Acute toxicity of widely used pharmaceuticals in aquatic species: Gambusia holbrooki, Artemia parthenogenetica and Tetraselmis chuii

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Ecotoxicology and Environmental Safety 61 (2005) 413–419 www.elsevier.com/locate/ecoenv

Acute toxicity of widely used pharmaceuticals in aquatic species: Gambusia holbrooki, Artemia parthenogenetica and Tetraselmis chuii B. Nunesa,b,, F. Carvalhoc, L. Guilherminoa,b a

ICBAS, Instituto de Cieˆncias Biome´dicas Abel Salazar, Departamento de Estudos de Populac- o˜es, Laborato´rio de Ecotoxicologia, Universidade do Porto, Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal b CIIMAR, Centro Interdisciplinar de Investigac- a˜o Marinha e Ambiental, Laborato´rio de Ecotoxicologia, Rua dos Bragas, 289, 4050-123 Porto, Portugal c REQUIMTE, Servic- o de Toxicologia da Faculdade de Farma´cia da Universidade do Porto, Rua Anı´bal Cunha, 164, 4050-047 Porto, Portugal Received 3 November 2003; received in revised form 18 August 2004; accepted 25 August 2004 Available online 5 November 2004

Abstract Pharmaceuticals are continuously dispersed into the environment as a result of human and veterinary use, posing relevant environmental concerns. This study evaluated the acute toxicity of three therapeutic agents (diazepam, clofibrate, and clofibric acid) and a detergent (sodium dodecyl sulfate; SDS) in three aquatic species, namely the euryhaline fish Gambusia holbrooki, the hypersaline crustacean Artemia parthenogenetica, and the marine algae Tetraselmis chuii. The ranking of 50% lethal concentrations (LC50) for the two animal species and 50% inhibitory concentration (IC50) for the algal species was, in decreasing order, clofibric acid4SDS4diazepam4clofibrate for G. holbrooki, clofibric acid4clofibrate4SDS4diazepam for A. parthenogenetica, and clofibric acid4clofibrate4SDS4diazepam for T. chuii. These differences show that the intrinsic nature of test organisms must be considered when evaluating the toxicity of these agents to aquatic ecosystems. r 2004 Elsevier Inc. All rights reserved. Keywords: Gambusia; Artemia; Tetraselmis; Pharmaceuticals; Detergent; LC50; Acute toxic effects

1. Introduction Therapeutic agents are a major class of chemical compounds, characterized by indiscriminate and continuous use and biological activity (Jones et al., 2002; Miao et al., 2002; Daughton and Ternes, 1999; HallingSørensen et al., 1998). In addition, some compounds exhibit resistance to metabolic degradation and lipophilicity, which results in their continued presence in the environment after therapeutic use. Pharmaceuticals can also cause synergistic toxic effects when in the presence of other compounds (Cleuvers, 2003). These intrinsic Corresponding author. Laborato´rio de Ecotoxocologia, Centro Interdisciplinar de Investigac- a˜o Marinha e Ambiental (CIIMAR), Rua dos Bragas, 289, 4050-123 Porto, Portugal. Fax: +351 22 3390608. E-mail address: [email protected] (B. Nunes).

0147-6513/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.08.010

features are the main reasons for considering pharmaceuticals potentially harmful, effective, and environmentally unfriendly compounds. High contamination values are described for several classes of therapeutic agents (Ku¨mmerer, 2001), which can consequently lead to acute effects over organisms. Recent decades have advanced our knowledge and awareness of issues related to environmental exposure to anthropogenic xenobiotics, with concomitant exertion of a variety of effects on biotic systems. Long-term consequences and implications for human populations are also matters of concern when considering the global circulation of man-made chemicals. Assessment of effects over the aquatic ecosystems is a major task, as these systems are the most impacted sites after the continuous dumping of heavy loads of environmental contaminants. A large number of bioindicators and test organisms (mainly

