A comparison of the chronic effects of human pharmaceuticals on two cladocerans, Daphnia magna and Ceriodaphnia dubia

Chemosphere 80 (2010) 1069–1074

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A comparison of the chronic effects of human pharmaceuticals on two cladocerans, Daphnia magna and Ceriodaphnia dubia L.A. Constantine a,*, D.B. Huggett b a b

Pfizer, Environmental Sciences, Pharmacokinetics, Dynamics and Metabolism Pfizer Global Research and Development, Groton, CT 06423, USA Department of Biological Sciences, University of North Texas, Denton, TX 76203, USA

a r t i c l e

i n f o

Article history: Received 13 January 2010 Received in revised form 29 April 2010 Accepted 7 May 2010 Available online 14 June 2010 Keywords: C. dubia D. magna Pharmaceuticals Environment Risk assessment

a b s t r a c t Scientific researchers and regulators are focusing attention on trace quantities of pharmaceuticals in wastewater effluents and surface waters, resulting in an increased level of concern regarding the potential environmental impact of these compounds. The current European regulatory guideline requires evaluation of the chronic effects of active pharmaceutical ingredients on Daphnia magna. Based on the life cycle of D. magna, chronic studies to establish survival and reproductive endpoints require a 21 d exposure period. A similar organism, Ceriodaphnia dubia, has a shorter life cycle and therefore survival and reproductive endpoints may be established following 7 d of exposure. No observed effect concentrations and lowest observed effect concentrations for survival and reproduction were obtained for D. magna and C. dubia following exposure to six human pharmaceuticals and two metabolites (i.e. celecoxib, linezolid, varenicline, sunitinib, Compound A, ziprasidone and the M1 and M4 metabolites of torcetrapib). These data were evaluated to determine whether one organism may be considered more sensitive. Survival and reproduction data obtained from the C. dubia study provide similar outcomes to D. magna when determining the predicted environmental concentration/predicted no effect concentration (PEC/PNEC) ratios for surface water. Based on these data, C. dubia may be used as a cost-effective alternative and representative invertebrate species when assessing the potential risk of human pharmaceuticals. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Human pharmaceuticals are routinely detected in wastewater, surface water and aquatic biota (Daughton and Ternes, 1999; Kolpin et al., 2002; Brooks et al., 2005; Kwon et al., 2009). Based on the use pattern of most human pharmaceuticals, it is proposed that the exposure of aquatic biota to these compounds will be long term (Daughton and Ternes, 1999). While the amount of chronic data in the peer-reviewed literature is increasing, much of these data focus on responses of aquatic organisms following acute, short term exposure (Carlsson et al., 2006). Use of acute data within pharmaceutical risk assessment scenarios has been questioned and the more recent regulatory guideline in the European Union (EU) has adopted chronic testing strategies (Ferrari et al., 2004; EMEA, 2006). In particular, understanding the potential effects of xenobiotics on invertebrates is a key component to assessing environmental risk. Daphnia sp. are of great importance to the ecosystem, serving as a food source for amphibians, fish and other aquatic organisms. The current EU ‘‘Guideline on the Environmental Risk Assessment of Medicinal Products for Human Use” (EMEA, 2006), requires a 21-d reproduc* Corresponding author. Tel.: +1 860 715 2729; fax: +1 860 715 8612. E-mail address: lisa.a.constantine@pfizer.com (L.A. Constantine). 0045-6535/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2010.05.009

tive study in Daphnia magna as per the OECD 211 (1998) protocol. D. magna, a freshwater invertebrate, is easily cultured in the laboratory and commonly used to evaluate the potential toxicity of xenobiotics, including pharmaceuticals, to aquatic invertebrates. Ceriodaphnia dubia, a common freshwater invertebrate similar to the D. magna, has been used extensively for similar testing within the United States due to their taxonomic similarity to D. magna, their prevalence in North American waters, the shorter duration required for chronic reproductive toxicity tests and their sensitivity to xenobiotics (Stewart and Konetsky, 1998). Despite these attributes, Ceriodaphnia are not typically recognized or recommended as an invertebrate species within the EU regulatory arena. An evaluation of the distribution and ecology of Ceriodaphnia shows a widespread abundance of various members of this genus on the continents of Asia, Europe and North America (Versteeg et al., 1997). Based on the prevalence of Ceriodaphnia in these geographical regions, use of Ceriodaphnia as a representative, alternative invertebrate species to assess the environmental effects of pharmaceuticals should be considered. Acute and chronic Daphnia sp. data compiled for various organic and inorganic compounds and wastewater effluents demonstrate essentially similar toxicological sensitivities between C. dubia and D. magna, which is not surprising due to their similar taxonomy (Versteeg et al., 1997). Assuming similar taxonomy plays a significant role in the correla-

