Title Chronic Effects Assessment and Plasma Concentrations of the β-blocker Propranolol in Fathead Minnows (Pimephales promelas) Authors Emma Giltrow

Title Chronic Effects Assessment and Plasma Concentrations of the β-blocker Propranolol in Fathead Minnows (Pimephales promelas)

Authors Emma Giltrow

, Paul D. Eccles

, Matthew J. Winter

, Paul J. McCormack

, Mariann Rand-Weaver

, Thomas H. Hutchinson

, John P. Sumpter

a

Institute for the Environment, Brunel University, Kingston Lane, Uxbridge, Middlesex, UB8 3PH, United Kingdom

b

Biosciences, School of Health Sciences and Social Care, Brunel University, Uxbridge, Middlesex, UB8 3PH, United Kingdom

c

AstraZeneca Safety, Health and Environment, Brixham Environmental Laboratory, Freshwater Quarry, Brixham, Devon, TQ5 8BA, United Kingdom

d

Natural Environmental Research Council, Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth, PL1 3DH, United Kingdom

* Corresponding author. Tel: +44 1895 266303; Fax: +44 1895 269761; E-mail address;

john.sumpter@brunel.ac.uk

Abstract

The presence of many human pharmaceuticals in the aquatic environment is now a worldwide concern, yet little is known of the chronic effects that these bioactive substances may be having on aquatic organisms. Propranolol, a non-specific beta adrenoreceptor blocker (β-blocker), is used to treat high blood pressure and heart disease in humans. Propranolol has been found in surface waters worldwide at concentrations ranging from 12 to 590 ng/L. To test the potential for ecologically relevant effects in fish in receiving waters, short-term (21d) adult reproduction studies were conducted, in which fathead minnows were exposed to concentrations propranolol hydrochloride [CAS number 318-98-9] ranging from nominal concentrations of free drug from 0.001 to 10 mg/L (measured concentrations typically from 78-130%). Exposure of fish to 3.4 mg/L (measured) over 3 days caused 100% mortality or severe toxicity requiring euthanasia. The most sensitive endpoints from the studies were a decrease in hatchability (with regard to the number of days to hatch) and a dose-related increase in female gonadal-somatic index (GSI), giving LOEC

hatchability

and LOEC

values of 0.1 mg/L. Dose related decreases in male weight were also observed, with LOEC

of 1.0 mg/L, and LOEC

values were 1.0 mg/L.

Collectively these data do not suggest that propranolol was acting as a reproductive toxin.

Propranolol plasma concentrations in male fish exposed to water concentrations of 0.1 and 1.0 mg/L 1

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were 0.34 and 15.00 mg/L respectively which constitutes 436 and 1546 % of measured water concentrations. These compare with predicted concentrations of 3.57 and 44.38 mg/L, and thus support the use of partition coefficient data models for predicting resultant fish plasma levels. In addition, propranolol plasma concentrations in fish exposed to water concentrations of 0.1 and 1.0 mg/L were greater than the human therapeutic plasma concentration and hence these data very strongly support the fish plasma model proposed by Huggett et al. (2003). (Huggett, D.B., Cook, J.C., Ericson, J.F., Williams, R.T. 2003. A theoretical model for utilizing mammalian pharmacology and safety data to prioritize potential impacts of human pharmaceuticals to fish. Human and Ecological Risk Assessment, 9, 1789-1799).

Keywords: β-blocker; Fathead minnow; Pharmaceutical; Propranolol; Fish plasma concentration

1. Introduction

Pharmaceuticals in the environment has become a growing concern after the realisation that ethinyl estradiol could be found in the aquatic environment at concentrations exceeding 0.5ng/L, the concentration at which vitellogenin induction can occur in male fish (Purdom et al., 1994). Since then advancement of analytical techniques have shown that there are in fact many pharmaceuticals in our rivers and surface waters, including the human beta adrenoreceptor blocker (-blocker) (Fent et al., 2006; Ternes 2001). The question that follows therefore, is are the concentrations of

pharmaceuticals found in the aquatic environment high enough to cause harm to the wildlife that are exposed to them?