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freshwater species) have been proposed in the past for the evaluation of ecotoxicity of anthropogenic compounds on aquatic environments. However, only scarce efforts were made for the proposal of seawater species as test organisms, and this may be extremely important for the assessment of environmental impact in particular aquatic systems, such as estuaries and ponds with high salt concentrations. The present study intended to calculate LC50 and IC50 values of three therapeutic agents (diazepam, clofibrate, and a metabolite of the latter, clofibric acid) and a widespread detergent (sodium dodecyl sulfate; SDS), on three species with different salinity preferences and from distinct trophic levels. We selected the unicellular marine algal species Tetraselmis chuii, the hypersaline crustacean Artemia parthenogenetica, and the euryhaline fish Gambusia holbrooki. T. chuii is characterized by high mobility; species of Tetraselmis are commonly used for aquaculture purposes and are considered to be a nutrient source suitable for crustaceans namely for several species of Artemia (Fa´bregas et al., 1996). Due to their role as primary producers in aquatic food webs, these species have a high ecological relevance, being thus of great scientific importance. A. parthenogenetica is one of the main components of hypersaline ecosystems and has long been considered a suitable test organism for high-salinity environments (Vanhaecke et al., 1981). Its natural characteristics (high prolificacy, short life cycle, and the possibility to be maintained under laboratory conditions) make A. parthenogenetica an ideal animal model for toxicity testing. G. holbrooki is a worldwidedistributed Poecilidea under temperate climate conditions. Its natural abundance, simplicity of capture, and ease of laboratory rearing make G. holbrooki a species suitable for tests under varying salinity conditions. Furthermore, this species is considered representative of the secondary consumers of the habitats into which they were introduced. Diazepam was selected due to its frequent use in human therapeutics. Diazepam is a benzodiazepine and acts on the central nervous system, exerting anxiolytic, sedative, and muscle relaxant effects. These pharmacological activities are a consequence of the enhancement of GABAergic transmission at benzodiazepine-sensitive GABAA receptors (Mohler et al., 1996). The presence of diazepam has been reported in concentrations up to 0.04 mg/L in effluents from a large number of German sewage treatment plants (Ternes, 1998). The presence of diazepam in the sewage system was a result of its exclusive human use. Clofibrate, despite its withdrawal from human therapeutics in most countries of Western Europe, is still thoroughly used in many other countries as a blood lipid regulator. Clofibrate exerts its pharmacological activity through activation of the nuclear peroxisome-proliferator-activated receptors (PPARs). Activation of PPARs

is common to a group of compounds generally designated fibrates. Clofibrate has been detected in effluents from sewage treatment plants in concentrations up to 0.8 mg/L (Andreozzi et al., 2003). Clofibric acid is the pharmacologically active derivative of clofibrate and several other fibrates, with an estimated persistence of 21 years in the environment (Buser et al., 1998). During passage through a sewage treatment plant, a loss of only 50% of the initial amount of clofibric acid was detected (Ternes, 1998). Due to these properties, clofibric acid has been found at concentrations of 270 ng/L in tap water (Heberer, 2002), 0.55 mg/L in surface waters of Swiss lakes (Buser et al., 1998), 1.6 mg/L in the majority of German sewage treatment plants (Ternes, 1998), 103 ng/L in Detroit River water (Boyd et al., 2003), 5 ng/L in effluents of Greek sewage treatment plants (Koutsouba et al., 2003), 18 ng/L in the estuary of the River Elba, and 0.28–1.35 ng/L in North Sea water (Weigel et al., 2002). Sodium dodecyl sulfate is a widely employed detergent, with applications in household products, industrial mixtures, and cleansing products in cosmetics. SDS is considered one of the world’s most commonly used synthetic surfactants, since it is found in liquid soaps and shampoos, bubble baths, bath and shower gels, and tooth pastes (Sirisattha et al., 2004). Its continuous input into the environment can be responsible for high concentrations of this compound in specific areas, particularly in human highly impacted sites by humans.

2. Material and methods 2.1. Test substances Clofibrate (hypolipidemic drug, CAS No. 637-07-0, purity 498%), clofibric acid (hypolipidemic drug, CAS No. 882-09-7, purity 497%) and diazepam (anxyolytic drug, CAS No. 439-14-5, purity 498%) were purchased from Sigma–Aldrich Chemical Co. SDS (detergent, CAS No. 151-21-3, purity 490%) was purchased from Merck–Schuchardt. 2.2. Algal growth inhibition test The green unicellular marine algae T. chuii was kindly provided by Dr. I. Varo´, from the Instituto de Acuicultura Torre de la Sal (IATS), CSIC, Castello´n, Spain. This species has been routinely cultivated in our laboratory under standardized abiotic conditions, such as medium composition (filtered Atlantic full-strength seawater with sodium chloride in a concentration of 33 g/L) supplemented with Agrocross Nutrileaf at 400 ml/L, temperature of 2571 1C, continuous aeration,