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tion observed between the chronic toxicity of D. magna and C. dubia for non-pharmaceutical compounds, one would expect similar correlations to be observed when testing pharmaceuticals under the same chronic protocols. The purpose of this study was to evaluate the relative responses of D. magna and C. dubia chronically exposed to six human pharmaceuticals; celecoxib, linezolid, varenicline, sunitinib, Compound A, ziprasidone and the M1 and M4 metabolites of torcetrapib, to determine if one organism is more sensitive or provided NOEC values that significantly altered the outcome of the risk analysis (i.e. PEC/PNEC analysis) associated with the substances tested. 2. Materials and methods 2.1. Pharmaceutical substances The purity of each of the pharmaceutical substances tested was determined to be >98%. Stock concentrations of each test substance were mixed in purified reagent water, dimethylformamide (DMF), or acetone and then diluted in exposure water such that the final concentration of co-solvent in the exposure vessels was <0.1%. The test substances evaluated in this study are listed below. Chemical properties are presented in Table 1.  Celecoxib (CAS# 169590-42-5), a selective inhibitor of the inducible form of the enzyme cyclooxygenase (COX-2), approved for symptomatic relief of osteoarthritis, rheumatoid arthritis and ankylosing spondylitis.  Linezolid (CAS# 165800-03-3), the first of a new class of antibiotics, the oxazolidinones, approved for the treatment of Grampositive bacterial infections, including those caused by resistant organisms.  Varenicline (CAS# 375815-87-5), a partial agonist with high affinity and high selectively for a4b2 nicotinic acetylcholine receptors, approved for smoking cessation treatment.  M1 metabolite (3,5-bis trifluoromethyl benzolic acid, CAS# 725-89-3) and M4 metabolite (2-quinolinecarboxylic acid, 6trifluoromethyl, CAS# 849818-58-2); human metabolites of torcetrapib. Torcetrapib inhibits cholesteryl ester transfer protein (CETP) transfer of cholesterol between high-density (HDL), non-high-density lipoproteins, low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL). The CETP pharmacological activity of the M1 and M4 metabolites (IC50 = 300 000 nM) is orders of magnitude lower than the parent compound torcetrapib (IC50 = 59 nM).  Sunitinib (CAS# 341031-54-7), inhibits receptor tyrosine kinases (RTK) involved in tumor proliferation and angiogenesis, specifically the platelet derived growth factor receptor (PDGFR), the vascular endothelial growth factor receptor (VEGFR), the

stem cell factor receptor (KIT), the Fms-like tyrosine kinase-3 receptor (FLT-3), and the tyrosine kinase receptor encoded by the ret proto-oncogene (RET). Approved for the treatment of advanced kidney cancer and gastrointestinal stromal tumors.  Compound A is currently under development and the mechanism of action is proprietary.  Ziprasidone (CAS# 138982-67-9), is a combined dopamine (D2) and serotonin (5HT2) receptor antagonist approved for the treatment of schizophrenia and acute bipolar mania. 2.2. Organisms and water All studies conducted to assess reproductive, survival and growth effects of the eight pharmaceutical test substances were initiated with organisms <24 h old. Organisms were obtained from breeding cultures at Springborn Smithers Laboratories in Wareham, MA. Test conditions, water quality measurements, and acceptability criteria were evaluated and determined to meet requirements as outlined in the OECD 211 and EPA 1002.0 methods, respectively. Water used in culturing and pharmaceutical exposures was groundwater with the following water quality characteristics: pH = 7.6–8.8; dissolved oxygen = 6.8–9.5 mg L 1; alkalinity = 56– 187; hardness = 80–80; Specific conductance = 290–600. Fluctuations in groundwater characteristics may be attributed to seasonal variations. 2.3. D. magna chronic studies The D. magna studies were conducted as per OECD Guideline for the Testing of Chemicals, D. magna Reproduction Test, Guideline 211 (1998). The purpose of the test was to assess the effect of substances on reproductive output of the water flea, D. magna, following a 21-d exposure under static-renewal conditions. The reproductive output of the daphnids was measured and a statistically determined no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) of the test organisms versus the control organisms were determined based on the number of offspring produced. The NOEC and LOEC, for the endpoints of survival (immobilization) and growth (total body length) were determined. When possible, a statistically estimated median effective concentration (EC50) for survival was determined. D. magna studies were conducted by exposing young female daphnia (<24 h of age) to the test substance in water at a range of concentrations over a 21-d period. At the end of the test, the total number of offspring produced at each exposure concentration and the controls was determined. The effect of the test substance on survival, growth and reproductive output was determined by comparing the survival, growth and total number of daphnids pro-