Propranolol and other human and animal pharmaceuticals reach the aquatic environment through a variety of routes. However, the majority of pharmaceuticals are present in the aquatic environment due to incomplete removal at sewage treatment works (STWs). In Germany it was found that 96 % of propranolol was removed from the influent compared to the effluent, yet the 4 % remaining is largely why propranolol has been found ubiquitously in rivers and streams in America and Europe at concentrations in the ng/L range, with maximum and median concentrations reaching 590 ng/L and 12 ng/L, respectively (Ashton et al., 2004; Huggett et al., 2003; Ternes, 1998).

There are many different classes of pharmaceuticals in the environment but this study and a companion study (Winter et al., 2008) focus solely on one group, the β-blockers. This is so that a comprehensive picture around one class of pharmaceuticals can be built up in order to try to establish some general principles that might be applicable to other classes of pharmaceuticals. A feature of the study was to apply the ‘fish plasma model’ as hypothesised by Huggett et al. (2003), 36

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which compares estimated or actual drug concentrations in fish plasma with human therapeutic plasma concentrations, in order to assess whether it is likely that environmental or experimental concentrations of a drug would produce therapeutic levels in fish. If the fish plasma model is found to be successful when applied to β-blockers, it might also be applicable to other groups of

pharmaceuticals.

β-blockers are grouped as one class of pharmaceuticals, and although they all act on beta-adrenergic receptors (β-ARs), they can differ greatly in their specificity and lipophilic properties (Owen et al., 2007). For example, propranolol has a relatively high log K

of 3.48, whereas atenolol has a considerably lower log K

of 0.23. A report by Winter et al. (2008) showed atenolol to have low chronic toxicity to fathead minnows. The results produced a 4 day embryo LOEC

value of >

10 mg/L, a LOEC

of >10 mg/L and the most sensitive endpoint was condition index, which value gave a LOEC

of 3.2 mg/L.

Published data show propranolol, out of all the β-blockers investigated, to be the most toxic to aquatic organisms. For example, invertebrate LC

values for metoprolol and propranolol range from 64 to >100 mg/L and 0.8 to 29.8 mg/L, respectively, showing that propranolol is harmful to invertebrates at much lower concentrations than metoprolol (Cleuvers, 2003; Huggett et al., 2002, Villegas-Navarro et al., 2003). In fish studies, the published data on propranolol are limited.

However, a report by Huggett et al. (2002) showed propranolol to be relatively toxic with LOEC

hatchability and egg production

values of 0.0005 mg/L. Hence the message is mixed, with most studies

suggesting that propranolol is not particularly toxic to aquatic organisms but some suggesting this is not the case.

2. Materials and Methods

2.1 Test design and concentration

The experimental design incorporated two separate replicate experiments. From an ethical standpoint, to conduct two smaller experiments, rather than one large experiment, was preferable because it meant that if no effect whatsoever was found in the first experiment, the second experiment need not be undertaken, hence reducing the number of fish used. Secondly, if a particular concentration of the test substance evoked a toxic effect, that particular concentration need not be repeated in the second experiment, hence less fish are put under duress. For statistical reasons, conducting two separate experiments allows the results of the two experiments to be compared, and if any effects occur and are repeatable, the results will be more robust. In order to compare atenolol and propranolol the experimental protocol was based on that used by Winter et al.

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(2008). Hence, as in Winter et al. (2008), the first experiment in this study incorporated a 10 mg/L treatment group to replicate concentrations that may be used if conducting experimental work for a environmental risk assessment (ERA).

2.2 Experimental procedure

A 21 day adult reproduction study was carried out using the protocol set out in Ankley et al. (2001) and Harries et al. (2000), later modified by Winter et al. (2008). Each experiment was carried out using a flow-through system in a room that contained two series of identical tanks.

