Mixture Toxicity Of The Anti-inflammatory Drugs Diclofenac .

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ARTICLE IN PRESSEcotoxicology and Environmental Safety 59 (2004) 309–315Mixture toxicity of the anti-inflammatory drugs diclofenac,ibuprofen, naproxen, and acetylsalicylic acidMichael Cleuvers Department of General Biology, Aachen University of Technology, KopernikusstraX e 16, D-52056 Aachen, GermanyReceived 15 May 2003; received in revised form 8 July 2003; accepted 19 July 2003AbstractThe ecotoxicity of the nonsteroidal anti-inflammatory drugs (NSAIDs) diclofenac, ibuprofen, naproxen, and acetylsalicylic acid(ASA) has been evaluated using acute Daphnia and algal tests. Toxicities were relatively low, with half-maximal effectiveconcentration (EC50) values obtained using Daphnia in the range from 68 to 166 mg L 1 and from 72 to 626 mg L 1 in the algal test.Acute effects of these substances seem to be quite improbable. The quantitative structure–activity relationships (QSAR) approachshowed that all substances act by nonpolar narcosis; thus, the higher the n-octanol/water partitioning coefficient (log Kow ) of thesubstances, the higher is their toxicity. Mixture toxicity of the compounds could be accurately predicted using the concept ofconcentration addition. Toxicity of the mixture was considerable, even at concentrations at which the single substances showed noor only very slight effects, with some deviations in the Daphnia test, which could be explained by incompatibility of the very steepdose–response curves and the probit analysis of the data. Because pharmaceuticals in the aquatic environment occur usually asmixtures, an accurate prediction of the mixture toxicity is indispensable for environmental risk assessment.r 2003 Elsevier Inc. All rights reserved.Keywords: Daphnia; Desmodesmus; Pharmaceuticals; Mixture toxicity; Concentration addition; QSAR; Narcosis1. IntroductionDuring the last several years many studies have shownpharmaceutical compounds to be present in measurableconcentrations in the aquatic environment (Kümmerer,2001; Daughton and Jones-Lepp, 2001; Daughton andTernes, 1999; Heberer, 2002; Halling-S rensen et al.,1998). Among the detected substances, nonsteroidalanti-inflammatory drugs (NSAIDs), including compounds used as analgesics, belong to one of the mostimportant groups of pharmaceuticals worldwide, withan estimated annual production of several kilotons. InGermany, for example, 87.5 million prescriptions forthese substances were written in 2001, with a transactionvolume of Bh1130 million (Schwabe and Paffrath,2001). Additionally, some of these drugs are purchasedwithout prescription, so that actual consumption iscertainly even higher. As a result of this high applicationamount as well as the drugs’ pharmacokinetics (half-life,urinary and fecal excretion, metabolism, etc.), analgesics Fax: 49-241-8022602.E-mail address: cleuvers@bio2.rwth-aachen.de.0147-6513/ - see front matter r 2003 Elsevier Inc. All rights reserved.doi:10.1016/S0147-6513(03)00141-6and anti-inflammatory drugs can reach detectableconcentrations in the environment. Stumpf et al.(1996) identified diclofenac (p1.59 mg L 1), ibuprofen(p3.35 mg L 1), and ASA (1.51 mg L 1) in sewage, andlower concentrations (0.01–0.5 mg L 1) in river water. Invery low doses (1–6 ng L 1), diclofenac and ibuprofencould be detected even in drinking water. Ternes (1998;Ternes et al., 1998) reported concentrations of diclofenac, ibuprofen, naproxen, ASA, and other compounds,some 41 mg L 1 in wastewater treatment plants andagain in lower concentrations in surface waters. InGermany, diclofenac and ibuprofen, consumed inquantities of B75 and 180 tons per year, respectively(Ternes, 2001), have been particularly recognized asimportant contaminants of the water cycle. In a screening-study of waters in Berlin, Germany, Heberer et al.(1998) found that among the most prevalent NSAIDswere diclofenac, ibuprofen, and propyphenazon. Inlong-term monitoring investigations of sewage andsurface water samples, diclofenac was identified as oneof the most important pharmaceutically active compounds present in the water cycle (Heberer et al., 2002).It was found in groundwater samples (Heberer et al.,