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and continuous cool-white fluorescent light (100 mE/m2/ s). All glass apparatuses, mediums, supplements, and air were previously sterilized. Initial cultures were cultivated in solid medium, to obtain bacteria-free algal suspensions. Solid medium was obtained after autoclave sterilization of agar-added seawater medium (proportion 1.5% m/v). Solid medium was aseptically transferred to transparent glass Petri dishes and submitted to continuous incident light and temperature of 2571 1C. After varying periods (1–2 weeks of light exposure), algal cultures were inoculated in liquid sterilized medium to obtain exponentially growing cultures. Incubation apparatuses were composed of 5000-mL glass Erlenmeyer flasks with perforated rubber stoppers, with aeration systems; air was sterilized with nitrocellulose filters (0.2 mm mean pore diameter). After growth to exponential phase, cultures were kept at 4 1C in the dark. The numbers of algal cells were quantified in the obtained suspensions using a hemocytometer. The following procedures, for determination of inhibitory concentration 50% values (IC50), were adapted from the guideline ‘‘Alga, Growth Inhibition Test’’ (OECD, 1984a). Mediums, glassware, supplements, and air were treated as described above. Algal tests were performed in incubation apparatuses composed of 500-mL glass Erlenmeyer flasks with perforated rubber stoppers. The perforations allowed insertion, in each flask, of one glass tube for aeration and two silicone tubes for air purge and withdrawal of samples. Several modifications were applied to the mentioned guideline, and these included a longer testing period (96 h exposure) and daily monitoring of parameters such as pH and temperature. The testing volume was 400 mL. For each compound, three replicates per concentration were used. One control was added per test, and an additional control was included when stock solutions were prepared using ethanol. Stock solutions of the tested substances were made with ultrapure water (in the cases of SDS, clofibric acid, and diazepam) and ethanol (in the case of clofibrate). The nominal concentrations of toxicants tested were 18.75, 37.5, 75, 150, and 300 mg/L for clofibrate, 200.9, 241.1, 289.4, 347.2, 416.7, and 500 mg/L for clofibric acid, 7.9, 11.85, 17.78, 26.67, and 40 mg/L for diazepam, and 9.375, 18.75, 37.5, 75, and 150 mg/L for SDS. Sampling was performed at 24 h intervals. Samples were withdrawn from the closed apparatuses under positive pressure, which prevented contamination. The algal concentrations were determined with a hemocytometer. Lugol solution was added to the samples (proportion 1:5) to prevent the natural movement of T. chuii cells. The obtained values were plotted against time (growth curves established a relationship of number of cells vs. time). Further procedures involved the calculation of corresponding areas under curve for each growth curve, according to the guideline 201 from

415

OECD (1984a): N1  N0 N 1 þ N 2  2N 0  t1 þ  ðt2  t1 Þ 2 2 N n1 þ N n2 N 0  ðtn  tn1 Þ; þ 2 where A is area, N0 is nominal number of cells/mL at time t0, N1 is measured number of cells/mL at t1, Nn is measured number of cells/mL at time tn, t1 is time of first measurement after beginning of test, and tn is time of nth measurement after beginning of test. The percentage inhibition of the cell growth at each test substance concentration (IA) is calculated as the difference between the area under the control growth curve (Ac) and the area under the growth curve at each test substance concentration (At) as A¼

