test medium pH is acceptable in the range of 6−8.5. However, recent OECD amendments 36 acknowledge the difficulty in testing ionizable compounds and provide guidance because there is potential for large variations in bioaccumulation of drugs over very small changes in pH (0.1 units ≈ 20% change in proton concentration) that could have significant implications for pharmaceutical risk assessment. In addition, we estimate that, during this study, we reduced the number of fish used in a conventional bioaccumulation study by 88%, thus adhering to the 3Rs principles.