Table 1 Compound properties. Compound Celecoxib Linezolid Varenicline Metabolite 1 Metabolite 4 Sunitinib

a b

Mechanism of action COX-2 inhibitor Antibacterial agent Nicotine agonist Parent compound, cholesteryl ester transfer protein (CETP) inhibitor

Compound A

Inhibits receptor tyrosine kinases (RTK) involved in tumor proliferation and angiogenesis Proprietary

Ziprasidone

Dopamine (D2) and serotonin (5HT2) receptor antagonist

Molecular weight

Log DpH

Water solubility (mg L 1)

pKa

7.4

381.38 337.35 361.35 258.08 241.11 532.57

3.53 0.55 0.817 1.25a 0.156 2.7

5 3200 132 000 11 000b 130 000b 50

11.1 1.8 9.2 3.34b 4.21b 8.95

–

1.62

800

467.42

2.12

75

5.33 8.5 6.68

Calculated. Advanced Chemistry Development, Inc., (ACD/Labs) – suite of predictors for physical chemical properties.

7–

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duced at each exposure concentration to that of the control group. Exposures were conducted using static renewals, three times per week with feeding following each renewal. Weekly exposure solution samples were collected before and after renewals and analyzed to confirm exposure concentrations of the pharmaceutical substance. For D. magna tests to be considered valid, the following performance criteria for the controls, as defined in OECD 211, were met:  the mortality of the parental daphnids did not exceed 20% at the end of the test;  the mean number of live offspring produced per parental daphnid surviving at the end of the test was P60. 2.4. C. dubia chronic studies The C. dubia studies were conducted based on US EPA, Methods for estimating the chronic toxicity of effluent and receiving waters to Freshwater Organisms, Test Method 1002.0 (US EPA, 1994). The purpose of the test was to assess the effect of test substances on reproductive output of the water flea, C. dubia, following a 7-d exposure under static-renewal conditions. The reproductive output of the daphnids was measured and a statistically determined no observed effect concentration (NOEC) of the test organisms versus the control organisms was determined based on the number of offspring produced. The lowest observed effect concentration (LOEC), and when possible, a statistically estimated median effective concentration (EC50), were determined for the endpoints of reproduction (number of offspring produced) and survival (immobilization). The C. dubia studies were conducted similarly to the D. magna studies, however the study duration was 7 d with daily renewals of exposure solutions. Exposure solution samples were collected before and after renewals and analyzed to confirm exposure concentrations of the test substance. For C. dubia tests to be considered valid, the following performance criteria, as defined in EPA 1002.0, were met:  the test organisms came from the same source;  the test was initiated with organisms that were <24 h old and these organisms were collected within an 8 h period;  P80 survival of the control organisms at test termination;  an average of P15 young per surviving female was produced in the controls at test termination;  P60% of surviving females in controls produced their third brood. 2.5. Analytical methods Compound specific analytical conditions are outlined in Supplementary Table S1. Unless otherwise noted, samples were analyzed using a Hewlett-Packard solvent pump 1050 or quaternary pump series 1100 equipped with a Hewlett-Packard Series 1050 or 1100 autosampler, vacuum degasser, a column heater, a variable wavelength UV/Vis detector and Hewlett-Packard ChemStation Version A.04.01, A.06.03 or A.09.01, for data acquisition (Hewlett-Packard, Inc.).