Thermostatically heated (25 ± 1 ºC) dechlorinated tap water from a header tank flowed through 6 flow meters into 6 mixing chambers, via medical grade silicon tubing, and from these the water went to 6 fish tanks through silicone rubber tubing. In total there were 36 fish tanks which each had a working volume of 10.5 litres. Each tank was aerated and received roughly 9 tank renewals of water per day. During the exposure period, stock concentrations of propranolol were pumped from 4L foil wrapped amber stock bottles through 0.16mm tubing to a peristaltic pump and out through peroxide cured silicon tubing to the mixing chambers.

Inside each fish tank, a half piece of PVC gutter was placed over a stainless steel mesh and glass base (Thorpe et al., 2007). Most eggs were collected from the underside of the tile, to which they had been attached by the fish. However, in the event that some eggs did not adhere to the tile, they would fall onto the mesh or into the glass base.

After a 21 day pre-exposure period followed by 21 days of exposure to propranolol-HCl, the fish were humanely terminated using an overdose of ethyl 3-aminobenzoate methanesulfonate (MS- 222). Plasma samples were pooled from each sex from each concentration for analysis of

propranolol concentration in the plasma. Wet weight, fork length, gonad weight and fatpad weight were recorded. The number of tubercles and the prominence of tubercles were determined using the qualitative scheme set out by Smith (1978).

2.3 Hatchability trails

To assess whether the eggs produced by each pair were viable (i.e. successfully produced live young), hatchability trials were undertaken. After the first successful spawning in the baseline period, fifty eggs were left on the tile from the next spawn and placed in a wire meshed cage that was submersed in a separate tank receiving the same concentration of propranolol (i.e. the

developing eggs were exposed to the same concentration of propranolol as were the adult fish that 106

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laid the eggs). The eggs were checked and counted daily and any abnormalities recorded and dead eggs removed.

2.4 Test substance

The test substance, pharmaceutical grade propranolol (DL-Propranolol hydrochloride, 1–

isopropylamino-3-(1-naphthyloxy) proprano-2-ol hydrochloride, 99 % pure, racemic mixture, CAS 318-98-9, to be referred to as propranolol), was obtained from AstraZeneca, Brixham (UK). In the first experiment, five concentrations of propranolol namely 0.001, 0.01, 0.1, 1.0 and 10 mg/L were used, together with a dilution water control (DWC). In the second experiment this was reduced to 4 concentrations of propranolol which were 0.001, 0.01, 0.1, 1.0 mg/L and DWC. In both

experiments, there were six tanks for each treatment group, i.e. four pairs of fathead minnow and 2 tanks for hatchability trials at each concentration. Because unexpected results occurred at 10 mg/L in the first experiment (see later), and because it was not intended to collect acute toxicity data for propranolol, the 10 mg/L concentration was not repeated in experiment 2, and so for the second experiment it was possible to collect data from five fathead minnow pairs in the control tanks.

2.5 Test species

The fish in both experiments were one year old fathead minnows, Pimephales promelas. They were virginal stock that had been bred at Brunel University. One male and one female were placed in each fish tank. All male fish used in the pair-breeding experiments exhibited secondary sexual characteristics prior to the start of the experiments. The fish were fed twice a day with frozen adult brine shrimp (Artemia salina) during the week and once a day during the weekend, and in addition, fish flakes were also fed once a day. The fish received a photoperiod of 16 hours of light followed by 8 hours of darkness, with a 20 minute dawn: dusk transition.

2.6 Analysis of water samples

Liquid chromatography was used for analyte separation with 0.1 % ammonia in water and 0.1 % ammonia in methanol over an elution gradient. Electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) was then used to quantify the analytes, which was carried out in the positive ionization mode (+) ESI. For increased selectivity, MS/MS with selected reaction monitoring (SRM) was used for detection of protonated propranolol ([M + H]

, 260.2 Da) and Xcalibur

software was used for data acquisition and processing.