ARTICLE IN PRESSM. Cleuvers / Ecotoxicology and Environmental Safety 59 (2004) 309–3153101997; Sacher et al., 2001) and sporadically even inraw or treated drinking water (Heberer et al., 2001a).Additional findings of diclofenac and ibuprofen havebeen reported, for example, from Swiss lakes and rivers(Buser et al., 1998, 1999), from Brazil (Stumpf et al.,1999), Spain (Farre et al., 2001), Greece (Heberer et al.,2001a), and the United States (Heberer et al., 2001b).In contrast to this large amount of analytical data,studies assessing the possible ecotoxicological effects ofdetected drug residues are sparse (Webb, 2001), andonly very few contain data about mixture toxicity(Cleuvers, 2002, 2003). These studies are very importantbecause drug residues found in the aquatic environmentusually occur as mixtures, not as single contaminants.Therefore, scientific assessment of risk to the aquaticenvironment should definitely consider this complexexposure situation. For this purpose, ecotoxicologistsuse concepts originally developed by pharmacologists inthe first half of the 20th century (Loewe and Muischnek,1926; Bliss, 1939) to predict the toxicity of mixtures. Inthe present study, the concept of concentration addition(Altenburger et al., 2000; Faust et al., 2001) was applied.Concentration addition is based on the idea of a similaraction of single compounds, which means in a strictsense that single substances should show the samespecific interaction with a molecular target site in theobserved test organism (Pöch, 1993). However, it hasbeen shown that the concept of concentration additionis also applicable to nonreactive, nonionized organicchemicals, which show no specific mode of action butwhose toxicity toward aquatic species is governed byhydrophobicity (Deneer et al., 1988; Van Loon et al.,1997). The nonspecific mode of action of such compounds is called narcosis or baseline toxicity (VanLeeuwen et al., 1992; Verhaar et al., 1992). The potencyof a chemical to induce narcosis is entirely dependent onits hydrophobicity, generally expressed by its log Kow :Mathematically, the concept of concentration addition for a mixture of n substances is described by(Berenbaum, 1985)nXi¼1ci¼ 1;EC xið1Þwhere ci represents the individual concentrations of thesingle substances present in a mixture with a total effectof x%, and EC xi are those concentrations of the singlesubstances that would alone cause the same effect x asobserved for the mixture. According to Eq. (1), theeffect of the mixture remains constant when onecomponent is replaced by an equal fraction of anequally effective concentration of another. As animportant point, concentration addition means thatsubstances applied at less than their individual ‘‘noobservable effect concentrations’’ (NOECs) can nevertheless contribute to the total mixture effect.The aim of this study was to create a broader basis forevaluating the ecotoxicological relevance and mode ofaction of mixtures of pharmaceutical compounds. Forthat purpose, the selected NSAIDs were applied inbiotests with the green alga Desmodesmus subspicatus(Chodat) Hegewald et Schmidt and the water fleaDaphnia magna Strauss.2. Materials and methods2.1. Test substancesAcetylsalicylic acid (2-(acetyloxy)benzoic acid; CASNo. 