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consideration human metabolism or potential degradation and/or removal during wastewater treatment. The PEC values for the M1 and M4 metabolites of torceptrapib were determined based on the PEC value for torcetrapib with adjustments made to account for 6% excretion as M1 and 29% excretion as M4, as determined in human metabolism studies. The predicted no effect concentration (PNEC) for each test compound was calculated using the NOEC value obtained for reproduction, with the exception of Sunitinib and Compound A (NOEC based on growth), divided by the standard assessment factor of 10. 2.7. Statistical analysis TOXSTATÒ version 3.5 (West and Gulley, 1996) was used to perform the statistical calculations to determine the NOEC and the LOEC for each test compound. The highest test concentration that elicited no statistically significant difference between the exposed organisms and the control was estimated to be the NOEC. The lowest test concentration that elicited a statistically significant difference between the exposed organisms and the control was estimated to be the LOEC. C. dubia survival data were evaluated using Fisher’s Exact Test. The reproduction data were tested for normality (Shapiro-Wilk’s Test or Chi-Square Test) and homogeneity of variance (Bartlett’s Test). If the data passed these two tests, the Williams’ Test was used to establish treatment level effects. If the data failed the test for normality and homogeneity of variance, the Dunn’s or Steel’s Many-One Rank Test (Sokal and Rohlf, 1981; Weber et al., 1989) was used. All statistical analyses were conducted at the 95% level of certainty except in the case of the Chi-Square Test and Bartlett’s Test, in which the 99% level of certainty was applied. For D. magna, the number of surviving daphnids in the control was compared to the number of surviving daphnids in each mean measured concentration using Fisher’s Exact Test (Weber et al., 1989). A t-test was conducted for reproduction and length to compare the performance of the control organisms with that of the solvent control organisms, as appropriate. If a statistically significant difference was detected between the two controls, only the solvent control was used in the statistical determination of the treatment effects. If no statistically significant difference was detected, the data from both controls were pooled and the pooled data were used to analyze for treatment effects. Chi-Square Test for normality (Weber et al., 1989) was used to compare the observed sample distribution with a normal distribution for all endpoints. If the data were not normally distributed, then a non-parametric procedure was used for subsequent analyses. As a check on the assumption of homogeneity of variance, data for each endpoint were analyzed using Bartlett’s Test (Sokal and Rohlf, 1981). If reproduction and growth data did not meet assumptions for homogeneity of variance, Wilcoxon’s Rank Sum Test (Weber et al., 1989) was used to determine reproduction effects, and Kruskal–Wallis’ Test was used to determine growth effects (Weber et al., 1989). The statistical analyses used to evaluate the correlation between the D. magna and C. dubia data presented were conducted using LabStats Excel, V5.R4.M0.

2.6. PEC/PNEC analysis

3. Results and discussion

An estimation of the predicted environmental concentration (PEC) for each test compound was calculated as described in the EU ‘‘Guideline on the Environmental Risk Assessment of Medicinal Products for Human Use” (EMEA, 2006). The PEC values used were considered a reasonable estimate of a worst-case environmental concentration resulting from patient use, as they did not take into

Good Laboratory Practices (GLP) were followed for all experiments, data generation and reporting. Mean measured aqueous test concentrations were P80% of nominal concentrations except for ziprasidone D. magna, which was 69%, (Table 2). Mean measured concentrations were used to calculate the EC50, LOEC and NOEC values.

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Table 2 Test concentrations. Compound

C. dubia

D. magna

Nominal test concentrations Celecoxib Linezolid Varenicline Metabolite 1 Metabolite 4 Sunitinib Compound A Ziprasidone

0.1–100% WAF of 8 mg L 0.94–30 mg L 1 0.31–10 lg L 1 6.3–100 mg L 1 4.4–70 mg L 1 0.10–3.2 mg L 1 0.016–1.0 mg L 1 0.25–32 lg L 1

1

std

% Mean measured

Nominal test concentrations

% Mean measured

0.0021–1.6 mg/La 100 108 99 100 81 99 80

0.026–1.0 mg L 1 6.3–100 mg L 1 2.5–80 lg L 1 6.3–100 mg L 1 4.4–70 mg L 1 0.031–1.0 mg L 1 0.0031–0.050 mg L 1.0–32 lg L 1

91 93 100 96 100 84 104 69

1

WAF – water accommodated fraction. a Mean measured of water accommodated fraction. % Mean measured not applicable.