2.7 Analysis of plasma samples 140

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All samples and standards were prepared by protein precipitation (PPT) by which 50 µL of plasma was added to acetonitrile (200µL) containing metoprolol internal standard (0.25 µg L

) in a filtration vial (0.45µm). After vortexing the filtrate transferred to a vial and diluted with 100 µL, 30:70 acetonitrile/water. Propranolol concentration was then measured using online solid-phase extraction/liquid chromatography (SPE/LC).

2.8 Statistical analyses

Data were checked for normal distribution and equal variance using the Kolmogorov-Smirnov test and Levene Median test, respectively. Statistical analyses of normally distributed data of equal variance were carried out either using a t-test, or a One Way Repeated Measures ANOVA to compare two sets of data or more than two data sets, respectively. If a statistical difference was found in the latter case, a Holm-Sidak post-hoc test was carried out to show which treatment groups were different. If the data were not of a normal distribution, a non-parametric Kruskal-Wallis One Way Analysis of Variance on Ranks test was performed, followed by a Dunn’s post hoc test. The data for 1.0 mg/L hatchability trials could not be analysed as there were fewer than two data-points from the exposure period

3. Results

3.1 Propranolol water concentration

Propranolol concentrations from the water chemistry analyses showed that the measured

propranolol concentration for each treatment was in the expected range, apart from the 10 mg/L tanks in experiment 1 (Table 1). This showed that there was no contamination of control tanks with propranolol. At a nominal concentration of 10 mg/L, only 34 % of the nominal dose was measured in the tanks. This may have been due to solubility issues, and/or uptake of the drug by these fish in the tanks. In all other treatments, the mean propranolol concentrations in experiments 1 and 2 ranged from 53 to 112 % and from 78 to 130 % of the nominal concentrations, respectively,

demonstrating that the measured concentrations were relatively close to the nominal concentrations.

Statistical analysis of the water chemistry results showed that there were no statistically significant differences in the mean results at each concentration between the two experiments, and so data from each experiment were combined to make one data set.

3.2 Fish mortality

During the three day transition period in experiment 1, during which the propranolol concentrations were expected to rise steadily towards the normal values, the fish in the 10 mg/L tanks (actual concentration 3.4 mg/L) died or had to be euthanized before the 21 day exposure period began. In 174

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all pairs the males became obviously ill before the females, and before termination showed a lack of orientation and were noted to be swimming on one side and nosing the bottom of the tank. At the 1.0 mg/L concentration on day 5 in experiment 1, two males started to show similar behaviour to the 10 mg/L fish and were euthanized. During experiment 2, the fish at the 1.0 mg/L concentration were closely observed during the exposure period. It was noted on day 2 that all the fish exposed to 1.0 mg/L propranolol had reduced appetites, as a noticeable quantity of uneaten food could be seen on top and around the spawning tiles. By day 4, two males were euthanized as they had started to swim on one side with an apparent loss of orientation and their females were terminated on day 5 as they also showed the same symptoms. The remaining fish at this concentration, despite having reduced appetites, showed no obvious signs of distress. On day 11 during experiment 2 of the exposure period, one of the males at the 1.0 mg/L concentration was found dead outside of the tank.

This was most unexpected, as no signs of distress had been observed in the days prior to this incident. The remaining fish being maintained at 1.0 mg/L were then culled after this event and sampling data and tissues collected. Hence, egg data were collected for only 11 days in experiment 2 from the 1.0 mg/L treatment group. Four other deaths occurred during the two experiments and in all cases it was because the female had become egg-bound. Data from fish that died during the exposure period were excluded from the baseline data.

3.3 Cumulative egg production

Table 2 shows a summary of egg production data during the baseline and exposure period from each experiment. During the baseline period of 21 days across both experiments, the total number of eggs per female ranged from 590 to 1074, and the mean number of eggs per female per

reproductive day ranged from 28 to 51. These data compare favourably to pair breeding assay data reported by Winter et al. (2008), where the number of eggs per female per reproductive day ranged from 27 to 50, but are lower than those reported by Thorpe et al. (2007) (80 to 93 eggs/female/day).