50-78-2) and the sodium salts of diclofenac (2-[(2,6dichlorophenyl)amino]benzeneacetic acid; CAS No.15307-79-6), ibuprofen (a-methyl-4-[isobutyl]phenylacetic acid; CAS No. 31121-93-4), and naproxen ((S)-6methoxy-a-methyl-2-naphtaleneacetic acid; CAS No.26159-34-2) were supplied in analytical grade bySigma-Aldrich (Taufkirchen, Germany). In tests withsingle compounds, substances were applied in sixconcentrations (1, 3.2, 10, 32, 100, and 320 mg L 1).For assessing mixture toxicity, a quarter of the calculated effect concentrations (EC5/4, EC10/4, EC20/4,EC50/4, and EC80/4) of each substance was used. If thesubstances follow the concept of concentration addition,according to Eq. (1) the combination effect of themixtures should add up to a total effect of B5%,10%, 20%, 50%, and 80%, respectively.2.2. Daphnia acute immobilization testsDaphnia tests were conducted following the EuropeanGuideline (Commission of the European Communities,1992) using the water flea D. magna.Daphnids were bred in ADaM, a culture mediumimitating natural freshwater (Klüttgen et al., 1994).Experiments were conducted at temperatures of2071 C and photoperiods of 16 h light/8 h dark(B20 mE s 1 m 2). As controls, 20 daphnids youngerthan 24 h were used, and each treatment was subdividedin four replicates, each containing 5 daphnids. Culturevolume was 50 mL. Immobility (the endpoint for effectcalculation) was observed after 24 and 48 h.2.3. Algal growth inhibition testsAlgae tests were conducted following the EuropeanGuideline (Commission of the European Communities,1993). I used the planktonic chlorococcale green alga D.subspicatus (formerly Scenedesmus subspicatus Chodat,SAG 86.81 UTEX 2594), obtained from the SAGSammlung von Algenkulturen at the University ofGöttingen, Germany. Initial cell densities were adjustedat 104 cells mL 1 using a calibration curve of chlorophyll

ARTICLE IN PRESSM. Cleuvers / Ecotoxicology and Environmental Safety 59 (2004) 309–315fluorescence (excitation at 460 nm; emission at 685 nm)versus cell number and appropriate dilution of preculture. The culture medium was prepared according tothe protocol using deionized water and analytical-gradechemicals. Algae were incubated at 2372 C undercontinuous white light (120 mE s 1 m 2) and were keptin suspension by continuous shaking (B80 rpm). Resultswere quantified in terms of average growth ratescalculated from cell numbers based on measurementsof chlorophyll fluorescence.2.4. Analysis of the mode of actionTo determine the mode of action of the observedsubstances, I used QSAR (Verhaar et al., 1992; Lipnick,1995; Schüürmann and Markert, 1998) for nonpolarnarcotic chemicals proposed for D. magna (Verhaaret al., 1995) (Eq. (2)) and for the chlorophyte Pseudokirchneriella subcapitata (Van Leeuwen et al., 1992)(Eq. (3)), whose sensitivity is seen to be comparable toD. subspicatus (Blanck et al., 1984). If the EC50 ofthe tested substances is as high as or even higherthan predicted by the equations, the compounds are311considered to show no specific mode of action, but to actby narcosis onlyLog EC50 ½mol L 1 ¼ 0:95 log Kow 1:32;ð2ÞLog EC50 ½mol L 1 ¼ 1:00 log Kow 1:23:ð3Þ2.5. Statistical evaluationAll calculations were conducted using the softwareToxRat (ToxRat Solutions, Aachen, Germany). ECxvalues were computed using probit analysis (dose–response fitting: normal sigmoid, maximum-likelihoodregression). NOECs were determined using Dunnett’smultiple t-test procedure; a ¼ 0:05; one-sided smaller.3. Results3.1. Single-compound toxicityTables 1 and 2 show the individual-effect concentrations and 95% confidence limits of the applied substancesin the algal growth inhibition test and the acute DaphniaTable 1Effect concentrations and 95% confidence limits of the drugs tested in the algal testSubstanceEC5 (mg L 1)EC10 (mg L 1)EC20 (mg L 1)EC50 (mg L 1)EC80 (mg L )n.d.: Not determinableTable 2Effect concentrations and 95% confidence limits of the drugs tested in the acute Daphnia testSubstanceEC5 (mg L 1)EC10 (mg L 1)EC20 (mg L 1)EC50 (mg L 1)EC80 (mg L �73.9)88.1(72.8–106.6)135.2(100.4–182.1)n.d.: Not determinable