The comparison of the survival NOEC values, D. magna versus C. dubia for each compound, varied by 64, with the exception of Celecoxib and Compound A (Table 3). D. magna was approximately 25 times more sensitive based on the NOEC values for celecoxib. Since the Compound A studies were conducted over different test concentration ranges, with no effects on survival at the highest concentrations, a comparison of the two species based on survival may not be directly comparable. For this reason, Compound A was not included in the statistical analysis. Definitive EC50 values could not be determined within many of these tests due to solubility limits or the lack of effects on 50% of the population at the maximum concentration tested. The comparison of the reproduction/growth NOEC and definitive LOEC values, D. magna versus C. dubia for each compound, varied by 65x (Table 3). Observations indicate the NOEC values for reproduction/growth are equal to or less than the NOEC values for survival in all studies conducted. Therefore, as the most sensitive endpoints, reproduction/growth may be considered most relevant for assessing the correlation between C. dubia and D. magna chronic data. Of particular interest is the nicotinic receptor agonist varenicline, which resulted in a reproductive/growth NOEC in the low lg L 1 range. Similar effects were observed following a 21 d exposure of D. magna to the organophosphate, paraoxon-methyl, a cholinesterase-inhibiting pesticide. Based on the number of neonates, the NOEC for reproduction was determined to be 0.3 lg L 1. (Duquesne et al., 2006). Whether these responses are associated with receptor activation is unknown, however an ortholog to the human nicotinic receptor has been detected in Daphnia sp. (Gunnarsson et al., 2008). D. magna and C. dubia data (Table 3) were converted to log (base 2) values and plotted against each other (Figs. 1 and 2). The probability of a false positive error (p-value) as derived from the paired

t-tests of the log NOEC values for D. magna and C. dubia for survival (n = 7) and reproduction/growth (n = 8) for the pharmaceutical compounds tested were 0.15 and 0.30, respectively. To further assess the robustness of the t-test results, a non-parametric Wilcoxon signed rank test was conducted and the p-values were 0.69 and 0.22 for the survival and reproduction/growth NOEC, respectively. These p-values reflect the error associated with rejecting the hypothesis that there is no difference between these chronic endpoints in D. magna versus C. dubia, when the hypothesis is true. Since the p-values are both >0.05, it may be concluded that there are no statistically significant differences in the chronic survival and reproduction/growth effects between D. magna and C. dubia for the substances tested. In the risk analysis frameworks utilized by FDA and EMEA, the toxicity data are adjusted by a safety factor that takes into account cross-species extrapolation (FDA, 1998; EMEA, 2006). As more data become available across multiple trophic levels, the degree of uncertainty in the extrapolation becomes lower and the safety factor is reduced. Based on the EMEA (2006) guidance, a safety factor of 10 is applied if chronic data from three species (fish, invertebrate, algae) are available. Since effects associated with the most sensitive endpoint of reproduction/growth differ by less than 10, which is the safety factor applied when calculating the PNEC value, one would assume the outcome of the risk analyses (PEC/ PNECD.magna versus PEC/PNECC.dubia ratios) of these eight compounds would be similar (Table 4). To confirm this assumption, the log PEC/PNEC values were plotted against each other (Fig. 3) and a paired t-test was conducted to compare the PEC/PNECD.magna and PEC/PNECC.dubia ratios. This test provided a p-value of 0.83. To further assess the robustness of the t-test result, a non-parametric Wilcoxon signed rank test was conducted and the p-value was determined to be 0.69. Since both of the p-values are >0.05, it

Table 3 Survival (NOEC and EC50) and reproduction (NOEC and LOEC) values for C. dubia and D. magna exposed to eight pharmaceuticals. Compound

Survival EC50 (mg L

Celecoxib Linezolid Varenicline Metabolite 1 Metabolite 4 Sunitinib Compound A Ziprasidone

Reproduction 1

)

NOEC (mg L

1

)

LOEC (mg L

1

)

NOEC (mg L

C. dubia

D. magna

C. dubia

D. magna

C. dubia

D. magna

>1.6 >31 >0.010 >100 >73 1.0 >1.1 >0.021

>0.89 >94 0.065 >95 >69 0.48 >0.052 >0.019

1.6 31 0.010 100 73 0.66 1.1 0.021

0.062 94 0.040 95 69 0.40 0.052 0.019

0.48 >31 0.005 >100 36 0.66 0.51 >0.021

0.14 47 0.020 >95 35 0.40 >0.052 0.019

EC50 = effect concentration in 50% of organisms tested. NOEC = no observed effect concentration. LOEC = lowest observed effect concentration. a Based on growth and/or reproduction.

a

1

)

C. dubia

D. magnaa

0.17 31 0.0027 100 18 0.32 0.26 0.021

0.062 24 0.010 95 18 0.21 0.052 0.011

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Log NOEC - Survival 10

Daphnia magna

5

0 -10

-5

0

5

10

-5

-10 Ceriodaphnia dubia

Log (base 2) 95% confidence for the mean difference -3.65 < 0.89 < 0.71 p value = 0.15, C. dubia is not significantly different from D. magna at 10% level in the 2-sided t-test for paired data p value = 0.69, C. dubia is not significantly different from D. magna at 10% level in the 2-sided Wilcoxon signed rank test for paired data Fig. 1. Log (base 2) plot of survival NOEC values For D. magna versus C. dubia following exposure to seven pharmaceuticals.