The mean number of eggs per spawning over the 21 days was between 147 and 238 in the baseline period. This range is higher than data reported by Winter et al. (2008) (103 to 161 eggs/spawn) and lower than that reported by Thorpe et al. (2007) (358 ± 14 eggs/spawn). In the 1.0 mg/L treatment group the data collected over 11 days during the baseline period show the number of eggs produced per day and per spawn in experiment 1 to be reduced compared to other treatment groups. Egg production in the exposure period was similar to that in the baseline period in the DWC group, and also in the groups exposed to 0.001, 0.01 and 0.1 mg propranolol/L. There was evidence of reduced egg production in response to exposure to 1 mg/L. This apparent effect was not significant in experiment 1 (in which low survival of the adult fish, and unexpected low egg production in the baseline period complicated any analysis) but was significant (p<0.05) in experiment 2. There was 209

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no statistically significant difference between baseline and exposure periods for any of the other treatments.

3.4 Body and organ size

Analyses of male and female wet weight data obtained at the end of the experiments showed that weight ranged from 2.433 to 5.680 g in male fish and from 0.811 to 2.490 g in female fish. The male wet weight data showed a dose-related decrease, in which there was a statistically significant difference between the DWC group and the 1.0 mg/L treatment group (p < 0.05). This trend was not seen in the female wet weight data, and there were no statistically significant differences between treatment groups. Male fork length ranged from 52 to 71 mm and female fork length ranged from 42 to 65 mm. No statistically significant differences were found between treatment groups.

Gonad weight in males ranged from 0.01 to 0.074 g and gonadal somatic index (GSI) ranged from 0.22 to 1.39 %. Figure 1A shows male GSI data, in which a statistically significant difference was found between the DWC and 0.01 mg/L treatment groups. Both the 0.001 mg/L and the 0.01 mg/L groups also had higher mean GSI values than the DWC group, however there was not a significant trend of increasing GSI with increasing propranolol concentration. Female gonad weight ranged from 0.01 to 0.357 g and female GSI ranged from 1.32 to 17.14 %. In contrast, female GSI data exhibited a dose-related effect, for as the concentration of propranolol increased, so did the GSI, as shown in Figure 1B. These female GSI data produced statistically significant differences between treatment groups DWC and 0.1 mg/L, DWC and 1.0 mg/L, and between 0.001 and 1.0 mg/L.

Figure 2 shows the weight of the fatpads from the male fish at each concentration of propranolol.

The male fatpad weight ranged from 0.090 to 0.477 g. As the concentration of propranolol increased from 0.01 mg/L, there was a dose-related decrease in the weight of the fatpad, and a statistically significant difference was found between treatment groups 0.01mg/L and 1.0 mg/L and also between groups 0.1 mg/L and 1.0 mg/L (p < 0.05). However, no treatment group showed a statistically significant difference when compared to the control group. The number of tubercles on the males ranged from 12 to 16, and the tubercle prominence score from 1 to 4 (Smith, 1978).

There were no statistically significant differences between treatment groups in either of these sets of data. No secondary sexual characteristics were observed in any female fish sampled.

Figure 3 shows the condition index (CI) of male and female fish in each treatment group. These data range from 1.21 to 1.99 in male fish and between 1.00 and 1.58 in female fish. A statistically 244

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significant difference exits between the male fish CI of the 0.001 mg/L and 1.0mg/L groups and between the CI of the 0.01 mg/L and 1.0 mg/L treatment groups. However, there were no treatment groups in which the CI was statistically significantly different from that of the control group. No statistically significant differences were found between treatment groups in the female data.

3.5 Hatchability

The data from the hatchability trials are summarized in Table 3. Data from the 1.0 mg/L group could not be statistically analysed because there were fewer than 2 data points. A high proportion of eggs (between 75 and 95 %) hatched. There was no evidence that exposure of eggs to propranolol affected the proportion of eggs that hatched. Time to hatch was unaffected by 0.001 and 0.01 mg propranolol/L (Table 3), but was significantly delayed (by one day) in eggs exposed to 0.1 mg propranolol/L. Time to hatch also appeared to be delayed (by 3 days) when eggs were exposed to 1 mg propranolol/L, but low sample number

compromised this apparent effect. In the 0.1mg/L treatment group there was an increase in the time to hatch between the baseline and exposure periods, which was statistically significant.