ARTICLE IN PRESSM. Cleuvers / Ecotoxicology and Environmental Safety 59 (2004) 309–315312test, respectively. The EU Directive 93/67/EEC (Commission of the European Communities, 1996) classifiessubstances according to the lowest measured EC50 valuein different risk classes. An EC50 o1 mg L 1 would entailthe classification, ‘‘very toxic to aquatic organisms’’; from1 through 10 mg L 1 ‘‘toxic to aquatic organisms’’, andfrom 11 through 100 mg L 1 ‘‘harmful to aquatic organisms’’. Substances with an EC504100 mg L 1 would notbe classified. On the basis of this scheme, the acutetoxicity of all tested substances is relatively low. Onlydiclofenac, with EC50 values of 68.0 mg L 1 in theDaphnia test and 71.9 mg L 1 in the algal test, andASA with an EC50 of 88.1 mg L 1 in the Daphnia testwould be classified as potentially harmful to aquaticorganisms. The lowest NOEC was 32 mg L 1 (ibuprofenand ASA in the algal test, naproxen in the Daphnia test;see Tables 4 and 5), and the lowest EC10 was 15.2 mg L 1(diclofenac in the Daphnia test; see Table 5).daphnids and algae. This is confirmed by a simpleQSAR approach using Eqs. (2) and (3), respectively. Allmeasured EC50 values are as high as or even higher thanthose predicted by the QSARs for narcosis, with theexception of that for ASA (Table 3).3.3. Mixture toxicityDrug concentrations applied in the mixtures areshown in Tables 4 and 5 in comparison to theirindividual NOECs for the Daphnia and the algal tests,respectively. In the algal test, mixture toxicity of theNSAIDs could be accurately predicted using the conceptof concentration addition (Fig. 1). The situation for theDaphnia test differed somewhat (Fig. 2). The first threetreatments (EC5, EC10, and EC20) showed no effect atThe high EC50 values obtained imply that no specificmode of action is responsible for the toxic effects onTable 3Measured EC50 values in comparison to those predicted in agreementwith the log Kow of the observed alicylic acidLog Kow4.43.53.31.22.3EC50 (mmol L 1)Predicted for narcosisMeasuredDaphniaAlgaeDaphnia Algae3.16 10 30.0230.0353.470.312.34 10 0.59—Inhibition of the average growth rate [%]1003.2. Mode of action and mixture toxicityMixture toxicity:PredictedMeasured80Single effects:DiclofenacIbuprofenNaproxenASA6040200EC5 /4EC10 /4EC20 /4EC50 /4EC80 /4Effect ConcentrationsFig. 1. Measured mixture toxicity of the NSAIDs tested as obtained inthe algal growth inhibition test in comparison to the individualtoxicities of the single compounds and the mixture toxicity predictedby the concept of concentration addition.Table 4Concentrations of the tested drugs applied in the mixture in the algal test in comparison to the individual NOECs of the single compoundsSubstanceEC5/4 (mg L 1)EC10/4 (mg L 1)EC20/4 (mg L 1)EC50/4 (mg L 1)EC80/4 (mg L 1)NOEC (mg L .1188.3241.929.7503210032Table 5Concentrations of the tested drugs applied in the mixture in the acute Daphnia test in comparison to the individual NOECs of the single compoundsSubstanceEC5/4 (mg L 1)EC10/4 (mg L 1)EC20/4 (mg L 1)EC50/4 (mg L 1)EC80/4 (mg L 1)NOEC (mg L 6.733.845753275

ARTICLE IN PRESSM. Cleuvers / Ecotoxicology and Environmental Safety 59 (2004) 309–315Immobilized daphnids [%]1008060Mixture toxicity:PredictedMeasuredSingle effects:DiclofenacIbuprofenNaproxenASA40200EC5 /4EC10 /4EC20 /4EC50 /4EC80 /4Effect ConcentrationsFig. 2. Measured mixture toxicity of the NSAIDs tested as obtained inthe acute Daphnia test in comparison to the individual toxicities of thesingle compounds and the mixture toxicity predicted by the concept ofconcentration addition.all, whereas mixture toxicity at the EC50 and the EC80doses were as high or even higher than that predicted byconcentration addition.4. DiscussionIn most cases, individual EC50 values of the testedcompounds were 4100 mg L 1. Therefore, based on thescheme used in the European Union, only diclofenacand ASA would be classified as potentially harmful toaquatic organisms. Regarding an effect threshold, thelowest NOEC was 32 mg L 1, which is 10,000- to100,000-fold higher than the highest detected