IA ¼

Ac  At  100: Ac

The calculated values of percentage of inhibition were used to calculate the inhibitory concentration 50 (IC50) using the probit method (Finney, 1971). 2.3. Crustacean 48-h test Parental parthenogenic cultures of A. parthenogenetica were maintained under standardized conditions of nutrients, temperature, photoperiod, medium changes and composition, aeration, and organic supplements. Photoperiod was 16 h L/8 h D, while temperature was kept at 2071 1C. Medium was composed of full strength Atlantic seawater, with a salinity of 3072 g/L. Nutrient supplies were composed of suspensions of T. chuii, with a final algal concentration of 6.37  105 cells/mL. An organic supplement was added to cultured animals (seaweed extract, Baird et al., 1989). The medium was enriched with a vitamin solution (thiamine, cianocobalamine, and biotin, in final concentrations of 1  105, 5  108, and 5  108 mg/L, respectively). Animals were cultured in 1000-mL recipients, in groups of 25–30 specimens per 800 mL of medium. Medium was changed three times per week. A clone of parthenogenic mature females allowed the collection of nauplii (less than 24 h), which were used in toxicity testing. Nauplii were isolated with glass Pasteur pipettes from the stock cultures and then immersed in the testing solutions. Tests were performed in 250-L glass flasks covered with Parafilm and filled to a volume of 100 mL of testing solution. The medium was fullstrength Atlantic seawater without nutrient supplies or organic supplements, with 10 specimens per replicate. No nutrients were added during the exposure period. Three replicates were used per concentration. Abiotic conditions were similar to those described above: seawater salinity was adjusted to 3072 g/L, photoperiod was 16 h L/8 h D, and temperature was kept at

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2071 1C, with continuous aeration. All assays included one control, and a supplementary control was added whenever a solvent was used to prepare the toxicant stock solution. LC50 values were calculated at 48 h using a total number of 20 specimens per concentration of tested compound. Test concentrations were 10, 20, 40, 80, 160, 320, and 640 mg/L for clofibrate, 10, 18, 32.4, 58.32, 104.98, 188.96, 340.12, and 612.22 mg/L for clofibric acid, 6.25, 12.5, 25, 50, and 100 mg/L for diazepam, and 3.98, 5.58, 7.81, 10.93, 15.31, and 21.43 mg/L for SDS. Values of pH, temperature, and percentage of dissolved oxygen were measured every 24 h, for test validation purposes. Mortality was adopted as the end point and considered to happen if immobilization continued for 10 s after gentle shaking. LC50 values were determined using the probit method (Finney, 1971). 2.4. Fish 96-h test G. holbrooki specimens were captured in the estuary of the Minho river (northern Portugal), at mean salinity of 6 g/L. This river is characterized by the absence of large sources of anthropogenic pollution (Ferreira et al., 2003). Fish were captured using hand nets and immediately transported to the laboratory facilities, in which they were kept in ASTM hard-water medium (ASTM, 1980) supplemented with 6 g/L sodium chloride. Abiotic conditions were photoperiod of 16 h L/8 h D, temperature of 2071 1C, and continuous aeration. Animals were fed with commercially available fish food (Sera Vipam flakes) and were kept in 70-L glass tanks. Both males and females, between 2 and 2.5 cm long, were used. Tests were performed accordingly to the

guideline Test No. 203: Fish, Acute Toxicity Test (OECD, 1984b), with modifications. Specimens were individually exposed in plastic bottles, previously used for human consumption of water, rinsed several times with deionized water. ASTM medium was used in volumes of 250 mL per replicate, equally supplemented with 6 g/L sodium chloride. Stock solutions of the tested compounds were prepared, using ultrapure water for SDS, clofibric acid, and diazepam. Clofibrate stock solution was prepared with ethanol. All assays included one control, with the addition of a supplementary control whenever a solvent was used to obtain the stock solutions. Seven replicates per concentration of compound were used. Test concentrations employed were 0.938, 1.875, 3.75, 7.5, and 15 mg/L for clofibrate, 362, 434, 521, 625, and 750 mg/L for clofibric acid, 5.21, 7.29, 10.2, 14.28, and 20 mg/L for diazepam, and 3.72, 5.21, 7.29, 10.2, 14.28, and 20 mg/L for SDS. The test period was 96 h; medium was changed after the first 48 h of exposure. Measurements of pH, temperature, and concentration of dissolved oxygen were performed every 24 h, for test validation purposes. Mortality was adopted as the end point and checked every 24 h. LC50 values were determined using the probit method (Finney, 1971). Summarized procedures, and additional information with regard to the performed tests are listed in Table 1.

3. Results LC50 and IC50 values of clofibrate, clofibric acid, diazepam, and SDS for the three species tested are summarized in Table 2. Diazepam was shown to be the more toxic compound for T. chuii, followed by SDS,

Table 1 Summarized methodology, endpoints, and guidelines followed in tests Test

Species

Exposure time (h)

Assessment end point

Standard guideline

Algae Crustacean Fish

T. chuii A. parthenogenetica G. holbrooki

96 48 96

Growth inhibition (IC50) (a) Mortality (LC50) (b) Mortality (LC50) (b)

OECD Test 201 OECD Test 202a OECD Test 203

(a) Concentration causing 50% inhibition of growth; (b) Concentration causing the death of 50% of the population tested. a Modified.