3.6 NOEC and LOEC values

Table 4 summarises the lowest observed effect concentrations (LOEC) and the no observed effect concentration (NOECs) for each of the endpoints that were measured. In summary, propranolol affected hatchability (with regard to the number of days it takes for eggs to hatch) and female GSI at 0.1 mg/L. These were the most sensitive endpoints. At 1.0 mg/L, propranolol significantly affected male survival and male wet weight, and at 3.4 mg/L propranolol significantly affected female survival.

3.7 Plasma propranolol concentration

Fish in the control tanks (DWC) had no measurable propranolol in their plasma, but as the concentration of propranolol increased in the water, the fish plasma propranolol concentration increased dramatically, as shown in Table 5. At exposure concentrations of 0.1 and 1.0 mg/L, the fish bioaccumulated propranolol in their blood, and this was especially obvious in male fish. At low propranolol concentrations (0.001 and 0.01 mg/L), fish plasma concentrations did not reach, or only just reached water concentrations, whereas at higher water concentrations (0.1 and 1.0 mg/L), plasma concentrations were higher than water concentrations. Fish exposed to propranolol concentrations of 0.1 and 1.0 mg/L for 21 days had plasma propranolol concentrations higher than the human therapeutic plasma concentration (H

PC).

4 Discussion 279

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The toxicity data obtained from the propranolol 21-day adult reproduction study reported here differed at least ten-fold from the comparable data obtained with atenolol (Winter et al., 2008).

Fathead minnows were able to tolerate high concentrations of atenolol (LOEC

>10 mg/L), unlike in this study, where propranolol caused acute toxicity at lower concentrations (LOEC

3.4 mg/L). The main reason for this could be due to differences in lipophilicity between the two β- blockers. Atenolol has a much lower log K

, compared to propranolol, and is expected therefore to less readily bioconcentrate. Indeed, plasma concentrations of atenolol in relation to the

corresponding measured water levels only ranged from 1.8 to 12.2 %, unlike plasma concentrations of propranolol, which reached up to 1550 % of the surrounding water concentration.

Before the fish in the 10 mg/L treatment group (actual concentration = 3.4 mg/L) were euthanized, they were observed to be very disorientated and swimming on one side or nosing the bottom of the tank. The loss of appetite together with the disorientation of the fish and lack of sexual behaviour indicates that propranolol may have been acting as a CNS toxin. The relatively high lipophilic nature of propranolol means it can easily move across the blood-brain barrier in organisms (Vu &

Beckwith 2007). Studies have shown that propranolol can be found in human brain tissue at concentrations ten to twenty six times higher than in plasma (Cruickshank & Neil-Dwyer, 1985;

Westerlund, 1985). This is a high degree of bioconcentration, especially when compared to brain concentrations of the hydrophilic β-blocker atenolol, which has a brain: plasma ratio of 0.2 (Cruickshank & Neil-Dwyer, 1985). Similar accumulation can also be found in the brains of rabbits and monkeys that were administered propranolol (Srivastava & Kapoor, 1983). Thus it is possible that in our experiments the fish exposed to 1.0 and 10 mg propranolol/L bioaccumulated enough propranolol within the CNS to cause the acute toxicity that occurred.

When Huggett et al. (2002) exposed Japanese medaka to propranolol, egg production was found to be decreased at 0.0005 mg/L, suggesting that propranolol was acting as a reproductive toxin. The effects found by Huggett et al. (2002), although statistically significant when compared with the controls, did not however exhibit a dose-response relationship. The data from this study do not agree with those reported in Huggett et al. (2002), as egg production was only affected at 1.0 mg/L, a 2000-fold higher concentration than that which appeared to inhibit egg production in medaka.