concentration of that compound in the aquatic environment. Infact, the ECx approach is generally more sensitive fordetecting low effects; this is because, on the one hand,NOEC is dependent on actually selected test concentrations, and on the other hand, to compute the NOECeach treatment was separately compared to the control.In contrast, using an ECx approach, all data points areused to determine effect concentrations. However, eventhe lowest measured EC10 (diclofenac, 15.2 mg L 1 inthe Daphnia test) is B10,000-fold higher than concentrations detected in the field. Thus, with respect to theresults obtained with daphnids and algae, acute effectsof these substances are very unlikely. If we keep in mindthat the tested substances are pharmaceuticals, strongacute effects caused by a specific mechanism mayactually not be expected, because such an acute toxicitywould surely diminish the pharmaceutical benefit of thecompound.Therefore, considering the situation in the field withmostly very low concentrations of pharmaceuticals, wemust assume that generally chronic effects are more313likely than acute effects, and there may be other speciesthat are more sensitive to these contaminants. Forexample, an earlier study (Cleuvers, 2003) indicated thatthe EC50 values of tested NSAIDs obtained using theduckweed Lemna minor were 5- to 10-fold lower thanthose obtained using the acute Daphnia or algal test. Forthat reason it was proposed to conduct Lemna testsroutinely in addition to other standard tests (Cleuversand Ratte, 2002). Additionally, it may be useful to useadditional benthic organisms, like freshwater gastropods or oligochaetes, to improve the environmental riskassessment.All drugs tested in this study are NSAIDs and areconsidered to have the same mode of action in humans.They inhibit the cyclooxygenases, the key enzymescatalyzing prostaglandin biosynthesis, which is partiallyinvolved in the genesis of pain and inflammation (Vaneand Botting, 1998; Vane, 1971). This inhibition isresponsible for the analgesic and anti-inflammatoryeffect of the NSAIDs. Among the other functions ofprostaglandins is their ability to cause contractions oratony of muscles in different organs, with effects onblood pressure and circulation. In addition, someprostaglandins act to protect cells in the gastrointestinaltract, and their inhibition by NSAIDs with thepossibility of internal bleeding is one of the mostimportant side effects of these pharmaceuticals. Severalpublications have reported on the occurrence ofprostaglandins in nonmammalian vertebrates (Bundy,1985; Nomura, 1988) such as fish, amphibians, andbirds, as well as in invertebrates such as corals, sponges,coelenterates, mollusks, crustaceans, insects, and inmarine algae and higher plants, where they areconsidered to carry out different functions, for example,in defense mechanisms of plants. Unfortunately, noliterature is available about the occurrence of prostaglandins in daphnids or freshwater algae, so that it isunclear whether the test species used in this study couldserve as a model for a specific effect of NSAIDs.Therefore, additional biotests with vertebrates like fishcould be a useful tool to assess environmental risk. Thehigh measured EC50 values imply that no specific modeof action is responsible for the toxic effects found indaphnids and algae. This is confirmed by QSAR, usingEqs. (2) and (3), respectively. One problem is that thelog Kow values of ibuprofen sodium and naproxensodium are not available in the literature. To allow acomparison

studies assessing the possible ecotoxicological effects of detected drug residues are sparse (Webb, 2001), and only very few contain data about mixture toxicity (Cleuvers, 2002, 2003). These studies are very important because drug residues found in the aquatic environment usually occur as mixtures, not as single contaminants.

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