Table 2 Calculated LC50 and IC50 values with confidence intervals (whithin parentheses) for the three chemicals tested Species

Clofibric acid (mg/L)

Clofibrate (mg/L)

Diazepam (mg/L)

SDS (mg/L)

T. chuii

318.2 (318.06–318.38) 87.22 (84.12–90.43) 526.5 (526.41–526.68)

39.7 (39.44–39.91) 36.6 (36.32–36.98) 7.7 (7.41–7.98)

16.5 (16.45–16.47) 12.2 (11.99–12.32) 12.7 (12.57–12.85)

30.2 (30.10–30.29) 12.2 (12.14–12.24) 15.1 (14.67–15.51)

A. parthenogenetica G. holbrooki

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80 70

90 80 70 Mortality (%)

90

Growth inhibition (%)

100

Clofibric acid Clofibrate Diazepam SDS

100

60 50 40 30

60

417

Clofibric acid Clofibrate Diazepam SDS

50 40 30

20

20

10

10

0

1

10

100

1000

Concentrations (mg/l)

0

0.1

1

10

100

1000

Concentrations (m g/l)

Fig. 1. Toxicity curves of the four tested compounds for T. chuii. Fig. 3. Toxicity curves of the four tested compounds for G. holbrooki.

Clofibric acid Clofibrate Diazepam SDS

100 90

Mortality (% )

80 70 60 50 40 30 20 10 0

1

10

100

1000

Concentrations (m g/l)

Fig. 2. Toxicity curves of the four tested compounds for A. parthenogenetica.

clofibrate, and clofibric acid. Similarly, tests with A. parthenogenetica showed that diazepam was the most toxic compound, followed by SDS, clofibrate, and clofibric acid. However, LC50 values calculated for G. holbrooki (summarized in Table 2) showed a different pattern. Clofibrate was the most toxic compound, followed by diazepam, SDS, and clofibric acid. Toxicity curves for all organisms exhibit a clear pattern of concentration dependence (Figs. 1–3).

4. Discussion An agreement of results was observed for all the tested species. The obtained patterns showed that clofibric acid was the least toxic compound for the three species, and highest toxicity was generally associated to diazepam (Figs. 1–3). Sensitivity in our research was also a parameter taken into account, and the three different test organisms showed distinct thresholds of

toxicity. In our study, Artemia revealed a comparative high sensitivity to clofibric acid, which may indicate a higher impact over environmentally exposed primary consumers. On the other hand, a highly refractory response was observed after exposure of the mosquitofish to this substance, which is reflected in the calculated LC50. In this study, clofibrate was considered to be moderately toxic to A. parthenogenetica and T. chuii and much more toxic to G. holbrooki. Clofibrate is not mentioned in the literature as a persistent and welldispersed compound, since it is readily metabolized after intake. Elimination of clofibrate occurs mainly via excretion of glucuronides of the acidic metabolite clofibric acid. The end of the therapeutic use of clofibrate in the European Union and in the United States of America contributed to a decrease in the number of studies concerning its disposal. Clofibrate concentrations are usually below the detection limits of the methodologies employed in monitoring programs of contamination of the aquatic environment by pharmaceutical companies. Given the order of magnitude of the calculated LC50’s, we can conclude that acute effects of clofibrate are not expected to occur, even for the worst scenarios of contamination. The presence of clofibrate-related compounds in the wild is mainly due to clofibric acid, as a consequence of the use of other fibrates as lipid regulators in human therapeutics. Comparisons between data related to clofibrate and clofibric acid showed a distinct expression of toxicity. Clofibric acid toxicity was a priori expected to be of lower magnitude due to metabolic transformation following the therapeutic administration and the pharmacological effects in humans. Much higher toxicity of the parent compound (clofibrate) was verified after acute exposure, since clofibrate was roughly 65 times more toxic than its metabolite (for the case of G. holbrooki).