Log NOEC Reproduction

Daphnia magna

10

5

0 -10

-5

0

5

10

-5

-10 Ceriodaphnia dubia

Log (base 2) 95% confidence for the mean difference -1.51 < 0.43 < 0.54 p value = 0.30, C. dubia is not significantly different from D. magna at 10% level in the 2-sided t-test for paired data p value = 0.22, C. dubia is not significantly different from D. magna at 10% level in the 2-sided Wilcoxon signed rank test for paired data Fig. 2. Log (base 2) plot of reproduction NOEC values For D. magna versus C. dubia following exposure to eight pharmaceuticals.

Table 4 PEC/PNEC values for C. dubia and D. magna. Compound

Celecoxib Linezolid Varenicline Metabolite 1 Metabolite 4 Sunitinib Compound A Ziprasidone

PEC/PNEC C. dubia

D. magna

0.059 0.002 0.033 0.0000018 0.00005 0.012 0.012 0.381

0.161 0.003 0.010 0.0000019 0.00005 0.018 0.058 0.727

may be concluded that there is no statistically significant difference between the risk analysis based on the PEC/PNECD.magna and the risk analysis based on the PEC/PNECC.dubia for the eight pharmaceutical substances evaluated. Within the scope of the studies conducted, irrespective of the mechanism of action associated with the eight substances tested, statistical evaluation of the data suggests that C. dubia may be considered a suitable surrogate for D. magna when assessing the chronic endpoints of reproduction/growth and survival. This outcome agrees with the data and conclusions published on non-pharmaceutical compounds (Versteeg et al., 1997). In the context of developing and applying chronic daphnia data, the pharmaceuti-

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Log PEC/PNEC Values 0 -15

-10

-5

Daphnia magna

-20

0 -5

-10

-15

-20

Ceriodaphnia dubia

Log (base 2) 95% confidence for the mean difference -1.04 < 0.48 < 1.25 p value = 0.83 PEC / PNEC is not significantly different from PEC / PNEC at 10% level in this 2-sided t-test for C. dubia D. magna paired data p value = 0.69, PEC / PNEC is not significantly different from PEC / PNEC at 10% level in the 2-sided Wilcoxon C. dubia D. magna signed rank test for paired data Fig. 3. Log (base 2) plot of PEC/PNEC values for D. magna versus C. dubia.

cals tested behaved similarly to other classes of compounds, providing no evidence to suggest that pharmaceuticals should be considered a distinct class of compounds in this regard. Use of C. dubia as a surrogate for D. magna provides comparable data within onethird the experimental time frame (7-d versus 21-d), at approximately one-third the cost, using significantly less test material. The widespread abundance of Ceriodaphnia on the continents of North America, Asia and Europe, further supports the use of C. dubia as an alternative and representative invertebrate species when assessing the potential risk of human pharmaceuticals to the environment. Acknowledgement Shibing Deng, Pfizer Global Research and Development, Groton, CT 06423, USA, for assistance with statistical evaluation and interpretation. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chemosphere.2010.05.009. References Brooks, B.W., Chambliss, C.K., Stanley, J.K., Ramirez, A., Banks, K.E., Johnson, R.D., Lewis, R.J., 2005. Determination of select antidepressants in fish from an effluent-dominated stream. Environ. Toxicol. Chem. 24 (2), 464–469. Carlsson, C., Johansson, A.-K., Alvan, G., Bergman, K., Kühler, T., 2006. Are pharmaceuticals potent environmental pollutants? Part I: environmental risk assessments of selected active pharmaceutical ingredients. Sci. Total Environ. 364 (1–3), 67–87. Daughton, C.G., Ternes, T.A., 1999. Pharmaceuticals and personal care products in the environment: agents of subtle change? Environ. Health Perspect. 107 (6), 907–938.

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