Surprisingly, fathead minnows (LOEC

= 1.0 mg/L, LOEC

= 3.4 mg/L) were much more acutely sensitive to propranolol than medaka (LC

= 24.3 mg/L) (Huggett et al., 2002).

The reason for this inter-species difference is presently unclear. The data from this study show that propranolol concentrations in the aquatic environment (maximum values = 590 ng/L, Ternes, 1998) and would not be expected to cause reproductive effects.

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When pharmaceuticals are taken by patients, the most relevant concentration that describes the quantity of drug needed to initiate a physiological response is the human plasma concentration of the drug. This is because it is the plasma containing the drug which comes into contact with the target site. Since a certain plasma concentration of pharmaceutical is required to affect target receptors and enzymes in humans, it has been hypothesised that approximately the same

concentration would be required to affect another species sharing the same target (Brown et al., 2007). This assumption has been used to describe concentrations of pharmaceuticals in the environment that may induce effects in receiving biota. One such model, the ‘fish plasma model’

proposed by Huggett et al. (2003) compares estimated (from the partition coefficient) or actual drug concentrations in fish plasma with human therapeutic plasma concentrations, in order to assess whether it is likely that environmental or experimental concentrations of a drug would produce therapeutic levels in fish (Brown et al., 2007). The effect ratio (ER) is calculated as shown below:

ER = Human therapeutic plasma concentration (H

PC)/ Fish steady state plasma concentration (F

PC)

The lower the ER (i.e. ≤ 1), the greater the potential there is for a pharmacological response to occur in fish (Huggett et al., 2003).

Due to the different physical properties of pharmaceuticals, some drugs are more likely to reach human plasma therapeutic levels from waterborne exposure of aquatic organisms than others.

Propranolol has a log K

of 3.48, and since it is relatively lipophilic it is likely to bioaccumulate in fish from exposure via water. The plasma propranolol concentrations in fathead minnows showed a dose-related relationship, in that as the concentration of propranolol in the water increased,

propranolol plasma concentrations also increased (Table 5). The measured F

PC concentration in the 0.1 and 1.0 mg/L treatment groups exceeded the water concentration of propranolol, showing bioaccumulation of propranolol. These data also agree with data by Wong et al. (1979), who established that propranolol plasma concentration and daily dose were significantly correlated. By using the ‘fish plasma model’, the predicted F

PC, as calculated by using log K

(Table 5) are within 1 order of magnitude to the measured F

PC, and hence the model does quite well in predicting the plasma concentrations of propranolol.

The ER values of male and female fathead minnows exposed to 0.1 and 1.0 mg/L of propranolol were less than 1; hence, the measured F

PC in the fish maintained at these concentrations was 349

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greater than the drug concentration in human plasma which is needed to elicit a therapeutic effect.

This could explain why fish exposed to concentrations of 0.1mg/L of propranolol, and higher, showed statistically significant effects from the control in some endpoints, and why propranolol at 3.4 mg/L was so toxic. These data support the fish plasma model and show that the read-across of information from humans to fish is plausible in that existing human pharmaco and toxicodynamic data could be used as a starting point to predict effects on organisms in the environment (at least those sharing targets with humans).

In summary, these data do not suggest that propranolol is acting as a reproductive toxin and

therefore do not agree with the findings of Huggett et al., 2002. The environmental concentrations of propranolol are not greater, or equal to, 0.1mg/L, which was the lowest concentration at which effects were observed, and so adult fathead minnows are not considered to be at risk from

propranolol in the aquatic environment. However, these data clearly support the fish plasma model, as effects were seen in treatment groups where the internal plasma concentration of propranolol exceeded the human therapeutic plasma concentration required to treat patients for hypertension (Huggett et al., 2003; Wong et al., 1979).

Acknowledgements

The authors would like to thank the European Union for funding this work as part of the ERAPharm project, contract no. 511135 (Knacker et al., 2005), and to NERC for providing studentship funding to PDE

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