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Similar patterns were found for T. chuii and A. parthenogenetica, but without such order of magnitude. Clofibric acid has been considered a nonhazardous (though environmentally persistent) compound, which is relatively harmless in the concentrations that have been found in the environment: our results are in agreement with earlier studies by different researchers (Henschel et al., 1997; Ferrari et al., 2003), simultaneously using different test organisms from various organizational and trophic levels. These authors showed a consistency in the lack of acute toxic effects of clofibric acid: Ferrari et al. (2003) reported acute ecotoxicity EC50 end points higher than 200 mg/L for clofibric acid (ceriodaphnid and daphnid tests). Henschel et al. (1997) reported EC50 values for Daphnia, fish embryo pulse rate, and ciliate proliferation around 100 mg/L; the same study also showed that cell cultures, fish embryos, and algae were sensitive to clofibric acid with EC50 values of 14, 86, and 89 mg/L, respectively. Nevertheless, the presence of clofibric acid in aquatic environments may not be absolutely negligible due to the possibility of endocrine disruption activity (through interference with cholesterol synthesis, Pfluger and Dietrich, 2001), which is particularly important with regard to long-term effects. Another factor of interest with regard to clofibric acid ecotoxicity is the possibility of toxicological interactions with residues of different classes of compounds. Clofibric acid has been shown to produce a synergistic toxic effect in the presence of other drugs, such as carbamazepine (Cleuvers, 2003). Acute toxicity following SDS exposure was comparatively high, and agreement between results obtained with different species was also verified (obtained LC50s and IC50 did not greatly differ from species to species). Again, sensitivity was higher when Artemia was used as the test organism. An extensive review concerning the effects of detergents was done by Cserha´ti et al. (2002), which focused on the biological effects of this class of compounds in the environment. SDS was referred as one of the most toxic compounds, even at concentrations of 50 ppm, for cyanobacterial species. Its presence at higher concentrations impaired the development of Lemna minor L. Earlier research data, referenced in the same work, involved the calculation of EC50’s for several crustacean species. This work showed different orders of sensitivity for SDS, but values were similar to those obtained in our study for A. parthenogenetica. High acute toxicity of SDS can be responsible for harmful effects on aquatic organisms, especially under the direct influence of large human populations, since SDS is widely used in domestic detergents and cosmetic products. On the other hand, effluents draining from health-related facilities are complex mixtures, frequently carrying large amounts of distinct biologically active components (Ku¨mmerer, 2001), including several detergents and disinfectants. The author states that the

efficacy of removal of these compounds in sewage treatment plants is reduced due to interactions among components of the complex sewage mixtures, resulting in high amounts being released into the environment. Even considering the possibility of biodegradation of SDS in the wild, the constant entry of this compound into the ecosystems can allow considerable levels of contamination. Alkyl sulfate surfactants are the most common components of domestic and industrial wastewaters and count among the most widely disseminated xenobiotics in streams and aquatic environments (Jer´ a´bkova´ et al., 1999). High concentrations (0.2–10 mg/L) of SDS were reported in irrigation fields loaded with wastewater (Dizer, 1990). Results such as the obtained LC50 and IC50 values show that acute toxicity is not an unlikely phenomenon and populations under the influence of heavily polluted water streams are likely to be targets for the exertion of lethal effects. Acute toxicity after diazepam exposure revealed that this is not a negligible compound with regard to toxicological effects. All three model species were similar in their responses to diazepam, with values ranging from an IC50 of 16.46 mg/L for T. chuii to an LC50 of 12.16 mg/ L for A. parthenogenetica. Actual concentrations found in aquatic ecosystems are far below the ones tested. Zuccato et al. (2000) reported values ranging from 0.7 to 1.2 ng/L in River Po (Italy). However, exposure to sublethal levels of diazepam may also be of concern, since it has been shown to induce aneuploidy, a severe condition that can generally result in birth defects, pregnancy wastage, and cancer (Aardema et al., 1998). The suitability of different test organisms for screening purposes in aquatic toxicology can be questioned. In this study, A. parthenogenetica seemed to be the most responsive species. These results, along with the simple and inexpensive routine procedures needed to maintain cultures of this crustacean, allow us to state that laboratory-reared A. parthenogenetica is a suitable test organism in ecotoxicological screening assays. Nevertheless, Artemia specimens are often considered a somewhat insensitive organism in ecotoxicological studies (Nascimento et al., 2000; Arau´jo and Nascimento, 1999). Additionally, Artemia inhabits only hypersaline environments, thus reducing the overall applicability of Artemia-based bioassays, since they cannot be considered a representative species for all saline environments. G. holbrooki was also a sensitive species but revealed high refractory response toward clofibric acid. T. chuii revealed an overall diminished responsiveness, with calculated IC50’s generally above the LC50’s calculated for the other species used in this study. The abovementioned findings, related to different magnitudes of toxic response and distinct sensitivities toward the mentioned compounds, underline the need for integrative evaluations of toxicity, with simultaneous use of different organisms, representative of different trophic levels.

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Acknowledgments The present work was partially funded by ‘‘Fundac- a˜o para a Cieˆncia e a Tecnologia’’ (Grant SFRH/BD/866/ 2000 and project ‘‘CONTROL’’: PDCTM/PP/MAR/ 15266/1999). We gratefully acknowledge Dr. Jonathan Wilson for his dedicated effort and valuable contributions during the preparation and revision of the present paper. References Aardema, M.J., Albertini, S., Arni, P., Henderson, L.M., KirschVolders, M., Mackay, J.M., Sarrif, A.M., Stringer, D.A., Taalman, R.D.F., 1998. Aneuploidy: a report of an ECETOC task force. Mutat. Res. 410, 3–79. Andreozzi, R., Raffaele, M., Nicklas, P., 2003. Pharmaceuticals in STP effluents and their solar photodegradation in aquatic environment. Chemosphere 50, 1319–1330. Arau´jo, M.M.S., Nascimento, I.A., 1999. Marine ecotoxicological tests: analysis of Sensitivity Responses. Ecotoxicol. Environ. Rest. 2 (1), 41–47. ASTM, 1980. Standard practice for conducting acute toxicity tests with fishes, macroinvertebrates and amphibians. Report E-790-80, American Society for Testing and Materials. Baird, D.J., Soares, A.M.V.M., Girling, A., Barber, I., Bradley, M.C., Calow, P., 1989. The long-term maintenance of Daphnia magna Straus for use in ecotoxicity tests, problems and prospects. In: Lokke, H., Tyle, H., Bro-Rasmussen, F. (Eds.), First European Conference on Ecotoxicology, Copenhagen, Conference Organising Committee, Lingby, Denmark, pp. 144–148 Boyd, G.R., Reemtsma, H., Grimm, D.A., Mitra, S., 2003. Pharmaceuticals and personal care products (PPCPs) in surface and treated waters of Louisiana, USA and Ontario, Canada. Total Environ. 311, 135–149. Buser, H.-R., Mu¨ser, M.D., Theobald, N., 1998. Occurrence of the pharmaceutical drug clofibric acid and the herbicide mecocrop in various Swiss lakes and in the North Sea. Environ. Sci. Technol. 32, 188–192. Cleuvers, M., 2003. Aquatic ecotoxicity of pharmaceuticals including the assessment of combination effects. Toxicol. Lett. 142, 185–194. Cserha´ti, T., Forga´cs, E., Oros, G., 2002. Biological activity and environmental impact of anionic surfactants. Environ. Int. 28, 337–348. Daughton, C.G., Ternes, T.A., 1999. Pharmaceuticals and personal care products in the environment: agents of subtle change? Environ. Health Perspect. 107, 907–938. Dizer, H., 1990. Influence of anionic detergents SDS and alkylbenzolsulfonate on adsorption and transport behavior of several enteretropic viruses in soil under stimulated conditions. Zentralbl. Hyg. Umweltmed. 190, 257–274. Fa´bregas, J., Otero, A., Morales, E., Cordero, B., Patin˜o, M., 1996. Tetraselmis suecica cultured in different nutrient concentrations varies in nutritional value to Artemia. Aquaculture 143 (2), 197–204. Ferrari, B., Paxe´us, N., Lo Giudice, R., Pollio, A., 2003. Ecotoxicological impact of pharmaceuticals found in treated wastewaters: study of carbamazepine, clofibric acid, and diclofenac. Ecotoxicol. Environ. Saf. 55, 359–370. Ferreira, J.G., Simas, T., Nobre, A., Silva, M.C., Shiffereggen, K., Lencart-Silva, J., 2003. Identification of sensitive areas and vulnerable zones in transitional and coastal Portuguese systems – Application of the United States National Estuarine

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Eutrophication Assessment to the Minho, Lima, Douro, Ria de Aveiro, Mondego, Tagus, Sado, Mira, Ria Formosa and Guadiana Systems. Ed. by Instituto da A´gua and Institute of Marine Research, Portugal. Finney, D.J., 1971. Probit Analysis, third ed. Cambridge Press, New York, NY 668pp. Halling-Sørensen, S., Nors Nielsen, S., Lanzky, P.F., Ingerslev, F., Holten Lu¨tzhøft, H.C., Jo¨rgensen, S.E., 1998. Occurrence, fate and effects of pharmaceutical substances in the environment—a review. Chemosphere 36, 357–393. Heberer, T., 2002. Occurrence, fate, and removal of pharmaceutical residues in the aquatic environment: a review of recent research data. Toxicol. Lett. 131, 5–17. Henschel, K.P., Wenzel, A., Diedrich, M., Fliedner, A., 1997. Environmental Hazard Assessment of Pharmaceuticals. Regul. Toxicol. Pharmacol. 25, 220–225. Jer´ a´bkova´, H., Kra´lova´, B., Na´hlı´ k, J., 1999. Biofilm of Pseudomonas C12B on glass support as catalytic agent for continuous SDS removal. Int. Biodeterior. Biodegradat. 44, 233–241. Jones, O.A.H., Voulvoulis, N., Lester, J.N., 2002. Aquatic environmental assessment of the top 25 English prescription pharmaceuticals. Water Res. 36, 5013–5022. Koutsouba, V., Heberer, T., Fuhrmann, B., Schmidt-Baumler, K., Tsipi, D., Hiskia, A., 2003. Determination of polar pharmaceuticals in sewage water of Greece by gas chromatography–mass spectrometry. Chemosphere 51, 69–75. Ku¨mmerer, K., 2001. Drugs in the environment: emission of drugs, diagnostic aids and disinfectants into wastewater by hospitals in relation to other sources—a review. Chemosphere 45, 957–969. Miao, X.-S., Koenig, B.G., Metcalfe, C.D., 2002. Analysis of acidic drugs in the effluents of sewage treatment plants using liquid chromatography–electrospray ionization tandem mass spectrometry. J. Chromatogr. A 952, 139–147. Mohler, H., Fritschy, J.M., Benke, D., Benson, J., Rudolph, U., Lu¨scher, B., 1996. In: Tanaka, C., Bowery, N.G. (Eds.), GABA: Receptors, Transporters and Metabolism. Birkhae¨user Verlag, Basel, pp. 157–171. Nascimento, A., Smith, D.H., Pereira, S.A., Sampaio de Arau´jo, M.M., Silva, M.A., Mariani, A.M., 2000. Integration of varying responses of different organisms to water and sediment quality at sites impacted and not impacted by the petroleum industry. Aquat. Ecosyst. Health Manage. 3, 449–458. OECD, 1984a. Test 201: alga, growth inhibition test. OECD Guidelines Testing Chem. 1(2), 1–14. OECD, 1984b. Test No. 203: Fish, Acute Toxicity Test. OECD Guidelines Testing Chem. 1(2), 1–9. Pfluger, P., Dietrich, D.R., 2001. Pharmaceuticals in the environment—an overview and principle considerations. In: Ku¨mmerer, K. (Ed.), Pharmaceuticals in the Environment. Springer, Heidelberg, Germany, pp. 11–17. Sirisattha, S., Momose, Y., Kitagawa, E., Iwahashi, H., 2004. Toxicity of anionic detergents determined by Saccharomyces cerevisiae microarray analysis. Water Res. 38, 61–70. Ternes, T.A., 1998. Occurrence of drugs in German sewage treatment, plants and rivers. Water Res. 32 (11), 3245–3260. Vanhaecke, P., Persoone, G., Claus, C., Sorgeloos, P., 1981. Proposal for a short-term toxicity test with Artemia nauplii. Ecotoxicol. Environ. Saf. 5, 328–387. Weigel, S., Kuhlmann, J., Huhnerfuss, H., 2002. Drugs and personal care products as ubiquitous pollutants: occurrence and distribution of clofibric acid, caffeine and DEET in the North Sea. Sci. Total Environ. 295, 131–141. Zuccato, E., Calamari, D., Natangelo, M., Fanelli, R., 2000. Presence of therapeutic drugs in the environment. Lancet 355, 1789–1790.